Highly water-soluble lutein / pea protein isolate complex and its preparation method and application

Abstract

The present invention belongs to the field of lutein processing technology, and specifically relates to a highly water-soluble lutein / pea protein isolate complex, a preparation method, and an application thereof. The preparation method comprises: (1) dissolving lutein in anhydrous ethanol to obtain a lutein solution; (2) dispersing pea protein isolate in ultrapure water, stirring to form a dispersion, adding a NaOH solution, and oscillating at a constant temperature for a certain time to completely dissolve the pea protein isolate; cooling to room temperature, and adding an HCl solution to adjust the pH value to obtain a pea protein isolate solution; (3) diluting the pea protein isolate solution to a target concentration and mixing it with the lutein solution in a certain volume ratio; oscillating at room temperature for a certain time, and freeze-drying to obtain a lutein / pea protein isolate complex. The lutein solubility in the lutein / pea protein isolate complex provided by the present invention reaches up to 48 μg / mL; the lutein can be used to prepare beverages, emulsified sausages, and high internal phase emulsion systems.

Description

Technical Field

[0001] The invention belongs to the technical field of lutein processing, and particularly relates to a highly water-soluble lutein / pea protein isolate complex and a preparation method and application thereof. Background Art

[0002] Lutein (Lutein, Lut) is a natural pigment widely found in vegetables, fruits and flowers. Its key role in improving cognition, treating age-related macular degeneration, and alleviating cardiovascular disease is gradually becoming increasingly prominent. Since the human body does not have the key enzyme for lutein biosynthesis, lutein must be obtained through dietary supplementation. However, dietary supplementation can only provide 10% of the lutein required by the human body daily. To ensure the nutritional health of the people, it is imperative to develop functional foods rich in natural lutein.

[0003] During food development, natural lutein, due to the long chromophores composed of conjugated double bonds in its structure, is chemically unstable and easily degraded by factors such as temperature, oxygen, pH, and light. Furthermore, lutein is a fat-soluble pigment, insoluble in water, and has low bioavailability, which significantly limits its application in areas such as food processing and medicine. Consequently, there are relatively few natural lutein products on the market, with the majority being lutein ester-related products, such as lutein ester compressed candies and lutein ester soft capsules. However, lutein esters and natural lutein differ significantly in structure, stability, functionality, and bioavailability. Consequently, maintaining the stability of natural lutein under processing conditions and improving its solubility have become important scientific issues that need to be addressed in the basic theoretical research for the development of lutein-rich foods.

[0004] Research reports indicate that lutein degradation can be partially controlled through delivery systems such as nanoparticles, liposomes, microcapsules, and emulsions. For example, Chinese patent CN115886122A, "A method and application for improving the stability of plant protein emulsions," demonstrates that plant protein emulsions can be used as carriers or delivery systems for lutein, thereby improving its stability. However, lutein emulsions tend to aggregate and eventually separate into oil and water phases when stored for extended periods or under heating, which can affect the sensory quality of food in practical applications. Furthermore, the use of liposomes, inclusion compounds, and self-microemulsions can improve lutein's stability and water solubility to varying degrees, but these also present challenges such as high excipient usage and shelf stability.

[0005] Protein can be combined with insoluble or hydrophobic nutrients through intermolecular interactions to improve the water dispersibility, stability and biological activity of these bioactive molecules. At present, Chinese patent CN108308615A has been reported to use corn peptide as a food functional ingredient carrier. The particle size of corn peptide-loaded lutein nanoparticles is less than 100nm, which can partially improve the solubility of lutein (more than 10 times), but there are still deficiencies in the solubility and release of active ingredients. Research reports show that the emulsification and antioxidant activity of lutein-red bean protein complexes can be improved by ultrasonically induced modification of red bean protein. However, ultrasound is a sound wave with a frequency higher than 20,000Hz and has a strong propagation ability in water. Red bean protein is highly hydrophobic and easily aggregates in the solution system, resulting in larger particles prepared by ultrasonically modified red bean protein-lutein complex, poor water solubility, which is not conducive to digestion and absorption, and it is difficult for lutein to exert its physiological function in the body; and the ultrasonic treatment operation process is relatively complicated and has high requirements for the actual operation process. It needs to undergo 200W ultrasonic treatment for 20 minutes, and the reaction time interval needs to be strictly controlled (treatment 5s, interval 5s) to prevent the occurrence of thermal effects.

[0006] Currently, pea protein isolate (PPI) is attracting increasing attention due to its bioaccessibility, high nutritional value, and low allergenicity. However, its functional properties, such as solubility, emulsification, gelation, foaming, and antioxidant capacity, are limited, making it unable to meet future food needs. Furthermore, 65-80% of PPI consists of 7S / 11S salt-soluble globulins, whose compact structure significantly limits its use as a carrier for bioactive substances such as polyphenols and curcumin. Therefore, PPI modification is needed to promote structural expansion and improve its functional properties to meet the requirements of the food industry. Protein modification methods include physical modification, restricted enzymatic hydrolysis, and glycosylation, but these methods are associated with high operating costs and complex systems. For example, Chinese invention patent CN115836706A discloses a method for improving the solubility and emulsification of pea protein isolate using acoustic resonance technology. A 20-minute acoustic resonance treatment significantly increased the solubility of pea protein, reaching 71.8%. However, acoustic resonance treatment has high requirements on equipment, takes up to 30 minutes to process, and consumes high energy. Summary of the Invention

[0007] In response to the above technical problems, the present invention provides a highly water-soluble lutein / pea protein isolate complex, a preparation method, and an application thereof. The preparation method provided by the present invention utilizes pea protein isolate as an exogenous protein to form a new lutein / pea protein isolate complex through intermolecular interactions, thereby improving the water solubility of lutein and maintaining the stability of lutein.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing a highly water-soluble lutein / pea protein isolate complex, the method comprising:

[0010] (1) Preparation of lutein solution: dissolving lutein in anhydrous ethanol to obtain a lutein solution;

[0011] (2) Preparation of pea protein isolate solution: pea protein isolate was dispersed in ultrapure water, stirred to form a dispersion, NaOH solution was added, and constant temperature oscillation was performed at a certain temperature for a certain time to completely dissolve the pea protein isolate; cooled to room temperature, and HCl solution was added to adjust the pH value to obtain a pea protein isolate solution;

[0012] (3) Preparation of lutein / pea protein isolate complex: The pea protein isolate solution is diluted to a target concentration and then mixed with the lutein solution in a certain volume ratio; the solution is shaken at room temperature for a certain period of time to obtain a lutein / pea protein isolate complex solution; and a highly water-soluble lutein / pea protein isolate complex (Lut-PPI complex) is obtained after freeze-drying.

[0013] Furthermore, in step (1), lutein is a fat-soluble pigment that is insoluble in water but soluble in organic solvents such as ethanol, ethyl acetate, and acetone. When lutein is dissolved in solvents such as ethyl acetate and acetone, it fails to fully dissolve in the pea protein isolate aqueous system. In the present invention, ethanol, which is miscible with water, is used as a solvent to dissolve the lutein to obtain a lutein solution with a concentration of 13-65 μM, thereby obtaining a stable and fully miscible lutein / pea protein isolate complex solution.

[0014] Furthermore, step (2) is specifically as follows:

[0015] Pea protein isolate is dispersed in ultrapure water and continuously stirred by magnetic force for 20-30 minutes to obtain a pea protein isolate dispersion. 0.1-1 mol / L NaOH solution is added to the pea protein isolate dispersion, the pH is adjusted to 11.5-12.0, and the mixture is shaken in a constant temperature oscillator at 83-87°C for 25-30 minutes to promote the structural unfolding of the pea protein and complete dissolution of the pea protein isolate. To prevent excessive denaturation of the pea protein isolate induced by heat treatment, the sample is quickly placed in ice water for 5-10 minutes to cool to room temperature, and the pH is adjusted to 7.4-7.8 with 0.1-1 mol / L HCl solution to obtain a pea protein isolate solution with a concentration of 5 mg / mL. Pea protein isolate is insoluble in water, and its water solubility is poor after treatment with alkaline pH alone, heat treatment, and ultrasonic modification. The pea protein isolate preparation method is effective through alkaline pH coupled with heat treatment, which solves the problem of protein precipitation when the pH is adjusted back to neutral.

[0016] Furthermore, in step (3), the lutein concentration in the lutein / pea protein isolate complex solution obtained is in the range of 13-65 μM, and the pea protein isolate concentration is in the range of 0.5-4.5 mg / mL.

[0017] A highly water-soluble lutein / pea protein isolate complex prepared by the above method, wherein the lutein / pea protein isolate complex is formed by spontaneous binding of lutein and pea protein isolate through hydrophobic interactions, wherein the lutein and pea protein isolate combine to form uniform and stable spherical nanoparticles, and the average particle size of the spherical nanoparticles is 20-40 nm;

[0018] The solubility of lutein in the lutein / pea protein isolate complex reaches a maximum of 48 μg / mL.

[0019] Furthermore, the solubility of lutein in the lutein / pea protein isolate complex freeze-dried powder is determined as follows:

[0020] 1.4 g of lyophilized powder of lutein / pea protein isolate complex was dissolved in 100 mL of water, 1.0 mL was added to a 5 mL centrifuge tube, and 3 mL of ethyl acetate was added. The mixture was thoroughly mixed for 30 seconds and allowed to stand for 30 minutes to allow complete separation. The absorbance of lutein in the upper organic phase was measured at 447 nm using a UV spectrophotometer. The lutein concentration, i.e., the solubility of lutein, was obtained using a standard curve of absorbance of lutein in ethyl acetate versus lutein concentration.

[0021] Furthermore, the binding rate of lutein and pea protein isolate in the lutein / pea protein isolate complex can reach up to 89.83%, that is, a maximum of 0.43g of lutein can be bound per 100g of protein;

[0022] The use of the highly water-soluble lutein / pea protein isolate complex according to claim 4 or 5, characterized in that the lutein / pea protein isolate complex is used to prepare beverages, emulsified sausages, and high internal phase emulsion systems.

[0023] Beneficial technical effects of the present invention:

[0024] (1) The preparation method provided by the present invention is based on the solubility characteristics of fat-soluble lutein and the modification technology of hydrophobic pea protein isolate. By utilizing the principle of "alcohol-water miscibility" and optimizing process parameters, a pea protein isolate-lutein complex preparation system with an optimal binding rate of 89.83% is constructed. In the lutein / pea protein isolate complex solution, the lutein concentration ranges from 13 to 65 μM, and the pea protein isolate concentration ranges from 0.5 to 4.5 mg / mL. This preparation method is simple and easy to operate, has a high binding rate, is economical and practical, is suitable for large-scale production, and has broad application prospects.

[0025] (2) The lutein-pea protein isolate complex prepared by the present invention has high water solubility, small particle size, strong stability, and uniform color. At present, the corn peptide-loaded lutein nanoparticles of Chinese patent CN108308615A can partially improve the solubility of lutein (more than 10 times), while the lutein-pea protein isolate complex of the present invention has good dispersibility in water, and the lutein solubility reaches 48μg / mL, which is more than 133 times higher; the lutein-pea protein isolate complex is uniformly distributed in the form of uniform and stable spherical nanoparticles with an average particle size of 20-40nm; the lutein-pea protein isolate complex significantly improves the color stability and antioxidant activity of lutein under acid and heat treatment conditions, and can be widely used in food, medicine and other fields, effectively expanding the application range of lutein.

[0026] (3) The present invention uses the interaction between lutein and protein to block the degradation pathway of lutein, and prepares a pea protein isolate-lutein composite nanoparticle product with high stability and good solubility, which significantly improves the added value of pea protein isolate and lutein. The research results may provide a new solution for improving the color deterioration of lutein during processing; and the materials used are all edible materials with high safety, and can be used as food and health products for direct consumption, avoiding the residue of organic compounds in traditional nanocarrier materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the standard curve for determining lutein concentration in the embodiment of the present invention;

[0028] Figure 2 This is a graph analyzing the binding rates of pea protein isolate and lutein at different concentrations in an embodiment of the present invention;

[0029] Figure 3A This is a diagram showing the results of a lutein solubility experiment in an embodiment of the present invention;

[0030] Figure 3B This is a graph showing the solubility test results of pea protein isolate in an embodiment of the present invention;

[0031] Figure 3C This is a graph showing the solubility test results of the Lut-PPI complex after redissolution in an embodiment of the present invention;

[0032] Figure 4 Graph showing the loss rate of Lut, PPI, and the total antioxidant capacity of Lut-PPI under acidic conditions in an embodiment of the present invention;

[0033] Figure 5A Graph showing the total antioxidant capacity of lutein before and after heat treatment in an embodiment of the present invention;

[0034] Figure 5BThis is a graph showing the loss rate of Lut, PPI, and the total antioxidant capacity of Lut-PPI under high temperature conditions in an embodiment of the present invention;

[0035] Figure 6 This is a comparison of the surface hydrophobicity of PPI and Lut-PPI in the examples of the present invention;

[0036] Figure 7 This is a comparison chart of infrared spectra of Lut, PPI and Lut-PPI in the examples of the present invention;

[0037] Figure 8A TEM image of PPI in an embodiment of the present invention;

[0038] Figure 8B This is a transmission electron microscope image of Lut-PPI in an embodiment of the present invention;

[0039] Figure 9 This is the binding energy diagram of Lut and PPI in the embodiment of the present invention; DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] On the contrary, the present invention covers any alternatives, modifications, equivalents, and solutions that fall within the spirit and scope of the present invention as defined by the claims. Furthermore, to facilitate a better understanding of the present invention, certain specific details are described in detail below in the detailed description of the present invention. Those skilled in the art will be able to fully understand the present invention without these details.

[0042] The present invention provides an embodiment of a method for preparing a highly water-soluble lutein / pea protein isolate complex, the preparation method comprising:

[0043] (1) Preparation of lutein solution: dissolving lutein in anhydrous ethanol to obtain a lutein solution;

[0044] (2) Preparation of pea protein isolate solution: pea protein isolate powder is dispersed in ultrapure water, stirred to form a dispersion, NaOH solution is added, and constant temperature oscillation is performed at a certain temperature for a certain time to completely dissolve the pea protein isolate powder; cooled to room temperature, HCl solution is added to adjust the pH value, and a pea protein isolate solution is obtained;

[0045] (3) Preparation of lutein / pea protein isolate complex: The pea protein isolate solution was diluted to a target concentration and then mixed with the lutein solution in a certain volume ratio; oscillated at room temperature for a certain period of time to obtain a lutein / pea protein isolate complex solution; and freeze-dried to obtain a lutein / pea protein isolate complex (Lut-PPI complex).

[0046] In step (1) of this embodiment, a lutein solution with a concentration of 13-65 μM is prepared. Lutein is a fat-soluble pigment that is insoluble in water but soluble in organic solvents such as ethanol, ethyl acetate, and acetone. When lutein is dissolved in solvents such as ethyl acetate and acetone, it fails to fully dissolve in the pea protein isolate aqueous system. Using ethanol, which is miscible with water, as a solvent to dissolve lutein, a stable and fully miscible lutein / pea protein isolate complex solution can be obtained.

[0047] In this embodiment, step (2) is specifically as follows:

[0048] The method comprises dispersing pea protein isolate powder in ultrapure water and continuously stirring the mixture by magnetic force for 20-30 minutes to obtain a pea protein isolate dispersion; adding a 0.1-1 mol / L NaOH solution to the pea protein isolate dispersion, adjusting the pH value to 11.5-12.0, and oscillating the mixture in a constant temperature oscillator at 83-87° C. for 25-30 minutes to promote the structural unfolding of the pea protein isolate and completely dissolve the pea protein isolate; and to prevent excessive denaturation of the pea protein isolate induced by heat treatment, rapidly placing the sample in ice water for 5-10 minutes to cool the sample to room temperature, and adjusting the pH value to 7.4-7.8 with a 0.1-1 mol / L HCl solution to obtain a pea protein isolate solution with a concentration of 5 mg / mL.

[0049] In step (3) of this embodiment, the lutein / pea protein isolate complex solution obtained has a lutein concentration range of 13-65 μM and a pea protein isolate concentration range of 0.5-4.5 mg / mL.

[0050] Specifically, in this example, a 0.5-4.5 mg / mL pea protein isolate solution was mixed with a 13-65 μM lutein solution in a 9:1 volume ratio and shaken at room temperature for 25-30 minutes to form a Lut-PPI complex solution. The resulting 45 Lut-PPI complex solution samples had lutein and pea protein isolate concentrations of 13, 26, 39, 52, and 65 μM and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg / mL, respectively.

[0051] The present invention also provides an embodiment of a highly water-soluble lutein / pea protein isolate complex, prepared using the above-described method. The present invention screens the optimal concentration of the lutein / pea protein isolate complex by measuring binding rate. The protective effect of the lutein / pea protein isolate complex on lutein stability is analyzed by measuring changes in the water solubility of the complex and the effects of exogenous processing conditions (heat, pH) on the color stability and total antioxidant capacity (T-AOC) of the complex, thereby providing a new approach to improving lutein stability. Table 1 lists the materials and reagents used.

[0052] Table 1 Materials and reagents

[0053]

[0054] Lutein / pea protein isolate binding rate determination and result analysis: The binding rate of lutein and pea protein isolate was determined by ethyl acetate extraction method. The main operations are as follows:

[0055] (1) Drawing of the standard curve: Add 1.0 mL of lutein standard solution with concentrations of 0, 13, 26, 39, 52, and 65 μM to a 5 mL centrifuge tube, then add 3 mL of ethyl acetate, mix thoroughly for 30 seconds, and let it stand for 30 minutes to allow it to completely separate. Use an ultraviolet spectrophotometer to measure the absorbance of lutein in the upper organic phase at 447 nm. Use ethyl acetate as a blank control to draw a standard curve, as shown in the following example: Figure 1 shown.

[0056] (2) Determination of free lutein concentration: 1.0 mL of each of the 45 prepared Lut-PPI complex samples (the concentrations of lutein and pea protein isolate were 13, 26, 39, 52, and 65 μM and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg / mL, respectively) was added to a 5 mL centrifuge tube, and 3 mL of ethyl acetate was added. The mixture was thoroughly mixed for 30 seconds and allowed to stand for 30 minutes to allow for complete separation. The absorbance of lutein in the upper organic phase was measured at 447 nm using an ultraviolet spectrophotometer. The standard curve of lutein absorbance in ethyl acetate - lutein concentration was calculated using the equation y = 0.0250x - 0.0178 (R 2 =0.9994) to obtain the free lutein concentration in the Lut-PPI complex solution. The binding rate of lutein to pea protein isolate was calculated using the following formula:

[0057]

[0058] Analysis of the binding rate of lutein / pea protein isolate complex: Lut-PPI complexes were prepared by adding different concentrations of pea protein isolate (0.5-4.5 mg / mL) to 13-65 μM lutein alcohol-soluble systems. Using the binding rate as an indicator, the highest binding rate was observed when lutein at a concentration of 26 μM combined with different concentrations of pea protein isolate. Figure 2 As shown, when lutein at a concentration of 26 μM was combined with different concentrations of pea protein isolate, the binding rate of lutein and pea protein isolate gradually increased with increasing protein concentration. However, when the protein concentration exceeded 3.5 mg / mL, the binding rate did not change significantly (p>0.05), and the optimal binding rate was 89.83%. The study found that with increasing protein concentration, the binding rate increased linearly within a certain range. However, higher concentrations of pea protein isolate may cause it to self-aggregate, thereby reducing its binding to lutein and weakening its protective effect on lutein, so the binding rate will not increase indefinitely. Therefore, the concentrations of lutein and pea protein isolate in the Lut-PPI complex were selected to be 26 μM and 3.5 mg / mL, respectively, under the conditions of optimal binding rate. That is, every 100 g of protein can bind 0.43 g of lutein. Lutein / pea protein isolate complexes were prepared at this concentration for subsequent studies.

[0059] Determination of water solubility of lutein / pea protein isolate complex: Dissolve 0.006g of untreated lutein powder, 1.394g of pea protein isolate powder, and 1.4g of optimal binding rate Lut-PPI complex freeze-dried powder in 100mL of ultrapure water. Centrifuge at 3000rpm / 10min, and measure the solubility of lutein and pea protein isolate in the supernatant. The determination method is as follows:

[0060] (1) Determination of lutein water solubility: 1.4 g of Lut-PPI complex lyophilized powder was dissolved in 100 mL of water, 1.0 mL was taken and added to a 5 mL centrifuge tube, and then 3 mL of ethyl acetate was added. The mixture was thoroughly mixed for 30 seconds and allowed to stand for 30 minutes to allow complete separation. The absorbance of lutein in the upper organic phase was measured at 447 nm using an ultraviolet spectrophotometer. The absorbance of lutein in ethyl acetate was calculated using a standard curve of lutein concentration (y = 0.0250x - 0.0178 (R 2 =0.9994), and the lutein concentration, i.e., the solubility of lutein, was obtained.

[0061] (2) Determination of water solubility of pea protein isolate: The content of pea protein isolate in the supernatant was determined by the Coomassie brilliant blue method according to the instructions of the total protein determination kit, and its solubility was calculated.

[0062] (3) Determination of water solubility of lutein and pea protein isolate in the composite system: The lyophilized powder of the Lut-PPI complex was dissolved in 100 mL of ultrapure water and centrifuged at 3000 rpm for 10 min. The supernatant was collected. The solubility of free lutein and pea protein isolate in the supernatant was determined according to the above method.

[0063] Analysis of water solubility of lutein / pea protein isolate complex: Figure 3A-3C As shown in Table 2, lutein alone or unmodified pea protein isolate were insoluble in water, while the Lut-PPI complex exhibited good water solubility and a clear, transparent solution, indicating that the interaction between lutein and pea protein isolate significantly improved lutein's water solubility. Solubility measurements revealed that the solubility of free lutein was 0.36 μg / mL, while that of the complex was 48 μg / mL, a 133-fold increase. This indicates that the combination of lutein and pea protein isolate effectively improved lutein's water solubility. When the lutein / pea protein isolate complex formed a new complex, the water solubility of the encapsulated lutein in the dry powder was significantly improved due to the protection of the pea protein isolate on the lutein during the freeze-drying process.

[0064] Table 2 Water solubility of lutein, pea protein isolate and Lut-PPI complex freeze-dried powder

[0065]

[0066] (4) Evaluation of the color and total antioxidant activity of lutein / pea protein isolate complex under exogenous conditions: Ultrapure water was added to the Lut ethanol solution at a ratio of Lut:water = 1:9 (v / v), and anhydrous ethanol was added to the PPI solution at a ratio of ethanol:PPI = 1:9 (v / v) to make the lutein concentration, pea protein isolate concentration and solvent composition in the solution the same as those of the Lut-PPI complex solution. The pH values ​​of the three groups of samples were adjusted to 2.0, 4.0, 7.0, and 10.0, respectively. After standing at 4℃ for 4 hours, the samples were centrifuged at 6000 rpm / 10 min. The color and total antioxidant activity (T-AOC) before and after pH treatment were measured. The three groups of samples were heated at 65℃ and 121℃ for 30 minutes, and then quickly cooled to room temperature after heating. The color and total antioxidant activity (T-AOC) before and after heat treatment were measured.

[0067] Color determination: A spectrophotometer was used to determine the apparent color of the sample before and after pH or heat treatment. A 4mm aperture was used for measurement and calibration was performed using a black and white plate suitable for the instrument. Color evaluation was performed using CIELab. The spectrophotometer displays lightness (L*), red (+a*) or green (-a*), yellow (+b*), or blue (-b*). ΔE, which describes the total color difference of the color change, can be obtained using the following formula:

[0068]

[0069] Where L0*, a0*, and b0* are the initial values ​​of L*, a*, and b*, representing the color coordinates of the untreated sample. The color coordinates of the sample after pH or heat treatment are L*, a*, and b*.

[0070] Determination of total antioxidant activity (T-AOC): The standard curve was drawn according to the instructions of the total antioxidant capacity (T-AOC) detection kit and the total antioxidant capacity of the three groups of samples before and after pH or heat treatment was determined.

[0071] Analysis of the Color Stability of Lutein / Pea Protein Isolate Complexes under Exogenous Conditions: Lutein exhibits poor stability under exogenous processing conditions, such as light and heat. Improving its color stability is crucial. Therefore, the present invention evaluated the protective effect of pea protein isolate on lutein color under heat and varying pH conditions by measuring color changes. As shown in Tables 3 and 4, pH and temperature can irreversibly affect lutein color.

[0072] As can be seen from Table 3, after pH treatment, the L*, a*, and b* values ​​of lutein alone all changed significantly (p<0.05), and the ΔE values ​​were all greater than 1. Under acidic conditions, the total color difference ΔE exceeded 2, indicating that the lutein had faded significantly. Generally, when the color difference value was greater than 2, it indicated that there was a significant difference between the two samples, indicating that acidic conditions had the greatest destructive effect on the color of lutein. This may be because lutein undergoes deesterification and cis / trans isomerization under acidic conditions, resulting in color loss, while it is relatively stable under neutral and alkaline conditions. Observing the values ​​of the Lut-PPI complex, except for the condition of pH 4, the total color difference ΔE values ​​under other acidic and alkaline conditions were all less than 1, with no obvious color change. This indicates that pea protein isolate has a significant protective effect on lutein color under acidic and alkaline conditions. The color loss is greater under pH 4 because pH 4 is the isoelectric point of pea protein isolate. At this time, pea protein isolate precipitates and cannot combine with lutein to form a complex, thus failing to function. It can be seen that in the processing process affected by pH factors, excluding the isoelectric point of pea protein isolate, the Lut-PPI complex has a significant protective effect on lutein color.

[0073] As can be seen from Table 4, after high-temperature treatment at 65°C or 121°C, the L*, a* and b* values ​​of lutein all decreased significantly, indicating that its brightness decreased and the color became lighter. At the same time, the ΔE values ​​were 1.48 and 2.58, respectively, indicating that high temperature had a significant effect on the color of lutein, among which the effect was greater at 121°C. The ΔE values ​​of the Lut-PPI complex decreased to 0.23 and 1.27, respectively, after heat treatment, indicating that pea protein isolate has a protective effect on the color loss of lutein during heat treatment. However, the color of lutein is relatively lost at 121°C. Therefore, in the processing of Lut-PPI complex, pasteurization is recommended for sterilization.

[0074] Table 3 Color analysis of Lut, PPI, and Lut-PPI complex before and after pH treatment

[0075]

[0076]

[0077] Table 4 Color analysis of Lut, PPI, and Lut-PPI composite before and after heat treatment

[0078]

[0079] Analysis of the total antioxidant activity of lutein / pea protein isolate complex under exogenous conditions: Figure 4 It can be seen that at pH 2, the T-AOC loss rates of lutein, pea protein isolate, and Lut-PPI complex were 81.68%, 58.28%, and 40.47%, respectively. The loss rate of Lut-PPI complex was significantly lower than that of the lutein solution alone and the pea protein isolate solution alone, indicating that the complex formed by the combination of the two is relatively stable and has a protective effect on the antioxidant capacity of lutein and pea protein isolate. However, at pH 4, the T-AOC loss rates of lutein, pea protein isolate, and Lut-PPI complex were all above 80%. This is also due to the precipitation of pea protein isolate due to its isoelectric point, which is unable to play a protective role for lutein, which is consistent with the results of color stability.

[0080] Depend on Figure 5A It can be seen that after lutein was treated at 65℃ and 121℃ for 30min, the T-AOC decreased from 0.6μmol / mL to 0.11μmol / mL and 0.13μmol / mL, respectively, indicating that heat treatment has a great influence on the T-AOC of lutein. Figure 5BAs shown in the data, after treatment at 65°C, the T-AOC loss rates of lutein, pea protein isolate and Lut-PPI complex were 81.17%, 48.76% and 38.60%, respectively; after treatment at 121°C, the T-AOC loss rates of lutein, pea protein isolate and Lut-PPI complex were 77.91%, 74.18% and 64.17%, respectively. It can be seen that the T-AOC loss rates of Lut-PPI complex under 65°C and 121°C conditions were significantly lower than those of lutein and pea protein isolate, indicating that heat treatment induced the structural expansion of pea protein isolate, promoted the combination of pea protein isolate and lutein, and thus enhanced the protective effect on the antioxidant activity of lutein. Therefore, the interaction of Lut-PPI complex under heat treatment conditions has a protective effect on the antioxidant capacity of both.

[0081] In summary:

[0082] (1) Complexes of pea protein isolate aqueous solution and lutein ethanol solution at different concentrations were prepared. Through binding rate analysis, the optimal complex composition with a binding rate of 89.83% was selected, which was 26 μmol / L lutein and 3.5 mg / mL pea protein isolate, that is, every 100 g of pea protein isolate could bind 0.43 g of lutein.

[0083] (2) In the Lut-PPI complex, pea protein isolate effectively increased the water solubility of lutein, and the solubility of lutein increased from 0.36 μg / mL to 48 μg / mL, which is 133 times the original solubility.

[0084] (3) The color and antioxidant activity of free lutein decreased significantly after acidic pH treatment. After lutein combined with pea protein isolate to form Lut-PPI complex, the color difference value ΔE at pH 2 decreased from 2.71 to 0.61, and the T-AOC loss rate decreased from 81.68% to 40.47%, indicating that the interaction between lutein and pea protein isolate has a protective effect on the color deterioration and antioxidant activity loss of lutein under acidic conditions.

[0085] (4) Lutein will deteriorate in color and decrease in antioxidant activity after heat treatment. The color difference values ​​ΔE after treatment at 65℃ and 121℃ were 1.48 and 2.58, respectively, indicating fading. After combining with pea protein isolate, the color difference values ​​ΔE decreased to 0.23 and 1.27, respectively, both showing good protective effects; the T-AOC loss rate decreased from 81.17% to 38.60% at 65℃ and from 77.90% to 64.16% at 121℃. Therefore, under the induction of mild heat treatment (65℃), the Lut-PPI complex has a more obvious protective effect on the color stability and antioxidant activity of lutein.

[0086] The present invention further reveals the interaction mechanism between 26 μM lutein and 3.5 mg / mL pea protein isolate, achieving an optimal binding rate of 89.83%. The structural and micromorphological changes of the complex were analyzed using surface hydrophobicity, ultraviolet absorption spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, and transmission electron microscopy. Molecular docking simulations were used to verify the spatial structure, binding sites, and interaction types of the new complex formed by lutein and pea protein isolate, aiming to provide theoretical support for clarifying the interaction between lutein and pea protein isolate.

[0087] Surface hydrophobicity: Surface hydrophobicity (H0) was determined using 8-anilino-1-naphthalenesulfonic acid (ANS) as a fluorescent probe. Lut-PPI complex solutions and Lut-free PPI solutions were diluted to 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Twenty microliters of 8.0 mM ANS solution was mixed with 2 mL of sample solution and allowed to react at room temperature for 10 minutes. The samples were then placed in a 96-well plate and fluorescence intensity was measured at 390 nm (excitation) and 470 nm (emission). H0 (H0 is expressed as the slope of the line) was obtained by linearly fitting the fluorescence intensity against PPI concentration.

[0088] Ultraviolet absorption spectra: The ultraviolet absorption spectra of PPI and Lut-PPI complex solutions with different concentrations were obtained by scanning in the wavelength range of 360-480 nm using a microplate reader.

[0089] Circular dichroism: PPI was diluted to 0.20 mg / mL, and the circular dichroism spectra of PPI and Lut-PPI were scanned in the 200-260 nm range using a circular dichroism spectrometer. Using a 10% ethanol solution as the background, the scan rate was 15 nm / min, and the response time was 0.5 s. The composition and specific content of the PPI secondary structure were fitted using CD pro software.

[0090] Fourier transform infrared spectroscopy: FT-IR spectra were obtained using a Fourier transform infrared spectrometer. Lut, PPI, and Lut-PPI freeze-dried powder were mixed with potassium bromide at a ratio of 1:100 (g:g), pressed into tablets, and measured with a resolution of 4 cm -1 , the signal-to-noise ratio is 50000:1, the cumulative scan is 32 times, and the wavelength range is 4000-400cm -1 .

[0091] Transmission electron microscopy: Dilute PPI and Lut-PPI samples to a PPI concentration of 0.02 mg / mL. Pipette a small amount of sample and pass it through a 300-mesh carbon grid for glow discharge. Then, incubate the sample on the grid for 1 minute. Remove the excess sample and stain with 1% uranyl acetate for 1 minute. Remove the excess stain and observe the sample at 100 kV.

[0092] Molecular docking simulation: The initial structure of Lut was obtained from PubChem (ID: 5281243), and the crystal structure of PPI (PDBID: 5XNL) was obtained from the Protein Data Bank (https: / / www.rcsb.org / ). Molecular docking was performed using Autodock Vina. The docking results were analyzed using Discovery Studio 4.5 (Accelrys Inc, San 205 Diego, CA, USA).

[0093] Effect of lutein on surface hydrophobicity of pea protein isolate: Surface hydrophobicity (H0) is one of the important indicators for measuring protein functional properties, which can reflect the number of hydrophobic groups on the protein surface and the aggregation tendency of protein molecules. 8-anilino-1-naphthalenesulfonic acid (ANS) is a fluorescent "hydrophobic probe" that can reflect the changes in the hydrophilicity / hydrophobicity of proteins. The changes in H0 values ​​of PPI and Lut-PPI composite systems are shown in Figure 2. Figure 6 As shown in the figure, after adding lutein, the H0 of PPI decreased significantly, indicating that the interaction between Lut and PPI reduced the surface hydrophobicity H0 of PPI. The possible reasons are: (1) there is a hydrophobic interaction between PPI and Lut, and the hydrophobic residues on the PPI surface become the binding sites of Lut, thereby reducing the possibility of binding with the ANS fluorescent probe; (2) when binding to PPI, the hydrophilic groups such as hydroxyl groups introduced by Lut increase the surface hydrophilicity of PPI; (3) the binding of Lut to PPI changes the protein conformation of PPI, thereby exposing its hydrophilic groups.

[0094] Lutein / pea protein isolate UV absorption spectrum analysis: As shown in Table 5, at low concentrations, Lut exhibits an absorption peak at 450 nm. As the concentration increases, a new absorption peak appears at 385 nm at 26 μM. This change in peak position indicates a change in Lut structure. Lut is highly hydrophobic and readily forms aggregates in hydrophilic environments. The free hydroxyl groups at both ends of the Lut molecule promote the formation of strongly coupled (H-type) Lut aggregates through the formation of intermolecular hydrogen bonds. Furthermore, the 450 nm absorption peak completely disappears in the 65 μM Lut-PPI complex, indicating that at this concentration, lutein exists primarily in an aggregated form. Therefore, in organic-aqueous systems, the interaction between lutein and protein macromolecules at least partially involves their aggregated form.

[0095] Table 5 Maximum absorption peak value of Lut-PPI (λ max )

[0096]

[0097] Circular dichroism analysis of lutein / pea protein isolate: Circular dichroism is a sensitive technique for monitoring conformational changes in individual proteins or proteins bound to ligands. To clarify specific changes in protein structure, CDpro software was used to fit the content of α-helices, β-sheets, β-turns, and random coils in the complex. As shown in Table 6, upon binding to Lut, the α-helical structure of the PPI decreased from 72.5% to 68.9%, while the β-sheets, β-turns, and random coils all increased slightly. This further confirms that the interaction between the PPI and Lut leads to structural unfolding and changes in its secondary structure.

[0098] Table 6 Changes in secondary structure before and after PPI complexation

[0099]

[0100] Fourier transform infrared spectroscopy analysis of lutein / pea protein isolate: Fourier transform infrared spectroscopy is often used to study the interaction between small molecules and proteins. Figure 7 As shown, at 1631cm -1 1039cm -1 The characteristic peaks of Lut were observed at 1722 cm-1 and 1732 cm-2, which represent the functional groups C=C and -OH of Lut respectively. -1 、1039cm -1 、967cm- 1 The characteristic peaks at do not appear in Lut-PPI, indicating that there is an interaction between Lut and PPI to form a complex.

[0101] Amide I band (1700-1600 cm -1 ) represents C=O stretching vibration; amide II band (1600-1500 cm -1 ) represents CN stretching and NH bending. The spectra of PPI are at 1650 and 1538 cm -1 The amide I and amide II bands are shown at the bottom. After adding Lut, the two absorption peaks move to 1647 and 1536 cm -1 , and the peak intensity decreases. The present invention shows the existence of interaction between the two and the change in the secondary structure of PPI, which is consistent with the results of circular dichroism. At the same time, the decrease in peak intensity indicates the presence of hydrophobic interaction between Lut and PPI, which is consistent with the surface hydrophobicity results.

[0102] Transmission electron microscopy analysis of lutein / pea protein isolate: Figure 8A 、 Figure 8BAs shown in the figure, the shape and size of PPI are irregular, and some proteins have aggregated. However, the Lut-PPI complex appears as spherical nanoparticles with an average particle size of 20-40nm, which is significantly smaller than that of individual PPIs, showing a smaller particle size and regular and uniform distribution.

[0103] Lutein / pea protein isolate molecular docking simulation: Molecular docking simulation was used to explore the interaction between Lut and PPI. The lower the binding energy, the greater the possibility of binding and the more stable the complex. The present invention uses binding energy analysis to find the site with the lowest binding energy, determine the amino acid composition near the site and its interaction type with lutein. Figure 9 As shown, the binding energy of Lut to different PPI sites ranges from -4.52 kcal / mol to 7.2 kcal / mol. The lowest binding energy is -7.2 kcal / mol, indicating that the binding process of Lut to PPI is energetically favorable, and therefore it was selected as the optimal site for subsequent studies.

[0104] By selecting the optimal binding site (with the lowest binding energy) as the target for investigation, we further analyzed the amino acid composition and interaction types surrounding the docking site. As shown in Table 7, amino acid residues around the PPI that interact with Lut include hydrophobic residues (e.g., Phe431, Ile183, and Met328), polar residues (e.g., Tyr161 and Asn191), and charged residues (e.g., His190). Phe431, Ile183, Ile326, Met328, Leu184, Tyr161, and His190 form hydrophobic interactions with Lut, consistent with the fluorescence quenching and surface hydrophobicity results, demonstrating that hydrophobic interactions drive the pea protein isolate / lutein interaction. Furthermore, other binding forces, such as π bonds, hydrogen bonds, and carbon-hydrogen bonds, contribute to the stability of the Lut-PPI complex.

[0105] Table 7 Amino acid residues and corresponding interaction forces of Lut binding to PPI

[0106]

[0107] In summary, the UV absorption intensity of the Lut-PPI complex increases with the increase of Lut concentration. At the same time, the absorption peak gradually shifts from 450 nm to 385 nm, indicating that with the increase of Lut concentration, its highly hydrophobic properties and the free hydroxyl groups at both ends of the molecule promote the formation of Lut strong coupling (H-type) aggregates through the formation of intermolecular hydrogen bonds. Therefore, in the 26 μM Lut-PPI complex, Lut exists in both dispersed and aggregated forms. Furthermore, circular dichroism, Fourier transform infrared spectroscopy, and transmission electron microscopy revealed that the interaction of 26μM Lut-PPI led to the unfolding of the PPI structure and changes in the secondary structure, showing that the α-helix structure of PPI in the Lut-PPI complex decreased, while the β-fold, β-turn, and random coil structures increased. At the same time, the absorption peaks corresponding to the amide I band and the amide II band shifted and the peak intensity decreased. In the 26μM Lut-PPI complex system, Lut and PPI formed uniform and stable spherical nanoparticles with an average particle size of 20-40nm, and their structure was denser and more stable.

[0108] The present invention also provides an application of a highly water-soluble lutein / pea protein isolate complex: the lutein / pea protein isolate complex is used to prepare a beverage. Sensory evaluation determined the formula of the Lut-PPI functional beverage to be 6.0 mg Lut, 1.4 g PPI, 1.5 g xylitol, 0.04 g citric acid, 5.0 g citrus fiber, 3.0 g goji berry powder, and 100 mL water. This beverage is bright yellow and uniform in color, has an antioxidant capacity of 220 ± 4.2 μmol / mL, a stability coefficient of 85.05%, and a centrifugal sedimentation rate of 2.46%. A shelf life prediction model was constructed, revealing that the theoretical shelf life of this beverage at temperatures of 4°C, 25°C, and 37°C is 217 days, 109 days, and 76 days, respectively.

[0109] This study addresses the issues of color deterioration, poor water solubility, and loss of bioactivity in lutein-rich products under exogenous processing conditions. Based on a novel approach to blocking lutein degradation through interactions between lutein and proteins, a lutein / pea protein isolate complex was prepared. The complex's color stability and antioxidant activity under exogenous processing conditions were analyzed, clarifying the interaction mechanism between lutein and pea protein isolate. Application strategies based on the pigment-protein interaction between the two in food systems were explored, laying a theoretical foundation for maintaining color stability and bioactivity in lutein-rich products under exogenous processing conditions. The research findings may provide new solutions to address color deterioration and bioactivity loss in lutein during processing, and are of great significance for promoting the processing and industrialization of lutein-related products.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a highly water-soluble lutein / pea protein isolate complex, characterized in that: The preparation method comprises: (1) Preparation of lutein solution: Dissolve lutein in anhydrous ethanol to obtain a lutein solution; (2) Preparation of pea protein isolate solution: pea protein isolate powder was dispersed in ultrapure water, stirred to form a dispersion, NaOH solution was added, and constant temperature oscillation was performed at a certain temperature for a certain time to completely dissolve the pea protein isolate; cooled to room temperature, HCl solution was added to adjust the pH value, and a pea protein isolate solution was obtained; (3) Preparation of lutein / pea protein isolate complex: 0.5-4.5 mg / mL pea protein isolate solution and 13-65 μM lutein solution were mixed in a volume ratio of 9:1 and shaken at room temperature for 25-30 min to obtain a lutein / pea protein isolate complex solution; after freeze-drying, a highly water-soluble lutein / pea protein isolate complex was obtained; the solubility of lutein in the lutein / pea protein isolate complex reached a maximum of 48 μg / mL, and the solubility of pea protein isolate in the lutein / pea protein isolate complex reached 11.3 mg / mL; In step (1), the lutein concentration in the prepared lutein solution is 13-65 μM; Step (2) is as follows: Dispersing pea protein isolate in ultrapure water and continuously stirring the solution by magnetic force for 20-30 minutes to obtain a pea protein isolate dispersion; adding a 0.1-1 mol / L NaOH solution to the pea protein isolate dispersion, adjusting the pH to 11.5-12.0, and oscillating the solution in a constant temperature oscillator at 83-87° C. for 25-30 minutes to promote structural expansion of the pea protein isolate and completely dissolve the pea protein isolate; To prevent excessive denaturation of pea protein isolate induced by heat treatment, the completely dissolved pea protein isolate solution was quickly placed in ice water for 5-10 minutes to cool to room temperature, and the pH value was adjusted to 7.4-7.8 with 0.1-1 mol / L HCl solution to obtain a pea protein isolate solution with a concentration of 0.5-4.5 mg / mL.

2. The method for preparing a highly water-soluble lutein / pea protein isolate complex according to claim 1, characterized in that: In step (3), the lutein / pea protein isolate complex solution obtained has a lutein concentration range of 13-65 μM and a pea protein isolate concentration range of 0.5-4.5 mg / mL.

3. A highly water-soluble lutein / pea protein isolate complex prepared by the method of claim 1, characterized in that: The lutein / pea protein isolate complex is formed by spontaneous binding of lutein and pea protein isolate through hydrophobic interactions. The lutein and pea protein isolate combine to form uniform and stable spherical nanoparticles, and the average particle size of the spherical nanoparticles is 20-40 nm. The solubility of lutein in the lutein / pea protein isolate complex freeze-dried powder reaches a maximum of 48 μg / mL, and the solubility of pea protein isolate in the lutein / pea protein isolate complex reaches 11.3 mg / mL.

4. The highly water-soluble lutein / pea protein isolate complex according to claim 3, characterized in that: The solubility of lutein in the lutein / pea protein isolate complex freeze-dried powder is determined as follows: Dissolve 1.4 g of lyophilized powder of lutein / pea protein isolate complex in 100 mL of water, take 1.0 mL and add it to a 5 mL centrifuge tube, then add 3 mL of ethyl acetate, mix thoroughly for 30 seconds, let it stand for 30 minutes, and completely separate the layers. Use an ultraviolet spectrophotometer to measure the absorbance of lutein in the upper organic phase at 447 nm. The lutein concentration, i.e., the solubility of lutein, is obtained through the absorbance value of lutein in ethyl acetate-lutein concentration standard curve.

5. The highly water-soluble lutein / pea protein isolate complex according to claim 3, characterized in that: The binding rate of lutein and pea protein isolate in the lutein / pea protein isolate complex can reach up to 89.83%, that is, every 100 g of pea protein isolate can bind up to 0.43 g of lutein.

6. Use of the highly water-soluble lutein / pea protein isolate complex according to any one of claims 3 to 5, characterized in that: The lutein / pea protein isolate complex is used in preparing beverages, emulsified sausages, and high internal phase emulsion systems.

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