Bacillus subtilis with high adhesiveness, selenium-rich bacillus subtilis powder prepared from bacillus subtilis and application of selenium-rich bacillus subtilis powder

Abstract

The invention relates to high-adhesiveness bacillus subtilis, selenium-rich bacillus subtilis powder prepared from the bacillus subtilis and application, the bacillus subtilis is named as bacillus subtilis C10, the bacillus subtilis is preserved on November 18, 2024, and the preservation number is CGMCC No.32669. The selenium-rich bacillus subtilis powder has the advantages that the selenium-rich bacillus subtilis powder is prepared from the bacillus subtilis, and the selenium-rich bacillus subtilis powder is prepared from the bacillus subtilis; the selenium-rich bacillus subtilis SC10 is prepared by fermenting bacillus subtilis C10 in a culture medium containing sodium selenite, the adhesion capacity of the selenium-rich bacillus subtilis SC10 is further improved, the freshness of fish is improved by utilizing the high-adhesion selenium-rich bacillus subtilis SC10, the quality guarantee period is prolonged, and the selenium-rich bacillus subtilis SC10 can be used as a fresh-keeping agent to be applied to fresh keeping of fish bodies. The selenium-enriched bacillus subtilis powder is added into the fish basal feed, so that the effects of improving the fish quality and prolonging the shelf life can be achieved. The method disclosed by the invention is green and safe, and effectively prolongs the shelf life of aquatic products while improving the flavor of fish meat.

Description

Technical Field

[0001] The present invention relates to the field of food biotechnology, and particularly to a highly adhesive Bacillus subtilis, a selenium-enriched Bacillus subtilis powder prepared therefrom, and applications thereof. Background Art

[0002] The traditional fish feed formulation design mainly focuses on the supply of basic nutrients (such as proteins, fats, carbohydrates, etc.) to meet the growth needs of fish. However, such feeds usually lack probiotics and other microecological agents, resulting in an easy imbalance in the intestinal microecology of fish. This imbalance will significantly reduce the activity of digestive enzymes, weaken the absorption efficiency of fish bodies for nutrients, not only affect the aquaculture efficiency, but also exacerbate the pollution of water bodies by unused feed residues. In addition, it will make the fish body vulnerable to the attack of pathogenic bacteria during the growth process, resulting in poor growth performance, and the subsequent storage process will also lead to a shortened shelf life due to the invasion of spoilage bacteria. Therefore, how to upgrade traditional fish feeds while improving fish growth performance, fish meat quality and preservation ability has become an urgent problem to be solved in modern aquaculture.

[0003] In recent years, the development of functional microecological agents has provided a new direction for the upgrading of aquaculture feeds. The application of probiotics (such as Bacillus and Lactobacillus) and their metabolites (postbiotics) can effectively regulate the intestinal flora and enhance the disease resistance of the host. Among them, Bacillus subtilis has become a research hotspot due to its characteristics such as high temperature resistance, gastric acid resistance, and production of digestive enzymes. However, its adhesion and colonization ability in the fish intestine still need to be improved, which limits its long-term probiotic effect. Therefore, developing new microecological agents with stronger colonization ability by improving the adhesion characteristics of strains not only helps to upgrade traditional fish feeds, but also helps to improve fish growth performance, enhance fish meat quality and extend the shelf life, which is of great significance for promoting the sustainable development of aquaculture. Summary of the Invention

[0004] The purpose of the present invention is to provide a highly adhesive Bacillus subtilis, a selenium-enriched Bacillus subtilis powder prepared therefrom, and the application of the selenium-enriched Bacillus subtilis powder in the preparation of a preservative for fish preservation or in the preparation of fish feeds.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A Bacillus subtilis has high adhesiveness. This strain is named Bacillus subtilis and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the deposit number CGMCC No. 32669.

[0007] The Bacillus subtilis is a Gram-positive bacterium, the spores are oval to columnar, located in the center or slightly off the bacterial body, the bacterial body does not swell after spore formation, the surface of the colony is rough and opaque, dirty white or slightly yellow, and often forms wrinkles; it is an aerobic bacterium that can utilize protein, various sugars and starch, and is VP-positive.

[0008] The application of the Bacillus subtilis in the preparation of selenium-enriched Bacillus subtilis powder.

[0009] A selenium-enriched Bacillus subtilis powder is obtained by culturing the Bacillus subtilis in a selenium-enriched fermentation broth medium and then centrifuging and freeze-drying the culture medium, wherein the selenium-enriched fermentation broth medium is prepared by adding sodium selenite to a sterilized LB medium.

[0010] The selenium-enriched Bacillus subtilis fermentation medium and fermentation conditions are as follows: LB is used as the basic medium, and the culture medium is activated to OD 600nm =1.0 (the concentration of bacterial solution is 1×10 8 CFU / mL) of Bacillus subtilis was inoculated into LB medium containing a certain concentration of sodium selenite at an inoculation rate of 2%, and cultured at 30°C and 200 rpm in a shaking incubator for 36 h.

[0011] The preparation method of the selenium-enriched Bacillus subtilis powder specifically comprises the following steps:

[0012] (1) Seed solution preparation: Take the Bacillus subtilis C10 single colony from the beef extract agar plate and inoculate it into LB liquid culture medium, and culture it at 30°C, 200 rpm, for 4 hours to obtain seed solution (the seed solution is Bacillus subtilis bacterial solution, and its OD 600nm =1.0 (the concentration of bacterial solution is 1×10 8 CFU / mL); wherein the formula of LB liquid culture medium is 5g yeast extract, 10g sodium chloride, 10g peptone, 1000mL distilled water, pH7.0; packaged, the liquid volume is one-third of the volume; sterilized for standby use;

[0013] (2) Preparation of selenium-enriched Bacillus subtilis fermentation broth: Sodium selenite was added to LB liquid culture medium to ensure that the selenium content in the system was 20 μg / mL, and the seed solution prepared in step (1) was inoculated at an inoculation amount of 2%; the culture was carried out at 30° C. and 200 rpm for 36 h to obtain selenium-enriched Bacillus subtilis fermentation broth;

[0014] (3) The selenium-enriched Bacillus subtilis fermentation broth obtained by culturing in step (2) is centrifuged at 8000 rpm for 10 min, the supernatant is removed, and the bacteria are freeze-dried to obtain selenium-enriched Bacillus subtilis powder.

[0015] Use of the described Bacillus subtilis in the preparation of fish feed.

[0016] Use of the described selenium-enriched Bacillus subtilis powder in the preparation of fish feed. The selenium-enriched Bacillus subtilis powder is added to the fish basal feed at an addition amount of 0.5×10 8 cfu / g.

[0017] Use of the described selenium-enriched Bacillus subtilis powder in fish farming. The method for farming fish using the selenium-enriched Bacillus subtilis powder is as follows:

[0018] (1) The selenium-enriched Bacillus subtilis powder is added to the fish basal feed at an addition amount of 0.5×10 8 cfu / g to obtain fish feed;

[0019] (2) The fish feed is fed to the fish twice a day, and the feeding amount is 2-3% of the fish body weight.

[0020] A fish feed containing the described selenium-enriched Bacillus subtilis powder.

[0021] The described fish feed includes fish basal feed and selenium-enriched Bacillus subtilis powder, and the selenium-enriched Bacillus subtilis powder is added to the fish basal feed at an addition amount of 0.5×10 8 cfu / g.

[0022] The fish in the present invention is mainly tilapia, and the corresponding feed is tilapia feed; the fish basal feed is the basal feed for tilapia farming, and its main components include soybean cake, wheat bran, fish meal, vitamins, inorganic salts and fish oil. The fish farming is tilapia farming. Although the fish used in the experiments of the present invention is tilapia, the selenium-enriched Bacillus subtilis powder prepared by the present invention can be used in the preparation of any fish feed and can be used to prepare a preservative for the preservation of any fish body.

[0023] Use of the described selenium-enriched Bacillus subtilis powder in the preparation of a preservative for fish body preservation.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] (1) The present invention provides a method for preparing selenium-enriched Bacillus subtilis. The Bacillus subtilis bacterial liquid (i.e., the described seed liquid) is inoculated into the selenium-enriched fermentation broth medium at an inoculation amount of 2% to obtain selenium-enriched Bacillus subtilis SC10, further improving the adhesion ability of Bacillus subtilis C10 so that it can be stably colonized. When used for fish body preservation, it can inhibit the adhesion and reproduction of spoilage bacteria.

[0026] (2) By adding the selenium-enriched Bacillus subtilis powder to the fish basal feed, the present invention simultaneously achieves the effects of improving fish quality and extending the shelf life, and has significant economic and social benefits.

[0027] (3) The method of the present invention is applied to fish bodies, being green, environmentally friendly, and efficient. Description of the Drawings

[0028] Figure 1 It is the colony morphology of Bacillus subtilis C10 of the present invention

[0029] Figure 2 It is the homology comparison result of Bacillus subtilis C10 of the present invention.

[0030] Figure 3 It is the diagram of the biofilm formation state and formation amount of the strain. Among them, Figure a is the diagram of the biofilm formation state, and Figure b is the formation amount. Detailed Embodiments

[0031] The following will describe the content of the present invention in detail with reference to the drawings of the specification and embodiments:

[0032] Example 1: Isolation, purification, and identification of Bacillus subtilis C10

[0033] A Bacillus subtilis provided by the present invention has high adhesiveness. This strain is named Bacillus subtilis C10 and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the deposit number CGMCC No. 32669.

[0034] The isolation method of Bacillus subtilis C10 is as follows:

[0035] In a sterile operation environment, the tilapia is dissected, and its intestine and gills are peeled off and placed in a sterilized glass homogenizer respectively. 10 mL of 0.85% sterile physiological saline is added, and homogenized thoroughly to prepare a sample homogenate. Subsequently, the homogenate is transferred to a sterilized test tube and mixed evenly, and the test tube mouth is sealed with a sealing film to ensure a sterile state. The test tube is placed in an 80°C water bath and heated for 20 min, taken out and allowed to cool to room temperature, and then 10-fold serial dilutions are performed. 100 μL of the original solution, 10-1, 10-2, and 10-3 dilution solutions are taken respectively and evenly spread on the beef extract peptone culture dishes. The spread culture dishes are placed in a 37°C constant temperature incubator and cultured for 24 h. After the culture is completed, select the culture dish with a suitable dilution degree with the number of colonies between 30 - 300 CFU. Pick the colonies with morphological characteristics suspected of being the target colonies from this culture dish and perform streak purification operations. Through multiple streak purifications, five strains of Bacillus subtilis including C10 are finally obtained after purification.

[0036] The colony morphology of Bacillus subtilis C10 is as Figure 1As shown, the colony morphology of Bacillus subtilis is characterized by circular, milky white, smooth or slightly rough surface, and regular edge opaque colonies with a colony diameter of 2-5 mm.

[0037] The molecular identification results of Bacillus subtilis C10 are as follows:

[0038] (1) DNA sequence

[0039] GGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGT

[0040] AACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACC

[0041] GGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAA

[0042] GGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAG

[0043] TTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGA

[0044] GAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC

[0045] GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG

[0046] AGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGT

[0047] TGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGG

[0048] TACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA

[0049] ATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTC

[0050] GCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGG

[0051] GAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTG

[0052] GAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACC

[0053] AGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAA

[0054] AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTA

[0055] AACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAG

[0056] CTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAA

[0057] ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT

[0058] TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTG

[0059] ACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTG

[0060] GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

[0061] GCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCAC

[0062] TCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0063] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG

[0064] GACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACA

[0065] AATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC

[0066] TGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCC

[0067] CGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAA (as shown in SEQ ID NO.1)

[0068] (2) Homology comparison: The results of homology comparison are as follows: Figure 2 As shown. Through NCBI BLAST comparison, the strain C10 has a 100% similarity with the corresponding sequences of Bacillus subtilis Y816 and Bacillus subtilis H20, and has a high degree of homology, confirming that the strain C10 is Bacillus subtilis.

[0069] In addition to Bacillus subtilis C10 (hereinafter referred to as C10), the inventors of the present invention also isolated multiple strains of bacteria according to the above method at the same time, namely Bacillus subtilis C6 (hereinafter referred to as C6), Bacillus subtilis C15 (hereinafter referred to as C15), Bacillus subtilis BSS15 (hereinafter referred to as B15), and Bacillus subtilis BSS18 (hereinafter referred to as B18). The inventors evaluated the adhesion effects of these bacteria, see Example 2 for details.

[0070] Example 2: Evaluation of the adhesion of Bacillus subtilis

[0071] Microbial adhesion is the first step in the formation of biofilms. The formation of biofilms can be used to characterize the adhesion of microorganisms. The inventors compared the biofilm-forming ability of five strains of Bacillus subtilis with reference to the crystal violet staining method. 200 μL of bacterial suspension (concentration of 1×10 8 CFU / mL). After 48 hours of static culture, rinse with PBS. Then, let the plate dry naturally at room temperature, then add 0.1% crystal violet staining solution, and set the staining time to 15 minutes. After the staining is completed, aspirate the staining solution and rinse to ensure that the excess stain is completely removed. After that, let the plate dry naturally again, add 95% ethanol to each well to fully dissolve the stained biofilm. For the control group, use an empty ELISA plate to eliminate background interference. Finally, use a multifunctional ELISA instrument to detect the OD value of each well.590nm The absorbance values under the following conditions are shown in the following table for the experimental results.

[0072] Biofilm formation amounts of different Bacillus subtilis strains

[0073]

[0074] Note: The differences of different lowercase letters in the same column of the table are significant (p < 0.05).

[0075] The biofilm formation ability of Bacillus subtilis C10 is significantly stronger than that of other strains. Therefore, the adhesion characteristics of Bacillus subtilis C10 are stronger. Thus, Bacillus subtilis C10 is preserved and further research is carried out on Bacillus subtilis C10.

[0076] Example 3: Preparation and application of selenium-enriched Bacillus subtilis powder

[0077] (1) Fermentation of the probiotic preparation

[0078] The fermentation of the probiotic preparation in this example generally includes the following steps:

[0079] 1. Preparation of selenium-enriched fermentation broth medium: Sodium selenite is added to the sterilized LB medium to ensure that the selenium content in the system is 20 μg / mL. The formula of the LB liquid medium is 5 g of yeast extract, 10 g of sodium chloride, 10 g of peptone, 1000 mL of distilled water, pH 7.0; it is dispensed, and the liquid loading amount is one-third of the volume; it is sterilized and reserved for use;

[0080] 2. Preparation of selenium-enriched Bacillus subtilis fermentation broth: Take the Bacillus subtilis C10 seed liquid activated to OD 600nm = 1.0 and inoculate it into the selenium-enriched fermentation medium at an inoculation amount of 2%, and culture it in a shaker at 30 °C and 200 rpm for 36 h.

[0081] Specifically, the preparation method of the selenium-enriched Bacillus subtilis powder includes the following steps:

[0082] (1) Preparation of seed liquid: Take the single colony of the above-mentioned Bacillus subtilis C10 from the beef extract agar plate and inoculate it into the LB liquid medium, and culture it at 30 °C and 200 rpm for 4 h to obtain the seed liquid (this seed liquid is the Bacillus subtilis bacterial liquid, and its OD 600nm = 1.0); among them, the formula of the LB liquid medium is 5 g of yeast extract, 10 g of sodium chloride, 10 g of peptone, 1000 mL of distilled water, pH 7.0; it is dispensed, and the liquid loading amount is one-third of the volume; it is sterilized and reserved for use;

[0083] (2) Preparation of selenium-enriched Bacillus subtilis fermentation broth: Sodium selenite was added to the LB liquid medium to ensure that the selenium content in the system was 20 μg / mL. At the same time, the seed liquid prepared in step (1) was inoculated with an inoculation amount of 2%; it was cultured at 30 °C and a rotation speed of 200 rpm for 36 h to obtain the selenium-enriched Bacillus subtilis fermentation broth;

[0084] (3) The selenium-enriched Bacillus subtilis fermentation broth obtained by culturing in step (2) was centrifuged at 8000 rpm for 10 min. After removing the supernatant, the thallus was freeze-dried to obtain the selenium-enriched Bacillus subtilis powder.

[0085] The selenium-enriched Bacillus subtilis prepared in the present invention was named Bacillus subtilis SC10.

[0086] In addition, referring to this method, the inventors of the present invention carried out selenium-enriched cultivation on another 4 kinds of bacteria isolated during the same period: Bacillus subtilis C6, Bacillus subtilis C15, Bacillus subtilis BSS15, and Bacillus subtilis BSS18, that is, replacing Bacillus subtilis C10 in the preparation of the seed liquid in step (1) with the other 4 kinds of bacteria, and obtaining the corresponding selenium-enriched bacteria according to similar technological steps, and evaluating the selenium-enriched ability of 5 strains of Bacillus subtilis. The specific measurement methods and measurement results are as follows:

[0087] Take the above 5 kinds of Bacillus subtilis bacterial solutions activated to OD 600nm = 0.5, and inoculate them into 100 mL of selenium-enriched fermentation broth medium at an inoculation amount of 2% respectively. Culture them in a constant temperature shaker at 37 °C and 200 rpm for 24 h. Then centrifuge at 8000 rpm for 10 min to take the supernatant, and indirectly obtain the selenium-enriched effects of different strains by measuring the content of residual selenium in the supernatant. The measurement results are shown in Table 1.

[0088] Table 1 Selenium enrichment rates (%) of different Bacillus subtilis

[0089]

[0090] Note: The differences of different lowercase letters in the same column of the table are significant (p < 0.05).

[0091] The measurement results show that the selenium enrichment rates of the five strains are all above 80%. Among them, the C10 strain shows the best selenium enrichment effect, and the selenium enrichment rate can reach 90%, which is significantly higher than the other four strains. Therefore, it was determined as the experimental strain for the later stage.

[0092] (2) Determination of the ability of thallus adhesion and antagonism against spoilage bacteria

[0093] The highly adhesive Bacillus subtilis strain C10 obtained from tilapia was used as the experimental strain. According to the above method, Bacillus subtilis C10 was cultured using a selenium-enriched fermentation broth medium to obtain selenium-enriched Bacillus subtilis SC10. The adhesion ability of selenium-enriched Bacillus subtilis SC10 was evaluated, including the mucus adhesion rate and the amount of biofilm formed, and the adhesion inhibitory effect of selenium-enriched Bacillus subtilis SC10 on spoilage bacteria (Pseudomonas and Shewanella) was analyzed.

[0094] Determination of the adhesion rate. Add 150 μL of fish intestinal mucus to a 96-well plate and incubate overnight at 4 °C (fixed for 18 h). Add 150 μL of the bacterial suspension labeled with FITC dye to the well plate. Incubate at a constant temperature of 30 °C for 1.5 h. Add sterile PBS to wash the well plate. After discarding the washing solution, add 150 μL of SDS (1%) solution and incubate the sample at a constant temperature of 60 °C for 1 h. Use a multifunctional microplate reader to detect the fluorescence intensity, with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. To ensure the accuracy of the results, each sample was detected six times in duplicate. In addition, SDS (1%) was set as the blank control group to eliminate experimental errors. The formula for calculating the adhesion rate is as follows:

[0095]

[0096] Determination of the biofilm. The method for biofilm determination can refer to Example 2.

[0097] (3) Feeding experiment

[0098] Preparation of the tilapia feed of the present invention: The prepared selenium-enriched Bacillus subtilis powder was added to the fish basal feed at an addition amount of 0.5×10 8 cfu / g to obtain the fish feed; among them, the basal feed used was the basal feed for tilapia farming, and the main components included soybean cake, wheat bran, fish meal, vitamins, inorganic salts, and fish oil.

[0099] Feeding:

[0100] The tilapia were grouped and fed according to different cases,

[0101] Case 1: Feeding the basal feed;

[0102] Case 2: Adding 1×10 8 cfu / g of Pseudomonas (LP-3) to the basal feed;

[0103] Case 3: Adding 0.5×10 8 cfu / g of Bacillus subtilis C10 and 0.5×10 8 cfu / g of Pseudomonas (C10+LP-3) to the basal feed;

[0104] Case 4: Add 0.5×10 8 cfu / g of selenium-enriched Bacillus subtilis SC10 and 0.5×10 8 cfu / g of Pseudomonas sp. (SC10 + LP-3);

[0105] Case 5: Add 0.5 mg / kg of sodium selenite and 0.5×10 8 cfu / g of Pseudomonas sp. (Na2SeO3 + LP-3).

[0106] When feeding tilapia, after a two-week adaptation period with normal feed (i.e., basal feed), group feeding is carried out according to different cases for 21 days. During the feeding period of each case, the water temperature is controlled at 27 - 29 °C, 50% of the culture water is changed every day, and an air pump is used to maintain oxygen. Feed the corresponding feed of the case twice a day (9:00 and 17:00), and the feeding amount is 2 - 3% of the tilapia body weight.

[0107] (4) Quality determination method

[0108] At the end of the feeding experiment, the fish are fasted for 24 h. Determine the growth performance of tilapia in different cases, and randomly select 3 tilapia from each case for dissection in a sterile environment, and take their fish meat for detecting crude protein and fat contents, volatile basic nitrogen and flavor indexes.

[0109] Determination of fish body growth performance. Catch each group of fish and place them on ice until the fish have no obvious reaction. Blot the water on the fish body surface with paper, weigh them, and calculate the weight gain rate, feed coefficient and condition factor according to the following formulas:

[0110] Weight gain rate (%) = (W t - W0) / W0 × 100%

[0111] Feed coefficient = (W t - W0) / W × 100%

[0112] Condition factor (g / cm 3 ) = (W t / L 3 ) × 100%

[0113] Among them, N1 and N2: the number of tilapia at the beginning and end of the feeding experiment; W0 and W t : the weight of tilapia at the beginning and end of the feeding experiment (g); W: the feed consumption in the feeding experiment (g); L: the body length of tilapia at the end (cm).

[0114] Determination of crude protein in fish meat. Referring to GB 5009.5-2016, the automatic Kjeldahl method was used to determine the crude protein content in fish meat. Weighed 2.00 g of fish meat into a digestion tube, added 0.4 g of copper sulfate, 6 g of potassium sulfate, and 20 mL of sulfuric acid, digested at 420 °C for 1 hour, and then cooled. Added 40 mL of sodium hydroxide, 25 mL of boric acid (receiving solution), and 10 mL of distilled water into the automatic Kjeldahl apparatus, set the reaction time for 3 minutes, and the operation was completed automatically. Titrated the receiving solution with a hydrochloric acid standard solution until it turned gray, which was the end point. Expressed by the formula:

[0115]

[0116] In the formula, V1 and V2 represent the volumes of hydrochloric acid consumed by the treatment group and the reagent blank, respectively (mL); c represents the concentration of the hydrochloric acid standard solution (mol / L); m represents the mass of the sample (g). F represents the coefficient for converting nitrogen to protein, 6.25; 100 represents the conversion coefficient.

[0117] Determination of crude fat in fish meat. Referring to GB 5009.6-2016, the Soxhlet extraction method was used to determine the crude fat content in fish meat. Weighed 2.00 g of fish meat into a filter paper cylinder, placed the filter paper cylinder into the extraction cylinder and connected it to the receiving bottle, added anhydrous ether to two-thirds of the condenser, heated by water bath for extraction, with a frequency of 6-8 times / h, until there were no oil spots. Recovered the ether, evaporated to dryness when there was 1-2 mL of the solvent left in the receiving bottle, dried at 100 °C for 1 h, cooled in a desiccator for 0.5 h, and then weighed. Repeated until a constant weight was achieved, and the difference was ≤2 mg. Expressed by the formula:

[0118]

[0119] In the formula: m0, m1, and m2 represent the mass of the receiving bottle, the mass of the receiving bottle and fat after constant weight, and the mass of the fish meat, respectively, with the unit of gram (g); 100 represents the conversion coefficient.

[0120] Determination of total volatile basic nitrogen (TVB-N). Referring to GB 5009.228—2016, 10.00 g of homogenized tilapia fish meat with different refrigeration times was taken and incubated with 75 mL of distilled water for 30 min. The supernatant after incubation was added into a digestion tube, 1.00 g of MgO was added, and at the same time, the digestion tube was connected to an automatic Kjeldahl apparatus for distillation, and the distillation time was 3 min. After distillation, it was titrated with a 0.01 mol / L hydrochloric acid solution to accurately determine the total volatile basic nitrogen content. And 10 mL of distilled water was used as a blank control. The absorbing solution used during the reaction was boric acid. Expressed by the formula:

[0121]

[0122] Wherein, V1 and V2 respectively represent the volumes of hydrochloric acid consumed by the treatment group and the reagent blank (mL); c represents the concentration of the hydrochloric acid standard solution (mol / L); m represents the mass of the sample (g).

[0123] Determination of volatile flavor substances. 2.00 g of fish meat sample, 5 mL of saturated NaCl solution and 200 μL of the standard substance 2-octanol with a concentration of 2 mg / L were placed in an extraction bottle. After equilibration at 60 °C in a water bath for 5 minutes, followed by 30 minutes of headspace adsorption. After extraction, desorption was carried out for 5 minutes. An HP-INNOWax capillary chromatographic column (specification: 30 m × 0.32 mm × 0.15 μm) was used. By comparing the peak area ratios of the flavor compounds to the internal standard, the concentration of the compounds was calculated. It is expressed by the formula:

[0124] Content of volatile flavor components (mg / L) = (A i / A) × c

[0125] Wherein, Ai and A respectively represent the peak areas of volatile compound i and the internal standard, and c represents the concentration of the internal standard (mg / L).

[0126] Experimental results:

[0127] (1) Effect of selenium enrichment on the adhesion characteristics of Bacillus subtilis

[0128] An in vitro immobilized mucus model was used and combined with fluorescence microscopy observation to evaluate the ability of Bacillus subtilis C10, selenium-enriched Bacillus subtilis SC10 and Na2SeO3 to adhere to fish intestinal mucus. The results are shown in Table 2. After selenium enrichment, the adhesion rate of Bacillus subtilis increased significantly; the same result was obtained by fluorescence microscopy observation. Through quantification, it was found that the fluorescence intensity of SC10 was significantly stronger than that of the other two groups. The biofilm formation status (a) and formation amount (b) of C10, SC10 and Na2SeO3 are as Figure 3 shown. C10 and SC10 formed a denser biofilm, while Na2SeO3 did not form a biofilm, and the biofilm formation amount of SC10 was significantly higher than that of C10. It indicates that selenium enrichment can significantly improve the adhesion characteristics of Bacillus subtilis.

[0129] Table 2 Adhesion ability of C10, SC10 and Na2SeO3 to fish intestinal mucus

[0130]

[0131] Note: The differences with different lowercase letters in the same column of the table are significant (p < 0.05).

[0132] (2) Ability of selenium-enriched Bacillus subtilis to antagonize spoilage bacteria

[0133] The adhesion inhibition rate of selenium-enriched Bacillus subtilis SC10 against spoilage bacteria (Pseudomonas LP-3 and Shewanella HR-15) was measured to analyze the adhesion and antagonistic effect of SC10 against spoilage bacteria. The results are shown in Table 3. After selenium enrichment, the adhesion inhibition rate of Bacillus subtilis against spoilage bacteria increased significantly. The adhesion inhibition rates of SC10 against LP-3 and HR-15 were 58% and 35% respectively. It indicates that after selenium enrichment, Bacillus subtilis exhibits a strong competitive adhesion ability and can effectively antagonize spoilage bacteria Pseudomonas and Shewanella.

[0134] Table 3 Adhesion inhibition rates of C10, SC10 and Na2SeO3 against spoilage bacteria (%)

[0135]

[0136] Note: The differences with different lowercase letters in the same column of the table are significant (p < 0.05).

[0137] (3) Comparison of fish body colonization and preservation effects

[0138] The implementation cases include

[0139] Case 1: Feeding basal diet;

[0140] Case 2: Adding 10 8 cfu / g of Pseudomonas (LP-3) to the basal diet;

[0141] Case 3: Adding 0.5×10 8 cfu / g of Bacillus subtilis C10 and 0.5×10 8 cfu / g of Pseudomonas (C10 + LP-3) to the basal diet;

[0142] Case 4: Adding 0.5×10 8 cfu / g of selenium-enriched Bacillus subtilis and 0.5×10 8 cfu / g of Pseudomonas (SC10 + LP-3) to the basal diet;

[0143] Case 5: Adding 0.5 mg / kg of sodium selenite and 0.5×10 8 cfu / g of Pseudomonas (Na2SeO3 + LP-3) to the basal diet.

[0144] The effects of different cases on the growth performance of tilapia are shown in Table 4. Adding Pseudomonas (Case 2) decreased the weight gain rate of tilapia. After adding Bacillus subtilis C10 (Case 3), the final weight, body length and weight gain rate were all increased. Moreover, the indexes of adding selenium-enriched Bacillus subtilis SC10 (Case 4) were significantly higher than those of Case 3, and it could also significantly reduce the feed coefficient and increase the body fatness of fish. The results of the nutritional components of tilapia meat are shown in Table 5. The contents of crude protein and crude fat in tilapia of Case 2 decreased, while the contents of nutritional components in the fish meat of Case 3 did not decrease; Case 4 significantly increased the contents of crude protein and fat in tilapia. Therefore, feeding selenium-enriched Bacillus subtilis (Case 4) can significantly improve the growth performance and nutritional components of fish body.

[0145] Table 4 Effects of five cases on the growth performance of tilapia

[0146]

[0147] Note: The differences with different lowercase letters in the same column of the table are significant (p < 0.05).

[0148] Table 5 Effects of five cases on the nutritional components of tilapia

[0149]

[0150] Note: The differences with different lowercase letters in the same column of the table are significant (p < 0.05).

[0151] The changes in the content of total volatile basic nitrogen (TVB-N) in tilapia during cold storage are shown in Table 6. At the 0th day of storage, the TVB-N content in the fish meat of Case 2 increased. Case 5 could relieve the spoilage situation and inhibit the accumulation of TVB-N, making it maintain the same content as that of Case 1; Case 3 could improve the increase of TVB-N, and Case 4 had the most significant improvement effect. After storage for 5 days, the TVB-N contents in Case 1 and Case 2 had exceeded the spoilage threshold (TVB-N = 30 mg / 100 g). The TVB-N contents in Case 3, Case 4 and Case 5 were effectively controlled within a reasonable range of freshness, and Case 4 had the best effect. The degree of spoilage from high to low was Case 2 > Case 1 > Case 5 > Case 3 > Case 4. Therefore, feeding Bacillus subtilis C10 (Case 3) can delay the spoilage process of fish body, and the fresh-keeping effect of selenium-enriched Bacillus subtilis SC10 (Case 4) is the most significant.

[0152] Table 6 Changes in TVB-N values (mg / 100 g) of fish meat during cold storage in five cases

[0153]

[0154] Note: The differences with different lowercase letters in the same column of the table are significant (p < 0.05), and the differences with the same letters are not significant (p > 0.05).

[0155] The main volatile substances of tilapia during refrigeration are shown in Table 7. Without any treatment (Case 1) and when Pseudomonas was added (Case 2), the main odoriferous representative substance 1-octen-3-ol monomer and hexanal, which contributes to a greasy and fatty smell, were mainly produced. However, the intervention of Bacillus subtilis C10 can effectively reduce the production of such spoilage characteristic flavor compounds and improve the fish flavor. Among them, the group treated with selenium-enriched Bacillus subtilis SC10 (Case 4) had the most significant effect.

[0156] Table 7 Main volatile compounds of tilapia fish meat at 5 days of storage in five cases (mg / L)

[0157]

[0158] Note: " / " indicates not detected.

[0159] The present invention proposes a method for enhancing the adhesion of strains through selenium-enriched fermentation, improving the functions of strain preservation and flavor, significantly improving the growth performance and nutritional components of fish, and delaying the spoilage of fish in a low-temperature environment. It can be applied to the refrigeration or chilled preservation process of farmed freshwater or seawater fish with Pseudomonas and Shewanella as the dominant spoilage bacteria. Equivalent changes and modifications made to the scope of the patent application shall fall within the scope covered by the present invention.

Claims

1. A Bacillus subtilis, characterized in that: It has high adhesiveness. This strain is named Bacillus subtilis C10 and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the deposit number CGMCC No. 32669.

2. Use of the Bacillus subtilis as claimed in claim 1 in the preparation of selenium-enriched Bacillus subtilis powder.

3. A selenium-rich Bacillus subtilis bacterial powder, characterized in that: It is obtained by culturing the Bacillus subtilis as claimed in claim 1 in a selenium-enriched fermentation broth medium and then centrifuging and freeze-drying; wherein, the selenium-enriched fermentation broth medium is prepared by adding sodium selenite to a sterilized LB medium.

4. The preparation method of the selenium-rich Bacillus subtilis bacterial powder according to claim 3, characterized in that: It comprises the following steps: (1) Seed liquid preparation: Take a single colony of the Bacillus subtilis C10 from a beef extract agar plate and inoculate it into an LB liquid medium, and culture it at 30 °C and 200 rpm for 4 h to obtain a seed liquid; wherein, the formula of the LB liquid medium is 5 g of yeast extract, 10 g of sodium chloride, 10 g of peptone, 1000 mL of distilled water, pH 7.0; aliquot, and the liquid filling amount is one-third of the volume; sterilize and reserve for use; (2) Preparation of selenium-enriched Bacillus subtilis fermentation broth: Add sodium selenite to the LB liquid medium to ensure that the concentration of sodium selenite is 20 μg / ml, and at the same time inoculate the seed liquid prepared in step (1) with an inoculation amount of 2%; culture it at 30 °C and a rotation speed of 200 rpm for 36 h to obtain a selenium-enriched Bacillus subtilis fermentation broth; (3) Centrifuge the selenium-enriched Bacillus subtilis fermentation broth obtained in step (2) at 8000 rpm for 10 min, remove the supernatant, and then freeze-dry the bacterial cells to obtain selenium-enriched Bacillus subtilis powder.

5. Use of the selenium-enriched Bacillus subtilis powder as claimed in claim 3 in the preparation of fish feed.

6. The application according to claim 5, wherein: Add the selenium-rich Bacillus subtilis bacterial powder to the fish basal diet at an addition amount of 0.5×10 8 cfu / g.

7. The application of the selenium-rich Bacillus subtilis bacterial powder according to claim 3 in fish farming, characterized in that: The method for culturing fish using the selenium-enriched Bacillus subtilis powder is as follows: (1) Add selenium-rich Bacillus subtilis powder to the fish basal diet at an addition amount of 0.5×10 8 cfu / g to obtain fish feed; (2) Feed the fish feed twice a day, and the feeding amount is 2-3% of the fish body weight.

8. A fish feed containing the selenium-enriched Bacillus subtilis powder as claimed in claim 3.

9. The fish feed according to claim 8, characterized in that: It includes fish basic feed and selenium-rich Bacillus subtilis powder, and the selenium-rich Bacillus subtilis powder is added to the fish basic feed at an addition amount of 0.5×10 8 cfu / g.

10. Use of the selenium-enriched Bacillus subtilis powder as claimed in claim 3 in the preparation of a preservative for fish body preservation.

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