微膠囊化對Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1耐熱及抗結腸炎機能性之影響

Abstract

本研究之目的乃開發可耐熱益生菌微膠囊，改善益生菌在高溫環境及儲存期間的存活率。研究中選用結蘭膠與褐藻酸鈉兩種食用膠與脫脂乳粉，混合成膠作為囊壁材質分別包覆益生菌Lactobacillus kefiranofaciens M1（M1）及Lb. mali APS1（APS1）。經耐熱試驗後選定囊壁材質添加比例濃度，結蘭膠、褐藻酸鈉與脫脂乳粉的比例分別是：1%、2%、10%。以此濃度製作微膠囊，冷凍乾燥後保存於4oC，測試其儲存期間、儲存後再經熱處理（水浴自25oC加熱至75oC，並放置1分鐘）與先經熱處理再經模擬腸胃環境之存活菌數。M1與APS1經包覆凍乾後的起始菌數分別為8.3和10.3 log CFU/g，，經8週的儲存後，M1和APS1菌數各能維持於6.6與9.7 log CFU/g以上；而儲存8週再加熱後，仍分別有5.8和9.2 log CFU/g的存活菌數；熱處理後再以模擬胃液處理，菌數平均下降0.3和0.5 log CFU/g；先加熱再以膽鹽處理則平均下降0.5與2.1 log CFU/g。 為了解益生菌經微膠囊化後，對菌株原有機能性之影響，因此以小鼠結腸炎模式評估經熱處理的益生菌微膠囊是否仍具有抗結腸炎的效果。結果發現包覆凍乾之M1（HCM1）與APS1（HCAPS1）可顯著降低糞便出血評分，減緩體重減輕及恢復結腸縮短之情形，且於組織切片觀察到HCAPS1可明顯舒緩腸道發炎。然而在小鼠盲腸內容物菌相分析中，內容物的乳酸桿菌與大腸桿菌群菌數比例在各組間並無差異。進一步探討HCM1與HCAPS1之抗腸炎可能機制，將益生菌微膠囊與人類腸道上皮細胞共培養，結果顯示HCM1與HCAPS1無法顯著提升跨上皮電阻值及趨化素CCL20的分泌量，由此推論加強上皮屏障可能並非HCM1與HCAPS1抗腸炎的可能機制。 由以上結果得知褐藻膠、結蘭膠與脫脂乳粉混合作為囊壁材質，具有提升菌株在儲存期間、高溫環境及模擬腸胃液中的存活率。另外在抗腸炎動物試驗當中，發現經熱處理的益生菌微膠囊仍可發揮其機能性，但其作用機制有待進一步研究探討。未來有機會應用在添加於溫熱飲品中的益生菌微膠囊，以提升產品的機能性。

Microbial viability is an important factor to exert health benefits, especially in host’s lower digestive tract. However, most of microorganisms cannot stand the adverse conditions like industrial heating processes and the digestive juice. Hence the purpose of the study was to develop heat-tolerant probiotic microcapsules to improve the survival of probiotics under heat treatment and the passage through the simulated gastrointestinal fluid (SGIF). The functionality of encapsulated probiotics was also evaluated. Gellan gum, sodium alginate and skim milk powder were incorporated as coating materials to encapsulate Lactobacillus kefiranofaciens M1 (M1) and Lb. mali APS1 (APS1) separately. The optimal proportion of gellan gum, sodium alginate and skim milk powder in microcapsules was determined by preliminary thermal-tolerant test. The bacterial counts in freeze-dried microcapsules stored at 4 oC were determined at different storage periods and heat treatments, followed by treatment of the SGIF. The initial bacteria counts of M1 and APS1 were 8.3 and 10.3 log CFU/g. The bacteria counts of M1 and APS1 after 8 weeks storage were 6.6 and 9.7 log CFU/g. After heat treatment, the bacteria numbers dropped to 5.8 and 9.2 log CFU/g. The viability of encapsulated M1 and APS1 were above 5.0 log CFU/g and 7.0 log CFU/g in SGIF test. On the contrary, the two free strains cannot detect viable bacteria counts after 8 weeks among these tests. Since both M1and APS1 demonstrated an anti-colitis effect in our previous study, we further investigated the anti-colitis effect of encapsulated these two strains in vivo and in vitro. Both heated encapsulated M1 and APS1 could significantly ameliorate the symptoms of DSS-induced colitis, including bleeding score, weight losing and colon length shortening, when compared with non-encapsulated groups in vivo. It is worth to notice that the distribution of lactobacilli and coliform in cecum was no difference among all experimental groups. We further studied the possible mechanism involved in the anti-colitis effect in vitro. The results indicated that both encapsulated strains could significantly increase the transepithelial electrical resistance (TEER) in Caco2 monolayer when compared with the blank group. However, the elevation of TEER value and CCL20 production was not found in the heated encapsulated strains. These findings revealed that encapsulated strains with heat-stable coating materials could elevate the viability during storage, heat resistance and simulated gastrointestinal conditions. Furthermore, the heat encapsulated strains demonstrated an anti-colitis effect in vivo. The heat-tolerant microcapsule might provide a high potential to apply the probiotics in warm drinks in the near future.