Stropharia rugoso-annulata polysaccharide and protein Maillard reaction product and preparation method thereof

Abstract

The invention discloses a Maillard reaction product of stropharia rugoso-annulata polysaccharide and protein and a preparation method of the Maillard reaction product. The preparation method of the Stropharia rugoso-annulata polysaccharide and protein Maillard reaction product comprises the following steps: S1, placing Stropharia rugoso-annulata powder in ethanol for reflux, and collecting Stropharia rugoso-annulata residues; s2, performing water extraction on the stropharia rugoso-annulata residues to obtain a crude polysaccharide extracting solution; s3, adding ammonium sulfate and tert-butyl alcohol into the crude polysaccharide extracting solution, preserving heat at a specific temperature, centrifuging, performing three-phase extraction, and separating each phase to collect a middle layer and a lower layer; s4, freeze-drying the middle layer to obtain stropharia rugoso-annulata protein powder; s5, inorganic salt in the lower layer is removed, and stropharia rugoso-annulata polysaccharide is obtained; s6, performing Maillard reaction on the stropharia rugoso-annulata protein powder and the stropharia rugoso-annulata crude polysaccharide to obtain a Maillard reaction product. The polysaccharide and the protein are simultaneously obtained through a three-phase method to be subjected to the Maillard reaction, and the emulsibility, the stability, the foamability and the oil absorption performance of the Maillard reaction product are all improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a Maillard reaction product of Stropharia rugosa polysaccharide and protein and a preparation method thereof. Background Art

[0002] Stropharia rugosoannulata, a basidiomycete in the family Strophariaceae, is also known as wine-red Stropharia, wrinkled Stropharia, or wrinkled Stropharia. It is one of the top ten mushroom varieties in the global mushroom market. Stropharia has a smooth, tender texture, a high yield, and is rich in nutrients. Existing research shows that Stropharia contains a variety of proteins, vitamins, and polysaccharides, which are beneficial to the human body. It has considerable edible and medicinal value and potential for development. Edible fungi are numerous and diverse in their functional properties. Therefore, the development of Stropharia polysaccharides has a positive impact on the development of the edible fungi industry. Currently, post-harvest processing of Stropharia mainly involves drying, pickling, canning, and manufacturing into primary products such as health foods, condiments, and snacks. These products have low added value, leaving significant room for industrial upgrading. To overcome the limitations of traditional processing models, it is urgent to explore deep processing methods for Stropharia and develop high-value-added products. It can not only fully tap the nutritional and medicinal value of Pleurotus ostreatus, improve the application value of Pleurotus ostreatus, enhance its market competitiveness, broaden the commercial use and value of Pleurotus ostreatus, but also promote the high-quality development of the Pleurotus ostreatus industry towards scale and high value.

[0003] The Maillard reaction refers to the reaction between the amino groups of amino acids and the carbonyl groups of reducing sugars. Because the Maillard reaction is a safe and environmentally friendly process, its use in modifying proteins and polysaccharides has attracted widespread attention from researchers both domestically and internationally. Plant protein hydrolysates primarily come from legumes and cereals, while animal protein hydrolysates primarily come from milk, meat, eggs, and aquatic products. Carbonyl-containing carbohydrates, such as reducing sugars like glucose, fructose, galactose, lactose, and maltose, as well as disaccharides, oligosaccharides, polysaccharides, and polysaccharides, can undergo the Maillard reaction with dietary proteins and protein hydrolysates / peptides. These carbohydrates are primarily derived from cereals, tubers, root vegetables, fungi, and dairy products, including rice, wheat, potatoes, fungus, fruits, vegetables, and milk.

[0004] The functional properties of Maillard reaction products of food-derived protein hydrolysates / peptides with carbohydrates primarily include solubility, emulsification activity and stability, foaming properties, gelation, water-binding capacity, and freeze-thaw stability. Current research on Maillard-reaction-based macromolecular modification primarily focuses on changes in the structural and functional properties of modified proteins. For example, whey protein-dextran conjugates exhibit improved emulsification and solubility under acidic pH conditions, while casein-dextrose conjugates exhibit excellent antioxidant activity. However, less research has been conducted on the functional properties of Maillard reaction products of other food-derived polysaccharides and proteins. Therefore, further exploration is needed to determine how to extract polysaccharides and proteins from Stropharia rugosa and produce Maillard products with superior functional properties. This research will not only promote the in-depth development and comprehensive utilization of Stropharia rugosa resources but also provide important insights for their application in systems such as food emulsions. Summary of the Invention

[0005] The present invention aims to provide a Maillard reaction product of Stropharia rugosoannulata polysaccharide and protein and a preparation method thereof. Compared with Stropharia rugosoannulata polysaccharide (SRP) and protein (SRD), the Maillard reaction product has improved emulsification and stability, foaming properties, and oil absorption properties. Moreover, the dry product is higher than the wet product, and the water holding capacity of the Maillard product is improved.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a Maillard reaction product of Stropharia rugosa polysaccharide and protein, comprising the following steps:

[0008] S1. placing the Stropharia rugosa powder in ethanol for reflux, and collecting the Stropharia rugosa residue;

[0009] S2. extracting the Stropharia officinalis residue with water to obtain a crude polysaccharide extract;

[0010] S3, adding ammonium sulfate and tert-butanol to the crude polysaccharide extract, keeping it warm at a specific temperature, and then centrifuging it to perform three-phase extraction, separating the phases to collect the middle layer and the lower layer;

[0011] S4, freeze-drying the middle layer to obtain Stropharia rugosa protein powder;

[0012] S5, removing the inorganic salts in the lower layer to obtain Stropharia rugosa polysaccharide;

[0013] S6. Performing a Maillard reaction on the Stropharia albumen powder and the Stropharia albumen crude polysaccharide to obtain a Maillard reaction product.

[0014] The method for preparing the Maillard reaction product of Stropharia capillaris polysaccharide and protein further comprises the following steps: in step S1, the particle size of the Stropharia capillaris powder is ≤80 mesh; as an example, the Stropharia capillaris powder is a powder obtained by drying Stropharia capillaris fruiting bodies at 45° C. under forced air for 24 hours and then grinding them;

[0015] In step S1, the material-liquid ratio of the Stropharia officinalis powder to ethanol is 1:20 g / mL, and the ethanol is added in the form of an aqueous solution with a mass concentration of 95% ethanol;

[0016] In the reflux step of step S1, the reflux time is 3 to 5 hours (eg, 5 hours), and is repeated 3 times.

[0017] The method for preparing the Maillard reaction product of Stropharia officinalis polysaccharide and protein further comprises: in the water extraction step of step S2, the solid-liquid ratio of the Stropharia officinalis residue to water is 1:30 g / mL, the water extraction temperature is 40-100°C, each extraction time is 1-2 hours, and after 2-3 extractions, the extracts are combined and concentrated to one-tenth of the extract volume. As an example, extraction is performed using 100°C boiling water for 2 hours, 2 extractions are performed, and the combined filtrates are concentrated under reduced pressure to a 30 mL solution for later use.

[0018] The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein further comprises the following steps: in step S3, 1.5 to 3.5 g (e.g., 3.5 g) of ammonium sulfate is added to every 10 mL of the crude polysaccharide extract;

[0019] In step S3, the volume ratio of the crude polysaccharide extract to the tert-butanol is 1:(0.6-2.5), such as 1:2.5;

[0020] In step S3, the specific temperature is 30-40° C. (e.g., 35° C.), and the holding time is 1 hour;

[0021] In step S3, the centrifugal speed is 4000 rpm / min, and the centrifugal time is 10 to 15 minutes (such as 10 minutes).

[0022] The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein further comprises the following steps: in step S5, the inorganic salts are removed by placing the lower layer in a dialysis bag with a molecular weight cut-off of 3500 Da.

[0023] In one embodiment of the present invention, in step S6, the Maillard reaction is prepared by a dry method.

[0024] Further preferably, in the dry preparation, the reaction temperature is 50-70° C. and the reaction humidity is 79%; and / or,

[0025] In the dry preparation, the reaction solvent is PBS buffer, pH=7.0; and / or,

[0026] In the dry preparation, the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:(1-3); and / or,

[0027] In the dry preparation, the reaction time is 50 to 70 minutes, such as 60 minutes; and / or,

[0028] After the dry preparation is completed, the method further comprises the following steps: placing the reactants in a dialysis bag with a molecular weight cut-off of 3500 Da to remove inorganic salts, and then freeze-drying.

[0029] More preferably, in the dry preparation, the reaction temperature is 55° C., the reaction time is 60 min, and the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:2.

[0030] In one embodiment of the present invention, the Maillard reaction is prepared by a wet method; preferably, in the wet preparation, the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:1, the reaction temperature is 90°C, and the reaction time is 2 hours; after the wet preparation is completed, the method further comprises the following steps: placing the reactants in a dialysis bag with a molecular weight cutoff of 3500Da to remove inorganic salts, and then freeze-drying.

[0031] In a second aspect, the present invention provides a Maillard reaction product of Stropharia rugosa polysaccharide and protein prepared by any of the methods described above.

[0032] The present invention has the following beneficial effects:

[0033] (1) The present invention uses Pleurotus ostreatus as the research object, obtains polysaccharides and proteins simultaneously through the three-phase method, and then prepares the Maillard products of SRP and SRD through the dry heat method and the wet heat method, and studies the effects of different preparation methods on the functional properties of the reactants. The results show that the grafting degree and browning degree of the dry heat method product are greater than those of the wet heat method product, and are significantly higher than those of SRP and SRD; the emulsification activity and stability, foaming property, and oil absorption of the products prepared by the two Maillard reaction methods are all improved compared with the original sample, and the dry method product is higher than the wet method product, and the water holding capacity of the Maillard products is improved; the ability of SRP and the two Maillard products to eliminate DPPH free radicals, ABTS free radicals, hydroxyl free radicals and reducing power increases with increasing concentration, and both are better than the scavenging and reducing abilities of SRD.

[0034] (2) The present invention uses response surface methodology to optimize the Maillard reaction conditions for optimal overall performance of the reaction product. The optimal process parameters optimized by response surface methodology are a reaction time of 60.22 min, a reaction temperature of 58.70°C, and a mass ratio of 1:2.15 for protein and polysaccharide from Stropharia officinalis. Under these conditions, the grafting degree is 22.15%. The actual experimental conditions were adjusted to a reaction temperature of 55°C, a reaction time of 60 min, and a mass ratio of 1:2. Three repeated tests were performed, and the average grafting degree was 22.35%. The actual value is close to the measured value, indicating that the model optimization results are good.

[0035] In summary, the present invention studies the functional properties of the two Maillard reaction products of Stropharia rugosa, which can not only promote the in-depth development and comprehensive utilization of Stropharia rugosa resources, but also provide an important reference for its application in food emulsions and other systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a comparison chart of the grafting degrees of the dry-heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet-heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0037] Figure 2 This is a comparison chart of the browning degree of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD and Stropharia albumen crude polysaccharide SRP at 420 nm.

[0038] Figure 3 This is a comparison chart of the emulsification activity and emulsification stability of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0039] Figure 4 This is a comparison chart of the foaming properties of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0040] Figure 5 This is a comparison chart of the water holding capacity of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0041] Figure 6This is a comparison chart of the oil absorption of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0042] Figure 7 This is a comparison chart of the DPPH radical scavenging abilities of the dry-heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet-heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0043] Figure 8 This is a comparison chart of the ABTS+ free radical elimination abilities of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0044] Figure 9 This is a comparison chart of the hydroxyl radical scavenging abilities of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0045] Figure 10 This is a comparison chart of reducing power determination of the dry heat Maillard reaction product SRP-SRDD prepared in Example 1 of the present invention, the wet heat Maillard reaction product SRP-SRDW prepared in Example 2, Stropharia albumen protein powder SRD, and Stropharia albumen crude polysaccharide SRP.

[0046] Figure 11 This is the effect of different reaction temperatures on the grafting degree of Stropharia rugosa protein and polysaccharide in the dry method preparation of Maillard reaction products in Example 3 of the present invention.

[0047] Figure 12 This is the effect of different reaction times on the grafting degree in the dry method preparation of the Maillard reaction product in Example 3 of the present invention.

[0048] Figure 13 This is the effect of different ratios of Stropharia rugosa protein and polysaccharide on the grafting degree in the dry method preparation of the Maillard reaction product in Example 3 of the present invention.

[0049] Figure 14 Response surface diagrams of the two-factor interaction on the grafting reaction in the dry preparation of Maillard reaction products in Example 3 of the present invention: reaction temperature-SPR:SRD (A), reaction temperature-reaction time (B), and SPR:SRD-reaction time (C). DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0051] Unless otherwise specified, the methods used in the following examples are all conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0052] The fresh Stropharia rugosa fruiting bodies in the following examples were purchased from Yucheng County, Shangqiu City, Henan Province.

[0053] The PBS buffer (0.1 mol / L, pH 7.0) in the following examples is prepared from two phosphates, Na2HPO4 and NaH2PO4, with a total phosphate concentration of 0.1 M.

[0054] The determination methods in the following examples are as follows:

[0055] (1) Determination of grafting degree

[0056] Preparation of o-phthalaldehyde (OPA) reagent: Dissolve 80.0 mg of OPA in 2.0 mL of methanol, then add 10 mL of 10% (w / w) sodium dodecyl sulfate (SDS), 50 mL of 0.1 mol / L borax solution, and 200 μL of β-mercaptoethanol in that order. Finally, dilute to 100 mL with distilled water and use immediately.

[0057] The o-phthalaldehyde method was used to determine free amino groups and calculate the degree of grafting. 2.0 mL of OPA reagent was placed in a test tube, 100 μL of sample was added, and the mixture was thoroughly mixed. After reacting in a 35°C water bath for 2 minutes, the absorbance was measured at 340 nm. Separately, 4.0 mL of OPA reagent was placed in a test tube, 100 μL of water was added, and the degree of grafting was calculated based on the change in absorbance.

[0058]

[0059] In formula (1), A0 and A1 represent the absorbance values ​​of the sample before and after the grafting reaction, respectively.

[0060] (2) Determination of browning degree

[0061] According to the literature "Xiong GY, Chen X, Zhang XX, et al. Process optimization and therelationship between the reaction degree and the antioxidant activity of Maillard reaction products of chicken liver protein hydrolysates [J]. Poultry Science, 2020, 99: 3733-3741.", an appropriate amount of sample solution was added to 1.0 mL of a diluent containing 10% (W / W) SDS and 50 mmol / L borax. After thorough stirring, the mixture was centrifuged at 4000 rpm for 5 minutes. The absorbance of the supernatant was measured at 420 nm, and the diluent was used as a reference.

[0062] (3) Determination of emulsification activity and emulsion stability

[0063] Weigh 100 mg of the reacted sample and dissolve it in 20 mL of pH 7.0 PBS buffer. Stir thoroughly. After reacting for 30 minutes, add 4.0 mL of peanut oil and homogenize at 8000 rpm for 1 minute. Immediately aspirate 200 μL of the emulsion from the bottom and quickly disperse it in 20 mL of 0.1% SDS solution. Measure the absorbance (A) of this dilution at a wavelength of 500 nm. The absorbance (A) is proportional to the interfacial area of ​​the emulsion droplets. Emulsification activity is expressed as ESI. After high-speed homogenization, let the sample stand for 15 minutes. Aspirate 100 μL of the emulsion from the bottom and quickly disperse it in 10 mL of 0.1% SDS solution. Measure the absorbance of the solution at a wavelength of 500 nm. ESI is used to express emulsion stability and is calculated according to the following formula:

[0064]

[0065] In formula (2) and formula (3), EAI is the emulsification area per gram of protein, m 2 / g; N is the dilution factor; φ is the fraction of the oil phase in the system, which is 0.2 in this experiment; C is the protein concentration, g / mL; L is the light path of the colorimetric cell, which is 1 cm in this experiment; A0 is the absorbance at time 0; At is the absorbance at time t.

[0066] (3) Foaming property measurement

[0067] Foamability was determined using the whipping method: the sample was dissolved in PBS buffer (pH 7.0) to form a 3% emulsion. 200 mL of the 3% emulsion was whipped (8000 rpm, 3 minutes) and the foam volume was measured. The mixture was allowed to stand for 30 minutes and the foam volume was measured again. The foamability index (FAI) was calculated using the following formula:

[0068]

[0069] In formula (4), V0 is the volume of the foam.

[0070] (4) Water holding capacity determination

[0071] Reference "Talja RA, Helén H, Roos YH, et al. Effect of type and content of binary polyol mixtures on physical and mechanical properties of starch-based edible films [J]. Carbohydrate Polymers, 2008, 71(2): 269-276." Take an appropriate amount of sample and place it in a 10mL dry centrifuge tube. Spread the sample evenly on the bottom. Slowly add distilled water to cover the sample. Mix thoroughly and let it stand for 30 minutes. Centrifuge at 4000 rpm for 10 minutes. Discard the supernatant, accurately weigh the precipitate and the total mass of the centrifuge tube. Water holding capacity is expressed as grams of water adsorbed per gram of sample. The calculation formula is as follows:

[0072]

[0073] In formula (5), m0 is the mass of the weighed sample, g; m1 is the total mass of the sample and the centrifuge tube, g; and m2 is the total mass of the sediment and the centrifuge tube after centrifugation, g.

[0074] (5) Oil absorption determination

[0075] Reference "Teng C, Xing B, Fan X, et al. Effects of Maillard reaction on the properties and anti-inflammatory, anti-proliferative activity in vitro of quinoa protein isolates[J]. Industrial Crops and Products, 2021, 174." Method: Weigh an appropriate amount of sample and place it in a 10mL dry centrifuge tube, spread the sample evenly on the bottom, slowly add 8mL of oil to cover the sample, mix the sample thoroughly, let it stand for 30 minutes, and centrifuge it at 4000rpm for 10 minutes. Discard the supernatant, accurately weigh the total mass of the precipitate and the centrifuge tube, and the oil absorption is expressed as the number of grams of oil absorbed per gram of sample. The calculation formula is as follows:

[0076]

[0077] In formula (6), m0 is the mass of the weighed sample, g; m1 is the total mass of the sample and the centrifuge tube, g; and m2 is the total mass of the sediment after centrifugation and the centrifuge tube, g.

[0078] (6) Determination of DPPH free radical scavenging ability

[0079] Reference "Zhang PZ, Wang L, Qian YY, et al. Influences of Extraction Methods on Physicochemical and Functional Characteristics of Three New Bulbil Starches from Dioscorea opposita Thunb.cv. Tiegun[J]. Molecules, 2019, 24(12)." The scavenging rate of the sample on DPPH free radicals was calculated according to the following formula:

[0080]

[0081] A0 is the absorbance of distilled water + DPPH solution; Ax is the absorbance of polysaccharide sample solution + DPPH solution; Ax0 is the absorbance of polysaccharide sample solution + anhydrous ethanol (same volume as DPPH solution).

[0082] (7) Determination of ABTS+ free radical scavenging ability

[0083] Reference "Wang L, Zhang PZ, Shen JW, et al. Physicochemical properties and bioactivities of original and Se-enriched polysaccharides with different molecular weights extracted from Pleurotus ostreatus [J]. International Journal of Biological Macromolecules, 2019, 141: 150-160.", the scavenging rate of the sample for ABTS free radicals was calculated according to the following formula:

[0084]

[0085] A0 is the absorbance value of the distilled water + ABTS mixture; Ax is the absorbance value of the polysaccharide sample solution + ABTS mixture; Ax0 is the absorbance value of the polysaccharide sample solution + PBS buffer.

[0086] (8) Determination of hydroxyl radical scavenging ability

[0087] Reference "Ruan Y, Niu CF, Zhang PZ, et al. Acid-Catalyzed Water Extraction of Two Polysaccharides from Artemisia argyi and Their Physicochemical Properties and Antioxidant Activities[J]. Gels, 2022, 8(1).", the scavenging rate of the sample for hydroxyl radicals was calculated according to the following formula:

[0088]

[0089] A0 is the absorbance value of distilled water + FeSO4 solution + salicylic acid·ethanol solution + H2O2 solution; Ax is the absorbance value of polysaccharide sample solution + FeSO4 solution + salicylic acid·ethanol solution + H2O2 solution; Ax0: absorbance value of polysaccharide sample solution + FeSO4 solution + salicylic acid·ethanol solution + distilled water.

[0090] (9) Reducing power determination

[0091] Reference: "Zhao ZW, Wang L, Ruan Y, et al. Physicochemical properties and biological activities of polysaccharides from the peel of Dioscorea opposita Thunb. extracted by four different methods [J]. Food Science and Human Wellness, 2023, 12(1): 130-139." The absorbance of each reaction was measured at 700 nm using a microplate reader. This absorbance value reflects the reducing ability of the sample; a larger absorbance value indicates a stronger reducing ability of the sample.

[0092] Example 1: Dry method for preparing Maillard reaction products

[0093] 1. Sample pretreatment

[0094] Dry the fruiting bodies of Stropharia rugosoium in a 45°C electric forced-air drying oven for 24 hours. Then grind them into a powder and pass them through an 80-mesh sieve. Take 10g of the powder and reflux it with 95% ethanol (1:20g / mL) for 5 hours, repeating three times. After the ethanol is completely evaporated from the residue, extract it with 300mL of distilled water at a 1:30 (w / v) ratio using boiling water for 2 hours. Repeat this step twice. Combine the filtrates and concentrate them under reduced pressure on a rotary evaporator to a 30mL solution for later use.

[0095] 2. Extraction of protein and crude polysaccharides

[0096] To 10 mL of the crude polysaccharide extract, add 35% (NH₄)₂SO₄ (w / v) (i.e., 3.5 g) and gently vortex. Then, add tert-butyl alcohol in a ratio of 1:2.5 (v / v). Maintain the mixture at 35°C for one hour. Next, centrifuge the mixture at 4000 rpm / min for 10 minutes to accelerate the three-phase extraction. After centrifugation, carefully separate the phases. The middle layer contains the protein (SRD), which is freeze-dried for later use. The lower layer, primarily composed of ammonium sulfate and polysaccharides, is dialyzed (3500 Da) to remove inorganic salts and then freeze-dried to yield Stropharia rugosa polysaccharide (SRP).

[0097] 3. Preparation of Maillard reaction products

[0098] The Stropharia protein powder (SRD) and Stropharia crude polysaccharide (SRP) prepared in step 2 above were dissolved in equal volumes of PBS buffer (i.e., 0.01 g of each was dissolved in 5 mL of PBS buffer) at a mass ratio of 1:1. After thorough stirring and dissolution, the mixture was placed in a desiccator (humidity 79%) containing a saturated potassium bromide solution (%) at the bottom. The oven temperature was adjusted to 60°C, and the mixture was placed in the desiccator. After reacting for 1 hour, the mixture was removed and rapidly cooled to room temperature to terminate the reaction. The reactants were dialysis-treated (3500 Da) to remove inorganic salts and then freeze-dried. The product was designated SRP-SRDD.

[0099] Example 2: Preparation of Maillard reaction products by wet method

[0100] 1. Sample pretreatment

[0101] Same as Example 1.

[0102] 2. Extraction of Protein and Crude Polysaccharides

[0103] Same as Example 1.

[0104] 3. Preparation of Maillard Reaction Products

[0105] The Stropharia protein powder SRD and Stropharia crude polysaccharide SRP prepared in the above step 2 were dissolved in the same volume of PBS buffer at a mass ratio of 1:1 (i.e., 0.01 g of each was dissolved in 5 mL of PBS buffer), stirred thoroughly and dissolved, and mixed evenly. After heating and stirring at 90°C for 2 h, the mixture was quickly cooled to room temperature to terminate the reaction. The resulting reactant was subjected to molecular dialysis (3500 Da) to remove inorganic salts and then freeze-dried. The product was recorded as SRP-SRDW.

[0106] The properties of the Maillard reaction products prepared in the above examples were measured.

[0107] 1. Grafting degree test

[0108] The binding of SRP and SRD depends largely on the Schiff base formed between the amino groups in the protein and the carbonyl groups in the reducing sugar. Therefore, the degree of Maillard reaction can be evaluated by measuring the content of free amino groups and calculating the degree of grafting (DG). The grafting degrees of SRP, SRD, SRP-SRDD and SRP-SRDW reactants are shown in Figure 2. Figure 1 As shown in the figure, the grafting degree of the dry heat method product is generally greater than that of the wet heat method. This is because the reaction temperature of the wet heat method, 90°C, is much higher than the reaction temperature of the dry heat method, 60°C. Although the increase in temperature will increase the reaction rate between reducing sugars and amino groups, high temperature will cause protein denaturation and aggregation, thereby limiting the glycosylation reaction.

[0109] 2. Functional property test

[0110] 1. Browning degree analysis

[0111] Melanoidin, one of the final products of the Maillard reaction, has a strong absorption capacity at a wavelength of 420 nm, so the degree of browning can be characterized by the absorbance at this wavelength. Figure 2 As shown, the grafting degree at 420 nm of the reaction products obtained by both the dry-heat method in Example 1 and the wet-heat method in Example 2 increased compared to the original samples. Furthermore, the browning degree of the dry-heat method was slightly higher than that of the wet-heat method, indicating that a Maillard reaction occurred in the system. During the dry-heat treatment, the SRP structure partially unfolded, and the protein's ε-amino groups combined with the carbonyl groups of the Stropharia officinalis polysaccharide to form intermediates. Simultaneously, some of these intermediates coagulated and polymerized to produce melanoidins, which increased the degree of browning.

[0112] 2. Emulsification activity and emulsification stability analysis

[0113] The emulsifying activity index (EAI) reflects the ability of proteins to quickly adsorb onto the surface of newly formed droplets and thus stabilize the water-oil interface. The emulsifying stability index (ESI) indicates the ability of an emulsion to maintain stability against various destabilizing factors over a long period of time. The emulsifying activity and emulsifying stability results of SRP and SRD after the Maillard reaction are shown in Figure 2. Figure 3 As shown, the emulsification properties of the products prepared using both Maillard reaction methods were improved compared to the original samples. The emulsification stability of the dry and wet products increased from 5.23 min (SRP) to 8.21 min (D) and 7.45 min (W), respectively, improving the emulsification stability of the SRP. Furthermore, the dry product exhibited higher emulsification activity than the wet product, indicating that the dry-heat combination possesses better emulsion stabilization capabilities.

[0114] 3. Foaming analysis

[0115] The changes in foaming properties before and after SRP and SRD Maillard reaction are as follows Figure 4 After dry and wet Maillard reactions, the foaming properties of SRP increased from 49.44% to 75.15% and 62.47%, respectively, representing increases of 1.52 and 1.26 times. Possible reasons for this improved foaming performance include: firstly, the solubility of the modified SRD is significantly enhanced, allowing the protein to diffuse toward the air-liquid interface, thereby enhancing its foaming ability; secondly, the introduction of hydrophilic hydroxyl groups into the modified SRD strengthens the intermolecular forces between the protein molecules, thereby forming a stable liquid film.

[0116] 4. Water holding capacity analysis

[0117] Depend on Figure 5It can be seen that among the four samples, the water holding capacity of SRD is similar to that of SRP. Among the Maillard products, the wet reaction product has the highest water holding capacity, and the water holding capacity of all samples increases after the Maillard reaction. This may be because the Maillard reaction destroys the highly regular and tightly packed protein peptide chains of SRD, making the peptide chains loose and irregular, thereby increasing water holding capacity. On the other hand, the introduction of SRP may also bring hydrophilic groups, and the increased content of hydrophilic groups in the solution may also increase water holding capacity.

[0118] 5. Oil absorption analysis

[0119] Oil absorption is another important functional property of protein, which mainly depends on the physical interception of oil by the protein molecular structure. The smaller the volume density of the protein, the greater the oil absorption. Oil absorption is also related to the hydrophobicity and structure of the protein. Figure 6 As shown in the figure, the oil absorption of Maillard dry and wet reaction products is enhanced compared with the samples before reaction, and the oil absorption of dry products is stronger than that of wet products.

[0120] In summary, the grafting degree of the dry-heat reaction product was generally greater than that of the wet-heat reaction product. The grafting degree of both dry-heat and wet-heat reaction products increased compared to the original sample, and the browning degree of the dry-heat reaction product was slightly higher than that of the wet-heat reaction product. The emulsification properties of the products prepared by both Maillard reaction methods were improved compared to the original sample, and the dry-heat reaction product had higher emulsification activity compared to the wet-heat reaction product. The foaming property of SRP increased from 49.44% to 75.15% and 62.47% after the dry and wet Maillard reactions, respectively, increasing by 1.52 and 1.26 times. Among the Maillard reaction products, the wet-heat reaction product had the highest water retention, and the water retention was improved after the Maillard reaction. The oil absorption of the Maillard dry and wet reaction products was enhanced compared to the original sample, with the dry-heat reaction product having stronger oil absorption than the wet-heat reaction product. The products prepared by the dry-heat reaction had higher emulsification stability than those prepared by the wet-heat reaction, and the emulsification properties of the products prepared by the dry and wet-heat reactions were improved compared to the original sample.

[0121] 6. Antioxidant activity analysis

[0122] 6.1 DPPH free radical scavenging ability

[0123] As shown in Figure 7, in the DPPH radical scavenging activity assay, as the concentration increased, the scavenging rates of SRP-SRDD reached a maximum of 99.2%, and those of SRP-SRDW reached a maximum of 97.1%. When the concentrations of SRP-SRDD and SSR-SRDD increased from 0 mg / mL to 0.5 mg / mL, the DPPH radical scavenging activity increased. SRP exhibited stronger DPPH radical scavenging activity than SRD, but weaker than SRP-SRDD and SRP-SRDW. This may be due to the fact that the functional groups within the conjugates generated by the Maillard reaction were fully exposed during heating, allowing them to react with DPPH radicals.

[0124] 6.2 Ability to scavenge ABTS free radicals

[0125] ABTS+ free radicals are reactive to most antioxidants, and discoloration reflects the ability of the antioxidant to inactivate the free radicals. Figure 8 As shown, in the ABTS free radical scavenging activity assay, the scavenging activity of SRP, SRP-SRDD, and SRP-SRDW increased from 0 mg / mL to 2 mg / mL, with SRP exhibiting greater ABTS free radical scavenging activity than SRP-SRDD and SRP-SRDW. SRD exhibited weaker ABTS free radical scavenging activity, with its ABTS free radical scavenging rate remaining largely unchanged with increasing concentration. This may be due to the ABTS free radical scavenging activity of Stropharia rugosa polysaccharides, which provide Maillard reaction products.

[0126] 6.3 Hydroxyl Radical Scavenging Ability

[0127] like Figure 9 As shown, SRD has a weak hydroxyl radical scavenging ability, with a scavenging rate of 30% at a concentration of 4 mg / mL. At a concentration of 4 mg / mL, SRP, SRP-SRDD, and SRP-SRDW achieved hydroxyl radical scavenging rates of 88%, 31%, and 30%, respectively. The hydroxyl radical scavenging abilities of SRP-SRDD and SRP-SRDW were enhanced compared to SRD, but weaker than those of SRP. This may be due to the high activity of Stropharia polysaccharides in scavenging hydroxyl radicals, with the reaction products of Stropharia polysaccharides and proteins providing electrons to eliminate hydroxyl radicals.

[0128] 6.4 Determination of reducing power

[0129] In the primary stage of the Maillard reaction, the heterocyclization and caramelization of sugars can form reduction products. Figure 10 As shown in the reducing power measurement, SRD has a weak reducing power. The absorbance of the SRD sample remained essentially unchanged as the concentration increased from 0.05 mg / mL to 4 mg / mL. However, the absorbances of SRP, SRP-SRDD, and SRP-SRDW increased with increasing concentration, reaching 0.363, 0.268, and 0.262, respectively. The reducing power of SRP-SRDD and SRP-SRDW was enhanced compared to that of SRD, but weaker than that of SRP. This may be due to the fact that the reduction products produced by the Maillard reaction caramelization consume reducing groups in the polysaccharide.

[0130] Example 3: Single factor experiment and response surface optimization experiment for dry preparation of Maillard reaction products

[0131] 1. Single-factor experiment

[0132] 1. Effect of temperature on Maillard reaction

[0133] The reaction time was fixed at 60 min, the ratio of raw materials was 1:1 between protein and polysaccharide, the reaction temperature conditions were changed (40, 50, 60, 70, 80℃), and the grafting degree of the product was measured to investigate the effect of extraction temperature on Maillard.

[0134] Effects of different reaction temperatures on the grafting degree of Stropharia rugosa protein and polysaccharide, such as Figure 11 As shown. Figure 11 It can be seen that the grafting degree of Stropharia rugosa polysaccharide and protein reached a maximum of 18.57% at 60℃, and the grafting degree showed a trend of first increasing and then decreasing with the increase of reaction temperature. In summary, the reaction temperatures of 50, 60, and 70℃ were selected for response surface optimization analysis.

[0135] 2. The influence of time on the Maillard reaction

[0136] The reaction temperature was fixed at 60℃, the ratio of raw materials was 1:1 of protein and polysaccharide, the reaction time was changed (30, 40, 50, 60, 70, 80 min), and the grafting degree of the product was measured to investigate the effect of extraction time on Maillard.

[0137] The effect of different reaction times on the grafting degree, such as Figure 12 As shown in the figure, the grafting degree between Stropharia rugosa polysaccharide and protein reached a maximum of 19.38% at 60 minutes. The grafting degree initially increased and then decreased with increasing reaction time. As reaction time increased, the Maillard reaction between the protein and the polysaccharide altered the protein's spatial structure and partially modified it, leading to a decrease in the grafting degree. Therefore, reaction times of 50, 60, and 70 minutes were selected for response surface optimization analysis.

[0138] 3. Effect of the ratio of protein and polysaccharide of Stropharia rugosa on grafting reaction

[0139] The reaction time was fixed at 60 min and the reaction temperature was kept constant at 60°C. The ratios of protein to polysaccharide were 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4 respectively. The grafting degree of the product was measured to investigate the effect of extraction temperature on Maillard.

[0140] Effects of different ratios of protein and polysaccharide on the grafting degree of protein and polysaccharide in Stropharia rugosodium. Figure 13 As shown in the figure, the grafting degree reaches the highest level of 20.57% when the ratio of polysaccharide to protein of Stropharia is 2. The grafting degree increases first and then decreases with the increase of the ratio of polysaccharide to protein of Stropharia. In summary, the ratios of protein to polysaccharide of Stropharia were selected as 1:1, 1:2, and 1:3 for response surface optimization analysis.

[0141] 2. Response Surface Optimization Experiment

[0142] Based on the results of single-factor experiments, the material-liquid ratio, reaction temperature and reaction time were used as reference factors. The Box-Behnken response surface experimental design was carried out using DesignExpert 13 software and the grafting degree of the Maillard reaction product as the response value. The results obtained from the experiment were analyzed to obtain the optimal predicted reaction parameters, and verification experiments were carried out.

[0143] Table 1. Box-Behnken design experimental factor levels

[0144]

[0145] The response surface experiment plan and results are shown in the following table.

[0146] Table 2. Response surface experiment plan and results

[0147]

[0148]

[0149] Analysis of Variance and Regression Equation: Based on the above research, the response surface experiment plan and results are shown in the table. Using reaction temperature (A), the ratio of Stropharia polysaccharide to protein (B), and reaction time (C) as independent variables, a multivariate regression analysis of the optimization results yielded a quadratic polynomial regression equation for the grafting degree (Y) and reaction conditions: Y = 22.06 - 0.64A + 0.60B + 0.26C - 0.03AB + 0.47AC - 0.68BC - 2.47A² - 2.01B² - 2.19C². The results of the response surface regression model analysis of variance are shown in the table. The model test value (P < 0.0001) indicates that the model is significant. The lack-of-fit term (P = 0.8405) is not significant, indicating that the regression model fits the actual experiment well and is both feasible and accurate. It can be seen from the table that R2=0.9787, R2Adj=0.9514, and the model fits the actual situation well. According to the experiment, A2, B2, and C2 all have extremely significant effects on the grafting degree (P<0.0001), and A, B, and the interaction term BC have significant effects (P<0.05).

[0150] Table 3. Analysis of variance of response surface regression equation

[0151] Sources of Difference sum of squares degrees of freedom mean square F-number P-value Model 79.61 9 8.85 35.8 <0.0001 A-Temperature 3.32 1 3.32 13.46 0.008 B-polysaccharide and protein ratio 2.91 1 2.91 11.77 0.011 C-Time 0.5233 1 0.5233 2.12 0.1889 AB 0.0048 1 0.0048 0.0195 0.8929 AC 0.8956 1 0.8956 3.63 0.0986 BC 1.87 1 1.87 7.59 0.0283 <![CDATA[A 2 ]]> 25.7 1 25.7 104.03 <0.0001 <![CDATA[B 2 ]]> 16.93 1 16.93 68.55 <0.0001 <![CDATA[C 2 ]]> 20.14 1 20.14 81.51 <0.0001 residual 1.73 7 0.247 Lack of Fit 0.2969 3 0.099 0.2763 0.8405 Pure error 1.43 4 0.3581 sum 81.34 16

[0152] Response surface analysis and optimization: the interaction between various factors affecting the grafting degree of protein and polysaccharide of Stropharia rugosa Figure 14The significance test of the regression equation coefficient showed that the reaction temperature, reaction time and the mass ratio of Stropharia rugosa polysaccharide to protein had a significant impact on the grafting degree. Figure 14 The interaction of various factors on the grafting reaction revealed that the influence of the four factors, according to F-value analysis, was as follows: reaction temperature (°C) > Stropharia capillaris polysaccharide to protein mass ratio (g / g) > reaction time (min). Response surface optimization experiments determined the optimal process parameters to be a reaction time of 60.22 min, a reaction temperature of 58.70°C, and a Stropharia capillaris protein to polysaccharide mass ratio of 1:2.15. Under these conditions, the grafting degree reached 22.15%. The actual experimental conditions were adjusted to a reaction temperature of 55°C, a reaction time of 60 min, and a mass ratio of 1:2. Three replicates were performed, and the average grafting degree was 22.35%. The actual values ​​were close to the measured values, indicating that the model optimization results were satisfactory.

[0153] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentration and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any variation, purposes or improvements of the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing a Maillard reaction product of Stropharia rugosa polysaccharide and protein, comprising the following steps: S1. placing the Stropharia rugosa powder in ethanol for reflux, and collecting the Stropharia rugosa residue; S2. extracting the Stropharia officinalis residue with water to obtain a crude polysaccharide extract; S3, adding ammonium sulfate and tert-butanol to the crude polysaccharide extract, keeping it warm at a specific temperature, and then centrifuging it to perform three-phase extraction, separating the phases to collect the middle layer and the lower layer; S4, freeze-drying the middle layer to obtain Stropharia rugosa protein powder; S5, removing the inorganic salts in the lower layer to obtain Stropharia rugosa polysaccharide; S6. Performing a Maillard reaction on the Stropharia albumen powder and the Stropharia albumen crude polysaccharide to obtain a Maillard reaction product.

2. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to claim 1, characterized in that: In step S1, the particle size of the Stropharia officinalis powder is ≤80 mesh; In step S1, the material-liquid ratio of the Stropharia officinalis powder to ethanol is 1:20 g / mL, and the ethanol is added in the form of an aqueous solution with a mass concentration of 95% ethanol; In the reflux step of step S1, the reflux time is 3 to 5 hours each time, and it is repeated 3 times.

3. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to any one of claims 1 to 2, characterized in that: In the water extraction step of step S2, the solid-liquid ratio of the Stropharia rugosa residue to water is 1:30 g / mL, the water extraction temperature is 40-100° C., each extraction time is 1-2 hours, and after 2-3 extractions, the extracts are combined and concentrated to one tenth of the extract.

4. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to any one of claims 1 to 3, characterized in that: In step S3, 1.5 to 3.5 g of ammonium sulfate is added to every 10 mL of the crude polysaccharide extract; In step S3, the volume ratio of the crude polysaccharide extract to the tert-butanol is 1:(0.6-2.5); In step S3, the specific temperature is 30-40°C and the holding time is 1 hour; In step S3, the centrifugal speed is 4000 rpm / min, and the centrifugal time is 10 to 15 minutes.

5. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to any one of claims 1 to 4, characterized in that: In step S5, the inorganic salts are removed by placing the lower layer in a dialysis bag with a molecular weight cut-off of 3500 Da.

6. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to any one of claims 1 to 5, characterized in that: In step S6, the Maillard reaction is prepared by a dry method.

7. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to claim 6, characterized in that: In the dry preparation, the reaction temperature is 50-70°C and the reaction humidity is 79%; In the dry preparation, the reaction solvent is PBS buffer, pH = 7.0; In the dry preparation, the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:(1-3); In the dry preparation, the reaction time is 50 to 70 minutes; After the dry preparation is completed, the method further comprises the following steps: placing the reactants in a dialysis bag with a molecular weight cut-off of 3500 Da to remove inorganic salts, and then freeze-drying.

8. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to claim 7, characterized in that: In the dry preparation, the reaction temperature is 55° C., the reaction time is 60 min, and the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:

2.

9. The method for preparing the Maillard reaction product of Stropharia rugosa polysaccharide and protein according to any one of claims 1 to 5, characterized in that: The Maillard reaction is prepared by a wet method; preferably, in the wet preparation, the mass ratio of the Stropharia albumen powder to the Stropharia albumen crude polysaccharide is 1:1, the reaction temperature is 90°C, and the reaction time is 2 hours; after the wet preparation is completed, the method further includes the following steps: placing the reactants in a dialysis bag with a molecular weight cutoff of 3500Da to remove inorganic salts, and then freeze-drying.

10. The Maillard reaction product of Stropharia rugosa polysaccharide and protein prepared by the method of any one of claims 1 to 9.

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