利用乳酸菌生產天然葉酸之探討

葉酸 (維生素B9) 為人類不可或缺之維生素，但人類無法自行合成。然而孕婦對葉酸之需求量高，其對於胎兒神經形成具有重要之影響，且益生菌對於健康益處良多具有其優勢，因此生產天然形式之葉酸成為目前研究趨勢。另外人類攝取很多的果汁以及新鮮水果，所以會產生大量的廢棄物 (種子和果皮)，其富含營養價值 (礦物質、抗氧化活性物質等)，可利用這些廢棄物 (種子和果皮) 可作為食品之配料以及增值之副產品。因此本研究之目的為利用百香果果皮及脫脂還原乳做為益生菌之受質，產生天然之葉酸之探討。本研究自生乳中分離出10株產酸能力較佳之乳酸菌菌株M1、M2、M5、M8、M9、M13、M14、M16、M17及M18；另外於牡蠣中分離出10株乳酸菌株K1-K10。然後將其分別接種至葉酸培養基 (Folic Acid Casei Medium, FACM) 中以35℃培養24hr後，再以微生物測定法將標準菌株Lactobacillus rhamnosus BCRC10940接種至上清液中培養，並以波長595 nm下測定吸光值後，再計算其葉酸含量。將挑選之菌株發酵於含不同比例之百香果果皮粉末之脫脂還原乳中，篩選出最適添加量以及最佳發酵菌株，進行後續之發酵乳製作、抗氧化、有機酸、食品衛生菌測定及儲藏性試驗。結果顯示，於脫脂還原乳之試驗中，以M13菌株之葉酸產量為最高1.01±0.15 ng/mL，其次為M18菌株0.91±0.03 ng/mL。整體而言，以脫脂還原乳作為發酵基質可促使乳酸菌株提升其葉酸產量，其中以M13菌株所生產之葉酸含量為最佳，其與葉酸培養基相較下葉酸含量可提升56%。經16S rDNA分別將其命名為Lactobacillus fermentum M13 (新名為Limosilactobacillus fermentum M13) 及Lactobacillus fermentum M18 (新名為Limosilactobacillus fermentum M18)，以及實驗室菌株Lactiplantibacillus plantarum 7-40做後續之實驗菌株。而M18發酵於脫脂還原乳中其百香果果皮粉末添加量為1%時，其發酵時間16小時具有最高之葉酸含量2.21±0.023 ng/mL。由於M18菌株於百香果果皮最適添加量中為最佳，因此以一次一因子之最佳條件發酵L. fermentum M18發酵乳，進行抗氧化能力之測定以及儲藏性試驗，其葉酸於儲藏性試驗中有下降之趨勢，於第9-15天維持為0.95±0.03 ng/mL至0.96±0.06 ng/mL，於抗氧化能力方面，將其以等比例稀釋，測定還原力之測定，其還原力為0.44±0.009，總酚含量為121.93±1.47 ppm，DPPH自由基清除能力為77.75±5.51%，類黃酮含量為74.25±2.08 ppm。將發酵乳置於4-8℃進行15天儲藏性試驗，每3天取樣一次，結果顯示總乳酸菌菌數皆維持8 Log CFU/mL以上，葉酸於儲存性試驗中，從第0天下降至第15天其葉酸含量趨於平穩為2.18±0.09 ng/mL-0.95±0.03 ng/mL。於儲藏期間其有機酸釋放至樣品中，而導致儲存期間D-蘋果酸乳酸、檸檬酸及琥珀酸上升至87.74±4.58、1,707.82±34.97、3,257.62±15.65及3,776.36±199.69 ppm，而還原力於儲藏期間無顯著性增加，但DPPH自由基清除能力以及總酚含量有顯著性增加。而在儲藏期間，食品微生物之檢測中大腸桿菌、大腸桿菌群、腸桿菌科及沙門氏桿菌皆為未檢出，皆符合發酵乳食品微生物之標準。未來亦可將 L. fermentum M18作為菌酛應用於葉酸之機能性產品開發。

關鍵字

葉酸；葉酸培養基；乳酸菌；脫脂還原乳；百香果皮； Lactobacillus fermentum M18

並列摘要

Folic acid (vitamin B9) is an essential vitamin for humans, but humans cannot synthesize it on their own. However, pregnant women have a high demand for folic acid, which has an important effect on fetal neurogenesis, and probiotics have their advantages for many health benefits, so the production of folic acid in its natural form has become a current research trend. In addition, humans consume a lot of fruit juice and fresh fruits, so they will produce a lot of waste (seeds and peels), which are rich in nutritional value (minerals, antioxidant active substances, etc.), and these wastes can be used. As a food ingredient and a value-added by-product. In this study, 10 lactic acid bacteria strains M1, M2, M5, M8, M9, M13, M14, M16, M17 and M18 with better acid-producing ability were isolated from raw milk. In addition, 10 lactic acid strains K1-K10 were isolated from raw oysters. Then they were respectively inoculated into folic acid medium (FACM) at 35℃ for 24 hours, and then the Lactobacillus rhamnosus BCRC10940 was inoculated into the supernatant by microbiological assay method, and the was measured at a wavelength of 595 nm, and calculate folic acid content. The selected strains were fermented in different ratios of passion fruit peel powder and skimmed reduced milk, and the most added amount and the best fermented strain were screened out, and the subsequent fermented milk production, antioxidant, organic acid and food hygiene bacteria were measured. The M18 strain 0.91±0.03 ng/mL. Overall, the use of skim reduced milk as the fermentation substrate can promote the lactic acid strains to increase their folic acid production, and the folic acid content produced by the M13 strain is the best, and the folic acid content can be increased by 56% compared with the folic acid medium. They were named Lactobacillus fermentum M13 (new name Limosilactobacillus fermentum M13) and Lactobacillus fermentum M18 (new name Limosilactobacillus fermentum M18) by 16S rDNA, and laboratory strains Lactiplantibacillus plantarum 7-40 were used as follow-up experimental strains. When M18 was fermented in the reduced milk with the addition of 1% passion fruit peel powder, the highest folic acid content was 2.21±0.023 ng/mL in the 16 hours. Then, the fermented milk was fermented under the best conditions of M18 fermentation, and the antioxidant capacity and storage test were carried out. The folic acid in the storage test showed a downward trend, and was maintained at 0.95±0.03-0.96±0.06 ppm on the 9-15 days, in terms of antioxidant capacity, it was diluted in equal proportions, and the determination of reducing power was determined. OD700 was 0.44±0.009, total phenolic content was 121.93±1.47 ppm, DPPH free radical scavenging ability was 77.75±5.51%, flavonoids The content is 74.25±2.08 ppm. The fermented milk was placed at 4-8°C for 15-day storage test, and the samples were taken every 3 days. The results showed that the total number of lactic acid bacteria remained above 8 Log CFU/mL, and its reducing power did not increase significantly, but the DPPH Radical scavenging capacity and total phenolic content were significantly. The results showed that the total number of lactic acid bacteria remained above 8 Log CFU/mL, and the folic acid in the storage test decreased from the 0 day. On the 15 day, the folic acid content was 2.18±0.09 ng/mL-0.95±0.03 ng/mL. During storage, organic acids were released into the samples, resulting in D-malate lactic acid, citric acid and succinic acid rising to 87.74±4.58, 1,707.82±34.97, 3,257.62±15.65 and 3,776.36±199.69 ppm during storage, and the reducing power was increased during storage There was no significant increase during the period, but the DPPH free radical scavenging ability and total phenolic content increased significantly. During the storage, Escherichia coli, Coliform, Enterobacteriaceae and Salmonella were all negative in the detection of food microorganisms, all of which met the standard of fermented milk food microorganisms. In the future, L. fermentum M18 can also be used in the development of functional products for folic acid.