乳酸菌廣佈於自然界，且長久以來被利用於發酵食品與健康保健食品的製造。為得到有益乳酸菌株並進一步進行多元化應用，本研究特進行乳酸菌株分離、鑑定與應用分析試驗。本研究從市售鮮乳中篩選出八株具凝乳反應，革蘭氏染色呈陽性，以及catalase試驗為陰性之類乳酸菌株。將八株類乳酸菌株再以乳酸菌快速鑑定套組（API 50CHL）進行菌種鑑定，分別得到與Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis及Pediococcus pentosaceus等乳酸菌株皆高於93% 鑑定相似度的結果。
為進一步確認所篩選類乳酸菌株之種源性，本研究挑選四株類乳酸菌（L1、Lac1、M43及M51）進行16S rDNA序列分析。所得到類乳酸菌株之16S rDNA序列經與GenBank資料庫中之核酸序列進行分析比對，可確定此四株類乳酸菌株分別為Lactobacillus sp. L1 , Pediococcus acidilactici Lac1 , Enterococcus faecium M43及Enterococcus sp. M51。
另外，為進行所篩選出之乳酸菌株做為保健性菌株之可行性評估，本研究分別進行四株乳酸菌之耐酸性、耐膽塩性、抗生素感受性試驗及抑制病原菌之能力等評估。試驗結果為四株乳酸菌株對酸及膽塩皆具耐受性，抗生素感受性試驗結果為對kanamycin及gentamcin皆具抗性，至於四株乳酸菌株之抑制病原菌能力則不顯著。 The lactic acid bacteria are widely distributed in the nature, and have been greatly used for a long time since they have useful characteristics. In recent years, the lactic acid bacteria were also applied in the development of healthy food. In this study, eight lactic acid bacteria-like strains were isolated from the fresh milk, and later it has been proved that these strains were lactic acid bacteria-like through examination. These bacteria were all positive in skim milk curdling, the same as in Gram stain but negative in catalase reaction. These eight lactic acid bacteria-like strains were further identified with API 50 CHL test kit. This experimentation tells us these strains have high similarities, which are all with Lactobacillus paracasei subsp. paracasei , Lactobacillus brevis and Pediococcus pentosaceus.
In order to identify the phylogeny hierarchy of the isolated bacteria, the 16S rRNA genes were identified in four strains. The amplification of the 16S rRNA gene were subjected by PCR tests with universal primers, and the PCR products were subjected to 16S rDNA sequencing analysis. The 16S rDNA sequences from the four strains were compared with the reference strains which were held in GenBank. The above show high similarities and the four isolates were Lactobacillus sp. L1, Pediococcus acidilactici Lac1, Enterococcus faecium M43 and Enterococcus sp. M51.
Moreover, further analyses were done to identify if the feasibility of the four lactic acid bacteria can be probiotics strains. The strain capabilities of tolerance to gastric juice and resistance to bile salt were done. The results showed that the four lactic acid bacteria can be both tolerant to gastric juice and resistant to bile salts. The antibiotic susceptibility and inhibition to pathogenic bacteria were tested. These four lactic acid bacteria were resistant to kanamycin and gentamicin, and no apparent inhibitions to pathogenic bacteria were found.
