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dissertation entitled

Ripening, Textural and Flavor Characteristics of Reduced
Fat Cheddar Cheese Mhde With Adjunct Cultures of
Lactobacillus casei LZA.

 

presented by

Mauro Mansur Furtado

has been accepted towards fulfillment
of the requirements for

Ph-D degree in EQQd Science

 

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/ Major professor /
John A, Partridge

Date magma/94

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RIPENING, TEXTURAL AND FLAVOR CHARACTERISTICS OF REDUCED FAT

CHEDDAR CHEESE MADE WITH ADJUNCT CULTURES OF

Iactobacillus casei LZA .

by

Mauro Mansur Furtado

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1994

ABSTRACT

RIPENING, TEXTURAL AND FLAVOR CHARACTERISTICS OF REDUCED FAT
CHEDDAR CHEESE MADE WITH ADJUNCT CULTURES OF

Lactobacillus casei LZA.

by

Mauro Mansur Furtado

Twenty-eight batches of cheese were made to evaluate the
use oftadjunct.cultureS»of.bactobacillus casei L2A.to increase
flavor intensity, improve texture and decrease the incidence
of bitterness in reduced fat Cheddar cheese. Cheeses were
made with different levels of live and heat shocked cells of
an adjunct culture of Lactobacillus casei LZA and a slow acid
producing culture of Lactococcus lactis var. lactis. Cheese
proteolysis was monitored.for 20 weeks through determinations
of pH 4.6 and 12% TCA-soluble nitrogen and SDS polyacrylamide
gel electrophoresis. Adhesiveness, cohesiveness, springiness
and hardness were measured by the Instron Universal Testing
Machine. A trained panel evaluated the same cheese textural
parameters, flavor intensity and development of tdtterness

after 8 months of ripening. Hydrophobic peptide extracts from

cheeses were analyzed by high performance liquid
chromatography on a reverse phase column.

Reduced fat Cheddar cheeses had a high moisture content
(average 49%), which probably enhanced Lactobacillus casei
activity. Cheeses made with heat-shocked Lactobacillus casei
presented the highest degree of proteolysis and the greatest
amount of 12% TCA-soluble nitrogen. In all cheeses, an-casein
was more extensively degraded than B-casein, especially when
heat shocked cells of Lactobacillus casei were used. Cheeses
made with live cells of Lactobacillus casei were more acidic
and, consequently, harder, less cohesive and less springy.
Hardness decreased in all cheeses during the first 8 weeks of
ripening as the result of the breakdown of the casein matrix.
A lack of typical Cheddar cheese flavor was noticed by the
sensory panel in all cheese samples. Increasing the
inoculation concentration of heat shocked cells of
Lactobacillus casei to the milk was the best approach to
reduce bitterness in the cheese. Chromatographic data showed
that the peptide fraction corresponding to a distinctly bitter
cheese had a high concentration of low molecular weight and
highly hydrophobic peptides. This peptide fraction was rich in
hydrophobic amino acids like phenylalanine and tyrosine. The
bitter taste intensity was somewhat reduced when heat shocked

cells of Lactobacillus casei were used as an adjunct culture.

ACKNOWLEDGMENTS

I would like to express my sincere appreciation and
thanks to my major professor Dr. John A. Partridge for his
guidance, encouragement and support throughout this study.
Appreciation is also expressed to the other members of the
committee, Drs. D. Smith, z. Ustunol and M. Zabik. I wish to
thank Dr. M. Bennink and Dr. W. Bergen for making available
the HPLC facilities used in this research. I also would like
to thank Dr. J. Gill for imparting to me algenerous measure of
his enthusiasm. for and. wide ,knowledge of the field of
Statistics. The writer also wishes to express his most sincere
appreciation to all of those who consented to participate in
the sensory evaluation panel.

I also want to express my deep appreciation and thanks to
our great friends Dr. Wanderley Calaca and his wife Saloma,
for their support and friendship. I will always have you as
"Friends for Life".

My' deep appreciation. and, gratitude are directed. to
Conselho de Desenvolvimento Cientifico e Tecnologico (CNPq,

Brazil), for the financial support provided during my studies.

iv

Finally and by no small measure lastly, I must thank
those who mean most, my familyu Without.my lovely wife, Vera,
it never would have happened. Her sacrifice, understanding,
encouragement, faith and patience were never ending. I would
like to thank my three children, Jonathan, Ariel, and Jessica
for the sacrifice they have made and the love and
encouragement they have provided. I will be forever thankful.
Thanks to my mother whose love and patience were a constant
motivation for me to complete my degree. My deepest gratitude

to my father, whose image of a man I have admired and loved.

I love you all.

TABLE OF CONTENTS

Page
LIST OF TABLES ..... ..... .............................. ix
LIST OF FIGURES .............. ..... .................... x
LIST OF APPENDIX TABLES ............ . . . . ............ . . . xiii

LIST OFAPPENDIX FIGURES 000......OOOOOOOOOOOOOOOOOOOOO xv

LISTOFABBREVIATIONSO...0.0.0....OOOOOOOOOCOOOOOOOOOO XVii

INTRODUCTION .00... OOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOO 1
LITERATURE REVIEW 00......OOOOOOOOOOOOOOOOOOOO0.0.0.... 5
1. Reduced Fat Cheese.. ........ .......... ....... .. 5

2. Texture Development During Ripening ...... ..... 15

2.1. Effect of Proteolysis on Texture ......... 17

2.2. Measurements of Textural Properties ...... 20

3. Flavor Development During Ripening ............ 27

3.1. Effect of Proteolysis on Flavor .......... 28
4. Bitter Peptides in Cheese ..................... 31
4.1. Mechanism of Bitter Flavor Formation
and Degradation .......................... 32
4.1.1. Role of Rennet in Cheese
Bitterness ........................ 41
4.1.2. Role of Starter Microorganisms in
Cheese Bitterness ................. 48
4.1.2.1. Extracellular Proteinase
Activity ................. 49
4.1.2.2. Peptidase Activity ....... 51

4.2. Source of Bitter Peptides ................ 55
4.3. Chemical and Structural Aspects of Bitter
Peptides ................................. 57
4.4. Control of Bitterness Level in Cheese .... 60
4.4.1. Control by Manufacturing
Adjustments ....................... 60

4.4.2. Control by Culture Selection ...... 61
4.4.3. The Use of Lactobacillus .......... 63

vi

CHAPTER

CHAPTER

Page
4.5. Peptide Separation ..... ...... ....... ..... 66

I - RIPENING CHARACTERISTICS OF REDUCED FAT
CHEDDAR CHEESE USING HEAT SHOCKED AND
LIVE Lactobacillus casei L2A AS ADJUNCT
CULTURE ................................... 69

Introduction ............ .............. ........ 69
Material and Methods .......................... 72
Culture Preparation ... ......... .......... 72
Cheesemaking ............................. 73

Compositional Analysis of Cheese ......... 74
Assessment of Proteolysis ................ 75

Nitrogen Fractions ... ..... . ......... 75

Ripening Indexes .. ....... ........... 75
Electrophoresis .............. ....... 75
Statistical Analysis ..................... 76
Results and Discussion ........................ 77
Composition of Cheese .................... 77
Cheese pH ................................ 79
Proteolysis .............................. 81
Electrophoretic Patterns ......... ........ 87
Conclusions ................................... 92

II - THE USE OF Lactobacillus casei L2A AS
ADJUNCT CULTURE AND ITS EFFECTS ON
FLAVOR AND TEXTURE 0F REDUCED FAT CHEDDAR

CHEESE 0.00.00... OOOOOOOOOOOOOOOOOOOOOOOOO 93
Introduction ................ . ................. 93
material and methads O O O O O O O O O O ........ O O O O O O O O 95

Rheological Measurements ................. 95

Sample Preparation ......... .... ..... 96

Setting of the Instron .............. 96
Determination of the Parameters ..... 97
Organoleptic Assessment .................. 99

Results and Discussion ...... .................. 101
Rheological Evaluation .. ............ ..... 101
Adhesiveness ........................ 101
Cohesiveness ..... ................... 103
Springiness ............ ....... ...... 105

Hardness .... ..... .. ...... ........... 105

Sensory Evaluation ....................... 109
Textural Characteristics ............ 109
Organoleptical Assessment ........... 112

Conclusions ................................... 115

vii

CHAPTER III - HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF

PEPTIDES AND AMINO ACIDS IN REDUCED FAT
CHEDDAR CHEESE WITH ADDED Lactobacillus

casei L2A00000......0.....00......0.000.

Introduction ... ...............................

Material andMethOds OOOOOOOOOOOOOOOOOOOOOOO
Organoleptic Assessment ...............

Peptide Extraction ...... ..... ...... ......

Peptide Analysis by HPLC ..............

Sample Preparation for HPLC .... .....

Solvents .........................

HPLC Analysis ....................

Amino Acid Analysis by HPLC ...........
Results and Discussion .....................

Sensory Evaluation .... ...... ... .........

HPLC Analysis of Cheese Peptides ......

Cheese Amino Acids ...... ........ . .......

CODCIuSions 00......0.0.0.0....0000000000.0.

SUMMARY AND CONCLUSION .. ................ ...........

APPENDIXI00.00000000000000000000.00.00.00.000.....

APPENDIX II 0.0.0.0000000000. ..... 0000......00000000

APPENDIX III .00.... ...... 0.000.000.0000 000000000000000

LIST OFREFERENCES 0.0.0.000......0.00.. 000000000000

viii

Page

117

117
119
119
120
120
120
122
122
124
125
126
128
137
141

142
145
147
156
174

178

Table

LIST OF TABLES

Page

Effect of rennet level on bitterness of
six month old cheese manufactured with
bitter strain Lactococcus cremoris .......... 44

Effect of the initial pH of the cheese
milk on the rennet content and the bitter
flavor of Gouda cheese ..... ..... ............ 46

Effect of the cooking temperature on rennet
retention and bitterness perception in Gouda
type Cheese ......OOOOOOOOOOOOO.....OOOOOOOOO 47

Composition of reduced fat Cheddar cheese

manufactured with adjunct culture of
Lactobacillus casei L2A ..................... 78

ix

Figure

LIST OF FIGURES

Page

Czulak’s hypothesis for the formation and
breakdown of bitter peptides in cheese ....... 34

Lowrie and Lawrence’s hypothesis for the
formation and breakdown of bitter peptides
in Cheese 0.0.000.0....0....0..00........0...0 36

Tentative scheme for the formation and
breakdown of bitter peptides in cheese ....... 39

Mechanism for the development of bitterness
in Gouda-type cheese ......................... 40

Utilization of milk protein for synthesis of
bacterial protein in lactic streptococci ..... 53

Changes in cheese pH throughout ripening of
reduced fat cheddar cheese made with adjunct
culture of Lactobacillus casei L2A ........... 80

Evolution of nitrogen fractions during the

ripening of reduced fat cheddar cheese made

with 1.5% of live and heat shocked

Lactobacillus casei L2A ...................... 82

Changes in the ripening indices during the

ripening of reduced fat Cheddar cheese made

with different levels of adjunct culture

of Lactobacillus casei L2A ................... 84

Percentage of water soluble nitrogen (WSN)
corresponding to TCA-soluble nitrogen (TCA-N)
after 20 weeks of ripening ................... 86

I

Figure Page

I-5 SDS-polyacrylamide gel electrophoretograms
of caseins in reduced fat Cheddar cheese at
1 week and 20 weeks of ripening ............... 88

I-6 Degradation of B-casein and afl-casein
throughout the ripening of reduced fat Cheddar
cheese made with 1.5% of live and heat shocked
Lactobacillus casei L2A .......... ..... ....... 89

I-7 Correlation between degradation of afl-casein
and increase in water soluble nitrogen (WSN)
throughout the ripening of reduced fat Cheddar
cheese . ........... ............. ........ ...... 91

II-1 Typical texture profile analysis (TPA) curve
obtained by the Instron Universal Testing
MaChine O......OOOOOOOOOOOOOOOOOO ..... 00.0.... 98

II-2 Textural definition of springiness ........... 100

II-3 Adhesiveness throughout the ripening of
reduced fat Cheddar cheese made with 1.5%
of live and heat shocked cells of
Lactobacillus casei L2A ..... . ........... ..... 102

II-4 Cohesiveness throughout the ripening of
reduced fat Cheddar cheese made with 1.5%
of live and heat shocked cells of
Lactobacillus casei L2A ..... ....... ...... .... 104

II-S Springiness throughout the ripening of
reduced fat Cheddar cheese made with 1.5%
of live and heat shocked cells of
Lactobacillus casei L2A ...................... 106

II-6 Hardness throughout the ripening of reduced
fat Cheddar cheese made with 1.5% of live
and heat shocked cells of Lactobacillus casei
L2A .......................................... 107

II-7 Textural characteristics of reduced fat
Cheddar cheese made with adjunct culture of
Lactobacillus casei L2A, as scored by the
sensory evaluation panel ..................... 110

II-8 Organoleptic characteristics of reduced
fat Cheddar cheese made with adjunct culture
of Lactobacillus casei L2A, as scored by the
sensory evaluation panel ............ ...... ... 113

xi

Figure Page

III-1 Extraction procedure used to separate the
hydrophobic peptide extract from reduced fat
Cheddar Cheese .00..0.0.0.0...000000000000.... 121

III-2 Evolution of bitterness during the ripening
of reduced fat Cheddar cheese made with
adjunct culture of Lactobacillus casei L2A,
as determined by trained sensory panel ....... 127

III-3 High performance liquid chromatography
elution patterns of standard peptides along
with hydrophobic extract of reduced fat
Cheddar cheese ............................... 129

III-4 High performance liquid chromatography elution
patterns of hydrophobic extracts of Control,
HSLC-1.5 and LLC-1.5 cheeses after 4 weeks of
ripening ...................................... 131

III-5 High performance liquid chromatography elution
patterns of hydrophobic extracts of Control,
HSLC-1.5 and LLC-1.5 cheeses after 20 weeks of
ripening ..................................... 132

III-6 Relative peak areas of peptide fractions
eluted from the high performance liquid
chromatography column ........ ......... . ...... 136

III-7 Chromatographic profiles of amino acid
standards 0.0.0......0.....0.0000.0.0.00...... 138

III-8 Relative distribution of amino acids in

peptide fractions of reduced fat Cheddar
cheese at 20 weeks of ripening. .............. 140

xii

I-6

I-7

LIST OF APPENDIX TABLES

Time schedule for making reduced fat Cheddar
cheese of high moisture content. ..............

Changes in cheese pH throughout the ripening
of reduced fat Cheddar cheese made with
different levels of adjunct culture of
Lactobacillus casei L2A. ......................

Soluble Nitrogen (SN) evolution throughout the
ripening of reduced fat Cheddar cheese made
with different levels of adjunct culture of
Lactobacillus casei L2A. ......................

Non-Protein Nitrogen (NPN) evolution throughout
the ripening of reduced fat Cheddar cheese made
with different levels of adjunct culture of

Lactobacillus casei L2A. ......................

Total Nitrogen, Ripening Extension Index and
Ripening Depth Index after 20 weeks of ripening
of reduced fat Cheddar cheese made with
different levels of adjunct culture of
Lactobacillus casei LZA. ......................

List of parameters used to scan the
electrophoretic gels in the Shimadzu
Dual-Wavelength Scanner. ............. ...... ...

Degradation of an-casein (peak area)
throughout the ripening of reduced fat Cheddar
cheese made with adjunct culture of
Lactobacillus casei L2A. ............ ...... ....

xiii

Page

147

148

149

150

151

152

153

Table

II-l

II-3

II-4

II-5

III-1

III-2

Page

Degradation of fl-casein (peak area)

throughout the ripening of reduced fat Cheddar

cheese made with adjunct culture of

Lactobacillus casei LZA. ...................... 154

Adhesiveness throughout the ripening of

reduced fat Cheddar cheese made with adjunct

culture of Lactobacillus casei L2A, as

measured by the Instron Universal Testing

Machine. ...................................... 156

Cohesiveness throughout the ripening of

reduced fat Cheddar cheese made with adjunct

culture of Lactobacillus casei L2A, as

measured by the Instron Universal Testing

Machine. ...................................... 157

Springiness throughout the ripening of

reduced fat Cheddar cheese made with adjunct

culture of Lactobacillus casei L2A, as

measured by the Instron Universal Testing

Machine. ...................................... 158

Hardness throughout the ripening of reduced

fat Cheddar cheese made with adjunct culture

of Lactobacillus casei L2A, as measured by the
Instron Universal Testing Machine.............. 159

Textural characteristics of reduced fat

Cheddar cheese made with adjunct culture of
Lactobacillus casei L2A, as scored by the

sensory evaluation panel. ..................... 160

Organoleptic characteristics of reduced fat

Cheddar cheese made with adjunct culture of
Lactobacillus casei L2A, as scored by the

sensory evaluation panel. ..................... 161

Distribution of peptides by fractions (% area)
after 20 weeks of ripening of reduced fat
Cheddar Cheese. 0............OOOOOOOOOOOOOOOOOO 174

Areas (x 1000, arbitrary units) of selected

peaks from chromatographic profiles of

peptides extracted from reduced fat Cheddar

cheese made with adjunct culture of

Lactobacillus casei L2A. ...................... 175

xiv

LIST OF APPENDIX FIGURES

Figure Page

I-1 Electrophoretic patterns throughout the
ripening of reduced fat Cheddar cheese made
with adjunct culture of Lactobacillus casei
LZA. ......... ...... . ........ .......... ....... 155

II-1 Consent form presented to panel members ...... 162

II-2 Sample form of questionnaire presented to the
panel to evaluate adhesiveness of reduced fat
Cheddar cheese during training sessions. ..... 163

II-3 Sample form of questionnaire presented to the
panel to evaluate cohesiveness of reduced fat
Cheddar cheese during training sessions. ..... 164

II-4 Sample form of questionnaire presented to the
panel to evaluate springiness of reduced fat
Cheddar cheese during training sessions. ..... 165

II-S Sample form of questionnaire presented to the
panel to evaluate hardness of reduced fat
Cheddar cheese during training sessions. ..... 166

II—6 Sample form of questionnaire presented to the
panel to evaluate bitterness of reduced fat
Cheddar cheese during training sessions. ..... 167

II-7 Sample form of questionnaire presented to the
panel to evaluate cheese flavor intensity of
reduced fat Cheddar cheese during training
sessions. ..... .. .............................. 168

II-8 Sample form of questionnaire presented to the
panel to evaluate adhesiveness of reduced fat
Cheddar cheese ............................... 169

Figure Page

II-9 Sample form of questionnaire presented to the
panel to evaluate cohesiveness of reduced fat
Cheddar cheese ............................... 170

II-10 Sample form of questionnaire presented to the
panel to evaluate springiness of reduced fat 171
Cheddar Cheese OOOOOOOOOOOOO........OOOOOOOOO.

II-11 Sample form of questionnaire presented to the
panel to evaluate hardness of reduced fat
Cheddar Cheese O.....OOOOOOOOOOO0.0.0.0000...O 172

II-12 Sample form of questionnaire presented to the
panel to evaluate flavor of reduced fat Cheddar
Cheese. ......OOOOOOOOI ...... ......OOOOOOOOOOO 173

III-1 Sample form of questionnaire presented to the
panel to evaluate bitterness of reduced fat
Cheddar cheese slurry during training
sessions. .................................... 176

III-2 Sample form of questionnaire presented to the

panel to evaluate bitterness of reduced fat
Cheddar cheese slurry. ....................... 177

xvi

LIST OF ABBREVIATIONS

Abbreviation key: USN = water soluble nitrogen; FDM = fat in
the dry matter; Tea-N = trichloroacetic acid soluble nitrogen;
LLC-O.5, LLC-1.0, LLC-1.5 = addition of 0.5, 1.0 and 1.5% of
live Lactobacillus casei LZA; Hana-0.5, HSLC-1.0, sane-1.5 =
addition of 0.5, 1.0 and 1.5% of heat shocked Lactobacillus

casei LZA; HPLC = high performance liquid chromatography.

xvii

INTRODUCTION

The growing consumer interest in low calorie food has led
the cheese industry to provide consumers with a large variety
of reduced fat cheese. However, reduced-fat cheese has been
frequently associated with lack of flavor and a tendency to
develop excessive off-flavors. The off-flavors typically noted
in reduced-fat cheese are bitter, brothy and meaty (Johnson
and Chen, 1991; Olson and Johnson, 1990b) , which are probably
imparted by specific peptides (Edwards and Kosikowski, 1983;
Harwalkar and Elliott, 1971; Olson and Johnson, 1990b;
Richardson and Creamer, 1973; Stadhouders et al. 1983; Visser,
1977b and 1977c) . In addition to off-flavor development,
texture and body defects have also been associated with
reduced-fat cheeses (Johnson and Chen, 1991 ; Olson, 1984;
Simard, 1991). The partial removal of fat from cheese has been
considered the main cause for the appearance of these defects
(Banks et al., 1989; El-Neshawy et a1. 1986; Hargrove, 1966b;
McGregor and White, 1990a; Olson and Johnson, 1990a).

Cheese is described as a three dimensional network formed
by casein in which the milk fat globules are embedded
(Rosenberg, 1992). The rest of the matrix is filled by a

1

2

continuous phase which is comprised of soluble proteins,
minerals, sugars, vitamins, minor components and microbial
flora. The stabilization of this multi-component structure is
sustained by numerous physical and chemical interactions. The
physicochemical interactions and microbial activity that occur
during cheesemaking and ripening determine the quality
attributes of the cheese. The delicate balance of the specific
concentrations of each of the cheese components is responsible
for the type and extent of these interactions.

To produce reduced-fat cheese, the traditional
manufacturing procedure must be changed (Banks et al., 1989;
Hargrove et al., 1966b; Madsen et al., 1970; Olson and
Johnson, 1990a) . As milk fat is reduced, cheese moisture must
be increased to improve flavor, body and texture properties of
reduced fat cheese (Simard, 1991) . The consequence of changes
in the balance of the cheese components may lead to the
occurrence of texture and flavor problems.

Fat is known to contribute to physical characteristics
giving opacity and influencing cheese rheology, and helps
solubilize many flavor compounds modifying their perception
and volatility, especially in the mouth. Fat also enhances
smoothness and mouthcoating and reduces cheese cohesiveness
(Degouy, 1993) . The removal of fat from cheese will cause the
formation of a more compact mesh in the protein network, with
great impact on several mechanical properties of the cheese

(McGregor and White, 1990a; Rosenberg, 1992) . The relatively

 

3

high water levels required for reduced fat cheese
manufacturing reduces firmness of the curd (Rosenberg, 1992).
Removal of fat, which is considered a precursor of many
flavor components, will also lead to a lack of typical flavor
in the final product. The oil/water interface in the cheese is
of critical importance for microbial growth. The reduction in
the fat level causes flavor changes by affecting the microbial
flora. Increased water content in the cheese also alters the
microbial growth patterns and enzyme activity, which may lead
to enhanced proteolysis and consequently to off-flavor
production (O’Donnell, 1993).

The choice of a suitable starter culture is of utmost
importance for production of a quality reduced-fat cheese. Not
all lactic starter cultures appear to be suitable for reduced
fat cheese (Olson, 1991). Evidence has demonstrated that slow
acid producing cultures with less proteolytic activity and
possessing specific debittering activities should be
preferable for low-fat cheese (Rosenberg, 1992). The use of
starter culture adjuncts such as Lactobacilli spp. may reduce
bitterness and improve flavor in reduced-fat cheese (Ardo and
Mansson, 1990; Bartels et al. 1987; El-Abboudi et al. 1991;
O'Donnell, 1993; Trepanier et a1. 1992).

The objective of this paper is to verify the impact of an
adjunct culture of lactobacilli on the texture and flavor of
reduced fat Cheddar cheese. A softening of the curd,

improvement in flavor, and elimination of the bitter flavor

4
commonly associated with reduced fat cheeses would increase

the consumption in this cheese category.

LITERATURE REVIEW

1. REDUCED FAT CHEESE

In the last two decades we have seen dramatic shifts
toward the consumption of low fat foods (Egbert et al. , 1991) .
The wish to avoid high calorie food to control body weight and
a possible connection between animal fats and circulatory
disorders have been the :main causes for this increased
consumption of low fat foods (Hargrove et al. 1966a; Madsen et
al. 1970; Simard, 1991).

According to Dexheimer (1992), a projection of sales of
low fat foods showed that this food category will hit $41
billion by 1995. Reduced fat cheese will account for an
estimated 9% of the retail cheese market valued at $6.7
billion. A survey conducted by Delta's New Products News
showed that cheese accounted for 23% of the total new non-
fat/low fat products introduced in the market in 1991. This

number increased to 36% in 1992 (Rosenberg, 1992).

6

To service the growing consumer market in low energy
food, several attempts have been made by cheese manufacturers
to develop a palatable reduced fat cheese (Simard, 1991).
Despite the increasing consumption in reduced fat cheeses,
several problems have been encountered in their production.
Lack of flavor, development of off-flavors (meaty-brothy,
bitter, and unclean), and body defects (hardness or softness,
weakness, and pasty) are the three main factors responsible
for the low quality of reduced fat cheeses when compared to
their full fat counterparts (Johnson and Chen, 1991). The lack
of the flavor masking effect of fat which greatly contributes
to cheese flavor makes the off-flavors more readily perceived
in reduced fat cheeses. The meaty/brothy flavors are generated
in cheese by the flavor precursors furanones and pyrazines,
produced by the browning type reaction between a-dicarbonyls
and amino acids. Unclean off-flavor is generated by Strecker-
type reactions which produce aldehydes and alcohols (Lindsay,
1991). Bitterness is caused by the accumulation of bitter
tasting peptides in cheese, formed by enzymatic attack on the
caseins (Emmons et al. 1962b; Lowrie and Lawrence, 1972;
Stadhouders, 1974; Sullivan and Jago, 1972; Sullivan et al.
1973).

Levels of fat in cheese have a direct impact on the
acceptability by consumers. Pat is the precursor of many
flavor compounds and influences the body and texture of

cheese. Total or partial removal of fat from cheese leads to

7
hard, tough, dry body and low flavor levels of the cheese
(McGregor and White, 1990a).

The use of traditional procedures to make reduced fat
cheese has led to the production of a very hard, rubbery and
tasteless cheese, even after extensive ripening (Banks et al.
1989; El-Neshawy et al. 1986; Hargrove et al. 1966b; Simard,
1991). To improve the flavor, body and texture of reduced fat
cheese, the traditional manufacturing procedure must be
changed (Banks et al. 1989; Hargrove et al. 1966b; Madsen et
al. 1970; Olson and Johnson, 1990a) and slow acid producing
starters associated with low proteolytic activity should be
chosen (Olson, 1991) . Several attempts have been made to
improve the quality of reduced fat cheeses, including changes
in the traditional manufacturing process (Banks et al. 1989,
Chen, 1991; Chen et al. 1992), milk preparation prior to
cheese manufacture (McGregor and White, 1990a and 1990b;
Metzger and Mistry, 1993), culture selection (Chen et al.
1992; Simard, 1991), and use of fat replacers (El-Neshawy et
al. 1986).

One way to improve the texture of reduced fat cheese is
to increase the water content in the curd. The moisture
content in reduced fat cheese must be higher than that
normally employed in full fat cheese to improve the textural
properties of the cheese (Banks et al. 1989; Simard, 1991).
Milk fat globules fill the interstitial spaces within the

protein network of the cheese (Rosenberg, 1992). Removal of

8

fat from cheese causes the formation of a more compact mesh in
the protein network, consequently yielding a hard cheese.
Increased water content occupies the space previously occupied
by fat, loosening the protein network with positive effects on
the textural properties of the cheese (Bryant, 1993) . However,
the relatively high moisture content required to promote the
desired textural characteristics in reduced fat cheeses may
also lead to excessive proteolysis and consequently to off-
flavor production. If lactic acid production is not controlled
during the manufacturing procedure, high moisture cheese
develops a very acid flavor and the body becomes weak and
pasty with age (Johnson and Chen, 1991). According to Degouy
(1993) , the key issues in reduced fat cheese manufacture
consist of the replacement of fat properties, binding of
water and control of the proteolytic activity to develop
flavor and avoid bitterness formation.

Increased moisture retention in the curd (44-45%) can be
achieved by cutting the curd using larger knives (1 cm instead
of 0.6 cm), applying a lower cooking temperature and milling
the curd at a higher pH (5.9 vs 5.2) than normally used for
full fat Cheddar cheese (Chen, 1991). A modification of the
standard Cheddar process to produce a low fat cheese with high
moisture content was implemented by Banks et al. (1989). They
developed a technique for production of a reduced fat Cheddar
cheese containing intermediate and low fat content. A

control Cheddar cheese with 37.9% moisture and 33.1% fat, an

9

intermediate fat Cheddar cheese with 42.9% moisture and 25.6%
fat and a low fat cheese with 47.2% moisture and 16.8% fat
were produced by decreasing cook temperature and reducing
stirring and cheddaring time. The scalding temperature was
reduced from 39°C (control cheese) to 37°C and 35°C in the
intermediate and low fat cheeses, respectively. The stirring
time for both the experimental cheeses was 30 minutes as
compared to 60 minutes for the control cheese. The control
cheese was cheddared for 90 minutes while both the
intermediate and lOW' fat, cheeses were cheddared for 60
minutes. They found that the flavor and physical properties of
the cheese with intermediate fat content was comparable to
those of regular Cheddar cheese, but the cheese with low fat
content was over-firm and did not develop an adequate Cheddar
flavor. According to Olson and Johnson (1990a) reduction of
the fat content by 33% yields acceptable cheese, but reduction
of fat over 50% results in cheese with poor flavor quality and
physical properties.

The effects of milk pasteurization temperatures, starter
culture and rennet levels, and drain and mill pH on the
quality of a 33% reduced fat Cheddar cheese were studied by
Chen (1992). Their' results showed, that. a jpasteurization
temperature of T7JWC produced a cheese with higher moisture
content than that made with milk pasteurized at 73.3%:. The
cheese made with a 2% starter level produced higher Cheddar

flavor intensity than cheese made with 0.5% of starter level,

10

but ‘the cheese jpresented. excessive acid off-flavors and
inferior body. Improved Cheddar flavor and body were obtained
when higher rennet levels were used, especially for young
cheeses. But over 6 months of ripening lower rennet level
cheeses were preferable because the cheeses presented fewer
off-flavors and. had. higher Cheddar flavor' quality. They
concluded that pH control is a critical factor in producing a
good quality 33% reduced fat Cheddar cheese. A pH of 6.37 at
drain increased the moisture content, flavor intensity and
body breakdown when compared, with a drain pH of 6.13.
Moisture content and flavor quality were also higher in
cheeses with higher mill pH.

The use of a lower cooking temperature or simply
elimination of cooking from the manufacturing process can also
be used to achieve higher moisture content in the cheese.
Elimination of the cooking temperature (45.9%: for 15
minutes), increased the moisture content in Mozzarella cheese,
causing a softening of the curd with consequent improvement in
the textural properties (Tunick et al. 1991).

According to Olson and Johnson (1990a), pH of the cheese
can also be controlled by washing the curd or partially
diluting the whey with water. Washing the curd before salting
Zhas the advantage of reducing the lactose and lactic acid
concentration in the cheese. Reducing the concentration of
lactose by washing the curd results in a higher pH at

drainage, thus increasing the water retention in the cheese

11
(Simard, 1991). However, this process resulted in large
quantities of salt-drippings being expelled during pressing
(Olson and Johnson, 1990a).

Milk homogenization appears to contribute to improved
texture and smoothness of reduced fat cheese. According to
Olson and Johnson (1990b), a softer and less elastic Cheddar-
type cheese was produced when the cheese milk containing 1.4%
fat was homogenized prior to manufacture. This result was in
accordance with Emmons et al. (1980) who, in addition to a
softer and less elastic cheese, found a slightly higher
moisture content in the cheese made with homogenized milk when
compared to non-homogenized milk. In addition to improvement
in texture, homogenization of cream or whole milk used to
standardize milk for reduced fat cheese production can improve
flavor, possibly due to the increased dispersion of fat
globules, which make them more accessible for enzyme reactions
(Hargrove et al. 1966b) . Homogenization pressures higher than
2,500 psi are not recommended. Use of high homogenization
pressures produces brittle and inferior quality cheese
(Hargrove et al. 1966b; Olson and Johnson, 1990b). Tunick et
al. (1991) also noticed adverse affects on the rheological and
melting properties of reduced fat Mozzarella cheese when high
homogenization pressure (17200 kPa) was applied to cheese

milk, as opposed to low homogenization pressure (10300 kPa).

12

When studying the effect of homogenization on the quality
of reduced-fat Cheddar cheese, Metzger and Mistry (1993)
verified that homogenization. of the cream. prior to the
standardization process improved the texture of the cheese.
They observed that in all treatments where skim milk or
standardized milk were submitted to homogenization, an
extensive curd shattering occurred in the cheese, but when
skim milk was standardized to 1.9% fat with the previously
homogenized cream, curd shattering did not occur.

Ultrafiltration (UP) is another' method. that can, be
applied to enhance the quality of reduced fat cheese.
According to McGregor and White (1990a), two advantages of UF
process for making cheese are increased retention of whey
proteins in the curd with positive effects on yield and
reduction of calcium.which favors the softness of the cheese
(McGregor and White, 1990a). Acidification and diafiltration
were performed along with the ultrafiltration process. The
advantages of these two treatments were an enhancement in the
removal of calcium from the protein matrix, reducing the
firmness and improving the body and texture of cheese, and a
reduction in the level of lactose in the retentate to a point
that allows better control of the pH. The results showed a
significant improvement in the flavor and body and texture of
the cheese manufactured from acidified and diafiltered UF
lowfat milk as compared with the cheese made from UF lowfat

milk without acidification and diafiltration.

13

Condensation of the milk prior to cheese making can be
another alternative to improve the quality of reduced fat
cheeses. Anderson et al. (1992) made reduced fat Cheddar
cheese from condensed skim milk standardized with cream. The
treatments employed in the experiment were: non-condensed
10.3% total solids (TS) as a control; condensed, 15.4% TS;
condensed, 18.3% TS and condensed, 22.2% TS. Cheeses made
with condensed milk with the highest level of total solids
were extremely firm and crumbly. The results suggested that
the condensed milk with 15.4% TS and 18.3% T8 were more
suitable for reduced fat cheese making.

Fat replacers have been added to standardized milk to
enhance the quality of reduced fat cheese. El-Neshawy et al.
(1986) carried out an experiment to improve the body and
organoleptical properties of low-fat Ras cheese. Carrageenan
and carboxymethylcellulose (CMC) were added to cheese milk
containing 1%, 1.5% and 2% fat to improve the water retention.
These compounds form complexes with milk proteins increasing
the water binding capacity, with positive effects on the
moisture retention of the curd. They found that cheese made
from milk containing no stabilizers was hard, tough and
deficient in flavor. The addition of stabilizer tolcheesetmilk
enhanced the softness and smoothness of low fat cheese,

improving body characteristics.

14

According to Olson and Johnson (1990a) the lactic starter
cultures have a pronounced effect on all cheeses, but
especially on reduced fat cheeses. In the manufacture of
reduced fat cheese, starter cultures possessing lower rate of
acid production, lower proteolytic activity (Olson, 1991;
Rosenberg, 1992), and debittering activity (Rosenberg, 1992)
should be preferable. Lactic starters commonly used for full
fat cheeses are frequently associated with meaty/brothy
flavors in reduced fat cheeses (Johnson and Chen, 1991).

The effects of four different starter cultures on the
ripening of reduced-fat cheese were evaluated by Chen et al.
(1992). They used Lactococcus lactis subsp. lactis and
cremoris, and two strains of Lactococcus salivarius subsp.
thermophilus. They verified that the cheese produced with S.
thermophilus were acceptable but exhibited less lactic acid
and flavor development, and also less protein breakdown. More
typical Cheddar cheese was produced with the two strains of
Lactococcus. The Lactococcus lactis subsp. lactis developed
lower proteinase and higher peptidase activity, while the
Lactococcus lactis subsp. cremoris developed higher proteinase
and lower peptidase activity.

The use of lactobacilli as a starter culture adjunct has
been successfully applied as a way to reduce bitterness and
improve flavor in cheese (El-Neshawy et al. 1986; El-Soda et
al. 1981; El-Soda, 1986; Hargrove et al. 1966b; Johnson et al.

1993; O'Donnell, 1993; Simard, 1991; Trepanier et al. 1991a,

15
1991b and 1992). Johnson et al. (1993) used Lactobacillus
helveticus CNRZ attenuated by spray-drying, freeze-drying or
freeze shocking in the manufacture of reduced fat cheese. They
found that proteolysis as measured by trichloroacetic acid
(TCA) and phosphotungstic acid (PTA) soluble nitrogen was
higher in the cheeses 'made ‘with attenuated lactobacilli
adjuncts than that in the control cheese. They also observed
that the addition of attenuated lactobacilli produced a cheese
with enhanced flavor and body without causing bitterness or
excessive softening of the curd.

El-Neshawy et al. (1986) demonstrated that the addition
of Lactobacillus casei at the ratio of 1:1 with S. lactis
subsp. lactis in combination with MnClz, enhanced the flavor
of Ras cheese. Hargrove et al. (1966b) also observed
improvement in the flavor quality of reduced fat Cheddar
cheese when Lactobacillus casei used as supplemental starter

was added to the cheese milk as a 0.5% inoculum.

2. TEXTURE DEVELOPMENT DURING RIPENING

The ripening of cheese is a complex process which
involves several biochemical changes, including the
fermentation of lactose, the degradation of proteins, and the
hydrolysis of fat (Law et al. 1992; Schormuller, 1968). All
these processes result in a gradual change in the cheese curd

from.a coarse structure to a more or less plastic homogeneous

16
substance (Schormuller, 1968) and in the development of the
aroma and taste that together constitute the typical cheese
flavor.

The casein comprises a group of phosphoproteins, composed
of four different fractions, «1,,- , a,2-, B- and x-casein (Eigel
et al. 1984) , in a proportion of 3:1:3:1 (Grappin et al.
1985) . In addition to the four principal gene products, there
are several minor proteins which originate mainly from post-
secretion proteolysis of the primary caseins by the action of
plasmin. These include the x-caseins and the proteose peptones
5 and 8-fast, all derived from the B-casein (Fox, 1992). The
four primary caseins differ within each fraction according to
the degree of phosphorylation and glycosylation (Grappin,
1985).

Proteolysis is considered the most important biochemical
event during cheese ripening and affects both flavor and
texture (Fox, 1989a). These biochemical changes are catalyzed
by enzymes, which may have their origin in coagulating agents,
starter cultures, the naturally occurring or contaminating
microbes in milk, or possibly the enzymes present in the
original milk (Desmazeaud and Gripon, 1977; Kamaly and Marth,
1989; Scolari et al. 1993). During this process, casein, the
major structural component of curd, is slowly broken down to
smaller compounds and the cheese loses its rubbery character,
acquiring the desired texture (Kamaly and Marth, 1989). The

rate, extent, and nature of these changes during cheese

17
ripening, as well as the amount and nature of the degradation
products depends upon the enzyme involved in the process and

the ripening conditions (Grappin et al., 1985).

2.1. Effect of proteolysis on texture

The pH and the ratio of intact casein to moisture are the
two main factors responsible for texture changes during cheese
ripening (Lawrence et al., 1987). Changes in textural
properties in different cheese varieties are due to
proteolytic activity on an- and B-casein which constitute the
cheese protein matrix (Bertola et al., 1992). Rennet is the
first proteolytic agent involved in casein breakdown
(Desmazeaud and Gripon, 1977). Proteolysis of afl-casein
rendering the polypeptide afl-I is considered the first phase
in texture development during ripening. This phase takes place
during the first two weeks of ripening, when the rubbery
texture of the young cheese is converted to a smoother body.
The cheese matrix is greatly weakened when only a single bond
in about 20% of the a,,-casein is cleaved by the coagulant
yielding the afl-I peptide. The second phase involves a slow
degradation of the rest of the au-casein and other caseins
with more gradual changes in the texture (Lawrence et al.

1987).

18

Beta-casein undergoes very little proteolysis in cheese
(Fox, 1989a and 1993; Phelan et al., 1973) but in solution it
is readily hydrolysed by the action of chymosin and other
rennets to yield B-I, B-II and B-III caseins (Visser and
Slangen, 1977). The intermolecular hydrophobic interactions
between the hydrophobic C-terminal region of B-casein, which
contains the chymosin susceptible sites for cleavage, is the
most plausible explanation for its high resistance to
hydrolysis by rennet in cheese (Fox, 1989a).

The rate of proteolysis is affected by several factors.
These include rennet and plasmin retention in the curd, salt
to moisture ratio, temperature of ripening, type of coagulant
used, and changes in pH (Lawrence et al. 1987).

Although most coagulant is lost in the whey at drainage,
some remains entrapped in the curd. The amount entrapped
depends upon the total quantity added to the cheese milk, the
pH of cheese milk and curd, cooking temperature and rate of
acid production by the starter culture (Visser, 1993) . Holmes
et al. (1977) verified that rennet retention in the curd is pH
dependent. The rennet retention increased as the pH of the
curd decreased. At lower pH, a greater proportion of (rd-casein
was hydrolysed.when calf rennet was used. An important.fact is
that the retention of microbial rennet in the curd, unlike
calf rennet, is not affected by changes in milk pH. The extent

of degradation of (rd-casein in the presence of microbial

19
rennet was the same irrespective of the initial pH of the milk
(Lawrence et al. 1987).

The proportion of plasmin retained in the cheese curd is
also important in proteolysis. Plasmin is a native milk
proteinase which is associated with the casein micelles (Fox,
1989b; Visser, 1993). Plasmin activity in cheese is evidenced
by the accumulation of 7-caseins (Creamer, 1975; Richardson
and Pearce, 1981) . Plasmin activity is very important in
cheese with high pH, such.as Swiss type cheeses and other high
cook varieties in which the coagulant is denaturated
(Matheson, 1981).

The effect of the salt to moisture ratio on the
susceptibility of a“- and B-casein to hydrolysis was
demonstrated in an experiment in which Cheddar cheese was made
with linear S/M gradients across a single 20 kg block (Thomas
and Pearce, 1981). They verified that the salt concentration
had a large effect on the rate of proteolysis of both a“- and
fl-casein. In one-month old cheese containing 4% S/M (salt in
moisture) approximately 5% of the au-casein and 50% of the B-
casein remained intact, whereas in the cheese containing 8%
S/M, 60% of the urn-casein and 95% of the B-casein was
unhydrolysed. A linear relationship between S/M and
proteolysis was found between the two extremes.

The hydrolysis of a,,- and B-casein is greatly affected by
temperature. A Cheddar cheese ripened at 15°C for 8 days

developed the same texture as a Cheddar cheese ripened at 10%:

20
for 15 days. When the temperature was increased to 20°C the
cheese became more brittle and less springy. Cheese
proteolysis correlated negatively with cheese firmness and
springiness, indicating softening of the cheese as the protein
framework was degraded (Lawrence et al. 1987).

Protein breakdown was reported to increase in cheese
with low acidity (Noomen, 1978). At higher pH (6.2), B-casein
was more quickly degraded than afl-casein whereas at lower pH
(5.4), ad-casein was more extensively degraded than B-casein.
At pH above 5.6, B-casein was degraded more than afl-casein,

presumably due to the action of increased plasmin activity.

2.2. Measurements of textural properties

Rheology is defined as the study of deformation and flow
of matter (Shoemaker et al. 1992). The rheological properties
of cheese affect eating quality (consistency in the mouth),
usage (cutting, grating, spreading, melting, etc), handling
and packaging (Walstra and Peleg, 1991).

Cheese exhibits both fluid and solid like properties and
is considered a viscolastic material (Konstance and Holsinger,
1992; Shoemaker et al., 1992). This implies that at short
times of deformation, with low strain levels, cheese behaves
as an elastic material, recovering its original shape when the
stress is removed. At longer times, cheese behaves as viscous

material, because most of the deformation remains after the

21
stress is removed (Walstra and Peleg, 1991). The yield stress,
the point where permanent deformation occurs (van-Vliet,
1991), is very small for the majority of the cheeses. Even a
small force applied.to the test piece can cause some permanent
deformation (Walstra and Peleg, 1991).

Caseins are the major structural component in cheese, but
the milk fat greatly contributes to product quality (Konstance
and Holsinger, 1992). The removal of milkfat by 50% or more in
Cheddar-type. cheeses. has led. to ‘unacceptable flavor and
physical properties (Olson and Johnson, 1990a). With the
increased demand for’ a :reduced fat Cheddar cheese that
simulates its full fat counterpart, evaluation of textural
properties has assumed even greater importance (Konstance and
Holsinger, 1992).

The caseins, fat and moisture are responsible for the
structure and rheological properties of the cheese (Prentice,
1992). Casein forms a continuous meshlike structure, giving
the "solid" nature of cheese and entrapping fat and.water. Fat
acts mainly as a filler. At low temperatures, fat
contributes to the rigidity of the casein matrix. When the
temperature is 20°C or above, fat is in a liquid form and has
little contribution to cheese firmness. Water moves freely in
the space between fat and casein and acts mainly as a low-
viscosity lubricant (Prentice, 1992). The viscoelastic
properties of cheese arise from the interaction among the

major components (Lee et al. 1992).

22

Most of studies on mechanical and textural properties of
cheeses have been obtained through uniaxial compression tests
performed by the Instron Universal Testing Machine (Instron
Corporation, Canton, MA) (Ak and Gunasekaran, 1992). The use
of the texture profile analysis (TPA) in two successive
compression cycles is the method most often used for food
analysis (Bertola et al., 1992).

The Instron Universal Testing Machine can be used to
measure multiple textural parameters, such as hardness,
fracturability, adhesiveness, cohesiveness, springiness,
gumminess and chewiness (Tunick and Nolan, 1992). There are a
wide array of test conditions to be considered when evaluating
the rheological properties of cheese, including specimen
dimensions, deformation rate, and surface friction and sample
lubrication (Sherman, 1989).

Culioli and Sherman (1976) studied the influence of
cheese maturity, test temperature, sample shape, sample height
and surface area on the force-compression behavior of Gouda
cheese in the Instron Universal Testing Machine. The force
required to achieve a given percent compression increased from
the center to the edge of the cheese block for both young and
mature cheese. The force :required. to achieve any' given
compression correlated inversely with temperature. The force
also decreased with increasing sample height. The sample shape

also had influence on the compression force. Cubic samples

23
required a higher force to achieve a given percent compression
when compared to cylindrical shapes.

The effect of sample lubrication and deformation rate on
the rheological properties of Cheddar cheese under uniaxial
compression test was investigated by Ak and Gunasekaran
(1992). Six different deformation rates were applied to both
lubricated and non-lubricated cheese samples. There was no
significant effect of lubrication on the parameters studied,
but the data from the non-lubricated tests had higher
coefficients of variation (CV = 31%) than those from the
lubricated test (CV = 13.5%) , suggesting that lubrication
gives more reproducible results. Chu and Peleg (1985) studied
the compressive behavior of selected food materials, including
process American cheese, with small height-to-diameter ratios.
Their results showed that the dimensional effect was more
pronounced when there was considerable friction at sample-
machine contact surfaces. They noticed that thinner specimens
appeared stiffer, as measured by deformability modulus.

Bertola et a1. (1992) analyzed rheological parameters
during ripening of Tybo Argentino cheese. They applied the
compression test in two successive cycles to determine
hardness, cohesiveness and adhesiveness. Two viscoelastic
parameters (elastic moduli and relaxation times) were also
determined. Hardness decreased and adhesiveness increased
during ripening whereas cohesiveness did not show any relevant

variation. Analysis showed degradation of a,,—casein was more

24

rapid than B-casein. They concluded that high concentrations
of intact (rd-casein contributed to the rigidity of the protein
framework whereas low concentrations contributed to the
increased elasticity and softening of the cheese. They found
good negative correlation between both water and TCA-soluble
nitrogen and concentration of intact a,,-casein with both
correlation coefficients higher than -0.88.

Force-compression tests were also performed on Cheddar
cheese to evaluate the starter culture effect on ripening
characteristics and rheological parameters (Amantea et al.,
1986). Six strains of bacteriophage-insensitive Lactococcus
cremoris and conventional Lactococcus cremoris were used in
the experiment. Rheological parameters were correlated to the
degree of proteolysis as measured by trichloroacetic acid
(TCA)-soluble nitrogen, age, pH and moisture content. They
found that the firmness decreased with increased moisture
content in.the cheese. They also verified that.the yield.point
of the force-compression curves, when permanent deformation
occurs (van-Vliet, 1991), decreased as the free amino groups
increased with age. Creamer and Olson (1982) also noted an
inverse correlation between yield force and the concentration
of intact al.,-casein in the cheese, suggesting that the texture
of the cheese softens during aging.

Based on the correlation between firmness and the
quantity of intact a,,-casein in the cheese (Walstra and van-

\Eliet, 1982), Lawrence et al. (1987) found an explanation for

25
the weakening of the cheese with age. The breakdown products
formed during casein breakdown were essentially water soluble
and therefore did not contribute to the protein matrix,
causing the softening of the cheese.

Green et al. (1981) used the Instron Universal Testing
Machine to study the texture of Cheddar cheese made from
concentrated milk. Firmness and cohesiveness of the cheese
increased as the concentration factor of the milk increased,
suggesting a more rigid structure in the concentrated milk
cheeses. Adhesiveness, which expresses the capacity of the
cheese particles to stick to a steel plate, decreased with
increased concentration of milk, probably due to the reduced
level of fat and lower proteolysis in the protein matrix of
the more concentrated milk cheeses. Elasticity was little
affected by increased milk concentration, therefore being less
affected by differences in composition, network structure and
extent of proteolysis.

Stampanoni and Noble (1991a and 1991b) also used the
Instron to study rheological parameters in cheese analogs made
with ‘varying’ amounts. of fat (10, 17.5 and 25%), sodium
chloride (0.5 and 2.0%) and citric acid (0.1 and 1.2%). They
verified that the cheeses with higher fat level was softer,
less springy and more cohesive and more adhesive than cheeses
with lower fat level. When salt or acid content.was increased,
firmness also increased while cohesiveness and springiness

decreased significantly.

26

The ultimate objective of rheological investigation on
cheese is the correlation of the measured instrumental
properties with sensory data (Konstance and Holsinger, 1992) .
Lee et al. (1978) evaluated rheological properties of
commercial cheeses representing a wide variety of texture
using‘ both instrumental. methods and sensory' panels. The
cheeses used in the experiment included Cream cheese,
Camembert, Mozzarella, Muenster, Swiss cheese, American
processed cheese, and. mild, sharp and. processed Cheddar
cheese. Hardness, elasticity, adhesiveness and chewiness as
assessed.by the Instron correlated.significantly with the same
parameters evaluated by the sensory panel.

Chen.et.al. (1979) also used the Texture Profile.Analysis
(TPA) curve obtained by the Instron to evaluate the parameters
of firmness, cohesiveness, chewiness and adhesiveness in 11
cheese varieties. They used a plunger (d.= 6.4) at a crosshead
speed of 2.5 cm/min. to penetrate the cheese samples to a
depth of 1.91 cm. The sample height was 2.54 cm. The same
samples were submitted to a trained panel and both
instrumental and sensory results were correlated. Each of the
four parameters evaluated by the trained panel correlated
significantly with the corresponding parameters measured by
the Instron, with the correlation coefficients ranging from

0.79 to 0.85.

27

3 . FLAVOR DEVELOPMENT DURING RIPENING

Despite intensive research done on Cheddar cheese flavor
over the past years, the agents responsible for flavor
formation and how they carry out their functions are poorly
understood. The reactions involved are very complex and there
is a wide spectrum of products formed, some in very low
concentration (Aston and Dulley, 1982).

More than 100 jpotential flavor compounds. have been
identified in over 800 different cheese varieties recognized
in the world. These flavor compounds result from the action of
microorganisms and enzymes on the carbohydrates, fat and
proteins present in the cheese. These components are broken
down to lactic acid, fatty acids and amino acids,
respectively, generating what is termed the primary step of
cheese ripening. These primary products, with exception of
fatty acids, contribute little to cheese flavor. The
characteristic cheese flavor is generated in the secondary
step by the action of microorganisms and added enzymes on the
primary products (Kristoffersen, 1973).

There appear to be only two facts on Cheddar cheese
flavor formation that.all investigators seem to.agree: milkfat
must be present in the cheese for flavor perception and lactic
starters must be used to develop typical Cheddar cheese flavor
(Lawrence and Gilles, 1987). The current concept which finds

much support is that Cheddar cheese flavor results from the

28

combination of many compounds, which, when present in proper
quantitative balance, give rise to a typical flavor. When an
excess or lack of one or more of the component compounds is
present, atypical flavor is produced (Aston and Dulley, 1982).

Harper (1959) separated the water soluble fraction
containing the flavor from. cheese into taste and aroma
fractions. The chemical compounds included in the taste
fraction included lactic acid, amino acids, non-volatile fatty
acids, keto acids, non-volatile amines, salt, and fragments of
proteins and fats. The aroma fraction included amines, fatty

acids, aldehydes, ketones, alcohols, esters, lbs and

mercaptans.

3.1. Effect of proteolysis on Flavor

During proteolysis, the paracasein and the minor proteins
are gradually converted to simpler nitrogenous compounds,
namely, proteoses, peptones, polypeptides, amino acids, and
ammonia (Singh and Ganguli, 1972). The rate of protein
degradation varies from cheese to cheese, but protein
hydrolysis to amino acids occurs to some extent in all cheese
varieties (Harper and Kristoffersen, 1956). McGugan et al.
(1979) suggested that highly degraded protein of aged cheese
produces more flavor compounds than does the protein of a mild
cheese. Sanders et al. (1946) found that proteolysis was much

more pronounced in cheese made from high bacterial count milk

29
than in that made from low bacterial count milk, but the
desirable flavor and quality of the cheese was directly
related to the quality of the milk and not to the rate and
extent of proteolysis.

Although the contribution of amino acids to cheese flavor
is very controversial, they appeared to provide an important
"background" flavor (Harper and Kristoffersen, 1956). Keeney
and Day (1957) suggested that amino acids are involved in
cheese flavor formation through the Strecker degradation
reaction (a reaction between amino acids and dicarbonyl
compounds). These reactions might occur in Cheddar cheese
ripening with subsequent formation of aldehydes which could
contribute to flavor. Griffith and Hammond (1989) also
demonstrated that a wide variety of compounds were produced by
reaction between dicarbonyls and amino acids with direct
impact on Swiss cheese. Kosikowski (1951) found a direct
relationship between the intensity of the flavor and the total
concentration of free amino acids in cheese. Erekson (1949)
reported a direct relation between cheese flavor and free
tryptophan content. Glutamic acid was found to correlate well
with flavor in cheese (Harper, 1959). Proline was found to
impart considerable flavor in Swiss cheese as well as glutamic
acid in provolone cheese (Harper and Kristoffersen, 1956).

Marked increases of amino acids coincide with optimum
flavor and body (Kosikowski, 1951). This has led some

investigators to use a specific amino acid as a measure of

30

ripening. Tyrosine results from the hydrolysis of low
molecular weight peptides. An appreciable amount of tyrosine
is liberated after the amount of soluble protein has reached
its maximum. Therefore, total tyrosine is an important
indicator of Cheddar cheese ripening (Silverman and
Kosikowski, 1955). They claimed that measurement of tyrosine
is a much more sensitive index of protein breakdown during
cheese ripening than is soluble protein. Phosphotungstic acid
(PTA)-soluble nitrogen levels were shown to be good estimates
of the total free amino acid levels in Cheddar cheese (Jarrett
et al. 1982).

Addition of Lactobacillus casei to enhance liberation of
amino acids has been reported. Bullock and Irvine (1956)
verified that amino acids were liberated more rapidly in
cheese made with a supplemental culture of Lactobacillus casei
than in that made with only the usual lactic starter. They
found that the amino acid content of the cheese with added
Lactobacillus casei and control cheese were similar at four
months, but at 8.5 months a higher level of amino acids
appeared in the inoculated cheese. Tittsler et al. (1948),
added Lactobacillus casei to cheese milk and they observed
that the resultant cheese was generally more acid, had a
higher level of flavor, and a shorter body. Improvements in
:flavor of cheese made from milk inoculated with Lactobacillus

casei were also noticed by Sherwood (1939).

31

4. BITTER PEPTIDES IN CHEESE

Bitterness is a flavor defect quite frequently
encountered in Cheddar and Gouda cheese. The accumulation of
bitter tasting peptides formed by the action of proteolytic
enzymes from rennet and starter culture on caseins cause the
defect (Emmons et al. 1962b; Lowrie and Lawrence, 1972;
Stadhouders, 1974; Stadhouders and Hup, 1975; Sullivan and
Jago, 1972; Sullivan et al. 1973). Czulak (1959) classified
bitter flavor defects into three types, according to the time
of first appearance and duration: Type I, Bitterness appears
within a month or two after manufacture and disappears in 2-4
months of subsequent ripening; Type II, The defect appears as
type 1 but persists indefinitely throughout the ripening; and
Type III, Bitterness appears in 3-5 months after manufacturing
and persists indefinitely throughout the ripening. In Cheddar
cheese, bitterness has long been recognized as a major defect,
although it can be found in other cheese varieties
(Stadhouders, 1979). According'totCzulak (1959), the first two
flavor defects are the most frequently encountered in Cheddar

type cheese.

32

4.1. Mechanism of bitter flavor formation and degradation

Formation and degradation of bitter peptides is
accomplished by the proteolytic enzymes present in the cheese.
The proteolytic system.involved in the cheese ripening process
is.quite complex and may include enzymes from.rennet (chymosin
and pepsin), proteinases from.rennet substitutes, proteinases
and peptidases from starter culture, proteinases and
peptidases from non-starter microorganisms and endogenous milk
jproteinases (Lemieux and.Simard, 1992). Many studies have been
conducted and several hypotheses have been established to
elucidate the complex mechanism by which bitter peptides are
formed and degraded in cheese.

The mechanism postulated by Czulak (1959) involves the
combined action of rennet and bacterial proteolytic enzymes.
The attack of proteinases from rennet on casein releases the
bitter peptides which are believed to be further hydrolized by
the peptidase system of non-bitter strains of lactic acid
bacteria. Bitter strains of starter culture lack the ability
to degrade bitter peptides which accumulate resulting in the
bitter taste.

Czulak ( 1959) suggested that pH development plays an
important role in the accumulation of bitter peptides in
cheese. Experimental data (Czulak and Hammond, 1958) have
shown that bitter flavor was most frequently found in cheese

where the pH was 5 or less and was seldom found in cheeses

33
with pH higher than 5.2 during the first week after
manufacture. Based on those data, Czulak (1959) postulated a
mechanism (Figure 1) which would explain the transient
bitterness in cheese.

The proteolytic activity of rennet enzymes is enhanced at
low pH. Chymosin has its maximum activity at pH 4.9-5.5,
whereas pepsin has its maximum activity at still lower pH.
Enhanced proteolytic activity at low pH may be compared to
that of a larger amount of rennet added to cheesemaking at
normal or higher pH. A large pool of polypeptides and bitter
tasting peptones are formed by this enzyme activity. The
activity of the bacterial proteinases is stimulated by the
breakdown of casein by rennet enzymes, but at low pH the
polypeptides and peptones are not degraded at a sufficient
rate and tend.to accumulate in the cheese, thus generating the
bitter taste.

As ripening progresses, more and more casein is broken
down, causing an upward shift in pH, due to the increase in
the number of amino groups formed. This increased pH favors
the proteinases from the starter culture, which have their
optimum at pH 6 or higher. Thus, the surplus of polypeptides
and peptones is broken down to amino acids and the bitter
flavor gradually disappears.

Czulak's hypothesis is supported by Emmons et al.
(1962b), who explained the appearance of Type I and Type II

bitterness. In Type I, bitter peptides accumulates as a result

34

CASEN j r ucrose I
Q
"MT—E14 Powpemoes j

Ii

Low pH j: I ucnc AGO j
mans REGuAR BREAKDOWN or POLYPE noes

BY LAOTC PROTEASES

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MTG)! G POLYPEPTIXS
SITTERNESS OOILD SE PRESENT

 

 

 

PROTEQYSIS (SLOW)

 

FORMATION OF AMINO GRGJPS
INCREASES 5 pH

 

 

PROTEQYSIS

| AMINOACIDS l..— SITTE RNESS DISAPPEARS

- RENNETISTI'E MRESW (PLUSLOWpH)
- PASTEURIZEO MLK m SE EVEN WORSE (LW BACTERIAL COUNT)

 

 

 

 

Figure 1 - Czulak's hypothesis for the formation and breakdown of bitter
peptides in cheese. Source: Lemieux and Simard (1992)

35

of bitter strain activity. A secondary flora of lactobacilli
may have provided a suitable peptidase activity for
degradation of the bitter peptides to non-bitter peptides and
amino acids, reducing the bitter flavor. In Type II,
bitterness persists due to a lack of secondary peptidase
activity. Although the mechanisms for Type I and Type II
bitterness were supported by Czulack’s hypothesis, the third
type of bitter flavor, which appears later in the ripening
process, was not readily explained.

In 1972, Lowrie and Lawrence (1972) formulated an
alternative hypothesis to that proposed by Czulak (1959) for
the formation/degradation of bitter peptides. In their
hypothesis, rennet had a secondary role in bitterness
formation and the starter proteinases were responsible for the
defect. In this hypothesis, the major role of rennet in
cheese ripening was the degradation of casein into high
molecular weight non-bitter peptides. Increasing the
concentration of rennet increased the concentration of those
polypeptides in the cheese. Subsequent degradation of those
peptides by starter proteinases led to an accumulation of low
molecular weight bitter peptides. Subsequent degradation of
those peptides by the peptidase system present in certain
strains of lactic acid bacteria led to a disappearance of
bitterness. The mechanism proposed by Lowrie and Lawrence

(1972) is shown in Figure 2.

36

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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: .---—- .
: @315!!!“ $3“: ENG
: . :
I
: |____, %2' ____1 :
I m... i l
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I
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'- ------ ~ 3%. - ----- 4
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manna

PEPTIIXSA'D

wasps

 

 

 

 

 

 

Figure 2 - Lowrie and Lawrence's hypothesis for the formation and
breakdown of bitter peptides in cheese. Source: Lemieux and Simard
(1992)

37

Findings from Czulak's (1959) and Lowrie and Lawrence's
(1972) hypothesis on the existence of bitter flavor in cheese
led Stadhouders and Hup (1975) to postulate a scheme for the
formation/degradation of bitter peptides. They concentrated
their studies on Gouda cheese and especially on the amount of
rennet retained in the curd. They suggested that the
development of bitter flavor may have been due to a
synergistic effect of bitter peptides produced by rennet and
those produced by the (proteinase system. of lactic acid
bacteria. They suggested that the bitter flavor was caused by
a disproportion between the production of bitter peptides from
casein by' rennet and their consequent breakdown by the
proteolytic enzymes of the starter culture. This disproportion
led to an accumulation of bitter peptides in cheese above the
threshold value with consequent appearance of the defect.

Under this concept both rennet and starter bacteria may
produce bitter peptides, aloneior'together. The ability of the
starter to do so will depend on its proteolytic system, type
(slow and fast variants) and.the number of viable cells, which
may be reduced by increasing cooking temperatures, phage
infection or the presence of antibiotics. The type of rennet
(animal or microbial) and its content (chymosin and pepsin)
play a major role in the formation of bitterness, which is
also affected by milk pH, cooking temperature and, of course,
amount of rennet used for setting the milk. Salt content also

plays an important role, as it affects proteolysis.

38

Although not proven, Stadhouders and Hup’s concept has
been supported by a large number of experimental results
(Lemieux and Simard, 1992). The formation and breakdown of
bitter peptides in cheese is shown in Figure 3.

The hypothesis of Lowrie and Lawrence (1972) was
developed for Cheddar cheese and does not seem to fit the
results obtained by Stadhouders and Hup's experiments on Gouda
cheese (1975). Gouda cheese has composition and ripening
conditions different from those of Cheddar cheese. Lemieux and
Simard (1992) believe that the proteolytic properties of the
starter bacteria may have been influenced by the high cooking
temperature and early salt addition to the curd during Cheddar
manufacturing.

Visser (1977a and 1977b) made aseptic Gouda cheese, in
which the action of rennet and starter culture could be
studied separately, to investigate the contribution of both
enzymatic systems on bitterness formation. He concluded that
rennet and bitter starter bacteria were independently able to
produce bitterness in Gouda cheese, although the starter
bacteria appeared to produce more bitterness than rennet.
Based on his results and under the conditions in the cheese,
non-bitter starter can degrade bitter peptides. The bitterness
observed in normal and aseptic Gouda cheese was due to
separate actions of rennet and bitter starter bacteria. A
mechanism proposed by Visser (1977a) for the development of

bitterness in Gouda-type cheese is given in Figure 4.

39

 

Reaction 1
proteases of the starter bacteria

—> bitter tides
rennet pep

A paracasein

. rennet . proteases of the starter bacteria , _
B paracasein --——-> polypeptides —> bitter DCDIIdCS

influenced by:
— starter — proteolytic system of the starter bacteria — proportion slow and fast variants
— (cooking temperatures)
— number of starter bacteria — cooking temperatures
— phage infection
— penicillin
— rennet -— type of rennet
-— content — cooking temperatures
— amount of rennet added to the cheese milk
-— pH during manufacture — amount of the starter
—- activity of the starter
-— milk
— activity of proteases —- salt content of the cheese
—— moisture content of the cheese
— pH of the cheese
- ripening temperature of the cheese

Reaction 2

, , proteolytic enzymes of the starter bacteria . ,
bitter peptides ‘> non-bitter peptides

influenced by:
—— the proteolytic system of the starter bacteria - starter bacteria may differ in proteolytic
system capable of degrading bitter peptides

 

 

 

Figure 3 - Tentative scheme for the formation and breakdown of bitter
peptides in cheese. Source: Stadhouders and Hup ( 1975)

40

 

 

 

 

 

 

 

 

 

 

 

 

PARACASEIN
bitter 5107196
starter 2“ "em“ 25 (milk protease)
rennet
"\ NON-BITT E R
a. - .J -- - d A
‘- ~23" POLY PEPTIDES

ren net % énennet

 

 

 

BITTER NON-Bl TTER
PEPTIDES PEPTIDES

 

 

 

 

non-bitter
starter starters

 

NON-BITTER
PEPT l DES
AMI NO ACIDS

 

 

 

 

 

 

Figure 4 - Mechanism for the development of bitterness in Gouda-type
Cheese. Source: Visser (1977) i-

41

4.1.1 - Role of rennet in cheese bitterness

Rennet is the first proteolytic agent involved in the
overall mechanism of casein breakdown in cheeses. The primary
function of rennet is to specifically cleave the Phe(105)-
Met(106) peptide bond of x-casein (Fox, 1988; Visser et al.;
1976). During this "primary" phase of milk clotting, the
hydrophilic macropeptide, consisting of the fragment 106-169,
is removed from x—casein. The "secondary" phase includes the
aggregation of the unsoluble para-x-casein (fragment 1-105)
with the other (destabilized) casein components under the
influence of Ca2+, resulting in the formation of a casein
clot. The "tertiary" phase of rennet action relates to a
subsequent slow general proteolysis of other peptide bonds in
casein. The requirements for suitable milk clotting enzymes
should combine a high and specific cleavage capacity towards

x-casein at the pH of milk with a low general proteolytic
activity during cheese ripening (Visser, 1981) .

In a study on aseptic and chemically acidified (Glucano-
Delta-Lactone) Cheddar cheese, O’Keeffe et al. (1976) found
that rennet was mainly responsible for the level of
proteolysis detected by gel electrophoresis, soluble Nitrogen
at: pH 4.6 and gel permeation. They also found that rennet
alone is capable of releasing free amino acids, but only a
limited range of free amino acids was produced at quantifiable

1eVels, which includes methionine, histidine, glycine, serine

and
(19‘

pm

dur
I v!
dur
Pei
is

CdE

mo:
5a
in
no

II

42
and glutamic acid. Similar results were reported by Visser
(1977b) who studied the contribution of rennet enzymes on
proteolysis and flavor development in Gouda cheese.

Rennet is of major importance in hydrolysis of afl-casein
during the initial ripening period forming a large peptide a“-
I which is further degraded.by rennet and by other proteinases
during subsequent ripening (Ledford et al., 1966). The ah-I
peptide has been reported in all studies on rennet action and
is the most easily formed product of rennet activity on a“-
casein (Grappin et al., 1985).

The action of rennet on B-casein is more variable and
more dependent upon manufacturing conditions such as pH and
salt concentration. B-casein is hydrolyzed by rennet at pH 6.5
into three major products of increased electrophoretic
mobilities, representing the large breakdown products B-I, 3-
II and B-IIIa (Grappin et al., 1985). According to Creamer
(1976), the weak hydrolysis of B-casein in the curd might be
explained by the association between the C terminal region of
B-casein and a,,-casein, which make the cleavage sites no
longer accessible to rennet action. Visser and Slangen (1977) ,
identified other bonds on B-casein that were cleaved by
rennet. Those bonds were indentified as the residues Len-163-
Ser-164 and Len-127-thr-128, with the latter giving rise to

the B-IIIb peptide.

 

pa

de

Ch

43

Proteinases other than rennet will also be able to
coagulate milk under suitable conditions, but most are too
proteolytic relative to their milk-clotting activity. Since
many of these preparations are more generally proteolytic
(less specific) than rennet, they could be important sources
of bitterness in cheese (Fox, 1988).

Additional considerations relative to the involvement of
rennet in cheese proteolysis and bitterness include the level
used in cheese manufacture and the retention of the enzymes in
the curd. Increased amount of calf rennet at cheesemaking may
result in an increase of bitter flavor in the cheese
(Stadhouders and Hup, 1975). The effects of rennet level on
bitterness is shown in Table 1.

Lawrence et al. (1972) showed that bitter flavor was
produced in cheese with high levels of rennet when bitter
starters were used for cheesemaking. Their results have shown
that when non-bitter starters are used in cheesemaking, a 3-
fold increase.over the normal concentration of calf rennet did
not lead to such a high concentration of bitter peptides that
the cheese proved to be bitter. They suggested, based on
Czulak’s hypothesis (Czulak, 1959), that the bitter peptides
formed by rennet action were degraded by peptidases from these
particular starters.

Rennet retention in the curd is pH and temperature
dependent (Stadhouders and Hup, 1975). When the initial pH of

Cheese milk is decreased, more rennet remains in the cheese

44

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.Hm>mH anauo:

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meme .mflu3oq "mouzom

 

 

 

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cHo nucoewxwm mo mmmcuduufln co Hd>ma accede no pummmm .H manna

45
and there is a great propensity for bitterness development
(Table 2).

Stadhouders and Hup (1975) suggested that the increased
rennet retention in the curd at lower pH may be caused by
changes in the calcium.phosphate/casein complex. Reduction of
colloidal calcium phosphate causes weakening of the protein
matrix which becomes more accessible for enzymatic attack,
with consequent release of bitter peptides. Holmes et al.
(1977) studied the distribution of milk clotting enzymes
between curd and whey during Cheddar cheese making and found
that the retention of rennet in the curd.is pH dependent, with
86% of the rennet activity retained in curd formed at pH 5.2
compared to approximately 31% in curd formed at pH 6.6.

According to Stadhouders and Hup (1975) the cooking
temperature also affected rennet retention and bitter flavor
development. Their results showed that at higher cooking
temperatures, less rennet was found in the cheese and the
bitter flavor was less intense. Those observations are shown
in Table 3. They also concluded that above cooking
temperatures of 35°C (BS-39°C) , in addition to the reduced
rennet content, the growth of conventionally bitter starters
is restricted by temperature with.aidecrease in the content of
their proteases and consequently in the reduced bitter flavor.
They also found that curd retention of microbial coagulants is
apparently lower than that of bovine rennet and is less pH

dependent.

46

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47

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.Amhmav as: one mumcsonpnum "condom.

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ohduonudcna dnu cfi musueumeamu mcwxooo 0:» Mo vomuum .n manna

48

Czulak (1959) proposed that rennet was directly involved
in bitterness through production of bitter peptides in cheese
which are rendered non-bitter to a varied degree by microbial
peptidases from starter microorganisms. When rennet or other
proteinases used.as coagulants are used at excessive levels or
retained in the curd at high levels, they could directly form
bitter peptides in cheese. However, a more indirect
involvement is the more generally accepted hypothesis (Visser,
1977b; Visser, 1981). Generally rennet proteolysis results in
formation of large peptide moieties from casein which may be
non-bitter but may become precursors to bitter peptides via

further proteolytic cleavage (Lawrence et al. 1972).

4.1.2 - Role of starter microorganisms in cheese

bitterness

The most important role in bitterness appears to be
played by the starter bacteria (Lawrence and Gilles, 1969;
Richardson and Creamer, 1973). Emmons.et al. (1960a, 1960b‘and
1962a) investigated the effect of manufacturing conditions on
the formation of bitterness in Cheddar cheese and found that
the starter microorganism was the main factor responsible for
the defect.

The relationship between the strain of starter bacteria
used in cheesemaking and the bitterness of the cheese produced

has been clearly established (Emmons et al., 1962a and 1962b).

49

The starter lactococci (formely known as streptococci) have
been classified as bitter or non-bitter according to whether
or not they produce bitter flavor in cheese. Lowrie et al.
(1972), verified that during cheese ripening, bitterness
appears to decrease in intensity very slowly in cheese made
with a non-bitter strains and not at all in those made with a
bitter strain.

The most commonly used starter microorganisms used in
cheese making belong to the group N lactococci. The
proteolytic enzyme system present in the lactic lactococci has
been investigated (Law and Kolstad, 1983 ; Visser, 1993).
According to Visser (1993) the proteolytic system of the
starter bacteria. is divided into extracellular and
intracellular proteinases and peptidases. The enzymatic
activity of proteinases is towards the hydrolysis of native
proteins whereas the peptidase cleaves peptides produced by

action of proteinases (Kamaly and Marth, 1989).

4.1.2.1 - The extracellular proteinase activity

The proteolytic action of extracellular proteinases of
starter lactococci are of considerable significance for flavor
development in cheese. They contribute to the initial
fragmentation of the native milk caseins initiating substrate
degradation for peptidases in the subsequent hydrolysis during

cheese ripening (Laan et al., 1989; Umemoto and Itoh, 1981).

50

The location of the extracellular proteinase activity
appears to be cell wall surface bound. Exterkate (1979)
suggested that the proteinases are probably located at the
periphery of the cell, based on the fact that size and charge
of casein molecules restricts their diffusion into the cell
wall. There were three different types of proteolytic activity
related to the cell wall proteinases, designated PI, PII, and
PIII. The PI, PII, and PIII proteinase activity had pH and
temperature optima of 5.8 and 40°C, 6.5 and 30°C, and 5.4 and
30°C, respectively (Exterkate, 1976b).

Much of the evidence for the involvement of
extracellular proteinase activity with bitterness comes from
cheese making trials where proteinase negative (prt’) and
proteinase positive (prt‘) mutant strains were used (Mills
and Thomas, 1980). Exterkate (1975 and 1976a) studied the
proteolytic activity of a slow lactic acid producing variant
of Lactococcus cremoris, so called prt‘, and verified the lack
of PI and P11 activities which were normally present in the
prt+ variants. These prt' mutants are often considered to be
non-bitter strains and generally reach lower total cell
populations in cheese and thus have less total
proteinase/peptidase activity compared to prt“ strains. The
lower total cell population of non-bitter strains was
explained by Lowrie et al. (1972) who showed that bitter
strains were relatively temperature insensitive at the normal

cooking temperature of 38°C, whereas the non-bitter strains

51
were inhibited at this temperature. Increased cell densities
in the cheese may lead to a high concentration of starter
proteinase and elevated levels of bitter peptides (Lowrie and
Lawrence, 1972). Lawrence and Gilles (1969) have demonstrated
that "slow" starters.never cause bitter cheese. Bitterness was
absent even when conditions for bitterness formation were
present, such as increased level of starter added to give a
high rate of acid production or when the salt in moisture
level and pH was such that bitterness would be certainly
present if a "fast" starter is used (Lawrence and Gilles,
1969; Stadhouders and.Hup, 1975). Non-bitter strains are less
proteolytic than bitter strains and would be expected to
degrade high molecular weight peptides at a slower rate
(Gordon and Speck, 1965a and 1965b; Martley and Lawrence,
1972). Lowrie et al.(1972) observed that bitter strains grew
more rapidly and reached higher cell population than non-
bitter strains prior to salting’ ‘during normal Cheddar

cheesemaking.

4.1.2.2 - Peptidase activity

During the fermentation process in cheese making, the
lactic acid bacteria grow and produce lactic acid and other
flavor-related metabolites, as well as peptides. During cheese
ripening the microbial cells autolyze with consequent release

of proteinases and peptidases into the cheese environment.

52
These enzymes develop important roles in subsequent
proteolysis, releasing amino acids and contributing to overall
cheese flavor development (Schmidt, 1990).

The (peptidase activity' of lactic acid bacteria are
predomintantly aminopeptidases with a wide range of
specificity (Law and Kolstad, 1983). Although peptidase
activity has been found in extracellular cell-free broth, on
the cell surface, and in the intracellular fractions (Schmidt,
1990), the major portion of its activity is located near the
outside surface of and in the membrane, which suggests that
peptide hydrolysis is a function of the membrane (Exterkate,
1981 and 1984). Unlike cell wall-associated.proteinases which
are in direct contact with the substrate when bacterial cells
are intact, the intracellular peptidase activity must be
released by cell lysis for peptidase activity to take place
(Mills and Thomas, 1980). A high cell density in the cheese
does not necessarily' mean. that. a. high concentration of
peptidase is available for degradation of bitter peptides
since cells may remain intact and substrate will therefore be
inaccessible to the intracellular enzymes (Thomas and Mills,
1981).

A scheme for the pathway of protein degradation by lactic
acid bacteria is given in Figure 5 (Visser, 1993). The
proteinases bound to the cell wall surface are thought to act
on casein fragments generated by rennet and by indigenous

proteinases. The possession of surface-bound proteinases

53

 

  
       
 

 

MEDIUM } _ _CEL‘L WALL: ‘ CYTOPLASM
:1 '1 molecular sieve 1' -€_'__,
I. " "
I,,
A
'lk //§\\ ' ' El 4 ::
mi _ P p
protein E l. peptides f ,. i a?)
I is; ' .. 5 :::. «>48
:3 I .r. .,
? \\ i: /, 5 68
\S ’ . E _‘ + .
ll" 3,1 :. amino acids
peptides—L‘s . ' " V
' , ""1? ‘
I - : _
l bacterial protein
l .....

 

 

 

 

Figure 5 - Utilization of milk protein for synthesis of bacterial protein in lactic
streptococci. Source: Thomas (1991)

54

allows lactic acid bacteria to hydrolyse large molecules, such
as milk proteins, to peptides which are then small enough to
diffuse into ‘the cell ‘wall. Those jpeptides are further
degraded by extracellular and intracellular peptidases located
at or outside the surface of the cytoplasmic membrance since
the size limit for peptide transport through the membrane is
four to 6 amino acid residues.

Sullivan et al. (1973) verified that the bitter peptides
isolated from casein, contain a high content of proline and N-
terminal pyrrolidonecarboxilic acid, formed by cyclization of
glutamine. This high content of proline and the presence of an
N-terminal pyrrolidonecarboxylyl residue would effectively
block normal aminopeptidase activity. Thus, the resistance of
these peptides to enzymic hydrolysis would tend to result in
their accumulation in cheese. They suggested that bitter
strains might differ from non-bitter strains on the basis of
whether the organism contained a pyrrolidonecarboxylyl
peptidase. The main conclusion from their study was that all
starter cultures, including bitter and non-bitter strains, had
cytoplasmatic aminopeptidases required for degradation of
bitter peptides to non-biter peptides. These peptidase
activities included an aminopeptidase of wide specificity, a
proline iminopeptidase and an aminopeptidase-P. What made the
difference in each strains ability to hydrolyze bitter
peptides, was the possession of pyrrolidone carboxylyl

peptidase by non-bitter strains, but not by bitter starters.

55
One may conclude that the cell wall-associated proteinase
system developed an important role in the formation of bitter
peptides and that the intracellular peptidases had an
important role in the removal of bitter tasting peptides

(Mills and Thomas, 1980).

4.2. The Source of Bitter Peptides

The source of bitter peptides in cheese has been the
subject of several studies (Guigoz and Solms, 1976; Hamilton
et al. 1974; Hodges et al. 1972; Huber and Klostermeyer, 1974;
Richardson and Creamer, 1973; Visser et al., 1983a and 1983b)
and 0:,— and B-casein appear to be the major sources (Schmidt,
1990). According to Schmidt (1990), the role of minor caseins,
whey proteins and the fat globule membrane-associated proteins
in the formation of bitterness has not received as much
attention as the major caseins fractions. Due to their
globular conformation, the whey proteins are much more
resistant to proteolysis than caseins (Fox, 1989b).

The casein micelles, formed in the milk system through
the association of casein fractions, possess an amphiphilic
Structure in which discrete regions of their primary structure
are comprised of mostly hydrophobic amino acid residues,
hdmereas other regions contain hydrophilic amino acid residues
(Schmidt, 1990; Swaisgood, 1982). All the bitter peptides

have one common characteristic, that is, lipid solubility or

56
hydrophobicity (Lemieux and Simard, 1992). This finding has
led to investigation of the hydrophobic portions of casein as
the main source of bitter peptides in cheese (Schmidt, 1990;
Swaisgood, 1982).

Schmidt (1990) pointed out three important points that
should be mentioned about the formation of bitter peptides
from caseins: 1) Most of the work done so far was based on the
addition of proteinases to model systems containing purified
casein fractions. The response of individual casein fractions
to proteolysis differs from that of micellar caseins present
in milk (Fox and Guiney, 1973; Phelan et al. 1973). Therefore,
the results obtained from individual casein systems and milk
or cheese systems may not be comparable; 2) During
cheesemaking, most of the peptides are lost in.the whey, thus,
peptides responsible for bitterness in cheese may be different
from those existing in a milk system; 3) A low level of
bitterness may be essential for the overall flavor of cheese.
The bitterness problem only appears when the concentration of
bitter peptides in cheese exceeds certain threshold levels.

Alpha,,- and B-casein are potential sources of bitter
peptides, since they have high average hydrophobicities, 1.17
and 1.33 Kcal/residue,:respectively (Visser, 1977b). According
to Exterkate (1976b) , the action of proteinases present in the
cell wall of the starter culture on the B-casein may be the
source of bitter peptides in cheese. Visser et al. (1983 and

1983a) concluded that the C-terminal portion of B-casein has

57
an extremely bitter taste and is the main source of bitter
peptides in Gouda cheese.

Phelan et al. (1973) and Creamer (1975) found that B-
casein is highly resistant to proteolysis in Cheddar cheese,
while afl-casein is extensively degraded during ripening. The
main reason for this resistance to proteolysis is due to

polymerization of B-casein which makes hydrolysis difficult.

4.3. Chemical and structural Aspects of Bitter Peptides

Bitter peptides are rich in hydrophobic amino acids and
are released from the casein molecules primarily by the action
of rennet and bacterial proteinases (Crawford and Zwaginga,
1977; Stadhouders, 1974 and 1979; Sullivan and Jago, 1972).
Fractionation of an extract of bitter Cheddar cheese on gel
filtration chromatography using Sephadex G-25 has shown that
the most bitter fraction occurs in the molecular range of
2000-4000 da (Richardson and Creamer, 1973). Pelissier et al.
(1974), Visser et al. (1975) and Wieser and Belitz (1976)
demonstrated that, because of their solubility, only
relatively small peptides have been found to be bitter.

Bitterness which can represent a problem for many other
foods, is reasonably well correlated with the degree of
molecule hydrophobicity. Matoba and Hata (1972) have related

bitterness to hydrophobicity and concluded that the presence

in
nc
mo
pe
be

bi'

hyc

to

58
of at least one hydrophobic side chain in a peptide molecule
is an essential requirement for bitter taste.

Matoba and Hata (1972) concluded that bitterness is
attributable to hydrophobic amino acids in peptides regardless
of amino acid sequence and regardless of the state of the
amino or carboxy terminal group. On the other hand, Shiraishi
et al. (1973) concluded that the presence of hydrophobic
groups, as well as the sequence of the amino acids, is
important for taste. From the literature reviewed there was
not a definitive statement as to which of these theories was
most plausible. But considering the large varieties of
peptides exhibiting bitterness, the overall hydrophobicity can
be considered as the most important determinant of peptide
bitterness.

Bitterness of peptides can be evaluated from the
hydrophobic property of their amino acid side chain, according
to the Q-rule proposed by Ney (1971). The Q-rule has been
accepted as the most important predictor of peptide
bitterness . According to the Q-rule , the average
hydrophobicity, Q, is the ratio of the sum of the amino acid
side chain hydrophobicities of a peptide to the number of

amino acid residues:

59
where Af = free energy of transfer of the side chains of the
amino acid residues (cal.mol"), n = number of amino acid
residues, Q = average hydrophobicity of a peptide.

Peptides with Q-values below 1300 cal. res’l are not
bitter, whereas peptides with Q-values higher than 1400 cal.
res‘l are bitter. No prediction can be made about the peptide
bitterness for Q-values between 1300 and 1400 cal. res“. The
Q-rule is valid only for molecular weights up to approximately
6000 da; above this limit peptides with Q-values higher than
1400 cal.res‘i are not bitter. Prediction of bitterness
threshold may be possible if the structure of a peptide
molecule is known.

The contribution of L-proline residues to bitterness has
become evident from the study of Shiraishi et al. (1973) and
Shinoda et al. (1986), on syntheticidi- and tripeptides. Amino
acids such as leucine, phenylalanine or tyrosine, which
present high hydrophobicity, cause marked peptide bitterness.
The bitterness is more intense when leucine and phenylalanine
are located at the C-terminus (Ishibashi et at. 1988).

Sullivan et al. (1973) verified that the bitter peptides
which have been isolated from casein, contain a high content
of‘ proline and in one instance, N-terminal
pyrrolidonecarboxilic acid, formed by cyclization of
glutamine. This high content of proline along the peptide
chain make the peptide resistant to normal endo- and

exopeptidase attack. The presence of an N—terminal

Pi‘

31!

Chi

pr i
bi‘

cul

USE

ten

Pro

60

pyrrolidonecarboxylyl residue would effectively block normal

aminopeptidase activity.
4.4. Control of Bitterness Level in Cheese

According to Sullivan et al. (1973), bitterness can only
be controlled by control of total proteinase activity in the
cheese. The increase in peptidase activity must be adequate to
prevent the initial formation of bitterness (accumulation of
bitter peptides) by bacterial proteinase. According to Jago
(1974), reduction of proteolytic activity can be achieved by
culture selection, use of mixed cultures, proper selection and
use of coagulants and adjusting manufacturing parameters (pH,

temperature, salt-in-moisture, etc.).

4.4.1 - Control by manufacturing adjustments

The amount of proteases in the curd can be controlled by
using minimal amounts of rennet and by limiting the starter
cell population (Lawrence et al. 1972; Lowrie et al. 1972b).
According to Jago (1974) the starter cell population in the
curd should be limited by growth repression at the cooking
temperature. At abnormally low cooking temperature (33°C) the
ruin-bitter strains reach high cell populations in the curd,

producing bitter cheese whereas the typical bitter strains are

61
more heat-resistant and reach higher cell populations at 37-
38°C (Lowrie et al. 1972).

Bitterness is markedly reduced by limiting the amount of
rennet in the curd. Lawrence and.Pearce (1972) pointed out the
amount of rennet added to cheese milk should be the minimum
necessary for coagulation within 30-40 minutes. At increased
setting temperature less rennet is spent for the coagulation
process, thus the setting temperature should be increased by
1-2%: to reduce the amount of rennet in the curd, reducing
consequently the chance of bitterness (Jago, 1974; Lawrence
and Gilles, 1969 and 1971).

A.high salt-in-moisture content reduces the incidence of
Ibitterness in cheese :manufactured. with. a bitter starter
(Lawrence and Gilles, 1969). A possible explanation for this
is given by Fox and Walley (1971) and Sullivan and Jago
(1972). They found that a high salt-in-moisture may inhibit
the degradation of B-casein by rennet and bacterial

proteinases.

4.4.2 - Control by culture selection

Lawrence and Gilles (1969) found that cheese was seldom
‘bitter when non-bitter strains were used as starter,
:regardless of the amount of rennet, salt or moisture present,
or the rate of acid production in the curd, or the final pH of

the cheese. On the other hand, they found that when bitter

62
strains were used, the cheese was invariably bitter except
when the salt-in-moisture and the pH of the cheese were
relatively high.

Bitter and non-bitter strains differ in their growth
characteristics. According to Exterkate and Stadhouders
(1971) , bitter strains grow rapidly under normal cheese-making
conditions and reach high cell populations in the cheese curd
prior to salting while the non-bitter strains are inhibited.at
the normal cooking temperature in cheesemaking. Based on their
results, one may conclude that all strains are potentially
capable of contributing directly to the formation of bitter
peptides in cheese.

Sullivan and Jago (1970) claimed that the major
difference ‘between. bitter' and 'non-bitter strains is the
possession of a pyrrolidone carboxylyl peptidase by non-bitter
strains, but not by the bitter strains. Therefore, non-bitter
starters are suitable for degradation of the bitter peptides
present in the cheese.

Certain starters resulted in bitter taste in cheese and
pairing bitter starters with non-bitter starters markedly
reduced bitter flavor development in Cheddar cheese (Lawrence
and Pearce, 1968; Creamer et al., 1970). The same conclusion
was achieved by Visser ( 1977a) when manufacturing Gouda

cheese .

63

The choice of suitable starter is therefore of utmost
importance to the control of bitter compounds formed in.cheese
during ripening. According to Olson and Johnson (1990b)
lactic starters may have to be scrutinized for proteolytic and
peptidolytic activities and profiles as they specifically
impact on bitterness formation. Preliminary studies have shown
that cultures with less proteolytic activity and slower acid
production produced a better cheese, with less bitterness than
did a more proteolytic culture (Olson, 1984 and 1991). The
starter cultures should be chosen based on their ability to
degrade the bitter peptides as rapidly as they are formed by
rennet and other proteinases, so that they never exceed the

threshold level for bitterness perception (Jago, 1974).

4.4.3 - The Use of Lactobacillus

The peptidolytic enzymes of Lactobacillus casei, used as
adjunct culture and its possible use as an agent to improve
the flavor and body and texture of cheeses have been studied
(El-Neshawy et al. 1986; El-Soda et al. 1981; El-Soda, 1986;
Hargrove, 1966b; Simard, 1991). According to Simard (1991)
and Laleye (1989), Lactobacillus casei possesses three
exopeptidases (a dipeptidase, an amino-peptidase, and a
specific carboxypeptidase) and one endopeptidase which
increase protein and peptide hydrolysis with positive effects

on texture. and flavor of Cheese. The action of those

64
peptidases on the bitter peptides is a desirable
characteristic of this microorganism and might improve
considerably the texture, body and flavor of the cheese.

Several treatments have been applied to Lactobacillus
species to improve body and flavor development in cheeses. El-
Soda et. al. (1981) demonstrated that Cheddar cheese added
with whole cells or cell free extracts from Lactobacillus
casei, showed an acceleration of ripening, with high values
for protein breakdown and volatile acid formation, but.a rapid
bitter flavor development was detected in cheese. Trepanier et
al. (1991a and 1991b) studied the addition of live cells and
cell homogenates of Lactobacillus casei to Cheddar cheese.
Their results showed 40% increase in flavor development, but
no difference in rheological properties was observed between
the control and the experimental cheese.

The addition of heat and freeze treated cultures of
Lactobacillus have shown promising results. Bartels et al.
(1987) verified a reduced bitterness and great improvement in
flavor intensity in Gouda cheese with freeze shocked
.Lactobacillus belveticus added. Trepanier et al. (1992)
carried out an experiment for accelerating the maturation of
Cheddar cheese by using heat treated Lactobacillus casei and
neutrase. A good quality Cheddar cheese, presenting 50%
increase in flavor, was produced when heat shocked cells were

added.to the cheese milk. However, high levels of bitterness

de'

CO]

sh:

th
de
to
to

CO

de
a1

SE

65
developed in the cheeses with heat shocked cells added in

combination with neutrase.

A study to establish adequate conditions for heat-
shocking cells of Lactobacilli was carried out by El-Abboud.et
a1. (1991). A treatment of 67°C/15 sec. was considered the
best for inactivating the microorganism without detrimental
effects to its enzymatic system. According to Law (1984), this
treatment prevents the bacteria from producing acid while
keeping intact their proteolytic system. El-Abboud et al.
(1991), suggested that the increased.proteolytic potential in
the cheese with.heat—shocked cells added may lead to a further
degradation of peptides responsible for bitterness. According
to Ardo and Mansson (1990), the heat treated cells are added
to the cheese ‘milk prior to .renneting and the enzymes
contribute to the development of a desirable flavor. The
aminopeptidase from Lactobacillus casei responsible for the
degradation of bitter peptides is highly heat resistant,
allowing for continued activity during cheese ripening. If
sufficient levels of such enzyme are present in the cheese,
the peptides which cause bitter taste are broken down to amino

acids.

66

4.5. Peptide Separation

The appearance of bitter flavor during the ripening of
cheese has long been found as being attributable to the
accumulation of bitter peptides. One way to gain information
as to the kind of peptides which contribute to the cause of
bitter taste, consists of isolation and chemical
characterization of the bitter fraction from the cheese.

Several methods are available for isolation of bitter
peptides from.cheese. All follow similar patterns based on the
hydrophobic nature of the bitter compounds. Due to their
relatively large content of hydrophobic side chain amino
acids, bitter peptides are lipid-soluble and easily
extractable with organic solvents (Sullivan and Jago, 1970).

Peptide separation can be accomplished by several
tehniques. The process usually consists of a primary
separation by gel permeation chromatography, followed by
separation by high performance liquid chromatography.
Sephadex gels have been extensively used for preliminary
separation of peptides from cheese extracts (Champion and
Stanley, 1982; Edwards and Kosikowski, 1983; Harwalkar and
Elliott, 1971; Hetting and Parmelee, 1965; Richardson and
Creamer, 1970 and 1973; Schalinatus and Behnke, 1975; Visser,
1977c; Visser et al. 1983a). Richardson and Creamer (1970 and
1973) used gel filtration chromatography on Sephadex G-25 of

an alcoholic extract of bitter Cheddar cheese to demonstrate

67
that most bitter fractions occurred in the molecular weight
range of 2000-4000 daltons.

Separation by gel permeation has some disadvantages. It
is a highly time consuming technique and suffers from lack of
resolution. Numerous peaks may be included within each
fractions and further separation is thus needed to separate
the individual components (Rank et al. 1985). This separation
can be accomplished by using paper and thin-layer
chromatography, high-voltage paper electrophoresis, ion-
exchange chromatography or by high-performance liquid
chromatography (HPLC) on a reversed-phase type column
(Champion and Stanley, 1982; Edwards and Kosikowski, 1983;
Gordon and Speck, 1965b; Harwalkar and Elliott, 1971; Hetting
and Parmelee, 1965; Huber and Klostermeyer, 1974; Schalinatus
and Behnke, 1975; Visser et al. 1983a).

Reversed-phase high-performance liquid chromatography for
profile studiesl of jpeptides produced. during’ ripening' of
Cheddar cheese was initially proposed by Cliffe et al. (1989)
and has been the method of choice for fractionation of bitter
peptides. Despite the initial cost of the equipment and
«difficulty in the interpretation of the data, HPLC procedures
allow speed of analysis and the resolution is comparable if
not better than two-dimensional gels (Rank et al. 1985) .
Champion and Stanley (1982) obtained 71 peaks when a bitter

extract from Cheddar cheese, previously fractionated on

68
Sephadex G-50, was applied to a reverse-phase HPLC column

using a liner gradient from 0 to 90% methanol in water.

Chapter 1

Ripening Characteristics of Reduced Pat Cheddar Cheese
Using Beat-Shocked and Live Lactobacillus casei L23 as

Adjunct Culture

INTRODUCTION

The last two decades have seen shifts toward the
consumption of low fat foods (Egbert et al., 1991). The wish
to avoid high calorie foods to control body weight and a
possible connection between animal fats and circulatory
disorders has been the main cause for this increased
consumption of low fat foods (Hargrove et al., 1966a; Madsen
et al., 1970; Simard, 1991). The growing consumer interest in
low calorie food has led the cheese industry to provide
consumers with a large variety of reduced fat cheese. Levels
of fat in cheese have a direct impact on the sensory
acceptability by consumers. Fat is the precursor of many
flavor compounds and influences body and texture of cheese
(McGregor and White, 1990a) . The use of traditional procedures

‘tolmake reduced fat cheese has led to the production of hard,

69

70

rubbery and tasteless cheese, even after extensive ripening
(Banks et al., 1989; El-Neshawy et al., 1976; Hargrove et al.,
1966b; Simard, 1991). To improve the flavor, body and texture
of reduced fat cheese, the traditional manufacturing procedure
has been modified (Banks et al., 1989; Hargrove et al., 1966b;
Madsen et al., 1970; Olson and Johnson, 1990), in addition to
changes in milk preparation prior to manufacture (McGregor
and White, 1990a and 1999b; Metzger and Mistry, 1993), culture
selection (Chen et al. 1992; Simard, 1991), or use of fat
replacers (El-Neshawy et al., 1986).

The lactic starter cultures greatly affect all cheeses,
but especially reduced fat cheeses (Olson and Johnson, 1990).
Due to the association of reduced fat cheese with high acid
levels caused by increased lactose concentration and a range
of off-flavors caused by proteolytic activity (Rosenberg,
1992), the choicezof a suitable starter for making reduced fat
cheese is an important factor to consider. Starter culture
possessing lower rates of acid production, lower proteolytic
activity’ (Olson, 1991; Rosenberg, 1992), and. debittering
activity (Rosenberg, 1992) is prefered. The use of
lactobacilli as starter culture adjunct has been successfully
applied as a way to reduce bitterness and improve flavor in
cheese (El-Neshawy et al., 1986; El-Soda, 1986; El-Soda et
al., 1981; Hargrove et al., 1966b; Johnson et al., 1993;
O'Donnell, 1993; Simard, 1991; Trepanier et al., 1991a, 1991b

and 1992). The peptidolytic enzymes of Lactobacillus casei,

71

used as adjunct culture and its possible use as an agent to
improve the flavor and body and texture of cheeses have been
studied (El-Neshawy et al. 1986; El-Soda, 1986; El-Soda et
al., 1981; Hargrove et al., 1966b; Simard, 1991). According
to Simard (1991), Lactobacillus casei possesses three
exopeptidases (a dipeptidase, an amino-peptidase, and a
specific carboxypeptidase) and one endopeptidase which
increase protein and peptide hydrolysis resulting in positive
effects on the texture and flavor of cheese.

During ripening, cheese body becomes weaker and smoother
due to the effects of the proteolytic breakdown of the
structural matrix. Increasing the peptidase activity during
ripening without changes in the balance of acid production
during the manufacturing process could be an interesting
approach to improve flavor and soften the body of reduced fat
cheese. Lactobacillus casei is a suitable microorganism for
that purpose as it possesses highly heat resistant
peptidolytic enzymes (Ardo and Mansson, 1990).

The objective of this work.is to evaluate the use of live
and heat shocked cells of Lactobacillus casei as an adjunct
culture in the manufacture of reduced fat Cheddar cheese.
Protein degradation (i.e., soluble nitrogen fractions,
breakdown. of a;- and. B-caseins) will be followed. during

ripening.

72

ILIIRILLB AID IBTIODB

Culture Preparation

The lactic starters used in this experiment were BIOLAC
DSS LF slow acid producing culture (Marschall Products,
Madison, WI) consisting of selected single strain of
Lactococcus lactis subsp. cremoris and Lactobacillus casei
subsp. casei L2A (Food Research Institute, Ottawa, Canada).
The culture was supplied in slant form and was subcultured
daily in All Purpose Tween (APT) broth (37°C/24 h) for one
week and then in 11.5% reconstituted non-fat dry milk (NFDM)
daily, at the same conditions, during the week preceeding
cheese manufacture.

The live Lactobacillus casei subsp. casei L2A was added
to the cheese milk before rennet addition. The heat shock
treatment was adapted from El-Abboudi et al. (1991) . Following
the normal incubation period, the culture was cooled to 4%:.
The heat shock treatment ‘was performed in a Spiratherm
(Cherry-Burrel Corporation, Cedar' Rapids, Iowa). The
Spiratherm unit was properly sanitized immediately before
using. The stainless steel coiled system (0.4 cm i.d.)
consisted of a heater followed by’a holding tube and a cooler.
The holding tube had a total volume of 210 ml. The pH of the
cooled culture was adjusted to 6.5 with sterile 1 M NaOH to
avoid precipitation during the heat treatment. A positive,

sanitary pump with variable speed (Waukesha Foundry Company

73
Inc. , Waukesha, WI) was used to pump the bacterial suspension
through the system. The flow rate through the apparatus was
set to 9.5 ml/sec to give a process time of 22 seconds. The
temperature applied to the culture was 67°C. Loss of heat was
prevented by covering the holding tube with insulating
material. Temperature was checked in both extremes of the
holding tube of the apparatus. After heat treatment, the

culture was stored at 4°C until use.

Cheesemaking

Twenty-eight batches of reduced fat Cheddar cheese were
manufactured from milk received by the Dairy Plant, Department
of Food Science and Human Nutrition at Michigan State
University. The milk was standardized to 1.5% fat with
skimmilk. All cheeses were manufactured from a single lot of
milk. Milk of each vat received the following treatments: In
the Control treatment, single strain of Lactococcus cremoris
subsp. cremoris ( 1% inoculum) was used as the starter. In
treatments LLC-0.5, LLC-1.0 and LLC-1.5, Lactococcus cremoris
subsp. cremoris ( 1% inoculum) plus live Lactobacillus casei
L2A, at 0.5%, 1.0% and 1.5% inoculum, respectively, were used.
In treatments HSLC-0.5, HSLC-1.0 and HSLC-1.5, Lactococcus
cremoris subsp. cremoris (1% inoculum) plus heat shocked
Lactobacillus casei L2A, at 0.5%, 1.0% and 1.5% inoculum,
respectively, were used. Four trials were conducted for each

treatment .

74

Reduced fat Cheddar cheese was manufactured based on the
process described by Wilster (1980) with modifications to
produce a high moisture reduced fat cheese (Chen, 1991). The
standardized milk was pasteurized at 75°C/18 s, cooled to
32.2°C, weighed (127.12 kg) and inoculated with starter
culture, 40 min prior to rennet addition (1257 ml Chymax-
Double Strength, Pfizer, Milwaukee, WI). After 30 min of
coagulation, the curd was cut with horizontal and vertical
knives (1.7 cm). After 20 min of stirring the curd was
gradually heated to 37JWC in approximately 15 min, followed
immediately by draining. Fifteen minutes later the curd was
cut in blocks which were turned every 15 min until a curd pH
of 5.6 was achieved. The curd was then milled, salted (0.3
kg/100 kg of milk), hooped and pressed (2.79 kg/cmfi) for 12 h.
After vacuum packing the cheeses were ripened at 11%: for 20
weeks. A complete time schedule to make reduced fat Cheddar
cheese of high moisture content is presented in Table I-l

(Appendix I).

Compositional Analysis of Cheese

Fat content of cheese samples were determined by the
Babcock method, as described by Marshall (1992) . Measurements
of pH were determined using a CORNING 150 pH Meter (Halstead
Essex, Medfield, MA). Moisture in cheese was determined by the

vacuum oven method (Marshall, 1992) .

75

Assessment of Proteolysis

Nitrogen Fractions.

Samples were taken from each block of fresh cheese and
after 4, 8, 12, 16 and 20 weeks of ripening. The citrate-
cheese extract described by Gripon et al. (1975), was used for
determination of all nitrogen fractions (Total Nitrogen (TN),
Water Soluble Nitrogen (WSN) and 12% Trichloroacetic Acid

(TCA-N) ) .

Ripening Indexes
The Ripening Extension Index (RBI) and Ripening Depth

Index (RDI) represented the ratio of WSN to TN and TCA-N to
TN, respectively, and were determined as described by Furtado

and Partridge (1988).

Electrophoresis

Proteins were extracted from cheese according to Tieleman
and Warthesen (1991) with modifications. A 0.2 g aliquot of
grated cheese was dissolved in a buffer containing the
following composition: 0.001M sodium phosphate (pH 7.2), 2%
SDS, 0.06M Tris-HCl, 9.6% glycerol, 4.8% B-mercaptoethanol,
and 0.01% bromophenol blue. The cheese buffer was heated for
40 min. at 100°C, and cooled before use.

Vertical SDS-polyacrylamide gel electrophoresis was
carried out in a Hoefer Model SE600 gel cell (Hoefer

Scientific Instruments, San Francisco, CA) following the

76
technique stated by Laemmli (1970) . The slab gels measured 160
x 180 x 1.5 mm. The acrylamide concentration was 10% and 4%
(w/v) in the separating and stacking gel, respectively.

The gels were scanned on the Shimadzu Dual—Wavelength TLC
Scanner Model CS-930 (Kyoto, Japan), provided with a DR-2 Data
Recorder. A zigzag scanning mode with a slit size of 6
(height) and 2 (width) was used to limit the beam size on the
sample surface. The scan width.was set to 13 mm. A sensitivity
of 2 (medium) was used during scanning. Gels were scanned at
a wavelength of 580 nm. The signal from the start point to the
end point of each peak was determined by the area signal,
integrated, and the relative areas of the individual protein
peaks were recorded. The initial peak areas were considered
100% of the individual proteins. The peak areas of the main
casein fractions are listed.in Appendix I (Table I-8 and I-9).
A complete list of parameters used during gel scanning are

listed in Table I-5 (Appendix I).

Statistical Analysis

A randomized split plot design with repeated measurements
was used in this experiment. The main plots consisted of the
treatments (cheese made with different cultures and levels),
split because each cheese characteristic was analyzed at
different points in time. Analysis of variance and contrasts

among treatment means were performed using the MSTATC computer

77

program (Crops and Soil Science, Michigan State University,
MI).

The linear model (Gill, 1986) for response variable Y
used in this experiment was:

25.: = M + '1'i + ij + pk + (TP),]: + Eb},

(i = 1...t, j = 1...n per i, k = 1...p),
where the terms are overall mean (M), treatment effect (T),
effect of cheese within treatment (C), period effect (P),
treatment x period interaction (TP), and residual (E). It was
assumed that EiT = 0, EKP = 0, EATP) = 0, and 2,,(TP) = 0. The
cheese term (C) represented error associated with treatment
differences, and variability among cheeses ordinarily was

substantial.
RESULTS AND DISCUSSION

Composition of Cheese

The average composition of the cheeses, determined
immediately after manufacture, is shown in Table I-l. The
legal limits in the U.S. for moisture and fat in dry matter
(FDM) contents of Cheddar cheese are 5 39% and 2 50%,
respectively (Kosikowski, 1978). The average moisture content
of the cheeses (49.65%) was considerably above the legal
limit, and the average FDM was lower (28.40%). There were no

differences (p<0.05) in moisture content between treatments.

The higher moisture content of reduced fat Cheddar cheese is

78

.Amo.ovsv

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.mucmauemuu Ham you mcofiucofianmu v u c
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a Hm.m~ m 6H.m¢ m mo.ea Houucoo
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.dmn women nsqnfierOuocn no ousuaso
vocsnce spas cousuoeusces omooco unococo new sconce» no soduwuomaoo i HIH canes

79
required to attain firmness approximating that of normal
Cheddar cheese (Banks et al., 1989). Cheeses with lower fat
content contain more protein per unit volume than full fat
cheeses and require more water to present similar body and
texture (Olson and Johnson, 1990). Higher water levels may
enhance proteolysis during ripening if the cheese salt content
is not increased. In the present study, all cheeses were

salted at the same level.

Cheese pH

The changes in pH of cheeses during ripening showed
similar patterns within treatments and no differences (p<0.05)
were found.among levels of addition of starter culture. Figure
I-l displays the changes in pH of cheeses made with addition
of 1.5% of adjunct culture of Lactobacillus casei L2A. The
complete set of pH changes during ripening is displayed in
Table I-2 (Appendix I).

As a result of gradual fermentation of residual lactose
by the starter culture, the pH of all cheese decreased during
the first 12 weeks of ripening. A greater level of residual
lactose was expected due to the high moisture content. A
similar result was observed by Lee et al. (1992) on reduced
fat Cheddar cheese. After 30 min of pressing, HSLC-1.5
cheeses had the highest pH (5.56), which was probably due to
the reduced lactose fermenting ability of the heat shocked

adjunct culture. The lowest pH was shown by the LLC-1.5

80

 

 

 

 

 

 

 

 

5'6 El
_ HSLG-l.5 #1
LLc.1.5 $1
5.5 —
5.4 —
E 5.3 i“
5.2 —
5.1 —
- “$8-
5 L l l l l I
0* 4 8 12 16 20

Age of Cheeses (weeks)

 

Figure I -1. Changes in cheese pH throughout ripening of reduced fat
Cheddar cheese made with adjunct culture of Lactobacillus casei L2A.
* 30 min. after beginning pressing.

81
cheese, which may be related to the greater acidification
capacity caused by the presence of live cells of.Lactobacillus
casei. The pH of the LLC cheeses was lower (p<0.05) than the
pH of the other two treatments during the first 12 weeks of
ripening. After 12 weeks, the pH of all cheeses started
increasing to reach 5.17 at 20 weeks. This increase in pH is
probably’ related. to. the accumulation. of basic compounds

resulting from the protein breakdown during ripening.

Proteolysis

Protein hydrolysis is considered a good index of cheese
ripening, as proteolysis of casein is the result of several
proteinase and peptidase activities. The results of WSN and
TCA-N analyses during the ripening of reduced fat Cheddar
cheese are given in Figure I-2. No differences (p<0.05) were
found among levels of addition of starter culture, thus only
the 1.5% level was presented. The evolution.of N fractions and
ripening indices during ripening for all cheeses are displayed
in Table I-3, I-4 and I-5 (Appendix I). There was a four fold
increase in WSN values for all treatments (Control, LLC-1.5
and HSLC-1.5) after 20 weeks of ripening (Figure I-2A). The
WSN for the HSLC-1.5 (0.93%) was higher (P<0.05) than that for
the LLC-1.5 (0.86%) at 20 weeks. In cheese, WSN consists
predominantly of medium and small peptides and free amino
tacids. Rennet is considered to be the main agent in producing

medium peptides which are further degraded to small peptides

82

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.8 -
z 0.6 '
E A
.\° 0.4 ~
0.2 Control
. HSLC-1.5 m
LLC-1.5 e1
0 l 4 l l l l
0 4 8 12 16 20
0.8
0.7 f
0.6
2. 0.5
< .
g 0.4 F B
°\ 03 ~
0.2 -
Como:
0.1 v HSLC-1.5 £1
LLC-1.5 .1
o l l l l l
0 4 8 12 16 20
Age of Cheeses (weeks))

 

 

 

Figure l-2. Evolution of nitrogen fractions during the ripening of reduced fat
Cheddar cheese made with 1.5% of live and heat shocked Lactobacillus casei
LZA A = WSN (water-soluble nitrogen); B = 12% TCA-N (T CA-soluble
nitrogen).

83

and amino acids by starter peptidases (Adda et al., 1982; Fox,
1993; Fox and Law, 1991; O'Keeffe et al., 1978). According to
Mulvihill and Fox (1977), the specificity of chymosin on a“-
casein is pH-dependent, the protein being optimally hydrolysed
to smaller peptides at pH 5.8. LLC-1.5 had a low' pH (Figure
I-l) throughout ripening and at 20 weeks had the least WSN.
The liberation of TCA-N during ripening is shown graphically
in Figure I-ZB. The levels of TCA-N increased as the cheese
aged. The most pronounced peptidolytic activity was shown by
the HSLC-1.5 treatment. The TCA-N’ increased from 0.1 to 0.75%
in 20 weeks and was higher (P<0.05) than that for ‘the Control
treatment, which increased from 0.1 to 0.68% in 20 weeks.
These results confirm the findings of other studies (Ardo and
Mansson, 1990; El-Abboudi et al., 1991; Law, 1984), indicating
that the heat-shocking of Lactobacillus casei released
aminopeptidases which were ultimately responsible for the
greater proteolysis associated with TCA-N observed in the
HSLC-1.5 treatment.

The results of the ripening indices at 20 weeks for the
three levels of adjunct cultures are presented in Figure I-3.
A Ripening Extension Index of 21 and 19% (Figure I-3A) were
found for the HSLC and LLC treatments, respectively,
independent of the level of inoculation with adjunct cultures
of Lactobacillus casei. No differences (p<0.05) were found
among the HSLC nor the LLC levels of treatments, but all HSLC

results were higher (P<0.05) than those observed for the

84

 

 

 

  

 

 

 

 

 

 

 

 

 

 

      
    

  

      

 

 

 

   

 

24

22 ' 2111 E”

20 _ _ ' 19.05
A 18.52 ' ' '
é . l \
m 18 - . A
O: 7.

16 ' ' .

\
14 -\
Control 0.5% 1.0% 1.5%

18

17 b 16.50 16.50 16.80

16 - r 15-6 15.70 321;". 15'”
é N N

14 — \§ §§

13 - §§ \:

12 5:272" §§ ?Jil .§§ i»

Control 0.5% 1.0% 1.5%
Treatment
UHSLC.LLC

 

 

 

Figure I - 3. Changes in the ripening indices during the ripening of reduced fat Cheddar
cheese made with different levels of adjunct culture of Lactobacillus casei L2A. A = REI
(Ripening Extension Index); B = RDI (Ripening Depth Index).

85

Control and LLC treatments. These results are supported by the
pH changes observed throughout ripening (Figure I-1). The LLC
had the lowest pH which likely decreased the proteinase
activity of the starter in the early stages of ripening. In
Figure I-3B, the changes in the Ripening Depth Index are
shown. Again, no differences (P<0.05) were found among
different levels of adjunct cultures added both for the HSLC
and LLC treatments, nor between the results of these
treatments within each level of inoculation with Lactobacillus
casei. However, the RDI for all of them were higher (P<0.05)
than that for the control. These results confirm that the
addition of heat-shocked cells of Lactobacillus casei increase
the aminopeptidase activity and the liberation of amino acids
from peptides, as indicated elsewhere (Ardo and Mansson, 1990 ;
El-Abboudi et al., 1991; Law, 1984). The peptidolysis was not
enhanced by a three-fold increase in the inoculation of
adjunct cultures. This finding is in agreement with that of
Law et al. (1993), who observed a similar result when studying
the contribution of lactococcal proteinases to proteolysis in
Cheddar cheese.

The results concerning the percentage of WSN
corresponding to TCA-soluble nitrogen after 20 wk of ripening
are presented in Figure I-4. The TCA-N’of LLC-1.5 and.HSLC-1.5
(81.11 and 83.39%, respectively) were similar (P<0.05), but
both were higher (P<0.05) than that (76,67%) observed for the

Control treatment. These results indicate that endogenous

% of TCA—N in WSN

86

 

 

E Control [[111]] LLC-1.5 E HSLC-1.5

 

85-

83.39

 

 

 

Treatments

Figure l - 4 - Percentage of water soluble nitrogen (WSN)
corresponding to TCA-soiuble nitrogen (T CA-N) after 20 weeks of
ripening.

87
enzymes from Lactobacillus casei have peptidase activity,
which can be enhanced when the aminopeptidases are freed from
the cell by the heat shock treatment.

These results are important considering that the
degradation of casein implicates the corresponding breakdown
of the protein matrix. The loosening of the cheese protein
matrix causes texture changes and increases the water binding

capacity, which are important to reduced fat cheeses.

Electrophoretic Patterns

Typical densitograms of the Control, HSLC-1.5 and LLC-1.5
treatments are shown in Figure I-5 (A, B, and C),
respectively. The electrophoretic patterns throughout
ripening are presented in Figure I-l (Appendix I). After 20
weeks of ripening a,,-casein was extensively degraded as
opposed to a slight degradation of B-casein. These
observations are confirmed by the data presented in Figure I-6
(A, B) which shows the degradation of both caseins throughout
ripening. As found by other authors (El-Abboudi et al., 1991;
Fox and Law, 1991; Law et al., 1992; Visser, 1993) B-casein
was quite resistant to rennet and bacterial enzymes during
ripening. As shown in Figure I-6A, B-casein was slowly broken
down during 20 weeks of cheese ripening and at the end 79.11,
70.43 and 82.14% of original value were still intact for
Control, HSLC-1.5 and LLC-1.5 treatments, respectively. The

Control and LLC-1.5 values did not differ, but both were

88

 

-casem

£2 ] a,—cascms
3:23:- fl-cascin
E
> } a,-caseins .

 

 

 

 

 

 

g i (a)
is \A m/L/M’V 0’)
1 A?! M

[\M ' (C)
(1) W (2)
Direction of Migration;

 

Figure I - 5 - SDS-Polyacrylamide gel electrophoretograms of caseins in reduced fat
Cheddar cheese at 1 week and 20 weeks (2) of ripening. (a) Control cheese; (0)
cheese made with heat shocked Lactobacillus casei L2A; (0) cheese made with live
Lactobacillus casei L2A.

89

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.E
G)
en
=3
“1 A
‘6
£9
.E
‘8
o\°
* HSLC-1.5 t
10? LLC-1.5
o l I l i l 1
0 4 8 12 16 20
B

% of intact or,l -casein

 

 

 

 

0 4 8 12 16 20
Age of Cheeses (weeks))

 

 

 

Figure l - 6 - Degradation of ficasein (A) and orfl-casein (8) throughout the
ripening of reduced fat Cheddar cheese made with 1.5% of live and heat shocked
Lactobacillus casei L2A.

90
higher than that for the HSLC-1.5 cheeses (P<0.05). The values
presented in Figure I-6B show that au-casein was extensively
broken down. Only 26.94, 21.31 and 35.30% of the original a“-
casein remained intact after 20 weeks of ripening, in the
Control, HSLC-1.5 and LLC-1.5 cheeses, respectively. These
values were different (p<0.05) from each other. These results
are in agreement with those found by Lau et al. (1991), Visser
and Groot-Mostert (1977) and Kaminarides et al. (1990), who
observed that 85, 90 and 77% of an-casein, respectively, had
been degraded at the end of the ripening of different cheeses.

As shown in Figure I-6 (A, B), both caseins were degraded
more in the HSLC cheese. This was likely the result of the
strong aminopeptidase activity of the heat shocked cells of
Lactobacillus casei L2A.

Figure I-7 (A, B, C) shows the plots of progressive a“-
casein degradation against the increase in WSN throughout
reduced.fat Cheddar cheese ripening; The production.of soluble
nitrogen correlated negatively (correlation coefficient of
-0.98, -0.97 and -0.99 for LLC-1.5, HSLC-1.5 and Control
treatments respectively) with the decrease in the amount of

intact afl-casein.

91

 

 

 

 

 

% of intact as,-casein

 

% of water-soluble nitrogen (WSN)

 

 

 

 

 

O 4 8 12 16 20

Ripening Time lweekel

D eel-casein X WSN

 

 

 

Figure l - 7. Correlation between degradation of al.,-casein and increase in water
soluble nitrogen (WSN) throughout the ripening of reduced fat Cheddar cheese. A
= LLC-1.5; B = HSLC-1.5; C = Control. R = Correlation Coefficient. -

92

CONCLUSIONS

The results of this experiment showed that the addition
of adjunct cultures of live and heat shocked Lactobacillus
casei L2A enhanced starter aminopeptidase activity in reduced
fat Cheddar cheese. The addition of live cells lowered the pH
of cheeses, resulting in inhibition of aminopeptidase activity
when compared to the HSLC treatment. The heat shock treatment
of Lactobacillus casei L2A cultures inhibited the acid
producing ability of the microorganism without destroying the
aminopeptidases. Therefore, higher levels of pH 4.6 and 12%

TCA-N were produced in HSLC cheeses.

Chapter 2

The Use of Lactobacillus casei L2A as Adjunct Culture and
its Effects on Flavor and Texture of Reduced Fat

Cheddar Cheese.

INTRODUCTION

Milkfat affects the textural and structural properties of
cheese. When fat is removed from the protein network, the
cheese becomes more compact and tends to present different
mechanical properties from a full fat cheese. Fat affects the
mouthfeel and palatability of the cheese and the ratio of
water to protein, which ultimately regulate firmness and
elasticity. As there is a growing concern about the
consumption of fat in the diet, much research has been devoted
to the making of reduced fat cheeses (Banks et al. 1989; Lee
et al., 1992; Lindsay, 1991; Olson and Johnson, 1990).
However, the use of traditional processes to make reduced fat
Cheddar results in cheeses with a variety of textural and
flavor defects. It is well established (Lawrence et a1. 1983)
that the texture and the flavor characteristics of a mature

cheese result mainly from the degradation of the cheese

93

94
proteins, which is in turn highly influenced by such factors
as moisture, the ratio of moisture to casein, salt and the
proteolytic and peptidolytic activities of the starter.
Reduced fat Cheddar cheese tends to have higher moisture
content than its regular counterpart (Simard, 1991). This is
an important feature as the role of water in the cheese is
quite broad: it influences enzymatic activity and enhances
protein hydrolysis, and may react chemically with the fat or
casein, and therefore affects the rheological properties of
the cheese. Some water is bound to protein, but most of it
occupies the space between the fat and the casein, acting as
a lubricant.between their surfaces (Prentice, 1992). The major
structural component of a cheese is a continuous protein
matrix, which is gradually broken down during ripening. This
breakdown is important.t0wcheese texture: as each peptide bond
is cleaved, two new ionic groups are generated and each of
them will compete for the available water in the system
(Creamer and Olson, 1982) which is gradually bound to the
newly formed peptides and amino acids. Proteolysis by rennet
is believed to be responsible for the softening of cheese
'texture early during ripening via the hydrolysis of afl-casein
(Fox, 1988; Noomen, 1983), a task which is accomplished later
by peptidases and aminopeptidases from the starter. Therefore,
the choice of starters plays a major role in determining the
final characteristics of cheese. As the development of off-

flavors (meaty-brothy, bitter and unclean) is a common problem

95

in reduced fat Cheddar cheese, the use of adjunct cultures
with higher peptidolytic activities has been extensively
studied. Live or heat-shocked cells of Lactobacillus casei
have been a main source of peptidolytic enzymes for Cheddar
cheese trials (Laleye, 1989; Simard, 1991; Trepanier et al.
1992), due to its recognized ability to promote extensive
peptides degradation during cheese ripening.

The common textural and off-flavors problems of reduced
fat cheese could be approached by the evaluation of changes in
the cheese moisture and enhancement of peptidolytic activity
during ripening. The objectives of the present work were to
evaluate the effects of moisture content and the use of
different levels of adjunct cultures of live and heat-shocked
Lactobacillus casei on the textural and rheological properties
and flavor characteristics of reduced fat Cheddar cheese, as
well as to relate the rheological information to the

degradation of afl-casein during cheese ripening.

MATERIALS AND METHODS

Rheological Measurements

Cheeses 4, 8, 12, and 16 weeks of age were analyzed for
texture properties by the Instron Universal Testing Machine,
model 1102, following the procedure described by Chen, A.H.

et al. (1979), with modifications. Four textural

96
characteristics, hardness, cohesiveness, adhesiveness, and

elasticity were measured.

Sample preparation

Cheese samples were kept in plastic bags at room
temperature overnight and cut in cubes 2.54 cm (height) x 2.54
cm (width) x 3.5 cm (length), immediately prior to the test.

The test was performed at a constant temperature of 22%L

Setting of the Instpon
An Instron Compression Load Cell (500 N), fixed to the

crosshead, was used to :make the compression tests. The
parameters hardness, cohesiveness and adhesiveness were based
on force-displacement measurements with the use of a plunger.
A plunger (0.6 cm diameter) was attached to the moving
crosshead, which was set to cycle mode at a constant speed of
2.5 cm/min in both upward and downward directions. A flat
holding plate was used to keep the cheese sample held on the
stationary horizontal bedplate of the machine, avoiding
sticking of the cheese on the plunger in its upward movement.
The penetration of the plunger into the cheese sample was set
to 1.91 cm, which corresponded to 75% of the height of the
sample.

A.bite was considered.as alcomplete cycle of one downward
and one upward movement of the plunger, which penetrated and

then was removed from the sample. To perform the tests, 2

97
bites were taken. The chart speed was set to 5 cm/min. A
typical texture profile analysis (TPA) curve obtained by the

Instron Universal Testing Machine is shown in Figure II-l.

Determination of the parameters

The parameters hardness, cohesiveness, and adhesiveness
were defined and determined from the TPA curves, according to
formulas described. by Friedman. et al. (1963). Hardness,
defined as the maximum force exerted on the sample, was
measured from the TPA as the height of the first bite.
Cohesiveness was measured as the ratio of the area, in
arbitrary units, under the second peak and the area under the
first peak (AZ/A1) . Adhesiveness was measured as the area (A3) ,
in arbitrary units, of the negative peak beneath the base line
of the TPA curve, which represented the work necessary to
remove the plunger from the cheese sample.

Areas under the peaks were calculated by using the cut
and weigh method. The chart paper was photocopied using the
same type of paper to reduce errors due to variations in.paper
density. The peaks were cut and weighed in a digital
analytical scale. The individual peak weights were recorded
and then converted to area units.

The parameter springiness was calculated using the same
settings. described. previously, except ‘that. the ‘test. was
performed in.a single compression step, with a flat plate with

4 cm diameter. In this procedure, springiness was expressed

98

 

 

 

P—l9.1cm .“s 19.1cm—+—19.1cm—~i

 

I
f; iFirst Bite Downward
B T Second Bite Downward
a:
2

A1 Hardness
i A’
0 ~—
A, DISTANCE

m . .
a First Bite Upward
O
u.

v

 

 

 

Figure ll -1. Typical texture profile analysis (T PA) curve obtained by the Instron Universal
Testing Machine.

99
as the ratio of the sample heights before and after the
compression. An illustration of how springiness was calculated

is shown in Figure II-2.

Organoleptic Assessment

The organoleptic evaluation of the cheese was carried out
by a trained taste panel of 13 members, including faculty and
graduate students from the Department of Food Science and
Human Nutrition. Cheeses were sampled at 32 weeks of age,
coded with three-digit numbers and four samples of each were
presented simultaneously to the panelists.

Four textural characteristics, hardness, cohesiveness,
adhesiveness, springiness were evaluated. A balanced Hedonic
Scale of 1-9 was used for rating each textural attribute. Two
flavor characteristics were evaluated: Cheddar cheese flavor
intensity and bitterness. A diluted quinine sulfate solution
and Cheddar cheeses with different degrees of bitterness were
submitted to the panelists during the training sessions for
characterization of bitter flavor. A balanced Hedonic Scale of
1-6 was used for rating the flavor intensity and bitterness
attributes. A scoreaof’6‘was attributed to a sample exhibiting
"very strong" flavor and a score of 1 to one exhibiting "not
observed" flavor. Analysis of variance and contrasts among
treatments means at the 95% level were performed using the
MSTATC computer program (Crops and Soil Science, Michigan

State University, MI). All sample forms of questionnaire

100

 

FLAT PLATE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cheese L Cheese
Sample Sample,

 

 

 

 

 

 

 

 

 

 

 

 

 

BEFORE COMPRESSION AFTER COMPRESSION

L

 

Springiness =

 

 

 

Figure ll - 2. Textural definition of springiness.
Source: Chen et. al. (1979)

101
presented to the panelists during training and judgement

sessions are in the Appendix II (Figures II-l thru II-12).

RESULTS AND DISCUSSION

Rheological Evaluation

As previously reported in Chapter 1, only the results of
the highest level of addition of adjunct culture of
Lactobacillus casei L2A (1.5%) will be discussed. Complete
data set for' adhesiveness, cohesiveness, springiness and
hardness are listed in the Appendix section, under Tables II-

1, II-2, II-3 and II-4, respectively.

Agpgsiyepess

Adhesiveness is defined as the work required to overcome
the attraction forces between the surface of the cheese and
the surface of the compression plate, as the plunger begins
its upstroke (van-Vliet, 1991; Zoon, 1991). The adhesiveness
of all cheeses decreased during ripening. No differences
(p<0.05) were found among the three treatments (Figure II-3).
These results may be related to changes in the structural
matrix of the cheese due to the breakdown observed in (1,,-

casein.

Adhesiveness (N.mm)

102

 

 

 

6 ,
Comet
. HSLC-1.5 #1
LLC-1.5 «3‘91

 

 

 

 

 

 

2.5 l 1 l l
4 8 12 16

Age of Cheeses (weeks)
Figure ll - 3 - Adhesiveness throughout the ripening of reduced fat

Cheddar cheese made with 1.5% of live and heat shocked cells of
Lactobacillus casei L2A.

103

C e iveness

Cohesiveness or crumbliness can be described as the
extent to which a food stuff can be deformed before it
ruptures (Zoon, 1991) or the resistance of a material to
external forces, tending to separate parts of the body (van-
Vliet, 1991). Figure II-4 depicts the results of cohesiveness
throughout ripening. The scores showed variations among
treatments, especially during the first eight weeks of
ripening. As the LLC cheeses had the lowest pH throughout
most of the ripening period, this may have affected the
mineral content of the curd. According to Lawrence et al.
(1983), the mineral content of a cheese serves as an
indication of the extent to which the structure of the casein
sub-micelles have been disrupted. The lower the pH of a
cheese, less calcium is retained in curd, which makes it more
crumbly, a characteristic that does not change much during
ripening. The LLC cheeses, which had the lowest pH (Figure I-
1, Chapter 1), were less cohesive (p<0.05) than both the HSLC-
1.5 and Control cheeses over the first.8 weeks of ripening. No
differences (p<0.05) were found among treatments after 12
weeks of ripening. The lower pH of the LLC-1.5 cheeses may
have made them shorter with fracturing at smaller deformation.
This is in agreement with the findings of Walstra and van-
Vliet (1982) who reported that more acidic cheeses require

less force to fracture.

104

 

 

 

 

 

 

 

 

 

 

0.7 .
Como
HSLC-1.5 #3
' LLC-1.5 $1
0.6 r
8 0.5 -
m
c
a)
.5 ®
a)
r:
o
0
0.2 ' ' 1 I
4 8 12 16

Age of Cheeses (weeks)

Figure II - 4 - Cohesiveness throughout the ripening of reduced fat
Cheddar cheese made with 1.5% of live and heat shocked cells of
Lactobacillus casei L2A.

105

Sppipginess

Springiness is defined as the textural property
manifested by a tendency to recover from a large deformation
after removal of the deforming force (van-Vliet, 1991). No
significant changes were observed during ripening, but the
mean scores attained by the LLC-1.5 cheeses were lower
(p<0.05) than those observed for the other cheeses (Figure
II-S). As discussed earlier, the.pH onLLC-1.5 cheeses was the
lowest from the beginning. This increased acidification
decreased the retention of calcium in the curd, which is a
critical factor in cheese texture. Springiness decreases
during ripening as the original rubbery texture of fresh
cheese is gradually changed to a smoother body, due to a“-

casein breakdown.

a ss

Hardness is defined as the force necessary to attain a
given deformation (van-Vliet, 1991) . The hardness of all
treatments decreased rapidly until eight weeks of ripening and
then the process slowed down (Figure II-6). Hardness of the
Control and HSLC-1.5 treatments were lower (p<0.05) than those
of the LLC treatment after 16 weeks of ripening. Again, this
may be related to the lower pH of cheeses made with live cells
of Lactobacillus casei L2A. The lactic acid produced probably
removed calcium from the paracaseinate, in the form of calcium

lactate, which is soluble in water and was lost in the whey.

Springiness (%)

106

 

1OO

 

 

 

90 *- ucrs $1

80—

 

 

 

 

50-

40-

30—

20*-

10“-

 

 

 

0 1 l l 1
4 8 12 16

Age of Cheeses (weeks)
Figure ll - 5 - Springiness throughout the ripening of reduced fat

Cheddar cheese made with 1.5% of live and heat shocked cells of
Lactobacillus casei L2A

Hardness (N)

22

20

18

..s
O)

..a
.h.

..a
N

10

107

 

 

HSLC-1.5 m
LLC-1.5 081

 

 

 

 

 

 

/
r
l 1 l l
4 3 12 16

Age of Cheeses (weeks)

Figure ll - 6 - Hardness throughout the ripening of reduced fat Cheddar
cheese made with 1.5% of live and heat shocked cells of Lactobacillus
casei L2A.

108
Therefore, these cheeses had a more demineralized casein
backbone, which rendered the cheese structure less pliable and
firmer. This is supported by Walstra and van Vliet (1982) who
also reported increased firmness in cheeses with low pH
values.

According to Lin et al. (1987), hardness is the most
important parameter of cheese texture with respect to consumer
preference and sensory perception. Moisture plays a decisive
role in the softening of cheese texture by enhancing protein
hydrolysis. All cheeses from these experiments presented
moisture content around 49.65%, which is considerably higher
than the values of traditional Cheddar cheese. The breakdown
products of casein are largely water soluble and cannot
contribute to the protein matrix. The electrophoretic results
have shown a gradual degradation of au-casein with a
correspondent increase in TCA-N throughout ripening. These
events seem to correlate with the observed decrease in cheese
hardness, as found by other authors (Amantea et al., 1986;
Bertola et al., 1992; Creamer and Olson, 1982; Lin et al.,
1987; Walstra.and van-Vliet, 1982). These results suggest.that
in the making of reduced fat Cheddar cheese, the degree of
proteolysis is of utmost importance as it can affect hardness
and.partially overcome the lack.of calciumiin the structure of
more acidic cheeses. The contribution of water is important,
due not only to its physical role, but also because the

presence of a greater amount of water facilitates the access

109
of microorganisms and peptidolytic enzymes to the casein

(Prentice, 1992) stimulating the phenomenon of proteolysis.

Sensory Evaluation

Textural characteristics

The seven experimental reduced fat Cheddar cheeses, all
fully ripened (eight months) were evaluated by a trained
sensory panel. The results of four textural characteristics
(adhesiveness, cohesiveness, springiness and hardness) are
shown in Figure II-7 (A, B, C and D, respectively). The
textural characteristics of all cheeses, as scored by the
sensory evaluation panel are displayed in Table II-5 (Appendix
II).

Adhesiveness in sensory evaluation is defined as the
force necessary to remove the food sample that adheres to the
mouth surface during the eating process (Chen et al., 1979).
The scores (Figure II-7A) for the HSLC at the 0.5 and 1.0%
levels of addition of adjunct culture, were slightly lower
than those of HSLC-1.5 and the Control and LLC cheeses, but no
differences (p<0.05) were found among all treatments. The
average score of all treatments (4.9) indicate that cheeses
were moderately adhesive.

Figure II-7B shows the scores of cohesiveness, which is
defined as the degree to which the cheese sample deforms
before rupturing (Chen et al., 1979). There were no

differences (p<0.05) among levels of addition of adjunct

110

 

Adhesiveness (A) Cohesiveness (B)

  
  

 

Score
0

 

17““ 0'“ Treatment

Springiness (C)

)9. l 4,“ guns.

   

1 2 3 4 5 0

Treelment neeenent

 

 

 

Figure ll - 7 - Textural characteristics of reduced fat Cheddar cheese made with adjunct culture of
Lactobacillus casei L2A, as scored by the sensory evaluation panel. 1 = Control; 2, 3, 4 = LLC at
0.5, 1.0 and 1.5% of addition, respectively; 5, 6, 7 = HSLC at 0.5, 1.0 and 1.5% of addition,
respectively.

111
cultures in the HSLC trial nor between the latter and the
Control cheeses, but both have shown cohesiveness scores
higher (p<0.05) than those of all three LLC cheeses. The
average score for the HSLC cheese was 4.8 which means a
moderately cohesive cheese. Cohesiveness is related to
crumbliness which means the tendency of a cheese to break down
into small, irregular particles, that was a clear
characteristic of LLC cheeses. As discussed earlier, these
cheeses were more acidic and had a weaker structure due to a
possible lower calcium content.

Springiness is understood as the rate at which the cheese
returns to its undeformed condition after the compression
force is removed (van-Vliet, 1991 ; Zoon, 1991) . The results of
the evaluation of springiness are shown in Figure II-7C.
Again, there were no differences (p<0.05) among the levels of
addition of adjunct culture of Lactobacillus casei LZA in the
HSLC experiment nor between the latter and the Control cheese,
but both have presented springiness scores higher (p<0.05)
than those observed for the LLC cheeses. These results are
probably related to the reduced degree of mineralization
commonly found in more acidic cheeses, as it is the case of
the LLC cheeses. These cheeses were made with the help of live
Lactobacillus casei cells which kept their normal fermentation
ability in addition to that of regular mesophile starter used
in the manufacturing process. As a result, the pH of the LLC

cheeses throughout ripening was lower than in both Control and

112
HSLC cheeses. The LLC cheeses springiness average score was
3.1, which rated them in between a moderately to a not springy
cheese.

In Figure II-7D, the average scores of hardness detected
by the panel, are shown. Hardness in sensory applications is
defined as the force required to penetrate the cheese with the
teeth or to compress it between the molar teeth (van-Vliet,
1991; Zoon, 1991) . No differences (p<0.05) in hardness existed

among the treatments after eight months of ripening.

Opgapoleptic Assessment

Scores resulted from the evaluation of Cheddar cheese
flavor intensity and bitterness are shown in Figure II-8 (A
and B, respectively) and II-6 (Appendix II). A traditionally
made full fat Cheddar, aged for eight months, was used as a
reference for the sensory evaluation. Figure II-8A, shows the
scores for Cheddar cheese flavor intensity, as given by the
trained panel. No differences (p<0.05) were detected among the
Control, LLC or HSLC treatments, but panelists found more
(p<0.05) Cheddar cheese flavor intensity in the full fat
sample (score of 3.7, rated as "distinct Cheddar cheese
flavor"). All the other cheeses presented an average score of
about 2.6 correspondent to a rating of "moderate Cheddar
cheese flavor." These results should be expected, as the
cheeses were made with a substantial reduction in their fat

content. It is widely accepted in the pertinent literature

113

 

Flavor Intensity (A)

 

Score

 

 

 

 

 

Treatment

 

 

 

Figure ll - 8 - Organoleptic characteristics of reduced fat Cheddar cheese made with
adjunct culture of Lactobacillus casei L2A, as scored by the sensory evaluation panel. 1 =
Control; 2, 3, 4 = LLC at 0.5, 1.0 and 1.5% of addition, respectively, 5. 6, 7 = HSLC at 0.5,
1.0 and 1.5% of addition, respectively; 8 = full fat Cheddar cheese.

114
(Banks et al., 1989; Jameson, 1990) that reduced fat cheeses,
despite being well accepted by diet conscious consumers, do
lack some distinct Cheddar cheese flavor.

Figure II-8B shows the scores for bitterness. Again, a
full fat Cheddar cheese was taken as a reference for the
panelists. Cheddar cheese made with 1.5% added live cells of
Lactobacillus casei and the full fat cheese had scores higher
(p<0.05) than the other cheeses. The results showed decreasing
bitterness perception when increasing the amount of heat
shocked cells of Lactobacillus casei were added to the milk.
The lowest score was 1.85 (HSLC-1.5), rated as "slightly
bitter cheese" as opposed to the highest score attributed to
LLC-1.5 treatment, which corresponded to a "somewhat bitter
cheese." These results confirm the debittering effect of
intracellular aminopeptidases of Lactobacillus casei (Laleye,
1989; Simard, 1991; Trepanier et al., 1992), which are able to
hydrolyse bitter peptides before they accumulate to a critical
level in cheese. Cheese made with the addition of live cells
of Lactobacillus casei scored poorly for bitterness, possibly
due to the low pH observed in the manufacturing and ripening
process. The low pH, which favors the proteolytic activity of
rennet enzymes, may have caused an increase in the production
of bitter’ peptides. The low' pH also inhibits peptidase
activity of the starter cultures allowing accumulation of
bitter peptides. These bitter peptides are broken down to non-

bitter compounds by Lactobacillus casei aminopeptidases,

115
dipeptidases and carboxypeptidases with an optima pH of 6.5,
7.6 and 7.2, respectively (Simard, 1991). When Lactobacillus
casei is heat shocked, the disrupted cells liberate these
enzymes which become entrapped in the cheese curd and

intensify the peptidolytic activity during ripening.

116

CONCLUSIONS

Reduced fat cheese with moderate Cheddar flavor was
attained. The results indicated that the level of addition of
adjunct culture used did.not influence the cheese texture. The
addition of live cells of Lactobacillus casei LZA brought
acidifying activity to the cheese, resulting in increased
solubilization of the calcium-phosphate complex. The altered
salt balance made the cheese harder, less cohesive and less

springy than the other cheeses.

Chapter 3

High Performance Liquid Chromatography of Peptides and Amino
Acids in Reduced Fat Cheddar Cheese With Added Lactobacillus

casei L2A.

INTRODUCTION

Degradation of casein during cheese ripening results in
the formation of a large variety of flavor compounds including
the bitter tasting peptides. The manifestation of bitterness
in cheese involves the accumulation of bitter peptides to an
extent that exceeds the flavor threshold. It is generally
assumed that bitter flavor is due to a disproportion between
the formation of bitter peptides by the proteases of rennet
and starters and their breakdown to smaller, non bitter
compounds, by aminopeptidases secreted by the starter bacteria
(Stadhouders and.Hup, 1975). These bitter peptides have a high
proportion of hydrophobic amino acid residues in their
structure and a molecular weight in the range of 1,000 to
12,000 daltons (Kamaly and Marth, 1989; Lemieux and Simard,
1992) . Due to its particular aminopeptidase system,

Lactobacillus casei is believed to exhibit debittering

117

118

activity (Habibi-Najafi and Lee, 1994) which enables that
microorganism to be used as an adjunct culture to reduce
bitterness in cheese. In the making of reduced fat Cheddar
cheese, the development of off-flavors, like bitter or meaty-
brothy, is a common problem, which has been approached by the
use of adjunct cultures possessing high peptidolytic activity
like the heat shocked cells of Lactobacilus casei (Simard,
1991; Trepanier’ et al., 1992). When. disrupted cells of
Lactobacillus casei are entrapped in the cheese curd,
aminopeptidases are easily released into the cheese matrix,
enhancing the degradation of polypeptides, many of them
bitter-tasting compounds.

Reverse-phase high.performance liquid chromatography for
profile studies of peptides produced during ripening of
Cheddar cheese was initially proposed by Cliffe et al. (1989)
and has been the method of choice for fractionation of bitter
peptides. Since bitter peptides are relatively rich in
hydrophobic amino acids, they exhibit higher retention on a
reverse phase system compared to more hydrophilic peptides.
Different fractions collected from the HPLC column can be
hydrolyzed and submitted to amino acid analysis which will
provide more information on the nature and amino acid
composition of those peptides. This work was undertaken to
evaluate the development of bitterness in reduced fat Cheddar
cheese made with live and heat shocked cells of Lactobacillus

casei and to study' the use of Ihigh performance liquid

In

(n

81

119
chromatography as a tool to characterize the main peptide

fractions involved in the formation of bitter taste.

NATERIALS AND NETNODS

Organoleptic Assessment

The organoleptic evaluation of the cheese was carried out
by a trained taste panel of 5 members, including faculty and
graduate students from the Department of Food Science and
Human Nutrition. Slurries (66% moisture) of freeze-dried
cheeses aged 4, 8, 12, 16 and 20 weeks were prepared, coded
with three-digit numbers and four replications of each were
presented simultaneously to the panelists. The temperature of
the samples during the sensory evaluation was 7%:.

A balanced Hedonic Scale of 1-10 was used for rating the
cheese for bitterness. A score of 10 was attributed to a
sample exhibiting "very strong" bitter flavor and a score of
one indicating "not observed" bitter flavor. A diluted quinine
sulfate solution and Cheddar cheeses with different degrees of
bitterness were submitted to the panelists during the training
sessions for characterization of bitter flavor. Analysis of
variance and contrasts among treatment means were performed
using the MSTATC computer program (Crops and Soil Science,
Michigan State University, MI). Sample forms of questionnaire
presented to the panelists during training and judgement

sessions are in the Appendix III (Figures III-1 and III-2).

120

Peptide Extraction

The peptides were extracted from the cheeses by the
Chloroform-methanol extraction method of Harwalkar and Elliott
(1971) as illustrated in Figure III-1, which extracts the more
hydrophobic peptides of cheese. The freeze-dried cheese (55
grams) was blended for five minutes in 250 ml of 2:1
chloroform-methanol solution. The mixture was filtered
(Whatman #4) under vacuum. The precipitate was re-extracted
twice with 100 ml of chloroform-methanol solution and all the
filtrates were pooled. Water equivalent to 20% of the pooled
filtrate volume was added to form a biphasic mixture in a
separatory funnel. After 12 hours, the bottom chloroform layer
containing mostly lipids was discarded. The top aqueous-
methanolic layer containing the peptides was filtered (Whatman
# 4). The white precipitate formed at the interface of the two
layers was discarded and the aqueous-methanolic phase was
collected, rotary evaporated to remove the methanol,
centrifuged and the clear supernatant containing the peptides

was freeze-dried for HPLC analysis.

Peptide Analysis by High-Performance Liquid Chromatography

§gpp1g Bpepgpgtion fog HPLC

The freeze-dried samples of the chloroform-methanol
extract from the cheeses were dissolved at 4 mg/ml in HPLC

grade water and filtered through a 0.45 pm Millipore filter

121

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cheese
l
Shredded and
freeze-dried
i
Blend in chloroform-methanol
(2:1 v/v)
Filter
I
F1
Residue Filtrate 1 a Add 0.2 volumes
1 of water
Blended in Separatory funnel
chloroform-methanol overnight
1 1
Filter Lower chloroform ilpper aqueous
layer containing methanolic layer
lipids
i i
Residue Filtrate 2 __).. White precipitate
l at interface
ii
Blended in Remove methanol
chloroform-methanol 1
Filter Centrifuge
10,000 g
i v i
Residue Filtrate 3 , White Clear
precipitate supernatant
Freeee dry

 

Figure Ill -1 - Extraction procedure used to separate the hydrophobic peptide extract

from reduced fat Cheddar cheese.

Adapted from Harwalkar and Elliott (1971)

 

122
prior to injection on the column. The samples were prepared
immediately before the runs to reduce any hydrolysis of the

peptides.

Splvents

All solvents used during the experiment were HPLC grade.
Water and acetonitrile were degassed for at least 30 minutes
and. 1.5 minutes, respectively. The solvents were mixed in the
required volumes, degassed for 1 min and then equilibrated to

room temperature.

P a sis

Chromatography was performed on a Waters liquid
chromatograph (Millipore Corporation, Milford, MA), consisting
of two 1M-6000 solvent. delivery units, an. M-660 Solvent
Programmer and a Rheodyne injector Model 7125 (Rheodyne,
Cotati, CA) coupled to a Waters 486 Tunable Absorbance
Detector (Millipore Corporation, Milford, MA) interfaced with
the SRI 8600-2000 Peaksimple II data system (SRI Instruments,
Torrance, CA).

All separations were carried out on a 3.9 x 150 mm, 5 pm.
100 A° reverse phase Delta-Pak column (Millipore Corporation,
Milford, MA), which was protected by a 1 mm ID x 2 cm guard-
column (Upchurch Scientific, Inc., Oak Harbor, WA), packed

with a reverse phase Perisorb RP-18, 30-40 pm (Upchurch

123
Scientific, Oak Harbor, Wa) . The detector was set at a
wavelength of 214 nm.

The peptide extract, filtered through a 0.45 um
Millipore filter, was injected into the 200 pl loading loop
and then onto the column. Eight tenths mg of peptide extract
was loaded onto the column for each analytical separation. A
linear gradient system was used to elute the peptides from the
column. Eluent A consisted of 0.1% trifluoroacetic acid in
water and eluent B consisted of 0.1% trifluoroacetic acid in
acetonitrile-water (90:10). The gradient of B increased from
10 to 45% over 93 minutes. A solvent flow rate of 1.0 ml per
ml was used. All HPLC runs were performed at room temperature
(22°C) .

The chromatogram was divided into four fractions, with
the purpose of separating more efficiently the eluted peptides
according to their expected hydrophobicity. Four injections
were made in order to collect sufficient material for amino
acid analysis. The cuts were assigned based on the
chromatographic area and upon the presence of certain
characteristic peaks, which allowed a more efficient
separation of the eluted peptides.

A solvent gradient of 10%-100% B over 10 minutes with 10
minutes holding’ at ‘the final solvent ‘volume ratio *were
performed between runs. This was designed to remove the last
traces of the sample from the column and re-equilibrate with

the initial aqueous solvent.

124

Amino Acid.Analysis by High-Performance Liquid Chromatography

The four peptide fractions collected from the Delta—Pak
reverse phase column were hydrolysed in 6 N HCl for 12 h prior
to derivatization. Determination of amino acid composition of
the peptide fractions was carried.out by HPLC, by derivatizing
the amino acids using phenyl-isothiocianate (PITC) to produce
phenyl-thiocarbamyl amino acid derivative (PIC-AA). The
derivatization reaction was performed as described in the Pico
Tag TM Amino Acid Analysis System Operators Manual (Waters
Chromatography Division, Millipore Corp. , Manual # 88140,
Milford, MA).

The chromatograph assembly (Waters Chromatography
Division, Millipore Corp. , Milford, MA) consisted of two
Waters 510 pumps, a 720 System Controller, a 730 Data Module,
a 440 Detector and a WISP-712 Injector. This equipment was
fitted with a 15 cm Waters Pico-Tag AA Hydrolysates Reverse-
Phase column. A binary gradient, as described in the manual
above, was used to elute the amino acids from the column. The
eluate system consisted of solvent A, 0.1 M NaAc/TEA pH 6.3
and solvent B, 60% acetonitrile/water. A flow rate of 1 ml/min
was used. The temperature was kept at 38°C during elution. A
sample volume of 15 ul of amino acid hydrolysate was injected

and the PIC-AA derivatives were detected at 254 nm.

125

However, as this work dealt with ripened Cheddar cheese,
one should expect a high content of free amino acids in the
peptide extract, resulting from extensive degradation of
casein. The presence of those amino acids would thus interfere
with the results of the amino acid composition of the peptides
eluted from the Delta-Pak column, leading to values higher
than expected. Previous work (Champion and Stanley, 1982) , has
shown that the free amino acids elute in the very beginning of
the chromatogram. Therefore, to identify and determine the
retention time of these free amino acids, a pool of amino acid
standards were injected in the Delta-Pak reverse phase column,
under the same conditions applied for the cheese peptide
extract. Four fractions were collected using the same cutting
time as already mentioned, derivatized with PITC, injected in
the PICO-TAG column, and eluted under 254 nm. This procedure
allowed identification of the fraction containing free amino

acids present in the peptide extract.

RESULTS AND DISCUSSION

For the same reason reported in Chapter 1, only the
results of the highest level of addition of adjunct culture of
Lactobacillus casei L2A (1.5%) will be discussed in this

section.

126

Sensory Evaluation

The scores for bitterness are shown in Figure III-2. The
slurry (66% moisture) from the cheese aged 20 weeks, treated
with live cells of Lactobacillus casei (LLC), was rated
between somewhat and distinctly bitter by the panelists, with
an average score of 4.8, which was higher (p<0.05) than the
scores given to the HSLC and Control treatments, both
considered only slightly bitter (3.30 in both cases). The
bitterness score for the LLC treatment increased gradually as
the cheese aged. As postulated by different authors (Kamaly
and Marth, 1989; Lemieux and Simard, 1992; Stadhouders and
Hup, 1975), bitter taste comes from peptides released mainly
by rennet chymosin, which is stimulated by lower pH. The
bitter flavor would disappear only if sufficient amounts of
starter aminopeptidases are present in the cheese to hydrolyze
the bitter peptides at.a rate faster than they are formed. The
LLC cheese had a low pH throughout most of ripening (Figure I-
1, Chapter 1). The increased rennet activity at lower pH may
have accounted for an increase in the production of low
molecular weight polypeptides, many of them hydrophobic and
potentially bitterness precursors. The LLC cheese was made
with a normal mesophilic culture plus the addition of live
cells of Lactobacillus casei L2A and, therefore, had a higher
starter population density as compared to the Control cheese.
High cell numbers in the curd may contribute to lowering the

pH and to an increased number of bacterial proteases (Law et

Bitterness Score

127

 

 

 

 

 

 

 

 

 

 

 

 

 

W
_I
F
El
r HSLC-1.5 #1
LLC-1.5 ‘91
l J J I l
4 8 12 16 20

Age of Cheeses (weeks)

Figure Ill - 2 - Evolution of bitterness during the ripening of reduced fat
Cheddar cheese made with adjunct culture of Lactobacillus casei L2A,
as determined by trained sensory panel.

128
al., 1992; Stadhouders and Hup, 1975) which contribute to the
initial degradation of aid-casein to medium size peptides, some

of them possibly bitter (Thomas and Pritchard, 1987).

HPLC Analysis of Cheese Peptides

The HPLC chromatograms presented here are representative
of each set of cheeses. The results were highly reproducible
between trials.

As a reference, a HPLC peptide standard mixture
containing five different peptides (Gly-Tyr, Val-Tyr-Val,
Methionine-enkephalin, Leucine-enkephalin and angiotensin II
(Sigma Chemical Co., St. Louis, M0) were run under the same
conditions alone and together with the hydrophobic extract.
The elution pattern of the peptide standard mixture is shown
in Figure III-3. They had a hydrophobicity (Q) of 1,435,
2,083, 1,364, 1,588, and 1,821 cal. res“, respectively, as
calculated by Ney's formula (Guigoz and Solms, 1976). It was
observed that the standard peptides did not elute strictly in
order of increasing hydrophobicity. Molecular weight (MW) may
have played a role also. The peptides apparently eluted in
order of increasing MW. Three of them (A.= gly-tyr, MW 238 da,
Q = 1,435 cal. res“, B = val-tyr-val, MW = 379 da, Q = 2,083
cal. res“, C = methionine-enkephalin, MW = 573 da, Q = 1,364
cal. res“) eluted between 4 and 32 min, in fraction 1, and

the other two (D = Leucine-enkephalin, MW = 555 da, Q = 1,588

129

 

 

 

 

 

 

 

 

 

 

Figure lll - 3. High performance liquid chromatography elution patterns of
standard peptides along with hydrophobic extract of reduced fat Cheddar
cheese. A = eg-tyr, B = val-tyr-val, C = methionine enkephalin, D = leucine
enkephalin and E = angiotensin II.

130

cal. res“, and E = angiotensin II, MW = 1,046 da, Q = 1,821
cal. res“) eluted between. 37 and. 42 'min., in the area
corresponding to fraction two. It is interesting to notice
that when a ratio MW/Q was calculated, with exception for
methionine enkenphalin, the values found increased along with
retention time (MW/Q = 0.17, 0.18, 0.42, 0.35, and 0.57 for
peaks corresponding to gly-tyr, val-tyr-val, methionine
enkenphalin, leucine enkenphalin, and angiotensin II,
respectively, as in Figure III-4). As found by Champion and
Stanley (1982), the HPLC separation of bitter peptides from
Cheddar cheese has shown that, in general, compounds of higher
molecular' weight. eluted. later' despite the. positive
relationship between retention time and hydrophobicity. The
authors suggest that the retention of peptides from the
hydrophobic extract is influenced by a combination of
molecular weight and hydrophobicity factors.

The chromatograms related to the hydrophobic extracts of
cheeses 4 and 20 weeks of age are shown in Figures III-4 and
III-5, respectively. As cheeses aged and casein was broken
down, great numbers of peptides were formed as seen by
comparing the peak areas recorded in both chromatograms. The
peak areas of selected peaks from the chromatograms are listed
in Table III-2 (Appendix III). After 4 weeks of ripening no
marked differences were observed among the peptide profiles of
treatments Control, HSLC-1.5 and LLC-1.5, with the exception

of peak G (Figure III-4). The area corresponding to peak G

131

 

 

(A)

 

 

(B)

 

 

 

 

FR-1 FR-Z FR-3 i FR-4 t

81H
314—»
l

Time (Min)

 

 

Figure lll - 4 - High performance liquid chromatography elution
patterns of hydrophobic extracts of Control (A), HSLC-1.5 (B), and

LLC-1.5 (C) cheeses after 4 weeks of ripening.
FR = Fraction Number

132

 

 

 

(A)

 

 

 

 

 

__-_.__... ...IL—

 

 

 

 

I °- I
I I
I
I i D
l l C

G ' I

I I (B)
I A BI

J.) I l
__ I I _

l“ I l
I I D l
I
l Bl I
I A I' I. I
I I |
I C I (C)

G: I
I

_-.._ I I

FR-1 i FR-Z _$_ FR-3 _¢_ FR-4 ;
35 62 80 95
Time (Min.)

 

 

Figure Ill - 5 - High performance liquid chromatography elution
patterns of hydrophobic extracts of Control (A), HSLC-1.5 (B), and
LLC-1.5 (C) cheeses after 20 weeks of ripening.

FR = Fraction Number.

133
(13.1) for the LLC-1.5 was smaller (p<0.05) than those for the
Control and.HSLC-1.5 cheeses (44.9 and 42.6, respectively). As
this cheese had a lower pH than the other two cheeses, one
could expect a more intense casein breakdown in early ripening
due to rennet proteases, as discussed earlier. After 20 weeks
of ripening, more peaks were detected in the area
corresponding to fraction 1. With the hydrophobic nature of
the reverse phase column, low molecular weight and weakly
hydrophobic peptides should elute first at the UV detector
outlet (Champion and Stanley, 1982). No other relevant
differences were observed between 4 and 20 weeks of ripening
in the chromatograms of the HSLC-1.5 and Control cheese
hydrophobic extracts, except for a slight decrease of peak C
(from 29.5 to 26.8 in the Control and from 35.6 to 33.0 in the
HSLC-1.5) and increase in the area of peak D (from 14.8 to
19.6 in the Control and from 11.3 to 19.4 in the HSLC-1.5), as
the possible results of gradual breakdown of larger
polypeptides to small units of varied hydrophobicity, which
may have accounted for the appearance of several peaks at the
beginning of the chromatograms in the region of fraction 1.
Some marked differences were detectable between the peptide
profiles of LLC-1.5 samples aged for 4 and 20*weeks. There was
a net decrease (p<0.05) in the area of peak C (from 24.2 to
20.7). An increase (p<0.05) in the area of peaks A (from 4.8
to 15.3) and B (from 7.3 to 12.5) was noticed, in the area

corresponding to fraction 2. These results pointed out a

134

possible accumulation of two peptides with molecular weight
and Q values greater than 1046 da and 1821 cal. res“, based on
the retention times of the standard peptides discussed
earlier. These peptides did not accumulate in the Control nor
in the HSLC-1.5 cheeses. It is well known that bitterness is
most intense in hydrophobic medium sized peptides (tri- to
hexa-) (Lemieux and Simard, 1991) with molecular weight of
less than about 1,400 da (Visser, 1977). The peptide side
chain hydrophobicity and, consequently, the total amino acid
hydrophobicity plays the most significant role in creating a
perception of bitter taste. Hydrophobicity from leucine,
phenylalanine, tyrosine, valine, proline, lysine and
tryptophan causes marked peptide bitterness (Lemieux and
Simard, 1991). These amino acids have hydrophobicity Q values
ranging from 1,500 to 3,000 cal.res“, and according to Ney's
rule.(Guigoz and Solms, 1976) all peptides having high.average
hydrophobicities, 1,400 cal.res“ or higher, are bitter in
taste.

As discussed earlier, the LLC cheeses had a more intense
bitter taste (Figure III-2) than the other cheeses. When
comparing the peaks in the area corresponding to fraction 2 of
the chromatograms in Figure III-5, the areas of peaks A (15.3)
and B (12.5) of the LLC-1.5 extract, are much larger (p<0.05)
than their counterparts in the Control (8.8 and 5.6,
respectively) and. HSLC (3.9 and 1.6, respectively). The

peptides with low molecular weight and medium to high

135
hydrophobicity, believed to be the more bitter tasting (Guigoz
and Solms, 1976; Lemieux and Simard, 1991), seem to be
concentrated in fraction 2, especially in the LLC-1.5 cheese.
The higher cell density of this treatment has resulted in the
accumulation of peptides A and B, which may have not been
hydrolyzed quick enough to non-bitter compounds.

The total area under the peaks on the chromatograms is
directly proportional to the number of peptide bonds present
in the hydrophobic extracts of the reduced fat Cheddar cheese.
The calculation of this area can shed some light on the
results concerning the elution profile of the LLC-1.5
hydrophobic extract after 20 weeks of cheese ripening. Figure
III-6 and Table III-1 (Appendix III) illustrates the relative
chromatographic peak area for the four fractions. These areas
represent the average of four injections of the hydrophobic
extract to the HPLC column. No difference (p<0.05) in relative
peak area was noticed in fractions 1, 3, and 4. Fraction 4
presented the highest relative peak area, which is explained
by the presence of high molecular weight peptides in that
region. In fraction 2, the relative peak area of the LLC-1.5
was larger (p<0.05) than those for the other two treatments.
The LLC-1.5 extract showed the highest relative peak area
(20.71) , as opposed to the HSLC-1.5 and Control extracts which
had both similar low relative peak areas (12.77 and 12.13,
respectively). These results suggest that in the LLC-1.5

trial, there was a more intense breakdown of high molecular

 

136

 

 

 

 

 

 

 

 

so 1
D LLC aHSLC DControl
40
E so é
2 §
s 6 ?
.p / /
% zo / / '
o: / /
/ /
/ /
é a
1 O é /
2 Z
/
/ X
o
1 2 3 4

Fraction Number

 

 

Figure Ill - 6 - Relative peak areas of peptide fractions eluted from
the high performance liquid chromatography column.

137
weight peptides (fraction 4), possibly highly hydrophobic, to
low molecular weight bitter tasting peptides of intermediate
hydrophobicity (Fraction 2) . These potentially bitter peptides
may have accumulated in the cheese due to faster formation
than degradation. This Cheddar cheese was more acidic than
the others and it is reasonable to think that a more
proteolytic activity by rennet enzymes at low pH, associated
with an inhibition of the starter proteinases and peptidases,
may have led.to an accumulation of lowwmolecular weight bitter

peptides during ripening.

Cheese Amino Acids

The elution profile of amino acid standards present in
Fraction 1, as they eluted from the Delta-Pak reverse phase
column, is shown in Figure III-7A. The retention times of
standard amino acids (Figure III-7B) and.the fact that no free
amino acids were found in fractions 2, 3 and 4, indicate that
all free amino acids eluted in fraction 1. This confirms the
results of Champion.and.Stanley (1982). Thus, the fractions 2,
3, and 4 collected from the elution of the hydrophobic extract
did not contain free amino acids from cheese ripening.

Figure III-8 (A, B and C) shows the relative distribution
of amino acids in the peptide fractions 2, 3 and 4, at 20
weeks of cheese ripening. According to the Ney's rule (Guigoz

and Solms, 1976), the following amino acids can be listed in

138

 

 

.16

13
ll 15

14

 

 

(A)

 

 

 

 

 

 

 

OJ

 

 

 

 

H
N
.. 3
A (N
-.——""’n '5
C738 .1
’%9
8:39
- 2.45
3433.5 1.
a ‘3
Q I! 21

 

 

l6

9 10 13
12 15

(B)

 

l4

 

 

 

 

ll 54

 

 

611'

58

a.
1.1?
II.

 

 

 

 

 

 

 

Figure Ill - 7 - Chromatographic profiles of amino acids standards. A =
amino acid standards eluted in the first fraction. B = amino acid present
in the standard mixture. 1 = asp, 2 = glu, 3 = Ser, 4 = gly, 5 = his, 6 = arg,

7 = thr, 8 = ala, 9 = pro, 10 = tyr, 11 = val, 12 = ile, 13 = Ieu, 14 = Nor-leu,

15 = phe and 16 = Iys.

139

decreasing order of hydrophobicity: Try (3,000 cal. res“),
Ile, Tyr, Phe, Pro, Leu, Val and Lys (1,500 cal.res“) . In
Figure III-8B, it is seen that the highest concentration of
amino acids originated from the hydrophobic peptide extract of
the cheese trials (fraction 3) were from glutamic acid,
proline and leucine. Leucine was the only one with a high
degree of hydrophobicity. A very similar pattern was observed
in the amino acids from peptides in the fraction four, for all
three cheeses (Figure III-8C).

As expected, based on the significant difference in
relative peak area and peptide profile observed earlier, the
main differences were observed in the distribution of amino
acids related to the peptides collected in fraction 2 (Figure
III-8A). As observed in the other two fractions there was an
important concentration of glutamic acid, proline and leucine
in the extract of all cheese trials. However, in the LLC-1.5
cheese extract, the amount of glutamic acid was lower (p<0.05)
than in the other two extracts. There was a net increase in
the concentration of highly hydrophobic amino acids like
tyrosine, phenylalanine, and lysine in all cheese extracts.
The concentration of phenylalanine and tyrosine in Fraction 2
was higher (p<0.05) in the peptide extract of the LLC-1.5
cheese than in those of the HSLC-1.5 and Control treatment. As
shown in Figure III-2, the former cheese was considered
somewhat or distinctly bitter by the sensory evaluation panel,

as the other two cheeses (HSLC—1.5 and Control) . These results

140

 

 

(A)

 

 

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2, B

de fract'
Fraction

t

In pep
. A=

IpenIng

ds

"10 ac

ion 4.

Fract

Figure Ill - 8 - Relative distribution of am

reduced fat Cheddar cheese at 20 weeks of r

Fraction 3, and C

141
indicate that the development of bitter flavor in reduced fat
Cheddar cheese is related to the presence of hydrophobic
peptides at certain levels. The amino acids estimated to be
involved in bitterness and related in this work, have been
reported elsewhere (Edwards and Kosikowski, 1983) as

precursors of bitter taste in Cheddar cheese.

CONCLUSIONS

The findings of this work.demonstrate that.reversed phase
HPLC is an adequate tool to evaluate and identify the
formation of bitter compounds in reduced fat Cheddar cheese.
It has been shown that peptides with an average hydrophobicity
above 1,400 cal.res“ and a molecular weight higher than 1,046
were involved in the formation of bitter taste, as they were
present in higher concentrations in the cheese which had a
distinct bitter flavor; The pooled fraction pertinent to these
peptides has shown a high concentration of hydrophobic and
potentially bitter amino acids like tyrosine, phenylalanine,

and lysine.

 

M
the
UV
a 1

adj

amo

tha

0f,-

Pro
3&3.

0f

Sig!

SUMMARY AND CONCLUSIONS

All cheeses had a high moisture (49.35%) and a low fat
(14.8%) content. Increasing the inoculation levels (0.5, 1.0
and 1.5%) of adjunct cultures did not seem to interfere with
the ripening characteristics of the cheeses. Cheeses made with
live cells of Lactobacillus casei L2A were more acidic and had
a lower pH throughout ripening. Cheeses made with heat shocked
adjunct cultures of Lactobacillus casei L2A presented the
highest degree of proteolysis as measured by the greatest
amount of pH 4.6 and 12% TCA-soluble nitrogen.

In all cheeses a,,-casein was more extensively broken down
than B-casein. In the cheese treated with heat shocked cells
of Lactobacillus casei L2A, the aminopeptidases liberated from
the disrupted cells seemed to have played an important role in
proteolysis; in this treatment only 21.31% and 70.43% of a“-
casein and B-casein, respectively, were left intact at the end
of ripening. The liberation of nitrogen correlated

significantly with the decrease in the amount of intact (2,,-

casein.

142

e'.
de
cl
L2

br

a1
as

to

co
of
III
to
961
C01
lil
bit
inc
Peg
Con

143

Cheeses made with live cells of Lactobacillus casei L2A
were harder and less cohesive and less springy than the other
cheeses. This was probably related to its low pH and,
eventually, to its low degree of mineralization. Hardness
decreased in all cheeses during ripening, especially in the
cheeses made with heat shocked cells of Lactobacillus casei
L2A. The softening of the cheese texture was related to the
breakdown of the casein matrix during ripening.

A lack of distinct Cheddar cheese flavor was observed in
all cheeses by the sensory evaluation panel; flavor was rated
as "moderate Cheddar cheese f lavor". Bitter flavor was related
to a high concentration of low molecular weight and highly
hydrophobic peptides. These peptides were found in greater
concentration in a pooled fraction of the hydrophobic extract
of cheese made with live cells of Lactobacillus casei L2A,
eluted from the chromatographic column. This cheese was found
to be "somewhat bitter". Chromatographic analysis of this
peptide fraction following derivatization has shown it to be
composed of higher concentration of hydrophobic amino acids
like phenylalanine, tyrosine and lysine than found in non-
bitter HSLC and Control fractions. These amino acids may
increase the potential bitterness of low molecular weight
peptides when present in their structure. It is finally
concluded that the incorporation of heat shocked cells of

Lactobacillus casei L2A to the manufacturing process of

144
reduced fat Cheddar cheese has potential as an approach to

controll the intensity of bitter flavor.

FUTURE RESEARCH

Utilization of the same protocol of this dissertation
using Cheddar cheese with varying degrees of fat and

moisture content;

Increase the levels of heat shocked cells of lactobacilli
added to the cheese milk in the manufacture of reduced

fat Cheddar cheese.

Utilization of proteinase-negative mesophilic lactococci
or thermophilic streptococci and lactobacilli to reduce

the formation of bitter compounds in cheese;

Study of the utilization of non-bitter strains possessing
pyrrolidone carboxylyl peptidase (PCP) activity in the

manufacture of reduced-fat Cheddar cheese;

Study of new natural products (globular proteins)

modified to act as fat-replacers in low fat cheeses (i.e.

Simplesse);

145

146
6. Study of milk indigenous proteinases (plasmin, thrombin,
etc.) in the development, accumulation and degradation of

bitter peptides in cheese;

 

APPENDICES

APPENDIX I

 

147

APPENDIX I

Table I-l - Time schedule for making reduced fat Cheddar cheese
of high moisture content.

 

 

 

 

ACHNTY
OPERATION TIME TEMPERATURE TA pH COMMENTS
(0F)

Add starter 0:00 90 .15 6.62" Marachall‘s Biolac
D55 1.? (1 5) and
i.- aai. (Iditmct)

Add rennet 0:40 90 .16 6.60“ Recombinant
Chmnitflhflflb
kg of Milk

Cut curd 1:15 90 .10 15.55" 1.7 cm him. slow
agitation

Start cooking 1:35 90 .10 6.50" Raise heat slowly

End cooking 1:50 100 .12 6.44’

uflrhnnmgvdmy

Cut 8: 1" Turn 2:05

Milling 6:35 5.6“

Salting 6:50 0.3 kgllm kg milk
3mflmasflhmn
apart.

Hooping 7:20

Start pressing 7:30 9 kg/30 min.

18 kg/ 12 hr.

9 - Whey pH

“-Curdpl'l

 

148

APPENDIX I

Table I-2 — Changes in cheese pH throughout the ripening of
reduced fat Cheddar cheese made with different levels of
ad'unct culture of Lactobacillus casei L2A.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

tpH after 30 minutes pressing.
iMeans with standard deviations in parentheses.
°b°d°Means in same column with different superscripts are
different (p<0.05).
n = 4 replications.

 

—~— ,_ =35 *—-————-—— ——-—-— main—=1

Week 01 Week 4 Week 8 Week 12 Week 16 Week 20
5.382‘ 5.22” 5.19” 5.06” 5.14” 5.20”
(0.04) (0.01) ((0.06) (0.08) (9.00) (0.01)
5.33” 5.19” 5.16” 5.05” 5.13” 5.18”
(0.01) (0.00) (0.08) (0.08) (0.00) (0.08)
5.31” 5.15” 5.12” 5.06” 5.13” 5.17”
(0.02) (0.01) (9.06)) ((0.04) (0.00) (0.06)
5.47” 5.28” 5.26” 5.17” 5.08” 5.14”
(0.02) (0.00) (0.04) (0.04) (0.05) (0.03)
5.45” 5.28” 5.26” 5.17” 5.11” 5.17”
(0.05) ((0.01) (0.01) ((0.03) (0.05) (0.04)
5.56” 5.30” 5.27” 5.15” 5.14” 5.17”
(0.15)f (0.00) (0.03) (0.02) (0.04) (0.03)
5.51” 5.29” 5.26” 5.13” 5.14” 5.17”

_(0.15) (0.00),): (0.01) (0.01) ._ (0.04) (0.01)

 

 

 

149

APPENDIX I

Table I-3 - Soluble Nitrogen (SN) evolution throughout the
ripening of reduced fat Cheddar cheese made with different
levels of adjunct culture of_Lactobacillus casei L2A.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cheese Week 0‘ Week 4 Week 8 Week 12 Week 16 Week 20
Luz-0.5 0.16”” 0.46”” 0.59” 0.68” 0.82” 0.85 ”
(0.01) (0.07) (0.01) (0.06) (0.02) (0.03)
the-1.0 0.17” 0.45”” 0.60” 0.68” 0.82” 0.89 ””
(0.01) (0.07) (0.01) (0.01) (0.01) (0.02)
LLC-1.5 0.19” 0.46”” 0.60” 0.70” 0.79” 0.87 ”
(0.04) (0.06) (0.04) (0.02) (0.03) (0.02)
ism-0.5 0.17” 0.42”” 0.57” 0.68” 0.78” 0.95”
(0.02) (0.01) (0.04) (0.03) (0.02) (0.01)
new-1.0 0.18” 0.40”” 0.59” 0.72” 0.79” 0.96”
(0.02) (0.01) (0.02) (0.02) (0.01) (0.02)
saw-1.5 0.19” 0.38 ” 0.57” 0.73” 0.81” 0.93””
(0.03) (0.01) (0.05) (0.02) (0.03) (0.02)
control 0.17” 0.49” 0.62” 0.72” 0.82” 0.88 ””
(0.02) (0.11) (0.03) (0.02) (0.03) 0.03)

 

 

1dayfollowing manufacture.

2Means with standard deviations in parentheses.

11 = 4 replicates.

abcMeans in same column with different superscripts are
different (p<0.05).

 

.Amo.ovcv

ucoudumao mun mumfluomummsm ucououuwo nufi3 :EsHOO 03cm cw mccozga
.mcowucowammu e n :

.momonucouon :« mcoflyow>oo cucoccuu nuw3 manozu

.oHSPOMMSCME mow3oHHou wooH

150

 

 

 

 

 

 

 

 

$6.3 $6.8 $6.8 $6.3 $28 36.8
. $6 .36 ..36 .36 .36 . oo6 Houucoo
26.3 36.3 26.3 628 86.3 86.3
....»6 .86 .86 .36 . 26 8666 ....Toomz
$6.3 36.8 $6.3 36.8 36.8 86.8
.36 .36 936 .36 o 36 ...86 6.738.
36.3 366V $6.3 36.8 36.3 36.8
I .36 .36 936 .86 ... o~6 . 666 86-32.
m $6.3 36.8 26.3 36.8 66.8 366V
.m. ..36 .36 . $6 .36 336 .36 .1735
E
p 366V 86.3 36.3 36.8 36.8 66.3
n 32.6 .36 ..36 .36 .. 36 8666 6.735
Amo.oc Amo.ov Amo.ov Amo.oe Amo.oe Aao.ov
32.6 .66 836 .36 .. 4.6.6 .336 86-6.3
om some 3 some me some 6 some 4 some .6 some 6.6er

 

 

 

 

 

 

 

 

 

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mao>ma accumuuflo suw3 coca undone uncoonu vow uncanny no moanemfiu
one 0.536535 63326 2sz cocoa»; 53981.82 1 TH canoe

151

APPENDIX I

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mum mumwuomummsm ucmumHMfio saw: canaoo mawm cfi mammza
mcowumoflammu ¢ u c

.m#mxomun :fl m:0wumw>mc oumncmum nuw3 mmuoow cams.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. nm.¢fl 2 No.6” .eo.e aomazoo
.mp.ma .mo.o~ . am.e m.Hnoqmm
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.me.oa .Ho.H~ 2¢m.¢ m.onoqmm
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wmmmo unawflomnouomn.mo musuaso pocsnuw mo mHm>mH ucmumuuwo zufl3,moma
mmmmno umocmzo umu cmozomu no mcficmmwu mo mxm03.o~ umumm xmccH sumac
onwcmmwm can meCH cowwcmuxm onwcmmfim .cmmouuflz Hmuoa u mIH manna

152

APPENDIX I

Table I-6 -List of parameters used to scan the electrophoretic
gels in the Shimadzu Dual-Wavelength Scanner.

 

Photo node Select

signal Proc.
Linearizer
Accum No.
Background correct
Signal Average

Output node Select
Ordinate
Abscissa
Output Select
Drift Line

Peak Detect
Mode
Drift Line
Signal
Sensitivity
Min. Area
Min. Width

Scan Width
"x"
Delta "Y"

Program Scan Parameters
Wavelength l
Wavelength 2
"X" Start Position
"Y" Start Position
"X" End Position
"Y" End Position
Lane Dist.

Total Lane

Abs. Transmission

Off
1

1
16

X8
X1

Curve & Print
On

Area
Med.
10

13
0.20

580

580
Variable
Variable
Variable

Variable

1

Variable

 

153

APPENDIX I

Table I-7 - Degradation of an-casein (peak area) throughout
the ripening of reduced fat Cheddar cheese made with adjunct

Control

 

43.00“
(1.13)

 

24.60‘
(1.41)

 

16.60‘
(0.14)

 

10.95b
(0.64)

 

9.95b

 

6.80“
(0.42)

 

 

 

 

 

 

lMeans (peak area) with standard deviations in parentheses.
n = 2 replications.

‘hueans in same row with different superscripts are different
(p<0.05).

Table I-8 - Degradation of B-casein (peak area) throughout the
ripening of reduced fat Cheddar cheese made with adjunct

154

APPENDIX I

culture of Lactobacillus casei L2A.

 

 

 

 

 

 

 

.s:========.========
Weeks Control HSLC
0 44.05“ 39.90b
4 43.25' 38.40'
3 41.05' 37.75'
12 39.85' 34.70'
16 35.50“ 29.00b
20 34.85b 28.10”

1Means (peak area) with standard deviations in parentheses.

 

n = 2 replications.

“Means in same row with different superscripts are different

(p<0.05).

 

 

 

155

APPENDIX I

 

 

ens-caseins
B-casein

a,-caseins
B-casein 7

at
a
v?

 

n:
u

al—caseins If“... -
. ;. . .4 o e
B-caseln he? mt tn?
5.. .. a

I 1

 

 

Figure l-1 - Electrophoretic patterns throughout the ripening of reduced fat
Cheddar cheese made with adjunct culture of Lactobacillus casei L2A.

APPENDIX II

 

Table II - l - Adhesiveness throughout the ripening of reduced
fat Cheddar cheese made with adjunct culture of Lactobacillus
casei L2A, as measured by the Instron Universal Testing
Machine.

156

APPENDIX II

 

 

 

    

 

 
 

 

 
 

 

 

 

  

 

 
 
 

 

   
 

 

'Mean scores with standard deviations in brackets.

 

 

4 WEEKS 8 WEEKS 12 WEEKS
LLC-0.5 4.29““ 4.27“ 3.24“
(0.76) (0.42) (0.25)
LLC“1.0 3.14 ' 3.37 m 3.48“
(0.93) (0.08) (0.31)
LLC-1.5 4.26“I 4.06“. 3.43“
(0.45) (0.72) (0.46)
HSLC-O e 5 4 e 16” 3 0 88m 2 e 94‘
(0.55) (0.24) (0.13)
HSLC'1.0 4.62“ 2.91 D 3.20“
(0.79) (0.41) (0.22)
HSLC’1.5 5.33“ 3.15 CD 3.34“
(0.25) (0.25) (0.32)
CONTROL 5 41“ 4.12” 3.50“
(0.82) ( 0.18) (0.29)

 

n = 3 replications.

”“Means in same column with different superscripts are

different (p<0.05).

 

 

 

 

Table II - 2 - Cohesiveness throughout the ripening of reduced
fat Cheddar cheese made with adjunct culture of Lactobacillus
casei L2A,
Machine.

 

157

APPENDIX II

as measured by the Instron Universal Testing

 

 

 

 

 

 

 

CHEESE 4 WEEKS
LLC'O.5 0e45l“
HLZO)
LLC‘1.0 0.47““
(0.11)
LLC’1.5 0.31.
(0.0m
HSLC-O . 5 O . 61“
(0.0”
HSLC'1.0 0.54“
“LOS)
HSLC'l. 5 O. 52“
(CAM)
CONTROL 0. 62“
(0.0“

 

'Mean scores with standard deviations in brackets.

 

n = 3 replications.

”‘Means

different (p<0.05).

 

 

 

 

 

in same column with different superscripts

Table II - 3 - Springiness throughout the ripening of reduced
fat Cheddar cheese made with adjunct culture of Lactobacillus
casei L2A,
Machine.

158

APPENDIX II

as measured by the Instron Universal Testing

 

   

 

 
 
 

 

 
 

 

 

 

  

 

  

4 warns
LLC-0.5 0.65"c
(0.03)
LLC-1.0 0.62 c
(0.01)
LLC-1.5 0.62 c
(0.02)
HSLC-0.5 0.69”
(0.03)
Herc-1.0 0.71“
(0.05)
HSLC-1.5 0.72*
(0.06)

 

    
 

CONTROL

'Mean scores with standard

 

n = 4 replications.

”“Means in same column

different (p<0.05).

 

 

deviations in brackets.

with different superscripts are

 

 

 

159

APPENDIX II

Table II - 4 - Hardness throughout the ripening of reduced fat
Cheddar cheese made with adjunct culture of Lactobacillus
casei L2A, as measured by the Instron Universal Testing
Machine.

 

 

 

 

 

 

HSLC31.O

 

HSLC-1.5

 

CONTROL

 

 

 

 

 

‘Mean scores with standard deviations in brackets.

n = 3 replications

“”TMeans in same column ‘with, different superscripts are
different (p<0.05).

160

APPENDIX II

Table II-S - Textural characteristics of reduced fat Cheddar
cheese made with adjunct culture of Lactobacillus casei L2A,

 

 

 

 

 

 

 

 

 

 

 

 

 

as scored by the sensory evaluation panel.

CHEESE annzszvnnnss conEsIvENEss SPRINGINBSS EARDNESS
LLC-0.5 4.968 3.77B 3.10a 5.13“
(0.30) (0.07) (0.07) (0.24)

LLC-1.0 5.00a 3.803 3.13B 4.48“
(0.21) (0.66) (0.77) (0.48)

LLC-1.5 5.10a 3.938 3.123 4.77“
(0.36) (0.58) (0.24) (0.50)

HSLC-0.5 4.62a 4.71“ 4.17“ 4.45“
(0.70) (0.60) (0.39) (0.62)

HSLC-1.0 4.44' 4.73“ 4.21“ 4.63“
(0.81) (0.27) (0.11) (0.71)

HSLC-1.5 4.94B 4.86“ 4.21“ 4.73“
(0.24) (0.24) (0.21) (0.55)

Control 5.158 5.07“ 4.12“ 4.86“
(0.54) (0.27) (0.37) (0.46)

 

 

lMean scores with standard deviations in brackets.

n = 4 replications

“WMeans in same column with different superscripts are
different (p<0.05).

Table II-6 -

161

APPENDIX II

Organoleptic characteristics of reduced fat
Cheddar cheese made with adjunct culture of Lactobacillus
casei L2A, as scored by the sensory evaluation panel.

 

 

 

 

 

 

 

 

 

 

 

 

CHEESE rnavon INTENSITY BITTBRNEBB

Control 2.43Lb 2.18°

(0.14) (0.23)
...-..5 35?31, ?.?33,
LLC- 1 . 0 (20 ‘63:) (20231023)
LLC-1.5 fgffig, €0?:;)
HSLC-0.5 (%Zigz €035?)
HSLC-1.0 f6?§;) fd?§;)
HSLC-1.5 fdfifg) 3033;)
Full Fat 305:2) ?0?Z:)

 

 

lMean scores with standard deviations in brackets.
n = 4 replications
“5°Means in same column ‘with, different superscripts

different (p<0.

05).

are

162

APPENDIX II

CONSENT FOR TASTE PANEL MEMBERS

Food Science and Human Nutrition Department
Michigan State University

Reduced-fat Cheddar cheese prepared from pasteurized low fat
milk, cultures (Lactococcus lactis subsp. c o s and
Lactobgcillug casei), rennet, calcium chloride, salt and
natural color.

I have read the
above list of ingredients and find none that I am allergic to.
I have also been informed on the nature of the research
(including experimental materials and procedures) which will
be used during the tasting session. I understand that the
taste panel will take 5-15 minutes each time. I agree to serve
on the taste panel which will be conducted on

, 1992. I understand that I am
free to withdraw my consent and to discontinue participation
in the panel at any time without penalty.

 

 

 

Signature

 

Date

Figure II-l - Consent form presented to panel members.

 

163

APPENDIX II

 

 

NAME: DATE:
Y E O SAMPLE: Cheese
CflABAQTEBISTIC STUDIED: Adhesiveness
IH§IBUCIIONS

Place sample between molars; chew seven times: press the
sample to the roof of mouth with the tongue. Evaluate the
force required to remove the sample from the roof of the mouth
with tongue. Rate samples from least adhesive to most
adhesive. Expectorate the sample; rinse mouth with water.

 

# 925 # 123 # 187

Least Adhesive

 

 

Most Adhesive

 

Figure II-2 - Sample form of questionnaire presented to the
panel to evaluate adhesiveness of reduced fat
Cheddar cheese during training sessions.

 

164

APPENDIX I I

 

 

NAME: DATE:
TXPE OE SAMPLE: Cheese

C CT 8 C STUDIED: Cohesiveness
IESTBUCIIOHS

Place sample between molars; compress fully; evaluate the
degree to which the sample deforms rather that crumbles,
breaks, or falls apart as cohesive. Rank the samples from
least cohesive to most cohesive. Expectorate the sample: rinse
mouth with water between samples.

# 963 # 789 f 435

Least Cohesive

 

 

Most Cohesive

 

Figure II-3 - Sample form of questionnaire presented to the
panel to evaluate cohesiveness of reduced fat
Cheddar cheese during training sessions.

165

APPENDIX II

 

 

NAME: DATE:
Y E OF SAMPLE: Cheese
CMAAAQIEBISIIC STUDIED: Springiness

I UC ONS

Place sample between molars; compress partially without
breaking the sample structure; release. Evaluate the degree to
which the sample returns.toiits original shape as springiness.
Rate the samples from least springy to most springy.
Expectorate the sample; rinse mouth with water.

# 354 # 123 # 858

Least Springy

 

 

Most Springy

 

Figure II-4 - Sample form of questionnaire presented to the
panel to evaluate springiness of reduced fat
Cheddar cheese during training sessions.

 

011“. M.‘— C— I— P h t r

166

APPENDIX II

MAME: DATE:
112E OE SAMPLE: Cheese
CMAAACTEAISTIC STUDIED: Hardness

 

IMSTBUCIIOMS

Place sample between molars; bite through once; evaluate for
hardness. Rank.the samples from least hard to most hard. Taste
the samples in the following order. Expectorate the sample;
rinse mouth with water.

# 925 # 789 # 187

Least hard

 

 

Most hard

 

Figure II-S - Sample form of questionnaire presented to the
panel to evaluate hardness of reduced fat
Cheddar cheese during training sessions.

167

APPENDIX II

NAME: DATE:
OF SAMP E: Cheese

QMABACTERISTIC STUDIED: Bitterness

 

INSTRUCTIONS

"Bitterness is somewhat distasteful, resembling the taste of
quinine or caffeine. The bitter sensation is somewhat delayed
in terms of its initial perception and tends to persist for
sometime after sample expectoration. Bitterness is observed by
a taste sensation that occurs at the base of the tongue"

Place sample into the mouth and chew it until a semiliquid
stage is reached. Roll the macerated sample in the mouth for
sufficient time to determine both taste and aroma. Rank the
sample from non bitter to most bitter. Expectorate the sample;
rinse mouth with water between samples.

Sample # 425 Sample # 421 Sample # 915

Non bitter
Moderately bitter
Most bitter

 

 

 

QQHMENI§=

Figure II-6 - Sample form of questionnaire presented to the
panel to evaluate bitterness of reduced fat
Cheddar cheese during training sessions.

N.

If“! It"!

Sa

CO]

168

APPENDIX II

NAME: DATE:

 

 

TXPE OP SAMPLE: Cheese
CHARACTERISTIC STUDIED: Cheddar Cheese Flavor Intensity

INSIRQCIIONS

"High-quality Cheddar cheese should possess the characteristic
Cheddar flavor, which is best described as clean, moderately
aromatic, nutty-like, and pleasantly acidic".

Place sample into the mouth and chew it until a semiliquid
stage is reached. Roll the macerated sample in the mouth for
sufficient time to determine both taste and aroma. Rank the
sample from imperceptible to extremely perceptible.
Expectorate the sample; rinse mouth with water between
samples.

Sample # 354 Sample # 789 Sample # 345

Imperceptible
Moderately perceptible
Extremely perceptible

 

 

 

COMMENTS:

Figure II-7 - Sample form of questionnaire presented to the
panel to evaluate cheese flavor intensity of
reduced fat Cheddar cheese during training
sessions.

if.“ I

 

 

169

APPENDIX II

 

 

NAME: DATE:
TYPE OF SAMPLE: Cheese
CMAPACTERISTIC STUDIED: Adhesiveness
IMSIRUCTIONS

Place sample between molars; chew seven times: press the
sample to the roof of mouth with the tongue. Evaluate the
force required to remove the sample from the roof of the mouth
with tongue. Place an.X next to the value which bestidescribes
the adhesiveness of the sample. Expectorate the sample; rinse
mouth with water.

Sample # 621

Not Adhesive

Moderately Adhesive

\Dmdmmbwwl‘

VePy Adhesive

COMM NTS:

Figure II-8 - Sample form of questionnaire presented to the
panel to evaluate adhesiveness of reduced fat
Cheddar cheese.

170

APPENDIX II

NAME: DATE:

 

 

IXPE QF SAMPLE: Cheese
QMAPACIEPLSTIC STUDIED: Cohesiveness

INSTRUCTIONS

Place sample between molars; compress fully: evaluate the
degree to which the sample deforms rather than crumbles,
breaks, or falls apart as cohesive. Place an X next to the
value which best describes the cohesiveness of the sample.

Note: When evaluating cohesiveness, remember, if the sample
falls apart easily, the sample is not cohesive and should get
a lower score.

Sample # 425

Mm

Modeiately Cohesive

\oooqasmeutoi-I

E.

0 EN S:

Figure II-9 - Sample form of questionnaire presented to the
panel to evaluate cohesiveness of reduced
fat Cheddar cheese.

 

171

APPENDIX II

 

 

NAME: DATE:
0 SAMPLE: Cheese
CHARACTERISTIC STUDIED: Springiness
T UCTIONS

Place sample between molars; compress partially without
breaking the sample structure. Place an X next to the value
which best describes the springiness of the sample.
Expectorate the sample: rinse mouth with water.

 

Sample # 852

_IE<2t_§p_£insx
____2

__3

_4
_5Wim
__6

__7

__8

_____ 9 Ve S ri

CO ENTS:

Figure II-10 - Sample form of questionnaire presented to the
panel to evaluate springiness of reduced fat
cheddar cheese.

‘I

172

APPENDIX II

 

 

NAME: DATE:
TXEE_QE_§AEELE: ChOOBO
QEAEACTEEISIIC STUDIED: Hardness
INSTPUQTIONS

Place sample between molars; bite through once; evaluate for
hardness. Place an X next to the value which best describes
the hardness of the sample. Expectorate the sample; rinse
mouth with water.

Sample # 642

Not Hard

Moderately Hard

\OmQChUI-thD-t

Ve Ha

QQEMEHI§=

Figure II-ll - Sample form of questionnaire presented to the
panel to evaluate hardness of reduced fat
Cheddar cheese.

 

173

APPENDIX II

 

 

NAME: DATE:
TYPE OF SAMPLE: Cheese
CMAPACTERISTIC STUDIED: Flavor

N UCT ONS

Place sample into the mouth and chew it until a semiliquid

stage is reached. Roll the macerated sample about in the mouth

for sufficient time to determine both taste and aroma.
Expectorate the sample. and. determine ‘the overall flavor
judgment. Rate the sample for bitterness and Cheddar flavor
intensity on the following scale:

QQNMENI§=

Sample # 280

BITTERNESS:

mmbUNH

Not observed
Slightly bitter
Somewhat bitter
Distinctly bitter
Strongly bitter

Very strongly bitter

CHEDDAR CHEESE FLAVOR TENSI Y:

O‘UIbUNH

No Cheddar cheese flavor

Slight Cheddar cheese flavor
Moderate Cheddar cheese flavor
Distinct Cheddar cheese flavor
Strong Cheddar cheese flavor

Very strong Cheddar cheese flavor

Figure II-12 - Sample form of questionnaire presented to the
panel to evaluate flavor of reduced fat

Cheddar cheese.

APPENDIX III

 

174

APPENDIX III

Table III-1 - Distribution of peptides by fractions (% area)
after 20 weeks of ripening of reduced fat Cheddar cheese.

 

 

 

 

 

 

 

 

 

 

 

‘Means with standard deviations in brackets.

n = 4 replications
“WMeans in same column with different superscripts are
different (p<0.05).

 

CHEESE FRACTION I FRACTION II FRACTION III FRACTION IV n
“1|
LLC-1.5 25.744 20.71B 19.85“ 33.70“
(2.58) (4.24) (1.46) (5.49) .
HSLC-1.5 26.41“ 12.774 21.00: 39.82“ i 1
(5.40) (2.71) (2.58) (8.91)
Control 26.81“ 12.13: 18.18“ 42.88“
(4.20) (1.89) (1.84) (7.38)

 

 

175

APPENDIX III

 

 

 

 

 

 

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176

APPENDIX III

NAME: DATE:
T F SAMPLE: Cheese

QMAPAQIEPISPLC STUDIED: Bitterness

 

 

IMSIBUCTIONS

"Bitterness is somewhat distasteful, resembling the taste of
quinine or caffeine. The bitter sensation is somewhat delayed
in terms of its initial perception and tends to persist for
sometime after sample expectoration. Bitterness is observed by
a taste sensation that occurs at the base of the tongue"

Place sample into the mouth. Roll it about in the mouth for
sufficient time to determine taste. Rank the sample from non
bitter to most bitter. Expectorate the sample; rinse mouth
with water between samples.

Sample # 280 Sample # 420 Sample # 580

Non bitter
Moderately bitter
Most bitter

 

 

 

COMM NT :

Figure III-1 - Sample form of questionnaire presented to the
panel to evaluate bitterness of reduced fat
Cheddar cheese slurry during training
sessions.

 

 

NAME:

177

APPENDIX III

DATE:

 

IZPE OF SAMPLE:
QMABACTERISTIC STUDIED:

STRUCTIONS

Place sample into the mouth and chew it until a semiliquid
state is reached. Roll the macerated sample about in the mouth
for sufficient time to determine taste. Expectorate the sample
and.determine the overall flavor judgment. Rate the sample for
bitterness on the following scale.
between samples.

OBS.

Sample
Sample
Sample
Sample

COMMENTS:

Figure III-2 - Sample form of questionnaire presented to the
panel to evaluate bitterness of reduced fat
Cheddar cheese slurry.

#
f
#
#

Cheese

not observed;

Bitterness

 

Rinse mouth with water

 

 

 

10 = very strongly bitter)
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10

 

 

 

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