CN109943502B - Probiotics, leavening agent and application in Chinese herbal medicine preparation - Google Patents
Probiotics, leavening agent and application in Chinese herbal medicine preparation Download PDFInfo
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Abstract
The invention discloses probiotics, a leavening agent and application thereof in a Chinese herbal medicine preparation, wherein the leavening agent comprises 3 probiotics, two kinds of bacillus have the effect of producing complex enzyme, and the other kind of lactobacillus has stronger stress resistance. The leaven of the invention can efficiently ferment Chinese herbal medicines of cinnamomum camphora and erysipelas, and comprises liquid fermentation and solid fermentation. The fermented Chinese herbal medicine can obviously enhance the inhibition effect on pathogenic vibrios, and is 2-4 times of that before fermentation. The invention has the effects of preventing and treating diseases when being applied to aquaculture, has good effect, no drug resistance and environmental protection.
Description
Technical Field
The invention relates to probiotics and a leavening agent, in particular to the probiotics, the leavening agent and application in Chinese herbal medicine preparations.
Background
The dry fruits of the cinnamomum camphora (Cinnamomum camphora) are spherical, brown black to purple black, have uneven surface and have a diameter of about 5-10 mm. The medicine is pungent in flavor, warm in nature and nontoxic, and has the effects of dispelling cold, eliminating dampness, promoting qi circulation, relieving pain, inhibiting bacteria, and treating vomiting and diarrhea, stomach cold, abdominal pain and toxic swelling. The Laggera pterodonta is dry aerial parts of Laggera pterodonta of Compositae. Collected in autumn when stem and leaf flourish. It is pungent, bitter and cold in nature. Has the effects of clearing away heat and toxic materials, relieving cough and eliminating phlegm.
Vibrio (Vibrio) is widely distributed in the bodies of seawater and marine animals in estuaries, gulfs, offshore areas. Vibriosis is a type of disease caused by vibrio, and is the most common bacterial disease of marine fishes. A common condition of vibriosis is superficial skin ulceration. The main fish and shellfish pathogenic bacteria are: vibrio alginolyticus, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio vulnificus, etc.; some Vibrio species can also cause human diseases, such as Vibrio alginolyticus, Vibrio vulnificus, etc.
A plurality of extracellular enzymes generated by the enzyme-producing bacillus after proliferation in water can decompose organic matters such as starch, protein, fat and the like in the culture water body and the bottom sediment, thereby achieving the effects of reducing the eutrophication of the culture water body and reducing the generation of the bottom sediment. Enterococcus faecium is a kind of lactobacillus with strong stress resistance, has good stress resistance (high temperature, acid-base, high salt and oxygen), good intestinal adhesion capability, and also has good tolerance to partial antibiotics.
The probiotics is used for fermenting the Chinese herbal medicines, and the enzymatic reaction of microbial enzymes can enable active substances of the Chinese herbal medicines to be more fully released or further metabolized and converted into ingredients with higher activity. The test of the ministry of health medicine shows that the fermented Chinese herbal medicine can exert the same drug effect only by 1/28 of the amount of water extract which is decocted, boiled and boiled traditionally. Modern microbial fermentation research shows that enzymes generated in the process of fermenting Chinese herbal medicines by microorganisms can digest plant cell walls, release active substances to a large extent, degrade toxic substances and generate new bioactive substances, so that the effective components of the Chinese herbal medicines are biologically converted, macromolecular substances in the Chinese herbal medicines are converted into small molecular substances which can be directly absorbed by intestinal tracts of animals, and the Chinese herbal medicines become novel medicines with quick absorption and quantitative curative effects. The fermentation of Chinese herbal medicines with a single strain often has the defects of single enzyme production type, weak enzyme production capacity and the like. Compared with single-bacterium fermentation, the mixed-bacterium fermentation can play the composite effects of multiple bacteria and enzymes to a greater extent, the fermentation efficiency is higher, the fermentation product is richer, and the mixed-bacterium fermentation has more potential than single-bacterium fermentation.
Disclosure of Invention
In order to solve the technical problems, the invention provides probiotics, a leavening agent and application of the probiotics and the leavening agent in a Chinese herbal medicine preparation, so as to achieve the purposes of effectively inhibiting vibrio diseases and applying the probiotics and the leavening agent in aquaculture.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a probiotic is classified and named as Bacillus subtilis BSX, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is the microorganism institute of China academy of sciences No.3, West Lu No.1, North Chen West Lu, No.1, Beijing, the preservation date is 2018, 4 and 2 days, the preservation number is CGMCC No.15541, and the DNA sequence is shown as SEQ ID No. 1.
A leaven, which is prepared from the Bacillus subtilis BSX (Bacillus subtilis) as claimed in claim 1, and Enterococcus faecium (Enterococcus faecium) Ef026 and Bacillus licheniformis (Bacillus licheniformis) BL3C, wherein the Enterococcus faecium (Enterococcus faecium) Ef026 and the Bacillus licheniformis (Bacillus licheniformis) BL3C are both preserved in the common microbiology center of the china microbiology collection management committee, the preservation site is the institute of microbiology of china institute of china academy of sciences No.3, north chen west way 1, north township, north prefecture, the preservation date is 2018, 4 months and 2 days, and the preservation numbers are respectively: CGMCC No.15542 and CGMCC No.15543, and the DNA sequences are shown in SEQ ID No.2 and SEQ ID No. 3.
In the scheme, the preparation method of the leavening agent comprises the following steps: the bacillus subtilis BSX and the bacillus licheniformis BL3C are cultured in a liquid LB culture medium at 37 ℃ for 12-16 hours, the enterococcus faecium Ef026 is cultured in a liquid MRS culture medium at 37 ℃ for 12-16 hours, and the bacillus subtilis BSX, the bacillus licheniformis BL3C and the enterococcus faecium Ef026 are cultured according to a bacteria activity ratio of 1-3: 1-3: 1-2 mixing.
In a further technical scheme, the preparation method of the leavening agent comprises the following steps: the bacillus subtilis BSX and the bacillus licheniformis BL3C are cultured in a liquid LB culture medium at 37 ℃ for 14 hours, the enterococcus faecium Ef026 is cultured in a liquid MRS culture medium at 37 ℃ for 15 hours, and the bacillus subtilis BSX, the bacillus licheniformis BL3C and the enterococcus faecium Ef026 are cultured according to a bacteria activity ratio of 1: 1: 1 and mixing.
The application of the leaven in Chinese herbal medicine preparation, wherein the Chinese herbal medicine preparation has the effect of inhibiting pathogenic vibrio.
In a further technical scheme, the specific method for preparing the liquid Chinese herbal medicine preparation by fermenting the leavening agent is as follows: the Chinese herbal medicines are crushed to 30-60 meshes, account for 5-15% of the total mass, water accounts for 65-75% of the total mass, a leavening agent accounts for 15-20% of the total mass, and the Chinese herbal medicines are fermented for 48-72 hours at 35-40 ℃.
In a further technical scheme, the specific method for preparing the solid Chinese herbal medicine preparation by fermenting the leavening agent comprises the following steps: the Chinese herbal medicine is crushed to 30-60 meshes and accounts for 10-15% of the total mass, the bran accounts for 20-25% of the total mass, the corn flour accounts for 10-15% of the total mass, the soybean meal accounts for 5-10% of the total mass, the ammonium sulfate accounts for 0.5-1% of the total mass, the monopotassium phosphate accounts for 0.2-0.25% of the total mass, the magnesium sulfate accounts for 0.05-0.1% of the total mass, the water accounts for 25-30% of the total mass, the leavening agent accounts for 10-15% of the total mass, and the fermentation is carried out at 30-35 ℃ for 72-96 hours.
In a further technical scheme, the Chinese herbal medicine is cinnamomum camphora or Laggera pterodonta.
Through the technical scheme, the 2 probiotics (bacillus subtilis BSX and bacillus licheniformis BL3C) provided by the invention can generate a strong compound enzyme system comprising protease, amylase, pectinase, ligninase, hemicellulase, cellulase and the like. Enterococcus faecium Ef026 has good effects in improving animal immunity, regulating intestinal microecological balance, improving nutrient absorption, reducing diarrhea rate, and reducing death rate. After the cinnamomum camphora and the prodigiosin are fermented by the microbial fermentation agent, the inhibition effect on pathogenic vibrios is improved by 2-4 times. The Chinese herbal medicine preparation provided by the invention has no risks such as antibiotic residue and drug tolerance, is environment-friendly, and has a wide application prospect in the field of aquaculture.
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.
FIG. 1 shows the colony morphology of three probiotics of the present invention;
FIG. 2 shows the inhibitory effect of the herbal formulation of the present invention on Vibrio parahaemolyticus;
FIG. 3 shows the inhibitory effect of the herbal formulation of the present invention on Vibrio harveyi;
FIG. 4 shows the inhibitory effect of the herbal medicine preparation of the present invention on Vibrio alginolyticus.
Note: in the figure, the letter L represents Lingdancao; x represents cinnamomum camphora; the middle numbers 0, 1 and 2 respectively represent blank groups, liquid fermentation and solid fermentation; the right 0, 2 and 4 respectively represent dilution times, 0 is undiluted, 2 is diluted by 2 times, and 4 is diluted by 4 times; "Water" is a negative control.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
1. Bacterial colony culture
Both bacillus subtilis BSX and bacillus licheniformis BL3C were activated in LB broth and inoculated to 5mL LB broth at 1% inoculum size. Enterococcus faecium Ef026 was activated in MRS broth and inoculated at 1% inoculum size to 5mL MRS broth. Culturing the bacterial liquid at 37 ℃ and 180rpm for 12-16 hours, performing gradient dilution on the bacterial liquid by 10 times by using sterile water, taking 100 mu L of bacterial liquid with each gradient concentration BSX and BL3C, coating the bacterial liquid on an LB solid culture medium with the diameter of 90 multiplied by 90mm, culturing the bacterial liquid at 37 ℃ for 12-16 hours, taking 100 mu L of bacterial liquid with each gradient concentration Ef026, coating the bacterial liquid on an MRS solid culture medium with the diameter of 90 multiplied by 90mm, culturing the bacterial liquid at 37 ℃ for 16-20 hours, and taking a culture dish with a proper concentration to observe the colony morphology, wherein the figure is shown in figure 1.
2. Determination of the enzyme-producing Activity of two Probiotics
The research on the enzyme production characteristics of the bacillus subtilis BSX and the bacillus licheniformis BL3C is carried out by utilizing an LB culture medium, a TCBS culture medium, a lignin degradation culture medium, a CMC (carboxymethyl cellulose) separation culture medium, a protein degradation culture medium, a starch degradation culture medium, a hemicellulose degradation culture medium and a pectin degradation culture medium. Activating bacillus subtilis BSX and bacillus licheniformis BL3C in LB liquid culture medium, culturing at 37 ℃ for 16 hours, then dropping 10 mu L of bacterial liquid on corresponding enzyme plates, culturing for one to two days, and then measuring the colony diameter and the hydrolysis ring diameter by dyeing and rinsing with a coloring agent or direct observation, wherein the results are shown in Table 1.
TABLE 1 Complex enzyme producing ability of Bacillus subtilis BSX and Bacillus licheniformis BL3C
3. Preparation of leavening agent
Activating bacillus subtilis BSX and bacillus licheniformis BL3C in an LB liquid culture medium, transferring the activated bacillus subtilis BSX and the bacillus licheniformis BL3C to 100mL of the LB liquid culture medium in an inoculation amount of 1%, and culturing for 14 hours; enterococcus faecium Ef026 was activated in MRS liquid medium, inoculated to 100mL MRS liquid medium at 1% inoculum size, and cultured for 15 hours to obtain seed solution. And (2) enabling the bacillus subtilis, the enterococcus faecium and the bacillus licheniformis which grow well in the logarithmic growth phase to grow according to the bacteria activity ratio of 1: 1: 1, preparing mixed fermentation strains.
4. Preparation of Chinese herbal medicine preparation
Example 1 preparation of liquid fermented Cinnamomum camphora preparation
(1) Chinese herbal medicine preparation prepared by liquid fermentation of cinnamomum camphora
Pulverizing fructus Cinnamomi Camphorae to 40 mesh, 15% of total mass, water 70%, inoculating 15% of the leaven prepared by above 3, and fermenting at 37 deg.C for 72 hr.
And (3) indication bacteria: vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and the like.
(2) Fermentation broth bacteriostasis experiment
The fermentation broth was sterilized by 0.22nm membrane filtration and diluted by two-fold dilution. Taking 100 mu L of indicator bacteria to coat on a TCBS solid culture medium, dripping 10 mu L of fermentation liquor and double-diluted fermentation liquor on the TCBS culture medium coated with the indicator bacteria, culturing overnight at 37 ℃, and observing the condition of a transparent ring.
The diameter of the transparent ring is about 10mm, the cinnamomum camphora fermentation liquor is diluted to four times, and the three types of indicator bacteria have inhibition effects on vibrio parahaemolyticus, vibrio alginolyticus and vibrio harveyi, the blank group only has antibacterial activity (shown in figures 2 to 4) on the stock solution, and the bacteria liquid used for fermentation does not have inhibition effects on pathogenic vibrio. The minimum pathogenic vibrio inhibiting concentration (MIC) of the cinnamomum camphora prepared by fermenting the mixed liquid of the bacillus subtilis BSX, the enterococcus faecium Ef026 and the bacillus licheniformis BL3C is about 0.0375 g/mL.
Example 2 preparation of solid fermented Cinnamomum camphora preparation
(1) The Chinese herbal medicine preparation is prepared by solid fermentation of the cinnamomum camphora fruit.
Crushing the cinnamomum camphora to 40 meshes, wherein the crushed cinnamomum camphora accounts for 15% of the total mass, the bran accounts for 20% of the total mass, the corn flour accounts for 10% of the total mass, the soybean meal accounts for 9% of the total mass, the ammonium sulfate accounts for 0.7% of the total mass, the monopotassium phosphate accounts for 0.25% of the total mass, the magnesium sulfate accounts for 0.05% of the total mass, the water accounts for 30% of the total mass, inoculating 15% of the leavening agent prepared in the step 3, and fermenting for 72 hours at 30 ℃. The indicator bacteria were as described in example 1.
(2) Antibacterial test after fermentation
Taking 1g of fermented Chinese herbal medicine preparation, fully dissolving with 9mL of sterile water, filtering and sterilizing with a sterilized 0.22nm membrane, and diluting by a two-fold dilution method. Taking 100 mu L of indicator bacteria to coat on a TCBS solid culture medium, dripping 10 mu L of fermentation liquor and double-diluted fermentation liquor on the TCBS culture medium coated with the indicator bacteria, culturing overnight at 37 ℃, and observing the condition of a transparent ring.
The diameter of the transparent ring is about 8mm, the cinnamomum camphora fruit solid is diluted to four times after fermentation to have an inhibition effect on vibrio parahaemolyticus, the cinnamomum camphora fruit solid is diluted to two times to have an inhibition effect on vibrio harveyi, the stock solution has an inhibition effect on vibrio alginolyticus, and only the stock solution in the blank group has an antibacterial activity (shown in figures 2 to 4).
Example 3 preparation of liquid fermented Lingdancao preparation
(1) Chinese herbal medicine preparation prepared by liquid fermentation of prodigiosin
Pulverizing herba Laggera into 30 mesh powder, accounting for 10% of the total mass, adding water accounting for 75% of the total mass, inoculating 3% of the mixed starter, and fermenting at 37 deg.C for 72 hr. The indicator bacteria were as described in example 1.
(2) Fermentation broth bacteriostasis experiment
The procedure is as in example 1. The diameter of the transparent circle is about 10mm, the Laggera pterodonta preparation has an inhibitory effect on parahaemolysis, has an inhibitory effect on Vibrio harveyi and Vibrio alginolyticus after being diluted twice, and has no pathogenic vibrio inhibitory activity in the blank stock solution (shown in figures 2 to 4). The minimum pathogenic vibrio inhibiting concentration (MIC) of the liquid fermented prodigiosin is about 0.05 g/mL.
Example 4 preparation of solid fermented Laggera pterodonta formulation
(1) Preparation of Chinese herbal medicine preparation by solid fermentation of prodigiosin
Crushing the prodigiosin into 30 meshes, wherein the powder accounts for 14% of the total mass, the bran accounts for 20% of the total mass, the corn flour accounts for 10% of the total mass, the soybean meal accounts for 10% of the total mass, the ammonium sulfate accounts for 0.7% of the total mass, the monopotassium phosphate accounts for 0.25% of the total mass, the magnesium sulfate accounts for 0.05% of the total mass, the water accounts for 30% of the total mass, inoculating 15% of the leavening agent prepared in the step 3, and fermenting for 72 hours at 30 ℃. The indicator bacteria were as described in example 1.
(2) Antibacterial test after fermentation
The procedure is as described in example 2. The diameter of the transparent circle is about 8mm, the Lingdancao has inhibitory effect on Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus after solid fermentation, and the blank group has no bacteriostatic activity (shown in figures 2 to 4). The minimum pathogenic vibrio inhibiting concentration (MIC) of the solid fermentation of the prodigiosin is about 0.1 g/mL.
4. Influence of liquid cinnamomum camphora seed fermentation preparation and Lingdancao fermentation preparation on quantity and growth index of pathogenic vibrio of Penaeus vannamei Boone respectively (1) laboratory culture conditions
Experiment water body 0.1m3300 tails of Penaeus vannamei P6 stage larvae are released, the pH value is 7.8, the illumination intensity is 5 multiplied by 100LX, and the salinity is 25 per mill. Each group feeds 0.6g of feed each time, four times each day, and the feeding time is respectively 8: 00, 11: 00, 16: 00, 22: 00. the experiments are divided into three groups, namely a control group and two experimental groups, namely a camphor group and a pteris pterodonta group, and each group is repeated three times. Feeding 0.6g of feed for each time in a control group; cinnamomum camphora dispensingFeeding the feed added with the cinnamomum camphora fermentation liquid with the concentration of 150mg/mL in the example 1; the prodigiosin group was fed with the feed supplemented with 100mg/mL of the prodigiosin fermentation broth of example 3 for one month continuously.
(2) Determination of pathogenic Vibrio
Every other day, 10 times and 100 times of culture water sample is diluted by sterilized seawater, then 0.1mL of TCBS coated plate is taken, 3 plates are paralleled, cultured for 24 hours at 30 ℃, and counted.
(3) Determination of growth index of penaeus vannamei boone
After 20 days of culture, measuring the growth index of the penaeus vannamei boone, and calculating the survival rate and the weight gain rate according to the following formula:
survival (%). survival number at harvest x 100/initial stocking number
Weight gain (%) (weight of 50 penaeus orientalis at harvest-weight of 50 penaeus orientalis at initial stocking) × 100/weight of 50 penaeus orientalis at initial stocking
(4) Influence of cinnamomum camphora liquid fermentation preparation on quantity of pathogenic vibrios in water body
After 8 days of adding the cinnamomum camphora fermenting preparation until the experiment is finished, the number of pathogenic vibrio of the experimental group is lower than that of the control group. After three repeated feeding for 20 days, the average value of pathogenic vibrio is reduced by 52.56%, 43.40% and 48.70% respectively compared with that of a control group.
(5) Influence of cinnamomum camphora liquid fermentation preparation on growth indexes of penaeus vannamei boone
1. Survival rate
The cinnamomum camphora fruit fermentation preparation with a certain concentration has obvious influence on the survival rate of the penaeus vannamei boone (P is less than 0.05); when the feed is put for 10 days, the survival rate is the highest and reaches 95.30 percent, which is increased by 18.13 percent compared with a control group.
2. Rate of body weight gain
When the administration is carried out for 10 days, the weight gain rate is highest and reaches 1340.16%, which is increased by 50.19% compared with the control group and is obviously higher than other groups (P < 0.05). Duncan multiple comparison results show that the using amount of the cinnamomum camphora fermenting preparation is 0.15-1.5 g/mL when the weight growth rate is the highest.
(6) Influence of Laggera pterodonta fermentation preparation on number of pathogenic vibrio in water body
After 8 days of adding the prodigiosin fermentation preparation until the end of the experiment, the number of pathogenic vibrio of the experimental group is lower than that of the control group. After three repeated feeding for 20 days, the average value of pathogenic vibrio is reduced by 41.45%, 41.38% and 45.52% respectively compared with the control group.
(7) Influence of Laggera pterodonta liquid fermentation preparation on growth index of Penaeus vannamei Boone
1. Survival rate
The effect of the prodigiosin fermentation preparation with a certain concentration on the survival rate of the penaeus vannamei boone is remarkable (P is less than 0.05); when the feed is put for 10 days, the survival rate is the highest and reaches 91.0 percent, which is increased by 10.65 percent compared with a control group.
2. Rate of body weight gain
When the administration is carried out for 10 days, the weight growth rate is highest and reaches 1096.64%, which is increased by 36.50% compared with the control group. Duncan multiple comparison results show that the using amount of the Laggera pterodonta fermenting preparation is 0.1-1 g/mL when the weight growth rate is the highest.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Qingdao Marine biological medicine research institute
QINGDAO BIOANTAI BIOTECHNOLOGY Co.,Ltd.
DALI JINMING ANIMAL MEDICINE INDUSTRY Co.,Ltd.
<120> probiotics, leavening agent and application in Chinese herbal medicine preparation
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1360
<212> DNA
<213> Bacillus subtilis BSX (Bacillus subtilis)
<400> 1
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gaacgtattc accgcggcat gctgatccgc gattactagc gattccagct tcacgcagtc 120
gagttgcaga ctgcgatccg aactgagaac agatttgtgg gattggctta acctcgcggt 180
ttcgctgccc tttgttctgt ccattgtagc acgtgtgtag cccaggtcat aaggggcatg 240
atgatttgac gtcatcccca ccttcctccg gtttgtcacc ggcagtcacc ttagagtgcc 300
caactgaatg ctggcaacta agatcaaggg ttgcgctcgt tgcgggactt aacccaacat 360
ctcacgacac gagctgacga caaccatgca ccacctgtca ctctgccccc gaaggggacg 420
tcctatctct aggattgtca gaggatgtca agacctggta aggttcttcg cgttgcttcg 480
aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg agtttcagtc 540
ttgcgaccgt actccccagg cggagtgctt aatgcgttag ctgcagcact aaggggcgga 600
aaccccctaa cacttagcac tcatcgttta cggcgtggac taccagggta tctaatcctg 660
ttcgctcccc acgctttcgc tcctcagcgt cagttacaga ccagagagtc gccttcgcca 720
ctggtgttcc tccacatctc tacgcatttc accgctacac gtggaattcc actctcctct 780
tctgcactca agttccccag tttccaatga ccctccccgg ttgagccggg ggctttcaca 840
tcagacttaa gaaaccgcct gcgagccctt tacgcccaat aattccggac aacgcttgcc 900
acctacgtat taccgcggct gctggcacgt agttagccgt ggctttctgg ttaggtaccg 960
tcaaggtacc gccctattcg aacggtactt gttcttccct aacaacagag ctttacgatc 1020
cgaaaacctt catcactcac gcggcgttgc tccgtcagac tttcgtccat tgcggaagat 1080
tccctactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggccgatca 1140
ccctctcagg tcggctacgc atcgttgcct tggtgagcca ttacctcacc aactagctaa 1200
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<211> 1367
<212> DNA
<213> Enterococcus faecium Ef026(Enterococcus faecium)
<400> 2
tcgggtgtta caaactctcg tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt 60
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cgttgtactt cccattgtag cacgtgtgta gcccaggtca taaggggcat gatgatttga 240
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cgagctgacg acaaccatgc accacctgtc actttgcccc cgaaggggaa gctctatctc 420
tagagtggtc aaaggatgtc aagacctggt aaggttcttc gcgttgcttc gaattaaacc 480
acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcaac cttgcggtcg 540
tactccccag gcggagtgct taatgcgtta gctgcagcac tgaagggcgg aaaccctcca 600
acacttagca ctcatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctccc 660
cacgctttcg agcctcagcg tcagttacag accagagagc cgccttcgcc actggtgttc 720
ctccatatat ctacgcattt caccgctaca catggaattc cactctcctc ttctgcactc 780
aagtctccca gtttccaatg accctccccg gttgagccgg gggctttcac atcagactta 840
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cttcactcac gcggcgttgc tcggtcagac tttcgtccat tgccgaagat tccctactgc 1080
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tcggctatgc atcgtggcct tggtgagccg ttacctcacc aactagctaa tgcaccgcgg 1200
gtccatccat cagcgacacc cgaaagcgcc tttcaaatca aaaccatgcg gtttcgattg 1260
ttatacggta ttagcacctg tttccaagtg ttatcccctt ctgatgggca ggttacccac 1320
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<210> 3
<211> 1369
<212> DNA
<213> Bacillus licheniformis BL3C (Bacillus licheniformis)
<400> 3
cctcaccgac ttcgggtgtt acaaactctc gtggtgtgac gggcggtgtg tacaaggccc 60
gggaacgtat tcaccgcggc atgctgatcc gcgattacta gcgattccag cttcacgcag 120
tcgagttgca gactgcgatc cgaactgaga acagatttgt gggattggct tagcctcgcg 180
gcttcgctgc cctttgttct gcccattgta gcacgtgtgt agcccaggtc ataaggggca 240
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tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt tgctgcagca ctaaagggcg 600
gaaaccctct aacacttagc actcatcgtt tacggcgtgg actaccaggg tatctaatcc 660
tgttcgctcc ccacgctttc gcgcctcagc gtcagttaca gaccagagag tcgccttcgc 720
cactggtgtt cctccacatc tctacgcatt tcaccgctac acgtggaatt ccactctcct 780
cttctgcact caagttcccc agtttccaat gaccctcccc gggttgagcc gggggctttc 840
acatcagact taagaaaccg cctgcgcgcg ctttacgccc aataattccg gacacgcttg 900
ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtgctttctg gktaggtacc 960
gtcaaggtac cgccctattc gaacggtact tgttcttccc taacaacaga gttttacgat 1020
ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca ttgcggaaga 1080
ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt gtggccgatc 1140
accctctcag gtcggctacg catcgttgcc ttggtgagcc gttacctcac caactagcta 1200
atgcgccgcg ggtccatctg taagtggtag ctaaaagcca ccttttataa ttgaaccatg 1260
cggttcaatc aagcatccgg tattagcccc ggtttcccgg agttatccca gtcttacagg 1320
caggttaccc acgtgttact cacccgtccg ccgctaacat cagggagca 1369
Claims (6)
1. The leavening agent is characterized by being prepared from bacillus subtilis BSX, enterococcus faecium Ef026 and bacillus licheniformis BL3C, wherein the enterococcus faecium Ef026 and the bacillus licheniformis BL3C are both preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is the microorganism institute of China academy of sciences No.3, West Lu No.1 institute of China, North Cheng, the south China area, Beijing City, the preservation date is 2018, 4 months and 2 days, and the preservation numbers are respectively: CGMCC No.15542 and CGMCC No.15543, and the DNA sequences are shown as SEQ ID number 2 and SEQ ID number 3;
the bacillus subtilis BSX is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is the microorganism research institute of China academy of sciences No.3 of Xilu No.1 of Beijing Korean, 4 months and 2 days in 2018, the preservation number is CGMCC No.15541, and the DNA sequence is shown as SEQ ID number 1.
2. A starter culture according to claim 1, wherein the starter culture is prepared by a process comprising: the bacillus subtilis BSX and the bacillus licheniformis BL3C are cultured in a liquid LB culture medium at 37 ℃ for 12-16 hours, the enterococcus faecium Ef026 is cultured in a liquid MRS culture medium at 37 ℃ for 12-16 hours, and the bacillus subtilis BSX, the bacillus licheniformis BL3C and the enterococcus faecium Ef026 are cultured according to a bacteria activity ratio of 1-3: 1-3: 1-2 mixing.
3. A starter culture according to claim 2, wherein the starter culture is prepared by the following process: the bacillus subtilis BSX and the bacillus licheniformis BL3C are cultured in a liquid LB culture medium at 37 ℃ for 14 hours, the enterococcus faecium Ef026 is cultured in a liquid MRS culture medium at 37 ℃ for 15 hours, and the bacillus subtilis BSX, the bacillus licheniformis BL3C and the enterococcus faecium Ef026 are cultured according to a bacteria activity ratio of 1: 1: 1 and mixing.
4. The application of the leavening agent in preparing a Chinese herbal medicine preparation is characterized in that the leavening agent of claim 1 is adopted, the Chinese herbal medicine preparation has the effect of inhibiting pathogenic vibrio, and the Chinese herbal medicine is cinnamomum camphora or Laggera pterodonta.
5. The use of a fermentation broth for the preparation of herbal preparations according to claim 4, wherein the fermentation broth is used for the preparation of liquid herbal preparations by fermentation according to the following specific method: the Chinese herbal medicines are crushed to 30-60 meshes, account for 5-15% of the total mass, water accounts for 65-75% of the total mass, a leavening agent accounts for 15-20% of the total mass, and the Chinese herbal medicines are fermented for 48-72 hours at 35-40 ℃.
6. The use of the fermentation agent in the preparation of the Chinese herbal medicine preparation as claimed in claim 4, wherein the specific method for preparing the solid Chinese herbal medicine preparation by fermentation by using the fermentation agent is as follows: the Chinese herbal medicine is crushed to 30-60 meshes and accounts for 10-15% of the total mass, the bran accounts for 20-25% of the total mass, the corn flour accounts for 10-15% of the total mass, the soybean meal accounts for 5-10% of the total mass, the ammonium sulfate accounts for 0.5-1% of the total mass, the monopotassium phosphate accounts for 0.2-0.25% of the total mass, the magnesium sulfate accounts for 0.05-0.1% of the total mass, the water accounts for 25-30% of the total mass, the leavening agent accounts for 10-15% of the total mass, and the fermentation is carried out at 30-35 ℃ for 72-96 hours.
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