臺灣博碩士論文加值系統

醃製食品，尤其是泡菜，是台灣人的日常食品之ㄧ。實際上，泡菜對於人體健康的隱藏效益已漸漸受大眾所關注。而醃製蔬菜與醃製水果目前最明顯的好處，就是它為人們，尤其是那些經濟落後國家，在寒冬提供了富有維他命的乾糧來源。已有些許文獻證實，泡菜可以促進消化，同時也可降低膽固醇。更有各種說法認為泡菜，尤指韓國泡菜kimchi，可以有效抵抗糖尿病，癌症，甚至嚴重急性呼吸道綜合症候群(SARS)。大部分的乳酸菌(LAB)被認為是可被人們食用的食品安全級(GRAS)菌株，且常被應用於常見的發酵食品上，尤其是泡菜更為常見。在泡菜的醃製過程中，提供了乳酸菌極佳的生長環境，而乳酸菌也同時提供了“酸味”予該醃製蔬菜，因此泡菜皆富有"酸味"。現今，已有許多人類或動物來源的乳酸菌文獻研究，卻少有其他相關來源的乳酸菌報導。因此，在此論文中，我們將事先分離自台灣台北醃製蔬菜的288株乳酸菌中挑選出17株被認為富有益生菌(probiotic)功能的菌株，測定其對巨噬細胞(RAW264.7)的刺激免疫功效。就如結果所示，這17株乳酸菌皆有對巨噬細胞的TH1型與TH2型細胞激素因子向下調節的功能，而其中以E1、E40、E43與E55菌株最為明顯。而我們將這4株乳酸菌分別以species-specific PCR以及API��50 CHL鑑定方法進行進一步菌種鑑定，得出結果分別為：L. plantanum E1 (API��50 CHL)、L. casei E40 (species-specific PCR)、L. casei E43 (species-specific PCR) 以及L. plantanum E55 (API��50 CHL)。進一步的測定結果指出，雖說活菌的向下免疫調節效能無論是在致敏或致發炎實驗系統中皆比熱致死乳酸菌株來得好些，但將菌體加熱致死基本上是不太會影響這四株乳酸菌株的向下免疫調節效能的。於是，我的結論是台灣台北的醃製泡菜食品中皆可以找到有益的乳酸菌株，且基於這些有益乳酸菌株的存在，使這些泡菜有益於過敏體質或嚴重發炎症的改善。同時認為，生吃這些泡菜的免疫效能可能會比煮過後的泡菜來得有效些。

Pickles are one of the daily foods of Taiwanese, especially the pickled vegetables. In fact, there is increasing interest in the potential health benefits of pickles. The most obvious benefit, especially in undeveloped economies, is that pickling fruit and vegetables allows crops to be preserved to supply a valuable source of vitamins over the scarce winter months. There is also some evidence that pickles can promote digestive health and lower cholesterol. All manner of claims have been made for pickled vegetables, such as kimchi, as a preventative for diabetes, cancer and even SARS. Most lactic acid bacteria (LAB) are generally regarded as safe (GRAS) for human consumption, and are widely used to prepare fermented dairy products, especially the pickled vegetables. The salting procedure of pickle fermentation provides a suitable environment for LAB to grow which impart the acid flavor to the vegetables. Recently, there are plenty of studies on LAB isolated from animal and human sources, but less from the other sources. So, we isolated 288 strains from pickled vegetables in Taiwan, Taipei before, and select 17 out of 288 strains, which are believed to have probiotic characteristic, and determined further their immuno-regulated effects to macrophage cells (RAW264.7). As the result in our studies, almost all of the 17 isolated LAB strains have the ability to down-regulate either TH1 or TH2 cytokines in the macrophage cells model experiment, especially the strains E1, E40, E43 and E55. In this study, we identified the Lactobacillus isolates by either using API��50 CHL or species-specific PCR assay, and the results are L. plantanum E1 (API��50 CHL), L. casei E40 (species-specific PCR), L.casei E43 (species-specific PCR) and L. plantanum E55 (API��50 CHL), respectively. The further determination result shown that, thermal death procedure has less influence to the down-regulation effect of these 4 LAB strains, although the living LAB strains got better down-regulation capability in either inflammation or allergy immune system. The conclusion is that there are probiotic LAB in Taipei, Taiwan pickled vegetables. As shown in this study, eating these pickled vegetables is either benefit for allergy or inflammation treatment. It seems that, eating uncooked pickled vegetables will enhance more immune functions than eating the cooked pickled vegetables.

TABLES OF CONTENTS

ENGLISH ABSTRACT.i
CHINESE ABSTRACTiii
ACKNOWLEDGEMENTS.v
TABLE OF CONTENTSvi
LIST OF FIGURES xi
LIST OF TABLES.xiii
NOMENCLATURExiiii
CHAPTER
I Introduction1
1.1 Motivation1
1.2 The definition of probiotics and Lactic acid bacteria (LAB) as probiotics.4
1.3 Potential benefits of LAB.5
1.3.1 Alleviation of lactose intolerance5
1.3.2 Immune enhancement5
1.3.3 Reducing inflammation6
1.3.4 Decrease in fecal enzymes and mutagenicity.7
1.3.5 Hypocholoesterolemic effect7
1.3.6 Lowering blood pressure8
1.3.7 Reduction of risk of disease.8
1.3.8 Improving mineral absorption11
1.4 LAB in fermented vegetable.11
1.5 Macrophage cell and its role in specific immunity.12
1.6 Cytokines14
1.6.1 Types of cytokines15
1.6.2 Mechanism of allergy and related cytokines17
1.6.3 Mechanism of inflammation and related cytokines.18
1.7 Immune regulation of LAB.20
1.8 Scope of the Present Study23
II Materials and Methods24 2.1 Materials .24
2.1.1 Lactic Acid Bacteria (LAB) strains.24
2.1.2 Cell line.24
2.1.3 Reagents.24
2.1.4 Apparatus.26
2.1.5 Kits27
2.2 Methods.27
2.2.1 LAB strain cultures and media27
2.2.2 Cell culture of macrophage cell line RAW264.7.28
2.2.3 Serum starvation and LPS activation of macrophage cell line RAW264.7.28
2.2.4 MTT assay.29
2.2.5 LAB stimulation and ELISA kit assay31
2.2.5.1 h-IFN-γ immunoassay kit.31
2.2.5.2 IL-6 and IL-12p40p70 immunoassay kit32
2.2.6 Identification system for Lactobacillus species: API��50 CHL system and species-specific PCR assay33
2.2.6.1 API��50 CHL fermentation assays34
2.2.6.2 Species-specific PCR assay35
III Results and Discussions.36
3.1 Preliminary analysis of RAW264.7.36
3.1.1 RAW264.7 cell adherent time and cell concentration selection.36
3.1.2 Serum-starvation time of RAW264.7 cell.37
3.1.3 LPS-activation and cytotoxicity of LPS to RAW264.7.37
3.2 Cytotoxicity assay of Lactic Acid Bacteria (LAB) strains to RAW264.7.38
3.3 Set up of allergy macrophage cells system and inflammation macrophage cells system.39
3.4 Effect of low dose LPS on IFN-gamma production by RAW264.7 cells stimulated by LAB strains.40
3.5 Effect of low dose LPS on IL-6 production by RAW264.7 cells stimulated by LAB strains41
3.6 Effect of low dose LPS on IL-12p40p70 production by RAW264.7 cells stimulated by LAB strains.41
3.7 Thermal death LAB strains.42
3.7.1 Effect of low dose LPS on IL-6 production by RAW264.7 cells stimulated by thermal death LAB strains42
3.7.2 Effect of low dose LPS on IL-12p40p70 production by RAW264.7 cells stimulated by thermal death LAB strains43
3.8 Identification results for the 4 immuno-functional LAB isolates44
V Conclusions.45
VI Recommendation for Future Studies.47
REFERENCES48
APPENDICES (or APPENDIXES).56


LIST OF FIGURES
Fig.A1 Determination of IL-5+ and IL-6+ cells in the lamina propria of the small intestines of mice receiving L. casei administration. 58
Fig.1. Microscope photography of macrophage RAW264.7 under 100x and 200x magnification. .59
Fig.2. Selection of macrophage RAW264.7 cell concentration. 60
Fig.3. MTT assay of macrophage RAW264.7 serum starvation time. 61
Fig.4. Different time and dose response of LPS on macrophage. 62
Fig.5. The cytotoxicity of LAB. .63
Fig.6. Comparison of starvation and LPS-activation of RAW264.7 at different time. 64
Fig.7. Effect of low dose LPS on IFN-gamma production by RAW264.7 cells stimulated by LAB strains. 65
Fig.8. Effect of low dose LPS on IL-6 production by RAW264.7 cells stimulated by LAB strains. 66
Fig.9. Effect of low dose LPS on IL-12p40p70 production by RAW264.7 cells co-cultured with LAB strains. 67
Fig.10. Effect of low dose LPS in IL-6 production by RAW264.7 cells stimulated by thermal death LAB strains. .68
Fig.11. Effect of low dose LPS on IL-12p40p70 production by RAW264.7 cells stimulated by thermal death LAB strains. .69
Fig.12. Electrophoresis patterns of the PCR-amplified DNA products. 70
Fig.13. API��50 CHL results. 71

LIST OF TABLES
Table A1 Species-specific primers for PCR identification assay. 56
Table A2 Characterized probiotic strains (partial list). .57
Table 1 Comparison results of LAB adherence to cytokines down-regulation effect. 72
Table 2 Strain identification of 4 LAB isolates. 73

Periodical or Thesis


1.Alvarez-Olmos, M. I., M. M. Barousse, L. Rajan, B. J. Van Der Pol, D. Fortenberry, D. Orr, and P. L. Fidel, Jr. 2004. Vaginal lactobacilli in adolescents: presence and relationship to local and systemic immunity, and to bacterial vaginosis. Sex. Transm. Dis. 31: 393–400.

2.Antonio, M. A. D., L. K. Rabe, and S. L. Hillier. 2005. Colonization of the rectum by Lactobacillus species and decreased risk of bacterial vaginosis. J. Infect. Dis. 192: 394–398.

3.Antonio, M. A. D., S. E. Hawes, and S. L. Hillier. 1999. The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J. Infect. Dis. 180: 1950– 1956.

4.Bloksma N., E. de Heer, M. Van Dijk, and M. Willers. 1979. Adjuvanticity of Lactobacilli. I. Differential effects of viable and killed bacteria. Clin. Exp. Immunol. 37: 367-375.

5.Burch LH, IV. Yang, GS. Whitehead, FG. Chao, KG. Berman, and DA. Schwartz. 2006. The transcriptional response to lipopolysaccharide reveals a role for interferon-gamma in lung neutrophil recruitment. Am J. Physiol Lung Cell Mol Physiol. 291(4): L677-82.

6.Collins, J. K., G. Thornton, and G. O. Sullivan. 1998. Selection of probiotic strains for human applications. Int. Dairy J. 8: 487–490.

7.Cebra J.J. 1999. Influences of microbiota on intestinal immune system development. Am. J. Clin. Nutr. 69: 1046–1051.

8.Cheigh, H. S., and K. Y. Park. 1994. Biochemical, microbiological, and mutritional aspects of kimchi (Korean fermented vegetable products). Crit Rev Food Sci Nutr 34: 175-203.

9.Corthesy B, S. Boris, P. Isler, C. Grangette, and A. Mercenier. 2005. Oral immunization of mice with lactic acid bacteria producing Helicobacter pylori urease B subunit partially protects against challenge with Helicobacter felis. J. Infect. Dis. 192(8): 1441-9.

10.De Simone C., B. Bianchi-Salvadori, R. Negri, M. Ferrazzi, L. Baldinelli, and P. Vesely. 1986. The adjuvant effect of yogurt on production of gamma-interferon by ConA-stimulated human peripheral blood lymphocytes. Nutr. Reports. Int. 33: 419-443.

11.Falsen, E., C. Pascual, B. Sjo¨de´n, M. Ohle´n, and M. Collins. 1999. Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int. J. Syst. Bacteriol. 49: 217–221.

12.Famularo G., C. De Simone, V. Pandey, AR Sahu, and G. Minisola. 2005. Probiotic lactobacilli: an innovative tool to correct the malabsorption syndrome of vegetarians? Med Hypotheses 65(6): 1132-5.

13.Griffiths J. K., and J. S. Daly. 992. Two cases of endocarditis due to Lactobacillus species: antimicrobial susceptibility, review, and discussion of therapy. Clin. Infect. Dis. 15: 250–255.

14.Haller, D., S. Blum, C. Bode, W. P. Hammes, and E. J. Schiffrin. 2000. Activation of human peripheral blood mononuclear cells by nonpathogenic bacteria in vitro: evidence of NK cells as primary targets. Infect. Immun. 68: 752–759.

15.Hashimoto S., Y. Seyama, T. Yokokura, and M. Mutai. 1985. Cytotoxic factor production by Kupffer cells elicited with Lactobacillus casei and Corynebacterium parvum. Cancer Immunol. Immunother. 20: 117-121.

16.Hirohashi N., and DC. Morison. 1996. Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 production in vitro. Infect. Immune 64(3): 1011-5.

17.Ho PS, J. Kwang, and YK Lee. 2005. Intragastric administration of Lactobacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production. Vaccine. 23(11): 335-42.

18.Horn C. 1999. The potential of probiotics. Food Rev. 11: 27-29.

19.Isolauri E., Y. Sutas, , P. Kankaanpaa, , H. Arvilommi , and S. Salminen. 2001. Probiotics: effects on immunity. Am. J. Clin. Nutr. 73: 444–450.

20.Jackson TG, GRJ. Taylor, AM. Clohessy, et al. 1999. The effects of the daily intake of insulin on fasting lipid, insulin and glucose concentrations in middle-aged men and women. Br. Nutr. 89: 23-30.

21.Kaila M, E. Isolauri, E. Soppi, E. Virtanen, S. Laine, and H. Arvilommi. 1992. Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr. Res, 32: 141-144.

22.Kaila, M., E. Isolauri, E. Virtanen, and H. Arvilommi. 1992. Preponderance of IgM from blood lymphocytes in response to infantile rotavirus gastroenteritis. Gut 33: 639–642.

23.Kalliomaki M, S. Salminen, T. Poussa, H. Arvilommi, and E. Isolauri. 2003. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet. 361: 1869-1871.

24.Kato I., T. Yokokura, and M. Mutai. 1983. Macrophage activation by Lactobacillus casei in mice. Microbiol. Immunol. 27: 611-618.

25.Kato I., T. Yokokura, and M. Mutai. 1984. Augmentation of mouse natural killer cell activity by Lactobacillus casei and its surface antigens. Microbiol. Immunol. 28: 209-217.

26.Kay AB. , S. Ying, V. Varney et al. 1991. Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects. J. Exp Med 173: 775–778.

27.Kirjavainen PV, SJ Salminen, and E. Isolauri. 2003. Probiotic bacteria in the management of atopic disease: underscoring the importance of viability. J. Pediatr .Gastroenterol Nutr. 36: 223-227.

28.Klein, G., E. Zill, R. Schindler, and J. Louwers. 1998. Peritonitis associated with vancomycin-resistant Lactobacillus rhamnosus in a continuous ambulatory peritoneal dialysis patient: organism identification, antibiotic therapy, and case report. J. Clin. Microbiol. 36: 1781–1783.

29.Lee, J.-S., C. O. Chun, M. C. Jung and 8 other authors. 1997. Classification of isolates originating from Kimchi using carbon-source utilization patterns. J Microb Biotechnol 7: 68-74.

30.Link-Amster, H., F. Rochat, K. Y. Saudan, O. Mignot, and J. M. Aeschlimann. 1994. Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake. FEMS Immunol. Med. Microbiol. 10: 55–63.

31.Lin, W-H, B. Yu, C-K Lin, and W-Z Huang. 2007. Immune effect of heat-killed mutistrain of Lactobacillus acidopilus against Salmonella typhimurium invasion to mice. Journal of Applied Maicrobiology 102: 22-31.

32.Maldonado Galdeano C., and G. Perdigon. 2006. The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity. Clin Vaccine Immunol. 13(2): 219-226.

33.Marcel B. R. 2000. Prebiotics and probiotics : are they functional foods? Am J. Clin. Nutr. 71(suppl): 1682S-7S.

34.Martin, H. L., B. A. Richardson, P. M. Nyange, L. Lavreys, S. L. Hillier, B. Chohan, K. Mandaliya, J. O. Ndinya-Achola, J. Bwayo, and J. Kreiss. 1999. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J. Infect. Dis. 180: 1863–1868.

35.Matsuzaki, T. 1998. Immunomodulation by treatment with Lactobacillus casei strain Shirota. Int. J. Food Microbiol. 41: 133–140.

36.McGroarty, J. A. 1993. Probiotic use of lactobacilli in the human female urogenital tract. FEMS Immunol. Med. Microbiol. 6: 251–264.

37.Meydani, S. N. and W. K. Ha. 2000. Immunologic effects of yogurt. Am. J. Clin. Nutr. 71: 861–872.

38.Mheen, T. I. and T. W. Kwon. 1984. Effect of temperature and salt concentration on Kimchi fermentation. Kor J Food Sci Technol 16: 443-450.

39.Miettinen M., J. Vuopio-Varkila, and K. Varkila. 1996. Production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria. Infect. Immun. 64: 5403–5405.

40.Mosmann T. 1983. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Meth, 65: 55-63.

41.Mosmann TR, RL. Coffman. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev. Immunol 7: 145–173.

42.Nigatu, A. 2000. Evaluation of numerical analyses of RAPD and API 50 CH patterns to differentiate Lactobacillus plantarum, Lact. fermentum, Lact. rhamnosus, Lact. sake, Lact. parabuchneri, Lact. gallinarum, Lact. casei, Weissella minor and related taxa isolated from kocho and tef. Journal of Applied Microbiology 89: 969-978.

43.Ouwehand AC, S. Salminen, and E. Isolauri. 2002. Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek. 82: 279-89.

44.Perdigon G., M.E. de Macias, S. Alvarez, G. Oliver, and H. de Ruiz. 1988. Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei and Lactobacillus acidophilus. Immunology 63: 17–23.

45.Pouwels PH, RJ Leer, and WJ Boersma. 1996. The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens. J. Biotechnol. 44(1-3): 183-92.

46.Reid G, J. Jass, MT Sebulsky, and JK McCormick. 2003. Potential uses of probiotics in clinical practice. Clin. Microbiol Rev. 16: 658-72.

47.Revaz, V., and D. Nardelli-Haefliger. 2005. The importance of mucosal immunity in defense against epithelial cancers. Curr. Opin. Immunol. 17: 175-179.

48.Roberfroid M. B. 2000. Prebiotics and probiotics: are they functional foods? Am. J. Clin. Nutr, 71(suppl): 1682s-7s.

49.Roberta V., M. Gadina, M. Chiariello, E. H. Chen, L. F. Stancato, J. S. Gutkind, and J. J. O’Shea. 2000. Importance of the MKK6/p38 pathway for interleukin-12–induced STAT4 serine phosphorylation and transcriptional activity. Immunobiology 96: 1844-52.

50.Romagnani S. 1994. Lymphokine production by human T cells in disease states. Annu. Rev. Immunol. 12: 227–257.

51.Saito I., K. Sato, Y. Horikawa, B. Jin, H. Tonioka, and T. Watanabe. 1983. Enhanced humoral antibody production and delayed type hypersensitivity response in mice by Lactobacillus casei. Hiroshima J. Med. Sci. 32: 223-225.

52.Salminen S., C. Bouley, and MC Boutron-Ruault, et al. 1998. Functional food science and gastrointestinal physiology and function. Br. J. Nutr, 80(suppl): S147-71.

53.Sanders ME. 1993. Summary of the conclusions from a consensus panel of experts on health attributes on lactic cultures: significance to fluid milk products containing cultures. J. Diary Sci., 76: 1819-28.

54.Sanders ME. 2000. Considerations for use of probiotic bacteria to modulate human health. J Nutr. 130: 384S-390S.

55.Scheppler L., M. Vogel, AW Zuercher, M. Zuercher, JE Germond, SM Miescher, and BM Stadler. 2002. Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle. Vaccine. 20(23-24): 2913-2920.

56.Schiffrin E. J., F. Rochat, H. Link-Amster, J. M. Aeschlimann, and A. Donnet- Hughes. 1995. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci. 78: 491–497.

57.Sharpe M., R. Hill, and S. Lapage. 1973. Pathogenic lactobacilli. J. Med. Microbiol. 6: 281-286.

58.So, M. H. and Y. B. Kim. 1995. Identification of psychrophilic lactic acid bacteria isolated from Kimchi. Kor J Food Sci Technol 27: 495-505.

59.Takeda K., S. Akaqis Takahashi, A. Onishi, H. Hanada, and C. A. Pinkert. 2002. Mitochondrial activity in response to serum starvation in bovine (Bos Taurus) cell culture. Cloning Stem Cells 4(3): 223-229.

60.Teixeira P., H. Castro, C. Mohacsi-Farkas, and R. Kirby. 1997. Identification of sites of injury in Lactobacillus bulgaricus during heat stress. Journal of Applied Microbiology 83(8): 219-226.

61.Tsai C. L. and T. R. Yan. 2002. Isolation and identification of lactic acid bacteria from fermented vegetables and analysis for use as probiotics. Thesis of Tatung University in partial fulfillment of the requirement for the degree of Master of Science in Bioengineering.

62.Viljanen, M., E. Pohjavuori, T. Haahtela, R. Korpela, M. Kuitunen, A. Sarnesto, O. Vaarala, and E. Savilahti. 2005.. Induction of inflammation as a possible mechanism of probiotic effect in atopic eczema-dermatitis syndrome. J Allergy Clin Immunol 115(6): 1254-1259.

63.Walter, J., G.W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D.M. Loach, K. Munro, and T. Alatossava. 2000. J. Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers. Applied and Environmental Microbiology: 297-303.

64.Wang Chao. 2004. Goat fetal fibroblasts frozen and in vitro culture. Journal of northwest sci-tech university of agriculture and forestry [Natural Science Edition] vol. 32(4):55-58.

65.Wang K. S., D. A. Frank, and J. Ritz. 2000. Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4. Blood, Vol 95 No. 10 pp.: 3183-3190.

66.Wing E. J., Barczynski L.K., and Boehmer S. M. 1983. Effect of acute deprivation on immune function in mice. I. Macrophages. Immunology 48: 543-50.

67.Yasutake N., M. Ohwaki, T. Yokokura, and M. Mutai. 1984b. Comparison of antitumor activity of Lactobacillus casei with other bacterial immunopotentiators. Med. Microbiol. Immunol. 173: 113-125.

68.Yueng, P.S.M., R. Cano, P.S. Tong, and M.E. Sanders. 1999. Comparison of API, 16S rDNA sequencing and fatty acid analysis as methods to speciate commercial probiotic bacteria. J. Dairy Sci. 82:6, abstract #D22.

69.Zegers ND, E. Kluter, H. van Der Stap, E. van Dura, P. van Dalen, M. Shaw, and L. Baillie. 1999. Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax. J. Appl. Microbiol. 87(2): 309-14.

70.Zhu Y. M., D. D. Pan, S. X. Zou, and W. H. Chen. 2005. Effect of yogurt with different storage times on the immune system in initiatively immuned mice. Food Science Vol. 26: 229.


Books

1.Charles A., Jr Janeway, T. Paul, W. Mark, and J. S. Mark. 2003. Immuno Biology: the immune system in health and disease– 6th edition. Churchill Livingsone.

2.Holzapfel WH and BJB Wood (eds.). 1998. The genera of lactic acid bacteria, 1st edition. London Blackie Academic & Professional.

3.Playfair J.H.L. 1996. Immunology at a glance– 6th edition. Blackwell Science.

4.Stamer, J. R. 1983. Lactic acid fermentation of cabbage and cucumbers. InIn H. J. Rehm and G. Reed (ed.), Biotechnology. Verlag Chemie, Weinheim, Germany: 365-378.


Other

1.Chazan D. 2005. Korean dish 'may cure bird flu'. USDA Canning guides, Volume 7.

2.Fuller, R. 1992. History and development of probiotics. In: Probiotics: The Scientific Basis,. Chapman & Hall, New York, NY.:p1-8.