Preparation method and application of lactobacillus hilgardii and traditional Chinese medicine fermentation liquor

Abstract

The invention relates to a preparation method and application of lactobacillus hilgardii and traditional Chinese medicine fermentation liquor, and belongs to the technical field of biological agriculture. The invention provides a lactobacillus hilgardii RX strain and a preparation method of the lactobacillus hilgardii RX strain. According to the traditional Chinese medicine fermentation liquor and the preparation method thereof, the traditional Chinese medicine raw materials are fermented by adopting the lactobacillus hirsuti, and the prepared traditional Chinese medicine fermentation liquor has a strong bacteriostatic effect on bacterial pathogenic bacteria of various bacterial diseases and has the advantages of being free of residues and pollution, wide in bacteriostatic spectrum, good in bacteriostatic effect, low in raw material cost and high in raw material utilization rate.

Description

Technical Field

[0001] The present invention relates to the technical field of biological agriculture, and in particular to a preparation method of a Lactobacillus hilgardii and traditional Chinese medicine fermentation liquid and application thereof. Background Art

[0002] The bacterial pathogens that cause bacterial diseases in aquaculture are diverse in species (or groups). Currently, the isolated and identified bacterial pathogens belong to multiple genera, including Pseudomonas, Edwardsiella, Aeromonas, Vibrio, Acinetobacter, Flexibacter, Pasteurella piscicidalis, Citrobacter, Streptococcus, and Flavobacterium. Bacterial pathogens can cause secondary and mixed infections in the normal flora of aquatic animals and water bodies, and multiple bacterial pathogens can cause the same disease. For example, outbreaks of freshwater fish can be caused by infections with bacterial pathogens such as Aeromonas sobria, Vibrio, and Yersinia ruckeri.

[0003] Existing technologies for bacterial diseases in aquaculture often use a combination of internal and external medications. Disinfectants such as quicklime and bleaching powder, as well as external medications such as chlorine, iodine, and bromine, can eliminate pathogenic bacteria in aquaculture water. Internal antibiotics such as florfenicol and enrofloxacin can provide targeted treatment based on the specific bacterial pathogens. However, these commonly used external and internal medications can remain in the water environment, causing toxic side effects to aquatic animals. Excessive or excessive use of antibiotics can also increase drug resistance in bacterial pathogens. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of Lactobacillus hilgeni and traditional Chinese medicine fermentation broth and its application, so as to solve the problems in the prior art of poor control effect on bacterial pathogens, serious water environment pollution and deterioration of aquatic product quality.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a Lactobacillus shilgardii, which is the Lactobacillus shilgardii RX strain, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 10, 2020, and the deposit number is CGMCC No. 19559.

[0007] The present invention also provides a method for preparing a traditional Chinese medicine fermentation broth, comprising the following steps:

[0008] (1) inoculating Lactobacillus hilgensis into a culture medium and culturing for 2 to 4 days to obtain a Lactobacillus hilgensis culture medium;

[0009] (2) The Lactobacillus hilarii bacterial solution, the Chinese medicine solution, and the culture solution are mixed and fermented for 2 to 4 days to obtain the Chinese medicine fermentation solution.

[0010] Preferably, the culture medium in steps (1) to (2) uses water as a solvent and includes the following components in the following concentrations: 20 to 30 g / L glucose, 5 to 15 g / L yeast extract, 2 to 10 g / L sodium acetate, 1 to 5 g / L diammonium hydrogen citrate, 0.01 to 0.05 g / L manganese sulfate, and 1 to 4 g / L potassium dihydrogen phosphate.

[0011] Preferably, the culture temperature in step (1) is 25-40°C;

[0012] The initial live bacteria concentration of the Lactobacillus hilarii liquid is 0.5-2.5×10 8 CFU / mL.

[0013] Preferably, the method for preparing the Chinese medicine liquid in step (2) comprises the following steps:

[0014] (a) Mix the raw material drug and water and soak for 30 to 90 minutes, decoct 1 to 2 times, filter, and obtain the filtrate to obtain an intermediate solution;

[0015] (b) concentrating the intermediate solution at 90-100° C. to obtain a Chinese medicine solution.

[0016] Preferably, the raw materials described in step (a) include Terminalia chebula, Coptis chinensis, and Schisandra chinensis;

[0017] The mass ratio of the terminalia chebula, coptis root and schisandra chinensis is 0.5-2:0.5-2:0.5-2;

[0018] The mass volume ratio of the raw material drug and water is 1g:1-7mL;

[0019] The soaking temperature is 20-30°C;

[0020] The temperature of each decoction is 90-100°C, and the time of each decoction is 20-40 minutes.

[0021] Preferably, the crude drug content of the traditional Chinese medicine solution in step (b) is 0.5 to 1.5 g / mL.

[0022] Preferably, the volume ratio of the mixture of the Lactobacillus hilgris liquid, the Chinese medicine liquid, and the culture medium in step (2) is 40-60:20-70:870-940;

[0023] The fermentation temperature is 25-40°C.

[0024] The present invention also provides a traditional Chinese medicine fermentation liquid prepared by the preparation method.

[0025] The present invention also provides the use of the Lactobacillus hilgardii and the traditional Chinese medicine fermentation liquid in preparing a preparation for preventing and / or treating bacterial diseases in aquaculture.

[0026] The present invention has the following technical effects and advantages:

[0027] The present invention uses Lactobacillus hilgensis to ferment traditional Chinese medicine raw materials to prepare traditional Chinese medicine fermentation liquid, which has a strong antibacterial effect on bacterial pathogens of various bacterial diseases such as Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, Vibrio parahaemolyticus, Aeromonas vernix, and Vibrio cholerae, with a wide antibacterial spectrum, and is superior to other probiotics and other traditional Chinese medicines;

[0028] The traditional Chinese medicine fermentation liquid of the present invention has low toxic and side effects on aquatic animals, leaves no residue and causes no pollution in aquaculture water, meets the requirements for sustainable development of the aquaculture industry, and has the advantages of good antibacterial effect, low raw material cost, simple preparation steps, and high raw material utilization rate, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the molecular biological identification result of RX strain;

[0030] Figure 2 The antibacterial effect of each Chinese medicine fermentation broth prepared in Comparative Examples 1 to 3 on bacterial pathogens, wherein A is the antibacterial effect of each Chinese medicine fermentation broth on Vibrio alginolyticus, B is the antibacterial effect of each Chinese medicine fermentation broth on Listonella anguillarum, C is the antibacterial effect of each Chinese medicine fermentation broth on Edwardsiella ictaluri, D is the antibacterial effect of each Chinese medicine fermentation broth on Aeromonas hydrophila, E is the antibacterial effect of each Chinese medicine fermentation broth on Vibrio parahaemolyticus, F is the antibacterial effect of each Chinese medicine fermentation broth on Aeromonas vernix, G is the antibacterial effect of each Chinese medicine fermentation broth on Vibrio cholerae, a is the Chinese medicine fermentation broth of Comparative Example 1, b is the Chinese medicine fermentation broth of Comparative Example 3, c is the Chinese medicine fermentation broth of Comparative Example 2, and d is sterile water;

[0031] Figure 3 The antibacterial effect of each Chinese medicine fermentation broth prepared in Example 2 and Comparative Examples 4 to 6 on bacterial pathogens, wherein A is the antibacterial effect of the Chinese medicine fermentation broth of Comparative Example 4 on bacterial pathogens, B is the antibacterial effect of the Chinese medicine fermentation broth of Comparative Example 5 on bacterial pathogens, C is the antibacterial effect of the Chinese medicine fermentation broth of Comparative Example 6 on bacterial pathogens, and D is the antibacterial effect of the Chinese medicine fermentation broth of Example 2 on bacterial pathogens;

[0032] Figure 4 The results are the diameters of the inhibition zones of various Chinese herbal fermentation broths against bacterial pathogens.

[0033] Preservation Instructions

[0034] Lactobacillus hilgardii is the Lactobacillus hilgardii RX strain, which is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2020, and the deposit number is CGMCC No. 19559. DETAILED DESCRIPTION

[0035] The present invention provides a Lactobacillus shilgardii, which is a Lactobacillus shilgardii RX strain, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 10, 2020, and the deposit number is CGMCC No. 19559.

[0036] The present invention also provides a method for preparing a traditional Chinese medicine fermentation broth, comprising the following steps:

[0037] (1) inoculating Lactobacillus hilgensis into a culture solution and culturing for 2 to 4 days, preferably 3 days, to obtain a Lactobacillus hilgensis culture solution;

[0038] (2) The Lactobacillus hilarii bacterial solution, the Chinese medicine solution, and the culture solution are mixed and fermented for 2 to 4 days, preferably 3 days, to obtain the Chinese medicine fermentation solution.

[0039] In the present invention, the culture solution described in steps (1) to (2) uses water as a solvent and includes the following components in the following concentrations: 20 to 30 g / L of glucose, preferably 25 g / L; 5 to 15 g / L of yeast extract, preferably 8 g / L; 2 to 10 g / L of sodium acetate, preferably 5 g / L; 1 to 5 g / L of diammonium hydrogen citrate, preferably 2 g / L; 0.01 to 0.05 g / L of manganese sulfate, preferably 0.02 g / L; and 1 to 4 g / L of potassium dihydrogen phosphate, preferably 2 g / L.

[0040] In the present invention, the culture temperature in step (1) is 25-40°C, preferably 35°C;

[0041] The initial live bacteria concentration of the Lactobacillus hilarii liquid is 0.5-2.5×10 8 CFU / mL, preferably 2×10 8 CFU / mL.

[0042] In the present invention, the method for preparing the Chinese medicine liquid in step (2) comprises the following steps:

[0043] (a) mixing the raw material drug and water and soaking for 30 to 90 minutes, preferably 60 minutes, boiling for 1 to 2 times, filtering, and collecting the filtrate to obtain an intermediate solution;

[0044] (b) concentrating the intermediate solution at 90-100° C. to obtain a Chinese medicine solution.

[0045] In the present invention, the raw materials described in step (a) include Terminalia chebula, Coptis chinensis, and Schisandra chinensis;

[0046] The mass ratio of the terminalia chebula, coptis chinensis and schisandra chinensis is 0.5-2:0.5-2:0.5-2, preferably 1:1:1;

[0047] The mass volume ratio of the raw material drug and water is 1g:1-7mL, preferably 1g:5mL;

[0048] The soaking temperature is 20-30°C, preferably 25°C;

[0049] The decoction is performed 1 to 2 times, preferably 2 times;

[0050] The temperature of each decoction is 90-100° C., preferably 98° C.; the time of each decoction is 20-40 minutes, preferably 30 minutes.

[0051] In the present invention, the crude drug content of the traditional Chinese medicine solution in step (b) is 0.5 to 1.5 g / mL, preferably 1 g / mL;

[0052] The concentration temperature is 90-100°C, preferably 98°C.

[0053] In the present invention, the volume ratio of the Lactobacillus hilgris liquid, the Chinese medicine liquid, and the culture medium in step (2) is 40-60:20-70:870-940, preferably 50:50:900;

[0054] The fermentation temperature is 25-40°C, preferably 35°C.

[0055] The present invention also provides a traditional Chinese medicine fermentation liquid prepared by the preparation method.

[0056] The present invention also provides the use of the Lactobacillus hilgardii and the traditional Chinese medicine fermentation liquid in preparing a preparation for preventing and / or treating bacterial diseases in aquaculture.

[0057] In the present invention, the bacteria include one or more of Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, Vibrio parahaemolyticus, Aeromonas veronii and Vibrio cholerae.

[0058] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] The bacterial species of the present invention: Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, and Vibrio parahaemolyticus are from Tianjin Fisheries Research Institute; Aeromonas veronii and Vibrio cholerae are from Tianjin Academy of Agricultural Sciences;

[0060] The instrument of the present invention: Oxford cup (Φ8nm×6nm×10nm) was purchased from Shanghai Sanshe Industrial Co., Ltd.

[0061] Example 1: Strain isolation and identification

[0062] (1) Strain isolation: Activated sludge from the bottom sediment of a white shrimp aquaculture pond in Beichen District, Tianjin, was inoculated into MRS liquid medium and enriched for 5 days to obtain a culture medium. The culture medium was gradient diluted and then applied to MRS solid medium containing calcium carbonate. Single colonies with larger hydrolysis zones were selected and subcultured for 5 consecutive times to obtain the RX strain.

[0063] (2) Strain identification: The RX strain was morphologically identified and Gram-stained. The genomic DNA of the RX strain was extracted and amplified by PCR and sequenced to obtain the 16S rDNA sequence of the RX strain (as shown in SEQ ID NO. 1). Homology analysis was performed using the NCBI-BLAST tool, and multiple sequence alignment was performed using MEGA7.0 software to construct a phylogenetic tree. The results are shown in Figure 1. Figure 1 shown.

[0064] SEQ ID NO.1:

[0065]

[0066] Morphological identification results showed that the colony surface of RX strain was moist and smooth, milky white in color, and Gram-positive. The cells of the strain were short rod-shaped, with a diameter of 0.5-0.8×2.0-4.0 μm, non-spore-forming, and facultatively anaerobic.

[0067] The results of molecular biological identification showed that the similarity between the RX strain and Lactobacillus hilgardii was as high as 99%. Combined with the morphological characteristics of the RX strain, the RX strain was determined to be Lactobacillus hilgardii.

[0068] Example 2: Preparation of Chinese medicine fermentation broth

[0069] (1) 25 g of glucose, 8 g of yeast extract, 5 g of sodium acetate, 2 g of diammonium citrate, 0.02 g of manganese sulfate, and 2 g of potassium dihydrogen phosphate were diluted to 1 L of water and sterilized at 121°C for 20 min to obtain a culture solution. Lactobacillus shilgardii RX strain was inoculated into the culture solution and anaerobically cultured at 35°C for 3 days to obtain a Lactobacillus shilgardii culture solution with an initial viable bacterial concentration of 2 × 10 8 CFU / mL;

[0070] (2) Take 100 g of Coptis chinensis, 100 g of Terminalia chebula, and 100 g of Schisandra chinensis and mix them as raw materials. Add 1500 mL of distilled water and soak at 25°C for 60 min. Boil at 100°C for 30 min and filter to obtain the residue and liquid I.

[0071] (3) Add distilled water to the medicinal residue in a mass volume ratio of 1 g:2 mL of medicinal residue and water, decoct at 100°C for 30 min, filter, and obtain the filtrate to obtain medicinal solution II; mix medicinal solution I and medicinal solution II, evaporate and concentrate at 100°C to a crude drug content of 1 g / mL to obtain a Chinese medicinal solution;

[0072] (4) Take 50 mL of Lactobacillus hilgeni liquid, 50 mL of Chinese medicine liquid and 900 mL of culture medium, mix them evenly, and ferment them at 35°C for 3 days to obtain Chinese medicine fermentation liquid.

[0073] Comparative Example 1: Preparation of Chinese medicine fermentation broth

[0074] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that, in this comparative example, the raw material drug was 100 g of Schisandra chinensis, and the amount of distilled water added in step (2) was 500 mL.

[0075] Comparative Example 2: Preparation of Chinese medicine fermentation broth

[0076] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that, in this comparative example, the raw material drug was 100 g of Terminalia chebula and the amount of distilled water added in step (2) was 500 mL.

[0077] Comparative Example 3: Preparation of Chinese medicine fermentation broth

[0078] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that in this comparative example, the raw material drug was 100 g of Coptis chinensis, and the amount of distilled water added in step (2) was 500 mL.

[0079] Comparative Example 4: Preparation of Chinese medicine fermentation broth

[0080] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that, in this comparative example, the raw materials were 150 g of Coptis chinensis and 150 g of Terminalia chebula, and the amount of distilled water added in step (2) was 1000 mL.

[0081] Comparative Example 5: Preparation of Chinese medicine fermentation broth

[0082] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that, in this comparative example, the raw materials were 150 g of Coptis chinensis and 150 g of Schisandra chinensis, and the amount of distilled water added in step (2) was 1000 mL.

[0083] Comparative Example 6: Preparation of Chinese medicine fermentation broth

[0084] The Chinese medicine fermentation broth was prepared according to the method described in Example 2, except that, in this comparative example, the raw materials were 150 g of Terminalia chebula and 150 g of Schisandra chinensis, and the amount of distilled water added in step (2) was 1000 mL.

[0085] Experimental Example 1: In vitro antibacterial experiment

[0086] Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, Vibrio parahaemolyticus, Aeromonas veronii, and Vibrio cholerae were activated to prepare bacterial pathogen solutions, and the concentration of viable bacteria was 10 8CFU / mL; 200 μL of each bacterial pathogen solution was applied to LB culture medium, and an Oxford cup was placed on each LB culture medium. 100 μL of the traditional Chinese medicine fermentation broth prepared in Example 2 and Comparative Examples 1 to 6 was injected into each Oxford cup, and sterile water was used as a control; after standing at room temperature for 3 hours, it was cultured at 32°C for 48 hours, and the diameter of the inhibition zone of each LB culture medium was measured. The results are as follows Figures 2-4 As shown in Tables 1 and 2.

[0087] Table 1 Antibacterial effect of Chinese medicine fermentation broth

[0088]

[0089] Table 2 Antibacterial effect of Chinese medicine fermentation broth

[0090]

[0091] The results showed that the Chinese medicinal fermentation broth prepared in Example 2 and Comparative Examples 1 to 6 had different degrees of antibacterial effect on various bacterial pathogens, among which the Chinese medicinal fermentation broth using Coptis chinensis, Terminalia chebula and Schisandra chinensis as raw materials had the best antibacterial effect on various bacterial pathogens.

[0092] As can be seen from the above examples, the present invention provides a method for preparing a Lactobacillus hilgensis and a traditional Chinese medicine fermentation broth and its application. The traditional Chinese medicine fermentation broth of the present invention can inhibit a variety of bacterial pathogens, such as Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, Vibrio parahaemolyticus, Aeromonas vernix, and Vibrio cholerae, and has the advantages of good antibacterial effect, low raw material cost, simple preparation steps, and high raw material utilization rate.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Lactobacillus hilgris, characterized in that The Lactobacillus shilgardii is the Lactobacillus shilgardii RX strain, which is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2020, and the deposit number is CGMCC No. 19559.

2. A method for preparing a Chinese medicine fermentation broth, characterized in that: The steps include: (1) inoculating Lactobacillus hilgensis into a culture medium and culturing for 2 to 4 days to obtain a Lactobacillus hilgensis culture medium; (2) mixing the Lactobacillus hilarii bacterial solution, the Chinese medicine solution, and the culture solution, and fermenting for 2 to 4 days to obtain the Chinese medicine fermentation solution; The Lactobacillus hilgensis described in step (1) is the Lactobacillus hilgensis described in claim 1.

3. The preparation method according to claim 2, characterized in that The culture solution described in steps (1) to (2) uses water as a solvent and includes the following components in the following concentrations: 20 to 30 g / L of glucose, 5 to 15 g / L of yeast extract, 2 to 10 g / L of sodium acetate, 1 to 5 g / L of diammonium hydrogen citrate, 0.01 to 0.05 g / L of manganese sulfate, and 1 to 4 g / L of potassium dihydrogen phosphate.

4. The preparation method according to claim 3, characterized in that The culture temperature in step (1) is 25-40°C; The initial live bacteria concentration of the Lactobacillus hilarii liquid is 0.5-2.5×10 8 CFU / mL.

5. The preparation method according to claim 2, characterized in that The method for preparing the Chinese medicinal liquid in step (2) comprises the following steps: (a) Mix the raw material drug and water and soak for 30 to 90 minutes, decoct 1 to 2 times, filter, and collect the filtrate to obtain an intermediate solution; (b) concentrating the intermediate solution at 90-100° C. to obtain a Chinese medicine solution.

6. The preparation method according to claim 5, characterized in that The raw materials described in step (a) include Terminalia chebula, Coptis chinensis, and Schisandra chinensis; The mass ratio of the terminalia chebula, coptis root and schisandra chinensis is 0.5-2:0.5-2:0.5-2; The mass volume ratio of the raw material drug and water is 1g:1-7mL; The soaking temperature is 20-30°C; The temperature of each decoction is 90-100°C, and the time of each decoction is 20-40 minutes.

7. The preparation method according to claim 6, characterized in that The crude drug content of the traditional Chinese medicine solution in step (b) is 0.5-1.5 g / mL.

8. The preparation method according to claim 2, characterized in that The volume ratio of the mixture of the Lactobacillus hilgris liquid, the Chinese medicine liquid and the culture solution in step (2) is 40-60:20-70:870-940; The fermentation temperature is 25-40°C.

9. The Chinese medicinal fermentation liquid prepared by the preparation method according to claims 2 to 8.

10. Use of the Lactobacillus hilgardii according to claim 1 and the traditional Chinese medicine fermentation liquid according to claim 9 in preparing a preparation for preventing and / or treating bacterial diseases in aquaculture.

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