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Durieux Simon-Applied Microbiology-9780792368588 PDF
Durieux Simon-Applied Microbiology-9780792368588 PDF
VOLUME 2
FOCUS ON BIOTECHNOLOGY
Volume 2
Series Editors
MARCEL HOFMAN
Centre for Veterinary and Agrochemical Research, Tervuren, Belgium
JOZEF ANNÉ
Rega Institute, University of Leuven, Belgium
Volume Editors
ALAIN DURIEUX and JEAN-PAUL SIMON
Institut Meurice, Brussels, Belgium
COLOPHON
Focus on Biotechnology is an open-ended series of reference volumes produced for
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de Chimie Industrielle a.s.b.l.
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Biotechnology. ECB9 has been supported by the Commission of the European
Communities, the General Directorate for Technology, Research and Energy of the
Wallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels Capital
Region.
Applied Microbiology
Volume 2
Edited by
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v
IN MEMORY
On September 25th, 1999, Professor Charles A. Masschelein died here in Belgium. To
the end, Professor Masschelein remained active in the brewing and biotechnology
industries and had, in fact, just recently returned from visiting a number of African
breweries.
That day, I lost a friend. Moreover, at his moment of passing, I understood that I
had lost my mentor in the field of applied microbiology and the world had lost one of
the truly great scientists of the 20th century.
Twenty years before, I joined Masschelein’s department of brewing science at the
Institut Meurice, CERIA. Masschelein was a leader, a dedicated spirit to R&D with an
acutely inquiring mind. He completely devoted himself to the training of people and
transfer of technology from the academic to the industrial world.
Since 1954, C.A. Masschelein’s vision, as a Belgian scientist, was to understand the
physiology of micro-organisms in their industrial environment. He taught this idea at
all levels ; to large numbers of students in Europe and internationally, but also to a large
number of engineers already in charge of fermentation plants around the world.
Masschelein’s major topic has been the brewery. In this sector he was the defender
of a strict understanding of biochemical and microbiological principles balancing this
with both the reality and progression of the industrial method and the traditional quality
of the final product he knew. For more than 35 years, he held a number of responsible
positions in the European Brewery Convention as well as contributed greatly to brewing
science worldwide.
Biotransformation was always a major driving force for Masschelein. He was able
to introduce this philosophy to R&D in the 1960s and apply the results to processes in
order to use micro-organisms to produce industrially significant materials. For this
reason too, C.A. Masschelein has always been capable of combining new developments
in molecular biology on one hand to new materials and processes on the other. This
challenge was such a reality for him that, when he retired as professor at CERIA, he was
responsible for setting up a company to develop new continuous brewing processes.
In July, 1999, during the Ninth European Congress of Biotechnology, C.A.
Masschelein gave his last lecture delivering his idea concerning the future of applied
microbiology in correlation with new generation of continuous fermentation processes.
As with all great scientists, Masschelein’s work will be remembered and referred to for
many decades in the future.
J.P. Simon
1
TABLE OF CONTENTS
IN MEMORY ................................................................................. 1
3
3.2 The influence of lactic acid bacteria starters on wine quality and their
selection ........................................................................................................... 41
3.3 Efficiency of malolactic starters ................................................................ 42
4. The future of starters for winemaking .............................................................. 43
5. Conclusion ........................................................................................................ 45
References ............................................................................................................ 45
5
References .......................................................................................................... 107
7
PART 5 - NOVEL APPLICATIONS .......................................... 163
8
1.3 Incorporation of radioactivity from labelled n-Hexadecane into mycelia .
....................................................................................................................... 186
1.4 Fluorescence measurements ..................................................................... 186
1.5 Analysis of fatty acids .............................................................................. 187
1.6 Investigations with GTP analogues .......................................................... 187
2. Results and discussion .................................................................................... 187
3. Conclusion ...................................................................................................... 190
References .......................................................................................................... 190
9
BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION ........... 239
Cahill, S.M., Upton, M.E., and McLoughlin. A.J. .................................................. 239
Abstract .............................................................................................................. 239
1. Introduction .................................................................................................... 240
2. Meat preservation ........................................................................................... 241
2.1 Biological fermentation ........................................................................... 241
2.2 Chemical acidification ............................................................................. 243
3. The application of encapsulation technology to meat preservation ................ 243
3.1. The application of encapsulation technology to a microbial fermentation
....................................................................................................................... 243
3.1.1. Encapsulation matrices and the encapsulation process ................... 244
3.1.2. The benefits of meat starter culture encapsulation .......................... 246
3.1.3. Commercial applications ................................................................. 247
3.2. The application of encapsulation technology to chemical acidification . 248
3.2.1. Encapsulation matrices and the encapsulation process ................... 248
3.2.2. The benefits of acidulant encapsulation .......................................... 249
3.2.3 Commercial availability ................................................................... 249
4. Control of emerging pathogens ...................................................................... 250
5. The application of encapsulation technology to bacteriocin delivery ............. 251
5.1 Bacteriocins ............................................................................................. 251
5.2 Nisin ......................................................................................................... 251
5.2.1 Encapsulation of nisin ...................................................................... 252
6. Conclusions and future work .......................................................................... 261
References .......................................................................................................... 261
10
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED
FOODS
Abstract
1. Introduction
Mould fermented foods play an important role, especially in Asian countries where the
production process for many foods include a fungal fermentation. Fungal species which
are found in Asian type foods belong to different genera: Aspergillus oryzae, A.
glaucus, Rhizopus chinensis, Neurospora intermedia or Monascus purpureus
(Hesseltine, 1983). In contrast to this situation in European countries mainly three
species from the genus Penicillium are used for the production of fermented foods, in
particular Penicillium camemberti for white cheeses, P. roqueforti for blue cheeses and
P. nalgiovense for mould fermented meat products. It has to be mentioned that P.
chrysogenum also can often be isolated from fermented meat products and is sometimes
used as a starter culture for meat products in some countries (Leistner, 1986).
13
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 13-29.
©2001 Kluwer Academic Publishers. Printed in the Netherlands.
ROLF GEISEN AND PAUL FARBER
Fungal starter cultures extensively contribute to the flavour and texture formation of the
mould ripened food product. The most important enzymatic activities in this respect are
proteases and lipases (Kinsella and Hwang, 1976). Proteases degrade proteins to
flavour active peptides or amino acids, whereas lipases hydrolyse triacyl glycerins to
free fatty acids and glycerol. The fatty acids can be converted into methylketones by
fungal lipoxygenases that also contribute to the flavour formation. These compounds are
especially important in the ripening of blue cheese (Fox and Law, 1991). But not only
these enzymatic processes play a role in flavour development. It has been shown by
Jacobsen and Hinrichsen (1997) that also volatile compounds produced by the fungus
contribute to this process. Another important feature of the fungal starter culture is the
competition against undesired microorganisms, which would otherwise cover the
surface of the fermented product. These microorganisms are mainly other fungal species
that could lead to a discoloration of the product or the production of undesired
secondary metabolites. In addition the competition of fungal starter cultures agai nst
undesired pathogenic or toxinogenic bacteria is of interest for microbiological safe
production of mould fermented foods. During the fermentation process the pH of the
product raises due to the metabolic activity of the fungus. Lactic acid that may be
produced by accompanying lactic acid bacteria is degraded and also proteins may be
hydrolysed to ammonia by the fungal starter culture. Both processes lead to an increase
in pH during the fermentation which enables the growth of pathogenic or toxinogenic
bacteria like Listeria monocytogenes or Staphylococcus aureus. Of course starter
cultures which would suppress the growth of these undesired bacteria would be
advantageous for improved food safety.
Before a fungal strain can be used as a starter culture it has to fulfil several
requirements (Tab. 1).
Table 1. Requirements for a fungal starter culture
----------------------------------------------------
It should not produce a mycotoxin
It should not produce another undesired secondary metabolite
It should produce the desired flavour changes in the product
It should be adapted to the food product
It should compete against undesired moulds
It should have antibacterial activity against pathogens
----------------------------------------------------
Not all the currently used strains fulfil all these requirements. Many species of the genus
Penicillium are able to produce toxic secondary metabolites, the mycotoxins. Even
some species currently used, as starter cultures are able to produce mycotoxins or other
undesired secondary metabolites. For this reason it is important that useful methods
14
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
exist, which enables the screening of strains for particular characteristics and that fungal
strains can be optimised according particular requirements.
2. Penicillium nalgiovense
P. nalgiovense was first described by Laxa (1932). The type culture was isolated from a
bohemian cheese. Strains of these species are mainly found on meat and meat products
(Leistner et al, 1989). But they can also be isolated as spoilage organisms from different
cheeses (Lund et al. 1995). P. nalgiovense is not able to produce any of the known
mycotoxins, according to Frisvad (1988). Until recently P. nalgiovense was regarded as
a fungal species with poor capability of producing secondary metabolites.
Because of morphological similarities P. nalgiovense has long been regarded as a
domesticated form of P. chrysogenum, the species which is industrially used for
penicillin production (Samson and van Reenen-Hoekstra, 1988). In a recent publication
Banke et al. reclassified the P. chrysogenum complex (Banke et al, 1997). According to
their results, based on isozyme analysis, the P. chrysogenum complex was separated
into 4 distinct species: P. chrysogenum, P. dipodomyis, P. flavigenum and P.
nalgiovense. Molecular data confirmed the high relationship between P. nalgiovense
and P. chrysogenum (Geisen, 1995). With several strains of P. nalgiovense a RAPD
analysis was performed. This type of analysis is a molecular typing technique, which
was developed by Williams et al. (1990). This technique is a modified PCR approach in
which an arbitrary primer is used. It generates specific patterns if always the same
reaction conditions are used. These patterns are related to the genotype of the analysed
strain. Differences in the pattern reflects differences in the genotype and give
information about the relatedness between different strains. Depending on the primer
used the patterns can be species- (Guthrie et al., 1992), subspecies- (Hamelin et al.,
1993) or even strain specific (Bidouchka et al. 1994). This approach revealed that P.
nalgiovense is a homogenous species, irrespective of the origin of the strains analysed
(Figure 1). The figure shows that all the patterns generated with a random primer were
identical. The same result was derived with a second primer. The only difference in the
pattern exhibited one strain (BFE 61). This was due with all primers used, indicating
that it might be a misclassified strain
To compare P. nalgiovense and P. chrysogenum at the molecular level, the same
approach was carried out with both species. Figure 2 shows the results of this analysis.
They clearly demonstrate, that P. nalgiovense and P. chrysogenum are two distinct
species, because the RAPD patterns of both species are different, however a subsequent
hybridisation analysis, in which the labelled RAPD products of P. nalgiovense were
hybridised to the RAPD-PCR products of P. chrysogenum revealed, that both species
has homologies at the nucleotide sequence level, which indicate the close relationship
between the species.
15
ROLF GEISEN AND PAUL FARBER
16
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
Another aspect also indicates the close relationship between both species. P.
chrysogenum is the industrial producer of penicillin and for P. nalgiovense the
capability to produce also penicillin has been described independently by Färber and
Geisen (1994) and Andersen and Frisvad (1994). All strains analysed were able to
produce this secondary metabolite. It was demonstrated by PCR (polymerase chain
reaction), that obviously the genes for penicillin production are very similar at the
nucleotide level between P. chrysogenum and P. nalgiovense. With primers deduced
from the sequences of the three penicillin biosynthetic genes of P. chrysogenum it was
possible to amplify the respective PCR fragments from P. nalgiovense. The PCR
products of both species were identical in length, indicating homology between the
genes (Figure 3).
Subsequent sequence analyses of the pcbC gene, the gene coding for isopenicillin
synthetase, revealed that both genes are 94% identical. It is known from the genes of P.
chrysogenum that they are clustered and all located on chromosome 4, the largest
chromosome (Fiero et al. 1993). To analyse this situation in P. nalgiovense an
electrophoretic karyotyping was carried out. This analysis revealed, that like P.
chrysogenum, P. nalgiovense has also 4 chromosomes of high molecular weight, but
17
ROLF GEISEN AND PAUL FARBER
18
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
There are two important points that must be fulfilled before this method can be used: 1.
a transformation system for the particular organism must be established to be able to
introduce DNA into the microorganism and 2. the gene to be targeted must be available.
If this is the case a central fragment of that gene is cloned into a transformation vector.
This plasmid is than transformed into the fungal strain. The resulting transformants are
screened for activity of the undesired gene. It is highly probable, that some of the
transformants are no longer active in the undesired feature. In that cases the
transforming DNA had integrated into the undesired gene by homologous
recombination. This integration leads to a situation in which the target gene is
duplicated and separated by vector sequences. However both copies of the gene are
inactive, as the first copy is truncated at the 3' end and the second copy is truncated at
the 5' end. This approach was adapted to P. nalgiovense. A transformation system for
that microorganism was available (Geisen, 1989). The central fragment of the penDC
gene was cloned into the transforming vector and introduced into P. nalgiovense. From
the resulting transformants several could be identified, which were no longer able to
produce penicillin. Figure 5 shows the results of the experiment.
19
ROLF GEISEN AND PAUL FARBER
20
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
These results indicate that optimised strains in contrast to the wild type are able to
suppress the growth of undesired bacteria, which may occur in mould fermented foods.
It was mentioned above, that especially proteases and lipases play a role in the flavour
formation of the fermented product. The change of proteolytic activity of strains either
by gene amplification or gene inactivation by the gene disruption approach can lead to
strains isogenic to the wild type, but with the differences in the proteolytic activity. This
in term can lead to strains with different fermentation properties. It is known from P.
roqueforti, that this species has different proteolytic systems. It produces extra and
21
ROLF GEISEN AND PAUL FARBER
intracellular proteases. (Stepaniak et al. 1980). The relative activity between the
particular proteases can differ from strain to strain (Paquet and Gripon, 1980). P.
roqueforti shows its highest proteolytic activity at acidic pH, which is in agreement with
our results, as obviously the acidic protease of P. nalgiovense is the main protease of P.
nalgiovense. From P. roqueforti an aspartic proteinase (Zevaco et al. 1973) and an
metallo proteinase (Gripon and Hermier, 1974) have been characterised. Much less is
known about the proteolytic system of P. nalgiovense. In an attempt to isolate genes for
proteases a gene bank of P. nalgiovense was constructed in an expression plasmid, For
this purpose the chromosomal DNA of P. nalgiovense have been isolated partially,
digested with an restriction enzyme and fragments of 4 - 5 kb were cloned into an E.
coli expression vector. Highly competent cells of E. coli were transformed with the
plasmid gene bank of P. nalgiovense. About 20.000 independent clones of E. coli, each
carrying a particular plasmid were screened for proteolytic activity by growing on
medium containing skim milk. Among these clones one could be identified, which
exhibited proteolytic activity. The detailed analysis of the clone revealed that it
contained a chromosomal DNA fragment from P. nalgiovense of 1.4 kb, which
obviously coded for a protease gene. According to Southern blot experiments the gene
occurs only in a single copy in the genome of P. nalgiovense. To verify that the cloned
fragment indeed codes for a protease gene from P. nalgiovense the plasmid was
modified and reintroduced into the P. nalgiovense wild type strain. From the resulting
transformants one could be identified with highly reduced proteolytic activity (Fig. 7)
Apparently in this strain a gene replacement has taken place. Subsequent Southern Blot
analysis support this possibility (Geisen, 1995). An analysis of the pH optimum of the
cloned protease, by comparing the proteolytic activity of the different strains on media
22
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
with skim milk and different pH revealed, that the cloned protease has its optimum at
acidic pH.
The described analysis demonstrated that a protease gene of P. nalgiovense has been
cloned, and that it can be used for influencing the proteolytic activity of a given strain,
either by gene replacement or by gene amplification.
3. Penicillium camemberti
P. camemberti was first described by Thom (1906). Some synonyms exist for this
species like P. caseicola, P. album, P. candidum or P. rogeri. P. camemberti is nearly
exclusively found in cheeses and cheese factories. This species is thought to be a
domesticated form of P. commune (Pitt et al. 1986). As already discussed above, one
important prerequisite for a fungal starter culture is its toxicological acceptability. All
strain of P. camemberti analysed so far, are however able to produce cyclopiazonic acid
(CPA) according to Frisvad (1988). CPA is a secondary metabolite with toxic activity in
animal experiments (Le Bars, 1979). CPA is toxic mainly against the liver, kidneys or
the pancreas. CPA can also be produced by P. camemberti on cheeses, especially at
higher storage temperatures. According to an assessment of the toxicity of this
mycotoxin in cheese, it is not a real health problem, however strains that would not
produce this toxic secondary metabolite would be advantageous.
About the genetics of CPA production nothing is known, so the gene disruption
approach as described above cannot be used with this species. For this reason it was
tried to isolate a CPA- mutant of P. camemberti by mutation/selection. CPA consists of
the amino acid tryptophane and the isoprenoid dimethylallypyrophosphate (Fig. 8).
For mutation, spores of P. camemberti were treated with nitrite. Single spore colonies
from the mutation experiment were plated out on agar plates and screened for the
production of CPA. From 5000 colonies two strains could be isolated with reduced CPA
production. One strain Cpa1 did not produce any detectable amounts of CPA, whereas
the other strain Cpa2 produced only very small amounts of CPA compared to the wild
type. The wild type was able to produce 34 µg CPA per gram mycelium (wet weight),
whereas the mutant Cpa2 produced only 0,8 µg, which is only 2% of the amount of the
wild type (Geisen and Leistner, 1990). However after TLC analysis Cpa2 accumulated a
new metabolite, which could not be identified in the wild type (Fig. 9).
23
ROLF GEISEN AND PAUL FARBER
The possibility exists that this might be an intermediate of the CPA biosynthetic
pathway, which accumulates just before the mutational block. To analyse this
possibility the mutant strain Cpa2, as well as the wild type strain were grown in the
presence of radioactively labelled 3H-tryptophan, which is a precursor of CPA. The
extracted metabolites of both the wild type and the mutant were separated by two-
dimensional TLC and subjected to autoradiography. The CPA spot of the wild type
gave a strong signal, however the new metabolite, which occurred in the mutant was not
labelled. The isolated spots were counted in a scintillation counter and the incorporated
radioactivity was quantified. This data show that the new metabolite has the same
background activity than the control. This results indicate that obviously the mutational
block must be located "before" the ligation of the isoprenoid moiety to the amino acid
tryptophane. This means that the mutation should be located in the part of the pathway
in which the isoprenoid moiety is produced. For that reason, the same experiment were
carried out, but in the presence of 14C acetate, which is the direct precursor of the
isoprenoids. The extracted secondary metabolites of the wild type and the mutant Cpa2,
which were grown on the labelled medium, were also subjected to two dimensional
TLC. This time a clear difference in the pattern of the separated labelled secondary
metabolites could be observed (Geisen, 1992). This indicates that a mutation, which
influences the production of isoprenoid precursors, is the reason for the inability of this
strain to produce CPA.
A physiological analysis showed that the mutated strain has the same growth
behaviour than the wild type strain.
24
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
4. Penicillium roqueforti
P. roqueforti was first described by Thom (1906). Until recently P. roqueforti was a
heterogeneous species of blue green sporulating fungi. Because of their phenotypic
differences they were first grouped to different species, but were combined into one
species by Raper and Thom (1949). Recently Boysen et al. (1996) divided the species
according to secondary metabolite production and to nucleotide sequence differences in
the ribosomal ITS regions into three different species: P. roqueforti, P. paneum and P.
carneum. P. roqueforti can produce PR toxin, marcfortines and fumigaclavine A; P.
carneum patulin, penitrem A and mycophenolic acid and P. paneum patulin,
botryodiploidin and other secondary metabolites. Beside its activity as starter culture P.
roqueforti is an important spoilage organism for different food and feed commodities,
like bread (Spicher and Isfort, 1987), cheese (Wendorf, 1993), fruits (Harwig, 1978) and
silage (Häggblom, 1990). Beside other mycotoxins most strains of P. roqueforti are able
to produce PR toxin (Scott et al. 1977; Wie and Liu, 1978; Lafont et al., 1979; Chang et
al. 1991). PR toxin is not stable in cheese and is degraded to the less toxic PR imine
(Siemens and Zawistowski, 1993). However strains which would not produce this
mycotoxin would be advantageous over the producing strains. PR toxin was hepato- and
nephrotoxic in studies with mice. According to Moulé et al. (1979) it inhibits the protein
biosynthesis in eukaryotic systems. PR toxin is a sesquiterpene (Fig. 10). Strains that
would not produce this mycotoxin would be advantageous over producing strains to be
used as starter cultures.
However screening for the PR toxin negative phenotype only by analysis of the PR
toxin producing ability by chromatographic methods do not guarantee that the identified
strains are indeed not able to produce PR toxin. The production of PR toxin is highly
dependent on the environmental conditions, which means that the identified strains
might be able to produce the toxin under other conditions.
25
ROLF GEISEN AND PAUL FARBER
42 + +
50 - -
53 + +
168 ++ +
169 ++ +
17- ++ +
172 nd +
208 ++ +
209 ++ +
210 ++ +
211 ++ +
215 ++ +
216 - +
219 ++ +
269 ++++ +
270 +++ +
A better way to screen for PR toxin negative strains, is to look for the presence of
certain genes coding for key enzymes in the biosynthetic pathway of the secondary
metabolite. If strains can be identified, which do not have a gene coding for an enzyme
in the biosynthetic pathway of PR toxin, they should not be able to produce PR toxin
under any condition, if no other metabolic shunt exists. One of the genes for a key
enzyme in the metabolic pathway to PR toxin has been cloned and sequenced (Proctor
26
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
and Hohn, 1993). It codes for the aristolochene synthase ( ari1 ). Aristolochene is a
precursor in the biosynthesis of PR toxin. The knowledge of the nucleotide sequence of
that gene can be used for a molecular screening method for strains that did not carry the
aril gene by PCR. Strains, which do not carry the ari1 gene, should not be able to
produce PR toxin. For these purpose primer sequences have been deduced from the
published sequence of the ari1 gene and different P. roqueforti strains have been tested
for the presence of the ari1 gene by using these specific primer in a PCR reaction. The
results are shown in Table 2. As can be seen in this table one strain could be identified
by this method, which apparently did not carry an ari1 gene. As it was possible that
only the primer binding sites may have been mutated in that gene, which also would
give rise to a negative PCR reaction, the results were confirmed by dot blot analysis.
This negative strain was also not able to produce PR toxin under the conditions used.
On the other hand another strain could be identified, which also did not produce PR
toxin, however which gave a positive result in the PCR reaction. These results suggest,
that this strain contains either a mutant gene, which is responsible for the non-producing
phenotype, or the strain may produce the toxin under other conditions. In any case the
strain with the negative PCR results should be preferred.
5. Conclusions
Fungal strains should be carefully selected according the specific need of a fermented
product. If it is not possible to find a strain that fulfils all the requirements, it can be
optimised by several methods. Either molecular methods can be used to introduce
additional characteristics to a strain or to inactivate specifically undesired features of a
particular strain. In cases were molecular data are not available, the conventional
mutation/selection approach may be used, however it has to be kept in mind that this
approach is less specific and that secondary mutations with undesired phenotypic
changes may occur. Screening for particular characteristics, based on molecular data is
preferable over conventional phenotypic screening, as the results are more reliable.
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production et nature du systeme proteolytique. Le lait 52, 497-514.
Guthrie, P.A.I., Magill, C.W., Frederiksen, R.A. and Odvody, G.N. (1992) Random amplified polymorphic DNA
Markers: A system for identifying and differentiating isolates of Colletotrichum graminicola. Phytopathology,
82,832-835.
Häggblom, P. (1990) Isolation of roquefortin C from feed grain. Appl. Environ. Microbiol, 56, 2924-2926.
Hamelin, R.C., Ouellete, G.B. and Bemier, L.: Identification of Gremmeniella abietina races with random
amplified polymorphic DNA makers. (1993) Appl. Environ. Microbiol,, 59, 1752-1755.
Harwig, J., Blanchfield, B.J. and Scott, P.M. (1978) Patulin production by Penicillium roqueforti Thom from
grape. Canadian Institute of Food Science and Technology Journal 11, 149-151.
Hesseltine, C.W. (1983) Microbiology of oriental fermented foods. Ann. Rev. Microbiol. 37, 575-601
Jacobsen, T. and Hinrichsen, L. (1997) Bioformation of flavour by Penicillium candidum, Penicillium
nalgiovense and Geotrichum candidum on glucose, peptone, maize oil and meat extract. Food Chemistry
3, 409-416.
Kriechbaum, M., H. J. Heilmann, F. J. Wientjes, M. Hahn, K. D. Jany, H. G. Gassen, F. Sharif and G.
Alaeddinoglu.(1989) Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus
niger. FEBS Lett. 255, 63-66.
Lafont, P., Debeaupuis, J., Gaillardin, M. and Payen, J. (1979) Production of mycophenolic acid by
Penicillium roqueforti strains. Appl. Environ. Microbiol. 37, 365-368.
Laxa, O. (1932) Überdie Reifung des Ellischauer Käses. Zentral.f. Bakt. 86, 160-165.
Le Bars, J. (1979) Cyclopiazonic acid production by Penicillium camembert Thom and natural occurrence of
this mycotoxin in cheese. Appl Environ Microbiol 38, 1052-1055.
Leistner, L. (1986) Mould-ripened foods. Fleischwirtschaft 66, 1-4.
Leistner, L., Geisen, R. and Fink-Gremmels, J. (1989) Mould-fermented foods of Europe: hazards and
developments, in eds: S. Natori, K. Hashimoto and Y. Ueno (eds.), Mycotoxins and Phycotoxins,
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Lund, F., Filtenborg, O. and Frisvad, J.C. (1995) Associated mycoflora of cheese. Food Microbiol. 12: 173-
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Marth et al (1987) Dairy Products, in L. R. Beuchat (eds.), Food and Beverage Mycology, Publishers, Van
Nostrand Reinhold, New York, pp. 175- 209.
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4543-4548.
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Pitt, J.I., Cruickshank, R.H. and Leistner, L. (1986) Penicillium commune, P. camemberti, the origin of white
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NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
Scott, P.M., Kennedy, P.B.C., Harwig, J. and Blanchfield, B.J (1977) Studies for conditions for production of
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29
STARTERS FOR THE WINE INDUSTRY
ALINE LONVAUD-FUNEL
Faculté d’œnologie - Université Victor Segalen Bordeaux 2,
351 Cours de la Libération, 33405 TALENCE Cedex
Abstract
Grape must is naturally seeded with yeasts and lactic acid bacteria. Both
microorganisms are needed : yeasts to carry out alcoholic fermentation, and bacteria to
degrade malic acid to lactic acid during malolactic fermentation. Indigenous microflora
is often sufficient. However, use of selected yeast and malolactic starters allow a better
control of the process. Strains are selected for their influence on the sensorial and
hygienic quality of wine.
1. Introduction
was not an accident but a second microbial transformation that improves wine quality.
Usually lactic acid bacteria develop after the end of alcoholic fermentation, i.e. during
and after the decline phase of yeasts. Thus they multiply in a medium which is not only
acidic but which also contains up to 13 % ethanol. They face very harsh conditions. The
addition of selected malolactic starters was first considered in the early 70s. However,
the success of such preparations was much more difficult than for yeast starters,
precisely because of the composition of wine. Therefore, progress has been slowed
down by many drawbacks, so today the state of the art for lactic acid bacteria starters is
approximately the same as it was two decades ago for yeasts. However, due to the
increasing knowledge on the physiology and metabolism of such bacteria, it is expected
that the gap will be soon filled. In the early 90s the first malolactic starter for direct
inoculation of wine was commercialised (Nielsen et al., 1996).
The demand for yeast starters is high both in the new and older wine producing
countries. Yeast producers are still selecting new starters on a wide scale, since the
variety of wines produced all over the world is great, and the potential ability of yeasts
seems greater than as used at present. As for lactic acid bacteria , the knowledge of their
influence on wine is just in its infancy and more needs to be discovered about their
adaptation to the wine medium to increase the success of malolactic starters.
After grape crushing, the must is naturally seeded with the microorganisms that are on
the surface of the berries and the cellar equipment. Soon after the grapes have been sent
to the fermentation tank or barrel, alcoholic fermentation normally starts with the
indigenous yeasts, Several yeast species can be isolated on the grapes and thus in the
grape must before fermentation: Candida sp., Kloeckera apiculata, Metschinikovia
pulcherrima, Torulaspora delbruekii, Schizosaccharomyces pombe and Saccharomyces
sp. K. apiculata, T delbrueckii and S. cerevisiae often predominate (Herraiz et al.,
1990). However, most of them quickly disappear except for Saccharomyces cerevisiae
that is the main agent of alcoholic fermentation (Fleet et al, 1984). According to the way
alcoholic fermentation is carried out defects may be induced such as off-flavours,
volatile acidity or even stuck fermentations. This might result in the predominance of
some undesirable yeast strains. The non-Saccharomyces species are mainly involved in
such problems. In addition, some Saccharomyces strains are not well–adapted to the
fermentation of sugar-rich medium and cannot complete the whole process. Some also
produce high levels of SO2 and acetic acid. This explains why spontaneous fermentation
can be considered as a hazardous phase. Today yeast starters are exclusively selected
strains of Saccharomyces cerevisiae.
The reasons for using yeast starters were established at the end of 19th century. The
experiments consisted in seeding the grape must by actively growing yeasts which were
taken from fermenting musts of good quality. The experimenters observed that the
quality of wine was consequently higher, and fermentation more regular and quick
(Brunet, 1903). In addition, Pasteur (1876) noted that if the same grape must was
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STARTERS FOR THE WINE INDUSTRY
inoculated by several distinct yeasts, the wines would also be distinct. The present
objectives of winemakers are exactly the same. The only difference between the two
centuries is that the selected strains today are individual clones while they were
« mixed » starters before, since there was no isolation procedure. However, 100 years
ago, wines were probably obtained by Saccharomyces sp. Now winemakers use single
strain starters in order to control the process more closely.
There are two reasons for using starters. One is to start the alcoholic fermentation
quickly after the harvest. Indeed, in some cases, and preferably at the beginning of the
winemaking the yeast population is too low (less than 104 CFU.ml-1). Multiplication up
to 106 and more takes several days especially if the temperature is low. During this time,
other microorganisms can develop : yeasts with oxidative metabolism and acetic acid
bacteria that take advantage of the presence of oxygen to produce volatile acidity and
many other defects. Thus, inoculation with starters at the concentration of 106 CFU.ml-1
prevents the growth of such microorganisms. The second reason for the winemaker to
use yeast starters is to improve the final phase of alcoholic fermentation. Indeed, grape
musts are so rich in sugar and sometimes so poor in essential nutrients that yeast cannot
survive long enough to ferment all sugars. Stuck fermentation is one of the major
problems in winemaking. The fermentative ability of the yeast cell decreases during
alcoholic fermentation due to its sensitivity to increasing ethanol and fatty acids. Both
compounds are toxic for yeast (Lafon-Lafourcade et al , 1984) and the target of this
effect is the membrane. Oenologists and winemakers have long known that aeration is
essential to improve alcoholic fermentation. This was also explained by the work of
Larue and Lafon-Lafourcade (1989) who demonstrated that the survival of yeast in a
fermenting must depends on the sterol composition of the membrane, which itself
depends on the O2 available during growth. This also explains why yeast starters that
are produced in aerobiosis can respond more efficiently to these conditions than
indigenous yeasts.
The addition to must of selected starters also named « active dry yeasts » has
become essential in white winemaking. It has replaced the former starters that were
prepared by multiplying yeasts from a spontaneous fermenting wine in highly sulfited
must to prevent bacterial growth. White grape must is not easily fermentable. Indeed,
after crushing it generally undergoes to several operations (decanting, clarification) to
improve its sensorial quality, and these make it poorer in nutrients and in natural
microflora. Therefore, it is necessary to add yeast very soon, once the must has been
clarified or adjusted to the adequate turbidity by addition of light solids (Ollivier et al ,
1987). Active dry yeasts are added to reach 106 CFU.ml-1, i.e. 10 to 15 g.hl-1 of the
commercial preparation. They are added just after the clarification of grape juice, after a
reactivation in grape must and water (v/v ; 1 :1) for 20 minutes at 40°C.
Yeast starters are less used, and in fact less necessary in red winemaking. The
operations that follow grape crushing do not deprive the medium of the natural
microflora, since all the parts of the berries skin, pulps and seeds are poured into the
vats. Moreover, the process includes several pumping over phases to extract the wine
colour, and some of these are done with aeration. It is only when the temperature is too
low or if rain has considerably washed the grapes that alcoholic fermentation for red
wines may take several days to start. In these cases active dry yeasts are necessary.
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ALINE LONVAUD-FUNEL
However, as winemakers are more and more aware of the difficulties encountered in
obtaining the alcoholic fermentation, they also use starters for red wines.
The second reason to use active dry yeasts is to restart uncompleted alcoholic
fermentation. When several g.l-1 of sugar remain unfermented, the wine is not finished
and is exposed to spoilage. Indeed during the active phase of fermentation, yeasts
inhibit bacteria, but since their population decreases and may lead to stuck fermentation,
lactic acid bacteria can develop. This induces one of the major accidents in winemaking
called « piqûre lactique » characterised by the heterolactic fermentation of sugars. The
result is a high volatile acidity. Therefore as soon as alcoholic fermentation slows down
a sufficient population of active yeasts must be added. The commercial active dry yeasts
need a preliminary preparation otherwise they cannot survive, since the medium is high
in ethanol and fatty acids. The reactivation procedure is conducted as follows in a
medium prepared with the wine itself. The alcohol content is reduced to 8-9 % with
water and adjusted to 15 g.l-1 of sugar. After sulfiting at 3 g/hl-1, 20 g.hl-1 of active dry
yeast are added. At 20°C growth occurs within several days and the fermentation is
monitored by determination of sugar concentration or density. When the sugar has just
totally disappeared, the yeasts are in the active growing phase. The starter is added at
the concentration of 5-10 % in the wine to be treated. The completion of alcoholic
fermentation still takes several days. The efficiency of such starters can be increased by
the preliminary addition, 24-48 hours before, of yeast cell walls. Lafon-Lafourcade et al
(1984) demonstrated that the preparation obtained from yeast cells by elimination of the
yeast cellular components - i.e. the yeast cell walls – enhances yeast survival in
fermenting must. It is now an authorised procedure to prevent or to treat stuck
fermentations. This regulatory property is mainly due to the capacity of cell walls to fix
and to remove yeast inhibitors such as fatty acids.
All the active dry yeasts available share the same basic characters ; i.e. good
multiplication rate, high yield of ethanol production, ability to complete fermentation of
sugars (resistance to ethanol and other toxic compounds such as pesticide residues),
adaptation to low and high temperatures, low production of volatile acidity of ethanol,
sulphur dioxide, sulphur hydrogen, foam, and high glycerol production. In addition, it
is preferable for them to be neutral towards the killer toxins or even producers of the
toxin, and not to be sensitive since they mightbehampered by indigenous killer strains
(Jacobs and van Vuuren, 1991). Finally, such selected strains must withstand the drying
process and recover their properties after storage.
These are the minimum requirements for selected yeast starters. However, among
the dozens of preparations available, one must choose the best adapted to the special
problem to be solved. They can be divided in several categories : quick starter yeasts,
those for restarting stuck alcoholic fermentation, others for « prise de mousse » of
sparkling wine, some for wines used to make spirits and finally yeasts aimed to develop
regional or aroma typicality. The first are selected for their resistance to the hostile
conditions of wine fermentation. However, more and more work is now underway to
find strains able to develop aromas and to optimise phenolic compound extraction in red
wine. « Primeur » red wines and white wines are mainly characterised by fruity and
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STARTERS FOR THE WINE INDUSTRY
floral aromas. Esters, especially shorter chain fatty acid ethyl esters and acetate esters,
are the main volatile components involved in the sensorial quality of wines, together
with carbonyl compounds such as acetaldehyde and diacetyl. They are produced by
yeast metabolisms (Henschke and Jiranek, 1993). The conditions of alcoholic
fermentation, notably temperature and the presence of solids through yeast metabolism
modifications change the final amount of volatile compounds.
However, the strain itself has a real impact (Vasconcelos et al., 1996), e.g. if it
produces two much of one of the volatile components. Some are revealed by yeast
activity on the aroma precursors found in grape berries (Darriet et al , 1991).
On the contrary, some yeasts may suppress the variety aromas ; they must be used only
for the fermentation of neutral grape varieties. Among many other compounds, yeast
can produce vinyl phenols from p-coumaric acid and ferulic acid in must. Above a
certain threshold these give the wine pharmaceutical odours. The enzymatic activity,
cinnamate decarboxylase, depends on the yeast strain (Figure 2) (Chatonnet et al, 1993).
The role of yeast in the production of varietal aromas is currently intensively
studied. The involvement of yeast in the release of terpenes which characterise grape
varieties such as muscat, gewürztraminer, riesling, etc.... and in that of norisoprenoids
that are very odorous, is not clear. More is known about the characteristic aroma of
35
ALINE LONVAUD-FUNEL
In red wines, the type and amount of polyphenols is very important. An aim of
winemaking is to obtain the highest amounts of the « good » polyphenols. It has also
been shown that yeast can influence the final composition in phenolic compounds and
thus the wine colour. This study led to the selection of two yeast strains that will be
dried and tested in a pilot-scale study (Arioli et al., 1996).
From such studies, companies producing active dry yeast for the wine industry can
make new preparations. However, winemakers must remember that the wine aroma
precursors are in the must. Therefore, the yeast has to be adapted to the grape must. The
aim is to reveal the aromas without excess, since the overproduction of some
components is undesirable.
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STARTERS FOR THE WINE INDUSTRY
Active dry yeasts are usually added to grape must containing indigenous microflora
originating from the grapes and the cellar equipment. This microflora is usually low in
white grape musts due to several operations aimed to eliminate solid materials from the
medium. The level of microflora is normally higher in red grape musts. However, until
recently it was not easy to assess the efficiency of the starter, i.e. it was difficult to
ascertain whether alcoholic fermentation was done by the starters or by the indigenous
yeast population. Today molecular methods are very reliable tools to monitor and to
assess the role of starters. Several methods can be used to differentiate a Saccharomyces
strain from another. The first method that was developed was the analysis of restriction
patterns generated by hydrolysis of mitochondrial DNA (mtDNA). Oenological strains
were successfully characterised by Dubourdieu et al (1987) and Hallet et al (1988). The
profile of the active dry yeast used as starter is compared to that obtained from the lees
sampled during or after alcoholic fermentation. If there is no extra band, this means that
the starter has grown and has replaced the original yeasts. However, in spite of its
reliability, this method is no longer used because it is unwieldy and time-consuming.
The analysis of karyotypes needs less preparation. It consists in a separation by
pulse field gel electrophoresis of the 16 yeast chromosomes. Since their size varies from
250 to 2500 kbp, this gives a profile for each strain . The preparation of samples is rapid
and easy, but the electrophoresis of such large DNA molecules requires special
electrophoresis equipment. Blondin and Vezhinet (1 988), then Frezier and Dubourdieu
(1991) applied this method for wine yeast strain identification during winemaking. Like
others, Roulland et al (1996) using this method reported that spontaneous alcoholic
fermentation is carried out by one or at the most two dominant strains in wine used to
make Cognac.
Finally PCR (Polymerase chain reaction) using repeated sequences as primers is
another very interesting method for the identification of strains during winemaking
(Ness et al, 1992). It is much more rapid than the other methods and can be used
directly on whole cells. The profile generated by specific amplification can distinguish
most of the strains very easily. Like the mtDNA RFLP profiles and the karyotypes, the
PCR patterns of the yeasts taken in lees are compared to those of the starter. They are
identical if the starter has really taken part in the process. By PCR it is shown that 90 %
of the population is derived from the starter when the profiles are identical.
Usually the starter chosen is added as soon as possible to the grape must to inhibit
and eliminate the indigenous yeasts. The chance of success is 90 % if the ratio
starter/indigenous yeast is 10. Some have recommended successively adding two
different starters in order to enhance wine complexity. A study was undertaken to verify
the role of both starters inoculated in the same vat (Meurgues et al., 1998). Molecular
typing by PCR of the yeasts taken at the end of alcoholic fermentation clearly showed
that the second starter, which was added two days after the first one, had completely
disappeared. As expected, addition at about 6.106 CFU.ml-1 of the second starter, i.e.
three times the normal dose, could not replace the active fermenting population of the
first starter which was about 108 CFU.ml-1. Thus, the addition of selected yeasts during
the course of alcoholic fermentation is of little use.
37
ALINE LONVAUD-FUNEL
Analysis of the diversity of yeast strains during alcoholic fermentation has also
demonstrated that a selected starter may be easily disseminated in a cellar in red wine
winemaking (Frezier and Dubourdieu,1991). While only the first vat filled was
inoculated, the presence of the starter was dominant even in vats filled several days after
and not inoculated (Table I).
Table I : Percentages of yeast strains isolated in fermenting grape musts inoculated or not with active dry
yeasts (after Frezier and Dubourdieu, 1991)
This means that in red winemaking mere cellar handling is sufficient to disseminate
yeasts. However, the use of massive concentrations of different starters allows their
settlement in several vats. In white winemaking, the conditions and the results may be
similar except when must is fermented in barrels, thus making dissemination less
possible. Progress in the identification of yeast strains has also led to real advances also
for controlling the production of starters themselves. This is obviously very important
since winemakers can now really monitor the role of the starters they have chosen
according to the type of wine.
Lactic acid bacteria must reach the population of 106 CFU.ml-1 in order to start
malolactic fermentation. Depending on the variety, the producing area and the degree of
maturity, the grape must contains about 2 to 8 g.l-1 of malic acid. In most cases all malic
acid must be transformed to lactic acid. The benefit is a softer taste and greater aroma
complexity. However, some white wines only need the partial degradation of malic acid
that results in deacidification while preserving the fruity aromas that normally disappear
with total malolactic fermentation. Bacteria must grow after alcoholic fermentation in
very hostile conditions due to ethanol, fatty acids, other inhibitors and acidic pH.
However, in normal conditions, some days after the yeasts have completely fermented
the sugars, bacteria multiply very quickly from 102-103 CFU.ml.-1 to more than 106
CFU.ml-1. The growth rate depends on the environmental conditions, the adaptation of
the bacteria to the medium and on the yeast responsible for alcoholic fermentation (
Lonvaud-Funel et al, 1988). Like yeasts, the indigenous lactic acid bacteria originate
from grapes and cellar equipment. In the beginning of winemaking up to eight to nine
species are usually identified. However after a few days, a natural selection occurs in
the fermenting must. At the end of alcoholic fermentation, the Oenococcus oeni species
38
STARTERS FOR THE WINE INDUSTRY
39
ALINE LONVAUD-FUNEL
New research in the early 90s was undertaken on the resistance of O. oeni to several
inhibitors such as ethanol and fatty acids. The focus was cell membrane studies since it
was thought that the membrane was the primary target of the inhibitors, which kill the
bacteria. Lonvaud-Funel and Desens (1990), then Garbay and Lonvaud-Funel (1 996)
demonstrated that the cultivation in media added with ethanol or fatty acids or other
stresses such as heat shock induced changes in the membrane composition. The most
striking feature was the overproduction of some membrane proteins and a decrease in
phospholipids. Such stressed cells were also more apt to survive in wine. Following
these results, the first O. oeni lyophilised preparation for direct inoculation in wine
became available in 1993 (Viniflora oenos, Chr. Hansen, Denmark) (Nielsen et al,
1996). The population is about 5-106 CFU.ml-1 and it grows or survives until the
complete transformation of malic acid (Figure 3). A few other preparations are now
available. But it is clear that the production of malolactic starters is much more difficult
than yeast starters for which the winemaker can chose amongst dozens of preparations.
The other problem when using a malolactic starter is the time of inoculation. While
there is no alternative for yeast starters, it has often been suggested that malolactic
starters can be used before, during or after alcoholic fermentation. Of course addition
before alcoholic fermentation avoids the inhibition of bacteria by wine components.
However, as for the indigenous bacteria, survival is readily inhibited by the active
growing yeasts. Moreover, if bacteria survive during alcoholic fermentation, they can
ferment sugar, in competition with yeasts. The real problem is not the loss of ethanol
but the production of volatile acidity which accompanies the heterolactic fermentation
of hexoses and pentoses by O. oeni. In addition, it has clearly been demonstrated that at
the end of alcoholic fermentation, at a time when yeast cells are losing their activity, too
many bacteria (over than 105-106 CFU.ml-1) tend to accelerate the yeast decline phase
Paraskevopoulos (1988). Bacterial enzymatic activity probably contributes to the
hydrolysis of the yeast cell wall. In such conditions the major problem is incomplete
alcoholic fermentation. This frequently occurs naturally when the indigenous bacterial
population grows too early (Ribereau-Gayon et al, 1998). At this time wine still
contains some 5-10 g.l-1 of fermentable sugars, a concentration high enough to produce
volatile acidity. Since the behaviour of the bacteria is completely unpredictable, it is not
recommended to add malolactic starters as long as the sugar have not been completely
fermented.
Figure 3A : Evolution of malic acid in a red wine inoculated or not by a malolactic starter.
(Nielsen et al , 1996)
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STARTERS FOR THE WINE INDUSTRY
Since the crucial problem of the survival of malolactic starters in wine has been at least
partially solved, studies now focus on the important question of their influence on wine
quality. Two aspects are now considered : hygiene and the sensory qualities.
For a long time oenologists considered lactic acid bacteria only as the agents of
malic acid degradation. Even if it is the principal biochemical reaction, bacteria also
metabolise many other wine components. The most studied has been citric acid that is
transformed into acetic acid and acetonic compounds. Only about a maximum 0.3 g/l of
citric acid are metabolised but this is enough to induce sensorial changes due to the
production of acetic acid (volatile acidity) and diacetyl which is very aromatic. Only a
few mg/l of the latter gives the wine a buttery aroma. The threshold is dependent on the
wine, and is less for white wines (= 4-5 mg/l) than for red wines (7-8 mg/l). Sensorial
analyses show that some judges prefer « buttery » wines, while others consider high
diacetyl concentrations as a defect. The other sensorial effects are characterised by the
production of fruity and floral notes. It is not definitively established that they depend
on the dominant bacterial strain. Numerous studies have compared wines inoculated by
distinct bacterial strains and have shown their individual influence (Henick-Kling T.,
1993). However, sensorial analyses rarely show significant differences. Moreover in
such experiments, the repetition of inoculation by a given strain may often give more
sensorial differences than different strains. This point remains unclear and such results
41
ALINE LONVAUD-FUNEL
show that malolactic fermentation has a real influence on wine aroma, but this is less
strain-dependent than alcoholic fermentation.
Other compounds are undesirable such as biogenic amines, which might be
responsible for allergies, and ethyl carbamate that is thought to be carcinogenic. For a
long time only non-malolactic bacteria (all species except O. oeni), and especially
Pediococcus sp. were thought to produce histamine. However O. oeni strains may be
involved. Such strains contain the hdc gene that codes for the histidine decarboxylase.
Surprisingly, it has been shown that many strains have this property (Coton et al,
1998). During and mainly after malolactic fermentation, tyramine and putrescine
(diaminobutane) also increase in some wines. However, until now the bacteria involved
have not been completely characterised. Another observation is that some wines do not
contain any biogenic amine, while others produced from year to year in the same cellar
always contain them. At least for histamine, it is concluded that the indigenous bacterial
flora that is established in the cellar comprises aminoacid decarboxylating strains.
Ethyl carbamate that is produced from precursors such as urea, carbamylphosphate
and citrulline, is also studied by food hygienists. In wine, urea is produced by yeast, and
citrulline is produced by lactic acid bacteria from arginine (Liu et al., 1994). Current
studies aim to study and characterise O. oeni that may utilise the arginine deiminase
pathway and increase the risk of ethyl carbamate in wine.
Therefore, the selection criteria for malolactic fermentation starters are based both
on the capacity of the strain to be prepared at the industrial level for survival in wine
and on some special metabolic pathways. Usually the strains are isolated from wines
during malolactic fermentation. The only species considered is here O. oeni. After
identification the first selection procedure includes sensitivity to ethanol and to pH. The
latter is certainly the most important parameter. Malolactic activity is not a real
selection marker since it is generally high for all strains. The ability to metabolise citric
acid is also a general trait of this species and must be considered as a positive character.
The other property to be considered is the inability to produce biogenic amines. Since
many studies are focusing on sensorial quality and secondary metabolisms, selection
will probably include more criteria in the future.
Unlike yeast starters, malolactic fermentation starters have been difficult to promote
because in the beginning they were very unreliable. Moreover, in numerous studies,
authors have concluded that they were efficient because malolactic fermentation
occurred after inoculation. Frequently, it was not clear if the malic acid is degraded by
the starter or by the indigenous bacteria, especially when malolactic fermentation starts
long after inoculation. This probably explains why the market for malolactic
fermentation starters is still unsure. Winemakers are not convinced of the effectiveness
of the starter for the above mentioned reasons.
Today, it is possible to verify the settlement and the role of added bacteria with
molecular methods, such as for active dry yeasts. The most relevant method is to
compare the genomic fingerprints of the bacteria taken at the end of malolactic
fermentation to those of the starters. Genomic studies of O. oeni have shown that each
strain can be distinguished from others by the electrophoretic pattern of digested DNA.
Restriction by rare cutting enzymes generates DNA fragments that are well separated by
42
STARTERS FOR THE WINE INDUSTRY
pulse field electrophoresis. Three enzymes are frequently used and are well adapted for
this species. Thus, the procedure consists in the cultivation on solid medium of bacteria
present in wine at the end of malolactic fermentation. The DNA of the whole biomass
(or individual colonies) is analysed. If the starter has really dominated the indigenous
microflora, the profile is strictly similar to that of the lyophilised bacteria. The presence
of additional bands proves that other bacteria were in the wine at the same level and
probably took part in the process (Daniel et al. 1993, Gindreau et al., 1997). Of course,
analyses of samples during the process can indicate whether the starter undergoes a real
competition with the indigenous bacteria. This advance is very new but very promising.
Indeed in none of the previous studies, notably those to analyse the sensorial impact of
starters, could the survival of the starter be assessed. Today, wine scientists have a
suitable method to verify the ability of the starter to induce malolactic fermentation
before the decision is taken to implement fastidious and difficult sensorial and fine
chemical analyses.
After malolactic fermentation sulphur dioxide is added to eliminate as far as possible
all the microorganisms and to protect wine from oxidation. However, in many wines of
relatively high pH, sulphur dioxide reduces its antimicrobial activity and the bacterial
population remains relatively high. Identification to the strain level by fingerprinting
has recently shown an interesting property of malolactic fermentation starters.
Comparison of populations in inoculated and non-inoculated wines proved that the
population remaining after sulfiting did not contain the starter strain at least at a
sufficient level to be detected by DNA fingerprinting. It was composed of the
indigenous lactic microflora (Millet and Lonvaud-Funel, unpublished results). Thus, if
this result was confirmed, it would mean that starters are eliminated soon after
malolactic fermentation. This is very positive, since it means that a given starter would
be very unlikely to be perpetual in the cellar.
The interest of winemakers for yeasts and bacteria starters is motivated by the potential
they offer to control the fermentation processes. Nowadays, active dry yeasts are widely
used. Winemakers in USA, Canada, South Africa, Australia, New Zealand were the first
users by the mid-1960s and it took about 10 years more for the European producers to
start with yeast inoculation. Malolactic starters came very later and the industrial
preparations for direct inoculation are very recent.
Yeasts are inoculated : i) to shorten the time between the harvest and the active
alcoholic fermentation which is crucial to prevent spoilage, ii) to avoid incomplete
fermentation, iii) more and more, to develop specific aromas. In fact, the application of
elementary oenological rules solves the second point at least. The judicious aeration of
fermenting must provide enough O2 for yeast to synthesise the indispensable sterols it
needs for survival (Lafon-Lafourcade, 1983). Temperature control also minimises the
problems. Nevertheless, it seems that more and more active dry yeasts are widely used
for safety, even if they are not really necessary. While they are indeed almost
indispensable in white wine production because of all the preliminary treatments of the
grape musts, this is not the case for red wines. More and more the principal objective of
the producer is to develop aromas during alcoholic fermentation. Of course, it is highly
43
ALINE LONVAUD-FUNEL
preferable that they are typical aromas characterising if possible not only the grape
variety but also the grape variety in its environment, i.e. marked by the « terroir »
Even if there is already a considerable choice of starters , much work is still
underway to improve selection. The increasing knowledge of the aroma compounds
revealed during alcoholic fermentation is of course the driving force of this activity.
Typicality related to wine variety is consistently influenced by the dominant strain
during alcoholic fermentation. Therefore, aroma quality could be enhanced by the
selection of the best-adapted strains in view of their enzymatic activities. However,
typicality is also related to many other factors, and the use of a given starter will not
necessarily result in the same wine in different wine areas.
This justifies the increasing attention of yeast selectors for the aromas produced by
candidate strains for starters. As in the early age of wine microbiology, some
oenologists have in recent years proposed using yeast species other than S. cerevisiae.
Indeed it is well known that the indigenous yeast microflora is composed of several
genera such as Candida, Rhodotorula, Brettanomyces, Torulopsis, Harsemaspora, and
Kloeckera. They are in variable proportions in the must and disappear due to their
sensitivity to ethanol and O2 depuration. However, some of them may survive relatively
late during the alcoholic fermentation. Their influence on wine aromas has been
demonstrated (Herraiz et al., 1990). The apiculate yeasts, Kloeckera apiculata and
Hanseniasporia guillermondii, were assayed associated to Saccharomyces cerevisiae
(Romano et al, 1992 ). They were found to greatly influence the composition of wine
and aromas. Thus there is now renewed interest in the non-Saccharomyces microflora,
and active dry yeast producers are studying their potential use. Raguiel et al (1998)
describe the results obtained with a T. delbruekii strain and a strain obtained by fusion
of S. cerevisiae and Schizosaccharomyces pombe characterised by its capacity to
degrade malic acid. While the latter was studied for deacidification, the former was
studied for its property to produce strawberry aroma. The authors conclude that the use
of the T. delbruekii strain mixed in suitable proportions with S. cerevisiae is interesting
and justifies further experiments in various environmental conditions.
Selection of yeasts as starter should also include, strains for deacidification and on
others which retain or even increase the initial acidity of grape must. These mainly
differ by their ability to degrade or produce more or less L-malic acid. The traditional
selection of yeast strains might also be replaced in the future by genetically modified
yeasts. Even if the use of such microorganisms is subjected to controversy several
research teams are already preparing this approach. The first work in the early 90s
concerned the transformation of S. cerevisiae with the malolactic bacterial gene. The
idea was to add to the yeast the malolactic activity which would allow the simultaneous
alcoholic fermentation and malic acid degradation (Ansanay et al., 1993, Denayrolles et
al, 1995, Volschenk et al, 1997 ). Following this research several other functions were
considered for cloning in the wine yeast : higher production of glycerol (Remize et al,
1999), of L-lactic acid (Dequin and Barre, 1994), secretion of proteolytic enzymes
(Lourens and Pretorius, 1996). However, the success and the demand for such yeasts
starters is very unpredictable.
As regards lactic acid bacteria starters, much future work will concern the
adaptability of inoculated bacteria to the stress conditions of wine. Even if great
progress has been made with the starters for direct inoculation, there are still too many
44
STARTERS FOR THE WINE INDUSTRY
unaccountable cases where the best ones fail. Today, the present results on aminoacid
decarboxylation and the production of ethylcarbamate can be added to the conventional
traits for the selection of new starters. Therefore, much more knowledge is necessary
regarding the sensorial influence of lactic acid bacteria at the strain level. If this is
demonstrated and considered relevant, then malolactic starters will also be selected for
these features.
5. Conclusion
The production of quality wine is tightly linked not only to the quality of grapes but also
to the microorganisms that completely change the composition of the medium where
they develop. The fate of grape juice is to promote the growth of yeasts and bacteria.
Alcoholic fermentation and malolactic fermentation spontaneously occur in most cases.
However, fewer winemaking accidents occur now than in the former times by
controlling yeasts and bacteria. Yeast starters are readily available and the choice gets
wider each year. However, the winemaker now has a new challenge: he must choose the
most suitable strain according to the grape must and the type of wine. This is not so
difficult, but there are many examples of bad choice. This is especially true when strains
produce too high concentrations of aromas ; this might finally lead to standardising
wine. It is preferable to choose the strain which express the best the wine typicality in
relation to the grape and its environment. Sometimes the best solution is to use a
« neutral » strain that does not mark the wine too heavily. The present interest of
researchers for non- Saccharomyces strains is very important since most reports
demonstrate that wine aroma complexity is enhanced by inoculation of such yeasts.
However, mixed cultures even from selected starters may be as difficult to control as
the indigenous microflora.
Finally, the most worrying problem is the dissemination and above all the
remanence of the starters in the cellar. It has already been shown that yeast starters recur
in a given cellar from year to year. Of course this does not prevent to use different
strains efficiently because they are massively added. However, this will surely suppress
the natural biodiversity of grape and wine microflora. At least for the moment, the
danger seems, much lower for lactic acid bacteria starters which seem to be unable to
survive after malolactic fermentation.
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Coton, E., Rollan, G., Bertrand, A., Lonvaud-Funel, A. (1998) Histamine producing lactic acid bacteria in
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Sciences de la Vigne et du Vin 25, 167-174.
Denayrolles, M., Aigle, M., Lonvaud-Funel, A. (1995) Functional expression in Saccharomyces cerevisiae of
Lactobacillus lactis mleS gene encoding the malolactic enzyme. FEMS Microbiology Letters 125, 37-44.
Dequin, S., Barre, P. (1994) Mixed lactic acid-alcoholic fermentation by Saccharomyces cerevisiae expression
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Fleet, G.H., Lafon-Lafourcade, S. and Riberéau-Gayon, P. (1984) Evolution of yeasts and lactic acid bacteria
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1038.
Frézier, V. and Dubourdieu, D. (1991) Incidence du levurage sur I’écologie des souches de Saccharomyces
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Garbay, S. and Lonvaud-Funel, A. (1996) Response of Leuconostoc oenos to environmental changes. J. Appl.
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Gerbaux, V., Cuinier, C., Barrère, C. and Berger, J.L. (1995) Bilan de cinq années d’essais d’ensemencement
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Gindreau, E., Joyeux, A., de Revel, G., Claisse, O., Lonvaud-Funel A. (1997) Evaluation de I’établissement
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47
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN
BREVIBACTERIUM FLAVUM: IMPACT OF STRINGENT RESPONSE IN
BACTERIAL CELLS
Abstract
1. Introduction
51
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 51–57.
©2001 Kluwer Academic Publishers. Printed in the Netherlands.
M.RUKLISHA, R.JONINA, L.PAEGLE AND G.PETROVICA
The aim of the present studies was to investigate the effect of stringent response in B.
flavum RC 115 cells on changes in the concentrations of most relevant internal
metabolites, required for lysine synthesis and bacterial lysine synthesis activity as its
consequence.
The microorganism used was B. flavum RC 115 - auxotroph for threonine and
methionine (Culture collection of the University of Latvia). Bacteria were cultivated
under batch conditions in fermenter (MBR, Switzerland) on a glucose/corn steep liquor
containing media (Ruklisha et al., 1995). Respiratory activity of the cell culture (QO2)
under fermentation conditions was monitored by oxygen balance method using gas-
analysing system to estimate difference between oxygen concentration in the inlet and
outlet gas of fermenter (Baburin et al., 1986). In some experiments bacteria, collected
from fermenter, were recultivated for one hour in flasks on a rotary shaker in slight
modified mineral medium described by Kiss and Stephanopoulos (1991) with 1.5-mM
threonine or without its complementation. Biomass, lysine (as lysine.HCl) and sugar
concentration in the medium, intracellular concentration of ppGpp, as well as
parameters of bacterial physiological activity and lysine biosynthesis (µ, h-1; qP, g
lysine .g cells-1. h-1 and YP/S, g lysine. g glucose-1) were estimated as described by
(Ruklisha et al., 1995). Pyruvate and NADPH were extracted from cells by the
respective procedures described by Weibel et al., 1974, Matin & Gottschal, 1976 and
measured by enzymatic methods. Intracellular lysine was extracted by a method
proposed by Erdman et al. (1994). Concentration of amino acids in cell extracts or
fermentation medium was assayed by HPLC (Shimadzu C-R4A chromatograph, Japan).
The samples were treated with 10% sulphosalicylic acid in order to remove proteins,
and filtered prior to analysis. The amino acids were quantified by automatic precolumn
derivatisation with o-phtalaldehyde, separated in the column (Nova Pak C 18, Waters,
Milford, Mass., USA) using a buffer/methanol gradient, and detected fluorometrically
using Shimadzu RF-530, fluorescence HPLC monitor (Japan).
52
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM
18-22 h increased up to 0.55 ± 0.04 g lysine .g glucose-1. The increase in the lysine
synthesis activity of cells was correlated with a sharp increase in ppGpp concentration
in cells (Fig.2A).
Figure 1. Changes in the rate of bacterial growth (µ) and lysine biosynthesis (qP) by B.
flavum RC 115 cells as well as changes in the concentration of threonine in growth medium
during batch cultivation.
Hence, it was assumed that the increase of lysine biosynthesis might be a consequence
of the stringent response in bacterial cells like changes in RNA synthesis and
modification of metabolic pathway functioning, changes in intracellular concentrations
of lysine precursors and others
53
M. RUKLISHA, R. JONINA, L. PAEGLE AND G. PETROVICA
Figure 2. Changes in the bacterial respiratory activity (QO2) and internal concentrations of
ppGpp, pyruvate as well as NADPH in B. flavum RC 115 cells during batch cultivation.
54
METABOLISM AND LYSINE BIOSYNTHESlS CONTROL IN BREVlBACTERIUM
The fact that with the increase of lysine synthesis concomitantly increased synthesis of
other NADPH-consuming amino acids, e.g. valine (Fig.3), proline and isoleucine
(data is not presented), confirmed the possible role of NADPH accumulation in cells on
amino acid biosynthesis under stringent response conditions. It might indicate that the
physiological meaning of the increase of biosynthesis of lysine and other
NADPH-consuming amino acids under stringent response conditions would be
NADP+ regeneration in cells.
Figure 3. Changes in the external concentrations of valine and lysine in the cell culture of
B. flavum RC 115 during batch cultivation.
Changes in the intracellular concentration of lysine and the specific rate of its
extracellular accumulation by B. flavum RC 115 under stringent response conditions
differed. An internal concentration of lysine increased in parallel with the increase of
bacterial growth rate but did not increase further under stringent response conditions
(Fig.4). On the contrary, the extracellular accumulation of lysine under the latter
conditions significantly enhanced (see: Fig. 1A). Hence, it was suggested that, under
stringent conditions, induced by threonine starvation, lysine export from B. flavum RC
115 cells possibly increased. The lysine export with the help of specific excretion
carriers by C. glutamicum had been proved by Erdman et al. (1994). A possible role of
the stringent response on lysine export activity was investigated using cells of different
growth rate values, collected in appropriate moments of batch cultivation. Cell culture
of each sample was divided in two parts; cells were separated from the medium by
centrifugation and transferred in two versions of fresh pre-warmed mineral media (with
or without threonine). Cell culture with an initial concentration of biomass 10 ± 1 g
cells.l-1 were recultivated in shake flasks for one hour. The results of short-term
experiments Metabolism and Lysine Biosynthesis Control in Brevibacterium flavum
showed that lysine as well as valine (the latter data are not presented) were accumulated
in the medium only as a result of threonine limitation (Table 1). The higher was the
55
M. RUKLISHA, R. JONINA, L. PAEGLE AND G. PETROVICA
initial rate of bacterial growth the higher increase in lysine synthesis was achieved
under conditions of threonine limitation.
Figure 4. Changes in the internal concentration of lysine in B. flavum RC 115 cells during
batch cultivation.
Hence the stringent response, induced in B. flavum RC 115 cells by threonine limitation,
probably resulted with the increase in lysine export activity. It might be assumed that a
mechanism exists, which favour increase the export of lysine from bacterial cells under
stringent response conditions. Rapid induction of the export of some amino acids from
bacterial cells as a consequence of stringent response was experimentally proved in
other bacteria. Data obtained by Burkovski et al. (1995) clearly showed that glutamate
export from Escherichia coli cells was induced under stringent response conditions and
this export was rel-A gene controlled. However direct role of the stringent response on
lysine export activity in corynebacteria should be further investigated.
Table 1 Effect of cell incubation in the medium with or without threonine on their specific
lysine synthesis activity (qP) *Cells collected at appropriate times of batch cultivation (see
Fig. IA) **Data represents average means ±SE of four experiments
Growth rate of initial cells* Incubation with .5 mM Incubation without threonine
(Time of collection, h) threonine
4. Conclusions
The data presented in this study showed that lysine biosynthesis by B. flavum RC 115
cells increased as a consequence of stringent response, induced by threonine limitation.
56
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM
It was explained as a result of NADPH accumulation in bacterial cells and probably that
of an increase in lysine export activity.
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57
MOLECULAR BREEDING OF ARMING YEASTS WITH HYDROLYTIC
ENZYMES BY CELL SURFACE ENGINEERING
Abstract
1. Introduction
Cell surface is crucial to the life of the cells since it is the interface between the inside
and the outside of the cells. It encloses the cells and maintains essential differences
between the cytosol and extracellular environment. Surface proteins are responsible for
most cell surface functions, serving as cell-cell adhesion molecules, specific receptors,
molecular recognising devices, catalytic enzymes, transport machinery, and so on.
Many surface proteins are bound through noncovalent or covalent interaction to the cell
surface structures. Cells have means of anchoring specific surface proteins and of
confining surface proteins to particular domains of the cell surface structure. Recently,
it has been partially clarified that how these protein molecules are targeted and localised
to the cell surface. Thus, it is possible to remake the functions of cell surface by taking
advantage of the known transport mechanisms of proteins to the cell surface. We have
been developing "Cell Surface Engineering" of yeast based on the techniques of genetic
engineering to endow the cells with novel functions by displaying target proteins on
their cell surface.
The cell surface display system is intended to be applied to bioindustrial processes
because it is required to be safe. In practical use, the most suitable microorganism is
the yeast S. cerevisiae, which has 'generally regarded as safe (GRAS)’ status and can be
used for food and pharmaceutical production. S. cerevisiae is a useful organism to
develop the cell surface expression system. It is also an advantageous cell as a host for
genetic engineering, since it enables folding and glycosylation of eukaryotic
heterologous proteins expressed and is easy to handle with ample genetic techniques.
Moreover, the yeast can be cultivated to a high cell density with a cheap medium.
S. cerevisiae lacks both amylolytic and cellulolytic activities and is unable to
ferment starchy or cellulosic materials, although they are the most abundant and
utilisable resources of plant origin (Barnett, 1976). In the conventional procedure to
convert starchy materials to ethanol at a high yield, the mash should be cooked at 140-
180°C prior to amylolysis and the energy consumed in this process results in high
production costs (Maiorella, 1985). To save energy in the cooking process, the
development of a non-cooking fermentation system using the enzymes, which
efficiently digest raw starch, has been led. Intensive research has been conducted to
construct further improved starch-utilising systems by introducing heterologous genes
encoding amylolytic enzymes into yeast cells by the genetic engineering for secretive
production of the enzymes. Construction of a yeast cell secreting glucoamylase from
R. oryzae has been attempted for application to fermentation of raw starch without
cooking (Ashikari et al., 1989). Glucoamylase from R. oryzae was an exo-type
amylolytic enzyme cleaving α-1,4-linked and α-1,6-linked glucose effectively from
starch. The surface-engineered yeast cell displaying glucoamylase on the cell surface
60
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
may be able to saccharify starch by glucoamylase on its cell wall and assimilate the
released glucose to proliferate and ferment (Fig. 1).
On the other hand, utilisation of cellulosic materials in fermentation has not been
realised successfully. Cellulose, consisting of glucose units linked together by 1,4-
glycosidic bonds, is the most abundant carbohydrate in the biosphere. An estimated
synthesis rate of cellulose is approximately 4x107 tons per year. Cellulose is the most
promising renewable carbon source that is available in a large quantity for a long-range
solution to resource problems of energy. However, yeast S. cerevisiae is unable to
utilise cellulosic materials in spite of its versatility in industrial fermentation.
Enzymatic hydrolysis of cellulose has the potential to surmount many of the drawbacks
of acid hydrolysis. The cellulase system of the anaerobic cellulolytic bacterium,
Clostridium thermocellum, was shown to naturally construct a discrete multi-enzyme
complex located on the cell surface (Lamed et al., 1983; Tokatlidis et al., 1991),
"cellulosomes", which is considered to help C. thermocellum to obtain the source of
carbon and energy efficiently by enzymatic degradation of cellulose on its cell surface.
An attempt to genetically display cellulolytic enzymes in their active form on the cell
surface has been tried to construct a novel cellulose-utilising yeast, S. cerevisiae (Murai
et al., 1997b, 1998b). As one of the target enzymes, carboxymethylcellulase (CM-
cellulase) from Aspergillus aculeatus classified as endo-1,4-β-D-glucan glucohydrolase
(endoglucanase) which cleaves the β-1,4-glycosidic linkage of cellulose was utilised.
Since CM-cellulase is an endo-type cellulase, enzymatic degradation of cellulose to
glucose requires synergistic hydrolysis by different types of cellulolytic enzymes. It is
predicted that short-chain cellooligosaccharides formed by the endo-action of CM-
cellulase is converted quickly to glucose by β-glucosidase (1,4-β-D-glucoside
61
MITSUYOSHI UEDA et al.
Figure 2. Structure of α-agglutinin and partial amino acid sequence of its C-terminal
region containing GPI anchor attachment signal sequence (A), and the molecular design of
cell surface-displayed enryme (B). A: arrows indicate the predicted cleavage site. The
predicted ω site is underlined.
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MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
To target heterologous proteins to the outmost surface of the glycoprotein layer of the
cell wall, molecular information of α-agglutinin was utilised (Murai et al., 1997a). The
anchoring signal of α-agglutinin was combined with the signal of the secreted
enzymes using genetic engineering techniques. Fig. 2 shows the general structure of
the gene for cell surface display of an enzyme. The C-terminal half of α-agglutinin
(320 amino acid residues) contains a glycosylphosphatidylinositol (GPI) anchor
attachment signal at the C-terminal end, like other cell surface proteins, and is utilised
as an anchoring domain for heterologous proteins since these proteins are covalently
linked with glucan.
The cell wall of S. cerevisiae is mainly composed of mannoproteins and β-linked
glucans (Fleet, 1991), and has a bi-layered structure consisting of an internal skeletal
layer of glucan, composed of β-1,3- and β-1, 6-linked glucose (Manners et al., 1973),
and a fibrillar or brush-like outer layer, which is composed predominantly of
mannoproteins (Horisberger and Vonlanthen, 1977). These proteins are linked to glucan
through covalent bond. Two types of mannoproteins are present in the cell wall of S.
cerevisiae (Klis, 1994; Cid et al., 1995). Mannoproteins loosely associated with the
cell wall through non-covalent bond are extractable with sodium dodecylsulfate (SDS).
The other type of mannoproteins are extractable by glucanase, which are released by p-
1, 3- or β-1, 6-glucanase digestion of the glucan layer of the cell wall but not by SDS
extraction (Fleet and Manners, 1977). Among these glucanase-extractable
mannoproteins on the cell surface of S. cerevisiae, the mating-type specific agglutinins
(Lipke and Kurjan, 1992) that mediate direct cell-cell adhesion between cells of
opposite mating type during mating, which are supposed to be located on the outermost
surface. Mating type a and α cells express a-agglutinin and a-agglutinin, respectively
(Terrance et al., 1987). α-Agglutinin is encoded by AGα1 gene (Lipke et al., 1989) and
interacts with the binding subunit of the agglutinin complex of a cells (Cappellaro et al.,
1991). A-agglutinin consists of a core subunit encoded by AGA1 gene (Roy et al.,
199 1) that is linked through disulfide bridges to a small binding subunit encoded by a
different gene AGA2 (Cappellaro et al., 1991). The structures of both a-agglutinin and
the core subunit of a-agglutinin are composed of a Secretion signal region, an active
region, a support region rich in serine and threonine, and a putative GPI anchor
attachment signals, and presumably a heavily O-glycosylated form (Wojciechowicz et
al., 1993).
The structure of GPI anchors is highly conserved among molecules from various
organisms (Ferguson and Williams, 1988). The core structure of the yeast GPI anchor
is similar to that found in other eucaryotes (Lipke et al., 1989); ethanolamine
phosphate(6) mannose(α1,2) mannose(α1,6 )mannose(α1,4)glucosamine-(α1,6)-
inositol phospho-lipid (Fig. 3). Many of cell surface proteins in yeast, for example,
Agα1 (Lipke et al., 1989), Aga1 (Roy et al., 1991), Flo1 (Watari et al., 1994), Sed1
(Hardwick et al., 1992), Cwp1, Cwp2, Tip1,Tir1/Srp1 (Van der Vaat et al., 1995), have
GPI anchors, which play important roles for surface expression of cell-surface proteins
and are essential for the viability of yeasts. These glycophospholipid moieties are
covalently attached to the C-termini of proteins and their primary function is to afford
the stable association of proteins with the membrane. GP1-anchored proteins contain
63
MITSUYOSHI UEDA et al.
hydrophobic peptides at their C termini. After the completion of protein synthesis , the
precursor protein remains anchored in the endoplasmic reticulum (ER) membrane by
the hydrophobic carboxyl-terminal sequence, with the rest of the protein in the ER
lumen. The hydrophobic carboxyl-terminal sequence is cleaved at the site and
concomitantly replaced with GPI anchor presumably by a transamidase. The
localisation of both α-agglutinin and α-agglutinin to the cell surface occurs through the
secretory pathway (Tohoyama and Yanagishima, 1987) (Fig, 3). Secreted proteins are
first translocated into the lumen of the ER, and then transported from the ER to the
Golgi apparatus and from there to the plasma membrane in membrane-enclosed vesicles
(Schekman, 1992). Fusion of the Golgi-derived secretory vesicles with the plasma
membrane releases the secreted proteins to the cell exterior. Post-translational
proteolytic modification of precursors of secretory peptides occurs in the late
compartments of the secretory pathway (trans cisternae of the Golgi apparatus and
secretory vesicles). α-Agglutinin was proposed to be further transported to the outside
of the plasma membrane through the general secretory pathway in a GPI-anchored form
and then released from the plasma membrane by phosphatidylinositol-specific
phospholipase C (PI-PLC) and transferred to the outmost surface of the cell wall (Lu et
al., 1994).
Figure 3. Transportation of a-agglutinin to cell surface (A) and the structure of GPI
anchor (B), A: ER, endoplasmic reticulum: PI-PLC, phosphatidylinositol-specific
phospholipase C. B: a, ethanolamine phosphate bridge; b, glycan; c, inositol. Man,
Mannose; GlcNH2, glucosamine.
64
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
65
MITSUYOSHI UEDA et al.
Figure 4. Structures ofplasmids for displaying of enzymes. pGA11 (A) and plGA1I (B), for
displaying of glucoamylase; pIAA1I (C), for displaying of α-amylase; pCMC11 (D), for
displaying of CM-cellulase (CMCase); pBG21 1 (E), for displaying of β-glucosidase.
66
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
67
MITSUYOSHI UEDA et al.
Figure 5. Aerobic cultivation of S. cerevisiae MT8-1 cells harbouring pGA11 (A) and
harbouring various in tegrative glucoamylase and α-amylase/α-agglutinin fusion genes (B)
in the medium containing starch as the sole source of carbon and energy, and aerobic
cultivation of arming cells in the medium containing cellobiose (C) and
cellooligosaccharides (D) as the sole source of carbon and energy. A, cell growth ( );
starch concentration ( ); ethanol produced ( ); glucoamylase activity in cell pellet (
); glucoamylase activity in culture medium ( ). B, cell growth of the cells harbouring no
plasmid ( ), pIGA11 ( ), pIAA ( ), and pIGA11/pIAA∆ ( ); the concentration of
starch in the culture medium of the cells harbouring no plasmid ( ), pIGA11 ( ), and
pIGA11/pIAA∆ ( ). C and D, symbols for cells: S. cerevisiae MT8-1 ( ); MT8-1
harbouring pCMC11 ( ); MT8-1 harbouring pBG211 ( ); MT8-1 harbouring pCMC11
and pBG211 ( ). C, Cell growth was monitored by absorbance of culture broth at 600
nm. D, Cell growth was monitored by counting colonies appeared on plates.
68
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
The arming cells with both enzymes were cultivated aerobically in the medium
containing cellobiose as the sole carbon source, and cell growth was monitored (Fig. 5).
Cells harbouring pBG211 and cells harbouring pCMC11 and pBG211 could grow on
cellobiose and reached the absorbance of about 2, which was a little lower than that in
the case of the culture on 1% glucose, while no growth on cellobiose was observed with
the control cells and cells harbouring pCMC11. The arming cells were cultivated
aerobically in the medium supplemented with cellooligosaccharides (approximately
11% (W/W) cellohexaose, 29% (W/W) cellopentaose, 33% (W/W) cellotetraose, 17%
(W/W) cellotriose, 4% (W/W) cellobiose, and less than 1% (W/W) glucose) as the sole
carbon source and the cell growth was monitored by counting colonies appeared on
YPD plates. The cells harbouring pBG211 and cells harbouring pCMC11 and pBG211
could grow on cellooligosaccharides employed, while no growth on
cellooligosaccharides was again observed with control cells and cells harbouring
pCMC11. Since β-glucosidase was reported to be capable of degrading
cellooligosaccharides with glucose units of 2 to 6 (Sakamoto et al., 1985), the
breakdown of cellooligosaccharides by β-glucosidase seemed to be sufficient to sustain
the growth of the yeast displaying this enzyme. However, the difference between the
growth of the cells harbouring pCMC11 and pBG211 and that of the cells harbouring
pBG211 suggested that the yeast strain co-displaying CM-cellulase and β-glucosidase
had the enhanced ability to degrade cellooligosaccharides.
69
MITSUYOSHI UEDA et al.
When lipase of R. oryzae was displayed as mentioned above, little activity was
observed on the cell surface. The reason of this phenomenon seemed to be derived from
the fact that the active site of lipase resides in the C-terminal domain of the molecule.
Fusion of the C-terminus of lipase with the 3'-half of α-agglutinin may inhibit the
access of substrates to the active site. Therefore, to insure the free movement of the
lipase molecule, a spacer of an appropriate length was inserted between these two
molecules. This system will be further proved to be effective for the cells to express the
lipase activity on the cell surface, since the length of the spacer seemed to be affected
the enzyme activity.
Schreuder et al. (1993, 1996) reported that they had succeeded in targeting α-
galactosidase from Cyamopsis tetragonoloba seeds, as a reporter enzyme, to the cell
wall of S. cerevisiae. However, the engineered S. cerevisiae cells described here are the
first example of yeast in which proteins were targeted to the cell surface and endowed
the cells with new beneficial properties. These surface-engineered yeast cells were
termed "Arming yeasts" (Anonymous, 1997). The displayed enzymes are also
regarded as a kind of self-immobilised enzymes on the cell surface, this phenomenon
being passed on to daughter cells as long as the genes are retained by the cells. This
display system could turn or remake S. cerevisiae into a novel and attractive
microorganism as a whole-cell biocatalyst by surface expression of various enzymes,
especially when target substrates are not able to be taken up by the cells, and will make
it possible to produce renewable self-immobilised biocatalysts.
Here, the combination of the cell surface display system with endowment of an
additional metabolic reaction to the yeast cells was performed by cell surface
engineering. Thus, the cell surface can be regarded as a new target for giving additional
characteristics of metabolic reactions. This research will open a new frontier of cellular
engineering not only in yeast but also in all living cells. From the viewpoint of
70
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
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73
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
Abstract
1. Introduction
analysis is to identify the best alternatives for genetic manipulation that may lead to an
increase of overall flux through a given pathway. The principles of metabolic flux
analysis have proven successful as a tool for pathway analysis, since the carbon
distribution within the cell may be evaluated (Stephanopoulos et al., 1998). Hence, the
study of flux control targets the cellular metabolism ‘locally’, whereas the metabolic
flux analysis serves as a ‘global’ cellular approach where the intracellular fluxes of a
given metabolic network are estimated. In this paper the concepts of metabolic pathway
analysis are described. Furthermore, examples of metabolic pathway analysis applied to
the glycolysis and the galactose metabolism of Saccharomyces cerevisiae will be
referred to for demonstrating the use of this analytical tool in metabolic engineering.
To elucidate which enzyme(s) that controls the flux through a given biochemical route,
it is necessary to quantify the control over the overall flux exerted by the individual
enzymes. The metabolic control analysis aims at calculating the flux control coefficients
(FCC’s) of the individual enzymatic steps in a given pathway in order to quantify which
enzyme(s) that is (are) responsible for the control over the flux. The FCC’s are
normalised to one and they describe the relative change in steady-state flux (Jk) through
reaction k caused by an infinitesimal change in enzymatic activity (vi) of enzyme i, as
depicted in equation 1 where CiJk designates the flux control coefficient. Thus, the closer
a FCC is to one, the more control over flux is exerted by the respective enzyme.
(1)
The FCC’s can directly be obtained from measurements of the flux at various enzymatic
activities or indirectly by calculation of the elasticity coefficients (εij) (equation 2), that
describe the relative change in enzymatic activity (vi) caused by an infinitesimal change
in the concentration of metabolite j. Assuming validity of the flux-control summation
theorem (equation 3) and the flux-control connectivity theorem (equation 4) (Kacser
and Burns, 1973), the elasticity coefficients can be applied for deduction of the FCC’s.
(2)
76
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
(3)
(4)
In contrary to the metabolic control analysis that relies on the kinetics of the pathway
examined, the metabolic flux analysis consists of a stoichiometric model where the
metabolic pathway fluxes that span a biochemical network can be estimated by applying
mass balances around the intracellular metabolites of the model i.e. metabolite
balancing, or by determination of the fractional enrichment when using labelled
substrate. By comparison of flux distributions, the metabolic flux analysis can serve as
an analytical tool for identification of physiological changes that arise from changing
the operating conditions or for comparison of the carbon distribution in different
mutants. Thus, the application of the metabolic flux analysis can help investigating the
cellular control of a certain biochemical branch point in order to clarify the rigidity of a
certain pathway node. Furthermore, information regarding alternative pathways may
also be obtained from metabolic flux analysis by including questionable pathways in the
stoichiometric model and then concluding the lack or existence of the given pathway by
virtue of its ability to satisfy the metabolite balances.
For the performance of metabolic flux analysis based on metabolite balancing a
complete stoichiometric model of the cell should be set up which includes J intracellular
reactions comprising K pathway metabolites. The mass balances of the K metabolites
are given in vector notation as depicted in equation 5 where Xmet designates the
concentration of the K metabolites and rmet designates the vector with all the volumetric
net rates of formation of the metabolites in the Jreactions:
77
SIMON OSTERGAARD, LISBETH OLSSON, JENS NIELSEN
(5)
Due to the high turnover of the metabolite pools, the change in metabolite
concentrations rapidly adjusts to a new level, and hence, pseudo steady-state may be
assumed (dXmet/dt = 0). Since the metabolite levels generally are very low, the dilution
of the metabolite pool due to biomass growth (described by the term is
negligible. This assumption is further supported by the fact that the fluxes affecting a
given metabolite pool are significant higher than the effect of dilution. Thus, rmet equals
zero, and as the rmet-vector may be written as the product between the stoichiometric
matrix GT comprising the stoichiometry of the J reactions organised in columns and the
v-vector containing the individual rates of the J reactions, equation 6 forms the basis for
metabolite balancing.
(6)
This equation represents K linear algebraic balances with J unknown parameters equal
to the number of pathway fluxes. This system has J-K degrees of freedom, and
consequently, when J-K reaction rates are measured, it is possible to obtain estimates
for all the pathway fluxes of the v-vector by solving the linear algebraic balances. To do
so, all the measured rates are collected in a new vector, vm, and the residual rates that
are to be calculated, are collected in another vector, vc. Furthermore, the stoichiometric
matrix GT is divided into two sub-matrices GmT and GcT which contain the stoichiometry
of the reactions to be measured and the stoichiometry of the residual reactions,
respectively. Hence, equation 6 may be written as equation 7, and by rearrangements of
this equation, the vector vc containing all the calculated reaction rates of the metabolic
network can be computed as shown in equation 8.
(7)
(8)
The use of metabolite balancing for estimation of intracellular fluxes may serve as a
valuable tool for determination of net fluxes within a metabolic network. Nonetheless,
this approach does not allow for flux determinations of reversible reactions and for the
quantification of flux ratios between biosynthetic pathways leading to the same
metabolite. To get information about the fluxes in these cases it is necessary to apply
labelled substrates, Metabolic flux analysis using labelled substrate approaches the
metabolism from an even further ‘microscopic’ view compared with metabolite
78
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
balancing as the pathway fluxes are estimated from balances around each individual
carbon atom in the intracellular metabolites. Hence, the method may also allow for
calculation of the flux ratio between two biosynthetic routes that lead to the same
metabolite, if the two biosynthetic pathways discriminate differently between the
individual carbon atoms (Sonntag et al., 1993). Likewise, the method may also be used
for estimation of reversible fluxes and not only net-fluxes as obtained from metabolite
balancing (Marx et al., 1996; Fiaux et al., 1999). Furthermore, the use of labelled
substrate may also help to elucidate compartmentation to gain information about the
location of certain biosynthetic reactions within the cell (Pasternack et al., 1994). With
additional biochemical knowledge regarding the cellular structure, an extended
metabolic model may be established describing the cellular metabolism even more
accurately. The concepts of metabolic flux analysis using labelled substrate(s) have
been extensively described in Wiechert and de Graaf (1 996).
Having established the structure of a kinetic and/or a stoichiometric model for the
performance of metabolic pathway analysis, it is necessary to obtain values for the
model parameters. For indirectly determination of the FCC’s of metabolic control
analysis, the intracellular metabolite levels of the all compounds included in the kinetic
model can be used, and to estimate the flux distribution of the metabolic network
established, the rates included in the vm-vector are needed. For the analysis of microbial
cells it is possible to use submerged fermentation experiments for analysis of the
cellular metabolism. Furthermore, through the use of continuous cultures – often
referred to as chemostats – it is possible to study the cellular metabolism at a steady
state. By controlling the volumetric feed rate of medium (F) fed to the bioreactor, and
keeping the volume constant by having a simultaneous out-flow of medium from the
bioreactor, the specific growth rate µ can be controlled at any value, since it equals the
dilution rate D at steady-state. This is observed from the mass-balance of biomass (X)
over the bioreactor as shown in equation 9, i.e. no accumulation of biomass ( dX/dt = 0).
(9)
Furthermore, the volumetric formation rates of the extracellular metabolites (rp) which
may be included in the vm-vector mentioned in section 2.2., are easily obtained when
measuring the concentration of the products in the bioreactor, since the volumetric
formation rates are calculated by multiplication of the dilution rate with the
concentration of the extracellular metabolites at steady-state. This is can be seen from
equation 10 which shows the mass balance of a given product P over the bioreactor
where dP/dt equals zero at steady-state.
79
SIMON OSTERGAARD, LISBETH OLSSON, JENS NIELSEN
(10)
Thus, from continuous operated cultivations, input parameters for the metabolic
pathway analysis such as concentrations of extracellular and intracellular metabolites
may be obtained in a highly reproducible fashion under preferable physiological
conditions regarding the specific growth rate. The continuous operated cultivations also
give the possibility to examine dynamic physiological changes of a cellular system
caused by specific perturbations. These perturbations could be obtained from a pulse of
the limiting component that is added to the bioreactor momentarily in order to follow
the cellular response regarding substrate uptake and product formation rates. Also a step
change in dilution rate may be of interest in order to study the dynamics of a culture
when the specific growth rate, consequently, increases or decreases from one level to
another. Furthermore, continuous cultivations may also be used for physiological
studies of metabolic regulation such as glucose control (Klein et al., 1998) exerted on
the metabolism of slow fermentable carbon sources. To establish repressible conditions
within the medium, a high sugar concentration should be obtained which can be
achieved by operation of the bioreactor under nitrogen-limited conditions.
The concepts described in the previous sections have been applied to Saccharomyces
cerevisiae which have provided additional information to the understanding of yeast
metabolism. Galazzo and Bailey (1990) established a kinetic model describing the
anaerobic fermentation pathway from glucose to ethanol, glycerol, and polysaccharides
in order to characterise the physiological response caused by a change in the external
pH in suspended and immobilised yeast. In vivo phosphorus-31 nuclear magnetic
resonance measurements for determination of the intracellular concentrations of
substrates and effectors in addition to the kinetic expressions of the model were used for
calculation of the FCC’s as described in section 2.1. From this study the authors found
that control over flux was mainly exerted by the glucose uptake in the suspended cells
regardless of the extracellular pH of 4.5 or 5.5 (FCC = 0.83 and FCC = 0.64,
respectively). At the latter pH, the ATPase membrane ion pump also exhibited
considerable control over ethanol production with a FCC of 0.24, since ATP is not
consumed as rapidly by the ATPase at pH 5.5 compared with pH 4.5 in order to
maintain the intracellular pH. In immobilised cells the glucose uptake has a lower
impact, however still considerable, on the ethanol production (FCC = 0.31 and FCC =
0.30 at pH 4.5 and pH 5.5, respectively) compared with suspended cells. The control
over flux in the immobilised cells was mainly exerted by the phosphofructokinase (FCC
= 0.50 and FCC = 0.33 at pH 4.5 and pH 5.5, respectively), but also in these cells the
ATPase exhibited considerable control over ethanol production with a FCC of 0.24
80
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
when grown at an extracellular pH of 5.5. This study demonstrated the use of metabolic
control analysis for explaining the physiological differences between suspended and
immobilised yeast cells. Hence, metabolic control analysis may be used for
identification of the best alternatives for genetic manipulation but also for description of
metabolic differences in a quantitative fashion.
Another strategy different from metabolic control analysis but also involving kinetic
modelling, was taken for pathway analysis of the glycolysis in S. cerevisiae (Rizzi et al.,
1997; Theobald et al., 1997). The kinetics of the glycolysis was examined in order to
understand the regulation of the yeast metabolism during dynamic conditions. Theobald
et al. (1997) established a rapid sampling technique to measure the dynamic response of
the glycolytic intermediates, co-metabolites, and extracellular metabolites of S.
cerevisiae caused by a physiological perturbation. In order to predict the changes of the
intracellular and extracellular metabolite levels during dynamic conditions, a kinetic
model of the individual enzymatic steps of the glycolysis was set up (Rizzi et al., 1997).
The model comprised rate equations for the individual enzymatic steps of the glycolysis
and these were included in material balances for the individual metabolites, the co-
metabolites, and the extracellular metabolites. The dynamic response that arose from a
glucose pulse added to a glucose-limited continuous grown culture of S. cerevisiae
operated at a dilution rate of 0.1 h-1, was simulated by solving the material balances for
all these metabolites. To fit the experimental observations of the glucose pulse, the
model was structured into a cytoplasmic and a mitochondrial compartment but with
translocation of the adenine nucleotides. The model was able to simulate the steady state
situation of the glucose-limited continuous cultivation and the transient response of the
metabolite levels after a glucose pulse. The work of Rizzi et al. (1997) and Theobald et
al. (1997) illustrates the use of continuous operated cultivations as a great tool for
kinetic analysis of microorganisms, and furthermore, the studies also demonstrate the
valuable use of mathematical modelling for pathway analysis in order to improve the
understanding of the yeast metabolism.
The GAL genes of S. cerevisiae encoding the enzymes of the galactose utilisation
pathway, serve as one of the best studied models of genetic regulation in eukaryotic
systems (reviewed by Johnston and Carlson, 1992, and Melcher, 1997). The GAL genes
are tightly regulated being induced by galactose and strongly repressed by glucose. The
positive tramcriptional activator protein encoded by the GAL4 gene induces the
expression of the structural GAL genes in the presence of galactose. The genes GAL6,
GAL80, and MIG1 encode the proteins responsible for the down-regulation of the
structural GAL genes that encode the enzymes responsible for uptake of extracellular
galactose and subsequent conversion of intracellular galactose to glucose-6-phosphate
(known as the Leloir pathway). GAL2, GAL1, GAL7, GAL10, and GAL5 constitute the
structural GAL genes encoding the following enzymes: galactose permease,
galactokinase, galactose- 1 -phosphate uridylyltransferase, UDP-glucose 4-epimerase,
and phophoglucomutase, respectively, which are shown in Figure 1.
81
SIMON OSTERGAARD, LISBETH OLSSON, JENS NIELSEN
Figure I The Leloir pathway containing the enzymes responsible for conversion of
galactose io glucose-6-phosphate See the text for specific nomenclature of the enzymes
The galactose metabolism is interesting to approach with the tools of metabolic pathway
analysis in order to investigate the remarkable physiological differences that exist
between glucose and galactose metabolism. Metabolic control analysis was used for
‘locally’ examination of the galactose utilisation pathway (Østergaard et al., 1998), and
metabolic flux analysis was carried out to elucidate the ‘global’ physiological effects on
the entire metabolism caused by deletion of the two regulatory genes GAL80 and MIG1.
To identify which enzymes control flux through the Leloir pathway, the FCC’s were
determined indirectly as described in section 2.1., and hence, a kinetic model of the
Leloir pathway was set up followed by calculation of the elasticity coefficients from the
rate expressions of the individual enzymatic reactions. The explicit values for the
elasticity coefficients were obtained from measurements of the intracellular
concentrations of the intermediates of the Leloir pathway from a galactose-limited
continuous cultivation operated at a dilution rate of 0.1 h-1, Thus, the FCC’s could be
obtained by use of the summation and the connectivity theorem as described in section
2.1. The only unknown variable of the model was the intracellular galactose
concentration, and hence, the FCC’s were computed as a function of the intracellular
galactose concentration as depicted in Figure 2.
According to intracellular measurements of the glucose concentration when the
extracellular concentration was at or below the affinity of the transport system (1-2
mM), the intracellular glucose concentration was less than 0.1 mM (Teusink et al.,
1998). Assuming the same to be the case for the galactose metabolism when the
galactose concentration in the bioreactor was 1 mM, the intracellular galactose
concentration was at or below 0.1 mM. Consequently, from this metabolic control
analysis it was concluded that the control over flux through the Leloir pathway is
mainly exerted by the galactose permease (Gal2) under the physiological conditions
examined. Experimental work is in progress to examine the results of this analysis in
order to gain more insight to the galactose metabolism of yeast.
82
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
The glucose and the galactose metabolism were also studied in nitrogen-limited
continuous cultivations to examine the physiological differences between these two
sugars. It was of interest to investigate the physiological role of the proteins encoded by
the MIG1 and the GAL80 gene which are strongly involved in glucose control exerted
on the galactose metabolism. Mig1 takes part of a protein complex comprising Ssn6,
Tup1, and Mig1 of which the latter directs the complex to a specific consensus motif on
the promoters of the target genes whereby transcription of the target genes does not
occur (Keleher et al., 1992; Treitel and Carlsson, 1995). Mig1-mediated glucose control
not only affects the GAL genes, but also the MAL genes responsible for maltose
utilisation and the SUC genes encoding invertase that hydrolyses sucrose. In contrary,
Gal80 only acts as a negative regulatory protein on the GAL genes by binding to the C-
terminal end of Gal4 which prevents transcriptional activation by this protein. The
specific sugar uptake rates of a mig1 gal80 double mutant strain obtained from nitrogen-
limited cultivations on glucose and galactose, respectively, were compared with the
corresponding specific sugar uptake rates obtained for the wild type strain (CEN.PK
113-7D).
A stoichiometric matrix of the aerobic yeast metabolism was established to estimate
the flux distributions of the wild type strain and the mig1 gal80 mutant strain from the
data obtained by the nitrogen-limited continuous cultivations on either glucose or
galactose. By measuring the uptake rates of glucose and galactose in addition to the
product formation rates of pyruvate, ethanol, acetate, succinate, glycerol, ammonium,
and the biomass composition, it was possible to compute the vc-vector as described in
section 2.2., and hence, obtain flux distributions over the metabolic network established.
The uptake rates of glucose and galactose of the wild type strain were 4.3 mmol/gDW/h
and 2.7 mmol/gDW/h, respectively, and the corresponding uptake rates of the mig1
83
SIMON OSTERGAARD, LISBETH OLSSON, JENS NIELSEN
gal80 mutant strain were 3.4 mmol/gDW/h and 3.8 mmol/gDW/h. Thus, the deletion of
the MIG1 and the GAL80 genes had a remarkable impact on the uptake rates of these
two sugars by decreasing the specific glucose uptake rate and increasing the specific
galactose uptake rate. Some of the fluxes estimated from these four cultivations are
shown in Figure 3.
Figure 3 Flux distributions (given in mmol/gDW/h) obtained for the wild type strain (left)
and the mig1 gal80 mutant strain (right) when grown in nitrogen-limited continuous
cultivations on glucose or galactose (italic). Only selected fluxes are shown for simplicity
The metabolic flux analysis provides valuable information about the cellular control of
the pyruvate branch point. It is observed that the flux entering the TCA-cycle from the
pyruvate node is constant for the two strains examined but it varies between glucose and
galactose. The flux entering the TCA-cycle was 1.6-1.7 mmol/gDW/h when the two
strains were grown on glucose, and 2.1-2.2 mmol/gDW/h for both these two strains
when grown on galactose. Hence, it is concluded that not only growth on glucose results
in overflow metabolism where the residual carbon above the maximum capacity
entering the TCA-cycle is directed towards acetaldehyde formation, and subsequently
ethanol formation, but also galactose exerts a similar response onto the metabolism.
Although both glucose and galactose enter the metabolism at the glucose-6-phosphate
node, the figures of the fluxes entering the TCA-cycle from the pyruvate node show that
the maximum capacity entering the TCA-cycle is dependent on the specific sugar
consumed. Thus, the metabolic flux analysis has demonstrated its potential by
identification and quantification of metabolic changes caused by genetic manipulation,
and this approach enabled us to study the control mechanism around the pyruvate node.
84
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
Acknowledgements
This work has been partly supported financially by the Danish Programme for Food
Technology II (project 2409) as well as by European Commission Framework IV “Cell
Factory” (contract BIO-CT95-0 107). Thanks to Kristian 0. Walløe for performance of
the nitrogen-limited cultivations on glucose and galactose.
References
Fiaux, J., Anderson, C.I.J., Holmberg, N., Bülow, L., Kallio, P.T., Szyperski, T., Bailey, J.E., and Wüthrich,
K. (1999) 13C NMR flux ratio analysis of Escherichia coli central carbon metabolism in micro-aerobic
bioprocesses, J. Am. Chem. Soc. 121, 1407-1408.
Galazzo, J.L., and Bailey, J.E. (1990) Fermentation pathway kinetics and metabolic flux control in suspended
and immobilised Saccharomyces cerevisiae, Enzyme Microb. Technol. 12, 162-172.
Heinrich, R., and Rapoport, T.A. (1974) A linear steady state treatment of enzymatic chains. General
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Johnston M., and Carlson, M. (1992) Regulation of carbon and phosphate utilisation, in E.W. Jones, J.R.
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expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., pp. 193-281.
Kaeser, H., and Burns J.A. (1973) The control offlux, Symp. Soc. Exp. Biol. 27, 65-104.
Keleher, C.A., Redd, M.J., Schultz, J., Carlson, M., and Johnson, A.D. (1992) Ssn6-Tup1 is a general
repressor of transcription in yeast, Cell 68,709-719.
Klein, C.J.L., Olsson, L., and Nielsen, J. (1998) Glucose control in Saccharomyces cerevisiae: the role of
MIG1 in metabolic functions, Microbiology 144,13-24.
Marx, A., de Graaf, A.A., Wiechert, W., Eggeling, L., and Sahm, H. (1996) Determination of the fluxes in the
central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy
combined with metabolite balancing, Biotechnol. Bioeng. 49, 111-129.
Melcher, K. (1997) Galactose metabolism in Saccharomyces cerevisiae: A paradigm for eukaryotic gene
regulation, in F.K. Zimmermann, K.-D. Entian (eds), Yeast sugar metabolism, Technomic Publishing Co.,
Inc. Lancaster, Basel, pp. 235-269.
Pastemack, L.B., Laude, D.A., and Appling, D.R. (1994) 13C NMR analysis of intercompartmental flow of
one-carbon unit into choline and purines in Saccharomyces cerevisiae, Biochemistry 33, 74-82.
Rizzi, M., Bakes, M., Theobald, U., and Reuss, M. (1997) In vivo analysis of dynamics in Saccharomyces
cerevisiae: II. Mathematical model, Biotechnol. Bioeng. 55, 592-608.
Sonntag, K., Eggeling, L., de Graaf, A.A., and Sahm, H. (1993) Flux partitioning in the split pathway of
lysine synthesis in Corynebacterium glutamicum, Eur. J. Biochem. 213, 1325-1331.
Stephanopoulos, G., Aristodou, A., and Nielsen, J. (1998) Metabolic engineering, San Diego: Academic
Press.
Teusink, B., Diderich, J.A., Westerhoff, H.V., Dam, K., Walsh, M.C. (1998) Intracellular glucose
concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport
rate by 50%, J. Bacteriol. 180, 556-562.
Theobald, U., Mailinger, W., Baltes, M., Rizzi, M., and Reuss, M. (1997) In vivo analysis of dynamics in
Saccharomyces cerevisiae: I Experimental observations, Biotechnol. Bioeng. 55, 305-316.
Treitel, M.A., and Carlson, M. (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator
protein, Proc. Natl. Acad. Sci. USA 92, 3132-3136
Wiechert, W., and de Graaf, A.A. (1996) In vivo stationary flux analysis by 13C labelling experiments, Adv.
Biochem. Eng. Biotechnol. 54, 109-154.
Østergaard, S., Olsson, L., and Nielsen, J. (1998) Metabolic control analysis of the Leloir pathway in
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85
EFFECT OF AERATION IN PROPAGATION ON SURFACE PROPERTIES OF
BREWERS’ YEAST
Abstract
1. Introduction
2.2 HYDROPHOBICITY
The hydrophobicity index of the cells was determined by their partitioning between a
hydrocarbon and aqueous phase, based on the method of Smart et al. (1995). The cells
were harvested by centrifugation, washed with distilled water, deflocculated with 2 mM
EDTA and rewashed twice with deionised water. The washed cells were re-suspended
to an OD660 of 0.6 – 0.75 in a phosphate-urea-MgSO4 (PUM) buffer at pH 7.1. The
PUM buffer consists of 2.22% (w/v) K2HPO4.3H2O, 0.726% (w/v) KH2PO4, 0.18%
(w/v) urea and 0.02% (w/v) MgSO4.7H2O. The optical density of the suspension was
90
AERATION AND SURFACE PROPERTIES OF BREWERS' YEAST
recorded and 2.4 ml was transferred to a wide mouth test tube. 0.2 ml xylene was added
to the test tube. The mixture was vortexed for 2 minutes, and allowed to stand for 15
minutes. The aqueous phase was removed with a Pasteur pipette and its optical density
determined. The hydrophobicity index was calculated as follows.
(1)
Zeta potential of the growing yeast suspensions was measured using a Malvern
ZetaSizer 4. Washed cells were suspended in 20 mM sodium acetate buffer solutions
with pH values between 2.1 and 6.6 prior to zeta potential measurement. The zeta
potential obtained was converted into surface charge density by using the Gouy
Chapman equation (2), which describes the decay in potential around a charged particle
(Hiemenz 1986).
(2)
(3)
where ψ is the surface potential of the yeast (V) and σ is the surface charge (µC.cm-2).
In this study it is recommended that the charge density at the plane of shear, rather than
zeta potential be used as an indicator of the surface charge of particles as this parameter
removes the differences in experimental conditions used by different research groups.
91
ANDREW ROBINSON AND SUSAN T. L. HARRISON
2.4 FLOCCULATION
3. Results
The biomass yield coefficients were calculated at the end of each propagation and
expressed as the dry mass of cells obtained per mass of total sugars used (g dry weight.g
carbohydrate ).
-1 These values are summarised in Table 1 for both the aerobic and near
anaerobic propagation of the four strains. The increased ratio of aerobic to anaerobic
yields are also presented.
The rate of increase of cells during propagation was modelled using the logistic
equation (Bailey and Ollis, 1986) (4). The integrated form of the equation (5) providing
the cell concentration profile during propagation from the initial cell concentration, x0,
has two parameters, β (ml.cell-1), the inverse of the stationary population concentration
and k (h-1), the rate constant.
(4)
(5)
92
AERATION AND SURFACE PROPERTIES OF BREWERS' YEAST
Since the final population size is known, the logistic equation can be solved to yield the
logistic rate constant. Figure 1 illustrates the prediction of the data by the logistic
equation. The rate constants obtained for each strain are summarised in Table 1. The
ratio of aerobic to anaerobic rate constants is compared.
Table 1. Comparison of biomass yield coefficients and cell production rates of aerobic
and near anaerobic yeast propagation in high gravity brewers’ yeast.
Figure 1. Cell concentration during aerobic and anaerobic propagation of SAB1 with
logistic equation fit to data. aerobic data, ______ aerobic logistic fit, anaerobic data -
--- - ---- anaerobic logistic fit)
3.3 HYDROPHOBICITY
The cell growth profiles and hydrophobicity of the cell surface during propagation are
shown for the four strains in Figure 2. The hydrophobicity of the cells was found to
increase concomitantly with cell growth under anaerobic conditions. The aerobically
grown yeast were less hydrophobic throughout propagation. Once the cells reached the
stationary phase, no further change in hydrophobicity was observed. The
hydrophobicity in the stationary phase of propagation is summarised in Table 2.
93
ANDREW ROBINSON AND SUSAN T. L. HARRISON
The zeta potential profiles of all strains measured across the pH range 2.1 to 6.6 varied
with phase of growth to approach a constant charge profile towards the onset of the
stationary phase. This is illustrated for the aerobic and anaerobic propagation of strain
SAB2 in Figure 3a and 3b. The zeta potential of the aerobically grown strains was
generally lower than the anaerobic equivalent, especially under stationary phase
conditions. The iso-electric points for the stationary phase aerobic yeast were generally
below the measured range of the assay, i.e. < pH 2. The charge profiles of the strains in
their stationary phase of propagation are presented in Figures 4a and 4b for the aerobic
and near anaerobic growth condition.
Figure 2. Increase in cell hydrophobicity with cell growth during propagation of strains
under aerobic and near anaerobic conditions. ((a) SAB5, (b) SAB1, (c) SAB1/96 and (d)
SAB2, aerobic hydrophobicity, _______ aerobic cell concentration, anaerobic
hydrophobicity ---------- anaerobic cell concentration).
Figure 3. Zeta potential profiles for (a) aerobic and (b) anaerobic growth condition of
SAB2 changing smoothly during propagation and reaching a stable profile by the end of
propagation. pre-exponential, X early-exponential, late exponential and stationary
phase)
94
AERATION AND SURFACE PROPERTIES OF BREWERS’ YEAST
Figure 4. Zeta potential of the four strains of yeast at the end of (a) aerobic (b) and
anaerobic propagation showing the different profiles observed. SAB1, SAB2,
SAB1/96 and SAB5).
3.5 FLOCCULATION
A typical absorbance profile for the flocculation assay is shown in Figure 5 illustrating a
change in absorbance from the initial 2.44 to 0.44 after 2 minutes. These absorbencies
correspond to cell concentrations of 97.6 and 3.9 million cells per ml. Based on cell
concentrations the extent of flocculation is 96.4%.
Figure 5. Flocculation assay absorbance profile for SAB5 grown under brewery
conditions.
The three strains of flocculent yeast (SAB1, SAB1/96 & SAB5) were found to be more
flocculent when grown under aerobic conditions as compared to propagation under
anaerobic conditions. It was noticed that the yeast only became flocculent towards the
end of propagation within the wort medium. This coincides with depletion of sugars,
which may cause inhibition of the lectin binding required for flocculation. Samples
taken during propagation were visually seen to be weakly flocculent in flocculation
buffer until the late exponential phase. This coincided with the flocculation behaviour of
95
ANDREW ROBINSON AND SUSAN T. L. HARRISON
the yeasts in situ. The extent of flocculation of yeast harvested in the stationary phase is
summarised in Table 2 for both aerobic and near anaerobic growth conditions. Table 2
includes the zeta potential values at pH 4.5, the same pH as the flocculation buffer, for
reference.
Table 2. Summary of cell hydrophobicity, surface charge and extent of flocculation
values for aerobic and anaerobic propagation of yeast.
4. Discussion
The biomass yield coefficients of the aerobic propagation were consistently 2 to 3 fold
higher than under anaerobic conditions. In addition, aerobic growth yielded higher
logistic growth rate constants. The increased growth rates and higher yields illustrate
that aerobic propagation is advantageous for the biomass production cycle of yeast in
breweries.
Before exploiting the potential advantage of aerobic propagation, the suitability of
yeast produced by aerobic propagation for anaerobic brewing must be assessed.
Brewing yeast is required to have good fermentative abilities as well as suitable
physical characteristics necessary in the brewing environment. The physical attributes
required include a suitable cell envelope structure and the yeasts’ ability to flocculate at
the end of fermentation. Here the surface charge, hydrophobicity and flocculation
potential of the commercial strains are assessed as a function of oxygen availability and
growth phase.
All flocculent strains considered in this study were more charged and yet also more
flocculent when grown aerobically over those grown anaerobically. This suggests that
no correlation between lower zeta potential and increased flocculence exists, contrary to
the prediction of the Derjaguin-Landau-Venvey-Overbeek (DLVO) theory. Smit et al.
(1992) showed that flocculation is most effective at a pH of 4.5. This is the approximate
final pH attained in fermentation at which the yeast flocculates and settles out of
suspension. It is therefore instructive to consider the yeasts’ surface charge at this pH
when studying flocculation. From this study it is concluded that the increased surface
charge associated with the aerobic propagation system does not lower flocculation in
these yeast strains. Previously Amory and Rouxhet (1988), on comparing the surface
charge between top and bottom cropping yeasts, showed the bottom strains to be more
charged (zeta potential of -35 mV, corresponding to a surface charge of -0.12 µC.cm-2)
than the top strains (zeta potential of -12 mV and charge of -0.04 µC.cm-2) at pH 4.0. In
many of the early studies, zeta potentials were not measured in a defined environment.
As these measurements were taken without any background electrolyte addition, even
96
AERATION AND SURFACE PROPERTIES OF BREWERS’ YEAST
the smallest leakage of ions from the cells could alter the potential observed
significantly. Bowen and Cooke (1989) determined zeta potential in wort hence surface
charge can not be calculated. Dengis et al. (1995) found surface charges between -0.15
and -0.21 µC.cm-2 at pH 5.2 for top and bottom fermenting strains of Saccharomyces
cerevisiae. Bowen and Ventham (1994) reported a decrease in zeta potential and
surface charge during fermentation of top and bottom cropping ale yeast as well as lager
yeast. The bottom ale yeast and lager yeast carried similar charge during fermentation,
decreasing from -12 to -7 mV (-0.39 to -0.23 µC.cm-2) on progression from exponential
to stationary phase. The top cropping ale yeast was slightly more charged throughout
fermentation, decreasing from -16 mV to -12 mV (-0.53 to -0.39 µC.cm-2). These zeta
potential measurements were made at the pH occurring during fermentation. Smart et
al. (1995) observed a decrease in surface charge at pH 4.0 (-42.6 to -32.5 mV and -1.54
to - 1.12 µC.cm-2) when yeast was stored under conditions of nutrient starvation. From
all these investigations, no clear correlation can be drawn between the flocculence or
location of flocculated yeast (top vs. bottom) and the surface charge of the yeast. Van
Hammersveld et al. (1994) calculated the bond strength predicted by the DLVO theory
of flocculation. On comparison, they found the experimentally determined bond
strength to be far higher and concluded that the additional bonding energy resulted from
specific biological interactions. However, the parameters within the theory changed
during fermentation in a manner that favoured flocculation predicted by DLVO. Speers
et al. (1993) concurs that the DLVO theory cannot explain flocculence within flocculent
yeast strains.
The inability of physicochemical interactions at the surface to correlate with
flocculation behaviour is further illustrated by trends in hydrophobicity determined in
this study. Here a greater oxygen availability lowered the hydrophobicity but also
increased the cells‘ flocculence. The hydrophobicity index of the yeast in the near
anaerobic propagation increased during the exponential phase of growth while that of
the aerobic propagation remained relatively low. The increase in hydrophobicity
observed at the end of exponential growth, also seen by Smit et al. (1992), was found to
coincide with the onset of flocculation. Smit et al. inferred that the observed
hydrophobicity was attributed to flocculation proteins (lectins). This was supported by a
reduction in both hydrophobicity and flocculation by protease activity. It can be
concluded from these studies that the yeast lectins may contribute to the cells’
hydrophobicity, but this is not the only contributing factor and hydrophobicity alone is
not responsible for flocculation.
The non-flocculent strain (SAB2) showed the similar trend whereby the anaerobic
propagation was less charged while at the same time being more hydrophobic than the
aerobically grown yeast. This strain was observed to have a very low level of
flocculence (<5%) as measured by the flocculation assay used here. For this strain, the
anaerobic propagation condition caused lower charge, higher hydrophobicity and was
more flocculent. This observation is consistent with the prediction of DLVO theory.
Hence it is postulated that the small extent of flocculation found in this yeast strain is a
result of the classical colloidal model while the flocculence observed in the flocculent
strains is governed by lectin binding. The dominance of lectin-mediated flocculation is
indicated.
97
ANDREW ROBINSON AND SUSAN T. L. HARRISON
For all strains considered in this study, the inter-strain variation of surface charge profile
and hydrophobicity was smaller than the variation generated by changes in oxygen
supply or the position within the growth phase. This highlights the necessity to
adequately quantify the oxygen availability during growth as well as the position in the
growth cycle at which comparisons are made when comparing these properties for
different strains or classes of yeast, viz. ale vs. lager or top vs. bottom fermenting yeast.
Flocculation in the three flocculent strains studied here appears to be caused by the
lectin/receptor mechanism described by Miki et al. (1982), as reported for other
commercial strains of Saccharomyces cerevisiae. Such flocculation has been shown to
require calcium ions and the sugars mannose and glucose inhibit the formation of flocs.
According to the classification system of Stratford and Assinder (l991), these strains
belong to the NewFlo phenotype owing to the inhibitory action of glucose. Bony et al.
(1998) linked the level of flocculation observed in yeast to the amount of the
flocculation lectin Flop at the surface of the cell. The protein was also detected in the
endoplasmic reticulum, indicating that it was secreted along the protein excretory
pathway. This pathway is known to be partially repressed under anaerobic conditions,
indicating that flocculation may be repressed under anaerobic conditions as shown here.
Although hydrophobicity and surface charge are not the main cause of yeast
flocculation (van Hammersveld et al. 1994), these may play a role. At low levels of
mixing, a sufficiently high surface charge could prevent the close interaction of
individual cells to permit lectin-receptor interactions and so limit flocculation.
5. Conclusions
Acknowledgements
The technical and financial support of The South African Breweries Ltd is gratefully
acknowledged. Further financial support of the NRF, including sponsorship of AR
through a postgraduate bursary is appreciated.
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AERATION AND SURFACE PROPERTIES OF BREWERS’ YEAST
References
Amory, D.E. and Rouxhet, P.G. (1988) Surface properties of Saccharomyces cerevisiae and Saccharomyces
carlsbergensis: chemical composition, electrostatic charge and hydrophobicity Biochim. Biophys. Acta
938, 61-70.
Bailey, J.S. and Ollis, D.F. (1986) Biochemical Engineering Fundamentals, McGraw-Hill, Singapore.
Bony, M., Barre, P. and Blondin, B. (1998) Distribution of the flocculation protein, Flop, at the cell surface
during yeast growth: the availability of Flop determines the flocculation level. Yeast 14,25-35.
Bowen, W.R. and Cooke, R.J. (1989) Studies of Saccharomyces cerevisiae during fermentation – an in vivo
lectrokinetic investigation. Biotech. Bioeng. 33, 706-715.
Bowen, W.R., Sabuni, H.A.M. and Ventham, T.J. (1992) Studies of the cell wall properties of Saccharomyces
cerevisiae during fermentation. Biotech. Bioeng. 40, 1309-1318.
Bowen, W.R. and Ventham, T.J. (1994) Aspects of yeast flocculation. Size distribution and zeta potential. J.
Inst. Brew. 100, 167-172.
Dengis, P.B., Nélissen, L.R. and Rouxhet, P.G. (1995) Mechanisms of yeast flocculation: comparison of top-
and bottom-fermenting strains. App. Environ. Microbiol. 61, 718-728.
Hiemenz, P.C. (1986) Principles of colloid and surface chemistry. Marcel Dekker Inc., New York.
Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L. (1982) Possible Mechanism for flocculation
interactions governed by gene FLO1 in Saccharomyces cerevisiae. J. Bacteriol. 150, 878-889.
Smart, K.A., Boulton, C.A., Hinchliffe, E. and Molzahn, S. (1995) Effect of physiological stress on the
surface properties of brewing yeasts, J. Am. Soc. Brew. Chem. 53, 33-38.
Smit, G, Straver, M.H, Lugtenberg, B.J. and Kijne, J.W. (1 992) Flocculence of Saccharomyces cerevisiae
cells in induced by nutrient limitation, with cell surface hydrophobicity as a major determinant. App.
Environ. Microbiol. 58, 3709-3714.
Speers, R.A., Durance, T.D., Tung, M.A. and Tou, J. (1993) Colloidal properties of flocculent and non-
flocculent brewing yeast suspensions. Biotechnol. Prog. 9,267-272.
Stratford, M. and Assinder, S. (1991) Yeast flocculation: Flo1 and NewPlo phenotypes and receptor structure.
Yeast 7, 557-574.
Van Hamersveld, E.H., van Loosdrecht, M.C.M. and Luyben, K.ch.A.M. (1994) How important is the
physicochemical interactions in the flocculation of yeast cells? Coll. Surf. B: Biointerfaces 2, 165-171.
99
EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND
PRODUCTION COUPLING OF LACTIC ACID BACTERIA
Abstract
1. Introduction
From the pioneer work of Luedeking and Piret (1959) lactic acid production have been
recognised as partially linked to growth:
(1)
where the constants A and B are the coefficients for growth- and non-growth-associated
production respectively.
However, as previously shown (Amrane and Prigent, 1997), the best way to analyse
growth and production coupling is to plot the amount of lactic acid produced (p - p0) vs.
the biomass formed (x - x0), instead of the graph of the specific production rate as a
function of the specific growth rate. The linear part of this plot corresponded to the
growth-associated production, and its slope was the coefficient for growth-associated
production (parameter A in equation 1), or the product on biomass yield YP/X.
The effect of some culture parameters on growth and production coupling have
been outlined in the available literature. Indeed, contradictory results concerning the
effect of culture pH can be found: according to some authors the uncoupling between
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©2001 Kluwer Academic Publishers. Printed in the Netherlands.
A. AMRANE AND Y. PRIGENT
growth and production increase at acidic pH (Roy et al, 1987; Venkatesh et al, 1993),
while Norton et al (1994) have obtained a very low value for the coefficient B for the
non-growth associated production (0.01) at pH 4.3. Moreover, it has also been reported
that nitrogen starvation was involved in this coupling (Turner and Thomas, 1975).
However, systematic analysis of the effect of the main fermentation parameters on
this linking is not at our knowledge available in the bibliography; this study will be the
aim of the present work.
2.1 MICROORGANISM
Lactobacillus helveticus strain milano used throughout this work was kindly supplied by
Dr A. Fur (Even Ltd, Ploudaniel, France). Stock cultures were maintained on 10% (w/v)
skim milk and deep frozen at -16°C. As required, these cultures were thawed and
reactivated by two transfers in 10% (w/v) skim milk (42°C, 24 h).
2.2 MEDIA
Batch culture was carried out in a 2-1 fermentor (SET 2M, Inceltech, Toulouse, France),
magnetically stirred (300 rpm), at 42°C. The pH was maintained at 5.9 by automatic
addition of 10 M NaOH. The mass of the NaOH solution added for pH control was
continuously recorded, allowing on-line calculation of the quantity of lactate anion
produced at a given pH at each time point; the observed standard deviation was ±1 g l-1,
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CULTURE PARAMETERS, GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA
Seed culture was carried out in a 0.25-1 laboratory-designed glass fermentor equipped
with a sterilisable combination glass electrode (Ingold, Paris, France), cotton plug filter,
magnetic stirrer, infrared lamp temperature control (set at 42°C), and an aseptic transfer
line.
In addition, the two fermentors were equipped with an aseptic recirculation loop
(Watson-Marlow 50 1 U peristaltic pump; Volumax, Montlouis, France) incorporating a
laboratory-made turbidimeter. As the turbidity was continuously recorded, the total
biomass could be calculated on-line after dry weight calibration; the observed standard
deviation was 10.2 g l-1.
Bacteria were precultivated by inoculating sterile culture medium with 0.8 % (v/v)
of the second skim milk transfer. Then, 1.6 1 of pasteurised culture medium was
inoculated with 0.2 1 seed culture (11% v/v), unless specified, and the reaction
proceeded.
The concentration of total (p) and undissociated (HL) lactic acid was calculated
using the Henderson-Hasselbach equation (pKA = 3.8), the lactate concentration (L-)
and the corresponding pH value:
(2)
and
(3)
At the end of both the preculture and culture, the final biomass, lactose, and lactic acid
concentrations were determined as previously described (Amrane and Prigent, 1994).
The effect of preculture conditions on the subsequent culture were first considered. As
observed in Fig.1a, the preculture medium seemed to have no effect on the coupling
between growth and production. Indeed, for both cultures carried out on RM medium
and inoculated at 11 % seed level, growth and production were associated until 20-25 g
l-1 lactic acid was produced, i.e. approximately half of the production; in the associated
part of the curve, the same product on biomass yield (YP/X = 4.7, corresponding to the
slope of the curve) was found for both batches. To examine the effect of inoculation
level, another run (2) was carried out on RM medium, but inoculated at 6.3 % (v/v)
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A. AMRANE AND Y. PRIGENT
instead of 11 % (additional pasteurised culture medium was fed to the fermentor to keep
the total volume constant), with cells precultivated on RM medium too. No effect of the
inoculation level appeared at the examination of Fig.1a: as above 20-25 g l-1 lactic acid
was produced by an associated mechanism and YP/X = 4.7.
Figure 1: (a) Product on biomass yield for cells precultivated for approximately 13 h: on
whey supplemented with 5 g l-1 yeast extract and inoculated at 11 % seed level, run 1 (A); on
RM and inoculated at 6.3% seed level, run 2 on RM and inoculated at 11 % seed level,
run 3 ; linear fitting (–). (b) Product on biomass yield for cells precultivated on RM for
13.3 h: run 3 (0); 7.5 h: run 4 4.7 h: run 5 (A) ; linear fitting for runs 3 (.....), 4 (–)
and 5 (–•–•).
From Fig.1b, it appeared that the slope YP/X for the plot (p - p0) vs. (x - x0) increased in
the following order for the three preculture duration tested: 7.5 h < 13.3 h < 4.7 h.
Therefore, and by comparison with growth and production kinetics (Amrane and
Prigent, 1998a), product on biomass yield increased when growth and production rates,
as well as maximum cellular concentration, decreased.
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CULTURE PARAMETERS, GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA
As observed in Fig.2, the product on biomass yield displayed a minimum value of 3.7
for 10 g l-1 yeast extract supplementation; as previously demonstrated by derivation of
growth and production parameters with respect to the YE concentration (Amrane and
Prigent, 1998b and 1999), this corresponded to the best performances at minimum cost.
The end of coupled production was arbitrarily defined as the point for which a 3 %
deviation from the oblique asymptotic line was observed. From this, the part played by
an associated mechanism in total production increased continuously with the yeast
extract (YE) supplementation, from 15 % for 5 g l-1 YE to 83 % for 30 g l-1 YE, i.e. an
almost total coupling between growth and production; this had to be brought together
with previous result demonstrating that increasing YE supplementation resulted in a
shift from a nitrogen limitation to a carbon one (Amrane and Prigent, 1998b).
Figure 2: Product on biomass yield for batch cultures of L. helveticus carried out on
clarified whey supplemented with a range ofyeast extract concentrations.
Figure 3: Product on biomass yield for batch cultures of L. helveticus carried out with
various initial lactic acid concentrations p0 (g l-1) : 0 –.–), 10 – –), 40 and
50 –)
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A. AMRANE AND Y. PRIGENT
The experiments displayed in Fig.3 were carried out on a largely nitrogen supplemented
medium (RM) to avoid any nitrogen and growth factors limitation. As observed, at a
given pH control (5.9), the proportion of lactic acid produced by an associated
mechanism was not a function of the lactate initially added, about 60 % of the total
production irrespective of the amount of lactic acid initially added; even if for 50 g 1-1 of
initial lactic acid, the critical concentration (83 g l-1, Amrane and Prigent, 1998c) was
achieved before a total lactose exhaustion.
From Fig.3 also, increasing initial lactic acid additions resulted in increasing product
on biomass yield, from 4.2 without initial addition to 15.0 for p0 = 50 g l-1.
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CULTURE PARAMETERS, GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA
4. Conclusions
For the optimisation of the bioconversion of lactose into lactic acid, high values of the
product on biomass yield YP/X were not a convenient criterion, since they correspond
to a low coupling between growth and production: indeed the fermentation time was
minimal, and the volumetric productivity of the bioreactor was maximal when both
processes were almost totally coupled.
Neither the composition of the seed culture medium nor the inoculation level had
significant effect on YP/X and the percentage of associated production of the subsequent
culture: among the preculture parameters, only the seed culture duration, i.e. the
physiological state of cells, had such an effect.
A high concentration of lactate in the culture medium (50 g l-1) resulted in a large
increase of YP/X when the total lactate concentration was close to the critical one
(83 g 1 1), but it had practically no effect on the coupling.
Acidic culture pHs, leading to high proportions of undissociated lactic acid, resulted
in a large increase of YP/X and a large drop of associated production. A similar
behaviour was observed under conditions of nitrogen limitation (yeast extract
concentration from 2 to 10 g l-1). Therefore it should be pointed out that concentrations
of yeast extract in excess of 10 g l-1 led to a significant increase of YP/X.
Acknowledgements
References
Amrane, A. and Prigent, Y. (1994) Mathematical model for lactic acid production from lactose in batch
culture : model development and simulation. Journal of Chemical Technology and Biotechnology 60,
241-246.
Amrane, A. and Prigent, Y. (1997) Growth and lactic acid production coupling for Lactobacillus helveticus
cultivated on supplemented whey: influence of peptidic nitrogen deficiency. Journal of Biotechnology 55,
1-8.
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A. AMRANE AND Y. PRIGENT
Amrane A. and Prigent Y. (1998a) Identification and experimental validation of a criterion allowing
prediction of cellular activity for preculture of lactic acid bacteria. Journal of Fermentation and
Bioengineering 85,328-333.
Amrane A. and Prigent Y. (1998b) Influence of yeast extract concentration on batch cultures of Lactobacillus
helveticus: growth and production coupling. World Journal of Microbiology and Biotechnology 14, 529-
534.
Amrane A. and Prigent Y. (1998c) Influence of an initial addition of lactic acid on growth, acid production
and their coupling for batch cultures of Lactobacillus helveticus. Bioprocess Engineering 19, 307-312.
Amrane A. and Prigent Y. (1999) Analysis of growth and production coupling for batch cultures of
Lactobacillus helveticus with the help of an unstructured model. Process Biochemistry 34, 1-10,
Fauquant, J., Viéco, A., Brulé, G. and Maubois, J.-L. (1985) Sweet whey clarification by heat-calcium
aggregation of residual fat material. Lait 65, 1-20 (in French).
Gätje, G. and Gottschalk, G. (1991) Limitation of growth and lactic acid production in batch and continuous
cultures of Lactobacillus helveticus. Applied Microbiology and Biotechnology 34, 446-449.
Luedeking R. and Piret E.L. (1959) A kinetic study of the lactic acid fermentation. Batch process at controlled
pH. Journal of Biochemistry, Microbiology and Technological Engineering 1, 393-412.
Norton S., Lacroix C. and Vuillemard J.C. (1994) Kinetic study of continuous whey permeate fermentation by
immobilised Lactobacillus helveticus for lactic acid production. Enzyme and Microbial Technology 16,
457-466.
Roy, D., Le Duy, A. and Goulef J. (1987) Kinetics of growth and lactic acid production from whey permeate
by Lactobacillus helveticus. Canadian Journal of Chemical Engineering 65, 597-603.
Turner, K. W. and Thomas, T. D. (1975) Uncoupling of growth and acid production in lactic Streptococci.
New Zealand Journal of Dairy. Science Technology 10, 162-167.
Venkatesh, K. V., Okos, M. R. and Wankat, P. C. (1993) Kinetic model of growth and lactic acid production
from lactose by Lactobacillus bulgaricus. Process Biochemistry 28, 23 1-241.
108
PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES
CEREVISIAE
Abstract
1. Introduction
The line between basic and applied research is nowhere more nebulous than in yeast
molecular biology and genetic engineering, and is usually traversed at will in our
laboratory's research on the genetic improvement of industrial yeast strains. One of the
focuses of our yeast strain development programme is to increase the efficiency of
fermentation and processing. By expressing numerous genes encoding extracellular,
polysaccharide-degrading hydrolases in wine, brewing and baking yeast strains, we
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©2001 Kluwer Academic Publishers. Printed in the Netherlands.
F F. BAUER AND I.S. PRETORIUS
Like most organisms, S. cerevisiae can perceive two types of extracellular signals:
signals required for intercellular communication (for example hormonal signals like the
pheromones a and α), and signals emanating from the environment. Environmental
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
111
F.F. BAUER AND I.S. PRETORlUS
substrate. The two phenotypes, filamentation and invasive growth, therefore make use
of the same signal transduction pathways, but can be observed independently in some
genetic backgrounds or in specific conditions. In this review, the terms pseudohyphal
differentiation or filamentation will always imply invasive growth, unless otherwise
stated.
Figure 1. Morphology of yeast-type and filament-type yeast cells and observed phenotypic
changes.
Numerous genes have been, and are still being, identified for playing a role in - or for
influencing directly or indirectly - the events associated with the dimorphic transition of
S. cerevisiae cells. Several systematic genetic approaches using different phenotypic
screens have been implemented to identify such genes. These approaches include the
isolation of genes which (i) induce pseudohyphae formation when present in multiple
copies (Gimeno and Fink; 1994), (ii) lead to elongated cell morphology (Blacketer et
al., 1995) or result in the suppression of filamentous growth when mutated (Mösch and
Fink, 1997), or (iii) are able to suppress such mutations (Lorenz and Heitman, 1998a).
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
Other genes have been identified through indirect approaches, for example through their
ability to induce the STA1-3 glucoamylase genes (Lambrechts et al., 1996b), which
were shown to be co-regulated with invasive and pseudohyphal growth. Finally,
numerous genes were linked to the process through general and systematic screening of
phenotypes resulting from mutations within known genes (Radcliffe et al., 1997;
Chandarlapaty and Errede, 1998; Edgington et al., 1999) or because of homologies with
known developmental regulators in other species (Gavrias et al. 1996).
Gene products participating in pseudohyphal differentiation can be divided into four
groups:
• Proteins that belong to evolutionarily conserved signal transduction modules,
including plasma membrane receptors and associated heterotrimeric and small
GTP binding proteins forming part of the signal sensing apparatus, as well as
intermediate components of signalling cascades, for example MAP kinase
cascades and second messengers like cAMP (adenosine 3', 5'-cyclic
monophosphate) and their effectors.
• Proteins involved in morphogenetic events.
• Transcriptional regulators.
• Downstream effectors of the signalling pathway.
In the following section, the role of the above-mentioned signal transduction modules
will be discussed in some detail.
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F.F. BAUER AND I.S. PRETORIUS
specific sensors for each of the nitrogen and carbon sources whose limitation results in
pseudohyphal or invasive growth. Several other permeases can be expected to play a
similar role and to specifically signal the presence or limited availability of their
substrates. However, only indirect evidence has been provided so far (Lorenz and
Heitman, 1998b).
No receptor sensing carbon source limitation, and which would be required for
pseudohyphal differentiation, has been identified, but the putative G-protein associated
receptor Gpr1p is a promising candidate (Xue et al., 1998; Yun et al., 1998).
Furthermore, two other putative glucose sensors have recently been identified by Ozcan
et al. (1998). The proteins Snf3p and Rgt2p have homologies with hexose transporters,
but apparently generate an intracellular signal without transporting glucose. As for
Gpr1p, however, no direct evidence for a relation between these sensors and
pseudohyphal growth has yet been presented.
3.1.2.1. The role of Gpa2p. Gpa2p was initially identified because of strong homology
with α-subunits of mammalian heterotrimeric guanine-nucleotide-binding proteins
(Nakafuku et al., 1988). These proteins are well-studied receptor-associated signal
transduction modules consisting of three subunits, α, β, and γ (reviewed in Neer, 1995).
Upon activation of the receptor, the a-subunit exchanges GDP for GTP, which results in
the dissociation of the heterotrimer. The signal is passed on by either the a-subunit or
the β−γ-subunits, which stay attached to each other.
Based on homology searches, the S. cerevisiae genome sequence revealed the
presence of only two Gα subunits, Gpa1p and Gpa2p. Gpa1p is the well-studied
a-subunit of the heterotrimeric G-protein associated with the pheromone receptors
Ste2p and Ste3p (reviewed in Kurjan, 1992) and has not been implicated in any other
signalling event so far. Gpa2p, on the other hand, has been implicated in a number of
events, in general related to the control of cAMP levels in the cell (Nakafuku et al.,
1988; Papasavvas et al., 1992; Lorenz and Heitman, 1997; Kübler et al., 1997;
Colombo et al., 1998). Several genetic interactions with the Ras proteins, in particular
the ability of overexpressed GPA2 to suppress the growth defects of temperature
sensitive ras mutants, and the non-viability of a strain carrying deletions of both RAS2
and GPA2 have also been reported (Nakafuku et al., 1988; Papasavvas et al., 1992).
Interestingly, whereas the β and χ subunits of Gpa1p had been easily isolated and have
been well studied, no such subunits associated with Gpa2p have thus so been identified.
GPA2 was more recently shown to play an essential role during pseudohyphal
differentiation in diploids (Lorenz and Heitman, 1997; Kübler et al., 1997). A
gpa2/gpa2 deletion strain is unable - or presents a very reduced ability - to grow in the
filamentous form, whereas a strain carrying a hyperactivated allele (GPA2Gly132Val)
shows enhanced pseudohyphal differentiation even on rich media. A dominant negative
allele (GPA2Gly299Ala) was shown to inhibit pseudohyphal growth. Unpublished data of
our laboratory indicate that Gpa2p affects invasive growth and filamentation similarly
in haploid strains.
Since heterotrimeric G-proteins are receptor-coupled signal transduction modules,
our current knowledge strongly favours the hypothesis that Gpa2p associates with
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
3.1.2.2. Ras2p regulates filamentation via two separate pathways. The first signal
transduction element to be associated with invasive and pseudohyphal growth was the
small GTP-binding protein, Ras2p. Results presented by Gimeno et al. (1992) showed
that the hyperactivated form of Ras2p, Ras2pVa119, stimulated both pseudohyphal and
invasive growth. Initial experiments suggested that this regulation occurred through the
- at this time - only recognised function of Ras2p, the control of adenylate cyclase
(Cyr1p) activity and consequently the regulation of cAMP concentration within the cell.
Results presented by Ward et al. (1995) strengthened the hypothesis of a cAMP
dependent regulation of the process. Indeed, overexpression of the PDE2 gene
(encoding, as mentioned above, a phosphodiesterase responsible for cAMP degradation)
inhibits pseudohyphal and invasive growth and suppresses the pseudohyphal growth
phenotype of the RAS2Va119 allele.
Concomitantly, Liu et al. (1993) presented evidence for the involvement of elements
of the mating specific MAP kinase cascade, in particular the MEKKK Ste20p, MEKK
Ste11p and MEK Ste7p, and the transcriptional activator Ste12p (Liu et al., 1993;
Roberts and Fink, 1994) in invasive and pseudohyphal growth regulation. The authors
showed that other elements of the pheromone signal transduction cascade, like the
pheromone receptors Ste2p and Ste3p, or the receptor-associated heterotrimeric G-
protein did not have any effect on pseudohyphal differentiation. Initially, no link
between Ras2p and the MAPK cascade was established. Mösch et al. (1996) later
presented evidence that the regulation by the MAPK cascade was dependent on Ras2p.
The transmission of the signal via Ras2p and the MAPK cascade was shown to be
cAMP independent, but dependent on the rho-like small G-protein, Cdc42p. The
dominant negative CDC42 allele, CDC42Ala118, is indeed able to block the transmission
of the constitutive filamentation signal created by the hyperactivated RAS2Va119 allele.
Both Ras2p and Cdc42p were shown to act upstream of the MAP kinase cascade
through epistasis analysis. In this case, modification of the activity of cAMP dependent
kinase (PkA) through the use of bcy1 mutants or the overexpression of Tpk1p did not
seem to influence filamentous growth (Mösch et al., 1996).
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F.F. BAUER AND I.S. PRETORIUS
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
117
F.F. BAUER AND I.S. PRETORIUS
Figure 3 Comparison between the pheromone response and the pseudohyphal growth
pathways Common elements of the two pathways are in bold italics, whereas elements that
impart MAPK specificity are highlighted by a grey background
The MEKKK Ste20p, MEKK Ste1 1p and MEK Ste7p were shown to be required for
efficient invasive growth, as was the transcription factor Ste12p (Liu et al., 1993). Other
genes which when mutated result in sterile phenotypes like STE50 were also shown to
be required for the regulation of filamentous growth (Ramezani et al., 1998). Deletion
of any of the above-mentioned genes results indeed in strongly reduced invasive and
filamentous growth, whereas hyperactivated alleles (where available) present enhanced
phenotypes for both characteristics.
Epistasis analysis showed that the kinase cascade receives the filamentation signal
via Ras2p and Cdc42p in a cAMP independent manner (Möschet al., 1996).
Several hypotheses, recently reviewed by Madhani and Fink (1998), could explain
how specific signals can result in specific responses while using, at least partially, the
same elements for transmission (Figure 3). Firstly, the MEK Ste7p activates two
different MAP kinases, Fus3p and Kss1p. Cook et al. (1996, 1997) and Madhani et al.
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(1997) showed that Kss1p is specifically required for pseudohyphal growth, whereas
Fus3p is specific for mating. Both are inhibiting the activity of their specific targets
when inactive (non-phosphorylated), which explains why initial genetic data based on
the study of deletion mutants investigating the pheromone signal transduction suggested
a partially redundant activity for the two kinases (reviewed in Herskowitz, 1995). A
second mechanism to achieve MAP kinase cascade specificity relies on the
combinatorial control of the transcription factor Stel 2p, which has to associate with
Tec1p to activate hyphae specific genes (Gavrias et al., 1996; Madhani and Fink, 1997).
A third potential mechanism to ensure MAP kinase specificity could reside in higher
order association of the different kinases, which would keep particular signal
transduction pathways physically separated and make cross activation impossible. This
possibility is clearly demonstrated by the specific recruitment of the MEKK Stel 1p to
both the mating pheromone pathway-specific scaffold protein Ste5p (Elion, 1995) and
to the HOG MAPK pathway-specific scaffold Pbs2p (Posas and Saito, 1997). Indeed,
besides being required for both the pheromone and pseudohyphal growth pathway,
Stel1p is necessary for the osmotic activation by the osmosensor Sho1p of the HOG
MAP kinase pathway (Posas and Saito, 1997). However, no pseudohyphal response-
specific scaffold protein has yet been identified. MAP kinase associated proteins might
play an additional role in ensuring signalling specificity. The filamentation specific
MAPK Kss1p has been shown to associate with two proteins, Dig1p and Dig2p (Cook
et al., 1997; Tedford et al., 1997). The genes were identified through a 2-hybrid
interaction screen and Dig1p co-immunoprecipitates with Kss1p in the nucleus. A
∆dig1/∆dig2 double mutant presents a constitutively invasive phenotype, but neither the
double nor the single mutants present any defect with regard to the pheromone response.
Both proteins also associate with Ste12p, the transcriptional activator regulated by both
MAP kinases Fus3p and Kss1p. The data suggest that Dig2p acts as a negative regulator
of invasive growth, and is directly regulated by Kss 1 p, which, as mentioned above, acts
as a strong repressor of invasive growth in its non-phosphorylated state (Cook et al.,
1997; Madhani et al., 1997). In addition, Bardwell et al. (1998b) show that Kss1p in its
inactive form can directly bind to Ste12p, offering a further possibility for direct
regulation of the protein.
The different elements that might contribute to MAP kinase specificity are
summarised in Figure 3.
The specific contribution of the MAPK cascade to the modifications observed
during pseudohyphal differentiation is unclear. However, both pheromone response and
low nutrient response result in some cellular modifications of a similar nature, in
particular with regard to polarised growth (see following paragraph) and changes in
cell-adhesion properties. Some of these changes might be depending on the common
elements in the two cascades.
3.1.3.3. Morphogenetic factors associate with the MEKKK Ste20p. The major
morphogenetic event observed during filamentation is the strong elongation of the
filament-forming cells. Cell elongation requires polarised growth, which in turn is
dependent on the organisation of the actin cytoskeleton (reviewed in Madden and
Snyder, 1998). Numerous results have been taken as evidence that the morphogenetic
reorganisation of the actin cytoskeleton during pseudohyphal growth is dependent on
the MEKKK Ste20p and its association with the rho-like GTPase, Cdc42p. Results
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F.F. BAUER AND I.S. PRETORIUS
show that both proteins have a more general role in the organisation of polarised growth
during the cell cycle (Evangelista et al., 1997; Leberer et al., 1992, 1997; Leeuw et al.,
1995, 1998; Mösch et al., 1996). Thermosensitive mutations in CDC42 result in large,
round, non-budding cells, indicating the requirement for Cdc42p for polarised growth
during bud emergence. Ste20p, on the other hand, directly associates with the actin
cytoskeleton via an adapter protein, Bem1p (Leeuw et al., 1995; reviewed in Madden
and Snyder, 1997). Ste20p also directly binds to Cdc42p, and deletion or mutation of
the Cdc42p-binding site blocks pseudohyphal differentiation (Peter et al., 1996; Leberer
et al., 1997). Further evidence for this more general role of Ste20p is provided by the
fact that the hyperactivated allele of STE11, STE1 1-4, only partially bypasses
filamentation defects of ste20 mutants, whereas mutations in STE11, STE7 or STE12 do
not block cell-elongation in a RAS2Va119 strain (Mösch et al., 1996). The morphogenetic
function of Ste20p and Cdc42p could explain at least in part the common requirement
of the MAP kinase cascade for both mating and pseudohyphal differentiation, since in
both cases a strong polarisation of the growth pattern is required, either to produce the
cellular extension called shmoo during mating or to produce elongated cells during
pseudohyphal differentiation. A second phenotypic change associated with both
pathway is the modification of cell-adhesion properties.
Other proteins that probably associate with Ste20p are the S. cerevisiae 14-3-3
proteins, Bmh1p and Bmh2p (Roberts et al., 1997). The authors provide evidence that
these proteins are required specifically for the transmission of the Ras2p dependent
signal to the MAP kinase cascade during pseudohyphal differentiation. However, the
significance of this finding and the exact role of these proteins during signal
transduction are as yet unclear.
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
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F.F. BAUER AND I.S. PRETORIUS
Besides the direct modulation of the activity of pre-existing enzymes, the main
molecular response to extracellular signals resides in the activation or repression of the
transcription of specific target genes. It is therefore not surprising that a large number of
genes whose deletion or overexpression affects the pseudohyphal differentiation
phenotype encode transcriptional regulators.
There are several obvious requirements for transcription factors to be active: It has
to be localised in the nucleus, bind to DNA or associate with DNA-binding proteins,
and has to be able to - directly or indirectly - interact with the basal transcription
apparatus, the RNA polymerase holoenzyme. Each of these requirements, i.e. nuclear
localisation, DNA binding or the interaction with other proteins, can be modulated by
the extracellular signal (reviewed in Hill and Treisman, 1995). In this review, we shall
focus separately on each of the transcription factors that have been shown to contribute
to the filamentous growth phenotype. Several of these factors had previously been
identified for their role in other, sometimes related, cellular processes, underlining again
the interconnected nature of all signalling processes.
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
3.2.2. Msn 1p and Mss11p: Central elements in the pseudohyphal growth pathway
MSN1 has first been identified as a multi-copy suppressor of snf1 mutants (MSN1)
(Estruch and Carlson, 1990). Other authors cloned the same gene as FUP1, an enhancer
of iron-limited growth of S. cerevisiae (Eide and Guarente, 1992), as PHD2, a multi-
copy inducer of pseudohyphal growth (Gimeno and Fink, 1994) and, in our laboratory,
as MSS10, a multi-copy suppressor of the repression exerted by STA10 on the STA1-3
glucoamylase-encoding genes, involved in starch metabolism (Lambrechts et al.,
1996b). MSN1 has been suggested to encode a transcriptional activator since multiple
copies of the gene enhance the transcription of several genes, most of which are
involved in nutrient utilisation. In addition, MSN1 has been shown to activate reporter
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F.F. BAUER AND I.S. PRETORIUS
gene expression if fused to the LexA DNA-binding domain (Estruch and Carlson,
1990).
MSS11, like MSN1, was identified as a suppressor of the STA10 dependent
phenotype and was shown to induce STA1-3 encoded glucoamylase expression when
present on a 2µ plasmid (Webber et al., 1997). The protein displays homologies with a
number of transcriptional activators and suppressors, such as S. cerevisiae Snf5p,
Ssn6-/Cyc8p and Drosophila NTF-1, and in particular with Flo8p, a protein initially
identified for activating genes involved in flocculation (Kobayashi et al., 1996).
Both MSN1 and MSS11 strongly activate the expression of MUC1, a gene required
for invasive growth and pseudohyphal differentiation, when present in multiple copies
(Lambrechts et al., 1996a; Webber et al., 1997). Recent data by Gagiano et al. (1999)
show that Mss11p is a central factor for the establishment of pseudohyphal and invasive
growth patterns. Genetic evidence indicates that Mss11p is required for both Ras2p
dependent, MAP kinase dependent and independent, signal transduction pathways.
Genetic evidence for Msn1p would suggest that the protein acts in a pathway
downstream of Ras2p, but independent of the MAP kinase cascade. Msn1p requires the
presence of Msn11p in order to induce filamentation, whereas Mss11p is able to induce
the pathway in the absence of Msn1p. Mss11p therefore seems to be situated
downstream of Msn1p (Gagiano et al., 1999).
3.2.3. Sf11p, Sok2p and Flo8p: Factors depending on the CAMP dependent kinase
Three transcriptional regulators apparently controlled by cAPK and regulating
filamentation have been identified, i.e. Sf11p, Sok2p and Flo8p.
SFL1 had initially been cloned as a suppressor of flocculation (Fujita et al., 1989)
since disruption of the gene leads to strong, constitutive flocculation in most strains.
Robertson and Fink (1998) showed that deletion of SFL1 also results in enhanced
pseudohyphal and invasive growth. Sf11p was shown to associate specifically with
Tpk2p, the cAMP dependent protein kinase that specifically activates pseudohyphal
growth. Interestingly, Sf11p has also been shown to interact and associate with the
Srb/mediator proteins, which are part of the RNA polymerase holoenzyme and are
thought to be responsible for transcriptional repression (Song and Carlson, 1998).
Tpk2p therefore has to inactivate Sf11p, which in turn would relieve the repressive
effects of the Srb proteins on the RNA polymerase holoenzyme and allow transcription
of flocculation and pseudohyphal growth specific genes. This mechanism would
provide the first evidence for a direct interaction of the cAMP dependent kinase with
factors associated with the RNA polymerase holoenzyme.
A second putative transcriptional repressor that regulates filamentation is encoded
by the SOK2 gene, which was cloned by Ward and Garret (1 994). The gene was isolated
as a dosage-dependent suppressor of conditional growth defects of
temperature-sensitive cAPK mutants. Ward et al. (1995) presented evidence for the
involvement of Sok2p in cAMP dependent regulation of filamentous growth by Sok2p.
Diploid sok2/sok2 mutants show enhanced pseudohyphal growth, indicating a
repressive activity of Sok2p. Since Sok2p presents important homologies with the
helix-loop-helix DNA-binding motive of Phd1p and other fungal regulators of
morphogenetic processes (Stoldt et al., 1997), it most probably directly represses the
expression of filamentation related genes. Unlike Sf11 p, which directly associates with
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
Tpk2p, no such association has yet been found for Sok2p, and its direct regulation by
cAPK has yet to be demonstrated. In addition, the ∆sok2 phenotype presents defects in
the regulation of genes involved in a number of other cAMP dependent metabolic
regulations, including glycogen accumulation (Ward et al., 1995). It therefore behaves
like a general regulator of cAMP dependent cellular responses, and is not specific for
the filamentation response.
A third transcriptional regulator identified as acting downstream of the cAMP signal
and which induces filamentation is Flo8p. Kobayashi et al. (1996) cloned FLO8
because of its ability to induce flocculation. The gene was later shown to be required for
pseudohyphal growth (Liu et al., 1996). Interestingly, the authors presented evidence
that a mutation within this gene was responsible for the inability of most laboratory
strains of S. cerevisiae to undergo the dimorphic switch from yeast-type to
filament-type cells. Through genetic evidence, Rupp et al. (1999) showed that Flo8p
positively activated pseudohyphal differentiation and that this activation was cAMP
dependent. The authors showed that Flo8p activates MUC1 by directly acting on several
sequences within the promoter of this gene.
3.2.4.1. Ash1p. Ash1p was first identified for its role in mating type switching, where it
is responsible for the daughter cell specific repression of the HO endonuclease. ASH1
mRNA is localised asymmetrically, close to the tip of large budded cells that have
undergone anaphase (Long et al., 1997; Takizawa et al., 1997). This polarised
localisation explains the asymmetric distribution of Ash1p between mother and
daughter cell and its daughter cell specific effect on mating type switching. Ash1p
contains a zinc-finger-like domain, and binds therefore probably directly to DNA to
regulate gene expression.
Chandarlapaty and Errede (1998) presented data suggesting that Ash1p was required
for pseudohyphal growth. Cells lacking the ASH1 gene did not form filaments, whereas
cells with multiple copies of the gene showed increased filamentation. Similar
observations were made with regard to invasive growth in haploids. Genetic evidence
places Ash1p in a pathway independent of the MAP kinase cascade but downstream of
Ras2p, similar to what was observed for Msn1p (Gagiano et al., 1999).
3.2.4.2. Phd1p. This gene was identified through the screening of a genomic library for
genes that, when present on a multiple copy plasmid, would enhance pseudohyphal and
invasive growth (Gimeno and Fink, 1994). Phd1p showed significant homology with
transcriptional regulators. However, deletion of PHD1 does not affect the ability of
S. cerevisiae to form pseudohyphae or to grow invasively, and its role in pseudohyphal
differentiation is unclear.
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F.F. BAUER AND I.S. PRETORlUS
required for these pathways, and which have been described in the previous section.
However, these regulators have mostly been cloned for specific roles in processes that
only have an indirect or not yet clearly understood connection with filamentation. For
example, from the above it is clear that a phenomenon like flocculation in liquid media
is closely linked to the filamentation response, and some factors like Flo8p are required
for both. In addition, some obvious phenotypic links between both cellular responses
exist. Both processes for example require modified cell adhesion properties, and both
seem to occur in media with limited amounts of nutrients. However, differences and
similarities between the two events have not yet been fully investigated.
The same is true for signal-dependent processes of polarised growth during both
mating and filamentation. The shared elements in both pathways might at least in part
be involved in aspects of cellular polarisation, but it is as yet not understood how signal
dependent specificity is imparted.
The data presented in this review could suggest that it is the combination of a
number of different factors in a specific context that leads to the implementation of the
filamentation response. If this is the case, specific effector genes and proteins of the
pseudohyphal response pathway should only respond to a specific, finely balanced
combination of all the pathways and factors described above. The gene coming closest
to the above expectation is MUC1/FLO11.
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
attachment of the cell to a substrate, a step that is thought to be required for efficient
invasion. Both hypotheses are not mutually exclusive.
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F.F. BAUER AND I.S. PRETORIUS
been investigated for separate and specific purposes, namely (i) the cell cycle signal
emanating from CDKs, (ii) the nutrient-linked cAMP signal and (iii) the MAPK signal
which might be required for the cell-cell attachment and cell growth-polarisation
processes. This combination of three major pathways again demonstrates that co-
ordination of all cellular compartments and of most molecular processes underlies
cellular differentiation processes.
A different perspective can be taken by simply considering the initial phenotypes, which
were used to identify most of the genes mentioned in this review. Some of the genes
were initially identified for their role in the pheromone response, other for phenotypes
linked to flocculation, abnormal cell cycle, abnormal stationary phase entry, starch
degradation, ammonium transport, etc., again demonstrating the interconnected nature
of apparently unlinked events.
How does this fundamental understanding of complex processes impact on the
potential use of yeast in industrial processes? Traditionally, S. cerevisiae has been used
for several types of food and beverage fermentations and for the production of alcohol.
For these traditional uses, industrial yeast has been optimised through centuries of
mostly unaware selection by winemakers, brewers, bakers and other users. This
unconscious optimisation process has lead to a number of S. cerevisiae strains
specifically adapted for specific tasks, be it baking, brewing , winemaking or any other
industrial use. However, from an industrial perspective, every single aspect of yeast
physiology could still be optimised. Fermentation efficiency, alcohol resistance, general
stress resistance, production of specific metabolites without production of additional,
unwanted substances - there probably are as many potential targets for strain
improvement as there are industrial users. It is in this field of strain improvement that
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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
recombinant DNA technology and metabolic engineering can provide its most
important contribution.
How can research of the type described in this review contribute to achieve such
strain improvement targets? The answer becomes clear when looking at the results
obtained with recombinant and metabolically engineered strains. These strains can
frequently meet a specific target, be it a higher production of a metabolite (e.g. glycerol)
or of extracellular proteins and enzymes. However, these strains have just as frequently
failed to make an industrial impact. One reason for this failure can be found in the
review above, and resides in the interconnection of all metabolic and signalling
pathways. This interdependence makes it extremely difficult to achieve specifically
improved yeast without modifying several other parameters. These secondary
modifications frequently result in undesirable by-products or have a negative influence
on general yeast physiology. It is therefore essential that we better understand the
underlying network of metabolic and signalling connections, which would allow us to
design mechanisms to avoid metabolic and physiological problems. Such integrated
approaches, which will take into account not only the specific parameter to be modified,
but also all potential consequences on associated or linked pathways, should result in
modified yeast with much higher industrial potential.
Besides this general argument for the scientific and industrial usefulness of the
research outlined in this review, there are several potential industrial benefits directly
associated with the study of pseudohyphal development. Some wanted -or unwanted-
properties of industrial yeast strains are indeed directly linked to this pathway. The
aspect retaining most attention is flocculation, which refers to the clumping of cells in
liquid media and to their consequent sedimentation (reviewed in Stratford, 1992).
Flocculation is due to the modification of cell-cell adhesion properties, and is of
importance in several industrial processes, for example during brewing or the
production of sparkling wine (reviewed in Hammond, 1995). The signal transduction
pathways leading to flocculation is similar, if not identical, to the filamentation
pathway, as can be seen by the number of genes shared by both (reviewed in Kron,
1997). Understanding filamentation should therefore allow us to design yeast with
specific flocculation abilities. Other direct applications might reside in the development
of new top-fermenting brewing and sherry yeast. Indeed, the ability of flor yeast to float
on the surface of the substrate (top-ferment) is thought to be due to a modified
implementation of the signal transduction pathways described in this review.
Acknowledgements
We thank the National Research Foundation (NRF) and the South African wine industry
(Winetech) for funding of our research.
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133
MICROBIAL PRODUCTION OF THE BIODEGRADABLE POLYESTER
POLY-3-HYDROXYBUTYRATE (PHB) FROM AZOTOBACTER
CHROOCOCCUM 6B: RELATION BETWEEN PHB MOLECULAR WEIGHT,
THERMAL STABILITY AND TENSILE STRENGTH
Abstract
PHB with a high molecular weight (Mw) was obtained from fermentor cultures of
Azotobacter chroococcum 6B. Mw was significantly affected by aeration rate, being
reduced from 2900 to 110 kDa at 0.25 and 2.5 vvm, respectively. Temperature at which
maximal weight loss was obtained (decomposition temperature) markedly depended on
Mw , obtaining a 40°C span between 100 and 1400 kDa. Tensile strength reached a
value of 215 MPa at a Mw of 1240 kDa, being reduced to nearly 40 MPa at the lower
tested Mw of 445 kDa.
impellers and baffles. Temperature was controlled at 30°C and initial pH was adjusted
to 7.0. The air flow rate was set between 0.25 and 2.5 vvm, Initial C/N ratio (molar
basis) was set between 7.3 and 138 (ammonium sulphate limitation and glucose excess)
and agitation speed was 200 rpm. Batch experiments were finished at 72 h. When
molasses Bm medium was utilised, glucose was replaced by sugar cane molasses (5%
w/v) (Tucumán, Argentina) as carbon source.
Cell growth was monitored by measuring the optical density at 610 nm. Cell dry weight
(cdw) was measured by weighing lyophilised cells harvested from 3 ml culture broth.
Residual glucose in the broth was determined by the enzymatic method using glucose
oxidase/peroxidase (Wiener Lab., Rosario, Argentina). PHB content and its composition
were determined by gas chromatography using 3HB standards (Braunegg et al, 1978).
Mw was measured viscosimetrically and by gel permeation chromatography (GPC)
using five serial Styragel columns of 102, 103, 104, 105 and 106A. Flow rate of
chloroform was 0.5 ml/min. A refractive index detector was used.. Differential scanning
calorimetry (DSC) studies were performed using a Mettler TA9000 calorimeter between
-60 and 250°C at 10°C/min. Thermogravimetric (TG) measurements were made with a
Mettler TA9900 apparatus from 25 to 800°C at 10°C/min. Tensile strength was
measured on PHB probes (obtained according to ASTM 1708-84) utilising an Instron
model 1122 tensiometer from slopes of strain-stress curves
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MICROBIAL PRODUCTION OF PHB
(kDa
fixation, 0.5 vvm, 100 rpm 180.9 n.d. 291 59 1420
C/N 7.3, 0.5 vvm, 100 rpm 177.8 3.9 268 82 1330
C/N 29,0.5 vvm, 100 rpm 178.8 4.3 261.7 73 1170
molasses 5%, 0.5 vvm, 100 173.9 n.d. 286 54 1420
rpm
C/N 138,2 vvm, 400 rpm 173.0 n.d. 250 55 150
C/N 69,2.5 vvm, 100 rpm 178.2 5.7 n.d. 62 110
n.d.: not determined. Tg , Tm and % crystallinity obtained from DSC scans. %
crystallinity=∆ Hm pHB (J/g°C)/∆ HmpHB100%crystalline a: Td:temperaturewheremaximal
weight loss is obtained.
PHB Mw was significantly affected by aeration rate under fermentor cultivation at a C/N
ratio of 69. Figure 1 depicts the influence of aeration rate on PHB Mw: maximum Mw
(2.900 kDa) was obtained at the lowest aeration rate experimented of 0.25 vvm, while
when increasing aeration rate Mw diminished to 110 kDa at 2.5 vvm. This confirms that
PHB is accumulated under oxygen-deficient conditions, then in such conditions 3-
hydroxybutyrate monomers are incorporated to the growing PHB chains, consequently
increasing Mw. An increased Mw leads to an improvement of polymer performance, as
lower Mw are not desirable from the point of view of plastics applications, for example,
thermal stability is much lower with low Mw plastics. Also a product of low molecular
mass suffer a sensitive loss of volatile degradation products as temperature is increased
(Scandola et al. 1988).
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QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S.
Figure 1. PHB molecular weight (Mw) and PHB content (XPHB) under different aeration
rates under fermentor culture (4 litres) with Burk’s modified medium (Bm) at a C/N ratio of
69 (mol glucose/mol ammonium sulphate) at 72 h culture. : Mw, o : XPHB (% dry
weight).
Mw (kDa) σ (MPa)
445 38
1000 113
1240 215
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MICROBIAL PRODUCTION OF PHB
suggesting that PHB of 1000 kDa creates the most convenient pore size and
microenvironment for cellulase retention and activity.
3. Conclusions
From the above results, we concluded that aeration rate should be carefully controlled
under fermentor cultivation of strain 6B for PHB production, as Mw is markedly
affected by dissolved oxygen tension, and consequently by aeration rate. PHB with Mw
higher than 1200-1300 kDa are desirable for obtaining a bioplastic with both high
tensile strength and thermal stability, in order to be successfully processed in industrial
applications.
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QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S.
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140
SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES
DETERMINED BY ISOTOPIC RATIO MASS SPECTROMETRY OF
BIOMARKERS
1. Introduction
One of the fundamental characteristics of the microbial world is its inherent metabolic
diversity. An extraordinary physiological flexibility, achieved during, at least, 3.5
billion years (Doolittle et al., 1996; Feng et al., 1997; Pace, 1997), allows
microorganisms not only to colonise all habitats which are inhabited by eucaryotes but
also to exploit a wide spectrum of extreme biotopes (e. g., hot springs, salt and soda
lakes, highly-polluted waste waters, etc.) which are inhospitable to higher organisms.
The ubiquitous distribution of bacteria coincides with the observation that
microorganisms, and their natural communities, are of fundamental importance for the
global cycling of organic matter (Pomeroy & Wiebe, 1988). The metabolic diversity is
reflected in the species diversity and, particularly, in the large evolutionary distances
observed between the main phylogenetic lineages of microorganisms (Woese, 1987). It
is currently estimated that probably only 1 % to 10 % of the microorganisms living in
the biosphere are known (i. e., have been isolated and characterised) (Bull et al., 1992).
Moreover, one should recognise that the vast majority of microorganisms present in
environmental samples currently cannot be cultivated in the laboratory. An important
question concerning the cultivable microorganisms arises as to their actual frequencies
and significance in the ecosystem (Amann et al., 1995). The problem is further
complicated due to the fact that, in nature microorganisms live in complex communities
whose compositions vary depending on the environmental conditions. The activities of
individual members of such communities are strongly influenced by the prevailing
conditions in the environment as well as the activities of the other members (Roszak &
Colwell, 1987). The structure and dynamics of natural microbial communities, i. e. the
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WOLF-RAINER ABRAHAM ET AL.
species compositions, interactions and activities over time and space, is poorly
understood. This is in marked contrast to the wealth of knowledge about the community
structure and dynamics of higher organisms like plants and animals. Due to the problem
of non-culturability of the majority of the bacteria, traditional, cultivation-based
methods of microbiology are often of very limited value (Staley, 1980; Atlas, 1984;
Brock, 1987; Hugenholtz et al., 1998). Rather it is important to develop new
experimental approaches, which do not need cultivation in order to clarify the relations
between structure and function of microbial communities. Several approaches have
been proposed to tackle the basic problems of determining microbial community
structure, most of them based on the molecular analysis of ribosomal RNA or rDNA
(Pace et al., 1986; Hugenholtz & Pace, 1996; Pace, 1996) and some on the analysis of
polar lipids (Tunlid & White, 1992). There is an urgent need for the elucidation of the
critical interactions taking place in microbial communities which determine important
physiological activities, in order to be able to develop a knowledge-based strategy for
the optimisation and control of biotechnological applications in the environment, and to
control their safety. To achieve this long-term goal a novel approach was developed -
the analysis of biomarkers by isotope ratio mass spectrometry (IRMS) - for tracking the
flux of carbon within microbial consortia. For this approach two components are
needed: i) biomarkers which are specific for bacterial taxa, i. e. genera, families, phyla,
and ii) a calibration of the degree of isotopic fractionation during the microbial
degradation of the substrates and the incorporation of their carbons into the biomarkers.
Polar lipids exhibit large structural diversity, an attribute that enables them to be used as
chemotaxonomic criteria. Some lipids are unique to certain taxa, whereas the relative
concentrations of lipids within a taxon provides additional taxonomic resolving power.
The taxonomic utility of lipids is proportional to the quality of the structural information
that is obtained, Hence, in addition to conventional methods of lipid analysis (e.g. 2-
dimensional thin layer chromatography with selective staining and chemical cleavage,
derivatisation and gas chromatographic (mass spectrometric, GC-MS) analysis of the
component parts), considerable effort is currently being invested in detection/structural
characterisation of lipids (e.g. Fourier transformed infrared spectrometry (FT-IR),
dynamic chemical ionisation and fast atom bombardment (tandem) mass spectrometry
(DCI- and FAB-(MS/)MS), two-dimensional nuclear magnetic resonance spectroscopy
(1H and 13C NMR)).
Because of novel technologies it is more and more possible to analyse intact lipids.
Additional to the already established chromatography mainly the Tandem-MS is used
for the analysis of intact lipids, During the last years we applied this unit successfully
for the study of several lipid mixtures and the structure elucidation of their components.
About 200 glyco-, sulfo- and phospholipids were identified for the first time as natural
products and their structures characterised. Furthermore it was possible to find
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SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES
145
WOLF-RAINER ABRAHAM ET AL.
Figure 1: Continued
Using fatty acids this approach was calibrated in pure culture studies in order to
investigate the relationship between the isotopic composition of certain fatty acids and
that of the bacterial diet. In this manner representative strains of bacteria and fungi
grown in a minimal medium with different carbon sources with known isotopic ratios
were examined. Looking at stable carbon isotopic ratios of palmitic acid which is
common in all higher organisms it was found that all microorganisms vary in their
incorporation of 13C from the substrate into this fatty acid. The rates of isotopic
discrimination in the various substrates and fractions of polar lipids were partially found
to be very different. Especially interesting was a high depletion of 13C of all
microorganisms grown on glucose and in contrast to that the high ranges of isotopic
discrimination of the different organisms grown on mannose. In most cases a significant
146
SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES
difference in the isotopic ratio between palmitic acid extracted from glycolipids and that
from phospholipids was observed (Abraham et al., 1998a).
The 13C of bacterial biomass is a parameter in microbial ecology gaining increasing
interest (Boschker et al., 1999). It is used for the identification of substrate utilisation of
bacteria, relying on the observation that bacteria grown on substrates with known
isotopic values produce biomasses which differ in their isotopic ratio of only 2 λ from
those of their substrates. Our studies determined isotopic differences of palmitic acid of
up to 7 λ, both depletion and enrichment (Abraham et al., 1998a). We found a much
larger range of isotopic fractionation for amino acids of up to 80 λ. These ranges were
dependent on the biomarker and the substrate, but also on the bacterial species. From
calibration studies in pure cultures we could derive some rules controlling the depletion
or enrichment of carbon isotopes in the individual biomarkers. This basic new
discoveries of the isotopic discrimination of individual genera of bacteria and fungi may
have a great potential to study the crucial microbial catalysts of the heterotrophic
dominant part of the carbon cycle in natural ecosystems.
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WOLF-RAINER ABRAHAM ET AL.
148
SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES
(Brückmannet al., 1998). Since no activity of cis -dienelactone hydrolase was detected
in the cell extract of Pseudomonas sp. MT1, another, unidentified hydrolase was
assumed to be the responsible transforming enzyme.
One application of this approach was the incorporation of carbon isotopes from
mixtures of substrates into the individual members of the 4-chlorosalicylate-degrading
consortium. The individual strains grew on a carbon mixture of yeast extract and 4-
chlorocatechol, the latter of which was enriched with 13C-4-chlorocatechol to δ500λ for
a better detection of incorporation levels. The isotopic ratios of two fatty acids were
determined for all strains and it was found that Alcaligenes xylosoxidans MT 3
incorporated 4-chlorocatechol the best, while Flavobacterium sp. MT 2 showed almost
no incorporation of this substrate. Both Pseudomonas strains showed intermediate
incorporation levels. This approach will help to elucidate the functional roles of the
individual members of the consortium in the mineralization of 4-chlorosalicylate.
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WOLF-RAINER ABRAHAM ET AL.
(MT3). The large and fast 13C-enrichment in C16:1 of Pseudomonas sp. MT4, in
relation to that of Pseudomonas sp. MT1 and MT3, revealed that protoanemonin,
excreted by Pseudomonas sp. MT1, is used as a carbon and energy source
predominantly by Pseudomonas sp. MT4.
Due to its low abundance it was not possible to determine the isotope ratios of fatty
acids from immunocaptured cells of Empedobacter sp. MT2. However, previous 13C-
tracer studies of the individual strains of the consortium in pure culture, using unlabeled
yeast extract and 0.2 mM [U-13C]4-chlorocatechol, revealed assimilation of 4-
chlorocatechol into the biomasses of the strains MT1, MT3 and MT4, but not
Empedobacter sp. MT2 (Pelz et al., 1997) (Figure 2). These data and the low abundance
of the strain suggest that it carries out a role as consumer of cell debris with no active
involvement in the degradation of 4-chlorosalicylate.
Although Pseudomonas sp. MT1 is able to grow alone in a chemostat on 4-
chlorosalicylate, a stable microbial consortium has been isolated for the degradation of
4-chlorosalicylate. Within the stable consortium, Pseudomonas sp. MT1 is the most
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SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES
abundant strain, which underlines its important functional role as the primary degrader
of 4-chlorosalicylate. Based on the observed high 13C-enrichments in fatty acids of
strains MT3 and MT4 with the metabolites [U-13C]4-chlorocatechol and -protoanemonin
the formation of a consortium can be explained by nutritional interactions of individual
strains. Based on the above results, the functional roles of the consortium members were
assigned (Fig 3). Using immunocapture and IRMS the sharing of substrates within the
microbial community was unravelled and individual community members were shown to
protect the whole consortium from critical intermediates such as 4-chlorocatechol and
protoanemonin by using them as carbon sources and hence, improving the degradation
of 4-chlorosalicylate by Pseudomonas sp. MT1 (Pelz et al., 1999).
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5. Outlook
Acknowledgement
We thank Dagmar Wenderoth, Tanja Jeschke, Birgit Jung, and Peter Wolff for their
excellent technical assistance. The International Atomic Energy Agency, Vienna
(Austria), is acknowledged for providing free reference materials for the calibration of
the IRMS. This work was supported by a grant of the German Federal Ministry for
Science, Education and Research (Project No. 03 19433C).
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154
A COMPARISON OF THE MECHANICAL PROPERTIES OF DIFFERENT
BACTERIAL SPECIES
Abstract
1. Introduction
The physical properties of cells e.g. cell size, shape and especially wall strength, are
highly species, strain and physiological state specific and will influence the relative
resistance of different microorganisms to disruption, which may be used to indirectly
infer the relative strength of microorganisms (Table 1, Table 2).
155
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 155–162.
©2001 Kluwer Academic Publishers. Printed in the Netherlands.
C. SHIU, Z. ZHANG AND C.R. THOMAS
Animal cells 7 7 7 7
Gram - bacilli & cocci 6 6 5 6
Gram + bacilli 5 4 (4) 5
Yeast 4 2.5 3 3.5
Gram + cocci 3 2.5 (2) 3.5
Spores 2 1 (1) 2
Mycelia (1)a 5 6 1
a
clogging of orifice may occur
The wall thickness of yeast (> 70 nm) is typically greater than that of Gram-positive
cocci (10 - 80 nm) yet the latter would appear to be the more resilient according to
Table 1. This may be because size has been observed to play an important part in
disruption, with larger cells showing increased susceptibility to rupture.
Table 2: Protein release from microorganisms in a high-pressure homogeniser (modified
from Keshavarz et al., 1987).
The most obvious role of the cell wall is in protecting the cell from mechanical damage,
the structure and composition of which contributes greatly to the strength of individual
bacteria as may' be seen from the fact that Escherichia coli (Gram-negative rod),
although similar in size to Bacillus subtilis (Gram-positive rod), is disrupted more easily
(Table 2). This is most likely due to the relatively thin peptidoglycan layer found in
Gram-negative bacteria (see Section 0).
Prokaryotic cells can be segregated into different groups on the basis of their cell wall
structure. The Archaebacteria possess cell walls but the components and physiology are
vastly different from that of eubacteria. A small fraction of eubacteria, the mollicutes,
lack an outer cell wall and are bounded only by their cytoplasmic membrane (these will
not be considered here). The eubacteria which do possess cell walls can be divided into
two major groups: Gram-positive or Gram-negative, which refer to the manner in which
eubacteria respond to the Gram stain.
Under the electron microscope, the Gram-positive cell wall appears as an amorphous
layer with an average thickness of 30 nm, although this value may vary from between
156
A COMPARISON OF THE MECHANICAL PROPERTIES OF BACTERIAL SPECIES
A number of researchers have demonstrated the flexibility of the bacterial cell wall
(Mendelson and Thwaites, 1989; Baldwin, 1988; Marquis, 1968; Ou and Marquis,
1970) but perhaps the simplest and most compelling evidence was that presented by
Isaac and Ware (1974) who embedded cells of different organisms in glycerol-gelatine
strips and stretched them on a rack. Spirillum was found to uncoil and stretch reversibly
to a rod three times the length of its original shape. The Gram-positive bacterium, B.
megaterium elongated to one quarter of its original length before becoming detached
from the gel matrix. E. coli showed considerable stretching and deformation - up to 100
% elongation - without detaching from the gel. With the Gram-positive coccus, S.
aureus, no elongation was observed prior to detachment from the gel matrix. These
results would appear to suggest the bacterial cell wall is far from being rigid and non-
flexible. With Gram-positive organisms, the thicker murein leads to increased resistance
to stretching while their Gram-negative counterparts show great flexibility in
comparison.
Without a doubt, the small size of individual bacteria has proved to be a
considerable obstacle in the development of a method for directly measuring the cell
mechanical properties. The approach of Thwaites and Mendelson (1985, 1989, 1991)
cleverly side-stepped this difficulty by conceiving a means to produce “bacterial thread”
up to 1 m long with 50 000 parallel cellular filaments, which can be subjected to similar
techniques applied to textile threads. Tensile tests were performed on bacterial threads
of B. subtilis mutant FJ7 to determine the cell wall properties.
The length and diameter of the filaments were found to depend on the humidity and
unsurprisingly so were the mechanical properties. The tensile strength and modulus of
elasticity at low relative humidity were 300 MPa and 13 GPa respectively.
Corresponding values when extrapolated to 100% humidity were found to be 13 MPa
and 30 MPa. Such values of strength indicated that cells in vivo should theoretically be
able to withstand turgor pressures of up to 2.6 MPa (Thwaites and Surana, 1991). It was
found that the stress/strain curves of unwashed preparations were similar to those with
the residual culture medium removed at roughly 18% higher relative humidity. The
most likely reason for this was that the unwashed cells were encased by the highly
hygroscopic dried medium and were therefore held in a much more humid environment
relative to the bulk atmosphere.
157
C. SHIU, Z. ZHANG AND C.R. THOMAS
In addition, the walls of the B. subtilis mutants were shown to exhibit what was
apparently viscoelastic behaviour since the load depended on the rate of strain and load
relaxation also occurred in the extended state. The time-scale for decay proved to vary
widely but based on the initial rate of decay, was found to occur on the order of minutes
(Thwaites and Mendelson, 1985). Viscoelastic phenomena in cell walls has major
implications for micromanipulation measurements; at high deformation rates, the cells
would appear to be stiffer and more brittle and vice versa (Thwaites and Mendelson,
1991). Such behaviour would need to be accounted for in the development of structural
models.
One major drawback to the technique is that it is limited to filament forming
mutants. Furthermore, the cells were not in their naturally hydrated condition when the
tests were carried out and the humidity has been shown to affect the thread mechanical
properties. Micromanipulation has several advantages over the latter technique in terms
of simplicity, versatility and more importantly, since mechanical properties are likely to
be affected by the external environment, the cells can be measured whilst suspended in
the growth medium.
1.4 MICROMANIPULATION
The apparatus (fig 1) was based on the rig used by Mashmoushy et al. (1998) for work
with yeast. Cells are squeezed using a 50 µm optic fibre probe, one end of which is
attached to a force transducer (Aurora Scientific Inc., Aurora, Canada) with paraffin
wax. The end of the probe which comes into contact with the bacteria is ground down to
leave a tip with a much smaller area, thus minimising alignment errors and incorrect
probe-cell contacts. The transducer is attached to a stage which is driven downwards by
a stepping motor, The force on the probe is measured by the transducer in terms of
voltage, which is converted to give the bursting force. Lighting is provided by a slit
illuminator from the side. Cells are positioned underneath the probe by moving the
stage. When E. coli was being measured, the image was recorded onto video tape for
later image analysis to determine the length of the cell.
158
A COMPARISON OF THE MECHANICAL PROPERTIES OF BACTERIAL SPECIES
Staphylococcus epidermis ATCC 5885 cells were grown on agar slopes at 30°C for 48
hours and then used to inoculate a McCartney bottle containing 10 ml of sterilised
nutrient broth (Oxoid, UK). Following incubation at 30°C over 24 hours, the broth was
used to inoculate a 500 ml volume shake flask containing 90 ml of sterilised nutrient
broth to start the batch fermentation. The flask was maintained at 30°C and 200 rpm in
an orbital shaker. The progress of the fermentation was followed by taking absorbance
measurements at 6.50 nm. Samples were taken from the rapid growth phase at t = 4, 5
and 6 hours after inoculation.
In preliminary fermentations, E. coli NCIMB 10214 had demonstrated a relatively
short exponential growth phase. Hence, continuous as opposed to batch fermentation
was used to provide physiologically identical samples. Medium of the following
composition was used for E. coli fermentation: (g/l of distilled water): KH2PO4, 13.6;
(NH4)2SO4, 4; MgSO4, 0.2; FeSO4 5x10-4; yeast extract, 5.0 and adjusted to pH 7.0
using 2.0 M KOH solution. Glucose solution at a final concentration of 10 g/l of
medium was made up and sterilised separately and only added to the medium before
inoculation.
Cells of E. coli were incubated at 37°C on agar slopes for 24 hours and used to
inoculate a shake flask containing 100 ml of complex medium. The culture was grown
159
C. SHIU, Z. ZHANG AND C.R. THOMAS
overnight for approximately 16 hours at 37°C and 200 rpm in an orbital shaker before
being used to seed 1 1 of medium in a 2 1 fermenter. The pH was controlled using 0.5 M
H2SO4 and 2.0 M ammonia solution. Once the cells had reached mid-exponential phase,
the continuous fermentation was started at a dilution rate of 0.5 h-1. Samples were
diluted using distilled water to a concentration of approximately 1 x 107 cells/ml.
Figure 2a and Figure 2b represent typical traces of the bursting response of individual S.
epidermis and E. coli cells respectively. Both traces have a similar form; contact
between the probe and the cell is made at point A after which the cell deforms until
rupture (point B). Following bursting of the cell, there is a decrease in force on the
probe until it reaches the slide at point D, leading to a significant increase in force.
Plots of bursting force versus (equivalent spherical) diameter for S. epidermis and E.
coli are shown in Figure 3a and Figure 3b respectively. In both cases, the large scatter
observed in bursting force is attributed to biological variation within the culture. Three
separate samples were taken (t = 4, 5 and 6 hours) and tested within one hour of
sampling. Statistical testing using ANOVA indicated a strong probability that the three
samples were from equivalent populations and hence they were treated as such. For cell
diameters ranging from 0.5 to 1.6 µm with an average value of 0.92 µm (standard error
0.03 µm), the bursting force for S. epidermis cells was found to vary from 3 to 34 µN
(standard error 0.8 µN) with an average value of 13.8 µN. Furthermore, simple linear
regression appeared to suggest some relationship between bursting force and cell size,
which was confirmed using an F-test with 95% confidence.
160
A COMPARISON OF THE MECHANICAL PROPERTIES OF BACTERIAL SPECIES
Corresponding values for E. coli ranged from 1 to 9 µNwith an average value of 3.6 µN
(standard error 0.4 µN) for cells with equivalent spherical diameters between 0.7 to 1.8
µm with an average value of 1.26 µm (standard error 0.05 µm). Simple linear regression
showed no relation between the bursting force and equivalent spherical diameter. This
was confirmed with an F-test at the 95% confidence level.
Figure 3: Plots of bursting force vs. (equivalent spherical) diameter for (a) S. epidermis and
(b) E. coli
The bursting force measurements obtained for S. epidermis and E. coli demonstrate the
usefulness of the micromanipulation technique for obtaining information on the
mechanical properties of bacteria. Linked with a suitable cell model, it should be
possible to estimate other mechanical properties. Biomechanics might be linked to cell
physiology by carrying out micromanipulation in conjunction with experiments to
determine cell wall composition, even using mutants known to lack certain cell wall
components.
161
C. SHIU, Z. ZHANG AND C.R. THOMAS
Bacterial species do not always grow as individual cells but in a range of different
morphologies. Micromanipulation techniques that have been developed for tensile
testing of fungi (Roberts, 1999) and filamentous bacteria (Stocks and Thomas, 1999)
might be adapted to pull apart bacteria that grown in chains of while larger probes might
be used to measure the bursting forces of bacteria that grow in clumps.
References
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Keshavarz, E., Hoare, M., and Dunnill, P. (1987) Biochemical engineering aspects of cell disruption. In
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Mashmoushy, H., Zhang, Z. and Thomas, C.R. (1998) Micromanipulation measurement of the mechanical
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thread biomechanics: assembly and material properties of cell walls. In Antibiotic Inhibition of Bacterial
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Symposium Society of General Microbiology 6, 150 -180.
Ou, L.-T. and Marquis, R.E. (1970) Electromechanical interactions in cell walls of Gram-positive cocci.
Journal of Bacterology 101, 92-101.
Roberts, A.D. (1999). Determination of Fusarium graminearium mechanical properties by
micromanipulation. PhD Thesis (submitted). The University of Birmingham.
Shockman, G.D., Barrett, J.F., (1983) Structure, junction and assembly of cell walls of Gram-positive
bacteria. Annual Review of Microbiology 37, 501-527.
Srinorakutara, T. (1997) Mechanical Strength of Yeasts. PhD Thesis (unpublished). The University of
Birmingham.
Stocks, S.M. and Thomas, C.R. (1999). The tensile strength of Saccharoployspora erythraea. European
Journal of Histochemistry (Proceedings of the first international conference on analysis of microbial cells
at the single cell level, Como, Italy, 1999).
Thwaites, J.J. and Mendelson, N.H. (1985) Biomechanics of bacterial walls: Studies of bacterial thread made
from Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America
82, 2163-2167.
Thwaites, J.J. and Surana, C. (1991) Mechanicalproperties of Bacillus subtilis cell walls: Effects of removing
residual culture medium. Journal of Bacteriology 173, 197-203.
Thwaites, J.J. and Mendelson, N.H. (1991) Mechanical behaviour of bacterial cell walls. Advances in
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162
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
Abstract
A strain of Kocuria rosea with keratinolytic activity was isolated. This Gram-positive
coccus-shaped bacterium was able to degrade whole feathers in 72 h at 40° C. In batch
culture, the optimum temperature for feather degradation, bacterial growth and protease
secretion was 40° C. Under these conditions, biomass and caseinolytic activity reached
3.2 g/l and 0.15 U/ml, respectively, after 36 h incubation. Extracellular protease
secretion was associated with the exponential growth phase. Feather degradation
reached 51 % in 72 h with a yield on biomass of 0.32 g/g. Enzymatic assay by
polyacrylamide-gelatine gels showed two bands with alkaline proteolytic activities in
the fermentation juice after 72 h of culture.
1. Introduction
show that it is not very digestible and the destruction of certain amino acids during the
process, such as cysteine, magnifies the imbalance of essential amino acids (Bhargava
& O’neil 1975; Baker et al. 1981).
Thus, there is a growing interest in alternative methods for the treatment of feathers to
improve the nutritional quality of the feather meal and to develop new and useful by-
products from feathers. The nutritional value of the feather meal might be improved by
microbial action, this results in a modification of the structure of keratin, altering the
resistance to the enzymes of the digestive tract. The literature reports that isolates of
Bacillus lichenformis (Williams et al. 1991) and Streptomyces pactum (Böckle et al
1995) grown on feather media were able to modify the digestibility and profiles of the
specific amino acids that were liberated. The bacterial cells incorporated into the
fermented feathers may contribute to some amino acids, hence improving their balance.
Also, feather as a fermentation substrate may be useful in order to obtain other
microbial products (proteolytic enzymes, carotenoid pigments, etc.).
We conducted a study on an isolate from the soil of a poultry-processing plant in
order to determine microbial growth, possible keratinolytic activity and optimum
feather degradation conditions in submerged fermentation. To our knowledge, this is the
first report describing the capacity of Kocuria rosea to degrade feathers.
Isolating microorganisms from nature is the microbiologists first step in screening for
natural microbial products. It is possible to isolate many different microorganisms by
employing enrichment techniques on the appropriate culture medium. One successful
approach for the discovery of new metabolites involves considering the characteristics
of the desired product and process development and using ecological approaches for
isolation and screening. Consideration of the ecological approaches to isolation can
provide a screen with both a large number and a wide variety of microorganisms to
examine for the product of interest. Even though microorganisms are highly adapted,
specific microbial types are associated with different niches within a variety of
ecosystems. Therefore, what is isolated from the defined ecosystem or habitat is a
reflection of the isolation procedures and of the conditions found in nature.
166
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
isolate, designated as LPB-3, was able to degrade feathers in 72 h at 40° C when isolated
cells were incubated on whole feathers suspended in saline medium.
The keratinolytic activity of LPB-3 isolate was evidenced by visual observation of
feather disintegration, as well as increasing turbidity of the culture due to growing cells
(Figure 2). The bacterium grew aerobically and was catalase positive. Additional
biochemical tests conducted on LPB-3 isolate in our laboratory were confirmed by the
Pasteur Institute of France, which characterised the isolate as Kocuria rosea
Figure 2. Degradation of feathers by isolate. The LPB-3 strain was cultivated in assay tubes
with whole feathers suspended in mineral medium (g/l): NH4CI, 0.5; NaCI, 0.5; K2HPO4,
0.3; KH2PO4, 0.4; MgCI2.6H2O, 0.1; 0.1 yeast extract; pH 7.5 at 40°Cfor 72 h. A, control;
B, 24 h; C, 48 h; D, 60 h: E, 72 h.
167
NEFEIDA COELLO AND LUIS VIDAL
Only limited information regarding Kocuria rosea is currently available. This Gram
positive micro-organism, which was classified in the past in the genus Micrococcus and
reclassified recently in the genus Kocuria (Stakenbrandt 1999, has been studied mainly
in relation to the structure and properties of the carotenoid pigments it produces
(Cooney 1981; Jagamadham 1991). Until now, the feather degradation capacity of K.
rosea has not been reported in the literature.
Cells of the LPB-3 isolate were grown on whole-feather basal medium, in which the
autoclave treatment time was progressively reduced. The isolate was able to degrade
feathers which had been autoclaved for only 5 min. Attempts to maintain the growth of
the isolate on non-steam-treated feathers were not successful,
Microscopic observations of the isolate showed a coccus (1.5 µm) which appeared
singly and in chains adsorbed mostly to the feathers, although a few microorganisms
swam freely (Figure 3A). The adsorption to the fermentation substrate is part of a
survival strategy of microbes in certain limiting environments in order to improve
feather degradation via proteolytic enzymes secretion. The ultrastructure of K. rosea
was generally similar to that of other Gram-positive bacteria. The cells were separated
from the surrounding medium by a microcapsule and a cell wall with a homogeneous
structure, approximately 20 nm thick (Figure 3B). The cytoplasmic membrane was
closely adjacent to the cell wall and the periplasmic space was poorly discernible. K.
rosea was characterised by a high ribosome density in the cytoplasm and the occurrence
of polyphosphate granules and glycogen accumulation was also observed. The nucleoid,
consisting of DNA fibrils, was located in the centre of the cell and as cell division
began, a septum was formed inside the cells. Transmission electron micrographs
showed in some cells the presence of two asymmetrical septa.
168
KOCURlA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
169
NEREIDA COELLO AND LUIS VIDAL
Figure 4. Effect of quantity of feathers. LPB-3 strain was cultivated at 40°Cfor 96 h in 500-
ml Erlenmeyer baffled flasks, containing IO g/l 20 g/l (0) and 30 g/l scissors-cut
feathers, suspended in mineral medium described earlier shaken at 75 rpm. Control non
inoculated . Samples were taken and filtered through a Büchnerfunnel with Whatman
paper 4. Afer removing cells by centrifugation supernatant was used for free amino groups
analysis using a ninhydrin method described by Rosen
(1957).
Figure 5. Growth of Kocuria rosea LPB-3 (O) and free amino groups concentration at
different temperatures in feather medium (20 g/l). Samples were taken at 72 h filtered
through a Büchner funnel with a Whatman paper 4. Filtrated was used to measure cell mass
density by turbidity (600 nm) andfree amino groups as described earlier.
170
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
4. Industrial applications
Industrially, feather waste is processed into hydrolysed feather meal using high
temperatures and pressure. It is utilised as supplemental source of protein in poultry
foodstuffs. However, the use of hydrolysed feathers meal, as a replacement protein is
limited. Morris and Balloun (1973) observed that the addition of 6 % hydrolysed feather
meal to diets of chickens caused a decreased body weight. Research showed that raw
feathers can be fermented in anaerobiosis with a Bacillus licheniformis strain (Williams
et al, 1991) to produce a fermented feather lysate that can be used as a replacement
protein up to 15 % in poultry foodstuff. The feather lysate obtained with K. rosea is
171
NEREIDA COELLO AND LUIS VIDAL
Figure 6. Growth -O- (A), proteolytic activity and residual feathers (B) of Kocuria
rosea LPB-3 in feather medium (20 g/l) at 40° C for 96 h and 75 rpm agitation. Residual
feathers are expressed as a percentage of dry weight of the residue obtained upon filtration
of the culture contents through a Whatman paper No. 4. Protease activity was assayed by
photometric method (280 nm) using casein as substrate based on the method of Kunitz
(1947) One unit of proteolytic activity was defined as the amount of enzyme which produced
an absorbance increase of 1.0 units within one min.
172
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
4.2. ENZYMES
Proteolytic enzymes are the most important industrial enzymes because of their wide
applications in the detergent, dairy, pharmaceutical, leather and food industry. Many
alkaline proteases are produced by bacteria and fungi and are also present in
mammalian tissues. Cultures of K. rosea in feather medium stimulate the secretion of at
least two alkaline proteases (Figure 7) with a broad tolerance to temperature (35-70 ° C)
and high long-term storage stability. These proteases degrade keratin of feathers to
produce short peptides and amino acids that can be used as carbon and nitrogen sources
for the microorganism. They could be useful in the dehairing stage of leather processing
and the formulation of dehairing lotion removers of cosmetic industry. Bovine and
porcine pancreatic trypsin have demonstrated to be effective in reducing the
requirement of lime, shortening the dehairing process time and lowering the waste
treatment costs (Ward, 1985). Thus, extensive genetic and physiological studies must be
carried out for a better understanding of the mechanism that controls keratinase
secretion in the wild-type strain of K. rosea in order to make this protease available for
industrial purposes.
4.3. PIGMENTS
Many microorganisms including algae, fungi and bacteria produce carotenoid pigments.
Industrially, they are used as a food colouring agents (e.g. margarine, cheese, and egg
173
NEREIDA COELLO AND LUIS VIDAL
products) and at a lesser extent as oral tanning agents in the cosmetic industry.
Cantaxanthin, a xantophyll carotenoid used as feeding additive together with
astaxanthin, have been used in the poultry industry to improve the colour of chicken and
egg yolk and for muscle pigmentation in farmed salmonids (Elmadfa, 1998).
Cantaxanthin is the major coloured carotenoid pigment biosynthesised in mesophilic K.
rosea strains (Cooney, 198 1). Further research concerning qualitative and quantitative
pigment content and factors influencing carotene biosynthesis in K. rosea must be done
to consider the microbiological pigment processes available.
Acknowledgements
The authors acknowledge Lic. C. Bernal and Dr. A. Bretaña for their helpful
contribution in enzyme gel assay and electron microscopy, respectively. This research
was supported by grants from Interamerican Development Bank and Consejo Nacional
de Ciencia y Tecnología (BTI-88), and Consejo de Desarrollo Científico y Humanístico
of Central University of Venezuela (09- 12-404497).
References
Atalo, K. and Gashe, B. (1993) Protease production by a thermophilic Bacillus species (p-001A) which
degrades various kinds of fibrous proteins. Biotechnology Letters 15, 1115-1156.
Baker, D. H., Blitenthal, R. C., Boebel, K. P., Czrnecki, G. L., Southern, L.L. and Willis, G. M. (1981)
Protein amino-acid evaluation of steam-processed feather meal. Poultry Science 60, 1865-1872.
Bhargava, K.K. and O’Neil, J. B. (1975) Composition and utilisation of poultry by-product and hydrolysed
feather meal in broiler diets. Poultry Science 54, 1511-1518.
Böckle, B., Galunsky, B. and Muller, R. (1995) Characterisation of a keratinolytic serine proteinase from
Streptomycespactum DSM 40530. Appl. Environ. Microbiol. 61, 3705-3710
Chattopadhyay, MK., Jagannadham, MV., Vairamani, M., and Shivaji, S. (1997) Carotenoid pigments of an
Antarctic psychrotrophic bacterium Micrococcus roseus: temperature dependant. biosynthesis, structure
and interaction with synthetic membranes. Biochemical and Biophysical Research Communications 239,
85-90.
Cooney, J.J., and Berry, R.A. (1981) Inhibition of carotenoid synthesis in Micrococcus roseus. Canadian
Journal of Microbiology 27, 421-425
Elmadfa, I. and Majchrzak D. (1998) Carotenoids and Vitamin A in fish. ZErnahrungswiss 37, 207-210
Jagannadham, M.V., Narayanan, K., Rao, C.M., and Shivaji, S. (1996) In vivo characteristics and localisation
of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic
spectroscopy. Biochemical and Biophysical Research Communications 227, 221-226.
Jagannadham, MV., Rao, VJ., and Shivaji, S. (1991) The major carotenoid pigment of a psychrotrophic
Micrococcus roseus strain: purification, structure and interaction with synthetic membranes. Journal of
Bacteriology 173, 7911-7917.
Kunitz, M. (1947) Crystalline soybean trypsin inhibitor II. General properties. Journal of General Physiology
30, 291-310.
Noval, J. and Nickerson, W. (1959) Decomposition of native keratin by Streptomyces fradiae. Journal of
Bacteriology 77, 251-263
Rosen, H. (1957). A modified Ninhydrin Colorimetric analysis for amino acids. Archives of Biochemistry and
Biophysics 67, 10-15.
Stackebrandt, E., Koch, C., Gvozdiak, O., and Shumann, P. (1995) Taxonomic dissection of the genus
Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen.
nov., and Micrococcus. International Journal of Systematic Bacteriology 4, 682-692.
Ward, O.P (1985). Proteolytic enzymes, in Comprehensive Biotechnology Moo-Young Editor, Pergamon
Press. Oxford, U.K. pp.790-817
174
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
Williams, C. M., Richter, C. S., Mackenzie, J. M., Jr. and Shih, J. (1990) Isolation, identification, and
characterisation of a feather-degrading bacterium. Applied and Environmental Microbiology 56, 1509-
1515.
Williams, C. M., Lee, C.G., Garlich, J.D. and Shih, J. (1991). Evaluation of a feather fermentation product,
feather lysate, as a feed protein. Poultry Science 70, 85-94.
Xiang Lin, Chung-Ginn Lee, Ellen S. Casale and Shih, J. (1992) Purification and Characterisation of a
Keratinase from a Feather-Degrading Bacillus licheniformis Strain. Applied and Environmental
Microbiology 58, 3271-3275.
175
COMPARISON OF PB2+ REMOVAL CHARACTERISTICS BETWEEN
BIOMATERIALS AND NON-BIOMATERIALS
Abstract
The order of Pb2+ removal capacities in chemical adsorbents were found as ion
exchange resin > zeolite > granular activated carbon (GAC) > powdered activated
carbon (PAC), while in biomass was Aureobasidium pullulans > Saccharomyces
cerevisiae > activated sludge. Although Pb2+ removal capacity (mg Pb2+/g) of the
activated sludge (30.9) was lower than those of ion exchange resin (167.7) and other
pure cultures of A. pullulans (170.4) and S. cerevisiae (95.3), it was higher than those of
other chemical adsorbents such as GAC (26.9), PAC (2.1), and zeolite (30.2). The initial
Pb2+ removal rates in chemical adsorbents were in the order of PAC > GAC > zeolite >
ion exchange resin, while in biomass was A. pullulans > activated sludge > S.
cerevisiae. The initial Pb2+ removal rate of activated sludge was higher than those of
GAC, zeolite, ion exchange resin and S. cerevisiae cells.
1. Introduction
There are numerous reports documenting the capability of pure cultures of bacteria
(Yong and Macaskie, 1997), algae (Leush et al., 1995), and fungi (Suh et al., 1998) to
remove heavy metal ions from solution. According to the report of Shumate and
Strandberg (1985), multi-species communities of bacteria removed silver equal to 32%
of the dry cell weight, which was considerably higher than those exhibited by pure
cultures of Pseudomonas maltophilia, Thiobacillus thiooxidans, and T. ferroxidans. The
important point was thus made that mixed microbial cultures could be more efficient in
removing heavy metal ions than pure cultures.
Removal of heavy metal ions by mixed cultures of activated sludge have been
studied for the last 40 years by many investigators (Ruchoft, 1949; Brown and Lester,
1982; Rudd et al., 1984) to evaluate the possibility for removing a number of heavy
metal ions.
177
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 177–183.
©2001 Kluwer Academic Publishers. Printed in the Netherlands.
DONG SEOG KIM AND JUNG HO SUH
In particular, Pb2+, well recognised for its detrimental effect on the environment where it
accumulates throughout the food web, is generated from mining, metal, dyestuff,
electric, and petroleum industries. Pb2+ was known to be easily removed by
microorganisms. Rossin et al. (1982), for example, pointed out that average heavy metal
ion removal with activated sludge was as low as 1% for Ni2+ and as high as 92% for
Pb2+. Equilibrium metal uptake values using freeze-dried Rhizopus arrhizus increased in
the order of Sr2+ < Mn2+ < Zn2+ < Cd2+ < Cu2+ < Pb2+, and were positively correlated
with the covalent index of the metal ions (Brady and Tobin, 1995). The order of
adsorption for Sargassaumfluitans biomass particles (Leush et al., 1995) was in the order
of Pb2+ > Cd2+ > Cu2+ > Ni2+ > Zn2+.
A major goal of this research is to examine the feasibility of activated sludge on Pb2+
removal, by comparison with two pure cultures (Saccharomyces cerevisiae and
Aureobasidium pullulans) and widely used chemical adsorbents (activated carbons, ion
exchange resin and zeolite).
2.1. MATERIALS
Activated carbon, which is used in a filtration plant, was made with charcoal and the
sizes of granular activated carbon (Sigma C2764, USA) and powdered activated carbon
(Sigma C5260, USA) were 4~8 mesh and 100~400 mesh, respectively. Cation exchange
resin (SK1B) with sulfonyl group was purchased from Sam Yang Co. (Korea). Zeolite
(Z3125) was obtained from Sigma Co. as a powder type, of which diameter was below
10 µm. Activated sludge was prepared from the secondary return-sludge from the
municipal wastewater treatment plant.
In the case of biomass, the prepared activated sludge or cell suspension was mixed with
an equal volume of the initial concentration of aqueous Pb(NO3)2 solution prepared in
twice the desired concentration. The pH values of the Pb2+ solution, biomass
178
PB2+ REM0VAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
suspensions, and the mixture were 3.0-4.0, 5.5-5.7 and 3.5-5.5, respectively. pH
adjustment was not conducted and no spontaneous metal precipitation was observed in
the prepared solutions. The experiments were carried out by employing 50 ml Pb2+
solution and 50 ml activated sludge or cell suspension in 300 ml conical flasks and
shake them on a rotary-shaker incubator at (150 rpm). Samples of 1.8 ml were
taken at the proper time period and centrifuged immediately (10,000 x g, 10 min). The
Pb2+ concentration in the supernatant was measured by atomic absorption spectrometry
(Perkin Elmer 3300, USA). The dry weight of biomass was obtained after drying at
105| to a constant weight. Removed Pb2+ amounts per dry weight of adsorptive
materials at equilibrium state (q) were calculated from Pb2+ mass balance yield : q (mg
Pb2+/g dry weight) = (Ci - Ce)/m. Where Ci and Ce are the Pb2+ concentrations (mg
Pb2+/l) in the supernatant at the initial and equilibrium state, respectively, and m is the
concentration of the dried activated sludge or cells (mg dry weight/l). The initial Pb2+
removal rate (ri) was measured by calculating the slope from the plot of the removed
Pb2+ amounts per dry weight (mg Pb2+/g dry weight) vs. time (min) at t=0.
Fig, 1. Typical time courses of Pb2+ removal by chemical adsorbents under various initial
Pb2+ concentrations: (a) granular activated carbon, (b) powdered activated carbon, (c) ion
exchange resin, (d) zeolite. Initial adsorbent concentrations (g/l) in (a), 1.0; (b), 2.0; (c),
1.0; (d) 1,0,
The order of Pb2+ removal capacity in chemical adsorbents was found as ion exchange
resin > zeolite > granular activated carbon (GAC) > powdered activated carbon (PAC)
(Fig. 1). On the comparison between the results of GAC and PAC, the Pb2+ removal
179
DONG SEOG KIM AND JUNG HO SUH
capacity of GAC was three times higher than that of PAC. However, the time required
to reach an equilibrium state with GAC was much longer than with PAC because GAC
has much larger specific surface area and pore diffusion resistance than PAC.
The increase in Pb2+ removal capacity according to the increase of initial Pb2+
concentration in biomass was similar to those of chemical adsorbents (Fig. 2). The Pb2+
removal capacity of the activated sludge was increased only 1.8 times (from 42 mg
Pb2+/g to 74 mg Pb2+/g) even though the initial Pb2+ concentrations increased by 6-fold
(from 37 mg/l to 228 mg/l).
Fig.2. Typical time courses of Pb2+ removal by biomass various initial Pb2+ concentrations:
(a) activated sludge, (b) A, pullulans, (c) S. cerevisiae. Initial biomass concentrations (g/l)
in (a), 1.0; (b). 0.8; (c), 0.8.
An interesting result was obtained by the two selected microorganisms. That is, in our
experimental range, as the initial Pb2+ concentrations were increased accurately 5.7
times (from 49 mg/l to 278 mg/l) and 6 times (from 16 mg/l to 96 mg/l) in A. pullulans
and S. cerevisiae, respectively, the Pb2+ removal capacities were increased about 5.7
times (from 53 mg Pb2+/g to 300 mg Pb2+/g) and 6 times (from 12 mg Pb2+/g to 72 mg
Pb2+/g), respectively. The Pb2+ removal in pure cultures of A. pullulans and S. cerevisiae
gave more favourable result than activated sludge.
Considering Pb2+ removal characteristics, it was found that the time required to
reach an equilibrium state in activated sludge and A. pullulans was independent on the
initial Pb2+ concentrations. But, in S. cerevisiae, the time was increased in response to
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PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
the initial Pb2+ concentration, and was much longer than those of the activated sludge
and A. pullulans. This may be caused by the difference in Pb2+ removal process. In other
words, most of the Pb2+ taken up by S. cerevisiae was deposited in the inner parts of the
cell at equilibrium state and the Pb2+ accumulation mechanism was divided into three
steps, involving metabolism-independent, -dependent and -independent (Suh et al,
1998a). On the contrary, A. pullulans used a different metabolism-independent Pb2+
accumulation process due to the existence of extracellular polymeric substances (EPS)
(Suh et al., 1998b). Therefore, it was suggested that Pb2+ penetration into inner cellular
regions may not occurred in the activated sludge because the trend in Pb2+ removal of
the activated sludge has a resemblance to that of A. pullulans. Nevertheless, the detailed
process is still in doubt because activated sludge is composed of a great number of
organisms and several organic materials such as extracellular polymeric substances and
cell flocs, etc.
When compared the Pb2+ removal capacity of chemical adsorbents with those of
biomass, the Pb2+ removal characteristics of activated sludge and A. pullulans marked
similar process to that of PAC. Moreover, S. cerevisiae had a similar removal process to
that of ion exchange resin. The ion exchange played an important role in the Pb2+
removal in S. cerevisiae but little occur in A. pullulans (data not shown). A similar
result was observed when the initial chemical adsorbents or biomass concentrations
were changed, too (data not shown).
Table 1. Comparison of adsorption models and the removed Pb2+ amounts
at q10 and q200 between the chemical absorbents and biomass
The Pb2+ removal capacity in chemical adsorbents, evaluating by q10, was in the order of
ion exchange resin > zeolite > GAC > PAC, while that of biomass was A. pullulans > S.
cerevisiae > activated sludge. A. pullulans showed remarkably higher Pb2+ removal
capacity than S. cerevisiae in q10 due to the effect of EPS formation. However, the q200
value of S. cerevisiae was slightly higher than that of A. pullulans. The terms of q10 and
q200 were the Pb2+ removal amounts (mg Pb2+/g) at the equilibrium concentrations of 10
mg/l and 200 mg/l, respectively. This interesting phenomenon was observed when the
181
DONG SEOG KIM AND JUNG HO SUH
initial Pb2+ concentration was very high or initial biomass concentration was extremely
low. This result may be caused by the difference in Pb2+ removal mechanisms, but
further study is required to reinforce this result.
In general, q10 may be more reasonable than q200 from the practical points of view. The
Pb2+ removal capacity of the activated sludge was relatively lower than those of other
biomass (A. pullulans and S. cerevisiae) or ion exchange resin, but higher than those of
other chemical adsorbents (GAC, PAC, and zeolite). This result implies the possible
application of the activated sludge on Pb2+ removal process.
The Pb2+ removal capacity of the activated sludge was around one to fifth and one to
third as low as those of A. pullulans and S. cerevisiae, respectively. However, the
utilisation of activated sludge on Pb2+ removal can be recommended, taking into
consideration of economical and practical aspects and the stability of operation system.
In heavy metal removal processes, not only the removal capacity but also the initial
removal rates are considered to be very important factors from the practical aspects of
reactor design and process optimisation. The initial Pb2+ removal rate in response to the
variation of initial Pb2+ concentration is shown in Fig. 3. The initial Pb2+ removal rate
was increased as initial Pb2+ concentration increased. However, it was almost
independent of initial Pb2+ concentration over a critical concentration. The Pb2+ removal
rates of activated sludge and A. pullulans were much higher than those of the chemical
adsorbents, but S. cerevisiae was not the case. The order of initial rate of Pb2+ removal
in adsorbents was found as PAC > GAC > zeolite > ion exchange resin (Fig. 3(a)).
Where, the low degree of resistance in pore diffusion of PAC might cause the highest
initial Pb2+ removal rate. Moreover, the ion exchange resin showed the lowest initial
Pb2+ removal rate because ion exchange is here main process rather than physical
adsorption.
Fig. 3. Comparison of the initial Pb2+ removal rates between (a) chemical adsorbents and
(b) biomass: Symbols in (a); (0) GAC, PAC, ion exchange resin, zeolite.
Symbols in (b); (0) activated sludge, A. pullulans, (A) S. cerevisiae.
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PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
On the other hand, the initial Pb2+ removal rate in biomass was in the order of A.
pullulans > activated sludge > S. cerevisiae, as shown in Fig. 3(b). The Pb2+ removal in
A. pullulans was very rapid, whereas that in S. cerevisiae was very slow. This is, as
mentioned earlier, because Pb2+ removal in A. pullulans was mainly achieved by
adsorption onto EPS around the cell surface and that in S. cerevisiae was caused by the
Pb2+ penetration into the inner cellular region. Therefore, by evaluating as initial Pb2+
removal rate, the activated sludge was placed in the middle of A. pullulans and S.
cerevisiae, and it can be recommended as an useful resource for the removal process of
Pb2+.
4. Conclusions
References
Brady, J.M. and Tobin, J.M. (1995) Binding of hard and soft metal ions to Rhizopus arrhizus biomass.
Enzyme Microb. Technol. 17, 791-796.
Brown, M.L. and Lester, J.N. (1982) Role of bacterial extracellular polymers in metal uptake in pure bacterial
culture and activated sludge-I Effects of metal concentration, Water Res. 16, 1539-1548.
Leusch, A., Holan, Z.R. and Volesky, B. (1995) Biosorption of heavy metals (Cd, Cu, Ni, Pb, Zn) by
chemically-reinforced biomass of marine algae. J Chem. Tech. Biotechnol. 62, 279-288.
Rossin, A.C., Sterritt, R.M. and Lester, J.N. (1982) The influence of process parameters on the removal of
heavy metals in activated sludge. Water, Air, andSoil Pollution 17, 185-198.
Ruchoft, C.C. (1949) The possibilities of disposal of radioactive wastes by biological treatment methods.
Sewage Works J. 21, 877-883.
Rudd, T., Sterritt, R.M. and Lester, J.N. (1984) Complexation of heavy metals by extracellular polymers in
the activated sludge. J Water Pollut. Control Fed. 56, 1260-1268.
Shumate II, S.E. and Strandberg, G.W. (1985) Accumulation of metals by microbial cells, in M. Moo-Young
(ed.), Comprehensive Biotechnology, Pergamon Press, New York, vol. 4, pp. 235 - 247.
Suh, J.H., Kim, D.S., Yun, J.W. and Song, S.K. (1998a) Process of Pb2+ accumulation in Saccharomyces
cerevisiae. Biotechnol. Left. 20, 153-156.
Suh, J.H., Yun, J.W. and Kim, D.S. (1998b) Comparison of Pb2+ accumulation characteristics between live
and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans. Biotechnol. Lett. 20,247-251.
Yong, P. and Macaskie, L.E. (1997) Removal of lanthanum, uranium and thorium from the citrate complexes
by immobilised cells of Citrobacter sp. in a flow-through reactor: implications for the decontamination of
solutions containing plutonium. Biotechnol. Lett. 19, 251-255.
183
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA
Abstract
Streptomyces strains from Kuwait oil field were isolated by selective inorganic media
containing the oil fractions as sole carbon and energy sources. Hydrocarbon utilisation
was measured by gas-liquid chromatography, and by the use of proper radioactively
labelled oil fraction. Streptomyces strains rapidly used n-alkanes, and incorporated them
to corresponding fatty acids of identical chain length. n-Alkane uptake was markedly
increased by specific GTP-binding protein activators. Fluorescence measurements of the
uptake of the hydrophobic diphenylhexatriene (DPH) showed significant difference
between oil-utilising and non-utilising strains.
Strains isolated from Kuwait desert oil fields (Barabás et al., 1995) were coded as KCC
(Kuwait Culture Collection). KCC26, KCC28, KCC30 and KCC42 were identified as
Streptomyces plicatus, KCC25 as Streptomyces griseoflavus. Strains KCC18, KCC33,
KCC36 and Khiran30 have not been taxonomically identified yet, they show, however,
the typical Streptomyces morphology in light microscope. The strains, their isolation,
the composition of starch-casein medium and their potential of utilising n-alkanes were
previously described (Barabás et al., 1995). S. griseus 2682 was used as an oil non-
utilising control (designated as ,,non-utilising”).
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A. Durieux and J-P. Simon (eds.), Applied Microbiology, 185–190.
©2001 KIuwer Academic Publishers. Printed in the Netherlands.
GY. BARABÁS ET AL
The biomass samples were obtained by growing the test organisms on suitable
conventional media at 27°C. The Streptomyces strain was cultivated on sterile
cellophane sheets covering the surface of starch-casein-agar medium: starch, 10g;
casein (Difco, vitamin free), 0.3g; KNO3, 2g; NaCl, 2g; K2HPO4, 2g; MgSO4.7H2O,
0.05g; CaCO3, 0.02g; FeSO4.7H2O, 0.01g; Bacto agar, 18g; soil extract (from 1% soil
suspension), 100 ml; distilled water to 1000 ml; pH 6.8. The cultures were incubated at
27°C for 5 d. The Streptomyces biomass samples were harvested by removing the
cellophane sheets.
For fluorescence measurement of diphenylhexatriene (DPH) uptake, n-hexadecane (C16)
utilising and non-utilising strains were cultivated in filtered soybean liquid medium
described previously for S. griseus 52-1 (Szabó et al., 1985). Cells were harvested after
36 h cultivation, washed twice with distilled water, resuspended in inorganic medium
(IM) with the following composition in g per litre: 0.85 NaNO3; 0.56 KH2PO4; 0.86
Na2HPO4; 0.17 K2SO4; 0.37 MgSO4.7H2O; 0.007 CaCl2.6H2O; 0.004 FeIIIEDTA; 2.5
ml of a trace element solution consisting of (g per litre): 2.32 ZnSO4.7H2O; 1.78
MnSO4.4H2O; 0.56 H3BO3; 1.0 CuSO4.5H2O; 0.39 Na2MoO3.2H2O; 0.66 KI; 1.0
EDTA; 0.4 FeSO4.7H2O; 0.004 NiCl2.6H2O.
Incubation was carried out at 27°C in a gyratory shaker at 300 rpm. The optical density
of the cell suspension was adjusted to 0.5 at 535 nm and 2% (V/V) of n- hexadecane
(C16) was added to IM.
The different potential of C16 utilising and non-utilising strains to take up hydrocarbons
was studied by fluorescence measurements. For this purpose, the hydrophobic
compound diphenylhexatriene (DPH) was selected as the test fluorescent material.
Fresh biomass samples of C16 utilising and non-utilising strains grown in filtered
soybean medium were resuspended in IM than the samples were supplemented with 90
µM DPH dissolved in n-hexadecane. Their fluorescence was examined in quartz cells in
a Perkin-Elmer MPF 44B spectrofluorimeter equipped with a thermostatic cell holder.
The DPH fluorescence of cells was excited at 355 nm and the emission was collected at
430 nm. The cell suspensions did not show significant autofluorescence in this
wavelength range. In experiments when the kinetics of DPH uptake was investigated,
186
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERlA
small aliquots (2ml; n=3) were taken from the culture flask from time to time, washed
and the DPH fluorescence (corrected for light scattering) was determined.
The fatty acid determinations were performed as described by Barabás et al., 1995.
0.5 g aliquots of mycelium precultivated in soy-bean medium for 36h were resuspended
in 25 ml aliquots of inorganic medium containing 10 mg n-hexadecane or n-octadecane.
The submerged cultures were supplemented with various amounts of GTPγS or AIF4-
and incubated at 30°C with a shaking frequency of 250 rpm. Biomass was harvested
after 6 hours by centrifugation and the hydrocarbon uptake of the cells was determined
from these samples as described in Barabás et al., 1995.
Streptomyces are soil bacteria occurring even under extreme conditions (hot desert, salt-
marsh area, alpine slopes, etc.). They are capable of hydrocarbon uptake and utilisation.
Uptake of oil as followed by incorporated diphenylhexatriene (DPH) from n-
hexadecane phase of the medium was significantly higher in the oil utilising strain than
in a non-utilising one (Table 1, Radwan et al., 1998). Fluorescence measurements with
DPH showed that the membrane characteristics of hydrocarbon-using strains
significantly differed from that of the non-using ones (Radwan et al., 1998).
Incorporation of the label from radioactive hexadecane into mycelia was also
measured in several oil degrading strains and one non-degrading one (Table 2). During
the 48-hour cultivation with labelled hydrocarbon the highest amount found to be
incorporated (into strain KCC18) was equivalent to about 3% of the total amount of the
radioactivity added to the culture. Other strains incorporated somewhat less amount, and
strain Khiran30, that served as a control, could not incorporate radioactivity.
Table 1 DPH fIuorescence incorporated frorn n-hexadecane phase by oil utilising (KCC 26)
and non-utilising (S griseus) strains
Time DPH fluorescence
(hour) (arbitrary units)
KCC 26 S. griseus
19 0.039 0.010
40 0.361 0.283
62 0.65 1 0.324
91 0.820 0.315
122 0.795 0.344
182 0.852 0.308
187
GY. BARABÁS ET AL
Fatty acid analysis showed that the incubation with n-alkanes resulted in an increase of
the fatty acids with chain length equivalent to those of the alkane substrates (Barabás et
al., 1995). The fatty acid 16:1 fraction increased from 17.4 to 29.9 in the presence of n-
hexadecane. Fatty acids of C18 appeared only in the presence of n-octadecane.
It is known that GTP-binding proteins play essential role in mediating cellular
responses to a wide variety of extracellular signals, such as hormones, growth factors,
neurotransmitters, chemical signals or light (Gilman, 1987, Taylor, 1990). These
proteins transmit signals via a GTP-dependent mechanism to the effector systems
(enzymes or ion channels) and thereby regulate these systems that often control
production of intracellular second messenger molecules. GTP-binding proteins (GBPs)
act as molecular switches, their activation is catalysed by a ligand activated receptor and
deactivation is established by the intrinsic GTPase activity of the GBP. According to
this mechanism, the GTP-bound form is the active complex, which returns to inactive
state by hydrolysing its GTP to GDP. The exchange of GDP to GTP and the rate of
GTP hydrolysis is regulated by specific regulatory proteins (Gilman, 1987, Itoh et al.,
1986, Taylor, 1990).
GTP-binding proteins were reported to be present in S. coelicolor A(3)2 and we have
shown the presence of these proteins in S. griseus and in several other Streptomyces
strains (Itoh et al., 1996, Penyige et al., 1992). Our previous results suggested that in S.
griseus A-factor - a γ-utyrolactone type autoregulator molecule produced by wild type
S. griseus cells and required for the normal differentiation process and antibiotic
production in the producer strain (Khokhlov et al., 1973) - could activate an intrinsic
GTPase activity present in the cellular membrane of S. griseus NRRL B-2682 (Penyige
et al., 1992).
The study of their role in different cellular processes was greatly enhanced by using
certain reagents, such as AIF4- or GTPγS. AlF4- mimics the effect of the γ-phosphate of
GTP on the inactive GDP-bound form of the protein, GTPγS is a non-hydrolysable GTP
analogue (Yatani et al., 1991).
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HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA
Table 3. The effect of AlF4- on n-hexadecane and n -octadecane uptake by oil utilising micro-organisms
0 50 75 100 125
KCC25 C16 7.2 10.0 12.5 12.0 11.6
C18 6.3 ND ND ND ND
KCC 28 C16 8.0 9.4 9.8 9.7 9.9
C18 5.2 4.9 5.3 7.8 10.2
KCC 33 C16 9.2 10.5 10.9 11.8 13.6
C18 7.2 ND 6.2 6.6 8.2
KCC 42 C16 7.6 12.3 ND 13.7 15.7
C18 10.3 ND 12.4 11.2 11.8
A. nicotiana C16 5.9 4.9 6.7 12.9 10.8
The results of the effect of GTPγS and AIF4- – stimulators of GBPs – on the uptake of
the hydrocarbon molecules (C16 and C18) are shown in Tables 3 and 4. These show that
the uptake of n-hexadecane (C16) and n-octadecane (C18) from the medium by
hydrocarbon utilising micro-organisms were enhanced by the addition of and
AIF4- although the rate of uptake was strain specific. Moreover, the magnitude of
uptake was, in most cases, directly proportional to the concentration of these effectors in
the medium. These results suggest that GBPs could fulfil important physiological
functions in Streptomyces strains.
With electron microscopy the hydrocarbon utilising strains were found to be
enriched in large less-electron dense areas as inclusions in the cytoplasm in n-alkane
189
GY. BARABÁS ET AL
containing media (Radwan et al., 1998). The oil utilising strains also eliminated
hydrocarbons from soil samples artificially impregnated with hydrocarbons in
laboratory experiments. Field experiments for oil bioremediation are in progress.
3. Conclusion
Streptomyces strains isolated from Kuwait oil fields actively utilised oil fractions and
crude oil, either in hydrocarbon containing inorganic media, or in the soil. Since these
strains are typical soil bacteria tolerating extreme conditions (hot desert, alpine slopes,
salt-marsh area) their practical application in bioremediation of oil pollution is
promising.
References
Barabás, Gy., Sorkhoh, N.A., Fardoon, F. and Radwan, S.S. (1995) n-Alkane-utilisation by oligocarbophilic
actinomycete strains from oil-polluted Kuwaiti desert soil. Actinomycetologica 9, 13-18.
Gilman, A. G. (1987) G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56,615-649.
Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H.,
Suzuki, K. and Kaziro, Y. (1986) Molecular cloning and sequence determination of cDNAs for a subunits
of the guanine nucleotide-binding proteins Gs, Gi and Go from rat brain. Proc. Nail. Acad. Sci. USA 83,
3776-3780.
Itoh, M., Penyige, A., Okamoto, S. and Ochi, K. (1996) Proteins that interact with GTP in Streptomyces
griseus and its possible implication in morphogenesis. FEMS Microbiol. Lett. 135, 311-316.
Khokhlov, A. S., Anisova, L.N., Tovarova, J.J., Kleiner, E.M., Kovalenko, O.S., Krasilnikova, O.S.,
Kornitskaya, E.Y. and Pliner, S.A. (1973) Effect of A-factor on growth of asporogeneous mutant of
Streptomyces griseus, not producing this factor. Z Allg. Microbiol. 13,647-655.
Penyige, A., Vargha, Gy., Ensign, J.C and Barabás, Gy. (1992) The possible role of ADP-ribosylation in
physiological regulation in Streptomyces griseus. Gene 115, 181-185.
Radwan, S.S., Barabás, Gy., Sorkhoh, N.A., Damjanovich, S., Szabó, I., Szöllôsi, J., Matkó, J., Penyige, A.,
Hirano, T. and Szabó, I.M. (1998) Hydrocarbon uptake by Streptomyces. FEMS Microbiol. Letters 169,
87-94
Szabó, I., Benedek, A. and Barabás, Gy. (1985) Possible role of streptomycin released from spore cell wall of
Streptomyces griseus. Appl. Environ. Microbiol. 50, 438-440.
Taylor, C. V. (1990) The role of G proteins in transmembrane signalling. Biochem. J. 272, 1-13.
Yatani, A,, and Brown, A.M. (1991) Mechanism of fluoride activation of G protein-gated muscarinic atrial
K+ channels. J. Biol. Chem. 266,22872-22877.
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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL
PATHOGENS: A REVIEW
Abstract
In most developed countries a sharp increase in foodborne intoxications occurs since the
last decade. Amongst the bacterial pathogens, the incidence rate is highest for
Campylobacter jejuni and Salmonella spp. Nucleid acid based identification and
detection methods have been developed for nearly all bacterial pathogens, based on
probes in hybridisation assays or primers in PCR, NASBA or RT-PCR assays. As
targets for molecular identification, virulence genes, the rRNA gene region or other
specific sequences can be used and several commercialised systems are already
available. For the (direct) detection of pathogens in food products, several problems
may be encountered: PCR inhibition by food components, contamination in sensitive
PCR assays, detection of living as well as dead cells. The latter problem can be solved
by using mRNA as amplification target, but for routine applications the combination of
a short culturing period with a less sensitive PCR is more suitable. Direct quantification
of pathogens is possible with quantitative competitive PCR using an internal standard or
with kinetic quantitative PCR (TaqMan or LightCycler commercial system).
In bacterial typing, distinct types, strains or clones within a pathogenic bacterial
species are differentiated which is important in epidemiological studies of foodborne
outbreaks but also in the “from stable to table” investigation of the whole food
production chain. Compared to the classical phenotypic typing techniques, molecular
typing techniques have several advantages such as general applicability and a high
discriminatory power. The currently available molecular techniques can be classified
according to their working principle in PCR-mediated typing techniques (RAPD, rep-
PCR), typing techniques combining PCR with restriction analysis (e.g.flaA typing of C.
jejuni), typing techniques based on chromosomal restriction fragment length
polymorphisms (e.g. ribotyping, pulsed field gel electrophoresis or PFGE), typing
techniques combining restriction digestion with selective amplification (AFLP), and
plasmid analysis. Both PFGE and AFLP are proposed as likely candidates for a uniform
definite molecular typing approach using appropriate software for cluster analysis and
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A. Durieux and J-P. Simon (eds.), Applied Microbiology, 193–238.
©2001 Kluwer Academic Publishers. Printed in the Netherlands.
M. HEYNDRICKX N. RIJPENS AND L. HERMAN
database storing of the fingerprints. For Salmonella, two typing levels can be proposed:
the first important level corresponds with the serovar level and the second level can be
performed by classical phage typing or molecular typing revealing clonal lineage or
strain level. Several molecular techniques have a serovar dependent discriminatory
power with the greatest challenge presented by the highly clonal serovar Salmonella
enteritidis.
1. Introduction
In the last decade a sharp increase in foodborne intoxications has been observed in most
developed countries. This may sound surprising because it is generally assumed that
hygiene at home during cooking and food storage and in manufacturing practices in the
food industry has greatly improved and is of a sufficiently high level in this high-
technology society. This phenomenon may be attributed to several factors such as the
increasing international trade in food and food and feed ingredients, international
tourism, climate changes, demographic changes in the population with a higher number
of elderly people, changing eating habits with a greater proportion of ready-to-eat foods
and mass catering facilities. The general trend is that the zoonoses, i.e. infectious
diseases caused by animal associated microorganisms such as Salmonella, are becoming
a particular increasing problem which is probably associated with the current bio-
industrial practice of mass production of animal products (e.g. poultry). In the food
industry, food processors and regulators are asked to establish, control and monitor
critical control points in the plant environment as essential part of the development and
use of a HACCP concept. It is likely that this type of approach will become more
important in the near future as food safety concerns increase in food processing and
distribution systems. Molecular detection and typing methods are powerful tools in the
whole food production chain for quality control and to unravel the prevalence and the
epidemiology of the foodborne pathogens.
In this paper a review is given of the molecular detection, identification and typing
techniques for foodborne bacterial pathogens in the last decade. Their possibilities and
eventually their associated problems, limitations and possible solutions are also
discussed with reference to own observations or those of other researchers. The term
“molecular” techniques is used here to denote those techniques which are “molecular
biology based”, and more specific those that make use of the nucleic acids (DNA or
RNA) in the bacterial cell.
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M. HEYNDRICKX N. RIJPENS AND L. HERMAN
Table I: Foodborne bacterial pathogens, nature of the disease and associated foods
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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
Table 1: Continued
Aandoeningen door een Netwerk van Laboratoria voor Microbiologie, 1997, Epidemiologische Trends 1983
1996
Wetenschappelijk Instituut Volksgezondheid - Louis Pasteur, Afdeling Epidemiologie, November 1998)
(http://www. iph.fgov. be/epidemio/epinl/plabnl/plabannl/index. htm).
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For molecular identification different regions of the bacterial DNA can be chosen as
target. For pathogenic organisms virulence determinants are frequently used and often
allow a very specific identification of a bacterial species (differentiation between C.
jejuni and C. coli, Gonzalez et al., 1997) or a serotype (specific identification of S.
enteritidis, Lampel et al., 1996 and of S. enteritidis and S. typhimurium, Soumet et al.,
1999). Some of these PCR identification systems (Gonzalez et al., 1997, Lampel et al.,
1996), however, were not sufficiently validated on field strains and have to be seen
more as an indication than as a real identification (Heyndrickx & Herman, personal
communication)
A lot of these virulence genes are located on plasmid DNA. When possible a
chromosomal localisation is preferred because of the instability of plasmids during lab
manipulations, This is seen for plasmid bearing Yersinia enterocolitica, where loss of
the virulence plasmid during enrichment and isolation complicates the detection of the
pathogen in food products (Bhaduri & Cottrell, 1997).
By PCR targeting virulence loci, pathogenic strains can be discriminated from non-
pathogenic strains belonging to the same species, which is important for identification
of pathogenic E. coli and Y. enterocolitica strains. Research on the use of PCR for the
identification of toxin genes (LT I, LT II, ST I and ST II for enterotoxigenic E. coli;
verotoxins, VT1, VT2 and virulence genes, eaeA, hly, virulence plasmid for
enterohaemorrhagic E. coli) is in full expanse (Chen et al., 1998; Tortorello et al., 1998;
Gilgen et al., 1998; Tsen et al., 1998; Radu et al., 1998). For the identification of
pathogenic Yersinia, primers, targeting the chromosomally encoded ail (adhesion-
invasion-locus) gene, can be used (Kwaga et al., 1992). Evaluation of these primers
showed no signal for any non-pathogenic Y. enterocolitica strain. The American and
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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
European pathogenic strains gave a positive reaction. All non-Y enterocolitica strains
reacted negatively with the exception of some Y. kristensenii strains. The amplicon
obtained for these strains was cloned and sequenced and it was found that the whole
coding region and the 259 bp upstream region of the ail gene as present in European
pathogenic Y. enterocolitica was found in these Y. kristensenii strains (Rijpens et al.,
1999b).
For many pathogens rRNA genes are targeted. The rRNA gene contains next to very
conserved parts also variable regions in which specific primers for a certain species can
be chosen. For some pathogens however, the variability is not sufficient for
discriminating particular species and the pathogen may only be identified to the genus
level. This is the case for Brucella (Herman & De Ridder, 1992; Romero et al., 1995)
and Campylobacter where C. jejuni, C. coli and C. lari were be differentiated
(Giesendorf et al., 1992, Linton et al., 1996).
A significant advantage of using rRNA as target is the high copy number (>104/cell)
allowing the use of rRNA probes for in situ hybridisation purposes (Nordentoft et al.,
1997, describing a 23S rRNA probe for Salmonella; Wagner et al., 1998, describing a
16S rRNA probe for L. monocytogenes).
Identification of foodborne pathogens by NASBA applies till now mostly rRNA as
target molecule. In one case the mRNA from the L. monocytogenes hlyA gene was
targeted (Blais et al., 1997). The RNA is amplified through the concerted action of
avian myeloblastosis virus reverse transcriptase (AMV-RT), T7 RNA polymerase and
RNase H (Fig 1). The reaction starts with a non-cyclic phase, in which a downstream
primer containing a tail-sequence of the T7 promoter anneals to the RNA. Through the
action of AMV-RT, cDNA is formed. The RNase H hydrolyses the RNA from the
RNA-DNA hybrid, which results in a single strand of DNA to which the upstream
primer can anneal. The AMV-RT, through its DNA polymerase activity, synthesises a
second DNA strand. The T7 RNA polymerase generates then single-stranded RNA
copies, which can serve as a template in a new cycle. For food pathogens, the
identification of C. jejuni, C. coli and C. lari (Uyttendale et al., 1994, 1995a) and L.
monocytogenes (Uyttendale et al., 199Sb) is described based on 16S rRNA probes. Also
the spacer region between the 16S rRNA and 23S rRNA genes is being used as target
for PCR. The spacer region is showing a considerable variation and is therefore suitable
for species identification. The spacer region offers the possibility to develop ‘multi-
pathogen’ tests allowing the simultaneous identification of different species. The line
probe assay (LiPA, Innogenetics, Belgium), has been developed for the simultaneous
detection of Listeria spp. and L. monocytogenes (Rijpens et al., 199.5) and recently
extended to all defined Listeria species (Rijpens & Innogenetics N.V., Belgium,
personal communication). The spacer region is amplified using primers targeting quasi
universally conserved sequences at the 3’ end of the 16S rRNA gene and the 5’ end of
the 23S rRNA gene. The amplification product is used in a reverse hybridisation assay
with a strip where different specific oligonucleotide probes are immobilised as parallel
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Figure 1. Schematic presentation of the NASBA method. Normal lines represent DNA, wavy
lines represent RNA.
In fewer cases a specific sequence with a known or unknown function is used as target
for the molecular identification. Examples of the use of known genes for PCR are: the
flaA and flaB (flagellin genes) and their spacer region for the identification of C jejuni
and C. coli (Kirk & Rowe, 1994; Wegmüller et al., 1993); the IS200 element for
Salmonella (Cano et al., 1993); the IS711 based multiplex-PCR system AMOS allowing
the discrimination of different Brucella spp. (Bricker & Halling, 1994). DNA sequences
with unknown function are used for the identification of Vibrio parahaemolyticus (Lee
et al., 1995) and Salmonella (ST11-ST15 amplicon, Aabo et al., 1993; Tsen et al.,
1994). The full sequence of the ST11-ST15 amplicon is recently published (Rijpens et
al., 1999a).
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For all important bacterial pathogens, DNA probes and primers are available through
the international literature (see above) and/or through commercialised systems. The
commercialised systems are summarised in Table 2.
Table 2: List of commercialised molecular identification and detection tests for foodborne
bacterial pathogens (taken from Berben, 1998)
The first 2 systems (AccuProbe and Gene-Trak) are using a specific oligonucleotide
probe with the rRNA as target molecule in a hybridisation assay. In the AccuProbe
system the probe is labelled with an acridinium ester and is used in a hybridisation
protection assay. When the DNA probe is hybridised to its target rRNA, the acridinium
is protected from chemical hydrolysis and can react with hydrogen peroxide under basic
conditions, to produce chemiluminescence. If the probe remains unbound, the ester band
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The other 4 systems (Probelia, BAX, GeneSTAR and TaqMan) are using PCR to
specifically amplify the target DNA but differ in their detection system. They provide
all PCR reagents in ready to use reaction mixes, including positive and negative
controls. The Probelia system uses an ELISA detection protocol in a microtiter plate
format. The BAX system detects the amplified product by gel electrophoresis or
temperature dependent fluorescence analysis using SY BR green I (L. monocytogenes:
Stewart & Gendel, 1998; Salmonella: Mrozinski et al., 1998, Bennett et al., 1998; Tige
et al.,1998, E. coli O157:H7, Johnson et al., 1998, Tseng & Ghandi, 1998). In the latter
case the fluorescence is monitored in the PCR tubes during an additional thermal cycle
consisting of a denaturation step and a product annealing step. The GeneSTAR system
uses fluorescent primers for PCR and hybridises the labelled PCR amplicon to
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As already mentioned, PCR opens a lot of possibilities for reliable and quick
identification of bacterial pathogens. By, PCR the DNA target is enzymatically
amplified and therefore could be applied to decrease the bacterial culturing time,
normally applied in the conventional detection methods. It is even possible to directly
detect the pathogen in the food product as is established for e.g. the detection of L.
monocytogenes (Herman et al., 1995) and Brucella (Rijpens et al., 1996) in raw milk.
However, when PCR is applied for the detection of pathogens in food products, some
problems could be encountered.
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When highly sensitive PCR protocols are used, one has to consider the possibility of
PCR contamination. A good lab hygiene protocol eventually combined with an anti-
carry over system can overcome most of the problems when a single 30 cycles PCR is
applied. More serious contamination problems can occur when very sensitive PCR
protocols are applied in routine laboratories.
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most abundant proteins, allowing a considerable increase in the sensitivity level. Third,
the function and the primary structure of the gene is conserved between living cells
allowing the modulation of the specificity of detection. Using the elongation factor
messenger as target a sensitive detection (detection level of 10 cells per ml of milk) was
obtained with a specificity for viable cells within about 6 to 8 min after heat treatment
(15 min at 65°C) (Vaitilingom et al., 1998). Although these results look promising, a lot
of research will have to be performed before RT-PCR could be used in routine
laboratory practice. The main problem is the reliable and constant destruction of spores
of DNA, which could trouble the correlation with the viability of the cells. Another
problem is the dependence of the sensitivity of the test on the absence of RNA
degrading enzymes during the RNA extraction procedure. This is especially important
because intensive DNase treatments could be necessary for sensitive detection systems
(Rijpens N., personal communication).
For routine application the combination of a short period of culturing (e.g. 16 h
during the night) with a less sensitive PCR detection is most often used to specifically
detect living cells by PCR (see protocols of all commercial PCR systems mentioned in
Table 1). Using the screening/Salmonella BAX method it was estimated that 104 cfu/ml
has to be present in the primary enrichment medium to be detected by PCR (Mrozinski
et al., 1998; Bennett et al., 1998). For the Listeria monocytogenes BAX system the
estimated number was 105- 106cfu/ml (Stewart & Gendel, 1998). Only when highly
contaminated (numbers of 104 to 105cfu/g or ml) products are investigated killed cells
could be detected using these combined protocols. Other advantages of inchding an
enrichment step are the possibility to use less specialised sample preparation methods,
the lower PCR sensitivity which decreases the problems of PCR contamination and the
easier validation of the PCR method which would correlate better with the conventional
method (see further).
As DNA based methods are used more frequently to detect bacterial pathogens in food
products, there is a great need for a thorough evaluation of the numerous protocols. This
is difficult because the inherent uncertainties of food analysis are coupled with new
variables introduced by the so called rapid methods. Evaluation of these methods is
easiest for these pathogens where the conventional method can be considered as a real
standard to which the results for the rapid methods can be compared. This is the case for
the detection of Salmonella. More problems are encountered when the conventional
detection method lacks sensitivity as is the case for e.g. L. monocytogenes and E. coli.
Artificial inoculation experiments of E. coli O157:H7 in ground beef revealed a
detection rate of 96.5% with the BAX for Screening/E. coli O157:H7 (commercial PCR
method) compared to 39% for the best cultural method (Johnson et al., 1998). Even the
use of a combination of 4 recovery methods yields an unacceptably low recovery rate
(67%). With the Probelia kit for detection of L. monocytogenes in raw milk cheeses a
high sensitivity is reached due to the application of a sensitive PCR (35 cycles). On a
total of 39 raw milk cheeses, 2 cheeses were found positive with the conventional
method, 14 with the Probelia method (Herman L., personal communication).
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Because the DNA based methods can reach a higher sensitivity in comparison with the
conventional method, it becomes very important to proof the validity of the positive
results and to make sure that no false positives are obtained. This is not so easy because,
unlike cultural methods, which almost always yield a viable pure culture isolate, the
PCR methods require lysis of the bacterial cell. Therefore, viable cells needed for test
confirmation can only be obtained by repeat analysis of the original enrichment medium
using cultural techniques. False positive results can especially be obtained when a very
sensitive PCR is used. This increases the possibility of PCR contamination and the
detection of a small number of killed cells. The absence of PCR contamination is not so
easy to proof. Obtaining a negative PCR control would not be sufficient to exclude the
possibility of a very small contamination during sample preparation and even PCR
preparation. It was experienced that even after plating of 10 ml of the enrichment
culture of L. monocytogenes no strain could be isolated from some PCR positive
samples (Rijpens N., personal communication). Only repeated culturing could validate
the positive result in this case.
For routine applications, the use of less sensitive PCR cycling programs should be
encouraged. This implies, at the time being, a limitation for the use of universal
protocols for detection of different food pathogens in one enrichment. Wang et al.
(1997) reported the possibility to detect up to 13 species of foodborne pathogens in
foods using an universal protocol of enrichment and detection. Such multi-analyte test,
however, implies the use of a very sensitive PCR cycling program (40 cycles) to obtain
a sufficient sensitivity.
In validation experiments, the rapid method is normally directly compared to the
conventional culturing method. Next to naturally contaminated samples different kinds
of spiking experiments are applied. Very often pure well growing bacterial cultures are
used for this purpose. Because bacterial pathogens present in foods are in a more
stressed condition the sensitivities of such experiments may be overestimated. This
could be overcome by an extra validation on naturally contaminated samples. For some
food products (e.g. dairy products for contamination with Salmonella), however, such a
validation is impossible because of the lack of possible samples. Methods could then be
evaluated using artificially stressed bacterial cells. For some bacterial pathogens they
are commercially available. Rijpens et al. (1999a) spiked the different dairy products
with an average of 5.9 stressed S. panama or S. typhimurium cells by using the reference
material prepared at the RIVM (National Institute of Public health and Environment,
Bilthoven, The Netherlands; in ‘t Veld et al., 1996). The results show that, even within
the group of dairy products, a differentiation of methods have to be made depending on
the specific group of products tested. For ice-cream and cheeses made from pasteurised
milk, PCR was applied after 16 h of preenrichment in buffered peptone water (BPW)
using immunomagnetic separation (IMS, using the Dynabeads anti- Salmonella, Dynal,
Oslo, Norway) and alkaline lysis as sample preparation method. For milk powder and
raw milk cheeses, the 16 h preenrichment in BPW was followed by IMS and a 4 h
enrichment in Rappaport-Vassiliadis broth. It is therefore clear that one have to be
careful to apply simplified universal protocols without proper validation on the specific
food products tested.
Because of the need of validation of the DNA based methods a number of validation
schemes have been developed in various countries throughout the world. In Europe
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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
A lack of a simple and reliable method for quantification of the PCR products has partly
hindered the use of PCR in routine food laboratories. Quantification of foodborne
pathogens by PCR is possible by an indirect method based on the Most Probable
Concept (MPN) and by direct quantification of the PCR product.
The MPN concept was developed for estimation of the number of organisms based
on the probability of getting positive and negative results in different dilutions of the
bacterial culture (Cochran W., 1995). MPN-PCR has first been described for
enumeration of specific micro-organisms in soil samples (Picard et al., 1992). The
detection limit in these experiments was quite high, as at least 103 target cells were
needed for positive results. Because of the inefficiency of a PCR of 30 cycles, this
underestimation is also encountered in food samples (Herman L., personal
communication). The application of a nested PCR protocol could improve the PCR
efficiency to about 20 cfu/ml, allowing comparable quantitative results for MPN-PCR
with plate counting methods (Mgntynen et al., 1997).
For direct quantification, quantitative competitive PCR can be used based on the co-
amplification of the target with a known concentration of an internal standard (IS) in
one reaction tube. In most cases, the IS shares the primer recognition sites with the
specific template. Both template and internal standard must be amplified with the same
efficiency and both end products must be analysed separately. Quantification is
performed by comparing the PCR signal of the specific template with the PCR signals
obtained for the known concentrations of the competitor. Wang & Hong (1999) used an
ELISA-competitive-PCR method to detect and quantify L. monocytogenes in milk
samples. Amplification of the target and the IS was performed in the presence of
fluorescein-dUTP. The labelled PCR products were hybridised with biotinylated probes,
specific for target and IS. The hybrids were bound to streptavidin-coated ELISA plates
to which an alkaline phosphatase-conjugated antibody to fluorescein was added in the
presence of substrate.
Kinetic quantitative PCR is another direct quantification method where the amount
of PCR products is followed during the PCR at several cycles. In the kinetic method, an
internal standard is not mandatory and an external scale is sufficient if the
reproducibility of the PCR is good. The PCR efficiency of each reaction is checked by
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M. HEYNDRICKX N. RIJPENS AND L. HERMAN
looking at the slopes of the increase in PCR products for the experimental samples and
for the external scale. If the slopes are parallel quantification is possible. Monitoring
PCR kinetics has become automated by different commercially available systems, The
TaqMan PCR detection system (Fig. 2, Perkin Elmer) depends on the irreversible
cleavage of the probe by the polymerase exonuclease activity. Quantification was
demonstrated for a pure L. monocytogenes culture and proved to be linear over a range
of 50 to 5. 105 cfu of L. monocytogenes (Batt C., 1997). The Lightcycler hybridisation
system (Roche Molecular Biochemicals, Mannheim, Germany) is based on the
hybridisation of 2 independent probes. Resonance energy is transferred from the donor
probe to the acceptor probe resulting in fluorescence emission (Fig. 3). Both
amplification and hybridisation reactions are performed together and the fluorescence is
automatically measured during the process.
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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
When stable typing data are stored in a database or alternatively, the bacterial isolates
themselves are collected for future typing analysis, the persistence and possible
evolution of certain strains or clones can be followed, which is of importance in the
investigation of the international spread of infectious agents. In the case of zoonotic
pathogens, this typing approach is useful to extend the epidemiological link from the
human infections to the main animal reservoirs (“from stable to table”) by thorough
investigation of each step in the whole food production chain (environment, farm,
slaughterhouse, distribution, retail, kitchen). In this way, the most critical points for
infection as well as for control or prevention can be identified, but it is also possible to
characterise the strains or clones with a changed or particular pathotype (virulence,
invasion) or which have acquired (higher) resistance to certain disinfecting products
(e.g. hypochlorous acid resistant Salmonella strains, Mokgatla et al., 1998) or
antibiotics (e.g. quinolone resistant Campylobacter, multiresistent Salmonella
typhimurium DT104).
4.1.2 Species-subspecies-variety-clone-strain-isolate
The basic unit of bacterial taxonomy is the species, defined as a group of strains,
including the type strain, sharing at least 70% DNA-DNA relatedness with 5°C or less ∆
Tm (with Tm the melting temperature of the hybrid). According to Vandamme et al.
(1996), “the bacterial species appears to be an assemblage of isolates which originated
from a common ancestor population in which a steady generation of genetic diversity
resulted in clones with different degrees of recombination, characterised by a certain
degree of phenotypic consistency and by a significant degree of DNA-DNA
hybridisation and over 97% of 16S rDNA sequence homology”. The latter authors
advocate the polyphasic taxonomic approach for bacterial classification as first step in
the delineation and description of species, which takes account of all possible variation
on the phenotypic and/or genotypic level. This polyphasic approach gives a higher
guarantee that intra-specific reliable and stable targets (e.g. on the 16S rDNA level) for
molecular identification can be found. Nevertheless, a polyphasic identification will
remain necessary for atypical isolates or isolates from new niches, which may belong to
unknown, related species. It must be stressed that a precise species identification is an
important and necessary step before any typing can be performed. A “List of bacterial
names with standing in nomenclature” is available on http://www-
sv.cict.fr/bacterio/index.html.
The classification of organisms below the species level is of particular interest for
epidemiology and typing. The only level with nomenclature status is the subspecies
(Wayne et al., 1987), while variety has no official rank. The designation “var” (e.g.
biovar, pathovar, serovar, phagovar) is commonly given to groups of strains which are
distinguished by certain characters. A strain is the basic working unit in the daily
laboratory practice, where the term denotes a pure culture, either fresh or stored in some
way. In the Bergey’s Manual of Systematic Bacteriology, a strain is described as the
“descendants of a single isolation in pure culture, and usually made up of a succession
of cultures ultimately derived from an initial single colony”. A useful definition for a
strain is also “an isolate or a group of isolates exhibiting phenotypic and/or genotypic
traits which are distinctive from those of other isolates of the same species’’ (Struelens
and members of the European Study Group on Epidemiological Markers, 1996). The
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meaning of the terms strain and isolate are thus synonymous in many cases. The term
clone has a meaning in population biology as denoted by Ørskov and Ørskov (1983) as
“bacterial cultures isolated independently from different sources, in different locations,
and perhaps at different times, but showing so many identical phenotypic and genetic
traits that the most likely explanation for this identity is a common origin”. For bacteria,
clonality has thus a somewhat broader meaning than for eukaryotic organisms, where
clones are definitely genetically identical organisms. In bacterial epidemiology, the
clone concept is of an even more pragmatic nature to denote isolates obtained during
clear-cut outbreaks and exhibiting the same phenotypic and/or genotypic features or to
denote organisms with common features (e.g. multiple antibiotic resistance) from
different geographic locations, so-called epidemic clones. The clonal relatedness
between strains is usually inferred from a numerical analysis of a polyphasic typing
approach combining phenotypic (e.g. multilocus enzyme electrophoresis or MEE of
metabolic enzymes) and genotypic methods (comparative sequence analysis or high-
resolution typing with pulsed field gel electrophoresis). Many pathogenic species are of
a clonal nature (e.g. Salmonella), whereas in other species genetic recombination occurs
resulting in horizontal gene exchange and/or a mosaic structure of recombined
segments. Because the evidence for clonality in bacteria is relative rather than absolute
(unless the whole genomes of different strains could be sequenced), it may be
recommended to speak only in the sense of “clonally related strains or strains of a clonal
lineage” or even “clone complex” when it is to be stated that strains have probably the
same origin as indicated by polyphasic typing.
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be used for numerical cluster analysis, which can be useful to delineate clonal
relatedness. In several cases, these molecular techniques are characterised by their
simplicity of performance, their reproducibility or their high resolution enabling the
differentiation between individual strains of a species. However, it must be said that not
all these interesting characteristics are automatically combined in one single technique.
In Table 3, a simple and pragmatic classification of the several molecular typing
techniques is given based on their basic working principles. Some indication is also
given of their complexity or simplicity, universal applicability (i.e. typability of all
bacterial species), reproducibility and resolution, which is a somewhat subjective matter
and for the latter sometimes pathogen-dependent.
Table 3: Currently used molecular typing techniques for foodborne pathogens and some of
their characteristics
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Table 3: Continued
4.1.3.1 PCR-mediated typing techniques. The PCR has without any doubt
revolutionised the molecular typing approach of bacteria and has also brought the
possibility of molecular typing within the reach of routine laboratories. A large variety
of PCR mediated typing techniques exists and some of them are listed in Table 3. The
most widely known and used techniques in this category are AP-PCR and RAPD,
developed independently by Welsh and McClelland (1990) and by Williams et al
(1990), respectively. Since there are no fundamental differences between both
techniques and the name RAPD is more frequently used, it is recommended that the
latter name is used throughout. RAPD is based on the random amplification of genome
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fragments lying between arbitrary sequences, which are used as targets for priming,
Usually, only one primer of 10 up to approximately 20 bases long is used in the PCR
working at a low annealing temperature (around 35ºC, but higher temperatures are also
used) and with a high MgCl2 concentration (up to 4 mM). As a consequence, these
reaction conditions will permit specific as well as non-specific primed amplification of
fragments. Another consequence is the generally low degree of reproducibility of this
kind of molecular typing. A technical review on RAPD typing in microbiology has been
done by Power (1996). The several experimental factors affecting the amplification
reaction and the reproducibility of RAPD have been reviewed by Tyler et al. (1997).
One of the important factors affecting the complexity of the banding pattern is the
primer selection. As a matter of fact, for every organism studied a suitable primer has to
be selected empirically. The most critical factor for reproducibility seems to be the
primer concentration to template DNA concentration. Tyler et al. (1997) have
formulated several recommendations for using arbitrary PCR:
• quantify DNA for each organism and each extraction method, and the use of
whole-cell extracts is to be avoided.
• primers should be screened for priming ability and reproducibility.
• quantify primers for every synthesis reaction.
• titrate DNA against primer concentration to reach an ideal primer/template
ratio.
• standardise the use of Taq DNA polymerase by maintaining the same supplier,
• titrate the Taq DNA polymerase against the primer/template ratio.
• standardise the MgCI2 concentration.
• use one thermocycler with a standard set of cycling conditions.
• run the appropriate control blanks to account for background.
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throughout the eubacterial kingdom. They contain a conserved inverted repeat to which
complementary consensus primers have been designed for PCR-typing (Versalovic et
al., 1991). The modular BOX-element is the first repetitive element found in a
Grampositive organism (Streptococcus pneumoniae) consisting of the subunits boxA,
boxB and boxC (Martin et al., 1992). Based on the widespread conservation of the
boxA subunit among diverse bacteria, a similar PCR-typing method has been designed
(Koeuth et al., 1995). Repetitive element primed PCR or rep-PCR is a general term to
collect at present the REP-PCR, ERIC-PCR and BOX-PCR typing methods, based on
the respective repetitive elements. In rep-PCR, amplicons are generated which contain
unique sequence chromosomal segments lying between the repetitive sequences. Unlike
RAPD, rep-PCR is considered not to be arbitrary because known conserved sequences
are used as priming targets at higher annealing temperatures (40°C for REP-PCR and
52°C for ERIC- and BOX-PCR) (Tyler et al., 1997). However, some authors (e.g.
Giesendorf et al., 1993) use only one of the repetitive element targeting primers at low
annealing temperatures, which resembles more a RAPD typing. We suggest that the
term rep-PCR typing and its different subdivisions should be reserved for the techniques
relying on the original procedures. Recently, some discordant notes have been observed
within the promising proposition of rep-PCR as a stringent PCR-typing approach with
reduced experimental variation and PCR artefacts and with the potential of storing the
patterns in databases for strain and species identification. Gillings and Holley (1997)
concluded that ERIC-PCR, using the standard annealing temperature of 52°C, does not
necessarily direct amplification from genuine ERIC sequences, and that this typing
technique applied to non-enterobacterial organisms must be regarded as a highly
reproducible variant of RAPD. A similar observation was made by us (Herman and
Heyndrickx, in press) with REP-PCR applied on the Grampositive UHT-resistant
Bacillus sporothermodurans. For Grampositives, this is not surprising since they do not
contain genuine REP- and ERIC- elements and it is suggested that the BOX-elements
are the preferable targets for these organisms (Tyler et al., 1997). With Salmonella, we
observed small differences (band intensities) in both REP- and ERIC-PCR patterns
between independent PCR-runs, but a considerable variation when primers were used
from 2 different sources (Heyndrickx M., unpublished results). For all rep-PCR
techniques, we recommend to adhere to the same recommendations as for RAPD and to
analyse strains with the same batches of primers and, if possible, within the same PCR
experiment. For a detailed protocol inclusive of the primer sequences for the different
rep-PCR techniques, we refer to http://www.msu.edu/~debruijn/loadr.html. A major
advantage of rep-PCR over RAPD however is the fact that a single primer set targeting
a known conserved and repetitive sequence can be used for both Gramnegative and
Grampositive organisms, whereas in RAPD a suitable primer with enough
discriminatory power has to be selected for any individual organism.
Finally, some PCR techniques aiming at other sequences occurring at multiple sites in
the bacterial genome may also be effective for typing. The 2 most important targets are
the tRNA sequences and the ribosomal spacer regions. tRNA sequences occur in multiple
copies dispersed throughout the genome and contain shared sequence motifs from
which outwardly directed primers can be derived. tRNA-PCR has more potential for
species identification, but in certain cases subgroups corresponding to the variety level
can be delineated (Seal et al., 1992). In many bacterial species multiple copies or alleles
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of the ribosomal operon are present (e.g. up to 10 copies in Bacillus). The spacer region
between the 16S and the 23S rRNA genes may vary in length between species, between
the rRNA alleles of a species and even between strains of a species as has been
demonstrated convincingly for Clostridium difficile (Gürtler, 1993). This variation in
length is in part due to the number and type of tRNA genes present in the spacer. A
universal bacterial identification and typing method is based on the PCR amplification
of the variable length 16S-23S rDNA spacer regions (Gürtler and Stanisich, 1996;
Jensen et al., 1993). This typing method has also been called PCR-ribotyping (Kostman
et al., 1992). The typing resolution of the ribosomal spacer based PCR technology can
be enhanced by additional restriction analysis to detect also sequence differences
between the spacers.
4.1.3.2 Typing techniques combining PCR with restriction analysis. After PCR
amplification of certain genes or gene fragments, the amplicons can be digested with
restriction enzymes and the resulting restriction fragments separated by agarose gel
electrophoresis to reveal sequence polymorphisms. This restriction analysis is necessary
because the targeted genes differ only in sequence and (usually) not in length (unless
insertions or deletions have occurred in the gene) between strains of a species. Protein
encoding genes showing intraspecific variability are used for this typing approach. A
good example is the flagellin gene typing of C. jejuni based on the restriction analysis
of the amplified flaA gene (Nachamkin et al., 1993). Since the amplicons are generally
up to 1500 bp long, tetracutter restriction enzymes (i.e. enzymes with a 4-base
recognition sequence) which cut theoretically after each 256 bp, are the best choice to
reveal enough restriction fragments and thus to reveal sequence polymorphisms. Also
enzymes with a longer recognition sequence, which contains only 4 defined bases and
for the rest undetermined bases (e.g. DdeI with the recognition sequence C^TNAG, with
N indicating an undetermined base), are suitable for this purpose. Information
concerning all currently known restriction enzymes is available at the daily updated
“Rebase” (http://rebase.neb,com/rebase/rebase.html). It must be noted that amplified
ribosomal DNA restriction analysis (ARDRA) is frequently categorised under the
typing techniques with potential of subspecies/strain differentiation. This is erroneous
since this technique relies on the 16S rRNA gene which is conserved amongst bacterial
species and is thus ideally suited for taxonomic and phylogenetic studies, but not for
molecular typing (Heyndrickx et al., 1996).
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fragments which together equal the size of the lost fragment, are generated. Deletions
and insertions affect the RFLP patterns obtained with all restriction enzymes because of
the change in size of a particular restriction fragment corresponding to the size of the
deletion or insertion. For RFLP analysis, hexacutter restriction enzymes (i.e. with a 6-
base recognition sequence) are normally used, but this still may result in >1000 bands
for a normal bacterial genome of 4x106 bp, which makes separation and interpretation
of the complex banding patterns very difficult. Several approaches (high-size or high-
frequency RFLP using tetracutters and low-size RFLP using hexacutters and
polyacrylamide gel electrophoresis, selective restriction fragment hybridisation, pulsed
field gel electrophoresis) have been developed to overcome this problem by reducing
the number of (visible) bands.
In the selective restriction fragment hybridisation (SRFH), the separated
chromosomal restriction fragments are transferred by Southern blotting on
nitrocellulose or on (more durable) nylon filters or membranes by capillary action or by
vacuum. The denatured membrane-bound DNA fragments are hybridised to a probe,
which can be either 32P-radioactive labelled or non-radioactive labelled with a
chemically modified nucleotide containing a hapten (e.g. digoxigenin- or DIG-dUTP of
Boehringer Mannheim, biotin-dATP). Only the fragments hybridising with the probe
are then revealed by autoradiography or with an anti-DIG or -biotin alkaline
phosphatase conjugate combined with a chemiluminescent or chromogenic substrate.
This hybridisation procedure results in simple and easy interpretable SRFH patterns.
Several factors influence the specificity of the hybridisation reaction such as the
stringency of the hybridisation temperature and several critical parameters (e.g. ionic
strength of the hybridisation solution, probe length and % G+C) affecting the Tm of the
hybrids formed. An essential element in SRFH analysis is the type of the probe used,
which can be either a single-stranded DNA or a RNA sequence. SRFH probes can be
categorised in random, directed and reiterated sequence probes (reviewed by Demezas,
1998), with the latter 2 being the most important ones. Reiterated sequence probes
include transposons, insertion sequences (IS) and duplicated genes. SRFH with
virulence/toxin probes, phage probes or IS-probes are very useful in the molecular
typing of certain foodborne pathogens. It must be noted that most of these SRFH
applications are named as RFLP techniques preceded by the name of the probe used,
e.g. IS200-RFLP and The most widely used SRFH application is based on
probes directed at the conserved ribosomal RNA genes and is named ribotyping
(Grimont and Grimont, 1986) or (erroneously) also restriction analysis of rRNA genes.
Ribotyping is universal applicable but its resolution is dependent on the number and the
spatial distribution of the rRNA copies on the bacterial chromosome. In the first place,
ribotyping is a useful taxonomic and identification technique and also a potential typing
technique for microorganisms containing several ribosomal operons (multiplicity of
rRNA operons in prokaryotes reviewed by Schmidt, 1998). The ribotyping technique
has been automated with the RiboPrinter™system (Qualicon Inc., Wilmington, DE,
USA). Starting from a pure colony, this automated system analyses 32 samples per day
in 4 batches of 8 samples, with the results from the first batch available in 8 hours.
The second approach to reduce the amount of fragments in RFLP analysis is
macrorestriction analysis (MRA) by pulsed field gel electrophoresis (PFGE). This
technique is based on the in situ restriction digestion of the intact unsheared bacterial
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4.1.3.5 Typing techniques based on plasmid analysis. Plasmids were the first targets for
molecular typing starting already in the mid-seventies and they are still very useful for
certain pathogenic bacteria such as Salmonella. Plasmid analysis starts with the specific
isolation of the plasmid DNA which is mostly accomplished by a modified procedure of
the alkaline lysis method, originally described by Kado and Liu (1981). In most rapid
plasmid preparations, chromosomal contamination is difficult to eliminate, but this
should not present a problem for interpretation since the chromosomal DNA appears as
a diffuse single band of the same length for all strains. The most simple technique is
plasmid profiling (PP) in which the size and number of the plasmids present in the
strains is investigated by agarose gel electrophoresis. An E. coli reference strain
harbouring several large plasmids of known size is very useful for exact molecular
weight determination in PP analysis. In some plasmid preparations, the same plasmid
may be present in multiple forms (the native supercoiled form, the open circular form
resulting from a cut in one DNA strand, and the linearised form resulting from a cut in
both DNA strands), each with its own electrophoretic mobility. Especially with smaller
plasmids, this may result in multiple bands for the same plasmid, which results in an
overestimation of the number of plasmids present. Another problem is the instability of
plasmids resulting in the possibility that a certain strain loses one or more of its
plasmids. On the other hand, plasmid DNA may undergo more rapid evolution than
chromosomal DNA because plasmid borne genes do not encode for essential cell
functions. It may thus be expected that plasmid DNA is less subjected to evolutionary
pressure and is more prone to sequence variations. Sequence polymorphisms between
identical sized plasmids are revealed by restriction analysis (plasmid REA).
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Molecular typing is a relatively young discipline but has already encountered many
changes and variations on the same theme, in which the PCR technology has played a
major role. It is the prospect that molecular typing of foodborne bacteria will increase
constantly in importance and will generate data, which can be exchanged between
different types of laboratories (veterinary, industrial, and clinical/public health
laboratories) and between countries. On the International Meeting on Bacterial
Epidemiological Markers in 1997 (IMBEM IV, Elsinore, Denmark), PFGE and AFLP
were proposed as the likely candidates for a high-resolution, standardised, definitive
typing approach in which patterns can be reproducibly generated, stored in databases
and exchanged via networks.
In the USA, PulseNet (http://www.cdc.gov/ncidod/dbmd/puIsenet/pulsenet.htm) is
launched as a National Molecular Subtyping Network for Foodborne Disease
Surveillance, permitting rapid comparison of PFGE fingerprints through an electronic
database at the Centers for Disease Control and Prevention (CDC).
Cost and speed are also important parameters for the implementation of a typing
system, which can benefit from new developments or applications. Another fragment
separation technique based on fluorescent labelling and capillary electrophoresis of
DNA fragments (e.g. with the ABI Prism 310 Genetic Analyzer, PE Applied
Biosystems) enables a higher level of automation, flexibility and throughput of samples.
It is also possible to directly reveal sequence polymorphisms (down to the level of point
mutations) with specialised electrophoresis techniques in which a denaturing gradient is
applied in the gel, either chemically in denaturing gradient gel electrophoresis (DGGE),
or in temperature gradient gel electrophoresis (TGGE) or alternatively in temporal
temperature gradient gel electrophoresis (TTGE). Equally sized fragments or amplicons
will stop migrating at different positions and hence will be separated in the denaturing
gradient depending on the melting temperature Tm of their sequence. PCR-TGGE and -
DGGE analysis is currently used in molecular microbial ecology (reviewed by Muyzer
and Smalla, 1998), but has potential for rapid molecular typing as well.
A further prospect is the deviation of molecular typing by DNA fingerprinting towards
sequencing data and hybridisation pattern data on DNA-chips. Sequence data are still
the preferred type of information because of their absolute nature and it is the challenge
of future molecular typing to identify suitable nucleic acid targets, which show enough
intraspecific variation within a single short sequencing run. DNA chips are high-density
oligonucleotide arrays which are used in fluorescent hybridisation analysis to screen
sequence variation in large sets of (amplified) DNA samples in a high throughput
manner. Simultaneous genotyping and species identification using hybridisation pattern
recognition analysis on DNA-chips has been demonstrated for mycobacteria (Gingeras
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et al., 1998). It is expected that whole genome sequencing data will provide new targets
(e.g. DNA repeats or genes) for molecular typing.
5.1 SALMONELLA
From Table 4, it can be deduced that Salmonella has been the subject of numerous
molecular typing studies. It is important to clarify first the rather confusing Salmonella
taxonomy. The genus Salmonella forms a single DNA homology group divided in seven
sub-groups. The seventh group is given the species rank as Salmonella bongori, while
the other 6 groups represent 6 subspecies of one species, which should carry the name
Salmonella choleraesuis (Skerman et al., 1980). Because of confusion with the name of
the serovar Choleraesuis, Le Minor and Popoff (1987) proposed the species name
Salmonella enterica instead, which is now used by almost every author, but has no
standing in nomenclature because not officially accepted by the Judicial Commission of
the International Committee on Systematic Bacteriology. Therefore, Euzéby (I 999) has
recently proposed to reject the name Salmonella choleraesuis and to designate
Salmonella enterica as neotype species. The official situation will thus be that
Salmonella contains 2 species, S. bongori and S. enterica, the latter being divided in 6
subspecies (Christensen et al., 1998). S. enterica subsp. enterica is the most important
subspecies containing most of the >2300 serovars. Three serovars of high clinical
importance have been given species rank: S. enteritidis, S. typhimurium and S. typhi.
Other serovars should not be considered as species and should be designated as in the
example: Salmonella enterica subsp. enterica ser. Hadar or (more practically)
Salmonella Hadar.
Two typing levels can be proposed for Salmonella. The serovar level is (and will
remain if only for historical reasons) a very important first typing level for daily
practice. Several molecular typing techniques (see Table 4) such as PCR-ribotyping
(Lagatolla et al., 1996), PCR-RFLP of the ribosomal operon (Shah and Romick, 1997),
ERIC-PCR (Van Lith and Aarts), REP-PCR (Heyndrickx M., unpublished results) and
AFLP (Aarts et al., 1998) seem to provide the possibility of serovar identification and
thus to replace the classical serotyping.
However, it should be noted that some serovars seem to be polyphyletic because of
recombinations in the genes encoding for the O- and H-antigens, which could be
reflected in different fingerprint patterns for some strains of these serovars (Hilton and
Penn, 1998).
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Table 4: Molecular typing techniques used for foodborne and animal associated
Salmonella serovars and some important literature references for each of them. References
are classified according to the sole typing technique involved or according to the most
studied or (if possible) the most discriminatory typing technique when several techniques
are involved. In the latter case, relevant information on the other typing techniques is given
as well. For more complete information on a specific technique or serovar, we refer to
further studies cited in the references.
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Table 4: Continued
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M. HEYNDRICKX N. RIJPENS AND L. HERMAN
Table 4: Continued
PFGE Echeita & Usera, 1998 detection of chromosomal rearrangements in S. typhi by PFGE
with I-CeuI; also ribotyping
Liebisch & Schwarz, superior discriminatory value of PFGE using XbaI, SpeI and NotI
1996a for epidemiologically unrelated S. enteritidis isolates; see also
plasmid profile, ribotyping and 6200-typing
Powell et al., 1995 derivation of S. enteritidis PT9a and 7 strains from a PT4 strain
revealed by PFGE and ribotyping; also ribotyping
Thong et al., 1998 investigation of S. enteritidis gastro-enteritis outbreak by PFGE
(Xbal, AvrII and SpeI)
Murase et al., 1995 evaluation of PFGE (BlnI, XbaI) for epidemiological analysis of
Salmonella outbreaks (S. typhimurium, S. Thompson, S.
enteritidis)
Murase et al., 1996 variations in PFGE patterns of S. enteritidis isolates from a food
poisoning
Wegener & Baggesen, tracing back of aS. Infantis outbreak to a single pig
1996 slaughterhouse and its supplier pig herds by PFGE (XBaI)
Buchrieser et al., 1997 subdivision of S. enteritidis PT4 and 8 strains by PFGE using NotI
and XbaI, supporting the hypothesis of a single clone pandemic
On & Baggesen, 1997 determination of 2 principal clonal lines of S. typhimurium in
Denmark by PFGE using XbaI and ribotyping; also ribotyping
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Table 4: Continued
PCR- Lagatolla et al., 1996 specific PCR profile for 7 Salmonella serovars (a.o. S. enteritidis),
ribotyping spacer region polymorphic for 3 serovars (a.o. S. typhimurium)
Shah & Romick, 1997 restriction analysis (HinfI) of ribosomal spacer regions for
Salmonella subspecies differentiation
rep-PCR Van Lith & Aarts, 1994 typing of Salmonella up to the serotype level with ERIC-PCR
Millemann et al., 1996 1 and 2 fingerprints obtained respectively for S. enteritidis and S.
typhimurium strains with ERIC-PCR; see also RAPD
Lopez-Molina et al., 1998 ERIC-PCR with 1 primer useful for differentiation between
Salmonella serotypes, not for S. enteritidis phage types; see also
RAPD
Beyer et al., 1998 REP-PCR and ERIC-PCR (with 1 primer) discriminates epidemic
S. Saintpaul strain from other strains, see also RAPD
AFLP Aarts et al., 1998 Salmonella serotype and phage type identification and
discrimination of strains, previously identified as identical by
other methods
The classical second level is phage typing, but this method has often a too low
discriminatory power for detailed outbreak investigations, a phage typing scheme is
only available for some serovars, and some isolates may be untypable. Being a
phenotypic method, phage typing can not be used for delineating phylogenetic
relationships. Another problem is phage conversion in some serovars, which may
disturb the study of epidemiological relationships (Rankin and Platt, 1995). Several
molecular typing techniques have the potential of second level typing, which
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corresponds with clonal lineage or strain level (see Table 4). For some techniques, the
discriminatory power or applicability is serovar dependent: e.g. IS200 typing gives the
highest discrimination in S. typhimurium (Olsen et al., 1997), but is not applicable for S.
Hadar (Weide-Botjes et al., 1998b). The demonstrated discriminatory power may also
depend on the set of isolates included: e.g. ribotyping has been reported as giving no or
little differentiation (Liebisch and Schwarz, 1996a) and as being slightly higher
discriminatory than PFGE (Thong et al., 1995) for S. enteritidis. One of the greatest
challenges is the molecular typing of S. enteritidis which is a highly clonal organism,
particularly within certain phage types (e.g. PT4 and 8). Currently, it seems that RAPD
gives the best discriminatory power for S. enteritidis isolates belonging to different or
even the same phage type (e.g. Fadl et al. 1995), provided that a suitable primer is
identified (e.g. Lin et al., 1996). Also PFGE using the enzyme combination XbaI - NotI
- SpeI (Liebisch and Schwarz, 1996a) and plasmid analysis (e.g. Millemann et al., 1995)
have been shown to be useful for this serovar. A combination of different molecular
typing techniques used under optimal conditions and eventually complemented by
phage typing, is highly recommended to trace isolates in epidemiological investigations
(Weide-Botjes et al., 1998a). S. typhimurium isolates usually show more genetic
variation and can be differentiated with several techniques (see Table 4). AFLP using an
EcoRI primer with 2 selective bases enabled phage type identification and
differentiation of strains, which were indistinguishable by other methods (Aarts et al.,
1998).
C. jejuni is one of the most common causes of sporadic gastro-enteritis with poultry
products being the most important vehicles for infection. C. jejuni is divided in the
subspecies jejuni and doylei, but the former subspecies is the most important one.
Classical typing schemes are the Penner heat-stable and Lior heat-labile serotyping
schemes and biotyping. Several molecular typing techniques have been developed or
evaluated for a higher and universal applicable discrimination of isolates. A PCR-RFLP
technique is based on the sequence heterogeneity of the flagellin gene flaA and is
referred to as flaA typing or profiling (Nachamkin et al., 1993) using mostly the
restriction enzymes DdeI and HinfI (Santesteban et al., 1996). Both the flaA and fla B
genes, occurring in tandem, can also be used as combined target for restriction analysis
(Ayling et al., 1996). Alternatively, a short (150 bp) variable region of theflaA gene
can be used as target in direct sequence analysis for epidemiologic investigations
(Meinersmann et al., 1997). Recently, evidence for intergenomic recombination
betweenflaA genes of different C. jejuni strains as well as intragenomic recombination
between the flaA and flaB genes within a strain has been deduced from mosaic flaA
gene structures, which has as important consequence that flagellin gene typing cannot
be used for long-term epidemiological monitoring and determination of clonal
relationships within Campylobacter populations (Harrington et al., 1997). This problem
could be overcome by combining the polymorphisms determined by PCR-RFLP of
several genetic loci as demonstrated by a multiplex PCR gene fingerprinting method
based on the variable gyrA and pflA genes (Ragimbeau et al., 1998).
It seems that flaA types are conserved across different serotypes and that flaA typing
is less discriminatory than PFGE and thus cannot be used as sole basis for grouping
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strains (Santesteban et al., 1996). PFGE was also shown to be the most discriminatory
of 3 typing methods (including ribotyping and phage typing) for C. jejuni strains of
different Penner serotypes and within a single heat stable serotype (Gibson et al., 1995),
and the most discriminatory of 4 typing methods (including fatty acid profile typing,
biotyping and serotyping) for C. jejuni and C. coli isolates from abattoirs (Steele et al.
1998). For the DNAse-positive strains of Lior biotype II, formaldehyde fixation of the
cells is necessary to perform PFGE (Gibson et al., 1994). Definition of clonal lineages
within C. jejuni with PFGE must be based on the patterns obtained with at least 2
restriction enzymes (e.g. SmaI and KpnI) (Gibson et al., 1997). PFGE typing of field
isolates from Finnish patients, chicken faecal samples and meat samples (Hänninen et
al., 1998), from Canadian meat processing plants (Steele et al., 1998) and from sporadic
cases of diarrhoeal disease in England (Owen et al., 1997) have all shown a high degree
of genomic diversity within C. jejuni. It is not unlikely that the observed large genetic
variation may at least partly be attributed to genomic instability within single strains as
the result of intragenomic recombinations or natural transformation by non-homologous
DNA and driven by environmental pressures (Wassenaar et al., 1998). In vitro
genotypic variation of C. coli induced by repeated subculturing has already been
observed by PFGE (On, 1998). PFGE patterns must therefore be carefully interpreted
and preferably combined with other (single locus) molecular typing techniques and/or
with numerical analysis in order to evaluate relationships between Campylobacter
strains. The same caution probably also applies to other whole-genome typing
techniques such as RAPD, which has been shown to have an excellent discrimination
ability within different Campylobacter species and Penner serotypes (Hernandez et al.,
1995; Madden et al., 1996). By RAPD typing, possible transmission routes (e.g.
environment, farmer’s footwear) of Campylobacter infection in well-defined settings
(successive broiler flocks) have been demonstrated (van de Giessen et al., 1998; Payne
et al., 1999). Because Campylobacter does not seem to be of a clonal nature, but rather
consists of genomic mosaics, it may be difficult or impossible to define epidemiological
links by molecular typing in less defined settings (e.g. different farms) as shown by
Weijtens et al. (1997). A SRFH technique with a probe based on the highly conserved
domain (HCD) of the tlpA gene, encoding for a methyl-accepting chemotaxis-like
protein from C. coli, has also been proposed as a molecular typing scheme which is not
likely to be subject to an extensive degree of genetic instability (Gonzalez et al., 1998).
L. monocytogenes causes a rare but severe disease in humans, which is in most if not all
cases caused by industrially processed food with an international distribution (e.g.
cheese). Serotyping is of limited epidemiological value as only 3 serovars (1/2a, 1/2b
and 4b) are causing most of the infections. Because of the large socio-economic impact,
the World Health Organisation (WHO) food safety unit in Geneva mandated in 1990 a
multicenter study on L. monocytogenes subtyping methods. The results of the first phase
are published in a special issue “Molecular typing of Listeria” of the International
Journal of Food Microbiology (Vol. 32, 1996). The conclusions of this study can be
summarised as follows (Bille and Rocourt, 1996):
227
M. HEYNDRICKX N. RIJPENS AND L. HERMAN
The serotype E. coli O157:H7 (motile) and O157:H- (non-motile variant), belonging to
the enterohaemorrhagic E. coli (EHEC) which are a subset of the verocytotoxic E. coli
(VTEC), stands as the type example of the so-called new emerging infectious diseases..
The diversity of strains in cattle and sheep, the major reservoirs of potential pathogenic
VTEC and of E. coli 0157 strains, has been investigated by PFGE (Kudva et al., 1997;
Beutin et al., 1997; Heuvelink et al., 1998). PFGE (using Xba I) has the highest
discrimination for E. coli 0157 (giving the best discrimination) (Grif et al., 1998) and is
therefore highly used in the epidemiological investigation of foodborne outbreaks,
228
MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
which have been linked in many cases to undercooked hamburgers (Barrett et al., 1994).
Ribotyping, on the contrary, is not able to discriminate between E. coli 0157 isolates
(Martin et al., 1996). In the USA, the PulseNet uses a standard PFGE procedure and an
electronic pattern database for E. coli 0157 (Anonymous, 1996). However, in the
interpretation of molecular typing results of E. coli 0157, 2 important phenomena must
be accounted for. Firstly, for the phylogenetically highly related E. coli 0157 isolates
from human infections which seem to belong to a single clone complex, PFGE has its
limitations because epidemiologically unrelated strains may differ in only a few
fragment bands making an unequivocal differentiation between outbreak and non-
outbreak related strains sometimes difficult (Böhm and Karch, 1992). Combination with
another typing method such as RAPD (Birch et al., 1996) or phage typing is therefore
highly advised (Grif et al., 1998). Secondly, E. coli 0157 can undergo rapid genotype
alteration in the course of infection, so-called clonal turnover, which may be caused by
the chromosomal integration or loss of verocytotoxin gene carrying bacteriophages
(Datz et al., 1996), and may result in the appearance of new PFGE patterns. SRFH
techniques with the use of Shiga-like (SLT) or verocytotoxins probes (SLT-RFLP)
(Samadpour, 1995) and bacteriophage λ probe (λ-RFLP) (Grimm et al., 1995) have
been shown to be very sensitive methods for interstrain differentiation. A PCR based
typing method based on the E. coli repetitive element IS3 has been developed as a rapid
screening method for the identification of unrelated E. coli O157:H7 isolates
(Thompson et al., 1998).
PFGE (using Sma I) (Liu et al., 1997) and RAPD methods (Nilsson et al., 1998) have
been developed for the discrimination of strains of the sporeformer Bacillus cereus.
The AFLP technique has been extensively evaluated for the genus Aeromonas (Huys et
al., 1996). The combination of different ribotyping procedures (classical ribotyping and
PCR-ribotyping) has been shown useful for strain discrimination in Yersinia
enterocolitica (Lobato et al., 1998).
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238
BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
Abstract
The fermentation process has long been used as a method of meat preservation. In order
to eliminate batch to batch variation the fermentation process must be standardised.
This, combined with problems associated with emerging pathogens such as
enterohaemorrhagic Escherichia coli has led to a re-examination of the process of
fermented meat production in order to ensure the production of a consistently high
quality and safe product. Encapsulation technology can be applied to meat
fermentations with the objectives of enhancing the existing methods of preservation and
in developing novel methods to combat the problem of emerging and re-emerging
pathogens. Encapsulation technology has been shown to be beneficial in the production
of fermented meats by both direct and indirect acidification. Indirect acidification
occurs after the addition of a starter culture to the meat and encapsulation technology
has been observed to enhance its activity upon its addition to meat. The success of
encapsulation would appear to be based on some form of spatial organisation involving
a) protection and b) controlled release. The creation of a microenvironment, which
provides the desired conditions or populations and the physical regulatory systems,
minimises the effect of fluctuations in the macroenvironment and protects the cells from
competition, predation and lysis. Controlled delivery from the microenvironment assist
the cells to adapt to the new macroenvironmental conditions and then release the
adapted cells under regulated conditions. Encapsulated acidulants are already in use in
the United States and their use is on the increase in Europe. Problems such as
discoloration and lack of binding associated with direct acidification can be overcome
by encapsulation, which allows the time and rate of acid release to be controlled.
Emerging pathogens are now challenging the antimicrobial hurdles present in a
fermented meat product. This has led to investigations into enhancing the safety of
existing manufacturing processes for fermented meats. Bacteriocins are antimicrobial
agents, which are naturally produced by lactic acid bacteria. However, their
ineffectiveness towards Gram-negative bacteria and their reduced activity in meat
products has excluded their use until now. Combining bacteriocins, for example nisin,
with other stresses enhances their activity towards Gram-negative pathogens.
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©2001 Kluwer Academic Publishers. Printed in the Netherlands.
CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.
Encapsulation in polymer gels facilitates the optimisation of conditions for in situ nisin
production and permits the controlled release of bacteriocins into the
macroenvironment. Therefore, this technology has the potential to facilitate the use of
bacteriocins produced by lactic acid bacteria in meat products as a means of overcoming
potential problems associated with emerging pathogens.
1. Introduction
The deterioration of food products is an inevitable process. However the rate at which
this process occurs is dependent on the food type, composition, formulation, packaging
and storage conditions (Gould, 1995). For centuries meat has been considered as a
highly valued and nutritious food (Shay and Egan, 1992). However, it is due to this
nutritious nature that meat is also a highly perishable product and when stored in air
spoils rapidly as a result of the growth of Gram-negative bacteria (Egan, 1983). Apart
from microbiological spoilage numerous chemical and biochemical changes occur
during the storage of meat, which limit its shelf life. While many foodstuffs, including
meat can now be preserved by refrigeration and freezing, the preservation of food has
long preceded the technical advances, which permit such methods of shelf-life
extension. Examples of the more traditional methods of preservation include heating,
drying, curing, smoking and fermentation. The high sensory and nutritious quality of
the resulting products has meant that, even in the face of modern technology, many of
these methods of preservation have not only survived but also retained a high level of
popularity. For example, pepperoni, a fermented meat product, has an annual
consumption rate of 370 million pounds in weight in the United States (Hinkens et al.,
1996).
While many methods of meat preservation exist, the focus of this article will be the
enhancement, using bioencapsulation technology, of meat preservation techniques based
on fermentation and acidification. Although meat preservation by microbial
fermentation is among the oldest forms of food preservation (Dillon and Cook, 1994),
dating back to about 1000 BC (Nychas and Arkoudelos, 1990), and also one of the most
successful means of prolonging the shelf-life, new challenges and research opportunities
continually arise. Recent food poisoning outbreaks associated with enterohaemorrhagic
Escherichia coli in fermented meat products (Anonymous, 1995b; Tilden et al., 1996)
have prompted many researchers to further examine the safety of these products and to
search for novel means of enhancing their safety. Also, as in any process, there is a
continual search for technical innovations, which will enhance quality and production
efficiency (Prochaska et al., 1998). Encapsulation, which has been described as an “old
yet new” technology (Pszczola, 1998) is one means by which advancement can be made
in the area of meat preservation.
Encapsulation technology has been used in the food industry for more than sixty
years with the encapsulation of flavourings by spray drying in the 1930’s being
considered the first application of this process (Reineccius, 1995). However, it has only
been adapted slowly and so can still be regarded as a developing technology within the
food sector. This is probably due to the requirement for low cost food grade
encapsulation materials by the food industry. Encapsulation technology in the food
sector is currently growing at a level of 30% annually in the US and one of the leading
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2. Meat preservation
Fermented meat products are defined as meats that are deliberately inoculated to ensure
sufficient acidification and controlled microbial activity to alter the product
characteristics (Bacus, 1986). Although this technology has been extensively
researched and reviewed (Leistner and Lucke, 1988; Hammes et al., 1990; Shay and
Egan, 1992; Campbell-Platt and Cook, 1995; Ricke and Keton, 1997) there continue to
exist areas for improvement. The general steps in a meat fermentation process are
outlined in Figure 1.
Examination of the steps in the production of a fermented meat product allows the
identification of potential problems in this process (Table 1). While many quality and
safety problems can be overcome by the implementation of a HACCP system, this does
not solve all problems, particularly those associated with the activity of the starter
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CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.
culture. Process control must combine optimisation of the conditions for acid
production with those for drying, flavour and texture development etc. and achieving
this may necessitate a compromise of the conditions. In relation to the starter culture
activity, it is not be possible to control important factors such as strain degeneration,
shelf-life stability and ecological competence by controlling the conditions in the
fermentation process. Encapsulation has been described as a ‘technology which can
solve certain problems which cannot be solved otherwise’ (Pszczola, 1998) and
therefore its potential in meat preservation, particularly in the area of starter culture
activity deserves some examination.
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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
While the direct addition of organic acids such as lactic acid would appear to be a faster
and more reliable means of preservation than microbial fermentation, their use has been
limited due to their instantaneous reaction with the meat which causes undesirable
colour and texture changes (Shay and Egan, 1992). A more successful alternative has
been the addition of glucono-delta-lactone (GDL) to lower the pH. After addition to the
meat the GDL is hydrolysed to gluconic acid bringing about a reduction in pH (Shay
and Egan, 1992). As the use of GDL does not involve the direct addition of an acid, the
problems associated with discoloration and textural changes are reduced. However, the
extent to which this reduction in undesirable characteristics occurs depends on the rate
at which the GDL is hydrolysed. Furthermore, once hydrolysed to gluconic acid, it can
be further metabolised to acetic acid and carbon dioxide which can add a bitter taste and
gas pockets to the meat (Kneissler et al., 1986). On the other hand, the application of
encapsulation technology to food acids permits the controlled or slow release of these
ingredients thus increasing their usefulness in meat preservation (Dziezak, 1988).
While the natural microflora in a meat system will, under suitable conditions, result in a
fermentation process (Cooke et al., 1987; Gibbs, 1987; Grombas, 1989), the
unreliability of this process has long been recognised. The practice of backslopping,
which involves initiating a new fermentation by the addition of some of the product of a
successful fermentation, was developed as a means of overcoming the uncertainty
(Gibbs, 1987; Grombas, 1989; Nout and Rombouts, 1992). Today, the production or
purchase of specially selected and prepared starter cultures is probably one of the most
important and also expensive parts of the fermentation process since a successful
fermentation is dependent on the addition of a viable and active inoculate. Therefore,
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status of alginate as a food grade additive (sodium alginate -E401, calcium alginate -
E404) (Anonymous, 1995a).
Carrageenans are polysaccharides, which are extracted from red seaweed of which
Chondrus crispus is the main source. There are three main types of carrageenan, kappa
(κ), lambda (λ) and iota (i) with κ-carrageenan the most widely used for cell
immobilisation (Willaert and Baron, 1996). Carrageenans are sulphated linear
polysaccharides of D-galactose and 3-6 anhydro-D-galactose with the various
carrageenans differing from one another in their content of 3-6 anhydro-D-galactose and
the number and position of ester groups (Trius and Sebranek, 1996). Gelation can be
achieved by cooling, due to the thermal properties of the gel, or by contact with a
solution of gel inducing reagents such as K+, NH4+, Ca2+, Cu2+, Mg2+, amines or water
miscible organic solvents (Chibata et al., 1987). This is a mild and simple process.
Carrageenan gel networks are formed during cooling by a series of polymer chain
associations to give rise to a 3-D helix framework. This framework is stabilised in the
presence of cations by the formation of additional bonds (Trius and Sebranek, 1996).
carrageenan gelation is dependant on cations but unlike alginate beads, carrageenan
beads are thermally reversible (Willaert and Baron, 1996).
Chitosan is another polysaccharide which is used for immobilisation but it is not as
widely employed a matrix as alginate or carrageenan. Chitosan is a partially
deacetylated chitin, which is formed by reacting chitin with a concentrated alkali
(Vorlop and Klein, 1987). It is a high molecular weight linear polymer consisting of
1→4 linked glucosamine and has a high nitrogen content (>7% w/w) (Vorlop and Klein,
1987; Willaert and Baron, 1996). Chitosan is soluble in organic acids and to a more
limited extent in mineral acids e.g. HCl. Gelation of chitosan occurs as a result of
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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
whose properties are influenced by the presence of polyols and skim milk, can result in
increased survival rates of meat starter cultures (Kearney, 1990).
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Methods of internal gelation, which involve the addition of the calcium source directly
into the alginate solution and controlling the availability of the calcium ions through the
use of calcium chelators (Monshipouri and Rice, 1995) or pH (Poncelet et al., 1992,
1995; Quong et al., 1998) have permitted the development of emulsification or
dispersion methods of encapsulation. This involves the addition of the alginate/calcium
mixture into a bath of oil and applying a shear in order to disperse the gel mixture
throughout the oil, thus producing spherical encapsulates. The use of a calcium-
chelating agent slows down the gelation process sufficiently to permit the dispersion of
the gel in the oil. The pH method involves the external addition of an acid to reduce the
pH and cause the release of calcium ions from the calcium source present. The acid is
added after the emulsification or dispersion step. While these techniques are suitable
for the large-scale production of alginate beads, an improvement of the encapsulation
technique is still required. The harvesting of the encapsulates from the oil has been
reported to be problematic (Friel, 1998). Also, a broad size distribution of the
encapsulates produced by this method has been observed (Poncelet et al., 1992; 1995).
In contrast to meat starter cultures, the encapsulation of acidulants has been extensively
studied and commercialised (Marchot, 1993). The application of encapsulation
technology to food acids has been spurred on by the fact that acidification is a valuable
means of food preservation (Davidson, 1997), even though the direct addition of acids
to many foods results in undesirable flavour and colour changes (Shay and Egan, 1992).
Also, the application of acids to a wide range of food products including meat, dairy
products, bakery products, desserts and canned fruits and vegetables (Dziezak, 1988;
Janovsky, 1993; Marchot, 1993) would appear to assure the commercial viability of
encapsulated acids.
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related to temperature but other mechanisms of release such as that caused by the
gradual intrusion of water are also being investigated.
Spray cooling and spray chilling involve the use of cool air to solidify the
encapsulate (Lamb, 1987). These two processes differ in the encapsulating material
used in that lipids with low melting points (32 – 42°C) are used in spray chilling while
spray cooling utilises lipids with a higher melting point (45 – 122°C) (Dziezak, 1988).
The acid/lipid mixture is extruded through heated nozzles into a low temperature
chamber where the lipid solidifies, entrapping the acid.
While spray chilling and cooling are suitable for liquid acids, air suspension coating
is used to coat or encapsulate a solid core material. The process involves suspending
the solid core material in a fluidised bed of air and spraying with the coating material
(Dziezak, 1988; Kondo, 1989). The thickness of the coating material can be controlled
by the length of time for which the coating is applied.
Centrifugal-suspension coating involves suspending the core particles in the liquid
coating and then introducing them onto a rotating disk (Sparks and Mason, 1990). This
results in the coating material forming a film around the core material. Another process
which has been described for acidulant encapsulation involved plating the particulate
acidulant onto calcium lactate and then coating with a molten edible lipid (Percel and
Perkins, 1985). The product of this process would have controlled release at a higher
temperature.
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using an encapsulated acid (Anonymous, 1998). The process involves the use of an
acidulant, which has been encapsulated in a material with a melting temperature of at
least 90°F. Encapsulated acids are available under brand names such as CAP-SHURE®
and DURKOTE® with some companies also offering the facility whereby they will
tailor-make encapsulates to meet the customers requirements. It must also be noted that
the cost of encapsulates compared to the free form is quite expensive, up to three times
that of the free acid. For example encapsulated lactic acid costs IR£7.00/kg compared
to IR£1.50/kg to IR£2.00/kg for the free form. Therefore, these encapsulates must offer
some unique functionality over the free acid in order to achieve and retain economic
viability.
The application of encapsulation to meat products is not confined to encapsulated
acids. Encapsulated salt is also used in meats (DeZarn, 1995). The use of encapsulated
salt permits the addition of extra salt to meat products without adversely affecting the
shelf life or the texture of the meat.
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While the successful application of nisin to meat preservation has so far been elusive, as
in the case of direct acidification, encapsulation technology may provide a means of
overcoming this problem.
5.1 BACTERIOCINS
5.2 NISIN
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CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.
nisin production and nisin immunity to industrial starter cultures (Hugenholtz and de
Veer, 1991). This would mean that many fermented food products could be protected
by nisin produced in situ without the fermentation being adversely affected. Extending
the spectrum of nisin activity to Gram-negative bacteria is also being examined. The
application of nisin in combination with food grade chelating agents is reported to
increase the inhibitory activity and inhibitory spectrum of nisin (Blackburn et al., 1989;
Stevens et al., 1991, 1992; Cutter and Siragusa, 1995a, b; Shefet et al., 1995). The use
of nisin in combination with other bacteriocins such as pediocin AcH has been reported
to demonstrate greater antibacterial activity against a greater number of Gram-positive
bacteria (Hanlin et al., 1993). Nisin in combination with lactate has been found to
reduce the numbers of Salmonella typhimurium attached to beef and nisin in
combination with EDTA has been reported to reduce the numbers of Escherichia coli
O157:H7 attached to beef (Cutter and Siragusa, 1995b).
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combination of nutrients and protective agents into the matrix, which enhances
inoculum survival (Heijnen et al., 1992). Formulation of a delivery system for a
microorganism by incorporating nutrient adjuncts into the bead microenvironment has
been previously reported. Including maize cob grits and wheat gluten in alginate beads
containing Aspergillus flavus spores enhanced the activity of the mould after field
release (Daigle and Cotty, 1995). The incorporation of a combination of skim milk and
bentonite resulted in the highest survival and colonisation rates of Pseudomonas
fluorescens inoculated into soil (van Elsas et al., 1992). Similarly with immobilised L.
lactis, co-entrapping a combination of nutrients and microenvironmental modifying
agents increased the activity of the immobilised cells. Thus, one of the obvious benefits
of immobilised cell technology is in the control and exploitation of the unique
microenvironment associated with gel entrapment and especially using this
microenvironment in the stabilisation of microbial cultures (Karel et al., 1985).
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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
5.2.1.2 The application ofpolymer gels in the controlled release of nisin. One of the
disadvantages of nisin production in an immobilised cell system is retention of the
bacteriocin within the bead. Microenvironmental manipulation was shown to increase
nisin yield, however, this was also accompanied by an increase in nisin retention within
the encapsulation matrix. Therefore, the release of nisin from polymer gels and how the
release could be controlled and manipulated were investigated. There are numerous
advantages to be gained by the controlled release of nisin to the macroenvironment such
as enhanced stability and protection from proteinase degradation thus, prolonging
activity.
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CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.
Figure 5: The release of nisin from polymer gel capsules. The release of nisin into liquid
medium DMI was followed over a one week period at 4°C and 200 rpm.
For many bioactive agents it is recognised that there is a need to prolong the duration of
activity and develop a means of more efficient utilisation of the bioactive agent
(Schacht et al., 1993). Within the area of pharmaceuticals, sustained release
formulations are now a relatively common method of delivering low molecular weight
drugs (Langer, 1990). The encapsulation of proteins and the development of slow
release formulations are viewed as enhancing the stability and thus the applications of
proteins in therapeutics (Putney and Burke, 1998). Natural polymers can serve as depot
systems in which the agent can be incorporated and from which it is subsequently
released over an extended and controllable period of time (Schacht et al., 1993).
Although the application of immobilisation or encapsulation technology in the
delivery of bacteriocins is a relatively new development (Degnan and Luchansky, 1992;
Kirby, 1993; Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997, 1998; Wan et al.,
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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
1997), the encapsulation of nisin in calcium alginate has been achieved with some
success. It has been observed to prolong the activity of the nisin and protect it from
degradation by proteolytic enzymes (Fang and Lin, 1995; Cutter and Siragusa, 1996,
1997; Wan et al., 1997). However, manipulation of the gel matrix in order to control
the release of nisin to the macroenvironment has not been reported. This, however, is
an important factor in the development of delivery systems for other bioactive agents,
such as therapeutic agents (Aydin and Akbuga, 1996; Berthold et al., 1996).
The influence of polymers on the release of nisin from the immobilisation matrix
has been examined with a view to developing a delivery system, which offered a
controlled mode of nisin release. Through exploitation of the polymer composition and
charge, immobilisation matrices were designed with the aim of producing encapsulates
which exhibited controlled release properties for the bacteriocin nisin. The preparation
of encapsulates from alginate and chitosan has been demonstrated as a means of
controlling the rate of nisin release, at least into a liquid macroenvironment (Figure 5).
The structure of these encapsulates (Figure 6) as well as the chemical properties of the
encapsulation matrix and the core material have been identified as factors with a role in
the mechanism of controlled release.
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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION
The modification of polymer beads by coating with a polymer of the opposite charge
has been previously reported (Huguet et al., 1996; Quong and Neufeld, 1998). It has
also been reported that better slow release properties can be achieved when chitosan or
polylysine are used to change the permeability of the alginate membrane (Nuissinovitch
et al., 1996). Chitosan is also acceptable for oral administration as well as being a good
matrix for sustained release properties (Chandy and Sharma, 1992, 1993). Alginate -
chitosan capsules can be produced as a result of complexing between two oppositely
charged polymers, alginate being polyanionic and chitosan being a polycationic
polymer (Huguet et al., 1996). Capsules have been prepared with an alginate core and a
chitosan coat and vice versa, and the effect of each polymer on the release properties of
the other evaluated. Low molecular weight counterions were included in the
preparation of these capsules (Knorr and Daly, 1988; Pandya and Knorr, 1991 ; Polk et
al., 1994; Chang et al., 1996; Hari et al., 1996; Nussinovitch et al., 1996) as this
stabilised the capsule structure. Although capsules can be produced solely from
alginate and chitosan (Vorlop and Klein, 1987) these were observed to be weak due to
narrow width of the capsule membrane compared to the overall diameter of the beads.
This may be overcome by producing capsules of very small diameter.
The release pattern from capsules was dependent on a number of factors. According
to Huguet and Dellacherie (1996), the release of materials encapsulated in alginate
beads coated with chitosan depends on 1) the molecular weight of the material, 2) the
chemical composition and 3) the conformation of the molecules. These factors can vary
depending on the composition of their environment.
Coating alginate beads with a polycation has been reported to drastically reduce
diffusion from the beads. Nussinovitch et al. (1996) reported that after 6 days only 50%
of the entrapped bovine albumin and 10% of entrapped gamma globulin was released
from alginate polylysine capsules. At the surface of the membrane a chemical reaction
occurs between the polycation and the polyanion and the strength of this membrane can
be controlled by the polycation used thus making it possible to tailor liquid-core beads
to specific aims (Nussinovitch et al., 1996). Chitosan is insoluble at a pH above 5.4 and
so, while it is unsuitable for biomolecules unstable at low pH values (Huguet at al.,
1996), this makes it a suitable gel for nisin encapsulation due to the high stability of
nisin at acidic pH values.
The molecular weight of nisin is 3,353 Da (Jung, 1991a, b), although, it often occurs
as stable dimers with a molecular mass of about 7,000 or tetramers of 14,000 Da
(Cheeseman and Berridge, 1957, 1959; Jarvis et al, 1968). While the rate of diffusion
decreases with increasing molecular weight (Martinsen et al., 1989), this should not
affect the diffusion of nisin due to its low molecular weight. Proteins with a molecular
weight of between 2,500 and 66,000 Da have been reported to diffuse readily from a
matrix (Nussinovitch et al., 1996).
The chemical nature of the environment, such as pH or ionic charge, is an important
factor in relation to nisin release as this determines how the bacteriocin reacts under
particular conditions. For example, the effect of pH on the solubility of nisin is well
known (Liu and Hansen, 1990). The pH is also an important factor in relation to the
physical structure of the encapsulating matrix. At high pH values chitosan forms an
insoluble precipitate while at acidic pH values an ionotrophic gel is formed (Vorlop and
Klein, 1987). The pH at which these capsules are prepared is an important factor in
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relation to entrapment and release. For example, if alginate - chitosan encapsulates are
prepared at a pH of about 5.5, the surface of the beads formed is highly negative and the
positively charged chitosan can establish a strong ionic interaction with the alginate
(Huguet et al., 1996). On the other hand, if the capsules are prepared at a very low pH
(~ 2) the alginate will have a very low content of negative charges and so cannot
interact strongly with the chitosan (Huguet et al., 1996). In the preparation of alginate
core capsules, the pH of the alginate was 5.48, which would optimise ionic interactions
with the chitosan. In the preparation of chitosan core capsules, the pH of the alginate gel
was 7.17 and so it was highly negatively charged and so there was an ionic interaction
between the two gels. While normally such a high pH would cause precipitation of the
chitosan (Vorlop and Klein, 1987), the high molecular weight of the alginate would
prevent its diffusion through the chitosan. Therefore, the chitosan would not be
completely exposed to the high pH. The membrane produced as a result of the ionic
interaction between the alginate and the chitosan appeared to be the important factor in
relation to nisin release. The absence of any visible interaction between these two
polymers in chitosan-alginate-tripolyphosphate (CAT) and alginate-tripolyphosphate-
chitosan-calcium chloride (ATCC) capsules and the observed differences in release
from these capsules compared to chitosan-calcium chloride-alginate (CCA) and
alginate-chitosan-calcium chloride (ACC) capsules highlights this fact. The inclusion
of tripolyphosphate in the alginate increased its pH to approximately 8.5. Exposing
chitosan to such an alkaline pH causes it to precipitate (Vorlop and Klein, 1987) and in
the case of ATCC and CAT capsules this is likely to have prevented any interaction
between the chitosan and the alginate. CCA capsules exhibited a very low level of nisin
release and structural differences were observed between the chitosan core in these
capsules and the chitosan coat in ACC capsules. This was likely to be due to the pH
factor, The chitosan core of CCA capsules contained calcium chloride, which diffused
outwards to stabilise the alginate coating. There is no influx of ions into the bead and
so the pH of the core remained low (< 5). This is a stable environment for nisin (Liu
and Hansen, 1990) and as the alginate coat will be of a higher pH than the nisin
containing core the nisin was probably retained in this part of the capsule resulting in a
low level of release.
The solubility, stability and biological activity of nisin are highly dependent on pH.
They drop sharply and continuously as the pH is increased and nisin is almost insoluble
at neutral and alkaline conditions (Liu and Hansen, 1990). How the pH affects the
charge of a protein will affect its release (Huguet and Dellacherie, 1996). Proteins
which are stored under pH conditions lower than their isoelectric point present a
positive charge and their release remains low (Huguet and Dellacherie, 1996). In this
study, the low level of nisin release from CCA capsules, which have a low internal pH,
reflected this.
Investigating the application of polymer gels in the development of a delivery
system for nisin has shown that by entrapment of the nisin-producing organism, L.
lactis, in a polymer matrix, conditions for cell activity can be optimised. This permitted
nisin production to be enhanced under both stress and non-stress macroenvironmental
conditions. Manipulating the composition of the entrapment matrix and modifying the
entrapment/encapsulation process, enabled the production of nisin encapsulates, from
which the release of the bacteriocin to the external environment could be controlled.
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Thus, it can be concluded that naturally occurring polymer gels can be successfully
applied to the development of a delivery system for nisin. The encapsulates which have
been developed for the delivery of L. lactis and nisin would now need to be evaluated in
a meat product. This would determine the full extent of the success of encapsulation in
polymer gels to nisin delivery.
The production of safe food is a difficult task and while there will always be the risk of
foodborne disease there is an ongoing endeavour to control or minimise the risks. Risk
managers are continually looking for new options to minimise the risks associated with
foods and this in turn encourages researchers to focus on developing these options.
Encapsulation of food ingredients is one of these options and the technology offers great
potential in the area of food preservation. The pharmaceutical/medical industry has
extensively developed and used this technology for the purposes of time-release, flavour
masking and improved stability of pharmaceutical formulations (DeZarn, 1995) and
continues to do so to great benefit, such as limiting the side effects of drugs to the
development of techniques for in situ insulin production for diabetics (Badwan et al.,
1985; Langer, 1990; Duncan, 1993; Willaert and Baron, 1996).
The encapsulation of food ingredients was once regarded as too expensive and
customised for use in the food industry. However, the development of the technology,
although relatively unsophisticated when compared to other fields, means that the use of
encapsulated products has increased. Encapsulated ingredients are being designed to
meet the needs of a specific application. In order to do this a variety of factors must be
considered such as the functional properties of the finished product, the type of
encapsulation material, the processing conditions and the desired release properties
(Pszczola, 1998).
It is widely recognised that much remains to be done in the area of encapsulation of
food ingredients or additives. The necessity to use safe and edible materials and the
associated cost continue to impede the evolution of the technology. However the
potential for using naturally occurring polymer gels such as alginate, carrageenan and
chitosan as encapsulation matrices has been demonstrated here and they may provide a
means of overcoming such impediments.
Within the area of meat preservation, emerging and re-emerging pathogens are
challenging the existing methods of preservation. This means that it is necessary to
seek out new solutions to these new problems. Encapsulation may provide us with a
means of finding solutions and adapting and enhancing existing methods of
preservation. The amount of basic laboratory research being carried out in this area is
quite extensive and perhaps the greatest challenge, which faces the application of
encapsulation technology to particular areas in the food sector such as meat
preservation, is transposing the techniques from the laboratory to the industry.
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266
INDEX
Acetate ............................................................................. 24, 35, 83, 91, 92
Acidification.................. .239, 240, 241, 243, 246, 247, 248, 249, 251, 265
Activated sludge ............................................................................. 178, 181
Active dry yeast........................................ 33, 34, 36, 37, 38, 42, 43, 44, 47
Aerobic propagation ......................................................................... 90, 98
Aeromonas ............................................................................ 194, 196, 229
Aeromonas hydrophila ........................................................................... 196
AFLP ................. 193, 211, 212, 218, 220, 221, 225, 226, 229, 232, 233
Alcaligenes sp ................................................................................ 147, 150
Alcoholic fermentation ... 31, 32, 33, 34, 36, 37, 38, 39, 40, 42, 43, 44, 45,
46
Alginate.................................................................................. 244, 259, 264
Amplified ribosomal DNA restriction analysis ............................. 215, 232
Antagonistic activity ......................................................................... 19, 20
Antibacterial activity ........................................................ 14, 20, 252, 263
Antibiotics .............................................................................................. 18
Antimicrobial ..................................................................................... 1, 263
AP-PCR .................................................................. 211, 212, 222, 223, 228
ARDRA .......................................................................................... 215, 232
Aristolochene synthase ............................................................................ 27
Arming yeasts ..................................................................................... 59, 70
Arthrobacter nicotiana .......................................................................... 189
Aspergillus aculeatus .................................................................. 59, 61, 72
Aspergillus niger ......................................................................... 20, 28, 72
Aspergillus oryzae ................................................................................... 13
Aureobasidium pullulans ....................................................... 177, I78, 183
Azotobacter chroococcum .............................................................. 135, 139
Bacilli ..................................................................................................... 156
Bacillus cereus ................................................................. 20, 196, 233, 234
Bacillus stearothermophilus ........................................................ 59, 66, 73
Bacillus subtilis............................................................. 156, 162, 195, 265
Bacteria.............................................................................. 38, 52, 103, 263
261
Bacterial biomechanics .......................................................................... 157
Bacterial pathogens 1, 193, 194, 195, 196, 198, 201, 203, 205, 206, 208,
210, 212, 229, 235, 23 7
Bacterial typing ..................................................................... 193, 208, 210
Bacteriocins............................................. 20, 239, 250, 251, 252, 256, 262
Biogenic amines ....................................................................................... 42
Biomarker .............................................................................................. 145
Biomaterials ........................................................................................... 262
Biomechanics ................................................................................. 161, 162
Biosorption ............................................................................................ 183
Blue cheese .................................................................................. 13, 14, 29
Botryodiploidin ........................................................................................ 25
Brevibacterium flavum ................................................................ 51, 55, 57
Brucella.................................................................. 199, 200, 203, 230, 235
Campylobacter 193, 194, 195, 196, 199, 201, 209, 210, 226, 227, 231,
232, 233, 234, 235, 236, 237, 238
Campylobacter jejuni 193, 196, 226, 231, 232, 233, 234, 235, 236, 237,
238
Carbon sharing ...................................................................................... 147
Carrageenan .......................................................................................... 245
Cell growth rates ..................................................................................... 92
Cell wall ........................................................................................... 72, 156
Cellulase ................................................................................................ 138
Chitosan ......................................................................... 245, 259, 262, 263
Citric .................................................................................................. 41, 42
Classical typing ..................................................................................... 210
Cloning .............................................................. 44, 73, 131, 132, 190, 247
Cloning of genes ...................................................................................... 21
Clostridium ............................................ 20, 61, 72, 73, 195, 196, 201, 215
Clostridium perfringens .................................................................. 20, 196
Community physiology........................................................................... 150
Competition......................................... 14, 40, 43, 203, 239, 243, 246, 263
Contamination ........................... 65, 89, 193, 203, 204, 205, 206, 219, 243
Cyclopiazonic acid., .......................................................................... .23, 28
Detection1, 46, 144, 148, 149, 152, 153, 193, 194, 198, 199, 201, 202,
203, 204, 205, 206, 207, 218, 223, 224, 229, 230, 231, 232, 233, 234,
235, 236, 237, 238
DGGE ............................................................................................ 220, 234
Diacetyl............................................................................................. 35, 41
268
DNA amplification ................................................................................. 207
DNA chips. ............................................................................................. 220
DNA fingerprints ................................................................................... 219
DPH....................................................................................... 185, 186, 187
Effect of aeration ........................................................................... 137, 139
Effector protein ...................................................................................... 125
Effector proteins .................................................................................... 125
Electrophoretic karyotyping .................................................................... 17
Empedobacter sp.................................................................... 14 7, 149, 150
Encapsulation239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,
251, 252, 254, 255, 256, 257, 259, 260, 261, 262, 263, 265
Encapsulation matrices ................................................................ 244, 248
Enterococcus................................................................................. 195, 197
Escherichia coli 0157 ................................... 196, 229, 235, 237, 252, 263
Ethylcarbamate ........................................................................................ 45
Fattyacids 14, 33, 34, 38, 40, 46, 146, 148, 149, 150, 151, 152, 185, 187,
188
FCC............................................................................ 76, 77, 79, 80, 82, 83
Feather ................................................................................................... 165
Fermentation 2, 13, 14, 18, 20, 21, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
42, 43, 44, 46, 47, 52, 57, 60, 61, 65, 67, 71, 72, 79, 80, 89, 90, 96, 98,
99, 102, 107, 108, 109, 159, 160, 165, 166, 168, 171, 173, 175, 239,
240, 241, 242, 243, 244, 247, 249, 250, 252, 262, 263, 264, 265
Fermented feather meal ......................................................................... 171
Filamentous growth ...................................................................... 131, 132
flaA typing ...................................................................................... 193, 226
Flavor ............................................................ 14, 21, 28, 47, 242, 248, 261
Flocculation .......................................................... 92, 95, 96, 98, 129, 130
Fluorescence measurement., ................................................. 185, 186, 187
Food 27, 28, 29, 45, 47, 85, 227, 230, 231, 232, 233, 234, 235, 236, 237,
238, 249, 261, 262, 263, 264, 265, 266
Food components ........................................................................... 193, 203
Fumigaclavine ......................................................................................... 25
GAC ....................................................................... 177, 179, 181, 182, 183
Galactose .......................................................................................... 83, 85
Galactose metabolism., ............................................................................ 85
Gene disruption ........................................................................... 18, 21, 23
Genetically modified yeasts ..................................................................... 44
Genomic instability ................................................................................ 227
269
Glucoamylase..................................................................................... 59, 60
Glucose oxidase .................................................................. 20, 28, 29, 136
GRAS status ..................................................................... 20, 250, 251, 262
GTP analogues ...................................................................................... 187
GTP-binding proteins ................................................................... 109, 188
HACCP ......................................................................................... 194, 241
Histamine ......................................................................................... 42, 194
Hydrocarbon utilisation ......................................................................... 185
Hydrolytic enzyme .................................................................................... 61
Hydrolytic enzymes .................................................................................. 61
Hydrophobicity ...................................................................... 90, 91, 93, 96
Identification 37, 38, 42, 46, 77, 81, 84, 145, 147, 153, 154, 166, 175,
193, 194, 198, 199, 200, 203, 209, 214, 216, 220, 221, 225, 226, 229,
231, 232, 233, 234, 236, 237, 241
Idiophase .................................................................................................. 18
Immunomagnetic separation ........................................................ 206, 235
IMS......................................................................................................... 206
Infection ......................................... 195, 209, 223, 226, 227, 229, 265, 266
Inhibitory activity............................................................................ 20, 252
Insertion sequences....................................................................... 216, 236
Ion exchange resin ................................................................................. 181
IS200 typing ................................................................... 222, 223, 226, 235
Isoprenoid ......................................................................................... 23, 24
Isotopic fractionation. ............................................................................ 146
ITS regions ............................................................................................... 25
Kinetic fermentation .............................................................................. 171
Kinetic studies .......................................................................................... 80
Kocuria rosea ........................................ 165, 166, 167, 168, 170, 172, 173
Lactic acid ......................................................................... 14, 38, 262, 263
Lactic acid bacteria 14, 31, 32, 34, 38, 39, 41, 42, 44, 45, 46, 108, 239,
247, 250, 251, 254, 263, 264
Lactobacillus helveticus ................................................. 101, 102, 107, 108
Lactococcus lactis .................................................... 45, 252, 262, 263, 264
Leloir pathway. ........................................................................... 81, 82, 85
Line probe assay .................................................................................... 199
LiPA ....................................................................................................... 199
Lipase ....................................................................................................... 70
Lipases ............................................................................................... 14, 21
270
Listeria monocytogenes 14, 20, 195, 196, 201, 205, 227, 230, 231, 232,
233, 234, 235, 236, 237, 238, 262, 263, 264, 266
LPB-3 .................................................................... 167, 168, 170, 172, 173
Luedeking and Piret ............................................................................... 101
Lysine ................................................................................................. 55, 57
Malic .................................................................... 31, 38, 39, 40, 41, 42, 44
Malolactic fermentation ............................................. 31, 38, 39, 42, 43, 45
Malolactic starters ............................................................................. 38, 43
MAP kinase cascade 109, 113, 115, 117, 119, 120, 124, 125, 127, 131,
133
Marcfortines ............................................................................................ 25
Meat....................................................................... 241, 243, 262, 264, 265
Meatpreservation.................................................................. 239, 240, 241
Mechanical properties ........................................................................... 162
Metabolic control analysis .................................................... 75, 76, 82, 85
Metabolic engineering. ............................................................................ 85
Metabolic flux .................................................................................... 77, 78
Metabolicflux analysis .................................................... 75, 77, 79, 82, 84
Metabolic pathway analysis .................................................. 75, 76, 80, 81
Methylketones .......................................................................................... 14
Microencapsulation ...................................................... 262, 263, 264, 265
Micromanipulation ....................................................................... 158, 162
Molecular breeding .................................................................................. 73
Molecular detection ........................................................................... 1, 194
Molecular identification ....................................... 193, 198, 200, 201, 209
Molecular typing 15, 193, 208, 210, 211, 212, 215, 216, 217, 219, 220,
221, 225, 226, 227, 229, 230, 231, 232, 233, 238
Molecular weight..... 17, 135, 137, 138, 217, 219, 245, 256, 259, 260, 262
Mould fermented foods ...................................................................... 14, 21
Mould fermented meat products .............................................................. 13
MUC1 ............................................................................ 124, 125, 126, 127
Mutation........................... 18, 23, 24, 27, 28, 120, 121, 125, 130, 131, 217
Mycophenolic acid. ........................................................................... 25, 28
Mycotoxin............................................................................... 14, 23, 25, 28
NADPH accumulation ...................................................................... 55, 57
n-Alkane ................................................................................................. 185
NASBA ............................................................... 193, 198, 199, 200, 204, 237
NASBA method ....................................................................................... 200
n-Hexadecane ........................................................................................ 189
271
Nisin............................................... 250, 251, 252, 262, 263, 264, 265, 266
Nucleic acid based identification methods ............................................ 198
0157
h7 ....................................................... 195, 201, 202, 205, 217, 228, 250
Oenococcus oeni ...................................................................................... 38
Oil bioremediation ................................................................................. 190
Outbreak ........ 21 7, 222, 223, 224, 225, 229, 231, 234, 235, 236, 237, 238
Outer membrane proteins ...................................................................... 145
PAC........................................................................ 177, 179, 181, 182, 183
Pathogenic E. Coli ................................................................................. 198
Patulin., .................................................................................................... 25
Pb2+ removal........................................ 177, 178, 179, 180, 181, 182, 183
PCBC gene ............................................................................................... 17
PCR15, 17, 26, 27, 37, 193, 198, 199, 200, 202, 203, 204, 205, 206, 207,
210, 211, 212, 213, 214, 215, 218, 220, 222, 225, 226, 229, 230, 231,
232, 233, 235, 236, 237, 238
PCR detection ................................................................ 203, 205, 208, 238
PCR ribotyping ...................................................................................... 233
Penicillin.................................................................... 15, 17, 18, 19, 27, 28
Penicillin biosynthetic genes ................................................................... 17
Penicillin production ......................................................................... 17, 27
Penicillium camemberti ......................................................... 13, 23, 28, 29
Penicillium chrysogenum.................................................................. 27, 28
Penicillium commune ............................................................................... 28
Penicillium nalgiovense.. ...................................................... 13, 15, 27, 28
Penicillium roqueforti........................................................... 25, 27, 28, 29
Penitrem A ............................................................................................... 25
Peptidoglycan ........................................................................................ 157
PFGE193, 211, 212, 216, 217, 220, 222, 223, 224, 225, 226, 228, 229,
230, 231, 234, 235, 236
Phage typing., ........................................................................................ 233
PHB........................................................................ 135, 136, 137, 138, 139
Pigments ................................................................................................ 173
Plasmid.......................................................................... 219, 230, 234, 235
Plasmidanalysis .................... 193, 212, 219, 222, 223, 224, 225, 226, 234
Polar lipids ................................................................................... 144, 146
PR imine............................................................................................. 25, 29
PR toxin ....................................................................................... 25, 26, 27
272
Primers 15, 17, 29, 37, 193, 198, 199, 201, 202, 213, 214, 218, 222, 232,
236, 238
Probes 46, 136, 153, 158, 162, 193, 198, 199, 201, 203, 207, 208, 216,
229. 231. 233. 235. 236
Propagation ............................................................................................. 90
Propagation conditions ........................................................................... 90
Protease ................................................................................. 171, 172, 174
Proteases .................................................................................... 14, 21, 173
Pseudomonas sp ....................................................... 57, 145, 147, 148, 150
Pulsedfield gel electrophoresis .................................... .193, 210, 216, 238
Quantification...................................................... 75, 78, 84, 193, 207, 234
rAPD15, 16, 27, 193, 211, 212, 213, 214, 220, 222, 223, 225, 226, 227,
228, 229, 232, 234, 235, 236, 238
rAPD analysis .......................................................................................... 15
rDNA...................................................................... 144, 147, 209, 215, 232
Reactivation ................................................................. 33, 34, 39, 244, 246
Relative resistance ................................................................................. 155
Repetitive elements ................................................................................ 213
REP-PCR............................... 193, 211, 214, 220, 222, 223, 225, 228, 231
Respiratory activity .................................................................................. 52
Restriction analysis........................ 193, 212, 215, 216, 219, 222, 225, 226
Restriction fragment length polymorphism ... 193, 211, 215, 230, 234, 236
RFLP................................................................ 37, 211, 215, 216, 228, 236
Rhizopus oryzae ....................................................................................... 59
Ribosomal RNA ...................................................................................... 204
Ribotyping 193, 211, 216, 222, 223, 224, 225, 226, 227, 228, 229, 231,
232, 233, 234, 235, 236, 23 7
rRNA .............. 145, 153, 193, 199, 201, 204, 215, 216, 230, 234, 235, 237
rRNA genes., .......................................................................................... 199
RT-PCR.................................................................................. 193, 198, 204
Saccharomyces cerevisiae. 32, 47, 59, 71, 72, 73, 97, 98, 99, 130, 131,
132, 133, 183
Salmonella 193, 194, 195, 196, 199, 200, 201, 202, 205, 206, 207, 209,
210, 214, 219, 221, 222, 223, 224, 225, 229, 230, 231, 232, 233, 234,
235, 236, 237, 238, 252, 262, 266
Salmonella sp ......................................................................................... 236
Salmonella typhimurium ................ 209, 234, 235, 236, 237, 238, 252, 266
Secondary metabolite ..................................... 14, 15, 17, 18, 23, 24, 25, 26
Sensitivity ............................................................................................... 203
273
Serotyping ............................................................................................. 227
Serovars ........................................................ 221, 222, 223, 225, 227, 228
Shigella ........................................................................... 194, 195, 197, 201
Signal transduction .......................................................................... 110, 113
Signal transduction modules .............................................................. 113
Spacer region ......................................................... 199, 200, 214, 225, 232
Spirillum ............................................................................................... 157
SRFH ............................................................................... 211, 216, 227, 229
Staphylococcus aureus ................................ 14, 20, 197, 201, 231, 234
Staphylococcus epidermis ............................................................. 155, 159
Starch degrading enzymes ............................................................... 127
Starter ................................................................................................... 243
Starter culture 13, 14, 18, 19, 20, 23, 25, 239, 241, 242, 243, 244, 246,
247, 248, 250, 251, 252, 254, 264, 265
Starters..................................................................................................... 11
Steady-state continuous cultivation ......................................................... 79
Streptococcus pyogenes ........................................................................ 197
Streptomyces......................... 166, 171, 174, 185, 186, 187, 188, 189, 190
Streptomyces griseoflavus ...................................................................... 185
Streptomyces plicatus ............................................................................ 185
Stuckfermentation ............................................................................ 32, 34
Substrate competition ............................................................................ 149
Surface charge ........................................................................................ 91
Surface properties ............................................................................... 99
Target for molecular identification .................................... 198, 199, 200
Target genes ................................................................................ 83, 122
Taxon specific biomarkers ............................................................. 144
Taxonomic relationships .................................................................... 15
Taxonomy ........................................................................ 123, 131, 132
TEC1 ...................................................................................... 123, 131, 132
Tensile strength.............................................. 135, 136, 138, 139, 157, 162
Texture ..................................................................... 14, 242, 243, 249, 250
TGGE............................................................................................. 220, 234
Thermal stubility .............................................................. 65, 136, 137, I39
TLC analysis ............................................................................................ 23
Toxin ........................................................................................................ 29
Transcriptional regulators ............................................................. 113, 122
Transformation., ............................................. 19, 20, 32, 40, 44, 111, 227
Transformation vector ............................................................................ 19
274
tRNA-PCR ........................................................................................ 211, 214
Tryptophane ...................................................................................... 23, 24
Two dimensional TLC .............................................................................. 24
Typing1, 37, 193, 194, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,
218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 238
Typing techniques .................................................................. 215, 218, 219
U-13 C...................................................................................... 148, 150, 151
Validation .................................................................................... 108, 205, 206
Viability of cells. .................................................................................... 204
Vibrio............................................................................. 195, 197, 200, 233
Volatile compounds .......................................................................... 14, 35
Whey....................................................................................................... 102
White cheese ..................................................................................... 13, 28
Wine .............................................................................................. 46, 109
Yeast starters .................................................................................... 33, 45
Yersinia.......................................... 194, 197, 198, 201, 229, 230, 233, 235
Yield coefficient ....................................................................................... 92
Zeolite ............................................................................................ I 78, 181
Zeta potential ......................................................................... 91, 94, 95, 96
275