LU501445B1 - Carboxymethyl Chitosan Schiff Base and its Preparation and Application - Google Patents

Abstract

The application discloses a carboxymethyl chitosan Schiff base and its preparation and application. The preparation includes: dissolving the self-made carboxymethyl chitosan with water; dissolving an appropriate amount of Perilla essential oil with ethanol and add it to carboxymethyl chitosan solution, and then carry out ultrasonic microwave treatment for 3.5-4.5 h; filtering, washing and drying are carried out successively to obtain carboxymethyl chitosan Schiff base. The application uses chitosan as raw material to carry out carboxymethylation of chitosan, which effectively improves the properties of chitosan insoluble in water and alkaline environment. On this basis, the natural antibacterial substance Perilla essential oil is used to carry out Schiff alkalization modification on carboxymethyl chitosan, so as to further improve the antibacterial property of chitosan while retaining its good water solubility. The carboxymethyl chitosan Schiff base of the application has good bacteriostatic effect and can be used to prepare bacteriostatic agents.

Description

DESCRIPTION HUS0T445 Carboxymethyl Chitosan Schiff Base and its Preparation and Application

TECHNICAL FIELD The application relates to the technical field of novel antibacterial materials, in particular to a preparation method of novel carboxymethyl chitosan Schiff base and its application.

BACKGROUND Perilla essential oil is a highly volatile aromatic oil obtained from Perilla leaves or straw by a specific extraction method. Perilla essential oil is warm and non-toxic. It has high antioxidant, antibacterial, anti-inflammatory, insecticidal, anti-cancer and depression activities. At present, the research and utilization of Perilla essential oil has attracted widespread attention in the field of scientific research. It is widely used in baked food, beverage, frozen dairy products, pudding, processed vegetables and soup. In addition, Perilla essential oil has been proved to be a more reliable and effective biological reagent than chemically synthesized food preservatives and pesticides. Therefore, Perilla essential oil has brought considerable economic benefits in the fields of medicine, food, cosmetics and chemical industry, and has great scientific research value and market development potential. As a natural cationic polysaccharide, chitosan has the characteristics of non toxicity, bacteriostasis, oxidation resistance, chemical modifiability and high reactivity. It is widely used in many fields such as chemical industry, medicine, food and biotechnology. However, chitosan has good solubility only in dilute acid solution and is insoluble in water and alkaline solution, the application range of chitosan is greatly limited.

SUMMARY The application modifies chitosan for its bacteriostasis and water solubility, so that chitosan can also be dissolved in alkaline solution, enhance its bacteriostatic effect and expand the application range of Chitosan on the basis of retaining its excellent bacteriostasis.

LL . . . LU501445 One of the purposes of the application is to provide a carboxymethyl chitosan Schiff base modified with chitosan, which has the structural formula shown in formula (1): > EMDCHÈOUE 8 ne ——" LuQe mnt em? # i x wp LH \ Sa at SA , n=30-300.

The second purpose of the application is to prepare a carboxymethyl chitosan Schiff base with excellent performance. The method has the advantages of simple equipment, simple process, mild action conditions and good repeatability. A preparation method of carboxymethyl chitosan Schiff base, comprising: (1) Chitosan is placed in 40-45% sodium hydroxide solution, the mass volume ratio of chitosan and sodium hydroxide is 1:23-28 (w/v g:mL), and stirred in ice water bath for 3.5-4.5 h; (2) add chloroacetic acid and isopropanol to the reaction solution obtained in step (1), the volume ratio of isopropanol to sodium hydroxide solution in step (1) is 1:0.8-1.2, and the mass volume ratio of chloroacetic acid and isopropanol is 2:23-28 (w/v g:mL), and then stir at room temperature for 7.5-8.5 h; (3) the reaction solution obtained in step (2) is precipitated, the supernatant is removed, the precipitate is washed with ethanol, then dissolved in water, and the pH is adjusted to 6.5-7.5;

(4) adding ethanol to the aqueous solution obtained in step (3) to precipitate HUS0T445 carboxymethyl chitosan, and finally filtering and drying to obtain carboxymethyl chitosan; (5) dissolving the carboxymethyl chitosan obtained in step (4) with water, the mass ratio of carboxymethyl chitosan to water is 2-4% (w/w), and heat and stir to dissolve carboxymethyl chitosan; (6) dissolve an appropriate amount of Perilla essential oil with ethanol and add it to the carboxymethyl chitosan solution obtained in step (5), and then carry out ultrasonic microwave treatment for 3.5-4.5 h; the mass ratio of carboxymethyl chitosan to Perilla essential oil is 1:6-8 (w/w). (7) The liquid after the reaction in step (6) is successively filtered, washed and dried to obtain carboxymethyl chitosan Schiff base.

Several optional modes are also provided below, but they are not additional limitations to the above overall scheme, but only further additions or optimization.

On the premise of no technical or logical contradiction, each optional mode can be combined separately for the above overall scheme or between multiple optional modes.

Optionally, the Perilla essential oil is extracted by the following method: after microwave ultrasonic synergistic extraction, it is extracted by distillation extraction; the conditions of the microwave ultrasonic synergistic mode are as follows: material liquid ratio 1 g: 5-8 mL, microwave power 930 W-940 W, ultrasonic power 240 W-250 W, microwave ultrasonic temperature 45°C-55°C, microwave ultrasonic time 4 min-5 min; the extraction time of the distillation extraction method is 5.5-6.5 h.

Further, the conditions of the microwave ultrasonic cooperative mode are as follows: material liquid ratio 1g: 6 mL, microwave power 933W, ultrasonic power 245W, microwave ultrasonic temperature 50°C, microwave ultrasonic time 4.5 min; the extraction time of the distillation extraction method is 6 h.

The material liquid ratio refers to the mass volume ratio of sun dried Perilla leaf powder to the extraction solution, wherein the extraction solution is a NaCl solution with a mass fraction of 3%.

Under the conditions of solid-liquid ratio 1g:6 mL, microwave power 933 W, HUS0T445 ultrasonic power 245 W, microwave ultrasonic temperature 50°C and microwave ultrasonic time 4.5 min, the extraction rate of Perilla essential oil is 2.34%. Optionally, the sodium hydroxide solution in step (1) is a 42% sodium hydroxide solution by mass fraction, the ratio of chitosan to sodium hydroxide solution is 1:25 (w/v), and stirred in an ice water bath for 4 h.

Optionally, the volume ratio of isopropanol in step (2) to sodium hydroxide solution in step (1) is 1:1, the ratio of chloroacetic acid to isopropanol is 2:25 (w/v), and the stirring time is 8 h.

Optionally, water dissolution in step (3) can be understood as dissolving all the precipitates.

Optionally, the ethanol in step (4) can be completely precipitated with carboxymethyl chitosan.

Optionally, the mass ratio of carboxymethyl chitosan to distilled water in step (5) is 3% (w/w). Optionally, in step (6), the mass ratio of carboxymethyl chitosan to Perilla essential oil is 1:7 (w/w), and the reaction time is 4 h.

Optionally, the ultrasonic microwave treatment conditions in step (6) are: temperature 65°C-75°C, microwave power 180 W-220 W, ultrasonic power 140 W-160 W.

Further, the temperature is 70°C, the microwave power is 200 W, and the ultrasonic power is 150 W.

Optionally, the chitosan in step (1) is chitosan with a molecular weight of 40-60 kDa.

Further, the chitosan is a chitosan with a molecular weight of 50 kDa.

The solubility of Schiff base prepared from chitosan with low molecular weight of 1 kDa is too high, which has a certain impact on its subsequent application in fresh-keeping materials; the solubility of Schiff base prepared from chitosan with high molecular weight of 100 kda is too low and the antibacterial effect is not obvious.

Therefore, chitosan with molecular weight of 40-60 kDa is preferred, and chitosan with molecular weight of 50 kDa is most preferred.

The application also provides a carboxymethyl chitosan Schiff base prepared by the HUS0T445 preparation method.

The application also provides the application of carboxymethyl chitosan Schiff base as aquatic product antifreeze or in the preparation of aquatic product antifreeze.

The application also provides the application of the carboxymethyl chitosan Schiff base as an antibacterial agent or in the preparation of an antibacterial agent.

Optionally, the bacteriostatic agent is a preparation for inhibiting the bacteria S. putrefaciens, E. coli or S. aureus.

Further, the bacteriostatic agent is a preparation for inhibiting Æ. coli.

For E. coli, the antibacterial effect of carboxymethyl chitosan Schiff base modified with Perilla essential oil is significantly higher than that modified with pure Perilla aldehyde, and its MIC value and MBC value are reduced by about 50% respectively.

Perilla essential oil is combined with carboxymethyl chitosan by Schiff base reaction.

In addition to the reaction between Perilla aldehyde, the main component of Perilla essential oil, and carboxymethyl chitosan, other aldehydes with less content in essential oil, such as citral, also react with carboxymethyl chitosan.

They have a certain synergistic effect, which makes the antibacterial activity of Schiff base stronger than that produced by the reaction of pure Perilla aldehyde, especially against E. coli.

Compared with the prior art, the present application has at least one of the following beneficial effects: (1) the main antibacterial component of Perilla essential oil is Perilla aldehyde.

In this patent, the aldehydes (mainly Perilla aldehyde) in Perilla essential oil are grafted onto carboxymethyl chitosan through Schiff base structure to successfully synthesize a new antibacterial substance carboxymethyl chitosan Schiff base; (2) the carboxymethyl chitosan Schiff base of the application has a large number of carboxymethyl and hydroxyl groups, which can combine with the free water in the fish meat cells, increase the bound water in the cells, reduce the generation of large ice crystals in the process of ice crystal generation, increase the water retention of fish meat, reduce the cooking loss rate of fish meat, and delay the denaturation of protein HUS0T445 in fish meat in the process of preservation, increased enzyme activity; (3) taking chitosan as raw material, carboxymethylation of chitosan effectively improved the properties of chitosan insoluble in water and alkaline environment. On this basis, the natural antibacterial substance Perilla essential oil is used to modify carboxymethyl chitosan by Schiff alkalization, so as to further improve the antibacterial activity of chitosan while retaining its good water solubility. Raw materials are natural green and come from a wide range of sources. The product has obvious antibacterial effect, simple preparation process and mild conditions, so it has a wide application prospect. (4) The Schiff base reaction combination of Perilla essential oil and carboxymethyl chitosan is used. In addition to the reaction between Perilla aldehyde, the main component of Perilla essential oil, and carboxymethyl chitosan (the principle of the main reaction is shown in FIG. 1), other aldehydes with less content in the essential oil, such as citral, also react with carboxymethyl chitosan. They have a certain synergistic effect, which makes the antibacterial activity of Schiff base stronger than that produced by the reaction of pure Perilla aldehyde. (5) Schiff base is synthesized by ultrasonic microwave assisted synthesis. Ultrasound can produce hole effect, improve the reaction environment, blur the phase interface, accelerate the reaction speed and improve the reaction yield, and greatly shorten the reaction time. On the one hand, microwave can rapidly raise the reaction system to the required temperature; on the other hand, it can reduce the reaction energy barrier and make the reaction easier in thermodynamics. (6) Carboxymethyl chitosan in the reaction is self-made in the laboratory and its purity can be guaranteed.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a reaction mechanism diagram of the main reaction of the application. FIG. 2 is the infrared characterization diagram of Embodiment 2 (in the figure, a is chitosan, B is carboxymethyl chitosan, C is Perillaldehyde carboxymethyl chitosan Schiff base, and D is Perilla essential oil carboxymethyl chitosan Schiff base).

FIG. 3 is the variation diagram of water holding capacity of Pseudosciaena crocea HUS0T445 during freeze-thaw cycle in embodiment 4. FIG. 4 shows the change of cooking loss rate of Pseudosciaena crocea during freeze-thaw cycle in embodiment 4. FIG. 5 is a diagram of TBA changes of Pseudosciaena crocea during freeze-thaw cycle in Embodiment 4. FIG. 6 is a diagram of the change of protein concentration of Pseudosciaena crocea during the freeze-thaw cycle of Embodiment 4. FIG. 7 shows the change of Ca**-ATPase activity of Pseudosciaena crocea during freeze-thaw cycle in Embodiment 4.

DESCRIPTION OF THE INVENTION The technical scheme in the embodiment of the application will be described clearly and completely below in combination with the accompanying figures in the embodiment of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all of the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the technical field of the application. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. If there is no definite concentration limit for the ethanol used in the following embodiments, it refers to absolute ethanol, and the reagents used in the experiment can be commercially available. Embodiment 1 (I) Extraction of Perilla essential oil: the volatile essential oil from Perilla leaves is extracted by microwave ultrasonic assisted simultaneous distillation extraction method. Under the conditions of material liquid ratio of 1:6 (mass volume ratio: g:mL,

CL . Ce . . . LU501445 material is Perilla leaf powder, liquid is NaCl solution with mass fraction of 3%), microwave power 933 W, ultrasonic power 245 W, microwave ultrasonic temperature 50°C, microwave ultrasonic time 4.5 min, the extraction rate of Perilla essential oil is

2.34%. The specific steps are as follows: Weigh 40 g of Perilla leaf powder with 40 mesh sieve, put it into a round bottom flask, add 3% NaCl solution according to the material liquid ratio of 1:6 (w/v, g: mLO0), and put it into a microwave ultrasonic combined extractor. The microwave power is 933 W, the ultrasonic power is 245 W, the microwave ultrasonic temperature is 50°C, and the microwave ultrasonic time is 4.5 min; then the essential oil is extracted by distillation extraction for 6 h. After extraction, dry the organic phase with anhydrous sodium sulfate, filter, spin steam, purify and concentrate to obtain Perilla essential oil for GC-MS analysis. The results are shown in Table 1: Table 1 GC-MS identification results of Perilla essential oil ; Percent Percen Serial i age Serial tage numb Compound name Compound name content number content er (%0) (%0) 1 2,2-Dimethyl butane 0.02 24 a-Farnesene 0.43 2 2,2,3-Trnmethylbutane 0.26 25 6-Dusonene 0.18 3 Methyl cyclohexane 0.16 26 Citronellol 0.13 4 2-Pinene 0.09 27 Perilla aldehyde 57.88 P-Pinene 0.11 28 2-Butyryl furan 1.86 6 D-limonene 3.70 29 Nerol 0.12 7 Octanol 0.11 30 Caryophyllene B 0.06 8 1-Octene-3-alcohol 0.48 31 Geraniol 0.04 9 6-Elemene 0.06 32 trans-Shisool 2.17 3R)-1-methylene-3- 1-1 alfa-copaene 0.10 33 (3R)-1-methylene-3-propy 0.19 -ene-2-yl cyclohexane 11 Benzaldehyde 0.49 34 P-Violone 0.06 12 Linalool 0.85 35 Caryophyllin 0.66 13 P-Flemene 0.09 36 Perilla Alcohol 0.89 S-(Z)-3,7,11-trimethyl-1,6,10 14 B-Caryophyllene 5.96 37 (Z)-3,7, 11 -trimethyl-L6,10 5, -dodecanetriene-3-ol Menthol 0.08 38 Epoxidized serpentine II 0.06 16 Trans-ß-Farnesene 2.28 39 Eucalyptus oleanol 0.23

17 a-Caryophyllene 1.43 40 Butyric acid 0.58 LU501445 18 een 0.16 41 2-Methoxy-4-vinylphenol 0.08 19 a-Terpineol 0.25 42 a-Bilberry solanesol 0.06 Geranylene D 1.44 43 Myristic ether 0.12 21 Trans-o-Limonene 8.93 44 Parsley camphor 2.16 22 Citral 1.11 45 Plant alcohol 1.41 23 Bicyclic permethrin 1.16 46 palmitic acid 0.37 (IT) Preparation of carboxymethyl chitosan: (1) chitosan (CS) is placed in 42% sodium hydroxide solution, the mass volume ratio of chitosan to sodium hydroxide is 1:25 (w/v), and stirred in ice water bath for 4 h; (2) add chloroacetic acid and isopropanol to the solution in step (1), the volume ratio of isopropanol to sodium hydroxide solution in step (1) is 1:1 (v/v), and the mass volume ratio of chloroacetic acid and isopropanol is 2:25 (w/v), and then stir at room temperature for 8 h; (3) precipitate the solution obtained in step (2), remove the supernatant, wash the precipitate with ethanol, then dissolve in water, and adjust the pH to 7 with concentrated hydrochloric acid, (4) add ethanol to the solution obtained in step (3) to precipitate carboxymethyl chitosan, and finally filter and dry to obtain carboxymethyl chitosan, which is recorded as CMC. (IIT) Preparation of carboxymethyl chitosan Schiff base: (5) dissolve the carboxymethyl chitosan in step (4) with distilled water, the ratio of carboxymethyl chitosan to distilled water is 3% (w/w), and heat and stir to dissolve carboxymethyl chitosan; (6) dissolve Perilla essential oil with ethanol (the volume ratio of ethanol to distilled water in step (5) is 5:1), add it to the carboxymethyl chitosan solution in step (5), and then carry out ultrasonic microwave treatment for 4 h; reaction conditions: temperature 70°C, microwave power 200 W, ultrasonic power 150 W, mass ratio of carboxymethyl chitosan to Perilla essential oil 1:7 (w/w); (7) the liquid after the reaction in step (6) is filtered, washed and dried to obtain carboxymethyl chitosan Schiff base, which is recorded as PEOSB.

Embodiment 2 10501405 Replace the Perilla essential oil in step (6) of Embodiment 1 with pure Perilla aldehyde, and the mass ratio of carboxymethyl chitosan to Perilla aldehyde is 1:7 (w/w). Other operation steps are the same as those in Embodiment 1, and the obtained modified product is recorded as PSB.

FIG. 2 shows the infrared characterization of chitosan (CS), carboxymethyl chitosan (CMC) (prepared in Embodiment 1), Perillaldehyde carboxymethyl chitosan Schiff base (PSB) (prepared in Embodiment 2) and Perilla essential oil carboxymethyl chitosan Schiff base (PEOSB) (prepared in Embodiment 1). As can be seen from FIG. 2, the main peaks of chitosan in a are C = O peak at 1676 em”! and amino N-H deformation vibration peak at 1,597 cm’, ring antisymmetric stretching vibration peak at 1,156 cm, C-OH absorption peak at 1,083 cm“! and pyran ring vibration peak at 891 cm“. In b, carboxymethyl chitosan has a strong absorption peak at 3,425 cm“, which is the result of the partial overlap of the characteristic absorption peaks of -OH and -NH.

Compared with chitosan, this characteristic peak is narrower.

Due to the substitution of -OH or -NH in the substituted CMC, the number of hydrogen bonds formed by association is reduced. 1,602 cm”! is the characteristic absorption peak of -NH>, which indicates that a large amount of -NH> on the chitosan molecular chain does not undergo carboxymethylation reaction.

The absorption peaks of 1,415 cm”! and 1,324 em”! are not superimposed, indicating that CMC does not contain N-C, that is, there is no -NH-CH, bond.

A weak absorption peak appears at 1,261 cm“, which is the characteristic absorption produced by the coupling of C-O stretching vibration and -OH in plane bending vibration in -CH:-COOH, indicating that the product molecule contains carboxymethyl.

The stretching vibration absorption peaks of C-O-C and C-O appeared at 1,149 cm! and 1,076 cm“, indicating that -OH on chitosan had carboxymethylation reaction.

The disappearance of 1,602 cm”! amino group in c and d and the appearance of C=N peaks of 1,641 cm”! and 1,635 cm”! indicate the formation of Schiff base.

Embodiment 3 Bacteriostatic test

MIC is the minimum inhibitory concentration, which refers to the minimum 7501445 concentration that can inhibit microbial growth.

MBC is the minimum bactericidal concentration, which refers to the minimum concentration that can kill the growth of microorganisms.

The tested bacteria S. putrefaciens, E. coli and S. aureus are inoculated into LB broth at 1:1,000, respectively.

S. putrefaciens 30°C, E. coli and S. aureus 37°C, 200 rmp shaking table, and the first activation is carried out within 12-16 h.

Then inoculated into LB broth at 1:100, S. putrefaciens 30°C, E. coli and S. aureus 37°C, and activated for the second time in a 200 rmp shaker to OD600 nm of 0.6. Then dilute the bacterial suspension with LB broth 1:1,000 to 10° CFU/mL.

Inoculate the above bacterial suspension at 1:100 into the sample broth diluted into different concentrations by double dilution method, and culture in a shaking table at a suitable temperature for 24 hours.

After culture, observe the clarification of the solution in the test tube with the naked eye.

The minimum concentration in the clarifying test tube is the MIC value of the sample solution to the tested bacteria.

According to the MIC value, take 200 pL solution from the solution clarification test tube, apply it to LB solid medium, culture at a suitable temperature for 24 hours, and observe whether there is colony growth on the medium.

Take the minimum sample concentration corresponding to the culture medium for the growth of sterile colonies as the MBC value of the tested bacteria.

The samples include chitosan (CS), carboxymethyl chitosan (CMC) (prepared in Embodiment 1), Perillaldehyde carboxymethyl chitosan Schiff base (PSB) (prepared in Embodiment 2) and Perilla essential oil carboxymethyl chitosan Schiff base (PEOSB) (prepared in Embodiment 1). The results are shown in Table 2: Table 2 bacteriostatic test results Tested strains rr MIC MBC S. aureus + + + + + + + - - CS E. coli + + + + + + + - - S. putrefaciens + + + + + + + - - CMC S. aureus - - - + + + + 4 8

E. coli - - - + + + + 4 8 LU501445 S. putrefaciens - - - - + + + 2 4 S. aureus - - - - + + + 2 4 PSB E. coli - - - - + + + 2 4 S. putrefaciens - - - - - + + 1 2 S. aureus - - - - + + + 2 4 PFOSB E. coli - - - - - + + 1 2 S. putrefaciens - - - - - + + 1 2 It can be seen from table 2 that the modified chitosan derivatives have good antibacterial properties.

For Æ. coli, the antibacterial effect of PEOSB is better than that of PSB and CMC, which are obtained by the reaction of pure Perilla aldehyde, and the mic and MBC are reduced by 50% respectively.

Therefore, PEOSB has a good prospect as a new antibacterial substance.

Embodiment 4 Antifreeze preservation experiment Fresh Pseudosciaena crocea is purchased from Xixi happy city store of Wumei shopping mall in Hangzhou.

The fish is 34.2 + 2.3 cm long, 8.1 + 0.8 cm wide and weighs 450 + 130 g.

Remove the internal organs of freshPseudosciaena crocea, wash and drain.

Each fish is cut into six pieces of roughly the same size.

The treated fish are divided into two groups and soaked in the prepared reagent (clean water and 1% PEOSB) for two hours (clean water as the control group). After soaking, drain the water, put it at -18°C and start the experiment according to the set freeze-thaw cycle.

A series of data are measured after the cycle bundle of 1, 3, 5 and 7. The cycle setting is shown in Table 3 below.

Table 3 freeze thaw cycle Freeze thaw -18°C frozen storage 4°C refrigeration times 1 20:00 on day 0-20:00 on day 1 20:00 on day 1-8:00 on day 2 2 8:00 on day 2-8:00 on day 3 8:00 on day 3-20:00 on day 3 3 20:00 on day 3-20:00 on day 4 20:00 on day 4-8:00 on day 5 4 8:00 on day 5-8:00 on day 6 8:00 on day 6-20:00 on day 6 20:00 on day 6-20:00 on day 7 20:00 on day 7-8:00 on day 8 6 8:00 on day 8-8:00 on day 9 8:00 on day 9-20:00 on day 9 20: 10-8: 7 20:00 on day 9-20:00 on day 10 0:00 on day | ; 8:00 on day

The change of water holding capacity of Pseudosciaena crocea during freeze-thaw 7501445 cycle is shown in FIG. 3; the change of cooking loss rate of Pseudosciaena crocea during freeze-thaw cycle is shown in FIG. 4; the changes of TBA of Pseudosciaena crocea during freeze-thaw cycle are shown in FIG. 5; the change of protein concentration of Pseudosciaena crocea during freeze-thaw cycle is shown in FIG. 6; the changes of Ca”*-ATPase activity of Pseudosciaena crocea during freeze-thaw cycle are shown in FIG. 7. As a derivative of chitosan, PEOSB not only retains the good physical and chemical properties of chitosan, but also increases the antibacterial activity.

PEOSB carries a large amount of -OH with good biodegradability and biocompatibility.

Therefore, it can combine with free water in fish cells and increase the bound water in cells.

In this way, the generation of large ice crystals can be reduced, the water retention of fish meat can be increased, and the cooking loss rate of fish meat can be reduced.

In terms of protein, large ice crystal particles will destroy the protein structure of fish meat, and then accelerate the change of protein.

PEOSB has a large amount of -OH, which can reduce the production of large ice crystals, delay the denaturation of protein in fish meat and improve enzyme activity during preservation.

PEOSB also has certain antioxidant properties.

It can also reduce and delay the fat oxidation of fish.

Therefore, the experimental results show that PEOSB can be used as a new antifreeze.

The above embodiments only express several embodiments of the application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent application.

It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the application, which belong to the protection scope of the application.

Therefore, the scope of protection of the patent application shall be subject to the appended claims.

Claims (10)

CLAIMS LU501445

1. A carboxymethyl chitosan Schiff base, characterized in that it has the structural formula shown in formula (1): © CHODRTDEN es” TH, , n=30-300.

2. An application of carboxymethyl chitosan Schiff base as claimed in claim 1 as an aquatic product antifreeze or in the preparation of aquatic product antifreeze.

3. A preparation method of carboxymethyl chitosan Schiff base according to claim 1, characterized by comprising: (1) chitosan is placed in 40-45% sodium hydroxide solution, the mass volume ratio of chitosan to sodium hydroxide is 1 g: 23-28 mL, and stirred in ice water bath for

3.5-45 h: (2) adding chloroacetic acid and isopropanol to the reaction solution obtained in step (1), the volume ratio of isopropanol to sodium hydroxide solution in step (1) is 1:0.8-1.2, and the mass volume ratio of chloroacetic acid and isopropanol is 2 g: 23-28 mL, and then stir at room temperature for 7.5-8.5 h; (3) the reaction solution obtained in step (2) is precipitated, the supernatant is removed, the precipitate is washed with ethanol, then dissolved in water, and the pH is adjusted to 6.5-7.5;

(4) adding ethanol to the aqueous solution obtained in step (3) to precipitate HUS0T445 carboxymethyl chitosan, and finally filtering and drying to obtain carboxymethyl chitosan; (5) dissolving the carboxymethyl chitosan obtained in step (4) with water, the mass ratio of carboxymethyl chitosan to water is 2-4%, and heating and stirring to dissolve carboxymethyl chitosan; (6) the Perilla essential oil is dissolved in ethanol and added to the carboxymethyl chitosan solution obtained in step (5), followed by ultrasonic microwave treatment for

3.5-4.5 h; the mass ratio of carboxymethyl chitosan to Perilla essential oil is 1:6-8; (7) the reaction solution after the reaction in step (6) is successively filtered, washed and dried to obtain carboxymethyl chitosan Schiff base.

4. The preparation method according to claim 3, characterized in that the Perilla essential oil is extracted by the following method: after microwave ultrasonic synergistic extraction, it is extracted by distillation extraction; the conditions of the microwave ultrasonic synergistic mode are as follows: material liquid ratio 1g: 5-8 mL, microwave power 930 W-940 W, ultrasonic power 240 W-250 W, microwave ultrasonic temperature 45°C-55°C, microwave ultrasonic time 4 min-5 min; the extraction time of the distillation extraction method is 5.5-6.5 h.

5. The preparation method according to claim 3, characterized in that in step (1), the sodium hydroxide solution is 42% sodium hydroxide solution, the mass volume ratio of chitosan and sodium hydroxide solution is 1g: 25 mL, and stirred in an ice water bath for 4 h; the volume ratio of isopropanol to sodium hydroxide solution in step (2) is 1:1, the mass volume ratio of chloroacetic acid to isopropanol is 2 g:25 mL, and the stirring time is 8 h; in step (5), the mass ratio of carboxymethyl chitosan to distilled water is 3%.

6. The preparation method according to claim 3, characterized in that the mass ratio of carboxymethyl chitosan to Perilla essential oil in step (6) is 1:7 and the reaction time is 4 h.

7. The preparation method according to claim 3, characterized in that the ultrasonic HUS0T445 microwave treatment conditions in step (6) are: temperature 65C-75°C, microwave power 180 W-220W, ultrasonic power 140 W-160 W.

8. The preparation method according to claim 3, characterized in that the chitosan in step (1) is chitosan with a molecular weight of 40-60 kDa.

9. An application of carboxymethyl chitosan Schiff base as claimed in claim 1 as bacteriostatic agent or in preparation of bacteriostatic agent.

10. The application according to claim 9, characterized in that the bacteriostatic agent is a preparation for inhibiting the bacteria S. putrefaciens, E. coli or S. aureus.