Selection and characterization of probiotic lactic acid bacteria and its impact on growth, nutrient digestibility, health and antioxidant status in weaned piglets

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分离获得高效的益生菌性乳酸菌（Lactic Acid Bacteria，LAB），并对其益生菌特性开展体外表征。为验证益生菌的宿主物种特异性，本研究观察了分离得到的LAB对早期断奶仔猪生长性能、营养物质利用、健康状况及抗氧化状态的影响。

本研究从5头28日龄健康仔猪的粪便中共分离得到30株LAB。所有分离株均为革兰氏阳性、球杆菌且过氧化氢酶阴性。在30株LAB分离株中，根据其对pH（3.0、4.0、7.0和8.0）及胆盐（0.075%、0.15%、0.3%和1.0%）的耐受性，筛选出20株候选菌株。其中14株因对甲苯的细胞表面疏水性较高（P<0.05，>45%），被选作进一步的体外益生菌特性研究。

该14株分离株可发酵27种不同的碳水化合物，但对邻硝基苯基-β-D-半乳糖苷（ONPG）、柠檬酸盐及丙二酸盐呈阴性反应；同时能够合成淀粉酶、蛋白酶、脂肪酶及植酸酶。它们对青霉素、阿奇霉素、林可霉素、克林霉素、红霉素、头孢噻吩及氯霉素敏感，而对环丙沙星、氧氟沙星、加替沙星、万古霉素及复方新诺明耐药。除3株分离株外，其余菌株对大肠杆菌（Escherichia coli）、肠炎沙门氏菌（Salmonella Enteritidis）、鼠伤寒沙门氏菌血清型（Salmonella ser. Typhimurium）、中间葡萄球菌（Staphylococcus intermedius）、产色葡萄球菌（Staphylococcus chromogenes）、奇异变形杆菌（Proteus mirabilis）、维氏气单胞菌（Aeromonas veonii）、支气管败血波氏杆菌（Bordetella bronchiseptica）及产酸克雷伯菌（Klebsiella oxytoca）具有拮抗活性（共培养抑制率>60%）。

菌株Lacp28对酸性pH及胆盐（最高可达0.3%）的耐受性、植酸酶活性、细胞表面疏水性、拮抗活性及共培养生长抑制效果（>80%）均为所有分离株中最高。与鸡源菌株相比，Lacp28可在体外强力黏附仔猪肠上皮细胞，进一步验证了其宿主特异性。因此，本研究通过16S rRNA系统发育分析，将Lacp28鉴定为乳酸片球菌（Pediococcus acidilactici）菌株FT28，同源性达100%（GenBank登录号：KU837245、KU837246及KU837247）。乳酸片球菌FT28被选为潜在益生菌候选株，用于断奶仔猪的体内功效评价。

本试验将36头28日龄杂交仔猪随机分为3组（每组设4个重复，每个重复3头仔猪），分别为基础日粮组（T0，无益生菌添加）、添加嗜酸乳杆菌（Lactobacillus acidophilus）NCDC15组（T1，常规乳品源益生菌）及添加乳酸片球菌FT28组（T2，猪源特异性益生菌）。试验结束时，选取体重相近的6头仔猪进行消化试验，以评估营养物质消化率。

试验结果表明，与对照组相比，添加两种益生菌均可显著改善（P<0.001）料重比（Feed Conversion Ratio，FCR），但对平均日增重及干物质采食量无显著影响。然而，与对照组及嗜酸乳杆菌添加组相比，饲喂乳酸片球菌FT28的仔猪其粗蛋白质及乙醚浸出物的表观消化率更高（P<0.01）。饲喂乳酸片球菌FT28的仔猪的白细胞计数（White Blood Cell，WBC）、红细胞计数（Red Blood Cell，RBC）、血清葡萄糖、总蛋白、白蛋白及球蛋白浓度均显著升高（P<0.05），且红细胞中的过氧化氢酶及超氧化物歧化酶活性也显著提升（P<0.05）。

综上，本研究分离得到的猪源特异性乳酸片球菌FT28具备优异的体外益生菌特性，与对照组及嗜酸乳杆菌NCDC15组相比，其可显著提升营养物质消化率、血液生化指标及抗氧化状态，因此是极具潜力的益生菌候选株。