fractionation and characterisation of a commercial yeast extract to facilitate acceleration of yogurt

203 Pages·2014·2.81 MB·English

FRACTIONATION AND
CHARACTERISATION OF A
COMMERCIAL YEAST EXTRACT TO
FACILITATE ACCELERATION OF
YOGURT FERMENTATION
by
ESTI-ANDRINE SMITH
Submitted in accordance with the requirements for the degree of
PHILOSOPHIAE DOCTOR (FOOD SCIENCE)
in the
Faculty of Natural and Agricultural Sciences
Department of Microbial, Biochemical and Food Biotechnology
University of the Free State
Supervisor: Dr J. Myburgh
Co-supervisors: Prof G. Osthoff
Dr M. de Wit
November 2013
DECLARATION
I declare that the thesis hereby submitted for the qualification Philosophiae Doctor in Food
Science at the University of the Free State is my own independent work and that I have not
previously submitted the same work for a qualification at another university or faculty. I
furthermoreconcede copyright of the thesistothe University of the Free State.
Esti-Andrine Smith
November 2013
ACKNOWLEDGEMENTS
I wish to thank the following:
Dr. J. Myburgh for his support, advice, guidance, patience and friendship;
Prof. G. Osthoff for his valuable advice, support and expertise;
Dr. M. de Wit for her contribution;
Proffs. E. van Heerden and J.C. du Preez for valuable advice and troubleshooting;
The staff and students at the Department of Microbial, Biochemical and Food Biotechnology,
University of the Free State, for their support and the use of infrastructure;
The staff at the Division of Food Science, University of the Free State, for their help and
advice;
My parents, Andréand Cathy Smith, for their continuous support and encouragement.
I could not have wished for better parents;
The Creator for giving me the perseverance, strength and gifts for completing this study.
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TERMINOLOGY USED IN THIS STUDY
The terms “positive control” and “yeast extract supplemented milk” are used interchangeably
in this study.
“Negative control” refers to yogurt containing no supplementations.
All instances where reference is made to the current study refer to this specific research
study.
During all experiments discussed in this study a positive control and a negative control were
used.
American Psychological Association (APA) 6th Edition Reference style was used throughout
this document.
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OUTCOMES OF THIS STUDY
Patents:
Myburgh, J., & Smith, E. (2012). Production of a fermented foodstuff. Application number
2012/01139. Johannesburg, RSA: DM Kisch Inc.
Publications submitted to International Journals:
Smith, E., & Myburgh, J. (2013). The optimisation of accelerated yogurt production. Journal
of Microbiology, Biotechnology and Food Sciences.
Smith, E., Myburgh, J., Osthoff, G. & de Wit, M. (2013). Characterisation of the active
component in yeast extract which decreases yogurt fermentation time. Journal of Dairy
Research.
iii
LIST OF TABLES
TABLE NUMBER TABLE TITLE PAGE
Table 1 Protein fraction (g/100 g protein) of cow’s milk 7
Table 2 Typical analysis (% w/w) of yeast extract LP0021 provided by
manufacturer (Oxoid) 42
Table 3 Typical Amino Acids Analysis (% w/w) of Yeast Extract LP0021
provided by manufacturer (Oxoid) 42
Table 4 Free amino acid content (mg/100 ml) of milk and yogurt 46
Table 5 Protein content of yeast extract fractions obtained during
ultrafiltration 95
Table 6 Mineral composition of the accelerating fraction (Fraction 4) 117
Table 7 Amino acid content of accelerating fraction (Fraction 4) expressed
in g/100 g (%) of total amino acids 119
Table 8 Solubility and pH of the amino acids present in the accelerating
fraction (Fraction 4) 130
Table 9 Amino acid combinations reported in literature to stimulate
Streptococcus thermophilusgrowth 132
Table 10 Determinations of metabolic rates of the positive and negative
controls 164
Table 11 Appearance of colonies observed on three different variations of
Elliker’s broth 166
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LIST OF FIGURES
FIGURE NUMBER FIGURE TITLE PAGE
Figure 1 Time profile of yogurt production 3
Figure2 Scheme for proto-cooperation of yogurt starter culture 17
Figure 3 Sugar metabolism by starter cultures via the Embden-Meyerhof-
Parnas (EMP) pathway 20
Figure4 The traditional process of yogurt making 21
Figure 5 The improved process of yogurt making 22
Figure 6 Main processing steps in the manufacture of set and stirred yogurt 25
Figure 7 Casein micelle and submicelle model 31
Figure 8 The effect of heating milk to 90 °C on the casein micelle 32
Figure 9 Changes evident in the casein micelle structure during
pasteurisation of milk 33
Figure 10 The production processof yeast extract 40
Figure 11 Ground starter culture (A) and starter culture as provided by
supplier (B). 67
Figure 12 The variation in control yogurts prepared with three different
batches of fresh milk 68
Figure 13 Control yogurts prepared with different batches of skim milk powder
69
Figure 14 The effect of different yeast extract concentrations on the yogurt
fermentation process 71
Figure 15 Yogurt fermentation using different volumes of substrate 72
Figure 16 Preparative and analytical fractionation of yeast extract 79
Figure 17 Schematic diagram of the ultrafiltration sequence of yeast extract
using 4 individual molecular weight cut-off membranes 81
Figure 18 Comparison of yogurt supplemented with dialysed yeast extract
(12-14 kDa membrane) with positive and negative controls. 92
Figure 19 Comparison of yogurt supplemented with dialysed yeast extract (3.5
kDa membrane) with positive and negative controls 93
Figure 20 Colour response curve for BSA using the Standard Test Tube
Protocol 94
Figure 21 Effects of fractions obtained during yeast extract ultrafiltration on
yogurt fermentation time 96
Figure 22 HPLC chromatogram of unfractionated yeastextract 101
v
Figure 23 HPLC chromatogram of the 30 kDa retentate fraction obtained by
ultrafiltration of yeast extract 101
Figure 24 HPLC chromatogram of the 30 kDa filtrate fraction obtained by
ultrafiltration of yeast extract 101
Figure 25 HPLC chromatogram of the 10 kDa retentate fraction obtained by
ultrafiltration of yeast extract 102
Figure 26 HPLC chromatogram of the 10 kDa filtrate fraction obtained by
ultrafiltration of YE 102
Figure 27 HPLC chromatogram of the 3 kDa retentate fraction obtained by
ultrafiltration of yeast extract 102
Figure 28 HPLC chromatogram of the 3 kDa filtrate fraction obtained by
ultrafiltration of yeast extract 103
Figure 29 HPLC chromatogram of the 1 kDa retentate fraction obtained by
ultrafiltration of yeast extract 103
Figure 30 HPLC chromatogram of the 1 kDa filtrate fraction obtained by
ultrafiltration of yeast extract 103
Figure 31 HPLC chromatogram of 1 kDa filtrate fraction obtained by
ultrafiltration of yeast extract with adapted acetonitrile conditions
5%-75%-5%. 104
Figure 32 HPLC chromatogram of the supernatant obtained by TCA
precipitation of 1 kDa filtrate fraction obtained by ultrafiltration of
yeast extract 104
Figure 33 HPLC chromatogram of the precipitate obtained by TCA
precipitation of 1 kDa filtrate fraction obtained by ultrafiltration of
yeast extract 104
Figure 34 Yogurt fermentations with HPLC pooled fractions of 1 kDa filtrate
sample 105
Figure 35 SDS-PAGE of the 9 fractions obtained during ultrafiltration of yeast
extract 107
Figure 36 Silver stained SDS-PAGE of the 9 fractions obtained during
ultrafiltration of yeast extract 108
Figure 37 Size exclusion chromatogram of yeast extract with 4 identified
regions 110
Figure 38 Yogurt fermentations with 4 individually pooled size exclusion
fractions 111
Figure 39 SDS-PAGE of the 4 fractions obtained during size exclusion
chromatography of yeast extract 113
Figure 40 Size exclusion chromatogram of the mineral solution, vitamin
solution and yeast extract 118
Figure 41 Size exclusion chromatograms of yeast extract, the amino acid
solution and the free amino groups present in yeast extract 119
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Figure 42 The effect of mineral- and vitamin solutions on the yogurt
fermentation process 136
Figure 43 The effect of amino acid solutions on the yogurt fermentation
process 136
Figure 44 The effect of various concentrations of the amino acid cocktail on
the yogurt fermentation process 138
Figure 45 Various amino acid cocktail concentrations with the initial pH
adjusted 139
Figure 46A The effect of individual amino acids on the yogurt fermentation
process 143
Figure 46B The effect of individual amino acids on the yogurt fermentation
process 143
Figure 46C The effect of individual amino acids on the yogurt fermentation
process 144
Figure 47 The effect of various combinations of the amino acids present in
amino acid cocktail on the yogurt fermentation process 148
Figure 48A Effect of combinations of free amino acids on yogurt fermentation
according to literature 151
Figure 48B Effect of combinations of free amino acids on yogurt fermentation
according to literature 152
Figure 49 Depiction of enumeration efficiency of Elliker’s lactic agar incubated
at 37°C for48 hours 166
Figure 50 Cell counts of yogurt starter bacteria in YE supplemented and
unsupplemented (negative control) yogurt 167
Figure 51A Cell counts of negative control yogurt 169
Figure 51B Cell counts of positive control yogurt 169
Figure 52 Effect of lactic acid addition to reconstituted skim milkon pH 173
Figure 53 pH profile of unsupplemented yogurt (negative control) in
comparison to yeast extract supplemented yogurt (positive control) 175
Figure 54 pH and lactic acid content of YE supplemented and the control
yogurt 177
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS I
TERMINOLOGY USED IN THIS STUDY II
OUTCOMES OF THIS STUDY III
LIST OF TABLES IV
LIST OF FIGURES V
CHAPTER 1
LITERATURE REVIEW 1
1.1INTRODUCTION 2
1.2 MILK AND MILK CONSTITUENTS 4
1.2.1 Proteins in milk 4
1.2.2 Enzymes in milk 7
1.3 STARTER BACTERIAUSED FOR YOGURT MANUFACTURE 12
1.3.1 Background 12
1.3.2Characteristicsof starter cultures 13
1.3.3 Role of starter cultures in acid production 14
1.3.4 Starter bacteria proto-cooperation and proteolytic ability 14
1.3.5 Folate production 17
1.3.6 Lactose utilisation 18
1.4 YOGURT MANUFACTURING PROCESS 21
1.4.1Traditional and improved yogurt processes 21
1.4.2Flavour and texture components in yogurt 26
1.4.3Problems encountered with final product 28
1.4.4Health aspects of yogurt 29
1.4.5Changes in protein structure during yogurt processing 30
1.4.6Physico-chemical mechanisms involved in the formation of yogurt gels 34
1.5 YEAST EXTRACT 36
1.5.1 General introduction 36
1.5.2 Background on yeast extract 37
1.5.3 Production process of yeast extract 38
1.5.4 Yeast extract composition 41
1.5.5Thesupplementation of different media with yeast extract 43
1.5.6Effect of yeast extract on bacterial growth 47