A method for preparing polypeptides and water-soluble sugars from low-temperature peanut cake meal

Abstract

The application belongs to the technical field of deep processing of agricultural products, and particularly relates to a method for co-production of peanut polypeptide and water-soluble sugar by using cold-pressed peanut cake meal, and specifically comprises the following steps: taking low-temperature peanut cake meal as raw material, crushing and deoiling, extruding and puffing, microwave-ultrasonic-assisted acidolysis, multi-enzyme hydrolysis, centrifugation, resin separation, ultrafiltration concentration and drying to obtain polypeptide and water-soluble sugar. The above preparation method has the advantages of high raw material utilization rate, high production efficiency and good product quality. The method improves the processing added value of the cold-pressed peanut cake meal, expands the application range of the cold-pressed peanut cake meal, and has positive social and economic value.

Description

Technical Field

[0001] This invention belongs to the field of deep processing technology of agricultural and sideline products, specifically relating to a method for co-producing peanut polypeptides and water-soluble sugars, and particularly to a method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal; the method prepares peanut polypeptides and water-soluble sugars through processes such as extrusion puffing, microwave-assisted acid hydrolysis, multi-enzyme hydrolysis, resin separation, membrane filtration, and drying. Background Technology

[0002] Peanuts are one of the world's important oilseed crops, and my country is a major producer and consumer of peanuts, ranking among the world's top producers. However, domestic peanut production falls short of demand, leading to a continuous increase in peanut imports. In 2024, imports of peanuts and peanut products reached 1.9184 million tons. Therefore, developing the peanut industry plays a positive role in ensuring my country's oil supply and market consumption.

[0003] Currently, peanuts in my country are mainly used for oil extraction (oil extraction accounts for about 50% of total peanut consumption). The oil extraction process produces peanut meal, with my country generating approximately 4.5 million tons of peanut meal annually. Depending on the peanut oil processing method, two types of peanut meal are produced: high-temperature peanut meal and low-temperature peanut meal. High-temperature peanut meal is a byproduct of producing aromatic peanut oil. Due to the high-temperature (usually above 160℃) roasting process, peanut protein undergoes severe denaturation and produces harmful components. Low-temperature peanut meal is a byproduct of producing low-temperature (usually around 70℃) pressed peanut oil. Because it is not subjected to high-temperature treatment, the peanut protein has a lower degree of denaturation and higher biological activity. Compared to high-temperature peanut meal, low-temperature peanut meal is of higher quality and contains fewer harmful components, making it more suitable for food processing. Studies have shown that low-temperature peanut meal is rich in nutrients, containing over 40% protein and about 30% carbohydrates (mainly dietary fiber). Effective utilization of low-temperature peanut meal not only helps increase my country's protein and carbohydrate supply but also increases the profits of oil processing enterprises and enriches consumer products.

[0004] Current methods for utilizing peanut meal primarily focus on peanut protein, with extraction mainly achieved through alkali dissolution and acid precipitation. Furthermore, the utilization of sugars in peanut meal is neglected. These processing methods not only increase subsequent pollution control investment but also pose safety risks to the product. Simultaneously, they fail to utilize sugar resources, reducing raw material utilization and impacting enterprise revenue. Therefore, it is necessary to develop a technology that can simultaneously utilize both protein and sugars in peanut meal. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the existing technology and provide a method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal. This method has the advantages of being green and environmentally friendly, efficient and fast, with high raw material utilization and good product quality.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal includes the following steps: 1) Preprocessing: After crushing and degreasing the low-temperature peanut meal, defatted low-temperature peanut meal powder is obtained. 2) Extrusion puffing: The defatted low-temperature peanut meal obtained in step 1) is added to acidic water and sealed and balanced at below 30°C for 8-12 hours. Then, it is extruded and expanded at 135-150°C with a screw speed of 300-400 rpm using a twin-screw extruder to obtain extruded and expanded defatted low-temperature peanut meal. 3) Microwave-ultrasound assisted acid hydrolysis: The extruded, defatted, and low-temperature peanut meal obtained in step 2) is pulverized, water is added to make the mass concentration of the meal powder 5~10% (w / w), the pH is adjusted to 0.5~1.5, microwave ultrasonic treatment is performed for 3~10 min, followed by water bath heating at 75~90℃ for 20~60 min to obtain hydrolysate; During the water bath heating process, the sample is sealed with nitrogen and the stirring speed is 100~300 r / min; 4) Multi-enzyme hydrolysis: Cool the hydrolysate obtained in step 3) to 35~55℃, adjust the pH to 2~4, add mixed enzymes and enzymatically hydrolyze for 60~180 min to obtain the enzymatic hydrolysate; 5) Resin separation: Neutralize the enzymatic hydrolysate obtained in step 4), centrifuge (3500~5000 r / min for 10~30 min), collect the supernatant, add the supernatant to a macroporous resin column, collect the first permeate, and load 1 bed volume (BV) of distilled water, collect the second permeate, and combine the two permeates; add the permeate to an ion exchange resin column, collect the first permeate, and load 1 bed volume of distilled water onto the ion exchange resin, collect the second permeate, combine the two permeates, concentrate under reduced pressure, and dry to obtain water-soluble sugar; The macroporous resin column was eluted with ethanol solution, the eluent was collected, and concentrated by rotary evaporation. In this study, both macroporous resin and ion exchange resin adsorption were performed using dynamic adsorption. The bed volume (BV) to feed volume ratio of macroporous resin was 1~2:1, the bed volume to feed volume ratio of ion exchange resin was 0.1~0.5:1, and the diameter-to-height ratio of the resin column was 1:3~12. The loading rate was 0.5~2.5 BV / h, and the elution rate was 0.5~3 BV / h. The vacuum concentration conditions for water-soluble sugars are as follows: heating temperature 65~85℃, vacuum degree -0.05 MPa ~ -0.09 MPa, and concentration to 20%~50% of the original volume; the water-soluble sugars are dried by rotating scraper drying, the sugar solution is preheated to 50~60℃, the surface temperature of the drum is controlled at 80~120℃, and the drum speed is set to 3~9 r / min. 6) Nanofiltration separation: The obtained concentrate was added to water to the initial enzymatic hydrolysate volume, concentrated by nanofiltration, and spray-dried to obtain the peptide.

[0007] Specifically, in step 1), the low-temperature peanut meal obtained by cold pressing after removing the red skin is pulverized to 40-100 mesh. After solvent extraction to remove oil, the residual oil content in the defatted low-temperature peanut meal powder is <1%. The solvent extraction uses diethyl ether, petroleum ether (boiling range 30-60 ℃), or n-hexane as the extractant, and the extraction is carried out for 6-10 h at an extraction temperature of 40-80 ℃. The solvent volume to material ratio is 4-12 L:1kg.

[0008] Specifically, in step 2), the acidic water used is weakly acidic electro-generated functional water with a pH of 2.7 to 5.0 and an effective chlorine concentration of 10 to 60 mg / L; the amount of acidic water added is 10 to 20% (w / w) of the mass of defatted low-temperature peanut meal powder.

[0009] Specifically, in step 3), the extruded, puffed, defatted, low-temperature peanut meal obtained in step 2) is pulverized to 60-140 mesh; the microwave and ultrasonic treatment conditions are as follows: microwave and ultrasound are used simultaneously, the ultrasonic frequency is 40±10 kHz, the ultrasound is a focused ultrasonic generator, and the ultrasonic power to meal mass ratio is 40-60 W: 1 kg; the microwave treatment is in constant temperature mode, and the temperature is controlled at 80-95℃; during the microwave and ultrasonic treatment, continuous stirring is carried out at a stirring speed of 100-300 r / min.

[0010] Specifically, in step 4), the mixed enzyme used consists of mixed enzyme A and mixed enzyme B. Mixed enzyme A consists of four or five enzymes selected from cellulase, hemicellulase, β-glucosidase, pectinase, and amylase, while mixed enzyme B consists of two or three enzymes selected from acidic protease, pepsin, and papain. The amount of mixed enzyme added is 0.1% to 1.0% of the hydrolysate mass. The mass ratio of mixed enzyme A to mixed enzyme B is 1 to 3:1.

[0011] Furthermore, in step 4), the enzyme addition order is as follows: first add mixed enzyme A, then add mixed enzyme B; the enzymatic hydrolysis time of mixed enzyme A alone accounts for 1 / 5 to 1 / 3 of the total time; in mixed enzyme A, the content of cellulase, pectinase and amylase is not less than 30%, 10% and 20% respectively by mass percentage.

[0012] Specifically, in step 5), the macroporous resin used is a mixture of weakly polar macroporous resin and moderately polar macroporous resin in a ratio of 2-5:1; the ion exchange resin is a mixture of anion exchange resin and cation exchange resin in a ratio of 1-2:1. The concentration of the ethanol solution used as the eluent is 50-80% (v / v). The macroporous resin column is eluted with 2-4 bed volumes of 50-80% ethanol solution, and then concentrated by rotary evaporation to 0.3-0.6 bed volumes. Weakly polar macroporous resins can be AB-8, DM130, ADS-8, etc.; moderately polar macroporous resins can be HPD-400, DM301, ADS-17, etc. Anion exchange resins can be 717, D301, YKW204, and cation exchange resins can be 732, D001, YKW801, etc.

[0013] Specifically, in step 6), nanofiltration concentrates the solution to 10%–25% of the initial enzymatic hydrolysate volume, and the nanofiltration membrane used has a molecular weight cutoff of 200–300 Da. The inlet air temperature for spray drying is 140–200°C, and the outlet air temperature is 60–100°C.

[0014] This invention provides polypeptides and water-soluble sugars prepared using the above-described preparation method.

[0015] The present invention also provides the use of the above-mentioned polypeptide in the preparation of antioxidant drugs or the use of the water-soluble sugar as a food additive.

[0016] Currently, the enzymatic hydrolysis of proteins into peptides and the conversion of water-insoluble sugars into water-soluble sugars are hot topics in agricultural product processing. Therefore, the enzymatic hydrolysis technology for degrading proteins and sugars in low-temperature peanut meal into peptides and water-soluble oligosaccharides is of research value. However, peanut proteins and sugars are nested and tightly connected, and proteins are mostly tightly folded, while water-insoluble sugars (especially cellulose) have a crystalline structure. These factors hinder the enzymatic hydrolysis reaction and limit the application of peanut meal co-production of peanut peptides and water-soluble sugars. This study found that extrusion puffing can destroy the structure of proteins and sugars, exposing enzyme cleavage sites and improving the efficiency of enzymatic hydrolysis. Microwave and ultrasound-assisted acid hydrolysis utilizes microwave energy to directly act on polar molecules (such as water molecules) and ions (such as H⁺) in the reaction system, causing them to vibrate and rotate at high speed in the microwave field. This generates intense friction and collisions at the molecular level, efficiently converting electromagnetic energy into heat energy, uniformly raising the temperature of the reaction system, providing sufficient activation energy for the acid hydrolysis reaction, and thus accelerating the reaction. In addition, ultrasound destroys the structure of proteins and polysaccharides through cavitation, increasing porosity and improving the acid hydrolysis rate. Multi-enzyme hydrolysis can target more enzymatic sites than single-enzyme hydrolysis, thus promoting mutual enhancement and improving hydrolysis efficiency. Therefore, the integrated innovation of the above methods to establish a method for co-producing peanut polypeptides and water-soluble sugars using low-temperature peanut meal is theoretically feasible. Furthermore, extrusion puffing, microwave-assisted ultrasonic hydrolysis, and multi-enzyme hydrolysis are all supported by appropriate equipment, making the development of the above technologies practically feasible.

[0017] This invention integrates extrusion puffing, microwave-ultrasound-assisted acid hydrolysis, multi-enzyme hydrolysis, and nanofiltration technologies. Extrusion puffing effectively breaks down the cellulose crystal structure, creating pores and exposing enzymatic hydrolysis sites, providing favorable conditions for subsequent acid and enzymatic hydrolysis. Microwave-ultrasound-assisted acid hydrolysis utilizes the "volume heating" effect of microwaves and the cavitation effect of ultrasound to lower the activation energy of acid hydrolysis and disrupt the structure of carbohydrates and proteins, thereby accelerating the acid hydrolysis reaction. Multi-enzyme hydrolysis utilizes the synergistic effect of carbohydrate hydrolases and proteolytic enzymes to mutually promote the destruction of carbohydrate and protein structures, driving the generation of water-soluble sugars and peptides. Nanofiltration removes free amino acids from peptides, achieving simultaneous separation and concentration. The integrated application of these technologies effectively improves production efficiency and raw material utilization while reducing production costs and pollution control costs, providing a new approach for the resource utilization of low-temperature peanut meal.

[0018] The method of this invention has advantages such as high raw material utilization, high production efficiency, and good product quality. This method increases the added value of cold-pressed peanut cake and expands its application range, thus possessing positive socio-economic value. Compared with existing technologies, the advantages and beneficial effects of this invention are as follows: 1) The method of the present invention simplifies the process flow, eliminates the protein extraction step, shortens the extraction time, improves production efficiency, and thus reduces production costs; 2) The method of the present invention effectively utilizes the sugar resources in peanut meal through microwave and ultrasound-assisted acid hydrolysis and multi-enzyme hydrolysis, thereby improving the utilization rate of peanut meal; 3) The method of this invention applies extrusion puffing and microwave-ultrasound assisted acid hydrolysis technology to the pretreatment of peanut cake, which effectively destroys the structure of protein and carbohydrate complex in low-temperature peanut cake, improves the enzymatic hydrolysis effect, increases the utilization rate of raw materials, and helps enterprises increase revenue; 4) The polypeptides prepared by the method of the present invention have antioxidant activity and increased sweetness of water-soluble sugars, and have broad application prospects. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0020] In the following examples, all raw materials used are common commercially available products that can be purchased directly, or can be prepared using conventional techniques in the art.

[0021] For example, the cellulase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 10,000 U / g; The hemicellulase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 5000 U / g; β-glucosidase was purchased from Amano Enzyme Preparations (Jiangsu) Co., Ltd. Shanghai Branch, with an enzyme activity of 1200 U / g. Pectinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 50,000 U / g; The amylase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 1500 U / g. The acidic protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 50,000 U / g. The pepsin was purchased from Shanghai Baoman Biotechnology Co., Ltd., with an enzyme activity of 3000 U / g; Papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g.

[0022] Weakly polar macroporous resins: AB-8 (Shanghai McLean Biochemical Technology Co., Ltd.), DM130 (Shanghai McLean Biochemical Technology Co., Ltd.), ADS-8 (Tianjin Guangfu Technology Development Co., Ltd.).

[0023] Medium polar macroporous resins: HPD-400 (Beijing Ruida Henghui Technology Development Co., Ltd.), DM301 (Jining Fangyu Chemical Co., Ltd.), ADS-17 (Beijing Solarbio Technology Co., Ltd.).

[0024] Anion exchange resins: 717 (Sinopharm Chemical Reagent Co., Ltd.), D301 (Tianjin Yunkai Resin Technology Co., Ltd.), YKW204 (Tianjin Yunkai Resin Technology Co., Ltd.).

[0025] Cation exchange resins: 732 (Sinopharm Chemical Reagent Co., Ltd.), D001 (Tianjin Yunkai Resin Technology Co., Ltd.), YKW801 (Tianjin Yunkai Resin Technology Co., Ltd.). Example 1

[0026] A method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal includes the following steps: 1) Preprocessing Take 10 kg of low-temperature peanut meal obtained by cold pressing after removing the red skin, grind it to 60 mesh, and then perform solvent extraction (hexane as the extractant, volume 80 L) at 75 ℃ for 6 h to obtain defatted low-temperature peanut meal powder (oil content 0.3%).

[0027] 2) Extrusion puffing The defatted low-temperature peanut meal obtained in step 1) was added to 1.5 kg of acidic electro-generated functional water (pH 3.0, effective chlorine concentration 30 mg / L), and after being sealed and balanced at 25 ℃ for 10 h, it was extruded and expanded in a twin-screw extruder at a screw speed of 350 rpm and an extrusion temperature of 140 ℃ to obtain extruded and expanded defatted low-temperature peanut meal.

[0028] 3) Microwave and ultrasonic-assisted acid hydrolysis The extruded, defatted, low-temperature peanut meal obtained in step 2) was pulverized to 80 mesh. Water was added to make the meal powder concentration 7% (w / w). After stirring evenly at 150 r / min, the pH was adjusted to 1 with HCl solution. Under continuous stirring, microwave and ultrasonic treatment were applied simultaneously for 10 min. The ultrasonic frequency was 40 kHz, and the ultrasonic power to meal mass ratio was 60 W:1 kg (i.e., ultrasonic power 600 W). The microwave treatment was performed in a constant temperature mode, with the temperature controlled at 85℃. The treated liquid was transferred to a reaction vessel and heated in a water bath at 85℃ for 40 min. During the water bath heating process, the vessel was sealed with nitrogen and stirred continuously at 150 r / min to obtain a hydrolysate.

[0029] 4) Multi-enzyme hydrolysis The hydrolysate was rapidly cooled to 45°C, and the pH was adjusted to 3 with HCl solution. 80 g of mixed enzyme A (composed of cellulase, hemicellulase, β-glucosidase, pectinase, and amylase, with concentrations of 40%, 10%, 10%, 10%, and 30% respectively) was added, and hydrolysis was carried out for 60 min. Subsequently, 80 g of mixed enzyme B (composed of acidic protease and pepsin in a 6:4 ratio) was added, and hydrolysis was carried out for 120 min to obtain the enzymatic hydrolysate. The amount of mixed enzyme added was 0.114% of the hydrolysate mass.

[0030] 5) Resin separation After neutralizing the enzymatic hydrolysate obtained in step 4) with NaOH solution, centrifuge at 4000 r / min for 15 min to obtain 125 L of supernatant. Load the supernatant onto a macroporous resin column (BV 125 L, composed of weakly polar macroporous resin DM130 and medium-polarity macroporous resin HPD-400 in a mass ratio of 4:1; column diameter 36 cm, height 180 cm) at a loading rate of 0.5 BV / h. During loading, collect the first permeate. Subsequently, add 125 L of distilled water at an elution rate of 1 BV / h to collect the second permeate. Combine the two permeates.

[0031] The permeate was added to an ion exchange resin column (70 L BV, composed of cation exchange resin D001 and anion exchange resin D301 in a 1:1 mass ratio, with a column diameter of 30 cm and a height of 140 cm). The first permeate was collected, followed by elution of the ion exchange resin column with 70 L of distilled water at a rate of 1 BV / h. The second permeate was collected, and the two permeates were combined. The column was then concentrated under reduced pressure at 80 °C and a vacuum of -0.08 MPa to 70 L (approximately 25% of the initial volume). The volume was then cooled to 60 °C and dried using a rotary scraper (drying temperature of 90 °C and drum speed of 5 r / min). 1.62 kg of water-soluble sugars were collected.

[0032] The macroporous resin column was eluted with 300 L of 75% (v / v) ethanol solution at a rate of 1.5 BV / h. The eluent was collected and concentrated to 40 L by rotary evaporation at 75 °C.

[0033] 6) Nanofiltration separation Add distilled water to the concentrate obtained in step 5) to the initial enzymatic hydrolysate volume, concentrate using nanofiltration (nanofiltration membrane molecular weight cutoff 300 Da) to 20% of the initial enzymatic hydrolysate volume, and spray dry (inlet air temperature 160 ℃, outlet air temperature 70 ℃) to obtain 1.89 kg of peptide. Example 2

[0034] A method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal includes the following steps: 1) Preprocessing Take 1 kg of low-temperature peanut meal obtained by cold pressing after removing the red skin, grind it to 80 mesh, then add 10 L of petroleum ether (boiling range 30~60℃) for solvent extraction, extract and reflux at 60℃ for 8 h, recover the solvent, and obtain 0.92 kg of defatted low-temperature peanut meal powder (oil content 0.2%).

[0035] 2) Extrusion puffing Add the defatted low-temperature peanut meal powder obtained in step 1) to 0.18 kg of acidic electro-generated functional water (pH 2.7, effective chlorine concentration 50 mg / L), seal and stabilize at 25 ℃ for 10 h, then feed it into a twin-screw extruder and extrude it at a screw speed of 400 r / min and 150 ℃ to obtain extruded and extruded defatted low-temperature peanut meal.

[0036] 3) Microwave and ultrasonic-assisted acid hydrolysis The 0.85 kg of extruded, defatted, low-temperature peanut meal obtained in step 2) was crushed to 120 mesh. Water was added to adjust the meal powder concentration to 5% (w / v). After stirring evenly at 200 r / min, the pH was adjusted to 1.5 with H2SO4 solution. Under continuous stirring, the mixture was added to a microwave ultrasonic extractor. Microwave and ultrasonic treatment were carried out at 200 r / min for 10 min with stirring. The microwave treatment was set to constant temperature mode, with the temperature set at 90 ℃, the ultrasonic frequency at 40 kHz, and the ultrasonic power to meal mass ratio at 50 W: 1 kg (i.e., ultrasonic power 50 W). The treated liquid was added to a sealed reaction vessel and stirred at 200 r / min. The mixture was heated in a 90 ℃ water bath for 60 min. During the water bath heating process, nitrogen was used to seal the vessel to obtain the hydrolysate.

[0037] 4) Multi-enzyme hydrolysis The hydrolysate obtained in step 3) was rapidly cooled to 50°C, and the pH was adjusted to 3.5 with HCl solution. 17.85 g of a mixed enzyme was added and hydrolyzed for 150 min. The mixed enzyme consisted of 11.9 g of mixed enzyme A and 5.95 g of mixed enzyme B. First, mixed enzyme A (composed of cellulase, β-glucosidase, pectinase, and amylase, with contents of 45%, 10%, 15%, and 30% respectively) was added and hydrolyzed for 30 min. Then, mixed enzyme B (composed of 60% acidic protease and 40% papain, by mass percentage) was added and hydrolyzed for 120 min to obtain the hydrolysate. The amount of mixed enzyme added was 0.105% of the hydrolysate mass.

[0038] 5) Resin separation The enzymatic hydrolysate obtained in step 4) was neutralized and centrifuged at 5000 r / min for 25 min. The supernatant was collected to obtain 16 L of supernatant. The supernatant was loaded onto a macroporous resin column (BV 24 L, macroporous column size 20 cm × 100 cm, column packed with weakly polar macroporous resin ADS-8 and medium polar macroporous resin ADS-17 in a mass ratio of 4:1) at a rate of 0.6 BV / h. The first permeate was collected. Subsequently, the macroporous resin column was eluted with 24 L of distilled water at a rate of 1 BV / h. The second permeate was collected. The two permeates were combined to obtain a total of 36 L.

[0039] The permeate was added to an ion exchange column (BV = 7.2 L, composed of anion exchange resin YKW204 and cation exchange resin YKW801 in equal mass ratio, column size 14 cm × 60 cm) at a rate of 2 BV / h. The first permeate was collected, and the column was then washed with 7.2 L of distilled water at a rate of 1 BV / h. The second permeate was collected, and the two permeates were combined to obtain a total of 38.5 L. The permeate was then concentrated to 8 L under reduced pressure at 75 °C and a vacuum of -0.08 MPa, cooled to 60 °C, and dried using a rotary scraper dryer at 90 °C and a drum speed of 6 r / min to obtain 0.132 kg of water-soluble sugars.

[0040] The macroporous resin column was eluted with 48 L of 70% (v / v) ethanol solution at 1 BV / h. The eluent was collected and concentrated to 12 L by rotary evaporation at 75 °C.

[0041] 6) Nanofiltration separation Add distilled water to the concentrate obtained in step 5) to the initial enzymatic hydrolysate volume, and concentrate using a nanofiltration membrane with a molecular weight cutoff of 200 Da to 15% of the initial enzymatic hydrolysate volume. Spray dry at an inlet air temperature of 180 ℃ and an outlet air temperature of 80 ℃ to obtain 0.148 kg of peptide. Example 3

[0042] A method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal includes the following steps: 1) Preprocessing Take 4 kg of low-temperature peanut meal obtained by cold pressing after removing the red skin, grind it to 100 mesh, then add 32 L of diethyl ether and extract by reflux at 40℃ for 10 h for solvent extraction, recover the solvent, and obtain 3.7 kg of defatted low-temperature peanut meal powder (oil content 0.25%).

[0043] 2) Extrusion puffing Add the defatted low-temperature peanut meal powder obtained in step 1) to 0.70 kg of acidic electro-generated functional water (pH 3.5, effective chlorine concentration 20 mg / L), seal and stabilize at 30℃ for 12 h, then add it to a twin-screw extruder and extrude at a screw speed of 380 r / min and 145℃ to obtain extruded and extruded defatted low-temperature peanut meal.

[0044] 3) Microwave and ultrasonic-assisted acid hydrolysis The extruded, defatted, low-temperature peanut meal obtained in step 2) was pulverized to 140 mesh. Water was added to adjust the meal powder concentration to 10% (w / v). After stirring at 200 r / min until homogeneous, the pH was adjusted to 0.6 with H2SO4 solution. The mixture was then transferred to a microwave ultrasonic extractor and subjected to continuous stirring. Microwave and ultrasonic treatment were performed at 250 r / min for 8 min. The microwave was operated at a constant temperature of 95 ℃, the ultrasonic frequency was 40 kHz, and the ultrasonic power to meal mass ratio was 50 W: 1 kg (ultrasonic power 200 W). The treated liquid was then added to a sealed reaction vessel and stirred at 200 r / min. The mixture was heated in a 90 ℃ water bath for 40 min, with nitrogen purging and sealing during the water bath heating process, to obtain the hydrolysate.

[0045] 4) Multi-enzyme hydrolysis The 38 L hydrolysate obtained in step 3) was rapidly cooled to 55°C, and the pH was adjusted to 3.0 with NaOH solution. 76 g of a mixed enzyme was added and hydrolyzed for 140 min. The mixed enzyme consisted of 38 g of mixed enzyme A and 38 g of mixed enzyme B. First, mixed enzyme A (composed of cellulase, hemicellulose, pectinase, and amylase, with contents of 35%, 15%, 15%, and 35% respectively) was added and hydrolyzed for 40 min. Then, mixed enzyme B (containing pepsin, acidic protease, and papain at mass percentages of 25%, 45%, and 30% respectively) was added and hydrolyzed for 100 min to obtain the hydrolysate. The amount of mixed enzyme added was 0.2% of the hydrolysate mass.

[0046] 5) Resin separation The enzymatic hydrolysate obtained in step 4) was neutralized and centrifuged at 4800 r / min for 30 min. The supernatant was collected to obtain 35 L of supernatant. The supernatant was loaded onto a macroporous resin column (BV 40 L, column packed with weakly polar macroporous resin AB-8 and medium polar macroporous resin DM301 in a mass ratio of 7:3, column size 20 cm × 120 cm) at 0.5 BV / h. The first permeate was collected. Then, the macroporous resin column was eluted with 40 L of distilled water at a rate of 1 BV / h. The second permeate was collected. The two permeates were combined to obtain a total of 70 L.

[0047] The permeate was added to an ion exchange column (BV = 20 L, composed of anion exchange resin 717 and cation exchange resin 732 in equal mass ratio, column size 20 cm × 80 cm) at a rate of 1 BV / h. After collecting the first permeate, the ion exchange column was washed with 20 L of distilled water at a rate of 1 BV / h, and the second permeate was collected. The two permeates were combined to obtain a total of 82 L. The permeate was then concentrated to 20 L under reduced pressure at 80 °C and a vacuum of -0.08 MPa, cooled to 55 °C, and dried using a rotary scraper dryer at 85 °C and a drum speed of 9 r / min to obtain 0.532 kg of water-soluble sugars.

[0048] 80 L of 65% (v / v) ethanol solution was used to elute the macroporous resin column at 1 BV / h. The eluent was collected and concentrated to 18 L by rotary evaporation at 80 °C.

[0049] 6) Nanofiltration separation Add distilled water to the concentrate obtained in step 5) to the initial enzymatic hydrolysate volume, concentrate using a nanofiltration membrane with a molecular weight cutoff of 200 Da to 22% of the initial enzymatic hydrolysate volume, and spray dry at an inlet air temperature of 175 ℃ and an outlet air temperature of 65 ℃ to obtain 0.703 kg of peptide.

[0050] Molecular weight distribution of peptides and water-soluble sugars, determination of peptide antioxidant activity and sweetness of water-soluble sugars.

[0051] In the following tests, the molecular weight distribution of peptides and water-soluble sugars was determined according to the following procedures.

[0052] 1) Determination of polypeptide molecular weight distribution.

[0053] 10 mg of peptide was dissolved in ultrapure water to prepare a 0.05 mg / mL peptide solution. After dissolution, the sample was filtered through a 0.22 μm filter membrane, and the supernatant was collected into a 250 μL inner liner tube. The molecular weight distribution of the peptide was determined by volume exclusion chromatography (SEC). The determination conditions were as follows: column temperature 25℃, flow rate 0.7 mL / min, injection volume 20 μL, column BioCore SEC-300 (5 μm, 7.8 × 300 mm), mobile phase 150 mmol phosphate mobile phase (8.99 g anhydrous sodium dihydrogen phosphate, 10.65 g anhydrous disodium hydrogen phosphate to 1 L water), and detection wavelength UV 214 nm. The markers used for molecular weight determination were immunoglobulin G (molecular weight 150,000 Da), bovine serum albumin (BSA) (molecular weight 66,000 Da), erythrochrome c (molecular weight 12,365 Da), small peptide VLSATDKTNVLAAWGKVGGNAPAFGAEALERM (molecular weight 3,246 Da), and tyrosine (181 Da). The molecular weight of the samples was calculated using a regression equation of marker molecular weight versus retention time, and then the percentage of peptides with different molecular weight distributions was calculated based on peak area. The results are shown in Table 1.

[0054] As shown in Table 1, there are significant differences in the molecular weight distribution of Examples 1-3. This indicates that the present invention can adjust the extraction conditions to prepare polypeptides with different compositions, confirming that the technology of the present invention can produce a variety of polypeptide products and has wide applicability.

[0055] Table 1. Molecular weight distribution of peanut polypeptides prepared in the examples

[0056] 2) Determination of molecular weight distribution of carbohydrates.

[0057] 10 mg of carbohydrates were dissolved in ultrapure water to prepare a 0.1 mg / mL solution. After dissolution, the sample was filtered through a 0.45 μm filter membrane, and the supernatant was collected into a sample vial for detection using high-performance gel permeation chromatography (HPGPC). Chromatographic conditions: TSKgel® G5000PWXL gel permeation column (7.8 mm × 300 mm), column temperature 20 ℃, 0.002 mol / L sodium dihydrogen phosphate (containing 0.05% NaN3) as the mobile phase, flow rate 0.6 mL / min, and differential refractive index detector for carbohydrate detection. The molecular weight of carbohydrates was calculated based on the retention time-molecular weight standard curve of Dextran standard. The results are shown in Table 2.

[0058] As shown in Table 2, the water-soluble sugars prepared in Examples 1-3 are mainly monosaccharides and oligosaccharides, indicating that the present invention has a strong ability to degrade sugars in peanut meal and has a significant effect.

[0059] Table 2. Molecular weight distribution of peanut water-soluble sugars prepared in the examples

[0060] 3) Determination of peptide antioxidant activity.

[0061] The antioxidant activity of the peptides was evaluated using DPPH and ABTS inhibition rates, and the assay methods followed GB / T 39100-2020. The results are shown in Table 3.

[0062] As shown in Table 3, all the peptides obtained in the examples have antioxidant activity, among which the peptide obtained in Example 3 has the strongest antioxidant activity. This indicates that the antioxidant activity of peptides can be regulated by adjusting the enzymatic hydrolysis conditions of the protease.

[0063] Table 3 Antioxidant activity of peanut peptides prepared in the examples

[0064] Note: The peptide concentration was 2 mg / mL during the assay.

[0065] 4) Determination of the sweetness of water-soluble sugars.

[0066] Sensory evaluation was used, with a 5% (w / v) sucrose aqueous solution as the baseline (sweetness 1.0). Subsequently, water-soluble sugars prepared in the examples were prepared into 0.1%-25% (w / v) solutions, and the evaluators conducted blind tests to compare and find the concentration of water-soluble sugars that were equal to the baseline sweetness. The relative sweetness was calculated using Formula 1.

[0067] Relative sweetness = sucrose concentration / concentration of sugars of equal sweetness (1).

[0068] Since the relative sweetness of glucose is 0.7, the sweetness of the water-soluble sugar prepared by this invention is close to that of glucose, and therefore it can be used as a sweetener in food systems.

[0069] Table 4. Relative sweetness of peanut water-soluble sugars prepared in the examples

[0070] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal, characterized in that, Includes the following steps: 1) Pretreatment: The low-temperature peanut meal is crushed and deoiled to obtain defatted low-temperature peanut meal powder; 2) Extrusion puffing: The defatted low-temperature peanut meal powder obtained in step 1) is added to acidic water and sealed and balanced at below 30°C for 8~12 h, and then extruded and puffed at 135~150°C to obtain extruded and puffed defatted low-temperature peanut meal. 3) Microwave and ultrasonic-assisted acid hydrolysis: The extruded, puffed, defatted, low-temperature peanut meal obtained in step 2) is crushed, water is added to make the mass concentration of the meal powder 5~10%, the pH is adjusted to 0.5~1.5, microwave ultrasonic treatment is performed for 3~10 min, followed by heating in a water bath at 75~90℃ for 20~60 min to obtain hydrolysate; 4) Multi-enzyme hydrolysis: Cool the hydrolysate obtained in step 3) to 35~55℃, adjust the pH to 2~4, add mixed enzymes and hydrolyze for 60~180 min to obtain the enzymatic hydrolysate; 5) Resin separation: Neutralize the enzymatic hydrolysate obtained in step 4), centrifuge, collect the supernatant, add the supernatant to a macroporous resin column, collect the first permeate, and load 1 bed volume of distilled water onto the ion exchange resin, collect the second permeate, and combine the two permeates; add the permeate to an ion exchange resin column, collect the first permeate, and load 1 bed volume of distilled water onto the ion exchange resin, collect the second permeate, combine the two permeates, concentrate under reduced pressure, and dry to obtain water-soluble sugar; The macroporous resin column was eluted with ethanol solution, the eluent was collected, and concentrated by rotary evaporation. 6) Nanofiltration separation: Add water to the obtained concentrate to the initial enzymatic hydrolysate volume, concentrate by nanofiltration, and spray dry to obtain peptides.

2. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 1), the low-temperature peanut cake obtained by cold pressing after removing the red skin is crushed to 40-100 mesh, and after solvent extraction to remove oil, the residual oil content in the defatted low-temperature peanut cake powder is <1%.

3. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 2), the acidic water used is weakly acidic electro-generated functional water with a pH of 2.7 to 5.0 and an effective chlorine concentration of 10 to 60 mg / L; the amount of acidic water added is 10 to 20% of the mass of defatted low-temperature peanut meal powder.

4. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 3), the extruded, puffed, defatted, low-temperature peanut cake obtained in step 2) is pulverized to 60-140 mesh. The microwave and ultrasonic treatment conditions are as follows: microwave and ultrasound are used simultaneously, the ultrasonic frequency is 40±10 kHz, and the ratio of ultrasonic power to cake mass is 40-60 W:1 kg; the microwave treatment is in constant temperature mode, and the temperature is controlled at 80-95℃; during the microwave and ultrasonic treatment, continuous stirring is carried out at a stirring speed of 100-300 r / min.

5. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 4), the mixed enzyme used consists of mixed enzyme A and mixed enzyme B. Mixed enzyme A consists of four or five of the following: cellulase, hemicellulase, β-glucosidase, pectinase, and amylase. Mixed enzyme B consists of two or three of the following: acidic protease, pepsin, and papain. The amount of mixed enzyme added is 0.1 to 1.0% of the mass of the hydrolysate. The mass ratio of mixed enzyme A to mixed enzyme B is 1 to 3:

1.

6. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 5, characterized in that, In step 4), the enzyme addition order is as follows: first add mixed enzyme A, then add mixed enzyme B; the enzymatic hydrolysis time of mixed enzyme A alone accounts for 1 / 5 to 1 / 3 of the total time; in mixed enzyme A, the content of cellulase, pectinase and amylase is not less than 30%, 10% and 20% respectively by mass percentage.

7. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 5), the macroporous resin used is a mixture of weakly polar macroporous resin and medium polar macroporous resin in a ratio of 2 to 5:1; the ion exchange resin is a mixture of anion exchange resin and cation exchange resin in a ratio of 1 to 2:1; the macroporous resin column is eluted with 2 to 4 bed volumes of 50 to 80% ethanol solution and concentrated by rotary evaporation to 0.3 to 0.6 bed volumes.

8. The method for preparing polypeptides and water-soluble sugars using low-temperature peanut meal as described in claim 1, characterized in that, In step 6), nanofiltration concentrates the solution to 10% to 25% of the initial enzymatic hydrolysate volume, and the nanofiltration membrane used has a molecular weight cutoff of 200 to 300 Da.

9. The polypeptide and water-soluble sugar prepared by any one of the preparation methods according to claims 1 to 8.

10. The use of the polypeptide of claim 9 in the preparation of antioxidant drugs or the use of the water-soluble sugar as a food additive.

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