食品中組織胺生產菌分子生物檢測法之研究

Development of molecular methods for the detection of histamine-forming bacteria in foods

龔賢鳳(Hsien-Feng Kung)

指導教授 ： 廖遠東 博士 ； 鄧德豐 博士

國立屏東科技大學/農學院/食品科學系所/博士(2009年)

https://doi.org/10.6346/NPUST.2009.00295

摘要

論文摘要內容：
本研究採集市售旗魚排、烏魚子、酸菜及豆豉樣品 (總計107件)，進行組織胺生成菌分離與鑑定，結果顯示自107件樣品中共分離出37株組織胺生產菌，其中自酸菜與豆豉樣品分離出的革蘭氏陽性菌 Staphylococcus capitis 皆為耐鹽性強組織胺生成菌；而旗魚排樣品分離之Enterobacter aerogenes、Raoultella ornithinolytica則為革蘭氏陰性強組織胺生成菌。為能快速有效檢測食品中組織胺生成菌，故進行分子生物檢測技術之開發研究。
針對革蘭氏陽性強組織胺生成菌，本研究設計引子對 kp1f/kp3r 進行 PCR 反應後可得專一性 433 bp 增幅片段，非組織胺生成菌皆不會產生 433 bp 增幅片段，將 S. capitis 接種於不同鹽度之虱目魚肉均質液時，PCR 反應可於低菌量時 (102 CFU/g ) 即產生明顯之433 bp專一性增幅片段。更進一步，本研究設計專一性 real-time PCR 引子 kp2f/kp2r 並以 SYBR green I 系統偵測樣品中革蘭氏陽性組織胺生產菌 S. capitis ，在食品中檢出極限是 101 CFU/g 菌量；當CT 為24〜25 時，菌量約為5.4〜6.0 log CFU/mL(g)，此時組織胺開始蓄積；當CT 值在17〜20，菌數含量在7.0〜7.75 log CFU/mL(g)，樣品中組織胺已大量生成至美國食品藥物管理局規定之中毒危害量 50 mg/100 g (500 ppm) 以上。以此分子生物檢測方法應用於28件市售水產品中組織胺生產菌之檢測並評估組織胺之危害，結果顯示2件鮪魚香腸樣品解鏈曲線分析得之Tm 值可確知其含革蘭氏陽性組織胺生產菌，由CT 值推估樣品中約含101 CFU/g 的菌量，在此菌量下組織胺尚未生成，故本研究開發之 real-time PCR 可在組織胺尚未生成蓄積前將樣品中革蘭氏陽性組織胺生產菌快速並正確的檢測出來，更可推估組織胺之含量，進而得知該食品之食用安全性。
在革蘭氏陰性組織胺生成菌快速檢測技術之研究，本研究針對所分離之革蘭氏陰性強組織胺生成菌設計出引子對 kt1/kt5r，進行 PCR 反應後，可得到 311 bp 之專一性增幅片段，在非組織胺生產菌則皆不會產生特異性放大片段。於旗魚肉均質液中時，此 PCR 法之檢測靈敏度為104 CFU/g，並可在組織胺產生前4小時偵測出組織胺生產菌。另外，將數株革蘭氏陰性組織胺生產菌組胺酸脫羧酶基因排列比對後設計專一性引子 N1-f/kt1-r，以 real-time PCR SYBR Green I 系統應用於食品中革蘭氏陰性組織胺生產菌之檢測研究。實驗顯示於水產魚肉中組織胺生產菌檢出靈敏度 2 ×102 CFU/g (CT 約34.76±0.11)；在魚肉貯藏實驗結果顯示 CT 值在24〜26，樣品組織胺開始蓄積之，當 CT 值低於20，樣品內組織胺可能已超過美國食品藥物管理局建議之中毒危害限量標準 500 ppm；以此 real-time PCR 法應用於市售水產品組織胺生產菌之檢測並評估組織胺之危害，結果顯示48件水產加工製品有14件於 real-time PCR 呈現正反應，解鏈曲線分析得之Tm 值確知其含革蘭氏陰性組織胺生產菌，由 CT (28.14-35.16) 推估樣品中含有約 102〜103 CFU/g 之組織胺生成菌，然在組織胺含量分析48件樣品均未檢出，此結果亦可顯示本研究設計之 real-time PCR 引子可在樣品組織胺尚未生成前即可偵測到組織胺生產菌之存在，同時分析評估組織胺含量，並建立簡單、快速預測水產品中組織胺危害的方法與實際應用。

關鍵字

組織胺 ； 組織胺酸脫羧酶 ； 組織胺生產菌 ； 聚合酶連鎖反應 ； 即時定量聚合酶連鎖反應

並列摘要

The contents of abstract in this thesis:
In this study, sailfish fillets, salted mullet roe products, mustard pickle products and douchi products (total 107 samples) were purchased and tested to isolate the histamine-forming bacteria. Thirty seven bacterial strains were isolated from all samples on a selective medium for histamine-forming bacteria. Among them, the gram-positive (G+) bacteria Staphylococcus capitis , isolated from mustard pickle and douchi samples, which were potent histamine-formers, while the gram-negative (G–) bacteria Enterobacter aerogenes and Raoultella ornithinolytica isolated from sailfish fillets were potent histamine-formers.
A polymerase chain reaction (PCR) assay targeting the gene encoding histidine decarboxylase (HDC) was developed for detecting gram-positive histamine-forming bacteria. Using a kp1f/kp3r primer set, the PCR assay yielded a 433 bp DNA amplification fragment from histidine decarboxylase of gram-positive histamine-forming bacteria. In contrast, none of the non- histamine producing strains produced an amplification product. Gram-positive histamine-forming bacteria was detected at levels of 102 CFU/g in milkfish homogenate inoculated with gram-positive histamine-forming bacteria after the PCR amplification.
A real time PCR primer set kp2f/kp2r was designed, based on the histidine decarboxylase gene sequence of gram-positive histamine-forming bacteria. The detection limit was 101 CFU/g. The 5.4〜6.0 log CFU/mL(g) levels of gram-positive histamine-forming bacteria (CT value:24-25) can be related to the detection of histamine concentrations by HPLC. However, The 7.0〜7.75 log CFU/mL(g) levels of gram-positive histamine-forming bacteria (CT value: 17-20) can be related to the histamine concentrations higher than 500 mg/100 g. While 28 seafood samples purchased at markets were analyzed by the real-time PCR method and HPLC, the results showed that two tuna sausage samples had 101 CFU/g of gram-positive histamine-forming bacteria at CT value 34.16-35.63, but non detection of histamine. In this respect, a good correlation between the histamine concentration and CT value can be used to predict a hazard of histamine accumulation in food, and the real-time PCR method may be a rapid, specific and highly sensitive technique for detecting potential gram-positive histamine-producing strains.
Under the optimized conditions, the PCR assay yielded a 311 bp DNA amplification fragment from histidine decarboxylase of gram-negative histamine-forming bacteria using kt1/kt5r primer set. In contrast, none of the non-histamine producing strains produced an amplification product. When the sensitivity of the assay was evaluated, gram-negative histamine-forming bacteria was detected at levels of 104 CFU/g in sailfish homogenate after the PCR amplification. Moreover, the hdc gene was detected 4 h earlier than HPLC detection of histamine in sailfish homogenates inoculated with histamine- forming bacteria.
A real time PCR primer set N1f/kt1r was designed based on the histidine decarboxylase gene sequence of gram-negative histamine-forming bacteria. The detection limit was 102 CFU/g. The CT value: 24-26 can be related to the detection of histamine concentrations by HPLC. However, The CT value＜20 can be related to the histamine concentrations higher than 50 mg/100 g. While 48 seafood samples purchased at markets were analyzed by the real-time PCR method and HPLC, the results suggested that 14 seafood samples had 102 〜103 CFU/g of gram-negative histamine-forming bacteria at CT value 28.14- 35.16, but non detection of histamine. Conclusively, the real-time PCR method to detect histamine producers before histamine accumulates will be an important tool for hazard analysis and monitoring of critical control point fish processing plants.

並列關鍵字

histamine ； histidine decarboxylase ； histamine-producing bacteria ； polymerase chain reaction (PCR) ； real time PCR

參考文獻

Andersson RE. 1988. Biogenic amines in lactic acid fermented vegetables. Lebensmittel-Wissenschaft und –Technologie 21: 68-9.

Antoine FR, Wei CI, Littell RC, Marshall MR. 1999. HPLC method for analysis of free amino acids in fish using o-Phthaldialdehyde precolumn derivatization. Journal of Agricultural and Food Chemistry 47: 5100-7.

AOAC. (1995) Fish and other marine products. In “Official Methods of Analysis of AOAC International”, Chap. 35, Cunniff, P. ed. pp. 16-7. AOAC International. Virginia, USA.

Arnold SH, Brown W. 1978. Histamine toxicity from fish products. Advances in Food Research 24: 113-54.

Arnold SH, Price RJ, Brown WD. 1980. Histamine formation by bacteria isolated from skipjack tuna, Katsuwonus pelamis. Bulletin of the Japanese Society of Scientific Fisheries 46: 991-5.