TWI816432B - A bacillus safensis strain providing beneficial effects on growth performance and health, and application thereof - Google Patents

Abstract

The present invention provides Bacillus safensis, which is deposited at the Biological Resource Preservation and Research Center of the Food Industry Development Institute in Hsinchu, Taiwan. The deposit date is June 10, 2011, and the deposit number is BCRC 911134. The present invention also provides a use of the Salfordella strain as a probiotic to feed aquaculture organisms, thereby promoting the nutritional metabolism and growth performance of aquaculture organisms, improving the intestinal microbial phase, and improving the innate immune function. Reduce the survival rate after pathogenic bacteria infection.

Description

A kind of Bacillus sulfaensis that enhances the growth and health benefits of fish and its application

The invention relates to Bacillus safensis , which can be used as a probiotic to feed aquaculture organisms, which can promote nutritional metabolism and growth performance, improve intestinal microbial phase, improve immune function, and reduce the survival rate after pathogenic bacteria infection. .

Due to the catastrophe of marine resources and the rapid increase in the world's population, the current catch of capture fisheries has reached the upper limit. In order to meet human food needs, the importance of aquaculture around the world has gradually increased in recent years. According to a 2018 report by the Food and Agriculture Organization of the United Nations (FAO), aquaculture accounts for 53% of total fishery production, with an output value of US$232 billion (see non-patent document 1).

Aquaculture operators often use antibacterial drugs in advance to prevent the occurrence of diseases. However, excessive use often leads to serious negative effects. Bacteria may develop resistance to the antibiotics, causing the antibacterial drugs to lose their inhibitory effect on pathogenic bacteria, or remain in the bodies of aquatic animals. It cannot be ruled out that it will have a significant impact on the ecological environment, thus causing quality and safety issues in aquaculture products. When people eat, they may also ingest and accumulate antibacterial drugs in their bodies, which may pose a risk of harm to humans. Therefore, it is necessary to find a method that can replace the use of antibacterial drugs, which has become the most important issue at present, and the use of probiotics is a method worthy of attention.

In the biological control method, the use of probiotics isolated from nature has less impact on the environment and can reduce the risk of the emergence of drug-resistant strains. It is one of the effective strategies to replace antibiotics in preventing diseases. In addition, the ability to fight various pathogenic bacteria is also one of the very important characteristics of probiotics. In the field of aquaculture, lactic acid bacteria and Bacillus are the most widely used. This is because the hydrolytic enzymes (such as phytase, fiberase, protease and xylanase) produced by these probiotics can improve nutrient utilization. And because it has been used on mammals for a long time, it is safe. In addition, Patent Documents 1 and 2 respectively disclose Chromobacterium aquaticum strain JS-1 (Chromobacterium aquaticum) and Rummeliibacillus stabekisii strain BST-01 (Rummeliibacillus stabekisii) that can be used as probiotics.

The aforementioned Bacillus spp. can produce endospores that are resistant to the low pH value of the gastrointestinal tract, and therefore is often used as a probiotic (see Non-Patent Document 2). Although the genus Bacillus has been widely used as a probiotic in aquaculture, so far, Bacillus safensis in this genus has not been used as a probiotic in aquaculture. Relevant disclosures about probiotics are still minimal. Although the study by Zhang et al. (see Non-Patent Document 3) mentions Salfobacillus spp., the probiotics used as dietary supplements for fermented feed products in their study are a mixture of multiple strains and were not separately discussed in this study. The effect of any Sulfobacterium as a probiotic, and this study only revealed the effect of the mixed probiotic on Penaeus vannamei. [Prior technical literature]

[Patent Document 1] Republic of China Patent Publication No. 202143852 [Patent Document 2] Republic of China Patent Publication No. 202022108

[Non-patent document 1] FAO, The State of World Fisheries and Aquaculture, Meeting the Sustainable Development Goals Rome, License: CC BY-NC-SA 3.0 IGO, 2018.

[Non-patent document 2] Kuebutornye, F., Abarike, E., & Lu, Y. (2019). A review on the application of Bacillus as probiotics in aquaculture. Fish & Shellfish Immunology, 87, 820-828.

[Non-patent document 3] Zhang, M.; Pan, L.; Fan, D.; He, J.; Su, C.; Gao, S.; Zhang, M. Study of fermented feed by mixed strains and their effects on the survival, growth, digestive enzyme activity and intestinal flora of Penaeus vannamei. Aquaculture 2021 530, 735703.

In view of the above problems, the purpose of the present invention is to provide a probiotic that can replace antibiotics and chemical agents. It is expected that it has the effect of regulating immune function and can be widely used in the prevention and treatment of various diseases. Furthermore, in order to pursue increased production and feed efficiency, it is also expected that the probiotics can have the effect of promoting the nutritional metabolism and growth performance of cultured organisms.

After in-depth research by the inventor of the present invention, it was discovered that Bacillus safensis NPUST1 (hereinafter also referred to as Bacillus safensis strain NPUST1 or NPUST1) isolated from the intestine of tilapia has the ability to secrete protease, amylase and lipase. With the ability of various hydrolytic enzymes such as enzymes, it has the potential to be used as a probiotic for aquaculture applications. Moreover, this strain can improve the innate immune performance of tilapia and strengthen its resistance to pathogenic bacteria.

Therefore, the present invention provides the following technical means.

A Bacillus safensis strain ( Bacillus safensis ), characterized by the fact that the Bacillus safensis strain is deposited at the Biological Resource Preservation and Research Center of the Food Industry Development Institute in Hsinchu, Taiwan. The deposit date is June 10, 2011, and the deposit number is BCRC 911134. .

Use of the aforementioned Salfobacillus strain as a feed additive, the feed additive being used for aquaculture fish.

In some embodiments, the aforementioned feed additives are used to improve the nutritional metabolism of aquaculture fish. In some implementation forms, the aforementioned feed additives are used to improve the growth performance of aquaculture fish. In some embodiments, the aforementioned feed additives are used to enhance the innate immunity of aquaculture fish.

In some implementation forms, the aforementioned improvement of nutritional metabolism of aquaculture fish refers to improvement of feed conversion rate (FE).

In some implementation forms, the aforementioned improvement of nutritional metabolism of aquaculture fish refers to improving the expression of nutritional metabolism-related genes. The aforementioned nutritional metabolism-related genes are glucokinase (GK) or glucose-6-phosphatase (glucose-6-phosphatase). phosphatase, G6Pase).

In some implementation forms, the aforementioned improvement of the growth performance of aquaculture fish refers to the improvement of body weight, specific growth rate (SGR) or the performance of growth-related genes. The aforementioned growth-related genes are growth hormone receptors (GHR). ) or type 1 insulin-like growth factor (insulin-like growth factor-1, IFG-1).

In some implementation forms, the aforementioned improvement of the innate immunity of aquaculture fish refers to the improvement of the expression of innate immunity-related genes. The aforementioned innate immunity-related genes are interleukin-1β (IL-1β), interleukin-1β, and interleukin-1β. Interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) or lysozyme (LYZ).

In some embodiments, the aforementioned feed additives are used to improve the disease resistance of aquaculture fish to Streptococcus S.iniaee.

In some embodiments, the feed additive is used to increase the proportion of probiotics in the intestinal microbial phase of aquaculture fish, and the probiotics are Bacillus licheniformis or Lactococcus lactis .

In some embodiments, the aforementioned feed additive is used to reduce the proportion of pathogenic bacteria in the intestinal microbial phase of aquaculture fish, and the aforementioned pathogenic bacteria are Aeromonas veronii , Aeromonas jandaei , Enterovibrio nigricans c or Enterovibrio coralii .

The use of the Salfordella strain NPUST1 of the present invention as a feed supplement in the aquaculture industry can improve the nutritional metabolism of aquaculture fish, promote feed conversion efficiency, and improve growth performance. In addition, when used as a feed supplement, the Salfordella strain of the present invention can promote innate immunity and thereby improve disease resistance against pathogenic bacteria. At the same time, the Salfordella strain of the present invention provides the effect of improving the intestinal microbial phase of fish, increasing the proportion of probiotics and reducing the proportion of pathogenic bacteria in the intestinal microbial phase.

The following discloses the embodiments of the present invention, which does not limit the present invention to be implemented in the following manner, but is intended to illustrate the details and implementation effects of the present invention.

[Screening of bacterial strains]

First, the intestinal tissues of six tilapia from local breeding ponds in Pingtung, Taiwan were sampled. After the intestinal tissues were homogenized, they were cultured in TSB medium at 28°C overnight, and then the culture was taken into another fresh medium. Incubate at 45°C for 2 days to induce sporulation of endosporogenic bacteria. This was followed by a 20-minute 80°C water bath to eliminate the non-endospore-producing bacteria and further isolate a single strain. Potential probiotics were screened from all isolates by culturing them on screening agar plates. After screening, a strain with strong protease, amylase, cellulase and xylanase activities was obtained, which may be a potential probiotic (refer to Figure 1 and Example 1 described below), so the strain was named NPUST1 strain, and the following biochemical characteristics and 16S rDNA genetic relationship identification were conducted by the Bioresource Collection and Research Center of Hsinchu Food Industry Development Research Institute in Taiwan.

[Identification of biochemical characteristics of bacterial strains]

The characterization results of the Salfordella strain NPUST1 showed that in terms of biochemical characteristics, the strain is an aerobic and motile endospore-producing Gram-positive bacillus. This strain has strong protease, amylase, cellulase and xylanase activities. At the same time, this strain also has catalase, β-galactosidase and gelatinase activities, and can ferment a variety of carbohydrates, including, for example, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol. D-cellobiose, D-maltose, D-sucrose, D-trehalose and D-tagatose.

The Sulfobacterium strain NPUST1 can secrete the above-mentioned enzymes, so it has a positive effect on improving the nutritional metabolism and growth performance of animals. This is because these enzymes help degrade macromolecules in the feed into forms that are easy to digest and absorb in the intestines. , thereby improving the utilization of nutrients in feed.

〔 16S rDNA sequence analysis results 〕

The 16S rDNA of NPUST1 was sequenced and compared with the known bacterial 16S rDNA sequence in the NCBI GenBank database. According to the comparison results, the sequence (GenBank accession number: OL477428, SEQ ID NO: 1) is identical to that of Sulfobacter sp. PgKB20 (GenBank accession number: CP043404.1) has 99% similarity. According to the BLAST results, NPUST1 is 99% similar to Bacillus safensis PgKB20. A genetic tree was established based on the 16S rDNA sequence (refer to Figure 2), and the genetic tree showed that NPUST1 belonged to Salfobacterium.

The following examples use tilapia as a test animal model to evaluate the effect of Salfordella strain NPUST1 as a probiotic fed to fish.

[Establishment of tilapia animal model]

In order to establish a tilapia experimental animal model, before the start of the experiment, tilapia were purchased from a local fish farm in Pingtung, Taiwan, and were adaptively raised in a 150L glass fish tank containing 120L fresh water for 7 days. The breeding facility has been certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). During the feeding process, 30% of the feeding water was replaced three times a week to maintain the feeding quality. During the adaptation period and the subsequent feeding test period, the feeding water environment conditions were as follows: water temperature was maintained at 28 ± 1 °C, pH was maintained at 6.7 ± 0.2, and dissolved oxygen was maintained at 6.1 ± 0.45 mg/L. The experimental protocols in each of the following examples were in compliance with the animal welfare regulations of the place where the experiment was conducted, and were approved by the Laboratory Animal Care and Use Committee of the National Pingtung University of Science and Technology (Approval Number: NPUST-107-063).

360 tilapia with an average weight of 0.55 ± 0.042g were randomly assigned into four groups: control group, G1 group, G2 group and G3 group. There are 90 fish in each group, each with three replicates and independently raised in a 120L glass fish tank (30 fish/tank), and each tank has an independent recirculation system. As shown in Table 1, in each experiment, the control group was fed basic diet feed, and the G1, G2, and G3 groups were fed different contents of NPUST1-containing feed, with the contents being 10 ⁵ CFU/g and 10 ⁶ CFU respectively. /g and 10 ⁷ CFU/g. Each group was fed twice a day, and the amount of feed was approximately 5% of the fish's body weight. During the trial, the animals were weighed every two weeks and the feeding amount was adjusted according to changes in body weight. During the test period, the fish tank was cleaned every day and uneaten feed was collected 1 hour after feeding to confirm the feed intake. After 8 weeks of feeding, the fish in each group were analyzed for growth performance values, digestive enzyme activities, innate immunity-related values, nutritional or immune-related gene expressions, and streptococcal challenge experiments and intestinal microbial phase analysis were conducted. .

Table 1 raw material Formula (g/Kg) control group G1 group G2 group G3 group Sarfordella strain NPUST1 0 10 ⁸ CFU 10 ⁹ CFU 10 ¹⁰ CFU fishmeal 50 50 50 50 soy flour 300 300 300 300 couscous 161 161 161 161 rice bran 300 300 300 300 α‐starch 60 60 60 60 cellulose 70 70 70 70 Skim milk 10 10 10 10 Soybean oil 30 30 30 30 mineral mixture 16 16 16 16 vitamin mixture 3 3 3 3 main ingredient crude protein 236.1 234.0 236.1 230.1 crude lipid 103.5 104.8 102.1 101.6 Moisture 96.8 96.2 93.8 96.5 Ash 65.9 64.9 66.1 64.2

[Preparation of Streptococcus bacterial liquid for test inoculation]

Regarding the Streptococcus iniae samples used as test pathogens in the examples described below, the preparation method is as follows: take out the stored Streptococcus strains from the -80°C freezer, and first absorb 0.5 mL of bacterial liquid. into 50 mL of TSB culture medium, and incubate at 28°C for 12 hours. Then add 1 mL of bacterial liquid to 100 mL of TSB culture medium, and activate at 28°C for 12 hours. The absorbance value OD600 nm of the culture solution was measured through routine experiments, and the serially diluted bacterial solution was cultured and the number of bacteria was calculated. Through the calculation results, a standard curve was established with the horizontal axis being the absorbance value OD600 nm and the vertical axis being the bacterial concentration (CFU/mL). In the examples described below, the bacterial solution for testing is prepared based on this standard curve.

[Preparation of nutritional supplement (Sulfobacterium strain NPUST1)] In each of the following examples, when Sulfobacterium strain NPUST1 is added to feed as a nutritional supplement, it is prepared in the following manner: Add Sulfobacterium strain NPUST1 to 100 mL TSB medium, incubate at 28°C and 175rpm for 16 hours. The culture solution was centrifuged at 4°C (6000×g, 15 minutes) to collect bacterial cells. The collected bacterial particles were washed three times with phosphate buffer saline (PBS) and then resuspended in sterile water to a concentration of 1×10 ⁹ CFU/mL. The suspension was added to the basal feed to prepare test feeds with different contents of Salfordella strain NPUST1. The raw materials and main component compositions of the aforementioned test feeds are shown in Table 1. The mineral mixture and vitamin mixture in Table 1 were purchased from Changxing Food Chemical Raw Materials Co., Ltd. (Taiwan). After the raw materials are thoroughly mixed, they are ground with a hammer mill and sieved with an 80 mesh screen (150 μm). The mixture was stirred with a mixer to form a hard mass, and the mass was formed into particles with a particle size of about 1 cm using a grinder (die diameter: 3 mm). After drying at 37°C until the moisture content is about 10%, store at 4°C for use in the tests described later. At the same time, during the feeding trial, counts were performed on the TSB medium plate every two weeks to monitor the survival rate of the strains in the feed.

[Analysis of Expression Levels of Genes Related to Nutritional Metabolism, Growth and Immune Function] The analysis of expression levels of genes related to nutritional metabolism, growth and immune function in each of the examples described below is performed by the following steps: In response to the analysis needs of each example, Collect blood, liver, spleen or head kidney tissue samples from the fish. RNA was extracted using a TriPure extraction kit (Roche, Germany). The RNA concentration was detected with an ultramicrovolume spectrophotometer. After detection, 1 μg of RNA was converted into cDNA using iScript ^TM cDNA Synthesis Kit (Bio-Rad, USA) and stored at -20°C. Then, quantitative PCR (qPCR) was performed using the corresponding primers of each indicator gene listed in Table 2 to detect the expression of nutritional metabolism, growth and immune-related genes. The aforementioned quantitative PCR uses the KAPA SYBR FAST qPCR Master Mix kit (KR0389, KAPA, USA). The PCR program conditions are set as follows: 60°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles. The cycle conditions are 95°C for 15 minutes. seconds, 60°C for 1 minute. The expression level of the elongation factor (EF-1α) gene was used as the internal control, and each gene was normalized to the relative expression level and expressed as the mean ± standard error (SE).

Table 2 Gene Primer sequence (5'→ 3') reference sequence Amplicon size (bp) SEQ ID NO: Glucokinase (GK) GCAGCGAGGAAGCCATGAAGA XM_003451020 101bp 2 GAGGTCCCTGACGACTTTGTGG 3 Glucose 6- phosphatase (G6Pase) AGCGCGAGCCTGAAGAAGTACT XM_003448671 107bp 4 ATGGTCCAGCAGGTCCACAT 5 growth hormone receptor (GHR) GAATACAAGTCCTTCCGGGCTAA AY973232 100bp 6 CTCATACTCCACACGCATCCA 7 Insulin-like growth factor type 1 (IGF-1) TGTCTGCCAGTAAGGATGTTCTTG EU272149 100bp 8 GGCTTTCCACGCCACTTAAC 9 Tumor necrosis factor-alpha (TNF-alpha) CCAGAAGCACTAAAGGCGAAGA AY428948 82bp 10 CCTTGGCTTTGCTGCTGATC 11 Interleukin-1β (IL-1β) TGTCGCTCTGGGCATCAA KJ574402 63bp 12 GGCTTGTCGTCATCCTTGTGA 13 Interleukin-8 (IL-8) CCTCGAGAAGGTGGATGTGAA GQ355864 100bp 14 CATGAGACCCAGGGCATCA 15 Lysozyme (LYZ) GCCTGACCGAACATGAGTCA LC012581 100bp 16 CACCAGCGGCTATTTATCTGAA 17 Elongation factor-1α (EF-1α) CCACACAGTGCCCATCTACGA EU887951 111bp 18 CCACGCTCTGTCAGGATCTTCA 19

The test data obtained in each example described below were statistically analyzed by one-way ANOVA. According to the analysis results, Tukey's multiple comparison test was further used to detect the significance of differences between groups (p<0.05). The cumulative survival rate in the streptococcal challenge test was analyzed by the Kaplan–Meier method. SAS software (SAS Computer Software, USA) was used for data analysis.

Example 1: Evaluation of enzyme activity of Sarfordella strain NPUST1

In this example, TSB was used as the basic culture medium, and 0.5 % skim milk (for protease test), 0.2% starch (for amylase test), 0.5% carboxymethyl cellulose (for cellulase test), 1% xylan (for xylanase test), made of Plate media for testing. After inoculating the Salfordella strain NPUST1 into each culture medium, culture it for 24 hours, and then follow the routine test steps to stain the amylase test medium with 5% iodine solution, and stain the cellulase test medium and xylanase test The culture medium was stained with 0.4% Congo red solution. In each culture medium that has been cultured for 24 hours, the transparent area around the inoculation hole will reflect whether the nutrients have been metabolized, and therefore can display the activity of each enzyme secreted by the strain.

Figure 1 shows the analysis results of the enzyme activity characteristics of the strain after 48 hours of culture. The culture media in the figure respectively represent: (a) protease test; (b) amylase test; (c) cellulase test; (d) xylanase test. In each culture medium, NC in the upper middle represents the negative control group ( Escherichia coli ), PC in the lower left represents the positive control group ( Bacillus amyloliquefaciens R8); and the lower right represents the Salfordella strain NPUST1 of the present invention.

The analysis results show that compared with the positive control group, the Salfordella strain NPUST1 of the present invention has similar protease activity and relatively high amylase, cellulase and xylanase activities.

Example 2: Effect of Sarfordella strain NPUST1 on improving growth performance

Based on the body weight (BW) and other values measured during the aforementioned feeding test, the weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) of the control group and G1~G3 groups were calculated. The calculation formula is as follows: Weight gain (WG) = final weight – initial weight Specific growth rate (SGR) = (ln final body weight - ln body weight) × 100/test duration (days) Feed conversion ratio (FE) = (final body weight – initial body weight)/feed intake Survival rate (SR)=100×(final number of test fish)/(initial number of test fish)

The growth performance values of each group after 8 weeks of feeding are shown in Table 3. The values in Table 3 are the mean ± standard error (SE) of three replicates. The superscript letters indicate whether there are significant differences between each group. The results showed that there was no significant difference in the survival rate between the groups, indicating that administration of the Sarfordella strain NPUST1 was harmless to tilapia. When fed the G2 and G3 groups containing 10 ⁶ CFU/g and 10 ⁷ CFU/g NPUST1 strains, the weight gain (WG) of tilapia was 0.87 ± 0.114 g and 1.02 ± 0.111 g respectively, which was significantly higher than that of the G1 group (0.71 ± 0.025 g) and 0.75± 0.020 g in the unfed control group. Similarly, the specific growth rate (SGR) and feed conversion rate (FE) of the G2 and G3 groups were also significantly higher than those of the control group. There were no significant differences in WG, FE and SGR between the G2 group and the G3 group. This result confirms that the strain of the present invention has the effect of promoting growth performance.

table 3 Growth performance value Dosage control group 10 ⁵ CFU/g(G1) 10 ⁶ CFU/g(G2) 10 ⁷ CFU/g(G3) Initial weight (g) 0.56± ^0.021a 0.54± ^0.016a 0.54± ^0.020a 0.56± ^0.017a Final weight (g) 1.31± ^0.004a 1.25± ^0.025a 1.41 ± ^0.095b 1.58 ± ^0.125b Weight gain (g) 0.75± ^0.020a 0.71± ^0.013a 0.87 ± ^0.114b 1.02 ± ^0.111b feed efficiency 0.33± ^0.010a 0.33± ^0.015a 0.39 ± ^0.044b 0.41 ± ^0.034b specific growth rate 2.41 ± 0.153 ^a 2.34± ^0.061a 2.81 ± ^0.471b 3.26 ± ^0.290b Survival rate (%) 97.8 ± 1.92 ^a 95.6 ± 3.85 ^a 96.7 ± 0.00 ^a 97.8 ± 1.92 ^a

Example 3: Effect of Salfordella strain NPUST1 on improving intestinal digestive enzyme activity

After 8 weeks of feeding, 6 tilapias from each group were randomly selected and sacrificed for dissection. 200g of intestinal tissue samples were collected, homogenized in 1 mL of PBS buffer, and centrifuged at 6000×g and 4°C for 10 minutes. Transfer the supernatant to a 1.7 mL centrifuge tube, place it on ice, and perform quantitative detection of supernatant protein and digestive enzyme activity. The detection methods of each item are as follows.

The total protein amount of the supernatant was determined by the Bradford method. First, 100 µg/mL BSA was prepared, and then serially diluted to 80, 60, 40, 20 and 0 µg/mL. Add 50 µL of the supernatant protein sample and BSA standard of each concentration into a 96-well plate, then add 200 µL of Bio-Rad protein concentration detection reagent that has been diluted 5 times, and react in a dark place for 10 minutes. Use a spectrophotometer to measure the absorbance value at OD 595 nm and prepare a standard curve. Substitute the absorbance value of the sample into the standard curve to calculate the protein content.

Determination of protease activity: First, prepare 100, 80, 60, 40, 20, 0 µg/mL tyrosine (L-tyrosine) solutions as standards, take 100 µL of tyrosine solutions of each concentration, and add 500 mM of 500 µL of sodium carbonate (Na ₂ CO ₃ ) solution and 100 µL of Folinol reagent (manufactured by Sigma-Aldrich, No. F-9252) diluted 5 times. After reacting for 10 minutes at 37°C, measure the absorbance value of OD660 nm with a spectrophotometer and prepare a standard curve. Prepare 0.65% casein solution with 50 mM Potassium phosphate solution, heat to dissolve on a hot plate at 80~90°C and cool to room temperature for later use. Then take 100 µL of intestinal supernatant. Mix evenly with 500 µL of 0.65% casein solution, react at 37°C for 10 minutes, add 500 µL of 110 mM trichloroacetic acid (TCA), and continue to react at 37°C for 30 minutes. The reaction is completed. Then filter with a 0.45 µm filter membrane. Take 200 µL of the filtrate, add 500 µL of 500mM sodium carbonate solution and 100 µL of 5-fold diluted folinol reagent, and react at 37°C for 10 minutes. After the reaction is completed, measure the absorbance value of OD660 nm with a spectrophotometer, and Substitute the values into the standard curve to calculate the tyrosine content of the sample. Protease activity unit (U) is defined as: an activity unit that produces 1 µmole of tyrosine per minute at 37°C.

Determination of amylase, cellulase and xylanase activities: First, prepare glucose solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL as standards, and take 100 µL of glucose solutions of each concentration and 100 µL Mix the DNS reagent evenly in a glass test tube and heat it in boiling water for 15 minutes. Then take 200 µL of the mixture into a 96-well plate. Use a spectrophotometer to measure the absorbance value of OD540 nm and prepare a standard curve. For the measurement of amylase activity, 1% soluble starch solution is used as the measurement matrix. 100 µL of intestinal supernatant and 100 µL of starch solution are evenly mixed, react at room temperature for 10 minutes, and then add 200 µL DNS reagent, and react in boiling water for 15 minutes. After cooling, measure the absorbance value of OD540 nm with a spectrophotometer, and substitute the measured value of the sample into the standard curve to calculate the glucose content. Amylase activity unit (U) is defined as an activity unit that can release 1 µmole of reducing sugar per minute. For the determination of cellulase activity, 1% carboxymethylcellulose (CMC) solution is used as the measurement matrix. 200 µL of intestinal supernatant, 1% carboxymethylcellulose solution and PBS buffer solution are taken and mixed evenly. React at 50°C for 15 minutes, then add 600 µL DNS reagent and react in boiling water for 15 minutes. After cooling, measure the absorbance value of OD540 nm with a spectrophotometer, and substitute the measured value of the sample into the standard curve to calculate the glucose content. Cellulase activity unit (U) is defined as an activity unit that can release 1 µmole of reducing sugar per minute. For the determination of xylanase activity, 1% xylose (Xylan) solution is used as the assay matrix. 100 µL of intestinal supernatant and 100 µL of 1% xylan solution are uniformly mixed and reacted at 55°C. 15 minutes, then add 200 µL DNS reagent and react in boiling water for 15 minutes. After cooling, measure the absorbance value of OD540 nm with a spectrophotometer, and substitute the measured value of the sample into the standard curve to calculate the glucose content. Xylanase activity unit (U) is defined as: one activity unit can release 1 µmole of reducing sugar per minute.

Lipase activity determination: Use sodium phosphate buffer (pH 7.0) containing 0.5% Triton-X 100 to prepare 0, 115, 230, 345, 460, 575 µM p-Nitrophenol solutions as standards, and take 200 µL of different concentrations. The standard solution was added to a 96-well plate, and the absorbance value at OD 405 nm was measured using a spectrophotometer and a standard curve was prepared. The xylanase activity was based on a 0.5 mM p-nitrophenyl butyrate solution in sodium phosphate buffer (pH 7.0). Add 10 µL sample and 190 µL reaction matrix to a 96-well plate and mix evenly. After reacting at 37°C for 30 minutes, measure the absorbance value of OD 405 nm. Substitute the measured value of the sample into the standard curve to calculate the p-Nitrophenol content. Lipase activity unit (U) is defined as: at 37°C, one unit of p-Nitrophenol can be hydrolyzed to produce 1 µmole per minute.

The intestinal digestive enzyme activity values (U/mg) of each group after 8 weeks of feeding are shown in Table 4. The values in Table 4 are the mean ± standard deviation (S.D.) of each group. The superscript letters indicate whether there are differences between each group. There are significant differences. The results showed that compared with the control group, the intestinal protease, amylase, and lipase activities of the G2 group and G3 group were significantly increased. And compared with the control group, the cellulase activity of the G3 group increased significantly. This example proves that using the strain of the present invention as a nutritional supplement for feed can improve the utilization rate of nutrients in the feed by fish, thereby improving the growth performance of fish.

Table 4 Activity(U/mg) control group G1 G2 G3 Protease 0.50± ^0.007a 0.51± ^0.009a 0.64 ± ^0.013b 0.68 ± ^0.005b amylase 0.15± ^0.003a 0.15 ± ^{0.08a b} 0.18 ± ^0.004b 0.18 ± ^0.009b cellulase 0.14± ^0.010a 0.15 ± ^{0.005a b} 0.16 ± ^{0.004a b} 0.18 ± ^0.025b Xylanase 0.10± ^0.001a 0.11± ^0.013a 0.11± ^0.007a 0.10± ^0.001a Lipase 0.03± ^0.003a 0.05 ± ^0.004b 0.05± ^0.003b 0.06± ^0.003c

Example 4: The effect of Sarfordella strain NPUST1 on improving the expression of glucose metabolism and growth-related index genes

After 8 weeks of feeding, 6 tilapias from each group were randomly selected and sacrificed for dissection, and the expression of glucose metabolism and growth-related genes in liver tissue was analyzed. The aforementioned glucose metabolism-related genes include glucokinase (GK) and glucose-6-phosphatase (G6Pase). The aforementioned growth-related genes include growth hormone receptor (GHR) and insulin-like growth factor-1 (IFG-1). The analysis results are shown in Figure 3. In Figure 3, the relative expression of genes in each group is expressed as the mean ± standard deviation (S.D.). The letters marked on the upper side of the long bar graph indicate whether there is a significant difference between each group.

The analysis results showed that compared with the control group, the expression amounts of GK, G6 Pase, GHR-1 and IGF-1 in the G2 group and G3 group were significantly increased. Moreover, the expression amounts of GK and G6 Pase in the G3 group were also significantly higher than those in the G2 group.

The liver plays an important role in glucose balance and metabolism. GK in the liver is the main hexonase, which converts glucose into glucose-6-phosphate, which is the primary step in glucose metabolism. The G6P enzyme in the liver releases glucose into the blood. On the other hand, the growth hormone (GH)-IGF-I axis is an important endocrine mechanism regulating the growth of vertebrates, and GHR in the liver receives GH signals from the subbrain glands, thereby triggering the liver to release IGF-1 into the circulation system. , thereby stimulating body growth. Therefore, this example proves that the strain of the present invention can promote the expression of these glucose metabolism and growth-related genes, thereby achieving the effect of promoting the nutritional metabolism and growth performance of reared fish.

Example 5: The effect of Salfordella strain NPUST1 on improving innate immune response. After 8 weeks of feeding, 6 tilapias from each group were randomly selected to collect their macrophages and blood samples to evaluate the performance of the following main indicators of innate immunity: Phagocytic activity (PA), respiratory burst activity (RB), superoxide dismutase activity (SOD), and lysozyme activity in the blood.

First, macrophages were separated by centrifugation with different concentrations of Percoll. The concentration selected in this experiment is 32%/51% (V/V). At the beginning, 32% and 51% Percoll were prepared separately, and then 2 mL of 32% Percoll was added to a 15 mL centrifuge tube, and then the injection The needle extends 2 mL of 51% Percoll into the bottom of the 15 mL centrifuge tube and slowly injects it. This allows 32% Percoll to be completely pressed on top of the 51% Percoll, causing a layering phenomenon. Then slowly drop 200 - 500 μL of head kidney sample from above, and finally use a 1 mL micropipette to draw 1 mL of 1 x PBS with pH 7.6 and slowly drip it in, pressing it on top of the head kidney sample to maintain different concentrations of Percoll. , head kidney samples and 1 x PBS were separated into distinct layers, and then the centrifuge tube was placed in a centrifuge and centrifuged at 400 x g for 30 minutes at 4°C. After centrifugation, if turbid material is found sandwiched between the two concentrations of Percoll, it means that the macrophages have been successfully separated. Use a micropipette to suck out the middle layer of macrophages and place it in a 1.7 mL microcentrifuge tube. Place on ice to maintain low temperature. Finally, take 10 μL from each sample and count on a hemocytometer, and dilute macrophages from different samples to the same power for subsequent experiments. The test and analysis methods for each of the aforementioned items are as follows.

Phagocytic activity (PA): This test needs to be conducted in a sterile operating table, and the entire process needs to be protected from light when operating fluorescent emulsion beads. Before starting the test, prepare fluorescent emulsion beads and iodide (PI). Take 15 mL of L-15 culture medium in a 15 mL centrifuge tube, then add 0.4 μL of fluorescent emulsion beads, and the iodide system is sterilized. It is prepared in sterile water with a concentration of 1 mg/mL. After the preparatory work is completed, add 300 μL of macrophage sample to the 12-well cell culture plate, let it sit for 1 hour to allow the phagocytes to adsorb in the holes, then discard the supernatant and add 300 μL of L-15 culture medium. Repeat washing three times. After washing, add 300 μL of prepared fluorescent emulsion beads, then wrap the 12-well cell culture plate with aluminum foil and let it stand for 2 hours. After the reaction is completed, add 300 μL of PBS and wash once, and remove most of the cells. Remove the uningested fluorescent emulsion beads, and then add 300 μL of 1% formaldehyde to fix the macrophages. After the reaction for 30 minutes, wash 2 to 3 times with 300 μL of PBS to wash away the remaining 1% formaldehyde, and then add 300 μL of Iodide staining was performed for 10 minutes. After staining, the cells were washed twice with 300 μL of PBS and left to air dry. Finally, the phagocytosis of phagocytes was observed under an Olympus IX50 fluorescence microscope. Calculate the number of 200 macrophages with the ability to phagocytose fluorescent emulsion beads, and calculate the phagocytic activity using the following formula.

Phagocytic activity (%) = [100 x (number of macrophages phagocytosing fluorescent emulsion beads) x (total number of macrophages) ^-1 ]

Respiratory blast (RB) activity: divided into control group and experimental group. First, take 100 μL of 0.2% Poly-L-Lysine and place it in a 96-well plate for 30 minutes. Then add 100 μL of macrophage sample, place it in a centrifuge and centrifuge at 300 x g for 20 minutes. Remove the supernatant. , add 100 μL of MCHBSS to the control group, add 100 μL of Zymosan A to the test group, and let stand for 30 minutes. After the reaction, remove the supernatant, repeat washing three times with 100 μL of MCHBSS, and then add 100 μL of 0.3% NBT for staining. 30 minutes, and then add 100 μL of 100% methanol to terminate the reaction. Shake slightly and remove the supernatant. Repeat three times with 100 μL of 70% methanol. After cleaning, remove the supernatant and place it on the table to air dry for about 30 minutes. Wait for 20 to 30 minutes, wait for air drying, then add 120 μL of 2 M KOH and 140 μL of Dimethyl Sulfoxide (DMSO) to dissolve the cytoplasmic formazan, let it stand for 2 minutes, and then measure the absorbance value OD630 nm with a spectrophotometer. In this experiment, those treated with the addition of the non-immunostimulant MCHBSS were regarded as the basal activity (BA), while those treated with the addition of the immune stimulant Zymosan A were regarded as the stimulated activity (SA). The difference between the two is the respiratory burst activity (RBA). ), used to express the increase or decrease in superoxide anion production produced by macrophages.

Superoxide dismutase (SOD) activity: First, take 200 μL of macrophage sample and place it in a 1.7 mL microcentrifuge tube. Add 500 μL of HBSS and mix evenly. Place it in a centrifuge and centrifuge at 400 x g at 4°C. After 20 minutes, remove the supernatant and repeat adding 500 μL of HBSS to clean the blood cells and centrifuge. This step needs to be repeated three times to clean the macrophages. After washing, add 150 μL of 1x PBS with pH 7.8 to suspend the macrophages. Break the blood cells with an ultrasonic cell crusher, then place them in a centrifuge and centrifuge them at 1500 x g for 10 minutes at 4°C. Remove the supernatant, pipet 100 μL into a 1.7 mL microcentrifuge tube, and then add 250 μL 0.15 M Phosphate Buffer (pH 7.8), 75 μL 0.13 M Methionine, 75 μL 1 mM Na2EDTA, 75 μL 0.63 mM NBT, and and 150 μL of 7.5 μM Riboflavin. Then place it in a 25°C incubator and react with light above 4000 Lux for 10 minutes. After the reaction, pipet 200 μL into a 96-well plate and measure the absorbance value OD560 nm with a spectrophotometer. Under these conditions, the unit of superoxide dismutase activity is defined as: the amount of enzyme required to inhibit the photochemical reduction of nitrotetrazolium blue chloride (NBT) by 50% is 1 SOD activity unit (U ). After quantifying the protein concentration of the sample using the Bradford method, calculate the SOD activity contained in each milligram of sample protein according to the formula below.

SOD activity formula: (blank control group – sample) / (blank control group / 2) x 6 / mg protein

Lysozyme activity in blood: Before starting the test, prepare 0.05 M Sodium Phosphate Buffer with pH 6.2. Use 0.05 M Sodium Phosphate Buffer with pH 6.2 to prepare 0.16 mg/mL lysozyme (Lysozyme, from chicken egg white, Sigma) and 0.2 mg/mL Micrococcus lysodeikticus (ATCC No. 4698, Sigma). Lysozyme was used as a standard substance for experimental testing. It was diluted to 0.08, 0.04, 0.02, 0.01, and 0 mg/mL. Take 10 μL of each standard substance and place it in a 96-well plate. Then add 200 μL of M. lysodeikticus and mix. The method of measuring immediately after adding the bacterial solution to the well is to use a spectrophotometer to measure the reaction value of the absorbance value OD530 nm at 0 minutes and 5 minutes. The activity of lysozyme is judged based on the difference in the absorbance value OD530 nm between 0 and 5 minutes. The difference value was made into a standard curve, and the linear regression method was used to calculate the lysozyme content in the serum sample. After the standard curve is completed, the blood samples can be measured. Take out the blood samples that have been placed in the refrigerator at 4°C for 24 hours, place them in a centrifuge and centrifuge at 3000 x g for 5 minutes at 4°C, and then absorb the serum from the upper layer. Place it in a new 1.7 mL microcentrifuge tube and place it on ice to proceed with the lysozyme activity analysis test, or store it in a -80°C refrigerator for later use. Take 10 μL of each serum sample after removal and place it in a 96-well plate. Then add 200 μL of M. lysodeikticus and mix. Use a spectrophotometer to measure the absorbance value OD530 nm at 0 minutes and 5 minutes by measuring immediately after adding the bacterial solution to each well. The reaction values are based on 0 to 5 minutes. The difference in the internal absorbance value OD530 nm is used to determine the activity of lysozyme in the serum. Finally, the absorbance value OD530 nm of each sample is substituted into the standard curve for calculation, and the lysozyme content in the serum can be obtained. Under these conditions, the unit of lysozyme activity is defined as: one unit of enzyme activity (U) can reduce the absorbance value OD530 nm by 0.001 per minute at room temperature. Therefore, based on the principle that lysozyme destroys the bacterial cell wall and thereby reduces the turbidity of the bacterial suspension, it can be measured by spectrophotometry. Similarly, after quantifying the protein concentration of the serum sample using the Bradford method, the lysozyme activity contained in each milligram of serum sample protein can be calculated.

The analysis results are shown in Figure 4. In Figure 4, the values of each of the aforementioned analysis items are expressed as mean ± standard deviation (S.D.). The letters marked on the upper side of the long bar graph indicate whether there are significant differences between each group. The analysis results showed that compared with the control group, the phagocytic activity (PA) and lysozyme activity in the blood of the G2 and G3 groups were significantly increased. And there was no significant difference between the G2 group and the G3 group. Compared with the control group, respiratory burst (RB) activity and superoxide dismutase (SOD) activity were significantly increased in the G1, G2 and G3 groups that were administered the Salfobacillus strain NPUST1. In addition, the superoxide dismutase (SOD) activity of the G2 group was significantly higher than that of the G1 group and G3 group.

The head kidney and spleen are the main lymphoid organs of fish and play an important role in regulating innate immunity. White blood cells in the head kidney, such as neutrophils and macrophages, engulf pathogens through phagocytosis. During the phagocytosis process, substances such as reactive oxygen species (ROS) are released to destroy the engulfed pathogens, and the release of these substances is reflected in the respiratory burst (RB). Superoxide dismutase (SOD) reacts to oxidative stress by catalyzing the decomposition of ^O2- into hydrogen peroxide and therefore serves as an indicator of the ability to protect against ROS damage. Lysozyme, on the other hand, destroys bacterial cell walls and inhibits the growth of pathogenic bacteria. This example proves that feeding the strain of the present invention can improve the above-mentioned main indicators of innate immunity of fish. That is, the strain of the present invention can improve the innate immunity of fish, and the dosage of 10 ⁶ CFU/g is The effect can be achieved.

Example 6: Effect of Sarfordella strain NPUST1 on improving immune-related index gene expression

After 8 weeks of feeding, 6 tilapias from each group were randomly selected and sacrificed for dissection, and the expression of immune-related genes in the head kidney and spleen tissues was analyzed. The aforementioned immune-related genes include interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and Lysozyme (LYZ). The analysis results are shown in Figure 5. In Figure 5, (a)~(d) are the relative expression amounts of genes in the head kidney, and (e)~(h) are the expression amounts in the spleen. Each group is expressed as the mean ± standard deviation (S.D.). The letters marked on the upper side of the long bar graph indicate whether there is a significant difference between each group.

The analysis results showed that compared with the control group, the expression amounts of IL-1β and IL-8 in the head kidneys of the G1, G2 and G3 groups were significantly increased, and the expression amounts of the G2 group were significantly higher than those of the G1 group. At the same time, compared with the control group and G1 group, the expression amounts of TNF-α and LYZ in the head kidney of the G2 group and G3 group were significantly increased. The expression amounts of these four immune-related genes in the spleens of the G2 and G3 groups were also significantly higher than those of the control group. And compared with the control group, the expression levels of IL-8 and LYZ in the spleen of the G1 group were also significantly higher. This example proves that the strain of the present invention can promote the expression of these immune-related genes, thereby achieving an immune-regulating effect on fish. In addition, there was no significant difference in performance between the G2 group and the G3 group.

IL-1β and TNF-α are pro-inflammatory cytokines that are induced in the early stages of pathogen infection to increase the survival rate of macrophages and increase leukocyte bactericidal activity by increasing ROS production during the phagocytosis process. IL-8 is a fish chemokine that attracts and activates neutrophils during inflammation. Therefore, this example can prove that the strain of the present invention has the effect of improving the expression of immune-related indicator genes. And the dosage of 10 ⁶ CFU/g is enough to achieve the effect of improving the expression of immune-related genes.

Example 7: Effect of Sarfordella strain NPUST1 on improving disease resistance

The aforementioned Streptococcus bacteria liquid was cultured in TSB medium for 24 hours, diluted with sterile water, and based on the weight of the tilapia fish in each group after two months of feeding, the number of bacteria corresponding to the body weight was injected intraperitoneally, so that each group The injection concentration for tilapia is 1 x 10 ⁵ CFU/g body weight. The treatment conditions of the negative control group were as follows: feeding the basal feed without Salfordella strain NPUST1, and injecting physiological saline instead. Each group was independently raised in three replicates in a 60L fish tank containing 40L fresh water (10 fish/tank), and the breeding temperature was 28±1°C. Observations were conducted every day for 7 days after injection, and the cumulative survival rate was recorded. Comparisons were made between the control group and the G1~G3 groups fed NPUST1-containing feed.

The survival rate results of each group are shown in Figure 6. Within 7 days after injection, the survival rate of the negative control group (injected with PBS) was maintained at 100%. In contrast, the cumulative survival rate of the control group (injected with streptococcal bacterial solution) decreased significantly after 2 days and remained at 43 ± 5.7% thereafter. The cumulative survival rates of the G2 group and G3 group were 60% and 73 ± 5.7% respectively, which were significantly increased compared with the control group, but there was no significant difference between the G2 group and the G3 group. This example confirms that feeding feed containing Salfordella strain NPUST1 can improve the immunity of tilapia against streptococcal infection, and a dose of 10 ⁶ CFU/g is sufficient to achieve this effect.

Example 8: Effect of Sarfordella strain NPUST1 on improving intestinal microbiome

After feeding the diet for 8 weeks, 4 tilapia were taken from each group, and the intestinal microbial genomic DNA was extracted using the EasyPure Genomic DNA Spin kit, and PCR was performed on the full-length fragment of the 16S gene (V1-V9 region). Use the KAPA HiFi HotStart ReadyMix kit for PCR reagents. The primers used in PCR are: forward primer (SEQ ID NO: 20), 5-terminal phosphate modification and barcode degradation, 5′-Phos/GCATCAGRGTTYGATYMTGGCTCA-3′; reverse primer (SEQ ID NO: 21), 5′ -Phos/GCAT-CRGYTACCTTGTTACGACTT-3′. The PCR program conditions are set as follows: 95°C for 3 minutes, followed by 20 to 27 cycles (depending on the sample). The cycle conditions are 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 60 seconds, and finally 72°C for 5 minutes. After DNA gel electrophoresis analysis confirmed that the fragment size was approximately 1500 bp, the PCR product was purified with AMPure PB beads. Carry out high-throughput NGS analysis: use the full-length 16S SMRTbell Library Preparation and Sequencing kit (PacBio) to build the library, and use a PacBio Sequel IIe system for sequencing. The mode is circular consensus sequence (CCS) mode to obtain the minimum HiFi reading with prediction accuracy of 0.9. After decoding, the CCS was further processed using the DADA2 algorithm to obtain amplicons with single nucleotide resolution. The DADA2 algorithm can resolve accurate amplicon sequence variants (ASVs) of the full-length 16S rRNA gene at single-nucleotide resolution with near-zero error rate. Use the distance matrix to perform principal coordinate analysis (PCoA) to obtain principal coordinates for visualizing complex and multidimensional data. For each representative sequence, the feature-classifier and classify-consensus-blast algorithms in QIIME2 are used to classify and annotate based on the information retrieved from the NCBI database.

A total of 259,936 nucleotide sequences were retrieved from a total of 16 samples in four groups. The QIIME pipeline was used to assign the verified sequences to 465 ASVs with a similarity of 97%, and a dilution curve was drawn. Simultaneously conduct principal component analysis and relative abundance analysis of the correlation between intestinal microorganisms in each group. The results are shown in Figure 8. In Figure 8, (a) is the dilution curve of each sample (rarefaction curve); (b) is the dilution curve of the intestinal microbial phase of each group; (c) is the intestinal microbial phase of each group Principal component analysis results of microbial composition; (d) is the relative abundance analysis results of different bacterial species in the intestines of each group. Group C in the figure is the control group.

The results show that the dilution curve is biased towards the plateau, which means that each group has reached sufficient sampling depth (coverage rate reaches 99.9%). According to the results of the principal component analysis, the intestinal microorganisms of the G1, G2 and G3 groups fed with the feed containing the Salfordella strain NPUST1 were closely clustered with each other, but all were separated from the control group. This means that the intestinal microorganisms of the G1, G2 and G3 groups fed with the Salfordella strain NPUST1 of the present invention were all separated from each other. It will cause changes in the microbial phase in the gastrointestinal tract of tilapia. The bacterial species with the highest relative abundance is Cetobacterium somerae , accounting for 60.72%, 60.28%, 61.05% and 68.19% of reads in the control group, G1, G2 and G3 groups respectively. Among the four groups, three dominant bacterial species are the same: Bacteroides luti , Plesiomonas shigelloides and Deefgea chitinilytica . The relative abundance of Bacteroides luti in the control group, G1, G2 and G3 groups was 22.23%, 18.58%, 20.50% and 21.69% respectively; the relative abundance of Plesiomonas shigelloide in the control group, G1, G2 and G3 groups was 3.20% respectively. , 6.03%, 5.27% and 4.45%; the relative abundance of Deefgea chitinilytica in the control group, G1, G2 and G3 groups were 3.31%, 3.10%, 3.19% and 2.34% respectively. There were no significant differences in the relative proportions of the four dominant bacterial species. In-depth analysis of bacterial species revealed significant changes in relative abundance, and the proportions of putative potential probiotics (including Sulforaphane) and pathogenic bacteria are shown in Table 5.

As shown in Table 5, compared with the control group fed basal feed, the abundance of Bacillus safensis was higher in the group fed the feed containing the Bacillus strain NPUST1 of the present invention, indicating that the strain of the present invention can indeed Colonizes in the gut of tilapia. In the G1, G2 and G3 groups, the abundance of potential probiotics Bacillus licheniformis and Lactococcus lactis was significantly higher than that in the control group. Relatively, compared with the control group, the abundance of potential pathogenic bacteria Aeromonas veronii, Aeromonas jandaei, Enterovibrio nigricans and Enterovibrio coralii in the G1, G2 and G3 groups were significantly lower. The results of this example show that feeding feed containing the Salfordella strain NPUST1 of the present invention can improve the intestinal microbial phase.

The use of the Salfordella strain NPUST1 of the present invention as a feed supplement in the aquaculture industry can improve the nutritional metabolism of aquaculture fish, promote feed conversion efficiency, and improve growth performance. In addition, when used as a feed supplement, the Salfordella strain of the present invention can promote innate immunity and thereby improve disease resistance against pathogenic bacteria. At the same time, the Salfordella strain of the present invention provides the effect of improving the intestinal microbial phase of fish, increasing the proportion of probiotics and reducing the proportion of pathogenic bacteria.

Therefore, the Salfordella strain NPUST1 of the present invention can effectively reduce production costs, shorten the growth time of aquaculture fish, increase the weight gain rate, and enhance disease resistance, so that future aquatic fish farming can be nutritional, economical, and environmentally friendly. direction development.

【Storage of Biological Materials】

Domestic storage information: TW Republic of China, Taiwan Hsinchu Food Industry Development Institute Biological Resource Preservation and Research Center, 2022/06/10, BCRC 911134

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          <![CDATA[<400> 19]]>
          ccacgctctg tcaggatctt ca 22
          <![CDATA[<210> 20]]>
          <![CDATA[<211> 24]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Artificial Sequence]]>
          <![CDATA[<220>]]>
          <![CDATA[<223> Forward primer used to amplify the full-length fragment of the 16S gene (V1-V9 region), 5-terminal phosphate modified and striped]]>
                 code degradation
          <![CDATA[<400> 20]]>
          gcatcagrgt tygatymtgg ctca 24
          <![CDATA[<210> 21]]>
          <![CDATA[<211> 24]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Artificial Sequence]]>
          <![CDATA[<220>]]>
          <![CDATA[<223> Reverse primer used to amplify the full-length fragment of the 16S gene (V1-V9 region), 5-terminal phosphate modified and bar-shaped]]>
                 code degradation
          <![CDATA[<400> 21]]>
          gcatcrgyta ccttgttacg actt 24
          
      

Claims (7)

A Bacillus safensis strain (Bacillus safensis), characterized by the fact that the Bacillus safensis strain is deposited at the Biological Resource Preservation and Research Center of the Food Industry Development Institute in Hsinchu, Taiwan. The deposit date is June 10, 2011, and the deposit number is BCRC 911134. . A use of the Salfordella strain described in claim 1 as a feed additive, the feed additive being used for aquaculture fish. The use as described in claim 2, wherein the aforementioned feed additive is used to improve the expression of nutritional metabolism-related genes of aquaculture fish, and the aforementioned nutritional metabolism-related genes are glucokinase (GK) or glucose 6-phosphatase (glucose -6-phosphatase, G6Pase). The use as described in claim 2, wherein the aforementioned feed additive is used to increase the body weight, specific growth rate or expression of growth-related genes of aquaculture fish, and the aforementioned growth-related genes are growth hormone receptors (GHR). ) or type 1 insulin-like growth factor (insulin-like growth factor-1, IFG-1). The use as described in claim 2, wherein the aforementioned feed additive is used to increase the proportion of probiotics in the intestinal microbial phase of aquaculture fish, and the aforementioned probiotics are Bacillus licheniformis or Lactococcus lactis. The use as described in claim 2, wherein the aforementioned feed additive is used to reduce the proportion of pathogenic bacteria in the intestinal microbial phase of aquaculture fish, and the aforementioned pathogenic bacteria are Aeromonas veronii, Aeromonas jandaei, Enterovibrio nigricansc or Enterovibrio coralii. A feed for tilapia, characterized by containing the Bacillus safensis strain described in claim 1.