enzyme technology in dairy

8/2/2019 Enzyme Technology in Dairy

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Click to edit Master subtitle style

4/15/12

Ezyme Technology

For Dairy Industry

Lecturer: Dr. L H NG P


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Group

3Nguyn Tt Hon V BTFTIU10208Nguyn Ngc Mai Trinh BTFTIU10215

L Th Hng Nhi BTFTIU10229

Hong H Thanh Hi BTFTIU10210

Trn Th Mai Hng BTFTIU10


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CONTENTS

INTRODUCTION INDIGENOUSENZYMES OF

BOVINE MILK

EXOGENOUSENZYMES USED IN THE

DAIRY INDUSTRY


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INTRODUCTI


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.In the dairy industry, enzymes are widely used inthe production of milk, cheeses, yogurt and other dairyproducts


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q Enzyme technology in the dairy industrycan be divided into two main areas:

1. INDIGENOUS ENZYMES in milk andtheir impact on dairy products

2. The use ofEXOGENOUS ENZYMESin the manufacture of dairy products.


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INDIGENOUS

ENZYMES OFBOVINE MILK


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INDIGENOUS ENZYMES OF BOVINEMILK


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INDIGENOUS ENZYMES OFBOVINE MILK

- Microorganisms in Bovine milk are a potential enzymatic source

have positive or negative impacts in dairy applications.- Most microorganisms do not survive pasteurization limited toproducts produced mainly from raw milk.- The microflora of raw cold stored milk is dominated by Gram-negative, nonlactose fermenting psychrotrophic bacteria, with more

than 70% of milk microflora consisting of Pseudomonas species.


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Approximately sixty indigenous enzymes are present inraw milk have been reported.

q Indigenous milk enzymes arise mainly from three potentialsources:

1. Blood

2. Secretary cell cytoplasm3. The milk fat globule membrane.q. Most indigenous enzymes are inactive in milk due to

inappropriate environmental conditions or lack of suitable

substrates Enzyme Technology and destroyed by pasteurizationto create a standardized product.q. Those indigenous enzymes that can be exploited to aid certain

dairy applications, or those that could have a major negative

impact in dairy applications if not controlled.


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LIPASES Lipase is an enzyme that catalyzes the formation or cleavage (

hydrolysis ) of fats ( lipids ). Low levels of lipolysis can have a marked

impact on flavor. For example: In general the lower chain

FFAs (C4 C8) impart rancid flavors, whilelonger chain FFAs impart soapy flavors.

The fat globules are surrounded by a thinmembrane called the milk fat globulemembrane (MFGM).

be damaged by agitation, homogenization,foaming, or poor handling, which can result inthe rapid interaction of enzyme and substrate,causing the development ofoff flavors.
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Lipoprotein Lipase It is a water soluble enzyme that

hydrolyzestriglycerides in lipoproteins. Indigenous LPL is present in milk via

leakage through the mammary cellmembrane from the blood where it is

involved in the metabolism of plasmatriglycerols.

LPL is activated by an apolipoprotein activator, apo-CII, and ispresent in high enough concentrations in milk to be of real dangerin terms of its spoilage capability.

LPL is susceptible to standard high temperature short time (HTST)pasteurization; however it is thought that it is not completelyinactivated and may be active in some milk products.
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Bacterial Lipases Extracellular lipases produced by psychrotrophic bacteria

have the potential to cause hydrolytic rancidity in milkand milk products as these dominate in typical cold bulkmilk storage conditions.

Lipases of psychrotrophicbacteria have varying heatstability and some can survive

pasteurization, however

lipolytic spoilage rarely occursin pasteurized milk.

Pseudomonas is the most frequentlyreported psychrotroph in raw milk


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Phospholipases

Phospholipase activity has been identified in milk LPLand in several bacteria.

Phospholipases are potentially very important in milkas they degrade the phospholipids of the MFGM,thereby making the fat susceptible to hydrolysis.

These enzymes are also quite heat stable and are notdestroyed by pasteurization.


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PROTEINASES

A few indigenous milk proteinases have beenidentified in bovine milk, but only two appear tobe of importance in the dairy industry:

1. Plasmin or Milk Alkaline Proteinase.

2. Cathepsin D or Acid Milk Proteinase.


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Proteinase Plasmin (EC 3.4.21.7) is an alkaline serine proteinase

and is secreted as plasminogen that is activated inblood and milk.

Its role in blood is to break

down blood clots and is a partof a complex systemconsisting of activators andinhibitors.

The concentration of plasmin and plasminogen inmilk increases with advancing lactation, mastiticinfection, and number of lactations.


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Plasmin has a high specificity for peptide bonds with a

carboxyl group supplied by lysine or arginine.

It has an optimumactivity at pH 7.5 and

35C, and still exhibitsabout 20% activity at5C.

It is active over a widepH range from pH 4 to9 and is quite heatstable.

LYSINE ARGININE


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Plasmin can have a positive or

negative effect on cheesemaking as it is involved inproteolysis during ripening.

It is also linked to poor curd

formation in cheese made fromlate lactation milks.

Plasmin is also associated with the generation of gluey and

bitter off flavors during storage of UHT milks and is a majorfactor affecting their shelf life.

It can accelerate cheese ripening by the activation of

plasminogen to plasmin in the cheese.


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Proteinase

Cathepsin D (EC 3.4.23.5) is a lysozomal enzyme thathas a similar specificity to chymosin, but has very poormilk clotting activity.

It is relatively heat labile as it is inactivated by 70Cfor 10 min, and may contribute to proteolysis incheese, but not significantly.

A a ine P osp omonoesterase


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A a ine P osp omonoesterase(Phosphatase)

v Phosphatase enzymes are ableto split specific phosporic acidesters into phosphoric acid andthe related alcohols.

v It has a pH and temperatureoptima differing from

physiological values; pH of9.8.

v The enzyme is destroyed byminimum pasteurizationtemperatures.

Alkaline


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v The enzyme is a glycoprotein and

has a lower optimum pH than that ofalkaline phosphatase (pH 5).

v Acid phosphatase hydrolysesaromatic phosphomonoesters.

v The enzyme is active against caseins.

v This enzyme affects casein micelleformation through dephosphorylation

of casein, which destroys interchainbridging via phosphate groupsbetween casein polypeptides andcalcium ions.

AlkalinePhosphomonoesterase(Phosphatase)


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LACTOPEROXIDASE

v Peroxidases have beenshown to be involvedin antibacterial

activity.v Lactoperoxidase

requires H2O2 andthiocyanate to causebacterial inhibition.

v The mode of action ofthis system has beenused to cold pasteurize

SO S


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LYSOZYMES

Lysozyme hydrolyses the(1-4)-linkage betweenmuramic acid and N-acetylglucosamine of

mucopolysaccharides of thebacterial cell wall.

Lysozyme is fairly heatstable in acid conditions, but

heat labile at pH 7 or above. Lysozyme is effectively an

antibacterial agent .


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XANTHINE OXIDASE It concentrated in the MFGM where

it is one of the principal proteins. All of the major milk proteins can

act as either activators or inhibitorsof xanthine oxidase.

The pH optimum is about 8.5 , theenzyme requires flavin,adenine,dinucleotide, othercofactors.

It in milk is associated with oxidativerancidity.

Xanthine oxidase has been

implicated as having bacteriostatic

GLUTAMYL TRANSPEPTIDASE


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- GLUTAMYL TRANSPEPTIDASE(Transferase)

-Glutamyl transpeptidase catalyses thetransfer of _ -glutamyl residues from - glutamyl-containing peptides.

It plays an important role in amino acidtransport in the mammary gland


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EXOGENOUS

ENZYMES USED IN THEDAIRY INDUSTRY


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-GALACTOSIDASE

-Galactosidases (commonly referred to as lactase),which hydrolyse lactose to glucose + galactose, areprobably the second most significant enzyme in dairytechnology


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Roles: Getting easy to consume milk and dairy products for

people who are lactose-intolerant

Limiting many defects in refrigerated foods such as

crystallization in dairy foods, development of sandy orgritty texture, and deposit formation.

LIPASE


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LIPASE

The principal application of lipases in dairy technology is incheese


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LIPASE

a

b

c

Rennet P t


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a combination ofthe milk clotting enzyme rennet andpregastriclipases, which contribute to the required flavor and aromadevelopment

Produced by grinding the semi-dry stomach of the dairy animal

into a paste + NaCl. Rennet pastes contains

chymosin which coagulates

the milk + a potent lipase +

pregastric esterase, which

leads to much lipolysis

during ripening.

Rennet Paste


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(PGE) The lipase in rennet paste, generally referred to as

pregastric esterase (PGE), is secreted by a gland at thebase of the tongue, which is stimulated by suckling; thesecreted lipase is washed into the stomach with theingested milk.

Pecorino Provolone


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The physiological significance: to assist in lipiddigestion in the neonate which has limited pancreaticfunction

Provide the sharp piquant flavorwhich is is primarily

due to short chain FFAs arising from the action of PGE For example : lipases that primarily release short chain

fatty acids lead to the development of a piquant, sharp,spicy, tangy flavour, while release of medium to long

chain fatty acids can lead to a soapy taste


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PGEs are regioselective for the Sn1 and Sn3 positions of

milk triglycerides and have a particular affinity for shortchain fatty acids at the Sn3 position.

This ensures the

release of the mostvolatile free fattyacids, which by theirnature have the

greatest impact onflavor and aromadifferences in thecheeses

Mi bi l Li


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Microbial Lipases

Microbial lipases are lipolytic enzyme preparations,which are derived from yeasts, molds, and bacteria

MOLDS


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MOLDS play a direct role in flavor and aroma development

The lipases originate from the mold Penicillium roquefortiused in the manufacture of the cheese. The cheeses are then

pierced using rods, which enables carbon dioxide to bereleased from the curd and oxygen to penetrate. This together

with the correct humidity, salt, and oxygen facilitatesgermination of the spores

LACTIC ACID


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LAB initiate the production of lactic acid, their primary

role in natural cheese making Secondary role: contribute to the cheese ripening

process by the release of cell wall or intracellularenzymes

LACTIC ACIDBACTERIA (LAB)

The lipolytic activity of these bacteriais very low, but lipase activity has

been identified in many LAB.nonstarter lactic acid bacteria

(NSLAB) which are predominant inthe microbiological content of manycheese varieties during ripeninghave a role in the level of lipolysis in

these cheeses

YEAS


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Yeasts contribute to lipolysis levels in these cheeses.

YEASTS

Geotrichum candidum is

commonly found on somecheese varieties beforesalting and is veryacylspecific for cis-9

unsaturated fatty acids


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RIPENINGv Cheese curd can be made from raw or pasteurized milk.

Raw milk cheese should be ripened for 60 days or more asa safeguard against pathogens.

Cheddar, where a mature cheese is typically 12 to 18months old, costs associated with ripening become a

major issue.

Methods for accelerated cheese


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Methods for accelerated cheeseripening

v

Non-enzymatic method Elevated ripening temperatures

High Pressure

v Enzymatic methods

Exogenous enzymes

Attenuated starter cultures Adjuncts

Genetic modification

Nonenzymatic Methods For


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Nonenzymatic Methods ForAccelerating Cheese Ripening

The first is elevated temperatureripening, which provides a betterenvironment for the enzymes towork,thereby accelerating ripening.Should care needs to be taken to

ensure microbial safety and toprevent textural defects.

The second method is high pressuretreatment, which still in its infancy,but may have the potential toaccelerate cheese ripening

Ri i U i E


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Ripening Using EnzymeTechnology

1. Exogenous Enzymes2. Commercial Enzymes for Accelerating Cheese Ripening3. Attenuated Starter Cultures and Adjuncts

Exogenous Enzymes


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Exogenous Enzymes

Adding increased amounts of enzymes during thecheese making process.

=> Quicker hydrolysis of the major milk components.=> Reducing its ripening time

i i b l d fl d t t


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ieving a balanced flavor and texture pro Using this technology has proven difficult.

Enhancing hydrolysis of the major casein proteinscauses:

1. Bitterness

2. Losses in yield3. Weakness in texture

Exogenous lipases


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Exogenous lipases

Lipolysis to be important in Cheddar cheeseflavor development. Small increases in the level of lipolysis can have

major negative sensory impacts.

=> The correct dosage level of lipase is critical forsuccess.

Blends of enzymes or enzyme


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Blends of enzymes or enzymecocktails

They are available to accelerate cheeseripening without negative effects.

However, problems can still occur with

unbalanced ripening because:1. The enzymes have not been added to the curd

uniformly.

2. Differences in production techniques amongdifferent factories.

Liposome technology


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Liposome technology

The enzymes would then fully partition withthe curd and not negatively affect yield.

It would be easy to use, but also ensure even

distribution of the enzyme in the curd. Encapsulating water soluble enzymes in the

aqueous phase between phospholipid layers in

liposome globules.

ro ems ex s e w posome


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ro ems ex s e w posometechnology

1. Maximizing enzyme encapsulation rates.

2. High costs due to the expense of using soya lecithinin the production of liposomes.

3. Increased moisture4. Changes in rheological characteristics of the cheese

5. Most soya lecithin is derived from genetically

modified soya which further limits its use in certaincountries.

ng exogenous enzymes a e


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ng exogenous enzymes a esalting stage

Adding exogenous enzymes at the saltingstage (milling) of the cheese making processoffers the best route for Cheddar cheese

production. It has shown an increase in the indices of

ripening.

Many different types of exogenous enzymeshave been added to different cheeses, despiteproblems associated with uneven enzymedistribution in the curd.

C i l E f A l ti Ch Ri i


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Commercial Enzymes for Accelerating Cheese Ripening

Few commercially successful enzymesystems to accelerate cheese ripeningexist.

Ex:

Attenuated Starter Cultures and Adjuncts


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q Cheese curd :retain up to 3 times the level of startertypically used in cheese making, therefore opening the

possibility of enabling extra starter culture, containingmore enzymes, to be added to the curd to accelerate itsripening.

q Using sublethal heat treatments or freeze-thawtreatments, which do not affect the enzyme activity of thestarter bacteria. Lactose-negative strains may also beused, as these occur naturally, because they cannot

produce acid they can also be used to accelerate cheeseripening.



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The advent of genetic modification (GM) has enhanced thepossibility of using cultures for the acceleration of cheeseripening. Not only has the taxonomy of cultures been

advanced but also their enzyme complement, activities,

ENZYME TECHNOLOGY IN CHEESE


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AS AN INGREDIENTq Cheeses have been used as ingredients in foods for years and used in

the retail, food service (catering), and industrial sectors.

q This aspect has driven suppliers to develop cost effective alternativesto using natural cheese in ingredient formulations.

q Natural cheese is an essential ingredient in many products, providing

flavor, aroma, color, texture, and functionality, but it can be quiteexpensive.

Fast Ripened Cheese


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Fast Ripened Cheese

q It s defined by their high moisture content and short ripening

times, which can be weeks rather than months.

q The enzymes and starter cultures used in their production aresimilar to those outlined in the accelerated cheese ripeningsection and are added at levels higher than that found in naturalcheese.


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q These cheeses are high moisture cheeses, which aretypically ripened at 15 to 20C to increase the rate ofenzyme reaction and thus hydrolysis .Once anacceptable flavor profile has been reached- stored

(shredded)

q A variation of this type of cheese involves shredding thecheese and adding enzymes, then reblending it in afashion similar to processed cheese, and ripening it athigh temperatures.

Enzyme Modified Cheese


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Enzyme Modified Cheese

Cheese with flavor enhanced by enzyme treatment

Typically lipolytic and proteolytic enzymes used

More intense flavors than naturally ripened cheese, with

values of greater than 30X noted in many examples


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Factors Influencing EMC Flavor

Starting material e.g. Fresh curd, immature cheese, cream,milk

Free fatty acids produced (using lipases)

Peptides and free amino acids (proteases and peptidases)

Reaction conditions (time, temperature, pH and enzymeused)


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EMC Preparation

Use of Lipases


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Extensively used to enhance the flavor of EMC, due toformation of fatty acids from milk fat

Short chain fatty acids are key component incheese flavor

Animal pregastric esterase widely used in naturalcheese and EMC production, but microbial lipases are

becoming more desirable

Use of Lipases

Lipases Reaction


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Lipases Reaction

Use of Proteases


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Used to improve taste of EMC Liberation of low-molecular peptides and amino acids

create umami or savory flavor

Produces less bitter notes (usually found due tohydrophobic amino acids within peptides)

Use of Proteases

Glutamin


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Converts glutamine into glutamate Glutamine is a flavorless amino acid, butconversion to glutamate can increase taste

intensity without addition of separateingredients or raising sodium content Glutaminase DAIWA SD-C100S acts on freeglutamine and can be used in combination withUmamizyme to improve EMC taste

Glutaminase

Substitute or Imitation


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Substitute or ImitationCheeseq These are cheese products which, as opposed to natural cheese, are not

made directly from milk. Two types of imitation cheese are producedin very high volumes: processed and analogue cheese. Neitherimitation cheese uses enzymes directly in its manufacture, but eithermay contain ingredients produced using enzymes.

PROTEINS


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PROTEINSHYDROLYSIS

q Enzyme hydrolysis is generally seen as a noninvasive technology,

and is an extrapolation of many natural processes. Two milkproteins, casein and whey, are used in the food industry forspecific flavor, functional, or nutritional applications. A widevariety of commercial enzymes are useful in the preparation ofhydrolysates that are very soluble, and stable at low pH and whenheated


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However, a major problem associated with protein hydrolysis,

particularly for casein and whey proteins, is the development ofbitterness, which is associated with the accumulation of hydrophobicpeptides

Generally, a number of key peptidase activities are important in

bitterness reduction. General aminopeptidase activity, which cleavessingle amino acids from the N-terminal of a peptide (Pep N and Pep Cactivity) is important. The removal of the amino acid proline from a

peptide can greatly reduce bitterness, because it alters the peptidesthree-dimensional structure, changing its solubility and increasing its

susceptibility to hydrolysis .


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Key peptidase activities involved in removing proline are prolinespecific peptidases such as: postprolyl dipeptidyl dipeptidase (Pep X),which releases N-terminal dipeptides from prolyl peptides; prolineaminopeptidase (Pep P), which exclusively hydrolyzes

the N-terminal amino acid from peptides that have proline in the secondposition; proline iminopeptidase (Pep I) which removes unsubstitutedN-terminal proline residues from tri- , oligo-, and polypeptides;prolinase (Pep R) which cleaves N-terminal Pro-X dipeptides; andprolidase (Pep Q) which hydrolyzes X-Pro dipeptides

LACTOSE HYDROLYSIS


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The Galactosidase can used accelerating cheeseripening. Hydrolysis of milk lactose for its use in ice

cream to prevent crystallization has been shown to besuccessful. The treatment of hydrolysed whey for animalfeed removes problems associated with crystallization oflactose on drying and makes the whey more useful as a

feed component

LACTOSE HYDROLYSIS

LYSOZYM


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Lysozyme is used in some Dutch, Swiss, and Italian cheese

varieties to prevent late gas blowing and off flavors causedby the growth of Clostridium tyrobutyricum. Mostcommercial lysozyme is derived from chicken egg white

LYSOZYMES

TRANSGLUTAMIN


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Transglutaminase (TGase) is an enzyme which catalyzes an

acyl-transfer reaction introducing a covalent cross linkbetween glutamine and lysine residues.

TGase has found applications throughout the food sectorparticularly in improving functional properties of variousproducts. The use of TGase in the dairy industry at present isgenerally limited to research.

TRANSGLUTAMINASE


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enzyme technology in dairy

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