食品中大腸桿菌平板計數檢驗方法之評估   全文下載

Evaluation of Test Methods for Colony-Count Technique of Escherichia Coli in Food

大腸桿菌為人類和其他溫血動物腸道中的正常菌種，常被作為監測飲用水、食品之衛生指標菌，如超過衛生標準限量，則表示食品遭受污染，食入受污染之產品可能引起腸胃道的不適，造成腹瀉、腹痛等症狀。大腸桿菌檢驗方法，衛生福利部已公告最確數計數法，惟因應產品輸銷歐盟之檢驗需求，尚需建立平板計數法。本研究目的旨在評估美國FDA/BAM（Bacteriological Analytical Manual）及ISO之大腸桿菌平板計數法，兩者皆是檢測大腸桿菌產生葡萄糖醛酸苷酶（β-glucuronidase）之特性，該酵素將培養基內含呈色原質4-甲基繖形酮-β-d-葡萄醣醛酸苷（4-methylumbelliferyl β-D-glucuronide, MUG）或5-溴-4-氯-3-吲哚-β-d-葡萄醣醛苷（5-bromo-4-chloro-3-indolyl-β-d-glucuronide, BCIG）水解產生螢光物質4-甲基繖形酮（4-methylumbelliferone, 4-MU）或呈色物質5-溴-4-氯-3-吲哚（5-bromo-4-chloro-3-indolyl），以計數平板培養基生長的典型菌落。經試驗後之靈敏度及專一性結果分別為：以不同血清型別的大腸桿菌54株進行靈敏度測試，其中只有1株O157為偽陰性；以氣單胞菌等非大腸桿菌46株進行專一性測試，其中有3株志賀氏桿菌為偽陽性。培養基經評估結果為：紫紅膽鹽乳糖瓊脂培養基／紫紅膽鹽乳糖瓊脂培養基-MUG（violet red bile lactose agar/violet red bile lactose agar-4-methylumbelliferyl β-D-glucuronide, VRBA/VRBA-MUG）及胰化蛋白腖-膽鹽-X-葡萄糖醛酸苷培養基（tryptone-bile X-glucuronide agar, TBX）培養基的菌數回收率均大於70%。以市售綜合果汁為基質進行大腸桿菌及混菌之添加試驗，結果大腸桿菌均與原添加菌數相當。另，抽樣檢驗市售產品20件進行大腸桿菌檢驗，其中TBX培養基共5件產品檢出大腸桿菌，而VRBA/VRBA-MUG培養基僅於1件產品檢出大腸桿菌。本評估方法結果作為修訂公告檢驗方法之依據。

Escherichia coli (E. coli) is widely distributed in the intestines of humans and warm-blooded animals. It is often used as a sanitary indicator for monitoring the quality of drinking water and food. The presence of E. coli is a strong indication of animal waste contamination. Consumption of contaminated products may cause gastrointestinal discomfort, diarrhea, abdominal pain and other symptoms. The traditional most probable number (MPN) method for E. coli count is an announced official method in Taiwan. However, in response to the inspection needs of products exported to the European Union, a plate count method is required. Therefore, this study aimed on the evaluation of two E. coli plate count methods including the US Food and Drug Administration/Bacteriological Analytical Manual (FDA/ BAM) and International Standard for Organization (ISO). Both methods were associated to the detection of β-glucuronidase activity from E. coli. The enzyme hydrolyzed 4-methylumbelliferyl β-D-glucuronide (MUG) or 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (BCIG) in the culture media, and produced fluorogenic (4-methylumbelliferone) or coloring (5-bromo-4-chloro-3-indolyl) substance making colonies visible. The sensitivity and specificity test results were as follows: the sensitivity test for 54 strains of different serotypes of E. coli, of which only one strain of O157 showed false negative. Forty-six strains of non-E. coli bacteria, such as Aeromonas were tested for specificity, among those 3 strains of Shigella showed false positive. The recovery tests in two culture media including violet red bile lactose agar/violet red bile lactose agar-4-methylumbelliferyl β-D-glucuronide (VRBA/VRBA-MUG) and tryptonebile X-glucuronide (TBX) agar medium, showed higher than 70%. An additional test in fruit juice matrix inoculated with E coli and mixed bacteria was carried out, and the results showed that the amount of E. coli was equivalent to the original inoculation. A survey consisted of 20 commercial products were applied for E. coli determination. The results showed that E. coli was detected in TBX and VRBA-MUG media from 5 products and 1 product, respectively. The evaluation data in this study would be used as reference for revising the current official method.