Dairy Applications for Transglutaminase

Ajinomoto Food Ingredients, LLC

Transglutaminase
Enzyme Basics

Transglutaminase

TG is an enzyme that covalently cross-links protein Found throughout nature (from microorganisms to mammals)

ACTIVA Transglutaminase

TG enzyme used by Ajinomoto is produced via fermentation by a GMO-free microorganism Marketed as TG preparations by Ajinomoto Food Ingredients (AFI) under the ACTIVA brand name

Linking Reaction

TG

Transglutaminase: Cross-linking Mechanism

Carbonyl Group of Glutamine Amino group of Lysine

| | GLN-C-NH2 + H-N-LYS || || || | | H O TG | | GLN-C-N-LYS NH3 || | || | | | OH

Forms permanent covalent bonds unaffected by freezing or cooking conditions

ACTIVA TG Temperature Impact

Activity and Stability over Temperature Range
100 Relative Activity (%) 80 60 40 20 0 0 10 20 30 40 50 60 70 Temperature (C) Relative Activity Stability

Temperature range: 32 to ~150F Optimal Temperature range: 115-130F (45-55C) (Specific denaturation temp depends on system composition)

ACTIVA TG pH Impact
pH Stability
120
Relative Activity (%

100 80 60 40 20 0 3 4 5 6
pH

10

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

Protein substrate specificity of TG

Food Protein Casein Milk Reaction Strength +++ + + + ++ ++ Collagen Meat Gelatin Myosin ++ +++ +++ +++
+++ reacts very well ++ reacts well + reacts depending on conditions - mostly does not react

a-Lactoalbumin b-Lactoglobulin Egg white Egg yolk

Egg Wheat

Actin Soybean

Reaction time and temperature: speed of reaction

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

Temperature 5 15 20 30 40

Time 240min 105min 70min 35min 20min

Functions & Benefits in Food Processing

Reaction Function
Increase gel strength

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

Cross-linking protein molecules

Provide food bond

Modify protein property

Specific ACTIVA Preparation Applications

ACTIVA RM/GS ACTIVA TI ACTIVA STG-MS

Bonding ACTIVA BF ACTIVA TI

Surimi Super Curd ACTIVA TI ACTIVA TI

Noodle / Pasta ACTIVA YG ACTIVA TI Dairy

Ham

Sausage

Tofu

Transglutaminase
Use in dairy products

Use of TG in Dairy Products

Casein protein has a high concentration of Glutamine and Lysine, making it a great substrate for TG activity
ACTIVA YG for use in situations where raw milk is being used in the process ACTIVA TI used when processed milk is being used

Can develop enhanced structure and handling characteristics

Use of Transglutaminase in Cheese Products

TG in Natural Cheese Applications

Good potential functionality in cheese manufacture Use of low levels of TG in formulation can offer enhanced yield and cost savings
Retain more whey protein Retain more moisture

Mozzarella Cheese Study

Examine use of TG incorporated into industrial process

TG either denatured, or active in final product Best use: 0.0625 units TG/gram protein
Undenatured process ~$1.68/ton cheese produced 0.46% increase in final yield

Results

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

Organoleptic and physical quality same or better than

control over study time (4 weeks)

TG in Cheese Sauce Applications

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

Melt Restriction Reduction of oiling off upon melting Enhanced elasticity Improved shred Replacement of texturizing ingredients

Milk protein: Whey powder, whey isolate

Study TG use in Cheese Sauce

Enhanced Melt restriction with dramatic reduction in oiling out

Control - Without TG

#101 - With TG (0.041% TI)

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

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

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

Study - TG use in Cheese Sauce

Melt Restriction and Moisture Data
Ajinomoto Analytical Testing Results v5
Formulation Number 3902 - 2001 Control 3902 - 2002 101 Cheese Pasteurized Process Cheese Sauce Pasteurized Process Cheese Sauce - 100% texturants TI Amount TI Amount
(units/gram) (percentage)

pH 5.28 5.30

Cheese Analysis Total Fat Moisture Protein 24.72 25.09 45.40 43.74 -----

Salt 2.61 2.87

Whey Analysis Milk Analysis Total Total Fat Fat Protein Protein -----------------

Melt Test (mm) 50 x 11 42 x 13

Control 0.2500

0.0000 0.0410

Pasteurized Process Cheese 3902 - 2003 102 Sauce - 50% texturants Pasteurized Process Cheese 3902 - 2004 103 Sauce - 25% texturants Pasteurized Process Cheese 3902 - 2005 104 Sauce - 0% texturants 3902 3902 3902 3902 3902 3902 3902 3902 - 5101 - 5102 - 5103 - 5104 - 5105 - 5106 - 5107 - 5108

0.2500

0.041

5.32

24.51

46.10

---

2.56

---

---

---

---

45 x 12

0.2500

0.0410

5.30

25.14

46.88

---

2.50

---

---

---

---

46 x 10

0.2500

0.0410 0.0000 0.0030 0.0050 0.0060 0.0000 0.0030 0.0050 0.0060

5.31 4.57 4.55 4.55 4.57 4.70 4.65 4.60 4.53 4.65 4.83 4.82 4.95

26.84 40.30 42.03 42.26 40.61 42.02 44.29 40.58 40.09 31.42 26.84 26.90 32.56

45.39 48.29 45.96 46.62 47.12 49.34 46.29 47.38 46.30 53.95 59.39 58.92 53.66

------------------6.25 4.98 5.30 5.17

2.56 -----------------

--2.62 1.28 1.40 1.47 --1.28 1.47 1.47

--16.26 3.32 4.91 4.39 --0.68 2.06 2.20

-------------------

-------------------

53 x 12 -----------------

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

Cream Cheese Control Cream Cheese 0.0200 Cream Cheese 0.0400 Cream Cheese 0.0500 Cream Cheese Control Cream Cheese 0.0200 Cream Cheese 0.0400 Cream Cheese 0.0500 Kraft Philadelphia Cream Chease Kraft Philly Chease Spread Best Choice Cream Cheese Spread Best Choice Cream Cheese

Melt test measurements are width vs. height

Study TG use in Cheese Sauce

Enhanced moisture holding characteristics while maintaining comparable textural attributes

46% moisture in #102 vs. 43% moisture in #101

Studies indicate similar cost savings benefits seen when used in spreadable/pourable cheese sauces ACTIVA TG has the potential for delivery of cost reduced cheese sauce-type products with enhanced physical characteristics

Use of Transglutaminase in Yogurt Products

TG use in Yogurt - Benefits

Increased gel strength/viscosity Improved creaminess Reduced syneresis Reduction of added dry matter and/or stabilizers Improved gelling capacity of reduced fat yogurt

ACTIVA Preparations for Dairy Products

ACTIVA TI

For use in pre-processed milk For use in raw milk

Raw milk contains TG inhibitor.

ACTIVA YG

YG has reducing agent

to eliminate inhibition

Yogurt Enhanced Gel Strength

2500 ViscosityPa 2000 1500 1000 500 0
-TG -TG 0.5 U/g p 1 U/g p

11 NFDM

10 NFDM

Powder milk(10, 11 NFDM, milk fat 2.7 A CTIVA TI (00.5, 1U/g protein) Incubation 25, 2 hours) Cooling after enzyme deactivation 70 Starter addition Cooling after fermentation Stirring Stirred yogurt sample Cooling O/NViscosity measurement

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

Study Reduction of NFDM

Yogurt - NFDM replacement by TG-YG
1200

1000

Viscosity (cps)

800

5% NFDM 4% NFDM 4% NFDM/0.3% YG 4% NFDM/0.6% YG 3% NFDM/0% YG 3% NFDM/0.6% YG

600

400

200

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

Potential Savings through NFDM Reduction

ACTIVA YG cost reduction calculation
NFDM $ 5.45 Added ACTIVA ACTIVA YG unit/g milk protein Reduced cost NFDM Dosage Reduced reduction cost / 1t milk WPC80

WPC80 Reduced cost / 1t milk

Total cost Total Reduced cost / 1 ton milk

0.05 0.1 0.2 0.3 0.6

0.2% 0.3% 0.7% 1.0% 2.0%

$ 9.08 $ 18.17 $ 36.33 $ 54.50 $ 109.00

0.2% 0.3% 0.7% 1.0% 2.0%

$ $ $ $ $

$ $ $ $ $

(7.62) (15.24) (30.48) (45.73) (91.45)

Shelf Life Textural Stability

Yogurt Viscosity
3000 2500 2000 cps 1500 1000 500 0
3.6% 3.5% 3.5% 3.5% Prot ein, 0 U Prot ein, 0 U Prot ein, 0.1 Prot ein, 0.2 TG TG U TG U TG 3.4% 3.4% 3.4% 3.3% Prot ein, 0 U Prot ein, 0.2 Prot ein, 0.3 Prot ein, 0.4 TG U TG U TG U TG

1 day 7 days 14 days 21 days 42 days

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

Enhanced Gelling with Lower MF

35 Breaking strengthg/cm2) 30 25 20 15 10 5 0 -TG -TG 1 u/g p 2 u/g p

MF 3.6

MF 1.0

Powder milk8.2 NFDM, 3.6% or 1.0 milk fat A CTIVA TI (0, 1, 2U/g protein) Incubation 25, 2 hours) Cooling after enzyme deactivation 70 Starter addition Stuffing Fermentation Set type yogurt Cooling O/N Breaking 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.

Transglutaminase
Regulations

TG: Use Levels Affirmed as GRAS

FOOD CATEGORY Proc Meat & Poultry Non-Standard & select Std Product Chicken Breast Products Processed Seafood Meat Analogs/Substitutes Natural Cheese/Processed Cheese Cream Cheese Refrigerated Yogurt Frozen Desserts USE Level as TG 65 ppm 100 ppm GMP* GMP* GMP* GMP* GMP* GMP*

* GRAS Notice 0095

Canada

Canada Gazette Part II vol. 138, No. 9 Apr 22, 2004

Non-standardized prepared fish products Simulated meat products Non-standardized cheese Processed cheese and cream cheese products Yogurt and Non-standardized frozen dairy desserts at GMP level

Transglutaminase
Ingredient Statements

Labeling of TG by itself: Current Regulations

Area Japan EU Category Food additive Remarks If enzyme is active, state "enzyme"

Processing aid No statement required State "enzyme", "TG enzyme" or "TGP enzyme" State "transglutaminase"

USA

GRAS

Canada

Food additive

USA

Transglutaminase preparations
Typical usage Binding Binding (strong bind, stable solution) Sausage, Ham Ham Ingredients (as shown on the label)

series
Allergen

Preparation

Ingredient statement in final products

ACTIVA RM

Sodium caseinate, Maltodextrin, Sodium caseinate, Maltodextrin, Enzyme Transglutaminase

Contains Milk

ACTIVA GS

Sodium Chloride, Gelatin, Gelatin not considered allergen Trisodium Phosphate, Salt, Gelatin, Trisodium None Maltodextrin, Transglutaminase, Phosphate, Enzyme Safflower Oil Maltodextrin, Transglutaminase
Maltodextrin, Enzyme None

ACTIVA TI

ACTIVA BF

Ammonium glutamate, Ammonium glutamate, Maltodextrin, Transglutaminase, Enzyme Safflower oil Lactose, Yeast extract, Maltodextrin, Safflower oil, Transglutaminase

None

ACTIVA YG

Yogurt, Cheese

Lactose, Yeast Extract, None Enzym e Lactose not considered allergen

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

Canada
Preparation

Transglutaminase preparations
Typical usage Ingredients (as shown on the label)
Ingredient statement in final products Sodium caseinate,

series
Allergen

ACTIVA RM

Binding

Sodium caseinate, Maltodextrin, Maltodextrin, Transglutaminase

Contains Milk

Transglutaminase

ACTIVA GS

Binding (strong bind, stable solution) Sausage, Ham Ham

Sodium Chloride, Gelatin, Salt, Gelatin, Trisodium Trisodium Phosphate, allergen Phosphate, Gelatin not considered None Maltodextrin, Transglutaminase, Transglutaminase Determining whether fish must Safflower Oil be on label
Maltodextrin, Transglutaminase

ACTIVA TI

Maltodextrin, Transglutaminase

None

ACTIVA BF

Ammonium glutamate, Ammonium glutamate, Maltodextrin, Transglutaminase, Transglutaminase Safflower oil Lactose, Yeast extract, Maltodextrin, Safflower oil, Transglutaminase

None

ACTIVA YG

Yogurt, Cheese

Lactose, Yeast Extract, Lactose not considered allergen None Transglutam inase

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

Thank you very much