由Kefir分離乳酸菌製作柳橙發酵飲品及其生理活性之探討

Abstract

本研究欲由kefir發酵乳中篩選出具有生理活性潛力且發酵柳橙果汁之能力良好，並發酵後能保持柳橙果汁抗氧化能力之菌株。研究中將活化後之kefir發酵乳利用MRS平板培養基培養，共挑選出生長較良好的32株菌株。根據乳酸菌的定義為革蘭氏陽性菌、桿菌或球菌、缺乏catelase活性等指標。測試其catalase活性、石蕊乳試驗以及格蘭氏染色鏡檢， 找出共12株乳酸菌菌株。以PCR-DGGE技術將重複菌株去除，可分出4群不同之乳酸菌株。利用16s rDNA片段鑑定法，可發現k1與Lactococcus lactis subsp. Lactis有98%的相似度。k2菌株與Lactobacillus plantarum，具有99%的相似度。而k3菌株和Lactobacillus kefiri，具有98%的相似程度。10 ℃下發酵48小時，所篩出菌株皆無法有效的發酵柳橙汁。於20 ℃及37 ℃下發酵48小時， k2、k3、都對柳橙汁具有良好的發酵能力。k2、k3菌株可於柳橙果汁中產生有機酸造成pH值下降，可以避免加工中腐敗菌的汙染。發酵過程中，k2、k3皆能於48小時的發酵取樣點下使柳橙汁提高其總酚類化合物含量。在20 ℃及37 ℃下，利用k2及k3菌株發酵柳橙汁，24小時以後，其發酵液之DPPH自由基清除能力較其餘三株菌株發酵柳橙汁較強。在37 ℃下發酵條件下，多數發酵樣品其總抗氧化活性皆稍低於未發酵之柳橙汁，且其經過發酵之柳橙發酵液之還原力並沒有提升的情況。k2、k3菌株於20 ℃及37 ℃下的發酵條件下，其有機酸生成特性，可賦予發酵飲品之良好風味。其安全性、加工合適性、機能性，皆可顯示k2、k3菌株具有成為具有良好發酵柳橙果汁能力的益生菌潛力。與林，2001之結果比較，可發現k2與k3菌株與B. longum、L. acidphilus、L. bulgaricus比較後，除了L. acidphilus之耐酸性較k2與k3菌株強外，其餘兩株之耐酸性皆較k2與k3菌株弱。k2與k3菌株會受到膽鹽的輕微抑制，但其還是具有膽鹽耐受能力，其耐受力等級皆為第2等級 (number of strains bile salt tolerant)，由上述可得知，由kefir發酵乳分離出之菌株k2及k3，對於柳橙具加工合適性，並且具有成為益生菌之潛力。

For this study, kefir fermented milk from screened with potential physiological activity and fermentation ability of a good orange juice, and fermentation can be maintained after the antioxidant capacity of orange juice the strains. Research will be activated after the use of fermented milk kefir flat MRS medium, were selected for better growth of 32 strains. According to the definition of lactic acid bacteria as Gram-positive bacteria, bacilli or cocci, such as the lack of activity indicators catelase. Tested for catalase activity, litmus milk test, as well as Gram-stain microscopy, to identify a total of 12 strains of lactic acid bacteria. To PCR-DGGE technology will remove duplication strains can separate 4 groups of different strains of lactic acid bacteria. Use of 16s rDNA fragment identification can be found k1 and Lactococcus lactis subsp. Lactis 98% of similarity. k2 strains and Lactobacillus plantarum, with 99% of similarity. K3 strain and the Lactobacillus kefiri, with 98% of the degree of similarity. 10 ℃ under a 48-hour fermentation, the strains were screened out of the fermentation of orange juice can not be effective. At 20 ℃ and 37 ℃ under a 48-hour fermentation, k2, k3, are fermented orange juice has a good capacity. k2, k3 strains can produce organic acids in orange juice resulted in decreased pH values, to avoid spoilage organisms in the processing of the pollution. Fermentation process, k2, k3 emerged within 48 hours of fermentation to make orange juice under the sampling point to increase its total content of phenolic compounds. At 20 ℃ and 37 ℃ using k2 and k3 orange juice fermentation, 24 hours later, the fermentation broth of DPPH free radical scavenging ability than the other three strains a stronger orange juice fermentation. Fermentation at 37 ℃ under the conditions of the majority of the fermentation samples of its total antioxidant activity are slightly lower than that of orange juice is not fermented, and the orange, after the fermentation broth of the reducing power and did not upgrade. k2, k3 strains in the 20 ℃ and 37 ℃ under the fermentation conditions, the organic acids generated features, will give a good flavor of fermented beverages. Their safety, processing suitability, functional, and can show that k2, k3 strains have become a good orange juice fermentation capacity of the potential of probiotics. And Lin, the results of 2001 compared to k2 and k3 can be found with the strains B. longum, L. acidphilus, L. bulgaricus comparison, in addition to acid resistance of L. acidphilus than k2 and k3 strong strains, the other two acid resistance of k2 and k3 are relatively weak strain. k2 and k3 strains will be a slight inhibition of bile salt, but it still has a salt tolerance, the tolerance levels are Level 2 (number of strains bile salt tolerant), from the above we can see that, by the fermentation of kefir Lactobacillus strains isolated from the k2 and k3, suitable for the processing of an orange, and has become a potential probiotic.