Bacillus natto and application thereof in preparation of functional feed for breeding of euryzonus sinensis

Abstract

The application provides a bacillus natto and application thereof in preparation of functional feed for Chinese mitten crab breeding, and belongs to the technical field of aquaculture. Bacillus natto HQ-1 has been preserved in China Center for Type Culture Collection on August 11, 2025, and the preservation number is CCTCC No. M 20251811. Research shows that the bacillus natto has a water product pathogenic bacteria inhibition effect, a toxin degradation capacity and a water body environment purification function which are significantly improved compared with the same strain. The functional feed prepared by fermentation of the strain contains bioactive ingredients such as probiotic live bacteria, free amino acids, antibacterial lipopeptides and polyglutamic acid, can improve the nutritional form of the feed, improve the body weight, disease resistance and feeding rate of the Chinese mitten crab, and help to reduce the content of harmful substances in the aquaculture water body and improve the water body environmental quality. The application provides a new technical approach for green, antibiotic-free and sustainable breeding of the Chinese mitten crab.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a strain of Bacillus natto and its application in the preparation of functional feed for mitten crab farming. Background Technology

[0002] Chinese mitten crab ( Eriocheir sinensis The Chinese mitten crab (also known as the hairy crab) is a highly representative high-value economic species in my country's aquaculture industry, widely favored by the market for its tender meat and delicious flavor. Statistics show that in 2024, the national annual output of Chinese mitten crabs reached 894,400 tons, with the entire industry chain exceeding 80 billion yuan in output value, effectively driving the coordinated development of related industries such as feed processing, aquaculture technology, and cold chain logistics. However, the current Chinese mitten crab farming still faces multiple challenges, including an unreasonable feed structure, frequent disease outbreaks, and deteriorating water quality, seriously restricting the improvement of farming efficiency and sustainable ecological development.

[0003] Research shows that currently less than 30% of aquaculture farmers use nutritionally complete and scientifically formulated high-quality compound feed. The vast majority still rely on traditional feeding methods using chilled fish and agricultural byproducts such as cornmeal and rapeseed cake. Chilled fish is not only costly and difficult to store, but it is also highly susceptible to spoilage. Feeding it to these fish can exacerbate eutrophication and bottom sediment deterioration, leading to the proliferation of pathogenic bacteria such as Vibrio and Aeromonas, significantly increasing the risk of outbreaks of disease. Furthermore, some raw materials are susceptible to mold contamination during storage, potentially producing mycotoxins such as aflatoxin and zearalenone. These toxins not only harm the liver health and immune function of crabs but also pose a potential threat to the safety of the final aquatic products for consumption.

[0004] Currently, most commercially available aquatic feeds are simply compounded from animal and plant-based raw materials, making it difficult to accurately match the nutritional needs and health control goals of different farmed organisms at different growth stages. Furthermore, due to the generally insufficient water stability of these feeds, the rate of dissolution after being added to water is high. Uneaten feed not only wastes resources but also easily becomes a carrier for pathogenic microorganisms to attach and proliferate in the aquaculture environment, indirectly inducing secondary infections, leading to large-scale mortality events and significant economic losses. For example, Chinese invention patent CN108634135A discloses a feed for mitten crabs, whose composition includes various aquatic animal meat pastes, corn flour, silage straw powder, garlic powder, and traditional Chinese medicine powder. While this formula meets basic nutritional needs to some extent, it fails to effectively address the problems of pathogenic bacteria growth and water pollution induced by the feed. If the feed is not consumed promptly after being added to the water, it can accelerate water quality deterioration, induce the accumulation of toxins in the water, and ultimately cause large-scale mortality of farmed organisms, thus limiting its practical application effectiveness.

[0005] In summary, there is an urgent need to develop a new type of aquatic feed that combines nutritional supply, immune enhancement, and water quality regulation. This feed should improve the feeding efficiency and disease resistance of Chinese mitten crabs while achieving multiple ecological benefits such as inhibiting pathogen reproduction, slowing down eutrophication, and reducing toxin residues in the water. This would support the green, antibiotic-free, and sustainable development of Chinese mitten crabs and other aquaculture species. Summary of the Invention

[0006] This invention aims to address the problems of insufficient feed functionality, low disease resistance, deterioration of aquatic environment, and toxin residues in raw materials during the breeding process of farmed organisms such as mitten crabs. It provides a new strain of Bacillus natto and its application in the preparation of functional feed for mitten crab farming, thereby overcoming the shortcomings of existing mitten crab feed.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention obtained a strain of Bacillus natto through systematic screening and functional evaluation. Compared with commercially available strains of the same species, this strain exhibits significantly improved inhibitory effects against aquatic pathogens, degrades toxins from various feed sources, and demonstrates stronger aquatic environment purification capabilities. This Bacillus natto strain and its secondary metabolites show significant inhibitory effects against various aquatic pathogens, including Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Aeromonas hydrophila. Bacillus natto can effectively degrade various toxins that are easily generated during feed storage, including zearalenone, aflatoxin, ochratoxin, vomitoxin, and citrinin. Bacillus natto and its secondary metabolites also have purification functions for aquatic environments, including ammonia nitrogen, nitrite, phosphorus, chemical oxygen demand (COD), and particulate matter.

[0009] The 16S rDNA gene of this strain has a nucleotide sequence length of 1399 bp, and its gene sequence is shown in SEQ ID NO:1. This strain is similar to Bacillus natto. B. natto JLCC513 showed high homology. Based on morphological observation, physiological and biochemical analysis, and identification results, this strain is identified as *Bacillus natto*, and is therefore classified and named *Bacillus natto*. B. natto HQ-1 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M 20251811, deposited on August 11, 2025, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, 430072, China.

[0010] Based on the aforementioned special properties of Bacillus natto, this application seeks protection for the application of the aforementioned Bacillus natto in aquaculture.

[0011] Furthermore, the aquaculture species are crustaceans, mollusks, or fish. Crustaceans include common farmed shrimp and crabs, mollusks include various freshwater and seawater shellfish, fish include various freshwater and seawater farmed fish, and crabs are mainly Chinese mitten crabs.

[0012] This invention provides the application of the above-mentioned Bacillus natto in the preparation of aquaculture microbial agents or functional feeds for aquaculture.

[0013] This invention provides an aquaculture microbial agent containing the aforementioned Bacillus natto.

[0014] Furthermore, the aquaculture microbial agent is a liquid fermentation agent, which is prepared by inoculating the aforementioned Bacillus natto into a fermentation medium and culturing it at 24-40 °C for 12-24 h. When the viable count in the bacterial solution is not less than 1.5 × 10⁻⁶, the fermentation process is complete. 8 Fermentation was stopped when the concentration of CFU / mL was reached, yielding the liquid fermentation agent. The fermentation medium was formulated as follows: molasses 5-10 g / L, crude glycerol 10-15 g / L, peptone 5-15 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 1-5 g / L, water as solvent, and pH 7.0-7.5. Preferably, the culture temperature was controlled at 37 °C, and the culture was carried out for 16 h until the viable cell count in the culture was not less than 1.5 × 10⁻⁶. 8 Fermentation was stopped when CFU / mL was reached.

[0015] Preferably, the inoculum amount of Bacillus natto is 5% to 15% (v / v), and the fermentation is carried out in a fermenter with a rotation speed of 150 to 300 rpm.

[0016] Preferably, the volume of liquid in the fermentation tank is 55%~70%, the aeration ratio is 0.8~1.2 vvm, and the initial pH is 7.0~8.0.

[0017] Preferably, the fermentation medium comprises the following components: molasses 10 g / L, crude glycerol 15 g / L, peptone 10 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 2 g / L, and the initial pH of the fermentation broth is adjusted to 7.2 using ammonia water.

[0018] This invention provides a functional feed for aquaculture, wherein the functional feed for aquaculture contains the aforementioned Bacillus natto or is obtained through fermentation of the aforementioned Bacillus natto.

[0019] This invention provides a method for preparing the aforementioned functional feed for aquaculture. The functional feed is obtained by fermenting the aforementioned Bacillus natto, and its fermentation substrate includes soybeans, corn, soybean meal, fishmeal protein, and wheat bran. This invention uses Bacillus natto as the fermentation strain and utilizes an agricultural by-product substrate with soybeans as the main component for solid-state fermentation to prepare a functional fermented feed suitable for aquatic animals, particularly for the farming of Chinese mitten crabs.

[0020] Furthermore, in the above-mentioned method for preparing functional feed for aquaculture, the fermentation substrate is soybean, corn, soybean meal powder, fish meal protein and wheat bran, with the following composition by weight: 50-55 parts soybean, 10-20 parts corn, 10-15 parts soybean meal powder, 10-15 parts fish meal protein, and 10-15 parts wheat bran; preferably, the composition is as follows: 50 parts soybean, 15 parts corn, 12.5 parts soybean meal powder, 12.5 parts fish meal protein, and 10 parts wheat bran.

[0021] Preferably, the fermentation substrate needs to be pulverized and sterilized. The method is to weigh and mix the fermentation substrate, including soybeans, corn, soybean meal powder, fish meal protein, and wheat bran, according to the proportion. After being fully pulverized and mixed by a homogenizer, the mixture is passed through a 30-50 mesh sieve to obtain the fermentation substrate of microbial agent. The fermentation substrate is placed in a boiling pot for high-temperature boiling sterilization for 1-1.5 hours. After cooling to below 40 °C, it is inoculated with Bacillus natto.

[0022] The Bacillus natto is prepared as a liquid fermentation agent or a solid agent with the same viable cell count as the liquid fermentation agent, and is mixed with the fermentation substrate. The viable cell count in the liquid fermentation agent is not less than 1.5 × 10⁻⁶. 8 The fermentation substrate and the liquid fermentation agent have a mass ratio of CFU / mL, which is (2~10):1, preferably 5:1.

[0023] Furthermore, the fermentation conditions are 30-40 °C for 1-3 days, with turning treatment at 4-6 h intervals; preferably, fermentation is carried out at 37 °C for 2 days, with turning treatment at 4 h intervals.

[0024] Preferably, the fermentation stirring speed is 150~200 rpm for 10~20 min, and aeration is turned on during the stirring process, with the aeration rate controlled at 0.8~1 vvm.

[0025] Furthermore, after the liquid fermentation inoculant is mixed with the fermentation substrate, the moisture content of the mixture is 50%~60%.

[0026] This invention provides the application of the above-mentioned functional feed for aquaculture or the functional feed for aquaculture prepared by the above-mentioned method for preparing functional feed for aquaculture in crab, shrimp and fish farming. Preferably, the crab is a Chinese mitten crab.

[0027] Preferably, when inoculating the Bacillus natto into the fermentation medium, the Bacillus natto needs to be activated by slant culture medium first, and then cultured in liquid seed culture medium to obtain seed liquid. The seed liquid is then inoculated into the fermentation medium for fermentation to prepare liquid fermentation agent.

[0028] Preferably, the activation of the bacterial strain involves inoculating Bacillus natto onto a slant culture medium, incubating it statically at 24-40 °C for 12-16 h, then picking a single colony and streaking it onto the slant culture medium again, and incubating it at 24-40 °C for 12-16 h to obtain the activated bacterial strain.

[0029] Preferably, the seed liquid is prepared by inoculating 3 loops of activated bacterial sludge into a shake flask under aseptic conditions, placing it on a shaker at a speed of 150-200 rpm, and culturing it at a temperature of 24-40 °C for 12-16 h to obtain the fermentation seed liquid.

[0030] Preferably, the slant culture medium is composed of: glucose 10-15 g / L, peptone 5-15 g / L, yeast extract 5-10 g / L, sodium chloride 5-15 g / L, agar powder 15-20 g / L, water as solvent, and pH value of 7.0-7.5. Preferably, the liquid seed culture medium consists of: molasses 10-15 g / L, peptone 5-15 g / L, potassium dihydrogen phosphate 1-5 g / L, water as solvent, and pH 7.0-7.5. Compared with the prior art, the present invention has the following beneficial effects: (1) This invention obtained a strain of Bacillus natto through systematic screening and functional evaluation. Compared with two commercially available strains of the same species, this strain has significantly improved inhibitory effects on aquatic pathogens, degrades various feed-derived toxins (zearalenone, aflatoxin, ochratoxin, vomitoxin, and citric acid), and exhibits stronger water environment purification functions. This Bacillus natto and its secondary metabolites have significant inhibitory effects on various aquatic pathogens, including Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Aeromonas hydrophila. This Bacillus natto and its secondary metabolites also have purification functions on ammonia nitrogen, nitrite, phosphorus, COD, and particulate matter in the aquatic environment, and can significantly improve the breeding environment of Chinese mitten crabs.

[0031] (2) Functional feed for aquaculture prepared by fermentation of Bacillus subtilis using soybeans, corn, soybean meal, fish meal protein, wheat bran and other substrates can greatly reduce the production cost of microbial agents. On the other hand, the functional feed is rich in bioactive components such as probiotics, soluble protein, antimicrobial lipopeptides, and polyglutamic acid.

[0032] (3) The functional feed for aquaculture of the present invention can not only effectively degrade toxins that are easily generated during feed storage, but also significantly improve the weight gain, resistance and feeding rate of hairy crabs. The feeding rate of hairy crabs is increased by about 28% to 30%, the weight of a single crab can be increased by 18% to 30%, and the disease rate is reduced by 50% to 65%. In addition, it can effectively purify the water quality and ecological environment of hairy crab farming, which is of great significance for the green, antibiotic-free and sustainable farming of hairy crabs.

[0033] This strain of Bacillus natto is classified and named Bacillus natto. B. natto HQ-1 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M 20251811, deposited on August 11, 2025. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, 430072, China. Attached Figure Description

[0034] Picture 1 Bacillus subtilis B. natto Agarose gel electrophoresis image of 16S rDNA of HQ-1.

[0035] Picture 2 Bacillus subtilis B. natto Phylogenetic tree of HQ-1.

[0036] Picture 3 Bacillus subtilis B. natto The inhibition zone effect of HQ-1 fermentation broth on Aeromonas hydrophila.

[0037] Picture 4 Bacillus subtilis B. natto Degradation curves of HQ-1 on zearalenone, vomitoxin, citric acid, aflatoxin, and ochratoxin. Detailed Implementation The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not and will not limit the invention as described in detail in the claims.

[0038] Other bacterial strains used in this invention: Vibrio harlequinae CGMCC 1.8773 ( Vibrio harveyi Purchased from China General Microbiological Culture Collection Center; Vibrio vulnificus CICC 21615 ( Vibrio vulnificus Vibrio parahaemolyticus CICC 23924 Vibrio parahaemolyticus Aeromonas hydrophila CICC 25017 Aeromonas Hydrophila ) and Pseudomonas aeruginosa CICC 21643 ( Pseudomonas aeruginosa All strains were purchased from the China Industrial Microbial Culture Collection Center; the Bacillus natto strains used for comparison were derived from Bacillus natto preparations from the Mizutani Shin brand and the Jiubang brand.

[0039] Example 1: Screening of Bacillus natto 1. Strain screening test The samples were sourced from handmade natto sold in Suqian City, Jiangsu Province. 2 g of natto from different regions was weighed and inoculated into 500 mL Erlenmeyer flasks containing enrichment medium (20% of the flask volume). The flasks were incubated at 80 °C for 10 min to select spore-forming bacteria. Enrichment culture was then carried out at 37 °C and 200 rpm for 24 h. 3 mL of the enriched solution was transferred to fresh enrichment liquid medium, and enrichment was repeated under the same conditions for another 24 h. This process was repeated once, for a total of three enrichment cycles. Under aseptic conditions, the third enrichment culture was diluted to 10⁻⁶. -6 10 -7 and 10 -8 Take 100 μL of each sample and spread it evenly on solid screening medium. Incubate at 37 °C for 12–16 h, preferably 15 h. Screen bacteria, prioritizing colonies with milky white or light yellow color and filamentous striations. Streak the colonies on plates to obtain purified colonies and preserve them. Use the agar plate diffusion method to screen for antagonistic bacteria against aquatic pathogens, using *Vibrio harveyi* and *Pseudomonas aeruginosa* as indicator bacteria. Activate the indicator bacteria with LB broth and then transfer them to fresh LB broth for overnight incubation, adjusting the bacterial density to 10⁻⁶. 7 CFU / mL, 100 μL of bacterial suspension was spread on LB solid medium, wells were punched, and 25 μL of overnight cultured and purified bacterial suspension was added. The culture was incubated at 37 °C for 12–16 h, preferably 15 h, and the diameter of the inhibition zone was measured and compared. The polyglutamic acid synthesis ability of the purified strain was evaluated by culturing on polyglutamic acid screening solid medium at 37 °C for 12–24 h and observing the filamentation phenomenon on the plate, preferably 18 h, and compared with commercial Bacillus natto.

[0040] The culture medium used has the following composition: (1) Enriched liquid culture medium (by mass): 2% sucrose, 0.5% yeast extract, 0.5% dipotassium hydrogen phosphate, pH 7.0~7.5; (2) Solid culture medium (by mass): 2% sucrose, 0.5% yeast extract, 0.5% dipotassium hydrogen phosphate, 2% agar powder, pH 7.0~7.5; (3) LB liquid medium (by weight): 1% peptone, 0.5% yeast extract, 1% sodium chloride, pH natural; (4) LB solid medium (by mass): 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder, pH natural; (5) Polyglutamic acid screening solid culture medium (by mass): sucrose 1%, sodium glutamate 2%, yeast powder 0.5%, magnesium sulfate 0.5%, agar powder 2%, pH 7.0~7.2.

[0041] 2. Screening test results The results of the secondary screening of the strains are shown in Table 1 below. As can be seen from the table, strain HQ-1 showed significant inhibitory effects against Vibrio harveyi and Pseudomonas aeruginosa, with inhibition zones reaching 16.4 mm and 14.4 mm respectively, and exhibiting significant filamentation. Furthermore, it was more effective than the two commercially available natto strains in inhibiting pathogenic bacteria and promoting polyglutamic acid production. After comprehensive consideration, strain HQ-1 was selected as the strain for subsequent fermentation of the feed.

[0042] Table 1. Secondary screening of Bacillus natto

[0043] Note: ++ indicates that the stringy phenomenon is more obvious, and + indicates that there is a stringy phenomenon.

[0044] Example 2 Bacillus natto B. natto Molecular biological identification of HQ-1 1. Test Methods Molecular biological identification was performed on the HQ-1 strain screened in Example 1 to clarify its systematic taxonomic position. The specific methods are as follows: Genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit. The genomic DNA was amplified by PCR using universal 16S rRNA gene primers. The amplified product was recovered and purified after detection by 1% agarose gel electrophoresis. The purified product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The sequence was submitted to the NCBi GenBank database, and homology analysis was performed using the BLAST program with known 16S rDNA sequences. A phylogenetic tree was constructed using MEGA software, and the complete 16S rRNA gene sequences of strain HQ-1 were compared with those of several closely related Bacillus strains.

[0045] 2. Test Results Purification results are as follows Picture 1 As shown, the full-length 16S rDNA nucleotide sequence obtained by sequencing is 1399 bp, and its sequence is shown in SEQ ID No. 1. Homology comparison results show that strain HQ-1 is related to the previously disclosed Bacillus natto. B. natto JLCC513 showed high homology, indicating that this strain belongs to Bacillus natto. A phylogenetic tree was constructed, revealing that strain HQ-1 is closely related to several other strains. Bacillus The 16S rDNA full sequence of the genus strain was compared, and the results are as follows: Picture 2 As shown in the results, it clusters with Bacillus natto in the same branch, with a clear phylogenetic position, supporting its classification as Bacillus natto. Combining the observations and measurements of the morphological characteristics (such as colony morphology, Gram staining reaction, spore formation, etc.) and physiological and biochemical properties of strain HQ-1 shown in Table 2, and based on a comprehensive assessment of morphological and physiological and biochemical characteristics, this strain was identified as Bacillus natto and named... Bacillus natto HQ-1.

[0046] Table 2 B. natto Physiological and biochemical characteristics of HQ-1

[0047] Note: + indicates a positive result, - indicates a negative result.

[0048] Example 3 Bacillus natto B. natto Antagonistic effects of HQ-1 on various aquatic pathogens 1. Test Methods Preparation process of Bacillus natto fermentation broth: ① Bacillus natto B. natto HQ-1 strain was inoculated onto an agar slant and incubated statically at 37 °C for 12 h. A single colony was then streaked onto the agar slant and incubated at 37 °C for 12 h to obtain activated strain for later use. ② The activated strain from the above step was taken and, under aseptic conditions, 3 loops of bacterial sludge were inoculated into a shake flask containing the seed culture. The flask was placed on a shaker at 150 rpm and incubated at 37 °C for 12 h to obtain the fermentation seed culture. ③ Under aseptic conditions, the seed culture was transferred to the fermentation medium at an inoculation rate of 10% (v / v) and placed on a fermenter at 150 rpm. The fermentation was carried out at 37 °C for 16–24 h until the viable cell count was not less than 1.5 × 10⁻⁶. 8 When the concentration of CFU / mL was reached, the expansion culture was stopped, and a fermentation broth rich in Bacillus natto was obtained, which is the liquid fermentation agent of Bacillus natto. Alternatively, a commercially available Bacillus natto strain was used as a control strain, and fermentation broth was prepared using the same method.

[0049] Select Bacillus natto B. nattoHQ-1 was used as the experimental strain to test its antagonistic effects against several common aquatic pathogens, including Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Aeromonas hydrophila, and compared with commercially available Bacillus natto. The antagonistic / antibacterial assay was conducted using the agar plate diffusion method. Different indicator bacteria were activated and cultured in their respective suitable media. Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus were cultured in TSB optimized medium, while Pseudomonas aeruginosa and Aeromonas hydrophila were cultured in LB medium. The activated indicator bacterial solutions were diluted to a concentration of 1 × 10⁻⁶. 6 After CFU / mL, the mixture was evenly spread onto the corresponding plates. After punching holes, HQ-1 overnight culture medium, commercial Bacillus natto overnight culture medium, or fermentation broth supernatant obtained by centrifugation at 1000 rpm for 5 min, or supernatant treated with proteinase K (10 U / mL, 37 °C for 1 h), was added. After incubation at 30 °C for 24 h, the diameter of the inhibition zone was measured.

[0050] The composition of the culture medium used for culturing aquatic pathogens in this embodiment is as follows: (1) TSB optimized medium: 5 g / L tryptone, 15 g / L peptone, 3 g / L soybean peptone, 1 g / L yeast extract, 4 g / L glucose, 5 g / L dipotassium hydrogen phosphate, 15 g / L sodium chloride, suitable for Vibrio harveyi, Vibrio vulnificus and Vibrio parahaemolyticus, and the culture temperature is 30 °C. (2) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, suitable for Pseudomonas aeruginosa and Aeromonas hydrophila, and the culture temperature is 30 °C.

[0051] The composition of the culture medium used for culturing Bacillus natto in this embodiment is as follows: (1) Slant culture medium: glucose 15 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, agar powder 20 g / L, solvent is water, pH value is 7.2; (2) Liquid seed culture medium: molasses 15 g / L, peptone 10 g / L, potassium dihydrogen phosphate 2 g / L, solvent is water, pH 7.2; (3) Liquid fermentation medium: molasses 10 g / L, crude glycerol 10 g / L, peptone 10 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 5 g / L, solvent is water, pH value is 7.2; 2. Test Results Picture 3 Bacillus natto B. natto The inhibition zone effect of HQ-1 fermentation broth on Aeromonas hydrophila is shown in the figure. Picture 3The results showed that the fermentation broth of Bacillus natto had a significant inhibitory effect on Aeromonas hydrophila. Table 3 shows that Bacillus natto... B. natto HQ-1 fermentation broth and its supernatant showed significant inhibitory effects on all five aquatic pathogens mentioned above, with particularly prominent inhibitory effects against Vibrio harveyi and Aeromonas hydrophila. Compared to two commercially available Bacillus natto strains, B. natto HQ-1 showed significantly higher inhibitory effects against five pathogenic aquatic bacteria in the supernatant than commercially available Bacillus natto, indicating that... B. natto HQ-1 has significant value in aquaculture applications and can effectively antagonize various aquatic pathogens. Treatment with proteinase K significantly reduced the inhibition zone by 40-60%, indicating that *Bacillus natto*... B. natto The antimicrobial lipopeptides produced by HQ-1 are important antibacterial active ingredients.

[0052] Table 3 B. natto Antagonistic effects of HQ-1 and its fermentation broth supernatant against other aquatic pathogens

[0053] Example 4 Bacillus natto B. natto HQ-1's effect on toxin degradation 1. Test Methods Culture of Bacillus natto B. natto HQ-1 and commercial Bacillus natto were inoculated into a 250 mL shake flask containing 40 mL of fermentation medium at a 20% (v / v) inoculation rate, following the seed culture preparation process in Example 3. 1 mg / L of each of the following single substances—zearalenone, aflatoxin, ochratoxin, vomitoxin, and citrinin—was added to the shake flask. After fermentation at 37 °C and 150 rpm for 48 h, the residual toxin content in the fermentation broth was determined by enzyme-linked immunosorbent assay (ELISA). A sample without inoculation but with only culture medium and toxins was used as a control.

[0054] 2. Test Results like Picture 4 As shown, Bacillus natto B. nattoHQ-1 exhibited excellent degradation capabilities against five common feed toxins (zearalenone, vomitoxin, citric acid, aflatoxin, and ochratoxin). After 48 hours of fermentation, the degradation rates of zearalenone, vomitoxin, citric acid, aflatoxin, and ochratoxin A by strain HQ-1 were 92%, 96%, 98%, 86%, and 80%, respectively. The degradation rates for vomitoxin and citric acid were the fastest and most significant. Further comparative experiments with commercially available Bacillus natto inoculants showed that HQ-1 improved the degradation rates of zearalenone, vomitoxin, citric acid, and aflatoxin by 29.6%, 14.3%, 42.0%, and 4.8%, respectively, compared to commercial strains; its degradation capacity for ochratoxin was comparable to that of commercial strains. These results indicate that strain HQ-1 possesses significant multi-toxin degradation capabilities, especially in removing common mycotoxins found in stored feed.

[0055] Example 5 Bacillus natto B. natto The purification effect of HQ-1 on wastewater from crab farming 1. Test Methods Preparation of Bacillus natto B. natto The fermentation broth of HQ-1 was prepared according to the process described in Example 3. A 10% (v / v) inoculum was added to each 250 mL Erlenmeyer flask containing 40 mL of wastewater from crab farming (initial parameters: ammonia nitrogen 12 mg / L, nitrite 0.2 mg / L, total phosphorus 0.5 mg / L, COD 80 mg / L, total suspended solids (TSS) 180 mg / L). The flasks were then incubated at 25 °C and 120 rpm with shaking for 24 h. Four control groups were established: a blank control (no inoculation, only an equal volume of basal culture medium was added), positive control 1 (20 mg / L commercial flocculant aluminum sulfate was added), positive control 2 (an equal volume of Bacillus natto fermentation broth was added), and positive control 3 (an equal volume of Bacillus natto fermentation broth was added). After the culture was completed, the samples from each treatment group were allowed to stand for 30 minutes. The supernatant from 2 cm above the liquid surface was taken and the TSS sedimentation rate was determined by gravimetric method. The concentrations of ammonia nitrogen, nitrite, total phosphorus and COD were detected using the corresponding standard test kits.

[0056] 2. Test Results Test results showed that Bacillus natto B. natto HQ-1 exhibits excellent water purification performance. Under simulated treatment conditions, the 24-hour removal rates for ammonia nitrogen, nitrite, total phosphorus, and COD reached 86.1%, 62.4%, 92.1%, and 75.3%, respectively. Compared with the fermentation broth of two commercial Bacillus natto strains used in the control group, B. nattoHQ-1 showed more significant degradation effects on key indicators such as ammonia nitrogen, total phosphorus, and COD. Furthermore, HQ-1 exhibited significant flocculation and sedimentation effects on suspended solids (TSS) in water, with removal efficiency approaching that of the 20 mg / L commercial flocculant aluminum sulfate treatment group, and significantly superior to two commercial natto bacteria preparation groups. This is presumably related to the polyglutamic acid produced by HQ-1 metabolism and its adsorption by the bacteria. These results fully demonstrate… B. natto HQ-1 has the potential to be applied in the purification of wastewater from crab farming. It can effectively reduce nitrogen and phosphorus pollution, alleviate the risk of eutrophication, and improve the quality of the aquaculture environment, making it valuable for promotion and application in the aquaculture field.

[0057] Table 4 B. natto The purification effect of HQ-1 on wastewater from crab farming

[0058] Example 6: Optimization of Raw Material Formulation for the Preparation of Functional Feeds from Bacillus natto 1. Test Methods (1) Formula optimization This embodiment optimizes the raw material composition of functional feed through multi-stage gradient formulation design, thereby enhancing the efficacy of Bacillus natto. B. natto The bioactivity and water purification performance of HQ-1 fermented feed were studied. In the formulation design, the protein content, palatability, and cost of the raw materials were considered. The five components—soybeans (30%~60%), corn (10%~30%), soybean meal (10%~15%), fishmeal protein (10%~15%), and wheat bran (5%~15%)—were optimized. The optimal formulation and fermentation process conditions were screened using the viable cell count of the fermentation product, the pH value at the fermentation endpoint, the diameter of the inhibition zone against Vibrio harveyi, the soluble sugar content, and the stringiness as evaluation indicators.

[0059] Table 5 Raw material formulation of functional feed

[0060] The specific implementation steps are as follows: Mix 1.5 kg of feed substrate with 0.5 kg of Bacillus natto in different proportions. B. natto The HQ-1 fermentation broth was mixed at a solid-liquid mass ratio of 3:1. Before mixing, the mixture was thoroughly pulverized using a homogenizer and filtered through a 40-mesh sieve. It was then sterilized by boiling in a kettle for 1 hour. After cooling to below 40 °C, it was mixed with the HQ-1 fermentation broth and placed under aerated fermentation conditions (1 vvm) at 37 °C. Stirring was performed every 4 hours (150 rpm for 15 minutes) for 3 days. Three parallel samples were set up for each group. Samples were taken after fermentation (on the 3rd day) to test the above-mentioned indicators.

[0061] (2) Determination of other components To further verify Bacillus natto B. natto The advantages of HQ-1 fermented feed were investigated. Control group 1 (Shuiguxin Aquatic Bacillus natto inoculant) and control group 2 (Jiubang Aquatic Bacillus natto inoculant) were set up. Both groups fermented the same formula substrate under the same fermentation conditions. Protein concentration was determined using a protein assay kit, toxin content was determined by enzyme-linked immunosorbent assay, starch content was determined by optical rotation, and total sugar was determined by phenol-sulfuric acid method.

[0062] 2. Test Results (1) The results of the formulation optimization are shown in Table 6. Among them, formulation D (mass ratio: soybean 50%, corn 15%, soybean meal powder 12.5%, fishmeal protein 12.5%, wheat bran 10%) showed the best performance in terms of viable bacteria count, inhibition zone diameter, stringiness, and endpoint pH; formulation E showed the highest performance in terms of soluble sugar content. A lower pH value helps to inhibit the proliferation of harmful bacteria and prolong the shelf life of functional feed. Considering both functionality and economy, formulation D was determined to be the optimal formulation.

[0063] Table 6. Fermentation of functional feeds with different formulation ratios

[0064] Note: ++ indicates that the stringy phenomenon is more obvious, and + indicates that there is a stringy phenomenon.

[0065] (2) Before fermentation, the substrate of formula D was tested and found to have a crude protein content of 374 g / kg dry feed, a soluble sugar concentration of 36.2 g / kg dry feed, a free amino acid content of 3.8 g / kg dry feed, and a starch content of 235 g / kg dry feed. The comparison results of other components of the functional feeds after fermentation with different Bacillus natto strains are shown in Table 7. Table 7 shows that Bacillus natto... B. natto The HQ-1 functional feed group outperformed the two commercially available microbial feed groups in all evaluation indicators, particularly in terms of viable cell count, soluble sugar content, inhibition zone diameter, and free amino acid content. Among these, the HQ-1 group showed significant differences compared to the two commercially available microbial feed groups. B. nattoAfter HQ-1 fermentation, the crude protein content in the functional feed decreased to 281 g / kg dry feed, a decrease of approximately 24.9%; at the same time, the starch content decreased from 235 g / kg dry feed to approximately 124 g / kg dry feed, with a degradation rate of approximately 47.2%; the soluble sugar content significantly increased from 36.2 g / kg dry feed to 82.5 g / kg dry feed, an increase of approximately 129.2%; and the free amino acid content increased from 3.8 g / kg to 17.2 g / kg dry feed, an increase of approximately 3.5 times. The above changes indicate that Bacillus natto HQ-1 can efficiently degrade starch and macromolecular proteins in feed during fermentation, converting them into small molecule nutrients such as soluble sugars and free amino acids. This significantly improves the palatability and nutrient utilization efficiency of the feed, which is more conducive to the feeding, digestion, absorption, and healthy growth of mitten crabs. Furthermore, the levels of zearalenone, aflatoxin, ochratoxin, vomitoxin, and citric acid in the feed were all reduced to below 8 ppb. Specifically, the levels of zearalenone, aflatoxin, vomitoxin, and ochratoxin were significantly lower than the relevant limits stipulated in the feed hygiene standards GB 13078-2017, GB 13078.2-2017, and GB 13078.3-2017. These results demonstrate that the screened Bacillus natto strain... B. natto The HQ-1 strain possesses excellent fermentation characteristics and potential for developing feed functions.

[0066] Table 7 Functional feeds fermented with different natto bacteria

[0067] Note: ++ indicates that the stringy phenomenon is more obvious, and + indicates that there is a stringy phenomenon.

[0068] Example 7: Optimization of inoculum size for preparing functional feed using Bacillus natto 1. Test Methods This embodiment aims to optimize Bacillus natto. B. natto The inoculum ratio of HQ-1 in the fermentation of functional feed was determined to enhance the bioactivity and water purification performance of the resulting fermented feed. Following the optimal preparation process of the fermentation broth in Example 3, fermented feeds were prepared with different solid-liquid ratios (i.e., the mass ratio of dry feed substrate to fermentation broth), setting five experimental groups with mass ratios of 10:1, 8:1, 5:1, 3:1, and 2:1. Each group of experiments was performed in triplicate.

[0069] The feed fermentation substrate was formulated by weight as follows: soybeans 50%, corn 15%, soybean meal powder 12.5%, fishmeal protein 12.5%, and wheat bran 10%. All raw materials were thoroughly homogenized using a homogenizer, passed through a 40-mesh sieve, and sterilized in a boiling kettle for 1 hour. After cooling to below 40°C, the mixture was added to the Bacillus natto fermentation broth according to the set solid-liquid ratio, with a total system weight controlled at 2 kg. Fermentation was carried out at 37°C. During fermentation, the mixture was stirred every 4 hours at 150 rpm for 15 minutes, with 1 vvm aeration to maintain good aerobic conditions. The fermentation cycle was 3 days, with samples taken on the 3rd day for testing. The impact of the inoculation ratio on the quality of the functional feed was comprehensively evaluated based on the pH value, viable cell count (CFU / g), inhibition zone diameter (for Vibrio harveyi), soluble sugar content, and the stringiness of the fermentation products.

[0070] 2. Test Results The experimental results are shown in Table 8. The fermented feed with a solid-liquid ratio of 5:1 showed the best overall performance in terms of viable cell count, pH value, inhibition zone diameter, and stringiness. Therefore, a dry-based feed substrate to fermentation broth mass ratio (w / w) of 5:1 was determined as the optimal ratio for Bacillus natto. B. natto The optimal inoculation ratio of HQ-1 in the preparation of functional feeds can significantly improve the biological quality of functional feeds and their application effect in aquaculture.

[0071] Table 8. Fermentation of functional feeds at different inoculation ratios

[0072] Note: Inoculation ratio indicates the mass ratio of dry feed substrate to fermentation liquid (w / w); ++ indicates that the stringing phenomenon is more obvious, + indicates that the stringing phenomenon exists, and – indicates that the stringing phenomenon does not exist.

[0073] Example 8: Optimization of fermentation temperature for preparing functional feed using Bacillus natto 1. Test Methods This embodiment aims to optimize Bacillus natto. B. natto HQ-1 is used as a fermentation temperature parameter in the preparation of functional feeds to improve the biological activity of fermented feeds and their water purification effect.

[0074] Following the fermentation broth preparation process described in Example 7, the same feed fermentation substrate formula and optimal inoculation ratio (dry feed substrate to fermentation broth mass ratio of 5:1) were used. The raw materials were homogenized, sieved through a 40-mesh sieve, sterilized by boiling in water for 1 hour, and cooled to below 40°C for later use. 1.5 kg of dry feed substrate was mixed with 0.3 kg of HQ-1 fermentation broth to form a fermentation system with a solid-liquid ratio of 5:1. Five different fermentation temperature gradients were set: 30°C, 33°C, 35°C, 37°C, and 40°C. Static fermentation was carried out for 3 days under the corresponding conditions, with stirring every 4 hours (150 rpm for 15 minutes) and continuous aeration at 1 vvm to ensure aerobic conditions. Each group had three parallel replicates. Samples were taken on day 3 of fermentation, and the following parameters were measured as evaluation indicators: pH value of fermentation product, viable cell count (in CFU / g), diameter of inhibition zone (for Vibrio harveyi), soluble sugar content, and stringiness of fermentation product.

[0075] 2. Test Results The results are shown in Table 9. The results indicate that under 37 °C conditions, the fermented feed exhibited better performance across all indicators. The viable cell count and inhibition zone diameter were significantly higher than in other temperature groups, and the soluble sugar content remained at a high level. The fermentation product showed obvious stringiness and good apparent viscosity, and the pH remained stable at around 4.6, which is beneficial for the survival and functional maintenance of probiotics. While the viable cell count and inhibition zone diameter were slightly higher at 40 °C than at 37 °C, the soluble sugar content was significantly reduced. Considering energy consumption costs, 37 °C was more economical. Taking into account fermentation activity, product quality, and energy consumption costs, 37 °C was determined to be the optimal temperature for fermentation. B. natto The optimal fermentation temperature for preparing functional feed using HQ-1.

[0076] Table 9. Fermentation of functional feeds under different temperature conditions

[0077] Note: Inoculation ratio indicates the mass ratio (w / w) of dry feed substrate to fermentation broth. ++ indicates more pronounced stringing, + indicates some stringing, and – indicates no stringing.

[0078] Example 9: Optimization of fermentation time for preparing functional feed using Bacillus natto 1. Test Methods This embodiment aims to optimize Bacillus natto. B. natto HQ-1 reduces the fermentation time during the preparation of functional feeds to enhance the bioactivity and water purification performance of the resulting fermented feeds. (Bacillus natto) Bacillus nattoThe preparation process of HQ-1 fermentation broth was carried out under the optimal conditions described in Example 8. The specific steps are as follows: 1.5 kg of feed fermentation substrate and 0.3 kg of Bacillus natto fermentation broth were mixed at a solid-liquid ratio of 5:1. The raw material ratio (mass ratio) of the feed fermentation substrate was: 50% soybeans, 15% corn, 12.5% ​​soybean meal powder, 12.5% ​​fishmeal protein, and 10% wheat bran. After homogenization and sieving through a 40-mesh sieve, the raw materials were sterilized at high temperature in a boiling kettle for 1 hour. After sterilization, the mixture was naturally cooled to below 40°C, and then the Bacillus natto fermentation broth was added for mixed fermentation. The fermentation process was carried out at 37°C, with stirring every 4 hours at a stirring rate of 150 rpm for 15 minutes, and continuous oxygen supply at a ventilation rate of 1 vvm to maintain good aerobic fermentation conditions. The total fermentation time was 3 days. Samples were taken for analysis on days 1, 2, and 3 of fermentation, and three replicates were set up for each of the three experiments. The effects of different fermentation times on feed quality were comprehensively evaluated using the pH value, viable cell count (CFU / g), inhibition zone diameter against Vibrio harveyi, soluble sugar content, and stringiness of the fermentation products as evaluation indicators.

[0079] 2. Test Results The test results are shown in Table 10. The results show that the fermented feed obtained on the second day of fermentation had the best overall indicators: the viable count reached 2.9 × 10⁻⁶. 9 The CFU / g feed concentration was reduced to approximately 4.6, the inhibition zone diameter against *Bacillus natto* reached 15.9 mm, the free amino acid content reached 16.2 g / kg dry feed, and the total soluble sugar content was 92.4 g / kg dry feed. Furthermore, the product exhibited good stringiness, indicating a high content of viscous components such as polyglutamic acid in its metabolites, demonstrating excellent adsorption and sedimentation capabilities. In conclusion, *Bacillus natto*... Bacillus natto HQ-1 achieves optimal feed bioactivity and functional indicators after fermentation at 37°C for 2 days, which is superior to treatments fermented for 1 or 3 days. Therefore, a fermentation time of 2 days is the ideal fermentation time parameter for this process.

[0080] Table 10. Performance indicators of functional feeds at different fermentation times.

[0081] Note: ++ indicates that the stringing phenomenon is more obvious, + indicates that there is stringing phenomenon, and – indicates that there is no stringing phenomenon.

[0082] Example 10: Implementation of functional feed prepared from Bacillus natto and fed to Chinese mitten crabs. 1. Test Methods In this embodiment, Bacillus natto is used. B. nattoA feeding experiment was conducted on Chinese mitten crabs using functional feed prepared with HQ-1 to evaluate its effects on crab growth, health, and meat protein content. The specific experimental procedure is as follows: Nine identical rearing ponds were prepared, and 80 male mitten crabs of uniform size (50-60 g) and health status were randomly introduced into each pond. Three ponds were randomly selected and fed functional feed (experimental group) twice daily, morning and evening. Control group 1 was fed feed substrate fermented with Shuiguxin bacteria agent, and control group 2 was fed feed substrate fermented with Jiubang bacteria agent. Other conditions, such as pH, temperature, and dissolved oxygen, were kept consistent with the experimental group. During the experiment, growth performance indicators of mitten crabs from the growth stage to adult stage were recorded in each pond, including: number of adult crabs, weight, crab meat protein content, crab roe percentage, and proportion of diseased individuals; feed intake of mitten crabs at the adult stage was monitored regularly; 10 mitten crabs were randomly harvested periodically, dissected, and their crab meat protein content was measured; water quality in the rearing ponds was tested every two weeks, including ammonia nitrogen, nitrite, total phosphorus, COD, and suspended solids.

[0083] 2. Experimental Results The results in Table 11 show that using Bacillus natto... B. natto The functional feed prepared with HQ-1 for raising mitten crabs showed significant improvements compared to two other aquatic Bacillus natto inoculants: a 18%–30% increase in adult crab numbers, a 7%–18% increase in average weight, a 28%–30% increase in feed intake, a 20%–30% increase in crab meat protein content, a more than 20% increase in the proportion of crab roe, and a 50%–65% reduction in disease incidence. Furthermore, it significantly improved water quality, reducing ammonia nitrogen by approximately 25%–35%, COD by approximately 40%–45%, total phosphorus by approximately 70%–80%, and suspended solids by approximately 30%. These results demonstrate that the strain of this invention has excellent effects in inhibiting aquatic pathogens, removing feed-source toxins, and purifying water, significantly improving the growth performance of mitten crabs and the quality of the aquaculture environment.

[0084] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content of the present invention should be considered within the scope of protection of the present invention.

[0085] Table 11 Statistical data on growth indicators of Chinese mitten crabs after being fed functional feed

Claims

1. A strain of Bacillus natto, characterized in that, The Bacillus natto is classified and named Bacillus natto. Bacillus natto HQ-1, the Bacillus natto described therein, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M 20251811 and deposit date of August 11, 2025.

2. The application of Bacillus natto as described in claim 1 in the preparation of functional feed or aquaculture microbial agents for aquaculture.

3. The application according to claim 2, characterized in that, The aquaculture species mentioned is the Chinese mitten crab.

4. An aquaculture microbial agent, characterized in that, Contains the Bacillus natto as described in claim 1.

5. The aquaculture microbial agent according to claim 4, characterized in that, The aquaculture microbial agent is a liquid fermentation agent, which is prepared by inoculating the Bacillus natto described in claim 1 into a fermentation medium and culturing it at 24-40 °C for 12-24 h. The viable count in the liquid fermentation agent is not less than 1.5 × 10⁻⁶. 8 Fermentation was stopped when the concentration of CFU / mL was reached, and the liquid fermentation agent was obtained. The fermentation medium was formulated as follows: molasses 5-10 g / L, crude glycerol 10-15 g / L, peptone 5-15 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 1-5 g / L, water as solvent, and pH 7.0-7.

5.

6. A functional feed for aquaculture, characterized in that, The functional feed for aquaculture is obtained by fermentation using the Bacillus natto strain described in claim 1.

7. The method for preparing the functional feed for aquaculture according to claim 6, characterized in that, The functional feed for aquaculture is obtained by fermenting the Bacillus natto described in claim 1. Its fermentation substrate consists of soybeans, corn, soybean meal powder, fishmeal protein, and wheat bran, and is composed of the following by weight: Soybeans 50-55, corn 10-20, soybean meal 10-15, fish meal protein 10-15, wheat bran 10-15; The Bacillus natto is the aquaculture bacterial agent described in claim 5 or has a live bacteria count of not less than 1.5 × 10⁻⁶. 8 A solid inoculum of CFU / g is mixed with the fermentation substrate, wherein the viable count of the liquid fermentation inoculum is not less than 1.5 × 10⁻⁶. 8 CFU / mL, wherein the mass ratio of the fermentation substrate to the liquid fermentation agent is (2~10):

1.

8. The method for preparing functional feed for aquaculture according to claim 7, characterized in that, The fermentation conditions are 30-40 °C for 1-3 days, with the material being turned over at 4-6 hour intervals.

9. The application of the functional feed for aquaculture prepared by the method of claim 6 or any one of claims 7-8 in the farming of crabs, shrimps, and fish, wherein the crab is a Chinese mitten crab.

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