CN114921111B - Preparation process and application of black rice pigment - Google Patents

Preparation process and application of black rice pigment Download PDF

Info

Publication number
CN114921111B
CN114921111B CN202210621827.9A CN202210621827A CN114921111B CN 114921111 B CN114921111 B CN 114921111B CN 202210621827 A CN202210621827 A CN 202210621827A CN 114921111 B CN114921111 B CN 114921111B
Authority
CN
China
Prior art keywords
blueberry
black rice
reaction
glycoside
blue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210621827.9A
Other languages
Chinese (zh)
Other versions
CN114921111A (en
Inventor
周建芹
刘江云
祁子严
周胜
闫兆威
张健
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou University
Original Assignee
Suzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou University filed Critical Suzhou University
Priority to CN202210621827.9A priority Critical patent/CN114921111B/en
Publication of CN114921111A publication Critical patent/CN114921111A/en
Application granted granted Critical
Publication of CN114921111B publication Critical patent/CN114921111B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/47—Addition of dyes or pigments, e.g. in combination with optical brighteners using synthetic organic dyes or pigments not covered by groups A23L5/43 - A23L5/46
    • A23L5/48—Compounds of unspecified constitution characterised by the chemical process for their preparation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a preparation process of a black rice pigment, which comprises the following steps: s1, weighing blueberry glycoside, neutral hydrophobic aliphatic amino acid and polyphenol oxidase, preparing a mixed aqueous solution, controlling the pH value of the solution to be 4-6, and reacting for 2-10 hours at a constant temperature of 40-80 ℃ under an aeration condition; s2, passing the reaction solution obtained in the step S1 through a macroporous resin chromatographic column, eluting by using water and 30% -80% ethanol in sequence, collecting the corresponding developed ethanol eluent, concentrating under reduced pressure, and drying to obtain the black rice pigment. The preparation process of the black rice pigment provided by the invention takes the blueberry glycoside extracted from the blueberry leaves as a raw material, takes amino acid as a substrate and takes polyphenol oxidase as a catalyst to synthesize the blue-to-blue black novel black rice pigment.

Description

Preparation process and application of black rice pigment
Technical Field
The invention relates to the technical field of foods and dyes, in particular to a preparation process and application of a black rice pigment.
Background
Oriental blueberry (Vaccinium bracteatum Thunb), also known as Vaccinium bracteatum, vaccinium, hedyotis, etc., is a evergreen shrub of Vaccinium genus of Vacciniaceae, and is widely spread in the low elevation mountain region of the Yangtze river. Oriental blueberry is a traditional Chinese medicinal material, and fruits, leaves and roots of Oriental blueberry can be used as medicines. In the south of China, every early in the lunar calendar, people can use the tender leaf juice of the blueberry to soak the glutinous rice or the water boiled by the tender leaf of the blueberry to soak the glutinous rice to prepare special delicious food, namely the blueberry, and the custom is inherited from the Tang dynasty. The black rice is black and bright, contains longan aroma, can stimulate appetite of people, and has the effects of strengthening spleen and tonifying kidney; the black rice can be continuously placed for a plurality of days without deterioration. Tang "Ben Cao Shi Yi" and Ming "Ben Cao gang mu" are recorded in relation to Wu Mian, wherein "Ben Cao gang mu" indicates that: wu Fan can promote the gastrointestinal function, tonify bone marrow, and can lighten the body, improve the eyesight, arrest spontaneous emission and beautify the face after long-term administration.
Compared with artificial pigment, natural pigment is derived from natural resources such as animals, plants, microorganisms and the like, has high biological safety and huge potential market demand, and is widely focused by researchers. Since blueberry leaves have been widely used for dyeing and have been proved to be excellent in effect, a method of extracting natural melanin from blueberry leaves has been a focus of attention in recent years. For example, chinese patent publication No. CN1939979a discloses a method for extracting natural melanin from blueberry leaves, which uses natural plant blueberry leaves as main raw material, and adopts enzyme preparation in combination with solvent to extract natural melanin. Chinese patent publication No. CN103468747a discloses a method for preparing blueberry leaf pigment by enzyme method, which uses fresh blueberry leaves as raw materials, and prepares blueberry leaf pigment by enzyme source preparation, precursor solution preparation, enzymatic reaction and drying steps in sequence. Chinese patent publication No. CN103756354a discloses an extraction process of blueberry leaf pigment, which uses blueberry leaves as raw materials, water as extraction solvent, and adopts ultrasonic-microwave auxiliary technology to extract and prepare blueberry leaf pigment. However, the dyeing process of blueberry leaves is a complex enzymatic reaction, and the effective components and mechanisms of dyeing the blueberry leaves are not clear at present. Moreover, the extraction methods all take blueberry leaves as raw materials, and the components in the fresh blueberry leaves are hundreds of seeds, so that the extraction efficiency of the blueberry pigment is low, the components are complex, the purity is low, and an effective quality control means is lacked, so that the further expansion application of the blueberry leaves is limited.
Disclosure of Invention
The invention aims to provide a novel black rice pigment synthesis process based on polyphenol oxidase catalysis, which takes blueberry glycoside extracted from blueberry leaves as a raw material, takes amino acid as a substrate and takes polyphenol oxidase as a catalyst to synthesize blue-to-blue black rice pigment.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a preparation process of a black rice pigment, which comprises the following steps:
s1, weighing blueberry glycoside, neutral hydrophobic aliphatic amino acid and polyphenol oxidase, preparing a mixed aqueous solution, controlling the pH value of the solution to be 4-6, and reacting for 2-10 hours at a constant temperature of 40-80 ℃ under an aeration condition;
s2, passing the reaction solution obtained in the step S1 through a macroporous resin chromatographic column, eluting by using water and 30% -80% ethanol in sequence, collecting the corresponding developed ethanol eluent, concentrating under reduced pressure, and drying to obtain the black rice pigment.
Further, in the step S1, the blueberry glycoside is extracted from blueberry leaves, and the purity of the blueberry glycoside is more than or equal to 60%.
Further, in step S1, the neutral hydrophobic aliphatic amino acid includes one or more of glycine, glutamine, valine, isoleucine, asparagine, alanine, leucine, and methionine. The neutral hydrophobic aliphatic amino acid, optionally, also includes derivatives or compositions of the amino acid salt, polypeptides containing the amino acid, and the like, having primary amino groups necessary for carrying out the synthesis reaction.
Further, in step S1, the polyphenol oxidase is tyrosinase. Tyrosinase is commercially available, preferably from mushrooms. The preferable ratio of the polyphenol oxidase to the blueberry glycoside is 20-60U/1 mg. The pH of the tyrosinase reaction solution is preferably 4.5 to 5.5. The tyrosinase reaction temperature is preferably 40-50 ℃.
Further, in the step S1, the reaction temperature and the reaction time are divided into two steps, wherein the reaction temperature in the first step is 40-50 ℃ and the reaction time is 2-5 h; the reaction temperature in the second step is 70-80 ℃ and the reaction time is 3-5 h.
The invention also provides the black rice pigment prepared by the preparation process, and the black rice pigment is blue or blue-black and can be used as a dye in the field of food additives.
The invention discovers for the first time that blueberry glycoside can react with 15 amino acids containing primary amino groups to synthesize novel blueberry blue pigment under the catalysis of polyphenol oxidase with monophenol substrate oxidation activity. The reactivity of different amino acids is different, and through detailed researches on the reaction mechanism and rules thereof, it is found that 8 aliphatic neutral amino acids are selected, and the black rice blue pigment can be efficiently synthesized under the catalysis of tyrosinase. The key preparation process parameters are further studied, and the invention is completed.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the blueberry glycoside extracted from blueberry leaves is used as a raw material, neutral hydrophobic aliphatic amino acid is used as a substrate, polyphenol oxidase is used as a catalyst, and the novel blue-to-blue-black blueberry pigment is efficiently synthesized, so that the application of blueberry leaf effective substances is expanded, and a scientific basis is provided for the industrialized application of blueberry pigment.
2. The black rice pigment prepared by the invention is synthesized from natural active substances, is safe and nontoxic, and can be used as an additive in food.
Drawings
FIG. 1 is the effect of reaction pH on tyrosinase-catalyzed synthesis of blueberry glycoside to blueberry;
FIG. 2 is a graph showing the effect of reaction temperature and time on tyrosinase-catalyzed synthesis of blueberry glycoside into blueberry blue;
FIG. 3 is the effect of tyrosinase levels on the conversion of blueberry to blueberry;
FIG. 4 is a graph showing the effect of glycine amount on the amount of black meal blue produced;
FIG. 5 shows the effect of reaction temperature and time on the amount of black meal blue produced;
FIG. 6 is a HPSEC assay of black meal blue (585 nm);
FIG. 7 is a UV diagram of black meal blue;
fig. 8 is an infrared spectrum of dark blue.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
Taking 2mL of 0.2M phosphate buffer solution (pH 6.0) of 0.8mg/mL blueberry glycoside, adding 72U of tyrosinase and 24mg of glycine, carrying out constant temperature reaction for 2h in a water bath at 50 ℃ under the aeration condition, and then heating to 80 ℃ to continue constant temperature reaction for 5h under the aeration condition, so as to prepare the blueberry (VBP) sample solution.
By adopting the method, 100mL of the black rice blue sample solution is amplified and prepared, an AB-8 macroporous resin column (32 multiplied by 3.0 cm) is adopted for loading, separation and purification are carried out, 300mL of deionized water and 200mL of 70% ethanol solution are sequentially used for eluting, the developed 70% ethanol solution eluents are collected, and the black rice blue pigment sample is obtained after merging and concentrating under reduced pressure until the black rice blue pigment sample is dried, and the black rice blue pigment sample is blue black powder.
In the above preparation process, the preparation and analysis and identification of blueberry glycoside (10-p-coumaroyl cytaraxoside) are carried out according to previous reports of the subject group (Zhao Jianping, et al, HPLC method for determining blueberry glycoside content in blueberry leaves, shanghai journal of Chinese medicine, 2017, 51 (10): 100-102), preferably, the purity is not less than 60%. The reference preparation method is as follows: weighing 100g of blueberry leaves, soaking and extracting for 4 times with 1L of methanol for 12 hours each time; mixing the leaching solutions, concentrating at 60deg.C under reduced pressure to near dryness, adding 0.5L deionized water, filtering, loading onto pretreated AB-8 column (column bed volume 2.0L), eluting with water (4.0L), 35% ethanol (4.0L), and 75% ethanol (4.0L) sequentially; collecting 75% ethanol eluate, concentrating under reduced pressure to near dryness, dissolving with 100mL 30% methanol under ultrasound, filtering, loading onto pre-equilibrated C18 column (column volume 350 mL), and sequentially eluting with 30%, 35%, 40%, 60% methanol (300 mL each); detecting by HPLC, mixing target product fractions (35%, 40%), concentrating under reduced pressure to dry to obtain blueberry glycoside (120 mg; purity 83.5%).
In the preparation process, tyrosinase (364U/mg, derived from fungus mushrooms) is purchased from Shanghai Ara Ding Shenghua technology Co., ltd; the other reagents such as amino acid are all analytically pure commercial products. Optionally, tyrosinase may also be selected from other polyphenol oxidases (Ployphenol oxidase, PPO), such as fresh leaves of vaccinium bracteatum and extracts thereof, which enzymes have the property of catalyzing monophenolic substrates. The reaction of the present preparation process may be carried out in a conventional aqueous solution, preferably phosphate buffer is optionally used to adjust the desired pH.
Example 2: investigation of amino acid species
Referring to example 1, 20 amino acids such as glycine were used to prepare a blue black rice sample solution. The black rice blue sample solution was subjected to ultraviolet-visible light full-band scanning (400 to 800 nm) using a UV-2600 ultraviolet-visible spectrophotometer (Shimadzu), and the maximum absorption wavelength and absorbance value A thereof were measured. See table 1.
It can be seen from Table 1 that the kind of amino acid has a significant effect on the synthesis of Wufanblue. For tyrosinase, the specific reaction law of catalytic synthesis of black rice blue is summarized as follows:
(1) Most amino acids can undergo this reaction, including aliphatic neutral, acidic, basic amino acids, and aromatic amino acids, suggesting that the reactive group is the primary amino group of the amino acid.
The blue color value of the black rice synthesized by 8 hydrophobic neutral amino acids is higher (A is more than 0.9), and the black rice is sequentially as follows:
Ile(1.375)>Val>Gln>Gly(1.035)>Asn>Ala>Leu>Met(0.907)
with Gly as a reference, the color value of the product of Ile, val, gln three amino acids is higher than Gly, suggesting that the color value of beta-CH 3 Substitution is beneficial to enhancing the color value of the product.
The blue color value of the black rice synthesized by 7 other amino acids is lower (A, 0.32-0.54), and the black rice is sequentially as follows:
Arg(0.538)>Phe>Lys>His>Tyr(0.416)>Glu>Asp(0.323)
wherein, the aromatic amino acid Phe has higher hydrophobic Phe color value compared with Tyr and His; compared with Gly, it is presumed that the aromatic amino acid has a larger steric hindrance and is unfavorable for color development. The color value of the basic amino acid Arg, lys, his product is basically similar; the products of the acidic amino acids Glu (isoelectric point, pI 3.22), asp (pI 2.77) are weaker in color number, presumably related to the appropriate pH range (4-6) for tyrosinase activity.
(2) No dark blue can be produced with 5 amino acids. The Cys reaction solution is colorless, and the amino acid is the only reducing agent, and PPO is an oxygen-requiring oxidoreductase, so that the amino acid cannot react as a PPO inhibitor. Ser, thr, pro, trp is brown, and is the conventional polyphenol enzymatic browning color catalyzed by PPO, and amino acid does not participate in synthesizing pigment. Wherein Ser and Thr contain nucleophilic-OH, and can react with o-quinone preferentially, so that the polymerization reaction of amino is inhibited; the Pro and Trp structures have only secondary amino groups, and subsequent amino acid polymerization reaction cannot be performed.
TABLE 1 absorbance values of blueberry glycoside and amino acid enzymatic Synthesis of blueberry blue (A max )
PPO is widely found in plants, animals, fungi and is classified into three major classes, monophenol oxidase (tyrosinase), bisphenol oxidase (catechol oxidase), and laccase, depending on the substrate. PPO is an oxidoreductase, and in the presence of oxygen, PPO can catalyze phenolic compounds to convert into quinone, and the quinone and amino acid and related proteins form melanin through polymerization reaction, so that tissue browning is further caused. Based on the above results, it is assumed that the reaction formula of PPO for catalyzing the synthesis of VBP-P by Vac is as follows, taking Gly as an example.
Oxidation of the hydroxy group of p-coumaric acid in vaccinium bracteatum glycosides to ortho-diphenol and further activation to ortho-quinone intermediates under PPO catalysisThe method comprises the steps of carrying out a first treatment on the surface of the The o-quinone intermediate can spontaneously perform complex polymerization reaction under the aerobic condition to form brown; in the presence of an amino acid such as glycine, the o-quinone intermediate is reacted with the-NH of the amino acid 2 The 1, 4-addition reaction and further the subsequent complex polymerization reaction occur, and finally the black rice pigment is formed. The neutral hydrophobic aliphatic amino acid, optionally, may also comprise derivatives or compositions of the amino acid salt, polypeptide/protein containing the amino acid, etc., having primary amino groups necessary for the synthesis reaction to proceed.
In conclusion, the invention discovers for the first time that PPO can catalyze the reaction of blueberry glycoside and amino acid to synthesize novel blueberry pigment. The reactivity of the different amino acids varies, and the reaction mechanism and the rule thereof are summarized above. 8 aliphatic neutral hydrophobic amino acids are selected, and the black rice blue pigment can be efficiently synthesized under the catalysis of tyrosinase.
Example 3: investigation of the parameters of the enzymatic reaction process
A single factor investigation of the synthesis of Wu-Mian blue was performed by the method of example 1, with one process parameter of the enzymatic reaction being adjusted during each set of experiments. The process parameters examined included pH (4.0,5.0,6.0,7.0,8.0), reaction temperature (40, 50, 60, 70, 80 ℃), reaction time (1, 2,3,4,5 h), amount of tyrosinase (18U, 36U, 72U, 145U, 290U), amount of glycine (6, 12, 24, 48, 60, 72 mg). In the reaction, the relative content of the black rice blue is expressed by detecting the absorbance value A of the black rice blue at 585nm by adopting UV-Vis; the conversion of blueberry glycoside is expressed as the percent remaining (RR) and is calculated as the relative percentage of the chromatographic peak area analyzed by HPLC.
(1) Influence of pH on catalytic reactions
As a result of examining the effect of pH on the enzymatic reaction, as shown in FIG. 1, the remaining percentage of blueberry glycoside was small in the pH range of 4 to 6, which is a preferred use range of tyrosinase.
(2) Influence of the temperature and time of the first enzymatic reaction
As shown in FIG. 2, the reaction can be completed within 1-2 hours with the minimum blueberry glycoside remaining at 40-50deg.C. When the reaction is carried out for 1 to 5 hours at the temperature of 60 ℃, the RR values of the blueberry glycoside are all stable at about 10 percent. When the reaction is carried out for 5 hours at 70 ℃, the RR value of the blueberry glycoside is about 18 percent. The result shows that the enzyme is stable in reaction for 1-5 h at 40-60 ℃.
(3) Effect of tyrosinase amount on blueberry glycoside conversion
The effect of tyrosinase levels on the residual percentage of vaccinium bracteatum glycosides at a fixed level (1.6 mg) is shown in FIG. 3. As can be seen from the figure, when the amount of tyrosinase is 18U, the RR value of blueberry glycoside is 57%; as the amount of enzyme increased, the RR value decreased significantly, and at a tyrosinase level of 145U, the blueberry glycoside conversion was complete (RR 1.84%). The dosage of the enzyme is selected to be in a proper condition range of 32-96U (corresponding to 20-60U/mg blueberry glycoside).
(4) Effect of Glycine usage on the amount of dark blue produced
As shown in FIG. 4, when the amount of the added glycine was 6 to 24mg at the time of fixing blueberry glycoside at 1.6mg, the absorbance of the product rapidly increased to the maximum value, so that the amount of added glycine was 10 to 24mg.
(5) Influence of the reaction temperature and time in the second step
In the research of the invention, the unexpected finding that the first step of enzymatic reaction is basically completed for 2 hours, and the second step of later temperature rise is favorable for further improving the completion degree of the black rice blue. As shown in fig. 5, the absorbance value of the product gradually increases with the increase of the temperature of the reaction system and the increase of the reaction time; the black rice blue is generated in a large amount at 70-80 ℃, the reaction speed is high, and the absorbance is basically stabilized at the highest value (A0.88) at 3-5 h. Therefore, the reaction temperature is preferably 80℃and the reaction time is preferably 3 to 5 hours.
In summary, when blueberry glycoside was immobilized at 1.6mg, the optimization conditions selected were: adding 24mg glycine and 32-96U tyrosinase, reacting in 0.2M phosphate buffer solution with pH value of 5, reacting for 2h at constant temperature of 50 ℃ in the first step, and continuously reacting for 3-5 h at the temperature of 70-80 ℃ in the second step.
Example 4: physical and chemical properties investigation of Wufanlan pigment
Taking the black rice blue sample (120 mg) described in the example 1, further separating and purifying by using a Sephadex G-15 (32 multiplied by 3.0 cm) column, eluting by using deionized water, collecting the target product eluent with color development, merging, concentrating under reduced pressure to dryness, and obtaining a purified black rice blue purified sample which is black powder (75 mg).
The following physicochemical property analysis was performed on the sample.
(1) HPSEC analysis
HPSEC analysis: the chromatographic condition is an Agilent 1260 information II liquid phase system, a TSK G2500 PWxl chromatographic column (7.8. 7.8 ID X300 mm,7 μm), and water isocratic elution is carried out for 30min; the flow rate is 0.5mL/min; column temperature 25 ℃; DAD detection wavelengths 585nm, 310nm and 210nm; the sample injection amount was 20. Mu.L.
Referring to fig. 6, the purity of the black meal blue was detected to be approximately 100%.
(2) Ultraviolet-visible spectral analysis
Ultraviolet-visible full-band scanning (400-800 nm) was performed on the black meal blue sample solution using a UV-2600 ultraviolet-visible spectrophotometer (Shimadzu). The maximum absorption wavelength of the dark blue sample was measured at 585nm, see FIG. 7.
Accurately weighing 10mg of black rice blue, and putting deionized water into a 100mL volumetric flask to fix the volume. Precisely 5mL was measured, the volume was set to 10mL with deionized water, and the absorbance A was measured at 585 nm. The average value of A was determined to be 0.453 (C=0.05 mg/mL), and the color value of the dark blue was calculated to be 90.5.
(3) Infrared spectrum
Infrared spectrum scanning of black rice blue (4000-400 cm) with Vertex 70-Hyperion 2000 Fourier transform infrared spectrometer -1 ) See fig. 8.
In FIG. 8, 3356cm -1 Is O-H bond stretching vibration of 2900cm -1 Is C-H bond stretching vibration of 1605cm -1 The bending vibration of the conjugated C-C bond was estimated to be 1281cm -1 1381cm -1 1078cm for C-N stretching vibration -1 Is the tensile vibration of C-O.
(4) In vitro antioxidant Activity assay
Referring to the relevant literature, the effects of black meal blue on two free radicals, ABTS and DPPH, were tested.
Precisely measuring 0.5mL of sample solution, adding 1.0mL of ABTS working solution, mixing, standing at 30 ℃ for 6min, measuring the absorbance Ai at 734nm, and measuring in parallel for 3 times. Calculated as follows:
radical clearance (%) = [1- (a) i －A j )/A 0 ]×100％
Wherein A is 0 To test the absorbance of the sample, A j The absorbance of the blank solvent is equal to that of the radical working fluid. Calculating a half-rate (IC) from a rate curve 50 ). The test results are shown in Table 2, and indicate that the Wufanlan has a remarkable scavenging effect on ABTS free radicals. IC for calculating and eliminating ABTS by using black rice blue and rutin as reference substance 50 The values were 0.0092, 0.1186mg/mL in this order.
Precisely measuring 0.5mL of sample solution, adding 1.0mL of 0.004% DPPH methanol solution, mixing, performing light-proof reaction at room temperature for 30min, measuring the light absorption value Ai at 517nm, and performing parallel measurement for 3 times. The clearance of DPPH free radical was calculated for each sample by the same method as the above formula, and the test results are shown in Table 3. The results in Table 3 show that the dark blue has a significant scavenging effect on DPPH free radicals. IC for calculating DPPH (digital PH) removal by black rice blue and rutin as reference substance 50 The values were 0.0141 mg/mL, 0.1216mg/mL in this order.
TABLE 2 clearance of dark blue to ABTS (734 nm)
TABLE 3 clearance of dark blue to DPPH (517 nm)
In summary, the invention provides a preparation process of black rice pigment, which utilizes blueberry glycoside extracted from blueberry leaves as a raw material, preferably amino acid as a substrate and polyphenol oxidase as a catalyst to synthesize novel blue-to-blue-black rice pigment. The invention also carries out intensive research on the amino acid enzymatic synthesis mechanism of the black rice blue, carries out investigation optimization and antioxidation activity detection on the preparation method and process of the black rice blue pigment, and provides scientific basis for developing new products of the black rice pigment and promoting the realization of innovative application thereof.
The preparation process of the invention is simple, and the obtained novel black rice pigment is safe and nontoxic, can be used as a food additive, and expands the application range of the black rice leaves.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (4)

1. A preparation process of a black rice pigment is characterized by comprising the following steps:
s1, weighing blueberry glycoside, neutral hydrophobic aliphatic amino acid and polyphenol oxidase, preparing a mixed aqueous solution, controlling the pH value of the solution to be 4-6, and reacting for 2-10 hours at a constant temperature of 40-80 ℃ under an aeration condition;
s2, passing the reaction solution obtained in the step S1 through a macroporous resin chromatographic column, eluting by using water and 30% -80% ethanol in sequence, collecting the corresponding ethanol eluent with color development, concentrating under reduced pressure, and drying to obtain the black rice pigment;
in step S1, the neutral hydrophobic aliphatic amino acid is selected from one or more of glycine, glutamine, valine, isoleucine, asparagine, alanine, leucine and methionine;
in the step S1, the reaction temperature and the reaction time are divided into two steps, wherein the reaction temperature of the first step is 40-50 ℃ and the reaction time is 2-5 hours; the second step of reaction is carried out at the temperature of 70-80 ℃ for 3-5 hours;
in the step S1, the polyphenol oxidase is tyrosinase;
in the step S1, the dosage ratio of the polyphenol oxidase to the blueberry glycoside is 20-60U: 1mg.
2. The process for preparing a black rice pigment according to claim 1, wherein in step S1, the blueberry glycoside is extracted from blueberry leaves and has a purity of not less than 60%.
3. The process for preparing a black rice pigment according to claim 1, wherein in step S1, the pH of the solution is 4.5 to 5.5.
4. A black rice pigment prepared by the preparation process according to any one of claims 1 to 3.
CN202210621827.9A 2022-06-02 2022-06-02 Preparation process and application of black rice pigment Active CN114921111B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210621827.9A CN114921111B (en) 2022-06-02 2022-06-02 Preparation process and application of black rice pigment

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210621827.9A CN114921111B (en) 2022-06-02 2022-06-02 Preparation process and application of black rice pigment

Publications (2)

Publication Number Publication Date
CN114921111A CN114921111A (en) 2022-08-19
CN114921111B true CN114921111B (en) 2024-04-05

Family

ID=82813442

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210621827.9A Active CN114921111B (en) 2022-06-02 2022-06-02 Preparation process and application of black rice pigment

Country Status (1)

Country Link
CN (1) CN114921111B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102747105A (en) * 2011-04-19 2012-10-24 浙江医药股份有限公司新昌制药厂 Method for preparing water-soluble plant melanin
CN103408964A (en) * 2013-08-15 2013-11-27 宜兴市红豆杉生态科技有限公司 Method for improving extraction yield of melanin in Vaccinium bracteatum Thunb by using polyphenol oxidase and tea polyphenol
CN107245251A (en) * 2017-06-07 2017-10-13 苏州大学 A kind of preparation method and application of blueberry leaf and its extract
CN109097413A (en) * 2018-08-01 2018-12-28 江南大学 A method of the different indigo pigments of tone are prepared based on Vaccinium bracteatum Thunb. Leaves
CN113383892A (en) * 2021-07-01 2021-09-14 苏州求是本草健康科技有限公司 Preparation method and application of dried leaves of oriental blueberry

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102747105A (en) * 2011-04-19 2012-10-24 浙江医药股份有限公司新昌制药厂 Method for preparing water-soluble plant melanin
CN103408964A (en) * 2013-08-15 2013-11-27 宜兴市红豆杉生态科技有限公司 Method for improving extraction yield of melanin in Vaccinium bracteatum Thunb by using polyphenol oxidase and tea polyphenol
CN107245251A (en) * 2017-06-07 2017-10-13 苏州大学 A kind of preparation method and application of blueberry leaf and its extract
CN109097413A (en) * 2018-08-01 2018-12-28 江南大学 A method of the different indigo pigments of tone are prepared based on Vaccinium bracteatum Thunb. Leaves
CN113383892A (en) * 2021-07-01 2021-09-14 苏州求是本草健康科技有限公司 Preparation method and application of dried leaves of oriental blueberry

Also Published As

Publication number Publication date
CN114921111A (en) 2022-08-19

Similar Documents

Publication Publication Date Title
Yang et al. Effect of methylation on the structure and radical scavenging activity of polysaccharides from longan (Dimocarpus longan Lour.) fruit pericarp
CN105431491B (en) From Geniposide or the colouring agent compound of the material containing Geniposide
CN109694886B (en) Green tea fermentation filtrate and preparation method and application thereof
Huang et al. Structural elucidation and osteogenic activities of two novel heteropolysaccharides obtained from water extraction residues of Cibotium barometz
KR20160089257A (en) Development of skin-care products Based on Antioxidative effect of Abeliophyllum distichum Nakai
Poomanee et al. Optimizing anthocyanins extraction and the effect of cold plasma treatment on the anti‐aging potential of purple glutinous rice (Oryza sativa L.) extract
TW201641100A (en) Use of 3'-hydroxygenistein for manufacturing composition to inhibit melanogenesis
CN114921111B (en) Preparation process and application of black rice pigment
Prasad et al. Exploring the nutritional profiling and health benefits of Palmyra palm haustorium
CN108619016B (en) Whitening and wrinkle-improving application of aquilaria sinensis
KR20130093371A (en) Novel preparation method of mulberry leaf extract for anti-hypertensive, anti-diabetic, and anti-aging and the product of the same
Qi et al. Metabolomic and transcriptomic analysis reveal high solar irradiance inhibited the melanin formation in persimmon fruit peel
Hsiao et al. Physicochemical properties and protective effects on UVA-induced photoaging in Hs68 cells of Pleurotus ostreatus polysaccharides by fractional precipitation
CN108578317B (en) Method for extracting active ingredients from bamboo leaves and skin care application of active ingredients
Yang et al. Salt interferences to metabolite accumulation, flavonoid biosynthesis and photosynthetic activity in Tetrastigma hemsleyanum
KR0168485B1 (en) The production of ginseng steamed red essence
CN114989324A (en) Preparation method and application of black garlic polysaccharide
JP7461470B2 (en) Rice peptide with whitening function and its preparation method
CN114409738A (en) Preparation method and application of multifunctional pinctada martensii source whitening peptide
KR20080034712A (en) Method of preparating composition comprising fermented soybean and peptide derived therefrom
KR20040108226A (en) Compositions of the cosmetic product containing the germinated black bean and/or black rice peptides that prepared by enzymatic hydrolysis
KR20190063600A (en) Composition for skin whitening comprising extract of stichopus japonicas red
KR102363902B1 (en) Manufacturing method of soy sauce and sogseong doenjang using mshroom mycelia
KR102557683B1 (en) Fermentation composition of sprouts of mountain-cultivated ginseng having increased ginsenoside Rg3 and Protopanaxadiol, Rutin, Chlorogenic acid and Essential amid acids by using Tricholoma matsutake mycelium and preparation method thereof
JP4223410B2 (en) Cosmetics

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant