Animal additive based on probiotic synergistic fermentation of hippophae rhamnoides residues and hermetia illucens protein and preparation method of animal additive

Abstract

The invention relates to the field of animal nutrition additives, and particularly discloses an animal additive based on probiotic synergistic fermentation sea buckthorn residue and hermetia illucens protein and a preparation method thereof, the animal additive comprises the following components by mass: 5%-10% of a filling auxiliary material, and the balance of a fermentation composite product; the sea-buckthorn residues and the hermetia illucens larvae can be fermented to form a double-substrate fermentation product, so that the double-substrate fermentation product has a synergistic nutrition effect, and can be used for remarkably promoting the weight gain condition of animals, improving spleen and thymus indexes and increasing serum IgG and IgM; the hermetia illucens protein is rich in lauric acid, antibacterial peptide and high-quality animal protein, so that the non-specific immunity and the anti-stress capability of an organism are enhanced, and the infection risk in the breeding process is reduced; the low-value sea-buckthorn residues and the hermetia illucens larvae are upgraded and converted into high-value feeding resources in a biological fermentation mode, the environmental pressure of processing by-products is relieved, the additional value of raw materials is increased, and the method conforms to resource circulation and environmental protection policy guidance.

Description

Technical Field

[0001] This invention relates to the field of animal nutritional additives, specifically to an animal additive based on the co-fermentation of sea buckthorn residue and black soldier fly protein by probiotics and its preparation method. Background Technology

[0002] With the rapid development of animal husbandry, traditional feed resources are facing the dual pressures of shortage and rising costs. Meanwhile, the overuse of antibiotics has led to increasingly serious problems with animal resistance and food safety, prompting a growing demand for research and development of green, safe, and efficient new feed additives. In recent years, the combination of natural plant resources and microbial technology has become an important direction for replacing antibiotics and improving animal health.

[0003] Sea buckthorn (Hippophae rhamnoides) is an economically valuable plant with both medicinal and edible properties. Its pomace, a major byproduct of sea buckthorn processing, remains rich in flavonoids, polyphenols, polysaccharides, organic acids, and other bioactive substances, possessing antioxidant, anti-inflammatory, and tissue-repair-promoting functions. However, untreated sea buckthorn residue suffers from poor taste, low palatability, and low bioavailability, limiting its widespread application in animal feed. Probiotic fermentation technology can effectively improve the physicochemical properties of sea buckthorn residue, enhance the release of active ingredients, and increase nutritional value and bioavailability, demonstrating significant development potential.

[0004] Black soldier fly (Hermetia illucens) is a sustainable high-protein insect resource. Its larvae are rich in protein, fat, and functional fatty acids, especially lauric acid, which has strong antibacterial and immune-enhancing effects. As a source of animal protein, black soldier fly has shown good potential to replace traditional fishmeal or blood meal in livestock and poultry farming.

[0005] Although there is already much research on the fermentation and development of sea buckthorn residue, which can improve animal immunity when used as a feed additive, the fermentation raw materials are often singular. There is little research on co-fermentation with other raw materials to enhance product efficacy. Furthermore, there is a lack of systematic research and product development on the co-fermentation of sea buckthorn residue and black soldier fly protein with probiotics for animal growth promotion and immune enhancement. The co-fermentation of these two materials presents several challenges. When co-fermenting sea buckthorn residue and black soldier fly protein, black soldier fly protein, as a high-density, sustainable animal protein source, is combined with sea buckthorn residue, which is rich in polyphenols, fiber, and complex polysaccharides. However, sea buckthorn residue contains a large amount of organic acids, while black soldier fly protein powder has an isoelectric point of approximately pH [missing information]. When the pH of the mixture is between 4.5 and 5.0, it is lower than the isoelectric point of the protein, causing the protein molecules to expand and expose hydrophobic groups, leading to irreversible aggregation, "clumping and stratification," and reducing the uniformity of subsequent processing. Furthermore, sea buckthorn residue is rich in polyphenols (such as proanthocyanidins and quercetin), whose phenolic hydroxyl groups can form hydrogen bonds or covalent bonds with the amino and thiol groups of black soldier fly protein, resulting in both a decrease in protein digestibility (due to the difficulty of protease action after complexation) and the loss of polyphenol antioxidant activity. Therefore, to solve these problems, this invention constructs a novel functional additive composition that comprehensively enhances animal nutrient absorption, antioxidant capacity, and immune function through a triple synergistic mechanism of "plant functional factors + probiotic fermentation + insect protein," demonstrating good industrial application prospects and promotional value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an animal additive based on the co-fermentation of seabuckthorn residue and black soldier fly protein by probiotics and its preparation method, so as to solve the problems of slow growth, low immune function and intestinal flora disorder that are common in animal breeding processes in the prior art due to the lack of mixed fermentation of seabuckthorn residue and black soldier fly protein.

[0007] This invention solves the above-mentioned technical problems. This invention provides an animal additive based on the co-fermentation of sea buckthorn residue and black soldier fly protein by probiotics, comprising the following components by weight: 5% to 10% filler excipients, and the remainder being fermentation compound products; The fermentation compound product is obtained by mixing and sterilizing sea buckthorn pomace and black soldier fly protein, adding a regulator for pre-fermentation, and then adding compound probiotics for anaerobic fermentation; the black soldier fly protein powder is selected from defatted black soldier fly larvae powder. The compound probiotics include Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus. The fermentation compound product is prepared by mixing sea buckthorn pomace with dried and sterilized black soldier fly protein powder at a mass ratio, sterilizing by high pressure steam at 115-125℃ for 20 minutes, cooling to room temperature, adding 2-4% of a regulator and stirring, then pre-fermenting, and then inoculating with 5%-10% of compound probiotics. The product is obtained by solid-state anaerobic fermentation at 65% humidity and 35-37℃ for 72 hours. The pre-fermentation involves inoculating 8%–12% of Bacillus licheniformis at 35–37°C for 10–12 hours; Bacillus licheniformis degrades polyphenols in advance, clearing away toxicity obstacles for the main fermentation microbiota. The defatting process involves washing black soldier fly larvae, microwaving them at 600-800W for 60-80 seconds, then adding a solution containing 0.1%-0.2% alkaline protease at a solid-liquid ratio of 2-5:11-15 g / mL to obtain a mixture. Next, glucose is added to the mixture at 6-10 wt% and the mixture is reacted at 60-65℃ for 2-4 hours. After drying, the product is obtained. This process increases the content of small molecule peptides, reduces active groups, and efficiently removes fat. The compound probiotics include Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus in a mass ratio of 3:2-4:3; the effective bacterial concentration of the strains is ≥10 billion CFU / g; Bacillus licheniformis can not only inhibit pathogenic bacteria, but also has excellent ability to degrade plant polysaccharides, while Bacillus amyloliquefaciens has the ability to degrade proteins. The enzymes secreted by Bacillus licheniformis and Bacillus amyloliquefaciens degrade anti-nutritional factors, while Bacillus mucilaginosus regulates intestinal pH, promotes the proliferation of beneficial bacteria in the intestine, and at the same time, the extracellular polysaccharides secreted reduce the oxidation of active substances; The filler material is corn flour doped with 3%-6% by mass of modified soybean phospholipid concentrate; The modified soybean phospholipid is prepared by taking food-grade concentrated soybean phospholipid, adding 3-5 times the amount of deionized water, stirring and dissolving at 50-60℃ to obtain a phospholipid emulsion, adding 8-12wt% alginate to the phospholipid emulsion, stirring and mixing, and then carrying out an electro-reaction for 0.1-0.2s to obtain the product. The electric field strength of the electro-reaction is 20-30 kV / cm; the modified phospholipid has high emulsifying activity, which can promote the interfacial fusion of black soldier fly protein and sea buckthorn fiber, and at the same time improve the absorption efficiency of fat-soluble vitamins in animals. The regulating agent is a carboxymethyl cellulose and an amino acid iron complex in a mass ratio of 2-4:1. Carboxymethyl cellulose improves the stability of active substances such as proteins and polyphenols through amidation and long-chain structure, and provides a weak buffer to improve the stability of probiotics. The amino acid iron complex can remove reactive oxygen species generated during fermentation and protect the active groups of polyphenols and peptides. This invention also provides a method for preparing an animal additive based on the co-fermentation of sea buckthorn residue and black soldier fly protein by probiotics. The specific process is as follows: the fermentation compound product and the filler are mixed in proportion to obtain a solid additive. The preparation method further includes the preparation of liquid additives, specifically: take the fermentation compound product, add deionized water at a solid-liquid ratio of 1:1-3, stir at a constant temperature of 40-50℃ for 20-30 minutes to obtain a slurry; send the slurry into a plate and frame filter press for filtration, and collect the initial filtrate; after repeatedly extracting and filtration the filter residue 2-3 times, combine the filtrates, filter and sterilize the filtrate to obtain the final product.

[0008] The beneficial effects of this invention are as follows: The animal additive provided by this invention involves the addition of a fermentation compound product, which is produced by defatting black soldier fly larvae using a defatting process and then fermenting it with sea buckthorn residue. By adding a regulating agent and successively carrying out pre-fermentation and anaerobic fermentation, anti-nutritional components can be removed, and the fermentation performance of the compound probiotics can be improved. This significantly increases the yield of soluble small peptides and absorbable amino acids, improves protein digestibility, and the esterases secreted by the compound probiotics enable the glycosidic bonds of sea buckthorn polyphenols to be directionally unchained, releasing free phenolic hydroxyl groups and converting them into more active phenolic derivatives, thereby enhancing antioxidant and immune-stimulating capabilities. The regulating agent can improve fermentation stability and efficiency and stabilize active ingredients. The dual-substrate fermentation product formed by the fermentation of sea buckthorn residue and black soldier fly larvae has a synergistic nutritional effect, which can significantly promote animal weight gain, increase spleen and thymus indices, and raise serum IgG and IgM levels. Black soldier fly protein is rich in lauric acid, antimicrobial peptides, and high-quality animal protein, which enhances the body's non-specific immunity and stress resistance, and reduces the risk of infection during the breeding process. Attached Figure Description

[0009] Figure 1 This is a tissue diagram of a mouse spleen. Detailed Implementation

[0010] The present invention will be further described in detail below through specific implementation examples. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0011] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0012] Example 1 A method for preparing an animal additive based on probiotic co-fermentation of sea buckthorn residue and black soldier fly protein A solid-state compound fermentation additive is obtained by mixing 90% of the fermentation compound product and 10% of the filler material. The fermentation compound product was prepared by mixing sea buckthorn pomace with dried and sterilized black soldier fly protein powder at a mass ratio of 1:1, sterilizing by high-pressure steam at 120°C for 20 minutes, cooling to room temperature, adding 3% (by mass) of a regulating agent and stirring, then inoculating with 10% Bacillus licheniformis and fermenting at 36°C for 11 hours, followed by inoculation with 8% compound probiotics and solid-state anaerobic fermentation at 36°C for 72 hours at 65% humidity. The regulating agent was a carboxymethyl cellulose and amino acid iron complex at a mass ratio of 3:1. The black soldier fly protein powder is selected from defatted black soldier fly larvae powder. The defatting process involves washing the black soldier fly larvae, microwaving them at 700W for 70s, then adding a solution containing 0.1% alkaline protease at a solid-liquid ratio of 4:13g / mL to obtain a mixture, then adding 8wt% glucose to the mixture and reacting at 60℃ for 3h, followed by drying. The compound probiotics include Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus in a mass ratio of 1:1:1. The filler is corn flour doped with 5% modified soybean phospholipid concentrate by mass. The modified soybean phospholipid is prepared by taking food-grade soybean phospholipid concentrate, adding 4 times the amount of deionized water, stirring and dissolving at 55°C to obtain a phospholipid emulsion, adding 10wt% alginate to the phospholipid emulsion, stirring and mixing, and then carrying out an electro-reaction for 15s. The electric field strength of the electro-reaction is 25kV / cm.

[0013] Example 2 A method for preparing an animal additive based on probiotic co-fermentation of sea buckthorn residue and black soldier fly protein Take the fermentation compound product, add deionized water at a solid-liquid ratio of 1:1, and stir at 40℃ for 20 minutes to obtain a slurry. Send the slurry to a plate and frame filter press for filtration and collect the initial filtrate. After repeatedly extracting and filtration the filter residue twice, combine the filtrates, filter and sterilize the filtrate to obtain the final product. The fermentation compound product was prepared by mixing sea buckthorn pomace with dried and sterilized black soldier fly protein powder at a mass ratio of 1:1, sterilizing by high-pressure steam at 115°C for 20 minutes, cooling to room temperature, adding 2% (by mass) of a regulating agent and stirring, then inoculating with 8% Bacillus licheniformis and fermenting at 35°C for 10 hours, followed by inoculation with 5% compound probiotics and solid-state anaerobic fermentation at 35°C for 72 hours at 65% humidity. The regulating agent was a carboxymethyl cellulose and amino acid iron complex at a mass ratio of 2:1. The black soldier fly protein powder is selected from defatted black soldier fly larvae powder. The defatting process involves washing the black soldier fly larvae, microwaving them at 600W for 60 seconds, then adding a solution containing 0.2% alkaline protease at a solid-liquid ratio of 2:11 g / mL to obtain a mixture, then adding 6 wt% glucose to the mixture and reacting at 60°C for 2 hours, followed by drying. The compound probiotics include Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus gelatinosa in a mass ratio of 3:2:3.

[0014] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not add sea buckthorn residue, and uses black soldier fly protein fermentation product instead of compound fermentation product, while other aspects remain unchanged.

[0015] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 uses an equal proportion of unfermented seabuckthorn residue-black soldier fly mixture instead of the compound fermentation product, while other aspects remain unchanged.

[0016] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that no modifier was added during the preparation of the fermentation complex in Comparative Example 3, while everything else remained the same.

[0017] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the filler in Comparative Example 4 is food-grade soybean concentrated lecithin instead of modified soybean lecithin, while everything else remains the same.

[0018] I. Analysis of the nutritional components of the examples and comparative examples Peptides: The OPA method was used for determination. An appropriate amount of sample extract was taken and reacted with o-phthalaldehyde (OPA) colorimetric reagent. The absorbance was measured at 340 nm using a spectrophotometer. A standard curve was plotted using glutamine as a standard, and the peptide content in the sample was calculated.

[0019] Total sugar: determined by the phenol-sulfuric acid method. After water extraction, phenol and concentrated sulfuric acid are added to the sample to react and generate a yellow compound. The absorbance is measured colorimetrically at 490 nm, and the content is calculated using glucose as the standard.

[0020] Total polyphenols: determined by the Folin-Ciocalteu method, in which polyphenols react with Folin reagent to form a blue complex, and the absorbance is measured at 765 nm. The content is calculated using gallic acid (GAE) as a standard.

[0021] Total flavonoids: Total flavonoids can be determined by the aluminum acetate method. After the sample is extracted with an appropriate solvent, it reacts with aluminum salt to form a yellow complex. The absorbance is measured at 415 nm. The content is calculated with rutin as the standard. The results are shown in Table 1.

[0022] Table 1 Nutritional composition of fermentation products Note: Different letters in the same row indicate significant differences (p<0.05), while the same letter indicates no significant differences.

[0023] As shown in Table 1, after fermentation, the black soldier fly protein underwent significant degradation through the fermentation action of probiotics, and the polypeptide content increased significantly. Nutrients such as polysaccharides, polyphenols, and flavonoids in the sea buckthorn residue also increased significantly under the action of probiotics, indicating that the fermentation process of this invention has a significant effect. The regulating agent can increase the content of probiotics and active ingredients.

[0024] II. Grouping and Feeding Design: To verify the effectiveness of the "probiotic synergistic fermentation of sea buckthorn residue and black soldier fly protein growth promoter" in promoting animal growth and enhancing immunity, the inventors conducted animal experiments based on SPF-grade male Kunming mice.

[0025] Experimental animals: SPF-grade male Kunming mice (KM), 3 weeks old, weighing 28-32g, were purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd. Fifty-six mice were randomly divided into 7 groups of 8 mice each. Control group: basal feed; Examples 1-2 and Comparative Examples 1-4: Basic feed + additives, with an addition amount of 80g / kg or 80mL / L; The experiment lasted for 15 days. Mice were allowed free access to food and water. Daily food intake and body weight were recorded. After the experiment, blood samples were collected and the mice were dissected for analysis of various indicators.

[0026] Test items: Nutritional components of the product: polypeptides, total sugars, total polyphenols, total flavonoids, etc. Growth performance indicators: body weight, daily weight gain, feed conversion ratio (FCR), etc. Organ indices: Spleen and thymus indices (organ mass / body weight × 100%). Pathological histology: Observe the morphology of liver and spleen tissues; Immune function indicators: serum levels of IgA, IgG, and IgM factors; Liver injury markers: serum ALT and AST levels; Spleen tissues of mice fed in Examples 1-2, Comparative Example 1, and the control group were as follows: Figure 1 As shown, from left to right, they are the control group, Example 1 group, Example 2 group, and Comparative Example 1 group. Figure 1 It can be seen that the spleens of mice in each experimental group were longer than those in the control group, and no lesions were observed on the surface. Similarly, no pathological damage was observed in the liver tissue.

[0027] The growth performance indicators, organ indices, immune function indicators, and liver damage indicators of mice are shown in Tables 2-5.

[0028] Table 2 Comparison of growth performance in mice Note: Different letters in the same row indicate significant differences (p < 0.05), while the same letter indicates no significant differences.

[0029] As shown in Table 2, the growth rate of mice in the solid compound additive group increased significantly, and both total weight gain and daily weight gain increased significantly compared with the control group. Furthermore, the feed conversion rate improved after the additive was used. Although the growth rate of mice in the liquid additive group was not significantly different from that of the control group, the feed conversion rate still improved to a certain extent. Therefore, the fermentation product has better economic viability in animal husbandry.

[0030] Table 3. Mouse organ index Note: Different letters in the same row indicate significant differences (p < 0.05), while the same letter indicates no significant differences.

[0031] As shown in Table 3, compared with the control group, the organ indices of the spleen and thymus of mice increased to a certain extent after consuming each fermentation product, indicating the activation and enhancement of immune organs and a certain positive immune enhancement effect. The effect was most obvious in the solid compound additive group of mice.

[0032] Table 4 Comparison of mouse immune indicators Note: Different letters in the same row indicate significant differences (p<0.05), while the same letter indicates no significant differences.

[0033] Table 4 shows that serum IgG levels increased after the use of the additive, and the IgM levels in the liquid-added group also showed a certain upward trend compared to the control group. This indicates that the mice treated with the additive showed a slight upward trend in both humoral and cellular immunity, demonstrating a certain immune-enhancing effect.

[0034] Table 5 Indicators of Liver Injury in Mice Interpretation of results: For mice, the normal range for ALT is 5-50 U / L and the normal range for AST is 80-200 U / L. The test results showed that the indicators were all within the normal range, and no liver damage was observed in the mice. This indicates that the additive does not cause damage to normal organisms and has high biosafety.

[0035] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics, characterized in that, It comprises the following ingredients by mass fraction: 5-10% of filling adjuvant, and the balance of fermentation composite product; The fermentation composite product is obtained by mixing and sterilizing seabuckthorn pomace and black soldier fly protein, adding a regulating agent for pre-fermentation, and then adding a composite probiotic for anaerobic fermentation; the black soldier fly protein powder is selected from defatted black soldier fly larvae powder; The composite probiotic comprises bacillus licheniformis, bacillus amyloliquefaciens, and bacillus mycoides; The filling adjuvant is corn flour doped with 3-6% of modified soybean concentrated phospholipid.

2. The animal additive based on the synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The preparation of the fermentation composite product comprises the following steps: uniformly mixing seabuckthorn pomace and black soldier fly protein powder that has been dried and sterilized according to a mass ratio, sterilizing the mixture by high-pressure steam at 115-125°C for 20 minutes, adding a regulating agent with a mass fraction of 2-4% after cooling to room temperature, then stirring and mixing, pre-fermenting, inoculating the composite probiotic with an inoculation amount of 5-10%, and then performing solid-state anaerobic fermentation under the conditions of humidity of 65% and temperature of 35-37°C for 72 hours.

3. The animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The pre-fermentation is performed by inoculating bacillus licheniformis with an inoculation amount of 8-12% and then fermenting at 35-37°C for 10-12 hours.

4. The animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The composite probiotic comprises bacillus licheniformis, bacillus amyloliquefaciens, and bacillus mycoides according to a mass ratio of 3:2-4:

3.

5. The animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The defatting treatment comprises the following steps: washing the black soldier fly larvae, then microwaving the larvae at 600-800W for 60-80s, adding a solution containing alkaline protease with a mass concentration of 0.1-0.2% to obtain a mixed solution according to a solid-liquid ratio of 2-5:11-15g / mL, then adding 6-10wt% of glucose to the mixed solution, and reacting at 60-65°C for 2-4h, and then drying to obtain the defatted black soldier fly larvae powder.

6. The animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The preparation of the modified soybean phospholipid comprises the following steps: taking food-grade soybean concentrated phospholipid, adding 3-5 times of deionized water, stirring and dissolving at 50-60°C to obtain a phospholipid emulsion, adding 8-12wt% of alginate to the phospholipid emulsion, stirring and mixing, and then performing electric reaction for 0.1-0.2s.

7. The animal additive based on probiotic synergistic fermentation of sea buckthorn residues and black soldier fly proteins according to claim 6, characterized by, The electric field strength of the electric reaction is 20-30kV / cm.

8. The animal additive based on synergistic fermentation of sea buckthorn residues with black soldier fly proteins with probiotics according to claim 1, characterized by, The regulating agent is a complex of carboxymethyl cellulose and amino acid iron according to a mass ratio of 2-4:

1.

9. A method for the preparation of an animal additive based on probiotic synergistically fermented sea buckthorn residues and black soldier fly proteins, for the preparation of an animal additive based on probiotic synergistically fermented sea buckthorn residues and black soldier fly proteins according to any one of claims 1 to 8, characterized in that, The specific process is as follows: mixing the fermentation composite product and the filling adjuvant according to the proportions to obtain a solid-state additive.

10. A method for preparing an animal additive based on probiotic synergistic fermentation of sea buckthorn residues with black soldier fly proteins according to claim 9, characterized in that, The preparation of the liquid-state additive comprises the following steps: taking the fermentation composite product, adding deionized water according to a solid-liquid ratio of 1:1-3, and stirring at 40-50°C for 20-30min to obtain a slurry; feeding the slurry into a plate-and-frame filter press to collect the initial filtrate; repeatedly extracting and filtering the filter residue for 2-3 times, combining the filtrates, and then filtering and sterilizing the filtrates to obtain the liquid-state additive.

Images