Some factors influencing the choice of lactic streptococci for use as starters in cheesemaking : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, New Zealand

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.

SOI"IE FACTORS INFLUENCING THE CHOICE

OF LACTIC STREPTOCOCCI FOR USE AS STARTERS IN CHEESEMAKING

A thesis pres ented in partial fulfilment of the requirements for the degr e e o f Doctor o f Philos ophy 1n Food Technology

at Mas s ey Univers jty , N ew Zealand.

Robert Jamies on Lowrie

1974

ABSTRACT

S trains of lactic s treptococci charact eri z ed as bit t er chees e s tart ers multipli ed rapidly during normal chees emaking and r eached high populations in the curd b efor e s al t ing . The multiplic ation o f s tart er s trains which are typically non­

b i t t er was inhib i t e d by the c ooking t emperatures us ed in normal ChedQar che e s emaking , even though ac i d produc t i o n c on­

t inued . The populations reached were subs tantially l ower than with bitt er s tart ers . I f manufacturing c onditi ons were alt ered s o that non-bit t er strains reached high numb ers in the curd , chees es were bitt er. C o Lvers ely , bitternes s was abs ent , or of reduc ed int ens ity, when growth of c onventional �y b i t t er s tarters was restri c t ed during chees e manufactur e ,

e ither by the us e of rai s e d c ooking t emperatures , or by

b ac t eri ophage attack. The results of 60 che e semaking trials with 1 0 s tarter s trains c onfirmed this as s oc iation b etwe en the s i z e of s tarter population reached during chees emaking and the pres enc e or abs enc e of bitterness .

A new model is proposed to acc ount f or the development o f b it t erne s s in Cheddar che es e . Unlike other schemes WLere the produc tion of b i tt er peptides is attribut ed directly t o r e nnet action on c as ein , the new hypothesis c o nsiders that the s t arter s train is respons ibl e for the format ion of bitt er­

flavoured peptides and do es not mer e ly act to remove them. All s tart ers are p o t entially able to produc e either b i t t er or non-bitter che es e , depending upon the c o nditi ons of manufac ture

(

inc luding bacteriophage devel opment

)

and , henc e , the p opula- t i on r8ached. The r espons e to s e l ec t ed manufac turing c on- ditions rather than any s ingl e differ enc e b etween particul ar s t arter s trains det ermines the likelihood of bitt erness

devel opment .

The key chees emaking trials were repeated us ing as eptic t echniques under c ontrolled bacterio logical c onditions .

Previ ous s tudi es of as eptically manufac tured chees e uti l i z ed s t art ers which gave l ittle Cheddar flavour o r exhib it ed

pronounc ed flavour defects . In this inves tigation , che es es

made as ept ically with Streptoc occus cremoris AM2 alone ,

poss es s ed full Cheddar flavour and no off-flavours provided that normal manufacturing proc edures were followed. Che es es made with Al'12 at a l ow c o oking temperature

(

^33°C

)

^{and ML8} che es es made at the normal c o oking· t emperature

(

^38°C

)

^were extremely bitter and lacking in Cheddar flavour. When appropr i at e leve ls of bact eriophage were pres ent during

manufactur e , bitt erness was abolished entirely from both the

ML8 and l ow-c ook AM2 chees es. There were acc ompanying increas e s in Cheddar flavour s c ores .

A s earch f or temperate b ac t eriophages and lys ogenic s trains in the l ac tic str eptoc occ i us ed as s tart ers in chees emaking showed that the oc currenc e of lys o geny or defective lys ogeny c ould b e r elative ly widespread. A t emperat e bact eri ophage was induc ible from Str.cremoris R1 by ultravio l e t irradiation or mitomyc in C treatment . Induc ed lys ates produc ed plaques on lawns of 3 c l o s ely related Str.cremoris s trains , AM1 , SK1 1 and us3 • Strain SK11 was r eaclily lys ogenised. Str . cr emori.s AM1 was the most r e l iab l e indicator s train , although the age

of the c ulture us e d for s e eding plat es was criti c al. Zones of lyeis , but no plaque format�on were obs erved on lawns of 9 adJiti ona l Str . cr emoris strains. S everal other s trains of b oth Str.cremoris and Str.lac t is were induc ibl e by either tr eatment but no indic at or s trains for the induc ed phages w ere found. The s i gnific anc e of lysogeny in che es emaking c annot yet b e. assess ed.

iv

PREFACE

Thes e studies were undertaken to investigat e the r o l e o f the s tart er s treptoc occ i in the devel opment of bitt erness and che es e flavour in Cheddar che es e . Becaus e o f the s eas onal nature of the work and long p eriods r equir ed to obs erve the

out c ome o f chees emaking experiment s a s ec ond pro j ect , the incidenc e of lys ogeny amongs t the s trains of start er s tr ept o­

cocc i , was undertak en c oncurrent ly with the chees e investiga­

t ions . Mos t previous studies had c onsidered only virul ent b acteriophaGes whereas temperat e bac t eriophages had b e en

virtually ignored . There was no c onfirmed r eport of lys ogeny in group N s treptoc occi when this investigation was c ommenc ed. I t was pos sib l e that s ome che es emaking charac t eris tic s of particular start er s trains were due t o their lys ogenic natur e . The results of c onventional che esemaking trials c arried out t o inves tigat e bitt erness development are r eport ed in

S e ction I . Sec tion II describ es experiments bas ed on che es e­

m aking using as eptic t echniques under c ontro l l ed bact erio­

l ogicel c onditions. The inve3t igations o f t emperate

bac t,�riophages and lys o geny in the start er streptoc occi are contained in Sec tion III.

V

ACKNOWLEDGEMENTS

I wish to thank the N ew Zealand Dairy Res earch Ins titute for the opportunity to undertake thes e investigations and for providing the facilities .

I am grateful to Dr R . C . Lawrenc e for his helpful advic e , c omments and enthus iasm in al l phas es of the proj ec ts and t o Pro f . E . L . Richards for overal l supervi s i on .

Almost a l l o f my c o l l e agues a t the Institut e c ontribut ed t o this study in some way. I n par t icular , thanks are due t o Dr L . E . Pea:c e for many valuab le discus s i ons , Mr J . Gilles for che es emaking , Mr M . F . Peb erdy for help in the as eptic che es emaking experiments , and Mrs P . E . Leyland and

MIBC . J . C l arke for t ec hnic al as s is tanc e.

Th e Elec tron Microsc opy Unit , Appl ied Biochemis try Divi s i o�,.

D . S . I . R . , Palmers ton Nor th c arri ed out the e l e c tron mic rosc opy .

vi

TABLE OF CONTENTS PREFACE

ACKNOWLEDGEMENTS LIST OF FIGURES LIST OF TABLES

Page N o . iv

V

ix

X

SECTION I THE GRO\r!TH OF LACTIC STREPTOCOCCI DURING CHEESEMAKING AND THE EFFECT ON BITTERNESS

DEVELOPMENT 1 1

INTRODUCTION 12

METHODS 1 3

S·cart ers and bac t eri ophages 13

Che e s e manufac tur e 14

Bitt erness evaluation 1 4

Laborat ory activity t es ts 15

Start er c o l ony c ounts 15

RESULTS 1 6

Laborat ory a c t ivity t e s t s 1 6

Che es emaking trials 1 8

F�avour evaluation 21

Bact eriophage infec tion during chees emaking 23

Additional experiment s 2 5

DISCUSSION 2 5

SECT ION II

Starter populations and b itterne s s devel op-

ment in Cheddar ch e e s e 2 5 A new hypoth e s i s to acc o unt for the develop-

ment of b i t t erness in Cheddar che es e 29 THE INFLUENCE OF BACTERIOPHAGE AND COOKING

TEMPERATURE ON FLAVOUR DEVELOPMENT IN CHEESE MANUFACTURED UNDER CORTROLLED BACTERIOLOGICAL CONDITIONS

INTRODUCTION

3 5 36 37 37 37 37 38 MATERIALS AND METHODS

Starters and bac t eri op�ages Milk supply

Chees e manufac ture

Bact eriologi c a l analys es

TABLE OF CONTENTS ( C ontd . ) C omp o s itional analys es

Flavour evaluati on RESULTS

Effec t ivenes s of as eptic chees emaking t echniques

Efiec t of star t er s train on flavour developm ent

Eff e c t of c ooking t emperature on flavour developm ent

Effec t of bac t eriophage o� flavour development

Start er survival DISCUSSION

SECTION III TEMPERNL'E BACTERIOPHAGEG AND LYSOGENY IN LACTIC STREPTOCOCCI USED AS STARTERS IN

CHEESEM.AKING INTRODUCTION

MATERIALS AND METHODS

Strains of lac tic stTeptococci Bac t eriophages

Media and growth of cultures C o l ony c ounts

Turbidity measur ements Ass ay of bac t eri ophages

Mitomyc in C tr eatment of cultures Ultraviolet irradiation of cultur e s Propagation of phage

C onc entration of phage r1 t Elec tron micro s c opy

RESULTS

Search for lys ogens

MC induction o f lys i s in S tr.cr emoris R1 UV induction o f lys i s in Str.cremoris R1 Lytic s pectrum det erminati ons

Charac t eristic s of strain R1 and its verificati on as a lys ogen

vii

Page No . 39 39 39 39 40 40

42 42 46

49 50 53 53 53 55 56 56 56 . 58 58 59 59 60 60 60 61 61 63 69

TABLE OF CONTENTS

(

^{C ontd .}

)

viii

Page N o . Stability of the lys ogenic charac t er of s train

R� with r epeat ed re-isolation and s ub-

c ulture 70

The indicator s train 7�

Attempted lys o g enis ation of other s trains 73

Lysogenisation of s train SK� � 73

Att empts to derive a c ured strain of R�

74

Charac t eristics of the induc ed r� t phage 77 Relationship b etwe en virul ent and t emperate

phages of Str.cremoris R� 78 Induc ibility of other s trains of Str.lactis

and Str . cremoris DISCUSSI ON

82 83 CONCLUSI ONS

APPENDIX I EXPERI I1ENTAL CHEESES : DETAILS OF MANUFACTURE AND ANALYSES

MATERIALS AND METHODS Milk s upply

Chees e manufac ture C ompos it i onal analys es Bac t eri o l o gical analys es

Express i on of s tart er c o l ony c ount data Flavour evaluat i on

LIST OF EXPERIMENTAL CHEESES

MANUFACTURING RECORDS AND ANALYSES REFERENCES

90

9�

92 92 92 92 93 93 94 9 5 97

� � 3

I1IST OF FIGURES

'1.

Growth of Str.cr e moris Al"12 and Str.lactis ML8 during laboratory act ivity t e s ts at

different coo king t emperatQres

2 . A model for bitternes s development in Cheddar chees e

3 . Starter co l ony counts in maturing Cheddar che es e

ix

Page No

30

made us ing as eptic t e chniques 4-5 4. Opt ical dens ity readings and colony counts in

M'1 6 broth cultures of Str.cremoris R1 57 5. Induc�ion o f lys is in Str.cr emoris R1 with

mitomycin C 62

6. Survival of Str.cremoris R1 and Str.lactis ML3

after exposure to UV irradiati on 64 7A. Induct ion of lys i s of Str.cremoris

�'1

^by

exposur e to UV irradiation

7B. Induction of lys i s of Str.cremoris R1 by exposure to UV irradiation

8 . Induction of sus p e cted SK1 1 lys ogens with mito mycin C

9 . The pres ence of phage r1 t i n a non-induced cultur e of Str.cremoris R1

'1 0 . Phage r1 t in a MC- induced culture of Str. cremoris R1

'1 ^•

PLATE

Bacteri ophages of Strepto co ccus cr emoris s train

R1 negatively s tained with neutral 2%

potas s ium phosphotungs tat e

65 . 66 76 79 80

8'1

X

LIST OF TABLES

Page N o I

II

Che es e manufactur e at low , normal and high c ooking t emperatures

Bitt ern e s s of che e s es graded 3 and 6 months aft er manufacture

I I I Chees e manufact ur e with Str . cremoris HP in IV

the pr es enc e of added b acteri ophage

Summary o f effects of manufacturing c onditions during chees e making on b itterness produc ti8n in che es es made with 1 0 different s tart er strains

V Influenc e of bac t eriophag e and c ooking t empera- t ur·e on bitt erness and Cheddar flavour

devel opment in che es es manufac tured under c ontro l l ed b ac t eri ol ogical c onditions VI Che es e manufacture under b ac t er i o l o gic ally

VII VIII IX

X

XI

XII

c ontr o l l e d c onditions in the pre s e nc e o f adde d bac t eri ophage

BaGteri ophages us ed in phage-s ens i t ivity t es t s Lyi:;ic s pectrum o f t emperat e and virulent

bac t eriophages o f Str.c r emoris s train R1 Effec t o f age , pH and suspending medium o f

strain AM1 s e ed cultur e s on t itres of induc ed r1 t phage

Phage s ens itivity of strain SK1 1 and suspec t e d lys o genic is o lat es

Induct i on of lys is in strains of Str.cremoris Induc tion of lys is in strains of Str.lactis

1 9 22 24

26

L�1

43

54

68

72

75

84

85