Dairy Applications for Dairy Applications for TransglutaminaseTransglutaminase

Ajinomoto Food Ingredients, LLC

TransglutaminaseTransglutaminaseEnzyme BasicsEnzyme Basics

TransglutaminaseTransglutaminase

TG is an enzyme that covalently crossTG is an enzyme that covalently cross--links links proteinprotein

Found throughout nature (from Found throughout nature (from microorganisms to mammals)microorganisms to mammals)

ACTIVAACTIVA®® TransglutaminaseTransglutaminase

TG TG enzyme used by Ajinomoto is produced enzyme used by Ajinomoto is produced via fermentation by a GMOvia fermentation by a GMO--free free microorganismmicroorganism

Marketed as Marketed as TG preparationsTG preparations by Ajinomoto by Ajinomoto Food Ingredients (AFI) under the Food Ingredients (AFI) under the ACTIVAACTIVA®® brand namebrand name

Linking ReactionLinking Reaction

TGTG

Transglutaminase: Cross-linking Mechanism

GLN-C-NH2 + H-N-LYSGLN-C-NH2 + H-N-LYS||

O ||

O

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TGTG

NH3GLN-C-N-LYSGLN-C-N-LYS|

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O H|| |

O H

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H |

H

γ Carbonyl Group of Glutamine ε Amino group of Lysine

Forms permanent covalent bonds unaffectedby freezing or cooking conditions

ACTIVAACTIVA®® TG Temperature ImpactTG Temperature Impact

Activity and Stability over Temperature Range

0

20

40

60

80

100

0 10 20 30 40 50 60 70

Temperature (C)

Rel

ativ

e A

ctiv

ity (%

)

RelativeActivityStability

Temperature range: 32 to ~150FOptimal Temperature range: 115-130F (45-55C)

(Specific denaturation temp depends on system composition)

ACTIVAACTIVA®® TG pH ImpactTG pH Impact

pH Stability

0

20

40

60

80

100

120

3 4 5 6 7 8 9 10

pH

Rel

ativ

e A

ctiv

ity (%

pH Range: 4-9Optimal pH: 6-7

Protein substrate specificity of TGProtein substrate specificity of TG

MilkCasein

a-Lactoalbuminb-Lactoglobulin

EggEgg whiteEgg yolk

Wheat

Meat

CollagenGelatinMyosinActin

Soybean

Food Protein Reaction Strength+++

++

+++

++++

++++++

-+++

+++ reacts very well

+ reacts depending on conditions

++ reacts well

- mostly does not react

Reaction time and temperature:Reaction time and temperature:speed of reactionspeed of reaction

Temperature Time5℃ 240min15℃ 105min20℃ 70min30℃ 35min40℃ 20min

Required time for the reactions at each temperature to the same extent as the reaction at pH6.0, 50℃ 10min

Functions & BenefitsFunctions & Benefits in Food Processingin Food Processing

Cross-linking protein molecules

Increase gel strength

Provide food bond

Modify protein property

Reaction Function Benefit-Texture improvement of gel based foods, such as sausage, surimi, yogurt, cheese and etc.-Cost reduction by reducing necessary protein levels

-Bonding meat pieces into desired size or shape. (portion control, ham)

-Reduced syneresis by improved water retention

-Smooth, thick texture

-Pasta texture improvement

ACTIVA® STG-MS

Super CurdACTIVA® TI ACTIVA® YG

ACTIVA® TI

Surimi Noodle / PastaBonding

Ham Sausage Tofu

Dairy

Specific ACTIVASpecific ACTIVA®® Preparation ApplicationsPreparation Applications

ACTIVA® TI

ACTIVA® TI

ACTIVA® RM/GS

ACTIVA® BFACTIVA® TI

TransglutaminaseTransglutaminaseUse in dairy productsUse in dairy products

Use of TG in Dairy ProductsUse of TG in Dairy Products

Casein protein has a high concentration of Casein protein has a high concentration of Glutamine and Lysine, making it a great Glutamine and Lysine, making it a great substrate for TG activitysubstrate for TG activity

ACTIVAACTIVA®® YG YG –– for use in situations where raw for use in situations where raw milk is being used in the processmilk is being used in the processACTIVAACTIVA®® TI TI –– used when processed milk is being used when processed milk is being usedused

Can develop enhanced structure and handling Can develop enhanced structure and handling characteristicscharacteristics

Use of Use of TransglutaminaseTransglutaminase in Cheese in Cheese ProductsProducts

TG in Natural Cheese ApplicationsTG in Natural Cheese Applications

Good potential functionality in cheese Good potential functionality in cheese manufacturemanufactureUse of low levels of TG in formulation can offer Use of low levels of TG in formulation can offer enhanced yield and cost savingsenhanced yield and cost savings

Retain more whey proteinRetain more whey proteinRetain more moistureRetain more moisture

Mozzarella Cheese StudyMozzarella Cheese StudyExamine use of TG incorporated into industrial Examine use of TG incorporated into industrial processprocess

TG either denatured, or active in final productTG either denatured, or active in final product

ResultsResultsBest use: 0.0625 units TG/gram proteinBest use: 0.0625 units TG/gram protein

UndenaturedUndenatured processprocess~$1.68/ton cheese produced~$1.68/ton cheese produced0.46% increase in final yield0.46% increase in final yield

9.2 lb increase per ton cheese produced9.2 lb increase per ton cheese produced~$17.00 savings/ton~$17.00 savings/ton

OrganolepticOrganoleptic and physical quality same or better than and physical quality same or better than control over study time (4 weeks)control over study time (4 weeks)

TG in Cheese Sauce ApplicationsTG in Cheese Sauce Applications

The addition of TG to a cheese sauce The addition of TG to a cheese sauce application can give distinct advantagesapplication can give distinct advantages

Melt RestrictionMelt RestrictionReduction of Reduction of ““oiling offoiling off”” upon meltingupon meltingEnhanced elasticityEnhanced elasticityImproved shredImproved shredReplacement of Replacement of texturizingtexturizing ingredientsingredients

Milk protein: Whey powder, whey isolateMilk protein: Whey powder, whey isolate

Study Study –– TG use in Cheese SauceTG use in Cheese Sauce

Enhanced Melt restriction with dramatic Enhanced Melt restriction with dramatic reduction in reduction in ““oiling outoiling out””

Control - Without TG

Avg: 0.5”high x2.25”x axis x2.5”y axis

#101 - With TG (0.041% TI)

Avg: 0.75”high x1.75”x axis x1.5”y axis

Melt test: 1” cubes heated covered at 400F for 7 minutes

Study Study -- TG use in Cheese Sauce TG use in Cheese Sauce Melt Restriction and Moisture DataMelt Restriction and Moisture Data

pH Fat MoistureTotal

ProteinSalt

Total Protein

FatTotal

ProteinFat

3902 - 2001Pasteurized Process CheeseSauce

Control 0.0000 5.28 24.72 45.40 --- 2.61 --- --- --- --- 50 x 11

3902 - 2002Pasteurized Process CheeseSauce - 100% texturants

0.2500 0.0410 5.30 25.09 43.74 --- 2.87 --- --- --- --- 42 x 13

3902 - 2003Pasteurized Process CheeseSauce - 50% texturants

0.2500 0.041 5.32 24.51 46.10 --- 2.56 --- --- --- --- 45 x 12

3902 - 2004Pasteurized Process CheeseSauce - 25% texturants

0.2500 0.0410 5.30 25.14 46.88 --- 2.50 --- --- --- --- 46 x 10

3902 - 2005Pasteurized Process CheeseSauce - 0% texturants

0.2500 0.0410 5.31 26.84 45.39 --- 2.56 --- --- --- --- 53 x 12

3902 - 5101 Cream Cheese Control 0.0000 4.57 40.30 48.29 --- --- 2.62 16.26 --- --- ---3902 - 5102 Cream Cheese 0.0200 0.0030 4.55 42.03 45.96 --- --- 1.28 3.32 --- --- ---3902 - 5103 Cream Cheese 0.0400 0.0050 4.55 42.26 46.62 --- --- 1.40 4.91 --- --- ---3902 - 5104 Cream Cheese 0.0500 0.0060 4.57 40.61 47.12 --- --- 1.47 4.39 --- --- ---3902 - 5105 Cream Cheese Control 0.0000 4.70 42.02 49.34 --- --- --- --- --- --- ---3902 - 5106 Cream Cheese 0.0200 0.0030 4.65 44.29 46.29 --- --- 1.28 0.68 --- --- ---3902 - 5107 Cream Cheese 0.0400 0.0050 4.60 40.58 47.38 --- --- 1.47 2.06 --- --- ---3902 - 5108 Cream Cheese 0.0500 0.0060 4.53 40.09 46.30 --- --- 1.47 2.20 --- --- ---

Kraft Philadelphia Cream Chease 4.65 31.42 53.95 6.25Kraft Philly Chease Spread 4.83 26.84 59.39 4.98Best Choice Cream Cheese Spread 4.82 26.90 58.92 5.30Best Choice Cream Cheese 4.95 32.56 53.66 5.17

TI Amount(units/gram)

Formulation Number

Ajinomoto Analytical Testing Results v5

CheeseWhey Analysis Milk AnalysisCheese Analysis

TI Amount(percentage)

Melt Test(mm)

A positive melt restriction is seen when TG is A positive melt restriction is seen when TG is used in combination with other used in combination with other texturantstexturants

Melt test measurements are width vs. heightMelt test measurements are width vs. height

103

101

102

Control

104

Enhanced moisture holding characteristics while Enhanced moisture holding characteristics while maintaining comparable textural attributesmaintaining comparable textural attributes

46% moisture in #102 vs. 43% moisture in #10146% moisture in #102 vs. 43% moisture in #101Studies indicate similar cost savings benefits Studies indicate similar cost savings benefits seen when used in spreadable/pourable cheese seen when used in spreadable/pourable cheese sauces sauces ACTIVAACTIVA®® TG has the potential for delivery of TG has the potential for delivery of cost reduced cheese saucecost reduced cheese sauce--type products with type products with enhanced physical characteristicsenhanced physical characteristics

Study Study –– TG use in Cheese SauceTG use in Cheese Sauce

Use of Use of TransglutaminaseTransglutaminase in Yogurt in Yogurt ProductsProducts

Increased gel strengIncreased gel strengthth/viscosity/viscosityImproved creaminess Improved creaminess Reduced syneresisReduced syneresisReduction of added dry matter and/or Reduction of added dry matter and/or stabilizersstabilizersImproved gelling capacity of reduced fat Improved gelling capacity of reduced fat yogurtyogurt

TG use in Yogurt TG use in Yogurt -- BenefitsBenefits

ACTIVAACTIVA®® Preparations for Dairy Preparations for Dairy ProductsProducts

ACTIVAACTIVA®® TITIFor use in preFor use in pre--processed milkprocessed milk

ACTIVAACTIVA®® YGYGFor use in raw milkFor use in raw milk

Raw milk contains TG inhibitor. YG has reducing agent Raw milk contains TG inhibitor. YG has reducing agent to eliminate inhibitionto eliminate inhibition

Yogurt Yogurt –– Enhanced Gel StrengthEnhanced Gel Strength

0

500

1000

1500

2000

2500

-TG -TG 0.5 U/g p 1 U/g p

Visco

sity（

ｍPa

・ｓ

） 10％ NFDM11％ NFDM

Powder milk(10Powder milk(10％％, 11, 11％％ NFDM, milk fat 2.7NFDM, milk fat 2.7％）％）↓↓ ACTIVA TI ACTIVA TI (0(0，，0.5, 1U/g protein)0.5, 1U/g protein)

Incubation Incubation （（2525℃℃, 2 hours), 2 hours)

↓↓Cooling after enzyme deactivation Cooling after enzyme deactivation （（7070℃℃））↓↓Starter additionStarter addition

↓↓Cooling after fermentationCooling after fermentation

↓↓StirringStirring

↓↓Stirred yogurt sampleStirred yogurt sample

↓↓Cooling O/NCooling O/N→→Viscosity measurementViscosity measurement

Viscosity is increased with TG, achieving equivalence at 1U per gram protein when treating 10% SNF addition to 11% SNFWith ACTIVA® YG, dosage should be 1/3 that of ACTIVA® TI when using raw milk

Study Study –– Reduction of NFDMReduction of NFDM

Yogurt - NFDM replacement by TG-YG

0

200

400

600

800

1000

1200

Vis

cosi

ty (c

ps) 5% NFDM

4% NFDM4% NFDM/0.3% YG4% NFDM/0.6% YG3% NFDM/0% YG3% NFDM/0.6% YG

Addition of ACTIVA® YG allows reduction of NFDM levels while achieving desired viscosification

Potential Savings through NFDM Potential Savings through NFDM ReductionReduction

ACTIVA YG cost reduction calculationNFDM WPC80

5.45$

Added Reduced cost Total costACTIVA NFDM WPC80 Total ACTIVA YG unit/g milk protein

Dosage reduction

Reduced cost / 1t milk

Reduced cost / 1t milk

Reduced cost / 1 ton milk

0.05 0.2% 9.08$ 0.2% -$ (7.62)$ 0.1 0.3% 18.17$ 0.3% -$ (15.24)$ 0.2 0.7% 36.33$ 0.7% -$ (30.48)$ 0.3 1.0% 54.50$ 1.0% -$ (45.73)$ 0.6 2.0% 109.00$ 2.0% -$ (91.45)$

Shelf Life Textural StabilityShelf Life Textural Stability

Yogurt Viscosity

0

500

1000

1500

2000

2500

3000

3.6%Protein, 0 U

TG

3.5%Protein, 0 U

TG

3.5%Protein, 0.1

U TG

3.5%Protein, 0.2

U TG

3.4%Protein, 0 U

TG

3.4%Protein, 0.2

U TG

3.4%Protein, 0.3

U TG

3.3%Protein, 0.4

U TG

cps

1 day7 days14 days21 days42 days

Addition of ACTIVA® YG allows a higher final viscosity through the end of shelf life.

Enhanced Gelling with Lower MFEnhanced Gelling with Lower MF

0

5

10

15

20

25

30

35

-TG -TG 1 u/g p 2 u/g p

Brea

king s

treng

th（

g/cm

2)

MF 3.6％ MF 1.0％Powder milkPowder milk（（8.28.2％％ NFDM, 3.6% or 1.0NFDM, 3.6% or 1.0％％ milk fatmilk fat））↓↓ ACTIVA TIACTIVA TI (0, 1, 2U/g protein)(0, 1, 2U/g protein)

Incubation Incubation （（2525℃℃, 2 hours), 2 hours)

↓↓Cooling after enzyme deactivation Cooling after enzyme deactivation （（7070℃℃））↓↓Starter additionStarter addition

↓↓StuffingStuffing

↓↓FermentationFermentation

↓↓Set type yogurtSet type yogurt

↓↓Cooling O/N Cooling O/N →→ Breaking strength measurementBreaking strength measurement

An increase in gelling character and creaminess can be obtained in low fat yogurt products through the use of TG without the necessity of thickening agents. With ACTIVA® YG, dosage should be 1/3 of ACTIVA® TI when using raw milk.

TransglutaminaseTransglutaminaseRegulationsRegulations

TG: Use Levels Affirmed as GRASTG: Use Levels Affirmed as GRAS

FOOD CATEGORY USE Level as TG

Proc Meat & Poultry Non-Standard & select Std Product Chicken Breast Products

65 ppm

100 ppm Processed Seafood GMP*

Meat Analogs/Substitutes GMP*

Natural Cheese/Processed Cheese GMP*

Cream Cheese GMP*

Refrigerated Yogurt GMP*

Frozen Desserts GMP*

* GRAS Notice 0095

CanadaCanada

Canada Gazette Part II Canada Gazette Part II vol. 138, No. 9vol. 138, No. 9

Apr 22, 2004Apr 22, 2004

NonNon--standardized prepared fish productsstandardized prepared fish products

Simulated meat productsSimulated meat products

NonNon--standardized cheesestandardized cheese

Processed cheese and cream cheese productsProcessed cheese and cream cheese products

Yogurt and NonYogurt and Non--standardized frozen dairy dessertsstandardized frozen dairy desserts

at GMP levelat GMP level

TransglutaminaseTransglutaminaseIngredient StatementsIngredient Statements

Labeling of TG by itself:Labeling of TG by itself:Current RegulationsCurrent Regulations

Area Category Remarks

Japan Food additive If enzyme is active, state "enzyme"

EU Processing aid No statement required

USA GRAS State "enzyme", "TG enzyme" or "TGP enzyme"

Canada Food additive State "transglutaminase"

TransglutaminaseTransglutaminase preparations preparations seriesseries

ACTIVA® RM BindingSodium caseinate, Maltodextrin, Transglutaminase

Sodium caseinate, Maltodextrin, Enzyme Contains Milk

ACTIVA® GS

Binding (strong bind, stable solution)

Sodium Chloride, Gelatin, Trisodium Phosphate, Maltodextrin, Transglutaminase, Safflower Oil

Salt, Gelatin, Trisodium Phosphate, Enzyme None

ACTIVA® TI Sausage, Ham Maltodextrin, Transglutaminase Maltodextrin, Enzyme None

ACTIVA® BF HamAmmonium glutamate, Maltodextrin, Transglutaminase, Safflower oil

Ammonium glutamate, Enzyme None

ACTIVA® YG Yogurt, Cheese

Lactose, Yeast extract, Maltodextrin, Safflower oil, Transglutaminase

Lactose, Yeast Extract, Enzyme None

AllergenPreparation Typical usage

Ingredients (as shown on the label)

Ingredient statement in final products

USAUSA

*Note: Information is for reference only. Product labeling must be determined by manufacturer.

Lactose not considered allergen

Gelatin not considered allergen

TransglutaminaseTransglutaminase preparations preparations seriesseries

ACTIVA® RM Binding Sodium caseinate, Maltodextrin, Transglutaminase

Sodium caseinate, Maltodextrin, Transglutaminase

Contains Milk

ACTIVA® GS

Binding (strong bind, stable solution)

Sodium Chloride, Gelatin, Trisodium Phosphate, Maltodextrin, Transglutaminase, Safflower Oil

Salt, Gelatin, Trisodium Phosphate, Transglutaminase

None

ACTIVA® TI Sausage, Ham

Maltodextrin, Transglutaminase Maltodextrin, Transglutaminase None

ACTIVA® BF HamAmmonium glutamate, Maltodextrin, Transglutaminase, Safflower oil

Ammonium glutamate, Transglutaminase None

ACTIVA® YG Yogurt, Cheese

Lactose, Yeast extract, Maltodextrin, Safflower oil, Transglutaminase

Lactose, Yeast Extract, Transglutaminase None

AllergenPreparation Typical usage

Ingredients (as shown on the label)

Ingredient statement in final products

CanadaCanada

*Note: Information is for reference only. Product labeling must be determined by manufacturer.

Lactose not considered allergen

Gelatin not considered allergen

Determining whether “fish” must be on label

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