CN113100257B

CN113100257B - Preparation method and application of natural quorum sensing inhibitor

Info

Publication number: CN113100257B
Application number: CN202110379239.4A
Authority: CN
Inventors: 于淑池; 周玲玲
Original assignee: Hainan Tropical Ocean University
Current assignee: Hainan Dafei Pharmaceutical Technology Co ltd
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-11-09
Anticipated expiration: 2041-04-08
Also published as: CN113100257A

Abstract

本发明属于食品贮藏保鲜技术领域，公开了一种天然群体感应抑制剂的制备方法及其应用，以大黄、黄芩、黄柏为原料，利用乙醇水溶液进行提取，得到三黄粉提取物，将所述三黄粉提取物经溶解、过滤、除菌后，即得天然群体感应抑制剂。本发明所用的三种中药来源丰富，提取物制备工艺简单、易操作，经实验首次证明，本发明所述三黄粉提取物具有抑制紫色杆菌CV026群体感应系统的活性，能够干扰水产品腐败希瓦氏菌的群体感应系统，抑制其致腐特性的表达，对细菌不会产生耐药性，无毒无害，同时该提取物能够有效抑制冷藏卵形鲳鲹贮藏过程中的腐败变质，最大程度延长卵形鲳鲹的保鲜时长，可用于水产品腐败菌天然群体感应抑制剂的开发及应用。The invention belongs to the technical field of food storage and fresh-keeping, and discloses a preparation method and application of a natural quorum sensing inhibitor. Rhubarb, Scutellaria baicalensis and Phellodendron phellodendri are used as raw materials, and ethanol aqueous solution is used for extraction to obtain Sanhuang powder extract. After the yellow powder extract is dissolved, filtered and sterilized, a natural quorum sensing inhibitor is obtained. The three kinds of traditional Chinese medicines used in the present invention are rich in sources, and the preparation process of the extract is simple and easy to operate. It is proved for the first time by experiments that the three yellow powder extract of the present invention has the activity of inhibiting the quorum sensing system of Bacillus purpureus CV026, and can interfere with the spoilage of aquatic products. The quorum sensing system of the bacteria inhibits the expression of its putrefaction characteristics, does not produce drug resistance to bacteria, is non-toxic and harmless, and at the same time, the extract can effectively inhibit the spoilage during the storage process of refrigerated ovoid pomfret. Extending the fresh-keeping time of oval pomfret can be used for the development and application of natural quorum sensing inhibitors of spoilage bacteria in aquatic products.

Description

Preparation method and application of natural quorum sensing inhibitor

Technical Field

The invention relates to the technical field of food storage and preservation, in particular to a preparation method and application of a natural quorum sensing inhibitor.

Background

China is a large country for producing and consuming aquatic products, the aquaculture yield of the aquatic products accounts for 70% of the total world production, however, the spoilage loss of fresh aquatic products is very serious, the average loss rate is about 15%, the microbial activity is the main cause of the spoilage of the aquatic products, only one or more specific microorganisms play a leading role in the spoilage process in a complex microbial phase, at the end point of a storage period, the microorganisms account for absolute advantages in quantity and proportion and are called as dominant spoilage bacteria or specific spoilage bacteria (SSOs), the growth and propagation of the dominant spoilage bacteria of the aquatic products are mainly related to the fish spoilage degree above the freezing temperature, and the dominant spoilage bacteria are closely related to the shelf life of the aquatic products, so that the dominant spoilage bacteria become the key point for controlling the quality of the aquatic products.

Trachinotus ovatus (arch of Trachinotus ovatus)Trachinotus ovatus) The golden pompano is a trachinotus genus of the Perciformes, is widely distributed in the southeast coastal areas such as Guangdong, Guangxi, Hainan and the like, is a newly increased marine economic fish species for culturing in recent years in China, and has the advantages that the culture yield of the golden pompano breaks through more than 15 million tons and the production value is nearly 100 million yuan according to data of the peak forum of the golden pompano industry in 2019; most of the dominant putrefying bacteria in the process of refrigerating marine products are Shewanella (Shewanella)Shewanellasp.), most of which are Shewanella putrefaciens (Shewanella putrefaciens) ((Hiwakia sp.)Shewanella putrefaciens). The laboratory earlier researches show that the dominant putrefying bacteria of the refrigerated trachinotus ovatus at 4 ℃ is also putrefying Shewanella (accession number U91555.1), the putrefying activity of the putrefying bacteria is strong, and the putrefying bacteria can reduce trimethylamine oxideBecomes trimethylamine, generates volatile gases such as hydrogen sulfide and the like, causes fishy and foul smell of aquatic products, can form a biofilm to make the surfaces of the aquatic products sticky, and shows a series of putrefaction characteristics. Shewanella putrefaciens is therefore one of the important factors that causes spoilage of cryopreserved seafood.

Most of the traditional antibacterial agents take substance synthesis or function realization for inhibiting cell walls, cell membranes or cell nuclei and the like of single bacteria as action targets, and finally achieve the purpose of bacteriostasis or sterilization. Under this survival pressure, bacteria mutate and evolve into resistant strains. Therefore, the search for alternative new bacteriostats and avoidance of drug resistance of bacteria becomes a focus of attention of researchers. Quorum Sensing (QS) is a communication mechanism among thallus cells, so that bacteria fight in a team mode, namely, microorganisms monitor population density of the bacteria through self-secretion or self-synthesis of self-induced signal molecules (AI), and when the density of the bacteria increases and the concentration of the secreted signal molecules reaches a certain threshold value, the bacteria can be combined with cytoplasmic receptor protein, so that the bacteria open a specific putrefactive gene expression mechanism with cell density dependence, such as bioluminescence, biofilm formation, harmful toxin generation, extracellular enzyme synthesis and other putrefactive characteristics, so that food is putrefactive, a food putrefactive flora response system is used as a target point, the expression of putrefactive factors is inhibited, and a new direction is provided for green preservation of food.

Under the premise of not interfering normal life activities, a Quorum Sensing Inhibitor (QSI) takes a quorum sensing system of bacteria as a target point to control the formation of a bacterial biofilm and the expression of a decay-causing factor, so that the decay property of spoilage bacteria is reduced, the drug resistance of the bacteria is not easy to induce, and the purposes of corrosion prevention and freshness preservation can be achieved. The quorum sensing inhibitors discovered at present mainly comprise two types of natural sources and artificial synthesis, wherein QSI from natural sources comprise extracts of marine algae, fruits and vegetables, Chinese herbal medicine plants and the like, have the characteristics of wide sources, low cost, high biological safety and the like, and are widely concerned by scholars at home and abroad. Compared with artificially synthesized QSI, the QSI from natural sources has low use concentration and low toxicity, does not hinder the normal growth of bacteria, plays a role in regulating and controlling a quorum sensing system of target cells at sub-bacteriostatic concentration, avoids the generation of bacterial drug resistance, and can reduce the decay-causing capacity of bacteria.

The Sanhuang powder serving as a veterinary drug in the market at present is prepared by compounding rhubarb, scutellaria and phellodendron bark (5: 3: 2), is used as a veterinary drug in the field of aquatic product cultivation, has the functions of clearing heat and removing toxicity, resisting bacteria and diminishing inflammation, can be used for preventing and treating bacterial diseases such as hepatobiliary diseases, gill rot, bleeding, enteritis, rotten skin and the like, and can enhance the disease-resistant immune function of organisms.

Therefore, how to provide a method for preparing a natural quorum sensing inhibitor by using three-yellow powder is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a preparation method of a natural quorum sensing inhibitor from Chinese herbal medicines and application of the natural quorum sensing inhibitor to inhibition of aquatic product putrescence Shewanella, wherein traditional Chinese medicinal materials including rhubarb, scutellaria and phellodendron are used as raw materials, and an ethanol extraction method, an acid dissolution method and an ethyl acetate extraction method are adopted to obtain a three-yellow-powder extract.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing natural quorum sensing inhibitor comprises extracting radix et rhizoma Rhei, Scutellariae radix, and cortex Phellodendri with organic solvent ethanol to obtain SANHUANG powder extract, dissolving the SANHUANG powder extract, filtering, and sterilizing to obtain natural quorum sensing inhibitor.

Preferably, in the above method for preparing a natural quorum sensing inhibitor, the preparation method comprises the following steps:

(1) pretreatment of raw materials: mixing rhubarb, scutellaria and phellodendron according to the proportion of 1:1.5-0.5:0.5-1.5, drying and crushing to obtain three-yellow powder;

(2) alcohol extraction: adding 45-75% ethanol solution into SANHUANG powder, leaching for several times, vacuum filtering to remove residue, and mixing filtrates;

(3) salt precipitation: adjusting the pH value of the filtrate to 6.0, adding aluminum salt and zinc salt into the filtrate for precipitation, centrifuging, and taking the precipitate of the lower layer;

(4) acid dissolution and extraction: adding hydrochloric acid into the precipitate for dissolving, carrying out vacuum filtration to obtain supernatant, and extracting the supernatant with ethyl acetate to obtain extract liquor;

(5) and (3) drying: evaporating, concentrating and drying the extract by a rotary evaporator to obtain a three-yellow powder extract;

(6) and (3) finished product: dissolving the three-yellow powder extract, filtering, and sterilizing to obtain the natural quorum sensing inhibitor.

In the preparation method, the steps of alcohol extraction, acid dissolution and extraction play an important role, different organic solvents have different polarities, and the active ingredients of the extract are different; particularly, the Sanhuang powder extract contains tannin components with larger polarity, and the ethanol water solution has good dissolving capacity on the tannin components; the ethyl acetate belongs to a medium-polarity organic solvent, can extract components such as tannin with strong antibacterial activity, is easier to remove when being evaporated under reduced pressure, and the plant ethyl acetate extract has strong antibacterial activity under an acidic condition, so the extraction process adopts acid dissolution and then extraction, and meanwhile, the ethyl acetate extract also has good thermal stability.

Preferably, in the above preparation method of a natural quorum sensing inhibitor, in the step (1), the drying temperature is 50 ℃ and the drying time is 24-48 h.

Preferably, in the above preparation method of a natural quorum sensing inhibitor, the mass-to-volume ratio of the tribasic yellow powder to the ethanol solution in step (2) is 1 g: (1.5-3) ml; the leaching temperature is 60-85 ℃, and the leaching time is 20-40 min.

Preferably, in the preparation method of the natural quorum sensing inhibitor, the mass ratio of the aluminum salt to the zinc salt in the step (3) is 1: 1-2.

Preferably, in the above preparation method of a natural quorum sensing inhibitor, the volume concentration of the hydrochloric acid in the step (4) is 12%, and the addition amount is 200 mL; the volume ratio of the supernatant to the ethyl acetate is 1: 2-4.

Preferably, in the above method for preparing a natural quorum sensing inhibitor, the pressure of evaporation and concentration in step (5) is 0.065MPa, and the temperature is 64 ℃; the drying temperature is 60-70 ℃.

The invention also discloses the natural quorum sensing inhibitor prepared by the method.

And discloses application of the natural quorum sensing inhibitor prepared by the method in regulating and controlling the decay-causing capability of Shewanella putrefaciens of aquatic products.

Preferably, in the application of one natural quorum sensing inhibitor in regulating and controlling the decay-causing capability of Shewanella putrefactive in aquatic products, the natural quorum sensing inhibitor has quorum sensing inhibitory activity on purple bacillus and has an inhibitory effect on the expression of decay-causing factors of Shewanella putrefactive.

Preferably, in the application of one natural quorum sensing inhibitor in regulating and controlling the putrescence ability of Shewanella putrefaciens in aquatic products, the minimum inhibitory concentration of the natural quorum sensing inhibitor on Shewanella putrefaciens is 2.3 mg/m L, and the Sanhuang powder extract can inhibit the quorum sensing activity of Shewanella putrefaciens at sub-inhibitory concentrations (0.5, 1.0, 1.5 and 2.0 mg/mL).

Also discloses application of the natural quorum sensing inhibitor prepared by the method in low-temperature fresh-keeping storage of aquatic products.

Preferably, the natural quorum sensing inhibitor is applied to low-temperature fresh-keeping storage of aquatic products, and the application of the natural quorum sensing inhibitor to the fresh-keeping of the aquatic products is the application of trachinotus ovatus in the low-temperature fresh-keeping storage of the aquatic products.

According to the technical scheme, compared with the prior art, the invention discloses a preparation method and application of a natural quorum sensing inhibitor, and the preparation method has the following beneficial effects:

(1) the raw materials used by the invention are three common traditional Chinese medicinal materials, the sources are rich, and the extraction process is simple and easy to operate;

(2) the prepared three-yellow powder extract can tolerate the extraction temperature of 60-85 ℃ during extraction, the drying temperature of the concentrate is 60-70 ℃, and the heat resistance is stronger compared with that of a common natural quorum sensing inhibitor; the precipitate is dissolved by 12% hydrochloric acid in the extraction process, which shows that the extract has acid stability and strong bacteriostatic activity and is beneficial to application and popularization;

(3) the three-yellow powder extract prepared by the invention has the activity of inhibiting a quorum sensing system of a reporter strain purple bacillus CV026, and the quorum sensing inhibition activity to Shewanella putrefaciens is shown as follows: the Shewanella putrefaction inhibitor has an inhibiting effect on quorum sensing such as formation of a biological membrane of Shewanella putrefaction, activity and clustering of extracellular enzymes, mobility and the like, and the quorum sensing is decay-causing factors, so that the Sanhuang powder extract can reduce decay-causing capacity of Shewanella putrefaction, does not generate survival pressure on Shewanella putrefaction, does not generate drug resistance, is a novel antibacterial substance based on the bacterial quorum sensing inhibiting effect, can be used in the field of preservation and preservation of aquatic products, can be used independently or used in combination with other preservatives to achieve the aim of preservation of the aquatic products, and has a good application prospect as a natural quorum sensing inhibitor.

(4) The prepared three-yellow powder extract has quorum sensing inhibition activity under the sub-antibacterial concentration, has low acting dosage and can greatly save the cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 shows the comparison of the antibacterial activity of the extracts of rhubarb, scutellaria and phellodendron bark and the extract of Sanhuang powder;

FIG. 2 is a comparison of the bacteriostatic activity of Sanhuang powder extracts in different proportions on Shewanella putrefaciens;

FIG. 3 shows the bacteriostatic activity of Sanhuang powder extract against Shewanella putrefaciens (note: A: Sanhuang powder extract at 9.2mg/mL (4 MIC); b: Sanhuang powder extract at 1.15mg/mL (0.5 MIC); c: methanol control)

FIG. 4 is the quorum sensing activity of Sanhuang powder extract on purple bacillus;

FIG. 5 is a graph showing the effect of Sanhuang powder extract on the growth curve of Shewanella putrefaciens;

FIG. 6 shows the effect of Sanhuang powder extract on the relative inhibition rate and bacterial liquid density of Shewanella putrefaciens biofilm formation (note: different letters indicate significant difference: (see: different letters) P< 0.05), the same below);

FIG. 7 is a graph showing the effect of Sanhuang powder extract on Shewanella putrefaction biofilm morphology;

FIG. 8 shows the qualitative determination results of the effect of SANHUANG powder extract on extracellular protease activity of Shewanella putrefaciens (note: a. negative control; b.0.5 mg/mL SANHUANG powder extract; c.1.0 mg/mL SANHUANG powder extract; d.1.5 mg/mL SANHUANG powder extract; e.2.0 mg/mL SANHUANG powder extract, the same applies below);

FIG. 9 shows the quantitative determination of the effect of Sanhuang powder extract on extracellular protease activity of Shewanella putrefaciens;

FIG. 10 is a graph showing the effect of Sanhuang powder extract on extracellular lipase activity of Shewanella putrefaciens;

FIG. 11 is a graph showing the inhibitory effect of Sanhuang powder extract on Shewanella putrefaciens cluster (A) and swimming motility (B);

FIG. 12 is a graph of the effect of Sanhuang powder extract on clustering and swimming;

figure 13 shows the effect of the phellinus igniarius extract on the sensory quality of refrigerated trachinotus ovatus;

FIG. 14 shows the effect of the trachinotus ovatus powder extract on the volatile basic nitrogen index of refrigerated trachinotus ovatus;

FIG. 15 shows the effect of Sanhuang powder extract on the total number of colonies of refrigerated trachinotus ovatus.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Mixing radix et rhizoma Rhei, Scutellariae radix, and cortex Phellodendri at a certain ratio (1: 1.5-0.5: 0.5-1.5), oven drying at 50 deg.C for 24 hr to dry, pulverizing with Retsch high quality tissue grinder (6.5 × 1000rpm, 6 min), and sieving with 80 mesh sieve to obtain SANHUANG powder; weighing 100g of powder, adding 75% ethanol solution at a ratio of 1:2.5, leaching at 75 deg.C for 35min, repeating twice, filtering to remove residue, mixing filtrates, adding AlCl₃(6g) And ZnCl₂(12g) Precipitating, centrifuging (3500 r/min, 15 min)) after the solution is layered and obvious precipitate appears, adding 200mL of 12% hydrochloric acid into the precipitate for dissolving, carrying out vacuum filtration to obtain supernatant, adding 2 times volume of ethyl acetate for extraction, concentrating the extract in a rotary evaporator (0.065 Mpa, 64 ℃), drying the concentrate in an oven at 60 ℃ to obtain the radix et rhizoma Rhei powder extract, dissolving with methanol when in use, preparing 30mg/mL stock solution, and filtering and sterilizing with a 0.22 mu m microporous filter membrane to obtain the natural quorum sensing inhibitor for later use.

Research on antibacterial effect of single extracts and combined extracts of rhubarb, scutellaria and phellodendron

The rhubarb extract, the scutellaria extract, the phellodendron extract and the phellodendron bark powder (1: 1: 1) extract are respectively prepared by the same extraction method, the antibacterial activity of Shewanella putrefaciens (separated from refrigerated trachinotus ovatus in the laboratory) is tested according to the concentration of the extract stock solution (50 mu L), the test result is shown in figure 1 by taking methanol as a control, and the result shows that the phellodendron bark powder extract has the strongest antibacterial activity, namely the rhubarb, the scutellaria and the phellodendron bark have a synergistic antibacterial effect after being mixed.

Screening of proportioning range of rhubarb, scutellaria and phellodendron bark

In order to determine the reasonable proportion of the rhubarb, the radix scutellariae and the golden cypress, the rhubarb, the radix scutellariae and the golden cypress are unfolded according to the proportion of 1:1.5:0.5, 1:1:1 and 1:0.5:1.5, an Oxford cup punching method is adopted, and the Shewanella putrefaciens is screened according to the concentration (50 mu L) of an extract stock solution, the test result is shown in figure 2, and figure 2 shows that the three proportion modes have bacteriostasis, but the bacteriostasis activity is strongest according to the equal proportion (1: 1: 1) of the rhubarb, the radix scutellariae and the golden cypress.

Research on Minimum Inhibitory Concentration (MIC) of Shiwa bacteria and purple bacillus by using Sanhuang powder extract

The experimental method comprises the following steps: determination of minimum inhibitory concentration of Shiwa bacteria by Sanhuang powder extract by plate coating method, purple bacillus CV026 (purchased from Beijing Baiohobowei biotechnology limited) and target Shewanella putida were respectively inoculated into LB broth culture medium (yeast extract 5g/L, tryptone 10g/L, NaCl 10g/L, p H7.2.2-7.4, sterilized at 121 deg.C for 30min, solid LB culture medium added with agar 20 g/L), and activated overnight at 28 deg.C and 160r/min for two times to logarithmic phase (OD)₅₉₅nm = about 1). Respectively sucking three yellow powder extract stock solutions with different dosages of 30mg/mL, adding the three yellow powder extract stock solutions into an LB agar culture medium cooled to about 40 ℃, uniformly mixing, pouring the mixture into a flat plate, and determining the minimum inhibitory concentration of purple bacillus, wherein the final concentrations of the three yellow powder extracts are respectively 0.525, 1.05, 2.1, 4.2 and 8.4 mg/mL; the final concentration of Shewanella putrefaciens was set to 0.575, 1.15, 2.3, 4.6, and 9.2mg/mL, and 100. mu.L of activated bacteria of purple bacillus and Shewanella putrefaciens were added dropwise to the center of the plate and applied uniformly. Taking a blank culture medium without adding the Sanhuang powder extract as a negative control, standing and culturing at a constant temperature of 28 ℃ for 24 hours, observing the growth condition of bacteria on each plate, and taking the mass concentration without bacteria growth as the minimum inhibitory concentration.

The experimental results are as follows: through observing the growth influence conditions of the three-yellow powder extract on purple bacillus CV026 and shewanella putrefaciens in different concentration gradient ranges, the MIC of the three-yellow powder extract on the purple bacillus CV026 is determined to be 2.1 mg/mL, the MIC on the shewanella putrefaciens is determined to be 2.3 mg/mL, the subsequent inhibition effect experiment of the three-yellow powder extract on the shewanella putrefaciens is carried out, the treatment concentration is carried out by taking the sub-antibacterial concentration, and the treatment concentration is specifically set to be 0.5mg/mL, 1.0mg/mL, 1.5mg/mL and 2.0 mg/mL.

Sanhuang powder extract antibacterial activity and quorum sensing activity detection

(1) Determination of bacteriostatic activity against Shewanella putrefaciens

The experimental method comprises the following steps: adding 15mL of LB solid culture medium into a sterile culture dish, and placing aside for later use after the LB solid culture medium is solidified; inoculating Shewanella putrefaciens bacterial liquid into a proper amount of unsolidified LB solid culture medium according to the inoculation amount of 2 percent, taking 1mL of the Shewanella putrefaciens bacterial liquid into the solidified LB solid culture medium, uniformly spreading the LB solid culture medium, clamping an oxford cup on the solidified solid culture medium by using tweezers after solidification, and slightly pressing the oxford cup to ensure that the oxford cup is well contacted with the culture medium; diluting 30mg/mL of Sanhuang powder extract into 1.15mg/mL (0.5MIC) and 9.2mg/mL (4MIC) concentrations by adopting a multiple dilution method, adding 50 mu L of Sanhuang powder extract into an Oxford cup, and adding methanol as a blank control group. The diameter of the zone of inhibition was measured after 24h of static culture at 28 ℃ and 3 per group were performed in parallel.

The experimental results are as follows: as shown in figure 3, when the concentration of the Sanhuang powder extract is 9.2mg/mL (4MIC), the Sanhuang powder extract has a remarkable bacteriostatic effect, and a transparent inhibition ring is obviously formed around the aperture of the Sanhuang powder extract, wherein the diameter of the Sanhuang powder extract reaches 3.33 mm; the concentration of the three-yellow powder extract is 1.15mg/mL (0.5MIC) and a control group, and no inhibition zone is generated around the three-yellow powder extract, which shows that the three-yellow powder extract with higher concentration (4MIC) has stronger antibacterial activity, and the sub-inhibition concentration of 0.5MIC has no inhibition effect on the growth of pseudomonas fluorescens.

(2) Determination of activity of three-yellow powder extract on induction inhibition of purple bacillus CV026 population

Purple bacterium (I)Chnomobacterium violaceum) CV026 reporter strain is a wild strainChromobacterium violaceumThe mini-Tn5 mutant of ATCC 31532, which is resistant to kanamycin, does not produce purplish rhzomorph per se and does not produce N-acyl homozygotesSerine lactone signal molecules (AHLs), when exogenous signal molecules AHLs exist, the existence of the exogenous signal molecules can be sensed, and purpurins are generated; the production of purpurin of purple bacillus CV026 is regulated by bacterial quorum sensing, and the quorum sensing inhibitor can inhibit the production of purpurin.

The experimental method comprises the following steps: determining the effect of SANHUANG powder extract on the production of purpurin of reporter strain CV026 by using reporter plate method, activating the purple bacteria CV026 of reporter strain overnight at 28 deg.C and 160r/min for 2 times, inoculating into LB broth containing 20 μ g/mL kanamycin at an inoculum size of 2%, culturing for 18 h, inoculating into LB agar medium (containing 20 μ g/mL C) cooled to about 40 deg.C at an inoculum size of 2%₆-HSL signal molecules), mixing uniformly, cooling and solidifying, punching by an Oxford cup, adding 100 mu L of 2.0mg/mL Sanhuang powder extract into the cup, standing and culturing at 28 ℃ for 24h, and observing the generation condition of the purpurin.

The experimental results are as follows: FIG. 4 is a graph showing the results of the three-yellow powder extract for inhibiting quorum sensing activity of purple bacillus, and it can be seen from FIG. 4 that after 2mg/ml of the three-yellow powder extract is added, a yellowish and opaque purple pigment inhibition zone (diameter 8.3 mm) appears around the round hole, which indicates that the three-yellow powder extract with sub-inhibitory concentration can inhibit the production of purple bacillus purpurin, has quorum sensing inhibition activity, but has no effect on the growth thereof.

Influence of Sanhuang powder extract on growth curve of Shewanella putrefaciens

The experimental method comprises the following steps: shewanella putrefaciens is activated twice overnight, inoculated into LB broth according to the inoculum size of 2%, and added with different dosages of Sanhuang powder extract to make the final concentration of 0.5, 1.0, 1.5 and 2mg/mL respectively, and LB broth without the Sanhuang powder extract is set as blank control group, and is subjected to shake cultivation at 28 deg.C for 160r/min, and OD is measured every 2h₆₀₀nm value, time (h) as abscissa, absorbance OD of bacteria liquid₆₀₀And nm is the ordinate to draw the bacterial growth curve.

The experimental results are as follows: FIG. 5 is a graph showing the experimental results of the effect of Sanhuang powder extract on the growth curve of Shewanella putrefaciens. As can be seen from FIG. 5, the three yellow powder extracts at different concentrations were obtained at the pre-treatment period (0-2 h)The Shewanella putrefaciens of the treatment group and the control group are in the slow growth process; when the time is 2-12h, the Shewanella in each treatment group and the Shewanella in the control group enter a rapid growth period; after 12h, the stationary phase was followed and the growth curve became gentle, overall, the OD of the control group growth₆₀₀The nm value is slightly higher, and the OD of the thallus growth is increased along with the increase of the concentration of the Sanhuang powder extract treatment group₆₀₀Gradually decreases, but the overall growth situation tends to be uniform overall without obvious difference (PTg is 0.05), and the result shows that the three-yellow powder extract has no influence on the growth of Shewanella putrefaciens under the sub-antibacterial concentration, and the antibacterial effect is mainly embodied by quorum sensing inhibition activity.

Determination of inhibition effect of Sanhuang powder extract on putrefactive Shewanella putrefactive property under sub-bacteriostatic concentration

(1) Determination of Effect on biofilm formation and morphology of Shewanella putrefaciens

(ii) measurement of influence on relative inhibition ratio of biofilm formation

The experimental method comprises the following steps: shewanella putrefaciens bacteria solution activated twice overnight was diluted with LB broth at a ratio of 1: 100. Adding 1 mL into a 1.5mL centrifuge tube, adding different dosages of radix Tripterygii Wilfordii powder extract to make the final concentrations of 0.5, 1.0, 1.5 mg/mL and 2.0 mg/mL respectively, and performing static culture at 28 deg.C for 48 h with sterile deionized water as negative control. Taking out, respectively taking 200 μ L of bacterial liquid, and placing in OD with enzyme labeling instrument₅₉₅And (3) measuring the density of the bacterial liquid at nm, pouring the bacterial liquid after the bacterial liquid density is measured, rinsing the planktonic bacteria for 3-5 times by using sterile water, drying for 35 min by using sterile air, adding 200 mu L of 0.1% (w/v) crystal violet, dyeing for 15min at room temperature, and washing the centrifugal tube by using sterile water until the water is clear. Then adding 1mL of 95% ethanol for dissolving, standing for 10min, measuring an absorbance value under the condition that an OD value of an enzyme-labeling instrument =595nm, and calculating the formation amount of the biological membrane according to the absorbance value. The biofilm relative inhibition rate was calculated as follows:

biofilm relative inhibition (%) = (OD)_control-OD_QSI/OD_control）×100%

In the formula: OD_QSIOD measured for inhibitor treated group₅₉₅The value of nm; OD_controlOD measured for negative control group₅₉₅And (5) nm value.

Experimental results, figure 6 shows the results of measuring the effect of the Sanhuang powder extract on the amount of Shewanella putrefaciens biofilm formation inhibition. The biofilm formation amount of Shewanella putrefaciens is reduced along with the increase of the concentration of the Sanhuang powder extract, the relative inhibition rate of the biofilm is gradually increased under the action of the Sanhuang powder extract with different concentrations (0.5, 1.0, 1.5 and 2.0mg/mL), and the inhibition rate is up to 35.1% when the concentration is 2.0 mg/mL. The three-yellow powder extract has obvious inhibition effect on the formation of the Shewanella putrefaciens biofilm under the sub-bacteriostatic concentration.

② detection of influence on form of biological membrane

The experimental method comprises the following steps: pretreating a glass slide (25.4 mm multiplied by 76.2 mm, thickness 1-2 mm): cleaning the glass slide, performing ultrasonic treatment in absolute ethyl alcohol for 30 min and in deionized water for 30 min, drying, and sterilizing for later use. The Shewanella after overnight activation is inoculated in LB broth according to the volume ratio of 1:100, 10 mL of bacterial liquid is added into a sterile culture dish, the three yellow powder extract is added to ensure that the final mass concentration is 0.5, 1.0, 1.5 and 2.0 mg/mL respectively, and the treated glass slide is immersed in the three yellow powder extract and cultured for 72 h at the constant temperature of 28 ℃. Meanwhile, a negative control group without adding the Sanhuang powder extract was set. And (3) taking out the glass slide by using a pair of tweezers, rinsing the glass slide for 3-5 times by using sterile water, placing the glass slide in a sterile culture dish, adding a proper amount of methanol for fixing for 15 min, adding a proper amount of 2% crystal violet for dyeing for 5 min, washing the glass slide for 5-7 times by using the sterile water, drying and fixing the glass slide by using sterile air, and observing and photographing and recording the glass slide by using an optical microscope oil mirror (100 x).

The experimental results are as follows: FIG. 7 shows the effect of the Sanhuangfen extract on the morphology of Shewanella putrefaciens biofilm at different concentrations, and it can be seen from FIG. 7 that the cell density and the cell mass size of the biofilm gradually decrease and the biofilm distribution gradually becomes dispersed and thin as the concentration of the Sanhuangfen extract increases. The negative control group (a) had the most dense and clumpy biofilm and was also darkest in color; the biomembrane of the 2.0mg/mL radix et rhizoma Rhei powder extract treated group is loose and sparse in distribution, thin in thickness and light in color. The inhibition effect on the Shewanella putrefaciens biological membrane is continuously enhanced along with the increase of the concentration of the Sanhuang powder extract, and the measurement result is matched with the quantitative measurement result of the biological membrane.

(2) Determination of inhibition of extracellular protease Activity of Shewanella putrefaciens

Qualitative determination of protease activity (hydrolysis loop method)

The experimental method comprises the following steps: skim milk agar plates were prepared, and 15% skim milk powder was dissolved in purified water and separately sterilized (108 ℃ C., 15 min). And (3) uniformly mixing 10 mL of skim milk with 90 mL of solid nutrient agar culture medium, solidifying and then punching by using an Oxford cup. Preparing overnight activated Shewanella putrefaciens bacterial suspension containing three yellow powder extracts with different concentrations (0.5, 1.0, 1.5 and 2.0mg/mL), adding 200 μ L bacterial liquid into the well, standing and culturing at 28 deg.C for 18-24 h, and observing the diameter of hydrolysis ring. After the casein is hydrolyzed by the protease, a clear circle appears around the colony, and the size of the clear circle indicates the level of the proteolytic activity.

The experimental results are as follows: FIG. 8 is a graph showing the results of experiments on the effect of Sanhuang powder extract on extracellular protease activity of Shewanella putrefaciens. The diameter of the proteolytic transparent ring can show that the inhibition effect of the three-yellow powder extract on the protease is strong and weak, and the diameter of the proteolytic ring of the negative control group is the largest and reaches 7.84 mm. The three-yellow powder extract has obvious inhibition effect on the extracellular protease activity of Shewanella putrefaciens, the protease hydrolysis ring is gradually reduced along with the increasing of the treatment concentration, the minimum value is only 0.76mm at 2.0mg/mL, and the inhibition rate can reach as high as 90.3%.

② quantitative determination of protease Activity

The experimental method comprises the following steps: shewanella putrefaciens was added to LB broth at an inoculum size of 2%, and the respective Sanhuang powder extracts were added to give final mass concentrations of 0.5, 1.0, 1.5 mg/mL and 2.0 mg/mL, and a negative control group without the Sanhuang powder extract was set. Centrifuging at 4 ℃ at 10000r/min for 10min, taking supernatant, filtering and sterilizing, operating according to the instruction of the alkaline protease activity detection kit, measuring absorbance at 680nm, and calculating protease activity:

in the formula: Δ A determination = A determination tube-A control tube; Δ A Standard = A Standard tube-A blank tube; cpr is the protein concentration of the sample solution, mg/mL.

The experimental results are as follows: FIG. 9 is a graph showing the results of quantitative determination of extracellular protease activity of Shewanella putrefaciens by using Sanhuang powder extract. FIG. 9 shows that the protease activity is gradually reduced and the inhibition effect is gradually increased with the increase of the concentration of the Sanhuang powder extract, the protease activity is only 0.189mg/mL at 2.0mg/mL, while the negative control reaches 0.512mg/mL, and the bacteriostasis rate reaches up to 63.08%.

(3) Determination of the Effect on the Lipase Activity of Shewanella putrefaciens

The experimental method comprises the following steps: shewanella putrefaciens was added to LB broth at an inoculum size of 2%, and Sanhuang powder extract was added to give final mass concentrations of 0.5, 1.0, 1.5 and 2.0 mg/mL, respectively, and a negative control group without the Sanhuang powder extract was set. Centrifuging at 4 deg.C at 10000r/min for 10min, collecting supernatant, filtering, sterilizing, and determining lipase activity with lipase detection kit (Nanjing institute of bioengineering research, product number A054). Lipase activity was calculated according to the following formula:

LPS viability (U/mL) = (A1-A2)/As × 0.425 μmol/L × (2.05mL/0.05mL) ÷ 10min ÷ 1000

In the formula: a1 OD measured after mixing the reagents and before heating in a 37 ℃ water bath₄₂₀Value, A2 OD measured after heating in a water bath at 37 ℃ for 10 minutes₄₂₀Value, As: OD of standard tube (0.45. mu. mol/L) concentration₄₂₀Value of

The experimental results are as follows: FIG. 10 is a graph showing the results of experiments on the effect of Sanhuang powder extract on the lipase activity of Shewanella putrefaciens, and FIG. 10 shows that the lipase activity of the negative control is as high as 0.485U/mL, gradually decreases with the increase of the concentration of Sanhuang powder extract, and the lipase activity of Shewanella putrefaciens treated with 2.0mg/mL of Sanhuang powder extract is the lowest, and is only 0.0986U/mL. The treated group differed significantly from the control group (PLess than 0.05), and the three-yellow powder extract has concentration dependence on the inhibition of extracellular lipase activity of putrefactive Shewanella, and has high inhibition rateTo 79.67%.

(4) Determination of Effect on Shewanella putrefaction clustering Property and swimming Properties

The experimental method comprises the following steps: a colony agar medium (1% peptone, 0.5% sodium chloride, 0.5% agar, and 0.5% glucose) and a migration agar medium (1% tryptone, 0.5% sodium chloride, and 0.3% agar) were prepared, and different amounts of the Triflola powder extract were added to the respective media to give final concentrations of 0.5, 1.0, 1.5, and 2mg/mL, respectively. After the flat plate is solidified, 5 mu L of overnight cultured bacterial liquid is taken and spotted in the center of the flat plate, the flat plate is dried by sterile wind, the flat plate is cultured in an incubator at 28 ℃ for 24h, the diameter of a diffusion ring is measured, the area of the diffusion ring is calculated, and the inhibition effect of the three-yellow powder extract on Shewanella putrefaciens clustering and swimming ability is judged according to the diameter.

The experimental results are as follows: FIGS. 11 and 12 are graphs showing the inhibitory effects of Sanhuang powder extract on Shewanella putrefaction colonization (A) and migration (B). FIGS. 11 and 12 show that the strains in the negative control treatment group have the strongest clustering and swimming abilities, and the clustering area reaches 228.9mm at the maximum²The diameter of the steel tube reaches 16.02 mm; the swimming area reaches 310.98mm²The swimming diameter reaches 19.9 mm. While the cluster area of the treated group of 2.0mg/mL of the Sanhuang powder extract was only 15.7mm²The swimming area is only 15mm². Shewanella putrefaciens migrates outward in the form of flagella, and its clustering and swimming characteristics are markedly reduced with the increase in the concentration of Sanhuang powder, which are inversely related. Each treatment group was significantly different from the control group (P< 0.05), indicating that the motor capacity of Shewanella putrefaciens is regulated by a quorum sensing system, and the fomes fomentarius extract interferes with the ability of Shewanella putrefaciens flagella to adhere to the contact surface, thereby inhibiting the putrefactive ability thereof. The inhibition rates of 2mg/mL of radix et rhizoma Rhei Palmati extract cluster and swimming area are 93.14% and 95.17% respectively, and the inhibition rates of cluster and swimming diameter are 72.09% and 78.04% respectively.

Application of trachinotus ovatus fresh-keeping extract based on quorum sensing inhibitor to fresh-keeping of refrigerated trachinotus ovatus

The preparation method of the phellodendron amurense rupr extract is the same as that in example 1, and the phellodendron amurense rupr extract is used for the fresh-keeping research of an aquatic product, namely trachinotus ovatus (commonly called golden pompanus, purchased in the litchi ditch market of the third city), and the specific implementation method is as follows:

preparing aseptic fish blocks: killing fresh and alive non-pathological trachinotus ovatus with ice water mixture, cleaning with sterile water, draining, wiping fish body with 75% ethanol, shearing fish meat inside ridge with sterile scissors to make fish block thickness about 1 cm and 40-50 g/block, and treating sterilized fish block with colony count less than 10²CFU/g;

preparation of Shewanella putrefaciens suspension: taking out and activating Shewanella putrefaciens preserved in a refrigerator at the temperature of-80 ℃, selecting a single colony to be inoculated in an LB liquid culture medium, placing the single colony in a shaking table at the temperature of 30 ℃ and the rotating speed of 160 rpm for constant-temperature shaking culture until the concentration of the bacterial suspension reaches 10⁸CFU/mL, centrifuged (12000 rpm, 30 min), discarded supernatant and diluted to 10 with sterile physiological saline⁶CFU/mL, spare.

Inoculation and storage fresh-keeping experiment: in a clean bench, the sterile trachinotus ovatus pieces are divided into 2 groups of 3 parallel groups, and the control group is immersed in Shewanella putrefaciens suspension (10)⁶CFU/mL), and 1 group were immersed in a shewanella putrefaciens suspension containing 2mg/mL of a saffron extract at a fish chunk to inhibitor volume ratio of 1:1, completely immersing the fish blocks in the water; taking out after 1-2min, sucking bacteria liquid on the surface of the fish blocks with sterile filter paper, taking out, draining, placing into a sterile self-sealing bag, storing in a refrigerator at 0-4 deg.C, taking out the inoculated fish blocks at intervals of 2 d, and performing sensory evaluation, total bacterial colony count and TVB-N index determination.

Influence of the extract of the powder of phellinus igniarius based on the quorum sensing inhibitor on sensory scores of trachinotus ovatus during refrigeration

The experimental method comprises the following steps: according to GB/T18108-2019 general rule of fresh seawater fish, a sensory evaluation scoring table (table 1) of the trachinotus ovatus is designed, four indexes of color, smell, tissue form and elasticity of the trachinotus ovatus are scored, the weight is 0.25, the sum is 1, and the score lower than 6 is a sensory critical point.

TABLE 1 sensory Scoring criteria for Trachinotus ovatus

The experimental results are as follows: a scoring panel was composed of 10 trained panelists, and sensory scoring was performed on the trachinotus ovatus blogs according to evaluation Table 1. The results are shown in FIG. 13, and it can be seen from FIG. 13 that the sensory scores of the fish fillets in the group 2 showed a gradual decrease as a whole with the prolongation of the storage time, but the sensory scores of the Sanhuang powder extract group were significantly higher than those of the control group: (P< 0.05). The control group is close to the putrefaction critical value at 4d, compared with the control group, the quality of the trachinotus ovatus mass treated by the trachinotus ovatus powder extract is better maintained, and reaches the sensory putrefaction point at 10d, which shows that the shelf life of the trachinotus ovatus mass can be prolonged by the treatment of the quorum sensing inhibitor, and the putrefaction end point is prolonged by 6 d.

Influence of the extract of the powder of phellinus igniarius based on the quorum sensing inhibitor on the colony count of trachinotus ovatus during refrigeration

The experimental method comprises the following steps: the total number of colonies is determined by the method of national standard GB 4789.2-2016. Taking 5g of samples every 2 days, stirring, adding 45m L sterile normal saline, shaking for 5min, performing gradient dilution on the supernatant, selecting a proper multiple to absorb 1m L, injecting into a PCA plate, shaking uniformly, repeating each gradient for 2 times, culturing at 30 ℃ for 48h, and counting.

The experimental results are as follows: the change in total colony count (TVC) reflects the degree of protein and amino acid catabolism, as defined by Al-Daqal et Al (Al-Daqal M, Bazara W A. Extension of shelf life of white and particulate shelf with organic acid salts and bifidobacteria [ J ]. Journal of Food Protection, 1999, 62(1): 51-56) that the upper limit of the total number of colonies edible to aquatic products is 6.0 lg (cfu/g), beyond that which is putrefaction. As can be seen from FIG. 14, the total number of colonies increased with the storage time, and both were 4d above the critical value, but there was no significant difference between the two, indicating that the Sanhuang powder extract did not inhibit the growth of Shewanella putrefaciens. The bacteriostatic action is reflected by quorum sensing inhibition.

Influence of the trachinotus ovatus powder extract on volatile basic nitrogen (TVB-N) in storage and preservation of trachinotus ovatus based on quorum sensing inhibitor

The experimental method comprises the following steps: 20g of fish meat samples are taken every 2 days to measure the content of volatile basic nitrogen, and the measuring method is carried out by referring to a semi-trace nitrogen determination method in a first method in national standard GB 5009.228-2016 (measuring volatile basic nitrogen in national standard food for food safety).

The experimental results are as follows: referring to the GB/T18108-2019 standard, the sample freshness TVB-N is not more than 15 mg/100g and is a high-grade product; the TVB-N is not more than 30 mg/100g, which is qualified. The result of the change of total volatile basic nitrogen value of the trachinotus ovatus during the refrigeration period is shown in figure 15, the volatile basic nitrogen value of the control group is 29.89mg/100g at 10 days, and the putrefaction end point is close to the putrefaction end point, so that the putrefaction end point of the control group is 10 days; the putrefaction end point of the experimental group treated by the three-yellow powder extract can reach 14 days, and compared with the control group, the accumulation amount of volatile basic nitrogen of the fish blocks treated by the quorum sensing inhibitor is reduced, so that the putrefaction period is prolonged by 4 days. The results show that the trachinotus ovatus putrefaction period can be effectively prolonged by the treatment of the trachinotus ovatus powder extract. This is probably due to the fact that the Sanhuang powder extract interferes with the quorum sensing system of Shewanella putrefaciens, and inhibits the degradation of fish meat by protease.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. For the scheme disclosed by the embodiment, the scheme corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

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.

Claims

1. a preparation method of a natural quorum sensing inhibitor, is characterized in that, with Rhubarb, Scutellaria baicalensis, Cortex Phellodendri as raw material, utilize ethanol aqueous solution to extract, obtain Sanhuang powder extract, by described Sanhuang powder extract through dissolving, filtering, After sterilization, a natural quorum sensing inhibitor is obtained;

The preparation method of the natural quorum sensing inhibitor specifically includes the following steps:

(1) Raw material pretreatment: Rhubarb, Scutellaria baicalensis, and Treats are mixed in a ratio of 1: 1.5 to 0.5: 0.5 to 1.5, dried and pulverized to obtain Sanhuang powder;

(2) Alcohol extraction: add 45-75% ethanol solution to the three yellow powder to extract, remove the residue by suction filtration, and combine the filtrates;

(3) Salt precipitation: adjust the pH of the filtrate to 6.0, then add aluminum salt and zinc salt to the filtrate for precipitation and centrifugation, and remove the lower sediment;

(4) Acid dissolution and extraction: add hydrochloric acid to the precipitate for dissolution, suction filtration under reduced pressure to obtain a supernatant, and extract the supernatant with ethyl acetate to obtain an extract;

(5) Drying: the extract is evaporated, concentrated and dried by a rotary evaporator to obtain the three yellow powder extract;

(6) Finished product: After dissolving, filtering and sterilizing the extract of Sanhuang powder, a natural quorum sensing inhibitor is obtained.

2 . The method for preparing a natural quorum sensing inhibitor according to claim 1 , wherein the drying temperature in step (1) is 50° C., and the drying time is 24 h to 48 h. 3 .

3 . The method for preparing a natural quorum sensing inhibitor according to claim 1 , wherein in step (2), the mass-volume ratio of Sanhuang powder to the ethanol solution is 1 g: (1.5-3) mL; 3 . The extraction temperature is 60-85℃, and the extraction time is 20-40min.

4 . The method for preparing a natural quorum sensing inhibitor according to claim 1 , wherein the mass ratio of the aluminum salt to the zinc salt in step (3) is 1:1-2. 5 .

5 . The preparation method of a natural quorum sensing inhibitor according to claim 1 , wherein the volume concentration of the hydrochloric acid in step (4) is 12%, and the addition amount is 200 mL; the supernatant and The volume ratio of the ethyl acetate is 1:2-4.

6 . The method for preparing a natural quorum sensing inhibitor according to claim 1 , wherein the evaporation concentration pressure in step (5) is 0.065 MPa, and the temperature is 64° C.; and the drying temperature is 60˜60° C. 7 . 70°C.

7. A natural quorum sensing inhibitor prepared by the method of any one of claims 1-6.

8. the application of the natural quorum sensing inhibitor prepared by the method of any one of claims 1-6 in regulating the spoilage ability of Shewanella spoilage of aquatic products, it is characterized in that, described natural quorum sensing inhibitory The agent has quorum-sensing inhibitory activity against Bacillus violaceum, and has inhibitory effect on the expression of spoilage factor of Shewanella putrefaction.

9 . The application of the natural quorum sensing inhibitor prepared by the method according to any one of claims 1 to 6 in the low-temperature preservation and storage of aquatic products. 10 .