Sargassum polyphenol epsilon-polylysine hydrochloride composite preservative and application thereof

Abstract

The invention relates to the technical field of refrigeration and preservation of aquatic products, and discloses a sargassum polyphenol epsilon-polylysine hydrochloride composite preservative and application thereof.The preservative is prepared by mixing sargassum polyphenol SP, epsilon-polylysine hydrochloride PLH and distilled water according to the ratio of 20 mg: 0.5 mg: 1 L. The SP is obtained through salting sargassum lyticum pretreatment, ethanol extraction and extraction and purification. The compound has a synergistic antibacterial effect (FICI = 0.06) on vibrio parahaemolyticus, and also can inhibit and remove a biofilm of the vibrio parahaemolyticus. The preservative is especially suitable for refrigeration and preservation of sea bass fillets at 4 DEG C, the sea bass fillets are soaked for 5 min according to the proportion of 1: 1 g / mL after specific pretreatment, the shelf life of the sea bass fillets can be prolonged from 6 d to 8 d, the increase of the total number of bacterial colonies can be inhibited, the TVB-N content can be reduced, it is ensured that the sea bass fillets still meet the edible standard after being refrigerated for 8 d, safety is high, cost is controllable, and the preservative is suitable for industrial application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquatic product cold storage and preservation, and particularly relates to a Sargassum muticum polyphenol epsilon-polylysine hydrochloride compound preservative and application. BACKGROUND

[0002] Fish products, as an important source of high-quality protein, essential omega-3 polyunsaturated fatty acids (such as EPA and DHA), and trace nutrients such as iodine, selenium, and vitamin D in the global dietary structure, occupy an irreplaceable position in human dietary nutrition balance. Among them, sea bass (Lateolabrax japonicus) is one of the fish varieties with great economic value in the world, and has a wide audience in the consumer market due to its rich protein content and unsaturated fatty acids.

[0003] However, the physicochemical properties of fish products make them prone to quality deterioration and spoilage after production: high moisture content, rich degradable nutrients, and near-neutral pH value create ideal conditions for the rapid proliferation of spoilage microorganisms, lipid oxidation reactions, and autolytic degradation processes. For sea bass, the rich nutritional ingredients in its muscle tissue become an excellent substrate for microbial reproduction, and the massive growth of microorganisms is the core inducement for the deterioration of sea bass quality, the shortening of shelf life, and the outbreak of foodborne diseases.

[0004] In the current field of fish product preservation technology, there are mainly three types of preservation methods: physical, chemical, and biological, but all have significant limitations:

[0005] Physical preservation technology: represented by low-temperature preservation, although widely used in aquatic product storage, ice crystals formed in low-temperature environments can damage the cell structure of aquatic products, leading to a decrease in the physicochemical properties of fish meat after thawing, loss of nutritional substances, and difficulty in balancing preservation effect and product quality;

[0006] Chemical preservation technology: by adding chemical preservatives to inhibit the growth of spoilage bacteria, although it has the advantages of high efficiency and convenient operation, drug residues, bacterial resistance, and the difficulty of controlling the amount of addition pose a major threat to food safety, severely restricting its application and promotion;

[0007] Biological preservation technology: using natural or microbially derived preservatives can avoid texture degradation caused by low-temperature storage, better preserve the natural flavor, texture, and nutritional ingredients of fish products, and reduce the environmental pollution risk caused by the use of chemicals, becoming a research hotspot in recent years.

[0008] Bio-preservatives can be divided into four categories according to their sources: plant (such as essential oils, polyphenols and other plant extracts), animal (such as chitosan, lactoferrin), microorganism (such as nisin, ε-polylysine hydrochloride) and enzyme (such as lysozyme), which have the characteristics of high safety and strong specificity, and are often used in combination to achieve enhanced efficacy, with broad application prospects. Among the microorganisms related to the preservation of sea bass, Vibrio parahaemolyticus is the main pathogen that causes seafood-related gastrointestinal illness worldwide. Its contamination is directly related to the consumption of raw or undercooked seafood, which can cause severe abdominal pain, watery diarrhea, nausea and vomiting, fever and other symptoms, and even sepsis in severe cases, posing a greater threat to people with weak immunity. More importantly, Vibrio parahaemolyticus can easily form a biofilm in food processing environments (such as equipment surfaces, pipelines, sinks), which is a bacterial community structure wrapped in a polysaccharide matrix secreted by itself. This structure can significantly enhance its resistance to environmental stress such as drying, temperature changes, disinfectants, and other environmental stressors. Conventional cleaning and disinfection procedures are difficult to completely remove, and it becomes a persistent and persistent source of contamination and cross-contamination breeding ground in the food processing chain. Therefore, effectively controlling the formation and removal of Vibrio parahaemolyticus biofilm is a key link to ensure the safety of sea bass and other aquatic products and extend their shelf life.

[0009] ε-polylysine hydrochloride (PLH) is a microbial-derived preservative obtained by purification from the controlled fermentation broth of Streptomyces diastatochromogenes. It has good inhibitory effect on a variety of foodborne pathogens, including Vibrio parahaemolyticus, and has been approved for use in food preservation by many countries. However, PLH has relatively weak antioxidant properties, and fish meat is prone to lipid oxidation during storage, leading to flavor deterioration and quality decline. This limitation restricts the independent application of PLH in fish product preservation.

[0010] Brown algae, as a plant resource with both medicinal and edible value, is rich in various bioactive components and has antioxidant, antibacterial, and radiation protection effects. Sargassum thunbergii is a major source of marine phenolic substances due to its abundance and easy accessibility. Sargassum polyphenol (SP), as the core active ingredient of Sargassum thunbergii, is mainly composed of polyphenols, brown algae tannins, flavonoids, and phytosterols. It exhibits significant antioxidant properties in various antioxidant evaluation systems and has good antibacterial ability against the pathogenic Vibrio parahaemolyticus. Given that SP has both antioxidant and antibacterial activities, and PLH has insufficient antioxidant properties, scientific combination of the two can achieve synergistic effects, enhance antibacterial effects, make up for the shortcomings of PLH's antioxidant properties, reduce the use of single preservatives, and reduce costs and potential risks. This is of great significance in solving the preservation problems of sea bass and other aquatic products and promoting the industrial application of bio-preservation technology. SUMMARY

[0011] The present application aims to solve the technical problems in the prior art that physical preservation easily damages the cell structure of fish meat, leading to quality degradation, chemical preservation has the risk of drug residues and bacterial resistance, and single biological preservative has short board (such as weak antioxidant activity of epsilon-polylysine hydrochloride and low antibacterial efficiency of sargassum polyphenol alone), and the prior art cannot effectively inhibit vibrio parahaemolyticus and its biofilm to cause easy spoilage of aquatic products, short shelf life and food safety risk.

[0012] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0013] A sargassum polyphenol epsilon-polylysine hydrochloride composite preservative is prepared by mixing sargassum polyphenol SP, epsilon-polylysine hydrochloride PLH and distilled water in a mass-volume ratio of 20 mg:0.5 mg:1 L.

[0014] The specific ratio is verified by experiments to realize efficient fusion of the two active ingredients while ensuring the preservation effect, and to avoid cost increase or potential quality impact caused by excessive use of a single component.

[0015] As a preferred embodiment, the preparation method of the sargassum polyphenol (SP) comprises the following steps:

[0016] S1, take salted Sargassum sp. and remove epiphytes and salt particles, rinse with clean water, dry at 40-50℃ for 12-16h, crush and pass through a 40-mesh sieve to obtain sargassum powder;

[0017] S2, add 56% ethanol by volume to the sargassum powder at a solid-liquid ratio of 1:40 g / mL, and extract in a 58℃ hot water bath for 5h;

[0018] S3, after extraction, use a Buchner funnel to filter the filtrate, concentrate and dry under reduced pressure at 45℃ to obtain a crude extract;

[0019] S4, dissolve the crude extract with 500mL distilled water, extract with equal volume of dichloromethane and ethyl acetate three times in turn, collect the ethyl acetate extract phase, and concentrate and dry under reduced pressure at 45℃ to obtain the sargassum polyphenol SP.

[0020] This preparation process can maximize the retention of active ingredients of sargassum polyphenol by precisely controlling temperature, time, solid-liquid ratio and extraction solvent sequence, and reduce impurity residues, providing a high-quality raw material basis for the antibacterial and antioxidant performance of the composite preservative.

[0021] As a preferred, the Sargassum polyphenol SP and the ε-polylysine hydrochloride PLH in the composite preservative have a synergistic antibacterial effect on Vibrio parahaemolyticus. The minimum inhibitory concentration (MIC) of the Sargassum polyphenol SP and the ε-polylysine hydrochloride PLH against Vibrio parahaemolyticus is determined by the micro-broth dilution method, and the MIC is 625 μg / mL and 15.63 μg / mL, respectively.

[0022] The fractional inhibitory concentration index (FICI) of the Sargassum polyphenol SP and the ε-polylysine hydrochloride PLH against Vibrio parahaemolyticus is determined by the micro-broth dilution method. When the concentration of the Sargassum polyphenol SP is 1 / 32 MIC and the concentration of the ε-polylysine hydrochloride PLH is 1 / 32 MIC, the FICI value is 0.06, and the synergistic antibacterial effect is the most significant. This synergistic effect not only improves the inhibition efficiency on Vibrio parahaemolyticus, but also reduces the use concentration of the single preservative, reduces the potential impact on the flavor and nutrition of aquatic products, and reduces the application cost.

[0023] As a preferred, the composite preservative has inhibitory and cleaning activities on Vibrio parahaemolyticus biofilm. When the Sargassum polyphenol SP with a concentration of 1 / 32 MIC is combined with the ε-polylysine hydrochloride PLH with a concentration of 1 / 32 MIC, the inhibition rate on the formation of Vibrio parahaemolyticus biofilm is 54.70±1.84%, and the cleaning rate on the mature biofilm is 30.44±0.98%. The inhibitory activity and the cleaning activity are significantly stronger than the activity when the Sargassum polyphenol SP or the ε-polylysine hydrochloride PLH is used alone (P<0.05). This characteristic enables the composite preservative to reduce the stubborn pollution of Vibrio parahaemolyticus in the food processing environment from the source, and further reduces the risk of corruption and cross-contamination of aquatic products caused by biofilm.

[0024] The application of a Sargassum polyphenol ε-polylysine hydrochloride composite preservative in the preservation of aquatic products.

[0025] This application expands the application scenarios of natural composite preservatives in the field of aquatic product processing, and provides a feasible technical solution for solving the preservation problem of perishable aquatic products such as sea bass.

[0026] As a preferred, the aquatic product is sea bass, and the specific application object is sea bass fillets, and the application scenario is the cold storage preservation of sea bass fillets. The specific application design for sea bass fillets not only considers the meat characteristics and corruption rules of sea bass, but also effectively balances the preservation effect and product quality retention through the adaptation of the cold storage scenario.

[0027] As a preferred, the application includes the following pretreatment steps: cleaning the ice-fresh sea bass, peeling, removing the head and internal organs, taking the back muscle, rinsing with sterile water, wiping the fillets with 75% ethanol, placing the fillets in a sterile tray with floating ice for ultraviolet disinfection for 20 minutes, and obtaining the fillets to be preserved.

[0028] The application method is to immerse the sea bass fillets in the composite preservative according to the ratio of 1:1 g / mL of mass to preservative volume, slowly stir during 5 min, and then drain and store in cold storage.

[0029] The standardized pretreatment and application steps ensure the standardization and repeatability of the operation, facilitate the large-scale popularization and application in aquatic product processing enterprises, and reduce the influence of human operation differences on the preservation effect.

[0030] As preferred, if the sea bass fillets to be preserved need to be inoculated with Vibrio parahaemolyticus, the inoculation method is as follows: the Vibrio parahaemolyticus is inoculated into MH broth, cultured at 28°C to the logarithmic phase, diluted to 10 6 CFU / mL with 0.85% sterile normal saline, and then the sea bass fillets to be preserved are immersed in the Vibrio parahaemolyticus suspension for 10 s and then drained. The inoculation method can simulate the possible Vibrio parahaemolyticus pollution scene in actual production, and provides a scientific experimental basis for verifying the preservation capacity of the composite preservative under the pollution risk.

[0031] As preferred, the temperature of cold storage is 4°C; the shelf life of the sea bass fillets treated by the composite preservative is prolonged from 6 d to 8 d under the cold storage condition of 4°C. The prolongation of the shelf life directly improves the market circulation period and commodity value of the sea bass fillets, and provides technical support for aquatic product enterprises to reduce post-production losses and ensure product supply stability.

[0032] As preferred, the composite preservative can effectively inhibit the increase of the total viable count (TVC) of the sea bass fillets during cold storage, and the total viable count of the sea bass fillets is still lower than the edible limit of 6.0 lgCFU / g after 8 d of cold storage at 4°C; and can significantly reduce the content of total volatile basic nitrogen (TVB-N), and the TVB-N value is 27.89 mgN / 100g after 8 d of cold storage, which meets the edible standard (≤30 mgN / 100g). The control of the two key indicators directly proves that the composite preservative can effectively delay the corruption process of the sea bass fillets, and ensure that the sea bass fillets still meet the food safety and edible quality requirements in the prolonged shelf life.

[0033] Compared with the prior art, the technical effects and advantages of the present application are as follows:

[0034] The present application effectively makes up for the functional shortcomings of the existing single preservative, and realizes the complementary advantages. Although the ε-polylysine salt has a certain inhibitory effect on pathogenic bacteria such as Vibrio parahaemolyticus, the antioxidant performance is weak, and it is difficult to cope with the lipid oxidation problem in fish meat storage; the sargassum polypenol has dual activities of antibiosis and antioxidation, and the two form a synergistic effect after compounding, which not only strengthens the antibiosis effect, but also makes up for the deficiency of the ε-polylysine salt in the aspect of antioxidation, and solves the problem of single function and limited effect of the single preservative.

[0035] The application can efficiently inhibit Vibrio parahaemolyticus and its biofilm, significantly reduce the foodborne safety risk and corruption risk of aquatic products. Vibrio parahaemolyticus is the main pathogenic bacteria causing corruption of sea bass and other aquatic products and causing foodborne diseases, and the biofilm formed by the bacteria is difficult to be removed by conventional techniques, while the composite preservative of the application can not only inhibit the growth of the bacteria, but also effectively inhibit the formation of the biofilm and remove the mature biofilm, thus reducing the pollution and corruption inducement from the source and ensuring the safety of aquatic products.

[0036] The application can better retain the quality of aquatic products and prolong the shelf life while improving the preservation effect. Compared with the physical preservation which easily damages the cell structure of fish meat and leads to loss of nutrients, and the chemical preservation which may affect the flavor, the composite preservative of the application can slow down the increase of the total number of colonies of fish meat, abnormal changes of pH value, accumulation of volatile base nitrogen and fat oxidation products, maintain the good sensory quality of fish meat, and greatly prolong the cold storage shelf life of sea bass and other aquatic products.

[0037] The application has high safety and controllable cost, and is more suitable for industrial application. The main components of the composite preservative, Sargassum polyphenol and epsilon-polylysine hydrochloride, are of natural origin, avoiding the risk of drug residue that may be brought by chemical preservatives, and being safer to use. At the same time, the synergistic effect of the two components can reduce the use amount of a single component, reduce the raw material consumption and the use cost under the premise of ensuring the preservation effect, and provide favorable conditions for the practical popularization of aquatic product preservation. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure is a graph of the change of total viable count (TVC) of sea bass fillets during cold storage according to the application;

[0039] Figure 2 Figure is a graph of the change of pH value of sea bass fillets during cold storage according to the application;

[0040] Figure 3 Figure is a graph of the change of volatile base nitrogen content (TVB-N) of sea bass fillets during cold storage according to the application;

[0041] Figure 4 Figure is a graph of the change of thiobarbituric acid reactant (TBARS) of sea bass fillets during cold storage according to the application;

[0042] Figure 5 Figure is a graph of the change of sensory evaluation of sea bass fillets during cold storage according to the application. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0044] The following will be described in detail Figures 1 to 5 The present application is further described in detail;

[0045] I. Preparation of experimental materials

[0046] (I) Raw materials and reagents

[0047] Core raw materials: salted Gracilaria lemaneiformis (need to remove epiphytes and surface salt particles in advance); ε-polylysine hydrochloride (PLH, food grade, purity meets industry application standards); fresh sea bass (body weight preferably 500-700 g, freshness meets subsequent experimental requirements).

[0048] Strains: Vibrio parahaemolyticus (standard strain): stored in a-80℃ refrigerator, and needs to be activated before use.

[0049] Culture medium and reagents: MH broth medium, plate count agar medium (PCA); dichloromethane, ethyl acetate, 56% ethanol (analytical pure); 0.85% sterile normal saline, phosphate buffer (PBS, pH 7.2-7.4); 0.1% crystal violet solution, 33% glacial acetic acid; TBA reagent (formula: 0.375 g 2-thiobarbituric acid and 15 g trichloroacetic acid are dissolved in 100 mL 0.25 mol / L HCl solution); volatile nitrogen (TVB-N) rapid test kit; dimethyl sulfoxide (DMSO, analytical pure); experimental water is distilled water (meets laboratory primary water standard).

[0050] (II) Instruments and equipment

[0051] Constant temperature drying oven: used for drying Gracilaria lemaneiformis, temperature control range 40-50℃, accuracy ±1℃;

[0052] Pulverizer and 40-mesh sieve: used for pulverizing and sieving Gracilaria lemaneiformis, to ensure uniform particle size of fine powder;

[0053] Constant temperature water bath: used for temperature control during extraction process, temperature range 0-100℃, accuracy ±0.5℃;

[0054] Büchner funnel and suction filtration device: used for filtering the extraction liquid, with a vacuum pump;

[0055] Rotary evaporator and vacuum drying oven: used for concentration and drying under reduced pressure, concentration temperature 45℃, vacuum degree ≤-0.09MPa;

[0056] 96-well cell culture plate and microplate reader: the microplate reader can measure the absorbance at 412nm, 532nm, 600nm, etc., with an accuracy of ±0.001A;

[0057] Sterile operating table: used for sterile operation to ensure no contamination during the experiment;

[0058] Constant temperature incubator: used for microbial culture, temperature control at 37℃ (Vibrio parahaemolyticus culture), 28℃ (Vibrio parahaemolyticus logarithmic phase culture), accuracy ±1℃;

[0059] pH meter: used to measure the pH value of the sample, with an accuracy of ±0.01pH;

[0060] High-speed centrifuge: used for centrifugal separation, maximum speed ≥10000r / min, centrifugal temperature can be controlled;

[0061] Homogenizer: used for homogenization of sea bass fillets, speed adjustable to ensure uniform homogenization.

[0062] II. Preparation of Sargassum polyphenol (SP)

[0063] (I) Pretreatment

[0064] Take the salted Gracilaria dura, manually remove the attached small marine organisms, impurities and surface residual salt particles, and rinse with flowing fresh water for 3 times (5 minutes each time) until the rinsing water has no salt taste. Put the washed Gracilaria dura into a constant temperature drying oven and dry at 40-50℃ for 12-16h (turn over every 4h to ensure uniform drying). Grind the dried Gracilaria dura with a grinder, pass the grinding product through a 40-mesh sieve, collect the Gracilaria dura powder that passes through the sieve, and seal it in a desiccator for later use.

[0065] (II) Extraction

[0066] On the sterile operating table, accurately weigh 100g of Gracilaria dura powder and put it into a 500mL round-bottom flask. According to the solid-liquid ratio of 1:40g / mL (i.e. the mass of Gracilaria dura powder to the volume of ethanol), add 4000mL of 56% ethanol solution to the flask. Place the flask in a 58℃ constant temperature water bath and assemble a condenser tube for reflux extraction. The extraction time is 5h (stir with a sterile glass rod every 30min to ensure that the powder and ethanol are in full contact).

[0067] (III) Purification

[0068] After the extraction is completed, cool the mixture to room temperature, filter it with a Buchner funnel (2 layers of qualitative filter paper), and collect the filtrate in a sterile conical flask.

[0069] The filtrate was transferred to a rotary evaporator and concentrated under reduced pressure at 45°C and a vacuum degree of ≤-0.09 MPa until the volume of the filtrate was concentrated to 1 / 10 of the original volume (about 400 mL).

[0070] The concentrated solution was transferred to a vacuum drying oven and dried at 45°C for 8-10 h to obtain the Sargassum polyphenol crude extract.

[0071] The crude extract was completely redissolved with 500 mL distilled water and transferred to a separatory funnel:

[0072] An equal volume (500 mL) of dichloromethane was first added, shaken for 10 min (shaking frequency 30 times / min), and allowed to separate into layers for 30 min, after which the lower dichloromethane phase (containing impurities) was discarded, and the upper water phase was retained; this operation was repeated 3 times.

[0073] An equal volume (500 mL) of ethyl acetate was added to the upper water phase, and similarly shaken for 10 min and allowed to separate into layers for 30 min, after which the upper ethyl acetate phase (containing Sargassum polyphenol) was collected; this operation was repeated 3 times, and all the ethyl acetate extraction phases were combined.

[0074] The combined ethyl acetate extraction phases were again concentrated under reduced pressure at 45°C using a rotary evaporator until no liquid drops were obtained, and then dried in a vacuum drying oven at 45°C for 6-8 h to obtain refined Sargassum polyphenol (SP), which was stored in a -20°C refrigerator in the dark and sealed.

[0075] III. Determination of key parameters of the composite preservative (MIC and FICI)

[0076] (1) Determination of the minimum inhibitory concentration (MIC) of SP and PLH against Vibrio parahaemolyticus by micro-broth dilution method

[0077] Bacterial liquid activation and preparation: Vibrio parahaemolyticus was taken out from a -80°C refrigerator, a single colony was picked up with a sterile inoculation loop, and inoculated into 150 mL MH broth medium, which was incubated at 37°C for 12 h with shaking (rotation speed 180 r / min); 1 mL of the primary bacterial liquid was inoculated into fresh 150 mL MH broth, which was incubated at 37°C for 6 h to reach the logarithmic growth phase; the logarithmic phase bacterial liquid was diluted with MH broth, and the concentration was corrected to 10 6

[0078] Sample solution preparation: MH broth was used as the solvent, and a series of concentrations of SP test solution (concentration range 10000 μg / mL-78.125 μg / mL) and PLH test solution (concentration range 250 μg / mL-1.953 μg / mL) were prepared by the double dilution method, with 3 parallel samples for each concentration; at the same time, MH broth containing an equal volume of DMSO (final concentration ≤1%) was prepared as a solvent control.​

[0079] MIC determination operation: In a sterile 96-well plate, 190 μL of activated bacteria suspension was added to each well, and 10 μL of different concentrations of SP or PLH test solution was added respectively, and mixed gently. Set up negative control wells (200 μL of MH broth) and positive control wells (190 μL of bacteria suspension + 10 μL of DMSO), and each control well was set in triplicate. After incubation at 37°C for 24 h, the absorbance at 600 nm was measured by an enzyme-labeled instrument, and the naked eye observation (clear for sterile growth, turbid for bacterial growth) was combined, and the "lowest sample concentration without bacterial growth" was defined as MIC.

[0080] Determination result: The MIC of SP against Vibrio parahaemolyticus was 625 μg / mL, and the MIC of PLH against Vibrio parahaemolyticus was 15.63 μg / mL.

[0081] (ii) Determination of fractional inhibitory concentration index (FICI) of SP and PLH against Vibrio parahaemolyticus by micro chessboard dilution method

[0082] Solution preparation: Prepare 10 6 mL of Vibrio parahaemolyticus suspension; based on MIC, prepare SP solution (gradient: MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC, 1 / 64MIC) and PLH solution (gradient: MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC) with serial dilution concentrations in MH broth.

[0083] Chessboard inoculation and culture: In a sterile 96-well plate, 50 μL of bacteria suspension was added to each well first; 25 μL of different concentrations of SP solution was added horizontally (transversely), and 25 μL of different concentrations of PLH solution was added vertically (longitudinally), so that the final volume in the well was 100 μL. Set up negative control wells (100 μL of MH broth) and positive control wells (50 μL of bacteria suspension + 50 μL of distilled water), and each combination and control was set in triplicate. Incubate at 37°C for 24 h.

[0084] FICI calculation and result determination:

[0085] Determine the combined MIC according to the MIC determination method, and calculate according to the formula: FICI = (MIC sp when combined / MIC sp when alone) + (MIC PLH when combined / MIC PLH when alone).

[0086] Determination standard: FICI < 0.5 is synergistic effect, 0.5 < FICI ≤ 1 is additive effect, 1 < FICI ≤ 2 is irrelevant effect, and FICI > 2 is antagonistic effect, and the growth of bacteria in the well is irrelevant effect.

[0087] The results of the determination are as follows: when the concentration of SP is 1 / 32 MIC (≈19.53 μg / mL) and the concentration of PLH is 1 / 32 MIC (≈0.488 μg / mL), the FICI value is 0.06, and the synergistic antibacterial effect is most significant. The concentration combination is the core component concentration of the composite preservative, and the specific results are shown in Table 1.

[0088] Table 1 FICI determination results table

[0089]

[0090] Note: "+" represents long bacteria, and "-" represents no bacteria.

[0091] Four, preparation of sargassum polyphenol / ε-polylysine hydrochloride composite preservative

[0092] According to the concentration at the minimum FICI (1 / 32 MIC SP ≈ 20 μg / mL, 1 / 32 MIC PLH ≈ 0.5 μg / mL), the amount is calculated based on 1 L of distilled water as the solvent: 20 mg of SP and 0.5 mg of PLH are accurately weighed and placed in a 1 L volumetric flask. An appropriate amount of distilled water is added, and ultrasonic dissolution is performed for 5 min (power 300 W, frequency 40 kHz). After ensuring complete dissolution, the volume is adjusted to 1 L with distilled water, and the mixture is shaken to obtain the SP / PLH composite preservative. The preservative is divided into sterile reagent bottles and stored at 4°C (storage period not exceeding 7 days).

[0093] Five, determination of the activity of the composite preservative against Vibrio parahaemolyticus biofilm (crystal violet staining method)

[0094] The crystal violet staining method is used to determine the inhibitory activity of SP, PLH, and SP / PLH against Vibrio parahaemolyticus biofilm formation and the clearance activity of mature biofilm.

[0095] (I) Determination of inhibitory activity against biofilm formation

[0096] Solution preparation: prepare 10 6 μL / mL of Vibrio parahaemolyticus suspension; prepare three test samples: 1 / 32 MIC SP solution, 1 / 32 MIC PLH solution, and 1 / 32 MIC SP + 1 / 32 MIC PLH mixture (i.e., composite preservative, diluted with DMSO to a final concentration of 100 times, ensuring that the final concentration of DMSO is ≤1%).

[0097] Inoculation and culture: 190 μL of bacterial suspension is added to each well of a 96-well plate, and then 10 μL of each of the three test samples is added, with three replicates for each. Positive control wells (190 μL of bacterial suspension + 10 μL of DMSO) and negative control wells (200 μL of MH broth) are also prepared. The plate is incubated at 37°C for 24 h.

[0098] Staining and determination: Discard the culture solution, rinse with 200 μL PBS twice, dry at room temperature for 30 min; add 200 μL 0.1% crystal violet to each well for 30 min, discard the dye and rinse with PBS once, dry again; add 200 μL 33% glacial acetic acid to dissolve for 30 min, measure the absorbance at 600 nm (absorbance of sample As, absorbance of positive control Ac) by microplate reader.

[0099] Inhibition rate calculation: according to the formula inhibition rate Y = [(A c –A S ) / A c ] x 100%.

[0100] (II) Mature biofilm removal activity determination

[0101] Mature biofilm preparation: add 190 μL 10 6 mL bacterial suspension to each well of a 96-well plate, and incubate at 37°C for 24 h to form mature biofilm.

[0102] Sample treatment and culture: discard the culture solution, rinse with PBS twice; add 190 μL MH broth + 10 μL sample to be tested to each well, and incubate at 37°C for 24 h; the subsequent staining, determination and removal rate calculation steps are the same as those in the inhibition activity determination.

[0103] Experimental results: 1 / 32 MIC SP + 1 / 32 MIC PLH had significantly stronger inhibition activity on Vibrio parahaemolyticus biofilm formation and removal activity on mature biofilm than the two substances alone (P < 0.05), and the specific results are shown in Table 2 below.

[0104] Table 2 Biofilm activity determination results

[0105]

[0106] Note: Different letters represent significant differences (P < 0.05).

[0107] Six, application of composite preservative in cold storage and preservation of sea bass fillets

[0108] (I) Sea bass fillet treatment

[0109] After cleaning the ice-fresh sea bass, remove the skin, head and internal organs, take the dorsal muscle, rinse it with sterile water, wipe the fillet with 75% ethanol, and place the fillet in a sterile tray with floating ice for ultraviolet disinfection for 20 minutes to obtain the sea bass fillet to be preserved.

[0110] If Vibrio parahaemolyticus needs to be inoculated: inoculate Vibrio parahaemolyticus into MH broth, incubate at 28°C to the logarithmic phase, and dilute to 10 6CFU / mL, soak the sea bass fillets to be preserved in the Vibrio parahaemolyticus suspension for 10 seconds and then drain.

[0111] Grouping: Sea bass fillets were randomly divided into two groups: a control group (soaked in sterile water) and a treatment group (soaked in a Sargassum polyphenol / ε-polylysine hydrochloride composite preservative). The treatment group was soaked in the composite preservative for 5 minutes at a ratio of sea bass fillet weight to preservative volume of 1:1 g / mL, with slow stirring during the soaking process; the control group was soaked in distilled water for the same amount of time.

[0112] Refrigerated storage: After draining the two groups of fish fillets, they were placed in specific sealing bags, the gas inside the bags was slightly removed, and the bags were labeled. They were then refrigerated at 4°C for 10 days. Every 2 days, one sea bass fillet from the control group and the treatment group was randomly selected to test the total bacterial count (TVC), pH value, volatile basic nitrogen (TVB-N), and thiobarbituric acid reactants (TBARS) content, and sensory evaluation was performed.

[0113] (II) Total bacterial count (TVC) determination

[0114] Test Method: The method was performed according to GB4789.2-2022 with slight modifications. In a sterile operating room, 5g of sea bass fillets were weighed and homogenized in 40mL of 0.85% sterile physiological saline for 2 minutes. The homogenate was then serially diluted 10-fold. 0.1mL of each appropriate dilution was spread onto approximately 20mL of sterile plate counting agar (PCA). Each dilution was repeated three times. The plates were incubated upside down at 37℃ for 24 hours, and colony counting was performed. Plates with a total colony count range of 30–300 were used for counting. The results are expressed as lgN (N is CFU / g of sample).

[0115] Results Analysis: Microbial detection data can be used as an evaluation benchmark for the refrigeration and preservation effect of sea bass fillets. When the total bacterial count of sea bass fillets is ≥6.01g CFU / g, they lose their commercial value and exceed the edible range. Figure 1 As shown, the total bacterial count in both groups of samples naturally increased with accumulated refrigeration time. After 6 days of refrigeration, the total bacterial count in the control group was 6.311g CFU / g, exceeding the upper limit of the standard; after 8 days of refrigeration, the total bacterial count in the treatment group was 5.471g CFU / g, still below the critical value, and only exceeded the limit on the 10th day. This indicates that the SP / PLH compound preservative significantly inhibited the increase in total bacterial count and extended the shelf life of sea bass fillets by at least 2 days.

[0116] (III) pH Measurement

[0117] Test method: Refer to the method of GB5009.237-2016. Accurately weigh 5 g of minced fish and place it in a 250 mL beaker, add 40 mL of neutral distilled water, stir well and stand for 30 min (shake constantly during this period), filter and measure the pH of the filtrate with a pH meter, repeat 3 times for each sample.

[0118] Result analysis: pH value can be used as a basis for judging the freshness of aquatic products. Fresh fish pH value is 6.5-6.8, sub-fresh fish is 6.9-7.0, and ≥7.0 indicates deterioration. As shown in Figure 2 , the pH value of sea bass fillets first decreased slightly and then increased as the refrigeration time increased. On the 4th day, the pH of both groups decreased to the lowest (due to the glycolysis of glycogen to produce organic acids and phosphoric acid); after 6 days of refrigeration, the pH value of the control group was 7.09, indicating that it had deteriorated; the pH of the treated group was still lower than 7.0 on the 8th day, and did not exceed the sub-fresh fish limit. This shows that the compound preservative can inhibit the decomposition of protein and amino acid and the reproduction of bacteria in refrigerated sea bass fillets to a certain extent.

[0119] (Four) Determination of volatile base nitrogen content (TVB-N value)

[0120] Test method: Use volatile base nitrogen content rapid test kit. Weigh 2 g of sea bass fillets into a plastic bowl, add 20 mL of sterile water, soak for 15 min, then take 1 mL of sample treatment liquid into a colorimetric tube, add 2 drops of TVB-N detection liquid, mix well, observe the color development within 2 min, measure the absorbance at 412 nm, and calculate the TVB-N value according to the ammonia standard curve (Y = 0.0267X - 0.1081, R 2 = 0.9967). The result is expressed in mgN / 100g of sample.

[0121] Result analysis: According to GB2733-2015 "National Food Safety Standard Fresh and Frozen Animal Aquatic Products", sea bass and other seawater fish TVB-N value <15 mgN / 100g is superior product, ≤30 mgN / 100g is qualified product. As shown in Figure 3 , the TVB-N value of both groups increased with storage time, but the treated group was always lower than the control group (P <0.05). The TVB-N value of the control group was 30.58 mgN / 100g after 6 days of storage, which exceeded the limit; the TVB-N value of the treated group was 23.20 mgN / 100g after 6 days of storage, and 27.89 mgN / 100g on the 8th day, which was still a qualified product. This shows that the compound preservative can kill part of the microorganisms and inactivate the enzymes, thereby inhibiting the production of nitrogen-containing substances and the increase of TVB-N content.

[0122] (Five) Determination of thiobarbituric acid reactant content (TBARS value)

[0123] Test method: 1 g of minced fish was accurately weighed into a test tube, and 5 mL of TBA reagent (0.375 g of 2-thiobarbituric acid and 15 g of trichloroacetic acid dissolved in 100 mL of 0.25 mol / L HC1 solution) was added. The test tube with a plug was placed in a boiling water bath for 10 min, and after color development, it was cooled to room temperature, centrifuged at 10,000 r / min for 10 min, and the supernatant was measured at 532 nm. Distilled water was used as a blank, and the absorbance value was multiplied by 2.77 to obtain the content of malondialdehyde (mg MDA / kg) in each kg of sample.

[0124] Result analysis: TBARS value is an important indicator for evaluating the degree of fat oxidation, and malondialdehyde (MDA) is the main degradation product of fat oxidation. As shown in Figure 4 , the TBARS content of both groups increased naturally during cold storage, but was always below the threshold value of 1 mg / kg. During 0-4 d of cold storage, the TBARS of both groups increased smoothly (the fish fat was relatively stable); the TBARS of the control group increased sharply from the 6th day, and the TBARS of the treated group was still significantly lower than that of the control group. This indicates that the composite preservative can effectively inhibit the oxidation and deterioration of fish fat and maintain the freshness of fish.

[0125] (VI) Sensory evaluation

[0126] Evaluation method: Based on GB2733-2015 "National Food Safety Standard Fresh and Frozen Animal Aquatic Products", the evaluation method was modified. A sensory evaluation team consisting of 5 people (2 men and 3 women, aged 25-45) was trained to have a unified standard, and scored from muscle tissue, color, and odor, and the average of the total score of the three scores was taken. The score range is 0 (completely rotten) to 10 (extremely fresh), with 5 as the acceptable limit, and the specific evaluation criteria are shown in Table 3.

[0127] Table 3 Sensory evaluation criteria for sea bass fillets

[0128]

[0129] Evaluation process: The sea bass fillets to be tested were placed in white porcelain dishes, numbered, and randomly presented to the evaluation team. The evaluators observed the color, judged the muscle elasticity by pressing, and smelled the odor in a disturbance-free environment, and independently scored to avoid communication.

[0130] Result statistics and analysis: The score sheets were collected, the average score of each sample was calculated, and the average of the total score was taken as the final sensory score. The results showed that at 2 d of cold storage, the sensory scores of both groups were ≥8; at 6 d of cold storage, the total score of the control group decreased to 5 (the critical value), and the total score of the treated group was still ≥6; at 8 d of cold storage, the total score of the control group was <5 (unacceptable), and the total score of the treated group was still ≥5; until the 10th day, the total score of the treated group was still <5. As shown in Figure 5As shown, with the extension of refrigeration time, the sensory scores of both groups decreased naturally, but the treatment group could better maintain the sensory quality of the sea bass fillets.

[0131] Seven, experimental conclusion

[0132] The sea tangle polyphenol / ε-polylysine hydrochloride complex preservative prepared in this example can significantly inhibit the growth and biofilm formation of Vibrio parahaemolyticus through the synergistic antibacterial effect (FICI=0.06) of SP and PLH; when applied to the preservation of sea bass fillets at 4°C, it can effectively slow down the increase of total bacterial count, pH value, TVB-N and TBARS content, maintain good sensory quality, and extend the shelf life of sea bass fillets from 6 days of the control group to 8 days. Both SP and PLH are of natural origin, which can reduce the dosage of a single preservative and the use cost, has high safety, and is suitable for practical preservation of aquatic products.

[0133] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing embodiments of the present application are described in detail, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement, within the spirit and principles of the present application, any modification, equivalent replacement, improvement, etc., should be included in the scope of protection of the present application.

Claims

1. A Sargassum polyphenol ε-polylysine hydrochloride composite preservative, characterized in that, It is prepared by mixing Sargassum polyphenols SP, ε-polylysine hydrochloride PLH and distilled water in a mass-volume ratio of 20 mg: 0.5 mg: 1 L.

2. The Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 1, characterized in that: The preparation method of the Sargassum polyphenol SP includes the following steps: S1. Take salted Sargassum fusiforme, remove epiphytes and salt particles, rinse with fresh water, dry at 40-50℃ for 12-16 hours, pulverize and pass through a 40-mesh sieve to obtain Sargassum fusiforme fine powder. S2. Add 56% ethanol (by volume) to the Sargassum powder at a material-to-liquid ratio of 1:40 g / mL, and extract in a hot water bath at 58°C for 5 hours. S3. After extraction, filter using a Buchner funnel, collect the filtrate, concentrate under reduced pressure at 45°C and dry to obtain the crude extract. S4. The crude extract was redissolved in 500 mL of distilled water and extracted three times with equal volumes of dichloromethane and ethyl acetate, respectively. The ethyl acetate extract was collected, concentrated under reduced pressure at 45 °C, and dried to obtain the Sargassum polyphenol SP.

3. The Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 1, characterized in that: The Sargassum polyphenols SP and ε-polylysine hydrochloride PLH in the compound preservative have a synergistic antibacterial effect against Vibrio parahaemolyticus. The minimum inhibitory concentrations (MICs) of Sargassum polyphenols SP and ε-polylysine hydrochloride PLH against Vibrio parahaemolyticus were determined to be 625 μg / mL and 15.63 μg / mL, respectively, by the micro-broth dilution method. The fractional inhibition concentration index (FICI) of Sargassum polyphenol SP and ε-polylysine hydrochloride PLH against Vibrio parahaemolyticus was determined by the micro-checkerboard dilution method. The FICI value was 0.06 when the concentration of Sargassum polyphenol SP was 1 / 32MIC and the concentration of ε-polylysine hydrochloride PLH was 1 / 32MIC.

4. The Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 1, characterized in that: The compound preservative has inhibitory and scavenging activity against Vibrio parahaemolyticus biofilm. When Sargassum polyphenol SP with a concentration of 1 / 32 MIC is combined with ε-polylysine hydrochloride PLH with a concentration of 1 / 32 MIC, the inhibition rate of Vibrio parahaemolyticus biofilm formation is 54.70±1.84%, and the scavenging rate of mature biofilm is 30.44±0.98%.

5. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to any one of claims 1-4 in the preservation of aquatic products.

6. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 5 in the preservation of aquatic products, characterized in that: The aquatic product in question is sea bass, specifically sea bass fillets, and the application scenario is the refrigeration and preservation of sea bass fillets.

7. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 6 in the preservation of aquatic products, characterized in that: The application includes the following pretreatment steps: After cleaning the chilled sea bass, remove the skin, head, and internal organs, take the back muscle, rinse it with sterile water, wipe the fish fillets with 75% ethanol, and place the fish fillets in a sterile tray with floating ice for ultraviolet disinfection for 20 minutes to obtain sea bass fillets to be preserved. The application method is to soak sea bass fillets in the compound preservative at a ratio of 1:1 g / mL (mass to volume of preservative) for 5 minutes, stirring slowly during the process, draining and then refrigerating.

8. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 7 in the preservation of aquatic products, characterized in that: If sea bass fillets are to be preserved, they must first be inoculated with Vibrio parahaemolyticus. The inoculation method is as follows: Inoculate Vibrio parahaemolyticus into MH broth, incubate at 28°C until the logarithmic phase, and then dilute with 0.85% sterile physiological saline to a final concentration of 10. 6 CFU / mL, soak the sea bass fillets to be preserved in the Vibrio parahaemolyticus suspension for 10 seconds and then drain.

9. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 7 in the preservation of aquatic products, characterized in that: The refrigeration temperature is 4℃; the shelf life of sea bass fillets treated with the compound preservative is extended from 6 days to 8 days under refrigeration conditions at 4℃.

10. The application of the Sargassum polyphenol ε-polylysine hydrochloride composite preservative according to claim 1 in the preservation of aquatic products, characterized in that: The compound preservative can effectively inhibit the increase of total bacterial count (TVC) in sea bass fillets during refrigeration. After refrigeration at 4℃ for 8 days, the total bacterial count of the sea bass fillets is still below the edible limit of 6.01 g CFU / g. It can also significantly reduce the content of volatile basic nitrogen (TVB-N). After refrigeration for 8 days, the TVB-N value is 27.89 mgN / 100g, which meets the edible standard (≤30 mgN / 100g).

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