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The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 10 9  cfu kg diet -1 . After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1 H nuclear magnetic resonance ( 1 H NMR) based metabolomic analysis. Results from the 1 H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp., (Copyright © 2020. Published by Elsevier Ltd.)

The probiotic efficiencies of the mixed probiotics containing Lactobacillus pentosus BD6, Lac. fermentum LW2, Bacillus subtilis E20, and Saccharomyces cerevisiae P13 for shrimp growth and health status improvement were better than those when using single probiotics. The probiotic mixture at a level of 10 8  colony-forming units (cfu) (kg diet) -1 and the diets containing BD6 and E20 at 10 9  cfu (kg diet) -1 significantly improved the growth and health status of shrimp, whereas the diets containing P13 or LW2 did not significantly affect the growth of shrimp. No significant difference in the carcass composition was recorded among the control and treatments. After 56 days of feeding, shrimp fed the diet containing the probiotic mixture (10 7 ∼10 9  cfu (kg diet) -1 ) had higher survival after injection with the V. alginolyticus, but 10 9  cfu (kg diet) -1 of single probiotics (except for S. cerevisiae P13) had to be administered to improve shrimp survival. The better disease resistance of shrimp in groups fed the probiotic mixture might have been due to increased phenoloxidase activity, respiratory bursts, and lysozyme activity of hemocytes. Therefore, we considered that the probiotic mixture could adequately provide probiotic efficiency for white shrimp, and a diet containing 10 8  cfu (kg diet) -1 probiotic mixture is recommended., (Copyright © 2018. Published by Elsevier Ltd.)

Bacillus subtilis E20-fermented soybean meal (FSBM) was found to produce antimicrobial peptides (AMPs) with great antimicrobial activity against Vibrio alginolyticus (VA) and V. parahaemolyticus (VP). Three AMPs were purified with a 5 kDa ultrafiltration, Sephadex G-15 column and reverse-phase high-performance liquid chromatography (RP-HPLC). The FSB-AMP, HTSKALLDMLKRLGK, identified by an RP-nano-ultrapure liquid chromatography (UPLC) electrospray ionization (ESI)-tandem mass spectroscopic (MS/MS) analysis exhibited the highest bactericidal activity against VA and VP compared to the others. The antimicrobial activity assessment indicated that FSB-AMP inhibited the growth of VA and VP with minimal inhibitory concentrations of 72.5 and 72.5 μM. Alterations in the morphology of VA were observed by scanning electronic microscopy, and membrane disruption of VA and VP was confirmed by fluorescent microscopy with propidium iodide staining. The FSB-AMP was then incorporated into the diet of white shrimp, Litopenaeus vannamei, and a protective effect in shrimp against VP infection was recorded as well as for shrimp fed a diet containing 15% fish meal replaced by B. subtilis E20-FSBM. Results demonstrated that B. subtilis E20-FSBM could be a biofunctional ingredient to prevent vibriosis in shrimp aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Six bacteria, including, Lactobacillus casei M15, Lac. plantarum D8, Lac. pentosus BD6, Lac. fermentum LW2, Enterococcus faecium 10-10, and Bacillus subtilis E20, and one yeast, Saccharomyces cerevisiae P13 were selected as probiotics for Asian seabass, Lates calcarifer, by tracking the growth performance and disease resistance of fish against Aeromonas hydrophila in the first trial. The probiotic efficiency screening results showed that B. subtilis E20 and Lac. pentosus BD6, and S. cerevisiae P13 and Lac. fermentum LW2 respectively improved either the growth performance or disease resistance. Therefore, these four probiotics were then selected to prepare a probiotics mixture, and this was incorporated in equal proportions into diets for Asian seabass at levels of 0 (control), and 10 6 (MD6), 10 7 (MD7), 10 8 (MD8), and 10 9 (MD9) colony-forming units (cfu) (kg diet) -1 . A synergistic effect of the combined probiotics was investigated in this study, and the probiotics mixture was able to improve both the growth performance and health status of fish. After 56 days of feeding, fish fed the MD9 diet had a higher final weight and percentage of weight gain. In addition, protein contents in the dorsal muscle of fish fed the MD8 and MD9 diets were significantly higher compared to the control. For the pathogen challenge test, fish fed the MD7, MD8, and MD9 diets had significantly lower cumulative mortalities after A. hydrophila infection compared to those of fish fed the control and MD6 diets, which might have been due to increased respiratory bursts, decreased superoxide dismutase activity in leucocytes, and increased phagocytic activity. Therefore, we considered that the probiotics mixture could adequately provide probiotic efficiency for Asian seabass, and the diet containing 10 9  cfu (kg diet) -1 probiotic mixture is recommended to improve the growth and health status of Asian seabass., (Copyright © 2016. Published by Elsevier Ltd.)

The aim of this study was to improve the growth performance, immune response and disease resistance of grouper, Epinephelus coioides by using probiotic, Bacillus subtilis E20. The percent weight gain (PWG) and feeding efficiency (FE) of grouper administered the probiotic B. subtilis E20 were calculated. Survival of B. subtilis E20 in the posterior intestines was determined using a specific primer pair of BPHYF/BPHYR, as were the non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus when fish were fed diets containing B. subtilis at 0 (control), 10(4), 10(6), and 10(8) colony-forming units (cfu) g(-1) up to 28 days. Results showed that grouper fed a diet containing B. subtilis at the levels of 10(4), 10(6), and 10(8) cfu g(-1) had significantly increased PGW (203.0%, 229.6%, and 238.0%) and FE (1.15, 1.20, and 1.22) compared to control (191.8% and 1.0), and these directly increased in a dose-dependent manner with B. subtilis concentrations. B. subtilis was able to survive in the fish's posterior intestines during the feeding period. The survival rate increased in grouper challenged with Streptococcus sp. or an iridovirus when the fish were fed B. subtilis at 10(4), 10(6), and 10(8) cfu g(-1) for 14 and 28 days, and it was higher at 28 days than at 14 days. After 28 days of feeding, the relative survival percentages of fish challenged with Streptococcus sp. and an iridovirus were 22.8, 40.9 and 45.5, and 21.7, 30.4, and 52.2, respectively. The phagocytic activity, respiratory bursts, and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH(50)) of fish fed diets containing B. subtilis at 10(4), 10(6) and 10(8) cfu g(-1) were significantly and dose-dependently higher than those of fish fed the control diet for 28 days. We therefore recommend dietary B. subtilis E20 administration of 10(4) - 10(8) cfu g(-1) to E. coioides to promote growth, and enhance immunity and resistance against Streptococcus sp. and an iridovirus. The best results were seen in the 10(8) cfu g(-1) group fed for 28 days., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, the probiotic, Bacillus subtilis E20, isolated from the human health food, natto, was used for white shrimp, Litopenaeus vannamei, larvae breeding to improve the larval survival rate and development by adding probiotic to the rearing water at (control), 10(8), and 10(9) cfu L(-1) salt water once every 3 days during the 14 days of breeding experiment. Thereafter, stress tolerance and immune status of postlarvae were evaluated. Shrimp larval development was significantly accelerated after adding the probiotic to the larval rearing water at a level of 10(9) cfu L(-1). The survival rate of larvae was significantly higher in the treatment with 10(9) cfu L(-1) compared to the control and the treatment with 10(8) cfu L(-1) after all larvae had metamorphosed to postlarvae. Adding the probiotic to the shrimp larvae rearing water produced a weak inhibition of bacterial growth by an analysis of the total bacterial count and presumptive Vibrio count. For stress tests, no postlarvae died when they were reared in water in which the temperature was decreased from 30 to 2 degrees C at a rate of 0.1 degrees C min(-1). Postlarvae had significantly lower cumulate mortality in the treatments with 10(8) and 10(9) cfu L(-1) compared to the control when they were suddenly exposed to fresh water and 60 per thousand salt water. A significant decrease in the cumulative mortality of postlarvae treated with the probiotic at a level of 10(9) cfu L(-1) was recorded after the sudden transfer to 300 mg L(-1) nitrite-N compared to the control and treatment with 10(8) cfu L(-1). The analysis of immune-related gene expressions showed that the gene expression of prophenoloxidase I, prophenoloxidase II, and lysozyme of larvae were significantly increased after being reared in probiotic-containing water at the levels of 10(8) and 10(9) cfu L(-1). However, no significant difference in serine proteinase or glutathione peroxidase gene expressions was recorded in this study. It is therefore suggested that 10(9) cfu L(-1) of probiotic, B. subtilis E20 adding to rearing water for shrimp larva breeding., (2010 Elsevier Ltd. All rights reserved.)

Effects of Bacillus subtilis E20 isolated from fermented soybean on immune parameters and the disease resistance of the white shrimp (Litopenaeus vannamei) after 98 days of B. subtilis E20 feeding were evaluated in this study. Shrimp fed B. subtilis E20-containing diets at concentrations of 10(6) (E206), 10(7) (E207), and 10(8) (E208)cfu kg(-1), respectively, had significantly increased survival rates of 13.3%, 16.7%, and 20%, compared to the control (fed no probiotic) after being challenged with Vibrio alginolyticus. There were no significant differences in the total hemocyte count, respiratory burst, or superoxide dismutase glutathione peroxidase among all treatments. Shrimp fed a higher concentration of the probiotic (E208) exhibited significant increases in phenoloxidase activity, phagocytic activity, and clearance efficiency compared to control shrimp. In addition, B. subtilis E20 showed a weaker inhibitory effect against the growth of Aeromona hydrophila with around a 0.3-cm inhibitory zone, but showed no inhibitory effects against other selected pathogens, such as white shrimp pathogens: V. alginolyticus and Vibrio vulnificus. These results suggest that the increased resistance of shrimp after B. subtilis E20 consumption occurs through immune modifications, such as increases in phenoloxidase activity, phagocytic activity, and clearance efficiency against V. alginolyticus.

Microencapsulation is an advanced technique used to improve the viability of probiotics and minimize sensitivity during processing, storage, and in the gastrointestinal environment. Two dietary treatments including a control and an encapsulated probiotic, Bacillus subtilis E20 (EP), were used to evaluate the efficacy in improving the intestinal microbiome of white shrimp, Penaeus vannamei, after a 60-feeding trial. The 16S rDNA next-generation sequencing (NGS) analysis indicated that shrimp fed the EP diet generated higher amplicon reads than shrimp fed the control diet. No significant differences were observed in the α-diversity index of the intestinal microbiota of shrimp that were fed the control and EP diet. At the phylum level, Proteobacteria was relatively abundant in the microbiota of shrimp fed both the control and EP diet. The treatment with EP increased the expression of Tenericutes, Bacteroidetes, and Firmicutes, more than the control. The PC analysis revealed that the EP diet altered the bacterial profile in shrimp's intestines into forming different clusters. Unique genera such as Luteolibacter, Simkaniaceae, Haemophilus, Pirellulaceae, Filomicrobium, Sphingomonas, and Erysipelotrichaceae UCG-003 along with well-known probiotic genera Bacillus and Lactobacillus were found in the intestine of shrimp fed the EP diet. The PCA eigenvector plots indicated a higher abundance of Bacillus in shrimp fed with EP diet, but a higher abundance of Vibrio in shrimp fed with control diet. These results suggest that encapsulated B. subtilis E20 can be beneficial to shrimp microbiota. [ABSTRACT FROM AUTHOR]

The purpose of the present study is to profile the microfloral structure in the intestines of the white shrimp, Litopenaeus vannamei, after being fed a control diet, in comparison with the diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide (AMP) isolated from B. subtilis E20‐FSBM (diets designated FSBMD and AMPD, respectively) for 60 days. Metagenomic data showed detection of eight phyla, 20 classes, 40 orders, 68 families and 96 genera. Despite no statistically significant difference, an evaluation of microbial diversity recorded higher species richness, Shannon–Weaver diversity index and evenness in the AMPD and FSBMD groups, compared to the control diet group. Venn diagrams showed that 58 of the operational taxonomic units (OTUs) were shared among the control, FSBMD and AMPD groups, but additional beneficial microbes were only found in the FSBMD and AMPD groups. In addition to the augmentation of beneficial bacteria in the FSBMD and AMPD groups, the abundance of potential pathogens, Vibrio and Flavobacterium, was lower in the gut of shrimp fed the FSBMD and AMPD. The results clearly suggest that the FSBMD and AMPD have the capability to change the microfloral structure of shrimp intestines and could be used for disease prevention in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

The aims of this study were to evaluate the probiotic efficiency of Bacillus subtilis E20 (109 colony‐forming units kg−1) on the growth and apparent digestibility coefficients (ADCs) of dry matter (DM), crude protein (CP), amino acids (AAs), and crude lipids (CLs) of the white shrimp, Litopenaeus vannamei. A control diet without probiotic supplementation was used. Shrimp exhibited significant increases in growth performance and feed utilization after being fed the test diet for 56 days. Shrimp were then used to evaluate the apparent nutrient digestibility using 0.1% Cr2O3 in the diet as an indicator. During the trial, faeces was collected from triplicate groups of shrimp twice daily. ADCs of CP and DM in the test diet significantly increased compared to those of the control diet. Although the ADCs of CLs did not significantly differ between the control and probiotic group, the ADC of CLs was higher in the probiotic group than in the control group. In addition, ADCs of most AAs in the test diet were significantly higher compared to those of the control diet. These results suggest that shrimp in the test group had significantly better growth performance, which was due to increases in nutrient digestibility and absorption. [ABSTRACT FROM AUTHOR]

An experiment was conducted to evaluate the potential of a Bacillus subtilis E20-fermented mixture ( FSFEM) containing soybean meal ( SBM) and Eisenia fetida earthworm meal ( EM) at a ratio of 4:1 to increase the methionine level in order to satisfy the methionine requirement of white shrimp, Penaeus vannamei in a diet with fish meal ( FM) completely replaced by mixtures. B. subtilis E20 fermentation improved the mixture's palatability and utilization based on better growth performance in comparison to shrimp fed FSEM (contains fermented SBM and EM at a ratio of 4:1) diets. FSFEM is a good substitute for FM. Maximal replacement levels of FM with FSFEM were 80% in a shrimp diet with 37% of crude protein and 7% of crude lipid based on weight gain and 100% based on feeding efficiency. In addition, shrimp fed experimental diets had no significant differences in survival after being challenged by Vibrio alginolyticus. It is suggested that B. subtilis E20- FSFEM has the potential to replace FM in cultured shrimp diets. [ABSTRACT FROM AUTHOR]

The potential of Bacillus subtilis E20-fermented soybean meal ( FSBM) as a partial alternative component of fish meal ( FM) in fed diets of orange-spotted grouper ( Epinephelus coioides) was evaluated in this study. An FM-based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal ( SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively, based on broken-line analyses. Histopathological changes in the liver and distal intestine, and significantly lower activity levels of digestive enzymes, including pepsin in the stomach and trypsin, chymotrypsin, amylase and lipase in the distal intestine, were found in fish fed a diet containing 30% of FM replaced by SBM. However, these parameters were improved by the substitution of FSBM. It is therefore believed that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM. [ABSTRACT FROM AUTHOR]

This study aimed to improve the nutritional value and utilization of soybean meal ( SBM) used as feed ingredient for shrimp by an approach of solid-state fermentation with Bacillus subtilis E20. The protein content and B. subtilis E20 proliferation significantly increased, as the initial moisture increased from 30% to 50% during fermentation. Compared with SBM, the protein content of FSBM increased by 19% after fermentation, accompanied by an increase of 18.75% in the total hydrolyzed amino acids. The free amino acid profile and content in FSBM also obviously increased by 374.9% compared with SBM. The FSBM is a good substitute for fish meal (FM) in a diet with 37% protein and 7% lipid content. The maximal replacement levels of FM in shrimp diet with SBM and FSBM were 37.42% and 61.67%, respectively, based on the feed efficiency estimated by a broken-line analysis. The B. subtilis E20- FSBM can be a potential protein source used as a replacement for FM in shrimp diet. [ABSTRACT FROM AUTHOR]

The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5–2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg−1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg−1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg−1, shrimp's growth performance and health status improved after being fed 107 CFU kg−1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg−1) was able to increase the growth performance and health status of shrimp. • Probiotic efficacy of Bacillus subtilis E20 was improved by encapsulation. • Encapsulated probiotic had better resistance against adverts conditions of gastrointestinal tract and storage. • Encapsulated probiotic in diet at a level of 107 CFU kg−1 were able to improve the growth and health status of shrimp. [ABSTRACT FROM AUTHOR]