A quorum sensing inhibitor and its application in the preservation of sturgeon

Abstract

The present application relates to the low-temperature food preservative and fresh-keeping technical field, and particularly provides a quorum sensing inhibitor and its application in sturgeon preservative and fresh-keeping, specifically, the fresh-keeping method of vacuum packaging sturgeon provided by the present application comprises the following steps: placing the sturgeon into a resveratrol solution with a concentration of 0.1 mM-0.5 mM at 0-4 DEG C, taking out and draining, and vacuum packaging.The present application utilizes the quorum sensing inhibition effect of resveratrol to provide an efficient, green and safe preservative and fresh-keeping technology for low-temperature storage food, especially sturgeon, without adding chemical preservatives.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antiseptic and fresh-keeping technology, and particularly relates to a quorum sensing inhibitor and application thereof in sturgeon antiseptic and fresh-keeping. BACKGROUND

[0002] Sturgeons are rich in nutrition, delicious, high in protein content and extremely low in fat content, and thus occupy an important position in people's daily diet. However, due to the high water content of sturgeons and the large amount of bacteria carried from the water environment of breeding and fishing, sturgeons are prone to spoilage during processing, circulation and storage, which is the biggest challenge for sturgeon cold chain transportation.

[0003] The growth and metabolism of microorganisms are important factors causing sturgeon spoilage. Dominant spoilage bacteria gradually occupy a dominant position and produce spoilage metabolites during food storage, which is the main factor leading to sturgeon spoilage.

[0004] Quorum sensing is a signal communication mechanism by which bacteria secrete signal molecules (also known as autoinducers) to regulate the expression of related genes and adapt to the environment. In recent years, studies have shown that food spoilage is related to quorum sensing. A large amount of accumulation of quorum sensing signal molecules is detected in the spoilage process of some foods such as milk, meat and vegetables, indicating that the quorum sensing system of microorganisms may be involved in the spoilage process.

[0005] Meanwhile, studies have found that enzymes related to food spoilage, such as proteases and lipases, are regulated by quorum sensing, which further confirms that food spoilage is regulated by quorum sensing. A large number of researchers have studied the relationship between the spoilage phenotype of specific microorganisms and quorum sensing by inhibiting or activating the quorum sensing system.

[0006] Currently reported quorum sensing inhibitors such as halogenated furanones and their derivatives have certain mutagenic and carcinogenic effects, and are not suitable for application in food antiseptic and fresh-keeping. Foodborne plant extracts or phytochemicals generally have the characteristics of stable chemical properties, low molecular weight and no harm to the human body, and also do not cause bacterial drug resistance problems, which are an important source of obtaining green, safe and effective quorum sensing inhibitors. SUMMARY

[0007] Resveratrol is a polyphenolic compound found in grapes, which has various beneficial physiological effects such as antioxidant activity, anti-inflammatory effect, cardiovascular protection, etc., and also has good edible safety.

[0008] However, whether resveratrol can inhibit the quorum sensing system of sturgeon spoilage bacteria and its application as a quorum sensing inhibitor in the antiseptic and fresh-keeping of vacuum-packaged fish meat has not been reported.

[0009] The application finds through a bacterial strain population sensing experiment that 0.1-0.3 mM resveratrol has a strong inhibitory effect on the population sensing of spoilage bacteria of vacuum-packed sturgeon.

[0010] Population sensing mainly regulates food spoilage by affecting the activity of bacterial extracellular proteases, the ability to produce volatile basic nitrogen, stress response, trimethylamine production, biogenic amine production, biofilm formation, etc. Based on the high correlation between population sensing and food spoilage, it can be considered that food spoilage is indeed regulated by population sensing, which also provides a new idea for food preservation, that is, using population sensing inhibitors to delay food spoilage deterioration.

[0011] Further, the application first finds that resveratrol in the range of (0.5 mM) has a population sensing inhibitory effect on A. salomonicida. The inhibitory effect of low-concentration resveratrol (0.5 mM) treatment on the microbial growth and sensory and physicochemical property deterioration of refrigerated vacuum-packed sturgeon fillets is similar to that of high-concentration resveratrol (5 mM), and it is speculated that the low-concentration resveratrol (0.5 mM) treatment reduces the spoilage ability of sturgeon spoilage bacteria by inhibiting the population sensing of the sturgeon spoilage bacteria, thereby delaying the quality deterioration of the sturgeon.

[0012] Based on this, the application provides a resveratrol preservative and a preservation method, to solve the short shelf life of vacuum-packed sturgeon at low-temperature cold freshness in the prior art, and to prolong the shelf life of vacuum-packed sturgeon.

[0013] In a first aspect, the application provides a preservation method for vacuum-packed sturgeon, the sturgeon being stored at 0-4 DEG C, the sturgeon being soaked in a preservative and preservation agent composed of 0.1-0.5 mM resveratrol solution for 15-40 min, and then being taken out and drained, and being vacuum-packed.

[0014] The dominant spoilage bacteria of low-temperature stored aquatic products are mainly determined by the packaging method, and are not related to the fish species. At present, aquatic processing products, especially pre-conditioned products, are mainly circulated and sold in the market in the form of vacuum packaging, and the dominant spoilage bacteria of vacuum-packed sturgeon meat are mainly Aeromonas.

[0015] In a second aspect, the application provides a preservative and preservation agent for vacuum-packed sturgeon, the sturgeon being stored at 0-4 DEG C, and the preservative and preservation agent being composed of 0.1-0.5 mM resveratrol solution.

[0016] Further, the preservative and preservation agent provided by the application is composed of 0.3 mM resveratrol solution.

[0017] In the preservative and preservation agent provided by the application, a food-grade resveratrol solvent is used as the solvent of the resveratrol solution; more specifically, a food-grade PEG400 is used as the solvent of the resveratrol solution.

[0018] Based on the above finding of quorum sensing inhibition, in a third aspect, the present application provides a quorum sensing inhibitor of Aeromonas microorganism consisting of 0.1-0.5 mM resveratrol solution. And provides the application of the above quorum sensing inhibitor in the quorum sensing inhibition of spoilage bacteria of aquatic products, the spoilage bacteria being Aeromonas salmonicida.

[0019] Preferably, the quorum sensing inhibitor consists of 0.3 mM resveratrol solution.

[0020] In a fourth aspect, the present application provides the application of the above preservative or the above quorum sensing inhibitor of Aeromonas microorganism in prolonging the shelf life of vacuum-packed fresh sturgeon.

[0021] The beneficial effects of the present application are:

[0022] The present application provides a new method for the preservation of vacuum-packed low-temperature food, especially sturgeon, by using the quorum sensing inhibition of resveratrol at low concentration without using any chemical preservative.

[0023] The method provided by the present application not only can significantly prolong the shelf life of vacuum-packed sturgeon blocks at 4℃, effectively reduce the accumulation of volatile basic nitrogen and thiobarbituric acid during storage, but also can delay the decline of sensory quality such as color and smell of fish meat. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The figure shows the change of total bacteria number during the 4℃ light-avoiding storage of vacuum-packed sturgeon blocks. A is 15% PEG400 (blank control group), B, C and D are 0.5 mM, 1 mM and 5 mM resveratrol treatment groups respectively.

[0026] Figure 2 The figure shows the change of sensory score during the 4℃ light-avoiding storage of vacuum-packed sturgeon blocks. A is 15% PEG400 (blank control group), B, C and D are 0.5 mM, 1 mM and 5 mM resveratrol treatment groups respectively.

[0027] Figure 3TVB-N changes of vacuum-packed sturgeon chunks during 4℃ storage in the dark. Among them, A is 15% PEG400 (blank control group), B, C, D are 0.5 mM, 1 mM, 5 mM resveratrol treatment groups, respectively.

[0028] Figure 4 TBARS changes of vacuum-packed sturgeon chunks during 4℃ storage in the dark. Among them, A is 15% PEG400 (blank control group), B, C, D are 0.5 mM, 1 mM, 5 mM resveratrol treatment groups, respectively.

[0029] Figure 5 Effect of different concentrations of resveratrol on the viable count of spoilage bacteria in the stationary phase.

[0030] Figure 6 Detection of AHLs signal molecule concentration of C.violaceum CV026 treated with different concentrations of resveratrol. Among them, A is the control group; B, C, D are 0.1 mM, 0.2 mM, 0.3 mM resveratrol-treated spoilage bacteria AHLs extract, respectively.

[0031] Figure 7 Effect of resveratrol on the biofilm production ability of A.salomonicida, CK in the figure is the control group, and lowercase letters abcd are the results of significance difference analysis, different letters represent P<0.05.

[0032] Figure 8 Effect of resveratrol on the protease activity of A.salomonicida, CK in the figure is the control group, and lowercase letters abcd are the results of significance difference analysis, different letters represent P<0.05.

[0033] Figure 9 Effect of resveratrol on the lipase activity of A.salomonicida, CK in the figure is the control group, and lowercase letters abcd are the results of significance difference analysis, different letters represent P<0.05. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0035] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0036] The materials, reagents and the like used in the following examples, unless otherwise specified, can be obtained commercially.

[0037] Example 1 Preparation, storage and sampling method of sturgeon fillets treated with resveratrol

[0038] 1. Preparation of resveratrol solution

[0039] Resveratrol was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. with a purity of 99%.

[0040] Resveratrol was prepared into 0.5 mM, 1 mM and 5 mM resveratrol solutions with 15% (w / v) polyethylene glycol 400 (PEG400) solution, and labeled as groups B, C and D, respectively. Another group was set without adding resveratrol as group A (control group), and was fully shaken and mixed.

[0041] 2. Sturgeon fillet treatment

[0042] Fresh sturgeon was washed with sterile water, beheaded, eviscerated, cut into pieces (3 cm*4 cm), skinned and washed again. The sturgeon fillets were randomly divided into four treatment groups: (A) 15% PEG400 (blank control group), (B) 0.5 mM resveratrol, (C) 1 mM resveratrol, (D) 5 mM resveratrol. The fillets were soaked in the respective treatment solutions at 4°C for 30 min, so that the surface of the fillets was in full contact with the soaking solution, and then taken out and drained for 15 s, so that the fillets did not drip water. Then the fillets were placed in vacuum packaging bags for vacuum packaging.

[0043] 3. Storage and sampling method

[0044] The treated samples were immediately taken out for analysis, and the data were recorded as day 0. The remaining fish meat was placed in a covered stainless steel basin and stored in a 4°C refrigerator. Sampling and analysis were performed on days 6, 10, 12, 14 and 18 of storage. Three bags were taken from each treatment group for determination, and the average value was taken.

[0045] Example 2 Analysis of microbial changes in different treatment groups of fish fillets

[0046] 25 g of sturgeon meat was homogenized in 225 mL of physiological saline (0.85%, w / v) for 60 s, and 10-fold gradient dilution was performed. Appropriate dilutions were selected for inoculation on PCA medium plates by the pour plate method, and three gradients were selected for inoculation, with two parallel plates for each gradient. The microbial analysis method was in accordance with GB 4789.2-2010 "Determination of Total Number of Bacteria in Food Microbiological Examination". The detection limit of the microbial detection method in this test was 10 CFU / g.

[0047] Figure 1The effects of resveratrol treatment on the total bacterial count of vacuum-packed sturgeon fillets stored at 4°C in the dark were described. The total bacterial count significantly increased in all groups during storage (P<0.05). The number of days it took for the total bacterial count to increase to 7 log CFU / g was as follows: Group A: 10-12 days; Groups B, C, and D: 14-18 days. On day 12, there was no significant difference in the total bacterial count between Groups D and C (P>0.05), but both were significantly lower than the other treatment groups (P<0.05). On day 14, the total bacterial count in Group D was significantly lower than the other treatment groups (P<0.05).

[0048] This indicates that resveratrol has an inhibitory effect on the growth of microorganisms in sturgeon fillets, and the higher the concentration, the stronger the inhibitory effect. Compared with the control group, the shelf life of fish fillets treated with 0.5mM, 1mM, and 5mM resveratrol was extended by about 4-6 days.

[0049] Example 3: Analysis of changes in the physicochemical and sensory properties of fish fillets treated with different methods

[0050] 1) Analysis of changes in sensory characteristics

[0051] Sensory evaluation of each treated sample was conducted using quantitative descriptive analysis (QDA). An evaluation panel of six trained sensory evaluators scored the samples according to the quality indicators in Table 1. Four scoring levels (0-3) and four parameters (texture, odor, color, and looseness) were used to evaluate the sensory characteristics of the sturgeon fillets.

[0052] Table 1 Sensory Evaluation Criteria for Sturgeon Fillets

[0053]

[0054]

[0055] Changes in sensory scores of sturgeon under different treatment conditions, as follows Figure 2 As shown in the figure. 0 points represent that the fish is completely fresh, and 6 points represent that the fish fillets have reached the sensory acceptable limit. The sensory scores of fish fillets in all treatment groups increased significantly during storage (P<0.05), indicating that the fish fillets were gradually spoiling. With increasing storage time, the fish fillets mainly showed signs of softening, increased water content, becoming less dense, and developing a stronger rancid smell, and the color of the fish fillets changed from white to yellowish.

[0056] The shelf life of sensory evaluation of group A was 10-12 days, and that of groups B, C and D was 14-18 days, which was basically consistent with the shelf life of microbial evaluation. Compared with the shelf life of sensory evaluation of the control group, the shelf life of groups B, C and D was prolonged by about 4 days, indicating that 0.5 mM, 1 mM and 5 mM resveratrol treatment could effectively delay the deterioration of the sensory quality of fish fillets. There was no significant difference in the sensory scores of samples in groups B, C and D during storage (P>0.05), indicating that 0.5 mM low-concentration resveratrol and 5 mM high-concentration resveratrol could achieve the same effect of sensory quality control.

[0057] 2) Change of total volatile basic nitrogen (TVB-N)

[0058] Determination by semi-micro nitrogen determination method in GB / T 5009.44-2003 "Analysis method for hygiene standards of meat and meat products". The brief description is as follows: 10 g of minced meat is weighed in 100 mL of water, shaken uniformly, soaked for 30 min and filtered. FOSS Kjeltec 2300 type Kjeldahl apparatus is used. The conical flask containing 10 mL of absorption liquid and 5 to 6 drops of mixed indicator liquid is placed at the lower end of the condenser tube, and the lower end is inserted into the liquid surface of the absorption liquid. 5 mL of sample liquid is accurately measured in the digestion tube, distillation is carried out, and after 5 min, the distillation is stopped. The absorption liquid is titrated with hydrochloric acid standard solution (0.01 mol / L), and the end point is blue purple. Distilled water is used as a blank.

[0059] Result calculation: the content of volatile basic nitrogen in the sample is calculated according to formula (1):

[0060] Formula (1): X = [(V1-V2) x c x 14] / (m x 5 / 100) x 100;

[0061] In formula (1):

[0062] X—content of volatile basic nitrogen in the sample, unit: milligrams per hundred grams (mg / 100g);

[0063] VI—volume of hydrochloric acid or sulfuric acid standard solution consumed by the sample liquid for determination, unit: milliliters (mL);

[0064] V2—volume of hydrochloric acid or sulfuric acid standard solution consumed by the reagent blank, unit: milliliters (mL);

[0065] c—actual concentration of hydrochloric acid standard solution, unit: moles per liter (mo1 / L);

[0066] 14—mass of nitrogen corresponding to 1.00 ml of hydrochloric acid standard titration solution (c(HCL)=1.000 mol / L), unit: milligrams (mg);

[0067] m—sample mass, unit: grams (g).

[0068] TVB-N value is a common evaluation index of fish freshness. TVB-N value is the basic nitrogenous substances such as amine and ammonia produced by bacteria through the degradation of protein decomposition. The TVB-N changes of sturgeon blocks under different treatment conditions are shown in Figure 3 Group A reached the sensory rejection point on the 12th day, and the TVB-N value was 31.08 mg / 100 g; the TVB-N values of groups B, C and D during storage were all lower than 22 mg / 100 g, which was lower than the upper limit of 35 mg / 100 g of fish spoilage acceptable by the European Commission. From the 12th to the 18th day of storage, the TVB-N values of groups B, C and D were significantly lower than those of group A (P<0.05), and there was no significant difference between groups B, C and D, indicating that 0.5 mM, 1 mM and 5 mM resveratrol treatment could effectively delay the accumulation of TVB-N in fish fillets, and 0.5 mM low-concentration resveratrol and 5 mM high-concentration resveratrol could achieve the same inhibitory effect on TVB-N accumulation.

[0069] 3) Changes in thiobarbituric acid reducing value (TBARS)

[0070] Take 5 g of sample in a mortar and add a small amount of quartz sand to grind it, then add 50 mL of 7.5% trichloroacetic acid (containing 0.1% EDTA), shake for 25 min, centrifuge at 10000 x g for 10 min, take the supernatant and centrifuge at 12000 x g for 1 min, take 5 mL of supernatant and add 5 mL of 0.02 mol / L thiobarbituric acid (TBA) solution, incubate in a 90°C water bath for 30 min. After cooling for 1 h, centrifuge at 10000 x g for 10 min, add 5 mL of chloroform to the supernatant, shake well, and after layering, take the supernatant and record the absorbance values at 532 nm and 600 nm, and perform reagent blank test at the same time. Repeat the experiment twice, and calculate the TBARS value using the following formula.

[0071]

[0072] In the formula: A 532 , A 600 represents the absorbance values at 532 nm and 600 nm; W represents the mass value of the sample.

[0073] TBARS can be used to reflect the degree of fish fat oxidation, and is a common evaluation index of fish freshness. The TBARS values of each treatment group during storage increased significantly (P<0.05), as shown in Figure 4The TBARS values of groups B, C and D were significantly lower than that of the control group A (P < 0.05) on the 12th to 18th day of storage, indicating that the treatment of 0.5 mM, 1 mM and 5 mM resveratrol can effectively delay the oxidation of fish fillets. On the 14th day of storage, the TBARS value of group D was slightly lower than that of groups B and C, but on the 12th and 18th days of storage, the TBARS values of groups B, C and D had no significant difference, indicating that low-concentration resveratrol (0.5 mM) and high-concentration resveratrol (5 mM) can achieve the same inhibitory effect on fish meat fat oxidation.

[0074] Example 4: Study on the quorum sensing inhibition effect of resveratrol

[0075] This example provides the process of exploring the quorum sensing inhibition effect of resveratrol on spoilage bacteria.

[0076] The three types of quorum sensing systems include: AHLs-mediated type I quorum sensing system, AI-2-mediated type II QS system, and AI-3 / epinephrine / norepinephrine-mediated type III QS system.

[0077] In the type I quorum sensing system, the signal molecule AHLs is synthesized under the control of the LuxI homologous gene, and the LuxR homolog is the receptor of the signal molecule. After binding with the signal molecule, it is activated and regulates the transcription of downstream genes. This example studies the effect of resveratrol on the type I quorum sensing system of Aeromonas salomonicida.

[0078] 1. Strain culture

[0079] Aeromonas salomonicida GMT3 is a dominant spoilage bacteria isolated from low-temperature vacuum-packaged spoiled sturgeon, which belongs to Aeromonas salmonicida. GMT3 was inoculated in PCA culture medium (5 g of tryptone, 2.5 g of yeast extract, 1 g of glucose, 1000 mL of distilled water, 1.5% of solid culture medium agar, and the pH was adjusted to 7.0±0.22), and cultured at 30°C with 200 rpm shaking.

[0080] The quorum sensing reporter bacterium Chromobacterium violaceum CV026 was cultured in LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of distilled water, and the pH was adjusted to 7.0, and 1.5% of solid culture medium agar was added), and kanamycin was added to a final concentration of 20 μg / mL during culture. The culture conditions were 30°C and 200 rpm shaking.

[0081] C. violaceum CV026 itself does not produce purple color, but is able to produce purple color when detecting exogenous quorum sensing signal molecules AHLs.

[0082] 2. Resveratrol concentration selection assay

[0083] PCA medium containing different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mM) of resveratrol were prepared, and the activated strain A. salomonicida GMT3 was inoculated into the PCA medium at 1%, and after uniform shaking, the bacterial solution was cultured at 30°C and 200 rpm for 12 h to determine the OD 600 nm At the same time, the bacterial solution in the stable growth phase was diluted by 10 times gradient with sterile normal saline, and three appropriate dilutions were selected for pour culture on PCA agar plates, and three parallel samples were prepared for each gradient, and the viable cell count in the stable growth phase was recorded.

[0084] 3. Effect of resveratrol on AHLs production by A. salomonicida GMT3

[0085] (1) AHL extraction

[0086] PCA medium containing 0.1 mM, 0.2 mM and 0.3 mM resveratrol was used as the treatment group, and PCA medium containing the same volume of DMSO was used as the control group. The activated spoilage bacteria strain was inoculated into 100 mL of PCA medium (inoculation amount was 1%), and each group of culture solution was uniformly shaken and cultured at 30°C and 200 rpm for 12 h, then centrifuged at 4°C and 12000 x g for 15 min, the supernatant was aspirated, and 0.22 μm filter paper was used to filter and sterilize. The filtrate was extracted with an equal volume of ethyl acetate (containing 0.1% formic acid). Shake well for 30 s, stand for 30 min, and completely separate the organic and aqueous phases. The upper organic phase was retained.

[0087] The bacterial fermentation supernatant was extracted three times, and the organic phases of the three extractions were combined and transferred to a rotary evaporator to evaporate most of the liquid. The rotary evaporation conditions were set to water bath temperature 30°C and rotation speed 60 r / min. Then the remaining liquid was blown dry with nitrogen, and after the liquid was completely evaporated, an appropriate amount of chromatographic grade methanol was added for dissolution, and the sample was transferred to a HPLC brown sample vial and stored at -20°C for standby.

[0088] (2) C. violaceum CV026 detects AHLs

[0089] When the sterilized LB medium is cooled to about 50℃, kanamycin is added to a final concentration of 20 μg / mL, and shaken to mix. After the LB medium is cooled and solidified, 10 μL of activated overnight C. violaceum CV026 reporter bacteria are spotted on an LB plate, a total of 4 bacterial samples are spotted, and 2 μL of AHL signal molecule extract from different resveratrol treatment groups is spotted 1 cm from the end of each bacterial sample. Then the plate is placed in a constant temperature incubator at 30℃ for 24 h, the plate is observed for changes, and photographs are taken for record.

[0090] 4. Experimental results

[0091] (1) Effect of resveratrol on the growth of sturgeon spoilage bacteria

[0092] The number of viable bacteria of strain GMT3 in the stationary phase was determined under different resveratrol treatment concentrations. Compared with the growth of the control group, the number of viable bacteria of strain GMT3 in the stationary phase was not significantly affected by 0.1, 0.2, and 0.3 mM resveratrol treatment (P > 0.05). The number of viable bacteria of strain GMT3 (log CFU / mL = 6.37) and OD 600 nm = 0.03 in the 0.5 mM resveratrol treatment group were not significantly different from the initial inoculum (log CFU / mL = 6.18) and OD 600 nm = 0.03 (P > 0.05), and the minimum inhibitory concentration (MIC) of resveratrol for strain GMT3 was 0.5 mM. The results are shown in Figure 5 .

[0093] The number of viable bacteria in the 0.3 mM and below resveratrol treatment groups was not significantly different from the control group (P > 0.05), so the resveratrol treatment concentration for the subsequent test was selected to be 0.1, 0.2, and 0.3 mM, which did not affect the normal growth of strain GMT3, to determine the quorum sensing inhibition effect.

[0094] (2) Quorum sensing inhibition effect of resveratrol on sturgeon spoilage bacteria

[0095] The effect of resveratrol treatment on the production of quorum sensing signal molecules AHLs by spoilage bacteria was detected using the quorum sensing reporter bacteria C. violaceum CV026. As shown in Figure 6 , the 0.1 mM, 0.2 mM, and 0.3 mM resveratrol treatment groups could not induce C. violaceum CV026 to produce purple Figure 6 , while the control group could induce C. violaceum CV026 to produce purple Figure 6A), i.e. AHLs in 0.1-0.3 mM resveratrol treatment group were lower than the minimum threshold to initiate C. violaceum CV026 quorum sensing, which indicated that 0.1-0.3 mM resveratrol treatment could significantly inhibit the production of quorum sensing signal molecules AHLs of sturgeon spoilage bacteria, combined with the fact that resveratrol had no significant effect on the growth of sturgeon spoilage bacteria in the range of 0.1-0.3 mM, it was concluded that resveratrol had an inhibitory effect on the quorum sensing system of sturgeon spoilage bacteria.

[0096] Example 5 Effect of resveratrol on spoilage-related phenotypes of A. salomonicida

[0097] In this example, A. salomonicida GMT3 was used as the research object to study the effect of resveratrol on spoilage-related phenotypes of A. salomonicida, including the effect of resveratrol on biofilm, protease activity, and lipase activity of A. salomonicida.

[0098] 1. Biofilm quantification by crystal violet staining (CV)

[0099] Biofilm is composed of prokaryotic or eukaryotic cells and extracellular matrix produced and accumulated by cells, such as proteins, polysaccharides, humic substances, extracellular DNA, and intercellular communication signal molecules, and forms a three-dimensional structure of membrane polymers. Microorganisms control the production of biofilm through quorum sensing, and promote spoilage by regulating the formation of biofilm, thereby increasing the risk of food safety and endangering human health.

[0100] Crystal violet staining is a method that uses crystal violet dye to bind to biofilm to achieve biofilm quantification. Currently, crystal violet staining is still the most commonly used biofilm quantification technique in microtiter plate assays.

[0101] Overnight cultured strain GMT3 was added to sterile 12-well plates at an inoculum of 1%, 2 mL of PCA medium containing different concentrations of resveratrol (0, 0.1, 0.2, and 0.3 mM) was added to each well, and labeled, with the 0 mM group as the control group, and three replicates for each treatment group. Incubate at 30°C for 48 h. The 12-well plates after incubation were gently washed with sterile ultrapure water for 3 times, air-dried at room temperature, added with 1 mL of 0.1% crystal violet staining for 30 min, the crystal violet was aspirated, washed with sterile ultrapure water until the eluent was colorless, air-dried at room temperature, and added with 2 mL of anhydrous ethanol per well for 20 min. Take 200 μL of ethanol crystal violet solution from each well to a 96-well plate and measure the absorbance at 595 nm.

[0102] The results showed that 0.1, 0.2, and 0.3 mM resveratrol treatment could significantly reduce (P<0.05) the biofilm production of strain GMT3Figure 7 ), the inhibition rates were 19.19%, 35.86% and 45.96% respectively compared with the control group. Many studies reported that bacterial biofilm was regulated by quorum sensing, indicating that resveratrol might inhibit the biofilm formation of the strain by inhibiting the quorum sensing system.

[0103] 2. Detection of protease activity

[0104] The activated strain GMT3 was inoculated in PCA medium containing different final concentrations (0, 0.1, 0.2 and 0.3 mM) of resveratrol at an inoculation amount of 1%, and cultured at 30°C with 200 rpm for 12 h, and the OD600 nm absorbance value of the bacterial solution was measured. The culture solution was centrifuged at 10000 x g for 10 min, and the supernatant was aspirated and filtered with a 0.22 μm inorganic filter membrane. 150 μL of the filtered supernatant was mixed with 250 μL of reaction solution (1% (w / v) casein, dissolved in 50 mmol / L, pH 7.5 sodium phosphate buffer), and incubated in a 37°C water bath for 3 h. 1.2 mL of trichloroacetic acid was added to terminate the reaction, and the mixture was allowed to stand at room temperature for 15 min, followed by centrifugation at 10000 x g for 10 min. 600 μL of the supernatant was mixed with 750 μL of 1 mol / L NaOH solution, and the absorbance at 440 nm was measured. The protease activity was determined by the ratio of (OD 440 nm ) and (OD 600 nm ).

[0105] Bacterial proteases secreted into the external environment hydrolyze proteins to provide nutrition for cell proliferation, leading to food spoilage. The effect of resveratrol on the protease activity of strain GMT3 is shown in Figure 8 . The protease activities of the groups treated with 0.1, 0.2 and 0.3 mM resveratrol were significantly lower than that of the control group (P<0.05), i.e., resveratrol could significantly inhibit the protease activity of strain GMT3. Some studies reported that the protease activity of bacteria was regulated by quorum sensing. Since 0.1-0.3 mM resveratrol had no significant effect on the growth of strain GMT3 Figure 6 , it was inferred that resveratrol might inhibit the protease activity of the strain by inhibiting the quorum sensing system.

[0106] 3. Detection of lipase activity

[0107] According to the detection instruction of Shanghai Tongwei lipase kit, the specific method is as follows: (1) dilution of standard: a standard curve (y=0.0031x+0.0556, R 2(2) sample loading: 50 μL per well, six parallels; (3) incubation: 37 °C for 30 min after sealing with sealing film; (4) washing: carefully remove the sealing film, discard the liquid, shake dry, add washing solution to each well, stand for 30 s, discard, repeat for 5 times, and pat dry; (5) enzyme loading: 50 μL per well except for blank wells; (6) incubation: operation as in (3); (7) washing: operation as in (5); (8) color development: add color developing agent A 50 μL to each well, then add color developing agent B 50 μL, shake gently, color development at 37 °C for 10 min in dark; (9) termination: add 50 μL of termination solution to each well to terminate the reaction (at this time, blue color turns to yellow color); (10) determination: zero the blank wells, measure the absorbance of each well at 450 nm. The determination should be performed within 15 min after the termination solution is added. The corresponding lipase concentration of the sample is calculated according to the standard curve.

[0108] Many microorganisms can produce more than one extracellular lipase, which can hydrolyze different long-chain fatty acids, especially short-chain fatty acids, leading to food spoilage. The lipase activity of strain GMT3 in the 0.1, 0.2 and 0.3 mM resveratrol treatment groups was significantly lower than that in the control group (P < 0.05), and the lipase activity of strain GMT3 was reduced to 55.96%, 40.53% and 40.49% of the control group, respectively. Figure 9 ).

[0109] Since 0.1-0.3 mM resveratrol had no significant effect on the growth of strain GMT3 ( Figure 6 ) while significantly inhibiting the lipase activity of strain GMT3, it was inferred that resveratrol might inhibit the lipase activity of strain GMT3 by inhibiting its quorum sensing system.

[0110] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a quorum sensing inhibitor in the manufacture of a sturgeon spoilage bacteria quorum sensing inhibition product, the spoilage bacteria being Aeromonas salmonicida Aeromonas salmonicida ; The quorum sensing inhibitor consists of a solution of 0.1-0.5 mM resveratrol.

2. A bacterium of the species Aeromonas salmonicida Aeromonas salmonicida Use of a quorum sensing inhibitor in extending the shelf life of vacuum-packed fresh sturgeon fish under low-temperature preservation conditions. The quorum sensing inhibitor consists of a solution of 0.1-0.5 mM resveratrol.

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