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

Introduction some undesirable properties are transferred, which

Microorganisms have been used for preparing food
products like bread, yoghurt or curd, alcoholic bever-
ages, cheese, etc, for a long time without even know-
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 foods.
Need for Improved Cultures
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,
*Tel: 022-24145156; Fax: 022-24245156
E-mail: j spai @foodbio.udct.ernet.in
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, which might
prove useful in industrial food production and proc-
essing.
Organic Acids by Microorganisms
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.
 PAl: APPLICATIONS OF MICRO-ORGANISMS
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
IN FOOD BIOTECHNOLOGY 383
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. lac tis, 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 important exo-
peptidases are classified as per their catalytic mecha-
nisms: serine, cysteine (thiol), metallo- and aspartic
(carboxyl) proteases. The serine proteases (PH, 9-11)
have no metal ion requirement and are resistant to
high temperature and oxidising agents. Such proper-
ties are further enhanced by protein engineering
making them useful in laundry detergents. They are
useful in production of fish-meal and protein hydroly-
sates from fish. Acid proteases, useful in cheese in-
dustry, are found less in bacteria than in moulds such
as Mucor from which commercial microbial rennet is
prepared. Similarly thiol proteases like papain are not
common in bacilli.
Fermentative Production of Amino Acids
Many amino acids are used in food industry
(Leuchtenberger, 1996), L-glutamate as flavour en-
hancer, glycine as sweetener, lysine and methionine
as food and feed additives, aspartate and phenylala-
nine for aspartame, an low-calorie sweetener, etc. The
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
cost less than Rs400 per kg. The two amino acids, not
needed in optically pure forms, are prepared by
chemical synthesis. Methionine can be utilised in dl-
or racemic form and glycine does not have d- and 1-
forms. Others are prepared either by fermentation or
by enzymatic catalysis.
Corynebacterium glutamicum is the most versatile
organism used commercially to prepare glutamate,
lysine, threonine, phenylalanine, etc. Escherichia,
Serratia, Bacillus, Hansenula, Candida, and Saccha-
romyces are also used in amino acid production, some
of them are genetically modified. Bacteria normally
do not accumulate large amounts of amino acids be-
cause of regulatory control over their synthesis. Mu-
tants have to be prepared by laborious mutagenesis
and selecting the mutant producing highest amount.
By using recombinant DNA technology, new produc-
ers can be developed rapidly by increasing limiting
enzyme activities, etc. (Eggeling & Sahm, 1999).
The genome analysis of producer strains is now be-
coming a useful tool. Entire sequence of the chromo-
 PAl: APPLICA nONS OF MICRO-ORGANISMS
somes of C. glutamicum and E. coli is available. It is
possible to compare mutants and identify mutations
necessary for overproduction of metabolites (Eik-
manns et ai, 1991; Miwa et ai, 1983). Genetic ma-
nipulations including transduction, transformation and
conjugation have been used in genetic study of these
bacteria. This has led to the understanding of regula-
tory mechanism for microbial metabolism at genes
level. Genetic engineering modification aimed at im-
proving amino acid production by these organisms
has not yet resulted in substantial increase in amino
acid production (Aiba et ai, 1980). Amplification of
genes coding for limiting enzymes might result in in-
creased amino acid production and is carried out by
multiple copy plasmids. There are problems of main-
taining stability unless selective pressure is exerted by
adding antibiotics in the media. A new system with
Mu recombinant phages can integrate several copies
into the host chromosomes showing stable accumula-
tion without antibiotic selection pressure.
Biotechnology of Dairy Products
Lactic acid bacteria (Lactobacillus, Leuconostoc,
Pediococcus, Bifidobacterium, and Lactococcus) have
been used to improve the flavour, texture, preserva-
tion and nutritive value of dairy as well as vegetable,
cereal and legume fermentation products including
yoghurt, buttermilk, cheese, pickled vegetables, idli,
etc. (Luchansky et ai, 1988; Wood, 1992). In addition,
some are even used as probiotics, which contribute to
the overall health of the user. In milk, the lactic acid
bacteria ferment lactose and other sugars. Some pro-
teases play role in the process along with the sugar
metabolising enzymes. Formation of these products as
well as compounds affecting flavour and texture gives
the typical pleasant aroma, taste and body to the
product. The metabolic activity also forms some use-
ful vitamins. Many lactic acid bacteria like L. aci-
dophilus and L. sake produce antimicrobial bacterio-
cins, which help in controlling unwanted microor-
ganisms. Molecular strategies are being studied. Ge-
netically engineered lactics with better fermentation
efficiency, better shelf-life, nutritional and sensory
properties for the product, etc. will be the target of
these studies (Lin & Savage, 1986).
When cheese, yoghurt, etc. are made, undesirable
contaminants can lead to poor flavour, low yield and
food poisoning. Lactic acid bacteria can be geneti-
cally engineered to grow faster than the contaminants,
as well as inhibit and destroy the growth of the con-
taminants including pathogens by producing antimi-
IN FOOD BIOTECHNOLOGY 385
crobial agents. The starter cultures have been modi-
fied to produce an antimicrobial agent, which destroys
cell walls of Listeria monocytogenes. Similar modifi-
cation can also be carried out to protect against or-
ganisms like Salmonella.
Alcoholic Fermentation using Improved Cultures
Yeast strain used in beer brewing is selected on the
basis of flavour and aroma, imparted by the strain
during fermentation. Flocculation, fermentation rate,
ethanol tolerance, osmotolerance, and oxygen re-
quirements are other important factors in considering
different strains. For commercial beer production,
mostly S. uvarum and S. cerevisiae have been com-
monly used. Earlier protoplast fusion, which used to
produce a fusion product from S. uvarum and S. di-
astaticus, was not only more rapid in fermenting but
utilised available sugars more completely. Many of
the desirable properties can be incorporated by ge-
netic modification (GMO). Though GMOs are not
being used commercially for brewing beer, but with
better understanding of genes controlling the proper-
ties of brewer's yeast and application of genetic engi-
neering, increasing efficiency and productivity at
minimum cost without affecting adversely the beer
quality will soon become a reality. Some work is also
carried out to develop zymocide resistant strain.
The studies to improve the distiller's yeast strain
include manipulation of alcohol dehydrogenase pro-
moter gene, leading to increased production of u-
amylase in S. cerevisiae (Ruohonen et ai, 1991),
cloning of regulatory genes into S. cerevisiae result-
ing in higher maltase activity thereby improved con-
version to ethanol (Rodicio & Zimmermann, 1985),
transforming amylase genes from S. diastaticus into S.
cerevisiae to enable latter to utilise dextrins, incorpo-
rating glucoamylase genes from R. oryzae and A.
awamori(Ashikari et ai, 1989). Most of the efforts
were directed towards faster and more complete con-
version of carbohydrates to ethanol.
Miscellaneous Microbial Products
Candida utilis has been used industrially in the
production of SCP for food and fodder, waste treat-
ment and the production of fine chemicals used as
flavour enhancers (Boze et ai, 1992). Among the
products useful in foods, besides SCP, are 5' -GMP &
5' -IMP, ethanol, ethylacetate, acetylaldehyde, amino
acids like serine, histidine, glutamic acid and lysine,
xylitol, etc. C. utilis does not possess enzymes to hy-
drolyse starch, cellulose or pectic substrates. Two-
386 INDIAN J BIOTECHNOL, JULY 2003
step dual fermentation can be carried out using C.
utilis with organisms like S. fibuliger, which produces
amylases and can be used in starch wastes, and T.
reesei, which has cellulases and can be used in cellu-
losic waste. Molecular genetics of C. utilis is not ade-
quately studied. Although some transformations have
been successfully carried out, no commercial strain
has been developed by GMO including protoplast
fusion. Since some of the enzymes are lacking in this
organisms, incorporation of genes encoding these en-
zymes would produce a desirable modified organism
with application in food industry.
Bacillus species have provided traditional biotech
products such as extracellular enzymes and insect
toxins. B. thuringiensis strains with toxicities against
a variety of pests have been exploited to the extent of
getting the genes inserted into food crops for success-
ful development of resistance against these pests.
Protein engineering and molecular technologies will
slowly replace screening programmes.
Future Applications of Biotechnology in Foods
At present, the large amounts of Genetically Modi-
fied (GM) Foods including soya beans, corn, toma-
toes, etc. as well as ingredients from GM organisms
are being used in most parts of the world. With wide
acceptance to biotechnology in food applications,
commercial interest would stimulate research in the
area. With genetic engineering techniques being used
to develop improved cultures, there will be marked
improvement in production and quality in addition to
many new applications of microorganisms.
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