Indian Journal of BiotechnologyVol 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 withfood related enzymes were the first products of modern biotechnology, followed by organic acids and amino acidproduction 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 tobacteriophages rather than yield improvement. Improvement in product characteristics including better nutritivequality will be the driving force of future research in food biotechnology.

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

IntroductionMicroorganisms 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 Pasteurshowed the role of microorganisms in spoilage andsubsequent elucidation that fermentation also involvesmicroorganisms. Once this fact was established, thescientists tried to isolate microorganisms, which weremore efficient in producing better products or im-provement of processes. Some species are useful fordevelopment of flavour unique to certain wines. Thustraditionally certain microorganisms were used insuch 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. Microbialtechniques (selection, isolation of pure culture, muta-tion, protoplast fusion, etc.), well developed by mid-dle of last century, were used advantageously in themaximum output of the desired product with mini-mum by-product formation. These techniques, how-ever, are slow in developing a microorganism havingthe desirable traits and very few undesirable proper-ties. Sometimes when protoplast fusion is carried out,

*Tel: 022-24145156; Fax: 022-24245156E-mail: j spai @foodbio.udct.ernet.in

some undesirable properties are transferred, whichhave to be slowly removed by further mutation. Allthis takes a long time and the results are not precise asmuch of the development is being performed empiri-cally.With the capabilities of modern biotechnology, the

scientists can now transfer desirable characteristics orgenes, without simultaneous transfer of other undesir-able genes (Hui & Khachatourians, 1995). Cut andpaste techniques, developed in genetic engineering,can incorporate only the desirable genes. Thus geneti-cally modified organism takes only a few monthscompared 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 isgiven. An attempt is also made to point out some de-sirable traits for commercial cultures, which mightprove useful in industrial food production and proc-essing.

Organic Acids by MicroorganismsCitric 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 valuemore than half a billion dollars. It is primarily used infoods. Some of the other acids produced in largequantities by fermentation are gluconic acid, lacticacid and ascorbic acid, each with production over50,000 tonnes per annum.

PAl: APPLICATIONS OF MICRO-ORGANISMS IN FOOD BIOTECHNOLOGY

Citric acid had been prepared from citrus fruits likelemon but now it is mostly produced by fermentationusing Aspergillus niger, in large corrosion resistantfermenters having stirrers. Some yeasts like Candidahave also been used to a smaller extent. A smalleramount is also made by older technique with surfacefermenters. In submerged culture, when environ-mental conditions are controlled, organisms grow intosmall pellets. Sugar from cane molasses is commonlyused in the medium, which needs to be controlled fortrace metals like iron, copper etc. Maintenance ofvery low pH avoids by-products formation. Highaeration rate is needed for higher yields. Conversionof glucose to product is high (70-90%) depending onthe strain, purity of carbohydrate raw material, andenvironmental conditions. By-product formation ofoxalic 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 havebeen undertaken on parasexual recombination,diploidization, and heterokaryon formation, etc. (Vis-ser, 1991). Although recombinant DNA technologyhas been reported for Aspergillus species, no reportsare available on using this technique for commercialcitric acid production. Genes cloned from A. niger forpyruvate kinase and phosphofructokinase will tre-mendously improve the commercial strains producingcitric acid.Lactic acid is another important acid produced by

fermentation, although an equal amount is alsochemically synthesised. The acid is mostly used forthe manufacture of emulsifiers and as additives infood industry. It has two enantiomers, L(+)- and D(-)-lactic acid. The L-lactic acid is involved in normalhuman metabolism which can selectively be producedby fermentation and this is used in food applications,whereas the chemical synthesis produces DL-lacticacid.Strains of Lactobacillus delbruckii, L. casei, L.

helveticus and L. acidophilus, employed in commer-cial fermentation, can ferment a medium containing12-15% sugar in 2 to 4 days with more than 90%yield (Kascak et al, 1996). Most lactobacilli cannotuse starch. L. amylophilus and L. amylovorus are ableto ferment starch to lactic acid (Zhang & Chery an,1991). The production of lactic acid or products likeethanol, acetic acid, etc. depends on the strains as wellas the substrate and environmental conditions (Cheng

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et al, 1991). Lactobacilli are very fastidious and re-quire many nutrients including nucleotides, aminoacids 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, improveproduct tolerance, use of simpler nitrogen, increasedisease 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 acidat high levels(97 -99%) with negligible by-productswhen the trace elements are controlled and sufficientmanganese is present. Glucose is converted to glu-cono delta-lactone by glucose oxidase enzyme. Thelactone hydrolyses to gluconate. Fermentation condi-tions are designed to maintain high levels of this en-zyme. Medium contains glucose with low levels ofphosphate and nitrogen to prevent excess growth.High aeration, temperature about 30°C and mildlyacidic pH produces gluconate rapidly. The pH ismaintained 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 improvedorganisms and fermentation (Kopetzski et al, 1989).

Microbial Enzymes in Food IndustryEnzymes 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 stilluse live cells as in leavening of bread and alcoholicfermentation (Godfrey & West, 1996). World marketfor enzymes, mostly microbial enzymes, is over 1.5billion dollars. Newer applications for enzymes are indetergents, textiles, paper & pulp, chemical industry,etc. However, the largest application (over 45% of thetotal enzyme produced), mostly bulk enzymes, is usedin foods (Ratledge & Kristiansen, 2001). Largestmarket 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 Rs2,35,000 or more per kg.

384 INDIAN J BIOTECHNOL, JULY 2003

Cheese is traditionally prepared using calf rennet, aprotease. In 60s and 70s, due to severe shortage ofcalf rennet, several substitutes from microorganisms,Rhizomucor miehei, Endothia parasitica and Rhizo-mucor pusillus, were chosen by mutation and selec-tion method. Recently, several genetically modifiedmicroorganisms(E. coli, K. lac tis, A. niger), whichcontain calf rennet gene, have been developed. Chy-mosin gene coding for calf rennet was taken from calfstomach cells and inserted into a plasmid, which wasinserted into microbial cells. The microorganismsstarted producing calf rennet (Madden, 1995). Theserennets by GMOs have been commercially producedsince 1980s and, in India, only the microbial rennet isbeing used since the ban on calf rennet.

Bacillus spp., mostly extracellular, are importantsource 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 isomeraseby B. coagulans, ~-glucanase by B. subtilis, etc. (Coc-concelli et al, 1991).

Amylases and related enzymes are mostly obtainedfrom Bacillus species (Svensson et al, 1991). The a-amylase, that hydrolyse internal 1-4 a-bonds of starchresulting in rapid reduction of viscosity of the sub-strate, is called liquefying enzyme and producesmostly maltohexaose or maltopentaose. This enzymefrom B. licheniformis is exceptionally thermostableand is used in starch processing. The enzyme from B.subtilis is referred to as saccharifying ~-amylase as itpredominantly produces maltose and glucose fromstarch. It shares limited sequence homology with theliquefying enzymes and is less thermostable. The cy-clodextrin glucanotransferases catalyse the hydrolysisof amylose to cyclic dextrins, a-, ~- and y-cyclodextrins, which have useful properties in foodindustry for stabilisation of volatile flavours, en-hancers, etc. These are also produced by a number ofBacillus sp. While amylases hydrolyse 1-4 linkages,pullulanase hydrolyse 1-6 a-linkages in pullulan andamylopectin. Whereas saccharifying amylases yieldmostly maltose, glucoamylase produces glucose.These are commercially produced mostly by fungalcultures but a few Bacillus species are also used (Coleet al, 1988).

The bacilli also produce proteases useful in foodindustry. 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 tohigh temperature and oxidising agents. Such proper-ties are further enhanced by protein engineeringmaking them useful in laundry detergents. They areuseful 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 suchas Mucor from which commercial microbial rennet isprepared. Similarly thiol proteases like papain are notcommon in bacilli.

Fermentative Production of Amino AcidsMany amino acids are used in food industry

(Leuchtenberger, 1996), L-glutamate as flavour en-hancer, glycine as sweetener, lysine and methionineas food and feed additives, aspartate and phenylala-nine for aspartame, an low-calorie sweetener, etc. Thetotal 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 about350,000 tonnes. The market is steadily growing at arapid rate of 5-10% per year. Amino acids producedin larger quantities are cheaper, glutamate being thecheapest followed by methionine and lysine. All threecost less than Rs400 per kg. The two amino acids, notneeded in optically pure forms, are prepared bychemical 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 orby enzymatic catalysis.

Corynebacterium glutamicum is the most versatileorganism used commercially to prepare glutamate,lysine, threonine, phenylalanine, etc. Escherichia,Serratia, Bacillus, Hansenula, Candida, and Saccha-romyces are also used in amino acid production, someof them are genetically modified. Bacteria normallydo not accumulate large amounts of amino acids be-cause of regulatory control over their synthesis. Mu-tants have to be prepared by laborious mutagenesisand selecting the mutant producing highest amount.By using recombinant DNA technology, new produc-ers can be developed rapidly by increasing limitingenzyme 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 IN FOOD BIOTECHNOLOGY

somes of C. glutamicum and E. coli is available. It ispossible to compare mutants and identify mutationsnecessary for overproduction of metabolites (Eik-manns et ai, 1991; Miwa et ai, 1983). Genetic ma-nipulations including transduction, transformation andconjugation have been used in genetic study of thesebacteria. This has led to the understanding of regula-tory mechanism for microbial metabolism at geneslevel. Genetic engineering modification aimed at im-proving amino acid production by these organismshas not yet resulted in substantial increase in aminoacid production (Aiba et ai, 1980). Amplification ofgenes coding for limiting enzymes might result in in-creased amino acid production and is carried out bymultiple copy plasmids. There are problems of main-taining stability unless selective pressure is exerted byadding antibiotics in the media. A new system withMu recombinant phages can integrate several copiesinto the host chromosomes showing stable accumula-tion without antibiotic selection pressure.

Biotechnology of Dairy ProductsLactic acid bacteria (Lactobacillus, Leuconostoc,

Pediococcus, Bifidobacterium, and Lactococcus) havebeen used to improve the flavour, texture, preserva-tion and nutritive value of dairy as well as vegetable,cereal and legume fermentation products includingyoghurt, buttermilk, cheese, pickled vegetables, idli,etc. (Luchansky et ai, 1988; Wood, 1992). In addition,some are even used as probiotics, which contribute tothe overall health of the user. In milk, the lactic acidbacteria ferment lactose and other sugars. Some pro-teases play role in the process along with the sugarmetabolising enzymes. Formation of these products aswell as compounds affecting flavour and texture givesthe typical pleasant aroma, taste and body to theproduct. 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 fermentationefficiency, better shelf-life, nutritional and sensoryproperties for the product, etc. will be the target ofthese studies (Lin & Savage, 1986).When cheese, yoghurt, etc. are made, undesirable

contaminants can lead to poor flavour, low yield andfood 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-

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crobial agents. The starter cultures have been modi-fied to produce an antimicrobial agent, which destroyscell walls of Listeria monocytogenes. Similar modifi-cation can also be carried out to protect against or-ganisms like Salmonella.

Alcoholic Fermentation using Improved CulturesYeast strain used in beer brewing is selected on the

basis of flavour and aroma, imparted by the strainduring fermentation. Flocculation, fermentation rate,ethanol tolerance, osmotolerance, and oxygen re-quirements are other important factors in consideringdifferent strains. For commercial beer production,mostly S. uvarum and S. cerevisiae have been com-monly used. Earlier protoplast fusion, which used toproduce a fusion product from S. uvarum and S. di-astaticus, was not only more rapid in fermenting bututilised available sugars more completely. Many ofthe desirable properties can be incorporated by ge-netic modification (GMO). Though GMOs are notbeing used commercially for brewing beer, but withbetter understanding of genes controlling the proper-ties of brewer's yeast and application of genetic engi-neering, increasing efficiency and productivity atminimum cost without affecting adversely the beerquality will soon become a reality. Some work is alsocarried 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 effortswere directed towards faster and more complete con-version of carbohydrates to ethanol.

Miscellaneous Microbial ProductsCandida utilis has been used industrially in the

production of SCP for food and fodder, waste treat-ment and the production of fine chemicals used asflavour enhancers (Boze et ai, 1992). Among theproducts useful in foods, besides SCP, are 5' -GMP &5' -IMP, ethanol, ethylacetate, acetylaldehyde, aminoacids like serine, histidine, glutamic acid and lysine,xylitol, etc. C. utilis does not possess enzymes to hy-drolyse starch, cellulose or pectic substrates. Two-

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step dual fermentation can be carried out using C.utilis with organisms like S. fibuliger, which producesamylases 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 havebeen successfully carried out, no commercial strainhas been developed by GMO including protoplastfusion. Since some of the enzymes are lacking in thisorganisms, incorporation of genes encoding these en-zymes would produce a desirable modified organismwith application in food industry.

Bacillus species have provided traditional biotechproducts such as extracellular enzymes and insecttoxins. B. thuringiensis strains with toxicities againsta variety of pests have been exploited to the extent ofgetting the genes inserted into food crops for success-ful development of resistance against these pests.Protein engineering and molecular technologies willslowly replace screening programmes.

Future Applications of Biotechnology in FoodsAt present, the large amounts of Genetically Modi-

fied (GM) Foods including soya beans, corn, toma-toes, etc. as well as ingredients from GM organismsare being used in most parts of the world. With wideacceptance to biotechnology in food applications,commercial interest would stimulate research in thearea. With genetic engineering techniques being usedto develop improved cultures, there will be markedimprovement in production and quality in addition tomany new applications of microorganisms.

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