Preparation method of double-bacterium fermented opuntia ficus-indica fermentation extract and application of double-bacterium fermented opuntia ficus-indica fermentation extract in cosmetics

Abstract

The invention relates to a preparation method of a double-bacterium fermented opuntia ficus-indica fermentation extract and application of the double-bacterium fermented opuntia ficus-indica fermentation extract in cosmetics, and belongs to the technical field of cosmetics. The invention provides a preparation method of a double-bacterium fermented opuntia ficus-indica fermentation extract and application of the double-bacterium fermented opuntia ficus-indica fermentation extract to cosmetics, and the preparation method roughly comprises the following steps: preparing opuntia ficus-indica stem slurry and rice milk, mixing the slurry and the rice milk, performing double-bacterium fermentation, centrifuging, retaining residues, and finally enriching supernate. According to the invention, food-grade opuntia ficus stem and rice are used for fermentation, and aspergillus oryzae and saccharomycetes are used for combined fermentation, so that the introduction of exogenous enzyme is avoided, macromolecular substances are fully decomposed through synergistic metabolism, the release rate of micromolecular active ingredients is increased, and finally, the effects of moisturizing, oxidation resistance, wrinkle resistance and the like of the product are further improved through enrichment.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and relates to a preparation method of a fermented extract of Opuntia ficus-indica by double bacteria fermentation and its application in cosmetics. Background Art

[0002] With the increasing complexity of skin problems caused by environmental pollution, increasing life pressure and irregular work and rest, consumers are no longer satisfied with basic moisturization, but rather purposefully choose skin care products. Functional skin care products have gradually become the mainstream of the market. Currently, there are numerous functional skin care products on the market, and raw materials such as Ganoderma lucidum and Dendrobium officinale are highly regarded for their high activity. However, the cost of such raw materials is relatively high. Products with low prices may have low active ingredient content, while products with high active ingredient content have relatively high prices, creating a consumption threshold and unable to meet the needs of most consumers. Therefore, functional products with low cost and high efficiency have always been pursued by the public. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method of a fermented extract of Opuntia ficus-indica by double bacteria fermentation and its application in cosmetics. The present invention uses edible Opuntia ficus-indica stems and rice for fermentation, and adopts a combined fermentation of two strains of Aspergillus oryzae and Saccharomyces cerevisiae, avoiding the introduction of exogenous enzymes. Through synergistic metabolism, macromolecular substances are fully decomposed, and the release rate of small molecular active ingredients is increased. Finally, the effects of moisturizing, antioxidant and anti-wrinkle of the product are further enhanced through enrichment.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A preparation method of a fermented extract of Opuntia ficus-indica by double bacteria fermentation, the preparation method comprising the following steps:

[0006] Step 1: Mix Opuntia ficus-indica stems with deionized water and beat to obtain a slurry, and process to obtain an Opuntia ficus-indica stem slurry;

[0007] Step 2: Cook the rice, cool it, and obtain a rice slurry;

[0008] Step 3: Mix the rice slurry with the Opuntia ficus-indica stem slurry to obtain a mixed solution, and perform double bacteria fermentation to obtain a fermentation product;

[0009] Step 4: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

[0010] Furthermore, in Step 1, the material-liquid ratio of Opuntia ficus-indica to deionized water is 1:3 - 5; the processing refers to high-pressure homogenization 3 times at a pressure of 150 MPa.

[0011] Further, the cooking in step two means that after mixing rice and deionized water at a liquid-to-solid ratio of 1:1.3 - 1.7, cook them at 100°C for 20 - 30 min.

[0012] Further, the mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry in step three is 1.05 - 1.13:1.34 - 1.42.

[0013] Further, the double-strain fermentation in step three specifically includes the following steps:

[0014] Step Z1: Inoculate the mixed liquid with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension, which respectively account for 0.6 - 1 wt% and 1.6 - 2.2 wt% of the total amount of the mixed liquid. Dropwise add a citric acid-sodium citrate buffer solution to adjust the pH to 6.2 - 6.6. Control the fermentation temperature at 28 - 32°C, control the dissolved oxygen content at 2.4 - 4.8 ppm, and ferment at a rotation speed of 150 - 250 rpm for 32 - 38 h to obtain a first fermentation intermediate product;

[0015] Step Z2: Dropwise add a citric acid-sodium citrate buffer solution to adjust the pH to 4.8 - 5.2. Control the fermentation temperature at 26 - 28°C, control the dissolved oxygen content at 0.5 - 0.7 ppm, adjust the rotation speed to 80 - 120 rpm, and ferment for 36 - 48 h to obtain a second fermentation intermediate product;

[0016] Step Z3: Add glucose and residue, which respectively account for 3.2 - 5 wt% and 0 - 1.8 wt% of the total amount of the second fermentation intermediate product, to the second fermentation intermediate product. Control the anaerobic amount < 0.04 ppm, continue to ferment for 20 - 30 h, and inactivate at a controlled temperature.

[0017] Further, the concentration of Aspergillus oryzae in the Aspergillus oryzae suspension in step Z1 is 0.8 - 1.2×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1 - 1.2×10 8 CFU / mL.

[0018] Further, the temperature-controlled inactivation in step Z3 means heating to 60 - 65°C at a rate of 2 - 3°C / min, keeping warm for 15 min, and then cooling to room temperature at a rate of 4 - 6°C / min.

[0019] Further, the residue in step four and step Z3 needs to be processed. The specific processing operation is to mix the residue and deionized water at a mass ratio of 1:4 - 5, use a citric acid-sodium citrate buffer solution to adjust the pH to 4.8 - 5.2, let it stand at 50 - 52°C for 2 - 3 h, inactivate at 80°C for 10 min, and filter through a 0.22 μm filter membrane to obtain it.

[0020] Further, the centrifugation described in Step 4 refers to centrifuging at a speed of 8000 - 12000 rpm for 20 - 30 min at 4°C; the enrichment refers to passing the supernatant through a 5-μm filter membrane, then filtering with a 10 - 12 kDa PES membrane, and finally filtering with a 1 - 3 kDa PES membrane.

[0021] Further, the fermented extract of Opuntia ficus-indica is applied to cosmetics at an addition amount of 5 - 10 wt%.

[0022] Advantages of the present invention:

[0023] (1) The present invention uses edible Opuntia ficus-indica stems and rice for fermentation, and adopts a combined fermentation of Aspergillus oryzae and Saccharomyces cerevisiae, avoiding the introduction of exogenous enzymes. Through synergistic metabolism, macromolecular substances are fully decomposed, the release rate of small-molecule active ingredients is increased, and finally the effects of moisturizing, antioxidant and anti-wrinkle of the product are further improved through enrichment.

[0024] (2) The Opuntia ficus-indica stems adopted in the present invention are rich in polysaccharides, amino acids and antioxidant active ingredients. The hydrolysis products and fermentation products of rice contain active ingredients such as glucosylceramide, polypeptides, small-molecule proteins, polysaccharides and kojic acid. The combination of the two can ensure the safety and efficacy of the raw materials.

[0025] (3) In the preparation process of the present invention, the Opuntia ficus-indica stems are preliminarily enzymolyzed first. During the fermentation process, Aspergillus oryzae further secretes composite enzymes such as cellulase, pectinase and amylase, thereby decomposing the macromolecular structure in the Opuntia ficus-indica stems, hydrolyzing rice starch at the same time, releasing the active ingredients in rice and participating in the fermentation, synergistically improving the efficacy; in addition, the residue obtained from the previous fermentation is introduced in the later stage of fermentation, and the endogenous enzymes are activated through autolysis, releasing nucleotides and short peptides, and enhancing the metabolic activity of the strains. Specific embodiments

[0026] To further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following combines examples to detail the specific embodiments, structures, features and their effects according to the present invention.

[0027] The Opuntia ficus-indica stems in all examples and comparative examples of the present invention are all directly purchased from the market, and are all purchased from Qingyuan Shanhe Ecological Agriculture Co., Ltd.; the rice is all directly purchased from the market, and is all purchased from Shandong Helaixiang Food Co., Ltd.; the citric acid-sodium citrate buffer solution is all directly purchased from the market, and is all purchased from Shanghai Yuanye Biotechnology Oil Phase Company, and the concentration of the citric acid-sodium citrate buffer solution when used is 0.1 mol / L.

[0028] The preparation method of the Aspergillus oryzae suspension is as follows: Inoculate Aspergillus oryzae in potato dextrose agar and culture at 30 °C for 72 h. Add 10 mL of normal saline to the slant, gently scrape to remove the mycelium, collect the suspension, centrifuge at 3000 rpm for 10 min, and then resuspend in normal saline to adjust to the required concentration. The Aspergillus oryzae is purchased from Guangzhou Zhenwei Microbiology Technology Co., Ltd.; the potato dextrose agar is purchased from Hubei Xinjiecheng Chemical Technology Co., Ltd.

[0029] The preparation method of the Saccharomyces cerevisiae is as follows: Take 10 g of yeast extract, 20 g of peptone, and 20 g of glucose, make up the volume to 1 L, sterilize at 121 °C for 20 min to obtain the YPD liquid medium. Pick a single colony from the YPD slant and inoculate it into 50 mL of the YPD liquid medium, culture with shaking at 28 °C and 180 rpm for 18 h, and then inoculate it into a fresh YPD liquid medium at an inoculation amount of 10 wt%, and culture with shaking at 28 °C and 180 rpm until the required concentration. The yeast extract is purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the peptone and glucose are purchased from Shanghai Macklin Biochemical Co., Ltd.

[0030] Example 1

[0031] A preparation method of a fermented extract of Opuntia ficus-indica by double bacteria fermentation, the preparation method comprising the following steps:

[0032] Step 1: Mix the Opuntia ficus-indica stem with deionized water and beat to obtain a slurry, and process to obtain an Opuntia ficus-indica stem slurry.

[0033] Step 2: Cook the rice and cool it to obtain a rice slurry.

[0034] Step 3: Mix the rice slurry with the Opuntia ficus-indica stem slurry to obtain a mixed liquid, and perform double bacteria fermentation to obtain a fermentation product.

[0035] Step 4: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

[0036] The material-liquid ratio of the Opuntia ficus-indica to deionized water in Step 1 is 1:3; the treatment refers to high-pressure homogenization 3 times at a pressure of 150 MPa.

[0037] The cooking in Step 2 refers to mixing the rice and deionized water at a material-liquid ratio of 1:1.3 and cooking at 100 °C for 20 min.

[0038] The mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry in Step 3 is 1.05:1.34.

[0039] The double bacteria fermentation in Step 3 specifically comprises the following steps:

[0040] Step Z1: Inoculate the mixed solution with Aspergillus oryzae suspension and Saccharomyces cerevisiae suspension, each accounting for 0.6 wt% and 1.6 wt% of the total amount of the mixed solution respectively. Add citric acid-sodium citrate buffer solution to adjust the pH to 6.2, control the fermentation temperature at 28 °C, control the dissolved oxygen content at 2.4 ppm, and ferment at a rotation speed of 150 rpm for 32 h to obtain the first fermentation intermediate product.

[0041] Step Z2: Add citric acid-sodium citrate buffer solution to adjust the pH to 4.8, control the fermentation temperature at 26 °C, control the dissolved oxygen content at 0.5 ppm, adjust the rotation speed to 80 rpm, and ferment for 36 h to obtain the second fermentation intermediate product.

[0042] Step Z3: Add glucose, each accounting for 3.2 wt% of the total amount of the second fermentation intermediate product, to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue to ferment for 20 h, and inactivate by controlling the temperature.

[0043] The concentration of Aspergillus oryzae in the Aspergillus oryzae suspension described in Step Z1 is 0.8×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1×10 8 CFU / mL.

[0044] The temperature-controlled inactivation described in Step Z3 means heating to 60 °C at a rate of 2 °C / min, holding for 15 min, and then cooling to room temperature at a rate of 4 °C / min.

[0045] The centrifugation described in Step Four means centrifuging at a rotation speed of 8000 rpm at 4 °C for 20 min; the enrichment means passing the supernatant through a 5-μm filter membrane, then filtering with a 10-kDa PES membrane, and finally filtering with a 1-kDa PES membrane.

[0046] The fermented extract of Opuntia ficus-indica is applied to cosmetics at an addition amount of 5 wt%.

[0047] Example 2

[0048] A preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation, the preparation method comprising the following steps:

[0049] Step One: Mix the Opuntia ficus-indica stem with deionized water and make a pulp to obtain an Opuntia ficus-indica stem pulp solution.

[0050] Step Two: Cook the rice, cool it, and obtain rice slurry.

[0051] Step Three: Mix the rice slurry with the Opuntia ficus-indica stem pulp solution to obtain a mixed solution, and perform double-bacteria fermentation to obtain a fermentation product.

[0052] Step Four: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

[0053] The material-liquid ratio of the Opuntia ficus-indica fruits in Step 1 to deionized water is 1:3.5; the treatment refers to high-pressure homogenization 3 times at a pressure of 150 MPa.

[0054] The cooking in Step 2 refers to mixing rice and deionized water at a material-liquid ratio of 1:1.4 and then cooking at 100 °C for 22 min.

[0055] The mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry in Step 3 is 1.07:1.36.

[0056] The double-bacteria fermentation in Step 3 specifically includes the following steps:

[0057] Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension, each accounting for 0.7 wt% of the total amount of the mixed solution. Add a citric acid-sodium citrate buffer solution to adjust the pH to 6.3, control the fermentation temperature at 29 °C, control the dissolved oxygen content at 3 ppm, and ferment at a rotation speed of 180 rpm for 34 h to obtain a first fermentation intermediate product;

[0058] Step Z2: Add a citric acid-sodium citrate buffer solution to adjust the pH to 4.9, control the fermentation temperature at 26 °C, control the dissolved oxygen content at 0.55 ppm, adjust the rotation speed to 90 rpm, and ferment for 38 h to obtain a second fermentation intermediate product;

[0059] Step Z3: Add glucose and residue, each accounting for 3.6 wt% and 0.5 wt% of the total amount of the second fermentation intermediate product, respectively, to the second fermentation intermediate product. Control the anaerobic amount <0.04 ppm and continue fermentation for 22 h, then control the temperature to inactivate.

[0060] The concentration of Aspergillus oryzae in the Aspergillus oryzae suspension in Step Z1 is 0.9×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1.06×10 8 CFU / mL.

[0061] The temperature-controlled inactivation in Step Z3 refers to heating to 62 °C at a rate of 2.2 °C / min, holding for 15 min, and then cooling to room temperature at a rate of 4.5 °C / min.

[0062] The residue in Step 4 and Step Z3 needs to be treated. The specific treatment operation is to mix the residue and deionized water at a mass ratio of 1:4.2, use a citric acid-sodium citrate buffer solution to adjust the pH to 4.9, stand at 50.6 °C for 2.3 h, inactivate at 80 °C for 10 min, and filter through a 0.22-μm filter membrane to obtain it.

[0063] The centrifugation described in Step 4 refers to centrifuging at 9000 rpm for 22 min at 4°C; the enrichment refers to passing the supernatant through a 5-μm filter membrane, then filtering with a 10-kDa PES membrane, and finally filtering with a 1-kDa PES membrane.

[0064] The fermented extract of Opuntia ficus-indica is applied to cosmetics at an addition amount of 6.5 wt%.

[0065] Example 3

[0066] A preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation, the preparation method comprising the following steps:

[0067] Step 1: Mix the Opuntia ficus-indica stem with deionized water and beat to obtain a slurry, and process to obtain an Opuntia ficus-indica stem slurry;

[0068] Step 2: Cook the rice, cool it to obtain a rice slurry;

[0069] Step 3: Mix the rice slurry and the Opuntia ficus-indica stem slurry to obtain a mixed solution, and perform double-bacteria fermentation to obtain a fermentation product;

[0070] Step 4: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

[0071] The material-liquid ratio of the Opuntia ficus-indica to deionized water in Step 1 is 1:4; the treatment refers to high-pressure homogenization 3 times at a pressure of 150 MPa.

[0072] The cooking in Step 2 refers to mixing the rice and deionized water according to a material-liquid ratio of 1:1.5, and cooking at 100°C for 25 min.

[0073] The mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry in Step 3 is 1.09:1.38.

[0074] The double-bacteria fermentation in Step 3 specifically comprises the following steps:

[0075] Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension respectively accounting for 0.8 wt% and 1.9 wt% of the total amount of the mixed solution, add a citric acid-sodium citrate buffer solution to adjust the pH to 6.4, control the fermentation temperature at 30°C, control the dissolved oxygen content at 3.6 ppm, and ferment at 200 rpm for 35 h to obtain a first fermentation intermediate product;

[0076] Step Z2: Add a citric acid-sodium citrate buffer solution to adjust the pH to 5, control the fermentation temperature at 27°C, control the dissolved oxygen content at 0.6 ppm, adjust the rotation speed to 100 rpm, and ferment for 42 h to obtain a second fermentation intermediate product;

[0077] Step Z3: Add glucose and residue accounting for 4.1 wt% and 0.9 wt% of the total amount of the second fermentation intermediate product respectively to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue fermentation for 25 h, and control the temperature to inactivate.

[0078] The concentration of Aspergillus oryzae in the Aspergillus oryzae suspension described in Step Z1 is 1×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1.1×10 8 CFU / mL.

[0079] The temperature-controlled inactivation described in Step Z3 means heating to 62.5 °C at a rate of 2.5 °C / min, holding for 15 min, and then cooling to room temperature at a rate of 5 °C / min.

[0080] The residue described in Step Four and Step Z3 needs to be processed. The specific processing operation is to mix the residue and deionized water according to a mass ratio of 1:4.5, adjust the pH to 5 using a citric acid-sodium citrate buffer solution, stand at 51 °C for 2.5 h, inactivate at 80 °C for 10 min, and filter through a 0.22 μm filter membrane to obtain it.

[0081] The centrifugation described in Step Four means centrifuging at 10000 rpm for 25 min at 4 °C; the enrichment means filtering the supernatant through a 5 μm filter membrane, then filtering with an 11 kDa PES membrane, and finally filtering with a 2 kDa PES membrane.

[0082] The Opuntia ficus-indica fermentation extract is applied to cosmetics at an addition amount of 7.5 wt%.

[0083] Example 4

[0084] A preparation method of an Opuntia ficus-indica fermentation extract by double-bacteria fermentation, the preparation method comprising the following steps:

[0085] Step One: Mix the Opuntia ficus-indica stem with deionized water to make a slurry, and process to obtain an Opuntia ficus-indica stem slurry;

[0086] Step Two: Cook the rice, cool it to obtain rice slurry;

[0087] Step Three: Mix the rice slurry and the Opuntia ficus-indica stem slurry to obtain a mixed solution, and perform double-bacteria fermentation to obtain a fermentation product;

[0088] Step Four: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain it.

[0089] The material-liquid ratio of the Opuntia ficus-indica to deionized water in Step One is 1:4.5; the processing means high-pressure homogenization 3 times at 150 MPa.

[0090] The cooking described in Step 2 means that after mixing rice and deionized water at a liquid-to-solid ratio of 1:1.6, cook them at 100 °C for 28 min.

[0091] The mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry described in Step 3 is 1.11:1.4.

[0092] The dual-strain fermentation described in Step 3 specifically includes the following steps:

[0093] Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension, each accounting for 0.9 wt% and 2.1 wt% of the total amount of the mixed solution, respectively. Dropwise add a citric acid-sodium citrate buffer solution to adjust the pH to 6.5, control the fermentation temperature at 31 °C, control the dissolved oxygen content at 4 ppm, and ferment at a rotation speed of 220 rpm for 32 - 38 h to obtain a first fermentation intermediate product;

[0094] Step Z2: Dropwise add a citric acid-sodium citrate buffer solution to adjust the pH to 5.1, control the fermentation temperature at 28 °C, control the dissolved oxygen content at 0.65 ppm, adjust the rotation speed to 110 rpm, and ferment for 45 h to obtain a second fermentation intermediate product;

[0095] Step Z3: Add glucose and residues, each accounting for 4.4 wt% and 1.2 wt% of the total amount of the second fermentation intermediate product, respectively, to the second fermentation intermediate product. Control the anaerobic amount < 0.04 ppm, continue to ferment for 28 h, and control the temperature to inactivate.

[0096] The residues described in Step 4 and Step Z3 need to be processed. The specific processing operation is to mix the residues and deionized water at a mass ratio of 1:4.7, use a citric acid-sodium citrate buffer solution to adjust the pH to 5.1, let it stand at 51.5 °C for 2.8 h, inactivate at 80 °C for 10 min, and filter through a 0.22 μm filter membrane to obtain the product.

[0097] The centrifugation described in Step 4 means centrifuging at 4 °C at a rotation speed of 11000 rpm for 20 - 30 min; the enrichment means that the supernatant is filtered through a 5 μm filter membrane, then filtered through a 12 kDa PES membrane, and finally filtered through a 3 kDa PES membrane.

[0098] The Opuntia ficus-indica fermentation extract is applied to cosmetics at an addition amount of 8 wt%.

[0099] Example 5

[0100] A preparation method of a dual-strain fermented Opuntia ficus-indica fermentation extract, the preparation method comprising the following steps:

[0101] Step 1: Mix the Opuntia ficus-indica stem and deionized water to make a slurry, and process it to obtain an Opuntia ficus-indica stem slurry;

[0102] Step 2: Steam the rice and then cool it to obtain rice slurry.

[0103] Step 3: Mix the rice slurry with the Opuntia ficus-indica stem slurry to obtain a mixed solution, and perform double-bacteria fermentation on the mixed solution to obtain a fermentation product.

[0104] Step 4: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

[0105] The material-liquid ratio of the Opuntia ficus-indica described in Step 1 to deionized water is 1:5; the treatment refers to high-pressure homogenization 3 times under a pressure of 150 MPa.

[0106] The steaming described in Step 2 refers to mixing the rice with deionized water at a material-liquid ratio of 1:1.7 and then steaming at 100 °C for 30 min.

[0107] The mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry described in Step 3 is 1.13:1.42.

[0108] The double-bacteria fermentation described in Step 3 specifically includes the following steps:

[0109] Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension, each accounting for 1 wt% of the total amount of the mixed solution, add a citric acid-sodium citrate buffer solution to adjust the pH to 6.6, control the fermentation temperature at 32 °C, control the dissolved oxygen content at 4.8 ppm, and ferment at a rotation speed of 250 rpm for 38 h to obtain a first fermentation intermediate product.

[0110] Step Z2: Add a citric acid-sodium citrate buffer solution to adjust the pH to 5.2, control the fermentation temperature at 28 °C, control the dissolved oxygen content at 0.7 ppm, adjust the rotation speed to 120 rpm, and ferment for 48 h to obtain a second fermentation intermediate product.

[0111] Step Z3: Add glucose and residue, each accounting for 5 wt% and 1.8 wt% of the total amount of the second fermentation intermediate product, respectively, to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue fermentation for 30 h, and control the temperature to inactivate.

[0112] The concentration of Aspergillus oryzae in the Aspergillus oryzae suspension described in Step Z1 is 1.2×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1.2×10 8 CFU / mL.

[0113] The temperature-controlled inactivation described in Step Z3 refers to heating to 65 °C at a rate of 3 °C / min, holding for 15 min, and then cooling to room temperature at a rate of 6 °C / min.

[0114] The residues described in Step 4 and Step Z3 need to be processed. The specific processing operation is as follows: Mix the residues and deionized water in a mass ratio of 1:5, adjust the pH to 5.2 with citric acid-sodium citrate buffer solution, let it stand at 52 °C for 3 h, inactivate at 80 °C for 10 min, and filter through a 0.22 μm filter membrane to obtain the product.

[0115] The centrifugation described in Step 4 refers to centrifuging at 12,000 rpm for 30 min at 4 °C; the enrichment refers to filtering the supernatant through a 5 μm filter membrane, then filtering with a 12 kDa PES membrane, and finally filtering with a 3 kDa PES membrane.

[0116] The fermented extract of Opuntia ficus-indica is applied to cosmetics at an addition amount of 10 wt%.

[0117] Example 6

[0118] On the basis of Example 3, remove the rice slurry and replace it with an equal weight of Opuntia ficus-indica stem slurry, and keep other conditions the same as in Example 3.

[0119] Comparative Example 1

[0120] On the basis of Example 3, keep other conditions the same and replace the double-strain fermentation with Aspergillus oryzae single-strain fermentation. The specific fermentation steps are as follows:

[0121] Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension accounting for 2.7 wt% of the total amount of the mixed solution, add citric acid-sodium citrate buffer solution to adjust the pH to 6.4, control the fermentation temperature at 30 °C, control the dissolved oxygen content at 3.6 ppm, and ferment at 200 rpm for 35 h to obtain the first fermentation intermediate product; the concentration of Aspergillus oryzae in the Aspergillus oryzae suspension is 0.98×10 8 CFU / mL;

[0122] Step Z2: Add citric acid-sodium citrate buffer solution to adjust the pH to 5, control the fermentation temperature at 27 °C, control the dissolved oxygen content at 0.6 ppm, adjust the rotation speed to 100 rpm, and ferment for 42 h to obtain the second fermentation intermediate product;

[0123] Step Z3: Add glucose accounting for 4.1 wt% of the total amount of the second fermentation intermediate product and residues accounting for 0.9 wt% to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue to ferment for 25 h, and control the temperature to inactivate.

[0124] Comparative Example 2

[0125] On the basis of Example 3, keep other conditions the same and replace the double-strain fermentation with Saccharomyces cerevisiae single-strain fermentation. The specific fermentation steps are as follows:

[0126] Step Z1: Inoculate the mixed solution with a suspension of Saccharomyces cerevisiae accounting for 2.7 wt% of the total amount of the mixed solution, add a citric acid-sodium citrate buffer solution to adjust the pH to 6.4, control the fermentation temperature at 30 °C, control the dissolved oxygen content at 3.6 ppm, and ferment at a rotation speed of 200 rpm for 35 h to obtain a first fermentation intermediate product; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 0.98×10 8 CFU / mL;

[0127] Step Z2: Add a citric acid-sodium citrate buffer solution to adjust the pH to 5, control the fermentation temperature at 27 °C, control the dissolved oxygen content at 0.6 ppm, adjust the rotation speed to 100 rpm, and ferment for 42 h to obtain a second fermentation intermediate product;

[0128] Step Z3: Add glucose accounting for 4.1 wt% and residues accounting for 0.9 wt% of the total amount of the second fermentation intermediate product to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue to ferment for 25 h, and control the temperature to inactivate.

[0129] Comparative Example 3

[0130] On the basis of Example 3, while keeping other conditions the same, change the parameters of temperature-controlled inactivation to: heat up to 62.5 °C at a rate of 5 °C / min, keep warm for 15 min, and then cool down to room temperature at a rate of 5 °C / min.

[0131] Comparative Example 4

[0132] On the basis of Example 3, while keeping other conditions the same, change the step of enriching the supernatant to: filter the supernatant through a 5-μm filter membrane and then filter it through a 2-kDa PES membrane.

[0133] 1. Patch test

[0134] Test subjects: Select 30 healthy volunteers with skin, half male and half female, aged between 20 and 55. All volunteers have no history of skin diseases, no known allergies to the test materials, and have not used any drugs or cosmetics that may affect skin reactions for at least 2 weeks before the test;

[0135] Test materials: Use the fermented extract of Opuntia ficus-indica prepared in Examples 1-5 as samples, and dissolve the samples in deionized water respectively to prepare sample solutions with a concentration of 7.5 wt%;

[0136] Test method: Drop 0.02 ± 0.005 ml of the sample solution onto the patch tester (with an area of 40 mm 2, in the depth of about 1 mm, attach it to the flexor side of the subject's forearm with a low-sensitization tape. After 24 hours, remove the patch tester, and observe the skin reactions at 0.5 h, 24 h, and 48 h after removal respectively. Record the results according to the skin reaction grading standard in the "Technical Specifications for Cosmetics Safety" (2015 Edition). Among them, the scoring and grading are divided into levels 0-4, and level 0 is a negative reaction. The test results are shown in Table 1.

[0137] Table 1 Patch test results

[0138]

[0139] As can be seen from Table 1, the fermented extract of Opuntia ficus-indica fruits obtained in the examples of the present invention is mild to the skin and has no adverse reactions during use.

[0140] 2. Moisturizing test

[0141] Test subjects: Select 30 healthy volunteers with skin, half male and half female, aged between 20-55. All volunteers have no history of skin diseases, no known allergies to the test materials, and have not used any drugs or cosmetics that may affect skin reactions for at least 2 weeks before the test;

[0142] Test materials: Use the fermented extracts of Opuntia ficus-indica fruits prepared in Examples 1-6 and Comparative Examples 1-4 as test samples, and dissolve the test samples in deionized water respectively to prepare sample solutions with a concentration of 7.5 wt%.

[0143] Test method: The subjects uniformly clean the inner sides of their forearms, select a 3×3 cm test area and make marks, apply the sample solution, and the dosage is 1 ml / cm 2 . Use a skin moisture tester (Corneometer, CM825) to measure the water content of the skin area to be tested before application and at 1 h, 3 h, and 5 h after application. Each area is measured in parallel 3 times. All testers are in an indoor space with constant temperature and humidity before and after the test. The same subject is completed by the same measurement personnel, and the average value is recorded as shown in Table 2 below.

[0144] Table 2 Test results of skin moisture content

[0145]

[0146] As can be seen from Table 2, the fermented extract of Opuntia ficus-indica fruits prepared in the present invention has good moisturizing effects.

[0147] 3. Determination of free radical scavenging rate by DPPH method

[0148] Test materials: Use the fermented extracts of Opuntia ficus-indica fruits prepared in Examples 1-6 and Comparative Examples 1-4 as test samples, and dissolve the test samples in deionized water respectively to prepare sample solutions with a concentration of 0.5%.

[0149] Test method: Take 0.5% concentration of the sample solution and place them into 96-well plates respectively. Add DPPH prepared with 100 μM methanol solution to make the total volume of the solution reach 200 μl. After standing at 37 °C for 30 min, measure at a wavelength of 560 nm. The calculation formula for the free radical scavenging rate is: Free radical scavenging rate (%) = (100 - A1 / A0) × 100, where A0 is the absorbance of the control group wells without adding the sample; A1 is the absorbance of the experimental group wells with the added sample; measure 3 times in parallel, and record the test results after taking the average value as shown in Table 3 below.

[0150] Table 3 Test results of free radical scavenging rate

[0151] Free radical scavenging rate / % Example 1 73.34 Example 2 76.28 Example 3 76.48 Example 4 76.37 Example 5 76.31 Example 6 70.59 Comparative Example 1 67.83 Comparative Example 2 60.74 Comparative Example 3 65.85 Comparative Example 4 71.72

[0152] As can be seen from Table 3, the fermented extract of Opuntia ficus-indica prepared by the present invention has good antioxidant effect.

[0153] 4. Anti-wrinkle test

[0154] Test subjects: Select 30 female volunteers with healthy skin, aged between 35 - 55. All volunteers have no history of skin diseases, no known allergies to the test materials, and have not used any drugs or cosmetics that may affect skin reactions within at least 2 weeks before the test;

[0155] Test materials: Use the fermented extracts of Opuntia ficus-indica prepared in Examples 1 - 6 and Comparative Examples 1 - 4 as samples, and dissolve the samples in deionized water respectively to prepare sample solutions with a concentration of 7.5 wt%.

[0156] Test method: Clean the face uniformly before the test. Mark a 3×3 cm test area on the cheek wrinkle area of the subject. Apply the sample evenly on this area, with a dosage of 1 ml / cm 2 , once in the morning and once in the evening, for 4 consecutive weeks. Use an Antera 3D skin analyzer to test the reduction in the surface wrinkle area of the test area. Record the average value of each group as shown in Table 4 below.

[0157] Table 4 Test results of the average reduction in surface wrinkle area

[0158] Average reduction in surface wrinkle area / % Example 1 14.2 Example 2 15.5 Example 3 16.2 Example 4 15.9 Example 5 15.7 Example 6 13.4 Comparative Example 1 12.1 Comparative Example 2 9.8 Comparative Example 3 13.6 Comparative Example 4 14.8

[0159] As can be seen from Table 3, the fermented extract of Opuntia ficus-indica prepared by the present invention has good anti-wrinkle effect.

[0160] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content without departing from the technical solution of the present invention. However, as long as it does not depart from the technical solution content of the present invention, any indirect modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of a fermented extract of Opuntia ficus-indica by double-strain fermentation, characterized in that: The preparation method comprises the following steps: Step 1: Mix the Opuntia ficus-indica stems with deionized water and beat them into a slurry, and then process to obtain the Opuntia ficus-indica stem slurry; Step 2: Cook the rice, and then cool it to obtain rice slurry; Step 3: Mix the rice slurry with the Opuntia ficus-indica stem slurry to obtain a mixed solution, and perform double-bacteria fermentation to obtain a fermentation product; Step 4: Centrifuge the fermentation product, retain the residue, and enrich the supernatant to obtain the product.

2. The preparation method of the fermented extract of Opuntia ficus-indica by double-bacteria fermentation according to claim 1, characterized in that: In Step 1, the material-liquid ratio of the Opuntia ficus-indica to deionized water is 1:3 - 5; the processing refers to high-pressure homogenization 3 times under a pressure of 150 MPa.

3. The preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation according to claim 1, wherein: The cooking in Step 2 refers to mixing the rice and deionized water according to a material-liquid ratio of 1:1.3 - 1.7, and then cooking at 100 °C for 20 - 30 min.

4. The preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation according to claim 1, characterized in that: In Step 3, the mass ratio of the rice slurry to the Opuntia ficus-indica stem slurry is 1.05 - 1.13:1.34 - 1.

42.

5. The preparation method of the fermented extract of Opuntia ficus-indica by double bacteria fermentation according to claim 1, characterized in that: The specific steps of the double-bacteria fermentation in Step 3 are as follows: Step Z1: Inoculate the mixed solution with an Aspergillus oryzae suspension and a Saccharomyces cerevisiae suspension, respectively accounting for 0.6 - 1 wt% and 1.6 - 2.2 wt% of the total amount of the mixed solution, add a citric acid-sodium citrate buffer solution to adjust the pH to 6.2 - 6.6, control the fermentation temperature at 28 - 32 °C, control the dissolved oxygen content at 2.4 - 4.8 ppm, and ferment at a rotation speed of 150 - 250 rpm for 32 - 38 h to obtain a first fermentation intermediate product; Step Z2: Add a citric acid-sodium citrate buffer solution to adjust the pH to 4.8 - 5.2, control the fermentation temperature at 26 - 28 °C, control the dissolved oxygen content at 0.5 - 0.7 ppm, adjust the rotation speed to 80 - 120 rpm, and ferment for 36 - 48 h to obtain a second fermentation intermediate product; Step Z3: Add glucose and residue, respectively accounting for 3.2 - 5 wt% and 0 - 1.8 wt% of the total amount of the second fermentation intermediate product, to the second fermentation intermediate product, control the anaerobic amount < 0.04 ppm, continue to ferment for 20 - 30 h, and control the temperature to inactivate.

6. The preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation according to claim 5, characterized in that: The concentration of Aspergillus oryzae in the Aspergillus oryzae suspension described in step Z1 is 0.8 - 1.2×10 8 CFU / mL; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1 - 1.2×10 8 CFU / mL.

7. The preparation method of a pear cactus fermentation extract by dual-bacteria fermentation according to claim 5, characterized in that: The temperature-controlled inactivation in Step Z3 refers to heating to 60 - 65 °C at a rate of 2 - 3 °C / min, holding for 15 min, and then cooling to room temperature at a rate of 4 - 6 °C / min.

8. The preparation method of a fermented extract of Opuntia ficus-indica by double-bacteria fermentation according to claim 1 or 5, characterized in that: The residues in Step 4 and Step Z3 need to be processed. The specific processing operation is to mix the residue and deionized water according to a mass ratio of 1:4 - 5, use a citric acid-sodium citrate buffer solution to adjust the pH to 4.8 - 5.2, stand at 50 - 52 °C for 2 - 3 h, inactivate at 80 °C for 10 min, and filter through a 0.22 μm filter membrane to obtain the product.

9. The preparation method of the fermented extract of Opuntia ficus-indica by double bacteria fermentation according to claim 1, characterized in that: The centrifugation in Step 4 refers to centrifuging at 4 °C at a rotation speed of 8000 - 12000 rpm for 20 - 30 min; the enrichment refers to filtering the supernatant through a 5 μm filter membrane, then filtering with a 10 - 12 kDa PES membrane, and finally filtering with a 1 - 3 kDa PES membrane.

10. Use of the fermented extract of Opuntia ficus-indica prepared by the preparation method of the fermented extract of Opuntia ficus-indica by dual-bacteria fermentation according to any one of claims 1-9, characterized in that: The Opuntia ficus-indica fermentation extract is applied to cosmetics at an addition amount of 5 - 10 wt%.

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