A kind of Aloe proferment pulp cosmetic and preparation method thereof and application
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Aloe proferment pulp cosmetic and preparation method thereof and application.
Background technology
Aloe (Aloe) is Liliaceae, perennial evergreen meat succulence herbaceous plant.Aloe is a kind of Important Economic plant integrating medicine and eat, keep healthy, improve looks and view and admire.The chemical composition of Aloe is quite complicated, finds in Aloe containing 80 plurality of active ingredients headed by aloin through studying for a long period of time.Main containing anthraquinone and glycoside, glycosides, naphthalenone, resin, organic acid, saccharide, protein, calcium oxalate, fiber, trace element etc.In beauty treatment and cosmetics, what play a major role is mucus composition to Aloe, and the natural wetting agent of polysaccharide material wherein and Amino acid profile, can supplement the moisture in skin, recover the function of collagen protein, prevent facial wrinkles, keep skin smooth, high resilience.Aloe arbutin in Aloe, Aloe crow are pungent, and (Aloeulcin) can treat gastric and duodenal ulcers, mucosa festers.Aloe arbutin can also prevent skin aging.China's Aloe resources enriches, for Application and Development Aloe provides advantage.Develop rapidly along with luxuriant growth cultivation industry and range of application is widened, to aloe nutritional composition and in medical care effect, research is also progressively deeply.Also constantly perfect to the extraction process of active aloe material, but extracting method is mainly based on homogenate extraction method, water extraction.
What existing active aloe thing extraction the most often adopted is water extraction: Aloe dry product adds deionized water after pulverizing, and get filtrate after vacuum filtration, filtrate concentrated on a rotary evaporator, last refrigerator cold-storage also uses alcohol settling, obtains product after centrifugal.But the method introduces Organic substance in leaching process, not only welding, and potential potential safety hazard is there is in the process of application.Therefore, in order to avoid the problems referred to above, need to provide a kind of method utilizing fermentation engineering to extract active aloe material.
Summary of the invention
The object of this invention is to provide a kind of Aloe proferment pulp cosmetic and preparation method thereof and application, the method utilizes fermentation engineering to extract active substance from Aloe, simple to operate, and owing to not adding any foreign substance, the Aloe fermented product prepared and Aloe fermentation oleo stock, to skin safe fanout free region, have higher whitening and activity of fighting against senium.
The preparation method of Aloe fermentation oleo stock provided by the invention, comprises the steps: Aloe oleo stock, water and zymocyte to be mixed to get initial system, and carries out fermentation culture to initial system, obtains described Aloe fermentation oleo stock.
In above-mentioned preparation method, in described initial system, the proportioning of described zymocyte, described Aloe oleo stock and water can be (18 ~ 35) mL:(15 ~ 30) g:(250 ~ 400) mL, described zymogenic concentration can be (10
6~ 10
9) CFU/mL; Particularly, in described initial system, the proportioning of described zymocyte, described Aloe oleo stock and water can (18 ~ 35) mL:(15 ~ 30) g:320mL; More specifically, in described initial system, the proportioning of described zymocyte, described Aloe oleo stock and water is 20mL:18g:320mL, and the concentration of described Aloe oleo stock can be 10
6~ 10
9cFU/mL.
In above-mentioned preparation method, the temperature of described fermentation culture can be 30 ~ 45 DEG C, specifically can be 35 DEG C; Time can be 24 ~ 38h, specifically can be 28h.
In above-mentioned preparation method, described zymocyte can be streptococcus, specifically can be streptococcus thermophilus Streptococcusthermophilus; Described Aloe oleo stock, from the blade of Aloe, is the slurry obtained after refiner pulverizing, specifically can be the slurry like material pulverizing 3 ~ 10min acquisition under 1000 ~ 1200r/min rotating speed.
In above-mentioned preparation method, described zymocyte exists with the form of its culture fluid or suspension; The pH value of described zymocyte culture fluid or described zymocyte suspension can be 6.5 ~ 7.5, specifically can be 7.0.
In above-mentioned preparation method, described method, after described fermentation culture, also comprises and carries out sterilizing, centrifugal and get the step of supernatant successively to fermentation liquid.
In above-mentioned preparation method, the condition of described sterilizing is as follows: sterilising temp can be 115 ~ 121 DEG C, specifically can be 115 DEG C; Sterilization time can be 15 ~ 25min, specifically can be 20min.
In above-mentioned preparation method, described centrifugal condition is as follows: centrifugal rotational speed can be 4000 ~ 6000r/min, concrete 4700r/min; Described centrifugation time can be 20 ~ 30min, specifically can be 25min; Described centrifugal radius can be 9cm.
Invention further provides a kind of Aloe fermentation oleo stock prepared by above-mentioned preparation method.
Above-mentioned Aloe fermentation oleo stock has following 1 in preparation)-4) at least one function cosmetics in application or directly as having following 1)-4) application in the cosmetics of middle at least one function, also in protection scope of the present invention:
1) restraint of tyrosinase is active;
2) skin-whitening;
3) DPPH free radical is removed;
4) defying age.
Above-mentioned 1) in application-4), described cosmetics include but not limited to: facial film, essence or cosmetic water.
The present invention has following beneficial effect:
(1) preparation method of Aloe fermented product provided by the invention (i.e. Aloe fermentation oleo stock) adopts fermentation engineering, by selecting suitable strain and fermentation condition, extracting and obtaining active substance from Aloe.On the one hand, farthest remain the active component in plant, and relative and traditional water extraction, avoid the introducing of organic solvent, safety and environmental protection; On the other hand, do not need to add the foreign substances such as enzyme, not only save production cost, and the maximized production stage that simplifies, enable this fermentation technique realize a large amount of production, suitability for industrialized production, and fully can ensure the stability of product quality.
(2) the indispensable natural component such as aminoacid, mineral of healthy skin is contained in Aloe fermentation oleo stock provided by the invention, and owing to not introducing foreign substance in preparation process, not containing any chemical composition, can directly use as the finished product of facial film or essence or cosmetic water, more natural than existing other products on the market, any negative interaction can not be caused to skin; And it is little to obtain component molecules amount, is easy to allow skin fully absorb.
(3) Aloe provided by the invention fermentation oleo stock, having stronger oxidation resistance can scavenging free radicals, promotes cellular metabolism, strengthen cell viability, improve the 26S Proteasome Structure and Function of body, improve body vitality, thus delay cell senescence, there is antidotal effect.
(4) Aloe provided by the invention fermentation oleo stock, can the activity of restraint of tyrosinase, and its whitening effect is equivalent to the effect of the arbutin of 0.44%, has certain white-skinned face function.
Accompanying drawing explanation
Fig. 1 is the active polypeptide molecular weight distribution HPLC collection of illustrative plates of Aloe fermentation oleo stock.
Fig. 2 is the Aloe fermentation oleo stock of variable concentrations and the relation curve of DPPH free radical scavenging activity.
Fig. 3 be 10% Aloe fermentation oleo stock and 1% arbutin to the comparison diagram of the suppression ratio of tryrosinase.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Fluid medium in following embodiment is by solute and solvent composition, and solvent is water, and the concentration in solute and liquid medium within thereof is: glucose 20g/L, peptone 20g/L, yeast extract 10g/L, pH value is 7.0.
YPD culture medium in following embodiment is by solute and solvent composition, solvent is water, concentration in solute and liquid medium within thereof is: yeast extract 1% (mass fraction), tryptone 2% (mass fraction), glucose 2% (mass fraction), pH value is 7.0.
Aloe oleo stock in following embodiment is aloe leaf is the slurry like material that in the refiner of 1000 ~ 1200r/min, pulverizing 3 ~ 10min obtains at rotating speed, and aloe leaf used is sold from market.
The ATCC of the streptococcus thermophilus Streptococcusthermophilus in following embodiment is numbered 19258, and the public can buy from ATCC and obtain.
Streptococcus thermophilus bacterium liquid in following embodiment prepares as follows:
1, the activation of strain
Picking streptococcus thermophilus (Streptococcusthermophilus) bacterium colony one ring, in fluid medium, puts into shaking table by actication of culture, obtains activating rear streptococcus thermophilus bacterium liquid.
2, the purification of strain
Bacterium liquid gradient dilution bed board after activation step 1 obtained, to obtain single bacterium colony, obtains streptococcus thermophilus after purification.
3, the amplification culture of strain
Strain inoculation after purification step 2 obtained, in YPD culture medium (pH value is 7.0), is cultivated in the shaking table of 25 DEG C, and when OD value=0.6 ~ 1.2, (strain is in logarithmic (log) phase, and concentration is 10 to obtain zymocyte bacterium liquid
6~ 10
9cFU/mL).
Embodiment 1, preparation Aloe fermentation oleo stock
Prepare Aloe fermentation oleo stock in accordance with the following steps
(1) streptococcus thermophilus fermentation bacterium bacterium liquid 20mL above-mentioned steps 3 obtained is inoculated in the Aloe oleo stock of 18g and the water of 320mL, obtains fermentation system;
(2) fermentation system is fermented 28 hours in 35 DEG C of shaking tables, obtain tunning;
(3) by tunning sterilizing 20min at 115 DEG C, make bacterium inactivation, obtain the tunning after sterilizing;
(4) by the centrifugal 25min under 4700r/min, centrifugal radius are the condition of 9cm of the tunning after sterilizing, abandon precipitation, collect supernatant, be Aloe fermentation oleo stock.
Embodiment 2, Aloe fermentation oleo stock are as the application in cosmetics
One, the character of Aloe fermentation oleo stock
The fermentation oleo stock of gained prepared by embodiment 1 is adopted activated carbon decolorizing, de-tastes.Its outward appearance is thick liquid, color is faint yellow to brown color.PH value 5.2-6.3, viscosity 200-500cP, solubility is admittedly containing thing content 1.5-5.0%, and total plate count is less than 50CFU/mL, detects without pathogenic bacterium.According to hygienic standards for cosmetics GB7916-87, cosmetics total number of bacteria is not higher than 1000CFU/mL, so this fermentation oleo stock meets cosmetics quality requirement.
Carry out component analysis to this Aloe fermentation oleo stock, protein detection method is with reference to GB5009.5-2010; Crude polysaccharides detection method is with reference to GB/T5009.8-2008; Flavone detection method is with reference to GB/T5009.124-2003; Total phenols detection method is with reference to GB/T8313-2008, and acquired results is as follows:
The Aloe fermentation oleo stock that the present invention obtains is containing protein 4.35g/kg, crude polysaccharides 15.36g/kg, total flavones 0.086g/kg (in rutin), total phenols 0.14g/kg.
Two, the safety of Aloe fermentation oleo stock detects
Human body patch test is mainly used for the zest detecting cosmetics finished product or raw material.The present invention carries out human closed formula patch test to the Aloe fermentation oleo stock that embodiment 1 obtains, and is intended to assess its potential skin irritation.
1, subjects
Select suitable volunteer 30 people, the range of age is at 18-60 year Stochastic choice.
2, test method
Taking 0.020g-0.025g solid sample or semisolid sample, to put into speckle examination device for subsequent use.Fluid sample 0.2mL to 0.025mL is dripped on filter paper, then filter paper is placed in speckle examination device.Each sample all arranges blank, in contrast speckle examination device hole, add the sample solvent with sample equivalent, as distilled water or olive oil (the present embodiment employing distilled water).
Human body back is elected at test position as, utilizes non-irritating adhesive tape speckle to be tried device and fixedly sticks in experimenter back.Test period continues 24h.In order to the accurate, credible of result of the test and science, test period volunteer as requested, speckle examination device can not be removed, recipient site also can not be made to contact water.Remove speckle examination device after 24h, after leaving standstill 30min, wait for that impression disappears, observe the reaction of skin.If result of the test is negative, then 24h and 48h after patch test is needed to observe once respectively again.
3, result of the test
Patch test results is as shown in table 1, and the implication that in table 1, each symbol represents is as follows: "-"=negative reaction; " ± "=suspicious reaction: only have faint erythema; "+"=weak positive reaction (erythematous response): erythema, infiltration, edema, can pimple be had; " ++ "=strong positive reaction (herpes reaction); Erythema, infiltration, edema, pimple, herpes; Reaction can exceed tested district; " +++ "=extremely strong positive reaction (reaction of amalgamation herpes); Obvious erythema, severe infiltration, edema, amalgamation herpes; Reaction exceeds tested district.
Criterion: according to cosmetics health specification 2007, occur in 30 routine experimenters that the number of 1 grade of skin adverse reaction is more than 5 examples, or occur that the number of secondary skin adverse reaction is more than 2 examples, or when there is any 1 example more than three grades or three grades skin adverse reactions, then judge that tested material has untoward reaction to human body, otherwise, be then considered as having no adverse reaction to human body.
The patch test results of the Aloe fermentation oleo stock that table 1, embodiment 1 obtain
As can be seen from Table 1: the Aloe fermentation oleo stock that embodiment 1 obtains does not produce suspicious reaction, illustrate that Aloe provided by the invention fermentation oleo stock all has safety, untoward reaction can not be brought to human body.
Three, the molecular size range of Aloe fermentation oleo stock detects
Molecular size range checks the key factor whether can carrying out Cutaneous permeation.The present invention measures the molecular size range of Aloe fermentation oleo stock prepared by embodiment 1 by high performance liquid chromatography (HPLC).Concrete steps are as follows:
1, the reagent of test and the preparation of solution
(1) mobile phase: acetonitrile: water: trifluoroacetic acid=30:70:0.1 (v/v/v).
(2) sample preparation: the sample take mobile phase as solvent compound concentration being 5mg/mL, then for sample introduction after filtering with microporous membrane (0.45 μm).
(3) preparation of standard sample: bovine serum albumin (Mr67000), B12 (Mr1335), oxidized form of glutathione (Mr614) are made into mixed mark, and the concentration of often kind of material is 5mg/mL.
2, test method
Standard substance by three kinds of known molecular amounts: bovine serum albumin (Mw67000), vitamin B
12(Mw1335) and oxidized form of glutathione (Mw614) by high performance liquid chromatograph (Waters company) reference literature " Wang Chengzhong etc. the research of polypeptide molecular weight distribution in protease hydrolyzed wheat germ protein and enzymolysis solution. grain processing; 2012:37 (2) " in method carry out HPLC analysis, draw the standard curve of molecular weight logarithm and elution time.According to gel column permeation chromatography principle, the material that polypeptide molecular weight is large is first by eluting out, and the logarithm of elution time and molecular weight is linear.After the same method the Aloe of preparation in embodiment 1 fermentation oleo stock is carried out HPLC analysis again, obtain the elution time of respective peaks, elution time is substituted into standard curve, the molecular size range of Aloe fermentation oleo stock prepared by embodiment 1 can be obtained.
Chromatographic condition: chromatographic column: TsKgel2000SWXL300mm × 7.8mm; Determined wavelength: UV280nm; Flow velocity: 1ml/min; Column temperature: 30 DEG C.
3, result of the test
The molecular weight of standard substance and the as shown in table 2 of elution time, be figure according to the logarithm of molecular weight and elution time and obtain standard curve: y=-2.4738x+17.656, R
2=0.9998.Wherein y is elution time (min); X is molecular weight logarithm.
The molecular weight of table 2, standard substance and elution time
Embodiment 1 prepare Aloe fermentation oleo stock HPLC collection of illustrative plates as shown in Figure 1: as can be seen from Figure 1,1 component peaks is mainly comprised in the collection of illustrative plates of Aloe fermentation oleo stock, elution time is 11.63min, elution time is substituted into standard curve: y=-2.4738x+17.656, the molecular weight calculating peak is 272.85Da, and accounts for 99.95% according to calculated by peak area Aloe fermentation oleo stock.It is generally acknowledged that molecular weight about 500Da bioactive peptide composition is more easily absorbed by the skin, so there is absorbable active polypeptide in Aloe fermented product.
Four, the antioxygenic property of Aloe fermentation oleo stock detects
DPPH is a kind of organic free radical of early stage synthesis, be commonly used to the hydrogen supply capacity assessing polyphenoils, it is highly stable in organic solvent, in purple, and have a characteristic absorption peak at 517nm place, when running into free radical scavenger, the lone pair electrons of DPPH are paired and make it fade, and namely diminish at the light absorption value of maximum absorption wave strong point.Therefore, the change by measuring light absorption value carrys out the elimination effect of assess sample to DPPH free radical.
Getting the Aloe fermentation oleo stock that not commensurability embodiment 1 prepares is dissolved in deionized water, and obtained concentration expressed in percentage by volume is a series of Aloes fermentation oleo stock liquid to be measured of 0.63%, 1.25%, 2.50%, 5.00% and 10.00%.
The specific experiment step of DPPH free radical scavenging experiment is:
(1) liquid to be measured and 2 × 10 of equal-volume (being generally 3mL) is got
-4the DPPH solution mixing (A of mol/L
1pipe);
(2) isopyknic dehydrated alcohol (determinand solvent) and 2 × 10 is got
-4the DPPH solution mixing (A of mol/L
2pipe);
(3) isopyknic dehydrated alcohol is got and liquid to be measured mixes (A
3pipe);
(4), after reacting 30min, under 517nm, A is surveyed
1, A
2, A
3pipe absorbance.
Clearance rate computing formula is: clearance rate (%)=[(A
2+ A
3)-A
1]/A
2(1)
With the concentration expressed in percentage by volume of Aloe fermentation oleo stock for abscissa, clearance rate is vertical coordinate, makes Aloe fermentation oleo stock to DPPH free radical scavenging effect curves, sees Fig. 2.
As seen from the figure, embodiment 1 gained Aloe fermentation oleo stock is to the IC of DPPH scavenging action
50=8.82%, illustrating that Aloe fermentation oleo stock has stronger oxidation resistance can scavenging free radicals, promotes cellular metabolism, strengthens cell viability, improve the 26S Proteasome Structure and Function of body, improve body vitality, thus delay cell senescence, play its antidotal effect.
Five, the white-skinned face function analysis of Aloe fermentation oleo stock
Tryrosinase is melanogenic key enzyme, which control the forming process of melanocyte, and the deposition of its level of activity to pigment plays a major role.Many whitenings of selling in the market, speckle dispelling product are all reach whitening function with restraint of tyrosinase, therefore are evaluate the leading indicator of whitening cosmetic to the power of tyrosinase inhibitory action.
Carried out the whitening function of assess sample by the impact of working sample on tryrosinase, be testing sample with 10% (v/v, solvent is deionized water) Aloe fermentation oleo stock, measure its inhibition to tyrosinase activity.
Table 2, solution preparation list
Solution |
C
1(mL)
|
C
2(mL)
|
T
1(mL)
|
T
2(mL)
|
TYR |
2 |
2 |
2 |
2 |
Sample |
0 |
0 |
2 |
2 |
PBS |
4 |
5 |
2 |
3 |
Tryrosinase |
1 |
0 |
1 |
0 |
Cumulative volume |
7 |
7 |
7 |
7 |
Note: C
1and T
1add 1mL tryrosinase, enzyme is lived as 100U/mL.
Configure each solution by table 2, in accordance with the following steps the maximum inhibition of tryrosinase in sample measured:
(1) C
2after pipe prepares and shakes up, heating in water bath 10min in 37 DEG C of water-baths, returns to zero under wavelength 475nm.
(2) C
1pipe solution prepares and shakes up, and after 37 DEG C of water-bath 10min, adds tryrosinase 1mL, continues water-bath 10 minutes, measures C
1absorbance.
(3) by (1) (2) same method, with T
2zeroing measures T
1absorbance.
(4) calculation sample is to the maximum inhibition T (%) of tryrosinase.
The computing formula of maximum inhibition T (%) is: T (%)=(C
1-T
1)/C
1× 100% (2)
Experiment prove, 10% Aloe fermentation oleo stock to the suppression ratio of tyrosinase activity be the arbutin of 43.81%, 1% (v/v, solvent is deionized water) to the suppression ratio 99.52% of tyrosinase activity, see Fig. 3.Namely the whitening effect of Aloe fermentation oleo stock is equivalent to the effect of the arbutin of 0.44%, has certain white-skinned face function.