Ezyme Technology For Dairy Industry

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Lecturer: Dr. L HNG PH

Group 3 BTFTIU10208 Nguyn Tt Hon V

Nguyn Ngc Mai Trinh L Th Hng Nhi Hong H Thanh Hi Trn Th Mai Hng BTFTIU10215 BTFTIU10229 BTFTIU10210 BTFTIU10

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CONTENTS

INTRODUCTION

INDIGENOUS ENZYMES OF BOVINE MILK

EXOGENOUS ENZYMES USED IN THE DAIRY INDUSTRY

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INTRODUCTIO

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

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Enzyme technology in the dairy industry can be divided into two main areas:

1. INDIGENOUS ENZYMES in milk and their impact on dairy products 2. The use of EXOGENOUS ENZYMES in the manufacture of dairy products.

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INDIGENOUS ENZYMES OF BOVINE MILK

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INDIGENOUS ENZYMES OF BOVINE MILK

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- Microorganisms in Bovine milk are a potential enzymatic source have positive or negative impacts in dairy applications. - Most microorganisms do not survive pasteurization limited to products produced mainly from raw milk. - The microflora of raw cold stored milk is dominated by Gramnegative, nonlactose fermenting psychrotrophic bacteria, with more than 70% of milk microflora consisting of Pseudomonas species.
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INDIGENOUS ENZYMES OF BOVINE MILK

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Approximately sixty indigenous enzymes are present in raw milk have been reported.
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Indigenous milk enzymes arise mainly from three potential sources: Blood Secretary cell cytoplasm The milk fat globule membrane. Most indigenous enzymes are inactive in milk due to inappropriate environmental conditions or lack of suitable substrates Enzyme Technology and destroyed by pasteurization to create a standardized product. 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. 4/15/12

1. 2. 3. q.

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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, while longer chain FFAs impart soapy flavors.

The fat globules are surrounded by a thin membrane called the milk fat globule membrane (MFGM). be damaged by agitation, homogenization, foaming, or poor handling, which can result in the rapid interaction of enzyme and substrate, causing the development of off flavors.

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Lipoprotein Lipase

It is a water soluble enzyme that hydrolyzes triglycerides in lipoproteins. Indigenous LPL is present in milk via leakage through the mammary cell membrane from the blood where it is involved in the metabolism of plasma triglycerols. LPL is activated by an apolipoprotein activator, apo-CII, and is present in high enough concentrations in milk to be of real danger in terms of its spoilage capability. LPL is susceptible to standard high temperature short time (HTST) pasteurization; however it is thought that it is not completely inactivated 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 milk and milk products as these dominate in typical cold bulk milk storage conditions.

Lipases of psychrotrophic bacteria have varying heat stability and some can survive pasteurization, however lipolytic spoilage rarely occurs in pasteurized milk.

Pseudomonas is the most frequently 4/15/12 reported psychrotroph in raw milk

Phospholipases

Phospholipase activity has been identified in milk LPL and in several bacteria. Phospholipases are potentially very important in milk as they degrade the phospholipids of the MFGM, thereby making the fat susceptible to hydrolysis. These enzymes are also quite heat stable and are not destroyed by pasteurization.
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PROTEINASES
A few indigenous milk proteinases have been identified in bovine milk, but only two appear to be of importance in the dairy industry:
1. 2.

Plasmin or Milk Alkaline Proteinase. Cathepsin D or Acid Milk Proteinase.

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Plasmin or Milk Alkaline Proteinase

Plasmin (EC 3.4.21.7) is an alkaline serine proteinase and is secreted as plasminogen that is activated in blood and milk. Its role in blood is to break down blood clots and is a part of a complex system consisting of activators and inhibitors. The concentration of plasmin and plasminogen in milk increases with advancing lactation, mastitic infection, 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 optimum activity at pH 7.5 and 35C, and still exhibits about 20% activity at 5C. It is active over a wide pH range from pH 4 to 9 and is quite heat stable.
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LYSINE

ARGININE

Plasmin can have a positive or negative effect on cheese making as it is involved in proteolysis during ripening. It is also linked to poor curd formation in cheese made from late lactation milks. Plasmin is also associated with the generation of gluey and bitter off flavors during storage of UHT milks and is a major factor affecting their shelf life. It can accelerate cheese ripening by the activation of plasminogen to plasmin in the cheese.
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Cathepsin D or Acid Milk Proteinase

Cathepsin D (EC 3.4.23.5) is a lysozomal enzyme that has a similar specificity to chymosin, but has very poor milk clotting activity. It is relatively heat labile as it is inactivated by 70C for 10 min, and may contribute to proteolysis in cheese, but not significantly.

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Alkaline Phosphomonoesterase (Phosphatase)

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Phosphatase enzymes are able to split specific phosporic acid esters into phosphoric acid and the related alcohols. It has a pH and temperature optima differing from physiological values; pH of 9.8. The enzyme is destroyed by minimum pasteurization temperatures.
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Phosphomonoesterase The enzyme is a glycoprotein and (Phosphatase)

has a lower optimum pH than that of alkaline phosphatase (pH 5). Acid phosphatase hydrolyses aromatic phosphomonoesters. The enzyme is active against caseins. This enzyme affects casein micelle formation through dephosphorylation of casein, which destroys interchain bridging via phosphate groups between casein polypeptides and calcium ions.
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Alkaline

LACTOPEROXIDASE
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Peroxidases have been shown to be involved in antibacterial activity. Lactoperoxidase requires H2O2 and thiocyanate to cause bacterial inhibition. The mode of action of this system has been 4/15/12 used to cold pasteurize

LYSOZYMES

Lysozyme hydrolyses the (1-4)-linkage between muramic acid and Nacetylglucosamine of mucopolysaccharides of the bacterial cell wall. Lysozyme is fairly heat stable 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 inhibitors of xanthine oxidase. The pH optimum is about 8.5 , the enzyme requires flavin, adenine,dinucleotide, other cofactors. It in milk is associated with oxidative rancidity. Xanthine oxidase has been implicated as having bacteriostatic 4/15/12

 - GLUTAMYL TRANSPEPTIDASE (Transferase)

-Glutamyl transpeptidase catalyses the transfer of _-glutamyl residues from glutamyl-containing peptides. It plays an important role in amino acid transport in the mammary gland

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EXOGENOUS ENZYMES USED IN THE DAIRY INDUSTRY

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-GALACTOSIDASE

-Galactosidases (commonly referred to as lactase), which hydrolyse lactose to glucose + galactose, are probably the second most significant enzyme in dairy technology

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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 or gritty texture, and deposit formation.

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LIPASE
The principal application of lipases in dairy technology is in cheese

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LIPASE b
c

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Rennet Paste

a combination of the milk clotting enzyme rennet and pregastric lipases, which contribute to the required flavor and aroma development 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.
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Pregastric Esterases (PGE)

The lipase in rennet paste, generally referred to as pregastric esterase (PGE), is secreted by a gland at the base of the tongue, which is stimulated by suckling; the secreted lipase is washed into the stomach with the ingested milk.

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Provolone

The physiological significance: to assist in lipid digestion in the neonate which has limited pancreatic function Provide the sharp piquant flavor which 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 short chain fatty acids at the Sn3 position. This ensures the release of the most volatile free fatty acids, which by their nature have the greatest impact on flavor and aroma differences in the cheeses
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Microbial Lipases

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

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MOLDS

play a direct role in flavor and aroma development The lipases originate from the mold Penicillium roqueforti used in the manufacture of the cheese. The cheeses are then pierced using rods, which enables carbon dioxide to be released from the curd and oxygen to penetrate. This together with the correct humidity, salt, and oxygen facilitates germination of the spores

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LACTIC ACID LAB BACTERIAof(LAB)their primary initiate the production lactic acid, role in natural cheese making
Secondary role: contribute to the cheese ripening process by the release of cell wall or intracellular enzymes

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The lipolytic activity of these bacteria is very low, but lipase activity has been identified in many LAB. nonstarter lactic acid bacteria (NSLAB) which are predominant in the microbiological content of many cheese varieties during ripening have a role in the level of lipolysis in these cheeses

YEAS TS Yeasts contribute to lipolysis levels in these cheeses.

Geotrichum candidum is commonly found on some cheese varieties before salting and is very acylspecific for cis-9 unsaturated fatty acids

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ACCELERATED CHEESE RIPENING

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Cheese curd can be made from raw or pasteurized milk. Raw milk cheese should be ripened for 60 days or more as a safeguard against pathogens. Cheddar, where a mature cheese is typically 12 to 18 months old, costs associated with ripening become a major issue.

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Methods for accelerated cheese ripening

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Non-enzymatic method
Elevated ripening temperatures High Pressure

Enzymatic methods
Exogenous enzymes Attenuated starter cultures Adjuncts Genetic modification

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Nonenzymatic Methods For Accelerating Cheese Ripening

The first is elevated temperature ripening, which provides a better environment for the enzymes to work,thereby accelerating ripening. Should care needs to be taken to ensure microbial safety and to prevent textural defects. The second method is high pressure treatment, which still in its infancy, but may have the potential to accelerate cheese ripening
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Accelerating Cheddar Cheese Ripening Using Enzyme Technology

1. 2. 3.

Exogenous Enzymes Commercial Enzymes for Accelerating Cheese Ripening Attenuated Starter Cultures and Adjuncts

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Exogenous Enzymes

Adding increased amounts of enzymes during the cheese making process.

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

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hieving a balanced flavor and texture pro

Using this technology has proven difficult. Enhancing hydrolysis of the major casein proteins causes: Bitterness Losses in yield Weakness in texture

1. 2. 3.

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Exogenous lipases

Lipolysis to be important in Cheddar cheese flavor development. Small increases in the level of lipolysis can have major negative sensory impacts.

=> The correct dosage level of lipase is critical for success.

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Blends of enzymes or enzyme cocktails

They are available to accelerate cheese ripening without negative effects. However, problems can still occur with unbalanced ripening because: The enzymes have not been added to the curd uniformly. Differences in production techniques among different factories.

1.

2.

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Liposome technology

The enzymes would then fully partition with the 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.

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Problems existed with Liposome technology

1. 2.

Maximizing enzyme encapsulation rates. High costs due to the expense of using soya lecithin in the production of liposomes. Increased moisture Changes in rheological characteristics of the cheese Most soya lecithin is derived from genetically modified soya which further limits its use in certain countries.

3. 4. 5.

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Adding exogenous enzymes at the salting stage

Adding exogenous enzymes at the salting stage (milling) of the cheese making process offers the best route for Cheddar cheese production. It has shown an increase in the indices of ripening. Many different types of exogenous enzymes have been added to different cheeses, despite problems associated with uneven enzyme distribution in the curd. 4/15/12

Commercial Enzymes for Accelerating Cheese Ripening Few commercially successful enzyme systems to accelerate cheese ripening exist. Ex:

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Attenuated Starter Cultures and Adjuncts

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Cheese curd :retain up to 3 times the level of starter typically used in cheese making, therefore opening the possibility of enabling extra starter culture, containing more enzymes, to be added to the curd to accelerate its ripening. Using sublethal heat treatments or freeze-thaw treatments, which do not affect the enzyme activity of the starter bacteria. Lactose-negative strains may also be used, as these occur naturally, because they cannot produce acid they can also be used to accelerate cheese ripening.

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Attenuated Starter Cultures and Adjuncts

The advent of genetic modification (GM) has enhanced the possibility of using cultures for the acceleration of cheese ripening. Not only has the taxonomy of cultures been 4/15/12 advanced but also their enzyme complement, activities,

ENZYME TECHNOLOGY IN CHEESE AS AN INGREDIENT

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Cheeses have been used as ingredients in foods for years and used in the retail, food service (catering), and industrial sectors.

Natural cheese is an essential ingredient in many products, providing flavor, aroma, color, texture, and functionality, but it can be quite expensive. This aspect has driven suppliers to develop cost effective alternatives to using natural cheese in ingredient formulations.
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Fast Ripened Cheese

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It s defined by their high moisture content and short ripening times, which can be weeks rather than months.

The enzymes and starter cultures used in their production are similar to those outlined in the accelerated cheese ripening section and are added at levels higher than that found in natural cheese.
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These cheeses are high moisture cheeses, which are typically ripened at 15 to 20C to increase the rate of enzyme reaction and thus hydrolysis .Once an acceptable flavor profile has been reached- stored (shredded) A variation of this type of cheese involves shredding the cheese and adding enzymes, then reblending it in a fashion similar to processed cheese, and ripening it at high temperatures.

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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 enzyme used)

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EMC Preparation

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Use of Lipases
Extensively used to enhance the flavor of EMC, due to formation of fatty acids from milk fat Short chain fatty acids are key component in cheese flavor Animal pregastric esterase widely used in natural cheese and EMC production, but microbial lipases are becoming more desirable

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Lipases Reaction

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Use of Proteases

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 to hydrophobic amino acids within peptides)

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Glutamin ase
Converts glutamine into glutamate Glutamine is a flavorless amino acid, but conversion to glutamate can increase taste intensity without addition of separate ingredients or raising sodium content Glutaminase DAIWA SD-C100S acts on free glutamine and can be used in combination with Umamizyme to improve EMC taste

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Substitute or Imitation TheseCheese which, as opposed to natural cheese, are not are cheese products made directly from milk. Two types of imitation cheese are produced
in very high volumes: processed and analogue cheese. Neither imitation cheese uses enzymes directly in its manufacture, but either may contain ingredients produced using enzymes.

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PROTEINS HYDROLYSIS Enzyme hydrolysis is generally seen as a noninvasive technology,

and is an extrapolation of many natural processes. Two milk proteins, casein and whey, are used in the food industry for specific flavor, functional, or nutritional applications. A wide variety of commercial enzymes are useful in the preparation of hydrolysates that are very soluble, and stable at low pH and when heated

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However, a major problem associated with protein hydrolysis, particularly for casein and whey proteins, is the development of bitterness, which is associated with the accumulation of hydrophobic peptides Generally, a number of key peptidase activities are important in bitterness reduction. General aminopeptidase activity, which cleaves single amino acids from the N-terminal of a peptide (Pep N and Pep C activity) is important. The removal of the amino acid proline from a peptide can greatly reduce bitterness, because it alters the peptides three-dimensional structure, changing its solubility and increasing its susceptibility to hydrolysis .

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Key peptidase activities involved in removing proline are proline specific peptidases such as: postprolyl dipeptidyl dipeptidase (Pep X), which releases N-terminal dipeptides from prolyl peptides; proline aminopeptidase (Pep P), which exclusively hydrolyzes the N-terminal amino acid from peptides that have proline in the second position; proline iminopeptidase (Pep I) which removes unsubstituted N-terminal proline residues from tri- , oligo-, and polypeptides; prolinase (Pep R) which cleaves N-terminal Pro-X dipeptides; and prolidase (Pep Q) which hydrolyzes X-Pro dipeptides

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LACTOSE HYDROLYSIS

The Galactosidase can used accelerating cheese ripening. Hydrolysis of milk lactose for its use in ice cream to prevent crystallization has been shown to be successful. The treatment of hydrolysed whey for animal feed removes problems associated with crystallization of lactose on drying and makes the whey more useful as a feed component

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LYSOZYM Lysozyme is used in some Dutch, Swiss, and Italian cheese ES blowing and off flavors caused varieties to prevent late gas
by the growth of Clostridium tyrobutyricum. Most commercial lysozyme is derived from chicken egg white

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TRANSGLUTAMIN Transglutaminase (TGase) is an enzyme which catalyzes an ASE

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

TGase has found applications throughout the food sector particularly in improving functional properties of various products. The use of TGase in the dairy industry at present is generally limited to research.
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Thank You
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