Method for producing compound protein through fermentation

Abstract

The invention discloses a method for producing compound protein by fermentation, which comprises the following steps: pretreating raw materials, inoculating microorganisms for fermentation, crushing and packaging to obtain fermented goose blood compound protein. The obtained protein can be efficiently degraded, and the proportion of flavor amino acid is remarkably increased; the inherent fishy smell of the goose blood is removed through thorough fermentation, and the fresh-keeping degree is high, so that the goose blood can be put into production as an antibiotic alternative scheme, and is green and environment-friendly.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization of livestock and poultry by-products and microbial fermentation technology, specifically to a method for fermenting goose blood using a compound microbial agent to prepare goose blood protein powder with high protein content, low fishy smell, and rich active ingredients. Background Technology

[0002] Poultry blood, due to its high protein, low fat, high amino acid content, rich mineral elements, and functional activity, is known as "liquid meat" and is often used as a feed component in livestock and aquaculture. It can partially replace plant or animal protein sources, especially the high lysine content, which allows it to effectively replace oilseed meals or fishmeal in feed, thereby increasing the content of limiting amino acids in complete feeds. Poultry blood mainly contains hemoglobin and serum albumin, as well as functional components such as superoxide dismutase, immunoglobulins, thrombin, and prothrombin. Poultry blood protein accounts for 15% of wet weight and over 90% of dry weight, containing 18 amino acids in good balance. However, the bloody taste of poultry blood protein reduces palatability and results in poor color and sensory performance in certain product categories, limiting its widespread and high-value utilization in the food industry.

[0003] Geese are herbivorous poultry, and their blood is rich in nutrients. Goose blood is a major byproduct of goose slaughtering, rich in protein (mainly hemoglobin), immunoglobulins, various amino acids, trace elements, and bioactive peptides, making it extremely nutritious. Current research on goose blood primarily focuses on its medical applications. Goose blood is believed to have detoxifying, softening and dissolving masses, invigorating qi and replenishing deficiency, warming the stomach and promoting salivation, and is particularly effective against esophageal cancer, stomach cancer, upper respiratory tract cancer, and liver cancer. It also has an adjunctive therapeutic effect on symptoms such as weakness, emaciation, and diabetes. From a traditional Chinese medicine perspective, goose blood is neutral in nature and salty in taste, penetrating deeply into the heart, liver, and stomach meridians. Modern research has found that goose blood is rich in immunoglobulins and anti-cancer factors, which can enhance human immunity and improve symptoms in cancer patients. However, current methods of utilizing goose blood are relatively crude, often involving simple drying to produce ordinary blood meal, which is then used as a feed additive. This traditional process has significant drawbacks: the high-temperature drying process easily leads to protein denaturation and reduced digestibility; it results in a strong fishy smell and poor palatability; the functional active ingredients are destroyed, resulting in low added value; and it is difficult to store, easily absorbing moisture and clumping. Aside from a small portion being processed into blood meal and blood tofu, the vast majority of the goose blood produced each year is not well utilized. Large quantities of goose blood are directly discharged into the environment, not only wasting valuable resources but also causing serious environmental problems.

[0004] Microbial fermentation technology offers an effective way to improve the above-mentioned problems. Microbial communities in nature exhibit various relationships, including neutrality, favoritism, cooperation, symbiosis, parasitism, competition, and antagonism. Among these, the positive interactions of favoritism, cooperation, and symbiosis inspire the use of mixed fermentation with different strains. Different microorganisms have different metabolic pathways, and their metabolic products have different synergistic effects, complementing each other and achieving effects that a single strain cannot. Microbial communities multiply rapidly in suitable environments, secreting large amounts of enzymes that partially decompose and transform macromolecular nutrients such as proteins and carbohydrates. Disulfide bonds in proteins that are difficult to open are broken, and large amounts of metabolic products accumulate. Through the metabolic action of specific microorganisms, macromolecular proteins in goose blood can be degraded, generating small peptides and free amino acids, improving digestibility and absorption; simultaneously removing the fishy smell and improving palatability; and potentially producing beneficial metabolites such as antimicrobial peptides and organic acids, enhancing the product's functionality and added value. However, current research on efficient and targeted fermentation strain combinations and processes for goose blood is insufficient, making it difficult to stably produce high-quality fermented goose blood protein products. Existing technology CN 106173272 A discloses a method for preparing animal blood protein peptides by fermentation. This method involves inoculating cultured Lactococcus lactis into fresh animal blood after anticoagulation treatment. After fermentation, whole blood protein peptides can be prepared, and these peptides can be further separated to prepare plasma protein peptides and hemoglobin peptides. This effectively overcomes the technical difficulties of fresh animal blood being easily perishable, requiring low-temperature control during storage and processing, and consuming large amounts of energy. Furthermore, after fermentation with Lactococcus lactis, the lactic acid nisin in the blood protein peptides can inhibit contamination by other bacteria, reducing antibiotic usage. Simultaneously, some proteins in the blood are hydrolyzed into short peptides, improving utilization. Existing technology CN 117186177 A discloses a duck hemoglobin peptide with uric acid-lowering activity and its preparation method, including the step of adding protease to duck hemoglobin to obtain uric acid-lowering peptides prepared by enzymatic hydrolysis of duck hemoglobin. This provides a new option for the deep processing of poultry blood in my country, enriches the variety of peptide products, and has significant economic and social benefits. 118805849A discloses a bioactive peptide composed of complex unsaturated fatty acids, which aims to solve the problem of declining growth performance and feed utilization caused by the lack of essential amino acids in plant protein sources such as soybean meal and fishmeal substitutes in traditional aquatic feed. This invention prepares low molecular weight bioactive peptides rich in unsaturated fatty acids from protein raw materials such as soybean meal, rapeseed cake, peanut meal and spray-dried chicken blood cell powder through specific fermentation, extraction and refining processes.

[0005] Therefore, it is of great significance to develop a microbial fermentation preparation method that can maximize the preservation of goose blood's nutrients, enhance its functional properties, and ensure stable and controllable processes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing highly active goose blood protein using compound microbial fermentation. This method, through the synergistic effect of a specific combination of microbial strains and fermentation processes, effectively degrades goose blood protein, removes fishy odor, and enriches active ingredients. The resulting product has high protein content, low fishy odor, and effective spoilage resistance, making it suitable as a high-quality feed protein source or functional food ingredient.

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

[0008] A method for preparing a biological composition, characterized by comprising the following steps:

[0009] 1) Raw material pretreatment: Take fresh goose blood, add food-grade sodium citrate at a content of 3-5g / kg, stir thoroughly, and pass through a 90-100 mesh sieve to obtain anticoagulant goose blood stock solution;

[0010] 2) Inoculation: Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into anticoagulated goose blood culture medium;

[0011] 3) Fermentation: Cultivate at 28-35℃ and 150-200r / min for 48-72h to obtain goose blood fermentation broth;

[0012] 4) Crushing and packaging: The fermented goose blood liquid is vacuum freeze-dried and passed through an 80-100 mesh sieve to obtain fermented goose blood complex protein.

[0013] Furthermore, the inoculation amount was 3.0% Bacillus subtilis and 0.5% Bacillus amyloliquefaciens.

[0014] Furthermore, the fermentation temperature is 30℃.

[0015] Furthermore, the fermentation pH was 6.8.

[0016] Furthermore, step 2) also includes inoculation with Aspergillus oryzae.

[0017] Furthermore, the Aspergillus oryzae, Bacillus subtilis, and Bacillus amyloliquefaciens have a synergistic effect in fermenting goose blood protein.

[0018] Furthermore, the inoculum amount of Aspergillus oryzae is 1.0%.

[0019] Another aspect of the present invention provides a fermented goose blood complex protein prepared by the aforementioned method.

[0020] Another aspect of the present invention provides a food product prepared from the aforementioned fermented goose blood complex protein.

[0021] Another aspect of the present invention provides a feed prepared from the aforementioned fermented goose blood complex protein.

[0022] The present invention discloses the following technical effects:

[0023] (1) Synergistic and efficient strains: The combination of Aspergillus oryzae with Bacillus subtilis and Bacillus amyloliquefaciens is scientifically formulated. Bacillus subtilis secretes abundant proteases (especially alkaline proteases), which efficiently hydrolyze large molecules of goose blood protein into small peptides and amino acids; together with Bacillus amyloliquefaciens and Aspergillus oryzae, the three work synergistically to achieve the goal of efficient protein degradation and a significant increase in the proportion of umami amino acids.

[0024] (2) Less fishy smell and better palatability: Fermentation completely removes the inherent fishy smell of goose blood. After fermentation, the product has a rich fermented aroma and is very palatable.

[0025] (3) High preservation: The fermented goose blood protein obtained by compound fermentation can protect it from spoilage to the greatest extent and can be put into production as an alternative to antibiotics, which is green and environmentally friendly. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] In this invention, sodium citrate is prepared as a 10% aqueous solution; for the determination of free amino acids, the sample pretreatment steps include: diluting the sample 6 times with 5% sulfosalicylic acid, adding 1.1694g of blank sample to 1.2ml of 6% sulfosalicylic acid, sonicating at 150W for 30min, placing in a 4℃ refrigerator for 2h, centrifuging at 12000r / min for 2min, filtering with a 0.22um filter membrane, and then analyzing. The analytical conditions were as follows: one sample analysis cycle was 60 min; separation column: elution buffer flow rate 0.5 ml / min, column temperature 70℃, column pressure 9.7 MPa; reaction column: ninhydrin and ninhydrin buffer flow rate 0.4 ml / min, column temperature 130℃, column pressure 1.05 MPa; when preparing the anticoagulated goose blood culture medium, goose blood stock solution was used as the sole nitrogen source, which contained: glucose 10 g / L, anticoagulated goose blood stock solution 10 g / L, potassium dihydrogen phosphate 0.3 g / L, disodium hydrogen phosphate dodecahydrate 4 g / L, CaCl2 0.05 g / L, magnesium sulfate heptahydrate 0.2 g / L, pH adjusted to 6.8. Other conventional alternative basal culture media in the art are also suitable for this invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Example 1: Optimization Experiment of Strains and Their Proportions

[0031] 1.1 Microbial fermentation experiment

[0032] 1.1.1 Sample Source:

[0033] Fresh goose blood: purchased from Qingyuan Jinyufeng Goose Industry Co., Ltd., the goose breed is Wuzong goose;

[0034] Microorganisms: Bacillus subtilis (CGMCC 1.1391), Bacillus amyloliquefaciens (

[0035] Bacillus amyloliquefaciens, CGMCC 1.403), Aspergillus oryzae (CGMCC 3.31);

[0036] 1.1.2 Determination Method

[0037] Protein content: determined by Kjeldahl method;

[0038] Amino nitrogen content: Formaldehyde titration (GB 5009.235-2016)

[0039] Soluble amino nitrogen content: TCA method (take 10 ml of goose blood fermentation broth, add 10 ml of 10% TCA solution, mix well, let stand for 30 min, centrifuge at 10000 rpm and 4℃ for 10 min, and determine the nitrogen content in the supernatant by Kjeldahl method).

[0040] Degree of hydrolysis determination: The degree of hydrolysis (DH) of a protein represents the extent to which peptide bonds are broken during the hydrolysis process. In this experiment, the degree of hydrolysis was used as an indicator of the production of polypeptides by microbial fermentation of goose blood meal. The total nitrogen content (C) before fermentation (mg / ml) and the soluble nitrogen content (B) and (A) before and after fermentation (mg / ml) were determined by the Kjeldahl method. The degree of hydrolysis (DH)% was calculated using the formula: Degree of hydrolysis (DH)% = (AB) / C × 100%.

[0041] 1.1.3 Test Methods

[0042] Activated Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into LB medium, and Aspergillus oryzae was inoculated into PDA medium; after incubation at 30℃ and 150 r / min for 24 h, the concentrations were measured to be 1.1 × 10⁻⁶. 7 cfu / ml, 1.5×10 7 cfu / ml and 1.0×10 6 CFU / ml was added to the anticoagulated goose blood stock solution culture medium at an inoculation rate of 3.0% (v:v) and cultured at 30℃ and 150r / min for 72h. The degree of hydrolysis was measured, and the results are shown in Table 1.

[0043] Table 1 Results of single-microbial fermentation

[0044]

[0045] This shows that after fermentation by Bacillus subtilis (CGMCC 1.1391), Bacillus amyloliquefaciens (CGMCC 1.403), and Aspergillus oryzae (CGMCC 3.31), the amino acid nitrogen content of goose blood increased by 3.39, 2.62, and 1.65 times, respectively, and the soluble protein content increased by 2.59, 1.71, and 1.14 times, respectively. These microorganisms can secrete proteases during fermentation. Goose blood protein is composed of tetrameric globin, hemoglobin, etc., and the proteases degrade macromolecules, leading to an increase in the content of soluble protein and amino protein.

[0046] Furthermore, the microbial fermentation ratio was experimentally determined, and the ratio content is shown in Table 2.

[0047] Table 2. List of Mixed Microbial Fermentation Ratios

[0048]

[0049]

[0050] The fermentation results were further determined using the methods described above, as shown in Table 3.

[0051] Table 3 Results of microbial fermentation

[0052]

[0053] This shows that the addition of Bacillus amyloliquefaciens, especially Aspergillus oryzae, has a significant synergistic effect on microbial fermentation. The optimal combination of inoculum amounts is 3.0% Bacillus subtilis, 0.5% Bacillus amyloliquefaciens, and 1.0% Aspergillus oryzae, which yields the best results in terms of the highest degree of hydrolysis, amino nitrogen content, and soluble nitrogen content.

[0054] The sensory evaluation results of Group 10 goose blood before and after fermentation are shown in Table 4.

[0055] Table 4 Sensory Evaluation

[0056]

[0057] This demonstrates that the fermentation of goose blood by mixed microorganisms reduces the bloody odor and produces a suitable aroma, resulting in a satisfactory outcome. Therefore, it is an ideal raw material for formulating fermented goose blood foods or feeds. Further single-factor and orthogonal optimization experiments revealed the optimal temperature parameters to be 30℃ and pH 6.8.

[0058] Example 2 Protein Property Determination

[0059] The free amino acid content of group 10 goose blood before and after fermentation in Example 1 was determined using a fully automated amino acid analyzer (Techcomp). The results were compared with the total free amino acid content and are shown in Table 5.

[0060] Table 5 Free amino acid content

[0061] Amino acid classification Before fermentation (mg / 100g) After fermentation (mg / 100ml) Essential amino acids 12.65 465.12 Umami amino acids 10.02 225.61 Total 58.33 871.46

[0062] The essential amino acids are lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, and valine; the umami amino acids are aspartic acid and glutamic acid. Therefore, after fermentation, the total amount of essential amino acids accounted for 53.37% of the total free amino acid content, an increase of 2.46 times compared to before fermentation; the content of umami amino acids accounted for 25.89% of the total free amino acid content, an increase of 1.51 times compared to before fermentation.

[0063] Example 3 Freshness determination

[0064] Volatile basic nitrogen (VBN) refers to the alkaline nitrogenous substances such as ammonia and amines produced during the spoilage process of animal-derived foods due to the decomposition of proteins by enzymes and bacteria. VBN accurately reflects the freshness of protein powder; the lower the VBN content, the higher the freshness. High VBN content indicates spoilage of the protein powder. Spoiled protein powder will destroy the nutritional components of blood plasma, contain pathogenic factors, and reduce the digestibility of the protein powder. The VBN values ​​of group 10 goose blood before and after fermentation were measured. The measurement method was based on GB 5009.228-2016, and the results are shown in Table 6.

[0065] Table 6 VBN Values

[0066]

[0067] It can be seen that, regardless of whether fermentation or not, the volatile basic nitrogen of goose blood gradually increases over time during storage. However, compared with the non-fermented group, the increase in VBN in fermented goose blood is significantly lower than that in the untreated group. This indicates that the microbial combination of the present invention has a preservation effect on fermented goose blood, which may be related to the production of antimicrobial peptides by fermentation metabolism.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for producing complex proteins through fermentation, characterized in that, Includes the following steps: 1) Raw material pretreatment: Take fresh goose blood, add food-grade sodium citrate at a content of 3-5g / kg, stir thoroughly, and pass through a 90-100 mesh sieve to obtain anticoagulant goose blood stock solution; 2) Inoculation: Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into anticoagulated goose blood culture medium; 3) Fermentation: Cultivate at 28-35℃ and 150-200r / min for 48-72h to obtain goose blood fermentation broth; 4) Crushing and packaging: The fermented goose blood liquid is vacuum freeze-dried and passed through an 80-100 mesh sieve to obtain fermented goose blood complex protein.

2. The method according to claim 1, characterized in that, The inoculation amount was 3.0% Bacillus subtilis and 0.5% Bacillus amyloliquefaciens.

3. The method according to claim 2, characterized in that, The fermentation temperature is 30℃.

4. The method according to claim 2, characterized in that, The fermentation pH was 6.

8.

5. The method according to claim 2, characterized in that, Step 2) also includes Aspergillus oryzae inoculation.

6. The method according to claim 5, characterized in that, The Aspergillus oryzae, Bacillus subtilis, and Bacillus amyloliquefaciens have a synergistic effect in fermenting goose blood protein.

7. The method according to claim 6, characterized in that, The inoculum size of Aspergillus oryzae was 1.0%.

8. The fermented goose blood complex protein prepared according to claim 1.

9. A food product, characterized in that, The food product is prepared by processing the fermented goose blood complex protein according to claim 8.

10. A feed, characterized in that, The feed is prepared by processing the fermented goose blood complex protein according to claim 8.

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