篩選本土性類胡蘿蔔素生產菌對類胡蘿蔔素最適化及生物活性探討

類胡蘿蔔素 (Carotenoids) 為一類重要之天然色素總稱，其中有1,000多種類胡蘿蔔素已被鑑定。而蝦紅素及β-胡蘿蔔素於動物飼料添加劑中扮演重要角色，其可提供生物生長之營養、降低疾病發生率、增加抗氧化能力及提升免疫功能等功用。然而利用微生物生產類胡蘿蔔素，具有生長週期短、易回收且不易受氣候影響等優勢，因此成為天然色素主要之研究趨勢。本研究第一部份自仙丹花中分離具有鮮豔色澤之酵母菌及國立屏東科技大學食品學系FS107實驗室保存之產色細菌，透過類胡蘿蔔素之測定作為菌株之初步篩選，再經由HPLC定性分析選定出具有可產生蝦紅素及β-胡蘿蔔素於之菌株，而後將此菌株進行基本生長特性探討，最後進行16S rDNA種鑑定。實驗第二部分為探討菌株提升類胡蘿蔔含量之最適發酵條件，先經由一次一因子法探討溫度、轉速、V8番茄培養基濃度及不同金屬鹽類 (CuSO4 • 5H2O、FeSO4 • 7H2O及ZnSO4 • 7H2O) 及其濃度之因子階層，而後利用反應曲面法 (Response surface methodology, RSM) 搭配Box-Behnken Desig (BBD) 實驗設計進行最適化之探討。第三部分應用上述最適化之條件培養完成後製備成類胡蘿蔔素粗萃物，並分析其抗氧化活性及抑菌試驗。結果顯示，本實驗自仙丹花選出33株產色酵母菌，經由類胡蘿蔔素含量測定後，初步篩選出5株色素含量大於881 μg/L之酵母菌並加入FS107實驗室之5株細菌，而後再經由色素鑑定，發現唯有一株細菌可產生蝦紅素及β-胡蘿蔔素於，該菌株進行16S rDNA種鑑定後將其命名為Williamsia serinedens O3Y。接著於RSM-BBD探討最適培養條件為W. serinedens O3Y接種於含有19.6%V8 medium及0.0093%金屬鹽類FeSO4 • 7H2O之YM培養基中，於30℃下以175 rpm振盪培養96小時，可得到最佳之類胡蘿蔔素產量1,273.23±44.28 μg/L。最後製備類胡蘿蔔素粗萃物進行抗氧化及抑菌分析，於抗氧化方面，類胡蘿蔔素粗萃物可表現出82.83±3.40%之自由基清除率，皆大於β-胡蘿蔔素 (60.74±3.00%) 及蝦紅素標準品 (16.66±5.01%)，而其IC50為1,748.73 ppm；還原力方面，類胡蘿蔔素粗萃物最佳之還原力數值為0.48±0.03與β-胡蘿蔔素標準品無顯著差異，但高於蝦紅素標準品43.75%。抑菌實驗方面，類胡蘿蔔素粗萃物對於S. aureus subsp. aureus BCRC10451及E. coli ATCC8739於24小時之抑菌圈大小分別為4.1±0.3 mm及5.6±0.5 mm，而其最小抑制濃度分別為5,000 ppm、3,000 ppm。綜合上述，W. serinedens O3Y生產之類胡蘿蔔素具有較佳之供氫及電子轉移之能力，而對革蘭氏陰性之病原菌具有較佳之抑菌效果。未來可將W. serinedens O3Y菌株生產之類胡蘿蔔素應用在飼料添加劑之開發，以提高畜產或水產動物之附加價值。

關鍵字

類胡蘿蔔素；抗氧化；反應曲面法；抑菌；金屬鹽類

並列摘要

Carotenoids are an important class of natural pigments, of which more than 1,000 carotenoids have been identified. Astaxanthin and β-carotene play an important role in animal feed additives, which can provide nutrients for biological growth, reduce the incidence of diseases, increase antioxidant capacity and enhance immune function. However, the use of microorganisms to produce carotenoids has the advantages of short growth cycle, easy recovery, and is not easily affected by climate, so it has become the main research trend of natural pigments.　In the first part of this study, bright-colored yeasts were isolated from Ixora x williamsii and color-producing bacteria which keep in the laboratory of FS107, Department of Food Science, National Pingtung University of Science and Technology. Qualitative analysis by HPLC selected a strain that can produce astaxanthin and β-carotene, and then the basic growth characteristics of this strain were discussed, and finally the 16S rDNA species was identified. The second part of the experiment was to explore the optimal fermentation conditions for the strains to increase the carotenoid content. First, the temperature, rotation speed, concentration of V8 tomato medium and different metal salts (CuSO4 • 5H2O, FeSO4 • 7H2O and ZnSO4 • 7H2O) and The factor hierarchies of its concentrations were then optimized using the response surface methodology (RSM) combined with the Box-Behnken desig (BBD) experimental design. In the third part, the crude carotenoid extract was prepared after the above-mentioned optimized conditions were cultivated, and its antioxidant activity and antibacterial test were analyzed.　The results showed that 33 color-producing yeast strains were selected from the Ixora x williamsii in this experiment. After the determination of carotenoid content, 5 strains of yeast with pigment content greater than 881 μg/L were preliminarily screened and 5 strains of bacteria from the FS107 laboratory were added. Through pigment identification, it was found that only one strain of bacteria can produce astaxanthin and β-carotene, and this strain was named Williamsia serinedens O3Y after 16S rDNA species identification. Then, the optimal culture conditions were discussed in RSM-BBD. W. serinedens O3Y was inoculated in YM medium containing 19.6% V8 medium, 0.0093% metal salt FeSO4 • 7H2O and incubated at 30°C with shaking at 175 rpm for 96 hours. The best carotenoid yield was 1,273.23±44.28 μg/L. Finally, the crude carotenoid extract was prepared for antioxidant and antibacterial analysis. In terms of antioxidant, the crude carotenoid extract showed a free radical scavenging rate of 82.83±3.40%, which was higher than that of β-carotene (60.74±3.00%). and astaxanthin standard (16.66±5.01%), and its IC50 is 1,748.73 ppm; in terms of reducing power, the best reducing power value of carotenoid crude extract is 0.48±0.03 and β-carotene standard has no significant difference, but 43.75% higher than astaxanthin standard. In the antibacterial experiment, the inhibition zone of the crude carotenoid extract for S. aureus subsp aureus BCRC10451 and E. coli ATCC8739 at 24 hours was 4.1±0.3 mm and 5.6±0.5 mm, respectively, and the minimum inhibitory concentrations were 5,000 ppm, 3,000 ppm.. In summary, the carotenoids produced by W. serinedens O3Y have better hydrogen-donating and electron-transfer capabilities, and have better bacteriostatic effect on Gram-negative pathogens. In the future, carotenoids produced by W. serinedens O3Y strain can be used in the development of feed additives to increase the added value of livestock or aquatic animals.