Extraction method for improving stability of lycium ruthenicum anthocyanin and application of extraction method

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to an extraction method for improving stability of lycium ruthenicum anthocyanin and application of the extraction method. Wherein the whole preparation process comprises the steps of pretreatment, ultrasonic extraction, enzymolysis, centrifugation, membrane filtration, macroporous adsorption resin purification, freeze-dried powder preparation and the like, in the preparation process, the influence of high-temperature treatment on the stability of the anthocyanin is reduced, introduction of impurities is also reduced, and the purity of the anthocyanin and the extraction rate of active ingredients are effectively improved. The lycium ruthenicum extract containing anthocyanidin is applied to functional food, active ingredients of anthocyanidin can be effectively protected from being affected by the external environment, the stability and bioavailability of anthocyanidin in the functional food are improved, the use scene of anthocyanidin is enriched, and the health of the functional food is improved. Healthier and more nutritional food choices are brought to consumers, and the development of the functional food industry is also promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and in particular relates to an extraction method for improving the stability of anthocyanin from black wolfberry and application thereof. Background Art

[0002] Black wolfberry, also known as black wolfberry, is the mature fruit of the black wolfberry species of the Solanaceae family and the Lycium genus. It is included in classic works such as "Four Medical Classics", "Crystal Pearl Materia Medica", and "Uyghur Medicine Records". It has the effects of lowering blood pressure, lowering blood lipids, protecting cardiovascular system, and improving immunity. Modern scientific research has found that black wolfberry is rich in a variety of bioactive ingredients, including proanthocyanidins, polysaccharides, phenolic acids, 18 essential amino acids and vitamins for the human body. It is a safe and non-toxic resource for both medicine and food. In recent years, black wolfberry and its extracts have been widely used in food, beverages, health care, and skin care.

[0003] Black wolfberry has a higher content of natural anthocyanins among all wild plants. Anthocyanins are a common natural pigment that is commonly found in most plants. They belong to flavonoid compounds and have good solubility. Anthocyanins have antioxidant, obesity prevention, anti-cancer, chronic disease prevention, antibacterial, anti-depression, protection of the body's nervous system, and reduction of DNA damage. Therefore, they have attracted much attention. However, anthocyanins are easily attacked by free radicals and reactive oxygen, which leads to their degradation.

[0004] At present, alcohol extraction or enzymatic hydrolysis is mainly used for the extraction and preparation of active ingredients of black wolfberry. Although alcohol extraction can extract anthocyanins, the efficiency is low. After extraction, a large amount of alcohol in the extract needs to be rotary evaporated, which takes a lot of time and easily causes anthocyanin degradation. Enzymatic extraction is prone to introduce new impurities, and failure to thoroughly remove impurities will lead to low purity of the extracted anthocyanins. Therefore, it is necessary to develop an extraction method and application that improves the stability of anthocyanins in black wolfberry to improve the quality requirements and application scenarios of anthocyanins. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings involved in the above-mentioned anthocyanin extraction method steps and provide an extraction method for improving the stability of black wolfberry anthocyanin. The present invention adopts the following technical solutions: A method for extracting anthocyanins from black wolfberry to improve their stability comprises the following steps: S1, pretreatment: rinse the frozen fresh black wolfberry and place it in a reaction container, add water to crush and pulp, and obtain black wolfberry slurry; Specifically, fresh black wolfberry after freezing can maintain a good cell structure to reduce enzyme activity and keep the ingredients stable, which is beneficial for subsequent extraction. After adding an appropriate amount of water, use a grinder or homogenizer to beat it into a slurry. Specifically, the ratio of fresh black wolfberry to pure water is 1:3 to 1:5. Water grinding and slurrying can destroy the cell wall of black wolfberry and release more active ingredients.

[0006] S2, ultrasonic extraction: adding ethanol to the black wolfberry slurry obtained in step S1, and performing ultrasonic-assisted extraction to obtain a first black wolfberry extract; Specifically, ethanol as a solvent can effectively extract a variety of active ingredients in black wolfberry. The cavitation and mechanical effects of ultrasound can be used to further break up the cell walls. Ultrasonic assistance can accelerate the diffusion of solutes into the solvent, thereby improving the extraction efficiency and facilitating separation without introducing too many impurities. By adding 75% ethanol, the ethanol concentration in the final solution reaches 40%-60%.

[0007] S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to between 3.5 and 6.0, adding at least three complex enzymes of acid protease, pectinase, cellulase, and β-glucanase to the first black wolfberry extract, and combining with ultrasonic extraction to obtain a second black wolfberry extract; Specifically, adjusting the pH value between 3.5 and 6.0 is conducive to the activity of the enzyme, and the use of compound enzymes can more comprehensively decompose the cell wall and increase the release rate of active ingredients. By combining ultrasonic extraction again, the extraction effect can be further enhanced.

[0008] Specifically, the polysaccharides in black wolfberry are usually combined with proteins and exist in the form of proteoglycans. Acidic proteases can effectively enzymatically hydrolyze these proteins combined with polysaccharides. They can break down proteins into small molecular peptide chains or amino acids, thereby destroying the structure of cell walls and cell membranes, making it easier for other enzymes to enter the cells and act, which helps to release polysaccharides and flavonoids. Therefore, in the process of polysaccharide extraction, by first using proteases to remove most of the proteins, the subsequent extraction steps can be optimized.

[0009] In addition, pectin is one of the main components of the cell wall of black wolfberry, and its presence will affect the juice yield and the release of nutrients. Pectinase can hydrolyze pectin, destroy the cell wall structure, and separate the water-soluble pectin and original gelatin layer between the pulp cells, which is conducive to the dissolution of soluble solids.

[0010] Furthermore, the cellulose in black wolfberry is also an important component of the cell wall. Cellulase can destroy the plant cell wall and hydrolyze cellulose into oligosaccharides or monosaccharides, further promoting the release of flavor and functional substances and improving the taste.

[0011] Specifically, β-glucan is a soluble dietary fiber with multiple biological activities. β-glucanase can hydrolyze β-glucan, break down the structure of the cell wall, and release the monosaccharides or oligosaccharides therein.

[0012] S4, centrifugal collection: centrifugally filter the second black wolfberry extract obtained in step S3, and collect the supernatant; Specifically, the rotation speed of the centrifugal collection process is 3000-5000 rpm and the time is 10-20 minutes. Centrifugal filtration can effectively remove solid impurities and retain the supernatant containing active ingredients.

[0013] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities to obtain black wolfberry filtrate; Specifically, the supernatant is subjected to membrane filtration treatment to further remove tiny impurities and particles, thereby improving the purity of the extract.

[0014] S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography to collect the purified product containing active ingredients; Specifically, the macroporous adsorption resin can selectively adsorb according to the properties of the components, thereby achieving purification, and the target components are gradually eluted using ethanol of different concentrations and the purified products containing active ingredients are collected, thereby obtaining a purified product with anthocyanins.

[0015] S7, preparation of freeze-dried powder: freeze-dry, crush and sieve the purified black wolfberry product in step S6 in sequence to obtain a black wolfberry extract containing anthocyanins.

[0016] Specifically, freeze drying can retain the active ingredients and natural structure of the extract, and crushing and sieving can obtain a uniform and fine powder product. Specifically, the freeze-dried powder is crushed and sieved through a 80-100 mesh sieve to ensure uniform particles.

[0017] Furthermore, in step S2, the pH value of the black wolfberry slurry is adjusted between 3.5-6.0, the extraction temperature is 30-50°C, the ultrasonic power is 200-500w, and the extraction time is 15-20min.

[0018] Specifically, 75% v / v ethanol solution is added to the black wolfberry slurry obtained in step S1 to ensure that the target component can be effectively extracted and the difficulty of subsequent solvent removal can be reduced. The pH value of the black wolfberry slurry is adjusted by citric acid and sodium hydroxide. Citric acid not only has the function of adjusting the pH value, but also has a certain antioxidant property, which can protect the active ingredients in the black wolfberry from oxidative loss.

[0019] Specifically, within the range of 30-50°C, the solubility of ethanol is moderate, which is conducive to the dissolution and extraction of active ingredients. At the same time, this temperature range can reduce the destruction of heat-sensitive components in black wolfberry. Within the range of 200-500w, ultrasound can generate enough energy to destroy the cell walls of black wolfberry and promote the release of active ingredients. On the contrary, too high power will cause overheating of the solvent and destruction of the active ingredients. Within the range of 15-20min, the extraction time can enable most of the active ingredients to be effectively extracted. On the contrary, too long an extraction time will lead to excessive evaporation of the solvent and degradation of the active ingredients.

[0020] Furthermore, in step S3, the first black wolfberry extract in step S2 is supplemented with pure water according to the solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.01-0.08% of the total mass of the first black wolfberry extract, the enzymolysis time is 60-100 min, the enzymolysis temperature is 25-50°C, and the ultrasonic frequency is 40-60kHz.

[0021] Specifically, in the pretreatment in step S1, the weight of fresh black wolfberry is weighed, and pure water is added to the black wolfberry slurry per gram to reach 35 ml of the first black wolfberry extract to ensure sufficient liquid medium to promote the enzymatic hydrolysis reaction. The appropriate solid-liquid ratio can ensure that the solvent fully infiltrates the black wolfberry, which is beneficial to the enzymatic hydrolysis reaction and the release of active ingredients.

[0022] Specifically, in step S2, part of ethanol is easily volatilized during heating and ultrasonic extraction, but the first black wolfberry extract still contains a higher ethanol concentration. When the ethanol concentration is higher, ethanol can produce a significant inhibitory effect on the activity of the enzyme. The ethanol molecules interact with the key amino acid residues in the enzyme molecules, causing the spatial structure of the enzyme to change, thereby affecting its catalytic activity. In addition, high concentrations of ethanol can also denature or precipitate the enzyme molecules, further reducing their activity. Due to the inhibitory effect of ethanol on enzyme activity, the reaction rate in the enzymolysis process can be reduced, causing the enzymolysis efficiency to decrease. If the enzymolysis efficiency decreases or the enzyme is inactivated, the quality, output and cost of the final product can be affected. By supplementing pure water, the ethanol concentration can be reduced to less than 20%, thereby reducing the impact on the activity of the complex enzyme.

[0023] In addition, the amount of enzyme added determines the speed and efficiency of the enzymatic hydrolysis reaction. The right amount of enzyme can accelerate the decomposition of the cell wall and promote the release of active ingredients. Specifically, complex enzymes can more effectively decompose polysaccharides in the cell wall and increase the dissolution rate of active ingredients such as anthocyanins, while too high an enzyme amount will lead to an increase in enzyme costs and the generation of unnecessary enzymatic hydrolysis byproducts.

[0024] In addition, the enzymatic hydrolysis time determines the extent of the enzymatic hydrolysis reaction. Appropriate enzymatic hydrolysis time can ensure that the cell wall is fully decomposed and the active ingredients are effectively released. On the contrary, too long enzymatic hydrolysis time will lead to reduced enzyme activity and degradation of active ingredients.

[0025] In addition, the enzymolysis temperature is a key factor affecting the activity of the enzyme. Within a suitable temperature range, the enzyme can maintain a high activity and promote the enzymolysis reaction. On the contrary, too high a temperature can lead to the loss of enzyme activity and the degradation of active ingredients.

[0026] Additionally, within the range of 40-60 kHz, ultrasound can generate sufficient energy to accelerate the enzymatic reaction and the release of active ingredients. On the contrary, too high a frequency can lead to overheating of the solvent and destruction of the active ingredients.

[0027] Specifically, the effective working pH range of acidic protease is 2.0-6.0, and the optimum pH range is 2.5-3.5; the effective working pH range of pectinase is 3.0-6.0, and the optimum pH range is 3.0-3.5; the effective working pH range of cellulase is 4.0-7.0, and the optimum pH range is 4.0-5.0; the effective working pH range of β-glucanase is 4.0-6.0, and the optimum pH range is 4.0-5.0; in addition, acidic protease, pectinase, cellulase, and β-glucanase will gradually lose their activity above 70°C. When the enzymatic hydrolysis is completed, the ultrasonic treatment is immediately stopped, and then the pH value is adjusted between 7.0 and 7.5 by adding sodium hydroxide solution to the second black wolfberry extract to reduce the continued action of the complex enzyme. Compared with the method of heating the mixed solution to 70-80°C and maintaining it for 10-15 minutes to inactivate the enzyme, adjusting the pH value can reduce the effect of the heating process on the thermal stability of anthocyanins. After the ultrasonic treatment is completed, the mixed solution is allowed to cool naturally or quickly cooled by a cold water bath, and then the second black wolfberry extract is collected.

[0028] Furthermore, in step S5, a ceramic membrane and an ultrafiltration membrane are first used for filtration, and then a nanofiltration membrane is used for concentration.

[0029] Specifically, the ceramic membrane has excellent chemical corrosion resistance and thermal stability, and can withstand high pressure and temperature. At the same time, the ceramic membrane has a uniform pore size distribution and high filtration accuracy, and can effectively remove large particle impurities and bacteria. In step S5, the ceramic membrane acts as the first filtration barrier, which can remove large particle impurities and suspended matter in the second black wolfberry extract, and provide clean feed liquid for subsequent membrane filtration and concentration processes. The pore size of the ceramic membrane is between 0.2μm and 1.0μm, the operating pressure is 0.5-1.0 MPa, and the flow rate is 10-50 L / m 2 / h, temperature is 25-40℃.

[0030] Specifically, the ultrafiltration membrane can retain macromolecular substances such as proteins and polysaccharides, while allowing small molecular solutes and solvents to pass through. The pore size of the ultrafiltration membrane is 0.01-0.1 microns, the operating pressure is 0.5-1.0 MPa, and the flow rate is 10-50 L / m 2 / h, temperature is 25-40℃.

[0031] Specifically, after the ceramic membrane filtration, the ultrafiltration membrane can further remove small molecular impurities and colloidal substances in the feed liquid, thereby improving the purity of anthocyanins. In addition, the ultrafiltration membrane can also selectively separate the active ingredients according to their molecular weight, facilitating the subsequent purification steps.

[0032] Specifically, nanofiltration membranes can intercept small molecular solutes and some ions, such as pigments, oligosaccharides, etc., while allowing solvents to pass through. The pore size of nanofiltration membranes is between 0.5-10 nanometers, the operating pressure is 1.0-3.0 MPa, and the flow rate is 10-50 L / m 2 / h, the temperature is 25-40℃, after the ultrafiltration membrane filtration, the nanofiltration membrane can concentrate the liquid to increase the concentration of anthocyanins. At the same time, the nanofiltration membrane can also remove some small molecular impurities and ions, further improving the purity of anthocyanins.

[0033] Furthermore, in step S6, purification is performed using macroporous adsorption resin HPD100.

[0034] Specifically, HPD100 has a suitable pore size distribution, which can accommodate molecules of different sizes and achieve selective separation of target compounds through molecular sieve effect. The surface of HPD100 has abundant functional groups, such as hydroxyl and carboxyl groups, which can interact with target compounds, such as hydrogen bonding and ion exchange, to achieve efficient adsorption. HPD100 has good chemical stability and thermal stability, and can maintain stable adsorption performance in a wide range of pH values ​​and temperatures.

[0035] In the pretreatment process, before using the macroporous adsorption resin HPD100, pretreatment such as washing and regeneration is required to remove impurities and residues in the resin to ensure the smooth progress of the purification process. Specifically, the HPD100 macroporous adsorption resin is first rinsed with deionized water to remove white floating matter, then the resin is soaked in anhydrous ethanol for 24 hours, then rinsed with deionized water until there is no alcohol smell, and finally soaked in 40 g / L hydrochloric acid solution for 5 hours, repeatedly rinsed with water until neutral, and then soaked in 40 g / L sodium hydroxide solution for 5 hours, washed with water until neutral, and set aside.

[0036] During the column packing process, the pretreated HPD100 resin is loaded into the chromatography column to ensure that the resin is filled evenly and to avoid the generation of bubbles and voids.

[0037] During the loading process, slowly add the black wolfberry filtrate that has been filtered through the membrane to the top of the chromatography column, control the flow rate to avoid disturbing the resin bed, and the resin will selectively adsorb the target compound. Specifically, the loading amount does not exceed 30% of the resin column volume, and the flow rate is 1-5mL / min.

[0038] During the elution process, different concentrations of 30%-95% ethanol aqueous solution were used for gradient elution, and the elution flow rate was 1-5mL / min. Different components were gradually eluted according to the difference in affinity between the active ingredients and the resin.

[0039] During the collection process, the eluate containing the target active ingredient is collected and subsequently processed, and the collected eluate will have a higher purity and concentration.

[0040] Specifically, elution starts from low concentration ethanol, first using 30% ethanol, then collecting the eluent of each gradient according to 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol, and performing detection, such as by HPLC, UV-Vis and other instruments, to determine the distribution of the target component, gradually increase the ethanol concentration until 95% ethanol, and continue to collect the eluent. According to the detection results, specifically, the eluent of 60-80% ethanol is collected, and the purified product is obtained by combining the eluent containing the target component.

[0041] Furthermore, the freeze drying in step S7 comprises the following steps: M1, pre-freeze the filtered black wolfberry purified product at low temperature, the pre-freezing temperature is controlled between -35 and -45°C, and the time is between 2-3 hours; Step M1 converts the free water and bound water in the purified product into ice crystals to prepare for subsequent sublimation drying. Specifically, too low a temperature will lead to a loss of product quality, while too short a time will not be able to completely freeze all the water.

[0042] M2, at a vacuum degree of 200-500 mTorr, a material temperature of -30℃--35℃, and an absolute pressure of 4-7 Pa, to remove ice crystals and some moisture in the material; The vacuum degree is controlled at 200-500 mTorr, which helps to effectively remove ice crystals and some moisture in the material at a lower temperature. The material temperature is about -30℃--35℃. In this temperature range, ice crystals can gradually sublimate without destroying the active ingredients in the material. The absolute pressure is about 4-7 Pa, which helps to control the evaporation rate of water during the drying process, thereby facilitating the sublimation of ice crystals and the removal of moisture. Under vacuum and low temperature conditions, ice crystals directly sublimate into water vapor and are discharged through the vacuum system. The drying time at this stage is between 12-24 hours to ensure that most of the ice crystals are removed.

[0043] M3. Gradually increase the material temperature from -30℃ to -35℃ to 30-35℃. The gradual increase in temperature within this temperature range helps to remove residual moisture and avoid damage to the material structure caused by rapid temperature changes. The vacuum degree is gradually reduced from 100-200 Pa to 50-100 Pa to remove residual moisture. As the temperature increases and the vacuum degree decreases, the residual moisture in the material gradually evaporates and is discharged through the vacuum system. During the analytical drying process, it is necessary to gradually increase the temperature and reduce the vacuum degree to avoid overheating of the product and the occurrence of oxidation reactions. The drying time at this stage is between 4-12 hours, so that all moisture is basically removed, thereby achieving the best drying effect.

[0044] Furthermore, between step S6 and step S7, the low-temperature vacuum concentration is also included, and vacuum low-temperature concentration is performed under the conditions of a temperature of 10-50° C. and a vacuum degree of -0.06 MPa to obtain a black wolfberry anthocyanin concentrate.

[0045] The process is carried out at a temperature of 10-50°C. This temperature range helps to maintain the stability of active ingredients such as black wolfberry anthocyanins, avoids ingredient degradation caused by high temperature, and ensures a sufficient water evaporation rate. By reducing the amount of solvent and increasing the concentration of the target ingredient, the vacuum degree is set to -0.06MPa. The lower pressure helps to lower the boiling point of the liquid, allowing water to evaporate at a lower temperature, thereby reducing the impact on heat-sensitive ingredients. By lowering the temperature and increasing the vacuum degree, the evaporation point of the solvent is reduced, thereby achieving the concentration of the black wolfberry purified product at a lower temperature, which helps to remove excess water, increase the concentration of the extract, and prepare for subsequent freeze-drying.

[0046] Since the concentration of the ethanol eluent in step S6 is relatively high, the purified product in step S6 is first concentrated. By reducing the ethanol content and the content of some water in the purified product, the conversion of free water and bound water in the purified product in step S7 into ice crystals can be accelerated.

[0047] Specifically, the product after low-temperature vacuum concentration has a lower water content, which helps to shorten the freeze-drying time and improve the drying efficiency. At the same time, due to the reduction of water, the stability and shelf life of the product may also be improved. Through low-temperature vacuum concentration, impurities and excess water can be further removed, the purity and concentration of black wolfberry anthocyanins can be increased, and the quality of the final product can be improved. The specific steps are: P1: Transfer the solution purified by macroporous adsorption resin HPD100 to the vacuum concentration equipment; ensure that the equipment is well sealed to prevent outside air from entering and affecting the vacuum degree.

[0048] P2: Turn on the vacuum pump and gradually reach the set vacuum degree of -0.06MPa. At the same time, start heating and control the temperature within the range of 10-50℃.

[0049] P3: During the concentration process, closely monitor the changes in temperature and vacuum degree, and adjust the heating power and the working status of the vacuum pump in time to ensure stable concentration conditions. As the water content decreases, the concentration of anthocyanins in black wolfberry gradually increases. It is necessary to prevent crystallization or burning caused by excessive concentration.

[0050] P4: When the concentrate reaches the predetermined volume or concentration, turn off the heating and vacuum pump, wait for the equipment to cool naturally to room temperature, and then take out the black wolfberry concentrate. At this time, a high concentration of black wolfberry anthocyanin solution is obtained, which is ready for subsequent freeze drying.

[0051] The entire preparation process of the present invention includes the steps of pretreatment, ultrasonic extraction, enzymolysis, centrifugation, membrane filtration, macroporous adsorption resin purification and freeze-dried powder preparation. During the preparation process, the influence of high temperature treatment on the stability of anthocyanins is reduced, the introduction of impurities is also reduced, and the purity of anthocyanins and the extraction rate of active ingredients are effectively improved.

[0052] Furthermore, another object of the present invention is to provide an application of an extraction method for improving the stability of anthocyanins in black wolfberry, wherein the black wolfberry extract containing anthocyanins is applied to functional food, comprising the following steps: N1. Place the freeze-dried black wolfberry powder into the fluidized bed equipment to prepare the wall material solution; the freeze-dried black wolfberry extract powder has high purity, high stability and good solubility, and the wall material can form a protective film to protect the black wolfberry extract from environmental factors such as oxygen, humidity and light. The wall material is dissolved in the solvent in a certain proportion to make a uniform wall material solution. When preparing the wall material solution, it is necessary to ensure that the wall material is completely dissolved to avoid the presence of particles or lumps.

[0053] N2. Spray the wall material solution evenly onto the fluidized black wolfberry powder through a nozzle to form a protective film. The fluidized bed uses airflow to suspend the powder particles in the air. In the fluidized bed, gaps are formed between the powder particles, so that the wall material solution can be evenly sprayed onto each particle. Specifically, the nozzle is set above the fluidized bed to spray the wall material solution at a certain angle and speed. Ensure that each powder can be covered by the wall material solution to form a uniform protective film.

[0054] N3. Pass hot air until the wall material is completely dry and solidified; the function of hot air is to accelerate the drying and solidification process of the wall material solution. While maintaining the fluidized state, pass hot air for drying. Set the appropriate temperature and air volume to promote the evaporation of water in the wall material solution so that the wall material gradually solidifies on the surface of the black wolfberry powder. At the same time, avoid thermal damage to the product. As the water evaporates, the wall material gradually forms a protective film on the surface of the black wolfberry powder particles. This protective film needs to be sufficiently dry and solidified to ensure that it can stably protect the black wolfberry extract.

[0055] N4. Repeat steps N2 and N3 until the coating thickness is achieved by spraying; after each spraying, ensure that the wall material is completely solidified before spraying again. Specifically, the coating thickness is between 20-200μm. A thicker coating layer can provide better protection, but it will also affect the solubility and taste of the product. If it is too thin, it may not achieve the expected protection effect.

[0056] N5. After coating, collect the coated black wolfberry powder. When the predetermined coating thickness is reached, turn off the hot air system and nozzle, and let the equipment cool naturally to room temperature. After coating, the coated black wolfberry powder can be separated from the fluidized bed using equipment such as a screen or cyclone separator. The coated black wolfberry powder can be stored in a dry, cool, dark place to maintain its stability and extend its shelf life. When preparing functional foods, the coated black wolfberry powder can be added to food as needed.

[0057] The black wolfberry extract containing anthocyanins provided by the present invention is applied to functional foods, which can effectively protect active ingredients such as anthocyanins from the influence of the external environment, and improve the stability and bioavailability of anthocyanins in the black wolfberry extract in functional foods. It not only enriches the use scenarios of the black wolfberry extract, brings healthier and more nutritious food choices to consumers, but also promotes the development of the functional food industry.

[0058] Specifically, by precisely controlling the coating thickness and wall material selection, the release characteristics of the product can be further optimized to meet different health needs and taste preferences. A black wolfberry extract powder coated with a protective film can be obtained, and the black wolfberry extract powder coated with a protective film can be directly used as an ingredient of functional food, or used for further food processing. The coating protective film can improve the stability of black wolfberry powder, prevent oxidation, extend the shelf life, and improve its performance in food processing, and can also improve its taste and flavor in functional foods. In addition, by precisely controlling the parameters in the coating process, such as spray rate, air inlet temperature, and coating time, the coating effect can be further optimized to meet the needs of different application scenarios.

[0059] Furthermore, the wall material includes at least two of modified β-glucan ester, modified gum arabic, modified β-cyclodextrin, and glutinous rice starch.

[0060] Specifically, modified β-glucan ester, which is a natural polysaccharide, may have enhanced solubility, stability and emulsification after modification. During the microencapsulation process, β-glucan ester can act as an effective protective film to prevent the black wolfberry extract from being affected by oxygen, humidity and light.

[0061] The preparation method of modified β-glucan ester is: Q1: Prepare octenyl succinic anhydride-ethanol solution, and adjust the pH value to 8-10 after mixing β-glucan with 95% ethanol solution; specifically, prepare octenyl succinic anhydride ethanol solution with 3-5 times the mass of ethanol. Then, mix β-glucan with 95% ethanol solution evenly, and adjust the pH value to 8-10 with sodium hydroxide solution and hydrochloric acid solution. This step can ensure that β-glucan is fully dissolved and reacts with octenyl succinic anhydride. The mass ratio of octenyl succinic anhydride to β-glucan is between 1:10 and 1:20.

[0062] Q2: While stirring, spray the octenyl succinic anhydride ethanol solution into the β-glucan ethanol solution to carry out esterification reaction; the reaction can be carried out in a microwave reactor to improve efficiency and energy saving. The reaction conditions are a temperature of 55°C to 85°C and a reaction time of 2-6 hours.

[0063] Q3: After the reaction is completed, the pH value is adjusted to neutral, washed and dried to obtain octenyl succinic anhydride β-glucan ester. After the reaction is completed, the product needs to be post-treated, neutralized to a neutral pH value using hydrochloric acid solution and sodium hydroxide solution, and then washed with ethanol to remove substances produced by side reactions and octenyl succinic anhydride that did not participate in the reaction. Finally, the product is dried to constant weight to obtain octenyl succinic anhydride β-glucan ester.

[0064] Specifically, the hydrophobic groups of octenyl succinic anhydride β-glucan ester can be close to each other to form a hydrophobic core, while the hydrophilic groups are in contact with water molecules to form the shell of the micelle. This structure can effectively wrap and protect anthocyanidins to prevent them from being destroyed by external factors such as light, heat, and oxygen, thereby improving the stability of anthocyanidins. The micellar structure of octenyl succinic anhydride β-glucan ester can increase the solubility of anthocyanidins, especially in aqueous media. This increase in solubility helps anthocyanidins to be better absorbed and utilized by the human body. The addition of octenyl succinic anhydride β-glucan ester can improve the taste of food, making it more delicate and smooth, thereby covering up some of the astringency or bitterness of anthocyanidins themselves.

[0065] Specifically, gum arabic is a natural gum with excellent film-forming properties and stability. After modification, its adhesion and solubility are improved, making it more suitable for microencapsulation coating. Modified gum arabic is mainly used to enhance the adhesion and water resistance of the coating layer, so that the black wolfberry extract can maintain good stability even in a humid environment.

[0066] The preparation method of modified gum arabic is as follows: T1. Prepare a gum arabic-ethanol solution, adjust the pH value to 9-11, weigh glyceryl monolaurate; the mass ratio of glyceryl monolaurate to gum arabic is between 1:10 and 1:20. Dissolve gum arabic in anhydrous ethanol, heat to 50-60°C to promote dissolution and form a uniform solution. Under stirring, slowly add an appropriate amount of alkaline catalyst sodium hydroxide solution and adjust the pH value to 9-11.

[0067] T2. While stirring, slowly add glyceryl monolaurate to evenly disperse it in the gum arabic solution, heat the mixture to 50-80°C, and react for 2-6 hours; the addition speed should be moderate to avoid excessive local concentration leading to agglomeration or precipitation.

[0068] T3. After the reaction is completed, adjust the pH value to neutral, wash, dry, seal and store for later use. After the reaction is completed, cool to room temperature. Add an appropriate amount of acid such as hydrochloric acid or acetic acid to neutralize the remaining alkaline catalyst and adjust the pH value to neutral. Wash the product with a large amount of deionized water or ethanol to remove unreacted monolaurin and solvent, filter the washed product, and dry it in a vacuum drying oven until it is completely dry. Collect the dried monolaurin modified gum arabic and seal and store for later use.

[0069] Specifically, monolaurin is a nonionic surfactant with good emulsifying and dispersing properties. When it is combined with gum arabic to form monolaurin modified gum arabic, anthocyanins can be more effectively dispersed in the system to prevent aggregation and precipitation between anthocyanin molecules. This emulsification and dispersion effect helps to reduce the degradation reaction of anthocyanins due to direct contact, thereby improving their stability. Monolaurin itself has an antibacterial effect. The monolaurin modified gum arabic formed by combining with gum arabic can inhibit the growth of microorganisms and reduce the degradation of anthocyanins, thereby extending the shelf life of anthocyanin products.

[0070] Specifically, β-cyclodextrin is a cyclic oligosaccharide with a cavity structure that can contain and stabilize a variety of compounds. The modified β-cyclodextrin has better solubility and inclusion capacity. The modified β-cyclodextrin is mainly used to improve the embedding efficiency and sustained-release performance of the coating layer, so that the black wolfberry extract can be more effectively absorbed and utilized in the human body.

[0071] The preparation method of modified β-cyclodextrin is: V1. Mix the β-cyclodextrin solution with the alkali solution, cool it to room temperature, slowly add propylene oxide in an ice bath, and continue stirring until the oil layer disappears; heat the sodium hydroxide solution to 60°C. While stirring, slowly add β-cyclodextrin until it is completely dissolved. Cool the solution to room temperature. Under ice bath conditions, add propylene oxide evenly within 3 hours while stirring. After the addition is completed, adjust to room temperature and continue stirring until the oil layer disappears. The molar ratio of propylene oxide to β-cyclodextrin is between 1:1 and 5:1. The concentration of β-cyclodextrin solution is 20-30%, the room temperature reaction temperature is 30-40°C, and after the β-cyclodextrin solution is mixed with the alkali solution, the sodium hydroxide accounts for 3% of the total mass.

[0072] V2. After the reaction is completed, adjust the pH of the solution to neutral, perform vacuum distillation, and obtain a concentrate; use hydrochloric acid to neutralize the solution to adjust the pH to 7.0. Perform vacuum distillation at 45°C to obtain a slurry.

[0073] V3. Use 95% ethanol to dissolve the concentrate, add sodium sulfate to precipitate it, and remove unreacted salts and other impurities by filtering; use 95% ethanol to dissolve the slurry, and stir to precipitate sodium sulfate. After filtering out the precipitate, the filtrate is again subjected to reduced pressure distillation at 45°C to obtain a concentrated slurry. After dissolving the concentrated slurry with water, it is dialyzed in running water for 3 hours, and is again subjected to reduced pressure distillation at 45°C to obtain the final product.

[0074] V4. Wash the product with acetone and dry it to obtain hydroxypropyl-β-cyclodextrin. Collect the dried hydroxypropyl-β-cyclodextrin and seal it for later use. The hydroxyl part of β-cyclodextrin is replaced by a hydroxypropyl group. This modification increases the hydrophilicity and solubility of cyclodextrin while maintaining its cavity structure. The hydrophilic outer layer of hydroxypropyl-β-cyclodextrin can also promote the dispersion of anthocyanins in water and improve the solubility of anthocyanins. Through inclusion complexation, anthocyanins can be more evenly dispersed in the solution, reducing degradation and precipitation caused by poor dissolution. Hydroxypropyl-β-cyclodextrin has a transdermal absorption promoting effect, which is beneficial for active ingredients such as anthocyanins to enter the body through the skin barrier. When anthocyanins exist in the form of hydroxypropyl-β-cyclodextrin inclusion complex, their intestinal absorption rate will be improved. The inclusion complex can protect anthocyanins from degradation by enzymes and microorganisms in the intestine, thereby increasing their stability and absorption rate in the intestine. In addition, hydroxypropyl-β-cyclodextrin has certain antioxidant properties, so it can enhance the antioxidant properties of anthocyanins to a certain extent and reduce the degradation of anthocyanins.

[0075] Specifically, glutinous rice starch is a natural plant starch with good viscosity and gelling properties. Glutinous rice starch is mainly used to improve the taste and texture of the coating layer, making the final product more delicate and smooth, while increasing the satiety of the product. In the microencapsulation process, glutinous rice starch acts as part of the wall material to provide additional protection and support.

[0076] Specifically, different wall material components have different functions and advantages. By using them in combination, they can exert synergistic effects and improve the effect of microencapsulation coating. The combination of multiple wall materials can form a stronger and more stable protective film to prevent the black wolfberry extract from being affected by environmental factors.

[0077] Optionally, in some embodiments, the mixing ratio of the wall materials, calculated by mass, can be: 20-30 parts of octenylsuccinic anhydride β-glucan ester, 20-30 parts of monolaurin modified gum arabic, 30-40 parts of hydroxypropyl-β-cyclodextrin, and 10-20 parts of glutinous rice starch.

[0078] Optionally, in some embodiments, the mixing ratio of the wall materials, calculated by weight, may be: 50-60 parts of octenylsuccinic anhydride β-glucan ester, 10-20 parts of monolaurin modified gum arabic, and 20-30 parts of glutinous rice starch.

[0079] Optionally, in some embodiments, the mixing ratio of the wall materials, calculated by weight, may be: 30-40 parts of octenylsuccinic anhydride β-glucan ester, 30-40 parts of hydroxypropyl-β-cyclodextrin, and 20-30 parts of glutinous rice starch.

[0080] Optionally, in some embodiments, the mixing ratio of the wall materials, calculated by weight, may be: 30-40 parts of hydroxypropyl-β-cyclodextrin, 30-40 parts of monolaurin modified gum arabic, and 20-30 parts of glutinous rice starch.

[0081] Optionally, in some embodiments, the mixing ratio of the wall materials, calculated by weight, may be: 30-40 parts of octenylsuccinic anhydride β-glucan ester, 30-40 parts of hydroxypropyl-β-cyclodextrin, and 30-40 parts of monolaurin modified gum arabic.

[0082] Furthermore, the concentration of the wall material solution is 10-30%, the ratio of black wolfberry extract to wall material is 1:1 to 1:5, and the air outlet temperature is 30-40°C.

[0083] Specifically, the concentration of the wall material solution is 10-30%. This concentration range ensures that the wall material solution has sufficient viscosity and stability to form a uniform protective film during the spraying process. The concentration of the wall material solution directly affects the thickness and uniformity of the coating layer. Too low a concentration may result in a coating layer that is too thin and unable to provide adequate protection; too high a concentration may result in a coating layer that is too thick, affecting the taste and solubility of the product. Specifically, in order to improve solubility and prevent cohesion precipitation, the wall material solution uses pure water as the dissolution medium and contains 10% ethanol to improve the stability of octenyl succinic anhydride β-glucan ester and monolaurin modified gum arabic.

[0084] Specifically, the mass fraction ratio of black wolfberry extract to wall material ranges from 1:1 to 1:5. A higher wall material ratio can provide a thicker protective layer and enhance the storage resistance and stability of the product; while a lower wall material ratio can reduce costs and maintain the natural properties of the product. A high ratio will result in the coating layer not being able to completely cover the black wolfberry extract, affecting its stability; a low ratio will waste wall materials and increase costs.

[0085] Specifically, an outlet air temperature of 30-40°C can effectively promote the evaporation of water in the wall material solution and solidify the wall material on the surface of the black wolfberry powder. The appropriate outlet air temperature can speed up the drying process and improve production efficiency. Too low a temperature may cause the drying speed to be too slow, affecting production efficiency; too high a temperature may cause heat damage to the black wolfberry extract, reducing product quality.

[0086] Furthermore, the inlet air temperature is 40-60°C, the spray rate is 10-50 mL / min, and the coating time is 1-4 hours.

[0087] Specifically, controlling the air inlet temperature between 40-60°C can ensure that the wall material can be dried evenly and quickly, while avoiding damage to the nutrients in the black wolfberry powder. If the temperature is too high, it may cause loss of nutrients in the black wolfberry powder or excessive carbonization of the wall material; if the temperature is too low, the drying speed will slow down, affecting the coating efficiency.

[0088] Specifically, an appropriate spray rate can ensure that the wall material solution is evenly sprayed onto the black wolfberry powder to form a uniform protective film. If the spray rate is too fast, the wall material will be unevenly deposited on the powder particles, forming a coating layer of varying thickness; if the spray rate is too slow, it will affect the coating efficiency and increase production costs. If the coating time is too short, the wall material may not be completely dried and solidified, affecting the coating effect; if the coating time is too long, it will increase production costs and energy consumption.

[0089] Compared with the prior art, the present invention has the following advantages: 1. The entire preparation process of the present invention includes the steps of pretreatment, ultrasonic extraction, enzymolysis, centrifugation, membrane filtration, macroporous adsorption resin purification and freeze-dried powder preparation. During the preparation process, the influence of high temperature treatment on the stability of anthocyanins is reduced, the introduction of impurities is also reduced, and the purity of anthocyanins and the extraction rate of active ingredients are effectively improved.

[0090] 2. The black wolfberry extract containing anthocyanins provided by the present invention is applied to functional foods, which can effectively protect the active ingredients of anthocyanins from the influence of the external environment, improve the stability and bioavailability of anthocyanins in functional foods, and not only enrich the use scenarios of anthocyanins, bring healthier and more nutritious food choices to consumers, but also promote the development of the functional food industry. DETAILED DESCRIPTION

[0091] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0092] The present invention will be further described below in conjunction with specific embodiments: A method for extracting anthocyanins from black wolfberry to improve their stability comprises the following steps: S1. Pretreatment: Rinse the frozen fresh black wolfberry and place it in a reaction container, add water to crush and pulp it to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water is 1:3 to 1:5.

[0093] S2, ultrasonic extraction: add ethanol to the black wolfberry slurry obtained in step S1, and combine with ultrasonic assisted extraction to obtain the first black wolfberry extract; add 75% ethanol to make the ethanol concentration in the final solution reach 40%-60%. Adjust the pH value of the black wolfberry slurry between 3.5-6.0, the extraction temperature is 30-50°C, the ultrasonic power is 200-500w, and the extraction time is 15-20min.

[0094] S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to between 3.5 and 6.0, adding at least three kinds of complex enzymes of acid protease, pectinase, cellulase and β-glucanase to the first black wolfberry extract, combining with ultrasonic extraction, to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.01-0.08% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 60-100 min, the enzymatic hydrolysis temperature is 25-50° C., and the ultrasonic frequency is 40-60 kHz.

[0095] When the enzymatic hydrolysis is completed, the ultrasonic treatment is immediately stopped, and the pH value is adjusted between 7.0 and 7.5 by adding sodium hydroxide to the second black wolfberry extract to reduce the continued action of the complex enzyme.

[0096] S4, centrifugal collection: centrifugally filter the second black wolfberry extract obtained in step S3, and collect the supernatant; The centrifugal collection process is performed at a speed of 3000-5000 rpm and takes 10-20 minutes.

[0097] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0098] The pore size of the ceramic membrane is between 0.2μm and 1.0μm, the operating pressure is 0.5-1.0 MPa, and the flow rate is 10-50 L / m 2 / h, temperature is 25-40℃. The pore size of ultrafiltration membrane is 0.01-0.1 micron, the operating pressure is 0.5-1.0 MPa, and the flow rate is 10-50L / m 2 / h, temperature is 25-40℃.

[0099] The pore size of the nanofiltration membrane is between 0.5-10 nanometers, the operating pressure is 1.0-3.0 MPa, and the flow rate is 10-50 L / m 2 / h, temperature 25-40℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, preparation of freeze-dried powder: freeze-dry, crush and sieve the purified black wolfberry product in step S6 in sequence to obtain a black wolfberry extract containing anthocyanins.

[0100] The freeze-dried powder was crushed and sieved through a 80-100 mesh sieve to ensure uniform particles.

[0101] Specifically, the basic operations are as follows: In step S6, in the pretreatment process, the HPD100 macroporous adsorption resin is first rinsed with deionized water to remove white floating matter, and then the resin is soaked in anhydrous ethanol for 24 hours, and then rinsed with deionized water until there is no alcohol smell, and finally soaked in 40 g / L HCl solution for 5 hours, repeatedly rinsed with water until neutral, and then soaked in 40 g / L NaOH solution for 5 hours, washed with water until neutral, and set aside.

[0102] During the column packing process, the pretreated HPD100 resin is loaded into the chromatography column.

[0103] During the loading process, the black wolfberry filtrate that had been filtered through a membrane to remove impurities was slowly added to the top of the chromatography column. The loading amount was 25% of the resin column volume, and the flow rate was 2 mL / min.

[0104] In the elution process, different concentrations of 30%-95% ethanol aqueous solution are used for gradient elution, and the elution flow rate is 3mL / min. Start elution from low concentration ethanol, first use 30% ethanol, then collect the eluent of each gradient according to 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol, and detect it. Use HPLC instrument to determine the distribution of target components, gradually increase the ethanol concentration until 95% ethanol, and continue to collect eluent. According to the test results, the 60-80% ethanol eluent containing the target component is combined to obtain a purified product.

[0105] The freeze drying in step S7 comprises the following steps: M1. Pre-freeze the filtered black wolfberry purified product at low temperature, with the pre-freezing temperature controlled at -40°C for 2.5 hours; M2, at a vacuum degree of 300 mTorr, a material temperature of -30°C, an absolute pressure of 6 Pa, and a drying time of 20 hours.

[0106] M3. Gradually increase the material temperature from -30°C to 32°C. Gradually reduce the vacuum degree from 150 Pa to 80 Pa, and the drying time is 8 hours.

[0107] Furthermore, between step S6 and step S7, the low-temperature vacuum concentration is also included, and vacuum low-temperature concentration is performed under the conditions of a temperature of 20° C. and a vacuum degree of -0.06 MPa to obtain a black wolfberry anthocyanin concentrate.

[0108] The specific steps are: P1: Transfer the solution purified by macroporous adsorption resin HPD100 to the vacuum concentration equipment; ensure that the equipment is well sealed to prevent outside air from entering and affecting the vacuum degree.

[0109] P2: Turn on the vacuum pump and gradually reach the set vacuum degree of -0.06MPa. At the same time, start heating and control the temperature within 20℃.

[0110] P3: During the concentration process, closely monitor changes in temperature and vacuum degree.

[0111] P4: When the concentrate reaches one-third of the volume of the original purified product solution, turn off the heating and vacuum pump, wait for the equipment to cool naturally to room temperature, and then take out the black wolfberry concentrate.

[0112] The black wolfberry extract containing anthocyanins is applied to functional food, comprising the following steps: N1. Place the freeze-dried black wolfberry extract into a fluidized bed device to prepare a wall material solution; the concentration of the wall material solution is 30%, and the ratio of the black wolfberry extract to the wall material is 1:3; the wall material solution uses pure water as a dissolving medium and contains 10% ethanol.

[0113] N2. Spray the wall material solution evenly onto the fluidized black wolfberry powder through a nozzle to form a protective film; the spray rate is 30 mL / min.

[0114] N3. Let in hot air until the wall material is completely dry and solidified; the inlet air temperature is 45℃ and the outlet air temperature is 35℃.

[0115] N4. Repeat steps N2 and N3 until the coating thickness reaches 80 μm.

[0116] N5. After coating, collect the coated black wolfberry powder. The coating time is 3 hours.

[0117] The preparation method of modified β-glucan ester is: Q1: Specifically, octenyl succinic anhydride is prepared into an octenyl succinic anhydride ethanol solution with 4 times the mass of ethanol. Then, 60 parts of β-glucan and 40 parts of 95% ethanol solution are mixed evenly by mass, and the pH value is adjusted to 9 with sodium hydroxide solution and hydrochloric acid solution. The mass ratio of octenyl succinic anhydride to β-glucan is 1:10.

[0118] Q2: While stirring, spraying the ethanol solution of octenylsuccinic anhydride into the ethanol solution of β-glucan to carry out esterification reaction; the reaction is carried out in a microwave reactor under the reaction conditions of temperature 70° C. and reaction time 4 hours.

[0119] Q3: After the reaction is completed, the solution is neutralized to a neutral pH value using hydrochloric acid solution and sodium hydroxide solution, and then washed with ethanol to remove the substances produced by the side reaction and the octenyl succinic anhydride that did not participate in the reaction. Finally, the product is dried to a constant weight to obtain octenyl succinic anhydride β-glucan ester.

[0120] The preparation method of modified gum arabic is as follows: T1. Weigh monolaurin; the mass ratio of monolaurin to gum arabic is 1:15. Dissolve 60 parts of gum arabic in 40 parts of anhydrous ethanol by mass, and heat to 50-60°C to promote dissolution and form a uniform solution. Slowly add an appropriate amount of alkaline catalyst sodium hydroxide solution under stirring, and adjust the pH value to 10.

[0121] T2. While stirring, slowly add glyceryl monolaurate to make it evenly dispersed in the gum arabic solution, heat the mixture to 60°C, and react for 4 hours; T3. After the reaction is completed, adjust the pH value to neutral, wash, dry, seal and store for later use. After the reaction is completed, cool to room temperature.

[0122] The preparation method of modified β-cyclodextrin is: V1. Mix the β-cyclodextrin solution with the alkali solution, cool it to room temperature, slowly add propylene oxide in an ice bath, and continue stirring until the oil layer disappears; heat the NaOH solution to 60°C. While stirring, slowly add a 25% β-cyclodextrin solution until it is completely dissolved. After the β-cyclodextrin solution is mixed with the alkali solution, NaOH accounts for 3% of the total mass. Cool the solution to room temperature. Under ice bath conditions, add propylene oxide evenly within 3 hours while stirring. After the addition is completed, adjust to room temperature and continue stirring. The room temperature reaction temperature is 35°C until the oil layer disappears. The molar ratio of propylene oxide to β-cyclodextrin is 3:1.

[0123] V2. After the reaction, HCl was used to neutralize the solution to a pH of 7. The solution was distilled under reduced pressure at 45°C to obtain a slurry.

[0124] V3. Use 95% ethanol to dissolve the concentrate, add sodium sulfate to precipitate it, and remove unreacted salts and other impurities by filtering; After filtering off the precipitate, the filtrate was distilled under reduced pressure at 45°C again to obtain a concentrated slurry. The concentrated slurry was dissolved with water and dialyzed with running water for 3 hours. The concentrated slurry was distilled under reduced pressure at 45°C again to obtain the final product.

[0125] V4. Wash the product with acetone and dry it to obtain hydroxypropyl-β-cyclodextrin. Collect the dried hydroxypropyl-β-cyclodextrin and seal it for future use.

[0126] Example 1 A method for extracting anthocyanins from black wolfberry to improve their stability, comprising the following steps: S1. Pretreatment: 300 g of frozen fresh black wolfberry was rinsed and placed in a reaction container, and water was added to crush and pulp to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water was 1:5.

[0127] S2, ultrasonic extraction: adding ethanol to the black wolfberry slurry obtained in step S1, and combining with ultrasonic-assisted extraction to obtain a first black wolfberry extract; adding 75% ethanol to make the ethanol concentration in the final solution reach 45%. The pH value of the black wolfberry slurry was adjusted to 3.5, the extraction temperature was 30°C, the ultrasonic power was 200w, and the extraction time was 20min.

[0128] S3, enzymatic extraction: adjust the pH value of the first black wolfberry extract in step S2 to between 3.5, add a complex enzyme of acid protease, pectinase, cellulase and β-glucanase to the first black wolfberry extract, and combine with ultrasonic extraction to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.05% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 60 min, the enzymatic hydrolysis temperature is 25°C, and the ultrasonic frequency is 60 kHz.

[0129] Calculated by weight, there are 20 parts of acid protease, 30 parts of pectinase, 35 parts of cellulase, and 15 parts of β-glucanase. When the enzymolysis is completed, the ultrasonic treatment is immediately stopped, and sodium hydroxide is added to the second black wolfberry extract to adjust the pH value to 7.5 to reduce the continued action of the complex enzyme.

[0130] S4, centrifugal collection: centrifuge and filter the second black wolfberry extract obtained in step S3 to collect the supernatant; centrifuge and collect at 3000 rpm for 20 minutes.

[0131] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0132] The pore size of the ceramic membrane is 0.2μm, the operating pressure is 0.5MPa, and the flow rate is 10L / m 2 / h, temperature is 25℃. The pore size of the ultrafiltration membrane is 0.01 micron, the operating pressure is 0.5MPa, and the flow rate is 10L / m 2 / h, temperature is 25℃.

[0133] The nanofiltration membrane has a pore size of 0.5 nanometers, an operating pressure of 1.0 MPa, and a flow rate of 10 L / m 2 / h, temperature is 25℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, freeze-dried powder preparation: freeze-dry, crush and sieve the purified black wolfberry product in step S6 to obtain a finished product.

[0134] The freeze-dried powder was crushed and sieved through an 80-mesh sieve to ensure uniform particles.

[0135] Among them, the macroporous adsorption resin purification in step S6, the freeze drying in step S7, the low-temperature vacuum concentration step between step S6 and step S7, the step of applying the black wolfberry extract containing anthocyanins to functional food, the preparation method of modified β-glucan ester, the preparation method of modified gum arabic, and the preparation method of modified β-cyclodextrin are shown in the basic operation. Calculated by mass, the mixing ratio of the wall material is: 40 parts of octenyl succinic anhydride β-glucan ester, 30 parts of hydroxypropyl-β-cyclodextrin, and 30 parts of monolaurin modified gum arabic.

[0136] Example 2 A method for extracting anthocyanins from black wolfberry to improve their stability, comprising the following steps: S1. Pretreatment: 300 g of frozen fresh black wolfberry was rinsed and placed in a reaction container, and water was added to crush and pulp to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water was 1:3.

[0137] S2, ultrasonic extraction: add ethanol to the black wolfberry slurry obtained in step S1, and combine with ultrasonic assisted extraction to obtain the first black wolfberry extract; add 75% ethanol to make the ethanol concentration in the final solution reach 40%. Adjust the pH value of the black wolfberry slurry to 6.0, the extraction temperature to 50°C, the ultrasonic power to 500w, and the extraction time to 15min.

[0138] S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to 6.0, adding a composite enzyme of acid protease, pectinase, cellulase, and β-glucanase to the first black wolfberry extract, and combining with ultrasonic extraction to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.08% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 100 min, the enzymatic hydrolysis temperature is 50°C, and the ultrasonic frequency is 40 kHz.

[0139] Calculated by weight, there are 20 parts of acid protease, 30 parts of pectinase, 35 parts of cellulase, and 15 parts of β-glucanase. When the enzymolysis is completed, the ultrasonic treatment is immediately stopped, and sodium hydroxide is added to the second black wolfberry extract to adjust the pH value to 7.5 to reduce the continued action of the complex enzyme.

[0140] S4, centrifugal collection: centrifuge and filter the second black wolfberry extract obtained in step S3 to collect the supernatant; the centrifugal collection is performed at 5000 rpm for 10 minutes.

[0141] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0142] The pore size of the ceramic membrane is 1.0 μm, the operating pressure is 1.0 MPa, and the flow rate is 50 L / m 2 / h, temperature is 40℃. The pore size of the ultrafiltration membrane is 0.1 micron, the operating pressure is 1.0 MPa, and the flow rate is 50 L / m 2 / h, temperature is 40℃.

[0143] The pore size of the nanofiltration membrane is between 10 nanometers, the operating pressure is 3.0 MPa, and the flow rate is 50 L / m 2 / h, temperature is 40℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, freeze-dried powder preparation: freeze-dry, crush and sieve the purified black wolfberry product in step S6 to obtain a finished product.

[0144] The freeze-dried powder was crushed and sieved through a 100-mesh sieve to ensure uniform particles.

[0145] Among them, the macroporous adsorption resin purification in step S6, the freeze drying in step S7, the low-temperature vacuum concentration step between step S6 and step S7, the step of applying the black wolfberry extract containing anthocyanins to functional food, the preparation method of modified β-glucan ester, the preparation method of modified gum arabic, and the preparation method of modified β-cyclodextrin are shown in the basic operation. Calculated by mass, the mixing ratio of the wall material is: 20 parts of octenyl succinic anhydride β-glucan ester, 30 parts of monolaurin modified gum arabic, 35 parts of hydroxypropyl-β-cyclodextrin, and 15 parts of glutinous rice starch.

[0146] Example 3 A method for extracting anthocyanins from black wolfberry to improve their stability, comprising the following steps: S1. Pretreatment: 300 g of frozen fresh black wolfberry was rinsed and placed in a reaction container, and water was added to crush and pulp to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water was 1:4.

[0147] S2, ultrasonic extraction: add ethanol to the black wolfberry slurry obtained in step S1, and combine with ultrasonic assisted extraction to obtain the first black wolfberry extract; add 75% ethanol to make the ethanol concentration in the final solution reach 50%. Adjust the pH value of the black wolfberry slurry to 5.0, the extraction temperature to 40°C, the ultrasonic power to 300w, and the extraction time to 18min.

[0148] S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to 5.0, adding a complex enzyme of acid protease, pectinase and cellulase to the first black wolfberry extract, combining with ultrasonic extraction, to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.01% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 70 min, the enzymatic hydrolysis temperature is 35°C, and the ultrasonic frequency is 50 kHz.

[0149] Calculated by weight, there are 30 parts of acid protease, 35 parts of pectinase, and 35 parts of cellulase. When the enzymolysis is completed, the ultrasonic treatment is immediately stopped, and sodium hydroxide is added to the second black wolfberry extract to adjust the pH value to 7.0 to reduce the continued action of the complex enzyme.

[0150] S4, centrifugal collection: centrifuge and filter the second black wolfberry extract obtained in step S3 to collect the supernatant; the centrifugal collection is performed at 4000 rpm for 15 minutes.

[0151] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0152] The pore size of the ceramic membrane is 0.5 μm, the operating pressure is 0.75 MPa, and the flow rate is 30 L / m 2 / h, temperature is 35℃. The pore size of the ultrafiltration membrane is 0.05 micron, the operating pressure is 0.75 MPa, and the flow rate is 30 L / m 2 / h, temperature is 35℃.

[0153] The nanofiltration membrane has a pore size of 3 nanometers, an operating pressure of 2.0 MPa, and a flow rate of 30 L / m 2 / h, temperature is 35℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, freeze-dried powder preparation: freeze-dry, crush and sieve the purified black wolfberry product in step S6 to obtain a finished product.

[0154] The freeze-dried powder was crushed and sieved through a 90-mesh sieve to ensure uniform particles.

[0155] Among them, the macroporous adsorption resin purification in step S6, the freeze drying in step S7, the low-temperature vacuum concentration step between step S6 and step S7, the step of applying the black wolfberry extract containing anthocyanins to functional food, the preparation method of modified β-glucan ester, and the preparation method of modified gum arabic are shown in the basic operation. Calculated by mass, the mixing ratio of the wall material is: 50 parts of octenyl succinic anhydride β-glucan ester, 20 parts of monolaurin modified gum arabic, and 30 parts of glutinous rice starch.

[0156] Example 4 A method for extracting anthocyanins from black wolfberry to improve their stability, comprising the following steps: S1. Pretreatment: 300 g of frozen fresh black wolfberry was rinsed and placed in a reaction container, and water was added to crush and pulp to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water was 1:3.

[0157] S2, ultrasonic extraction: add ethanol to the black wolfberry slurry obtained in step S1, and combine with ultrasonic assisted extraction to obtain the first black wolfberry extract; add 75% ethanol to make the ethanol concentration in the final solution reach 55%. Adjust the pH value of the black wolfberry slurry to 4.5, the extraction temperature to 35°C, the ultrasonic power to 400w, and the extraction time to 18min.

[0158] S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to between 3.5 and 6.0, adding a composite enzyme of pectinase, cellulase and β-glucanase to the first black wolfberry extract, combining with ultrasonic extraction, to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.03% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 90 min, the enzymatic hydrolysis temperature is 45° C., and the ultrasonic frequency is 40 kHz.

[0159] Calculated by weight, there are 40 parts of pectinase, 40 parts of cellulase, and 20 parts of β-glucanase; when the enzymolysis is completed, the ultrasonic treatment is immediately stopped, and sodium hydroxide is added to the second black wolfberry extract to adjust the pH value to 7.0 to reduce the continued action of the complex enzyme.

[0160] S4, centrifugal collection: centrifugally filter the second black wolfberry extract obtained in step S3, and collect the supernatant; The centrifugation was performed at 3000 rpm for 20 minutes.

[0161] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0162] The pore size of the ceramic membrane is between 0.8μm, the operating pressure is 0.8MPa, and the flow rate is 20 L / m 2 / h, temperature is 30℃. The pore size of the ultrafiltration membrane is 0.08 micron, the operating pressure is 0.8MPa, and the flow rate is 20 L / m 2 / h, temperature is 30℃.

[0163] The nanofiltration membrane has a pore size of 8 nanometers, an operating pressure of 2.5 MPa, and a flow rate of 20 L / m 2 / h, temperature is 30℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, freeze-dried powder preparation: freeze-dry, crush and sieve the purified black wolfberry product in step S6 to obtain a finished product.

[0164] The freeze-dried powder was crushed and sieved through an 80-mesh sieve to ensure uniform particles.

[0165] Among them, the macroporous adsorption resin purification in step S6, the freeze drying in step S7, the low-temperature vacuum concentration step between step S6 and step S7, the step of applying the black wolfberry extract containing anthocyanins to functional food, the preparation method of modified β-glucan ester, and the preparation method of modified β-cyclodextrin are shown in the basic operation. Calculated by mass, the mixing ratio of the wall material is: 40 parts of octenyl succinic anhydride β-glucan ester, 30 parts of hydroxypropyl-β-cyclodextrin, and 30 parts of glutinous rice starch.

[0166] Example 5 A method for extracting anthocyanins from black wolfberry to improve their stability, comprising the following steps: S1. Pretreatment: 300 g of frozen fresh black wolfberry was rinsed and placed in a reaction container, and water was added to crush and pulp to obtain black wolfberry slurry; the ratio of fresh black wolfberry to purified water was 1:5.

[0167] S2, ultrasonic extraction: add ethanol to the black wolfberry slurry obtained in step S1, and combine with ultrasonic assisted extraction to obtain the first black wolfberry extract; add 75% ethanol to make the ethanol concentration in the final solution reach 60%. Adjust the pH value of the black wolfberry slurry to 5.5, the extraction temperature to 35°C, the ultrasonic power to 300w, and the extraction time to 15min.

[0168] S3, enzymatic extraction: the pH value of the first black wolfberry extract in step S2 is adjusted to 5.5, and a composite enzyme of acid protease, pectinase, cellulase and β-glucanase is added to the first black wolfberry extract, and ultrasonic extraction is performed to obtain a second black wolfberry extract; the first black wolfberry extract in step S2 is supplemented with pure water at a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.06% of the total mass of the first black wolfberry extract, the enzymatic hydrolysis time is 80 min, the enzymatic hydrolysis temperature is 35° C., and the ultrasonic frequency is 60 kHz.

[0169] Calculated by weight, there are 30 parts of acid protease, 20 parts of pectinase, 25 parts of cellulase, and 25 parts of β-glucanase. When the enzymolysis is completed, the ultrasonic treatment is immediately stopped, and sodium hydroxide is added to the second black wolfberry extract to adjust the pH value to 7.5 to reduce the continued action of the complex enzyme.

[0170] S4, centrifugal collection: centrifugally filter the second black wolfberry extract obtained in step S3, and collect the supernatant; The centrifugation was performed at 5000 rpm for 10 minutes.

[0171] S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities, thereby obtaining black wolfberry filtrate; firstly, the filtrate is filtered using a ceramic membrane and an ultrafiltration membrane, and then concentrated using a nanofiltration membrane.

[0172] The pore size of the ceramic membrane is 0.3μm, the operating pressure is 0.6MPa, and the flow rate is 40 L / m 2 / h, temperature is 35℃. The pore size of the ultrafiltration membrane is 0.03 micron, the operating pressure is 0.6MPa, and the flow rate is 40 L / m 2 / h, temperature is 35℃.

[0173] The nanofiltration membrane has a pore size of 5 nanometers, an operating pressure of 1.5 MPa, and a flow rate of 40 L / m 2 / h, temperature is 35℃ S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography, purified by macroporous adsorption resin HPD100, and the purified product containing active ingredients is collected; S7, freeze-dried powder preparation: freeze-dry, crush and sieve the purified black wolfberry product in step S6 to obtain a finished product.

[0174] The freeze-dried powder was crushed and sieved through a 100-mesh sieve to ensure uniform particles.

[0175] Among them, the macroporous adsorption resin purification in step S6, the freeze drying in step S7, the low-temperature vacuum concentration step between step S6 and step S7, the step of applying the black wolfberry extract containing anthocyanins to functional food, the preparation method of modified gum arabic, and the preparation method of modified β-cyclodextrin are shown in the basic operation. Calculated by mass, the mixing ratio of the wall material is: 40 parts of hydroxypropyl-β-cyclodextrin, 40 parts of monolaurin modified gum arabic, and 20 parts of glutinous rice starch.

[0176] Comparative Example 1 The main difference between Comparative Example 1 and Example 2 is that the low-temperature vacuum concentration step between step S6 and step S7 is omitted in Comparative Example 1, and the remaining steps and component parameters of Comparative Example 1 are the same as those of Example 2.

[0177] Comparative Example 2 The main difference between Comparative Example 2 and Example 2 is that the pH value is not adjusted in step S2 ultrasonic extraction and step S3 enzymatic extraction of Comparative Example 2, and the remaining steps and component parameters of Comparative Example 2 are the same as those of Example 2.

[0178] Comparative Example 3 The main difference between Comparative Example 3 and Example 2 is that the enzymatic extraction in step S3 of Comparative Example 3 uses a composite enzyme formed by pectinase and cellulase, and the remaining steps and component parameters of Comparative Example 3 are the same as those of Example 2.

[0179] Comparative Example 4 The main difference between Comparative Example 4 and Example 2 is that in step S3 of enzymatic extraction in Comparative Example 4, pure water is not added, and complex enzyme is directly added. The remaining steps and component parameters of Comparative Example 4 are the same as those of Example 2.

[0180] Comparative Example 5 The main difference between Comparative Example 5 and Example 2 is that after the enzymatic hydrolysis and extraction in step S3 of Comparative Example 5 is completed, the pH value is not adjusted, the mixed solution is heated to 70-80°C and maintained for 10-15 minutes to inactivate the enzyme, and the remaining steps and component parameters of Comparative Example 5 are the same as those of Example 2.

[0181] Comparative Example 6 The main difference between Comparative Example 6 and Example 2 is that ordinary filter paper is used for membrane filtration and impurity removal in step S5 of Comparative Example 6, and the remaining steps and component parameters of Comparative Example 6 are the same as those of Example 2.

[0182] Comparative Example 7 The main difference between Comparative Example 7 and Example 2 is that the macroporous adsorption resin purification in step S6 is omitted in Comparative Example 7, and the black wolfberry concentrate obtained in the low-temperature vacuum concentration step is subjected to freeze-dried powder preparation in step S7. The remaining steps and component parameters of Comparative Example 7 are the same as those of Example 2.

[0183] Comparative Example 8 The main difference between Comparative Example 8 and Example 2 is that the wall material used in Comparative Example 8 has a mixing ratio of 60 parts of β-cyclodextrin and 40 parts of glutinous rice starch calculated by weight. The remaining steps and component parameters of Comparative Example 8 are the same as those of Example 2.

[0184] Comparative Example 9 The main difference between Comparative Example 9 and Example 2 is that in step N3 of Comparative Example 9, hot air is introduced until the wall material is completely dried and solidified; the inlet air temperature is 80° C. and the outlet air temperature is 70° C. The coating time is 1 hour. The remaining steps and component parameters of Comparative Example 9 are the same as those of Example 2.

[0185] Comparative Example 10 The main difference between Comparative Example 10 and Example 2 is that the spray drying method is used in Comparative Example 10 to coat the wall material on the freeze-dried black wolfberry to improve its stability. The remaining steps and component parameters of Comparative Example 10 are the same as those of Example 2.

[0186] The steps of spray drying are: A. Mix the freeze-dried black wolfberry powder with the wall material in a ratio of 1:3, and use an appropriate amount of water to dissolve the mixture to form a uniform solution or suspension.

[0187] B. Send the mixed liquid into the spray drying tower, and atomize the liquid into fine droplets through a high-pressure pump or compressed air. The atomized droplets quickly evaporate water under the action of hot air to form solid particles. The feed rate is 100 mL / min, the inlet air temperature is 170℃, the outlet air temperature is 75℃, and the atomization pressure is 20 bar.

[0188] C. Collect products: The dried particles are collected from the bottom of the spray drying tower and cooled.

[0189] The above embodiments and comparative examples were subjected to performance tests, wherein the test results are shown in Table 1 and Table 2, and the test method is as follows: 1. The anthocyanin content in the black wolfberry extract was determined by the hydrochloric acid-methanol method. The black wolfberry extracts in Examples 1-5 and Comparative Examples 1-9 were prepared into solutions of appropriate concentrations. A certain volume of the solution was added to a stoppered test tube, and then 10 mL of a 2% hydrochloric acid-methanol solution was added for immersion. The solution was placed in a dark place for 30 min, and the absorbance at 520 nm and 700 nm was measured using a UV-visible spectrophotometer to calculate the anthocyanin yield and anthocyanin extraction rate.

[0190] 2. The coated black wolfberry extract was placed in a light (100 lux) environment at room temperature of 25°C for 30 days, and the retention rate of anthocyanins was measured and calculated.

[0191] 3. The coated black wolfberry extract was placed at room temperature of 25°C in a dark environment for 30 days to measure and calculate the retention rate of anthocyanins.

[0192] 4. The coated black wolfberry extract was placed at 60°C for 2 hours to measure and calculate the retention rate of anthocyanins.

[0193] Table 1: Performance test results of Examples 1-5

[0194] Table 2: Performance test results of comparative examples 1-10

[0195] In Example 1-Example 5, the entire preparation process of Example 1-Example 5 includes the steps of pretreatment, ultrasonic extraction, enzymolysis, centrifugation, membrane filtration, macroporous adsorption resin purification and freeze-dried powder preparation. From the anthocyanin extraction rate and anthocyanin yield in Table 1, it can be seen that during the preparation process, the effect of high temperature treatment on the stability of anthocyanins is reduced, and the introduction of impurities is also reduced, which effectively improves the purity of anthocyanins and the extraction rate of active ingredients. The black wolfberry extract containing anthocyanins provided by Example 1-Example 5 is applied to functional foods. From the embedding rate, light retention rate, light-proof retention rate, and 60°C retention rate in Table 1, it can be seen that the wall material can effectively protect active ingredients such as anthocyanins from the influence of the external environment, improve the stability and bioavailability of anthocyanins in functional foods, and not only enrich the use scenarios of anthocyanins, but also bring healthier and more nutritious food choices to consumers, but also promote the development of the functional food industry.

[0196] As can be seen from Table 2, compared with Example 2, Comparative Example 1 omits the low-temperature vacuum concentration step between step S6 and step S7. In Example 2, low-temperature vacuum concentration is used to shorten the freeze-drying time in step S7 and improve the drying efficiency, further remove impurities and excess water, improve the purity and concentration of black wolfberry anthocyanins, and help improve the quality of the final product. Therefore, in terms of yield, Comparative Example 1 is slightly lower than Example 2.

[0197] Compared with Example 2, the pH value is not adjusted in step S2 ultrasonic extraction and step S3 enzymatic extraction of Comparative Example 2. Since the acid protease, pectinase, cellulase, and β-glucanase are not in the optimal working pH range, it is not conducive to the activity of the composite enzyme. The composite enzyme cannot efficiently decompose the cell wall and increase the release rate of the active ingredients, resulting in a decrease in the final anthocyanin extraction rate of Comparative Example 2.

[0198] Comparative Example 3 Compared with Example 2, the enzymatic extraction of step S3 of Comparative Example 3 uses a composite enzyme formed by pectinase and cellulase. The acidic protease of the present invention can effectively enzymatically hydrolyze proteins combined with polysaccharides. It can decompose proteins into small molecular peptide chains or amino acids, thereby destroying the structure of cell walls and cell membranes, making it easier for other enzymes to enter the cell interior to act, and helping to release polysaccharides and flavonoids. In the polysaccharide extraction process, by first using protease to remove most of the protein, the subsequent polysaccharide extraction steps can be optimized. β-glucanase helps to destroy the cell wall and release nutrients. β-glucan is a soluble dietary fiber with multiple biological activities. β-glucan is decomposed by β-glucanase, destroying the structure of the cell wall and releasing the monosaccharide or oligosaccharide therein. Comparative Example 3 Conventional pectinase and cellulase can only destroy the structure of part of the cell wall and cell membrane and release part of the internal substances, so the final anthocyanin extraction rate of Comparative Example 3 will be lower than that of Example 2.

[0199] Comparative Example 4 Compared with Example 2, the enzymatic extraction in step S3 of Comparative Example 4 is not supplemented with pure water, and the composite enzyme is directly added. Although ethanol will volatilize during the treatment process of step S2, the first black wolfberry extract still contains a high ethanol concentration. When the ethanol concentration is high, ethanol will have a significant inhibitory effect on the activity of the enzyme. The ethanol molecules will interact with the key amino acid residues in the enzyme molecules, causing the spatial structure of the enzyme to change, thereby affecting its catalytic activity. The ethanol in Example 2 will be reduced to about 10%, which can reduce the impact on the activity of the composite enzyme. Therefore, the anthocyanin extraction rate of Example 2 is higher than the extraction rate of Comparative Example 4.

[0200] Compared with Example 2, in Comparative Example 5, the pH value is not adjusted after the enzymatic hydrolysis and extraction in step S3 of Comparative Example 5, and the mixed solution is heated to 70-80°C and maintained for 10-15 minutes to inactivate the enzyme. Since anthocyanins are sensitive to temperature, the high-temperature heating process will cause some anthocyanins to degrade, thereby resulting in a decrease in the anthocyanin extraction rate of Comparative Example 5.

[0201] Comparative Example 6 Compared with Example 2, after the supernatant of Example 2 is filtered through the ceramic membrane, the ultrafiltration membrane can further remove small molecular impurities and colloidal substances in the feed liquid, thereby improving the purity of the black wolfberry extract. After the ultrafiltration membrane is filtered, the nanofiltration membrane can concentrate the feed liquid to improve the concentration of the black wolfberry extract. At the same time, the nanofiltration membrane can also remove some small molecular impurities and ions, further improving the purity of the black wolfberry extract. The ordinary filter paper of Comparative Example 6 has a poor interception effect, which will increase the subsequent macroporous adsorption resin purification time and increase the pressure of the macroporous adsorption resin purification, thereby reducing the anthocyanin extraction rate of Comparative Example 6.

[0202] Comparative Example 7 Compared with Example 2, Comparative Example 7 omits the macroporous adsorption resin purification in step S6, and the black wolfberry concentrate obtained in the low-temperature vacuum concentration step is subjected to freeze-dried powder preparation in step S7. The macroporous adsorption resin in Example 2 can selectively adsorb according to the properties of the components, thereby achieving purification, and gradually eluting the target components with ethanol of different concentrations and collecting the purified products containing active components. Comparative Example 7 lacks the classified collection of the purified products containing active components, which will lead to an increase in non-target products, and the increase in impurities will lead to a decrease in the anthocyanin extraction rate of Comparative Example 7.

[0203] Comparative Example 8 Compared with Example 2, the wall material used in Comparative Example 8 is calculated by weight, and the mixing ratio of the wall material is 60 parts of β-cyclodextrin and 40 parts of glutinous rice starch. The modified β-glucan ester, modified gum arabic, modified β-cyclodextrin and glutinous rice starch of the present invention can be used in combination to exert the synergistic effect between them and improve the effect of microencapsulation coating. The combined use of multiple wall materials can form a stronger and more stable protective film to prevent the black wolfberry extract from being affected by environmental factors. Specifically, the hydrophobic groups of octenyl succinic anhydride β-glucan ester can be close to each other to form a hydrophobic core, while the hydrophilic groups are in contact with water molecules to form the shell of the micelle. This structure can effectively wrap and protect anthocyanins to prevent them from being destroyed by external factors such as light, heat, and oxygen, thereby improving the stability of anthocyanins. Modified gum arabic can more effectively disperse anthocyanins in the system to prevent aggregation and precipitation between anthocyanin molecules. This emulsification and dispersion helps to reduce the degradation reaction of anthocyanins due to direct contact, thereby improving its stability. Hydroxypropyl-β-cyclodextrin has a certain antioxidant property, so it can enhance the antioxidant property of anthocyanins to a certain extent. Therefore, when placed under light, placed away from light, and placed at 60°C, Example 2 has a higher retention rate.

[0204] Compared with Example 2, in Comparative Example 9, step N3, hot air is introduced until the wall material is completely dried and solidified; the inlet air temperature is 80°C, and the outlet air temperature is 70°C. The coating time is 1 hour. During the drying process, due to the high inlet air temperature and outlet air temperature, part of the anthocyanin is degraded, and at the same time, the coating time is shortened, resulting in part of the wall material not completely fitting the anthocyanin core material, and immediately bonding with the sprayed wall material, resulting in insufficient coating connection strength and reduced embedding rate. Therefore, when placed in light, away from light, and at 60°C, the retention rate of Comparative Example 9 is lower than that of Example 2.

[0205] Compared with Example 2, Comparative Example 10 adopts spray drying method to coat the wall material on the freeze-dried black wolfberry. During the drying process, due to the high inlet and outlet air temperatures, part of the anthocyanins are degraded. In addition, the mixed liquid is sent into the spray drying tower, which will cause part of the wall material to fail to completely coat the anthocyanin core material, and the embedding rate is reduced. When placed in light, placed away from light, and placed at 60°C, the retention rate of Comparative Example 10 is lower than that of Example 2.

[0206] The above is a preferred embodiment of the present invention. Relevant personnel in this technical field may make several improvements and modifications without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting anthocyanins from black wolfberry to improve their stability, characterized in that: The following steps are involved: S1, pretreatment: rinse the frozen fresh black wolfberry and place it in a reaction container, add water to crush and pulp, and obtain black wolfberry slurry; S2, ultrasonic extraction: adding ethanol to the black wolfberry slurry obtained in step S1, and performing ultrasonic-assisted extraction to obtain a first black wolfberry extract; S3, enzymatic extraction: adjusting the pH value of the first black wolfberry extract in step S2 to between 3.5 and 6.0, adding at least two complex enzymes of acid protease, pectinase, cellulase, and β-glucanase to the first black wolfberry extract, and combining with ultrasonic extraction to obtain a second black wolfberry extract; S4, centrifugal collection: centrifugally filter the second black wolfberry extract obtained in step S3, and collect the supernatant; S5, membrane filtration to remove impurities: the supernatant obtained in step S4 is subjected to membrane filtration to remove remaining impurities to obtain black wolfberry filtrate; S6, macroporous adsorption resin purification: the black wolfberry filtrate in step S5 is subjected to column chromatography to collect the purified product containing active ingredients; S7, preparation of freeze-dried powder: freeze-dry, crush and sieve the purified black wolfberry product in step S6 in sequence to obtain a black wolfberry extract containing anthocyanins.

2. The method for extracting anthocyanins from black wolfberry to improve their stability according to claim 1, characterized in that: In step S2, the pH value of the black wolfberry slurry is adjusted between 3.5-6.0, the extraction temperature is between 30-50° C., the ultrasonic power is 200-500w, and the extraction time is 15-20min.

3. The method for extracting anthocyanins from black wolfberry according to claim 1, characterized in that: In step S3, the first black wolfberry extract in step S2 is supplemented with pure water according to a solid-liquid ratio of fresh black wolfberry to the first black wolfberry extract of 1:35 (g:mL), the amount of enzyme added is 0.01-0.08% of the total mass of the first black wolfberry extract, the enzymolysis time is 60-100 min, the enzymolysis temperature is 25-50° C., and the ultrasonic frequency is 40-60 kHz.

4. The method for extracting anthocyanins from black wolfberry to improve their stability according to claim 1, characterized in that: In step S5, the ceramic membrane and the ultrafiltration membrane are first used for filtration, and then the nanofiltration membrane is used for concentration.

5. The method for extracting anthocyanins from black wolfberry to improve their stability according to claim 1, characterized in that: In step S6, purification is performed using macroporous adsorption resin HPD100.

6. The method for extracting anthocyanins from black wolfberry for improving stability according to claim 1, characterized in that: The freeze drying in step S7 comprises the following steps: M1. Pre-freeze the filtered black wolfberry purified product at low temperature, with the pre-freezing temperature controlled between -35 and -45°C for 2-3 hours; M2, at a vacuum degree of 200-500 mTorr, a material temperature of -30℃--35℃, and an absolute pressure of 4-7 Pa, to remove ice crystals and some water in the material; M3. Gradually increase the material temperature to 30-35°C and reduce the vacuum degree from 100-200 Pa to 50-100 Pa to remove residual moisture.

7. The method for extracting anthocyanins from black wolfberry to improve their stability according to claim 1, characterized in that: Between step S6 and step S7, low-temperature vacuum concentration is also included, and vacuum low-temperature concentration is performed under the conditions of a temperature of 10-50° C. and a vacuum degree of -0.06 MPa to obtain a black wolfberry concentrate.

8. The use of a method for improving the stability of anthocyanins from black wolfberry according to any one of claims 1 to 7, characterized in that: The black wolfberry extract containing anthocyanins is applied to functional food, comprising the following steps: N1. Place the freeze-dried black wolfberry extract into a fluidized bed device to prepare a wall material solution; N2, spraying the wall material solution evenly onto the fluidized black wolfberry extract through a nozzle to form a protective film; N3, pass hot air until the wall material is completely dry and solidified; N4, repeat steps N2 and N3 until the spraying reaches the coating thickness; N5. After coating, collect the coated black wolfberry extract.

9. The use of the method for improving the stability of anthocyanins from black wolfberry according to claim 8, characterized in that: The wall material comprises at least two of modified β-glucan ester, modified gum arabic, modified β-cyclodextrin and glutinous rice starch.

10. The use of the method for extracting anthocyanins from black wolfberry to improve their stability according to claim 8, characterized in that: The concentration of the wall material solution is 10-30%, the ratio of black wolfberry extract to wall material is between 1:1 and 1:5, the inlet air temperature is 40-60°C, the outlet air temperature is 30-40°C, the spray rate is 10-50 mL / min, and the coating time is 1-4 hours.

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