Green preparation process of grape seed procyanidine by fusing fermentation and adsorption elution

Abstract

The invention relates to the technical field of procyanidine separation and preparation, and discloses a green preparation process of grape seed procyanidine by fusing fermentation and adsorption elution, which comprises the following steps: pretreating raw materials; fermentation: taking lactobacillus rhamnosus as a strain, and obtaining fermented grape seeds after fermentation of the grape seeds and the lactobacillus rhamnosus is finished; ohmic heating: placing the fermented grape seeds in an electric field, heating, and then cooling to room temperature; extracting with a deep eutectic solvent; and adsorption and elution. According to the invention, lactobacillus rhamnosus is fermented in a specific mixed culture medium, and biological enzyme generated by fermentation can decompose cell walls and intercellular substances and destroy cell structures, so that procyanidine is fully exposed and released. In the subsequent ohmic heating step, material resistance is utilized to generate heat, the cell structure is further destroyed, procyanidine diffusion is promoted, and the extraction rate is greatly increased through cooperation of the two.

Description

Technical Field

[0001] The present invention relates to the technical field of proanthocyanidin separation and preparation, and particularly to a green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption-elution. Background Art

[0002] As a natural compound with important biological activities, proanthocyanidins exhibit significant effects in many aspects such as antioxidant, anti-inflammatory, and cardiovascular protection. In terms of antioxidant, it can effectively scavenge free radicals in the body and prevent cells from being damaged by oxidative stress. This property enables proanthocyanidins to be used as natural antioxidants in the food field to extend the shelf life of food and maintain the color, flavor, and nutritional components of food. In the health product industry, products rich in proanthocyanidins are highly favored, which helps to enhance human immunity and delay the aging process. In the cosmetics field, proanthocyanidins can inhibit the activity of tyrosinase, reduce melanin production, achieve the effect of whitening and lightening spots, and at the same time, due to its antioxidant ability, it can prevent skin photoaging and maintain skin elasticity and luster. In the pharmaceutical industry, a large number of studies have shown that proanthocyanidins have potential preventive and therapeutic effects on cardiovascular diseases, such as reducing blood lipids, inhibiting platelet aggregation, and improving vascular endothelial function, showing broad medicinal prospects.

[0003] Traditional extraction techniques for grape seed proanthocyanidins mostly use solvent extraction methods, such as directly soaking grape seeds with organic solvents such as ethanol and acetone. There are also those using supercritical fluid extraction methods, which utilize the special solubility of fluids such as carbon dioxide in the supercritical state to extract proanthocyanidins. Although these methods can obtain proanthocyanidins to a certain extent, there are obvious deficiencies in the extraction rate. Due to the complex structure of grape seeds, proanthocyanidins are wrapped inside the cell wall, and traditional methods are difficult to fully break the structure, resulting in a large amount of proanthocyanidins unable to be effectively dissolved out, and the extraction rate is low.

[0004] From the perspective of cost and energy consumption, traditional solvent extraction methods often require a large amount of organic solvents, which not only increases the raw material cost, but also makes the subsequent solvent recovery and treatment process cumbersome and energy-consuming. Although the supercritical fluid extraction method can achieve a higher purity extraction, it requires special high-pressure equipment, with high equipment investment costs and extremely high energy consumption during operation. In order to improve the extraction rate of proanthocyanidins, increase the output per unit of raw material, and at the same time reduce costs and energy consumption, it is urgent to develop a more efficient and economical extraction method.

[0005] Therefore, we propose a green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption-elution. Summary of the Invention

[0006] The present invention mainly solves the technical problems existing in the above-mentioned prior art, and provides a green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption-elution.

[0007] To achieve the above object, the present invention adopts the following technical solution, a green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption-elution, comprising the following steps:

[0008] Step 1: Raw material pretreatment. Collect non-mildewed grape seeds, first place them in clean water for full cleaning, and the cleaning duration is 20 - 30 minutes; after cleaning, perform a draining operation, and then soak the grape seeds in sterile water for 60 - 90 minutes; after soaking, transfer the grape seeds to an environment of 40 - 60 °C for drying until reaching a constant weight state, and then use a pulverizer to pulverize the dried grape seeds and pass through a 40-mesh sieve to obtain grape seed powder with uniform particle size;

[0009] Step 2: Fermentation. Use Lactobacillus rhamnosus as the strain, and prepare a fermentation medium in advance. This fermentation medium is composed of YPD medium and MRS medium mixed at a volume ratio of 1:1. Among them, YPD medium contains 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose, and MRS medium contains 10 g / L of peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 20 g / L of glucose, 5 g / L of sodium acetate, 2 g / L of K2HPO4, 0.5 g / L of MgSO4, 0.3 g / L of MnSO4, and 1 mL of Tween 80. The mixed medium is sterilized at 121 °C for 20 minutes; inoculate the pretreated grape seeds and Lactobacillus rhamnosus into the fermentation medium at a mass ratio of 20:1, and then place the inoculated fermentation medium in an environment of 30 °C and perform shaking culture at a shaking speed of 110 r / min for 48 - 60 hours. During this period, perform a stirring operation every 8 hours. After fermentation, obtain fermented grape seeds;

[0010] Step 3: Ohmic heating. Place the fermented grape seeds in an electric field environment with an electric field strength of 60 - 75 V / cm and a power frequency of 50 Hz, heat to 60 - 70 °C, and maintain for 60 - 100 seconds, and then cool to room temperature;

[0011] Step 4: Deep eutectic solvent extraction. Weigh the hydrogen bond acceptor and hydrogen bond donor at a molar ratio of 1:4 in a round-bottom flask, place the round-bottom flask in a 70 °C water bath for rotary evaporation until a viscous solution state that is transparent and without crystals is formed, that is, obtain the deep eutectic solvent; mix the grape seeds cooled to room temperature with the deep eutectic solvent at a solid-liquid ratio of 1:30, homogenize with a disperser at 6000 - 8000 r / min at room temperature for 2 - 3 minutes, then pressurize the mixed material with a pressure pump, control the pressure at 0.5 - 1 MPa, and then place it in a 60 °C water bath for extraction for 60 - 80 minutes. After cooling to room temperature, perform suction filtration to remove the filter residue, and remove the moisture by rotary evaporation with a rotary evaporator to obtain the crude proanthocyanidins;

[0012] Step 5: Adsorption and elution step. Prepare the crude proanthocyanidins into an aqueous solution with a concentration of 1.5 - 2.5 mg / ml by adding water, mix it with macroporous resin at a mass ratio of 1:3 - 1:5, put it into a shaker at 25 - 30 °C and oscillate and adsorb for 24 - 30 hours at an oscillation speed of 110 r / min. After the oscillation ends, filter to remove the unadsorbed crude proanthocyanidins aqueous solution, then rinse the resin with distilled water 3 - 5 times, and the water consumption for each rinse is 2 - 3 times the volume of the resin; subsequently, elute with ethanol with a volume fraction of 70%, control the elution flow rate at 1 - 2 BV / h, collect the eluate, perform reduced pressure distillation on the eluate to recover ethanol, and finally obtain a high-purity proanthocyanidins product.

[0013] Preferably, when cleaning grape seeds in Step 1, use flowing clear water and use a stirring device to stir and assist cleaning at a speed of 100 - 150 r / min.

[0014] Preferably, when performing oscillating culture in Step 2, use a constant temperature oscillating shaker, and the temperature can be controlled at 30 °C ± 1 °C.

[0015] Preferably, the method of cooling to room temperature in Step 3 is natural cooling, and the cooling time is controlled within 1 - 2 hours.

[0016] Preferably, when performing rotary evaporation of the deep eutectic solvent in Step 4, the vacuum degree of the rotary evaporator used is controlled at 0.08 - 0.09 MPa.

[0017] Preferably, the distilled water after rinsing the resin in Step 5 is collected for detection, and when the impurity content in the rinsing liquid is detected to be lower than 0.05%, stop rinsing.

[0018] Preferably, when performing reduced pressure distillation to recover ethanol in Step 5, the distillation temperature is controlled at 50 - 60 °C, and the vacuum degree is controlled at 0.06 - 0.07 MPa.

[0019] Beneficial effects

[0020] The present invention provides a green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption elution. It has the following beneficial effects:

[0021] (1) This green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption elution uses Lactobacillus rhamnosus to ferment in a specific mixed culture medium (YPD and MRS are mixed at a ratio of 1:1). The biological enzymes produced by fermentation can decompose cell walls and intercellular matrix substances, destroy cell structures, and make proanthocyanidins fully exposed and released. In the subsequent Ohmic heating step, heat is generated by the resistance of the material, further destroying the cell structure and promoting the diffusion of proanthocyanidins. The two work together to greatly improve the extraction rate.

[0022] (2) The green preparation process of grape seed proanthocyanidins by integrating fermentation and adsorption-elution. Fermentation and ohmic heating simplify the subsequent extraction process, shorten the time, reduce the solvent consumption and the operation time of equipment, and lower the energy consumption cost. Moreover, fermentation does not require complex equipment, reducing the equipment investment. Detailed implementation manners

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] The green preparation process of grape seed proanthocyanidins by integrating fermentation and adsorption-elution includes the following steps:

[0025] Raw material pretreatment: First, collect non-mildewed grape seeds. Place the collected grape seeds in clean water for thorough cleaning. The cleaning duration is controlled within 20 - 30 minutes. Use flowing clean water and a stirring device to stir at a speed of 100 - 150 r / min to assist in cleaning, removing contaminants such as impurities and dust attached to the surface. After cleaning, perform a draining operation. Subsequently, soak the grape seeds in sterile water for 60 - 90 minutes to allow the grape seeds to absorb water and swell appropriately, which is beneficial for the release of subsequent components. After soaking, transfer the grape seeds to an environment at 40 - 60 °C for drying until reaching a constant weight state to remove moisture for convenient subsequent pulverization operation. Then use a pulverizer to pulverize the dried grape seeds and pass through a 40-mesh sieve to obtain grape seed powder with uniform particle size, making the grape seed particle sizes uniform.

[0026] Fermentation step: In this step, Lactobacillus rhamnosus is selected as the strain, and the fermentation medium is prepared in advance. The fermentation medium is composed of YPD medium and MRS medium mixed at a volume ratio of 1:1. Among them, the YPD medium contains 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose, and the MRS medium contains 10 g / L of peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 20 g / L of glucose, 5 g / L of sodium acetate, 2 g / L of K2HPO4, 0.5 g / L of MgSO4, 0.3 g / L of MnSO4, and 1 mL of Tween 80. The mixed medium is sterilized at 121 °C for 20 minutes to kill miscellaneous bacteria and ensure the purity of the fermentation environment. The pretreated grape seeds and Lactobacillus rhamnosus are inoculated into the fermentation medium at a mass ratio of 20:1. Subsequently, the inoculated fermentation medium is placed in an environment of 30 °C, and a constant temperature shaking incubator is used to shake and culture at an oscillation speed of 110 r / min for 48 - 60 hours, and the stirring operation is carried out every 8 hours during this period. During the fermentation process, Lactobacillus rhamnosus grows and reproduces by using the nutrients in the fermentation medium, and at the same time ferments the grape seeds. On the one hand, through the metabolic activities of microorganisms, the content of proanthocyanidins in grape seeds can be increased; on the other hand, the fermentation process can destroy some structures of grape seeds, making the internal proanthocyanidins more easily released, creating favorable conditions for the subsequent extraction step.

[0027] Ohmic heating: The fermented grape seeds are placed in an electric field environment with an electric field strength of 60 - 75 V / cm and a power supply frequency of 50 Hz. Using the principle of ohmic heating, heat is generated inside the fermented grape seeds, heated to 60 - 70 °C, and maintained for 60 - 100 seconds. Subsequently, natural cooling is used to cool to room temperature, and the cooling time is controlled within 1 - 2 hours. Ohmic heating can further destroy the seed coat and cell wall structure of grape seeds, making the proanthocyanidins in cells more easily released from the cells, increasing the dissolution rate of proanthocyanidins, and improving the subsequent extraction efficiency.

[0028] Deep eutectic solvent extraction: First, prepare the deep eutectic solvent. Weigh the hydrogen bond acceptor and hydrogen bond donor, choline chloride and urea, according to a molar ratio of 1:4 and place them in a round-bottom flask. Put the round-bottom flask in a 70°C water bath for rotary evaporation. When using a rotary evaporator, control the vacuum degree at 0.08 - 0.09 MPa until a viscous solution state that is transparent and free of crystals is formed, thus obtaining the deep eutectic solvent. Mix the grape seeds cooled to room temperature with the deep eutectic solvent according to a solid-liquid ratio of 1:30, and homogenize them for 2 - 3 minutes at 6000 - 8000 r / min using a disperser at room temperature to fully mix the grape seeds and the deep eutectic solvent. Then, pressurize the mixed material using a pressure pump, control the pressure at 0.5 - 1 MPa, and place it in a 60°C water bath for extraction for 60 - 80 minutes to fully dissolve the proanthocyanidins in the deep eutectic solvent. After cooling to room temperature, perform suction filtration to remove the filter residue, obtain the filtrate containing proanthocyanidins, and remove the water by rotary evaporation using a rotary evaporator to obtain the crude proanthocyanidin product. The deep eutectic solvent has good solubility and selectivity, can effectively extract proanthocyanidins from grape seeds, and its green and environmentally friendly characteristics conform to the green preparation concept of the present invention.

[0029] Adsorption and elution: Prepare an aqueous solution of the crude proanthocyanidin product with a concentration of 1.5 - 2.5 mg / ml, mix it with macroporous resin according to a mass ratio of 1:3 - 1:5, and place it in a shaker at 25 - 30°C and oscillate at an oscillation speed of 110 r / min for adsorption for 24 - 30 hours to fully adsorb the proanthocyanidins on the macroporous resin. After the oscillation ends, filter to remove the unadsorbed aqueous solution of the crude proanthocyanidin product, and then rinse the resin 3 - 5 times with distilled water. The amount of water used for each rinse is 2 - 3 times the volume of the resin. Detect the rinse solution, and stop rinsing when the impurity content in the rinse solution is detected to be less than 0.05% to ensure the removal of impurities adsorbed on the macroporous resin. Subsequently, elute with ethanol with a volume fraction of 70%, control the elution flow rate at 1 - 2 BV / h (BV is the resin bed volume), collect the eluate, perform vacuum distillation on the eluate to recover ethanol, control the distillation temperature at 50 - 60°C, and control the vacuum degree at 0.06 - 0.07 MPa to finally obtain a high-purity proanthocyanidin product. Through the adsorption and elution steps, impurities in the crude proanthocyanidin product can be further removed, the product purity can be improved, and the requirements for high-purity proanthocyanidins in different fields can be met.

[0030] Example 1

[0031] A green preparation process of grape seed proanthocyanidins integrating fermentation and adsorption elution, comprising the following steps:

[0032] Raw material pretreatment: First, collect mold-free grape seeds. Place the collected grape seeds in clean water and wash them thoroughly for 20 minutes. Use flowing clean water and a stirring device to stir at a speed of 100 r / min to assist in washing, removing contaminants such as impurities and dust adhering to the surface. After washing, perform a draining operation, and then soak the grape seeds in sterile water for 60 minutes to allow the grape seeds to absorb water and swell appropriately, which is beneficial for the release of subsequent components. After soaking, transfer the grape seeds to an environment at 40 °C for drying until a constant weight state is reached, removing moisture to facilitate subsequent crushing operations. Then use a crusher to crush the dried grape seeds and pass them through a 40-mesh sieve to obtain grape seed powder with a uniform particle size, making the grape seed particles of uniform size.

[0033] Fermentation step: In this step, Lactobacillus rhamnosus is selected as the strain, and the fermentation medium is prepared in advance. The fermentation medium is composed of YPD medium and MRS medium mixed at a volume ratio of 1:1. Among them, the YPD medium contains 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose, and the MRS medium contains 10 g / L of peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 20 g / L of glucose, 5 g / L of sodium acetate, 2 g / L of K2HPO4, 0.5 g / L of MgSO4, 0.3 g / L of MnSO4, and 1 mL of Tween 80. The mixed medium is sterilized at 121 °C for 20 minutes to kill miscellaneous bacteria and ensure the purity of the fermentation environment. Inoculate the pretreated grape seeds and Lactobacillus rhamnosus into the fermentation medium at a mass ratio of 20:1, and then place the inoculated fermentation medium in an environment at 30 °C. Use a constant temperature shaking incubator and shake at an oscillation speed of 110 r / min for 48 hours, and perform a stirring operation every 8 hours during this period. During the fermentation process, Lactobacillus rhamnosus utilizes the nutrients in the fermentation medium to grow and reproduce, and at the same time ferments the grape seeds. On the one hand, through the metabolic activities of microorganisms, the content of proanthocyanidins in grape seeds can be increased; on the other hand, the fermentation process can destroy part of the structure of grape seeds, making the internal proanthocyanidins more easily released, creating favorable conditions for subsequent extraction steps.

[0034] Ohmic heating: Place the fermented grape seeds in an electric field environment with an electric field strength of 60 V / cm and a power supply frequency of 50 Hz. Using the principle of ohmic heating, heat is generated inside the fermented grape seeds, heated to 60 °C, and maintained for 60 seconds, and then cooled to room temperature by natural cooling, with the cooling time controlled within 1 hour. Ohmic heating can further destroy the seed coat and cell wall structure of grape seeds, making the proanthocyanidins inside the cells more easily released from the cells, increasing the dissolution rate of proanthocyanidins, and improving the subsequent extraction efficiency.

[0035] Deep eutectic solvent extraction: First, prepare the deep eutectic solvent. Weigh the hydrogen bond acceptor and hydrogen bond donor, choline chloride and urea, according to a molar ratio of 1:4 and place them in a round-bottom flask. Then, put the round-bottom flask into a 70°C water bath for rotary evaporation. When using a rotary evaporator, control the vacuum degree at 0.08 MPa until a viscous solution that is transparent and free of crystals is formed, thus obtaining the deep eutectic solvent. Mix the grape seeds cooled to room temperature with the deep eutectic solvent at a solid-liquid ratio of 1:30, and homogenize them for 2 minutes at 6000 r / min using a disperser at room temperature to ensure full mixing of the grape seeds and the deep eutectic solvent. Then, pressurize the mixed material using a pressure pump, control the pressure at 0.5 MPa, and place it in a 60°C water bath for extraction for 60 minutes to fully dissolve the proanthocyanidins in the deep eutectic solvent. After cooling to room temperature, perform suction filtration to remove the filter residue, obtaining a filtrate containing proanthocyanidins, and remove the water by rotary evaporation using a rotary evaporator to obtain the crude proanthocyanidin product. The deep eutectic solvent has good solubility and selectivity, can effectively extract proanthocyanidins from grape seeds, and its green and environmentally friendly characteristics conform to the green preparation concept of the present invention.

[0036] Adsorption and elution: Prepare an aqueous solution of the crude proanthocyanidin product with a concentration of 1.5 mg / ml by adding water, and mix it with macroporous resin at a mass ratio of 1:3. Place it in a shaker at 25°C and shake and adsorb for 24 hours at an oscillation speed of 110 r / min to fully adsorb the proanthocyanidins on the macroporous resin. After the oscillation ends, filter to remove the unadsorbed aqueous solution of the crude proanthocyanidin product, and then rinse the resin 3 times with distilled water, with the amount of water used for each rinse being 2 times the volume of the resin. Detect the rinse solution, and stop rinsing when the impurity content in the rinse solution is detected to be lower than 0.05% to ensure the removal of impurities adsorbed on the macroporous resin. Subsequently, elute with ethanol with a volume fraction of 70%, control the elution flow rate at 1 BV / h (BV is the resin bed volume), collect the eluate, perform vacuum distillation on the eluate to recover ethanol, control the distillation temperature at 50°C, and control the vacuum degree at 0.06 MPa to finally obtain a high-purity proanthocyanidin product. Through the adsorption and elution steps, impurities in the crude proanthocyanidin product can be further removed, the product purity can be improved, and the requirements for high-purity proanthocyanidins in different fields can be met.

[0037] Example 2

[0038] A green preparation process for grape seed proanthocyanidins integrating fermentation and adsorption elution, comprising the following steps:

[0039] Raw material pretreatment: First, collect mold-free grape seeds. Place the collected grape seeds in clean water and wash them thoroughly for 30 minutes. Use flowing clean water and a stirring device to stir at a speed of 150 r / min to assist in cleaning, removing contaminants such as impurities and dust attached to the surface. After cleaning, perform a draining operation. Subsequently, soak the grape seeds in sterile water for 90 minutes to allow the grape seeds to absorb water and swell appropriately, which is beneficial for the release of subsequent components. After soaking, transfer the grape seeds to an environment at 60 °C for drying until they reach a constant weight state, removing moisture to facilitate subsequent crushing operations. Then use a crusher to crush the dried grape seeds and pass them through a 40-mesh sieve to obtain grape seed powder with uniform particle size, making the grape seed particles of uniform size.

[0040] Fermentation step: In this step, Lactobacillus rhamnosus is selected as the strain, and the fermentation medium is prepared in advance. The fermentation medium is composed of YPD medium and MRS medium mixed at a volume ratio of 1:1. Among them, the YPD medium contains 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose, and the MRS medium contains 10 g / L of peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 20 g / L of glucose, 5 g / L of sodium acetate, 2 g / L of K2HPO4, 0.5 g / L of MgSO4, 0.3 g / L of MnSO4, and 1 mL of Tween 80. The mixed medium is sterilized at 121 °C for 20 minutes to kill miscellaneous bacteria and ensure the purity of the fermentation environment. Inoculate the pretreated grape seeds and Lactobacillus rhamnosus into the fermentation medium at a mass ratio of 20:1. Subsequently, place the inoculated fermentation medium in an environment at 30 °C and use a constant temperature shaking incubator to shake and culture at an oscillation speed of 110 r / min for 60 hours, and perform a stirring operation every 8 hours during this period. During the fermentation process, Lactobacillus rhamnosus uses the nutrients in the fermentation medium to grow and reproduce, and at the same time ferments the grape seeds. On the one hand, through the metabolic activities of microorganisms, the content of proanthocyanidins in grape seeds can be increased; on the other hand, the fermentation process can destroy part of the structure of grape seeds, making the internal proanthocyanidins more easily released, creating favorable conditions for the subsequent extraction step.

[0041] Ohmic heating: Place the fermented grape seeds in an electric field environment with an electric field strength of 75 V / cm and a power supply frequency of 50 Hz. Using the principle of ohmic heating, heat is generated inside the fermented grape seeds, heated to 70 °C, and maintained for 100 seconds. Subsequently, cool them to room temperature by natural cooling, and control the cooling time within 2 hours. Ohmic heating can further destroy the seed coat and cell wall structure of grape seeds, making the proanthocyanidins inside the cells more easily released from the cells, increasing the dissolution rate of proanthocyanidins, and improving the subsequent extraction efficiency.

[0042] Deep eutectic solvent extraction: First, prepare the deep eutectic solvent. Weigh the hydrogen bond acceptor and hydrogen bond donor choline chloride and urea according to a molar ratio of 1:4 and place them in a round-bottom flask. Then, put the round-bottom flask into a 70°C water bath for rotary evaporation. When using a rotary evaporator, control the vacuum degree at 0.09 MPa until a transparent and crystal-free viscous solution state is formed, thus obtaining the deep eutectic solvent. Mix the grape seeds cooled to room temperature with the deep eutectic solvent at a solid-liquid ratio of 1:30 and homogenize them for 3 minutes at 8000 r / min at room temperature using a disperser to fully mix the grape seeds and the deep eutectic solvent. Then, pressurize the mixed material using a pressure pump with the pressure controlled at 1 MPa, and then place it in a 60°C water bath for extraction for 80 minutes to fully dissolve the proanthocyanidins in the deep eutectic solvent. After cooling to room temperature, perform suction filtration to remove the filter residue, obtaining a filtrate containing proanthocyanidins, and remove the water by rotary evaporation using a rotary evaporator to obtain the crude proanthocyanidin product. The deep eutectic solvent has good solubility and selectivity, can effectively extract proanthocyanidins from grape seeds, and its green and environmentally friendly characteristics conform to the green preparation concept of this invention.

[0043] Adsorption and elution: Prepare an aqueous solution of the crude proanthocyanidin product with a concentration of 2.5 mg / ml, mix it with macroporous resin at a mass ratio of 1:5, and place it in a shaker at 30°C and shake and adsorb at an oscillation speed of 110 r / min for 30 hours to fully adsorb the proanthocyanidins on the macroporous resin. After the oscillation ends, filter to remove the unadsorbed aqueous solution of the crude proanthocyanidin product, and then rinse the resin 5 times with distilled water, with the amount of water used for each rinse being 3 times the volume of the resin. Detect the rinse solution, and stop rinsing when the impurity content in the rinse solution is detected to be lower than 0.05% to ensure the removal of impurities adsorbed on the macroporous resin. Subsequently, elute with ethanol with a volume fraction of 70%, control the elution flow rate at 2 BV / h (BV is the resin bed volume), collect the eluate, perform vacuum distillation on the eluate to recover ethanol, control the distillation temperature at 60°C, and control the vacuum degree at 0.07 MPa to finally obtain a high-purity proanthocyanidin product. Through the adsorption and elution steps, impurities in the crude proanthocyanidin product can be further removed, the product purity can be improved, and the requirements for high-purity proanthocyanidins in different fields can be met.

[0044] Example 3

[0045] Raw material pretreatment: Collect 500 g of non-mildewed grape seeds, place them in clean water, and use flowing clean water and a stirring device to stir and wash at a speed of 120 r / min for 25 minutes to fully remove surface impurities. After washing, drain the water, soak them in sterile water for 75 minutes, and then transfer them to a 50°C environment to dry to constant weight. Crush them using a crusher and pass through a 40-mesh sieve to obtain grape seed powder.

[0046] Fermentation step: Mix YPD medium (containing 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) and MRS medium (containing 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L K2HPO4, 0.5 g / L MgSO4, 0.3 g / L MnSO4, 1 mL Tween 80) at a volume ratio of 1:1. The mixed medium is sterilized at 121 °C for 20 minutes. Inoculate 20 g of Lactobacillus rhamnosus and 500 g of pretreated grape seed powder into the fermentation medium, place it in a constant temperature shaking incubator, and incubate at 30 °C with an oscillation speed of 110 r / min for 54 hours. Stir every 8 hours to obtain fermented grape seeds.

[0047] Ohmic heating: Place the fermented grape seeds in an electric field environment with an electric field strength of 65 V / cm and a power frequency of 50 Hz, heat to 65 °C, maintain for 80 seconds, and then cool naturally to room temperature. The cooling time is about 1.5 hours.

[0048] Deep eutectic solvent extraction: Weigh choline chloride and urea in a molar ratio of 1:4 in a round-bottom flask, place it in a 70 °C water bath for rotary evaporation. The vacuum degree of the rotary evaporator is controlled at 0.085 MPa until a transparent and viscous deep eutectic solvent without crystals is formed. Mix the grape seeds cooled to room temperature with the deep eutectic solvent at a solid-liquid ratio of 1:30, homogenize with a disperser at 7000 r / min for 2 minutes at room temperature, then pressurize with a pressure pump to 0.8 MPa, and extract in a 60 °C water bath for 70 minutes. After cooling to room temperature, filter by suction to remove the filter residue, and remove the water by rotary evaporation with a rotary evaporator to obtain the crude proanthocyanidins.

[0049] Adsorption and elution: Prepare an aqueous solution with a concentration of 2 mg / ml of the crude proanthocyanidins, mix it with macroporous resin at a mass ratio of 1:4, place it in a shaker at 28 °C and oscillate at an oscillation speed of 110 r / min for adsorption for 27 hours. After the oscillation ends, filter, wash the resin 4 times with distilled water, and the water consumption for each wash is 2.5 times the volume of the resin. Detect the washing solution and stop washing when the impurity content is less than 0.05%. Elute with ethanol with a volume fraction of 70%, control the elution flow rate at 1.5 BV / h, collect the eluate, and recover ethanol by vacuum distillation under the conditions of a distillation temperature of 55 °C and a vacuum degree of 0.065 MPa to obtain a high-purity proanthocyanidin product. After detection, the yield of the proanthocyanidin product is 15.6 g and the purity is 92.5%.

[0050] Comparative Example 1

[0051] Raw material pretreatment: The same as Example 3, collect 500 g of non-mildewed grape seeds for pretreatment to obtain grape seed powder.

[0052] Fermentation step: Change the fermentation time to 48 hours, and keep the other conditions the same as in Example 3. Inoculate Lactobacillus rhamnosus and grape seed powder into the fermentation medium, and culture them with shaking at 30 °C for 48 hours to obtain fermented grape seeds.

[0053] Ohmic heating: The same as in Example 3, perform Ohmic heating treatment on the fermented grape seeds and then cool them to room temperature.

[0054] Deep eutectic solvent extraction: Change the composition ratio of the deep eutectic solvent. Weigh choline chloride and urea according to a molar ratio of 1:3 to prepare the deep eutectic solvent, and keep the other extraction steps the same as in Example 3 to obtain the crude proanthocyanidins.

[0055] Adsorption and elution: The same as in Example 3, perform adsorption and elution operations on the crude proanthocyanidins to obtain high-purity proanthocyanidin products.

[0056] After detection, the yield of the proanthocyanidin product is 14.2 g, and the purity is 90.3%. Compared with Example 3, after shortening the fermentation time and changing the composition ratio of the deep eutectic solvent, both the yield and purity of the proanthocyanidins have decreased, indicating that appropriate fermentation time and deep eutectic solvent composition have an important impact on the preparation effect.

[0057] Comparative Example 2

[0058] Raw material pretreatment: Still collect 500 g of non-mildewed grape seeds, and perform cleaning, soaking, drying and sieving according to the method of Example 3 to obtain grape seed powder.

[0059] Fermentation step: Raise the fermentation temperature to 32 °C and adjust the fermentation time to 60 hours. Keep the other fermentation conditions the same as in Example 3. Inoculate Lactobacillus rhamnosus and grape seed powder into the fermentation medium, and culture them with shaking at 32 °C for 60 hours to obtain fermented grape seeds.

[0060] Ohmic heating: The same as in Example 3, perform Ohmic heating treatment on the fermented grape seeds and then cool them to room temperature.

[0061] Deep eutectic solvent extraction: Adjust the solid-liquid ratio to 1:35, and keep the other extraction conditions the same as in Example 3. Mix the grape seeds cooled to room temperature with the deep eutectic solvent according to the new solid-liquid ratio for extraction to obtain the crude proanthocyanidins.

[0062] Adsorption and elution: The same as in Example 3, perform adsorption and elution operations on the crude proanthocyanidins to obtain high-purity proanthocyanidin products. After detection, the yield of the proanthocyanidin product is 16.1 g, and the purity is 93.2%. This example shows that appropriately increasing the fermentation temperature, prolonging the fermentation time and adjusting the solid-liquid ratio can, to a certain extent, improve the yield and purity of the proanthocyanidins, further verifying the feasibility and stability of the present invention under different conditions, and better preparation effects can be obtained by optimizing the conditions.

[0063] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A green preparation process of grape seed proanthocyanidins by integrating fermentation and adsorption elution, characterized in that: The following steps are involved: Step 1: Pre-treatment of raw materials: collect grape seeds without mildew, wash them thoroughly in clean water for 20-30 minutes, drain them after washing, and then soak them in sterile water for 60-90 minutes. After soaking, transfer the grape seeds to a 40-60°C environment for drying until constant weight is reached, and then use a grinder to crush the dried grape seeds and pass them through a 40-mesh sieve to obtain grape seed powder with uniform particle size. Step 2: fermentation, using Lactobacillus rhamnosus as the strain, preparing a fermentation medium in advance, the fermentation medium is a mixture of YPD medium and MRS medium in a volume ratio of 1:1, wherein the YPD medium contains 10 g / L yeast powder, 20 g / L peptone, and 20 g / L glucose, and the MRS medium contains 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L K2HPO4, 0.5 g / L MgSO4, 0.3 g / L MnSO4, and 801 mL Tween, and the mixed medium is sterilized at 121°C for 20 minutes; The pretreated grape seeds and Lactobacillus rhamnosus are inoculated into a fermentation medium at a mass ratio of 20:1, and then the inoculated fermentation medium is placed in an environment of 30° C. and cultured at a shaking speed of 110 r / min for 48-60 hours, during which the medium is stirred every 8 hours, and fermented grape seeds are obtained after the fermentation is completed; Step 3: Ohmic heating, placing the fermented grape seeds in an electric field environment with an electric field strength of 60-75V / cm and a power supply frequency of 50Hz, heating to 60-70°C, and maintaining for 60-100 seconds, and then cooling to room temperature; Step 4: Extraction with low eutectic solvent: weigh hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:4 into a round-bottom flask, place the round-bottom flask in a 70°C water bath for rotary evaporation, until a transparent, crystal-free viscous solution is formed, thereby obtaining a low eutectic solvent; mix the grape seeds cooled to room temperature with the low eutectic solvent at a solid-liquid ratio of 1:30, homogenize at room temperature with a disperser at 6000-8000 r / min for 2-3 minutes, then pressurize the mixture with a pressure pump, control the pressure at 0.5-1 MPa, and then extract it in a 60°C water bath for 60-80 minutes, cool it to room temperature, perform suction filtration to remove the filter residue, and remove water with a rotary evaporator to obtain crude proanthocyanidins; Step 5: adsorption and elution step, adding water to the crude proanthocyanidin to prepare an aqueous solution with a concentration of 1.5-2.5 mg / ml, mixing it with a macroporous resin at a mass ratio of 1:3-1:5, placing it in a shaker at 25-30° C. and oscillating at a speed of 110 r / min for 24-30 hours, filtering and removing the unadsorbed crude proanthocyanidin aqueous solution after the oscillation, and then washing the resin with distilled water for 3-5 times, and the amount of water used for each washing is 2-3 times the volume of the resin; then eluting with 70% volume fraction of ethanol, the elution flow rate is controlled at 1-2 BV / h, collecting the eluate, and performing reduced pressure distillation on the eluate to recover ethanol, and finally obtaining a high-purity proanthocyanidin product.

2. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: When washing the grape seeds in step 1, flowing clean water is used, and a stirring device is used to stir and assist washing at a speed of 100-150 r / min.

3. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: During the shaking culture in step 2, the shaker used is a constant temperature shaking shaker, and the temperature can be controlled at 30°C ± 1°C.

4. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: The method of cooling to room temperature in step 3 is natural cooling, and the cooling time is controlled within 1-2 hours.

5. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: When the low eutectic solvent is rotary evaporated in step 4, the vacuum degree of the rotary evaporator used is controlled at 0.08-0.09 MPa.

6. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: The distilled water after washing the resin in step 5 is collected and tested. When the impurity content in the washing liquid is detected to be lower than 0.05%, the washing is stopped.

7. The green preparation process of grape seed proanthocyanidins by fusion fermentation and adsorption elution according to claim 1, characterized in that: When the ethanol is recovered by reduced pressure distillation in step 5, the distillation temperature is controlled at 50-60° C. and the vacuum degree is controlled at 0.06-0.07 MPa.