Co-encapsulation of Bacillus aryabhattai CKNJH11 with Algae-derived Polysaccharides Enhances Growth Performance and Immune Response in Asian Seabass (Lates calcarifer)

Co-encapsulation of Bacillus aryabhattai CKNJH11 with Algae-derived Polysaccharides Enhances Growth Performance and Immune Response in Asian Seabass (Lates calcarifer) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-encapsulation of Bacillus aryabhattai CKNJH11 with Algae-derived Polysaccharides Enhances Growth Performance and Immune Response in Asian Seabass ( Lates calcarifer ) Waraporn Appamano, Orathai Dangsawat, Sarayut Onsanit, Rapeewan Sowanpreecha, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6715276/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The application of Bacillus species as probiotics in aquaculture has been widely documented, showing notable benefits in growth performance, feed efficiency, and immune response. In this study, Bacillus aryabhattai CKNJH11, isolated from shrimp pond sediment, was evaluated for its probiotic potential through 16S rRNA sequencing and comprehensive in vitro assays. The strain exhibited strong probiotic traits, including high acid tolerance (survival at pH 2.0), bile salt resistance (up to 5% for 6 h), and significant inhibition of biofilm formation by Pseudomonas aeruginosa and Vibrio parahaemolyticus (> 50% reduction). Safety evaluations confirmed the absence of hemolytic activity and susceptibility to common antibiotics, supporting its suitability for aquaculture use. An eight-week feeding trial involving 120 barramundi ( Lates calcarifer ) fingerlings compared four dietary treatments: control (no probiotics), free spores, alginate-encapsulated spores, and spores co-encapsulated with sodium alginate and red seaweed polysaccharides. Fish receiving the co-encapsulated probiotic diet exhibited significantly enhanced growth (final body length: 8.05 ± 0.11 cm; weight: 60.67 ± 1.98 g) and improved survival. Immunological analyses showed reduced Vibrio spp. in the gastrointestinal tract, along with elevated white and red blood cell counts and hemoglobin levels, indicating enhanced immune status. These results suggest that co-encapsulation of B. aryabhattai CKNJH11 with algae-derived polysaccharides improves probiotic efficacy and stability, offering a promising approach to enhance growth, immunity, and productivity in aquaculture systems. Aquaculture and Mariculture Bacillus aryabhattai Spore-forming polysaccharides Immunological response Gastrointestinal microbiota Pathogen resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Barramundi (Lates calcarifer), also known as Asian seabass, is a key aquaculture species in Southeast Asia and is widely cultivated in countries such as Australia, Thailand, and Indonesia due to its high physiological adaptability, rapid growth rate, and efficient reproduction [ 1 , 2 ]. In Thailand, production rose markedly by 186%, from 16,500 tons in 2014 to 47,200 tons in 2019, contributing approximately 15% of the country’s total seafood output [ 3 ]. This growth is largely driven by increasing consumer demand for larger, high-quality fish products [ 4 ]. The sustainability of aquaculture hinges on several critical factors, including the use of genetically improved broodstock, effective management of water quality and disease, and optimized nutrition strategies [ 5 ]. However, intensification of farming practices has led to heightened stress and increased disease susceptibility in fish populations [ 6 ], necessitating a shift from conventional antibiotic use toward more sustainable health management approaches [ 7 , 8 ]. Among these, functional feeds containing medicinal plants, probiotics, and enzymes have shown considerable promise [ 9 – 11 ]. The overuse of antibiotics in aquaculture has raised serious concerns, such as environmental contamination, development of antimicrobial-resistant pathogens, suppression of host immunity, disruption of the gut microbiota, and food safety risks [ 12 ]. Consequently, alternative strategies, particularly those involving probiotics, phytogenic compounds, and enzyme-based additives, are being increasingly adopted [ 13 , 14 ]. Notably, Bacillus species have gained attention due to their spore-forming ability, environmental resilience, and multifunctional benefits, including enhanced growth performance, immunomodulation, improved nutrient assimilation, and stress mitigation [ 15 ]. Aquatic sediments are a rich source of Bacillus strains adapted to nutrient-depleted and competitive environments, often exhibiting antimicrobial peptide production, extracellular enzyme secretion, and immunostimulatory properties [ 16 , 17 ]. A species of emerging interest is Bacillus aryabhattai , a Gram-positive, spore-forming bacterium recognized for its environmental robustness and antipathogenic activity [ 18 , 19 ]. For instance, strain LSG3-7, isolated from the gut of Rhynchocypris lagowskii , effectively inhibited the adhesion of Aeromonas hydrophila and A. veronii , exhibited no hemolytic activity, and remained susceptible to most tested antibiotics [ 20 ]. Similarly, strain TBRC8450 was shown to enhance gut microbial balance, immune responses, and antioxidant defenses in Pacific white shrimp ( Litopenaeus vannamei ) [ 21 ]. Despite these promising results, the application of B. aryabhattai in finfish aquaculture, particularly in L. calcarifer , remains underexplored. In addition to probiotics, macroalgae-derived supplements, especially red algae like Gracilaria fisheri , are recognized for their prebiotic properties, primarily due to their richness in bioactive polysaccharides. These compounds promote intestinal health and immunity by serving as fermentable substrates for beneficial microbes, resulting in the production of short-chain fatty acids [ 22 , 23 ]. Given the potential synergistic effects of combining probiotics and prebiotics, this study aims to evaluate the probiotic potential of B. aryabhattai strain CKNJH11, isolated from shrimp pond sediment. Furthermore, it investigates the effects of co-encapsulation with sodium alginate and G. fisheri -derived polysaccharides on the growth performance, gut microbiota, and immune response of L. calcarifer . 2. Materials and methods 2.1 Soil sampling and bacterial isolation Sediment samples were collected from shrimp ponds located in Phunphin District, Surat Thani Province, Thailand (coordinates: 9°02'53.3"N, 99°09'43.9"E), following the protocol outlined by [ 24 ]. Briefly, 5 g of sediment was suspended in 45 mL of sterile saline solution (0.85% w/v NaCl), and the mixture was incubated at 80°C for 10 minutes to selectively enrich spore-forming bacteria. The suspensions were then serially diluted ten-fold and spread onto nutrient agar (NA) plates for the isolation of Bacillus spp. The plates were incubated at 37°C for 24 hours, after which morphologically distinct colonies were selected and purified by repeated streaking on fresh NA plates. The purified isolates were preserved at − 80°C in sterile 20% (v/v) glycerol for subsequent analyses. 2.2 Characterization of probiotic properties of B. aryabhattai CKNJH11 2.2.1 Bacterial identification and phylogenetic analysis Preliminary identification of bacterial isolates was conducted through Gram staining and biochemical tests, including catalase, oxidase, indole production, and starch hydrolysis, using EMPARTA® test kits (Merck Life Science), as described by [ 25 ]. solates showing potential probiotic traits were further identified through 16S rDNA sequencing. Genomic DNA was extracted using the Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer’s instructions. The 16S rDNA gene was amplified using the universal primers 20F (5′-GAG TTT GAT CCT GGC TCA G-3′) and 1500R (5′-GTT ACC TTG TTA CGA CTT-3′) according to the protocol of [ 26 ]. Pairwise alignment of the resulting 16S rDNA sequences was performed using the BLASTn tool available on the National Center for Biotechnology Information (NCBI) website ( https://www.ncbi.nlm.nih.gov ; accessed May 11, 2024) to determine the closest taxonomic identity. For phylogenetic analysis, 16S rDNA sequences of the CKNJH11 isolate and closely related Bacillus species were retrieved from the NCBI database. Multiple sequence alignment was conducted using MAFFT v7.525 [ 27 ] with default parameters. A phylogenetic tree was then constructed using the Maximum Likelihood (ML) method implemented in IQ-TREE v2.3.4 [ 28 ],, with 1000 bootstrap replications to evaluate the robustness of the inferred clades. Bootstrap values were indicated at the corresponding nodes, and branch lengths reflected the genetic distances among sequences. The phylogenetic tree was visualized using the Interactive Tree of Life (iTOL) web-based tool ( https://itol.embl.de ). 2.2.2 Sporulation efficiency Bacillus aryabhattai CKNJH11 was cultured on Difco Sporulation Medium (DSM; BD Diagnostics, USA) and incubated at 30°C for 72 hours to induce sporulation. Sporulation efficiency was assessed using a dual-staining protocol involving 5% malachite green and 0.1% safranin. After staining, spore suspensions were washed with sterile distilled water and centrifuged at 8000 × g for 10 minutes at 4°C. To eliminate residual vegetative cells, the suspensions were heat-treated at 80°C for 30 minutes. Viable spore counts were subsequently determined by plating serial dilutions on nutrient agar (NA; Himedia, India) and incubating at 37°C for 24 hours, following the method described by [ 29 ]. Sporulation efficiency was expressed as the percentage of viable spores relative to the initial cell count. 2.2.3 pH, bile salt, and saline tolerance The tolerance of B. aryabhattai CKNJH11 spores to extreme pH conditions was evaluated by incubating them in sterile pepsin solutions (Sigma-Aldrich, Singapore) adjusted to pH 1.0, 2.0, and 3.0 at 37°C for 2 hours. Post-incubation, viability was assessed using the standard plate count method on NA plates, followed by incubation at 37°C for 24 hours. Bile salt tolerance was tested by exposing spore suspensions to bile salt solutions (Himedia, India) at concentrations of 0%, 2.5%, 5.0%, 7.5%, and 10%. After 2 hours of exposure at 37°C, viable cell counts were determined using the standard plate count method on NA plates. Salinity tolerance was assessed by culturing the spores on NA plates supplemented with NaCl at concentrations of 0%, 0.5%, 1%, 2%, and 3%. Cultures were incubated in a shaking incubator at 37°C for 24 hours. Viability was then determined using the standard plate count method, with plates incubated for an additional 24 hours at 37°C. 2.2.4 Pathogenic bacteria biofilm inhibition The biofilm inhibition assay was performed following the method described by [ 30 ], with minor modifications. Bacillus aryabhattai CKNJH11 was cultured in tryptone soy broth (TSB; Himedia, India) and incubated at 37°C with shaking at 150 rpm for 72 hours. After incubation, the culture was centrifuged at 8000 × g for 10 minutes at 4°C, and the supernatant was filtered through a 0.2-µm syringe filter to obtain a cell-free supernatant. The bacterial concentration of the culture was adjusted to 10⁸ CFU/mL. For the biofilm inhibition assay, 100 µL of the bacterial suspension and 100 µL of the cell-free supernatant were co-inoculated into each well of a sterile 96-well microtiter plate. The plate was incubated at 37°C for 24 hours to allow biofilm formation. Following incubation, non-adherent cells were gently removed, and the wells were washed three times with sterile 1× phosphate-buffered saline (PBS, pH 7.1; Himedia, India). Biofilms were stained with 0.1% (w/v) crystal violet for 20 minutes at room temperature. Excess stain was removed by washing the wells three times with PBS. To solubilize the retained dye, 95% ethanol was added to each well, and the plate was incubated at 4°C for 30 minutes. The absorbance at 630 nm was measured using a microplate reader (SPECTROstar® Nano, BMG LABTECH, Ortenberg, Germany) to quantify biofilm biomass. Lower absorbance values indicated greater inhibition of biofilm formation. 2.2.5 Safety assessment of B. aryabhattai CKNJH11 2.2.5.1 Hemolytic activity assessment Hemolytic activity was evaluated according to the protocol described by [ 31 ]. Bacillus aryabhattai CKNJH11 was streaked onto sheep blood agar plates (Hardy Diagnostics, USA) and incubated at 37°C for 48 hours. Hemolysis was assessed by examining the appearance and type of halo around bacterial colonies: a greenish halo indicated α-hemolysis, a clear halo denoted β-hemolysis, and the absence of any halo signified non-hemolytic activity. 2.2.5.2 Antibiotic susceptibility The antibiotic susceptibility of B. aryabhattai CKNJH11 was determined using the disc diffusion method as described by [ 32 ]. The isolate was cultured in Lysogeny Broth (LB; Himedia, India) at 37°C, and four antibiotics were tested: ampicillin (10 µg), tetracycline (30 µg), amoxicillin (30 µg), and cloxacillin (30 µg) (all from Himedia, India). For the assay, bacterial suspensions were spread evenly onto Mueller-Hinton Agar (MHA) plates (Himedia, India). Antibiotic discs were then placed on the surface of the inoculated plates, which were incubated at 37°C for 24 hours. After incubation, the diameters of the inhibition zones were measured and interpreted as follows: susceptible (S) ≥ 20 mm; moderately susceptible (M) = 12–19 mm; and resistant (R) ≤ 12 mm. 2.4 Experimental design and diet preparation 2.4.1 Experimental fish A total of 120 barramundi ( L. calcarifer ) fingerlings, with an average body weight of 13.50 ± 0.35 g and an average length of 10.53 ± 0.12 cm, were obtained from the Phang Nga Coastal Aquaculture Research and Development Center (Phang Nga, Thailand; coordinates: 8°25'12.7"N, 98°14'34.5"E). Prior to the start of the experiment, the fish were acclimatized for two weeks under standardized conditions and fed a commercial control diet (Higade 9006T; Charoen Pokphand Foods, Bangkok, Thailand). 2.4.2 Extraction of polysaccharides from G. fisheri Polysaccharides were extracted from dried G. fisheri seaweed collected from Thung Sai Chai, Chaiya District, Surat Thani Province, Thailand. Twenty grams of seaweed were soaked in a 6% sodium hydroxide (NaOH) solution at 28°C for 5 hours to facilitate cell wall breakdown. After soaking, the seaweed was thoroughly rinsed with distilled water and cut into 1.0–1.5 cm segments in preparation for hot-water extraction. A total of 1500 mL of distilled water was added, and the pH was adjusted to between 6.2 and 6.8. The mixture was then boiled for 1.5 hours with occasional stirring. After boiling, the extract was filtered through two layers of fine cloth and allowed to cool to room temperature. The filtrate was subsequently frozen at − 20°C for approximately 24 hours. The resulting frozen agar was dried in an oven at 60°C until a constant weight was achieved. The dried agar was then ground into a fine powder using a blender and stored at room temperature for further use in feed formulation. 2.4.3 Experimental design Probiotic diets were prepared by incorporating B. aryabhattai CKNJH11 spores, which were obtained through nutrient exhaustion in Difco Sporulation Medium (DSM). Spores were subsequently purified and quantified according to the method described by [ 33 ], and incorporated into the feed at a final concentration of 1 × 10⁶ CFU/g. A total of 120 barramundi fingerlings were randomly assigned to twelve 25-liter tanks, with 10 fish per tank. The feeding trial employed a completely randomized design with four dietary treatments, each consisting of three replicates: D1 : Control diet (no probiotic supplementation) D2 : Diet supplemented with free (non-encapsulated) spores D3 : Diet supplemented with spores encapsulated in sodium alginate D4 : Diet supplemented with spores co-encapsulated with red algal polysaccharides and sodium alginate Encapsulation was performed by adding the probiotic-alginate mixture dropwise into a 0.45 M calcium chloride solution and allowing bead formation for 45 minutes. The resulting gel beads were collected via filtration using filter paper, rinsed with sterile distilled water to remove residual calcium ions, and then dried at 50–70°C until a stable consistency was achieved. The feeding trial lasted eight weeks. Fish were fed twice daily (at 09:00 and 17:00) at a rate of 3% of their body weight. All tanks were aerated continuously using air stones connected to a central air compressor. Water quality parameters, including total ammonia (NH₃), nitrite (NO₂⁻), pH, salinity, and total solids, were monitored daily to maintain optimal rearing conditions. 2.5 Growth performance At the end of the 8-week feeding trial, all surviving fish were individually weighed. Growth performance was assessed using the following parameters: weight gain (WG), average daily growth (ADG), feed conversion ratio (FCR), and survival rate (SR). These parameters were calculated using the formulas below: Weight gain (WG, g) = Final weight – Initial weight Average daily growth (ADG, g/day) = (Final weight – Initial weight) / number of days Feed conversion ratio (FCR) = Total feed intake / weight gain Survival rate (SR, %) = (Final number of fish / Initial number of fish) × 100 2.6 Bacterial detection in the intestine of L. calcarifer Three fish per tank (n = 9 per treatment group) were randomly selected for intestinal bacterial analysis following previously described protocol [ 34 ]. Using sterile dissection tools, the abdominal cavity of each fish was opened, and approximately 5 g of midgut tissue was aseptically collected. The samples were homogenized and suspended in 9 mL of sterile 0.85% (w/v) saline. Serial dilutions were prepared for bacterial enumeration. Selective media were used to isolate specific bacterial genera: Vibrio spp. on Thiosulfate–Citrate–Bile Salts–Sucrose (TCBS) agar Aeromonas spp. on Starch Ampicillin Agar (SAA) Pseudomonas spp. on Pseudomonas Isolation Agar (PIA) B. aryabhattai on Mannitol Egg Yolk Polymyxin (MYP) agar Total bacterial counts were expressed as colony-forming units (CFU) per gram of intestinal content, based on colony growth on the respective selective media. 2.7 Hematological parameters At the conclusion of the feeding trial, three fish per tank were randomly selected (n = 9 per treatment) for hematological analysis. Fish were anesthetized using clove oil at an appropriate concentration, and blood samples were collected from the caudal vein using heparinized syringes. The samples were transferred into K₂-EDTA-coated VACUETTE® tubes for analysis. Hematological parameters, including total white blood cell (WBC) count, total red blood cell (RBC) count, hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), were evaluated following the methods described in previous studies [ 35 , 36 ]. 2.8 Statistical analysis Data normality was assessed using the Shapiro–Wilk test. Differences among treatments were evaluated using ANOVA, with significant differences between means identified using the LSD test at a 95% confidence level. These analyses were conducted using IBM SPSS Statistical software (IBM Corp. Released 2023. IBM SPSS Statistics for Windows, Version 29.0.2.0, Armonk, NY: IBM Corp). All data were presented as mean ± standard error (SEM). Correlations between growth parameters were analyzed using Pearson’s linear correlation coefficient ( r ), computed with the ggplot2 package in R v3.4.4. The formula for Pearson’s correlation at a confidence interval of 95% is expressed as follows: $$\:r\:=\frac{\sum\:\left(x\:-\:{m}_{x\:}\right)\:\left(y\:-{m}_{y}\right)}{\sqrt{{\sum\:\left(x-{m}_{x}\right)}^{2}\sum\:{(y-{m}_{y)}}^{2}}}$$ where r is the correlation coefficient; x and y are the variables being compared; mx and my are the means of x and y , respectively. 3. Results 3.1 Identification of isolate CKNJH11 The results of physiological and biochemical analyses indicated that the isolate produced catalase and amylase and demonstrated the ability to hydrolyze starch. The strain also tested positive for sporulation, confirming its capacity to form spores efficiently. Microscopic examination of the spore suspensions revealed that 90–99% of the cells had sporulated, with green-stained spores and red-stained vegetative cells observed using dual staining. The isolate was negative for both cytochrome oxidase activity and indole production, suggesting the absence of tryptophan degradation (Table 1 ). Gram staining revealed that the bacterium was bacilli-shaped and retained a purple coloration under light microscopy, indicating it is a Gram-positive rod (Table 2 ). Based on 16S rDNA sequencing and phylogenetic analysis, the isolate was identified as B. aryabhattai , showing the highest sequence similarity to B. aryabhattai B8W22 (Fig. 1 ). 3.2 Analysis of B. aryabhattai CKNJH11 as probiotic The isolated strain B. aryabhattai CKNJH11 exhibited strong acid tolerance, maintaining survival rates of 64.88 ± 0.40% at pH 2.0, 84.13 ± 2.31% at pH 3.0, and 97.85 ± 0.35% at pH 4.0 over a 6-hour incubation period. The strain also showed considerable resistance to bile salts, with survival rates of 84.62 ± 0.78% at 1%, 84.43 ± 0.73% at 2.5%, and 78.06 ± 8.12% at 5% concentrations. Sporulation efficiency was notably high, reaching 95.65 ± 1.11% (Table 1 ). The cell-free supernatant of B. aryabhattai CKNJH11 significantly inhibited biofilm formation by P. aeruginosa , V. parahaemolyticus , and S. aureus , with inhibition rates of 58.27 ± 3.17%, 59.89 ± 6.49%, and 31.91 ± 6.25%, respectively. Additionally, the strain demonstrated robust salinity tolerance, maintaining survival rates above 90.42 ± 9.65% across a salinity range of 5 to 30 ppt (Table 1 ). 3.3 B. aryabhattai CKNJH11 safety analysis The hemolytic activity assay (Fig. 2 ) showed that the indicator strains S. aureus , B. subtilis , and P. aeruginosa produced distinct clear zones of hemolysis on blood agar, confirming their hemolytic potential. In contrast, colonies of the test strain B. aryabhattai CKNJH11 displayed no observable hemolytic zones, indicating the absence of hemolytic activity. This finding supports the strain’s safety for aquaculture use, as it suggests a lack of hemolytic virulence factors. Antibiotic susceptibility testing revealed that CKNJH11 was sensitive to ampicillin, tetracycline, and cloxacillin, while showing moderate susceptibility to amoxicillin (Table 1 ). These results further support the biosafety of B. aryabhattai CKNJH11 for potential probiotic application in aquaculture systems. 3.4 Growth performance After the 8-week feeding trial, fish fed diets supplemented with B. aryabhattai CKNJH11 exhibited significantly enhanced growth performance compared to the control group (Table 3 ). The WG in groups D2, D3, and D4 ranged from 45.59 ± 3.24 to 60.67 ± 1.98 g, while the ADG ranged from 0.76 ± 0.05 to 1.01 ± 0.03 g/day. Both WG and ADG were significantly higher in all probiotic-treated groups than in the control group D1 (p < 0.05), with the highest values observed in group D4. The FCR also differed significantly among treatments (p < 0.05). The lowest FCR, indicating the most efficient feed utilization, was recorded in group D4 (5.67 ± 0.18), whereas the control group (D1) had the highest FCR (8.13 ± 0.37). Survival rates across all groups ranged from 93.33–100%, with no statistically significant differences (p > 0.05). Table 3 Effects of dietary supplementation with B. aryabhattai CKNJH11 on the growth performance of L. calcarifer after an 8-week feeding trial. Parameters Experimental diets D1 D2 D3 D4 Initial length (cm) 10.61 ± 0.25 10.67 ± 0.32 10.38 ± 0.60 10.48 ± 0.17 Final length (cm) 16.71 ± 0.35 c 17.12 ± 0.34 b 17.45 ± 0.08 b 18.53 ± 0.05 a Initial weight (g) 13.35 ± 0.55 13.16 ± 0.62 14.00 ± 0.99 13.51 ± 0.34 WG (g) 38.60 ± 1.34 c 45.59 ± 3.24 b 48.99 ± 1.45 b 60.67 ± 1.98 a ADG (day) 0.67 ± 0.02 c 0.76 ± 0.05 b 0.81 ± 0.02 b 1.01 ± 0.03 a FCR (g/g) 8.13 ± 0.37 c 7.10 ± 0.44 b 7.07 ± 0.49 b 5.67 ± 0.18 a SR (%) 93.33 ± 5.77 96.66 ± 5.77 100.00 ± 0.00 100.00 ± 0.00 D1: control diet with no probiotics; D2: diet supplemented with free spore probiotics; D3: diet with spore probiotics encapsulated in sodium alginate; D4: diet with spore probiotics co-encapsulated with polysaccharide and sodium alginate. Values are expressed as mean ± SEM (n = 3). Different superscript letters within the same row indicate statistically significant differences between treatments (p < 0.05). Linear regression analysis revealed strong positive correlations between diet supplementation and growth parameters, including WG (r = 0.975), ADG (r = 0.960), and survival rate (SR; r = 0.944). In contrast, FCR exhibited a strong negative correlation (r = − 0.947), further indicating the beneficial effects of probiotic supplementation on growth performance and feed efficiency in L. calcarifer (Fig. 3 ). 3.5 Detection of bacterial species in the intestine of L. calcarifer The experimental results revealed significant differences in intestinal bacterial colonization between the treatment groups and the control group. Vibrio spp. incapable of sucrose fermentation were significantly reduced in all probiotic-treated groups, with bacterial counts ranging from log 2.25 ± 1.95 to 3.57 ± 0.33 CFU/g, compared to log 3.97 ± 0.29 CFU/g in the control group (p < 0.05) (Fig. 4 ). Similarly, Vibrio spp. capable of sucrose fermentation also exhibited lower counts in the treated groups, ranging from log 3.43 ± 0.40 to 3.59 ± 0.25 CFU/g, in contrast to log 4.46 ± 0.21 CFU/g observed in the control group (p < 0.05). Notably, Aeromonas spp. and Pseudomonas spp. were not detected in any of the experimental groups, including the control. In contrast, B. aryabhattai CKNJH11 was detected exclusively in the intestines of fish from groups D3 and D4, where the probiotic was encapsulated. Quantitative analysis showed concentrations of log 3.93 ± 0.04 CFU/g in group D3 and log 3.75 ± 0.39 CFU/g in group D4, while the strain was absent in the control group. These findings indicate successful colonization and potential competitive exclusion of pathogenic bacteria by the probiotic strain. 3.6 Hematological parameters The hematological profiles of L. calcarifer across the four dietary treatments indicated that fish fed diets supplemented with B. aryabhattai CKNJH11 spores exhibited improved hematological indices compared to the control group. The RBC counts in groups D2, D3, and D4 ranged from 4.28 ± 0.30 to 4.97 ± 0.16 × 10⁶ cells/mL. Similarly, WBC counts were elevated in the supplemented groups, ranging from 23.62 ± 2.89 to 24.50 ± 4.56 × 10³ cells/mL. The highest levels of Hb and Hct were recorded in group D4, with values of 8.13 ± 0.40 g/dL and 53.60 ± 33.05%, respectively. However, MCH and MCHC did not differ significantly among the treatment groups (p > 0.05) (Fig. 5 ). 3.7 Water quality Water quality parameters were monitored throughout the experimental period. pH levels exhibited a slight upward trend across all treatment groups (D1–D4), ranging from 7.14 ± 0.52 to 7.23 ± 0.94, though these differences were not statistically significant (p > 0.05) (Fig. 6 A). Salinity values remained relatively stable, ranging from 24.67 ± 0.49 to 25.00 ± 0.00 ppt, with no significant differences observed among treatments (p > 0.05) (Fig. 6 B). In contrast, notable differences were observed in nitrite and ammonia concentrations. Nitrite levels ranged from 0.60 ± 0.46 to 0.93 ± 0.49 mg/L, while ammonia concentrations varied between 0.05 ± 0.02 and 0.17 ± 0.03 mg/L. The D4 group exhibited the lowest levels of both nitrite and ammonia, showing significant reductions compared to the control group (p 0.05) (Fig. 6 E). 4. Discussion In recent years, stakeholders in the aquaculture industry have made significant strides toward sustainability by adopting clean and hygienic farming practices. Key initiatives include the reduction of antibiotic use and the incorporation of alternative supplements aimed at enhancing the overall health of aquatic animals [ 37 , 38 ]. In this context, the present study sought to isolate and evaluate the probiotic potential of Bacillus aryabhattai strain CKNJH11, derived from shrimp pond sediments. The study provides a comprehensive characterization of the strain’s morphological, biochemical, and functional properties. In vitro assessments demonstrated that B. aryabhattai CKNJH11 possesses robust probiotic traits suitable for aquaculture applications. Physiological and biochemical analyses confirmed hallmark Bacillus features, including catalase and amylase production, efficient starch hydrolysis, and a high sporulation efficiency (90–99%), which are essential for survival and stability under stressful environmental conditions [ 39 , 40 ]. Molecular identification through 16S rDNA sequencing and phylogenetic analysis revealed a close genetic relationship to B. aryabhattai B8W22, supporting its taxonomic classification. The strain exhibited excellent tolerance to simulated gastrointestinal conditions, maintaining high survival rates from 64.9% at pH 2.0 to 97.9% at pH 4.0 over a 6-hour period. Additionally, it remained viable in the presence of bile salts up to 5%, suggesting its capability to colonize the gastrointestinal tract of fish. Its resilience across a salinity range of 5–30 ppt further underscores its adaptability to diverse aquaculture systems. Notably, the cell-free supernatant of CKNJH11 significantly inhibited biofilm formation by common aquaculture pathogens, including P. aeruginosa , V. parahaemolyticus , and S. aureus , indicating the production of antimicrobial compounds that may suppress pathogenic colonization [ 41 , 42 ]. Importantly, safety evaluations revealed that the strain is non-hemolytic and susceptible to several commonly used antibiotics, minimizing potential risks associated with virulence or antibiotic resistance. These findings collectively highlight the promise of B. aryabhattai CKNJH11 as a safe and effective probiotic candidate for application in sustainable aquaculture. Dietary supplementation with Bacillus spp. has consistently been associated with enhanced growth performance and health status across various aquaculture species, including Oreochromis niloticus [ 43 , 44 ], Litopenaeus vannamei [ 45 , 46 ], Labeo chrysophekadion [ 47 ], Clarias gariepinus [ 48 , 49 ], and Apostichopus japonicus [ 50 ]. In the present study, Lates calcarifer fed diets supplemented with B. aryabhattai CKNJH11 exhibited significantly improved performance in weight gain, daily weight gain, feed conversion ratio, and survival rate compared to the control group. These enhancements are largely attributable to the ability of Bacillus spp. to produce extracellular bioactive compounds, such as enzymes and vitamins, that improve nutrient digestibility and stimulate innate immune responses [ 15 ]. Additionally, Bacillus probiotics are known to modulate the gut microbiota, thereby contributing to improved nutrient assimilation and growth outcomes [ 51 , 52 ]. Hematological assessments further confirmed the beneficial effects of B. aryabhattai CKNJH11 supplementation. Treated fish exhibited elevated red and white blood cell counts, with the highest values observed in group D4. These findings indicate enhanced hematopoiesis and immune function. Previous studies have demonstrated that probiotic supplementation increases immune cell populations, including macrophages, lymphocytes, granulocytes, and neutrophils, in fish, mirroring responses seen in higher vertebrates [ 44 ]. Probiotics exert immunomodulatory effects by interacting with key immune cells (e.g., monocytes, macrophages, and lymphocytes), enhancing the host’s innate immune response [ 53 , 54 ]. For example, in rainbow trout, probiotic supplementation significantly elevated RBC and WBC levels [ 55 ], while similar trends have been reported in major carp and rainbow trout following dietary inclusion of B. subtilis [ 56 , 57 ]. An other study also reported increased WBC and lymphocyte levels in juvenile cobia fed indigenous Bacillus isolates ( B. spp. RCS1 and B. cereus RCS3), reinforcing the probiotic’s immunostimulatory potential [ 58 ]. The superior performance observed in group D4 can be attributed to the dual-encapsulation approach using sodium alginate and red algae-derived polysaccharides. Sodium alginate forms a protective gel matrix that shields probiotic spores from gastric degradation, enhancing delivery to the intestinal tract [ 59 ]. Concurrently, polysaccharides act as prebiotics, supporting a favorable gut environment and contributing to immune modulation [ 60 ]. This co-encapsulation strategy not only enhances probiotic viability during gastrointestinal transit but also improves microbial colonization, thereby amplifying the probiotic’s effects on host health and performance. Microbial analyses revealed significant changes in the intestinal microbiota of treated fish. Notably, Vibrio spp. populations, both sucrose-fermenting and non-fermenting, were significantly reduced in probiotic-treated groups (p < 0.05), consistent with previous studies demonstrating the antagonistic activity of probiotics against Vibrio pathogens in aquaculture [ 61 ]. The complete absence of Aeromonas spp. and Pseudomonas spp. in all experimental groups suggests a potential selective exclusion effect by the probiotic, possibly through competitive colonization or antimicrobial metabolite production [ 15 ]. Moreover, the detection of B. aryabhattai exclusively in the intestines of probiotic-treated fish confirms successful colonization and highlights its potential to confer health benefits through modulation of the intestinal microbial community. In addition to growth and immune-related benefits, the study also demonstrated that B. aryabhattai CKNJH11 supplementation contributed to improved water quality. The D4 group exhibited significantly reduced ammonia and nitrite levels, aligning with previous findings that Bacillus spp. can enhance water quality by metabolizing nitrogenous waste [ 62 , 63 ]. These environmental benefits further support the application of B. aryabhattai CKNJH11 as a sustainable probiotic, promoting both fish health and ecological balance in aquaculture systems. Collectively, these findings demonstrate that dietary inclusion of B. aryabhattai CKNJH11, especially when co-encapsulated with prebiotic polysaccharides, can significantly enhance growth performance, immune status, intestinal health, and environmental quality in L. calcarifer culture. This integrated approach holds promise for sustainable and health-conscious aquaculture practices. 5. Conclusion This study demonstrated the probiotic potential of B. aryabhattai CKNJH11 in improving the health and productivity of L. calcarifer . The strain exhibited key probiotic characteristics, including non-hemolytic activity, high sporulation efficiency, and strong tolerance to salinity and acidic conditions. Dietary supplementation with B. aryabhattai CKNJH11 significantly enhanced growth performance, feed efficiency, and hematological parameters, with the most pronounced effects observed in fish receiving co-encapsulated spores. Furthermore, probiotic-treated groups showed a marked reduction in gut-associated pathogenic Vibrio spp. and improved water quality through decreased nitrite and ammonia concentrations. Collectively, these findings support the application of B. aryabhattai CKNJH11 as a safe, effective, and environmentally beneficial probiotic for sustainable aquaculture. Declarations Acknowledgements This research was partially supported by Chiang Mai University, Chiang Mai, Thailand. Additional support was provided by the Program in Agricultural Science and Technology, Faculty of Innovative Agriculture, and the Fishery Establishment Project at Prince of Songkla University, Surat Thani Campus, Surat Thani, Thailand. We also acknowledge the Scientific Laboratory and Equipment Center, Office of Surat Thani Campus, Prince of Songkla University, for their assistance. Author Contributions Waraporn Appamano : Methodology, Investigation, Writing - Original Draft. Orathai Dangsawat : Methodology, Investigation. Sarayut Onsanit : Methodology, Investigation Rapeewan Sowanpreecha : Methodology, Investigation. Phatthanaphong Therdtatha: Writing - Review & Editing. Tran Hoang Trieu Quan : Writing - Review & Editing. Thi Hang Ho : Writing - Review & Editing. Luu Tang Phuc Khang : Writing - Review & Editing. Papungkorn Sangsawad : Writing - Review & Editing, Formal Analysis, Supervision. Nguyen Dinh-Hung: Formal Analysis, Writing - Original Draft, Writing - Review & Editing. Nguyen Vu Linh : Investigation, Methodology, Validation, Software, Data Curation, Formal Analysis, Writing - Review & Editing. Patima Permpoonpattana : Conceptualization, Validation, Resources, Supervision, Project administration, Funding acquisition, and Writing - Review & Editing. Conflicts of interest The authors declare that there are no conflicts of interest. Ethic of animal use All experiments in this study were performed in accordance with the relevant guidelines and regulations. The experimental protocols were approved by the Institutional Animal Care and Use Committee, Prince of Songkla University (Approval Ref. Number 90/2566). All the procedure of the study is followed by the ARRIVE guidelines. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. 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Linh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBACPmYGNjCDHy4kQUALG0yLZANUhIegFgaoFoMDRGth5z32mLeNIc/4+Ok0CYYaOwZ76QYCWpj50o2BWorNzuRuk2A4lszAI3OAkBYeM8mZbQyJ227wArWwHQA6LIFILZtngLT8I1KLxEeglg0SQC2MbcRq+XBOInHGmdzNFol9yTw8Nwho4ec/YyaRUGaT2N9+duOND9/s5NhnENACBdC4ACrmIUr9KBgFo2AUjAL8AACLETIr1ukE0QAAAABJRU5ErkJggg==","orcid":"","institution":"Chiang Mai University","correspondingAuthor":true,"prefix":"","firstName":"Nguyen","middleName":"Vu","lastName":"Linh","suffix":""},{"id":459820550,"identity":"320794e6-214a-49e6-9e8c-cb9d89036f41","order_by":11,"name":"Patima Permpoonpattana","email":"","orcid":"","institution":"Prince of Songkla University","correspondingAuthor":false,"prefix":"","firstName":"Patima","middleName":"","lastName":"Permpoonpattana","suffix":""}],"badges":[],"createdAt":"2025-05-21 10:07:21","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6715276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6715276/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83275650,"identity":"ffc03846-3cc9-4af1-b2c3-504066b6cada","added_by":"auto","created_at":"2025-05-22 08:58:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114198,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 showing its evolutionary relationship with other \u003cem\u003eBacillus \u003c/em\u003espp. retrieved from the NCBI database. The tree was constructed using the Neighbor-Joining method with 1000 bootstrap replications in MEGA 11 and PAUP* 4.0b. Bootstrap values are shown at the nodes, and the scale bar represents genetic distance.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/c4f7c9e9549deea57436e653.png"},{"id":83275651,"identity":"b86d63af-b13a-4d15-921e-55409f0ed9dc","added_by":"auto","created_at":"2025-05-22 08:58:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98344,"visible":true,"origin":"","legend":"\u003cp\u003eHemolytic activity of bacterial strains on sheep blood agar. Distinct clear zones indicate β-hemolysis, greenish zones indicate α-hemolysis, and the absence of zones indicates non-hemolytic activity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/e9c3e0a6bba23b8a329635f3.png"},{"id":83275861,"identity":"15a12699-fa92-4f6a-b516-86abddd21ada","added_by":"auto","created_at":"2025-05-22 09:06:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90563,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation coefficients of (A) weight gain (WG), (B) average daily gain (ADG), (C) feed conversion ratio (FCR), and (D) survival rate (SR) in \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 dietary treatments. Probiotic concentrations are expressed in CFU/g of diet. Data are presented as mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/5fa875ca9fa45cd1508965fd.png"},{"id":83275654,"identity":"f187ea73-df12-4617-8b71-0be00f22a6fc","added_by":"auto","created_at":"2025-05-22 08:58:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67024,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dietary \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 supplementation on intestinal bacterial populations (log CFU/g) in four treatment groups: D1 (control), D2 (free spore probiotics), D3 (encapsulated spores in sodium alginate), and D4 (co-encapsulated spores with polysaccharide and sodium alginate). V(G): \u003cem\u003eVibrio\u003c/em\u003e spp., green colonies; V(Y): \u003cem\u003eVibrio\u003c/em\u003e spp., yellow colonies; A: \u003cem\u003eAeromonas\u003c/em\u003e spp.; P: \u003cem\u003ePseudomonas\u003c/em\u003e spp.; B: \u003cem\u003eB. aryabhattai\u003c/em\u003e; NS: non-significant; ND: not detected. Different letters indicate significant differences between treatments (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/6a911e2d9467099a4a5d41a2.png"},{"id":83276622,"identity":"a9e2bcbe-d01b-4d51-b9db-a13c93c572f6","added_by":"auto","created_at":"2025-05-22 09:14:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":65217,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameters of \u003cem\u003eL. calcarifer\u003c/em\u003e after dietary administration of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11: (A) red blood cell count, (B) white blood cell count, (C) hemoglobin concentration, (D) hematocrit, (E) mean corpuscular hemoglobin (MCH), and (F) mean corpuscular hemoglobin concentration (MCHC). Values are expressed as mean ± SEM (n = 3). Different letters indicate significant differences (p \u0026lt; 0.05); \"ns\" denotes non-significant differences.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/289272a0f35fb1c7c3819582.png"},{"id":83275658,"identity":"a4f3b486-e96d-41b5-a939-5d0b7395868b","added_by":"auto","created_at":"2025-05-22 08:58:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49273,"visible":true,"origin":"","legend":"\u003cp\u003eWater quality parameters measured during the feeding trial with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11: (A) pH, (B) salinity, (C) nitrite, (D) ammonia, and (E) total dissolved solids (TDS). Values are expressed as mean ± SEM (n = 3). Different letters indicate significant differences among treatments (p \u0026lt; 0.05); \"ns\" denotes non-significant differences.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/5a518ee466b0ade0e329c3e2.png"},{"id":83276940,"identity":"968de76b-1fc7-463e-bf7d-898bf4059c3d","added_by":"auto","created_at":"2025-05-22 09:22:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1754510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/01bbc03e-7462-45cd-855b-804db41c21d8.pdf"},{"id":83275860,"identity":"9173cb31-db14-46d8-a495-923748673921","added_by":"auto","created_at":"2025-05-22 09:06:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":440014,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6715276/v1/739bcf55a777b8e736fc96cd.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCo-encapsulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBacillus aryabhattai\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e CKNJH11 with Algae-derived Polysaccharides Enhances Growth Performance and Immune Response in Asian Seabass (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLates calcarifer\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBarramundi (Lates calcarifer), also known as Asian seabass, is a key aquaculture species in Southeast Asia and is widely cultivated in countries such as Australia, Thailand, and Indonesia due to its high physiological adaptability, rapid growth rate, and efficient reproduction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In Thailand, production rose markedly by 186%, from 16,500 tons in 2014 to 47,200 tons in 2019, contributing approximately 15% of the country\u0026rsquo;s total seafood output [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This growth is largely driven by increasing consumer demand for larger, high-quality fish products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The sustainability of aquaculture hinges on several critical factors, including the use of genetically improved broodstock, effective management of water quality and disease, and optimized nutrition strategies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, intensification of farming practices has led to heightened stress and increased disease susceptibility in fish populations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], necessitating a shift from conventional antibiotic use toward more sustainable health management approaches [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among these, functional feeds containing medicinal plants, probiotics, and enzymes have shown considerable promise [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe overuse of antibiotics in aquaculture has raised serious concerns, such as environmental contamination, development of antimicrobial-resistant pathogens, suppression of host immunity, disruption of the gut microbiota, and food safety risks [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, alternative strategies, particularly those involving probiotics, phytogenic compounds, and enzyme-based additives, are being increasingly adopted [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, \u003cem\u003eBacillus\u003c/em\u003e species have gained attention due to their spore-forming ability, environmental resilience, and multifunctional benefits, including enhanced growth performance, immunomodulation, improved nutrient assimilation, and stress mitigation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aquatic sediments are a rich source of \u003cem\u003eBacillus\u003c/em\u003e strains adapted to nutrient-depleted and competitive environments, often exhibiting antimicrobial peptide production, extracellular enzyme secretion, and immunostimulatory properties [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A species of emerging interest is \u003cem\u003eBacillus aryabhattai\u003c/em\u003e, a Gram-positive, spore-forming bacterium recognized for its environmental robustness and antipathogenic activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For instance, strain LSG3-7, isolated from the gut of \u003cem\u003eRhynchocypris lagowskii\u003c/em\u003e, effectively inhibited the adhesion of \u003cem\u003eAeromonas hydrophila\u003c/em\u003e and \u003cem\u003eA. veronii\u003c/em\u003e, exhibited no hemolytic activity, and remained susceptible to most tested antibiotics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, strain TBRC8450 was shown to enhance gut microbial balance, immune responses, and antioxidant defenses in Pacific white shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite these promising results, the application of \u003cem\u003eB. aryabhattai\u003c/em\u003e in finfish aquaculture, particularly in \u003cem\u003eL. calcarifer\u003c/em\u003e, remains underexplored.\u003c/p\u003e \u003cp\u003eIn addition to probiotics, macroalgae-derived supplements, especially red algae like \u003cem\u003eGracilaria fisheri\u003c/em\u003e, are recognized for their prebiotic properties, primarily due to their richness in bioactive polysaccharides. These compounds promote intestinal health and immunity by serving as fermentable substrates for beneficial microbes, resulting in the production of short-chain fatty acids [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Given the potential synergistic effects of combining probiotics and prebiotics, this study aims to evaluate the probiotic potential of \u003cem\u003eB. aryabhattai\u003c/em\u003e strain CKNJH11, isolated from shrimp pond sediment. Furthermore, it investigates the effects of co-encapsulation with sodium alginate and \u003cem\u003eG. fisheri\u003c/em\u003e-derived polysaccharides on the growth performance, gut microbiota, and immune response of \u003cem\u003eL. calcarifer\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Soil sampling and bacterial isolation\u003c/h2\u003e \u003cp\u003eSediment samples were collected from shrimp ponds located in Phunphin District, Surat Thani Province, Thailand (coordinates: 9\u0026deg;02'53.3\"N, 99\u0026deg;09'43.9\"E), following the protocol outlined by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, 5 g of sediment was suspended in 45 mL of sterile saline solution (0.85% w/v NaCl), and the mixture was incubated at 80\u0026deg;C for 10 minutes to selectively enrich spore-forming bacteria. The suspensions were then serially diluted ten-fold and spread onto nutrient agar (NA) plates for the isolation of \u003cem\u003eBacillus\u003c/em\u003e spp. The plates were incubated at 37\u0026deg;C for 24 hours, after which morphologically distinct colonies were selected and purified by repeated streaking on fresh NA plates. The purified isolates were preserved at \u0026minus;\u0026thinsp;80\u0026deg;C in sterile 20% (v/v) glycerol for subsequent analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization of probiotic properties of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Bacterial identification and phylogenetic analysis\u003c/h2\u003e \u003cp\u003ePreliminary identification of bacterial isolates was conducted through Gram staining and biochemical tests, including catalase, oxidase, indole production, and starch hydrolysis, using EMPARTA\u0026reg; test kits (Merck Life Science), as described by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. solates showing potential probiotic traits were further identified through 16S rDNA sequencing. Genomic DNA was extracted using the Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer\u0026rsquo;s instructions. The 16S rDNA gene was amplified using the universal primers 20F (5\u0026prime;-GAG TTT GAT CCT GGC TCA G-3\u0026prime;) and 1500R (5\u0026prime;-GTT ACC TTG TTA CGA CTT-3\u0026prime;) according to the protocol of [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Pairwise alignment of the resulting 16S rDNA sequences was performed using the BLASTn tool available on the National Center for Biotechnology Information (NCBI) website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed May 11, 2024) to determine the closest taxonomic identity. For phylogenetic analysis, 16S rDNA sequences of the CKNJH11 isolate and closely related \u003cem\u003eBacillus\u003c/em\u003e species were retrieved from the NCBI database. Multiple sequence alignment was conducted using MAFFT v7.525 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with default parameters. A phylogenetic tree was then constructed using the Maximum Likelihood (ML) method implemented in IQ-TREE v2.3.4 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e],, with 1000 bootstrap replications to evaluate the robustness of the inferred clades. Bootstrap values were indicated at the corresponding nodes, and branch lengths reflected the genetic distances among sequences. The phylogenetic tree was visualized using the Interactive Tree of Life (iTOL) web-based tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Sporulation efficiency\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus aryabhattai\u003c/em\u003e CKNJH11 was cultured on Difco Sporulation Medium (DSM; BD Diagnostics, USA) and incubated at 30\u0026deg;C for 72 hours to induce sporulation. Sporulation efficiency was assessed using a dual-staining protocol involving 5% malachite green and 0.1% safranin. After staining, spore suspensions were washed with sterile distilled water and centrifuged at 8000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. To eliminate residual vegetative cells, the suspensions were heat-treated at 80\u0026deg;C for 30 minutes. Viable spore counts were subsequently determined by plating serial dilutions on nutrient agar (NA; Himedia, India) and incubating at 37\u0026deg;C for 24 hours, following the method described by [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Sporulation efficiency was expressed as the percentage of viable spores relative to the initial cell count.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 pH, bile salt, and saline tolerance\u003c/h2\u003e \u003cp\u003eThe tolerance of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 spores to extreme pH conditions was evaluated by incubating them in sterile pepsin solutions (Sigma-Aldrich, Singapore) adjusted to pH 1.0, 2.0, and 3.0 at 37\u0026deg;C for 2 hours. Post-incubation, viability was assessed using the standard plate count method on NA plates, followed by incubation at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003eBile salt tolerance was tested by exposing spore suspensions to bile salt solutions (Himedia, India) at concentrations of 0%, 2.5%, 5.0%, 7.5%, and 10%. After 2 hours of exposure at 37\u0026deg;C, viable cell counts were determined using the standard plate count method on NA plates.\u003c/p\u003e \u003cp\u003eSalinity tolerance was assessed by culturing the spores on NA plates supplemented with NaCl at concentrations of 0%, 0.5%, 1%, 2%, and 3%. Cultures were incubated in a shaking incubator at 37\u0026deg;C for 24 hours. Viability was then determined using the standard plate count method, with plates incubated for an additional 24 hours at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Pathogenic bacteria biofilm inhibition\u003c/h2\u003e \u003cp\u003eThe biofilm inhibition assay was performed following the method described by [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with minor modifications. \u003cem\u003eBacillus aryabhattai\u003c/em\u003e CKNJH11 was cultured in tryptone soy broth (TSB; Himedia, India) and incubated at 37\u0026deg;C with shaking at 150 rpm for 72 hours. After incubation, the culture was centrifuged at 8000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C, and the supernatant was filtered through a 0.2-\u0026micro;m syringe filter to obtain a cell-free supernatant. The bacterial concentration of the culture was adjusted to 10⁸ CFU/mL.\u003c/p\u003e \u003cp\u003eFor the biofilm inhibition assay, 100 \u0026micro;L of the bacterial suspension and 100 \u0026micro;L of the cell-free supernatant were co-inoculated into each well of a sterile 96-well microtiter plate. The plate was incubated at 37\u0026deg;C for 24 hours to allow biofilm formation. Following incubation, non-adherent cells were gently removed, and the wells were washed three times with sterile 1\u0026times; phosphate-buffered saline (PBS, pH 7.1; Himedia, India).\u003c/p\u003e \u003cp\u003eBiofilms were stained with 0.1% (w/v) crystal violet for 20 minutes at room temperature. Excess stain was removed by washing the wells three times with PBS. To solubilize the retained dye, 95% ethanol was added to each well, and the plate was incubated at 4\u0026deg;C for 30 minutes. The absorbance at 630 nm was measured using a microplate reader (SPECTROstar\u0026reg; Nano, BMG LABTECH, Ortenberg, Germany) to quantify biofilm biomass. Lower absorbance values indicated greater inhibition of biofilm formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Safety assessment of B. aryabhattai CKNJH11\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.1 Hemolytic activity assessment\u003c/h2\u003e \u003cp\u003eHemolytic activity was evaluated according to the protocol described by [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eBacillus aryabhattai\u003c/em\u003e CKNJH11 was streaked onto sheep blood agar plates (Hardy Diagnostics, USA) and incubated at 37\u0026deg;C for 48 hours. Hemolysis was assessed by examining the appearance and type of halo around bacterial colonies: a greenish halo indicated α-hemolysis, a clear halo denoted β-hemolysis, and the absence of any halo signified non-hemolytic activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.2 Antibiotic susceptibility\u003c/h2\u003e \u003cp\u003eThe antibiotic susceptibility of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 was determined using the disc diffusion method as described by [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The isolate was cultured in Lysogeny Broth (LB; Himedia, India) at 37\u0026deg;C, and four antibiotics were tested: ampicillin (10 \u0026micro;g), tetracycline (30 \u0026micro;g), amoxicillin (30 \u0026micro;g), and cloxacillin (30 \u0026micro;g) (all from Himedia, India). For the assay, bacterial suspensions were spread evenly onto Mueller-Hinton Agar (MHA) plates (Himedia, India). Antibiotic discs were then placed on the surface of the inoculated plates, which were incubated at 37\u0026deg;C for 24 hours. After incubation, the diameters of the inhibition zones were measured and interpreted as follows: susceptible (S)\u0026thinsp;\u0026ge;\u0026thinsp;20 mm; moderately susceptible (M)\u0026thinsp;=\u0026thinsp;12\u0026ndash;19 mm; and resistant (R)\u0026thinsp;\u0026le;\u0026thinsp;12 mm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental design and diet preparation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Experimental fish\u003c/h2\u003e \u003cp\u003eA total of 120 barramundi (\u003cem\u003eL. calcarifer\u003c/em\u003e) fingerlings, with an average body weight of 13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 g and an average length of 10.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 cm, were obtained from the Phang Nga Coastal Aquaculture Research and Development Center (Phang Nga, Thailand; coordinates: 8\u0026deg;25'12.7\"N, 98\u0026deg;14'34.5\"E). Prior to the start of the experiment, the fish were acclimatized for two weeks under standardized conditions and fed a commercial control diet (Higade 9006T; Charoen Pokphand Foods, Bangkok, Thailand).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Extraction of polysaccharides from G. fisheri\u003c/h2\u003e \u003cp\u003ePolysaccharides were extracted from dried \u003cem\u003eG. fisheri\u003c/em\u003e seaweed collected from Thung Sai Chai, Chaiya District, Surat Thani Province, Thailand. Twenty grams of seaweed were soaked in a 6% sodium hydroxide (NaOH) solution at 28\u0026deg;C for 5 hours to facilitate cell wall breakdown. After soaking, the seaweed was thoroughly rinsed with distilled water and cut into 1.0\u0026ndash;1.5 cm segments in preparation for hot-water extraction. A total of 1500 mL of distilled water was added, and the pH was adjusted to between 6.2 and 6.8. The mixture was then boiled for 1.5 hours with occasional stirring. After boiling, the extract was filtered through two layers of fine cloth and allowed to cool to room temperature. The filtrate was subsequently frozen at \u0026minus;\u0026thinsp;20\u0026deg;C for approximately 24 hours. The resulting frozen agar was dried in an oven at 60\u0026deg;C until a constant weight was achieved. The dried agar was then ground into a fine powder using a blender and stored at room temperature for further use in feed formulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Experimental design\u003c/h2\u003e \u003cp\u003eProbiotic diets were prepared by incorporating \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 spores, which were obtained through nutrient exhaustion in Difco Sporulation Medium (DSM). Spores were subsequently purified and quantified according to the method described by [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and incorporated into the feed at a final concentration of 1 \u0026times; 10⁶ CFU/g.\u003c/p\u003e \u003cp\u003eA total of 120 barramundi fingerlings were randomly assigned to twelve 25-liter tanks, with 10 fish per tank. The feeding trial employed a completely randomized design with four dietary treatments, each consisting of three replicates:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eD1\u003c/b\u003e: Control diet (no probiotic supplementation)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eD2\u003c/b\u003e: Diet supplemented with free (non-encapsulated) spores\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eD3\u003c/b\u003e: Diet supplemented with spores encapsulated in sodium alginate\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eD4\u003c/b\u003e: Diet supplemented with spores co-encapsulated with red algal polysaccharides and sodium alginate\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eEncapsulation was performed by adding the probiotic-alginate mixture dropwise into a 0.45 M calcium chloride solution and allowing bead formation for 45 minutes. The resulting gel beads were collected via filtration using filter paper, rinsed with sterile distilled water to remove residual calcium ions, and then dried at 50\u0026ndash;70\u0026deg;C until a stable consistency was achieved.\u003c/p\u003e \u003cp\u003eThe feeding trial lasted eight weeks. Fish were fed twice daily (at 09:00 and 17:00) at a rate of 3% of their body weight. All tanks were aerated continuously using air stones connected to a central air compressor. Water quality parameters, including total ammonia (NH₃), nitrite (NO₂⁻), pH, salinity, and total solids, were monitored daily to maintain optimal rearing conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Growth performance\u003c/h2\u003e \u003cp\u003eAt the end of the 8-week feeding trial, all surviving fish were individually weighed. Growth performance was assessed using the following parameters: weight gain (WG), average daily growth (ADG), feed conversion ratio (FCR), and survival rate (SR). These parameters were calculated using the formulas below:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eWeight gain (WG, g)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Final weight \u0026ndash; Initial weight\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAverage daily growth (ADG, g/day)\u003c/b\u003e = (Final weight \u0026ndash; Initial weight) / number of days\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFeed conversion ratio (FCR)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Total feed intake / weight gain\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSurvival rate (SR, %)\u003c/b\u003e = (Final number of fish / Initial number of fish) \u0026times; 100\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Bacterial detection in the intestine of \u003cem\u003eL. calcarifer\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThree fish per tank (n\u0026thinsp;=\u0026thinsp;9 per treatment group) were randomly selected for intestinal bacterial analysis following previously described protocol [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Using sterile dissection tools, the abdominal cavity of each fish was opened, and approximately 5 g of midgut tissue was aseptically collected. The samples were homogenized and suspended in 9 mL of sterile 0.85% (w/v) saline. Serial dilutions were prepared for bacterial enumeration.\u003c/p\u003e \u003cp\u003eSelective media were used to isolate specific bacterial genera:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eVibrio\u003c/em\u003e spp. on Thiosulfate\u0026ndash;Citrate\u0026ndash;Bile Salts\u0026ndash;Sucrose (TCBS) agar\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eAeromonas\u003c/em\u003e spp. on Starch Ampicillin Agar (SAA)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003ePseudomonas\u003c/em\u003e spp. on Pseudomonas Isolation Agar (PIA)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eB. aryabhattai\u003c/em\u003e on Mannitol Egg Yolk Polymyxin (MYP) agar\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eTotal bacterial counts were expressed as colony-forming units (CFU) per gram of intestinal content, based on colony growth on the respective selective media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Hematological parameters\u003c/h2\u003e \u003cp\u003eAt the conclusion of the feeding trial, three fish per tank were randomly selected (n\u0026thinsp;=\u0026thinsp;9 per treatment) for hematological analysis. Fish were anesthetized using clove oil at an appropriate concentration, and blood samples were collected from the caudal vein using heparinized syringes. The samples were transferred into K₂-EDTA-coated VACUETTE\u0026reg; tubes for analysis. Hematological parameters, including total white blood cell (WBC) count, total red blood cell (RBC) count, hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), were evaluated following the methods described in previous studies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData normality was assessed using the Shapiro\u0026ndash;Wilk test. Differences among treatments were evaluated using ANOVA, with significant differences between means identified using the LSD test at a 95% confidence level. These analyses were conducted using IBM SPSS Statistical software (IBM Corp. Released 2023. IBM SPSS Statistics for Windows, Version 29.0.2.0, Armonk, NY: IBM Corp). All data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). Correlations between growth parameters were analyzed using Pearson\u0026rsquo;s linear correlation coefficient (\u003cem\u003er\u003c/em\u003e), computed with the \u003cem\u003eggplot2\u003c/em\u003e package in R v3.4.4. The formula for Pearson\u0026rsquo;s correlation at a confidence interval of 95% is expressed as follows:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:r\\:=\\frac{\\sum\\:\\left(x\\:-\\:{m}_{x\\:}\\right)\\:\\left(y\\:-{m}_{y}\\right)}{\\sqrt{{\\sum\\:\\left(x-{m}_{x}\\right)}^{2}\\sum\\:{(y-{m}_{y)}}^{2}}}$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere \u003cem\u003er\u003c/em\u003e is the correlation coefficient; \u003cem\u003ex\u003c/em\u003e and \u003cem\u003ey\u003c/em\u003e are the variables being compared; \u003cem\u003emx\u003c/em\u003e and \u003cem\u003emy\u003c/em\u003e are the means of \u003cem\u003ex\u003c/em\u003e and \u003cem\u003ey\u003c/em\u003e, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Identification of isolate CKNJH11\u003c/h2\u003e\n \u003cp\u003eThe results of physiological and biochemical analyses indicated that the isolate produced catalase and amylase and demonstrated the ability to hydrolyze starch. The strain also tested positive for sporulation, confirming its capacity to form spores efficiently. Microscopic examination of the spore suspensions revealed that 90\u0026ndash;99% of the cells had sporulated, with green-stained spores and red-stained vegetative cells observed using dual staining. The isolate was negative for both cytochrome oxidase activity and indole production, suggesting the absence of tryptophan degradation (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Gram staining revealed that the bacterium was bacilli-shaped and retained a purple coloration under light microscopy, indicating it is a Gram-positive rod (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on 16S rDNA sequencing and phylogenetic analysis, the isolate was identified as \u003cem\u003eB. aryabhattai\u003c/em\u003e, showing the highest sequence similarity to \u003cem\u003eB. aryabhattai\u003c/em\u003e B8W22 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Analysis of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 as probiotic\u003c/h2\u003e\n \u003cp\u003eThe isolated strain \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 exhibited strong acid tolerance, maintaining survival rates of 64.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40% at pH 2.0, 84.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31% at pH 3.0, and 97.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35% at pH 4.0 over a 6-hour incubation period. The strain also showed considerable resistance to bile salts, with survival rates of 84.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78% at 1%, 84.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73% at 2.5%, and 78.06\u0026thinsp;\u0026plusmn;\u0026thinsp;8.12% at 5% concentrations. Sporulation efficiency was notably high, reaching 95.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11% (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The cell-free supernatant of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 significantly inhibited biofilm formation by \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, with inhibition rates of 58.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17%, 59.89\u0026thinsp;\u0026plusmn;\u0026thinsp;6.49%, and 31.91\u0026thinsp;\u0026plusmn;\u0026thinsp;6.25%, respectively. Additionally, the strain demonstrated robust salinity tolerance, maintaining survival rates above 90.42\u0026thinsp;\u0026plusmn;\u0026thinsp;9.65% across a salinity range of 5 to 30 ppt (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 safety analysis\u003c/h2\u003e\n \u003cp\u003eThe hemolytic activity assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that the indicator strains \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e produced distinct clear zones of hemolysis on blood agar, confirming their hemolytic potential. In contrast, colonies of the test strain \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 displayed no observable hemolytic zones, indicating the absence of hemolytic activity. This finding supports the strain\u0026rsquo;s safety for aquaculture use, as it suggests a lack of hemolytic virulence factors.\u003c/p\u003e\n \u003cp\u003eAntibiotic susceptibility testing revealed that CKNJH11 was sensitive to ampicillin, tetracycline, and cloxacillin, while showing moderate susceptibility to amoxicillin (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These results further support the biosafety of \u003cem\u003eB. aryabhattai\u003c/em\u003eCKNJH11 for potential probiotic application in aquaculture systems.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Growth performance\u003c/h2\u003e\n \u003cp\u003eAfter the 8-week feeding trial, fish fed diets supplemented with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 exhibited significantly enhanced growth performance compared to the control group (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The WG in groups D2, D3, and D4 ranged from 45.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24 to 60.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 g, while the ADG ranged from 0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 to 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 g/day. Both WG and ADG were significantly higher in all probiotic-treated groups than in the control group D1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the highest values observed in group D4. The FCR also differed significantly among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The lowest FCR, indicating the most efficient feed utilization, was recorded in group D4 (5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18), whereas the control group (D1) had the highest FCR (8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37). Survival rates across all groups ranged from 93.33\u0026ndash;100%, with no statistically significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of dietary supplementation with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 on the growth performance of \u003cem\u003eL. calcarifer\u003c/em\u003e after an 8-week feeding trial.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eExperimental diets\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eD1\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eD2\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInitial length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinal length (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInitial weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWG (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADG (day)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFCR (g/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eD1: control diet with no probiotics; D2: diet supplemented with free spore probiotics; D3: diet with spore probiotics encapsulated in sodium alginate; D4: diet with spore probiotics co-encapsulated with polysaccharide and sodium alginate. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3). Different superscript letters within the same row indicate statistically significant differences between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eLinear regression analysis revealed strong positive correlations between diet supplementation and growth parameters, including WG (r\u0026thinsp;=\u0026thinsp;0.975), ADG (r\u0026thinsp;=\u0026thinsp;0.960), and survival rate (SR; r\u0026thinsp;=\u0026thinsp;0.944). In contrast, FCR exhibited a strong negative correlation (r = \u0026minus;\u0026thinsp;0.947), further indicating the beneficial effects of probiotic supplementation on growth performance and feed efficiency in \u003cem\u003eL. calcarifer\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Detection of bacterial species in the intestine of \u003cem\u003eL. calcarifer\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe experimental results revealed significant differences in intestinal bacterial colonization between the treatment groups and the control group. \u003cem\u003eVibrio\u003c/em\u003e spp. incapable of sucrose fermentation were significantly reduced in all probiotic-treated groups, with bacterial counts ranging from log 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 to 3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 CFU/g, compared to log 3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 CFU/g in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, \u003cem\u003eVibrio\u003c/em\u003e spp. capable of sucrose fermentation also exhibited lower counts in the treated groups, ranging from log 3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 to 3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 CFU/g, in contrast to log 4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 CFU/g observed in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eNotably, \u003cem\u003eAeromonas\u003c/em\u003e spp. and \u003cem\u003ePseudomonas\u003c/em\u003e spp. were not detected in any of the experimental groups, including the control. In contrast, \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 was detected exclusively in the intestines of fish from groups D3 and D4, where the probiotic was encapsulated. Quantitative analysis showed concentrations of log 3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 CFU/g in group D3 and log 3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 CFU/g in group D4, while the strain was absent in the control group. These findings indicate successful colonization and potential competitive exclusion of pathogenic bacteria by the probiotic strain.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Hematological parameters\u003c/h2\u003e\n \u003cp\u003eThe hematological profiles of \u003cem\u003eL. calcarifer\u003c/em\u003e across the four dietary treatments indicated that fish fed diets supplemented with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 spores exhibited improved hematological indices compared to the control group. The RBC counts in groups D2, D3, and D4 ranged from 4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 to 4.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u0026times; 10⁶ cells/mL. Similarly, WBC counts were elevated in the supplemented groups, ranging from 23.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89 to 24.50\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56 \u0026times; 10\u0026sup3; cells/mL. The highest levels of Hb and Hct were recorded in group D4, with values of 8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 g/dL and 53.60\u0026thinsp;\u0026plusmn;\u0026thinsp;33.05%, respectively. However, MCH and MCHC did not differ significantly among the treatment groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Water quality\u003c/h2\u003e\n \u003cp\u003eWater quality parameters were monitored throughout the experimental period. pH levels exhibited a slight upward trend across all treatment groups (D1\u0026ndash;D4), ranging from 7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 to 7.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94, though these differences were not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Salinity values remained relatively stable, ranging from 24.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 to 25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 ppt, with no significant differences observed among treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eIn contrast, notable differences were observed in nitrite and ammonia concentrations. Nitrite levels ranged from 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 to 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 mg/L, while ammonia concentrations varied between 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/L. The D4 group exhibited the lowest levels of both nitrite and ammonia, showing significant reductions compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Total dissolved solids (TDS) ranged from 24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;8.88 to 27.88\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53 mg/L across all treatments, with no statistically significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, stakeholders in the aquaculture industry have made significant strides toward sustainability by adopting clean and hygienic farming practices. Key initiatives include the reduction of antibiotic use and the incorporation of alternative supplements aimed at enhancing the overall health of aquatic animals [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this context, the present study sought to isolate and evaluate the probiotic potential of \u003cem\u003eBacillus aryabhattai\u003c/em\u003e strain CKNJH11, derived from shrimp pond sediments. The study provides a comprehensive characterization of the strain\u0026rsquo;s morphological, biochemical, and functional properties.\u003c/p\u003e \u003cp\u003eIn vitro assessments demonstrated that \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 possesses robust probiotic traits suitable for aquaculture applications. Physiological and biochemical analyses confirmed hallmark \u003cem\u003eBacillus\u003c/em\u003e features, including catalase and amylase production, efficient starch hydrolysis, and a high sporulation efficiency (90\u0026ndash;99%), which are essential for survival and stability under stressful environmental conditions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Molecular identification through 16S rDNA sequencing and phylogenetic analysis revealed a close genetic relationship to \u003cem\u003eB. aryabhattai\u003c/em\u003e B8W22, supporting its taxonomic classification.\u003c/p\u003e \u003cp\u003eThe strain exhibited excellent tolerance to simulated gastrointestinal conditions, maintaining high survival rates from 64.9% at pH 2.0 to 97.9% at pH 4.0 over a 6-hour period. Additionally, it remained viable in the presence of bile salts up to 5%, suggesting its capability to colonize the gastrointestinal tract of fish. Its resilience across a salinity range of 5\u0026ndash;30 ppt further underscores its adaptability to diverse aquaculture systems. Notably, the cell-free supernatant of CKNJH11 significantly inhibited biofilm formation by common aquaculture pathogens, including \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, indicating the production of antimicrobial compounds that may suppress pathogenic colonization [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Importantly, safety evaluations revealed that the strain is non-hemolytic and susceptible to several commonly used antibiotics, minimizing potential risks associated with virulence or antibiotic resistance. These findings collectively highlight the promise of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 as a safe and effective probiotic candidate for application in sustainable aquaculture.\u003c/p\u003e \u003cp\u003eDietary supplementation with \u003cem\u003eBacillus\u003c/em\u003e spp. has consistently been associated with enhanced growth performance and health status across various aquaculture species, including \u003cem\u003eOreochromis niloticus\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], \u003cem\u003eLabeo chrysophekadion\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eClarias gariepinus\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and \u003cem\u003eApostichopus japonicus\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the present study, \u003cem\u003eLates calcarifer\u003c/em\u003e fed diets supplemented with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 exhibited significantly improved performance in weight gain, daily weight gain, feed conversion ratio, and survival rate compared to the control group. These enhancements are largely attributable to the ability of \u003cem\u003eBacillus\u003c/em\u003e spp. to produce extracellular bioactive compounds, such as enzymes and vitamins, that improve nutrient digestibility and stimulate innate immune responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, \u003cem\u003eBacillus\u003c/em\u003e probiotics are known to modulate the gut microbiota, thereby contributing to improved nutrient assimilation and growth outcomes [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Hematological assessments further confirmed the beneficial effects of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 supplementation. Treated fish exhibited elevated red and white blood cell counts, with the highest values observed in group D4. These findings indicate enhanced hematopoiesis and immune function. Previous studies have demonstrated that probiotic supplementation increases immune cell populations, including macrophages, lymphocytes, granulocytes, and neutrophils, in fish, mirroring responses seen in higher vertebrates [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Probiotics exert immunomodulatory effects by interacting with key immune cells (e.g., monocytes, macrophages, and lymphocytes), enhancing the host\u0026rsquo;s innate immune response [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. For example, in rainbow trout, probiotic supplementation significantly elevated RBC and WBC levels [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], while similar trends have been reported in major carp and rainbow trout following dietary inclusion of \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. An other study also reported increased WBC and lymphocyte levels in juvenile cobia fed indigenous \u003cem\u003eBacillus\u003c/em\u003e isolates (\u003cem\u003eB. spp.\u003c/em\u003e RCS1 and \u003cem\u003eB. cereus\u003c/em\u003e RCS3), reinforcing the probiotic\u0026rsquo;s immunostimulatory potential [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe superior performance observed in group D4 can be attributed to the dual-encapsulation approach using sodium alginate and red algae-derived polysaccharides. Sodium alginate forms a protective gel matrix that shields probiotic spores from gastric degradation, enhancing delivery to the intestinal tract [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Concurrently, polysaccharides act as prebiotics, supporting a favorable gut environment and contributing to immune modulation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This co-encapsulation strategy not only enhances probiotic viability during gastrointestinal transit but also improves microbial colonization, thereby amplifying the probiotic\u0026rsquo;s effects on host health and performance.\u003c/p\u003e \u003cp\u003eMicrobial analyses revealed significant changes in the intestinal microbiota of treated fish. Notably, \u003cem\u003eVibrio\u003c/em\u003e spp. populations, both sucrose-fermenting and non-fermenting, were significantly reduced in probiotic-treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with previous studies demonstrating the antagonistic activity of probiotics against \u003cem\u003eVibrio\u003c/em\u003e pathogens in aquaculture [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The complete absence of \u003cem\u003eAeromonas\u003c/em\u003e spp. and \u003cem\u003ePseudomonas\u003c/em\u003e spp. in all experimental groups suggests a potential selective exclusion effect by the probiotic, possibly through competitive colonization or antimicrobial metabolite production [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, the detection of \u003cem\u003eB. aryabhattai\u003c/em\u003e exclusively in the intestines of probiotic-treated fish confirms successful colonization and highlights its potential to confer health benefits through modulation of the intestinal microbial community.\u003c/p\u003e \u003cp\u003eIn addition to growth and immune-related benefits, the study also demonstrated that \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 supplementation contributed to improved water quality. The D4 group exhibited significantly reduced ammonia and nitrite levels, aligning with previous findings that \u003cem\u003eBacillus\u003c/em\u003e spp. can enhance water quality by metabolizing nitrogenous waste [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. These environmental benefits further support the application of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 as a sustainable probiotic, promoting both fish health and ecological balance in aquaculture systems.\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that dietary inclusion of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11, especially when co-encapsulated with prebiotic polysaccharides, can significantly enhance growth performance, immune status, intestinal health, and environmental quality in \u003cem\u003eL. calcarifer\u003c/em\u003e culture. This integrated approach holds promise for sustainable and health-conscious aquaculture practices.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated the probiotic potential of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 in improving the health and productivity of \u003cem\u003eL. calcarifer\u003c/em\u003e. The strain exhibited key probiotic characteristics, including non-hemolytic activity, high sporulation efficiency, and strong tolerance to salinity and acidic conditions. Dietary supplementation with \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 significantly enhanced growth performance, feed efficiency, and hematological parameters, with the most pronounced effects observed in fish receiving co-encapsulated spores. Furthermore, probiotic-treated groups showed a marked reduction in gut-associated pathogenic \u003cem\u003eVibrio\u003c/em\u003e spp. and improved water quality through decreased nitrite and ammonia concentrations. Collectively, these findings support the application of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 as a safe, effective, and environmentally beneficial probiotic for sustainable aquaculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by Chiang Mai University, Chiang Mai, Thailand. Additional support was provided by the Program in Agricultural Science and Technology, Faculty of Innovative Agriculture, and the Fishery Establishment Project at Prince of Songkla University, Surat Thani Campus, Surat Thani, Thailand. We also acknowledge the Scientific Laboratory and Equipment Center, Office of Surat Thani Campus, Prince of Songkla University, for their assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWaraporn Appamano\u003c/strong\u003e: Methodology, Investigation, Writing - Original Draft. \u003cstrong\u003eOrathai Dangsawat\u003c/strong\u003e: Methodology, Investigation. \u003cstrong\u003eSarayut Onsanit\u003c/strong\u003e: Methodology, Investigation \u003cstrong\u003eRapeewan Sowanpreecha\u003c/strong\u003e: Methodology, Investigation. Phatthanaphong Therdtatha:\u0026nbsp;Writing - Review \u0026amp; Editing. \u003cstrong\u003eTran Hoang Trieu Quan\u003c/strong\u003e: Writing - Review \u0026amp; Editing. \u003cstrong\u003eThi Hang Ho\u003c/strong\u003e: Writing - Review \u0026amp; Editing. \u003cstrong\u003eLuu Tang Phuc Khang\u003c/strong\u003e: Writing - Review \u0026amp; Editing. \u0026nbsp;\u003cstrong\u003ePapungkorn Sangsawad\u003c/strong\u003e: Writing - Review \u0026amp; Editing, Formal Analysis, Supervision. \u003cstrong\u003e\u0026nbsp;Nguyen\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Dinh-Hung:\u0026nbsp;\u003c/strong\u003eFormal Analysis, Writing - Original Draft, Writing - Review \u0026amp; Editing. \u003cstrong\u003eNguyen Vu Linh\u003c/strong\u003e: Investigation, Methodology, Validation, Software, Data Curation, Formal Analysis, Writing - Review \u0026amp; Editing. \u003cstrong\u003ePatima Permpoonpattana\u003c/strong\u003e: Conceptualization, Validation, Resources, Supervision, Project administration, Funding acquisition, and Writing - Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic of animal use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments in this study were performed in accordance with the relevant guidelines and regulations. The experimental protocols were approved by the Institutional Animal Care and Use Committee, Prince of Songkla University (Approval Ref. Number 90/2566). All the procedure of the study is followed by the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGhanei-Motlagh R, Gharibi D, Mohammadian T, Khosravi M, Mahmoudi E, Zarea M, Menanteau-Ledouble S, El-Matbouli M (2021) Feed supplementation with quorum quenching probiotics with anti-virulence potential improved innate immune responses, antioxidant capacity and disease resistance in Asian seabass (Lates calcarifer). 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Aquaculture 503:347\u0026ndash;356\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Chiang Mai University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacillus aryabhattai, Spore-forming polysaccharides, Immunological response, Gastrointestinal microbiota, Pathogen resistance","lastPublishedDoi":"10.21203/rs.3.rs-6715276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6715276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe application of \u003cem\u003eBacillus\u003c/em\u003e species as probiotics in aquaculture has been widely documented, showing notable benefits in growth performance, feed efficiency, and immune response. In this study, \u003cem\u003eBacillus aryabhattai\u003c/em\u003e CKNJH11, isolated from shrimp pond sediment, was evaluated for its probiotic potential through 16S rRNA sequencing and comprehensive in vitro assays. The strain exhibited strong probiotic traits, including high acid tolerance (survival at pH 2.0), bile salt resistance (up to 5% for 6 h), and significant inhibition of biofilm formation by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (\u0026gt;\u0026thinsp;50% reduction). Safety evaluations confirmed the absence of hemolytic activity and susceptibility to common antibiotics, supporting its suitability for aquaculture use. An eight-week feeding trial involving 120 barramundi (\u003cem\u003eLates calcarifer\u003c/em\u003e) fingerlings compared four dietary treatments: control (no probiotics), free spores, alginate-encapsulated spores, and spores co-encapsulated with sodium alginate and red seaweed polysaccharides. Fish receiving the co-encapsulated probiotic diet exhibited significantly enhanced growth (final body length: 8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 cm; weight: 60.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 g) and improved survival. Immunological analyses showed reduced \u003cem\u003eVibrio\u003c/em\u003e spp. in the gastrointestinal tract, along with elevated white and red blood cell counts and hemoglobin levels, indicating enhanced immune status. These results suggest that co-encapsulation of \u003cem\u003eB. aryabhattai\u003c/em\u003e CKNJH11 with algae-derived polysaccharides improves probiotic efficacy and stability, offering a promising approach to enhance growth, immunity, and productivity in aquaculture systems.\u003c/p\u003e","manuscriptTitle":"Co-encapsulation of Bacillus aryabhattai CKNJH11 with Algae-derived Polysaccharides Enhances Growth Performance and Immune Response in Asian Seabass (Lates calcarifer)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 08:58:29","doi":"10.21203/rs.3.rs-6715276/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08463b1d-36ea-477f-afff-12579d30b9f1","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48845294,"name":"Aquaculture and Mariculture"}],"tags":[],"updatedAt":"2025-05-22T08:58:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 08:58:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6715276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6715276","identity":"rs-6715276","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}