Red onion fermentation liquor with effects of resisting inflammatory aging and whitening as well as preparation method and application of red onion fermentation liquor

Abstract

The invention belongs to the technical field of biological products, and particularly relates to an eleutherine americana fermentation liquor with the effects of resisting inflammatory aging and whitening as well as a preparation method and application of the eleutherine americana fermentation liquor. The preparation method comprises the following steps: respectively activating and propagating compound strains to obtain a seed solution, adding sterilized red onion powder and the seed solution of the compound strains into a liquid culture medium, and carrying out fermentation culture to obtain a fermentation solution; inactivating and filtering the fermentation liquor, and taking filtrate, so as to obtain the eleutherine americana fermentation liquor. The compound strains comprise penicillium oxalicum, aspergillus sydowii and rhizopus. According to the invention, symbiotic fermentation is carried out on the compound strain and iris eleutherine according to a specific ratio, and the strain can be better matched with an eleutherine plant, so that the release of an eleutherine naphthoquinone derivative is remarkably enhanced, and the prepared eleutherine eleutherine fermentation liquor has good effects of resisting oxidation, inhibiting inflammatory aging and whitening, and has a good application prospect. The method can be widely applied to the fields of cosmetics, food health-care products and the like, and has social significance and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and specifically relates to a red onion fermentation liquid with anti-inflammatory, aging and whitening effects, and a preparation method and application thereof. Background Art

[0002] Red onion (Eleutherine plicata) is a plant of the genus Eleutherine in the family Iridaceae, and is a traditional folk medicinal plant of ethnic minorities in southwestern regions such as Yunnan and Guangxi. Studies have shown that the red onion of the family Iridaceae has a variety of pharmacological effects, such as vasodilation, antibacterial, antifungal, antiviral, anti-inflammatory analgesic, antioxidant, antitumor, and a series of activities such as inhibition of tyrosinase activity. For example, patents CN108671127B and CN103110761B use ethanol aqueous solution for multiple extractions, and decompression and recovery of ethanol to no alcohol taste. The resulting red onion extract extract has a brain protective effect of reducing cerebral infarction after hypoxic-ischemic brain damage in newborn mice, and enhances the sexual vitality function of normal male rats. However, due to unclear understanding of these specific active ingredients, the extraction and separation process is too simple, and the content of active ingredients in the extract is uncertain, which cannot meet the needs of large-scale industrial production.

[0003] According to the pharmacological activity screening, the red shallot extract of Iridaceae has many aspects of pharmacological activity, so more and more scholars are studying the active ingredients of red shallot of Iridaceae. According to literature reports, the chemical composition of red shallot of Iridaceae mainly contains compounds such as naphthols, naphthoquinones, anthraquinones, glucosides, phenolic acids, steroids, etc. In recent years, research has focused on 39 compounds (identified substances) of naphthols, naphthoquinones, and anthraquinones. CN109820925A provides a preparation method and application of total anthraquinones of red shallots. After pure water extraction and concentration, the total anthraquinone substances of red shallots are obtained by macroporous resin adsorption, ethanol elution, and semi-preparative chromatography column separation, which has antibacterial and vasodilatory effects. Patents CN103651356A, CN111303182A, and CN116535451A provide methods for separating and purifying iso-onion methyl, naphthoquinone dimer, and naphthol dimer. The onion powder is extracted with methanol, and then extracted with petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence to obtain different polar parts, which are then separated by silica gel column chromatography, and gradient elution and recrystallization are used to obtain different active ingredients of onion. Since the monomer components that have been separated and identified in onion are limited, and it is difficult to obtain monomer components with sufficient experimental amounts, the research on the chemical composition and pharmacological activity of onion is still in its early stages. Combined with literature research, naphthoquinone derivatives in onion, onion methyl, onion ethyl, and onion isoethyl, have significant anti-inflammatory, antioxidant, and melanin-inhibiting effects. Therefore, it is necessary to develop a high value-added processing and extraction method for onion of Iridaceae.

[0004] However, so far, there has been no systematic research on the processing and utilization of Iridaceae red onions at home and abroad, and the extraction technology of Iridaceae red onions is not mature enough. The methods of using water, methanol or ethanol for extraction and chromatographic column separation and chromatography have low production capacity and efficiency, resulting in the inability of Iridaceae red onion products to be applied and promoted on a large scale in the market. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a red onion fermented liquid with anti-inflammatory aging and whitening effects, and a preparation method and application thereof. By adopting specific composite bacteria and fermentation process conditions, the red onion fermented liquid with good antioxidant, anti-inflammatory aging and whitening effects is obtained, thereby improving its nutritional utilization value, expanding its application range in the fields of food or cosmetics, and having important economic and social value.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing shallot fermentation liquid, comprising the following steps:

[0008] (1) activating and expanding the composite strains to obtain seed liquid, then adding the sterilized shallot powder and the seed liquid of the composite strains into a liquid culture medium, fermenting and culturing to obtain a fermentation liquid;

[0009] (2) inactivating the fermentation liquid of step (1), filtering, and taking the filtrate to obtain the shallot fermentation liquid;

[0010] The composite strain comprises Penicillium oxalicum, Aspergillus polymorpha and Rhizopus.

[0011] The oxalicum penicillium and polytoxin Aspergillus described in the present invention are both endophytic fungi with antibacterial activity separated from the leaves of red onions of the Iridaceae family. They can produce pectinase and cellulase under certain fermentation conditions, hydrolyze the plant cell walls of red onions to break them, thereby efficiently releasing the active ingredients of red onion naphthoquinone derivatives, and hydrolyzing cellulose into oligopolysaccharides and glucose; Rhizopus is an endophyte in the roots of red onions of the Iridaceae family, and is one of the common industrial ethanol-producing fungi. It can convert glucose into ethanol under micro-aerobic conditions, thereby increasing the solubility and extraction rate of red onion naphthoquinone derivatives in the fermentation liquid.

[0012] Through experimental research, it was found that the present invention uses the above-mentioned three specific composite strains to carry out symbiotic fermentation of red onions. The three strains cooperate with each other and can better adapt to the red onion plants, thereby significantly enhancing the release of red onion naphthoquinone derivatives, significantly improving the content of active ingredients, and further improving its nutritional utilization value.

[0013] Preferably, the concentrations of Penicillium oxalicum, Aspergillus polymorpha and Rhizopus in the seed solution are all 1×107 -1×10 9 CFU / mL.

[0014] Preferably, the total inoculation amount of the composite strain is 4-10% of the total volume of the liquid culture medium; the inoculation ratio of the Penicillium oxalicum, Aspergillus niger and Rhizopus spp. is Penicillium oxalicum: Aspergillus niger: Rhizopus spp. = (1-3): (1-3): (2-4).

[0015] Preferably, the inoculation ratio of the Penicillium oxalicum, Aspergillus niger and Rhizopus spp. is Penicillium oxalicum: Aspergillus niger: Rhizopus spp.=2:1:3.

[0016] Experimental research found that when Penicillium oxalicum, Aspergillus niger and Rhizopus spp. were used in the above-mentioned optimal ratio to carry out symbiotic fermentation of shallot powder, the content of active ingredients in the shallot fermentation liquid - isoallione B, allione B and allione A - was the highest, thus exerting the best antioxidant, anti-inflammatory aging and whitening effects.

[0017] Preferably, in step (1), the fermentation culture conditions are: microaerobic fermentation, aeration volume of 0.1-0.3 vvm, fermentation at 25-32°C for 72-96 hours, rotation speed of 100-300 r / min, and pH of 4.0-6.0.

[0018] Experimental research found that when the above-mentioned preferred fermentation process conditions are used for red onion fermentation, it is more conducive to the metabolism of the three specific composite strains. The red onion cell walls are hydrolyzed by high-yield enzymes such as pectinase and cellulase, thereby effectively releasing red onion naphthoquinone derivatives and increasing their solubility in the fermentation broth, thereby significantly improving the extraction efficiency of the active ingredients in red onions.

[0019] Preferably, the liquid culture medium comprises the following components: 10-30 g of bean dregs, 10-30 g of bagasse, 3-10 g of lemon juice, and water is added to 1 kg.

[0020] The bean dregs and bagasse are crushed and passed through a 50-80 mesh sieve; the bean dregs contain a large amount of protein, dietary fiber, amino acids, vitamins, minerals and other nutrients, which provide a natural nitrogen source for the growth of the strain; the rich polysaccharide components in bagasse are used to provide a natural carbon source for the growth of the strain; and the citric acid in natural lemon juice is used to regulate the pH of the culture medium; the fermentation medium prepared by compounding the above components can fully meet the growth and metabolic needs of the three composite strains, and is more conducive to the release and extraction of active ingredients in red onions.

[0021] Preferably, the mass of the shallot powder is 1-5% of the mass of the liquid culture medium. The particle size of the shallot powder is 100 mesh.

[0022] Preferably, in step (2), the specific method of filtering is: performing coarse filtration and fine filtration in sequence, wherein the coarse filtration includes using a plate and frame filter or a diatomaceous earth filter, and the fine filtration includes using a horizontal centrifuge or a ceramic membrane filter.

[0023] Preferably, the coarse filtration uses a plate-and-frame filter, and the fine filtration uses a plate-and-frame filter. The filtration area of ​​the plate-and-frame filter is 1 m 2 The filtration accuracy is 1.2μm, and the filtration accuracy of the ceramic membrane filter is 0.45μm.

[0024] In the step (2), after taking the filtrate, a preservative is added to obtain the shallot fermentation liquid;

[0025] Preferably, the preservative includes at least one of 1,3-butylene glycol, 1,2-hexanediol, pentylene glycol, p-hydroxyacetophenone, ethylhexylglycerin and caprylhydroxamic acid.

[0026] Preferably, the preservatives include 1,3-butanediol, 1,2-hexanediol and p-hydroxyacetophenone; the mass percentages of the preservatives in the fermentation broth are: 2%-10% of 1,3-butanediol, 0.3%-1% of 1,2-hexanediol and 0.3%-1% of p-hydroxyacetophenone.

[0027] In a second aspect, the present invention provides shallot fermentation liquid obtained by the preparation method.

[0028] In a third aspect, the present invention provides the use of the shallot fermentation liquid in the preparation of cosmetics or health products.

[0029] The cosmetics in the present invention can be common cosmetics such as toner, lotion, essence, cream, facial mask, eye mask, etc., as well as food and health products.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention utilizes the endophytic bacteria of shallots of the Iridaceae family: Penicillium oxalicum, Aspergillus polydophorus and Rhizopus to carry out symbiotic fermentation with shallots of the Iridaceae family, so that the bacteria and the shallot plants can be better adapted to each other, thereby significantly enhancing the release of naphthoquinone derivatives of shallots; the fermentation process is simple and does not use any organic solvent; at the same time, bean dregs, bagasse and lemon juice are used as culture media, and the shallot fermentation liquid is obtained by fermentation using natural plant culture media and endophytic bacteria. The raw materials are green, safe and available, and can be manufactured and promoted on a large scale;

[0032] (2) The present invention obtains a shallot fermentation liquid rich in allium acetone, allium acetone, and allium isoacetone, which has the effects of anti-oxidation, scavenging intracellular active oxygen, inhibiting anti-inflammatory aging, and whitening. It can be widely used in cosmetics or food health products, and has social significance and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatograms are of the shallot fermentation broth (A) and the standard mixed sample (B) prepared in Example 1, wherein 1 is isoallione B, 2 is allione B, and 3 is allione A. DETAILED DESCRIPTION

[0034] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0035] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0036] The oxalicum Penicillium described in the present invention is the Po-5 strain of Penicillium Oxalicum, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M2010350 and a deposit date of December 14, 2010; the polydole Aspergillus is polydole Aspergillus sydowii, which was deposited in the General Microbiological Center of the China General Microbiological Culture Collection Administration (CGMCC) on September 18, 2012 with a deposit number of CGMCC No. 6577; and the root mold is the root mold Q303 strain (Rhizopus Sp.), which is a common sugar-substituting strain and has a strain number of GIM3.132.

[0037] The above strains were commercially available from Beijing Biobo Biotechnology Co., Ltd.

[0038] Example 1

[0039] This embodiment provides a method for preparing shallot fermentation liquid, comprising the following steps:

[0040] (1) Preparation of seed solution: The preserved strains of Penicillium oxalicum, Aspergillus niger, and Rhizopus spp. were taken out from a -80°C refrigerator and cultured on plates for three generations, each for 12 h. The revived strains were then cultured on a shaking table. The final concentrations of Penicillium oxalicum, Aspergillus niger, and Rhizopus spp. in the seed solution were 1×10 8 CFU / mL;

[0041] During the shaking culture, the liquid culture medium includes the following components: 20 g of bean dregs, 30 g of sugarcane bagasse, 5 g of lemon juice, supplemented with deionized water to 1 L, and an initial pH of 5.0; the culture conditions are: aeration 0.3 vvm, temperature 28°C, culture time 24 h, and rotation speed 150 r / min;

[0042] (2) Liquid fermentation process: the bulb of shallot of Iridaceae is dried and crushed to 100 mesh, and then sterilized at 0.1 MPa pressure and 120° C. for 20 min, and then 5% of shallot powder is added to the liquid culture medium with the same proportion as above, the addition amount of Penicillium oxalicum seed liquid is 2%, the addition amount of Aspergillus polydorum seed liquid is 1%, and the addition amount of Rhizopus seed liquid is 3%, and fermentation is performed to obtain a fermentation liquid;

[0043] The fermentation process conditions are: microaerobic fermentation, ventilation volume 0.1vvm, fermentation temperature 28°C, rotation speed 150r / min, and fermentation time 72h;

[0044] (3) Inactivation: After the fermentation is completed, the fermentation liquid is inactivated at high temperature, and the optimal temperature is 90°C for 30 min;

[0045] (4) Filtration: The filtration equipment used includes a coarse filter plate and frame filter with a filtration area of ​​1m 2 , the filtration accuracy is 1.2μm, and then through the precision filtration ceramic membrane filter, the filtration accuracy is 0.45μm, to obtain the fermentation filtrate;

[0046] (5) Preparation: Then, a preservative is added to the fermented filtrate, wherein the preservative ingredients and contents are 3% 1,3-butanediol, 0.8% 1,2-hexanediol and 0.3% p-hydroxyacetophenone, to obtain the shallot fermentation liquid.

[0047] Example 2

[0048] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0049] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 1% of Penicillium oxalicum, 1% of Aspergillus polymorpha and 2% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0050] Example 3

[0051] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0052] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 1% of Penicillium oxalicum, 1% of Aspergillus polymorpha and 4% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0053] Example 4

[0054] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0055] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 2% of Penicillium oxalicum, 2% of Aspergillus polymorpha and 2% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0056] Example 5

[0057] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0058] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 1% of Penicillium oxalicum, 2% of Aspergillus polymorpha and 3% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0059] Example 6

[0060] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0061] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 3% of Penicillium oxalicum, 3% of Aspergillus polymorpha and 4% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0062] Example 7

[0063] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0064] The fermentation time is different. The fermentation time in this embodiment is 96 hours, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0065] Example 8

[0066] The present embodiment provides a method for preparing shallot fermentation liquid, which is different from that of Example 1 in that the liquid culture medium comprises the following components during shaking culture: 20 g of bean dregs, 30 g of sugarcane bagasse, 10 g of lemon juice, supplemented with deionized water to 1 L, with an initial pH of 4.0, and the remaining fermentation processes and post-fermentation treatment conditions are the same.

[0067] Example 9

[0068] This embodiment provides a method for preparing shallot fermented liquid, which is different from that of embodiment 1 in that:

[0069] The fermentation process conditions are as follows: oxygen ventilation volume 0.3 vvm, fermentation temperature 28°C, rotation speed 150 r / min, fermentation time 72 h. The rest of the fermentation process and post-fermentation treatment conditions are the same.

[0070] Comparative Example 1

[0071] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0072] The composite strain and inoculation amount ratio are different, and the strain and inoculation amount are: 6% of Penicillium oxalicum, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0073] Comparative Example 2

[0074] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0075] The composite strain and inoculation amount ratio are different, and the strain and inoculation amount are: 6% of Aspergillus polymorpha, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0076] Comparative Example 3

[0077] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0078] The composite strain and inoculation amount ratio are different, and the strain and inoculation amount are: Rhizopus 6%, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0079] Comparative Example 4

[0080] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0081] The composite strain and inoculation ratio are different, and the strain and inoculation ratio are: 3% of Penicillium oxalicum and 3% of Aspergillus polymorpha, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0082] Comparative Example 5

[0083] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0084] The composite strain and inoculation amount ratio are different, and the strain and inoculation amount are: 3% of Penicillium oxalicum and 3% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0085] Comparative Example 6

[0086] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0087] The composite strain and inoculation amount ratio are different, and the strain and inoculation amount are: 3% of Aspergillus polymorpha and 3% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0088] Comparative Example 7

[0089] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0090] The inoculation ratio of the composite strain is different, and the inoculation ratio of the strain is: 0.5% of Penicillium oxalicum, 4% of Aspergillus polymorpha and 1.5% of Rhizopus, and the other fermentation processes and post-fermentation treatment conditions are the same.

[0091] Comparative Example 8

[0092] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0093] The fermentation medium is different. The liquid medium used in the fermentation of this comparative example includes the following components: 30 g of oligofructose, 10 g of peptone, 10 g of yeast powder, 1.5 g of MgSO4·7H2O, 2 g of K2HPO4, 0.05 g of MnSO4·4H2O, and 1 L of deionized water, with an initial pH of 7.0. The rest of the fermentation process and post-fermentation treatment conditions are the same.

[0094] Comparative Example 9

[0095] This comparative example provides a method for preparing shallot fermented liquid, which is different from Example 1 only in that:

[0096] The fermentation process conditions are: anaerobic fermentation, fermentation temperature of 28°C, rotation speed of 150r / min, and fermentation time of 72h. The remaining fermentation process and post-fermentation treatment conditions are the same.

[0097] Comparative Example 10

[0098] This comparative example provides a method for preparing a shallot extract, which differs from Example 1 only in that:

[0099] This comparative example adopts a conventional pure water extraction method to prepare shallot extract, specifically, the shallot of Iridaceae is dried and crushed to 100 mesh, then sterilized at 0.1Mpa pressure and 120°C temperature for 20min, then 5% of shallot powder is added to deionized water, and extracted at 80°C, 150r / min uniform stirring, condensation reflux conditions for 4h to obtain shallot extract. The subsequent filtering and blending treatment conditions are the same.

[0100] Effect Example 1

[0101] In this effect example, the shallot fermented liquid prepared in Examples 1-9 and Comparative Examples 1-9 and the shallot extract prepared in Comparative Example 10 were used as samples, and the contents of allium A, allium B, and isoallium B therein were determined. The specific method is as follows:

[0102] Phenomenex Luna C18 column (250mm×4.6mm, 5μm) was used; the mobile phase was acetonitrile (A)-water (B) gradient elution (0min→10min, 40%A→50%A; 10min→15min, 50%A→80%A; 15min→22min, 80%A→40%A), flow rate 1mL / min, detection wavelength 254nm, column temperature 25℃, injection volume 10μL. Reference substance stock solution preparation: Accurately weigh reference substances 5mg of iso-onion B, 10mg of onion B, and 10mg of onion A into the same 100mL brown volumetric flask, dissolve and dilute to the mark with ethanol, shake well, and obtain mixed solutions with concentrations of 5, 10, and 10mg / 100g, respectively. Under the above chromatographic conditions, the retention times of iso-allionein, allionein B and allionein A were 11.7, 12.5 and 14.2 min, respectively. Figure 1 According to the above method, the contents of isoallione B, allione B and allione A in Examples 1-9 and Comparative Examples 1-10 were tested respectively, and the results are shown in Table 1.

[0103] Table 1

[0104]

[0105] From the results in Table 1, it can be seen that the contents and total amount of isoallione B, allione B, and allione A in the shallot fermentation broth obtained by the specific composite strain preparation method of Examples 1-9 using the present invention are significantly higher than those in Comparative Examples 1-10, proving that the combination of mixed strains of Penicillium oxalicum, Aspergillus polydorus and Rhizopus is most effective in the release of shallot naphthoquinone derivatives, and when the composite strain is inoculated with an optimal ratio of 2% Penicillium oxalicum, 1% Aspergillus polydorus and 3% Rhizopus for symbiotic fermentation of shallots, the active ingredients of shallot naphthoquinone derivatives in the obtained shallot fermentation broth are optimal. When the strains described in Comparative Examples 1-6 only use one or any two of Penicillium oxalicum, Aspergillus niger and Rhizopus, the contents of isocarpine B, carmine B and carmine A in the obtained fermented product are significantly reduced, indicating that the three specific strains described in the present invention cooperate with each other during symbiotic fermentation and can better adapt to the carmine plant, thereby synergistically enhancing the content of active ingredients in the fermented product, and none of the three is dispensable. When the inoculation ratio of the composite strain described in Comparative Example 7 exceeds the specified range of the present invention, the content of active ingredients in the obtained carmine fermented liquid is also reduced. The above results show that the specific composite strains described in the present invention can better exert the release effect of the active ingredients in carmine when they are within the specified range, and significantly improve the extraction efficiency. In addition, conventional fermentation medium is used to prepare carmine fermented liquid in Comparative Example 8 , the contents of isoallione B, allione B and allione A in the obtained fermented product also decreased, indicating that when the culture medium with the specific components and the initial pH of the composite strain are used under slightly acidic conditions, it is more conducive to the release and extraction of active ingredients in shallots; Comparative Example 9 adopts anaerobic fermentation, while Penicillium oxalicum, Aspergillus polydophorus and Rhizopus are all aerobic bacteria, and thus the activity of the strains is poor under anaerobic conditions; Comparative Example 10 adopts conventional hydrothermal extraction method to prepare shallot extract, wherein the contents of isoallione B, allione B and allione A of naphthoquinone derivatives of shallot are significantly lower than those in the embodiment, indicating that the fermentation process using the composite strain of the present invention is significantly better than the conventional hydrothermal extraction method in extracting the active ingredients of naphthoquinone derivatives in shallots, and is more conducive to large-scale promotion and application.

[0106] Effect Example 2

[0107] 1. MTT analysis:

[0108] Cell culture: Human skin keratinocytes (HaCaT) were cultured in DMEM high-glucose medium containing 10% FBS in a cell culture incubator at 37°C, with an air content of 95% (V / V) and a CO2 content of 5% (V / V). The medium was changed once every 2 days during the cell culture period.

[0109] MTT test: HaCaT cells were cultured at 10 4The cells were inoculated at a density of 1 / well in a 96-well cell culture plate, and 100 μL of test samples of different concentrations were added to each well. The cells were incubated in an incubator for 24 hours, and the cell morphology and characteristics were observed under a phase contrast inverted microscope. 100 μL of cell culture fluid was added to each well of the blank control group; 100 μL of culture fluid containing the test substance at the corresponding concentration was added to each well of the sample group; and the zero adjustment group had no cell inoculation in the wells, and only 100 μL of cell culture fluid was added.

[0110] After 24 hours of incubation, discard the culture medium, add 100 μL of the prepared MTT solution (0.5 mg / mL) to each well, incubate in the incubator for 4 hours, remove the supernatant, and add 150 ml / well of DMSO. After shaking on a shaker at a constant temperature (37°C) for 10 minutes, measure the OD value (OA570nm) with an enzyme marker, and calculate the relative cell viability (Viability) of each group according to the following formula:

[0111]

[0112] In the formula, OD TA is the test value of the sample group, OD Blank is the test value of the withering group, OD NC This is the test value of the blank control group.

[0113] The results are shown in Table 2.

[0114] Table 2

[0115]

[0116] The MTT test results show that the red onion fermentation liquid of the present invention has no obvious cytotoxicity to cells within the test concentration range of 10%; the cell survival rate is greater than 75% according to the USP toxicity grading method, and the cytotoxicity level is 0 or 1, wherein the cytotoxicity of Examples 3, 6 and 7 is slightly stronger than that of other examples, which may be because Rhizopus produces ethanol by fermentation under anaerobic conditions, and the high content of Rhizopus or the long fermentation time will lead to an increase in ethanol content, thereby increasing the irritation to cells. While the cell survival rate of Comparative Example 3 is lower than 75% at the 10% test concentration, and the cytotoxicity level is 2, and there is a certain cytotoxicity, which may be because the content of Rhizopus is too high, which will lead to an increase in ethanol content, thereby increasing the irritation to cells. According to the cell MTT test of the samples prepared in the above embodiments and comparative examples, the efficacy is further evaluated.

[0117] 2. Test for removing intracellular reactive oxygen species (ROS):

[0118] Ultraviolet radiation will increase the reactive oxygen species in cells, leading to skin inflammation and increased skin melanin. The present invention uses UVA-induced cell model: human skin keratinocytes (HaCaT) are digested and made into cell suspension, which is then inoculated into a 96-well plate with 1.0×10 5 -2.0×10 5 The cells were cultured using DMEM medium. After 24 hours of culture, the medium was replaced, and a blank group (PBS buffer was added, no light was irradiated), a control group (PBS buffer was added, light was irradiated), and an experimental group (samples of Examples 1-9 and Comparative Examples 1-10 were added, respectively, in an amount of 1% and 3% of the amount of the cell culture medium, i.e., diluted with PBS buffer) were set up. The culture was continued for 24 hours, and then UVA light was given to the control group and the experimental group (the light dose was 6-10 J / cm 2 After the illumination, the cell culture medium was removed, the cells were washed three times with PBS, 100 μL of 10 μmol / L DCFH-DA was added to each well, and the cells were placed in a CO 2 Incubate in an incubator for 20 minutes. After incubation, wash with PBS three times, then add serum-free DMEM culture medium again, use a fluorescence microplate reader to detect ROS content at an excitation wavelength of 488nm and an emission wavelength of 525nm. The relative level of cellular ROS is the ratio of the OD value of the experimental group (or the OD value of the control group) to the OD value of the blank group. The specific results are shown in Table 3.

[0119] Table 3

[0120]

[0121]

[0122] As can be seen from Table 3, ultraviolet irradiation will cause an increase in intracellular reactive oxygen species. Compared with the normal intracellular ROS level in the blank group, the relative content of intracellular ROS increased to 155.3% after ultraviolet irradiation. The intracellular ROS level was significantly reduced in the sample group prepared using 1% or 3% of the embodiment and the comparative example, indicating that the red onion fermentation liquid and the extract have antioxidant properties, can remove intracellular ROS, and reduce the damage of light damage. Further analysis of the data of the embodiment and the comparative example shows that the red onion fermentation liquid prepared by the embodiment 1-9 has a lower relative level of ROS in HaCaT cells after light damage than the comparative example group, indicating that the red onion fermentation liquid prepared by the process method of the present invention has better antioxidant properties, can remove intracellular reactive oxygen species, and reduce the damage of light damage. Among them, when 3% of the red onion fermentation liquid prepared in Example 1 is added, the relative level of ROS in HaCaT cells after light damage is as low as 102.2%, which is close to the level of normal cells. The content of intracellular ROS in Comparative Example 10 was the highest, indicating that the effect of the red onion extract prepared by the water extraction method in removing intracellular ROS was significantly worse than that of the red onion fermentation liquid prepared by the fermentation process of the present invention.

[0123] 3. Test for inhibiting the release of inflammatory factors:

[0124] As we age, a pro-inflammatory state appears in the body, which is named inflammatory aging. Inflammageing is a sterile, low-grade chronic inflammation that gradually increases with age. It is not only a sign of aging, but also can lead to health decline, physical weakness, and aging-related diseases. We systematically evaluate and analyze the efficacy of the red onion fermented liquid described in this patent in inhibiting inflammatory aging.

[0125] Human skin keratinocytes (HaCaT) cells were cultured as described above. The original culture medium in the wells was discarded, and 30 μg / mL of lipopolysaccharide was added to each well: the blank group was complete culture medium, the model group was lipopolysaccharide group, and the sample group contained lipopolysaccharide and samples at concentrations of 0.5%, 1%, and 3%, and cultured for 24 hours. Replace with fresh culture medium and culture for 24 hours. Aspirate the culture medium, centrifuge to obtain the supernatant, and use the ELISA method to test the TNF-α, IL-6, and IL-1β content in the supernatant. The relative levels of intracellular TNF-α, IL-6, and IL-1β are the ratios of the content in the experimental group to that in the blank group. The specific results are shown in Tables 4-6.

[0126] Table 4 TNF-α relative content (%)

[0127]

[0128] Table 5 IL-6 relative content (%)

[0129]

[0130]

[0131] Table 6 IL-1β relative content (%)

[0132]

[0133] As shown in Tables 4-6, LPS lipopolysaccharide can cause a significant increase in intracellular inflammatory factors. Compared with the normal intracellular inflammatory factor levels in the blank group, the relative contents of intracellular TNF-α, IL-6, and IL-1β increased to 195%, 212%, and 190% after LPS treatment, respectively. The sample groups prepared using 0.5%, 1%, and 3% of the embodiments and comparative examples showed that the intracellular TNF-α, IL-6, and IL-1β levels were significantly reduced, indicating that the red onion fermentation liquid and extract can inhibit the release of cytokines. Further analysis of the data of the embodiments and comparative examples shows that the relative contents of inflammatory factors TNF-α, IL-6, and IL-1β after the red onion fermentation liquid prepared in Examples 1-9 acted on the cells were lower than those in the comparative example group, indicating that the red onion fermentation liquid prepared by the process method of the present invention has better inhibitory effect on cell inflammatory response. Among them, when 3% of the red onion fermentation liquid prepared in Example 1 was added, the relative levels of TNF-α, IL-6, and IL-1β in cells were as low as 102.2%, 106.1%, and 103%, which are close to the normal cell levels. The content of intracellular inflammatory factors in the water extraction method of Comparative Example 10 was the highest, indicating that the effect of the red onion extract prepared by the water extraction method in inhibiting inflammatory response was significantly worse than that of the red onion fermentation liquid prepared by the fermentation process of the present invention.

[0134] 4. Test to promote the production of type I collagen:

[0135] Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen is synthesized intracellularly by dermal fibroblasts and secreted to the extracellular space. Under the action of terminal procollagen peptidase, the terminal peptide is separated and polymerized to form collagen fibers. Human dermal fibroblasts can be used as a cell model to study the effect of cosmetics on improving the content of type I collagen. By measuring the upregulation rate of type I collagen content after administration of the test substance, the blank control and the test substance, the efficacy of the test substance in promoting collagen synthesis is evaluated. It is often used as one of the indicators for in vitro evaluation of the firming and anti-wrinkle effects of cosmetics. The specific principle is: after type I collagen specifically binds to the collagen antibody coated on the ELISA plate, it binds to the anti-type I collagen antibody with a substrate label. After the substrate is catalyzed by the enzyme, a colored product is generated. The content of type I collagen is positively correlated with the depth of the color of the colored product. The optical density (OD value) is measured at a wavelength of 450nm using an ELISA instrument to calculate the content of type I collagen.

[0136] The specific test methods are as follows:

[0137] (1) Cell culture: Prepare cell suspension 24 h before the experiment and inoculate the cell suspension into a 96-well cell culture plate, with 0.1 mL per well and 5000 cells per well, and culture for 24 h;

[0138] (2) Exposure: Discard the original culture medium in the wells, add 0.15 mL of test samples of different concentrations to each well, and the concentrations of red onion fermentation liquid are 1% and 3%, respectively, and incubate in an incubator for 24 h;

[0139] (3) Sample collection: After centrifugation, the supernatant was collected and used fresh or stored at -20°C for later use;

[0140] (4) ELISA test: Follow the instructions of the kit and measure the absorbance at a wavelength of 450 nm on an ELISA reader.

[0141] The relative content of type I collagen (CoL-I) was calculated according to the following formula:

[0142] Relative content of type I collagen (%) = (Col content of sample test group / Col content of blank control group)

[0143] ×100%

[0144] The specific results are shown in Table 7.

[0145] Table 7

[0146]

[0147] The effects of different addition amounts of shallot fermentation broth and shallot extract on the synthesis of type I collagen are shown in Table 7. As can be seen from Table 7, the relative content of type I collagen in cells acted on by the shallot fermentation broth prepared in the embodiment of the present invention is higher than that of the fermentation broth and extract prepared in the comparative example, and has good effects of activating cells to secrete collagen and firming and anti-wrinkle. Among them, Example 1 has the best effect. When 1% is added, the relative content of type I collagen in cells can be increased by 70%, and when 3% is added, the relative content of type I collagen in cells can be increased by 110%. The content of type I collagen in cells by the water extraction method of comparative example 10 is the lowest, indicating that the effect of the shallot extract prepared by the water extraction method in promoting type I collagen is significantly worse than that of the shallot fermentation broth prepared by the fermentation process of the present invention.

[0148] 5. Tyrosinase inhibition test:

[0149] Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, which mainly affects the production of melanin by affecting the conversion of tyrosine to DOPA and the oxidation of DOPA to DOPAQUINONE. The principle is that tyrosine or DOPA is converted into DOPAQUINONE under the action of tyrosinase. This reaction is a color reaction. The inhibition rate of tyrosinase activity by different brightening agents can be determined by colorimetry.

[0150] (1) Reagents: tyrosinase (25 kU); L-dopa, ≥98%; α-arbutin, ≥99%

[0151] (2) Reagent preparation:

[0152] Solution A (0.05 mol / L Na2HPO4 solution): weigh 7.099 g of disodium hydrogen phosphate and add distilled water to 1000 mL;

[0153] Solution B (0.05 mol / L KH2PO4 solution): weigh 6.803 g of potassium dihydrogen phosphate and add distilled water to 1000 mL;

[0154] Preparation of 0.05mol / L PBS (pH 6.8): Mix 50ml of solution A and 50ml of solution B;

[0155] Tyrosinase solution: Prepared with pH 6.8 PBS buffer, 100 U / mL, prepared immediately before use;

[0156] DOPA solution: weigh 0.04 g DOPA and dissolve it in 40 mL pH 6.8 PBS buffer and store in dark;

[0157] α-Arbutin: pH 6.8 PBS buffer was diluted sequentially to a 100 μM solution.

[0158] (3) Instruments and equipment: analytical balance (accurate to 0.0001 g); 1 L volumetric flask; 50 mL measuring cylinder; 1000 μL pipette; constant temperature water bath; UV-visible spectrophotometer.

[0159] (4) Determination steps: Sample, 0.05 M PBS (pH 6.8), and tyrosinase solution were added to the test tube in sequence. The composition (mL) of the reaction solution is shown in Table 8. Incubate in a 37°C water bath for 10 min, add 2 mL of DOPA solution, react for 5 min, and measure the absorbance at 475 nm;

[0160] The calculation formula is: tyrosinase inhibition rate (%) = [1-(ODC-ODD) / (ODA-ODB)] × 100%;

[0161] in:

[0162] ODA——absorbance value of enzyme solution control group;

[0163] ODB——Absorbance value of enzyme solution control blank group;

[0164] ODC——absorbance value of sample group;

[0165] ODD——Absorbance value of blank control of sample group.

[0166] The specific results are shown in Table 9.

[0167] Table 8

[0168]

[0169]

[0170] Table 9

[0171]

[0172] The results are shown in Table 9. Adding 5% of the fermented shallot broth prepared in Examples 1-9 can significantly inhibit tyrosinase, with an inhibition rate of more than 70%; adding 10% of the fermented shallot broth can achieve an inhibition rate of more than 95% on tyrosinase, that is, it has a good skin-lightening effect; adding 5% of the fermented shallot broth prepared in Comparative Examples 1-9 has an inhibition rate of more than 50% on tyrosinase; and adding 10% of the fermented shallot broth has an inhibition rate of more than 70% on tyrosinase. According to the results, the inhibition rate of tyrosinase activity in Examples 1-9 is significantly higher than that in Comparative Examples 1-10, that is, the inhibition rate of tyrosinase in Examples 1-9 is at least 15% higher than that in Comparative Examples. The raw material prepared by the water extraction method in Comparative Example 10 has the worst inhibitory effect on tyrosinase, and the inhibition rate of tyrosinase in the amount of 10% added is only 50.4%, indicating that the inhibitory effect of the shallot extract prepared by the water extraction method on tyrosinase is significantly worse than that of the shallot fermented broth prepared by the fermentation process of the present invention.

[0173] From the above results, it can be seen that the composition and proportion of the composite strains used for fermenting shallots provided by the present invention, and the configuration of the fermentation medium have a great influence on the content of the active ingredients-onion A, onion B and onion isoethyl, as well as the effects of clearing intracellular ROS, inhibiting inflammatory factors, inhibiting tyrosinase activity and promoting collagen production. Selecting a single strain or an inappropriate strain ratio or the configuration of the liquid culture medium will reduce the content of the active ingredients and the efficacy in the final fermentation product.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing shallot fermentation liquid, characterized in that: The following steps are involved: (1) activating and expanding the composite strains to obtain seed liquid, then adding the sterilized shallot powder and the seed liquid of the composite strains into a liquid culture medium, fermenting and culturing, and obtaining a fermentation liquid; (2) inactivating the fermentation liquid of step (1), filtering, and taking the filtrate to obtain the shallot fermentation liquid; The composite strain comprises Penicillium oxalicum, Aspergillus polymorpha and Rhizopus.

2. The preparation method according to claim 1, characterized in that The concentrations of Penicillium oxalicum, Aspergillus polymorpha and Rhizopus in the seed solution were all 1×10 7 -1×10 9 CFU / mL.

3. The preparation method according to claim 1, characterized in that: The total inoculation amount of the composite strain is 4-10% of the total volume of the liquid culture medium; The inoculation ratio of the Penicillium oxalicum, Aspergillus niger and Rhizopus spp. is Penicillium oxalicum: Aspergillus niger: Rhizopus spp. = (1-3): (1-3): (2-4).

4. The preparation method according to claim 3, characterized in that: The inoculation ratio of the Penicillium oxalicum, Aspergillus niger and Rhizopus spp. is Penicillium oxalicum: Aspergillus niger: Rhizopus spp.=2:1:

3.

5. The preparation method according to claim 1, characterized in that: In the step (1), the fermentation culture conditions are: microaerobic fermentation, aeration volume of 0.1-0.3 vvm, fermentation at 25-32°C for 72-96 hours, rotation speed of 100-300 r / min, and pH of 4.0-6.

0.

6. The preparation method according to claim 1, characterized in that: The liquid culture medium comprises the following components: 10-30 g of bean dregs, 10-30 g of sugarcane bagasse, 3-10 g of lemon juice, and water is added to 1 kg.

7. The preparation method according to claim 1, characterized in that: The mass of the shallot powder is 1-5% of the mass of the liquid culture medium.

8. The preparation method according to claim 1, characterized in that: In the step (2), the specific method of filtering is: performing coarse filtration and fine filtration in sequence, wherein the coarse filtration includes using a plate and frame filter or a diatomaceous earth filter, and the fine filtration includes using a horizontal centrifuge or a ceramic membrane filter.

9. The shallot fermented liquid obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the shallot fermented liquid according to claim 9 in the preparation of cosmetics or health products.

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