Evaluation of genetically modified Lactobacillus reuteri Pg 4 containing xylanase gene as probiotic for poultry

本研究目的為探討由雞隻腸道篩選之乳酸桿菌Lactobacillus reuteri Pg 4 菌株，經轉殖聚木醣酵素(xylanase)基因後(以TX-Pg 4稱之)，評估作為家禽益生菌之可行性。首先以in vitro方法分析TX-Pg 4耐酸能力及吸附能力。結果顯示，TX-Pg 4菌株於PBS (pH 2.0)中培養1~3小時後，其菌數仍維持108 CFU/mL以上，且具吸附嗉囊上皮細胞之特性。此等特性與未轉殖之Lact. Pg 4菌株無明顯差異。以比色法測定其聚木醣酵素之活性，分別含胞外酵素1.91±0.37 U/mL，及胞內酵素2.51±0.25 U/mL；所含之酵素亦能以酵素擴散圈法測得。進一步以乾燥之菌末（109 CFU/g）添加0.1%於含小麥之肉雞飼糧，利用400隻肉雞進行飼養試驗。結果顯示，添加TX-Pg 4菌株之處理組顯著改善前期雞隻之體增重(699 vs. 664 g，P< 0.05)，但對後期生長性狀並無顯著效果。處理組全期雞隻腸道內容物之乳酸桿菌菌數均較對照組高，然無顯著差異。處理組全期雞隻之盲腸總揮發性脂肪酸(total volatile fatty acids)含量，均顯著較對照組高(3週齡時為189.35 vs. 140.45μmole/g；6週齡時為198.0 vs. 171.45 μmole/g，P< 0.05)。由於供試驗之TX-Pg 4菌株具有分泌聚木醣酵素之能力，可以水解小麥所含之聚木醣，因此在處理組前期雞隻之十二指腸內容物黏度亦有較對照組為低之趨勢(P<0.1)，但後期並無顯著差異。處理組之雞隻於生長後期亦顯示其排泄物之氨濃度顯著較對照組低(22.69 vs. 36.83 mM/g，P< 0.05)。進一步利用species-specific PCR檢測處理組雞隻之食糜，可檢測出TX-Pg 4菌株之存在。綜上所述，顯示轉殖xylanase之 Lact. reuteri Pg 4菌株可存活於雞隻腸道中，對餵予含小麥飼糧之雞隻具有增加前期體重，提高盲腸內容物之VFA濃度，及降低排泄物中氨濃度，因此TX-Pg 4可作為兼具分泌酵素之益生菌候選菌株。The aim of this study was to evaluate the probiotic characterization of xylanase gene transferred Lactobacillus reuteri Pg 4 strain(TX-Pg 4), which isolated form GI tract of poultry. According to the in vitro assay, the Pg 4 and TX-Pg 4 strain showed the ability to survive after one and three hours under incubating in PBS (pH 2.0). In addition, Pg 4 and TX-Pg 4 strains could adhere to epithelium cell of crop of broiler in the adhesive ability assay. After incubated in MRS broth for 8 hours, the viable counts of TX-Pg 4 strain was up to 1010 CFU/mL. It showed that there was no different of probiotic characterics between Pg 4 and TX-Pg 4. By colorimeteric method and enzyme radial diffusion method, TX-Pg 4 showed the xylanase activity (Intracellulal and extracellual: 2.78 and 1.91 U/mL), but Pg 4 was undetable. In order to study the effect of the TX-Pg 4 strain supplement in the diet on the growth performance of broiler under normal environment. We process the TX-Pg 4 culture powder(1010 CFU/g)and supplemented in wheat-base diet. The results showed that Lactobacillus counts in the content of ileum and caecum of TX-Pg 4 group were higher than the control group on 21 and 42 day-old of broilers. And improved the body weight (P<0.05) on 21 day-old. By colony-PCR methods, we also confirmed the TX-Pg 4 exact in the content of GI of broiler.The concentration of total VFA and lactic acid of caecum content of treatment group had significantly higher than control group (P<0.05), the mucosa histology of the ileum of treatment group had the same result. The concentration of ammonia of digesta of caecum of treatment group also lower than control group(P<0.05). So as in the excreta. To summarize, after evaluated by in vitro and in vivo assay, the TX-Pg 4 strain could survive in the broiler GI tract with activity of xylanase. TX-Pg 4 strain powder supplement in barley diet could decrease digesta viscosity and significantly improved the body weight (P