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Critical Reviews in Food Science and Nutrition

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Application of enzymes in food processing

Jennylynd James , Benjamin K. Simpson & Dr. Maurice R. Marshall

To cite this article: Jennylynd James , Benjamin K. Simpson & Dr. Maurice R. Marshall (1996)Application of enzymes in food processing, Critical Reviews in Food Science and Nutrition, 36:5,437-463, DOI: 10.1080/10408399609527735

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Critical Reviews in Food Science and Nutrition, 36(5):437-463 (1996)

Application of Enzymes in Food ProcessingJennylynd James and Benjamin K. SimpsonDepartment of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University,21111 Lakeshore Rd., Ste-Anne de Bellevue, Quebec, Canada, H9X 3V9

Referee: Dr. Maurice R. Marshall, Dept. of Food Science and Human Nutrition, Pesticide Research Lab., Univ. of Florida,Gainesville

ABSTRACT: Enzymes offer potential for many exciting applications for the improvement of foods. There is still,however, a long way to go in realizing this potential. Economic factors such as achievement of optimum yieldsand efficient recovery of desired protein are the main deterrents in the use of enzymes. Changing values in societywith respect to recombinant DNA and protein engineering technologies and the growing need to explore allalternative food sources may in time make enzyme applications more attractive to the food industry. Research iscontinuing on the commercially viable enzymes in use today to improve various properties such as thermostabili-ties, specificities, and catalytic efficiencies. New and unique enzymes continue to be developed for use inenzymatic reactions to produce food ingredients by hydrolysis, synthesis, or biocatalysis. An aggressive approachis needed to open new opportunities for enzyme applications that can benefit the food industry.

KEY WORDS: enzymes, applications, food industry.

I. INTRODUCTION

Enzymes are biological catalysts that act tospeed up chemical reactions. They are specific,effective in small amounts, and react under mildconditions of temperature and pH. Enzymes arederived from natural sources and may be readilyinactivated after a desired transformation has takenplace.1

Unlike inorganic catalysts, enzymes are highlyspecific, catalyzing the transformation of only asingle substrate or the splitting of a small group ofclosely related compounds or a specific bond.This minimizes byproduct formation in large-vol-ume reactions. The capacity of enzymes to reactunder mild conditions of temperature and pH (upto 100°C and pH 3 to 10) achieves a reduction inenergy costs. Low usage levels make enzymeseconomical and practical for commercial applica-tion.1 Because they are derived from plants, ani-mals, or microbial sources, enzymes are perceivedas natural, nontoxic food components and arepreferred over chemical aids as food-processingaids by consumers.2 Based on these properties,enzymes find numerous applications in industry.Reaction times for industrial enzymes vary from

1040-8398/96/$.50© 1996 by CRC Press, Inc.

a few minutes to up to several days. During ca-talysis, the enzyme forms a transient complexwith the substrate that may decay to form freeenzyme and product (or substrate).1 Thousands ofenzymes, each capable of catalyzing a uniquereaction, have been identified, and many are im-portant to the food industry today.

II. TRADITIONAL USES OF ENZYMES INTHE FOOD INDUSTRY

A. Early Enzyme Use

Enzymes have been used since ancient timesin an empirical manner, and special knowledgehas been handed down from generation to genera-tion of craftsmen, housewives, and chefs. Oneexample is the use of the mucous membrane ofweaning calves to preserve and improve the qual-ity of milk for human consumption.3 Other ex-amples of early uses of enzymes include the ad-dition of saliva to starchy products in preparationof fermented liquors, preparation of malted bar-ley and moldy bran for starch saccharification,fermentation of grape juice to wine, conversion of

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milk to curds and whey in containers made ofanimal stomachs, meat tenderization with papainin unripe papaya fruit, baking of bread with yeastby ancient Egyptians, and use of molds to makevarious oriental fermented foods.4"6

The discovery of enzymes of the digestivetract was yet another milestone in the impact ofenzymes on food products and the food industry.A few historical moments in food-related enzy-mology are illustrated in Table 1. Technical en-zyme preparations have been in use since thebeginning of this century. Beer had been stabi-lized with papain preparations since about 1911.Pectinases were used to improve the pressingquality of Concord grapes and for the clarifica-tion of fruit juices in the 1930s.7 The first genera-tion of enzyme preparations was unspecific, be-cause no accurate knowledge of substrate norenzyme activity was available. Manufacturers,however, have been constantly trying to improvethe specificity of technical enzymes. Table 2 listssome of the early enzyme-related patents in thefood industry. Takamine (1914) of Japan was thefirst to patent an enzyme from the microorganismAspergillus oryzae.4 Prior to 1960, the functionand structure of enzymes was not too clear, butmany applications had been found in the foodindustry (Table 3).

B. Current Enzyme Applications

Common use of enzymes in the food industryincludes ingredient production and texture modi-fication with industrial applications such as the

production of high-fructose corn syrup, beverageclarification, brewing, baking, production of low-lactose milk, and meat tenderization. However, itis rare that the consumer would use enzymesdirectly.7 Many food enzymes are used to degradevarious biopolymers. Their specificity and highreaction rates under mild reaction conditions arefavored over competing chemical treatments. In-dustrial food enzymes fall into three main groups:hydrolases, oxidoreductases, and isomerases. Bulkenzymes such as proteases, amylase, glucoamy-lase, pectinases, and cellulases (all hydrolases)have been produced using mainly two microbialgenera: Bacillus and Aspergillus. Extracellularenzymes released by these microorganisms cansimply be concentrated from the spent culturebroth after removal of cells at the end of theproduction phase.3

Newer uses of microbial enzymes are emerg-ing within the meat, fish, plant protein, and veg-etable oil sectors. Their share of the total marketfor food enzymes is still quite small. Unfamiliar-ity with enzymes among potential users and lackof well-defined application areas of great eco-nomic significance are the main reasons for theslow advancement of enzymes in food produc-tion.8

1. Enzymes and Biotechnology

The application of microbial enzymes has itsroots in Asia, while in North America and West-ern Europe it was more usual to use enzymesfrom plants and animals.9 Microbial enzymes have

TABLE 1Early Discovery of Enzymes

Year

1831183318361837184618561897

Enzyme

Ptyalin (amylase)Diastase (amylase)Pepsin activityEmulsin activityInvertase activityTrypsin activityEnzymes convertingglucose to ethanol

Scientist

LeuchsPayen and PersozSchwannLeibig and WholerDubonfantCorvisartBuchner

Adapted from Neidleman, S. L, Food Technol., 45(1), 88, 1991.

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TABLE 2

Some

Year

1894

1906

1911

1915

1917

1922

1923

1932

1933

1937

Early Enzyme Patents in the Food

Enzyme

Amylases

Amylases

Malt protease

ProteasesPepsinPapainBromelinYeast proteaseAmylasesAmylases

Amylases

Invertase

Amylases,protease,lipase

Amylases,papain

AmylasesInvertase

Proteases

U.S. patent no.

525,823

836,699

991,560995,820

995,823995,824995,825997,826997,873

1,129,3871,513,640

1,513,641

1,437,816

1,460,736

1,854,353-5

1,858,8201,919,675

2,077,477-9

Industry

Inventor

J. Takamine

J. Takamine

J. TakamineL. Wallerstein

S. FranknelI. Pollak

A. Boidin

H. S. Paine andJ. Hamilton

J. Takamine

M. Wailerstein

R.DouglasL. Wallerstein

L. Wallerstein

Title

Process of Making DiastaticEnzyme

Diastatic Substance and Method ofMaking Same

EnzymeBeer and Method of PreparingBeer

Preparation of Use in BrewingMethod of Treating Beer or AleMethod of Treating Beer or AleMethod of Treating Beer or AleMethod of Treating Beer or AleManufacture of DiastaseDiastase Preparation and Method ofMaking Same

Malt Extract and Method of MakingSame

Process for Preparing Fondant orChocolate Soft Cream Center

Enzymatic Substance and Processof Making Same

Method of Making ChocolateSyrups

Process of Preparing PectinInvertase Preparation andMethod of Making Same

Process of Chillproofing andStabilizing Beers and Ales

Adapted from Neidleman, S. L, Food Technol., 45(1), 88, 1991.

largely been considered by the North Americanfood industries as merely substitutes for the tradi-tional enzyme sources and not as processing aids.

Progress made in sterile technology as well asdevelopment of antibiotic fermentations and masscultivation of single organisms have laid the foun-dation for technical production of microbial en-zymes.3 Introduction of pectinases for the clarifi-cation of fruit juices in the 1930s was probablythe first example of industry's acceptance of mi-crobial enzymes for an application in which en-zyme technology had not been used previously.8

The commercial use of isolated microbial en-zymes, on a large scale, was enhanced in the1960s following the inclusion of alkaline pro-teases in washing powders. Many of the bulk ofenzymes used industrially today are of microbial

origin, because, unlike plants and animals, micro-organisms multiply rapidly and are not subject tothe same political and agricultural policies thatregulate the production of livestock for slaugh-ter.10-11

Enzyme technology involves a great deal ofoverlap with the fields of fermentation and tissueculture technology, because these methods arealso used for enzyme production. In fermentationtechnology and plant tissue culture, whole organ-isms are used. The multistep chemical conver-sions catalyzed by these processes require en-zymes acting in sequence. Complex cofactors maybe required. Traditional enzyme technology onlyrequires one or two chemical reactions. Thus,instead of the inconvenience of using whole or-ganisms, the enzyme is extracted from the plant,

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TABLE 3Pre-1960 Applications of Enzymes in the Food Industry

Enzyme

Amylase andprotease

AmylaseProteaseGlucose oxidase

AmylaseAmylaseAmylasePectinase

Source3

2,322,31,2,32

2,32,322

Pectinase and hemicellulase 2ProteaseInvertaseRenninLipaseCatalase

ProteaseProteaseLactase

Glucose oxidaseAmylaseGlucose oxidasePectinaseProtease

InvertaseAmylase andamyloglucosidase

Pectinase

1,2,32111,2

11,2,32

22,3 .222,3

22

2

Food product

Baked food

Beer

Carbonated drinks

Cereals

Chocolate

CondimentsConfectionaryDairy

Distilled beveragesEggFruit juiceMeat

MolassesStarch

Wine

Application

Bread and cracker baking

MashingChillproofinglOxygen removal

Precooked baby foodBreakfast foodSyrupBean fermentationconcentrate

Flavor ingredientsSoft center, fondantsCheese productionCheese favoringMilk sterilization with hydrogenperoxide

Off-flavor prevention in milkProtein hydrolysates in milkWhey concentrates, ice cream,frozen desserts, milk concentrates

Oxygen removal from dried milkMashingGlucose removalClarification, filtrationMeat tenderizing, casing tenderizingcondensed fish solubles

High-test molassesCorn syrup

Pressina. clarification, filtration

a 1 = animal, 2 = microbiological, 3 = plant.

Adapted from Neidleman, S. L, Food Technol., 45(1), 88, 1991.

animal, or microbial source, purified, and the iso-lated form used to catalyze a reaction.7

Recombinant DNA methods are having a sig-nificant impact on enzyme technology. Some ofthe benefits include increased enzyme productionand improvement of enzyme properties such asthermostability and ability to operate outside thenormal pH range. Even though plants and animalsmay serve as sources of several useful enzymes,their use as sources of industrial enzymes may belimited by availability, cost, and various political, •

economic, and environmental factors. However,this limitation may be obviated by isolating theenzyme from a plant or animal source and ampli-fying it in a microorganism. Methods that havebeen employed include screening from nature,mutagenesis, genetic engineering and cloning tech-niques, polymerase chain reaction (PCR), proteinengineering, and chemical derivatization.

The three main cloning techniques employedinclude "shotgun" cloning, cDNA cloning, andsynthetic DNA cloning. "Shotgun" cloning is rela-

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tively simple to carry out because the gene se-quence coding for a particular protein does nothave to be known. The disadvantages of thismethod, however, are that a very specific selec-tion method is required to identify recombinantstrains, genes that contain introns would not beaccurately expressed, and where prokaryotes areused to produce enzymes, these microorganismswould have to recognize foreign DNA and ex-press it. There is no problem of introns beingtranslated in cDNA cloning. Selection methodstend to be simpler than those of "shotgun" clon-ing and less laborious. However, mRNA requiredfor production of cDNA may not be available insufficient quantities. The cloning of cDNA is tech-nically more difficult to carry out and codon choicemay not be optimal for the host cell. SyntheticDNA cloning has the advantage that the sequenceof gene promoters and ribosomal binding sitescan be optimized and changed. Thus, highly spe-cific mutations and enzyme production are pos-sible. The disadvantage here, however, is that thegene sequence must be elucidated first and thereis usually a problem with degeneracy.12

PCR has been applied in all cloning tech-niques. This process has been used to generatespecific sequences of cloned double-stranded DNAfor use as probes, to generate probes specific foruncloned genes by selective amplification of par-ticular segments of cDNA, to generate libraries ofcDNA from small amounts of mRNA, to generatelarge amounts of DNA for sequencing, and for theanalysis of mutations. The main limitation of thismethod, however, is the high rate of misincorpor-ation because Taq DNA polymerase lacks theediting function of other polymerases like theKlenow fragment in Escherichia coli (Pol I). PCRis still a very useful tool that can be applied inprotein engineering and the production of benefi-cial food enzymes.13

DNA technology and its means of improvingenzyme production using cloning and expression,with protein engineering techniques to form uniqueenzymes, have led to the making of new enzymesbased on specific or desired functional needs. Ifthe three-dimensional structure or primary aminoacid sequence is known, the process becomeseven more feasible.14 The development of suc-cessful commercial products for food-processingenzymes and additives by genetic engineering

technology requires a concerted approach of ge-netic manipulation, protein engineering, and pro-cess development contributed by scientists andengineers from diversified disciplines, as seen inFigure 1.

Genetic engineering has made it possible toisolate particular genes coding for enzymes, fromorganisms whose genetics are unknown. Using invitro recombination these genes can be introducedinto microorganisms that have been used in foodpreparation for centuries, with subsequent syn-thesis of the novel gene products (enzymes).Genetic engineering methods provide the oppor-tunity to increase gene dose and so affect productyield. Plant and animal enzymes may be pro-duced more efficiently when cloned into a micro-bial cell and should be produced under highlyregulated conditions.15

Protein engineering involves protein crystal-lography, interactive computer graphics, databases,and other forms of commercial modeling to gen-erate knowledge of the relationship among se-quence, three-dimensional structure, and function.The necessary sequence changes for novel prop-erties are suggested and carried out by site-di-rected mutagenesis to alter one or a few specificamino acids in a molecule. The progress made inthe genetic engineering and protein engineeringstages are only useful when a large-scale processhas been developed to cultivate the "new" organ-isms in a fermenter and to purify a large enoughquantity of active product for product formulationand toxicology testing. Optimization of condi-tions for the cloned gene products is an importantstep in translating labscale experiments into com-mercial success.16

Enzymes of biotechnological interest that havebeen cloned fall into two main categories: thosethat have applications in vitro in some commer-cial processes and enzymes involved in biosyn-thetic pathways leading to commercially usefulproducts. Polysaccharases and proteases are themain groups of genetically engineered enzymesapplied commercially. The ot-amylases of severalspecies of bacilli have been cloned and in somecases partially or totally sequenced. Fungalglucoamylase genes from Aspergillus awamoriand A. niger have been cloned. In both casescDNA clones were prepared as probes and thegenes identified using purified mRNAs that en-

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1. Target newproteinproducts

3. Geneticengineering

Cloning Expression

5. Small-scalefermentationand purification

6. Pilot-scalefermentationand purification

10. Commercialproduction

11. Marketing

2. Justify newproperties ofexisting products

4. Proteinengineering

7. Productformulation

8. Toxicologystudies

9. FDAacceptance

FIGURE 1. Flowchart for commercial development of recombinant food-processing enzymes and food additives.(Adapted from Lin, Y. L, Food Techno!., 41(10), 104, 1987.)

coded the glucoamylases. Cellulases from bothbacterial and fungal sources have been cloned.There are reports of cloning of genes from organ-isms that code for protease production. One ex-

ample is the cloning of a thermostable neutralprotease gene from Bacillus stearothermophilusand its expression in B. subtilis. The gene codingfor calf chymotrypsin (rennin), a proteolytic en-

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zyme of great commercial importance, has beencloned and sequenced in Escherichia coli using atryptophan promoter from the host bacterium. Thereader is referred to the review by Peberdy15 forfurther information on genetic engineering in re-lation to enzymes.

2. Bulk Enzymes and Immobilization

Traditionally, enzymes were used in bulkform, for example, specific proteases for hydroly-sis of casein in cheese production. Through massproduction of secreting cells, extracellular en-zymes can be concentrated from the spent culturebroth after removal of the cells at the end of theproduction phase. Enzymes thus became avail-able at low cost and were used only once and thendiscarded or left in the product in a denaturedform. This is a simple approach to enzyme use notrequiring complex equipment or technical staff.Bulk enzymes are distributed for use either asconcentrated liquids or as dry powders.

Alternatively, the enzyme may be immobi-lized, that is, constrained one way or anotherwithin the confines of a solid support to permit itto be reused from one operation to another.17

Other advantages of enzyme immobilization in-clude: (1) a reduction in the need for huge quan-tities of enzymes thus reducing costs, (2) the abil-ity to reuse the enzyme many times more thanwhen in the soluble form, (3) no residual enzymeleft in the product, (4) increased stability of theenzyme (5) improved enzyme behavior (pH op-tima shifted to more advantageous pH on immo-bilization to certain supports), (6) the possibilityof a continuous process, which permits betterquality control of the product, (7) less additionalprocessing, (8) lower labor costs, and (9) advan-tageous use of multiple enzyme systems. Theprocedure has been explored since the mid-1960s.Substrate can now be continuously added to asupport as product is removed.

Solid supports used for enzyme immobiliza-tion include polysaccharides, inorganic supports,fibrous proteins, synthetic polymers, hydrogels,and hollow fibers. Enzymes can be constrained orheld to these supports by adsorption, covalentattachment, crosslinking, entrapment, microencap-sulation, or by other methods.17 Examples of

polysaccharide supports include diethylaminoethyl(DEAE)-Sephadex, which provides a polycationic,weakly basic anion exchange group. Enzymeshaving a relatively high content of acidic aminoacids remain firmly bound to DEAE-cellulose orDEAE-Sephadex even at high substrate concen-tration as long as certain physical conditions suchas ionic strength and pH are maintained.

Some of the disadvantages of enzyme immo-bilization include: (1) the cost of immobilization,(2) requirement for highly skilled personnel, (3)unique sanitation and toxicology problems, and(4) loss of enzyme activity; for example,aminoacylase from Aspergillus oryzae adsorbedon DEAE-Sephadex lost 40% of its activity overa 32-d period when used at 50°C for continuoushydrolysis of acetylated L-methionine.17

Chitin is another carbohydrate support mate-rial used in the immobilization of enzymes, asdescribed with glucose isomerase and glucoamy-lase attached by simple adsorption. The deacetyl-ated form of chitin is chitosan and this polymerhas a large percentage of free amino groups.Chitosan has been used along with gluteraldehydeto prepare immobilized acid-tolerant lactose.17

Porous glass, alumina, nickle oxide on nicklescreen, silica alumina impregnated with nickeloxide, porous ceramics, and sand are some of theinorganic materials to which enzymes have beenattached. Inorganic supports have the followingadvantages: (1) structural stability over varyingconditions, (2) excellent flow properties in reac-tors, (3) inertness to microbial attack or attack byenzymes, (4) ease of adaptability to various par-ticle shapes and sizes, and (5) good regenerationcapability.

General methods used to immobilize enzymesinclude adsorption, covalent binding, and entrap-ment. For a very informative discourse on immo-bilized enzymes in the food industry, the reader isreferred to the review article by Pitcher.18 Theinfluence of the use of enzyme immobilization onthe food industry is illustrated by the corn starchindustry, where glucose isomerase is immobi-lized for the production of high-fructose cornsyrup. This syrup has become a very competitivesweetener in the food industry.19

It is also possible to immobilize whole cellsinstead of isolated enzymes. This approach hasseveral advantages: (1) it avoids the lengthy and

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expensive operations of enzyme purification, (2)there is preservation of the enzyme environmentand reduction of its degradation, (3) sophisticatedtransformations are performed more easily, espe-cially those requiring several enzymes and cofac-tor regeneration, and (4) a multipurpose catalystmay be provided.19 However, there are limitationsto the approach, namely, (1) rate limitation due todiffusion of substrate and products through thecell wall, and (2) undesirable reactions of thesubstrate or product, causing a loss of specificity.Continuous processes are favored by cell immo-bilization.

C. Dairy Industry

For quite some time, rennet obtained from thestomach of calves was the only technical enzymeused in cheese making, apart from the specific useof esterases from sheep and goat gland extracts.Demand for cheese and dairy products is on theincrease worldwide. The dairy industry has re-sponded to these demands through mechanizationand consolidation and by taking advantage ofimprovements and new technology. Microbiologi-cal proteases from Mucor or Endothia parasiticahave been used as substitutes for animal rennet,which is becoming scarce. However, microbio-logical rennin has been found to be less specificthan pure chymosin.7

Pepsin and chymosin-like enzymes from harpseal and cold-temperature-adapted fish specieshave been shown to be able to curdle milk, butsimilarly to the microbial enzymes, are relatively

less specific. The introduction of microbialchymosin produced by a genetically engineeredorganism is considered the most significant futurebreakthrough in microbial rennet production.8 Im-provements are needed in the area of shortenedcheese ripening time and use of the waste productwhey.

1. Microbial Proteinases in the DairyIndustry

Microbial rennet substitutes derived fromMucor miehei, M. pusillus and Escherichiaparasitica are utilized in countries such as Hol-land and the U.S. Commercial products derivedfrom these rennet substitutes are listed in Table 4.Mucor rennets are marketed as liquids stabilizedwith salts and permitted preservatives. The en-zyme from E. parasitica is less stable and isusually marketed in a solid form. Religious andethnic regulations against the use of animal-de-rived enzymes have encouraged the search foralternatives to calf rennet in the United States.European countries have a larger supply of calfrennet and are in the practice of using variousblends of calf rennet and bovine or porcine pep-sin.21

In Cheddar cheese manufacture, the rennet isadded to milk at a temperature of 31 °C. The milk-clotting enzyme is added at a rate of 150 to 200ml/100 kg of milk, and clotting occurs within 10to 15 min. A temperature of 35°C is used in Swisscheese manufacture. Chymosin is used as the stan-dard of evaluation for other milk clotting en-

TABLE 4Commercial Microbial Rennets

Microbial source

Mucor pusillusMucor mieheiMucor mieheiMucor mieheiAspergillus nigerMucor mieheiMucor miehei

Supplier

Dairyland Food LaboratoriesG.B. Fermentation IndustriesChr. Hansen's LaboratoriesMiles LaboratoriesMiles LaboratoriesPfizer, Inc.Novo Laboratories

Product name

EmporaseFromaseHannilaseMarzymeMilenzymeMorcurdRennilase

Adapted from Ward, O. P., Microbial Enzymes and Biotechnology, Fogarty, W. M.,Ed., Applied Science Publishers, New York, 1983, 251.

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zymes. Comparative trials indicate that microbialenzymes from M. miehei compare favorably withrennet in cheese making with respect to curd yieldand firmness. Various types of cheeses preparedby microbial rennet were judged satisfactory byorganoleptic tests. However, it has been reportedthat microbial rennets produce more bitter prod-ucts, particularly in young cheeses.21 Most otherproteolytic enzymes cause extensive digestion ofmilk proteins that leads to unacceptable flavor,poor texture, and reduced yield.21

Studies of acceleration of the complex bio-chemical reactions in cheese ripening have fo-cused on proteinases, with less emphasis on li-pases and peptidases, in hard/semihard internallybacterially ripened cheeses. Accelerated ripeningdid not occur with increased rennet levels. Use ofan optimal level of Neutrase reduced the ripeningby approximately 50%, but the product thus ob-tained had a softer body and was more brittle thancheese of the same age.22 A combination ofNeutrase and streptococcal cell-free extract (CFE)gave better results with increased proteolysis, butno increased flavor intensity. This combined en-zyme preparation is now being produced com-mercially by Imperial Biotechnology of London,U.K., as a product called Accelase and is to beused in large-scale commercial cheese-makingtrials.22

2. Lactases

Lactose is a disaccharide composed of glu-cose and galactose residues linked together by anoc-(l—»4) glycosidic bond. It is a major compo-nent of milk and appears in whey during cheesemanufacture. Lactose is less sweet and less solublethan its component sugars and is thus of little usecommercially, because it needs to be broken downinto its component sugars.

It is estimated that 70% of the world popula-tion is deficient in intestinal lactase (p-galactosi-dase), the enzyme necessary to digest lactose.Certain individuals from various ethnic groups inthe U.S., namely, African-Americans (45 to 81%),Mexican-Americans (47 to 74%), Asian-Ameri-cans (65 to 100%), Native Americans (50 to 75%),and people of northwestern European ancestry (6

to 25%), have been found to be lactose intolerant.Thus, one-third of the U.S. population may bepresumed to have some form of lactose intoler-ance.23

The enzyme lactase, which coverts lactoseinto its subunits (glucose and galactose), can thusfind useful commercial applications in the foodindustry. The development of processes involv-ing soluble or immobilized lactase has been veryslow, possibly due to the high cost of the enzymein correlation to the value of the substrates.9 Thepotential for enzymatic modification of lactosehas been recognized since the 1950s, but produc-tion of lactose-modified dairy products becamepossible only with the commercial developmentof processes for the isolation of the enzyme frommicrobial sources. Lactases generally used forlactose hydrolysis are extracted from yeast suchas Kluveromyces lactis (K. marxianus) and K.fragilis or from fungi such as Aspergillus nigerand A. oryzae. These lactases differ widely intheir properties, especially in their pH optima.23-24

A dairy-modified lactose-reduced milk isavailable on the market. It is well accepted de-spite the slightly sweeter taste brought about byhydrolysis. One promising process is the TetraLacta System, developed by Tetra Pak Interna-tional AB. In this process milk is sterilized by anultrahigh-temperature (UHT) treatment, afterwhich it is mixed with sterile enzyme solution inlow concentrations. Hydrolysis of lactose thentakes place during storage of the packed milk(1 to 2 weeks — over 80% conversion of lac-tose).8

Lactase capsules were introduced by theKremers Urban Co., Millwaukee, WI and Lactaid,Inc. began commercial marketing of tablets tohealth professionals in 1985. Lactase-containingtablets are now available for both consumers andhealth professionals. Each tablet contains (5-D-galactosidase from A. oryzae and is taken with ameal. For example, in 1990 Winthrop ConsumerProducts, Division of Sterling Drugs, New York,NY introduced a chewable tablet known as DairyEase.23

Lactase has been employed in the manufac-ture of ice cream. Partial replacement of the skimmilk powder and sugar with hydrolyzed sweetwhey in ice cream formulae gives a smoother

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texture and an acceptable flavor. The possibilityof producing low-fat (3 to 5%) ice cream mixesthat are comparable in flavor and texture withordinary ice cream (10% fat) is being investi-gated.8 Complete replacement of sucrose in icecream mix with hydrolyzed-lactose syrup de-pressed the freezing point slightly.

In general, when cheeses are prepared fromhydrolyzed-lactose milk, a faster rate of acid de-velopment is observed during the initial stages ofcheese making, thereby reducing manufacturingtime. Faster ripening time for flavor and texturedevelopment has been observed, as well as highercheese yield. Reported disadvantages, however,include off-flavor development, sweet taste, fail-ure of curd to knit properly during cheddaring,and crumbly body.23

3. Future of Enzymes in the DairyIndustry

Genetic engineering offers the advantage ofdirected manipulation of microorganisms toachieve significant improvements on existing pro-cesses. Research progress has been slow in thearea of diary products for the following reasons:(1) higher intrinsic value of pharmaceutical tar-gets, (2) difficulty of culturing in the laboratory— dairy starter organisms have complex nutri-tional growth requirements, (3) few selectionmarkers, and (4) lack of secretion of proteins andother substances.

In the dairy industry, genetic engineering tech-nology has targeted the cloning of starter cultureorganisms with genes coding for long-term resis-tance to bacteriophage infection and the elimina-tion of culture rotations. Improved rates of acidproduction and substrate use have also been im-portant areas of research. Formation of culturesthat give new or improved textures and flavors,with accelerated flavor development, have beentargeted. Production of antimicrobial compoundsthat reduce spoilage and inhibit harmfulmicroorgansisms is an area of major concern.Above all, fermentation-based production of mi-crobial calf chymosin will prove to have enor-mous economic savings.

The ability to use the full potential of geneticengineering in lactic streptococci was initiatedwhen Kondo and McKay (1982) reported suc-cessful transformation of plasmid DNA in Strep-tococcus lactis protoplasts; however, the frequencyof transformation was low. Using a polyethyleneglycol-induced uptake system, an increase in ef-ficiency of about 300-fold was achieved.25

Also important was polyethylene glycol-in-duced transfection of protoplasts of Streptococ-cus lactis subsp. diacetylactis. The efficiency ofgenetic exchange reported was 50,000 transfec-tants per microgram of phage DNA. By shuttlingplasmid vectors between organisms, such asEscherichia coli or Bacillus subtilis, and the strep-tococci, the vector permits chromosomal frag-ments to be cloned and manipulated in the well-developed E. coli or B. subtilis system and thentransferred to the streptococci for further study.25

Several research groups have developed recombi-nant DNA techniques for the introduction of theprochymosin gene from calf stomach cells intomicroorganisms such as E. coli or Saccharomy-ces cerevisiae. Table 5 gives a partial list of com-panies with patent applications filed on microbialcalf chymosin. One problem experienced withrecombinant proteins produced in E. coli is theformation of soluble granules that are catalyti-cally inactive, packed inside the cell. These inclu-sion bodies of protein present major problems inindustrial scale-up and enzyme recovery.

D. Protein Modification

Food protein modification using microbialenzymes has played an important role in breadmanufacture. Strong gluten of low extensibilityreduce the volume of the baked product. Thissituation is remedied by using fungal proteinasesto achieve limited, specific gluten degradation.7

Enzymatic hydrolysis of proteins has been ap-plied to improve their whipping capacity or emul-sifying power. In the confectionary industry, pro-tein modification using microbial enzymes hasbeen used in the production of foaming agentsand specialty protein hydrolysates for dieteticpurposes. The development of industrial protein

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TABLE 5Companies and Investigators Who Have PublishedResearch, Have Patent Applications Filed, or HavePatents on Microbial Calf Chymosin

Company or investigator

T. Beppu, U. of TokyoCelltech, U.K.Codon, CACollaborative ResearchGenencorGenexGist BrocadesUnilever

Microorganism

E. coliE. coli and S. cerevisiaeE. coliE. coli and S. cerevisiaeE. coli and B. subtilisE. coliKluveromycesE. coli

From Barach, J. T., Food Techno),, 39(10), 73, 1985. Withpermission.

hydrolysis processes was hampered by the forma-tion of bitter-tasting peptides. Examples of en-zyme-modified proteins are discussed below.

1. Soy Protein Hydrolysates/HealthFoods

The treatment of soya flour or isolated soyprotein with proteases produces proteins withmodified functional properties, such as increasedisoelectrical solubility and improved whippingcapacity, foam stability, and emulsifying capac-ity.7 Soy protein hydrolysates have great potentialas ingredients in low-pH foods such as beverageswhere applications depend on a soluble productthat does not cause problems of gelation, turbid-ity, and off-flavor to the food produced. The majorproblem encountered with soy protein hydroly-sates is the problem of bitter peptide formation.The degree of bitterness is related to the degree ofhydrolysis. A compromise thus has to be estab-lished.20

The production of isoelectric, soluble soyaprotein hydrolysate (ISSPH) and the decoloriza-tion of the blood cell fraction of slaughterhouseblood are both processes that use Bacilluslicheniformis protease (Alcalase®), a product usedin the detergent industry. The neutral proteasefrom B. amyloliquefaciens (Neutrase) is used in

the production of soya milk for increasing itsprotein content.8 Traditionally, soy milk is pro-duced by initial soaking of the beans for at least3 h, depending on water temperature. The beansare then ground in water to bean ratio of 10 to 1.The slurry is filtered and the resulting milk heatedto boiling for about 30 min to improve its nutri-tional value and flavor. The product has a typicalbean flavor caused by enzymatic lipid oxidationduring the wet grinding process.26'27

It was found that enzymes improved the yieldof soy milk compared with traditional procedures.The neutral protease Neutrase gave superior re-sults at neutral pH. Olsen and Alder-Nissen27

devised a method whereby hydrolysates of soyaprotein could be produced, removing compoundsresponsible for bitter off-flavors. Figure 2 givesthe structures of precursors and off-flavor com-pounds in soybean. "Cooked soybean" off-flavorresults from the presence of phenolic acids suchas syringic, vanillic, ferulic, gentistic, salicyclic,p-coumaric, and chlorogenic acids. Ferulic andp-coumaric acids have been found to be precursorsfor the soybean off-flavor compounds, which wereidentified as 4-vinyl-guaiacol and 4-vinyl-phe-nol.

Soya white flakes are washed at pH 4.5 withwater in a four-step extraction process to producea concentrate with a low level of beany off-fla-vors. The concentrate is immediately hydrolyzed

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HC=C-COOH C=CH2

OH

p-cou marie acid

OH

4-vinyI-phenol

HC=C-COOH HC=CH2

OCH3 OCH3

4-vinyl-guaiacol

FIGURE 2. Precursors and off-flavors from soybean. (From Olsen, H. S. and Adler-Nissen.27 With permission.)

with the alkaline protease Alcalase to a specifieddegree of hydrolysis. Hydrolysis is terminated bylowering the pH and the supernatant is then re-covered by centrifugation.28

E. The Starch Industry

1. High-Fructose Corn Syrup Production

The method of acid hydrolysis of starch form-ing glucose was developed by Russian chemistKirchoff in 1811, and de Saussure in 1815 estab-lished the basis for the commercial production ofsyrups and crude sugars. Although acid-convertedcorn syrup was useful in many applications, the

lack of sweetness relative to that of sucrose andthe formation of reversion products limited theuse of glucose syrups. The introduction of enzy-matic processes with liquefying bacterial amylaseand saccharifying amyloglucosidase today allowsyields of up to approximately 90% glucose. Theuse of immobilized glucose isomerase has madeit possible to produce isomerase syrup with highfructose content.7 A large percent of microbialenzymes produced are utilized in the starch in-dustry. The large-scale process of high-fructosesyrup production was developed in the mid-1970susing Novo's commercial preparations of ther-mostable a-amylase (for the initial degradation ofstarch to dextrins) and immobilized glucoseisomerase (for converting glucose to fructose).

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The second process step, the saccharification ofdextrins to glucose, is carried out by soluble glu-coamylase from Aspergillus niger. Immobilizedglucoamylase has proved inefficient because themaximum relative glucose content obtained islower than that obtained with soluble glucoamy-lase.8-29

High-fructose corn syrup (HFCS) has becomean established product for the corn wet millingindustry, and almost every major corn processorin the U.S. has an HFCS plant in operation. HFCSis used as a less expensive direct replacement ofsucrose.30 A simplified flow diagram for the pro-duction of HFCS starting from starch was de-scribed by Bucke.32 The need for multiple treat-ment during HFCS production arises from thenature of starch itself. Starch consists of two frac-tions. The amylose fraction consists of long, un-branched chains of D-glucose units linked by oc-(1—»4) bonds. In contrast, the amylopectin fractionis highly branched and has both a-(l->4) and oc-(1—>6) bonds.31 The main challenge in the earlysaccharification steps is that a-amylases act mainlyon 1—>4 bonds so that addition of a debranchingenzyme like pullulanase or isoamylase, whichspecifically attack a-(l—>6) glycosidic bonds, isnow widely practiced.31 Commercial preparationof debranching enzymes exhibiting the right com-bination of pH and temperature, activity, and sta-bility has begun.

The heart of the process is the enzymaticisomerization step using immobilized glucoseisomerase. Two enzymes, oc-amylase andglucoamylase, are used in a soluble form to hy-drolyze starch. Syrup containing typically 90 to95% dextrose is fed to the isomerization reactorsystem. A series of pretreatment steps such asseparation of residual protein, carbon treatment,and ion exchange are needed to condition the feedsyrup for isomerization. A series of posttreatmentsteps, including evaporation, are needed to obtainthe end product.30'32

The amylase from B. amyloliquefaciens, whichhas a temperature optimum of approximately 70°C,was initially used to liquefy starch to form dex-trin. This enzyme is also used today in brewingand for the starch breakdown in brandy manufac-ture. Maize starch production requires tempera-

tures in excess of 100°C for complete starch di-gestion. There was thus the need for bacterialamylases with high-temperature stability, such asthat from B. licheniformis, which has optimumworking temperature 90°C.

2. Maltose Production

Amyloglucosidase from A. niger and Rhizo-pus is used for saccharification of dextrins toglucose, which is then isomerized to fructose inhigh-fructose syrup production. The reaction isslow: 45 to 60 h at pH < 5.0 and temperature60°C.7 Exoacting maltogenic a-amylase has beendescribed. It is also the first near commercialexample of an industrial enzyme produced bygenetically engineered organisms. In trials, moreglucose was obtained than with plant P-amylases.

The demand for high-maltose syrups in Japanis growing. Most Japanese prefer the much mildersweet flavor of maltose to the very sweet flavor ofsucrose. Therefore, a lot of high-maltose syrupinstead of sucrose has been recently used for,making cakes and soft drinks.33 By treating starchpaste with P-amylase, maltose may be obtained ina yield of about 80%, with 20% of dextrins beingproduced simultaneously. Maltose syrups pro-duced from corn starch are of two types. One typeis 30 to 50% maltose and 6 to 10% glucose (high-maltose syrup) corresponding to 43 to 49 dex-trose equivalent (DE) corn syrups (Table 6). Theother is 30 to 40% maltose and 30 to 50% glucose(fermentable syrup) representing 65 to 70 DE(Table 6).

Maltose production can be divided into theproduction procedure for high-maltose syrup andthe fermentable syrup. For high-maltose syrupproduction, two common procedures used are (I)the acid-enzyme conversion process, and (2) themultiple-enzyme process. For the first process, amaltose-producing saccharifying enzyme such asbarley P-amylase is added and the residual en-zyme is deactivated by heating. In the multiple-enzyme precursor, starch is gelatinized and thepreliminary starch depolymerization is broughtabout by enzymes such as fungal a-amylase orbacterial a-amylase instead of acid. Barley

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TABLE 6Compositional Data of Syrups

% Saccharide, carbohydrate basis

Sample

Corn syrup,AC

Corn syrup,AC

Corn syrup,AC

Corn syrup,AC

Corn syrup,HM, DC

Corn syrup,HM, CD

Corn syrup,DC

Corn syrup,DC

Corn syrup,DC, E

a Data suppliedb Sum of DP* „,

Dextroseequivalent

27

36

42

55

43

49

65

70

95

by cooperating

DP,

9

14

20

31

8

9

39

47

92

member companies

DP2

9

12

14

18

40

52

31

27

4

of the CRA.

DP3

8

10

12

12

15

15

7

5

1

DP4

7

9

9

10

7

1

5

5

1

DP5

7

8

8

7

2

2

4

4

DP6

6

7

7

5

2

2

3

3

DP7

54

40

30

17

26

19

11

9

2 b

Note: DP = degree of polymerization; AC = acid conversion, DC = dual conversion (acid-enzyme), HM = maltose,E = enzyme conversion.

From Maeda, H. and Tsao, G. T., Process Biochem., 14(7), 2, 1979. With permission.

p-amylase is then added to continue the conver-sion. Microbial P-amylase has been known to bevery stable in supply and economical to use.

A. oryzae a-amylase is reported to be themost effective of many amylases tested. StaleyCorn Processing, Inc. has applied a debranchingenzyme for the production of high-maltose syrupof 80% maltose content as a commercial product.Barley P-amylase and microbial pullulanase havebeen used as debranching enzymes. It was foundthat B. cereus var. mycoides accumulated largeamounts of p-amylase and pullulanase in the brothat the same time when cultivated with meat ex-tract and mild casein as the nitrogen sources.These enzymes find great industrial applications.33

F. Brewing Industry

Traditionally, manufacture of beer is dividedinto stages: a main fermentation stage and a matu-

ration stage. During the main fermentation, thebulk of fermentable sugars in the wort (an aque-ous extract of malted barley) is metabolized intoethanol, while during maturation, flavor, carbondioxide content, haze stability, attenuation, andother properties of the beer are adjusted care-fully.34-35 Proteinases and exopeptidases presentin the malt cause the increase in soluble nitrogencompounds required for the brewing process. Ifunmalted grain is used, the missing proteolyticactivity must be supplemented by exogenous pro-tease preparations. In selecting proteases, the fol-lowing conditions must be observed: (1) high-temperature stability at 55 to 60°C, (2) optimumactivity between pH 6 to 7, (3) insensitivity tonatural proteinase inhibitors, and (4) activity atvery low protein concentrations. Traditionally,this function was carried out using papain.7

In beer manufacture, proteinases act to in-crease the actual yield of wort and the level of oc-amino nitrogen in the wort, and to remove chill

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haze from beer during finishing. It is agreed thata level of 140 to 180 mg of a-amino nitrogen perliter of wort is required for normal fermentation.20

Beer contains complexes of proteins withcarbohydrates and tannin, which are soluble inthe newly pasteurized product, but tend to be-come less soluble with time as the beer is cooled.The complexes are insoluble, but do not precipi-tate. They instead form unacceptable turbidity inthe beer. This turbidity is called chill haze. Thenature of chill haze and its formation vary de-pending on the type and source of barley malt hopadjuncts and processing variables used in the par-ticular brewing (5 to 70% proteins, 1 to 55%tannins, and 3 to 80% carbohydrates).35 In NorthAmerica, where beer is chilled before serving, theproblem is unavoidable; thus, chill proofing orprevention of chill haze formation is required.

Examples of haze-removing material areadsorbents such as bentonite, wood chips, silica,nylon powder, and activated carbon. These solidparticles would have to be removed at the end ofthe process, thus requiring additional costs forfiltering. There would also be a reduction in yield

because some final product remains trapped inthe filters. Around 1914, the Wallerstein Com-pany used proteolytic enzymes to hydrolyze pro-teins involved in haze formation. For example,papain has been immobilized on chitin and beerpassed over columns of the enzyme for chill proof-ing.34-35

The brewing industry is quite conservative inthe use of microbial enzymes; however, the en-zyme acetolactate decarboxylase isolated fromseveral microorganisms has been applied to cata-lyze the degradation of oc-acetolactic acid toacetoin. During beer maturation processes, theoxidative transformation of a-acetolactic acid intodiacetyl and the subsequent reduction of diacetylto acetoin by reductases from the yeast are usu-ally rate limiting for the overall maturation pro-cess (Figure 3). Acetoin is a major flavor compo-nent in beer and thus its formation is critical inbeer production.

Godtfredsen et al.34 determined the occur-rence of cc-acetolactate decarboxylases amonglactic acid bacteria. Streptococcus diacetylactisstrain FD-64-D was found to generate a decar-

- Acetolacticacid

Diacetyl

AcetolactateDecarboxylase

o

Diacetylreductase

OH

H H

OH

2,3-Butanediol

Acetoinreductase

H

OHAcetoin

FIGURE 3. Key reactions in diacetyl generation in and removal from beer. (Adapted from Godtfredsen, S. E.,Rasmussen, A. M., Ottesen, M., et al., Appl. Microb. Biotechnol., 20, 23, 1984.)

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boxylase exhibiting a satisfactory activity and anexcellent stability at the pH prevailing in beer andwort. Lactobacillus casei DSM 2546 produced adecarboxylase with improved solubility charac-teristics and also exhibited satisfactory activityand stability at low beer pH. Based on these supe-rior qualities, the enzyme from L. casei was evalu-ated for solubility for pilot-scale production. Thus,acetolactate decarboxylase production from lac-tobacilli was not considered to be profitable eventhough it has GRAS status, because it requires toomuch zinc for stabilization. Therefore, more suit-able bacterial sources are required for the produc-tion of acetolactate decarboxylase.8

Microbial enzymes are being used in the pro-duction of low-calorie beer. The main differenceof the "lite" beers compared with "normal" beersis that "lite" beers are relatively lower in calories(i.e., ~ 15 to 50% lower). Brewing techniques in"lite" beer formation include a low original grav-ity and a complete fermentation. One simple pro-duction method is to dilute a regular beer. How-ever, this may upset flavor balance. A morecomplex technique includes the use of dextrose asan adjunct to reduce the residual carbohydratecontent of the beer or addition of amyloglucosidaseto wort to achieve the same results. However,with the enzyme technique, it is difficult to re-move all residual enzyme activity in the finalbeer, which leads to a distinctly sweeter producton the market. Glucoamylases and fungal p-amy-lase are used commercially for this purpose.8

G. Lipid Modification

Lipases are produced by plants, animals, andmicroorganisms. Plant lipases have not foundcommercial applications, but microbial and ani-mal lipases have been used successfully in pro-ducing novel ingredients.36 Bacteria of the generaPseudomonas, Achromobacter, and Staphylococ-cus, as well as yeasts (i.e., Candida and Torulopis),and molds (i.e., Rhizopus, Aspergillus, andGeotricum) have been shown to produce lipases.

Glycerol ester hydrolases or lipases are en-zymes that hydrolyze tri-, di-, and monoglyceridespresent at an oil-water interface. Although thereaction seldom goes to completion, hydrolysis of

a triglyceride by lipase can yield monoglyceridesand free fatty acids. Lipases have been used forthe interesterification of triglycerides, but thesereactions are commonly carried out using inor-ganic catalysts in the oil and fat industry. Com-pared with inorganic catalysts, lipases are moresubstrate specific, that is, they will catalyze theinteresterification of only certain types of fattyacids in particular positions in the triglycerides.8

Vegetable and animal fats are commonly usedas substrates for lipolysis. The ease with which fator oil is hydrolyzed depends on the lipase itself.The substrate, however, has to be emulsified be-cause a true lipase by definition acts at the oil-water interface. Glycerol esters are the preferredsubstrates.

Microbial lipases may be divided into twogroups, depending on their positional specificity.There is a nonspecific group that releases fattyacids from all three positions of the glycerol moi-ety. The nonspecific lipases also cause completebreakdown of triglycerides to free fatty acids andglycerol. The second group is specific in thathydrolysis takes place at the Snl and Sn3 posi-tions preferentially to give free fatty acids and thedi- and monoglycerides as reaction products.36

One good example of this is the system pioneeredby Unilever Research in which one of the majorlipids of palm oil, 1,3-dipalmitoyl-2-oleyl glyc-erol (POP), is transformed into more valuablecompounds. One or both of the palmitic acidresidues are substituted with stearic acid to formpalmitoyl-2-oleyl-3-steroyl glycerol and steroyl-2-oleyl-3-steroyl glycerol, that is, POS and SOS,respectively.32

These lipids (POS and POP) are the maincomponents of cocoa butter.8 In this process theenzyme is immobilized and hydrated before use,and the substrate is a particular grade of palm oil,mixed with tristearin or stearic acid in an organicsolvent. These reactions may be termed transes-terification: a process used in the oil industry tomodify the composition and hence the physicalproperties (crystallinity and melting characteris-tics) of triglyceride mixtures. In this process achemical catalyst is used to promote acyl migra-tion between glyceride molecules so that productsconsist of glyceride mixtures in which the fattyacyl groups are randomly distributed among the

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glyceride molecules. The process can also beapplied to mixtures of triglycerides and free fattyacid, resulting in the formation/production ofinteresterified triglycerides randomly enrichedwith the added fatty acid.37

Controlled lipolysis has been applied in thegeneration of short-chain fatty acids, which inturn are oxidized into carbonyl compounds suchas methyl ketones, which are responsible for milkaroma. Numerous procedures describing the ap-plications of various lipolytic enzymes have beenpatented, and a few examples of these patentedprocedures are summarized in Table 7. Differentlipolytic enzymes yield differing amounts of prod-ucts with the same substrate; for example, theaddition of lamb gastric extracts combined withlamb pregastric esterase in the manufacture ofItalian cheese leads to the development of thepicante flavor of Romano and Provolone.36 Inclu-

sion of pregastric calf esterase and fungal esteraseresulted in typical Fontina cheese flavor. In theU.S., effective August 13, 1974 enzyme-modi-fied cheese became an acceptable optional ingre-dient in processed cheese, processed cheese food,or processed cheese spread.36

1. Production of ̂ -Monoglycerides

Monoglycerides are used widely in the foodindustry as emulsifiers. They can be produced bythe base-catalyzed hydrolysis of triglycerides at ahigh temperature. This chemical process, how-ever, is not specific and a statistical distribution ofvarious glycerides (mono-, di-, and triglycerides)and glycerol are obtained. Enzyme-derivedmonoglycerides are formed using a method oflipase-catalyzed alcoholysis of triglycerides. These

TABLE 7Patents Pertaining to use of Lipolytic Enzymes in Food IngredientManufacture or Processing

Application

Modification of fat in milk

Use in Italian cheese

Use in American cheese

Enzyme-modified milk powder

Lipolyzed milkLipolyzed milk fat "buttery"flavor.

Lipolyzed milk fat "culturedcream" flavors

Lipolyzed milk fat "bluecheese" flavors

Lipolyzed milk fat "cheese-like" flavors

Use in yogurt

Country

U.S.U.S.U.S.U.S.

U.K.CanadaCanadaU.S.U.S.

U.S.U.S.JapanU.K.JapanCanadaU.S.

U.S.

U.S.U.S.U.S.U.K.U.S.Japan

Patent no.

1,966,4603,469,9332,531,3292,794,743

1,326,516911,248911,2503,650,7682,531,329

2,794,7432,638,4183,187/701,251,27272^15108912,9053,469,993

3,469,993

2,965,4923,100,1533,072,4881,326,5163,780,1823107/71

Year of issue

1930196919501957

19711971197119721950

1957195319701971197219711972

1969

196019631963197119731971

From Kilara, A., Process Biochem., 20(4), 35, 1985. With permission.

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monoglycerides possess good emulsification char-acteristics due to the P position of the fatty acid,as opposed to the a position in the chemicallyderived compound.38

2. Fatty Acids and Health

Polyunsaturated fatty acids have been thefocus of research activities for a number of yearsbecause of their potential health benefits. Veg-etable oils such as coconut oil are made up mostlyof medium-chain fatty acids. Corn and soybeanoil contain mostly long-chain polyunsaturated fattyacids, while palm oil has about equal amounts oflong-chain saturated and polyunsaturated fattyacids. Polyunsaturates in large amounts may in-crease cancer risk. Hydrogenation converts poly-unsaturated fats to saturated fatty acids or iso-meric trans forms. Both of these are believed toraise serum cholesterol levels and are thus unde-sirable with respect to adverse effect of high cho-lesterol levels to human health.39

Omega-3 fatty acids are esterified mainly atthe C2 position of glycerol in the triglycerides offish oils. The health benefits of omega-3 fattyacids, eicosapentaenoic acid and docosahexaenoicacid in fish oil, have been the subject of activeresearch in recent years. Enrichment for theseacids can be carried out using a method of lipase-catalyzed alcoholysis.38 For example, Menhadenoil (which contains about 25% omega-3 fatty ac-ids), when hydrolyzed by lipase and followed byseparation of the esters, cholesterol, and frac-tional crystallization, formed product with 60 to70% omega-3 fatty acids.

H. Fruit and Juice Processing

Fruit and juice processors use enzymes for avariety of benefits, which include: increased juiceyields, improved efficiency of juice filtration,improved juice stability for concentration, en-hanced juice clarity, reduced juice bitterness, andincreased rate of fruit dehydration.1 Enzymes usedmost often in juice processing are pectinase, cel-lulase, hemicellulase, amylase, and arabinase.Naringinase and limonase have been used to hy-

drolyze naringin and limonin, the bitter compoundsfound in grapefruit juice. Biomacerase may beused in the production of stable purees from awide variety of fruits and vegetables, such ascarrots, tomatoes potatoes, celery, and pears.Biopectinase ML is specifically formulated tohydrolyze pectin, cellulose, and hemicellulosemore extensively than traditional pectinase prod-ucts.40

Aspergillus, Rhizopus, and Trichoderma arethe chief fungi used as sources of enzymes toincrease juice yield and reduce juice viscosity.Structural polysaccharides that interfere with juiceextraction, filtration, clarification, and concentra-tion are broken down by pectinase, cellulase, andhemicellulase. Macerating enzymes are also usedto extract juice from citrus fruits and some tropi-cal fruits (such as pineapple) that have a greatdeal of juice trapped in the pulp. One example isthe treatment of papaya with two macerating en-zymes. This achieves a 12 to 30% increase injuice yield.1 It is now customary to treat applemash with enzymes prior to pressing. This servesto get rid of a clouding agent called araban, whichcomes from the cell walls. Araban consists of alinear chain of oc-l,5-arabinofuranosyl residueswith a-1-3 branches. Debranching arabinosidasesand endoarabinase are needed to break downaraban and prevent precipitation.41

III. POTENTIALLY NEW OR INCREASEDUSES OF ENZYMES IN THE FOODINDUSTRY

A. Inulinase

The importance of fructose because of itscomparative sweetness vs. sucrose has stimulateda search for alternative sources of fructose. Vari-ous plants of the compositae family, especiallythe Jerusalem artichoke or topinambour, containup to 16% inulin, the fructose polymer in whichfructofuranose units are joined by a (3-1,2 linkageas opposed to furosans, which have an a-2,6 gly-cosidic bond. Jerusalem artichokes can be grownas a high-yield crop. Inulinase, an enzyme pro-duced industrially from Aspergillus species, canalmost completely hydrolyze inulin (up to 99%)

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at pH 4.5 and 60°C.5 The enzyme has not been putinto use because of political decisions that haveprevented the financing for European farmers togrow these crops.8

B. Marine Food Quality Assessment

The application of enzyme methods to ma-rine food analysis provides an objective, inexpen-sive, and rapid approach to seafood quality. Ifthese methods are adapted to a visual basis, en-zyme analysis could be carried out anywhere,dockside or aboard ships. Enzyme analysis ofmarine foods can be used for quality assessmentat critical points where this type of determinationhas an impact for grading and purchasing.42

The compound hypoxanthine, a metabolicbyproduct of ATP breakdown, is used as a mea-sure of fish quality. Using the enzyme xanthineoxidase, which converts hypoxanthine to uric acid,followed by differential spectrophotometry in theUV region, fish freshness can be determined. The

K value is used in the determination of fish fresh-ness, where K is defined as:

[HXR] + [HX]K =

where HXR = inosine, Hx = hypoxanthine, IMP =inosine monophosphate, all degradation productsof ATP. If K = 1, then [IMP] = 0, thus denotingpoor-quality fish. A K value of 0.2 or lower de-notes high-quality fish.

Rapid visual enzyme methods were designedfor convenience aboard ship, dockside, or in afood-processing plant. A strip of absorbent paperis soaked in resazurin, xanthine oxidase, and abuffer. When the enzyme test strip is moistenedwith flounder extracts and allowed to react for 5min at room temperature, the color changes fromblue to pink, and the intensity is correlated with alaboratory colorimeter. Diamines developed afterthe hypoxanthine peak can also be used to deter-mine the onset of spoilage (Figure 4).42

HYPOXANTHINE URIC ACID

DIP

Xanthine Oxidase(Oxidized)

Xanthine Oxidase(Reduced)

(oxidized)DIP(reduced)

DIP = 2,6-dichlorophenol

FIGURE 4. The reduction of DIP by xanthine oxidase in the presence of hypoxanthine. (Adapted from Jahns, F. D.and Rand, A. G., in Ory, R. L and St. Angelo, A. J., Eds., Enzymes in Food and Beverage Processing, AmericanChemical Society, Washington, D.C., 1978, 266.)

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C. Baking Industry

Some of the functions of enzymes in bakedproducts include: flour quality improvement, re-tardation of staling, dough improvement, and moreefficiently machinability. Enzymes are also usedto enhance bread crust color, bleach flour, im-prove the quality of high fiber baked products,and reduce the phytate content in whole-grainformulations. Fungal and bacterial enzymes avail-able for use in bakery processing include a- andP-amylases, proteases, amyloglucosidases,pentosanase, glucanase, and phytase. The mostimportant of these are amylases and proteases.1

Bakers have added a surfactant to bread toretard staling. These compounds reduce the ten-dency for retrogradation by complexing with boththe amylose and amylopectin fractions of starch.A novel approach is the addition of oc-amylasedirectly to flour for the conversion of starch tomaltose for yeast fermentation and for retardingstaling. Addition of oc-amylase is believed to cleavea few bonds in the soluble region of the starch,leaving the insoluble regions separated by regionsthat can bend. Another theory in the retardation ofstaling is that the enzyme is thought to shorten thechain length of amylopectin from 19 to 21 units to12 to 15 units, which reduces its tendency toretrograde.1

Enzymes for the baking and milling indus-tries may be used to control dough properties andimprove the quality of the finished product. Theenzymes Milenzyme® fungal protease, Clarase®,HT-proteolytic, bromelain, and Tenase® act onthe raw material to enhance leavening action andmodify the protein structure, thus contributing toflavor development. Milenzyme fungal protease,derived from Aspergillus oryzae, is used in mak-ing yeast-raised breads. It hydrolyzes the interiorbonds of gluten and modifies the extensibilityproperties of dough.40

Doughs containing high levels of the wheatprotein gluten show improved machinability andbetter baked quality when formulated with pro-teases. Sodium bisulfite, a reducing agent, hadbeen used to weaken gluten by hydrolyzing thedisulfide linkages in the protein. However, thiscompound was found to destroy vitamin B (thia-mine). Instead of chemical agents, bacterial pro-

tease, an endoenzyme that weakens the glutenwithout affecting the nutritional quality of theflour, is now used. The enzyme acts on the innerpeptide bonds of gluten and results in reducedelasticity and improved extensibility of thedough.40 HT-proteolytic is a protease derived fromBacillus subtilis that is useful for conditioninghigh-protein flours in crackers, cookies, pizzacrust, and other specialty baked products. It soft-ens the dough for improved extensibility withreduced elasticity. The enzyme is effective at pHrange of 5.0 to 9.5 and up to 60°C.

Fungal proteases are added to bread formula-tions to improve loaf symmetry and uniformity,to improve grain and texture, and to provide asofter crumb. Fungal proteases contain both exo-and endoenzyme activities. Amino acids liberatedby the exoenzyme activity during hydrolysis ofgluten react with sugars in the Maillard reaction.This improves flavor and crust color, contributingto yeast growth and ability to form gas.1

Pentosanase incorporated into bread formulationsbreaks down the pentosans, yielding easier-to-handle doughs and finished products with a largerloaf volume.

D. Fat Degradation

Fat concentrations in excess of 0.025% havea detrimental effect on the whipping properties ofegg white, which normally contains about 0.04%fat. Fat in egg white may be eliminated usinglipase MAP 10, which is derived from Mucor andrecommended for use at the rate of 1 kg/45,000lb. Compared with pancreatic lipase, which iscurrently being used by many egg processors,MAP 10 has Kosher status and does not containprotease, which affects the egg flavor.40

E. Bacterial Detection and Elimination

Enzymes can now be used for the detection ofbacterial contamination in dairy, meats, fish, poul-try, and other food products. This is accomplishedby an instrument called the Catalasemeter. Itmeasures, within minutes, the amount of catalaseenzyme in a sample. This relative catalase value

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can be correlated with a specific level of bacteriaor abnormality in a culture.40

1. Antimicrobial Enzymes

Enzymes have been demonstrated to play animportant role in extending the shelf life of foodsby preventing the growth of spoilage organisms.This can be achieved by several methods:43 (1)functioning to deprive the problem organism(s)of a necessary nutrient; (2) production of reactionproducts harmless to humans or animals, but bac-teriostatic or bactericidal in nature; (3) destruc-tion of an outer membrane, cell wall, or cell mem-brane component, causing a change in permeabilityor a physical disruption of the cell wall, resultingin death; or (4) inactivation of an essential en-zyme by a "killer enzyme".

An example of an enzyme that deprives anorganism of a necessary nutrient is glucose oxi-dase-catalase, which, in the presence of excessglucose, depletes oxygen, preventing the growthof obligate aerobes. Oxidases that generate H2O2

in a system that does not contain catalase or per-oxidases are lethal, because of the residual H2O2

present and the depletion of oxygen. Xanthineoxidase, found in milk, may also generate toxicH2O2 in the absence of catalase. Xylitol is beingused in toothpaste as an anticariogenic ingredient.Xylitol is converted to xylitol-5-phosphate by theenzyme xylitol phosphorylase. This phosphatecannot be further metabolized by Streptococcusmutans, a cariogenic organism, thus killing theorganism.43

Enzymes that attack cell walls, causing open-ing up and wall permeability, can cause cell death.Examples include proteases, lysozyme, chitinases,peroxidase, various hydrolases, phenolases, andphosphatases. Crude trypsin, combined withlysozyme and lysolecithin, is reported to lyse E.coli and Shigella flexner.39

Cell wall degradation using bacteriolytic en-zymes is a novel method of coping with bacterialcontamination in food. Bacterial cell wall has asugar backbone consisting of iV-acetyl-muramicacid and N-acetyl glucosamine linked togetherthrough (3-1—>4 bonds. The sugar backbone iscross-linked by peptides to form a peptidoglycan

layer. Some bacteriolytic enzymes hydrolyze the|3-l->4 bond, while some hydrolyze the peptidebonds. A few new bacteriolytic enzymes havebeen developed as replacements for lysozymefound in egg white (N-acetyl-muramidase). Theeffectiveness of three bacteriolytic enzymes com-pared to that of lysozyme is presented in Table 8.Bacteriolytic enzyme SP417 produced byNocardiopsis dassonvillei lyses Gram-positive aswell as Gram-negative bacteria, including Sta-phylococcus aureus and Pseudomonas aeruginosa.Enzyme SP417 produced by Bacillus pabuli dif-fers from SP416 in that it lyses Gram-negativeorganisms, including Escherichia coli, Vibrioparahaemolyticus, and Salmonella arizona.44

Bacteriolytic enzyme SP455 produced bystrain N. dassonvillei is active on only a fewbacteria, in particular, P. aeruginosa. Bacteriolyticenzymes may be used in the food industry toextend shelf life of unprocessed or little-processedfoods. They may be used to control the process offermented food production, such as cheese orsalami, and act as a processing aid in releasingintracellular products from bacteria. Disinfectionof equipment, packaging material, and processwater can all implement the action of bacteri-olytic enzymes.44

"Killer enzymes" or "antienzyme enzymes"fall into two main groups: proteases and sulfhy-dryl oxidases for -SH enzymes. Other importantenzymes include those able to inactivate cytosolicaspartate aminotransferase. Mutastein, a proteinproduced by Aspergillus terreus, has been shownto be an effective inhibitor of cyclodextringlucosyltransferase and other oc-glucosyl-trans-ferases and to prevent dextran by Streptococcusmutans.43

F. Proteases

Acid proteases are available commercially toperform many, varied functions, such as degrada-tion of haze-forming proteins in fruit juices, hy-drolysis of gelatin, preparation of casein hydroly-sates, and the digestion of soya protein.

There has been an upsurge of interest in pro-teases from aquatic species because of their spe-cial properties. Proteases used in industry have

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TABLE 8Comparison of Bacteriolytic Enzymes

Activity3

SP416 SP417 SP455 LysozymeMicroorganism

Gram-positiveBacillus cereusBacillus subtilisCorynebacterium liquefaciensLactobacillus plantarumListeria inocuaMicrococcus kristinaeMicrococcus luteusMicrococcus sedentariusStaphylococcus aureusStreptococcus lactisStreptococcus faecalisStreptococcus faecium

Gram-negativeCampylobacter fetusEnterobacter aerogenesEscherichia coliPseudomonas aeruginosaSalmonella arizonaSerratia marcescensVibrio parahaemolyticus

a SP416 was dosed at 160 mg/l, SP417 at 8 mg/l, SP455 at 1600 mg/l, and pure lysozyme at 1000 mg/l. Thereactions were run at pH 7 for 20 min at 30°C. Enzyme activity (lysis) was measured as decrease in turbidityof the bacterial suspension at 660 nm. 0 indicates less than 10% lysis; +, 11 to 25% lysis; ++, 26 to 50%lysis; +++, more than 50% lysis; and nd, not determined.

From Neilsen, H. K., Food Techno!., 45(1), 102, 1991. With permission.

000

00000

00++++++++

+++

000

++

+nd00

nd0++++00++

000

++00nd00

nd000000

been mainly derived from plant, animal, and mi-crobial sources. Marine proteases have beenunderutilized for the following reasons:45 (1) rela-tively few studies have been carried out on ma-rine proteases; there is thus a lack of informationabout their potential; (2) fluctuations exist in sup-ply due to seasonal nature of source material; and(3) personnel are reluctant to work with fish offalbecause of the odor.

There is a large pool of diversified speciesadapted to a variety of habitat conditions in themarine environment. Genetic variations withinspecies as well as adaptation to different environ-mental conditions have resulted in differences inthe properties of proteases. These unique proper-ties can be applied in the food industry in manyareas. For example, trypsin has been isolated frommany species, including Greenland cod, Atlanticcod, and cunner fish. Compared with bovine

trypsin, the turnover number of fish trypsins isone order of magnitude higher for both amidaseand esterase reactions. Fish chymotrypsins werealso found to have a higher rate of reaction and tohydrolyze more peptide bonds in various proteinsubstrates than mammalian trypsin from beefpancreas.45

Pepsins from cold-water fish exhibit a lowArrhenius activation energy, low-temperatureoptimum, and low thermal stability compared withwarm-habitat and warm-blooded animals. Fishpepsins are also resistant to autolysis at low pH.These pepsins are inactivated at relatively mildheat treatment, a useful property in cheese manu-facture after the milk clotting reaction is com-plete.45

Fish proteases have traditionally been appliedin the seafood industry to reduce viscosity ofstickwater in the fish meal industry. They are

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used for preparation of fish protein hydrolysatesfor food or animal feed and fermentation pro-cesses, depending on endogenous proteases. Re-cent applications include the removal of skin andscales, the hydrolysis of membranes and othersupportive tissue that surrounds roe, roe sacks,and other tissues, and recovery of pigment andflavor extracts.45

G. Artificial Sweetener: Aspartame

Aspartame is a dipeptide of the methyl esterL-aspartic acid linked to L-phenylalanine. Aspar-tame is marketed under the brandname Nutrasweet(Nutrasweet, Inc., Mount Prospect, IL). Aspar-tame may be used in the body similarly to anyother protein, but its caloric value is insignificant.Individuals suffering from the condition phenyl-ketonuria (absence of the enzyme phenylalanine4-monooxygenase) should avoid aspartame andproducts that include it as an ingredient.46

The key step in aspartame production is theformation of the peptide bond between the a-carboxyl group of L-aspartic acid and the aminogroup of L-PheOMe. Aspartame can be chemi-cally produced by the reaction of carbobenzoxy-L-aspartyl anhydride with L-PheOMe, with thereaction taking place at the ot-carbonyl (Figure 5).There is a 7% byproduct from the reaction withthe P-carbonyl. Phenylserine is used as the build-ing block in the enzymatic phenylalanine-sparingroute to aspartame production. Enzymatic synthe-sis of aspartame starting with phenylserine me-thyl ester is outlined in Gross.38 The metallopro-tease used is thermolysin, which catalyzes thedipeptide bond formation in organic solvent suchas ethyl acetate with a 93% dipeptide yield.38

Then oc-aspartame is obtained in a single step bycatalytic hydrogenofysis with a yield of 85%.

Enzymatic synthesis of aspartame has theadvantage of regiospecificity where no protectionof the P-carbonyl of oc-L-aspartic acid is neces-sary. An inexpensive racemic mixture of isomersof phenylserine methyl ester can be used as start-ing material because the reaction is highly ste-reospecific. Only the oc-erythro isomer ofphenylserine methyl ester reacts with aspartic acid(Figure 5).38 In the chemical synthesis of aspar-tame the reaction is first complicated by blocking

of the amino group of carbobenzoxy-L-aspartylanhydride. Reaction of this anhydride withL-PheOMe is not specific because the amino groupof L-PheOMe can react with either the a- orP-carbonyl group of the anhydride to form twostereoisomers. It is thus necessary to have a finalpurification step to remove the P-aspartame (-7%)present, thus giving low yields and increasingproduction costs.

IV. OTHER POTENTIAL ENZYMEAPPLICATIONS

A. Food Quality

Enzymes are useful in determining several ofthe quality indices of foods. Some of these arelisted in Table 9. Substrate kits and simple assaytechniques need to be developed for routine ap-plication of these indices in the quality control offoods. In order to change the solubility and func-tional properties of proteins, enzymes can be usedspecifically to modify the side-chain amino acidresidues in proteins or to hydrolyze peptide bonds.Proteolytic enzymes can form bitter peptides,which is one problem experienced in the cheeseindustry. Enzymes may be used in the removal oftoxic and antinutritive components. Plants suchas cassava, lima beans, sorghum, linseed, andkernels of almonds have high levels of cyano-genic glycosides. HCN is liberated from the cya-nogenic glycosides by the action of the thio-glycosidase, this compound being able to reactwith cytochrome oxidase, a key respiratory en-zyme. Proper cooking would distill the HCN;however, more research is needed on the use ofthioglycosidases in reducing toxicity.

Oligosaccharides (e.g., raffinose, stachyose,and verbascose) found in beans cannot be hydro-lyzed in the human small intestines because of theabsence of cc-galactosidase. Thus, the use of beansin human diet is limited by the development offlatulence caused by the presence of these undi-gested oligosaccharides. There is a need to de-velop a-galactosidase from microbial sources toaid in digestion of bean oligosaccharides, perhapsby pretreatment of the beans.47

Nucleic acid content in single-cell proteincontinues to be a major deterrent in its use for

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NH2

°<Aspartame

and B-Aspartame HCL

(3-Z-Aspartame

L-phenylalanine methylester

L-Aspartjc acid

o

FIGURE 5. Chemical synthesis of aspartame. Z = CO2CH2Ph. (Adapted from Gross, A., Food Techno]., 45(1), 96,1991.)

human consumption. The nucleic acid is knownto crystallize out as uric acid in the joints causingthe condition gout. Nucleases need to be devel-oped to find a solution to this problem.

B. Protein Supplementation

Apart from removal of toxic substances andimprovement of functional properties, nutritionalquality can be improved by supplementation withlimiting amino acids. Japan has been one of theworld leaders in amino acid production since 1969.

Systems have been developed using aminoacylaseimmobilized to DEAE-cellulose on a commercialscale to resolve D- and L-mixtures of methionine,valine, tryptophan, and phenylalanine.4647

The protein in the human body consists of L-amino acids that are continuously taken up withthe food. Pure L-amino acids infusion solutionsare used in artificial feeding to supply the neces-sary protein structural elements. These are ob-tained in special bioreactors with the aid of en-zymes. By a method of enzyme immobilization,the catalyst can be retained in a reaction vesselwith continuous flow through. The adult human

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TABLE 9Indices of Food Quality

Index

Proper heat treatment

Freezing and thawing

Bacterial contamination

Maturity

Freshness

Sprouting

Color

Significant enzyme

PeroxidaseAlkaline phosphatasep-Acetylglucosaminidase

Malic enzymeGlutamate oxaloacetatetransaminase

Acid phosphataseCatalase

Sucrose synthetasePectinase

LysolecithinaseXanthine oxidase

AmylasePeroxidase

Polyphenol oxidaseSuccinic dehydrogenase

Food product

Fruits and vegetablesMilk, dairy products, hamEggs

OystersMeat

Meat, eggsBeans

PotatoesPears

FishHypoxanthine content of fish

FlourWheat

Coffee, wheatMeat

Adapted from Whitaker, J. R., Enzymes. The Interface between Technology and Economics, Danehy, J. P. andWolnak, B., Eds., Marcel Dekker, New York, 1980, 53.

requires about 60 g of protein per day. L-Aminoacids are used as food supplements and in infu-sion solutions on a large scale.48 Stereoselectivityis one of the important characteristics of enzymes.They catalyze reactions with only one enantio-meric and diastereomeric pair of chiral compounds,although some enzymes such as racemases areexceptional in that respect. Enzymatic processeshave been developed for optically active aminoacids that are important as nutrients and startingmaterials in the pharmaceutical industry.

C. Oxidoreductases

Oxidoreductases are being employed as cata-lysts in food systems to convert cholesterol to thenontoxic coprostanol. If this process is fully de-veloped, it will serve to replace the current ap-proach of cholesterol extraction by steam distilla-tion and supercritical fluid extraction.

0. Invertase

Several enzymes have found application inthe food industry, although on a small scale. Onesuch example is invertase. Invertase is used formaking candies with a soft center. The enzyme isadded to sucrose candy that is covered with choco-late. With time, the sucrose is partially hydro-lyzed to glucose and fructose. This makes thecandy soft.8

V. CONCLUSIONS

Change in availability of energy and protec-tion of the environment may make some enzy-matic processes more attractive. Active researchis being carried out to make improvements indairy products. The following are being devel-oped: Streptococcus lacti with significantly im-proved resistance to bacteriophage; lactic acid

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bacteria with improved ability to produce lacticacid, especially under adverse conditions of tem-perature and salt level; single-strain starters foryogurt, Italian cheese, and buttermilk, uniqueyogurt cultures that produce biopolymers for tex-ture and as sweeteners; fermentations based onsource of microbial calf rennet; and industriallyimportant recombinant DNA products from thelactic acid bacteria.40

Recombinant DNA techniques have aug-mented the potential for new developments in thefood enzymology area. Some specific examplescited point out these developments:40 (1) thermo-stable bacterial a-amylase suited for maltose syrupproduction is being produced by Novo; (2) re-combinant chymosin is being investigated, amongothers, by Genencor, Genex, Dairyland FoodLaboratories, and companies in Japan; (3) accel-erated cheese ripening systems are being per-fected by Miles under the commercial name NaturAge; (4) heat lability of microbial rennets is beingimproved to approach the heat lability of calfchymosin; (5) glucoamylase expressed by recom-binant DNA is being studied by CPC Interna-tional; (6) Cetus Corporation is trying to cloneglucoamylo-cellulase and oxidase to better break-down cellulose and glucose.

The aim now is to produce enzymes that offertechnological advantages over currently availableenzymes, but do not appreciably alter the sensoryattributes of the products. Processes using thenew generation of enzymes will realize cost sav-ings due to the engineered technologically desir-able properties. At this stage there is a generalunderstanding of how enzymes work at the mo-lecular level. It is hoped that DNA technologywill lead to the development of enzymes that canperform what is now considered "impossible",for example, manufacture of Cheddar cheese fromsoyprotein and production of beer from any car-bohydrate source within 1 or 2 days.

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2. Simpson, B. K. and Haard, N. F., Cold-adaptedenzymes from fish, in Food Biotechnology, Knorr, D.,Ed., Marcell Dekker, New York, 1987, 495.

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