CN105296541A - Oat fermentation liquor, preparation method thereof and application of oat fermentation liquor as cosmetic raw material - Google Patents
Oat fermentation liquor, preparation method thereof and application of oat fermentation liquor as cosmetic raw material Download PDFInfo
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Abstract
The invention discloses an oat fermentation liquor, a preparation method thereof and application of the oat fermentation liquor as a cosmetic raw material. The preparation method of the oat fermentation liquor includes the step of inoculating enterococcus faecium into a culture medium containing oat for fermentation culture so as to obtain a fermentation product, namely oat fermentation liquor. Through experiments, commercially-available edible oatmeal can be selected as a biotransformation object, so that quality safety of the oat raw material is guaranteed; a probiotic, namely the enterococcus faecium, serves as a fermentation strain and is used for subjecting the oat to fermentation biotransformation to obtain the oat fermentation liquor, and the oat fermentation liquor contains the probiotic and nutrition of plant active substances and has the effects which cannot be achieved by individual use of the probiotic or plant active substances. The oat fermentation liquor as a biotransformation liquor is high in safety, has excellent anti-aging, antioxidative and whitening effects and can be applied to cosmetics.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of oat fermented liquid and preparation method thereof and the application as cosmetic material.
Background technology
Oat is one of ancient raise crop originating from China, will become the third-largest staple food grain of the mankind; Oat has reducing blood-fat, auxiliary hyperglycemic, improves the functions such as immunizing power.Oat is the cereal served many purposes, and can be used for the aspects such as feed, medicine, industrial raw material and makeup, cereals nutrient enriches, and not only kind is many for the function composition that it contains, and content is also very high.The effects such as oat has moisturizing in makeup, anti-oxidant, anti-ageing, anti-quick, antipruritic.Its effective constituent comprises beta-glucan, protein polypeptide, and phenols, alkaloid, VITAMIN etc.Such as a kind of method promoting cell regeneration disclosed in patent documentation 1 (USpatentno.US5888521).A kind of composition of this patented invention includes hydroxycarboxylic acid and oat extract, and finds that this composition can promote cell regeneration.
Probiotic bacterium is generally acknowledged useful to human body, and has a large amount of amino acid in its fermented liquid, natural harmless as cosmetics additive.Current a lot of well-known skin care product are all using probiotics fermention product as active additive component, such as the AdvancedNightRepair of Estee Lauder (EsteeLauder) with the addition of bifidobacterium fermentation product, the general red sun-proof foundation emulsion of releiving of Clinique (Clinique) with the addition of lactobacillus fermentation product, the patent composition Pitera of SKII brand.Pitera is a kind of liquid that a kind of yeast being called Saccharomycopsis is extract during the fermentation, has excellent moistening and special moisture-keeping functions.
Such as lactobacillus-fermented rice extracting solution disclosed in patent documentation 2 (USpatentno.US8071115B2) is as the manufacture method of skin care product active ingredient.Describe in patent documentation 1 in skin care product and add lactobacillus-fermented rice extracting solution and can change hair quality, bright color, and cosmetic emulsion can be made more stable.
And for example a kind of preparation method obtaining cosmetic raw materials from lactobacillus fermentation product disclosed in patent documentation 3 (USpatentno.US5324515).Lactobacillus cell lysate is included in preparation method.And cosmetic raw materials that document describes its invention has oxidation resistant effect, and can repair the DNA of skin damage, improve the immunizing power of skin.
Summary of the invention
The object of this invention is to provide a kind of preparation method of oat fermented liquid.
Method provided by the invention, comprise the steps: to be inoculated into by faecium in the substratum containing oat, fermentation culture, obtains tunning, namely obtains oat fermented liquid.
In aforesaid method, the described substratum containing oat is made up of oat, MRS liquid nutrient medium and water, and the mass percentage of described oat in the described substratum containing oat is 1.25%-5%; The volumn concentration of described MRS liquid nutrient medium in the described substratum containing oat is 83.3%;
The above-mentioned substratum containing oat is prepared as follows: be that the ratio of 1:5 arrange and mix with volume ratio by the oat liquid of 7.5% (mass percentage) and MRS liquid nutrient medium, formation contains the substratum of oat.
MRS liquid nutrient medium is made up of the material of following final concentration and water: 20g/L glucose, 2g/L ammonium sulfate, 2g/L disodium citrate, 5g/L sodium-acetate, 0.1g/L magnesium sulfate heptahydrate, 0.05g/L tetra-water manganous sulfate, 2g/L dipotassium hydrogen phosphate, 15g/L yeast powder, 10g/L peptone.
Described faecium inoculates with the form of faecium nutrient solution.
Above-mentioned faecium nutrient solution is cultivated for being inoculated in seed culture medium by faecium, obtains faecium nutrient solution.
Above-mentioned seed culture medium is MRS liquid nutrient medium;
Described culture condition is 37 DEG C, 100r/min, shaking culture 20 hours.
In aforesaid method, the described mass percentage containing the oat in the substratum of oat is 1.25%;
Described inoculation is by 1.5 × 10
9cfu/ml faecium nutrient solution is inoculated in the substratum containing oat, makes the density OD of the described faecium in fermentation initial system
600be 0.5.
Above-mentioned fermentation initial system be the access of described faecium nutrient solution described containing in the substratum of oat time fermenting container in total material;
In aforesaid method, the temperature of described fermentation culture is 30-40 DEG C, and the time of described fermentation culture is 24h-50h; The best of described fermentation culture or actual temp are 37 DEG C, and the best or the concrete time of described fermentation culture are 30 hours;
In described fermentation culture, the pH value of fermentation system is 6.2-7.0, and in described fermentation culture, the pH value of fermentation system is specially 6.8;
The concentration of the glucose of described tunning is no more than 10g/L;
The mode of described fermentation culture for stirring fermentation culture, and adds glucose in fermentation culture process, and makes the final concentration of glucose in fermentation system be no more than 10g/L, is specially 0.5-10g/L.。
In aforesaid method, described method also comprises the steps: after obtaining tunning, collects supernatant liquor; Again by degerming for described supernatant liquid filtering.
Described collection supernatant liquor is centrifugal, described centrifugal be centrifugal 0.5 hour of 14000rpm;
Described filtration adopts filter membrane, and the aperture of described filter membrane is 0.22 μm.
In aforesaid method, described oat has following characteristic: water soluble dietary fiber content is 4.3%, and protein content is 15.2%;
Described faecium is GIMCC numbering: the faecium of GIM1.388.
The oat fermented liquid prepared by aforesaid method.
Above-mentioned oat fermented liquid is also the scope of protection of the invention in the preparation application had in following A or B characteristic product;
A, anti-ageing, whitening and/or anti-oxidant;
Tyrosinase activity and/or elimination free radical in melanic synthesis in B, promotion cell proliferation, T suppression cell, T suppression cell.
In above-mentioned application, described product is makeup or test kit.
In above-mentioned application, described anti-ageing by promoting that cell proliferation embodies; Described cell specifically adopts l cell Balb/c3T3;
Described whitening is embodied by tyrosinase activity in melanic synthesis in T suppression cell and/or T suppression cell; Described cell is melanoma b16-F10 cell;
Described anti-oxidant by eliminating free radical embodiment.
Above-mentioned oat fermented liquid is as cosmetic material or the application prepared in makeup.
Above-mentioned oat fermented liquid following 1)-4) application at least one:
1) cell proliferation is promoted; Described cell is l cell (Balb/c3T3 cell)
2) melanic synthesis in T suppression cell; Described cell is melanoma b16-F10 cell;
3) tyrosinase activity in T suppression cell; Described cell is melanoma b16-F10 cell;
4) free radical is eliminated.
Experiment of the present invention proves, the present invention chooses commercially available edibility rolled oats as bio-transformation object, ensures the quality and safety of oat raw material; Adopt probiotic bacterium faecium as fermented bacterium, fermentative bioconversion is carried out to oat and obtains oat fermented liquid, the nutrition of probiotic bacterium and vegetable active thing can be had concurrently.Therefore, bioconversion broth security of the present invention is high, and has the anti-ageing, anti-oxidant of excellence, and whitening function, may be used in makeup.
There is no particular limitation can to coordinate the makeup kind of oat fermented liquid of the present invention.
Accompanying drawing explanation
Fig. 1 is the impact of oat fermented liquid on melanin content relative in cell
Fig. 2 is the impact of oat fermented liquid on intracellular tyrosine enzyme Relative biological activity
Fig. 3 is the resistance of oxidation of the Trolox equivalent of oat fermented liquid
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, oat conversion fluid is oat fermented liquid.
The preparation of embodiment 1, oat fermented liquid
The configuration of the substratum one, containing oat
Choose commercially available edibility rolled oats as bio-transformation object, its water soluble dietary fiber content is 4.3%, and protein content is 15.2%.
Preparation 7.25% (mass percentage) oat liquid, is dissolved in rolled oats in water, 121 DEG C of sterilizing 20min.
MRS liquid nutrient medium is made up of the material of following final concentration and water: 20g/L glucose, 2g/L ammonium sulfate, 2g/L disodium citrate, 5g/L sodium-acetate, 0.1g/L magnesium sulfate heptahydrate, 0.05g/L tetra-water manganous sulfate, 2g/L dipotassium hydrogen phosphate, 15g/L yeast powder, 10g/L peptone, pH regulator to 7.0,121 DEG C of sterilizing 20min.
Substratum containing oat is made up of oat, MRS liquid nutrient medium and water, and wherein oat is being 1.25% containing the mass percentage in the substratum of oat; The volumn concentration of MRS liquid nutrient medium in the described substratum containing oat is 83.3%.
The above-mentioned substratum containing oat is prepared as follows: be that the ratio of 1:5 arrange and mix with volume ratio by the oat liquid of 7.5% (mass percentage) and MRS liquid nutrient medium, formation contains the substratum of oat.
Two, the preparation of faecium nutrient solution
Bacterial classification: (Guangdong Microbes Inst Culture Collection, GIMCC numbers faecium: GIM1.388).
Plate culture medium: MRS nutrient agar, containing 2.0% agar, pH is 6.5.
Shake-flask seed substratum is MRS liquid nutrient medium, and pH is 6.2 ~ 6.5.
Draw oblique line inoculation faecium on plate culture medium with transfering loop, cultivate 18 for 37 DEG C and littlely to occur up to faecium bacterium colony.3 colony inoculations are got in shake-flask seed substratum (250ml triangle bottled MRS liquid nutrient medium 150ml) from flat board, 37 DEG C, 100r/min, shaking culture 20 hours, obtains faecium nutrient solution and (is about 1.5 × 10
9cfu/ml).
Three, the acquisition of oat fermented liquid
Faecium nutrient solution is inoculated in the substratum containing oat, makes the density of the initial faecium of fermentor tank be OD
600value is 0.5, carries out stirring fermentation culture, obtain tunning in 1L fermentor tank, by tunning with 16000 × g centrifugal 0.5 hour, and then degerming with the filter membrane of 0.22 μm, collect filtrate, be oat fermented liquid.
The condition of above-mentioned fermentation culture is 37 DEG C, and 100rpm, pH are 6.8, cultivates 30 hours.
Automatically regulate pH with 4M sodium hydroxide in fermenting process, pH is controlled 6.8.After the 20g/L glucose in initial medium has been consumed, adopt the method for the fed-batch fermentation adding glucose in fermentor tank.Concentration as feed supplement glucose is 50%, and the concentration of adding glucose in glucose secondary fermentation tank (fermentation system) is no more than 10g/L (0.5 ~ 10g/L), makes the content of glucose in final oat fermented liquid may not exceed 10g/L.
The evaluation of embodiment 2, oat fermented liquid
1, the evaluation of the anti-aging effects of oat fermented liquid
Oat fermented liquid anti-aging effects is evaluated by the effect of its on cell proliferation, adopts l cell (Balb/c3T3 cell) as the reacting cells detected.In cell tests, reaction substrate MTS is become red water-soluble Xing formazan by cell desaturase, its amount formed becomes positive correlation with living cells metabolize rate and degree of cell proliferation, carry out the situation of reacting cells propagation at 490nm place absorption peak finally by it, thus evaluate the senile-resistant efficacy of oat fermented liquid, specific as follows:
By the normal saline dilution 10 times of the above-mentioned oat fermented liquid prepared by embodiment 1 with 0.1% bovine serum albumin, then get the first row that 200 μ l put test 96 hole microtest plates into, add the normal saline dilution liquid of 100 μ l0.1% bovine serum albumins toward the secondary series in dilution plate to the 12 row.Extract 100 μ l from first row and put into secondary series, get 100 μ l and put into the 3rd row after mixing, serial dilution is until the 11 row according to this.100 μ l are got in every hole in the 11 row after dilution well throw aside.12 is classified as no specimen blank (physiological saline of 0.1% bovine serum albumin).
On another block 96 orifice plate, every hole all inoculate 50 μ l also have 10000 l cells (Balb/c3T3 cell,
cCL-163
tM) enchylema, substratum is the DMEM substratum not adding serum.
Using the oat fermented liquid that diluted (with the physiological saline of 0.1% bovine serum albumin as diluted oat fermented liquid, make the volumn concentration of oat fermented liquid be 5%, 2.5%, 1.25%) and no specimen blank correspondence get 50 μ l and add on above-mentioned cell 96 orifice plate, put into 37 DEG C of constant temperature CO2gas incubator cultivations about 44 ~ 48 hours.
Add in cell plate by MTS/PMS reagent, every hole is 20 μ l, then puts into 37 DEG C of constant temperature CO2gas incubator 4 hours, measures the absorbancy of 490nm wavelength by microplate reader.Then using 0.1% bovine serum albumin physiological saline as the absorbancy of blank group for 1, calculate the relative value of the amount of viable cell of other experimental example, evaluate cel l proliferation.
Result is as shown in table 1,
Table 1 is the impact of oat fermented liquid on cell proliferation
| Sample | 490nm absorbancy | Relative cell proliferation multiple | |
| 1 | 0.1% bovine serum albumin physiological saline | 0.163±0.007 | 1.00 |
| 2 | 5% containing the substratum of oat | 0.169±0.008 | 1.04 |
| 3 | 5% oat fermented liquid | 0.641±0.017 | 3.93 |
| 4 | 2.5% oat fermented liquid | 0.636±0.024 | 3.90 |
| 5 | 1.25% oat fermented liquid | 0.536±0.014 | 3.29 |
As seen from Table 1, the fermented liquid after faecium bio-transformation oat and oat fermented liquid, compared with the substratum containing oat of non-microbe conversion, have excellent cel l proliferation.Show that oat fermented liquid has senile-resistant efficacy.
2, the evaluation of the whitening function of oat fermented liquid
Oat fermented liquid white-skinned face function is by the relative content of intracellular melanin, and the Relative biological activity of intracellular tyrosine enzyme is evaluated.
1) mensuration of intracellular melanin relative content
Take the logarithm melanoma b16-F10 cell (ATCC ° of number:CRL-6475 in vegetative period
tM), adjustment cell density is 6 × 10
4/ 1.8ml, is inoculated in 6 well culture plates, and each hole of experimental group adds the oat fermented liquid prepared by embodiment 1 of 200 μ l different concns, and the diluent pH of sample is the PBS damping fluid of 7.4, and positive controls is arbutin, and negative control group is the PBS of 200 μ l.Be placed in saturated humidity, 37 DEG C, 5%CO
272h is cultivated in incubator.
Suck substratum, wash 2 times with PBS, add 0.25% pancreatin 100 μ l and digest, basis of microscopic observation cellular form, add 1ml perfect medium and stop digestion, collect Digestive system and manage in 1.5mlEP.Centrifugal 3000 × g, 5min, remove supernatant, washs 1 time with PBS, adds 500 μ lPBS (getting 100 μ l for surveying albumen), centrifugal, removes supernatant, then add 10% trichoroacetic acid(TCA) 100 μ l.Centrifugal, remove supernatant, add 1molL
-1naOH solution (comprising 10%DMSO) 120 μ l, water-bath 80 DEG C, to dissolving completely, proceeds to enzyme plate, measures every hole 405nm absorbance A value immediately with microplate reader.
For surveying 100 μ l cells of albumen, centrifugal, remove supernatant.Add 10% trichoroacetic acid(TCA) 100 μ l, centrifugal, remove supernatant.Resolution of precipitate is at the 1NNaOH solution (comprising 5%SDS) of 40 μ l.Add the distilled water of 960 μ l again.Testing sample carries out determining the protein quantity according to BCA test kit.
According to the method for calculation of intracellular melanin synthesis relative content, calculate the relative content of B16 cell, in order to evaluate the white-skinned face function of oat fermented liquid.
Result as shown in Figure 1, can be found out, B16 cell relative content in oat fermented liquid T suppression cell.
2) intracellular tyrosine activity determination method
Get above-mentioned 1) application of sample cultivates 6 orifice plates of 72h, after collecting cell, 1 time is washed with PBS, every hole adds 1%TritonX-100/PBS solution 250 μ l, puts rapidly-80 DEG C of frozen 30min, and room temperature thawing subsequently makes the complete cracking of cell, centrifuging and taking supernatant liquor 100 μ l after 37 DEG C of pre-temperature, add 1% levodopa (L-DOPA) solution 10 μ l, 37 DEG C of reaction 2h, measure 405nm absorbance A value.The method of the total protein content BCA of cell measures.Concrete grammar is: get above-mentioned lysate, and the experimental technique according to BCA test kit carries out protein determination.Calculate intracellular tyrosine enzyme relative reactivity, evaluate oat fermented liquid to the suppression of tyrosinase activity.
Result as shown in Figure 2, tyrosinase activity in oat fermented liquid T suppression cell.
Find out according to Fig. 1 and Fig. 2, oat fermented liquid can the synthesis of obvious check melanin, and has very strong restraining effect to intracellular tyrosine enzyme, and its effect can reach the effect that certain density arbutin plays.
3, the evaluation of the antioxygenation of oat fermented liquid
Measure the resistance of oxidation of the oat fermented liquid prepared by embodiment 1, mainly by the resistance of oxidation of the Trolox equivalent of TEAC method analysis and research oat fermented liquid.Employing test kit is the OxiSelect of CellBiolabs company of the U.S.
tMtotalAntioxidantCapacity (TAC) AssayKit.
Result as shown in Figure 3, can be found out, oat fermented liquid has the ability eliminating free radical, and the oat fermented liquid of 12.5% is equivalent to the resistance of oxidation of the Trolox of 0.1mM.
Claims (10)
1. a preparation method for oat fermented liquid, comprise the steps: to be inoculated into by faecium in the substratum containing oat, fermentation culture, obtains tunning, namely obtains oat fermented liquid.
2. method according to claim 1, is characterized in that:
The described substratum containing oat is made up of oat, MRS liquid nutrient medium and water, and the mass percentage of described oat in the described substratum containing oat is 1.25%-5%.
3. method according to claim 1 and 2, is characterized in that: the described mass percentage containing the oat in the substratum of oat is 1.25%.
4., according to described method arbitrary in claim 1-3, it is characterized in that:
Described faecium inoculates with the form of faecium nutrient solution;
The temperature of described fermentation culture is 30-40 DEG C, and the time of described fermentation culture is 24h-50h;
In described fermentation culture, the pH value of fermentation system is 6.2-7.0;
The mode of described fermentation culture for stirring fermentation culture, and adds glucose in fermentation culture process, and makes the final concentration of glucose in fermentation system be no more than 10g/L;
The concentration of the glucose of described tunning is no more than 10g/L.
5., according to described method arbitrary in claim 1-4, it is characterized in that:
Described method also comprises the steps: after obtaining tunning, collects supernatant liquor; Again by degerming for described supernatant liquid filtering, obtain oat fermented liquid.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
Described oat has following characteristic: water soluble dietary fiber content is 4.3%, and protein content is 15.2%.
7. the oat fermented liquid prepared by claim 1-6.
8. the application had in following A or B characteristic product prepared by oat fermented liquid according to claim 7;
A, anti-ageing, whitening and/or anti-oxidant;
Tyrosinase activity and/or elimination free radical in melanic synthesis in B, promotion cell proliferation, T suppression cell, T suppression cell.
9. application according to claim 8, is characterized in that: described product is makeup or test kit.
10. application according to claim 8 or claim 9, is characterized in that:
Described anti-ageing by promoting that cell proliferation embodies;
Described whitening is embodied by tyrosinase activity in melanic synthesis in T suppression cell and/or T suppression cell;
Described anti-oxidant by eliminating free radical embodiment.
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| CN113018239A (en) * | 2021-03-30 | 2021-06-25 | 山东花物堂生物科技有限公司 | Oat fermentation extract and preparation method and application thereof |
| CN113018239B (en) * | 2021-03-30 | 2023-01-17 | 山东花物堂生物科技有限公司 | Oat fermentation extract and preparation method and application thereof |
| CN113559045A (en) * | 2021-08-26 | 2021-10-29 | 浙江辰海生命科学有限公司 | Oat bran fermentation product, skin external preparation containing oat bran fermentation product, and preparation method and application of oat bran fermentation product |
| CN114159370A (en) * | 2021-12-29 | 2022-03-11 | 山东花物堂生物科技有限公司 | Application of oat fermentation extract in preparation of cosmetics |
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