利用體外及小鼠試驗篩選促進乳牛健康之潛力益生菌組合

Abstract

益生菌的精準餵食方法可以解決益生菌菌株、宿主及其微生物組中固有的異質性。實驗室先前研究發現，Bifidobacterium longum 和 Ruminococcus flavefaciens 在腸道中的相對豐富度對牛隻的健康有顯著的影響。然而，目前 R. flavefaciens 並非行政院農委會所認可的飼料添加劑。先前有研究顯示餵食牛隻 Saccharomyces cerevisiae 可以顯著提高其瘤胃中 R. flavefaciens 的相對豐富度。因此，S. cerevisiae 和 B. longum 可能是促進牛隻健康之潛力益生菌。 首先，我們從健康仔牛糞便中分離出乳酸菌及從水果中分離出 S. cerevisiae。從仔牛糞便中分離出共 57 株乳酸菌，並透過 16S rRNA 基因序列進行定序。 初步篩選乳酸菌菌株的條件為具有抑制牧場常見之病原菌。結果顯示，共 10 個菌株分别具有抑制 Salmonella enterica 及 Staphylococcus aureus 的能力。但其中只有 6 株乳酸菌目前是被行政院農委會認可的飼料添加劑。接續使用 RAW264.7 细胞在脂多糖刺激之下與乳酸菌菌株共培養作為篩選潛力益生菌株。結果發現，Ligilactobacillus salivarius K108 具有同時抑制兩個病原菌及免疫調節的能力。此外，從水果中分離出57株酵母菌，經由 18S-26S ITS來進行鑑定。結果顯示其中 14 株被鑑定為 S. cerevisiae。進一步進行瘤胃體外發酵試驗來進行篩選 S. cerevisiae。在 24 小時的體外發酵過程中，S. cerevisiae T15 表現出較高的 pH 值和產生較多的微生物菌體蛋白。另外，本實驗室先前分離出 5 株 B. longum，其中 B. longum APL 30具有較好的免疫調節和抑制病原菌之能力。在進行益生菌單株篩選後，將 B. longum APL 30、L. salivarius K108 和 S. cerevisiae T15 組合並檢測其對免疫調節及瘤胃體外發酵之影響。結果發現，細胞在LPS 刺激之下，組合益生菌顯示出較高的 IL-10 和較低的 TNF-α 分泌量。在 24 小時瘤胃體外發酵後，有較高的丁酸產生；而在 48 小時瘤胃體外發酵後，有較高的丙酸和较低的乙酸/丙酸比例。 接著，使用 DSS 誘導小鼠結腸炎模式來釐清組合益生菌對抗發炎的能力。結果顯示，組合益生菌有能力緩解 DSS 誘導造成的下痢、糞便潛血及腸道通透性等症狀。給予高劑量組合益生菌可以增加抗發炎細胞素 IL-10 的分泌量、提高腸道中 Bifidobacterium 的菌數及腸道中丙酸的含量。此外，給予低劑量組合益生菌可以降低腸道中 Clostridium perfringens 的菌數。未來還需在乳牛場進行試驗，以進一步驗證此混合菌株對乳牛健康益處的影響。

A targeted method towards probiotics could effectively tackle the natural diversity present in probiotic strains, the hosts, and their microbiomes. Our previous research found that the abundance of Bifidobacterium longum and Ruminococcus flavefaciens in intestinal tracts positively affects dairy cattle health. However, R. flavefaciens is not yet allowed to be applied as a feed supplement by the Council of Agriculture, Taiwan. Several studies indicated that supplementation of Saccharomyces cerevisiae could significantly increase R. flavefaciens relative abundance. Thus, S. cerevisiae and B. longum might be potential probiotics for promoting dairy cows’ health. First, we aimed to isolate lactic acid bacteria (LAB) from healthy dairy calf feces and S. cerevisiae from fruits. Fifty-seven LAB were isolated and identified through the sequencing of 16S rRNA gene from calves’ feces. Isolates with the ability to inhibit common pathogens in dairy farms as the preliminary screening criteria. The results showed that 10 strains have the ability to inhibit Salmonella enterica and Staphylococcus aureus, respectively. Only 6 strains of LAB were allowed to be applied as feed supplements by the Council of Agriculture, Taiwan. In the in vitro screening, RAW264.7 cells stimulated by lipopolysaccharide (LPS) were used as a model. We found that Ligilactobacillus salivarius K108 has the ability to inhibit pathogens and immunomodulation. Besides, 57 isolates were obtained from fruits, using the 18S-26S ITS region to identify them. The results showed that 14 strains were confirmed as S. cerevisiae. We further evaluated the functionality of S. cerevisiae by in-vitro rumen fermentation. S. cerevisiae T15 showed higher pH value and microbial crude protein (MCP) production in 24-hour period fermentation. Additionally, 5 strains of B. longum were previously isolated in our lab, and B. longum APL 30 showed better immunomodulatory and anti-pathogen properties. After single strain screening, the combination of B. longum APL30, L. salivarius K108, and S. cerevisiae T15 was tested for their effects on immunomodulatory and in-vitro rumen fermentation. The probiotic mixture showed a higher IL-10 production and lower TNF-α when stimulated by LPS, higher butyrate after 24-hour in-vitro rumen fermentation, and a higher propionate and lower acetate/propionate ratio after 48-hour in-vitro rumen fermentation. Moreover, to clarify the ability of anti-inflammatory of the probiotic mixture, the induction of colitis using DSS was carried out in the mouse model. The findings indicated that the probiotic mixture has the ability to alleviate symptoms such as diarrhea, fecal bleeding, and gut leakage, which were induced by DSS. Administration of a high-dosage probiotic mixture has been shown to enhance the secretion of the anti-inflammatory cytokine IL-10, increase the bacterial count of Bifidobacterium, and elevate the level of propionic acid in the colon content of DSS-induced mice. However, administration of a low-dosage probiotic mixture has been shown to decrease the bacterial count of Clostridium perfringens. The field trial in dairy farms is also needed to verify the health benefits in dairy cattle.