Optimal culture conditions for pigment production of yeast isolated from Citrus paradisi

面對逐漸擴大的天然色素市場，開發具有安全營養、保健效用、著色功能之天然食用色素是目前主要的研究趨勢，其中以紅色系天然色素開發進展較快。以微生物生產色素具週期短、易回收、不受氣候影響等優勢，而利用發酵真菌生產天然色素最適化條件主要在於環境溫度、pH值、碳源、氮源的控制，試驗期望未來可應用在蔬果廢棄物作為培養基值來降低色素生產成本，研究自蔬果：澎湖仙人掌、番茄、胡蘿蔔、蘋果、葡萄柚、檸檬中，以平板培養基純化分離出產色菌落，其中篩得之一個產色菌落形態呈現圓球狀，菌落直徑約3~4 mm，邊緣整齊無菌絲，菌落些微隆起，菌面濕潤有光澤，為不透明橘粉紅色。經基因鑑定，擴增ITS1和ITS4區域片段序列分析比對後，確立菌株為膠紅酵母(Rhodotorula mucilaginosa)的一種，所萃色素經光譜掃描初步定性其主要為為八氫番茄紅素所組成，為一株具有生產類胡蘿蔔素潛力之真菌。本研究欲探討Rhodotorula mucilaginosa的最適條件，經試驗固定接種量0.1%、轉速125 rpm搖瓶培養，確立進入stationary phase後24 小時為最佳色素收菌時間，控制條件溫度於27.5℃、initial pH value 5、碳源為15 g/L Glucose、氮源為2.5 g/L Yeast extract時，具高色素產量1469.29 (μg/L)。經最適條件培養至最高色素產量後，試驗以短時高溫37℃逆境六小時、短時低溫10℃逆境六小時和持續以低溫22.5℃培養，發現短時溫度變化逆境無法有效提升菌體色素產量的累積，而培養72小時候，再以27.5℃持續培養，可提升色素產量最高至1533.27 (μg/L)，並延長維持色素之高產量並減緩菌體色素的降解，於未來發酵生產上可供大量生產色素的參考依據。Expanding demands on natural pigment have driven the development of nutritive and functional natural food colorants. In among of that, red pigment has achieved rapid development nowadays. Production of pigments by fungi is characterized by various benefits such as short cycle, high recovery, climate-unaffected, optimal conditions on growth can be determined on temperature, pH, carbon and nitrogen sources. This study has utilized fruit and vegetable wastes as substrates for colorant production including wastes Penghu cactus, tomato, carrot, apple, grapefruit, lemon. Different color formation colonies with diameter 3-4 mm, and further classified according their appearances on mycelia, size, glossiness and colors were screened by plating. After verification on the ITS1 and ITS4 region of the screened colonies, it was classified as a species similar to Rhodotorula mucilaginosa. This strain is found highly to produce carotenoids. We have further optimized the condition for its growth. After inoculation 0.1%, a shaking condition of 125 rpm was carried out to reach 24 hour stationary phase under 27.5℃, pH 5.0 substrate, glucose 15 g/L and yeast extract at 2.5 g/L, highest colorant production was determined at1469.29 μg /L. The obtained colorant was further tested under short time high temperature (STHT) at 37℃, 6 hours, short time low temperature (STLT) at 10℃, 6 hours, and 22.5℃ conditions. It was discovered that temperature change during short time was unable to increase colorant production, however, a 22.5℃ would found to increase colorant production as high to 1533.27μg/g, this condition also found to prolong the production and reduce colorant degradation. This could be potential for bulk colorant production in near future