A study of the effects of plane of nutrition on bovine milk proteins, with particular emphasis on the individual whey proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University

(1)

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

(2)

EMPHASIS ON THE INDIVIDUAL WHEY PROTEINS

A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF AGRICULTURAL SCIENCE IN ANIMAL

SCIENCE AT MASSEY UNIVERSITY

ROBERT MACAULAY GRAY

1977

(3)

SUi'lMARY

The

work in this thesis consists of

a short term study of the effects of

p

_la

ne of nutrition on the synthesis and

secretion of m

i

_lk

proteins

by dairy cows, •·l'i th particular e

m

pha

sis on the individual wh

_ey p

ro

^t_e

ins.

The treatments c

o

m

pri

s

ed

a hieh and

low

pl

an

e of nut

r

i

ti

o

n and were imposed by strictly controlled stocking

_{rates of}_{the ex}

p

_erime

n

_t

a

_l

animals.

Six sets of identical twin covrs

_were

split

_b

e

_twee

n

_{the two}

treatment groups.

The technique

of discontinuous gel

electrophoresis was

_{used for}

the

separation of t

he whey proteins

and a

_method

(

^called

"Proportional Subdivision") was developed for

the qua

n

titat

iv

e a

na

ly

si

_s of

the

individual proteins. The

four major whey

pr

_ot

eins (

p lactoglobulin, ii.

a. la

c

tal

b

um

in ,

^bovine

serum albumin and immunoglobulins) \oTere

i

s

olat

ed and

_qua

n

t

i

_fi

ed

.

1 ) . The treatments v1ere deemed effective as

j

u

^dge

d

by the differences which

were measured in

_{the c}

oncent

rat

ion

_s

and yields

_of

the major

milk fractions. The yields of milk,

_fat

and protein were significantly (P < 0.01)

_affe

cte

d by treatment a

s

_wa

s

_t

h

_e

pr

_otei

n

percentage measured in skim milk, but

not in whole milk. Fat

p

ercen

tage did

not

alter

sig

nif

i

ca

n

tly

(P

_>

0.05).

2). The

mas

t

iti

s

status of

the animals was monitored using the Wisconsin Mastitis

_T

e

_st

.

^It ^was

possible

^therefore

to show

^that

Mastitis infections

were

not influencing

the responses to treatment.

3).

Total

whey and casei

n proteins, each expressed as a percentage of

total

protein, did not

alter

(P

_>

0.05). The caseins, expressed as a percentage of whole

_milk_differed

s

ignif

i

_c^a

n

_tly

(P

<

0.01)

_between

t

re

atme

nts

whereas

the

total whey prote

in

s ex

pr

es

se

d similarly

did not

(P

>

0.05).

_The

yields of both

_classes

of

_proteim

altered significantly

(P

<

0.01) with

_t_r_ea

t

m

e

nt

.

4).

_The

c

o

nc

e

ntr

at

i

ons of

the

Hhey p

r

o

t

ei

ns

which are synthesised in

the

_mammary

gland (

j3

l

_act

o

_gl

o

_bu

lin

_and a.

lactalbumin) d

_e

cr

_e

a

_sed

with

t

_he

low plane of nutrition,

_but

only

_{the d}

if

f

er

_e_n

c

_e

between treatments in

a.

la

_ctalbum

i

n reached

sig nificance (P

<

0.05).

_The

yields

of both these p

_rote

ins

_were _re

du

_c_e

d significantly (P < 0.01)

by

the

_low

plane treatment.

(4)

5).

The concentrations of the proteins entering the mammary gland pre-formed from the blood stream (Bovine Serum Albumin and the Immunoglobulins) moved in the opposite direction to the mammary synthesised proteins in response to the low plane treatment. The difference between treatments (increase in concentration under the low plane of nutrition) \'ras highly significant

(P

<

0.01)

for Bovine Serum Albumin, but failed to reach significance for the Immunoglob�lins.

The change in concentration was sufficient to decrease the yield of·

Bovine Serum Albumin under the high plane relative to the low plane treatment. The yield of Immunoc;lobulin was not altered significantly by the treatments

(P

_>

0.05).

6).

The separation of the individual

p

lactoglobulin proteins (A and B) in the CO\o{S heterozygous (AB) for ,3 lactoglobulin indicated that no significant changes occurred

(P > 0.05)

in the concentration of the proteins beh1een treatments. Similarly, the ratio of the two proteins (A/B) did not alter significantly

(P

_>

0.05)

with treatment.

The relevance of these results is discussed both in relation to the synthesis and secretion of milk proteins a nd in relation to observations on the influence of the effect of protein composition on the manufacturing properties of milk.

(5)

ACKNOWLEDGEMENTS

I gra t e fully a cknowled ge the gene r o s i t y of the Vic tor ian Depar tment of Agr i culture in granting me leave to und e r t ake t h i s work .

I would like to expr e s s my s incere appr e c i a t ion f o r t he guidance , suppo r t and d i r e c t ion g iven me by Dr . D . D.S . Ma ckenzie throughout h i s superv i s ion o f t h i s proj e c t .

I am indeb ted to Dr . D . News tead and Dr . L. Creamer o f the Da i ry Research Ins t i t u t e , Palme r s t on Nor th , for their a s s i s tance and adv i c e in developing the ana ly t ic al methods , f o r the use of equ ipment and ma t e r ial be longing to the i r labora t o r i e s and f o r their help in

inte rpeting resu l t s .

iv.

I am g ra t e f u l t o Professor D . S . Flux o f the Da i ry Husband ry

Depar tmen t , Ma s s ey Univer s i t y, fo r prov i s ion o f the f ield and laboratory f a c i l i t i e s requ ired by this proje c t . Also to the s t a f f o f the Da i ry Husb andry Depar tment who a s s i s t ed w i t h the plann ing o f t he exper iment and to Dr . G . F . Wil son for a rranging f ie l d fac ili t ie s , I expre s s my

thanks .

Thanks are also due t o the Farm Mana ger o f Mas s ey Unive r s i t y's No . 3 Da i ry Uni t , Mr . Alan Lowe and h i s s taff (Mr . �ob S c o t t ) f o r t he i r

a s s i s tance and pa t ience i n t h e co llect ion o f samples and for their competence in manag ing the expe r imental anima l s .

Spec ial thanks are extended t o Mr . Tony Pleasants fo r h i s advice w i th the compu t e r handling o f the data and its s ta t i s t ic a l ana lys i s .

The r eady and e f f i c ient a s s i s tance of the Lib rar ian (Mr . D . L . Jenkins and h i s s ta f f a t Massey i s also gra t e f ully ac knowledged .

(6)

I am also indeb t e d for the advice and a s s i s tance tendered by numerous colleagues and fri end s dur ing the c ourse of this proj e c t .

I wish to expr ess my thanks to Mr s . Margaret Booker for the typ ing of this the s i s under unusually d i f f i cult c ir c umstanc e s .

Finally w i th gra t i t ud e , I wish to thank my w i f e Robyn fo r her a s s i s t ance with all pha s e s of this thesis , includ ing her unwaver ing pa t ience and mo ral suppo r t .

The f inanc ial a s s i s tanc e o f the D.J. McGowan S c holar ship i s also acknowledged .

(7)

2 : 2 : 1 Separat ion of I ndividual Whey P r o t eins 43 2 : 2 : 2 Quan t i f i c a t ion of the Separa t ed P ro t e i ns 4 4 2 : 2 : 2 : 1 Ac rylamide Concen t ra t ion 4 6 2 : 2 : 2 : 2 Method o f D e s t a in ing G e l s 4 6 2:2:2:3 Time of Elec t ropho re t ic Dest aining 4 9 2 : 2:2 : 4 S epara t i on of In t ergrato r Response

Uni t s o f Ove r l a p p ing Peaks 5 1 2:2:2 : 5 Comparison o f Methods of

Quanti fying P ro t e in Band Un i t s 5 3 2 : 2 : 3 De t ermina t ion o f T o t a l Whey P r o t e in

Concen t ra t ion 56

(9)

2:2:4 Ma s titis

2:2:4: 1 Dir e c t Me thods 2:2:4:2 I ndir e c t Me thods 2:2:5 Milk Fat

2:2:6 Milk Pro t e in

5 7 5 7 58 5 9 59

CHAPTER 3 Ma t e r i a l s and Methods 60

3: 1

3:2 3 : 3

3:4

3:5

Exp e r iment a l Design

Anima l s (Expe rimental Uni t s ) Exp erimental Procedures

3:3: 1 Field Work

3:3:2 Samp ling and Anal ysis o f the Maj o r Milk Parame t e r s

3:3:2: 1 Milk Yie l d

3:3:2:2 Milk Fat and Pro t ein 3:3:2:3 Ma s ti tis

Ana l y s i s o f the Whey Pro t ein Component s 3:4: 1 Prepara tion o f Whey

3:4:2 P ro t ein S eparat ion 3:4:3 P r o t ein Quan t i f i c a t ion

3:4:3:1 S t andard Curves of D

y

e-binding by

6 0 60

63 63

6 3 6 3 6 5 6 5

6 5 6 5 6 6 7 1

P r o te in s in Gel s 7 2

3:4:3:2 S t andard Curve s o f Dye-binding by P ro t e ins in the Spectropho tomet ric

Me t hod 7 3

Total Whey and Casein P ro t e ins Conc entration 7 4

(10)

4:2

Eff e c t s 7 5

4:1:2 Examina t ion o f the Ef f e c t o f Trea tmen t s on

Milk Y i e l d and Gross Mil k Compos i t ion 7 7 4:1:3 I n f l uence o f Mas t i t is o n Trea tment Eff ec t s 8 6

4: 1:3: 1 E f f e c t o f Ba s a l Level o f I n f e c t ion and Changes in S t a t u s over Time 86 4: 1:3:2 E f f e c t of I so l a t e d S u s p i c ious and

P o s i t ive WMT S c o r e s 9 0 E f f ec t o f P l ane o f Nut r i t ion on t h e Conc entrat ion

and the Y i e l d of Milk P ro t e ins 9 3

4:2: 1 Effe c t o n Maj o r Milk Pro t ein C l a s s e s 9 3 t+: 2:2 E f f e c t o n the Maj o r Ind ividua l Whey Pro t ins 1 0 1

CHAPTER 5 Discuss ion 1 1 8

5: 1 E f f e c t o f Treatment on To t a l Milk Pro t e in 1 1 8 5:2 E f f e c t o f Trea tmen t on the Ma j o r C las s e s o f Milk

P r o t e ins 1 20

5:3 The E f f e c t o f Trea tmen t on Ind ividual Whey P r o t eins 1 2 2 5:3:1 Mammary S ynthes ized Whey P r o t e],ns 1 2 2 5:3:2 Whey P r o t eins o r igina t ing i n the B l o od s t r eam 1 2 3

CHAPTER 6 Conclus ions 1 2 6

B IBLI OGRAPHY 1 2 9

APPEND I CES 1 4 4

(11)

Figure

1 : 1 1:2 1:3

4: 1 4:2

4:3 4:4

4:5 4:6 4:7

4:8

4:9

4. 1 0

L I S T OF FIGURES

Dist ribu tion of Milk Proteins

Diagramma tic s umma ry of the p ro c e s s of P rot ein Biosynthesis .

E f f e c t of Mas titis on the p ropor tions of S erum Albumin and Immunoglobulin in the Total Whey Pro t ein .

E f f e c t o f P lane of Nu t ri tion on Milk Yield E f f ec t o f P lane of Nut ri tion on Milk Fat P e r c en tage

E f f e c t o f P lane of Nut rition on Milk Fat Yie ld Ef f e c t of P lane o f Nu trition on Tot a l Milk P rotein Con t ent , exp ressed as a p e r c en tage of Whole Milk .

E f f e c t of P l ane of Nu t rition on Tot a l Milk Prot ein Yie l d .

E f f e c t of P lane of Nut rition on To tal Milk P rotein as a percen t age o f Skim Milk .

Mean Wisconsin Ma s titis Tes t S cores (± S . E . ) for Individual Cows and Trea tment Groups ove r t he Pre limina ry and Trea tment pe riods .

Ef f e c t of Plane of Nut rition on Tot a l Whey P roteins exp r e s s ed as ^a p e rc entage of Who l e Mil k .

Ef f e c t o f P lane o f Nut rition on the Total Non Whey Pro t eins exp re s s e d as a perc entage of Whol e Mil k .

E f f e c t of P lane of N u t rition on Tot a l Whey Proteins exp ressed as a p e r c entage of Tot a l Prot ein .

4: 1 1 E f f e c t of P lane of Nu t rition on the Tot a l Non Whey P ro t eins exp res sed as a p e r c entage o f Tot a l P rotein .

4 . 1 2 E f f e c t of P lane of Nu t ri tion on Tota l Whey Protein Yie l d .

x.

5

1 4

3 3 7 9

8 0 8 1

82 8 3 84

89

9 5

9 6

9 7

98

99

(12)

4 : 1 3 E f f e c t o f P lane o f Nutrit ion o n T o t a l Non Whey Pro t ein Yie l d .

4:14 E f f e c t o f P lane o f Nut rit ion on a Lac ta lbumin Concen t ra tion .

4 : 15 E f f ect o f P l ane o f Nu t r i t ion on a La c t albumin Y i e l d .

4 : 1 6 E f f e c t o f Plane o f Nut rit ion on Bov ine Se rum Alb umin Concen tration .

4 : 17 E f fe c t o f P lane o f Nu t r i t ion on Bovine Serum Al bumin Y i e l d .

4:18 Ef f e c t o f P l ane o f Nu t r i t ion on Immunoglobulin Conc en t r a tion .

4 : 1 9 E f f e c t o f Plane o f Nu t ri t i on on Immunoglobulin Y ie l d .

4 : 20 E f f ec t o f P lane o f Nu t rit ion on B Lac toglobul in Concen t r a t ion .

4 : 21 4:22

4 : 2 3

4:24

5: 1

E f f e c t o f P lane o f Nut rition on B Lac t oglobulin Yie l d .

E f f e c t o f P l ane o f Nut rition on the Concen t ration of B Lactoglobulin A .

Eff e c t o f P lane o f Nut r i t ion on t he Concen t ra tion o f B Lac toglobu l in B .

Eff e c t o f Plane o f Nu t r i t ion on the Re lat ive P ropo r tion o f B Lactoglobulin Var ian ts (A

/

B)· E f f ec t o f P lane o f Nut rit ion on M i lk S NF cont ent .

1 00 1 0 3 1 04 1 0 5 1 06

1 0 7

1 08 1 09

110

1 1 1 112 1 1 3

1 1 9

(13)

Pla t e 3: 1

3:2a

3: 2aii 3:2bi

3:2bii

3 :2ci

3: 2cii

3:2di

3:2dii

4: la 4; lb

4: le

4:2a 4:2b 4:2c

LIST OF PLATES

App a r a t us fo r Polyac rylamide Discontinuou s Gel E l e c t ropho r esis (PAGE) , f e a t u ring 2 X 12 gel

t anks . 64

Purity o f Immunoglobulin used f o r t he p repara tion

of Dye-Binding S t andard Curves 67

S�an Trace and I n t e r g r a t o r Plot of t he

Immunoglobu lin S t andard. 67

Puri t y o f Bovine S erum Albumin used f o r the

prep a r a tion of Dye-Binding S t�ndard Curves 68 Scan Trace and I n t e r g r a t o r P l o t of t he Bovine

Serum Albumin S t andard . 68

Puri t y o f Alpha l a c t a lbumin used for the

preparation of Dye-Binding S t andard Curves. 6 9 S c a n T r a c e and Int ergr a to r P l o t o f the Al pha

Lac t a lbumin S t anda rd . 6 9

Purity o f t h e B e t a Lac t oglob u l i n used for the

p r e p a r a tion of Dye-Binding S tandard Cu rves . 7 0 S can Trace and In t e r g r a t o r P l o t o f the B e t a

Lac t og lob u lin S t and ard . 7 0

Whey P ro t ein Separa tion o b t ained by PAGE . l l4 S c a n Tr a c e and I n t e r g r a t o r P l o t o f the Gel

shown in 4:la (H . P . ) 1 1 5

S can Trace and Int e r g r a t o r P l o t of the Gel

shown in 4: 1 a (L . P . ) 1 1 5

Whey P r o t ein Separa tion o b t ained by PAGE . 1 1 6 S c an Trace and In t e r gra tor Plo t o f the Gel

shown in 4:2a (H.P.) 1 1 7

S c an Trace and I n t e r g r a t o r Plo t o f the Ge l

shown in 4:2a ( L . P.) 1 1 7

xii.

(14)

Tab l e 1 : 1 1:2 2: 1 2:2 2:3

2:4

3: 1 4: l

4:2 4:3

4:4 4:5 4:6 4:7

L I ST OF TABLES

Gene t i c Polymor phs o f the Maj or Milk Prot�ins

Origin of s ome of the Minor Milk Prot e ins Comp a r i son of Two Me t hods of Ge l De s ta ining E f f e c t o f P r o longed El e c t rophore t ic Des t a ining on P r o t e in Con c en t r a t ion

Re l a t ionsh ip b e tween t he peak propo r t ion f rom two me thods o f separa t ing these

� �:� !

overl app ing peaks .

Compa rison o f the Sources o f Va r i a t ion wit h in each o f two methods o f Es t ima t ing S Lgb

Concen t r a t ion f rom the same samp l e s De s c r ip t ion o f Exp e r imen t a l An ima l s

Ana l y s i s o f t ime o f ons e t o f Treatment E f f ec t s o n Mean Milk Y i e l d - f ir s t day o f t rea tmen t .

I n f l uen c e o f P l ane of Nutri t ion on Milk Y i eld and Compo s i t ion .

WMT scores o f indivi dual cows samp l e s a t the a f t e rnoon mi lking .

Ana lysis o f individual cow WMT scores over t ime . E f f e c t o f iso l a t e d h igh WMT scores on milk yield and compos i t ion .

E f f e c t o f P lane o f Nu t r i t ion on the Y ie l d and

6 9 48 5 0

5 2

5 5 6 2 7 6 8 5

8 8 9 1 9 2

Concen tra t ion o f t he Major Milk Pro t e i n C l a s s e s . 94 E f f e c t o f P l ane of Nut r i t ion on the Conc ent r a t ion and Yield of the Major Whey Pro t e ins . 1 0 2

A study of the effects of plane of nutrition on bovine milk proteins, with particular emphasis on the individual whey proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

SCIENCE AT MASSEY UNIVERSITY

split

oncent

possible

lactalbumin) d

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

B IBLI OGRAPHY 1 2 9

LIST OF PLATES

L I ST OF TABLES