利用醬油粕以耐鹽菌生產天然四氫嘧啶及其在皮膚抗老化功效評估

Abstract

四氫嘧啶(1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, ectoine)為環狀胺基酸，對微生物在高滲透壓下具保護功能，屬於一種相容性溶質，其可支持細胞膨壓，並維持體內酵素的穩定性，因此如四氫嘧啶等物質能幫助嗜鹽菌(halophile)在高鹽的環境下存活。四氫嘧啶具有改善皮膚乾燥與彈性及防止紫外線照射所引起的細胞損傷等功能，目前應用於化妝品方面，具輔助增加保濕、抗UV及抗老化等功效。四氫嘧啶通常以微生物培養進行量產，在生產上主要以高鹽濃度環境刺激嗜鹽菌而大量累積後，再以低鹽濃度使菌體因滲透壓差釋放的循環方式萃取。本實驗以Halomonas beimenensis耐鹽菌株利用醬油粕整合培養生產四氫嘧啶，進一步利用甲醇、陽離子交換(cation exchange)及活性碳(activated carbon)脫色(decolorization)等步驟建立純化分離四氫嘧啶之製程，平均純度目前可達85.1%。在皮膚抗老化評估方面，採用LPS誘導巨噬細胞(macrophage)之抗發炎模式、黑色素(melanin)含量抑制模式及TNF-α誘導纖維母細胞(fibroblast)之皮膚抗老化模式進行，結果顯示，純化後之四氫嘧啶對細胞沒有毒性，且於1至1000 mg·L-1的濃度下具有能抑制一氧化氮(nitric oxide)含量與減少黑色素生成之效果，而於100 mg·L-1濃度下，四氫嘧啶對膠原蛋白(collagen)含量有較高之延緩效果。本研究建立利用醬油粕(soy sauce residue)以耐鹽菌生產四氫嘧啶純化製程，來幫助減少為生產四氫嘧啶所需要的大量鹽分，在廢棄物再利用上具有潛力，且可應用於化妝品方面，以期增進農業副產物之附加價值。

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, which is easily soluble in water and can accumulate in cells in large quantities without affecting cell metabolism. It is a compatible solute, which has a protective function that helps microbes against under high osmotic pressure. Thus it can maintain the osmotic pressure balance exo- and endo-cellular and offer the stability of the enzyme in the body. Therefore, halophilic or salt-tolerant bacteria can survive under high salt environment due to compatible solute. There is an increasing demand in the production and purification of ectoine at present, especially for cosmetic and healthcare applications. Ectoine has been mainly mass-produced by microorganisms. In the way of production, microbes accumulate a large amount of ectoine mainly by high salt concentration, and the osmotic pressure difference is caused by low salt concentration. This study used salt tolerant strain Halomonas beimenensis to integrate soya pomace of fermentation techniques. Methanol, cation exchange, and activated carbon were used to establish a process for purification of ectoine. The average purity was up to 85.1%. The purified ectoine was evaluated on anti-inflammatory, whitening and anti-aging effects with model of LPS-induced macrophages, melanocytes and TNF-α-induced fibroblasts. The results showed that the purified ectoine was not toxic to cells. It is potential to inhibit nitric oxide levels and melanin content at 1 to 1000 mg·L-1 of ectoine. And at 100 mg·L-1 ectoine, it has a great effect to delay collagen loss. Agricultural by-products that added value of the use will help the utilization and innovation of waste resources and create a new agricultural industry value chain.