Chitinase-producing bacillus albidus a01 and application thereof in fermentation of solid waste of shrimps and crabs

Abstract

This invention discloses a chitinase-producing Bacillus albus A01 and its application in the fermentation of shrimp and crab solid waste. The chitinase-producing Bacillus albus is named... Bacillus albus A01, with accession number CCTCC: M 20252571; the 16S rDNA gene sequence of this chitinase-producing Bacillus albus strain A01 is shown in SEQ ID NO.1. The fermentation agent of this strain can effectively degrade the chitin content in shrimp and crab solid waste, and improve the nutritional value of shrimp and crab fermented powder through solid-state fermentation. This invention solves the problem of unsatisfactory fermentation effects of existing feed fermentation strains, and has great application potential for improving the feed nutritional quality of shrimp and crab fermented powder and developing feed resources.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a chitinase-producing Bacillus albus A01 and its application in the fermentation of shrimp and crab solid waste. Background Technology

[0002] With the rapid development of fisheries, the scale of shrimp and crab farming and processing has been continuously expanding. In recent years, the total annual output of shrimp and crabs has reached 10 million tons. After removing the edible parts, shrimp and crabs generate a large amount of solid waste, including heads, shells, and tails. This solid waste is unavoidable in the production and consumption process. However, this waste has enormous potential for environmental protection and resource utilization. The solid waste in shrimp and crabs contains approximately 50% protein, 20% chitin, and valuable components such as the natural antioxidant astaxanthin. These components are tightly bound together in the solid waste and cannot be effectively digested and absorbed by animals. Therefore, how to comprehensively utilize the solid waste in shrimp and crabs is an urgent problem to be solved. At the same time, how to degrade chitin, which is difficult for animals to digest and absorb, into high-value chitin oligosaccharides is also one of the current research hotspots.

[0003] Currently, the main methods for treating solid waste from shrimp and crabs are disposal or simple landfilling, which not only causes serious environmental pollution but also wastes valuable resources. Existing technologies for the resource utilization of solid waste mainly include physical, chemical, and biological methods, each with the following characteristics: 1. Physical methods for treating solid waste can lead to complex processes.

[0004] 2. Chemical methods, which use strong acids and alkalis to treat solid waste, can extract chitin, but they suffer from problems such as harsh reaction conditions, high energy consumption, large amounts of acid and alkali wastewater, low product purity, and impaired biological activity, which do not conform to the trend of green and environmentally friendly development.

[0005] 3. The biological method mainly utilizes chitinase produced by chitinase-producing strains to degrade chitin in solid waste, and at the same time utilizes proteases produced by the metabolism of the strains to degrade proteins. It has the advantages of mild reaction conditions, environmental friendliness, high product activity, and high resource utilization rate, making it an ideal direction for the resource utilization of solid waste.

[0006] Therefore, solid waste can improve its feed value, increase nutrient utilization, and provide a better solution for solid waste treatment through microbial solid-state fermentation. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a chitinase-producing Bacillus albus A01 and its application in the fermentation of shrimp and crab solid waste. This invention screened a chitinase-producing Bacillus albus A01 from flowerpot soil, which exhibits a significant degradation effect on chitin in shrimp and crab solid waste.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a chitinase-producing Bacillus albus A01, wherein the chitinase-producing Bacillus albus is named Bacillus albus A01, with accession number CCTCC: M 20252571.

[0009] Furthermore, the 16S rDNA sequence of the chitinase-producing Bacillus albus A01 is shown in SEQ ID NO: 1.

[0010] The present invention also provides an application of the chitinase-producing Bacillus albus A01 in reducing the chitin content and improving the nutritional value of shrimp and crab solid waste.

[0011] The present invention also provides a chitinase-producing Bacillus albus fermentation agent, wherein the fermentation agent comprises the chitinase-producing Bacillus albus A01 at a concentration of 3.85 × 10⁻⁶. 7 ~10 8 CFU / mL.

[0012] The present invention also provides a method for preparing the aforementioned fermentation agent, comprising the following steps: (1) Activate the chitinase-producing Bacillus albus A01, and then inoculate the activated strain into beef extract peptone liquid culture medium and shake to culture, which is the first-stage seed culture; (2) Inoculate the primary seed liquid into the liquid fermentation medium, shake and culture to obtain the secondary seed liquid, which is the chitinase-producing Bacillus albus fermentation agent.

[0013] Furthermore, in step (1), the culture conditions are: 30℃ shaking culture for 20-24 h; In step (2), the inoculation amount of the primary seed liquid is 4-6% of the weight of the liquid fermentation medium, and the culture conditions are 30℃ shaking culture for 20-24 h.

[0014] This invention also provides an application of the above-mentioned chitinase-producing Bacillus albus fermentation agent in the preparation of shrimp and crab fermented powder with low chitin content and high nutritional value using shrimp and crab solid waste as raw materials.

[0015] This invention also provides a method for preparing shrimp and crab leavening powder with low chitin content and high nutritional value, comprising the following steps: (1) Crush, sieve, and autoclave the shrimp and crab solid waste to obtain solid fermentation substrate; set aside for later use; (2) Weigh the solid fermentation substrate and water at a material-to-liquid ratio of 1:0.6~1.0, mix well, add the above-mentioned chitinase-producing Bacillus albus fermentation agent, mix well, and obtain a solid fermentation initial liquid with a pH of 3.0~7.0; carry out solid fermentation for 2~5 days at a temperature of 25~30℃ and under shaking conditions to obtain shrimp and crab fermentation powder, wherein the inoculation amount of fermentation agent is 6~10ml for every 100g of solid fermentation substrate.

[0016] In practice, shrimp and crab solid waste consists of any one or more of the head, shell, and tail.

[0017] Furthermore, the solid-state fermentation substrate to water ratio is 1:0.8. The optimal inoculum size for the fermentation agent is 8 ml per 100 g of solid-state fermentation substrate; the initial pH of the solid-state fermentation broth is 5.0. The solid-state fermentation temperature was 30℃, and the culture time was 3 days.

[0018] The present invention also provides an application of the shrimp and crab fermented powder prepared by the method described above in the preparation of yellow catfish feed as a substitute for fish meal.

[0019] The beneficial effects of this invention are: 1. This invention screened and obtained a functional strain with stable chitinase production ability, namely, chitinase-producing Bacillus albus A01. This strain can continuously secrete chitinase with high specific activity during the solid-state fermentation of shrimp and crab solid waste. By measuring the chitinase activity in the supernatant after solid-state fermentation, its specific activity can reach 38.23 U / mg. The results show that the strain has good chitin hydrolysis potential, providing a reliable enzymatic basis for subsequent substrate degradation and nutrient conversion.

[0020] 2. Using the chitinase-producing Bacillus albus A01 obtained by screening in this invention to ferment shrimp and crab solid waste, the chitin in the solid waste is converted into other substances, while the nutritional value of the solid waste is improved. During the fermentation process, the crude protein is increased, and some large molecular proteins in the solid waste are converted into low molecular proteins and peptides. This shows that the method of this invention can effectively improve the structural composition and potential digestibility and utilization of solid waste raw materials.

[0021] 3. The method of this invention for fermenting solid waste features mild processing conditions, a simple process, and low equipment requirements. It eliminates the need for strong acids and alkalis, avoiding chemical residues and environmental pollution, thus demonstrating excellent safety and environmental friendliness. The fermented solid waste raw materials are more suitable for use as aquatic feed, improving the efficiency of nutrient digestion and absorption by aquatic animals, reducing feed production costs, and playing a significant practical role in promoting the resource utilization of aquaculture by-products, alleviating the shortage of high-quality protein resources, and achieving green and sustainable aquaculture. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of Bacillus albus A01, which produces chitinase, as described in Example 1.

[0023] Figure 2 This is a phylogenetic tree diagram of Bacillus albus A01, which produces chitinase, as described in Example 1.

[0024] Figure 3 N-acetylglucosamine (GlcNAc) OD 540 Standard curve of values.

[0025] Figure 4 Figure showing the effect of different solid-liquid ratios on the solid-state fermentation of chitinase-producing Bacillus albus A01. Different letters (a, b, c) indicate significant differences between treatment groups (P<0.05).

[0026] Figure 5 The figure shows the effect of different inoculum amounts on the solid-state fermentation effect of chitinase-producing Bacillus albus A01.

[0027] Different letters (a~d) indicate significant differences between treatment groups (P<0.05).

[0028] Figure 6 Figure 1 shows the effect of different initial pH values ​​of solid-state fermentation broth on the solid-state fermentation effect of chitinase-producing Bacillus albus A01. Different letters (a~e) indicate significant differences between treatment groups (P<0.05).

[0029] Figure 7 Figure showing the effect of different solid-state fermentation temperatures on the solid-state fermentation effect of chitinase-producing Bacillus albus A01. Different letters (a~d) indicate significant differences between treatment groups (P<0.05).

[0030] Figure 8 Figure showing the effect of different solid-state fermentation culture times on the solid-state fermentation effect of chitinase-producing Bacillus albus A01. Different letters (a~d) indicate significant differences between treatment groups (P<0.05).

[0031] Figure 9 This is a thin-layer chromatogram of shrimp and crab baking powder.

[0032] Figure 10 SDS-PAGE analysis of protein molecular weight changes in shrimp and crab fermentation powder. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0034] The culture medium used and prepared in this embodiment Chitin screening medium: 20 mL colloidal chitin, 0.03 g KH2PO4, 0.07 g K2HPO4, 0.001 g FeSO4, 0.05 g MgSO4, 0.01 g NH4Cl, 0.01 g NaCl, 2.0 g agar, add distilled water to a final volume of 100 mL, pH 7.0±0.2, sterilize at 121℃ for 20 min; used for screening target strains.

[0035] Liquid fermentation medium: 2.0 g fructose, 0.7 g KH2PO4, 0.3 g K2HPO4, 0.6 g MgSO4, 0.5 g yeast extract, 4.0 g beef extract, 4.0 g peptone, add distilled water to a final volume of 100 mL, pH 7.0±0.2, sterilize at 121℃ for 20 min; used for shake-flask fermentation of the inoculum.

[0036] Beef extract peptone liquid culture medium: 0.3 g beef extract, 1.0 g peptone, 0.5 g NaCl, add distilled water to a final volume of 100 mL, pH 7.0±0.2, sterilize at 121℃ for 20 min; used for seed culture.

[0037] Beef extract peptone solid medium: 0.3 g beef extract, 1.0 g peptone, 0.5 g NaCl, add distilled water to a final volume of 100 mL, pH 7.0±0.2, sterilize at 121℃ for 20 min; used for seed culture.

[0038] Solid fermentation medium: 15.0 g sterilized shrimp shells, 10.8 mL distilled water, 1.2 mL bacterial strain; fermented shrimp shells were then used for chitin determination.

[0039] Example 1 Isolation and identification of chitinase-producing Bacillus albus A01 1. Isolation of chitinase-producing Bacillus albus A01 (1) Source: The fungal strains were collected from the soil in flower pots at the Flower Practice Teaching Base of Huazhong Agricultural University.

[0040] (2) The method for isolating chitinase-producing Bacillus albus includes the following steps: Step S1: Collect potted soil from the flower practice teaching base of Huazhong Agricultural University, grind and crush it, mix it evenly, put it into a sterilized bag, and refrigerate it for later use; Step S2: Weigh 1.0 g of the ground and pulverized mixture obtained in step S1 and place it in a 15 mL centrifuge tube containing 9 mL of sterile water, and shake at 180 r / min for 180 min. Step S3: Serially dilute the bacterial culture medium obtained in step S2 to 10⁻⁶. -2 10 -3 10 -4 Each gradient was multiplied by 30 μL and spread onto chitin selection medium, and incubated at 30°C inverted for 3 days. Step S4: Based on the size of the clear zone of different colonies on the plate, select single colonies with strong chitinase clear zone activity and streak them on beef extract peptone solid medium for preservation.

[0041] 2. Identification of chitinase-producing Bacillus albus A01 (1) Identification of colony morphology and physiological and biochemical characteristics The colony morphology and physiological and biochemical characteristics of strain A01 isolated in Example 1 were observed.

[0042] like Figure 1 As shown: the strain is round, smooth, and has neat edges.

[0043] (2) Molecular biological identification The strain A01 isolated in Example 1 was subjected to 16S rDNA molecular biological identification. Genomic DNA was extracted from single colony cultures and used as a template. PCR amplification was performed using the bacterial 16S universal primer 27F / 1492R. The product was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequence (as shown in SEQ ID NO.1) was compared with known sequences in the GenBank database using BLAST analysis, and a phylogenetic tree was constructed.

[0044] Based on the nucleotide and amino acid comparison results of strain A01, it can be seen that strain A01 is related to... Bacillus albus It has a high degree of matching, and its physiological characteristics are highly similar to those of Bacillus albus. Figure 2 It was named Bacillus albopictus, a chitinase-producing bacterium. Bacillus albusA01; deposited on November 17, 2025, with accession number CCTCC: M20252571; deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China, postal code 430072.

[0045] Example 2 The preparation method of chitinase-producing Bacillus albus fermentation agent 1 includes the following steps: Step S1: First, scrape 2-3 loops of the chitinase-producing Bacillus albus A01 preserved in beef extract peptone solid medium and inoculate it into beef extract peptone liquid medium. Incubate at 30℃ with shaking for 24 hours to obtain the first-stage seed culture.

[0046] Step S2: The primary seed culture is inoculated into the liquid fermentation medium at an inoculation volume of 6% of the liquid fermentation medium weight. The mixture is then cultured at 30℃ with shaking for 24 hours to obtain the secondary seed culture, which is the chitinase-producing Bacillus albus fermentation agent 1. The concentration of chitinase-producing Bacillus albus A01 in fermentation agent 1 is 3.85 × 10⁻⁶. 7 CFU / mL.

[0047] Example 3 The method for preparing shrimp and crab fermented powder with low chitin content and high nutritional value based on the above-mentioned fermentation agent 1 includes the following steps: (1) Crush the shrimp and crab solid waste (taking marine shrimp as an example) with a crusher, pass it through a 60-mesh sieve, and sterilize it at 121℃ for 20 minutes to obtain solid fermentation substrate; set aside for later use; (2) Weigh the solid fermentation substrate and water at a material-to-liquid ratio of 1:0.6~1.0, mix well, add the above-mentioned chitinase-producing Bacillus albus fermentation agent 1, mix well, and obtain a solid fermentation initial liquid with a pH of 3.0~7.0; carry out solid fermentation for 2~5 days at a temperature of 25~30℃ and under shaking conditions to obtain shrimp and crab fermentation powder, wherein the inoculation amount of fermentation agent is 6~10ml for every 100g of solid fermentation substrate.

[0048] Single-factor optimization of preparation conditions for shrimp and crab fermentation powder based on the above-mentioned shrimp and crab solid waste. 1. A method for calculating the specific activity of chitinase in shrimp and crab baking powder, including the following steps: Step S1: Add 5-10 times the volume of 50 mmol / L sodium acetate buffer (pH 5.5-6.0) to the shrimp and crab fermentation powder, mix well, and extract by shaking at 4℃ for 30-60 min; obtain the extract; centrifuge the extract at 10000 r / min at 4℃ for 10-15 min, and collect the fermentation supernatant as crude enzyme solution; Step S2: Take two test tubes and label them 1 (sample group) and 2 (control group) respectively (make three replicates according to the actual situation). Add 1.0 mL of fermentation supernatant to tube 1 and 1.0 mL of inactivated fermentation supernatant to tube 2 (control group). Add 0.5 mL of 2% colloidal chitin substrate and 0.5 mL of phosphate buffer (50 mmol / L) at pH 7.0. Shake well and react in a 37°C water bath for 30 min. Then, quickly boil to terminate the reaction and centrifuge at 12000 r / min for 5 min. Obtain the supernatant separately. Step S3: Replace with new test tubes (continue to be numbered 1 and 2 (control group)), add 1 mL of the supernatant corresponding to the number, add 1.5 mL of DNS solution, boil in a water bath for 5 min, and then cool with running water; obtain the test sample and control sample. Step S4: Establish a standard curve for reducing sugars using the 3,5-dinitrosalicylic acid (DNS) method. The specific steps are as follows: (1) Preparation of standard solutions Weigh N-acetylglucosamine (GlcNAc), prepare a standard stock solution with a mass concentration of 1 mg / mL, and dilute it stepwise with distilled water to prepare a series of standard solutions with different concentration gradients. (2) Color reaction Take 0.5 mL of each concentration standard solution, add 1.5 mL of DNS reagent, mix well, and heat in a boiling water bath for 5 min to allow a color reaction to occur. (3) Absorbance measurement After the reaction is complete, the mixture is rapidly cooled to room temperature. If necessary, distilled water is added to bring the volume to a final volume. The absorbance is then measured at a wavelength of 540 nm using a spectrophotometer. (4) Plotting the standard curve A standard curve was plotted with the concentration of the standard solution on the x-axis and absorbance on the y-axis. Figure 3 And perform linear regression to obtain the regression equation y=0.3871x+0.0262; Step S5: Measure the OD of the two samples. 540 The corresponding N-acetyl-D-glucosamine value was calculated based on the standard curve, and then the enzyme activity of the test samples was calculated separately.

[0049] An enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of N-acetyl-D-glucosamine from the substrate in 1 minute under the given reaction conditions.

[0050] Step S6: Take another test tube, add 10 μL of the fermentation supernatant obtained in step S1, add an equal volume of BCA working solution, and incubate at 37℃ for 30 min. After incubation, measure the OD. 562The value is used to calculate the protein concentration.

[0051] The specific activity of chitinase is calculated by dividing the measured enzyme activity by the corresponding protein concentration, in units of U / mg.

[0052] 2. Single-factor condition optimization 2.1 Effect of the feed-to-liquid ratio on fermentation efficiency With the preparation method of shrimp and crab fermentation powder remaining basically unchanged (temperature 30℃, pH 5.0, culture time 3d), the solid fermentation substrate to water ratio was set to 1:0.4, 1:0.6, 1:0.8, 1:1.0, and 1:1.2, respectively, to prepare different shrimp and crab fermentation powders. Then, the specific activity of chitinase was calculated according to the above method.

[0053] The results are as follows Figure 4 It can be seen that when the material-to-liquid ratio is set to 1:0.6, 1:0.8, and 1:1.0 respectively, the specific activity of chitinase in the extract of shrimp and crab fermentation powder is 26.07~30.12 U / mg, and the specific activity is the highest when the material-to-liquid ratio is 1:0.8, which is 30.12 U / mg. Therefore, the optimal material-to-liquid ratio for solid-state fermentation of substrate is 1:0.8.

[0054] 2.2 Effect of inoculum size on fermentation efficiency Under the condition that the method for preparing shrimp and crab fermentation powder remains basically unchanged (fermentation conditions of 30℃, pH 5.0, culture time of 3 days, and material-to-liquid ratio of 1:0.8), the inoculum amount of fermentation agent in 100g of solid fermentation substrate is set to 2ml, 4ml, 6ml, 8ml, and 10ml, respectively, to prepare different shrimp and crab fermentation powders. Then, the specific activity of chitinase is calculated according to the above method.

[0055] The results are as follows Figure 5 As shown: In every 100g of solid fermentation substrate, the inoculum amount of fermentation agent is 6ml~10ml, and the specific activity of chitinase in the extract of shrimp and crab fermentation powder is 34.08~38.23U / mg; and in every 100g of solid fermentation substrate, the specific activity is the highest when the inoculum amount of fermentation agent is 8ml, that is, 38.23U / mg. Therefore, the optimal inoculum amount of fermentation agent in every 100g of solid fermentation substrate is 8ml.

[0056] 2.3 Effect of initial solid-state fermentation broth on fermentation efficiency Under the condition that the preparation method of shrimp and crab fermentation powder remains basically unchanged (fermentation conditions of 30℃, pH 5.0, culture time of 3 days, and material-to-liquid ratio of 1:0.8, the optimal inoculum amount of fermentation agent is 8ml per 100g of solid fermentation substrate), the pH value of the initial solid fermentation liquid was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively, and different shrimp and crab fermentation powders were prepared. The specific activity of chitinase was calculated using the above-mentioned method.

[0057] The results are as follows Figure 6 It can be seen that when the pH of the initial solid-state fermentation liquid is 3.0~7.0, the specific activity of chitinase in the extract of shrimp and crab fermentation powder is 10.54~16.92U / mg, and the specific activity of chitinase is the highest at pH 5.0, which is 16.92U / mg. Therefore, the optimal pH of the initial solid-state fermentation liquid is 5.0.

[0058] 2.4 Effect of solid-state fermentation temperature on fermentation efficiency With the basic preparation method of shrimp and crab fermented powder remaining unchanged (fermentation conditions of material-to-liquid ratio of 1:0.8, optimal inoculum volume of fermentation agent of 8 ml per 100 g of shrimp and crab solid waste, initial pH of solid fermentation liquid of 5.0 and culture time of 3 days), solid fermentation temperature was set to 25℃, 30℃, 35℃, 37℃ and 40℃ respectively, and different shrimp and crab fermented powders were prepared. Then, the specific activity of chitinase was calculated according to the above method.

[0059] The results are as follows Figure 7 It can be seen that the specific activity gradually increases from 25℃ to 30℃, and reaches its highest value of 34.06U / mg at 30℃. After 30℃, the specific activity decreases. Therefore, the optimal fermentation temperature for solid-state fermentation of shrimp and crab solid waste is 30℃.

[0060] 2.5 Effect of solid-state fermentation culture time on fermentation efficiency With the preparation method of shrimp and crab fermented powder remaining basically unchanged (fermentation conditions of material-to-liquid ratio of 1:0.8, optimal inoculum volume of fermentation agent of 8 ml per 100 g solid fermentation substrate, initial solid fermentation liquid pH of 5.0 and fermentation temperature of 30℃), solid fermentation culture time was set to 1, 2, 3, 4, 5 and 6 days respectively; different shrimp and crab fermented powders were prepared respectively, and the specific activity of chitinase was calculated according to the above method.

[0061] The results are as follows Figure 8It can be seen that when the solid-state fermentation culture time of the substrate is 2 to 5 days, the specific activity of chitinase in the extract of shrimp and crab fermentation powder is 10.69 to 19.47 U / mg, and the specific activity is the highest when the culture time is 3 days, which is 19.47 U / mg; therefore, the optimal culture time for solid-state fermentation is 3 days.

[0062] Conclusion: Through single-factor optimization, the optimal conditions for the degradation of shrimp and crab solid waste were determined to be: a material-to-liquid ratio of 1:0.8, an optimal inoculum volume of 8 ml per 100 g of solid fermentation substrate, an initial pH of 5.0 in the solid fermentation broth, a fermentation temperature of 30℃, and a culture time of 3 days. These conditions significantly increased chitinase activity and resulted in the best degradation effect. This indicates that the above conditions are the preferred fermentation conditions.

[0063] Example 4 Comparison of chitinase-producing ability of Bacillus albus A01 with other Bacillus species Strain A01 was fermented with other Bacillus strains under the same culture conditions (material-to-liquid ratio of 1:0.8, optimal inoculum size of 8 ml per 100 g solid fermentation substrate, initial pH of 5.0, fermentation temperature of 30°C, and culture time of 3 days). The tested strains included Bacillus cereus (…). Bacillus cereus B1, Bacillus subtilis ( Paenibacillus albilobatus E1.

[0064] The results showed that different strains all possessed a certain ability to produce chitinase, but the enzyme activities differed significantly. The specific activity of chitinase in the extract of shrimp and crab fermentation powder treated by strain A01 reached 38.23 U / mg, significantly higher than that of Bacillus cereus B1 (9.65 U / mg) and Bacillus subtilis E1 (11.48 U / mg) (P < 0.05). Statistical analysis indicated that strain A01 exhibited a stronger chitinase synthesis capacity under the same culture conditions.

[0065] The above results indicate that the chitinase-producing Bacillus albus A01 obtained by screening in this invention has excellent enzyme production performance, and its chitinase activity is significantly higher than that of the tested control strain, indicating that this strain has good application potential in the biotransformation of chitin resources and the fermentation utilization of shrimp and crab solid waste.

[0066] Example 5 A method for preparing fermented shrimp and crab starter powder with low chitin content and high nutritional value includes the following steps: (1) The solid waste of marine shrimp is crushed by a crusher and passed through a 60-mesh sieve. It is then sterilized at 121°C for 20 minutes to obtain solid fermentation substrate; it is ready for use. (2) Add 30g of solid fermentation substrate and 24g of distilled water to the fermentation glass bottle, mix well, then add 2.4ml of the above-mentioned chitinase-producing Bacillus albus fermentation agent 1, mix well, and obtain a solid fermentation initial liquid with pH 5.0; carry out solid fermentation for 3 days at a temperature of 30℃ and under shaking conditions to obtain shrimp and crab fermentation powder 1, and store it in a desiccator.

[0067] Performance testing and analysis of the above-mentioned shrimp and crab baking powder 1 1. Thin-layer chromatographic analysis of shrimp and crab baking powder 1 a. Weigh 1.0 g of shrimp and crab fermentation powder 1, add 10 mL of sterile distilled water, and extract by shaking at 30 ℃ and 180 rpm for 2 h to fully dissolve the soluble chitin degradation products in the sample. Centrifuge the extract at 8000 rpm and 4 ℃ for 10 min, filter through a 0.22 μm microporous membrane, and use it as the sample for TLC analysis.

[0068] b. Use silica gel plates as the stationary phase in thin-layer chromatography. The developing solvent is prepared by mixing n-propanol, deionized water, and ammonia in a volume ratio of 70:30:1. Spotting volume is 2–5 μL per spot, with a spot spacing of 8–10 mm. Place the thin-layer plate in the developing tank and develop. When the solvent front is 6–10 cm from the bottom of the plate, remove it and allow it to air dry. Then, spray the plate with an aniline–diphenylamine–phosphoricacid reagent for color development and heat at 150 °C for 5–10 min. Use N-acetyl-D-glucosamine, chitobiose, and chitotriose as standard references.

[0069] The results are as follows Figure 9 It can be seen that after the shrimp and crab baking powder 1 is developed, it forms multiple clearly distinguishable color spots. The migration position of the main spots is basically consistent with the Rf range of N-acetyl-D-glucosamine and chitosan oligosaccharide standards, indicating that the main components of shrimp and crab baking powder 1 are oligosaccharides and monosaccharides produced by chitin hydrolysis.

[0070] 2. Nutritional index testing of shrimp and crab baking powder 1 a. Crude protein content was determined according to GB / T 6432-2018.

[0071] b. The crude ash content was determined according to GB / T 6438-2007.

[0072] c. Crude fat content was determined according to GB / T 6433-2025.

[0073] After fermentation by chitinase-producing Bacillus albus A01, the nutritional indicators of shrimp and crab solid waste are shown in Table 1. The crude protein and crude ash content of shrimp and crab fermentation powder 1 increased by 4.60% and 7.90%, respectively, while the crude fat content decreased by 16.24%. This indicates that the fermentation process can significantly improve the nutritional composition of shrimp and crab solid waste. At the same time, the microbial degradation of chitin structure promotes the release of bound protein, improves protein utilization, and effectively reduces fat content to enhance product stability and storage performance.

[0074] Table 1. Effects of chitinase-producing Bacillus albus A01 fermentation on nutrient composition in shrimp and crab solid waste. 3. SDS-PAGE analysis of protein molecular weight changes in shrimp and crab yeast powder 1 Weigh 0.5 g of shrimp and crab fermentation powder 1, add 2.0 mL of buffer (Tris-HCl (pH 8.0), 1% (w / v) SDS, DTT, PMSF) to extract soluble proteins, and then perform SDS-PAGE analysis. Electrophoresis was performed using a 12% polyacrylamide separating gel system, stained and developed with Coomassie Brilliant Blue. The distribution of protein bands in different samples was compared and analyzed using a standard protein molecular weight marker as a reference.

[0075] The results are as follows Figure 10 It can be seen that the protein band distribution in shrimp and crab fermentation powder 1 underwent significant changes. Compared with before fermentation, the protein bands that were originally concentrated in the medium and high molecular weight range were significantly weakened or partially disappeared, while multiple new bands appeared in the low molecular weight range and showed a continuous distribution characteristic. This indicates that some large molecular weight proteins were decomposed and transformed into smaller molecular weight protein or polypeptide components during fermentation. This demonstrates that the use of chitinase-producing Bacillus albus fermentation agent in this invention can effectively improve the molecular weight structure of shrimp and crab solid waste, which is beneficial to improving the bioavailability and application value of the raw materials.

[0076] In summary, this invention provides a chitinase-producing Bacillus albus strain A01 and its application in shrimp shell fermentation. The fermentation agent of strain A01 can increase the protein content in shrimp and crab fermented powder, reduce the chitin content in shrimp and crab solid waste, and hydrolyze it to produce oligosaccharides and monosaccharides, increasing the small molecule protein components. The nutritional value of the prepared shrimp and crab fermented powder is significantly improved. The prepared shrimp and crab fermented powder can partially replace fishmeal in yellow catfish feed, showing good potential for feed development. This invention solves the problem of unsatisfactory fermentation effects of existing feed fermentation strains and has significant application potential for improving the nutritional quality of shrimp and crab solid waste and developing feed resources.

[0077] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chitinase-producing Bacillus albopictus A01, characterized in that: The chitinase-producing Bacillus albus was named Bacillus albus A01, with accession number CCTCC: M 20252571.

2. The chitinase-producing Bacillus albus A01 according to claim 1, characterized in that: The 16S rDNA sequence of the chitinase-producing Bacillus albus A01 is shown in SEQ ID NO.

1.

3. The application of the chitinase-producing Bacillus albus A01 as described in claim 1 in reducing the chitin content and improving the nutritional value of shrimp and crab solid waste.

4. A chitinase-producing Bacillus albopictus A01 fermentation agent, characterized in that: The fermentation agent comprises Bacillus albus A01, a chitinase-producing bacterium as described in claim 1, at a concentration of 3.85 × 10⁻⁶. 7 ~10 8 CFU / mL.

5. A method for preparing the fermentation agent as described in claim 4, characterized in that: Includes the following steps: (1) Activate the chitinase-producing Bacillus albus A01, and then inoculate the activated strain into beef extract peptone liquid culture medium and shake to culture, which is the first-stage seed culture; (2) Inoculate the primary seed liquid into the liquid fermentation medium, shake and culture to obtain the secondary seed liquid, which is the chitinase-producing Bacillus albus fermentation agent.

6. The preparation method according to claim 5, characterized in that: In step (1), the culture conditions are 30℃ shaking culture for 20~24 h; In step (2), the inoculation amount of the primary seed liquid is 4-6% of the weight of the liquid fermentation medium, and the culture conditions are 30℃ shaking culture for 20-24 h.

7. The application of the chitinase-producing Bacillus albus fermentation agent as described in claim 4 in the preparation of shrimp and crab fermented powder with low chitin content and high nutritional value using shrimp and crab solid waste as raw material.

8. A method for preparing shrimp and crab leavening powder with low chitin content and high nutritional value, characterized in that: Includes the following steps: (1) Crush, sieve, and autoclave the shrimp and crab solid waste to obtain solid fermentation substrate; set aside for later use; (2) Weigh the solid fermentation substrate and water at a material-to-liquid ratio of 1:0.6~1.0, mix them well, and then add the chitinase-producing Bacillus albus fermentation agent prepared by the method described in claim 5. Mix well to obtain a solid fermentation initial liquid with a pH of 3.0~7.

0. Carry out solid fermentation for 2~5 days at a temperature of 25~30℃ and under shaking conditions to obtain shrimp and crab fermentation powder. The amount of fermentation agent inoculated for each 100g solid fermentation substrate is 6~10ml.

9. The method according to claim 8, characterized in that: The solid-state fermentation substrate to water ratio is 1:0.

8. The inoculum size of the fermentation agent is 8 ml per 100 g of solid fermentation substrate; the initial pH of the solid fermentation broth is 5.

0. The solid-state fermentation temperature was 30℃, and the culture time was 3 days.

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