Indian Journal of Biotechnology

Vol 2, July 2003, pp 382-386

Applications of Microorganisms in Food Biotechnology

J S Pai*

Department of Food and Fermentation Technology, Institute of Chemical Technology, University of Bombay, Matunga, Mumbai
400019, India

Received 28 November 2002; accepted 20 February 2003

Strain improvement of microorganisms in food products has been slow as isolation and mutation are time-consuming
and labour-intensive. Hybridization also is slow as unwanted traits have to be bred out. Applications with food related
enzymes were the first products of modern biotechnology, followed by organic acids and amino acid production by
microorganisms. Food fermentation applications such as fermented dairy products and alcoholic bev-erages have also
shown good possibility for using GMOs for improved fermentation performance and resistance to bacteriophages
rather than yield improvement. Improvement in product characteristics including better nutritive quality will be the
driving force of future research in food biotechnology.

Keywords: genetically engineered microorganisms, acids, enzymes, dairy fermentation, alcoholic fermentation

ages, cheese, etc, for a long time without even know-

Introduction
Microorganisms have been used for preparing food
products like bread, yoghurt or curd, alcoholic bever-
ing their involvement in fermentation. Louis Pasteur
showed the role of microorganisms in spoilage and
subsequent elucidation that fermentation also
involves microorganisms. Once this fact was
established, the scientists tried to isolate
microorganisms, which were more efficient in
producing better products or im-provement of
processes. Some species are useful for development
of flavour unique to certain wines. Thus traditionally
certain microorganisms were used in such fermented which might prove useful in industrial food
foods. production and proc-essing.

Need for Improved Cultures Organic Acids by Microorganisms

When large-scale commercialisation of such prod-
ucts occurred, there was a need to increase the
pro-duction to meet the increasing demands.
Microbial techniques (selection, isolation of pure
culture, muta-tion, protoplast fusion, etc.), well
developed by mid-dle of last century, were used
advantageously in the maximum output of the
desired product with mini-mum by-product
formation. These techniques, how-ever, are slow in
developing a microorganism having the desirable
traits and very few undesirable proper-ties.
Sometimes when protoplast fusion is carried out,
Citric acid is the most important organic acid pro-
duced by fermentation with an estimated annual
pro-duction of about half a million tonnes with the
value more than half a billion dollars. It is primarily
used in foods. Some of the other acids produced in
large quantities by fermentation are gluconic acid,
lactic acid and ascorbic acid, each with production
over 50,000 tonnes per annum.

*Tel: 022-24145156; Fax: 022-24245156 E-mail: j spai

@foodbio.udct.ernet.in

some undesirable properties are transferred, which

have to be slowly removed by further mutation. All
this takes a long time and the results are not
precise as much of the development is being
performed empiri-cally.

With the capabilities of modern biotechnology, the

scientists can now transfer desirable
characteristics or genes, without simultaneous
transfer of other undesir-able genes (Hui &
Khachatourians, 1995). Cut and paste techniques,
developed in genetic engineering, can incorporate
only the desirable genes. Thus geneti-cally
modified organism takes only a few months
compared to a few years of laboratory work by
tradi-tional methods. This communication presents
devel-opments related to foods, wherein a review
of geneti-cally modified microorganisms useful in
foods is given. An attempt is also made to point out
some de-sirable traits for commercial cultures,

PAl: APPLICATIONS OF MICRO-ORGANISMS IN FOOD BIOTECHNOLOGY

383
Citric acid had been prepared from citrus fruits like
lemon but now it is mostly produced by
fermentation using Aspergillus niger, in large
corrosion resistant fermenters having stirrers.
Some yeasts like Candida have also been used to
a smaller extent. A smaller amount is also made by
older technique with surface fermenters. In
submerged culture, when environ-mental
conditions are controlled, organisms grow into
small pellets. Sugar from cane molasses is
commonly used in the medium, which needs to be
controlled for trace metals like iron, copper etc.
Maintenance of very low pH avoids by-products
formation. High aeration rate is needed for higher
yields. Conversion of glucose to product is high
(70-90%) depending on the strain, purity of
carbohydrate raw material, and environmental
conditions. By-product formation of oxalic or
gluconic acid can be reduced by strict con-trol of
growth conditions (Roehr et al, 1996).
A. niger strains have been developed by
mutagene-sis and screening, for higher productivity
and adapt-ability to industrial fermenters. Some
studies have been undertaken on parasexual
recombination, diploidization, and heterokaryon
formation, etc. (Vis-ser, 1991). Although
recombinant DNA technology has been reported
for Aspergillus species, no reports are available on
using this technique for commercial citric acid
production. Genes cloned from A. niger for
pyruvate kinase and phosphofructokinase will tre-
mendously improve the commercial strains
producing citric acid.
Lactic acid is another important acid produced by
fermentation, although an equal amount is also
chemically synthesised. The acid is mostly used for
the manufacture of emulsifiers and as additives in
food industry. It has two enantiomers, L(+)- and
D(-)-lactic acid. The L-lactic acid is involved in
normal human metabolism which can selectively
be produced by fermentation and this is used in
food applications, whereas the chemical synthesis
produces DL-lactic acid.
Strains of Lactobacillus delbruckii, L. casei, L.
helveticus and L. acidophilus, employed in commer-
cial fermentation, can ferment a medium containing
12-15% sugar in 2 to 4 days with more than 90%
yield (Kascak et al, 1996). Most lactobacilli cannot
use starch. L. amylophilus and L. amylovorus are
able to ferment starch to lactic acid (Zhang & Chery
an, 1991). The production of lactic acid or products
like ethanol, acetic acid, etc. depends on the strains
as well as the substrate and environmental conditions
(Cheng
et al, 1991). Lactobacilli are very fastidious and re-
quire many nutrients including nucleotides, amino
acids and vitamins provided by yeast extract or
pep-tone.
Mutants can be generated spontaneously or by
mutagenic agents to give higher conversion or
con-centration of lactic acid. For commercial and
eco-nomical production of lactic acid, the further
im-provements will expand substrate range,
improve product tolerance, use of simpler nitrogen,
increase disease resistance and control LID
isomer ratio (Demirci & Pometto, 1992).
Gluconic acid is prepared by fermentation using
mostly A. niger and less commonly Acetobacter (Glu-
conobacter) suboxidans and some Penicillium spe-
cies(Milsom & Meers, 1985). A. niger produces acid
at high levels(97 -99%) with negligible by-products
when the trace elements are controlled and sufficient
manganese is present. Glucose is converted to glu-
cono delta-lactone by glucose oxidase enzyme. The
lactone hydrolyses to gluconate. Fermentation condi-
tions are designed to maintain high levels of this en-
zyme. Medium contains glucose with low levels of
phosphate and nitrogen to prevent excess growth.
High aeration, temperature about 30°C and mildly
acidic pH produces gluconate rapidly. The pH is
maintained by adding CaC03 to produce calcium glu-
conate whereas NaOH gives sodium gluconate. A.
niger gene for glucose oxidase has been cloned into
S. cerevisiae, A. niger and A. nidulans to yield
improved organisms and fermentation (Kopetzski et
al, 1989).

Microbial Enzymes in Food Industry

Enzymes have been used in foods such as leaven-

ing of bread, fermentation of fruit juices or malt,
clot-ting of milk for cheese, etc. Purified enzymes
are be-ing used mostly in food industry although
some still use live cells as in leavening of bread
and alcoholic fermentation (Godfrey & West, 1996).
World market for enzymes, mostly microbial
enzymes, is over 1.5 billion dollars. Newer
applications for enzymes are in detergents, textiles,
paper & pulp, chemical industry, etc. However, the
largest application (over 45% of the total enzyme
produced), mostly bulk enzymes, is used in foods
(Ratledge & Kristiansen, 2001). Largest market is
for rennet (25%) followed by glucoamylase (20%),
a-amylase (16%) and glucose isomerase (15%).
Bulk enzymes normally cost less (Rs235-1400/kg)
whereas speciality enzymes may cost Rs 2,35,000
or more per kg.
384 INDIAN J BIOTECHNOL, JULY 2003
Cheese is traditionally prepared
using calf rennet, a protease. In
60s and 70s, due to severe
shortage of calf rennet, several
substitutes from microorganisms,
Rhizomucor miehei, Endothia
parasitica and Rhizo-mucor
pusillus, were chosen by mutation
and selec-tion method. Recently,
several genetically modified
microorganisms(E. coli, K. lactis,
A. niger), which contain calf
rennet gene, have been
developed. Chy-mosin gene
coding for calf rennet was taken
from calf stomach cells and
inserted into a plasmid, which
was inserted into microbial cells.
The microorganisms started
producing calf rennet (Madden,
1995). These rennets by GMOs
have been commercially
produced since 1980s and, in
India, only the microbial rennet is
being used since the ban on calf
rennet.
Bacillus spp., mostly extracellular,
are important source of stable
enzymes for use in food industry.
These degrade substrate into
smaller molecules, which are
easily absorbed by the bacteria.
Some in-dustrial enzymes
produced by Bacillus sp. are
Pullu-lanase by B.
acidopullulyticus, a-amylase by
B. amy-loliquefaciens and B.
licheniformis, glucose isomerase
by B. coagulans, ~-glucanase by
B. subtilis, etc. (Coc-concelli et al,
1991).
Amylases and related enzymes are
mostly obtained from Bacillus
species (Svensson et al, 1991).
The a-amylase, that hydrolyse
internal 1-4 a-bonds of starch
resulting in rapid reduction of
viscosity of the sub-strate, is called
liquefying enzyme and produces
mostly maltohexaose or
maltopentaose. This enzyme from
B. licheniformis is exceptionally
thermostable and is used in starch
processing. The enzyme from B.
subtilis is referred to as
saccharifying ~-amylase as it
predominantly produces maltose
and glucose from starch. It shares
limited sequence homology with the
liquefying enzymes and is less
thermostable. The cy-clodextrin
glucanotransferases catalyse the
hydrolysis of amylose to cyclic
dextrins, a-, ~- and y-cyclodextrins,
which have useful properties in
food industry for stabilisation of
volatile flavours, en-hancers, etc.
These are also produced by a
number of Bacillus sp. While
amylases hydrolyse 1-4 linkages,
pullulanase hydrolyse 1-6 a-
linkages in pullulan and
amylopectin. Whereas
saccharifying amylases yield mostly
maltose, glucoamylase produces
glucose. These are commercially
produced mostly by fungal cultures
but a few Bacillus species are also
used (Cole et al, 1988).
The bacilli also produce
proteases useful in food industry.
Proteases are divided into exo-
and endo-
acting types. The industrially cost less than Rs400 per kg. The
important exo-peptidases are two amino acids, not needed in
classified as per their catalytic optically pure forms, are prepared
mecha-nisms: serine, cysteine by chemical synthesis.
(thiol), metallo- and aspartic Methionine can be utilised in dl-or
(carboxyl) proteases. The serine racemic form and glycine does
proteases (PH, 9-11) have no not have d- and 1-forms. Others
metal ion requirement and are are prepared either by
resistant to high temperature and fermentation or by enzymatic
oxidising agents. Such proper- catalysis.
ties are further enhanced by
protein engineering making them
useful in laundry detergents. They
are useful in production of fish- Corynebacterium glutamicum is the
most versatile organism used
meal and protein hydroly-sates
commercially to prepare glutamate,
from fish. Acid proteases, useful
lysine, threonine, phenylalanine,
in cheese in-dustry, are found
etc. Escherichia, Serratia, Bacillus,
less in bacteria than in moulds Hansenula, Candida, and Saccha-
such as Mucor from which romyces are also used in amino
commercial microbial rennet is acid production, some of them are
prepared. Similarly thiol genetically modified. Bacteria
proteases like papain are not normally do not accumulate large
common in bacilli. amounts of amino acids be-cause
of regulatory control over their
synthesis. Mu-tants have to be
prepared by laborious mutagenesis
Fermentative Production of and selecting the mutant producing
Amino Acids highest amount. By using
recombinant DNA technology, new
produc-ers can be developed
rapidly by increasing limiting
Many amino acids are used in enzyme activities, etc. (Eggeling &
food industry (Leuchtenberger, Sahm, 1999).
1996), L-glutamate as flavour en-
hancer, glycine as sweetener,
lysine and methionine as food
and feed additives, aspartate and The genome analysis of producer
strains is now be-coming a useful
phenylala-nine for aspartame, an
tool. Entire sequence of the
low-calorie sweetener, etc. The
chromo-
total commercial production of all
the amino acids (chemical and
enzymatic) is over 1.6 million
tonnes, of which glutamate is
almost 1 million tonnes, fol-lowed
by lysine and methionine with
each about 350,000 tonnes. The
market is steadily growing at a
rapid rate of 5-10% per year.
Amino acids produced in larger
quantities are cheaper, glutamate
being the cheapest followed by
methionine and lysine. All three
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