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 timeconsuming 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 beverages 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
Microorganisms have been used for preparing food
products like bread, yoghurt or curd, alcoholic beverages, cheese, etc, for a long time without even knowing 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 improvement 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 products occurred, there was a need to increase the production to meet the increasing demands. Microbial
techniques (selection, isolation of pure culture, mutation, protoplast fusion, etc.), well developed by middle of last century, were used advantageously in the
maximum output of the desired product with minimum by-product formation. These techniques, however, are slow in developing a microorganism having
the desirable traits and very few undesirable properties. Sometimes when protoplast fusion is carried out,
*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 empirically.
With the capabilities of modern biotechnology, the
scientists can now transfer desirable characteristics or
genes, without simultaneous transfer of other undesirable genes (Hui & Khachatourians, 1995). Cut and
paste techniques, developed in genetic engineering,
can incorporate only the desirable genes. Thus genetically modified organism takes only a few months
compared to a few years of laboratory work by traditional methods. This communication presents developments related to foods, wherein a review of genetically modified microorganisms useful in foods is
given. An attempt is also made to point out some desirable traits for commercial cultures, which might
prove useful in industrial food production and processing.
Organic Acids by Microorganisms
Citric acid is the most important organic acid produced by fermentation with an estimated annual production 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.

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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 environmental 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 control of growth conditions (Roehr et al, 1996).
A. niger strains have been developed by mutagenesis and screening, for higher productivity and adaptability to industrial fermenters. Some studies have
been undertaken
on parasexual
recombination,
diploidization, and heterokaryon formation, etc. (Visser, 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 tremendously 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 commercial 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

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383

et al, 1991). Lactobacilli are very fastidious and require many nutrients including nucleotides, amino
acids and vitamins provided by yeast extract or peptone.
Mutants can be generated spontaneously or by
mutagenic agents to give higher conversion or concentration of lactic acid. For commercial and economical production of lactic acid, the further improvements 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 (Gluconobacter) suboxidans and some Penicillium species(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 glucono delta-lactone by glucose oxidase enzyme. The
lactone hydrolyses to gluconate. Fermentation conditions are designed to maintain high levels of this enzyme. Medium contains glucose with low levels of
phosphate and nitrogen to prevent excess growth.
High aeration, temperature about 30C and mildly
acidic pH produces gluconate rapidly. The pH is
maintained by adding CaC03 to produce calcium gluconate 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 leavening of bread, fermentation of fruit juices or malt, clotting of milk for cheese, etc. Purified enzymes are being 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 (Rs2351400/kg) whereas speciality enzymes may cost Rs
2,35,000 or more per kg.

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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 Rhizomucor pusillus, were chosen by mutation and selection method. Recently, several genetically modified
microorganisms(E. coli, K. lac tis, A. niger), which
contain calf rennet gene, have been developed. Chymosin 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.

acting types. The industrially

important
exopeptidases are classified as per their catalytic mechanisms: 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 properties are further enhanced by protein engineering
making them useful in laundry detergents. They are
useful in production of fish-meal and protein hydrolysates from fish. Acid proteases, useful in cheese industry, 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.

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 industrial enzymes produced by Bacillus sp. are Pullulanase by B. acidopullulyticus, a-amylase by B. amyloliquefaciens and B. licheniformis, glucose isomerase
by B. coagulans, ~-glucanase by B. subtilis, etc. (Cocconcelli et al, 1991).

Fermentative Production of Amino Acids

Amylases and related enzymes are mostly obtained

from Bacillus species (Svensson et al, 1991). The aamylase, that hydrolyse internal 1-4 a-bonds of starch
resulting in rapid reduction of viscosity of the substrate, 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 cyclodextrin glucanotransferases catalyse the hydrolysis
of amylose to cyclic dextrins, a-, ~- and ycyclodextrins, which have useful properties in food
industry for stabilisation of volatile flavours, enhancers, 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-

Many amino acids are used in food industry

(Leuchtenberger, 1996), L-glutamate as flavour enhancer, glycine as sweetener, lysine and methionine
as food and feed additives, aspartate and phenylalanine 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, followed 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 dlor racemic form and glycine does not have d- and 1forms. 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 Saccharomyces are also used in amino acid production, some
of them are genetically modified. Bacteria normally
do not accumulate large amounts of amino acids because of regulatory control over their synthesis. Mutants have to be prepared by laborious mutagenesis
and selecting the mutant producing highest amount.
By using recombinant DNA technology, new producers can be developed rapidly by increasing limiting
enzyme activities, etc. (Eggeling & Sahm, 1999).
The genome analysis of producer strains is now becoming a useful tool. Entire sequence of the chromo-

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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 (Eikmanns et ai, 1991; Miwa et ai, 1983). Genetic manipulations including transduction, transformation and
conjugation have been used in genetic study of these
bacteria. This has led to the understanding of regulatory mechanism for microbial metabolism at genes
level. Genetic engineering modification aimed at improving 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 increased amino acid production and is carried out by
multiple copy plasmids. There are problems of maintaining 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 accumulation 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, preservation 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 proteases 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 useful vitamins. Many lactic acid bacteria like L. acidophilus and L. sake produce antimicrobial bacteriocins, which help in controlling unwanted microorganisms. Molecular strategies are being studied. Genetically 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 genetically engineered to grow faster than the contaminants,
as well as inhibit and destroy the growth of the contaminants including pathogens by producing antimi-

IN FOOD BIOTECHNOLOGY

385

crobial agents. The starter cultures have been modified to produce an antimicrobial agent, which destroys
cell walls of Listeria monocytogenes.
Similar modification can also be carried out to protect against organisms 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 requirements are other important factors in considering
different strains. For commercial beer production,
mostly S. uvarum and S. cerevisiae have been commonly used. Earlier protoplast fusion, which used to
produce a fusion product from S. uvarum and S. diastaticus, was not only more rapid in fermenting but
utilised available sugars more completely. Many of
the desirable properties can be incorporated by genetic modification (GMO). Though GMOs are not
being used commercially for brewing beer, but with
better understanding of genes controlling the properties of brewer's yeast and application of genetic engineering, 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 promoter gene, leading to increased production of uamylase in S. cerevisiae (Ruohonen et ai, 1991),
cloning of regulatory genes into S. cerevisiae resulting in higher maltase activity thereby improved conversion to ethanol (Rodicio & Zimmermann, 1985),
transforming amylase genes from S. diastaticus into S.
cerevisiae to enable latter to utilise dextrins, incorporating glucoamylase genes from R. oryzae and A.
awamori(Ashikari
et ai, 1989). Most of the efforts
were directed towards faster and more complete conversion of carbohydrates to ethanol.
Miscellaneous Microbial Products
Candida utilis has been used industrially in the
production of SCP for food and fodder, waste treatment 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 hydrolyse starch, cellulose or pectic substrates. Two-

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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 cellulosic waste. Molecular genetics of C. utilis is not adequately 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 enzymes 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 successful 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 Modified (GM) Foods including soya beans, corn, tomatoes, 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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