從克弗爾粒中篩選具降膽固醇能力之潛力益生菌株並探討其可能之機制

Abstract

克弗爾是源自於高加索山的傳統發酵乳，為乳酸菌及酵母菌同質共生之克弗爾粒發酵而成，具有酸味、氣泡的口感及些許的酒精風味。先前研究證實，本實驗室之新竹組克弗爾，於餵食正常飼糧及高膽固醇飼糧之兩試驗組別中，皆具有降低倉鼠血液中總膽固醇、非高密度脂蛋白（non-high density lipoprotein）膽固醇及肝臟中總膽固醇之能力。故本研究由新竹組克弗爾粒中，篩選降膽固醇能力較佳之菌株，調查其可能之膽固醇移除機制，並且檢測其潛力益生菌之相關特性。 本研究由新竹組克弗爾粒中分離出38株乳酸菌，利用體外試驗檢測其膽固醇移除能力，加以篩選出具較高移除能力之菌株24、26及30，進一步利用16S rDNA定序進行鑑定。結果顯示，此三菌株皆為Lactobacillus plantarum。接下來檢測菌株於活細胞、休眠細胞（懸浮於0.85%氯化鈉溶液）及死細胞（高壓蒸氣滅菌）狀態之膽固醇移除能力，以瞭解其可能之膽固醇移除機制。結果顯示，三株菌於休眠細胞狀態皆具有移除能力，然於死細胞狀態僅有菌株26表現出高於20%之膽固醇移除能力。此研究指出菌株細胞壁直接吸附膽固醇，為可能移除機制之一。胞外多醣體（exopolysaccharide, EPS）與膽固醇移除能力具有關聯性，而菌株24、26及30之EPS產量分別為270、268及231 mg/L，三株菌之產量無顯著性差異。除此之外，考量到菌株開發為益生菌之可能，本研究同時測試菌株於腸道環境之耐受能力及貼附能力，結果顯示三菌株皆有高膽鹽耐受能力，而於酸處理環境下，菌數約降低1-2個對數值，然而此三菌株並未表現出良好的細胞貼附能力。 綜合上述，由克弗爾粒中所分離之Lb. plantarum 24、26及30具有膽固醇移除之機能性，為潛力益生菌株，而此三菌株之可能移除機制為物理性吸附及高產EPS之特性。

Kefir is a traditional fermented milk which originated in the Caucasus Mountains. This beverage, fermented by kefir grains, a symbiotic culture of yeasts and lactic acid bacteria, has sour, carbonated and slightly alcoholic flavor. In our previous study, we found that Hsin-Chu kefir maintained in our laboratory could lower hamsters’ total cholesterol and non-high density lipoprotein (N-HDL) cholesterol level in serum on both cholesterol-free and cholesterol-enriched diets groups. Total cholesterol concentration in liver on different diets was significantly decreased in Hsin-Chu kefir group as well. In the present study, we further screened the strains from Hsin-Chu kefir grains with high cholesterol removing abilities. The possible cholesterol removing mechanisms and potential characteristics of probiotics were also investigated. Thirty-eight lactic acid bacteria strains were isolated from Hsin-Chu kefir grain and evaluated their cholesterol removing abilities in vitro. Three strains, 24, 26 and 30, demonstrating the highest cholesterol removing rate, were selected. We further identified the selected strains using 16S rDNA sequencing. Three strains are all Lactobacillus plantarum. For investigating the possible mechanisms, we evaluated cholesterol removing ability of resting cells（suspended in 0.85% sodium chloride solution）and dead cells（autoclaved）. Three strains in resting condition showed cholesterol removing ability, whereas, only strain 26 possessed 20% cholesterol removing ability in dead condition. The findings suggested that the cell wall binding might be one of mechanisms. Exopolysaccharide（EPS）also associated with reducing cholesterol. We then examined EPS producing abilities of the selected strains in MRS broth. The EPS levels of strains 24, 26 and 30 were 270, 268 and 231 mg/L, respectively. There are no significant differences among three strains. Additionally, for further application as probiotics, the tolerance in intestinal condition and adherence ability of the selected strains were also investigated. Results indicated the three strains possessed good bile salt tolerance. They also could survive under acid condition with about 1-2 log reduction. On the contrary, the strains didn’t demonstrated a good cell adherence ability. In conclusion, Lactobacillus plantarum strains 24, 26 and 30 isolated from kefir grains possessed cholesterol removing abilities. Physical binding and high EPS production might be the possible mechanisms associating with this functionality. This three strains can be potential probiotics strains. Key words: Cholesterol removing, Lactobacillus plantarum, Kefir grains, Probiotics