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This study investigated the effects of supplementation of low-temperature probiotics isolated from the intestines of olive flounder on the growth performance, digestibility, and regulation of intestinal microbiota and the expression of genes related to growth, immunity, and apoptosis in olive flounder. Bacteria showing high growth at approximately 15-20 °C, which is the temperature of olive flounder culture, were isolated and confirmed to be Pseudomonas species through 16S rRNA gene sequence analysis. Whole-genome sequencing revealed that the strain has a 6,195,122 bp single circular chromosome and a guanine-cytosine content of 59.9%. In the feeding trial, supplementation with 1 × 10 8  CFU/g of the isolate strain positively modulated growth performances, digestive enzyme activity, and gut microbiota composition of olive flounder. RT-qPCR for the comparison of growth, immunity, and apoptosis-related gene expression levels showed no significant differences between the groups. Therefore, the isolated host-associated low-temperature probiotics improved the growth performance of olive flounder by causing positive changes in digestive activity and intestinal microbial composition without affecting host gene expression., (© 2024. The Author(s).)

The purpose of this study was to characterize the bacteria isolated from rockfish intestines and to investigate the effects of feed supplementation in rockfish aquaculture. Bacillus sp. KRF-7 isolated from the intestine of rockfish (Sebastes schlegelii) was demonstrated to be safe based on in vitro tests confirming the absence of hemolysis, cytotoxicity, and genes with toxigenic potential. In a feeding trial, providing a supplemental diet of 1 × 10 8  CFU g -1 Bacillus sp. KRF-7 was observed to positively alter the weight gain, specific growth rate, feed conversion ratio, and protein efficiency ratio of juvenile rockfish. KRF-7 supplementation showed positive regulation of nonspecific immune parameters, such as superoxide dismutase, lysozyme activity, and myeloperoxidase activity. This analysis also revealed a change in the composition of the intestinal microbiota at the phylum level from Proteobacteria to Firmicutes. In both the kidney and spleen, the expression levels of IL-10, NF-κB, and B cell activating factors in the KRF-7-supplemented group were significantly increased compared to those in the control group. Therefore, this study verified the safety of KRF-7 isolated from the intestine of rockfish and suggests that dietary supplementation with KRF-7 enhances the growth performance of rockfish and has beneficial effects on the regulation of the intestinal microbiota and immune response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder's skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 10 8 -10 9  CFU kg -1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In recent years, Bacillus spp. have garnered attention as probiotic supplements in aquafeed owing to the production of heat stable and low pH resistant spores. Herein, we isolated and characterized an autochthonous Bacillus licheniformis KCCM 43270 from the intestine of olive flounder (Paralichthys olivaceus) for supplementation in low-fishmeal extruded aquafeeds. The KCCM 43270 was screened based on amylase, protease, cellulase, and lipase as well as non-hemolytic activities. The isolate was able to grow in carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) when used as a single carbon source in the minimal nutrient M9 medium. The KCCM 43270 spores displayed complete survival in acid (pH 2.5) and bile (0.3%, w/v) for 3 hr, strong biofilm formation, and nearly 50% adhesion with intestinal mucus. The spores of the isolate also showed significant survival ability at 80, 90, 100°C for 60, 30, and 1 min, respectively. In addition, the spores in a blend of SBM complex carrier showed significant heat stability at 120°C for 5 min and under different drying conditions. Furthermore, the spores also survived the extrusion process during low-fishmeal aquafeed manufacturing, implying the potential application of B. licheniformis KCCM 43270 in aquafeed industry., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 10 8  CFU g -1 ) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1β, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 10 8  CFU mL -1 ), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis., (Copyright © 2019. Published by Elsevier Ltd.)

To identify and quantify the effects of a combination of dietary 1 × 10 8 CFU/g Lactococcus lactis subsp. lactis I2 (LI 2 ) and 0.1% β-glucooligosaccharides (BGO) on the growth and immunity of olive flounder ( Paralichthys olivaceus ), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6). Results demonstrated the synbiotic diet had significantly better ( p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level ( p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 10 8 CFU/ml), the synbiotically fed group exhibited significantly higher ( p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI 2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP 1 , 2 × 10 9  CFU kg -1 diet), single-probiotics 2 (Bacillus licheniformis; SP 2 , 2 × 10 9  CFU kg -1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP 1 , 2 × 10 9  CFU kg -1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP 2 , 2 × 10 9  CFU kg -1 diet) and oxytetracycline (OTC) at 5 g OTC kg -1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP 1 , MP 1 and MP 2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP 2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP 1 , MP 1 and MP 2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP 1 , SP 2 , MP 1 and MP 2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP 1 and MP 2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP 1 , SP 2 , MP 1 , MP 2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Prebiotics, probiotics, and synbiotics are considered natural functional food ingredients and an alternative feeding strategy for immunomodulation and antibiotic eradication. Olive flounder (Paralichthys olivaceus) is commercially cultured in China, Japan, and Korea. Antibiotics used in flounder aquaculture produce antibiotic-resistant pathogens and residual effects on human. Prebiotics are non-digestible oligosaccharides, fermented by intestinal microbiota to produce short-chain fatty acids that bind G protein receptors. Probiotics are usually incorporated into the diet at a certain concentration to alter the intestinal microbial population through colonization or implantation. Synbiotics are combinations of prebiotics and probiotics that produce synergistically better benefits than separately. Previous studies on olive flounder have revealed that various types of immunostimulants, prebiotics, probiotics, synbiotics, and potentially immunoreactive feed additives can increase growth and feed utilization; cellular and humoral immunity; immune gene expression; blood/serum biochemistry; and infectious-disease resistance. Additive concentrations, their activity, and viability after feed storage and in the intestine, and interactions with the intestinal microbial community are major limiting factors. This review summarizes and discusses changes in growth and feed utilization, innate immunity, biochemical parameters, gene transcription, and disease protection in olive flounder after diet supplementation with various types of additives. It also indicates areas needing greater attention for future research. [ABSTRACT FROM AUTHOR]

(1,3)(1,4)-β- d -glucan has been determined to have various beneficial effects due to its unique structure. β-glucooligosaccharides (β-GOS), which are hydrolysates of barley (1,3)(1,4)-β- d -glucan, provide a useful prebiotic material for selective growth of probiotic bacteria. In this study, recombinant β-1,3-1,4-glucanase (Bg1314) from Bacillus sp. SJ-10 (KCCM 90078) was immobilized on porous silica using glutaraldehyde as a crosslinking reagent to achieve efficient production of β-GOS. We investigated the effects of factors such as the amounts of enzyme and glutaraldehyde, reaction temperature, and pH on catalytic activity. Enzyme activity decreased sharply at high concentrations of glutaraldehyde, likely due to the reaction of glutaraldehyde with lysine residues at the catalytic site of Bg1314, because lysine-substituted Bg1314 retained its activity under the same conditions. Immobilized Bg1314 protein (ImBg1314) was stable over a wide range of pH and could be stored long term at 4 °C. The optimal conditions of ImBg1314 were similar to those of Bg1314. However, the optimal temperature of ImBg1314 differed from that of Bg1314. The products were β-GOS composed of 3- O -β-cellobiosyl- d -glucose and 3- O -β-cellotriosyl- d -glucose. After ImBg1314 was reused for 10 cycles, it retained 42% of its initial catalytic activity. This study showed that the Imbg1314 applied economical production of β-GOS. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the effect of dietary differences on intestinal microbial population alteration and its relationship to immune-related gene expression in juvenile olive flounder (Paralichthys olivaceus). Initially, flounder were divided into three groups and supplemented with fishmeal diet (FM), fishmeal replaced by plant protein (termed low-fishmeal (LFM)), and Bacillus sp. SJ-10 probiotic-inoculated LFM (Pro) for 4 weeks. At the start (Initial group) and after completing the feeding trial, intestines were subjected to microbiota analysis and immune-related gene expression. The results of the intestinal microbiota analysis demonstrated that the FM and LFM groups' microbial richness (Chao1, ACE, and Jackknife) and diversity (Shannon and Simpson) did not differ significantly (P >.05) compared to the Initial group. However, the Pro group exhibited increased (P <.05) richness compared to all other groups, but its diversity was lower than that of the Initial group. A similar alteration phenomenon was also found in community composition between these two groups. At the phylum level, the most abundant phylum was Proteobacteria for all groups, but the second most abundant phylum in the Initial and Pro groups was Bacteroidetes, whereas in the FM and LFM groups, it was Firmicutes. An additional analysis of genus-level community composition and prediction of functional composition analysis showed differences among the groups. In the intestine, the transcription of interleukin (IL)-10 and IL-1β was increased more in the LFM and Pro groups compared to the FM group. IL-6 was increased only in the Pro group, and tumor necrosis factor-α (TNF-α) gene expression did not differ significantly among the groups. Therefore, we concluded that dietary changes affect intestinal microbiota composition and immune-related gene expression; in particular, diets containing probiotics can lead to greater change during the same feeding period. • Dietary changes affect intestinal microbiota composition and immune-related gene expression of olive flounder. • Probiotic have a greater impact on intestinal microbial diversity and composition than changes in fishmeal content. • Feed composition changes and probiotic supplements can manipulate the intestinal microbiota of olive flounder. [ABSTRACT FROM AUTHOR]