A fermentation strain composition, fermentation oil and preparation method and application thereof

Abstract

The present invention belongs to the technical field of microbial fermentation, and specifically relates to a fermented strain composition, fermented oil, and a preparation method and application thereof. The raw materials of the fermented oil include oil, bumblebee Candida aurea and a second strain; the second strain is selected from at least one of Staryi Lipozyme, Saccharomyces cerevisiae, Rhodosporidium involucrata, Ganoderma lucidum and Cicadae Convolvulus. The preparation method thereof comprises first adding oil to a culture medium, inoculating bumblebee Candida aurea and a second strain, and fermenting; after the fermentation is completed, sterilizing and filtering to obtain the oil. The present invention provides a combination of fermented strains of oil raw materials that can improve the stability of oil and significantly reduce the amount of oil emulsifier used to improve the transdermal absorption effect of oil as an active substance carrier, and provides a basis for its application in cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a fermentation strain composition, fermentation oil, and a preparation method and application thereof. Background Art

[0002] Whether it is natural animal or plant oils or synthetic oils, the crude oil contains a certain amount of free fatty acids, and the content of free fatty acids is further affected by freshness and extraction process. Free fatty acids are the decomposition products of triglycerides. They are chemically active and unstable. They are very easy to oxidize when in contact with oxygen, light, metal ions, etc. Once the oxidation reaction of oil is started, it will quickly cause fatty acid rancidity, resulting in the generation of unpleasant odors and lipid oxidation products that are harmful to human health.

[0003] Therefore, the free fatty acid content of oil products must be reduced to below safety standards during use and storage.

[0004] There are two common methods to remove free fatty acids from oils and fats. One is alkali refining, which removes free fatty acids by alkali treatment; or esterification, which converts free fatty acids into triglycerides to reduce the content of free fatty acids. Due to the serious environmental pollution caused by alkali refining and deacidification, its use has been gradually reduced. The second is physical and chemical methods, which mainly include steam distillation and liquid-liquid extraction. Steam distillation is used to remove odorous substances and free fatty acids, liquid-liquid extraction is used for decolorization and removal of free fatty acids, and physical deacidification is to separate and remove free fatty acids under high temperature and high vacuum conditions. The deacidification temperature is generally above 240°C, which is not suitable for some oil raw materials containing heat-sensitive oil components.

[0005] Therefore, it is urgent to develop a new method for preparing fermented oil with low fatty acid content. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a fermentation strain composition, fermentation oil, and a preparation method and application thereof.

[0007] In order to achieve the purpose of the present invention, the technical solution adopted is as follows:

[0008] A fermented bacterial composition comprises Candida bumblebee and a second bacterial species, wherein the second bacterial species is selected from one of Lipomyces staryi, Saccharomyces cerevisiae, Rhodosporidium involucrata, Ganoderma lucidum and Cicadae concolor.

[0009] Preferably, the strain number of the brewer's yeast is: CGMCC 2.3880, purchased from the China General Microbiological Culture Collection Center;

[0010] Preferably, the strain number of the red winter spore yeast is CICC 31300, purchased from the China Industrial Microbiological Culture Collection Center;

[0011] Preferably, the bumblebee Candida species number is ATCC 22214, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.;

[0012] Preferably, the strain number of the Starch yeast is: CGMCC 2.2291, purchased from the China General Microbiological Culture Collection Center;

[0013] Preferably, the strain number of the Ganoderma lucidum is CICC 14029, purchased from the China Industrial Microbiological Culture Collection Management Center;

[0014] Preferably, the strain number of the cicada fungus is: CFCC 81170, which is purchased from the China Forestry Microbial Culture Collection Management Center.

[0015] Preferably, the concentration of the bumblebee Candida and the second bacterial species is 0.5×10 8 -5×10 9 CFU / ml, preferably 1×10 8 -5×10 9 CFU / ml.

[0016] Preferably, the bumblebee Candida and the second bacterial species are cultured in seed culture medium respectively and fermented at 30° C. to corresponding concentrations.

[0017] Preferably, the seed culture medium is composed of 0.5-2.0 g of glucose, 0.5-2.0 g of peptone (supplied by Beijing Aoboxing Company), 0.1-1 g of yeast powder (supplied by Beijing Aoboxing Company) and 100 mL of pure water; after preparation, it is sterilized; the sterilization temperature is 115-125°C, and the sterilization time is 13-35 min.

[0018] The second object of the present invention is to provide the use of the above fermented bacteria composition in reducing the free fatty acid content of oils and fats, and / or the saponification value of oils and fats, and / or promoting the transdermal absorption of active substances.

[0019] The third object of the present invention is to provide a fermented oil with a low fatty acid content, wherein the raw materials of the fermented oil include oil and a fermentation strain composition;

[0020] Preferably, the oil is selected from jojoba seed oil or squalane.

[0021] The fourth object of the present invention is to provide a method for preparing the fermented oil with low fatty acid content, comprising the following steps:

[0022] (1) adding oil to the culture medium, inoculating the fermentation bacteria composition, and fermenting; the oil is selected from jojoba seed oil or squalane;

[0023] (2) After fermentation, sterilize and filter to obtain the product.

[0024] Preferably, the mass of the oil in step (1) is 20%-80% of the mass of the culture medium, and the inoculation amount of the fermentation strain composition is 0.5-2% of the volume of the culture medium.

[0025] Preferably, the fermentation temperature in step (1) is 26-30°C and the fermentation time is 24-144h;

[0026] Preferably, the sterilization temperature in step (2) is 115-125°C, and the sterilization time is 13-35 minutes.

[0027] Preferably, the composition of the culture medium in step (1) includes the following components by weight percentage: 0.5-5% glucose, 0.1-10% peptone, 0.1-1% yeast powder, 0.001-0.01% potassium dihydrogen phosphate, and the rest is water;

[0028] After the culture medium is prepared, it is sterilized at a temperature of 115-125° C. and a sterilization time of 13-35 minutes.

[0029] Preferably, in step (1), the oil is added and then sterilized, and then inoculated after being placed at room temperature; and in step (2), the mixture is filtered and separated using a separatory funnel.

[0030] The fifth object of the present invention is to provide the use of the fermented oil with low fatty acid content or the fermented oil prepared by the above preparation method as a carrier in promoting the transdermal absorption of active components.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The fermentation strain combination of the present invention was used to ferment two representative oils. The fermented oil products obtained were compared with the oil products fermented by a single strain and unfermented crude oil. The acid value of the mixed strain fermented oil products was significantly reduced, the free fatty acid content was significantly reduced, the oil oxidation stability was significantly increased, and the transdermal absorption effect was significantly enhanced. This shows that after fermentation by the fermented strain combination of the present invention, the free fatty acid content in the oil was significantly reduced and the stability was significantly optimized.

[0033] (2) The results of the transdermal absorption experiment showed that the oil fermented by the strain of the present invention has better skin permeability than the unfermented oil and is more easily absorbed by the skin.

[0034] (3) The present invention provides a combination of fermented strains of oil raw materials that can improve the stability of oil and significantly reduce the amount of oil emulsifier used to improve the transdermal absorption effect of oil as an active substance carrier, providing a basis for its application in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the HPLC detection spectrum of paeonol standard. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments. The raw materials used in the following examples are all commercially available conventional raw materials.

[0037] The squalane and jojoba seed oil mentioned in the following examples include but are not limited to these two oils, and are suitable for synthetic oils represented by squalane and natural plant oils represented by jojoba seed oil. Both oils were purchased by this laboratory, and the supplier was Guangzhou Shuangbao Daily Chemical Trading Co., Ltd., and the raw material specifications were cosmetic grade.

[0038] The following strains used in the present invention are all commercially available conventional strains.

[0039] The strain number of brewer's yeast is CGMCC 2.3880, which was purchased from China General Microbiological Culture Collection Center.

[0040] The strain number of red winter spore yeast is CICC 31300, which was purchased from China Industrial Microbiological Culture Collection Center.

[0041] The strain number of bumblebee Candida albicans is ATCC 22214, which was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0042] The strain number of Staryi's oleaginous yeast is CGMCC 2.2291, which was purchased from the China General Microbiological Culture Collection Center.

[0043] The strain number of Ganoderma lucidum is CICC 14029, which was purchased from the China Industrial Microbiology Culture Collection Center.

[0044] The strain number of cicada fungus is: CFCC 81170, which was purchased from the China Forestry Microbial Culture Collection Management Center.

[0045] The basic culture medium is composed of 3% glucose, 1% peptone, 0.5% yeast powder, 0.005% potassium dihydrogen phosphate, and the remainder is ultrapure water, and is obtained after sterilization at 121°C for 20 minutes.

[0046] The strains in the following specific embodiments are cultured in seed culture medium (or in other culture medium suitable for the growth of the strains), and fermented at 30°C to the corresponding concentration. The seed culture medium is composed of 1.0g of glucose (purchased from Xilong Chemical), 1.0g of peptone (Beijing Aoboxing), 0.5g of yeast powder (Beijing Aoboxing), and 100 mL of pure water; sterilized after preparation. The sterilization temperature is 120°C and the sterilization time is 20min.

[0047] Example 1

[0048] Fermentation strain combination: Bombus spp. Candida albicans and Saccharomyces cerevisiae, the strain concentration of Bombus spp. Candida albicans is 1×10 9 CFU / ml, the concentration of Saccharomyces cerevisiae strains is 1×10 9 CFU / ml.

[0049] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at an addition amount of 50% (mass percentage), sterilized and placed at room temperature, inoculated with 1.8% (volume ratio) of Candida bumblebee and 0.2% (volume ratio) of Saccharomyces cerevisiae, fermented at 28°C, cultured for 100h under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20min as a whole, filtered through a 0.45μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0050] Example 2

[0051] Fermentation strain composition: Bombus spp. Candida albicans and Rhodosporidium spp., the strain concentration of Rhodosporidium spp. was 1×10 9 CFU / ml, the concentration of bumblebee Candida species is 1×10 9 CFU / ml.

[0052] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at a dosage of 50%, sterilized and placed at room temperature, inoculated with bumblebee Candida albicans and 0.2% (volume ratio) red winter spore yeast at an inoculation rate of 1.8% (volume ratio), fermented at 28°C, cultured for 100 hours under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0053] Example 3

[0054] Fermentation strain combination: Bombus spp. Candida albicans and Lipomyces staryi, with the strain concentration of Lipomyces staryi being 1×10 9 CFU / ml, the concentration of bumblebee Candida species is 1×10 9 CFU / ml.

[0055] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at an addition amount of 50%, sterilized and placed at room temperature, inoculated with 1.8% (volume ratio) Candida bombus and 0.2% (volume ratio) Lipozyme Staryi at an inoculation amount of 1.8%, fermented at 28°C, cultured for 100 hours under the basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20 minutes as a whole, filtered through a 0.45 μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0056] Example 4

[0057] Fermentation strain combination: Bumblebee Candida and Ganoderma lucidum, the strain concentration of Bumblebee Candida is 1×10 9 CFU / ml, Ganoderma lucidum is obtained by inoculating activated Ganoderma lucidum into seed culture medium at 1% (volume ratio), culturing at 24°C, 170rpm for 10 days until its bacterial balls grow full and fill the seed culture medium.

[0058] Select undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples, add 50% of them into the basic culture medium, sterilize and place them at room temperature, inoculate them with bumblebee Candida albicans and 1% Ganoderma lucidum at an inoculation rate of 1.8% (volume ratio), ferment at 28°C, and culture for 100 hours under basic culture conditions. After the fermentation is completed, sterilize the whole at 121°C for 20 minutes, filter through a 0.45μm filter plate, and separate the fermentation product with a separatory funnel.

[0059] Example 5

[0060] Fermentation strain combination: Bumblebee Candida and Cicadae, Bumblebee Candida strain concentration 1×10 9 CFU / ml.

[0061] The cicada fungus is prepared by inoculating the activated cicada fungus into a seed culture medium at 1% (volume ratio), and culturing the culture medium at 30°C and 140rpm for 5 days until the fungus ball grows fully and fills the seed culture medium.

[0062] Unspoiled, clarified and stable squalane raw materials and jojoba seed oil samples were selected and added into the basal culture medium at an addition amount of 50%. After sterilization, they were placed at room temperature, and inoculated with 1.8% bumblebee Candida albicans and 1% cicada fungus according to the volume ratio. The fermentation temperature was 28°C and the culture was carried out under the basic culture conditions for 100 hours. After the fermentation was completed, the whole was sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and the fermentation product was separated by a separatory funnel.

[0063] Example 6

[0064] Fermentation strain combination: Bombus spp. Candida albicans and Saccharomyces cerevisiae, with a strain concentration of 1×10 9CFU / ml, the concentration of Saccharomyces cerevisiae strains was 5×10 9 CFU / ml.

[0065] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at an addition amount of 20%, sterilized and placed at room temperature, inoculated with bumblebee Candida albicans and 0.2% (volume ratio) Saccharomyces cerevisiae at an inoculation amount of 1.8% (volume ratio), fermented at 26°C, cultured for 24 hours under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0066] Example 7

[0067] Fermentation strain combination: Bombus spp. Candida albicans and Saccharomyces cerevisiae, with a strain concentration of 1×10 9 CFU / ml, the concentration of Saccharomyces cerevisiae strains is 1×10 8 CFU / ml.

[0068] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at an addition amount of 80%, sterilized and placed at room temperature, inoculated with bumblebee Candida albicans and 0.2% (volume ratio) Saccharomyces cerevisiae at an inoculation amount of 1.8% (volume ratio), fermented at 30°C, cultured for 144 hours under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0069] Comparative Example 1

[0070] Fermentation strain: Rhodosporidium sporei, strain concentration is 1×10 9 CFU / ml.

[0071] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected and added into the basic culture medium at a dosage of 50%. After sterilization, they were placed at room temperature and inoculated with 2% (volume ratio) red winter spore yeast. The fermentation temperature was 28°C and the culture was carried out under the basic culture conditions for 100 hours. After the fermentation was completed, the whole was sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and the fermentation product was separated by a separatory funnel.

[0072] Comparative Example 2

[0073] Fermentation strain: Bumblebee Candida, strain concentration is 1×10 9 CFU / ml.

[0074] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at a dosage of 50%, sterilized and placed at room temperature, inoculated with bumblebee Candida albicans at an inoculation rate of 2% (volume ratio), fermented at 28°C, cultured for 100 hours under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20 minutes, filtered through a 0.45 μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0075] Comparative Example 3

[0076] Fermentation strain: Saccharomyces cerevisiae, strain concentration is 1×10 9 CFU / ml.

[0077] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected and added into the basal culture medium at a dosage of 50%. After sterilization, they were placed at room temperature, and brewer's yeast was inoculated at an inoculation rate of 2% (volume ratio). The fermentation temperature was 28°C and the culture was carried out under the basic culture conditions for 100 hours. After the fermentation was completed, the whole was sterilized at 121°C for 20 minutes, filtered through a 0.45μm filter plate, and the fermentation product was separated by a separatory funnel.

[0078] Comparative Example 4

[0079] Fermentation strain: Starch yeast, strain concentration is 1×10 9 CFU / ml.

[0080] Undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples were selected, added into the basal culture medium at an addition amount of 50%, sterilized and placed at room temperature, inoculated with Staryi sebaceous yeast at an inoculation amount of 2% (volume ratio), fermented at 28°C, cultured for 100h under basic culture conditions, and after the fermentation was completed, sterilized at 121°C for 20min as a whole, filtered through a 0.45μm filter plate, and separated by a separatory funnel to obtain the fermentation product.

[0081] Comparative Example 5

[0082] The fermented strain is a single Ganoderma lucidum, which is consistent with the strain culture method of Ganoderma lucidum in Example 4.

[0083] Select undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples, add them to the basic culture medium at an addition amount of 50%, sterilize and place at room temperature, inoculate Ganoderma lucidum at an inoculation amount of 10% (volume ratio), ferment at 28°C, culture for 100 hours under basic culture conditions, and after the fermentation is completed, sterilize the whole at 121°C for 20 minutes, filter through a 0.45μm filter plate, and separate the fermentation product through a separatory funnel. In Example 4, Ganoderma lucidum is a block fungus and mycelium, and the concentration ratio with Bombus spp. is close to 1:5, so the inoculation amount of Ganoderma lucidum is set to 10%, which is equivalent to the inoculation amount of Bombus spp. in Comparative Example 2.

[0084] Comparative Example 6

[0085] Fermentation strain: The fermentation strain is a single cicada fungus, which is consistent with the cicada fungus cultivation method of Example 5.

[0086] Select undeteriorated, clarified and stable squalane raw materials and jojoba seed oil samples, add them to the basic culture medium at an addition amount of 50%, sterilize and place at room temperature, inoculate cicada fungus at an inoculation amount of 10% (volume ratio), ferment at 28°C, culture for 100 hours under basic culture conditions, sterilize at 121°C for 20 minutes after fermentation, filter through a 0.45 μm filter plate, and separate the fermentation product through a separatory funnel. In Example 5, cicada fungus is a block fungus and mycelium, and the concentration ratio with bumblebee Candida is close to 1:5, so the cicada fungus inoculation amount is set to 10%, which is equivalent to the bumblebee Candida inoculation in Comparative Example 2.

[0087] Comparative Example 7 (the only difference from Example 1 is the fermentation temperature)

[0088] Select unspoiled, clarified and stable squalane raw materials and jojoba seed oil samples, add them into the basic culture medium at a rate of 50%, sterilize them and place them at room temperature, and inoculate Candida albicans at an inoculation rate of 1.8% (volume ratio) (the strain concentration is 1×10 9 CFU / ml and 0.2% Saccharomyces cerevisiae (strain concentration 1×10 9 CFU / ml), the fermentation temperature was 24℃, and the culture was carried out under the basic culture conditions for 100h. After the fermentation was completed, the whole was sterilized at 121℃ for 20min, filtered through a 0.45μm filter plate, and the fermentation product was separated by a separatory funnel.

[0089] Experiment 1: Oil acid value assessment and antioxidant test

[0090] With reference to GB5009.229-2016, the free fatty acid content of oils was determined by evaluating the acid value of oils, and the antioxidant property of oils was determined using the 892 Rancimat Oil Oxidation Stability Analyzer.

[0091] 1.1 Evaluation of oil acid value (free fatty acid detection)

[0092] The detection method refers to the national standard GB5009.229-2016, "National Food Safety Standard-Determination of Acid Value in Food" in the first method: cold solvent indicator titration method.

[0093] According to this method, the acid value of the unfermented crude oil-based jojoba oil in the laboratory is 3.8 mgKOH / g, and the acid value of squalane oil is 0.34 mgKOH / g. The results of the changes in the acid value of jojoba seed oil and squalane after fermentation with different bacterial strains are shown in Table 1.

[0094] Table 1 Changes in acid value of oil products after jojoba seed oil and squalane fermentation

[0095]

[0096] The test results show that compared with each comparative example, the acid value of the fermented oil sample prepared by the bacterial strain combination described in Examples 1-5 is significantly reduced, indicating that the combined fermentation process provided by the present invention can significantly reduce the acid value of oils and fats, and Example 1 has the best effect. The acid value of fermented jojoba seed oil is reduced by 63.16% compared with unfermented jojoba seed oil, reduced to 1.4, and the acid value of fermented squalane is reduced by 64.70% compared with unfermented squalane, reduced to 0.12. It shows that the fermentation bacterial strain combination of the present invention has the effect of significantly reducing the free fatty acid content of oils and fats and reducing the acid value of oils and fats.

[0097] 1.2 Oil oxidation stability test

[0098] According to GB / T21121-2007 standard method 16, Rancimat892 (Swiss Metrohm) oil oxidation stability tester was used.

[0099] Measurement parameters: temperature 120.0±0.1℃; injection volume 3.0±0.2g; gas flow 20L / h; Deon-153 water (conductivity value <5 S / cm) 60 ml.

[0100] Experimental results:

[0101] According to this method, the oxidation reaction time of squalane in the unfermented jojoba seed oil in the laboratory was 22.75 and 33.04 respectively. The results of the changes in the oxidation time of oil after fermentation are shown in Table 2.

[0102] Table 2 Changes in oxidation time of oil products after jojoba seed oil and squalane fermentation

[0103]

[0104] The results of the determination show that compared with each comparative example, the oxidation time of the fermented oil samples prepared by the bacterial combination described in Examples 1-5 is significantly increased, indicating that the combined fermentation process provided by the present invention can significantly improve the oxidizability of oils and fats, and Example 1 has the best effect. The oxidation time of fermented jojoba seed oil is increased by 22.42% to 27.85h compared with unfermented jojoba seed oil, and the oxidation time of fermented squalane is increased by 32.87% to 43.90h compared with unfermented squalane. It shows that the fermentation bacterial combination has the effect of significantly increasing the oxidation time of oils and fats and enhancing the oxidation stability of oils and fats.

[0105] Experiment 2: Determination of Saponification Value of Oils and Fats

[0106] With reference to GB / T5534-2008, the saponification value of oils and fats was determined. Based on the saponification value determination results, the emulsification effect of fermented oils and fats was compared with that of unfermented oils and fats to determine the optimal fermentation bacteria combination.

[0107] 2.1 Determination of saponification value

[0108] Referring to GB / T5534-2008, method a, potassium hydroxide-aluminum sheet-ethanol distillation method, was selected.

[0109] Experimental results:

[0110] Determined by this method, the saponification values ​​(KOH) of the unfermented jojoba seed oil and squalane in the laboratory are 175 mg / g and 232 mg / g respectively. The saponification value changes of the samples of the embodiments and comparative examples before and after are shown in Table 3.

[0111] Table 3 Changes in saponification value of jojoba seed oil and squalane fermentation oil products

[0112]

[0113] The test results show that compared with the single strain fermentation product of the comparative example, the saponification value of the fermented oil sample prepared by the strain combination described in Examples 1-5 is significantly improved, indicating that the combined fermentation process provided by the present invention can significantly improve the saponification value of oils and fats, and Example 1 has the best effect. The saponification value of fermented jojoba seed oil is increased by 117.14% compared with the unfermented jojoba seed oil, and increased to 380 mg / g. The oxidation time of fermented squalane is increased by 61.21% compared with the unfermented squalane, and increased to 374 mg / g. It shows that the fermentation strain combination has the effect of significantly improving the saponification value of oils and fats and enhancing the hydrophilicity of oils and fats.

[0114] Experiment 3: Testing the effect of oil as a carrier to promote the transdermal absorption of active substances

[0115] 3.1 Test skin preparation

[0116] The whole mouse abdominal skin was purchased and cut into 1.77 cm pieces manually. 2 The cut skin was washed in 0.1 mol / L PBS solution for about 30 min, taken out and dried with filter paper. After processing, it was wrapped with tin foil and stored at -20 ℃ for later use.

[0117] 3.2 Transdermal test

[0118] The transdermal absorption permeation diffusion device uses a Franz diffusion cell. A 30% ethanol PBS solution is added to the receiving cell, and a water bath is turned on. After the temperature is constant, the prepared skin (the stratum corneum is on top) is placed on the Franz diffusion instrument, and after confirming that the lower layer of the skin is in seamless contact with the receiving solution, it is fixed, and the skin surface is wiped clean with filter paper, and 0.5 g of the sample (0.5% by mass of paeonol is added to the fermented oil of Examples 1-5 and Comparative Examples 1-7) is taken and applied to the stratum corneum to ensure close contact with the skin.

[0119] Take 0.5 mL of the receiving solution at 2h, 4h, 6h, 8h, and 24h, and add the same volume of fresh receiving solution after each sampling, and be careful to prevent bubbles from entering. After sampling, filter through a 0.25μm microporous filter membrane, use high-performance liquid chromatography HPLC to test, record the data, and use the standard curve method to determine the content, and accumulate the transdermal amount Q and transdermal percentage W at each point;

[0120] Calculation method:

[0121]

[0122] Where Cn is the sample concentration measured at the nth sampling point (μg / mL); is the sample concentration measured at the i-th (i=n-1) sampling point; 8 is the total volume of the receiving solution (mL); 1 is the sampling volume (mL); 1.766 is the effective skin penetration area (cm 2 ).

[0123] 3.3HPLC content detection method and results

[0124] Instrument model: Agilent 1260; Chromatographic column: Eclipse XDB-C18 (4.6mm×250mm, 5μm);

[0125] Injection volume: 10 μL, flow rate: 1 mL / min, detection wavelength: 274 nm;

[0126] Column temperature: 30°C; mobile phase: pure water and methanol;

[0127] Test drug: The reference substance selected was paeonol standard, which was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Methanol, acetonitrile and formic acid were all chromatographically pure, and other reagents were of analytical grade.

[0128] Methodological investigation

[0129] Stability test: The test solution was taken and sampled at 0, 2, 4, 8, 12, and 24 h for detection. The RSDs of the relative retention time and relative peak area of ​​the chromatographic peaks were both less than 2.0%, indicating that the test solution had good stability within 24 hours.

[0130] Precision test: Take the test solution and inject it 6 times continuously. The RSD of the relative retention time and relative peak area of ​​each main chromatographic peak is less than 2.0%, indicating that the precision of the instrument is good.

[0131] Repeatability test: Take the same batch of samples, prepare 6 test solutions, and measure them separately. The RSD of the relative retention time and relative peak area of ​​the main chromatographic peaks are less than 2.0%, indicating that the repeatability of this method is good.

[0132] According to the above method, the laboratory unfermented jojoba seed oil, squalane and each embodiment, the comparative sample oil as the active substance carrier transdermal absorption effect was tested. The experimental results of jojoba seed oil and fermented jojoba seed oil are shown in Table 4, the experimental results of squalane and fermented squalane are shown in Table 5, and the HPLC detection spectrum of paeonol standard is shown in Table 6. Figure 1 , where the standard curve equation is y=87221x-5.2, R 2 =0.9994.

[0133] 3.4 Test results:

[0134] Table 4 Detection results of the cumulative transdermal penetration of paeonol by jojoba seed oil in each group (μg / cm 2 )

[0135]

[0136] The test results show that compared with the unfermented jojoba seed oil, the transdermal absorbability of the fermented jojoba seed oil is improved to varying degrees, indicating that the fermentation treatment has an improving effect on the transdermal absorbability of the jojoba seed oil sample. Compared with the single strain fermentation products of Comparative Examples 1, 2, 3, 4, 5, and 6, the transdermal absorbability of the fermented jojoba oil sample prepared by the strain combination described in Examples 1-5 is significantly improved, indicating that the combined fermentation process provided by the present invention can significantly improve the transdermal absorbability of oils and fats, and Example 1 has the best effect. Compared with the unfermented jojoba seed oil, the transdermal absorption amount of the fermented jojoba seed oil is 141.21 μg / cm after 6 hours of absorption. 2 Increased to 183.82 μg / cm 2 , an increase of 30.17%, indicating that the fermentation bacteria combination has a significant effect on improving the transdermal absorption of oil and promoting the absorption of effective substances.

[0137] Table 5 The results of the test on the transdermal absorption of paeonol by squalane oil in each group (μg / cm 2 )

[0138]

[0139] The measurement results show that compared with unfermented squalane, the transdermal absorbability of fermented squalane is improved to varying degrees, indicating that the fermentation treatment has an improving effect on the transdermal absorbability of squalane samples. Compared with the single strain fermentation products of Comparative Examples 1, 2, 3, 4, 5, and 6, the transdermal absorbability of the fermented jojoba oil samples prepared by the strain combination described in Examples 1-5 is significantly improved, indicating that the combined fermentation process provided by the present invention can significantly improve the transdermal absorbability of oils.

[0140] After fermentation treatment, the transdermal absorbability of the fermented oil samples prepared in each embodiment and comparative example is improved to varying degrees compared with the unfermented oil, among which the transdermal absorbability of the fermented oil prepared by the combination of fermented strains is significantly higher than that of the oil fermented by a single strain, indicating that the combined strain fermentation treatment has a significant promoting effect on the transdermal absorbability of oil.

[0141] In Example 6-Example 7, a strain combination of Candida bombus and Saccharomyces cerevisiae was used to ferment squalane raw materials and jojoba seed oil samples. The difference from Example 1 is the strain concentration of Saccharomyces cerevisiae. Then, according to the above strain concentration, the fermentation process parameters were adaptively adjusted. The fermentation principle was the same as that in Example 1, and the strain combination of the present invention was used to ferment squalane raw materials and jojoba seed oil raw materials, so that the acid value of the fermented oil product was reduced, the oxidative stability of the oil was increased, and the transdermal absorption was enhanced.

[0142] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A fermented oil with a low fatty acid content, characterized in that: The raw materials of the fermented oil include oil and a fermented bacterial composition; the oil is selected from jojoba seed oil or squalane; the fermented bacterial composition is composed of Candida bombus and a second bacterial species, and the second bacterial species is selected from one of Lipomyces staryi, Saccharomyces cerevisiae, Rhodosporidium involucrata, Ganoderma lucidum and Cicadae concolor; The method for preparing the fermented oil with low fatty acid content comprises the following steps: (1) adding oil to the culture medium, inoculating the fermentation strain composition, and fermenting; the oil is selected from jojoba seed oil or squalane; (2) After fermentation, sterilize and filter to obtain the product.

2. The fermentation bacteria composition according to claim 1, characterized in that The concentration of the bumblebee-grown Candida and / or the second bacterial species is 0.5×10 8 -5×10 9 CFU / ml.

3. A method for preparing a fermented oil with a low fatty acid content according to claim 1, characterized in that: The steps include: (1) adding oil to the culture medium, inoculating the fermentation strain composition, and fermenting; the oil is selected from jojoba seed oil or squalane; (2) After fermentation, sterilize and filter to obtain the product.

4. The preparation method according to claim 3, characterized in that: In step (1), the mass of the oil is 20%-80% of the mass of the culture medium, the inoculation amount of the fermentation strain composition is 0.5-2% of the volume of the culture medium, and the concentrations of the bumblebee Candida and the second strain are both 1×10 8 -5×10 9 CFU / ml.

5. The preparation method according to claim 3, characterized in that: The fermentation temperature in step (1) is 26-30°C, and the fermentation time is 24-144 hours; the sterilization temperature in step (2) is 115-125°C, and the sterilization time is 13-35 minutes.

6. The preparation method according to claim 3, characterized in that: The composition of the culture medium in step (1) includes the following components by weight percentage: 1-5% glucose, 0.1-10% peptone, 0.1-1% yeast powder, 0.001-0.01% potassium dihydrogen phosphate, and the rest is water; after adding oil in step (1), sterilization is carried out, and inoculation is carried out after being placed at room temperature; after filtering in step (2), separation is carried out using a separatory funnel to obtain the obtained product.

7. Use of the fermented oil with low fatty acid content according to claim 1 or the fermented oil prepared by the preparation method according to any one of claims 3 to 6 as a carrier in promoting transdermal absorption of active ingredients.

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