WO2022147873A1

WO2022147873A1 - Avocado oil-in-water liquid fermented product, and preparation method therefor and use thereof

Info

Publication number: WO2022147873A1
Application number: PCT/CN2021/075386
Authority: WO
Inventors: 李周欣; 蒋旭峰; 张鑑锋; 温文忠; 杜养标
Original assignee: 青蛙王子（福建）婴童护理用品有限公司; 江苏瑞霆生物科技有限公司
Priority date: 2021-01-11
Filing date: 2021-02-05
Publication date: 2022-07-14
Also published as: CN112708652B; CN112708652A

Abstract

Provided are a method for preparing an avocado oil-in-water liquid fermented product and the use thereof in cosmetics. The fermented product effectively converts proteins, nucleic acids, polysaccharides, etc. into a small-molecule water-soluble fermentation system that can be easily absorbed by the skin, and moreover, avocado oil is biologically refined by means of using a surfactant of probiotics and is encapsulated. The fermented product has a relatively strong anti-lipid peroxidation effect and cell drying damage repair effect. The lipid peroxidation inhibition rate of 2.5% avocado oil-in-water liquid fermented product is 23.29%, and the effect is close to 176 ppm V _C; and the repair rate of 1.25% avocado oil-in-water liquid fermented product on drying damaged cells is close to that of 10% glycerol. The fermented product is green and safe, non-hemolytic, has no adverse reactions on skin and is non-irritating. New development and uses of avocado is good for increasing the economic application value of the avocado in cosmetics.

Description

A kind of avocado oil-in-water liquid fermentation product and preparation method and application thereof

technical field

The invention relates to an avocado oil-in-water liquid fermentation product and a preparation method and application thereof, belonging to the field of fermentation and the technical field of skin care products.

Background technique

The skin is an important organ of the human body with a complex tissue structure and is closely connected with other organs of the human body, acting as a barrier to protect the human body from external stimuli and damage. The body functions of infants and young children have not yet fully developed, and the skin tissue is very different from that of adults. Due to the delicate skin and poor development of the stratum corneum, its protective function is weaker than that of adults, and it is easily damaged and microorganisms are easily invaded, so it is prone to eczema, allergies, and dryness. And so many skin problems. In addition, pregnant women are also prone to various skin problems under the influence of progesterone, such as dry skin, pigmentation, allergies, acne, etc., basic care is essential. Pregnant women have an increasing demand for safe and effective skin care products, and the development of mild, naturally fermented cosmetics has become an important direction to solve the skin problems of pregnant women.

Fruits, vegetables, grains and other plants are fermented to produce various aroma substances, and the flavor of the raw materials themselves is improved. In addition, microbial metabolism produces extracellular enzymes such as pectinase and cellulase, which can promote the rupture of plant cells and release active ingredients. Microbial metabolism can also produce proteases, etc., which can effectively degrade macromolecular substances into small molecular substances, which are easier for the human body to absorb, thereby enhancing the efficacy of drugs. Based on the above advantages, as well as the characteristics of high quality, low price and stable quality of fermented products, they are now widely used in people's food and daily necessities, such as yeast fermented products and lactic acid bacteria fermented products for moisturizing and anti-aging daily necessities.

Avocado, also known as avocado, avocado, avocado, cream fruit, is a fruit with high nutritional value. Avocado is rich in unsaturated fatty acids, vitamins, sterols, protein and some mineral elements, and has the reputation of "forest butter". Eating avocado on a regular basis can help soften and moisturise the skin, as well as help shrink pores and make the skin look smoother and firmer. Avocados are rich in vitamins and vegetable oils, which moisturize the skin and strengthen the skin's moisturizing properties; vitamins can soothe the skin, prevent aging, and to a certain extent prevent the early formation of wrinkles, delay the speed of skin aging, and prevent premature wrinkles. Avocado is rich in nutrients, which can soothe and repair sensitive skin and strengthen the skin barrier. At present, there is no research report on the use of avocado fermented raw materials to prepare effective moisturizing and alleviating sensitive cosmetics.

In the past, the cosmetic raw materials derived from avocados were mostly avocado oil extracted by chemical and physical processes, and other proteins, polysaccharides, nucleic acids, water-soluble vitamins, and minerals contained in avocados were discarded. For example, CN108815072A discloses a fermented puree of a plant composition and its preparation method and application. Although avocado is used, it actually removes the peel and pulp to get the avocado pit, and uses the avocado pit, golden chamomile and Honeysuckle flower petals, Aloe vera mesophyll, ginseng and Panax notoginseng roots are prepared as plant composition filtrate, and the compound bacterial liquid of Lactobacillus delbrueckii subsp. bulgaricus and natural Saccharomyces cerevisiae is used to ferment the plant composition filtrate to prepare the composition Fermented puree; the invention only uses the avocado pit as the raw material, and abandons the rich oil in the avocado pulp, and the fat (fruit oil) content in the avocado pulp accounts for about 20%, which is a natural moisturizing ingredient. Further, how to utilize the fat (fruit oil) in the avocado pulp is also a major problem at present. The traditional biological fermentation method is mostly carried out in the water phase. This traditional fermentation process is not compatible with avocado oil, and it is difficult for avocado oil to exist in it stably; other avocado fermentation processes are mostly aimed at the preservation of avocado pulp, and the fermented products It is semi-solid and cannot be used in the production of cosmetic raw materials.

Therefore, it is necessary to develop a method for maximizing the utilization of the nutrients of avocado as a cosmetic raw material.

SUMMARY OF THE INVENTION

In order to solve the problem of maximizing the use of avocado nutrients and using them as cosmetic raw materials, the method of the present invention was developed. The oil-in-water liquid fermentation product of avocados of the present invention effectively converts proteins, nucleic acids, polysaccharides, etc. The small molecule water-soluble fermentation system uses the surfactant of probiotics to bio-refine the avocado oil and encapsulate it. That is, the fermentation process is used to convert the avocado into a stable mixed liquid fermentation product with polypeptides, amino acids, oligosaccharides, water-soluble microbial minerals, etc. as the water phase, and avocado oil wrapped with surfactants as the lipid phase. Nutrients are maximized. The avocado oil-in-water liquid fermented product obtained by the invention can be used for preparing cosmetics, has the effects of superior moisturizing performance, enhancing skin barrier, and food source, safety is more secure, and pregnant women and infants can use it with confidence.

The first object of the present invention is to provide a preparation method of avocado oil-in-water liquid fermentation product, the method comprising: inoculating a microbial compound bacterial agent into a fermentation substrate with avocado as the main component, and fermenting and culturing to prepare ; Described microbial compound bacterial agent is the mixed bacterial agent that comprises the saccharomyces fascicularis and Lactobacillus pentosus. In the fermentation process, yeast enzymes are used to degrade polysaccharides and proteins, and the natural oils of avocados are retained; the surfactants produced by lactic acid bacteria are used to coat oil droplets.

The Saccharomycopsis fibuligera is a high-yielding protease strain screened from liquor fermented grains, and has been deposited in the General Microbiology Center of the China Microorganism Culture Collection on December 21, 2020, and is preserved The serial number is CGMCC NO.21512, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The Lactobacillus pentosus (Lactobacillus pentosus) was obtained by screening from liquor fermented grains, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection on December 21, 2020, and the preservation number is CGMCC NO.21511. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Preferably, the avocado includes avocado pulp and a fruit pit; optionally, the whole avocado fruit is obtained after cleaning, removing the stems and breaking the walls of fresh avocados.

In one embodiment, the inoculation of the compound bacterial agent is to inoculate a liquid bacterial agent, which is the addition of 5%-15% by volume of the complex microbial inoculum to the fermentation substrate. Optionally, the inoculum of the composite microbial inoculum is 10%.

In one embodiment, the compound bacterial agent is a mixed bacterial agent of Saccharomyces spp. and Lactobacillus pentosus, wherein the volume ratio of Yeast saccharomyces pouches and Lactobacillus pentosus seed liquid is 1 :0.5-1:2. Optionally, the volume ratio is 1:1.

In one embodiment, the fermentation is at 28-32°C for 25-30h. Optionally, ferment at 30°C for 27h.

In one embodiment, the fermentation substrate is 80-120 g·L ^-1 of avocado and 15-25 g·L ^-1 of glucose. Optionally, avocado 100g·L ^-1 and glucose 20g·L ^-1 .

In one embodiment, the fermentation culture is carried out in a ventilated environment.

In one embodiment, the preparation method comprises the following steps:

(1) the avocado is pulverized, sterilized, and mixed with glucose to obtain a mixed solution;

(2) Adding compound microbial bacterial liquid to the sterilized fermentation substrate, and fermenting at 30°C for 27 hours; the ventilation rate was 4 sL·min ^-1 when the fermentation process was carried out in the fermenter.

(3) Use ceramic membrane filtration to collect the filtrate, which is the avocado oil-in-water liquid fermentation product.

The second object of the present invention is to provide the avocado oil-in-water liquid fermentation product prepared according to the above method.

The third object of the present invention is to provide a strain of Saccharomycopsis fibuligera suitable for whole fruit fermentation of avocado, which has been deposited in the General Microorganism Center of the China Committee for the Preservation of Microorganisms on December 21, 2020. The serial number is CGMCC NO.21512, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The fourth object of the present invention is to provide a strain of Lactobacillus pentosus suitable for whole fruit fermentation of avocado, which has been deposited in the General Microorganism Center of the China Committee for the Preservation of Microorganisms on December 21, 2020, with a deposit number of It is CGMCC No. 21511, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The fifth object of the present invention is to provide a composite inoculum suitable for whole fruit fermentation of avocado, the composite inoculum is the saccharomyces saccharomyces cerevisiae with the preservation number of CGMCC NO.21512 and the pentose with the preservation number of CGMCC NO.21511 Lactobacillus mixture.

The sixth object of the present invention is to provide the preparation method of the above-mentioned avocado oil-in-water liquid fermentation product or the application of the avocado oil-in-water liquid fermentation product or the above-mentioned composite bacterial agent in the preparation of cosmetics.

In one embodiment, the cosmetic may be a toner, a serum, a lotion, a cream, or the like.

In one embodiment, by selecting suitable strains to ferment the avocado, an oil-in-water liquid fermented product of avocado is prepared, which has good resistance to lipid peroxidation, cell drying and damage repair capabilities, and has good beauty. skin, skin care effect.

The seventh object of the present invention is to provide the preparation method of the above-mentioned avocado oil-in-water liquid fermentation product or the application of the avocado oil-in-water liquid fermentation product or the above-mentioned composite bacterial agent in the preparation of products with antioxidative and moisturizing effects.

Preservation of biological material:

Saccharomycopsis fibuligera Z1-9, classified as Saccharomycopsis fibuligera, has been deposited in the General Microbiology Center of the China Commission for the Collection of Microorganisms on December 21, 2020, and the deposit number is CGMCC NO.21512. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Lactobacillus pentosus C-7, classified as Lactobacillus pentosus, has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on December 21, 2020, the preservation number is CGMCC NO.21511, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Beneficial effects:

In the avocado oil-in-water liquid fermentation product of the present invention, the fat content can reach 0.11 g·100 ^-1 ·g ^-1 , the protein content can reach 1813 ppm, the amino acid content can reach 635 ppm, the total phenolic content can reach 148 ppm, and the total sugar content can reach 1813 ppm. Up to 4.68 g·L ^-1 ; fatty acid composition is 8.50% palmitoleic acid, 20.78% palmitic acid, 9.81% linoleic acid, 52.06% oleic acid, 7.96% oleic acid, 0.89% stearic acid; peptide concentration is 9.68± 0.22g·L ^-1 , rich in small molecule oligopeptides; rich in important components of natural moisturizing factor (NMF), among which, the concentration of alanine is 102.02ppm, the concentration of glutamic acid is 78.09ppm, and the concentration of aspartic acid is 78.09ppm. The acid concentration is 49.46ppm; the lipid peroxidation inhibition rate of 2.5% avocado oil-in-water liquid fermentation product is 23.29%, and the effect is close to 176ppm VC; the repair of dry damaged cells by 1.25% avocado oil-in-water liquid fermentation product is close to 10% glycerol 2.5% and 5.0% avocado oil-in-water liquid fermentation products can improve the cell repairing effect of dry damage by 27.35% and 29.83% respectively; green and safe, no hemolysis, no adverse reactions to the skin, no irritation.

The oil-in-water liquid fermented product of the avocado of the present invention can be used as a cosmetic raw material for the preparation of cosmetics such as toner, essence, lotion or face cream, and has the functions of anti-oxidation and moisturizing.

Description of drawings

Figure 1 is a comparison of peptide concentrations;

Figure 2 is the comparison result of the sample fat morphology;

Figure 3 is the sample appearance comparison;

Fig. 4 is the graph after centrifugation of unfermented sample;

Fig. 5 is the GC-MS spectrum of the sample after fermentation;

Fig. 6 is the comparison result of the molecular weight distribution of sample peptide;

Fig. 7 is the amino acid concentration comparison before and after fermentation;

Fig. 8 is the anti-lipid peroxidation effect of avocado oil-in-water liquid fermentation product;

Figure 9 shows the repairing effect of avocado oil-in-water liquid fermentation products on drying damaged cells (n=3); Note: * indicates a significant difference (p<0.05) with the drying damage protection effect of 10% glycerol, determined by ANOVA-Dunnett Calculated.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but they are not intended to limit the present invention.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The avocados in the following examples are from commercially available, specifically California avocados.

Saccharomycopsis fibuligera used in the embodiment was screened in liquor fermented fermented grains, and the preservation number was CGMCC NO.21512; Lactobacillus pentosus was screened in liquor fermented grains, and the preservation number was CGMCC NO.21511.

In the following examples, the formula of YPD liquid culture medium is: 10.0 g of yeast powder, 20.0 g of peptone, 20.0 g of glucose, pH 6.5-6.7, supplemented with 1000 mL of deionized water. YPD solid medium is YPD liquid medium supplemented with 2% agar powder. After the medium components were prepared, they were sterilized at 121 °C for 20 min.

In the following examples, the formula of MRS liquid medium is: peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, diammonium hydrogen citrate 2.0g, glucose 20.0g, Tween 80 1.0g, sodium acetate trihydrate 5.0g g, K ₂ HPO ₄ ·3H ₂ O 2.0g, MgSO ₄ ·7H ₂ O 0.58g, MnSO ₄ ·H ₂ O 0.25g, pH 6.2～6.6, make up 1000mL of deionized water. MRS solid medium is MRS liquid medium supplemented with 2% agar powder. After the medium components were prepared, they were sterilized at 121 °C for 20 min.

Example 1 High-throughput breeding of yeast with high protease production

In the process of liquor fermentation, a high-yielding protease-encapsulating yeast has been screened. The process is as follows.

The formula of the solid medium for strain screening is: peptone 20.0g, yeast powder 1.0g, skim milk 10.0g, glucose 5.0g, KH ₂ PO ₄ 1.0g, NaH ₂ PO ₄ 2.4g, agar 20.0g, pH 6.8, make up Ionized water 1000mL. After the medium components were prepared, they were sterilized at 121 °C for 20 min.

1. Primary screening of strains

Dissolve 0.1 g of liquor fermented grains in 1 mL of sterile physiological saline, add glass beads and shake evenly. After 10-fold dilution, 200 μL of the mixed solution was taken and spread on the solid medium for strain selection, and cultured at 30° C. for 2 d.

2. Rescreening of strains

The strains showing a transparent circle were picked, and after isolation and purification, a single colony was picked and placed in YPD liquid medium, and cultured for 2 days under shaking conditions of 200r·min ^-1 and 30°C. The fermentation broth was centrifuged at 10,000 rpm for 10 min and the supernatant was taken to measure the protease activity.

Determination of protease activity: adopt GB/T 23527-2009 "Protease Preparation" method. Mix 1 mL of crude enzyme solution with 1 mL of substrate (casein solution) evenly, react at 40°C for 10 min, then add 2 mL of trichloroacetic acid to stop the reaction, and centrifuge at 12000 rpm for 3 min. Add 5 mL of Na ₂ CO ₃ and 1 mL of Folin-phenol reagent to 1 mL of the supernatant, mix evenly, and keep at 40 °C for 20 min, and measure the absorbance at a wavelength of 660 nm using a UV-Vis spectrophotometer. The group that first added trichloroacetic acid to terminate the reaction was used as the control group. The enzymatic activity is defined as follows: 1 mL of the test solution, in the environment of 40°C, can catalyze the production of casein by the required amount of enzyme equivalent to 1 μg of tyrosine per minute, that is, one enzymatic activity unit (U).

3. Screening results of protease-producing bacteria

Several strains of protease-producing strains were obtained by screening from liquor fermented grains, and their protease activity was determined. The results are shown in Table 1. By comparing the enzymatic activities of the fermentation broths of these strains, a high protease-producing strain Z1-9 was screened out, and the protease activity of this strain reached 82.1 U·mL ^-1 in the preliminary screening.

Table 1 Screening results of protease-producing bacteria

4. Identification of strains

(1) Colony characteristics and cell morphology

The colonies of strain Z1-9 were round, white, fluffy, raised, with irregular edges. The morphological results were observed by microscope, and it was found that the isolated and screened strain cells were nearly elliptical, with different sizes, and some were budding and dividing.

(2) Molecular biological identification

The strain Z1-9 was inoculated into YPD medium, cultured for 1 d, and the total DNA of the strain was extracted as a PCR template. Yeast ITS universal primers were used for PCR amplification, and the selected universal primers were ITS1 (nucleotide sequence: 5'-TCCGTAGGTGAACCTGCGG-3', sequence: SEQ ID NO: 1) and ITS4 (nucleotide sequence: 5'-TCCTCCGCTTATTGATATGC) -3', sequence as SEQ ID NO: 2). PCR amplification conditions: 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, cycle 30 times; 72°C for 10 min. PCR amplification products were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing after passing the 1% agarose gel inspection. The sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database for BLAST comparison, and the strain was found to be Saccharomycopsis fibuligera.

The strain Z1-9 was preliminarily identified as Saccharomycopsis fibuligera by comprehensive colony morphological characteristics, physiological and biochemical characteristics and ITS sequence analysis. The strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, with the preservation number of CGMCC NO.21512.

Example 2 Screening of biosurfactant strains

Liquor fermentation is a mixed-bacteria fermentation process, in which abundant microorganisms adapt to each other and work synergistically during the long-term domestication process. The microorganisms screened in the same environment can better synergize. Therefore, the screening of surfactant-producing strains was performed from the growth environment of the yeast of Example 1.

Biosurfactants discovered so far include many different species, such as glycolipids, lipopeptides, polysaccharide-lipid complexes, phospholipids, fatty acids, and neutral lipids. Different surfactants contain different components, and not all surfactants are suitable for the encapsulation of avocado oil. Therefore, the screening of surfactant-producing strains is carried out with the medium supplemented with avocado oil, and the process is as follows.

Avocado oil liquid medium: avocado oil 8.0g, peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, diammonium hydrogen citrate 2.0g, glucose 20.0g, K ₂ HPO ₄ 3H ₂ O 2.0g, MgSO ₄ · 7H ₂ O 0.58g, MnSO ₄ ·H ₂ O 0.25g, pH 7.0, make up 1000 mL of deionized water. After the medium components were prepared, they were sterilized at 121 °C for 20 min.

1. Plate separation

Take 3.5 g of the sample and add it to a triangular flask containing 35 mL of sterile physiological saline, add glass beads and shake evenly to prepare a wine grain suspension. The fermented grains suspension was inoculated into the avocado oil liquid culture medium with 5% (V/V) inoculation amount, and shaken cultured at 37°C. It was spread on MRS solid plate medium and incubated at 37°C for 2 days.

2. Shake flask primary screening

The single colonies obtained from the primary screening were respectively inoculated into the avocado oil liquid medium, shaken and cultured at 37°C, 200 r·min ^-1 shake flasks, and the strains with good avocado oil emulsifying effect were selected to obtain 4 strains.

3. Rescreening by oil drain ring method

The four strains with avocado oil emulsifying effect were inoculated into MRS liquid medium, and cultured in shake flasks at 37°C, 200 r·min ^-1 for 2 d. After centrifugation, the diameter of the oil discharge circle of the supernatant was measured.

Drain ring method: the fermentation broth was centrifuged at 4000 rpm for 10 min, and the supernatant was taken for later use. Take a petri dish with a diameter of 9 cm, add 30 mL of distilled water, and drop 0.2 mL of avocado oil on the water surface. After an oil film is formed, add 0.2 mL of the fermentation broth supernatant diluted 2 times to the center of the oil film to form a stable oil drain. Measure the diameter of the oil drain ring.

4. Screening results of oil drain ring method

By comparing the diameter of the oil discharge circle of the four strains obtained by the primary screening, and comparing their ability to produce biosurfactants, the results of the re-screening are shown in Table 2. Compared with other strains, strain C-7 had the largest diameter of the oil discharge ring of the fermentation broth and produced the most biosurfactants.

Table 2 Screening results of biosurfactant strains

5. Identification of strains

(1) Colony characteristics and cell morphology

The colonies of strain C-7 were tiny round, white, raised, with neat edges. The morphological results were observed by microscope, and it was found that the isolated and screened strain cells were rod-shaped, short-chain-shaped, and had no spores.

(2) Molecular biological identification

The strain C-7 was inoculated into MRS medium, cultured for 1 d, and the total DNA of the strain was extracted as a PCR template. PCR amplification was carried out using bacterial 16S universal primers, and the selected universal primers were 27F (the nucleotide sequence was 5'-AGAGTTTGATCMTGGCTCAG-3', the sequence was as SEQ ID NO: 3) and 1492R (the nucleotide sequence was 5'-TACGGHTACCTTGTTACGACTT) -3', sequence as SEQ ID NO: 4). PCR amplification conditions: 94°C for 10 min, 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, cycle 30 times; 72°C for 10 min. PCR amplification products were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing after passing the 1% agarose gel inspection. The sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database for BLAST comparison, and the strain was found to be Lactobacillus pentosus.

The strain C-7 was preliminarily identified as Lactobacillus pentosus by comprehensive colony morphological characteristics, physiological and biochemical characteristics and 16S sequence analysis. The strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, with the preservation number of CGMCC NO.21511.

The preparation of embodiment 3 avocado oil-in-water liquid fermentation product

1. Preparation of saccharomyces cerevisiae seed solution

Under sterile conditions, a single colony of Saccharomycopsis fibuligera Z1-9 was picked and inoculated into YPD liquid medium. 30 ℃, 220r·min ^-1 shaking culture for 24h, when the bacterial volume reached 10 ⁸ CFU·mL ^-1 , the seed liquid was obtained.

2. Preparation of Lactobacillus pentosus seed solution

Under sterile operating conditions, a single colony of Lactobacillus pentosus C-7 was picked and inoculated into MRS liquid medium. 37 ℃, 200r·min ^-1 shaking culture for 24h, when the bacterial volume reached 10 ⁸ CFU·mL ^-1 , the seed liquid was obtained.

3. Preparation

Wash the fresh avocados with tap water, then wash and drain them with deionized water, and remove the stems; add the avocados to a wall breaker, add 3 times the mass ratio of deionized water, and crush until there are no visible large particles of solids.

4. Preparation of fermentation substrate

Avocado 100g·L ^-1 , glucose 20g·L ^-1 . Sterilize at 121 °C for 20 min to obtain fermentation substrate, which is then used after cooling. Among them, glucose is sterilized and added into the system alone.

5. Inoculation and fermentation

To the sterilized slurry, add a 10% volume ratio of composite microbial inoculum, and ferment at 30°C for 27 hours; the composite microbial inoculum is from yeast Z1-9 and Lactobacillus pentosus C-7. The mixed inoculum is obtained by composing the saccharomyces cerevisiae seed liquid and the Lactobacillus pentosus seed liquid in a volume ratio of 1:1; when the fermentation process is carried out in a fermentation tank, the ventilation rate is 4sL·min ^-1 .

6. Microfiltration

Use ceramic membrane filtration to collect the filtrate, which is the avocado oil-in-water liquid fermentation product.

Comparative Example 1 Avocado Extract

The only difference from Example 3 is that the composite microbial inoculum was not inoculated.

Comparative Example 2

The only difference from Example 3 is that the yeast Z1-9 was fermented by single use.

Comparative Example 3

The only difference from Example 3 is that Lactobacillus pentosus C-7 is used alone for fermentation.

Comparative Example 4

The only difference from Example 3 is that the yeast CICC 33226 was used instead of the yeast of the present invention, and it was mixed with the Lactobacillus of the present invention for fermentation.

Among them, CICC 33226 was purchased from China Industrial Microbial Culture Collection and Management Center, and it was found that it could produce protease by the transparent circle method.

Comparative Example 5

The difference from Example 3 is that the yeast CICC 1717 is used instead of the yeast of the present invention, and is mixed with the Lactobacillus of the present invention for fermentation.

Among them, CICC 1717 was purchased from China Industrial Microorganism Culture Collection and Management Center, and it was found that it could produce protease by transparent circle method.

Comparative Example 6

The difference from Example 3 is that Saccharomyces cerevisiae CICC 1210 is used instead of the yeast of the present invention, and is mixed with the Lactobacillus of the present invention for fermentation.

Among them, Saccharomyces cerevisiae CICC 1210 was purchased from the China Industrial Microorganism Culture Collection and Management Center, and it was found that it could produce protease by the transparent circle method.

Comparative Example 7

The difference from Example 3 is that Lactobacillus pentosus CICC 24202 is used instead of the Lactobacillus of the present invention, which is mixed with the yeast of the present invention for fermentation.

Among them, Lactobacillus pentosus CICC 24202 was purchased from China Industrial Microorganism Culture Collection and Management Center.

Comparative Example 8

The difference from Example 3 is that Lactobacillus casei CICC 20252 is used instead of the Lactobacillus of the present invention, which is mixed with the yeast of the present invention for fermentation.

Among them, Lactobacillus casei CICC 20252 was purchased from China Industrial Microorganism Culture Collection and Management Center. It has been reported that Lactobacillus casei can produce biosurfactants (for details, see references: Ferreira A, Vecino X, Ferreira D, et al. Novel cosmetic formulations containing a biosurfactant from Lactobacillus paracasei. 2017, 155:522-529. ).

Embodiment 4 Detection of avocado fermented products

From dimensions such as polypeptide content, fruit oil encapsulation, active substance content, fatty acid composition, polypeptide molecular weight distribution, etc., the avocado oil-in-water liquid fermented products prepared in Example 3 and the avocado fermented products obtained in Comparative Examples 1-8 were measured.

1. Comparison of peptide content

1. Preparation of the sample

The oil-in-water liquid fermentation product of avocado prepared in Example 3 and Comparative Examples 1-8 were used as samples to be tested.

2. Determination of total peptide content

Refer to the trichloroacetic acid method combined with the Lowry Method to determine the total polypeptide content in the sample:

Take 2.5 mL of the sample to be tested, add 2.5 mL of 10% trichloroacetic acid aqueous solution, mix evenly on a vortex mixer, let stand for 20 min, and then centrifuge at 3500 r·min ^-1 for 10 min to remove protein precipitation. The supernatant was taken and the concentration of total peptide was determined according to the Lowry Method.

3. Comparison of total polypeptide content of samples

The comparison results of the total polypeptide content of the samples are shown in Figure 1. The polypeptide concentration of Comparative Example 1 without microbial fermentation treatment was 874.03±18.46ppm. The highest content of polypeptide after fermentation is Example 3 of the present invention, and its concentration is 1330.64±36.90 ppm. Compared with the extract obtained in Comparative Example 1 without fermentation treatment, the polypeptide concentration is increased by 52%.

The polypeptide concentration of Comparative Example 3 was similar to that of unfermented Comparative Example 1, indicating that Lactobacillus pentosus C-7 fermentation alone could not hydrolyze proteins in avocado into small molecular polypeptides.

Compared with the unfermented comparative example 1, the polypeptide concentrations of Comparative Examples 4 to 6 are increased, but far less than the yeast used in Example 3. It is indicated that the yeast Z1-9 of the present invention is more suitable for the avocado fermentation system of the present invention in which the whole utilization of the avocado pulp and pits is carried out.

The polypeptide content of Comparative Example 8 is lower than that of Example 3, the Lactobacillus casei CICC 20252 affects the effect of yeast Z1-9, and the Lactobacillus casei CICC 20252 cannot replace the Lactobacillus pentosus C-7 of the present invention .

2. Contents of avocado oil

1. Preparation of the sample

The avocado oil-in-water liquid fermentation product prepared in Example 3 of the present invention and Comparative Example 1, Comparative Example 2, and Comparative Example 7 were used as samples to be tested.

2. Nile red staining

Nile red can bind to lipids and emit a fluorescent detection signal. The samples were stained with Nile red, and the fat morphology was observed under a laser confocal microscope.

3. Comparison of the microscopic state of avocado oil

It can be seen from Figure 2 that, compared with the unfermented Comparative Example 1, the oil droplets of Example 3 are more uniformly distributed, and the encapsulation effect of the avocado oil is the best, and an oil-in-water microstructure can be formed. The avocado oil of Comparative Example 2 is not encapsulated, indicating that the single use of the yeast Z1-9 for fermentation cannot encapsulate the fruit oil in the avocado. The avocado oil of Comparative Example 7 is not contained, indicating that Lactobacillus pentosus CICC 24202 cannot replace the Lactobacillus pentosus C-7 of the present invention, and Lactobacillus pentosus C-7 is more suitable for the avocado fermentation system of the present invention.

3. Determination of active substance content of avocado oil-in-water liquid fermentation products

The oil-in-water liquid fermented product of avocado prepared in Example 3 of the present invention was used as the sample to be tested.

Refer to the Lowry Method to determine the total protein content in the sample: prepare bovine serum albumin solutions of different concentrations to draw a standard curve. 0.5 mL of bovine serum albumin solution was added to 5 mL of alkaline copper solution. After standing at room temperature for 20 min, 0.5 mL of a 50% Folin phenol reagent was added to the above solution. After standing at room temperature for 30min, select the wavelength of 650nm to measure the absorbance. Taking the absorbance as the ordinate and the concentration of the bovine serum albumin solution (unit: ppm) as the abscissa to draw the standard curve: y=0.0008x+0.0125, R ² =0.9956. Determine the absorbance of the sample to be tested according to the above method, and substitute it into the standard curve to calculate the protein content.

Refer to the ninhydrin chromogenic method to determine the total amino acid content in the sample: prepare glutamic acid solutions of different concentrations to draw a standard curve. Add 2 mL of glutamic acid solution, 0.5 mL of PBS buffer, and 0.5 mL of 2% ninhydrin solution to the test tube, and heat in a boiling water bath for 20 min. Select the wavelength of 570nm to measure the absorbance. Taking the absorbance as the ordinate and the concentration of the glutamic acid solution (unit: ppm) as the abscissa to draw the standard curve: y=0.0002x ² -0.0086x+0.0372, R ² =0.9991. Measure the absorbance of the sample to be tested according to the above method, and substitute it into the standard curve to calculate the total amino acid content.

Determination method of total phenolic content of samples: prepare gallic acid solutions of different concentrations to draw a standard curve. 100 μL of gallic acid solution, 1.0 mL of Folinphenol, 1.0 mL of 10% Na ₂ CO ₃ were added to the test tube, and the reaction was carried out at room temperature for 90 min. Select the wavelength of 760nm to measure the absorbance. Taking the absorbance as the ordinate and the concentration of the gallic acid solution (unit: ppm) as the abscissa, the standard curve was drawn: y=0.0075x+0.0396, R ² =0.999. Measure the absorbance of the sample to be tested according to the above method, and substitute it into the standard curve to calculate the total phenolic content.

Determination method of total sugar content of samples: prepare glucose solutions of different concentrations to draw a standard curve. Add 1.0 mL of 5% phenol aqueous solution, 1.0 mL of glucose solution, and 5.0 mL of concentrated sulfuric acid to the test tube, shake well, and react in boiling water for 25 min. Select the wavelength of 490nm to measure the absorbance. Taking the absorbance as the ordinate and the concentration of the glucose solution (unit: g·L ^-1 ) as the abscissa, the standard curve was drawn: y=7.2226x-0.0262, R ² =0.997. Measure the absorbance of the sample to be tested according to the above method, and substitute it into the standard curve to calculate the total sugar content.

The active substance content of the oil-in-water liquid fermentation product of avocado is shown in Table 3.

The active substance content of the avocado oil-in-water liquid fermentation product obtained by table 3 embodiment 3

As can be seen from Table 3, the avocado oil-in-water liquid fermented product obtained in Example 3 is rich in protein, amino acids, phenols and sugars, indicating that the fermentation method of the present invention can obtain the fermentation puree of high active ingredients.

Fourth, fat comparison

1. Preparation of the sample

The avocado oil-in-water liquid fermentation product prepared in Example 3 of the present invention and the avocado extract prepared in Comparative Example 1 were used as samples to be tested.

2. Determination of total fat content

Refer to the second method of GB 5009.6-2016 to determine the total fat content in the sample:

Weigh about 10g of the sample to be tested, accurate to 0.001g, put it in a 50mL test tube, and add 10mL of hydrochloric acid. Put the test tube into a water bath at 70-80°C, and stir with a glass rod every 5-10 minutes until the sample is completely digested, about 40-50 minutes.

Remove the test tube, add 10 mL of ethanol, and mix. After cooling, the mixture was transferred into a 100 mL measuring cylinder with stopper, and the test tube was washed several times with 25 mL anhydrous ether, and poured into the measuring cylinder together. After all the anhydrous ether was introduced into the graduated cylinder, stopper and shake for 1 min, carefully open the stopper, release the gas, plug it again, let it stand for 12 min, carefully open the stopper, and rinse the stopper and the fat attached to the mouth of the graduated cylinder with anhydrous ether. Let stand for 10 to 20 minutes, until the upper liquid is clear, aspirate the supernatant into a conical flask with constant weight, add 5 mL of anhydrous ether into a stoppered measuring cylinder, shake, and after standing, still suck out the upper ether, into the original conical flask.

Recover anhydrous ether, evaporate to dryness on a water bath when 1-2 mL of solvent remains, dry at 100±5°C for 1 hour, cool in a desiccator for 0.5 hours and weigh. Repeat the above operation until constant weight (until the difference between the two weighings does not exceed 2 mg).

The fat content in the sample is calculated as follows:

where:

X——The content of fat in the sample, the unit is g·100 ^-1 ·g ^-1 ;

m ₁ ——the content of the receiving bottle and fat after constant weight, the unit is g;

m ₀ ——the mass of the receiving bottle, the unit is g;

m ₂ — the mass of the sample, in g;

3. Comparison of total fat content of samples

The results are shown in Table 4.

Table 4 Comparative results of the total fat content of the samples

As can be seen from Table 4, the fat content in the fermented puree obtained in Example 3 after fermentation treatment of avocado was 0.11 g·100 ⁻¹ ·g ⁻¹ , and the fat content in the extract obtained in Comparative Example 1 without fermentation treatment. It is 0.15g·100 ^-1 ·g ^-1 , indicating that the avocado oil (fat) is still retained after fermentation.

4. Comparison of sample fat stability

After the sample to be tested is placed at 4°C for 1 week, the appearance is shown in Figure 3.

It can be seen from Fig. 3 that the extract obtained in Comparative Example 1 (left) without fermentation treatment was placed for one week and the fat was precipitated, while the fat of the fermented puree obtained in Example 3 (right) after fermentation treatment of avocado was more stable, and the fat was not stable. Precipitation occurs. Lactobacillus pentosus C-7 in the mixed bacterial agent produces biosurfactant in the fermentation process, which encapsulates fat, makes the fermentation liquid more stable and uniform, and ensures that the most valuable components can exert their moisturizing effect. It is indicated that the fermentation method of the present invention can obtain products with more stable components, which is a more scientific and effective method.

5. Analysis of fatty acid composition

1. Preparation of the sample

The avocado oil-in-water liquid fermentation product prepared in Example 3 of the present invention and the avocado extract prepared in Comparative Example 1 were centrifuged at 5000 × g for 10 min.

As shown in Figure 4, after centrifugation of the unfermented sample, there is a layer of oily substances on the liquid surface, and the fat is not encapsulated and is easy to precipitate. The fermented sample oil is coated with surfactant, which is not easy to separate out, and the upper layer is free of oil after centrifugation.

Take the lower layer of liquid as the sample to be tested.

2. Lipid extraction

Take 50 mL of the liquid to be tested and add 50 mL of dichloromethane to extract the lipid of the sample to be tested. The dichloromethane phase was taken, and the dichloromethane was evaporated to dryness to obtain an oil sample.

3. Lipid saponification

Add 25 mL of 1.2 mol·L ^-1 KOH solution to the above oil sample, condense and reflux at 65°C for 2 h; cool the reaction solution, extract 3 times with 10 mL of n-hexane; take the water phase.

4. Fatty acid methylation

Add 10 mL of hydrochloric acid and 2 mL of methanol to the above aqueous phase, and condense and reflux at 70 °C for 1 h; cool the reaction solution, extract 3 times with 10 mL of n-hexane, and evaporate to dryness; add 15 mL of anhydrous methanol, 1-2 drops of concentrated sulfuric acid, and condense and reflux at 80 °C 20 min; cooling the reaction solution, adding 10 mL of saturated NaCl aqueous solution and 10 mL of n-hexane, and extracting; taking the n-hexane phase, and diluting to 2 mL with n-hexane.

5. Determination of fatty acids

The fatty acid composition of the above samples was analyzed by GC-MS. Chromatographic conditions: Chromatographic column: HP-88 elastic quartz capillary column (100m×0.25mm, 0.25μm); the heating program is the initial temperature of 150°C for 2min, and the temperature is increased to 210°C at 2°C·min ^-1 , and the temperature is 50°C·min ^-1 was heated to 250°C for 10min. The temperature of the injection port was 250°C, the injection volume was 1 μL, the total flow rate was 35.6 mL·min ^-1 , the carrier gas was N ₂ , and the split ratio was 50:1. Qualitative and quantitative analysis: qualitative analysis was performed using NIST 14 and Wiley 9.0 mass spectrometry database, and the relative percentage of each component was calculated by area normalization method.

6. Content comparison

In the unfermented sample, oil was precipitated after centrifugation, there was no lipid in the lower layer liquid, and no fatty acid was detected by GC-MS. The GC-MS profile of the post-fermentation sample is shown in Figure 5.

The fatty acid composition of avocado oil-in-water liquid fermentation product and its relative percentage are shown in Table 5. The fat content of the oil-in-water liquid fermentation product of avocado was measured in the early stage to be 0.15 g·100 ^-1 ·g ^-1 . According to the relative percentage content of fatty acids, the concentration was estimated, as shown in Table 5.

The fatty acid composition and content of table 5 avocado oil-in-water liquid fermentation product

The most abundant oil-in-water liquid fermented product of avocado is oleic acid, accounting for 52.06% of the fatty acid composition, with a concentration of 572.66ppm. Oleic acid can inhibit the activity of the capsaicin receptor TRPV1, thereby reducing the pain and itching response to external stimuli (for details, see references: Morales-Lázaro Sara L, Llorente Itzel, Sierra-Ramírez Félix, et al. Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch. 2016, 7:13092.). Furthermore, oleic acid and essential fatty acids in avocados promote wound healing and help reduce inflammation during the healing process (for details see References: Sales-Campos Helioswilton, Souza Patricía Reis de, Peghini

Crema, et al. An overview of the modulatory effects of oleic acid in health and disease. 2013, 13(2):201-10.). In addition, stearic acid, palmitic acid, oleic acid, and linoleic acid in avocado all have inhibitory effects on the signaling molecule AI-2 (for details, see references: KWWidmer, KASoni, MEHume, et al. Identification of Poultry Meat-Derived Fatty Acids Functioning as Quorum Sensing Signal Inhibitors to Autoinducer‐2 (AI-2). 2007, 72(9):M363-M368.). AI-2 is a signaling molecule for bacterial interspecies information exchange and a compound that plays a key role in bacterial cell quorum sensing. These fatty acids in avocado further inhibit the growth of pathogenic microorganisms by inhibiting quorum sensing.

6. Comparison of molecular weight distribution of peptides

1. Preparation of the sample

2. Peptide molecular weight distribution analysis

The sample to be tested was filtered with a 0.22 μm microporous membrane, and its molecular weight distribution was determined by high-performance liquid space exclusion chromatography (SEC-HPLC) under the following conditions: the chromatographic column was TSK gel 2000 SWXL (300mm×7.8mm); The temperature was 30° C.; the mobile phase was acetonitrile:water:trifluoroacetic acid=45:44:0.1 (volume ratio); the flow rate was 0.5 mL·min ⁻¹ ; the detection wavelength was 220 nm.

3. Comparison of molecular weight distribution of sample peptides

The comparison results of the molecular weight distribution of the sample peptides are shown in Figure 6.

As can be seen from FIG. 6 , compared with the extract obtained in Comparative Example 1 without fermentation treatment, the oligopeptides of the fermented puree obtained in Example 3 after fermentation treatment of avocado increased. The specific molecular weight and percentage are shown in Table 6 and Table 7.

The peptide molecular weight distribution of the avocado oil-in-water liquid fermentation product obtained in Table 6 Example 3

The peptide molecular weight distribution of the avocado extract that table 7 comparative example 1 obtains

Comparing Table 6 and Table 7, it is found that the peptides with molecular weights of 1077, 650 and 288 are increased in the oil-in-water liquid fermentation product of avocado compared with the extract. In addition, the content of peptides with a molecular weight of 184 was also significantly increased. There is a relatively good affinity between peptides and skin cells. Compared with natural active substances such as nucleic acids and proteins, peptides are easily soluble in water, have small molecular weight, and are easily absorbed and utilized. In terms of skin and other functions, the performance of active polypeptides is far superior to that of proteins with larger molecules, and it has relative safety and stability. This result indicates that fermentation can degrade avocado proteins into small molecular oligopeptides, making them easier to absorb.

Seven, amino acid content comparison

1. Preparation of the sample

2. Determination of amino acid content

The content of free amino acids in the samples was detected using a high performance liquid chromatograph and a Diamonsil C18 (250mm×4.6mm, 5μm) chromatographic column. Pre-test samples were subjected to OPA-FMOC pre-column derivatization. Mobile phase A was 27.6 ^{mmol·L -1} of sodium acetate-triethylamine-tetrahydrofuran (50:0.11:2.5, volume ratio), and the pH was adjusted to 7.2 by using acetic acid. Mobile phase B was 80.9 mmol·L ^-1 sodium acetate-methanol-acetonitrile mixture (1:2:2, volume ratio), and the pH was adjusted to 7.2 by using acetic acid. The UV detection wavelength was 338 nm, the flow rate was 1 mL·min ^-1 , the column temperature was 40 °C, and the injection volume was 10 μL.

3. Comparison of amino acid content of samples

The comparison results of the amino acid content of the samples are shown in Figure 7.

Alanine is a humectant and an important component of Natural Moisturizing Factor (NMF). The concentration of alanine in the extract obtained in Comparative Example 1 without fermentation treatment was 12.04 ppm, accounting for 2.67% of the total amino acids; the concentration of alanine in the fermented puree obtained in Example 3 of the avocado after fermentation treatment was 102.02ppm, accounting for 21.62% of total amino acids. Compared with the unfermented sample, the concentration of alanine increased by 8.48 times after fermentation, which is the amino acid with the highest content in the fermentation broth.

Embodiment 4 Application of avocado oil-in-water liquid fermentation product in cosmetics

The efficacy and safety of the avocado oil-in-water liquid fermentation product prepared in Example 3 in the preparation of cosmetics were analyzed.

1. Anti-lipid peroxidation

1. Preparation of the sample

The avocado oil-in-water liquid fermented product prepared by the embodiment of the present invention 3 was used as the sample to be tested, and 176ppm VC was used as a negative control.

2. Anti-lipid peroxidation test

Add 1 mL of 50 mM PBS (pH 7.4), 1 mL of 2% lecithin solution, 1 mL of sample solution, and 1 mL of 5 mM EDTA-Fe(II) to the sample tube in sequence, and incubate in a water bath at 37 °C for 45 min.

To the solution was added a mixture of 2 mL of thiobarbituric acid (TBA, 20%) and trichloroacetic acid (TCA, 1%), mixed well, heated in boiling water for 15 min, and immediately cooled with an ice-water bath. After sufficient cooling, the clear liquid was taken, and the absorbance was measured at 535 nm. The lipid peroxidation inhibition rate was calculated according to the following formula.

where:

A——The absorbance of the sample group;

C——The control group replaced the sample with its solvent, absorbance;

B ₁ ——Blank group 1 does not add EDTA-Fe(Ⅱ) on the basis of the control group, the absorbance

B ₂ ——When the sample contains lipid, blank group 2 needs to be set additionally, that is, lecithin is not added on the basis of the sample group, and the absorbance

3. Anti-lipid peroxidation effect of avocado oil-in-water liquid fermentation products

Figure 8 shows the anti-lipid peroxidation efficacy of avocado oil-in-water liquid fermentation products. The lipid peroxidation inhibition rate of 2.5% avocado oil-in-water liquid fermentation product (the liquid fermentation product of Example 3 is diluted to 2.5% by volume with deionized water, the same below), 176ppm V _C (ascorbic acid) is 23.29 ± 2.67%. acid; vitamin C), the lipid peroxidation inhibition rate was 23.35±3.26%, indicating that the lipid peroxidation inhibition effect of 2.5% avocado oil-in-water liquid fermentation product was close to 176ppm V _C .

2. Repairing effect of dry cell damage

1. Experimental materials

Cell line: HaCaT human immortalized keratinocytes

Reagents: SDS (sodium dodecyl sulfate), DMEM medium, DMEM complete medium (added with 10% FBS fetal bovine serum and 1% double antibody), PBS buffer, glycerol (positive control), test samples, MTT, DMSO.

2. Cell culture

1) Cell culture

The cultured cells were digested, centrifuged, counted with a hemocytometer, diluted at a concentration of 1.5×10 ⁵ ·mL ^-1 and added to a 96-well plate, and 100 μL of cell fluid was added to each well to reach 15,000 cells per well. Pay attention to pipetting during the plating process to reduce the influence of cell sedimentation. Place the 96-well plate in a 37°C, 5% _CO2 incubator for 24-30h.

2) Addition of samples

The medium in the 96-well plate was discarded and washed 1-2 times with PBS. To each well was added 100 [mu]L of solid sample pre-dissolved in PBS. The low-concentration samples were diluted proportionally, and PBS was added first and then the high-concentration sample solution was added. The blank group and the injury group were added with 100 μL of PBS solution. Incubate in the incubator for 30 minutes.

At least three parallel wells were set for each sample concentration, and six parallel wells were set for the blank group and the damaged group.

3) Addition of SDS

After the incubation time, 100 μL of 50 μg·mL ^-1 SDS in PBS was added to each well to make the final concentration 25 μg·mL ^-1 . The blank group was added with the same amount of PBS solution. Place the 96-well plate in the incubator for 20 min.

4) Determination of cell viability

After the incubation, discard the liquid in the well plate. Wash 1-2 times with PBS solution. 150 μL of 0.5 mg·mL ^-1 MTT-medium solution was added to each well. After 4 h of incubation, the solution was discarded, and DMSO was added to dissolve the formazan crystals. After shaking in the microplate reader for 10min, the absorbance value was measured at 490nm, and the data was processed as the cell viability value%.

3. The repairing effect of avocado oil-in-water liquid fermentation products on dry damaged cells

The repairing effect of avocado oil-in-water liquid fermentation products on dry damaged cells is shown in Figure 9. 1.25% avocado oil-in-water liquid fermented product can repair dry damaged cells close to 10% glycerol. The 2.5% and 5.0% avocado oil-in-water liquid fermentation products can improve the cell repairing effect of drying damage by 27.35% and 29.83%, respectively.

3. Safety evaluation of avocado oil-in-water liquid fermentation products

1. Red blood cell hemolysis test

(1) Experimental materials

Porcine red blood cells (2%, Beijing Bolsi Technology Co., Ltd.);

PBS: 0.27 g (2 mM) KH ₂ PO ₄ , 3.58 g (10 mM) Na ₂ HPO ₄ , 8 g NaCl and 0.2 g KCl were dissolved in 1 L of deionized water, and the pH was adjusted to 7.4 with hydrochloric acid.

(2) Experimental method

Whole hemolysis control group: water + porcine red blood cells

Negative control group: PBS+porcine red blood cells

Sample group: avocado oil-in-water liquid fermentation product + pig red blood cells

Hemolysis test: The sample was dispersed in PBS or normal saline, the red blood cells and the sample were mixed evenly in a ratio of 2:3, incubated at 37°C for 10min, centrifuged at 10000r·min ^-1 for 3min, and the supernatant was taken to test the absorbance at 540nm. The calculation formula of hemolysis rate is: HD%=(A _{540 sample-} A _540PBS )/(A _{540 water} -A _540PBS )×100%

Set 5 points above and below the HD ₅₀ , set the standard curve with the sample concentration as the abscissa and HD as the ordinate, and calculate the HD ₅₀ .

(3) HD ₅₀ of avocado oil-in-water liquid fermentation product

The oil-in-water liquid fermented product of avocado has no hemolysis at the maximum experimental concentration (90%, a certain amount of PBS needs to be added to the liquid to remove hemolysis caused by the difference in osmotic pressure inside and outside the cell), so HD ₅₀ >90%.

2. Human skin patch experiment (closed type)

(1) Experimental method

According to the method specified in the 2015 Cosmetics Technical Specifications.

Volunteer requirements: 30 normal subjects (20-40 years old), no clinical unhealed inflammatory skin disease, no scars, pigments, atrophy, port-wine stains or other blemishes in the skin to be tested. Investigators who have not participated in other clinical trials, those who have no highly sensitive constitution, and those who have not performed patch tests in the past month.

Sample set: 40% avocado oil-in-water liquid fermented product

Negative control group: deionized water

Add each of the above solutions to the filter paper attached to the patch tester and place it in the patch tester, the dosage is about 0.020-0.025mL, and apply the patch tester with the test substance to the side of the forearm with non-irritating tape. Gently press the palm of the hand to make it evenly applied to the skin for 24 hours. 30 minutes after removing the test object, the skin reaction was observed after the indentation disappeared. If the result is negative, observe the test again 24h and 48h after the patch test to record the reaction result.

(2) Results

Table 8 Experimental results of human skin patch (closed type)

As shown in Table 8, all the 30 subjects in the human patch experiment had grade 0 reactions. According to the 2015 Cosmetic Hygiene Standard, 40% of the oil-in-water liquid fermented products of avocado did not cause adverse reactions to human skin. It can be seen from this that , the product has high human safety.

3. Human skin patch experiment (open)

(1) Experimental method

Sample set: 40% avocado oil-in-water liquid fermented product

Negative control group: deionized water

The sample dosage is about 0.020-0.025 mL, the test site is the curved side of the hand forearm, and the test area is 3×3 cm ² . Apply twice a day for 7 days.

(2) Results

Table 9 Results of human skin patch experiment (open)

As shown in Table 9, all the 30 subjects in the human patch experiment had grade 0 reactions. According to the 2015 Cosmetic Hygiene Standard, 40% of the avocado oil-in-water liquid fermented products did not cause adverse reactions to human skin. It can be seen that , the product has high human safety.

4. Chick embryo chorioallantoic membrane blood vessel test

(1) Experimental materials

Chicken embryos: SPF grade White Leghorn chicken, purchased from Beijing Merial Weitong Laboratory Animal Technology Co., Ltd.

Culture conditions: incubator temperature 37.5±0.5°C, relative humidity 55%-70%.

Reagents and controls: positive control (PC1): 1.0% SDS solution;

Positive control (PC2): 0.1mol/L NaOH solution;

Negative control (NC): 0.9% NaCl solution;

Test Sample (TA): Avocado oil-in-water liquid fermentation product.

(2) Experimental method

CAM preparation: buy 0d-old chick embryos, 50-60g, turn them once a day during the incubation process, check the developmental status of the chick embryos when hatching to 5d-old, and peel off the egg shell at 6 days to expose the white egg membrane; use tweezers carefully The intima is removed, ensuring that the vascular membrane is not damaged.

Formal test: at least 6 chick embryos in each group, take 0.1 mL of the test substance as it is and directly drop it on the surface of CAM, observe the reaction of CAM, and record the time of each toxic effect within 5 minutes, accurate to seconds, including bleeding, Coagulation and vascular melting were three kinds of reactions, and the degree of reaction was recorded. The same procedure was used for positive and negative controls.

Data analysis: Record the time and response degree of each detection end point, and calculate the stimulus score (IS) using the following formula. The results are kept to two decimal places:

sec H, sec L, and sec C represent the mean time (seconds) observed on the CAM membrane to begin bleeding, vascular fusion, and coagulation, respectively.

Table 10 Evaluation of results of stimulus scoring method

(3) Results

Table 11 The results of the chicken embryo chorioallantoic membrane blood vessel test

According to the judgment standard of SN/T 2329-2009, the 100% avocado oil-in-water liquid fermented product in the sample group has no irritation, while the 1.0% SDS solution and 0.1mol/L NaOH solution in the control group have severe irritation, and the 0.9% NaCl solution has no irritation.

Claims

A preparation method of avocado oil-in-water liquid fermentation product, characterized in that, the method comprises: inoculating a microbial compound bacterial agent into a fermentation substrate with avocado as a main component, and fermenting and culturing to prepare; the microbial compound bacteria The agent is a mixed bacterial agent containing Saccharomyces fascicularis and Lactobacillus pentosus.
The method according to claim 1, wherein the Saccharomycopsis fibuligera has been deposited in the General Microbiology Center of the China Microorganism Culture Collection on December 21, 2020, and the deposit number is CGMCC NO.21512; The Lactobacillus pentosus has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee on December 21, 2020, and the deposit number is CGMCC NO.21511.
The method according to claim 1, wherein the avocado comprises avocado pulp and fruit stone; alternatively, the compound bacterial agent inoculation is an inoculation of a liquid bacterial agent, which is added to the fermentation substrate in a volume ratio of 5%- 15% compound microbial inoculum.
The avocado oil-in-water liquid fermentation product prepared according to the method described in any one of claims 1-3.
A strain of Saccharomycopsis fibuligera suitable for whole-fruit fermentation of avocado has been deposited in the General Microbiology Center of the China Microorganism Culture Collection on December 21, 2020, with the preservation number of CGMCC NO.21512 and the deposit address It is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
A strain of Lactobacillus pentosus suitable for whole fruit fermentation of avocado has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee on December 21, 2020, the preservation number is CGMCC NO.21511, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
The compound inoculum suitable for whole avocado fermentation, the compound inoculum is the mixed inoculum of the yeast with the preservation number of CGMCC NO.21512 and the Lactobacillus pentosus with the preservation number of CGMCC NO.21511.
Application of the preparation method of the oil-in-water liquid fermented product of any one of claims 1-3 or the oil-in-water liquid fermented product of claim 4 or the composite bacterial agent of claim 7 in the preparation of cosmetics .
The application according to claim 8, wherein the cosmetic is any one of toner, essence, lotion or face cream.
The preparation method of the oil-in-water liquid fermented product of any one of claims 1-3 or the oil-in-water liquid fermented product of claim 4 or the composite bacterial agent of claim 7 has antioxidant, Moisturizing effect of the product in terms of application.