冷凍乾燥對乳酸菌存活率與其主要生理特性之影響

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Title:	冷凍乾燥對乳酸菌存活率與其主要生理特性之影響
Other Titles:	Effects of Freeze-Drying on the Survival and Major Physiological Characteristics of Lactic Acid Bacteria
Authors:	林世偉 Lin, Shih-Wei
Contributors:	施宗雄 Shih, Chung-Hsung 東海大學畜產與生物科技學系
Keywords:	乳酸菌;冷凍乾燥;保護劑;存活率 lactic acid bacteria;freeze-drying;protectant;survival
Date:	2003
Issue Date:	2011-05-19T06:22:14Z (UTC)
Abstract:	本實驗目的在探討保護劑進行冷凍乾燥製作成凍乾菌?，藉由其生理及生化特性之測定，以了解冷凍乾燥添加不同保護劑對乳酸菌之影響，並探究自製凍乾乳酸菌?之可行性，其結果期能提供日後相關研究與利用之參考。試驗一為冷凍乾燥對乳酸菌生理生化特性之影響，其試驗結果如下：1.添加10％ NFSM為保護劑之L. acidophilus冷凍乾燥後菌數明顯低於乾燥前（p<0.05），其餘各組則無明顯的變化（p>0.05）。2.冷凍及乾燥皆有使菌體對NaCl、lysozyme及oxgall等物質敏感性增加的趨勢，其中以單一L. bulgaricus組及S. thermophilus＋L. bulgaricus混合組有較佳的耐受性表現；添加10％ NFSM+5％ glycrol為保護劑組，則有提高S. thermophilus及L. acidophilus的耐受性。3.冷凍乾燥菌體經培養於含2％乳糖液態培養基中，其中以添加保護劑之B. longum及L. acidophilus＋B. longum經冷凍乾燥後β-galactosidase活性測定有明顯比冷凍乾燥前增加（p<0.05）。4.冷凍乾燥前後菌?試製醱酵乳完成醱酵時間之變化，以添加10％ NFSM為保護劑之S. thermophilus、L. acidophilus及B. longum與添加10％ NFSM+5％ glycerol為保護劑之B. longum完成醱酵時間顯著比對照組長（p<0.05）。5.冷凍乾燥前後菌?試製醱酵乳菌數之變化，無論添加何種保護劑與何種菌?組乳酸菌數皆可達到108 CFU/ml以上，符合CNS 3058凝態醱酵乳菌數（>107 CFU/ml）之標準。6.冷凍乾燥前後菌?試製醱酵乳之β-galactosidase活性，除了添加保護劑之B. longum醱酵乳中β-galactosidase活性則顯著比對照組低（p<0.05）外，其餘各組則無明顯的變化（p>0.05）。7.冷凍乾燥菌體細胞溶出物試驗，有添加保護劑組菌體細胞溶出物中並無發現任何蛋白質存在，但菌體直接進行冷凍乾燥後則會出現一分子量約為46 kDa的蛋白質存在。試驗二為冷凍乾燥菌?貯存期間之變化，將冷凍乾燥菌?貯存於25℃下四個月，每月採樣測定其乳酸菌生理與生化特性，其試驗結果如下：1.冷凍乾燥菌?各組菌數均有隨著貯存時間增長而有減少之趨勢。但其中以L. bulgaricus、S. thermophilus＋L. bulgaricus及L. acidophilus＋B. longum混合組有較高的存活菌數，而添加10% NFSM+5% glycerol為保護劑則可提高S. thermophilus及L. acidophilus的存活菌數。2.冷凍乾燥菌?經NaCl、lysozyme及oxgall試驗發現，各組菌體均隨時間增長而有敏感性增加的現象，顯示乾燥菌體有細胞壁穩定性及細胞膜通透性的改變。3.冷凍乾燥菌體貯存期間細胞溶出物試驗，添加保護劑之冷凍乾燥菌?貯存四個月後細胞溶出物各組皆可發現一分子量約為33 kDa的蛋白質存在，這可能因受到貯存環境的影響，使得菌體細胞壁上蛋白質結構變得不穩定，因此容易被解離出來。 Abstract The aims of this study were to research the effects of different protectants for lactic acid bacteria through the treatment of their physical and chemical characteristics and to find out the possibility of self-made freeze-dried starters on the basis of the following experiments. The purpose of the first part experiments was to study the effects of freeze-drying for lactic acid bacteria. Single strain and mixed strains bacteria groups were examined the differences of the physiological and biochemical properties before and after freeze drying. The results of the first part study were as follows: (1) The viable counts of L. acidophilus after freeze-drying was lower than that before freeze-drying (p<0.05) and the others were no differences (p>0.05). (2) In the test of NaCl, lysozyme and oxgall, the results represented that freezing and drying had significantly increased the bacteria sensitivity to these materials. The bacteria strains such as L. bulgaricus and S. thermophilus+L. bulgarius presented better performances in tolerance. Furthermore, adding 10% NFSM+5% glycerol as protectants increased the tolerance of S. thermophilus and L. acidophilus. (3) The freeze-dried B. longum and L. acidophilus+B. longum with protectants were incubated in MRS broth with 2% lactose. After freeze-drying, the activity of β-galactosidase increased significantly (p<0.05). (4) The changes of freeze-dried bacteria in the finish-up time of freeze-dried S. thermophilus, L. acidophilus, B. longum with 10 % NFSM and B. longum with 10% NFSM+5% glycerol as protectants were significantly longer than the control (p<0.05). (5) Regards to the changes of freeze-dried bacteria on the viable counts of fermented skim milk before and after freeze-drying, the lactic acid bacteria population reached more than 108 CFU/ml, and achieved the criterion of CNS 3058 (>107 CFU/ml). (6) According to the study of the changes of freeze-dried bacteria in the β-galactosidase activity of fermented skim milk before and after freeze-drying, we understood the activity of β-galactosidase of freeze-dried B. longum with protectants were significantly lower than the control (p<0.05) and the others were no differences (p>0.05). (7) The results of SDS-PAGE for the cell extracted from normal and dried cells with protectants indicated that no protein existed. However, the molecular weight of protein in freeze-dried cells without protectants was 46 kDa. The second part of the experiment was to detect the changes of freeze-dried bacteria during the storage under 25℃ for four months and to analyze the physiological and biochemical properties of all samples every month during storage.The results of the second study were indicated that (1) During the storage of freeze-dried bacteria, the viable counts declined with time. The survival rate of freeze-dried L. bulgaricus, S. thermophilus+L. bulgaricus and L. acidophilus+B. longum represented better than the others. Furthermore, adding 10% NFSM+5% glycerol as protectants increased the survival rate of S. thermophilus and L. acidophilus. (2) According to the results of the freeze-dried bacteria treated with NaCl, lysozyme and oxgall during the storage, the bacteria sensitivity to those chemicals was increased with time. This suggested that the storage would affect the stability of cell wall and the permeability of cell membrane. (3) The bacteria cells stored with protectants for four months were extracted for SDS-PAGE experiment. The results indicated that the 33 kDa proteins existed which suggested that the freeze-dried bacteria might be affected by the storage environment. The proteins of bacteria cell wall was unstable, therefore the proteins were extracted easily.
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