臺灣天然發酵火腿中菌種之鑑定與熟成時期菌相分布之研究

Abstract

從天然發酵肉製品中分離及鑑定的自發性菌元預期能夠控制發酵過程並穩定產品的食用安全性。本研究的目的是從試製之傳統台灣天然發酵火腿 (Taiwanese naturally fermented ham, TNFH) 鑑定乳酸菌 (lactic acid bacteria, LAB) 以及凝固酶反應陰性球菌 (coagulase-negative cocci, CNC)，結合傳統微生物培養方式以及分子技術包括聚合酶連鎖反應—變性梯度膠體電泳 (polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE) 及DNA定序，可深入研究於熟成期不同階段，菌相動態之消長變化。完成鑑菌後，再測試每一菌種之特性。結果發現，從TNFH樣品中，總共分離出12株LAB及3株葡萄球菌；在樣品的表面，Lactobacillus sakei及Staphylococcus saprophyticus為優勢菌種，然而在最終產品中，Leuconostoc mesenteroides 及 Carnobacterium divergens成為數量最多的菌種。除此之外，Microbacterium spp.、Carnobacterium spp.、Enterobacter spp.、 Brochothrix spp.、Enterococcus spp. 和Bacillus spp. 於熟成前期也被鑑定，然而樣品內部之菌數於此時期幾近於零。在經過約30日的熟成期後，樣品內部之菌相與表面之菌相結果已達一致。從TNFH樣品中所分離出來的菌種中，Lc. lactis subsp. lactis、Lb. sakei、Lc. lactis subsp. cremoris、Staph. xylosus被認為最有潛力作為製作發酵肉品之初始發酵菌元。 結果顯示，與其它地區生產之發酵肉品相比，台灣天然發酵火腿具有相當特殊的發酵菌相。結合培養方式以及分子技術包括PCR-DGGE及DNA定序，能夠有效鑑定菌種以及菌相的分布，以達開發適用於台灣發酵火腿之發酵菌元的目的。

Isolation and identification of the autochthonous starter from a naturally fermented meat allows control of the fermentation process and promises microbiological safety for this specialty. Thus the purpose of this study was to identify the lactic acid bacteria and coagulase-negative cocci present in a traditional Taiwanese naturally fermented ham (TNFH) and to study the microbial dynamics at different ripening stages; the approach was a combination of conventional microbiological cultivation, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. The characteristics of each isolated strains were also investigated. In total, twelve different strains of lactic acid bacteria and three Staphylococcus strains were identified in the TNFH samples. The bacterial ecology on the surface of the samples was mainly characterized by the stable presence of Lactobacillus sakei and Staphylococcus saprophyticus; nonetheless Leuconostoc mesenteroides and Carnobacterium divergens were the most abundant bacteria found in the final product. In addition, Microbacterium spp., Carnobacterium spp., Enterobacter spp., Brochothrix spp., Enterococcus spp., and Bacillus spp. were also present at the beginning of the ripening, but few bacteria were found at the center of the TNFH samples during the early ripening stages. However, after 30 days of ripening, the microbial ecology at the center of the TNFH samples paralleled that of the surface. Results of characterization of each strains isolated from TNFH indicated that Lc. lactis subsp. lactis, Lb. sakei, Lc. lactis subsp. cremoris and Staph. xylosus may use as starter cultures for manufacturing fermented meat. Our findings showed that the naturally fermented ham in Taiwan demonstrated a very different ecology when comparing with other fermented meat products. This study also confirmed that combination of cultivation, PCR-DGGE and DNA sequencing can be used to effectively describe the microbial communities of Taiwanese naturally fermented ham. This information could help us to design autochthonous starter cultures.