CN114196592A - Lactobacillus casei R and fermentation method and application thereof - Google Patents
Lactobacillus casei R and fermentation method and application thereof Download PDFInfo
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- CN114196592A CN114196592A CN202111648951.6A CN202111648951A CN114196592A CN 114196592 A CN114196592 A CN 114196592A CN 202111648951 A CN202111648951 A CN 202111648951A CN 114196592 A CN114196592 A CN 114196592A
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of microorganisms, and relates to lactobacillus casei, in particular to lactobacillus casei R and a fermentation method and application thereof. The classification name of the lactobacillus casei R is lactobacillus caseiLactobacillus casei(ii) a The preservation number is CGMCC No. 23438; the preservation unit is China general microorganism strain preservation and management center; the preservation time is 2021, 9 months and 17 days. Compared with the prior art, the invention provides a method for fermenting pulsatilla chinensis powder by using lactobacillus casei. After fermentation, the macromolecular substances in the traditional Chinese medicines are fermented into micromolecular substances, which indicates that the lactobacillus casei R breaks in the fermentation processThe cell wall of the traditional Chinese medicine is damaged, so that the effective medicinal components in the traditional Chinese medicine are released and decomposed into small molecular substances which are easier to absorb and exert the medicinal effect, the effective components in the fermentation liquor are obviously increased, and the antibacterial property of the traditional Chinese medicine is obviously improved.
Description
Technical Field
The invention belongs to the field of microorganisms, and relates to lactobacillus casei, in particular to lactobacillus casei R and a fermentation method and application thereof.
Background
In recent years, the problem of drug resistance is becoming more serious due to the abuse of antibiotics, and the problem of drug residues in livestock and poultry food is also receiving wide attention. The stopping of the production of commercial feed containing growth-promoting drug feed additives (except traditional Chinese medicines) by feed production enterprises has become a rule to be followed by feed enterprises; therefore, research on a novel, green, non-drug resistant, and therapeutically effective prophylactic and therapeutic drug or feed additive to replace antibiotics is urgent.
Probiotic fermented traditional Chinese medicines have attracted attention in recent years, and are widely used because of their advantages of being natural and green, free of pollution, free of drug resistance and the like. The lactobacillus is the dominant strain in the normal flora of human and animal intestinal tracts, has a plurality of physiological functions beneficial to the health of human and animal organisms, and can generate a plurality of acidic substances to inhibit the growth of some common intestinal pathogenic bacteria. Patent 201710138380.9 discloses a lactobacillus buchneri strain and its silage starter; the strain has excellent oxidation resistance and acid resistance, is heterotypic fermentation lactobacillus and can generate a large amount of acetic acid; patent 201710137699.X discloses a lactobacillus plantarum strain and a silage starter thereof, wherein the lactobacillus plantarum strain is homotype lactobacillus fermentum, can generate a large amount of lactic acid, rapidly reduces pH, is acid-resistant, and has strong bacteriostatic activity on staphylococcus aureus, bacillus cereus, salmonella, escherichia coli and saccharomycetes. Therefore, these strains are not suitable for fermentation of Chinese medicinal materials.
The traditional Chinese medicine as the feed additive has been used and recorded 1000 years ago in China, and although the traditional Chinese medicine preparation is used in clinic, the traditional Chinese medicine preparation has the problems of complex active ingredients, difficult purification, poor palatability, low conversion efficiency of livestock and poultry, poor effect and the like, and the wide use of the traditional Chinese medicine preparation as the feed additive is hindered. According to a large amount of researches on traditional Chinese medicine preparations, the method for processing the traditional Chinese medicine becomes a novel traditional Chinese medicine processing method by utilizing microbial fermentation. The microbial fermentation is to decompose macromolecular substances in traditional Chinese medicines or forage grass by using metabolites generated in the proliferation process of microorganisms, decompose plant cell walls by using the enzyme production or acid production of the microorganisms, promote the release of effective components, and in addition, in the fermentation process of strains, certain metabolites can be influenced by the components in the traditional Chinese medicines to generate changes, so that various bacteriostatic and antiviral secondary metabolites can be generated.
Therefore, the search for a probiotic with good fermentation effect to improve the efficacy of the traditional Chinese medicine and inhibit the propagation of pathogenic bacteria in the intestinal tract is of great significance.
Disclosure of Invention
The invention provides a lactobacillus casei R and a fermentation method and application thereof, and the lactobacillus casei R is used for obtaining a fermentation product with higher drug effect and stronger bactericidal capacity.
The technical scheme of the invention is realized as follows:
lactobacillus casei R, wherein the classification name of the lactobacillus casei R is lactobacillus caseiLactobacillus casei(ii) a The preservation number is CGMCC No. 23438; the preservation unit is China general microorganism strain preservation and management center; the preservation time is 2021, 9 months and 17 days; the storage address is No. three of Xilu-Hayao-Beijing, Chaoyang, Beijing.
The lactobacillus casei R is used for improving the antibacterial property of the pulsatilla chinensis.
Further, the antibacterial property of the pulsatilla chinensis powder is improved by fermenting the pulsatilla chinensis powder with lactobacillus casei R.
The method for fermenting the pulsatilla chinensis powder by using the lactobacillus casei R comprises the following steps:
(1) culturing a seed solution: inoculating lactobacillus casei R into MRS liquid culture medium for culture to obtain seed liquid;
(2) and (3) amplification culture: inoculating the seed liquid in the step (1) into an MRS liquid culture medium for fermentation culture to obtain a fermentation liquid;
(3) the traditional Chinese medicine decoction: adding Pulsatilla chinensis powder into ultrapure water, soaking for a period of time, decocting and concentrating to obtain a concentrated solution, and autoclaving to obtain Pulsatilla chinensis powder decoction;
(4) and (3) mixing the fermentation liquor obtained in the step (2) and the pulsatilla chinensis decoction obtained in the step (3) in proportion, and then standing and culturing to obtain a fermentation product.
Preferably, the culturing condition in the step (1) is culturing at 37 ℃ for 24 h.
Preferably, the inoculation ratio of the seed liquid in the step (2) is 1% of the volume of the MRS liquid culture medium; the fermentation culture condition is that the culture is carried out for 24 hours at 37 ℃.
Preferably, the concentration of the pulsatilla chinensis powder in the ultra-pure water in the step (3) is 0.02 g/mL; the concentration of the pulsatilla chinensis powder in the concentrated solution is 1 g/mL; autoclaving condition is 121 deg.C for 30 min.
Preferably, the volume ratio of the fermentation liquid in the step (2) to the pulsatilla chinensis decoction in the step (3) in the step (4) is 1: 1; the conditions for static culture were 37 ℃ for 24 hours.
The fermentation product obtained by the method.
The fermentation product is applied to preparing bacteriostatic drugs, and the bacteria are staphylococcus aureus, salmonella or escherichia coli.
The invention has the following beneficial effects:
1. the invention provides application of the Lactobacillus casei (Lactobacillus casei) R strain in increasing effective components of traditional Chinese medicines after the traditional Chinese medicines are fermented. The fermented components can produce stronger application for inhibiting the growth of staphylococcus aureus, salmonella and escherichia coli. Compared with the prior art, the invention provides lactobacillus casei, a method for fermenting pulsatilla chinensis powder by using the lactobacillus casei and application of the lactobacillus casei. After fermentation, macromolecular substances in the traditional Chinese medicine are fermented into micromolecular substances, which shows that Lactobacillus casei (Lactobacillus casei) R destroys cell walls of the traditional Chinese medicine in the fermentation process, so that effective medicinal components in the traditional Chinese medicine are released and decomposed into micromolecular substances which are easier to absorb and exert medicinal effects, the effective components in the fermentation liquor are obviously increased, and the antibacterial property of the traditional Chinese medicine is obviously improved.
2. After the lactobacillus casei is used for fermenting the pulsatilla chinensis powder, the inhibition zone for staphylococcus aureus is as high as 19.95mm, the inhibition zone for salmonella is as high as 19.97mm, the inhibition zone for escherichia coli is as high as 16.80mm, and the pulsatilla chinensis powder presents high sensitivity level, and the single pulsatilla chinensis powder does not have antibacterial activity. The Chinese pulsatilla root powder fermented by the lactobacillus casei has unexpected technical effects.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a general optical microscopic view of Lactobacillus casei R.
FIG. 2 is a biological phylogenetic tree diagram of Lactobacillus casei R.
FIG. 3 is a comparison of normal optical microscope images of Lactobacillus casei R fermented Pulsatilla chinensis powder before and after fermentation.
FIG. 4 is a comparison of the bacteriostatic effect of Lactobacillus casei R fermented Pulsatilla chinensis powder before and after fermentation.
FIG. 5 is a linear regression equation chart of Pulsatilla chinensis powder standard.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The formula of the MRS liquid culture medium used by the invention is as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of beef powder, 2g/L of dipotassium phosphate trihydrate, 5g/L of sodium acetate trihydrate, 0.1% of Tween 80, 2g/L of triammonium citrate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 8g/L of lactose and 6.2-6.6 of pH; and (3) sterilization conditions: 121 ℃, 100kPa, 30 min.
The MRS solid culture medium used in the embodiment of the invention has the following formula: 1.5% agar was added on the basis of MRS liquid medium.
Example 1: isolation, screening and characterization of Lactobacillus casei R
1.1 sampling: in 12 months and 10 days in 2020, Zhengzhou city in Henan province, a tester Lishuiqi collects silage samples from a cattle farm;
1.2 isolation and screening: weighing 1g silage, cutting into pieces, adding into 250mL conical flask, adding 99mL sterile PBS, shaking thoroughly, standing, adding 1mL supernatant into 9mL PBS, sequentially diluting by 10 times, sucking 10 times-4、10-5、10-6And coating 100 mu L of diluent in an MRS solid culture medium, culturing at 37 ℃ for 24h, picking suspected colonies, inoculating the suspected colonies in the MRS liquid culture medium, and streaking the suspected colonies onto an MRS solid plate for subsequent tests after static culture.
2. Identification
2.1 the biochemical characteristic test identification of the strain R, the identification result is shown in the table 1:
TABLE 1 Biochemical Property test results
In Table 1, "+" indicates a positive reaction.
2.2 molecular biological characterization of Strain R
2.2.1 extraction of DNA of Strain R
2.2.2 the activated strain is amplified by extracting DNA by using a bacterial genome kit. The 16S rRNA amplification primer is a universal primer 27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-TACGGCTACCTTGTTACGACTT-3', and performing PCR amplification reaction by taking the total DNA of R as a template;
the PCR reaction conditions are 94 ℃ for 3min, 94 ℃ for 15 s; 53 ℃ for 15s, 30cycles, 72 ℃ for 30s, 72 ℃ for 5 min. The PCR product is subjected to a 1.5% agarose nucleic acid gel electrophoresis test, the band is about 1500bp, and the PCR product is purified and sent to the department of Ongjingke biology company for sequencing.
2.2.3 sequencing results are shown in SEQ ID No.1, BLAST analysis of the 16s sequence was performed on the NCBI website, several reference sequences were downloaded from GenBank and phylogenetic trees were constructed using Megalign, as shown in FIG. 2. Finally, the strain is identified as lactobacillus casei R, the morphological characteristics of the colony are shown in figure 2, the strain is delivered to China general microbiological culture collection and management center for collection in 2021, 9 months and 17 days, and the collection number is CGMCC No. 23438.
Example 2: lactobacillus casei (Lactobacillus casei) R fermented Chinese pulsatilla powder
1. Preparing a fermentation liquid:
1.1 preparing an MRS liquid culture medium;
1.2 seed liquid culture: inoculating the Lactobacillus casei (Lactobacillus casei) R strain into an MRS liquid culture medium, standing, fermenting and culturing to obtain a seed solution, wherein the fermentation culture conditions are as follows: culturing at 37 deg.C for 24 hr;
1.3 amplification culture: inoculating 1% of seed liquid by volume percentage into an MRS liquid culture medium, standing, fermenting and culturing to obtain fermentation liquid, wherein the fermentation culture conditions are as follows: culturing at 37 deg.C for 24 hr.
2. Lactobacillus casei (Lactobacillus casei) R fermented Chinese pulsatilla powder
2.1 Chinese medicine decoction: weighing 20g of Chinese pulsatilla root powder, adding 1000mL of ultrapure water, soaking for 3h, decocting to 20mL, then decocting to 121 ℃, and sterilizing at high pressure for 30min to obtain 1g/mL of Chinese pulsatilla root powder decoction;
2.2 fermentation product: mixing the obtained fermentation liquor and the Chinese pulsatilla root decoction according to the weight ratio of 1:1 proportion, and then the mixture is kept still and cultured for 24 hours at 37 ℃.
Example 3: detection and microscopic comparison of content of components before and after fermentation of Pulsatilla chinensis powder by Lactobacillus casei R
3.1 test methods: detecting the content change of effective components before and after fermentation by using a high performance liquid chromatograph, repeating the test for three times, and taking an average value. Substituting the peak area in the detection result into a regression line equation and then carrying out significant difference analysis.
3.2 the results are shown in FIG. 5, and the data before and after fermentation in each test group are shown in Table 2:
TABLE 2 results of HPLC for detecting the change of effective components before and after fermentation of Pulsatillae radix
Test results show that after the Chinese pulsatilla root powder is fermented by Lactobacillus casei R, the effective components of the Chinese pulsatilla root powder are remarkably improved (P is less than 0.01).
As shown in figure 3, the macromolecular substances in the traditional Chinese medicine are changed into small molecular substances after fermentation, which shows that Lactobacillus casei (Lactobacillus casei) R destroys the cell wall of the traditional Chinese medicine in the fermentation process, so that the components in the traditional Chinese medicine are released and decomposed into smaller granular substances.
3. Bacteriostatic test before and after lactobacillus casei R fermentation of pulsatilla chinensis powder
3.1 test methods: exploring the bacteriostatic condition before and after fermentation by Oxford cup method, firstly, taking the final concentration of 1 × 107Coating 60 mu L of CFU/mL staphylococcus aureus, salmonella and escherichia coli, wherein the thickness of the plate is 20-25 mm; after the oxford cup is punched, the adding amount of the decoction/fermentation liquid is 60-80 mu L. After the decoction/fermentation broth is added into the pores, the diameter is measured after the mixture is subjected to static culture at 37 ℃ for 24 hours, the experiment is repeated three times, and the average value is taken.
3.2 test results
As shown in fig. 4, the bacteriostatic properties of the pulsatilla chinensis powder before and after fermentation are significantly changed: the Chinese pulsatilla root powder has no bacteriostatic zone before fermentation; after the fermentation of Lactobacillus casei (Lactobacillus casei) R, no pathogenic bacteria grow around the fermentation product, and an obvious bacteriostatic zone appears.
The zone of inhibition evaluation criteria are shown in table 3:
TABLE 3 evaluation criteria for zone of inhibition
The data before and after fermentation are shown in table 4:
TABLE 4 bacteriostatic diameters before and after fermentation of Pulsatilla chinensis powder by Lactobacillus casei R
As can be seen from the above table, the fermentation product has an inhibition zone of 19.95mm for Staphylococcus aureus, 19.97mm for Salmonella, and 16.80mm for Escherichia coli, and shows high sensitivity level, while the single Pulsatilla chinensis powder does not have antibacterial activity. The Chinese pulsatilla root powder fermented by the lactobacillus casei has unexpected technical effects.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> Henan university of agriculture
<120> lactobacillus casei R and fermentation method and application thereof
<141> 2021-12-31
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 848
<212> DNA
<213> Lactobacillus casei
<400> 1
ggatgggctg ctatgatgca gtcgaacgag ttctcgttga tgatcggtgc ttgcaccgag 60
attcaacatg gaacgagtgg cggacgggtg agtaacacgt gggtaacctg cccttaagtg 120
ggggataaca tttggaaaca gatgctaata ccgcatagat ccaagaaccg catggttctt 180
ggctgaaaga tggcgtaagc tatcgctttt ggatggaccc gcggcgtatt agctagttgg 240
tgaggtaatg gctcaccaag gcgatgatac gtagccgaac tgagaggttg atcggccaca 300
ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat 360
ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa ggctttcggg tcgtaaaact 420
ctgttgttgg agaagaatgg tcggcagagt aactgttgtc ggcgtgacgg tatccaacca 480
gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc 540
cggatttatt gggcgtaaag cgagcgcagg cggtttttta agtctgatgt gaaagccctc 600
ggcttaaccg aggaagcgca tcggaaactg gaaaacttga gtgcagaaga ggacagtgga 660
actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc 720
tgtctggtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac 780
cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt 840
gccgcagc 848
Claims (10)
1. A lactobacillus casei R is characterized in that: the classification name of the lactobacillus casei R is lactobacillus caseiLactobacillus casei(ii) a The preservation number is CGMCC No. 23438; the preservation unit is China general microorganism strain preservation and management center; the preservation time is 2021, 9 months and 17 days; the storage address is No. three of Xilu-Hayao-Beijing, Chaoyang, Beijing.
2. Use of the Lactobacillus casei R as set forth in claim 1 for improving the antibacterial activity of Pulsatilla chinensis.
3. Use according to claim 2, characterized in that: the antibacterial property of the pulsatilla chinensis powder is improved by fermenting the pulsatilla chinensis powder with lactobacillus casei R.
4. The method for fermenting pulsatilla chinensis powder by using lactobacillus casei R as claimed in claim 1, which is characterized by comprising the following steps:
(1) culturing a seed solution: inoculating lactobacillus casei R into MRS liquid culture medium for culture to obtain seed liquid;
(2) and (3) amplification culture: inoculating the seed liquid in the step (1) into an MRS liquid culture medium for fermentation culture to obtain a fermentation liquid;
(3) the traditional Chinese medicine decoction: adding Pulsatilla chinensis powder into ultrapure water, soaking for a period of time, decocting and concentrating to obtain a concentrated solution, and autoclaving to obtain Pulsatilla chinensis powder decoction;
(4) and (3) mixing the fermentation liquor obtained in the step (2) and the pulsatilla chinensis decoction obtained in the step (3) in proportion, and then standing and culturing to obtain a fermentation product.
5. The method of claim 4, wherein: the culture condition in the step (1) is culture at 37 ℃ for 24 h.
6. The method of claim 4, wherein: the inoculation proportion of the seed liquid in the step (2) is 1 percent of the volume of the MRS liquid culture medium; the fermentation culture condition is that the culture is carried out for 24 hours at 37 ℃.
7. The method of claim 4, wherein: the concentration of the Chinese pulsatilla root powder in the ultra-pure water in the step (3) is 0.02 g/mL; the concentration of the pulsatilla chinensis powder in the concentrated solution is 1 g/mL; autoclaving condition is 121 deg.C for 30 min.
8. The method of claim 4, wherein: the volume ratio of the fermentation liquid in the step (2) to the pulsatilla chinensis decoction in the step (3) in the step (4) is 1: 1; the conditions for static culture were 37 ℃ for 24 hours.
9. A fermentation product obtainable by the process of any one of claims 4 to 8.
10. Use of a fermentation product according to claim 9 in the preparation of a bacteriostatic medicament, wherein: the bacteria are staphylococcus aureus, salmonella or escherichia coli.
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