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EFFECT OF HIGH-CONTENT

ISOMALTOOLIGOSACCHARIDES ON THE

GROWTH OF BIFIDOBACTERIA

Ya-Ting Shu

Prof. Dey-Chyi Sheu

Thesis for Master of Science

Department of Bioengineering

Tatung University

June 2004

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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

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I

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, 37C. 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.

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II

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

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III

glucosyltransferase

58% 99%

2 1.3 5%

37C 100rpm

pH HPLC

72panose, tertesaccharides

isomaltose glucose maltoseB. adolescentis CCRC 14609

37.41 g/L

B. bifidum CCRC 11844B. breve CCRC 11846

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CONTENTS

page

Abstract ..........................................................................................I

......................................................................................IIICONTENTS ................................................................................. 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 -16

glucosidase .......................................................................... 15

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

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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

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VII

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 byB. adolescentis CCRC 14607 .............. 101

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

fermentation. byB. adolescentis CCRC 14607 ............. 102

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

fermentation byB. 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

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VIII

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 37C 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 37C 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

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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 37C

by eight bifidobacteria in MRS medium with glucose being


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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

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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

byB. bifidum CCRC 11844 in a 2-L jar fermenter. .................... 51



fermentation byB. breve CCRC 11846 in a 2-L jar fermenter... 53


concentratiosns, and acid concentrations during the

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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 byB. pseudocatenulatum CCRC 15467 in a 2-L

jar fermenter. ............................................................................... 59

Figure 3.12 Changes of viable counts during 72 h of fermentation at 37C


substituted by 5 % (w/v) of high-content IMO........................... 61

Figure 3.13 Changes of OD600 during 72 h of fermentation at 37C 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

37C by eight bifidobacteria in MRS medium with glucose

being substituted by 5 % (w/v) of high-content IMO................. 67

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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 37C 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



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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 37C by eight bifidobacteria in

MRS medium with glucose being substituted by 5 % (w/v) of

high-content IMO. ...................................................................... 83

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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

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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 andEnterobacferiaceae.

The prebiotic approach to increasing beneficial bacteria in the colon

potentially provides some advantages over the probiotic strategy. Consumed

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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.

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Figurg 1.1 Proposed mechanisms of prebiotic action to improve human

health.

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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 colonis the multiplicity of different carbon sources to which they have

access. A range ofdietary 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

thehuman 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 toshort-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 microfloracontributes 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

saccharolyticorganisms belong to the genera Bacteroides,Bifidobacteriiim,

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Eiibacterium,Lactobacillus, and Clostridium. Any fermentable dietary

component that arrivesundigested 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, andlarge polysaccharides,

all with a variety of sugar compositions and glycosidiclinkages. Such a

diverse range of chemical structures would be expected to providean equally

diverse range of effects on the colonic microflora. However, almost allof

these food ingredients are claimed as prebiotics on the basis of selectively

stimulating the proliferation of the bifidobacteria in the colon. Such food

ingredientshave 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

withmany saccharolytic species is not clear. The ability to efficiently utilize

such a variety of substrates indicates that bifidobacteria possess an array of

glycosidases, makingthem 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). Itappears that bifidobacteria are one of the most efficient

groups in the colonic microflora at utilizing many non-digestible

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carbohydrates providing them with acompetitive advantage and allowing

them to proliferate relative to other specieswhen prebiotics are consumed.

Their tolerance to the acid micro-environmentproduced as a consequence of

prebiotic fermentation and SCFA production mayalso contribute to their

selective proliferation.

1.1.2 OligosaccharidesOver the last two decades a range of non-digestible oligosaccharides

(NDO's) have been developed, initially as low calorie, low cariogenic,

sucrose substitutes for useas bulking agents in foods. As the effects of these

oligosaccharides on the colonicmicroflora was investigated, it emerged that

some had the potential to increasebifidobacteria in the colon. The ability to

act as a prebiotic has become a marketingedge for these products and has

promoted research into the ability of oligosaccharidesto induce beneficial

changes in the composition and metabolism of the colonicmicroflora.

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 sugarsusing transglycosylases.

The one exception is soybean-oligosaccharides which aredirectly extracted.

The products produced for food applications are not chemically

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homogeneous but are mixtures containing oligosaccharides with different

numbers of monosaccharide moieties and often different glycosidic

linkages. Currently thereare nine different types of NDO's produced

commercially, predominantly m Japanand Europe (Crittenden and Playne,

1996; Playne and Crittenden, 1996). Almost all of these are claimed by

their manufacturers to bebifidogenic. 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--glucosyl-maltose), isomaltotriose, and tetrasaccharides, are

glucosyl saccharides with -(16) 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 -(16) glucosidic linkages, commercial IMO syrup is generally

accepted as a mixture of glucosyl saccharides containing both -(16) and

-(14) glucosidic linkages (Yun et al., 1994).

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Figure 1.2 Molecular structure of IMO.

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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).

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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).

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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

37-41C; minimum growth temperature 25-28C maximum 43-45C.

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.

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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).

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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

areB. 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).

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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 -16 glucosidase

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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 -16 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

-16 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.

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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.

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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)

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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

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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

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Fermentor: CHIN-CHI MBR. Co., Ltd., CMF-5 fermentor

EYELA, JAR FEREMENTOR MBF

Filter: Whatman, Syringe Filter (diamteter 13 mm, pore size 0.2 m )

High-performance Data Acquisition Card:

A/D and D/A transformation cardGenie 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

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HPLC detector: JASCO, RI930

Waters 401, Differential Refractometor

HPLC column: Merck, Lichroapher

100 NH2(5m)

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..

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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 30C for 2-3 days. For the fermentation in shake

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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 30C 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 m and the exhaust gas

was passed through a solution of 5% (w/v) copper (II) sulfate pentahydrate

(CuSO45H2O). 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

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the following conditions: temperature, 30C; 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 70C until syrup containing 50%

(w/v) solid was obtained. A scheme of the procedures in the experiments

was shown in Figure 2.1.

Cultivation ofS. cerevisiae WP500 on agar plate

Incubation at 30C for 2-3 days

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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

Shaking culture

Batch fermentation (2-L scale) in 5-L fermenter

Shaking cultivation at 180 rpm

and 30C for 48 h

Temperature, 30C; 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, vacuumevaporation

Stock at 4C

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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 37C 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.

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Bifidobacteria cultures were always anaerobically incubated at 37C 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, 37C; 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,

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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 (5m); mobile

phase, acetonnitrile: H2O (75:25), flow rate, 1.0 ml/min; RI detector, JASCO

RI-930, and temperature of column, 35C. 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, 35C.

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 l of diluted samples were distributed on MRS agar plates and

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then incubated in an anaerobic jar. Colonys of bifidobacteria grown on MRS

agar plate were counted after an anaerobic incubation at 37C for 2 days.

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Figure 2.2 Scheme of large scale fermentation by bifidobacteria.

Cultivation of the freeze-dried culture on MRS agar plate

in anaerobic jar

Culture: MRS broth covered with liquid paraffin

Cultivation at 37C for 24 h

Batch fermentation in 2-L fermenter

Temperature, 37C; agitation, 100 rpm;anaerobic.

Analysis of viable count, OD600 and sugar components

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Figure 2.3 The schematic diagram of the system for batch frmentation by

yeast.

pH controller

Pump

DO meter

Air in

Air out

5N NaOH

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Figure 2.4 The schematic diagram of the system for batch fermentation by

bifidobacteria.

pH controller

Pump

ORP meter

5N NaOH

Sampling tube

liquid paraffin

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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.

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Figure 2.5 A display of the original ADVENTECH GENIE strategy of

fermentation (mainboard).

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Figure 2.6 A display of original ADVENTENCH GENIE strategy for

fermentation (connecting system).

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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 specieswere 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).

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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.

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Time (h)

0 5 10 15 20

S

ugarconcentration(g/l)

0

10

20

30

40

50

60

OD

600

0

10

20

30

40

50

60

G G2 G3

G4

IG2

IG3P 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, 30C; agitation, 400 rpm;aeration, 1 v.v.m; pH, automatically controlled at 5.0, initial

concentration of IMO, 200 g/L (w/v).

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Retention time (min)

0 10 20 30 40

mV

0

500

1000

1500

2000

2500

a

b

c

d

e

fg

a. glucose

b. maltosec. Isomaltosed. Maltotriosee. Panosef. Isomaltotrioseg. Tetrasaccharides

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 m) under the following conditions: detector,Waters 410 differential refractometer; mobile phase, acetonitrile/water

75:25; and flow rate of mobile phase, 1 ml/min.

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Retention time (min)

0 10 20 30 40

mV

0

500

1000

1500

2000

2500

c. Isomaltosee. Panosef. Isomaltotrioseg. Tetrasaccharides

c

e

gf

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 m) under the following conditions: detector,Waters 410 differential refractometer; mobile phase, acetonitrile/water

75:25; and flow rate of mobile phase, 1 ml/min.

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Table 3.1 Depletion of maltose and glucose in IMO by Saccharomyces

cerevisiae WP500 during fermenter.

The fermentation was carried out in a 5-L jar fermenter under the

following conditions: working volume, 2 L; temperature, 30C; agitation,400 rpm; aeration, 1 v.v.m; pH, automatically controlled at 5.0, initial

concentration of IMO, 200 g/L.

Concentration (g/L) G G 2 IG 2 G 3 P IG 3 G 4 him o/im o0hr 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

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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

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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

glucosidase. G was produced after h 20, because IMO were markly

hydrolyzed by 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.

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Time(h)

0 20 40 60

Sugarconcentration(g/l)

0

5

10

15

20

25

Acidconcentratio

n(g/l)

0

5

10

15

20

25

30

G G2

G3

G4

IG2

IG3PAL

Time(h)

0 20 40 60

OD

600

0

2

4

6

8

10

12

14

16

18

20

log(cfu/ml)

7.5

8.0

8.5

9.0

9.5

10.0

ORP

-300

-250

-200

-150

-100

-50

0

log(cfu/ml)OD600 ORP

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.

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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 glucosidase. G and G2 was produced at 20 h,

because IMO were markly hydrolyzed by 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.

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Time (h)

0 20 40 60

Sugarconcentration

(g/l)

0

5

10

15

20

25

Acidconcentration(g/l)

0

2

4

6

8

10

Time (h)

0 20 40 60

OD

600

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

log(cfu/ml)

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

ORP

-300

-250

-200

-150

-100

-50

0

50

100


G G2

G3

G4

IG2

IG3PAL


and acid concentrations during the fermentation by B. longum


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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-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 thatB. 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.

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Time(h)

0 20 40 60

Sugarconcentration

(g/l)

0

5

10

15

20

25


0

5

10

15

20

25

Time(h)

0 20 40 60

OD

600

0

2

4

6

8

10

12

14

16

18

log(cfu/ml)

7.8

8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

ORP

-350

-300

-250

-200

-150

-100

-50

0


G G2

G3

G4

IG2

IG3PAL


and acid concentrations during the fermentation by B. bifidum


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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 didnt 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.

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Time (h)

0 20 40 60


0

5

10

15

20

25

Acidconcentration

(g/l)

0

1

2

3

4

5

time(hr)

0 20 40 60

OD

600

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

log(cfu/ml)

3

4

5

6

7

8

9

ORP

-250

-200

-150

-100

-50

0

50

100

150


G G2

G3

G4

IG2

IG3PAL


and acid concentrations during fermentation by B. bifidum


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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 didnt 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.

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Time (h)

0 20 40 60


0

5

10

15

20

25


0

1

2

3

4

Time (h)

0 20 40 60

OD

600

0.5

1.0

1.5

2.0

2.5

3.0

log(cfu/ml)

7.6

7.8

8.0

8.2

8.4

8.6

8.8

9.0

ORP

-300

-250

-200

-150

-100

-50

0

50


G G2

G3

G4

IG2

IG3PAL


and acid concentrations during the fermentation by B. breve


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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 glucosidases. G and G2 began to

increased at 16 h, because IMO were markly hydrolyzed by 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 14607used 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.

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Time (h)

0 20 40 60

Sugarconcentra

tion(g/l)

0

5

10

15

20

25


0

5

10

15

20

25

30

Time (h)

0 20 40 60

OD

600

0

5

10

15

20

log(cfu/ml)

7.6

7.8

8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

9.6

ORP

-300

-250

-200

-150

-100

-50


G G2

G3

G4

IG2

IG3PAL

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.

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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.

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time(hr)

0 20 40 60

Sugarconcentration

(g/l)

0

5

10

15

20

25

Acidconcentration

(g/l)

0

10

20

30

40

time(hr)

0 20 40 60

OD

600

0

5

10

15

20

25

log(cfu/ml)

7.8

8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

9.6

9.8

ORP

-400

-300

-200

-100

0

100


G G2

G3

G4

IG2

IG3PAL


concentrations, and acid concentrations during the fermentation

byB. adolescentis CCRC 14609 in a 2-L jar fermenter.

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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 ofglucosidases. 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.

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Time (h)

0 20 40 60


0

5

10

15

20

25

Acidconcentration

(g/l)

0

5

10

15

20

25

30

35

Time (h)

0 20 40 60

OD

600

0

2

4

6

8

10

12

log(cfu/ml)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

ORP

-80

-60

-40

-20

0

20

40

60

80

100


G G2

G3

G4

IG2

IG3PAL


and acid concentrations during the fermentation by B.

pseudocatenulatum CCRC 15467 in a 2-L jar fermenter.

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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.

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Time (h)

0 20 40 60

log(cfu/ml)

4

6

8

10

B. adolescentisCCRC 14607

B. breveCCRC 11846

B. pseudocatenulatumCCRC 15476B. adolescentisCCRC 14609

B. longumCCRC 14602

B. bifidumCCRC 11844

B. longumCCRC 14634


Figure 3.12 Changes of viable counts during 72 h of fermentation at 37C by

eight bifidobacteria in MRS medium with glucose being

substituted by 5 % (w/v) of high-content IMO.

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Table 3.2 The maximum viable counts of bifidobacteria during fermentation

at 37C in MRS medium with glucose being substituted by 5 %

(w/v) of high-content IMO.

Maximumviable count Ferm entation tim e(log cfu/ml) (h)

14634 9.56 16

14602 8.87 12

14615 9.32 30

11844 7.78 1211846 8.87 36

14607 9.47 16

14609 9.71 24

15476 9.34 24

B. adolescentis

B. pseudocatenulatum

CCRC

B. longum

B. bifidum

B. breve

B. longum

B. bifidum

Species

B. adolescentis

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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.

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Time (h)

0 20 40 60

OD

600

0

5

10

15

20


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.13 Changes of OD600 during 72 h of fermentation at 37C by eight

bifidobacteria in MRS medium with glucose being substituted

by 5 % (w/v) of high-content IMO.

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Table 3.3 The maximum optical densities of bifidobacteria during



Maximum optical density Fermentation time

(600nm) (h)

14634 18.2 32

14602 3.5 20

14615 15.6 30

11844 2.1 1611846 2.5 12

14607 18.3 36

14609 19.3 16

15476 10.4 52

B. longum

B. bifidum

CCRC

B. longum

B. bifidum

B. breve

B. adolescentis

B. pseudocatenulatum

Species

B. adolescentis

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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

glucosidase. It can hydrolyze IMO. B. longum CCRC 14634,B. longum

CCRC 14602, B. bifidum CCRC 14615,B. adolescentis CCRC 14607 andB.

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 37C by eight

bacteria in MRS broth with glucose being substituted by 5 % (w/v) of

high-content IMO were shown in Figure 3.14.

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Time (h)

0 20 40 60

Gc

oncentration(g/l)

0

1

2

3

4

5

6


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.14 Changes of glucose concentration during fermentation at 37C



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During the fermentation by these bifidobacteria, initial maltose (G2)

levels varied from 0.0027 to 0.0257 g/L. Bifidobacterium were shown to

possess 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 37C

with eight bacteria in MRS broth with glucose being substituted by 5 % (w/v)

of high-content IMO were shown in Figure 3.15.

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time (hr)

0 20 40 60

G2concentration(g/l)

0

2

4

6

8

10


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.15 Changes of maltose concentration during fermentation at 37C



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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 andB. 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 37C

by eight bacteria in MRS broth with glucose being substituted by 5 % (w/v)

of high-content IMO were shown in Figure 3.16.

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Time (h)

0 20 40 60

IG2concentration(g/l)

0

2

4

6

8

10

12

14


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.16 Changes of isomaltose concentration during fermentation at


being substituted by 5 % (w/v) of high-content IMO.

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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 didB. 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 37C with eight bifidobacteria in MRS

medium with glucose being substituted by 5 % (w/v) of high-content IMO

were shown in Figure 3.17.

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time (hr)

0 20 40 60

Pconcentration(g/l)

0

5

10

15

20

25


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.17 Changes of panose concentratiom during fermentation at 37C



.

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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 37C by eight bifidobacteria in MRS

medium with glucose being substituted by 5 % (w/v) of high-content IMO

were shown in Figure 3.18.

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Time (h)

0 20 40 60

G4concentration(g/l)

0

5

10

15

20


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.18 Changes of tetrasaccharides concentration during fermentation at


being substituted by 5 % (w/v) of high-content IMO.

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Changes of total IMO concentration during fermentation at 37C 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 byB. 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.

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Time (h)

0 20 40 60

TotalIMO

concentration(g/l)

0

10

20

30

40

50

60


B. breveCCRC 11846


B. longumCCRC 14602


B. longumCCRC 14634


Figure 3.19 Changes of total IMO concentration during fermentation at 37C



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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

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