Preparation method of 11-seed fermentation oil and fermentation liquor with multiple effects

Abstract

The invention provides a preparation method of 11-seed fermentation oil and fermentation liquor with multiple effects, and belongs to the technical field of fermentation. The method comprises the following steps: S1, preparing recombinant saccharomycetes; s2, jointly fermenting lactobacillus, recombinant saccharomycetes and 11 seed oil; s3, separating an oil phase and a water phase, and taking the oil phase; and S4, dehydrating the oil phase to obtain the 11-seed fermentation oil. And S5, further refining the water phase to obtain the 11-seed fermentation liquor. The 11-seed fermented oil prepared by transforming a microbial metabolic network is rich in dihydroquercetin, and has multiple effects of resisting oxidation, resisting wrinkles, tightening, repairing cells and the like. In the fermentation process of the 11-seed fermentation oil, 11-seed fermentation liquor can be obtained, and the fermentation liquor has multiple effects of resisting oxidation, relieving, moisturizing, resisting wrinkles, tightening and the like.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a method for preparing 11-seed fermented oil and fermented liquid with multiple functions. Background Art

[0002] As consumers' demand for health and beauty products grows, natural, efficient and multifunctional cosmetic ingredients are gaining more and more attention. Vegetable oils are widely used in cosmetics due to their natural properties and nourishing effects on the skin. However, vegetable oils have a complex chemical structure and contain high molecular weight compounds synthesized from unsaturated fatty acids and glycerol, which limits their ability to be fully absorbed by the skin and affects the feel and efficacy of the product. In addition, as people further pursue functional skin care, how to enhance the efficacy of vegetable oils has become a research topic with innovative significance.

[0003] Dihydroquercetin is a scarce resource in the world and an extremely precious ingredient for medicine and functional food. It is mainly found in yew, yellow fir and larch. Because yew and yellow fir have been listed as endangered tree species and logging is prohibited, and larch is only distributed in eastern Siberia, Russia, northeastern Mongolia, northeastern China and North Korea, and has a long growth cycle, the resources available for production are extremely scarce and scarce. Due to the scarcity of dihydroquercetin sources, it also leads to its high selling price. The most important thing is that dihydroquercetin has excellent antioxidant properties, which is far superior to vitamin C, vitamin E and ergothioneine, and dihydroquercetin has good stability and biosafety.

[0004] In view of the demand for functional active substances in plant oils and the super antioxidant properties of dihydroquercetin, enriching plant oils with dihydroquercetin will effectively enhance the efficacy and value of plant oils. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for preparing 11-seed fermented oil and fermented liquid with multiple functions. The 11-seed fermented oil prepared by the present invention is rich in dihydroquercetin by transforming the microbial metabolic network, and has multiple functions such as anti-oxidation, anti-wrinkle and firming, and cell repair. In the fermentation process of the 11-seed fermented oil, an 11-seed fermented liquid can also be obtained, and the fermented liquid has multiple functions such as anti-oxidation, soothing, moisturizing, and anti-wrinkle and firming.

[0006] The technical solution of the present invention is as follows: A method for preparing eleven-seed fermented oil and fermented liquid with multiple functions, the method comprising the following steps: S1. preparing a recombinant yeast; S2, fermenting lactobacillus, recombinant yeast and 11 seed oil together; S3, separating the oil phase and the water phase, and taking the oil phase; S4, dehydrating the oil phase to obtain the 11-seed fermented oil; S5. Further purifying the aqueous phase to obtain the 11-seed fermentation liquid.

[0007] Preferably, the method for preparing the recombinant yeast in step S1 is to express the flavonoid reductase having an amino acid sequence as shown in SEQ ID No. 1 in Saccharomyces cerevisiae.

[0008] Furthermore, the brewing yeast was screened from the soil near the larch in Daxinganling.

[0009] Preferably, the 11 seed oil in step S2 is composed of white meadowfoam seed oil, sunflower seed oil, wheat germ oil, borage seed oil, filbert seed oil, echium plantago seed oil, macadamia seed oil, jojoba seed oil, peony seed oil, bilberry seed oil and perilla seed oil.

[0010] Preferably, the fermentation process in step S2 comprises the following steps: M1: Activate the recombinant yeast and lactobacillus separately and prepare seed solution; M2: inoculating the recombinant yeast into the yeast fermentation medium for pre-fermentation; inoculating lactobacillus into the lactobacillus fermentation medium for pre-fermentation; M3: After pre-fermentation with recombinant yeast, Sophora japonica extract was added to the yeast fermentation system for continued fermentation and culture; after pre-fermentation with lactobacillus, 11 seed oil was added to the lactobacillus fermentation system for continued fermentation and culture; M4: After the recombinant yeast and lactobacillus continue to ferment separately, the yeast fermentation system and the lactobacillus fermentation system are mixed and fermented together.

[0011] Preferably, the preparation method of the recombinant yeast seed solution in step M1 is: inoculating the recombinant yeast into a yeast seed culture medium, and culturing at 25-37° C. for 12-24 h; the yeast seed culture medium is composed of: 10-30 g / L peptone, 5-15 g / L yeast extract, and 15-25 g / L glucose; The preparation method of the lactobacillus seed liquid is as follows: inoculating lactobacillus into a lactobacillus seed culture medium, and culturing for 12-24 hours at 25-37°C; the lactobacillus seed culture medium is composed of: 5-15 g / L peptone, 2-10 g / L beef powder, 2-10 g / L yeast powder, 1-10 g / L glucose, 1-5 g / L dipotassium hydrogen phosphate, 2-10 g / L sodium acetate, 1-10 g / L triammonium citrate, and 0.1-1 g / L magnesium sulfate.

[0012] Preferably, the yeast fermentation medium in step M2 is composed of: 10-22 g / L rice, 18-23 g / L soybean extract, and 8-11 g / L glucose; the recombinant yeast pre-fermentation condition is culturing at 25-42°C for 12-144 h; The lactobacillus fermentation medium comprises: 10-30 g / L glucose, 10-30 g / L soybean extract, 5-20 g / L corn flour, and 10-40 g / L whey powder; the lactobacillus pre-fermentation condition is fermentation culture at 25-40° C. for 4-48 h; During pre-fermentation, the inoculation amount of the recombinant yeast seed solution is 0.5-10%, and the inoculation amount of the lactobacillus seed solution is 0.5-10%.

[0013] Preferably, the mass volume ratio of the added amount of the Sophora japonica extract to the recombinant yeast pre-fermentation liquid in step M3 is 0.5-5 g / L, and the recombinant yeast continues to ferment for 6-96 h; The amount of the 11 seed oil added is 10-900% of the weight of the initial lactobacillus culture medium, and the lactobacillus continues to ferment for 3-10 hours.

[0014] Preferably, the co-fermentation temperature in step M4 is 25-42° C. and the fermentation time is 12-96 h.

[0015] Preferably, the method for separating the oil phase and the water phase in step S3 comprises at least one of static separation, centrifugal separation and membrane separation.

[0016] Preferably, the method for dehydrating the oil phase in step S4 comprises at least one of vacuum dehydration, anhydrous sodium sulfate dehydration, and anhydrous calcium chloride dehydration; The method for further refining the aqueous phase in step S5 is to remove impurities in the aqueous phase using activated carbon, ceramic membrane and / or resin.

[0017] Furthermore, the process of further refining the aqueous phase in step S5 comprises the following steps: (1) Add activated carbon to the aqueous phase and stir at 25-60°C for 0.5-6 h; (2) Separating and removing the activated carbon, the resulting water system is further treated with a ceramic membrane to remove bacteria and residues; (3) The obtained ceramic membrane clear liquid is further refined using an ultrafiltration membrane system; (4) The obtained ultrafiltration membrane clear liquid is further refined using resin; (5) adjusting the pH of the refined liquid to 5-7 to obtain 11-seed fermentation liquid; The amount of activated carbon added in step (1) is 0.1-10% of the aqueous phase; The separation process in step (2) includes but is not limited to centrifugal filtration, plate and frame filtration and filter bag filtration; The pore size of the ceramic membrane in step (2) is 10-200 nm; The ultrafiltration membrane in step (3) has a molecular weight cutoff of 3-50 kD.

[0018] The beneficial technical effects of the present invention are: 1. The present invention obtains a recombinant yeast capable of producing dihydroquercetin by screening brewer's yeast from the vicinity of larch and reconstructing the metabolic network of brewer's yeast.

[0019] 2. The combination of 11 seed oils can well exert the active effects of plant oils, and how to further enhance the efficacy of 11 seed oils so that 11 seed oils have multiple effects on the basis of fully exerting the efficacy of plant oils is a more important issue. The present invention ferments 11 seed oils using recombinant yeast and lactobacillus, and the prepared 11 seed fermented oil is rich in the precious active substance dihydroquercetin.

[0020] 3. The antioxidant capacity of the 11-seed fermented oil prepared by the present invention is more than 4 times higher than that of the 11-seed crude oil. The antioxidant capacity test of the 11-seed fermented oil shows that the 11-seed fermented oil has very good performance for different types of free radicals and active oxygen types, proving that the 11-seed fermented oil has comprehensive antioxidant capacity. At the same time, the 11-seed fermented oil obtained by the present invention also has anti-wrinkle, firming and DNA repair effects.

[0021] 4. The present invention creatively adds Sophora japonica extract after pre-fermentation with recombinant yeast. The quercetin contained in the Sophora japonica extract can be used as a substrate for the production of dihydroquercetin, so that the final fermented oil contains dihydroquercetin; and the addition of Sophora japonica extract is beneficial to the growth of microorganisms, thereby increasing the content of dihydroquercetin in the final 11-seed fermented oil and the efficacy of the 11-seed fermented oil.

[0022] 5. The present invention can prepare 11-seed fermented oil and obtain 11-seed fermented liquid at the same time. The 11-seed fermented liquid contains not only beneficial ingredients produced by microbial fermentation, but also dihydroquercetin. The finally obtained 11-seed fermented liquid has multiple effects such as anti-oxidation, soothing, moisturizing, anti-wrinkle and firming. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The yeast screening plate of Example 1; Figure 2 This is the plasmid map for expressing flavonoid reductase in Example 2; Figure 3 The results of the determination of dihydroquercetin content in 11-seed crude oil and 11-seed fermented oil in Test Example 1; Figure 4 The results of total flavonoids content determination of 11-seed crude oil and 11-seed fermented oil in Test Example 2; Figure 5 The results of total phenol content determination of 11-seed crude oil and 11-seed fermented oil in Test Example 2; Figure 6 This is a comparison of the DPPH radical scavenging ability of 11-seed fermented oil and 11-seed crude oil in Test Example 3; Figure 7 The scavenging rate of ABTS free radicals by 11-seed fermented oil at different concentrations in Test Example 4 was determined; Figure 8 The scavenging rate of hydroxyl radicals by 11-seed fermented oil at different concentrations in Test Example 5 was determined; Fig. 9 The scavenging rate of superoxide anion free radicals by 11-seed fermented oil of different concentrations in Test Example 6 was determined; Fig.10 The PTIO free radical scavenging rate of 11-seed fermented oil at different concentrations in Test Example 7 was determined; Fig.11 This is a summary diagram of the determination of ROS content in keratinocytes in Test Example 8; Fig.12 This is a graph showing the relative content of ROS in Test Example 8; Fig.13 The results of the determination of the content of Collagen I after incubation for 12 hours in Test Example 9; Fig.14 This is a summary of the immunofluorescence staining results of Collagen I after 24 h of incubation in Test Example 9; Fig.15 This is a graph showing the relative integrated optical density (IOD) results of Collagen I content after 24 h of incubation in Test Example 9; Fig.16 This is a summary of the 8-OHdG immunofluorescence staining results in Test Example 10; Fig.17 This is a graph showing the relative integrated optical density (IOD) results of 8-OHdG immunofluorescence staining in Test Example 10; Fig.18 This is the DPPH free radical scavenging rate test of the 11-seed fermentation liquid in Test Example 11; Fig.19 This is the comparison of hyaluronidase inhibition rate of blank group / 11 seed fermentation liquid in test example 12; Fig. 20 This is a bar graph of the moisture retention rate of the 11-seed fermented liquid (diluted 25%) at different times in Test Example 13; Fig.21 This is a bar graph of the moisture retention rate of the 11-seed fermented liquid (diluted 50%) at different times in Test Example 13; Fig. 22 This is a comparison of the collagenase inhibition rate of the blank group / 11 seed fermentation broth in Test Example 14. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0025] Example 1: Screening of dihydroquercetin-tolerant strains The present invention prepares a strain capable of producing dihydroquercetin and ferments it with 11 seed oil to enhance the efficacy of 11 seed oil. To this end, a strain capable of tolerating dihydroquercetin is first screened. Considering that dihydroquercetin is mainly derived from larch, soil samples are taken from the roots of larch around Mohe in Daxing'anling for screening. The specific screening method is: (1) Pipette 1 mL of activated soil suspension into 20 mL of enrichment medium and incubate in a shaker at 30°C and 200 rpm for 1 day. The formula of the enrichment medium (1 L) is: 2 g glucose, 0.2 g yeast extract, 0.2 g NH4NO3, 0.3 g KH2PO4, 0.5 g Na2HPO4·12H2O, and 0.05 g MgSO4·7H2O.

[0026] (2) Take the enriched culture medium from 1 day of culture and dilute it by 10 4 -10 5 times, 100 μL was spread on a YPD solid plate containing 5 g / L dihydroquercetin, and inverted in a constant temperature incubator at 30°C for 24 h. Figure 1 The composition of the YPD solid plate is: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, and 20 g / L agar.

[0027] (3) Select the large colonies, streak them onto YPD solid plates for later use, and prepare glycerol tubes for storage at -80°C.

[0028] (4) The strain obtained by screening was subjected to ITS determination, and the strain obtained by screening was found to be Saccharomyces cerevisiae through sequence comparison.

[0029] Example 2: Construction of recombinant yeast A recombinant yeast capable of producing dihydroquercetin was constructed using the Saccharomyces cerevisiae screened in Example 1. The steps for constructing the recombinant yeast were as follows: The amino acid sequence of the flavonoid reductase shown in SEQ ID No. 1 was codon optimized and gene synthesis was performed by Shanghai Biotech. The TDH1 promoter, flavonoid reductase and CYC1 terminator were integrated into the plasmid pRS426. The resulting plasmid map is shown in Figure 2 The obtained plasmid was transformed into the competent cells of Saccharomyces cerevisiae obtained in Example 1, thereby obtaining a recombinant yeast for the production of 11-seed fermented oil.

[0030] Example 3: Preparation of 11-seed fermented oil (1) The recombinant yeast and lactobacillus prepared in Example 2 (purchased from Chr. Hansen) were inoculated into yeast seed culture medium and lactobacillus seed culture medium, respectively, and the fermentation conditions were both 30° C. and cultured for 24 h to obtain seed solutions of recombinant yeast and lactobacillus, respectively.

[0031] The yeast seed culture medium is composed of: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0032] The lactobacillus seed culture medium is composed of: 7 g / L peptone, 5 g / L beef powder, 4 g / L yeast powder, 2 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 4 g / L sodium acetate, 2 g / L triammonium citrate, and 0.2 g / L magnesium sulfate.

[0033] (2) The recombinant yeast seed liquid prepared in step (1) was transferred into a yeast fermentation medium at a 1% inoculum amount. The yeast fermentation medium was composed of: 18 g / L rice, 20 g / L commercially available soybean extract, and 10 g / L glucose. The medium was cultured at 30° C. for 24 h to obtain a recombinant yeast pre-fermentation liquid.

[0034] (3) The lactobacillus seed liquid prepared in step (1) was transferred into a lactobacillus fermentation medium at a 1% inoculum rate, wherein the lactobacillus fermentation medium was composed of 18 g / L glucose, 20 g / L soybean extract, 10 g / L corn flour, and 20 g / L whey powder, and cultured at 30° C. for 8 h to obtain a lactobacillus pre-fermentation liquid.

[0035] (4) Add 2 g / L Sophora japonica extract (purchased from Shaanxi Guanchen Biology) to the yeast pre-fermentation liquid obtained in step (2), and continue fermentation at 30°C for 24 h.

[0036] (5) Adding 11 seed oil in an amount of 200% by weight of the initial lactobacillus culture medium to the lactobacillus pre-fermentation liquid obtained in step (3), and continuing the fermentation culture at 30° C. for 6 h. The 11 seed oil is composed of meadowfoam seed oil, sunflower seed oil, wheat germ oil, borage seed oil, filbert seed oil, echium plantaginum seed oil, macadamia seed oil, jojoba seed oil, peony seed oil, bilberry seed oil, and perilla seed oil.

[0037] (6) The yeast fermentation broth and the lactobacillus fermentation broth obtained in step (4) and step (5) are mixed and co-fermented at 25° C. for 12 h.

[0038] (7) After fermentation, the obtained fermentation product was removed from the bacteria using a ceramic membrane. The mixture was further centrifuged at 10,000 r / min for 10 min and the upper oil phase was collected.

[0039] (8) Add anhydrous sodium sulfate to the obtained oil phase and centrifuge at 10,000 r / min for 10 min to obtain the finished 11-seed fermented oil.

[0040] Example 4: Preparation of 11-seed fermentation broth On the basis of Example 3, the lower aqueous phase obtained by centrifugation in step (7) was collected, 1% activated carbon was added, and the mixture was stirred at 50° C. for 2 h. The activated carbon was further removed by centrifugation, and the clear liquid was collected.

[0041] The clear liquid obtained in step (1) is passed through a ceramic membrane to further remove bacteria and residues, the pore size of the ceramic membrane used is 50 nm, and the clear liquid of the ceramic membrane is collected; The clear liquid obtained in step (2) is further purified by ultrafiltration membrane, the molecular weight cut-off size of the ultrafiltration membrane used is 5 kD, and the clear liquid is collected; The clear solution obtained in step (3) is purified by using a cation exchange resin and then an anion exchange resin; The pH of the obtained refined liquid was adjusted to 5.2 to obtain an 11-seed fermentation liquid.

[0042] Test Example 1: Determination of dihydroquercetin content in 11-seed fermented oil The content of dihydroquercetin in the 11-seed fermented oil and the 11-seed crude oil prepared in Example 3 was determined by high performance liquid chromatography. Figure 3 As shown in the figure, it can be seen that dihydroquercetin was not detected in the 11-seed crude oil, while the dihydroquercetin content in the 11-seed fermented oil was 1086 mg / kg.

[0043] Test Example 2: Test of total flavonoids and total polyphenols in 11-seed fermented oil The total flavonoids and total polyphenols contents of the 11-seed crude oil and the 11-seed fermented oil prepared in Example 3 were measured respectively. The results were as follows: Figure 4and Figure 5 As shown. Figure 4 It can be seen that the total flavonoids content in 11-seed fermented oil is 4036 mg Rutin / kg, while the total flavonoids content in 11-seed crude oil is only 1363 mg Rutin / kg. The total flavonoids content in 11-seed fermented oil is 2.98 times that of 11-seed crude oil. Figure 5 It can be seen that the total polyphenol content in 11-seed fermented oil is 1.59 times that of 11-seed crude oil.

[0044] Test Example 3: Antioxidant Capacity Test of 11-seed Fermented Oil - DPPH Free Radical Scavenging Experiment 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517 nm. When there is a free radical scavenger, it pairs with its single electron and weakens the light absorption of DPPH ethanol solution. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the strength of its antioxidant activity.

[0045] The DPPH free radical scavenging ability of the 11-seed fermented oil and the 11-seed crude oil prepared in Example 3 was measured respectively. The results are as follows: Figure 6 As shown in the figure, the DPPH radical scavenging rate of 11-seed fermented oil is 87.1%, while that of 11-seed crude oil is only 20.2%. The DPPH radical scavenging capacity of 11-seed fermented oil is 4.3 times that of 11-seed crude oil, indicating that 11-seed fermented oil has outstanding antioxidant capacity.

[0046] Test Example 4: Antioxidant Capacity Test of 11-seed Fermented Oil - ABTS Free Radical Scavenging Experiment The ABTS free radical scavenging rate test evaluates the antioxidant activity by measuring the ability of antioxidants to scavenge ABTS free radicals. ABTS free radicals are a blue-green pigment with a maximum absorption wavelength of 734 nm. When antioxidants are present, their absorption intensity decreases, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the strength of its antioxidant activity.

[0047] The ABTS free radical scavenging ability of the 11-seed fermented oil prepared in Example 3 at different concentrations after being dissolved in ethanol was measured. The results are as follows: Figure 7 As shown in the figure, when the concentration of 11-seed fermented oil is 0.66%, it shows significant antioxidant capacity, and as the concentration of 11-seed fermented oil increases, the ABTS free radical scavenging rate gradually increases, showing an obvious concentration-dependent relationship.

[0048] It should be noted that when using the t-test two-tailed test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p < 0.05 is indicated by #, and p < 0.01 is indicated by ##; the significance of the sample group and PC group compared with the NC group is indicated by *, p < 0.05 is indicated by *, p < 0.01 is indicated by **, and p < 0.001 is indicated by ***. When there is no star * in the figure, it means that p is less than 0.05 or 0.01, and only the minimum data symbol of the p value is marked in the figure. Subsequent test examples 5-14 are applicable, and the meaning of the pound sign and asterisk in the figure will not be repeated.

[0049] Test Example 5: Antioxidant Capacity Test of 11-seed Fermented Oil - Hydroxyl Radical Scavenging Experiment Hydroxyl free radical (·OH) is one of the most active reactive oxygen species in the body, which can mediate many physiological changes and damage biological molecules such as proteins, nucleic acids, lipids, etc., leading to damage to cell structure and function, and then causing diseases. 2+ Hydroxyl radicals are generated by Fenton reaction and Fe 2+ Oxidized to Fe 3+ , resulting in the red phenanthroline-Fe 2+ Oxidized to colorless phenanthroline-Fe 3+ , making o-phenanthroline-Fe 2+ The maximum absorption peak at 536 nm disappears, and the change in absorbance at 530-540 nm can be measured by a spectrophotometer. The change in the content of hydroxyl free radicals can be calculated based on this, and the hydroxyl free radical scavenging rate or scavenging capacity of the sample can be calculated.

[0050] The hydroxyl radical scavenging ability of the 11-seed fermented oil prepared in Example 3 at different ethanol concentrations was measured. The results are as follows: Figure 8 As shown in the figure, when the concentration of 11-seed fermented oil is 0.66%, it shows significant antioxidant capacity, and as the concentration of 11-seed fermented oil increases, the hydroxyl radical scavenging rate gradually increases, showing an obvious concentration-dependent relationship.

[0051] Test Example 6: Antioxidant Capacity Test of 11-seed Fermented Oil - Superoxide Anion Radical Scavenging Rate Determination Experiment Superoxide anion free radicals are a type of free radical produced during the metabolism of organisms. They have strong oxidative ability and can attack biological macromolecules such as lipids, proteins, nucleic acids, etc., causing damage to cell structure and function. Therefore, the ability to scavenge superoxide anion free radicals is an important indicator for evaluating antioxidant performance. The determination method is the pyrogallol self-oxidation method, which is simple to operate and sensitive. Pyrogallol self-oxidizes under alkaline conditions to produce O2· - , O2· -It can accelerate the self-oxidation rate of pyrogallol and generate colored intermediates. The accumulation of intermediates has a good linear relationship with time at a certain time. The colored products have strong light absorption at 325nm. - It can inhibit the self-oxidation reaction and prevent the accumulation of intermediate products, thereby achieving the purpose of removing superoxide anions.

[0052] The superoxide anion radical scavenging ability of the 11-seed fermented oil prepared in Example 3 at different ethanol concentrations was measured. The results are as follows: Fig. 9 As shown in the figure, when the concentration of 11-seed fermented oil is 0.66%, it shows significant antioxidant capacity, and as the concentration of 11-seed fermented oil increases, the superoxide anion radical scavenging rate gradually increases, showing an obvious concentration-dependent relationship.

[0053] Test Example 7: Antioxidant Capacity Test of 11-seed Fermented Oil - PTIO Free Radical Scavenging Rate Determination Experiment PTIO free radical is an oxygen free radical that is similar to free radicals in the human body, so it is used to detect the ability to scavenge reactive oxygen species. The absorbance of PTIO free radicals at a specific wavelength decreases as they are scavenged, and the absorbance is usually measured at 557 nm. The change in absorbance at 557 nm can be measured by a spectrophotometer, and the change in the content of PTIO free radicals can be calculated based on this, and the PTIO free radical scavenging rate or scavenging ability of the sample can be calculated.

[0054] The PTIO free radical scavenging ability of the 11-seed fermented oil prepared in Example 3 at different ethanol concentrations was measured. The results are as follows: Fig.10 As shown in the figure, when the concentration of 11-seed fermented oil is 0.66%, it shows significant antioxidant capacity, and as the concentration of 11-seed fermented oil increases, the PTIO free radical scavenging rate gradually increases, showing an obvious concentration-dependent relationship.

[0055] Test Example 8: Antioxidant capacity test of 11 seed fermented oil - UVB stimulated keratinocyte ROS content determination experiment The skin is often exposed to a variety of external stimuli such as ultraviolet rays, air pollutants, toxic chemicals or physical mechanical damage. These exposure factors will directly or indirectly lead to an increase in the content of reactive oxygen species (ROS) in the skin, destroying the balance of the skin's internal environment and causing oxidative stress. The long-term cumulative effect will lead to skin inflammation, aging (photoaging) and even cancer. Therefore, effective antioxidant measures are important for maintaining skin health. This test is based on the human immortalized keratinocyte (HaCaT) model. An in vitro oxidative damage cell model is established by inducing the production of ROS through UVB irradiation. The antioxidant capacity and anti-wrinkle and firming ability of the sample to be tested are evaluated by detecting the inhibitory ability of the 11-seed fermented oil prepared in Example 3 on ROS.

[0056] The ROS content of keratinocytes was measured using the reactive oxygen species detection kit. Fig.11 and Fig.12 As shown. Fig.11 and Fig.12 It can be seen that based on the UVB-stimulated keratinocyte (HaCaT) model, the ROS content of 11-seed fermented oil dissolved in DMSO solution at concentrations of 0.0125%, 0.025% and 0.05% (V / V) was significantly reduced, and there was a statistical difference compared with the NC group (p<0.01), indicating that the sample 11-seed fermented oil has antioxidant capacity and anti-wrinkle and firming effects.

[0057] Through the antioxidant capacity tests of Test Example 3, Test Example 4, Test Example 5, Test Example 6, Test Example 7 and Test Example 8 on 11-seed fermented oil, it can be seen that the 11-seed fermented oil has very good performance for different types of free radicals and active oxygen types, proving that the 11-seed fermented oil prepared by the present invention has comprehensive antioxidant capacity.

[0058] Test Example 9: Anti-wrinkle and firming efficacy test - Immunofluorescence detection of type Ⅰ collagen in human fibroblasts Collagen I is the main protein in the extracellular matrix of the skin, which forms highly ordered collagen fibers, providing structural support and elasticity for the skin. Mature Collagen I can self-assemble into collagen fibers, which are the key to skin firmness and elasticity. With age, the content of Collagen I in the skin will gradually decrease, causing the skin to lose elasticity and firmness, thus forming wrinkles. Therefore, by promoting the synthesis of Collagen I, the elasticity of the skin can be enhanced and the formation of wrinkles can be reduced. This test uses human fibroblasts as the test system, and uses immunofluorescence technology to detect changes in the expression level of type I collagen in human fibroblasts to evaluate whether the 11-seed fermented oil prepared in Example 3 has anti-wrinkle and firming effects.

[0059] Different concentrations of samples were incubated with UVA-irradiated human fibroblasts, and the changes in the content of Collagen I were measured at 12 h and 24 h of treatment. The results of the 12-hour Collagen I content measurement are shown in Figure 2. Fig.13 The results of the 24-hour Collagen I content determination are shown in Fig.14 and Fig.15 As shown. Figure 13-15 It can be seen that regardless of whether the 11-seed fermented oil was used to treat UVA-treated human fibroblasts for 12 h or 24 h, based on the UVA-stimulated human fibroblast model, when the concentration of the sample 11-seed fermented oil in the DMSO solution was 0.0125%, 0.025% and 0.05% (V / V), the content of type I collagen (Collagen Ⅰ) in human fibroblasts was significantly increased, and there was a statistical difference compared with the NC group (p<0.01), indicating that the sample 11-seed fermented oil has anti-wrinkle and firming effects.

[0060] Test Example 10: DNA damage repair test - UVB stimulated keratinocyte 8-OHdG protein content detection Certain specific damage products can be formed during DNA damage, and DNA damage can be assessed by detecting damage products (γ-H2AX, 8-OHdG, etc.). Among the DNA oxidative damage products, 8-hydroxydeoxyguanosine (8-OHdG), as a hydroxyl adduct of deoxyguanosine (dG) in DNA, has been recognized as the biomarker that best reflects the degree of DNA oxidative damage in vivo because of its large formation amount and few influencing factors. This test uses UVB to stimulate human immortalized keratinocytes (HaCaT) to simulate light damage, and uses immunofluorescence technology to detect changes in the content of cell DNA damage product 8-OHdG to evaluate the effect of 11-seed fermented oil prepared in Example 3 on repairing DNA damage.

[0061] The results of the test are as follows Fig.16 and Fig.17 As shown. The results show that based on the UVB-stimulated human immortalized keratinocyte (HaCaT) model, the 11-seed fermented oil at concentrations of 0.0125%, 0.025% and 0.05% (V / V) in DMSO solution significantly reduced the content of cell barrier-related protein 8-hydroxydeoxyguanosine (8-OHdG), which was statistically different from the NC group (p<0.01), indicating that the 11-seed fermented oil has the effect of repairing DNA damage.

[0062] Based on the efficacy evaluation results of test examples 2-10, 11-seed fermented oil has good antioxidant capacity, excellent anti-wrinkle and firming effects, and outstanding DNA repair effects.

[0063] Test Example 11: Antioxidant Capacity Test of 11-seed Fermentation Broth 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH for short) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517 nm. When a free radical scavenger is present, it pairs with its single electron and weakens the light absorption of the DPPH ethanol solution. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the strength of its antioxidant activity. The DPPH free radical scavenging rate of the 11-seed fermentation broth prepared in Example 4 is shown in the figure. Fig.18 As shown, when the sample was a 25% water dilution, the free radical scavenging rate was 8.21%; when the sample was a 50% water dilution, the free radical scavenging rate was 32.63%, indicating that the 11-seed fermentation liquid has antioxidant effects.

[0064] Test Example 12: Soothing effect test of 11 seed fermentation liquid The soothing effect is mainly characterized by evaluating the inhibition rate of the test sample on hyaluronidase. High molecular weight hyaluronic acid plays an important role in regulating skin repair and can significantly reduce inflammatory reactions. However, the degradation products of hyaluronic acid are one of the causes of skin inflammatory reactions. The degradation reaction of hyaluronic acid depends on the activity of hyaluronidase. Hyaluronidase is also involved in the process of tissue inflammation and is a participant in type I allergic reactions. It participates in the reaction in the third stage of allergic reactions, leading to mast cell degranulation. Therefore, the inhibition of hyaluronidase activity can be used as an indicator of the anti-inflammatory and anti-allergic reaction of the sample. The experimental principle of inhibiting hyaluronidase is that hyaluronidase catalyzes the reaction with the enzyme substrate, and the absorbance changes after the active substance is added. The size of the absorbance change reflects the inhibition rate of the hyaluronidase inhibitor. The higher the hyaluronidase inhibition rate, the stronger the soothing effect of the sample.

[0065] The test results of the 11-seed fermentation liquid prepared in Example 4 are shown in Fig.19 When the samples were 25% water dilution and 50% water dilution, the hyaluronidase inhibition rates were 10.76% and 19.87% respectively; compared with the blank group, the hyaluronidase inhibition rate of the sample group was better than that of the blank group, and the statistical difference p value was 0.000, which was significantly different (p < 0.05). This shows that the 11-seed fermented liquid has a soothing effect.

[0066] Test Example 13: Moisturizing effect test of 11 seed fermentation liquid The gravimetric method for determining the moisturizing rate of moisturizing cosmetics is also known as the in vitro weighing method. Generally, glass plates, bionic materials or medical breathable tapes are used as sample carriers. The use of cosmetics is simulated by directly applying the sample on the carrier. The carrier with the sample to be tested is placed under constant temperature and humidity conditions for a certain period of time, and the mass of the sample before and after is weighed. The moisturizing rate is calculated according to the formula. The gravimetric method for determining the moisturizing rate of moisturizing cosmetics is based on the fact that different moisturizer molecules in cosmetics have different forces on water molecules, and their ability to absorb and retain moisture is also slightly different.

[0067] When the 11-seed fermented liquid prepared in Example 4 was diluted by 25%, the moisture retention test results at different times were as follows: Fig. 20 As shown in the figure, as time goes by, the moisturizing rates of the sample group and the control group (blank, positive) gradually decrease. The moisturizing rate of the test sample group at each time point (30min / 1h / 2h / 4h) is better than that of the blank control group, and p < 0.05 is statistically significant. When the sample is diluted by 50%, the moisturizing rate test results at different times are shown in Fig.21 As shown in the figure, as time goes by, the moisturizing rates of the sample group and the control group (blank, positive) gradually decrease. The moisturizing rate of the test sample group at each time point (30min / 1h / 2h / 4h) is better than that of the blank control group, and p < 0.05 is statistically significant. In summary, 11-seed fermented liquid has moisturizing effect.

[0068] Test Example 14: Anti-wrinkle and firming efficacy test of 11-seed fermented liquid The anti-wrinkle / firming effect can be characterized by evaluating the inhibition rate of the test sample on collagenase. Collagen is a biopolymer synthesized by animal fibroblasts. It is a white, opaque, unbranched fibrous protein. Collagen has a unique fibrous structure and good biocompatibility and resistance to oxidative stress. It can effectively improve the structure of the skin epidermis and dermis, promote the synthesis of collagen in the skin, and make the skin elastic and firm. The collagen in the skin is mainly type I collagen; the three-strand helix structure of collagen can be hydrolyzed by collagenase to form gel polypeptides, which affects the loss of skin collagen to a certain extent. Therefore, inhibiting the activity of collagenase reduces the loss of collagen to a certain extent.

[0069] The experimental principle of inhibiting collagenase is that type 1 collagenase catalyzes the reaction with the enzyme substrate. After adding the active substance, the absorbance changes, and the size of the absorbance change reflects the inhibition rate of the collagenase inhibitor. The anti-wrinkle / firming effect is mainly characterized by evaluating the inhibition rate of the test sample on collagenase. The higher the collagenase inhibition rate value, the stronger the anti-wrinkle / firming effect of the sample. The test results of the 11-seed fermentation liquid prepared in Example 4 are shown in Fig. 22 When the sample was diluted with 25% water, the inhibition rate of elastase was 64.02%; when the sample was diluted with 50% water, the inhibition rate of elastase was 68.25%. Compared with the blank group, the inhibition rate of collagenase in the sample group was better than that in the blank group, and the statistical difference p value was 0.000, which was significantly different (p < 0.05). In summary, 11-seed fermented liquid has anti-wrinkle / firming effects.

[0070] It can be seen from the efficacy evaluation results of comprehensive test examples 11-14 that the 11-seed fermentation liquid prepared by the present invention has antioxidant, soothing, moisturizing and anti-wrinkle and firming effects.

[0071] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing 11-seed fermented oil and fermented liquid with multiple functions, characterized in that: The method comprises the following steps: S1. preparing a recombinant yeast; S2, fermenting lactobacillus, recombinant yeast and 11 seed oil together; S3, separating the oil phase and the water phase, and taking the oil phase; S4, dehydrating the oil phase to obtain the 11-seed fermented oil; S5. Further purifying the aqueous phase to obtain the 11-seed fermentation liquid.

2. The preparation method according to claim 1, characterized in that: The preparation method of the recombinant yeast in step S1 is to express the flavonoid reductase with an amino acid sequence as shown in SEQ ID No. 1 in Saccharomyces cerevisiae.

3. The preparation method according to claim 1, characterized in that: The 11 seed oils in step S2 are composed of white meadowfoam seed oil, sunflower seed oil, wheat germ oil, borage seed oil, filbert seed oil, echium plantago seed oil, macadamia seed oil, jojoba seed oil, peony seed oil, bilberry seed oil and perilla seed oil.

4. The preparation method according to claim 1, characterized in that: The fermentation process in step S2 comprises the following steps: M1: Activate the recombinant yeast and lactobacillus separately and prepare seed solution; M2: inoculating the recombinant yeast into the yeast fermentation medium for pre-fermentation; inoculating lactobacillus into the lactobacillus fermentation medium for pre-fermentation; M3: After pre-fermentation with recombinant yeast, Sophora japonica extract was added to the yeast fermentation system for continued fermentation and culture; after pre-fermentation with lactobacillus, 11 seed oil was added to the lactobacillus fermentation system for continued fermentation and culture; M4: After the recombinant yeast and lactobacillus continue to ferment separately, the yeast fermentation system and the lactobacillus fermentation system are mixed and fermented together.

5. The preparation method according to claim 4, characterized in that: The preparation method of the recombinant yeast seed solution in step M1 is: inoculating the recombinant yeast into the yeast seed culture medium, and culturing at 25-37° C. for 12-24 h; The yeast seed culture medium is composed of: 10-30 g / L peptone, 5-15 g / L yeast extract, and 15-25 g / L glucose; The preparation method of the lactobacillus seed liquid is as follows: inoculating lactobacillus into a lactobacillus seed culture medium, and culturing at 25-37° C. for 12-24 h; The lactobacillus seed culture medium comprises: 5-15 g / L peptone, 2-10 g / L beef powder, 2-10 g / L yeast powder, 1-10 g / L glucose, 1-5 g / L dipotassium hydrogen phosphate, 2-10 g / L sodium acetate, 1-10 g / L triammonium citrate and 0.1-1 g / L magnesium sulfate.

6. The preparation method according to claim 4, characterized in that: The yeast fermentation medium in step M2 is composed of: 10-22 g / L rice, 18-23 g / L soybean extract, and 8-11 g / L glucose; The recombinant yeast pre-fermentation condition is to culture at 25-42°C for 12-144 hours; The lactobacillus fermentation medium comprises: 10-30 g / L glucose, 10-30 g / L soybean extract, 5-20 g / L corn flour, and 10-40 g / L whey powder; The lactobacillus pre-fermentation condition is to ferment and culture at 25-40°C for 4-48 hours; During pre-fermentation, the inoculation amount of the recombinant yeast seed solution is 0.5-10%, and the inoculation amount of the lactobacillus seed solution is 0.5-10%.

7. The preparation method according to claim 4, characterized in that: In step M3, the mass volume ratio of the added amount of the Sophora japonica extract to the recombinant yeast pre-fermentation liquid is 0.5-5 g / L, and the recombinant yeast continues to ferment for 6-96 h; The amount of the 11 seed oil added is 10-900% of the weight of the initial lactobacillus culture medium, and the lactobacillus continues to ferment for 3-10 hours.

8. The preparation method according to claim 4, characterized in that: The co-fermentation temperature in step M4 is 25-42° C. and the fermentation time is 12-96 h.

9. The preparation method according to claim 1, characterized in that: The method for separating the oil phase and the water phase in step S3 includes at least one of static separation, centrifugal separation, and membrane separation.

10. The preparation method according to claim 1, characterized in that: The method for dehydrating the oil phase in step S4 includes at least one of vacuum dehydration, anhydrous sodium sulfate dehydration, and anhydrous calcium chloride dehydration; The method for further refining the aqueous phase in step S5 is to use activated carbon, ceramic membrane and / or resin to remove impurities in the aqueous phase.

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