CN112708652B - Avocado oil-in-water liquid state fermentation product and preparation method and application thereof - Google Patents

Abstract

The invention discloses a preparation method of a avocado oil-in-water liquid state fermentation product and application of the avocado oil-in-water liquid state fermentation product in cosmetics. The fermentation product effectively converts protein, nucleic acid, polysaccharide and the like into a micromolecular water-soluble fermentation system which is easy to absorb by skin, and simultaneously utilizes the surfactant of probiotics to biologically refine the avocado oil and carry out entrapment. The fermented product has strong lipid peroxidation resisting effect and cell drying injury repairing effect, wherein the lipid peroxidation inhibition rate of the 2.5% avocado oil-in-water liquid fermented product is 23.29%, and the effect is close to 176ppm V _C (ii) a 1.25% avocado oil-in-water liquid fermentation product repairs dry damaged cells with approximately 10% glycerol. The fermented product is green and safe, has no hemolytic effect, no adverse reaction on skin, and no irritation. The invention is a novel development and utilization of avocado, and is beneficial to improving the cosmetic quality of avocadoThe economic application value in (1).

Description

Avocado oil-in-water liquid state fermentation product and preparation method and application thereof

Technical Field

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

Background

The skin is an important organ of the human body, has a complex tissue structure, is closely connected with other organs of the human body, plays a role of a barrier and protects the human body from external stimulation and injury. The body functions of infants are not developed completely, skin tissues are different from those of adults, and because the skin is tender and has poor stratum corneum development, the protection function of the infants is weaker than that of adults, the infants are easy to be damaged, and microorganisms are easy to invade, so that a plurality of skin problems such as eczema, allergy, dryness and the like are easy to occur. In addition, the pregnant women are affected by the progestational hormone, and various skin problems such as dry skin, color spots, allergy, acne and the like are easy to occur, and basic care is essential. Pregnant and lying-in women have increasingly increased demands for safe and effective skin care products, and the development of mild and natural fermentation cosmetics becomes an important direction for solving the skin problems of the pregnant and lying-in women.

The plants such as fruits, vegetables and grains are fermented to produce various fragrant substances, and the flavor of the raw materials is improved. In addition, the microorganism metabolizes to produce extracellular enzymes such as pectinase and cellulase, which can promote the rupture of plant cells and release effective components. The microbial metabolism can also produce protease and the like, effectively degrade macromolecular substances into micromolecular substances, and are easier to absorb by a human body, so that the drug effect is enhanced. Based on the advantages and the characteristics of high quality, low price and stable quality of the fermented product, the yeast fermented product and the lactobacillus fermented product are widely used in food and daily necessities of people, such as the yeast fermented product and the lactobacillus fermented product which are used in moisturizing and anti-aging daily necessities.

Avocado, also known as avocado, butter fruit, is a very high nutritional value fruit. Avocado is rich in unsaturated fatty acids, vitamins, sterols, proteins and some mineral elements, and has the reputation of "forest butter". Frequent eating of the avocado can help to soften and moisten the skin, and can help to shrink pores, so that the skin looks smoother and firmer. The avocado contains abundant vitamins and vegetable oil, and the oil moistens the skin and strengthens the moisture retention of the skin; the vitamins can relieve skin, prevent aging, prevent the generation of wrinkles in advance to a certain extent, delay the aging speed of skin and prevent the generation of wrinkles in advance. The avocado is rich in nutrition, and has effects of relieving and repairing sensitive muscle and enhancing skin barrier. At present, no research report is available for preparing cosmetics which can effectively preserve moisture and relieve sensitivity by using the fermented raw materials of avocados.

The cosmetic raw materials from the conventional avocado are mostly avocado oil extracted by chemical and physical processes, and other proteins, polysaccharide nucleic acid, water-soluble vitamins and minerals contained in the avocado are discarded. For example, CN108815072A discloses a plant composition fermented raw pulp, a preparation method and an application thereof, wherein although avocado is used, the fruit peel and the pulp are actually removed to obtain avocado kernels, the avocado kernels, golden chamomile, honeysuckle flower petals, aloe barbadensis leaf pulp, ginseng and roots of pseudo-ginseng in a specific ratio are prepared into a plant composition filtrate, and the plant composition filtrate is fermented by using a compound bacterial liquid of lactobacillus delbrueckii subsp bulgaricus and natural saccharomyces cerevisiae to prepare the composition fermented raw pulp; the invention only uses the kernel of the avocado as the raw material, discards the rich grease in the avocado flesh, and the fat (fruit oil) content in the avocado flesh accounts for about 20 percent and is a natural moisture-keeping component. Further, how to utilize the fat (fruit oil) in the flesh of the avocado is a big problem at present. The traditional biological fermentation method is mostly carried out in a water phase, the traditional fermentation process is incompatible with the avocado oil, and the avocado oil is difficult to stably exist in the avocado oil; the fermentation process of other avocados is mainly directed at the preservation of avocado flesh, and the fermented product is semisolid and cannot be used for the production of cosmetic raw materials.

Therefore, there is a need to develop a method for maximizing the utilization of the nutrients of avocados as a raw material for cosmetics.

Disclosure of Invention

In order to solve the problems of maximally utilizing the nutrient substances of the avocado and using the nutrient substances as cosmetic raw materials, the method disclosed by the invention is developed, and the avocado oil-in-water liquid fermentation product disclosed by the invention can be used for effectively converting proteins, nucleic acids, polysaccharides and the like into a small-molecule water-soluble fermentation system which is easy to absorb by skin, and meanwhile, the surfactant of probiotics is used for biologically refining the avocado oil and carrying the avocado oil. Namely, the avocado is converted into a stable mixed liquid fermentation product which takes polypeptide, amino acid, oligosaccharide, water-soluble microbial minerals and the like as a water phase and takes avocado oil coated by a surfactant as a lipid phase by utilizing the fermentation process, so that the maximization of the nutrient substances of the avocado is realized. The avocado oil-in-water liquid fermentation product obtained by the invention can be used for preparing cosmetics, has the effects of superior moisturizing performance and skin barrier enhancement, is more guaranteed in food source and safety, and can be used for pregnant women, lying-in women and infants without worry.

The first object of the present invention is to provide a method for preparing a avocado oil-in-water liquid fermentation product, which comprises: inoculating the microbial composite inoculant into a fermentation substrate taking avocado as a main component for fermentation culture to obtain the microbial composite inoculant; the microbial compound microbial inoculum is a mixed microbial inoculum containing the strain coated yeast and lactobacillus pentosus. Degrading polysaccharide and protein by using a yeast enzyme system in the fermentation process, and retaining the natural grease of the avocado; the oil droplets are coated with a surfactant produced by lactic acid bacteria.

The sacculus-covering yeast (Saccharomyces fibuligera) is a yeast with high protease yield obtained by screening from fermented grains of white spirit, is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC NO. 21512) within 21 days 12 months 2020, and has the preservation address of No. 3 Xilu 1 Beicheng Shangyang district in Beijing.

The Lactobacillus pentosus is obtained by screening fermented grains of white spirit, is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) within 21 days 12 months 2020, has the preservation number of CGMCC NO.21511, and has the preservation address of No. 3 North Lu No. 1 Chen of the sunward district in Beijing city.

Preferably, the avocado comprises avocado flesh and a stone; optionally, cleaning fresh avocado, removing pedicel, and breaking cell wall.

In one embodiment, the inoculation of the complex microbial inoculum is to inoculate a liquid microbial inoculum, and 5-15% of the complex microbial inoculum by volume is added into a fermentation substrate. Optionally, the inoculation amount of the complex microbial agent is 10%.

In one embodiment, the composite microbial inoculum is a mixed microbial inoculum of the strain capsule composite yeast and lactobacillus pentosus, wherein the volume ratio of the strain capsule composite yeast seed solution to the lactobacillus pentosus seed solution is 1. Optionally, the volume ratio is 1.

In one embodiment, the fermentation is at 28-32 ℃ for 25-30h. Alternatively, fermentation was carried out at 30 ℃ for 27h.

In one embodiment, the fermentation substrate is avocado 80-120 g.L ^-1 15-25 g.L of glucose ^-1 . Alternatively, avocado 100 g.L ^-1 Glucose 20 g.L ^-1 。

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

In one embodiment, the method of making comprises the steps of:

(1) Pulverizing avocado, sterilizing, and mixing with glucose to obtain a mixed solution;

(2) Adding the compound microbial liquid into the sterilized fermentation substrate, and fermenting for 27h at the temperature of 30 ℃; has been fermentedWhen the process is carried out in a fermentation tank, the ventilation rate is 4 sL.min ^-1 。

(3) Filtering by using a ceramic membrane, and collecting filtrate to obtain the avocado oil-in-water liquid state fermentation product.

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

The third purpose of the invention is to provide a Saccharomyces fibuligera strain suitable for whole-fruit fermentation of avocado, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC NO. 21512) within 21 months in 2020, and the preservation address is No. 3 Xilu No. 1 Beijing of the rising district of the Chaoyang district of Beijing.

The fourth purpose of the invention is to provide Lactobacillus pentosus (Lactobacillus pentosus) suitable for fermenting whole avocado, which is preserved in the common microorganism center of China general microbiological culture Collection Center (CCM) in 12 months and 21 days in 2020, with the preservation number of CGMCC NO.21511 and the preservation address of No. 3 of Xilu No. 1 of morning in the area of rising Yang in Beijing.

The fifth purpose of the invention is to provide the composite microbial inoculum suitable for fermenting the whole avocado, which is a mixed microbial inoculum of the Saccharomycopsis fibuligera with the preservation number of CGMCC NO.21512 and the Lactobacillus pentosus with the preservation number of CGMCC NO.21511.

The sixth purpose of the invention is to provide a preparation method of the avocado oil-in-water liquid fermentation product or an application of the avocado oil-in-water liquid fermentation product or the composite microbial inoculum in the aspect of preparing cosmetics.

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

In one embodiment, the avocado is fermented by selecting a proper strain to prepare an avocado oil-in-water liquid fermentation product, which has good lipid peroxidation resistance, cell drying damage repair capacity and good skin beautifying and protecting effects.

The seventh purpose of the invention is to provide a preparation method of the avocado oil-in-water liquid fermentation product or an application of the avocado oil-in-water liquid fermentation product or the composite microbial inoculum in the preparation of products with antioxidant and moisturizing effects.

Biological material preservation:

the capsule-covering yeast Z1-9 is classified as capsule-covering yeast Saccharomyces fibuligera, which is preserved in China general microbiological culture Collection center (CGMCC) on 21 days 12 months 2020, with the preservation number of CGMCC NO.21512 and the preservation address of No. 3 of Xilu No. 1 Beichen of the sunward area in Beijing.

Lactobacillus pentosus C-7, classified as Lactobacillus pentosus, has been deposited in China general microbiological culture Collection center (CGMCC) at 21.12.2020, with the deposition number of CGMCC NO.21511 and the deposition address of Beijing province No. 3, west Lu No. 1, beicheng Yang district.

Has the advantages that:

in the avocado oil-in-water liquid state fermentation product, the fat content can reach 0.11 g.100 ^-1 ·g ^-1 The protein content can reach 1813ppm, the amino acid content can reach 635ppm, the total phenol content can reach 148ppm, and the total sugar content can reach 4.68 g.L ^-1 (ii) a The fatty acid composition was 8.50% palmitoleic acid, 20.78% palmitic acid, 9.81% linoleic acid, 52.06% oleic acid, 7.96% vaccenic acid, 0.89% stearic acid; peptide concentration 9.68. + -. 0.22 g.L ^-1 Rich in small molecular oligopeptides; the composition is rich in important components of Natural Moisturizing Factor (NMF), wherein the concentration of alanine is 102.02ppm, the concentration of glutamic acid is 78.09ppm, and the concentration of aspartic acid is 49.46ppm; the lipid peroxidation inhibition rate of the 2.5 percent avocado oil-in-water liquid fermentation product is 23.29 percent, and the effect is close to 176ppmVC;1.25% avocado oil-in-water liquid fermentation product has approximately 10% glycerol for repairing dry damaged cells; 2.5 percent and 5.0 percent of avocado oil-in-water liquid fermentation products can respectively improve the cell repair effect of the dryness injury by 27.35 percent and 29.83 percent; green and safe, has no hemolytic effect, causes no adverse reaction to skin, and has no irritation.

The avocado oil-in-water liquid fermentation product can be used as a cosmetic raw material, is used for preparing cosmetics such as toner, essence, emulsion or cream and the like, and has the functions of resisting oxidation and preserving moisture.

Drawings

FIG. 1 is a comparison of polypeptide concentrations;

FIG. 2 is a comparison of the fat morphology of samples;

FIG. 3 is a comparison of appearance of samples;

FIG. 4 is a graph after centrifugation of an unfermented sample;

FIG. 5 is a GC-MS spectrum of a sample after fermentation;

FIG. 6 is a comparison of the molecular weight distributions of peptides from samples;

FIG. 7 is a comparison of amino acid concentrations before and after fermentation;

FIG. 8 shows the lipid peroxidation resistance of avocado oil-in-water liquid fermentation products;

FIG. 9 shows the repairing effect of avocado oil-in-water liquid fermentation product on dry damaged cells (n = 3); note: * Indicating a significant difference in dry damage protection effect from 10% glycerol (p < 0.05) as calculated by ANOVA-Dunnett.

Detailed Description

The present invention will be described in further detail with reference to examples, but the present invention is not limited thereto.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

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

The capsule coated yeast (Saccharomyces fibuligera) used in the embodiment is screened from fermented grains of white spirit, and the preservation number is CGMCC NO.21512; lactobacillus pentosus (Lactobacillus pentosus) is screened from fermented grains of white spirit, and the preservation number is CGMCC NO.21511.

In the following examples, the YPD liquid medium formulation was: 10.0g of yeast powder, 20.0g of peptone, 20.0g of glucose, 6.5-6.7 of pH and 1000mL of deionized water. The YPD solid culture medium is YPD liquid culture medium added with 2% agar powder. The culture medium components are prepared and sterilized at 121 deg.C for 20min.

The following embodimentsIn the examples, the formulation of the MRS liquid medium is as follows: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast powder, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0g of tween, 5.0g of sodium acetate trihydrate and K ₂ HPO ₄ ·3H ₂ O 2.0g，MgSO ₄ ·7H ₂ O 0.58g，MnSO ₄ ·H ₂ 0.25g of O, 6.2 to 6.6 of pH and 1000mL of deionized water. The MRS solid culture medium is prepared by adding 2% agar powder into MRS liquid culture medium. The prepared components of the culture medium are sterilized at 121 ℃ for 20min.

EXAMPLE 1 high throughput Breeding of high protease producing Yeast

A strain of Saccharomycopsis fibuligera for producing protease with high yield is obtained by screening in the fermentation process of Chinese liquor as follows.

The solid culture medium for strain screening comprises the following components: peptone 20.0g, yeast powder 1.0g, skimmed milk 10.0g, glucose 5.0g, KH ₂ PO ₄ 1.0 g，NaH ₂ PO ₄ 2.4 g, agar 20.0g, pH 6.8, make up to 1000mL of deionized water. The prepared components of the culture medium are sterilized at 121 ℃ for 20min.

1. Preliminary screening of bacterial strains

Dissolving 0.1g of fermented grains of white spirit in 1mL of sterile physiological saline, adding glass beads, and uniformly oscillating. Diluting 10 times, spreading 200 μ L of the mixed solution on strain screening solid culture medium, and standing at 30 deg.C for 2d.

2. Rescreening of bacterial strains

Selecting strains with transparent circles, separating and purifying, selecting single colony, inoculating into YPD liquid culture medium, and culturing at 200 r.min ^-1 And cultured for 2d under shaking at 30 ℃. Centrifuging the fermentation liquid at 10000rpm for 10min, and collecting supernatant to determine protease activity.

Determination of protease activity: the method of GB/T23527-2009 protease preparation is adopted. 1mL of the crude enzyme solution was mixed with 1mL of the substrate (casein solution) uniformly, reacted at 40 ℃ for 10min, then 2mL of trichloroacetic acid was added to terminate the reaction, and the mixture was centrifuged at 12000rpm for 3min. 5mLNa was added to 1mL of the supernatant ₂ CO ₃ And 1 mLFolin-phenol reagent, uniformly mixing, keeping the temperature at 40 ℃ for 20min, and measuring the absorbance value at the position with the wavelength of 660nm by adopting an ultraviolet-visible spectrophotometer. The group in which the reaction was terminated by adding trichloroacetic acid first was used as a control group. Enzyme activity is defined as follows: 1mL of the solution to be tested, and in the environment of 40 ℃, the enzyme amount required by catalyzing casein to generate and is equal to 1 mu g of tyrosine per minute, namely one enzyme activity unit (U).

3. Screening results for protease-producing bacteria

A plurality of strains producing protease are obtained by screening from fermented grains of white spirit, the protease activity of the strains is measured, and the result is shown in table 1. By comparing the enzyme activities of the fermentation liquids of the strains, a high-yield protease strain Z1-9 is screened, and the primary screening protease activity of the strain reaches 82.1 U.mL ^-1 。

TABLE 1 screening results of protease-producing bacteria

4. Identification of the strains

(1) Bacterial colony characteristics and bacterial morphology

Bacterial colonies of the strain Z1-9 are round, white, villous, raised and irregular in edge. The shape result of microscope observation shows that the separated and screened strain cells are nearly elliptical and have different sizes, and part of the strain cells are budding and splitting.

(2) Molecular biological assay

Inoculating the strain Z1-9 to YPD culture medium, culturing for 1d, and extracting the total DNA of the strain as a PCR template. The yeast ITS universal primer is adopted for PCR amplification, and the selected universal primer is ITS1 (the nucleotide sequence is 5 'TCCGTAGGTGAACCTGCGG-3', the sequence is shown as SEQ ID NO: 1) and ITS4 (the nucleotide sequence is 5 'TCCTCCGCTTATTGATGC-3', the sequence is shown as SEQ ID NO: 2). PCR amplification conditions: cycling for 30 times at 94 ℃ for 5min,94 ℃ for 30s,55 ℃ for 30s, and 72 ℃ for 1 min; 10min at 72 ℃. The PCR amplification product is passed through 1% agarose gel test and then sent to Suzhou Jinzhi Biotechnology limited for sequencing. Sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database for BLAST alignment and the strain was found to be Saccharomyces fibuligera.

The strain Z1-9 is preliminarily identified as the saccharomyces cerevisiae (Saccharomyces fibuligera) by combining the colony morphological characteristics, physiological and biochemical characteristics and ITS sequence analysis. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC NO.21512.

Example 2 screening of biosurfactant-producing strains

The white spirit fermentation is a mixed fermentation process, wherein abundant microorganisms are adapted to each other in a long-term domestication process and play a role in a synergistic manner. The microbes screened in the same environment can better perform synergistic action. Therefore, screening of the surfactant-producing strains was performed from the growth environment of the yeast of example 1.

The biosurfactants currently found include many different classes, such as glycolipids, lipopeptides, polysaccharide-lipid complexes, phospholipids, fatty acids, neutral lipids, and the like. Different surfactants have different ingredients, and not all surfactants are suitable for inclusion in shea butter. Therefore, the surfactant-producing strains were screened in a medium supplemented with shea butter as follows.

Avocado oil liquid medium: 8.0g of avocado oil, 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast powder, 2.0g of diammonium hydrogen citrate, 20.0g of glucose and K ₂ HPO ₄ ·3H ₂ O 2.0g，MgSO ₄ ·7H ₂ O 0.58g，MnSO ₄ ·H ₂ O0.25g, pH 7.0, make up 1000mL of deionized water. The culture medium components are prepared and sterilized at 121 deg.C for 20min.

1. Plate separation

Adding 3.5g of sample into a triangular flask containing 35mL of sterile physiological saline, adding glass beads, and uniformly oscillating to obtain fermented grain suspension. Inoculating the fermented grain suspension into a avocado oil liquid culture medium in an inoculation amount of 5% (V/V), performing shake culture at 37 ℃, properly diluting the culture solution, coating the diluted culture solution on an MRS solid plate culture medium when the emulsification phenomenon of the culture medium in a triangular flask is observed, and performing constant-temperature culture at 37 ℃ for 2d.

2. Shaking bottle prescreening

Inoculating the single colony obtained by primary screening to avocado oil liquid culture medium respectively, and culturing at 37 deg.C for 200r min ^-1 Shake flask shake culture, select the good bacterial strain of emulsification of avocado oil, get 4 bacterial strains.

3. Oil discharge ring method double screen

Inoculating 4 strains with avocado oil emulsifying effect into MRS liquid culture medium respectively, and culturing at 37 deg.C for 200r min ^-1 Shake flask for shake culture for 2d, and measure the diameter of oil drain ring of supernatant after centrifugation.

Oil draining method: and (4) centrifuging the fermentation liquor at 4000rpm for 10min, and taking supernatant for later use. Taking a culture dish with the diameter of 9cm, adding 30mL of distilled water, dropwise adding 0.2mL of avocado oil on the water surface, dropwise adding 0.2mL of fermentation liquid supernatant diluted by 2 times into the center of the oil film after the oil film is formed, and measuring the diameter of the oil discharge ring after a stable oil discharge ring is formed.

4. Results of oil-ring method

The oil drain diameters of the 4 strains obtained by the primary screening were compared, the amounts of the biosurfactant-producing ability thereof were compared, and the results of the secondary screening are shown in Table 2. Compared with other strains, the strain C-7 has the largest diameter of the oil discharge ring of the fermentation liquid and the largest amount of biosurfactant.

TABLE 2 screening results of biosurfactant-producing strains

5. Identification of strains

(1) Bacterial colony characteristics and bacterial morphology

The bacterial colony of the strain C-7 is tiny round, white, convex and neat in edge. The shape result of the strain cells obtained by separation and screening is observed by a microscope, and the strain cells are rod-shaped, short-chain-shaped and spore-free.

(2) Molecular biological assay

Inoculating the strain C-7 to an MRS culture medium, culturing for 1d, and extracting the total DNA of the strain as a PCR template. The bacterial 16S universal primer is used for PCR amplification, and the universal primers are 27F (the nucleotide sequence is 5 '-AGAGTTTGATCTMTGGCTCAG-3', the sequence is shown as SEQ ID NO: 3) and 1492R (the nucleotide sequence is 5 '-TACGGTATTGATCTT-3', the sequence is shown as SEQ ID NO: 4). PCR amplification conditions: circulating for 30 times at 94 deg.C for 10min, 55 deg.C for 1min, and 72 deg.C for 2 min; 10min at 72 ℃. The PCR amplification product is passed through 1% agarose gel test and then sent to Suzhou Jinzhi Biotechnology limited for sequencing. Sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database for BLAST alignment and the strain was found to be Lactobacillus pentosus.

The strain C-7 is preliminarily identified as Lactobacillus pentosus (Lactobacillus pentosus) by combining the colony morphological characteristics, physiological and biochemical characteristics and 16S sequence analysis. The strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.21511.

Example 3 preparation of avocado oil-in-water liquid fermented product

1. Preparation of buckled membrane yeast seed liquid

Under aseptic conditions, single colonies of Saccharomyces cerevisiae Z1-9 were picked and inoculated into YPD liquid medium. 30 ℃ and 220r min ^-1 Shake culturing for 24h until the bacterial amount reaches 10 ⁸ CFU·mL ^-1 And then seed liquid is obtained.

2. Preparation of Lactobacillus pentosus seed liquid

Under aseptic operation conditions, single colony of Lactobacillus pentosus (Lactobacillus pentosus) C-7 is picked and inoculated into MRS liquid medium. 37 ℃ and 200r min ^-1 Shake culturing for 24h until the bacterial amount reaches 10 ⁸ CFU·mL ^-1 Then, the seed liquid is obtained.

3. Stock preparation

Cleaning fresh avocados with tap water, cleaning with deionized water, draining, and removing pedicels; adding the avocado into a wall breaking machine, adding deionized water with the mass ratio of 3 times, and crushing until no large visible solid particles exist.

4. Preparation of fermentation substrate

Avocado 100 g.L ^-1 Glucose 20 g.L ^-1 . Sterilizing at 121 deg.C for 20min to obtainAnd cooling the fermentation substrate for later use. Wherein glucose is separately sterilized and added to the system.

5. Inoculating fermentation

Adding 10% of compound microbial agent by volume into the sterilized slurry, and fermenting at 30 deg.C for 27h; the composite microbial agent is a mixed microbial agent of the strain capsule and membrane-covered yeast Z1-9 and lactobacillus pentosus C-7, and is prepared by mixing strain capsule and membrane-covered yeast seed liquid and lactobacillus pentosus seed liquid according to the volume ratio of 1; the ventilation rate is 4 sL.min when the fermentation process is carried out in a fermentation tank ^-1 。

6. Microfiltration

Filtering by using a ceramic membrane, and collecting filtrate to obtain the avocado oil-in-water liquid fermentation product.

Comparative example 1 avocado extract

The only difference from example 3 is that: the compound microbial inoculum is not inoculated.

Comparative example 2

The only difference from example 3 is that: the yeast Z1-9 is used singly for fermentation.

Comparative example 3

The only difference from example 3 is: the fermentation is carried out by using the lactobacillus pentosus C-7 alone.

Comparative example 4

The only difference from example 3 is that: the yeast CICC 33226 of the invention is used to replace the yeast of the invention and is mixed with the lactobacillus of the invention for fermentation.

Wherein, the Saccharomycopsis fibuligera CICC 33226 is purchased from China industrial microorganism strain preservation management center and can produce protease by a transparent circle method.

Comparative example 5

The difference from example 3 is that: the yeast CICC 1717 with the envelope of capsules is used for replacing the yeast of the invention and is mixed with the lactobacillus of the invention for fermentation.

Wherein, the Saccharomycopsis fibuligera CICC 1717 is purchased from China center for culture Collection of industrial microorganisms, and can produce protease by a transparent circle method.

Comparative example 6

The difference from example 3 is that: saccharomyces cerevisiae CICC 1210 is used for replacing the yeast of the invention and is mixed with the lactobacillus of the invention for fermentation.

Wherein the saccharomyces cerevisiae CICC 1210 is purchased from China industrial microorganism strain preservation management center and can produce protease by a transparent circle method.

Comparative example 7

The difference from example 3 is that: lactobacillus pentosus CICC 24202 is used to replace the lactobacillus of the invention, and is mixed with the yeast of the invention for fermentation.

Wherein the lactobacillus pentosus CICC 24202 is purchased from China center for preservation and management of industrial microorganism strains.

Comparative example 8

The difference from example 3 is that: lactobacillus paracasei CICC 20252 is used instead of the Lactobacillus of the invention, and mixed with the yeast of the invention for fermentation.

Wherein the Lactobacillus paracasei CICC 20252 is purchased from China center for culture Collection of industrial microorganisms. Lactobacillus paracasei has been reported to produce biosurfactants (see in particular references: ferreira A, vecino X, ferreira D, et al. Novel cosmetic formulations a biosurfactant from Lactobacillus paracasei.2017,155: 522-529.).

Example 4 testing of avocado fermentation products

From dimensions such as polypeptide content, loading condition of the avocado oil, active matter content, fatty acid component, molecular weight distribution of the polypeptide and the like, the avocado oil-in-water liquid fermentation product prepared in example 3 and the avocado fermentation products obtained in comparative examples 1 to 8 are measured.

1. Comparison of polypeptide content

1. Preparation of the test specimens

The avocado oil-in-water liquid fermentation product prepared in example 3 and comparative examples 1 to 8 were used as samples to be tested.

2. Total peptide assay

The total polypeptide content in the sample was determined with reference to the trichloroacetic acid Method in combination with the Lowry Method:

2.5mL of sample to be tested is taken, and 2.5mL of 10% trichloroMixing acetic acid water solution on vortex mixer, standing for 20min, and then 3500 r.min ^-1 Centrifuging for 10min, and removing protein precipitate. Supernatants were taken and the concentration of total peptide was determined according to the Lowry Method.

3. Comparison of Total polypeptide content of samples

A comparison of the total polypeptide content of the samples is shown in FIG. 1. The concentration of the polypeptide of comparative example 1 without microbial fermentation treatment is 874.03. + -. 18.46ppm. The highest polypeptide content after fermentation is example 3 of the present invention, with a concentration of 1330.64. + -. 36.90ppm, which is a 52% increase over the extract obtained in comparative example 1 without fermentation treatment.

The concentration of the polypeptide of comparative example 3 is similar to that of unfermented comparative example 1, indicating that fermentation using lactobacillus pentosus C-7 alone cannot hydrolyze the protein in avocado to small molecule polypeptides.

The polypeptide concentrations of comparative examples 4 to 6 were increased compared to the unfermented comparative example 1, but were far less than the yeast used in example 3. The Saccharomycopsis fibuligera Z1-9 is more suitable for the avocado fermentation system for fully utilizing the avocado pulp and the kernel.

The polypeptide content of comparative example 8 was lower than that of example 3, and Lactobacillus paracasei CICC 20252 affected the exertion effect of the Fungiella fibuligera Z1-9, and Lactobacillus paracasei CICC 20252 could not replace Lactobacillus pentosus C-7 of the present invention.

2. Avocado oil loading conditions

1. Preparation of the test specimens

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

2. Nile Red dyeing

Nile red can bind to lipid materials 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 microscopic State of avocado oil

As can be seen from FIG. 2, compared with the comparative example 1 without fermentation, the oil droplets of the example 3 are distributed more uniformly, the loading effect of the shea butter is the best, and an oil-in-water microstructure can be formed. The shea butter of comparative example 2 is not encapsulated, indicating that the fruit oil in shea butter cannot be encapsulated by using the envelope-covering yeast Z1-9 for fermentation alone. The shea butter of comparative example 7 is not entrapped, which shows that Lactobacillus pentosus CICC 24202 can not replace Lactobacillus pentosus C-7 of the invention, and Lactobacillus pentosus C-7 is more suitable for the shea butter fermentation system of the invention.

3. Method for measuring active matter content of avocado oil-in-water liquid fermentation product

The avocado oil-in-water liquid fermentation product prepared in the embodiment 3 of the invention is used as a sample to be tested.

Total protein content in the samples was determined with reference to Lowry Method: bovine serum albumin solutions of different concentrations were prepared to draw a standard curve. 0.5mL of bovine serum albumin solution was added to 5mL of basic copper solution. After standing at room temperature for 20min, 0.5mL of 50% by mass Folin's phenol reagent was added to the above solution. Standing at room temperature for 30min, selecting 650nm wavelength, and measuring absorbance. A standard curve is plotted with the absorbance as the ordinate and the concentration of bovine serum albumin solution (unit: ppm) as the abscissa as follows: y =0.0008x +0.0125, and R2=0.9956. And (4) measuring the absorbance of the sample to be measured according to the method, and substituting the absorbance into the standard curve to calculate the protein content.

And (3) determining the content of the total amino acids in the sample by referring to a ninhydrin color development method: glutamic acid solutions of different concentrations were prepared to draw a standard curve. 2mL of glutamic acid solution, 0.5mL of PBS buffer solution and 0.5mL of 2% ninhydrin solution were added to the test tube, and the mixture was heated in a boiling water bath for 20min. The wavelength of 570nm was selected and the absorbance was measured. A standard curve was plotted with the absorbance as the ordinate and the concentration of glutamic acid solution (unit: ppm) as the abscissa as: y =0.0002x ² -0.0086x+0.0372，R ² =0.9991. And (4) measuring the absorbance of the sample to be measured according to the method, and substituting the absorbance into the standard curve to calculate the total amino acid content.

The method for measuring the total phenol content of the sample comprises the following steps: preparing gallic acid solutions with different concentrations to draw a standard curve. Adding 100 μ L gallic acid solution, 1.0mL forskolin phenol, 1.0mL 10% Na in test tube ₂ CO ₃ Reacting at normal temperature for 90min. Measuring with a wavelength of 760nmAnd (5) determining the absorbance. A standard curve was plotted with the absorbance as the ordinate and the concentration of gallic acid solution (unit: ppm) as the abscissa, and was: y =0.0075x+0.0396 ² =0.999. And (4) measuring the absorbance of the sample to be measured according to the method, and substituting the absorbance into the standard curve to calculate the total phenol content.

The method for measuring the total sugar content of the sample comprises the following steps: glucose solutions of different concentrations were prepared to draw a standard curve. 1.0mL of 5% aqueous phenol solution, 1.0mL of glucose solution, and 5.0mL of concentrated sulfuric acid were added to a test tube, shaken well, and reacted in boiling water for 25min. The absorbance was measured at 490 nm. The absorbance was plotted as the ordinate against the concentration of the glucose solution (unit: g. L) ^-1 ) The standard curve is plotted for the abscissa as: y =7.2226x-0.0262, R2=0.997. And (4) measuring the absorbance of the sample to be measured according to the method, and substituting the absorbance into the standard curve to calculate the total sugar content.

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

TABLE 3 avocado oil-in-water liquid fermentation product active content obtained in example 3

As can be seen from Table 3, the avocado oil-in-water liquid fermentation product obtained in example 3 is rich in protein, amino acids, phenols and sugars, which indicates that the fermentation method of the present invention can obtain a fermentation raw stock with high active ingredients.

4. Fat contrast

1. Preparation of the test specimens

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

2. Determination of the Total fat content

The total fat content in the sample was determined by reference to the second method of GB 5009.6-2016:

about 10g of sample to be tested is weighed to 0.001g, placed in a 50mL test tube, and 10mL of hydrochloric acid is added. The test tube is put into a water bath with the temperature of 70-80 ℃, and the glass rod is used for stirring once every 5-10 min until the sample is completely digested, which lasts for about 40-50 min.

The tube was removed, 10mL of ethanol was added, and mixed. After cooling, the mixture was transferred into a 100mL stoppered graduated cylinder, and the tube was washed several times with 25mL dry ether and poured into the graduated cylinder. After the anhydrous ether is completely introduced into the measuring cylinder, adding the plug, shaking for 1min, carefully opening the plug, discharging gas, plugging, standing for 12min, carefully opening the plug, and washing the plug and fat attached to the opening of the measuring cylinder with the anhydrous ether. Standing for 10-20 min, sucking supernatant liquid out of a cone with constant weight when the upper liquid is clear, adding 5mL of anhydrous ether into a measuring cylinder with a plug, shaking, standing, sucking the upper layer of ether out of the cone, and putting the cone into the original cone.

And recovering the anhydrous ether, evaporating to dryness on a water bath when 1-2 mL of the solvent remains, drying at 100 +/-5 ℃ for 1h, cooling in a dryer for 0.5h, and weighing. The above procedure was repeated until the weight was constant (until the difference between the two weighings did not exceed 2 mg).

The fat content of the sample is calculated as follows:

in the formula:

x-the fat content in the sample, in g.100 ^-1 ·g ^-1 ；

m ₁ -receiving the content of bottles and fat after constant weight in g;

m ₀ -receiving the mass of the bottle in g;

m ₂ -mass of the sample in g;

3. comparison of the Total fat content of the samples

The results are shown in Table 4.

Table 4 comparison of total fat content of samples

As can be seen from Table 4, avocado is processedThe fat content in the fermented puree obtained in example 3 after fermentation treatment was 0.11g 100 ^-1 ·g ^-1 The extract obtained in comparative example 1 without fermentation treatment had a fat content of 0.15g 100 ^-1 ·g ^-1 It is stated that the shea butter (fat) remains after fermentation.

4. Comparison of sample fat stability

The appearance of the test sample after being left at 4 ℃ for 1 week is shown in FIG. 3.

As shown in FIG. 3, the extract obtained in comparative example 1 (left) without fermentation treatment has fat precipitation after standing for one week, while the fermented puree obtained in example 3 (right) after the avocado is subjected to fermentation treatment has more stable fat and has no precipitation phenomenon. The lactobacillus pentosus C-7 in the mixed microbial inoculum generates a biosurfactant in the fermentation process, and entraps fat, so that the fermentation liquor is more stable and uniform, and the most valuable components are ensured to exert the moisturizing effect. The fermentation method of the invention can obtain products with more stable components, and is a more scientific and effective method.

5. Analysis of fatty acid composition

1. Preparation of the test specimens

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 Xg for 10min.

As shown in FIG. 4, after the unfermented sample is centrifuged, a layer of grease substances exists on the liquid surface, and the fat is not entrapped and is easy to separate out. And the fermented sample oil is wrapped by the surfactant, so that the oil is not easy to separate out, and the upper layer does not contain oil after centrifugation.

And taking down the liquid of the layer as a sample to be detected.

2. Lipid extraction

50mL of the liquid to be tested is taken and 50mL of dichloromethane is added to extract the lipid of the sample to be tested. And (4) taking a dichloromethane phase, and evaporating dichloromethane to obtain an oil sample.

3. Lipid saponification

25mL of 1.2 mol. L was added to the oil sample ^-1 Condensing and refluxing the KOH solution at 65 ℃ for 2 hours; cooling the reaction solution, and extracting with 10mL of n-hexane for 3 times; taking the water phase.

4. Methyl esterification of fatty acids

Adding 10mL of hydrochloric acid and 2mL of methanol into the water phase, and condensing and refluxing for 1h at 70 ℃; cooling the reaction solution, extracting for 3 times by using 10mL of normal hexane, and volatilizing to be dry; adding 15mL of anhydrous methanol and 1-2 drops of concentrated sulfuric acid, and carrying out condensation reflux for 20min at 80 ℃; cooling the reaction solution, adding 10mL of saturated NaCl aqueous solution and 10mL of normal hexane, and extracting; taking the n-hexane phase, and using the n-hexane to fix the volume to 2mL.

5. Determination of fatty acids

The fatty acid composition of the above samples was analyzed by GC-MS. Chromatographic conditions are as follows: and (3) chromatographic column: HP-88 elastic quartz capillary column (100 m × 0.25mm,0.25 μm); the temperature raising program is that the initial temperature is kept for 2min at 150 ℃, and the temperature is kept for 2 ℃ min ^-1 Heating to 210 deg.C, 50 deg.C/min ^-1 Heating to 250 deg.C and keeping for 10min. The sample inlet temperature is 250 ℃, the sample injection amount is 1 mu L, and the total flow rate is 35.6 mL/min ^-1 The carrier gas is N ₂ (ii) a The split ratio is 50: 1. Qualitative and quantitative analysis: qualitative analysis was performed using NIST 14 and wiley9.0 mass spectrum database, and the relative percentages of each component were calculated using area normalization.

6. Content comparison

And separating out grease from an unfermented sample after centrifugation, wherein no grease exists in the lower layer liquid, and no fatty acid is detected by GC-MS. The GC-MS spectrum of the sample after fermentation is shown in FIG. 5.

The fatty acid composition and relative percentage of the avocado oil-in-water liquid fermentation product are shown in table 5. The fat content of the avocado oil-in-water liquid fermentation product is measured at the early stage and is 0.15 g.100 ^-1 ·g ^-1 The concentrations were estimated based on the relative percentage of fatty acids, see table 5.

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

The avocado oil-in-water liquid fermentation product contains oleic acid at the maximum content of 52.06 percent of the fatty acid composition and has the concentration of 572.66ppm. Oleic acid inhibits the activity of the capsaicin receptor TRPV1, thereby alleviating pain and itching associated with external stimuli (see in particular references: morales-L zaro Sara L, llorente Itzel, sierra-Rami i rez F elix, et al. Inhibition of TRPV1 channels by energy absorption apparatus omega-9 fatty acid reduction pain and issue.2016, 7. Furthermore, oleic acid and essential fatty acids in avocados promote wound healing and help reduce inflammation during healing (see in particular references: sales-Campos Helioswitton, souza Patrica Reis, peghini

Crema, et al, an overview of modular effects of oleic acid in height 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 Signal molecule AI-2 (see in particular references: K.W.Widmer, K.A.Soni, M.E.Hume, et al.identification of Poultry mean-Derived Fatty Acids functional as Square Sensing Signal Inhibitors to Autoinducer-2 (AI-2). 2007,72 (9): M363-M368.). AI-2 is a signaling molecule for information exchange between bacterial species and is 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. Polypeptide molecular weight distribution comparison

1. Preparation of the test specimens

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

2. Peptide molecular weight distribution analysis

The sample to be tested was filtered using a 0.22 μm microfiltration membrane and its molecular weight distribution was determined by high performance liquid size exclusion chromatography (SEC-HPLC) under the following conditions: the column was TSK gel 2000SWXL (300 mm. Times.7.8 mm); the column temperature is 30 ℃;the mobile phase is acetonitrile: water: trifluoroacetic acid = 45; the flow rate was 0.5 mL/min ^-1 (ii) a The detection wavelength was 220nm.

3. Comparison of sample peptide molecular weight distribution

The results of the comparison of the molecular weight distributions of the peptides of the samples are shown in FIG. 6.

As can be seen from FIG. 6, the oligopeptide content of the fermented puree obtained in example 3 after fermenting avocado was increased as compared to the extract obtained in comparative example 1 without fermentation treatment. Specific molecular weights and percentages are shown in tables 6 and 7.

TABLE 6 peptide molecular weight distribution of avocado oil-in-water liquid fermentation product obtained in example 3

TABLE 7 peptide molecular weight distribution of avocado extract obtained in comparative example 1

Comparing tables 6 and 7, it was found that peptides having molecular weights of 1077, 650, 288 were added to the avocado oil-in-water liquid fermented product compared to the extract. In addition, the content of peptide having a molecular weight of 184 was also significantly increased. Compared with high molecular natural bioactive substances such as nucleic acid, protein and the like, the polypeptide is easy to dissolve in water, has small molecular weight, is easy to absorb and utilize, has far better performance than protein with larger molecules in the aspects of whitening, antioxidation, skin repair and other efficacies, and has relative safety and stability. The results show that the fermentation can degrade the protein of the avocado into small-molecule oligopeptide, so that the oligopeptide is easier to absorb.

7. Comparison of amino acid content

1. Preparation of the test specimens

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

2. Determination of amino acid content

The content of free amino acids in the sample was measured by using a high performance liquid chromatograph, and a Diamonsil C18 (250 mm. Times.4.6 mm,5 μm) column. Pre-test samples were subjected to OPA-FMOC pre-column derivatization. The mobile phase A is 27.6 mmol.L ^-1 Sodium acetate-triethylamine-tetrahydrofuran (50, volume ratio). The mobile phase B is 80.9 mmol.L ^-1 The sodium acetate-methanol-acetonitrile mixture (1. The ultraviolet detection wavelength is 338nm, and the flow rate is 1 mL/min ^-1 The column temperature was 40 ℃ and the amount of sample was 10. Mu.L.

3. Comparison of amino acid content of samples

A comparison of the amino acid content of the samples is shown in FIG. 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 12.04ppm, which was 2.67% of the total amino acids; the concentration of alanine in the fermented puree obtained in example 3 after the avocado is subjected to fermentation treatment is 102.02ppm and accounts for 21.62% of the total amino acids. Compared with the unfermented sample, the alanine concentration after fermentation is improved by 8.48 times and is the amino acid with the highest content in the fermentation liquor.

Example 4 use of avocado oil-in-water liquid fermentation product in cosmetics

The avocado oil-in-water liquid fermented product prepared in example 3 was analyzed for efficacy, safety, and the like in the preparation of cosmetics.

1. Anti-lipid peroxidation

1. Preparation of the test specimens

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

2. Anti-lipid peroxidation experiment

To the sample tube were added 1mL of 50mM PBS (pH 7.4), 1mL of 2% lecithin solution, 1mL of sample solution, and 1mL of 5mM EDTA-Fe (II) in this order, followed by incubation in a water bath at 37 ℃ for 45min.

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

In the formula:

a-absorbance of the sample set;

c-the control group changes the sample to its solvent, absorbance;

B ₁ blank 1 No EDTA-Fe (II) was added to the control, absorbance

B ₂ When the sample contains lipid, blank group 2 is additionally arranged, namely lecithin is not added on the basis of the sample group, and the absorbance

3. Avocado oil-in-water liquid fermentation product with anti-lipid peroxidation effect

The efficacy of the avocado oil-in-water liquid fermentation product against lipid peroxidation is shown in figure 8. The lipid peroxidation inhibition rate of 2.5% avocado oil-in-water liquid fermentation product (the liquid fermentation product of example 3 is diluted with deionized water to 2.5% by volume, the same is applied below) is 23.29 + -2.67%, 176ppm V _C The lipid peroxidation inhibition rate of (ascorbic acid; vitamin C) is 23.35 +/-3.26%, which shows that the lipid peroxidation inhibition effect of the 2.5% avocado oil-in-water liquid fermentation product is close to 176ppm V _C 。

2. Repairing effect of cell desiccation and damage

1. Experimental materials

Cell line (b): haCaT human immortalized keratinocytes

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

2. Culture of cells

1) Culture of cells

The cultured cells were digested, centrifuged, and counted using a hemocytometer in a 1.5X 10 format ⁵ Each is mL ^-1 The concentration of (2) was diluted and added to a 96-well plate, and 100. Mu.L of cell liquid was added to each well to reach 15000 cells per well. Blowing and beating are taken care of in the plate laying process, and the influence caused by cell sedimentation is reduced. Placing 96-well plate at 37 deg.C, 5% ₂ Culturing in an incubator for 24-30 h.

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 a solid sample previously dissolved with PBS. The low-concentration sample is diluted according to a proportion, and PBS is added firstly and then the high-concentration sample solution is added. The blank and lesion groups were added with 100. Mu.L of PBS solution. Incubate in incubator for 30 min.

At least three parallel wells are provided for each sample concentration, and 6 parallel wells are provided for the blank group and the lesion group.

3) Addition of SDS

After the incubation time was completed, 100. Mu.L of 50. Mu.g.mL was added to each well ^-1 SDS into PBS to a final concentration of 25. Mu.g.mL ^-1 . The blank was added with an equal amount of PBS solution. The 96-well plate was placed in an incubator and incubated for 20 minutes.

4) Determination of cell viability

After incubation was complete, the liquid in the well plate was discarded. Washing was performed 1 to 2 times using a PBS solution. 150 μ L of 0.5 mg/mL was added to each well ^-1 MTT-Medium solution (1). After incubation for 4h, the solution is discarded, and DMSO is added to dissolve the formazan crystals. And (3) measuring the absorbance value at 490nm after shaking for 10min in a microplate reader, and processing the data into cell viability values%.

3. Repairing effect of avocado oil-in-water liquid fermentation product on dry damaged cells

The repairing effect of the avocado oil-in-water liquid fermentation product on dry damaged cells is shown in figure 9. 1.25% avocado oil-in-water liquid fermentation product repairs approximately 10% glycerol on dry damaged cells. The cell repair effect of the avocado oil-in-water liquid fermentation products with the concentration of 2.5 percent and 5.0 percent on drying damage can be respectively improved by 27.35 percent and 29.83 percent.

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

1. Haemolysis test of erythrocytes

(1) Experimental Material

Pig red blood cells (2%, beijing Boersui technologies, inc.);

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

(2) Experimental method

Control group of total hemolysis: water + porcine red blood cells

Negative control group: PBS + porcine red blood cells

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

Hemolysis test: the sample was dispersed in PBS or physiological saline, and the red blood cells and the sample were mixed uniformly at a ratio of 2 ^-1 Centrifuging for 3min, and taking supernatant to test absorbance at 540 nm. The hemolysis rate is calculated as: HD% = (a) _{540 sample} -A _540PBS )/(A _{540 Water} -A _540PBS )×100％

In HD ₅₀ Setting 5 points up and down, setting standard curve with sample concentration as abscissa and HD as ordinate, calculating HD ₅₀ 。

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

The avocado oil-in-water liquid fermentation product has no hemolytic effect under the maximum experimental concentration (90 percent, a certain amount of PBS needs to be added into liquid to remove hemolysis caused by osmotic pressure difference between inside and outside cells)Thus HD ₅₀ >90％。

2. Human body skin patch experiment (closed type)

(1) Experimental method

According to the method specified in 2015 cosmetic specifications.

The volunteer requires: 30 (20-40 years old) normal subjects, patients who do not have clinical unhealed inflammatory dermatosis, judges that the skin to-be-tested part has no scar, pigment, atrophy, port nevus flammeus or other flaws, does not participate in other clinical test researchers, patients who have no high-sensitivity physique, and patients who have not been subjected to a spot test in the last month.

Sample group: 40% avocado oil-in-water liquid fermentation product

Negative control group: deionized water

The solutions are added on a filter paper sheet attached to a spot tester and placed in the spot tester, the dosage is about 0.020-0.025 mL, the spot tester added with the tested object is pasted on the curved side of the forearm by using a non-irritant adhesive tape, and the patch is evenly pasted on the skin by lightly pressing with the palm for 24 hours. And (5) removing the tested substance spot tester for 30min, and observing skin reaction after the indentation disappears. If the result is negative, the reaction result is recorded by observing once more at 24h and 48h after the patch test.

(2) Results

TABLE 8 human skin patch test results (closed type)

As shown in table 8, 30 human plaque experimental subjects showed a grade 0 reaction, and according to the regulation of 2015 cosmetic hygiene specifications, 40% avocado oil-in-water liquid fermentation product did not cause adverse reactions to human skin, and thus the product was found to have high human safety.

3. Human body skin spot test (open type)

(1) Experimental methods

According to the method specified in 2015 cosmetic specifications.

Volunteers require: 30 (20-40 years old) normal subjects, patients who do not have clinical unhealed inflammatory dermatosis, judges that the skin to-be-tested part has no scar, pigment, atrophy, port nevus flammeus or other flaws, does not participate in other clinical test researchers, patients who have no high-sensitivity physique, and patients who have not been subjected to a spot test in the last month.

Sample group: 40% avocado oil-in-water liquid fermentation product

Negative control group: deionized water

The dosage of the sample is about 0.020-0.025 mL, the tested part is the curved side of the forearm, and the test area is 3 multiplied by 3cm ² . Applied twice daily for 7 consecutive days.

(2) Results

TABLE 9 human skin Patch test results (open type)

As shown in table 9, 30 subjects in the human body patch test all have a grade 0 reaction, and 40% of the avocado oil-in-water liquid fermentation product does not cause adverse reactions on human skin according to the specification of 2015 cosmetic hygiene specifications, so that the product has high human safety.

4. Chick embryo chorioallantoic membrane blood vessel test

(1) Experimental Material

Chicken embryo: SPF-grade White Lai Hangzhou chickens (White Leghorn chicken) were purchased from Experimental animals technology, inc., meiliya Viton, beijing.

The culture conditions are as follows: the temperature of the incubator is 37.5 +/-0.5 ℃, and the relative humidity is 55-70%.

Reagents and controls: positive control (PC 1): 1.0% SDS solution;

positive control (PC 2): 0.1mol/LNaOH solution;

negative Control (NC): 0.9% NaCl solution;

test sample (TA): the avocado oil-in-water liquid fermentation product.

(2) Experimental methods

Preparation of CAM: purchasing 50-60 g of chick embryos of 0d age, turning over once a day in the hatching process, checking the development state of the chick embryos when the chick embryos are hatched to 5d age, and stripping off eggshells to expose white egg membranes when the chick embryos are hatched for 6 days; the intima is carefully removed with forceps to ensure that the vascular membrane is not damaged.

And (3) formal test: at least 6 embryos per group, 0.1mL of the test substance as received was directly dropped onto the CAM surface, the CAM reaction was observed and the time to each toxic effect within 5min of action was recorded to the nearest second, including 3 reactions of bleeding, clotting and vessel thawing, and the extent of the reaction was recorded. The same procedure was used for the positive and negative controls.

And (3) data analysis: recording the time and degree of response at each detection endpoint, calculating the stimulation score (IS) using the following formula, and retaining the last two decimal points:

sec H, sec L and sec C represent the average time (seconds) at which bleeding, vessel thawing and clotting were observed on the CAM membrane, respectively.

TABLE 10 evaluation of stimulation scoring results

(3) Results

TABLE 11 chick embryo chorioallantoic membrane blood vessel test results

According to the criteria of determination of SN/T2329-2009, the sample group 100% avocado oil-in-water liquid fermented product was not stimulated, the control group 1.0% SDS solution and 0.1mol/LNaOH solution had severe stimulation, and 0.9% NaCl solution was not stimulated.

SEQUENCE LISTING

<110> frog prince (Fujian) baby nursing products Co Ltd

Jiangsu Ruiting Biotechnology Co.,Ltd.

<120> avocado oil-in-water liquid state fermentation product, and preparation method and application thereof

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Claims (9)

1. A method for preparing a avocado oil-in-water liquid fermentation product, comprising: inoculating the microbial compound bacteria agent into a fermentation substrate taking avocado as a main component for fermentation culture to obtain the microbial compound bacteria agent; the microbial compound microbial inoculum is a mixed microbial inoculum containing the strain capsule coated yeast and lactobacillus pentosus;

the sacculus-covering yeast (Saccharomyces fibuligera) is preserved in China general microbiological culture Collection center (CGMCC) in 21 days 12 and 12 months 2020, and the preservation number is CGMCC NO.21512;

the Lactobacillus pentosus (Lactobacillus pentosus) is preserved in the common microorganism center of China general microbiological culture Collection center at 21.12.2020, with the preservation number of CGMCC NO.21511;

the avocado comprises avocado flesh and kernels.

2. The method according to claim 1, wherein the inoculation of the complex microbial inoculum is to inoculate a liquid microbial inoculum, and 5-15% of the complex microbial inoculum by volume is added into a fermentation substrate.

3. The avocado oil-in-water liquid fermentation product prepared by the method of claim 1, wherein the method comprises:

(1) Preparation of Saccharomycopsis fibuligera seed liquid

Selecting single colony of Saccharopolyspora caput-covering yeast CGMCC NO.21512 under aseptic operation condition, and inoculating into YPD liquid culture medium; 30 ℃,220 r.min ^-1 Shake culturing for 24h until the bacterial amount reaches 10 ⁸ CFU·mL ^-1 Then, obtaining seed liquid;

(2) Preparation of Lactobacillus pentosus seed liquid

Under the aseptic operation condition, picking a single colony of lactobacillus pentosus CGMCC NO.21511, and inoculating the single colony into an MRS liquid culture medium; 37 ℃ and 200r min ^-1 Shake culturing for 24h until the bacterial amount reaches 10 ⁸ CFU·mL ^-1 Then, obtaining seed liquid;

(3) Stock preparation

Cleaning fresh avocados with tap water, cleaning with deionized water, draining, and removing pedicels; adding the avocado into a wall breaking machine, adding deionized water in an amount which is 3 times of the mass ratio, and breaking until no large visible solid particles exist;

(4) Preparation of fermentation substrate

Avocado 100 g.L ^-1 Glucose 20 g.L ^-1 (ii) a Sterilizing at 121 deg.C for 20min to obtain fermentation substrate, and cooling; wherein the glucose is separately sterilized and added into the system;

(5) Inoculating fermentation

Adding 10% of compound microbial agent by volume into the sterilized slurry, and fermenting at 30 deg.C for 27h; the composite microbial agent is a mixed microbial agent of a sacculus complex membrane yeast CGMCC NO.21512 and a lactobacillus pentosus CGMCC NO.21511, and is prepared by mixing sacculus complex membrane yeast seed liquid and lactobacillus pentosus seed liquid according to a volume ratio of 1; the ventilation rate is 4 sL.min when the fermentation process is carried out in a fermentation tank ^-1 ；

(6) Microfiltration

Filtering by using a ceramic membrane, and collecting filtrate to obtain the avocado oil-in-water liquid state fermentation product.

4. The composite microbial inoculum is a mixed microbial inoculum of the Saccharum covering yeast with the preservation number of CGMCC NO.21512 and the Lactobacillus pentosus with the preservation number of CGMCC NO.21511.

5. Use of the method for the preparation of a avocado oil-in-water liquid fermented product according to any one of claims 1 to 2 for the preparation of a cosmetic product.

6. Use of the avocado oil-in-water liquid fermented product of claim 3 for the preparation of a cosmetic product.

7. Use of the complex microbial agent of claim 4 for the preparation of cosmetics.

8. The use according to claim 7, wherein the cosmetic is any one of a toner, a serum, a lotion or a cream.

9. The preparation method of the avocado oil-in-water liquid fermentation product as claimed in any one of claims 1 to 2 or the use of the avocado oil-in-water liquid fermentation product as claimed in claim 3 or the composite microbial agent as claimed in claim 4 for preparing cosmetic products with antioxidant and moisturizing effects.

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