Preparation of modified eucommia seed meal protein and application of modified eucommia seed meal protein in stabilizing xanthophyll in red date juice

Abstract

The invention relates to preparation of modified eucommia seed meal protein and application of the modified eucommia seed meal protein in stabilizing xanthophyll in red date juice. According to the method, the eucommia seed meal protein is modified by adopting a dielectric barrier discharge low-temperature plasma technology, modification of the eucommia seed meal protein is controlled by adjusting plasma discharge voltage and discharge time, and optimal modification conditions are screened. Then adopting a layer-by-layer self-assembly technology, preparing a multi-layer emulsion from eucommia seed meal protein and chitosan to embed the xanthophyll, realizing effective load, and evaluating the stability of the xanthophyll in the red date juice. The xanthophyll in the prepared red date juice beverage is good in thermal stability and storage stability, the product form is novel, the use of a traditional chemical emulsifier is avoided, and the consumption demand of modern consumers on green and healthy products is met.

Description

Technical Field

[0001] The invention relates to modification of eucommia seed meal protein by low-temperature plasma technology and application of the modification in stabilizing a lutein-rich red date juice system, belonging to the technical field of functional food composite nanoparticles and healthy beverage preparation. Background Art

[0002] As a water-soluble system, fruit and vegetable juice lacks fat-soluble nutrients, which leads to a large defect in its nutritional balance. Adding fat-soluble nutrients, such as ω-3 polyunsaturated fatty acids, lutein, linolenic acid, vitamin E, etc., to fruit and vegetable juice can not only improve the problem of nutritional deficiency in fruit and vegetable juice, but also meet the nutritional needs of special populations. Red dates are rich in vitamins, trace elements and polysaccharides, and have the effects of blood tonic, blood pressure reduction, enhancing human immunity, protecting the liver, calming the nerves, etc. Lutein is a pigment that exists naturally in plants and cannot be synthesized in the human body. Lutein has a variety of biological functions, has a strong antioxidant effect, can help remove free radicals in the body, reduce and repair the damage of free radicals to cells, and has the effect of beautifying and delaying aging; it can reduce cholesterol in the blood, reduce blood viscosity, and prevent and relieve atherosclerosis; lutein is an important component of the retina of the eye. Eating lutein can improve vision and protect the eyes, and can also resist the damage of ultraviolet rays and blue light to the retina, and has the effect of preventing cataracts. At present, lutein has been widely used in food, medicine and cosmetics. Developing a healthy drink rich in lutein red date juice will have broad market prospects. However, since most of the lutein is an unsaturated structure, the water solubility and stability of lutein are poor, and it is very easy to degrade and inactivate, which greatly affects its efficacy. It is still difficult to add it to the complex water system red date juice and ensure its stability during storage.

[0003] Eucommia seed oil is a new resource food that contains a variety of functional nutrients. However, during its production, a large amount of seed meal is discarded, which not only pollutes the environment, but also leads to the loss of a large number of nutrients and functional factors. The protein content of Eucommia seed meal is as high as 35.98%, containing 18 kinds of amino acids, and the content of essential amino acids for the human body accounts for 32.3% of the total amino acids. It is a new source of plant protein, but its high recycling value has not been paid attention to. Studies have found that some plant proteins have poor functional properties such as solubility and emulsification, which limits their application in the food industry. Modification of plant protein can improve its functional properties and meet the needs of the food industry. At present, there are few reports on the modification of Eucommia seed meal protein. There are only reports on the treatment of Eucommia seed meal protein with ultra-high pressure microfluidization, and the structure and properties of Eucommia seed meal protein have been changed accordingly.

[0004] At present, the methods of protein modification include enzymatic hydrolysis, glycosylation, microwave, ultrasound, freezing, etc. These modification methods provide references for the high-value utilization of proteins, but they have disadvantages such as low efficiency, reagent residues, high cost, and large losses. In recent years, low-temperature plasma (CP) has attracted much attention in the field of modified plant proteins due to its non-thermal, safe, environmentally friendly and high efficiency. During the discharge process, CP can produce a large number of high-energy active particles, which bombard the protein surface, induce the covalent bond breakage of the protein surface, increase the roughness, and change the microstructure. At the same time, CP treatment can induce protein depolymerization and change the secondary structure, and the solubility, emulsification, water retention and oil retention are all improved. At present, there are no reports on the modification of Eucommia ulmoides seed meal protein using low-temperature plasma technology and using it as a carrier to load nutrients. Summary of the invention

[0005] On the one hand, the present invention provides a method for modifying eucommia seed meal protein by low-temperature plasma, which is applied to the loading of lutein and added to red date juice, thereby solving the problems of poor water solubility, easy degradation and difficulty in stabilizing lutein in red date juice. Meanwhile, the use of traditional chemical emulsifiers can be avoided, thereby meeting consumers' demand for green and healthy food.

[0006] On the other hand, the present application provides a method for preparing red date juice, which adopts low-temperature plasma technology to modify eucommia seed meal protein, and then prepares a lutein primary emulsion by ultrasonic emulsification technology based on the principle of layer-by-layer self-assembly technology, and then adds chitosan solution to the primary emulsion to prepare a lutein multilayer emulsion, and finally adds it to red date juice to prepare a red date juice health drink rich in lutein.

[0007] Specifically, a method for modifying eucommia seed meal protein comprises the following steps: taking eucommia seed meal protein, preparing a protein solution, then treating the protein solution with dielectric barrier discharge low-temperature plasma, and obtaining plasma-modified eucommia seed meal protein powder by freeze-drying.

[0008] Furthermore, the modified eucommia seed meal protein preparation method comprises: low-temperature plasma treatment voltage is 5kv-20kv, treatment time is 5s-120s, and discharge distance is 0.5-3cm.

[0009] Furthermore, the preferred voltage is 10kv-20kv; 12kv-18kv; 13kv-17kv; 14kv-16kv, the preferred treatment time is 5s-100s; 5s-80s; 5s-60s; 5s-50s; 5s-40s; 5s-30s; 5s-20s; 10s-20s, and the preferred discharge distance is 0.5-3cm; 0.5-2.5cm; 0.5-2cm; 0.5-1.5cm.

[0010] On the other hand, the present application provides a method for preparing a lutein multilayer emulsion, the preparation method comprising: dispersing the above-mentioned low-temperature plasma-modified eucommia seed meal protein in a liquid to obtain an eucommia seed meal protein solution, mixing lutein with eucommia seed oil as an oil phase, adding the eucommia seed meal protein solution to the oil phase for emulsification, adding a chitosan solution, and stirring to prepare the lutein multilayer emulsion.

[0011] Furthermore, the emulsification method includes ultrasonic emulsification, high-speed stirring emulsification and high-pressure homogenization emulsification.

[0012] The weight ratio of lutein to eucommia seed oil is 1:5-100; preferably 1:5-90; preferably 1:5-80; preferably 1:5-70; preferably 1:5-60; preferably 1:5-50; preferably 1:10-40; preferably 1:10-30; preferably 1:15-25.

[0013] Furthermore, the eucommia seed meal protein solution is added to the oil phase for ultrasonic treatment to prepare a primary emulsion, and the chitosan solution is slowly added dropwise to the primary emulsion.

[0014] Further, chitosan solution is added, and after thorough mixing, the pH of the mixture is adjusted to 3-7; preferably to 3-6; preferably to 4-6.

[0015] Furthermore, the concentration of low-temperature plasma modified eucommia seed meal protein is 0.25%-2%, the amount of lutein-containing oil phase added is 1%-10%, the amount of chitosan added is 0.05%-0.3%, the ultrasonic power is 200W-1200W, and the ultrasonic time is 5min-20min.

[0016] In another aspect of the present application, a method for preparing lutein-rich red date juice is provided, which is obtained by adding the above-mentioned lutein multilayer emulsion into red date juice.

[0017] Furthermore, the weight ratio of the lutein multilayer emulsion to red date juice is 1:5-50; preferably 1:5-40; preferably 1:5-30; preferably 1:5-25; preferably 1:10-20.

[0018] Furthermore, the lutein multilayer emulsion is added to red date juice and homogenized by a high-pressure homogenizer at a pressure of 15-30 MPa for 1-3 times.

[0019] 1. Plasma treatment exposes the internal groups of Eucommia seed meal protein, which is more conducive to improving the interfacial stability of the protein compared with the unmodified protein.

[0020] 2. During the plasma discharge process, high-energy particles impact the protein in eucommia seed meal, causing moderate changes in the secondary and tertiary structures of the protein. Compared with the unmodified protein, the plasma-modified eucommia seed meal exposes more active sites and improves its water adsorption capacity. At the same time, the non-polar amino acids on the protein surface are exposed, which enhances the interaction between oil and protein and improves the oil holding capacity.

[0021] 3. Through the layer-by-layer self-assembly technology, eucommia seed meal protein and chitosan were used to prepare a multilayer emulsion of lutein. The stability of lutein was improved by non-covalent bonds, especially in the acidic red date juice environment. This can solve the shortcomings of free lutein, such as low water solubility, poor stability, and easy degradation and inactivation.

[0022] The protein of Eucommia seed meal is modified to improve its emulsifying properties and amphiphilicity, and enhance its ability to load functional factors to meet the needs of the food industry, opening up a new way for the utilization of Eucommia seed meal waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effect of plasma treatment voltage on water and oil holding capacity of Eucommia ulmoides seed meal protein; Figure 2 Effect of plasma treatment voltage on the emulsification and emulsion stability of eucommia seed meal protein; Figure 3 Effect of plasma treatment time on water and oil holding capacity of Eucommia ulmoides seed meal protein; Figure 4 Effect of plasma treatment time on the emulsification and emulsion stability of eucommia seed meal protein; Figure 5 Effects of different concentrations of eucommia seed meal protein on the lutein embedding rate in colostrum; WG: unmodified eucommia seed meal protein, GX: plasma-modified eucommia seed meal protein;

[0024] Figure 6 Effect of different oil phase addition amounts on lutein embedding efficiency in primary emulsion; Figure 7 Effects of different concentrations of eucommia seed meal protein on the thermal stability of lutein in red jujube juice; YL: free lutein, WG: unmodified eucommia seed meal protein, GX: plasma-modified eucommia seed meal protein;

[0025] Figure 8 Effects of different concentrations of eucommia seed meal protein on the storage stability of lutein in red jujube juice; YL: free lutein, WG: unmodified eucommia seed meal protein, GX: plasma-modified eucommia seed meal protein;

[0026] Fig. 9 Effects of different chitosan concentrations on the thermal stability of lutein in red date juice; Fig.10 Effect of different chitosan concentrations on the storage stability of lutein in red jujube juice. DETAILED DESCRIPTION

[0027] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0028] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0029] Example 1: Effects of different plasma discharge voltages on the structural properties of eucommia seed meal protein

[0030] 1. Preparation of Eucommia ulmoides seed meal protein The Eucommia seed meal was crushed and passed through an 80-mesh sieve to obtain Eucommia seed meal powder. The Eucommia seed meal powder was taken, and a NaOH solution with a pH of 9.5 was added at a solid-liquid ratio of 1:10, extracted at 50°C for 2 hours, centrifuged at 8000r / min for 20 minutes, the supernatant was taken and the pH was adjusted to 4.6 with a 1mol / L HCl solution, and the precipitate was taken after standing overnight, washed with water to a pH of 7.0, and freeze-dried to obtain Eucommia seed meal protein.

[0031] 2. Low-temperature plasma modification of Eucommia ulmoides seed meal protein Eucommia seed meal protein was taken and prepared into a 50 mg / mL protein solution with distilled water, and then the protein solution was treated with dielectric barrier discharge low-temperature plasma. The plasma discharge distance was set to 1 cm, the discharge time was 30 s, and the discharge voltage was 5-20 kV to study the effects of different discharge voltages on the functional properties of Eucommia seed meal protein. After low-temperature plasma treatment, plasma-modified Eucommia seed meal protein powder was obtained by freeze drying and stored at -4 °C for further use.

[0032] 3. Determination of functional properties of Eucommia ulmoides seed meal protein (1) Measurement of water and oil holding capacity Accurately weigh 1 g of plasma-treated eucommia seed meal protein in a centrifuge tube, add 20 mL of deionized water and soybean oil respectively, mix well and let stand for 30 min, centrifuge at 10000 r / min for 20 min, remove the supernatant, and measure the mass after water and oil absorption. The water / oil holding capacity of the sample is characterized by the ratio of the mass of absorbed water / oil to the mass of protein before water / oil absorption.

[0033] (2) Emulsification property measurement Eucommia seed meal proteins modified under different conditions were dispersed in phosphate buffer (0.1 M, pH 7.4). The samples were mixed with soybean oil at a mass ratio of 3:1 and then homogenized in a high-speed dispersing homogenizer at 12,000 rpm for 2 min. 50 μL of the emulsion was mixed with 5 mL of SDS solution (0.1%, w / v) at 0 min and 10 min, respectively, and the absorbance values ​​were measured at 500 nm. The emulsifying activity (EAI) and emulsifying stability (ESI) were calculated using formulas (1) and (2), respectively: EAI(m2 / g)=(2×2.303×A0×N) / (C×θ×φ×10000) (1) ESI(%)=A 10 / A0×100 (2) Among them, A0 is the absorbance value at 0 min, A 10 is the absorbance value at 10 min, N is the dilution factor, C is the concentration of the protein solution (g / mL), θ is the volume fraction of the oil phase in the emulsion, and φ is the optical diameter.

[0034] 4. Test results The water-holding capacity and oil-holding capacity of Eucommia ulmoides seed meal protein increased with the increase of plasma treatment voltage ( Figure 1 ), when the plasma treatment voltage is 15kv, the water holding capacity and oil holding capacity reach the maximum, which are 5.36g / g and 3.21g / g respectively. When the voltage continues to increase, the water holding capacity and oil holding capacity of Eucommia seed meal protein begin to decrease. This is because high-energy particles hit the protein during the plasma discharge process, causing the secondary and tertiary structures of the protein to change, exposing more active sites and improving the adsorption capacity of water. At the same time, the non-polar amino acids on the surface of the protein are exposed, which enhances the interaction between oil and protein and improves the oil holding capacity. However, when the discharge voltage is too large, the water holding capacity and oil holding capacity will decrease again. This may be because when the voltage is too high, the protein will form aggregates and the internal groups cannot combine well with water and oil.

[0035] After plasma treatment, the emulsification and emulsion stability of Eucommia ulmoides seed meal protein were significantly improved ( Figure 2 ), and reached the maximum at 15kv, which are 65.46m 2 / g and 82.2%, proving that proper plasma treatment can loosen the spatial structure of the protein, moderately denature the protein, expose the internal hydrophobic groups, and allow the protein to unfold at the oil-water interface to form a relatively stable network structure and interfacial membrane, thereby enhancing the emulsification.

[0036] Example 2: Effects of different plasma discharge times on the structural properties of eucommia seed meal protein 1. Preparation of Eucommia ulmoides seed meal protein The steps are the same as in Example 1.

[0037] 2. Low-temperature plasma modification of Eucommia ulmoides seed meal protein Eucommia seed meal protein was taken and prepared into a 50 mg / mL protein solution with distilled water, and then the protein solution was treated with dielectric barrier discharge low-temperature plasma. The plasma discharge distance was set to 1 cm, the discharge voltage was 15 kV, and the discharge time was 5-120 s to study the effects of different discharge times on the functional properties of Eucommia seed meal protein. After low-temperature plasma treatment, plasma-modified Eucommia seed meal protein powder was obtained by freeze drying. It was stored at -4 °C for further use.

[0038] 3. Determination of functional properties of Eucommia ulmoides seed meal protein (1) Measurement of water and oil holding capacity The steps are the same as in Example 1.

[0039] (2) Emulsification property measurement The steps are the same as in Example 1.

[0040] 4. Test results The water-holding capacity and oil-holding capacity of Eucommia ulmoides seed meal protein increased with the extension of plasma treatment time ( Figure 3 ), the water holding capacity reaches the maximum when the plasma treatment time is 30s, which are 5.39g / g and 3.26g / g respectively. This is because high-energy particles hit the protein during the plasma discharge, causing the secondary and tertiary structures of the protein to change, exposing more active sites and improving the adsorption capacity for water. At the same time, the non-polar amino acids on the surface of the protein are exposed, which enhances the interaction between oil and protein and improves the oil holding capacity. However, when the discharge time is too long, the water holding capacity and oil holding capacity will decrease. This may be because when the discharge time is too long, the protein will form aggregates and the internal groups cannot combine well with water and oil.

[0041] After plasma treatment, the emulsification and emulsion stability of Eucommia ulmoides seed meal protein were significantly improved ( Figure 4 ), and reached the maximum at 30s of discharge, which were 65.37m 2 / g and 81.9%, proving that proper plasma treatment can loosen the spatial structure of the protein, moderately denature the protein, expose the internal hydrophobic groups, and allow the protein to unfold at the oil-water interface to form a relatively stable network structure and interfacial membrane, thereby enhancing the emulsification.

[0042] The modified Eucommia ulmoides seed meal protein used for encapsulating oil phase lutein is suitable for selecting the modification conditions with the best oil holding and emulsifying properties. In summary, the plasma discharge voltage of 15 kV and the discharge time of 30 s are selected as the best modification conditions for Eucommia ulmoides seed meal protein.

[0043] Example 3: Effect of different protein addition amounts on lutein embedding efficiency in primary emulsion

[0044] 1. Preparation of Eucommia ulmoides seed meal protein-lutein colostrum The best plasma discharge conditions in Example 1 and Example 2 were selected, i.e., a discharge voltage of 15 kV and a discharge time of 30 s to treat eucommia seed meal protein, and then the modified eucommia seed meal protein was dispersed in a phosphate buffer (0.1 M, pH 7.4), stirred for 1 h, and eucommia seed meal protein solutions with concentrations of 0.25%, 0.5%, 0.75%, 1.0%, and 1.25% were obtained. After stirring, the solution was hydrated overnight at 4 ° C. Lutein was weighed and added to eucommia seed oil according to a mass ratio of 1:20 to prepare eucommia seed oil rich in lutein as an oil phase.

[0045] The preparation of Eucommia seed meal protein-lutein primary emulsion was carried out by ultrasonic emulsification. 5% oil phase was added to Eucommia seed meal protein solutions of different concentrations. Primary emulsion was prepared under the conditions of ultrasonic 5 s, 5 s interval, ultrasonic power adjusted to 800 W, and time for 10 min in an ice-water bath.

[0046] 2. Determination of lutein embedding rate in colostrum Weigh an appropriate amount of Eucommia seed meal protein-lutein emulsion, add ethanol, stir thoroughly and centrifuge to remove free lutein in the supernatant, repeat this step several times until the supernatant is colorless. Place the precipitate in distilled water, ultrasonicate for 15 min to release the embedded lutein in the water, then dissolve the embedded lutein in the distilled water with ethanol, centrifuge to remove impurities, and use ultraviolet spectrophotometry to determine the absorbance value of the embedded lutein. The detection wavelength is 445nm, and the lutein content is calculated using the standard curve. The embedding rate calculation formula is as follows: E=m1 / m2*100%; Where: E is the lutein retention rate, m1 is the embedded lutein content, and m2 is the total lutein content.

[0047] 3. Test results like Figure 5As shown in the figure, compared with the unmodified Eucommia seed meal protein (embedding rate is 63.4%), the plasma-modified Eucommia seed meal protein has a higher embedding rate of lutein, and with the increase of the amount of modified protein added, the embedding rate of lutein also increases. When the concentration of modified Eucommia seed meal protein reaches 0.75%, the embedding rate reaches the highest 84.7%. With the increase of protein addition, the embedding rate no longer continues to increase. This may be because at this oil phase addition, the 0.75% protein addition can reach the maximum degree of embedding.

[0048] Example 4: Effect of different oil phase addition amounts on lutein embedding efficiency in primary emulsion

[0049] 1. Preparation of Eucommia ulmoides seed meal protein-lutein colostrum The best plasma discharge conditions in Example 1 and Example 2, i.e., a discharge voltage of 15 kV and a discharge time of 30 s were selected to treat the Eucommia seed meal protein, and then the modified Eucommia seed meal protein was dispersed in a phosphate buffer (0.1 M, pH 7.4) and stirred for 1 h to obtain an Eucommia seed meal protein solution with a concentration of 0.5%. After stirring, the solution was hydrated overnight at 4 ° C. Lutein was weighed and added to Eucommia seed oil at a ratio of 1:20 to prepare Eucommia seed oil rich in lutein as an oil phase.

[0050] The preparation of Eucommia seed meal protein-lutein primary emulsion was carried out by ultrasonic emulsification. 2.5%, 5%, 7.5%, 10%, and 12.5% ​​oil phase were added to Eucommia seed meal protein solutions of different concentrations. The primary emulsion was prepared in an ice-water bath with ultrasonic power adjusted to 800 W for 5 s, intermittent for 5 s, and time for 10 min.

[0051] 2. Determination of lutein embedding rate in colostrum Weigh an appropriate amount of Eucommia seed meal protein-lutein emulsion, add ethanol, stir thoroughly and centrifuge to remove free lutein in the supernatant, repeat this step several times until the supernatant is colorless. Place the precipitate in distilled water, ultrasonicate for 15 min to release the embedded lutein in the water, then dissolve the embedded lutein in the distilled water with ethanol, centrifuge to remove impurities, and use ultraviolet spectrophotometry to determine the absorbance value of the embedded lutein. The detection wavelength is 445nm, and the lutein content is calculated using the standard curve. The embedding rate calculation formula is as follows: E=m1 / m2*100%; Where: E is the lutein retention rate, m1 is the embedded lutein content, and m2 is the total lutein content.

[0052] 3. Test results like Figure 6As shown in the figure, when the amount of oil phase added is 2.5%, the lutein embedding rate reaches the maximum, which is 84.55%. When the amount of oil phase added is 5%, there is no significant change. As the amount of oil phase added continues to increase, the embedding rate decreases. This may be due to the limited embedding capacity of protein. When the amount of oil phase added is too much, it exceeds the embedding capacity of protein, resulting in incomplete embedding of the oil phase. Therefore, it is more appropriate to choose 5% as the amount of oil phase added.

[0053] Example 5: Effect of different protein addition amounts on the stability of lutein-rich red date juice

[0054] 1. Preparation of Eucommia ulmoides seed meal protein-lutein colostrum The best plasma discharge conditions in Example 1 and Example 2 were selected, i.e., the discharge voltage was 15 kV, and the discharge time was 30 s to treat the eucommia seed meal protein, and then the modified eucommia seed meal protein was dispersed in a phosphate buffer (0.1 M, pH 7.4), stirred for 1 h, and eucommia seed meal protein solutions with concentrations of 0.25%, 0.5%, 0.75%, 1.0%, and 1.25% were obtained. After stirring, the solution was hydrated overnight at 4 ° C. Lutein was weighed and added to eucommia seed oil at a ratio of 1:20 to prepare eucommia seed oil rich in lutein as an oil phase.

[0055] The preparation of Eucommia seed meal protein-lutein primary emulsion was carried out by ultrasonic emulsification. 5% oil phase was added to Eucommia seed meal protein solutions of different concentrations. Primary emulsion was prepared under the conditions of ultrasonic 5 s, 5 s interval, ultrasonic power adjusted to 800 W, and time for 10 min in an ice-water bath.

[0056] 2. Preparation of Eucommia ulmoides seed meal protein-lutein multilayer emulsion Weigh 2g of chitosan, dissolve it in an acetic acid buffer solution at pH 3.0, and stir it thoroughly under a magnetic stirrer to dissolve it to prepare a chitosan solution. Dilute the primary emulsion 5 times with a phosphate buffer solution at pH 7.0, and then slowly add it dropwise to the 0.2% chitosan solution under stirring. After it is fully mixed, adjust the pH of the mixture to 5, and continue stirring for 20 minutes to prepare an emulsion.

[0057] 3. Preparation of red date juice Select red dates with bright colors, full flesh and integrity, clean them and remove the pits to obtain pitted red dates. Add the pitted red dates into water at a mass ratio of 1:3-1:5, soak for 8 hours and then beat them. Add 0.05 g·L -1 The composite enzyme (cellulase and pectinase mass ratio is 2.5:1, and then the red date pulp is enzymatically clarified and filtered to obtain the supernatant. 5% white sugar and 0.045% citric acid are added to the supernatant for preparation, and 0.01% potassium sorbate is added as a preservative, and stirred evenly to obtain red date juice.

[0058] 4. Preparation of lutein-rich red date juice According to the eucommia seed meal protein modification method described in Example 1, the eucommia seed meal protein was modified under the conditions of a plasma discharge voltage of 15 kV and a discharge time of 30 s, and then the eucommia seed meal-lutein multilayer emulsion was obtained according to the low-temperature plasma modified eucommia seed meal protein loaded with lutein emulsion preparation method described in Example 3. The emulsion was added to red date juice at a ratio of 1:15, homogenized by a high-pressure homogenizer at a pressure of 25 MPa for 3 times, and then pasteurized and filled to obtain a red date juice beverage rich in lutein.

[0059] 5. Determination of thermal stability of lutein-rich red date juice Lutein-rich red date juice was stored at 4°C, 25°C, and 60°C for 24 hours to examine the effect of processing temperature on lutein and its emulsion stability. After the preparation of the red date juice, 0h and 24h after storage, appropriate amounts of red date juice samples were taken for determination, where 0h sampling was used to determine the lutein embedding rate, and 24h sampling was used to determine the lutein retention rate. The lutein determination steps are as follows: take an appropriate amount of red date juice sample and add ethanol, stir thoroughly and centrifuge at 3500 r / min to remove the free lutein in the supernatant, and repeat this step several times until the supernatant is colorless. Place the precipitate in distilled water, ultrasonicate for 15 min to release the retained lutein in the water, then dissolve the lutein in the distilled water with ethanol, centrifuge to remove impurities, and use ultraviolet spectrophotometry to determine the absorbance value of the retained lutein, with a detection wavelength of 445nm. Use the standard curve to calculate the embedded lutein content, and the embedding rate calculation formula is as follows: K=m1 / m2*100%; Where: K is the lutein retention rate, m1 is the retained lutein content, and m2 is the embedded lutein content.

[0060] 6. Storage stability test of lutein-rich red date juice Lutein-rich red date juice was stored in the dark at 25°C. After the preparation of the red date juice, 0h and after storage for 1d, 5d, and 10d, appropriate amounts of red date juice samples were taken for determination. The sampling at 0h was to determine the lutein embedding rate, and the sampling at other time points was to determine the lutein retention rate. The determination steps of lutein are as follows: take an appropriate amount of red date juice sample and add ethanol. After sufficient stirring, centrifuge at 3500 r / min to remove the free lutein in the supernatant. Repeat this step several times until the supernatant is colorless. Place the precipitate in distilled water, ultrasonicate for 15 min to release the retained lutein in the water, then dissolve the lutein in the distilled water with ethanol, centrifuge to remove impurities, and use ultraviolet spectrophotometry to determine the absorbance value of the retained lutein. The detection wavelength is 445nm. The embedded lutein content is calculated using the standard curve. The embedding rate calculation formula is as follows: K=m1 / m2*100%; Where: K is the lutein retention rate, m1 is the retained lutein content, and m2 is the embedded lutein content.

[0061] At the same time, the retention rates of unmodified Eucommia seed meal protein (added at 1%) and modified Eucommia seed meal protein with different concentrations and free lutein were compared.

[0062] 7. Test results like Figure 7 As shown in the figure, after 24h of storage, the free lutein content in the red date juice decreased significantly, and the retention rates at 4 °C, 25 °C and 60 °C were 78.5%, 69.2% and 51.08%, respectively. The retention rates of lutein encapsulated with unmodified Eucommia ulmoides seed meal protein at 4 °C, 25 °C and 60 °C were 79.8%, 74.6% and 62.1%, respectively. When plasma-modified Eucommia ulmoides seed meal protein was used, the retention effect of lutein became better and better with the increase of protein addition. When the concentration of modified protein was 1%, the retention effect reached the highest, and the retention rates at 4 °C, 25 °C and 60 °C were 88.7%, 84.85% and 77.4%, respectively. In addition, the lower the temperature, the higher the retention rate of lutein. It shows that the emulsion prepared by plasma-modified Eucommia ulmoides seed meal protein can effectively inhibit the degradation of lutein at high temperature, which can effectively maintain the stability of lutein during food processing.

[0063] Figure 8The effect of storage time on the stability of lutein in red date juice was demonstrated, and the retention rate of lutein gradually decreased with the increase of storage time. After 10 days of testing, the retention rate of free lutein was only 31.4%. In contrast, the retention rate of lutein in the unmodified Eucommia seed meal protein-lutein multilayer emulsion was 42.6%, and the plasma-modified Eucommia seed meal protein-lutein multilayer emulsion showed the highest lutein retention rate. With the increase of protein concentration, the retention first increased and then decreased. The lutein retention rate was the highest at 1% addition, which was 80.1% and 63.1% at 5d and 10d, respectively. This shows that the plasma-modified Eucommia seed meal protein-loaded lutein multilayer emulsion has a significant effect on enhancing the stability of lutein under conventional storage conditions and can maintain its stability in red date juice. Considering the effect of protein addition on encapsulation efficiency and stability, it is determined that the addition amount of modified Eucommia seed meal protein is more appropriate to be 1%.

[0064] Example 6: Effect of different chitosan addition amounts on the stability of lutein-rich red date juice

[0065] 1. Preparation of Eucommia ulmoides seed meal protein-lutein colostrum The steps are the same as step 1 of Example 5, except that the added amount of protein is 0.5%, the added amount of oil phase is 5%, and the added amounts of chitosan are 0.05%, 0.1%, 0.15%, 0.2%, and 0.3%, respectively.

[0066] 2. Preparation of Eucommia ulmoides seed meal protein-lutein multilayer emulsion The steps are the same as step 2 in Example 5.

[0067] 3. Preparation of red date juice The steps are the same as step 3 in Example 5.

[0068] 4. Preparation of lutein-rich red date juice The steps are the same as step 4 in Example 5.

[0069] 5. Determination of thermal stability of lutein-rich red date juice The steps are the same as step 5 of Example 5.

[0070] 6. Storage stability test of lutein-rich red date juice The steps are the same as step 6 of Example 5.

[0071] 7. Test results like Fig. 9As shown in the figure, with the increase of chitosan concentration, the retention rate of lutein in red date juice first increased and then decreased. When the chitosan addition was 0.05%, the retention rates of lutein in red date juice after storage for 24 hours were 81.3%, 74.2% and 63.1% at 4 °C, 25 °C and 60 °C, respectively. When the chitosan addition was increased to 0.2%, the retention rate of lutein reached the maximum, and the retention rates at 4 °C, 25 °C and 60 °C were 86.6%, 81.2% and 73.8%, respectively. Fig.10 As shown in the results, the retention rate of lutein in red date juice decreased with the extension of storage time. The retention rate of lutein in red date juice also increased with the increase of chitosan concentration. This may be because the positively charged chitosan can form a polyelectrolyte complex with the negatively charged primary emulsion through electrostatic adsorption, forming a completely dense second layer on the interface of the primary emulsion, thereby preparing a multilayer emulsion. When the chitosan concentration is low, the amount of positively charged chitosan is insufficient to form a complete covering film on the surface of the primary emulsion, which causes aggregation and bridging flocculation between the emulsion droplets, resulting in poor droplet stability. It can be seen from the results that when the chitosan concentration reaches 0.2%, the stability of lutein in red date juice reaches the maximum. When the chitosan concentration continues to increase, the stability of lutein no longer increases, indicating that a stable emulsion can be formed at a chitosan concentration of 0.2%, thereby maintaining the stability of lutein in red date juice. Therefore, the optimal amount of chitosan is determined to be 0.2%.

Claims

1. A method for modifying eucommia seed meal protein, characterized in that: The method comprises the following steps: Taking eucommia seed meal protein, preparing a protein solution, then treating the protein solution with dielectric barrier discharge low-temperature plasma, and obtaining plasma-modified eucommia seed meal protein powder by freeze drying; The low-temperature plasma treatment voltage is 10kv-20kv, the treatment time is 5s-120s, and the discharge distance is 0.5-3cm.

2. A modified eucommia seed meal protein, characterized in that: Prepared by the method described in claim 1.

3. A method for preparing a lutein multilayer emulsion, characterized in that: The preparation method comprises: dispersing the modified eucommia seed meal protein according to claim 2 in a liquid to obtain an eucommia seed meal protein solution, mixing lutein with eucommia seed oil as an oil phase, adding the eucommia seed meal protein solution to the oil phase for emulsification, adding a chitosan solution, and stirring to prepare a lutein multilayer emulsion.

4. The method according to claim 3, characterized in that The emulsification method includes ultrasonic emulsification, high-speed stirring emulsification or high-pressure homogenization emulsification.

5. The method according to claim 3, characterized in that: The weight ratio of the lutein to the eucommia seed oil is 1:5-100.

6. The method according to claim 3, characterized in that The eucommia seed meal protein solution is added to the oil phase for ultrasonic treatment to prepare a primary emulsion, and the chitosan solution is slowly added dropwise to the primary emulsion.

7. The method according to claim 3, characterized in that After adding chitosan solution and mixing thoroughly, adjust the pH of the mixture to 3-7; the concentration of modified eucommia seed meal protein is 0.25%-2%, the amount of lutein-containing oil phase added is 1%-10%, the amount of chitosan added is 0.05%-0.3%, the ultrasonic power is 200W-1200W, and the ultrasonic time is 5min-20min.

8. A lutein multilayer emulsion, characterized in that: The method is described in claim 3.

9. A method for preparing red date juice, comprising adding the lutein multilayer emulsion according to claim 8 into red date juice.

10. The method according to claim 9, characterized in that The lutein multilayer emulsion is added to red date juice and homogenized by a high-pressure homogenizer at a pressure of 15-30 MPa for 1-3 times.

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