作为被批评的抗生素的替代品,益生菌因其环保性质和增强宿主生长和免疫力的能力而被采用。然而,报告表明,市售益生菌无效,因为大多数来自非鱼类来源;因此,这项研究是为了分离和表征新的芽孢杆菌。来自混合石斑鱼(Epinephelusfuscoguttatus‰×Epinepheluslanceolatus‰)的肠道,作为潜在的益生菌。根据它们的形态和生化特性以及16SrRNA测序同源性分析进行分离和表征,随后进行了为期4周的体内生物安全喂养试验,以确定对分离株非致病性的影响,增长,肠粘膜微绒毛经扫描电镜(SEM)分析。四种芽孢杆菌。菌株,即,B.velezensis菌株PGSAK01(登录号OQ726606),B.stercoris菌株PGSAK05(登录号OQ726607),B.velezensis菌株PGSAK17(登录号OQ726601),和枯草芽孢杆菌菌株PGSAK19(登录号OQ726605),在当前的研究中进行了鉴定和表征。菌株显示出有希望的益生菌特性,例如显示出更高的粘附能力,更高的耐热性,对0.5%的胆汁显示出更高的生存能力,较低的pH耐受性,γ-溶血活性,和多物种特征。在测试的24种抗生素中,分离株对21株敏感,而PGSAK01菌株对呋喃唑酮抗生素具有抗性。没有一个分离株显示拥有i)编码产肠毒素(hblA,HBLC,hbld,nheA,nheB,和entFM)和催吐剂(谷物合成酶基因,ces)基因,和ii)链霉素抗性基因(增值税),氨苄青霉素抗性基因(mecA和bla),和万古霉素抗性基因(VanB)。然而,PGSAK01和PGSAK17菌株显示具有tekK,猫,和抗ant(4')-Ia(腺苷酰转移酶)(PGSAK01除外)基因。所有分离株对病原菌无乳链球菌表现出更好的抗菌作用,S.iniae,哈维氏弧菌,和溶藻V.体内生物安全性试验涉及将杂种石斑鱼分为五种(平均体重32±0.94g),即,该组饲喂分离物补充剂的基础日粮(对照),其余四组在基础日粮中添加1×108CFU/g单株PGSAK01、PGSAK05、PGSAK17和PGSAK19。在研究结束时,显著更高的WGR,K(PGSAK01组除外),VSI;溶菌酶(PGSAK01组除外),超氧化物歧化酶,总抗氧化活性,碱性磷酸酶活性;高密度肠粘膜绒毛(基于扫描电子显微镜分析);与对照组相比,在分离的治疗组中观察到明显较低的丙二醛水平,支持在自动聚集和细胞表面疏水性测试中获得的结果。这项工作的结果提供了发人深省的目标;因此,将探索涉及广泛基因组测序和功能注释分析的研究,以提供对其作用机制和潜在健康益处的深不可测的见解,进一步建立了四个芽孢杆菌菌株PGSAK01、PGSAK05、PGSAK17和PGSAK19在益生菌领域和功能性食品中的潜在作用。
As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) which could serve as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties, and 16S rRNA sequencing homology analysis. A subsequent 30-day in vivo biosafety feeding trial was conducted to ascertain isolates\' non-pathogenicity, as well as their effects on fish growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5 % bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, while all isolates showed susceptibility to 21, the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4\')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The in vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32 ± 0.94 g), namely, the group fed the basal diet void of isolate\'s supplementation (control), and the remaining four groups fed the basal diet with 1 × 108 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), total antioxidant activity, alkaline phosphatase, superoxide dismutase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work\'s results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains\' (PGSAK01, PGSAK05, PGSAK17, and PGSAK19) potential role in probiotic fields and functional foods.