Black truffle osmanthus fragrans lactobacillus fermentation filtrate as well as preparation method and application thereof

Abstract

The invention relates to a perigord truffle osmanthus fragrans lactic acid bacteria fermentation filtrate and a preparation method and application thereof, and belongs to the technical field of fermentation filtrate, the preparation method of the fermentation filtrate comprises the following steps: S1, strain culture; s2, pretreating the sweet-scented osmanthus and the perigord truffle; s3, preparing fermentation filtrate. Osmanthus fragrans, perigord truffle, hydrolyzed soybean and soybean oil rice koji extract are creatively combined to serve as a culture medium, lactobacillus is used for fermentation, then microfiltration is conducted, and fermentation filtrate is obtained; during the period, appropriate fermentation conditions and parameters are adjusted in the research and development process of fermentation filtrate, the optimal culture medium condition is found out, papain and bromelain are compounded for primary hydrolysis in the preparation process of hydrolyzed soybean serving as one of raw materials, and then flavourzyme is adopted for secondary hydrolysis; the obtained fermentation product filtrate is high in oxidation resistance and light damage protection capability, and the fermentation product filtrate contains a relatively strong unique fresh and sweet smell.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation filtrates, and specifically relates to a black truffle osmanthus lactic acid bacteria fermentation filtrate and a preparation method and application thereof. Background Art

[0002] With the continuous growth of consumer demand for green, safe and effective cosmetics, the cosmetics industry has undergone countless changes and derivations, and its core competitiveness has gradually condensed in the two aspects of safety and efficacy. Therefore, safe and effective cosmetic raw materials and products have good prospects in the market. Nowadays, the application of bio-fermentation technology has gradually entered the field of cosmetics, and it has good applications in moisturizing, whitening, anti-aging, soothing and other effects. Bio-fermentation technology utilizes certain unique functions of microorganisms, adopts modern bioengineering technology, and produces products on an industrial scale or directly applies microorganisms to product terminals. At present, bio-fermentation technology has become a research and development hotspot and application direction in the field of cosmetics, greatly improving the safety and efficacy of cosmetics.

[0003] Lactobacillus fermentation products are easy to obtain and have been favored by many products in recent years. Among the components of lactobacillus fermentation products, lactic acid is the largest, followed by proteins, enzymes, vitamins, organic matter, etc. Lactic acid is not only an important component of yogurt, but also has certain skin care value. It is an α-hydroxy acid like fruit acid, which can soften the stratum corneum and promote the regeneration of epidermal cells. Because its molecular weight is larger than that of fruit acid, lactic acid penetrates the skin more slowly, so it is less irritating than fruit acid.

[0004] The normal metabolism of lactic acid bacteria will first decompose the protein, sugar and fat in the culture medium, and degrade the macromolecular components in osmanthus and black truffle into small molecular peptides and essential amino acids that can be directly absorbed. The fermentation smell of lactic acid bacteria itself is relatively weak, which can retain the smell of osmanthus and black truffle to the maximum extent, so that the sweet smell of osmanthus and the fresh fragrance of black truffle form a unique sweet smell. At present, the products with added fermentation raw materials on the market are increasing day by day, but due to the limitation that the fermentation smell of the fermentation itself is relatively unpleasant, the fermentation liquid is relatively turbid, and there are certain restrictions on the amount of addition and the type of product used. Therefore, in addition to functionality, the product is also required to have a special aroma.

[0005] In summary, it is of great practical significance to develop a fermentation filtrate that has whitening, UV resistance and fragrance. Summary of the invention

[0006] The invention aims to provide a black truffle osmanthus lactic acid bacteria fermentation filtrate and a preparation method and application thereof. The fermentation product filtrate is obtained by fermenting a culture medium of osmanthus, black truffle and soybean with lactobacillus and then performing microfiltration. In addition to the fragrance of osmanthus and the fresh fragrance of black truffle, the fermentation filtrate also has whitening, anti-oxidation and anti-UVA / UVB effects. Therefore, it has certain potential in cosmetics and can be used in functional external products of skin care products or sunscreen products, thereby solving the problems of poor whitening, anti-UV and fragrance of the fermentation filtrate in the prior art.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A method for preparing black truffle sweet osmanthus lactic acid bacteria fermentation filtrate, specifically comprising the steps:

[0009] S1, bacterial strain culture: hydrolyzed soybean, glucose and sterilized water are mixed uniformly, temperature-controlled sterilization is carried out to obtain lactic acid bacteria culture medium, plant lactobacillus is inoculated in the lactic acid bacteria culture medium and temperature-controlled culture is carried out to obtain lactic acid bacteria strains, which are standby;

[0010] S2, pre-treating sweet osmanthus and black truffle:

[0011] S21, crushing sweet osmanthus into powder to obtain powdered sweet osmanthus, which is set aside; chopping fresh black truffle to obtain chopped black truffle, which is set aside;

[0012] S22, mixing the chopped black truffle and soybean oil rice koji extract evenly and oil-sealing them at a controlled temperature, and crushing and homogenizing the mixture after the oil sealing to obtain a pretreated black truffle;

[0013] S3, preparing fermentation filtrate:

[0014] S31, mixing powdered osmanthus, pretreated black truffle, glucose, sodium chloride, hydrolyzed soybean, soybean oil rice koji extract and sterile water to obtain a culture medium, sterilizing the culture medium under temperature control, inoculating lactic acid bacteria strains, and fermenting under temperature control to obtain a fermentation product;

[0015] S32, sterilizing the fermented product by controlling the temperature, cooling, adjusting the pH, and filtering through a microfiltration membrane to obtain a finished product of the black truffle osmanthus lactic acid bacteria fermentation filtrate.

[0016] Osmanthus fragrans is a flower of the Oleaceae family. It has a thin and weak pedicel, very short filaments, and extremely fragrant flowers. It has a good promoting effect on the activity of fibroblasts. Combined with its antioxidant properties, it has a skin revitalizing effect and can be used in anti-aging cosmetics. The extract inhibits the production of dihydrotestosterone, that is, it inhibits the secretion of male hormones. Combined with its anti-inflammatory properties, it has a good preventive and therapeutic effect on hair loss, acne, etc. caused by high male hormones.

[0017] Black truffle is a kind of fungus that grows mostly under pine trees and oak trees. This fungus is generally black in color and is produced in abundance in Jiangxi Province of my country. Black truffle is rich in various active ingredients, has antioxidant functions, eliminates free radicals, and can be used in anti-aging cosmetics. It can inhibit the activity of tyrosinase and has the effect of whitening the skin.

[0018] Soybean is an annual herbaceous plant of the genus Glycine max in the Leguminosae family. It is rich in antioxidants such as isoflavones and polyphenols, which can neutralize free radicals and reduce the damage of oxidative stress to cells. It can inhibit the production of inflammatory mediators and reduce inflammatory responses.

[0019] As a preferred technical solution of the present invention, the usage ratio of the hydrolyzed soybean, glucose and sterile water in step S1 is 202-205g:9-11g:1L.

[0020] As a preferred technical solution of the present invention, the mass ratio of Lactobacillus plantarum to the lactic acid bacteria culture medium in step S1 is 1-1.5:50.

[0021] Furthermore, the total viable count of the plant lactobacillus is 1.24×10 11 CFU / g, purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.

[0022] As a preferred technical solution of the present invention, the temperature of the temperature-controlled sterilization treatment in step S1 is 95-100°C and the time is 30 minutes; the temperature of the temperature-controlled culture is 23-25°C and the time is 72 hours.

[0023] As a preferred technical solution of the present invention, the osmanthus described in step S21 includes dried osmanthus and fresh osmanthus in a mass ratio of 1:3.

[0024] As a preferred technical solution of the present invention, the mass ratio of the black truffle and the soybean oil rice koji extract in step S22 is 1:1.

[0025] As a preferred technical solution of the present invention, the preparation method of the soybean oil rice koji extract described in step S22 is:

[0026] The rice koji and soybean oil are mixed evenly, oil-sealed under controlled temperature, and then sterilized under controlled temperature to obtain a soybean oil and rice koji extract.

[0027] Furthermore, the raw material of the rice koji includes any one of rice, polished round-grained rice and starch.

[0028] Furthermore, the mass ratio of the rice koji to soybean oil is 1-1.2:3.

[0029] Furthermore, the temperature of the temperature-controlled oil seal is 40° C., and the time is 72 hours; the temperature of the temperature-controlled sterilization treatment is 121° C., and the time is 30 minutes.

[0030] Furthermore, the method for preparing the rice koji comprises: soaking the rice koji raw material under temperature control, draining, steaming until each grain is distinct, cooling, adding Aspergillus oryzae, stirring evenly, fermenting, and drying the fermented rice koji raw material under temperature control to obtain the rice koji.

[0031] Furthermore, the mass ratio of the rice koji raw material to Aspergillus oryzae is 20-22:1.

[0032] Furthermore, the temperature of the temperature-controlled dipping is 23-25°C, and the time is 24 hours; the conditions of the fermentation are: temperature 28-30°C, humidity 90-95%, and time 24 hours; the temperature of the temperature-controlled drying is 75-80°C, and the time is 6-8 hours.

[0033] As a preferred technical solution of the present invention, the temperature of the temperature-controlled oil seal in step S22 is 40° C. and the time is 72 hours.

[0034] As a preferred technical solution of the present invention, the mass ratio of the powdered osmanthus, pretreated black truffle, glucose, sodium chloride, hydrolyzed soybeans, soybean oil and rice koji extract and sterile water in step S31 is 22-26:3-5:2-2.5:2-2.5:5-6:5-6:56-60.

[0035] As a preferred technical solution of the present invention, the mass ratio of the culture medium to the lactic acid bacteria strain in step S31 is 1:0.05.

[0036] As a preferred technical solution of the present invention, the temperature of the temperature-controlled sterilization in step S31 is 121° C. and the time is 30 minutes; the temperature of the temperature-controlled fermentation is 22-24° C. and the time is 7 days.

[0037] As a preferred technical solution of the present invention, the preparation method of the hydrolyzed soybean is:

[0038] A1. Grind and cold-press fresh soybeans to obtain soybean powder, mix the soybean powder and water evenly, adjust the pH once, oscillate, centrifuge at a controlled speed, filter, take the filtrate and adjust the pH twice, let stand, centrifuge at a controlled speed, filter again, take the precipitate and adjust the pH three times to obtain pretreated soybean powder, and mix the pretreated soybean powder and deionized water evenly to obtain a mixed solution A;

[0039] A2. Add papain and bromelain to the mixed solution A for primary temperature-controlled hydrolysis, then add flavor protease for secondary temperature-controlled hydrolysis to obtain a hydrolyzate, centrifuge the hydrolyzate, filter the supernatant with a microporous membrane, and obtain hydrolyzed soybeans.

[0040] Furthermore, in step A1, the mass ratio of soybean powder to water is 1:15-20; the first pH adjustment is to adjust pH=8.5 using sodium bicarbonate; the oscillation time is 50 min; the speed of the controlled centrifugation is 3800-4000 r / min, and the time is 15 min; the second pH adjustment is to adjust pH=4.5 using acetic acid; the standing time is 20-30 min; the third pH adjustment is to adjust pH=7.0 using sodium bicarbonate.

[0041] Furthermore, the dosage ratio of the pretreated soybean powder and deionized water in step A1 is 1 g:10-12 mL.

[0042] Furthermore, the mass ratio of the mixed solution A in step A2 to papain, bromelain and flavor protease is 1:0.001-0.002:0.002-0.003:0.02-0.03.

[0043] Furthermore, the enzyme activity of the papain is 204,000 U / g, purchased from Shandong Huiheng Biotechnology Co., Ltd.; the enzyme activity of the bromelain is 52,000 U / g, purchased from Shandong Huiheng Biotechnology Co., Ltd.; the enzyme activity of the flavor protease is 20,000 U / g, purchased from Hubei Xinyuhong Biomedicine Technology Co., Ltd.

[0044] Furthermore, the temperature of the temperature-controlled hydrolysis in step A2 is 55-60°C, and the time is 2 hours; the temperature of the secondary temperature-controlled hydrolysis is 50-55°C, and the time is 2 hours.

[0045] As a preferred technical solution of the present invention, the temperature of the temperature-controlled sterilization in step S32 is 121°C and the time is 30 minutes; the cooling is cooling to room temperature; the pH adjustment is adjusting the pH to neutral using sodium dihydrogen phosphate; and the microfiltration membrane is a 0.22μm microfiltration membrane.

[0046] A fermentation filtrate prepared by the method for preparing the black truffle and osmanthus lactic acid bacteria fermentation filtrate.

[0047] An application of the fermentation filtrate is specifically used in cosmetics.

[0048] Beneficial effects of the present invention:

[0049] (1) The present invention provides a black truffle osmanthus lactic acid bacteria fermentation filtrate, specifically, the osmanthus, black truffle, hydrolyzed soybean and soybean oil rice koji extract are innovatively combined as a culture medium, fermented with lactobacillus and then microfiltered; during the research and development of the fermentation filtrate, the present invention adjusts the appropriate fermentation conditions and parameters, finds the optimal culture medium conditions, and in the preparation process of hydrolyzed soybean as one of the raw materials, papain and bromelain are compounded for primary hydrolysis, and then flavor protease is used for secondary hydrolysis. The fermentation product filtrate obtained in this way has high antioxidant and light damage protection capabilities, and the fermentation product filtrate contains a relatively strong unique fresh and sweet smell.

[0050] (2) The present invention also provides a microfiltration step in the process of preparing the black truffle osmanthus lactic acid bacteria fermentation filtrate, so that the fermentation filtrate is clearer and the application range of the fermentation filtrate product is improved.

[0051] (3) Tyrosinase plays an important role in melanin production and is the main cause of skin pigmentation and skin darkening. The present invention combines osmanthus, black truffle, hydrolyzed soybean and soybean oil rice koji extracts to inhibit tyrosinase activity with the components contained in osmanthus, and the high antioxidant and UVA / UVB-induced skin protection effects of black truffles, so that the obtained fermentation filtrate product has excellent antioxidant efficacy and tyrosinase activity inhibition. Hydrolyzed soybeans and soybean oil rice koji can effectively assist osmanthus and black truffles to make the fermentation filtrate product have a prominent aroma, which can effectively expand the scope of application and audience of the fermentation filtrate product. DETAILED DESCRIPTION

[0052] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The enzymatic activity of papain used in the embodiment of the present invention is 204,000 U / g, purchased from Shandong Huiheng Biotechnology Co., Ltd.; the enzymatic activity of bromelain used is 52,000 U / g, purchased from Shandong Huiheng Biotechnology Co., Ltd.; the enzymatic activity of flavor protease used is 20,000 U / g, purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd.; the total viable count of Lactobacillus plantarum used is 1.24×10 11 CFU / g, purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.; the above will not be repeated in the following.

[0054] Example 1

[0055] Prepare hydrolyzed soybeans, the preparation method of the hydrolyzed soybeans is:

[0056] A1. Fresh soybeans were crushed and cold-pressed to obtain soybean powder, and the soybean powder and water were mixed at a mass ratio of 1:15, and the pH was adjusted to 8.5 with sodium bicarbonate, shaken for 50 minutes, centrifuged at a speed of 4000 r / min for 15 minutes, filtered, and the filtrate was adjusted to pH 4.5 with acetic acid, allowed to stand for 25 minutes, and then centrifuged at a speed of 3800 r / min for 15 minutes, filtered again, and the precipitate was adjusted to pH 7.0 with sodium bicarbonate to obtain pretreated soybean powder, and the pretreated soybean powder and deionized water were mixed at a dosage ratio of 1 g:12 mL to obtain a mixed solution A;

[0057] A2, adding papain and bromelain to the mixed solution A, controlling the temperature at 57.5°C for hydrolysis for 2 hours, and then adding flavor protease, controlling the temperature at 55°C for hydrolysis for 2 hours to obtain a hydrolyzate, centrifuging the hydrolyzate, filtering the supernatant with a microporous membrane, and obtaining hydrolyzed soybeans;

[0058] The mass ratio of the mixed solution A to papain, bromelain and flavor protease is 1:0.001:0.0025:0.03;

[0059] A method for preparing black truffle sweet osmanthus lactic acid bacteria fermentation filtrate, specifically comprising the steps:

[0060] S1, bacterial culture: the above-mentioned hydrolyzed soybean, glucose and sterilized water are mixed in an amount ratio of 202g:10g:1L, and the mixture is sterilized at 95°C for 30min to obtain a lactic acid bacteria culture medium, and plant lactobacillus is inoculated in the lactic acid bacteria culture medium and cultured at 23°C for 72h to obtain a lactic acid bacteria strain, which is then used for standby use;

[0061] The mass ratio of the plant lactobacillus to the lactic acid bacteria culture medium is 1.25:50;

[0062] S2, pre-treating sweet osmanthus and black truffle:

[0063] S21, crushing osmanthus into powder to obtain powdered osmanthus for standby use; the osmanthus comprises dried osmanthus and fresh osmanthus in a mass ratio of 1:3;

[0064] Chop the fresh black truffle into small pieces to obtain chopped black truffles, and set aside;

[0065] S22, mixing the chopped black truffle and soybean oil rice koji extract in equal quantities and sealing with oil at 40° C. for 72 hours, crushing and homogenizing after the sealing to obtain pretreated black truffle;

[0066] The preparation method of the soybean oil rice koji extract is as follows: rice koji and soybean oil are uniformly mixed in a mass ratio of 1:3, and the mixture is oil-sealed at 40° C. for 72 hours, and then sterilized at 121° C. for 30 minutes to obtain the soybean oil rice koji extract;

[0067] The preparation method of the rice koji comprises the following steps: controlling the temperature of starch to 25° C. and soaking it for 24 hours, draining and steaming it until each grain is distinct, cooling it, adding Aspergillus oryzae and stirring it evenly, fermenting it, and controlling the temperature of the fermented rice koji raw material to 78° C. and drying it for 8 hours to obtain the rice koji;

[0068] The mass ratio of the rice koji raw material to Aspergillus oryzae is 22:1; the fermentation conditions are: temperature 28° C., humidity 95%, and time 24 h;

[0069] S3, preparing fermentation filtrate:

[0070] S31, powdered osmanthus, pretreated black truffle, glucose, sodium chloride, the hydrolyzed soybean, soybean oil rice koji extract and sterile water are mixed uniformly in a mass ratio of 22:5:2:2.3:5:5.5:58 to obtain a culture medium, the culture medium is sterilized at 121° C. for 30 min, lactic acid bacteria are inoculated, and the culture medium is fermented at 24° C. for 7 days to obtain a fermentation product;

[0071] The mass ratio of the culture medium to the lactic acid bacteria strain is 1:0.05

[0072] S32, sterilize the fermented product at 121°C for 30 minutes, cool it, adjust the pH to neutral with sodium dihydrogen phosphate, filter it through a 0.22 μm microfiltration membrane, and obtain a finished product of black truffle osmanthus lactic acid bacteria fermentation filtrate.

[0073] Example 2

[0074] Prepare hydrolyzed soybeans, the preparation method of the hydrolyzed soybeans is:

[0075] A1. Fresh soybeans were crushed and cold-pressed to obtain soybean powder, and the soybean powder and water were mixed at a mass ratio of 1:17.5, and the pH was adjusted to 8.5 with sodium bicarbonate, shaken for 50 minutes, centrifuged at a controlled speed of 3900 r / min for 15 minutes, filtered, and the filtrate was adjusted to pH 4.5 with acetic acid, allowed to stand for 20 minutes, and then centrifuged at a controlled speed of 3900 r / min for 15 minutes, filtered again, and the precipitate was adjusted to pH 7.0 with sodium bicarbonate to obtain pretreated soybean powder, and the pretreated soybean powder and deionized water were mixed at a dosage ratio of 1 g:10 mL to obtain a mixed solution A;

[0076] A2, adding papain and bromelain to the mixed solution A, controlling the temperature at 55°C for hydrolysis for 2 hours, and then adding flavor protease, controlling the temperature at 50°C for hydrolysis for 2 hours to obtain a hydrolyzate, centrifuging the hydrolyzate, and filtering the supernatant with a microporous membrane to obtain hydrolyzed soybeans;

[0077] The mass ratio of the mixed solution A to papain, bromelain and flavor protease is 1:0.002:0.003:0.02;

[0078] A method for preparing black truffle sweet osmanthus lactic acid bacteria fermentation filtrate, specifically comprising the steps:

[0079] S1, bacterial culture: the above-mentioned hydrolyzed soybean, glucose and sterilized water are mixed in an amount ratio of 205g:9g:1L, and the mixture is sterilized at 100°C for 30min to obtain a lactic acid bacteria culture medium, and plant lactobacillus is inoculated into the lactic acid bacteria culture medium and cultured at 24°C for 72h to obtain a lactic acid bacteria strain for standby use;

[0080] The mass ratio of the plant lactobacillus to the lactic acid bacteria culture medium is 1.5:50;

[0081] S2, pre-treating sweet osmanthus and black truffle:

[0082] S21, crushing osmanthus into powder to obtain powdered osmanthus for standby use; the osmanthus comprises dried osmanthus and fresh osmanthus in a mass ratio of 1:3;

[0083] Chop the fresh black truffle into small pieces to obtain chopped black truffles, and set aside;

[0084] S22, mixing the chopped black truffle and soybean oil rice koji extract in equal quantities and sealing with oil at 40° C. for 72 hours, crushing and homogenizing after the sealing to obtain pretreated black truffle;

[0085] The preparation method of the soybean oil rice koji extract is as follows: rice koji and soybean oil are uniformly mixed in a mass ratio of 1.1:3, and the mixture is oil-sealed at 40° C. for 72 hours, and then sterilized at 121° C. for 30 minutes to obtain the soybean oil rice koji extract;

[0086] The method for preparing the rice koji comprises: soaking the rice at a temperature of 25° C. for 24 hours, draining and steaming the rice until each grain is distinct, cooling the rice, adding Aspergillus oryzae, stirring the mixture evenly, fermenting the mixture, and drying the fermented rice koji at a temperature of 75° C. for 6 hours to obtain the rice koji;

[0087] The mass ratio of the rice koji raw material to Aspergillus oryzae is 20:1; the fermentation conditions are: temperature 29° C., humidity 90%, and time 24 h;

[0088] S3, preparing fermentation filtrate:

[0089] S31, powdered osmanthus, pretreated black truffle, glucose, sodium chloride, the hydrolyzed soybean, soybean oil rice koji extract and sterile water are mixed uniformly in a mass ratio of 24:3:2.5:2.5:6:5:60 to obtain a culture medium, the culture medium is sterilized at 121° C. for 30 min, lactic acid bacteria are inoculated, and the culture medium is fermented at 22° C. for 7 days to obtain a fermentation product;

[0090] The mass ratio of the culture medium to the lactic acid bacteria strain is 1:0.05

[0091] S32, sterilize the fermented product at 121°C for 30 minutes, cool it, adjust the pH to neutral with sodium dihydrogen phosphate, filter it through a 0.22 μm microfiltration membrane, and obtain a finished product of black truffle osmanthus lactic acid bacteria fermentation filtrate.

[0092] Example 3

[0093] Prepare hydrolyzed soybeans, the preparation method of the hydrolyzed soybeans is:

[0094] A1. Fresh soybeans were crushed and cold-pressed to obtain soybean powder, and the soybean powder and water were mixed at a mass ratio of 1:20, and the pH was adjusted to 8.5 with sodium bicarbonate, shaken for 50 minutes, centrifuged at a controlled speed of 3800 r / min for 15 minutes, filtered, and the filtrate was adjusted to pH 4.5 with acetic acid, allowed to stand for 30 minutes, and then centrifuged at a controlled speed of 4000 r / min for 15 minutes, filtered again, and the precipitate was adjusted to pH 7.0 with sodium bicarbonate to obtain pretreated soybean powder, and the pretreated soybean powder and deionized water were mixed at a dosage ratio of 1 g:11 mL to obtain a mixed solution A;

[0095] A2, adding papain and bromelain to the mixed solution A, controlling the temperature at 60°C for hydrolysis for 2 hours, and then adding flavor protease to control the temperature at 52.5°C for hydrolysis for 2 hours to obtain a hydrolyzate, centrifuging the hydrolyzate, filtering the supernatant with a microporous membrane, and obtaining hydrolyzed soybeans;

[0096] The mass ratio of the mixed solution A to papain, bromelain and flavor protease is 1:0.0015:0.002:0.025;

[0097] A method for preparing black truffle sweet osmanthus lactic acid bacteria fermentation filtrate, specifically comprising the steps:

[0098] S1, bacterial culture: the above-mentioned hydrolyzed soybean, glucose and sterilized water are mixed in an amount ratio of 203.5g:11g:1L, and the mixture is sterilized at 97.5°C for 30min to obtain a lactic acid bacteria culture medium, and plant lactobacillus is inoculated in the lactic acid bacteria culture medium and cultured at 25°C for 72h to obtain a lactic acid bacteria strain, which is then used for standby use;

[0099] The mass ratio of the plant lactobacillus to the lactic acid bacteria culture medium is 1:50;

[0100] S2, pre-treating sweet osmanthus and black truffle:

[0101] S21, crushing osmanthus into powder to obtain powdered osmanthus for standby use; the osmanthus comprises dried osmanthus and fresh osmanthus in a mass ratio of 1:3;

[0102] Chop the fresh black truffle into small pieces to obtain chopped black truffles, and set aside;

[0103] S22, mixing the chopped black truffle and soybean oil rice koji extract in equal quantities and sealing with oil at 40° C. for 72 hours, crushing and homogenizing after the sealing to obtain pretreated black truffle;

[0104] The preparation method of the soybean oil rice koji extract is as follows: rice koji and soybean oil are uniformly mixed in a mass ratio of 1.2:3, and the mixture is oil-sealed at 40° C. for 72 hours, and then sterilized at 121° C. for 30 minutes to obtain the soybean oil rice koji extract;

[0105] The method for preparing the rice koji comprises: soaking the polished round-grained rice at a temperature of 24° C. for 24 hours, draining, steaming until each grain is distinct, cooling, adding Aspergillus oryzae, stirring evenly, fermenting, and drying the fermented rice koji raw material at a temperature of 80° C. for 7 hours to obtain the rice koji;

[0106] The mass ratio of the rice koji raw material to Aspergillus oryzae is 21:1; the fermentation conditions are: temperature 30° C., humidity 93%, and time 24 h;

[0107] S3, preparing fermentation filtrate:

[0108] S31, powdered osmanthus, pretreated black truffle, glucose, sodium chloride, the hydrolyzed soybean, soybean oil rice koji extract and sterile water are mixed uniformly in a mass ratio of 26:4:2.3:2:5.5:6:56 to obtain a culture medium, the culture medium is sterilized at 121° C. for 30 min, lactic acid bacteria are inoculated, and the culture medium is fermented at 23° C. for 7 days to obtain a fermentation product;

[0109] The mass ratio of the culture medium to the lactic acid bacteria strain is 1:0.05

[0110] S32, sterilize the fermented product at 121°C for 30 minutes, cool it, adjust the pH to neutral with sodium dihydrogen phosphate, filter it through a 0.22 μm microfiltration membrane, and obtain a finished product of black truffle osmanthus lactic acid bacteria fermentation filtrate.

[0111] Test Example 1

[0112] Sensory odor test: The finished product of the black truffle osmanthus lactic acid bacteria fermentation filtrate obtained in Example 1-3 was used as a test sample, and 10 trained professional tasters performed a sensory evaluation, specifically evaluating the osmanthus flavor, black truffle flavor, sweetness, sourness, saltiness, flavor, and unpleasant odor of the fermentation filtrate. The scoring gradient is divided into five levels, from high to low: very high, relatively high, ordinary, relatively low, and very low. The number of assessors in each level and the results are shown in Table 1.

[0113] Table 1

[0114]

[0115] As can be seen from Table 1, the professional tasters generally felt that the odors of osmanthus and black truffle of the test samples were well preserved and had unique flavors; that is, the finished product of the black truffle and osmanthus lactic acid bacteria fermentation filtrate prepared by the present invention can be accepted by most people.

[0116] Test Example 2

[0117] In vitro antioxidant activity assay:

[0118] (1) DPPH scavenging activity:

[0119] DPPH Spectrophotometry

[0120] Take 2mL of vitamin C with different concentrations of 0.004-0.300mg / mL in a stoppered test tube, add 2mL of the prepared DPPH· solution, mix well, seal and let stand in a 25℃ water bath for 30min, pour into a cuvette with a light path of 1cm, and measure its absorbance Ai at 517nm. At the same time, measure the absorbance Aj of 2mL vitamin C plus 2mL ethanol, and the absorbance A0 of 2mL DPPH· solution plus 2mL double distilled water to calculate the scavenging rate of vitamin C for DPPH·

[0121] The DPPH inhibition rate was calculated according to the following formula: DPPH inhibition rate / % = (B + CA) / B × 100%;

[0122] Where:

[0123] A: absorbance at 517.0 nm after the test solution reacts with the DPPH solution;

[0124] B: absorbance at 517.0 nm after the reaction between solvent and DPPH solution;

[0125] C: absorbance at 517.0 nm after the test solution reacts with the solvent;

[0126] The results are shown in Table 2.

[0127] (2) ABTS+ scavenging activity:

[0128] The finished product of the black truffle sweet-scented osmanthus lactic acid bacteria fermentation filtrate obtained in Example 1-3 was used as a test sample to detect the ABTS+ removal effect of the test sample. The specific detection method is as follows:

[0129] ABTS [2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonate)], or [2,2'-azobis-(3-ethylbenzthiazoline-6-sulfonic acid)], generates a stable blue-green cationic free radical ABTS+ after being oxidized by active oxygen. The ion solution containing ABTS+ free radicals turns blue-green after oxidation. When a free radical scavenger is added, it reacts with ABTS+ and causes the reaction system to fade. The antioxidant activity of the free radical scavenger can be evaluated by detecting the change in absorbance of the ABTS+ free radical at the maximum absorption wavelength (734nm). The smaller the absorbance, the stronger the scavenging ability of the free radical scavenger.

[0130] The ABTS+ inhibition rate was calculated according to the following formula: ABTS+ inhibition rate / % = (B+CA) / B×100%;

[0131] Where:

[0132] A represents the absorbance at 734.0 nm after the test solution reacts with the ABTS solution;

[0133] B represents the absorbance at 734.0 nm after the reaction between the solvent and the ABTS solution;

[0134] C represents the absorbance at 734.0 nm after the test solution reacts with the solvent. The ABTS+ inhibition rate is calculated according to the following formula: ABTS+ inhibition rate / % = (B+CA) / B×100%;

[0135] Where:

[0136] A represents the absorbance at 734.0 nm after the test solution reacts with the ABTS solution;

[0137] B represents the absorbance at 734.0 nm after the reaction between the solvent and the ABTS solution;

[0138] C represents the absorbance at 734.0 nm after the test solution reacts with the solvent.

[0139] (3) UVA / UVB absorption capacity determination:

[0140] The finished product of black truffle osmanthus lactic acid bacteria fermentation filtrate obtained in Example 1-3 was used as a test sample, and the ultraviolet-visible spectrophotometer was used to absorb ultraviolet light in the UVB region of 280-320nm and the UVA region of 320nm-400nm of the test sample. The formula is

[0141] A = lg(1 / R);

[0142] Where: R: absorbance;

[0143] The results are shown in Table 2.

[0144] (4) Determination of iron reducing ability

[0145] The FRAP method was used to detect the iron reducing capacity of the finished product of the black truffle osmanthus lactic acid bacteria fermentation filtrate obtained in Examples 1-3. A standard curve was prepared by replacing the test sample with a 0.10-2.00 mmol / L FeSO4 standard solution. The standard curve equation was Y=0.3438X+0.194, and its correlation coefficient was r 2 =0.9931. Results Taking 1.0 mmol / LFeSO4 as the standard, the test sample reaching the same absorbance value is one Trolox equivalent; the results are shown in Table 2.

[0146] Table 2

[0147]

[0148]

[0149] As can be seen from Table 2, the finished product of the black truffle and osmanthus lactic acid bacteria fermentation filtrate prepared by the present invention has an inhibition rate of DPPH higher than 85%, an inhibition rate of ABTS+ higher than 90.00%, a UVA absorption rate of not less than 95%, and a UVB absorption rate of not less than 90%, indicating that the finished product of the black truffle and osmanthus lactic acid bacteria fermentation filtrate prepared by the present invention has excellent antioxidant ability, a strong potential for reducing oxidative stress reaction in the body, and an outstanding absorption effect on UVA / UVB.

[0150] Test Example 3

[0151] Tyrosinase monophenol oxidase activity assay:

[0152] The finished product of black truffle osmanthus lactic acid bacteria fermentation filtrate obtained in Example 1-3 was used as a sample. Tyrosine was used as a substrate and arbutin was used as a positive control to determine the inhibitory activity of the sample on tyrosinase monophenol oxidase. The experiment was divided into a blank group, a positive control group, a sample group and a background group (n=3). No sample was added to the blank group, and no tyrosinase solution was added to the background group. 50 μL of L-tyrosine (0.5 mmol / L) and 100 μL of sample solution were added to the remaining groups in turn, mixed and incubated at 37°C for 5 minutes. Subsequently, 100 μL of tyrosinase solution (250 U / mL) was added, mixed and incubated at 37°C for 15 minutes, and the absorbance value was measured at 475 nm. The inhibition rate calculation formula is as follows:

[0153] Inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100

[0154] Where: A1: absorbance of sample solution with enzyme added;

[0155] Sample control group A2: 50 μL PBS replaced with 50 μL tyrosinase;

[0156] Blank group A3: 100 μL PBS replaced 100 μL sample solution;

[0157] Blank control group A4: 100 μL of sample solution was replaced by 100 μL of PBS, and 100 μL of tyrosinase was replaced by 100 μL of PBS;

[0158] The results are shown in Table 3.

[0159] Table 3

[0160]

[0161]

[0162] As can be seen from Table 3, the finished product of the black truffle osmanthus lactic acid bacteria fermentation filtrate prepared by the present invention has excellent tyrosinase inhibitory activity performance. Tyrosinase plays an important role in the production of melanin and is the main cause of skin pigmentation and skin darkening. Therefore, the finished product of the fermentation filtrate prepared by the present invention can effectively inhibit the activity of tyrosinase, that is, it has excellent whitening effect.

[0163] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0164] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing black truffle osmanthus lactic acid bacteria fermentation filtrate, characterized in that: Specifically include the following steps: S1, bacterial strain culture: hydrolyzed soybean, glucose and sterilized water are mixed uniformly, temperature-controlled sterilization is carried out to obtain lactic acid bacteria culture medium, plant lactobacillus is inoculated in the lactic acid bacteria culture medium and temperature-controlled culture is carried out to obtain lactic acid bacteria strains, which are standby; S2, pre-treating sweet osmanthus and black truffle: S21, crushing sweet osmanthus into powder to obtain powdered sweet osmanthus, which is set aside; chopping fresh black truffle to obtain chopped black truffle, which is set aside; S22, mixing the chopped black truffle and soybean oil rice koji extract evenly and oil-sealing them at a controlled temperature, and crushing and homogenizing the mixture after the oil sealing to obtain a pretreated black truffle; S3, preparing fermentation filtrate: S31, mixing powdered osmanthus, pretreated black truffle, glucose, sodium chloride, hydrolyzed soybean, soybean oil rice koji extract and sterile water to obtain a culture medium, sterilizing the culture medium under temperature control, inoculating lactic acid bacteria strains, and fermenting under temperature control to obtain a fermentation product; S32, sterilizing the fermented product by controlling the temperature, cooling, adjusting the pH, and filtering through a microfiltration membrane to obtain a finished product of the black truffle osmanthus lactic acid bacteria fermentation filtrate.

2. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 1, characterized in that: In step S1, the usage ratio of the hydrolyzed soybean, glucose and sterile water is 202-205 g:9-11 g:1 L; the mass ratio of the plant lactobacillus and the lactic acid bacteria culture medium is 1-1.5:

50.

3. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 1, characterized in that: The mass ratio of the black truffle and the soybean oil rice koji extract in step S22 is 1:

1.

4. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 1, characterized in that: The preparation method of the soybean oil rice koji extract in step S22 is: uniformly mixing rice koji and soybean oil, oil-sealing under temperature control, and then sterilizing under temperature control to obtain the soybean oil rice koji extract; The mass ratio of the rice koji to soybean oil is 1-1.2:

3.

5. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 4, characterized in that: The method for preparing the rice koji comprises the following steps: temperature-controlled soaking of the rice koji raw material, draining, steaming until each grain is distinct, cooling, adding Aspergillus oryzae, stirring evenly, fermenting, and temperature-controlled drying of the fermented rice koji raw material to obtain the rice koji.

6. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 5, characterized in that: The mass ratio of the rice koji raw material to Aspergillus oryzae is 20-22:1; the temperature of the temperature-controlled dipping is 23-25° C., and the time is 24 hours; the conditions of the fermentation are: temperature 28-30° C., humidity 90-95%, and time 24 hours; the temperature of the temperature-controlled drying is 75-80° C., and the time is 6-8 hours.

7. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 1, characterized in that: In step S31, the mass ratio of the powdered osmanthus, pretreated black truffle, glucose, sodium chloride, hydrolyzed soybeans, soybean oil and rice koji extract and sterile water is 22-26:3-5:2-2.5:2-2.5:5-6:5-6:56-60; the mass ratio of the culture medium and the lactic acid bacteria strain is 1:0.

05.

8. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 1, characterized in that: The preparation method of the hydrolyzed soybean is: A1. Grind and cold-press fresh soybeans to obtain soybean powder, mix the soybean powder and water evenly, adjust the pH once, oscillate, centrifuge at a controlled speed, filter, take the filtrate and adjust the pH twice, let stand, centrifuge at a controlled speed, filter again, take the precipitate and adjust the pH three times to obtain pretreated soybean powder, and mix the pretreated soybean powder and deionized water evenly to obtain a mixed solution A; A2. Add papain and bromelain to the mixed solution A for primary temperature-controlled hydrolysis, then add flavor protease for secondary temperature-controlled hydrolysis to obtain a hydrolyzate, centrifuge the hydrolyzate, filter the supernatant with a microporous membrane, and obtain hydrolyzed soybeans.

9. The method for preparing a black truffle osmanthus lactic acid bacteria fermentation filtrate according to claim 8, characterized in that: The mass ratio of the soybean powder and water in step A1 is 1:15-20; the dosage ratio of the pretreated soybean powder and deionized water in step A1 is 1g:10-12mL; the mass ratio of the mixed solution A and papain, bromelain and flavor protease in step A2 is 1:0.001-0.002:0.002-0.003:0.02-0.

03.

10. A fermentation filtrate prepared by the method for preparing black truffle and osmanthus lactic acid bacteria fermentation filtrate according to any one of claims 1 to 9.

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