Bacillus subtilis 12-10-2 and application thereof in synergistic preparation of fish oil and fish sauce

Abstract

The invention discloses bacillus subtilis 12-10-2 and application thereof in synergistic preparation of fish oil and fish sauce, and belongs to the technical field of microbial fermentation. The preservation number of the bacillus subtilis (Bacillus subtilis) 12-10-2 is CGMCC (China General Microbiological Culture Collection Center) No.34966. The bacillus subtilis (Bacillus subtilis) 12-10-2 can be used for preparing the bacillus subtilis. The bacillus subtilis 12-10-2 can be used for efficiently hydrolyzing protein in a high-salt environment. The bacillus subtilis 12-10-2 is used for performing bacterium-adding fermentation on the snakehead processing leftovers, so that the fish oil and the fish sauce can be synergistically prepared, and the obtained product is high in yield and good in quality. A new microbial resource is provided for high-value utilization of snakehead processing leftovers, the problems of environmental pollution and resource waste caused by improper treatment can be solved, a new technical support is provided for establishment of a preparation process of high-quality fish oil and fish sauce, and the method has a wide market application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a Bacillus subtilis 12-10-2 and its application in the synergistic preparation of fish oil and fish sauce. Background Technology

[0002] Snakehead fish (Channa argus), also known as blackfish, snakehead fish, or black carp, belongs to the genus Channa in the family Channidae of the order Perciformes. Snakehead fish are favored by consumers for their delicious and nutritious flesh, and their strong adaptability and rapid growth make them an important species for aquaculture. Snakehead fish meat is tender and boneless, retains its elasticity even after prolonged cooking, and its dense muscle fibers allow it to maintain its shape even when sliced ​​thinly, making it a preferred ingredient for dishes such as pickled fish and boiled fish. With the steady growth of the market for pickled fish and boiled fish, the snakehead fish fillet processing industry has developed rapidly. However, the processing of snakehead fish into fillets generates a large amount of processing waste, including fish heads, skin, scraps of fish meat, bones, and internal organs. These wastes are generally processed directly into animal feed, resulting in low added value. Therefore, solving the problem of high-value utilization of snakehead fish processing waste is a pressing issue for the snakehead fish industry.

[0003] Snakehead fish processing by-products are rich in lipids and can be used to produce fish oil. Traditional fish oil production often uses a cooking method; however, high temperatures accelerate the oxidation of unsaturated fatty acids in the fish oil, leading to a decline in quality. Furthermore, the high-temperature treatment process discards nutrients such as proteins in the waste liquid, polluting the environment and wasting resources. This invention utilizes microbial fermentation, which can fully utilize the lipids and proteins in snakehead fish processing by-products to efficiently produce high-quality fish oil and fish sauce without causing environmental pollution or resource waste, and has broad market application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus subtilis 12-10-2 strain and its application in the co-production of fish oil and fish sauce, thereby solving the problems existing in the prior art. This invention screened and obtained a Bacillus subtilis 12-10-2 strain capable of efficiently hydrolyzing proteins in a high-salt environment. Using this Bacillus subtilis 12-10-2 for the fermentation of snakehead processing by-products, the co-production of fish oil and fish sauce can be achieved, resulting in products with high yield and good quality. This invention provides a new microbial resource for the high-value utilization of snakehead processing by-products, solving the problems of environmental pollution and resource waste caused by improper handling, and providing new technical support for the establishment of high-quality fish oil and fish sauce preparation processes, with broad market application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a Bacillus subtilis 12-10-2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2025, with accession number CGMCC No. 34966.

[0007] The present invention also provides an application of the above-mentioned Bacillus subtilis 12-10-2 in the preparation of microbial fermentation agents.

[0008] The present invention also provides a microbial fermentation agent, wherein the microbial agent comprises the above-mentioned Bacillus subtilis 12-10-2 and / or its metabolites.

[0009] The present invention also provides the application of the above-mentioned Bacillus subtilis 12-10-2 or the above-mentioned microbial fermentation agent in the synergistic preparation of fish oil and fish sauce.

[0010] The present invention also provides a method for synergistically preparing fish oil and fish sauce, wherein the above-mentioned Bacillus subtilis 12-10-2 or the above-mentioned microbial fermentation agent is used to ferment the processing waste of snakehead fish. After the fermentation is completed, the upper liquid is collected as fish oil and the lower liquid is collected as fish sauce.

[0011] Furthermore, the step of pre-treating the snakehead processing waste material before adding bacteria for fermentation includes the following steps;

[0012] The pretreatment step is as follows: mince the snakehead processing waste, add water at a ratio of 1-2g:1-2mL, and adjust the salt concentration to 5wt%-15wt%.

[0013] Furthermore, during the fermentation process, the final concentration of Bacillus subtilis 12-10-2 in the fermentation system is 10. 6 -10 8 CFU / g.

[0014] Furthermore, the fermentation temperature is 25-35℃, and the time is 3-30 days.

[0015] The present invention also provides a fish oil prepared by the above method.

[0016] The present invention also provides a fish sauce prepared by the above method.

[0017] The present invention discloses the following technical effects:

[0018] This invention screened and obtained a Bacillus subtilis strain 12-10-2 capable of hydrolyzing proteins in a high-salt environment. Using this Bacillus subtilis strain 12-10-2 for fermentation of snakehead processing by-products allows for the synergistic production of fish oil and fish sauce, resulting in high yields and good quality products. The fermentation with Bacillus subtilis strain 12-10-2 rapidly decomposes and destroys the protein structure in snakehead processing by-products, promoting rapid and automatic separation of lipids from water-soluble components such as proteins and amino acids. High-quality fish oil and fish sauce can be obtained through simple separation and extraction. Compared to traditional cooking methods, fermentation with Bacillus subtilis strain 12-10-2 significantly increases fish oil yield and reduces fish oil oxidation; the proteins in the by-products decompose rapidly, and the amino acid nitrogen content of the fish sauce is significantly increased.

[0019] This invention uses snakehead processing by-products as raw materials to produce fish oil and fish sauce, which can realize the high-value utilization of snakehead processing by-products, solve the problems of environmental pollution and resource waste caused by improper processing, and provide new technical support for the establishment of high-quality fish oil and fish sauce preparation processes, and has broad market application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Phylogenetic tree of strain 12-10-2;

[0022] Figure 2 The results show the protease activity of strain 12-10-2 at different salt concentrations. Detailed Implementation

[0023] 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.

[0024] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0025] 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.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0029] Example 1

[0030] Potential microorganisms were screened and isolated from fish sauce samples fermented for 12 months at Shantou Fish Sauce Factory Co., Ltd. in Guangdong Province. The screening process is as follows:

[0031] Fish sauce fermentation broth at different dilutions was spread onto screening media (10 g / L peptone, 5 g / L yeast extract, 20 g / L agar, 30 g / L skim milk powder, and 100 g / L sodium chloride) and incubated at 37°C for 3 days. Single colonies producing a clear zone were selected and purified by continuous streaking on LB agar plates to obtain the target strain 12-10-2. Molecular biological identification of this strain was performed. The 16S rRNA gene of the strain was sequenced using universal bacterial primers. The sequencing results are shown in SEQ ID NO.1, and a molecular phylogenetic tree was constructed. The phylogenetic tree is shown below. Figure 1 As shown, the strain was identified as Bacillus subtilis.

[0032] SEQ ID NO.1:

[0033]

[0034] The strain 12-10-2 was cultured in LB medium containing different salt concentrations, and its protease activity was determined using the Folin-Ciocalteu method. The results are as follows: Figure 2 As shown, this strain has the ability to produce proteases under high salt conditions. The protease activity is highest at a salt concentration of 5%, and it can still maintain 43.7% of the protease activity under the unsalted condition at a salt concentration of 15%, indicating that this strain has the potential to hydrolyze proteins under high salt conditions.

[0035] The strain 12-10-2 was named Bacillus subtilis 12-10-2 and was deposited on June 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34966. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] Example 2

[0037] Bacillus subtilis 12-10-2 was cultured at 35℃ for 1 day, centrifuged at 12000g for 10 min at 4℃, and then resuspended.

[0038] Mince the snakehead fish by-products in a meat grinder. Add water at a ratio of 2:1 (g:mL) and 5wt% salt. 8 Add Bacillus subtilis 12-10-2 at a concentration of CFU / g (raw material), ferment at 25℃ for 3 days, let stand for 1 hour after fermentation, collect the upper liquid as fish oil, and the lower liquid as fish sauce.

[0039] Example 3

[0040] Bacillus subtilis 12-10-2 was cultured at 35℃ for 1 day, centrifuged at 12000g for 10 min at 4℃, and then resuspended.

[0041] Mince the snakehead fish processing waste in a meat grinder, add water at a material-to-liquid ratio of 1:1 (g:mL), add salt at 10wt%, and so on. 6 Add Bacillus subtilis 12-10-2 at a concentration of CFU / g (raw material), ferment at 30℃ for 3 days, let stand for 1 hour after fermentation, collect the upper liquid as fish oil, and the lower liquid as fish sauce.

[0042] Example 4

[0043] Bacillus subtilis 12-10-2 was cultured at 35℃ for 1 day, centrifuged at 12000g for 10 min at 4℃, and then resuspended.

[0044] Mince the snakehead fish processing waste in a meat grinder, add water at a material-to-liquid ratio of 1:2 (g:mL), add salt at 15wt%, and add 10... 8 Add Bacillus subtilis 12-10-2 at a concentration of CFU / g (raw material), ferment at 25℃ for 15 days, let stand for 1 hour after fermentation, collect the upper liquid as fish oil, and the lower liquid as fish sauce.

[0045] Example 5

[0046] Bacillus subtilis 12-10-2 was cultured at 35℃ for 1 day, centrifuged at 12000g for 10 min at 4℃, and then resuspended.

[0047] Mince the snakehead fish by-products in a meat grinder. Add water at a ratio of 2:1 (g:mL), and salt at 15wt%. 7 Add Bacillus subtilis 12-10-2 at a concentration of CFU / g (raw material), ferment at 35℃ for 6 days, let stand for 1 hour after fermentation, collect the upper liquid as fish oil, and the lower liquid as fish sauce.

[0048] Comparative Example 1

[0049] Fish oil and fish sauce are prepared using a steaming method. The specific preparation process is as follows:

[0050] The by-products of snakehead fish processing are put into a meat grinder and minced. Water is added at a ratio of 2:1 (g:mL) and mixed evenly. The mixture is then extracted in a 90℃ constant temperature water bath for 40 minutes with constant stirring. After standing and cooling for 1 hour, the upper liquid is fish oil and the lower liquid is fish sauce.

[0051] Experimental Example 1

[0052] The quality of the fish oil and fish sauce prepared in Examples 2-5 and Comparative Example 1 was determined using the following methods:

[0053] (1) Determination of fish oil extraction rate

[0054] The fat content of snakehead processing by-products was determined according to the "Soxhlet extraction method" in GB 5009.6—2025 "National Food Safety Standard - Determination of Fat in Food". The fish oil extraction rate was calculated based on the percentage of fat extracted by cooking or fermentation relative to the total fat content in the snakehead processing by-products.

[0055] (2) Determination of acid value of fish oil

[0056] The determination of acid value in fish oil is based on the "cold solvent indicator titration method" in GB 5009.229—2025 "National Food Safety Standard: Determination of Acid Value in Food".

[0057] (3) Determination of peroxide value of fish oil

[0058] The determination of peroxide value in fish oil shall be performed in accordance with the "indicator titration method" in GB 5009.227—2023 "National Food Safety Standard for Determination of Peroxide Value in Food".

[0059] (4) Determination of iodine value in fish oil

[0060] The determination of iodine value in fish oil shall be in accordance with GB / T 5532—2022 "Determination of Iodine Value in Animal and Vegetable Oils".

[0061] (5) Determination of amino acid nitrogen content in fish sauce

[0062] The determination of amino acid nitrogen in fish sauce is based on the "acidity meter method" in GB 5009.235—2016 "National Food Safety Standard: Determination of Amino Acid Nitrogen in Food".

[0063] The results are shown in Table 1. As can be seen from Table 1, after fermentation, the lipids and proteins in the snakehead processing by-products were fully utilized through the metabolic action of Bacillus subtilis. Specifically, compared with the cooking method (Comparative Example 1), the fish oil extraction rate in Example 2 increased by 15.2%, and the quality of the fish oil was significantly improved. Specifically, the acid value and peroxide value of the fish oil decreased by 23.1% and 70.9%, respectively, while the iodine value increased by 8.5%. The amino acid nitrogen content in the fish sauce increased by 1.1 times after fermentation. In Example 3, the fish oil extraction rate increased by 13.9%, and the quality of the fish oil was significantly improved. Specifically, the acid value and peroxide value of the fish oil decreased by 40.7% and 50.7%, respectively, while the iodine value increased by 10.2%. The amino acid nitrogen content in the fish sauce increased by 1.9 times after fermentation. In Example 4, the fish oil extraction rate increased by 15.7%, and the quality of the fish oil was basically the same as that of the cooking method. The amino acid nitrogen content in the fish sauce increased by 2.4 times after fermentation. In Example 5, the extraction rate of fish oil was increased by 11.7%, and the quality of fish oil was significantly improved. Specifically, the acid value and peroxide value of the fish oil decreased by 12.1% and 36.6%, respectively, while the iodine value increased by 5.9%. After fermentation, the amino acid nitrogen content in the fish sauce increased by 2.0 times.

[0064] Table 1. Effects of different treatments on the quality of fish sauce and fish oil

[0065]

[0066] 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 Bacillus subtilis 12-10-2 strain, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2025, with accession number CGMCCNo.34966.

2. The application of Bacillus subtilis 12-10-2 as described in claim 1 in the preparation of microbial fermentation agents.

3. A microbial fermentation agent, characterized in that, The microbial agent includes Bacillus subtilis 12-10-2 as described in claim 1 and / or its metabolites.

4. The application of Bacillus subtilis 12-10-2 as described in claim 1 or the microbial fermentation agent as described in claim 3 in the synergistic preparation of fish oil and fish sauce.

5. A method for synergistically preparing fish oil and fish sauce, characterized in that, Using Bacillus subtilis 12-10-2 as described in claim 1 or the microbial fermentation agent as described in claim 3, the by-products of snakehead fish processing are fermented with bacteria. After fermentation, the upper liquid is collected as fish oil and the lower liquid is collected as fish sauce.

6. The method according to claim 5, characterized in that, Before the addition of bacteria for fermentation, the process includes a step of pre-treating the snakehead processing waste. The pretreatment step is as follows: mince the snakehead processing waste, add water at a ratio of 1-2g:1-2mL, and adjust the salt concentration to 5wt%-15wt%.

7. The method according to claim 5, characterized in that, During the fermentation process, the final concentration of Bacillus subtilis 12-10-2 in the fermentation system is 10. 6 -10 8 CFU / g.

8. The method according to claim 5, characterized in that, The fermentation process is carried out at a temperature of 25-35℃ for 3-30 days.

9. A fish oil prepared by the method according to any one of claims 5-8.

10. A fish sauce prepared by the method according to any one of claims 5-8.

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