CN115124630B - Chitosan derivative and preparation method and application thereof - Google Patents
Chitosan derivative and preparation method and application thereof Download PDFInfo
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- CN115124630B CN115124630B CN202110181835.1A CN202110181835A CN115124630B CN 115124630 B CN115124630 B CN 115124630B CN 202110181835 A CN202110181835 A CN 202110181835A CN 115124630 B CN115124630 B CN 115124630B
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 186
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 40
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 claims abstract description 26
- OEIJRRGCTVHYTH-UHFFFAOYSA-N Favan-3-ol Chemical group OC1CC2=CC=CC=C2OC1C1=CC=CC=C1 OEIJRRGCTVHYTH-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229940026509 theaflavin Drugs 0.000 claims abstract description 10
- WMBWREPUVVBILR-GHTZIAJQSA-N (+)-gallocatechin gallate Chemical compound O([C@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-GHTZIAJQSA-N 0.000 claims abstract description 8
- LVJJFMLUMNSUFN-UHFFFAOYSA-N gallocatechin gallate Natural products C1=C(O)C=C2OC(C=3C=C(O)C(O)=CC=3)C(O)CC2=C1OC(=O)C1=CC(O)=C(O)C(O)=C1 LVJJFMLUMNSUFN-UHFFFAOYSA-N 0.000 claims abstract description 8
- IPMYMEWFZKHGAX-UHFFFAOYSA-N Isotheaflavin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C(C1=C2)=CC(O)=C(O)C1=C(O)C(=O)C=C2C1C(O)CC2=C(O)C=C(O)C=C2O1 IPMYMEWFZKHGAX-UHFFFAOYSA-N 0.000 claims abstract description 7
- UXRMWRBWCAGDQB-UHFFFAOYSA-N Theaflavin Natural products C1=CC(C2C(CC3=C(O)C=C(O)C=C3O2)O)=C(O)C(=O)C2=C1C(C1OC3=CC(O)=CC(O)=C3CC1O)=CC(O)=C2O UXRMWRBWCAGDQB-UHFFFAOYSA-N 0.000 claims abstract description 7
- IPMYMEWFZKHGAX-ZKSIBHASSA-N theaflavin Chemical compound C1=C2C([C@H]3OC4=CC(O)=CC(O)=C4C[C@H]3O)=CC(O)=C(O)C2=C(O)C(=O)C=C1[C@@H]1[C@H](O)CC2=C(O)C=C(O)C=C2O1 IPMYMEWFZKHGAX-ZKSIBHASSA-N 0.000 claims abstract description 7
- 235000014620 theaflavin Nutrition 0.000 claims abstract description 7
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 22
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- 229940030275 epigallocatechin gallate Drugs 0.000 abstract description 32
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- 238000007254 oxidation reaction Methods 0.000 abstract description 13
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 abstract description 12
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- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 abstract description 5
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 abstract description 5
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 abstract description 5
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- 238000000034 method Methods 0.000 description 11
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
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- CHHHXKFHOYLYRE-STWYSWDKSA-M potassium sorbate Chemical group [K+].C\C=C\C=C\C([O-])=O CHHHXKFHOYLYRE-STWYSWDKSA-M 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of chitosan materials, and discloses a chitosan derivative, a preparation method and application thereof. The chitosan derivative comprises a flavan-3-ol structure and a chitosan structure; the flavan-3-ol structure is connected with the chitosan structure through an ionic bond; the flavan-3-ol structure is selected from at least one of catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, anthocyanin or theaflavin. The chitosan derivative has good antibacterial property and oxidation resistance, particularly has good high-temperature stability, for example, after being treated at the temperature of more than 120 ℃, the chitosan derivative still has good antibacterial property, and even the mildew resistance is further improved. In addition, the solubility of the chitosan derivative is not less than 1g/100g of water at 25 ℃.
Description
Technical Field
The invention belongs to the field of chitosan materials, and particularly relates to a chitosan derivative, a preparation method and application thereof.
Background
Chitin is a naturally occurring polymer formed from β -1,4 glycosidic linkages linking N-acetyl-D-glucosamine and D-glucosamine. It can be processed to achieve partial or complete removal of acetyl groups and to obtain a polymer called chitosan. Most commercially available chitosan is obtained by first extracting chitin from fishery by-products (e.g., shrimp or crab shells) and then deacylating the chitin with alkali or acid.
Chitosan is known to have a variety of biological properties, including its antibacterial activity, and thus can be applied to the food or pharmaceutical industry. Chitosan is a food additive that has been approved in the national standard GB2760-2014, with specific functions of thickener and coating agent. However, the following problems exist in the application process of chitosan: first, poorly soluble in water; second, chitosan is used as an antimicrobial agent, and a higher concentration is needed to have an antimicrobial effect, so that compared with other antimicrobial agents, the chitosan has high application cost and lower benefit. Therefore, in the specific application process of chitosan, improvement on chitosan is needed to meet the actual production requirement.
The prior related art describes a preparation method of chitosan derivatives, and the chitosan-polyphenol conjugate is prepared, so that the antibacterial property, the oxidation resistance and the water solubility of the chitosan-polyphenol conjugate are improved relative to those of chitosan. However, the chitosan and the polyphenol conjugate are connected through covalent bonds, catalytic enzymes (such as laccase) are required to be introduced in the preparation process, or non-food-grade chemical reagents (such as hydrogen peroxide) are required to be introduced in the preparation process, the preparation process is relatively complex, the cost is high, and the antibacterial property, the oxidation resistance or the stability are not good. This provides a certain barrier to the application of chitosan.
Therefore, it is desirable to provide a novel chitosan derivative which has not only good antibacterial and antioxidative properties but also good stability, contributing to the application of the chitosan derivative.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides the chitosan derivative, the preparation method and the application thereof, and the chitosan derivative has good antibacterial property and oxidation resistance, particularly has good high-temperature stability, for example, after being treated at the temperature of more than 120 ℃, the chitosan derivative still has good antibacterial property, and even the antibacterial property is further improved. In addition, the water solubility of the chitosan derivative is obviously improved compared with that of chitosan.
The invention is characterized in that: the chitosan derivative is a salt formed by electrostatic interaction between hydroxyl on flavan-3-ol and amino on a side chain of chitosan. The preparation process of the chitosan derivative requires chitosan-acid salt as an important reaction intermediate product, and then the flavan-3-alcohol group replaces an anionic group formed by acid in the chitosan-acid salt to generate chitosan-flavan-3-alkoxide, and the hydroxyl group in the flavan-3-alcohol reacts with the amino group in the chitosan to form an ionic bond. In the prior art, the chitosan acid salt derivative reacts with hydrogen ions in the organic acid and hydroxyl groups of chitosan side chains to generate covalent bond conjugate derivatives.
The first aspect of the present invention provides a chitosan derivative.
Specifically, a chitosan derivative comprises a flavan-3-ol structure and a chitosan structure; the flavan-3-ol structure is connected with the chitosan structure through an ionic bond.
The chitosan derivative is a salt, the chitosan structure serves as positive ions, the flavan-3-ol structure serves as negative ions, and the hydroxyl in the flavan-3-ol and the amino in the chitosan have electrostatic interaction, so that the salt is formed.
Preferably, the flavan-3-ol structure is selected from at least one of catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, anthocyanin, or theaflavin.
Preferably, the chitosan structure has a degree of deacetylation of 50-100%; further preferably, the chitosan structure has a degree of deacetylation of 80-99%.
Preferably, the chitosan derivative has a molecular weight of 1kDa to 5000kDa; further preferably, the chitosan derivative has a molecular weight of 1kDa to 2500kDa; more preferably 120kDa to 1000kDa.
Preferably, the chitosan derivative is selected from at least one of chitosan-catechin salt, chitosan-epicatechin salt, chitosan-epigallocatechin salt, chitosan-epicatechin gallate salt, chitosan-epigallocatechin gallate salt, chitosan-gallocatechin gallate salt, or chitosan-theaflavin salt.
Preferably, the structural formula of the chitosan-epigallocatechin gallate isWherein, the value of n is 1-10000. The epigallocatechin gallate and the chitosan are connected through an ionic bond (the chitosan-epigallocatechin gallate salt can also be any hydroxyl group on the epigallocatechin gallate structure and amino groups in the chitosan structure undergo electrostatic interaction to form an ionic bond).
In a second aspect, the present invention provides a method for preparing a chitosan derivative.
Specifically, the preparation method of the chitosan derivative comprises the following steps:
mixing chitosan with acid liquor, heating to obtain chitosan-acid salt, adding flavan-3-ol, stirring, and reacting to obtain the chitosan derivative.
Preferably, the molecular weight of the chitosan is from 1kDa to 4000kDa; further preferably, the molecular weight of the chitosan is from 1kDa to 1500kDa; more preferably 120kDa to 840kDa.
Preferably, the deacetylation degree of the chitosan is 50-100%; further preferably, the chitosan has a degree of deacetylation of 80-99%.
Preferably, the acid solution is selected from a solution of hydrochloric acid, formic acid, lactic acid, citric acid or acetic acid.
Preferably, the volume concentration of the acid liquor is 0.5-10%; further preferably, the volume concentration of the acid solution is 2-8% (where the volume concentration of the acid solution refers to the volume concentration of the acid in the mixture formed by mixing the acid solution with chitosan).
Preferably, the mass volume ratio of the chitosan to the acid liquor is (5-20) g (5-30) mL; further preferably, the mass volume ratio of the chitosan to the acid liquor is (8-15) g (10-25) mL.
Preferably, the pH value of the mixture formed by mixing the chitosan and the acid liquor is 1-6; further preferably, the pH is 2-5.
Preferably, the heating temperature is 30-300 ℃; further preferably, the temperature of the heating is 50-100 ℃.
Preferably, the heating time is 10-500 minutes; further preferably, the heating time is 20 to 200 minutes.
Preferably, the mass ratio of the addition amount of the flavan-3-ol to the chitosan-acid salt is (0.1-5): 1; further preferably, the mass ratio of the addition amount of the flavan-3-ol to the chitosan-acid salt is (0.5-4): 1.
Preferably, the temperature of the reaction is 30-300 ℃; further preferably, the temperature of the reaction is 50-100 ℃.
Preferably, the reaction time is 10 to 500 minutes; further preferably, the reaction time is 20 to 200 minutes. The addition of flavan-3-ol, the proper reaction temperature and the proper reaction time of the chitosan-acid salt enable the prepared chitosan derivative to have better antibacterial property, and particularly, the prepared chitosan derivative can still keep good antibacterial property after being treated at a high temperature (for example, 120 ℃).
Preferably, a preparation method of the chitosan derivative comprises the following steps:
mixing chitosan with acid liquor, heating, removing impurities for the first time to obtain chitosan-acid salt, adding flavan-3-ol, stirring, reacting, and removing impurities for the second time to obtain the chitosan derivative.
Preferably, the specific process of the first impurity removal is as follows: the mixture obtained after heating was dried, and then subjected to dialysis purification (commercially available cellulose filter membrane, pore size was adjusted according to molecular weight requirements, and dialysis purification was performed with ultrapure water at room temperature for 3 to 7 days) to remove the excess unreacted acid. Helping to reduce impurities in chitosan-acid salt.
Preferably, the specific process of the second impurity removal is as follows: the mixture obtained after the reaction was dried and then subjected to dialysis purification to remove the excess unreacted flavan-3-ol. Is beneficial to reducing impurities in the chitosan derivative, thereby improving the antibacterial property and the oxidation resistance of the chitosan derivative.
More preferably, a method for preparing a chitosan derivative comprises the steps of:
mixing chitosan with acid liquor, heating, drying, removing impurities for the first time to obtain chitosan-acid salt, mixing the chitosan-acid salt with a solvent, adding flavan-3-ol, stirring, reacting, drying, removing impurities for the second time, and obtaining the chitosan derivative.
Preferably, the second impurity removal is followed by drying and grinding to obtain powdery chitosan derivative. Facilitating the use of chitosan derivatives.
The specific mode of the drying can be air drying, freeze drying, spray drying or hot drying.
In a third aspect the invention provides the use of a chitosan derivative.
The chitosan derivative is applied to preparation of medicines, foods, cosmetics or paints.
Preferably, the coating is an antimicrobial coating.
Preferably, the food or cosmetic is a food or cosmetic having good antibacterial and antiseptic effects.
Compared with the prior art, the invention has the following beneficial effects:
(1) The chitosan derivative is a salt formed by electrostatic interaction between hydroxyl on flavan-3-ol and amino on a side chain of chitosan. The preparation process of the chitosan derivative requires chitosan-acid salt as an important reaction intermediate product, and then the flavan-3-alcohol group replaces an anionic group formed by acid in the chitosan-acid salt to generate chitosan-flavan-3-alkoxide, and the hydroxyl group in the flavan-3-alcohol reacts with the amino group in the chitosan to form an ionic bond. In the prior art, the chitosan acid salt derivative reacts with hydrogen ions in the organic acid and hydroxyl groups of chitosan side chains to generate covalent bond conjugate derivatives. Therefore, the chitosan derivative provided by the invention has better water solubility (the solubility is not less than 1g/100g of water at 25 ℃, for example, 1g/100g of water to 12g/100g of water), has good antibacterial property and oxidation resistance, particularly has good high-temperature stability, and still has good antibacterial property even further improved mycotic property after being subjected to treatment at a temperature of more than 120 ℃.
(2) The chitosan derivative prepared by the invention has good high-temperature stability, good antibacterial property and oxidation resistance, so that the chitosan derivative can be widely applied to medicines, foods and cosmetics.
Drawings
FIG. 1 is a Fourier infrared spectrum of the chitosan-epigallocatechin gallate prepared in example 1.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1: preparation of chitosan derivative (chitosan-epigallocatechin gallate)
A preparation method of chitosan derivative (chitosan-epigallocatechin gallate) comprises the following steps:
selecting chitosan with molecular weight of 280kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding acetic acid (the volume concentration of acetic acid is 2%, namely, the volume concentration of acetic acid in a mixture formed by chitosan, water and acetic acid is 2%), mixing to form a mixture, heating the mixture at 50 ℃ for 1 hour, performing 24-hour freeze-drying treatment after heating is completed, grinding to obtain powder, performing dialysis purification treatment on the powder to remove excessive unreacted acetic acid, and performing freeze-drying and grinding treatment again to obtain chitosan-acetate powder;
weighing 10g of chitosan-acetate powder, dissolving in 1L of water, adding 10g of epigallocatechin gallate, reacting under stirring at 70 ℃ for 1.5 hours under a closed condition (under the closed condition to help avoid volatilization of acetic acid), freeze-drying for 24 hours, grinding, performing dialysis purification to remove the unreacted epigallocatechin gallate, and freeze-drying and grinding to obtain the chitosan derivative (chitosan-epigallocatechin gallate).
FIG. 1 is a Fourier infrared spectrum of the chitosan-epigallocatechin gallate obtained in example 1 (the ordinate in FIG. 1 is "transmissibility" and the abscissa in "Wave number" is "Wave number", and the abscissa in "cm" is shown -1 "; CHIT represents chitosan, CHIT-AC represents chitosan-acetate, CHIT-FLAV represents chitosan-epigallocatechin gallate, FLAV represents epigallocatechin gallate). As can be seen from FIG. 1, 1556cm of CHIT-AC -1 The peak is carboxylate anions COO - ，1408cm -1 The peak is the stretching mode of carboxylate anions, and 1693cm of FLAV and CHIT-FLAV -1 And 1606cm -1 Peak, representing 1230cm in C=C double bond in epigallocatechin gallate benzene ring, FLAV and CHIT-FLAV -1 The peak represents the C-O bond in the hydroxyl group of epigallocatechin gallate.
The intermediate chitosan-acetate and the product chitosan-epigallocatechin gallate obtained in this example 1 were subjected to structural analysis by fourier infrared spectroscopy, and the spectra thereof are shown in fig. 1. From fig. 1, it can be seen that chitosan-acetate is an important intermediate in the synthesis step, and subsequently, the acetic acid group is replaced by an epigallocatechin gallate group, forming a chitosan-epigallocatechin gallate salt having different structural characteristics from those of chitosan, epigallocatechin gallate salt, and chitosan acetate salt.
The structural formula of the chitosan is as follows:(since the molecular weight of the chitosan selected is 280kDa, n is also defined herein).
The structural formula of the chitosan-acetate is as follows:(since the molecular weight of the chitosan selected is 280kDa, n is also defined herein).
The structural formula of the chitosan-epigallocatechin gallate salt is as follows:(since the molecular weight of the chitosan selected is 280kDa, n is also defined herein).
Example 2: preparation of chitosan derivatives (chitosan-catechin salts)
A method for preparing a chitosan derivative (chitosan-catechin salt), comprising the following steps:
selecting chitosan with molecular weight of 100kDa and deacetylation degree of 80% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 8g of chitosan, dissolving in 1L of water, adding hydrochloric acid (the volume concentration of hydrochloric acid is 3%), mixing to form a mixture, heating the mixture at 40 ℃ for 40 minutes, performing freeze-drying treatment for 28 hours after heating, grinding to obtain powder, performing dialysis purification treatment on the powder to remove excessive unreacted hydrochloric acid, and then performing freeze-drying and grinding treatment again to obtain chitosan-hydrochloride powder;
10g of chitosan-hydrochloride powder is weighed and dissolved in 1L of water, then 15g of catechin is added, the mixture is stirred and reacted for 2 hours under a closed condition at 75 ℃, then the mixture is subjected to freeze-drying and grinding treatment for 24 hours, then dialysis and purification are carried out, unreacted complete catechin is removed, and then the freeze-drying and grinding treatment are carried out, so that the chitosan derivative (chitosan-catechin salt) is obtained.
Example 3: preparation of chitosan derivatives (chitosan-epicatechin salts)
A method for preparing a chitosan derivative (chitosan-epicatechin salt), comprising the steps of:
selecting chitosan with molecular weight of 1000kDa and deacetylation degree of 70% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 12g of chitosan, dissolving in 1L of water, adding acetic acid (the volume concentration of the acetic acid is 4%), mixing to form a mixture, heating at 60 ℃ for 50 minutes, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted acetic acid, and freeze-drying and grinding again to obtain chitosan-acetate powder;
10g of chitosan-acetate powder was weighed and dissolved in 1L of water, then 11g of epicatechin was added, and the mixture was stirred and reacted for 2 hours under a closed condition at 70℃and then subjected to lyophilization and grinding treatment for 24 hours, followed by dialysis purification to remove the epicatechin which was not completely reacted, and then subjected to lyophilization and grinding treatment to obtain a chitosan derivative (chitosan-epicatechin salt).
Example 4: preparation of chitosan derivative (chitosan-epigallocatechin salt)
A preparation method of chitosan derivative (chitosan-epigallocatechin salt) comprises the following steps:
selecting chitosan with molecular weight of 1100kDa and deacetylation degree of 75% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding acetic acid (the volume concentration of the acetic acid is 2.5%), mixing to form a mixture, heating at 70 ℃ for 70 minutes, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted acetic acid, freeze-drying and grinding again to obtain chitosan-acetate powder;
10g of chitosan-acetate powder was weighed and dissolved in 1L of water, then 18g of epigallocatechin was added, and the mixture was stirred and reacted for 1 hour under a closed condition at 80℃and then subjected to lyophilization and grinding treatment for 24 hours, followed by dialysis purification to remove unreacted epigallocatechin, and then subjected to lyophilization and grinding treatment to obtain a chitosan derivative (chitosan-epigallocatechin salt).
Example 5: preparation of chitosan derivative (chitosan-epicatechin gallate)
A preparation method of chitosan derivative (chitosan-epicatechin gallate) comprises the following steps:
selecting chitosan with molecular weight of 1200kDa and deacetylation degree of 85% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding formic acid (the volume concentration of the formic acid is 2%) to mix to form a mixture, heating at 75 ℃ for 75 minutes, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted formic acid, and freeze-drying and grinding again to obtain chitosan-formate powder;
10g of chitosan-formate powder is weighed and dissolved in 1L of water, then 20g of epicatechin gallate is added, the mixture is stirred and reacted for 1.5 hours under a closed condition at 85 ℃, then the mixture is subjected to freeze-drying and grinding treatment for 24 hours, then dialysis and purification are carried out, the epicatechin gallate which is not completely reacted is removed, and then the mixture is subjected to freeze-drying and grinding treatment, thus obtaining the chitosan derivative (chitosan-epicatechin gallate).
Example 6: preparation of chitosan derivative (chitosan-gallocatechin gallate)
A preparation method of chitosan derivative (chitosan-gallocatechin gallate) comprises the following steps:
selecting chitosan with molecular weight of 1500kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding lactic acid (the volume concentration of lactic acid is 2%), mixing to form a mixture, heating at 75 ℃ for 75 minutes, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted lactic acid, freeze-drying and grinding again to obtain chitosan-lactate powder;
weighing 10g of chitosan-lactate powder, dissolving in 1L of water, adding 22g of gallocatechin gallate, stirring and reacting for 2 hours at 90 ℃ under a closed condition, performing freeze-drying and grinding treatment for 24 hours, performing dialysis and purification to remove unreacted complete gallocatechin gallate, and performing freeze-drying and grinding treatment to obtain the chitosan derivative (chitosan-gallocatechin gallate).
Example 7: preparation of chitosan derivative (chitosan-theaflavin salt)
A method for preparing a chitosan derivative (chitosan-theaflavin salt), comprising the following steps:
selecting chitosan with molecular weight of 100kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding citric acid (the volume concentration of lactic acid is 2.5%) and mixing to form a mixture, heating at 80 ℃ for 70 minutes, performing 24 hours freeze-drying treatment after heating is completed, grinding to obtain powder, performing dialysis purification treatment on the powder to remove excessive unreacted citric acid, and performing freeze-drying and grinding treatment again to obtain chitosan-citrate powder;
weighing 10g of chitosan-citrate powder, dissolving in 1L of water, adding 13g of theaflavin, stirring and reacting for 2 hours at 90 ℃ under a closed condition, freeze-drying and grinding for 24 hours, dialyzing and purifying to remove the unreacted complete theaflavin, and freeze-drying and grinding to obtain the chitosan derivative (chitosan-theaflavin salt).
Example 8: preparation of chitosan derivative (chitosan-epigallocatechin gallate)
A preparation method of chitosan derivative (chitosan-epigallocatechin gallate) comprises the following steps:
selecting chitosan with molecular weight of 180kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding acetic acid (the volume concentration of the acetic acid is 2.5%), mixing to form a mixture, heating at 80 ℃ for 1 hour, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted acetic acid, and freeze-drying and grinding again to obtain chitosan-acetate powder;
weighing 10g of chitosan-acetate powder, dissolving in 1L of water, adding 6g of epigallocatechin gallate, stirring and reacting for 1.5 hours at 70 ℃ under a closed condition, performing freeze-drying and grinding treatment for 24 hours, performing dialysis and purification, and performing freeze-drying and grinding treatment to obtain the chitosan derivative (chitosan-epigallocatechin gallate).
Example 9: preparation of chitosan derivatives (chitosan-anthocyanin salts)
A method for preparing chitosan derivative (chitosan-anthocyanin salt), comprising the following steps:
selecting chitosan with molecular weight of 200kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding lactic acid (the volume concentration of lactic acid is 2.5%), mixing to form a mixture, heating at 85 ℃ for 2 hours, freeze-drying for 24 hours after heating is finished, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted lactic acid, and freeze-drying and grinding again to obtain chitosan-lactate powder;
weighing 10g of chitosan-lactate powder, dissolving in 1L of water, adding 15g of anthocyanin, stirring and reacting for 2 hours at 70 ℃ under a closed condition, performing freeze-drying and grinding treatment for 24 hours, performing dialysis and purification, and performing freeze-drying and grinding treatment to obtain the chitosan derivative (chitosan-anthocyanin salt).
Example 10
Example 10 was different from example 1 only in that the reaction temperature of chitosan-acetate and epigallocatechin gallate in example 10 was 110℃and the reaction time was 1.5 hours, and the other steps were the same as in example 1.
Comparative example 1 (preparation method of conjugated product of chitosan-epigallocatechin gallate in the prior art)
Selecting chitosan with molecular weight of 280kDa and deacetylation degree of 88% (through nuclear magnetic resonance hydrogen spectrum verification), weighing 10g of chitosan, dissolving in 1L of water, adding acetic acid (the volume concentration of acetic acid is 2%, namely, the volume concentration of acetic acid in a mixture formed by chitosan, water and acetic acid is 2%), mixing, stirring overnight to obtain a chitosan solution, taking 100mL of the chitosan solution, adding 1mol/L hydrochloric acid to adjust pH to 3.5, adding 1mL of 0.5mol/L hydrogen peroxide and 0.025g of ascorbic acid, stirring at 40 ℃ for 1 hour, adding 1g of epigallocatechin gallate, stirring at 40 ℃ for reacting for 12 hours, then performing 24 hours of freeze-drying and grinding treatment, then performing dialysis purification, removing the epigallocatechin gallate which is not completely reacted, and then performing freeze-drying and grinding treatment to obtain the chitosan-epigallocatechin gallate conjugated product.
Product effect test
1. Antibacterial effect test
The chitosan derivatives prepared in examples 1 to 7, 10 and comparative example 1 were tested against bacteria (including gram-positive bacteria including staphylococcus aureus, bacillus cereus and lactobacillus plantarum, and gram-negative bacteria including escherichia coli and pseudomonas aeruginosa), yeasts (malassezia furfur and candida albicans) and molds (aspergillus niger and penicillium italicum) (sample inoculation concentration unit is ppm, and concentration gradient is 4000ppm, 2000ppm, 1000ppm and 500 ppm).
The cultured system was a commercially available nutrient broth (model 022010, available from Guangdong CycloKai microorganism Co., ltd.) and had a pH of 6, the bacteria were cultured at 36℃for 7 days, the yeasts and molds were cultured at 28℃for 7 days, and the results are shown in Table 1.
Table 1: antibacterial Effect (data in Table 1 indicate MIC in ppm)
Remarks: in Table 1 "/" indicates no bacteriostatic effect.
As can be seen from Table 1, the chitosan derivatives prepared in examples 1 to 7 and example 10 of the present invention have better antibacterial effect than comparative example 1. In contrast, the conjugated chitosan-epigallocatechin gallate product prepared in comparative example 1 had no antibacterial effect on mold. From the data of example 1 and example 10, it can be seen that the temperature at which chitosan-acetate reacts with epigallocatechin gallate has a certain effect on the antibacterial effect of the chitosan derivative produced. The antibacterial effect of the chitosan derivative prepared in the remaining examples was similar to that of example 1.
2. Stability test
The chitosan derivative prepared in example 1 was tested for antibacterial effect after being treated under different temperature conditions, and further used for testing the stability of the chitosan derivative prepared in example 1. The system and conditions of the culture were the same as above (the bacteria were cultured at 36℃for 7 days, and the yeasts and molds were cultured at 28℃for 7 days). The different temperature conditions are specifically divided into: normal temperature 25 ℃ for 1 hour, high pressure wet heat treatment (101 KPa,121 ℃,15 minutes), normal pressure water bath 121 ℃ for 1 hour, and normal pressure oil bath 180 ℃ for 1 hour. The antibacterial results are shown in table 2.
Table 2: antibacterial Effect (data in Table 2 indicate MIC in ppm)
As can be seen from Table 2, after the treatment under conditions of 1 hour of high-pressure wet heat treatment, 1 hour of normal-pressure water bath 121 ℃ treatment, 1 hour of normal-pressure oil bath 180 ℃ treatment and the like, the antibacterial effect of the chitosan derivative prepared in the example 1 on gram-positive bacteria, gram-negative bacteria and yeast is kept consistent with that of the chitosan derivative prepared in the normal-temperature 25 ℃, which shows that the chitosan derivative prepared in the example 1 has good high-temperature stability. In addition, the inventors have unexpectedly found that the chitosan derivative prepared in example 1 has unexpectedly enhanced antibacterial effect against mold after the treatment under conditions such as high-pressure wet heat treatment for 1 hour, normal-pressure water bath treatment for 1 hour at 121 ℃ and normal-pressure oil bath treatment for 1 hour at 180 ℃ and thus has unexpectedly achieved a technical effect.
The chitosan derivatives prepared in the remaining examples have similar high temperature stability as the chitosan derivatives prepared in example 1 above.
3. Oxidation resistance test
The chitosan derivatives prepared in examples 1 to 6 of the present invention were tested for oxidation resistance (the oxidation resistance was measured by DPPH radical scavenging rate, DPPH means 1, 1-diphenyl-2-trinitrophenylhydrazine), and the results are shown in Table 3.
Table 3: oxidation resistance effect
| Sample of | DPPH radical scavenging Rate (%) |
| Chitosan | 0 |
| Catechin | 54 |
| Example 2 | 50 |
| Epicatechin | 68 |
| Example 3 | 59 |
| Epicatechin gallate | 78 |
| Example 5 | 65 |
| Epigallocatechin (EGCG) | 73 |
| Example 4 | 61 |
| Epigallocatechin gallate | 84 |
| Example 1 | 72 |
| Gallocatechin gallate | 74 |
| Example 6 | 60 |
| Comparative example 1 | 73 |
As can be seen from Table 3, the chitosan derivatives prepared in examples 1-6 still have a good oxidation resistance.
4. Application effect in food preservation
The chitosan derivative prepared in the example 1 is taken and tested for application effect in food preservation.
The experimental procedure was as follows:
(1) Thawing frozen beef, cutting into blocks, wherein each block is about 300-400g;
(2) The experimental groups, in particular 5 groups, are blank group, chitosan group, epigallocatechin gallate group, chitosan derivative group prepared in example 1 and potassium sorbate group respectively,
(3) Preparing saline solution with the mass concentration of 2%, respectively adding chitosan (the concentration of chitosan in the saline solution is 3000 ppm), epigallocatechin gallate (the concentration of epigallocatechin gallate in the saline solution is 3000 ppm), the chitosan derivative prepared in the example 1 (the concentration of the chitosan derivative prepared in the example 1 in the saline solution is 3000 ppm), potassium sorbate (the concentration of potassium sorbate in the saline solution is 75 ppm), recording the original pH value of the saline solution by the potassium sorbate group, adding citric acid to adjust the pH value to 5.0, adding potassium sorbate, stirring uniformly to be favorable for exerting the antibacterial effect of the potassium sorbate), heating in a pot, putting 2 large beef blocks, stewing with small fire for about 1 hour after boiling with strong fire, and taking beef soft but not suitable for dispersing;
(4) Cutting the cooked beef into small blocks of about 20g, filling each 2 blocks into a bag, directly sealing the bags in a heat sealing way, and sealing each group into 14 bags;
(5) Refrigerating, placing, testing the total number of colonies on the 0 th day, taking 2 parallel samples for each detection, and testing the total number of colonies on the 1 st, 2 nd, 3 rd, 4 th and 7 th days;
(6) The test was repeated twice and the final result was based on the average of the two independent test data.
The results of the above-mentioned monitoring of the total number of colonies are shown in Table 4.
Table 4: colony count (Unit: log cfu/mL)
As can be seen from Table 4, after 7 days of storage of the food, the total number of colonies in the blank group had reached 7.96log cfu/mL, the potassium sorbate group was 4.12log cfu/mL, and the chitosan derivative group obtained in example 1 could also be maintained at 1.02log cfu/mL. Therefore, the chitosan derivative prepared in the embodiment 1 has good anti-corrosion and fresh-keeping effects. The chitosan derivative prepared by the other embodiments also has similar preservative and fresh-keeping effects.
The addition of chitosan and epigallocatechin gallate alone to the above food does not have good antibacterial properties.
In addition, the chitosan derivative prepared by the invention has good high-temperature resistance stability and good antibacterial property and oxidation resistance, so that the chitosan derivative can be widely applied to medicines, cosmetics or paints.
5. Dissolution effect
The solubility of the chitosan derivative prepared in the embodiment 1 of the invention in water is 10g/100g water at room temperature of 25 ℃, the solubility of chitosan is less than 0.01g/100g water, and the solubility of the chitosan-epigallocatechin gallate conjugate product prepared in the comparative example 1 is less than 0.01g/100g water.
Claims (2)
1. A chitosan derivative comprising a flavan-3-ol structure and a chitosan structure; the flavan-3-ol structure is connected with the chitosan structure through an ionic bond;
the flavan-3-ol structure is gallocatechin gallate or theaflavin;
the preparation method of the chitosan derivative comprises the following steps:
selecting chitosan with molecular weight of 1500kDa and deacetylation degree of 88%, weighing 10g of chitosan, dissolving in 1L of water, adding lactic acid, mixing, wherein the volume concentration of lactic acid is 2%, forming a mixture, heating at 75 ℃ for 75 minutes, freeze-drying for 24 hours after heating, grinding to obtain powder, dialyzing and purifying the powder to remove excessive unreacted lactic acid, freeze-drying again, and grinding to obtain chitosan-lactate powder;
weighing 10g of chitosan-lactate powder, dissolving in 1L of water, adding 22g of gallocatechin gallate, stirring and reacting for 2 hours at 90 ℃ under a closed condition, performing freeze-drying and grinding treatment for 24 hours, performing dialysis and purification to remove unreacted complete gallocatechin gallate, and performing freeze-drying and grinding treatment to obtain chitosan derivatives;
or selecting chitosan with molecular weight of 100kDa and deacetylation degree of 88%, weighing 10g of chitosan, dissolving in 1L of water, adding citric acid to mix, wherein the volume concentration of the citric acid is 2.5%, forming a mixture, heating for 70 minutes at 80 ℃, performing freeze-drying treatment for 24 hours after heating is completed, grinding to obtain powder, performing dialysis purification treatment on the powder to remove excessive unreacted citric acid, and performing freeze-drying and grinding treatment again to obtain chitosan-citrate powder;
weighing 10g of chitosan-citrate powder, dissolving in 1L of water, adding 13g of theaflavin, stirring and reacting for 2 hours at 90 ℃ under a closed condition, freeze-drying and grinding for 24 hours, dialyzing and purifying to remove the unreacted complete theaflavin, and freeze-drying and grinding to obtain the chitosan derivative.
2. Use of the chitosan derivative of claim 1 in the preparation of a pharmaceutical, food, cosmetic or paint.
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