Hydrolyzed procyanidine freeze-dried powder and preparation method thereof

Abstract

The invention relates to the field of functional additives for daily chemicals, and particularly discloses hydrolyzed procyanidine freeze-dried powder and a preparation method thereof. The hydrolyzed procyanidine freeze-dried powder is prepared by freeze-drying and comprises the following raw materials in parts by weight: 0.1-0.2 part of hydrolyzed procyanidine, 25-35 parts of carnosine, 10-20 parts of a stabilizer, 0.1-0.3 part of acetyl hexapeptide-8 and 45-55 parts of water. The stabilizer is selected from at least one of mannan, trehalose, mannitol, cane sugar, betaine, whey protein, PVP (Polyvinyl Pyrrolidone), PEG (Polyethylene Glycol), glycine, histidine, leucine, sodium alginate, beta-cyclodextrin, maltodextrin and chitosan. The hydrolyzed procyanidine freeze-dried powder has good storage stability, can keep activity for a long time, is still stable after being dissolved in water for a long time, and has less activity loss.

Description

Technical Field

[0001] This application relates to the field of functional additives for daily chemical use, and more specifically, it relates to a hydrolyzed proanthocyanidin freeze-dried powder and its preparation method. Background Technology

[0002] In the food industry, consumer demand for healthy and functional foods is growing, making the application of natural functional ingredients a research hotspot. Hydrolyzed proanthocyanidins are natural polyphenolic compounds with various biological activities such as antioxidation, anti-inflammation, and antibacterial properties. They can effectively eliminate superoxide anion free radicals and hydroxyl free radicals, participate in the metabolism of phosphoric acid and arachidonic acid and protein phosphorylation, protect lipids from oxidative damage, and have soothing and anti-aging effects. They have broad application prospects in the fields of food, medicine, and cosmetics.

[0003] However, hydrolyzed proanthocyanidins are extremely sensitive to temperature and readily undergo degradation reactions at high temperatures, leading to the destruction of their molecular structure, a significant reduction or even complete loss of their biological activity, and a weakening or disappearance of their antioxidant, anti-inflammatory, and antibacterial functions. This diminishes their value for applications in food, pharmaceuticals, and cosmetics. Furthermore, high temperatures can alter the color of hydrolyzed proanthocyanidins, causing discoloration. This makes conventional processing methods such as high-temperature drying, sterilization, baking, and heat concentration unsuitable for the production and preservation of hydrolyzed proanthocyanidin products. Additionally, hydrolyzed proanthocyanidins are unstable in water; their molecular structure readily undergoes hydrolysis and oxidation upon contact with water. Prolonged dissolution in water leads to a continuous decrease in the content of active substances, a significant reduction in biological activity, and even complete loss, severely limiting their industrial application. In actual production and storage, even with conventional protective measures such as low temperatures and protection from light, it is difficult to effectively prevent the attenuation of their activity in aqueous solutions, resulting in products failing to achieve expected efficacy, shortened shelf life, and increased production costs. Summary of the Invention

[0004] In order to improve the bioactivity of hydrolyzed proanthocyanidin products and avoid the reduction of activity caused by prolonged dissolution in water, this application provides a lyophilized hydrolyzed proanthocyanidin powder and its preparation method.

[0005] In the first aspect, this application provides a hydrolyzed proanthocyanidin freeze-dried powder, which adopts the following technical solution: A hydrolyzed proanthocyanidin freeze-dried powder, produced by freeze-drying, comprises the following raw materials in parts by weight: Hydrolyzed proanthocyanidins 0.1-0.2 parts, carnosine 25-35 parts, stabilizer 10-20 parts, acetyl hexapeptide-8 0.1-0.3 parts, water 45-55 parts; The stabilizer is selected from at least one of mannan, trehalose, mannitol, sucrose, betaine, whey protein, PVP, PEG, glycine, histidine, leucine, sodium alginate, β-cyclodextrin, maltodextrin and chitosan.

[0006] By adopting the above technical solution, freeze-drying is used to prepare freeze-dried powder. Freeze-drying technology sublimates the water in the material under low temperature and vacuum conditions, achieving drying without undergoing a high-temperature process. This effectively avoids the degradation of hydrolyzed proanthocyanidins caused by high temperatures, maintaining their biological activity for a long time. However, ice crystals inevitably form during the freeze-drying process. These ice crystals can mechanically damage the molecular structure of hydrolyzed proanthocyanidins and the microstructure of the product during their growth, potentially altering their physicochemical properties and affecting product quality and stability. Therefore, at least one of the following components, such as mannan, trehalose, betaine, and sucrose, is used as a stabilizer. The stabilizer can overcome the structural damage caused by ice crystal formation. Moreover, after freeze-drying, free water is removed, weakening the solvation effect of water molecules on anthocyanin molecules, reducing the reaction rate of hydrolysis and oxidation, improving its storage stability, and reducing the degradation rate. Furthermore, after the freeze-dried powder is reconstituted, the stabilizer can stabilize the conformation of anthocyanin molecules, inhibit degradation, improve the stability of anthocyanin aqueous solutions, and reduce anthocyanin degradation.

[0007] Optionally, the stabilizer is mannan.

[0008] By adopting the above technical solutions, mannan, a neutral polysaccharide, contains hydroxyl groups that can replace water molecules on the surface of anthocyanin molecules, forming a stable hydration layer. This prevents molecular aggregation or conformational changes caused by freeze-drying dehydration. Furthermore, it can reduce collisions and oxidation reactions between anthocyanin molecules through steric hindrance, while also inhibiting the disordered growth of ice crystals. The hydrophilic long chains of mannan can encapsulate anthocyanin molecules, reducing their self-aggregation in solution or their contact area with oxygen. At the same time, mannan itself has a certain free radical scavenging ability (such as hydroxyl radicals), which can synergistically inhibit the oxidative degradation of anthocyanins. Mannan increases the viscosity of aqueous solutions, reduces the diffusion rate of anthocyanin molecules, and reduces degradation induced by factors such as light and heat. The gel-like matrix formed after freeze-drying maintains the encapsulation state of anthocyanins during reconstitution, prolonging the stability period.

[0009] Optionally, the stabilizer comprises chitosan and mannan in a mass ratio of 1:3-5.

[0010] By adopting the above technical solution, chitosan molecules contain a large number of amino and hydroxyl groups. The hydroxyl groups can form hydrogen bonds with the phenolic hydroxyl groups of anthocyanins, reducing the damage to the anthocyanin molecular structure caused by ice crystal growth during freeze-drying. Moreover, it can inhibit the degradation of anthocyanins through electrostatic adsorption. After freeze-drying, chitosan forms a glassy matrix, which encapsulates anthocyanins in a rigid network, reducing oxidation or degradation caused by molecular motion. Chitosan can maintain the pH stability of the system under acidic conditions, while anthocyanins are more stable in acidic environments. At the same time, the positively charged chitosan forms a complex with anthocyanins, inhibiting oxidative degradation and enhancing the stability of aqueous solutions. During freeze-drying, chitosan inhibits ice crystal growth and forms a porous matrix, which accelerates the release of anthocyanins and maintains their molecular integrity upon reconstitution.

[0011] When chitosan and mannan are combined, chitosan forms an electrostatic complex with anthocyanins. Mannan then encapsulates the complex through hydrogen bonds and steric hindrance, forming a multi-layered protective structure that reduces the influence of the external environment. Mannan and chitosan are cross-linked through hydrogen bonds, reducing the exposure of free hydroxyl groups and lowering the adsorption sites for water molecules. Mannan preferentially adsorbs some moisture, indirectly reducing the hygroscopicity of chitosan.

[0012] Optionally, the stabilizer also includes succinylated rice glutenin, with a mass ratio of chitosan to succinylated rice glutenin of 1:1-2.

[0013] By adopting the above technical solution, succinylated rice gluten undergoes an acylation reaction between succinic anhydride and the amino groups in rice gluten, introducing carboxyl groups and changing the protein's charge from neutral to negative. The carboxyl groups of succinylated gluten dissociate into -COO-, resulting in an overall negative charge and a lower isoelectric point. Under a pH of 4.5-6, the amino groups of chitosan and the -COO- groups of succinylated rice gluten form ion pairs, forming a polyelectrolyte complex through electrostatic adsorption. This encapsulates hydrolyzed proanthocyanidins, reducing direct contact between anthocyanins and moisture and oxygen. Furthermore, the residual positive charge of chitosan on the surface of the electrostatic complex inhibits the contact between anthocyanins and dissolved oxygen, improving their stability, preventing anthocyanin precipitation, and extending shelf life. Furthermore, the hydrophobic region (unmodified amino acid residues) of succinylated rice gluten binds to the benzene ring of anthocyanin through hydrophobic interactions. Simultaneously, the electrostatic interactions between succinylated rice gluten and chitosan enhance overall stability and improve the encapsulation effect of anthocyanins. The porous structure of the electrostatic complex inhibits the growth direction of ice crystals, reduces mechanical damage to anthocyanins from ice crystals, and mitigates physical destruction. The hydroxyl groups of chitosan form hydrogen bonds with anthocyanins, fixing the molecular structure and thus protecting the anthocyanins through freeze-drying. The charge interaction of chitosan complements the network structure of mannan: chitosan first fixes anthocyanins through electrostatic adsorption, and then mannan wraps the complex with long chains to form a core-shell protective layer.

[0014] Optionally, the molecular weight of the chitosan is 50-100 kDa.

[0015] By adopting the above technical solutions, chitosan with the above molecular weight has a moderate density of amino groups, which can be firmly electrostatically bound to anthocyanins under acidic conditions. At the same time, the molecular chain length can form a moist network structure, inhibiting ice crystal growth and anthocyanin aggregation, inducing the formation of fine and uniform ice crystals, and reducing mechanical damage. If the molecular weight of chitosan is too low, it cannot form a complete network encapsulation, and the rigidity after freeze-drying is weak. If the molecular weight is too high, the molecular chains are severely entangled, which easily leads to precipitation, low porosity after freeze-drying, and slow reconstitution release rate.

[0016] Secondly, this application provides a method for preparing hydrolyzed proanthocyanidin freeze-dried powder, using the following technical solution: A method for preparing hydrolyzed proanthocyanidin freeze-dried powder includes the following steps: Hydrolyzed proanthocyanidins were mixed with carnosine, stabilizer, acetyl hexapeptide-8 and water, stirred evenly, and the pH was adjusted to 4.5-6 to obtain the material. The material is refrigerated at 4-5℃ for 2-3 hours, and then freeze-dried under a vacuum of 20-50Pa for 34-36 hours to obtain freeze-dried powder.

[0017] By adopting the above technical solution, the raw materials are mixed evenly and the pH value is adjusted. At this pH value, hydrolyzed proanthocyanidins are not prone to discoloration and degradation. Then, they are refrigerated to make the material uniform in color and prevent ice crystals from being generated and destroying the anthocyanins due to rapid cooling.

[0018] Optionally, the freeze-drying process comprises: heating from -40°C to -30°C for 90 minutes and holding for 5 hours; then heating from -30°C to -10°C for 2 hours and holding for 5 hours; heating from -10°C to 0°C for 2 hours and holding for 10 hours; heating from 0°C to 5°C for 30 minutes and holding for 8 hours; heating from 5°C to 10°C for 30 minutes and holding for 3 hours; heating from 10°C to 20°C for 30 minutes and holding for 2 hours; and finally heating from 20°C to 40°C for 30 minutes and holding for 1 hour.

[0019] By adopting the above technical solution and using an appropriate freeze-drying time, the material can be completely freeze-dried and will not change color under light.

[0020] Optionally, before refrigerating the material, a 15-20 wt% silk fibroin ethanol solution is added to the material and mixed evenly. The amount of silk fibroin ethanol solution added is 4-8 wt% of the total weight of the material.

[0021] By adopting the above technical solution, the silk fibroin fiber ethanol solution is mixed with the material and then refrigerated and freeze-dried. After freeze-drying, a silk fibroin aerogel with a three-dimensional porous network structure is formed. This structure can act as a skeleton during the freeze-drying process, providing a physical barrier protection for anthocyanins, preventing ice crystal damage, and inhibiting the migration of anthocyanin molecules. Moreover, the encapsulation of silk fibroin aerogel can reduce oxidation reactions and reduce the attack of external factors on anthocyanins. In addition, after the aerogel swells, it forms colloidal particles that can increase the viscosity of the solution, thereby increasing the viscosity of the anthocyanin aqueous solution, reducing the Brownian motion of anthocyanin molecules, reducing their degradation, and inhibiting intermolecular aggregation and precipitation. At the same time, the aerogel can also reduce the hygroscopicity of the freeze-dried powder and increase the moisture-proof effect.

[0022] Optionally, after the material is freeze-dried, it is immersed in a treatment solution and air-dried. The treatment solution is a beeswax emulsion containing stearic acid and beeswax, with a mass ratio of beeswax emulsion to stearic acid of 4:1.

[0023] By employing the above technical solution, the freeze-dried powder, especially with the addition of silk fibroin aerogel, has a multi-structured interior that easily absorbs moisture. The freeze-dried product is then immersed in a stearic acid and beeswax microemulsion. Stearic acid, a long-chain fatty acid, contains hydrophobic alkyl chains and hydrophilic carboxyl groups in its molecular structure. It can chemically bond with polar groups on the surface of the freeze-dried powder (such as the hydroxyl groups of silk fibroin aerogel, the phenolic hydroxyl groups of anthocyanins, and the hydroxyl groups of chitosan). The alkyl chains are oriented to form a hydrophobic layer, thus preventing external moisture from contacting the hydrophilic groups inside the freeze-dried powder and reducing hygroscopicity. Therefore, stearic acid penetrates into the pores, forming a hydrophobic layer and even filling tiny pores, reducing moisture penetration channels and contact with oxygen, thus reducing anthocyanin degradation. The beeswax microemulsion has adhesive properties, increasing the firmness of the hydrophobic coating, enhancing the adhesion between stearic acid and the surface of the freeze-dried powder, preventing detachment, and also possessing a certain hydrophobic-hydrophilic balance, which can regulate surface wettability.

[0024] Optionally, the concentration of the beeswax emulsion is 1-2%.

[0025] By adopting the above technical solution, if the beeswax emulsion has a high concentration and high viscosity, it will affect the uniformity of coating.

[0026] In summary, this application has the following beneficial effects: 1. Since this application uses at least one of mannan, betaine, trehalose, etc. as a stabilizer, the hydrolyzed proanthocyanidins are mixed and then refrigerated and freeze-dried to produce hydrolyzed anthocyanin freeze-dried powder. The stabilizer can bind with active ingredients such as proteins, enzymes, and nucleic acids through hydrogen bonds during the freeze-drying process, reduce the structural damage caused by ice crystal formation and dehydration, maintain its biological activity, avoid the activity reduction caused by high temperature, and at the same time avoid the reduction or even loss of the biological activity of anthocyanin aqueous solution.

[0027] 2. In this application, mannan and chitosan are preferred as stabilizers, which can form a multi-layer protective structure for hydrolyzed proanthocyanidins, reduce the influence of the external environment, and further enhance the storage stability of the freeze-dried powder. In addition, the combination of succinylated rice gluten with mannan and chitosan can reduce the degradation of anthocyanins by dissolved oxygen, extend the shelf life, and reduce hygroscopicity.

[0028] 3. The method of this application, by adding silk fibroin ethanol solution to the material before refrigeration, forms silk fibroin aerogel during freeze-drying, which can reduce ice crystal damage, reduce oxidative degradation, and enhance the moisture-proof effect of freeze-dried powder. Detailed Implementation

[0029] The following embodiments provide a further detailed description of this application.

[0030] Example 1: Preparation of succinylated rice gluten: Rice gluten was dissolved in deionized water to form a 30 wt% dispersion. The pH was adjusted to 8.5 with 0.5 mol / L sodium hydroxide and maintained stable. The protein was hydrated for 30 min. Succinic anhydride was added to the dispersion at a ratio of 30% (by mass of rice gluten). The pH was adjusted to a constant value with 2 mol / L sodium hydroxide solution at 50 °C. The reaction was allowed to proceed for 120 min. After the reaction was complete, the reaction solution was transferred, and the reaction was terminated by an ice-water bath. The solution was then placed in a dialysis bag for 48 h to remove unreacted succinic anhydride and salt ions. Dialysis was performed every 4 hours. Replace the dialysis distilled water once per hour. Freeze-dry at -50℃ and 0.05MPa to obtain succinylated rice glutenin. Rice glutenin is extracted using the alkali dissolution and acid precipitation method. Rice is pulverized and ground into powder, passed through an 80-mesh sieve, and dissolved in distilled water at a solid-liquid ratio of 1:10 (g / ml). The pH is adjusted to 12 with 1 mol / L sodium hydroxide solution, stirred for 4 hours, and then centrifuged at 10000g for 15 minutes at 4℃. The supernatant is collected, and the pH is adjusted to 4.5 with 1 mol / L hydrochloric acid to obtain the precipitate. The precipitate is washed twice with distilled water, the pH is adjusted to 7, and then freeze-dried to obtain rice glutenin. Example

[0031] Example 1: A hydrolyzed proanthocyanidin freeze-dried powder, comprising 50g water, 0.1g hydrolyzed proanthocyanidins, 30g carnosine, 0.2g acetyl hexapeptide-8 and 15g stabilizer. The stabilizer includes chitosan and mannan in a mass ratio of 1:3. The chitosan is selected from Hefei Bomei Biotechnology, with a molecular weight of 50kDa and catalog number B15051. The mannan is konjac mannan, selected from Jiangsu Weizhirun Biotechnology, catalog number 01. The hydrolyzed proanthocyanidins are selected from Xi'an Huaxing Biotechnology, with a single product catalog number of HWLMHQS25. The carnosine is selected from Shaanxi Hengruikang, with a catalog number of GHZ-0409-06.

[0032] The above method for preparing hydrolyzed proanthocyanidin lyophilized powder includes the following steps: Hydrolyzed proanthocyanidins, carnosine, acetyl hexapeptide-8, mannan, and water were mixed and stirred at 2000 rpm until a purple-red transparent liquid was obtained. The pH was adjusted to 5.5 with a 1% acetic acid solution. Fill the vial with the material, lightly place the rubber stopper on the mouth of the vial, and refrigerate at 5°C for 2 hours. Turn on the freeze dryer and set the temperature to -40℃. Transfer the vials from the refrigerator to the freeze dryer. Set the freeze-drying degree to -40℃, raise the temperature to -30℃ for 90 minutes, and hold for 5 hours. Then raise the temperature from -30℃ to -10℃ for 2 hours and hold for 5 hours. Raise the temperature from -10℃ to 0℃ for 2 hours and hold for 10 hours. Raise the temperature from 0℃ to 5℃ for 30 minutes and hold for 8 hours. Finally, raise the temperature from 5℃ to 1℃... Start at 0℃, raise the temperature for 30 minutes, and hold for 3 hours. Raise the temperature from 10℃ to 20℃, raise the temperature for 30 minutes, and hold for 2 hours. Finally, raise the temperature from 20℃ to 40℃, raise the temperature for 30 minutes, and hold for 1 hour. Run the freeze dryer according to the above procedure, turn on the vacuum pump to 50 Pa, and after freeze drying, press the rubber stopper into the vial to seal the vial. Turn off the vacuum pump to allow air to enter the freeze drying chamber, turn off the freeze drying equipment, and remove the product.

[0033] Example 2: A hydrolyzed proanthocyanidin freeze-dried powder, comprising 55g water, 0.2g hydrolyzed proanthocyanidins, 35g carnosine, 0.3g acetyl hexapeptide-8, and 20g stabilizer. The stabilizer comprises chitosan and mannan in a mass ratio of 1:4. The chitosan is selected from Hefei Bomei Biotechnology, with a molecular weight of 50kDa and catalog number B15051. The mannan is konjac mannan, selected from Jiangsu Weizhirun Biotechnology, catalog number 01. The hydrolyzed proanthocyanidins are selected from Xi'an Huaxing Biotechnology, with a single product catalog number of HWLMHQS25. The carnosine is selected from Shaanxi Hengruikang, with a catalog number of GHZ-0409-06.

[0034] The above method for preparing hydrolyzed proanthocyanidin lyophilized powder includes the following steps: Hydrolyzed proanthocyanidins, carnosine, acetyl hexapeptide-8, mannan, and water were mixed and stirred at 1000 rpm until a purple-red transparent liquid was obtained. The pH was then adjusted to 6 with a 1% acetic acid solution. Fill the vial with the material, lightly place the rubber stopper on the mouth of the vial, and refrigerate at 4°C for 3 hours. Turn on the freeze dryer and set the temperature to -40℃. Transfer the vials from the refrigerator to the freeze dryer. Set the freeze-drying degree to -40℃, raise the temperature to -30℃ for 90 minutes, and hold for 5 hours. Then raise the temperature from -30℃ to -10℃ for 2 hours and hold for 5 hours. Raise the temperature from -10℃ to 0℃ for 2 hours and hold for 10 hours. Raise the temperature from 0℃ to 5℃ for 30 minutes and hold for 8 hours. Finally, raise the temperature from 5℃ to 1℃... Start at 0℃, raise the temperature for 30 minutes, and hold for 3 hours. Raise the temperature from 10℃ to 20℃, raise the temperature for 30 minutes, and hold for 2 hours. Finally, raise the temperature from 20℃ to 40℃, raise the temperature for 30 minutes, and hold for 1 hour. Run the freeze dryer according to the above procedure, turn on the vacuum pump to 20 Pa, and after freeze drying, press the rubber stopper into the vial to seal the vial. Turn off the vacuum pump to allow air to enter the freeze drying chamber, turn off the freeze drying equipment, and remove the product.

[0035] Example 3: A hydrolyzed proanthocyanidin freeze-dried powder, comprising 45g water, 0.1g hydrolyzed proanthocyanidins, 25g carnosine, 0.1g acetyl hexapeptide-8 and 10g stabilizer. The stabilizer includes chitosan and mannan in a mass ratio of 1:5. The chitosan is selected from Hefei Bomei Biotechnology, with a molecular weight of 50kDa and catalog number B15051. The mannan is konjac mannan, selected from Jiangsu Weizhirun Biotechnology, catalog number 01. The hydrolyzed proanthocyanidins are selected from Xi'an Huaxing Biotechnology, with a single product catalog number of HWLMHQS25. The carnosine is selected from Shaanxi Hengruikang, with a catalog number of GHZ-0409-06.

[0036] The above method for preparing hydrolyzed proanthocyanidin lyophilized powder includes the following steps: Hydrolyzed proanthocyanidins, carnosine, acetyl hexapeptide-8, mannan, and water were mixed and stirred at 1500 rpm until a purple-red transparent liquid was obtained. The pH was then adjusted to 4.5 with a 1% acetic acid solution. Fill the vial with the material, lightly place the rubber stopper on the mouth of the vial, and refrigerate at 4°C for 3 hours. Turn on the freeze dryer and set the temperature to -40℃. Transfer the vials from the refrigerator to the freeze dryer. Set the freeze-drying degree to -40℃, raise the temperature to -30℃ for 90 minutes, and hold for 5 hours. Then raise the temperature from -30℃ to -10℃ for 2 hours and hold for 5 hours. Raise the temperature from -10℃ to 0℃ for 2 hours and hold for 10 hours. Raise the temperature from 0℃ to 5℃ for 30 minutes and hold for 8 hours. Finally, raise the temperature from 5℃ to 1℃... Start at 0℃, raise the temperature for 30 minutes, and hold for 3 hours. Raise the temperature from 10℃ to 20℃, raise the temperature for 30 minutes, and hold for 2 hours. Finally, raise the temperature from 20℃ to 40℃, raise the temperature for 30 minutes, and hold for 1 hour. Run the freeze dryer according to the above procedure, turn on the vacuum pump to 20 Pa, and after freeze drying, press the rubber stopper into the vial to seal the vial. Turn off the vacuum pump to allow air to enter the freeze drying chamber, turn off the freeze drying equipment, and remove the product.

[0037] Example 4: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the stabilizer is betaine.

[0038] Example 5: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the stabilizer is trehalose.

[0039] Example 6: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the stabilizer is mannan.

[0040] Example 7: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the chitosan has a molecular weight of 15 kDa and is selected from Hefei Bomei Biotechnology Co., Ltd., with the product number B11151.

[0041] Example 8: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the stabilizer also contains succinylated rice gluten protein prepared in Preparation Example 1, and the stability includes chitosan, mannan and succinylated rice gluten protein in a mass ratio of 1:3:2.

[0042] Example 9: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that the stabilizer also contains succinylated rice gluten protein prepared in Preparation Example 1, and the stability includes chitosan, mannan and succinylated rice gluten protein in a mass ratio of 1:3:1.

[0043] Example 10: A hydrolyzed proanthocyanidin freeze-dried powder, differing from Example 9 in that, before refrigeration, a 20wt% silk fibroin ethanol solution is added to the material, mixed evenly, and then refrigerated and freeze-dried. The amount of silk fibroin ethanol solution added is 8wt% of the total weight of the material. The silk fibroin ethanol solution is prepared by mixing silk fibroin and ethanol. The silk fibroin is prepared by dissolving silk fibroin in formic acid to obtain a spinning solution with a concentration of 25wt%. Electrospinning is performed, and the solution is cut into 1cm×1cm fragments. The fragments are pulped by a high-speed shearing machine for 30 minutes, and then ethanol is added to prepare a 20wt% silk fibroin ethanol solution. The spinning voltage is 20kV, the receiving distance is 15cm, and the feed speed is 0.1ml / h. The silk fibroin is selected from Tianben Biotechnology (Shaanxi), and the product code is tb-0625-035.

[0044] Example 11: A hydrolyzed proanthocyanidin freeze-dried powder, differing from Example 9 in that, before refrigeration, a 15wt% silk fibroin ethanol solution is added to the material, mixed evenly, and then refrigerated and freeze-dried. The amount of silk fibroin ethanol solution added is 4wt% of the total weight of the material. The silk fibroin ethanol solution is prepared by mixing silk fibroin and ethanol. The silk fibroin is prepared by dissolving silk fibroin in formic acid to obtain a spinning solution with a concentration of 25wt%. Electrospinning is performed, and the solution is cut into 1cm×1cm fragments. The fragments are then pulped by a high-speed shearing machine for 30 minutes, and ethanol is added to prepare a 20wt% silk fibroin ethanol solution. The spinning voltage is 20kV, the receiving distance is 15cm, and the feed speed is 0.1ml / h. The silk fibroin is selected from Tianben Biotechnology (Shaanxi), and the product code is tb-0625-035.

[0045] Example 12: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 11 in that, after freeze-drying, the freeze-dried product is immersed in a treatment solution at 65°C for 2 minutes, then removed and air-dried. The mass ratio of the treatment solution to the freeze-dried product is 1:20. The treatment solution contains beeswax emulsion and stearic acid in a mass ratio of 4:1. The beeswax emulsion is prepared by uniformly mixing 20g beeswax, 2g Tween-80 and 980g water at 65°C.

[0046] Comparative Example Comparative Example 1: A hydrolyzed proanthocyanidin freeze-dried powder, which differs from Example 1 in that no stabilizer was added.

[0047] Performance testing Hydrolyzed anthocyanin lyophilized powder was prepared according to the methods in the examples and comparative examples, and its performance was tested according to the following methods.

[0048] 1. Lyophilization Protection: Take the same mass of hydrolyzed anthocyanin lyophilized powder prepared in Examples 1-12 and Comparative Example 1, and adjust the volume to 10 ml with buffer solutions of pH 1 and pH 4.5 respectively. Screen for the maximum absorption wavelength using a full-wavelength scan and measure the anthocyanin sample under that wavelength condition. Simultaneously, measure the absorbance of the anthocyanin sample at 700 nm. The anthocyanin content is calculated using the following formula: Q = (A / εL) × M × D × (V / W), where Q is the anthocyanin content (mg / g), and A is the absorbance difference. The calculation formula is [(A... max -A 700 ) PH1.0 -(A max -A 700 ) PH4.5 M is the molecular weight of anthocyanin (calculated as cyanidin-3-glucoside, 449.2 g / mol), D is the dilution factor, ε is the molar absorptivity (26900 L / (mol·cm)), L is the optical path of the cuvette (1 cm), V is the test liquid volume (mL), and W is the sample mass (g). The detection results are recorded in Table 1.

[0049] 2. Hygroscopicity: Place the weighing bottle in a 105℃ oven to dry for 2 hours, cool to room temperature and weigh (recorded as M1). Take about 1.0g of anthocyanin lyophilized powder (accurate to 0.1mg), spread it evenly in the weighing bottle and weigh (recorded as M2). Place the weighing bottle in a desiccator (without capping) and place it in a constant temperature and humidity chamber (controlled at 25℃±1℃, 75% RH±1%) for 24 hours. Remove the weighing bottle, cap it and weigh it immediately (recorded as M3). Calculate the hygroscopic rate: (M3-M2) / (M2-M1)×100%. Record the test results in Table 1.

[0050] Table 1 As can be seen from Examples 1-3 and the data in Table 1, chitosan and mannan, as stabilizers, can effectively protect the formation of hydrolyzed proanthocyanidins during freeze-drying, reduce the damage of ice crystals to anthocyanins, and reduce freeze-drying losses.

[0051] In Example 4, betaine was used as a stabilizer; in Example 5, trehalose was used; and in Example 6, mannan was used alone. Compared with Example 1, the anthocyanin content in the hydrolyzed proanthocyanidin freeze-dried powder prepared in Examples 4-6 was reduced, indicating that using betaine or trehalose alone as stabilizers, or using mannan as a stabilizer, is not as effective as using a combination of mannan and chitosan for freeze protection.

[0052] In Example 7, chitosan with a reduced molecular weight was used in combination with mannan as a stabilizer, but its antifreeze protection effect was reduced.

[0053] In Examples 8 and 9, succinylated rice gluten was added to the stabilizer. As can be seen from the data in Table 1, the anthocyanin content in the hydrolyzed proanthocyanidin freeze-dried powder prepared in Examples 8 and 9 increased, indicating that the antifreeze protection effect of succinylated rice gluten on anthocyanins was increased, and the hygroscopicity of the freeze-dried powder decreased.

[0054] Compared with Example 9, Examples 10 and 11 added a silk fibroin fiber ethanol solution before refrigeration, and formed a silk fibroin aerogel after freeze-drying, thereby reducing ice crystal damage, increasing anthocyanin content, and improving the moisture-proof and hygroscopic properties of the freeze-dried powder.

[0055] Compared with Example 11, the freeze-dried powder in Example 12 was treated by impregnating it with stearic acid and beeswax emulsion, which showed that the freeze-dried powder had better moisture-proof and damp-proof effects.

[0056] In Comparative Example 1, no stabilizer was used. Hydrolyzed proanthocyanidins were mixed with carnosine and other ingredients, refrigerated, and then freeze-dried. The anthocyanin content in the resulting freeze-dried hydrolyzed proanthocyanidins powder decreased significantly.

[0057] 3. Storage stability: The high-temperature environment stability and aqueous solution environment stability of the hydrolyzed proanthocyanidin freeze-dried powder were tested according to the following methods, and the test results were recorded in Table 3.

[0058] (1) Room temperature stability: The hydrolyzed proanthocyanidin lyophilized powders prepared in the examples and comparative examples were placed in an environment of 25°C and 75% RH for three months. The antioxidant activity was determined by the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals. The specific method was as follows: 2 mL of the sample solution to be tested was mixed with 5 mL of 0.1 mmol / L DPPH / ethanol solution. After thorough shaking and mixing, the mixture was protected from light for 30 min. The absorbance of the supernatant at a wavelength of 517 nm was measured by ELISA. An equal amount of ethanol was used to replace the blank control. The DPPH free radical scavenging activity was calculated according to the following formula: R 测试后样品 = (A0-A1) / A0×100%, where A0 is the absorbance value of the blank control group; A1 is the absorbance value of the test sample; R 测试前样品 = (A0-A1) / A0×100%, where A0 is the absorbance value of the blank control group; A1 is the absorbance value of the test sample; then, according to the DPPH free radical scavenging rate decrease rate under high temperature environment = (R 测试前样品 -R 测试后样品 ) / R 测试前样品 ×100%.

[0059] (2) Aqueous solution stability: The prepared hydrolyzed proanthocyanidin lyophilized powder was made into a 1 mg / ml solution and stored at 25°C. After one month, the rate of decrease in antioxidant activity was detected. The test method was the same as the method for determining high-temperature stability, according to the formula: DPPH free radical scavenging rate decrease rate = (R... 测试前样品 -R 测试后样品 ) / R 测试前样品 Calculate the rate of decrease in antioxidant capacity after 1 month of storage of the aqueous solution by ×100%.

[0060] Table 2 As can be seen from the data in Table 2, the lyophilized powder prepared by freeze-drying using chitosan and mannan as stabilizers showed a small decrease in DPPH free radical scavenging rate after long-term storage at room temperature, indicating that anthocyanin degradation was less and the powder was more stable. Moreover, after being prepared into an aqueous solution, the antioxidant effect decreased slowly after long-term storage, indicating good stability.

[0061] In Example 4, betaine was used as a stabilizer; in Example 5, trehalose was used; and in Example 6, mannan was used alone. Compared with Example 1, the freeze-dried powders prepared in Examples 4-6 showed decreased storage stability at room temperature or in aqueous solution.

[0062] Compared with Example 1, the molecular weight of chitosan in Example 7 is lower, indicating that the stability of the freeze-dried powder prepared in Example 7 is lower than that in Example 1.

[0063] Compared with Example 1, Examples 8 and 9 also added succinylated rice gluten. As shown in Table 2, the freeze-dried powder prepared has increased stability when stored at room temperature. When stored in an aqueous solution, it can avoid the reduction of anthocyanin activity due to prolonged dissolution in water.

[0064] Compared with Example 9, Examples 10 and 11 added silk fibroin fiber ethanol solution before refrigeration and formed silk fibroin aerogel after freeze-drying, which shows that the storage stability of the freeze-dried powder is increased.

[0065] Compared to Example 11, Example 12 involves immersing the lyophilized powder in a stearic acid and beeswax emulsion. As shown in Table 2, the lyophilized powder prepared in Example 12 exhibits strong antioxidant capacity in aqueous solution and good stability during room temperature storage.

[0066] No stabilizer was added in Comparative Example 1. Compared with Example 1, the storage stability of the lyophilized powder prepared in Comparative Example 1 was significantly reduced.

[0067] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A hydrolyzed proanthocyanidin freeze-dried powder, characterized in that, It is made by freeze-drying and includes the following raw materials in parts by weight: Hydrolyzed proanthocyanidins 0.1-0.2 parts, carnosine 25-35 parts, stabilizer 10-20 parts, acetyl hexapeptide-8 0.1-0.3 parts, water 45-55 parts; The stabilizer is selected from at least one of mannan, trehalose, mannitol, sucrose, betaine, whey protein, PVP, PEG, glycine, histidine, leucine, sodium alginate, β-cyclodextrin, maltodextrin and chitosan.

2. The hydrolyzed proanthocyanidin freeze-dried powder according to claim 1, characterized in that: The stabilizer is mannan.

3. The hydrolyzed proanthocyanidin freeze-dried powder according to claim 1, characterized in that: The stabilizer comprises chitosan and mannan in a mass ratio of 1:3-5.

4. The hydrolyzed proanthocyanidin freeze-dried powder according to claim 3, characterized in that: The stabilizer also includes succinylated rice glutenin, with a chitosan-to-succinylated rice glutenin mass ratio of 1:1-2.

5. The hydrolyzed proanthocyanidin freeze-dried powder according to claim 3, characterized in that: The molecular weight of the chitosan is 50-100 kDa.

6. The method for preparing the hydrolyzed proanthocyanidin freeze-dried powder according to any one of claims 1-5, characterized in that: Includes the following steps: Hydrolyzed proanthocyanidins were mixed with carnosine, stabilizer, acetyl hexapeptide-8 and water, stirred evenly, and the pH was adjusted to 4.5-6 to obtain the material. The material is refrigerated at 4-5℃ for 2-3 hours, and then freeze-dried under a vacuum of 20-50Pa for 34-36 hours to obtain freeze-dried powder.

7. The method for preparing hydrolyzed proanthocyanidin freeze-dried powder according to claim 6, characterized in that: The freeze-drying process is as follows: heating from -40℃ to -30℃ for 90 minutes and holding for 5 hours; then heating from -30℃ to -10℃ for 2 hours and holding for 5 hours; heating from -10℃ to 0℃ for 2 hours and holding for 10 hours; heating from 0℃ to 5℃ for 30 minutes and holding for 8 hours; heating from 5℃ to 10℃ for 30 minutes and holding for 3 hours; heating from 10℃ to 20℃ for 30 minutes and holding for 2 hours; and finally heating from 20℃ to 40℃ for 30 minutes and holding for 1 hour.

8. The method for preparing hydrolyzed proanthocyanidin freeze-dried powder according to claim 6, characterized in that: Before refrigerating the material, add a 15-20 wt% silk fibroin ethanol solution to the material and mix well. The amount of silk fibroin ethanol solution added is 4-8 wt% of the total weight of the material.

9. The method for preparing hydrolyzed proanthocyanidin freeze-dried powder according to claim 6, characterized in that: After the material is freeze-dried, it is immersed in a treatment solution and air-dried. The treatment solution is a beeswax emulsion containing stearic acid and beeswax, with a mass ratio of beeswax emulsion to stearic acid of 4:

1.

10. The method for preparing hydrolyzed proanthocyanidin freeze-dried powder according to claim 9, characterized in that: The concentration of the beeswax emulsion is 1-2%.

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