Some aspects of the microbiology of cheese ripening investigated using aseptic manufacturing techniques : 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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OF CHEESE RIPEI�II�G I�·IVESTIGATED USING

ASEPTIC t1ANUFACTURING TECH�IIQUES

A thes i s presented in partial fulfilment o f the requirements for the degree of Doctor of Phi l o sophy in Food Technology at

Mas s ey Univers i ty Kei th Wil l i s Turner

Augus t 1988

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NAME AND ADDRESS DATE

MASSEY UNIVERSilt LlSUA�

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ABSTRACT

The effect of non- s tarter lactic acid bacteria ( NSLAB ) on the texture and flavour development in cheese was inves tigated us ing Cheddar cheese matured in impermeable cheese barrier bags . Cheeses made normally in open vats ( and contaminated with adventitious NSLAB) were compared with NSLAB - free cheese s .

To produce NSLAB - free chee s e s , two totally enclosed

cheese -making vats were des i gned and cons tructed . Thes e vats were s terilized by soaking overnight with Iodophor , then s team flushed for 1 hour . This procedure was suffic ient to exc lude NSLAB but did no t produce complete s teril ity as evidenced by the growth of bac i l lus - type organisms in UHT - treated milk incubated in the ster i l ized vats .

The thermal death characteris tics o f representative s trains of NSLAB showed that mos t species would no t survive milk pas teurization temperatures . However , two spec ies ,

Lactobac illus casei var casei and Leuconos toc lac t i s were sufficiently res i stant that , if present as the dominant flora of a raw milk , they could survive into the curd . Therefore , for asep tically manufactured cheeses , the milk was obtained by careful mi lking of the cows , to avo id NSLAB contamination . In the pasteurized vat - milks , the total bacterial count was

routinely less than 1 cfujmL .

i i i

A panel of tas ters was trained to determine the texture and flavour of the cheeses us ing attribute scal ing technique s . Six textural and 5 flavour attributes were defined , and the intens ities of these were monitored in the cheeses at vari ous ages up to 9 months .

The aseptic chees e -making procedure effect ively e l iminat e d NSLAB contamination from the cheese s . However , when che e s e s i n bags were matured for long periods , a surface flora of adventitious NSLAB developed , apparently by contamination during sampling . Despite all s ampl ing precaut ions , the only method found to s atis factorily prevent thi s contamination was waxing of the cheese surface s prior to bagging . I t was a l s o observed that i n adventitious ly contaminated chee s e s , the count of NSLAB in the surface 1-2 mm was at least 10 t imes the count in the remainder of the chees e .

The texture and flavour of Cheddar cheeses made without NSLAB and matured at 10 C for 9 months c ould not be distinguished from

cheeses with adventitious NSLAB matured under the same c onditions . In equivalent cheeses matured at lSC , the textures were again identical but the cheeses with NSLAB had greater intens i t i e s of sharpnes s and sulphide than the cheeses without NSLAB . Thus , s tarter alone appears to be the predominant contributor o f thos e compounds which produce Cheddar cheese flavour and NSLAB , al though pres ent , do not normally contribute to flavour production .

In a final part , the growth and c itrate uti l iz ing capab i li t i e s of three leucono s toc organisms were determined in a washed- curd

cheeses . Again , NSLAB - free cheeses were made s ince there are

currently no effective means of differentiating between leucono s toc and NSLAB organisms , and NSLAB organisms can also utilize c i trate.

While all three strains were capab l e of rap i d growth to leve ls around 107 cfu/mL in RSM , only one grew s ignificantly in the chee s e s . Thi s s train was capable o f fermenting c i trate in the abs ence of a carbohydrate energy s ource and removed the c it rate present in brine - salted cheeses within 1 month at e i ther 10 C o r 15 c .

ACKNOWLEDGEMENTS

I would l ike to thank a number of people without whose as s i s tance the research reported in this the s i s would have been almos t impos s ible .

V

Firstly to Dr . P . S . Robertson , Director o f the N . Z . Dairy Research Ins titute , my grateful thanks for arranging the S taff Development Fellowship . Thank you also to my supervis ors , Dr . J. LeLievre , Dr . R . C . Lawrence , and Mr . R . Benne tt who gui ded me through the frus trating times of negative results .

To Mr . I . Horley who turned the concepts o f the asep tic vats into specifications and drawings , Mr . C . Ros s i ter who turned the drawings into phys ical plant , and to Mr . A . Mathes on , who created the computer controller , thank you for your

perseverance, skill and dedication to a j ob wel l done . Thanks to the s taff of the Cheese Technology Section ,

D . P . D . C . who a s s i s ted with the manufacture of open vat che e s e and t o the s taff of the Analytical Chemistry Section , NZDRI , who analysed the fat levels in the cheese .

The help of Dr . H . R . Cooper and the s taff o f the Product Use and Evaluat ion Section , NZDRI , in the s ensory evaluation o f the cheese i s gratefully acknowledge d , as i s the as s i s tance o f the s taff of the Appl ied Mathematics Section , whos e ass is t ance with data manipulati on for the computerized s tatis t ical

analys is was invaluable .

I would espec ially l ike to thank the following people whos e ass is tance and friendship throughout the length of the proj ect was e specially valuable . To Debb ie Whi te and Tania Burdan , laboratory Technic ians in the Cheese Techno logy Sect ion , thank you for your skilful assis tance , espec ially in thos e rather mundane but important areas like plate counting . To Dr . Frank Martley , I have particularly valued your support whi ch was s o cons tant and encouraging . Our friendship will remain s trong .

Als o , to my many friends in the N . Z . D . R . I . who have helpe d i n thos e numerous little ways too many t o mention

individually , thank you . We both know the value of your ass is s tance .

Finally , no married s tudent can succeed without the selfless support of spouse and family . Thank you Kathryn for coping with all the s train and s tress , the early mornings and the

late evenings . Thi s has been a labour of love , which has not gone unnoticed or unapprec iated . And to Chr i s topher , who a t the age o f three thought that a good game was to s i t a t the computer and type a the s i s , may yours be eas ier than mine!

Abs tract

Acknowledgements Table of Contents Lis t of Figures Lis t o f Tables Lis t of Plates

TABLE OF CONTENTS

Literature Review and General Introduction Part I . Methodology

Page i i

V

vii x i x i i

XV

1

I . A . l Des ign and Commis s ioning of the Aseptic Vats 1 2

I . A . l . l Introduction 1 2

I . A . l . 2 Des ign Criteria Se lec ted 13 I . A . l . 3 Description of the Plant 13 I . A . l . 4 Plant S ter i l ization 1 9 I . A . l . 5 Steril i ty of the Plant 2 1 I . A . l . 6 Pasteurization and Milk Sampl ing 23

I . A . l . 7 Cheese Manufacture 2 4

I . A 1 . 8 Vat Cleaning 2 8

I . A . l . 9 Dehooping , Bagging and Maturing 2 9 I . A . l . lO Compos ition of the Cheeses 29 I . A . l . ll Efficiency of the Des ign 3 2 I . A . 2 Manufacture o f NSLAB - free Cheeses 33

I . A . 2 . 1 Background 33

I . A . 2 . 2 Experimental Approach 33

I . A . 2 . 3 Milk Composition 3 4

I . A . 2 . 4 Cheese Compos i tion 3 4

I . A . 2 . 5 Bacterial Counts i n the Cheeses 36

I . A . 2 . 6 Conclus ions 3 6

I . A . 3 A Microbiological Specification for Milk

for Aseptic Cheesemaking 3 8

I . A . 3 . 1 Introduction 3 8

I . A . 3 . 2 Methods 3 9

I . A . 3 . 3 Results 40

I . A . 3 . 3 . 1 Target Level for the Des truction

of NSLAB 40

I . A . 3 . 3 . 2 Survival Fol l owing Heating for 15 s 41 I . A . 3 . 3 . 3 Thermal Death Characteris tics of

Selected S trains . 42

I . A . 3 . 3 . 4 Conditions Required to Obtain

"NSLAB - free " milk . 43 I . A . 4 Aseptic Milking and the Proof of NSLAB - free

Conditions 45

I . A . 4 . 1 Aseptic Milking Procedures . 45

I . A . 4 . 1 . 1 Methods 45

I . A . 4 . 2 Bacterial Counts in " S lurrie s " Made

from Aseptically Drawn Milk 49

I . A . 4 . 2 . 1 Slurry Manufacture 49 I . A . 4 . 2 . 2 Bacterial Counts in the " S lurries " 5 1

I . A . 4 . 3 Conclus ions 5 2

I . B Methods for Texture and Flavour Analys i s of Cheeses

I . B . l Introduction

I . B . 2 Details of the Conditions for Sample I . B . 3

I . B . 4 I . B . 5 I . B . 6

Assessment

Selecti on o f the Pane l is ts Panel Training

Scale Comparison Data Analys i s

Part I I . The Effect of NSLAB on Cheddar Cheese Ripening 5 3 5 3 5 5 5 6 5 7 5 8 5 9

I I . 1 Literature Review 6 5 '

I I . l . l Occurance , Source and Growth of the

NSLAB Flora 6 5

1 1 . 1 . 2 Metabolic Capab i li ty of NSLAB 6 7 1 1 . 1 . 3 Aims o f thi s Research Proj ect 70

1 1 . 2 Methods 7 2

1 1 . 2 . 1 Culture His tory and Maintenance 7 2 11 . 2 . 2 Temperature Optimum Determination 7 3

1 1 . 2 . 3 Cheesemaking Methods 7 3

11 . 2 . 3 . 1 Milk Supply 7 3

11 . 2 . 3 . 2 Aseptic Cheesemaking 7 3 11 . 2 . 3 . 3 Open Vat Manufacturing Methods 7 4 11 . 2 . 3 . 4 Waxing of Cheeses 7 5

I I . 2 . 4 S lurry Manufacture 7 6

I I . 2 . 5 Sampl ing o f Cheeses 7 6

1 1 . 2 . 6 Colony Counts 7 7

1 1 . 2 . 7 Chemical Analyses 78

1 1 . 2 . 8 Texture and Flavour Assessment 80 1 1 . 3 Trials with Cheeses Matured in Bags 8 1

1! . 3 . 1 Comparison o f Cheeses Made in the Aseptic

Vats with Cheeses Made in Open Vats 8 1 1! . 3 . 1 . 1 S tatis tical Approach 8 1 11 . 3 . 1 . 2 Cheese Comp o s i t ion 8 2 I I . 3 . 1 . 3 Bacterial Counts 84 1! . 3 . 1 . 4 Texture and Flavour in the

Cheeses hel d for 6 months at 10

and 15 C 8 5

1 ! . 3 . 1 . 5 Texture and Flavour o f Chees e s

Held for 9 months at 1 0 C 90 1! . 3 . 2 Source of NSLAB in Control Aseptic Vat

Cheeses 9 1

I I . 3 . 3 Conclus ions 9 4 I I . 3 . 3 . 1 Phys ical and Bacterial Compos i t ion 9 4 I I . 3 . 3 . 2 Texture and Flavour 9 4 I I . 3 . 3 . 3 Surface Contamination 9 6 II . 4 Trials with Waxed Cheeses

I I . 4 . 1 S tatistical Approach I I . 4 . 2 Cheese Compos ition I I . 4 . 3 Bacterial Counts I I . 4 . 4 Texture and Flavour

I I . 4 . 4 . 1 Texture II . 4 . 4 . 2 Flavour I I . 4 . 5 Conclus ions

I I . 5 Selection and Growth Characteri s tics of NSLAB for Addition to Cheeses I I . 5 . 1 Introduction

I I . 5 . 2 Characteristics of NSLAB Cultures I I . 5 . 2 . 1 Homolactic Lactobaci l l i

I I . 5 . 2 . 2 Heterofermentative Lactobaci l l i I I . 5 . 2 . 3 Pediococci

I I . 5 . 3 Growth Characteristics in Mi lk I I . 5 . 4 Procedures for the Inoculation of

NSLAB - free Curd with NSLAB Organisms I I . 5 . 5 Growth of NSLAB Organisms Del iberately

Added to Aseptically Manufactured Curd I I . 5 . 5 . 1 Initial Trial

I I . 5 . 5 . 2 The Effect of NSLAB Addition on Texture and Flavour

I I . 5 . 5 . 2 . 1 Statis tical Approach I I . 5 . 5 . 2 . 2 Cheese Compos ition I I . 5 . 5 . 2 . 3 Bacterial Counts

I I . 5 . 5 . 2 . 4 Texture and Flavour of

II . 5 . 6 Conclus ions

Cheeses Held for 6 months 10 and 15 C

I I . 6 Discuss i on and Conclus ions

I I . 6 . 1 Manufacturing Conditions Required to Produce NSLAB - free Cheeses

I I . 6 . 2 The Effect of NSLAB on the Maturation o f Cheddar Cheeses

I I . 6 . 3 Pointer s for Further Research

at

Part III . The Growth of Leuconostoc Organisms In Cheese III . l �iterature Review

9 8 9 8 9 8 100 101 1 0 1 102 105

108 108 109 109 110 1 1 2 1 1 3 1 1 4 1 1 7 1 1 7 1 1 9 1 1 9 1 2 0 1 2 0

1 2 2 1 2 5 1 2 7 1 2 7 1 2 9 1 3 4

1 3 7

I I I . 2 Methods 141

I I I . 2 . 1 Microb iological Methods 141 I I I . 2 . 1 . 1 Cul ture History and Maintenance 141 I I I . 2 . 1 . 2 Colony Counting 141

I I I . 2 . 1 . 4 Growth Sugar Determinations 142 I I I . 2 . 1 . 5 Growth in RSM 143

I I I . 2 . 2 Chemical Analyses 143

I I I . 2 . 3 Citrate Utilization S tudies 144 I I I . 2 . 4 Asep t ic Vat Cheesemaking 145

I I I . 3 Results 148

I I I . 3 . 1 Characterization of Leuconos toc I s o l ates 148 I I I . 3 . 2 The Effect of Glucose and pH on Citrate

Utilization by Leuconos toc Lc83 149 I I I . 3 . 3 Growth and Citrate Utilization by

Leuconos toc Isolates in Gouda - type

Cheeses 150

I I I . 3 . 3 . 1 S tatis tical Approach 150 I I I . 3 . 3 . 2 Cheese Composition 1 5 1 I II . 3 . 3 . 3 Bacterial Counts in the Vat Milks

and the Chees e s 1 5 2

I I I . 3 . 3 . 4 C itrate Disappearance from

Cheeses containing Leuconos tocs 153 I I I . 4 D is cuss ion and Conclus ions 154

Bib l iography 1 5 9

Figure 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 .

9 . 10 . 11 .

12 .

LIST OF FIGURES

Follow ing page Front and s ide elevations of the aseptic vat uni t ,

plan view and frame detail . 15

S ide rear and p lan views of details o f each aseptic

vat body . 1 5

Details o f all mountings on the aseptic vat bodies 15 Agi tator des ign and mounting detail . 15 P iping diagram for the aseptic vat unit . 15 Detail of the ancilliary cheese making equipment

for the aseptic vats . 15

Swabbing pattern to determine the s teril i ty o f the

s teril ized aseptic vats . 2 2

Growth of NSLAB organisms i n control cheeses made in the aseptic vats from non- asep tically drawn

milk . 36

Thermal death of L . plantarum 8014 in milk . 41 Z -values of two representative NSLAB cul tures . 43 Counts of s tarter , NSLAB and micrococcus organisms

in " s lurries " made under s trict aseptic laboratory c onditions from aseptically drawn mi lk . 5 1 Counts o f s tarter and NSLAB organisms i n aseptically

made cheeses and in open vat cheeses made from

milk from the s ame farm . 84

13 . S tarter and NSLAB counts in aseptic and open vat chees e s matured either rinded and waxed or waxed

and b agged . 100

14 . Threeway interaction means for fruity , sharp and sulphide attributes of the flavours o f aseptic and open vat cheeses matured e i ther r inded and

waxed or waxed and bagged . 104

15 . Growth and pH changes of pediococcus isolate 3 9 sequentially subcultured in RSM a t 3 0 C every 24

hours. 113

16 . Growth of two strains of pediococci in MRS , RSM

and RSM fortified with yeast extract . 114 1 7 . Growth of 6 NSLAB i sloates in aseptically

manufactured Cheddar chees e s . 117

18 . Growth of deliberately added NSLAB in aseptically

manufactured cheeses . 118

1 9 . S tarter counts in aseptically manufac tured cheeses

with deliberately added NSLAB . 120

20 . Counts of del iberately added NSLAB organi sms in

aseptically manufactured cheeses . 120

21 . The rate of growth of three leuconos toc i s olates

in RSM at 30 C . 149

2 2 . The effect of glucose and pH on the util ization of

c i trate by Lc83 . 150

23 . The c ounts of three leucono s toc s trains ino culated into aseptically manufac tured cheeses . 152 24 . The disappearance of c i trate from aseptically

manufac tured cheeses inoculated with one o f

three leuconos toc organisms . 153

i . Des ign criteria for aseptic cheesemaking vats . 14 i i . The microb iological counts and changes in pH o f UHT

treated mi lk incubated in the s teri l ized aseptic

vats . 22

i i i . Chee semaking protocol for Cheddar cheesemaking in the

aseptic vats . 2 5

iv . The compos ition of milk , whey and 1 day cheeses

made in the aseptic vats and in the open vats . 31 v . Bacteri al counts in 1 3 raw milks collected from the

bulk vat of one town- supply dairy farm , and in the s ame mi lks after pas teur ization . 3 5 vi . Mean 1 day composi tion of 15 cheeses made in the

asep t ic vats from non- asep tically drawn milk . 36 vii . Thermal death of NSLAB cul tures inoculated into RSM

and held for 1 5 s at temperatures between 5 0 C

and 80 C . 42

vi i i . Mean bacterial counts from 45 aseptically drawn raw milks collected over three manufacturing seasons . 48 ix . Samples of reformed cheeses used for pane l i s t

screening . ₅₈

x . Definitions o f the attributes used by the Cheddar

cheese panel . ₆₁

xi . Copy of the score sheet used by the panel for scoring

the cheese attributes . ₆₂

xii . Chemical compos ition of cheeses made in the aseptic

and open vats . 8 3

xi i i . F-values and estimates of s i gnificance o f texture and flavour attributes of cheeses made in the aseptic and open vats and he ld for 6 months at

10 C and 15 C . 8 6

xiv . Significant means of the differences in texture and flavour attributes of chees e s made in the aseptic and open vats and held for 6 months at 10 C and

1 5 c . 87

xv . Interaction means of the differences in texture and flavour attributes of cheeses made in the aseptic and open vats and held for 6 months at 10 C and

1 5 c. 8 8

xvi . Threeway interactions for the differences in texture and flavour attributes of cheeses made in the aseptic and open vats and held for 6 months at

1 0 C and 15 C ^. 8 9

xvi i . F-values and e s timates o f s i gnificance of texture and flavour attributes of cheeses made in the aseptic and open vats and held for 9 months at

10 c . 9 0

xvii i . S i gnificant means of the differences i n texture and flavour attributes of cheeses made in the aseptic and open vats and held for 9 months at 10 C . 9 1

xix . NSLAB counts during the maturing of two cheeses made in the aseptic vats and in s lurries made from

the ir curds when 1 day o ld . 9 2

xx . Interior and exterior NSLAB counts of 9 month

chees e s . 9 3

xxi . Chemical compos ition of cheeses made in the aseptic and open vats during the comparison with waxed

cheeses . 9 9

xxi i . The change in pH o f aseptic and open vat cheeses waxed and matured either bagged or rinded at

10 C and at 1 5 C . 1 00

xxi i i . F-values and es timates of s i gnificance of texture and flavour attributes of cheeses made in the aseptic and open vats and matured either waxed

or bagged held for 6 months at either 10 or 15 C . l02 xxiv . Means of the s i gnificant differences in texture

and flavour attributes o f cheeses made in the aseptic and open vats and matured either waxed and bagged or waxed and r inded and held for

6 months at either 10 or 15 C . 1 0 3 xxv . The differential effect of type o f manufacture

(aseptic or open vat ) with either temperature or time of maturation on the sharpnes s of

cheese . 104

xxvi . Temperature*time interaction means for fruitines s , sulphide and sharpnes s o f cheeses made in the aseptic and open vats and matured either waxed or bagged held for 3 and 6 months at e i ther

10 or 15C . 1 0 5

xxvi i . Growth characteristics of various homolactic lactobacillus cultures originally isolated from

Cheddar cheeses . 1 10

xxvi i i . Growth characteristics o f various heterolactic lactobacillus cultures original ly isolated from

Cheddar cheeses . 1 1 1

xxix . Growth characteristics o f various pediococcus

cultures originally isolated from Cheddar cheese s . l l 2 xxx . Reproduc ib ility of counts o f NSLAB cultures grown

for 48 h in RSM at 30 C . 1 1 5

xxx i . Effect o f growth medium o f NSLAB ( Pe 3 9 ) and s ite

of inoculation on the count in cheese . 1 1 6 xxx i i . Generation times ( in days ) for NSLAB del iberately

added to cheese . 1 1 8

xxx i i i . Chemical compos iti on o f cheeses made i n the aseptic vat with and without added NSLAB

cultures . 1 2 0

xxxiv . F-values and estimates o f s ignificance of texture and flavour attributes of cheeses made in the aseptic vat with added NSLAB cultures and held

for 6 months at 10 C and 15 C . 1 2 2 xxxv . S ignificant means of the differences in texture

and flavour attributes of cheeses made in the aseptic vat with added NSLAB cultures and held

for 6 months at 10 C and 1 5 C . 1 2 3

aseptic vat with added NSLAB cultures and held

for 6 months at 10 C and 15 C . 124 xxxvii . Threeway interact i ons for the difference s in

texture and flavour attributes o f cheeses made in the aseptic and open vats and held for 6 months at 10 C and 15 C . 1 2 5 xxxvi i i . Cheesemaking protocol for Gouda cheesemaking in

the aseptic vats . 1 46

xxxix . Growth o f various leuconos toc i s o lates in MRS

agar , with different growth sugar s . 149 xl . Composition of " Gouda" cheeses made in the

aseptic vats . 1 5 1

LIST OF PLATES

Following

Plate page

1 . Front view of the aseptic vat unit ins talled in

the dairy plant . 19

2 . Rear view of the aseptic vat unit ins talled in

the dairy plant . 1 9