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Molecular Identification of Lactic Acid Bacteria and Characterization as well as Functional Evaluation
lactic acid bacteria
28-day feeding toxicity study
polymerase chain reaction
|摘要:||乳酸菌廣泛用於食品和飼料，其分子檢測和機能性的研究廣受探討。本研究主要分為兩大部分，第一部分為雙歧桿菌分子鑑定與分類；第二部分為研究室篩選之乳酸菌進行特性與機能性評估。在分子鑑定方面，針對16S rRNA和16S-23S ITS基因序列設計特異性引子組，可以分別檢測Bifidobacterium angulatum、B. animalis、B. breve、B. dentium、B. minimum、B. adolescentis和B. asteroides。使用國外文獻之引子組Lm3/P0，從336株分離自嬰兒、成人糞便和發酵產品之乳酸菌，檢測出44株Bifidobacterium菌株，並發現3株為B. adolescentis，5株為B. angulatum，1株B. animalis和2株B. minimum。
在抑制幽門桿菌之機能性評估方面，本研究為從嬰兒糞便篩選出抗幽門桿菌之乳酸菌Enterococcus faecium TM39。其具有吸附人類上皮細胞Int 407、結腸上皮細胞Caco-2、人類子宮頸癌上皮細胞HeLa和人類胃癌細胞TSGH 9201之能力，以及具耐酸耐膽鹽能力。TM39上清液在體外試驗可明顯抑制幽門桿菌及其尿素酶活性，其抑制物質除乳酸外，還包含一些目前還未知的物質。TM39乳酸菌菌體與上清液可降低幽門桿菌吸附TSGH 9201細胞。在TM39毒性試驗方面，體外試驗證明TM39菌株不具vancomycin抗性和對人類胃癌細胞TSGH 9201及人類腸道上皮細胞Int-407不具侵入性。分別連續28天餵食TM39不同濃度1 × 1012, 5 × 1011和2 × 1010 CFU/ kg，結果證實大鼠的行為、生長、飲食、飲水、血液分析、臨床血清生化分析、器官重量和組織病理觀察並無顯著影響。因此，TM39菌株可安全用於抗幽門桿菌之用途上。
在抑制沙門氏菌侵入之機能性方面，豬腸分離之L. acidophilus LA5和雞腸分離之L. fermentum LF33，對Int-407、Caco-2和BALB/ c小鼠腸道上皮細胞皆具吸附能力。植物來源乳酸菌皆不會吸附Int-407和Caco-2細胞，其中L. plantarum BCRC10069對BALB/ c小鼠之腸道上皮細胞具有吸附能力。所以，在抑制沙門氏菌侵入Int-407、小鼠肝和脾之感染能力時，乳酸菌LA5、LF33和LP69皆可抑制。在LA5毒性試驗方面，連續28天餵食Wistar大鼠三組不同濃度乳酸菌，分別為2.1×1011、1.05×1011和4.2×109 CFU/ kg大鼠體重。實驗結果證明Wistar大鼠攝取大量乳酸菌L. acidophilus LA5也不會有任何顯著的毒性產生。|
Probiotic products from lactic acid bacteria (LAB) are widely used as human food and animal feed supplements. Molecular identification and potential evaluation are widely used methods. Two major topics were included in this study, one was the development of identification methods for Bifidobacterium spp., and the other was evaluation of the potential for lactic acid bacteria strains screened in this study. For molecular identification, we developed specific polymerase chain reaction (PCR) primers from 16S rRNA or 16S-23S ITS, and used of them for the identification of lactic acid bacteria species include Bifidobacterium angulatum, B. animalis, B. breve, B. dentium, B. minimum, B. adolescentis and B. asteroides. 44 Bifidobacterium strains including 3 B. adolescentis, 5 B. angulatum, 1 B. animalis and 2 B. minimum strains were identified from 336 LAB strains isolated from infant, adult stool and ferment product using Lm3/P0 and other specific primers. On evaluation of the potential of antagonist effect against Helicobacter pylori, a strain of Enterococcus faecium TM39 from infant feces was screened for anti-H. pylori use. The screening process was based on the capability of LAB strains to adhere to the human intestinal epithelial cell (Int 407), colonial enterocyte-like Caco-2 cell, human cervical epithelioid carcinoma cell (HeLa), and human gastric carcinoma cell (TSGH 9201), and the capability to tolerate acid and bile salts. The spent culture supernatant (SCS) of a selected strain TM39, ie, TM39-SCS, was found significantly inhibitory to the viability of H. pylori in vitro. It also inhibited the urease activity of H. pylori in vitro. For these antagonistic effects, in addition to pH and lactic acid, some factors in TM39-SCS might play the major role. Treatment of H. pylori with the SCS or cells of strain TM39 significantly reduced its binding to TSGH 9201 cells. For the toxicity study, we demonstrated that strain TM39 is not vancomycin resistant and is not invasive to TSGH and Int-407 cells in vitro. Cells of strain TM39 were oral dietary administrated at concentrations of 11012, 51011 and 21010 CFU/ kg to the rat for 28 days. There were no adverse effects on the general conditions and behavior, growth, feed and water consumption, hematology, clinical chemistry values, organ weights and histopathologic analysis. Results of this study demonstrated that consumption of strain E. faecium TM39 was not associated with any obvious signs of toxicity in Wistar rats even following consumption of large quantities. Therefore, the newly isolated E. faecium strain TM39 is with potential for anti-H. pylori use. Strain L. acidophilus LA5 from porcine intestine and strain L. fermentum LF33 from poultry caecum, were found adherent to the Int-407 and Caco-2 cells, the intestinal epithelium of their host as well as BALB/c mice. On the other hand, all the strains from vegetable origins were unable to adhere to the Int-407 and Caco-2 cells. One strain from vegetable origin, ie, strain L. plantarum LP69, however, was able to adhere to the mice intestinal epithelial cells. On the antagonistic activity against Salmonella invasion to Int-407 cells and the mice livers and spleens, LAB strains LA5, LF33 and LP69 were found to have inhibitory effect. For the toxicity of the strain L. acidophilus LA5 in Wistar rats assayed by oral administration at concentrations of 2.11011, 1.051011 and 4.2109 CFU/ kg for 28 days, there were no adverse effects on the general condition and behavior, growth, feed and water consumption, hematology, clinical chemistry values, organ weights and histopathologic analysis. Results of this study demonstrated that consumption of strain L. acidophilus LA5 was also not associated with any obvious signs of toxicity in Wistar rats even following consumption of large quantities.
|Appears in Collections:||食品暨應用生物科技學系|
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