Breeding method for improving musk secretion of forest musk deer

Abstract

The invention belongs to the technical field of special economic animal breeding, and particularly discloses a breeding method for improving musk secretion of forest musk deer. According to the method, 0.5%-1.0% of a functional feed additive is added into daily concentrated feed of the forest musk deer to reduce the diarrhea rate of the forest musk deer and improve the musk yield of the forest musk deer, the additive is prepared by taking bacillus subtilis and lactobacillus plantarum as fermentation strains through a two-stage temperature control solid-state fermentation process, the icariin content in a fermentation product is larger than or equal to 2.5 mg / g, and the viable count is larger than or equal to 108 CFU / g. Through verification, 0.5%-1.0% of the additive is added into daily concentrated feed of the forest musk deer, the yield of the forest musk deer is increased by 89.43%-109.49% compared with that of a control group, and the diarrhea rate is remarkably reduced to 5.44%-8.06% from 40.17%. The method is stable in process, low in cost and suitable for popularization and application of large-scale forest musk deer breeding, and has important significance in improving the economic benefit of the musk industry and the animal health level.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of special economic animal breeding, and particularly relates to a breeding method for improving musk secretion of forest musk deer. BACKGROUND

[0003] Male forest musk deer starts secreting musk at the age of 1 year, and the peak period of musk secretion is concentrated in May to June every year, and lasts for about 5 to 7 days. During this period, the food intake of male musk deer decreases by 90% to 95%, the testis and scent sac are significantly enlarged, the epithelial cells of the scent gland are columnar, and the initial musk is produced by the "apical secretion" mode. After mixing with the lipids secreted by the sebaceous glands, the initial musk matures in the scent sac to form mature musk within 2 months, and the content of muscone, the core component of the musk, directly affects the market value. Studies have shown that extending the peak period of musk secretion by one day can increase the yield of musk by 12% to 15%, but the existing breeding technology is difficult to accurately regulate the musk secretion cycle.

[0004] The nutritional level of feed and intestinal health are crucial for nutrient absorption. Lactobacillus plantarum, as a feed additive, can regulate the balance of intestinal flora, enhance the barrier function, and improve the absorption rate of key nutrients such as protein and vitamins in animals, providing the material basis for musk secretion. However, a single Lactobacillus plantarum cannot meet the multiple demands of bacteria inhibition, digestion promotion, and immune regulation at the same time.

[0005] As a traditional aphrodisiac and fertility-promoting traditional Chinese medicine, the main active ingredient of icariin in Epimedium brevicornum is broken down into aglycone after fermentation, and the bioavailability is increased by 3 to 5 times, which can more efficiently regulate hormone secretion through the hypothalamic-pituitary-gonadal axis, providing physiological stimulation for musk secretion. However, direct addition of traditional Chinese medicines such as Epimedium brevicornum has problems such as low bioavailability and insufficient release of active ingredients.

[0006] The current breeding system has not been able to break through the technical problems of "low nutrient absorption efficiency - difficulty in releasing active ingredients of traditional Chinese medicine", and there is an urgent need to develop a forest musk deer breeding method that can promote the health of intestinal flora and enhance nutrient absorption while improving the efficient conversion of active ingredients of traditional Chinese medicine, in order to improve the musk secretion of forest musk deer. SUMMARY

[0007] To solve the above technical problems, the present application uses a solid-state fermentation medium containing Epimedium to prepare a functional feed additive containing icariin active substances by solid-state fermentation, which is used in forest musk deer breeding to increase musk yield; the solid-state fermentation product prepared by composite fermentation contains rich Lactobacillus plantarum and Bacillus subtilis, and the functional feed additive prepared by using the solid-state fermentation product can promote the health of intestinal flora of forest musk deer and indirectly increase musk yield. The experiment proves that the functional feed additive provided by the present application can significantly increase musk secretion when added to the daily concentrate of forest musk deer, and the present application provides a new technical solution for increasing musk secretion of forest musk deer.

[0008] In one aspect, the present application provides a breeding method for increasing musk secretion of forest musk deer, which adds 0.5% to 1.0% of a functional feed additive to the daily concentrate of forest musk deer to increase musk secretion, wherein the functional feed additive is obtained by solid-state fermentation of a solid-state medium containing Epimedium, and the fermentation strains used in the solid-state fermentation are Bacillus subtilis and Lactobacillus plantarum, the preservation number of the Bacillus subtilis is CGMCC 1.9086, and the preservation number of the Lactobacillus plantarum is CICC 20261.

[0009] Further, in the breeding method, the mass ratio of Epimedium powder to wheat bran powder in the solid-state fermentation medium is 3:7 to 4:6, and 0.5% to 1.0% of yeast extract is contained.

[0010] Further, in the breeding method, the wheat bran powder is obtained by drying after high-pressure steam treatment.

[0011] Further, in the breeding method, the wheat bran powder is 100 to 200 mesh.

[0012] Further, in the breeding method, the mass ratio of Lactobacillus plantarum to Bacillus subtilis in the solid-state fermentation is 5 to 6:4 to 5.

[0013] Further, in the breeding method, the content of icariin in the solid-state fermentation product is 2.58 mg / g to 2.87 mg / g.

[0014] Further, in the breeding method, the viable cell count of the Lactobacillus plantarum and the Bacillus subtilis in the solid fermentation product is ≥1×10 8 CFU / g.

[0015] Further, in the breeding method, the solid fermentation comprises two stages.

[0016] The first stage is that the fermentation temperature is 32-33℃, and the fermentation time is 20-24h.

[0017] The second stage is that the fermentation temperature is 35-36℃, the culture medium is stirred every 12h, and the fermentation time is 70-72h.

[0018] Further, in the breeding method, the addition amount of the functional feed additive is 0.5%-1.0% of the daily concentrated feed of the forest musk deer, and the feeding period covers the 30 days before the peak of musk secretion to the end of musk secretion.

[0019] Finally, the feed containing the functional feed additive prepared by the breeding method is also provided, and the mass percentage of the functional feed additive in the feed is 0.5%-1.0%. The functional feed additive is obtained by solid fermentation of a solid culture medium containing Epimedium, and the fermentation strains used in the solid fermentation are Bacillus subtilis and Lactobacillus plantarum. The preservation number of the Bacillus subtilis is CGMCC 1.9086, and the preservation number of the Lactobacillus plantarum is CICC 20261.

[0020] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages:

[0021] (1) The present application uses Bacillus subtilis and Lactobacillus plantarum to solid-ferment Epimedium, so that the content of the active ingredient icariin is 2.58-2.87mg / g, and the bioavailability is significantly improved.

[0022] Icariin regulates hormone secretion through the hypothalamus-pituitary-gonadal axis, and improves the secretion amount of musk of forest musk deer. The musk yield of forest musk deer added with the functional feed additive provided by the present application is much higher than that of the group without fermented Epimedium and the blank control group.

[0023] (2) The Lactobacillus plantarum and Bacillus subtilis synergistically regulate the intestinal flora balance, enhance the intestinal barrier function, and reduce the pathogenic bacteria colonization. The cumulative diarrhea rate of the forest musk deer fed with the functional feed additive prepared by the method is only 5.44% to 8.06%, which is far lower than 40.17% of the blank control group and 37.28% of the Epimedium group, and far lower than the cumulative diarrhea rate of 34.17% to 36.28% of the forest musk deer fed with the Bacillus subtilis single strain fermentation group.

[0024] (3) The Lactobacillus plantarum and Bacillus subtilis are complexly fermented with Epimedium as a substrate, the product prepared from the functional feed additive is fed to the forest musk deer, and the musk yield of the forest musk deer is increased by 89.43% to 109.49% compared with the blank control, which indicates that the product of the complex fermentation of the Lactobacillus plantarum and Bacillus subtilis and icariin synergistically act, and greatly improve the musk yield of the forest musk deer.

[0025] The functional feed additive used in the breeding method provided by the application is prepared by combining microbial fermentation and nutrition regulation, breaks through the bottleneck of “low nutrient absorption efficiency-difficult release of active components of traditional Chinese medicine” in the prior art, has the advantages of improving the musk yield and reducing the diarrhea rate of the forest musk deer, and has a wide industrial application prospect. DETAILED DESCRIPTION

[0026] The technical solutions of the application will be described below in combination with examples, but the application is not limited to the following examples.

[0027] In order to enable those skilled in the art to better understand the technical solutions of the application and implement the same, the application will be further described below in combination with specific examples, but the examples are not used as limitations to the application.

[0028] The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified; and the reagents and materials described are all commercially available unless otherwise specified.

[0029] Bacillus subtilis, preservation number: CGMCC 1.9086, purchased from the China General Microbiological Culture Collection Center.

[0030] Lactobacillus plantarum CICC 20261, purchased from the China Industrial Microbial Culture Collection Management Center.

[0031] Example 1

[0032] This example is for preparing a functional feed additive.

[0033] Preparation of the solid-state fermentation medium:

[0034] Epimedium was dried in a 60℃ drying oven until constant weight, and was ground by a grinder to pass through a 100 mesh sieve to obtain Epimedium powder. Wheat bran was treated by high-pressure steam at 180℃ for 5 min, and was then dried in a 60℃ drying oven until constant weight. The wheat bran was ground to pass through a 100 mesh sieve to obtain wheat bran powder. The Epimedium powder and the wheat bran powder were mixed at a mass ratio of 3:7, and 0.5% yeast extract, 0.05% MgSO4 and 0.02% MnSO4 were added to obtain a solid-state fermentation medium.

[0035] Preparation of the compound fermentation seed liquid:

[0036] Lactobacillus plantarum was inoculated into MRS solid medium, and was cultured in a 34℃ incubator for 24 h. Single colonies were picked and streaked on solid medium. After continuous activation for 2 times, single colonies were inoculated into MRS liquid medium, and were cultured in a 34℃ incubator for 18 h to obtain a final concentration of 2×10 6 CFU / mL, i.e. a Lactobacillus plantarum seed liquid.

[0037] Bacillus subtilis was activated in LB solid medium, and single colonies were inoculated into LB liquid medium. The medium was incubated at 35℃ and 200 rpm / min for 20 h to obtain a final concentration of 2×10 6 CFU / mL, i.e. a Bacillus subtilis seed liquid.

[0038] The Lactobacillus plantarum seed liquid and the Bacillus subtilis seed liquid were mixed at a mass ratio of 6:4 to obtain a compound fermentation seed liquid.

[0039] Solid-state fermentation of Epimedium:

[0040] The solid-state fermentation medium was added with an equal amount of water, and was mixed. The compound fermentation seed liquid was added at 5% of the total mass of the solid-state fermentation medium, and was mixed. The medium was fermented at 32℃ for 24 h to complete the first-stage fermentation. The fermentation temperature was adjusted to 35℃ to enter the second-stage fermentation. The medium was stirred every 12 h during the second-stage fermentation. The fermentation was performed for 72 h. The concentrations of Lactobacillus plantarum and Bacillus subtilis were determined by plate counting to ensure that the concentrations of Lactobacillus plantarum and Bacillus subtilis were both above 10 8 CFU / g. The content of icariin in the fermentation product was detected by high performance liquid chromatography after the fermentation was completed. The content of icariin was 2.58 mg / g.

[0041] The fermentation product was vacuum dried at 45℃, and was mixed with trehalose at a mass ratio of 98:2 to obtain a functional feed additive.

[0042] Example 2

[0043] This example is for preparing a functional feed additive.

[0044] Solid-state fermentation medium preparation:

[0045] The Epimedium grandiflorum was dried in a 60℃ drying oven until constant weight, and was crushed by a pulverizer to pass through a 200-mesh sieve to obtain Epimedium grandiflorum powder. The wheat bran was treated with high-pressure steam at 180℃ for 5 min, and then was dried in a 60℃ drying oven until constant weight. The wheat bran was crushed to pass through a 200-mesh sieve to obtain wheat bran powder. The Epimedium grandiflorum powder and the wheat bran powder were mixed at a mass ratio of 4:6, and 1.0% of yeast extract, 0.06% of MgSO4 and 0.04% of MnSO4 were added to obtain a solid-state fermentation medium.

[0046] Composite fermentation seed liquid preparation:

[0047] The Lactobacillus plantarum was inoculated into MRS solid medium, and was cultured in a 34℃ incubator for 24 h. Single colonies were picked and streaked on solid medium, and were continuously activated for 2 times. Single colonies were inoculated into MRS liquid medium, and were cultured in a 32℃ incubator for 20 h. The bacterial concentration was measured, and the bacterial liquid was diluted to a final Lactobacillus plantarum concentration of 2×10 6 CFU / mL, which was the Lactobacillus plantarum seed liquid.

[0048] LB solid medium was used to activate Bacillus subtilis. Single colonies were inoculated into LB liquid medium, and were incubated at 36℃ and 250 rpm / min for 24 h. The bacterial concentration was measured, and the final Bacillus subtilis concentration was 2×10 6 CFU / mL, which was the Bacillus subtilis seed liquid.

[0049] The Lactobacillus plantarum seed liquid and the Bacillus subtilis seed liquid were mixed at a mass ratio of 5:5 to obtain a composite fermentation seed liquid.

[0050] Epimedium grandiflorum solid-state fermentation:

[0051] The solid-state fermentation medium was added, and an equal amount of water was added to mix. The composite fermentation seed liquid was added at 8% of the total mass of the solid-state fermentation medium to mix. The fermentation was carried out at 33℃ for 20 h to complete the first-stage fermentation. The fermentation temperature was adjusted to 36℃ to enter the second-stage fermentation. The medium was stirred every 12 h during the second-stage fermentation. The fermentation was carried out for 70 h. The concentrations of Lactobacillus plantarum and Bacillus subtilis were determined by plate counting to ensure that the concentrations of Lactobacillus plantarum and Bacillus subtilis were both above 10 8 CFU / g. After the fermentation was completed, the content of icariin in the fermentation product was detected by high-performance liquid chromatography. The content of icariin was 2.87 mg / g.

[0052] The fermentation product was vacuum dried at 45℃, and was mixed with trehalose at a mass ratio of 98:2 to obtain a functional feed additive.

[0053] Comparative Example

[0054] The functional feed additive comparative agent was prepared by using Bacillus subtilis single strain.

[0055] Preparation of solid-state fermentation medium:

[0056] Epimedium was dried in a 60°C drying oven until constant weight, and then ground through a 200-mesh sieve using a grinder to obtain Epimedium powder. Wheat bran was treated with high-pressure steam at 180°C for 5 min, and then dried in a 60°C drying oven until constant weight. The wheat bran powder was obtained by passing it through a 200-mesh sieve. The Epimedium powder and the wheat bran powder were mixed in a mass ratio of 3:7, and 1.0% yeast extract, 0.06% MgSO4, and 0.04% MnSO4 were added and mixed uniformly to obtain the solid-state fermentation medium.

[0057] Preparation of fermentation seed liquid:

[0058] LB solid medium was used to activate Bacillus subtilis. Single colonies were inoculated into LB liquid medium, and incubated at 36°C and 250 rpm / min for 24 h. The bacterial concentration was measured, and the final concentration of Bacillus subtilis in the culture was adjusted to 2×10 6 CFU / mL, which was the Bacillus subtilis seed liquid.

[0059] Solid-state fermentation of Epimedium:

[0060] The solid-state fermentation medium was added, and an equal amount of water was added to mix it uniformly. Bacillus subtilis seed liquid was added at a concentration of 8% of the total mass of the solid-state medium. The medium was stirred every 12 h during fermentation, and the fermentation was carried out at 36°C for 72 h. After fermentation, the concentration of Bacillus subtilis was determined by plate counting method. The concentration of Bacillus subtilis reached 10 8 CFU / g. After fermentation, the content of icariin in the fermentation product was detected by high-performance liquid chromatography, and the content of icariin was 1.17 mg / g.

[0061] The fermentation product was vacuum dried at 45°C, and then mixed with trehalose at a mass ratio of 98:2 to obtain the functional feed additive comparative agent.

[0062] Example 3

[0063] This example evaluated the effect of different feed on the secretion of musk in forest musk deer.

[0064] Select 80 adult male forest musk deer, randomly divided into 8 groups, 10 forest musk deer in each group, test group 1, using the functional feed additive prepared in example 1, the functional feed additive is added to 0.5% of the daily concentrate feed;Test group 2 uses the functional feed additive prepared in example 1, the functional feed additive is added to 1.0% of the daily concentrate feed;Test group 3 is the functional feed additive prepared in example 2, the functional feed additive is added to 0.5% of the daily concentrate feed;Test group 4 is the functional feed additive prepared in example 2, the functional feed additive is added to 1.0% of the daily concentrate feed;Test group 5 is the functional feed additive prepared in the comparative example, the contrast agent is the additive, and the addition amount is 0.5% of the daily concentrate feed;Test group 6 is the functional feed additive prepared in the comparative example, the contrast agent is the additive, and the addition amount is 1.0% of the daily concentrate feed;Test group 7 uses unfermented epimedium as functional feed, and the unfermented epimedium additive is added to 1.0% of the daily concentrate feed;At the same time, the blank control group is set, and the blank control group is not added to the functional feed group.

[0065] The test was from April 2024 to September 2024, and during the test period, a fixed observer observed the feces of each group of forest musk deer every day, and observed once in the morning and once in the evening. The diarrhea judgment criteria are as follows:

[0066] Feces grading system (1-5 points system):

[0067] 1 point: hard formed stool (normal)

[0068] 2 points: soft formed stool (mildly abnormal)

[0069] 3 points: paste stool (moderate diarrhea)

[0070] 4 points: water-like stool (severe diarrhea)

[0071] 5 points: bloody stool / mucus stool (pathological diarrhea)

[0072] When the feces grade is ≥3 points, it is counted into the diarrhea statistics. The cumulative diarrhea rate of each group during the test period is calculated, and the calculation formula is as follows:

[0073]

[0074] Note: Multiple diarrhea of the same forest musk deer within 24 hours is considered as one diarrhea event, regardless of the number of defecation times, which is counted as one diarrhea, and 180 days of data are counted within 6 months. Taking 10 forest musk deer in each group as an example, the total observation head times of the group within 6 months are 1800. The test results are shown in table 1.

[0075] Table 1 cumulative diarrhea rate of forest musk deer with different treatments

[0076] Test group Number of diarrhea heads Cumulative diarrhea rate Test group 1 109 6.06％ Test group 2 145 8.06％ Test group 3 98 5.44％ Test group 4 121 6.72％ Test group 5 653 36.28％ Test group 6 615 34.17％ Test group 7 671 37.28％ Blank control group 723 40.17％

[0077] As shown in Table 1, compared with the blank control group without adding any functional additive, the daily fine feed added with unfermented Epimedium herb for feeding the musk deer, the diarrhea rate of the musk deer slightly decreased, which might be related to the anti-inflammatory and antibacterial functions of the Epimedium herb; the cumulative diarrhea rate of the contrast agent of the functional feed additive prepared by using Bacillus subtilis single strain in the test group 5 and the test group 6 was slightly decreased compared with the blank control, and there was a dose relationship, which indicated that Bacillus subtilis had an improvement effect on the intestinal tract of the musk deer, and could reduce the occurrence of diarrhea of the musk deer, but the effect was effective.

[0078] Compared with the blank control, the cumulative diarrhea rate of the musk deer in the test group 1 to the test group 4 fed with the functional feed additive provided by the application was greatly decreased, and compared with the test group 5 and the test group 6, the cumulative diarrhea rate of the musk deer was also greatly decreased, which indicated that the Lactobacillus plantarum and Bacillus subtilis and their metabolites contained in the test group 1 to the test group 4 had a synergistic effect, and could improve the intestinal flora of the musk deer, improve the resistance of the musk deer to pathogenic bacteria, and greatly reduce the occurrence of diarrhea.

[0079] When the test was completed, the musk deer musk was collected, the musk yield was recorded, and the moisture was removed by vacuum drying to prepare dry musk for comparison of the musk yield, and the average yield of musk in each group was calculated, and the musk yield improvement rate was evaluated compared with the blank control group, and the results are shown in Table 2.

[0080] Table 2 Musk yield of musk deer in different treatments

[0081] Test group Average musk yield (g) Musk yield improvement rate (%) Test group 1 24.13 100.92 Test group 2 22.75 89.43 Test group 3 25.16 109.49 Test group 4 23.61 96.59 Test group 5 12.98 8.08 Test group 6 13.11 9.16 Test group 7 12.64 5.25 Blank control group 12.01 -

[0082] As shown in Table 2, compared with the blank control, the musk yield of the test group 1 to the test group 7 was increased to different degrees, which indicated that icariin was related to the secretion of musk by the musk deer, and icariin had a certain yield-increasing effect on the secretion of musk by the musk deer, and the results of the test group 5, 6 and 7 showed that there might be a dose-dependent relationship between icariin and the secretion of musk by the musk deer within a certain range, but the effect of icariin on the secretion of musk by the musk deer was limited. The musk yield of the musk deer in the test group 5 to 7 was far lower than that of the musk deer in the test group 1 to 4, so it could be seen that the Epimedium herb fermented by Lactobacillus plantarum and Bacillus subtilis and the metabolites thereof might have a synergistic effect with icariin, and greatly improved the musk yield of the musk deer.

[0083] The results of comparison between the test group 1 and the test group 2, and comparison between the test group 3 and the test group 4 show that the musk yield increasing rate of the musk produced by the functional feed additive of Example 1 and the functional feed additive of Example 2 at 0.5% is higher than that at 1.0%. It is shown that the functional feed additive prepared from the product of the compound fermentation of Epimedium with Lactobacillus plantarum and Bacillus subtilis is not the higher the addition amount, the higher the musk yield of the forest musk deer, which indicates that the compound fermentation of Bacillus subtilis and Lactobacillus plantarum and the synergistic effect between the compound fermentation products are different from the characteristics of the dose relationship between the single strain fermentation product and the musk yield of the forest musk deer. The synergistic effect of the compound fermentation product is the highest for the musk yield increasing rate of the musk produced by the functional feed additive of Example 2 at 0.5%, which is 109.49% compared with the blank control, and is the best addition amount of the present application.

[0084] The basic principles, main features and advantages of the present application are described above. The above examples and descriptions are only for describing the preferred embodiments of the present application, and the present application is not limited by the above examples. Various changes and improvements to the technical solutions of the present application made by those skilled in the art without departing from the spirit and scope of the present application shall fall within the scope of protection of the present application.

Claims

1. A breeding method for increasing musk secretion in forest musk deer, characterized in that, Adding 0.5% to 1.0% of a functional feed additive to the daily concentrated feed of the musk deer can increase its musk secretion. The functional feed additive is obtained by solid-state fermentation on a solid culture medium containing Epimedium. The fermentation strains used in the solid-state fermentation are Bacillus subtilis and Lactobacillus plantarum. The preservation number of Bacillus subtilis is CGMCC 1.9086, and the preservation number of Lactobacillus plantarum is CICC 20261.

2. The aquaculture method according to claim 1, characterized in that, The solid-state fermentation medium contains epimedium powder to wheat bran powder in a mass ratio of 3:7 to 4:6, and contains 0.5% to 1.0% yeast extract.

3. The breeding method according to claim 2, characterized in that, The wheat bran powder is obtained by drying wheat bran after high-pressure steam treatment.

4. The aquaculture method according to claim 3, characterized in that, The wheat bran powder is 100-200 mesh.

5. The aquaculture method according to claim 1, characterized in that, In solid-state fermentation, the mass ratio of Lactobacillus plantarum to Bacillus subtilis is 5-6:4-5.

6. The aquaculture method according to claim 1, characterized in that, The content of icariin in the solid-state fermentation product is 2.58 mg / g to 2.87 mg / g.

7. The aquaculture method according to claim 1, characterized in that, The viable counts of *Lactobacillus plantarum* and *Bacillus subtilis* in the solid-state fermentation product are both ≥1×10⁻⁶. 8 CFU / g.

8. The aquaculture method according to claim 1, characterized in that, The solid-state fermentation includes two stages: First stage: Fermentation temperature is 32-33℃, fermentation time is 20-24 hours; Second stage: Fermentation temperature is 35-36℃, the culture medium is turned over once every 12 hours, and the fermentation time is 70-72 hours.

9. The aquaculture method according to any one of claims 1 to 8, characterized in that, The amount of the functional feed additive added is 0.5% to 1.0% of the daily concentrated feed of the musk deer, and the feeding cycle covers 30 days before the peak of musk secretion and until the end of musk secretion.

10. A feed containing a functional feed additive for implementing the breeding method of claim 1, characterized in that, The functional feed additive in the feed is 0.5% to 1.0% by mass. The functional feed additive is obtained by solid-state fermentation on a solid culture medium containing Epimedium. The fermentation strains used in the solid-state fermentation are Bacillus subtilis and Lactobacillus plantarum. The preservation number of Bacillus subtilis is CGMCC 1.9086, and the preservation number of Lactobacillus plantarum is CICC 20261.

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