CN105432766A - Quorum-sensing inhibitor of aquatic Pseudomonas fluorescens and preparation method thereof - Google Patents

Abstract

The invention relates to an aquatic product Pseudomonas fluorescens quorum sensing inhibitor and a preparation method thereof. The quorum sensing inhibitor can be applied to the development of composite preservatives for aquatic products; the preparation method uses paeonol as a raw material, pretreats paeonol, adds an organic solvent, and then performs ultrasonic-assisted vibration extraction, filtering and collecting filtrate, the filtrate is rotary evaporated to obtain a crude extract, then absolute ethanol is added for alcohol precipitation, the filter residue is removed by suction filtration, the filtrate is rotary evaporated to obtain a paeonol extract, and the paeonol extract is freeze-dried to obtain Cortex paeonol extract, the cortex paeonol extract is the quorum sensing inhibitor. The source of raw materials to be used in the present invention is abundant, and the preparation method is simple; the paeonol extract in the present invention can interfere with the quorum sensing system of Pseudomonas fluorescens in aquatic products, block the expression of its putrefaction factors, and can be used for fresh-keeping of aquatic products And the purpose of compound preservative.

Description

Quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products and preparation method thereof

technical field

The invention belongs to the technical field of food storage and preservation, and in particular relates to a quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products and a preparation method thereof.

Background technique

Fresh aquatic products are rich in nutrients and water, the muscle tissue is fragile, the content of soluble protein is high, and the pH value is close to neutral. However, fresh aquatic products are prone to corruption without effective fresh-keeping treatment. The main causes of spoilage of aquatic products include the action of enzymes, microorganisms and fat oxidation. Among them, microorganisms grow and reproduce by decomposing nutrients such as proteins, lipids, and sugars, causing corruption of aquatic products, resulting in huge economic losses and serious public health problems. The spoilage of aquatic products is not caused by all microbial activities, but specific spoilage bacteria play a key role. Pseudomonas is the dominant spoilage bacteria of aquatic products, most of which are Pseudomonas fluorescens. The activity of Pseudomonas fluorescens can produce bad odor, which makes the aquatic products sensory unacceptable. In addition, Pseudomonas fluorescens is also one of the conditional pathogens of fish, often causing red skin disease.

In recent years, the discovery of quorum sensing (QS) system in spoiled food provides a new direction for the study of food spoilage mechanism. QS participates in the regulation of various living habits and various physiological processes of microorganisms, such as bioluminescence, synthesis of antibiotics, formation of biofilm, expression of virulence factors, and the phenomenon of biogroup swimming. Existing studies have shown that QS is involved in the process of food spoilage and regulates the activities of decomposing enzymes such as protein, fat, pectin, and chitin related to food spoilage. Therefore, inhibiting the expression of spoilage factors by interfering with the quorum sensing system of food spoilage bacteria provides a new direction for food preservation.

Blocking the generation of the bacterial QS phenomenon by different means is called quorum sensing inhibition or interference. Quorum sensing inhibitors (Quorum sensing inhibitors, QSI) are mainly divided into two categories: QSI of natural origin and synthetic QSI. However, natural QSI has low concentration and low toxicity, which is a hot research topic at present. At present, the natural QSIs discovered by research at home and abroad include extracts of marine algae, fruits and vegetables, and Chinese herbal plants, such as wakame, broccoli, pineapple, emblical, windmill, and laurel. In addition, curcumin in turmeric, gingerol, andrographolide, apigenin and cinnamaldehyde in ginger also have QSI activity. However, these QSI inhibitory effects mainly focus on the pathogenicity and putrefaction of Pseudomonas aeruginosa, Escherichia coli and Serratia marcescens, etc., and the inhibition of putrefactive Pseudomonas fluorescens has not been reported yet.

Cortex paeonol, as a kind of traditional Chinese medicinal material, is cultivated in many parts of my country. It has the effects of clearing away heat and cooling blood, promoting blood circulation and dispelling blood stasis, and has little toxic and side effects. At present, the patents on paeonol mostly focus on the extraction of its active ingredients and its application in the medical field. However, the application of Dan cortex extract in the aspect of quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products has not been publicly reported so far.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention discloses a quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products and a preparation method thereof. The Chinese herbal medicine Cortex paeonol is used as a raw material, and the cortex paeonol extract is obtained through a series of treatments. After verification, The paeonol extract has an inhibitory effect on the quorum sensing of Pseudomonas fluorescens, by interfering with the quorum sensing system of Pseudomonas fluorescens in aquatic products, blocking the expression of its putrefaction factors, it can be used for fresh-keeping and compounding of aquatic products Development of preservatives.

The present invention is achieved through the following technical solutions:

A method for preparing a quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products. The preparation method uses paeonol as a raw material, adds an organic solvent, and utilizes ultrasonic-assisted oscillation extraction technology to obtain a paeonol extract. The paeonol extract is The substances include gallic acid and paeonol; the paeonol extract is a quorum sensing inhibitor, which can reduce the spoilage ability of Pseudomonas fluorescens, and is used for preservation of aquatic products and compound preservatives Developed; tested, gallic acid and paeonol have inhibitory activity on quorum sensing of Pseudomonas fluorescens.

Further, the preparation method includes the steps of paeonol pretreatment, ultrasonic-assisted vibration extraction, alcohol precipitation and freeze-drying.

Further, the preparation method specifically includes: taking paeonol as a raw material, pretreating paeonol to obtain paeonol powder, adding an organic solvent and performing ultrasonic-assisted vibration extraction, filtering and collecting the filtrate, and carrying out the filtrate Rotary evaporation to obtain a crude extract, then adding absolute ethanol for alcohol precipitation, suction filtration to remove the filter residue, rotary evaporation of the filtrate to obtain a paeonol extract, and freeze-drying the paeonol extract to obtain a paeonol extract.

Further, the specific steps of the preparation method:

(1) Cortex paeonol pretreatment: dry cortex paeonol in a constant temperature incubator at 30-40°C, then pulverize to obtain cortex paeonol powder;

(2) Ultrasonic-assisted vibration extraction: the paeonol powder treated in step (1) is mixed with the organic solution according to a solid-liquid ratio of 1:6-1:10, first ultrasonically extracted for 10-15min, and then extracted by vibration 3-6h, remove the residue by suction filtration, and collect the filtrate; the organic solution is any one of methanol solution, ethanol solution and acetone solution, and the concentration of the methanol solution is 60%-90% (W/V), so The concentration of the ethanol solution is 50%-90% (W/V), and the concentration of the acetone solution is 60%-80% (W/V);

(3) Alcohol precipitation: the filtrate collected in step (2) is subjected to rotary evaporation to obtain a crude extract; absolute ethanol is added to the crude extract for alcohol precipitation, and the alcohol precipitation time is 8-14h, wherein, The volume ratio of the crude extract to the absolute ethanol is 1:5-10; suction filtration is carried out after the alcohol precipitation is completed, the filter residue is removed, and the filtrate is subjected to rotary evaporation to obtain the paeonol extract;

(4) Freeze-drying: freeze-drying the paeonol extract obtained in step (3) to obtain the paeonol extract.

Further, in step (2), the ultrasonic power of the ultrasonic extraction is 200-300W, and the conditions of the oscillation extraction are: temperature 30-40°C, oscillation speed 150-180r/min.

A preparation method of a composite antistaling agent, the composite antistaling agent includes a quorum sensing inhibitor, the preparation method of the composite antistaling agent first judges the freshness of aquatic products, and then determines the composite preservative according to the freshness of the aquatic products The formula of antistaling agent, the concrete steps of the preparation method of described composite antistaling agent are as follows:

1) Comprehensive evaluation of aquatic products: Comprehensive scoring is carried out according to the color, smell, tissue shape, and tissue elasticity of aquatic products to obtain a comprehensive scoring value. The highest score of the comprehensive scoring value is 5 points and the lowest score is 1 point, and according to The comprehensive scoring value divides the aquatic products into three grades of fresh, relatively fresh and stale; since the stale grade aquatic products are no longer suitable for consumption, only the fresh and relatively fresh aquatic products are added with the Said compound antistaling agent to keep fresh;

2) Preparation of composite preservative raw materials and solvents: the raw materials for the composite preservative are: the quorum sensing inhibitor, chitosan and tea polyphenols; the solvent for the composite preservative is: 0.1mol/L acetic acid solution Mixed solution with 0.2mol/L sodium chloride solution;

3) Determination of the compound antistaling agent formula: according to the comprehensive evaluation result of the aquatic product in step (1), when the aquatic product is classified as a fresh grade, the formula of the compound antistaling agent is: by weight ratio, the 10 parts of quorum sensing inhibitor, 1-2 parts of the chitosan, 1-2 parts of the tea polyphenol, 30-60 parts of the composite preservative solvent; when the aquatic product is classified as a relatively fresh grade , the formula of the composite preservative is: by weight, 10 parts of the quorum sensing inhibitor, 4-5 parts of the chitosan, 3-5 parts of the tea polyphenol, and the solvent of the composite preservative 20-30 servings.

Further, in the comprehensive evaluation of aquatic products in step (1), the basis for dividing the aquatic products into three grades of fresh, relatively fresh and not fresh according to the comprehensive scoring value is: the aquatic products classified as fresh grades correspond to The comprehensive score value is 4-5 points, including 4 points; the comprehensive score value corresponding to the fresher grade aquatic products is 3-4 points, including 3 points but not including 4 points; classified as stale grade The comprehensive score value corresponding to the aquatic products is less than 3 points, 3 points are not included.

Beneficial technical effect of the present invention:

(1) The source of raw materials to be used in the present invention is abundant, and the preparation method is simple;

(2) The prepared paeonol extract has quorum sensing inhibitory activity, can effectively interfere with the quorum sensing system of Pseudomonas fluorescens in aquatic products, and can also be used alone or in combination with other fresh-keeping agents to achieve the purpose of fresh-keeping aquatic products;

(3) The quorum sensing inhibitory activity of the paeonol extract of the present invention to Pseudomonas fluorescens is manifested in: the formation of the biofilm of the paeonol extract to Pseudomonas fluorescens, the production of protease, and groups such as clustering and swimming Sensing all have inhibitory effect, and the above-mentioned quorum sensing is a putrefaction factor, therefore, the paeonol extract can reduce the putrefaction ability of Pseudomonas fluorescens.

Description of drawings

Fig. 1 is a flow chart of the steps for the preparation method of a quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products;

Fig. 2 is the total number of bacterial colonies of control group and Danpi treatment group fish meat under different storage time;

Fig. 3 is the pH value of control group and Danpi treatment group fish meat under different storage time;

Fig. 4 is the TBA (thiobarbituric acid) value of control group and paeonol treatment group fish meat under different storage time;

Figure 5 shows the TVB-N (volatile base nitrogen) values of fish meat in the control group and the Danpi treatment group under different storage times.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

On the contrary, the invention covers any alternatives, modifications, equivalent methods and schemes within the spirit and scope of the invention as defined by the claims. Further, in order to make the public have a better understanding of the present invention, some specific details are described in detail in the detailed description of the present invention below. The present invention can be fully understood by those skilled in the art without the description of these detailed parts.

Example 1

Dried paeonol in a constant temperature incubator at 30-40°C, crushed into powder to obtain paeonol powder, mixed the paeonol powder with 75% (W/V) methanol solution according to a material-to-liquid ratio of 1:8, First ultrasonic extraction for 10 minutes, then vibration extraction for 6 hours, the ultrasonic power of the ultrasonic extraction is 200-300W, the conditions of the vibration extraction are: temperature 30-40°C, vibration speed 150-180r/min; then remove the residue by suction filtration, Collect the filtrate; obtain the obtained filtrate by rotary evaporation to obtain a crude extract; add absolute ethanol to the crude extract for alcohol precipitation, and the alcohol precipitation time is 8h, wherein the crude extract and the anhydrous The volume ratio of ethanol is 1:5; suction filtration is carried out after the alcohol precipitation is completed, the filter residue is removed, and the filtrate is subjected to rotary evaporation to obtain a paeonol extract; the paeonol extract is freeze-dried to obtain the paeonol extract ; In the process of preparing the paeonol extract, the present embodiment first utilizes ultrasonic-assisted vibration extraction to improve the extraction rate of the paeonol extract; and the paeonol drying temperature and vibration extraction temperature are all set at 30- 40 ℃, guaranteed the activity of the paeonol extract of preparation, prevented because temperature was too high, the inhibitory effect of the quorum sensing of the paeonol extract material of extraction is affected to the aquatic product Pseudomonas fluorescens; The activity of the prepared paeonol extract is protected.

Resuspend the obtained dry powder of paeonol extract in deionized water, pass through a 0.22 μm sterile filter membrane to sterilize; resuspend the paeonol extract with a mass concentration of 0.39-6.25 mg/mL after sterilization The reporter strain Violet Bacillus CV026 was used for detection, that is, after Violet Bacillus CV026 was activated twice overnight, it was inoculated into fresh LB broth containing 20 μg/mL kanamycin according to the inoculation amount of 2%, and cultured with shaking at 150r/min for 16-18h. Mix it with 100mL of LB medium containing 10μmol/LC4-HSL, pour it on the plate, punch holes with a hole punch with a diameter of 6mm after solidification, add different concentrations of resuspension into the holes, and observe the results after incubation at 30°C for 12 hours. The control group was sterile water. The detection results showed that the paeonol extract could inhibit the production of its purple pigment, indicating that the paeonol extract had quorum sensing inhibitory activity on the reporter strain Violaceum violaceum CV026, and the inhibition rate was up to 89.30%.

Among them, C4-HSL is a signal molecule that can freely enter and exit the cell wall and cell membrane of fungi. When its concentration reaches a certain value, it can initiate the expression of related virulence factors and the formation of biofilms by combining with genes in the bacteria. A key factor in the formation of extracellular biofilms.

To determine the effect of paeonol extract on the biofilm of Pseudomonas fluorescens, that is, after Pseudomonas fluorescens is activated twice, inoculate it into fresh LB broth according to 1% inoculated amount, and add 200 μL to each well of a 96-well plate Bacteria solution and 50 μL of paeonol extract with different concentrations make the final concentration 1.56-6.25mg/mL. The negative control was sterile LB broth. After culturing at 28°C for 48 hours, remove the culture medium in the wells, wash with 250 μL sterilized distilled water for 3-5 times, stain with 250 μL 0.4% crystal violet for 5 min, wash with sterile distilled water for 3 times, and dry at 60°C After oven drying for 10 min, the 96-well plate was taken out, 250 μL of 95% ethanol was added to each well, and after standing for 10 min, the OD value was measured with a microplate reader at a wavelength of 595 nm. The results showed that the paeonol extract could effectively inhibit the biofilm of Pseudomonas fluorescens, and the inhibition rate was up to 71.74%.

To measure the influence of paeonol extract on the production of Pseudomonas fluorescens protease, the skimmed milk agar plate was made with 10% skimmed milk powder and 1.5% agar powder, and sterilized at 105°C for 15 minutes. When the culture medium drops to about 40°C, invert the plate. After the plate is solidified, punch holes with a hole puncher with a diameter of 6 mm, and then add sterile medium and Pseudomonas fluorescens solution activated twice overnight into the air. , Bacterial fluid+inhibitor and bacterial fluid+C4-HSL, cultured at 28°C for 24h, and measured the diameter of the proteolytic circle. The results showed that the paeonol extract could effectively inhibit the production of Pseudomonas fluorescens extracellular protease, and the inhibition rate was up to 59%.

Determine the effect of paeonol extract on the clustering and swimming of Pseudomonas fluorescens, that is, the crude extract of paeonol with different concentrations is resuspended in deionized water, and then filtered through a 0.22 μm filter membrane, and cooled to 40 ℃ clustered agar medium (1% peptone, 0.5% sodium chloride, 0.5% agar and 0.5% glucose) and swimming agar medium (1% tryptone, 0.5% sodium chloride and 0.3% agar) mixed Evenly, pour the plate, so that the final concentration of paeonol extract in the plate is 1.56-6.25mg/mL. Add 5 μL of Pseudomonas fluorescens solution activated twice overnight to the center of the cooled plate, dry it with sterile air, and incubate at a constant temperature of 28°C for 48 hours to observe the migration of Pseudomonas fluorescens. Negative control without crude extract. It was found that with the increase of the concentration of paeonol extract, the diameters of Pseudomonas fluorescens clusters and migration zones gradually decreased, reaching a significant level compared with the control group. The highest inhibition rate of flocking movement was 60.71%, and the inhibition rate of swimming was 40.15%.

Example 2

Dried paeonol in a 30-40°C constant temperature incubator, crushed into powder to obtain paeonol powder, mixed the paeonol powder with 75% (W/V) methanol solution according to the ratio of material to liquid at 1:10, First ultrasonic extraction for 15 minutes, then vibration extraction for 3 hours, the ultrasonic power of the ultrasonic extraction is 200-300W, the conditions of the vibration extraction are: temperature 30-40°C, vibration speed 150-180r/min; then remove the residue by suction filtration, Collect the filtrate; obtain the obtained filtrate by rotary evaporation to obtain a crude extract; add absolute ethanol to the crude extract for alcohol precipitation, and the alcohol precipitation time is 8h, wherein the crude extract and the anhydrous The volume ratio of ethanol is 1:10; suction filtration is carried out after the alcohol precipitation is completed, the filter residue is removed, and the filtrate is subjected to rotary evaporation to obtain a paeonol extract; the paeonol extract is freeze-dried to obtain the paeonol extract .

The obtained dried paeonol extract powder was resuspended in deionized water, and sterilized by passing through a 0.22 μm sterile filter membrane. The paeony cortex extract resuspension with a mass concentration of 6.25 mg/mL after sterilization was detected with Aeromonas hydrophila (isolated from spoilage turbot), and its effect on Aeromonas hydrophila organisms was determined. Effects on envelope formation, protease production, clustering and motility. The result shows that the inhibitory rate of described paeonol extract to Aeromonas hydrophila reaches 54.32%, and the inhibitory rate to the extracellular protease activity of Aeromonas hydrophila reaches 42.3%, and the group movement of Aeromonas hydrophila The inhibition rate of swimming and migration was 46.4% and 22.81%, respectively.

Example 3

The preparation method of the paeonol extract used in this embodiment is the same as in Example 1, and it is used as the fresh-keeping research of aquatic product-turtfish, and the specific research method is as follows:

Fresh turbot was crushed and frozen to death, the fish meat was taken out, washed with sterile water and blotted dry with filter paper, the sterile fish pieces were respectively added to the prepared Pseudomonas fluorescens bacteria solution (control group) and Pseudomonas fluorescens Soak in bacterial solution + paeonol extract for 1min (paeonol extract treatment group), take it out immediately, blot dry with filter paper, sub-package, store at 4°C, measure the total number of bacterial colonies, pH value, TVB-N value and TBA of fish meat value.

It can be seen from Figure 2 that there was no significant difference in the total number of bacterial colonies between the paeonol extract treatment group and the control group, indicating that the paeonol extract did not inhibit the growth of Pseudomonas fluorescens.

After the sudden death of fish, glycogen in the body produces lactic acid through glycolysis, which reduces the pH value of the muscle. As the freshness decreases, the protein is broken down, producing volatile base-based compounds like ammonia and trimethylamine, which are mostly alkaline, and eventually cause the pH of the fish to rise. As can be seen from Figure 3, in the two experimental groups, the pH value presents a trend of first decreasing and then increasing, and the pH value of the paeonol extract treatment group is always lower than that of the control group, thereby illustrating that the paeonol extract can delay microbial metabolism , so that its freshness can be maintained for a long time, and the decomposition of protein in fish meat can be delayed.

TBA (thiobarbituric acid) value: Thiobarbituric acid value (TBAvalue) is an effective method for detecting oil oxidation products, and it has been widely used to determine fatty foods, especially meat and aquatic products. The degree is an important indicator to judge the degree of fat oxidation of fish meat. The larger the TBA value, the more fat oxidation, as can be seen from Figure 4, the TBA value of the paeonol extract treatment group is significantly lower than the control group, so the paeonol extract can inhibit the oxidation of fat in fish.

TVB-N (volatile basic nitrogen) value: volatile basic nitrogen (TVB-N) is the ammonia produced by the decomposition of protein due to the joint action of endogenous enzymes and bacteria in the muscle during storage of animal food And basic nitrogen-containing substances such as amines. The larger the TVB-N value, the more protein degradation, and the more serious the fish meat corruption. It can be seen from Figure 5 that the paeonol extract can effectively inhibit protein degradation.

However, when paeonol extract is used as a preservative, the mechanism of preservation is different from that of traditional preservatives, which mainly achieve the purpose of preservation by inhibiting the growth of bacteria. The inhibitor is to interfere with the production of spoilage factors of spoilage bacteria, reduce its spoilage ability, and achieve the purpose of preservation without affecting the growth of spoilage bacteria. This preservation mechanism can prevent the emergence of drug resistance and is a new type of biological preservation. agent.

Example 4

A method for preparing a composite antistaling agent. The composite antistaling agent is prepared by applying the quorum sensing inhibitor of the Pseudomonas fluorescens described in aquatic products. The preparation method of the composite antistaling agent first judges the freshness of the aquatic product through evaluation. degree, then determine the formula of the composite antistaling agent according to the freshness of the aquatic product, the aquatic product in the present embodiment is mackerel, purchased from the seafood market.

The concrete steps of the preparation method of described composite antistaling agent are as follows:

1) Comprehensive evaluation of Spanish mackerel: Cut the purchased mackerel into pieces for subsequent use; the evaluation is by a specially trained evaluation team (5 people), according to the color and luster and smell of mackerel pieces according to the evaluation table of table 1 mackerel , histological form, tissue elasticity are carried out comprehensive score, obtain comprehensive score value, as shown in table 1, the highest score of described comprehensive score value is divided into 5 minutes and the lowest score is divided into 1 minute, and according to described comprehensive score value, described Spanish mackerel Fish is divided into three grades of fresh, relatively fresh and stale; since the stale grade mackerel is no longer suitable for consumption, so only the fresh and relatively fresh two grades of mackerel are added with the composite antistaling agent for fresh-keeping treatment;

According to the comprehensive score value, the basis for dividing the aquatic product into three grades of fresh, relatively fresh and not fresh is: the comprehensive score value corresponding to the aquatic product classified as fresh grade is 4-5 points, including 4 points The comprehensive score value corresponding to the aquatic products classified as relatively fresh grades is 3-4 points, including 3 points but not including 4 points; the composite score value corresponding to the aquatic products classified as stale grades is lower than 3 points, excluding 3 points.

After the above evaluation, the Spanish Spanish mackerel in this embodiment has a comprehensive score of 3.5 points, which belongs to the fresher grade.

Table 1: Evaluation table for mackerel pieces

2) Preparation of composite preservative raw materials and solvents: the raw materials for the composite preservative are: the quorum sensing inhibitor, chitosan and tea polyphenols; the solvent for the composite preservative is: 0.1mol/L acetic acid solution Mixed solution with 0.2mol/L sodium chloride solution;

Chitosan and tea polyphenols are traditional antistaling agents, which can inhibit the growth of spoilage bacteria; among them, chitosan has good film-forming properties and strong antibacterial properties, and its antibacterial mechanism: small molecule chitosan can wear Penetrate the cell wall and cell membrane into the cell, affecting DNA replication and protein synthesis, while macromolecular chitosan forms a polymer membrane outside the cell, hindering the metabolism of microorganisms, and eventually causing the death of bacteria, and the amino groups in chitosan, etc. The group can chelate with metal ions, inhibit the intake of microelements of microorganisms, reduce the combination of trace elements and nutrients necessary for growth, and affect the metabolism of microorganisms; tea polyphenols have a wide range of antibacterial properties, which are effective against Gram-positive cocci Bacillus, Gram-negative, aerobic cocci and bacilli, facultative anaerobic bacilli, cocci and bacilli all have obvious inhibitory effects. Antibacterial mechanism: The phenolic hydroxyl group in tea polyphenols hydrogen bonds with the amino or carboxyl groups of protein molecules in bacteria, and the benzene ring can also hydrophobically bond with proteins. This multi-point combination leads to the obstruction of microbial metabolism, and tea polyphenols destroy bacteria The structure of the cell membrane.

The selection of the composite preservative is not arbitrary, and the selected solvent of the composite preservative can ensure the full dissolution of chitosan. The dissolution conditions are: stirring with magnetic force for about half an hour, up to 2 hours, without heating, the chitosan is completely dissolved, and there will be no precipitation after standing overnight, and the stability is good.

3) Determination of compound antistaling agent formula: According to the comprehensive evaluation result of the aquatic product in step (1), Spanish mackerel is classified as fresher grade; after the aquatic product dies, its quality change can be divided into 4 stages: stiff → Relief of stiffness → Autolysis → Corruption. The deterioration of aquatic products is mainly due to the deterioration of quality caused by the proliferation of microorganisms, mainly in the autolysis stage and the spoilage stage. Therefore, when the aquatic product is at a relatively fresh level, the aquatic product is already in the primary stage of the autolysis stage or the spoilage stage, and the number of microorganisms in the aquatic product has begun to multiply in large numbers. In order to achieve a good fresh-keeping effect, the composite preservative must be increased The total content of chitosan and tea polyphenols with bactericidal effect in the medium, so that the total content of said chitosan and tea polyphenols is not lower than the content of paeonol extract (the quorum sensing inhibitor); set the present embodiment The formula of the compound antistaling agent raw material described in is: by weight, 10 parts of the quorum sensing inhibitor, 4-5 parts of the chitosan, 3-6 parts of the tea polyphenol, the compound preservative 30 parts of solvent.

However, if the aquatic product is at a relatively fresh level, its quality is in the stage of stiffening and unstiffening, and the microorganisms in the aquatic product have not multiplied in large numbers. In the composite preservative for aquatic products used at this time, the quorum sensing inhibitor should be increased correspondingly. A good fresh-keeping effect can be achieved. Although the inhibitor cannot reduce the number of flora, a good fresh-keeping level can be achieved by keeping the flora at the current level. In this case, the formula of the composite antistaling agent raw material can be set as: by weight, 10 parts of the quorum sensing inhibitor, 4 parts of the chitosan, 3 parts of the tea polyphenol, the Composite preservative solvent 30 parts.

Claims (6)

1. a preparation method of the quorum sensing inhibitor of Pseudomonas fluorescens of aquatic products, it is characterized in that, described preparation method takes paeonol as raw material, adds organic solvent, utilizes ultrasonic assisted vibration extraction technology, obtains paeonol extract , the paeonol extract includes gallic acid and paeonol; the paeonol extract is a quorum sensing inhibitor, which can reduce the spoilage of Pseudomonas fluorescens and is used in aquatic products Development of fresh-keeping and compound preservatives. 2. the preparation method of the quorum sensing inhibitor of a kind of aquatic product Pseudomonas fluorescens according to claim 1, is characterized in that, described preparation method comprises paeonol pretreatment, ultrasonic assisted vibration extraction, alcohol precipitation, freeze-drying step. 3. according to the preparation method of the quorum sensing inhibitor of a kind of aquatic product Pseudomonas fluorescens described in claim 2, it is characterized in that, described preparation method is specifically: take paeonol as raw material, described paeonol is carried out pretreatment treatment to obtain paeonol powder, add an organic solvent and perform ultrasonic-assisted vibration extraction, filter and collect the filtrate, and perform rotary evaporation on the filtrate to obtain a crude extract, then add absolute ethanol for alcohol precipitation, and remove the filter residue by suction filtration. The filtrate was rotary evaporated to obtain a paeonol extract, which was freeze-dried to obtain a paeonol extract. 4. according to the preparation method of the quorum sensing inhibitor of a kind of aquatic product Pseudomonas fluorescens described in claim 3, it is characterized in that, the concrete steps of described preparation method: (1) Pretreatment of paeonol: drying paeonol in a constant temperature incubator at 30-40°C, and then crushing to obtain paeonol powder; (2) Ultrasonic-assisted oscillation extraction: mix the paeonol powder treated in step (1) with the organic solution according to the ratio of solid to liquid 1:6-1:10, first extract ultrasonically for 10-15min, and then extract by oscillation 3-6h, remove the residue by suction filtration, and collect the filtrate; the organic solution is any one of methanol solution, ethanol solution and acetone solution, the concentration of the methanol solution is 60%-90%, and the concentration of the ethanol solution 50%-90%, the concentration of the acetone solution is 60%-80%; (3) Alcohol precipitation: The filtrate collected in step (2) is subjected to rotary evaporation to obtain a crude extract; absolute ethanol is added to the crude extract for alcohol precipitation, and the alcohol precipitation time is 8-14h, wherein, The volume ratio of the crude extract to the absolute ethanol is 1:5-10; suction filtration is carried out after the alcohol precipitation is completed, the filter residue is removed, and the filtrate is subjected to rotary evaporation to obtain the paeonol extract; (4) Freeze-drying: freeze-drying the paeonol extract obtained in step (3) to obtain the paeonol extract. 5. A method for preparing a quorum sensing inhibitor of Pseudomonas fluorescens in aquatic products according to claim 4, characterized in that, in step (2), the ultrasonic power of the ultrasonic extraction is 200-300W, and the The conditions for vibration extraction are: temperature 30-40°C, vibration speed 150-180r/min. 6. A preparation method of a composite antistaling agent, said composite antistaling agent comprising a quorum sensing inhibitor, characterized in that the preparation method of said composite antistaling agent first judges the freshness of aquatic products, and then according to the freshness of said aquatic products, Degree to determine the formula of the composite preservative, the concrete steps of the preparation method of the composite preservative are as follows: 1) Comprehensive evaluation of aquatic products: according to the comprehensive evaluation of the color, smell, tissue shape and tissue elasticity of aquatic products, the aquatic products are divided into three grades: fresh, relatively fresh and not fresh; 2) Preparation of raw materials and solvents for the composite preservative: the raw materials for the composite preservative are: the quorum sensing inhibitor, chitosan and tea polyphenols; the solvent for the composite preservative is: 0.1mol/L acetic acid solution Mixed solution with 0.2mol/L sodium chloride solution; 3) Determination of the compound antistaling agent formula: according to the comprehensive assessment results of the aquatic product in step (1), when the aquatic product is classified as fresh, the formula of the compound antistaling agent is: by weight ratio, the 10 parts of quorum sensing inhibitor, 1-2 parts of the chitosan, 1-2 parts of the tea polyphenol, 30-60 parts of the composite preservative solvent; when the aquatic product is classified as a relatively fresh grade , the formula of the composite preservative is: by weight, 10 parts of the quorum sensing inhibitor, 4-5 parts of the chitosan, 3-5 parts of the tea polyphenol, and the solvent of the composite preservative 20-30 servings.