Properties of some animal derived milk coagulating enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University

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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.

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A thesis presented in partial fulfilment of the requirements for the deeree

of

DOCTOR OF HHJJOSOPHY in

FOOD TECHUOLOGY

HASSEY UTHVEHSIJ:Y

Neil Harvcy Cle.rke

197?

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ABSTHACT

Extracts of milk cosgula·l;il'lg

cnzJrrnes were ohte.ined,

ac1.ult bovir.te frotn the abomasurn£� of pasi;ure fed. er-:�:t;-tle and lmnb rennet fron1 the abD'�'asu:·.,s of partially r·ilk fed.

lan;bs r.�lau.e;hterecl between three ond six months of <-l:<j8, Both r·enncts wc�re. ^r in the pres�:mce of sod:i.•J_;·" cl.1J Gr-1 rlE: ^t moE:t stable et ·i �°C or low��j:• at pH

4. 7

for arlul t bovin.e and p�I 4. 1 for 1a·�b. Heat treat•,:fmt in ⁿ soc.iu'·'1

chlori(1e solu:t;j on

(

²⁰⁰

,.-;1?'/�l)

at f58°C for SO ·YJill:..ttes

destroyed les::; than

35��

of the activi-ty of et=:tch rennet.

Under the sa··ne co.r:tditions calf rennet v1as completely inactivated.

1'he cheese:nakint� properties of adiJ.lt bovine a.nd lano rcnn8ts were compared with calf rennet, Adult bovi�o 8nd calf rennE:Jts responded. similarly t;o cheJtgcR of pH hcl\:<�veJ' lamb ronnat appeared J.E.'ss act:Lve tl12..1� CB.lf X'E!nnet F1.t

milk I'J;)f 3. cu.rd ·[,t:::o sion :net1·�od. The opt:i.n,u:11 tEon(p<'::(;-�:ti'J.I"c' for m:i.lk coaenJating activity was

40°C

f0r ad1�li; bov-ine and ca.J.:f renne-Gs but only 30°C for lam.b ren.net wb e;.'}

measured in Cf'toeinate solution. 'l'hc cu:<:>d tension of milk coagulated v.-i th the three ^rennets incl�aat;ecl wi·iih i:;ir-:.e but bo·t;h adult bovine and lGJi1b rcn.nets appeared 1;1ore

sensitive to 1v;ilk (;aJ.ciu.:r. levels than calf

rennet.

^Whole

casein,

^a(.�-,

!3-

and k-casei.ns wl3re hydrolyBec1 in a

'"

similar

r,anner

^by the three rennets. Adult bovine rem"le-G was the ·roost proJlieolytic on whole and

�-

case:\.ns while

calf

rennet hydrolysed o.8- and k-caseins more rapidly then t!1e other ronnets.

Two pepsins, of similar mnino acid compositions, were isolated, one from CJ.rlul t bovine reimet and one from lamb rer:.net ^which also contained a rennin. All thre

e

enzymes were purified so that lamb rennin and. ^pepsin each produced only a single band. on polyacrylamide gel

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electro:phorests but the adult bovine p<:!psin prort'.l_ce:l two bai:H1.s and appeared to be het;eroceneous.

Cheese made w:i.·t;h ar1n1t bovine, la�,b ^{or ;:_1,}

50/50

t•liYt:nre of ca1f -adult bovine rt;nnt!t s wE::ro cornpe.rerJ wi t::l cheese rnac1e w_i_l;h ceJ.f renne·t anr]_ :fGu.rv1 to be si.�d.Jar �-n mmndact'l).ri.nt:;

charac:teristics,

fl8.V01.1X a1H�. bod;�,r <:�:J.ter

-:·'rl·r'•"P _\.! ... ... -::.C..\.J.�.Ir� .t. ^,��_..., ___ i ^x "'o·n.t�l"' •. , - · ·· ·=--- c.�i-o,...,, :.) .J I D�t:.>"-N""" ' (.1,t_, ···-'- 17 "r I "-'

13°"'

\_ • •

�E-!J elec-�T·opho-retoc:;"r·�:)·'·1s of ti1ese c:heese -::J.fte1 0'.:(� t.;o twelve !'>C>Dths fO:tor::tt;e aol; i)°C :3hOi':8d thc':lG GrH� nilk c.o::1_�,__1_ls.nt 'r·w.0 no effect Ofl. the e:a::;;ein rlecrgr1a-t ior!.

;_Yeoii.ucts pJ.�o"\uce·:l in ^the cheese

F:1thon�1l

t}Je rate l)f

Perci1J.oric ?..ci·� �''as :fouri•�. to be a ,soo-:� prot;c:i.n

prer_·i _pit

a.r1t for-

Cf\..lJ:' 1.c hi;1,g ca�ei 11-rc r' :•.e f; r<��;J.::t im1s c:.r�r.J. v·;as

uti J.iz.e c�

in

⁸ ·etho �:;_ v.,r>i eh v:8:� r:;eveloper] for ac�:R;;ri'•�

ilk C08{,1.ll8l;j;1(j E'ct·i.vit;{. Rer ^..r•f;t Vi2.s nc-:_,·:c��,,� to so·hu··

coscinate l:Oltrl:i'l.O'l. 2nr) Oi'.t� ^-.iln.ri>3 le.ter the reE�c:ticu.

q_\J.enc:her1. with

per-chlorie e.cid. The qu�:;tit�r of

pe:;,Ytic:.es h�n:Lr�)lysed

^fro·' the cHsein \'rus •r:e<:1.surec1. at

217

^ll'' �tcJ/i. '1!(-l.S

directly pl'or;ortion8.1 �o rennet c-tcti vi.ty over' a 1i "11i t 8(1 ran.::;e of act i vit�{.

A

method

for

rel'llovine; mtwoprot eins, the

major

irnpuri t;sr in

rennet,

^was developed and a corn'•8rcial

scale

plant· com�issioned. A diethylaminoethyl ^cellulose based resin, equilibrated w i^th a citrate buffer, retained the rennet enz�r·�1es v1hile the !11UCoproteins pe.ssed throue;h uninhibited.. The enz�rnes were eluted with

t

^he sa.,e buffer 111arle 1 ^•

0 H

^with so dill:·.., chloTid.e. The eluen-tj VJ8.S

more B.ct i ve than the orieinal rennet, was Cr>Jstal clear in appearance a.nrl re"'ained so durine tv.,relve ^...,onths

storaee when no!""·ally Y··ucoproteins would have

precipitated out of solution to for� a cloud in the ren;.1et.

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ACKIWvVLE DG EH ENT S

I extend. P1Y appreciation to the New Zealand Co-opera·ti

ve

^Rennet Compan;>r I;imi ted

for

maki.ng

tbe

opportunity and funds available for this study.

I wish to acknowledge with ·t;he1.nks the assistan.ee received from

the

^following ^people;

Professor E.L. Rich ard_s for being an understanding supervisor.

D:r. R

�c.

I,awrence for his consultations and assistance with equipment.

Drs. I, .K. Crea"'(!r and

B. C.

Richard:::.;on for their advice on protein chernist:r·y and for the a'"tlino acid

analyses.

Jiiessrs

J.

^Gill ^es ^a::1d

0. �r. Free

^se for the :nanufacture ancl stora8^e of cheese.

Miss M.A. Hu�phries for flavour and body asses�nent of cheese.

Hr. H.W. Legg ^and the laboratory staff of the new Zealand Rennet Company for rennet ^assays.

Judi th, my wife, for her support;.

All those other people who in any way contributed to the completion of this study.

iv

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ACIGl'OWLEDGEMENTS

JJIST OF FIGTJRES LIST OP TABJJES

•

• •

INTRODUC'.r ION

CHAFTKR

II

REmmT ASSAYS REVIEW

OF

LITERATURE ^•

The coagula·tion of milk Rennet Assays

METHODS ^• ^• Rennet source

•

• •

•

• •

•

Precipi tc..:tnt s for casein-rennet reaction mixtures ^•

Assay RESULTS •

•

• •

Precipitants for casein-rennet

Assay DISCUSSION

reaction mixtures

• e

• • •

•

CHAPrER I I I ENZY1-t2 PURIFICATION

REVIEV/

OF LIT.t:."'RATURE

• •

Enzyme purification

HETHODS ^• ^• • •

Ion exchange chromatography Gel chromatography •

Enzyme purification •

•

Electrophoresis of purified enzymes Commercial rennet purification •

RESULTS ^• ^• ^• ^• ^•

Rennet enzymes purification

Enzyme purity and homogeneity ^• Commercial rermet purifi cation •

DISCUSSION

• • • • •

•

c

•

page

iv viii

X

11

14 14 14 19 22 22

22 24

25

25 25 30

32 32 3 2 35 3 5 35 36 37 37 39 39 4 3 43 49

V

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CHAP'rER

^IV BNZYHB PROP£-:RTIBS R.SVI�W OF r.rr i:.'RATURE ^• ^e

Enz�ne properties •

Hydrolysis of casein fractions

METHODS ^c ^• ^� ^• ^• ^•

Effect ^of :pH en rennet stability Effect of heat on rennet stability

• •

..

0

• •

•

51 51 51 54 58 58 58 Variations of rennet activity with pH ^�

59

Effect of temperature on reru1et activity 60 Curd tension measurements

Proteolysis of whole casein Casein fractionation •

•

Hydrolysis of casein fractions Molecul<:4,r weight determination Amino acid analysis • •

RESULTS

• • • • • •

Effect of pH on rennet stability

• •

•

Effect of hea·t on rennet stabiJ.i ty

VariatioDs of rennet activity with pH ^• Effect of temperature on

rennet

^activity

Effect of time

on

curd tension ^• ^• Effect of �� on curd tension

Effect of calcium concentration on curd tension • •

Proteolysis of whole casein Casein fractionation •

•

Hydrolysis of casein fractions 11olecular weight determinations

Amino acid composition •

DISCUSSION

• • • •

CHEESE HANUFACTtJRE •

REVIE'.V OF LIT

.2,'RAT'(JRE ^• ^• ^• Cheese manufacture in New

Zealand

• •

•

66

72 72

75 75 78 78 85 87 90

95 95 95

vi

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v:'Li

MEr.rHODS

^• ^• ^• ^• ^• ^• ^• ^•

98

Che

es

e

manufacture ^• ^• ^•

98

C

he

e

se flavour

and body

assessment •

98 Casein degradation in cheese

0

99

RESULTS • • •

100

Cheese

manufacture ^• ^•

100

Cheese

flavour and body

assessment

^•

100 Casein degradation in cheese

^•

104

DISCUSSION

• • • e

107

.cJ;IAprEE

^V�

COf�CLUSIONS

^• • • •

110

.AP.I;ENDIX I

C

he

e

se flavour assessment chart • •

113

REFERENCES

_• _• _• _• _• _• _•

114

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viii

LIST OF 1-'IGURBS

Figure Page

1. The comparison of 0. ]0-M perchloric acid

(

^o

) ,

0.12-H (

_e

) and Oc 73-H ( 6 ) trichloroacetic acids as precipitants for casein-rennet reaction mixtures using adult bovine (A), calf (B) and lamb (C)

2.

renne·ii s. ^· ^• ^• ^" ^• ^• ^• The comparison of

A217 and

_RU

/

^ml

for

(A), calf

(B)

^and ^lamb

(C) rennets.

•

adult bovine

-

.

3.

Elution patterns of adul·� bovine

(A) and

lamb (:S) rennets from ion exchange chromatography on

DEAE

26 28

cellulose. ^• ^• ^• ^• ^• ^•

40

4.

Elution patterns of adult bovine (A), la

m

_b

1

(B) and lamb

2 (C) enzymes from gel chromatography on Sephadex G-100 gel. • • • • 5. Electrophoretic pattern of adult bovine ( 1),

• 41

Lamb

1 (2) and lamb 2 (3) purified enzyme solutions. 44 6.

The elution pa·ttern of calf rennet from a

commercial DBAE ion exchange colv�n. ^• ^• 46

7.

Elution patterns of traditional

(A) and purified

(B)

calf rennets from ion exchange chromatography

on DEAE cellulose. ^• ^• ^• ^•

48

8.

The effect of pH on the stability of adult bovine

(A),

^calf

(B)

^and

lamb (C)

rennets at ionic strengths of

0.04 (3, (o ), 20, (e) and 30, ( t::,) °C) and

o.86 (30, (A) 0c). • • • • • 68

9. The effect of heat on the stability of adult; bovine

(

^o^{) , calf}

( e )

^and

lamb (

^!::.

)

rennets in

200 mg/ml (A) and 17 mg/ml (B) sodium chloride s olutions. 69 10.

The variation of milk coagulating activity with pH

for adult bovine

( o )

^{, calf}

(

^e

)

^{and lamb}

(

^A ⁾

11

•

12.

rennets.

^• ^• ^• ^• ^• ^• ^•

The effect of temperature on the milk coagulating activity of adult bovine

( o ) ,

^calf

( •

^{) and}

lamb

( 4 ) rennet s . • • • • •

The effect of time on the curd tension of

milk

coagulated by adult bovine

( o ) , calf ( e ) and

lamb

( � )

rennet s . ^• ^• ^• ^•

•

70

71

73

(10)

ix

13.

The effect of pH on the curd tension of milk coagu.lated by adult bovine (

o

^{) , calf}

(e

⁾

and lamb ( t. ) rennets. ^• ^• ^•

14.

The effect of calcium concentration on the curd.

tension of milk coagu.lated by

adu1:� bovine (

^o

)

^,

74

calf

(

^o⁾ ^and ^{lamb (}⁶) rel'l..net so ^c ^• •

76

15.

The proteolysis of \'vhole casein by adul·� bovine

(o

⁾^, calf($) and

1EW'1lb (!:.)

rennets.

16. Elution

pattern of whole acid casein from ion

17.

exchange chromatography on DEAE eellulose.

Elution pattern of whole acid casein from gel chromatography on Sephadex

G-1

00 gel. ^•

18.

Alkaline polyacrylamide gel electrophoresis of

•

Cl - casein hydrolysed for

o, 1, 4, 15, 30, 6o,

s -

120

and

240

minutes at pH

6. 5

by adult bovine (A),

77

79 80

calf (B)

and lamb (C) r

e

ⁿⁿ^e^t^s

.

^• ^• ^•

81 19.

Alkaline polyacrylamide gel electrophoretoe;rams

of �-casein h;ydrolysed for o,

1, 4, 15, 30, 6o, 120

Rnd

240

minutes at pH

6.5

_by adult bovine (A), calf

(B)

and lamb (C) rennet s. ^• ^• ^•

83 20.

Acid polyacrylamide gel electrophoretograms of

k-casein hydrolysed for

0, 15, 30

_and

60

_mfnutes

by adult bovine (A), calf

(B)

and lamb

(C)

rennets.84

21.

-The comparison between the logarithm of the

molecular weights and the elution volumes from a Sephad.ex

G-1 00

gel column for bovine seru_m albumin

( o

) , ovalbumin

( e),

myoglobin ( � ) and cytochrome

c (

^•

)

^. ^. ^• ^• ^• ^•

86

22.

Alkaline polyacrylamide gel electrophoretograms of cheese, after one, three, six, nine and twelve months storage at

60c,

manufactured with adult bovine (A), calf

(B),

lamb

(C)

and mixture

(D)

rennets. • • • • • • • •

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X

LIST OF TABLES

Table

Page

I A regre s s ion e quation and standard deviation for the

comparison

^of ^A217 ^c�d

RU/ml

betwe e n 0.01 and

0.09 RU/ml

for adult

bovine, calf

and lamb rennet s . • • • • •

II The amino acid composit ion of bovine pe psinogen,

27

lamb rennins A and

B

and c alf prorennin . 53

III

Mole cular we ight e st imations for adult bovine, lamb 1 and lamb 2 enzyme s from ge l

c

h

^roma^t^ograp

h

y me asurement s. ^• ^•

IV Amino ac i d composit ion of adult bovine, lamb 1

and lamb

2

enzymes . • • • •

V The frequency of t he differenc e betwe en flavour s core s of che e se

m

ade

with

^adult

bovine (B),

lamb

(L)

or mixture

(N)

^rennets ^compared ^with

the corre sponding che e s e made with

calf (C)

85

88

rennet . • • • • • • • 101

VI Normal variable value s t e sting the

hypotheses

that

che e se made

with

^{c alf} re1met was preferre d in half of the comparisons with che e s e made with adult bovine, lamb or mixture renne t s .

VII The average score s of flavour charact e rist i c s for che e se made with adult bovine, c alf, lamb or mixture rennet s . ^• ^• ^• VIII The percent age of judgement s allot t e d to e ach

102

103

body score of che ese manufacture d with adult bovine, calf, lamb or mixture rennet s . ^• 104