CN111084273B - Bupleurum overground part fermentation product, and preparation method, monitoring method and application thereof - Google Patents

Abstract

According to the invention, lactobacillus plantarum is adopted to ferment the aerial parts of the bupleurum for different time, and in the fermentation process of the aerial parts of the bupleurum, the content of 3 flavones (rutin, quercetin-3-O-beta-L-arabinoside, isoquercitrin) and DL-3-phenyl-2-hydroxy acrylic acid is increased along with the prolongation of the fermentation time, so that the bupleurum has time dependence; the antibacterial activity of the overground part of the bupleurum subjected to fermentation is enhanced along with the increase of the fermentation time, and the antibacterial effect on aquatic pathogenic bacteria is obviously stronger than that of non-aquatic pathogenic bacteria and has high sensitivity; therefore, the compound can be used for replacing antibiotics in feed and applied to aquaculture. In addition, the content of the components in the fermentation broth can be dynamically determined by adopting a proper biological sample treatment method and a UPLC-MS-MS method established in the invention.

Description

Bupleurum overground part fermentation product, and preparation method, monitoring method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to a novel application of lactobacillus plantarum fermented bupleurum aerial parts in preparation and application thereof in aquatic pathogenic bacteria.

Background

With the development of aquaculture industry, the risk of infection of aquatic animals with various diseases is greatly increased. In order to prevent and treat aquatic animal diseases, promote animal growth and development and improve feed utilization rate, a feed producer adds antibiotics as feed additives into feed to be used as growth promoters and medicines. However, with the long-term and large-scale use of antibiotic additives, part of antibiotics can accumulate in aquatic animals, and cause drug residues, so that consumers are indirectly transferred and influenced, and the human health and the environmental safety are seriously influenced. The quality safety of aquatic products has become a hot spot of great concern in the healthy and sustainable development of aquaculture.

The abuse of antibiotics in aquaculture mainly produces two negative effects: firstly, drug resistance is formed, diseases cannot be cured, and a large number of animals die and double losses of drug investment are caused. The use of antibiotics can cause the bacterial colony in the breeding environment to form drug resistance, once pathogenic bacteria have acquired drug resistance to antibiotics shared by people and livestock, after people are infected with the bacteria, the originally effective antibiotic curative effect can be reduced or even ineffective, so that the human loses body resistance to certain diseases. Secondly, veterinary drug residues; antibiotics which can only be used for breeding animals can enter human bodies through food chains, and can cause harm to the human bodies and influence human health. Meanwhile, antibiotics can damage micro balance in the culture environment and pollute the environment; but also can inhibit the immune function of the cultured animals.

Therefore, the development of a low-toxicity, safe, effective and anti-disease feed additive which is not easy to generate drug resistance replaces the existing antibiotics and is a problem which needs to be solved urgently at present.

Because antibiotics are mostly used as feed additives in the current aquatic animal cultivation, the antibiotics can be transmitted through a food chain, and the health of people is endangered. The research shows that the spray-dried plasma protein powder, chinese herbal medicine, probiotics, prebiotics, egg yolk antibody and the like can be used as feed additives instead of antibiotics, wherein the Chinese herbal medicine is a treasure which inherits the Chinese medical treasury for more than four thousand years, has double functions of nutrition and disease treatment, and has the advantages of low toxicity, wide resources and difficult drug resistance. Is popular with farmers and also attracts attention of researchers.

Bupleurum is derived from dry root (Bupleurum scorzonerifolium Willd.) of Bupleurum scorzonerifolium (Bupleurum chinense DC.) of Umbelliferae, and widely distributed in northeast, north China, northwest, east China, hubei and Sichuan etc., and is a common traditional Chinese medicine, and has application history of over 2000 years in China, which is first recorded in Shennong Ben Cao Jing, yuan Ming Hu and Di Lau, listed as top grade, and easily named as Bupleurum scorzonerifolium in Song dynasty, and is collected by 2015 edition pharmacopoeia of China, and is classified into 'Nanfu' and 'North Bupleurum' according to characters. Bupleurum root is pungent and bitter in flavor and slightly cold in nature, has the effects of dispelling heat, soothing liver, relieving depression, lifting yang qi and the like, and is mainly used for treating symptoms such as cold and fever, cold and heat going forward, malaria, irregular menstruation, uterine prolapse and the like. Modern pharmacological researches have shown that bupleurum root has the effects of relieving fever, easing pain, calming, resisting inflammation, protecting liver, promoting bile flow, resisting bacteria, resisting viruses, resisting tumors and the like. The root of bupleurum is generally used as a medicinal part, and the aerial parts are discarded, so that the aerial parts of bupleurum are wasted greatly. Research reports show that the stems, leaves, flowers, fruits and underground partial roots of bupleurum root are rich in flavonoid components, the contents of different parts are different, and the total flavone content of the overground parts is far higher than that of the underground parts according to the literature report. Literature researches show that the bupleurum water extract has obvious inhibition effect on staphylococcus aureus and typhoid bacillus, and can reduce the death rate of acute abdominal infection of mice caused by the typhoid bacillus; in addition, the ethanol extract of the aerial parts of the bupleurum root has very remarkable antibacterial effect on staphylococcus epidermidis, shigella flexneri, escherichia coli and streptococcus pyogenes.

Along with the wide application of microbial fermentation technology, emerging microorganisms and traditional Chinese medicines are fermented together gradually to become a method for improving the active ingredients of the traditional Chinese medicines. The microorganism can generate secondary metabolites such as phenyllactic acid in the fermentation process with the traditional Chinese medicine, and the secondary metabolites interact with the active ingredients of the traditional Chinese medicine to play a role of synergism and toxicity reduction. Meanwhile, various active enzymes produced by microorganisms are beneficial to the release of active ingredients of the traditional Chinese medicine.

Disclosure of Invention

The invention aims at relating to a new application of lactobacillus plantarum fermented bupleurum aerial parts in preparation and application thereof in aquatic pathogenic bacteria.

The specific technical scheme is as follows:

the invention provides a preparation method of bupleurum overground part fermentation products, which comprises the following steps:

preparing an extract of aerial parts of bupleurum chinense: reflux extracting aerial parts of radix bupleuri with water, concentrating, and drying to obtain aerial parts of radix bupleuri extract;

culturing lactobacillus plantarum: inoculating the preserved lactobacillus plantarum into an MRS solid culture medium by a streaking method, and inoculating the strain into an MRS broth culture medium in an inoculum size of 10% to prepare a standby lactobacillus plantarum mother solution;

preparing a fermentation liquid: dissolving aerial parts of radix bupleuri extract with MRS culture medium to obtain extract solution, inoculating lactobacillus plantarum, and incubating in a constant temperature shake incubator to obtain aerial parts fermentation product of radix bupleuri.

Preferably, the step of preparing the bupleurum aerial part extract comprises:

weighing 100g of aerial parts of bupleurum, pulverizing, sieving, adding water according to a liquid-to-material ratio of 10:1, soaking for 2h, heating and refluxing for 2h, filtering and collecting filtrate, extracting twice, combining the filtrates, recovering solvent under reduced pressure at 40 ℃, freeze-drying to obtain medicinal material extract, and placing in a refrigerator for standby.

According to another aspect of the present invention, there is also provided an aerial part fermentation product of bupleurum prepared according to the preparation method as described above.

According to another aspect of the present invention, there is also provided the use of an aerial part fermentation product of bupleurum prepared according to the preparation method as described above, as an aquatic feed additive.

Preferably, the bupleurum root overground part fermentation product has an antibacterial effect on aquatic pathogenic bacteria.

Preferably, the aquatic pathogenic bacteria include aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus, and vibrio harveyi.

According to another aspect of the present invention, there is also provided a method for dynamically monitoring the aerial parts of bupleurum fermentation product prepared according to the preparation method as described above, by monitoring the content changes of 8 flavone compounds and DL-3-phenyl-2-hydroxypropionic acid in the fermentation product at different times using a liquid chromatography-mass spectrometry method.

Preferably, the flavone compound comprises kaempferol-3-O-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, vetch tail glycoside, quercetin-3-O-beta-L-arabinoside, kaempferol.

The bupleurum overground part fermentation product and the preparation method, the monitoring method and the application thereof have the following beneficial effects:

the aerial parts of the bupleurum have rich flavonoid compounds, have antibacterial effect, are mostly discarded as waste, pollute the environment and cause waste of a large amount of resources, so the aerial parts of the bupleurum can be developed, resources can be saved, the aerial parts can be developed into aquatic feed additives of alternative antibiotics, and the development of non-medicinal parts of the bupleurum is also an improvement;

the compound UPLC-MS-MS detection condition established by the invention has good linearity, high sensitivity and strong specificity; under the condition, the extraction chromatogram of each component to be detected in the bupleurum aerial fermentation sample has no interference with the internal standard peak, the peak shape is good, and the base line is stable;

experiments prove that the content of DL-3-phenyl-2-hydroxypropionic acid is obviously increased in the process of fermenting aerial parts and bacterial liquid of the bupleurum, and the increase rate of DL-3-phenyl-2-hydroxypropionic acid in the aerial parts of the bupleurum is higher than that of the fermented bacterial liquid, so that the fermentation of the aerial parts of the bupleurum can stimulate lactobacillus plantarum to produce more DL-3-phenyl-2-hydroxypropionic acid; in addition, in 8 flavone compounds measured from the aerial parts of the fermented bupleurum, rutin, quercetin-3-O-beta-L-arabinoside and isoquercitrin have higher content, and the content is increased along with the fermentation time, so that the content of other flavone is basically unchanged;

the inventor finds that the overground part of the fermented bupleurum has different inhibition effects on 9 pathogenic bacteria through in vitro bacteriostasis experiments, and along with the increase of the fermentation time, the diameter of a bacteriostasis zone of the bupleurum increases, which indicates that the fermentation can promote the bacteriostasis activity of the bupleurum; in the group of the overground parts of the fermented bupleurum, the antibacterial effect on aquatic pathogenic bacteria (aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus and vibrio harveyi) is obviously stronger than that of non-aquatic pathogenic bacteria (staphylococcus aureus, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and bacillus subtilis), and the antibacterial effect is higher in sensitivity; after each group of fermentation for 48 hours, the antibacterial effect on aquatic pathogenic bacteria is shown as follows, the antibacterial activity of the overground part of the fermented bupleurum is strongest, and then the overground parts of the fermented bacterial liquid, kanamycin and bupleurum are fermented, and PBS is a negative control and does not show activity; the overground part of the fermented bupleurum and the bacterial fermentation liquid have higher sensitivity; therefore, the compound can be used for replacing antibiotics in feed and is suitable for aquaculture.

Drawings

Fig. 1 is an ion mass spectrometry fragment, wherein 1: kaempferol-3-O-rutinoside; 2: isoquercitrin; 3: quercetin; 4: isorhamnetin; 5: rutin; 6: wild-type uropein; 7: quercetin-3-O-beta-L-arabinoside; 8: kaempferol; 9: DL-3-phenyl-2-hydroxypropionic acid; 10: calycosin (internal standard);

FIG. 2 is an MRM chromatogram, wherein A: blank samples; b: fermenting aerial parts of bupleurum for 48 hours; c: standard (50 μg/mL) added to the matrix; 1: kaempferol-3-O-rutinoside; 2; isoquercitrin; 3: quercetin; 4: isorhamnetin; 5: rutin; 6: wild-type uropein; 7: quercetin-3-O-beta-L-arabinoside; 8: kaempferol; 9: DL-3-phenyl-2-hydroxypropionic acid; 10: calycosin (internal standard);

fig. 3 shows the dynamic content change (n=6) of the component to be measured in the fermentation broth.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The general technical scheme of the invention is as follows:

the specific technical scheme is as follows:

(1) Preparation of bupleurum aerial part extract: reflux-extracting aerial parts of radix bupleuri with water, concentrating, and drying to obtain aqueous extract of aerial parts of radix bupleuri.

(2) Preparation of test drugs: setting a blank control group, a bupleurum aerial part fermentation group, a zymophyte liquid group and a bupleurum aerial part control; samples were taken at time points 0,9, 18, 48h, respectively, for further analysis and experimentation.

(3) Dynamic monitoring of chemical components: establishing UPLC-MS-MS conditions of 8 flavone compounds (kaempferol-3-O-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, wild-tail glycoside, quercetin-3-O-beta-L-arabinoside, kaempferol) and DL-3-phenyl-2-hydroxypropionic acid, measuring the content of each component in fermentation liquid in a fermentation sample, and dynamically monitoring the content change of each component in different fermentation time of the aerial parts of bupleurum.

(4) In vitro bacteriostasis experiment: because the invention has high content of flavonoid components in the aboveground part liquid of bupleurum and reports that DL-3-phenyl-2-hydroxypropionic acid is produced in the fermentation process and has antibacterial activity, the invention selects five common non-aquatic pathogenic bacteria (staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and bacillus subtilis) and four common aquatic pathogenic bacteria (aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus and vibrio harveyi), tests the in-vitro antibacterial activity of the aboveground part of bupleurum at different fermentation times and examines the capability of the bupleurum to replace aquatic antibiotics.

Example 1 preparation of fermentation broth samples

1. Reagents and apparatus

Lactobacillus plantarum (ACCC 11095, lactobacillus plantarum) was given to hong kong university laboratory; MRS culture medium (lot number 20190226) is purchased from Haibo biotechnology limited company of Qingdao high-tech industrial park, liquid culture medium is prepared according to the formula, and 2% agar is added into solid culture medium; the materials used were taken from the county of Xin Jing county, shanxi province and identified as the aerial parts of Bupleurum (North Bupleurum, bupleurum chinense DC.) by the professor Dong Tingxia of the university of hong Kong science and technology.

2. Experimental method

2.1. Preparation of Bupleurum root overground part extract

Weighing 100g of aerial parts of bupleurum, pulverizing, sieving, adding water according to a liquid-to-material ratio of 10:1, soaking for 2h, heating and refluxing for 2h, filtering and collecting filtrate, extracting twice, combining the filtrates, recovering solvent under reduced pressure at 40 ℃, freeze-drying to obtain medicinal material extract, and placing in a refrigerator for standby.

2.2. Culture of Lactobacillus plantarum

The preserved lactobacillus plantarum is inoculated in MRS solid culture medium by streaking method and cultured for 24 hours at 37 ℃. The strain was inoculated in an inoculum size of 10% in MRS broth for 24 hours at 37 ℃. Preparing the lactobacillus plantarum mother solution for standby.

2.3 preparation of fermentation broths

Weighing an appropriate amount of extract of aerial parts of bupleurum, dissolving with MRS culture medium, and preparing into extract solution with final concentration of 30 mg/mL. Inoculating lactobacillus plantarum according to 10% (v/v), incubating in a constant temperature shaking incubator at 37 ℃ to obtain groups of overground parts of the fermented bupleurum, respectively taking out the groups after fermentation for 0h,9h,18h and 48h, and preserving the groups at-80 ℃ for later use. And a blank control group, a zymophyte liquid group and a bupleurum overground part group are simultaneously arranged (the inoculated lactobacillus plantarum mother liquid is replaced by MRS).

Example 2 dynamic quantitative detection of fermentation broth Components at different times Using the method of UPLC-MS-MS

1. Reagents and apparatus

1.1 reagents

Kaempferol-3-O-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, wild-type tail glycoside, quercetin-3-O-beta-L-arabinoside, DL-3-phenyl-2-hydroxypropionic acid, kaempferol and calycosin, which are provided by the laboratory of Chinese medicine research and development center of hong Kong university, are subjected to HPLC-DAD and C13 nuclear magnetic detection, and the purity is more than 98%.

1.2 instruments

PerkinElmer instruments limited PerkinElmer ultra-high performance liquid chromatography triple quadrupole mass spectrometers (PerkinElmer a-30 chromatograph, Q-sight type mass spectrometer); shake incubator of Shanghai know Chu instruments limited company; himac CR 22G low-speed high-capacity centrifuge; sartorius BSA224 electronic balance (Max 22g, d=0.1 mg).

2. Experimental method

2.1 preparation of reference solution and internal Standard solution thereof

Accurately weighing appropriate amounts of kaempferol-3-O-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, wild-type tail glycoside, quercetin-3-O-beta-L-arabinoside, kaempferol, DL-3-phenyl-2-hydroxypropionic acid, placing in a 10mL volumetric flask, and diluting with methanol to scale. Shaking uniformly to prepare a reference substance mother solution with the concentration of 1mg/mL, taking a proper amount of each solution, and mixing to prepare a reference substance mixed stock solution with the concentration of 100 mug/mL. And putting a proper amount of Calycosin 10mg into a volumetric flask, diluting with methanol to a scale, preparing a stock solution of 1mg/mL, and finally diluting into an internal standard solution of 10 mug/mL.

2.2 preparation of standard curve

Diluting the reference substance mixed standard solution with methanol to form a series of standard solutions with mass concentrations of 0.19,0.39,0.78,1.56,3.13,6.25, 12.50, 25.00, 50.00 and 100.00 mug/mL; 100uL of MRS medium blank substrate was added to prepare a series of standard substrate working solutions with concentrations of 0.1,0.19,0.39,0.78,1.56,3.13,6.25, 12.50, 25.00, 50.00. Mu.g/mL.

2.3 treatment of test samples

200 mu L of fermentation samples at different time points are sucked, 50 mu L of internal standard solution is added, the mixture is fully mixed for 30s by shaking, 800 mu L of methanol is added, the mixture is further vibrated for 30s, and then the mixture is centrifuged at 12000rpm, and the supernatant is sucked for standby.

2.4 detection conditions for UPLC-MS-MS analysis

Chromatographic conditions: chromatographic column: waters ACQUITY UPLC BEH C ₁₈ (2.1 mm. Times.50 mm,1.7 μm); column temperature: 30 ℃; mobile phase: aqueous 0.1% formic acid-acetonitrile; the gradient elution procedure is shown in table 1; flow rate: 0.1mL/min; sample injection amount: 1 mul.

TABLE 1 UPLC gradient elution procedure

Time (min)	Formic acid water	Acetonitrile
			0	90	10
4	60	40
			6	10	90
7	10	90
			8	90	10

Mass spectrometry conditions: ion source: electrospray ion source (ESI); detection mode: esi+, MRM multiple reaction monitoring mode; capillary voltage (electro spray voltage): 5500V; atomizing gas temperature (nebulizer gas): 450 ℃; mass spectrometry interface temperature (HSID): 280 ℃. The MRM mass spectrum analysis parameters of the components to be detected are shown in Table 2, and the ion mass spectrum fragments are shown in FIG. 1.

Mass spectral parameters of the compounds of table 2

Note that: * Quantification of ions

3 results of experiments

3.1 linear relationship investigation

The working solution containing the matrix which has been prepared is treated according to the sample treatment method of "2.3" in this example. According to the chromatographic mass spectrometry item of '2.4' in the embodiment, the concentration of the reference substance series working solution is taken as an abscissa (X), and the ratio of the peak area of the reference substance and the internal standard is taken as an ordinate (Y), so that the standard curves of 9 to-be-detected objects are obtained. The results in this example are shown in Table 3, and the results show that the detection method has good linearity, the widest linear range is between 0.19 and 50 mug/mL, the quantitative limit range is between 0.19 and 1.56 mug/mL, and the detection limit is between 0.1 and 0.78 mug/mL.

3.2 dynamic detection of the content of the to-be-detected Components in the fermentation broth

200. Mu.L of each group of samples for 0h,9h,18h and 48h of fermentation in example 1 was taken and treated according to the sample treatment method in this example. Analysis was performed according to the condition "2.4" in this example, the MRM chromatogram thereof is shown in FIG. 2, and the dynamic content change of the component to be measured in the fermentation broth is shown in FIG. 3.

The results of FIG. 2 show that under the condition of UPLC-MS-MS adopted in the experiment, the extract chromatograms of all the components to be detected (kaempferol-3-O-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, wild-type astragaloside, quercetin-3-O-beta-L-arabinoside, kaempferol and DL-3-phenyl-2-hydroxypropionic acid) of the bupleurum root overground part fermented 48h samples have no impurity peak interference with internal standard peaks (calycosin), and have good peak shape and stable base line.

The result of figure 3 shows that the rutin content is highest compared with the flavone content in the process of fermenting the aerial parts of the bupleurum for 0-48 hours; secondly, quercetin-3-O-beta-L-arabinoside and isoquercitrin are added, the content of the isoquercitrin is steadily increased along with the increase of fermentation time (rutin is increased from 607.68 mug/mL to 855.70 mug/mL, quercetin-3-O-beta-L-arabinoside is increased from 108.43 mug/mL to 154.75 mug/mL, isoquercitrin is increased from 44.27 mug/mL to 63.73 mug/mL), and the content of other flavones is almost unchanged. In the group of the aerial parts of the fermented bupleurum and the fermentation broth, the content of DL-3-phenyl-2-hydroxypropionic acid is linearly increased in the fermentation process of 0-48 hours, the time dependency is shown, and the increase rate of the DL-3-phenyl-2-hydroxypropionic acid (4.44 mug/mL is increased to 66.57 mug/mL) in the aerial parts of the fermented bupleurum is higher than that of the fermentation broth (2.91 mug/mL is increased to 51.25 mug/mL), so that the fermentation of the aerial parts of the bupleurum can stimulate lactobacillus plantarum to produce more DL-3-phenyl-2-hydroxypropionic acid. And for the aerial parts of bupleurum without lactobacillus plantarum fermentation, the contents of flavone and DL-3-phenyl-2-hydroxy acrylic acid have no obvious change in 0-48 h.

TABLE 3 Standard curve of the components to be measured

Conclusion 4

The established UPLC-MS-MS analysis method for simultaneously detecting 8 flavones and DL-3-phenyl-2-hydroxypropionic acid in the overground part of the fermented bupleurum has simple operation, high sensitivity and strong specificity.

DL-3-phenyl-2-hydroxy propionic acid is also called DL-3-phenyl lactic acid and phenyllactic acid, and is a novel antibacterial substance discovered at present, and the novel antibacterial substance is characterized by small molecular weight, wide antibacterial spectrum, high stability, and better solubility and stability of phenyllactic acid compared with other biological preservatives. The aerial parts of bupleurum root can enhance the lactobacillus plantarum to produce DL-3-phenyl-2-hydroxypropionic acid in the fermentation process. In addition, the content of partial flavone in the aerial parts of the fermented bupleurum is increased (rutin, quercetin-3-O-beta-L-arabinoside and isoquercitrin), and the next step is to measure the antibacterial activity in vitro.

EXAMPLE 3 in vitro antibacterial Activity study of fermentation broths

1 reagents and apparatus

The culture medium is the same as that of the embodiment 1, and staphylococcus aureus is treated14917,Staphylococcus aureus), E.coli (>10536，Escherichia coli) Pseudomonas aeruginosa (/ -)>10145,Pseudomonas aeruginosa) Klebsiella pneumoniae (A.pneumoniae)>700603,Klebsiella pneumoniae), bacillus subtilis (Reddish)>11774,Bacillus subtilis) Aeromonas hydrophila (>7966,Aeruginosa hydrophila), edwardsiella tarda (A.tarda)>15947,Delayed Edwards) Vibrio alginolyticus (>33787,Vibrio alginolyticus), vibrio harveyi (a. K)>BAA1117, vibrio alginolyticus), all obtained from the Shenzhen institute of science and technology university of hong Kong; the U.S. bio-tec microplate detector, other instrument example 2, is listed.

2 Experimental methods

2.1 preparation of bacterial suspension

The colony to be tested is inoculated in MRS culture medium, and is subjected to shaking culture for 24 hours at 180rpm at 37 ℃, and the culture medium is utilized to prepare bacterial suspension with certain concentration for standby.

2.2 determination of bacteriostatic Activity

Five common non-aquatic pathogens (Staphylococcus aureus, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, bacillus subtilis) and four aquatic pathogens (Aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus and Hash) after overnight cultureVibrio) bacterial solution diluted to OD _595nm About 0.3 (density of 10) ⁸ cfu/mL), and then diluted 100-fold to 10-fold with PBS ⁶ cfu/mL. 100. Mu.L of the bacterial liquid was applied to an agar medium and smeared uniformly, oxford cups were placed on the agar medium and firmly attached, and 20. Mu.L of each group of fermentation broth samples prepared in example 1, PBS (negative control), and 0.5mg/mL of kanamycin as a positive control were added to oxford cups. The dishes were placed in an incubator for 12 hours, and then the diameter of the transparent zone of inhibition was measured.

3 results of experiments

3.1 in vitro bacteriostatic Activity of fermentation broth

As shown in table 4, the greater the antibacterial zone, the stronger the antibacterial ability, the higher the antibacterial sensitivity of the drug, by measuring the size of the transparent antibacterial zone to reflect the strength of the antibacterial activity; the overground part of the fermented bupleurum shows inhibitory effects on 9 pathogenic bacteria to different degrees. Along with the extension of fermentation time, the diameter of the bacteriostasis ring is also increased, which indicates that the fermentation process can enhance the bacteriostasis activity. The antibacterial effect of the fermented bupleurum group on aquatic pathogenic bacteria (aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus and vibrio harveyi) is obviously stronger than that of non-aquatic pathogenic bacteria (staphylococcus aureus, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and bacillus subtilis), and the fermented bupleurum group has better effect on aquatic pathogenic bacteria than other groups and has higher sensitivity (diameter is more than 15 mm). In addition, the fermented bacterial liquid also has good effect of inhibiting aquatic pathogenic bacteria at 48 hours and is stronger than positive control kanamycin and aerial parts of bupleurum. Although the antibacterial effect of the aerial parts of the bupleurum root is not obviously changed along with the increase of the incubation time, the aerial parts of the bupleurum root have a certain inhibition effect on aquatic pathogenic bacteria, but are weaker than the aerial parts of the bupleurum root, fermentation broth and kanamycin group.

Conclusion 4

An oxford cup method in-vitro bacteriostasis experiment shows that after each group is fermented for 48 hours, the antibacterial effect on aquatic pathogenic bacteria is shown as follows, the antibacterial activity of overground parts of the fermented bupleurum is strongest, and then the overground parts of the fermented bupleurum are fermented bacterial liquid, kanamycin and the overground parts of the bupleurum are fermented, and PBS negative comparison does not show activity; and the aerial parts of the fermented bupleurum and the bacterial fermentation liquid have higher sensitivity. Therefore, the overground part of the fermented bupleurum is expected to replace antibiotics in feed and be applied to aquaculture.

Table 4 influence of fermentation broths on the size of zone of inhibition of different species (n=3)

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes or modifications may be made by those skilled in the art within the scope of the appended claims without affecting the spirit of the invention. The embodiments of the present application and features in the embodiments may be combined with each other arbitrarily without conflict.

Claims (2)

1. A method for preparing an aerial part fermentation product of bupleurum as an aquatic feed additive, which is characterized by comprising the following steps:

preparing an extract of aerial parts of bupleurum chinense: reflux extracting aerial parts of radix bupleuri with water, concentrating, and drying to obtain aerial parts of radix bupleuri extract;

culturing Lactobacillus plantarumLactobacillus plantarum) ACCC11095: inoculating the preserved lactobacillus plantarum into an MRS solid culture medium by a streaking method, and inoculating the strain into an MRS broth culture medium in an inoculum size of 10% to prepare a standby lactobacillus plantarum mother solution;

preparing a fermentation liquid: dissolving aerial parts of radix bupleuri with MRS culture medium to obtain extract solution, inoculating lactobacillus plantarum, and incubating in a constant temperature shake incubator to obtain aerial parts fermentation product of radix bupleuri;

the bupleurum root overground part fermentation product has an antibacterial effect on aquatic pathogenic bacteria when used as an aquatic feed additive;

the aquatic pathogenic bacteria comprise aeromonas hydrophila, edwardsiella tarda, vibrio alginolyticus and Vibrio harveyi.

2. The method of preparing according to claim 1, wherein the step of preparing the extract of aerial parts of bupleurum comprises:

weighing 100g of aerial parts of bupleurum, pulverizing, sieving, adding water according to a liquid-to-material ratio of 10:1, soaking for 2h, heating and refluxing for 2h, filtering and collecting filtrate, extracting twice, combining the filtrates, recovering solvent under reduced pressure at 40 ℃, freeze-drying to obtain medicinal material extract, and placing in a refrigerator for standby.