Centre of Advanced Faculty Training in Animal Nutrition, ICAR-Indian Veterinary Research Institute, Izatnagar, UP, India.
Division of Epidemiology, ICAR-Indian Veterinary Research Institute, Izatnagar, UP, India.
PLoS One. 2018 Mar 8;13(3):e0192978. doi: 10.1371/journal.pone.0192978. eCollection 2018.
The present study was aimed to develop an effective probiotic lactic acid bacteria (LAB) from piglet feces and in vitro characterization of probiotic properties. To confirm host-species specificity of probiotics, the efficacy of isolated LAB on growth, nutrient utilization, health and antioxidant status was observed in early weaned piglets. A total of 30 LAB were isolated from feces of five healthy piglets (28d old). All isolates were Gram positive, cocco-bacilli and catalase negative. Out of thirty LAB isolates, twenty were shortlisted on the basis of their tolerance to pH (3.0, 4.0, 7.0 and 8.0) and bile salts (0.075, 0.15, 0.3 and 1.0%). Whereas, fourteen isolates were selected for further in vitro probiotic characterization due higher (P<0.05) cell surface hydrophobicity to toluene (>45 percent). These isolates fermented twenty-seven different carbohydrates but were negative for ONPG, citrate and malonate. Also enabled to synthesize amylase, protease, lipase and phytase. They were sensitive to penicillin, azithromycin, lincomycin, clindamycin, erythromycin, cephalothin and chloramphenicol and resistant to ciprofloxacin, ofloxacin, gatifloxacin, vancomycin and co-trimoxazole. Except three isolates, all showed antagonistic activity (>60% co-culture activity) against Escherichia coli, Salmonella Enteritidis, Salmonella serotype (ser.) Typhimurium, Staphylococcus intermedius, Staph. chromogenes, Proteus mirabillis, Areomonas veonii, Bordetella bronchioseptica and Klebsialla oxytoca. The isolate Lacp28 exhibited highest tolerance to acidic pH and bile salts (up to 0.3%), phytase activity, cell surface hydrophobicity, antagonistic activity and co-culture assay (>80% growth inhibition). Host specificity of Lacp28 was further confirmed by heavy in vitro adhesion to pig intestinal epithelium cells compared to chicken. Hence, Lacp28 was selected and identified by phylogenetic analysis of 16S rRNA as Pediococcus acidilactici strain FT28 with 100% similarity (GenBank accession nos. KU837245, KU837246 and KU837247). The Pediococcus acidilactici FT28 was selected as potential probiotic candidature for in vivo efficacy in weaned pigs. Thirty-six crossbred piglets (28d) were randomly distributed into three groups (four replicates of three each) namely, basal diet without probiotics (T0) or with Lactobacillus acidophilus NCDC15 (conventional dairy-specific probiotic; T1) or Pediococcus acidilactici FT28 (swine-specific probiotic; T2). At end of the experiment, six piglets of similar body weight were selected to conduct digestion trial for estimation of nutrient digestibility. Results of the study indicated that supplementation of both probiotics improved (P<0.001) FCR compared to control without significant effect in average daily gain and DM intake. However, the apparent digestibility of crude protein and ether extract was better (P<0.01) in pigs fed P. acidilactici FT28 compared control and L. acidophilus fed groups. The total WBC and RBC count, serum glucose, total protein, albumin and globulin concentration was higher (P<0.05) in P. acidilactici FT28 fed group with better (P<0.05) catalase and superoxide dismutase activity measured in erythrocyte. It is concluded that species-specific Pediococcus acidilactici FT28 isolated with potential in vitro probiotic properties and also hold probiotic candidature by showing the potential capabilities with higher nutrient digestibility, heamato-biochemical and antioxidant status compared to control and Lactobacillus acidophilus NCDC15.
本研究旨在从仔猪粪便中开发出一种有效的益生菌乳酸菌(LAB),并对其体外益生特性进行表征。为了确认益生菌的宿主特异性,观察分离的 LAB 对早期断奶仔猪生长、营养利用、健康和抗氧化状态的影响。从 5 只健康仔猪(28 日龄)的粪便中分离出 30 株 LAB。所有分离株均为革兰氏阳性、球菌杆菌和过氧化氢酶阴性。在 30 株 LAB 分离株中,有 20 株根据其对 pH(3.0、4.0、7.0 和 8.0)和胆盐(0.075、0.15、0.3 和 1.0%)的耐受性进行了筛选。而 14 株分离株由于较高的细胞表面疏水性(甲苯>45%)而被选为进一步的体外益生菌特性研究。这些分离株发酵 27 种不同的碳水化合物,但对 ONPG、柠檬酸和丙二酸呈阴性。还能够合成淀粉酶、蛋白酶、脂肪酶和植酸酶。它们对青霉素、阿奇霉素、林可霉素、克林霉素、红霉素、头孢噻吩和氯霉素敏感,对环丙沙星、氧氟沙星、加替沙星、万古霉素和复方新诺明耐药。除了 3 株分离株外,所有分离株对大肠杆菌、肠炎沙门氏菌、沙门氏菌血清型(血清)鼠伤寒、中间葡萄球菌、产色葡萄球菌、奇异变形杆菌、魏氏梭菌、支气管败血波氏杆菌和产酸克雷伯菌均表现出拮抗活性(共培养活性>60%)。分离株 Lacp28 对酸性 pH 和胆盐(高达 0.3%)、植酸酶活性、细胞表面疏水性、拮抗活性和共培养试验表现出最高的耐受性(>80%的生长抑制)。Lacp28 的宿主特异性通过与鸡相比,其在体外对猪肠上皮细胞的高粘附性进一步得到证实。因此,Lacp28 通过 16S rRNA 的系统发育分析被鉴定为戊糖片球菌 FT28,相似度为 100%(GenBank 登录号为 KU837245、KU837246 和 KU837247)。戊糖片球菌 FT28 被选为在断奶仔猪中进行体内功效的潜在益生菌候选物。36 头杂交仔猪(28 日龄)随机分为三组(每组 4 个重复,每个重复 3 头),即基础日粮中不含益生菌(T0)或添加嗜酸乳杆菌 NCDC15(常规乳制品专用益生菌;T1)或戊糖片球菌 FT28(猪专用益生菌;T2)。在试验结束时,选择 6 头体重相似的仔猪进行消化试验,以估计养分消化率。研究结果表明,与对照组相比,添加两种益生菌均显著改善了 FCR(P<0.001),但对平均日增重和 DM 摄入量无显著影响。然而,与对照组和嗜酸乳杆菌喂养组相比,戊糖片球菌 FT28 喂养组的粗蛋白和乙醚提取物的表观消化率更好(P<0.01)。与对照组和嗜酸乳杆菌喂养组相比,戊糖片球菌 FT28 喂养组的总白细胞和红细胞计数、血清葡萄糖、总蛋白、白蛋白和球蛋白浓度更高(P<0.05),红细胞中的过氧化氢酶和超氧化物歧化酶活性也更高(P<0.05)。结论是,从仔猪粪便中分离出的戊糖片球菌 FT28 具有潜在的体外益生菌特性,并且通过显示出更高的营养物质消化率、血液生化和抗氧化状态的潜力,与对照组和嗜酸乳杆菌 NCDC15 相比,也具有益生菌候选物的潜力。