高壓處理 (HPP) 是一種非熱殺菌技術，藉由高壓使微生物之營養細胞失去活性。而Bacillus coagulans之內生孢子可忍受高溫及高壓，其可作為加工食品及飼料添加劑之益生菌。本研究的目的是探討B. coagulans BC1、BC3、BC5內生孢子之益生菌特徵 (耐酸性、耐膽鹽、酵素活性、疏水性及自凝集性)、其於高壓食品之應用以及產孢條件之優化。B. coagulans之內生孢子於不同pH (pH 2, 2.5, 3) 及不同濃度膽鹽 (0.3, 0.5, 1%) 之環境下24小時後測試其之存活率，B. coagulans BC1、BC3、BC5之內生孢子於pH 2之環境下靜置24小時後其孢子存活率超過50%; B. coagulans BC1、BC3、BC5之內生孢子於1%膽汁鹽之環境下靜置24小時後其孢子存活率高於75%。在酵素活性試驗中，三株B. coagulans皆具有活性較高之β-半乳糖苷酶、α-葡萄糖苷酶、α-半乳糖苷酶，且不具有β-葡萄醣醛酸酶 (具致癌性之酵素)。於高壓處理試驗中，Escherichia coli ATCC 8739、Lactobacillus plantarum 7-40、Salmonella enterica subsp. enterica BCRC 10747皆於600 MPa之環境下5分鐘後失去活性，而B. coagulans其內生孢子於600 MPa之環境下10分鐘後仍維持106 CFU/mL，其中B. coagulans BC1其內生孢子之存活率高達92%。於儲存試驗中，三株B. coagulans內生孢子添加於榛果可可醬經600 MPa環境下5分鐘處理後儲存於4℃環境下50天，三株B. coagulans內生孢子皆於4℃環境下50天後仍可維持105 CFU/mL以上。B. coagulans BC1之內生孢子較耐高壓，將其作為實驗菌株進行孢子產生之最適化，以三項變因子 (溫度、震盪程度及pH) 應用於反應曲面法 (RSM) 之Central Composite Design (CCD)，探討生產高孢子數之最適化條件，最適化生產內孢子條件為43.41℃、pH 6.78及124.41 rpm，於最適條件下B. coagulans BC1產孢量可達到107 CFU Log/mL以上。綜合上述，B. coagulans之內生孢子具耐酸性、耐膽鹽、穩定性及耐高壓之特性，而BC1其內生孢子耐高壓能力較佳，其於高壓食品中具有穩定性，且能被大量生產，因此其具有潛力成為高壓處理食品之益生菌。

High Pressure Processing (HPP) is a non-thermal food preservation technology which can inactivate the vegetative cell through the high pressure. However, endospores of Bacillus coagulans (spore forming probiotics) can tolerate high temperature and high pressure, and it is used to serving as probiotic in processing food and feed additives. The target of this study is exploring the probiotic characteristics (acid tolerance, bile salt tolerance, enzymatic activity, hydrophobicity, auto-aggregation) of B. coagulans BC1, BC3, BC5 endospores and the application of its endospores in high pressure processing foods and the optimization of spore production. B. coagulans endospores are tested at pH 2, 2.5, 3 and in 0.3, 0.5, 1% bile salt for 24 hours, and their survival rates will be calculated. For processes in pH 2 and 1% bile salt in 24 hours, the three strains of B. coagulans endospores have survived more than 50% in pH 2 for 24 hours, and their survival rates are over 75% in 1% bile salt for 24 hours. In enzymatic activity test, three B. coagulans have highly active β-galactosidase, α-glucosidase, α-galactosidase, and they are negative for β-glucuronidase activity which was known as a carcinogenic enzyme. In high pressure processing treatment, the vegetative cell of Escherichia coli ATCC 8739、Lactobacillus plantarum 7-40、Salmonella enterica subsp. enterica BCRC 10747 were inactivated totally at 600 MPa for 5 minutes. However, B. coagulans endospores can maintain 106 CFU/mL at 600 MPa for 10 minutes. In storage test, three B. coagulans spores in cocoa paste which is processed at 600 MPa for 10 minutes were stored at 4℃ for 50 days, their spore can maintain 105 CFU/mL at 4℃ for 50 days. B. coagulans BC1 which can produce the spore with the higher tolerance for high pressure would be selected as the experimental strain to do process of spore yield. The three factor (temperature, pH, agitation) were applied on central composite design (CCD) of response surface methodology (RSM) to explore the optimized condition of sporulation. The results were shown that the best condition of sporulation of B. coagulans BC1 in BC broth was 43.41℃ and pH 6.78 and 124.41 rpm, and it could produce above 107 CFU/mL. Above all, B. coagulans spores had the tolerance to acid, bile salt, pressure, and B. coagulans BC1 spores had best pressure tolerance in three strains. It was stable in the HPP food, and it could be mass production. Therefore, B. coagulans BC1 could be potential probiotic food additives in HPP-treatment food product.

莫博欽。2009。凝孢乳酸菌最適化培養條件探討。嘉南藥理科技大學生物科技研究所碩士論文。
Abdhul, K., M. Ganesha, S. Shanmughapriya, S. Vanithamani, M. Kanagavel, K. Anbarasu, and K. Natarajaseenivasan. 2015. Bacteriocinogenic potential of a probiotic strain B. coagulans [BDU3] from Ngari. International journal of biological macromolecules 79: 800-806.
Abriouel, H., C. M. A. P. Franz, N. B. Omar1 and A. Galve. 2011. Diversityand applications of B. bacteriocins. Federation of european microbiological societies microbiology reviews 35: 201-232.
Anisha, G. S. 2017. 16-α-Galactosidases. In: Current Developments in Biotechnology and bioengineering. Pandey, A., S. Negi, and C. R. Soccol (eds.), Elsevier press, 369-394.
Arora, S., R. Rani, and S. Ghosh. 2018. Bioreactors in solid state fermentation technology: Design, applications and engineering aspects. Journal of biotechnology 269: 16-34.
Batalha, I. L., P. Keb, E. Tejeda-Montes, S. Uddin, C. F. Walle, and G. Christie. 2017. Dipicolinic acid as a novel spore-inspired excipient for antibody formulation. International journal of pharmaceutics 526: 332-338.
Binns, N. 2013. Probiotics and prebiotics: mechanisms of actin. In: probiotics, prebiotics and the gut microbiota. Retrieved on Jun. 16, 2017, from the World Wide Web: https://reurl.cc/dVgmD.
Black, E. P., P. Setlow, A. D. Hocking, C. M. Stewart, A. L. Kelly, and D. G. Hoover. 2007. Response of Spores to High-Pressure Processing. Comprehensive reviews in food science and food safety 6: 104-119.
Casula, G., and S. M. Cutting. 2002. Bacillus probiotics: spore germination in the gastrointestinal tract. Applied and environmental microbiology 68: 2344-2352.
Cavazzoni, V., and A. Adami. 1993. Biomass production, preservation and characteristic of a strain of Bacillus coagulans usable as probiotic. Microbiologie-aliments-nutrition 11: 93-100.
Chanalia, P., D. Gandhi, P. Attri, and S. Dhanda. 2018. Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis. Bioorganic chemistry 77: 176-189.
Cregenzán-Alberti, O., C. Arroyo, A. Dorozko, P. Whyte, and J. G. Lyng. 2017. Thermal characterization of Bacillus subtilis endospores and a comparative study of their resistance to high temperature pulsed electric fields (HTPEF) and thermal-only treatments. Food control 73: 1490-1498.
Cruywagen, C. W., I. Jordan, and Venter L. 1995. Effect of Lactobacillus acidophilus supplementation of milk replacer on preweaning performance of calves. J. Dairy science 79: 483-486.
Daelman, J., A. Vermeulen, T. Willemyns, R. Ongenaert, L. Jacxsens, M. Uyttendaele, and F. Devlieghere. 2013. Growth/no growth models for heat-treated psychrotrophic Bacillus cereus spores under cold storage. International journal of food microbiology 161: 7-15.
Das, S., S. Kharkwal, S. K. Pandey, and R. Sen. 2010. Multi-objective process optimization and integration for the sequential and increased production of biomass, lipase and endospores of a probiotic bacterium. Biochemical engineering journal 50: 77-81.
Elshaghabee, F. M. F., N. Rokana, R. D. Gulhane, C. Sharma, and H. Panwar. 2017. Bacillus as potential probiotics: status, concerns, and future perspectives. Frontiers in microbiology 1490: 1-15.
Evelyn, and F. V. M. Silva. 2016. Modeling the inactivation of psychotropic Bacillus cereus spores in beef slurry by 600 MPa HPP combined with 38-70℃: Comparing with thermal processing and estimating the energy requirements. Food and bioproducts processing 99: 179-187.
Fan, L., F. Hou, A. I. Muhammad, L.V. Ruiling, R. B. Watharkar, M. Guo, T. Ding, and D. Liu. 2018. Synergistic inactivation and mechanism of thermal and ultrasound treatments against Bacillus subtilis spores. Food research international 116: 1094-1102.
Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). 2001. Health and nutritional properties and guidelines for evaluation. Retrieved on Nov. 5, 2018, from the World Wide Web: https://reurl.cc/1g7Vm.
Fijan, S. 2014. Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature. International journal of environmental research and public health 11: 4745-4767.
Fuller, R. 1989. Probiotics in man and animals. Journal of applied bacteriology 66: 365-378.
Fuller, R. 2004. What is a probiotic? Biologist 51: 232.
Gauvry, E., A. Mathot, I. Leguérinel, O. Couvert, F. Postollec, V. Broussolle, and L. Coroller. 2016. Knowledge of the physiology of spore-forming bacteria can explain the origin of spores in the food environment. Research in microbiology 168: 369-378.
Giffel, M.C. T., R. R. Beumer, P. E. Granum, and F. M. Rombouts. 1997. Isolation and characterization of Bacillus cereus from pasteurized milk in household refrigerators in the Netherlands. International journal of food microbiology 34: 307-318.
Glaser, R., and J. Venus. 2018. Co-fermentation of the main sugar types from a beechwood organosolv hydrolysate by several strains of Bacillus coagulans results in effective lactic acid production. Biotechnology reports 18: 245.
Grand View Research, Inc. 2016. Probiotics market analysis by application (probiotic foods & beverages (dairy products, non-dairy products, cereals, baked food, fermented meat products, dry food), probiotic dietary supplements (food supplements, nutritional supplements, specialty nutrients, infant formula), animal feed probiotics), by end-use (human probiotics, animal probiotics) and segment forecast to 2024. Retrieved on Jun. 16, 2017, from the World Wide Web: https://reurl.cc/EzLqR.
Haberbeck L. U., C. A. S. Riehl, B. C. M. Salomão, and G. M. F. Aragão. 2012. Bacillus coagulans spore inactivation through the application of oregano essential oil and heat. Lebensmittel-wissenschaft and technologie-food science and technology 46: 267-273.
Hamilton-Miller, J. M. T., G. R. Gibson, and W. Bruck. 2003. Some insights into the derivation and early uses of the word ‘probiotic’ British journal of nutrition 90: 845.
Honda, H., G. R. Gibson, S. Farmer, D. Keller, and A. L. McCartney. 2011. Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota. Anaerobe 17: 36-42.
Hu, Y., W Chen, C Liu, P. K. Ouyang, and W. Yang 2014. Analysis of Physiological and Biochemical Characteristics of Bacillus. In: Production and application of Bacillus probiotics. Y. Hu (eds.), Beijing chemical industry publishing press, 35-39.
Huang, H., H. Lung, B. B. Yang, and C. Wang. 2014. Responses of microorganisms to high hydrostatic pressure processing. Food control 40: 250-259.
Huang, H., S. Wu, J. Lu, Y. Shyu, and C. Wang. 2017. Current status and future trends of high-pressure processing in food industry. Food control 72: 1-8.
Hyronimus, B., C. L. Marrec, and M. C. 1998. Urdaci Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4. Journal of applied microbiology 85: 42-50.
Hyronimus, B., C. L. Marrec, A. H. Sassi, and A. Deschamps 2000. Acid and bile tolerance of spore-forming lactic acid bacteria. International journal of food microbiology 61:193-197.
Iannitti, T., and B. Palmieri. 2010. Therapeutical use of probiotic formulations in clinical practice. Clinical nutrition 29: 701-725.
Iqbal, M. Z., M. I. Qadir, T. Hussain, K. H. Janbaz, Y. H. Khan, and B. A. Pak. 2014. Probiotics and their beneficial effects against various diseases. Journal of pharmaceutical sciences 27: 405-415.
Isolauri, E., S. Rautava, and M. Kalliomaki. 2002. Role of probiotics in food hypersensitivity. Current opinion in allergy and clinical immunology 2: 263-271.
Jafari, M., A. M. Mortazavian, H. Hosseini, F. Safaei, A. M. Khaneghah, and A. S. Sant'Ana. 2017. Probiotic Bacillus: Fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores. Food research international 95: 46-51.
Jao, C., S. Huang, S. Wu, and K. Hsu. 2011. The study on SFLAB GanedenBC30 viability on baking products during storage. Food science 1: 1601-1609.
Kaur, I. P., K. Chopra, and A. Saini. 2002. Probiotics: potential pharmaceutical applications. European Journal of Pharmaceutical Sciences 15:1-9.
Kechagia, M., D. Basoulis, S. Konstantopoulou, D. Dimitriadi, K. Gyftopoulou, N. Skarmoutsou, and E. M. Fakiri. 2013. Health Benefits of probiotics: A Review. Retrieved on Nov. 5, 2018, from the World Wide Web: http://dx.doi.org/10.5402/2013/481651.
Keller, D., S. Farmer, A. McCartney, and G. Gibson. 2010. Bacillus coagulans as a probiotic. Food science and technology bulletin: functional foods 7: 103-109.
Khurana M., and M. V. Karwe. 2009. Numerical Prediction of Temperature Distribution and Measurement of Temperature in a High Hydrostatic Pressure Food Processor. Food bioprocess technol 2: 279-290.
Kmet, V., H. J., Flint and R. J. Wallace. 1993. Probiotics and manipulation of rumen development and function. Archives of animal nutrition 44: 1-10.
Kovac, K., M. Diez-Valcarce, M. Hernandez, P. Raspor and D. Rodríguez-Lázaro. 2010. High hydrostatic pressure as emergent technology for the elimination of foodborne viruses. Trends in food science and technology 21: 558-568.
Laughon, B. E., S. A. Syed, and W. J. Loesche. 1982. API ZYM System for Identification of Bacteroides spp., Capnocytophaga spp., and Spirochetes of oral origin. Journal of clinical microbiology 15: 97-102.
Lee, S., J. Lee, Y. Jin, J. Jeong, Y. H. Chang, Y. Lee, Y. Jeong, and M. Kim, 2017. Probiotic characteristics of Bacillus strains isolated from Korean traditional soy sauce. LWT-food science and technology 79: 518-524.
Leroy, F., G. Falony, and L. Vuyst. 2008. Latest Developments in Probiotics. In: Meat biotechnology, F. Toldra (eds.), Brussels, Belgium: Springer, 220.
Lilley, D. M., and R. H. Stillwell. 1965. Probiotics: growth promoting factors produced by microorganisms. Science 147: 747-748.
Lucas, R., M. J. Grande, H. Abriouel, M. Maqueda, N. B. Omar, E. Valdivia, M. Martínez-Cañamero, and A. Gálvez. 2006. Application of the broad-spectrum bacteriocin enterocin AS-48 to inhibit Bacillus coagulans in canned fruit and vegetable foods. Food and chemical toxicology 44: 1774-1781.
Ministry of Health and Welfare (MOHW). 2001. Notice of the ministry of health on printing and approving the regulations for the health foods of fungi and probiotics (衛生部關於印發真菌類和益生菌類保健食品評審規定的通知).Retrieved on Oct. 31, 2018, from the World Wide Web: https://reurl.cc/Dv3kj.
Naidu, A. S., W. R. Bidlack, and R. A. Clemens. 1999. Probiotic spectra of lactic acid bacteria (LAB). Critical reviews in food science and nutrition 39: 13-126.
Nakamura, L. K., Blumenstock I., and D. Claus. 1988. Taxonomic study of Bacillus coagulans Hammer 1915 with a proposal for Bacillus srnithii sp. Parker nov. International journal of systematic bacteriology 38:1.
Parker, R. B. 1974. Probiotics: the other half of the antibiotic story. Animal nutrition and health 29: 4-8.
Patterson, M. F. 2005. Microbiology of pressure-treated foods. Journal of applied microbiology 98: 1400-1409.
Pereira, G. V. M., B. O. Coelho1, A. I. M. Júnior, V. Thomaz-Soccol, and C. R. Soccol. 2018. How to select a probiotic? A review and update of methods and criteria. Biotechnology advances 36: 2060-2076.
Prado, F. C., J. L. Parada, A. Pandey, and C. R. Soccol. 2008. Trends in non-dairy probiotic beverages. Food research international 41:111-123.
Rajoka, M. S. R., H. M. Mehwish, M. Siddiq, Z. Haobin, J. Zhu, L. Yan, D. Shao, X. Xu, and J. Shi. 2017. Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. LWT-Food science and technology 84: 271-280.
Ray, B. 2006. Stress adaptation. In: Fundamental food microbiology, B. Ray (eds.), The United States: CRC press, 105-111.
Roselli, M., A. Finamore, M. Britti, P. Bosi, I. Oswald, and E. Mengheri. 2005. Alternatives to in-feed antibiotics in pig production: evaluation of probiotics, zinc oxide or organic acids as protective agents for the intestinal mucosa: a comparison of in vitro and in vivo results. Animal research 54: 203-218.
Sadegh, A. 2016. In vitro assessment of some probiotic properties of Lactobacillus fermentum isolated from pickled garlic. Journal of food quality and hazards control 3: 67-72.
Schaafsma, G. 1996. State-of-the-art concerning probiotic strains in milk products. The international dairy federation-reports and newsletters 5: 23-24.
Schillinger, U., R. Geisen, and W. H. Holzapfel. 1996. Potential of antagonistic microorganisms and bacteriocins for the biological preservation of foods. Trends in food science and technology 71: 58-64.
Sella, S., L. Vandenberghe, and C. Soccol. 2014. Life cycle and spore resistance of spore-forming Bacillus atrophaeus. Microbiological research 169: 931-939.
Sen, R., and K. Babu. 2005. Modeling and optimization of the process conditions for biomass production and sporulation of a probiotic culture. Process biochemistry 40: 2531-2538.
Setlow, P. 2018. Spore resistance properties. P, Setlow. (eds.), The United States: Society for microbiology press, 1-14.
Shehata, M. G., S. A. E. Sohaimy, M. A. El-Sahn, and M. M. Youssef. 2016. Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Annals of agricultural science 61: 65-75.
Simone, C. 2018. The unregulated probiotic market. Clinical gastroenterology and hepatology 17: 809-817.
Smelt, J. P. P. M. 1998. Recent advances in the microbiology of high pressure processing. Trends in food science and technology 9: 152-158.
Song, D., S. Ibrahim and S. Hayek. 2012. Recent Application of probiotics in food and agricultural science. In: Probiotics, E. C. Rigobelo (eds.), InTech, 3-4.
Soni, A., I. Oey, P. Silcock, and P. Bremer. 2016. Bacillus Spores in the food industry: A review on resistance and response to novel inactivation technologies. Institute of food technologist 15: 1139-1148.
Spinosa, M. R., T. Braccini, E. Ricca, M. D. Felice, L. Morelli, G. Pozzi, M. R. Oggionia. 2000. On the fate of ingested Bacillus spores. Research in microbiology 151: 361-368.
Taiwan food and drug administration (TFDA). 2013. Methods of test for food microorganisms test of standard plate count (Aerobic Plate Count) of amendment to the announcement of No. 1021950329 of the Ministry of Food. Retrieved on Jun. 18, 2019, from the World Wide Web: https://reurl.cc/V1rW6.
Tallapragada, P., B. Rayavarapu, P. P. Rao, N. N. Ranganath, P. P. Veerabhadrappa. 2018. Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification. Journal of genetic engineering and biotechnology 16: 357-362.
Todd, R. K., 2001. The probiotic concept. In: Food Microbiology, P. D. Michael, R. B. Larry, and J. M. Thomas (eds.), The United States of America: American Society for Microbiology Press, 128-139.
United State Food and Drug Administration (USFDA). 2015. Determination of the generally recognized as safe (GRAS) status of Bacillus coagulans unique is-2 for use in food. Retrieved on Apr. 9, 2018, from the World Wide Web: https://reurl.cc/MZN5p.
United States Department of Agriculture. (2012). High pressure processing (HPP) and inspection program personnel (IPP) verification responsibilities. Retrieved on Apr. 14, 2019, from the World Wide Web: https://reurl.cc/NxDvx.
United States Patent Certificate (USPC), Inc. 2013. Bacillus coagulans GBI-30, 6086. Retrieved on Oct. 30, 2018, from the World Wide Web: https://reurl.cc/Gdd6A.
Vecchi, E. D., and L. Drago. 2006. Lactobacillus sporogenes or Bacillus coagulans: misidentification or mislabelling? International journal of probiotics and prebiotics 1: 3-10.
Vercammen, A., B. Vivijs, I. Lurquin, and C. W. Michiels. 2012. Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce. International journal of food microbiology 152: 162-167.
Vinderola, G., M. Gueimonde, C. Gomez-Gallego, L. Delfederico, and S. Salminen. 2017. Correlation between in vitro and in vivo assays in selection of probiotics from traditional species of bacteria. Trends in food science and technology 68: 83-90.
Wang, Y., W. Cao, J. Luo, and Y. Wan. 2018. Exploring the potential of lactic acid production from lignocellulosic hydrolysates with various ratios of hexose versus pentose by Bacillus coagulans IPE22. Bioresource technology 261: 342-349.
Watson, R., and V. Preedy. 2016. Probiotics, prebiotics, and synbiotics. Elsevier inc. 1: 75-82.
Wolken, W. A. M., J. Tramper, and M. J. Werf. 2003. What can spores do for us? Trends in biotechnology 21: 338-345.
Zeng, L., H. Ding, X. Hu, G. Zhanga, and D. Gong. 2019. Galangin inhibits α-glucosidase activity and formation of non-enzymatic glycation products. Food chemistry 271: 70-79.
Zhang, X., B. X. Hu, H. Ren, and J. Zhang. 2018. Composition and functional diversity of microbial community across a mangrove-inhabited mudflat as revealed by 16S rDNA gene sequences. Science of the total environment 633: 518-528.
Zimmermann, M., D. Schaffner, and G. Aragão. 2013. Modeling the inactivation kinetics of Bacillus coagulans spores in tomato pulp from the combined effect of high pressure and moderate temperature. Lebensmittel-wissenschaft and technologie-food science and technology 53: 107-112.
Zion Market Research, Inc. 2018. Global Probiotics Market Will Reach USD 65.87 Billion by 2024. Retrieved on Nov. 20, 2018, from the World Wide Web: https://reurl.cc/1o4nW.