Bifidobacteria ( 1 : Bifidobacteria, 2 : Bifidobacteria, 3 : Bifidobacteria, : Bifidobacteria ) : : : : : : : : : : : :. : : Bifidobact

Transcription

1 7 ] 1996 B ifidobacteria. : : : ( ) :

2 Bifidobacteria ( 1 : Bifidobacteria, 2 : Bifidobacteria, 3 : Bifidobacteria, : Bifidobacteria ) : : : : : : : : : : : :. : : Bifidobacteria 1 : Bifidobacteria 2 : Bifidobacteria 3 : Bifidobacteria : Bifidobacteria - 1 -

3 . Bifidobacteria,, cholesterol bifidobacteria : Bifidobacteria bifidobacteria. bifidobacteria B. bifid um Bb-11 B. bifidum K-7, MRS broth whey : Bifidobacteria. bifidobacteria. bifidobacteria,, bifidobacteria : B. bifidum. B. bifidum B. bifidum. 4. : Bifidobacteria. cholesterol bifidobacteria

4 bifidobacteria bifidobacteria. cholesterol : B. bifidum Bb-11 bifidobacteria B. bifidum K-7 bifidobacteria bifidobacteria. Bifidobacteria bifidobacteria, bifidobacteria yeast bifidobacteria MRS broth. B. bifidum Bb-11 B. bifidum K-7,, air atomization sodium alginate poly-l-lysine microparticles : bifidobacteria air-atomization. sodium alginate CaCl2 bifidobacteria, ( ) bifidobacteria :, in vitro bifidobacteria B cell, B cell B. bifidum, B. bifidum, B. bifidum, B. bifidum - 3 -

5 B. bifidum cytokine. 4. : 16S rdna bifidobacteria. Bifidobacteria,,, : bifidobacteria B. bifidum Bb-11 B. bifidum K-7 sodium alginate poly-l-lysine microparticles air atomization,. bifidobacteria B. bifidum K-7 L-cystein e HCl 0.05% MRS broth whey B. bifidum Bb-11. B. bifidum K-7 MRS broth B. bifidum K % B. bifidum Bb % B. bifidum K B. bifidum Bb , ph 2. Whey whey protein isolate(wpi) yeast extract 0.75% MRS broth B. bifidum K ph 5.20, 0.33% , B. bifidum Bb-11 ph 4.03, 0.64% %, yoghurt starter culture B-3 yoghurt,,., yoghurt 200 B. bifidum Bb-11 B. bifidum K-7 5g 4-20, 16, ph. B. bifidum Bb-11 B. bifidum K ph bifidobacteria - 4 -

6 bifidobacteria milk. B. bifidum Bb-11 B. bifidum K-7 ph % 16 ph T.A 1.07% 1.02% B. bifidum Bb-11 4 B. bifidum K-7 8. B. bifidum Bb-11 B. bifidum K ph bifidobacteria.. 2 : bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing m. Starter culture 0.5g, Yeast extract 0.5g, Glycerol 5.0g, NaHSO3 0.5g, Mg3(PO4)21.0g. slurry bifidobacteria culture cfu/g bifidobacteria 107 cfu/g. bifidobacteria ph. bifidobacteria cfu/g. bifidobacteria cfu/g cfu/g. bifidobacteria bfidobacteria bifidobacteria culture bifidobacteria. lactic acid bacteria, enterobacteriaceae staphylococci. bifidobacteria frequency

7 bifidobacteria.. 3 : B. bifidum spleen B cell. B cell cytokine (TGF- 1 IL-5) IgA. B cell polyclonal activator LPS. B. bifidum mesenteric lymph node (MLN) B cell IgA. B. bifidum spleen B cell Peyer's patch (PP) B cell MLN IgA. B. bifidum PP MLN B cell IgA, IgA. B. bifidum B. bifidum. B. bifidum B. bifidum. B. bifidum MLN cell TGF- 1 mrna. TGF- 1 IgA, bifidobacteria, IgA.. : 16S rdna ribotyping, PCR 16S rdna SDS-PAGE bifidobacteria PCR 16S rdna. Bifidobacteria RAPD SDS-PAGE. B. infantis B. bifidum bifidobacteria. B. bifidum K-7. B. bifidum. B. bifidum Bb-11 40,000 Caco2 cell B. bifidum K-7. B. bifidum K-7 bifidobacteria clostridia.. B. bifidum Bb-11 B. bifidum Bb-11 cholesterol B. bifidum Bb-11 cholesterol

8 . Whey bifidobacteria bifidobacteria bifidobacteria bifidobacteria, sodium alginate poly-l-lysine microparticles.. Bifidobacteria,.., bifidobacteria... bifidobacteria. Bifidobacteria

9 SUMMARY Subject 1: Development of Culturing Method of Bifidobacteria and value-added dairy products 1. Purpose of Study: In order to develop the value-added dairy products containing either B. bifidum Bb-11 or B. bifidum K-7 bifidobacteria-loaded microparticles, 2. Results and Conclusion: Both bifidobacteria were massively grown and stimulated at optimal condition then micrparticulated by air atomization with sodium alginate poly-l-lysine. To evaluate the employed bifidobacteria, the B. bifidum K-7, selected by this collaborative research project was compared to the commercial B. bifidum Bb-11. The growth of B. bifidum K-7 and B. bifidum Bb-11 in MRS broth containing L-cysteine HCl 0.05% were respectively and at 24 hours, indicates no difference. However, in the culture based on whey, the growth of B. bifidum K-7 was at 24 hours which was inferior to of B. bifidum Bb-11. Therefore, both bifidobacteria were employed in the experiment to develop value-added dairy products. The experimental 12.7% sterilized market skim milk was simulated with commercial skim milk powder. The experimented fermented milk was cultured with commercial yoghurt starter culture B-3. Finally the experimental ice cream was made from commercial ice cream mix. After individual addition 5g of either B. bifidum Bb-11 and B. bifidum K-7 microparticles into each 200 of the experimental 12.7% skim milk, fermented milk and ice cream mix, the skim milk and fermented milk were stored at 4 while the ice cream was stored at -20. The number of live bacteria, ph and titratable acidity in each product were measured every 4 days during 16 days of storage. During the storage of bifidobacteria added experimental skim milk, the initial number of live B. bifidum Bb-11 and B. bifidum K-7 were respectively and then changed to and after 16 days, indicates relatively good survivability. And the ph and titratable acidity were also nearly unchanged for 16 days. This result proved that the commercial production of so called bifidobacteria containing milk is technically feasible. In the fermented milk containing B. bifidum Bb-11 and B. bifidum K-7 microparticles, the initial ph was 4.95 and the initial titratable acidity was 0.8 %. At 16 days, the ph and the titratable acidity in the fermented milk containing B. bifidum Bb-11 were 4.62 and 1.07% while those in the fermented milk containing B. bifidum K-7 were 4.60 and 1.02%, respectively. However, the number of live B. bifidum - 8 -

10 Bb-11 and B. bifidum K-7 were zero at 4 days and 8 days of storage. In the ice cream containing B. bifidum Bb-11 and B. bifidum K-7 microparticles, the initial number of live bacteria were and , but at 16 days, those were and respectively. This showed no difference in number of live bacteria through the storage period. In addition, the ph and the titratable acidity were almost similar. Based on the results, it is likely that ice cream containing bifidobacteria microparticles also can be made. Subject 2: Preparation and Evaluation of Bifidobacteria-loaded microparticles 1. Purpose of Study: To develop bifidobacteria-loaded alginate poly-l-lysine m icroparticles using air atomization method. 2. Results and Conclusion: The optimal survival of bifidobacteria loaded in alginate poly-l-lysine microparticles during freeze-drying process was established. Thereafter, the survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles was widely investigated as a function of ph, dissolution time, media and storage condition. Finally, the changes of microorganisms such as bifidobacteria, lactic acid bacteria, enterobacteriaceae and staphylococci in feces after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles to human volunteers were investigated in addition to physical status of feces. The mean size of bifidobacteria-loaded alginate poly-l-lysine microparticles was ranged between um. The bifidobacteria-loaded alginate poly-l-lysine microparticles were prepared by changing concentration of alginate solution (1.5%), spraying air pressure (0.75 bar) and delivery rate of alginate solution (8 ml/min). The bifidobacteria-loaded alginate poly-l-lysine microparticles were irregularly and roughly spherical in shape but had wrinkled surface. The bifidobacteria-loaded alginate poly-l-lysine microparticles were small sized and free flowable. The survival of bifidobacteria loaded in alginate poly-l-lysine microparticles was optimized when the formulation containing 0.5% yeast extracts, 5% glycerol, 0.5% NaHSO3and 1% Mg3(PO4)2was used. The moisture content was also important to influence survival of Bifidobacteria during the freeze-drying process. The optimal freeze-drying time was estimated around 14 h. The survival of bifidobacteria loaded in alginate poly-l-lysine microparticles was highly dependent on dissolution media, ph and incubation time. When the - 9 -

11 bifidobacteria was immobilized with alginate or even poly-l-lysine treatment, the survival of bifidobacteria was highly enhanced in the gastric fluid for 120 min, showing over 107 cfu/g. The bifidobacteria-loaded alginate poly-l-lysine microparticles maintain their survival over 107cfu/g after storage for 16 weeks. Bifidobacteria-loaded alginate poly-l-lysine microparticles significantly increased survival of bifidobacteria in fecal microflora in human beings when compared to bifidobacteria cultures as a control. It was evident that the microencapsulation improved resistance of bifidobacteria to gastric acid. However the survival of fecal lactic acid bacteria, enterobacteriaceae and staphylococci did not change significantly after ingestion of bifidobacteria products during the study period. Bifidobacteria-loaded alginate poly-l-lysine microparticles had a tendency to increase the frequency of stools. But the amount of stools was not changed. The viscosity of stools was also significantly decreased during the study periods. From these findings, bifidobacteria-loaded alginate poly-l-lysine microparticles could be use to deliver bifidobacteria to the colon without losing their viability in low ph conditions. The current microencapsulation method using air atomization technique provides an alternative to entrap bifidobacteria without using harmful organic solvents. The bifidobacteria-loaded alginate poly-l-lysine microparticles could be also used in the dairy products to deliver bifidobacteria to the colon without losing viability significantly in the low ph condition. Subject 3: Effect of Bifidobactria on Intesitinal Immune Function 1. Purpose of Study: Bifidobacterium is regarded to be stimulatory for overall host immune responses. However, its exact role in the systemic and mucosal immunity is largely unkown. This study was performed to determine the role of bifidobacterium in the systemic and mucosal antibody response 2. Results and Conclusion: We first examined the direct modulatory effect of bifidobacterium on the synthesis of antibodies by murine spleen B cells. Whole spleen B cells were cultured with Bifidobacterium bifidum or Clostridium perfringens (Welch's bacilli, negative control) and antibody synthesis was measured by ELISA and ELISPOT assay. B. bifidum, but not C. perfringens, substantially increased the total secretion of major Ig isotypes as well as the number of IgA-secreting cells. In addition, B. bifidum increased the proliferation of spleen cells by 3-fold while C. perfringens had little to diminishing effect on it. These results indicate that B

12 bifidum increased Ig synthesis through its mitogenic influence on B cells. Further, B. bifidum induced spleen B cells to be reactive to TGF- 1 and IL-5, resulting in the increase of surface IgA expression ( 3-fold) and total IgA production (>20 fold) but not IgM and IgG2a isotypes. As much as seen in spleen B cells, B. bifidum increased IgA production by mesenteric lymph node (MLN) B cells. In addition, peroral immunization with B. bifidum enhanced number of IgA secreting cells in MLN. Subsequently, we found that this modulating effect of B. bifidum is owing to its cell wall components. Further, it was found that perorally administered alginate-encapsulated B. bifidum is more potent in the increase of IgA expression by MLN B cells than unencapsulated B. bifidum. Finally, we examined the effect of B. bifidum on cytokine expression. Peroral immunization of alginate-encapsulated B. bifidum increased mrna level of TGF- 1 in MLN cells. Taken together, these studies indicate that B. bifidum can act as a LPS-like polyclonal activator for B cells. Furthermore, the fact that bifidobacterium enables B cells to respond to TGF- 1 and IL-5 for the IgA production has important implications in the primary defense against pathogens in the gastrointestinal tract. Subject 4: In Vivo biological activity of Bifidobacteria 1. Purpose of Study: The purpose of the present study is two fold. 1) To develop the molecular biological methods to identify and differentiate species and strains of bifidobacteria. 2) To examine physiological characteristics of bifidobacteria which are important as probiotics and the in vivo effects of bifidobacteria and microencapsulation on fecal bacteria and serum cholesterol 2. Results and Conclusion: 16S rdna ribotyping, restriction pattern of PCR-amplified 16S rdna, and SDS-PAGE of whole cell protein could be used to identify species of bifidobacteria. B. infantis strains and B. bifidum strains were tolerant to acid and bile acid, respectively. Commercial cultures of bifidobacteria were tolerant to both acid and bile acid. B. bifidum K-7 were tolerant to both acid and bile acid. All B. bifidum strains adhered to Caco2 cell better than other bifidobacteria. The protein with molecular weight of 40,000 which bound to Caco2 cell seemed to mediate adherence of B. bifidum Bb-11. Feeding of rat with milk and fermented milk containing B. bifidum K-7 decreased counts of clostridia and increased counts of bifidobacteria in feces. Microencapsulation of B. bifidum K-7 did not affect the bacterial counts of

13 the feces. There was no change in serum cholesterol due to feeding of B. bifidum K-7. Feeding of rat with milk containing microencapsulated B. bifidum Bb-11 and fermented milk decreased serum cholesterol but feeding with B. bifidum Bb-11 without microencapsulation did not. CONTENTS Chapter. Introduction Section 1. Purpose of study Section 2. Scope of study Chapter. Development of Culturing Method of Bifidobacteria and Value-added Dairy Products Section 1. Effects of casein hydrolyzates fractionated by molecula r weight on the growth of Bifidobacterium bifidum Bb Section 2. Growth of Bifidobacterium bifidum K-7 and stability of bifidobacteria microparticles in dairy products- 39 Section 3. References Chapter. Preparation and Evaluation of Bifidobacteria-loaded Microparticles Section 1. Introduction Section 2. Materials and methods Section 3. Results and discussion Section 4. Summary Section 5. References Chapter. Effects of Bifidobacteria on Intesitinal Immune Function Section 1. Introduction Section 2. Materials and methods Section 3. Results and discussion Section 4. Summary Section 5. References

14 Chapter. In Vivo Biological Activity of Bifidobacteria Section 1. Molecular biological identification of differentiation of bifidobacteria Section 2. Physiological characteristics and mechanisms of bifidobacteria adhesion ability to Caco2 cells Section 3. In vivo biological activity of microencapsulated bifidobacteria Section 4. References

15 Bifidobacteria 1 Bifidobacterium bifidum Bb B. bifidum Bb-11 B.bifidum K-7 Bifidobacteria Bifidobacteria Bifidobacteria Bifidobacteria 1 Bifidobacteria Bifidobacteria Bifidobacteria

16 - 15 -

17 1 1. Bifidobacteria,, cholesterol. bifidobacteria bifidobacteria bifidobacteria. bifidobacteria bifidobacteria. air atomization bifidobacteria sodium alginate poly-l-lysine microparticles, bifidobacteria bifidobacteria. 2 1 bifidobacteria bifidobacteria 2 bifidobacteria bifidobacteria microparticles,. 2 1 bifidobacteria bifidobacteria, bifidobacteria microparticles. 3 bifidobacteria bifidobacteria. in vitro bifidobacteria B cell, B cell B. bifidum, B.bifidum, B. bifidum, B. bifidum B. bifidum cytokine. bifidobacteria, bifidobacteria cholesterol

18 2 Bifidobacteria 1 Bifidobacterium bifidum Bb ,,,. peptide,. Kehagias (1977) casein peptide B. bifidum,. bifidobacteria (Poch Bezkorovainy, 1988, 1991; Petschow Talbott, 1991), Poch Bezkorovainy(1988)

19 casein bifidobacteria, trypsin -casein casein bifidobacteria (Poch Bezkorovainy, 1991). Proulx (1992) casein peptide bifidobacteria, Cheng Nagasawa(1984) 1,100 2,300Da peptide Norris, B. breve B. infantis. Azuma (1984) -casein chymosin pepsin bifidobacteria -casein.,,, bifidobacteria. -lactoglobulin, -lactalbumin, immuno globulin, serum albumin, proteose-peptone,. cheese 86 90% (Yamauchi, 1992). bifidobacteria B. bifidum Bb Bifidobacteria bifidobacteria Bifidobacterium bifidum Bb-11(Chr. Hansen's Lab., Denmark). B. bifidum Bb-11 L-cysteine HCl MRS 2.. B. bifidum Bb-11 Whey powder ( ), milk protein hydrolysate(mph) casein hydrolysate(ch) ( ), -casein, -casein, -casein, trypsin Sigma (St. Louis, U.S.A). whey powder 6.5% (ph 7.0, 1N NaOH ), (Beckman J2-21, England) 9,000rpm 5. whey L-cysteine HCl 0.05% yeast extract 0.75%, milk protein hydrolysate(mph) casein hydrolysate(ch),

20 trypsin 10,000Da, 10,000 3,000Da, 3,000Da -, -, -casein 1%(w/v) 0.25, 0.5, % B. bifidum Bb-11 1%, Trypsin casein -, -, -Casein M phosphate buffer(ph 8.0) , trypsin 1% 125 (1437U) ( : = 1:200, wt/wt), 40 6, , -, -Casein trypsin 2 10,000Da, 10,000 3,000Da, 3,000Da. trypsin Centriprep-10(Amicon, Inc., USA) 3,000 g 40 (Union 32R, Hanil Sci. Korea) 10,000Da 10,000Da, 10,000Da Centricon-3(Amicon, Inc., USA) 7,500 g 2 3,000Da fraction 3,000Da (Fig.1). Waddell (A215/A225 ) ( Uvikon 942, Kontron Co. Italy). ( / ) = (A215-A225) 144., ph ph Delta 350 ph (Mettler, England), (titratable acidity, TA) Marth (1978)., 9 9 1% phenolphtalein ph N NaOH ( ). Lactobacillus MRS agar(difico Lab., USA) L-cysteine HCl(Sigma Chem. Co. U.S.A.) 0.05% , MRS broth 1/2, L-cysteine HCl 0.05%,. 1 petridish MRS agar, (BBl Gas Pak 100 Anaerobic Jar Becton Dickinson Microbio. systems, Cockeysville, MD. U.S.A) Gas Pak 100TM Anaerobic Envelops (Becton Dickinson Microbio. systems, Cockeysville, MD. U.S.A)

21 Casein Dissolved in 0.1M phosphate buffer(ph 8.0) 1% trypsin treatment (40 / 6hr) (enzyme : protein = 1 : 200 wt/wt) Inactivation (85 90 /10min) Centriprep-10 (MWCO : 10,000Da) centrifuge (3,000 g / 40min) retentate permeate Centricon-3 (MWCO : 3,000Da) centrifuge (7,500 g / 2hr) retentate permeate Mw>10,000Da Mw<3,000Da 10,000Da>Mw>3,000Da Fig.1. Procedure for the fractionation of -, -, - casein hydrolyzates by ultrafiltration after trypsin digestion. 3.. B. bifidum Bb-11 whey MPH(Milk protein hydrolysate) CH(Casein hydrolysate) 0.25, 0.5, % B. bifidum ph,. MPH ph (Table 1)

22 ph, MPH ph, 24 ph. Table 1. Changes of ph and titratable acidity of whey medium supplemented with milk protein hydrolyzate during the growth of B. bifidum Bb-11. Incubation time (hr) Control ph T.A. (%) Milk protein hydrolyzates 0.25% 0.5% 0.75% 1.0% T.A. ph ph T.A. T.A. ph ph T.A. (%) (%) (%) (%) , 1% (Table 1). 24, %., 12, 1% %,, 0.5%. MPH 0.75% 1% (Fig.2), MPH B. bifidum

23 2 Fig.2. Changes of viable cell counts during the growth of B. bifidum Bb-11 in whey medium supplemented with milk protein hydrolyzate. CH ph (Table 2) % 0.75%, 24 ph, ph. (Table 2) %,, 1% 0.75% %, 0.75%. 24, (Fig. 3). CH B. bifidum 12 24, B. bifidum. Table 2. Changes of ph and titratable acidity of whey medium supplemented with casein hydrolyzate during the growth of B. bifidum Bb

24 Casein hydrolyzates Control Incubation 0.25% 0.5% 0.75% 1.0% time (hr) T.A T.A T.A T.A T.A ph ph ph ph ph (%) (%) (%) (%) (%) Fig.3. Changes of viable cell counts during the growth of B. bifidum Bb-11 in whey medium supplemented with casein hydrolyzate.. Casein -Casein, -casein, -casein 40 6 trypsin 10,000Da, 10,000 3,000Da, 3,000Da Table 3. Table 3,,. -, - -Casein trypsin 10,000Da, 3,000 10,000Da 3,000Da, 1%(w/v) B. bifidum

25 ph,. ph -casein 24 10,000 3,000Da 3,000Da ph (Table 4), -casein (Table 5). -Casein 12 ph, 10,000Da ph (Table 6). Table 3. Protein concentration of three fractions with different molecular weight of -, - and -casein hydrolyzates by trypsin. ( / ) Molecular weight > 10,000 Da 10,000 3,000 Da 3,000 Da > -casein casein casein Table 4. Changes of ph and titratable acidity of whey medium supplemented with different molecular weights of -casein hydrolyzates during the growth of B. bifidum Bb-11. Incubation time (hr) Control >10,000Da 3,000 10,000Da <3,000Da ph T.A.(%) ph T.A.(%) ph T.A.(%) ph T.A.(%) Table 5. Changes of ph and titratable acidity of whey medium supplemented with different molecular weights of -casein hydrolyzates during the growth of B. bifidum Bb

26 Incubation time (hr) Control >10,000Da 3,000 10,000Da <3,000Da ph T.A.(%) ph T.A.(%) ph T.A.(%) ph T.A.(%) Table 6. Changes of ph and titratable acidity of whey medium supplemented with different molecular weights of -casein hydrolyzates during the growth of B. bifidum Bb-11. Incubation time (hr) Control >10,000Da 3,000 10,000Da <3,000Da ph T.A.(%) ph T.A.(%) ph T.A.(%) ph T.A.(%) B. bifidum Bb-11. -Casein 24 10,000Da, 10,000 3,000Da 3,000Da, 3,000Da (Table 4). -Casein 3,000Da (Fig.5). -Casein 10,000Da, 3,000Da

27 Fig.4. Changes of viable cell counts during the growth of B. bifidum Bb-11 in whey medium supplemented with different molecular weights of -casein hydrolyzates. Fig.5. Changes of viable cell counts during the growth of B. bifidum Bb-11 in whey medium supplemented with different molecular weights of -casein hydrolyzates. -casein B. bifidum Bb-11 12, 24 10,000Da,, 3,000Da 10,000 3,000Da (Fig.4). -Casein 12, 24 (Fig.5). -Casein

28 , 3,000Da 10,000Da,. 24 B. bifidum 10,000Da (Fig.6). Fig.6. Changes of viable cell counts during the growth of B. bifidum Bb-11 in whey medium supplemented with different molecular weights of -casein hydrolyzates. Whey B. bifidum Bb-11 (MPH, CH). 0.5% bifidobacteria,, bifidobacteria Park Heo(1995)., bifidobacteria. bifidobacteria, Rose (1963) pepsin, trypsin, papain bifidobacteria, Kehagias (1977), Jao (1978) casein casein bifidus factor, N-acetyl-glucosamine. casein bifidobacteria (Proulx Gaurhier. 1992) Klaver (1993) bifidobacteria casein, bifidobacteria. MPH CH 600Da

29 (Mcdonald, 1990). Trypsin 3 casein B. bifidum Bb-11, - and -casein 3,000Da. 1,100 2,300Da peptide bifidobacteria Cheng Nagasawa(1984) casein peptide peptide bifidobacteria Proulx (1992) 5,000Da peptide bifidobacteria Poch Betskorobainy(1991). Bouhallab (1993) casein tryptic hydrolysates (MP) peptide bifidobacteria, MP 2,000Da peptide., Poch Betzkorovainy(1991) trypsin -casein casein, -casein -, -casein 3,000Da peptide B. bifidum. Bezkorovainy Topouzian (1981) B. bifidum var. pennsylvanicus 10,000Da, B. bifidum, B. infantis B. breve 10,000Da 10,000Da bifidobacteria. 4. Whey Bifidobacterium bifidum Bb-11 protein hydrolysate (MPH) casein hydrolysate (CH), -, -, -casein trypsin ( > 10,000Da, 10,000 3,000Da, < 3,000Da)., MPH CH, 1%,,. casein 3,000Da, 3,000 10,000Da, 10,000Da B. bifidus Bb-11, -casein -casein (3,000Da ), -casein 10,000Da ph

30 , 2 B. bifidum Bb-11 B. bifidum K-7 Bifidobacteria 1. Bifidobacteria (Gibson, 1994 ; Misra, 1995; Moore 1995), (Link, 1994 ; Schiffrin 1995), (Gri ll, 1995 ; Kampmann, 1994 ; Singh, 1997 ; Van't, 1989) cholesterol (Seki, 1978) (Bouhnik Y, 1996). bifidobacteria. ph 2.0 bifidobacteria. (conway, 1987 ; Floch, 1971 ; Midtvedt, 1974). Mayer(1948) bifidobacteria bifidobacteria bifidobacteria. bifidobacteria B. bifidum Bb-11 B. bifidum K-7,, sodium alginate poly-l-lysine microparticles air atomization,,. 2.. Bifidobacteria

31 bifidobacteria B. bifidum Bb-11(Chr. Hansen's, Denmark) B. bifidum K-7 L-cysteine HCl(Sigma) MRS broth (Beckman J2-21, England) 3,000rpm 10.. Bifidobacteria MRS broth L-cysteine 0.05% ph 6.8. Whey whey powder 6.5% ph 1N NaOH 6.8 (Beckman J2-21, England) 9,000rpm 5,. whey 0.59%, 0.38%, 3.74%, 0.35%, 94.90%, B. bifidum K-7 Ammonium citrate 0.2%, Sodium Acetate 0.5% Magnesium sulfate 0.01% Manganese sulfate 0.005%.. ( ) 12.7%, yoghurt starter culture B-3 (S. salivarius s ubsp. thermophilus, L. delbrueckii subsp. bulgaricus, Chr. Hansen's, Denmar k ) yoghurt, (, ) (Sani-Serv A4071-E. U.S.A).. bifidobacteria B. bifidum Bb-11 B. bifidum K-7 sodium alginate poly-l-lysine air atomization,, 200 bifidobacteria 5g, 4, -20, (0, 4, 8, ) ph,.. ph, ph Mettler Delta 350, (titratable acidity, TA) Marth (1978) 9 1% phenolphtalein, ph N NaOH ( ). Bifidobacteria NPNL MRS agar. NPNL Neomycin sulfate 2g, Paromomycin sulfate 4g, Nalidixic acid 0.3g, Lithium chloride 60g m membrane filter

32 4. MRS agar L-cysteine HCl 0.05%, % NPNL MRS agar, MRS broth 1/2 L-cysteine HCl 0.05%. 0, 4, 8, ml 4. 1 MRS agar BBL Gas Pak 100TM Anaerobic Systems (Cockeysville, MD. U.S.A) B. bifidum Bb-11 B. bifidum K-7 bifidobacteria B. bifidum K-7 MRS broth whey B. bifidum Bb-11. B. bifidum Bb-11 B. bifidum K-7 MRS broth L-cysteine HCl 0.05% 37 24, ph Fig.1, Fig.2 Fig. 3. Fig.1. Growth effects of B. bifidum K-7 and B. bifidum Bb

33 in MRS broth. Fig.2. Changes of ph during the growth of B. bifidum K-7 and B. bifidum Bb-11 in MRS broth. Fig.3. Changes of titratable acidity during the growth of B. bifidum K-7 and B. bifidum Bb-11 in MRS broth. B. bifidum K-7 MRS broth B. bifidum K % B. bifidum Bb %, Fig.1 12 B. bifidum K B. bifidum

34 Bb B. bifidum K B. bifidum Bb ph 2. MRS broth L-cysteine HCl 0.05% B. bifidum K-7 B. bifidum Bb-11. Whey L-cysteine HCl 0.05% B. bifidum K ph 5.20 B. bifidum Bb-11 ph 4.03, B. bifidum K % B. bifidum Bb %, B. bifidum K , B. bifidum Bb MRS broth whey B. bifidum Bb-11 B. bifidum K-7 ph,. (Fig.4, Fig.5 Fig.6) Fig.4. Growth effects of B. bifidum K-7 and B. bifidum Bb-11 in Whey-based medium

35 Fig.5. Changes of ph during the growth of B. bifidum K-7 and B. bifidum Bb-11 in Whey-based medium. Fig.6. Changes of titratable acidity during the growth of B. bifidum K-7 and B. bifidum Bb-11 in Whey-based medium. B. bifidum K-7 whey whey whey protein isolate(wpi) 0.75%, yeast extract 0.75% B. bifidum K-7 B. bifidum Bb-11.. bifidobacteria

36 Acidophilus milk bifidobacteria bifidobacteria milk bifidobacteria ph bifidobacteria. bifidobacteria 12.7% 121 1, B. bifidum Bb-11 B. bifidum K-7 5g 4 16 (0, 4, 8 16 ) bifidobacteria, ph. B. bifidum Bb-11 B. bifidum K % 4 Fig.7. Fig.7. Changes of viable cell counts during the storage of milk added bifidobacteria microparticles B. bifidum Bb-11 B. bifidum K-7 B. bifidum Bb B. bifidum K B. bifidum Bb B. bifidum K B. bifidum Bb-11 B. bifidum K-7. B. bifidum Bb-11 B. bifidum K-7 bifidobacteria B. bifidum K-7 B. bifidum Bb-11. bifidobacteria. bifidobacteria

37 , 4 B. bifidum Bb-11 B. bifidum K-7 16 bifidobacteria. B. bifidum Bb-11 B. bifidum K % 4, ph Fig.8. Fig.8. Changes of ph during the storage of milk added bifidobacteria microparticles Fig.8 B. bifidum Bb-11 B. bifidum K-7 ph B. bifidum Bb-11 ph 6.5 B. bifidum K ph B. bifidum K-7 B. bifidum Bb-11 ph 16 ph bifidobacteria milk ph % B. bifidum Bb-11 B. bifidum K-7 4, (T.A) Fig

38 Fig.9. Changes of titratable acidity during the storage of milk added bifidobacteria microparticles B. bifidum Bb-11 B. bifidum K %, 8 B. bifidum Bb % B. bifidum K % 16 B. bifidum Bb % B. bifidum K %. ph bifidobacteria, bifidobacteria milk. 4 4 B. bifidum Bb % B. bifidum K %. 12.7% bifidobacteria (Gilliland, 1979), (Petterson, 1983), bifidobacteria ph bifidobacteria (, 1998). 2 bifidobacteria

39 bifidobacteria yoghurt starter culture B-3 (S. salivarius subsp. thermophilus, L. delbrueckii subsp. bulgaricus, Chr. Hansen's, Denmark ) yoghurt, yoghurt 200 B. bifidum Bb-11 B. bifidum K-7 5g 4, 16 (0, 4, 8, ) bifidobacteria, ph. S. salivarius subsp. thermophilus, L. delbrueckii subsp. bulgaricus 2 yoghurt bifidobacteria 4, Fig.10 Fig.10. Changes of viable cell counts during the storage of fermented milk added bifidobacteria microparticles Fig.10 B. bifidum Bb-11 B. bifidum K B. bifidum Bb B. bifidum K , yoghurt bifidobacteria B. bifidum K-7 B. bifidum Bb-11 B. bifidum K-7 B. bifidum Bb-11. B. bifidum Bb-11 B. bifidum K-7 4, ph Fig

40 Fig.11. Changes of ph during the storage of fermented milk added bifidobacteria microparticles Yoghurt B. bifidum Bb-11 B. bifidum K-7 ph 4.75, 8 B. bifidum Bb B. bifidum K , 16 B. bifidum Bb B. bifidum K Bifidobacteria 4 ph bifidobacteria. B. bifidum Bb-11 B. bifidum K-7 Fig

41 Fig.12. Changes of titratable acidity during the storage of fermented milk added bifidobacteria microparticles B. bifidum Bb-11 B. bifidum K-7 0.8%, 8 B. bifidum Bb % B. bifidum K %, 16 B. bifidum Bb % B. bifidum K %. bifidobacteria ph.. bifidobacteria ice cream frozen yoghurt.. (, ) (Sani-Serv A4071-E. U.S.A.) bifidobacteria. 200 B. bifidum Bb-11 B. bifidum K-7 5g , 4, 8, bifidobacteria, ph. bifidobacteria Fig.13 B. bifidum Bb B. bifidum K , 4 B. bifidum Bb B. bifidum K B. bifidum K-7 B. bifidum Bb-11,

42 16 B. bifidum Bb B. bifidum K B. bifidum K-7 B. bifidum Bb-11. bifidobacteria bifidobacteria. bifidobacteria. Fig.13. Changes of viable cell counts during the storage of ice added bifidobacteria microparticles cream B. bifidum Bb-11 B. bifidum K-7-20, 16 ph, Fig.14 Fig.15. B. bifidum Bb-11 B. bifidum K-7 ph , 16 B. bifidum Bb B. bifidum K bifidobacteria ph

43 Fig.14. Changes of ph during the storage of ice cream added bifidobacteria microparticles Bifidobacteria B. bifidum Bb-11 B. bifidum K % 16 B. bifidum Bb % B. bifidum K % bifidobacteria. Fig.15. Changes of titratable acidity during the storage of ice cream

44 added bifidobacteria microparticles 4. bifidobacteria B. bifidum Bb-11 B. bifidum K-7 sodium alginate poly-l-lysine microparticles air atomization. bifidobacteria B.bifidum K-7 L-cysteine HCl 0.05% MRS broth whey B.bifidum Bb-11. B.bifidum K-7 MRS broth B. bifidum K % B. bifidum Bb % B. bifidum K B. bifidum Bb , ph 2. Whey whey protein isolate(wpi) yeast extract 0.75% MRS broth B.bifidum K ph 5.20, 0.33% , B.bifidum Bb-11 ph 4.03, 0.64% %, yoghurt starter culture B-3 yoghurt,,., yoghurt 200 B. bifidum Bb-11 B. bifidum K-7 5g 4-20, 16, ph. B. bifidum Bb-11 B. bifidum K ph bifidobacteria bifidobacteria milk. B. bifidum Bb-11 B. bifidum K-7 ph % 16 ph T.A 1.07% 1.02% B. bifidum Bb-11 4 B. bifidum K-7 8. B. bifidum Bb-11 B. bifidum K ph bifidobacteria

45 3 1. Azuma, N., K., Yamauchi and T. Mitsuoka Bifidus growth-promoting activity of a glycomacropeptide derived from human -casein. Agric. Biol. Chem. 48(8): Bezkorovainy, A. and N. Topouzian Bifidobacterium bifidus var. pennsylvanicus growth promoting activity of human milk casein and its derivatives. Int. J. Biochem. 13: Bouhallab, S., C. Favrot and J. L. Maubois Growth-promoting activity of tryptic digest of caseinomacropeptide for Lactococcus lactis subsp. lactis. Lait 73: Bouhnik Y, B. Flourie, c. Andrieux, N. Bisetti, F. Brie and J.C. Rambaud Effects of bifidobacterium sp. fermented milk ingested with or without inulinon colonic bifidobacteria and enzymatic activities in healthy humans. Eur.J.Clin.Nutr. 50 : Cheng, C. C. and Nagasawa, T Effect of peptides and amino acids produced by Lactobacillus casei in milk on the acid production of bifidobacteria. Jpn. J. Zootech. Sci. 55: Desjardins, M. L., D. Roy, and J. Goulet. 1990a. Growth of bifidobacteria and their enzyme profiles. J. Dairy Sci. 73: Desjardins, M. L., D. Roy, C. Toupin, and J. Goulet. 1990b. Uncoupling of growth and acids production in Bifidobacterium spp. J. Dairy Sci. 73: El-Soda, M., A. Macedo, and N. F. Olson The peptide hydrolase system of Bifidobacterium species. Milchwissenschaft Gibson, G.R. and X. Wang Regulatory effect of bifidobacteria on the growth of other colonic bacteria. J.Appl.Bacteriol. 77 : Grill, J.P., J. Crociani and J. Ballongue Effect of bifidobacteria on nitrites and nitrosamines. Appl. Microbiol. 20: Hukins, R. W., and N. L. Nannen ph homeostasis in lactic acid bacteria. J. Dairy Sci. 76: Jao, V. C., E. M. Milkolajcik, and P. M. T. Hansen, Growth of Bifidobacterium bifidum var. pennsylvanicus in laboratory media supplemented with amino sugars and spent broth from E. coli. J. Food Sci. 43: Kampmann, E, A. Goldbohm, V.D.Brandt and P. Veer Fermented dairy products,

46 calcium and colorectal cancer in the Netherlands Cohort Study. Cancer Res. 54 : Kehagias, C., Y. C., Jao,E. M., Mikolajick and P. M. T. Hansen Growth response of Bifidobacterium bifidum to a hydrolytic product isolated from bovine casein. J. Food Sci. 42: Klaver, A. M., F. Kingma and H. Weewkamp Growth and survival over Bifidobacteria in milk. Neth. Milk Dairy J. 47: Link, A.H., F. Rochat, K.Y. Saudan, O. Mignot and J. M. Aeschlimann Modulation of a specific humoral immune response and change in intestinal flora mediated through fermented milk intake. Immunol. Med. Microbiol. 10 : Marth, E. H Standard methods for the examination of dairy products. Americ. Public Health Assoc. Washington, DC Marteau, P., P. Pochart, B. Flourie, P. Pellier, L. Sanots, J. F. Desjeux, and J. C. Rambaud Effect of chronic ingestion of a fermented dairy product containing Lactobacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the colonic flora in humans. Am. J. Clin. Nutr. 52, Mcdonald, L. C., H. P. Fleming, and H. M. Hassen Acid tolerance of Leuconost oc mesenteroides and Lactobacillus plantarum. Appl. Enviro n. Microbiol. 56: Misra, A.K. and P.k. Kuila Antimicrobial substances from Bifidobacterium bi fidum. Indian J. Dairy Sci. 48: Modler, H. W., R. C. Mc Kellar, H. D. Goff, and D. A. Mackie Using ice cream as a mechanism to incorporate Bifidobacteria and fructooligosaccharides into the human dite. Cult. Dairy Prod. J. 25: 4-9. Moore, W.E and L.H. Moore Intestinal floras of populations that have a high of coion cancer. Appl. Environ. Microbiol. 61: Park, H. K., and T. R. Heo Studies on the characteristics of Bifidobacterium spp. for the Industrial use. Korean, J. Food Sci. Ani. Resour. Vol.15(2): Petschow, B. W., and R. D. Talbott Response of Bifidobacterium sp -ecies to gr owth promoters in human and cow milk. Pediatr Res. 29: Poch, M., and A. Bezkorovainy Growth-enhancing supplements for varous species of the genus. Bifidobacterium. J. Dairy Sci. 71: Poch, M., and A. Bezkorovainy Bovine milk -casein trypsin digest is a growth enhancer for the genus Bifidobacterium. J. Agric Food Chem. 39: Proulx, M., S. F. Gauthier and D. Roy Utilisation d'hydrolysats enzymatiques de caseine pour la croissance des bifidobacteries. Lait 72:

47 27. Reuter, G Bifidobacteria cultures as components of yoghurt-like products. Bifidobacteria Microflora 9: Rose, C. S. and P. György Bifidus factor II for growth of Lactobacillus bifidus. Proc. Soc. Exp. Bio. N.Y. 112(4): Van't, V.P., J. Dekker, J.W.J. Lamers, F.J.Kok, E.G.Schouten, H. Brants, F. Sturmans and R.J.J Hermus Consumption of fermented milk products and breast cancer : A case-cotrol study in the Netherlands. Cancer Res. 49 : Schiffrin, E.J., F. Rochat, H.A. Link, J.M. Aeschlimann and A.H. Donnet Immunomodulation of human blood cells following the ingestion of lactic acid bacteria J. Dairy Sci. 78: Seki, M., M. Igarashi, Y. Fukuda, S. Simamura, T.Kawashima and K. Ogasa The effect of bifidobacterium cultured milk on the "regularity" among aged group. Nutr. Food. 4 : Singh, j., A. Rivenson, M. Tomita, S. Shimamura, N. Ishibashi and B.S Reddy Bifidobacteria longum, a lactic acid-producing intestinal bacterium inhibits coion cancer and modulates the intermediate biomarkers of coiol carcinogenesis. Carcinogenesis. 18 : Yamauchi, K Biologically functional proteins of milk and peptides derived from milk proteins. Bulletin of the IDF. 272: Yoshigawa, M., H. Suganuma, A. Shiata, H. Usui, K. Kurahashi, T. Mizumoto, Y. Suitani, and K. Kashimoto In peptides chemistry 1993". Y. Okata(Editor), Protein Rerch Foundation, Osaka, Yoshihama, M., E. Mochizuki, S. Mitsuhashi, and K. Ahiko, Studies on application of galactosyl lactose for infant formula.. Effects of galactosyl lactose on intestinal bacterial flora of newborn infantis, Reports of researich lab. Technical research institute, snow brand milk products Co.,Ltd, 78:

48 3 Bifidobacteria 1 Bifidobacteria,,, cholesterol. Bifidobacteria. Bifidobacteria. Bifidobacteria,. Bifidobacteria. sodium alginate. Sodium Alginate. mm. alginate m mm bead Bifidobacteria. Nozzle Bifidobacteria. Bifidobacteria Bifidobacteria.... Bifidobacteria. Bifidobacteria cholesterol,

49 anti-mutagenicity,. Bifidobacteria. Bifidobacteria Bifidobacteria. Bifidobacteria Bifidobacteria. air-atomization ( ) sodium alginate CaCl2 Bifidobacteria, ( ) (Bifidobacteria lactic acid bacteria, enterobacteriaceae staphylococci) Bifidobacteria. ( ), Bifidobacteria,, in vitro in vivo. 1.,,, Bifidobacteria.. 2 Bifidobacteria in vitro /,. 3 Bifidobacteria in vivo

50 2 1. Bifidobacteria Chr. Hansen Lab.(Denmark) B.bifidum Bb-11 B.bifidum K-7 MRS broth whey (Beckman J-21, England) 3,000rpm % Sodium alginate stirring autoclave 30. Bifidobacteria Start culture sodium alginate. 30 stirring homogeneous. Peristaltic pump air atomization device 8ml/min 0.2M CaCl2 15 CaCl2 curing 0.02% poly-l-lysine 5 cross-linking. washing pre-freezing 56, 8mmtorr 14.,,, alginate polylysine. 3. Bifidobacteria microcapsules SEM(scanning electron micrograph), Laser particle size.. 4. Bifidobacteria, yeast extract, cryoprotectant (glycerol, lactose), (ascorbic acid, NaHSO3), (Mg3(PO4)2)

51 . 5. Bifidobacteria 1g (0.5% yeast extract) 200ml (ph= ) (ph= ). 1ml MRS gradient MRS-Agar colon formed unit.. MRS-agar 1) Bifidobacteria 5.5% MRS 0.05% Yeast extract Paraffin autoclave 15. 2) MRS-agar 5.5% MRS, 1.5% agar 0.05% Cystein.HCl autoclave g NaCl 15g Yeast extract 2900ml. 7.4% HCl ph ml. 20.4g KH2PO4(MW ) 15g Yeast extract 2900ml. 1N NaOH ph ml.. Poly-lysine Bifidobacteria Bifidobacterium cultures, Bifidobacteria alginate poly-lysine 1 g (0.5% yeast extract) (0.05% cysteine.hcl) 200 ml (ph=1.5) ml 2.5 % MRS gradient 10-8 MRS agar 37, colon formed unit.. ph Bifidobacteria Phosphate buffer (ph=7.4) 0.1M HCl ph 6.8, 4.0,

52 1.5 ph. Bifidobacteria 1g 200ml (0.5% yeast extract 0.05% cysteine.hcl ) N NaOH ph 6.8 Bifidobacteria ph.. (ph=1.5) Bifidobacteria Bifidobacteria 1 g (0.5% yeast extract) (0.05% cysteine.hcl) 200 ml (ph=1.5) 37. 1ml 2.5 % MRS gradient N NaOH ph 6.8 MRS agar 37, colon formed unit Bifidobacteria.. Bifidobacteria Bifidobacteria petri dish 4. sampling 12 Bifidobacteria. 6. Bifidobacteria in vivo. 8 group control group 2. Bifidobacterium bifidum cultures Bifidobacteria gelatin capsule cfu/g, fecal sample, , 7, Bifidobacteria, Lactic acid bacteria, Enterobacteriaceae Staphylococci

53 -Bifidobacteria (g) BL agar 58 Sodium propionate 15 Paromomycin 0.05 Neomycin 0.1 Lithium chloride 3 Water added 1000 ml Bifidobacteria BS medium 37, colony Bifidobacteria colony. -Lactic acid bacteria (g) Lactobacilli MRS broth 55 Agar 15 NaN3 0.2 Water added 1000 ml 121, Lactic acid bacteira MRS agar medium 37, colony. -Enterobacteriaceae EMB agar Water added (g) 27 g 1000 ml Enterobacteriaceae EMB agar medium 37, 48 colony. -Staphylococci (g) Baird-parker agar base 63 Bacto EY Tellurite 50 (ml) Water 950 (ml)

54 Baird-parker agar base 950 ml Bacto EY Tellurite 50ml. 55. Staphylococci Baird-parked agar medium colony.. Bifidobacteria Bifidobacteria (frequency), 1. Table 1. Score and criterion for evaluation of characteristics of stool before, during and after taking Bifidobacteria-loaded microparticles Scores Frequency of stools/day Amount of stools Viscosity of stools 1 irregular highly reduced very soft 2 half reduced soft 3 one normal normal 4 two increase hard 5 three highly increased very hard

55 3 1.Bifidobacteria Air atomization Fig.1. Air atomization Fig.2. Bifidobacteria particle. sodium alginate 1.2% 1.5%, 2%. Bifidobacteria loading sodium alginate. 30ml 200ml sodium alginate 50ml/200ml 10ml/200ml. Sodium alginate 8ml/min 12ml/min. Air pressure atomization device sodium alginate atomization device CaCl2. Air pressure

56 Fig.1. Diagram of air atomization device

57 Fig.2. Surface morphology of Bifidobacteria-loaded microparticles using scanning electron microscope Bifidobacteria sodium alginate 2.0% 1.5%, 1.2%. Peristaltic pump sodium alginate Bifidobacteria atomization device 8ml/min, 12ml/min (50%) 4ml/min.. Bifidobacteria (50%) air pressure. loading

58 Bifidobacteria. Bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing 15. poly-l-lysine 0.02%. slurry Table 2. Table 2. Processing parameters for the preparation of Bifidobacteria-loaded No. Alginate conc. (%) alginate-polylysine microparticles and their size distribution Air pressure (bar) Delivery rate (ml/min) Culture loading (%) Size ( m) 1 1.2% % % % % % % % % The 0.02 % poly-l-lysine was invariably used to cross-link Bifidobacteria-loaded algiante microparticles. 2. Bifidobacteria

59 Bifidobacteria. 1.5g. Bifidobacteria croyprotectant. 5% glycerol 2.5% lactose. yeast extract Bifidobacteria 0.5%. antioxidants. NaHSO3 ascorbic acid. 0.5% NaHSO3. Ascobic acid alginate spraying CaCl2 aggregation. Mg3(PO4)2 neutralizer 1%. Table 3. Formulation for the optimization of survival of Bifidobacteria loaded in alginate poly-l-lysine microparticles during freeze-drying process Cryoprotectant Antioxidant Neutralizer Survival Yeast Culture rate extract Glycerol Lactose NaHSO3 Ascorbic Mg3(PO4)2 acid Log cfu/g F1 0.5 g F2 1.5 g F3 2.5 g F4 1.5 g 0.5 % F5 1.5 g 2.5 % F6 1.5 g - 5 % F6 1.5 g - 10 % F8 1.5 g % F9 1.5 g % F g % F g % F g % F g % F g % F g % Alginate solution (1.5%) was used invariably used. Bifidobacteria Table

60 Table 4. Optimal condition for the survival of Bifidobacteria loaded in alginate poly-l-lysine microparticles during freeze-drying process Alginate Start culture Yeast extract Glycerol NaHSO3 Mg3(PO4)2 H2O 1.5g 0.5 g 0.5g 5.0g 0.5g 1.0g 100 ml Fig Fig.3. Effect of freeze-drying time on the survival of bifidobacteria loaded in alginate poly-l-lysine microparitcles. Bar represents the standard deviation. 3. Bifidobacteria. 1) Polylysine Bifidobacteria

61 Bifidobacteria ph. air-atomization Bifidobacteria alginate-polylysine alginate. Bifidobacteria. Bifidobacteria alginate polylysine Fig.4. Bifidobacteria culture cfu/g 103cfu/g alginate polylysine Bifidobacteria cfu/g cfu/g. alginate ph polylysine. Bifidobacteria 120. Fig.4. Comparison of survival of bifidobacteria loaded in alginate and alginate poly-l-lysine microparticles after 120 min treatment in simulated gastric fluid (without pepsin, ph 1.5). ap<0.01, compared to bifidobacteria culture in simulated gastric fluid; *, the survival was less than 103cfu/g

62 2) ph Bifidobacteria ph bifidobacteria Fig.5. Bifidobacteria ph ph. ph 6.8 ph 1.5. alginate polylysine bifidobacteria ph ph.. Fig.5. Effect of ph on the survival of bifidobacteria loaded in alginate poly-l-lysine microparticles. Bar represents standard deviation. ap<0.05, compared to ph 1.5 and ph ) (ph=1.5) Bifidobacteria Bifidobacteria. Bifidobacteria Fig (108cfu/g) ( ) 107 cfu/g ( ) 107 cfu/g. gastric emptying

63 Bifidobacteria. ph. Fig.6. Survival of bifidobacteria loaded in alginate poly-l-lysine microparticles in simulated gastric fluid (without pepsin, ph 1.5) as a function of time. Bar represents the standard deviation.. Bifidobacteria Bifidobacteria (4 ) Fig.7. Bifidobacteria cfu/g cfu/g cfu/g. 20 ( cfu/g). 20 Bifidobacteria

64 Fig.7. Stability of bifidobacteria-loaded alginate poly-l-lysine microparticles during storage in a refrigerator at 4. Bar represents the standard deviation. 4. Bifidobacteria in vivo. Bifidobacteria Bifidobacteria Fig.8. Bifidobacteria culture Bifidobacteria. Bifidobacteria ( ) 108cfu/g Bifidobacteria 3, , ( 108cfu/g). Bifidobacteria Lactic acid bacteria, Enterobacteriaceae Staphylococci Fig.9, Bifidobacteria Bifidobacteria

65 Fig.8. Changes of bifidobacteria in feces after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles to human subjects. Bar represents the standard deviation. ap<0.05, compared to before study period

66 Fig.9. Changes of lactic acid bacteria in feces after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles to human beings. Bar represents the standard deviation

67 Fig.10. Changes of enterobacteriaceae in feces after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles to human beings. Bar represents the standard deviation

68 Fig.11. Changes of staphylococci in feces after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles to human beings. Bar represents the standard deviation.. Bifidobacteria frequency, Fig.12. Bifidobacteria frequency. Bifido bacteria

69 Fig.12. Effect of bifidobacteria-loaded microparticles on the characteristics of stools. Bifidobacteria Table 5. Table 5. Physical characteristics of stools after oral administration of bifidobacteria-loaded alginate poly-l-lysine microparticles

70 before after stop Frequency A group 1.00± ± ±0.75 B group 0.71± ± ±0.34 Amount A group 2.75± ± ±0.50 B group 3.25± ± ±0.50 Viscosity A group 3.00± ± ±0.58 B group 3.25± ± ±0.00 A group; Bifidobacteria cultures were orally given. B group, Bidobacteria-loaded alginate poly-l-lysine microparticles were orally given

71 4 Bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing m. 0.5 g Start culture, 0.5g Yeast extract, 5.0g Glycerol, 0.5g NaHSO3,1.0g Mg3(PO4)2. slurry Bifidobacteria culture cfu/g Bifidobacteria 107 cfu/g. Bifidobacteria ph. Bifidobacteria cfu/g. Bifidobacteria cfu/g cfu/g. Bifidobacteria Bfidobacteria Bifidobacteria culture Bifidobacteria. Lactic acid bacteria, Enterobacteriaceae Staphylococci. Bifidobacteria frequency. Bifidobacteria

72 5 1. Bodmeier, R., Chen, H. and Paeratakul, O., A novel approach to the oral delivery of micro- or nanoparticles, Pharm. Res., 6(5), (1989) 2. Goh, J. S., Kwon, I. K. and Kim, Y. O., Studies on the growth of Bifidobacterium bifidum ATCC in milk, Kor. J. Dairy Sci., 8(1), (1986) 3. Goh, J. S., Kwon, I. K., Ahn, J. K. and Yoon, Y. H., Fermentation of milk by Bifidobacteria, and improvement of usability of Bifidobacterium bifidum ATCC by lactic acid bacteria for the fermentation of milk, Kor. J. Dairy Sci., 9(4), (1987) 4. Goh, J. S., Kwon, I. K., Ahn, J. K. and Yoon, Y. H., Fermentation of milk by Bifidobacterium bifidum ATCC ; Conversion of milk constituents by fermentation, Kor. J. Anim. Sci., 30(10), (1988) 5. Kim, D. H. and Han, M. J., Inhibition of intestinal bacterial enzymes by lactic acid bacteria,yakhak Hoeji, 39(2), (1995) 6. Kim, H. S., Kamara, B. C., Good, I. C. and Enders, G. L., Method for the preparation of stabile microencapsulated lactic acid bacteria, J. Ind. Microbiol., 3, (1988) 7. Krueter, J. and Liehl, E., Long-term studies microencapsulated and adsorbed influenza vaccine nanoparticles, J. Pharm. Sci., 70(4), (1981) 8. Kwok, K. K., Groves, M. J. and Burgess, D. J., Production of 5-15 diameter anginate-polylysine microcapsules by an air-atomization technique, Pharm. Res., 8(3), (1991) 9. Lee, B.-J. and Min, M.-G., Preparation and release characteristics of polymer-reinforced and coated alginate beads. Arch. Pharm. Res., 18(3), , 1995b. 10. Lim, F. and Moss, R. D., Microencapsulation of living cells and tissues, J. Pharm. Sci., 70(4), (1981) 11. Lin, S. Y., Ayres, J. W., Winkler, W. and Sandine, W. E., Lactobacillus effects on cholesterol ; In vitro and In vivo results, J. Dairy Sci., 72, (1989) 12. Lin, S. Y. and Kao, Y. H., Tablet formulation study of spray-dried sodium diclofenac enteric-coated microcapsules, Pharm. Res., 8(7), (1991)

73 4 Bifidobacteria 1 Bifidobacteria, (Tisser, 1899). Elie Metchnikoff Bifidobacteria (Mechnikoff, 1908), bifidobacteria (Chandan, 1989). microflora (Bullen et al., 1977), mitogenic (Simone et al., 1992), (Duffy et al., 1993 ; Yasui et al., 1996). (Gilliland et al., 1989; Klaver et al., 1990). bifidobacteria (Fernandes et al., 1990). Bifidobacteria macrophage lymphocyte (Hatcher et al.,1993),, spleen Peyer's patch (PP) mitogenic response (Kado-oka et al., 1991) natural killer cell (Igarashi et al., 1990).. Perdigon (1991) bacteria host mucosal immunity. Lactobacillus GG, human Lactobacillus strain, rotavirus IgA response (Kaila et al., 1992). virus mucosal surface dietary lactic acid bacteria.,, (Hanson Brandtzaeg, 1989),, systemic immunity. S-IgA (Holmgren et al., 1992). lympoid organ B cell. PP, mesenteric lymph node (MLN), Lamina propria (LP) (McGhee et al., 1992 ; Russell et al., 1991). PP M cell PP (Owen et al., 1990) B cell

74 IgA B cell. IgA+ B cell B cell MLN, thoracic duct LP homing (Stollman et al., 1989) IgA B cell plasma cell IgA (McGhee et al., 1992 a : Briere et al., 1994 a). IgA secretory component. IgA. IgA class 80. (Kagnoff, 1987). IgA (Lambert et al., 1974). LPS B cell TGF- 1 B cell IgA. TGF- 1 IL-2 Il-5 IgA (Kim Kagnoff et al., 1990)., bifidobacterium bifidobacteira B cell. spleen B cell bifidobacteira ELISA flow cytometry bifidobacteira thymidine uptake. Peyer's patch mesenteric lymph node B cell bifidobacteira in vitro. bifidobacteria TGF-beta1 IL-5. 2 bifidobacteria B cell. 1. Bifidobacteria spleen B cell. in vitro bifidobacteria spleen cell 7 ELISA ELISPOT assay. spleen cell Peyer's patch mesenteric lymph node cell ELISPOT assay. bifidobacteria B cell IgA cytokine IL-5 TGF-beta1 ELISA

75 bifidobacteria. 3 Bifidobacteria., bifidobacterium alginate bifidobacterium 4 BALB/C spleen, MLN, PP. bifidobacterium, bifidobacterium cell spleen, MLN IgA.. T cell macrophage bifidobacteira spleen cell RT-PCR cytokine. B cell polyclonal activator LPS T cell polyclonal activator ConA spleen cell cytokine. bifidobacteria macrophage T cell cytokine. bifidobacteria T cell macrophage

76 2 1. BALB/C Bifidobacterium bifidum Bifidobacterium bifidum MRS. MRS 0.5% L-cystein, HCl 0.1% sodium azide Liquid paraffin 1.5 ml B. bifidum washing. MRS parpffin oil 400g 10. bacteria HBSS cell/well. RPMI Encapsulated bacterial cell counting Bifidobacteria 5 ml 30 ml suspension rpm 5. 2 ml 100 ml tube. tube 0.01 M PBS with 0.1% yeast extract. tube 37 shaker 4. capsule 50 ml tube 1000 rpm.. 4. Bifidobacterium bifidum cell Encapsulated Bifidobacterium bifidum bifidobacterium powder 0.01 M PBS 1 108cells/ml powder 0.1 g 0.01 M PBS 5 ml tip blunted feeding needle. Bifidobacterium bifidum cell ethyl ether retroorbital bleeding

77 .. PBS. 25G needle 2 ml soybean trypsin inhibitor 0.1 /ml lavage solution NaCl 25 nm, NaSO4 40 mm, KCl 10 mm, NaHCO3 20 mm, Polyethylene glycol ; average M.W mm ml EDTA solution 50 mm centrifuge tube. sample PBS 6 ml vortaxing 650 g 10. sample 3 ml 95% ethanol 30 phenylmethylsulfonyl fluoride PMSF, 100 mm 4 14,000 g ml 20 PMSF 20 1% sodium azide ) Spleen cell spleen cell Murray et al. (1987). spleen PBS needle forcep g ammonium chloride solution 5. ammonium chloride solution 5 HBSS suspension. suspension HBSS washing. RPMI FBS. 2) MLN cells MLN PBS needle forcep. suspension 500g 5. HBSS washing RPMI FBS. 3) PP cells PP cell Frangakis et al. (1982). PP PBS. PP RPMI-1640 Dispase 50 ml stirring. supernatant 30 ml 500 g HBSS

78 washing 10 FBS-RPMI ml resuspension, Dispase RPMI Isotype-specific ELISA Goat anti-mouse isotype spesific antibody 1.2 /ml sodium bicarbonate buffer ph well plate coating. 4 overnight 0.5% gelatin 37 1 blocking. PBST 3 well 50 standard protein 0.5% gelatin 37 1 plate 3 horseradish peroxidase-conjugated goat anti-mouse antibody 1.2 /ml PBST 3, 3 2 substrate 2,2-azino-bis 3-ethylbenzhiazolin -6-sulfuric acid nm ELISA reader. 6. ELISPOT assay isotype-specific antibody-secreting cell ELISPOT assay. Anti-mouse isotype-specific antibody 0.05 M sodium bicarbonate buffer 1.2 / nitrocellulose bottomed polystyrene 96-well microplates coating (Costar, Cambridge, MA). 4 overnighat incubation 0.05 Tween-20 PBS (PBST) 1 gelatin solution blocking. PBST RPMI-1640 well PBST 3 Biotin-conjugated gote anti-mouse Ig isotype 0.5 gelatin 1:1000 well incubation. PBST 3 Avidin-peroxidase 1:1000 well incubation. plate PBST AEC (3-amino-9-ethyl carbazole) 15 spot counting. 7. RNA B cell, cells TRIzol (Gibco) 1 lysis chloroform 4, 12, aqueous phase tube 500 isopropyl alcohol 10 4, 12, ( RNA) 75% cold ethanol 7,500 5 DEPC-H2O 50. RNA reverse transcription UV Spectrophotometer A260/A280 RNA purity

79 500ng/ DEPC-H2O. 8. RT-PCR. Reverse Transcription RNA 10 mm Tris, 50 mm KCl, 5 mm MgCl2, 1 mm datp, dctp, dgtp dttp, 20 U RNasin (ribonuclease inhibitor ; Promega), 0.1 oligo (dt)15 (Boehringer Mannheim) 50 U M-MLV reverse transcriptase (BRL) PCR tube RNA reverse transcription heat-inactivation 4.. Primer 1) -actin primer 3' - TGACGGGGTCACCCACACTGTGCCCATCTA 5' - CTAGAAGCACTGCGGTGGACGATGGAGGG 2) TGF- 1 primer 3' - CTGCTCCACCTTGGGCTTGCGACCCAC 5' - CGGGGCGACCTGGGCACCATCCATGAC 3) TNF- primer 3' - TACAGGCTTGTCACTCGAATT 5' - ATGAGCACAGAAAGCATGATC 4) IL-2 primer 3' - CTTATGTGTTGTAAGCAGGAGGTAC 5' - CCTGAGCAGGATGGAGAATTACAG 5) IL-4 primer 3' - GCATGGTGGCTCAGTACTACG 5' - CTTCTTTCTCGAATGTACCAGG 6) IL-5 primer 3' - AGGTAATCCAGGAACTGCCTCGTC 5' - ATGAGCACAGTGGTGAAAGAGACC 7) IL-6 primer 3' - CTGTTAGGAGAGCATTGGAAATTG 5' - GAACGATAGTCAATTCCAGAAACC 8) IL-10 primer 3' - TCATTCATGGCCTTGTAGACAC 5' - AGCTGGACAACATACTGCTAACC 9) IL-12 primer

80 3' - TGGAGCACTCCCCATTCCATT 5' - AGAGGTGGACTGGACTCCCGA 10) IFN- primer 3' - GGCTGGATTCCGGCAACA 5' - CGCTACACACTGCATCTTGG. Polymerase Chain Reaction RT cdna products PCR buffer (10 mm Tris, 50 mm KCl, 2 mm MgCl2, 200 um datp, dctp, dgtp dttp) specific 5' 3 primer 25 pmol PCR. PCR temperature profile 95 1 denaturation, 55 1 annealing, 72 1 extension 35 cycle. PCR products 2% agarose gel. 9. Flow cytometry 500 g dimethyl sulfoxide in sucrose-citrate buffer (Becton Dickinson, San Jose, CA, USA) % BSA 0.1% sodium azide PBS 2, 200 PBS FITC goat anti-iga (Becton Dickinson, San Jose, CA, USA) 25 / % BSA 0.1% sodium azide PBS 2 4, FACScan (Becton Dickinson, Mountain View, CA, USA) IgA. 10. (retroorbital bleeding). ether. heparin. 4 overnight 100 g 3 microtube B. bifidum C. perfringens 96 well plate spleen cell (5 105 cells/well) Ci/ 3[H thymidine. cell harvester (Inotech IH-110, Switzerland), liquid scintillation counter (Wallac 1410, Pharmacia, Finland)

81 3 1. B. bifidum C. perfringens.. colony counting direct counting. Table 1 direct counting colony counting. direct counting. 2. in vitro Bifidobacteria B cell. B. bifidum spleen B cell B. bifidum spleen B cell. B. bifidum. BALB/C mouse spleen whole cell cells/ 96-well culture plate seeding, 5 103, 5 104, 5 105, 5 106, cells/ bifidobacteria LPS (12.5 / ) 37, 5% CO2incubator. 7 ELISA., cells/ bifidobacteria IgA, IgM, IgG1 isotype (Fig.1). LPS cells/ bifidobacteria LPS (Fig.2). bifidobacteria B cell. negative control Clostridium perfringens ( ) bifidobacteria Table 1. Comparison of bacterial viability between colony and direct counting method

82 B. bifidum C. perfring ens Method Dilution factor No. of cells Colony counting (CFU/ ) Direct count ing (cells / ) Colony counting method: B. bifidum and C. perfringens were serially diluted in culture media. Cell suspension was poured into MRS agar plates and covered with melted agar media. After mixing of bacterial cell suspension, it was transferred to anaerobic jar. The anaerobic jar was vaccumed and incubated at 37. Colonies were counted after 3 days of incubation. The data are the means of average number of duplicate samples. Direct counting method: Bacterium was washed with 0.01 M PBS and serially diluted in culture media. 5 of bacterial cell suspension was transferred on Petroff-Hausser bacteria counter and covered with cover glass. Then, cells were counted using inverted microscope. The data are the means of average number of duplicate samples. A B

83 C Fig. 1. Effect of B. bifidum on the secretion of Igs by murine whole spleen cells in the presence of LPS. Whole spleen cells were cultured with B. bifidum or C. perfringens (none, 1 103, 1 104, 1 105, 1 106, 1 107) in 96-well plates. LPS (12.5 / ) was added to culture on day 0. After 7 days of culture, supernatants were obtained and antibody response was measured by ELISA. Data are means of triplicate samples. Vertical bars represent SEM. C. perfringens ( ); B. bifidum ( ). A B C

84 Fig.2. B. bifidum alone stimulates whole spleen cells to secrete Igs. whole spleen cells were cultured as described in Figure 1 without LPS. Data are the means of triplicate samples. Vertical bars represent SEM. C. perfringens ( ); B. bifidum ( ). (Fig. 1, 2). B. bifidum B cell polyclonal activator LPS mitogen.. Fig.3, BB control C. perfringens B cell. B. bifidum B cell.. Bifidobacteria spleen whole cell 5, 1 106cells/ seeding ELISPOT., bifidobacteria IgA isotype 3. B. bifidum.. B. bifidum B cell cytokine TGF- 1 in vitro IgA class switch IgA (Kim and Kagnoff, 1990). IL-5 B cell cytokine. Bifidobacteria B cell TGF- 1 IL-5 B cell. spleen cell (5 105cell/well) media, B. bifidum, TGF- 1+IL-5, B+T+5 7, ELISA Ig. IgA IgG2b B. bifidum B+T+5 (Fig.5). IgM, IgG2a, IgG1 (Fig.6). B. bifidum LPS polyclonal activator. B cell surface IgA, IgA BB B cell TGF- 1 IL-5 (Fig.7)

85 A B Fig.3. B. bifidum enhances the proliferation of whole spleen cells. whole spleen cells were cultured with B. bifidum at a density of cells/well with or without LPS (12.5 / ) in 96-well culture plates and incubated for 4 days. Wells were pulsed for 18 h with 5 Ci/ of 3[H thymidine. Cells were harvested and filtered through glass fiber filters and counted in a liquid scintillation counter. A) In the presence of LPS. B) In the absence of LPS

86 Fig.4. B. bifidum increases number of IgA secreting cells. Spleen B cells (5 105 cells/well) were incubated with B. bifidum (1 107 cells/well) for 5 days. Number of IgA secreting cells were enumerated by ELISPOT assay as described in Materials and Methods. Data are means of duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars respresent SEM. A B

87 Fig.5. B. bifidum induces B cells to responsive to TGF- 1 and IL-5 resulting in increase of IgA and IgG2b synthesis. Spleen B cells (5 105cells/well) were cultured with B. bifidum (1 107 bacteria/well). Cells were stimulated with TGF- 1 (0.2 ng/ ) or IL-5 (0.5 ng/ ) on day 0. After 7 days of culture, supernatants were obtained and antibody response was measured by ELISA. Data are means of duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars represent SEM. B, B. bifidum; T, TGF- 1; 5, IL

88 Fig.6. Effect of B. bifidum on the synthesis of IgM, IgG1 and IgG2a by spleen B cells in the presence of TGF- 1 and IL-5. Culture conditions were the same as described in Fig. 5. Data are means of duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars represent SEM. B, Bifidobacterium bifidum; T, TGF- 1; 5, IL

89 Fig.7. Effect of B. bifidum on the expression of surface IgA by murine spleen B cells in the presence of TGF- 1 and IL-5. Culture conditions were the same as described in Figure 5. Cells were harvested after 4 days of culture and analyzed by FACS following staining with FITC-conjugated anti-mouse IgA antibody. T, TGF- 1; 5, IL

90 3. B cell B. bifidum. Mesenteric Lymph Node (MLN) B cell B. bifidum spleen B cell Ig. B. bifidum. MLN B cell. spleen B cell IgA IgM. IgG1 (Fig.8). B. bifidum surface IgA LPS (Fig.9). TGF- 1 IL-5 spleen B cell IgA, IgM IgG1 (Fig.10). B. bifidum B cell IgA.. Peyer's Patch (PP) B cell B. bifidum PP B cell IgA. C. perfringens. BB IgM IgG1 (Fig.11). MLN B cell, spleen B cell. B cell. spleen B cell B. bifidum PP B cell TGF- 1 IL-5 IgA (Fig.12).. PP B cell IgA TGF- 1 IgA isotype switching

91 Fig. 8. B. b i f i d u m stimulated murine MLN B cells to secrete IgA and IgM antibody. MLN cells (5 105 cells/well) were cultured with bacteria/well of B. bifidum or C. perfringens in a 96-well culture plate on day 0. Supernatants were harvested after 7 days of culture and Ig secretion was measured by ELISA. Data are the means duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars represent SEM

92 Fig.9. Bifidobacterium increased siga expression by murine MLN B cells. MLN cells were cultured with LPS (12.5 / ) or B. bifidum (1 107bacteria/well) at 5 105cells/well in a 96-well culture plate on day 0. After 4 days of culture, cells were harvested and analyzed with FITC-conjugated anti-mouse IgA antibody using FACS

93 Fig.10. Effect of TGF- 1 and IL-5 on Ig synthesis of murine MLN B cells by bifidobacterium. MLN cells were cultured with B. bifidum or C. perfringens (1 107bacteria/well) and/or TGF- 1 (0.2 ng/ ) and IL-5 (0.5 ng/ ) at per well in a 96-well culture plate on day 0. Supernatant were harvested after 7 days of culture and Ig secretion was measured by ELISA. Triplicate wells were pooled and the data are means of duplicate samples. B, B. bifidum; C, C. perfringens; T, TGF- 1; 5, IL-5. Vertical bars represent SEM

94 Fig.11. B. bifidum stimulated murine PP B cells to secrete IgA antibody. Culture conditions were the same as described in Fig.8. Supernatants were harvested after 7 days of culture. IgA, IgM, and IgG1 were assayed by ELISA. Data are the means of duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars represent SEM

95 F i g Effect of TGF- 1 and IL-5 on Ig synthesis of murine PP B cells by bifidobacterium. Culture conditions were the same as described in Fig.10. Supernatants were harvested after 7 days of culture. IgA, IgM, and IgG2b were assayed by ELISA. Data are the means duplicate samples. Each sample contains 2-fold diluted wells (n=3). Vertical bars represent SEM. 4. B. bifidum. B. bifidum B. bifidum B. bifidum. B. bifidum. B. bifidum C. Perfringens BB ELISA. B.bifidum BB control C. Perfrigens (Fig.13) (Fig.14). B. bifidum adjuvant.. B. bifidum spleen, MLN, PP

96 Bifidobacteria. ELISPOT assay (Fig.15). spleen B. bifidum, MLN IgA. PP B. bifidum IgG1, IgM control C. Perfringens. B. bifidum PP MLN B cell IgA, IgA. 5. Bifidobacteria B cell IgA B. bifidum B. bifidum B. bifidum. BALB/c spleen cell LPS 7. ELISA Fig.13. Effect of peroral administration of B. bifidum o n antigen-specifi c antibody responses in intestinal lavages. B. bifidum or C. perfringens (1010 cells/mouse) was administered every other day for 4 weeks. Lavages was collected as described in materials and Methods. Antigen-specific antibody responses was measured by ELISA

97 Fig.14. Effect of peroral administration of B. bifidum on antigen-specific antibody responses in the sera. B. bifidum or C. perfringens (1010cells/mouse) was administered every other day for 4 weeks. Sera was collected as described in materials and Methods. Antigen-specific antibody responses was measured by ELISA

98 Fig.15. Effect of peroral administration of B. bifidum on number of antigen non-specific Ig secreting cells. B. bifidum or C. perfringens (1010 cells/mouse) was administered every other day for 4 weeks. Number of antigen-specific antibody secreting cells was enumerated by ELISPOT assay. (Fig.16). LPS. LPS 2 5 B. bifidum cell IgA IgM. B. bifidum. 6. Bifidobacteria bacteria alginate B.bifidum B.bifidum (1 108 cells/day). 4 ELASA (Fig.17). B.bifidum B.bifidum cell, (non-feeding) IgA 2.5, 5. IgA B. bifidum IgA, control 3. B. bifidum IgA

99 B. bifidum. ELISPOT A- 1 assay (Fig.18). A- 2 MLN B cell, IgA B. bifidum B. bifidum cell B. bifidum 7. MLN B cell IgG B. bifidum B.bifidum cell,. Spleen B cell IgA B- 1 B- 2 B. bifidum B. bifidum cell, 9, 3, IgG 4.3, 3.4. B. bifidum IgA. C- 1 C- 2 D- 1 D- 2 Fig.16. Effect of bifidobacterial cells or its culture supernatants on IgA and IgM synthesis by murine spleen B cells. B cells were cultured with bifidobacterial cells (A and B panel) or its culture supernatants (C and D panel) in the presence of LPS and absence of LPS. Antigen non-specific antibody responses was measured by ELISA. D represents the incubation date of bacterial culture

100 Fig.17. Effect of peroal administration of B. bifidum on antigen-nonspecific antibody response in intestinal lavage and the sera. Male and female BALB/C mice were administered perorally with bifidobacterium cell (1 108 cells/ml) for 4 weeks for every other day. After 4 weeks, intestinal fluid and serum were obtained from mouse and antibody response was measured by ELISA assay. Fig.18. Effect of peroral administration of Encapsulated B. bifidum on the mouse

101 number of a n t i g e n nonspecific Ig s e c r e t i n g cells. MLN and Spleen were i s o l a t e d f o l l o w i n g p e r o r a l administration cells per well were incubated in a nitrocellulose-bottomed 96 well plate for 4h and IgA secreting cell and IgG secreting cell number were determined by ELISPOT assay as described in Material and Methods. Data are the means duplicate samples. Each sample contains 2-fold diluted wells

102 7. Bifidobacteria cytokine. Bifidobacteria cytokine cytokine Bifidobacteria mrna B cell T cell A macrophage cytokine. Bifidobacteria Peyer's patch (PP) mesenteric lymph node (MLN) mrna RT-PCR. mrna control -actin RT-PCR. Fig.19 mrna.. TGF- 1 mrna. PP Bifidobacteria TGF- 1 mrna. MLN. MLN IgA Fig. 18. TGF- 1 IgA cytokine. Bifidobacteria, IgA.. B. bifidum spleen cell cytokine B. bifidum cytokine. T helper cell IL-2, IFN-, IL-4, IL-5, IL-6, macrophage IL-1, IL-10, IL-12. cytokine B. bifidum BB. B. bifidum B cell polyclonal activator LPS, T cell polyclonal activator Con A. IL-2 mrna B. bifidum (Fig.20)

103 A. IL- 2/IL- 4 B. IL- 5/IL- 6 C. IL- 10/IL- 12 D. IFN- r B Fig.19. Detection of TGF- 1 mrna by RT-PCR. Appropriate samples were prepared from mice administered with Bifidobacterium alone or encapsulated Bifodobacterium. A) mrna level of TGF- 1. B) mrna level of -actin. Lane 1; conrol (PP), Lane 2; Bifidobacterium alone (PP), Lane 3; encapsulated Bifodobacterium (PP), Lane 4; conrol (MLN), Lane 5; Bifidobacterium alone (MLN), Lane 6; encapsulated Bifodobacterium (MLN)

104 Fig.20. Detection of mrna of various cytokines by RT-PCR. Mouse spleen cells (2 106 cells/well) were stimulated with LPS, ConA or B. bifidum for 2 days. Lane 1, spleen cells only; Lane 2, spleen cells + LPS; Lane 3, spleen cells + ConA; Lane 4, spleen cells+ B. bifidum. IL-10 LPS mrna. IFN- IL-12 B. bifidum. B. bifidum T helper cell macrophage cytokine. cytokine

105 4 B. bifidum. B. bifidum. B. bifidum. B. bifidum spleen B cell. B cell cytokine (TGF- 1 IL-5) IgA. B cell polyclonal activator LPS. B. bifidum mesenteric lymph node (MLN) B cell IgA. B. bifidum spleen B cell Peyer's patch (PP) B cell MLN IgA. B. bifidum PP MLN B cell IgA, IgA. B. bifidum B. bifidum. B. bifidum B. bifidum. B. bifidum MLN cell TGF- 1 mrna. TGF- 1 IgA, Bifidobacteria, IgA

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109 5 Bifidobacteria 1 Bifidobacteria 1. Bifidobacteria (Robin and Tamine, 1990; Fuller, 1992). bifidobacteria.,,,,,. fingerprinting. Bifidobacteria bifidobacteria (Biviati, 1982; Roy Ward, 1990). DNA/DNA hybridization(scardovi, 1971) (Biviati, 1982; Roy, 1994). DNA probe primer dot blot polymerase chain reaction(pcr) (Chareris, 1997). Yamamoto (1992) 16S rrna B. adolescentis, B. bifidum, B. breve, B. infantis, B. longum DNA probe. Bifidobacterium genus 16S rrna probe (Frothingham, 1993; Langendijk, 1995).,, phage, -. sodium dodecyl sulfate-polyacrylamide gel electrophoresis(sds-page) polypeptide (Pot, 1993; Tsakalidou, 1992). genome DNA

110 (, 1995), SmaI DNA genome DNA pulsed field gel electrophoresis DNA band (Roy, 1996; Tanskanen, 1990). Random primer polymerase chain reaction DNA band random amplified polymorphic DNA(RAPD) (Cancilla, 1992;, 1994;, 1997). 16S rrna 23S rrna probe restricton fragment length polymorphism(rflp) ribotyping M13 probe RFLP (Mangin, 1994;, 1997), bifidobacteria ribotyping PCR 16S rdna, SDS-PAGE RAPD. 2.. Bifidobacteria (KCTC) American Type Culture Collection(ATCC) 15 2 (Table 1). 5 bifidobacteria Bb-11, Bb-12L, Bb-12S, ABT-4, ABT-B, SJ Bifidobacteria. Table 1. Type strains of bifidobacteria used in the study

111 Species Strain Synonym Source B. adolescentis KCTC 3216 ATCC Intestine of adult B. adolescentis KCTC 3352 ATCC Intestine of adult B. adolescentis KCTC 3416 ATCC Intestine of adult B. bifidum KCTC 3202 ATCC Stool of breast-fed infant B. bifidum KCTC 3357 ATCC Human feces B. bifidum KCTC 3418 ATCC Intestine of infant B. bifidum KCTC 3476 ATCC Lactobacillus bifidus subsp. pensylvancum B. breve KCTC 3220 ATCC Intestine of infant B.breve KCTC 3419 ATCC Intestine of infant B. breve KCTC 3461 ATCC Intestine of infant B. infantis KCTC 3127 ATCC Intestine of infant B. infantis KCTC 3249 ATCC Intestine of infant B. infantis KCTC 3270 ATCC Intestine of infant B. infantis KCTC 3368 ATCC Intestine of infant B. infantis KCTC 3460 ATCC Intestine of infant B. longum KCTC 3466 DSM Calf feces B. longum ATCC Intestine of adult B. longum ATCC Intestine of infant. Bifidobaceria bifidobacteria Lactobacilli MRS agar(difco) NPNL(nalidixic acid, 15 / ; paromomycin sulphate, 125 / ; neomycin sulphate 10 / ; lithium chloride 300 / ). bifidobacteria BSI agar Beeren's agar. colony Y, V colony TPY agar(biviati, 1991) colony.. Bifidobacteria Bifidobacteria TPY broth anaerobic catalyst(difco) anaerobic jar(difco) 90% H2 10% CO2 anaerobic indicator strip(difco) % glycerol DNA Bifidobacteira Versalovic (1991) genomic DNA. TPY broth 24 7,000rpm

112 bifidobacteria. 1 1M NaCl 50mM TE (50mM Tris, 50mM EDTA, ph 7.8) mM TE (10mM Tris, 1mM EDTA, ph 7.8) 25 mutanolysin(5,000 unit/ 10mM TE ) 25 RNase A(6mg/ml 10mM Tris, 15mM NaCl, ph 7.8) SDS(10%) 30 proteinase K (10mg/ml, 10mM TE ) Phenol(0.4 ) 1. phenol chloroform(0.4 ) 2. 10mM TE M sodium acetate(ph 5.2) 0.8 ethanol DNA 70% ethanol 10mM TE. DNA RNase A proteinase K SDS phenol chloroform 3M sodium acetate(ph 5.2) ethanol DNA.. PCR bifidobacteria 16S rdna probe PCR 20 PCR PCR PreMix(50mM Tris-HCl; ph 8.3, 10mM KCl, 1.5mM MgCl2, 250 M dntp, 1U Taq polymerase) (Bioneer) 2 primer( 25 M), 1 bifidobacteria DNA(20ng), 17 H2O PCR. Perkin-Elmer thermal cycler 94 1, 60 1, 72 2, 30cycles. primer bifidobacteria 16S rrna 9bp 31bp 5'-GGTTCGATTCTGGCTCAGGATG-3' oligonucleotide primer 1351bp 1372bp 5'-TACAAGGCCCGGGAACGCATTC-3' oligonucleotide (Leblond-Bourget, 1996). PCR DNA 1364bp. Digoxigenin labeling 16S rdna probe 200 M dttp 130 M dttp 70 M digoxigenin-11-dutp(boehringer Mannaheim).. Southern blot EcoRI 20 1 bifidobacteria DNA, 10unit EcoRI (Promega), 2 10 reaction buffer % agarose gel 1 TAE (40mM Tris-acetate, 1mM EDTA) 30V 24. DNA marker HindIII/EcoRI(Promega). agarose gel depurination, denaturation, neutralization (Sambrook, 1989). gel positively-charged nylon membrane(boehringer Mannaheim) (Hoeffer) blotting UV crosslinker(uvp) DNA nylon membrane. Nylon

113 membrane DNA digoxigenin-labeled 16S rdna probe hybridization prehybridization (50% formamide, 5 SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent(boehringer Mannaheim) 42 2 prehybridization 200 digoxigenin-labeled 16S rdna hybridization. 0.1% SDS 2 SSC nylon membrane DIG luminescent detection kit(boehringer Mannheim) X-ray film(agfa Curix RP-1).. PCR 16S rdna PCR 20 PCR PCR PreMix(50mM Tris-HCl; ph8.3, 10mM KCl, 1.5m M MgCl2, 250 M dntp, 1U Taq polymerase; Bioneer) 2 forward primer(5'-10-g GTTCGATTCTGGCTCAGGATG-31-3')(25 M) reverse primer (5'-1376-TACAAGGCCCGGGAACGCATTC ')(25 M), 1 Template DNA(20ng), 17 H2O PCR. Perkin-Elmer thermal cycler 94 1, 60 1, 72 2, 30cycles. PCR 16S rdna RsaI, HhaI, AluI, HaeIII, MboI, DdeI. PCR 2.5% agarose gel (Nusieve : agarose = 3 : 1) 1 TAE buffer. DNA 0.5 ethidium bromide.. RAPD OPB-07(Operon) random primer PCR DNA agarose gel. PCR 20 PCR PCR PreMix(50mM Tris-HCl; ph 8.3, 10mM KCl, 1.5mM MgCl2, 250 M dntp, 1U Taq polymerase) (Bioneer) mM MgCl2(1.5mM), 1 OPB-07 primer(l M)(Operon), 1 Template DNA(20ng) 16.5 H2O PCR. Perkin-Elmer thermal cycler cycles. 1.5% agarose gel, 0.5 TBE buffer, 70V 4. DNA ethidium bromide.. SDS-PAGE Glucose lactose TPY broth bifidobacteri a Vibra Cell-VCX 600(Sonics and Materials Inc.) 37% amplitude 5 5 cycle 30 sonication. Sonication. SDS-PAGE sample buffer % polyacrylamide gel s

114 eparating gel. silver staining(pharmac ia). bovine serum albumin(66,000), egg albumin(45,000), glyceraldehyde-3-phosphate dehydrogenase(36,000), carbonic anhydrase(29,000), tryps in inhibitor(20,100) -lactalbumin(14,200) ( ). 3.. Ribotyping bifidobacteria Bifidobacterium 16S rrna 1364bp digoxigenin-labeled probe PCR ribotyping. EcoRI bifidobacteria genomic DNA agarose nylone membrane blot DNA digoxigenin-labeled 16S rdna probe hybridize nucleotide band pattern (Fig. 1;Table 2). Fig Chemiluminescence detection of EcoRI-digested DNA from Bifidobacterium strains

115 hybridized with digoxigenin-labeled 16S ribosomal DNA probe amplified in PCR. A, B. bifidum KCTC 3418; B, B, bifidum KCTC 3357; C, B. bifidum KCTC 3476; D, B. longum KCTC 3466; E. B. adolescentis KCTC 3416; F, B. adolescentis KCTC 3216; G, B. adolescentis KCTC 3352; H, B. breve KCTC 3461; I, B. breve KCTC 3220; J, B. infantis KCTC 3270; K. B. infantis KCTC 3460; L. B. infantis KCTC 3368; M. B. infantis KCTC 3127; N, Bifidobacterium spp. Bb-11; O, Bifidobacterium spp. Bb-12L; P, Bifidobacterium spp. Bb-12S. The numbers at left indicate the base pair of the nucleotides in HindIII/EcoRI marker. Table 2. The nucleotides detected by hybridization of EcoRI-digested DNA from Bifidobacterium strains probed with digoxigenin-labeled 16S ribosomal DNA amplified with PCR. Species Strain Length of nucleotide (base pair) B. adolescentis KCTC ,500 6,600 3,700 2,900 1,250 KCTC ,500 6,600 3,700 2,900 KCTC ,600 5, ,700 2,900 B. bifidum KCTC ,700 5,600 3,900 3,700 KCTC ,700 4,000 3,700 KCTC ,600 3,900 3,700 B. breve KCTC ,200 3,700 3,200 2,200 KCTC ,200 3,700 3,200 2,200 B. infantis KCTC ,200 3,700 2,400 1,800 1,250 KCTC ,200 3,700 3,100 2,400 1,250 KCTC ,200 3,700 3,100 2,400 1,250 KCTC ,200 3,700 3,100 2,400 1,250 B. longum KCTC ,100 3,700 3,500 3,400 1, Bifidobacterium Bb-11 9,700 6,600 3,900 3,700 sp. Bb-12L 9,700 6,600 3,900 3,700 Bb-12S 5,200 3,700 3,200 2,200 1,600 1,450 Bifidobacterium band 3,700bp B. adolescentis band 6,600bp 2,900bp B. bifidum 3 band 9,700bp B. breve band 4,200bp, 3,700bp 2,200bp

116 B. infantis band 4,200bp, 2,400bp, 1,250bp. B. infantis B. longum 4,200bp band. B. longum KCTC 3466 band 3,700bp band band. Bb-11 Bb-12L B. bifidum KCTC 3418 B. bifidum KCTC 3357 band B. bifidum. Bb-12S Bifidobacterium 3,700bp band band. B. bifidum KCTC 3357 genomic DNA template DNA 16S rdna PCR digoxigenin-labeled probe ribotyping. probe Bifidobacterium 16S rrna. Bifidobacterium genus band band. Ribotyping probe.. PCR 16S rdna bifidobacteria PCR primer 16S rdna DNA 1367bp DNA 1400bp. bifidobacteria DNA (Fig. 2). PCR 16S rdna AluI, HaeIII, HhaI, MboI RsaI DNA (bp) (Fig. 2). HaeIII MboI 6 bifidobacteria. HaeIII A, B, C, D E F. MboI. MboI D 380bp, 190bp, 100 bp DNA 1400bp. 1400bp 100bp DNA DNA. bifidobacteria (Table 3, 4). Bifidobacteria. B. adolescentis AluI MboI A B. bifidum HaeIII HhaI C, B. breve AluI D MboI C D, B. infantis MboI E F. HaeIII MboI A B

117

118

119 Fig.2. Restriction pattern of PCR-amplified 16S rdna of bifidobacteria M : pgem marker, A, B, C, D, E, and F : Restrction pattern (Table 3) Table 3. DNA fragment size and restriction pattern of PCR-amplified 16s rdna digested with restriction endonuclease

120 Restriction Endonuclease DNA fragment size (bp) Restriction pattern AluI A B C D HaeIII A B C D E F HhaI A B C D MboI A B C D E F RsaI A B Table 4. Classification of restriction pattern of PCR-amplified 16S rdna from the type culture of Bifidobacterium

121 Strains AluI HaeIII HhaI MboI RsaI B. adolescentis KCTC 3216 A A A A A B. adolescentis KCTC 3352 A B B A A B. adolescentis KCTC 3416 A B A A A B. bifidum KCTC 3202 B C C B A B. bifidum KCTC 3357 B C C B A B. bifidum KCTC 3418 C C C B A B. bifidum KCTC 3476 C C C B A B. breve KCTC 3220 D A A C A B. breve KCTC 3416 D A D D A B. breve KCTC 3419 D A A C A B. infantis KCTC 3127 C D A E A B. infantis KCTC 3249 C A A F B B. infantis KCTC 3270 C A A F B B. infantis KCTC 3368 C A A F B B. infantis KCTC 3460 C A A F B B. longum KCTC 3466 C A A B B B. longum ATCC C A A B B B. longum ATCC C A A B B (Table 5) (Table 6). Bifidobacterium Bb-11 Bb-12L B. bifidum B. bifidum Bifidobacterium Bb-12S, ABT4, ABTB, SJ-1 5. BSI agar K-2, K-4, K-5, K-7 K-10 Beeren's agar K-C B. bifidum Beeren's agar K-A, K-B, K-E, K-H, K-J B. adolescentis. K-3, K-C, K-I. PCR 16S rdna bifidobacteria 16S rdna

122 . Table 5. Identification of commercial culture of Bifidobacterium based on restriction pattern of PCR-amplified 16S rdna Culture name AluI HaeIII HhaI MboI RsaI Identification Bb-11 C C C B A B. bifidum Bb-12L C C C B A B. bifidum Bb-12S A E A C B Not identified ABT4 A E A C B Not identified ABTB A E A C B Not identified SJ-1 A E A C B Not identified Table 6. Identification of wild strain of Bifidobacterium isolated from human stool based on restriction patterns of PCR-amplified 16S rdna

123 Selective agar Culture name AluI HaeIII HhaI MboI RsaIII Identification BSI agar K-2 C C C B A B. bifidum K-3 C F A D B Not identified K-4 C C C B A B. bifidum K-5 C C C B A B. bifidum K-7 C C C B A B. bifidum K-10 C C C B A B. bifidum Beeren's K-A A A A A A B. adolescentis agar K-B A A A A A B. adolescentis K-C C C C B A B. bifidum K-E A A A A A B. adolescentis K-G C F A D B Not identified K-H A A A A A B. adolescentis K-I C F A D B Not identified K-J A A A A A B. adolescentis. SDS-PAGE bifidobacteria Bifidobacterium TPY cell sonicate SDS-PAGE (Fig 3). B. bifidum KCTC 3418 B. bifidum ,000, 69,000, 63,000, 60,000, 56,000, 54,000, 50,000, 47,000, 45,000, 21,000, 20,000 polypeptide B. adolescentis KCTC 3416 KCTC ,000, 62,000, 61,000, 20,000, 14,000 polypeptide B. breve KCTC 3461 B. breve KCTC 3419 polypeptide band B. breve KCTC 3220 B. breve 29,000 polypeptide polypeptide. B. infantis KCTC 3249 B. infantis KCTC 3460 polypeptide band

124 Fig.3. SDS-PAGE of cell proteins of bifidobacteria. S. Molecular marker; A. B. bifidum KCTC 3418; B. B. bifidum KCTC 3357; C. B. adolesecntis KCTC 3416; D. B. adolescentis KCTC 3216; E. B. breve KCTC 3461; F. B. breve KCTC 3419; G. B. breve KCTC 3220; H. B. infantis KCTC 3249; I. B. infantis KCTC Tsakalidou (1992) L. delbrueckii subsp. bulgaricus S. salivarius subsp. thermophillus cell SDS-PAGE polypeptide. Leuconostoc mesenteroides SDS-PAGE L. mesenteroides 3 subspecies. bifidobacteria SDS-PAGE polypetide band polypeptide.. RAPD Bifidobacterium genomic DNA random amplified DNA polymorphism(rapd) (Fig. 4). RAPD DNA band B. bifidum 3 750bp, 590bp, 300bp band. B. breve 3 340bp band B. infantis 340bp 300bp band. B. adolescentis band. B. infantis KCTC 3249 B. infantis KCTC 3270 band pattern B. infantis ATCC

125 15697 band pattern. Fig. 4. Agarose gel electrophoresis of random-amplified DNA products of bifidobacteria. S. 100bp DNA ladder marker; A, B. bifidum KCTC 3418; B. B. bifidum KCTC 3357; C. B. bifidum KCTC 3476; D. B. longum KCTC 3466; E. B. adolescentis KCTC 3416; F. B. adolescentis KCTC 3216; G. B. adolescentis KCTC 3352; H. B. breve KCTC 3461; I. B. breve KCTC 3419; J. B. breve KCTC 3220; K. B. infantis KCTC 3249; L. B. infantis KCTC 3270; M. B. infantis KCTC 3460; N. B. infantis KCTC RAPD 7-10bp random primer annealing MgCl2 stringency PCR DNA band fingerprinting (Welsh McClelland, 1990). (1994) bp degenerated primer 28 annealing PCR DNA pattern. (1997) S. salivarius subsp. thermophilus L. delbrueckii subsp. bulgraicus L. acidophilus band. Tyler (1997) RAPD