yieldmax customer jan2010

YieldMAX™ PL - enhancing Fat Recovery in Mozzarella / Pizza Cheese

2010 YieldMAX in pasta filata2

Background

Cost reduction by enhancing fat and protein retention an ongoing focus in the cheese industryMuch effort has concentrated on designing optimal cheese making equipment and process parameters, e.g. cutting timeDespite these efforts, fat and protein losses are observed especially after cutting, during early stage of stirring and during stretchCheese yield may be increased by protein concentration. However, this approach may lead to impaired flavour, texture and melting propertiesThe aim of this overview is to explain about the novel enzyme technology using a phospholipase YieldMAX™ PL to increase cheese yield and thereby production economy in the manufacturing of Pasta Filata type cheese

Business case on YieldMAX in Mozzarella/Pizza cheese


Where does the value of YieldMAX come from

YieldMAX modifies part of the fat globule membraneby acting on the phospholipidsAs a result more fat is retained in the cheeseRecovery of protein also increasesWater binding capacity is improved Altogether yield increases about 2%

YieldMAX results in more cheese from thesame amount of milk


What are the economic benefits of increasing fat recovery in pizza cheese manufacturing

Higher cheese yieldMore cheese from the same milk

Higher capacity of plant

Milk fat has the highest value when used in cheeseHigher recovery of fat Option to take more sweet cream out of cheese milk

Sweet cream has higher value than whey cream

Lower volume of whey creamRecovery of whey fat often lower than milk fat in cheeseUsing whey cream increases risk of bacteriophagesHigh usage may influence cheese quality negatively

Much less fat in stretching waterFat from stretching water is non-kosher


Example of economic benefits of using YieldMAX in 1m lbs milk/day pizza cheese production

Benefit of using YieldMAX360 days/year

Element Q ValueExtra cheese 792,000# 1,1m$

YieldMAX cost 570,000$

Gross earnings 539,000$

Gross return on investment 95%

Gross earning at cheese price of 1,6$/lbs

697,000$

Cheese price wheregross RoI is 50%

1.08$/lbs

Constants used

Cheese price 1.4$/lbs

Standard cheese yield

11%

Fat in cheese milk

2,6%

Yield benefit 2%

Milk volume/day

1 million lbs

Pilot plant and industrial validation of concept value


How to use YieldMAX™ PL

Simple additionAdd to cheese milk 20 min before renneting

While filling vatDosage (LMPS)

5 units per g of fat1.5 l per 25,000 kg milk≈45 fl oz per 55,000# milk

Up to 10x dilution gives no loss in activity YieldMAX™ does not affect the coagulation.


Pilot plant cheese trial design

Cheese type: Pizza 40+Variable: Addition of YieldMAX™ PL, 20 min

before rennetingAddition rate: 5 u per g fatVats per day: 4 vats: 2 control and 2 experimentalNumbers of days: 4

Cheese yield std. deviation between control vats = 56 g from 150 kg of milk


Effect on cheese - Pilot scale Mozzarella trial (LMPS)

VariableUntreated

(n = 8)With YieldMAX

Difference %

Fresh curds yield, % 10.80 + 1.1

Cheese yield a, % 10.35 + 3.2

Fat in whey, % 0.132 - 4.3

Fat in cooking water, % 0.300 - 2.1

Moisture in cheese, % 47.12 + 1.9

DM yield b, % 46.43 + 1.5

Fat recovery, % 87.20 + 0.4

Protein recovery, % 76.69 + 1.7a Cheese weight divided by milk weight. b Cheese dry matter divided by milk dry matter.

Lilbæk H.M .. ... .2006. J. D.... S... 89 .11. 4114.4125

P...... ........... .........

U......... ......... .. ....

.......

1H.NMR ..... ......

........ ..... .......

..... ..........

L... ... .... .. .... ...

.......... .....

Statistically significant


Cheese Functionality - (Confocal Laser Scanning Microscopy)

Fresh cheese, prior to stretching

3 days

14 days

28 days

Control YieldMAX™

No significant difference in image analysis with respect to

size and numbers of fat globule unitsdegree of coalescencecoarseness of protein network

Microstructure shows swelling of casein network and diminishing of whey pockets during storage

Meltability, stretchability and browning similar in control and treated cheeses (Ref: Lilbaek et al. 2006 Journal Dairy Science


Cheese Control YieldMAX SEM Difference P-value

Phospholipid retention, % 45.4 79.3 3.2 +75 % <0.001

Whey Control YieldMAX

Total phospholipids, µMSphingomyelin, µM

183.052.4

68.446.4

Lysophospholipids preferentially retained

Correlation between PL content and water content !

H.... .. ... 1985 J D.... S.. 68. 1603.1607

D.... .. ... 1988 J F... S.. 63. 1018.1023

T..... .. ... 2001 L... 81. 429.442

G...........L.... .. ... 2006 J D.... S.. 89. 454.467

Retention of phospholipids in cheese increased


Standard whey fat flow diagram

Milk in Cheese out

Mozzarella without YieldMAX™ PL

Cheese vat Stretching

Whey, 0.2-0.3% fat

Cooking water,2-4% fat

Whey, 0.05% fatWhey cream(25-30% fat)


YieldMAX whey fat flow diagram

Less whey cream to handle – Less fat in defatted whey

Milk in Cheese out

Mozzarella with YieldMAX™ PL

Cheese vat Stretching

Whey, (<0.2-0.3% fat)

Cooking water,(1-3% fat)

Whey, < 0.05% fatWhey cream(25-30% fat)Less volume


Improved whey fat separation

YieldMAX PL treatment

% Fat in whey after centrifugation

95 % Confidence interval

+ 0,031 (0,028;0,033)

- 0,040 (0,038;0,042)

Impact of YieldMax PL treatment of cheese milk on separability of whey from small scale (150 L cheese milk) mozzarella cheese production. Values in brackets are 95% confidence intervals.


Lower recovery of whey fatEffect of recirculation of whey cream

Whey cream fat is to a lesser extend in globular form

More susceptible to oxidationNon globular fat will form large pods of on surface

Less whey fat is retained in cheese lowering the overall fat retentionQuality issues

Recirculation of phageMoisture retention Flavour defects at higher dosage levels

% whey fat recovered

% total fat recovered

% FDM % yield

93.0 93,0 55,2 10,54

80,0 92,4 55,0 10,51

60,0 91,4 54,8 10,44

40,0 90,5 54,6 10,38

Based on whey cream fat as 5% of total fat: Milk 3.88% fat, 2.49% caseinCheddar with 38% moisture

Uni. of Wisconsin, October 1997


Industrial validation – effect on cheese

Yield data from industrial trials (≥500 cheese vats)

% yieldincrease

95%interval

Recipe 1 1,4 (0,3; 2,4)

Recipe 2 0,7 (-0,5; 1,9)

Recipe 3 2,1 (0,1; 4,0)

Recipe 4 (high fat) 3,8 (1,6; 6,0)

Significant increases seen infat in dry mattermoisture in cheesefat retentionprotein retention in two recipes

H...... E .. ... .2006. T.. A......... J. .. D.... T.... 61 .2. 179.182


Industrial validation - effects on whey fat

Significant decrease in whey fatTypical pilot plant data shows decrease from 0.22 to 0.19%9-10% decrease in industrial trialsSame effects seen on all tested recipes

Whe

y fa

t %LS

Mea

ns0,2

0,25

0,3

0,35

0,4

0,45

Recipe 1Recipe 2Recipe 3Recipe 4

Control pLipase

Treatment

LS Means Plot


Industrial validation - Effects on mixer/molder water

Fat recovery in cheese increased – industrial dataLess fat/protein loss in cooking water

Treated vats


Effects on process - foaming

YieldMAX addition may lead to some foam formation

To be addressed depending on individual circumstancesSpraying of water dropletsMinimizing air incorporation during processing (plant design)Ultrasound treatmentOther anti foaming methods

Untreated Treated


Summarizing the current knowledge -

A suggested mechanism for cheese yield increase

MilkC.....

W...

+ YieldMAX™ PL

. 1.3.


Process step Effect of YieldMAX™ PL

Incubation of milk with phospholipase

• Conversion of phospholipids to lysophospholipids• Release of lysophospholipids from fat globules





Coagulation • Complexation of lysophospholipids with casein






Curd cutting, stirring, scalding

• Lysophospholipids act as an emulsifier- protect fat globules from rupture - re-emulsify fat “leaked out” from damaged globules








Whey drainage • Lysophospholipid – casein complexes retained in the curd

• Less un-emulsified liquid fat is lost in the whey








Whey drainage • Lysophospholipid – casein complexes retained in the curd

• Less un-emulsified liquid fat is lost in the whey

Curd stretching • Lysophospholipid acts as an emulsifier- protect fat globules from breaking- re-emulsify fat “leaked out” from damaged globules- reduce syneresis more water is retained in cheese

Activity of YieldMAX in the milk


Phopsholipids in milk products

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

Milk

Skimmed

milk

Cream 40

%Butt

er

Butterm

ilk

Anhyd

rous m

ilk fat

% w

/w % in product% in solids

Based upon data fra Walstra et.al 2006. Dairy Science and Technology CRC Press


Total amount in milk 0.02 – 0.04 % w/wIn fresh raw milk ∼80% is on the fat globule surfaceLipoprotein aggregates/vesicles in milk plasma

50 – 400 ..

1 – 10 µm

Phospholipids in milk


Phospholipids in milk


Phospholipids in milk (mol-%)

√ Phosphatidyl ethanolamine (PE) 22-49 %

√ Phosphatidyl choline PC) 21-40 %

√ Phosphatidyl inositol (PI) 4-11 %

√ Phosphatidyl serine (PS) 2-12 %

÷ Sphingomyelin (SM) 18-32 %

Note! Sphingomyelin is not hydrolyzed.


Hydrolysis of milk phospholipids:catalyzed by YieldMAX™ Phospholipase A1

Example of a substrate: Phosphatidyl choline

P........... L............... . F....

....

Y....MAX PL

H

2

0

YieldMAX PL(PLA1)

OO

O

O

OPO

O-

ON

+

Hydrolyzes ester bonds in long-chain fatty acids


Phospholipids can be extracted from milk and analyzed by HPLC

Almost complete depletion of phosphatidylethanolamine and phosphatidylcholine

Development of long chain free fatty acids can be observed

Lysophospholipids are detected in whey, amount corresponds to approx. 5 % of content in hydrolyzed whole milk - 95% remains in cheese matrix

Sphingomyelin is not hydrolyzed by YieldMAX™ PL

0

25000

50000

75000

100000

125000

150000

0 5 10 15 20Time, min

mAU

PE

PCSph

0

25000

50000

75000

100000

125000

150000

0 5 10 15 20

Time, min

mA

U

PE PC

LPE

LPC

Sph

Untreated control

Phospholipase treated

The YieldMAX™ PL action


DH% in whole milk vs. incubation time YieldMAX PL dose 5 or 50 LEU/g fat

Max. Degree of hydrolysis 65-80%

0

10

20

30

40

50

60

70

80

0 5 10 15 20Time (h)

DH

% PE (5 Units /g fat)

PC (5 Units /g fat)

PE (50 Units /g fat)

PC (50 Units /g fat)


Phospholipids are in the fat globules -Lysophospholipids are in serum phase

∼ 50.60 . PC.PE

W.... ....

S... ....

∼ 50.60 . .. ............ PC.PE∼ 100 . ................. LPC.LPE

.Y....MAX

PC LPC

PE LPE

Lilbæk H.M .. ... .2007. J. A..... F... C.... I. .....

C.......... .......... C..........

..........


Fat globule stability unchanged

Whole milk particle size distribution

Particle diameter [µm]

0,01 0,1 1 10 100 1000

Rel

ativ

e di

strib

utio

n [%

]

0

2

4

6

8

10

12

14

16

B.

A.U........

Y....MAX

Phospholipase Control

Fat globules in cheese curd

Documentation on whey streamand casein

Positive effect on whey processing

No effect seen on whey protein fractions or their ratio

No effect seen on casein


Improved processing capabilities of YieldMAX whey

Whey contains less fat due to better separation properties of YieldMAX whey

Data from processing confirm thisLower fat in whey means easier processingLower fat means easier membrane filtration for WPI

Improved functionality of WPIsLower means less risk of oxidative flavor changesReplace fat with low value lactose in WPC

YieldMAX reduces surface tension of the wheyTheoretical explanation on functionality of lyso-phospholipidsProven in laboratory model Customer references

Longer runs between CIP on UFLess fouling


Method:Wilhelmy plate

Sample: Skim milk, separated from treated whole milk

General phenomenon:Surface tension is also reduced upon phospholipase treatment of:

-Cream

-Skim milk

-WPC-solution

-Milk phospholipid

suspension

-In whey from treated

cheese milk

Surface tension is reduced by YieldMAX

Control, no enzymePLA1 F. venenatum (YieldMAX)PLA2 Porcine pancreasPLC B. cereus


YieldMAX has high specificity

Enzyme heat labile - ≥90% of the enzyme is destroyed at low pasteurization of the whey(72°C/160°F, 20 sec)

Side activities removedAmylase and glucoamylases absent according to specsConformity checks also for ß-galactosidase, lipase and proteolytic activity

No effect on whey protein fractions

K-sorbate

Na-benzoateEnzyme


Products incubated with YieldMAX™ PL

3 different commercial whey protein concentrates (WPC)2 WPC from Mozzarella trials with or without use of YieldMAX™ PL4 different commercial whey protein isolates

Sample Whey protein (powder products)

123456789

Whey Protein Concentrate I (34%)Whey Protein Isolate WPC Trial (from Mozzarella production using YieldMAX™ PL)WPC Control (from Mozzarella production)Bovine Lactoferrin ILactoferrin / LactoperoxidaseBioactive Whey ConcentrateBovine Lactoferrin IIWhey Protein Concentrate II


Experimental setup 0.4 g Whey Protein

40 mL Buffer* 200 µL 5% Na-Azid

20 mL A

10 mL B

10 mL C

Mixing WPS

Incubation at 35°C for 21 hr

+ 5 µL YieldMAX PL

+ 5 µL HT YieldMAX PL

SEC SDS-Page

Detect proteins between 2.5 to 200 kDa

Detect proteinsless than 2000 kDa(optimal <200 kDa)


SDS-gel on WPC80

No appearance of new bands

No reduction of bands intensity

No effect observed of YieldMAX™ PL incubation25

Samples: WPC80 with and without YieldMAX™

45

10 ul 20 ul

kDa

116

66,2

35

18,414,4

Trea

ted

13-3

-06

Con

trol

14-3

-06

Trea

ted

21-3

-06

Con

trol

22-3

-06

Trea

ted

13-3

-06

Con

trol

14-3

-06

Trea

ted

21-3

-06

Con

trol

22-3

-06


Bovine Lactoferrin I & Lactoferrin/Lactoperoxidase

SECNo new peaks observedSame level of peak areasNo differences

1 % solutions of products

Bovine Lactoferrin I

-0,5

0

0,5

1

1,5

2

2,5

3

0 20 40 60 80

Time (min)

Abs

orba

nce

220

nm

WPS5

+YieldMAX

+HT YieldMAX

MwSTD

-0,5

0

0,5

1

1,5

2

2,5

3

0 20 40 60 80

Time (min)

Abs

orba

nce

220

nm

WPS7

+YieldMAX

+HT YieldMAX

MwSTD

Lactoferrin/Lactoperoxidase


Effect on casein

1. Sodium caseinate (from Sigma, C-8654), αs1-casein and β-casein were solubilised in 50 mM sodium phosphate buffer, pH 6.4, containing 0.025 % sodium azide. Protein concentration of the solutions were 5 mg/ml2. YieldMAX™ PL (Batch No. 2663736) was added to the solutions at a concentration of 0.05 % (v/v), and samples were incubated for 21 hours at 35ºC.3. Control reactions without YieldMAX™ PL addition were also included.4. Samples were analyzed using SDS-PAGE


SDS-PAGEcaseins incubated with YieldMAX™ PL

1: Molecular weight standards2: Sodium caseinate standard3: Sodium caseinate incubated without YiedMAX PL4: Sodium caseinate incubated with YieldMAX PL5: as1-casein standard6: as1-casein incubated without YieldMAX PL7: as1-casein incubated with YieldMAX PL8: b-casein standard9: b-casein incubated without YieldMAX PL10: b-casein incubated with YieldMAX PL

kDa

116.0

66.2

45.0

35.0

25.0

18.4

14.4

1 32 54 76 8 109

=> No hydrolysis of the caseins by YieldMAX™ PL


Summary

YieldMAX™ PL incubated with different whey protein and casein products under optimal conditions

No differences observed fromSDS-Page Gel ElectrophoresisSize Exclusion Chromatography

No effect of YieldMAX™ PL detected towards different whey protein or casein fractions


Regulatory overviewUSA

FDA notified GRAS accepted June 2004YieldMAX™ PL is a GRAS substance,covered by GRAS Notice GRN000142

(Canada – Health Canada Approval)

The Interim Marketing Authorization (IMA) for YieldMax™ PL was published in Gazette I on August 5, 2006. YieldMAX™ PL is legal for sales in Canada from that date.

Allergen statusNo allergens as defined by regulatory authorities in EU and USA

Dietary statusYieldMAX™ PL is certified Kosher Pareve

GMO StatusYieldMAX™ PL is an enzyme from Fusarium venenatum produced with a strain of Aspergillus oryzae. By current EU rules, YieldMAX™ PL should not be GM labelled


Conclusion

YieldMAX™ PL successfully applied in Pasta Filata cheese makingeasy to implement

Significant economic benefits through yield increasesignificant increased fat retentionincreased protein retentionhigher fat in dry matter (at unchanged milk fat content)higher moisture content (at unchanged parameters)

No detrimental effects found on whey products

No functional changes in cheese

yieldmax customer jan2010

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