Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Mucor rennin, is now being used in a significant

proportion of the dairy industry worldwide, and a

recombinant chymosin (the main rennet enzyme)

will be produced on a large scale. The application of

genetic engineering, particularly recombinant DNA

technology, is in fact having a major impact on the

development of new sources of industrial enzymes for

food industry, even if in western countries there is an

increasing tendency among consumers not to approve

the application of genetically engineered organisms to

food and food ingredients.

0005 Selected examples of enzymes used in food process-

ing are mentioned below, and some of these will be

discussed in detail:

0006 Meats are tenderized using the enzymes papain,

ficin, and bromelain.

0007 Invertase is added to the fondant centers of choc-

olate-covered cherries. This enzyme hydrolyzes

some sucrose, thereby decreasing the number of

sugar crystals in the fondant, and softening the

chocolate center.

0008 Glucose isomerase is used to convert glucose to

the sweeter fructose. This process is carried on

commercially with an immobilized enzyme system.

0009Pectinases are used commercially to break down

pectin molecules in certain fruit juices such as apple

juice in order to avoid a hazy appearance; clear,

sparkling juice is the final product.

0010Rennet is the crude extract of the protease rennin.

It is widely used to clot milk in the making of

cheese.

0011An enzyme system involving glucose oxidase and

catalase has been used to remove traces of oxygen

in tightly packaged food products. This system is

also used to remove small amounts of glucose from

white eggs before drying them. Browning may

occur during storage if white eggs contain even

small quantities of glucose.

Use in Tenderizing Meats

The Structure of Meat

0012It has been established beyond doubt that the consist-

ency of meat is governed by two principal factors: the

quantity and the properties of the muscle tissue

collagen and the mechanically contractile state of

the muscle. (See Meat: Structure.)

0013Collagen fiber consists of triple-helix tropocollagen

molecules that are linked by covalent bonds, providing

a structure of high mechanical tenacity. In muscle

tissue collagen both thermolabile and thermostable

bonds have been identified. The thermolabile linkages

are prevalent in the collagen of young animals, and are

readily broken when the meat is cooked. In older

animals the thermostable bonds predominate, with

the result that the meat has harder collagen fibers

when cooked, i.e., tougher meat. Both the amount of

collagen and the type of bond are thus important in

determining meat consistency. A further important

factor is the state of contraction of the muscle protein.

0014Once the animals have been slaughtered, chemical

bonds form between the thick and the thin filaments

tbl0001 Table 1 Sources of major industrial enzymes

Enzyme Process Source

Proteases – alkaline Detergents Bacterial

Proteases – acid Cheese-making Animal, fungal

Proteases – neutral Various Bacterial

Amylases Starch industry Bacterial, fungal

Isomerases High-fructose syrups Fungal

Pectinases Wine, beer, condiments Fungal

Lipases Various Bacterial, fungal,

animal

From Cowan DA (1994) Industrial enzymes. In: Moses V and Cope RE (eds)

Biotechnology, pp. 311–340. UK: Harwood Academic Publisher with

permission.

tbl0002 Table 2 Various sources of enzymes

General source Specific source Advantage/disadvantages Example

Animals Human blood No longer a viable source. Available in vast quantities Pancreatic lipase

Bovine, porcine, and ovine tissues Enzymes are often of low stability Rennin

Purification often complex Trypsin

Plants Leaves, fruit Purification can be a problem Papain

Microorganism Bacterial, algal, fungal, and yeast Large-scale production Protease

Technology advanced Amylase

Purification often simple Lipase

High yields

Wide variety of unusual enzymes available

Limited range of microorganism approved for use in

food industry

From Cowan DA (1994) Industrial enzymes. In: Moses V and Cope RE (eds) Biotechnology, pp. 311–340. UK: Harwood Academic Publisher with

permission.

ENZYMES/Uses in Food Processing 2127

that make up the mobile part of the muscle. It is these

bonds that make the muscle hard and reduce its

stretching properties. Hardness is most noticeable

in highly contracted muscle, where a compact and

inelastic structure forms.

0015 In order to make it tender, meat is subjected to

‘ripening’ (‘hanging’) once rigor mortis (a stiffness

of the muscle resulting from glycolysis) ceases. The

process is widely exploited to make the meat not only

tender, but also tasty and more digestible. This pro-

cess takes place when the carcass is cut into sides and

quarters. Hanging entails keeping the sides for several

days in controlled temperature and humidity cham-

bers. The mode of action of the tenderizing process

during this ripening and conditioning is not entirely

understood. It is suggested that during this period

there is a partial dissolution of the Z-lines, whose

function is to anchor the thin filaments (myofibrils)

of the muscle tissue. During such a process, dissoci-

ation of the thin filaments from the Z-lines leaves the

entire muscle structure more stretchable and tender.

However, the partial degradation of the Z-lines is

due to the action of a specific proteolytic enzyme,

which has been given the name of calcium-activated

sarcoplasmatic factor (CASF). (See Meat: Structure.)

Artificial Tenderizing

0016 The normal tenderization process thus seems to

involve one or more specific proteases present in the

muscle cells; and it is reasonable to expect that the

process can be reproduced artificially with the use

of proteases. This practice was already in use among

the pre-Columbian inhabitants of the Americas.

According to Hernan Corte

s, the conquistador of

Mexico, these people would wrap up pieces of meat

in papaya leaves. The papain (a proteolytic enzyme)

naturally present in the leaves acted on the structure

of the meat, making it tender.

0017 Artifical tenderization is achieved by resort to vari-

ous sulfhydryl proteases of vegetable origin, such as

papain (from the papaya plant), bromelain (from

pineapple), and ficin (from the fig tree) or microbial

proteases. Factors such as pH, the concentration of

the enzyme, and temperature also influence the

enzyme action and the degree of tenderization.

0018 Since the enzymes employed in this field are in-

active at low temperature (0–10



C) but increase in

activity as the temperature rises (65–80



C), becom-

ing inactive again at 82



C, the tenderization process,

for all practical purposes, begins and ends with

cooking. The problem, then, is to cause the enzyme

to act in such a way as to produce a controlled and

homogeneous tenderizing, since excessive action

during the cooking process would produce a formless

lump of tissues.

0019Tenderization by enzymes is achieved through the

action of the enzyme on the connective tissue (colla-

gen and elastin) of the meat, and cannot be compared,

in terms of its mechanism, with the natural tenderiz-

ing process described earlier.

0020Enzymes come in powder form, for dusting on the

surface of the meat, or in liquid form; in this case the

meat is either immersed in solutions of known con-

centrations or (a more effective method) by injecting

the enzyme into the blood stream a short time before

slaughtering. This is known as pretenderizing. The

method in this case relies on the fact that (1) the

animal’s vascular system offers an excellent means

of distribution among the body tissue; (2) the heart

is an efficient pump; (3) blood acts as a diluent and

insures uniform distribution of the enzyme; and (4)

the enzymes used are safe food ingredients.

0021The time elapsed between pretenderizing and

slaughtering – from 2 to 20 min – allows adequate

distribution in the tissues. The amount of solution

will vary with the weight, age, and sex of the animal.

The enzyme does not act immediately upon introduc-

tion into the animal, since the temperature is below

optimum, but begins to do so when the meat is

cooked.

0022Apart from the difficulty of achieving homoge-

neous distribution throughout the tissues, artificial

tenderizing with enzymes is further limited by the

fact that the enzymes used are of low specificity, in

that they act on both connective tissue (collagen and

elastin) and on contractile (myofibrillary, muscle

structure) protein. These two aspects make it difficult

to control the tenderization process.

0023To overcome these constraints, collagenases are

now starting to be used. Crude collagenases are highly

specific proteases which act on native or denaturated

collagen but not on muscle tissue, so that the

dissolution of the actomyosin and Z-line proteins is

restricted, while the connective tissue network is

weakened. Collagenases are obtained from aerobic

bacteria (Achromobacter spp.) and Clostridium

strains. The outlook is promising for their application

in this field.

Use in Hydrolyzing Starch

0024The conversion of native starch, which is the basic

constituent of many vegetable products, such as

maize, and potatoes, to soluble sugars is the most

important application of enzymes in food industry;

and in fact the process has almost entirely supplanted

that of acid hydrolysis for glucose production. (See

Starch: Structure, Properties, and Determination.)

0025In the last 30 years, as new enzymes have become

available, starch hydrolysis technology has been

2128 ENZYMES/Uses in Food Processing

transformed. There has been a big move away from

acid and today the vast majority of starch hydrolysis

is performed using enzymes. Furthermore, in the

1970s, an enzyme technique made possible the pro-

duction of syrup sweeter than glucose – high-fructose

syrup. The production of this syrup has significantly

boosted the growth of the starch industry in certain

countries.

0026 Depending on the enzymes used, syrups with dif-

ferent compositions and physical properties can be

obtained from the starch (Table 3). The dextrose

equivalent (DE) value is used as an indication of the

degree of hydrolysis of a syrup. The DE value of starch

is zero, and that of dextrose is 100. Syrup with DE

values from 35 to 43 are still widely produced by acid

hydrolysis, despite the drawbacks mentioned here.

However, due to the formation of byproducts, it is

difficult to produce low- and high-DE syrups of high

quality in this way.

0027 There are essentially five groups of enzymes in-

volved in the hydrolysis of starch: the endo-amylases

(Enzyme Commission (EC) 3.2.1.1) act primarily on

the a-1,4 linkages, and products of reactions are

oligosaccharides of varying chain lengths.

0028 The exo-amylases (amyloglucosidases or gluco-

amylases; EC 3.2.1.3) act on the same substrates as

endoamylases, but they are also able slowly to cleave

a-1,6 linkages. They act externally on substrate

bonds from the nonreducing end and produce low-

molecular-weight products.

0029The debranching enzymes (such as pullulanase, EC

3.2.1.41, and isoamylase, EC 3.2.1.68) act exclu-

sively on the a-1,6 linkages.

0030A fourth group, the isomerases (EC 5.3.1.5), act on

glucose syrups to convert them to fructose syrup.

0031Finally, cyclodextrin glycosyltransferase (CGTase,

EC 2.4.1.19) is an enzyme capable of hydrolysing

starch to a series of nonreducing cyclomaltooligo-

saccharides referred to as cyclodextrins.

0032There are three basic steps in enzymatic starch con-

version to glucose: gelatinization, liquefaction, and

saccharification. In the first step, the raw starch is

heated to above 40



C to disrupt the starch granules

and expose the polymer to enzymatic action (Figure 3).

In its native granular form, starch resists attack from

hydrolytic enzymes, so that the application of these

(enzyme hydrolysis) calls for acid or enzyme pretreat-

ment in order to bring about depolymerization and

consequent reduction in the viscosity typical of gelat-

inized starch. The starch slurry (pH 6.5) is gelatinized

by cooking at high temperature with a-amylase. The

gelatinized starch is then liquefied by thermostable

a-amylase to a soluble dextrin hydrolysate with a DE

of 5–15. After liquefaction, the pH of slurry is adjusted

tbl0003 Table 3 Classification of enzymes involved in the commercial production of starch syrups, maltodextrins, and cyclodextrins

Type Common name Source Substrate specificity Optimum

pH Temperature (



Endoamylase Bacterial amylase Acillus subtilis a-1,4-Glycosyl 6.0 65–70

Bacillus licheniformis a-1,4-Glycosyl 5.0–7.0 90

Fungal a-amylase Aspergillus oryzae a-1,4-Glycosyl 4.5 50–60

Exoamylase Amyloglucosidase A. niger a-1,4-Glycosyl 4.0–5.0 60

a-1,6-Glycosyl

Bacterial b-amylase Bacillus spp. a-1,4-Glycosyl 5.0 55–60

Clostridium spp. a-1,4-Glycosyl 5.5–6.0 75–85

a-1,6-amylase Pullulanase Klebsiella aerogenes a-1,6-Maltotryosyl 5.0 60

Isoamylase Pseudomonas spp. a-1,6-Heptasacch 4.0 50–55

Exo-a-amylases Exomaltotriohydrolase Streptomyces griseus a-1,4-Glycosyl

B. subtilis a-1,4-Glycosyl

Exomaltotetrahydrolase B. cir culans a-1,4-Glycosyl

Pseudomonas stutzeri a-1,4-Glycosyl

Exomaltopentahydrolase Pseudomonas spp. a-1,4-Glycosyl

Exomaltoexahydrolase B. subtilis a-1,4-Glycosyl

B. cir culan s a-1,4-Glycosyl

Isomerase Glucose isomerase B. cir c ulans Aldo/keto pentose 8.2 65

Aldo/keto hexose

Glucanotransferase Cyclodextrinase B. macerans a-1,4-Glycosyl 5.0 50

Thermoanaerobacter a-1,4-Glycosyl 4.5 90

Endo-/exocellulase Fungal cellulase Trichoderma reesei b-1,4-Glycosyl

Endoprotease Bacterial protease B. lic heniformis 6.5 90

B. subtilis 6.5 65.70

Data from Guzman-Maldonado H and Paredes-Lopez O (1995) Amylolytic enzymes and products derived from starch: a review. Critical Reviews in Food

Science and Nutrition 35: 373–403.

ENZYMES/Uses in Food Processing 2129

to 4.5 and the temperature lowered to 60



C. Amylo-

glucosidase is now added and the saccharification re-

action is allowed to continue for 48–96 h in a stirred

tank until the maximum level of glucose is reached.

0033 Replacing soluble amyloglucosidase with its immo-

bilized form allows for using the same enzyme batch

several times in order to shorten the hydrolysis period

and to apply a continuous process. However, the use of

immobilized amyloglucosidase is less efficient in the

production of glucose syrups than the soluble enzyme,

giving a maximum DE value of 96% instead of 98%.

The reason for this lower maximum DE is the increased

formation of isomaltose and the decreased hydrolysis

of the high-molecular-weight oligosaccharides.

0034 The carbohydrate composition of typical glucose

syrup is as follows:

0035 94–98% glucose

0036 1–3% maltose

00370.3–0.5% maltotriose

00381–2% higher saccharides.

It is possible, in industrial processing, to raise the

sweetening power of glucose syrup by converting

part of the glucose to fructose by isomerization with

immobilized glucose isomerase obtained from a

selected strain of Bacillus coagulans. This catalyzes

the conversion of the glucose syrup in a fructose syrup

containing at least 42% fructose.

0039Finally, it is worth mentioning cyclodextrins, which

have been studied for nearly a century but not utilized

for food applications until the 1990s because they

were too expensive and there was insufficient infor-

mation to declare them nontoxic. Now, their use is

permitted in a variety of food applications.

0040In a so-called nonsolvent process a reactor is filled

with starch and the selected enzyme under sterile

conditions (because no chemicals are present to pre-

vent microorganism growth); after the completion of

the reaction, heat or acid inactivates the CGTase, and

water is evaporated.

0041Cyclodextrins can stabilize volatile materials, such

as flavors and spices, and can also stabilize emulsions

of fats and oils, shielding them from oxidation and

preventing rancidity. They can be used to debitter

citrus juices, and can be widely used in the solubiliza-

tion of organic compounds. Their market is still

limited, because of their cost, but a novel production

of cyclodextrins in transgenic potatoes has been

developed at lower production costs.

Use in Baking

0042The use of wheat and other cereals as a source of flour

for baking (particularly in the production of bread,

probably the major staple food item in the western

world) provides a demonstration of a natural enzym-

atic process which can be artificially modified to

optimize product quality. Amylases are a natural con-

stituent of flour. The levels of amylase activity in flour

are strongly influenced by the climatic conditions in

which the grain was grown. A high amylase content,

the result of a moist climate, results in high dextrin

production during baking. The resulting dough has

low water retention and tends to be ‘sticky,’ while the

loaf is crumbly and dry. A dry environment yields

flour with a low amylase content. As a consequence,

the dextrin yield is low, fermentative gas production

is poor and loaf size is small. (See Bread: Breadmak-

ing Processes.)

0043Normal flour, milled from sound wheat, contains

significant amounts of b-amylase, but little or no

a-amylase. b-Amylase can produce some maltose to

aid fermentation without the presence of a-amylase,

Starch suspension

Gelatinization

(30−40% w/v; pH 6.5;

; α-amylase)

(110−140  C; 5−10 min)

Liquefaction

DE < 15

DE > 96

(α-amylase; 90−95  C)

Saccharification

(amyloglucosidase;

pullulanase; pH 4.5; 55−60 C,

48−96h)

Filtration and carbon

purification

Ion-exchanger

Evaporation

Glucose syrup

Steam

fig0003 Figure 3 Production of glucose syrups. DE, dextrose equiva-

lent.

2130 ENZYMES/Uses in Food Processing

but the amount produced is relatively small. The

supplementation of dough with a-amylase affects

the functional properties of the dough and may deter-

mine characteristics that are critical for automated

manufacturing processes. Largely for this reason,

a-amylase, sometimes in conjunction with protease,

is commonly used by the baking industry.

0044 Malted barley has traditionally served as the pri-

mary source of added a-amylase. This was the prac-

tice years ago when bread formulations were quite

lean and contained relatively little sugar. Today, the

primary reason for adding amylases to baked prod-

ucts is to improve the processing conditions as well as

the overall quality of the baked product. Although

barley malt is still used, supplementation with fungal

and, to a lesser degree, bacterial amylases has become

more common.

0045 Cereal amylases remain active until the tempera-

ture reaches about 70



C. They can cause some

additional starch hydrolysis after the starch has gel-

atinized. Thus, cereal amylases, whether added to the

flour (as a supplement) or native (due to sprouting),

can produce excessive hydrolysis in high-moisture

bread products if too much is present in the formu-

lation.

0046 Bacterial amylases (from Bacillus species) are gen-

erally the most heat-stable (> 100



C); some remain

active all the way through the bread baking cycle.

They must be considered to be most problematic in

bread production since they can continue to hydro-

lyze the starch throughout the baking process. In fact,

some bacterial amylases are capable of continuing

working even after baking and packaging and while

sitting on the grocery shelf. Excessive starch hydroly-

sis results in poor loaf characteristics, including low

loaf volume and dense, gummy crumb texture. The

heat stability of bacterial a-amylase is of less concern

in products such as cookies and crackers, which have

a higher baking temperature and contain little water.

0047 Fungal a-amylases, usually from Aspergillus ory-

zae, A. niger, A. awamori, or species of Rhizopus,is

normally used to supplement the amylolytic activity

in flour. Enzymes from these sources can raise the

levels of fermentable monosaccharides and disacchar-

ides of dough from a native level of 0.5% to concen-

trations that promote yeast growth. The sustained

release of glucose and maltose by added fungal and

endogenous enzymes provides the nutrients essential

for yeast metabolism and gas production during

panary fermentation. The A. oryzae a-amylase is

sometimes favored for baking applications since this

fungal enzyme is heat-labile at 60–70



C and does not

survive the baking process. Its thermolability prevents

enzymatic action on the gelatinized starch in the fin-

ished loaf, which would cause a soft or sticky crumb.

0048Even proteolytic action on the gluten component is

vital to the quality of the product. The protein com-

ponent makes up a relatively small percentage of the

flour, compared to starch, but it is responsible for the

formation of gluten, the unique viscoelastic protein

network peculiar to wheat. Because gluten protein is

primarily responsible for the mixing and sheeting

behavior of dough, it is an obvious candidate for

enzymatic modification by protease. In particular,

supplementation with proteases helps to break down

the gluten protein so that the dough is softer and more

extensible. Since cereals usually have low proteinase

levels, supplementation with fungal (A. oryzae) pro-

teinase is routine. The more thermostable bacterial

proteases are generally used only in biscuit and pizza

dough where the higher cooking temperature rapidly

inactivates the enzymes and prevents overproteolysis

and stickiness. While most commercially available

proteases contain relatively nonspecific endo- and

exoproteolytic activity, the units of activity will have

been determined on nongluten substrate protein. This

can make it difficult to choose the more efficient

enzyme to hydrolyze the gluten protein in a particular

process. The ability of any protease to hydrolyze the

gluten effectively must be determined by experimen-

tation in an appropriate formulation and processing

scheme. Because the majority of the effects desired on

wheat doughs involve altering the viscosity or rhe-

ology of the dough, an endoprotease is more effective

than an exoprotease. Furthermore, protease treat-

ment improves crumb characteristics, and the im-

proved flow properties of the dough contribute to

better, more uniform product shape.

0049Enzymes in the group of disulfide reductase/disul-

fide isomerase are being investigated as possible

contributors to controlled and effective gluten modi-

fication.

0050Finally, it was seen that, in bread making, treat-

ments with fungal (or bacterial) enzymes lower the

viscosity of bread dough, improving the ease of ma-

nipulation by manual workers or machines. Further-

more, favorable effects on taste and crust properties

are observed, and the storage characteristic of breads

change, giving a product with a softer, more com-

pressible crumb that firms more slowly and keeps

longer.

Use in Juice Clarifying

The Fruit Juice Production Process

0051With certain fruit juices, an initial phase consists of

the transformation of plant tissue into a semifluid

system made up of cells, fragment of cell walls, and

cellular liquid, and a second phase where the juices

ENZYMES/Uses in Food Processing 2131

are extracted by mechanical separation (pressing,

straining, centrifugation).

0052 Once extracted, the juice can be clarified further.

Where problems arise in the production process,

especially where demand is for limpid (clear) juices,

they are for the most part due to the presence of cell

wall material, of variable composition, during the

ripening process – cellulose, hemicellulose and, at a

high degree of esterification, pectin (polymethylgalac-

turonic acid). In unripe fruit, pectin is present in an

insoluble form whereas, on ripening, it is in part

broken down into a more soluble form and the fruit

becomes soft.

0053 As a result of this partial solubility, some of the

pectin passes into the fruit juice, which becomes

viscous and hard to separate from the pulp. This ex-

plains the low yield from extraction of the juice and of

its soluble components, and the fact that juice so

extracted is difficult to filter and clarify.

Enzymes in Fruit Juice Clarification

0054 The use of enzymes (Table 4) to overcome the diffi-

culties described is becoming more widespread today

with a twofold aim (Figure 4):

0055 In the treatment of the ground pulp – to raise

the juice yield, to improve the extraction rate for

certain flavor and color components, and to obtain

a partial or total liquefaction of the tissue (for

homogenized foods, nectars, juice, and pulp con-

centrates);

0056 In the treatment of the juice – to reduce viscosity

and facilitate concentration, to clarify in order to

obtain a limpid product; and to increase the speed

of filtration and thus prevent the formation of pre-

cipitates in the juice.

Commercial pectolytic enzymes, commonly referred

to as pectinases, are a mixture of pectin esterase (EC

3.1.1.11), which triggers the deesterification of the

pectin to pectic acid and methanol; polygalacturonase

(EC 3.2.1.15), which hydrolyzes the internal bond of

pectic acid; and pectin lyase (EC 4.2.2.10), that splits

the glycosidic bonds or either pectate or pectin. These

enzymes are chiefly produced from molds of the

Aspergillus genus. They are used to clarify apple and

grape juices and wine. All these preparations contain

traces of other enzymes as well, such as cellulase,

amylase, protease, and xylanase: when added to the

product they bring about the hydrolysis of the soluble

pectin and the elimination of its colloidal properties.

The process is accompanied by the flocculation and

then the removal by filtering or centrifugating of any

particles so formed. Traditionally, the industrial util-

ization of pectic enzymes in fruit juice processing has

been conducted in conventional batch reactors using

soluble enzymes. Unfortunately, after each cycle of

operations the enzymes cannot be recovered for fur-

ther use, and are inevitably present in the final prod-

uct, altering organoleptic properties. In this context,

the immobilization of pectolytic enzymes has proven

to be very advantageous for continuous processing.

0057Another aspect of using enzymes in fruit juice pro-

cessing is wine-making; the ideal enzyme prepar-

ations for wine-making are different from those for

fruit juice processing. In fruit juice processing the

enzymes are inactivated very shortly after they have

done their job, for example by pasteurization. In wine-

making no such heat treatment is imparted. The

enzymes therefore maintain their activity over

a longer period, and side activities may adversely in-

fluence wine quality during storage. During wine-

making, enzymatic reactions begin during the ripening

of the grapes and continue through the harvest, alco-

holic and malo-lactic fermentations, clarification, and

even after bottling. The wine-makers still have the

power to influence a few of the reactions with the

tools of pectinolytic and glycosidic enzymes. These

speed up the natural process of wine-making, make

the fullest use of facilities and equipment, and improve

the quality of wine. Pectic enzymes (from fungi, typic-

ally A. niger, Penicillium notatum or Botrytis cinerea)

are used to reduce haze or gelling of grape juice at

various stages of the wine-making process. A b-gluca-

nase prepared from a selected strain of Trichoderma is

now being used in wine-making as an adjuvant for

filtering and clarifying purposes of wines made from

grapes attacked from the fungus B. cinerea. In fact, the

Botrytis fungus produces b-glucans (polymers of glu-

cose with a high molecular weight), which pass into

the wine. These large molecules hinder clarification

and rapidly clog filters.

0058Finally, an improvement of our knowledge of grape

composition and of Saccharomyces cerevisiae would

allow the creation of a ‘superyeast strain’ by genetic

manipulation to depectinize the grape must, to

extract and release specific components such as

color (especially in the case of red wine) or aromas,

or to make the malo-lactic fermentation by heterol-

ogous cloning of enzymes from other microorganism.

Use in Cheese Production

Milk Clotting

0059The entire cheese-making process is based on milk

clotting. Clotting can be brought about by natural

or induced acidification to pH 4.6 (the isoelectric

point for casein) or by enzyme action (Figure 5).

The preparation most frequently used for this pur-

pose is rennet, a crude proteolytic extract obtained

2132 ENZYMES/Uses in Food Processing

from the fourth stomachs of calves, piglets, lambs,

and kids and containing 85–95% chymosin and

10–15% pepsin.

0060 In milk, primary soluble proteins are the whey

proteins a-lactalbumin and b-lactoglobulin. The insol-

uble proteins found in the colloidal particles include

casein micelles. k-Casein is a calcium-insensitive pro-

tein which forms a protective layer around the cal-

cium-sensitive caseins (a

1-, a

2-, b-, and g-), resulting

in stable casein micelles.

0061 Chymosin, or rennin (EC 3.4.23.4), triggers the

clotting of milk once the equilibrium of charges in

the casein micelle has been destabilized. It causes the

cleavage of k-casein at the Phe 105–Met 106 bond,

which results in a release of hydrophilic glycopeptide

which passes into the whey, while the para-k-casein

remains in the micelles, forming the coagulum needed

to produce cheese. (See Milk: Physical and Chemical

Properties.)

Milk-Clotting Enzymes

0062With the marked expansion in world cheese produc-

tion registered since the 1960s, it has become increas-

ingly difficult to obtain stomachs of young ruminants

tbl0004 Table 4 Classification of pectic enzymes acting on pectins or pectic acids

ECsuggested name Commonname EC number Substrate Actionpattern

Deesterifying enzymes

Polymethylgalacturonate esterase (PMGE) Pectinesterase 3.1.1.11 Pectin Random

Depolymerizing enzymes

Hydrolases

Endopolygalacturonase (endo PG) Polygalacturonase 3.2.1.15 Pectate Random

Exopolygalacturonase 1 (exo PG1) Polygalacturonase 3.2.1.67 Pectate Terminal

Exopolygalacturonase 2 (exo PG2) Polygalacturonase 3.2.1.82 Pectate Penultimate bonds

Endopolymethylgalacturonase (endo PMG) Pectin hydrolase Pectin Random

Exopolymethylgalacturonase (exo PMG) Pectin hydrolase Pectin Terminal

Lyases

Endopolygalacturonate lyase (endo PGL) Pectate lyase 4.2.2.2 Pectate Random

Exopolygalacturonate lyase (exo PGL) Pectate lyase 4.2.2.9 Pectate Penultimate bonds

Endopolymethylgalacturonate lyase (endo PMGL) Pectin lyase 4.2.2.10 Pectin Random

Exopolymethylgalacturonate lyase (exo PMGL) Pectin lyase Pectin Terminal

Pectic enzymes acting on oligogalacturonates have not been included in this table because they are not very abundant and are of little interest for

industrial pectin degradation. Enzymes have been classified and named according to the Enzyme Commission (EC) (IUPAC-IUB recommendations).

Data from Alkorta I, Garbisu C, Liama MJ and Serra JL (1998) Industrial applications of pectic enzymes: a review. Process Biochemistry 33: 21–28.

Processing

Sorting

Pitting

Peeling

Blanching

Crushing

Enzyming

Screening

Homogenizing

Concentration

Freezing

Pressing

Cloudy juice

Enzyme

Concentration

Concentrated juice

Filtration

Washing

(a)

(b)

Filtration

FillingPasteurization

Clear juice

(c)

fig0004 Figure 4 Use of enzymes in the preparation of (a) juices, (b) homogenates, and (c) clear juices.

ENZYMES/Uses in Food Processing 2133

from which chymosin can be extracted. In the USA in

particular, a general reduction in the number of calves

available for slaughter has reduced the supply of

rennet, increased the price, and stimulated interest

in developing rennet substitutes. Several rennet sub-

stitutes have been developed, including bovine rennet

from adult cows, fungus proteinase, and other pro-

teolytic enzymes. However, they have a much greater

level of nonspecific proteolytic activity, and higher

thermostability, which more completely degrade the

milk proteins to peptides, leading to a reduction in

yield and poor flavor development in some types of

cheese. Consequently, there have been numerous at-

tempts to produce chymosin in microorganisms.

0063 Several of these substitute preparations (Table 5)

are a mixture of rennet and pepsin but newly

developed recombinant DNA techniques have led to

their use in cloning the calf chymosin gene in Escher-

ichia coli. Recombinant chymosin contains only the

protein resulting from the expression of the sequence

of one gene and thus contains one variant only. Patents

have been taken out for the production of milk-

clotting enzymes from Endotia parasitica, Mucor

pusillus var. Lindt and Mucor miehei. Varieties of

cheese have been made from recombinant chymosin

and evaluated in comparison with cheese produced

using the natural enzyme. No significant differences

could be detected between them, with regard to the

recovery of milk solids, the rate of proteolysis during

ripening, or in the characteristics of the final cheese

product. Under US law, these enzymes may be used for

making any type of cheese.

Cheese Ripening

0064 Although some varieties of cheese are consumed

fresh, most varieties that have been subjected to

rennet clotting are ripened for periods ranging from

4 weeks to 2 years, the actual duration being in an

approximately inverse relation to the humidity con-

tent. During the ripening process, a series of chemical

and biological changes takes place, in the course of

which proteins, lipids, and residual lactose break

down into primary and secondary products, such as

peptides and fatty acids (lactic, propionic, etc.).

0065In the right combination, all these components

converge to produce the characteristic flavor of the

respective cheeses. The cost entailed in the ripening

operation has a considerable influence on the cost of

the finished product. Accordingly, if it is possible to

shorten the ripening time without adversely affecting

quality, the capital outlay required can be contained,

not to mention the risk of freak retarded fermentation

phenomena. (See Fermented Milks: Types of Fer-

mented Milks; Other Relevant Products.)

Accelerated Ripening Enzymes and Use of Lipases

0066The ripening time can be reduced by:

0067adding, during the manufacturing process,

enzymes which are not produced by lactic bacteria;

0068introducing additional lactic bacteria (as starters)

or extracts of lactic bacteria (as starters) or extracts

of lactic bacteria cells;

0069using slurry techniques.

Directly incorporating enzymes into the milk under-

going treatment can be costly but has the advantage

of insuring their homogeneous distribution. Prote-

ases, peptidases, lipases, and decarboxylases are

used for this purpose (Table 6). While the ripening

of certain varieties (blue, Parmesan, etc.) is markedly

influenced by lipase action, proteolysis is in all types

of cheese the primary biochemical event in determin-

ing flavor and texture. Neutral proteases of bacterial

origin such as neutrase (a metal protease, effective

in speeding up the ripening process whether or not

inserted into positively charged liposomes) or, again,

a serine protease obtained from Brevibacterium

linens (the main component of surface microflora on

cheese), have proved to yield an acceptable product

even if elasticity is somewhat sacrificed at times.

Moreover, if the crude enzyme containing the protease

(but amino peptidase as well) is used, better results

can be achieved in terms of the release of peptides and

amino acids without any bitter taste developing.

0070Studies on food and analytical-grade commercial

lipase have shown that these have a different specifi-

city in the hydrolysis of short-chain fatty acids and a

different specificity in the release of the free fatty

acids among them. In fact, depending on the chain-

length specificity of a given lipase, its addition to a

milk product may enhance the flavor of the cheese,

Milk

Whey

Coagulum

Rennet or clotting enzymes

Rennet and microbial enzymes

or added enzymes

Curd

(fat + protein)

Mature cheese

'Green' cheese ripening

fig0005 Figure 5 Flow diagram for cheese manufacturing.

2134 ENZYMES/Uses in Food Processing

accelerate the cheese ripening, or assist in the prepar-

ation of enzyme-modified cheese (EMC), an import-

ant commercial flavor used in the USA for the

manufacture of dips, sauces, dressings, and crackers.

EMC is produced from cheese curd by the addition of

lipases at elevated temperatures, increasing the con-

tent of free fatty acids about 10-fold. Depending on

the chain-length specificity of the lipases used, there is

either predominant liberation of short-chain fatty

acid (C4–C6), which results in a sharp, tangy flavor,

or of medium- and long-chain fatty acids (>C12),

which convey a soapy taste and are readily metabol-

ized to other flavor ingredients. Addition of lipase to

pasteurized cows’ milk can generate flavors similar to

goat, sheep, or raw milk if used with the appropriate

microbial consortia.

0071Currently, lipase technology is so advanced that the

production of compounds such as flavors or emulsi-

fiers are regarded as an old application. Commer-

cially available lipases are usually derived from

microorganisms, but since the advent of genetic en-

gineering techniques, an increasing number of lipases

are being commercially manufactured from recom-

binant bacteria and yeast. Some industrial applica-

tions of microbial lipases are reported in Table 7.

Use in Brewing

0072Traditionally, beer is produced by mixing crushed

barley malt and hot water in a large circular vessel

called a mash copper. This process is called ‘mashing.’

Besides malt, other starchy cereals such as maize,

sorghum, rice, and barley, or pure starch itself, are

added to the mash. These are known as adjuncts.

After mashing, the mash is filtered in a lauter tun.

The liquid, known as ‘sweet wort,’ is then run off to

the copper, where it is boiled with hops. The ‘hopped

wort’ is cooled and transferred to the fermenting

vessels where yeast is added. After fermentation, the

so-called ‘green beer’ is matured before the final

filtration and bottling. (See Beers: Biochemistry of

Fermentation.)

0073This is a much-simplified account of how beer is

made. Brewing can quite happily proceed without

any added enzymes, relying entirely on the natural

enzymes from the ingredients and the yeast to

tbl0005 Table 5 Enzymes in milk clotting

Enzyme IUBnumber enzyme classification (EC) Type and source

Calf, Piglet, and lamb rennet (chymosin) 3.4.23.4 (A) Stomach

Mammalian rennet

pepsin A 3.4.23.1 (A) Mammalian stomach

Pepsin B 3.4.23.2

Gastricsin 3.4.23.3

Chymosin 3.4.23.4

Pig (porcine pepsin) 3.4.23.1 (A) Pig stomach

Blends of two or more animal-derived enzymes (A)

Blends of animal-derived/microbial-derived enzymes (A) þ (M)

Microbial coagulant 3.4.23.6 (M) Mucor miehei

Microbial coagulant 3.4.23.6 (M) Mucor pusillus Lindt

Microbial coagulant 3.4.23.6 (M) Endotia parasitica

Microbial coagulant 3.4.23.6 (M) Penicillum janthinellum

Microbial coagulant 3.4.23.6 (M) Rhizopus chinensis

Microbial coagulant 3.4.23.6 (M) Aspergillus niger

A, animal; M, microbial.

IUB, International Union of Biochemistry; EC, Enzyme Classification.

tbl0006 Table 6 Enzymes in accelerated cheese ripening

Enzyme IUB number

enzyme

classification

(EC)

Typeand source

Lipase 3.1.1.3 (A) Edible forestomach tissue

of calf, kid, or lamb

Microbial lipase 3.1.1.3 (M) Aspergillus niger

Microbial lipase 3.1.1.3 (M) Aspergillus oryzae

Microbial lipase 3.1.1.3 (M) Mucor mihei

Lactase 3.2.1.23 (M) Saccharomyces lactis

Kluyveromyces spp.

Escherichia coli

Microbial serine

proteinase

3.4.21.14 (M) Aspergillus niger

Neutral proteinase 3.4.24.4 (M) Bacillus subtilis

Neutral proteinase 3.4.24.4 (M) Aspergillus oryzae

Proteinase 3.4 (M) Penicillium camemberti

Proteinase 3.1.1.1 (A) Pancreatic tissue

(M) Micrococcus caseolyticus

Proteinase 3.4.24.4 (M) Bacillus subtilis

Aspergillus niger

A, animal; M, microbial.

IUB, International Union of Biochemistry; EC, Enzyme Classification.

ENZYMES/Uses in Food Processing 2135

produce good-quality beer. From time to time, how-

ever, problems occur that cannot be easily identified

or rectified. Enzymes can often provide a useful diag-

nostic tool, giving the immediate remedy as well as

suggesting the long-term solution. Table 8 shows

some of the problems that can occur during brewing

and recommends some of the possible enzyme treat-

ments that can be used to solve the problem. It also

serves as a summary of the application of enzymes in

brewing, together with Figure 6, which shows where

the appropriate enzymes are usually used in the

brewing process. Supplementation can in fact occur

at various stages during the process. During the initial

step of decoction and mash formation, in which prob-

ably 80% of the original starch is converted into fer-

mentable sugars (glucose, maltose, maltotriose),

bacterial and fungal a-amylases are often added, to-

gether with bacterial and fungal glucanases. The latter

are important in the removal of b-glucans, b-1,3, and

b-1, 4-linked polymers, which are viscous gum-like

substances. Failure to hydrolyze the glucans intro-

duces problems in the efficiency of wort filtration

and in the later production of hazes. Further amylase

and glucanase addition may occur during the filtra-

tion of the wort. During subsequent fermentation,

particularly for low-calorie beers, where soluble poly-

saccharide levels need to be minimized, fungal a-

amylases and glucoamylases are added.

0074A typical example of the use of enzymes to resolve a

brewing problem is when a haze appears in the bright

filtered beer (this includes the enigmatic invisible

haze, which cannot be detected by the naked eye,

but is recorded as a machine haze). The haze is usually

ascribed to three probable causes: proteins, starch/

polysaccharides, or nonstarch polysaccharides. It

may result from other factors such as the passage of

diatomaceous earth, oxalic acid crystals, microorgan-

isms, or filter fibers, which can be easily identified

under a microscope or using an enzyme system.

In fact, adding b-glucanase, papain, and fungal

tbl0007 Table 7 Industrial application areas for microbial lipases

Industry Effect Product

Dairy Hydrolysis of milk fat Flavor agents

Cheese ripening Cheese

Modification of butter fat Butter

Bakery Flavor improvement and shelf-life prolongation Bakery products

Beverages Improved aroma Beverages

Food dressing Quality improvement Mayonnaise, dressings, and whipped toppings

Health food Transesterification Health foods

Meat and fish Flavor development and fat removal Meat and fish products

Fat and oil Transesterification Cocoa butter, margarine

Hydrolysis Fatty acids, glycerol, mono-, and diglycerides

Modified from Godtfredesen SE (1993) Lipases. In: Nagodawithana T and Redd G (eds) Enzymes in Food Processing, pp. 205–214. California: Academic

Press.

tbl0008 Table 8 A brewer first-aid kit

Location Symptom Remedy

Cereal cooker Glutinous starch Add heat-stable bacterial a-amylase

Retrograded starch

Mash mixer Enzyme-deficient malt Add bacterial a-amylase

Filtration problems

High malt glucans Add heat-stable fungal b-glucanase

Poor extract recovery

High adjunct brewing Add neutral proteinase

Fermentation Starch in beer Add fungal a-amylase

Require highly attenuated beers Add amyloglucosidase or pullulanase and b-amylase

Rapid diacetyl removal Add a-acetolactate decarboxylase

Maturation and filtration Low sweetness Add amyloglucosidases

Fuel for secondary fermentation

Chill haze Add papain

Bottling Poor resistance to staling in bottle Add immobilized glucose oxidase in the crown liner

Partially reproduced from O’Rourke T (1996) Brewing. In: Godfrey T and West SI (eds) Enzymology, pp. 103–131. London: MacMillan Press with

permission.

2136 ENZYMES/Uses in Food Processing