CN115607477A

CN115607477A - Method for maintaining thermal stability of phycocyanin under acidic condition

Info

Publication number: CN115607477A
Application number: CN202211299312.8A
Authority: CN
Inventors: 曾名湧; 尹梓浩; 王梦薇; 李崴
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2023-01-17
Anticipated expiration: 2042-10-24
Also published as: CN115607477B

Abstract

The invention discloses a method for maintaining the thermal stability of phycocyanin under an acidic condition, which comprises the following steps: (1) Adding phycocyanin and whey protein isolate into water, wherein the mass ratio of the phycocyanin to the whey protein isolate is 1; (2) adjusting the pH value of the mixed solution to 10-11, and fully and uniformly mixing; then adjusting the pH value to 5-6, separating out protein precipitate at the moment, and centrifuging to obtain precipitate; (3) Re-dissolving the precipitate in water, adding chitosan into the solution, mixing, and freeze drying to obtain thermostable phycocyanin. The heat-stable phycocyanin obtained by the method has heat stability under an acidic condition (pH = 3), does not precipitate or fade, has high activity, and can be applied to or used for preparing food additives and cosmetic additives. The invention has great application prospect in the fields of food, cosmetics and the like and has higher economic value.

Description

Method for maintaining heat stability of phycocyanin under acidic condition

Technical Field

The invention relates to a method for maintaining thermal stability of phycocyanin under an acidic condition, and belongs to the technical field of natural pigment preparation.

Background

Color is critical to the organoleptic properties of food products and consumer acceptance, and most food products and beverages require the addition of food colors to effect color changes in addition to the food raw materials and colors produced during processing. The food color refers to a dye or substance that generates color when added to food or drink, and is classified into natural color and synthetic color. Compared with natural pigments, the artificially synthesized pigments have the advantages of low cost, high color intensity, good color stability and the like, and are widely used in foods and beverages. However, the existing research shows that the compounds have side effects, medium and long-term toxicity, high-frequency potential health problems and the like on the health of organisms. Therefore, the preparation of a natural pigment with excellent physicochemical properties is important for ensuring the safety of foods and beverages.

Phycocyanin is a natural protein extracted from microalgae, is the only natural blue pigment approved by China for use in food, and is also approved by the U.S. food and drug administration as a natural blue colorant. Researches show that the phycocyanin has activities of antioxidation, anti-inflammation, antibiosis and the like, and can be applied to functional foods and cosmetics. However, phycocyanin is unstable under acidic conditions (especially at pH = 3), and proteins denature at temperatures above 60 ℃, resulting in loss of color and functional activity, which limits its use in foods, beverages, and cosmetics. Therefore, the research of a method capable of keeping stable color and activity of the phycocyanin under acidic and high-temperature conditions has great significance for the development of the practical application of the phycocyanin.

Phycocyanin is composed of monomers formed by alpha subunits and beta subunits, and the monomers are polymerized together to form trimers, hexamers or decamers. Each subunit of phycocyanin has linear tetrapyrrole chromophores (constrained to each subunit by thioether linkages) that create the unique blue color of phycocyanin, and changes in the chromophore structure lead to protein discoloration and loss of antioxidant activity. Thus, compounds that react with the tetrapyrrole structure, such as oxygen, free radicals and acids, all lead to the denaturing degradation of phycocyanin. The protein structure stability can effectively protect chromophore, so that factors which can influence the protein stability can influence the stability of phycocyanin.

Disclosure of Invention

Aiming at the prior art, the invention provides a method for maintaining the thermal stability of phycocyanin under an acidic condition, and the method has great economic value and scientific and technological significance for the fields of food and cosmetics.

The invention is realized by the following technical scheme:

a method for maintaining the thermal stability of phycocyanin under acidic conditions, comprising the steps of:

(1) Adding phycocyanin and whey protein isolate into water, wherein the mass ratio of the phycocyanin to the whey protein isolate is 1; the concentration of the phycocyanin in the mixed solution is 8.0-12.0 mg/mL, preferably 10.0mg/mL;

(2) Adjusting the pH value of the mixed solution to 10-11, and fully and uniformly mixing; then adjusting the pH value of the mixed solution to 5-6, separating out protein precipitate at the moment, and centrifuging to obtain precipitate;

(3) Redissolving the precipitate in water, wherein the concentration of the protein in the solution is 1.0-2.0 mg/ml; adding chitosan into the solution to make the concentration of the chitosan be 0.6-1.0 mg/ml, uniformly mixing (at the moment, the pH value of the solution is 2.7-3.7), and freeze-drying to obtain the compound of Phycocyanin (PC) -lactalbumin (WPI) -Chitosan (CS), namely the WPI-PC-CS compound, which is called as heat-stable phycocyanin.

Further, the mass ratio of the phycocyanin to the whey protein isolate is 2, 1, 2, 1.

Further, the chitosan is dissolved by a proper amount of hydrochloric acid before being added, and the concentration of the chitosan is 4.0-5.0 mg/ml; then adding the chitosan solution. Further, it was dissolved in 0.01mol/l hydrochloric acid.

The heat-stable phycocyanin obtained by the method has the advantages of heat stability under an acidic condition (pH = 3), no precipitation, no fading and high activity, and can be applied to or used for preparing food additives and cosmetic additives.

The method for maintaining the thermal stability of the phycocyanin under the acidic condition adopts a protein coprecipitation method to change the isoelectric point of the phycocyanin, then compounds the coprecipitated phycocyanin with a chitosan solution, prevents the degradation of a tetrapyrrole ring in the phycocyanin by maintaining the structural stability and electrostatic interaction of the protein and the like, thereby ensuring that the phycocyanin has better stability and oxidation resistance under the acidic condition, and can be used as an additive for preparing foods and beverages, such as bubble water, yoghourt, cheese, ice cream and the like. The invention has great application prospect in the fields of food, cosmetics and the like and has higher economic value.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.

Drawings

FIG. 1: and (3) an apparent image comparison schematic diagram of the heat-stable phycocyanin solution, wherein I, II, III, IV and V respectively represent heat-stable phycocyanin formed by respectively forming PC/WPI =1/6, PC/WPI =1/4, PC/WPI =1/2, PC/WPI =1/1 and PC/WPI =2/1 in the mass ratio of the coprecipitated protein.

FIG. 2 is a schematic diagram: the chromaticity variation statistical chart of the heat-stable phycocyanin solution before and after the sterilization treatment, wherein (A), (B), (C), (D) and (E) respectively represent heat-stable phycocyanin formed by respectively having mass ratios of co-precipitated proteins of PC/WPI =1/1, PC/WPI =1/2, PC/WPI =1/4, PC/WPI =1/6 and PC/WPI = 2/1.

FIG. 3: potential distribution of heat stable phycocyanin solution (PC/WPI = 1/2) under different pH conditions is shown schematically.

FIG. 4: potential distribution of the heat stable phycocyanin solution at pH =3 is shown.

FIG. 5: mean particle size distribution of heat stable phycocyanin solution at pH =3.

FIG. 6: particle size distribution of heat-stable phycocyanin solution (pH = 3) before and after sterilization treatment is schematically shown, wherein a, B, C, D, E respectively represent heat-stable phycocyanin formed by respectively setting mass ratios of co-precipitated proteins as PC/WPI =1/2, PC/WPI =1/1, PC/WPI =1/4, PC/WPI =1/6, and PC/WPI = 2/1.

FIG. 7: fluorescence spectra of a thermostable phycocyanin solution (pH = 3) before and after sterilization treatment are shown in the following schematic diagrams, wherein a, B, C, D, and E respectively represent thermostable phycocyanins formed by co-precipitated proteins with mass ratios of PC/WPI =1/1, PC/WPI =1/2, PC/WPI =1/4, PC/WPI =1/6, and PC/WPI =2/1, respectively.

FIG. 8: DPPH clearance profile of thermostable phycocyanin solution (PC/WPI = 1/2) at different pH conditions.

FIG. 9: DPPH clearance profile of heat stable phycocyanin solutions (pH = 3) before and after sterilization treatment.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The instruments, reagents and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and the detection methods mentioned in the following examples are all conventional experimental methods and detection methods known in the art.

Phycocyanin used in the examples of the present invention was purchased from spirulina maxima co, nja, of fuqing, under the trade name spirulina phycocyanin, and the trade name was 13021990.99 (customs code). Whey protein isolate was purchased from Shanghai-derived leaf Biotech, inc. under the trade designation whey protein isolate, and is available under the trade designation CAS #84082-51-9. Chitosan is available from Beijing Solay technologies, inc., under the trade name Chitosan, with the trade name Cat # C8320.

Research for experimental maintenance of heat stability of phycocyanin

The phycocyanin treatment method comprises the following steps:

(1) Mixing phycocyanin and whey protein isolate, adding the mixture into deionized water, and setting 5 experimental groups, wherein the mass ratio of the co-precipitated phycocyanin to the whey protein isolate in the experimental groups is 1:6. 1; the concentration of phycocyanin in the mixture was 10.0mg/mL.

(2) Continuously mixing at 200rpm for 30min, adjusting pH of the mixture to 10.50 (adjusted with 0.01mol/l sodium hydroxide solution), and continuously mixing at 200rpm for 1h; then, the pH of the mixture was adjusted to 6.0 (adjusted with 0.01mol/l hydrochloric acid) and protein precipitated, and the precipitate was obtained by centrifugation at 6000 g.

(3) Redissolving the precipitate in deionized water (the concentration of protein in the redissolved solution is 1.0 mg/ml), adding chitosan (the chitosan is dissolved in a small amount of 0.01mol/l hydrochloric acid in advance and then added) to the solution to ensure that the concentration of the chitosan is 0.8mg/ml, uniformly mixing (the pH of the solution is 2.7-3.7 at the pH, the chitosan has strong positive charge), standing for 12h at the temperature of 4 ℃, and freeze-drying to obtain the compound of Phycocyanin (PC) -lactalbumin (WPI) -Chitosan (CS), namely the WPI-PC-CS compound, which is called as heat-stable phycocyanin.

(4) The obtained heat-stable phycocyanin was dissolved in deionized water to obtain a solution having a concentration of 1.0mg/ml, and the pH of the solution was adjusted to 3.0 (adjusted with 0.01mol/l hydrochloric acid).

(5) And (3) sterilizing the solution by respectively adopting 4 sterilization modes, which respectively comprise the following steps: pasteurizing at 60 deg.C for 30 min; pasteurizing at 75 deg.C for 15 min; high temperature short time sterilization (HTST) at 90 ℃ for 3 min; ultra high temperature flash sterilization (UHT) at 100 deg.C for 30 s. Obtaining the sterilized phycocyanin solution.

(II) apparent image of phycocyanin solution after sterilization treatment

The phycocyanin solution obtained in the step (5) was placed in a 5mL centrifuge tube, and the apparent change in color was observed before and after sterilization, and the results are shown in FIG. 1.

Fig. 1 shows the overall color difference before and after sterilization of phycocyanin solutions (pH = 3.0) of 5 experimental groups. As can be seen from FIG. 1, the phycocyanin solutions treated in 5 experimental groups exhibited excellent apparent chromaticity retention in simulated heating at 60 ℃, 75 ℃,90 ℃ and 100 ℃. The color difference between each experimental group was not significant after the heat sterilization treatment, and the change in the color was smaller as the proportion of whey protein isolate was increased.

It is known that general phycocyanin precipitates at pH =3.0 and denatures and discolors under heating at 60 ℃. However, after the above treatment according to the present invention, no protein precipitate was formed in all experimental groups, indicating that the PC/WPI co-precipitation effectively changed the isoelectric Point (PI) of phycocyanin.

(III) chroma of phycocyanin solution after sterilization treatment

The colorimetry was performed using a konica minolta CR400 hand-held colorimeter calibrated using standard white tiles prior to measurement.

Phycocyanin solutions (2.5 ml) before and after sterilization treatment of 5 experimental groups were added to 6-well polystyrene plates and placed on a white background for colorimetric testing. The L (brightness), a (red green) and b (yellow blue) spatial color values were determined using the CIE scale and the measurements are shown in fig. 2, where positive a values indicate red, negative a values indicate green for the sample, positive b values indicate yellow, and negative b values indicate blue for the sample.

As can be seen from FIG. 2, the phycocyanin solution before and after the sterilization treatment has a certain variation in b value, but the overall variation degree is not significant and fluctuates within a small range. The significance analysis shows that the phycocyanin solution can have a certain blue preservation rate under the pasteurization condition, the preservation rate is more than 80%, and the blue preservation rate of the phycocyanin solution can reach more than 90% under the conditions of high-temperature short-time sterilization and ultrahigh-temperature instant sterilization, which indicates that the phycocyanin solution is stable to heat.

(IV) potential and particle size measurement of thermostable phycocyanin

Measurement of dynamic light scattering particle size: the intensity of the scattered light was measured at 90 deg. using a dynamic light scattering instrument equipped with a helium-neon laser and a temperature controlled cell holder, with the incident beam.

Preparing a sample solution, placing the solution in a testing dish of a Nano-ZS-90 type laser particle size analyzer, and measuring at 25 ℃ to represent the potential and the particle size distribution of the Nano particles so as to preliminarily judge the potential, the particle size and the distribution of the Nano particles in the solution. The pH of the sample was adjusted to 3.0, 5.0, 7.0, and 9.0 (adjusted with 0.01mol/l hydrochloric acid or 0.01mol/l sodium hydroxide solution) in step (4) using solutions of 5 experimental groups.

The potential distribution of experimental group iii (mass ratio of coprecipitated phycocyanin to whey protein isolate of 1. As can be seen from fig. 3, protein co-precipitation effectively changed the PI of phycocyanin, allowing PI to move to around pH = 6. And under different pH conditions, the phycocyanin solution presents different appearance colors, and when the pH is less than 5, the solution presents transparent blue, which is closely related to the potential.

Potential distribution of the solutions of 5 experimental groups at pH =3 is shown in fig. 4. As can be seen from FIG. 3, the potential of the thermostable phycocyanin is between +40mV and +60mV at pH =3, and the magnitude of the potential is related to the PC/WPI ratio, with the larger the PC ratio, the higher the potential of the resulting thermostable phycocyanin solution. The potential is closely related to the stability of the protein and the tetrapyrrole ring in the PC, the higher potential enables the complex to have higher electrostatic repulsion, and the electrostatic interaction between the molecules of the complex is relatively stronger, so that the structural stability of the complex is better.

The particle size distribution of the solutions of the 5 experimental groups at pH =3 is shown in fig. 5. As can be seen from fig. 5, when the co-precipitated protein PC/WPI is 2, 1; and the addition of the chitosan strengthens electrostatic repulsion among particles, prevents self-aggregation among the particles, and is important for the thermal stability of the phycocyanin solution. When the PC/WPI is 1, the coprecipitated protein particles increase significantly, as more WPI is encapsulated around the PC, resulting in an increase in particle size, but this may increase the thermal stability of the PC more.

The particle size distribution of the phycocyanin solution (pH = 3) of 5 experimental groups before and after the simulated pasteurization (75 ℃, 15 min) condition is shown in fig. 6. As can be seen from FIG. 6, the aggregation of the thermostable phycocyanin is not caused by heating, and the structure of the thermostable phycocyanin solution remains stable, which indicates that the composite structure of the co-precipitated protein-chitosan has strong stability, and can effectively protect the stability of the PC structure to prevent color fading.

(V) intrinsic fluorescence Spectroscopy

To further understand the conformational changes of thermostable phycocyanin under acid-heat conditions, the intrinsic fluorescence spectrum of thermostable phycocyanin under acid-heat conditions was determined. Intrinsic fluorescence refers to the fluorescence emission of Trp residues in PC, which is very sensitive to protein fold-unfold transitions.

Fluorescence was measured on a Shimadzu RF-6000 spectrophotometer (Shimadzu) using a 1cm quartz cuvette. Intrinsic fluorescence of tryptophan (Trp) residues in PC was obtained by excitation at 295nm and emission was monitored over a range of 310-400 nm; the excitation/emission slit width was 5nm, and the results are shown in FIG. 7.

Aggregation or precipitation of PC will bury Trp in the hydrophobic core of the protein, thereby separating Trp from the surrounding hydrophilic solvent, resulting in a blue-shift of intrinsic Trp fluorescence; if PC is buried in the aggregates, the exposed Trp in the solution will be less, resulting in a sharp decrease in fluorescence intensity. Interestingly, in all experimental groups, the peak position of the fluorescence spectrum did not undergo a red shift or a blue shift, but only changed in fluorescence intensity, indicating that the protein undergoes a weak structural stretching change and aggregation during the heating process of the thermostable phycocyanin (WPI-PC-chitosan complex), which also confirms the apparent stability image of the micronized phycocyanin. When the PC/WPI in the coprecipitated protein is 2, 1 and 2. Higher WPI content results in a relatively loose structure which can cause aggregation or stretching of the protein structure upon heat treatment. Meanwhile, the change of the fluorescence spectrum of the micronized phycocyanin solution heated by HTST and UHT is more similar to that of the control group, which shows that the change of the protein under the heating condition is smaller, and the stability of the phycocyanin can be maintained better.

(VI) measurement of Oxidation resistance

Phycocyanin has excellent oxidation resistance, and thus has a wide application range in functional foods and cosmetics.

The DPPH radical scavenging results of the heat-stable phycocyanin solution are shown in fig. 8 and 9. The results in fig. 8 and 9 show that the heat-stable phycocyanin still has high oxidation resistance, but as the proportion of WPI in the co-precipitated protein increases, the oxidation resistance of the phycocyanin tends to decrease. When the PC/WPI is 1. The reason may be that the decrease in the concentration of PC leads to a decrease in the amount of WPI-PC-chitosan complex formed in the solution, whereas PC alone has poor stability under acidic conditions, so the solution has reduced resistance to oxidation. The DPPH radical clearance of the solution after heat treatment of the samples was not significantly changed at PC/

WPI

1 and 1. And when the PC/WPI is 1, 4, 1, 6, 2. The reason may be that the number of complexes formed is small, whereas PC alone has poor stability at pH =3, thus resulting in poor antioxidant thermal stability of the solution. At pH =5, the DPPH radical clearance of the thermostable phycocyanin solution is highest, at pH =3, the DPPH radical clearance of the solution is slightly reduced, and at pH =7 or 9, the DPPH radical clearance of the solution is reduced to a greater extent, i.e., the oxidation resistance is poor. The reason may be that WPI-PC-chitosan complexes are more stable under acidic conditions, while more amino acid or small peptide groups with reducing and antioxidant properties are exposed to the complexes compared to pH =3, pH = 5. Combined with the apparent images of heat-stable phycocyanin before and after heating (fig. 1), analysis shows that the heat-stable phycocyanin can maintain the stability of chromaticity under the acid-heat condition, and meanwhile, the protein oxidation resistance is not influenced under different heat sterilization conditions.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. A method for maintaining the thermal stability of phycocyanin under acidic conditions, comprising the steps of:

(1) Adding phycocyanin and whey protein isolate into water, wherein the mass ratio of the phycocyanin to the whey protein isolate is 1; the concentration of phycocyanin in the mixed solution is 8.0-12.0 mg/mL;

(2) Adjusting the pH value of the mixed solution to 10-11, and fully and uniformly mixing; then adjusting the pH value of the mixed solution to 5-6, separating out protein precipitate, and centrifuging to obtain precipitate;

(3) Re-dissolving the precipitate in water to obtain solution with protein concentration of 1.0-2.0 mg/ml; adding chitosan into the solution to make the concentration of the chitosan be 0.6-1.0 mg/ml, uniformly mixing, and freeze-drying to obtain the protein, namely the heat-stable phycocyanin.

2. The method of claim 1, wherein the phycocyanin is thermostable under acidic conditions: the mass ratio of the phycocyanin to the whey protein isolate is (2, 1).

3. The method of claim 1, wherein the phycocyanin is thermostable under acidic conditions: dissolving the chitosan with a proper amount of hydrochloric acid before adding, wherein the concentration of the chitosan is 4.0-5.0 mg/ml; then adding the chitosan solution.

4. A thermostable phycocyanin produced by the method for maintaining the thermostability of phycocyanin under acidic conditions as claimed in any one of claims 1 to 3.

5. Use of the heat stable phycocyanin of claim 4 as or in the preparation of a food additive.

6. Use of the heat stable phycocyanin of claim 4 as or in the preparation of cosmetic additives.