CN113476379A

CN113476379A - Fermented product of lupin for cosmetic and its preparation method

Info

Publication number: CN113476379A
Application number: CN202110989266.3A
Authority: CN
Inventors: 张佳婵; 王昌涛; 赵丹; 王冬冬; 李萌
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2021-10-08

Abstract

The present disclosure provides a method for preparing a lupin fermented product useful for cosmetics, comprising: mixing a proper amount of lupin bean powder and water, sterilizing, and cooling to obtain a fermentation substrate, wherein the mass ratio of the lupin bean powder to the water is 0.5-5: 100; and (3) inoculating saccharomycetes and/or lactic acid bacteria to the fermentation substrate for fermentation treatment, and then sterilizing and separating to obtain the lupin fermentation liquor. The method comprises the following steps of carrying out liquid fermentation on lupin powder by using saccharomycetes and/or lactic acid bacteria, and extracting active substances in the lupin; especially, the first fermentation is carried out by using saccharomycetes, and the second fermentation is carried out by using lactic acid bacteria, so that the safety of the product is improved. The lupin fermentation product prepared according to the present disclosure can be applied to cosmetics, and can improve the antioxidant property of the cosmetics.

Description

Fermented product of lupin for cosmetic and its preparation method

Technical Field

The disclosure belongs to the technical field of biological fermentation, and particularly relates to a lupin fermentation product for cosmetics and a preparation method thereof.

Background

Lupine (scientific name: Lupinus miranthus Guss.) is commonly called Lu Bing Hua. The stem and leaf are mainly used for green feeding and grazing, also can be made into good silage, and can be used as good feed for pig and milk cow, and the seed is concentrated feed with high protein content, and can be used as green manure and covering crop, also can be used as honey source and ornamental plant.

Lupinus albus is a kind of Lupinus belonging to Leguminosae, and its seed extract is rich in various proteins and amino acids, and rich in anti-wrinkle vegetal peptide, and can inhibit activity of sarcoplasmic metalloprotease, thereby preventing damage to collagen, elastin, and fibronectin, effectively supporting these proteins, and preventing skin relaxation and wrinkle. But the extraction method thereof is less disclosed.

The natural active substance can be extracted by water extraction, organic solvent extraction, ultrasonic extraction, microwave extraction, supercritical fluid extraction, and microbial fermentation. In the face of such numerous extraction methods, how to screen a method more favorable for extracting active substances remains a technical problem faced by researchers in the field. Fermentation refers to a process in which people prepare microbial cells themselves, or direct metabolites or secondary metabolites, by virtue of the life activities of microorganisms under aerobic or anaerobic conditions, and is widely used in the food industry, the biological and chemical industries. The extraction of nutrients by fermentation technology has been reported, but the extraction effect is very different according to the fermentation substrate and the fermentation process conditions. The method depends on researchers to continuously explore more fermentation substrate combinations and fermentation methods to prepare more products with better effects so as to meet the requirements of consumers.

The invention has application number of 202010743613.3, and is named as 'a preparation method of probiotic fermented lupin fermented product', and the technical scheme is as follows: step 1, pretreating lupin seeds; step 2, adding cellulase into the lupin seed slurry, hydrolyzing, filtering, heating, adding sweetener, supplementing with water, homogenizing, pasteurizing, and cooling; step 3, inoculating a direct vat set starter in the lupin feed liquid, uniformly stirring, fermenting at constant temperature, and cooling; and 4, uniformly stirring, sealing and placing at the temperature below 6 ℃, or performing homogenization and pasteurization, and performing sterile filling. The scheme adopts direct vat set starter composed of lactobacillus to ferment lupin seeds, not only utilizes abundant protein in lupin seeds, but also retains most dietary fiber and other nutritional ingredients, and converts into nutritional and healthy novel food, and has relatively simple process, low cost and no waste. However, the resulting product is a food product and its performance for use in cosmetics is unknown.

There is no report of the use of lupin fermented extracts in the cosmetic field at present.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. It should be understood that this summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In view of the above-mentioned drawbacks of the prior art, it is an object of the present disclosure to provide a lupin fermented product useful for cosmetics, which has good safety and antioxidant efficacy, and a method for preparing the same.

According to a first aspect of the present disclosure, there is provided a method for preparing a fermented lupin product useful for cosmetics, comprising:

mixing proper amount of lupin powder and water, sterilizing, and cooling to obtain a fermentation substrate, wherein the mass ratio of the lupin powder to the water is 0.5-5: 100 (such as 1:100, 1.5:100, 2:100, 2.5:100, 3:100, 3.5:100, 4:100, 4.5:100 and the like);

and (3) inoculating saccharomycetes and/or lactic acid bacteria to the fermentation substrate for fermentation treatment, and then sterilizing and separating to obtain the lupin fermentation liquor.

In the above method for producing a fermented lupin, as a preferred embodiment, the fermentation treatment comprises: firstly, inoculating saccharomycetes to the fermentation substrate to carry out primary fermentation culture; and inoculating lactic acid bacteria for secondary fermentation culture after sterilization treatment.

In the above method for preparing fermented lupin, as a preferred embodiment, the lupin powder has a particle size of 100 mesh.

In the above method for preparing a lupin fermented product, as a preferred embodiment, the yeast is Saccharomyces cerevisiae YWY-1, the storage unit is China general microbiological culture Collection center (CGMCC), the address is Beijing, West Luo No.1 Hospital, Ind.1, the postal code is 100101, the storage date is 2019, 03 and 27 days, and the storage number is CGMCC No. 17452.

In the above method for producing a fermented lupin, as a preferred embodiment, the lactic acid bacteria are at least one selected from the group consisting of: lactobacillus delbruuchii subsp. bulgaricus; lactobacillus buchneri (Lactobacillus buchneri); bifidobacterium bifidum (Bifidobacterium bifidum); more preferably, the lactic acid bacterium is Lactobacillus delbruuchii subsp. In practice, it may be selected from at least one of the following: lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus delbruuchii subsp. bulgaricus) with the collection number of CGMCC 1.16075, available from CGMCC; lactobacillus buchneri (Lactobacillus buchneri) with preservation number of CGMCC 1.15607, available from CGMCC; bifidobacterium bifidum (CGMCC 1.5029) with preservation number can be purchased from CGMCC.

In the above method for preparing fermented lupin, as a preferred embodiment, the concentration of the yeast liquid for inoculation is 10⁴-10⁹CFU/mL (e.g., 10)⁵CFU/mL、10⁶CFU/mL、10⁷CFU/mL、10⁸CFU/mL, etc.), the volume ratio of the yeast liquid to the fermentation substrate is 5% -20% (such as 8%, 10%, 12%, 15%, 18%, etc.).

In the above method for preparing fermented lupin, as a preferred embodiment, for inoculationThe concentration of the lactic acid bacteria liquid of (2) is 10⁴-10⁹CFU/mL (e.g., 10)⁵CFU/mL、10⁶CFU/mL、10⁷CFU/mL、10⁸CFU/mL, etc.), the ratio of the volume of the lactic acid bacteria liquid to the volume of the fermentation substrate is 5% to 20% (e.g., 8%, 10%, 12%, 15%, 18%, etc.).

In the above method for preparing fermented lupin, as a preferred embodiment, in the fermentation treatment, if yeast is used, the fermentation culture temperature is 20-28 ℃ (for example, 22 ℃, 24 ℃, 26 ℃ and the like), the fermentation culture time is 30-50h (for example, 32h, 35h, 40h, 45h, 48h and the like), and the rotation speed is 100-180r/min (for example, 110r/min, 120r/min, 130r/min, 140r/min, 150r/min, 160r/min, 170r/min and the like); if lactic acid bacteria are used, the fermentation culture temperature is 37-45 deg.C (such as 38 deg.C, 40 deg.C, 42 deg.C, 44 deg.C, etc.), the fermentation culture time is 6-16h (such as 7h, 8h, 10h, 12h, 14h, 15h, etc.), and standing culture.

In the above method for preparing fermented lupin, as a preferred embodiment, the temperature of the sterilization treatment is 100-.

In the above method for producing a fermented lupin, as a preferred embodiment, the method further comprises: performing preservative treatment on the lupin fermentation liquor; more preferably, the preservative treatment is to add 2.3% by mass of ethylene glycol and 0.5% by mass of pentanediol after the lupin fermentation broth is cooled to 45 ℃ or lower, i.e. to add 2.3% by mass of ethylene glycol and 0.5% by mass of pentanediol relative to the lupin fermentation broth.

In the above method for producing a fermented lupin, as a preferred embodiment, the separation treatment is a centrifugation method; more preferably, the centrifugation speed is 4500r/min-6000r/min (such as 4800r/min, 5000r/min, 5200r/min, 5500r/min, 5800r/min, etc.), and the centrifugation time is 30min-60min (such as 35min, 40min, 45min, 50min, 55min, etc.).

In the above method for producing a fermented lupin, as a preferred embodiment, the method further comprises: drying the lupin fermentation liquid to finally obtain lupin fermentation dry powder; more preferably, the drying treatment may be spray drying, vacuum freeze drying, or the like.

According to a second aspect of the present disclosure, there is also provided a fermentation product prepared by the above method, including fermentation broth, fermentation dry powder, and the like.

According to a third aspect of the present disclosure, there is also provided the use of the above-mentioned fermented product in the preparation of a cosmetic; preferably, the cosmetic may be a mask, essence, toner, emulsion, etc.

The lupin fermentation product prepared by the method disclosed by the invention contains active substances such as polysaccharide, polypeptide and the like, has good antioxidation and free radical scavenging effects, and can be added into various fermentation products including but not limited to: facial mask, essence, toner, and lotion.

The method comprises the following steps of carrying out liquid fermentation on lupin powder by using saccharomycetes and/or lactic acid bacteria, and extracting active substances in the lupin; especially, the first fermentation is carried out by using saccharomycetes, and the second fermentation is carried out by using lactic acid bacteria, so that the safety of the product is improved. The lupin fermented product prepared according to the present disclosure can be applied to cosmetics and can improve the antioxidant property of the cosmetics.

The lupin fermentation product prepared by the method has excellent safety, can be directly used as a facial mask liquid, an essence, a toner and other finished cosmetics, and has no side effect on skin.

Drawings

FIG. 1 shows HPLC profiles of three standard samples, bovine serum albumin (Mw67000) and vitamin B₁₂(Mw1335), oxidized glutathione (Mw 614);

FIG. 2 is a plot of the relative log molecular mass of each standard sample material versus HPLC retention time;

FIG. 3 is an HPLC chromatogram of a blank sample (lupin medium);

FIG. 4 is an HPLC chromatogram of a sample obtained by fermenting lupin alone with the yeast of example 1;

FIG. 5 is an HPLC chromatogram of a sample obtained by fermenting lupin alone with lactic acid bacteria according to example 2;

FIG. 6 is an HPLC chromatogram of a sample obtained by two-step fermentation of lupin with yeast lactic acid bacteria in example 3.

Detailed Description

The technical solutions of the present disclosure will be described below with reference to exemplary embodiments.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

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

The lupin flour in the examples described below is commercially available.

The yeast in the following examples is Saccharomyces cerevisiae YWY-1, which is owned by the applicant's laboratory and has been submitted to China general microbiological culture Collection center (CGMCC for short) for preservation with the preservation date of 2019, 03 and 27 days and the preservation number of CGMCC No. 17452.

The lactic acid bacterium in the following examples was Lactobacillus delbrueckii subsp.Bulgaricus (Lactobacillus delbruuchii) with a collection number of CGMCC 1.16075 and purchased from CGMCC.

The seed culture medium of yeast and lactobacillus is yeast extract peptone glucose culture medium (YPD culture medium) and lactobacillus culture medium (MRS culture medium), respectively. Culturing yeast in a constant-temperature shaking incubator at 28 deg.C for 48 hr at 150r/min to obtain yeast liquid with concentration of about 10⁸CFU/mL. Standing and culturing lactobacillus in a constant temperature shaking incubator at 45 deg.C for 24 hr to obtain lactobacillus bacterial liquid with concentration of about 10 for inoculation¹⁰CFU/mL。

EXAMPLE 1 Yeast isolated fermentation

1) Mixing Lupinus Polyphyllus powder with deionized water at a ratio of 1:100, placing in a triangular flask, shaking, sterilizing in a high pressure steam sterilizing pot at 121 deg.C for 20min, and cooling to obtain Lupinus Polyphyllus culture medium (fermentation substrate);

2) inoculating yeast liquid into lupin culture medium according to volume ratio of 8%, and fermenting in constant temperature shaking incubator at 28 deg.C and rotation speed of 150r/min for 48 hr;

3) after fermentation, centrifuging, filtering to obtain supernatant, centrifuging at 5500rpm for 45min, and sterilizing the supernatant in a high-pressure steam sterilizer at 110 deg.C for 30 min;

4) after sterilization, the supernatant was left at room temperature, cooled to below 45 ℃, and preservative system, i.e. 2.3 wt.% ethylene glycol and 0.5 wt.% pentanediol, was added.

EXAMPLE 2 lactic acid bacteria fermentation alone

2) inoculating lactobacillus liquid into a lupin culture medium according to the volume ratio of 10%, and standing and fermenting for 8h in a constant-temperature shaking incubator at the temperature of 45 ℃;

3) after sterilization, the mixture is left at room temperature, cooled to below 45 ℃, and added with a preservative system, namely 2.3 wt.% of ethylene glycol and 0.5 wt.% of pentanediol.

Example 3 sequential fermentation of Yeast and lactic acid bacteria

3) after fermentation, placing into a high-pressure steam sterilization pot for sterilization, wherein the sterilization temperature is 110 ℃, and the sterilization time is 30 min;

4) cooling to below 45 ℃ after sterilization is finished, inoculating lactobacillus liquid according to the volume proportion of 10%, and standing and fermenting for 8 hours in a constant-temperature shaking incubator at the temperature of 45 ℃;

5) after fermentation, centrifuging, filtering to obtain supernatant, centrifuging at 5500rpm for 45min, and sterilizing the supernatant in a high-pressure steam sterilizer at 110 deg.C for 30 min;

6) after sterilization, the mixture is left at room temperature, cooled to below 45 ℃, and added with a preservative system, namely 2.3 wt.% of ethylene glycol and 0.5 wt.% of pentanediol.

Performance test

Analysis of physicochemical Properties of fermentation broth

The physical and chemical properties of the lupin fermentation broth obtained in example 1 were analyzed. The appearance of the bacterial colony is yellow liquid, the pH value is 5.3, the solid content is 2.9 percent, the total number of the bacterial colonies is less than 50CFU/mL, and no pathogenic bacteria are detected.

The physical and chemical properties of the lupin fermentation broth obtained in example 2 were analyzed. The appearance of the bacterial colony is light yellow liquid, the pH value is 5.5, the solid content is 3.2 percent, the total number of the bacterial colonies is less than 50CFU/mL, and no pathogenic bacteria are detected.

The physical and chemical properties of the lupin fermentation broth obtained in example 3 were analyzed. The appearance of the bacterial colony is yellow liquid, the pH value is 5.4, the solid content is 3.2 percent, the total number of the bacterial colonies is less than 50CFU/mL, and no pathogenic bacteria are detected. According to the cosmetic hygienic standard GB7916-87, the total number of bacteria in the cosmetics is not higher than 1000CFU/ml, so that the lupin fermentation liquor meets the requirements of cosmetic quality.

The lupin fermentation broths obtained in examples 1 to 3 were subjected to the composition analysis, and the results were as follows:

TABLE 1.1 comparison of the physico-chemical properties of the fermentation broths obtained in examples 1 to 3

	Example 1	Example 2	Example 3	Pre-fermentation blank control
					Polypeptide content mg/ml	4.55	4.11	3.62	3.43
Standard deviation of	0.27	0.05	0.24	0.38
					Polysaccharide content mg/mL	3.33	4.25	3.59	3.43
Standard deviation of	0.01	0.13	0.07	0.38

As can be seen from table 1.1: the content of polysaccharide after fermentation is increased, and the content of polypeptide is increased.

In addition, the molecular weight of the polypeptides of examples 1 to 3 was also measured.

The molecular weight of the polypeptide is determined as follows:

1. chromatographic conditions are as follows: 1) a chromatographic column: tsk gel 2000SW_XL300mm multiplied by 7.8 mm; 2) mobile phase: 0.05mol/L phosphate buffer (pH 7) +0.3mol/L NaCl; 3) detection wavelength: UV 220 nm; 4) flow rate: 1 ml/min; 5) column temperature: 25 ℃; 6) sample preparation: a sample with a concentration of 5mg/ml is prepared by using a mobile phase as a solvent, and is filtered by a microporous membrane (0.45 mu m) and then is injected. 7) Standard samples: bovine serum albumin (Mr 67000), B12(Mr 1335) and oxidized glutathione (Mr 614) were mixed to obtain a mixed standard, and the content of each substance was 5 mg/ml.

2. And (3) standard curve preparation: three standards were prepared at 5mg/ml and standard curves were generated according to the chromatographic conditions described above, as shown in FIG. 1. FIG. 1 shows HPLC profiles of three standard samples, bovine serum albumin (Mw67000) and vitamin B₁₂(Mw1335), oxidized glutathione (Mw614), column model TSKge 12000 SW_XL(gel column), therefore, according to the principle of gel permeation chromatography, the substance with large molecular weight is eluted first, and from fig. 1, it can be seen that the elution times corresponding to the appearance of the elution peaks are respectively 6.84min,10.12min and 12.40min, and then, according to the relative molecular mass logarithm values of the three standard samples (see table 1.2), the relationship curves of the relative molecular mass logarithm of the three standard samples and the elution time of the standard sample by HPLC are made (see fig. 2).

TABLE 1.2 molecular weight and elution time of the standard

The correlation between the log values of the molecular weights of the standard samples and the elution times can be seen in Table 1.2. Taking the logarithm of the molecular weight as the ordinate and the elution time as the abscissa, a straight line was drawn using three coordinate points (5.377, 4.826), (9.3, 3.125), (10.419, 2.788), and this straight line was the standard curve, as shown in fig. 2.

The regression equation of the straight line is solved by adopting a least square method as follows: y is-0.4119 x +7.0253, and the regression coefficient R2 is 0.9966. From the regression coefficient R of the equation²0.9966, the standard curve linear relation of the mixed standard sample is better, and the accuracy of calculation can be improved. In the formula, x represents elution time, and y represents logarithm of molecular weight. Thus, when the sample is subjected to HPLC analysis, the corresponding molecular weight can be calculated from the time of appearance of the elution peak of each substance in the elution profile, and the results are shown in Table 1.3 and FIGS. 3-6.

TABLE 1.3 measurement results of the molecular weights of the polypeptide in the blank samples and the samples obtained in examples 1 to 3

As can be seen from table 1.3: the molecular weight of the lupin fermentation broth obtained by fermenting lupin fermentation broth with yeast alone obtained in example 1, the lupin fermentation broth obtained by fermenting lupin fermentation broth with lactic acid bacteria alone obtained in example 2, and the lupin fermentation broth obtained by fermenting lupin with yeast lactic acid bacteria obtained in example 3 in two steps become single, the molecular weight becomes small, and transdermal absorption is easier.

Secondly, safety detection of fermentation liquor

The human body patch test is mainly used for detecting the irritation of the final cosmetic product or raw materials. The lupin fermentation broths obtained in examples 1 to 3 were subjected to a closed patch test in human body for the purpose of evaluating their potential skin irritancy.

1. Test object

Suitable volunteers were selected for 30 persons, and were randomly selected in the age range of 18-60 years.

2. Test method

0.02mL to 0.025mL of the liquid sample (i.e., the lupin broth) was added dropwise to the filter paper sheet, which was then placed in the plaque tester. A blank control is set for each sample, and an equal amount of sample solvent, such as distilled water or olive oil (distilled water is used in this example), is added to the control chamber.

The test part is selected as the back of a human body, and the spot tester is fixedly attached to the back of the testee by using a non-irritant adhesive tape. The test period lasted 24 h. In order to ensure the accuracy, credibility and scientificity of test results, the volunteers cannot remove the spot tester or make the tested part contact water according to the requirements during the test. And removing the spot tester after 24h, standing for 30min, waiting for the indentation to disappear, and observing the reaction of the skin. If the test result is negative, the test needs to be observed once more at 24h and 48h after the patch test.

3. Test results

The patch test results are shown in table 2, and each level in table 2 indicates the following: grade 0 is a negative reaction. Grade 1 ═ suspicious response; only faint erythema. Grade 2 ═ weakly positive reaction (erythema reaction); erythema, infiltration, edema, and possibly pimples. Grade 3 ═ strong positive response (herpes response); erythema, infiltration, edema, pimples, herpes; the reaction may be beyond the test area. Grade 4 ═ very strong positive response (fusogenic herpes response); obvious erythema, severe infiltration, edema, and fusional herpes; the reaction goes beyond the test area.

TABLE 2 Patch test results of fermentation broths obtained in examples 1 to 3

As can be seen from table 2: the lupin fermentation liquid obtained by fermenting the lupin fermentation liquid separately with the yeast obtained in the example 1 and the lupin fermentation liquid obtained by fermenting the lactobacillus separately with the yeast obtained in the example 3 both do not produce suspicious reactions, and the lupin fermentation liquid obtained by fermenting the lupin fermentation liquid separately with the lactobacillus obtained in the example 2 has slight reactions, which indicates that the lupin fermentation liquid provided by the invention has high safety and does not bring adverse reactions to human bodies.

Thirdly, detecting the oxidation resistance of the fermentation liquor

DPPH is an early synthesized organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, is very stable in organic solvents, is purple in color, and has a characteristic absorption peak at 517nm, when encountering a radical scavenger, the lone pair of DPPH is paired to discolor it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.

The lupin fermentation broth prepared in example 1-example 3 was used as the test solution, and vitamin C was used as the positive control. The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

(1) taking the same volume (3mL) of the solution to be detected and 2X 10^-4mixing (A) with a solution of DPPH in mol/L₁A tube);

(2) taking equal volume (3mL) of absolute ethanol (solvent of the test substance) and 2X 10^-4mixing (A) with a solution of DPPH in mol/L₂A tube);

(3) mixing the same volume (3mL) of anhydrous ethanol with the solution to be detected (A)₃A tube);

(4) after 30min of reaction, A was measured at 517nm₁Pipe, A₂Pipe, A₃Tube absorbance values.

The clearance calculation formula is: clearance (%) - (A)₂+A₃)-A₁]/A₂. See table 3 for specific results.

TABLE 3 DPPH radical scavenging test results for fermentation broths obtained in examples 1 to 3

	Example 1	Example 2	Example 3	Pre-fermentation blank control
					DPPH radical clearance rate	37.92％	70.79％	58.32％	48.34％

As can be seen from Table 3, the DPPH radical clearance of the fermentation broths obtained in examples 2 and 3 was increased after the fermentation.

Fourth, detecting cytotoxicity of fermentation liquor

Human Skin Fibroblasts (HSF) were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1X 10)⁵U/L penicillin, 100mg/L streptomycin). Cells were grown at 37 ℃ with 5% CO₂In the incubator with saturated humidity, when the cell fusion reaches more than 85%, the cells are digested and passaged with 0.05% pancreatin. Human dermal fibroblasts in good logarithmic growth phase were seeded in a cell culture plate for experiment.

The MTT method is used for detecting the influence of fermentation liquor on the survival rate of human skin fibroblasts. The method comprises the following specific steps: cells in the logarithmic growth phase were digested and the digestion was stopped with DMEM containing serum. Counting with cell counting plate, adjusting cell suspension concentration to 5 × 10⁴one/mL, the cell suspension was inoculated into a 96-well plate at a rate of 100. mu.L/well, at 37 ℃ with 5% CO₂Incubate under conditions overnight. Removing culture solution, adding filtered and sterilized fermentation broth, making 6 multiple wells per sample, and culturing for 24 hr. After the completion of the culture, 100. mu.L of a mixed solution of MTT solution (5mg/mL) and DMEM (v/v, 1: 5) was added to each well and incubated for 4 hours. Removing the medium and adding to each wellAfter incubation with 150. mu.L of DMSO at 37 ℃ for 10min, the absorbance (denoted A) of the experimental group at 490nm was read. The cell-free treated group (cell suspension was replaced with serum-free DMEM, and the rest was the same) was set as a blank control group and designated as B. The cell control group (sample solution was replaced with serum-free DMEM, and the rest of the procedure was the same) was designated as C.

The calculation formula of the cell viability is as follows: cell viability (%) - (a-B)/(C-B) × 100;

see table 4 for specific results.

TABLE 4 results of MTT cell assay of fermentation broths prepared in examples 1 to 3

	Pre-fermentation blank control	Example 1	Example 2	Example 3
					Cell viability%	55.02	73.69	81.49	84.22
Standard deviation std	3.283018	7.212286	3.605484	5.230078

As can be seen from Table 4, the fermentation broths obtained in the examples all have improved cell viability compared to those before fermentation, and among them, the fermentation broths obtained by the two-strain fermentation in example 3 have the highest cell viability.

Finally, it is also noted that, in the present disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

While the disclosure has been disclosed above by the description of specific embodiments thereof, it should be understood that various modifications, improvements or equivalents of the disclosure may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are intended to be included within the scope of the present disclosure as claimed.

Claims

1. A method for preparing fermented product of lupin useful for cosmetics, comprising:

mixing a proper amount of lupin bean powder and water, sterilizing, and cooling to obtain a fermentation substrate, wherein the mass ratio of the lupin bean powder to the water is 0.5-5: 100;

2. The method of claim 1, wherein the fermentation treatment comprises:

firstly, inoculating saccharomycetes to the fermentation substrate to carry out primary fermentation culture; inoculating lactobacillus after sterilization treatment for secondary fermentation culture;

preferably, the lupin flour has a particle size of 100 mesh.

3. The method according to claim 1 or 2, wherein the yeast is Saccharomyces cerevisiae YWY-1, the preservation date is 2019, 03 and 27 days, and the preservation number is CGMCC No. 17452; preferably, the concentration of the yeast liquid for inoculation is 10⁴-10⁹CFU/mL, wherein the volume ratio of the yeast liquid to the fermentation substrate is 5-20%.

4. The method according to any one of claims 1 to 3, wherein the lactic acid bacteria are at least one of the following species: lactobacillus delbruuchii subsp. bulgaricus; lactobacillus buchneri (Lactobacillus buchneri); bifidobacterium bifidum (Bifidobacterium bifidum);

preferably, the lactic acid bacteria are at least one of the following species: lactobacillus delbrueckii subsp. bulgaricus with a collection number of CGMCC 1.16075; lactobacillus buchneri (Lactobacillus buchneri) with the preservation number of CGMCC 1.15607; bifidobacterium bifidum (CGMCC 1.5029) with preservation number;

preferably, the Lactobacillus is Lactobacillus delbruuchii subsp.bulgaricus with the preservation number of CGMCC 1.16075;

preferably, the concentration of the lactobacillus bacteria liquid for inoculation is 10⁴-10⁹CFU/mL, the volume ratio of the lactobacillus liquid to the fermentation substrate is 5-20%.

5. The method according to any one of claims 1 to 4, wherein in the fermentation treatment, if yeast is used, the temperature of the fermentation culture is 20 ℃ to 28 ℃, the time is 30 to 50 hours, and the rotation speed is 100-; if lactic acid bacteria are adopted, the temperature of fermentation culture is 37-45 deg.C, the time is 6-16h, and standing culture is performed.

6. The method according to any one of claims 1 to 5, wherein the temperature of the sterilization treatment is 100 ℃ and 121 ℃ for 15 to 30 min.

7. The production method according to any one of claims 1 to 6, wherein the separation treatment is performed by a centrifugation method; more preferably, the centrifugal rotating speed is 4500r/min-6000r/min, and the centrifugal time is 30min-60 min.

8. The method of any one of claims 1-7, further comprising: performing preservative treatment on the lupin fermentation liquor; more preferably, the preservation treatment is the addition of 2.3 wt.% ethylene glycol and 0.5 wt.% pentanediol after the lupin fermentation broth has cooled to below 45 ℃.

9. The method of any one of claims 1-8, further comprising: drying the lupin fermentation liquid to finally obtain lupin fermentation dry powder; more preferably, the drying process is spray drying or vacuum freeze drying.

10. A fermentation product produced by the method of any one of claims 1-9.