Mulberry leaf protein extraction method, mulberry leaf protein extract, and application thereof

Abstract

The present invention relates to the field of plant protein extraction. Disclosed are a mulberry leaf protein extraction method, a mulberry leaf protein extract, and an application thereof. The extraction method comprises: pulverizing a dried mulberry leaf, adding water and fully wetting same, sequentially performing cellulase hydrolysis, protease-assisted extraction, and aqueous extraction to obtain a mulberry leaf protein crude extract, and then separating, purifying, and drying the mulberry leaf protein crude extract to obtain a mulberry leaf protein extract.

Description

A method for extracting mulberry leaf protein, mulberry leaf protein extract and its application Technical Field

[0001] This invention belongs to the field of plant protein extraction, specifically, it relates to a method for extracting mulberry leaf protein, a mulberry leaf protein extract, and its applications. Background Technology

[0002] Leaf protein, also known as green protein concentrate, refers to protein products extracted from the stems and leaves of green plants (Leaves Protein Concentrates, abbreviated as LPC). Proteins in green plant stems and leaves can be divided into two categories: solid proteins and soluble proteins. Solid proteins are found in the green precipitate separated after crushing and pressing, mainly including insoluble chloroplast, mitochondrial structural proteins and nucleoproteins, and cell wall proteins. Soluble proteins are found in the supernatant after crushing, pressing, and centrifugation, including soluble portions of cytoplasmic proteins and mitochondrial proteins, as well as chloroplast matrix proteins. Leaf protein is a concretion of these soluble proteins.

[0003] Plant leaf protein is a functional protein containing 17-18 amino acids, including all eight essential amino acids for the human body. Its balanced amino acid composition largely aligns with the adult amino acid pattern recommended by the Food and Agriculture Organization of the United Nations, making it highly nutritious for consumption. Furthermore, unlike animal muscle protein which contains saturated fatty acids and cholesterol, plant leaf protein is cholesterol-free, making it an important supplement or substitute for protein in the human diet.

[0004] Mulberry leaves are from a perennial deciduous tree belonging to the Moraceae family, native to China. *Morus alba* L. is listed in the Chinese Pharmacopoeia and is considered both a food and a medicinal plant in China. Mulberry leaves contain 21-27% protein, a relatively high content among plant leaf proteins, and contain all 18 amino acids, with essential amino acids accounting for 43% of the total amino acid mass fraction, making it a high-quality protein resource. Furthermore, my country is the largest mulberry-producing country, and mulberry leaves are the main product of mulberry cultivation, accounting for as much as 64% of the above-ground production. Therefore, mulberry leaves have a significant advantage in my country in terms of planting resources. In addition, the raw materials for leaf protein products on the market are mainly alfalfa leaves, duckweed, and *Euphorbia milii*, indicating that the development value and potential of mulberry leaf-based protein products is enormous.

[0005] Commercially available protein powders are primarily marketed as nutritional supplements, with little research into their other specific functions. Furthermore, most protein powders are mainly composed of whey protein or a blend of whey and soy protein, resulting in a limited variety. Therefore, this invention provides a mulberry leaf protein powder that not only functions as a nutritional supplement but also offers "muscle-building and fat-loss" benefits, thus supplementing and developing the protein powder market.

[0006] Currently, most methods for extracting plant leaf proteins include direct heating, organic solvent extraction, ultrasonic extraction, salting out, fermentation, and enzymatic methods. However, each method has its own drawbacks, such as low protein content, poor solubility, unsuitability for large-scale industrial production, and high cost. For example:

[0007] Chinese patent application CN103387599A discloses a process for extracting mulberry leaf protein. The process first pre-treats fresh mulberry leaves and homogenizes them. The homogenate is then filtered, and the filtrate and filter cake are collected separately. The filter cake is resuspended and then extracted and filtered again with water, enzymes, and iron salts. The filtrates from both extractions are combined, the pH of the solution is adjusted to 2.0-4.5, a calcium salt solution is added, and after standing, the precipitate is collected by centrifugation. The precipitate is then resuspended and spray-dried to obtain the mulberry leaf protein. This technique requires the use of strong acids, which can affect the activity or properties of the protein, and the introduction of iron and calcium salts increases the difficulty of subsequent purification.

[0008] Chinese patent application CN104543613A discloses a method for preparing mulberry leaf protein powder, including raw material pretreatment, ultrafine grinding, extrusion puffing, enzymatic extraction, alkali-soluble protein, acid-precipitated protein, enzymatic solubilization, and drying to obtain highly soluble mulberry leaf protein powder. This technology involves complex steps, high extraction costs, and conditions such as strong acid (acid precipitation of protein) and high temperature (extrusion temperature 60-80℃) can affect the activity or properties of the protein.

[0009] Chinese patent application CN114989242A discloses a method for extracting mulberry leaf protein. The method involves first preparing a suspension of mulberry leaf powder, followed by microwave heat treatment. Simultaneously, cellulase, after ultrasonic pretreatment, is added to the mulberry leaf suspension to degrade cellulose, with continuous ultrasonic-assisted degradation. After degradation, sodium hydroxide solution is added for further alkaline extraction of mulberry leaf protein. This technique requires microwave heat treatment and ultrasonic assistance; temperature-sensitive and easily decomposed products are easily altered, thus losing their activity. Furthermore, this technique is relatively unsuitable for large-scale industrial production due to its high cost. Therefore, a method for extracting mulberry leaf protein that consumes less heat, has lower production costs, and lower water consumption would be beneficial for industrial production.

[0010] In view of this, the present invention is proposed. Summary of the Invention

[0011] The technical problem this invention aims to solve is to overcome at least one of the shortcomings of existing technologies: a method for extracting mulberry leaf protein, a mulberry leaf protein extract, and its applications. The extraction method of this invention has low water consumption, low heat energy consumption, and low production costs, which is conducive to industrial production. The mulberry leaf protein extract prepared by this invention has a branched-chain amino acid content comparable to whey protein, and thus has the effects of fat reduction and muscle building.

[0012] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a method for extracting mulberry leaf protein, comprising the following steps:

[0014] (1) Pretreatment: Pulverize the dried mulberry leaves to obtain mulberry leaf powder;

[0015] (2) First extraction: Add water to mulberry leaf powder, add cellulase under certain temperature conditions, stir and extract; add alkaline solution to control the pH of the system, add protease, and continue extraction;

[0016] (3) Second extraction: Add water, cool down, and extract;

[0017] (4) Centrifuge the extract obtained in (3), take the supernatant to obtain crude mulberry leaf protein extract, then separate and purify it, dry it to obtain mulberry leaf protein extract.

[0018] The extraction method of this invention involves pulverizing dried mulberry leaves, soaking them thoroughly in water, and then sequentially performing enzymatic hydrolysis with cellulase, enzymatic hydrolysis with protease, and water extraction to obtain a crude mulberry leaf protein extract. This crude extract is then separated, purified, and dried to obtain the mulberry leaf protein extract. In this method, enzymatic hydrolysis is used in the first extraction, resulting in a mild reaction. The specificity of the enzyme allows it to act directly on the active ingredients, minimizing the generation of impurities and avoiding damage to substrates. The second extraction involves directly adding water for water extraction, which improves the extraction rate of mulberry leaf protein and offers high cost-effectiveness.

[0019] The extraction method of this invention does not require any ultrasonic or microwave assistance or high-temperature treatment throughout the process. This allows for higher bioactivity in many volatile and decomposable products, achieving an extraction rate of 55-70% for mulberry leaf protein, demonstrating high extraction efficiency. Furthermore, it reduces energy consumption and costs. The extraction method of this invention features a simple production process, low equipment requirements, and ease of operation, making it suitable for industrial-scale production.

[0020] In a further embodiment, in step (2), the protease includes chymotrypsin;

[0021] In a further embodiment, in step (2), the protease includes, in addition to chymotrypsin, one or more of alkaline protease, neutral protease, and trypsin.

[0022] In the extraction method of mulberry leaf protein of the present invention, it has been found through multiple experiments that when the protease used contains chymotrypsin, the mulberry leaf protein extract obtained has a high content of branched-chain amino acids, which can be comparable to whey protein, and has the effect of reducing fat and increasing muscle.

[0023] In this invention, the protease can be chymotrypsin alone, or a combination of two or more proteases, including chymotrypsin. When using a combination of multiple proteases, they can be added all at once, or sequentially according to time or after adjusting the pH of the system. There is no restriction on the order of adding chymotrypsin.

[0024] In a further step, in step (2), when adding the protease, the pH of the system is controlled to be 7.0-10.0.

[0025] In a preferred embodiment, when using neutral protease and / or trypsin, the pH of the system is controlled to be 7.0-7.5;

[0026] In a preferred embodiment, when alkaline protease and / or chymotrypsin are used, the pH of the system is controlled to be 9.0-10.0.

[0027] A further approach is to adjust the pH of the system by adding an alkaline solution;

[0028] Preferably, the alkaline solution includes a NaOH solution.

[0029] In a further embodiment, in step (2), the amount of protease added is 0.5-5.0% of the mass of mulberry leaf powder;

[0030] In a further step, in step (2), after adding protease, extraction is carried out for 4-6 hours.

[0031] In a further embodiment, in step (2), the amount of cellulase added is 0.5-2.0% of the mass of mulberry leaf powder;

[0032] In a further step, in step (2), cellulase is added at a temperature of 50-60℃ and then stirred for 1-2 hours for extraction.

[0033] In a further step, in step (2), 8-12 times the mass of water is added to the mulberry leaf powder.

[0034] Through experiments, this invention found that the pulverized dried mulberry leaves can be fully soaked under a material-to-water ratio of 1:8, and subsequent experimental operations can be carried out. However, excessive extraction solvent is not conducive to the results of subsequent experiments and reduces the efficiency of later industrial production. Therefore, the material-to-water ratio parameter is controlled at 1:8-12.

[0035] In a further step, in step (3), the water added is 2-6 times the mass of the mulberry leaf powder;

[0036] Preferably, in step (3), the temperature is lowered to 20-40℃. The temperature of the liquid can be controlled by adding water, which is beneficial to maintaining the activity of mulberry leaf protein under subsequent room temperature centrifugation conditions.

[0037] Preferably, in step (3), after cooling, extraction is performed for 0.5-1h.

[0038] The extraction method of this invention incorporates a secondary direct extraction. In existing methods, when multiple extractions are performed, the suspension from the first extraction is typically filtered or centrifuged, and the filter residue or precipitate is used as the raw material for the second extraction. However, in this invention, water is directly added to the first extraction for the second extraction without filtration or centrifugation. Studies have shown that this method not only improves the extraction rate of mulberry leaf protein but also offers extremely high cost-effectiveness.

[0039] On the one hand, the secondary extraction of this invention is a simple water extraction, without the use of other reagents; and direct water extraction, compared to secondary water extraction of filter residue or precipitate, does not require additional operational steps (such as filtration, centrifugation, etc.), making it simple and feasible. On the other hand, compared to primary extraction, adding secondary extraction increases the extraction rate of mulberry leaf protein to varying degrees. This increased extraction rate is due to minimal cost and simplified manual operation, thus offering high cost-effectiveness. Furthermore, experimental comparisons show that direct secondary extraction achieves a comparable extraction rate to secondary water extraction of filter residue or precipitate, but simplifies the operation and is more suitable for continuous industrial production.

[0040] In a further embodiment, in step (1), the moisture content of the dried mulberry leaves is 5-10%;

[0041] Preferably, the moisture content of the dried mulberry leaves is 5-7%.

[0042] Compared to traditional processes that require fresh mulberry leaves, this invention uses dried mulberry leaves as raw material, eliminating the limitations of fresh mulberry leaf production and storage. This eliminates the influence of time and external factors on extraction, facilitating large-scale production. Controlling the moisture content of the dried mulberry leaves in this invention helps standardize leaf quality and ensure raw material consistency. Excessive moisture content leads to easy spoilage and poor storage; insufficient moisture content increases drying time and energy consumption.

[0043] A further method involves pulverizing dried mulberry leaves and passing them through a 60-80 mesh sieve to obtain mulberry leaf powder.

[0044] In a further step, in step (4), the centrifugation speed is 3000-5000 r / min and the centrifugation time is 20-40 min.

[0045] In a further step, step (4) involves separating and purifying the crude mulberry leaf protein extract, including desalting and concentrating it by ultrafiltration to obtain a concentrated mulberry leaf protein solution.

[0046] In a further embodiment, in step (4), the drying can be carried out using existing conventional methods, such as freeze-drying the mulberry leaf protein concentrate to obtain mulberry leaf protein extract.

[0047] Secondly, the present invention provides a mulberry leaf protein extract, wherein the amino acid composition of the mulberry leaf protein extract has an essential amino acid / total amino acid mass percentage ≥30%, an essential amino acid / non-essential amino acid mass percentage ≥43%, and branched-chain amino acids account for ≥20% of the total amino acid mass.

[0048] Preferably, the mass percentage of essential amino acids to total amino acids is ≥35%, and the mass percentage of essential amino acids to non-essential amino acids is ≥54%.

[0049] The FAO / WHO specifies that the ideal amino acid ratio in a protein should meet the following requirements: the ratio of essential amino acids to total amino acids (E / T) should be around 0.4, and the ratio of essential amino acids to non-essential amino acids (E / N) should be above 0.6. The preferred embodiment of this invention yields mulberry leaf protein with E / T and E / N values ​​that meet or closely approximate the requirements for ideal proteins.

[0050] Preferably, in the mulberry leaf protein extract, glycine accounts for ≥7% of the total amino acids by mass.

[0051] Preferably, in the mulberry leaf protein extract, alanine accounts for ≥7% of the total amino acid mass.

[0052] Branched-chain amino acids (BCAAs) promote muscle growth, while glycine and alanine promote muscle growth and repair. The mulberry leaf protein extract of this invention has a high content of BCAAs, comparable to whey protein; it also has relatively high levels of glycine and alanine.

[0053] Furthermore, the mulberry leaf protein extract described in this aspect is obtained by the extraction method described in the first aspect.

[0054] Thirdly, the present invention provides an application of mulberry leaf protein extract in the preparation of fat-reducing products and / or muscle-building products, wherein the amino acid composition of the mulberry leaf protein extract contains branched-chain amino acids accounting for ≥20% of the total amino acids by mass.

[0055] Preferably, the glycine content in the mulberry leaf protein extract is ≥7% by mass of the total amino acids;

[0056] Preferably, the alanine content in the mulberry leaf protein extract is ≥7% of the total amino acids by mass.

[0057] Branched-chain amino acids (BCAAs) refer to leucine, valine, and isoleucine. These amino acids promote anabolism (muscle growth) in two specific ways: ① by promoting insulin release, and ② by promoting growth hormone release. The most important BCAA is leucine, the precursor to ketoisocaproic acid (KIC) and hemoglobin (HMB). KIC and HMB can increase muscle mass, reduce fat, and provide nutrients to the body. Glycine and alanine are also two amino acids that promote muscle growth and repair.

[0058] The mulberry leaf protein extract of this invention has a high percentage of branched-chain amino acids, surpassing that of hydrolyzed soybean protein and even comparable to whey protein; the percentages of glycine and alanine are also higher than those of hydrolyzed soybean protein and whey protein. Mouse model experiments have shown that the mulberry leaf protein extract of this invention has muscle-building and fat-reducing effects.

[0059] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0060] 1. The extraction method of the present invention uses dried mulberry leaves as raw material for extraction, which is different from the traditional process that requires fresh mulberry leaves. Therefore, the influence of time and external factors on the extraction can be eliminated.

[0061] 2. The extraction method of the present invention uses water as the extraction solvent, which is safe and harmless. Moreover, the present invention reduces the amount of water used to extract mulberry leaf powder by about 2 times compared with the traditional mulberry leaf extraction process.

[0062] In traditional mulberry leaf protein extraction processes, the material-to-water ratio of mulberry leaf powder is generally above 1:10, and sometimes even reaches 1:20 or higher. Compared to traditional extraction processes, this invention achieves sufficient saturation of mulberry leaf powder at a material-to-water ratio of 1:8, allowing for subsequent experimental operations. Excessive extraction solvent is detrimental to subsequent experimental results and reduces the efficiency of later industrial production; therefore, the material-to-water ratio is controlled at 1:8-12. This reduces water consumption, which is beneficial for later industrial production.

[0063] 3. The extraction method of the present invention uses an enzymatic method in the first extraction. Cellulase is used to degrade mulberry leaves first, and then the pH of the system is adjusted and protease is added. This reaction is mild, and the specificity of the enzyme allows it to act directly on the effective components without producing many impurities. It can also avoid damage to substances other than the substrate.

[0064] This invention does not require any ultrasonic or microwave assistance or high-temperature treatment, thus enabling many volatile and decomposable products to have relatively higher biological activity while reducing heat energy consumption and costs. The extraction rate of this invention reaches 55-70%, demonstrating high extraction efficiency.

[0065] 4. The extraction method of this invention adds a second direct extraction. In existing methods, when multiple extractions are performed, the suspension from the first extraction is usually filtered or centrifuged, and the filter residue or precipitate is used as the raw material for the second extraction. However, in this invention, water is directly added to the first extraction for the second extraction without filtration or centrifugation. Studies have found that this method not only improves the extraction rate of mulberry leaf protein but also has extremely high cost-effectiveness.

[0066] 5. The extraction method of the present invention has a simple production process, low requirements for equipment, and is easy to operate, which greatly reduces the production cycle and cost and is conducive to realizing industrialized production.

[0067] 6. The mulberry leaf protein extract prepared by the extraction method of this invention has a relatively complete amino acid composition, containing 7 of the 8 essential amino acids for adults, and its amino acid composition is similar to that of hydrolyzed soybean protein and whey protein. The ratio of essential amino acids to total amino acids and the ratio of essential amino acids to non-essential amino acids meet or approach the requirements of ideal protein.

[0068] In the extraction method of mulberry leaf protein of the present invention, the protease used contains chymotrypsin. The resulting mulberry leaf protein extract has a high percentage of branched-chain amino acids, exceeding the content of hydrolyzed soybean protein and even comparable to whey protein. The percentages of glycine and alanine are also higher than those of hydrolyzed soybean protein and whey protein. Mouse model experiments have shown that the mulberry leaf protein extract of the present invention has muscle-building and fat-reducing effects.

[0069] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0070] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0071] Figure 1 shows the test results of the gripping force of the mice in each group in Experiment Example 1 of this invention.

[0072] Among them, # indicates P < 0.05 compared with the blank group, and ** indicates P < 0.01 compared with the model group.

[0073] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0075] Detection methods

[0076] The mulberry leaf protein extracts obtained in the embodiments and control examples of this invention were pretreated with acid hydrolysis (hydrolysis with 2M trifluoroacetic acid for 8 hours), and the protein content was determined using the free amino acid method. Specifically, high-performance liquid chromatography (HPLC) was used, and the detection steps were referenced from Agilent AdvanceBio Amino Acid Analysis (AAA). The key steps and conditions are as follows:

[0077] 1. Preparation of test solution: Mix 50 μL of internal standard stock solution (10 nMoles / μL aqueous solution of valine and sarcosine) with 950 μL of sample.

[0078] 2. Three series of mixed amino acid standard working solutions were prepared using the Agilent Amino Acid Auxiliary Kit and amino acid standard solutions: 90 pMoles / μL, 225 pMoles / μL, and 900 pMoles / μL.

[0079] 3. Chromatographic conditions:

[0080] Column: Aglient ZORBAX Rapid Separation High-Throughput Eclipse Plus C18, 3.0*50mm, 1.8μm

[0081] Column temperature: 40℃

[0082] Detectors: Diode array detector (DAD) and fluorescence detector (FLD)

[0083] Detection wavelengths: DAD detection wavelengths: 338nm and 262nm; FLD detection wavelengths: excitation wavelength 230nm, emission wavelength 450nm and excitation wavelength 266nm, emission wavelength 305nm.

[0084] Mobile phases: Mobile phase A: 10 mM Na₂HPO₄ and 10 mM Na₂B₄O₇, pH 8.2; Mobile phase B: Acetonitrile: Methanol: Water = 45:45:10 (volume ratio)

[0085] Mobile phase flow rate: 0.85 mL / min

[0086] The gradient elution conditions are as follows:

[0087] 4. Online derivatization of OPA and FMOC: The autosampler is used to arrange the ampoules containing the derivatization reagents as follows: Ampoule 1 contains borate buffer, ampoule 2 contains OPA, ampoule 3 contains FMOC, and ampoule 4 contains injection diluent.

[0088] 5. Determination method: Inject the mixed standard series working solutions of the three amino acids into the HPLC system, and inject the test solution under the same chromatographic conditions. The test solution is quantified by peak area.

[0089] The protein extraction rate of the embodiments and control examples of the present invention is calculated as: (protein content in the extract / protein content in mulberry leaf powder) * 100%.

[0090] The yield of mulberry leaf protein extract in the embodiments and comparative examples of the present invention is: (mass of mulberry leaf protein extract / mass of raw mulberry leaf powder) * 100%.

[0091] Example 1

[0092] Take 15g of dried mulberry leaf powder (5.5% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the solution to 55℃. Then add 0.8% cellulase (based on the mass of mulberry leaf powder) and hydrolyze for 1 hour. Next, add sodium hydroxide solution to adjust the pH of the system to 10.0, and add 1.0% chymotrypsin (based on the mass of mulberry leaf powder) for extraction for 5 hours. Add 30g of pure water, cool the solution to 30℃, and extract for 30 minutes. Centrifuge at 3000 rpm for 20 minutes, collect the supernatant, and determine the protein extraction rate using the free amino acid method; it is 56.34%. After desalting, concentrating, and drying the supernatant, 3.77g of mulberry leaf protein extract is obtained, with an extract yield of 25.13%.

[0093] Example 2

[0094] Take 15g of dried mulberry leaf powder (6.0% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and heat the mixture to 55℃ while stirring. Then add 0.8% cellulase and hydrolyze for 1 hour. Next, add sodium hydroxide solution to adjust the pH of the system to 10.0, and add 0.6% chymotrypsin and 1.0% alkaline protease. Extract for 4 hours. Add 30g of pure water and cool the mixture to 30℃. Extract for 30 minutes, then centrifuge at 3000 rpm for 20 minutes. Collect the supernatant and determine the protein extraction rate using the free amino acid method; it is 55.94%. After desalting, concentrating, and drying the supernatant, 3.99g of mulberry leaf protein extract is obtained, with an extract yield of 26.60%.

[0095] Example 3

[0096] Take 15g of dried mulberry leaf powder (5.5% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the solution to 55℃. Add 0.7% cellulase and hydrolyze for 1 hour. Add sodium hydroxide solution to adjust the pH of the system to 7.0, then add 1% neutral protease and hydrolyze for 3 hours. Add sodium hydroxide solution again to adjust the pH of the system to 9.0. Add 0.6% chymotrypsin and 0.6% alkaline protease and extract for 1 hour. Add 30g of pure water and cool the solution to 30℃. Extract for 30 minutes, then centrifuge at 3000r / min for 20 minutes. Collect the supernatant and determine the protein extraction rate using the free amino acid method; it is 67.89%. After desalting, concentrating, and drying the supernatant, 5.41g of mulberry leaf protein extract is obtained, with an extract yield of 36.07%.

[0097] Example 4

[0098] Take 15g of dried mulberry leaf powder (5.2% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the solution to 55℃. Add 0.7% cellulase and hydrolyze for 1 hour. Add sodium hydroxide solution to adjust the pH of the system to 7.0, then add 1% neutral protease and 0.3% trypsin, and hydrolyze for 2 hours. Add sodium hydroxide solution again to adjust the pH of the system to 9.0, then add 0.3% chymotrypsin and 1% alkaline protease, and extract for 2 hours. Add 30g of pure water, cool the solution to 30℃, and extract for 30 minutes. Centrifuge at 3000r / min for 20 minutes, collect the supernatant, and determine the protein extraction rate using the free amino acid method: 62.20%. After desalting, concentrating, and drying the supernatant, 5.71g of mulberry leaf protein extract is obtained, with an extract yield of 38.07%.

[0099] Comparative Example 1

[0100] Take 15g of dried mulberry leaf powder (5.5% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the solution to 55℃. Add 0.7% cellulase and hydrolyze for 1 hour. Add sodium hydroxide solution to adjust the pH of the system to 7.0, then add 1% neutral protease and hydrolyze for 3 hours. Add sodium hydroxide solution again to adjust the pH of the system to 9.0, then add 1.2% alkaline protease and extract for 1 hour. Add 30g of pure water and cool the solution to 30℃. Extract for 30 minutes, then centrifuge at 3000 rpm for 20 minutes. Collect the supernatant, and determine the protein extraction rate using the free amino acid method: 65.78%. After desalting, concentrating, and drying the supernatant, 5.28g of mulberry leaf protein extract was obtained, with an extract yield of 35.20%.

[0101] Comparative Example 2

[0102] Take 15g of dried mulberry leaf powder (5.2% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the solution to 55℃. Add 0.8% cellulase and hydrolyze for 1 hour. Add sodium hydroxide solution to adjust the pH of the system to 7.0, then add 1% neutral protease and 0.3% trypsin, and hydrolyze for 2 hours. Add sodium hydroxide solution again to adjust the pH of the system to 9.0, then add 1.5% alkaline protease and extract for 2 hours. Add 30g of pure water, cool the solution to 30℃, and extract for 30 minutes. Centrifuge at 3000 rpm for 20 minutes, collect the supernatant, and determine the protein extraction rate using the free amino acid method: 65.20%. After desalting, concentrating, and drying the supernatant, 5.82g of mulberry leaf protein extract was obtained, with an extract yield of 38.80%.

[0103] Comparative Example 3

[0104] The difference from Example 2 is that the secondary extraction method is different.

[0105] Take 15g of dried mulberry leaf powder (6.0% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and while stirring, heat the mixture to 55℃. Add 0.8% cellulase and hydrolyze for 1 hour. Then add sodium hydroxide solution to adjust the pH of the system to 10.0. Add 0.6% chymotrypsin and 1.0% alkaline protease, and extract for 4 hours. Centrifuge at 3000 rpm for 20 minutes and collect the supernatant. Add 30g of pure water to the precipitate, cool the mixture to 30℃, and extract for 30 minutes. Centrifuge at 3000 rpm for 20 minutes and collect the supernatant. Combine the two supernatants, and determine the protein extraction rate using the free amino acid method; it is 56.15%. After desalting, concentrating, and drying the supernatant, 4.01g of mulberry leaf protein extract is obtained, with an extract yield of 26.73%.

[0106] Comparative Example 4

[0107] The difference from Example 3 is that ultrasonic pretreatment is added.

[0108] Take 15g of dried mulberry leaf powder (5.5% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and pre-treat the mulberry leaf powder using ultrasound. Set the ultrasound power to 600W, the temperature to 30℃, and the time to 20min. While stirring, raise the temperature of the liquid to 55℃, add 0.7% cellulase, and hydrolyze for 1h. Add sodium hydroxide solution to make the pH of the system 7.0, then add 1% neutral protease, and hydrolyze for 3h. Add sodium hydroxide solution again to make the pH of the system 9.0, then add 0.6% chymotrypsin and 0.6% alkaline protease, and extract for 1h. Add 30g of pure water, cool the liquid to 30℃, and extract for 30min. Centrifuge at 3000r / min for 20min, collect the supernatant, and determine the protein extraction rate using the free amino acid method, which is 58.37%. After desalting, concentrating, and drying the supernatant, 5.01g of mulberry leaf protein extract is obtained, with an extract yield of 33.40%.

[0109] Comparative Example 5

[0110] The difference from Example 3 is that microwave pretreatment is added.

[0111] Take 15g of dried mulberry leaf powder (5.5% moisture content) that has been pulverized and passed through a 60-mesh sieve, add 120g of pure water, and pretreat the mulberry leaf powder using microwave pretreatment at a power of 700W for 12 minutes. Heat the mulberry leaf suspension to 55℃, add 0.7% cellulase, and hydrolyze for 1 hour. Add sodium hydroxide solution to adjust the pH to 7.0, then add 1% neutral protease and hydrolyze for 3 hours. Add sodium hydroxide solution again to adjust the pH to 9.0, then add 0.6% chymotrypsin and 0.6% alkaline protease, and extract for 1 hour. Add 30g of pure water, cool to 30℃, and extract for 30 minutes. Centrifuge at 3000 rpm for 20 minutes, collect the supernatant, and determine the protein extraction rate using the free amino acid method; the rate is 43.42%. Desalt, concentrate, and dry the supernatant to obtain 5.99g of mulberry leaf protein extract, with an extract yield of 39.93%.

[0112] The main conditions, protein extraction rate, protein extract quality, and extract yield of Examples 1-4 and Comparative Examples 1-5 are shown in the table below:

[0113] Table 1

[0114] The amino acid composition of the mulberry leaf protein extracts prepared in Examples 1-4 and Comparative Examples 1-2 was determined by amino acid analysis and is shown in Table 2 below.

[0115] Table 2. Amino acid composition of each embodiment or comparative example. Note: Superscript a indicates essential amino acids for adults, and superscript b indicates branched-chain amino acids. EAA: Essential amino acids; NEAA: Non-essential amino acids; TAA: Total amino acids; BCAA: Branched-chain amino acids.

[0116] Results analysis:

[0117] 1. As can be seen from Table 2, the mulberry leaf protein extracted in Examples 1-4 of this invention has a relatively complete range of amino acids, including 7 of the 8 essential amino acids for adults, and its amino acid composition is similar to that of hydrolyzed soybean protein and whey protein.

[0118] The FAO / WHO specifies that the ideal amino acid ratio in a protein should meet the following requirements: the essential amino acid ratio (E / T) should be around 40% of the total amino acids, and the essential amino acid / non-essential amino acid ratio (E / N) should be above 0.6. Table 2 shows that the E / T and E / N values ​​of the mulberry leaf proteins extracted in Examples 1-4 are close to or meet the requirements for ideal proteins.

[0119] Branched-chain amino acids (BCAAs) refer to leucine, valine, and isoleucine. These amino acids promote anabolism (muscle growth) in two specific ways: ① by promoting insulin release, and ② by promoting growth hormone release. The most important BCAA is leucine, the precursor to ketoisocaproic acid (KIC) and hemoglobin (HMB). KIC and HMB can increase muscle mass, reduce fat, and provide nutrients to the body. Whey protein has a high BCAA content, around 26%.

[0120] As shown in Table 2, the mulberry leaf protein samples from Examples 1-4 contain more than 22% branched-chain amino acids. In comparison, the hydrolyzed soybean protein contains 11.70%, and whey protein contains 25.41%. The proportion of branched-chain amino acids in the mulberry leaf protein from Examples 1-4 is comparable to that of whey protein. While the proportion of branched-chain amino acids in the mulberry leaf protein prepared by the extraction process in Comparative Examples 1-2 is not as high as that of whey protein, it is higher than that of hydrolyzed soybean protein.

[0121] The glycine and alanine content in Examples 1-4 was also higher than that in the comparative soy hydrolyzed protein and whey protein. Glycine and alanine are also two types of amino acids that promote muscle growth and repair.

[0122] 2. Combining the results in Table 1 and Table 2, we can see that:

[0123] The extraction methods of Examples 1-4 of this invention achieve a protein extraction rate of 56-68%. Examples 1 and 2 employ a single enzymatic hydrolysis, achieving a protein extraction rate of approximately 56%; Examples 3 and 4 employ a two-stage enzymatic hydrolysis, achieving a protein extraction rate of over 62%. Therefore, the extraction method of this invention not only yields mulberry leaf protein with an amino acid composition meeting ideal protein requirements but also achieves a high protein extraction rate.

[0124] Compared to Examples 1-4, the method in Comparative Examples 1-2 differs in the type of protease used; the protease used does not contain chymotrypsin. Although the protease extraction rate is higher in Comparative Examples 1-2, the branched-chain amino acid content in the prepared mulberry leaf protein is lower than that in whey protein.

[0125] Compared to Example 2, the method in Comparative Example 3 differs in that the liquid after the first extraction is centrifuged, and the filter residue or precipitate is used as the raw material for the second extraction. The protein extraction rates of Example 2 and Comparative Example 3 are basically the same. This indicates that the simplified process of directly adding water for the second extraction based on the first extraction in the extraction method of the present invention is feasible. This method not only improves the extraction rate of mulberry leaf protein but also has extremely high cost-effectiveness.

[0126] Compared with Example 3, Comparative Example 4 added a step of ultrasonic pretreatment of mulberry leaf powder, but the protein extraction rate decreased instead of increasing, indicating that ultrasonic pretreatment has no promoting effect on the extraction method of the present invention.

[0127] Compared with Example 3, Comparative Example 5 added a step of microwave pretreatment of mulberry leaf powder, but the protein extraction rate decreased, indicating that microwave pretreatment did not promote the extraction method of the present invention.

[0128] Based on the above data, an efficacy experiment was designed to verify the "muscle building and fat reduction" efficacy of the mulberry leaf protein of this invention.

[0129] Example 1: Evaluation of the fat-reducing efficacy of mulberry leaf protein extract

[0130] Experimental animals: 6-week-old male C57BL / 6J mice;

[0131] Experimental Methods: Mice were fed a high-fat diet (D12492) containing 60 kcal% fat for four weeks to establish a high-fat model. Mice showing significant weight gain were selected and randomly divided into three groups of eight mice each: model group, experimental group 1 (mulberry leaf protein extract prepared in Example 1), and experimental group 2 (mulberry leaf protein extract prepared in Comparative Example 1). Eight mice were fed a normal diet (without high-fat modeling) as a control group. All four groups of mice were fed normally for eight weeks. Mice in experimental groups 1 and 2 were given 500 mg / kg of the corresponding mulberry leaf protein extract daily, respectively. The model group and control group were given the same amount of the corresponding solvent (both were physiological saline; the required amount of extract was dissolved in physiological saline and administered by gavage). Weight was measured before the experiment, at week 4, and at week 8. After the experiment, liver weight, abdominal fat weight, and subcutaneous fat weight were measured.

[0132] result:

[0133] 1. The test results of the weight change of mice in each group are shown in Table 3.

[0134] Table 3

[0135] The symbol marked with ## indicates that the p-value is less than 0.01 compared to the blank group, and the symbol marked with ** indicates that the p-value is less than 0.01 compared to the model group.

[0136] As shown in Table 3, there was no significant difference in the initial weight of the mice. At weeks 4 and 8, compared with the model group, the weight of mice in group 1 was significantly reduced (P < 0.01), while group 2 did not show any weight loss effect.

[0137] 2. The results of measuring the liver weight, abdominal fat weight, and subcutaneous fat weight of mice in each group are shown in Table 4.

[0138] Table 4

[0139] The symbol marked with ## indicates that the p-value is less than 0.01 compared to the blank group, and the symbol marked with ** indicates that the p-value is less than 0.01 compared to the model group.

[0140] Compared with the control group, the abdominal fat weight and subcutaneous fat weight of mice in the model group were significantly increased (P < 0.01). Compared with the model group, the liver weight, abdominal fat weight and subcutaneous fat weight of group 1 were significantly decreased (P < 0.05). There were no significant changes in liver, abdominal fat and subcutaneous fat weight in group 2.

[0141] Experiment Example 2: Evaluation of the efficacy of mulberry leaf protein extract in preventing muscle atrophy in mice

[0142] Experimental animals: 10-week-old male C57BL / 6J mice;

[0143] Experimental Methods: Thirty-two mice were randomly divided into four groups of eight each: a control group, a model group, experimental group 1 (mulberry leaf protein extract prepared in Example 1), and experimental group 2 (mulberry leaf protein extract prepared in Comparative Example 1). Mice in the model group and experimental groups 1-2 were housed in pairs per cage. After weighing, mice were anesthetized by intraperitoneal injection of Sutacetin 50 (0.05 ml / 10 g). After anesthesia took effect, the mice's tails were fixed to the top of the cage at a 30° angle to the ground. Once the animals had fully recovered from anesthesia, the suspension angle was adjusted to allow them to fully extend their legs and ensure their hind limbs did not touch the ground. The overall health of the mice and whether the tail cap had fallen off were observed daily. Mice were suspended for 14 days, with free access to food and fresh pure fruit jelly provided daily to ensure adequate water intake. Tail color and the mice's vital signs were observed daily in the morning and afternoon. Administration of medication began 14 days before model establishment and continued for 28 days. Mice in experimental groups 1-2 were administered the corresponding mulberry leaf protein extract at 500 mg / kg daily. The model group and the control group were given the same amount of the corresponding solvent (both solvents were physiological saline; during feeding, the required amount of extract was dissolved in physiological saline and then administered by gavage). The grip strength of the limbs was measured using an electronic balance to weigh and record the body weight of the four groups of mice. The grip strength of the mice's limbs was measured using a mouse grip strength meter (measured 3 times, and the average value was taken), and the grip strength / body weight ratio (g / g) was calculated. At the end of the experiment, the skeletal muscles of the mouse's hind limbs (gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, and quadriceps femoris) were collected, weighed, and the skeletal muscle wet weight / body weight ratio (mg / g) was calculated.

[0144] result:

[0145] 1. The results of the limb grip strength test in each group of mice are shown in Figure 1. Compared with the blank group, the limb grip strength of the model group mice was significantly reduced (P < 0.05). Compared with the model group, the mulberry leaf protein extract in group 1 significantly increased the limb grip strength (P < 0.01). The mulberry leaf protein extract in group 2 had no significant effect on the limb strength of the animals.

[0146] 2. The test results of hind limb skeletal muscle in each group of mice are shown in Table 5.

[0147] Table 5

[0148] A # indicates P < 0.05 compared to the blank group, and an * indicates P < 0.05 compared to the model group.

[0149] Compared with the control group, the hind limb skeletal muscle mass of mice in the model group was significantly reduced (P < 0.05). Compared with the model group, the mass of the gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, and quadriceps femoris muscles in group 1 was significantly increased (P < 0.05). There was no significant change in hind limb muscle mass in experimental group 2.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for extracting protein from mulberry leaves, characterized in that, Includes the following steps: (1) Pretreatment: Pulverize the dried mulberry leaves to obtain mulberry leaf powder; (2) First extraction: Add water to mulberry leaf powder, add cellulase under certain temperature conditions, stir and extract; add alkaline solution to control the pH of the system, add protease, and continue extraction; (3) Second extraction: Add water, cool down, and extract; (4) Centrifuge the extract obtained in (3), take the supernatant to obtain crude mulberry leaf protein extract, then separate and purify it, dry it to obtain mulberry leaf protein extract.

2. The method for extracting mulberry leaf protein according to claim 1, characterized in that, In step (2), the protease includes chymotrypsin; Preferably, the protease further includes one or more of alkaline protease, neutral protease, and trypsin.

3. The method for extracting mulberry leaf protein according to claim 2, characterized in that, In step (2), when adding the protease, the pH of the system is controlled to be 7.0-10.0; Preferably, when using neutral protease and / or trypsin, the pH of the system is controlled at 7.0-7.5; Preferably, when using alkaline protease and / or chymotrypsin, the pH of the system is controlled to be 9.0-10.

0.

4. The method for extracting mulberry leaf protein according to any one of claims 1-3, characterized in that, In step (2), the amount of protease added is 0.5-5.0% of the mass of mulberry leaf powder; Preferably, in step (2), after adding the protease, extraction is carried out for 4-6 hours.

5. The method for extracting mulberry leaf protein according to any one of claims 1-3, characterized in that, In step (2), the amount of cellulase added is 0.5-2.0% of the mass of mulberry leaf powder; Preferably, in step (2), after adding cellulase at a temperature of 50-60℃, the mixture is stirred and extracted for 1-2 hours. Preferably, in step (2), 8-12 times the mass of water is added to the mulberry leaf powder.

6. The method for extracting mulberry leaf protein according to any one of claims 1-3, characterized in that, In step (3), the water added is 2-6 times the mass of the mulberry leaf powder; Preferably, in step (3), the temperature is lowered to 20-40℃; Preferably, in step (3), after cooling, extraction is performed for 0.5-1h.

7. The method for extracting mulberry leaf protein according to any one of claims 1-3, characterized in that, In step (1), the moisture content of the dried mulberry leaves is 5-10%; Preferably, the moisture content of the dried mulberry leaves is 5-7%.

8. The method for extracting mulberry leaf protein according to any one of claims 1-3, characterized in that, In step (4), the centrifugation speed is 3000-5000 r / min and the centrifugation time is 20-40 min.

9. A mulberry leaf protein extract, characterized in that, The amino acid composition of the mulberry leaf protein extract has an essential amino acid / total amino acid mass percentage ≥30% and an essential amino acid / non-essential amino acid mass percentage ≥43%. Branched-chain amino acids account for ≥20% of the total amino acid mass; Preferably, the mass percentage of essential amino acids to total amino acids is ≥35%, and the mass percentage of essential amino acids to non-essential amino acids is ≥54%. Preferably, in the mulberry leaf protein extract, glycine accounts for ≥7% of the total amino acids by mass. Preferably, in the mulberry leaf protein extract, alanine accounts for ≥7% of the total amino acids by mass. Preferably, the mulberry leaf protein extract is prepared using the extraction method described in any one of claims 1-8.

10. The use of the mulberry leaf protein extract as described in claim 9 in the preparation of fat-reducing products and / or muscle-building products.

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