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1.
Three gut lactobacilli from piglets (Lactobacillus plantarum L 5, Lactobacillus paracasei L 81, Lactobacillus fermentum L 670) and Lactobacillus casei subsp. pseudoplantarum L.c.) from a calf were examined by microtitre plate binding assay for their lectin-like binding activity after their cultivation on Rogosa agar and in MRS broth. Three ECM (extracellular matrix) molecules (fetuin, porcine fibronectin and porcine mucin) were selected for this assay. Additionally, the effect of heparin on the binding of these three ECM molecules by Lactobacillus strains in microtitre plates was tested. Moreover, haemagglutination tests with pig, cattle, sheep, and hen erythrocytes were performed. However, none of the four Lactobacillus strains examined did react with any of the erythrocytes tested. The differences between individual strains were observed in their binding to immobilised ECM molecules. The best adherent was the Lactobacillus plantarum L5, however, the other three strains showed also good ECM binding. With regard to an influence of cultivation medium on lectin-like binding activity, binding of all ECM molecules was expressed in Lactobacillus paracasei L 81 to significantly higher degree (P < 0.001) after cultivation on Rogosa agar than in MRS broth. Similarly, strains Lactobacillus fermentum L 670 and Lactobacillus casei subsp. pseudoplantarum L.c. displayed significantly higher (P < 0.001) binding of fibronectin and mucin after growth on Rogosa agar in comparison with MRS broth cultivation. However, no significant (on fetuin and fibronectin binding) or opposite effect (on mucin binding) of cultivation medium was observed in Lactobacillus plantarum L 5 strain. The influence of cultivation medium on fetuin binding by Lactobacillus fermentum L 670 was also not significant while Lactobacillus casei subsp. pseudoplantarum L.c. bound fetuin significantly better (P < 0.01) after growth on Rogosa agar. Heparin pretreatment increased the binding of the ECM molecules by the Lactobacillus fermentum L 670 strain significantly (P < 0.001 or P < 0.05) with the exception of porcine fibronectin when the strain was cultivated in MRS broth. This result is important especially in the connection with the previous observations that heparin decreased ECM binding of enteropathogens as staphylococci or clinical enterococcal isolates. Following up on some earlier strain characteristics, these results indicate that the selected lactobacilli are probably suitable for probiotic purposes. 相似文献
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De Angelis M Siragusa S Caputo L Ragni A Burzigotti R Gobbetti M 《Veterinary microbiology》2007,123(1-3):133-144
Lactobacillus sp. are important inhabitants of the intestines of animals. They are also largely used as probiotics for both humans and animals. To exert beneficial effects, lactobacilli have to survive through the gastrointestinal transit. Based on bile-salt resistance, pH tolerance, antimicrobial activity and heat resistance, Lactobacillus plantarum 4.1 and Lactobacillus reuteri 3S7 were previously selected and used as probiotic additives in pelleted feeding trials. Both strains were fed to pigs (sows and piglets) at a cell number of ca. 10(10) viable cells per day. A polyphasic approach, comprising growth on selective media, Biolog System analysis, 16S rRNA gene sequencing and RAPD-PCR typing, was used to identify and differentiate L. plantarum 4.1 and L. reuteri 3S7 from other faecal Lactobacillus sp., L. plantarum 4.1 and L. reuteri 3S7 had the capacity to survive during the gastrointestinal transit and were found in the feaces at a cell number of 6-8 log cfu/g. Their persistence was shown after 6 days from the administration. Compared to untreated pigs, the administration of L. plantarum 4.1 and L. reuteri 3S7 significantly (P<0.05) decreased the population of Enterobacteriaceae. Besides, the beta-glucuronidase activity of all pigs decreased of ca. 23.0% during administration. The findings of this study showed that L. plantarum 4.1 and L. reuteri 3S7 have the potential to be used as probiotic additives in pelleted feed for pigs. 相似文献
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对国内外市售酸奶中添加的乳酸杆菌益生菌进行分离鉴定,对其产H2O2量及与宫颈癌HeLa细胞黏附能力进行分析,并初步判定酸奶中的乳酸杆菌是否对女性生殖道健康具有潜在的保健作用.实验共分离鉴定出10株乳酸杆菌,其中,嗜酸性乳杆菌5株、干酪乳杆菌1株、瑞士乳杆菌1株、鼠李糖乳杆菌1株、植物乳杆菌1株和德式乳杆菌1株.只有瑞士乳杆菌产H2O2量较高且与HeLa细胞的黏附作用较强,其余乳酸杆菌产H2O2的能力偏低.大部分国内外市售酸奶产品中添加的益生菌不具备抑制女性生殖道感染有关的生理生化特性,只有瑞士乳杆菌可能对女性生殖系统的健康具有保护作用. 相似文献
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以益生菌植物乳杆菌LIP-1为研究对象,MRS基础培养基为对照,通过改变培养基的成分及其含量（碳源、氮源、碳氮总量及碳氮比、缓冲盐）,研究培养基成分的变化对植物乳杆菌LIP-1高密度培养的活菌数以及冷冻干燥后存活率的影响。结果表明：通过优化工艺和配方确定的最佳培养基组成为碳源（蔗糖）添加量33.35 g/L、复合氮源（酵母粉、大豆蛋白胨、酪蛋白胨质量比3∶3∶5）添加量10.80 g/L、缓冲盐（乙酸钠、磷酸氢二钾、柠檬酸钠质量比2.5∶1∶1）添加量14.85 g/L（即0.13 mol/L）;用优化后 相似文献
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Influences of quorum‐quenching probiotic bacteria on the gut microbial community and immune function in weaning pigs 
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下载免费PDF全文 Jonggun Kim Jaepil Kim Younghoon Kim Sangnam Oh Minho Song Jee Hwan Choe Kwang‐Youn Whang Kwang Hyun Kim Sejong Oh 《Animal Science Journal》2018,89(2):412-422
The aim of this study is to investigate the dynamic gut microbial diversity in weaning swine after administering feed supplemented with probiotic bacteria that specifically inhibit the activity of quorum molecules. Initially, the universal quorum molecule autoinducer‐2 (AI‐2) bioassay results indicated that AI‐2 activity was profoundly inhibited in enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the presence of Lactobacillus acidophilus strain 30SC cell extract, although the growth of EHEC was not affected. Based on plate counting results, bacterial community analysis revealed a specific reduction in coliforms compared to the control, whereas the population of lactobacilli increased in weaning swine in in vivo trials. Supplementation with L. acidophilus strain 30SC did not affect the counts of other communities, such as total aerobes and yeast/mold. In addition, PCR‐denaturing gradient gel electrophoresis analysis showed a significant difference in the 16S rRNA gene products after administering L. acidophilus strain 30SC. Selected bands were sequenced, and most of them were identified as uncultured bacterium clones or a Lactobacillus‐ and Bifidobacterium‐specific community. Therefore, our results indicate that quorum‐quenching probiotic bacteria can significantly modulate the gut microbiota of swine and these beneficial effects can contribute to the improvement of performance and health in the gastrointestinal tract of weaning pigs. 相似文献
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以具有降血脂益生特性的植物乳杆菌LIP-1为研究对象,通过在培养基中添加不同浓度CaCl2,探究Ca2＋对菌株冷冻干燥抗性及耐热性的影响,并比较未加钙菌粉、加钙菌粉和未加钙市售菌粉在不同热饮中的活菌数。结果表明：与未添加Ca2＋对照组相比,培养基中添加0.5 mmol/L Ca2＋不仅可以显著提高菌株的冷冻干燥存活率（P＜0.05）,并可以改善其耐热性;与市售菌粉相比,加钙菌粉在热饮中的植物乳杆菌LIP-1存活率显著提高,探究其内在机制发现,加钙菌粉的植物乳杆菌LIP-1细胞膜与细胞壁损伤程度显著低于其他组别（P＜0.05）,同时细胞膜不饱和脂肪酸含量显著升高。上述结果表明,在培养基中添加Ca2＋能够显著提高植物乳杆菌LIP-1对冷热的抗性。 相似文献
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《畜牧与生物技术杂志(英文版)》2017,(4)
Background: The beneficial effects of Lactobacillus probiotics in animal production are often strain-related.Different strains from the same species may exert different weight-gain effect on hosts in vivo. Most lactobacilli are selected based on their in vitro activities, and their metabolism and regulation on the intestine based on strainrelated characters are largely unexplored. The objective of the present study was to study the in vivo effects of the three lactobacilli on growth performance and to compare the differential effects of the strains on the faecal microbiota and ileum mucosa proteomics of piglets.Methods: Three hundred and sixty piglets were assigned to one of four treatments, which included an antibioticstreated control and three experimental groups supplemented with the three lactobacilli, L. salivarius G1-1, L. reuteri G8-5 and L. reuteri G22-2, respectively. Piglets were weighed and the feed intake was recorded to compare the growth performance. The faecal lactobacilli and coliform was quantified using quantitative PCR and the faecal microbiota was profiled by denaturing gradient gel electrophoresis(DGGE). The proteomic approach was applied to compare the differential expression of proteins in the ileum mucosa.Results: No statistical difference was found among the three Lactobacillus-treated groups in animal growth performance compared with the antibiotics-treated group(P 0.05). Supplementation of lactobacilli in diets significantly increased the relative 16 S rRNA gene copies of Lactobacillus genus on both d 14 and d 28(P 0.05).,and the bacterial community profiles based on DGGE from the lactobacilli-treated groups were distinctly different from the antibiotics-treated group(P 0.05). The ileum mucosa of piglets responded to all Lactobacillus supplementation by producing more newly expressed proteins and the identified proteins were all associated with the functions beneficial for stabilization of cell structure. Besides, some other up-regulated and down-regulated proteins in different Lactobacillus-treated groups showed the expression of proteins were partly strain-related.Conclusions: All the three lactobacilli in this study show comparable effects to antibiotics on piglets growth performance. The three lactobacilli were found able to modify intestinal microbiota and mucosa proteomics. The regulation of protein expression in the intestinal mucosa are partly associated with the strains administrated in feed. 相似文献
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本试验旨在研究饲粮中添加植物乳杆菌(Lactobacillus plantarum)和非淀粉多糖(NSP)复合酶对断奶仔猪生长性能、粪便微生物菌群及血清指标的影响,从而达到代替抗生素的目的。试验采用单因素完全随机设计,选择28日龄健康断奶仔猪192头,平均体重为(7.15±0.36)kg,随机分为4个组,每组6个重复,每个重复8头猪。对照组(CT组)饲喂在基础饲粮中添加40 mg/kg杆菌肽锌的饲粮;试验组分别饲喂在基础饲粮中添加1 g/kg植物乳杆菌(PT组)、1 g/kg NSP复合酶(ZT组)、1 g/kg植物乳杆菌+1 g/kg NSP复合酶(PZT组)的饲粮。试验期35 d。结果表明:1)各组间断奶仔猪的试验末重、平均日增重、平均日采食量及料重比均无显著差异(P0.05)。2)各组间断奶仔猪粪便中乳酸菌数量、大肠杆菌数量及乳酸菌/大肠杆菌均无显著差异(P0.05)。但与对照组相比,PZT组断奶仔猪粪便中乳酸菌数量和乳酸菌/大肠杆菌有增加趋势(0.05≤P0.10),大肠杆菌数量有降低趋势(0.05≤P0.10)。3)各组间断奶仔猪的血清生化指标、免疫指标均无显著差异(P0.05)。由此可知,饲粮中复合添加植物乳杆菌和NSP复合酶可促进肠道菌群平衡,有益于仔猪健康生长,从而达到替代抗生素的目的。 相似文献
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应用高效液相色谱法(high performance liquid chromatography,HPLC)和SOS显色反应测定36株益生菌脱除4-硝基喹啉-1-氧化物(4-NQO)基因毒性的能力,同时用HPLC对它们的图谱进行比较分析。研究发现,基因毒性清除率高的菌株有1株植物乳杆菌、5株双歧杆菌、干酪乳杆菌代田株、3株芽孢杆菌、2株酵母菌、7株唾液乳杆菌和一株保加利亚乳杆菌。同属于一个菌属的各个菌株基因毒性清除率之间差异不显著。图谱分析发现,只有唾液乳杆菌才产生P1,即益生菌脱除4-NQO基因毒性具有菌株特异性。研究还发现益生菌转化4-NQO为4-HAQO的能力是决定益生菌脱除4-NQO基因毒性能力大小的关键。 相似文献
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《动物营养（英文）》2015,(3)
A total of 294 one-day-old Cobb broiler chickens were used to investigate the effects of four Lactobacillus strains on gut microbial profile and production performance. The six dietary treatments, each with 7 replicates were: 1) basal diet(negative control), 2) one of four strains of Lactobacillus(tentatively identified as Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus salivarius and an unidentified Lactobacillus sp.) and 3) basal diet with added zinc-bacitracin(ZnB, 50 mg/kg). Results showed that the addition of probiotic Lactobacillus spp. to the feed did not significantly improve weight gain, feed intake and feed conversion rate(FCR) of broiler chickens raised in cages during the 6-week experimental period,but tended to increase the number of total anaerobic bacteria in the ileum and caeca, and the number of lactic acid bacteria and lactobacilli in the caeca; and to significantly increase the small intestinal weight(jejunum and ileum). Furthermore, all 4 probiotics tended to reduce the number of Enterobacteria in the ileum, compared with the control treatments. The probiotics did not affect the p H and the concentrations of short chain fatty acids(SCFA) and lactic acid in both the ileum and caeca. 相似文献
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Two probiotics of different ecological origin, Enterococcus faecium NCIMB 10415 and Bacillus cereus var. toyoi, were chosen as model organisms. Feed for sows during gestation/lactation and for piglets pre-/postweaning was supplemented with either of these probiotics. To evaluate the effect of different starting points of E. faecium NCIMB 10415 initiations the probiotic was administered to piglets of sows, which have not received the probiotic, from birth onwards or just postweaning. Here we report the impact of these variants on probiotic distribution in the gut, on the gut microbiota, on diarrhea and on performance. Both probiotic strains were detected immediately after the start of the supplementation in feces of sows and piglets. The vertical transfer of both probiotic strains with sow feces to piglets could be demonstrated already before suckling piglets had access to the supplemented diets. Both probiotics were recovered from all intestinal segments of piglets. The dominant autochthonous colonic microbiota of young piglets as revealed by denaturing gradient gel electrophoresis was more similar within than between treatment groups (control vs. probiotic). Both probiotics reduced the incidence of postweaning diarrhea (p < 0.05). For the E. faecium probiotic the relative magnitude of this effect was largely independent of dietary probiotic concentration or starting time of supplementation. Significant overall influence on piglet performance was observed only with the B. cereus probiotic. 相似文献
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Qi Wang Qian Sun Renli Qi Jing Wang Xiaoyu Qiu Zuohua Liu Jinxiu Huang 《Journal of animal physiology and animal nutrition》2019,103(6):1908-1918
This study investigated the effect of Lactobacillus plantarum strain 299v on gut health in suckling piglets. Sixty newborn piglets were assigned to control and probiotic treatments, with three litters per treatment (ten piglets/litter). From days 1 to 20 of life, piglets were orally administered a placebo of 0.1% peptone or 1.0 × 1010 CFU L. plantarum 299v daily. Six piglets per treatment were sacrificed on day 20, and intestinal tissues (including duodenum, jejunum, ileum and colon) and the intestinal contents from colon segments were collected. The results demonstrated that piglets treated with L. plantarum 299v had a lower diarrhoea incidence than the controls. L. plantarum 299v administration significantly increased the ratio of the villus height to the crypt depth in the jejunum and ileum, as well as the mRNA expression of jejunal occludin and ileal zonula occludens 1 (ZO‐1). The L. plantarum treatment also increased the mRNA abundance of porcine β‐defensin 2 (pBD2) and pBD3 in the jejunum and ileum and of toll‐like receptors (TLRs), such as TLR2, TLR4, TLR6 and TLR9 in the ileum, and significantly upregulated the mRNA abundances of ileal pBD1 and colonic TLR4. Additionally, the L. plantarum 299v treatment significantly changed the structure of the colonic microbiota, as evidenced by the obvious increases in the relative abundances of the phyla Firmicutes and Actinobacteria and of the genus Lactobacillus. Our findings indicate that L. plantarum 299v facilitates the gut health of suckling piglets, probably by improving the intestinal morphology and intestinal barrier function and by modifying the structure of the gut microbiota. 相似文献
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本试验旨在筛选自然发酵苜蓿青贮的优势菌种,应用于高水分苜蓿青贮的调制,研究不同微生物及组合对高水分苜蓿青贮品质的影响。试验先调制自然发酵苜蓿,青贮30 d后,从中筛选优势菌种并进行鉴定。根据优势菌种筛选结果,组合了9组发酵菌种用于苜蓿青贮的调制,分别为:以植物乳杆菌与乳酸片球菌为发酵菌种,活菌数比例分别为1∶1、1∶2和2∶1,编号为1、2、3组;以植物乳杆菌、枯草芽孢杆菌和乳酸片球菌为发酵菌种,活菌数比例分别为2∶1∶1、1∶1∶1和4∶1∶1,编号为4、5、6组;以植物乳杆菌、解淀粉芽孢杆菌和乳酸片球菌为发酵菌种,活菌数比例分别为2∶1∶1、1∶1∶1和4∶1∶1,编号为7、8、9组;以不加任何菌种的自然苜蓿青贮作为对照。每组3个重复,发酵60 d后,对苜蓿青贮进行感官评定,并测定氨态氮/总氮(NH3-N/TN)、总酸含量和pH。结果表明:用MRS、LB和NA 3种培养基从自然发酵苜蓿青贮中筛选到微生物菌种31株,对其中18株进行了分子鉴定,主要菌种为植物乳杆菌9株、乳酸片球菌1株、粪肠球菌2株、各种芽孢杆菌6株。感官评分结果表明乳酸菌(植物乳杆菌和乳酸片球菌)比例大的各组评分等级显著高于对照组和添加了添加枯草芽孢杆菌的各组(P<0.05)。对于NH3-N/TN、总酸含量和pH等指标,乳酸菌比例较低的2、5、8和对照组与其余各组差异显著(P<0.05)。综上所述,植物乳杆菌和乳酸片球菌是苜蓿青贮主要菌种,用于苜蓿青贮有利于青贮品质的提高。枯草芽孢杆菌会影响苜蓿青贮的气味,解淀粉芽孢杆菌为菌种会改善苜蓿青贮风味,但比例太高会增加NH3-N含量。 相似文献
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Koenen ME Kramer J van der Hulst R Heres L Jeurissen SH Boersma WJ 《British poultry science》2004,45(3):355-366
1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised. 相似文献
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《动物营养（英文）》2015,(3)
Four delivery routes, via, feed, water, litter and oral gavage, were examined for their efficacy in delivering a novel probiotic of poultry origin, Lactobacillus johnsonii, to broilers. Seven treatments of 6 replicates each were allocated using 336 one-day-old Cobb broiler chicks. The treatments consisted of a basal diet with the probiotic candidate, L. johnsonii, added to the feed, and three treatments with L. johnsonii added to the drinking water, sprayed on the litter, or gavaged orally. In addition, a positive control treatment received the basal diet supplemented with zinc-bacitracin(Zn B, 50 mg/kg). The probiotic strain of L.johnsonii was detected in the ileum of the chicks for all four delivery routes. However, the addition of L.johnsonii as a probiotic candidate did not improve body weight gain, feed intake and feed conversion ratio of broiler chickens raised on litter during the 5-week experimental period regardless of the route of administration. The probiotic treatments, regardless of the routes of delivery, affected(P 0.05) the pH of the caecal digesta and tended(P = 0.06) to affect the p H of the ileal digesta on d 7, but the effect disappeared as the birds grew older. All probiotic treatments reduced the number of Enterobacteria in the caeca on d 21, and tended(P 0.054) to reduce it in the ileum and caeca on d 7 and in the ileum on d 21 compared with the controls. The probiotic also tended to increase the number of lactic acid bacteria and lactobacilli in the ileum and caeca on d 7, but this trend was not evident on d 21. The trend appeared most pronounced when the probiotic was delivered orally or via litter. The probiotic also decreased(P 0.05) the population of Clostridium perfringens rapidly from an early age to d 21 in the caeca, leading to a3-fold decrease in the number of C. perfringens between d 7 and 21. It also showed that the probiotic treatment presented the lowest number of C. perfringens in the caeca. Delivery of the probiotic through feed, water and litter increased(P 0.01) the weight of the pancreas on d 21, but the probiotic did not affect other morphometric parameters of the gut. Furthermore, the probiotic did not affect the p H and the concentrations of short chain fatty acids and lactic acid in either the ileum or caeca. 相似文献
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Monica Modesto M. Rosaria D'Aimmo Ilaria Stefanini Paolo Trevisi Sara De Filippi Luisa Casini Maurizio Mazzoni Paolo Bosi Bruno Biavati 《Livestock Science》2009,122(2-3):248-258
The study addresses the first two weeks of piglet weaning and is aimed at i) identifying new probiotic strains of Bifidobacterium spp. and non-digestible oligosaccharides (NDO) able to increase the level of autochthonous bifidobacteria in the gut, and ii) finding the best probiotic doses, and probiotic-NDO combinations for piglet growth and gut health.Initially, on a total of 60 pigs we administered 12 strains of 4 bifidobacteria species taken from our collection of strains of different ecological origin. We found the most promising probiotic candidates to be two strains of B. animalis subsp. lactis (M 354 and Ra 18) and one strain of B. choerinum (Su 891), as these led to an increase in the number of viable bifidobacteria in the caecum. In a second trial, a different set of 64 piglets were fed a diet containing a galacto-oligosaccharide (1%) from milk whey, and two fructo-oligosaccharides (4%) from chicory inulin and sugar beet (SbFOS). There was virtually no effect on the hindgut microbiota, except for SbFOS at 4% which tended to increase the endogenous bifidobacteria. Then, in a third trial employing 128 weaning pigs, the probiotic strains Ra 18 and Su 891 were tested individually, at different doses (0; 107; 109; 1011 per pig per day) and combined with 0% or 2% SbFOS supplementation. It was found that Ra 18 improved the live weight growth linearly, while the Su 891 did not. The FOS supplementation showed no interaction with the Ra 18 or Su 891 probiotics for growth performance and no effect on this response criterion.Our selection procedure has highlighted that B. animalis subsp. lactis Ra 18, at 1011 CFU per pig per day, appears to be the best probiotic choice for improved growth performance in weaning piglets. 相似文献
19.
Nasrin Noohi Moslem Papizadeh Mahdi Rohani Malihe Talebi Mohammad R. Pourshafie 《动物营养（英文）》2021,7(1):119
Considering the importance of the poultry industry and the increasing interest in alternative growth promoters, probiotics are considered as a potential candidate for use in the poultry industry. In this study, Lactobacillus species were isolated from 21 rectal swabs of 11 healthy 6-day-old and 10 healthy 21-day-old chickens and their fecal and feed samples. The isolates were characterized and their probiotic characteristics, including resistance to gastric acid and bile salts, biofilm formation and adherence to epithelium or mucus, amylase and protease activity and production of inhibitory compounds, were assessed. From 31 acid and bile resistant lactobacilli, only 2 Lactobacillus brevis and 1 Lactobacillus reuteri strains showed significant probiotic properties. These isolates indicated detectable attachment to Caco-2 cells and significant antibacterial activities against Gram-positive and Gram-negative pathogens. Additionally, phenotypic and genotypic diversity of lactobacilli isolates were studied by Phene Plate (PhP) system (PhP-LB) and random amplified polymorphic DNA (RAPD)-PCR, respectively. PhP-LB results of 24 L. brevis isolates showed a high phenotypic variation among the isolates. In comparison, results of RAPD-PCR highlighted a low diversity. Therefore, it seems that combination of the 2 techniques (PhP and RAPD-PCR) could result in a significant discriminatory power than each of them used alone. 相似文献