CN113698473A - Phycocyanin stability and acidolysis modification research method thereof - Google Patents
Phycocyanin stability and acidolysis modification research method thereof Download PDFInfo
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- CN113698473A CN113698473A CN202110930326.4A CN202110930326A CN113698473A CN 113698473 A CN113698473 A CN 113698473A CN 202110930326 A CN202110930326 A CN 202110930326A CN 113698473 A CN113698473 A CN 113698473A
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Abstract
The invention discloses a research method for stability and acidolysis modification of phycocyanin, belonging to the technical field of preparation and application of natural pigments; a method for researching stability and acidolysis modification of phycocyanin comprises taking phycocyanin as raw material, systematically researching influence factors of stability of phycocyanin, and researching thermal degradation rule thereof by thermal degradation kinetics; then, the phycocyanin is modified by a certain modification means and the stability change of the phycocyanin is researched, so that the change of the internal structure before and after the modification of the phycocyanin is discussed. The invention starts from two aspects of thermal instability and acid instability, successfully explores the method for improving the thermal stability and the acid stability of the phycocyanin, and provides a new idea for the practical application and popularization of the phycocyanin.
Description
Technical Field
The invention relates to the technical field of preparation and application of natural pigments, in particular to a research method for stability and acidolysis modification of a phycocyanin.
Background
Spirulina is an aquatic plant with high nutritive value and biological utilization value, and is commonly used in the fields of animal feed and cosmetics, and is rich in high-quality protein and natural pigment components, so that it has attracted much attention in the field of food. The spirulina mainly contains phycocyanin, allophycocyanin and phycoerythrin; the phycocyanin (also called phycocyanin) is water-soluble plant protein, and can be divided into the following according to the source: C-PC (obtained from blue algae), R-PC (obtained from red algae) and R-PCII (obtained from symbiotic cocci); the phycocyanin is one of two natural blue pigments allowed to be used in GB 2760-2014 as a natural pigment with a health care function. The pigment protein complex formed by connecting the tetrapyrrole chromophore and the prosthetic group protein has unique color and rich nutrition, has multiple physiological functions of oxidation resistance, tumor resistance, inflammation resistance and the like, and has wide development and application prospects.
However, the phycocyanin is unstable under high temperature and acidic environment, is easy to fade and generates precipitates, so the application of the phycocyanin in food and cosmetics, especially acidic food is severely restricted, in order to solve the problems, the invention provides a research method for stability and acidolysis modification of the phycocyanin, the phycocyanin is taken as a research object, the stability influence factors of the phycocyanin are studied in detail, and a method for improving the stability of the phycocyanin is researched from two aspects of thermal instability and acid instability, so that a new idea is provided for practical application and popularization of the phycocyanin.
Disclosure of Invention
The invention aims to provide a research method for stability and acidolysis modification of phycocyanin, which takes phycocyanin as a research object, examines the stability influence factors in detail, starts from two aspects of thermal instability and acid instability, explores a method for improving the stability of phycocyanin and provides a new idea for practical application and popularization of phycocyanin.
In order to achieve the purpose, the invention adopts the following technical scheme:
a research method for stability and acidolysis modification of phycocyanin comprises the following steps:
s1, accurately weighing a certain amount of phycocyanin by taking the phycocyanin as a raw material to prepare a phycocyanin solution with a proper concentration;
s2, taking the storage rate of the pigment as an index, combining the chromaticity change, systematically researching the influence of environmental factors and additive types on the stability of the phycocyanobilin solution experimental group, and analyzing the influence factors of the stability of the phycocyanobilin;
s3, placing the phycocyanin solution into a test tube with a plug, adding a protein protective agent into the test tube of the experimental group, and fully shaking and uniformly mixing;
s4, placing the test tubes of the experimental group treated in the S3 in water bath at 65 ℃ for 1h, taking out, and immediately cooling with ice water;
s5, measuring the absorbance value A of each experimental group in S4, calculating the pigment storage rate according to the absorbance value A, further analyzing the influence of the protein protective agent on the thermal stability of the phycocyanin, and simultaneously further researching the thermal degradation rule of the phycocyanin and the influence of the effective protective agent on the dynamics and thermodynamic parameters of the thermal degradation process of the phycocyanin;
s6, designing a single-factor and orthogonal experiment, optimizing the algae blue pigment acidolysis process to obtain an acidolysis product, researching the characteristics of the acidolysis product, and simultaneously further adding polyphenol as a color enhancer to research the stability of the product;
s7, characterizing the primary phycocyanin, the acidolysis modified phycocyanin obtained in the S6 and the internal structure change of the complex of the primary phycocyanin and the polyphenol through a Fourier infrared, laser particle size distribution instrument, a circular dichroism spectrum and an atomic force microscope;
and S8, based on the characterization information obtained in S7 and combined with the experimental contents in S1-S6, finishing and analyzing to obtain the stability of the phycocyanin and the acidolysis modification research method thereof.
Preferably, the environmental factors and the types of additives mentioned in S2 specifically include temperature, pH, light, metal ions, redox agents and food additives; the calculation formula of the pigment preservation rate is as follows:
in the formula, A1Denotes the final absorbance value, A0The initial absorbance value is indicated.
Preferably, when the thermal degradation rule of the phycocyanin and the influence of an effective protective agent on the dynamics and thermodynamic parameters of the thermal degradation process of the phycocyanin are researched, in order to further research the mechanism of the protective agent for improving the thermal stability of the phycocyanin, the phycocyanin solutions added with different protective agents are respectively heated at 65 ℃, 70 ℃, 75 ℃ and 80 ℃, samples are taken every 5min, the content of the phycocyanin is determined after the solutions are rapidly cooled, and the result of the phycocyanin solution without the protective agent under the same heat treatment condition is used as a blank control; drawing a thermal degradation curve to accord with a first-level dynamic model
ln(p/p0)＝-kt
In the formula: p0 is initial concentration of phycocyanin, mg/mL; p is the concentration of the blue algae after heating for t time, mg/mL; k is the first order reaction rate constant, min-1(ii) a t is heating time;
the half-life period is calculated by
t1/2＝ln2/k
Thermodynamically, the relationship between the chemical reaction rate constant and the temperature change is usually expressed by an arrhenius equation, the logarithm of the thermal degradation rate constant and the reciprocal of the corresponding absolute temperature are respectively regressed to form an arrhenius diagram, and the activation energy Ea can be calculated according to the slope of the obtained regression line:
lnk＝lnA-Ea/RT
other thermodynamic parameters can be calculated by combining Eying transition state theory
△H＝Ea-RT
△S＝R[lnA-ln(kB/hp)-lnT]
△G＝△H-T△S
In the formula: a is pre-dactylic factor, min-1(ii) a R is a gas constant of 8.314J/mol.K; t is the temperature in Kelvin, K; kB is Boltzmann constant; hp is the Planck constant; ea is activation energy, J/mol; Δ H is the enthalpy of activation, J/mol; delta S is activation entropy, J/mol.K; delta G is Gibbs free energy, J/mol.
Preferably, the single-factor and orthogonal test mentioned in S6 specifically includes the following steps:
a1, accurately weighing a proper amount of phycocyanin, mixing the phycocyanin with hydrochloric acid in proportion, and placing at room temperature for magnetic stirring for acidolysis reaction;
a2, stirring and carrying out acidolysis on the mixed solution in the S1 for a corresponding time according to experimental needs, and adding a proper amount of purified water to dilute the mixed solution after the specified time is reached so as to stop acidolysis reaction;
a3, treating the acidolysis reaction time, the mass ratio of the phycocyanin to the acid and the hydrochloric acid concentration mentioned in A1-A2 as experimental factors by using the experimental factors as single-factor experimental variables or adopting an orthogonal method, and repeating the operations in A1-A2;
a4, placing the diluted mixed solution on a centrifuge for centrifugal treatment, taking the precipitate for redissolving with water, adjusting the pH value to be neutral, cooling and drying to obtain acidolysis modified phycocyanin, and measuring the quality of the product;
a5, preparing pigment solution with pH of 2.5 with certain concentration from acidolysis modified phycocyanin obtained in A4, measuring chromatic aberration and scanning at full wavelength, and measuring the product quality and chromatic aberration b*The value is used as an index to determine the optimal acidolysis time.
Preferably, the characterization of the internal structural changes of the proto-phycocyanin and the acidolysis modified phycocyanin obtained in S6 and the polyphenol complex thereof mentioned in S7 specifically includes amino acid determination and evaluation of the acidolysis modified phycocyanin, particle size determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, microstructure determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, fourier infrared determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, and circular dichroism determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, wherein the amino acid evaluation adopts a Bano calculation method, and the calculation formula is as follows:
AAS＝Ax/As×100％
wherein AAS refers to amino acid score; ax refers to the amount of certain amino acid contained in the protein to be detected, mg/g; as refers to the amount of an amino acid in the FAO/WHO scoring scale, mg/g.
Compared with the prior art, the invention provides a research method for stability and acidolysis modification of phycocyanin, which has the following beneficial effects:
(1) starting from factors influencing the stability of the phycocyanin, the invention adopts a spectrophotometry to establish an evaluation method taking the storage rate of the pigment as an index; the influence of temperature, pH, illumination, metal ions and redox on the stability of the phycocyanin is researched, the temperature and the pH which have the greatest influence on the preservation rate of the phycocyanin are researched, the phycocyanin is kept below 40 ℃ and pH 4-6 as much as possible, and the influence in an environment with strong illumination is avoided;
(2) starting from the thermal stability of the phycocyanin, researching the influence of polyhydroxy compounds (saccharides and polyhydric alcohols) on the thermal stability of the phycocyanin by adopting a kinetic method and combining thermodynamic parameters, researching the thermal degradation kinetic rule of the phycocyanin, and analyzing by combining a fluorescence spectrum, the invention proves that the protective effect of the saccharides and the polyhydric alcohols on the phycocyanin under the heating condition is combined with the prosthetic group protein part of the phycocyanin by hydrogen bonds, thereby protecting the stability of the prosthetic group protein structure;
(3) aiming at the acid stability of the phycocyanin, the invention carries out acidolysis modification on the phycocyanin so as to solve the problems of instability and precipitation of the phycocyanin under the acidic condition, and the optimal technology of acidolysis of the phycocyanin is determined through experimental research; meanwhile, the properties of the acidolysis modified phycocyanin and the complex of the phycocyanin and polyphenol are further researched, the thermal stability of the phycocyanin is effectively improved, and the DPPH clearance rate, the total reducing power and the ABTS clearance rate of the acidolysis modified phycocyanin are all improved;
(4) the invention characterizes the change of the internal structure of the acidolysis modified phycocyanin and the compound of the acidolysis modified phycocyanin and polyphenol, researches prove that the acidolysis modification has little response to the relative content of amino acids of the phycocyanin, the first limiting amino acid is lysine, and the researches find that the phycocyanin after acidolysis modification has better solubility in an acid solution.
In conclusion, the invention takes the phycocyanin as a research object, carries out detailed investigation on the stability influence factors of the phycocyanin, and successfully explores a method for improving the thermal stability and the acid stability of the phycocyanin from the aspects of thermal instability and acid instability, thereby providing a new idea for the practical application and popularization of the phycocyanin.
Drawings
FIG. 1 is a schematic overall flow chart of a research method for stability and acidolysis modification of phycocyanin provided by the invention.
FIG. 2 is a schematic view of the process flow of acidolysis modified phycocyanin preparation in example 2 of the study method for stability and acidolysis modification of phycocyanin of the present invention;
FIG. 3 is a schematic diagram of a phycocyanin solution before and after acidolysis modification in example 2 of the research method for stability and acidolysis modification of phycocyanin according to the present invention;
FIG. 4 is a color representation diagram of an acidolysis modified phycocyanin solution in example 2 of the research method for stability and acidolysis modification of phycocyanin provided by the present invention;
FIG. 5 is a schematic view of the process flow of the preparation of the complex in example 2 of the research method for stability and acid hydrolysis modification of phycocyanin according to the present invention;
FIG. 6 is a schematic diagram of a material object of the stability of phycocyanin and its acidolysis modification developed under different pH conditions before and after compounding in example 2 of the research method for its stability and acidolysis modification;
FIG. 7 is a schematic diagram showing the effect of temperature on the stability of pc-AHpc (A), pc-AHpc-TA (B), and pc-AHpc-RA (C) in example 2 of the research method for stability and acid hydrolysis modification of phycocyanin provided in the present invention;
FIG. 8 is a schematic diagram showing the effect of temperature on the color development effect of phycocyanin in example 2 of the method for studying stability and acid hydrolysis modification of phycocyanin according to the present invention;
FIG. 9 is a schematic diagram showing the effect of sunlight irradiation (A) and ultraviolet irradiation (B) on the stability of phycocyanin complex in example 2 of the study method of stability and acid hydrolysis modification of phycocyanin according to the present invention;
FIG. 10 is an Atomic Force Microscope (AFM) chart of pc (A), AHpc (B), TA-AHpc (C), and RA-AHpc (D) in example 3 of the present invention;
FIG. 11 is an infrared spectrum of pc, AHpc, TA-AHpc, RA-AHpc (left), TA-AHpc, RA-AHpc, TA, RA (right) in example 3 of the method for studying stability and acid hydrolysis modification of phycocyanin according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1:
please refer to fig. 1;
a research method for stability and acidolysis modification of phycocyanin comprises the following steps:
s1, accurately weighing a certain amount of phycocyanin by taking the phycocyanin as a raw material to prepare a phycocyanin solution with a proper concentration;
s2, taking the storage rate of the pigment as an index, combining the chromaticity change, systematically researching the influence of environmental factors and additive types on the stability of the phycocyanobilin solution experimental group, and analyzing the influence factors of the stability of the phycocyanobilin;
the environmental factors and the types of additives mentioned in S2 specifically include temperature, pH, light, metal ions, redox agents and food additives; the formula for calculating the pigment preservation rate is as follows:
in the formula, A1Denotes the final absorbance value, A0Represents the initial absorbance value;
s3, placing the phycocyanin solution into a test tube with a plug, adding a protein protective agent into the test tube of the experimental group, and fully shaking and uniformly mixing;
s4, placing the test tubes of the experimental group treated in the S3 in water bath at 65 ℃ for 1h, taking out, and immediately cooling with ice water;
s5, measuring the absorbance value A of each experimental group in S4, calculating the pigment storage rate according to the absorbance value A, further analyzing the influence of the protein protective agent on the thermal stability of the phycocyanin, and simultaneously further researching the thermal degradation rule of the phycocyanin and the influence of the effective protective agent on the dynamics and thermodynamic parameters of the thermal degradation process of the phycocyanin;
S3-S5 are respectively heating the phycocyanin solution added with different protective agents at the conditions of 65 ℃, 70 ℃, 75 ℃ and 80 ℃ respectively, sampling every 5min, rapidly cooling and then determining the content of the phycocyanin, and taking the result of the phycocyanin solution without the protective agent under the same heat treatment condition as blank control; drawing a thermal degradation curve to accord with a first-level dynamic model
ln(p/p0)＝-kt
In the formula: p0 is initial concentration of phycocyanin, mg/mL; p is the concentration of the blue algae after heating for t time, mg/mL; k is the first order reaction rate constant, min-1(ii) a t is heating time;
the half-life period is calculated by
t1/2＝ln2/k
Thermodynamically, the relationship between the chemical reaction rate constant and the temperature change is usually expressed by an arrhenius equation, the logarithm of the thermal degradation rate constant and the reciprocal of the corresponding absolute temperature are respectively regressed to form an arrhenius diagram, and the activation energy Ea can be calculated according to the slope of the obtained regression line:
lnk＝lnA-Ea/RT
other thermodynamic parameters can be calculated by combining Eying transition state theory
△H＝Ea-RT
△S＝R[lnA-ln(kB/hp)-lnT]
△G＝△H-T△S
In the formula: a is pre-dactylic factor, min-1(ii) a R is a gas constant of 8.314J/mol.K; t is the temperature in Kelvin, K; kB is Boltzmann constant; hp is the Planck constant; ea is activation energy, J/mol; Δ H is the enthalpy of activation, J/mol; delta S is activation entropy, J/mol.K; Δ G is Gibbs free energy, J-mol；
S6, designing a single-factor and orthogonal experiment, optimizing the algae blue pigment acidolysis process to obtain an acidolysis product, researching the characteristics of the acidolysis product, and simultaneously further adding polyphenol as a color enhancer to research the stability of the product;
the single factor and orthogonal test mentioned in S6 specifically includes the following steps:
a1, accurately weighing a proper amount of phycocyanin, mixing the phycocyanin with hydrochloric acid in proportion, and placing at room temperature for magnetic stirring for acidolysis reaction;
a2, stirring and carrying out acidolysis on the mixed solution in the S1 for a corresponding time according to experimental needs, and adding a proper amount of purified water to dilute the mixed solution after the specified time is reached so as to stop acidolysis reaction;
a3, taking the acidolysis reaction time, the mass ratio of the phycocyanin to the acid and the hydrochloric acid concentration mentioned in A1-A2 as experimental factors, treating the experimental factors as single-factor experimental variables or by adopting an orthogonal method, and repeating the operations in A1-A2;
a4, placing the diluted mixed solution on a centrifuge for centrifugal treatment, taking the precipitate for redissolving with water, adjusting the pH value to be neutral, cooling and drying to obtain acidolysis modified phycocyanin, and measuring the quality of the product;
a5, preparing pigment solution with pH of 2.5 with certain concentration from acidolysis modified phycocyanin obtained in A4, measuring chromatic aberration and scanning at full wavelength, and measuring the product quality and chromatic aberration b*Determining the optimal acidolysis time by taking the value as an index;
s7, characterizing the primary phycocyanin, the acidolysis modified phycocyanin obtained in the S6 and the internal structure change of the complex of the primary phycocyanin and the polyphenol through a Fourier infrared, laser particle size distribution instrument, a circular dichroism spectrum and an atomic force microscope;
the characterization of the change of the internal structures of the proto-algal blue pigment and the acidolysis modified algal blue pigment obtained in the step S7 and the compound of the protolysis modified algal blue pigment and the polyphenol compound thereof specifically comprises the measurement and evaluation of amino acid of the acidolysis modified algal blue pigment, the measurement of the particle size of the acidolysis modified algal blue pigment and the polyphenol compound thereof, the measurement of the microstructure of the acidolysis modified algal blue pigment and the polyphenol compound thereof, the Fourier infrared measurement of the acidolysis modified algal blue pigment and the polyphenol compound thereof and the circular dichroism measurement of the acidolysis modified algal blue pigment and the polyphenol compound thereof, wherein the amino acid evaluation adopts a Bano calculation method, and the calculation formula is as follows:
AAS＝Ax/As×100％
wherein AAS refers to amino acid score; ax refers to the amount of certain amino acid contained in the protein to be detected, mg/g; as refers to the amount of a certain amino acid in FAO/WHO scoring standard, mg/g;
and S8, based on the characterization information obtained in S7 and combined with the experimental contents in S1-S6, finishing and analyzing to obtain the stability of the phycocyanin and the acidolysis modification research method thereof.
Starting from factors influencing the stability of the phycocyanin, the invention adopts a spectrophotometry to establish an evaluation method taking the storage rate of the pigment as an index; the influence of temperature, pH, illumination, metal ions and redox on the stability of the phycocyanin is researched, the temperature and the pH which have the greatest influence on the preservation rate of the phycocyanin are researched, the phycocyanin is kept below 40 ℃ and pH 4-6 as much as possible, and the influence in an environment with strong illumination is avoided; starting from the thermal stability of the phycocyanin, researching the influence of polyhydroxy compounds (saccharides and polyhydric alcohols) on the thermal stability of the phycocyanin by adopting a kinetic method and combining thermodynamic parameters, researching the thermal degradation kinetic rule of the phycocyanin, and analyzing by combining a fluorescence spectrum, the invention proves that the protective effect of the saccharides and the polyhydric alcohols on the phycocyanin under the heating condition is combined with the prosthetic group protein part of the phycocyanin by hydrogen bonds, thereby protecting the stability of the prosthetic group protein structure; aiming at the acid stability of the phycocyanin, the invention carries out acidolysis modification on the phycocyanin so as to solve the problems of instability and precipitation of the phycocyanin under the acidic condition, and the optimal technology of acidolysis of the phycocyanin is determined through experimental research; meanwhile, the properties of the acidolysis modified phycocyanin and the complex of the phycocyanin and polyphenol are further researched, the thermal stability of the phycocyanin is effectively improved, and the DPPH clearance rate, the total reducing power and the ABTS clearance rate of the acidolysis modified phycocyanin are all improved; the invention characterizes the change of the internal structure of the acidolysis modified phycocyanin and the compound of the acidolysis modified phycocyanin and polyphenol, researches prove that the acidolysis modification has little response to the relative content of amino acids of the phycocyanin, the first limiting amino acid is lysine, and the researches find that the phycocyanin after acidolysis modification has better solubility in an acid solution. In conclusion, the invention takes the phycocyanin as a research object, carries out detailed investigation on the stability influence factors, and successfully explores the method for improving the thermal stability and the acid stability of the phycocyanin from the aspects of thermal instability and acid instability, thereby providing a new idea for the practical application and popularization of the phycocyanin.
Example 2:
referring to FIGS. 2-9, the process flow of the acid hydrolysis modified phycocyanin preparation process is as follows, based on example 1, but different from that shown in FIG. 2:
1.1 weighing a proper amount of phycocyanin, mixing the phycocyanin and hydrochloric acid according to the mass ratio of 1:3, placing the mixture at room temperature for magnetic stirring for acidolysis, selecting the concentration of the hydrochloric acid to be 12mol/L, reacting for 2.5h, adding a proper amount of water for dilution to stop the reaction, placing the mixture in a centrifuge for 5000r/min for centrifugation for 30min, taking the precipitate, redissolving the precipitate with water, adjusting the pH value to be neutral, and freeze-drying to obtain acidolysis modified phycocyanin;
1.2 the acidolysis modified phycocyanin obtained by the method has better solubility under an acidic condition, particularly still presents bright blue under the condition of pH2.0-3.0, and has obviously improved thermal stability and light stability (as shown in figure 3);
1.3 the color and stability of the acidolysis modified phycocyanin obtained by the method are obviously improved in the environment with the pH value of 2.0-4.0, and the color is not good under other pH conditions (as shown in figure 4, wherein A-I are respectively pH values of 2.0-10.0);
2.1 compounding the proto-algal blue pigment, the acidolysis modified algal blue pigment and the specifically optimized polyphenol in the aqueous solution to form a compound of the proto-algal blue pigment, the acidolysis modified algal blue pigment and the polyphenol, wherein the compound has higher solubility and better color generation effect under an acidic condition, enhances the stability to acid, heat and light, and can keep good solubility and brilliant blue under the condition of pH2.0-10.0. The method is characterized by comprising the following steps of preferably and fully mixing protophycocyanin, acidolysis modified phycocyanin and tannic acid in a mass ratio of 30:5:1-3:2:1, protophycocyanin, acidolysis modified phycocyanin and rosmarinic acid in a mass ratio of 30:5:2-3:2:2 (note that protophycocyanin, protophycocyanin-acidolysis modified phycocyanin-tannic acid compound and protophycocyanin-acidolysis modified phycocyanin-rosmarinic acid compound are respectively expressed by pc, pc-AHpc-TA and pc-AHpc-RA);
2.2 preparing the compound according to the process shown in FIG. 5, it was found that the compound can maintain deeper blue color and stronger stability under the condition of pH2.0-10.0 (as shown in FIG. 6, A-I are pH2-10, respectively);
2.3 compared with original phycocyanin and acidolysis modified phycocyanin, the compound prepared in 2.2 has obviously improved acid stability, thermal stability and light stability (as shown in fig. 6-8);
2.4, the contents of 2.3 and 6-8 are combined to obviously obtain the compound aqueous solution which has higher computational stability, thermal stability and light stability, can well overcome a plurality of instability defects of the original phycocyanin, and has important significance for popularization and application of the phycocyanin.
In conclusion, the invention adopts a hydrochloric acid moderate acidolysis method to accurately modify the phycocyanin, determines the optimal modification process by investigating the color generation effect of the phycocyanin under different acidolysis conditions, and utilizes the optimized polyphenol color increasing and protecting effects, namely: the original algae blue pigment, the acidolysis modified algae blue pigment and the polyphenol are compounded according to a reasonable proportion, and the stable brilliant blue of the algae blue pigment compound is kept under the condition of the full range of pH 2.0-pH10.0 by utilizing the interaction among the original algae blue pigment, the acidolysis modified algae blue pigment and the polyphenol through optimization and compounding, and the thermal stability and the light stability of the algae blue pigment compound are also obviously improved.
Example 3:
referring to fig. 10-11, based on the example 1-2, but different from the example 1-2, the changes of the internal structures of the prototheca blue pigment, the acidolysis modified phycocyanin and the polyphenol complex thereof were characterized by fourier infrared, laser particle size distribution instrument, atomic force microscope, and the specific operations included:
(3.1) measurement and evaluation of amino acids in acid hydrolysis-modified phycocyanin
The amino acid composition and content of the phycocyanin and the acidolysis modified phycocyanin are determined by referring to GB/T5009.124-2016. The amino acid evaluation adopts a Bano calculation method, and the formula is as follows:
AAS＝Ax/As×100％
wherein AAS refers to amino acid score; ax refers to the amount of certain amino acid contained in the protein to be detected, mg/g; as refers to the amount of an amino acid in the FAO/WHO scoring scale, mg/g.
As shown in Table 1, the algal blue pigment contains 17 amino acids (no tryptophan detected) before and after acidolysis modification, and is abundant and reasonable in species. Compared with the original algae blue pigment, the relative content of the amino acid of the acidolysis modified algae blue pigment is not changed greatly, and the proportion of isoleucine, methionine and leucine in the essential amino acid is increased. The two substances contain glutamic acid and essential amino acid, and leucine.
As seen from Table 2, the scores of the other amino acids of the original phycocyanin except valine, methionine, cystine and lysine are all larger than 100, which shows that the content of the amino acids of the phycocyanin is rich and can meet or even far exceed the requirement of human body on the necessary amino acids, and the scores of the amino acids of the acidolysis modified phycocyanin are all larger than the scores of the original phycocyanin. The first limiting amino acid of the original algae blue pigment and the acidolysis modified algae blue pigment are lysine, and the amino acid scores are 72.09 and 79.70 respectively.
TABLE 1 relative amino acid content (%)
TABLE 2 amino acid scores for phycocyanin and acidolysis-modified phycocyanin
(3.2) determination of particle size of acidolysis modified phycocyanin and its complex with polyphenol
Preparing citric acid-sodium citrate buffer solution with pH of 2.5, preparing 0.1% solution from crude phycocyanin, acidolysis modified phycocyanin, tannin-phycocyanin compound, and rosmarinic acid-phycocyanin compound, stirring thoroughly until completely dissolved, and measuring on a particle size analyzer. Water is used as a dispersing agent, the refractive index is 5% -8%, and the refractive index of a medium is 1.53.
The particle sizes of the primary phycocyanin, the acidolysis-modified phycocyanin, and the complexes thereof with tannic acid and rosmarinic acid were measured by a particle size analyzer, and the results are shown in table 3. The D50 of the proto-phycocyanin is 24.46 μm in the acid environment with the pH value of 2.5, the D50 is reduced to 13.46 μm after the acidolysis modification, and the D4, 3 and D3, 2 are reduced, which shows that the volume and the surface area of the phycocyanin are reduced after the acidolysis modification, the particles are smaller in the solution, and the dissolution state of the phycocyanin in the acid solution can be improved after the acidolysis modification. The particle size increased after the addition of both tannic acid and rosmarinic acid, indicating that the two polyphenols and the acidolysis-modified phycocyanin may be combined by hydrogen bonds or hydrophobic interactions, thereby increasing the particle size. TA-AHpc has a larger particle size than RA-AHpc, which is likely to be associated with the large molecular weight of tannic acid, more binding sites and easier association with proteins. This is consistent with the tendency of molecular weight to change as measured by gel electrophoresis.
TABLE 3 analysis of particle size distribution of pc, AHpc, TA-AHpc, RA-AHpc
Note: d10、D25、D50、D75、D90Respectively, means that particles having a particle diameter smaller than this value account for 10%, 25%, 50%, 75%, 90% of the total particles. D [4,3]]Refers to the weighted average of particle size over particle volume; d3, 2]Refers to the weighted average of particle size over particle surface area.
(3.3) determination of microstructure of acidolysis modified phycocyanin and complex of phycocyanin and polyphenol
And respectively dripping the diluted proto-phycocyanin, the acidolysis modified phycocyanin and the polyphenol compound solution thereof into the middle of the surface of the newly opened mica sheet, and standing at room temperature for natural air drying. And fixing the mica sheet on a scanning table of an atomic force microscope for scanning, and observing the surface appearance of the phycocyanin after different treatments.
Fig. 10 shows atomic force microscope photographs of a primary algae blue pigment, an acid hydrolysis modified algae blue pigment, a tannic acid-acid hydrolysis modified algae blue pigment, and a rosmarinic acid-acid hydrolysis modified algae blue pigment, respectively, in which the primary algae blue pigment has a long-chain structure, and the chain thickness is about 1nm as observed in the wanghiping experiment. After acid hydrolysis, the long-chain structure of the phycocyanin is shortened by 'shearing' the phycocyanin by hydrochloric acid, the phycocyanin is evenly laid on a substrate, and the thickness of a chain is changed to 0.6nm, which further proves that the hydrochloric acid hydrolysis has certain destructive effect on the phycocyanin, the molecular weight of the phycocyanin is reduced, the structure is changed, and the analysis result is consistent with the analysis result of polyacrylamide gel electrophoresis; the TA-AHpc structure is observed, and the TA is added to ensure that the acidolysis modified phycocyanin is re-aggregated, disordered short chains are aggregated into clusters, the thickness is increased to 5-10 nm, RA has weak polymerization effect on the acidolysis modified phycocyanin, part of RA is aggregated into an irregular shape of about 2.5nm, and the rest part of RA is still dispersed in a short chain shape. This phenomenon is probably related to the fact that tannic acid has large molecular weight and many binding sites, and thus the binding capacity of acidolysis modified phycocyanin is stronger. This phenomenon indicates that the interaction between polyphenol and acidolysis modified phycocyanin may be related to the color-increasing and color-protecting effects of the polyphenol on acidolysis modified phycocyanin, and the polyphenol recombines the acidolysis modified phycocyanin to make the structure of the pigment more stable, thereby improving the stability of the acidolysis modified phycocyanin.
(3.4) acidolysis modified phycocyanin and compound Fourier infrared determination of phycocyanin and polyphenol
Measuring original algae blue pigment, acidolysis modified algae blue pigment and polyphenol compound by using potassium bromide tabletting method, wherein scanning range is 4000cm-1～400cm-1Resolution of 4cm-1And 32 scans were performed.
Phycocyanin and phycocyanin acidThe modified phycocyanin and its complex with polyphenol has infrared spectrum shown in FIG. 9, and the peak position of phycocyanin modified by acidolysis is shifted and the intensity is reduced at 1750cm-1In the short wavelength range below, the peak pattern significantly changes. Stretching vibration of N-H in amide group is 3100-3400 cm-1The absorption peak of (A) is related, and is generally considered to be 3400-3440 cm-1The vicinity is an amide A belt which is related to N-H stretching vibration; 1600-1700 cm-1The corresponding amide I band is mainly caused by protein skeleton peptide chain C ═ O stretching vibration and H-O-H bending vibration, and is a sensitive area for protein secondary structure change; 1500-1600 cm-1Corresponding to the amide II band, the vibration of the C-N, C-H bond is reflected; 1200-1360 cm-1Belonging to the amide III band, often associated with a C-O, C-O-C bond. The content of the crude algae blue pigment is 3500-3100 cm-1There is a large absorption peak, and the peak strength becomes weak after modification by acidolysis, which is related to the stretching vibration of N-H. The acidolysis modified phycocyanin is 3080cm-1A small peak is added nearby, which indicates that the acidolysis causes more N-H to appear in the phycocyanin, and the acidolysis is reacted to destroy the peptide bond breakage of the protein part of the phycocyanin. After acidolysis of phycocyanin, the band shape and the intensity of amide I are obviously changed, which shows that the secondary structure is changed and is 1530cm-1Nearby, the characteristic absorption peak of the acidolysis modified phycocyanobilin amide II band is widened, which indicates that N-H bending and C-H stretching vibration occur. As can be seen from the figure, compared with the original algae blue pigment, the characteristic absorption peaks of the acid hydrolysis modified algae blue pigment in the amide I band, the amide II band and the amide III band are weakened and shift rightward, which shows that after acid hydrolysis, partial amide groups are converted into carboxyl groups, the total absorbance of amino acid residues is changed, and the secondary structure of the protein is changed.
The infrared spectrum of the modified phycocyanin after acidolysis before and after the addition of the tannic acid and the rosmarinic acid is found to be 3100-3400 cm-1The peak pattern and intensity were varied, indicating the presence of hydrogen bonding interactions, and was found to be 3400cm after reaction against the polyphenol blank (FIG. 11)-1The characteristic absorption peaks at the left and right represent the contraction vibration of the phenolic group or hydroxyl group of polyphenol, and the reaction with acidolysis modified phycocyanin appears right shift, which indicates that the hydroxyl group or phenolic group of two polyphenols is ginsengAnd reacting. In the range of 1200-1300 cm-1After the nearby acidolysis modified phycocyanin is compounded with polyphenol, the absorption peak intensity is enhanced, which indicates that the two react to form C-N bonds. And is in the range of 1600-1700 cm-1The number and the position of absorption peaks of the TA-AHpc and RA-AHpc compounds are changed, which shows that the addition of polyphenol can obviously change the secondary structure of the acidolysis modified phycocyanin protein part.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A research method for stability and acidolysis modification of phycocyanin is characterized by comprising the following steps:
s1, accurately weighing a certain amount of phycocyanin by taking the phycocyanin as a raw material to prepare a phycocyanin solution with a proper concentration;
s2, taking the storage rate of the pigment as an index, combining the chromaticity change, systematically researching the influence of environmental factors and additive types on the stability of the phycocyanobilin solution experimental group, and analyzing the influence factors of the stability of the phycocyanobilin;
s3, placing the phycocyanin solution into a test tube with a plug, adding a protein protective agent into the test tube of the experimental group, and fully shaking and uniformly mixing;
s4, placing the test tubes of the experimental group treated in the S3 in water bath at 65 ℃ for 1h, taking out, and immediately cooling with ice water;
s5, measuring the absorbance value A of each experimental group in S4, calculating the pigment storage rate according to the absorbance value A, further analyzing the influence of the protein protective agent on the thermal stability of the phycocyanin, and simultaneously further researching the thermal degradation rule of the phycocyanin and the influence of the effective protective agent on the dynamics and thermodynamic parameters of the thermal degradation process of the phycocyanin;
s6, designing a single-factor and orthogonal experiment, optimizing the algae blue pigment acidolysis process to obtain an acidolysis product, researching the characteristics of the acidolysis product, and simultaneously further adding polyphenol as a color enhancer to research the stability of the product;
s7, characterizing the primary phycocyanin, the acidolysis modified phycocyanin obtained in the S6 and the internal structure change of the complex of the primary phycocyanin and the polyphenol through a Fourier infrared, laser particle size distribution instrument, a circular dichroism spectrum and an atomic force microscope;
and S8, based on the characterization information obtained in S7 and combined with the experimental contents in S1-S6, finishing and analyzing to obtain the stability of the phycocyanin and the acidolysis modification research method thereof.
2. The method as claimed in claim 1, wherein the environmental factors and additives mentioned in S2 include temperature, pH, light, metal ions, redox agents and food additives; the calculation formula of the pigment preservation rate is as follows:
in the formula, A1Denotes the final absorbance value, A0The initial absorbance value is indicated.
3. The method for researching the stability and the acidolysis modification of the phycocyanin as claimed in claim 1, wherein S3-S5 is characterized in that when researching the thermal degradation rule of the phycocyanin and the influence of an effective protective agent on the kinetic and thermodynamic parameters of the thermal degradation process of the phycocyanin, in order to further research the mechanism of the protective agent for improving the thermal stability of the phycocyanin, the phycocyanin solutions added with different protective agents are respectively heated at 65 ℃, 70 ℃, 75 ℃ and 80 ℃, samples are taken every 5min, the content of the phycocyanin is determined after rapid cooling, and the result of the phycocyanin solution without the protective agent under the same heat treatment condition is used as a blank control; drawing a thermal degradation curve to accord with a first-level dynamic model
ln(p/p0)＝-kt
In the formula: p0 is initialThe concentration of the phycocyanin is mg/mL; p is the concentration of the blue algae after heating for t time, mg/mL; k is the first order reaction rate constant, min-1(ii) a t is heating time;
the half-life period is calculated by
t1/2＝ln2/k
Thermodynamically, the relationship between the chemical reaction rate constant and the temperature change is usually expressed by an arrhenius equation, the logarithm of the thermal degradation rate constant and the reciprocal of the corresponding absolute temperature are respectively regressed to form an arrhenius diagram, and the activation energy Ea can be calculated according to the slope of the obtained regression line:
lnk＝lnA-Ea/RT
other thermodynamic parameters can be calculated by combining Eying transition state theory
△H＝Ea-RT
△S＝R[lnA-ln(kB/hp)-lnT]
△G＝△H-T△S
In the formula: a is pre-dactylic factor, min-1(ii) a R is a gas constant of 8.314J/mol.K; t is the temperature in Kelvin, K; kB is Boltzmann constant; hp is the Planck constant; ea is activation energy, J/mol; Δ H is the enthalpy of activation, J/mol; delta S is activation entropy, J/mol.K; delta G is Gibbs free energy, J/mol.
4. The method as claimed in claim 1, wherein the single-factor and orthogonal test mentioned in S6 comprises the following steps:
a1, accurately weighing a proper amount of phycocyanin, mixing the phycocyanin with hydrochloric acid in proportion, and placing at room temperature for magnetic stirring for acidolysis reaction;
a2, stirring and carrying out acidolysis on the mixed solution in the S1 for a corresponding time according to experimental needs, and adding a proper amount of purified water to dilute the mixed solution after the specified time is reached so as to stop acidolysis reaction;
a3, treating the acidolysis reaction time, the mass ratio of the phycocyanin to the acid and the hydrochloric acid concentration mentioned in A1-A2 as experimental factors by using the experimental factors as single-factor experimental variables or adopting an orthogonal method, and repeating the operations in A1-A2;
a4, placing the diluted mixed solution on a centrifuge for centrifugal treatment, taking the precipitate for redissolving with water, adjusting the pH value to be neutral, cooling and drying to obtain acidolysis modified phycocyanin, and measuring the quality of the product;
a5, preparing pigment solution with pH of 2.5 with certain concentration from acidolysis modified phycocyanin obtained in A4, measuring chromatic aberration and scanning at full wavelength, and measuring the product quality and chromatic aberration b*The value is used as an index to determine the optimal acidolysis time.
5. The method as claimed in claim 1, wherein the characterization of the original phycocyanin and the change of the inner structure of the acidolysis modified phycocyanin and the polyphenol complex thereof obtained in S6 mentioned in S7 specifically includes amino acid determination and evaluation of the acidolysis modified phycocyanin, particle size determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, microstructure determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, fourier infrared determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, and circular dichroism determination of the acidolysis modified phycocyanin and the polyphenol complex thereof, wherein the amino acid evaluation adopts a Bano calculation method, and the calculation formula is as follows:
AAS＝Ax/As×100％
wherein AAS refers to amino acid score; ax refers to the amount of certain amino acid contained in the protein to be detected, mg/g; as refers to the amount of an amino acid in the FAO/WHO scoring scale, mg/g.
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| 任顺成等: "藻蓝色素稳定性的研究", 河南工业大学学报(自然科学版), vol. 41, no. 6, 30 November 2020 (2020-11-30), pages 1 - 7 * |
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