CN109181352B - Method for modifying black sesame melanin into water-soluble melanin - Google Patents

Method for modifying black sesame melanin into water-soluble melanin Download PDF

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CN109181352B
CN109181352B CN201810945484.5A CN201810945484A CN109181352B CN 109181352 B CN109181352 B CN 109181352B CN 201810945484 A CN201810945484 A CN 201810945484A CN 109181352 B CN109181352 B CN 109181352B
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melanin
black sesame
water
sesame melanin
treatment
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CN109181352A (en
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高莉
杨柳
解军
赵英虎
王芳
史楠
贾万利
王林变
王瑞琪
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North University of China
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B68/00—Organic pigments surface-modified by grafting, e.g. by establishing covalent or complex bonds, in order to improve the pigment properties, e.g. dispersibility or rheology
    • C09B68/40—Organic pigments surface-modified by grafting, e.g. by establishing covalent or complex bonds, in order to improve the pigment properties, e.g. dispersibility or rheology characterised by the chemical nature of the attached groups
    • C09B68/42—Ionic groups, e.g. free acid
    • C09B68/425—Anionic groups
    • C09B68/4257—Carboxylic acid groups

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Abstract

The invention relates to the field of melanin solubility modification methods. A method for modifying black sesame melanin into water-soluble melanin comprises the steps of mixing amino acid, water and black sesame melanin, carrying out modification reaction under inert atmosphere nitrogen, and then separating to obtain the water-soluble melanin, wherein the mass ratio of the black sesame melanin to the amino acid to the water is 1g: (0.5-3) g: (50-80) mL, wherein the amino acid is one of L-arginine, DL-arginine or DL-histidine. The invention overcomes the problem that the black sesame melanin is not suitable for preparing oral preparations, food pigment additives, coloring agents, medicines, health products and the like because of being insoluble in water.

Description

Method for modifying black sesame melanin into water-soluble melanin
Technical Field
The invention relates to the field of melanin solubility modification methods, in particular to a method for modifying black sesame melanin into water-soluble melanin.
Background
Melanin (Melanin) is ubiquitous in the biological world, is one of pigments with wide sources in currently known biological pigments, and is a general term for a class of phenolic or indole biological macromolecular pigments with complex and diverse structures. According to the difference in structure, it can be classified into three types of eumelanin (eumelanins), pheomelanins (pheomelanins) and allomelanin (allomelanins). Melanin has many unique functional properties, and can be used not only as a class of photo-protecting agents, anti-radiation agents, chelating agents, biological antioxidants and immune promoters, but also as a class of biological semiconductor materials. In recent years, researches also find that the melanin has the effects of resisting snake venom, resisting cancers, inhibiting HIV replication, treating Parkinson's disease and the like, and has wide application prospects in the fields of medicines, cosmetics, foods, electronics and the like.
However, most melanin is insoluble in water and most organic solvents, and is only soluble in alkaline solutions and precipitated in acidic solutions, and the application of the melanin in the fields of food, medical treatment and the like is limited due to the special properties of the melanin. The alkali-soluble melanin is modified into water-soluble melanin, so that the development and application of the water-soluble melanin are facilitated, the biological function of the water-soluble melanin is fully exerted, and the water-soluble melanin has positive significance for expanding the application field and the application range of the melanin. Currently, the market for melanin is dominated by chemical synthesis.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to solve the defects in the prior art and provide a method for modifying black sesame melanin into water-soluble melanin.
The technical scheme adopted by the invention is as follows: a method for modifying black sesame melanin into water-soluble melanin comprises the steps of mixing amino acid, water and black sesame melanin, carrying out modification reaction under inert atmosphere nitrogen, and then separating to obtain the water-soluble melanin, wherein the mass ratio of the black sesame melanin to the amino acid to the water is 1g: (0.5-3) g: (50-80) mL, wherein the amino acid is one of L-arginine, DL-arginine or DL-histidine.
As a preferred mode: the modification reaction is carried out under the condition of stirring in the inert atmosphere of nitrogen, the stirring speed is 200-800rpm, the time is 5-20min, and the temperature is 25-37 ℃.
As a preferred mode: the separation process comprises the following steps of carrying out centrifugal treatment on the modified product to obtain a supernatant, wherein the centrifugal treatment conditions are as follows: rotating speed: 8000- & lt12000 r/min, time: 5-10 min; and (3) carrying out dialysis treatment on the supernatant, and then carrying out freeze drying, wherein the conditions of the dialysis treatment are that the molecular interception amount of a dialysis bag is 1000-2000D, the dialysis time is 36-48h, and the conditions of the freeze drying are that the temperature is minus 40 ℃ to minus 45 ℃, and the pressure is 5-13 Pa.
As a preferred mode: the preparation method of the black sesame melanin comprises the following steps:
a. mixing black sesame with alkali liquor, and then carrying out ultrasonic treatment to obtain a first mixed solution;
b. filtering the first mixed solution obtained in the step a to obtain a filtrate product;
c. c, regulating the pH value of the filtrate product obtained in the step b to 1.0-3.0 by using acid liquor, then carrying out centrifugal treatment, and carrying out vacuum freeze drying on the precipitate obtained by centrifugal treatment to obtain a black sesame melanin crude product;
d. c, dissolving the crude black sesame melanin product obtained in the step c in alkali liquor, adjusting the pH to 1.0-3.0 by using acid liquor, and then performing centrifugal treatment to obtain a primary treated crude black sesame melanin product;
e. d, performing acidolysis treatment on the primary treated black sesame melanin crude product obtained in the step d by using an acid solution, and centrifuging to obtain a secondary treated black sesame melanin crude product;
f. e, washing and centrifuging the secondary-processed black sesame melanin crude product obtained in the step e by adopting an organic solvent to obtain a tertiary-processed black sesame melanin crude product;
g. and f, washing the black sesame melanin treated for three times in the step f, and then carrying out vacuum freeze drying to obtain the pure black sesame melanin.
As a preferred mode: in the step a, the alkali liquor is 1-3mol/L NaOH solution, and the mass ratio of the black sesame to the volume of the alkali liquor is 1g: (1-5) mL, wherein the ultrasonic treatment condition is that the frequency is 40-60Hz and the time is 40-180 min;
the filtration mode in the step b is suction filtration;
in the step c, the acid solution is 2-8mol/L hydrochloric acid, the rotation speed of centrifugal treatment is 8000-12000r/min, the time of centrifugal treatment is 5-10min, and the conditions of vacuum freeze drying are as follows: the temperature is 60 ℃ below zero to 80 ℃ below zero, and the pressure is 4-15 Pa;
in the step d, the alkali liquor is 1-3mol/L NaOH solution, and the ratio of the mass of the black sesame melanin crude product to the volume of the alkali liquor is 1g: (1-5) mL, wherein the acid solution is 2-8mol/L hydrochloric acid, the rotation speed of the centrifugal treatment is 8000-12000r/min, and the time of the centrifugal treatment is 5-10 min;
the acid solution in the step e is 2-8mol/L hydrochloric acid, and the ratio of the mass of the black sesame melanin crude product to the volume of the acid solution in one-time treatment is 1g: (1-5) mL, wherein the acidolysis treatment is carried out at 90-100 ℃ for 60-180 min;
and f, washing by sequentially adopting chloroform, ethyl acetate and ethanol, wherein the ratio of the mass of the crude black sesame melanin product subjected to secondary treatment to the volume of each organic solvent is 1g (5-40) mL, and the centrifugation condition is as follows: the rotating speed is 8000-;
and g, the vacuum drying condition in the step g is that the temperature is 60 ℃ below zero to 80 ℃ below zero, and the pressure is 4-15 Pa.
The invention has the beneficial effects that: the invention can effectively modify the black sesame melanin into water-soluble melanin, thereby overcoming the problem that the black sesame melanin is not suitable for preparing oral preparations, food pigment additives, coloring agents, medicines, health care products and the like because of being insoluble in water, and having positive significance for expanding the application range of the black sesame melanin.
Drawings
FIG. 1 is a graph showing the effect of different amino acids on the modification of black sesame melanin;
FIG. 2 is a graph showing the relationship between the mass ratio of black sesame melanin to L-arginine and the modification rate;
FIG. 3 is a graph showing the relationship between the ratio of the black sesame melanin to DL-arginine and the modification rate;
FIG. 4 is a graph showing the relationship between the ratio of the black sesame melanin to DL-histidine and the modification rate;
FIG. 5 is an infrared spectrum of black sesame melanin;
FIG. 6 is an IR spectrum of L-arginine/melanin;
FIG. 7 is an IR spectrum of DL-arginine/melanin;
FIG. 8 is an IR spectrum of DL-histidine/melanin.
Detailed Description
The present invention will be further described with reference to specific examples, which are provided for the purpose of illustration and are not intended to be limiting.
Mixing amino acid, water and black sesame melanin, reacting under inert atmosphere nitrogen, and separating to obtain water-soluble melanin. Wherein the volume ratio of the mass of the black sesame melanin to the mass of the amino acid to the water is 1g: (0.5-3) g: (50-80) mL, wherein the amino acid is L-arginine, DL-arginine or DL-histidine.
Weighing 30g of black sesame, dissolving the black sesame in 1-3mol/L (preferably 2 mol/L) of NaOH solution, wherein the mass ratio of the black sesame to the NaOH solution is 1g: (1-5) mL (preferably 1g: 3 mL), ultrasonic conditions of 40-60Hz (preferably 60 Hz), 200-400W (preferably 200W), and extraction time of 40-180min (preferably 120 min) to obtain a first mixed solution.
And carrying out suction filtration on the first mixed solution to obtain a filtrate product.
And (3) regulating the pH value of the obtained filtrate product with acid liquor, then carrying out centrifugal treatment, and carrying out vacuum freeze drying on the obtained precipitate to obtain a freeze-dried product, namely a crude product of black sesame melanin. The acid solution is 2-8mol/L (preferably 6 mol/L) hydrochloric acid solution, the pH value is adjusted to be 1.0-3.0 (preferably 2.0), standing is carried out until melanin is precipitated, and the supernatant is removed by using a high-speed centrifuge 8000-12000r/min (preferably 9000 r/min) and centrifugation for 5-10min (preferably 5 min), so as to obtain the precipitate. Vacuum freeze drying at-60 deg.C to-80 deg.C under 4-15Pa by vacuum freeze dryer to obtain crude product of black sesame melanin, and storing at 4 deg.C.
Dissolving the crude black sesame melanin in an alkali liquor, adjusting the pH value with an acid liquor, and then carrying out centrifugal treatment, wherein the ratio of the mass of the crude black sesame melanin to the volume of the alkali liquor is 1g: (1-5) mL (preferably 1g: 2 mL), wherein the alkali solution is 1-3mol/L (preferably 2 mol/L) NaOH solution, namely, the crude black sesame melanin is re-dissolved in 1-3mol/L NaOH solution, the acid solution is 2-8mol/L (preferably 6 mol/L) hydrochloric acid, the pH value is adjusted to 1.0-3.0 (preferably 2.0) by using 2-8mol/L hydrochloric acid solution, the solution is centrifuged at 12000r/min (preferably 9000 r/min) at 8000-.
And performing acidolysis treatment on the primary treated black sesame melanin crude product by using acid liquor, and then washing and centrifuging to obtain a secondary treated black sesame melanin crude product. Wherein the acid solution is 2-8mol/L (preferably 6 mol/L) HCl solution, and the ratio of the mass of the black sesame melanin crude product treated once to the volume of the acid solution is 1g: (1-5) mL (preferably 1g: 3 mL), heating to 90-100 deg.C (preferably 100 deg.C), performing acidolysis in water bath for 60-180min (preferably 90 min) to remove impurities such as protein, carbohydrate and lipid, removing hydrochloric acid, centrifuging, washing melanin precipitate with water until the water washing solution contains no Cl-。
And washing and centrifuging the crude product of the black sesame melanin subjected to the secondary treatment by adopting an organic solvent to obtain the crude product of the black sesame melanin subjected to the tertiary treatment. Washing with chloroform, ethyl acetate and ethanol sequentially, repeating the step for more than 3 times (preferably 3 times), wherein the ratio of the mass of the crude black sesame melanin to the volume of each organic solvent in the secondary treatment is 1g (5-40) mL (preferably 1g:10 mL), and centrifuging under the following conditions: the rotating speed is 8000-12000r/min (preferably 9000 r/min), and the time is 5-10min (preferably 5 min).
Mixing the mass of the secondary-treated black sesame melanin crude product and the volume of chloroform according to the proportion of 1g: shaking 10mL sufficiently, centrifuging at 9000r/min for 5min to remove supernatant, collecting solid, and repeating the step for 3 times until the supernatant is colorless; and mixing the mass of the solid obtained in the previous step with the volume of ethyl acetate according to the proportion of 1g: shaking 10mL sufficiently, centrifuging at 9000r/min for 5min to remove supernatant, collecting solid, and repeating the step for 3 times until the supernatant is colorless; and finally, mixing the mass of the solid obtained in the last step with the volume of the absolute ethyl alcohol according to the proportion of 1g: shaking 10mL sufficiently, centrifuging at 9000r/min for 5-10min to remove supernatant, collecting solid, i.e. crude product of black sesame melanin processed for three times, repeating the step for 3 times, and obtaining colorless supernatant.
And washing the crude black sesame melanin processed for the third time, and then carrying out vacuum freeze drying to obtain the required pure black sesame melanin. Can be washed for many times, and is freeze-dried in vacuum at-60-80 deg.C and 4-15Pa pressure by vacuum freeze dryer to obtain high-purity black sesame melanin, which can be stored at 4 deg.C. The black sesame melanin with higher purity is prepared through the steps. In the subsequent modification process of the black sesame melanin, the mass ratio of the black sesame melanin to the L-arginine can be 1 (0.5-3); the mass ratio of the black sesame melanin to the DL-arginine can be 1 (0.5-3); the mass ratio of the black sesame melanin to the DL-histidine can be 1 (0.5-3).
The modification is carried out under the conditions of stirring in inert gas nitrogen, the stirring speed is 200-800rpm, the time is 5-20min, and the temperature is 25-37 ℃.
The separation step comprises the following steps of carrying out centrifugal treatment on the modified product to obtain a supernatant, wherein the centrifugal treatment conditions are as follows: the rotating speed is 8000-; and (3) carrying out dialysis treatment on the supernatant, and then carrying out freeze drying, wherein the dialysis treatment conditions comprise: the molecular cut-off of the dialysis bag is 1000-2000D, the dialysis time is 36-48h, and the freeze-drying conditions comprise: the temperature is between 40 ℃ below zero and 45 ℃ below zero, and the pressure is 5-13 Pa.
Through the technical scheme, the black sesame melanin can be effectively modified into the water-soluble melanin, so that the problem that the black sesame melanin is not suitable for preparing oral preparations, food pigment additives, coloring agents, medicines, health-care products and the like because of insolubility in water is solved, and the black sesame melanin modification method has positive significance for expanding the application range of the black sesame melanin.
Example 1
In this embodiment: adding 0.9g of L-arginine into 30mL of distilled water for dissolving, accurately weighing 0.6g of black sesame melanin, respectively feeding the black sesame melanin into a 100mL three-port reactor, switching on a condenser tube and a mechanical stirring device, mechanically stirring at the rotating speed of 200rpm under the atmosphere of inert gas nitrogen at the temperature of 25 ℃, taking out after mixing for 5min, centrifuging for 5min at 11000r/min, and collecting supernatant. And then putting the supernatant into a dialysis bag with the molecular cut-off of 1000D for dialysis for 48 hours, taking out the dialyzed.
Example 2
In this embodiment: adding 0.8g of DL-arginine into 48mL of distilled water for dissolving, accurately weighing 0.4g of black sesame melanin, respectively feeding the materials into a 100mL three-port reactor, connecting a condensation pipe and a mechanical stirring device, mechanically stirring and mixing at 25 ℃ under the atmosphere of inert gas nitrogen at 200rpm for 5min, taking out, centrifuging at 11000r/min for 5min, collecting supernatant, putting the supernatant into a dialysis bag with the molecular cut-off of 1000D for dialysis for 48h, taking out, and drying a finished product by a vacuum freeze dryer under the conditions of-40-45 ℃ and the pressure of 5-13Pa to obtain the solid finished product of the water-soluble black sesame melanin.
Example 3
In this embodiment: dissolving 1g of DL-histidine in 80mL of distilled water, accurately weighing 0.4g of black sesame melanin, respectively feeding the materials into a 100mL three-port reactor, connecting a condensation tube and a mechanical stirring device, mechanically stirring and mixing at 25 ℃ and 200rpm under the atmosphere of inert gas nitrogen for 6min, taking out, centrifuging at 11000r/min for 5min, and collecting the supernatant. And then putting the supernatant into a dialysis bag with the molecular cut-off of 1000D for dialysis for 48 hours, taking out the dialyzed.
Comparative example
The preparation process of the water-soluble melanin comprises the following steps: 0.5g of each of L-arginine, DL-histidine, L-methionine and L-asparagine was dissolved in 40mL of distilled water to give 5 parts of a mixture, and another 40mL of distilled water was prepared as a control without adding an amino acid to give 6 parts, which were charged into 6 100mL three-port reactors. And accurately weighing 6 parts of black sesame melanin, 0.5g of each part, respectively feeding the black sesame melanin into the 6 100mL three-port reactors, connecting a condensation pipe and a mechanical stirring device, and mechanically stirring and mixing for 8min at the temperature of 30 ℃ at 500rpm under the atmosphere of inert gas nitrogen. Then taking out, centrifuging for 10min at 10000r/min, collecting supernatant, diluting with the same times, and measuring OD500Higher absorbance values indicate better dissolution. FIG. 1 shows pairs of different amino acidsThe black sesame melanin modification effect is shown. As shown in FIG. 1, the melanin modified with L-arginine, DL-arginine, and DL-histidine has the highest absorbance, and OD500Values of 0.327, 0.320, 0.18, respectively; and the melanin OD modified by methionine and asparagine500The values are 0.099 and 0.094, respectively, and the modification effect is inferior to that of melanin modified by L-arginine, DL-arginine and DL-histidine.
And (3) putting the supernatant obtained after centrifugation in the step into a 1000D dialysis bag for dialysis for 48 hours, taking out, and carrying out vacuum freeze drying by using a vacuum freeze dryer at the temperature of between 40 ℃ below zero and 45 ℃ below zero and under the pressure of 5 to 13Pa to obtain the solid finished product of the water-soluble black sesame melanin. The solubility experiment of the obtained solid finished product water-soluble black sesame melanin shows that the water-soluble effects of L-arginine/melanin, DL-arginine/melanin and DL-histidine/melanin are better than those of L-methionine/melanin and L-asparagine/melanin.
To further determine the mass ratio of L-arginine, DL-arginine and DL-histidine modified black sesame melanin, the following experiments were performed:
verification example 1
Taking 0.15 g, 0.3g, 0.45g, 0.6g, 0.75g and 0.9g of L-arginine, respectively adding 6 parts of 50mL of distilled water for dissolution, accurately weighing 6 parts of black sesame melanin, wherein each part is 0.3g, respectively feeding the black sesame melanin and the L-arginine in a 100mL three-port reactor (the mass ratio of the black sesame melanin to the L-arginine is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 and 1: 3), measuring OD (optical density) after diluting the obtained 6 parts of solid finished water-soluble black sesame melanin by the same times as example 1, and then measuring the OD500The value is obtained. FIG. 2 is a graph showing the relationship between the ratio of black sesame melanin to L-arginine and the modification ratio according to an exemplary embodiment. As shown in FIG. 2, the amount of the modified melanin dissolved increases when the amount of L-arginine added increases, and the modified melanin can be completely dissolved and OD can be increased when the mass ratio is 1:2.5500The value is 0.600, the maximum is reached, and the amount of L-arginine is increased continuously, so that the OD is obtained500The value no longer increases but decreases instead. Thus, the black sesame melanin and the L-arginine are presentThe mass ratio is preferably 1: 2.5.
Verification example 2
Taking 0.15 g, 0.3g, 0.45g, 0.6g, 0.75g and 0.9g of DL-arginine, respectively adding 6 parts of 50mL of distilled water for dissolution, then accurately weighing 6 parts of black sesame melanin, wherein each part is 0.3g, respectively feeding the black sesame melanin and the DL-arginine in a 100mL three-port reactor (the mass ratio of the black sesame melanin to the DL-arginine is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 and 1: 3), measuring OD (optical density) after diluting the obtained 6 parts of solid finished water-soluble black sesame melanin by the same times as example 1, and then measuring the OD500The value is obtained. FIG. 3 is a graph illustrating the relationship between the ratio of black sesame melanin to DL-arginine in terms of mass and modification rate according to an exemplary embodiment. As shown in FIG. 3, the amount of the modified melanin dissolved was increased when the amount of DL-arginine added was increased, and the modified melanin was completely dissolved at a mass ratio of 1:2, OD500The value reached a maximum of 0.539, and the OD was determined by continuing to increase the amount of DL-arginine500The value no longer increases but decreases instead. As can be seen, the mass ratio of the black sesame melanin to the DL-arginine is preferably 1:2.
Verification example 3
Taking 0.15 g, 0.3g, 0.45g, 0.6g, 0.75g and 0.9g of DL-histidine, respectively adding 6 parts of 50mL of distilled water for dissolution, then accurately weighing 6 parts of black sesame melanin, wherein each part is 0.3g, respectively feeding the black sesame melanin and the DL-histidine in a 100mL three-port reactor (the mass ratio of the black sesame melanin to the DL-histidine is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 and 1: 3), measuring OD (optical density) after diluting the obtained 6 parts of solid finished water-soluble black sesame melanin by the same times as example 1, and then measuring the OD500The value is obtained. FIG. 4 is a graph illustrating the relationship between the ratio of black sesame melanin to DL-histidine by mass and the modification rate according to an exemplary embodiment. As shown in FIG. 4, when the amount of DL-histidine added was increased, the amount of the modified melanin dissolved was increased, and when the mass ratio was 1:1.5, the melanin was completely dissolved and OD was increased500The value is 0.446, the maximum is reached, and the amount of DL-histidine is increased continuously, then the OD500The value no longer increases but decreases instead. As can be seen, the mass ratio of black sesame melanin to DL-histidine is preferably 1: 1.5.
Determination of solubility
The black sesame melanin before and after modification is respectively dissolved in different solutions, and the solubility and the solution color are observed. Table 1 shows the solubility results for black sesame melanin, L-arginine/melanin, DL-arginine/melanin, and DL-histidine/melanin. Wherein more plus signs indicate higher solubility and more minus signs indicate lower solubility. As shown in Table 1, before modification, the black sesame melanin was slightly soluble in water (+ -), dissolved in 1mol/L NaOH aqueous alkaline solution and Dimethylsulfoxide (DMSO) solution (+++), and precipitated (- -) in an acidic solution (1 mol/L HCl). After amino acid modification, L-arginine/melanin, DL-arginine/melanin and DL-histidine/melanin can be dissolved in distilled water (+++), and have good water solubility. Fig. 5 is a graph showing the results of the solubility of black sesame-melanin, L-arginine-melanin, and DL-histidine-melanin provided by an exemplary embodiment.
TABLE 1
Figure 565197DEST_PATH_IMAGE001
Characterization of Water soluble Melanin
Respectively mixing 2mg black sesame melanin before and after modification with 400mg dried KBr, tabletting, and performing by using FT-IR6700 Fourier transform infrared spectrometer at 4000-400 cm-1Infrared spectrum measurement is carried out in the interval.
Infrared spectroscopy can characterize the major functional groups in the melanin structure. Fig. 5 is an infrared spectrum of black sesame melanin provided in an exemplary embodiment. As shown in FIG. 5, the infrared spectrum of black sesame melanin is a series of broad and strong absorption peaks like other natural melanin, and shows the characteristics of biomacromolecule compounds with complex structures, and each broad peak is the result of superposition of various functional group vibrations and lacks of characteristics. The characteristic absorption of the black sesame pigment is mainly as follows: (1) at 3408cm-1Has strong resonance absorption and is formed by-OH and-NH2And this region also belongs to the stretching vibration of-COOH. (2) The absorption is mainly 2900cm between 2940 and-1nearby (2923 cm)-1) Exist relativelyWeak absorption peak, reflecting C-H stretching vibration of alkane structure. (3) At 1618 cm-1The strong absorption peak caused by the vibration of the aromatic ring skeleton is the vibration absorption of the aromatic ring skeleton C = C, which indicates that the benzene ring exists in the molecular structure of the black sesame melanin. (4) 1125-1000 cm-1Where (1075.62 cm)-1) Absorption peak of (2), partially consisting of C-CH3The bending and the skeleton vibration absorption take part in and are formed. (5) At 865-660 cm-1Absorption within the range is weaker, indicating that the aromatic ring is substituted, forming a conjugated system with a lower aromatic hydrogen content. In summary, black sesame melanin may have functional groups of: -OH, -NH2、-COOH、C-H、C=C、C-CH3。
FIG. 6 is an IR spectrum of L-arginine/melanin provided by an exemplary embodiment. As can be seen from the comparative analysis of FIGS. 5 and 6, the black sesame melanin before and after modification has the characteristic absorption peak common to most of melanin, and L-arginine/melanin is 3200 cm-1、1405.42 cm-1、1020 cm-1、622.87 cm-1A characteristic absorption peak of L-arginine is formed; compared with black sesame melanin, at 1432.08 cm-1、1267.99 cm-1The intensity of the peak at L-arginine/black was reduced, probably due to the change in the oscillation of the group after modification with L-arginine. These results indicate that L-arginine has a better modifying effect on black sesame melanin.
Fig. 7 is an infrared spectrum of DL-arginine/melanin provided by an exemplary embodiment. As can be seen from the comparative analysis of FIGS. 5 and 7, the black sesame melanin before and after modification has DL-arginine/melanin at 1020 cm in addition to the characteristic absorption peak common to most of melanin-1A characteristic absorption peak of DL-arginine; and 1432.08 cm of black sesame melanin compared with black sesame melanin-1.The peak of (A) is shifted to the right to 1404.05cm-1At 1269.67cm-1Peak intensity was reduced, 480cm-1The peak disappeared, probably due to the DL-arginine modified group vibration change. These results indicate that DL-arginine has a better modifying effect on black sesame melanin.
FIG. 8 is an exemplary illustrationThe comparative analysis of the IR spectrum of DL-histidine/melanin provided in the example in FIG. 5 and FIG. 8 shows that the DL-histidine/melanin before and after modification has characteristic absorption peak common to most melanin, and is 1020 cm-1、625.55 cm-1A characteristic absorption peak of DL-histidine; and 1432.08 cm compared with black sesame melanin-1The peak of (A) is shifted to the right to 1405.14cm-1It is likely that the group was changed due to the vibration of the group after the modification with DL-histidine. These results indicate that DL-histidine has a better modifying effect on black sesame melanin.

Claims (5)

1. A method for modifying black sesame melanin into water-soluble melanin is characterized by comprising the following steps: mixing amino acid, water and black sesame melanin, reacting under inert atmosphere nitrogen, and separating to obtain water-soluble melanin, wherein the amino acid is one of L-arginine, DL-arginine or DL-histidine, and the mass ratio of the black sesame melanin to the L-arginine to the water is 0.3 g: 0.75 g: 50 mg; the volume ratio of the mass of black sesame melanin, the mass of DL-arginine and water is 0.3 g: 0.6 g: 50 ml; the volume ratio of the mass of black sesame melanin, the mass of DL-histidine to water is 0.3 g: 0.45 g: 50 ml.
2. The method of modifying black sesame melanin to water-soluble melanin according to claim 1, wherein: the reaction conditions are that the reaction is carried out under stirring, the stirring speed is 200-800rpm, the time is 5-20min, and the temperature is 25-37 ℃.
3. The method of modifying black sesame melanin to water-soluble melanin according to claim 1, wherein: the separation process comprises the following steps of carrying out centrifugal treatment on the modified product to obtain a supernatant, wherein the centrifugal treatment conditions are as follows: rotating speed: 8000- & lt12000 r/min, time: 5-10 min; and (3) carrying out dialysis treatment on the supernatant, and then carrying out freeze drying, wherein the conditions of the dialysis treatment are that the molecular interception amount of a dialysis bag is 1000-2000D, the dialysis time is 36-48h, and the conditions of the freeze drying are that the temperature is minus 40 ℃ to minus 45 ℃, and the pressure is 5-13 Pa.
4. The method of modifying black sesame melanin to water-soluble melanin according to claim 1, wherein: the preparation method of the black sesame melanin comprises the following steps:
a. mixing black sesame with alkali liquor, and then carrying out ultrasonic treatment to obtain a first mixed solution;
b. filtering the first mixed solution obtained in the step a to obtain a filtrate product;
c. c, regulating the pH value of the filtrate product obtained in the step b to 1.0-3.0 by using acid liquor, then carrying out centrifugal treatment, and carrying out vacuum freeze drying on the precipitate obtained by centrifugal treatment to obtain a black sesame melanin crude product;
d. c, dissolving the crude black sesame melanin product obtained in the step c in alkali liquor, adjusting the pH to 1.0-3.0 by using acid liquor, and then performing centrifugal treatment to obtain a primary treated crude black sesame melanin product;
e. d, performing acidolysis treatment on the primary treated black sesame melanin crude product obtained in the step d by using an acid solution, and then centrifuging to obtain a secondary treated black sesame melanin crude product;
f. e, washing and centrifuging the secondary-processed black sesame melanin crude product obtained in the step e by adopting an organic solvent to obtain a tertiary-processed black sesame melanin crude product;
g. and f, washing the black sesame melanin treated for three times in the step f, and then carrying out vacuum freeze drying to obtain the pure black sesame melanin.
5. The method of modifying black sesame melanin to water-soluble melanin according to claim 4, wherein:
in the step a, the alkali liquor is 1-3mol/L NaOH solution, and the mass ratio of the black sesame to the volume of the alkali liquor is 1g: (1-5) mL, wherein the ultrasonic treatment condition is that the frequency is 40-60Hz and the time is 40-180 min;
the filtration mode in the step b is suction filtration;
in the step c, the acid solution is 2-8mol/L hydrochloric acid, the rotation speed of centrifugal treatment is 8000-12000r/min, the time of centrifugal treatment is 5-10min, and the conditions of vacuum freeze drying are as follows: the temperature is 60 ℃ below zero to 80 ℃ below zero, and the pressure is 4-15 Pa;
in the step d, the alkali liquor is 1-3mol/L NaOH solution, and the ratio of the mass of the black sesame melanin crude product to the volume of the alkali liquor is 1g: (1-5) mL, wherein the acid solution is 2-8mol/L hydrochloric acid, the rotation speed of the centrifugal treatment is 8000-12000r/min, and the time of the centrifugal treatment is 5-10 min;
the acid solution in the step e is 2-8mol/L hydrochloric acid, and the ratio of the mass of the black sesame melanin crude product to the volume of the acid solution in one-time treatment is 1g: (1-5) mL, wherein the acidolysis treatment is carried out at 90-100 ℃ for 60-180 min;
and f, washing by sequentially adopting chloroform, ethyl acetate and ethanol, wherein the ratio of the mass of the crude black sesame melanin product subjected to secondary treatment to the volume of each organic solvent is 1g (5-40) mL, and the centrifugation condition is as follows: the rotating speed is 8000-;
and g, the vacuum drying condition in the step g is that the temperature is 60 ℃ below zero to 80 ℃ below zero, and the pressure is 4-15 Pa.
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