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FACTORS THAT INFLUENCE VIABILITY
0F BIFIDOBACTERIA IN MILK

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Han-Seung Shin

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FACTORS THAT INFLUENCE VIABILITY OF BIFIDOBACTERIA IN MILK

BY

Han-Seung Shin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1997

ABSTRACT

FACTORS THAT INFLUENCE VIABILITY OF BIF‘IDOBACTERIA IN MILK
By

Han—Seung Shin

The overall goal of this research was to investigate the viability of
bifidobacteria in several commercial dairy products and enhance growth and
viability of bifidobacteria in milk to be consistent with clinical studies on health
benefits. In the first part of the study, viability of bifidobacteria in commercial
A / B milk (containing both bifidobacteria and Lactobacillus acidophilus) and two
brands of yogurt was investigated. The viability of bifidobacteria in these
products maintained at 106 cfu/ ml or g during refrigerated storage.

The second part of this study involved investigating the effect of
oligosaccharides and inulin on growth and viability of selected bifidobacteria
strains. Effect of Streptococcus salivarius subsp. thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus in lowering redox potential to enhance growth and
viability of bifidobacteria co-cultured with these organisms was also studied.
FOS was most effective (p<0.0S) in enhancing growth and viability of
bifidobacteria investigated whereas inulin was the least effective. Growth of
bifidobacteria was more affected by the pH of the system than redox potential.

The third part of this study involved conditions identified above in the
manufacture of yogurt. The initial bifidobacteria counts in yogurt were well
above >108 cfu / g. The viability of bifidobacteria in yogurt manufactured with
our condition were higher (p<0.05) than yogurt manufactured according to the

manufacturer directions with two commercial starter culture blends.

Dedicated to My parents, Dr. Hyosun Shin and Dr. Yangja Y00

and my sisters, Haeseung and Yeonseung

u.-

ACKNOWLEDGMENTS

I wish to express my heart felt gratitude to my advisor, Dr.
Zeynep Ustunol for kind help, inspiring guidance, consistent
encouragement, understanding she gave me and critical evaluation
of the whole manuscript.

Genuine appreciation is extended to the members of the
research guidance committee, Dr. James J. Pestka and Dr. Melvin
T. Yokoyama for their help and direction. For many helpful
information and technical assistance, I owe a special gratitude to
Dr. Jong Hwa Lee.

I am especially thankful to Jay Chick for his willingness to help
and friendship. Thanks are also due to my entire lab partners, Dr.
Hookil Jung, Jay Chick, Seongjoo Kim, Julie Hazard and Heather
Vachon offered their invaluable help and encouragement. I also
appreciate all the help of a number of faculty and staff members in
the Department of Food Science and Human Nutrition.

Finally my deepest gratitude goes to my parents and sisters for
great emotional, financial support, interest and concern throughout

my study.

TABLE OF CONTENTS

Page
LIST OF TABLES ----------------------------------------------------------- vii
LIST OF FIGURES ---------------------------------------------------------- ix
1. INTRODUCTION --------------------------------------------------------- 1
II. LITERATURE REVIEW ------------------------------------------------ 3
2.1 Fermented dairy products and lactic acid bacteria -------------- 3
2.2 Bifidobacteria: discovery and introduction ------------------------ 7
2.3 Physiology and metabolism of bifidobacteria --------------------- 8
2.4 Therapeutic effects of bifidobacteria ------------------------------- 12
2.5 Bifidobacteria in dairy products ------------------------------------ 16
2.6 Viability of bifidobacteria in dairy products ----------------------- 17
2.7 Growth factors of bifidobacteria ------------------------------------- 20
2.8 Redox potential and bifidobacteria -------------------------------- 25
III. MATERIALS AND METHODS ---------------------------------------- 28
3.1 Viability of bifidobacteria in commercial dairy products ------- 28
3.1.1 Sampling of commercial dairy products ----------------------- 28
3.1.2 Enumeration of bifidobacteria and lactic acid bacteria ------ 29
3.2 Effect of oligosaccharides and inulin on growth and viability of
bifidobacteria in fermented milk ------------------------------------- 30
3.2.1 Culture preparation ------------------------------------------------ 30
3.2.2 Effect of oligosaccharides and inulin on growth of commercial
bifidobacteria --------------------------------------------------------- 30
3.2.3 Effect of oligosaccharides and inulin on viability of bifidobacteria
during refrigerated storage ----------------------------------------- 32
3.2.4 HPLC assay --------------------------------------------------------- 32
3.2.5 Standard curve and HPLC chromatograms of acetic acid
and lactic acid ------------------------------------------------------- 34
3.3 Redox potential of NDM cultured with S. salivan'us
subsp. thermophilus and L. delbrueckii subsp. bulgan'cus
and its effect on growth of bifidobacteria ------------------------- 38
3.3.1 Selective medium for lactic acid bacteria ----------------------- 38
3.3.2 Screening L. delbrueckii subsp. bulgaricus and
S. salivarius subsp. thermophilus --------------------------------- 38
3.3.3 Culture preparation ----------------------------------------------- 39
3.3.4 Determining redox potential -------------------------------------- 4O

3.3.5 Growth of bifidobacteria co-cultured with lactic acid ---------- 41
3.4 Viability of bifidobacteria in yogurt manufactured using

commercial yogurt starter cultures and selected condition ------ 41
3.5 Statistical analysis ---------------------------------------------------- 42
IV. RESULTS AND DISCUSSION --------------------------------------- 44
4.1 Evaluation of media for selective enumeration of

bifidobacteria ------------------------------------------------------------- 44
4.2 Viability of bifidobacteria and lactic acid bacteria in

commercial A /B milk --------------------------------------------------- 46
4.3 Viability of bifidobacteria and lactic acid bacteria in commercial

yogurt ------------------------------------------------------------------- 49
4.4 Effect of oligosaccharides and inulin on growth of

bifidobacteria ------------------------------------------------------- 56
4.5 Effect of oligosaccharides on viability of bifidobacteria during

refrigerated storage ------------------------------------------------------ 60
4.6 Effect of oligosaccharides and inulin on the production

of acetic acid and lactic acid by Bifidobacterium sp. -------------- 61
4.7 Screening media for selective enumeration of L. delbrueckii

subsp. bulgaricus and S. salivarius subsp. thermophilus ------- 64
4.8 Screening L. delbrueckii subsp. bulgaricus and

S. salivarius subsp. thermophilus --------------------------------- 66

4.9 Growth of bifidobacteria co-cultured with L. delbrueckii
subsp. bulgaricus NCK 231 and S. salivarius subsp.

thermophilus St- 133 ------------------------------------------------- 69
4.10 The conditions to enhance growth and viability of

bifidobacteria in yogurt --------------------------------------------- 81

V. CONCLUSIONS ------------------------------------------------------ 85

VI. LIST OF REFERENCES ---------------------------------------------- 86

vi

Table

Table l .

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Page
Evaluation of media for selective enumeration
of bifidobacteria ------------------------------------------------ 45
The pH of commercial A / B milk during
refrigerated storage -------------------------------------------- 48
The pH of commercial brand A yogurt during
refrigerated storage -------------------------------------------- 52
The pH of commercial brand B yogurt during
refrigerated storage -------------------------------------------- 54
Effect of oligosaccharides and inulin on growth of
commercial Bifidobacterium sp. in 12% NDM -------------- 57
Effect of oligosaccharides and inulin on viability of
commercial Bifidobacterium sp. in 12% NDM after
4 weeks of refrigerated storage ------------------------------ 59
Effect of oligosaccharides and inulin on acetic acid
and lactic acid production by Bifidobacterium sp.
in 12% NDM ---------------------------------------------------- 62
Screening media for selective enumeration of
L. delbrueckii subsp. bulgaricus and S. salivarius
subsp. thermophilus -------------------------------------------- 65
The redox potential (Eh7) of 12% NDM cultured
with S. salivarius subsp. thermophilus ---------------------- 67

Table 10. The pH of 12% NDM cultured with L. delbrueckii

subsp. bulgaricus or S. salivarius subsp. thermophilus --- 68

Table 11. Redox potential (Eh7) and pH of NDM co-cultured

with Bifidobacten'um sp. Bf-l, L. delbrueckii subsp.
bulgaricus NCK231 and S. salivarius subsp.
thermophilus St- 133 ------------------------------------------- 71

Table 12. Redox potential (Eh7) and pH of NDM co-cultured

with B. adolescentis M 101-4, L. delbrueckii subsp.

vii

bulgaricus NCK231 and S. salivan’us subsp.
thermophilus St-133 -------------------------------------------- 72

Table 13. Growth of Bifidobacten'um sp. Bf-1 in 12% NDM
co—cultured with L. delbrueckii subsp. bulgaricus
NCK 231 and S. salivarius subsp. thermophilus
St-133 ------------------------------------------------------------ 75

Table 14. Growth of B. adolescentis M101-4 in 12% NDM
co-cultured with L. delbmeckii subsp. bulgaricus
NCK231 and S. salivarius subsp. thermophilus
St-133 ------------------------------------------------------------ 76

Table 15. Growth of S. salivarius subsp. thermophilus
St-133 in 12% NDM co-cultured with
L. delbrueckii subsp. bulgaricus NCK 231
and Bifidobacterium sp. Bf-l --------------------------------- 77

Table 16. Growth of S. salivarius subsp. thermophilus
St-133 in 12% NDM co-cultured with
L. delbrueckii subsp. bulgaricus NCK 231
and B. adolescentis M 101-4 ---------------------------------- 78

Table 17. Growth of L. delbrueckii subsp. bulgaricus
NCK 231 in 12% NDM co-cultured with
S. salivarius subsp. thermophilus St-133 and
Bifidobacterium sp. Bf—l -------------------------------------- 79

Table 18. Growth of L. delbrueckii subsp. bulgaricus NCK
231 in 12% NDM co-cultured with S. salivarius
subsp. thermophilus St-133 and B. adolescentis
M 101-4 ---------------------------------------------------------- 80

Table 19. Viability of bifidobacteria in yogurt during
refrigerated storage ------------------------------------------- 83

Table 20. The pH of yogurt during refrigerated storage ------------ 84

viii

LIST OF FIGURES

Figure Page
Figure 1. Metabolic pathway of Bifidobacterium sp. -------------- 10
Figure 2. Oxygen dissimilation in Bifidobacterium sp. ---------- 1 1

Figure 3. Beneficial effects of bifidobacteria on human
health ....................................................... 13

Figure 4. Typical HPLC chromatogram of lactic acid
and acetic acid produced by bifidobacteria
cultured in 12% NDM with fructooligosaccharide,

galactooligosaccharide and inulin ------------------------ 35
Figure 5. Acetic acid standard curve -------------------------------- 36
Figure 6. Lactic acid standard curve --------------- _ ----------------- 3 7

Figure 7. Viability of (A) bifidobacteria and (B) lactic acid
bacteria in commercial A /B milk during 18 days
of refrigerated storage at 5°C ------------------------------ 47

Figure 8. Viability of (A) bifidobacteria and (B) lactic acid
bacteria in commercial brand A yogurt during
6 weeks of refrigerated storage at 5°C -------------------- 51

Figure 9. Viability of (A) bifidobacteria and (B) lactic acid
bacteria in commercial brand B yogurt during
6 weeks of refrigerated storage at 5°C ------------------- 53

I. INTRODUCTION

Bifidobacteria are inhabitants of the human intestine and well
adapted for metabolism in the gastrointestinal tract of humans.
Bifidobacteria were first isolated from feces of breast-fed infants in
1899 by Tissier at the Pasteur Institute in Paris (Tamine et al., 1995).
Bifidobacteria has received much attention recently due to studies
on their health promoting effects. Thus, there is an increasing
interest in incorporating bifidobacteria into foods, particularly dairy
products. Currently, There are more than 70 dairy products
containing bifidobacteria produced world wide (Ventling and Mistry,
1993)

Some health benefits of bifidobacteria include maintaining a
normal intestinal microflora balance (Yoshioka et al., 1983),
improving lactose tolerance of milk products (Gilliland, 1989),
promoting anti-tumorigenic activity (Fernandes and Shahani, 1990),
reducing serum cholesterol levels (Homma, 1988), and synthesis of B-
complex vitamins (Hughes and Hoover, 1991).

Dairy products have been used as a medium to reintroduce
viable populations of bifidobacteria into the GI tract of both children
and adults (Hughes and Hoover, 1991). Maintaining viability of

bifidobacteria in the carrier food prior to consumption is thought to

be necessary for their health promoting effect (Samona and Robinson,
1991)

Several clinical studies have shown significant benefits, which
were observed upon ingestion of approximately 109--1O10
organisms/d (Sanders et al., 1996). However, viability of
bifidobacteria in dairy products has been very variable. Because of
the low pH of fermented dairy product and aerobic conditions of
production of dairy products, viability of bifidobacteria in dairy
products has not been satisfactory (Dinakar and Mistry, 1994).
Many published studies on viability of bifidobacteria were not done
using commercial strains, or strains that have been shown to
provided health benefits. In addition, several growth promoting
factors investigated for bifidobacteria are not food grade or approved
for use in dairy products and they can not be incorporated into
commercial dairy products. Thus, these studies are not directly
relevant to the dairy industry. The purpose of my study was to
investigate the viability of bifidobacteria in commercial dairy
products and determine factors that enhance and maintain viability
that are commercially feasible using strains of bifidobacteria and

lactic acid bacteria that may have positive health benefits.

II. LITERATURE REVIEW

2.1 Fermented dairy products and lactic acid bacteria

Milk is an excellent medium to support the growth of many
microorganisms 'and to produce numerous fermented dairy
products. Fermented milks, like yogurt, were available thousands of
years ago, and recently there has been increasing interest in
consumption of fermented dairy foods (Mutukumira, 1995).
Fermented milk products have several important advantages, such
as a means of preserving food, providing better taste, increasing
digestibility, allowing for production of a variety of foods, and
providing several health benefits (Kroger et al., 1989; Marshall,
1993). Milk from domestic mammals such as cows, buffalo, sheep,
goats, horses, camels, and yaks has been used to make traditional
fermented milk products around the world, which include a variety
of cheeses, butter milk, kefir, yogurt, kumiss, taette, acidophilus
milk, tarhana and other products (Driessen and de Boer, 1989).
Since Mechnikoff (1908) at the Pasteur Institute proposed that
ingestion of fermented milks offer health benefits and longevity in
humans, fermented dairy products have been the subject of much

speculation (Hoover, 1993). Tamine and Robinson (1988) reported

that consumption of fermented milk per capita in the US. was 3.8
kg in 1987; consumption has rapidly increased.

Production of dairy products such as buttermilk, sour cream,
yogurt and cheese require controlled fermentation. The starter
culture used in dairy products is important in determining product
type, character, and quality. Fermentation of milk is primarily
accomplished by lactococci and lactobacilli, which breakdown
lactose to lactic acid. In addition, other parallel or post-
fermentation reactions produce compounds distinctive of fermented
foods. Two groups of lactic organisms have been used typically in
fermented dairy foods. Thermophilic organisms which include
Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius
subsp. thermophilus, Lactobacillus helveticus, and Lactobacillus
acidophilus have an optimum temperature of growth around 37-
45°C. These organisms are used for manufacture of products such
as yogurt, Bulgarian buttermilk, and skyr. Mesophilic organisms,
which include Lactococcus lactis subsp. lactis, Lactococcus lactis
subsp. cremoris, Leuconostoc sp. Lactobacillus kefir, and
Lactobacillus casei have an optimum temperature of around 30°C.
These organisms are used for manufacture of products such as
cheeses, cultured butter milk, and fermented milks (Marshall,

1993). Mesophilic organisms such as Leuconostoc sp. metabolize

citrate to diacetyl, acetate, and CO2, which are responsible for the
flavor or aroma of fermented dairy products (Tamine and Robinson,
1988)

Yogurt is the traditional and one of the most popular fermented
dairy products in many countries. Standard of identity require that
all yogurts in the US. are manufactured using S. salivarius subsp.
thermophilus and L. delbrueckii subsp. bulgaricus as starter cultures
(Tamine and Deeth, 1980). S. salivarius subsp. thermophilus is a
gram-positive and nonmotile organism. It has been classified as a
facultative anaerobic microorganism and its optimum growth
temperature is 40°C to 45°C. S. salivarius subsp. thermophilus
produces lactic acid and small quantities of volatile acids such as
formic, acetic, propionic, butyric, isovaleric, and caproic acids
(Marshall, 1993; Tamine and Robinson, 1988; T amine and Deeth,
1980)

L. delbrueckii subsp. bulgaricus is a gram-positive, nonmotile
organism; it has a slender rod shape with rounded ends. It has
been classified as a facultative anaerobic microorganism and has an
optimum growth temperature of 40°C to 43°C. L. delbrueckii subsp.
bulgaricus produces lactic acid and small quantities of carbonyl
compounds and ethanol. The most important carbonyl compounds

include acetaldehyde, acetone, butanone-2 and trace of acetoin.

Both L. delbrueckii subsp. bulgaricus and S. salivarius subsp.
thermophilus have been classified as homofermentive bacteria,
which use the glycolytic pathway for glucose fermentation (Jay,
1992). The homolactic bacteria possess enzymes such as aldolase
and hexose isomerase but lack phosphoketolase, which is found in
heterolactic bacteria. In homolactic fermentation, glucose results in
2 moles of lactic acid and a net gain of 2 ATP per mole glucose
consumed (Jay, 1992; Tamine and Deeth, 1980). Tamine and Deeth
(1980) reported a symbiotic relationship between L. delbrueckii
subsp. bulgan'cus and S. salivarius subsp. thermophilus during their
growth. Mixed cultures of L. delbrueckii subsp. bulgaricus and S.
salivarius subsp. thermophilus in milk produced more acid than
single strain cultures (Marshall, 1987; Tamine and Deeth, 1980). L.
delbrueckii subsp. bulgaricus provided essential growth
requirements such as glycine and valine for stimulation of S.
salivarius subsp. thermophilus (T amine and Robinson, 1988). S.
salivarius subsp. thermophilus stimulated growth of L. delbrueckii
subsp. bulgaricus by removing oxygen, which lead to production of
toxic hydrogen peroxide (Tamine and Deeth, 1980).

Organisms such as L. acidophilus, L. casei, L. delbrueckii subsp.
lactis, and Lactococcus lactis subsp. cremoris have been used in

various commercial dairy products as adjunct cultures (Driessen

and de Boer, 1989; Kim, 1988). They are normal inhabitants of the
human intestinal tract, they would not disturb the normal intestinal
flora (Martin and Chou, 1992). Recently, Bifidobacterium sp. was
recognized as possible dietary adjunct and several researchers have
reported on their beneficial effects on human health (Laroia and
Martin, 1990; Hughes and Hoover, 1991). Although requirement for
an adjunct cultures has not been clearly established, it is suggested
that they should be normal inhabitants of the intestinal tract, they
should be metabolically active and maintain viability in the carrier
food and should survive the gastrointestinal tract (Tamine and

Robinson, 1988).

2.2 Bifidobacteria: Discovery and introduction

Bifidobacteria are gram-positive, nonspore-forming, and
nonmotile cells that often stain irregularly with methylene blue
(Scardovi, 1986). Although the organism is classified as being
anaerobic, some species can tolerate low levels of 02. The optimum
growth temperature for bifidobacteria is 37°C to 41°C, and the
optimum pH for growth is 6.5 to 7.0 (Tamine et al., 1995).

Bifidobacteria produces both acetic and lactic acid via an
unusual glucose-metabolizing system that results in a ratio of 3:2

acetatezlactate as the primary metabolites (Scardovi and Trovatelli,

1965). In addition, small amounts of formic acid and ethanol are
often produced by bifidobacteria fermentation (Scardovi, 1986). In
the 8th edition of "Bergey's Manual" (Scardovi, 1986), twenty-four
species of bifidobacteria have been defined. Those species of
Bifidobacterium typically colonize the human GI tract are B. bifidum,
B. infantis, B. breve, B. longum, B. adolescentis, and B. catenulatum.
The other species occurring in the intestinal tract of various animals
and insects (Scardovi, 1981).

The taxonomy of bifidobacteria has changed since bifidobacteria
were first isolated. Many of the species groupings are
heterogeneous, and the entire genera are being reexamined using
several methods. Currently, there is no test that allows the
determination of the origin and classification based on the
taxonomy of the strains. Many bifidobacteria species are isolated
from both animal and human flora, with human bifidobacteria
species have been more extensively studied than the animal species

(Scardovi, 1986).

2.3 Physiology and metabolism of bifidobacteria
The pathway for the metabolism of carbohydrates by
Bifidobacterium sp. differs from that of homo and hetero-

fermentative lactic acid bacteria. Figure 1 shows the metabolic

pathway used by Bzfidobacterium sp. Hexoses are degraded by the
fructose-6-phosphate pathway (Modler et al., 1990). Fructose-6-
phosphate phosphoketolase (F6PPK) is found in Bifidobacterium sp.,
whereas there is no fructose—1,6-bisphosphate aldolase, which is
present in homofermentive lactic acid bacteria (Scardovi, 1986).
The fermentation of two moles of glucose leads to the production of
three moles of acetate and two moles of lactate when pyruvate is
converted to lactate by L(+) lactate dehydrogenase. The pathway
involves the splitting of pyruvate to form formate and acetyl
phosphate, which is reduced to form ethanol (Tamine et al., 1995).
Oxygen dissimilation by Bifidobacterium sp. is shown in Figure 2.
Oxygen toxicity to Bifidobacterium sp. results from metabolism of
various compounds such as superoxide and hydrogen peroxide.
The degree of tolerance to oxygen by bifidobacteria depends on the
species and the culture medium (Tamine et al., 1995). Some strains
grow in the presence of oxygen without accumulating hydrogen
peroxide, whereas other strains exhibit limited growth and
accumulation of hydrogen peroxide. Also, some strains appear to
be intolerant to oxygen and grow only when the redox potential is

lowered. The presence of oxygen prevents growth because of the

IO

2ATP 2Gucose

 

 

 

 

 

1
2ADP
Fructose-6-P
3ADP 3ATP
3v\ Erythrose-4—P l 82
cetyl-P 3Acetate
2Acetyl-P
Glyceraldehyde—3—P Sedoheptulose-3-P \7/:
Ribose- 5- P Xylulose-S-P *2Glyceraldehyde—3—P
Xylulose-S-P 2Pi
2NADH;
) 11 Pyruvic acid
Ribulose-S-P 2NADH2 >106 4ADP
2NAD 4ATP
I
2L(+)lactate

Figure 1. Metabolic pathway of Bzfidobacterium sp. 1 = hexokinase and
glucose-6-phosphate isomerase; 2 = fructose-6-phosphate
phosphocetolase; 3 = transaldolase; 4 = transketolase; 5 = ribose-5-
phosphate isomerase; 6 = ribulose-5-phosphate epimerase; 7 = xylulose-5-
phosphate phosphocetolase; 8 = acetate kinase; 9 = homofermentative
pathway enzymes; 10 = L(+) lactate dehydrogenase; 11 = phosphoroclastic
enzyme (Tamine et al., 1995).

ll

 
    
  
     

F6PPK pathway

   

/ NADH\

OZNADH oxidase “220 NADH peroxidase

02\ OAperoriide

dismutase

H20

     

 

P1202

Figure 2. Oxygen dissimilation in Bifidobacterium sp. (Ballongue,

1993i

12

difficulty in establishing a suitable redox potential (Klaver et al.,
1993). NADH oxidase and NADH peroxidase are enzymes involved
in oxygen utilization. The strains that are sensitive to oxygen have
low NADH oxidase and NADH peroxidase activity, resulting in an

accumulation of toxic hydrogen peroxide.

2.4 Therapeutic effects of bifidobacteria

Bifidobacteria have received much attention recently due to data
accumulating on their health promoting effects (Hoover, 1993).
Bifidobacteria account for 92% of the intestinal flora of breast-fed
infants, but only 20% in bottle-fed or weaned infants (Hori, 1984).
The beneficial effects of bifidobacteria on human health are
summarized in Figure 3. Bifidobacteria have been reported to be
effective inhibitors of gut pathogens due to their ability to produce
acetate, lactate and small amounts of formate from carbohydrate
catabolism. These organic acids and the lowering pH inhibit gram-
negative microorganisms such as Escherichia coli, Staphylococcus
aureus, Shigella dysenteriae, and Samonella typhi (Samona et al.,
1996)

Bifidin is a compound isolated from B. bifidum; it has been
reported to have antibacterial activity against Micrococcus flavus

and Staphylococcus aureus (Kanbe, 1992).

      
 
    
   
   
   

Anti-microbial

effect
iii Reduction o' I
serum cholesterol

   

Anti-tumor
effect

Bifidobacteria

     
    
     
 

swag...
vitamin

Improve
lactose
utilization

    

Anti-diarrhea
effect

Figure 3. Beneficial effects of bifidobacteria on human health.

14

The improvement of lactose tolerance of milk products is another
important health benefit of bifidobacteria.

Lactose intolerance is the result of insufficient amounts of B-
galactosidase in the small intestine (Martini et al., 1991; Modler et
al., 1990; Savaiano and Levitt, 1987).

Bifidobacteria possess high levels of B-galactosidase activity,
which is released during digestion of dairy products containing
bifidobacteria (Martini et al., 1991; Savaiano and Levitt, 1987).
However, Martini et al. (1991) reported that not all bifidobacteria
strains provided sufficient microbial B—galactosidase activity to
improve lactose digestion and tolerance. Differences in the activity
of B-galactosidase among bifidobacteria strains may cause a
variation in lactose digestion.

It has been reported that bifidobacteria reduced the source of
procarcinogens or enzymes, such as B-glucuronidase, azoreductase,
and nitroreductase, that lead to their formation (Hawkins, 1993;
Tamine et al., 1995). Sekine et al. (1985) reported that B. infantis
ATCC 15697 significantly suppressed tumor growth in mice. Also,
K00 and Rao (1991) reported that proliferation of liver tumors
significantly decreased when B. longum was present in the intestinal

flora.

15

Reduction of serum cholesterol levels is another benefit of
bifidobacteria. The role that bifidobacteria cultures may play in
lowering serum cholesterol is not yet understood. In rat models,
serum cholesterol was lowered by feeding bifidobacteria in a
mechanism that may involve HMG-CoA reductase (Homma, 1988).
Gilliland and Walker (1990) reported that bifidobacteria produce
HMG, which inhibits HMG-CoA reductase involved in the synthesis
of cholesterol. Jaspers et al. (1984) reported that both orotic acid
and uric acid produced during fermentation of cultured dairy
products lowered the serum cholesterol levels in humans.

Synthesis of vitamins is another benefit of bifidobacteria.
Hawkins (1993) reported that bifidobacteria produced thiamine,
riboflavin, vitamin K and vitamin Be. Deguchi et al. (1985) also
observed that B. longum produced vitamin 86 and 82. Thus, dairy
foods containing bifidobacteria help in meeting the requirements for
B-vitamin consumption (Hawkins, 1993).

Anti-diarrhea effect of bifidobacteria has been reported as
beneficial effect of bifidobacteria. Hotta et al. (1987) showed that
normal functions of children suffering from diarrhea were restored
more rapidly after the ingestion of milk fermented with B. breve.
Tojo et al., (1987) reported that feeding of bifidobacteria containing

dairy products has been used to treat diarrhea infections in

16

Japanease children. Homma (1988) also reported that ingestion of

milk fermented with B. longum helped prevent diarrhea in humans.

2.5 Bifidobacteria in dairy products

Because of the health benefits discussed above, there is
increasing interest in incorporating Bifidobacterium sp. into
fermented dairy products as a medium to reintroduce viable
populations of bifidobacteria into the GI tracts of children and
adults. Currently, more than 70 different dairy products containing
bifidobacteria are produced worldwide, including sour cream, butter
milk, yogurt, powered milk, cultured milk, ice-cream, cheese, and
other frozen desserts (Ventling and Mistry, 1993). The most
commonly employed bifidobacteria strains in dairy products are B.
bifidum, B. infantis, and B. longum. Because these species are of
human origin, they have an affinity to colonize the human intestine
(Ishibashi and Shimamura, 1993). Before 19803, the use of
bifidobacteria in food products was largely limited to foods and
beverages intended for therapeutic treatment (Hawkins, 1993). In
the 19703, technology began to catch up with the objective of
delivering viable bifidobacteria to commercial dairy products
(Driessen and de Boer, 1989). Today, dairy products containing

bifidobacteria have become as common in the US. as they are in

17

Japan and EurOpe. The incorporation of bifidobacteria into dairy
products in addition to the nutritional benefits also provides for
better taste, milder, less sour and bitter products.

Manufacturing problems do occur in cultured dairy products
containing bifidobacteria more often than they do in traditional
cultured dairy products. These problems occur because cultivation
of bifidobacteria in milk is more difficult than with other
microorganisms. More aseptic working conditions are needed
because of longer incubation times and slower acidification. Also,
bifidobacteria are classified as anaerobic microorganisms, although
some species are able to tolerate oxygen. Thus, oxygen toxicity is
an important and critical problem in commercial dairy processing.
Inoculum and starter culture amounts are much larger, and
bifidobacteria lose their viability more quickly at low pH values
during storage (Hoover, 1993; Ishibashi and Shimamura, 1993).
For the development of dairy product containing bifidobacteria, not
only the health benefits and taste, but also the viability of

bifidobacteria is very important.

2.6 Viability of bifidobacteria in dairy products
Sanders (1993); Salminen and Deighton (1992) recently

reviewed the various clinical benefits of consuming viable

l8

bifidobacteria. Although scientific opinions regarding the
significance of viability in the therapeutic efficacy of lactic acid
bacteria and bifidobacteria remain divided, the public expects
fermented dairy products to contain viable organisms at the time of
consumption. Clinical studies have shown significant clinical
benefits are observed upon ingestion of approximately 109 - 1010
organisms/d (Sanders et al., 1996). Viability of bifidobacteria in
commercial dairy products is not consistent with clinical data
available. The National Yogurt Association has established
standards for lactic acid bacteria, which is at least 109 cfu/ g for
refrigerated yogurt and 107 cfu/ g for frozen yogurt at the time of
manufacturing for the Seal Program in order to promote the
importance of live and active cultures. No standards have been
established for bifidobacteria. France has regulations on viable
culture numbers in fermented dairy products, requiring 21x 108
cfu/ml. Japan, South Korea, and Poland have regulations pertaining
to viability of cultures in fermented dairy products, which is 21 x
10° - l x 107 cfu/ml of Viable cultures (Orihara et al., 1992). In the U.
8., states such as California and Oregon have already adopted
regulations pertaining to Viability of cultures in dairy foods, which is
2 x 106 cfu/ ml of viable lactic cultures (Sanders et al., 1996). Other

states are also expected to adopt regulations regarding viability of

19

cultures in fermented foods. Increasing viability of lactic acid
bacteria and bifidobacteria in dairy products has been the focus of
much research (Hekmat and McMahon, 1992; Ibrahim and
Bezkorovainy, 1994; Medina and Jordano, 1994; Poch and
Bezkorovainy, 1988). It requires skill, knowledge, and advanced
technologies to maintain a satisfactory level of viable bifidobacteria
in dairy products for their probiotic effects (Kurmann et al., 1992).
There are many factors, which influence the viability of
bifidobacteria. These include the strain of bifidobacteria used in
milk, milk solids content, pH, storage temperature, presence and
content of sugars, culturing conditions, and individual
manufacturing conditions.

Medina and Jordano (1994) reported on the bifidobacteria count
of fermented milk produced in Spain stored at 7°C. They observed a
92.6% decrease in bifidobacteria count when the product was
expired. Biavati et al. (1992) observed that the viability of
bifidobacteria in skim milk at pH 4.0 and 4°C decreased by more
than 90% after 15 days. Modler et al., (1990) reported that the
viability of bifidobacteria is affected by a low pH environment
however some strains of bifidobacteria showed an acid tolerance at
pH 4.0. Blanchette et al. (1996) reported on the viability of

Bifidobacterium infantis ATCC 27920 in creamed cottage cheese.

20

They observed a decrease of 2-4 log cycles during 15 days of storage
at 4°C. Hekmat and McMahon (1992) reported that ice—cream may
serve as a good vehicle for delivering viable bifidobacteria. Their
study indicated that ice-cream mix fermented with L. acidophilus
and B. bifidum maintained viable cell counts after 17 weeks of
storage at -29°C. After fermented mix was frozen, L. acidophilus and
B. bifidum counts were 1.5x108 cfu/ml and 2.5x108 cfu/ml,
respectively. Seventeen weeks after frozen storage, counts of L.
acidophilus and B. bifidum in the ice—cream were decreased by two
log cycles to 4x106 cfu/ ml and by one log cycle to 1x107 cfu/ ml,
respectively. Shah et al. (1995) reported on initial bifidobacteria
counts in five brands of commercial yogurt. In two of the five
brands of yogurt, counts were 106-107 cfu/ g, and in the ramaining
three they were <103 cfu/ g, indicating significant variability in
counts in similar products. They also reported that all products
showed a constant decline in bifidobacteria and lactic acid bacteria

counts during storage.

2.7 Growth factors of bifidobacteria
Research also has been conducted to optimize growth
conditions for the various strains of bifidobacteria used in dairy

products (Desjardins and Roy, 1990; Dinakar and Mistry, 1994;

21

Dubey and Mistry, 1996; Hughes and Hoover, 1995). Growth-
promoting factors have been investigated to increase the viability of
bifidobacteria for significant clinical benefits (Driessen, 1988;
Hidaka et al., 1986; Ibrahim and Bezkorovainy, 1994; Pooh and
Bezkorovainy, 1988). Some growth-promoting factors that have
been tested include N—acetylglucosamine (Jao et al., 1978), casein
(Nicholas et al., 1974), carrot juice (Rasic and Kurmann, 1983),
porcine mucine (Modler et al., 1990), lactulose (Nagendra et al.,
1995), oligosaccharides (Yun, 1996), and inulin (Yamazaki and
Dilawri, 1990). To enhance the growth of bifidobacteria, trace
elements (Bezkorovainy et al., 1986) and vitamins (Deguchi et al.,
1985) also have been studied as nutrient requirements.
N-acetylglucosamine has been identified as a required substance
for growth of B. bifidum var. pennsylvanicus and has been named
“bifidus factor I” (Tamine et al., 1995). N-acetylglucosamine is
found in human and cow’s milk (Jao et al., 1978). Casein also has
been shown to have growth-promoting activity and is known as
“bifidus factor II”. Casein from human milk enhanced the growth of
various strains of bifidobacteria without any treatment and became
much more effective after treatment with chymosin or pepsin

(Azuma et al., 1984).

22

Carrot root extract also has been found to contain a bifidus
factor identified as a precursor of coenzyme A which is water soluble
and heat resistant (Rasic and Kurmann, 1983; Tamine et al., 1995).
Various studies have reported that growth of bifidobacteria was
promoted more in human milk than cow’s milk (Hidaka et al., 1991;
Homma, 1988; Petschow and Talbott, 1990). Lower buffering
capacity in human milk (Bullen et al., 1977), because of its lower
protein and mineral contents and the presence of lactoferrin and
transferrin (Roberts et al., 1992), could be responsible for the better
growth of bifidobacteria in human milk. Human milk also contains
nucleotides such as cytidine—S—phosphate that are not found in
cow’s milk. These nucleotides have been considered as bifidus
factors, which promote the establishment of bifidobacteria in the
human intestinal tract.

Lactulose is a disaccharide composed of one molecule each of
galactose and fructose, and its ingestion has been reported to
stimulate growth of bifidobacteria in the large intestine (Azuma et
al., 1984; Rasic and Kurmann, 1983; Roy and Goulet, 1991).
However, other studies have shown that several strains of other
intestinal bacteria can also utilize lactulose, but to a lesser extent

than bifidobacteria (Azuma et al., 1984; Roy and Goulet, 199 1).

23

A number of studies (Crittenden and Playne, 1996; McKellar
and Modler, 1989; Yazawa and Tamura, 1982; Yazawa et al., 1978)
have been undertaken to identify oligosaccharides that can be
utilized by bifidobacteria. Fructooligosaccharides, trans—galactosyl-
oligosaccharides, 4’-galactosyl-lactose and other oligosaccharides
also have been reported as bifidogenic factors, resulting in the
proliferation of human intestinal bifidobacteria (Hidaka et al., 1986).
B. breve and B. infantis selectively use raffinose, stachyose, and
inulin, but these sugars are not used by E. coli, L. acidophilus, and
S. faecalis. Oligosaccharides that are not digested in the small
intestine reach the large intestine where they become available for
degradation by the indigenous bifidobacteria in the colon (Yun,
1996). Yazawa et al. (1978) reported on various ingestible
oligosaccharides useful for increasing the number of intestinal
bifidobacteria. Generation times of B. breve and B. infantis have
been found to be as rapid with these sugars as with glucose or
lactose (Hidaka et al., 1991, Hayakawa et al., 1990).
Fructooligosaccharide consists of polymers of D-fructose linked by a
B(2—>1) bond and terminated with a D-glucose linked to fructose by
a a(1——>2) linkage. Fructooligosaccharides with a degree of

polymerization (DP) between 3 and 5 have been synthesized from

24

sucrose. Inulin is polymers with a DP more than 30 (McKellar and
Modler, 1989; Yun, 1996).

McKellar and Modler (1989) reported that B. adolescentis ATCC
15703, B. longum ATCC 15707, and B. thermophilum ATCC 25525
have the ability to metabolize short chain fructooligosaccharide with
a DP between 3 and 5. Yazawa and Tamura (1982) reported that
inulin was selectively utilized by B. infantis. Yazawa and Tamura
(1982) and Yazawa et al. (1978), reported that fructooligosaccharide
and inulin stimulated the growth of bifidobacteria. Although
bifidobacteria have been shown to metabolize single and complex
sugars, growth factors must be added to milk to promote their
growth (Crittenden and Playne, 1996; Yun, 1996).

Oligosaccharides are water soluble and 0.3 to 0.6 times as sweet
as sucrose. However, sweetness of oligosaccharides is affected by
their chemical structure, molecular weight, and the levels of mono
and disaccharides in the oligosaccharides. The low sweetness of
oligosaccharides may be desirable in dairy products because they
enhance flavors, increase solids thus may improve texture and
lower water activity (Crittenden and Playne, 1996; Spiegel et al.,

1994; Yun, 1996).

25

2.8 Redox potential and bifidobacteria

Another significant factor affecting the viability of bifidobacteria
is oxygen. It has been known that microorganisms show different
degrees of sensitivity to redox potential (oxidation-reduction
potential; Eh) of their growth medium (Jay, 1992). Redox potential
is a measure of the tendency of a given system to donate electrons
or to accept electrons. The redox potential of a given system is
determined by measuring the electrical potential difference between
that system and a standard hydrogen electrode (Singleton, 1987).

Redox potential (Eh) can be calculated using the following equation:

RT Activity of reduced species
Eh = Eo — loge
nF Activity of oxidized species

 

E0 = Constant characteristic potential
R = Gas constant (8.31 J / mol/ degree abs.)
T = Absolute temperature

n = Number of electrons

Redox potential, Eh is measured using a platinum electrode and
is expressed in millivolts (mv). Strict aerobic organisms require a
high positive Eh, between 300 and 500 mv for their growth.
Microaerophilic organisms require an Eh between 100 and 300 mv.

Facultative anaerobic organisms such as Bifidobacterium require a

26

Eh between —200 and 200 mv for their growth (Jay, 1992). Oxygen
toxicity is an important and critical problem because
Bifidobacterium sp. are facultative anaerobes (Kim, 1988). Reuter
(1989) suggested applying a S. salivarius subsp. thermophilus strain
with high oxygen consuming ability to enhance the viability of
bifidobacteria in yogurt type products. S. salivarius subsp.
thermophilus is a homofermentative facultative anaerobic
microorganism. During anaerobic fermentation, S. salivarius subsp.
thermophilus catabolize 1 mol of glucose to 2 mol of lactate through
the glycolytic pathway, but in aerobic metabolisms glucose or
pyruvate leads to the formation of acetate, a-acetolactate, acetoin,
and diacetyl in addition to lactic acid production. The following
equation shows the aerobic metabolism of S. salivarius subsp.

thermophilus associated with consumption of oxygen:

NADH oxidase
NADH + H” + 02 —) NAD + H202

Pyruvate oxidase
Pyruvate + 1/202 —) Acetate + C02

NADH generated in the aerobic metabolism is consumed in the
reaction catalyzed by NADH oxidase. Aerobic 02 uptake has also
been observed with lactobacilli, but to a lesser extent (Teraguchi et

al., 1987; Tinson et al., 1982). Okonogi et al. (1986), in a patent,

27

reported that due to the high oxygen uptake of Streptococcus
salivarius subsp. thermophilus, it provided a suitable environment
for bifidobacteria and greatly enhanced viability of bifidobacteria.
Developing or selecting oxygen and acid resistant bifidobacteria
strains would be another possible means of improving their
viability.

The purpose of the following study was to determine the viability
of bifidobacteria in commercial dairy products and investigate the
role of ~food grade oligosaccharides, inulin, and redox potential in
enhancing viability of bifidobacteria that may have enhanced health
benefits. The final part of this study will involve manufacture of a

yogurt product that is consistent with clinical studies.

III. MATERIALS AND METHODS

3.1 Viability of bifidobacteria in commercial dairy products.
3.1.1 Sampling of commercial dairy products.

Commercial A/B milk (containing L. acidophilus and
bifidobacteria) and two brands of yogurt (containing Lactobacillus
delbrueckii subsp. bulgan'cus, Streptococcus salivan'us subsp.
thermophilus and bifidobacteria) were obtained from retail outlets in
the Michigan area and stored at 5°C. All products claimed to contain
viable bifidobacteria and lactic acid bacteria. Milk was evaluated 9, 6
and 3 days prior to its expiration date, at its expiration date and 3, 6,
and 9 days after its expiration date. Whereas, yogurts were evaluated
3, 2 and 1 week prior to their expiration dates, at their expiration date
and 1, 2 and 3 weeks past the expiration date. Samples were mixed
well and aseptically removed from each container and diluted by
mixing lml of milk or 1g yogurt with 99ml of 0.1% (w/v) bacto
peptone (Difco, Detroit, MI) and subsequent serial dilutions were
made. Another sample was collected for pH measurements. The pH
of the products was determined at each sampling point. Unopened
A/ B milk and two brands of yogurt were used each sampling point

for the enumeration and pH determination.

28

29

3.1.2 Enumeration of bifidobacteria and lactic acid bacteria.
The first part of this research involved screening selective media
to enumerate bifidobacteria in commercial dairy products. Various
media were screened for selective enumeration of bifidobacteria.
Brain heart infusion agar, modified Columbia agar, RCA and MRS
agar containing 5% (w/v) lactose and 5% (v/v) NPNL antibiotic
solution were evaluated. MRS agar containing 5% (w/v) lactose and
5% (v/v) NPNL antibiotic solution was most successful in
enumerating bifidobacteria and inhibiting all other lactic acid
bacteria. Thus, in the following studies bifidobacteria were
enumerated using MRS agar (Difco) containing 5% (w/v) lactose and
5% (v/v) NPNL antibiotic solution. NPNL was prepared by mixing
60g of LiCl (Sigma, St. Louis, MO), 4g of paromomycin sulphate
(Sigma), 2g of neomycin sulphate (Sigma), 0.3g of nalidixic acid
(Sigma) in 1 liter of demineralized water. The mixture was filter-
sterilized (0.22um) prior to adding to MRSL. The inoculated plates
were incubated anaerobically at 37°C for 48hr using Gas Pak®
(Becton Dickinson Co., Cockeysville, MD). Lactic acid bacteria were
enumerated using MRS agar containing 5% (w/v) lactose. The
inoculated plates were incubated aerobically at 37°C for 72hr. The
colonies were counted using a Quebec colony counter (Fisher

Scientific, Pittsburgh, PA). Bifidobacteria and lactic acid bacteria

30

count were determined by phenotype characteristics as presented in
the 8th edition of Bergey’s Manual of Determinative Bacteriology

(Scardovi, 1986).

3.2 Effect of oligosaccharides and inulin on growth and viability
of bifidobacteria in fermented milk.
3.2. 1 Culture preparation.

Commercial strains of Bifidobacterium Bf-l and Bf—6 from Sanofi
Bio-Industries (Waukesha, WI) were selected in this research
because they have been shown to stimulate immune function via
altered cytokine secretion by leukocytes within the gastrointestinal
immune compartment in in vitro studies (Marin et al., 1997). Each
bifidobacteria culture was cultured and subcultured anaerobically
in MRS medium (Difco) containing 5% (w/v) lactose (MRSL) at 37°C
for 48hr using Gas Pak® (Becton Dickinson Co.). Cultures were
centrifuged 15 min at 1000 x g at 4°C and resuspended in 12%
(w/v) pasteurized (70°C, 30 min) non-fat dry milk (NDM; Difco) at a

5% (v/ v) level.

3.2.2 Effect of oligosaccharides and inulin on growth of
bifidobacteria.
Frutooligosaccharide (F08) and inulin were supplied from

Rhone-Poulenc Inc. (Cranbury, NJ). Galactooligosaccharide (GOS)

31

was supplied from Samyang Genex Co., Ltd. (Seoul, Korea). They
were added at 0.5, 1, 3, and 5% (w/v) level to 12% (w/v) NDM. The
controls had no oligosaccharides or inulin added. Each sample was
pasteurized at 70°C for 30min. Tubes inoculated with the cultures
prepared above were incubated anaerobically as described
previously at 37°C for 48hr. A sample was taken at 6hr intervals
and diluted (1:10, v/v) with 0.2% EDTA (pH 12.0) and turbidity was
measured at 640 nm as described by Hughes and Hoover (1995).
Uninoculated NDM was used as the blank for turbidity
measurement. Specific growth rate (u) for each culture was

calculated using the following equation (Roy and Goulet, 1991):

In X2 -1I‘l X1

 

t1-t2

X2 and X1 are the cell density at time t2 and t1. Mean doubling time

(Td) was calculated as:

In 2
Ta:

32

Another sample was collected for pH measurements. The pH of
culture samples was also monitored at 6hr intervals for 48hr.

3.2.3 Effect of oligosaccharides and inulin on viability of
bifidobacteria during refrigerated storage.

Each bifidobacteria sample was cultured anaerobically at 37°C
for 48 hr with or without oligosaccharides and inulin as previously
described. The samples were stored at 5°C for 4 weeks. One ml
each of the bifidobacteria sample was diluted with 99ml of 0.1%
(w/v) bacto peptone (Difco) and subsequent serial dilutions were
made. Bifidobacteria were enumerated using MRSL agar. The
inoculated plates were incubated anaerobically at 37°C for 48hr
using Gas Pak® (Becton Dickinson Co.). The colonies were counted
as described previously. Percent viability of each culture sample was
calculated as follows:

% viability = (cfu at 4 week storage) / (initial cfu before storage) x

100

3.2.4 HPLC assay.

Culture activity was determined by end products of
fermentation (lactic acid and acetic acid) using HPLC (High
Performance Liquid Chromatography). The HPLC system (Waters

Associates, Inc., Milford, MA) available in our laboratory consist of a

33

M-45 solvent delivery system, a 486 UV/Vis tunable absorbance
detector and a 730 data module. The UV detector, set at 220nm,
was used for quantification of organic acids. An Aminex HPX-87H
Column (300 mm x 7.8 mm, Bio-Rad Laboratories, Richmond, CA).
and guard column with disposable cartridges H+ (Bio-Rad
Laboratories) maintained at 65°C was used for the analysis. A
mobile phase of 0.009N H2804 filtered through a 0.45 pm
membrane filter (Millipore Corp., Bedford, MA) and degassed by
vacuum was used at a flow rate of a 0.6 ml/min.. The wavelength
for the detection of organic acid set at 220 nm was optimized and
organic acid was quantitated (Bouzas et al., 1991). Standard
solution of organic acids (lactic acid and acetic acid; Sigma) was
prepared to establish elution times and calibration curves.

NDM fermented with two strains of bifidobacteria in 5% of FOS,
GOS, or inulin were prepared for the HPLC analysis using the
method described by Dubey and Mistry (1996). One hundred
microliters of 15.8N HN03 and 14.9ml of 0.009N H2804 were added
to 1.5ml of sample and centrifuged at 5000 x g for 10 min. The
supernatant was filtered using Whatman #1 filter paper, 0.22 pm
membrane filter (Millipore Corp., Bedford, MA) and eluted through a
reversed phase Supelclean tube (Supelco Inc., Bellefonte, PA) and

stored in HPLC vials at -20°C until the HPLC analysis. Figure 4

34

shows a typical HPLC chromatogram of lactic acid and acetic acid
produced by bifidobacteria cultured in 12% NDM with 5% F08,

G08, and inulin.

3.2.5 Standard curves and HPLC chromatograms of acetic acid
and lactic acid.

Acetic acid had a retention time of 12 min (Figure 4). A
standard curve was made using five different standard acetic acid
solutions (2.5, 5, 10, 20 and 40 mmol/L). Figure 5 shows the
standard curve for acetic acid determination (R2 = 0.99).
Concentration of acetic acid (mmol/ L) in the samples was calculated
using the following relationship:

Peak area = -56254.5 + (22021.6 x acetic acid (mmol/L)).

Lactic acid had a retention time of 10 min (Figure 4). A
standard curve was made using five different standard acetic acid
solutions (2.0, 2.2, 3.0, 3.7 and 4.4 mmol/L). Figure 6 shows the
standard curve for lactic acid determination (R2 = 0.99). The
concentration of lactic acid (mmol/L) was in the samples calculated
using the following relationship:

Peak area = -95772 + (79723.8 x lactic acid (mmol/L)).

35

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”6"..-

 

 

 

Peak area

36

 

 

 

1e+6
Peak area = -56254.5 + (22021.6 x Acetic acid (mmollL))
R2 = 0.997
8e+5 —
6e+5 ~
4e+5 —
26+5 n
O
O
0910 r I I r I I I I I I

 

0 4 8 12 16 20 24 28 32 36 40

Concentration of acetic acid (mmol/L)

Figure 5. Acetic acid standard curve using different standard

acetic acid solutions (25, 5, 10, 20 and 40 mmol/ L).

Peak area

37

 

 

3e+5
Peak area = -95772 + (79723.8 x Lactic acid (mmollL))
R2 = 0.985
2e+5 —
1e+5 a .
O
Oe+0 Tr I I T I I

 

 

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Concentration of lactic acid (mmol/L)

Figure 6. Lactic acid standard curve using different standard

lactic acid solutions (2.0, 2.2, 3.0, 3.7 and 4.4 mmol/L).

38

3.3 Redox potential of NDM cultured with Streptococcus
salivarius subsp. thermophilus and Lactobacillus delbrueckii
subsp. bulgaricus and its effect on growth of bifidobacteria.
3.3.1 Selective medium for lactic acid bacteria

The first part of this research involved screening selective media
to enumerate the specific lactic acid bacteria of interest in this study.
Various media were screened for selective enumeration of S.
salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus.
Lee’s agar, Streptosel agar, Azide dextrose agar, ST agar and M17
agar were evaluated for selective enumeration of S. salivarius subsp.
thermophilus. MRS agar containing oxgall and modified RCA
(Reinforced Clostridial agar) were evaluated for selective
enumeration of L. delbrueckii subsp. bulgaricus. RCA was modified
by supplementing with 2% bacto agar (Difco) and adjusting pH to
5.0 after autoclaving. M17 agar was shown to be the most
successful in enumerating S. salivarius subsp. thermophilus and
modified RCA for selective enumeration of L. delbrueckii subsp.

bulgaricus.

3.3.2 Screening L. delbrueckii subsp. bulgaricus and S.
salivarius subsp. thermophilus.

Initially, S. salivarius subsp. thermophilus and L. delbrueckii

39

subsp. bulgaricus were screen for their ability to provide optimum pH
and redox potential conditions for growth of bifidobacteria. S.
salivarius subsp. thermophilus St—52, St-113, St-133, St-134, L.
delbrueckii subsp. bulgaricus Lr-28, Lr-78, Lr-79 from Sanofi Bio-
Industries (Waukesha, WI) and NCK231 from North Carolina State
University were screened. S. salivan'us subsp. thermophilus St-133
was selected because of its ability to reduce redox potential faster
than other strains. L. delbrueckii subsp. bulgaricus NCK 231 was
selected because it produced the most suitable amount of acid in

NDM.

3.3.3 Culture preparation.

Bifidobacterium Bf—l and S. salivarius subsp. thermophilus St-
133 was provided from Sanofi Bio-Industries (Waukesha, WI). L.
delbrueckii subsp. bulgaricus NCK 231 was supplied by North
Carolina State University and B. adolescentis M 101-4 was provided
from Japan Bifidus Foundation (Tokyo, Japan). S. salivarius subsp.
thermophilus St-133 and L. delbrueckii subsp. bulgaricus NCK231
were cultured and subcultured in MRS medium (Difco) containing
5% (w/v) lactose at 37°C for 48hr. Bifidobacterium Bf-l and B.
adolescentis M 101-4 were cultured and subcultured anaerobically

in MRS medium (Difco) containing 5% (w/v) lactose at 37 °C for 48hr

40

using Gas Pak® (Becton Dickinson Co.). Cultures were centrifuged
15min at 1000 x g at 4°C and resuspended in 12% (w/v)

pasteurized (90°C, 10 min) NDM (Difco).

3.3.4 Determining redox potential.

Twelve percent (w/v) pasteurized NDM cultured with or without
S. salivarius subsp. thermophilus St 133 and/ or L. delbrueckii
subsp. bulgaricus NCK 231 was inoculated with Bifidobacterium Bf-
1 or B. adolescentis M101-4 to a final ratio of 1:1, 1:2, 2:1, 1:1:1,
1:2:1, 1:122, or 2:1:1 (based on cfu). These ratios were obtained
after determining cells counts of each pure cultures cultured in
MRSL and appropriate volumes were transferred into NDM to have
a total inoculum at a 5% (v/v) level. Redox potential of NDM
samples was monitored at 12h intervals for 48h using a platinum
electrode (Corning Incorp., New York). Each sample was
equilibriated at 25°C for 10 min prior to determining redox
potential. The electrode was submerged into the sample and mV
value was measured. The Eh7, the redox potential of system
standardized to pH 7 and 25°C, was calculated by a formula from
Montville and Conway (1982) by substituting the correction factor
59mV/ pH at 25°C (Montville and Conway, 1982). Thus, the

formula derived was: Eh7 = Eh measured - 59(7.00 - pH measured).

41

At each interval, a separate sample was collected for the pH

measurement.

3.3.5 Growth of bifidobacteria co-cultured with lactic acid
bacteria.

Growth of bifidobacteria co-cultured with lactic acid bacteria
was monitored at 12h intervals for 48h. Inoculated samples were
incubated at 37°C for 48h. Bifidobacteria were enumerated using
anaerobic incubation of MRS agar containing 5% (w/v) lactose and
5% (v/v) NPNL antibiotic solution. S. salivarius subsp. thermophilus
was enumerated using M17 agar, and L. delbrueckii subsp.
bulgaricus was enumerated using modified RCA as described

previously.

3.4 Viability of bifidobacteria in yogurt manufactured using
commercial yogurt starter cultures and selected condition.

S. salivarius subsp. thermophilus and L. delbrueckii subsp.
bulgaricus used in this section were selected for pH and redox
potential conditions for growth of bifidobacteria as described
previously. S. salivarius subsp. thermophilus St—133 and L.
delbrueckii subsp. bulgaricus NCK 231 were cultured and

subcultured as described previously. Bifidobacterium Bf-l and Bf—6

42

were cultured and subcultured anaerobically as described
previously. Cultures were centrifuged 15 min at 1000 x g at 4°C
and resuspended in 12% (w/v) pasteurized (90°C, 10 min) NDM
(Difco) containing 5% (w/v) fructooligosaccharide which previously
have been determined to be optimum concentration of
fructooligosaccharide to enhance the growth and viability of
bifidobacteria. Twelve percent (w/v)' pasteurized NDM containing
fructooligosaccharide cultured with S. salivarius subsp.
thermophilus St-133 and L. delbrueckii subsp. bulgaricus NCK 231
was inoculated with Bifidobacterium Bf—l to a final ratio of 4:1:2
(Bifidobacterium: L. delbrueckii subsp. bulgaricus: S. salivarius subsp.
thermophilus; based on cfu) which previously have been determined
to be terms of redox potential and acid production. Inoculated
samples were incubated at 37°C for 8hr and stored at 5°C for 15
days. Viability of bifidobacteria was monitored at 5 days intervals
for 15 days. Yogurt containing bifidobacteria manufactured using
commercial yogurt starter cultures obtained from Chr. Hansen’s

Laboratories Inc. (Milwaukee, WI) was used as a control.

3.5 Statistical analysis.
In 3.1., three batches of milk and yogurt were purchased at

three different times. In 3.2, 3.3 and 3.4 analysis were conducted

43

in triplicates. All experiments were replicated three times in a
randomized design: Statistical analysis was done using Sigma Stat
1.0 (Jandel Corp., San Rafael, CA). Appropriate comparisons were
made using Student—Newman—Keuls test for multiple comparisons.

A p< 0.05 was considered statistically significant.

IV. RESULTS & DISCUSSION

A. Viability of bifidobacteria in commercial dairy products.
4.1 Evaluation of media for selective enumeration of
bifidobacteria.

Table 1 shows the evaluation of various media for selective
enumeration of bifidobacteria. Brain heart infusion agar did not
inhibit S. salivarius subsp. thermophilus and Columbia agar did not
inhibit L. delbmeckii subsp. bulgaricus. RCA inhibited both
bifidobacteria and S. salivarius subsp. thermophilus. It was
concluded that they would not be suitable for selective enumeration
of bifidobacteria from dairy products such as yogurt. MRS agar
containing 5% (w/v) lactose and 5% (v/v) NPNL antibiotic solution
was the most successful in inhibiting growth of lactic acid bacteria
and selective enumeration of bifidobacteria. Wijsman et al. (1989)
reported that N PNL agar gave the highest recovery of bifidobacteria in
dairy products compare to MSB agar and Bifidobacterium medium.
Bifidobacteria grown on selective medium were more irregular shaped
compare to when grown on non-selective medium. Samona and
Robinson (1991) suggested that presence of inhibitors such as
antibiotics caused alterations in morphology of bifidobacteria. MRSL
+ NPNL were used for selective enumeration of bifidobacteria in the

following studies.

44

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4.2 Viability of bifidobacteria and lactic acid bacteria in
commercial A] B milk

Figure 7 shows the viability of bifidobacteria and lactic acid
bacteria in commercial A/ B milk. Although, a significant decrease
(p< 0.05) in the bifidobacteria counts were observed at 3 days prior
to product expiration, viability of bifidobacteria in commercial A/ B
milk remained above 106 cfu/ ml until the product expired. Six days
after the expiration date, bifidobacteria counts dropped below 10°
cfu/ ml. Lankaputhra et al. (1996) observed that viability of B.
infantis in 12% skim milk at pH 4.3 were decreased by 30% after 12
days of storage at 4°C and more than 82% after 24 days at the same
temperature. Medina and Jordano (1994) reported on the
bifidobacteria count of fermented milk produced in Spain that was
stored at 7°C. They observed a 92.6% decrease in bifidobacteria
count when the product was expired. In our study, we observed a
70.7% reduction in bifidobacteria count at the time of expiration.
Comparing to Medina and Jordano’s study, our product was not a
fermented milk product. The pH of this product remained at 6.6 or
above during the investigation (Table 2). Thus, acid injury to the
organism was prevented. L. acidophilus counts had decreased by

37.0% in the A/ B milk at expiration time and the numbers were

47

107

 

106 L

cfulml milk

 

 

 

Days from expiration date

 

film
an)
a:

“no:
—lm
~10)

cfulml milk
3:.

 

 

 

Days from expiration data

Figure 7. Viability of (A) bifidobacteria and (B) lactic acid bacteria in commercial
A/B milk during 18 days of refrigerated storage at 5°C. 0 = Expiration day, -9, -6,
-3 = days prior to product expiration, 3, 6. 9. = days past expiration. Bars with

different letters are significantly different (p< 0.05)

48

Table 2. The pH of commercial A/B milk during refrigerated storage.

 

Days from product expiration1 (pH?)
-6 6601-0028
-3 6.60i-0.08°l
0 6.58:0.04‘al
3 6.60:0.04a
6 6.60:0.01a
9 6.56:0.01‘=1

 

10 = Product expiration day, -9, -6, and -3 = days prior to product
expiration, 3, 6, and 9 = days past expiration.

2Means with the same superscript are not significantly different
(p>0.05). Means with standard deviations; n=3 for all treatment.

49

above 10° cfu/ ml, however this change was not significantly during

the duration of this study (Figure 7).

4.3 Viability of bifidobacteria and lactic acid bacteria in
commercial yogurt

Figure 8 shows the viability of bifidobacteria and lactic acid
bacteria in commercial brand A yogurt. Although a significant
decrease (p<0.05) was observed one week past product expiration
day, viability of bifidobacteria in this product remained above 10°
cfu/ g, 2 weeks past the product expiration. It was only 3 weeks
after the product expired that the counts were below 106 cfu/ g.
Lactic acid bacteria counts were maintained above 107 cfu / g during
the duration of the study although a significant decline (p<0.05) was
observed on the expiration day of the product. In brand A yogurt,
bifidobacteria and lactic acid bacteria counts decreased 64.3% and
87.0%, respectively when the product expired. The pH of brand A
yogurt during refrigerated storage is shown in Table 3.

Figure 9 shows the viability of bifidobacteria and lactic acid
bacteria in commercial brand B yogurt. Viability of bifidobacteria in
this product steadily declined during refrigerated storage. This
decline was significant (p<0.05) at 1 week prior to product

expiration and again at the date of expiration. However,

50

bifidobacteria counts remained above 106 cfu/ g until 2 weeks past
product expiration. Lactic acid bacteria count in brand B yogurt
although declined significantly (p<0.05) at 1 week prior to product
expiration the counts were above 10° cfu/ g during the duration of
the study. Bifidobacteria and lactic acid bacteria counts decreased
88.0% and 65.0%, respectively on the expiration day. The pH of
brand B yogurt during refrigerated storage is shown in Table 4.
Brand B yogurt had lower lactic acid bacteria counts than brand A.
Difference in results may be due to different volume of inoculum,
different processing environment, and differences in lactic acid
bacteria strains between the two brands of yogurt.

Laroia and Martin, (1991) reported that viability of B. bifidum
was very poor in low-pH (3.9—4.6) frozen fermented dairy desserts.
However, Modler et a1 (1990) reported that some strains of
bifidobacteria showed acid tolerance at pH 4.0. Blanchette et al.
(1996) reported on manufacturing creamed cottage cheese with B.
infantis ATCC 27920G and count of B. infantis in cottage cheeses
was 107 cfu/ g after the cream dressing was fermented by B. infantis.
Viability of bifidobacteria in creamed cottage cheese was decreased
2-4 log cycles during storage for 15 days at 4°C. Hekmat and
McMahon (1992) reported that ice cream may serve as a good

vehicle for delivering bifidobacteria having potential health benefits

51

 

cfulg yogurt

 

cfulg yogurt

 

weeks from expiration date

Figure 8. Viability of (A) bifidobacteria and (B) lactic acid bacteria in
commercial brand A yogurt during 6 weeks of refrigerated storage at 5°C.
0 = Expiration day, -3, -2, -1 = weeks prior to product expiration, 1, 2, 3, =
weeks past expiration. Bars with different letters are significantly different

(p< 0.05)

52

Table 3. The pH of commercial brand A yogurt during
refrigerated storage.

 

 

Weeks from product expiration1 (sz)
-2 4.23:0.01at
— 1 4.21i0.02a
0 4.23i0.02a
1 4.211‘001a
2 4. 18:0.01b
3 4. 17:0.01b

 

10 = Product expiration day, -3, -2, and -1 = weeks prior to
product expiration, 3, 2, and 1 = weeks past expiration.

2Means with different superscripts are significantly different (p<
0.05). Means with standard deviations; n=3 for all treatment.

53

 

108 A

t a
a
a
o:
o
>- 107— °
2’ c
:1
M- C
o
C
106—
C

 

 

 

.3 .2 -1 0 1 2 3
weeks from expiration date

cfulg yogurt

 

weeks from expiration date

Figure 9. Viability of (A) bifidobacteria and (B) lactic acid bacteria in
commercial brand B yogurt during 6 weeks of refrigerated storage at 5°C. 0
= Expiration day, -3, -2, -l = weeks prior to product expiration, l, 2. 3, =
weeks past expiration. Bars with different letters are significantly different

(p<0.05)

54

Table 4. The pH of commercial brand B yogurt during

refrigerated storage.

 

 

Weeks from product expiration1 (pH2)
" -577" _._ 4.20:0.01—QTMT
-2 4.20i0.01a
-1 4.19i0.00°l
0 4.1910008
1 4.17J_r0.01b
2 4.17:0.01b
3 4.16i0.01b

 

10 = Product expiration day, -3, -2, and -1 = weeks prior to
product expiration, 3, 2, and 1 = weeks past expiration.

2Means with different superscripts are significantly different (p<
0.05). Means with standard deviations; n=3 for all treatment.

55

to humans. Their study indicated that ice cream mix fermented
with L. acidophilus and B. bifidum had higher numbers of viable cell
after 17 weeks of storage at -29°C. After freezing of the fermented
mix, L. acidophilus and B. bifidum count were 1.5x108 cfu/ ml and
2.5x108 cfu / ml, respectively. Seventeen weeks after frozen storage,
counts of L. acidophilus and B. bifidum were decreased by two log
cycle to 4x 106 and by one log cycles to 1x107 cfu/ml, respectively.
Shah et al. (1995) reported on the initial bifidobacteria counts to
be 10°-107 cfu/ g in two of five brands of yogurt they studied and
other three brands of yogurt had counts < 103 cfu/ g. Samples of five
brands of commercial yogurt were obtained directly from the
processors within two to three days of production. They also
reported that all products showed a constant decline in the
bifidobacteria and lactic acid bacteria counts during storage. There
appears to be a significant variation in counts among products.
Although significance of viability in receiving health benefits of
lactic acid bacteria have not been clearly established in the
scientific literature, USA, France, Japan, South Korea and Poland
already have regulation for viable culture numbers in fermented dairy
product which range 2106 - 108 cfu/ ml. In the US. these standards
are used for the Seal Program to promote live and active cultures.

Individual states such as California and Oregon state have also

56

adopted specific regulations pertaining to viability of cultures in
dairy products consistent with the national standard (Sanders et al.,
1996). There appears to be a great variation in viability of cultures
among products. Sanders et al., (1996) reported that clinical
studies have shown significant clinical benefits are observed upon
ingestion of approximately 109 - 1010 organisms/d. Viability of
cultures in dairy products may or may not be consistent with
clinical studies. Thus, next part of this research focused on
enhancing growth of bifidobacteria in dairy foods and maintaining

its viability during refrigerated storage.

4.4 Effect of oligosaccharides and inulin on growth of
bifidobacteria.

Table 5 shows the mean doubling times of Bifidobacterium sp.
Bf—l and Bf—6 in 12% NDM in the presence of various
concentrations of oligosaccharides and inulin. Mean doubling time
was used as a measure of specific growth rate for each culture.
FOS showed the highest growth-promoting activity (p<0.05) on both
strains of bifidobacteria when 21% was added as evident by the
mean doubling time. Growth of Bifidobacterium sp. Bf—l and Bf-6
was stimulated (p<0.05) when 23% of GOS was added to NDM prior

to inoculation. In case of inulin, growth of Bifidobacterium sp. Bf-l

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58

and Bf—6 was stimulated (p<0.05) only when 25% of inulin was
added. Bifidobacteria strains utilized inulin slowly, which deficient
in inulinase necessary for their metabolism (Yamazaki and Dilawri,
1990). Inulinase is an enzyme which splits off fructose moieties
from certain sugars displaying a fructose unit at the terminal B-2,1
position (Vandamme and Derycke, 1983). Fructooligosaccharide
can be characterized as polymers of D-fructose joined by (3(2—>1)
linkages and terminated with a D-glucose molecule linked to
fructose by an a(1—>2) bond as in sucrose (Kosaric et al., 1984).
Hidaka et al. (1986) reported that some bifidobacteria produce
enzymes, which hydrolyzed fructooligosaccharide (FOS) efficiently.
This is consistent with our study in that FOS was utilized most
effectively with the two strains of bifidobacteria studied and showing
the highest growth-promoting activity on bifidobacteria. However,
the degree of polymerization (DP) of the fructooligosaccharide is also
important. DP of fructooligosaccharide used in this study was
between 2 and 7. Gibson and Roberfroid (1995) reported that
maximum activity was obtained with short chains of
fructooligosaccharide with DP of between 3 and 5. However, Dubey
and Mistry (1996) reported that 0.5% of FOS did not stimulated the
growth of bifidobacteria in infant formulas. Difference in results

may be due to different composition of infant formulas and NDM,

59

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60

different concentration of FOS used, and differences in
bifidobacteria strains between the two studies.

4.5 Effect of oligosaccharides on viability of bifidobacteria
during refrigerated storage.

Table 6 shows the viability of two commercial bifidobacteria
after 4 weeks of refrigerated storage. The initial viability of
bifidobacteria was calculated to be 100% for both strains of
bifidobacteria. Bifidobacterium sp. Bf-l and Bf-6 in control 12%
N DM exhibited marked drop in viability of approximately 90% after
4 weeks of refrigerated storage at 5°C. Only 11.6 i 1.6% and 9.3 i
1.7% of Bifidobacterium sp. Bf-l and Bf—6 respectively remaining
viable, after 4 weeks of refrigerated storage at 5°C. The viability of
both strains of bifidobacteria was enhanced significantly (p<0.05)
when 23% of FOS or GOS was added. However, concentration of
5% was needed for inulin to show a significant effect (P<0.05) on
viability of Bifidobacterium sp. Bf-l. Inulin had no effect on viability
of Bifidobacterium sp. Bf—6. The preparation of freshly autoclaved
12% skim milk as a medium, followed by inoculation with a large
number of cells, incubation under anaerobic condition, and
presence of good carbon sources, may have contributed to
enhancing growth and maintaining good viability through

refrigerated storage. Viabilities observed in this study was better

61

than those reported by Lee et al. (1996), probably due to commercial
strains used in this study, which probably had better acid and
oxygen tolerance, and perhaps better utilization of oligosaccharides
and inulin. Modler et al. (1990) reported that FOS had no effect on
viability of bifidobacteria in ice cream. In their study, FOS was
mixed into ice cream and stored at —17°C. Bifidobacteria probably

didn’t have the opportunity to utilize FOS.

4.6 Effect of oligosaccharides and inulin on the production of
acetic acid and lactic acid by Bifidobacterium sp.

Culture activity of commercial bifidobacteria was determined by
measuring fermentation end products (lactic acid and acetic acid)
by HPLC. Table 7 shows the production and ratio of acetic and
lactic acid by Bifidobacterium sp. Bf-l and Bf—6. Acetic acid
production ranged from 6.2 mmol/ L (in 12% NDM; control) to 28.7
mmol/L (FOS) for Bifidobacterium sp. Bf—l. For Bifidobacterium sp.
Bf-6, acetic acid production ranged from 10.3 mmol/ L (in 12%
NDM; control) to 15.8 mmol/L (FOS). Lactic acid production on the
other hand ranged from 4.9 mmol/ L (in 12% NDM; control) to 15.5
mmol/L (FOS) for Bifidobacterium sp. Bf—l. For Bifidobacterium sp.
Bf-6, lactic acid production ranged from 5.7 mmol/L (in 12% NDM;

control) to 11.7 mmol/ L (FOS). Acetic acid and lactic acid

62

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production was stimulated significantly (p<0.05) in Bifidobacterium
sp. Bf—l and Bf-6 when FOS or GOS were added to NDM. Whereas,
inulin only stimulated (p<0.05) acetic acid production in
Bifidobacterium sp. Bf-l and did not stimulate lactic acid production
in either strain of bifidobacteria. The theoretical ratio of acetic acid to
lactic acid by bifidobacteria is reported to be 3:2 (Roy and Goulet,
1991; Tamine et al., 1995). Organic acid in dairy products is
important in monitoring culture activity, understanding microbial
metabolism, and in determining quality of milk products (Bouzas et
al., 1991). Fermented dairy products have various proportions of
lactic acid ranging from 0.9 - 6%, which has been reported to develop
the characteristic flavor and texture, and the inhibition of certain
pathogenic bacteria in dairy products (Driessen and de Boer, 1989;
Fernandez-Garcia and McGregor, 1994). Acetic acid in fermented
dairy products also has antimicrobial effects (Samona et al., 1996).
However, high acetic acid concentrations in dairy products are not
typically desirable from a quality stand point. The mean ratio of
acetic acid and lactic acid in this study ranged from 1.24 to 1.85, and
no difference in the ratios were observed between the treatments
except for Bifidobacterium sp. Bf-l grown with FOS (Table 7).
Samona et al. (1996) reported that an imbalanced ratio of acetic acid

and lactic acid could contribute to risk of a vinegar over lactic acid

63

production was stimulated significantly (p<0.05) in Bifidobacterium
sp. Bf-l and Bf—6 when FOS or GOS were added to NDM. Whereas,
inulin only stimulated (p<0.05) acetic acid production in
Bifidobacterium sp. Bf-l and did not stimulate lactic acid production
in either strain of bifidobacteria. The theoretical ratio of acetic acid to
lactic acid by bifidobacteria is reported to be 3:2 (Roy and Goulet,
1991 ; Tamine et al., 1995). Organic acid in dairy products is
important in monitoring culture activity, understanding microbial
metabolism, and in determining quality of milk products (Bouzas et
al., 1991). Fermented dairy products have various proportions of
lactic acid ranging from 0.9 - 6%, which has been reported to develop
the characteristic flavor and texture, and the inhibition of certain
pathogenic bacteria in dairy products (Driessen and de Boer, 1989;
Fernandez-Garcia and McGregor, 1994). Acetic acid in fermented
dairy products also has antimicrobial effects (Samona et al., 1996).
However, high acetic acid concentrations in dairy products are not
typically desirable from a quality stand point. The mean ratio of
acetic acid and lactic acid in this study ranged from 1.24 to 1.85, and
no difference in the ratios were observed between the treatments
except for Bifidobacterium sp. Bf—l grown with FOS (Table 7).
Samona et al. (1996) reported that an imbalanced ratio of acetic acid

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M 17 agar was shown to be the most successful in enumerating of S.
salivarius subsp. thermophilus and modified RCA was shown to be
the most successful for the selective enumeration of L. delbrueckii

subsp. bulgaricus (Table 8).

4.8 Screening L. delbrueckii subsp. bulgaricus and S.
salivarius subsp. thermophilus.

S. salivarius subsp. thermophilus St-52, St-113, St-133, St-134,
L. delbrueckii subsp. bulgaricus Lr—28, Lr-78, Lr-79, NCK231 were
screened for their ability to provide optimum pH and redox potential
conditions for growth of bifidobacteria. S. salivarius subsp.
thernwphilus St 133 showed the ability to reduce redox potential
faster than other strains (Table 9). It has been shown that some
strains of S. salivarius subsp. thermophilus have high oxygen
consuming ability thus reducing redox potential suitable for growth
of bifidobacteria (Okonogi et al., 1986; Reuter, 1989; Tinson et al.,
1982; Teraguchi et al., 1987). L. delbrueckii subsp. bulgaricus is
typically an acid producer. L. delbrueckii subsp. bulgaricus NCK 231
was selected because it produced the most suitable amount of acid
in 12% NDM (Table 10). Most strains of bifidobacteria are very
sensitive to the low pH (s 4.6) however, Rasic and Kurmann (1983)

reported that some strains of B. bifidum showed acid tolerance to

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pH 4.0. Although, acid tolerance varies from strain to strain, pH
4.6 — 4.0 might be the lowest pH value to prevent acid injury to

growth of bifidobacteria.

4.9 Growth of bifidobacteria co-cultured with L. delbrueckii
subsp. bulgaricus NCK 231 and S. salivarius subsp.
thermophi lus St- 133.

Table 11 and 12 show the EH7, redox potential of the NDM
standardized to pH 7 and 25°C that has been co-cultured with L.
delbrueckii subsp. bulgaricus NCK 231, S. salivarius subsp.
thermophilus St-133 and Bifidobacterium sp. Bf-1 or B. adolescentis
M101-4, respectively over a 24 hr incubation period. EH7 of NDM
cultured with S. salivarius subsp. thermophilus St-133 alone was
lowest (p<0.05) both at 12 and 24 hr compared to Eh7 of NDM,
NDM cultured with L. delbrueckii subsp. bulgaricus NCK 231 or
bifidobacteria alone. Co-culturing bifidobacteria with S. salivarius
subsp. thermophilus St-133 was more effective in lowering redox
potential (p<0.05) than co—culturing with L. delbrueckii subsp.
bulgaricus NCK 231. When co-cultured, a ratio of 1:2
(bifidobacteria to S. salivarius subsp. thermophilus St-133) provided
the lowest (p<0.05) Eh7 after 24 hr of incubation in both strains of

bifidobacteria (Table 11 and 12). When either strains of

70

bifidobacteria was co-cultured with L. delbrueckii subsp. bulgaricus
NCK 231 and S. salivarius subsp. thermophilus St-133, the most
effective ratio for lowering Eh7 was 1:1:2 (when S. salivarius subsp.
thermophilus St-133 was inoculated at twice the concentration of L.
delbrueckii subsp. bulgaricus NCK 231 and bifidobacteria). The Eh7
at 36 and 48h were similar to that of 24h for all treatments. Thus,
these results are not provided in the tables. Okonogi et al. (1986)
reported that novel strain of S. salivarius subsp. thermophilus has
high oxygen utilization and this ability can enhance viability of
bifidobacteria. This patent strains of S. salivarius subsp.
thermophilus M-8202, M-8203, M-8204, and M-8205, which have
high oxygen uptake ability could be utilized to reduce fermentation
cost of bifidobacteria in commercial culture processing. During
anaerobic fermentation, S. salivarius subsp. thermophilus
catabolizes lmol of glucose to 2mol of lactate through the glycolytic
pathway, but in aerobic metabolism glucose or pyruvate leads to
the formation of acetate, a-acetolactate, acetoin, and diacetyl in
addition to lactic acid production. NADH generated in aerobic
metabolism is consumed in the reaction catalyzed by NADH
oxidase. Aerobic 02 uptake was also observed with lactobacilli but
to a lesser extent (Tinson et al., 1982; Teraguchi et al., 1987).

Bifidobacteria are classified as anaerobes thus, certain precautions

71

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73

are required to prevent the toxic effects of oxygen when
Bifidobacterium sp. are cultivated for industrial application (De
Vries and Stouthamer, 1989).

Table 11 and 12 also show the pH variation of 12% NDM when
each bifidobacteria strain was co-cultured with both S.salivarius
subsp. thermophilus St-133 or L. delbrueckii subsp. bulgaricus NCK
231. Tinson et al. (1982) reported that the rate of acid development
by mixed starter cultures, S. salivarius subsp. thermophilus and L.
delbrueckii subsp. bulgaricus, was greater than that of single strain.
In our study, pH was primarily influenced by the inoculum level of
L. delbrueckii subsp. bulgaricus.

Table 13 and 14 show the growth of Bifidobacterium sp. Bf-l
and B. adolescentis M101-4 in 12% NDM when it was co-cultured
with L. delbrueckii subsp. bulgaricus NCK 231 and S. salivarius
subsp. thermophilus St-133. For both strains of bifidobacteria
there were no differences observed in the cfu of bifidobacteria when
cultured alone compared to bifidobacteria co—cultured with L.
delbrueckii subsp. bulgaricus NCK 231 and S. salivarius subsp.
thermophilus St-133. Co-culturing bifidobacteria with lactic acid
bacteria although provided the initial low Eh7 in NDM, this was not
significant to enhance the growth of bifidobacteria due to the low

pH of the system. It appears that growth of bifidobacteria is more

74

strongly influenced by pH than Eh7. De Vries and Stouthamer
(1989) reported that although the sensitivity to oxygen varies
between strains of Bifidobacterium sp., redox potential is related to
the growth of Bifidobacterium sp. Our results are consistent with
Klaver et al. (1993) who reported that low redox potential but more
importantly proper pH change in the early phase of incubation are
required for good growth of bifidobacteria. Growth of
Bifidobacterium sp. in NDM was related to pH more so than Eh7.
Table 15 and 16 show the growth of S. salivarius subsp.
thermophilus St-133 in 12% NDM when it was co—cultured with L.
delbrueckii subsp. bulgaricus NCK 231 and Bifidobacterium sp. Bf-l
or B. adolescentis M101-4. There were no differences observed in
the cfu of S. salivarius subsp. thermophilus St-133 when cultured
alone compared to S. salivarius subsp. thermophilus St-133 co-
cultured with L. delbrueckii subsp. bulgaricus NCK 231 and
Bifidobacterium sp. Bf-l or B. adolescentis M101-4. Table 17 and
18 show the growth of L. delbrueckii subsp. bulgaricus NCK 231 in
12% NDM when it was co-cultured with S. salivarius subsp.
thermophilus St-133 and Bifidobacterium sp. Bf-l or B. adolescentis
M101-4. There were no differences observed in the cfu of L.
delbrueckii subsp. bulgaricus NCK 231 when L. delbrueckii subsp.

bulgaricus NCK 231 cultured alone compared to co-cultured with S.

75

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81

salivarius subsp. thermophilus St-133 and Bifidobacterium sp. Bf—l
or B. adolescentis M101-4. This was similar to the data on growth

of bifidobacteria.

4.10 The conditions to enhance growth and viability of
bifidobacteria in yogurt.

Based on the results discussed previously, in this study the
conditions determine above were combined to enhance growth and
viability of bifidobacteria in yogurt. The culture blend selected was
a ratio of 4: 1: 2 of Bifidobacterium sp.: Lactobacillus delbrueckii
subsp. bulgaricus: Streptoccus salivarius subsp. thermophilus. These
cultures were inoculated into 12% NDM supplemented with 5%
F08. Two commercial yogurt culture blends containing
bifidobacteria PY-3 and PY-58 were used as controls. The initial
count of bifidobacteria under our conditions was 1.56x108 cfu/ g.
The initial counts of bifidobacteria manufactured using commercial
yogurt starter cultures PY-3 and PY-58, were 6.67x106 cfu/ g and
7 .43x 108 cfu/ g, respectively.

To investigate the viability of bifidobacteria under our
conditions, the percent viability of bifidobacteria during 15 days of
refrigerated storage at 5°C was determined. After 5 days of storage,

74 i 4.0% of bifidobacteria remained viable in the product

82

manufactured under our conditions. Whereas, in yogurts
manufactured with PY-3 and PY—58, 61 i 9.5% and 60 i 6.5%,
respectively of the organisms were viable. These difference,
however, were not statistically significant (Table 19). After 10 days
of refrigerated storage, the 47 i 11.1% of the bifidobacteria were
viable in the yogurt product produced under our conditions. This
was higher (p<0.05) than 28 i1.8 and 31 i 3.1 % which was the
viability of bifidobacteria remaining in yogurts manufactured with
PY-3 and PY-58, respectively. After 15 days of cold storage, the
viability of bifidobacteria in the yogurt product manufactured with
our conditions was 35 i 2.0%. This was more than double the
viability observed in yogurts manufactured with PY-3 and Py—58,
which had 15 i 2.1% and 17 i 3.6% viability respectively remaining
(Table 19). The pH of our product remained higher (p<0.05) than pH
of yogurt produced with the two commercial cultures (Table 20).
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and viability maintained during refrigerated storage by proper
selection of all strains of organisms, by proper their ratio and
inoculum levels, by providing suitable growth factors in the milk and
properly maintaining pH and redox potential in the milk medium.
Our results may be more consistent with the doses 1

recommended to receive health benefit of these organisms.

83

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V. CONCLUSIONS

<1> Our results show that the viability of bifidobacteria in
commercial milk (pH ~ 6.6) and two brands of commercial yogurt
(pH ~ 4.2) remained above 106 cfu/ml or g, respectively.

<2> The growth of Bifidobacterium sp. Bf—l and Bf—6 in NDM were
stimulated by FOS>GOS>Inulin. Both F08 and GOS had similar
effects on maintaining viability of Bifidobacterium sp. in N DM during
4 weeks of refrigerated storage. Both F08 and GOS stimulated
lactic acid and acetic acid production by Bifidobacterium sp. Among
the carbon sources tested, inulin was the least effective in
stimulating lactic acid and acetic acid production.

<3> Although S. salivarius subsp. thermophilus St-133 was effective
at lowering Eh7 of NDM media, the growth of Bifidobacterium sp. in
skim milk was influenced more by pH than Eh7.

<4> Growth and viability of bifidobacteria in milk was enhanced by
proper conditions such as strain selection, Optimizing inoculum
levels, using suitable growth factors and proper monitoring of pH

and redox potential.

85

REFERENCES

Azuma, N., K. Yamauchi, and T. Mitsuoka. 1984. Bifidus growth-
promoting activity of a glycomacropeptide derived from human
casein. Agric. Biol. Chem. 48: 2159.

Ballongue, J. 1993. Bifidobacteria and probiotic action. Ch. 13 in
Lactic Acid Bacteria, S. Salminen and A. Wright (Ed.), p. 357.
Marcel Deker, Inc., New York

Bezkorovainy, A., N. Topouzian, and R. M. Catchpole. 1986.
Mechanisms of ferric and ferrous iron uptake by Bifidobacterium
bzfidum var. pennsylvanicus. Clin. Physiol. Biochem. 4: 150.

Biavati, B., T. Sozzi, P. Mattarelli, and L. D. Trovatelli. 1992.
Survival of bifidobacteria from human habitat in acidified milk.
Microbiologica 15:197.

Blanchette, L. D. Roy, G. Belanger, and S. F. Gauthier. 1996.
Production of cottage cheese using dressing fermented by
bifidobacteria. J. Dairy Sci. 79: 8.

Bouzas, J ., C. A. Kantt, F. Bodyfelt, and J. A. Torres. 1991.
Simutaneous detection of sugars and organic acids in cheddar
cheese by high performance liquid chromatography. J. Food Sci.
56: 276.

Bullen, C. L., P. V. Tearle, and M. G. Stewart. 1977. The effect of
“humanized” milks and supplemented breast feeding on the fecal
flora of infants. Br. Med. J. 16: 403.

Crittenden, R. C., and M. J. Playne. 1996. Production, properties
and applications of food-grade oligosaccharides. Trends in Food
Sci. Technol. 7: 345.

Deguchi, Y., T. Morishita, and M. Mutai. 1985. Comparitive studies
on synthesis of water-soluble vitamins among human species of

bifidobacteria. Agric. Biol. Chem. 49: 13.

Desjardins, M., and D. Roy. 1990. Growth of bifidobacteria and their
enzyme profiles. J. Dairy Sci. 73: 299.

86

87

De Vries, W., and A. H. Stouthamer. 1989. Factors determing the
degree of anaerobiosis of Bifidobacterium strains. Arch.
Microbiol. 65: 275.

Dinakar, P., and V. V. Mistry. 1994. Growth and viability of
Bifidobacterium bifidum in Cheddar cheese. J. Dairy Sci. 77:
2854.

Driessen, F. M. 1988. New technology developments in the
manufacture of fermented milk. Int. Dairy Fed. Bull. 27: 129.

Driessen, F. M., and R. de Boer. 1989. Fermented milks with
selective intestinal bacteria. Neth. Milk Dairy J. 43: 367.

Dubey, U. K., and V. V. Mistry. 1996. Effect of bifidogenic factors on
growth characteristics of bifidobacteria in infant fomulas. J.
Dairy Sci. 79: 1156. ‘

Fernandes, C. F., and K. M. Shahani. 1990. Anticarcinogenic
and immuno logical properties of dietary lactobacilli. J. Food
Protect. 53: 704.

Fernandez-Garcia, E., and J. U. McGregor. 1994. Determination of
organic acids during the fermentation and cold storage of
yogurt. J. Dairy Sci. 77: 2934.

Gibson, G. R., and M. B. Roberfroid. 1995. Dietary modulation of
the human colonic microbiota: introducing the concept of
prebiotics. J. Nutr. 125: 1401.

Gilliland, S. E. 1989. Acidophilus milk products. J. Dairy Sci. 72:
2483.

Gilliland, S. E., and D. K. Walker. 1990. Factors to consider when
selecting a culture of Lactobacillus acidophilus as a dietaty
adjunct to produce a hypocholesterolemic effect in humans. J.
Dairy Sci. 73: 905.

Hawkins, S. M. 1993. Bifidobacteria in dairy products. Cult. Dairy
Prod. J. 28: 16.

88

Hayakawa, K., J. Mizutani, K. Wada, T. Masai, I. Yoshihara, and T.
Mitsuoka. 1990. Effect of soybean oligosaccharides on human
faecal flora. Microbial Ecology in Health and Disease 3: 293.

Hekmat, S., and D. J. McMahon. 1992. Survival of Lactobacillus
acidophilus and Bifidobacterium bifidum in ice cream for use as a
probiotic food. J. Dairy Sci. 75: 1415.

Hidaka, H., T. Eida, T. Takizawa, T. Tokunaga, and Y. Tashiro.
1986. Effects of frutooligosaccharides on intestinal flora and
human health. Bifidobacteria Microflora. 5: 37.

Hidaka, H., Y. Tashiro, and T. Eida. 1991. Proliferaton of
bifidobacteria by oligosachharides and their useful effect on
human health. Bifidobacteria Microflora. 10: 65.

Homma, N. 1988. Bifidobacteria as a resistance factor in human
beings. Bifidobacteria Microflora. 7: 35.

Hoover, D. 1993. Bifidobacteria: activity and potential benefits. Food
Technol. 47: 120.

Hori, S. 1984. Characteristics of "Mil-Mil E", a Bifidobacterium
fermented milk. J. Dairy Sci. (Suppl. 1). 46: 424.

Hotta, M., Y. Sato, S. Iwata, N. Yamashita, K. Sunakawa, T. Oikawa,
R. Tanaka, K. Watanabe, M. Yamjima, S. Sekiguchi, S. Arai, T.
Sakurai, and M. Mutai. 1987. Clinical effects of Bifidobacterium
preparations on pediatric intractable diarhea. Keio J. Med. 36:
298.

Hughes, D. B., and D. G. Hoover. 1991. Bifidobacteria: their
potential use in American dairy
products. Food Technol. 45: 74.

Hughes, D. B., and D. G. Hoover. 1995. Viability and enzymatic
activity of bifidobacteria in milk. J. Dairy Sci. 78: 268.

Ibrahim, S. A., and A. Bezkorovainy. 1994. Growth—promoting
factors for Bifidobacterium longum. J. Food Sci. 59: 189.

Ishibashi, N., and S. Shimamura. 1993. Bifidobacteria: research and
development in Japan. Food Technol. 47: 126.

89

Jao, Y. C., E. M. Mikolajcik, and P. M. T. Hansen. 1978. Growth of
Bifidobacterium bifidum var. pennsylvanicus in laboratory media
supplemented with amino sugars and spent broth from
Escherichia coli. J. Food Sci. 43: 1257.

Jaspers, D. A., L. K. Massey, and L. O. Luedecke. 1984. Efect of
consuming yogurts prepared with 3 culture strains on human
serum lipoproteins J. Food Sci. 49: 1178.

Jay, J. M. 1992. Fermented foods and related products of
fermentation. Ch. 16 in Modern Food Microbiology, 4th Ed.,
p.378. Chapman 8:. Hall, New York.

Kanbe, M. 1992. Uses of intestinal lactic acid bacteria and health.
Ch. 13 in Functions of Fermented Milk, Y. Nakazawa and A.
Hosono (Ed.), p. 289. Elsevier Applied Science, London.

Kim, H. S. 1988. Characterization of lactobacilli and bifidobacteria
as applied to dietary adjuncts. Cultured Dairy Products J. 23: 6.

Klaver, F. A. M., F. Kingma, and A. H. Weerkamp. 1993. Growth and
survival of bifidobacteria in milk. Netherlands Milk and Dairy J.
47: 151.

K00, M., and A. V. Rao. 1991. Long-term effect of bifidobacteria and
neosugar on precursor lesions of colonic cancer in CFl mice.
Nutr. Cancer 16: 249.

Kosaric, N., G. P. Cosentino, and A. Wieczorek. 1984. The Jerusalem
artichoke as an agricultured crop. Biomass. 5: 1.

Kroger, M., J. A. Kurmann, and J. L. Rasic. 1989. Fermented milks
-past, present and future. Food Technol. 43: 92.

Kurmann, J. A., Rasic, J. L., and M. Kroger. 1992. Encyclopedia of
Fermented Fresh Milk Products, Van Nostrand Reinhold, New
York.

Lankaputhra, W. E. V., N. P. Shah, and M. L. Britz. 1996. Survival
of bifidobacteria during refrigerated storage in the presence of
acid and hydrogen peroxide. Milchwissenschaft. 51: 65.

90

Laroia, S., and J.H. Martin. 1990. Effect of pH on survival of
Bifidobacterium bifidum and Lactobacillus acidophilus in frozen
fermented dairy desserts. Cult. Dairy Prod. J. 26: 13.

Lee, J. H., H. S. Shin, J. J. Pestka, and Z. Ustunol. 1996. Effect of
oligosaccharides and inulin on growth and viability of
bifidobacteria in fermented milk. J. Dairy Sci. 79 (Suppl. 1): 124.

Marin, M. L., J. H. Lee, J. Murtha, Z. Ustunol, and J. J. Pestka.
1997. Differential cytokine production in clonal macrophage and
T cell lines cultured with bifidobacteria. Accepted to J. Dairy Sci.

Marshall, V. M. 1987. Fermented milks and their furture trends. J.
Dairy Res. 54: 559. '

Marshall, V. M. 1993. Starter cultures for milk fermentation and
their characteristics. J. Society of Dairy Technol. 46: 49.

Matalon, M. E., and W. E. Sandine. 1986. Improved media for
differentiation of rods and cocci in yogurt. J. Dairy Sci. 69:
2569.

Martin, J. H., and K. M. Chou. 1992. Selection of bifidobacteria for
use as dietary adjuncts in cultured dairy foods. Cult. Dairy Prod.
J. 27: 21.

Martini, M. C., E. C. Lerebours, W. Lin, S. K. Harlander, N. M.
Berrada, J. M. Antoine, and D. A. Savaiano. 1991. Strains and
species of lactic acid bacteria in fermented milks: effect on in
vivo lactose digestion. Am. J. Clin. Nutr. 54: 1041.

McKellar, R. C., and H. W. Modler. 1989. Metabolism of
fructooligosaccharides by Bifidobacterium sp. Appl. Microbiol
Biotechnol. 31: 537.

Medina, L. M., and R. Jordano. 1994. Survival of constitutive
microflora in commercially fermented milk containing
bifidobacteria during refrigerated storage. J. Food Prot. 56: 731.

Metchnikoff, E. 1908. The Prolongation of Life, 1st edition. G. P.
Putnams sons, New York.

Modler, H. W., R. C. McKellar, and M. Yaguchi. 1990. Bifidobacteria
and bifidogenic factors. Can. Inst. Food Technol. 23: 29.

91

Montville, T. J., and L. K. Conway. 1982. Oxidation—reduction
potentials of canned foods and their ability to support
Clostridium botulinum toxigenesis. J. Food Sci. 47: 1879.

Mutukumira, A. N. 1995. Review of traditionally fermented milk in
some sub-saharan countries. Cult. Dairy Prod. J. 30: 6.

Nagendra, R., Viswanatha, S., Arun Kumar, S., Krishna B., and
Venkat, S. 1995. Effect of feeding milk formula containing
lactulose to infants on fecal bifidobacterial flora. Nutr. Res.
15:15.

Nicholas, J. H., Bezkorovainy, A., and Landau, W. 1974. Human
colostral whey M-l glycoprotein and their L. bifidus var. pen.
growth promoting activities. Life Sci. 14:967.

Okonogi, S., T. Kawashima, T. Kudo, Y. Yuguchi, H. Hiramatsu, S.
Teraguchi, and T. Yaeshima. 1986. Culture containing a viable
cell mass of bifidobacteria and lactic acid bacteria. US. Patent
4,588,595.

Orihara, 0., and I. Sakauchi and Y. Nakazawa. 1992. Types and
standards for fermented milks and lactic drinks. Ch. 1 in
Functions of Fermented Milk, Y. Nakazawa and A. Hosono (Ed.),
p. 1. Elsevier Applied Science, London.

Petschow, B. W., and R. D. Talbott. 1990. Growth promotion of
Bifidobacterium species by whey and casein fraction from human
and bovine milk. J. Clin. Microbiol. 28: 287.

Poch, M., and A. Benzkorovainy. 1988. Growth-enhancing
supplements for various species of the genus Bifidobacterium. J.
Dairy Sci. 71: 3214.

Rasic, J., and J. Kurmann. 1983. Bifidobacteria and Their Role,
Birkhauser Verlag Basel, Basel, Switzerland.

Reuter, G. 1989. Bifidobacterium characteristics and classification.
in Fermented Milks: Current Research, p. 49. Association
International des Fabricants de Yogurts, Paris, France.

92

Roberts, A. K., R. Chierici, G. Sawatzki, M. J. Hill, S. Volpato, and V.
Vigi. 1992. Supplementation of an adapted formula with bovine
lactoferrin: 1. Effect on the infant faecal flora. Acta Paediatr. 81:
1 19.

Roy, M. D., and J. Goulet. 1991. B-galactosidase and proteolytic
activities of bifidobacteria in milk: a preliminary study.
Milchwissenschaft 46: 11.

Salminen, S., and M. Deighton. 1992. Lactic acid bacteria in the gut
in normal and disordered states. Dig. Dis. 10: 227.

Samona, A., and R. K. Robinson. 1991. Enumeration of
bifidobacteria in dairy products. J. Soc. Dairy Technol. 44: 64.

Samona, A., and R. K. Robinson and S. Marakis. 1996. Acid
production by bifidobacteria and yogurt bacteria during
fermentation and storage of milk. Food Microbiology 13: 275.

Sanders, M. E. 1993. Effect of consumption of lactic cultures on
human health. Adv. Food Nut. Res. 37: 67.

Sanders, M. E., D. C. Walker, K. M. Walker, K. Aoyama, and T. R.
Klaehammer. 1996. Performance of commercial cultures in fluid
milk applications. J. Dairy Sci. 79: 943.

Savaiano, D. A., and M. D. Levitt. 1987. Milk intolerance and
microbe-containing dairy foods. J. Dairy Sci. 70: 397.

Scardovi, V., and L. D. Trovatelli. 1962. The fructose-6-phosphate
shunt as peculiar pattern of hexose degradation in the genus
Bifidobacterium. Ann. Microbiol. 15: 19.

Scardovi, V. 1981. The genus Bifibacterium. in The Prokaryotes, M.
P. Starr, H. Stolp, H.G. Truper, A. Balows, and HG. Schlegel
(Ed.), Vol. 2, p. 1951. Springer-Verlog, New York.

Scardovi, V. 1986. The genus Bifidobacterium. in Bergev’s Manual of
Systematic Bacteriolog, J. G. Holt (Ed.), Vol. 2, p. 1418.
Williams and Wilkins, Co., Baltimore.

Sekine, K., T. Toida, M. Saito, M. Kuboyama, T. Kawashima, and Y.
Hashimoto. 1985. A new morphologically characterized cell wall
preparation from Bifidobacterium infantis with higher efficacy on

93

the regression of an established tumor in mice. Cancer Res. 45:
1300.

Shah, N.P., W.E.V. Lankaputhra, M.L. Britz, and W.S.A. Kyle. 1995.
Survival of Lactobacillus acidophilus and Bifidobacterium bifidum
in commercial yogurt during refrigerated storage. Int. Dairy J. 5:
515.

Singleton, P. 1987. Dictionary of Microbiology and Molecular Biology
2nd ed. John Wiley 85 Sons Ltd. New York.

Spiegel, J. E., R. Rose, P. Karabell, V. H. Frankos, and D. F.
Schmitt. 1994. Safety and benefits of fructooligosaccharides and
food ingredients. Food Technol. 48: 85

Tamime, A. Y., Marshall, V. M. E., and R. K. Robinson. 1995.
Microbiological and technological aspects of milks fermented by
bifidobacteria. J. Dairy Res. 62: 151.

Tamine, A. Y., and R. K. Robinson. 1988. Fermented milks and their
future trends. J. Dairy Res. 55: 281.

Tamine, A. Y., and H. C. Deeth. 1980. Yogurt: technology and
biochemistry. J. Food Prot. 43: 939.

Teraguchi, S., J. Ono, I. Kiyosawa, and S. Okonogi. 1987. Oxygen
uptake activity and aerobic metabolism of Streptococcus
thermophilus STH450. J. Dairy Sci. 70: 514.

Tinson, W., M. F. Ratcliff, A. J. Hillier, and G. R. Jago. 1982.
Metabolism of Streptococcus thermophilus. 3. Influence on the
level of bacterial metabolites in cheddar cheese. Aust. J. Dairy
Technol. 37: 17.

Tojo, M., T. Oikawa, Y. Morikawa, N. Yamashita, S. Iwata, Y. Satoh,
J. Hanada, and R. Tanaka. 1987. The effects of Bifidobacterium
breve administration on Campylobacter enteritis. Acta Paediatr.
Jpn.29:160.

Vandamme, E. J., and D. G. Derycke. 1983. Microbial inulinase:
fermentation process, properties, and applications. Adv. Appl.
Microbial. 29: 139.

94

Ventling B. L., and V. V. Mistry. 1993. Growth characterization of
bifidobacteria in ultrafiltered milk. J. Dairy Sci. 76: 962.

Wijsman, M. R., J. L. P.M. Hereijgers, and J. M. F. H. de Groote.
1989. Selective enumeration of bifidobacteria in fermented milk
products. Neth. Milk Dairy J. 43: 395.

Yamazaki, H., and N. Dilawri. 1990. Measurement of growth of
bifidobacteria on inulofrutosaccharides. Letters in Appl.
Microbiol. 10: 229.

Yazawa, K., and Z. Tamura. 1982. Search for sugar sources for
selective increase of bifidobacteria. Bifidobacteria Microflora. 1:
39.

Yazawa, K., K. Imai, and Z. Tamura. 1978. Oligosaccharides and
polysaccharides specifically utilizable by bifidobacteria. Chem.
Pharm. Bull. 26: 3306.

Yoshioka, H. Y., Iseki, K, and K. Fujita. 1983. Development and
differences of intestinal flora in the neonatal period in brest-fed
and bottle-fed infants. Pediatrics. 72: 317.

Yun, J. W. 1996. Fructooligosaccharides-occurrence, preparation,
and application. Enzyme and Microbial Technol. 19: 107.

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