阅读说明：本技术 一种用于抑制腐败希瓦氏菌的抗菌剂及其制备方法和在南美白对虾保鲜剂制备中的应用 (Antibacterial agent for inhibiting Shewanella putrefaciens, preparation method thereof and application of antibacterial agent in preparation of litopenaeus vannamei preservative ) 是由 王靖 王鸿飞 许凤 邵兴锋 韦莹莹 于 2019-09-26 设计创作，主要内容包括：本发明公开了一种用于抑制腐败希瓦氏菌的抗菌剂及其在南美白对虾保鲜剂制备中的应用,特点是该抗菌剂为费菜总黄酮,所述的费菜总黄酮主要含有槲皮甙和没食子酸,还含有少量的山奈酚、槲皮素和异槲皮苷,其中槲皮甙的含量为35-50%总重量,没食子酸的含量为30-40%总重量,上述用于抑制腐败希瓦氏菌的抗菌剂在南美白对虾保鲜剂制备中的应用,该南美白对虾保鲜剂由以下原料及其重量份数组成：费菜总黄酮4份、蓝莓叶多酚2份、N-乙酰-L-半胱氨酸3.2份和水1000份,优点是能有效防治南美白对虾黑变腐败,提高其货架期。(The invention discloses an antibacterial agent for inhibiting Shewanella putrefaciens and an application thereof in preparation of a Penaeus vannamei preservative, which is characterized in that the antibacterial agent is general fern, the general fern mainly contains quercitrin and gallic acid, and also contains a small amount of kaempferol, quercetin and isoquercitrin, wherein the content of the quercitrin is 35-50% of the total weight, and the content of the gallic acid is 30-40% of the total weight, the antibacterial agent for inhibiting Shewanella putrefaciens is applied in the preparation of the Penaeus vannamei preservative, and the Penaeus vannamei preservative is composed of the following raw materials in parts by weight: 4 parts of sedum aizoon total flavone, 2 parts of blueberry leaf polyphenol, 3.2 parts of N-acetyl-L-cysteine and 1000 parts of water, and has the advantages of effectively preventing and treating the blackening and the putrefaction of the penaeus vannamei boone and prolonging the shelf life of the penaeus vannamei boone.)

1. An antibacterial agent for inhibiting Shewanella putrefaciens is characterized in that the antibacterial agent is herba Fimbristylis Dichotomae total flavone mainly containing quercetin and gallic acid, and also containing small amount of kaempferol, quercetin and isoquercitrin, wherein the content of quercetin is 35-50% of total weight, and the content of gallic acid is 30-40% of total weight.

2. A method for preparing the antibacterial agent for inhibiting shewanella putrefaciens according to claim 1, characterized by comprising the steps of:

(1) extracting with ethanol under ultrasonic wave to obtain extractive solution;

(2) concentrating the extracting solution obtained in the step (1), and adding absolute ethyl alcohol;

(3) treating the solution obtained in the step (2) at low temperature, and taking supernatant;

(4) concentrating the supernatant obtained in the step (3), adsorbing by adopting a macroporous resin method, discarding the supernatant, resolving by using an ethanol solution, and taking the supernatant;

(5) concentrating the supernatant obtained in the step (4) to obtain the sedum aizoon total flavone extract, and freeze-drying to obtain the finished product.

3. The method of claim 2, wherein the step of preparing an antibacterial agent for inhibiting Shewanella putrefaciens comprises the steps of:

(1) taking 1000 g of dried, crushed and 60-mesh-screened herba sedi aizoon powder, adding 40L of 70% ethanol solution, homogenizing, performing ultrasonic-assisted extraction for 60 min at 60 ℃, then extracting for 2 h at 60 ℃, and filtering to obtain a supernatant;

(2) concentrating the supernatant obtained in the step (1) under reduced pressure, recovering the solvent, heating to 45-50 ℃, and adding absolute ethyl alcohol with the volume twice that of the concentrated solution after the concentration is finished;

(3) treating the solution obtained in the step (2) at a low temperature of 4 ℃ for 24 hours, filtering, removing precipitates, and taking supernatant;

(4) concentrating the supernatant obtained in the step (3) under reduced pressure, recovering the solvent, and heating to 45-50 ℃; adding the concentrated solution into a 20% ethanol solution, diluting the concentrated solution into a sedum aizoon extract solution with the flavone concentration of 2.5 mg/mL, then adding macroporous resin which is treated by acid, alkali and alcohol until the solution is completely swelled, statically adsorbing for 24 hours, filtering, discarding filtrate, adding 80% ethanol solution with the same volume as the 20% ethanol solution for analysis, and filtering to obtain supernatant after the analysis is finished, wherein the ratio of the sedum aizoon extract solution to the macroporous resin is 10 mL: 1g of a compound;

(5) and (4) carrying out reduced pressure concentration on the supernatant obtained in the step (4), recovering the solvent, heating to 45-50 ℃ to obtain a concentrated solution, namely the herba sedi aizoon total flavone, and freeze-drying for later use.

4. The detection method of the antibacterial agent for inhibiting Shewanella putrefaciens is characterized by comprising the specific steps of dissolving freeze-dried powder of the total flavonoids of Ficus williamsii prepared according to claim 3 in methanol, filtering the solution through a 0.22-micron filter membrane to obtain a sample solution, and detecting the components of the total flavonoids of Ficus williamsii by using an ultra-high performance liquid chromatography-electrospray mass spectrometry, wherein the chromatographic column is an angionut Zorbax XDB-C18 column, the mobile phase A is methanol and the mobile phase B is 0.1% formic acid solution, the flow rate is 0.8 mL/min, the sample injection amount is 10 muL, the column temperature is 25 ℃, the detection wavelength is 254 nm, the linear gradient elution program is 0-5min, 70% -30% A, 5-18 min, 30% -10% A, the ion source is ESI, the scanning range is 100 ~ 1200 m/z, the capillary voltage is 2.50 KV, the cone hole voltage is 50.00V, the cone hole flow rate is 50V/h, the gas flow rate is 2-10% A, the ion source extraction temperature is 250: 250H, and the solvent is removed.

5. Use of the antibacterial agent for inhibiting Shewanella putrefaciens according to claim 1 in the preparation of a preservative for Penaeus vannamei Boone.

6. The use of the antibacterial agent for inhibiting Shewanella putrefaciens in the preparation of the preservative for Penaeus vannamei according to claim 5, wherein the antibacterial agent comprises: the preservative for the penaeus vannamei boone comprises the following raw materials in parts by weight: 4 parts of sedum aizoon total flavone, 2 parts of blueberry leaf polyphenol, 3.2 parts of N-acetyl-L-cysteine and 1000 parts of water.

Technical Field

The invention relates to an antibacterial agent for Shewanella putrefaction, in particular to an antibacterial agent for inhibiting Shewanella putrefaction and application thereof in preparation of a Penaeus vannamei preservative.

Background

Penaeus vannamei Boone is popular with consumers because of its rich nutrition and tender meat quality, however, Penaeus vannamei Boone is a perishable food that is extremely susceptible to bacterial attack and spoilage during fishing, transportation, processing and storage. Especially after death, under the action of polyphenol oxidase (PPO) in vivo, blackening is easy to occur, and the black tea can reach the unacceptable level of consumers in sense, thus causing great resource waste. The Shewanella putrefaction is a facultative anaerobic gram-negative motor bacillus serving as a dominant putrefaction bacterium in the putrefaction and deterioration process of the Penaeus vannamei Boone, is widely distributed in the nature, has extremely strong tolerance to salinity and temperature in a growth environment, generates metabolites with strong putrefaction odor and peculiar smell, and is a main source of the putrefaction odor of the Penaeus vannamei Boone. Shewanella putrefaciens has been reported to develop resistance to antibiotics such as kanamycin, cephalosporins, tetracyclines, etc. due to abuse of antibiotics, and the feasibility of using antibiotics in aquatic environments or in preservation processes is decreasing. The cumulative effect of chemical fungicides on food is forcing humans to forego this mode of controlling microbial growth. At present, researchers focus on natural products which are not easy to generate drug resistance and are harmless to human bodies.

The fresh-keeping of the penaeus vannamei boone generally utilizes physical, chemical, biological and other methods to ensure the freshness of the penaeus vannamei boone to the maximum extent. The traditional preservation methods such as low-temperature preservation and chemical preservative preservation cannot meet the requirements of people due to the limitations of the methods on the antibacterial activity and safety, and the biological preservative is applied to the preservation of penaeus vannamei boone more and more due to the strong antibacterial activity and high safety and the fact that the original flavor and quality of food cannot be changed in the using process.

Disclosure of Invention

The invention aims to solve the technical problem of providing an antibacterial agent for inhibiting Shewanella putrefaction and application thereof in preparation of a preservative for penaeus vannamei, wherein the preservative can effectively prevent the penaeus vannamei from blackening and rotting and improve the shelf life of the penaeus vannamei.

The technical scheme adopted by the invention for solving the technical problems is as follows:

1. an antibacterial agent for inhibiting Shewanella putrefaciens is prepared from herba Fimbristylis Dichotomae total flavone mainly containing quercetin and gallic acid, and also containing small amount of kaempferol, quercetin and isoquercitrin, wherein the content of quercetin is 35-50% of total weight, and the content of gallic acid is 30-40% of total weight.

2. A method for preparing an antibacterial agent for inhibiting Shewanella putrefaciens, comprising the steps of:

(1) extracting with ethanol under ultrasonic wave to obtain extractive solution;

(2) concentrating the extracting solution obtained in the step (1), and adding absolute ethyl alcohol;

(3) treating the solution obtained in the step (2) at low temperature, and taking supernatant;

(4) concentrating the supernatant obtained in the step (3), adsorbing by adopting a macroporous resin method, discarding the supernatant, resolving by using an ethanol solution, and taking the supernatant;

(5) concentrating the supernatant obtained in the step (4) to obtain the sedum aizoon total flavone extract, and freeze-drying to obtain the finished product.

The method comprises the following specific steps:

(1) taking 1000 g of dried, crushed and 60-mesh-screened herba sedi aizoon powder, adding 40L of 70% ethanol solution, homogenizing, performing ultrasonic-assisted extraction for 60 min at 60 ℃, then extracting for 2 h at 60 ℃, and filtering to obtain a supernatant;

(2) concentrating the supernatant obtained in the step (1) under reduced pressure, recovering the solvent, heating to 45-50 ℃, and adding absolute ethyl alcohol with the volume twice that of the concentrated solution after the concentration is finished;

(3) treating the solution obtained in the step (2) at a low temperature of 4 ℃ for 24 hours, filtering, removing precipitates, and taking supernatant;

(4) concentrating the supernatant obtained in the step (3) under reduced pressure, recovering the solvent, and heating to 45-50 ℃; adding the concentrated solution into a 20% ethanol solution, diluting the concentrated solution into a sedum aizoon extract solution with the flavone concentration of 2.5 mg/mL, then adding macroporous resin which is treated by acid, alkali and alcohol until the solution is completely swelled, statically adsorbing for 24 hours, filtering, discarding filtrate, adding 80% ethanol solution with the same volume as the 20% ethanol solution for analysis, and filtering to obtain supernatant after the analysis is finished, wherein the ratio of the sedum aizoon extract solution to the macroporous resin is 10 mL: 1g of a compound;

(5) and (4) carrying out reduced pressure concentration on the supernatant obtained in the step (4), recovering the solvent, heating to 45-50 ℃ to obtain a concentrated solution, namely the herba sedi aizoon total flavone, and freeze-drying for later use.

3. A detection method of an antibacterial agent for inhibiting Shewanella putrefaciens comprises the following specific steps: dissolving the prepared herba Fimbristylis Dichotomae total flavone powder with methanol, freeze drying, filtering with 0.22 μm filter membrane, and detecting herba Fimbristylis Dichotomae total flavone components by ultra high performance liquid chromatography-electrospray mass spectrometry:

the chromatographic column is an angiont Zorbax XDB-C18 column, and the mobile phase is an A phase: methanol and phase B: 0.1% formic acid solution, flow rate of 0.8 mL/min, sample amount of 10 μ L, column temperature of 25 deg.C, detection wavelength of 254 nm, and linear gradient elution procedure: 0-5min, 70% -30% A; 5-18 min, 30% -10% A;

ESI of ion source, scanning range of 100 ~ 1200 m/z, capillary voltage of 2.50 KV, taper hole voltage of 50.00V, taper hole gas flow rate of 50L/h, extraction voltage of 2.00V, ion source temperature of 125 deg.C, desolventizing gas temperature of 250 deg.C, and desolventizing gas flow rate of 400L/h.

The antibacterial agent for inhibiting Shewanella putrefaciens is applied to the preparation of the litopenaeus vannamei preservative.

The preservative for the penaeus vannamei boone comprises the following raw materials in parts by weight: 4 parts of sedum aizoon total flavone, 2 parts of blueberry leaf polyphenol, 3.2 parts of N-acetyl-L-cysteine and 1000 parts of water.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a natural biological antibacterial agent capable of obviously inhibiting Shewanella putrefaciens, and in-vitro experiments prove that the herba sedi aizoon total flavonoids have the characteristics of strong bacteriostasis, high effect, high safety, nutrition and the like.

2. The preparation process of the sedum aizoon total flavone extract provided by the invention can effectively enrich the total flavone, and the extraction rate is about 10%; the method has the advantages of high content of total flavonoids, high purity of about 67%, simple and easy control, and low process cost.

3. The invention applies the sedum aizoon total flavonoids to the application of aquatic product preservation for the first time, designs a biological preservative taking the sedum aizoon total flavonoids as main raw materials according to the biochemical characteristics of penaeus vannamei boone, the sedum aizoon total flavonoids in the preservative has synergistic interaction on blueberry leaf polyphenols and N-acetyl-L-cysteine, and when the sedum aizoon total flavonoids and the N-acetyl-L-cysteine are mixed for use, the preservative has the effect of delaying the putrefaction and the blackening of the penaeus vannamei boone and has better using effect than a single use.

Drawings

FIG. 1 is a liquid chromatography analysis chart of the total flavonoids of sedum aizoon prepared by the method of the present invention;

FIG. 2 is a graph showing the inhibition of Shewanella putrefaciens bactericidal activity of the Philadelphia total flavonoids prepared by the method of the present invention at different times;

FIG. 3 is a flow peak diagram of the herba Fimbristylis Dichotomae total flavone prepared by the method of the present invention for measuring the intracellular active oxygen content of Shewanella putrefaciens, wherein the abscissa is fluorescence intensity and the ordinate is cell number;

FIG. 4 is a flow-type peak diagram of calcium ion content test in Shewanella putrefaciens cells by using herba Fimbristylis Dichotomae total flavonoids prepared by the method of the present invention, wherein the abscissa is fluorescence intensity and the ordinate is cell number.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

First, experiment method

1. The method for measuring the content of the total flavonoids in sedum aizoon comprises the following steps:

measuring flavone content with aluminum chloride method by using quercetin as reference, preparing standard solution and standard kojiDrawing a line: accurately weighing 0.0224 g of standard substance, and diluting to a volume of 100 mL by using 70% ethanol solution, wherein the concentration of the standard solution is 0.224 g.L^-1. Precisely transferring 0, 0.2, 0.4, 0.5, 0.6, 0.8 and 1mL of standard solution into a 10mL colorimetric tube, adding 1mL of 2wt% aluminum chloride solution respectively, and fixing the volume to scale marks by using 70% ethanol solution to obtain standard solutions with mass concentrations of 0, 0.00448, 0.00896, 0.01120, 0.01344, 0.01792 and 0.02240 g.L^-1. Mixing, standing for 20 min, and measuring absorbance at 275 nm. Plotting the mass concentration of the standard solution as abscissa and the light absorption value A as ordinate, and performing linear regression analysis to obtain a standard curve of y =34.631 x +0.0016, R²=0.9993。

And (3) measuring the content of the total flavonoids of sedum aizoon: respectively sucking 1mL of sedum aizoon total flavone extracting solution, using 70% ethanol solution to fix the extracting solution in a 100 mL volumetric flask, respectively sucking 1mL of extracting solution with constant volume in a 10mL colorimetric tube, using 70% ethanol solution as blank control, respectively adding 1mL of 2wt% aluminum chloride solution, using 70% ethanol to fix the volume to a scale mark, uniformly mixing, standing for 20 min, then adjusting zero by using a blank tube, measuring a light absorption value at 275 nm, and obtaining the content of sedum aizoon total flavone in the extracting solution according to a standard curve.

2. Determination of soluble sugar leakage:

respectively adding a certain amount of prawn preservative into bacterial suspension of Shewanella putrefaciens cultured to logarithmic phase, and continuously culturing in a shaking table (120 r/min, 30 ℃), wherein the treatment time points are as follows: 0. and centrifuging 4 mL of bacterial liquid for 10min at 3000 g after 3 h, taking 1mL of supernatant, adding 4 mL of anthrone sulfate solution with the mass concentration of 0.2%, uniformly mixing in an ice bath, boiling in a water bath for 10min, rapidly cooling, and measuring the light absorption value at 620 nm. A standard curve was plotted using glucose as a standard, and the content was expressed in. mu.g/mL.

3. Determination of genetic material leakage

The ultraviolet spectrophotometer method is adopted to measure the content of DNA and RNA macromolecules in the Shewanella putrefaction bacterial suspension and slightly modifies the content. Respectively adding a certain amount of prawn preservative into the bacterial suspension of Shewanella putrefaciens cultured to logarithmic phase, and placing in a shaking table (120 r/min, 30 r/min)C) at the following treatment time points: 0. centrifuging for 3 hr to obtain 5 mL bacterial liquid, centrifuging at 3000 g for 10min, collecting supernatant, measuring light absorption value at 260 nm, and drawing A₂₆₀The variation of the value with time.

4. Determination of soluble protein:

respectively adding a certain amount of prawn preservative into bacterial suspensions of Shewanella putrefaciens cultured to logarithmic phase, and treating at the following treatment time points: 0. and centrifuging 4 mL of bacterial liquid at 3000 g for 10min after 3 h, taking 1mL of supernatant, measuring the content of soluble protein in the supernatant by using a Coomassie brilliant blue method, standing for two minutes, and measuring the light absorption value at 595 nm. Bovine serum albumin was used as a standard to draw a standard curve, and the content was expressed in μ g/mL.

Detailed description of the preferred embodiment

1. Extraction method of herba Fimbristylis Dichotomae total flavone

(1) Taking 1000 g of dried, crushed and 60-mesh-screened herba sedi aizoon powder, adding 40L of 70% ethanol solution, homogenizing, performing ultrasonic-assisted extraction for 60 min at 60 ℃, then extracting for 2 h at 60 ℃, and filtering to obtain a supernatant;

(2) concentrating the supernatant obtained in the step (1) under reduced pressure, recovering the solvent, heating to 45-50 ℃, and adding absolute ethyl alcohol with the volume twice that of the concentrated solution after the concentration is finished;

(3) treating the solution obtained in the step (2) at a low temperature of 4 ℃ for 24 hours, filtering, removing precipitates, and taking supernatant;

(4) concentrating the supernatant obtained in the step (3) under reduced pressure, recovering the solvent, heating to 45-50 ℃, and measuring the content of flavone in the crude herba sedi aizoon extract according to the absorbance value at the wavelength of 275 nm by adopting an aluminum chloride method after the concentration is finished; adding the concentrated solution into a 20% ethanol solution, diluting the solution into a sedum aizoon extract solution with the flavone concentration of 2.5 mg/mL, adding AB-8 macroporous resin which is treated by acid, alkali and alcohol to be completely swelled, statically adsorbing for 24 hours, filtering, discarding filtrate, adding 80% ethanol solution with the same volume as the 20% ethanol solution, resolving, filtering to obtain supernatant, and recovering macroporous resin, wherein the proportion of the sedum aizoon extract solution to the macroporous resin is 10 mL: 1g of a compound;

(5) and (4) carrying out reduced pressure concentration on the supernatant obtained in the step (4), recovering the solvent, heating to 45-50 ℃ to obtain a concentrated solution, namely the herba sedi aizoon total flavone, freezing and drying for later use, and measuring the content of the total flavone. Through multiple tests, the content of the sedum aizoon total flavone in the extracting solution is 60-80%.

2. Component analysis method of sedum aizoon total flavonoids

Extracting herba Sedi Aizoon total flavone with the above method, dissolving lyophilized herba Sedi Aizoon total flavone powder with methanol, and filtering with 0.22 μm filter membrane to obtain sample solution. Detecting the components of the sedum aizoon total flavonoids by adopting an ultra-high performance liquid chromatography-electrospray mass spectrometry method:

the chromatographic column was an angiont Zorbax XDB-C18 column (4.6X 150 mm, 5 μm), the mobile phase was methanol (A) and 0.1% formic acid solution (B), the flow rate was 0.8 mL/min, the sample size was 10 μ L, the column temperature was 25 ℃ and the detection wavelength was 254 nm. The linear gradient elution procedure was: 0-5min, 70% -30% A; 5-18 min, 30% -10% A.

ESI of ion source, scanning range of 100 ~ 1200 m/z, capillary voltage of 2.50 KV, taper hole voltage of 50.00V, taper hole gas flow rate of 50L/h, extraction voltage of 2.00V, ion source temperature of 125 deg.C, desolventizing gas temperature of 250 deg.C, and desolventizing gas flow rate of 400L/h.

As shown in FIG. 1, the detection and analysis show that the total flavonoids of herba Fimbristylis Dichotomae mainly contain quercetin (retention time of 8.07 min) and gallic acid (retention time of 2.26 min), wherein the content of quercetin is 35% ~ 50% by weight at the highest, and gallic acid is 30% ~ 40% by weight at the next highest.

Detailed description of the invention

1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Diluting the herba sedi total flavonoids prepared in the first embodiment by a two-fold dilution method, adding the herba sedi total flavonoids into a nutrient agar culture medium, uniformly mixing, respectively pouring into culture dishes, after solidification, coating 0.1 mL of putrefactive Shewanella bacteria suspension cultured to logarithmic phase into each culture dish, taking sterile water as a control, culturing at 30 ℃ for 24 h, observing the growth condition of bacterial colonies, and taking the minimum concentration of the completely aseptically grown herba sedi total flavonoids as the minimum inhibitory concentration. Continuing to culture at 30 deg.C, and taking minimum value of herba Sedi Aizoon flavone concentration grown in completely sterile colony as minimum bactericidal concentration after 48 hr. The experimental results are shown in tables 1 and 2 below,

TABLE 1 MIC of Philadelphia flavonoid against Shewanella putrefaciens

。

TABLE 2 Megasphaera-flavonoid MBC against Shewanella putrefaciens

Note: -means no bacterial growth; + indicates the presence of bacterial growth (number of colonies ≦ 20); + indicates that there is bacterial growth, bacteria clumping.

As can be seen from the table, the minimum inhibitory concentration of the total flavonoids of sedum aizoon to Shewanella putrefaciens is 0.675 mg/mL, and the minimum bactericidal concentration is 2.5 mg/mL, which indicates that the total flavonoids of sedum aizoon under lower concentration has obvious inhibitory effect, and is obviously lower than the minimum inhibitory concentration of the total flavonoids of moringa leaves to escherichia coli, namely 6mg/mL and 10 mg/mL.

2. Determination of bactericidal activity of sedum aizoon total flavonoids on Shewanella putrefaciens

Adding herba Fimbristylis Dichotomae total flavone into Shewanella putrefaction suspension to make flavone content reach minimum bactericidal concentration MBC value, and using sterile water as control group. Shaking table culture at 30 deg.C and 120r/min, sucking 1ml of mixed solution at selected time points of 0, 2, 4, 6, 8 and 10 h, performing gradient dilution with sterile water, inoculating into nutrient agar plate, culturing at 37 deg.C for 48 hr, counting the plate, and drawing curve with colony number of 30-300 CFU, abscissa as time, and ordinate as log value of bacteria number.

As shown in figure 2, the change of the viable bacteria residual amount of the herba Fimbristylis Dichotomae total flavonoids after acting on Shewanella putrefaction at different time is illustrated, which shows that the herba Fimbristylis Dichotomae total flavonoids have obvious bactericidal effect on Shewanella putrefaction, and the previous experiments prove that the higher the concentration of the herba Fimbristylis Dichotomae flavonoids is, the better the bactericidal effect is. As can be seen from FIG. 2, the bactericidal activity of the fisetin increased with the increase in the action time. Under the concentration of 1 MBC, the sterilization rate of the sedum aizoon flavone to Shewanella putrefaciens reaches more than 99.9 percent after 2 hours, and the later-period antibacterial effect reaches a stable level.

3. Effect of Total Flavonoids of Philippine on intracellular endogenous ROS of Shewanella putrefaciens

Adding a certain amount of herba Fimbristylis Dichotomae total flavonoids prepared in the first embodiment into Shewanella putrefaciens suspension cultured at logarithmic phase to make final concentration of herba Fimbristylis Dichotomae flavonoids reach 1.0 MIC and 1.0 MBC, adding phosphate buffer (0.1M, pH 7.2) as control group, and culturing in shaker (120 r/min, 30 deg.C). After 0 and 1 hour of culture, sampling, centrifuging for 10min at 3000 g, washing with PBS for three times, taking thalli with the same volume, adding a DCFU-DA fluorescent probe with the final concentration of 10 mu mol/L, and reacting for 30 min at 37 ℃ in a dark place. Intracellular ROS, i.e. endogenous ROS levels, are detected using a flow cytometer.

ROS are thought to be involved in aging, apoptosis or canceration of cells, and the generation harm is mainly caused by the fact that ROS can cause rapid oxidation of various biological macromolecules in cells, DNA, RNA, lipid, protein and the like in organisms are sensitive to ROS, and excessive ROS can cause damage to the substances. Wherein, lipid substances are the main objects of oxygen stress injury, and free radicals directly react with polyunsaturated fatty acids on cell membranes to cause lipid peroxidation, thereby causing the reduction of cell membrane fluidity, changing the characteristics of the cell membranes and further damaging cell membrane proteins. In addition, DNA is also a main attack object of ROS, and the ROS can damage basic groups and sugar components in the DNA, so that the double-stranded structure of the DNA is damaged, the normal replication of the DNA is influenced, and the occurrence probability of mutation is increased. The reaction between the bacteria and important biological macromolecules in vivo after excessive ROS generation is toxic to cells, and the normal survival of the bacteria can be influenced. In FIG. 3, the abscissa is fluorescence intensity and the ordinate is cell number, the peak migration represents the ROS level, and the shift to the right indicates the intracellular ROS content. As can be seen from FIG. 3, after the treatment of the general flavone of Ixeris chinensis, the intracellular ROS content of the putrefactive Shewanella fungi is greatly exploded, so that the membrane lipid of the cell membrane is overoxidized, the MDA content is increased, and genetic substances such as DNA and the like are oxidized and damaged to different degrees.

4. Effect of herba Sedi Aizoon Total Flavonoids on intracellular Malondialdehyde (MDA) content of Shewanella putrefaciens

Adding a certain amount of herba Fimbristylis Dichotomae total flavonoids prepared in the first embodiment into Shewanella putrefaciens suspension cultured at logarithmic phase to make final concentration of herba Fimbristylis Dichotomae total flavonoids reach 1.0 MIC, adding phosphate buffer (0.1M, pH 7.2) as control group, and culturing in shaker (120 r/min, 30 deg.C). At the following treatment time points: 0. taking 5 mL of bacterial liquid for 1 hour and 3 hours respectively, centrifuging (2000 g, 10 min), collecting thalli, resuspending with phosphate buffer solution, crushing with a cell crusher, centrifuging (8000 g, 10 min), taking supernatant, and measuring; 1.0 mL of the supernatant was added to 5 mL of trichloroacetic acid solution (5%) and mixed, 2.0mL of the mixture was transferred and added to 2.0mL of TBA solution (0.67%), followed by boiling water bath for 30 min and ice water cooling. The light absorption values at 450 nm, 532 nm and 600nm were measured respectively, and the MDA content was calculated, the results are shown in Table 3 below,

TABLE 3 Effect of Philadelphia total flavones on the intracellular Malondialdehyde (MDA) content of Shewanella putrefaciens

Malondialdehyde (MDA) is the end product of lipid peroxidation of cell membranes, and the amount of MDA in a cell reflects the degree of lipid peroxidation damage to the cell. Compared with a control group, the MDA content in the bacterial cells after the treatment of the sedum aizoon total flavonoids is obviously increased and is obviously increased along with the time, which shows that after the sedum aizoon total flavonoids stimulate Shewanella putrefaciens, ROS in the bacterial cells explode, further a large degree of oxidative damage in the bacterial cells is caused, the physiological activities such as cell metabolism are influenced, and further the bacteria die.

5. Influence of herba Fimbristylis Dichotomae total flavone on calcium ion content in Shewanella putrefaciens cells

Taking a certain amount of bacterial suspension cultured to Shewanella putrefaciens in logarithmic phase, washing with PBS buffer solution for three times, centrifugally collecting cells, adding Fluo-3 with the final concentration of 5 MuM, an AM fluorescent probe, reacting for 20 min in a dark place at 37 ℃, adding HBSS diluent with 5 times of volume, continuously culturing for 40 min, then washing with HEPES diluent for three times, adding herba sedi aizoon total flavonoids after the reaction is finished so that the final concentration of the herba sedi aizoon flavonoids reaches 1.0 MIC and 1.0 MBC, culturing for 0 and 3 h, sampling, loading on a machine, and detecting the change of the intracellular calcium ion concentration by adopting a flow cytometer.

As can be seen from FIG. 4, the peak appeared after the treatment with total flavonoids of Cauliflower was significantly shifted to the right, which indicates that the intracellular calcium ion concentration of Shewanella putrefaciens was significantly increased after the treatment with total flavonoids of Cauliflower. Research shows that intracellular ROS burst is caused after the herba Fimbristylis Dichotomae total flavone stimulates cells, so that calcium ion channels on plasma membranes and calcium pools in the cells are activated, calcium ions flow in the plasma membranes from the outside of the plasma membranes, and calcium ions are transported to cytosol by being released from intracellular calcium pools, so that related downstream signals are excited. After the calcium ion concentration is increased, the calcium ion can precipitate with phosphate radical in cells, the phosphate radical is necessary for energy and substance metabolism of the cells, and in addition, the high calcium ion concentration can cause the expression of genetic substances in the cells to be changed, thereby leading to the death of the bacterial cells.