Lactobacillus kefiranofaciens M1對不同壓力之反應及其於噴霧乾燥和冷凍乾燥後存活之探討

Abstract

本研究之目的為探討Lactobacillus kefiranofaciens M1對不同環境壓力包括熱、冷、酸、膽鹽、氯化鈉、乙醇和過氧化氫之適應性和交叉保護反應。測定壓力適應對L. kefiranofaciens M1生理特性 (生長、酸化活性、β-半乳糖苷酶活性、胞外多醣體產量、脂肪酸組成、蛋白質表現和菌體形態) 之影響。此外，測試壓力適應和未適應之菌體於噴霧乾燥、冷凍乾燥及後續貯存與模擬消化期間之存活。試驗中亦對噴霧和冷凍乾燥L. kefiranofaciens M1粉末之水分含量和形態及其於不同復水培養基之復甦情形進行探討。 結果顯示，除氯化鈉 (0.1 M) 適應外，熱 (37°C)、冷 (20°C)、酸 (pH 5.0)、膽鹽 (0.05%)、乙醇 (4%) 和過氧化氫 (100 ppm) 適應均可誘導L. kefiranofaciens M1之同源壓力忍受性。膽鹽、鹽、乙醇和過氧化氫適應可增加菌體之耐熱性 (52°C)；酸、膽鹽、鹽、乙醇或過氧化氫適應可賦予菌體耐冷性 (-20°C)；膽鹽和乙醇適應可增加耐酸性 (pH 3.0)；所有壓力適應均可誘導對膽鹽 (0.2%) 之交叉保護作用；冷、乙醇或過氧化氫適應可提升對氯化鈉 (3.0 M) 和乙醇 (20%) 之抵抗力；熱、酸或膽鹽適應可導致菌體過氧化氫 (1000 ppm) 忍受性之提升。 壓力適應組菌體相較於未適應組菌體，具有相同之生長、酸化活性和胞外多醣體產量。熱和酸適應組菌體較未適應組菌體呈現較高之β-半乳糖苷酶活性。除冷適應外，所有壓力適應可增加L. kefiranofaciens M1菌體之飽和脂肪酸總量，減少不飽和脂肪酸總量。二維電泳分析顯示熱、冷、酸、膽鹽、氯化鈉、乙醇和過氧化氫適應處理分別改變菌體15、21、21、17、22、20和17種蛋白質之表現量，這些壓力蛋白質可被一個以上之壓力誘導，主要與壓力反應、蛋白質生合成、能量和醣類代謝功能相關。此外，與未適應組菌體相較，壓力適應組菌體在後續同源壓力挑戰下呈現較輕微之細胞表面傷害。 另外，試驗發現酸和膽鹽適應可增加L. kefiranofaciens M1於噴霧和冷凍乾燥後之存活。大致上，酸和膽鹽適應組菌體相較於未適應組菌體於後續貯存於-20、4和25℃及暴露模擬胃液 (pH 2.0) 和腸液 (pH 8.0) 期間可保持較高之存活菌數。此外，噴霧乾燥L. kefiranofaciens M1粉末呈現球狀，而冷凍乾燥粉末則呈現不規則狀。噴霧和冷凍乾燥粉末之水分含量分別為6.84和1.97%。試驗中所試8種復水培養基對噴霧和冷凍乾燥菌體呈現不同之復甦效果，其中菌體在0.85% NaCl、0.1% peptone、磷酸鹽緩衝液 (phosphate-buffered saline, PBS)、analog man (AM) buffer和10% 重組脫脂牛乳 (reconstituted skim milk, RSM) 中相較在dd H2O、maximum recovery diluent (MRD) 和20% sucrose中，可具有較高之復甦菌數。

The objective of this study was to investigate the adaptive and cross-protective responses of Lactobacillus kefiranofaciens M1 to various environmental stresses, including heat, cold, acid, bile salts, sodium chloride, ethanol and hydrogen peroxide. The effects of stress adaptation on the physiological characteristics (growth, acidification activity, β-galactosidase activity, exopolysaccharide production, fatty acid composition, protein expression and cell morphology) of L. kefiranofaciens M1 were evaluated. Furthermore, survival of stress-adapted and non-adapted cells during spray drying, freeze drying and subsequent storage as well as simulated gastrointestinal digestion was examined. The moisture content and morphology of spray-dried and freeze-dried L. kefiranofaciens M1 powders and their recoveries in different rehydration media were also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat (37°C), cold (20°C), acid (pH 5.0), bile salts (0.05%), ethanol (4%) and hydrogen peroxide (100 ppm) stresses, except for sodium chloride (0.1 M), could induce homologous tolerance. Adaptation to bile salts, sodium chloride, ethanol and hydrogen peroxide increased heat (52°C) tolerance. Acid, bile salts, sodium chloride, ethanol or hydrogen peroxide adaptation provided protection against cold (-20°C) stress. Adaptation to bile salts and ethanol led to increased resistance to acid (pH 3.0) challenge. All stress adaptation induced cross-protection against bile salts (0.2%). Cold, ethanol or hydrogen peroxide adaptation enhanced the resistance against sodium chloride (3.0 M) and ethanol (20%) challenges. Pretreatment with heat, acid or bile salts caused an increase in hydrogen peroxide (1000 ppm) tolerance. All stress-adapted cells showed similar growth, acidification activity and exopolysaccharide production when compared with non-adapted cells. Heat- and acid-adapted cells exhibited higher β-galactosidase activity than non-adapted cells. All treatments of stress adaptation, except for cold adaptation, increased the content of saturated fatty acids and decreased the content of unsaturated fatty acids in cells of L. kefiranofaciens M1. Two-dimensional SDS-PAGE analysis revealed that the expression levels of 15, 21, 21, 17, 22, 20 and 17 proteins were altered by heat, cold, acid, bile salts, sodium chloride, ethanol and hydrogen peroxide treatments, respectively. Several stress proteins involved in stress response, protein biosynthesis, energy and carbohydrate metabolism were induced by more than one treatment. In addition, stress-adapted cells of L. kefiranofaciens M1 showed a relatively minor cell surface damage under subsequent homologous stress challenge compared with their non-adapted cells. On the other hand, it was found that adaptation to acid and bile salts increased the survival of L. kefiranofaciens M1 after spray drying and freeze drying. In general, acid- and bile salt-adapted cells maintained higher bacterial counts during subsequent storage at -20, 4 and 25℃ and exposure to simulated gastric (pH 2.0) and intestinal juices (pH 8.0) than non-adapted cells. Moreover, spray-dried L. kefiranofaciens M1 powders appeared globular in shape while freeze-dried powders had angular shapes. The moisture content of spray- and freeze-dried powders was 6.84 and 1.97%, respectively. Eight rehydration media tested showed different capacities for recovery of spray- and freeze-dried L. kefiranofaciens M1. Among them, 0.85% NaCl solution, 0.1% peptone solution, phosphate-buffered saline (PBS), analog man (AM) buffer and 10% reconstituted skim milk (RSM) yielded greater recovery counts of L. kefiranofaciens M1 than double distilled water (dd H2O), maximum recovery diluent (MRD) and 20% sucrose solution.