A method for efficiently preparing earthworm functional liquid by using earthworm intestinal endogenous probiotics and enzymes and earthworm functional liquid
By synergistic fermentation of endogenous probiotics and enzymes in the earthworm gut, an earthworm functional liquid rich in amino acids is prepared, which solves the problems of high cost and health risks of amino acid supplementation in existing technologies and achieves a highly efficient animal nutrition supplementation effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
- Filing Date
- 2025-08-11
- Publication Date
- 2026-06-26
AI Technical Summary
Existing technologies for directly supplementing amino acids into feed are costly and pose health risks, and lack synergistic effects. Bio-fermentation technology has not yet been fully utilized in livestock farming.
Earthworm functional liquid was prepared by synergistic fermentation of endogenous probiotics (Bacillus belyssus XY1, Priestella megaterium and Lactococcus lactis) and enzymes (protease, lipase, earthworm fibrinolytic enzyme and amylase) in the earthworm gut. It is rich in amino acids and enzyme active ingredients.
It significantly increases the total amino acid and small peptide content in earthworm functional fluid, enhances the effect of animal nutrition supplementation, and promotes animal growth.
Abstract
Description
Technical Field
[0001] This application relates to the field of bio-fermentation technology, specifically to a method for efficiently preparing earthworm functional liquid using endogenous probiotics and enzymes in the earthworm gut, and the earthworm functional liquid itself. Background Technology
[0002] With the technological development of the aquaculture industry, the feed for livestock has been transformed from traditional extensive farming to precision nutrition regulation. For example, feed formulations are optimized through technologies such as genomics and microbiome, or functional additives (such as enzyme preparations and probiotics) are developed to improve digestibility and nutritional value of feed.
[0003] For example, prior art 1: Chinese patent application 200910213358.1 discloses a total mixed feed pellet product for mutton sheep and its preparation method. The total mixed feed pellet product is prepared by the following method: a compound trace element premix is produced using various trace element raw materials and inorganic carriers; a compound vitamin premix is produced using various vitamin raw materials, antioxidants and organic carriers; other additive premixes are produced using amino acids, fatty acids, metabolic regulators and carriers; according to the compound premix formula, the aforementioned compound trace element premix, compound vitamin premix, other additive premixes and other materials are separately metered and mixed to produce a compound additive premix; then, according to the production formula, each main raw material and the compound additive premix are separately metered, mixed evenly and made into pellets and packaged.
[0004] The specification of prior art 1 states: "The formula of the compound additive premix D is: 20 parts of compound trace element premix A, 5 parts of compound vitamin premix B, 10-30 parts of other additive premix C, 30 parts of dicalcium phosphate, 10-20 parts of limestone powder, and 5-15 parts of corn gluten powder or soybean meal powder; wherein the formula of other additive premix C1 by weight is: 0.5-5.0 parts of choline, 2.5-8.0 parts of amino acids, 1.0-4.0 parts of fatty acids, 3.0-7.0 parts of urea, 5.0-7.0 parts of salt, 1.0-2.5 parts of metabolic regulator, and 5.0-9.5 parts of carrier."
[0005] Existing technology 1 promotes animal growth by directly supplementing trace elements, vitamins, amino acids, fatty acids, and other nutrients; however, directly supplementing amino acids in feed may have certain drawbacks:
[0006] (1) Directly supplementing large amounts of amino acids results in high costs, and high-purity synthesized amino acids may be accompanied by harmful residues, thus posing health risks to animals;
[0007] (2) Directly adding amino acids lacks synergistic promoting effect, and additional active ingredients such as enzymes, organic acids and growth factors are needed to achieve synergistic effect.
[0008] Therefore, bio-fermentation technology has become a better direction for providing amino acids. However, how to use bio-fermentation technology to supplement amino acids in animal husbandry is still in the exploratory stage and requires further in-depth research. Summary of the Invention
[0009] One of the objectives of this application is to provide a method for efficiently preparing earthworm functional liquid using endogenous probiotics and enzymes in the earthworm gut. The earthworm functional liquid prepared by this method is rich in amino acids. It can be used to ferment feed or added to feed as an additive to effectively supplement various amino acids, thereby promoting animal development and growth.
[0010] Another objective of this application is to provide an earthworm functional liquid, which is prepared using the method of this application that utilizes endogenous probiotics and enzymes in the earthworm gut to efficiently prepare earthworm functional liquid. It is rich in amino acids, and because it is obtained through biological fermentation, it contains active ingredients such as enzymes, which can effectively supplement the nutrients needed by animals.
[0011] To achieve the above objectives, this application provides a method for efficiently preparing earthworm functional fluid using endogenous probiotics and enzymes from the earthworm gut, comprising the following steps:
[0012] Step 1: Activate and expand Bacillus belye XY1, Priestella megaterium and Lactococcus lactis respectively to obtain Bacillus belye XY1 bacterial suspension, Priestella megaterium bacterial suspension and Lactococcus lactis bacterial suspension;
[0013] Step 2: Inoculate the earthworm slurry with Bacillus vesicle XY1, Priestella megaterium, Lactococcus lactis, and compound enzyme to obtain earthworm functional liquid.
[0014] The Bacillus velezensis XY1, classified and named Bacillus velezensis, with accession number GDMCCNO.64017, accession date November 14, 2023, is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0015] The *Priestia megaterium* species described is classified as *Priestia megaterium*, with accession number GDMCCNO.66160, accession date of April 18, 2025, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0016] The lactococcus lactis subsp. lactis, classified and named, has the accession number CGMCC NO.34336, the accession date is April 25, 2025, and it is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0017] The complex enzyme includes at least one of protease, lipase, earthworm fibrinolytic enzyme, and amylase.
[0018] In the technical solution of this application, those skilled in the art can obtain Bacillus belyssus XY1 bacterial suspension, Priestella megaterium bacterial suspension and Lactococcus lactis bacterial suspension through conventional activation and expansion treatment methods.
[0019] Preferably, the specific operation of step 1 is as follows:
[0020] Step 11: Inoculate Bacillus belye XY1, Priscilla megaterium and Lactococcus lactis at an inoculation rate of 1% into 10ml of liquid culture medium to activate them and obtain Bacillus belye XY1 activation solution, Priscilla megaterium activation solution and Lactococcus lactis activation solution.
[0021] Step 12: Inoculate Bacillus belye XY1 activation solution, Priscilla megaterium activation solution and Lactococcus lactis activation solution into 50ml of liquid culture medium at an inoculation rate of 2% respectively for expansion culture treatment to obtain Bacillus belye XY1 bacterial suspension, Priscilla megaterium bacterial suspension and Lactococcus lactis bacterial suspension.
[0022] Preferably, step 2 involves inoculating earthworm slurry with 1-5% Bacillus belye XY1 bacterial solution, 1-5% Priestella megaterium and Lactococcus lactis bacterial solution, 1-5% Lactococcus lactis bacterial solution and 1-5% compound enzyme, and fermenting at 30-35℃ and pH 5.0-6.5 for 6-12 hours to obtain earthworm functional liquid.
[0023] The earthworm slurry is obtained by grinding earthworms. Specifically, the earthworm slurry is prepared by washing earthworms to remove mud, putting them into a grinder, and grinding them at 3000 r / min for 10-15 minutes at room temperature to obtain the earthworm slurry.
[0024] This application also discloses an earthworm functional liquid, which is prepared by the above-mentioned method of efficiently preparing earthworm functional liquid using endogenous probiotics and enzymes in the earthworm intestine.
[0025] Beneficial effects
[0026] Compared with the prior art, this application has at least the following advantages:
[0027] Earthworm functional liquid was obtained by synergistic fermentation of Bacillus vesiculosus XY1, Priestella megaterium, Lactococcus lactis and compound enzymes, which significantly increased the content of total amino acids and small peptides, and could further enhance the effect of earthworm functional liquid. Detailed Implementation
[0028] The present application will be further described below with reference to embodiments, but this does not constitute any limitation on the present application. Any limited modifications made within the scope of the claims of the present application shall still be within the scope of the claims of the present application.
[0029] To illustrate the technical content of this application in detail, the following description is provided in conjunction with the embodiments.
[0030] In the following examples and comparative examples, the Bacillus velezensis XY1, classified as Bacillus velezensis, with accession number GDMCC NO.64017, accession date November 14, 2023, is deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0031] The *Priestia megaterium* species described is classified as *Priestia megaterium*, with accession number GDMCCNO.66160, accession date of April 18, 2025, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0032] The lactococcus lactis subsp. lactis, classified and named, has the accession number CGMCC NO.34336, the accession date is April 25, 2025, and it is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0033] The protease, lipase and amylase were all purchased from Guangzhou Huiwang Biotechnology Co., Ltd.
[0034] Earthworm fibrinolytic enzyme was purchased from Lanzhou Waterles Biotechnology Co., Ltd.
[0035] The earthworm slurry is prepared as follows: earthworms are cleaned and mud is removed, then put into a grinder and ground at 3000 r / min for 15 minutes at room temperature to obtain earthworm slurry.
[0036] Example 1
[0037] An earthworm functional fluid is prepared using the following steps:
[0038] Step 1: Inoculate Bacillus belye XY1, Priscilla megaterium, and Lactococcus lactis at a 1% inoculation rate into 10 ml of liquid culture medium to activate them, thus obtaining activated solutions of Bacillus belye XY1, Priscilla megaterium, and Lactococcus lactis.
[0039] Step 2: Inoculate Bacillus belye XY1 activation solution, Priscilla megaterium activation solution and Lactococcus lactis activation solution into 50ml of liquid culture medium at an inoculation rate of 2% respectively for expansion treatment to obtain Bacillus belye XY1 bacterial suspension, Priscilla megaterium bacterial suspension and Lactococcus lactis bacterial suspension;
[0040] Step 3: Inoculate earthworm slurry with 3% Bacillus belye XY1 bacterial solution, 2% Priestella megaterium bacterial solution, 2% Lactococcus lactis bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0041] Example 2
[0042] The process is largely the same as in Example 1, except that step 3 is changed to: inoculating 5% Bacillus belye XY1 bacterial solution, 3% Priestella megaterium bacterial solution, 1% Lactococcus lactis bacterial solution, 0.5% protease and 0.5% lipase into earthworm slurry and fermenting at 35°C and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0043] Example 3
[0044] The process is largely the same as in Example 1, except that step 3 is changed to: inoculating 1% Bacillus belye XY1 bacterial solution, 1% Priestella megater bacterial solution, 4% Lactococcus lactis bacterial solution, 1% protease, 1% lipase, 1% earthworm fibrinolytic enzyme and 1% amylase into earthworm slurry and fermenting at 35°C and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0045] Comparative Example 1
[0046] An earthworm functional fluid is prepared using the following steps:
[0047] Step 1: Inoculate Bacillus belye XY1 at a 1% inoculum into 10 ml of liquid culture medium to activate it and obtain Bacillus belye XY1 activated solution;
[0048] Step 2: Inoculate 2% of the activated Bacillus belye XY1 solution into 50ml of liquid culture medium for expansion treatment to obtain Bacillus belye XY1 bacterial suspension;
[0049] Step 3: Inoculate earthworm slurry with 7% Bacillus vesicle XY1 bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0050] Comparative Example 2
[0051] An earthworm functional fluid is prepared using the following steps:
[0052] Step 1: Inoculate *Priscilla megaterium* at a 1% inoculum into 10 ml of liquid culture medium to activate it and obtain *Priscilla megaterium* activation solution.
[0053] Step 2: Inoculate the activated solution of Priestella megaterium at a 2% inoculation rate into 50 ml of liquid culture medium for expansion culture to obtain Priestella megaterium bacterial suspension;
[0054] Step 3: Inoculate earthworm slurry with 7% Priestella megaterium bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0055] Comparative Example 3
[0056] An earthworm functional fluid is prepared using the following steps:
[0057] Step 1: Inoculate Lactococcus lactis at a 1% inoculum into 10 ml of liquid culture medium to activate it and obtain Lactococcus lactis activated solution;
[0058] Step 2: Inoculate the activated lactococcus lactis solution at a 2% inoculation rate into 50ml of liquid culture medium for expansion culture to obtain lactococcus lactis bacterial suspension;
[0059] Step 3: Inoculate 7% Lactococcus lactis bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase into earthworm slurry and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0060] Comparative Example 4
[0061] An earthworm functional fluid is prepared using the following steps:
[0062] Step 1: Inoculate Bacillus belye XY1 and Priestella megaterium at a 1% inoculation rate into 10 ml of liquid culture medium to activate them and obtain Bacillus belye XY1 activation solution and Priestella megaterium activation solution, respectively.
[0063] Step 2: Inoculate Bacillus belye XY1 activation solution and Priscilla megaterium activation solution at an inoculation rate of 2% into 50ml of liquid culture medium for expansion treatment to obtain Bacillus belye XY1 bacterial suspension and Priscilla megaterium bacterial suspension.
[0064] Step 3: Inoculate earthworm slurry with 4.2% Bacillus belye XY1 bacterial solution, 2.8% Priestella megater bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0065] Comparative Example 5
[0066] An earthworm functional fluid is prepared using the following steps:
[0067] Step 1: Inoculate Bacillus belye XY1 and Lactococcus lactis at a 1% inoculation rate into 10 ml of liquid culture medium for activation to obtain Bacillus belye XY1 activation solution and Lactococcus lactis activation solution, respectively.
[0068] Step 2: Inoculate Bacillus belye XY1 activation solution and Lactococcus lactis activation solution at an inoculation rate of 2% into 50ml of liquid culture medium for expansion culture to obtain Bacillus belye XY1 bacterial suspension and Lactococcus lactis bacterial suspension;
[0069] Step 3: Inoculate earthworm slurry with 4.2% Bacillus vesicle XY1 bacterial solution, 2.8% Lactococcus lactis bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0070] Comparative Example 6
[0071] An earthworm functional fluid is prepared using the following steps:
[0072] Step 1: Inoculate *Priscilla megaterium* and *Lactococcus lactis* at a 1% inoculation rate into 10 ml of liquid culture medium to activate them and obtain *Priscilla megaterium* activation solution and *Lactococcus lactis* activation solution, respectively.
[0073] Step 2: Inoculate the activated solutions of *Priscilla megaterium* and *Lactococcus lactis* at a rate of 2% into 50 ml of liquid culture medium for expansion culture to obtain *Priscilla megaterium* and *Lactococcus lactis* bacterial solutions.
[0074] Step 3: Inoculate earthworm slurry with 3.5% Priestella megaterium bacterial solution, 3.5% Lactococcus lactis bacterial solution, 1% protease, 1% earthworm fibrinolytic enzyme and 1% amylase and ferment at 35℃ and pH 6.5 for 9 hours to obtain earthworm functional liquid.
[0075] Amino acid content detection method: Detected according to GB / T 18246-2019 method.
[0076] Method for detecting small peptide content in functional liquids: High performance liquid chromatography (HPLC)
[0077] The amino acid content of the earthworm functional liquids of Examples 1-3 and Comparative Examples 1-3 was tested according to the above detection method, and the results are shown in Table 1.
[0078] Table 1. Results of amino acid content detection in earthworm functional fluids of Examples 1-3 and Comparative Examples 1-3
[0079] type Small peptide content (%) Total amino acid content (%) Example 1 65.52±2.05 72.14±2.29 Example 2 80.91±2.76 85.43±2.53 Example 3 58.68±2.01 68.07±2.11 Comparative Example 1 38.45±1.14 45.46±1.62 Comparative Example 2 35.66±1.25 42.84±1.87 Comparative Example 3 38.07±1.06 44.09±1.64 Comparative Example 4 50.08±1.55 60.35±2.02 Comparative Example 5 52.23±1.77 62.11±2.15 Comparative Example 6 46.62±1.84 55.71±2.03
[0080] According to the results in Table 1:
[0081] According to the data from Examples 1-3, the technical solution of this application can ferment earthworm functional liquid with high content of amino acids and small peptides.
[0082] According to the data from Comparative Examples 1-3, the total amino acid and small peptide contents of earthworm functional liquids obtained by fermenting with compound enzymes using Bacillus belye XY1, Priestella megaterium, or Lactococcus lactis alone are comparable, with Priestella megaterium being weaker and Lactococcus lactis being stronger. However, when Bacillus belye XY1, Priestella megaterium, and Lactococcus lactis were combined and fermented in Example 1, the total amino acid and small peptide contents of the earthworm functional liquid were significantly increased. This indicates that the combination of Bacillus belye XY1, Priestella megaterium, and Lactococcus lactis has a synergistic effect in increasing amino acid content, and can further improve the total amino acid and small peptide contents of earthworm functional liquids.
[0083] Meanwhile, according to the data from Comparative Examples 4-6, although the earthworm functional liquid obtained by fermenting earthworm slurry with any two of Bacillus belysium XY1, Priscilla megaterium, and Lactococcus lactis can further increase the total amino acid and small peptide content of earthworm functional liquid compared with single strains, its improvement effect is significantly lower than that of the technical scheme of Bacillus belysium XY1, Priscilla megaterium, and Lactococcus lactis used in Example 1. In particular, the technical scheme of Comparative Example 6 using Priscilla megaterium and Lactococcus lactis only showed a slight improvement compared with Comparative Example 3, further verifying that the combination of Bacillus belysium XY1, Priscilla megaterium, and Lactococcus lactis can produce a synergistic effect in increasing the total amino acid content.
[0084] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.
Claims
1. A method for efficiently preparing earthworm functional fluid using endogenous probiotics and enzymes from the earthworm gut, characterized in that, Includes the following steps: Step 1: Activate and expand Bacillus belye XY1, Priestella megaterium and Lactococcus lactis respectively to obtain Bacillus belye XY1 bacterial suspension, Priestella megaterium bacterial suspension and Lactococcus lactis bacterial suspension; Step 2: Inoculate the earthworm slurry with Bacillus vesicle XY1, Priestella megaterium, Lactococcus lactis, and compound enzyme to obtain earthworm functional liquid. The Bacillus velezensis XY1, classified and named Bacillus velezensis, with accession number GDMCCNO.64017, accession date November 14, 2023, is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The *Priestia megaterium* species described is classified as *Priestia megaterium*, with accession number GDMCCNO.66160, accession date of April 18, 2025, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The lactococcus lactis subsp. lactis, classified and named, has the accession number CGMCC NO.34336, the accession date is April 25, 2025, and it is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The complex enzyme includes at least one of protease, lipase, earthworm fibrinolytic enzyme, and amylase.
2. The method for efficiently preparing earthworm functional fluid using endogenous probiotics and enzymes from the earthworm gut according to claim 1, characterized in that, The specific operation of step 1 is as follows: Step 11: Inoculate Bacillus belye XY1, Priscilla megaterium and Lactococcus lactis at an inoculation rate of 1% into 10ml of liquid culture medium to activate them and obtain Bacillus belye XY1 activation solution, Priscilla megaterium activation solution and Lactococcus lactis activation solution. Step 12: Inoculate Bacillus belye XY1 activation solution, Priscilla megaterium activation solution and Lactococcus lactis activation solution into 50ml of liquid culture medium at an inoculation rate of 2% respectively for expansion culture treatment to obtain Bacillus belye XY1 bacterial suspension, Priscilla megaterium bacterial suspension and Lactococcus lactis bacterial suspension.
3. The method for efficiently preparing earthworm functional fluid using endogenous probiotics and enzymes from the earthworm gut according to claim 1, characterized in that, The specific operation of step 2 is as follows: 1-5% Bacillus belye XY1 bacterial solution, 1-5% Priestella megaterium and Lactococcus lactis bacterial solution, 1-5% Lactococcus lactis bacterial solution and 1-5% compound enzyme are inoculated into earthworm slurry and fermented at a temperature of 30-35℃ and pH 5.0-6.5 for 6-12 hours to obtain earthworm functional liquid.
4. An earthworm functional liquid, characterized in that, The earthworm functional liquid was prepared using the method described in any one of claims 1-3, which utilizes endogenous probiotics and enzymes from the earthworm gut for efficient preparation.