Extraction method and application of mulberry leaf protein

Abstract

The invention relates to the technical field of functional protein extraction, in particular to a mulberry leaf protein extraction method and application thereof. The extraction method comprises the following steps: dissolving mulberry leaf powder in water, and adjusting the pH value to be greater than or equal to 8 to obtain a mulberry leaf powder aqueous solution; carrying out digestion after ultrasonic crushing, and centrifuging to take supernatant as a crude extract; and adjusting the pH value of the crude extract to be less than or equal to 6, and centrifuging to take the precipitate. The method for extracting the mulberry leaf protein is provided by combining an alkali dissolution method and an ultrasonication method, the extraction rate of the mulberry leaf protein and the oxidation resistance of the extracted mulberry leaf protein can be effectively improved by combining the alkali dissolution method and the ultrasonication method, and the method has important application value for functional development of the mulberry leaf protein.

Description

Technical Field

[0001] The present invention relates to the technical field of functional protein extraction, and particularly relates to a method for extracting mulberry leaf protein and its application. Background Art

[0002] Mulberry leaves are the main product of Morus ( Morus alba L ) and are mainly used as feed for silkworms and raw materials for traditional Chinese medicine. However, due to limited utilization channels, there is a huge waste of mulberry leaf resources. Therefore, in recent years, the development and utilization of mulberry leaf resources have become a research hotspot. Mulberry leaves contain rich active substances, such as flavonoids, alkaloids, polyphenols and polysaccharides. Research shows that these small-molecule active substances have functions such as lowering blood sugar, enhancing immunity and intestinal barrier function. However, the crude protein with a protein content of up to 24.7% in dry mulberry leaves is often ignored, and current research mainly focuses on its nutritional value, and the research on its activity is relatively limited.

[0003] Some studies have shown that plant proteins also exhibit good biological activities after extraction and purification. However, currently, conventional plant protein extraction methods are all difficult to extract mulberry leaf protein from mulberry leaves well. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for extracting mulberry leaf protein and its application.

[0005] The alkali solution-acid precipitation method is a method for extracting proteins by utilizing the principle that proteins have a higher solubility in alkaline solutions and undergo isoelectric point precipitation in an acidic environment. The ultrasonic disruption method utilizes the dispersion effect of ultrasonic waves in liquids to generate cavitation, thereby achieving the disruption of solid particles or cell tissues in the liquid. However, when using the ultrasonic disruption method or the alkali solution-acid precipitation method alone to extract mulberry leaf protein, the protein extraction rate is low and the antioxidant activity of the extracted mulberry leaf protein is low. The present invention ingeniously combines the ultrasonic disruption method and the alkali solution-acid precipitation method. After alkalization, ultrasonic treatment and extraction are carried out, and then acidification precipitation is carried out, which not only significantly improves the extraction efficiency of mulberry leaf protein and the antioxidant activity of the protein, but also has the advantages of low extraction cost and short process time compared with the traditional enzymatic method for extracting plant proteins. It provides new ways and technologies for the development and utilization of mulberry leaf resources.

[0006] In the first aspect, the present invention provides a method for extracting mulberry leaf protein, comprising: Dissolving mulberry leaf powder in water and adjusting the pH to ≥8 to obtain an aqueous solution of mulberry leaf powder; Performing ultrasonic disruption and then extraction and centrifugation to obtain the supernatant as the crude extract; Adjusting the pH of the crude extract to ≤6 and centrifuging to obtain the precipitate.

[0007] Further, in the aqueous solution of mulberry leaf powder, the ratio of the mulberry leaf component to water is 1:(10 - 30), and the pH = (10 - 12).

[0008] Preferably, in the aqueous solution of mulberry leaf powder, the ratio of the mulberry leaf component to water is 1:(21 - 30).

[0009] Preferably, the pH of the crude extract is adjusted to ≤5.

[0010] Further, the conditions for ultrasonic disruption include: ultrasonic time of 4 - 6 min and ultrasonic power of 120 - 330 w.

[0011] Preferably, the ultrasonic power is 210 - 250 w.

[0012] Further, the extraction is carried out at 20 - 40°C for 20 - 100 minutes.

[0013] Preferably, the extraction is carried out at 25 - 35°C for 50 - 70 minutes.

[0014] Further, after centrifuging to obtain the precipitate, the following steps are also included: washing, resuspending and adjusting the pH to neutral to obtain the mulberry leaf protein extract, and freeze - drying after purification to obtain mulberry leaf protein.

[0015] Further, the purification includes: Dialyzing the mulberry leaf protein extract in water with a molecular weight cut - off of 2 - 5 kDa, and the volume ratio of the mulberry leaf protein extract to water is 1:(30 - 70).

[0016] Preferably, deionized water is used, and the deionized water is changed every 6 h for the first 12 h before dialysis and then every 12 h.

[0017] In a second aspect, the present invention provides a mulberry leaf protein prepared by the aforementioned extraction method.

[0018] In a third aspect, the present invention provides a product comprising the aforementioned mulberry leaf protein, and the product is a drug, a health product, a cosmetic, a food or a feed additive.

[0019] In a fourth aspect, the present invention provides the application of the aforementioned extraction method in any of the following: i) Producing mulberry leaf protein, ii) Producing mulberry leaf protein feed, iii) Producing antioxidant products, where the products are drugs, health products, cosmetics, foods or feed additives, iv) Improving the antioxidant performance of mulberry leaf protein.

[0020] The antioxidant properties described in the present invention include: the scavenging efficiency for ABTS·, DPPH·, OH· (reactive nitrogen, reactive oxygen, and hydroxyl radicals).

[0021] The present invention has the following beneficial effects: The present invention combines the ultrasonic fragmentation method and the alkali dissolution - acid precipitation method for the first time to extract mulberry leaf protein. The extraction rate of mulberry leaf protein obtained by this process is significantly improved. Under optimized conditions, the extraction rate can reach 34.12%, which is significantly higher than 19.65% before optimization. In addition, the in - vitro scavenging rates of the mulberry leaf protein extracted under optimized conditions for ABTS·, DPPH·, and OH· at a concentration of 0.4 mg / ml reach 100.47%, 74.92%, and 34.9% respectively, which are higher than the antioxidant activities of mulberry leaf proteins extracted by other processes in the prior art.

[0022] The mulberry leaf protein extraction process provided by the present invention has the advantages of high extraction rate, environmental protection, low cost, etc. The extracted mulberry leaf protein has the function of significantly scavenging free radicals in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is the graph showing the effect of extraction temperature on the extraction rate of mulberry leaf protein in the single - factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention; in the figure, the data columns marked with the same lowercase letters indicate no significant difference ( p >0.05), and different lowercase letters indicate significant difference ( p <0.05). If there is no annotation in other figures, it is the same as this.

[0025] Figure 2 It is the graph showing the effect of pH value of the extraction solution on the extraction rate of mulberry leaf protein in the single - factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention.

[0026] Figure 3 It is the graph showing the effect of extraction time on the extraction rate of mulberry leaf protein in the single - factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention.

[0027] Figure 4 It is the graph showing the effect of solid - liquid ratio on the extraction rate of mulberry leaf protein in the single - factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention.

[0028] Figure 5It is a graph showing the effect of ultrasonic time on the extraction rate of mulberry leaf protein in the single-factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention.

[0029] Figure 6 It is a graph showing the effect of ultrasonic power on the extraction rate of mulberry leaf protein in the single-factor optimization experiment for mulberry leaf protein extraction provided in Example 3 of the present invention.

[0030] Figure 7 It is a response surface diagram of two-factor interaction for the response surface optimization of mulberry leaf protein extraction provided in Example 4 of the present invention.

[0031] Figure 8 It is a graph of the experimental results of the in vitro antioxidant activity of mulberry leaf protein provided in Example 4 of the present invention; where a is a schematic diagram of the scavenging rate of DPPH free radicals by mulberry leaf protein at different concentrations; b is a schematic diagram of the reducing power of mulberry leaf protein at different concentrations; c is a schematic diagram of the scavenging rate of ABTS free radicals by mulberry leaf protein at different concentrations; d is a schematic diagram of the scavenging rate of hydroxyl free radicals by mulberry leaf protein at different concentrations; the data columns in the figure marked with capital letters and small letters represent the difference comparison between different concentrations of the same sample, and different letters represent significant differences ( p <0.05), * represents the difference comparison between different samples at the same concentration, and * represents significant differences ( p <0.05). Detailed implementation manners

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0033] The experimental methods involved in the following examples, unless otherwise specified, are all conventional methods in the art. For example, they can be referred to the experimental manuals in the art or carried out according to the conditions recommended in the manufacturer's instructions.

[0034] The experimental materials and reagents involved in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] Example 1 This example provides an extraction process for mulberry leaf protein, including the following steps: Weigh a certain mass of mulberry leaf powder, add an appropriate amount of deionized water, and then adjust the pH value to 9 with 0.5M NaOH solution and 0.5M HCl solution to make the final solid-liquid ratio reach 1:20. Vortex and mix well. After ultrasonic disruption at 260 w for 5 min, place it in a water bath. Set the extraction temperature at 40 °C and the extraction time at 40 min. After the extraction is completed, centrifuge at 6000×g for 10 min, and take the supernatant, which is the mulberry leaf protein extract. Adjust the mulberry leaf protein extract to pH = 4.0 with dilute hydrochloric acid, let it stand for 20 min, and then centrifuge at 8000×g for 10 min. Discard the supernatant, wash the precipitate with deionized water, repeat three times, then redissolve with deionized water and adjust to neutral. Dialyze against deionized water for 48 h using a dialysis bag with a cut-off molecular weight of 3.5 kDa to remove salts, and change the dialysis fluid regularly during this period. After dialysis, obtain mulberry leaf protein powder by freeze-drying and store it at -20 °C.

[0036] Example 2 Determination of Protein Content in Mulberry Leaf Protein Extract The total protein content of mulberry leaf powder was determined by the Kjeldahl method according to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Foods". The protein content of the protein extract was determined by the Bradford method, and the method can be briefly described as follows: Dilute the bovine serum albumin (BSA) standard solution into different concentration gradients with PBS solution, add the protein extract diluted by an appropriate multiple and the diluted BSA standard solution into a 96-well plate, add 20 μL to each well, and then add 200 μL of 1×G250 dye. After standing at room temperature for 3 min, measure the OD 600 . Referring to the BSA standard curve, the protein content of the protein extract can be calculated. Calculate the extraction rate of mulberry leaf protein according to the following formula: Extraction rate of mulberry leaf protein = (protein concentration of mulberry leaf protein extract × volume of mulberry leaf protein extract × dilution factor) / (mass of mulberry leaf powder × total protein content of mulberry leaf powder) × 100%.

[0037] Example 3 Single-Factor Optimization of Mulberry Leaf Protein Extraction Process 1. Optimization of Mulberry Leaf Protein Extraction Temperature Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as a solid-liquid ratio of 1:20, pH of the extraction solution = 9, extraction temperature of 40 °C, ultrasonic power of 260 w, ultrasonic time of 5 min, and extraction time of 40 min. Under the condition of keeping other conditions unchanged, the effects of extraction temperature (20 °C, 30 °C, 40 °C, 50 °C, 60 °C) on the extraction rate of mulberry leaf protein were investigated. Each treatment was repeated 3 times.

[0038] The effect of extraction temperature on the extraction rate of mulberry leaf protein is as Figure 1 shown. From Figure 1It can be seen that within the range of extraction temperature from 20°C to 30°C, the extraction rate of mulberry leaf protein is positively correlated with the extraction temperature, and reaches the maximum value of 20.30 ± 2.01% at 30°C. Within the range of 30°C to 60°C, with the increase of the extraction temperature, the extraction rate of mulberry leaf protein decreases significantly ( p < 0.01). Therefore, 30°C is selected as the optimal extraction temperature for mulberry leaf protein.

[0039] 2. Optimization of the pH value of the mulberry leaf protein extraction solution Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as the solid-liquid ratio of 1:20, the pH value of the extraction solution = 9, the extraction temperature of 40°C, the ultrasonic power of 260w, the ultrasonic time of 5min, and the extraction time of 40min. Under the condition of keeping other conditions unchanged, the effects of the pH value of the extraction solution (8, 9, 10, 11, 12) on the extraction rate of mulberry leaf protein were investigated respectively. Each treatment was repeated 3 times.

[0040] The effects of the pH value of the extraction solution on the extraction rate of mulberry leaf protein are as Figure 2 shown. It can be Figure 2 seen that within the range of pH = 8 to pH = 12, the extraction rate of mulberry leaf protein increases with the increase of the pH value of the extraction solution. However, the use of a large amount of alkali during the alkali dissolution process will also cause problems such as increased production costs and environmental protection. Therefore, considering comprehensively, pH = 11 is selected as the optimal pH value of the extraction solution for mulberry leaf protein.

[0041] 3. Optimization of the extraction time of mulberry leaf protein Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as the solid-liquid ratio of 1:20, the pH value of the extraction solution = 9, the extraction temperature of 40°C, the ultrasonic power of 260w, the ultrasonic time of 5min, and the extraction time of 40min. Under the condition of keeping other conditions unchanged, the effects of the extraction time (20min, 40min, 60min, 80min, 100min) on the extraction rate of mulberry leaf protein were investigated respectively. Each treatment was repeated 3 times.

[0042] The effects of the extraction time on the extraction rate of mulberry leaf protein are as Figure 3 shown. It can be Figure 3 seen that when the extraction time is increased from 20min to 60min, the extraction rate of mulberry leaf protein increases significantly ( p < 0.05), and reaches 29.51 ± 0.52% at 60min, and then levels off. 60min is selected as the optimal extraction time for mulberry leaf protein.

[0043] 4. Optimization of the solid-liquid ratio for mulberry leaf protein extraction Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as follows: solid-liquid ratio of 1:20, pH of the extraction solution of 9, extraction temperature of 40 °C, ultrasonic power of 260 w, ultrasonic time of 5 min, and extraction time of 40 min. Under the condition of keeping other conditions unchanged, the effects of solid-liquid ratio (1:10, 1:15, 1:20, 1:25, 1:30) on the extraction rate of mulberry leaf protein were investigated. Each treatment was repeated 3 times.

[0044] The effect of the solid-liquid ratio on the extraction rate of mulberry leaf protein is as Figure 4 shown. From Figure 4 it can be seen that when the solid-liquid ratio was 1:20, the extraction rate of mulberry leaf protein reached the maximum value of 26.40 ± 0.51%, and then tended to level off. Therefore, 1:20 was selected as the optimal solid-liquid ratio for the extraction of mulberry leaf protein.

[0045] 5. Optimization of ultrasonic time for the extraction of mulberry leaf protein Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as follows: solid-liquid ratio of 1:20, pH of the extraction solution of 9, extraction temperature of 40 °C, ultrasonic power of 260 w, ultrasonic time of 5 min, and extraction time of 40 min. Under the condition of keeping other conditions unchanged, the effects of ultrasonic time (3 min, 4 min, 5 min, 6 min, 7 min) on the extraction rate of mulberry leaf protein were investigated. Each treatment was repeated 3 times.

[0046] The effect of ultrasonic time on the extraction rate of mulberry leaf protein is as Figure 5 shown. From Figure 5 it can be seen that when the ultrasonic time increased from 3 min to 5 min, the extraction rate of mulberry leaf protein increased significantly ( p < 0.05). However, when the ultrasonic time increased from 5 min to 7 min, the extraction rate of mulberry leaf protein was negatively correlated with the ultrasonic time. Therefore, 5 min was selected as the optimal ultrasonic time for the extraction of mulberry leaf protein.

[0047] 6. Optimization of ultrasonic power for the extraction of mulberry leaf protein Referring to the extraction process of mulberry leaf protein in Example 1, the basic extraction conditions were set as follows: solid-liquid ratio of 1:20, pH of the extraction solution of 9, extraction temperature of 40 °C, ultrasonic power of 260 w, ultrasonic time of 5 min, and extraction time of 40 min. Under the condition of keeping other conditions unchanged, the effects of ultrasonic power (130 w, 195 w, 260 w, 325 w, 390 w) on the extraction rate of mulberry leaf protein were investigated. Each treatment was repeated 3 times.

[0048] The effect of ultrasonic power on the extraction rate of mulberry leaf protein is as Figure 6 shown. From Figure 6 it can be seen that within the power range of 160 w to 260 w, the extraction rate of mulberry leaf protein increased with the increase of ultrasonic power, and then decreased. Therefore, 260 w was selected as the optimal ultrasonic power for the extraction of mulberry leaf protein.

[0049] In summary, based on the results of single-factor experiments, four influencing factors, namely the material-liquid ratio (A), extraction temperature (B), ultrasonic time (C), and ultrasonic power (D), were selected as response factors to conduct subsequent optimization.

[0050] 7. Response Surface Optimization of Mulberry Leaf Protein Extraction Process According to the results of single-factor optimization experiments, four significantly influencing factors were selected: material-liquid ratio, extraction temperature, ultrasonic power, and ultrasonic time. Taking the extraction rate of mulberry leaf protein as the response value, Design Expert 10.0 software was used to further conduct a Box-Behnken design of a response surface experiment with 4 factors and 3 levels to optimize the mulberry leaf protein extraction process. The experimental factors and levels are shown in Table 1; the Box-Behnken design and results are shown in Table 2; the variance analysis is shown in Table 3; the response surface optimization two-factor interaction response surface diagram is shown in Figure 7 。

[0051] As can be seen from Table 3, this model is extremely significant ( p < 0.01), and the lack-of-fit term is not significant ( p > 0.05). The fitted equation is: Y = 31.76 + 4.58×A + 0.27×B - 3.38×C - 4.64×D - 0.90×AB + 2.25×AC + 1.44×AD + 0.57×BC + 0.086×BD - 4.11×CD - 2.87×A 2 - 2.95×B 2 - 4.50×C 2 - 3.59×D 2 ,R 2 = 0.9134, and the experimental error is small. Therefore, this model can be used to analyze and predict the protein extraction rate. At the same time, through variance analysis, it can be obtained that among them, factors A, C, D, CD, A 2 、B 2 、C 2 、D 2 have extremely significant effects on the extraction rate of mulberry leaf protein ( p < 0.01). At the same time, by comparing the F values, it can be concluded that the order of the influence of each factor on the extraction rate of mulberry leaf protein is A material-liquid ratio > D ultrasonic power > C ultrasonic time > B extraction temperature. By Figure 7It can be seen that the stronger the interaction between factors is on the corresponding value, the steeper the corresponding response surface is, and vice versa, the flatter it is. Among them, the response surface between C and D is the steepest, which also reflects that the interaction between C and D has a stronger influence on the response value than the interaction between other factors.

[0052] Finally, through Box-Behnken optimization analysis, the optimal process conditions for extracting mulberry leaf protein by ultrasonic-assisted alkali solution-acid precipitation method were obtained as follows: solid-liquid ratio 1:23.55 (g / mL), extraction temperature 30.62 °C, ultrasonic time 4.88 min, ultrasonic power 235.19 w. Under these conditions, the predicted protein extraction rate was 33.96%. For the convenience of verification, the verification test conditions were modified to a solid-liquid ratio of 1:24 (g / ml), an extraction temperature of 30 °C, an ultrasonic time of 4.9 min, and an ultrasonic power of 235 w. Under these conditions, the extraction rate of mulberry leaf protein was 34.12%, which was 73.64% higher than the extraction rate of 19.65% before optimization.

[0053] Example 4 Determination of antioxidant activity of mulberry leaf protein 1. Determination of ABTS (active nitrogen) free radical scavenging rate Weigh an appropriate mass of mulberry leaf protein and dissolve it in deionized water and dilute it to 0.05 - 0.4 mg / mL (measured by Bradford method for protein concentration). Mix 7 mM ABTS and 2.4 mM potassium persulfate in equal volumes and react in the dark at room temperature for 16 h to obtain ABTS free radical solution. Then dilute the ABTS free radical solution with PBS (pH = 7.4) to OD 734 = 0.7 ± 0.02 to obtain ABTS working solution. Then add 300 μL of the sample to 2 mL of ABTS working solution and react in the dark at room temperature for 10 min to measure OD 734 = A1. The blank group and the control group were replaced with ABTS working solution and sample with equal volumes of deionized water respectively, and OD 734 = A0, A were measured respectively, with ascorbic acid as the positive control. Calculate the ABTS free radical scavenging rate according to the following formula: ABTS active nitrogen free radical scavenging rate = (1 - (A1 - A0) / A) × 100%.

[0054] 2. Determination of DPPH oxygen free radical scavenging rate Weigh an appropriate mass of mulberry leaf protein and dissolve it in deionized water and dilute it to 0.05 - 0.4 mg / mL (measured by Bradford method for protein concentration). Prepare 0.2 mM DPPH working solution with absolute ethanol. Add 1 mL of the sample to 2 mL of DPPH working solution, shake well and react in the dark at room temperature for 30 min to measure OD 517=A1. The blank group used ethanol with an equal volume to replace the DPPH solution, and the OD was measured. 517 =A0. The control group used deionized water with an equal volume to replace the sample, and the OD was measured. 517 =A. Ascorbic acid was used as the positive control. The ABTS radical scavenging rate was calculated according to the following formula: DPPH oxygen radical scavenging rate = (1 - (A1 - A0) / A) × 100%.

[0055] 3. Determination of hydroxyl radical scavenging rate Weighed an appropriate mass of mulberry leaf protein, dissolved it in deionized water and diluted it to 0.05 - 0.4 mg / mL (measured by Bradford method for protein concentration). Added 1.5 mL of salicylic acid (1.8 mM), 2 mL of ferrous sulfate (1.8 mM), and 1 mL of hydrogen peroxide (6 mM) to 1 mL of the sample in sequence. After fully shaking and mixing, reacted at 37 °C for 30 min, then centrifuged at 3000 rpm / min for 5 min, and took the supernatant to measure the OD of the sample. 510 =A1. The blank group and the control group used deionized water with an equal volume to replace salicylic acid and the sample respectively, and measured the OD respectively. 510 =A0, A. Ascorbic acid was used as the positive control. The hydroxyl radical scavenging rate was calculated according to the following formula: Hydroxyl radical scavenging rate = (1 - (A1 - A0) / A) × 100%.

[0056] 4. Determination of reducing power Reducing power refers to the ability of a substance to lose electrons or accept hydrogen ions in a reduction reaction. In a chemical reaction, a reducing agent accepts oxygen or loses hydrogen or electrons, thereby reducing another substance. Reducing power is an important factor in chemical reactions, which can affect the reaction rate and direction. Usually, the stronger the reducing power of a substance, the easier it is to be oxidized, which also means that the antioxidant ability of this substance is stronger.

[0057] During the determination, weighed an appropriate mass of mulberry leaf protein, dissolved it in deionized water and diluted it to 0.05 - 0.4 mg / mL (measured by Bradford method for protein concentration). Took 1 mL of the sample and added 2.5 mL of sodium carbonate buffer solution (0.2 M, pH = 6.6) and 2.5 mL of 1% (w / v) potassium ferricyanide. After fully shaking and mixing, reacted at 50 °C for 20 min. Then added 2.5 mL of trichloroacetic acid (10%, w / v) and centrifuged at 3000 pm / min for 10 min. Took 2.5 mL of the supernatant, added an equal volume of deionized water and 0.5 mL of FeCl3 (0.1%, w / v), mixed evenly and placed at room temperature for 10 min, and measured the OD. 600 , with ascorbic acid as the positive control. The reducing power of the sample is proportional to the OD. 600 It is proportional.

[0058] The results are shown in Figure 8 , and it can be seen from Figure 8 that mulberry leaf protein exhibits excellent chemical antioxidant ability, and there is a positive dose correlation with concentration in the range of 0.1 - 0.4 mg / mL. At the same time, at a concentration of 0.05 mg / mL, the reducing power and hydroxyl radical scavenging rate of mulberry leaf protein are comparable to those of the positive control ascorbic acid. At a concentration of 0.4 mg / mL, the ABTS radical scavenging rate of mulberry leaf protein is comparable to that of the positive control. In particular, at a concentration of 0.1 mg / mL, the hydroxyl radical scavenging rate of mulberry leaf protein is even significantly better than that of the positive control ( p < 0.05), reaching 20.80 ± 0.12%. The experiment shows that mulberry leaf protein has strong chemical antioxidant ability.

[0059] In summary, the present invention uses ultrasonic fragmentation-assisted alkali solution-acid precipitation method to extract mulberry leaf protein. Through single-factor experiments and Box-Behnken response surface method experimental design and optimization, the optimal process for extracting mulberry leaf protein is finally obtained: liquid-to-material ratio 1:24 (g / mL), extraction temperature 30 °C, ultrasonic time 4.9 min, ultrasonic power 235 w, pH of extraction solution = 11, extraction time 60 min. Under these conditions, the extraction rate of mulberry leaf protein can reach 34.12%. It is 73.64% higher than the extraction rate of 19.65% before optimization, and significantly higher than 16.06% of the ultrasonic-cellulase combined-assisted method, 5.56% of the ultrasonic extraction combined with ultrafiltration method, 9.85% of the ultrasonic cell disruption method, and 5.18% of the ultrasonic-assisted salting-out method (calculated based on mulberry leaf powder) in the same type of mulberry leaf protein extraction research. In addition, the in vitro scavenging rates of ABTS·, DPPH·, and OH· of the mulberry leaf protein extracted by this process reach 100.47%, 74.92%, and 34.94% at a concentration of 0.4 mg / ml, which are higher than those in the same type of mulberry leaf protein antioxidant research. Therefore, the present invention not only optimizes the extraction efficiency of mulberry leaf protein, but also improves the antioxidant ability of the extracted mulberry leaf protein, which has great value for the utilization of mulberry leaf protein resources.

[0060] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for extracting mulberry leaf protein, characterized in that, Comprising: Dissolve mulberry leaf powder in water and adjust the pH to ≥8 to obtain an aqueous solution of mulberry leaf powder; After ultrasonic disruption, perform extraction and centrifugation to obtain the supernatant as the crude extract; Adjust the pH of the crude extract to ≤6 and centrifuge to obtain the precipitate.

2. The extraction method according to claim 1, wherein In the aqueous solution of mulberry leaf powder, the ratio of mulberry leaf components to water is 1:(10 - 30), and the pH = (10 - 12).

3. The extraction method according to claim 1 or 2, characterized in that, The conditions for the ultrasonic disruption include: ultrasonic time of 4 - 6 min and ultrasonic power of 120 - 330 w.

4. The extraction method according to any one of claims 1 to 3, characterized in that, The extraction is carried out under the condition of 20 - 40°C for 20 - 100 minutes.

5. The extraction method according to any one of claims 1-4, characterized in that After the centrifugation to obtain the precipitate, it further includes: washing, resuspending and adjusting the pH to neutral to obtain the mulberry leaf protein extract, and freeze-drying after purification to obtain mulberry leaf protein.

6. The extraction method according to claim 5, wherein The purification includes: Place the mulberry leaf protein extract in water for dialysis with a molecular weight cut-off of 2 - 5 kDa, and the volume ratio of the mulberry leaf protein extract to water is 1:(30 - 70).

7. A mulberry leaf protein, characterized in that, Prepared by the extraction method according to any one of claims 1 - 6.

8. A product, characterized in that, The product includes the mulberry leaf protein according to claim 7, and the product is a drug, health product, cosmetic, food or feed additive.

9. Use of the extraction method according to any one of claims 1 - 6 in any of the following: i) Producing mulberry leaf protein, ii) Producing mulberry leaf protein feed, iii) Producing antioxidant products, where the products are drugs, health products, cosmetics, foods or feed additives, iv) Improving the antioxidant performance of mulberry leaf protein.

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