自臺灣黏質發酵乳分離鑑定產胞外多醣乳酸菌應用於製造及改善低脂發酵乳之缺陷

Isolation and identification of exopolysaccharide-producing lactic acid bacteria from Taiwanese ropy fermented milk for low-fat fermented milk production

張毓純(Yu-Chun Chang)

指導教授 ： 陳明汝

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803207

摘要

因現今消費者為了控制卡路里攝取量而購買低脂乳製品，然而去除乳製品中的脂肪會造成一些質地、色澤和風味上的缺陷。在商業上會利用添加多醣類物質作為安定劑 (stabilizer) 或水合劑 (water-binding agent) 改善低脂產品的質地，然而隨著消費水平的增長以及食安議題的延伸，消費者會偏向購買較少或是無食品添加劑的「潔淨標示」 (clean label) 產品。因此希望利用乳酸菌 (lactic acid bacteria, LAB) 於發酵期間自體產生並釋放到產品中的胞外多醣 (exopolysaccharide, EPS) 去提升低脂發酵乳製品的品質，開發出潔淨標示的產品。根據乳酸菌胞外多醣生產能力可以分為產黏 (ropy) 乳酸菌和不產黏 (nonropy) 乳酸菌，其中產黏乳酸菌所產生的胞外多醣在提升產品的質地上有較好的能力。臺灣黏質發酵乳 (Taiwanese ropy fermented milk, TRFM) 是本實驗室長期繼代及保存的發酵乳製品，質地十分黏稠且延展力強。由於質地和風味與傳統的北歐發酵牛奶viili相似，亦被稱為Taiwanese viili。在本研究中擬自臺灣黏質發酵乳中分離出產黏乳酸菌，進而利用其產生的胞外多醣之特性去製作低脂發酵乳。
首先自臺灣黏質發酵乳中篩選出40個單一分離菌落，利用革蘭氏染色法去除革蘭氏陰性細菌和酵母，留下34個分離菌落 (isolate) 。將分離菌落接種在MRS培養基中後，以震盪後懸浮的型態選擇了13個產黏的單一分離菌落。使用隨機增幅多態性DNA聚合酶連鎖反應 (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR) 、16S rRNA序列以及API50進一步分類和鑑定，利用隨機增幅多態性DNA聚合酶連鎖反應的結果將13個分離菌落分成兩組，根據16s rRNA基因分析以及API 50 CHL的結果將菌株鑑定為Lactococcus lactis subsp. cremoris，並且命名為Lactococcus lactis subsp. cremoris APL15 (APL15) 和Lactococcus lactis subsp. cremoris APL16 (APL16) 。由於APL15以及APL16這兩株菌在胞外多醣產量上沒有顯著差異 (P>0.05) ，因此以這兩株菌皆進行第二部分實驗低脂發酵乳的製作。
在低脂發酵乳生產的部分，將低脂牛奶中接種1% APL15以及APL16，於26°C發酵24小時，分析物理特性和微生物菌數。結果顯示APL15和APL16可以在24小時內使發酵乳酸鹼值達pH 4.3，酸度大於0.7%，與市售產品的酸鹼值相近；且兩種樣品中的活菌均高於109 CFU/g，符合國家標準CNS3058活菌發酵乳的菌數（每毫升菌數高於107 CFU）。離水性以及流變能力的測定的結果也顯示APL15、APL16和添加安定劑的商業發酵乳產品沒有顯著差異 (P>0.05) ，證明產黏乳酸菌所產生的胞外多醣可以作為低脂發酵乳的安定劑。在酸及膽鹽耐受性測中，pH 3和膽鹽0.5%是APL15和APL16的致死條件；當pH達2.5、膽鹽濃度1%時APL15和APL16皆無法存活，未來可利用適應性實驗或是微膠囊包覆的形式使通過人體腸胃道的菌數提升。在產品保存試驗中組別APL15號APL16在pH值、酸度以及離水性在保存期間並無顯著差異 (P>0.05) ，然而菌數在十四天皆顯著降低 (P<0.05) 且低於107 CFU/g。冷凍乾燥後兩種菌株菌粉中的菌數都高於108 CFU/g，保存兩個月後仍然可以進行發酵乳製作，且以凍乾菌粉製作之發酵乳酸鹼值、可滴定酸度以及發酵乳中菌數與以凍管製作的產品並無太大差異。
根據上述研究結果，產黏乳酸菌所產生的胞外多醣可以減少或代替商業安定劑。

關鍵字

臺灣傳統黏質發酵乳 ； 潔淨標示 ； 胞外多醣 ； 乳酸菌 ； 低脂

並列摘要

Low-fat dairy products with reduced calories and fat might have the good market potential to meet the consumers’ need. However, removal of fat in dairy products creates several physical defects including poor texture, black color development and lack of flavor. During the process, the addition of polysaccharides could perform as stabilizers or water-binding agents to increase fermented dairy properties. Additionally, the consumers are preferring to buy “clean label” products with less artificial food additives. Exopolysaccharides (EPS) from lactic acid bacteria (LAB) are widely used in the dairy products such as yogurt, cheese, and butter. Based on their EPS-producing ability, LAB could be classified into ropy and nonropy. Ropy starter cultures could produce EPS, a natural water-binding agent, which improves moisture retention and reduces whey off in dairy products. Taiwanese ropy fermented milk (TRFM) is a dairy product showing great extension ability and viscosity. It is also referring to Taiwanese viili due to the similarity of texture and flavor to a traditional Nordic fermented milk, viili. In this study, we would like produce a fermented milk with ropy LAB, isolated from TRFM.
A total of 40 isolates were selected from Taiwanese ropy fermented milk. After Gram staining test to remove Gram-negative bacteria and yeast, 34 isolates were remained. Isolates were then selected according to the flame-like suspension in MRS broth. These isolates were further typed and identified using random amplified polymorphic DNA-polymerase chain reaction PCR (RAPD-PCR), 16S rRNA sequencing and API CHL 50. Results indicated that 13 isolates were categorized into 2 different groups based on their RAPD-PCR profiles. All of them were identified as Lactococcus lactis subsp. cremoris using 16s rRNA gene analysis and API50. One strain was picked from each group and compared their EPS production. No significant difference was found between Lactococcus lactis subsp. cremoris APL15 (APL15) and Lactococcus lactis subsp. cremoris APL16 (APL16) in EPS production (P>0.05). Therefore, both strains were selected for fermented milk production.
For fermented milk production, 1% APL15 and APL16 isolated from TRFM were inoculated in low fat milk at 26°C for 24 hours. Physiochemical and microbial properties were analyzed. Results indicated that both samples with APL15 and APL16 could reach pH 4.3 and acidity 0.7% in 24 hours, which is a necessary condition for a commercial fermented milk product. Viable cell counts in both samples were higher than 109 CFU/g, an important characteristic for probiotic products to provide health benefits on human beings. The results of syneresis and rheology tests also showed that no difference was found among samples and commercial fermented milk product with stabilizer, indicating EPS from LAB can act as stabilizer in products. For gastrointestinal stresses test, pH 3 and bile salt 0.5% were lethal conditions for APL15 and APL16. Stress adaptation and microencapsulation might improve survival rate under human gastrointestinal stresses. After freeze drying, the bacterial counts for both strains were higher than 108 CFU/g.
This study showed that it was possible to reduce or replace commercial stabilizers in the low-fat fermented milk production with EPS producing LAB.

並列關鍵字

Taiwanese ropy fermented milk ； clean label ； exopolysaccharide ； lactic acid bacteria ； low fat

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