Method for preparing insect protein peptide by using bacillus velezensis JAAS-22

Abstract

The invention discloses a method for preparing insect protein peptide by utilizing bacillus velezensis JAAS-22. The method comprises the following steps: (1) performing amplification culture on a strain of the bacillus velezensis JAAS-22 to obtain a bacillus velezensis JAAS-22 fermentation culture solution; (2) homogenizing insect larvae in a colloid mill to obtain insect pulp; (3) uniformly mixing the insect pulp with the bacillus velezensis JAAS-22 fermentation culture solution, and carrying out enzymolysis fermentation to obtain an insect pulp enzymolysis fermentation product; and (4) dialyzing the enzymatic hydrolysis fermentation product of the insect pulp to obtain the insect protein peptide with the molecular weight less than or equal to 30 kDa. According to the method, the bioactive peptide with stable and good antibacterial activity can be efficiently prepared.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a method for preparing insect protein peptides using Bacillus belye JAAS-22. Background Technology

[0002] Insect resource utilization technology is currently a research hotspot in the fields of organic waste treatment and sustainable protein development. In particular, housefly (Musca domestica) larvae (fly maggots) and black soldier fly (Hermetia illucens) larvae, due to their strong ecological transformation capabilities, can efficiently utilize kitchen waste, livestock manure, and agricultural byproducts for assimilation and growth, converting them into high-value-added insect protein. Insect protein is therefore considered a strategic new protein source for addressing global protein resource scarcity and replacing traditional fishmeal and soybean meal.

[0003] However, the current state of deep processing and utilization of fly larvae and black soldier fly protein mainly faces the following technical challenges and deficiencies: 1. The application level is rudimentary, and the product functions are limited. Currently, the commercial application of insect protein mainly focuses on primary processing. The mainstream technical route involves washing, drying (e.g., hot air drying, microwave drying), and mechanically pulverizing harvested insects to obtain whole insect powder or defatted insect powder, which is then directly added as a feed ingredient. Although studies have shown that this primary product can, to some extent, replace some fishmeal or soybean meal in the diet, improving the growth performance and gut health of farmed animals, it essentially remains at the stage of protein raw material substitution. Due to the lack of targeted intervention and separation of the active ingredients in the protein, the resulting product has a single function and cannot accurately meet the differentiated needs of high-end aquaculture, pet food, and functional food additives.

[0004] 2. Traditional processing techniques damage endogenous functional activity. Fly larvae and black soldier flies have developed unique innate immune systems through long-term evolution, resulting in a natural accumulation of various functional peptides, such as antimicrobial peptides (AMPs), anti-inflammatory peptides, and antioxidant peptides. However, existing conventional processing methods have significant drawbacks: Physical damage: Traditional high-temperature drying processes can easily destroy the spatial structure of active substances such as heat-sensitive antimicrobial peptides, causing them to lose their biological activity.

[0005] Inefficient extraction: Conventional crushing and simple water extraction methods are insufficient to destroy the cell walls of insects and the active ingredients that are firmly bound to proteins. As a result, a large number of high-value-added endogenous functional peptides are discarded with the insect residue and fail to enter the final product, causing a great waste of valuable bioactive resources.

[0006] 3. Lack of technical means for targeted enzymatic hydrolysis and controlled release. To achieve the transformation from "crude protein" to "functional bioactive peptides," modern biotechnology often employs enzymatic hydrolysis. However, existing enzymatic hydrolysis technologies for insect proteins are still immature: they mostly follow the enzymatic hydrolysis processes used for plant or fish proteins, failing to screen for specific enzyme systems tailored to the unique amino acid composition and peptide bond structure of fly larvae and black soldier fly proteins.

[0007] 4. Lack of industrial-scale integrated processes When preparing functional insect bioactive peptides (bioactive peptides derived from fly larvae and black soldier fly larvae), existing technologies lack a set of efficient, controllable, and industrializable preparation processes, making it difficult to transform these insect protein resources into protein peptide products with stable and clearly defined functions.

[0008] In summary, developing a process for the efficient preparation of stable and functionally defined bioactive peptides derived from fly larvae and black soldier fly larvae has become an urgent technical problem to be solved. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing insect protein peptides using Bacillus belyssus JAAS-22, which can efficiently prepare bioactive peptides with stable and good antibacterial activity.

[0010] The technical solution adopted by this invention to solve its technical problem is: A method for preparing insect protein peptides using Bacillus belyssus JAAS-22 includes the following steps: (1) The strain of Bacillus belyss JAAS-22 was expanded and cultured to obtain the fermentation broth of Bacillus belyss JAAS-22; (2) Homogenize the insect larvae in a glue mill to obtain insect pulp; (3) Mix the insect slurry with the Bacillus vesicle JAAS-22 fermentation culture medium and carry out enzymatic hydrolysis fermentation to obtain the insect slurry enzymatic hydrolysis fermentation product; (4) The insect larvae fermentation product was subjected to dialysis to obtain insect protein peptides with a molecular weight ≤ 30 kDa.

[0011] The inventors discovered a new Bacillus belyssus JAAS-22 through research. In-depth research revealed that the fermentation culture product of Bacillus belyssus JAAS-22 (i.e., its secreted protease) can be used as an enzyme catalyst to enzymatically hydrolyze specific insect larval proteins, thereby significantly increasing the content of antibacterial peptides (molecular weight ≤ 30 kDa) in the hydrolysate, thus significantly enhancing the antibacterial activity of the obtained functional peptides.

[0012] Bacillus belye JAAS-22 is classified as follows: Bacillus velezensisThe sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 10, 2023, with accession number GDMCC No: 63724. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0013] Step (1) is as follows: Single colonies of Bacillus belyss JAAS-22 were picked and inoculated into LB liquid medium and placed in a constant temperature incubator at a temperature of 32~37℃, a stirring speed of 100~200rpm, and a culture time of 20~36h to obtain Bacillus belyss seed culture. The Bacillus belysin seed culture was inoculated into a fermenter containing LB liquid medium at a volume ratio of 1% to 5%. The culture temperature was 32 to 40°C, the stirring speed was 100 to 400 rpm, the air ventilation rate was 0.5 to 2 vvm, and the culture time was 20 to 30 h to obtain the Bacillus belysin JAAS-22 fermentation culture.

[0014] In step (3), 30-80 mL of Bacillus belye fermentation culture medium is added to every 1 kg of insect slurry.

[0015] In step (3), the fermentation temperature of the enzymatic hydrolysis fermentation is 30~45℃ and the fermentation time is 12~36 h.

[0016] The insect larvae are fly maggots or black soldier fly larvae.

[0017] The insect larvae are 4-6 day old larvae.

[0018] In step (4), the enzymatic hydrolysis fermentation product of insect insect pulp is subjected to dialysis treatment to obtain insect protein peptides with a molecular weight of 3~10 kDa.

[0019] The present invention also provides the application of the above-mentioned insect protein peptides as raw materials for the preparation of biological antibacterial agents.

[0020] The beneficial effects of this invention are: This invention utilizes the fermentation culture product of Bacillus belyssus JAAS-22 (i.e., its secreted protease) as an enzyme catalyst to enzymatically hydrolyze insect larval proteins, thereby significantly increasing the content of antibacterial peptides (peptides with a molecular weight ≤ 30 kDa) in the hydrolysate, and thus significantly enhancing the antibacterial activity of the obtained functional peptides, specifically as follows: This invention utilizes Bacillus belye JAAS-22 to enzymatically ferment fly maggot slurry, resulting in a protein peptide (≤ 30 kDa) content 1.5 times that of the untreated group and a free amino acid content 1.2 times that of the untreated group; the inhibition zone diameters against Staphylococcus aureus and Vibrio parahaemolyticus are 1.68 times and 1.4 times that of the untreated group, respectively.

[0021] This invention utilizes Bacillus belye JAAS-22 to enzymatically ferment black soldier fly larvae slurry. The protein peptide (≤ 30 kDa) content of the untreated group is 1.53 times that of the untreated group, and the free amino acid content is 1.69 times that of the untreated group. The inhibition zone diameters against Staphylococcus aureus and Escherichia coli are 1.27 times and 1.35 times that of the untreated group, respectively. Attached Figure Description

[0022] Figure 1 This is a diagram of the protein hydrolysis rings of Bacillus belyssus JAAS-22; Figure 2 Example 6 shows the determination of the inhibition zone diameter of fly larvae protein peptides against pathogens: CK1, untreated fly larvae protein peptides; YF1, fly larvae protein peptides from the Bacillus belyssus JAAS-22 fermentation group; negative control: sterile water; positive control: chloramphenicol aqueous solution. Figure 3 Example 7 shows the determination of the inhibition zone diameter of black soldier fly protein peptides against pathogens: CK2, untreated black soldier fly protein peptides; HF2, black soldier fly protein peptides from the Bacillus belyss JAAS-22 fermentation group; negative control: sterile water; positive control: chloramphenicol aqueous solution. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0024] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0025] Example 1: Isolation, identification and fermentation broth preparation of Bacillus belyssus JAAS-22 strain Obtaining, isolating and purifying JAAS-22 strain Bacillus belye JAAS-22 was isolated in February 2023 from the black soldier fly breeding pilot plant of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province. Its colony morphology is described as follows: milky white colonies with a dry, sticky texture and irregular edges.

[0026] Molecular identification of JAAS-22 strain Single colonies were picked from JAAS-22 pure culture plates and inoculated into LB liquid medium, and cultured overnight at 37°C. The bacterial cells were collected by centrifugation at 10,000 rpm for 1 min, and the JAAS-22 genome was extracted as a template for 16S rDNA fragment amplification. The PCR product was then sequenced.

[0027] The PCR amplification products were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The sequenced gene fragments were compared with the NCBI database for homology. The comparison revealed that strain JAAS-22 shared 99.78% homology with *Bacillus velezensis*. Therefore, based on the strain's morphology and 16S rDNA (SEQ ID No. 1), strain JAAS-22 was identified as a new subspecies of *Bacillus velezensis*, and named *Bacillus velezensis* JAAS-22. The taxonomic name of strain JAAS-22 is *Bacillus velezensis*, and it was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 10, 2023, with accession number GDMCC No: 63724.

[0028] SEQ ID No. 1:

[0029] Example 2 Preparation of fermentation broth for Bacillus belyssus JAAS-22 strain Bacillus belye JAAS-22 was streaked onto LB agar plates and incubated at 37°C for 24 h to obtain activated single colonies. Single colonies of Bacillus belye JAAS-22 were picked and inoculated into LB liquid medium at 37°C with a shaking incubator speed of 200 rpm for 20 h to obtain a seed culture of Bacillus belye JAAS-22. This seed culture was then inoculated at a volume ratio of 2% into a fermenter containing LB liquid medium at 37°C with a stirring speed of 300 rpm and an air aeration rate of 1 vvm for 28 h to obtain the fermentation broth of Bacillus belye JAAS-22.

[0030] Bacillus belyssus JAAS-22 protein hydrolysis zone Take a small amount of *Bacillus belyssiensis* JAAS-22 bacterial suspension and lightly spot it onto a protease selection plate (protease selection plate formulation: 20 g / L skim milk, 20 g / L agar). Incubate at 30°C upside down for 24 h. Observe and measure the diameter of the proteolysis zone of *Bacillus belyssiensis* JAAS-22. Figure 1 The results showed that the diameter of the protein hydrolysis zone of Bacillus belyss JAAS-22 was 24±0.2 mm.

[0031] Determination of total protease activity in Bacillus belysinus JAAS-22 The total protease activity of Bacillus belyss JAAS-22 bacterial culture was determined using the total protease activity kit from Suzhou Greens Biotechnology Co., Ltd., and the enzyme activity was 5.27 azocasein / h / mg.

[0032] Example 3: Shake-flask fermentation of fly larvae by Bacillus belyssus JAAS-22 strain Preparation of seed culture of fly larvae in shake flask fermentation experiment Bacillus subtilis (purchased from Beijing Bio-Biotech Co., Ltd., product number: bio-67659), Bacillus methyltrophicus (purchased from Beijing Bio-Biotech Co., Ltd., product number: bio-113590), Bacillus belyss JAAS-22, and Bacillus belyss JSBV63 (provided by Jiangsu Academy of Agricultural Sciences, CGMCC No. 24638, see Chinese Patent CN117286050A) were inoculated into LB liquid medium at 37℃ and 200 rpm for 24 h to obtain seed culture of Bacillus belyss JAAS-22, Bacillus subtilis, Bacillus belyss JSBV63, and Bacillus methyltrophicus. The total protease activity kit of Suzhou Greens Biotechnology Co., Ltd. was used to determine the protease activity of each strain in the seed culture. The results are shown in Table 1.

[0033] Table 1. Protease activity of strains .

[0034] Shake-flask fermentation experiment of fly larvae pulp Weigh 3 kg of fresh five-day-old fly larvae and prepare fly larvae slurry in a gel mill. Weigh 150.0 g of fly larvae slurry into 500 mL Erlenmeyer flasks, and weigh a total of 20 portions. Sterilize at 121℃ for 15 min.

[0035] The experiment on fermented fly larvae slurry included an untreated group and a microbial treatment group, with three parallel experiments. The untreated group was treated with 5% (v / w, 5 mL water per 100 g), fermented at 37℃, in a shaker at 200 rpm for 24 h. The microbial treatment group was treated with 5% (v / w, 5 mL seed culture per 100 g), fermented at 37℃, in a shaker at 200 rpm for 24 h. Finally, different groups of fly larvae slurry fermentation broths were obtained.

[0036] Dialysis bags for dialysis of fly larvae protein peptides Dialysis bag pretreatment: Cut the dialysis bag with a molecular weight cutoff of 30 kDa into 11 cm long segments. Before use, boil the segments with EDTA (pH=8.0) to remove surface impurities. After removing the dialysis bag, rinse it thoroughly with distilled water to ensure that no cleaning agent or impurities remain on the surface of the dialysis bag.

[0037] Dialysis and determination of protein peptide (≤ 30 kDa) content: Take the maggot fermentation broth and place it into a dialysis bag, securing both ends with dialysis clamps or cable ties. Place the dialysis bag containing the maggot broth into a beaker, add 50 mL of distilled water, ensuring the bag is completely submerged. Dialyze for 36 hours, changing the distilled water every 12 hours. After dialysis, mix the three dialysis solutions and determine the protein peptide content using the BCA method. The results are shown in Table 2.

[0038] Determination of viable bacteria count in fly maggot fermentation broth: Take 1 mL of the fly larvae fermentation broth and place it into a 15 mL sterile centrifuge tube. Add 9 mL of sterile water, mix well, and then serially dilute to 10⁻⁶ liters. 4 The appropriate amount of diluted solution was spread onto LB solid medium and cultured at 37℃ for 18 hours. The number of viable bacteria was observed and recorded. The results are shown in Table 2. The results in Table 2 show that, compared with other Bacillus species, the yield of ≤30 kDa protein peptides by Bacillus JAAS-22 of this invention was significantly increased, and the effect was the best.

[0039] Table 2 Results of shake-flask fermentation experiment of fly larvae .

[0040] Example 4: Enzymatic hydrolysis preparation process of fly maggot slurry using Bacillus belye JAAS-22 4.1 Preparation of fly larvae slurry Weigh 15 kg of fresh five-day-old fly larvae and prepare fly larvae paste in a glue mill.

[0041] 4.2 Fermentation of fly maggot slurry by Bacillus belye Weigh 2.5 kg of fly larvae slurry and add it to a 5 L fermenter. Then add 125 mL of Bacillus belyssioides JAAS-22 fermentation broth (prepared in Example 2) and mix well. Maintain the fermenter temperature at 30℃ and ferment for 24 h to obtain the fly larvae slurry fermentation broth from the Bacillus belyssioides JAAS-22 fermentation group. Separately weigh 2.5 kg of fly larvae slurry and add it to a 5 L fermenter. Then add 125 mL of pure water and mix well. Maintain the fermenter temperature at 30℃ and ferment for 24 h to obtain the fly larvae slurry fermentation broth from the untreated group.

[0042] 4.3 Dialysis of fly larvae protein peptides (≤ 30 kDa) using dialysis bags 4.3.1 Dialysis bag pretreatment: Cut the dialysis bag with a molecular weight cutoff of 30 kDa into 11 cm long segments. Before use, boil the segments with EDTA (pH=8.0) to remove surface impurities. After removing the dialysis bag, rinse it thoroughly with distilled water to ensure that no cleaning agent or impurities remain on the surface of the dialysis bag.

[0043] 4.3.2 Dialysis and determination of protein peptide (≤ 30 kDa) content: Take out the fly larvae fermentation broth obtained in step 4.2, put the fermentation broth into a dialysis bag, and clamp both ends with dialysis clips or cable ties. Place the dialysis bag containing the larvae broth into a beaker, add 50 mL of distilled water, ensuring that the dialysis bag is completely immersed in the distilled water, and dialyze for 36 h, changing the distilled water every 12 h. After dialysis, mix the dialysis solutions three times, and determine the protein peptide content using the BCA method. The free amino acid content is determined using a free amino acid content kit (purchased from Suzhou Gres Biotechnology Co., Ltd.). The results are shown in Table 3. The results in Table 3 show that, compared with the untreated group, the fly larvae protein peptide (≤ 30 kDa) and free amino acid content in the Bacillus belyss JAAS-22 fermentation group of this invention are significantly increased.

[0044] Table 3. Content of fly larvae protein peptides and free amino acids in the untreated group and the Bacillus belysin JAAS-22 fermentation group. .

[0045] Example 5: Enzymatic hydrolysis preparation process of black soldier fly larvae spores by Bacillus belysinia JAAS-22 5.1 Preparation of Black Soldier Fly Powder Weigh 15 kg of fresh five-day-old black soldier fly larvae and prepare black soldier fly larvae slurry in a glue mill.

[0046] 5.2. Fermentation of black soldier fly larvae pulp by Bacillus belye JAAS-22 Weigh 5.0 kg of black soldier fly larvae slurry and add it to a 10 L fermenter. Then add 250 mL of Bacillus belyssioides JAAS-22 fermentation broth and mix well. Maintain the fermenter temperature at 30℃ and ferment for 24 h to obtain the fermentation broth of black soldier fly larvae slurry from the Bacillus belyssioides JAAS-22 fermentation group. Separately weigh 5.0 kg of black soldier fly larvae slurry and add it to a 10 L fermenter. Then add 250 mL of pure water and mix well. Maintain the fermenter temperature at 30℃ and ferment for 24 h to obtain the fermentation broth of black soldier fly larvae slurry from the untreated group.

[0047] 5.3 Dialysis of black soldier fly protein peptides (≤ 30 kDa) using dialysis bags 5.3.1 Dialysis bag pretreatment: Same as in Example 4.

[0048] 5.3.2 Dialysis and determination of protein peptide (≤ 30 kDa) content: Take out the black soldier fly larvae fermentation broth obtained in step 5.2, put the fermentation broth into a dialysis bag, and clamp both ends with dialysis clips or cable ties. Place the dialysis bag containing the larvae broth into a beaker, add 50 mL of distilled water, ensuring that the dialysis bag is completely immersed in the distilled water, and dialyze for 36 h, changing the distilled water every 12 h. After dialysis, mix the dialysis solutions three times, and determine the protein peptide content using the BCA method. The free amino acid content is determined using a free amino acid content kit (purchased from Suzhou Gres Biotechnology Co., Ltd.). The results are shown in Table 4. The results in Table 4 show that, compared with the untreated group, the content of black soldier fly protein peptides (≤ 30 kDa) and free amino acids in the Bacillus belyss JAAS-22 fermentation group of this invention is significantly increased.

[0049] Table 4. Content of black soldier fly protein peptides and free amino acids in the untreated group and the Bacillus belysinus JAAS-22 fermentation group. .

[0050] Example 6: Detection of antibacterial activity of fly larvae protein peptides 6.1 Preparation of fly maggot plasma protein peptides In Example 4, the protein peptides (≤ 30 kDa) isolated from the fermentation broth of fly maggots in the untreated group and the Bacillus vesicles JAAS-22 fermentation group were further freeze-dried to obtain fly maggot protein peptides.

[0051] 6.2 Determination of the antibacterial activity of fly larvae protein peptides The negative control was sterile water; chloramphenicol was used as the positive control, prepared as a 0.5 mg / mL aqueous solution. The Staphylococcus aureus bacterial culture was determined to be 5.0 × 10⁻⁶. 7 The CFU / mL concentration and the Vibrio parahaemolyticus bacterial concentration were 1.0 × 10⁻⁶. 7 CFU / mL, appropriate amounts of Staphylococcus aureus bacterial suspension were evenly spread on LB agar plates, and Vibrio parahaemolyticus bacterial suspension was evenly spread on TSB plates (tryptic soy peptone agar). Oxford cups (7.5 mm in diameter) were gently placed on LB plates, and 100 μL of fly larvae protein peptides were added to the Oxford cups. The untreated group had a protein peptide labeled CK1, and the Bacillus belyss JAAS-22 fermentation group had a peptide labeled YF1. The plates were incubated at 37℃ for 10–16 h, and the diameter of the inhibition zone was observed and measured. The results are shown in Table 5.

[0052] Experimental Results: In vitro antibacterial test of fly maggot protein peptides. The antibacterial effect is as follows: From Figure 2It can be seen that inhibition zones appeared in both the untreated group and the Bacillus vesicularis JAAS-22 fermentation group. The inhibition zones against Staphylococcus aureus in the untreated group and the Bacillus vesicularis JAAS-22 fermentation group were 9.5±0.1 mm and 22±0.2 mm, respectively. The inhibition zones against Vibrio parahaemolyticus in the untreated group and the Bacillus vesicularis JAAS-22 fermentation group were 10±0.2 mm and 24±0.2 mm, respectively. The experimental results show that the antibacterial properties of fly maggot slurry were significantly improved after enzymatic fermentation with Bacillus vesicularis JAAS-22.

[0053] Table 5 Antibacterial activity of fly larvae protein peptides .

[0054] Example 7: Detection of antibacterial activity of black soldier fly protein peptides 7.1 Preparation of antimicrobial peptides from black soldier fly larvae plasma In Example 5, the protein peptides (≤ 30 kDa) isolated from the fermentation broth of black soldier fly larvae in the untreated group and the Bacillus baileyi JAAS-22 fermentation group were further freeze-dried to obtain black soldier fly protein peptides.

[0055] 7.2 Determination of the antibacterial activity of black soldier fly protein peptides The negative control was sterile water; chloramphenicol was used as the positive control, prepared as a 0.5 mg / mL aqueous solution. The effective bacterial culture for Staphylococcus aureus was determined to be 1.0 × 10⁻⁶. 8 The CFU / mL concentration and the Escherichia coli bacterial concentration were 5.0 × 10⁻⁶. 7 CFU / mL, appropriate amounts of Staphylococcus aureus and Escherichia coli bacterial suspensions were evenly spread onto LB solid medium. Oxford cups (7.5 mm in diameter) were gently placed on LB plates, and 100 μL of black soldier fly protein peptides were added to the Oxford cups. The untreated group contained protein peptides labeled CK2, and the Bacillus belyss JAAS-22 fermentation group contained HF2. The plates were incubated at 37℃ for 10–18 h, and the diameter of the inhibition zone was observed and measured, as shown in Table 6.

[0056] Experimental Results: In vitro antibacterial tests were conducted on black soldier fly protein peptides. The antibacterial effect is as follows: From Figure 3It can be seen that inhibition zones appeared in both the untreated group and the Bacillus vesiculosus JAAS-22 fermentation group. The inhibition zones against Staphylococcus aureus in the untreated group and the Bacillus vesiculosus JAAS-22 fermentation group were 18±0.1 mm and 23±0.1 mm, respectively, while the inhibition zones against Escherichia coli in the untreated group and the Bacillus vesiculosus JAAS-22 fermentation group were 14±0.2 mm and 19±0.2 mm, respectively. In the experiment, it was found that although the inhibition zone in the blank group was larger, the color of the inhibition zone was light, indicating that it was not completely inhibited, while the inhibition zone in the fermentation group was bright, indicating that the antibacterial effect was obvious. The experimental results show that the antibacterial properties of black soldier fly protein peptides were significantly improved after enzymatic hydrolysis and fermentation with Bacillus vesiculosus JAAS-22.

[0057] Table 6. Antibacterial activity of black soldier fly protein peptides .

[0058] Example 8: Enzymatic hydrolysis preparation process of Bacillus belyssus JAAS-22 from fly maggot slurry The difference between this embodiment and Example 4 (Bacillus belyss JAAS-22) in the enzymatic hydrolysis preparation process is as follows: Preparation of fly maggot slurry Weigh 15 kg of fresh 4-day-old fly larvae and prepare fly larvae paste in a glue mill.

[0059] Bacillus vesiculosus fermented fly maggot slurry Weigh 2 kg of fly larvae slurry and add it to a 5 L fermenter. Then add 60 mL of Bacillus belysin JAAS-22 fermentation culture and mix well. Control the temperature of the fermenter at 35℃ and ferment for 36 h to obtain the fly larvae slurry fermentation broth from the Bacillus belysin JAAS-22 fermentation group.

[0060] Example 9: Enzymatic hydrolysis preparation process of Bacillus belyssus JAAS-22 from fly maggot slurry The difference between this embodiment and Example 4 (Bacillus belyss JAAS-22) in the enzymatic hydrolysis preparation process is as follows: Preparation of fly maggot slurry Weigh 15 kg of fresh 6-day-old fly larvae and prepare fly larvae paste in a glue mill.

[0061] Bacillus vesiculosus fermented fly maggot slurry Weigh 2 kg of fly larvae slurry and add it to a 5 L fermenter. Then add 160 mL of Bacillus belysin JAAS-22 fermentation culture and mix well. Control the temperature of the fermenter at 45℃ and ferment for 12 h to obtain the fly larvae slurry fermentation broth from the Bacillus belysin JAAS-22 fermentation group.

[0062] Example 10: Enzymatic hydrolysis preparation process of Bacillus belyssus JAAS-22 from fly maggot slurry The difference between this embodiment and Example 4 (Bacillus belyss JAAS-22) in the enzymatic hydrolysis preparation process is as follows: By changing the molecular weight cutoff setting of the dialysis bag, protein peptides with molecular weights of 3-10 kDa were finally obtained.

[0063] Example 11: Enzymatic hydrolysis preparation process of black soldier fly larvae spores by Bacillus belysin JAAS-22 The difference between this embodiment and Example 5 is the enzymatic hydrolysis preparation process of Bacillus belyssioides JAAS-22 from black soldier fly larvae slurry: Preparation of black soldier fly larvae slurry Weigh 15 kg of fresh 4-day-old black soldier fly larvae and prepare black soldier fly larvae slurry in a glue mill.

[0064] Bacillus belye JAAS-22 fermentation of black soldier fly larvae plasma Weigh 5.0 kg of black soldier fly larvae slurry and add it to a 10 L fermenter. Then add 150 mL of Bacillus belysin JAAS-22 fermentation culture and mix well. Control the temperature of the fermenter at 35℃ and ferment for 36 h to obtain the fermentation broth of black soldier fly larvae slurry from the Bacillus belysin JAAS-22 fermentation group.

[0065] Example 12: Enzymatic hydrolysis preparation process of Bacillus belysin JAAS-22 from black soldier fly larvae spores The difference between this embodiment and Example 5 is the enzymatic hydrolysis preparation process of Bacillus belyssioides JAAS-22 from black soldier fly larvae slurry: Preparation of black soldier fly larvae slurry Weigh 15 kg of fresh 6-day-old black soldier fly larvae and prepare black soldier fly larvae slurry in a glue mill.

[0066] Bacillus belye JAAS-22 fermentation of black soldier fly larvae plasma Weigh 5.0 kg of black soldier fly larvae slurry and add it to a 10 L fermenter. Then add 400 mL of Bacillus belysin JAAS-22 fermentation culture and mix well. Control the temperature of the fermenter at 45℃ and ferment for 12 h to obtain the fermentation broth of black soldier fly larvae slurry from the Bacillus belysin JAAS-22 fermentation group.

[0067] Example 13 Preparation of fermentation broth for Bacillus belyssus JAAS-22 strain Bacillus berleis JAAS-22 was streaked onto LB agar plates and incubated at 37°C for 24 h to obtain activated single colonies. Single colonies were then picked and inoculated into LB liquid medium at 32°C with a shaker speed of 100 rpm for 36 h to obtain a seed culture of Bacillus berleis JAAS-22. This seed culture was then inoculated at a volume ratio of 5% into a fermenter containing LB liquid medium at 32°C with a stirring speed of 100 rpm and an air aeration rate of 0.5 vvm for 30 h to obtain the fermentation broth of Bacillus berleis JAAS-22.

[0068] Example 14 Preparation of fermentation broth for Bacillus belyssus JAAS-22 strain Bacillus berleis JAAS-22 was streaked onto LB agar plates and incubated at 37°C for 24 h to obtain activated single colonies. Single colonies were then picked and inoculated into LB liquid medium at 35°C with a shaker speed of 150 rpm for 30 h to obtain a seed culture of Bacillus berleis JAAS-22. This seed culture was then inoculated at a volume ratio of 1% into a fermenter containing LB liquid medium at 40°C with a stirring speed of 400 rpm and an air ventilation rate of 2 vvm for 20 h to obtain the fermentation broth of Bacillus berleis JAAS-22.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for preparing insect protein peptides using Bacillus belye JAAS-22, characterized in that, Includes the following steps: (1) The strain of Bacillus belyss JAAS-22 was expanded and cultured to obtain the fermentation broth of Bacillus belyss JAAS-22; (2) Homogenize the insect larvae in a glue mill to obtain insect pulp; (3) Mix the insect slurry with the Bacillus vesicle JAAS-22 fermentation culture medium and carry out enzymatic hydrolysis fermentation to obtain the insect slurry enzymatic hydrolysis fermentation product; (4) The insect larvae fermentation product was subjected to dialysis to obtain insect protein peptides with a molecular weight ≤30kDa.

2. The method according to claim 1, characterized in that, Bacillus belye JAAS-22 is classified as follows: Bacillus velezensis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 10, 2023, with accession number GDMCC No: 63724.

3. The method according to claim 1, characterized in that, Step (1) is as follows: Single colonies of Bacillus belyss JAAS-22 were picked and inoculated into LB liquid medium and placed in a constant temperature incubator at a temperature of 32~37℃, a stirring speed of 100~200rpm, and a culture time of 20~36h to obtain Bacillus belyss seed culture. The Bacillus belysin seed culture was inoculated into a fermenter containing LB liquid medium at a volume ratio of 1% to 5%. The culture temperature was 32 to 40°C, the stirring speed was 100 to 400 rpm, the air ventilation rate was 0.5 to 2 vvm, and the culture time was 20 to 30 h to obtain the Bacillus belysin JAAS-22 fermentation culture.

4. The method according to claim 1, characterized in that, In step (3), 30-80 mL of Bacillus belye fermentation culture medium is added to every 1 kg of insect slurry.

5. The method according to claim 1, characterized in that, In step (3), the fermentation temperature of the enzymatic hydrolysis fermentation is 30~45℃ and the fermentation time is 12~36h.

6. The method according to claim 1, characterized in that, The insect larvae are fly maggots or black soldier fly larvae.

7. The method according to claim 1, characterized in that, The insect larvae are 4-6 day old larvae.

8. The method according to claim 1, characterized in that, In step (4), the enzymatic hydrolysis fermentation product of insect insect pulp is subjected to dialysis treatment to obtain insect protein peptides with a molecular weight of 3~10kDa.

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