An oat glucan-phenolic acid graft copolymer, a preparation method thereof, and application as an antioxidant and antibacterial liquid

Abstract

The present invention provides an oat glucan-phenolic acid graft copolymer, a preparation method thereof, and an application as an antioxidant antibacterial solution, belonging to the technical field of food preservation. Mainly using phenolic acid and oat glucan as reaction substrates, through the N,N'-carbonyldiimidazole method, under the condition of assisted vacuum ultrasound, an oat glucan-phenolic acid graft copolymer is prepared. The synthesis process of the present invention is simple, and vacuum ultrasound can effectively shorten the synthesis time and improve the synthesis efficiency. At the same time, the oat glucan-phenolic acid graft copolymer prepared by the N,N'-carbonyldiimidazole method has good antioxidant and antibacterial activities, and is used for the preservation of chilled meat, which can effectively extend the shelf life of chilled meat and retain its nutritional components.

Description

Technical Field

[0001] The present invention relates to an oat glucan-phenolic acid graft copolymer for fresh chilled meat preservation, a preparation method thereof, and an application as an antioxidant antibacterial liquid. The present invention belongs to the technical field of food preservation. Background Art

[0002] Fresh chilled meat, also known as chilled meat, refers to meat whose central temperature is reduced to (0°C - 4°C) without ice crystal formation in an environment below 0°C. It is widely popular due to its affordable price and rich nutrition. However, it is easily contaminated by microorganisms during slaughter, processing, and sales, and its quality such as color and odor is also prone to change, resulting in problems such as a short shelf life, which restricts the further development of the fresh chilled meat industry. Among them, fresh chilled rabbit meat, with its advantages of high protein, high lysine, high digestibility, and low fat, low cholesterol, and low calories, has seen a surge in market demand. To meet consumers' high requirements for freshness, safety, and nutrition, preservation technology has become a key challenge.

[0003] Phenolic acid is a natural antioxidant with rich sources and is often used for fresh chilled meat preservation. However, its application is often limited by factors such as poor water solubility, too fast metabolism rate, low bioavailability, and poor stability. In order to make it better play its physiological effects, be transported to the target site, and improve its stability and bioavailability during food processing and storage, phenolic acid can be grafted onto sugar molecules with good biocompatibility and biodegradability through a graft copolymerization reaction. For example, CN117567664A discloses a chitosan oligosaccharide phenolic acid derivative, a preparation method thereof, and an application. A graft copolymerization reaction is carried out between phenolic acid and chitosan oligosaccharide. The phenolic acid is gallic acid or p-coumaric acid, the activation time of the phenolic acid is 12 h, and chitosan oligosaccharide is added for a graft reaction for 12 h. After the reaction, a chitosan oligosaccharide phenolic acid derivative is obtained, which can obtain a chitosan oligosaccharide phenolic acid derivative that can inhibit fungi. However, the graft reaction time of this technology is relatively long and the production efficiency is low.

[0004] Oat glucan is a compound with excellent properties such as rheological properties, gelling properties, stability, processing properties, safety, and biocompatibility. It has strong biological activity and is also used as an antioxidant component in the preservation of meat products. For example, CN112006085A discloses a composite color protection and antioxidant coating solution for meat products, a preparation method and applications thereof. The raw materials of the coating solution include gallocatechin, gallic acid ester, yeast β-glucan, ascorbic acid, chitosan and water; the preparation method is as follows: dissolve yeast β-glucan in water, add gallocatechin, gallic acid ester and ascorbic acid to the yeast β-glucan solution, and then add chitosan. Soak the cut meat in the composite color protection and antioxidant coating solution or spray the composite color protection and antioxidant coating solution on the surface of the meat, then drain, and then package and store in sequence. This technology improves the color protection and antioxidant effect of meat products and plays a role in extending the shelf life of products. Although this composite color protection and antioxidant coating solution can play a good role in preserving meat products, it must use a large amount of effective components for compounding. It is difficult to obtain good preservation effects by using components such as yeast β-glucan or gallic acid ester alone, which limits the application of glucan-based bioactive substances in the preservation of chilled fresh meat. At the same time, commercially available oat glucan is also greatly limited in its application in the field of meat product preservation due to its inherent high viscosity characteristics and poor water solubility. Summary of the Invention

[0005] Aiming at the deficiencies in the existing chilled fresh meat preservation technology, such as low production efficiency of graft copolymers and poor preservation effect of single components, the present invention provides a preparation method of an oat glucan-phenolic acid graft copolymer and its application as an antioxidant and antibacterial solution, providing a new technical solution for the preservation of chilled fresh meat products.

[0006] A preparation method of an oat glucan-phenolic acid graft copolymer, comprising the following steps:

[0007] (1) Under vacuum conditions, dissolve phenolic acid and equimolar N,N'-carbonyldiimidazole (CDI) in anhydrous dimethyl sulfoxide (DMSO) at room temperature, stir and intermittently ultrasonic under heating conditions until phenolic acid and CDI are completely dissolved in DMSO;

[0008] (2) Maintain the vacuum conditions, add oat glucan powder to the solution obtained in step (1), heat and intermittently ultrasonic, and react to synthesize an oat glucan-phenolic acid graft copolymer solution;

[0009] (3) Add at least twice the volume of isopropanol to the solution obtained in step (2) and mix to make the formed oat glucan-phenolic acid graft copolymer precipitate, and centrifuge to recover the precipitate; the precipitate is granular;

[0010] (4) Wash the precipitate obtained in step (3) with isopropanol to remove unreacted phenolic acids and CDI;

[0011] (5) Dissolve the precipitate obtained in step (4) in distilled water, add absolute ethanol, centrifuge after alcohol precipitation, and then freeze-dry to obtain the oat glucan-phenolic acid graft copolymer.

[0012] Optionally, in step (1), the heating temperature is 55 - 65 °C, the reaction time is 3.5 - 4.5 h, assisted by intermittent ultrasonic conditions, ultrasonic for 1 h, intermittent for 1 h, and the ultrasonic power is 400 - 500 W. Preferably, the heating temperature is 55 - 65 °C, the reaction time is 4 h, assisted by intermittent ultrasonic conditions, ultrasonic for 1 h, intermittent for 1 h, and the ultrasonic power is 450 W. The vacuum condition described in steps (1) and (2) means that the vacuum degree is less than or equal to -0.095 MPa. Optionally, the vacuum degree is -0.150 MPa to -0.095 MPa.

[0013] The present invention has no strict requirements for the dosage of DMSO in step (1), as long as the phenolic acid, equimolar N,N'-carbonyldiimidazole (CDI), and the oat glucan powder subsequently added to the reaction liquid can be completely dissolved in DMSO. It is recommended to use 18 - 22 mL of DMSO per 1 g of phenolic acid.

[0014] In the present invention, where the actual temperature is not specifically stated, room temperature refers to 20 - 35 °C.

[0015] In step (2), the heating temperature is 85 - 95 °C, the reaction time is 3.5 - 4.5 h, and intermittent ultrasonic treatment is carried out during the reaction. Intermittent ultrasonic treatment means stopping ultrasonic treatment for 1 h after every 1 h of ultrasonic treatment at an ultrasonic power of 400 - 500 W, and repeating this process multiple times. Preferably, in step (2), the reaction temperature is 90 °C, the reaction time is 4 h, and intermittent ultrasonic treatment is carried out during the reaction, that is, intermittent for 1 h after every 1 h of ultrasonic treatment at an ultrasonic power of 450 W.

[0016] The mass ratio of the phenolic acid used in step (1) to the oat glucan powder added in step (2) is 20:8 - 12, and the preferred mass ratio is 20:10.

[0017] Optionally, the centrifugation conditions in step (3) are 4500 - 5500 r / min and the centrifugation time is 10 - 20 min. Preferably, the centrifugation conditions in step (3) are 5000 r / min and the centrifugation time is 15 min.

[0018] Preferably, in step (5), absolute ethanol is added for alcohol precipitation, and the concentration of ethanol in the solution should not be lower than 70% (v / v). The alcohol precipitation time is 18 - 22 h. Preferably, the concentration of ethanol in the solution is 70% (v / v), and the alcohol precipitation time is 20 h. Optionally, the centrifugation conditions in step (5) are 8000 - 12000 r / min, and the centrifugation time is 10 - 20 min. Preferably, the centrifugation is carried out at 10000 r / min for 15 min. The freeze-drying time is at least 24 h to remove moisture.

[0019] The antioxidant and antibacterial liquid prepared with the oat glucan-phenolic acid graft copolymer described in the present invention as the active ingredient can preserve fresh meat such as rabbit meat, beef, chicken, mutton, duck meat, pork, etc.

[0020] Preferably, the oat glucan powder used is modified oat glucan powder, which is a water-soluble higher oat glucan powder formed by modifying the commercially available oat glucan powder, that is, modified oat glucan powder. The β-glucan content of the oat glucan powder raw material is 70 wt% - 80 wt%. It is a white to light yellow odorless powder, and more than 90 wt% of the powder can pass through a 80-mesh sieve, and the moisture content is less than 5 wt%.

[0021] The preparation method of the modified oat glucan powder includes the following steps:

[0022] A. Add oat glucan to deionized water at 90 °C and stir until dissolved;

[0023] B. Add hydrochloric acid to step A and stir and react at 90 °C to hydrolyze oat glucan under acidic conditions. After the reaction is completed, cool to room temperature;

[0024] C. Add sodium hydroxide solution to the solution in step B to adjust the pH value of the solution to neutral;

[0025] D. Add absolute ethanol to the solution in step C to precipitate the modified oat glucan. After alcohol precipitation, centrifuge, and then freeze-dry to obtain the modified oat glucan powder.

[0026] Preferably, the concentration of hydrochloric acid in step B is 3 mol / L, and the concentration of hydrochloric acid in the reaction system obtained after adding hydrochloric acid is 0.5 mol / L, and the reaction time is at least 5 h.

[0027] Preferably, the concentration of the sodium hydroxide solution in step C is 1.0 mol / L, and making the solution pH neutral means that the pH value of the solution is 7.0.

[0028] Preferably, anhydrous ethanol is added in step D, and the concentration of ethanol in the solution should be not less than 70% (v / v). The alcohol precipitation time is 20 h, the centrifugation condition is 10,000 r / min, the centrifugation time is 15 min, and the freeze-drying time is 24 h.

[0029] Oat glucan has abundant active hydroxyl groups, which make it easy to be modified under specific conditions, such as degradation, carboxymethylation, phosphoric acid esterification, etc., so as to enhance its solubility, viscosity, antioxidant and antibacterial abilities. In the present invention, oat glucan is used as the raw material. First, a modified oat glucan with higher water solubility is prepared by an acid degradation method. Subsequently, phenolic acid is grafted onto the modified oat glucan by an N,N'-carbonyldiimidazole-mediated method to synthesize a modified oat glucan-phenolic acid graft copolymer, and the antioxidant activity and antibacterial activity of the obtained graft copolymer are significantly enhanced. The vacuum ultrasonic-assisted technology is adopted in the present invention, which greatly shortens the activation time of phenolic acid and the time of the chemical reaction. The prepared modified oat glucan-gallic acid graft copolymer is made into an antioxidant and antibacterial solution for rabbit meat, which will help to better solve the problem of easy spoilage and deterioration of rabbit meat products during storage and circulation, thereby improving the food safety level. At the same time, it will also effectively address the problem of food waste caused by the too short shelf life of rabbit meat products, and promote the rational utilization and conservation of resources.

[0030] The present invention also provides the application of the modified oat glucan-phenolic acid graft copolymer, which can be used as an effective component or one of the effective components in an antioxidant and antibacterial solution for fresh chilled meat preservation, and the antioxidant and antibacterial solution is used for the preservation of fresh chilled meat such as rabbit meat. The modified oat glucan-phenolic acid graft copolymer is formulated into an antioxidant and antibacterial solution with a concentration of 1‰ - 5‰, and the solvent is deionized water. Preferably, the formula of the antibacterial solution is: calculated by mass, it contains 2 parts of the modified oat glucan-phenolic acid graft copolymer and 998 parts of deionized water, that is, the modified oat glucan-phenolic acid graft copolymer is formulated into a 2‰ antioxidant and antibacterial solution.

[0031] Preferably, the using methods of the antioxidant and antibacterial solution for the preservation of rabbit meat and other meats include the dipping method, the coating method, and the spraying method.

[0032] The dipping method is as follows: remove the impurities on the surface of the meat product, and then immerse the meat in the antioxidant and antibacterial solution, and take it out to dry after 2 min.

[0033] The coating method is as follows: evenly coat the antioxidant and antibacterial solution on the surface of the fresh chilled meat to make the fresh chilled meat covered with a very thin film solution.

[0034] The spraying method is as follows: put the antioxidant and antibacterial solution into a sprayer and evenly spray it on the surface of the fresh chilled meat.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] The present invention uses an acid degradation method to convert oat glucan raw materials into modified oat glucans with better water solubility. This transformation can significantly improve the solubility and viscosity characteristics of oat glucan molecules, and can greatly enhance the grafting efficiency between glucan and phenolic acids during the grafting reaction of oat glucan and phenolic acids.

[0037] In order to accelerate and optimize the hydrolysis reaction of oat glucan and the graft copolymerization process of oat glucan and phenolic acids, the present invention adopts a vacuum ultrasonic assistance technology, which can significantly shorten the reaction time, reduce the activation reaction time of phenolic acids to 4 h, and reduce the combination reaction time to 4 h.

[0038] The antioxidant and antibacterial liquid prepared by the present invention uses only the oat glucan-phenolic acid graft copolymer as the active ingredient. Even at a low content of the active ingredient, it can quickly form a protective film on the surface of chilled meat, effectively isolate the external environment, reduce the natural evaporation of moisture, and at the same time build a strong microbial barrier to reduce the invasion of harmful microorganisms such as bacteria and molds, inhibit the growth of microorganisms, and significantly extend the shelf life of chilled meat. The modified oat glucan-gallic acid graft copolymer prepared by the present invention has a high scavenging activity against DPPH free radicals, and the scavenging rate is 65.1%.

[0039] The present invention expands the application method of oat glucan and phenolic acids in meat preservation. It can achieve good preservation effects with only a single active ingredient and can be produced efficiently, which is expected to provide more choices for improving the quality of meat preservation by using compounding processes in the future. Description of the Drawings

[0040] Figure 1 1H NMR spectrum of oat glucan-phenolic acid.

[0041] Figure 2 Effect of ultrasonic power on grafting rate.

[0042] Figure 3 Sensory index score results of rabbit meat.

[0043] Figure 4 Effect of juice loss rate of rabbit meat under different conditions.

[0044] Figure 5 Effect of TVB-N of rabbit meat under different conditions.

[0045] Figure 6 Effect of pH of rabbit meat under different conditions.

[0046] Figure 7 Effect of total bacterial count of rabbit meat under different conditions. Detailed Description of the Invention

[0047] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0048] Example 1

[0049] Determination of the structure and antioxidant activity of oat glucan-phenolic acid graft copolymers:

[0050] (1) The structural properties were analyzed using 1 1H NMR. Caffeic acid (CA), gallic acid (GA), oat glucan (OG), modified oat glucan (OGH), oat glucan-caffeic acid graft copolymer (CDI-CAOH), modified oat glucan-caffeic acid graft copolymer (CDI-CAOGH), oat glucan-gallic acid graft copolymer (CDI-GAOH), and modified oat glucan-gallic acid graft copolymer (CDI-GAOGH) were dissolved in 0.5 mL of D2O and 0.1 mL of DMSO, and their nuclear magnetic resonance proton spectra were measured using a Bruker spectrometer at an operating frequency of 500 MHz. The results are as Figure 1 shown.

[0051] From Figure 1 it can be seen that OG and OGH showed multiple peaks at δ 3.2 - 3.9 ppm, representing the hydrogen protons on C-2, C-3 to C-6. Caffeic acid showed proton signals at δ 7.38 ppm (H-d), δ 7.05 ppm (H-a), δ 6.99 ppm (H-b), δ 6.80 ppm (H-c), and δ 6.20 ppm (H-e). New peaks appeared at 6.21 - 7.32 ppm in the caffeic acid graft copolymer, which were the methyl protons of caffeic acid. Compared with the signal peaks of the control group, the hydroxyl peaks between 3.2 - 3.9 ppm in the caffeic acid graft copolymer were significantly enhanced, indicating that CA might interact with OG / OGH molecules by forming hydrogen bonds, confirming that GA was successfully grafted onto OG / OGH. In addition, the binding of phenol and glucan was formed by weak CH-π bonds between aromatic residues and the glucopyranose ring of glucan. GA had only one peak at 6.99 ppm (H-a of the benzene ring). The spectrum of the gallic acid graft copolymer contained all the peaks of oat glucan. However, a new peak appeared around 7.1 ppm, which was related to the H of the aromatic ring on the glucan chain, once again confirming that GA was successfully grafted onto OG / OGH.

[0052] (2) To determine the scavenging activity of oat glucan-phenolic acid graft copolymers against DPPH radicals, the specific method is as follows: CDI-CAOH / CDI-CAOGH / CDI-GAOH / CDI-GAOGH were all prepared in ultrapure water at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml. CA / GA were dissolved in methanol and prepared at the same concentration gradient. Then, 100 μL of the test sample was mixed with 100 μL of 0.1 mmol / L DPPH methanol. The solution was placed in a 96-well plate and kept dark at room temperature for 30 min. The absorbance of the resulting solution was measured at 517 nm using a Synergy H1 microplate reader. In the formula, A0 is the absorbance value of the blank control (ultrapure water), A1 is the absorbance value of the DPPH solution sample, and A2 is the absorbance value of the ultrapure water sample. Vc was used as a positive antioxidant reference in the DPPH radical scavenging test.

[0053]

[0054] Results of the DPPH radical scavenging activity of oat glucan-phenolic acid graft copolymers: The DPPH radical scavenging activities of oat glucan-caffeic acid graft copolymer (CDI-CAOH), modified oat glucan-caffeic acid graft copolymer (CDI-CAOGH), oat glucan-gallic acid graft copolymer (CDI-GAOH), and modified oat glucan-gallic acid graft copolymer (CDI-GAOGH) were 31.09%, 46.38%, 25.75%, and 65.1% respectively. The results showed that the synthesized modified oat glucan-gallic acid graft copolymer had the best antioxidant activity, reaching 65.1%.

[0055] Example 2

[0056] Effect of vacuum ultrasonic assistance on the synthesis of oat glucan-phenolic acid graft copolymers

[0057] During the grafting reaction of oat glucan and phenolic acid, the grafting reaction process was assisted by intermittent ultrasonic conditions. Under ultrasonic conditions, there was a cavitation effect. Ultrasonic waves generated a local negative pressure area in the liquid, forming tiny bubbles or cavities. The bubble nuclei vibrated under the action of ultrasonic waves and quickly closed to generate strong shock waves and microjets, increasing the movement frequency and speed of liquid molecules, making it easier for phenolic acid molecules to penetrate into the interior of oat glucan and promoting the progress of the grafting reaction.

[0058] (1) Effect of ultrasonic power on grafting:

[0059] Under vacuum conditions (vacuum degree -0.095 MPa), gallic acid and equimolar CDI were dissolved in DMSO at room temperature and stirred under heating conditions until gallic acid and CDI were completely dissolved in DMSO; maintaining a vacuum degree of -0.095 MPa, modified oat glucan was added to the resulting solution. Under the conditions of a modified oat glucan concentration of 5 mg / mL, a mass ratio of modified oat glucan to gallic acid of 2:1, a reaction time of 4 h (ultrasonic treatment for 1 h, intermittent for 1 h), and an ultrasonic temperature of 90 °C, the effect of different ultrasonic powers on the grafting degree of the modified oat glucan-gallic acid graft copolymer was investigated. The powers used are as Figure 2 shown.

[0060] It can be seen from Figure 2 that: the grafting degree continuously increases with the increase of ultrasonic power; when it reaches 450 W, the grafting degree is the largest, which is 22.3%; when the power continues to increase, the grafting degree shows a downward trend. This indicates that within a certain reaction time, ultrasonic power can accelerate the grafting reaction between oat glucan and phenolic acid.

[0061] (2) Influence of intermittent ultrasound and continuous ultrasound on grafting

[0062] In the present invention, intermittent ultrasound is used to assist the grafting reaction, with ultrasonic treatment for 1 h and intermittent for 1 h, alternating the ultrasonic working time and the intermittent time. Compared with continuous ultrasound, it reduces the temperature rise of the reaction system. This method saves energy, and the ultrasonic intermittent time can promote the homogenization between material molecules and strengthen the reaction between media, thereby improving the reaction efficiency.

[0063] Under vacuum conditions (vacuum degree -0.095 MPa), gallic acid and equimolar CDI were dissolved in DMSO at room temperature and stirred under heating conditions until gallic acid and CDI were completely dissolved in DMSO; maintaining a vacuum degree of -0.095 MPa, modified oat glucan was added to the resulting solution. Under the conditions of a modified oat glucan concentration of 5 mg / mL, a mass ratio of modified oat glucan to gallic acid of 2:1, a reaction time of 4 h, an ultrasonic temperature of 90 °C, and an ultrasonic power of 450 W, it was divided into two groups. One group used intermittent ultrasound assistance, with ultrasonic treatment for 1 h and intermittent for 1 h, and the reaction ended after 4 h. The other group used continuous ultrasound for 4 h to investigate the effect of different ultrasonic methods on the grafting degree of the modified oat glucan-gallic acid graft copolymer. After the 4-h reaction ended, the grafting rate of the intermittent ultrasound group was 23.75%, and the grafting rate of the continuous ultrasound group was 14.7%. The grafting rate of the intermittent ultrasound group was significantly better than that of the continuous ultrasound group.

[0064] From the verification test of the influence of ultrasonic power on grafting and the verification test of the influence of intermittent ultrasound and continuous ultrasound on grafting, by comparing the grafting rates of the intermittent ultrasound group, it can be seen that they all reached more than 23% of the grafting rate, obtaining a relatively high grafting rate.

[0065] In the following examples, the "oat glucan" refers to commercially available oat glucan, which has a β-glucan content of 70 wt% - 80 wt%, is a white to light yellow odorless powder, more than 90 wt% of the powder can pass through an 80-mesh sieve, and the moisture content is less than 5 wt%; the "modified oat glucan" in the following examples refers to a modified oat glucan with better water solubility obtained from "oat glucan" as the raw material.

[0066] Example 3 Modified Oat Glucan - Gallic Acid Graft Copolymer Antioxidant and Bacteriostatic Liquid

[0067] This example provides a modified oat glucan - gallic acid graft copolymer antioxidant and bacteriostatic liquid, and this modified oat glucan - gallic acid antioxidant and bacteriostatic liquid is composed of the following raw materials: 2 parts of modified oat glucan - gallic acid graft copolymer and 998 parts of deionized water.

[0068] In this example, the surface impurities of the rabbit meat sample were removed, and then the rabbit meat was immersed in the antioxidant and bacteriostatic liquid and taken out to dry after 2 min.

[0069] In this example, the preparation method of the modified oat glucan - gallic acid antioxidant and bacteriostatic liquid is: dissolving the modified oat glucan - gallic acid graft copolymer in deionized water.

[0070] The preparation method of the said modified oat glucan - gallic acid graft copolymer is:

[0071] (1) Add 25 g of oat glucan to 500 mL of deionized water at 90 °C and stir for 2 h, then add 100 mL of 3 mol / L HCl to make the concentration of the reaction system 0.5 mol / L, continue to stir the reaction at 90 °C for 5 h, then cool it to room temperature, adjust the pH value of the solution to 7.0 with 1.0 mol / L NaOH, add absolute ethanol to make its final concentration 70% (v / v), perform alcohol precipitation for 20 h and then centrifuge at 10000 r / min for 15 min, and finally obtain the modified oat glucan by freeze-drying for 24 h. The modified oat glucan has better water solubility compared with the raw material oat glucan.

[0072] (2) Under vacuum conditions (vacuum degree -0.095 MPa), dissolve 6 g of gallic acid and an equimolar amount of CDI in anhydrous DMSO (120 mL) at room temperature, place it in a reaction kettle, keep the mixture at 60 °C, assisted by intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W), and continuously stir (300 r / min) for 4 h to complete the activation of gallic acid. Secondly, add 3 g of modified oat glucan powder to the activated gallic acid solution respectively, and treat it for 4 h under the conditions of 90 °C, vacuum degree -0.095 Mpa, assisted by intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W) to synthesize the copolymer. After the reaction, mix the reaction solution with twice the volume of isopropanol, and recover the precipitate by centrifuging at 5000 r / min for 15 min. Wash the obtained precipitate twice with isopropanol to remove unreacted gallic acid and CDI. Dissolve the precipitate in 50 mL of distilled water, add anhydrous ethanol to make the final ethanol concentration 70% (v / v), precipitate with alcohol for 20 h and then centrifuge at 10000 r / min for 15 min. Finally, the precipitate is freeze-dried for 24 h to obtain the final modified product - modified oat glucan-gallic acid graft copolymer.

[0073] Example 4 Antioxidant and Bacteriostatic Solution of Oat Glucan-Gallic Acid Graft Copolymer

[0074] This example provides an antioxidant and bacteriostatic solution of oat glucan-gallic acid graft copolymer. The oat glucan is commercially available and unmodified. The antioxidant and bacteriostatic solution of oat glucan-gallic acid is composed of the following raw materials: 2 parts of oat glucan-gallic acid graft copolymer and 998 parts of deionized water.

[0075] In this example, remove the surface impurities of the rabbit meat sample, then immerse the rabbit meat in the antioxidant and bacteriostatic solution, and take it out and dry it after 2 min.

[0076] The preparation method of the antioxidant and bacteriostatic solution of oat glucan-gallic acid in this example is: dissolve the oat glucan-gallic acid graft copolymer in deionized water.

[0077] The preparation method of the said oat glucan-gallic acid is:

[0078] Under vacuum conditions (vacuum degree -0.095 MPa), 6 g of gallic acid and an equimolar amount of CDI were dissolved in anhydrous DMSO (120 mL) at room temperature, placed in a reaction kettle, and the mixture was maintained at 60 °C, supplemented with intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W), and continuously stirred (300 r / min) for 4 h to complete the activation of gallic acid. Secondly, 3 g of oat glucan powder was added to the activated gallic acid solution respectively, and it was treated for 4 h at 90 °C, vacuum degree -0.095 Mpa, supplemented with intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W) to synthesize the copolymer. After the reaction was completed, the reaction solution was mixed with twice the volume of isopropanol, and the precipitate was recovered by centrifugation at 5000 r / min for 15 min. The obtained precipitate was washed twice with isopropanol to remove unreacted gallic acid and CDI. The precipitate was dissolved in 50 mL of distilled water, and anhydrous ethanol was added to make the final concentration of ethanol 70% (v / v). After alcohol precipitation for 20 h, it was centrifuged at 10000 r / min for 15 min, and finally the precipitate was freeze-dried for 24 h to obtain the final modified product - oat glucan-gallic acid graft copolymer.

[0079] Example 5 Modified Oat Glucan-Caffeic Acid Graft Copolymer Antioxidant and Bacteriostatic Liquid

[0080] This example provides a modified oat glucan-caffeic acid graft copolymer antioxidant and bacteriostatic liquid, which is composed of the following raw materials: 2 parts of modified oat glucan-caffeic acid graft copolymer and 998 parts of deionized water.

[0081] The preparation method of the modified oat glucan-caffeic acid antioxidant and bacteriostatic liquid in this example is: dissolving the modified oat glucan-caffeic acid graft copolymer in deionized water.

[0082] In this example, the surface impurities of the rabbit meat sample were removed, and then the rabbit meat was immersed in the antioxidant and bacteriostatic liquid and taken out to dry after 2 min.

[0083] The preparation method of the modified oat glucan-caffeic acid is as follows:

[0084] (1) 25 g of oat glucan was added to 500 mL of deionized water at 90 °C and stirred for 2 h, then 100 mL of 3 mol / L HCl was added to make the concentration of the reaction system 0.5 mol / L, and the reaction was continued to stir at 90 °C for 5 h, then it was cooled to room temperature, and the pH value of the solution was adjusted to 7.0 with 1.0 mol / L NaOH, anhydrous ethanol was added to make the final concentration 70% (v / v), after alcohol precipitation for 20 h, it was centrifuged at 10000 r / min for 15 min, and finally the precipitate was freeze-dried for 24 h to obtain modified oat glucan.

[0085] (2) Under vacuum conditions (vacuum degree -0.095 MPa), 6 g of caffeic acid and an equimolar amount of CDI were dissolved in anhydrous DMSO (120 mL) at room temperature, placed in a reaction kettle, and the mixture was maintained at 60 °C, supplemented with intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W), and continuously stirred (300 r / min) for 4 h to complete the activation of caffeic acid. Secondly, 3 g of modified oat glucan powder was added to the activated caffeic acid solution respectively, and the mixture was treated for 4 h at 90 °C, vacuum degree -0.095 Mpa, supplemented with intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W) to synthesize the copolymer. After the reaction, the reaction solution was mixed with twice the volume of isopropanol, and the precipitate was recovered by centrifugation at 5000 r / min for 15 min. The obtained precipitate was washed twice with isopropanol to remove unreacted caffeic acid and CDI. The precipitate was dissolved in 50 mL of distilled water, anhydrous ethanol was added to make the final concentration of ethanol 70% (v / v), and after alcohol precipitation for 20 h, it was centrifuged at 10000 r / min for 15 min. Finally, the precipitate was freeze-dried for 24 h to obtain the final modified product - modified oat glucan-caffeic acid graft copolymer.

[0086] Example 6 Oat Glucan-Caffeic Acid Graft Copolymer Antioxidant and Bacteriostatic Liquid

[0087] This example provides an oat glucan-caffeic acid graft copolymer antioxidant and bacteriostatic liquid, which is composed of the following raw materials: 2 parts of oat glucan-caffeic acid graft copolymer and 998 parts of deionized water.

[0088] The preparation method of the oat glucan-caffeic acid antioxidant and bacteriostatic liquid in this example is: dissolving the oat glucan-caffeic acid graft copolymer in deionized water.

[0089] In this example, the surface impurities of the rabbit meat sample were removed, and then the rabbit meat was immersed in the antioxidant and bacteriostatic liquid and taken out to dry after 2 min.

[0090] The preparation method of the said oat glucan-caffeic acid is as follows:

[0091] Under vacuum conditions (vacuum degree -0.095 MPa), 6 g of caffeic acid and an equimolar amount of CDI were dissolved in anhydrous DMSO (120 mL) at room temperature, placed in a reaction kettle, and the mixture was maintained at 60 °C, assisted by intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W), and continuously stirred (300 r / min) for 4 h to complete the activation of caffeic acid. Secondly, 3 g of oat glucan powder was added to the activated caffeic acid solution respectively, and the mixture was treated for 4 h at 90 °C, vacuum degree -0.095 Mpa, assisted by intermittent ultrasound (ultrasound for 1 h, intermittent for 1 h, power 450 W) to synthesize the copolymer. After the reaction, the reaction solution was mixed with twice the volume of isopropanol, and the precipitate was recovered by centrifugation at 5000 r / min for 15 min. The obtained precipitate was washed twice with isopropanol to remove unreacted caffeic acid and CDI. The precipitate was dissolved in 50 mL of distilled water, and anhydrous ethanol was added to make the final concentration of ethanol 70% (v / v). After alcohol precipitation for 20 h, it was centrifuged at 10000 r / min for 15 min. Finally, the precipitate was freeze-dried for 24 h to obtain the final modified product - oat glucan-caffeic acid graft copolymer.

[0092] Comparative Example 1

[0093] The difference between Comparative Example 1 and Example 3 is that in Example 3, the grafting reaction was carried out in a vacuum and ultrasound-assisted manner, while in Comparative Example 1, the grafting reaction was carried out in a non-vacuum and ultrasound-assisted manner, and the others were the same.

[0094] Comparative Example 2

[0095] The difference between Comparative Example 2 and Example 3 is that in Example 3, the grafting reaction was carried out in a vacuum and ultrasound-assisted manner, while in Comparative Example 2, the grafting reaction was carried out in a vacuum and non-ultrasound-assisted manner, and the others were the same.

[0096] Comparative Example 3

[0097] The difference between Comparative Example 3 and Example 3 is that in Example 3, the grafting reaction was carried out in a vacuum and ultrasound-assisted manner, while in Comparative Example 3, the grafting reaction was carried out in a non-vacuum and non-ultrasound-assisted manner, and the others were the same.

[0098] The grafting rates of the products obtained in Comparative Examples 1 - 3 and Example 3 were measured by experiments. The results showed that the grafting rate of Example 3 was the highest, reaching 23.52%. The grafting rates of Comparative Examples 1 - 3 were 19.23%, 15.36%, and 13.75% respectively. It can be seen that under vacuum and ultrasound-assisted conditions, the reaction grafting rate can be effectively improved.

[0099] In addition, from the grafting rate comparison of the verification test on the influence of ultrasonic power on grafting in Example 3 and the verification test on the influence of intermittent ultrasound and continuous ultrasound on grafting, it can be seen that the grafting rate reaches over 23% in all cases. It can be seen that the preparation method of the present invention has good repeatability, and the grafting rates obtained from multiple tests do not vary significantly.

[0100] Performance study of oat glucan-phenolic acid graft copolymer antioxidant and antibacterial solution for fresh rabbit meat preservation:

[0101] Apply the oat glucan-phenolic acid graft copolymer antioxidant and antibacterial solution prepared in Examples 3 to 6 to fresh rabbit meat preservation. The test steps are as follows:

[0102] Select fresh rabbit meat and place it in a 4°C refrigerator for cold storage until its central temperature drops below 10°C; sterilize the used knives and chopping boards in a high-pressure steam sterilizer in advance and irradiate them with ultraviolet light for 15 minutes. In a sterile operating table, divide the rabbit meat into portions of 200 g for standby. Randomly divide the processed rabbit meat into 4 portions per group, with a total of two groups, where:

[0103] Blank group: The rabbit meat is not treated with anything;

[0104] Coating group: Immerse the processed rabbit meat samples in the antioxidant and antibacterial solution, take them out after 2 minutes, and let them air dry naturally at room temperature;

[0105] Then wrap and cover the rabbit meat in the blank group and the coating group with PE plastic wrap, place it in a 4°C refrigerator for 6 days, and measure the sensory score, juice loss rate, pH value, volatile basic nitrogen index, and total number of colonies of the samples on the 0th, 1st, 2nd, 3rd, 4th, 5th, and 6th days.

[0106] Sensory evaluation

[0107] Sensory evaluation is the most intuitive evaluation method for meat products. Select 20 food professional practitioners in the laboratory to score the chilled fresh rabbit meat on a 100-point scale, and conduct sensory evaluation on the meat samples from aspects such as color, smell, and tissue state, where color, tissue state, smell, and viscosity each account for 25 points. The evaluation criteria are based on GB / T 17239-2022 "Fresh, Frozen Rabbit Meat and By-products", as shown in Table 1 specifically, and the scoring criteria are shown in Table 2 specifically.

[0108] Table 1 Sensory evaluation criteria for chilled fresh rabbit meat

[0109]

[0110] Table 2 Sensory scoring table for chilled fresh rabbit meat

[0111]

[0112]

[0113] Figure 3 Results showed that under laboratory conditions, at the end of the experiment, the samples in the blank group had a reddish-brown color, a strong pungent odor, and the meat was inelastic, indicating spoiled meat; while the samples treated in the coating group had a dark red color and a slight ammonia smell. The sensory index scoring results of the rabbit meat showed that the sensory evaluation index of the group of Example 3 was significantly better.

[0114] Determination of juice loss rate

[0115] The juice loss was calculated by measuring the difference in the mass of the meat chunks at each sampling and the mass of the meat chunks before storage, and expressed as a mass fraction.

[0116] Figure 4 Results showed that under laboratory conditions, the juice loss rates of the samples in both the coating group and the blank group showed a gradually increasing trend with the prolongation of time. However, the juice loss rate of the blank group was significantly higher than that of the coating group, and the juice loss rate of Example 3 in the coating group was the lowest, indicating that the modified oat glucan-gallic acid graft copolymer in Example 3 had a good effect on reducing the juice loss rate of rabbit meat.

[0117] Determination of total volatile basic nitrogen (TVB-N)

[0118] The TVB-N value was determined by the automatic Kjeldahl distillation method according to GB 5009.228-2016 Determination of Total Volatile Basic Nitrogen in Foods.

[0119] Add 10 mL of boric acid solution and 5 drops of mixed indicator solution into the receiving flask, and insert the lower end of the condenser into the liquid surface. Accurately pipette 10.0 mL of the filtrate, pour it into the reaction chamber from the small glass cup, wash the small glass cup with 10 mL of water and let it flow into the reaction chamber, then tightly plug the rod-shaped glass stopper. Then inject 5 mL of magnesium oxide suspension into the reaction chamber, immediately cover the glass stopper tightly, and add water to the small glass cup to prevent air leakage. Clamp the screw clamp and start distillation. After distilling for 5 min, move the receiving flask of the distillate so that the liquid surface is away from the lower end of the condenser, and distill for another 1 min. Then rinse the outside of the lower end of the condenser with a small amount of water and remove the receiving flask of the distillate. Titrate to the end point with a standard titration solution of hydrochloric acid or sulfuric acid (0.0100 mol / L). Use a mixed indicator solution of 1 part methyl red ethanol solution and 5 parts bromocresol green ethanol solution, and the end point color is purple-red. Use a mixed indicator solution of 2 parts methyl red ethanol solution and 1 part methylene blue ethanol solution, and the end point color is blue-violet. At the same time, do a reagent blank.

[0120] According to the provisions of GB / T 5009.44-2003 Methods of Analysis for Hygienic Standards of Meat and Meat Products, when the TVB-N value of meat and meat products is less than or equal to 15 mg / 100 g, it is of first-class freshness; when it is greater than 15 mg / 100 g but does not exceed 25 mg / 100 g, it is of secondary freshness; when the TVB-N value is greater than 25 mg / 100 g, it is spoiled meat.

[0121] Figure 5 Show that: Under laboratory conditions, the initial content of TVB-N in the samples was 10.15 mg / 100 g. During refrigeration, the TVB-N in each group showed a gradually increasing trend. The final content of TVB-N in the blank group reached 27.50 mg / 100 g, which was deteriorated meat; the final content of TVB-N in the coating group was between 19.3 - 23.4 mg / 100 g, all of which were of secondary freshness. Among them, the TVB-N content in the coating group with the antioxidant and antibacterial liquid containing the modified oat glucan-gallic acid graft copolymer (Example 3) was the lowest, indicating that the modified oat glucan-gallic acid graft copolymer had the best effect in inhibiting the spoilage of rabbit meat.

[0122] Determination of pH

[0123] After homogenizing the rabbit meat, take 10 g and put it in a beaker, add 90 mL of neutral distilled water, shake for 30 min, centrifuge at 4000 r / min for 20 min, then take the supernatant and measure it with a pH meter. The measuring head of the pH meter needs to be rinsed before each measurement, and then the next measurement is carried out. Take the average and record it.

[0124] According to the regulations in GB / T9695.5 - 2008 "Determination of pH Value of Meat and Meat Products", meat with a pH between 5.8 - 6.2 is fresh meat; meat with a pH between 6.3 - 6.6 is sub-fresh meat; meat with a pH above 6.7 is deteriorated meat.

[0125] Figure 6 Show that: Under laboratory conditions, during the experiment, the pH value of chilled rabbit meat in each group increased from 5.91 to between 6.55 - 7.02. The pH value of the blank group was as high as 7.02, which was deteriorated meat; the pH value of the experimental group was between 6.55 - 6.68, which was sub-fresh meat; thus, it can be seen that the pH of the treated chilled rabbit meat increased more slowly and stably compared to the untreated ones.

[0126] Determination of total number of colonies

[0127] Take about 25 g of test samples from different meat samples respectively, cut them into pieces, place them in different small beakers, add 225 mL of sterile water to each beaker, and then homogenize the sample test solution using a homogenizer. Then, according to "GB 4789.2 - 2022 National Food Safety Standard Microbiological Examination of Foods - Determination of Total Number of Colonies", count the homogenized sample test solution by the conventional method and record the data.

[0128] Figure 7 Show that: Under laboratory conditions, the total number of colonies in the blank group exceeded the standard on the 6th day, reaching 6.56 log(CFU*g -1 )), and the other groups did not exceed 6 log(CFU*g -1) The total number of colonies in the blank group was higher than that of other coating groups during the same period, indicating that the oat glucan-phenolic acid graft copolymer had a good inhibitory effect on microorganisms on the surface of meat products. At the same time, the coating group using the modified oat glucan-gallic acid graft copolymer had the smallest total number of colonies on the 6th day, indicating that its antioxidant and antibacterial liquid had the best effect.

[0129] In summary, the antioxidant and antibacterial liquid containing the oat glucan-phenolic acid graft copolymer has a positive effect on extending the shelf life of rabbit meat.

Claims

1. A preparation method of an oat glucan-phenolic acid graft copolymer, characterized in that, It includes the following steps: (1) Under vacuum conditions, dissolve phenolic acid and equimolar CDI in DMSO at room temperature, stir and intermittently sonicate under heating conditions until the phenolic acid and CDI are completely dissolved in DMSO; (2) Maintain the vacuum conditions, add oat glucan powder to the solution obtained in step (1), heat and intermittently sonicate to synthesize an oat glucan-phenolic acid graft copolymer solution; (3) Add isopropanol to the solution obtained in step (2) to mix and make the oat glucan-phenolic acid graft copolymer formed by the reaction precipitate, and centrifuge to recover the precipitate; (4) Wash the precipitate obtained in step (3) with isopropanol; (5) Dissolve the precipitate obtained in step (4) in distilled water, add absolute ethanol, centrifuge after alcohol precipitation, and then freeze-dry to obtain an oat glucan-phenolic acid graft copolymer; The oat glucan powder is modified oat glucan powder, and the preparation method of the modified oat glucan powder includes the following steps: A. Add oat glucan to deionized water at 90 °C and stir until dissolved; B. Add hydrochloric acid to step A, stir and react at 90 °C to hydrolyze oat glucan under acidic conditions, and cool to room temperature after the reaction is completed; C. Add sodium hydroxide solution to the solution in step B to adjust the pH value of the solution to neutral; D. Add absolute ethanol to the solution in step C to precipitate the modified oat glucan, centrifuge after alcohol precipitation, and then freeze-dry to obtain the modified oat glucan powder; In step B, the concentration of hydrochloric acid is 3 mol / L, and the concentration of hydrochloric acid in the reaction system obtained after adding hydrochloric acid is 0.5 mol / L, and the reaction time is at least 5 h; in step C, the concentration of sodium hydroxide solution is 1.0 mol / L; in step D, add absolute ethanol to make the concentration of ethanol in the solution not less than 70% (v / v), and the alcohol precipitation time is 20 h.

2. The preparation method of the oat glucan-phenolic acid graft copolymer according to claim 1, characterized in that, In step (1), the heating temperature is 55 - 65 °C, the reaction time is 3.5 - 4.5 h, intermittent sonication means intermittent for 1 h every 1 h of sonication, the sonication power is 400 - 500 W, and the vacuum degree is -0.150 MPa to -0.095 Mpa; in step (2), the reaction temperature is 85 - 95 °C, the reaction time is 3.5 - 4.5 h, intermittent sonication means intermittent for 1 h every 1 h of sonication, the sonication power is 400 - 500 W, and the vacuum degree is -0.150 MPa to -0.095 MPa.

3. The preparation method of the oat glucan-phenolic acid graft copolymer according to claim 1, characterized in that, The mass ratio of the phenolic acid used in step (1) to the oat glucan powder added in step (2) is 20:8 - 12.

4. The preparation method of the oat glucan-phenolic acid graft copolymer according to claim 1, characterized in that The centrifugation conditions in step (3) are 4500 - 5500 r / min, the centrifugation time is 10 - 20 min, and the amount of isopropanol used is at least twice the volume of the solution obtained in step (2).

5. The preparation method of the oat glucan-phenolic acid graft copolymer according to claim 1, wherein In step (5), add absolute ethanol for alcohol precipitation to make the concentration of ethanol in the solution not less than 70% (v / v), and the alcohol precipitation time is 18 - 22 h; the centrifugation conditions in step (5) are 8000 - 12000 r / min, and the centrifugation time is 10 - 20 min.

6. An oat glucan-phenolic acid graft copolymer obtained by using the preparation method according to any one of claims 1 to 5.

7. Use of the oat glucan-phenolic acid graft copolymer according to claim 6, characterized in that, Prepare an antioxidant and antibacterial solution with oat glucan-phenolic acid graft copolymer at a concentration of 1‰ to 5‰, using deionized water as the solvent.

8. Use of the oat glucan-phenolic acid graft copolymer according to claim 7, characterized in that, Prepare an antioxidant and antibacterial solution with oat glucan-phenolic acid graft copolymer at a concentration of 2‰, using deionized water as the solvent.

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