YieldMAX Customer Jan2010

Yi el dMAXPL - enhanci ng Fat Recovery i n
Mozzarel l a / Pi zza Cheese

2010 Yi el dMAX i n past a f i l at a 2
Background
Cost r educt i on by enhanci ng f at and pr ot ei n r et ent i on an ongoi ng
f ocus i n t he cheese i ndust r y
Much ef f or t has concent r at ed on desi gni ng opt i mal cheese maki ng
equi pment and pr ocess par amet er s, e. g. cut t i ng t i me
Despi t e t hese ef f or t s, f at and pr ot ei n l osses ar e obser ved especi al l y
af t er cut t i ng, dur i ng ear l y st age of st i r r i ng and dur i ng st r et ch
Cheese yi el d may be i ncr eased by pr ot ei n concent r at i on. However ,
t hi s appr oach may l ead t o i mpai r ed f l avour , t ext ur e and mel t i ng
pr oper t i es
The ai m of t hi s over vi ew i s t o expl ai n about t he novel enzyme
t echnol ogy usi ng a phosphol i pase Yi el dMAXPL t o i ncr ease cheese
yi el d and t her eby pr oduct i on economy i n t he manuf act ur i ng of Past a
Fi l at a t ype cheese
Busi ness case on Yi el dMAX i n
Mozzarel l a/ Pi zza cheese
Where does t he val ue of Yi el dMAX come f rom
Yi el dMAX modi f i es par t of t he f at gl obul e membr ane
by act i ng on t he phosphol i pi ds
As a r esul t mor e f at i s r et ai ned i n t he cheese
Recover y of pr ot ei n al so i ncr eases
Wat er bi ndi ng capaci t y i s i mpr oved
Al t oget her yi el d i ncr eases about 2%
Yi el dMAX r esul t s i n mor e cheese f r om t he
same amount of mi l k
What are t he economi c benef i t s of i ncreasi ng f at
recovery i n pi zza cheese manuf act uri ng
Hi gher cheese yi el d
Mor e cheese f r om t he same mi l k
Hi gher capaci t y of pl ant
Mi l k f at has t he hi ghest val ue when used i n cheese
Hi gher r ecover y of f at
Opt i on t o t ake mor e sweet cr eam out of cheese mi l k
Sweet cr eam has hi gher val ue t han whey cr eam
Lower vol ume of whey cr eam
Recover y of whey f at of t en l ower t han mi l k f at i n cheese
Usi ng whey cr eam i ncr eases r i sk of bact er i ophages
Hi gh usage may i nf l uence cheese qual i t y negat i vel y
Much l ess f at i n st r et chi ng wat er
Fat f r om st r et chi ng wat er i s non- kosher
Exampl e of economi c benef i t s of usi ng Yi el dMAX
i n 1m l bs mi l k/ day pi zza cheese product i on
Benef i t of usi ng Yi el dMAX
360 days/ year
El ement Q Val ue
Ext r a cheese 792, 000# 1, 1m$
Yi el dMAX cost 570, 000$
Gr oss ear ni ngs 539, 000$
Gr oss r et ur n on i nvest ment 95%
Gr oss ear ni ng at cheese
pr i ce of 1, 6$/ l bs
697, 000$
Cheese pr i ce wher e
gr oss RoI i s 50%
1. 08$/ l bs
Const ant s used
Cheese pr i ce 1. 4$/ l bs
St andar d
cheese yi el d
11%
Fat i n cheese
mi l k
2, 6%
Yi el d benef i t 2%
Mi l k
vol ume/ day
1 mi l l i on l bs
Pi l ot pl ant and i ndust ri al
val i dat i on of concept val ue
How t o use Yi el dMAXPL
Si mpl e addi t i on
Add t o cheese mi l k 20 mi n
bef or e r ennet i ng
Whi l e f i l l i ng vat
Dosage ( LMPS)
5 uni t s per g of f at
1. 5 l per 25, 000 kg mi l k
45 f l oz per 55, 000# mi l k

Up t o 10x di l ut i on gi ves no l oss i n
act i vi t y
Yi el dMAXdoes not af f ect t he
coagul at i on.
Pi l ot pl ant cheese t ri al desi gn
Cheese t ype: Pi zza 40+
Var i abl e: Addi t i on of Yi el dMAXPL, 20 mi n
bef or e r ennet i ng
Addi t i on r at e: 5 u per g f at
Vat s per day: 4 vat s: 2 cont r ol and 2 exper i ment al
Number s of days: 4
Cheese yi el d st d. devi at i on
bet ween cont r ol vat s = 56 g
f r om 150 kg of mi l k
Ef f ect on cheese - Pi l ot scal e Mozzarel l a t ri al (LMPS)
Vari abl e
Unt reat ed
(n = 8)
Wi t h Yi el dMAX
Di f f erence %
Fresh curds yi el d, % 10. 80 + 1. 1
Cheese yi el d
a
, % 10. 35 + 3. 2
Fat i n whey, % 0. 132 - 4. 3
Fat i n cooki ng wat er, % 0. 300 - 2. 1
Moi st ure i n cheese, % 47. 12 + 1. 9
DM yi el d
b
, % 46. 43 + 1. 5
Fat recovery, % 87. 20 + 0. 4
Prot ei n recovery, % 76. 69 + 1. 7
a
Cheese weight divided by milk weight .
b
Cheese dry mat t er divided by milk dry
mat t er.
Lilbk

St at i st i cal l y si gni f i cant
Cheese Funct i onal i t y - (Conf ocal
Laser Scanni ng Mi croscopy)
Fresh cheese,
pri or t o st ret chi ng
3 days
14 days
28 days
Cont rol Yi el dMAX
No si gni f i cant di f f er ence i n
i mage anal ysi s wi t h r espect t o
si ze and number s of f at
gl obul e uni t s
degr ee of coal escence
coar seness of pr ot ei n
net wor k
Mi cr ost r uct ur e shows swel l i ng
of casei n net wor k and
di mi ni shi ng of whey pocket s
dur i ng st or age
Mel t abi l i t y, st r et chabi l i t y and
br owni ng si mi l ar i n cont r ol and
t r eat ed cheeses ( Ref : Li l baek et al .
2006 Jour nal Dai r y Sci ence
Cheese Control YieldMAX SEM Difference P-value
Phospholipid retention, % 45.4 79.3 3.2 +75 % <0.001
Whey Control YieldMAX
Total phospholipids, M
Sphingomyelin, M
183.0
52.4
68.4
46.4
Lysophosphol i pi ds pr ef er ent i al l y r et ai ned
Cor r el at i on bet ween PL cont ent and wat er cont ent !

Ret ent i on of phosphol i pi ds i n cheese i ncreased
St andard whey f at f l ow di agram
Mi l k i n Cheese out
Mozzar el l a wi t hout Yi el dMAXPL
Cheese vat St r et chi ng
Whey,
0. 2- 0. 3% f at
Cooki ng wat er,
2- 4% f at
Whey, 0. 05% f at
Whey cr eam
( 25- 30% f at )
Yi el dMAX whey f at f l ow di agram
Less whey cream t o handl e Less f at i n def at t ed whey
Mi l k i n Cheese out
Mozzar el l a wi t h Yi el dMAXPL
Cheese vat St r et chi ng
Whey,
( <0. 2- 0. 3% f at )
Cooki ng wat er,
( 1- 3% f at )
Whey, < 0. 05% f at
Whey cr eam
( 25- 30% f at )
Less vol ume
Improved whey f at separat i on
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 f at
Ef f ect of reci rcul at i on of whey cream
Whey cr eam f at i s t o a l esser
ext end i n gl obul ar f or m
Mor e suscept i bl e t o
oxi dat i on
Non gl obul ar f at wi l l f or m
l ar ge pods of on sur f ace
Less whey f at i s r et ai ned i n
cheese l ower i ng t he over al l f at
r et ent i on
Qual i t y i ssues
Reci r cul at i on of phage
Moi st ur e r et ent i on
Fl avour def ect s at hi gher
dosage l evel s
%whey f at
recovered
%t ot al f at
recovered
%FDM %yi el d
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 cr eam f at as 5% of t ot al f at :
Mi l k 3. 88% f at , 2. 49% casei n
Cheddar wi t h 38% moi st ur e
Uni . of Wi sconsi n, Oct ober 1997
Indust ri al val i dat i on ef f ect on cheese
Yi el d dat a f r om i ndust r i al t r i al s ( 500 cheese vat s)
% yi el d
i ncrease
95%
i nt erval
Reci pe 1 1, 4 (0, 3; 2, 4)
Reci pe 2 0, 7 (-0, 5; 1, 9)
Reci pe 3 2, 1 (0, 1; 4, 0)
Reci pe 4 (hi gh f at ) 3, 8 (1, 6; 6, 0)
Si gni f i cant i ncr eases seen i n
f at i n dr y mat t er
moi st ur e i n cheese
f at r et ent i on
pr ot ei n r et ent i on i n t wo r eci pes

Indust ri al val i dat i on - ef f ect s on whey f at
Si gni f i cant decr ease i n whey f at
Typi cal pi l ot pl ant dat a shows decr ease f r om 0. 22 t o 0. 19%
9- 10% decr ease i n i ndust r i al t r i al s
Same ef f ect s seen on al l t est ed r eci pes
W
h
e
y

f
a
t

%
L
S

M
e
a
n
s
0,2
0,25
0,3
0,35
0,4
0,45
Recipe 1
Recipe 2 Recipe 3
Recipe 4
Control pLipase
Treatment
LS Means Plot
Indust ri al val i dat i on - Ef f ect s on mi xer/ mol der wat er
Fat r ecover y i n cheese i ncr eased i ndust r i al dat a
Less f at / pr ot ei n l oss i n cooki ng wat er
Treated vats
Ef f ect s on process - f oami ng
Yi el dMAX addi t i on may l ead t o some f oam f or mat i on
To be addr essed dependi ng on i ndi vi dual ci r cumst ances
Spr ayi ng of wat er dr opl et s
Mi ni mi zi ng ai r i ncor por at i on dur i ng pr ocessi ng ( pl ant desi gn)
Ul t r asound t r eat ment
Ot her ant i f oami ng met hods
Unt reat ed Treat ed
Summari zi ng t he current knowl edge -
A suggest ed mechani sm f or cheese yi el d
i ncrease
Milk

+ Yield PL

Pr ocess st ep Ef f ect of Yi el dMAXPL
Incubat i on of mi l k
wi t h phosphol i pase
Conversi on of phosphol i pi ds t o l ysophosphol i pi ds
Rel ease of l ysophosphol i pi ds f rom f at gl obul es
Coagul at i on Compl exat i on of l ysophosphol i pi ds wi t h casei n
Cur d cut t i ng,
st i r r i ng, scal di ng
Lysophosphol i pi ds act as an emul si f i er
- prot ect f at gl obul es f rom rupt ure
- re-emul si f y f at l eaked out f rom damaged gl obul es
Cur d cut t i ng,
st i r r i ng, scal di ng
Whey dr ai nage Lysophosphol i pi d casei n compl exes ret ai ned i n t he
curd
Less un-emul si f i ed l i qui d f at i s l ost i n t he whey
Process st ep Ef f ect of Yi el dMAXPL
Curd cut t i ng, st i rri ng,
scal di ng
Whey drai nage Lysophosphol i pi d casei n compl exes ret ai ned i n t he curd
Less un-emul si f i ed l i qui d f at i s l ost i n t he whey
Curd st ret chi ng Lysophosphol i pi d act s as an emul si f i er
- prot ect f at gl obul es f rom breaki ng
- reduce syneresi s more wat er i s ret ai ned i n cheese
Act i vi t y of Yi el dMAX i n t he mi l k
Phopsholipids in milk products
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
M
i
l
k
S
k
i
m
m
e
d

m
i
l
k
C
r
e
a
m

4
0
%
B
u
t
t
e
r
B
u
t
t
e
r
m
i
l
k
A
n
h
y
d
r
o
u
s

m
i
l
k

f
a
t
%

w
/
w
% in product
% in solids
Based upon data f ra Walstra et.al 2006.
Dairy Science and Technology CRC Press
Tot al amount i n mi l k 0. 02 0. 04 % w/ w
In f r esh r aw mi l k 80% i s on t he f at gl obul e sur f ace
Li popr ot ei n aggr egat es/ vesi cl es i n mi l k pl asma

m
Phosphol i pi ds i n mi l k
Phosphol i pi ds i n mi l k
Phosphol i pi ds i n mi l k (mol -%)
Phosphat i dyl
et hanol ami ne ( PE)
22- 49 %
Phosphat i dyl chol i ne PC) 21- 40 %
Phosphat i dyl i nosi t ol ( PI)

4- 11 %
Phosphat i dyl ser i ne ( PS) 2- 12 %
Sphi ngomyel i n ( SM) 18- 32 %

Not e! Sphi ngomyel i n i s not hydr ol yzed.
Hydrol ysi s of mi l k phosphol i pi ds:
cat al yzed by Yi el dMAXPhosphol i pase A1
Example of a substrate: Phosphatidyl choline

H
YieldMAX PL
(PLA1)
O
O
O
O
O
P
O
O
-
O
N
+
Hydr ol yzes est er bonds i n l ong- chai n f at t y aci ds
Phosphol i pi ds can be ext r act ed f r om
mi l k and anal yzed by HPLC
Al most compl et e depl et i on of
phosphat i dyl et hanol ami ne and
phosphat i dyl chol i ne
Devel opment of l ong chai n f r ee f at t y
aci ds can be obser ved
Lysophosphol i pi ds ar e det ect ed i n
whey, amount cor r esponds t o
appr ox. 5 % of cont ent i n hydr ol yzed
whol e mi l k - 95% r emai ns i n cheese
mat r i x
Sphi ngomyel i n i s not hydr ol yzed by
Yi el dMAXPL
0
25000
50000
75000
100000
125000
150000
0 5 10 15 20
Time, min
m
A
U
PE
PC
Sph
0
25000
50000
75000
100000
125000
150000
0 5 10 15 20
Time, min
m
A
U
PE PC
LPE
LPC
Sph
Untreated control
Phospholipase treated
The Yi el dMAXPL act i on
DH% i n whol e mi l k vs. i ncubat i on t i me
Yi el dMAX PL dose 5 or 50 LEU/ g f at
Max. Degree of hydrol ysi s 65-80%
0
10
20
30
40
50
60
70
80
0 5 10 15 20
Time (h)
D
H
% PE (5 Units /g fat)
PC (5 Units /g fat)
PE (50 Units /g fat)
PC (50 Units /g fat)
Phosphol i pi ds are i n t he f at gl obul es -
Lysophosphol i pi ds are i n serum phase

LPC

Lilbk

Fat gl obul e st abi l i t y unchanged
Whol e mi l k part i cl e si ze di st ri but i on

Particle diameter [m]
0,01 0,1 1 10 100 1000
R
e
l
a
t
i
v
e

d
i
s
t
r
i
b
u
t
i
o
n

[
%
]
0
2
4
6
8
10
12
14
16
B.
A.

Phosphol i pase
Cont r ol
Fat gl obul es i n cheese curd
Document at i on on whey st ream
and casei n
Posi t i ve ef f ect on whey pr ocessi ng
No ef f ect seen on whey pr ot ei n f r act i ons or t hei r r at i o
No ef f ect seen on casei n
Improved processi ng capabi l i t i es of Yi el dMAX
whey
Whey cont ai ns l ess f at due t o bet t er separ at i on pr oper t i es of
Yi el dMAX whey
Dat a f r om pr ocessi ng conf i r m t hi s
Lower f at i n whey means easi er pr ocessi ng
Lower f at means easi er membr ane f i l t r at i on f or WPI
Impr oved f unct i onal i t y of WPIs
Lower means l ess r i sk of oxi dat i ve f l avor changes
Repl ace f at wi t h l ow val ue l act ose i n WPC
Yi el dMAX r educes sur f ace t ensi on of t he whey
Theor et i cal expl anat i on on f unct i onal i t y of l ysophosphol i pi ds
Pr oven i n l abor at or y model
Cust omer r ef er ences
Longer r uns bet ween CIP on UF
Less f oul i ng
Met hod:
Wilhelmy plat e
Sample:
Skim milk, separat ed from t reat ed
whole milk
General phenomenon:
Surface t ension is also reduced upon
phospholipase t reat ment of:
- Cream
- Skim milk
- WPC- solut ion
- Milk phospholipid
suspension
- I n whey from t reat ed
cheese milk
Surf ace t ensi on i s reduced by Yi el dMAX
Cont rol, no enzyme
PLA1 F. venenat um ( YieldMAX)
PLA2 Porcine pancreas
PLC B. cereus
Yi el dMAX has hi gh speci f i ci t y
Enzyme heat l abi l e - 90% of t he
enzyme i s dest r oyed at l ow
past eur i zat i on of t he whey
( 72C/ 160F, 20 sec)
Si de act i vi t i es r emoved
Amyl ase and
gl ucoamyl ases
absent accor di ng
t o specs
Conf or mi t y checks al so f or -
gal act osi dase,
l i pase and pr ot eol yt i c act i vi t y
No ef f ect on whey pr ot ei n f r act i ons
K- sor bat e
Na- benzoat e Enzyme
Product s i ncubat ed wi t h Yi el dMAXPL
3 di f f er ent commer ci al whey pr ot ei n concent r at es (WPC)
2 WPC f r om Mozzar el l a t r i al s wi t h or wi t hout use of Yi el dMAXPL
4 di f f er ent commer ci al whey pr ot ei n i sol at es
Sampl e Whey prot ei n (powder product s)
1
2
3
4
5
6
7
8
9
Whey Pr ot ei n Concent r at e I ( 34%)
Whey Pr ot ei n Isol at e
WPC Tr i al ( f r om Mozzar el l a pr oduct i on usi ng Yi el dMAXPL)
WPC Cont r ol ( f r om Mozzar el l a pr oduct i on)
Bovi ne Lact of er r i n I
Lact of er r i n / Lact oper oxi dase
Bi oact i ve Whey Concent r at e
Bovi ne Lact of er r i n II
Whey Pr ot ei n Concent r at e II
Experi ment al set up

0.4 g Whey Protein
40 mL Buffer*
200 L 5% Na-Azid
20 mL
A
10 mL
B
10 mL
C
Mixing
WPS
Incubation at 35C for 21 hr
+ 5 L
YieldMAX PL
+ 5 L HT
YieldMAX PL
SEC
SDS-Page
Det ect pr ot ei ns bet ween
2. 5 t o 200 kDa
Det ect pr ot ei ns
l ess t han 2000 kDa
(opt i mal <200 kDa)
SDS-gel on WPC80
No appear ance of new
bands
No r educt i on of bands
i nt ensi t y
No ef f ect obser ved of
Yi el dMAXPL i ncubat i on
25
Samples: WPC80 with and without YieldMAX
45
10 ul
20 ul
kDa
116
66,2
35
18,4
14,4
T
r
e
a
t
e
d
1
3
-
3
-
0
6
C
o
n
t
r
o
l
1
4
-
3
-
0
6
T
r
e
a
t
e
d
2
1
-
3
-
0
6
C
o
n
t
r
o
l
2
2
-
3
-
0
6
T
r
e
a
t
e
d
1
3
-
3
-
0
6
C
o
n
t
r
o
l
1
4
-
3
-
0
6
T
r
e
a
t
e
d
2
1
-
3
-
0
6
C
o
n
t
r
o
l
2
2
-
3
-
0
6
Bovi ne Lact of erri n I & Lact of erri n/ Lact operoxi dase
SEC
No new peaks obser ved
Same l evel of peak ar eas
No di f f er ences
1 %sol ut i ons of pr oduct s
Bovine Lactoferrin I
-0,5
0
0,5
1
1,5
2
2,5
3
0 20 40 60 80
Time (min)
A
b
s
o
r
b
a
n
c
e

2
2
0

n
m
WPS5
+YieldMAX
+HT YieldMAX
MwSTD
-0,5
0
0,5
1
1,5
2
2,5
3
0 20 40 60 80
Time (min)
A
b
s
o
r
b
a
n
c
e

2
2
0

n
m
WPS7
+YieldMAX
+HT YieldMAX
MwSTD
Lactoferrin/Lactoperoxidase
Ef f ect on casei n
1. Sodi um casei nat e ( f r om Si gma, C- 8654) , s
1
- casei n and - casei n wer e
sol ubi l i sed i n 50 mM sodi um phosphat e buf f er , pH 6. 4, cont ai ni ng 0. 025 %
sodi um azi de. Pr ot ei n concent r at i on of t he sol ut i ons wer e 5 mg/ ml
2. Yi el dMAXPL ( Bat ch No. 2663736) was added t o t he sol ut i ons at a
concent r at i on of 0. 05 % ( v/ v) , and sampl es wer e i ncubat ed f or 21 hour s at
35C.
3. Cont r ol r eact i ons wi t hout Yi el dMAXPL addi t i on wer e al so i ncl uded.
4. Sampl es wer e anal yzed usi ng SDS- PAGE
SDS-PAGE
casei ns i ncubat ed wi t h Yi el dMAXPL
1: Mol ecul ar wei ght st andar ds
2: Sodi um casei nat e st andar d
3: Sodi um casei nat e i ncubat ed wi t hout Yi edMAX PL
4: Sodi um casei nat e i ncubat ed wi t h Yi el dMAX PL
5: a
s1
- casei n st andar d
6: a
s1
- casei n i ncubat ed wi t hout Yi el dMAX PL
7: a
s1
- casei n i ncubat ed wi t h Yi el dMAX PL
8: b- casei n st andar d
9: b- casei n i ncubat ed wi t hout Yi el dMAX PL
10: b- casei n i ncubat ed wi t h Yi el dMAX PL
kDa
116.0
66.2
45.0
35.0
25.0
18.4
14.4
1 3 2 5 4 7 6 8 10 9
=> No hydr ol ysi s of t he casei ns by Yi el dMAXPL
Summary
Yi el dMAXPL i ncubat ed wi t h di f f er ent whey pr ot ei n and
casei n pr oduct s under opt i mal condi t i ons
No di f f er ences obser ved f r om
SDS- Page Gel El ect r ophor esi s
Si ze Excl usi on Chr omat ogr aphy
No ef f ect of Yi el dMAXPL det ect ed t owar ds di f f er ent whey
pr ot ei n or casei n f r act i ons
Regul at ory overvi ew
USA
FDA not i f i ed GRAS accept ed June 2004
Yi el dMAXPL i s a GRAS subst ance,
cover ed by GRAS Not i ce GRN000142
( Canada Heal t h Canada Approval )
The Int er i m Mar ket i ng Aut hor i zat i on ( IMA) f or Yi el dMaxPL was publ i shed
i n Gazet t e I on August 5, 2006. Yi el dMAXPL i s l egal f or sal es i n Canada
f r om t hat dat e.
Al l ergen st at us
No al l er gens as def i ned by r egul at or y aut hor i t i es i n EU and USA
Di et ary st at us
Yi el dMAXPL i s cer t i f i ed Kosher Par eve
GMO St at us
Yi el dMAXPL i s an enzyme f r om Fusar i um venenat um pr oduced wi t h a
st r ai n of Asper gi l l us or yzae. By cur r ent EU r ul es, Yi el dMAXPL shoul d
not be GM l abel l ed
Concl usi on
Yi el dMAXPL successf ul l y appl i ed i n Past a Fi l at a cheese maki ng
easy t o i mpl ement
Si gni f i cant economi c benef i t s t hr ough yi el d i ncr ease
si gni f i cant i ncr eased f at r et ent i on
i ncr eased pr ot ei n r et ent i on
hi gher f at i n dr y mat t er ( at unchanged mi l k f at cont ent )
hi gher moi st ur e cont ent ( at unchanged par amet er s)
No det r i ment al ef f ect s f ound on whey pr oduct s
No f unct i onal changes i n cheese

YieldMAX Customer Jan2010

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YieldMAX Customer Jan2010

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Yi el dMAXPL - enhanci ng Fat Recovery i n

Mozzarel l a / Pi zza Cheese

45 f l oz per 55, 000# mi l k

Phosphat i dyl chol i ne PC) 21- 40 %

Phosphat i dyl i nosi t ol ( PI)

Phosphat i dyl ser i ne ( PS) 2- 12 %

Sphi ngomyel i n ( SM) 18- 32 %

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