Bacillus coagulans之益生菌特性及其內生孢子在高壓處理食品應用之探討

The study on probiotic characteristics of Bacillus coagulans and application of its endospore in high pressure processing food

劉揚千(Liu Yang-Chien)

指導教授 ： 邱秋霞

國立屏東科技大學/國際學院/食品科學國際碩士學位學程/碩士(2019年)

https://doi.org/10.6346/NPUST201900099

摘要

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。

關鍵字

Bacillus coagulans ； 益生菌 ； 內生孢子 ； 高壓處理

並列摘要

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.

並列關鍵字

Bacillus coagulans ； probiotic ； endospore ； high pressure processing

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