Preparation method of hawthorn fermented filtrate as well as product and application of hawthorn fermented filtrate

Abstract

The invention provides a preparation method of hawthorn fermented filtrate as well as a product and application thereof, the preparation method comprises the following steps: (1) mixing hawthorn, first compound enzyme and water, performing first enzymolysis reaction, adding second compound enzyme, performing second enzymolysis reaction, and performing enzyme deactivation after the second enzymolysis reaction is finished to obtain hawthorn zymolyte; the first compound enzyme comprises cellulase and pectinase; the second compound enzyme comprises laccase and beta-glucosidase; and (2) inoculating an activated strain into a culture medium containing the hawthorn zymolyte, fermenting, and sterilizing and carrying out solid-liquid separation to obtain the hawthorn fermented filtrate. The laccase and the beta-glucosidase synergistically decompose macromolecular active matters of the haws, strains are further inoculated for fermentation, various effective components such as amino acids, polypeptides, vitamins, organic acids and minerals are generated, the content of flavonoid aglycones is remarkably increased, and the prepared haw fermentation filtrate has excellent anti-oxidation, whitening and brightening effects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a preparation method of hawthorn fermentation filtrate, a product thereof and an application thereof. Background Art

[0002] Hawthorn fermentation filtrate is a natural antioxidant and skin conditioner, with excellent antioxidant, skin brightening, soothing and repairing, and skin microecological regulation effects.

[0003] CN116473890A discloses the application of raspberry, oat and hawthorn fermentation products in repair products. The preparation method of the fermentation product specifically includes the following steps: S1, washing raspberries, oats and hawthorns, and then putting them into a wall breaking machine according to the mass ratio to crush them to obtain a mixture; S2, adding pectinase and cellulase to the mixture obtained in step S1, combining with ultrasonic extraction, and performing enzymolysis in a water bath at 25-40°C for 20-30 minutes to obtain an enzymolysis product; S3, high-temperature sterilizing the enzymolysis product in step S2, and putting it into a fermentation tank after cooling; S4, inoculating the fermentation bacteria into the fermentation tank in step S3, sealing the fermentation tank and fermenting to obtain the product.

[0004] CN113332354A discloses an anti-glycation and anti-aging fermentation composition and a preparation method thereof, the preparation method comprising the following steps: (1) crushing chamomile flowers, houttuynia cordata, emblica chinensis and hawthorn, sieving to a mesh size of 100-600 mesh, adding water to mix evenly, and then adding an acid-base regulator to adjust the pH to 4.0-5.0 to obtain a mixed solution; (2) adding an enzyme preparation to the mixed solution for enzymolysis at a temperature of 40-70°C and a time of 90-240 min, and adding an acid-base regulator to adjust the pH to 5.0-6.0 after the enzymolysis is completed to obtain an enzymatic solution; (3) extracting the enzymatic solution at a temperature of 80-102°C for 10-90 min to obtain an extract; (4) adding the fermentation bacteria powder to the extract for fermentation at a temperature of 15-45°C and a time of 36-96 h to obtain a fermentation solution, and separating the fermentation solution into a solid-liquid separation to obtain a fermentation clear solution.

[0005] However, the existing traditional extraction process has a low retention rate of hawthorn active ingredients and limited transdermal absorption effect, and the actual efficacy of hawthorn fermentation filtrate is subject to certain restrictions. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a preparation method of hawthorn fermentation filtrate and its product and application. The hawthorn fermentation filtrate prepared by the method of the present invention contains a variety of small molecule active ingredients, the content of flavonoid aglycones is significantly improved, it has a high skin absorption rate and product stability, and exerts excellent antioxidant and whitening and brightening effects.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing hawthorn fermentation filtrate, the preparation method comprising:

[0009] (1) mixing hawthorn, a first complex enzyme, and water to perform a first enzymatic hydrolysis reaction, then adding a second complex enzyme to perform a second enzymatic hydrolysis reaction, and then inactivating the enzymes to obtain a hawthorn hydrolyzate; the first complex enzyme comprises cellulase and pectinase; and the second complex enzyme comprises laccase and β-glucosidase;

[0010] (2) inoculating activated bacteria into a culture medium containing hawthorn enzymatic hydrolysate, performing fermentation, sterilizing after completion, and performing solid-liquid separation to obtain the hawthorn fermentation filtrate.

[0011] Hawthorn is rich in flavonoids, which mainly exist in the form of flavonoid glycosides and have poor absorption. The present invention is designed to carry out two enzymatic hydrolysis reactions: cellulase and pectinase are used to break down the hawthorn pulp tissue skeleton to release the contents, and laccase and β-glucosidase are used to further synergistically decompose the macromolecular compounds to obtain a hawthorn hydrolysate. The hawthorn hydrolysate is used as a matrix to inoculate activated bacteria for fermentation, producing a variety of effective ingredients such as amino acids, polypeptides, vitamins, organic acids, and minerals. Hawthorn flavonoids can be fully converted into flavonoid aglycones that are easy to absorb. The prepared hawthorn fermentation filtrate has excellent antioxidant and whitening and brightening effects.

[0012] Preferably, in step (1), the mass volume ratio of hawthorn to water is 1:(5-10) g / mL.

[0013] The specific point values in (5-10) can be 5, 5.2, 5.5, 5.8, 6, 6.3, 6.5, 6.7, 7, 7.5, 8, 8.5, 9, 9.5 or 10, etc.

[0014] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0015] Preferably, the mass ratio of the hawthorn and the first complex enzyme is (90-110):(0.2-1).

[0016] The specific point values in (90-110) can be 90, 92, 95, 98, 100, 103, 105, 107, 108 or 110, etc.

[0017] The specific point values in (0.2-1) can be 0.2, 0.22, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1, etc.

[0018] Preferably, the mass ratio of the cellulase to the pectinase is (2-4):(1-3).

[0019] The specific point values in (2-4) can be 2, 2.1, 2.2, 2.3, 2.5, 2.8, 3, 3.2, 3.5, 3.8 or 4, etc.

[0020] The specific point values in (1-3) can be 1, 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.2, 2.5, 2.8 or 3, etc.

[0021] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0022] Preferably, the temperature of the first enzymatic hydrolysis reaction is 40-50°C, for example, it can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C, etc.; the time is 0.8-2h, for example, it can be 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h or 2h, etc.

[0023] Preferably, the pH value of the first enzymatic hydrolysis reaction is 4.5-5.5, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

[0024] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0025] Preferably, in step (1), the mass ratio of the hawthorn and the second complex enzyme is (90-110):(0.05-0.5).

[0026] The specific point values in (90-110) can be 90, 92, 95, 98, 100, 103, 105, 107, 108 or 110, etc.

[0027] The specific point values in (0.05-0.5) can be 0.05, 0.08, 0.1, 0.12, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0028] Preferably, the mass ratio of the laccase to the β-glucosidase is (0.5-2):(1-3).

[0029] The specific point values in (0.5-2) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.7, 1.8 or 2, etc.

[0030] The specific point values in (1-3) can be 1, 1.1, 1.2, 1.5, 1.6, 1.8, 2, 2.2, 2.5, 2.8 or 3, etc.

[0031] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0032] Preferably, the temperature of the second enzymatic hydrolysis reaction is 35-45°C, for example, it can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C; the time is 30-40min, for example, it can be 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min or 40min.

[0033] Preferably, the pH value of the second enzymatic hydrolysis reaction is 4.5-6.0, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.5, 5.8 or 6.0.

[0034] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0035] Preferably, the second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment.

[0036] Preferably, the parameters of the pulsed ultrasonic treatment include: a working time of 4-6s, for example, 4s, 4.1s, 4.2s, 4.5s, 4.8s, 5s, 5.2s, 5.5s, 5.8s or 6s; an interval of 2-4s, for example, 2s, 2.1s, 2.2s, 2.3s, 2.5s, 2.7s, 3s, 3.2s, 3.5s, 3.8s or 4s; a frequency of 30-50kHz, for example, 30kHz, 32kHz, 35kHz, 38kHz, 40kHz, 43kHz, 45kHz, 47kHz or 50kHz; a power of 200-400W, for example, 200W, 210W, 220W, 250W, 280W, 300W, 320W, 350W, 380W or 400W.

[0037] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0038] In the present invention, the second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment, which can increase the enzymatic hydrolysis reaction rate of laccase and β-glucosidase. The cavitation effect generated by the pulsed ultrasonic treatment under specific parameters significantly improves the contact efficiency between the enzyme and the substrate, promotes the efficient conversion of flavonoid glycosides into flavonoid aglycones, and enhances the antioxidant and whitening and brightening effects of the hawthorn fermentation filtrate.

[0039] Preferably, in step (1), the enzyme inactivation method includes microwave treatment.

[0040] Preferably, the frequency of the microwave treatment is 2000-3000 MHz, for example, it can be 2000 MHz, 2100 MHz, 2200 MHz, 2300 MHz, 2400 MHz, 2500 MHz, 2600 MHz, 2700 MHz, 2800 MHz, 2900 MHz or 3000 MHz; the power is 600-800 W, for example, it can be 600 W, 620 W, 650 W, 680 W, 700 W, 730 W, 750 W, 770 W or 800 W; the time is 60-120 s, for example, it can be 60 s, 62 s, 65 s, 68 s, 70 s, 75 s, 80 s, 85 s, 90 s, 100 s, 110 s or 120 s.

[0041] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0042] The use of microwave treatment to inactivate enzymes can not only remove the effects of enzymes on subsequent reactions and ensure safety, but also maintain the activity of effective components in hawthorn fermentation filtrate and reduce thermal damage to the active components by 30-40%.

[0043] Preferably, in step (2), the mass percentage of the hawthorn hydrolysate in the culture medium is 20-40%, for example, it can be 20%, 21%, 22%, 25%, 28%, 30%, 32%, 35%, 37% or 40%, etc.

[0044] Preferably, the culture medium for activating the strain contains 0.1-5% (for example, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.3%, 1.5%, 1.7%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%) of hawthorn hydrolysate by mass.

[0045] Hawthorn hydrolysate is added to the culture medium as a growth promoter to activate the strain, which can advance the logarithmic growth phase of the strain by 3.5-4.5 hours. Compared with the strain activated by the basal culture medium, it has stronger reaction activity during the fermentation culture stage and higher conversion efficiency of flavonoid aglycones.

[0046] Preferably, the culture medium comprises any one of MRS medium, PYG medium or TPY medium, or a combination of at least two of them.

[0047] Preferably, the culture medium is MRS medium.

[0048] Preferably, in step (2), the inoculation amount of the strain is 2-20%, for example, it can be 2%, 2.2%, 2.5%, 2.8%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, etc.

[0049] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0050] Preferably, the bacterial species include Bifidobacterium and / or Lactobacillus plantarum.

[0051] Preferably, the bacterial species include Bifidobacterium and Lactobacillus plantarum.

[0052] The bifidobacterium and the lactobacillus plantarum in the present invention can synergistically improve the bioconversion rate of flavonoid aglycones, thereby enhancing the antioxidant and whitening and brightening effects of the hawthorn fermentation filtrate.

[0053] Preferably, the ratio of the viable counts of Bifidobacterium and Lactobacillus plantarum is (1-10):(1-10).

[0054] The specific point values in the first one (1-10) can be 1, 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0055] The specific point values in the second one (1-10) can be 1, 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0056] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0057] Preferably, in step (2), the fermentation includes a first stage fermentation and a second stage fermentation.

[0058] The conditions of the first stage fermentation include: temperature 28-30°C (for example, 28°C, 28.1°C, 28.2°C, 28.5°C, 28.8°C, 29°C, 29.2°C, 29.5°C, 29.7°C or 30°C), time 22-26h (for example, 22h, 22.2h, 22.5h, 22.8h, 23h, 23.5h, 24h, 24h). .5h, 25h, 25.5h or 26h, etc.), pH value 6.0-6.5 (for example, it can be 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, etc.), dissolved oxygen 4.5-5.5% (for example, it can be 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4% or 5.5%, etc.).

[0059] The conditions of the second stage fermentation include: initial fermentation at 32-34°C (for example, 32°C, 32.1°C, 32.2°C, 32.5°C, 32.8°C, 33°C, 33.3°C, 33.5°C, 33.7°C or 34°C, etc.) maintained for 10-14h (for example, 10h, 10.2h, 10.5h, 10.7h, 11h, 11.3h, 11.5h, 11.8h, 12h, 12.5h, 13h, 13.5h or 14h, etc.), subsequent fermentation at 25-28°C (for example, 25°C, 25.2°C, 25.5°C, 25.8°C, 26°C, 26.5°C, 27°C, 27.5°C or 28°C, etc.) maintained for 2 2-26h (for example, it can be 22h, 22.2h, 22.5h, 22.8h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h or 26h, etc.), the dissolved oxygen decreases linearly from 4.5-5.5% (for example, it can be 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4% or 5.5%, etc.) to 0.8-1.2% (for example, it can be 0.8%, 0.83%, 0.85%, 0.88%, 0.9%, 0.92%, 0.95%, 0.98%, 1%, 1.05%, 1.1%, 1.15% or 1.2%, etc.).

[0060] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0061] During the first stage of fermentation, the bacteria proliferate rapidly and the active ingredients are initially converted based on the hawthorn enzymatic hydrolysate; during the second stage of fermentation, the initial fermentation maintains the metabolic activity of the bacteria and further converts the products of the first stage of fermentation. The subsequent fermentation enhances the induction of secondary metabolism of the bacteria through gradient changes in temperature, time, and dissolved oxygen, enhances the bioconversion rate of flavonoid aglycones, and improves the antioxidant and whitening and brightening effects of the hawthorn fermentation filtrate.

[0062] Preferably, during the second stage fermentation, silk peptide is also added at the beginning of subsequent fermentation.

[0063] Preferably, the mass percentage of the silk peptide in the fermentation system is 0.1-1%, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0064] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0065] Silk peptide is a metabolic promoter of the strain. When added at the beginning of the subsequent fermentation of the second stage of fermentation, it can significantly increase the production of secondary metabolites of the strain, produce more small molecule active ingredients that are easy to absorb, and at the same time enhance the bioconversion rate of flavonoid aglycones, thereby improving the antioxidant and whitening and brightening effects of the hawthorn fermentation filtrate.

[0066] Preferably, in step (2), the sterilization temperature is 60-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C; the time is 10-20 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc.

[0067] Preferably, after the sterilization, the product is rapidly cooled to 4-10°C (for example, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, etc.) within 2-5min (for example, 2min, 2.min, 2.5min, 2.8min, 3min, 3.5min, 4min, 4.5min or 5min, etc.).

[0068] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0069] In the present invention, the product is rapidly cooled by a plate heat exchanger immediately after sterilization to terminate the biochemical reaction and protect the heat-labile active ingredients in the product.

[0070] Preferably, in step (2), the solid-liquid separation method includes centrifugation and / or filtration.

[0071] Preferably, the centrifugal speed is 10000-14000g, for example, it can be 10000g, 10500g, 11000g, 11500g, 12000g, 12500g, 13000g, 13500g or 14000g, etc.; the time is 5-15min, for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min, etc.

[0072] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0073] Preferably, the solid-liquid separation further includes a purification step.

[0074] Preferably, the purification process comprises: collecting the filtrate, performing tangential flow filtration and / or separation with a macroporous adsorption resin.

[0075] In a second aspect, the present invention provides a hawthorn fermentation filtrate prepared by the preparation method according to the first aspect.

[0076] In a third aspect, the present invention provides a use of the hawthorn fermentation filtrate as described in the second aspect in cosmetics, wherein the cosmetics include any one of lotion, emulsion, cream, essence, spray, mask, sunscreen, shampoo, shower gel, conditioner or hair mask.

[0077] Compared with the prior art, the present invention has the following beneficial effects:

[0078] In the present invention, laccase and β-glucosidase synergistically decompose the macromolecular active substances of hawthorn, and further inoculate bacteria for fermentation to produce effective ingredients such as various amino acids, polypeptides, vitamins, organic acids and minerals, significantly increasing the content of flavonoid aglycones. The obtained hawthorn fermentation filtrate has excellent antioxidant and whitening and brightening effects. DETAILED DESCRIPTION

[0079] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0080] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0081] The sources of materials used in the following specific embodiments are as follows:

[0082] Cellulase was sourced from Hubei Yuying Biotechnology Co., Ltd., pectinase was sourced from Hubei Yuying Biotechnology Co., Ltd., laccase was sourced from Shanghai Huashang Xiangyang Biotechnology Co., Ltd., and β-glucosidase was sourced from Beijing Biolab Technology Co., Ltd. Bifidobacterium was sourced from the Shanghai Collection of Microorganisms, strain Bifidobacterium animalis BB-12, strain number SHMCC (SHBCC) D24674; Lactobacillus plantarum was sourced from DuPont Danisco Biotechnology, strain number LP-115; and silk peptide was sourced from Suzhou Soho Biomaterials Technology Co., Ltd.

[0083] MRS medium was prepared by weighing 10 g / L peptone, 8 g / L beef extract powder, and 4 g / L yeast extract as nitrogen sources and growth factors, 20 g / L glucose as a carbon source, 2 g / L dipotassium hydrogen phosphate and 2 g / L triammonium citrate as a buffer system, 5 g / L sodium acetate for selective inhibition of bacteria, 0.21 g / L magnesium sulfate and 0.04 g / L manganese sulfate as trace elements, and 1 g / L Tween 80 as a surfactant to enhance nutrient absorption. All components were dissolved in ultrapure water, and the pH was adjusted to 6.5 with 1 M NaOH. The medium was sterilized by high-pressure steam at 121°C for 20 min. After cooling to room temperature, the medium was filtered through a 0.22 μm sterile filter membrane to remove pyrogens.

[0084] Example 1

[0085] This embodiment provides a method for preparing hawthorn fermentation filtrate, the preparation method comprising:

[0086] (1) Wash fresh hawthorn with water to remove impurities, remove the core and crush into particles, freeze-dry for later use. Mix 1000g freeze-dried hawthorn, 3g cellulase, 2g pectinase and 7L water, react at pH = 5.0 at 45℃ for 1h to carry out the first enzymatic hydrolysis reaction; then add 1g laccase and 2g β-glucosidase, react at pH = 5.0 at 40℃ for 35min to carry out the second enzymatic hydrolysis reaction. The second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment. The parameters of the pulsed ultrasonic treatment are set as follows: working time 5s, rest time 3s, frequency 40kHz, power 300W. After the enzymatic hydrolysis reaction is completed, microwave treatment is carried out at 2500MHz, 700W for 90s to inactivate the enzyme and obtain hawthorn hydrolysate.

[0087] (2) Bifidobacterium and Lactobacillus plantarum were inoculated into MRS medium containing 1% hawthorn hydrolysate, and the dissolved oxygen was maintained at 3% by nitrogen replacement. The culture was carried out at 37°C for 18 h to obtain activated strains. 3% activated Bifidobacterium and 2% activated Lactobacillus plantarum were inoculated into MRS medium containing 30% hawthorn hydrolysate (the bacterial solution concentration was 1×10 10 CFU / mL). The first stage of fermentation was carried out: maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermentation at 29°C for 24 hours. The second stage of fermentation was carried out: fermentation was first carried out at 33°C for 12 hours, then 0.5% silk peptide was added, and fermentation was carried out at 27°C for 24 hours, with the dissolved oxygen content linearly decreasing from 5.0% to 1.0%. After the fermentation was completed, sterilization was carried out at 65°C for 15 minutes, and then rapid cooling was carried out to 4°C for 5 minutes. The fermentation product was centrifuged at 12000g for 10 minutes, and the liquid was collected. A 0.2μm pore size zirconia ceramic membrane assembly was selected, and tangential flow filtration was performed at a constant pressure of 0.25MPa and a temperature of 25°C at a flow rate of 3m / s to collect the liquid to obtain the hawthorn fermentation filtrate.

[0088] Example 2

[0089] This embodiment provides a method for preparing hawthorn fermentation filtrate, the preparation method comprising:

[0090] (1) Wash fresh hawthorn with water to remove impurities, remove the core and crush into particles, freeze-dry for later use. Mix 1000g of freeze-dried hawthorn, 4g of cellulase, 3g of pectinase and 10L of water, react at pH = 5.5 at 40°C for 2h to carry out the first enzymatic hydrolysis reaction; then add 2g of laccase and 3g of β-glucosidase, react at pH = 4.5 at 45°C for 30min to carry out the second enzymatic hydrolysis reaction. The second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment, and the parameters of the pulsed ultrasonic treatment are set as follows: working time 4s, rest time 4s, frequency 30kHz, power 400W. After the enzymatic hydrolysis reaction is completed, microwave treatment is carried out at 2000MHz and 800W for 60s to inactivate the enzyme and obtain hawthorn hydrolysate.

[0091] (2) Bifidobacterium and Lactobacillus plantarum were inoculated into MRS medium containing 1% hawthorn hydrolysate, and the dissolved oxygen was maintained at 3% by nitrogen replacement. The culture was carried out at 37°C for 18 h to obtain activated strains. 2% activated Bifidobacterium and 3% activated Lactobacillus plantarum were inoculated into MRS medium containing 40% hawthorn hydrolysate (the bacterial solution concentration was 1×10 10 CFU / mL). The first stage of fermentation was carried out: maintaining a pH of 6.5, a dissolved oxygen content of 5.5%, and fermentation at 28°C for 26 hours. The second stage of fermentation was carried out: fermentation was first carried out at 32°C for 14 hours, then 0.8% silk peptide was added, and fermentation was carried out at 28°C for 22 hours, with the dissolved oxygen content linearly decreasing from 5.5% to 1.2%. After the fermentation was completed, sterilization was carried out at 70°C for 10 minutes, and then rapid cooling was carried out to 6°C over 5 minutes. The fermentation product was centrifuged at 12,000g for 10 minutes, and the liquid was collected. Tangential flow filtration was performed using a 0.2μm pore size zirconia ceramic membrane assembly at a constant pressure of 0.25MPa and a temperature of 25°C at a flow rate of 3m / s to collect the liquid to obtain the hawthorn fermentation filtrate.

[0092] Example 3

[0093] This embodiment provides a method for preparing hawthorn fermentation filtrate, the preparation method comprising:

[0094] (1) Wash fresh hawthorn with water to remove impurities, remove the core and crush into particles, freeze-dry for later use. Mix 1000g freeze-dried hawthorn, 2g cellulase, 1g pectinase and 5L water, react at pH = 4.5 at 50℃ for 0.8h to carry out the first enzymatic hydrolysis reaction; then add 0.5g laccase and 1g β-glucosidase, react at pH = 6.0 at 35℃ for 40min to carry out the second enzymatic hydrolysis reaction. The second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment, and the parameters of the pulsed ultrasonic treatment are set as follows: working time 6s, rest time 2s, frequency 50kHz, power 200W. After the enzymatic hydrolysis reaction is completed, microwave treatment is carried out at 3000MHz and 600W for 120s to inactivate the enzyme and obtain hawthorn hydrolysate.

[0095] (2) Bifidobacterium and Lactobacillus plantarum were inoculated into MRS medium containing 1% hawthorn hydrolysate, and the dissolved oxygen was maintained at 3% by nitrogen replacement. The culture was carried out at 37°C for 18 h to obtain activated strains. 1% activated Bifidobacterium and 1% activated Lactobacillus plantarum were inoculated into MRS medium containing 20% hawthorn hydrolysate (the bacterial solution concentration was 1×10 10 CFU / mL). The first stage of fermentation was carried out: maintaining a pH of 6.0, a dissolved oxygen content of 4.5%, and fermentation at 30°C for 22 hours. The second stage of fermentation was carried out: fermentation was first carried out at 34°C for 10 hours, then 0.2% silk peptide was added, and fermentation was carried out at 25°C for 26 hours, with the dissolved oxygen content linearly decreasing from 4.5% to 0.8%. After the fermentation was completed, sterilization was carried out at 60°C for 20 minutes, and then rapid cooling was carried out to 8°C over 5 minutes. The fermentation product was centrifuged at 12,000g for 10 minutes, and the liquid was collected. Tangential flow filtration was performed using a 0.2μm pore size zirconia ceramic membrane assembly at a constant pressure of 0.25MPa and a temperature of 25°C at a flow rate of 3m / s to collect the liquid to obtain the hawthorn fermentation filtrate.

[0096] Example 4

[0097] This embodiment provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (1), the second enzymatic hydrolysis reaction is not carried out under pulsed ultrasonic treatment, and the remaining steps remain unchanged.

[0098] Example 5

[0099] This embodiment provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (1), the second enzymatic hydrolysis reaction is carried out under ordinary ultrasonic treatment, and the parameters of ordinary ultrasonic treatment are set to: working time 8s, frequency 40kHz, power 300W, and the remaining steps remain unchanged.

[0100] Example 6

[0101] This embodiment provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (2), Bifidobacterium is not inoculated, and the reduced amount thereof is proportionally distributed to Lactobacillus plantarum, and the remaining steps remain unchanged.

[0102] Example 7

[0103] This embodiment provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that in step (2), Lactobacillus plantarum is not inoculated, and the reduced amount thereof is proportionally distributed to Bifidobacterium, and the remaining steps remain unchanged.

[0104] Example 8

[0105] This embodiment provides a method for preparing a hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (2), "performing a first-stage fermentation: maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermenting at 29°C for 24 hours; performing a second-stage fermentation: first fermenting at 33°C for 12 hours, then adding 0.5% silk peptide, and fermenting at 27°C for 24 hours, and linearly reducing the dissolved oxygen content from 5.0% to 1.0%" is replaced by "adding 0.5% silk peptide, maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermenting at 29°C for 60 hours", and the remaining steps remain unchanged.

[0106] Example 9

[0107] The present embodiment provides a method for preparing a hawthorn fermentation filtrate, which differs from the embodiment 1 only in that: in step (2), the steps of "performing a first-stage fermentation: maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermenting at 29°C for 24 hours; and performing a second-stage fermentation: first fermenting at 33°C for 12 hours, then adding 0.5% silk peptide, and fermenting at 27°C for 24 hours, and linearly reducing the dissolved oxygen from 5.0% to 1.0%" are replaced with "performing a first-stage fermentation: first fermenting at 33°C for 12 hours, then adding 0.5% silk peptide, and fermenting at 27°C for 24 hours, and linearly reducing the dissolved oxygen from 5.0% to 1.0%; and performing a second-stage fermentation: maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermenting at 29°C for 24 hours" are replaced with the steps of ..." are replaced with the steps of "performing a second-stage fermentation: maintaining a pH value of 6.2, a dissolved oxygen content of 5.0%, and fermenting at 29°C

[0108] Example 10

[0109] This embodiment provides a method for preparing a hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (2), "performing a second-stage fermentation: first fermenting at 33°C for 12 hours, then adding 0.5% silk peptide, fermenting at 27°C for 24 hours, and linearly reducing the dissolved oxygen from 5.0% to 1.0%" is replaced by "performing a second-stage fermentation: first adding 0.5% silk peptide, fermenting at 27°C for 24 hours, and linearly reducing the dissolved oxygen from 5.0% to 1.0%, and then fermenting at 33°C for 12 hours", and the other steps remain unchanged.

[0110] Example 11

[0111] This embodiment provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that in step (2), silk peptide is not added during the fermentation process, and the other steps remain unchanged.

[0112] Comparative Example 1

[0113] This comparative example provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (1), laccase is not added, and its reduced amount is proportionally distributed to β-glucosidase, and the other steps remain unchanged.

[0114] Comparative Example 2

[0115] This comparative example provides a method for preparing hawthorn fermentation filtrate, which differs from Example 1 only in that: in step (1), β-glucosidase is not added, and its reduced amount is proportionally distributed to laccase, and the other steps remain unchanged.

[0116] Test Example 1

[0117] Fresh hawthorn was washed to remove impurities, de-cored, crushed into granules, and freeze-dried to produce hawthorn freeze-dried powder for later use. The hawthorn fermentation filtrates prepared in Examples 1-11 and Comparative Examples 1-2 were freeze-dried to produce hawthorn fermentation filtrate freeze-dried powder for later use. 0.2 g of hawthorn freeze-dried powder and the hawthorn fermentation filtrate freeze-dried powder prepared in Examples 1-11 and Comparative Examples 1-2 were respectively weighed into a 25 mL volumetric flask, 10 mL of 80% ethanol solution was added, and the mixture was shaken evenly. Ultrasonic extraction was performed at 50°C for 30 minutes, and the mixture was centrifuged at 3000 rpm for 15 minutes. 1 mL of the supernatant was taken and diluted 5-fold with 80% methanol. The mixture was filtered through a 0.45 μm filter membrane before injection. The flavonoid glycoside and flavonoid aglycone content in the sample was determined by high performance liquid chromatography using the following procedures: chromatographic column: SB-C18 column (4.6 mm × 250 mm, 5 μm); column temperature: 35°C; flow rate: 1.0 mL / min; injection volume: 20 μL; UV detector wavelength: 260 nm; mobile phase: 0.2% glacial acetic acid + methanol; gradient elution program: 0-5 min, methanol from 20% to 25%; 5-25 min, methanol from 25% to 80%; 25-30 min, methanol from 80% to 20%, elution complete. Flavonoid aglycone conversion rate (%) = (flavonoid aglycone content of hawthorn fermentation filtrate freeze-dried powder - flavonoid aglycone content of hawthorn freeze-dried powder) / (flavonoid glycoside content of hawthorn fermentation filtrate freeze-dried powder - flavonoid glycoside content of hawthorn freeze-dried powder) × 100%.

[0118] The test results are shown in Table 1. The hawthorn fermentation filtrate prepared by the method of the present invention contains a high content of flavonoid aglycones. Laccase and β-glucosidase synergistically promote the conversion of macromolecular compounds. Pulsed ultrasonic treatment assists in improving the enzymatic hydrolysis efficiency and accelerating the conversion of flavonoid aglycones. Bifidobacterium and Lactobacillus plantarum synergistically ferment to further improve the bioconversion rate of flavonoid aglycones. By regulating the two-stage fermentation conditions, the production of multiple active ingredients can be induced.

[0119] Table 1

[0120]

[0121]

[0122] Test Example 2

[0123] According to the "Cosmetics - Free Radical (DPPH) Scavenging Experimental Method (T / SHRH006-2018)", the DPPH scavenging rates of the hawthorn fermentation filtrates prepared in Examples 1-11 and Comparative Examples 1-2 were respectively detected: a 96-well plate was used, 3 replicates were set for each group, and the system was 200 μL; after the hawthorn fermentation filtrates prepared in Examples 1-11 and Comparative Examples 1-2 were freeze-dried, water was added to prepare a test sample solution with a final concentration of 10%, 100 μL of the test sample solution was taken, and 100 μL of 0.1 mM DPPH solution was added to the reaction system; 100 μL of distilled water + 100 μL of 0.1 mM DPPH solution was used as the control group; after the reaction system was constructed, it was shaken in the dark for 10 minutes, and the absorbance at 520 nm was measured using a microplate reader to calculate the DPPH scavenging rate, DPPH scavenging rate (%) = [(control group absorbance - sample group absorbance) / control group absorbance] × 100%.

[0124] The test results are shown in Table 2. The hawthorn fermentation filtrate prepared by the method of the present invention has a high DPPH clearance rate and excellent antioxidant effect. Laccase and β-glucosidase synergistically promote the conversion of macromolecular compounds, and pulsed ultrasonic treatment assists in improving their enzymatic hydrolysis efficiency, significantly improving the DPPH clearance rate; Bifidobacterium and Lactobacillus plantarum synergistic fermentation further improve the DPPH clearance rate, and regulating the two-stage fermentation conditions can induce the production of multiple active ingredients and enhance the antioxidant effect.

[0125] Table 2

[0126]

[0127]

[0128] Test Example 3

[0129] Preparation of solution: ① Phosphate buffer: Take 250 mL of 0.2 M potassium dihydrogen phosphate solution, add 118 mL of 0.2 M sodium hydroxide solution, and dilute to 1000 mL with deionized water; ② Tyrosine solution: Weigh 27.17 mg of L-tyrosine, dissolve it in phosphate buffer and dilute to a 100 mL volumetric flask to prepare a 1.5 mM L-tyrosine solution, which is prepared for immediate use; ③ Tyrosinase solution: Weigh 18.5 mg of tyrosinase (25 KU, Solebol), add 12.5 mL of phosphate buffer and dilute to 2 KU / mL, aliquot and freeze; dilute to 200 U / mL with phosphate buffer before use; ④ Test sample solution: After freeze-drying the hawthorn fermentation filtrates prepared in Example 1-11 and Comparative Example 1-2, add water to prepare a solution with a final concentration of 10%.

[0130] Under dark conditions, 50 μL of phosphate buffer, 50 μL of test sample solution, 50 μL of tyrosine solution, and 50 μL of tyrosinase solution were added respectively, and the mixture was incubated at 37°C for 10 min in a microplate reader. The absorbance of the sample group was measured at 475 nm. Phosphate buffer was used instead of the test sample as a control group. In addition, A0 was 50 μL of tyrosine solution + 100 μL of buffer + 50 μL of tyrosinase solution, and A1 was 50 μL of tyrosine solution + 150 μL of buffer. The tyrosinase inhibition rate was calculated as follows: Tyrosinase inhibition rate (%) = [1-(sample group-control group) / (A0-A1)] × 100%.

[0131] The test results are shown in Table 3. The hawthorn fermentation filtrate prepared by the method of the present invention has a high tyrosinase inhibition rate and excellent whitening and brightening effects. Laccase and β-glucosidase synergistically promote the conversion of macromolecular compounds. Pulsed ultrasonic treatment assists in improving the enzymatic hydrolysis efficiency and significantly improves the tyrosinase inhibition rate. Bifidobacterium and Lactobacillus plantarum synergistically ferment to further improve the tyrosinase inhibition rate. Regulating the two-stage fermentation conditions can induce the production of multiple active ingredients and enhance the whitening and brightening effects.

[0132] Table 3

[0133] Test samples Tyrosinase inhibition rate (%) Example 1 78.5 Example 2 77.2 Example 3 76.8 Example 4 55.3 Example 5 52.7 Example 6 63.4 Example 7 61.9 Example 8 48.2 Example 9 50.6 Example 10 46.8 Example 11 68.3 Comparative Example 1 35.7 Comparative Example 2 33.9

[0134] The present invention uses the above-described embodiments to illustrate the preparation method of hawthorn fermentation filtrate, its product, and its applications. However, the present invention is not limited to these embodiments, and implementation of the present invention is not necessarily dependent on these embodiments. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions of raw materials for the present product, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0135] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0136] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing hawthorn fermentation filtrate, characterized in that: The preparation method comprises: (1) mixing hawthorn, a first complex enzyme, and water to perform a first enzymatic hydrolysis reaction, then adding a second complex enzyme to perform a second enzymatic hydrolysis reaction, and then inactivating the enzymes to obtain a hawthorn hydrolyzate; the first complex enzyme comprises cellulase and pectinase; and the second complex enzyme comprises laccase and β-glucosidase; (2) inoculating activated bacteria into a culture medium containing hawthorn enzymatic hydrolysate, performing fermentation, sterilizing after completion, and performing solid-liquid separation to obtain the hawthorn fermentation filtrate.

2. The preparation method according to claim 1, characterized in that In step (1), the mass volume ratio of hawthorn to water is 1:(5-10) g / mL; Preferably, the mass ratio of the hawthorn and the first complex enzyme is (90-110):(0.2-1); Preferably, the mass ratio of the cellulase to the pectinase is (2-4):(1-3); Preferably, the temperature of the first enzymatic hydrolysis reaction is 40-50°C and the time is 0.8-2h; Preferably, the pH value of the first enzymatic hydrolysis reaction is 4.5-5.5; Preferably, in step (1), the mass ratio of the hawthorn and the second complex enzyme is (90-110):(0.05-0.5); Preferably, the mass ratio of the laccase to the β-glucosidase is (0.5-2):(1-3); Preferably, the temperature of the second enzymatic hydrolysis reaction is 35-45°C and the time is 30-40 minutes; Preferably, the pH value of the second enzymatic hydrolysis reaction is 4.5-6.0; Preferably, the second enzymatic hydrolysis reaction is carried out under pulsed ultrasonic treatment; Preferably, the parameters of the pulse ultrasonic treatment include: working time 4-6s, rest time 2-4s, frequency 30-50kHz, power 200-400W.

3. The preparation method according to claim 1 or 2, characterized in that In step (1), the enzyme inactivation method includes microwave treatment; Preferably, the microwave treatment has a frequency of 2000-3000 MHz, a power of 600-800 W, and a time of 60-120 s.

4. The preparation method according to any one of claims 1 to 3, characterized in that In step (2), the mass percentage of the hawthorn enzymatic hydrolysate in the culture medium is 20-40%; Preferably, the culture medium for activating the strain contains 0.1-5% by weight of hawthorn enzymatic hydrolysate; Preferably, the culture medium comprises any one of MRS medium, PYG medium or TPY medium, or a combination of at least two thereof; Preferably, the culture medium is MRS medium.

5. The preparation method according to any one of claims 1 to 4, characterized in that In step (2), the inoculation amount of the bacterial strain is 2-20%; Preferably, the bacterial species include Bifidobacterium and / or Lactobacillus plantarum; Preferably, the ratio of the viable counts of Bifidobacterium and Lactobacillus plantarum is (1-10):(1-10).

6. The preparation method according to any one of claims 1 to 5, characterized in that In step (2), the fermentation includes a first stage fermentation and a second stage fermentation; The conditions of the first stage fermentation include: temperature 28-30°C, time 22-26h, pH 6.0-6.5, dissolved oxygen 4.5-5.5%; The conditions of the second stage fermentation include: initial fermentation at 32-34°C for 10-14 hours, subsequent fermentation at 25-28°C for 22-26 hours, and dissolved oxygen linearly reduced from 4.5-5.5% to 0.8-1.2%; Preferably, during the second stage fermentation, the method further comprises adding silk peptide at the beginning of subsequent fermentation; Preferably, the mass percentage of the silk peptide in the fermentation system is 0.1-1%.

7. The preparation method according to any one of claims 1 to 6, characterized in that In step (2), the sterilization temperature is 60-70°C and the time is 10-20 minutes; Preferably, after the sterilization, the mixture is rapidly cooled to 4-10°C within 2-5 minutes.

8. The preparation method according to any one of claims 1 to 7, characterized in that In step (2), the solid-liquid separation method includes centrifugation and / or filtration; Preferably, the centrifugal speed is 10000-14000g and the time is 5-15min; Preferably, the solid-liquid separation further includes a purification step; Preferably, the purification process comprises: collecting the filtrate, performing tangential flow filtration and / or separation with a macroporous adsorption resin.

9. The hawthorn fermentation filtrate prepared by the preparation method according to any one of claims 1 to 8.

10. The use of hawthorn fermentation filtrate in cosmetics according to claim 9, characterized in that: The cosmetics include any one of lotion, emulsion, cream, essence, spray, mask, sunscreen, shampoo, shower gel, conditioner or hair mask.

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