Etd 0824104 182901

高純度異麥芽寡糖對比菲德氏菌增值的影響
EFFECT OF HIGH-CONTENT
ISOMALTOOLIGOSACCHARIDES ON THE
GROWTH OF BIFIDOBACTERIA
研究生：徐雅亭（Ya-Ting Shu）
指導教授：許垤棊（Prof. Dey-Chyi Sheu）
大同大學
生物工程研究所
碩士論文
Thesis for Master of Science

Department of Bioengineering
Tatung University
中華民國九十三年六月
June 2004
EFFECT OF HIGH-CONTENT
ISOMALTOOLIGOSACCHARIDES ON THE
GROWTH OF BIFIDOBACTERIA
THESIS
Submitted to the Graduate School
Of
Tatung University
In Partial Fulfillment of the Requirement for
The Degree of Master of Science in Bioengineering
by
Ya-Ting Shu, B. S. Biol.
Taipei, Taiwan
Republic of China
2004
Abstract
Bifidobacteria can promote human health. Bifidobacteria are regularly
found in human large intestine and colon, where digestible sugars such as
glucose, sucrose and maltose, are not attainable. Isomaltooligosaccharides
(IMO), a function sugar, was made from maltose via a reaction catalyzed by
glucosyltransferase. IMO is preprobiotic, being able to enhance the growth
of bifidobacteria. High-content IMO can be produced, by treating
commercial IMO syrup with yeast, thereby digestible sugar including
maltose and glucose are depleted. In this way, the content of IMO increases
from 58% to 99% on a dry weight basis. In a 2-L jar fermenter,
bifidobacteria were cultured in 1.3 L broth in the presence of 5% (w/v)
high-content IMO. The fermentation conditions were as following: anaerobic
culture, overlaid with a layer of liquid paraffin; stir rate, 100rpm;
temperature, 37°C. During fermentation, turbidity, viable count, ORP, pH,
acetate, lactate and sugar components were determined periodically. During
72 h of fermentation, most of the bifidobacteria used in this study metabolize
primarily panose, tertesaccharides and isomaltose and thus produced G and
IG2. B. adolescentis CCRC 14609 consumed IMO much faster than other
strains did and produced the maximum lactic acid of 37.41 g/L. But B.
I
bifidum CCRC 11844 and B. breve CCRC 11846 did not grow well and
consumed isomaltooligosaccharides poorly.
The present investigation using such oligosaccharides which was free
of digestible sugars was much more significant than past research, in which
oligosaccharides accompanied with large amounts of digestible sugars were
used. The utilization of various IMO components by eight bifidobacteria was
clearly understood.
Key words: bifidobacteria, high-content isomaltooligosaccharides, probiotics
II
中文摘要
比菲德氏菌存在於人體的大腸和結腸中，消化性糖無法到達此
處。然而，有一些寡糖成分，具有非消化性，可到達大腸和結腸，進
而資化比菲德氏菌。異麥芽寡糖，是以麥芽糖為基質，利用
glucosyltransferase 催化反應生成，為機能性寡糖，是一種益生源，對於
比菲德氏菌的生長具有很好的促進作用。利用酵母菌去除市售異麥芽寡
糖中的消化性糖，亦即麥芽糖及葡萄糖，使寡糖的純度由 58%提高至 99%
以上。在 2 公升醱酵槽中，用 1.3 公升的培養基，添加 5%的高純度異麥
芽寡糖，在礦油覆蓋下的厭氧條件、於 37°C、攪拌速度 100rpm，觀察
比菲德氏菌的生長。醱酵過程中，監測培養液的混濁度、生菌數、氧化
還原電位、pH 值、醋酸與乳酸濃度，以及用 HPLC 分析寡糖成分的變
化。
在 72 小時醱酵中，比菲德氏菌主要代謝 panose, tertesaccharides 和
isomaltose，因而產生 glucose 和 maltose。B. adolescentis CCRC 14609 利
用異麥芽寡糖的速度為最快速且乳酸的產量 37.41 g/L 相較於其他菌株
為最大。而 B. bifidum CCRC 11844 和 B. breve CCRC 11846 則在高純度
異麥芽寡糖裡生長緩慢，因此寡糖的代謝較其他的菌株為差。由於此寡
糖不含消化性糖，所以這樣的研究比前人利用含有大量消化性糖的寡糖
的研究更具有意義。因此可以了解比菲德氏菌對於異麥芽寡糖中不同成
III
分的利用情形，也可以瞭解不同的比菲德氏菌利用寡糖的差異。
關鍵字：比菲德氏菌、異麥芽寡糖、益生源
IV
CONTENTS
page
Abstract ..........................................................................................I
中文摘要 ......................................................................................III
CONTENTS ................................................................................. V
LIST OF TABLES ...................................................................VIII
LIST OF FIGURES ..................................................................... X
CHAPTER 1. INTRODUCTION................................................ 1
1.1 Probiotics ........................................................................................... 1
1.1.1 Types of prebiotics and their effect on intestinal
microecology......................................................................... 5
1.1.2 Oligosaccharides ...................................................................... 7
1.1.3 Isomaltooligosaccharides ......................................................... 8
1.2 Bifidobacteria................................................................................... 12
1.2.1 Nutrition ................................................................................. 13
1.2.2 Carbohydrate matabolism ...................................................... 14
1.2.3 Extracellullar dextranase and intracellular α-1→6
glucosidase .......................................................................... 15
1.3 The effect of prebiotic in vitro experiments .................................... 16
V
CHAPTER 2. MATERIALS AND METHODS ....................... 20
2.1 Materials 20
2.1.1 Microorganisms ..................................................................... 20
2.1.2 Instruments............................................................................. 20
2.1.3 Chemicals............................................................................... 22
2.2 Methods 23
2.2.1 Depletion of digestible sugar in commercial IMO by
Saccharomyces cerevisiae WP500 ..................................... 23
2.2.2 Utilization of high-content IMO during batch fermentation
by bifidobacteria ................................................................. 26
2.3 Control of fermentation.................................................................... 34
CHAPTER 3. RESULTS AND DISCUSSION ......................... 37
3.1 Depletion of digestible sugar in commercial IMO by
Saccharomyces cerevisiae WP500 ................................... 37
3.2 Bacth fermentation by bifidobacteria .............................................. 43
3.2.1 The batch fermentation by individual bifidobacterium ......... 43
3.3.2.1 Viable counts ................................................................ 60
3.3.2.2 Optical density ............................................................. 63
3.3.2.3 Change of sugar components during fermentation ...... 66
VI
3.3.2.4 Organic acids................................................................ 78
3.3.2.5 The ORP ....................................................................... 86
CHAPTER 4. CONCLUSION................................................... 89
REFERENCES ........................................................................... 91
APPENDICES............................................................................. 98
Appendix 1: The data of bifidobacetria ................................................. 98
Appendix 2: The HPLC diagram of the IMO ........................................ 99
Appendix 3: The HPLC diagram of the organic acid .......................... 100
Appendix 4: HPLC analysis of high-content IMO before
fermentation by B. adolescentis CCRC 14607 .............. 101
Appendix 5: HPLC analysis of high-content IMO after 72 hours of
fermentation. by B. adolescentis CCRC 14607 ............. 102
Appendix 6: HPLC analysis of lactic acid and acetic acid before
fermentation by B. adolescentis CCRC 14607 .............. 103
Appendix 7: HPLC analysis of lactic acid and acetic acid after 72
hours of fermentation. by B. adolescentis CCRC
14607 .............................................................................. 104
VII
LIST OF TABLES
page
Table 1.1 Overview of the evolution of in vitro fermentation experiments
with non-digestible oligosaccharides (NDO), leading to the
assumption that NDO are prebiotic compounds .............................. 19
Table 3.1 Depletion of maltose and glucose in IMO by Saccharomyces
cerevisiae WP500 during fermenter................................................. 42
Table 3.2 The maximum viable counts of bifidobacteria during
fermentation at 37°C in MRS medium with glucose being
substituted by 5 % (w/v) of high-content IMO................................ 62
Table 3.3 The maximum optical densities of bifidobacteria during
Table 3.4 The concentrations of acetic acid, lactic acid and
acetate-to-lactate ratio during fermentation at 37°C by eight
bifidobacteria in MRS medium with glucose being substituted by
5 % (w/v) of high-content IMO. ...................................................... 85
Table 3.5 The time-points at which mixima of ORP, depletion of IMO
and maxima of viable count being achieved during the 72-h
fermentation. .................................................................................... 87
VIII
Table 3.6 The maximum viable counts, the concentrations of total acid
(acetic acid and lactic acid), acetate-to-lactate ratio and the
concentrations of depleted of IMO during fermentation at 37°C
by eight bifidobacteria in MRS medium with glucose being
IX
LIST OF FIGURES
Figurg 1.1 Proposed mechanisms of prebiotic action to improve human
health............................................................................................. 4
Figure 1.2 Molecular structure of IMO. ......................................................... 9
Figure 2.1 Scheme of depletion of digestible sugar in IMO by yeast. ......... 26
Figure 2.2 Scheme of large scale fermentation by bifidobacteria. ............... 31
Figure 2.3 The schematic diagram of the system for batch frmentation by
yeast. ........................................................................................... 32
Figure 2.4 The schematic diagram of the system for batch fermentation
by bifidobacteria. ........................................................................ 33
Figure 2.5 A display of the original ADVENTECH GENIE strategy of
fermentation (mainboard). .......................................................... 35
Figure 2.6 A display of original ADVENTENCH GENIE strategy for
fermentation (connecting system). ............................................. 36
Figure 3.1 Depletion of maltose and glucose in IMO during fermentation
by Saccharomyces cerevisiae WP500......................................... 39
Figure 3.2 HPLC analysis of commercial IMO before fermentation........... 40
Figure 3.3 HPLC analysis of IMO after 24 hours of fermentation. ............. 41
Figure 3.4 Time courses of ORP, OD600, viable count, sugar
X
concentrations, and acid concentrations during the
fermentation by B. longum CCRC 14634 in a 2-L jar
fermenter. .................................................................................... 45
fermentation by B. longum CCRC 14602 in a 2-L jar
fermenter. .................................................................................... 47
fermentation by B. bifidum CCRC 14615 in a 2-L jar
fermenter. .................................................................................... 49
concentrations, and acid concentrations during fermentation
by B. bifidum CCRC 11844 in a 2-L jar fermenter..................... 51
fermentation by B. breve CCRC 11846 in a 2-L jar fermenter... 53
concentratiosns, and acid concentrations during the
XI
fermentation by B. adolescentis CCRC 14607 in a 2-L jar
fermenter. .................................................................................... 55
fermentation by B. adolescentis CCRC 14609 in a 2-L jar
fermenter. .................................................................................... 57
fermentation by B. pseudocatenulatum CCRC 15467 in a 2-L
jar fermenter. ............................................................................... 59
Figure 3.12 Changes of viable counts during 72 h of fermentation at 37°C
substituted by 5 % (w/v) of high-content IMO........................... 61
Figure 3.13 Changes of OD600 during 72 h of fermentation at 37°C by
eight bifidobacteria in MRS medium with glucose being
substituted by 5 % (w/v) of high-content IMO........................... 64
Figure 3.14 Changes of glucose concentration during fermentation at
37°C by eight bifidobacteria in MRS medium with glucose
being substituted by 5 % (w/v) of high-content IMO................. 67
XII
Figure 3.15 Changes of maltose concentration during fermentation at
Figure 3.16 Changes of isomaltose concentration during fermentation at
Figure 3.17 Changes of panose concentratiom during fermentation at
Figure 3.18 Changes of tetrasaccharides concentration during
fermentation at 37°C by eight bifidobacteria in MRS medium
with glucose being substituted by 5 % (w/v) of high-content
IMO............................................................................................. 75
Figure 3.19 Changes of total IMO concentration during fermentation at
Figure 3.20 Changes of lactic acid concentration during fermentation at
XIII
Figure 3.21 Changes of acetic acid concentration during fermentation at
Figure 3.22 Changes of total acid concentration (acetic acid and lactic
acid) during fermentation at 37°C by eight bifidobacteria in
MRS medium with glucose being substituted by 5 % (w/v) of
high-content IMO. ...................................................................... 83
XIV
CHAPTER 1. INTRODUCTION
1.1 Probiotics
The human gastrointestinal tract constitutes a complex microbial
ecosystem comprising several hundred different species of bacteria. The
colon in particular is densely populated with in excess of 1011 bacteria per
gram of contents. These organisms and their metabolic activities are not inert
to the human host and can have both positive and negative impacts on health.
Within the intestinal microflora are bacterial species believed to benefit the
host and some that are potentially pathogenic. The balance of this ecosystem
is dynamic and may be adversely altered by aging, medication, stress, diet
and other environmental factors. The maintenance of a community of
bacteria which contains a predominance of beneficial species and minimal
putrefactive (protein degrading) processes is believed to be important for
maintaining intestinal health.
Two separate approaches exist to increase the number of
health-promoting organisms in the gastrointestinal tract. The first is the oral
administration of live, beneficial microbes. These organisms, termed
probiotics, have to date been selected mostly from lactic acid bacteria and
bifidobacteria that form part of the normal intestinal microflora of humans.
Bifidobacteria in particular are one of the dominant genera present in the
colon of healthy individuals (Mitsuoka, 1978). Since these organisms are
1
indigenous to the colon, a second strategy to increasing their numbers is to
supply those already present in the intestine with a selective carbon and
energy source that provides them with a competitive advantage over other
bacteria in the ecosystem. That is, to selectively modify the composition of
the microflora using dietary supplements. These selective dietary
components were named "prebiotics" in 1995 by Gibson and Roberfroid who
defined them as non-digestible food ingredients that beneficially affect the
host by selectively stimulating the growth and/or activity of one or a limited
number of bacteria in the colon, that can improve the host health.
To provide a fermentable substrate for bacteria in the colon, the first
requirement of a prebiotic is that it has to be at least partially undigested and
unabsorbed in the small intestine. Once it reaches the colon the prebiotic
must selectively promote the growth and/or stimulate the metabolic activity
of health-promoting bacteria and not bacteria with deleterious effects on
health. Through this alteration in gastrointestinal composition and
metabolism the prebiotic may induce systemic effects beneficial to health.
Bifidobacteria and lactobacilli are generally regarded as desirable genera and
are the major target organisms of prebiotics. It is also considered a desirable
property of a prebiotic if it can lead to the reduction of putrefactive and
potentially pathogenic organisms such as clostridia and Enterobacferiaceae.
The prebiotic approach to increasing beneficial bacteria in the colon
potentially provides some advantages over the probiotic strategy. Consumed
2
probiotic bacteria must survive transit through the hostile conditions in the
stomach and then adapt quickly to their new environment. In the colon they
must compete for nutrients and colonisation sites against an established
microflora with species that already occupy the available physical and
metabolic niches. Host-individual/bacterial-strain specificities may also exist,
hindering the establishment of new exogenous strains in the gastrointestinal
tract. An ecological study of the strains of bifidobacteria and lactobacilli in
individuals over a 12 month period failed to detect strains common to the
subjects (McCartney et al., 1996). In contrast to probiotics, prebiotics target
commensal bacteria specific to the host, and with effective colonisation sites.
These endogenous bacteria are also unlikely to cause immunological
problems associated with the intake of foreign antigens (Gibson et al., 1997).
Prebiotics offer not only the potential to increase the numbers of beneficial
bacteria, but also their metabolic activity through the supply of fermentable
substrate. This increase in metabolic activity is central to many of the
currently proposed mechanisms of health promotion by prebiotics (Figure
1.1). Probiotics are also limited mainly to "fresh products" and careful
attention must be paid to technological aspects of maintaining sufficient
levels of viable bacteria in the food delivery system.
3
Figurg 1.1 Proposed mechanisms of prebiotic action to improve human
health.
4
1.1.1 Types of prebiotics and their effect on intestinal microecology
One of the main reasons for the great variety of bacterial species found
in the colon is the multiplicity of different carbon sources to which they have
access. A range of dietary non-starch polysaccharides, resistant starches,
undigested sugars, oligosaccharides and proteins are fermented by the
microflora. In addition, endogenous sources such as mucus glycoproteins,
sloughed epithelial cells, and products from the metabolism of other
microflora components are available (Cummings and Macfarlane, 1997). Of
these it is the non-digested dietary carbohydrates that provide the principal
substrates for colonic bacterial growth. The amount of carbohydrate reaching
the human colon varies considerably with diet but ranges between 20-60
g/day (Cummings and Macfarlane, 1997). Carbohydrates entering the colon
are fermented by the microflora predominantly to short-chain fatty acids
(SCFAs), essentially acetate, propionate, and butyrate; lactate; and to the
gases carbon dioxide, methane, and hydrogen (Cummings and Macfarlane,
1997). The SCFAs produced are rapidly absorbed and metabolised by the
host. In this way the microflora contributes to salvaging energy from dietary
components that would otherwise be lost to the host by excretion from the
digestive tract (Cummings and Macfarlane, 1997).
The majority of bacterial species in the colon are saccharolytic and
can contribute to carbohydrate fermentation. However, the dominant
saccharolytic organisms belong to the genera Bacteroides, Bifidobacteriiim,
5
Eiibacterium, Lactobacillus, and Clostridium. Any fermentable dietary
component that arrives undigested in the colon has the potential to act as a
prebiotic. To date almost all prebiotics described and all those produced
commercially have been carbohydrates. These range from small sugar
alcohols and disaccharides, to oligosaccharides, and large polysaccharides,
all with a variety of sugar compositions and glycosidic linkages. Such a
diverse range of chemical structures would be expected to provide an equally
diverse range of effects on the colonic microflora. However, almost all of
these food ingredients are claimed as prebiotics on the basis of selectively
stimulating the proliferation of the bifidobacteria in the colon. Such food
ingredients have therefore also been referred to as "bifidogenic" or "bifidus
factors".
The exact mechanisms by which such a chemically diverse range of
carbohydrates preferentially stimulate one particular genus in a population
with many saccharolytic species is not clear. The ability to efficiently utilize
such a variety of substrates indicates that bifidobacteria possess an array of
glycosidases, making them nutritionally versatile and allowing them to adapt
and compete in an environment with changing nutritional conditions.
Interestingly, most bifidobacteria grow more rapidly on prebiotic
fructo-oligosaccharides than on glucose (Gibson and Wang, 1994a; Wang
and Gibson, 1993). It appears that bifidobacteria are one of the most efficient
groups in the colonic microflora at utilizing many non-digestible
6
carbohydrates providing them with a competitive advantage and allowing
them to proliferate relative to other species when prebiotics are consumed.
Their tolerance to the acid micro-environment produced as a consequence of
prebiotic fermentation and SCFA production may also contribute to their
selective proliferation.
1.1.2 Oligosaccharides
Over the last two decades a range of non-digestible oligosaccharides
(NDO's) have been developed, initially as low calorie, low cariogenic,
sucrose substitutes for use as bulking agents in foods. As the effects of these
oligosaccharides on the colonic microflora was investigated, it emerged that
some had the potential to increase bifidobacteria in the colon. The ability to
act as a prebiotic has become a marketing edge for these products and has
promoted research into the ability of oligosaccharides to induce beneficial
changes in the composition and metabolism of the colonic microflora.
Oligosaccharides are usually defined as glycosides that contain
between three and ten sugar moieties. They are produced commercially
using enzymatic processes involving either the hydrolysis of
polysaccharides or synthesis from smaller sugars using transglycosylases.
The one exception is soybean-oligosaccharides which are directly extracted.
The products produced for food applications are not chemically
7
homogeneous but are mixtures containing oligosaccharides with different
numbers of monosaccharide moieties and often different glycosidic
linkages. Currently there are nine different types of NDO's produced
commercially, predominantly m Japan and Europe (Crittenden and Playne,
1996; Playne and Crittenden, 1996). Almost all of these are claimed by
their manufacturers to be bifidogenic. The fructo-oligosaccharides (FOS),
transgactosyl-oligosacchandes (TOS), and soybean oligosaccharides have
been the more thoroughly researched, and so far have the best documented
evidence of prebiotic effects in humans.
1.1.3 Isomaltooligosaccharides
Isomaltooligosaccharides (IMO), such as isomaltose, panose
(62-O-α-glucosyl-maltose), isomaltotriose, and tetrasaccharides, are
glucosyl saccharides with α-(1→6) glucosidic linkages (Figure 1.2). They
occur naturally in various fermented foods and sugars in honey (Takaku
1988). Although, in a strict sense, IMO means glucosyl saccharides with
only α-(1→6) glucosidic linkages, commercial IMO syrup is generally
accepted as a mixture of glucosyl saccharides containing both α-(1→6) and
α-(1→4) glucosidic linkages (Yun et al., 1994).
8
Figure 1.2 Molecular structure of IMO.
9
IMO syrup has many favorable properties for application to the food
industry. It shows low viscosity, high moisture-retaining capacity, and low
water activity convenient in controlling microbial contamination. Besides, it
tastes mildly sweet that is about 0.4-0.5 times less than sucrose, a
conventional sweetener. IMO is hardly utilized by yeast and hydrolyzed by
fungal glucoamylases. Furthermore, IMO is very stable under acidic
conditions and at elevated temperatures. Because of these favorable
properties, IMO syrup is used in bakery, confectionery, soft drink, and
several other food-processing industries (Kanno 1990; Yatake 1993;
Nakakuki 1995).
The utilization of IMO-900R, commercial production, by various
intestinal bacterial species was examined in in vitro and in vivo experiments.
The data showed that isomaltose, isomaltotriose, panose, and IMO-900R
were utilized by bifidobacteria classified as beneficial intestinal bacteria, but
they were not utilized by Escherichia coli or the Clostridum species which
were unfavorable for their producing putrefactive substances in the digestive
tract. Consequently, the ingestion of IMO could improve intestinal flora,
relieve the constipation, and suppress the formation of putrefactive products
(Kanno 1990; Komoto et al., 1988; Yatake 1993; Komoto et al., 1991).
10
Another benefit of IMO is the dental caries inhibitory effect. It is well
known that Streptococcus mutans, a species of bacteria commonly existed in
the mouth, has been implicated in humans as a causative causative agent of
dental caries. After intake of sucrose-containing foodstuffs, S. mutans secrete
lactic acid and utilize sucrose for the glucosyltransferase (GTase) of the S.
mutans to synthesize the water-insoluble glucans, which are thought to be
the main cause of carious tooth. If we use IMO to replace sugar, it can
significantly inhibit not only the synthesis of the water-insoluble glucans
from sucrose, but also the sucrose-dependent adherence of these cells to a
glass surface. Thus, suffering for dental caries is avoidable (Kanno 1990;
Yatake 1993; Komoto et al., 1991; Nakakuki 1995).
The other beneficial effects of IMO proved in clinical studies are the
activation of immune response, helping people to resist disease, also
improving the functions of the livers and kidneys and metabolism of liqids.
In addition, IMO is used successfully to stimulate the growth of many
livestock such as piglets, calves among the animal husbandry, and improve
the feed efficiency (Kanno 1990).
11
1.2 Bifidobacteria
Rods of various shapes: short, regular, thin cells with pointed ends,
coccoidal regular cells, long cells with slight bends or protuberances or with
a large variety of branchings; pointed, slightly bifurcated club-shaped or
spatulated extremities; single or in chains of many arrangements. Colonies
smooth, convex, entire edges, cream to white, glistening and of soft
consistency. Gram-positive, non-acid-fast; nonspore-forming, nonmotile.
Cells often strain irregularly with methylene blue. Anaerobic; some species
can tolerate O2 only in the presence of CO2. Optimum growth temperature
37-41°C; minimum growth temperature 25-28°C maximum 43-45°C.
Optimum pH for initial growth 6.0-7.0: no growth at 4.5-5.0 or 8.0-8.5.
Saccharoclastic, acetic acid and lactic acid are formed primarily in the
molar ratio of 3:2. CO2 is not produced (except in the degradation of
gluconate). Small amounts of formic acid, ethanol and succinic acid are
produced. Butyric and propionic acid are not produced. Glucose is degraded
exclusively and characteristically by the fructose-6-phosphate shunt in which
fructose-6-phosphoketolase (F6PPK-EC 4.1.2.22) cleaves
fructose-6-phosphate into acetylphosphate and erythrose-4-phosphate. End
products are formed though the sequential action of transaldolase (EC
2.2.1.2), transketolase (EC 2.2.1.1), xylulose-5-phosphate phosphoketolase
(EC 4.1.2.9) and enzymes of EMP acting on glyceraldehyde-3-phosphate.
12
Additional acetic and formic acid may be formed through a cleavage of
pyruvate.
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49, NADP+- or
NAD+-dependent) generally not determinable.
Catalase-negative except that B. indicum and B. asteroides are
catalase-pasitive when growth in the presence of air with or without added
hemin.
Ammonium is generally utilized as a source of nitrogen.
The G + C content of DNA (Bd or Tm) varies from 55-67 mol%.
The organisms occur in the intestine of man, varies animals and honey
bees; found also in sewage and human clinical material.
1.2.1 Nutrition
Since its first isolation from human infants’ feces (Gyorgy, 1953) and
its designation as Lactobacillus bifidus var. pennsylvanicus (Gyorgy and
Rose, 1995), this organism, the growth of which is stimulated by human
milk, has been the object of numerous nutritional studies designed either to
properties of the bifidus factor(s) present in human milk, or to find a
substitute for it (Poupard et al., 1973; Yoshioka et al., 1968; Nakamura and
Tamura, 1972; Nichols et al., 1974; Gyorgy et al., 1974; Yazawa and
Tamura., 1978; Beerens et al., 1980).
13
Bifidobacteria are able to utilize ammonium salts as sole source of
nitrogen. This finding, reported first by Hassinen et al. (1951), is valid for
most species of the genus, but B. suis, B. magnum, B. choerinum and B.
cuniculi will not grow without organic nitrogen (Matteuzzi and Emaldi,
1978). The species which grow without organic nitrogen excrete
considerable amounts of varies amino acids into the medium: e.g. B. bifidum
can produce up to 150 mg/Liter threonine. Other active amino acid producers
are B. thermophilum, B. adolescentis, B. dentium, B. animalis and B. infantis.
The amino acids generally produced in the largest amounts are alanine,
valine and aspartic acid (Matteuzzi and Emaldi, 1978).
Analog-resistant mutants were obtained from B. thermophilum
showing increased production of isoleucine and valine (Matteuzzi et al.,
1976; Crociani et al., 1977).
1.2.2 Carbohydrate matabolism
The fermentation of hexose occurs in the genus Bifidobacterium
through the following sequence of reactions (bifid shout) (Scardovi and
Trovatelli, 1965; De Vries et al., 1967; Veerkamp, 1969b).
14
The theoretical ratio of acetate 1.5: lactate 1.0 is scarcely ever found
in growing cultures of bifidobacteria: phosphoroclastic cleavage of some
pyruvate to formic acid and acetyl phosphate and reduction of acetyl
phosphate to ethanol can often alter the fermentation balance in favor of the
production of acetate and some formic acid and ethanol. (De Vries and
Stouthamer, 1968; Lauer and Kandler, 1976).
1.2.3 Extracellullar dextranase and intracellular α-1→6 glucosidase
15
Although several species of molds have been shown to produce
extracellular dextranases, very few bacterial species have been reported to
produce this type of enzyme. Hydrolysis of dextran by the dextranase from
the latter source was unusual in that it did not liberate glucose or isomaltose,
which are the main products from in action of all other known dextranases
on dextrarn. Instead, the enzyme hydrolyzed dextran, by random cleavage
of the α-1→6 glucosidic links, to a mixture of isomaltotriose, isotetraose,
isopentaose, and higher isomalto dextrins. All extracts, prepared from a
rumen strain of bifidobacteria grown on dextran, were shown to contain an
α-1→6 glucosidase (Bailev and Roberton, 1962; Bailey and Clarke, 1959;
Hehre and Sery, 1952).
1.3 The effect of prebiotic in vitro experiments
These experiments concern fermentations carried out in small
bioreactors inoculated with pure bacterial strains, reconstituted mixtures or
faecal slurries. Variables such as temperature, pH and nutrient composition
are carefully controlled. This technique allows study of the influence of
NDO on the bacterial composition of mixed bacterial faecal slurries as well
as the fermentation of NDO by pure bacterial cultures.
16
The different compartments of the colon can be simulated with
continuous culture experiments with (sequences of) chemostats (Macfarlane
et al. 1998). One limitation of this type of experiment is that metabolites are
not withdrawn from the environment as they are in the colon. The
accumulation of these products affects the equilibrium of many ongoing
biochemical processes (inhibition). It is not possible to quantify the influence
of the artificial environment of a bioreactor. Nevertheless, the relative
comparisons that can be studied are considered important tools in prebiotic
research, as they allow the demonstration (qualitatively) of certain
mechanisms, leading to the formulation of hypotheses that can be tested by
well-designed human feeding studies.
In the ENDO project, an in vitro gnotoxenic (bacterial flora with
known composition) fermentation chemostat proved to be a suitable model
for studying bacterial interactions. Table 1 summarizes the in vitro
microflora experiments.
Consensus. The results obtained from in vitro fermentation
experiments (batch and continuous cultures inoculated either with faecal
slurries or pure cultures) are important in prebiotic investigations,
particularly when integrated with information from human in vivo studies.
17
Combination of these data provides valuable insight into the mechanisms of
prebiotic action. In vitro models alone cannot be considered adequate for
study of the complex ecosystem of the colon.
As a result of the discussion of the available scientific data, the
authors propose the following definition: ‘A prebiotic effect is a
food-induced increase in numbers and/or activity predominantly of
bifidobacteria and lactic acid bacteria in the human intestine’. This definition
is an adapted version of that of Gibson & Roberfroid (1995) and implies that
besides bifidobacteria and lactic acid bacteria, some (‘selectivity’) other
bacteria may be stimulated. However, the definition incorporates the
bifidobacteria and the lactic acid bacteria because they are considered good
biomarkers of a well-balanced intestinal flora. The health aspect is omitted
from the definition, because to date no information is available which could
support such a statement. (Jan et al.1999)
18
Table 1.1 Overview of the evolution of in vitro fermentation experiments
with non-digestible oligosaccharides (NDO), leading to the
assumption that NDO are prebiotic compounds
19
CHAPTER 2. MATERIALS AND METHODS
2.1 Materials
2.1.1 Microorganisms
Bifidobacterium longum CCRC14634, Bifidobacterium longum CCRC
14602, Bifidobacterium bifidum CCRC 14615, Bifidobacterium bifidum
CCRC 11844, Bifidobacterium breve CCRC 11846 Bifidobacterium
adolescentis CCRC 14607, Bifidobacterium adolescentis CCRC 14609 and
Bifidobacterium pseudocatenulatum CCRC 15476 were obtained from the
Culture Collection and Research Center, Food Industry Research and
Development Institute (FIRDI), Hsin-Chu, Taiwan, Republic of China.
2.1.2 Instruments
Autoclave: Tomin Medical Equipment Co., Ltd., Speedy autoclave
high-pressured steam sterilizer
Tomyseiko Co., Ltd., Tokyo. Japan., Autoclave model: SS-325
Balance: Swiss Quality, Precisa 500M-2000C
Centrifuge: Hitachi, model: HIMAC CF15D2
DO meter: Firstek Scientfic, DO CONTROLLER DC 100
DO sensor: Mettler-Toledo AG, InPro 6000 Series O2 Sensors
20
Fermentor: CHIN-CHI MBR. Co., Ltd., CMF-5 fermentor
EYELA, JAR FEREMENTOR MBF
Filter: Whatman, Syringe Filter (diamteter 13 mm, pore size 0.2 µm )
High-performance Data Acquisition Card:
A/D and D/A transformation card、Genie v.3.0 Software (Advantech,
U.S.A.)
Incubation shaker: Hotech, Orbital Shaker Incubator, Model-705
PC: 586-PC
ADVENTECH PCLD-8115 BOARD

ADVENTECH PCL-818L
ADVENTECH PCLD-885
pH meter: SUNTEX, pH/ORP Controller PC-310
pH sensor: Mettler-Toledo InPro® 3030/225 pH
Pump: Pharmacia, Peristatic Pump model: P-1
Watson Marlow 313S
Spectrophotometer: Trendtop Scientific corp., Spectrophotometer Model:
UV-300
Oven: RISEN VOLTS116, U.S.A.
ORP meter: SUNTEX, pH/ORP Controller PC-310
ORP sensor: Mettler-Toledo Pt4805 -SC-DPAS-K8S/225 Redox
21
HPLC detector: JASCO, RI930
Waters 401, Differential Refractometor
HPLC column: Merck, Lichroapher® 100 NH2(5μm)
TRANSGENOMIC, ICSep ICE-ION-300
HPLC pump: JASCO, PU1580
2.1.3 Chemicals
1. Malt extract was purchased form Difco.
2. Idustrial Yeast Extract was purchased from MDBio, Inc., France.
3. Agar was purchased from Difco.
4. Man Rogosa Sharpe (MRS) broth was purchased from Difco.
5. Proteose peptone (no.3) and Beef extract was purchased from Difco.
6. Sodium Acetate was purchased from Merck.
7. Dipotassium phosphate, Manganese Sulfate, Polysorbate 80,
Ammonium Acetate, Magnesium Sulfate and Liquid paraffin was
purchased from Wako.
8. IMO was purchased from TAIWAN FRUCTOSE CO. LTD.
9. Glucose was purchased from Italy.
10. Charcoal activated GR was purchased from Merck..
22
11. Amberlite MB-150 was purchased from Sigma.
2.2 Methods
2.2.1 Depletion of digestible sugar in commercial IMO by
Saccharomyces cerevisiae WP500
culture mdia
A. Agar plate:
It consisted of the following ingredients (%, w/v): yeast extract 0.3, malt
extract 0.3, glucose 1, peptone 0.5, Agar 1.5.
B. Shaking culture medium:
It consisted of the following ingredients (%, w/v): yeast extract 1, malt
extract 1.
C. Fermentation medium:
It consisted of the following ingredients (%, w/v): yeast extract 1,
commercial IMO 20.
Cultivation of Saccharomyces cerevisiae WP500
The Saccharomyces cerevisiae WP500 used in the experiment was
grown on YM agar plate at 30°C for 2-3 days. For the fermentation in shake
23
culture or in jar fermenter, a liquid seed culture was prepared as the
following procedures. Yeast cells on agar plate was pick up and inoculated in
a 500-ml Erlenmeyer flask containing 200 ml of liquid medium composed of
1% (w/v) yeast extract and 1% (w/v) malt extract. This was then cultivated
in shaking incubator at 180 rpm and 30°C for 48 h. This was consequently
the seed culture for the fermentation carried out in 5-L fermenter.
Batch fermentation in a 5-L fermenter
Laboratory scale fermentation was performed in a 5-L (working
volume 2L) CMF-5 fermenter (Figure 2.3). The pH was automatically
controlled above 5.0 by pulsing the addition of 5 N NaOH. The dissolved
oxygen (DO) was measured by polarographic oxygen electrode. The inlet
gas was filtered through a filter with pore size of 0.2 µm and the exhaust gas
was passed through a solution of 5% (w/v) copper (II) sulfate pentahydrate
(CuSO 4 •5H 2 O). Data logging measurements were recorded with the
RT-DAS program (real-time data acquisition system), (Genie Systems,
ADVENTECH). IMO solution put in the fermenter and yeast extract put in
an Erlenmeyer flask were autoclaved separately. After cooling, the yeast
extract was added in the fermenter. The fermentation was carried out under
24
the following conditions: temperature, 30°C; agitation, 400 rpm; aeration, 2
v.v.m. with air. Foam was controlled by addition of antifoaming agent.
Fermentation was carried out under these conditions for approximately 24 h.
Sampling for analysis of sugar components was performed at 4-h intervals.
Purification of isomaltooligosaccharides
The IMO solution in jar fermenter was filtered out via a ceramic MF
system. The remaining yeast cells could be reused if necessary. After mixed
with 1% (w/v) actived carbon powder and stirred for 2 h, the IMO solution
was filtered through a MF membrane. Then 0.5% (w/v) of Amberlite
MB-150 was added to this IMO solution. After stirred for 2 h the IMO
solution was filtered through a filter paper. Concentration of IMO solution
was carried out in a vacuum evaporator at 70°C until syrup containing 50%
(w/v) solid was obtained. A scheme of the procedures in the experiments
was shown in Figure 2.1.
Cultivation of S. cerevisiae WP500 on agar plate
Incubation at 30°C for 2-3 days
25
Shaking culture
Shaking cultivation at 180 rpm

and 30°C for 48 h
Batch fermentation (2-L scale) in 5-L fermenter
Temperature, 30°C; agitation, 400 rpm;

aeration, 2 v.v.m. with air; pH controlled
at 5.0
Filtration via ceramic MF system, treatment with active

carbon and ion exchange resin, filtration, vacuum
evaporation
Stock at 4°C
Figure 2.1 Scheme of depletion of digestible sugar in IMO by yeast.
2.2.2 Utilization of high-content IMO during batch fermentation by
bifidobacteria
26
Culture mdia
A. Culture medium:
Bifidobacteria was precultured in MRS broth with a cover of liquid
paraffin.
B. Fermentation medium:
It consisted of the following ingredients (%, w/v): proteose peptone NO3
1, beef extract 1, yeast extract 0.5, ploysorbate 80 0.1, ammonium citrate
0.2, sodium acetate 0.5, magnesium sulfate 0.01, manganese sulfate 0.005,
dipotassium phosphate 0.2, given amounts of high-content isomalto-
oligosaccharides and liquid parafirn.
Activating and culturing bifidobacteria
The freeze-dried culture was spread on an agar plate with culture media
and incubated in an anaerobic jar at 37°C for 1-3 days. After inoculated in
50ml MRS broth in a 100 ml bottle, bifidobacteria thrived, developed a
white ring in liquid culture. It must be activated several times in the way for
the best growth activity to be achieved. Five ml of this bifidobacteria culture
was transferred into a 100-ml culture medium in a 250 ml bottle. After a
24-h cultivation, the culture was used as an inoculum for the 5-L ferementer.
27
Bifidobacteria cultures were always anaerobically incubated at 37°C either
in anaerobic jar or in a liquid medium covered with liquid paraffin.
Batch fermentation
Laboratory scale fermentation was performed in a 2-L (working volume
1.3 L) Eyela fermenter (Figure 4). The pH was measured by an pH electrode
and was controlled above 6.0 by pulsing the addition of 5 N NaOH. The
oxidation-reduction potential (ORP) was measured by an ORP electrode. The
culture medium was covered with liquid paraffin, resulting in an anaerobic
environment. Data logging measurements were recorded with the RT-DAS
program (real-time data acquisition system) (Genie Systems, ADVENTECH).
The fermenter with 2 L culture medium which was covered with a layer of
liquid paraffin, was autoclaved at 110℃ for 40 min. The culture medium was
MRS broth except the 2% (w/v) glucose was replaced by 5% (w/v)
high-content isomaltooligosaccharides. After cooling, 100 ml of
bifidobacterium culture were inoculated. The fermentation was carried out
anaerobically under the following conditions: temperature, 37°C; agitation,
100 rpm; time approximately, 72 h. Sampling was done at 4 h intervals in the
first 24 h period and at 8 h intervals in the followed 48 h period. Viable count,
28
OD600 and sugar components were analyzed. A scheme of procedures in the
experiments was shown in Figure 2.2.
Analysis of oligosaccharides and organic acids by HPLC
Analysis of oligosaccharides was performed by HPLC under the
following conditions: column, Merck Lichrospher® 100 NH2 (5µm); mobile
phase, acetonnitrile: H2O (75:25), flow rate, 1.0 ml/min; RI detector, JASCO
RI-930, and temperature of column, 35°C. Analysis of organic acid was
performed by HPLC under the following conditions: column,
TRANSGENOMIC ICSep ICE-ION-300; mobile phase, 0.01N, H2SO4; flow
rate, 0.3 ml/min; RI detector, Waters 401 Differential Refractometor, and
temperature of column, 35°C.
Observation of growths of bifidobacteria
The growths of bifidobacteria were observed by determing the OD600
and the viable count (log cfu/ml) of the culture medium. The viable counts
were presented by log cfu/ml. Samples of the culture medium were diluted
stepwisely in 100-fold order (normally above 106) with 0.85% saline solution.
Ten and 100 µl of diluted samples were distributed on MRS agar plates and
29
then incubated in an anaerobic jar. Colonys of bifidobacteria grown on MRS
agar plate were counted after an anaerobic incubation at 37°C for 2 days.
30
Cultivation of the freeze-dried culture on MRS agar plate
in anaerobic jar
Culture: MRS broth covered with liquid paraffin
Cultivation at 37°C for 24 h
Batch fermentation in 2-L fermenter
Temperature, 37°C; agitation, 100 rpm;

anaerobic.
Analysis of viable count, OD600 and sugar components
Figure 2.2 Scheme of large scale fermentation by bifidobacteria.
31
DO meter
pH controller
Air in
Air out
Pump
5N NaOH
Figure 2.3 The schematic diagram of the system for batch frmentation by
yeast.
32
ORP meter
pH controller
Sampling tube
Pump
liquid paraffin
5N NaOH
Figure 2.4 The schematic diagram of the system for batch fermentation by
bifidobacteria.
33
2.3 Control of fermentation
The fermentation was controlled by Genie system with the software and
the hardware being purchased from Advantech Co. All signals of the
measures of pH, DO and ORP were transmitted through PCLD-8115, a
signal-collecting board, and then to PCL-818L data acquisition card. This
control system was communicated by Genie program installed in a Microsoft
compatible PC with the system of Win31, Win95 or Win98. PCLD-885 relay
board served as switches for driving the peristaltic pumps to feed the
solution of glucose, xylose, alkali or acid onto the culture medium in jar
fermentation. As shown in Figure 2.5, an program of ADVENTECH GENIE
was written for this study and it provided the monitoring of pH, DO and
ORP and the control of the feeding.of NaOH or substrate solution during
fermentation. A display screen associated with the strategy was shown in
Figure 2.6.
34
Figure 2.5 A display of the original ADVENTECH GENIE strategy of
fermentation (mainboard).
35
Figure 2.6 A display of original ADVENTENCH GENIE strategy for
fermentation (connecting system).
36
CHAPTER 3. RESULTS AND DISCUSSION
3.1 Depletion of digestible sugar in commercial IMO by Saccharomyces
cerevisiae WP500
One ml of seed culture as previously described was inoculated in a
500-ml Erlenmeyer flask containing 200 ml of liquid medium composed of
0.5% (w/v) yeast extract and 10% (w/v) commercial IMO. This was then
cultivated in shaking incubator at 30℃ for 5 days. The sugar composition
was analyzed daily. Ten yeast species−were used in the fermentation and the
sugar depletion abilities of them were compared. Saccharomyces cerevisiae
WP500 had the highest activity and completed the reaction more quickly
than other strains (data not shown).
Consequently, Saccharomyces cerevisiae WP500 was used in the
fermentation in a 5-L jar fermenter. Time course of the fermentation was
shown in Figure 3.1. During fermentation, glucose and maltose decreased
concurrently. The reaction completed in 24 hours. The IMO concentration
sustained at a constant level even at hr 40. After treatment with active carbon
and Amberlite MB-150, a IMO content, up to 99.5% on a dry weight basis
was obtained. The HPLC analysis of carbohydrates was shown in Figure 3.2
(after 24 hours of fermentation) and Figure 3.3 (fermentation for 24 hours).
37
So many derived peaks manifested the diversity of GOS components, which
also made this analysis difficult. But it is clear that the peaks of glucose and
maltose completely diminished after a 24-h fermentation.
38
60 60
50 50
Sugar concentration (g/l)
40 40
OD600
30 30
20 20
10 10
0 0
0 5 10 15 20
Time (h)
G G2 G3 IG2
P IG3 G4 OD600
Figure 3.1 Depletion of maltose and glucose in IMO during fermentation by
Saccharomyces cerevisiae WP500.
G: glucose; G2: maltose; IG2: isomaltose; G3: maltotriose; P: panose; IG3:
isomaltotriose; G4: tetrasaccharides; OD600: optical density at 600nm
The reaction was carried out in a 5-L jar fermenter under the following
conditions: working volume, 2 L; temperature, 30°C; agitation, 400 rpm;
aeration, 1 v.v.m; pH, automatically controlled at 5.0, initial
concentration of IMO, 200 g/L (w/v).
39
2500
a. glucose
a b. maltose
2000 c. Isomaltose
d. Maltotriose
e. Panose
1500
f. Isomaltotriose
mV
g. Tetrasaccharides
1000
b e
500
c
d g
f
0
0 10 20 30 40
Retention time (min)

Figure 3.2 HPLC analysis of commercial IMO before fermentation.
Analysis of carbohydrates was performed by HPLC on an NH2 column
(250 × 4 mm, particle size 5 µm) under the following conditions: detector,
Waters 410 differential refractometer; mobile phase, acetonitrile/water
75:25; and flow rate of mobile phase, 1 ml/min.
40
2500
c. Isomaltose
2000 e. Panose
f. Isomaltotriose
g. Tetrasaccharides
1500
mV
1000
e
500
c
g
f
0
0 10 20 30 40
Figure 3.3 HPLC analysis of IMO after 24 hours of fermentation.
Analysis of carbohydrates was performed by HPLC on an NH2 column
(250 × 4 mm, particle size 5 µm) under the following conditions: detector,
Waters 410 differential refractometer; mobile phase, acetonitrile/water
75:25; and flow rate of mobile phase, 1 ml/min.
41
Table 3.1 Depletion of maltose and glucose in IMO by Saccharomyces
cerevisiae WP500 during fermenter.
Concentration (g/L) G G2 IG2 G3 P IG3 G4 himo/imo

0hr 44.19 30.96 18.65 6.64 58.33 4.33 30.52 0.58
24hr 0.00 0.05 14.98 0.40 44.01 4.30 23.12 0.99
The fermentation was carried out in a 5-L jar fermenter under the
following conditions: working volume, 2 L; temperature, 30°C; agitation,
400 rpm; aeration, 1 v.v.m; pH, automatically controlled at 5.0, initial
concentration of IMO, 200 g/L.
42
3.2 Bacth fermentation by bifidobacteria
3.2.1 The batch fermentation by individual bifidobacterium
Bifidobacteria are regularly found in human large intestine and colon,
where digestible sugars such as glucose, sucrose and maltose, are not
attainable. The high-content oligosaccharides which do not include the
digestible sugars can reach there and stimulate the growth of bifidobacteria.
Bifidobacteria can promote human healthy. Because these high-content
oligosaccharides are free of digestible sugar, the investigation of enhancing
the growth of bifidobacteria with such oligosaccharides must be more
significant than the previous researches, in which commercial
oligosaccharides including large amounts of digestible sugars were used.
The high-content IMO with various concentrations were added to a
1.3-L bifidus culture in a 2-L jar fermenter. The fermentation was carried out
under an anaerobic condition with gentle stirring. Optical densities and
viable counts of the fermentation broth were monitored. Furthermore, the
utilization of each sugar component including glucose (G), maltose (G2),
isomaltose(IG2), maltotriose (G3), panose (P), isomaltotriose (IG3) and
tetrasaccharide (G4) was analyzed by HPLC throughout the fermentation
process. Other parameters such as ORP, pH and the acetate to lactate ratio
43
are also determined.
During a 72-h fermentation, ORP declined from –39.8 mv to –264 mv
in the period of h 0~13. After that it gradually increased. During the
exponential growth period, OD600 increased from 0.57 (at h 0) to 18.16 (at h
32). The viable counts increased dramatically from h 0 to h 12 with a
maximum value of 9.56 log cfu/ml. After h 32 the viable count declined.
Bifidobacterium primary metabolize P, G4 and IG2. P decreased quickly at
frist, and followed by G4. It revealed that this bifidobacterium contained
α–glucosidase. G was produced after h 20, because IMO were markly
hydrolyzed by α –glucosidase. At h 56 of the fermentation the
concentrations of acetic acid and lactic acid were 25.47 g/L and 24.27 g/L,
respectively. It manifested that B. longum CCRC 14634 used high-content
IMO very well and thus grew very well too. Large amounts of acetic acid
and lactic acid were concurrently produced. The time-course fermentation
was shown in Figure 3.4.
44
10.0 20 0
18
-50
9.5 16
14
-100
9.0 12
log(cfu/ml)
OD600
ORP
10 -150
8.5 8
-200
6
8.0 4
-250
2
7.5 0 -300
0 20 40 60
Time(h)
OD600 log(cfu/ml) ORP
25 30
25
20
Acid concentration (g/l)

20
15
15
10
10
5
5
0 0
0 20 40 60
Time(h)
G G2 IG2
G3 P IG3
G4 L A
Figure 3.4 Time courses of ORP, OD600, viable count, sugar concentrations,
and acid concentrations during the fermentation by B. longum
CCRC 14634 in a 2-L jar fermenter.
45
In the 72-h fermentation, ORP declined from 56 mv to –254 mv in the
period of h 0.5~19. However, the exponential growth period was h 0~20, the
OD600 varying from 0.51 to 3.48. The viable counts increased dramatically
during the first period of fermentation and reached its maximum of 8.87 log
cfu/ml a h 12. After that the viable count declined gradually and became
below 106 at h 48. The tendency of the growth of bifidobacteria based on the
viable counts was similar to that from the OD600 data. Bifidobactera
metabolized primarily P, G4 and IG2. P, G4 and IG2 declined rapidly,
especially P decreased much more quickly in the first 24 h. It reveald that
this bifidobacterium has α–glucosidase. G and G2 was produced at 20 h,
because IMO were markly hydrolyzed by α–glucosidase. The acetic acid
maxima of 8.09 g/L (at 72 h), was higher than that of lactic acid of 6.92 g/L
(at 40 h). It revealed that B. longum CCRC 14602 utilize high-content IMO
very well and thus grew very well too. Large amounts of acetic acid and
lactic acid were concurrently produced. The time-course fermentation was
shown in Figure 3.5.
46
9.5 4.0 100
9.0 3.5 50
8.5 3.0 0
8.0 2.5 -50

log(cfu/ml)
OD600
ORP
7.5 2.0 -100
7.0 1.5 -150
6.5 1.0 -200
6.0 0.5 -250
5.5 0.0 -300

0 20 40 60
Time (h)
25 10
20 8

15 6
10 4
5 2
0 0
0 20 40 60
Time (h)
G G2 IG2
G3 P IG3
G4 L A
and acid concentrations during the fermentation by B. longum
47
During a 72-h fermentation, ORP declined from -11 mv to –319 mv in
the period of h 0~22, and then increased very slowly. However, the
exponential growth period was h 0~30, the OD600 varying from 0.35 to 15.62.
The viable counts rose quickly from h 0 and reached its maximum of 9.32
log cfu/ml at h 11. After h 30 the viable counts declined. The tendency of the
growth of bifidobacteria based on the viable counts was similar to that from
the OD600 data. P, G4 and IG2 started to be comsumed at h 4, h 8 and h 16,
respectively. It revealed that this bifidobacterium has interestingly high
activity of α-glucosidases. The enzyme can hydrolyze IMO into G and G2.
G2 increased and reached maximum value of 9.54 g/L, and then decreased.
G increased and reached 7 g/L. After 8 h all of the acids increased. At h 72,
the concentration of acetic acid and lactic acid were 23.0 g/L and 21.8 g/L,
respectively. It revealed that B. bifidum CCRC 14615 used high-content IMO
very well and thus grew very well too. Large amounts of acetic acid and
lactic acid were concurrently produced. The time-course fermentation was
shown in Figure 3.6.
48
9.4 18
9.2 16 0
14 -50
9.0
12
8.8 -100
log(cfu/ml)
10
OD600
ORP
8.6 -150
8
8.4 -200
6
8.2 -250
4
8.0 2 -300
7.8 0 -350
0 20 40 60
Time(h)
25 25
20 20

15 15
10 10
5 5
0 0
0 20 40 60
Time(h)
G G2 IG2
G3 P IG3
G4 L A
and acid concentrations during the fermentation by B. bifidum
49
During a 72-h fermentation, ORP declined from 40 mv to -231 mv.
The greatest change occurred during the period of h 0.5~13. During the
exponential growth period, OD600 increased from 0.7 (at h 4) to 2.6 (at h 12).
The viable counts increased dramatically from h 4 to h 8 the maximum value
being 7.85 log cfu/ml. The tendency of the growth of bifidobacteria based on
the viable counts was similar to that from the OD600 data. Levels of all sugar
components declined very slowly. Acetic acid increased rapidly during h
4-20, but lactic acid didn’t increased. It revealed that B. bifidum CCRC
11844 used high-content IMO poorly. This strain picked up only a small
fraction of IG2 and P. The time-course fermentation was shown in Figure
3.7.
50
9 2.2 150
2.0
100
8
1.8
50
1.6
7
0
1.4
log(cfu/ml)
OD600
ORP
6 1.2 -50
1.0
-100
5
0.8
-150
0.6
4
-200
0.4
3 0.2 -250
0 20 40 60
time(hr)
25 5
20 4

15 3
10 2
5 1
0 0
0 20 40 60
Time (h)
G G2 IG2
G3 P IG3
G4 L A
and acid concentrations during fermentation by B. bifidum
51
In a 72-h fermentation, ORP declined from 25 mv to -270 mv. The
greatest change occurred in the period of h 0~30. The exponential growth
period was from h 4 to h 12 and the OD600 varied from 0.87 to 2.5. The
viable counts increased dramatically in the frist 12 h with a maximum value
of 8.87 log cfu/ml. The tendency of the growth of this bifidobacterium based
on the viable counts was similar to that from the OD600 data. Levels of all
sugar components declined very slowly, lactic acid increased rapidly during
h 4-12. However, and acetic acid didn’t increased. It revealed that B. breve
CCRC 11846 used high-content IMO poorly. This strain picked up only a
small fraction of P. The fermentation data were shown in Figure 3.8.
52
9.0 3.0 50
8.8 0
2.5
8.6 -50
2.0
log(cfu/ml)
8.4 -100
OD600
ORP
8.2 -150
1.5
8.0 -200
1.0
7.8 -250
7.6 0.5 -300

0 20 40 60
Time (h)
25 4
20
3

15
10
1
5
0 0
0 20 40 60
Time (h)
G G2 IG2
G3 P IG3
G4 L A
and acid concentrations during the fermentation by B. breve
53
In the 72-h fermentation, ORP declined from -66 mv to –279 mv. The
greatest change occurred in the period frist 19 h. The exponential growth
period was h 0~36 and the OD600 varied from 0.67 to 18.28. The viable count
rose rapidly from h 0 and increased stably to 9.47 log cfu/ml at h 16. After 44
h the viable counts declined and was becoming below 108 at h 56. The
tendency of the growth of bifidobacteria based on the viable counts was
similar to that from the OD600 data. P, G4 and IG2 declined rapidly. It
revealed that this bifidobacterium has α−glucosidases. G and G2 began to
increased at 16 h, because IMO were markly hydrolyzed by α–glucosidase.
All acid concentrations increased apparently during the fermentation. At h 68,
the concentrations of acetic acid and acetic acid were 28.4 g/L and 25.88 g/L,
respectively. B. adolescentis CCRC 14607 used high-content IMO very well
and thus grew very well too. Large amounts of acetic acid and lactic acid
were concurrently produced. The time-course fermentation was shown in
Figure 3.9.
54
9.6 20 -50
9.4
9.2 -100
15
9.0
8.8 -150
log(cfu/ml)
OD600
ORP
8.6 10
8.4 -200
8.2
5
8.0 -250
7.8
7.6 0 -300
0 20 40 60
Time (h)
25 30
25
20

20
15
15
10
10
5
5
0
0
0 20 40 60
Time (h)
G G2 IG2
G3 P IG3
G4 L A
Figure 3.9 Time courses of ORP, OD600, viable count, sugar concentratiosns,
and acid concentrations during the fermentation by B.
adolescentis CCRC 14607 in a 2-L jar fermenter.
55
In the 72-h fermentation, ORP declined from 18 mv to -361 mv in the
period of h 1~12. After that it gradually increased. The exponential growth
period was in the first 12 h and OD600 varied from 1.08 to 19.28. The viable
counts rose rapidly from h 0 and reached its maximumm of 9.71 log cfu/ml
at h 24. After 24 h the viable counts declined gradually. The tendency of the
growth of bifidobacteria based on viable counts was similar to that from the
OD600 data. B. adolescentis CCRC 14602 metabolized P, G4 and IG2
dramatically in the frist 16 h. After 40 h, all IMO component were depleted
completely, except that 2 g/L of IG2 was left behind. Lactic acid increased
dramatically in the first 20 h and reached its maximum of 37.41 g/L at 64 h.
However, concentration of acetic acid remain as low as 6.92 g/L, even after
72 h of fermentation. B. adolescentis CCRC 14609 almost did not product
acetic acid. B. adolescentis CCRC 14609 used high-content IMO very well
and thus grew very well too. Large amounts of acetic acid and lactic acid
were concurrently produced. The time-course fermentation was shown in
Figure 3.10.
56
9.8 25 100
9.6
9.4 20 0
9.2
9.0 15 -100
log(cfu/ml)
OD600
ORP
8.8
8.6 10 -200
8.4
8.2 5 -300
8.0
7.8 0 -400
0 20 40 60
time(hr)
25 40
20
30

15
20
10
10
5
0 0
0 20 40 60
time(hr)
G G2 IG2
G3 P IG3
G4 L A
concentrations, and acid concentrations during the fermentation
by B. adolescentis CCRC 14609 in a 2-L jar fermenter.
57
In the 72-h fermentation, ORP declined from 75 mv to -72 mv in the
period of h 8~25. The exponential growth period was h 4~28 and OD600
varied from 0.3 to 9.26. The viable counts rose rapidly after 12 h and reached
its maximum of 9.3 log cfu/ml at h 20. After 24 h the viable count declined
and became under 106 at h 56. P, IG2 and G4 decreased slowly in the first 16
h. But in the period of h 16~36 they delined dramatically. Bifidobacterium
has interesting high activity of α−glucosidases. The enzyme can hydrolyze
IMO. G2 increased and reached a maximum value of 5.4 g/L at h 28, and
then decreased gradually. At 60 h, the concentration of lactic acid and acetic
acid were 29 g/L and 27 g/L, respectively. B. pseudocatenulatum CCRC
15476 used high-content IMO very well and thus grew very well too. Large
amounts of acetic acid and lactic acid were concurrently produced. The
time-course fermentation was shown in Figure 3.11.
58
9.5 12 100
80
9.0
10
60
8.5
8 40
log(cfu/ml)
8.0 20
OD600
ORP
6
7.5 0
4 -20
7.0
-40
2
6.5
-60
6.0 0 -80
0 20 40 60
Time (h)
25 35
30
20

25
15
20
15
10
10
5
5
0 0
0 20 40 60
Time (h)
G G2 IG2
G3 P IG3
G4 L A
and acid concentrations during the fermentation by B.
pseudocatenulatum CCRC 15467 in a 2-L jar fermenter.
59
3.3.2.1 Viable counts
In order to exert positive health effects, the microorganisms need to be
viable, active and abundant in the concentration of at least 106 cfu/g in the
commercial product throughout the specified shelf life according to Samona
and Robinson (1991) and Vinderola. (2000). Most of these eight
bifidobacteria grew so well to reach hight viable counts as above-stated.
Among them, B. longum CCRC 14602, B. bifidum CCRC 11844, and B.
breve CCRC 11846 did not grow very well in MRS broth with glucose being
substituted with 5 % (w/v) of high-content IMO. Most of the eight
bifidobacteria achieved 109 cfu/ml, except B. longum CCRC 14602, B.
bifidum CCRC 11844, and B. breve CCRC 11846. The maximum viable
counts of there bifidobacteria were shown in Table 3.2. Most strains have
viable counts above 9 log cfu/ml at h 12~24 during the fermentation. B.
adolescentis CCRC 14609 showed the maximum viable count at h 24 during
the fermentation. B. longum CCRC 14602, B. bifidum CCRC 11844 and B.
pseudocatenulatum CCRC 15476 had a maximum viable counts of 7 log
cfu/ml at h 44~72 during the fermentation.
60
10
8
log(cfu/ml)
0 20 40 60
Time (h)
B. longum CCRC 14634 B. breve CCRC 11846
B. longum CCRC 14602 B. adolescentis CCRC 14607
B. bifidum CCRC 14615 B. adolescentis CCRC 14609
B. bifidum CCRC 11844 B. pseudocatenulatum CCRC 15476
Figure 3.12 Changes of viable counts during 72 h of fermentation at 37°C by
substituted by 5 % (w/v) of high-content IMO.
61
Table 3.2 The maximum viable counts of bifidobacteria during fermentation
at 37°C in MRS medium with glucose being substituted by 5 %
(w/v) of high-content IMO.
Maximum viable count Fermentation time

Species CCRC (log cfu/ml) (h)
B. longum 14634 9.56 16
B. longum 14602 8.87 12
B. bifidum 14615 9.32 30
B. bifidum 11844 7.78 12
B. breve 11846 8.87 36
B. adolescentis 14607 9.47 16
B. pseudocatenulatum 15476 9.34 24
62
3.3.2.2 Optical density
Another way for growth observation is the optical density. The
wavelength of the optical density for bifidobacteria is 600 nm. B. longum
CCRC 14602, B. bifidum CCRC 11844, and B. breve CCRC 11846 grew
slowly in MRS broth with glucose being substituted by 5 % (w/v) of
high-content IMO, and their maximum OD600 value was lower than 4. The
optimum optical density of each bifidobacterium were shown in Table 3.3.
Almost all strains gave their maximum values of OD600 value in the period of
h 16~24 h during fermentation. B. adolescentis CCRC 14607, B.
adolescentis CCRC 14609 and B. longum CCRC 14634 manifested the
fastest growth among these strains, and the maximum optical density value
of 18 was during 72-h of fermentation.
63
20
15
OD600
10
0
0 20 40 60
Time (h)
Figure 3.13 Changes of OD600 during 72 h of fermentation at 37°C by eight
bifidobacteria in MRS medium with glucose being substituted
by 5 % (w/v) of high-content IMO.
64
Table 3.3 The maximum optical densities of bifidobacteria during
Maximum optical density Fermentation time

Species CCRC (600nm) (h)
B. longum 14634 18.2 32
B. longum 14602 3.5 20
B. bifidum 14615 15.6 30
B. bifidum 11844 2.1 16
B. breve 11846 2.5 12
B. pseudocatenulatum 15476 10.4 52
65
3.3.2.3 Change of sugar components during fermentation
The high-content IMO consisted mainly of the following ingredients
⎯ isomaltose (large amounts), maltotriose, panose (large amounts),
isomaltotriose, tetrasaccharides (large amounts) ⎯ and, if any, a minor
amounts of glucose and maltose.
During the fermentation by these bifidobacteria, initial glucose (G)
levels varied from 0 to 0.182 g/L. Bifidobacterium were shown to possess
α–glucosidase. It can hydrolyze IMO. B. longum CCRC 14634, B. longum
CCRC 14602, B. bifidum CCRC 14615, B. adolescentis CCRC 14607 and B.
adolescentis CCRC 14609 glucose decreased during 72 h of fermentation in
MRS broth with glucose being substituted by 5 % (w/v) of high-content IMO
B. longum CCRC 11844, B. breve CCRC 11846 and B. pseudocatenulatum
CCRC 15476 made no significant change in glucose leve during 72 h of
fermentation. By B. adolescentis CCRC 14609 increased and reached its
maxmum at h 8, and then decrease slowly, being completely depleted at h 36.
Changes of glucose concentration during fermentation at 37°C by eight
bacteria in MRS broth with glucose being substituted by 5 % (w/v) of
high-content IMO were shown in Figure 3.14.
66
6
5
G concentration (g/l)
0
0 20 40 60
Time (h)
Figure 3.14 Changes of glucose concentration during fermentation at 37°C
67
During the fermentation by these bifidobacteria, initial maltose (G2)
levels varied from 0.0027 to 0.0257 g/L. Bifidobacterium were shown to
possess α–glucosidase. It can hydrolyze IMO. During the fermentation by
B. longum CCRC 11844, maltose increased and reached its maximum at h 16,
and then declined gradually. The similar behavior was found when B.
pseudocatenulatum CCRC 15476 was used, except the maximum
concentration of maltose appeared at h 28. During the fermentation by B.
longum CCRC 14602 and B. adolescentis CCRC 14607, maltose increasd
from h 20 to h 72 reached maximum values of 9 g/L and 2.9 g/L,
respectively. Changes of maltose concentration during fermentation at 37°C
with eight bacteria in MRS broth with glucose being substituted by 5 % (w/v)
of high-content IMO were shown in Figure 3.15.
68
10
8
G2 concentration (g/l)
0
0 20 40 60
time (hr)
Figure 3.15 Changes of maltose concentration during fermentation at 37°C
69
Isomaltose (IG2) ranked the third amount in the high-content IMO, the
initial concentrations ranging from 9.12 to 11.48 g/L. Isomaltose was
available for most of these bifidobacteria, except B. breve CCRC 11846. B.
bifidum CCRC 14615 depleted isomaltose completely at h 72. B. longum
CCRC 11844 consumed only small fraction (~14%) of the initial IG2. B.
longum CCRC 14634 and B. longum CCRC 14602 consumed nearly 80% of
the initial IG2. B. adolescentis CCRC 14609, B. adolescentis CCRC 14607
and B. pseudocatenulatum CCRC 15476 consumed more then 90% of the
initial IG2. Changes of isomaltose concentration during fermentation at 37°C
by eight bacteria in MRS broth with glucose being substituted by 5 % (w/v)
of high-content IMO were shown in Figure 3.16.
70
14
12
IG2 concentration (g/l)
10
0
0 20 40 60
Time (h)
Figure 3.16 Changes of isomaltose concentration during fermentation at
being substituted by 5 % (w/v) of high-content IMO.
71
Panose (P) was a main component in high-content IMO, initial
concentrationS ranging from 21.78 to 23.80 g/L. Panose decreased during 72
h of fermentation by all of these strains, but B. bifidum CCRC 11844
consumed only a small fraction of initial panose (from 22.57 to 21.10 g/L),
and so did B. breve CCRC 11846 (from 21.78 to 19.15 g/L). B. adolescentis
CCRC 14609, B. bifidum CCRC 14615 and B. longum CCRC 14602
depleted isomaltose completely. It took different time for the difidobactetia
to deplete isomaltose: B. longum CCRC 14602 h 56, B. adolescentis CCRC
14609 h 32 and B. bifidum CCRC 14615 at h 54. Changes of panose
concentration during fermentation at 37°C with eight bifidobacteria in MRS
medium with glucose being substituted by 5 % (w/v) of high-content IMO
were shown in Figure 3.17.
72
25
20
P concentration (g/l)
15
10
0
0 20 40 60
time (hr)
Figure 3.17 Changes of panose concentratiom during fermentation at 37°C
73
Tetrasaccharides (G4) ranked the second amount in the high-content
IMO, initial concentrations ranging from 16.12 to 14.04 g/L. B. bifidum
CCRC 11844 and B. breve CCRC 11846 used tetrasaccharides poorly. B.
adolescentis CCRC 14609 and B. bifidum CCRC 14615 depleted
tetrascaccharides completely at h 48 and h 54, respectively. B. longum
CCRC 14602 consumed 50% of initial G4. B. longum CCRC 14634, B.
adolescentis CCRC 14607 and B. pseudocatenulatum CCRC 15476
consumed more than 80% of initial G4. Changes of tetrasaccharides
concentration during fermentation at 37°C by eight bifidobacteria in MRS
medium with glucose being substituted by 5 % (w/v) of high-content IMO
were shown in Figure 3.18.
74
20
15
G4 concentration (g/l)
10
0
0 20 40 60
Time (h)
Figure 3.18 Changes of tetrasaccharides concentration during fermentation at
75
Changes of total IMO concentration during fermentation at 37°C with
eight bacteria in MRS medium with glucose being substituted by 5 % (w/v)
of high-content IMO were shown in Figure 3.19. The total IMO decreased
markedly. Especially by B. adolescentis CCRC 14609 total IMO was nearly
depleted a h 40. But B. bifidum CCRC 11844 and B. breve CCRC 11846
consumed IMO very poorly even after a 72-h fermentation. B. longum
CCRC 14602 consumed 50% of initial IMO. The other four strains
consumed 85% of initial IMO.
76
60
50
Total IMO concentration (g/l)
40
30
20
10
0
0 20 40 60
Time (h)

Figure 3.19 Changes of total IMO concentration during fermentation at 37°C
77
3.3.2.4 Organic acids
The amounts of lactic acid produced during the 72-h fermentation
Each strain produced lactic acid throughout the fermentation, the
greasteat productivity being in the fast cell-growth period. But B. bifidum
CCRC 11844 and B. breve CCRC 11846 produced only a small amount of
acetic acid during the 72-h fermentation. Amoung these bifidobacteria, B.
adolescentis CCRC 14609 produced the maximum lactic acid of 37.41 g/L.
B. longum CCRC 14634, B. bifidum CCRC 14615, B. adolescentis CCRC
14607 and B. pseudocatenulatum CCRC 15476 produced 18~28 g/L of lactic
acid. B. longum CCRC 14602 produced only 6 g/L of lactic acid.
Productions of lactic acid concentration during fermentation at 37°C by eight
bifidobacteria in MRS broth with glucose being substituted by 5 % (w/v) of
high-content IMO were shown in Figure 3.20.
78
40
Lactic acid concentration (g/l)
30
20
10
0
0 20 40 60
time (hr)
Figure 3.20 Changes of lactic acid concentration during fermentation at 37°C
79
The amount of acetic acid produced during the 72-h fermentation
Each strain produced lactic acid throughout the fermentation, greastest
productivity being in the fast cell-growth period. Amoung these
bifidobacteria, both B. adolescentis CCRC 14607 and B. pseudocatenulatum
CCRC 15476 produced the maximum lactic acid of nearly 28 g/L. But B.
bifidum CCRC 11844, B. breve CCRC 11846 and B. adolescentis CCRC
14609 produced only small amounts of acetic acid even after 72 h of
fermentation. B. longum CCRC 14634 and B. bifidum CCRC 14615
produced more than 20 g/L of acetic acid during fermentation. Productions
of acetic acid concentration during fermentation at 37°C with eight
bifidobacteria in MRS medium with glucose being substituted by 5 % (w/v)
of high-content IMO was showed in Figure 3.21.
80
30
25
Acetic acid concentration (g/l)
20
15
10
0
0 20 40 60
Time (h)
Figure 3.21 Changes of acetic acid concentration during fermentation at
81
Changes of total acid concentration (acetic acid and lactic acid) during
fermentation at 37°C by eight bifidobacteria in MRS medium with glucose
being substituted by 5 % (w/v) of high-content IMO were shown in Figure
3.22. Total acids increased markedly, eapecially B. pseudocatenulatum
CCRC 15476. B. pseudocatenulatum CCRC 15476 producing the maximum
amount of nearly 55 g/L during the 72-h fermentation. However, B. bifidum
CCRC 11844 and B. breve CCRC 11846 produced acids less than 6 g/L.
Total acids produced by these strains varied from 5.46 to 57.04 g/L. The
more rapid was the metabolism of sugars, the higher amount of total acid
was produced.
82
60
50
Total acid concentration (g/l)
40
30
20
10
0
0 20 40 60
Time (h)
Figure 3.22 Changes of total acid concentration (acetic acid and lactic acid)
during fermentation at 37°C by eight bifidobacteria in MRS
medium with glucose being substituted by 5 % (w/v) of
high-content IMO.
83
For most of these strains, the acetate-to-lactate ratio approximated to 1.
However, B. adolescentis CCRC 14609 produced much lactic acid but acetic
acid. So the acetate-to-lactate ratio was much higher than that of other
bifidobacteria. The concentrations of lactic acid, acetic acid and the
acetate-to-lactate ratio during fermentation at 37°C by eight bifidobacteria in
MRS medium with glucose being substituted by 5 % (w/v) of high-content
IMO were shown in Table 4.
84
Table 3.4 The concentrations of acetic acid, lactic acid and acetate-to-lactate
ratio during fermentation at 37°C by eight bifidobacteria in MRS
medium with glucose being substituted by 5 % (w/v) of
high-content IMO.
Species CCRC Acetic acid (g/L) Lactic acid (g/L) A:L

B. longum 14634 25.47 24.27 1.05:1
B. longum 14602 8.10 6.79 1.19:1
B. bifidum 14615 22.98 21.80 1.05:1
B. bifidum 11844 4.57 3.23 1.41:1
B. breve 11846 3.55 2.38 1.49:1
B. adolescentis 14607 28.41 25.89 1.10:1
B. adolescentis 14609 2.85 37.41 0.08:1
B. pseudocatenulatum 15476 27.24 29.80 0.91:1
85
3.3.2.5 The ORP
During the fermentation with a medium containing D-xylose, glucose
and other nutrients,it has been found that the ORP of the culture medium
declined gradually as glucose in the culture medium was being used and the
ORP would then fluctuate as glucose became completely depleted. If more
glucose were added, however, the ORP would decline again (Sheu et al.,
2003).
During 72-h of fermentation by bifidobacteria, IMO was being used,
the ORP always declined. However, when the IMO was ceased to be
consumed the ORP would fluctuate. Table 3.5 showed the time-points at
which mixima of ORP, depletion of IMO and maximum of viable counts
were achiened during the 72-h fermentation by bifidobacteria. Therefore,
ORP-Time rose gradually indicate IMO was utilized at the last phase and
viable bifidobacteria counts leave the exponential phase and get into the
stationary phage. This behavior was also found during the anaerobic
fermentation by bifidobacteria (S. L. Her et. al.,2004)
86
Table 3.5 The time-points at which mixima of ORP, depletion of IMO and
maxima of viable count being achieved during the 72-h
fermentation.
Minima of ORP Depletion of IMO Mixima of viable count

Species CCRC (Time-point,h) (Time-point,h) (Time-point,h)
B. longum 14634 12.25 - 16
B. longum 14602 20.00 20 12
B. bifidum 14615 20.75 34 30
B. bifidum 11844 12.00 - 12
B. breve 11846 30.25 12 36
B. adolescentis 14607 20.75 24 16
B. adolescentis 14609 12.50 8 24
B. pseudocatenulatum 15476 30.00 24 24
87
Table 3.6 The maximum viable counts, the concentrations of total acid
(acetic acid and lactic acid), acetate-to-lactate ratio and the
concentrations of depleted of IMO during fermentation at 37°C by
Maximum
viable count Total acid A:L Depleted of IMO
Species CCRC (log cfu/ml) (g/l) (g/l)
B. longum 14634 9.56 49.74 1.05:1 43.48
B. longum 14602 8.87 14.89 1.19:1 24.79
B. bifidum 14615 9.32 44.78 1.05:1 42.09
B. bifidum 11844 7.78 7.80 1.41:1 4.22
B. breve 11846 8.87 5.93 1.49:1 2.13
B. adolescentis 14607 9.47 54.30 1.10:1 41.68
B. adolescentis 14609 9.71 40.26 0.08:1 49.18
B. pseudocatenulatum 15476 9.34 57.04 0.91:1 42.04
88
CHAPTER 4. CONCLUSION
In the present work, the growth of cell and the utilization of IMO
during fermentation by eight bifidobacteria including Bifidobacterium
longum CCRC14634, Bifidobacterium longum CCRC 14602,
Bifidobacterium bifidum CCRC 14615, Bifidobacterium bifidum CCRC
11844, Bifidobacterium breve CCRC 11846, Bifidobacterium adolescentis
CCRC 14607, Bifidobacterium adolescentis CCRC 14609, and
Bifidobacterium pseudocatenulatum CCRC 15476 were investigated. The
fermentation medium was MRS medium with glucose being replaced by 5%
(w/v) high-content IMO, which was free of digestible sugars. Most of the
bifidobacteria used in this study metabolize primarily P, G4 and IG2 and
thus produced G and IG2. It revealed that these bifidobacteria had
α–glucosidase activity, so that IMO were apparently hydrolyzed. As IMO
was being consumed, the ORP of the culture broth declined, until most of the
IMO was depleted, and then it would increase very slowly. The decreasing
of ORP reflected the consuming of IMO and the growth of bifidobacteria.
Excepting B. longum CCRC 14602, B. bifidum CCRC 11844 and B. breve
CCRC 11846, other bifidobacteria grew very well and could reach a high
viable count of 9 log cfu/ml during fermentation. And excepting B. bifidum
CCRC 11844, B. breve CCRC 11846 and B. pseudocatenulatum CCRC
15476, the changes of OD600 were always consistent with the changes of
89
viable count. B. adolescentis CCRC 14609 consumed IMO much faster than
other strains and produced the maximum lactic acid of 37.41 g/L.
90
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APPENDICES
Appendix 1: The data of bifidobacetria
Strains CCRC ATCC Source

Bifidobacterium bifidum 11844 - adult intestine
Bifidobacterium breve 11846 15700 infant intestine
Bifidobacterium longum 14602 15697 infant intestine
Bifidobacterium adolescentis 14607 15704 adult intestine
Bifidobacterium adolescentis 14609 15706 adult intestine
Bifidobacterium bifidum 14615 29521 infant feces
Bifidobacterium longum 14634 15708 infant intestine
Bifidobacterium pseudocatenulatum 15476 27919 infant feces
98
Appendix 2: The HPLC diagram of the IMO
2500
a. glucose
a b. maltose
2000 c. Isomaltose
d. Maltotriose
e. Panose
1500
f. Isomaltotriose
mV
g. Tetrasaccharides
1000
b e
500
c
d g
f
0
0 10 20 30 40
Analysis of carbohydrates was performed by HPLC on an NH2 column (250
× 4 mm, particle size 5 µm) under the following conditions: detector, Waters
410 differential refractometer; mobile phase, acetonitrile/water 75:25; flow
rate of mobile phase, 1 ml/min; and temperature of column, 40°C.
99
Appendix 3: The HPLC diagram of the organic acid
250
Acetic acid
200
Lactic acid
150
100
mV
50
-50
-100
0 10 20 30 40
Analysis of organic acid was performed by HPLC on a TRANSGENOMIC
ICSep ICE-ION-300 column under the following conditions: RI detector,
Waters 401 Differential Refractometor; mobile phase, 0.01N H2SO4; flow
rate of mobile phase, 0.3 ml/min; and temperature of column, 35°C.
100
Appendix 4: HPLC analysis of high-content IMO before fermentation
by B. adolescentis CCRC 14607
1000
a. Isomaltose
b. Panose
800
c. Isomaltotriose
d. Tetrasaccharides
600
mV
400
b
200
a
c d
0 10 20 30 40
410 differential refractometer; mobile phase, acetonitrile/water 75:25; and
flow rate of mobile phase, 1 ml/min.
101
Appendix 5: HPLC analysis of high-content IMO after 72 hours of
fermentation. by B. adolescentis CCRC 14607
1000
800
600
mV
400
200
0 10 20 30 40
410 differential refractometer; mobile phase, acetonitrile/water 75:25; and
flow rate of mobile phase, 1 ml/min.
102
Appendix 6: HPLC analysis of lactic acid and acetic acid before
fermentation by B. adolescentis CCRC 14607
400
a. Lactic acid
300 b. Acetic acid
200
mV
100
a b
0
0 10 20 30 40
103
Appendix 7: HPLC analysis of lactic acid and acetic acid after 72 hours
of fermentation. by B. adolescentis CCRC 14607
400
300
200
mV
100
0 10 20 30 40
104

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高純度異麥芽寡糖對比菲德氏菌增值的影響

指導教授：許垤棊（Prof. Dey-Chyi Sheu）

Thesis for Master of Science

Ya-Ting Shu, B. S. Biol.

Bifidobacteria can promote human health. Bifidobacteria are regularly

glucose, sucrose and maltose, are not attainable. Isomaltooligosaccharides

glucosyltransferase. IMO is preprobiotic, being able to enhance the growth

of bifidobacteria. High-content IMO can be produced, by treating

commercial IMO syrup with yeast, thereby digestible sugar including

from 58% to 99% on a dry weight basis. In a 2-L jar fermenter,

bifidobacteria were cultured in 1.3 L broth in the presence of 5% (w/v)

high-content IMO. The fermentation conditions were as following: anaerobic

culture, overlaid with a layer of liquid paraffin; stir rate, 100rpm;

temperature, 37°C. During fermentation, turbidity, viable count, ORP, pH,

acetate, lactate and sugar components were determined periodically. During

72 h of fermentation, most of the bifidobacteria used in this study metabolize

primarily panose, tertesaccharides and isomaltose and thus produced G and

consumed isomaltooligosaccharides poorly.

The present investigation using such oligosaccharides which was free

oligosaccharides accompanied with large amounts of digestible sugars were

used. The utilization of various IMO components by eight bifidobacteria was

Key words: bifidobacteria, high-content isomaltooligosaccharides, probiotics

糖中的消化性糖，亦即麥芽糖及葡萄糖，使寡糖的純度由 58%提高至 99%

以上。在 2 公升醱酵槽中，用 1.3 公升的培養基，添加 5%的高純度異麥

芽寡糖，在礦油覆蓋下的厭氧條件、於 37°C、攪拌速度 100rpm，觀察

還原電位、pH 值、醋酸與乳酸濃度，以及用 HPLC 分析寡糖成分的變

在 72 小時醱酵中，比菲德氏菌主要代謝 panose, tertesaccharides 和

isomaltose，因而產生 glucose 和 maltose。B. adolescentis CCRC 14609 利

用異麥芽寡糖的速度為最快速且乳酸的產量 37.41 g/L 相較於其他菌株

為最大。而 B. bifidum CCRC 11844 和 B. breve CCRC 11846 則在高純度

LIST OF TABLES ...................................................................VIII

LIST OF FIGURES ..................................................................... X

1.1 Probiotics ........................................................................................... 1

1.1.1 Types of prebiotics and their effect on intestinal

1.1.2 Oligosaccharides ...................................................................... 7

1.1.3 Isomaltooligosaccharides ......................................................... 8

1.2.1 Nutrition ................................................................................. 13

1.2.2 Carbohydrate matabolism ...................................................... 14

1.2.3 Extracellullar dextranase and intracellular α-1→6

1.3 The effect of prebiotic in vitro experiments .................................... 16

2.1.1 Microorganisms ..................................................................... 20

2.2.1 Depletion of digestible sugar in commercial IMO by

Saccharomyces cerevisiae WP500 ..................................... 23

2.2.2 Utilization of high-content IMO during batch fermentation

2.3 Control of fermentation.................................................................... 34

CHAPTER 3. RESULTS AND DISCUSSION ......................... 37

3.1 Depletion of digestible sugar in commercial IMO by

Saccharomyces cerevisiae WP500 ................................... 37

3.2 Bacth fermentation by bifidobacteria .............................................. 43

3.2.1 The batch fermentation by individual bifidobacterium ......... 43

3.3.2.1 Viable counts ................................................................ 60

3.3.2.2 Optical density ............................................................. 63

3.3.2.3 Change of sugar components during fermentation ...... 66

3.3.2.5 The ORP ....................................................................... 86

Appendix 1: The data of bifidobacetria ................................................. 98

Appendix 2: The HPLC diagram of the IMO ........................................ 99

Appendix 3: The HPLC diagram of the organic acid .......................... 100

Appendix 4: HPLC analysis of high-content IMO before

fermentation by B. adolescentis CCRC 14607 .............. 101

Appendix 5: HPLC analysis of high-content IMO after 72 hours of

fermentation. by B. adolescentis CCRC 14607 ............. 102

Appendix 6: HPLC analysis of lactic acid and acetic acid before