CN114317616A - Preparation process of fungus fermentation product and cosmetics - Google Patents
Preparation process of fungus fermentation product and cosmetics Download PDFInfo
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- CN114317616A CN114317616A CN202111493934.XA CN202111493934A CN114317616A CN 114317616 A CN114317616 A CN 114317616A CN 202111493934 A CN202111493934 A CN 202111493934A CN 114317616 A CN114317616 A CN 114317616A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a preparation process of a fungus fermentation product and cosmetics, wherein the preparation process comprises the following steps: pulverizing fungi, and sieving to obtain fungi powder; carrying out mutagenesis on an original yeast strain and screening out a recombinant strain, wherein the recombinant strain has acetic acid tolerance and/or furfural tolerance; performing activation culture on the recombinant strain to obtain a yeast seed solution; adding the fungus powder and the yeast seed liquid into a liquid fermentation culture medium for fermentation to obtain a fermentation liquid; and centrifuging the fermentation liquor, filtering and sterilizing to obtain the fungus fermentation product. The invention can greatly improve the resource utilization rate and the nutrition added value of fungi, and the product has better free radical scavenging capacity and melanin generation inhibiting capacity, thereby improving the effects of aging resistance, oxidation resistance and whitening.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a preparation process of a bacteria fermentation product and cosmetics.
Background
The structure of the skin includes the epidermis, dermis, and subcutaneous fat. The dermis contains collagen, elastin, and other fibrous tissues. People face computers and mobile phone electronic equipment for a long time, the electronic equipment is easy to generate static electricity to attract dust or tiny particles to be attached to the surface of skin, and in addition, work and rest are irregular, the epidermis is easy to dry, and melanin precipitation is aggravated. With the age, the epidermis layer becomes thinner gradually, the metabolism of the skin is slowed down, the moisturizing factor in the dermis layer is reduced, the function of elastic fiber and collagen fiber in the dermis layer is reduced, the elasticity is weakened, and wrinkles appear; in addition, the active biological enzyme in the human body is reduced, a large number of free radicals destroy the cells of the human body, and the process of skin aging is accelerated.
Matsutake is the fruiting body of Tricholoma matsutake of Tricholomataceae, is mainly produced in northeast China and in coniferous and broadleaf forest areas with elevation of 3000m at the junction of Guizhou, Yunnan and Sichuan, and is called the king of edible fungi. Because the growth of the tricholoma matsutake requires unique environment and climate conditions, the tricholoma matsutake can not be completely cultivated artificially so far, only semi-artificial cultivation can be realized, the tricholoma matsutake belongs to the second-level endangered protected species of China, and the tricholoma matsutake is a precious natural wild medicinal fungus in the world. The matsutake is a large-scale fungus with fleshy fruit body, not only can be used as a delicious dish, but also has various health care and medicinal values. According to the literature, the tricholoma matsutake has the effects of strengthening the body, tonifying the kidney and strengthening yang, benefiting the intestines and stomach, regulating qi, reducing phlegm, expelling parasites and the like. Modern medicine shows that tricholoma matsutake also has the effects of treating diabetes and inhibiting cancer cell proliferation.
The microbial fermentation technology can switch the nutrients of the plants into small molecules which can be absorbed by the skin, thereby playing the role of skin care. The technology has mild reaction conditions, simple and safe equipment operation and no environmental pollution. At present, the microbial fermentation technology has been popularized to a certain extent in the application of skin care products, such as Pitera, i.e. tectorial membrane yeast fermentation products, bifidobacterium fermentation products, lactobacillus fermentation products and the like.
However, the existing tricholoma matsutake and tricholoma matsutake fermentation products thereof have poor oxidation resistance, anti-aging and whitening effects, and cannot be widely applied to various cosmetics.
Disclosure of Invention
The invention aims to solve at least one of the problems in the prior related art to a certain extent, and therefore, the invention provides a preparation process of a fungus fermentation product, which greatly improves the resource utilization rate and the nutrition added value of fungi, and simultaneously has better free radical scavenging capacity and melanin generation inhibiting capacity, thereby improving the effects of aging resistance, oxidation resistance and whitening.
The invention also provides a cosmetic with the preparation process of the fungus fermentation product.
According to the preparation process of the bacteria fermentation product, the preparation process is realized by the following technical scheme:
a preparation process of a fungus fermentation product comprises the following steps:
s1, pulverizing fungi, and sieving to obtain fungi powder;
s2, carrying out mutagenesis on the original yeast strain and screening out a recombinant strain, wherein the recombinant strain has acetic acid tolerance and/or furfural tolerance;
s3, carrying out activated culture on the recombinant strains to obtain yeast seed liquid;
s4, adding the fungus powder and the yeast seed liquid into a liquid fermentation culture medium for fermentation to obtain fermentation liquid;
s5, centrifuging the fermentation liquor, filtering and sterilizing to obtain the fungus fermentation product.
Further, the screening of recombinant strains after mutagenesis of the original yeast strain comprises:
s21, carrying out chemical mutagenesis and screening on the original yeast strain to obtain a first mutant strain with acetic acid tolerance and a second mutant strain with furfural tolerance;
s22, subjecting the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation;
s23, and obtaining the recombinant strain through screening.
Further, the subjecting of the original yeast strain to chemical mutagenesis and screening includes: culturing an original yeast strain in a liquid culture medium, centrifugally collecting thalli, and washing with a phosphate buffer solution to obtain a heavy suspension bacterial liquid; adding a heavy suspension bacterium solution, a phosphate buffer solution and diethyl sulfate into a 15mL sterilized centrifugal tube, performing vortex oscillation and uniform mixing, culturing for 20-40 min, adding a sodium thiosulfate solution to terminate the reaction, and obtaining a reaction bacterium solution after mutagenesis; and (3) diluting the reaction bacterial liquid, coating the diluted reaction bacterial liquid on an inhibitor tolerance screening solid culture medium for culture, and then screening the inhibitor tolerance.
Further, the volume ratio of the resuspended bacterial liquid to the phosphate buffer solution to the diethyl sulfate is 40-60: 40-55: 1.
Further, the subjecting of the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation comprises: extracting total genomic DNA of the first mutant strain and the second mutant strain for transformation to obtain a yeast liquid; and carrying out centrifugal collection and heavy suspension treatment on the yeast liquid to obtain competent cells.
Further, the centrifugal collection and heavy suspension treatment of the yeast strain liquid comprises the following steps: centrifugally collecting primary thalli from the saccharomycete liquid, and then carrying out heavy suspension treatment on the primary thalli by using a lithium acetate-dithiothreitol mixed solution; and centrifuging again to collect the secondary thalli, and then washing with precooled sterile water.
Further, the subjecting of the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation further comprises: mixing the total genomic DNA of the first mutant strain and the second mutant strain in equal amount, and adding diethyl sulfate for culturing to obtain DNA to be transformed; the competent cells and the DNA to be transformed are mixed according to the volume ratio of 2:1 and are placed in an electric transformation cup for electric shock treatment.
Further, in step S3, the activating culture of the recombinant strain specifically includes: inoculating the recombinant strain into a seed culture medium, and performing shaking culture on a shaking table at the temperature of 28-30 ℃ and at the speed of 180-200 r/min for 16-20 h.
Further, the volume ratio of the recombinant strain to the seed culture medium is 5-10; the seed culture medium comprises 10-30 g/L of glucose, 5-15 g/L of yeast extract and 10-30 g/L of peptone.
Further, in step S4, the liquid fermentation medium includes 10-20 g/L of fungi including Tricholoma matsutake and 10-30 g/L of carbon source including at least one of sucrose, maltose, glucose, fructose, lactose, galactose, mannitol and maltitol.
According to the cosmetic provided by the above, the cosmetic is realized by the following technical scheme:
cosmetic comprising a process for the preparation of a bacterial fermentation product as described above.
Compared with the prior art, the invention at least comprises the following beneficial effects:
1. according to the invention, the fungus is used as a substrate, the recombinant strain improved by the original yeast strain is used as a strain, and fermentation, centrifugation and filtration sterilization are carried out to obtain a fungus fermentation product, so that the resource utilization rate and the nutrition added value of the fungus are greatly improved, and compared with a direct extraction method, the addition of an organic solvent and an enzyme is avoided, so that the method is safe and environment-friendly, and the cost can be saved;
2. the fungus fermentation product is rich in flavone, polyphenol, polypeptide, various amino acids and other components, can effectively promote division and proliferation of human skin fibroblasts (NHDFs), promote synthesis of type I collagen of the human skin fibroblasts, and has good free radical scavenging capacity and melanin generation inhibiting capacity, so that the fungus fermentation product can improve the effects of aging resistance, oxidation resistance and whitening, and can be widely applied to various cosmetics.
Drawings
FIG. 1 is a flow chart of a process for producing a fermentation product of fungi according to example 1 of the present invention;
FIG. 2 is a graph of the concentration of fungus fermentation products in accordance with the present invention as a function of ABTS free radical clearance in example 1;
FIG. 3 is a graph showing the relationship between the concentration of fungus fermentation products and the relative cell growth ability in example 1 of the present invention;
FIG. 4 is a graph of the concentration of fermentation products of fungi versus the relative amount of type I collagen in example 1 of the present invention;
FIG. 5 is a graph showing the relationship between the concentration of fermentation products of fungi and the relative content of B16-F10 melanin in example 1 of the present invention.
Detailed Description
The present invention is illustrated by the following examples, but the present invention is not limited to these examples. Modifications to the embodiments of the invention or equivalent substitutions of parts of technical features without departing from the spirit of the invention are intended to be covered by the scope of the claims of the invention.
The experimental methods used in the following examples are not specifically described, and are all conventional methods. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
Referring to fig. 1, this example provides a process for preparing a fermentation product of bacteria, which comprises the following steps:
s1, pulverizing fungi, and sieving to obtain fungi powder;
specifically, the fungi comprise Tricholoma matsutake, and the Tricholoma matsutake is directly crushed or is firstly cut and then crushed, and is then screened by a 70-100 mesh screen to obtain Tricholoma matsutake powder which is used as a fermentation substrate. The tricholoma matsutake is made into tricholoma matsutake powder, so that the cell wall of the tricholoma matsutake is damaged, the enzymatic activity conversion efficiency is favorably improved, the fermentation effect is ensured, and the quality of the tricholoma matsutake fermentation product is ensured.
S2, carrying out mutagenesis on the original yeast strain KN-1, and screening a recombinant strain, wherein the recombinant strain has acetic acid tolerance and/or furfural tolerance;
specifically, the primary yeast strain KN-1 is preferably Saccharomyces cerevisiae, which is purchased from China center for Industrial culture Collection of microorganisms (CICC). The saccharomyces cerevisiae is subjected to mutagenesis so as to realize the improvement of the saccharomyces cerevisiae. Then, a target object, namely the recombinant strain KN-1-24 is obtained through screening, and the recombinant strain KN-1-24 has acetic acid tolerance and/or furfural tolerance. In the embodiment, the recombinant strain KN-1-24 has two functions of acetic acid tolerance and furfural tolerance, and the recombinant strain KN-1-24 can grow better on an inhibitor tolerance screening solid culture medium containing 1-8 g/L of acetic acid and 0.1-1.5 g/L of furfural.
Inoculating the recombinant strain KN-1-24 and the original yeast KN-1 into 10mL YEPD liquid culture medium, and culturing for 20h at 30 ℃ under the condition of 200 r/min; transferring 10% inoculum size to 50mL YEPD liquid culture medium, culturing at 30 deg.C and 200r/min for 20h, transferring 10% inoculum size to 100mL inhibitor-tolerant fermentation culture medium and cellulose hydrolysate fermentation culture medium respectively, culturing at 30 deg.C for 3 times, and sampling at regular time to detect various fermentation performance indexes. Taking out 1mL of fermentation liquid at different time points of fermentation, shaking, diluting properly, centrifuging the diluted liquid at 8000r/min for 5min, and measuring ethanol and glucose (g/L) with SBA-40C type biosensor analyzer. The results show that: after fermentation for 12h, the sugar utilization rate and the ethanol production capacity of the recombinant strain KN-1-24 are higher than those of the original yeast KN-1; in the cellulose hydrolysate, compared with the original yeast KN-1, the ethanol yield of the recombinant strain KN-1-24 is 9.8 percent, and the fermentation period is shortened by 6 hours; after fermentation for 36h, the glucose content in the medium of the recombinant strain KN-1-24 was substantially completely consumed, whereas the residual sugar content in the medium of the original yeast KN-1 was 34 g/L. Therefore, the improved recombinant strain KN-1-24 has higher sugar utilization rate and ethanol production capacity, can realize shortening of the fermentation period and remarkably improve the fermentation efficiency.
S3, carrying out activated culture on the recombinant strains to obtain yeast seed liquid;
s4, adding the fungus powder and the yeast seed liquid into a liquid fermentation culture medium for fermentation to obtain fermentation liquid;
s5, centrifuging the fermentation liquor, filtering and sterilizing to obtain the fungus fermentation product.
Specifically, the centrifugation and filtration sterilization of the fermentation liquor specifically comprises the following steps: centrifuging the fermentation liquor at 8000r/min for 20min to remove residues and collect supernatant; then filtering and sterilizing by a fine filtering paper board with the diameter of 1.2 mu m and a polyether sulfone filter element with the diameter of 0.22 mu m to obtain the fungus fermentation product.
The main functional components and contents of the fungus fermentation products are detected, and the results are shown in table 1. The detection method of the proteins in table 1 refers to GB 5009.5-2010; the detection method of the crude polysaccharide refers to GB 5009.8-2008; the detection method of total flavone (in rutin) refers to DB13T 385-1988; the detection method of polyphenol refers to GB/T8313-2008; the amino acid detection method is described in GB/T5009.124-2003.
TABLE 1 Main effective components and contents of fungus fermentation product
Therefore, the fungus including tricholoma matsutake is used as a substrate, and the recombinant strain improved by the original yeast strain is used as a strain to perform fermentation, centrifugation and filtration sterilization to obtain a fungus fermentation product, so that the resource utilization rate and the nutritional added value of the fungus are greatly improved; compared with a direct extraction method, the method avoids the addition of organic solvents and enzymes, is safe and environment-friendly, and can save cost; the fermented product of the three fungi is rich in flavone, polyphenol, polypeptide and various amino acids.
The following performance tests are carried out on the fungus fermentation products:
(1) and evaluating the antioxidant effect of the fungus fermentation product.
The scavenging capacity of the fungus fermentation product on various active oxygen (hydroxyl free radical, superoxide free radical, hydrogen peroxide and the like) is detected by a total antioxidant capacity detection kit (ABTS method).
Preparing fungus fermentation products (samples) into solutions to be detected with different concentrations (1.25%, 2.50%, 5.00%, 10.0%, 20.0% (v/v)), preparing a reaction system in a 96-well plate according to the addition amount of each reagent in the table 2, uniformly mixing, and arranging 3 multiple wells and 1 background control well for each concentration.
TABLE 2 ABTS radical scavenging test reaction System
And after incubation for 2-6 min at room temperature, reading the absorbance OD value at 734nm, and calculating the clearance rate of the sample on ABTS free radicals.
The sample has an ABTS free radical clearance (%) [ (C1-C2) - (T1-T2) ]/(C1-C2) × 100%
In the formula: c1-blank ABTS system absorbance value; c2-blank absorbance value without ABTS system; t1-sample set has ABTS system absorbance value; t2-sample set Absorbance value without ABTS system.
TABLE 3 ABTS free radical scavenging test results analysis (Mean + -SD)
As can be seen from Table 2, the fungus fermentation product (i.e. sample) has the ability to scavenge ABTS free radicals, the more strongly the fungus fermentation product can scavenge the free radicals as the concentration of the sample increases, and 75.21 + -1.20% of the ABTS free radicals can be scavenged by 20% (v/v) of the fungus fermentation product, which indicates that the fungus fermentation product has better antioxidant activity, see FIG. 2.
(2) And (4) evaluating the anti-aging effect of the fungus fermentation product.
2.1 evaluation of in vitro skin repair and regeneration efficacy (cell proliferation potency of NHDFs)
The relative proliferation capacity of human skin fibroblasts (NHDFs) was examined by the MTT method.
Cells in logarithmic growth phase were collected at a cell density of 6X 103One well was inoculated to a 96-well plate containing 100. mu.L of culture medium per well. In an incubator (37 ℃, 5% CO)2) After 24h of medium culture, the samples were set from low to high at 7 dosing concentrations of 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.0% (v/v), respectively, with 1% FBS as a positive control and untreated cells as a negative control, and 4 replicate wells were set for each group.
Adding the drug into an incubator (37 ℃, 5% CO)2) The culture was continued for 48h, and then MTT assay was performed with no MTT treatment as background control, 1 background control was set for each concentrationThe absorbance OD value was read at 490/603nm using MTT-treated wells without cells as a blank, and the average OD value of the blank should be less than 0.1. The relative proliferative capacity of the cells was calculated according to the following formula.
Relative proliferation capacity (%) × 100% (administration well OD-administration background OD)/(solvent control well OD-solvent control background OD).
TABLE 4 analysis of cell proliferation assay results (action 48h)
As is clear from Table 4, the fermentation products of fungi (i.e., samples) have the ability to promote the proliferation of human skin fibroblasts (NHDFs). Compared with a negative control, when the concentration of the sample is about 0.3%, the proliferation of the NHDFs cells can be obviously promoted, and the proliferation capacity of the NHDFs cells is stronger along with the increase of the concentration of the sample; when the concentration of the sample reached 10%, the proliferation potency of NHDFs was promoted to 4.47. + -. 0.25-fold of that of the negative control, as shown in FIG. 3.
2.2 in vitro tightening anti-wrinkle efficacy assessment (collagen I content)
The type I collagen content was determined by ELISA.
Human skin fibroblasts (NHDFs) in logarithmic growth phase were collected at a cell density of 6X 104One/well was inoculated into 12-well plates. In an incubator (37 ℃, 5% CO)2) After 24h of medium incubation, samples were added at a concentration of 10% (v/v) based on cytotoxicity results, with 50. mu.g/mL ascorbic acid (VC) as a positive control, untreated cells as a negative control, and reaction system reagents and solvents as solvent controls, and 3 replicate wells were set for each group.
Adding the drug into an incubator (37 ℃, 5% CO)2) Culturing for 48h, and collecting the culture solution. And (3) detecting the content of the type I collagen in the culture solution by adopting a Col IELISA kit, and calculating the relative content of the type I collagen relative to a solvent control.
Collagen content of negative control well
TABLE 5 analysis of type I collagen relative content results (Mean + -SD)
From table 5, based on the results of the test for promoting the synthesis of human skin fibroblast type I collagen, the fungus fermentation product (i.e. sample) has certain effects of tightening and anti-wrinkle, and the sample with the concentration of 10% (V/V) can promote the synthesis capacity of type I collagen to be increased to 6.87 ± 0.79 times of that of the negative control, which is obviously higher than that of the positive control, as shown in fig. 4.
(3) And evaluating the whitening effect of the fungus fermentation product.
The melanoma cells of mice in the logarithmic growth phase (B16-F10) were collected at a cell density of 8X 105One/bottle was inoculated into a T25 cell culture flask. In an incubator (37 ℃, 5% CO)2) After 24h of medium incubation, samples at a concentration of 10% (v/v) were added, 1% ascorbyl glucoside (AA2G) as a positive control and untreated cells as a negative control, with 3 replicates each, based on cytotoxicity results.
Adding the drug into an incubator (37 ℃, 5% CO)2) The culture is continued for 48h, the cells are collected by digestion with 0.25% pancreatin and counted, an equal number of cells are centrifuged, the supernatant is discarded, 0.5mL of 1M NaOH containing 10% DMSO is added, the mixture is placed in a water bath at 80 ℃ for 30min, the supernatant is centrifuged and added to a 96-well plate, the 1M NaOH containing 10% DMSO is used as a blank, the absorbance value is read at 475nm, and the relative content of the melanin in the cells is calculated.
Relative content (%) of melanin in cells was (administration hole OD-blank hole OD)/(control hole OD-blank hole OD) × 100%.
TABLE 6 relative melanin content results analysis (Mean + -SD)
As can be seen from table 6, the test results are shown in table 6, based on the results of the experiment for inhibiting melanin synthesis, the samples had better whitening effect, and the samples at the concentration of 10% (V/V) inhibited the melanin production by 44.01 ± 0.77%, as shown in fig. 5.
Therefore, the performance detection results of the fungus fermentation products show that the fungus fermentation products can effectively promote division and proliferation of human skin fibroblasts (NHDFs), promote synthesis of type I collagen, and have good free radical scavenging capacity and melanin generation inhibiting capacity, so that the effects of resisting aging, resisting oxidation and whitening can be improved, and the fungus fermentation products can be widely applied to various cosmetics.
Further, in step S2, the screening of recombinant strains after mutagenesis of the original yeast strain comprises:
s21, carrying out chemical mutagenesis and screening on the original yeast strain to obtain a first mutant strain with acetic acid tolerance and a second mutant strain with furfural tolerance;
specifically, the original yeast strain KN-1 was cultured in a yeast-extracted peptone glucose (YEPD) liquid medium for 22 hours until the logarithmic phase of bacterial growth to obtain a bacterial solution, and the following chemical mutagenesis experiment was performed with this material.
5mL of the cell suspension was centrifuged at 5000r/min for 5min to collect the cells, and washed 2 times with 0.2mol/L phosphate buffer (PB, pH 5.8) to resuspend the cells in 2.5mL of phosphate buffer to obtain a resuspended cell suspension, the liquid medium was removed by centrifugation and two washes, the cells were washed thoroughly, and resuspended in an appropriate buffer.
Add 500. mu.L of resuspended, 490. mu.L of phosphate buffer and 10. mu.L of diethyl sulfate to a 15mL sterilized centrifuge tube and vortex and mix to start the chemical mutagenesis reaction of yeast strains. Optionally, the volume ratio of the resuspended bacteria liquid, the phosphate buffer and the diethyl sulfate is 40-60: 40-55: 1. In this example, the volume ratio of the resuspended bacterial liquid, the phosphate buffer and the diethyl sulfate was 50:49: 1.
Culturing for 25-45 min at 30 ℃ and 200r/min, adding 25% sodium thiosulfate solution with the volume 5 times of that of the yeast strain to terminate the reaction, and obtaining the reaction bacterial liquid after mutagenesis, thereby realizing the first mutation of the original yeast strain KN-1.
Diluting the reaction bacterial liquid, coating the diluted reaction bacterial liquid on an inhibitor tolerance screening solid culture medium, culturing the reaction bacterial liquid at the constant temperature of 30 ℃ for 48 hours, and then screening the inhibitor tolerance, wherein the inhibitor comprises acetic acid and furfural.
Therefore, chemical mutagenesis is carried out on the original yeast strain KN-1, and a first mutant strain KN-1-8 and a second mutant strain KN-1-16 with acetic acid tolerance and a second mutant strain KN-1-3 and a second mutant strain KN-1-5 with furfural tolerance are obtained through inhibitor tolerance screening, wherein the first mutant strain can grow normally on an inhibitor tolerance screening solid culture medium containing 1-8 g/L acetic acid at the highest level, and the second mutant strain can grow normally on an inhibitor tolerance screening solid culture medium containing 0.1-1.5 g/L furfural at the highest level.
S22, subjecting the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation;
s23, obtaining the recombinant strain through inhibitor tolerance screening.
Specifically, after the mutagenesis and/or genetic transformation of genome DNA, a certain volume of sorbitol solution is immediately added and gently mixed to maintain the activity of thalli; transferring to a 5.0mL centrifuge tube, adding 1mLYEPD liquid culture medium, and incubating at 30 deg.C for 2 h; after the incubation is finished, centrifuging at 8000r/min for 3min to collect cells, and suspending the cells in 1mL of sterile ultrapure water; the recombinant strain KN-1-24 is uniformly coated on an inhibitor tolerance screening solid culture medium containing acetic acid and furfural with certain concentration, and the recombinant strain KN-1-24 which grows better on the inhibitor tolerance screening solid culture medium containing acetic acid of 1-8 g/L and furfural of 0.1-1.5 g/L is obtained through screening.
Therefore, the original yeast strain KN-1 is mutated for the first time to screen a first mutant strain with acetic acid tolerance and a second mutant strain with furfural tolerance, and then the first mutant strain and the second mutant strain are mutated for the second time to screen a recombinant strain KN-1-24 which grows better on an inhibitor tolerance screening solid culture medium of 1-8 g/L acetic acid and 0.1-1.5 g/L furfural at the same time, so that the improved recombinant strain is effectively ensured to have both acetic acid tolerance and furfural tolerance.
Inoculating original microzyme KN-1, a first mutant strain KN-1-8, a first mutant strain KN-1-16, a second mutant strain KN-1-3, a second mutant strain KN-1-5 and a recombinant strain KN-1-24 into 10mL YEPD liquid culture medium, culturing under the same condition, respectively transferring 10% of inoculum size into 100mL inhibitor tolerant fermentation culture medium and cellulose hydrolysate fermentation culture medium, setting 3 times of repetition for each condition, culturing under the condition of 30 ℃, and regularly sampling to detect various fermentation performance indexes. Taking out 1mL of fermentation liquid after fermenting for 36h, shaking, diluting properly, centrifuging the diluted liquid 8000r/min for 5min, and measuring ethanol and glucose (g/L) with SBA-40C type biosensor analyzer, with the detection results shown in Table 7.
TABLE 7 comparison of sugar utilization and ethanol production Capacity for each sample
As can be seen from Table 7, the sugar utilization rate and ethanol production capacity of the recombinant strain KN-1-24 are higher than those of the original yeast and the first and second mutant strains, and the sugar utilization rate and ethanol production capacity of the first mutant strain KN-1-16 are inferior to those of the recombinant strain KN-1-24. Therefore, the recombinant strain KN-1-24 has higher sugar utilization rate and ethanol production capacity, can shorten the period required by later-stage fermentation, and greatly improves the fermentation efficiency.
Further, in step S22, subjecting the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation includes:
s221, extracting total genomic DNA of the first mutant strain and the second mutant strain for transformation to obtain a yeast liquid;
specifically, extracting total genomic DNA of a first mutant strain KN-1-16 and a second mutant strain KN-1-3 by adopting an Ezup column type yeast genomic DNA extraction kit; and culturing 50mL of the strain until the logarithmic phase of the growth of the strain is reached, and converting to obtain a yeast liquid, wherein the yeast liquid is a mutant strain KN-1-16 and a second mutant strain KN-1-3.
S222, carrying out centrifugal collection and heavy suspension treatment on the yeast liquid to obtain competent cells.
Specifically, after centrifugally collecting primary thalli from the yeast liquid, carrying out heavy suspension treatment by using a lithium acetate-dithiothreitol mixed solution; and centrifuging again to collect the secondary thalli, washing with precooled sterile water, and suspending in precooled sorbitol solution to obtain competent cells. Optionally, pre-cooled sterile water is washed by sterile water at about 4 ℃; the pre-cooled sorbitol solution is a sorbitol solution at about 4 deg.C.
In this embodiment, first-order bacteria are collected by centrifugation at 5000r/min at 4 ℃ for 5min, resuspended in an equal volume of precooled lithium acetate-dithiothreitol mixed solution (the volume ratio of lithium acetate-dithiothreitol is preferably 7-11: 1), and treated for 20 min; and centrifuging at 5000r/min at 4 ℃ for 5min to collect thalli, washing with precooled sterile water for 2 times, finally suspending in 1mL of precooled 1mol/L sorbitol solution to obtain competent cells, and placing on ice for later use to preserve the activity of the competent cells. Therefore, the mixed solution of lithium acetate-dithiothreitol is removed by collecting the thalli through secondary centrifugation, the thalli are fully washed, and the volume of the bacteria solution is concentrated, so that relatively pure competent cells are obtained.
Further, in step S22, the subjecting the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation further comprises:
s223, mixing the total genomic DNA of the first mutant strain and the second mutant strain in equal amount, and adding diethyl sulfate for culturing to obtain the DNA to be transformed;
specifically, after equal amounts of total genomic DNA of a first mutant strain KN-1-16 and a second mutant strain KN-1-3 are taken and mixed, diethyl sulfate is added to enable the final volume fraction of the diethyl sulfate to be 1%, then the mixture is incubated at 37 ℃ for 1h, and DNA to be transformed is obtained after the culture is completed and is reserved.
S224, mixing the competent cells and the DNA to be transformed according to the volume ratio of 2:1, and placing the mixture in an electric transformation cup for electric shock treatment.
Specifically, 80. mu.L of competent cells were mixed with 20. mu.L of DNA to be transformed, and transferred to a 100. mu.L electric transformation cell, followed by electric shock at a voltage of 2.0kV to denature the mixture.
Therefore, competent cells are prepared by culturing the first mutant strain KN-1-16 and the second mutant strain KN-1-3, then the same amount of total genomic DNA of the first mutant strain and the second mutant strain is mixed and cultured to prepare DNA to be transformed, finally the competent cells and the DNA to be transformed are mixed according to the volume ratio of 2:1 and treated by electric shock, and the total genomic DNA of the first mutant strain and the second mutant strain is introduced into the competent cells.
Further, in step S3, the activating culture of the recombinant strain specifically includes: inoculating the recombinant strain KN-1-24 into a seed culture medium, and shake culturing for 16-20 h at 28-30 ℃ and 180-200 r/min in a shaking table. Optionally, the volume ratio of the recombinant strain KN-1-24 to the seed culture medium is 5-10. The seed culture medium comprises 10-30 g/L of glucose, 5-15 g/L of yeast extract and 10-30 g/L of peptone.
Further, in step S4, the liquid fermentation medium includes 10-20 g/L of fungi including Tricholoma matsutake and 10-30 g/L of carbon source including at least one of sucrose, maltose, glucose, fructose, lactose, galactose, mannitol and maltitol.
The embodiment also provides a cosmetic, which is prepared by the preparation process of the fungus fermentation product, so that the fungus fermentation product is prepared by the preparation process of the fungus fermentation product, is rich in flavone, polyphenol, polypeptide, various amino acids and other components, can effectively promote division and proliferation of human skin fibroblasts (NHDFs), promotes synthesis of type I collagen by the NHDFs, has good free radical scavenging capacity and melanin generation inhibiting capacity, can improve anti-aging, anti-oxidation and whitening effects, and can be widely applied to various cosmetics.
Comparative example 1
This comparative example differs from the examples in that step S2 is different, i.e., substeps S22 to step S23 in step S2 are omitted, thereby making the selection of recombinant strains different in step S2. In this comparative example, the recombinant strains were the first mutant strains KN-1-8, KN-1-16.
Comparative example 2
The comparative example is different from comparative example 1 in that the recombinant strains are a second mutant strain KN-1-3 and a second mutant strain KN-1-5.
The fungus fermentations prepared in examples and comparative examples 1-2 were tested for ABTS free radical clearance, relative cell proliferation, type I collagen synthesis and relative melanin inhibition under the same test conditions, and the results are shown in Table 8.
TABLE 8 comparison of results of detection indexes of fermented products of different fungi
As can be seen from Table 8, the fungus fermentations prepared in the examples have better ABTS free radical scavenging rate, cell relative proliferation capacity, type I collagen synthesis capacity and melanin relative inhibition rate than the fungus fermentations prepared in the comparative examples 1-2 under the same test conditions.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (11)
1. A preparation process of a fungus fermentation product is characterized by comprising the following steps:
s1, pulverizing fungi, and sieving to obtain fungi powder;
s2, carrying out mutagenesis on the original yeast strain and screening out a recombinant strain, wherein the recombinant strain has acetic acid tolerance and/or furfural tolerance;
s3, carrying out activated culture on the recombinant strains to obtain yeast seed liquid;
s4, adding the fungus powder and the yeast seed liquid into a liquid fermentation culture medium for fermentation to obtain fermentation liquid;
s5, centrifuging the fermentation liquor, filtering and sterilizing to obtain the fungus fermentation product.
2. The process of claim 1, wherein the step of mutagenizing the original yeast strain to select the recombinant strain comprises:
s21, carrying out chemical mutagenesis and screening on the original yeast strain to obtain a first mutant strain with acetic acid tolerance and a second mutant strain with furfural tolerance;
s22, subjecting the first mutant strain and the second mutant strain to genomic DNA mutagenesis and/or genetic transformation;
s23, obtaining the recombinant strain through screening.
3. The process of claim 2, wherein the subjecting the original yeast strain to chemical mutagenesis and screening comprises:
culturing an original yeast strain in a liquid culture medium, centrifugally collecting thalli, and washing with a phosphate buffer solution to obtain a heavy suspension bacterial liquid;
adding a heavy suspension bacterium solution, a phosphate buffer solution and diethyl sulfate into a 15mL sterilized centrifugal tube, performing vortex oscillation and uniform mixing, culturing for 20-40 min, adding a sodium thiosulfate solution to terminate the reaction, and obtaining a reaction bacterium solution after mutagenesis;
and (3) diluting the reaction bacterial liquid, coating the diluted reaction bacterial liquid on an inhibitor tolerance screening solid culture medium for culture, and then screening the inhibitor tolerance.
4. The process for preparing a bacterial fermentation product according to claim 3, wherein the volume ratio of the resuspended bacterial liquid, the phosphate buffer and the diethyl sulfate is 40-60: 40-55: 1.
5. The process of claim 2, wherein the mutagenesis and/or genetic transformation of genomic DNA of the first mutant strain and the second mutant strain comprises:
extracting total genomic DNA of the first mutant strain and the second mutant strain for transformation to obtain a yeast liquid;
and carrying out centrifugal collection and heavy suspension treatment on the yeast liquid to obtain competent cells.
6. The process of claim 5, wherein the centrifuging, collecting and resuspending the yeast liquid comprises:
centrifugally collecting primary thalli from the saccharomycete liquid, and then carrying out heavy suspension treatment on the primary thalli by using a lithium acetate-dithiothreitol mixed solution;
and centrifuging again to collect the secondary thalli, and then washing with precooled sterile water.
7. The process of claim 5 or 6, wherein the mutagenesis and/or genetic transformation of genomic DNA of the first mutant strain and the second mutant strain further comprises:
mixing the total genomic DNA of the first mutant strain and the second mutant strain in equal amount, and adding diethyl sulfate for culturing to obtain DNA to be transformed;
the competent cells and the DNA to be transformed are mixed according to the volume ratio of 2:1 and are placed in an electric transformation cup for electric shock treatment.
8. The process of claim 1, wherein in step S3, the step of activating the recombinant strain comprises: inoculating the recombinant strain into a seed culture medium, and performing shaking culture on a shaking table at the temperature of 28-30 ℃ and at the speed of 180-200 r/min for 16-20 h.
9. The process for preparing a bacterial fermentation product according to claim 8, wherein the volume ratio of the recombinant strain to the seed culture medium is 5-10; the seed culture medium comprises 10-30 g/L of glucose, 5-15 g/L of yeast extract and 10-30 g/L of peptone.
10. The process of claim 1, wherein in step S4, the liquid fermentation medium comprises fungi 10-20 g/L and carbon source 10-30 g/L, wherein the fungi comprises Tricholoma matsutake, and the carbon source comprises at least one of sucrose, maltose, glucose, fructose, lactose, galactose, mannitol, and maltitol.
11. Cosmetic product, characterized in that it comprises a process for the preparation of a bacterial fermentation product according to any one of claims 1 to 10.
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