以生物反應器擴大培養並優化機能性臺灣藜發酵乳製品及其抗衰老功效評估

Abstract

臺灣藜 (Chenopodium formosanum Koidz.) 又名 Djulis，是臺灣原生種假穀物，又被稱為「穀物界紅寶石」，因具有甜菜苷、多酚與黃酮類化合物使其具有優異之抗氧化能力。本研究目的是利用臺灣藜與在地優質乳源，結合乳酸菌以液態生物反應器 (發酵槽) 建構量產製程，並評估其於線蟲之抗衰老潛力。本研究以小量生產之臺灣藜發酵乳製品 (100 mL) 為控制組，藉由發酵槽進行擴大液態發酵。為增進機能性成分含量，以游離黃酮含量為指標進行發酵條件優化，階段式探討轉速、發酵溫度與發酵時間，開發五公升級臺灣藜發酵乳製品。首先於一公升液態發酵槽中加入鮮乳並添加 7.5 % 之臺灣藜、以 10^7 CFU/mL 個別接種Lactiplantibacillus plantarum subsp. plantarum BCRC 11697、Lactobacillus delbrueckii subsp. bulgaricus BCRC 10696 與 Streptococcus salivarius subsp. thermophilus BCRC 14085進行培養，而後以游離黃酮為指標確立最佳發酵條件為 40 °C、20 rpm、21 hr。以此優化條件進行五公升擴大液態發酵，並針對各營養組成、抗營養因子與機能性成分進行比較：於機能性成分，游離黃酮含量達 0.75±0.07 mg QE/g，顯著高於控制組並提升 30 %。游離酚含量達 1.54±0.05 mg GAE/g，顯著高於控制組；而結合酚含量則下降至 1.65±0.10 GAE/g，顯著下降 16 %。去醣基異黃酮daidzein含量達 2.17±0.35 μg/g，相較控制組顯著提升 47 %；游離胜肽含量達 3.27±0.44 mg SE/g，為控制組之 1.08 倍。於一般成分分析，粗脂肪含量顯著高於控制組 12 %。於抗營養因子，植酸則下降至 3.02±0.19 mg/g，相較控制組顯著下降 41 %。而於儲藏性試驗中，臺灣藜發酵乳製品之總乳酸菌數隨存放時間增加而逐漸下降，並於 14 ~ 21 天趨於平緩穩定維持在 9 log CFU/mL 左右。於感官品評試驗中，受試者對於臺灣藜發酵乳製品之接受度與喜好程度皆低於市售無糖穀物優格。而後以秀麗隱桿線蟲為模式生物，評估臺灣藜發酵乳製品之乙醇萃取物的抗衰老潛力與功效。臺灣藜發酵乳製品乙醇萃取物於氧化壓力抵禦能力提升線蟲的存活率至 56.68±5.22 %，顯著高於控制組並提升 15.24 %；而平均生命週期增加了 4.57 %，然於統計上無顯著差異；衰老相關指標-咽部抽動速率為 266.47±10.79 pumps/min，顯著提高至控制組的1.14倍。綜上所述，經優化之發酵條件能使五公升擴大液態發酵之臺灣藜發酵乳製品的機能性成分含量與不亞於小量生產組別 (100 mL)，成功建立擴大生產製程，其萃取物也具有抵抗氧化壓力與延長線蟲健康壽命之潛力，透過後續產品風味與質地的改良，期望能成為具抗衰潛力之新型產品。

Chenopodium formosanum Koidz., also known as "Djulis", which is a pseudocereal native in Taiwan. It is also known as the "Ruby of Cereals "and has an excellent antioxidant capacity due to its betanin, polyphenols and flavonoids. The aim of this study was to use Djulis and local high-quality milk sources, combined with lactic acid bacteria to construct a mass production process in a liquid state bioreactor (fermentation tank), and evaluated its anti-aging potential in C. elegans. In this study, we took 100 mL small-scale production of fermented Djulis dairy products (FDDP) as the control group, and the enlarged liquid fermentation was carried out by fermentation tank. To increase the content of functional components, this study took the content of free flavonoids as target to optimize the fermentation conditions. By adjusting the stirring speed, fermentation temperature and fermentation time step by step, 5 L FDDP was developed. First, fresh milk and 7.5 % of Djulis were added to a 1 L liquid fermentation tank. Lactiplantibacillus plantarum subsp. plantarum BCRC 11697, Lactobacillus delbrueckii subsp. bulgaricus BCRC 10696 and Streptococcus salivarius subsp. thermophilus BCRC 14085 were inoculated at 10^7 CFU/mL for culture, respectively. The results indicated that the optimal condition of liquid fermentation was at 40 °C, 20 rpm, 21 hr. Based on this optimal condition, 5 L production was carried out, and various nutritional components, anti-nutritional factors and functional components were compared. For functional components, free flavonoids reached 0.75±0.07 mg QE/g, which was significantly higher than the control group and increased 30 %. Free phenols reached 1.54±0.05 mg GAE/g, which was significantly higher than the control group; while bound phenols decreased to 1.65±0.10 GAE/g, a significant decrease of 16 %. The content of daidzein was 2.17±0.35 μg/g, which was 47 % higher than the control group. Free peptide content reached 3.27±0.44 mg mg SE/g, which was 1.08 times higher than the control group. In proximate analysis, crude fat content was significantly higher than the control group by 12 %. The anti-nutritional factor-phytic acid decreased to 3.02±0.19 mg/g, which was 41% lower than the control group. In the storage test, the total lactic acid bacterial count of FDDP gradually decreased with the increase of storage time, and then tended to be stable and maintained at about 9 log CFU/mL from 14 to 21 days. In sensory evaluation, the acceptability and preference of FDDP were lower than those of commercially sugar-free cereal yogurt. Then, C. elegans was used as a model organism to evaluate the anti-aging potential and efficacy of ethanol extracts of FDDP. The ethanol extracts of FDDP improved the survival rate of C. elegans to 56.68±5.22 % in the resistance to oxidative stress, which was significantly higher than the control group and elevated 15.24 %. The mean life span of C. elegans increased by 4.57 %, but there was no statistically significant difference. Aging related index: pharyngeal pumping rate was 266.47±10.79 pumps/min, significantly increased to 1.14 times of the control group. To sum up, the optimal fermentation condition could make the functional components of 5 L FDDP no less than 100 mL small-scale production. The enlarged production process has been successfully established, and the extracts of FDDP also has the potential to resist oxidative stress and prolong the healthspan of C. elegans. Through the improvement of the flavor and texture of FDDP, it is expected to become a new product with anti-aging potential.