利用本土耐鹽菌自醬油粕生產天然四氫嘧啶及抗發炎功效評估

The Evaluation of Anti-inflammatory Activities on the Natural Tetrahydropyrimidine (Ectoine) Production from Soy Sauce Residue by Halomonas beimenensis

利用分離自台南沿海地區之北門單胞鹽菌（Halomonas beimenensis sp. nov.）新物種作為嗜鹽微生物發酵並生產純化四氫嘧啶（ecotine）之研究。H. beimenensis為一種耐鹽菌，其可以在體內產生相容性溶質（compatible solute）調整滲透壓，因此可於高鹽的環境中生存，此相容性溶質為一種稱為ectoine的化合物，此種化合物具水和特性，目前被認為具有開發為保養品的潛力。本研究期望利用發酵培養技術並建立穩定之生產模式，研發出有效、低成本的含鹽廢棄物轉換生成美粧原料技術，並可解決含鹽副產物造成的問題，有助於廢棄物資源利用與創新。以醬油粕取代標準培養基進行探討，將水與醬油粕以1:2的比例混合，菌體在接種後的36小時進入平穩期。利用調整成分後的醬油粕完全取代標準培養基，並用兩階段饋料培養（two-step fed-batch）法生產ectoine，最大產量為1.42 g．L-1。結合標準培養基及醬油粕培養基，於two-step fed-batch中第一階段依然使用標準培養基進行，而第二階段則改用高鹽度的醬油粕，ectoine最大產量達2.22 g．L-1。將生產之ectoine進行細胞功效評估，利用LPS誘導RAW264.7細胞模式來評估細胞存活率與抗發炎之效果，ectoine可維持細胞正常生長狀態且有效抑制一氧化氮含量的釋放，降低發炎細胞激素TNF-α、IL-6與IL-1β之含量，並可抑制iNOS酵素與COX-2之基因表現，本研究結果，利用醬油粕生產ectoine可以降低生產成本並具有商業化生產應用之價值。同時，ectoine是天然的細胞保護劑，同時具有多項功能，未來相當具有開發多功能化粧品之潛力。

In this study, Halomonas beimenensis, a novel halophilic microorganism isolated from the coastal areas of Tainan, was used to produce and purify ectoine via fermentation. H. beimenensis is a salt-tolerant bacterium that can produce a compatible solute in the body, which allows it to adjust its osmotic pressure and survive in a highsalt environment. This compatible solute is a compound called ectoine. As this compound has hydration properties, it is now considered to have potential for the development of skin care products. This study will improve a combined fermented process to establish a stable production model and develop effective, low-cost key valueadded technologies to convert salt-containing waste for beauty materials, and solve the problem caused by saltladen waste. The ecotine yield was 1.42 g·L-1 in trials using soy sauce residue instead of basal medium, where water and soy sauce residue were mixed at a 1:2 ratio. In the two-step fed-batch method, the first stage was carried out using basal medium, and the second stage was carried out using soy meal culture, resulting in an ectoine yield of 2.22 g·L-1. RAW264.7 cells were used to assess cell viability and NO anti-inflammatory. Ectoine can help maintain normal cell growth and does not promote an inflammatory response. In B16F10 melanocytes, ectoine concentrations of 1–1000 ppm did not result in cytotoxicity. At concentrations of 100 and 1000 ppm, the efficiency for the inhibition of melanin production was 28.86% and 25.70%, respectively. Ectoine has the most obvious effect on inhibiting the production of nitric oxide and the inhibition rate was up to 86%,which has the same trend as the expression of nitric oxide synthase (iNOS).As the result, this study speculated that edamame leaves might regulate nitric oxide production by inhibiting iNOS. In addition, the expression level of COX-2 gene was also significantly reduced. The results of LPS-induced production of TNF-α, IL-1β, and IL-6 in RAW264.7 cells showed that 70% methanol extract and ethanol extract could significantly reduce the production of TNF-α, IL-6, and IL-1β. This optimized condition could reduce the cost of production and improve the commercial production value. Ectoine is a natural cytoprotective agent, and has several functions and a promising potential for the development of multi-functional cosmetic products.