7 ] 1996 B ifidobacteria. : 1. 10 2. 2 1999. 10. 27. : : ( ) :
Bifidobacteria ( 1 : Bifidobacteria, 2 : Bifidobacteria, 3 : Bifidobacteria, : Bifidobacteria ). 1999. 10. 27. : : : : : : : : : : : :. : : Bifidobacteria 1 : Bifidobacteria 2 : Bifidobacteria 3 : Bifidobacteria : Bifidobacteria - 1 -
. Bifidobacteria,, cholesterol bifidobacteria.. 1. 1 : Bifidobacteria bifidobacteria. bifidobacteria B. bifid um Bb-11 B. bifidum K-7, MRS broth whey. 2. 2 : Bifidobacteria. bifidobacteria. bifidobacteria,, bifidobacteria. 3. 3 : B. bifidum. B. bifidum B. bifidum. 4. : Bifidobacteria. cholesterol bifidobacteria. - 2 -
bifidobacteria bifidobacteria. cholesterol.. 1. 1 : 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. 2. 2 : bifidobacteria air-atomization. sodium alginate CaCl2 bifidobacteria, ( ) bifidobacteria. 3. 3 :, in vitro bifidobacteria B cell, B cell B. bifidum, B. bifidum, B. bifidum, B. bifidum - 3 -
B. bifidum cytokine. 4. : 16S rdna bifidobacteria. Bifidobacteria,,,.. 1.. 1 : 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 37 24 B. bifidum K-7 1.33% B. bifidum Bb-11 1.84% B. bifidum K-7 1.4 109 B. bifidum Bb-11 9.0 109, ph 2. Whey whey protein isolate(wpi) yeast extract 0.75% MRS broth B. bifidum K-7 37 24 ph 5.20, 0.33% 1.7 108, B. bifidum Bb-11 ph 4.03, 0.64% 1.9 109. 12.7%, 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-7 1.6 107 2.0 105 16 5.8 105 4.6 103 ph bifidobacteria - 4 -
bifidobacteria milk. B. bifidum Bb-11 B. bifidum K-7 ph 4.75 0.8% 16 ph 4.62 4.60 T.A 1.07% 1.02% B. bifidum Bb-11 4 B. bifidum K-7 8. B. bifidum Bb-11 B. bifidum K-7-20 4.1 106 4.0 105 16 4.1 105 3.1 104. ph bifidobacteria.. 2 : bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing 15.. 80-90 m. Starter culture 0.5g, Yeast extract 0.5g, Glycerol 5.0g, NaHSO3 0.5g, Mg3(PO4)21.0g. slurry -37 2-55 14. bifidobacteria culture 120 103 cfu/g bifidobacteria 107 cfu/g. bifidobacteria ph. bifidobacteria 30 60 90 120 107cfu/g. bifidobacteria 4 2 109-1010cfu/g 16 107cfu/g. bifidobacteria bfidobacteria bifidobacteria culture bifidobacteria. lactic acid bacteria, enterobacteriaceae staphylococci. bifidobacteria frequency. - 5 -
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. 2. - 6 -
. Whey bifidobacteria bifidobacteria bifidobacteria bifidobacteria, sodium alginate poly-l-lysine microparticles.. Bifidobacteria,.., bifidobacteria... bifidobacteria. Bifidobacteria. - 7 -
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 1.3 109 and 6.3 109 at 24 hours, indicates no difference. However, in the culture based on whey, the growth of B. bifidum K-7 was 1.7 108 at 24 hours which was inferior to 1.9 109 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 1.6 107and 2.0 205then changed to 5.8 105and 4.0 103after 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 -
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 4.1 106and 4.0 105, but at 16 days, those were 4.1 105 and 3.1 104respectively. 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 80.1-92.8 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 -
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. - 10 -
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 - 11 -
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 ---------------------------------- 21 Section 2. Scope of study ------------------------------------ 21 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-11 --------- ---------------------------- 23 Section 2. Growth of Bifidobacterium bifidum K-7 and stability of bifidobacteria microparticles in dairy products- 39 Section 3. References ---------------------------------------- 59 Chapter. Preparation and Evaluation of Bifidobacteria-loaded Microparticles Section 1. Introduction -------------------------------------- 63 Section 2. Materials and methods ----------------------------- 66 Section 3. Results and discussion ---------------------------- 73 Section 4. Summary ------------------------------------------- 93 Section 5. References ---------------------------------------- 95 Chapter. Effects of Bifidobacteria on Intesitinal Immune Function Section 1. Introduction -------------------------------------- 97 Section 2. Materials and methods ---------------------------- 101 Section 3. Results and discussion --------------------------- 108 Section 4. Summary ------------------------------------------ 136 Section 5. References --------------------------------------- 137-12 -
Chapter. In Vivo Biological Activity of Bifidobacteria Section 1. Molecular biological identification of differentiation of bifidobacteria -------------------------------- 142 Section 2. Physiological characteristics and mechanisms of bifidobacteria adhesion ability to Caco2 cells --- 164 Section 3. In vivo biological activity of microencapsulated bifidobacteria ----------------------------------- 174 Section 4. References --------------------------------------- 193-13 -
1 1 ----------------------------------- 21 2 ----------------------------------- 21 2 Bifidobacteria 1 Bifidobacterium bifidum Bb-11 ------------------ 23 2 B. bifidum Bb-11 B.bifidum K-7 Bifidobacteria ---------- 39 3 ------------------------------------------- 59 3 Bifidobacteria 1 ----------------------------------------------- 63 2 ---------------------------------------- 66 3 ---------------------------------------- 73 4 ----------------------------------------------- 93 5 ------------------------------------------- 95 4 Bifidobacteria 1 ----------------------------------------------- 97 2 --------------------------------------- 101 3 --------------------------------------- 108 4 ---------------------------------------------- 136 5 ------------------------------------------ 137 5 Bifidobacteria 1 Bifidobacteria ------------------------------------- 142 2 Bifidobacteria ------------------------------------- 164 3 Bifidobacteria - 174 4 ------------------------------------------ 193-14 -
- 15 -
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. - 16 -
2 Bifidobacteria 1 Bifidobacterium bifidum Bb-11 1..,,,. peptide,. Kehagias (1977) casein peptide B. bifidum,. bifidobacteria (Poch Bezkorovainy, 1988, 1991; Petschow Talbott, 1991), Poch Bezkorovainy(1988) - 17 -
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-11. 2.. 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), - 18 -
trypsin 10,000Da, 10,000 3,000Da, 3,000Da -, -, -casein 1%(w/v) 0.25, 0.5, 0.75 1%. 121 15 B. bifidum Bb-11 1%, 37 24.. Trypsin casein -, -, -Casein 250 0.1M phosphate buffer(ph 8.0) 30 40 5, trypsin 1% 125 (1437U) ( : = 1:200, wt/wt), 40 6, 85 10. -, -, -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 ) 215 225 ( 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 8.4 0.1N NaOH ( ). Lactobacillus MRS agar(difico Lab., USA) L-cysteine HCl(Sigma Chem. Co. U.S.A.) 0.05% 121 15, 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) 37 48. - 19 -
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, 0.75 1% B. bifidum ph,. MPH ph (Table 1) 12-20 -
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. (%) (%) (%) (%) 0 7.00 0.04 7.00 0.04 7.00 0.04 7.00 0.04 7.00 0.04 6 5.87 0.13 5.68 0.16 6.02 0.12 6.23 0.12 5.98 0.18 12 4.80 0.35 4.49 0.40 4.57 0.41 4.70 0.38 4.63 0.46 18 4.28 0.52 4.25 0.55 4.27 0.56 4.23 0.68 4.26 0.65 24 4.11 0.66 4.00 0.82 3.96 0.94 4.10 0.78 4.11 0.83, 1% (Table 1). 24, 12 0.5%., 12, 1%. 24 0.25%,, 0.5%. MPH 0.75% 1% (Fig.2), MPH B. bifidum. - 21 -
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) 12 0.25% 0.75%, 24 ph, ph. (Table 2) 12 0.5%,, 1% 0.75%. 24. 12 0.5%, 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-11. - 22 -
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 (%) (%) (%) (%) (%) 0 7.00 0.03 7.00 0.03 7.00 0.03 7.00 0.03 7.00 0.03 6 6.35 0.10 6.23 0.13 6.46 0.15 6.35 0.18 6.27 0.18 12 4.91 0.28 4.76 0.33 5.34 0.26 4.87 0.35 5.23 0.31 18 4.53 0.47 4.29 0.54 4.15 0.76 4.16 0.86 4.22 0.83 24 4.21 0.61 3.98 0.83 4.03 0.84 4.10 0.86 4.11 0.91 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 - 23 -
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 6.49 7.03 6.01 -casein 6.45 3.67 2.51 -casein 258.0 0.02 6.49 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.(%) 0 6.80 0.04 6.80 0.04 6.80 0.04 6.80 0.04 6 5.8 0.10 5.85 0.11 5.70 0.12 5.49 0.14 12 4.86 0.23 4.82 0.23 4.71 0.27 4.61 0.28 18 4.33 0.43 4.34 0.43 4.25 0.47 4.21 0.48 24 4.05 0.65 4.06 0.65 4.02 0.67 4.01 0.68 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-11. - 24 -
Incubation time (hr) Control >10,000Da 3,000 10,000Da <3,000Da ph T.A.(%) ph T.A.(%) ph T.A.(%) ph T.A.(%) 0 6.80 0.04 6.80 0.04 6.80 0.04 6.80 0.04 6 5.88 0.10 5.97 0.10 5.78 0.11 5.66 0.13 12 4.86 0.23 4.93 0.22 4.75 0.26 4.70 0.29 18 4.33 0.43 4.37 0.42 4.25 0.48 4.24 0.50 24 4.05 0.65 4.10 0.60 4.04 0.65 4.03 0.66 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.(%) 0 6.80 0.04 6.80 0.04 6.80 0.04 6.80 0.04 6 5.88 0.10 6.13 0.09 5.81 0.11 5.60 0.13 12 4.86 0.23 5.19 0.18 4.79 0.25 4.70 0.27 18 4.33 0.43 4.49 0.36 4.27 0.46 4.25 0.48 24 4.05 0.65 4.20 0.53 4.04 0.64 4.04 0.65 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. - 25 -
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 12-26 -
, 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 - 27 -
(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 - 28 -
, 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 - 29 -
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, 12 16 ) ph,.. ph, ph Mettler Delta 350, (titratable acidity, TA) Marth (1978) 9 1% phenolphtalein, ph 8.4 0.1N NaOH ( ). Bifidobacteria NPNL MRS agar. NPNL Neomycin sulfate 2g, Paromomycin sulfate 4g, Nalidixic acid 0.3g, Lithium chloride 60g 1. 0.22 m membrane filter - 30 -
4. MRS agar L-cysteine HCl 0.05%, 121 15 5% NPNL MRS agar, MRS broth 1/2 L-cysteine HCl 0.05%. 0, 4, 8, 12 16 1 100ml 4. 1 MRS agar 37 48 BBL Gas Pak 100TM Anaerobic Systems (Cockeysville, MD. U.S.A) 3. 3.. 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-11-31 -
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 37 24 B. bifidum K-7 1.33% B. bifidum Bb-11 1.84%, Fig.1 12 B. bifidum K-7 1.3 109 B. bifidum - 32 -
Bb-11 6.3 109 24 B. bifidum K-7 1.4 109 B. bifidum Bb-11 9.0 109 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-7 37 24 ph 5.20 B. bifidum Bb-11 ph 4.03, B. bifidum K-7 0.33% B. bifidum Bb-11 0.64%, B. bifidum K-7 1.7 108, B. bifidum Bb-11 1.9 109. 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. - 33 -
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 - 34 -
Acidophilus milk bifidobacteria bifidobacteria milk bifidobacteria ph bifidobacteria. bifidobacteria 12.7% 121 1, 4 200 B. bifidum Bb-11 B. bifidum K-7 5g 4 16 (0, 4, 8 16 ) bifidobacteria, ph. B. bifidum Bb-11 B. bifidum K-7 12.7% 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-11 1.6 107 B. bifidum K-7 2 105 16 B. bifidum Bb-11 5.8 105 B. bifidum K-7 4.6 103 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 - 35 -
, 4 B. bifidum Bb-11 B. bifidum K-7 16 bifidobacteria. B. bifidum Bb-11 B. bifidum K-7 12.7% 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 6.55 4 B. bifidum Bb-11 ph 6.5 B. bifidum K-7 6.48 16 2 ph 6.45. B. bifidum K-7 B. bifidum Bb-11 ph 16 ph bifidobacteria milk ph. 4 16 12.7% 121 1. B. bifidum Bb-11 B. bifidum K-7 4, (T.A) Fig.9. - 36 -
Fig.9. Changes of titratable acidity during the storage of milk added bifidobacteria microparticles B. bifidum Bb-11 B. bifidum K-7 2 0.17%, 8 B. bifidum Bb-11 0.19% B. bifidum K-7 0.20% 16 B. bifidum Bb-11 0.20% B. bifidum K-7 0.21%. ph bifidobacteria, bifidobacteria milk. 4 4 B. bifidum Bb-11 0.18% B. bifidum K-7 0.19%. 12.7% 121 1.. bifidobacteria (Gilliland, 1979), (Petterson, 1983), bifidobacteria ph bifidobacteria (, 1998). 2 bifidobacteria. - 37 -
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, 12 16 ) 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-7 5.6 105 2.0 105 B. bifidum Bb-11 4 103 B. bifidum K-7 8 1.1 103, 12 103. 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.11. - 38 -
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-11 4.63 B. bifidum K-7 4.60, 16 B. bifidum Bb-11 4.62 B. bifidum K-7 4.60. Bifidobacteria 4 ph bifidobacteria. B. bifidum Bb-11 B. bifidum K-7 Fig.12. - 39 -
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-11 0.97% B. bifidum K-7 1.01%, 16 B. bifidum Bb-11 1.07% B. bifidum K-7 1.02%. 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 -20 16 0, 4, 8, 12 16 bifidobacteria, ph. bifidobacteria Fig.13 B. bifidum Bb-11 4.1 106 B. bifidum K-7 4 105, 4 B. bifidum Bb-11 4.9 105 B. bifidum K-7 5.0 105 B. bifidum K-7 B. bifidum Bb-11, 12 2-40 -
16 B. bifidum Bb-11 4.1 105 B. bifidum K-7 3.1 104 B. bifidum K-7 B. bifidum Bb-11. bifidobacteria -20 16 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 2 6.75, 16 B. bifidum Bb-11 6.72 B. bifidum K-7 6.78. bifidobacteria ph. - 41 -
Fig.14. Changes of ph during the storage of ice cream added bifidobacteria microparticles Bifidobacteria B. bifidum Bb-11 B. bifidum K-7 0.11% 16 B. bifidum Bb-11 0.11% B. bifidum K-7 0.12% bifidobacteria. Fig.15. Changes of titratable acidity during the storage of ice cream - 42 -
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 37 24 B. bifidum K-7 1.33% B. bifidum Bb-11 1.84% B. bifidum K-7 1.4 109 B. bifidum Bb-11 9.0 109, ph 2. Whey whey protein isolate(wpi) yeast extract 0.75% MRS broth B.bifidum K-7 37 24 ph 5.20, 0.33% 1.7 108, B.bifidum Bb-11 ph 4.03, 0.64% 1.9 109. 12.7%, 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-7 1.6 107 2.0 105 16 5.8 105 4.6 103 ph bifidobacteria bifidobacteria milk. B. bifidum Bb-11 B. bifidum K-7 ph 4.75 0.8% 16 ph 4.62 4.60 T.A 1.07% 1.02% B. bifidum Bb-11 4 B. bifidum K-7 8. B. bifidum Bb-11 B. bifidum K-7-20 4.1 106 4.0 105 16 4.1 105 3.1 104. ph bifidobacteria. - 43 -
3 1. Azuma, N., K., Yamauchi and T. Mitsuoka. 1984. Bifidus growth-promoting activity of a glycomacropeptide derived from human -casein. Agric. Biol. Chem. 48(8):2159-2162. 2. Bezkorovainy, A. and N. Topouzian. 1981. Bifidobacterium bifidus var. pennsylvanicus growth promoting activity of human milk casein and its derivatives. Int. J. Biochem. 13:585. 3. Bouhallab, S., C. Favrot and J. L. Maubois. 1993. Growth-promoting activity of tryptic digest of caseinomacropeptide for Lactococcus lactis subsp. lactis. Lait 73:73-77. 4. Bouhnik Y, B. Flourie, c. Andrieux, N. Bisetti, F. Brie and J.C. Rambaud. 1996. Effects of bifidobacterium sp. fermented milk ingested with or without inulinon colonic bifidobacteria and enzymatic activities in healthy humans. Eur.J.Clin.Nutr. 50 : 269-273 5. Cheng, C. C. and Nagasawa, T. 1984. Effect of peptides and amino acids produced by Lactobacillus casei in milk on the acid production of bifidobacteria. Jpn. J. Zootech. Sci. 55:339-349. 6. Desjardins, M. L., D. Roy, and J. Goulet. 1990a. Growth of bifidobacteria and their enzyme profiles. J. Dairy Sci. 73:299-307. 7. Desjardins, M. L., D. Roy, C. Toupin, and J. Goulet. 1990b. Uncoupling of growth and acids production in Bifidobacterium spp. J. Dairy Sci. 73:1478-1484. 8. El-Soda, M., A. Macedo, and N. F. Olson. 1992. The peptide hydrolase system of Bifidobacterium species. Milchwissenschaft. 47. 87-90. 9. Gibson, G.R. and X. Wang. 1994. Regulatory effect of bifidobacteria on the growth of other colonic bacteria. J.Appl.Bacteriol. 77 : 412-420. 10. Grill, J.P., J. Crociani and J. Ballongue. 1995. Effect of bifidobacteria on nitrites and nitrosamines. Appl. Microbiol. 20: 328-330. 11. Hukins, R. W., and N. L. Nannen. 1993. ph homeostasis in lactic acid bacteria. J. Dairy Sci. 76: 2354. 12. Jao, V. C., E. M. Milkolajcik, and P. M. T. Hansen, 1978. Growth of Bifidobacterium bifidum var. pennsylvanicus in laboratory media supplemented with amino sugars and spent broth from E. coli. J. Food Sci. 43:1257-1260. 13. Kampmann, E, A. Goldbohm, V.D.Brandt and P. Veer. 1994. Fermented dairy products, - 44 -
calcium and colorectal cancer in the Netherlands Cohort Study. Cancer Res. 54 : 3186-3190. 14. Kehagias, C., Y. C., Jao,E. M., Mikolajick and P. M. T. Hansen. 1977. Growth response of Bifidobacterium bifidum to a hydrolytic product isolated from bovine casein. J. Food Sci. 42:146-150. 15. Klaver, A. M., F. Kingma and H. Weewkamp. 1993. Growth and survival over Bifidobacteria in milk. Neth. Milk Dairy J. 47:151 16. Link, A.H., F. Rochat, K.Y. Saudan, O. Mignot and J. M. Aeschlimann. 1994. Modulation of a specific humoral immune response and change in intestinal flora mediated through fermented milk intake. Immunol. Med. Microbiol. 10 : 55-64. 17. Marth, E. H. 1978. Standard methods for the examination of dairy products. Americ. Public Health Assoc. Washington, DC 20036. 18. Marteau, P., P. Pochart, B. Flourie, P. Pellier, L. Sanots, J. F. Desjeux, and J. C. Rambaud. 1990. 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, 685-688. 19. Mcdonald, L. C., H. P. Fleming, and H. M. Hassen. 1990. Acid tolerance of Leuconost oc mesenteroides and Lactobacillus plantarum. Appl. Enviro n. Microbiol. 56:2120. 20.Misra, A.K. and P.k. Kuila. 1995. Antimicrobial substances from Bifidobacterium bi fidum. Indian J. Dairy Sci. 48: 612-614. 21. Modler, H. W., R. C. Mc Kellar, H. D. Goff, and D. A. Mackie. 1990. 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. 1995. Intestinal floras of populations that have a high of coion cancer. Appl. Environ. Microbiol. 61: 3202-3207. 22. Park, H. K., and T. R. Heo. 1995. Studies on the characteristics of Bifidobacterium spp. for the Industrial use. Korean, J. Food Sci. Ani. Resour. Vol.15(2):139-149. 23. Petschow, B. W., and R. D. Talbott. 1991. Response of Bifidobacterium sp -ecies to gr owth promoters in human and cow milk. Pediatr Res. 29:208-213. 24. Poch, M., and A. Bezkorovainy. 1988. Growth-enhancing supplements for varous species of the genus. Bifidobacterium. J. Dairy Sci. 71: 3214-3221. 25. Poch, M., and A. Bezkorovainy. 1991. Bovine milk -casein trypsin digest is a growth enhancer for the genus Bifidobacterium. J. Agric Food Chem. 39: 73-77. 26. Proulx, M., S. F. Gauthier and D. Roy. 1992. Utilisation d'hydrolysats enzymatiques de caseine pour la croissance des bifidobacteries. Lait 72:393-404. - 45 -
27. Reuter, G. 1990. Bifidobacteria cultures as components of yoghurt-like products. Bifidobacteria Microflora 9: 107-118. 28. Rose, C. S. and P. György. 1963. Bifidus factor II for growth of Lactobacillus bifidus. Proc. Soc. Exp. Bio. N.Y. 112(4):923-926. 29.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. 1989. Consumption of fermented milk products and breast cancer : A case-cotrol study in the Netherlands. Cancer Res. 49 : 4020-4023 30. Schiffrin, E.J., F. Rochat, H.A. Link, J.M. Aeschlimann and A.H. Donnet. 1995. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria J. Dairy Sci. 78: 491-497. 31. Seki, M., M. Igarashi, Y. Fukuda, S. Simamura, T.Kawashima and K. Ogasa. 1978. The effect of bifidobacterium cultured milk on the "regularity" among aged group. Nutr. Food. 4 : 379-387 32. Singh, j., A. Rivenson, M. Tomita, S. Shimamura, N. Ishibashi and B.S Reddy. 1997. Bifidobacteria longum, a lactic acid-producing intestinal bacterium inhibits coion cancer and modulates the intermediate biomarkers of coiol carcinogenesis. Carcinogenesis. 18 : 833-841 33. Yamauchi, K. 1992. Biologically functional proteins of milk and peptides derived from milk proteins. Bulletin of the IDF. 272: 51. 34. Yoshigawa, M., H. Suganuma, A. Shiata, H. Usui, K. Kurahashi, T. Mizumoto, Y. Suitani, and K. Kashimoto. 1994. In peptides chemistry 1993". Y. Okata(Editor), Protein Rerch Foundation, Osaka, 157-160. 35. Yoshihama, M., E. Mochizuki, S. Mitsuhashi, and K. Ahiko, 1982. 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, 78:33-37. - 46 -
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, - 47 -
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. - 48 -
2 1. Bifidobacteria Chr. Hansen Lab.(Denmark) B.bifidum Bb-11 B.bifidum K-7 MRS broth whey (Beckman J-21, England) 3,000rpm 10. 2. 2% 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 -38 2 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) - 49 -
. 5. Bifidobacteria 1g (0.5% yeast extract) 200ml (ph=6.8 0.1) (ph=1.4 0.1). 1ml MRS gradient MRS-Agar 48-72 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 15.. 6.0g NaCl 15g Yeast extract 2900ml. 7.4% HCl ph 1.4 0.1. 3000ml. 20.4g KH2PO4(MW. 136.09) 15g Yeast extract 2900ml. 1N NaOH ph 6.8 0.1. 3000ml.. Poly-lysine Bifidobacteria Bifidobacterium cultures, Bifidobacteria alginate poly-lysine 1 g (0.5% yeast extract) (0.05% cysteine.hcl) 200 ml (ph=1.5) 37 120. 1ml 2.5 % MRS gradient 10-8 MRS agar 37, 48-72 colon formed unit.. ph Bifidobacteria Phosphate buffer (ph=7.4) 0.1M HCl ph 6.8, 4.0, 2.0-50 -
1.5 ph. Bifidobacteria 1g 200ml (0.5% yeast extract 0.05% cysteine.hcl ) 37 120. 0.1N 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 10-8 0.1N NaOH ph 6.8 MRS agar 37, 48-72 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. 5.0 109cfu/g, 1 2 30. fecal sample, 4. 3 7 3, 7, 14 5 10.. Bifidobacteria, Lactic acid bacteria, Enterobacteriaceae Staphylococci 4. - 51 -
-Bifidobacteria (g) BL agar 58 Sodium propionate 15 Paromomycin 0.05 Neomycin 0.1 Lithium chloride 3 Water added 1000 ml 121 20. 55. Bifidobacteria BS medium 37, 48-72 colony Bifidobacteria colony. -Lactic acid bacteria (g) Lactobacilli MRS broth 55 Agar 15 NaN3 0.2 Water added 1000 ml 121, 20. 55. Lactic acid bacteira MRS agar medium 37, 48-72 colony. -Enterobacteriaceae EMB agar Water added (g) 27 g 1000 ml 121 20. 55. Enterobacteriaceae EMB agar medium 37, 48 colony. -Staphylococci (g) Baird-parker agar base 63 Bacto EY Tellurite 50 (ml) Water 950 (ml) - 52 -
Baird-parker agar base 950 ml. 121 15. Bacto EY Tellurite 50ml. 55. Staphylococci Baird-parked agar medium 37 48 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 - 53 -
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. - 54 -
Fig.1. Diagram of air atomization device - 55 -
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 - 56 -
Bifidobacteria. Bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing 15. poly-l-lysine 0.02%. slurry -37 2-55 14. 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% 1.5 8 1.5 90.2 30.3 2 1.5% 1.5 8 1.5 86.7 28.7 3 2.0% 1.5 8 1.5 80.1 21.6 4 1.5% 1.0 8 1.5 88.1 30.8 5 1.5% 2.0 8 1.5 83.0 30.5 6 1.5% 1.5 4 1.5 81.4 28.2 7 1.5% 1.5 12 1.5 92.8 26.9 8 1.5% 1.5 8 0.5 81.4 30.3 9 1.5% 1.5 8 2.5 91.4 27.1 The 0.02 % poly-l-lysine was invariably used to cross-link Bifidobacteria-loaded algiante microparticles. 2. Bifidobacteria - 57 -
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 - - - - - - 7.56 0.11 F2 1.5 g - - - - - - 7.91 0.12 F3 2.5 g - - - - - - 7.67 0.13 F4 1.5 g 0.5 % - - - - - 9.25 0.08 F5 1.5 g 2.5 % - - - - - 8.89 0.17 F6 1.5 g - 5 % - - - - 8.35 0.08 F6 1.5 g - 10 % - - - - 7.99 0.12 F8 1.5 g - -- 2.5 % - - - 8.90 0.09 F9 1.5 g - -- 5 % - - - 8.25 0.11 F10 1.5 g - - - 0.5 % - - 8.73 0.20 F11 1.5 g - - - 1% - - 8.19 0.15 F12 1.5 g - - - - 0.25% - 8.51 0.15 F13 1.5 g - - - - 0.5% - 8.61 0.22 F14 1.5 g - - - - - 1% 8.15 0.19 F15 1.5 g - - - - - 2% 8.04 0.09 Alginate solution (1.5%) was used invariably used. Bifidobacteria Table 4. - 58 -
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.3. 14.. 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 - 59 -
Bifidobacteria ph. air-atomization Bifidobacteria alginate-polylysine alginate. Bifidobacteria. Bifidobacteria alginate polylysine Fig.4. Bifidobacteria culture 120 2.85 109cfu/g 103cfu/g alginate polylysine Bifidobacteria 2.62 107 cfu/g 3.46 107 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. - 60 -
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 2.0. 3) (ph=1.5) Bifidobacteria Bifidobacteria. Bifidobacteria Fig.6. 30 60 (108cfu/g) 90 120 (6.92 2.45) 107 cfu/g (4.47 2.75) 107 cfu/g. gastric emptying 90-61 -
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 2 109-1010cfu/g. 4 16 9.12 107cfu/g 1.66 107 cfu/g. 20 (6.0 105cfu/g). 20 Bifidobacteria. - 62 -
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 (2.51 1.62) 108cfu/g Bifidobacteria 3, 7 14 6.0 2.2, 7.2 2.19 5.5 3.2 ( 108cfu/g). Bifidobacteria Lactic acid bacteria, Enterobacteriaceae Staphylococci Fig.9, 10 11. Bifidobacteria Bifidobacteria 3. - 63 -
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. - 64 -
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. - 65 -
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. - 66 -
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. - 67 -
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. - 68 -
before after stop Frequency A group 1.00±0.00 1.34±0.75 0.88±0.75 B group 0.71±0.34 1.13±0.63 0.71±0.34 Amount A group 2.75±0.50 2.50±0.58 2.75±0.50 B group 3.25±0.50 3.25±0.96 2.75±0.50 Viscosity A group 3.00±0.82 2.25±0.50 2.50±0.58 B group 3.25±0.50 2.00±0.00 3.00±0.00 A group; Bifidobacteria cultures were orally given. B group, Bidobacteria-loaded alginate poly-l-lysine microparticles were orally given. - 69 -
4 Bifidobacteria sodium alginate 1.5%, 8 ml/min, 0.75 bar 7 curing 15.. 80-90 m. 0.5 g Start culture, 0.5g Yeast extract, 5.0g Glycerol, 0.5g NaHSO3,1.0g Mg3(PO4)2. slurry -37 2-55 14. Bifidobacteria culture 120 103 cfu/g Bifidobacteria 107 cfu/g. Bifidobacteria ph. Bifidobacteria 30 60 90 120 107 cfu/g. Bifidobacteria 4 2 109-1010cfu/g 16 107cfu/g. Bifidobacteria Bfidobacteria Bifidobacteria culture Bifidobacteria. Lactic acid bacteria, Enterobacteriaceae Staphylococci. Bifidobacteria frequency. Bifidobacteria. - 70 -
5 1. Bodmeier, R., Chen, H. and Paeratakul, O., A novel approach to the oral delivery of micro- or nanoparticles, Pharm. Res., 6(5), 413-417 (1989) 2. Goh, J. S., Kwon, I. K. and Kim, Y. O., Studies on the growth of Bifidobacterium bifidum ATCC 11863 in milk, Kor. J. Dairy Sci., 8(1), 48-56 (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 11863 by lactic acid bacteria for the fermentation of milk, Kor. J. Dairy Sci., 9(4), 211-219 (1987) 4. Goh, J. S., Kwon, I. K., Ahn, J. K. and Yoon, Y. H., Fermentation of milk by Bifidobacterium bifidum ATCC 11863 ; Conversion of milk constituents by fermentation, Kor. J. Anim. Sci., 30(10), 618-630 (1988) 5. Kim, D. H. and Han, M. J., Inhibition of intestinal bacterial enzymes by lactic acid bacteria,yakhak Hoeji, 39(2), 169-174 (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, 253-257 (1988) 7. Krueter, J. and Liehl, E., Long-term studies microencapsulated and adsorbed influenza vaccine nanoparticles, J. Pharm. Sci., 70(4), 367-371 (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), 341-344 (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), 183-188, 1995b. 10. Lim, F. and Moss, R. D., Microencapsulation of living cells and tissues, J. Pharm. Sci., 70(4), 351-354 (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, 2885-2899 (1989) 12. Lin, S. Y. and Kao, Y. H., Tablet formulation study of spray-dried sodium diclofenac enteric-coated microcapsules, Pharm. Res., 8(7), 919-924 (1991) - 71 -
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 - 97 -
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. 3. 1 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 4 2 1. 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. - 98 -
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. - 99 -
2 1. BALB/C 8-12. 2. Bifidobacterium bifidum Bifidobacterium bifidum MRS. MRS 0.5% L-cystein, HCl 0.1% sodium azide Liquid paraffin 1.5 ml 5 37. 1-5 B. bifidum washing. MRS parpffin oil 400g 10. bacteria HBSS 2. 1 105 cell/well. RPMI-1640-2 0. 3. Encapsulated bacterial cell counting Bifidobacteria 5 ml 30 ml suspension. 1000 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. 4 2.. 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. 1 500... 4 7 ethyl ether retroorbital bleeding. 4 2-20. - 100 -
.. 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. 3350 48.5 mm. 10 2 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 30. 2 ml 20 PMSF 20 1% sodium azide 15-20.. 1) Spleen cell spleen cell Murray et al. (1987). spleen PBS needle forcep. 5. 500 g 5. 0.83 ammonium chloride solution 5. ammonium chloride solution 5 HBSS suspension. suspension HBSS washing. RPMI-1640 10 FBS. 2) MLN cells MLN PBS needle forcep. suspension 500g 5. HBSS washing RPMI-1640 10 FBS. 3) PP cells PP cell Frangakis et al. (1982). PP PBS. PP RPMI-1640 Dispase 50 ml 37 30 stirring. supernatant 30 ml 500 g HBSS - 101 -
washing 10 FBS-RPMI-1640 2ml resuspension, Dispase RPMI 1640 3. 5. Isotype-specific ELISA Goat anti-mouse isotype spesific antibody 1.2 /ml sodium bicarbonate buffer ph 9.2 96 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 50 37 1. PBST 3, 3 2 substrate 2,2-azino-bis 3-ethylbenzhiazolin -6-sulfuric acid 50 10-15 405 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 200 1 37 blocking. PBST RPMI-1640 well 1.5 105 37 4. PBST 3 Biotin-conjugated gote anti-mouse Ig isotype 0.5 gelatin 1:1000 well 150 37 1 incubation. PBST 3 Avidin-peroxidase 1:1000 well 150 1 30 incubation. plate PBST AEC (3-amino-9-ethyl carbazole) 15 spot counting. 7. RNA B cell, 0.5 1 107cells TRIzol (Gibco) 1 lysis 5 200 chloroform 4, 12,000 15. aqueous phase tube 500 isopropyl alcohol 10 4, 12,000 10. ( RNA) 75% cold ethanol 7,500 5 DEPC-H2O 50. RNA reverse transcription UV Spectrophotometer A260/A280 RNA purity - 102 -
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 2 37 60 reverse transcription 95 10 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 - 103 -
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) 1. 0.5% BSA 0.1% sodium azide PBS 2, 200 PBS 5 105 1 106 FITC goat anti-iga (Becton Dickinson, San Jose, CA, USA) 25 / 4 30. 0.5% 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 -20. 11. B. bifidum C. perfringens 96 well plate spleen cell (5 105 cells/well) 4. 18 5 Ci/ 3[H thymidine. cell harvester (Inotech IH-110, Switzerland), liquid scintillation counter (Wallac 1410, Pharmacia, Finland). - 104 -
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 2.5 106 cells/ 96-well culture plate seeding, 5 103, 5 104, 5 105, 5 106, 5 107 cells/ bifidobacteria LPS (12.5 / ) 37, 5% CO2incubator. 7 ELISA., 5 106 5 107cells/ bifidobacteria IgA, IgM, IgG1 isotype (Fig.1). LPS 5 106 5 107cells/ bifidobacteria LPS (Fig.2). bifidobacteria B cell. negative control Clostridium perfringens ( ) bifidobacteria Table 1. Comparison of bacterial viability between colony and direct counting method. - 105 -
B. bifidum C. perfring ens Method Dilution factor No. of cells 1 106 1.29 108 2.09 108 Colony counting (CFU/ ) 1 107 1.25 108 3.18 108 1 108 2.5 108 7.05 108 1 2 108 7.96 108 Direct count ing (cells / ) 10 1.48 108 6.37 108 100 1.25 108 7.87 108 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 - 106 -
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 - 107 -
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). - 108 -
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 5 105 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. - 109 -
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 - 110 -
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-5. - 111 -
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-5. - 112 -
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-5. - 113 -
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. - 114 -
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 1 107 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. - 115 -
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. - 116 -
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 5 105 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. - 117 -
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. - 118 -
F i g. 1 2. 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 4 2. 4 BB ELISA. B.bifidum BB control C. Perfrigens (Fig.13) (Fig.14). B. bifidum adjuvant.. B. bifidum spleen, MLN, PP - 119 -
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. - 120 -
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. - 121 -
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 4 2. 1 500 (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. - 122 -
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. - 123 -
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 - 124 -
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. 1.5 105 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. - 125 -
7. Bifidobacteria cytokine. Bifidobacteria cytokine cytokine Bifidobacteria mrna 1. 2 3 4 5 6 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). - 126 -
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 1 2 3 4 1 2 3 4 1 2 3 4 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) - 127 -
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. - 128 -
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. - 129 -
5 1. Andre, C., Lambert, R., Bazin, H., and Heremans, J.F. 1974. Interference of oral immunization with the intestinal absoption of heterologous albumin. Eur. J. Immunol. 4:701 2. Beagley, K.W., Eldridge, J.H., Kiyono, H., Everson, M.P., Koopman, W.J., Honjo, T., and McGhee, J.R. 1988. Recombinant murine IL-5 induces high rate IgA synthesis in cycling IgA-positive Peyer's patch B cells. J. Immunol. 141:2035-2042 3. Brandtzaeg, P., Sollid, L.M., Thrane, P.S., Kvale, D., Bjerke, K., Scott, H., Kett, K., and Rognum, T.O. 1988. Lymphoepithelial interactions in the mucosal immune system. Gut. 29:1116-1130 4. Bullen, C.L., P.C. Terle, M.G. Srewart. 1977. The effect of humanized milks and supplemented breast feeding on the fecal flora of infants. Br. Med. J. 16, 403 5. Chabarri, F.J., M. De Paz, and M. Nunez. 1988. Cryptotective agents for frozen concentrated starters from non-bitter Streptococcus lactis strains. Biotechnol. Lett. 10, 11 6. Chandan, R.C. 1989. Yogurt : Nutritional and Health properties, ed. National Yogurt Association, McLean, Virginia. 7. Coffman, R.L., Lebman, D.A., and Shrader, B. 1989. Transforming growth factor- specifically enhances IgA production by lipopolysaccharide stimulated murine B lymphocytes. J. Exp Med. 170:1039-1044 8. Craig, S.W., and Cebra, J.J. 1971. Peyer's patches: An enriched source of precursors of IgA-producing immunocytes in the rab. bifidumit. J. Exp Med. 134:188-200 9. Czerkinsky, C.C., Nilsson, L., Nygren, H., Ouchterlony, O., and Tarkousky, A. 1983. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody secreting cells. J. Immunol Methods. 65:109. 10. De Aizpurua, H.J., and Russel-Jones, G.J. 1988. Identification of Classes of Proteins that Provoke an Immune Response upon Oral Feeding. J. Exp Med. 167:440-451 11. De Simone, R., R. Vesely, B. Negri, B.S. Salvadori, A. Zanzoglu, Cilli, and L. Lucci. 1987. Immunopharmacol. Immunotoxicol. 9, 87-100 12. Duffy, L.C., M.A. Zielezny, T.M. Riepenhoff, D. Dryja, A.S. Sayahtaheri, E. Griffiths, D. Ruffin, H. Barrett, J. Rossman, and P.L. Ogra. 1993. Effectiveness of B. bifidium in experimentally induced MRV infection: dietary implications in formulars for newborns. Endocrine Regulations. 27, 223-229 13. Fernandes, C.F., Shahani, K.M. 1990. J. Food. Prot. 53:704-710 - 130 -
14. Gilliland, S.E. 1989. Acidophilus Milk Products: A Review of Potential Benefits to Consumers. J. Dairy Sci. 72:2483-2494 15. Harriman, G.R., Kumimoto, D.Y., Elliott, J.F., Paetkau, V., and Strober, W. 1988. The role of IL-5 in IgA B cell differentiation. J. Immunol. 140:3033-3039 16. Kagnoff, M.F. 1987. In physiology of the Gastrointestinal Track, and 2nd Ed. pp 1699-1728. Raven, New York. 17. Kehrl, J., Roberts, H.A.B., Wakefield, L.M., Jakowlew, S., Sporn, M.B., and Fauci, A.S. 1986. Transforming growth factor is an important immunomodulatory protein for human B lymphocytes. J. Immunol. 137:3855 18. Kehrl, J.H., Wakefield, L.M., Roberts, A.B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M.B., and Fauci, A.S. 1986. Production of transforming growth factor by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037 19. Kim, P.H., and Kagnoff, M.F. 1990. Transforming growth factor- 1 is a costimulator for IgA production. J. Immunol. 144(9):3411-3416 20. Klaver, F.A.M., and van der Meer, R. 1993. Appl. Environ Microbiol. 59:1120-1129 21. Lebman, D.A., Lee, F.K., and Coffman, R.L. 1988. The effects of IL-4 and IL-5 on the IgA response by murine Peyer's patch B cell subpopulations. J. Immunol. 141:2050-2056 22. Lebman, D.A., Nomura, D.Y., Coffman, R.L., and Lee, F.D. 1990. Molecular characterization of germ-line immunoglobulin A transcripts produced during transforming growth factor -induced isotype switching. Proc. Natl. Acad. Sci. USA. 87:3962 23. Matsuoka, M., Yoshida, K., Usuda, S., and Sakano, H. 1990. Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell. 62:135 24. McIntyre, T.M., Klinman, D.R., Rothman, P., Lugo, M., Dasch, J.R., Mond, J.J., and Snapper, C.M. 1993. J. Exp Med. 177:1031 25. Murray, P.D., McKenzie, D.T., Swain, S.L., and Kagnoff, M.F. 1987. Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139:2669. 26. Nagaoka, M., S. Hashimoto, T. Watanabe, T. Yokokura, and Y. Mori. 1994. Anti-ulcer effects of lactic acid bacteria and their cell wall polysaccharides. Biol. Pharm. Bull. 17, 1012-1017. 27. Ogra, P.L., Mestecky, J., Lamm, M.E., Strober, W., McGhee, J.R., and Bienenstock, J. 1994. Handbook of Mucosal Immunology. ed. pp 373-390, 415-424. Academic Press, Inc. 28. Owen, R.L., and Ermak, T.H. 1990. Structural specialization for antigen uptake and processing in the digestive track. Spring Semin. Immunopathol. 12:139 29. Pestka, J.J. 1993. Food, diet and gastrointestinal immune fuction. In (Kinsella, J. ed. Advances in Food and Nutrition Research. Academic Press (in press)) 30. Roberts, A.B., Sporn, M.B., Assoian, R.K., Smith, J.M., Roche, N.S., Wakefield, - 131 -
L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H., and Fauci, A.S. 1986. Transforming growth factor type : rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci. USA. 83:4167 31. Sasaki, T., S. Fukami, and S. Namioka. 1994. Enhancement of cytotoxic activity of lymphocytes in Mice by oral administration of peptidoglycan (PG) derived from Bifidobacterium thermophilus. J. Vet. Med. Sci. 56, 1129-1133. 32. Sekine, K., Ohta, J., Onishi, M., Tatsuki, T., Shimokawa, Y., Toida, T., Kawashima, T., and Hashimoto, Y. 1995. Analysis of Antitumor Properties of Effector Cells Stimulated with a Cell Wall Preparation (WPG) of Bifidobacterium infantis. Biol. Pharm. Bull. 18(1):148-153 33. Sonoda, E., Matsumoto, R., Hitoshi, Y., Ishii, T., Sugimoto, M., Araki, S., Tominaga, A., Yamaguchi, N., and Takatsu, K. 1989. Transforming growth factor induces IgA production and acts additively with interleukin 5 for IgA production. J. Exp Med. 170:1415 34. Stokes, C.R., Newby, T.J., Huntley, D.P., and Bourne, F.J. 1979. The Immune response of mice to bacterial antigens given by mouth. Immunology. 38:497-502 35. Stokes, C.R., Soothill, J.F., and Turner, M.W. 1975. Immune exclusion is a function of IgA. Nature (London) 255:745 36. Tahri K., Grill J.P., and Schneder, F. 1996. Bifidobacteria strain behavior toward cholesterol: coprecipitation with bile salts and assimilation. Curr Microbiol. 33(3):187-193 37. Takahashi, T., Oka, T., Iwana, H., Kuwata, T., and Yamamoto, Y. 1993. Immune Response of Mice to Orally Administered Lactic Acid Bacteria. Biosci. Biotech. Biochem. 57(9):1557-1560 38. Tissier. 1899. 2a reaction chromophile d Echerich et le Bacteriym Coli. C. R. Soc Biol. 51, 943 39. Tsunawaki, S., Sporn, M., Ding, A., and Nathan, C. 1988. Deactivation of macrophages by transforming growth factor-. Nature (Lond.). 334:260 40. Yasui, H., and Ohwaki, M. 1991. Enhancement of Immune Response in Peyer's patch Cells Cultured with Bifidobacterium breve. J. Dairy Sci. 74:1187-1195 41. Yasui, H., Nagadka, N., Hayakawa, K. 1996, Augmentation of anti-influenza virus hemagglutinin antibody production by Peyer's patch cells with Bifidobacterium breve YIT 4064. Clin Diagn Lab Immunol. 1(2):244-246 - 132 -
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 - 142 -
(, 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-1. 15 Bifidobacteria. Table 1. Type strains of bifidobacteria used in the study - 143 -
Species Strain Synonym Source B. adolescentis KCTC 3216 ATCC 15703 Intestine of adult B. adolescentis KCTC 3352 ATCC 15706 Intestine of adult B. adolescentis KCTC 3416 ATCC 15740 Intestine of adult B. bifidum KCTC 3202 ATCC 29521 Stool of breast-fed infant B. bifidum KCTC 3357 ATCC 35914 Human feces B. bifidum KCTC 3418 ATCC 15696 Intestine of infant B. bifidum KCTC 3476 ATCC 11863 Lactobacillus bifidus subsp. pensylvancum B. breve KCTC 3220 ATCC 15700 Intestine of infant B.breve KCTC 3419 ATCC 15701 Intestine of infant B. breve KCTC 3461 ATCC 15698 Intestine of infant B. infantis KCTC 3127 ATCC 25962 Intestine of infant B. infantis KCTC 3249 ATCC 15697 Intestine of infant B. infantis KCTC 3270 ATCC 15697 Intestine of infant B. infantis KCTC 3368 ATCC 27920 Intestine of infant B. infantis KCTC 3460 ATCC 15702 Intestine of infant B. longum KCTC 3466 DSM 20097 Calf feces B. longum ATCC 15707 Intestine of adult B. longum ATCC 15708 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) 37 24. 10% glycerol -70.. DNA Bifidobacteira Versalovic (1991) genomic DNA. TPY broth 24 7,000rpm 10-144 -
bifidobacteria. 1 1M NaCl 50mM TE (50mM Tris, 50mM EDTA, ph 7.8) 0.45 10mM 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) 37 1. 30 SDS(10%) 30 proteinase K (10mg/ml, 10mM TE ) 55 2. Phenol(0.4 ) 1. phenol chloroform(0.4 ) 2. 10mM TE 0.4 40 3M sodium acetate(ph 5.2) 0.8 ethanol -20 2. 2 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 37 4. 1% 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 - 145 -
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 42 24 hybridization. 0.1% SDS 2 SSC 15 2. 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- 1355-3')(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) 1.5 20mM MgCl2(1.5mM), 1 OPB-07 primer(l M)(Operon), 1 Template DNA(20ng) 16.5 H2O PCR. Perkin-Elmer thermal cycler 94 1 37 1 72 1 35cycles. 1.5% agarose gel, 0.5 TBE buffer, 70V 4. DNA ethidium bromide.. SDS-PAGE Glucose lactose TPY broth 37 24 bifidobacteri a 2. 25 10 Vibra Cell-VCX 600(Sonics and Materials Inc.) 37% amplitude 5 5 cycle 30 sonication. Sonication. SDS-PAGE sample buffer 100 5. 13% polyacrylamide gel s - 146 -
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. 1. 108 Chemiluminescence detection of EcoRI-digested DNA from Bifidobacterium strains - 147 -
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 3216 8,500 6,600 3,700 2,900 1,250 KCTC 3352 8,500 6,600 3,700 2,900 KCTC 3416 6,600 5,100 3.900 3,700 2,900 B. bifidum KCTC 3202 9,700 5,600 3,900 3,700 KCTC 3357 9,700 4,000 3,700 KCTC 3418 9.700 5,600 3,900 3,700 B. breve KCTC 3220 4,200 3,700 3,200 2,200 KCTC 3461 4,200 3,700 3,200 2,200 B. infantis KCTC 3127 4,200 3,700 2,400 1,800 1,250 KCTC 3270 4,200 3,700 3,100 2,400 1,250 KCTC 3368 4,200 3,700 3,100 2,400 1,250 KCTC 3460 4,200 3,700 3,100 2,400 1,250 B. longum KCTC 3466 4,100 3,700 3,500 3,400 1,200 900 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 - 148 -
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. - 149 -
- 150 -
- 151 -
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 - 152 -
Restriction Endonuclease DNA fragment size (bp) Restriction pattern AluI 870 340 A 610 340 220 110 B 610 340 220 150 110 C 610 220 160 150 110 D HaeIII 330 230 190 160 80 A 330 260 160 90 80 B 330 300 165 160 80 C 330 260 230 160 90 80 D 290 285 210 160 E 330 260 200 160 80 F HhaI 480 370 290 260 A 480 370 270 260 B 480 370 290 C 480 370 260 160 130 D MboI 590 540 A 570 400 100 80 B 540 380 190 100 C 590 540 380 190 100 D 900 370 E 420 380 190 120 90 F RsaI 470 370 250 155 150 A 640 370 250 150 B Table 4. Classification of restriction pattern of PCR-amplified 16S rdna from the type culture of Bifidobacterium - 153 -
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 15707 C A A B B B. longum ATCC 15708 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 - 154 -
. 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 - 155 -
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 3357 78,000, 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 3216 786,000, 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. - 156 -
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 3460. 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 3-14. 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 - 157 -
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 3368. RAPD 7-10bp random primer annealing MgCl2 stringency PCR DNA band fingerprinting (Welsh McClelland, 1990). (1994) 17-27 bp degenerated primer 28 annealing PCR DNA pattern. (1997) S. salivarius subsp. thermophilus L. delbrueckii subsp. bulgraicus L. acidophilus band. Tyler (1997) RAPD. 4. - 158 -