CS / SA / PPC hydrogel loaded with lactic acid bacteria source quorum sensing quenching enzyme as well as preparation method and application of CS / SA / PPC hydrogel

Abstract

The invention provides a CS / SA / PPC hydrogel loaded with a lactic acid bacteria source quorum sensing quenching enzyme as well as a preparation method and application of the CS / SA / PPC hydrogel. The preparation method comprises the following steps: adding sodium alginate and pachymaran into a chitosan acetic acid aqueous solution to prepare a chitosan / sodium alginate / pachymaran solution; then adding glycerol to prepare a mixed solution; adding a lactic acid bacteria source quorum sensing quenching enzyme into the mixed solution to prepare a hydrogel solution, and finally drying and forming to obtain the hydrogel. The CS / SA / PPC hydrogel loaded with the lactic acid bacteria source quorum sensing quenching enzyme has excellent mechanical properties, can remarkably reduce the TVB-N value, the TBA value and the total number of bacterial colonies in the salmon storage process, delays muscle tissue degradation, prolongs the shelf life of salmon to 12 days or above, and ensures the eating quality and safety of salmon meat.

Description

TECHNICAL FIELD

[0001] The application provides a CS / SA / PPC hydrogel loaded with a lactic acid bacterial source quorum quenching enzyme and a preparation method and application thereof, and belongs to the technical field of hydrogels. BACKGROUND

[0002] As a high-value aquatic product, salmon is rich in protein and unsaturated fatty acids, which are prone to microbial contamination (such as Pseudomonas fluorescens and Hafnia alvei) and lipid oxidation during storage, which seriously limits its shelf life. Existing salmon preservation technologies mainly include the following: Chemical preservation technology: by adding potassium sorbate, nitrite and other chemical preservatives to inhibit microbial growth, but there is a risk of residue, which may affect food safety and consumer health, and cannot effectively delay fish muscle tissue degradation and lipid oxidation, and long-term use may cause consumer concerns.

[0003] Physical preservation technology: such as low-temperature refrigeration, modified atmosphere packaging, etc. Low temperature can slow down microbial reproduction, but cannot completely inhibit the growth of psychrophilic spoilage bacteria (such as Shewanella putrefaciens); modified atmosphere packaging has high cost and strict requirements for packaging sealing, and once the packaging is damaged, the preservation effect will decrease sharply, and it is also difficult to prevent the formation and accumulation of microbial biofilm.

[0004] Natural polymer preservation material: existing preservation films / hydrogels based on natural polysaccharides such as chitosan and sodium alginate have good biocompatibility and biodegradability, but have the following structural and performance defects: Single antibacterial mechanism: only relying on the cationic bactericidal effect of polysaccharides (such as the amino group of chitosan combining with bacterial cell membranes), the inhibition effect on microorganisms that have formed biofilms is poor, because the extracellular polysaccharide and protein matrix of the biofilm will hinder the contact between the active site of the polysaccharide and the bacteria; Uncontrolled release of active substances: if active ingredients (such as essential oils, enzyme preparations) are loaded, the active substances may be "burst released" due to the loose network structure of the hydrogel, which may affect the flavor of the fish meat at an early stage, and the insufficient concentration at a later stage may lead to preservation failure; Mechanical and swelling performance imbalance: single polysaccharide or simple compound hydrogel, such as pure chitosan hydrogel, has poor toughness and is prone to rupture, while sodium alginate hydrogel has a high swelling rate and may collapse due to excessive water absorption during storage, which cannot be attached to the surface of salmon to play a role.

[0005] The disadvantages of the prior art are essentially due to the lack of an integrated structure design of the preservation material for "targeted inhibition of microbial quorum sensing - long-term release of active ingredients - stable adhesion protection": either no quorum sensing inhibition function, unable to break the formation mechanism of microbial biofilm; or insufficient cross-linking degree of hydrogel network, leading to uncontrolled release of active substances and weak mechanical properties, making it difficult to meet the preservation needs of salmon for long periods (more than 10 days) at low temperature. SUMMARY

[0006] Therefore, the application provides a CS / SA / PPC hydrogel loaded with lactic acid bacteria-derived quorum quenching enzyme, a preparation method and applications thereof. The CS / SA / PPC hydrogel loaded with lactic acid bacteria-derived quorum quenching enzyme can significantly reduce the TVB-N value, TBA value and total bacterial count of salmon during storage, delay muscle tissue degradation, extend the shelf life of salmon to more than 12 days, and ensure the food quality and safety of fish meat.

[0007] The application is implemented by using the following technical solutions: A preparation method of a CS / SA / PPC hydrogel loaded with lactic acid bacteria-derived quorum quenching enzyme, comprising the following steps: (1) Dissolve chitosan in an acetic acid aqueous solution to obtain a chitosan acetic acid aqueous solution; (2) Add sodium alginate and Pachyman to the chitosan acetic acid aqueous solution, and fully stir to obtain a chitosan / sodium alginate / Pachyman solution; (3) Add glycerol to the chitosan / sodium alginate / Pachyman solution and continue to stir to obtain a mixed solution; (4) Add lactic acid bacteria-derived quorum quenching enzyme to the mixed solution, and stir uniformly to obtain a hydrogel solution; (5) Pour the hydrogel solution into a mold for drying and molding to obtain the CS / SA / PPC hydrogel loaded with lactic acid bacteria-derived quorum quenching enzyme.

[0008] Preferably, in step (2), the mass concentration of chitosan in the chitosan / sodium alginate / Pachyman solution is 1.5%; the mass concentration of sodium alginate is 1.5%; and the mass concentration of Pachyman is 1.5%. The concentration of acetic acid in the acetic acid aqueous solution is 1%.

[0009] More preferably, in step (3), the mass concentration of glycerol in the mixed solution is 5%.

[0010] Preferably, in step (4), the concentration of lactic acid bacteria-derived quorum quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0011] In some cases, the preparation method of the chitosan / sodium alginate / pachyman solution of the present application can also add a sodium alginate aqueous solution and a pachyman aqueous solution to the chitosan acetic acid aqueous solution, and prepare it after stirring.

[0012] Preferably, the lactic acid bacteria source quorum quenching enzyme loaded is the penicillin V acylase (LpPVA enzyme) synthesized by Lactiplantibacillus plantarum YP4-1-2. The Lactiplantibacillus plantarum YP4-1-2 is a Lactiplantibacillus plantarum isolated from the intestinal tract of fresh paralabrax fish by the previous research group, and is preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC No: 62035, the preservation date November 4, 2021, and the preservation address 5th floor, No. 59, Building, 100, Middle Liangma Street, Guangzhou. The LpPVA enzyme of the present application can be prepared by one of the following methods: Method 1: 50 mL Lactiplantibacillus plantarum YP4-1-2 culture was centrifuged at 10000 rpm for 5 min at 4 ℃, the supernatant was discarded, and the bacterial pellet was washed twice with PBS (pH 6.5) solution, then resuspended in 5 mL PBS (pH 6.5) solution. The bacterial suspension was treated by intermittent ultrasonic disruption (ultrasonic on 1 s and off 2 s; power 320 W) for 5 min in an ice bath, then centrifuged at 10000 rpm for 30 min at 4 ℃. The supernatant was filtered through a 0.22 μm filter membrane, and then 40% ammonium sulfate was added to the supernatant according to the ammonium sulfate fractionation precipitation table. After standing overnight, the precipitate was centrifuged. The precipitate after centrifugation was resuspended with PBS buffer solution. Vacuum freeze-drying was performed for 12 h, and the powder was collected.

[0013] Method 2: The LpPVA enzyme-loaded E. coli was cultured overnight at 20 ℃, the bacterial pellet was collected by centrifugation, resuspended and disrupted with a buffer solution (8 M Urea, 50 mM Tris, 300 mM NaCl, 0.1% TritonX 100, pH8.0) prepared in advance, and the supernatant was collected by centrifugation. The supernatant was incubated on the column filler after equilibrating the Ni-NTA column with Binding Buffer (8 M Urea, 50 mM Tris, 300 mM NaCl, pH 8.0), and the effluent was collected. The column was washed and equilibrated again with Binding Buffer. The protein sample was eluted and collected with Elution Buffer (8 M Urea, 50 mM Tris, 300 mM NaCl, 20 mM Imidazole, pH 8.0) containing 20 mM Imidazole. Vacuum freeze-drying was performed for 12 h, and the powder was collected.

[0014] The application also protects the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared by the above method.

[0015] The application of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source in food preservation, specifically, the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source is used as a preservative film for salmon preservation.

[0016] Compared with the prior art, the application has the following beneficial effects: The CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source has a three-dimensional uniform porous structure, and the 48 h swelling rate is more than 201.4%; the LpPVA enzyme can be released for more than 120 h in a long-acting manner in the pH range of 6.0-8.0. The toughness and tensile fracture strength of the hydrogel prepared in the application are improved by more than 25% compared with pure CS / SA / PPC hydrogel. The hydrogel prepared in the application has excellent antibacterial properties, and the inhibition rate of the hydrogel on the biofilm of Pseudomonas fluorescens and Hafnia alvei is as high as 79.82%; the inhibition rate of the hydrogel on the metabolic activity of the biofilm is 68.70%; the inhibition rates of the hydrogel on extracellular polysaccharide and extracellular protein are 83.11% and 50.51%, respectively; the hydrogel can effectively destroy the formation and accumulation of microbial biofilm, and can be widely used in the field of food preservation. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The SEM image of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared in Example 1 of the application is shown in the figure; Figure 2 The SEM image of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared in Example 2 of the application is shown in the figure; Figure 3 The SEM image of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared in Example 3 of the application is shown in the figure; Figure 4 The SEM image of the CS / SA / PPC hydrogel prepared in Comparative Example 1 is shown in the figure; Figure 5 The SEM image of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared in Comparative Example 2 is shown in the figure; Figure 6 The SEM image of the CS / SA / PPC hydrogel loaded with the quorum quenching enzyme of lactic acid bacteria source prepared in Comparative Example 2 is shown in the figure. DETAILED DESCRIPTION

[0018] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred embodiments of the present application are further described in detail below with examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0019] Example 1 A CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum quenching enzyme is prepared as follows: (1) Chitosan is dissolved in 1% (v / v) acetic acid aqueous solution to obtain a chitosan acetic acid aqueous solution; (2) Sodium alginate and Pachyman are added to the chitosan acetic acid aqueous solution, and after sufficient stirring, a chitosan / sodium alginate / Pachyman solution is obtained, wherein the mass concentration of chitosan in the chitosan / sodium alginate / Pachyman solution is 1.5%; the mass concentration of sodium alginate is 1.5%; and the mass concentration of Pachyman is 1.5%; (3) Glycerol is added to the chitosan / sodium alginate / Pachyman solution for continued stirring to obtain a mixed solution, and the mass concentration of glycerol in the mixed solution is 5%; (4) A lactic acid bacteria-derived quorum quenching enzyme is added to the mixed solution, and after uniform stirring, a hydrogel solution is obtained; (5) The hydrogel solution is poured into a mold for drying and molding to obtain a CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum quenching enzyme, wherein the concentration of the lactic acid bacteria-derived quorum quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0020] Example 2 A CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum quenching enzyme is prepared as follows: (1) Chitosan is dissolved in 1% (v / v) acetic acid aqueous solution to obtain a chitosan acetic acid aqueous solution; (2) Sodium alginate and Pachyman are added to the chitosan acetic acid aqueous solution, and after sufficient stirring, a chitosan / sodium alginate / Pachyman solution is obtained, wherein the mass concentration of chitosan in the chitosan / sodium alginate / Pachyman solution is 3%; the mass concentration of sodium alginate is 1.5%; and the mass concentration of Pachyman is 1.5%; (3) Glycerol is added to the chitosan / sodium alginate / Pachyman solution for continued stirring to obtain a mixed solution, and the mass concentration of glycerol in the mixed solution is 7.5%; (4) A lactic acid bacteria-derived quorum quenching enzyme is added to the mixed solution, and after uniform stirring, a hydrogel solution is obtained; (5) The hydrogel solution is poured into a mold and dried to obtain a CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme, wherein the concentration of lactic acid bacteria quorum sensing quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0021] Example 3 A CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum sensing quenching enzyme is prepared by the following method: (1) Dissolve chitosan in a 1% (v / v) aqueous acetic acid solution to obtain a chitosan-acetic acid aqueous solution; (2) Sodium alginate and Poria cocos polysaccharide are added to the chitosan acetic acid aqueous solution and stirred thoroughly to obtain a chitosan / sodium alginate / Poria cocos polysaccharide solution, wherein the mass concentration of chitosan in the chitosan / sodium alginate / Poria cocos polysaccharide solution is 1.5%; the mass concentration of sodium alginate is 1.5%; and the mass concentration of Poria cocos polysaccharide is 3%. (3) Add glycerol to the chitosan / sodium alginate / Poria cocos polysaccharide solution and continue stirring to obtain a mixed solution, wherein the mass concentration of glycerol in the mixed solution is 2.5%; (4) Add lactic acid bacteria quorum sensing quenching enzyme to the mixed solution, stir evenly to obtain hydrogel solution; (5) The hydrogel solution is poured into a mold and dried to obtain a CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme, wherein the concentration of lactic acid bacteria quorum sensing quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0022] Comparative Example 1 A CS / SA / PPC hydrogel is prepared as follows: (1) Dissolve chitosan in a 1% (v / v) aqueous acetic acid solution to obtain a chitosan-acetic acid aqueous solution; (2) Sodium alginate and Poria cocos polysaccharide are added to the chitosan acetic acid aqueous solution and stirred thoroughly to obtain a chitosan / sodium alginate / Poria cocos polysaccharide solution, wherein the mass concentration of chitosan in the chitosan / sodium alginate / Poria cocos polysaccharide solution is 1.5%; the mass concentration of sodium alginate is 1.5%; and the mass concentration of Poria cocos polysaccharide is 1.5%. (3) Add glycerol to the chitosan / sodium alginate / Poria cocos polysaccharide solution and continue stirring to obtain a mixed solution, wherein the mass concentration of glycerol in the mixed solution is 5%; (4) Pour the mixed solution into a mold for drying and molding to obtain CS / SA / PPC hydrogel.

[0023] Comparative Example 2 A CS / SA hydrogel loaded with a lactic acid bacterial source quorum quenching enzyme is prepared by the following method: (1) Dissolve chitosan in 1% (v / v) acetic acid aqueous solution to obtain a chitosan acetic acid aqueous solution; (2) Add sodium alginate to the chitosan acetic acid aqueous solution, and after fully stirring, obtain a chitosan / sodium alginate solution, wherein the mass concentration of chitosan in the chitosan / sodium alginate solution is 1.5%; and the mass concentration of sodium alginate is 1.5%; (3) Add glycerol to the chitosan / sodium alginate solution and continue to stir to obtain a mixed solution, wherein the mass concentration of glycerol in the mixed solution is 5%; (4) Add a lactic acid bacterial source quorum quenching enzyme to the mixed solution, and after uniform stirring, obtain a hydrogel solution; (5) Pour the hydrogel solution into a mold for drying and molding to obtain a CS / SA hydrogel loaded with a lactic acid bacterial source quorum quenching enzyme, wherein the concentration of the lactic acid bacterial source quorum quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0024] Comparative Example 3 A CS / PPC hydrogel loaded with a lactic acid bacterial source quorum quenching enzyme is prepared by the following method: (1) Dissolve chitosan in 1% (v / v) acetic acid aqueous solution to obtain a chitosan acetic acid aqueous solution; (2) Add pachyman to the chitosan acetic acid aqueous solution, and after fully stirring, obtain a chitosan / pachyman solution, wherein the mass concentration of chitosan in the chitosan / pachyman solution is 1.5%; and the mass concentration of pachyman is 1.5%; (3) Add glycerol to the chitosan / pachyman solution and continue to stir to obtain a mixed solution, wherein the mass concentration of glycerol in the mixed solution is 5%; (4) Add a lactic acid bacterial source quorum quenching enzyme to the mixed solution, and after uniform stirring, obtain a hydrogel solution; (5) Pour the hydrogel solution into a mold for drying and molding to obtain a CS / PPC hydrogel loaded with a lactic acid bacterial source quorum quenching enzyme, wherein the concentration of the lactic acid bacterial source quorum quenching enzyme in the hydrogel solution is 0.1 mg / mL.

[0025] The hydrogels prepared in Examples 1-3 and Comparative Examples 1-3 are cut into film materials with a thickness of 1.0 mm, and the morphological structure, mechanical properties, and fluid characteristics are detected, as follows: I. Morphology and structure: From Figure 1 and Figure 2As can be seen, the hydrogel prepared in Example 1 of the present application has a three-dimensional continuous porous structure, and the pore size is uniform, and the pore size is more delicate compared with the CS / SA / PPC hydrogel without enzyme loading in Comparative Example 1.

[0026] II. Mechanical properties The mechanical properties of the above hydrogel film materials were tested by a texture analyzer, as shown in Table 1.

[0027] Table 1 Tensile strength g Elastance g-sec Example 1 224.40 50.500 Example 2 161.81 29.554 Example 3 146.26 25.287 Comparative Example 1 181.81 39.554 Comparative Example 2 153.67 15.456 Comparative Example 3 196.26 38.287 III. Rheological properties The above hydrogel film materials were detected by a rotary rheometer, as shown in Table 2.

[0028] Table 2 Storage modulus (G') Pa Elastic modulus (G'') Pa Example 1 26240 7320 Example 2 12560 7932 Example 3 13640 6020 Comparative Example 1 25060 7295 Comparative Example 2 20840 6485 Comparative Example 3 19600 7140 As can be seen from Table 2, the hydrogel material prepared in the present application has a storage modulus (G') always greater than the elastic modulus (G'') in the frequency range of 0-15 Hz, showing typical gel solid properties, and the G' and G'' values are significantly higher than those of the CS / SA / PPC hydrogel in Comparative Example 1, indicating that the LpPVA enzyme promotes the crosslinking between polysaccharides and improves the stability of the gel network.

[0029] The hydrogels prepared in Examples 1-3 and Comparative Examples 1-3 were cut into 1.0 mm thick film materials, and the above film materials were used to wrap and preserve salmon, and the specific processing process was as follows: Firstly, fresh salmon was cut into 3x3x3 cm fish pieces, sterilized by ultraviolet lamp (wavelength 254 nm) irradiation for 30 min, and the initial microorganisms on the surface were removed; Then, the same size film material was tightly wrapped on the surface of the salmon pieces, ensuring that the hydrogel film material was completely attached to the fish meat without air bubbles remaining Finally, the wrapped salmon pieces were stored in a 4℃ refrigerated environment, and the TVB-N value, TBA value, total number of colonies (TVC), pH value and muscle tissue morphology of the fish meat were detected regularly. At the same time, the QS inhibition test of Chromobacterium violaceum CV026 was carried out on the salmon after 12 days of storage, and the specific results are shown in Table 3.

[0030] Table 3 Fluorescent Pseudomonas and Hafnia alvei dual species biofilm inhibition % Biofilm metabolic activity inhibition % Extracellular polysaccharide inhibition % Extracellular protein inhibition % Example 1 85.91 72.70 92.73 53.89 Example 2 75.52 55.53 74.56 35.13 Example 3 80.36 59.43 84.73 42.77 Comparative Example 1 8.39 9.82 4.73 4.89 Comparative Example 2 77.09 65.91 72.46 41.69 Comparative Example 3 80.53 64.80 83.72 41.47 After 12 d of storage at 4℃, the TVC, TVB-N value and TBA value of the above salmon were detected, as follows: TVC lg CFU / g TVB-N mg / 100g TBA mg MDA / kg Example 1 6.30 28.62 0.74 Example 2 11.11 39.62 1.81 Example 3 10.01 39.76 1.75 Comparative Example 1 13.30 40.02 1.81 Comparative Example 2 9.51 35.74 1.74 Comparative Example 3 TVC lg CFU / g TVB-N mg / 100g TBA mg MDA / kg 9.61 36.33 1.82 The embodiments described above are only part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

Claims

1. A method for preparing a CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum sensing quenching enzyme, characterized in that, Includes the following steps: (1) Dissolve chitosan in an aqueous acetic acid solution to obtain a chitosan-acetic acid aqueous solution; (2) Sodium alginate and Poria cocos polysaccharide were added to the chitosan acetic acid aqueous solution and stirred thoroughly to obtain a chitosan / sodium alginate / Poria cocos polysaccharide solution; (3) Add glycerol to the chitosan / sodium alginate / Poria cocos polysaccharide solution and continue stirring to obtain a mixed solution; (4) Add lactic acid bacteria quorum sensing quenching enzyme to the mixed solution, stir evenly to obtain hydrogel solution; (5) Pour the hydrogel solution into a mold and dry it to obtain a CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme.

2. The method for preparing CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme as described in claim 1, characterized in that, In step (2), the chitosan / sodium alginate / Poria cocos polysaccharide solution has a chitosan concentration of 1.5%; the sodium alginate concentration is 1.5%; the Poria cocos polysaccharide concentration is 1.5%, and the acetic acid concentration in the acetic acid aqueous solution is 1%.

3. The method for preparing CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme as described in claim 2, characterized in that, The mass concentration of glycerol in the mixed solution in step (3) is 5%.

4. The method for preparing CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme as described in claim 2, characterized in that, The concentration of lactic acid bacteria quorum sensing quenching enzyme in the hydrogel solution described in step (4) is 0.1 mg / mL.

5. The method for preparing CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme as described in claim 1, characterized in that, The loaded lactic acid bacteria quorum sensing quenching enzyme is a penicillin V acylated enzyme synthesized by Lactobacillus plantarum YP4-1-2.

6. A CS / SA / PPC hydrogel loaded with a lactic acid bacteria-derived quorum sensing quenching enzyme, characterized in that, It is prepared using the method described in any one of claims 1 to 5.

7. The application of the CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme as described in claim 6 in food preservation.

8. The application as described in claim 7, characterized in that, The CS / SA / PPC hydrogel loaded with lactic acid bacteria quorum sensing quenching enzyme was used as a preservation film for salmon.

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