CN108553403A

CN108553403A - A kind of purposes of thermus thermophilus and saccharomycete combined fermentation product

Info

Publication number: CN108553403A
Application number: CN201810358318.5A
Authority: CN
Inventors: 廖筝筝; 聂菁; 李维; 孙培文
Original assignee: Shanghai Yi Chemical Co Ltd
Current assignee: Shanghai Yi Chemical Co Ltd
Priority date: 2018-04-20
Filing date: 2018-04-20
Publication date: 2018-09-21
Anticipated expiration: 2038-04-20
Also published as: JP6793692B2; JP2019189596A; CN108553403B

Abstract

The invention discloses a kind of Thermophilic Bacteria and the tunnings of saccharomycete co-fermentation culture in the purposes for making anti-inflammatory, skin moisture-keeping or skin are dispelled on yellow reagent, wherein the Thermophilic Bacteria and yeast co-fermentation method include：The liquid under low temperature, acid condition first is carried out to yeast and once or repeatedly increases bacterium fermentation, allows it to be in exponential phase of growth, then allows the temperature of zymotic fluid after low temperature becomes high temperature, addition thermus thermophilus progress secondary fermentation, the condition of fermentation is alkaline condition.Some active constituents of thermus thermophilus can be significantly improved, the content for more especially applying to the active constituent of skin care formulation is increased or activity is improved, and is dispelled significant effect on Huang especially for mild anti-inflammatory, moisturizing and skin.

Description

A kind of purposes of thermus thermophilus and saccharomycete combined fermentation product

Technical field

The invention belongs to the fermentation raw material fields for skin nursing, more particularly to thermus thermophilus and saccharomycete combination hair The method of ferment and application.

Background technology

Biofermentation raw material includes that the application of cosmetic field is more and more in skin care formulation, nearly 2 years ferment makeup Product receive pursuing for consumer.Fermentation is the process of pure biology, the intermediate addition without any chemical components.Fermentation allows activity Substance is more prone to dissolve out, and extracts whole active constituents by fermenting, will produce new nutrient, allow nutritional ingredient more Add equilibrium, makes nutritional ingredient small molecule, activity is stronger, can be quickly absorbed by the skin.Itself is containing many in fermentative microorganism Enzyme and trace element have supplement synergistic effect.Meet expectation of the consumer for " natural, healthy, efficient " skin care item.

Saccharomycete is to apply the bacterial strain most in cosmetics fermentation raw material field at present, and yeast extract has natural, peace Entirely, potent moisture-keeping efficacy.In yeast cells contain abundant natural moisturizing factor (NMF), as amino acid, peptides, sodium, potassium, The mineral elements such as magnesium.Wherein, amino acid accounts for the 40% of natural moisturizing factor (NMF), and the mineral elements such as sodium, potassium, magnesium account for natural guarantor The 12% of the wet factor (NMF).Yeast cell wall main component includes yeast β-(1,3)/(1,6)-glucan and mannosan.Ferment Female β-(1,3)/(1,6)-glucan contains a large amount of hydrophilic radical, can absorb moisture or lock keratoderma moisture.Ferment Female distinctive molecular structure of mannosan can lock hydrone, have the function that long-acting moisturizing.Such as Chinese patent application discloses The Chinese patent of CN105434319A, CN107184411A, CN10655182A are disclosed using yeast tunning as makeup The raw material of product carries out making cosmetics.

Thermus thermophilus (Thermus Thermophilus) is a kind of thermoduric bacteria, can generate the superoxides of thermal stability Mutase (SOD) and B family vitamin.Superoxide dismutase can be by 0₂It is oxidized to H₂0₂, organism is avoided to be damaged by oxidation The effect of wound plays skin resistance free radical and encroaches on, anti-aging.B family vitamin can mitigate scytitis reaction, resist day The damage of light promotes a part for cytothesis, B family vitamin or many enzymes and auxiliary enzyme molecular structure, can promote amino The metabolism of acid is to keep skin health.In Chinese Patent Application No.:It is also revealed using thermus thermophilus in 2017107329367 Tunning as skin matrix, but the skin matrix is other than the tunning of thermus thermophilus, further includes recombined human Source collagen, but this application only refers only to the tunning of thermus thermophilus, but do not disclose how to ferment.Example again Such as, biological enzyme sunscreen composition is also disclosed in Chinese patent application, application number 200710031387, wherein in the composition Including thermus thermophilus biological enzyme and provide ratio.But general thermus thermophilus is all produced by fermentation, enzyme Activity or enzyme yield there is no disclose, and this enzyme with it is other at subassembly when, stability is also one Key factor, contains enzyme after all in sunscreen product, whether enzyme inactivates under ultraviolet light, if the activity with enzyme is not known yet.

All it is that single bacterium kind carries out after being respectively completed fermentation, then carries out two kinds of zymotic fluids and blent in existing traditional technology. Different floras and enzyme is dissolved into the counteracting reaction occurred together, can lose a large amount of flora and enzymatic activity, often practical effect Fruit is unsatisfactory.This just needs to be improved existing fermentation process and product, it is desirable to obtain more active materials, and reduce Independent tunning mixes mutual effect neutralization effect.

Invention content

To solve the above problems, the present invention proposes thermus thermophilus and saccharomycete combined fermentation process, it is thermophilic to reach raising The bioactivity of hot Thermus tunning enriches the practical application of thermus thermophilus tunning, especially improves thermophilic dwell Hot bacterium tunning mild anti-inflammatory, moisturizing and barrier reparation and dispel yellow party face the effect of, meet people to cosmetics " day So, health, it is efficient " pursuit.

Group of the present invention has been surprisingly found that a variety of bacterium are carried out controllability fermentation by more bacterium symbiotic fermentations, not only can ferment Type is more beneficial to enzyme, moreover it is possible to which it is more times of common single strain fermentation quantity to make the quantity of every fermentoid output, and enzyme is more, to people The catalysis healthcare function of body is stronger, although having occurred yeast extract and thermus thermophilus tunning in the market, All it is to be added in cosmetics as single product, opens up saccharomycete and thermus thermophilus is combined fermentation and is used in cosmetics It is a new issue.

Group of the present invention is found surprisingly that, allows yeast and Thermophilic Bacteria co-incubation, by special culture process, Ke Yirang Yeast plays the role of induced activation to the culture of Thermophilic Bacteria, and the activity than individually cultivating the product obtained by Thermophilic Bacteria has significantly It improves, and the activity of the culture products of yeast itself does not have much influence.Which enhances the active materials of Thermophilic Bacteria The increased activity of release or active material.

Although individually culture can generate some beneficial active materials to Thermophilic Bacteria, the amount of this active material is very It is few, and activity is not high.If yeast and Thermophilic Bacteria co-incubation are allowed, by special cultural method, it is found that can significantly carry The activity of the product of the active material of high Thermophilic Bacteria, especially some active materials significantly increases.

On the one hand, the method that the present invention provides the tunning of culture Thermophilic Bacteria, this method comprises the following steps：

(1) S. cervisiae under medium temperature, acid condition is first subjected to one or many fermentations, saccharomycete is increased Bacterium；

(2) etc. saccharomycete be in the exponential phase later stage temperature is increased again after 0 day or 1 day after, inoculation is thermophilic to dwell Hot bacterium simultaneously carries out second of fermentation under alkaline condition, obtains tunning.

In some preferred modes, Zengjing Granule at least twice carried out to yeast, either at least 3 times or at least 4 times, 5 times, 6 Zengjing Granules, to allow yeast to be in exponential phase.

In some preferred modes, also carries out primary before carrying out mixed fermentation to thermus thermophilus or repeatedly increase bacterium Culture.

In some preferred modes, this method further includes step (3), carries out degerming to step (2) zymotic fluid, decoloration is dispelled Taste filters impurity elimination, obtains thermus thermophilus and saccharomycete combined fermentation product.

In some preferred modes, the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese industrial microorganism Culture presevation administrative center, preserving number CICC1596.

In some preferred modes, thermus thermophilus HB27 used herein is purchased from American Type Culture Collection center (ATCC), preserving number is BAA-163.

In some modes, Low- temperature culture is carried out to yeast, the temperature is 20 DEG C -30 DEG C.

In some modes, acid culture is carried out to yeast, the pH value is 4.0-5.0.

In some modes, saccharomycete starts to increase temperature after being in 0 day exponential phase later stage or 1 day.Preferably, it rises High-temperature is to 55 DEG C -75 DEG C or 60 DEG C -65 DEG C.Or 60 DEG C, 65 DEG C, 70 DEG C, 58 DEG C, 61 DEG C.It is increased to high temperature from low temperature Time can generally be reached by a few minutes, heat up for example, by using electrically heated method.

In some modes, increase temperature after 0 day or 1 day, after start to be inoculated with Thermophilic Bacteria, while inoculation after 1 day The pH value for starting to adjust culture is alkalinity, pH value 7.0-8.5.

In some modes, the condition of secondary fermentation is that ventilatory capacity maintains 5m³/h/50L-10m³/h/50L,100rpm- The number of days of 200rpm, fermentation are 1-3 days.

In some modes, the expansion cultural method of the yeast includes the following steps：(1) it is taken on superclean bench Mono- rings of saccharomyces cerevisiae CICC1596 on test tube slant, 250mL triangle of the access equipped with 50mL culture solutions (pH is 4.8 ± 0.2) In bottle, 200rpm (per minute turn 200 turns), 30 DEG C of culture 10h or so, thalline is in exponential phase；(2) logarithm will be in give birth to Long-term thalline is inoculated into the 2.5L triangular flasks equipped with 2L (pH be 4.8 ± 0.2), inoculum concentration be 10% (percentage by volume, The Yeast Cultivation liquid in exponential phase of 200 milliliters of steps (1)), 200rpm, 30 DEG C of culture 10h or so；(3) by step (2) the 3L strains after cultivating are inoculated into the first order seed 50L tanks equipped with 30L zymotic fluids (pH is 4.8 ± 0.2), ventilatory capacity dimension It holds in 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.(4) 30L bacterium solutions are transferred to the secondary seed 500L equipped with 300L In tank.Ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.

On the other hand, the present invention provides a kind of method, and this method comprises the following steps：

Step 1：Zengjing Granule is carried out to S. cervisiae and thermus thermophilus respectively；

Wherein, it is as follows to increase bacterium method for S. cervisiae：

(1) mono- rings of saccharomyces cerevisiae CICC1596 on test tube slant, access is taken to be cultivated equipped with 50mL on superclean bench In the 250mL triangular flasks of liquid (pH is 4.8 ± 0.2), 200rpm (per minute turn 200 turns), 30 DEG C of culture 10h or so, at thalline In exponential phase；

(2) thalline in exponential phase is inoculated into the 2.5L triangular flasks equipped with 2L (pH is 4.8 ± 0.2), is connect Kind amount be 10% (percentage by volume, the Yeast Cultivation liquid in exponential phase of 200 milliliters of steps (1)), 200rpm, 30 DEG C Cultivate 10h or so；

(3) the 3L strains after cultivating step (2) are inoculated into the first order seed equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) In 50L tanks, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so；

(4) 30L bacterium solutions are transferred in the secondary seed 500L tanks equipped with 300L.Ventilatory capacity maintains 5m³/h/50L, 200rpm, 30 DEG C of culture 10h or so；

It is as follows that 1.2 thermus thermophilus increase bacterium method：

Take thermus thermophilus HB27 frozen stock solutions 2mL accesses equipped with 2L culture solutions on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flasks in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thalline is in logarithmic growth After phase, 30L bacterium solutions are inoculated into the 500L two level 500L seeding tanks of 300L culture solutions；

Step 2:It is as follows to the one time fermentation method of yeast：

It is as follows to the one time fermentation method of yeast：By the yeast in the secondary seed tank of 300L (after step (4)) Bacteria culture fluid is transferred in the 5000L fermentation tanks equipped with 2400L culture solutions (pH is 4.8 ± 0.2), and ventilatory capacity maintains 5m³/h/ 50L, 200rpm, 30 DEG C of culture 14h or so；

Step 3:Secondary fermentation

One time fermentation (step 2) culture-liquid temp is promoted from 30 DEG C to after 65 DEG C, temperature is (0 after being increased to 65 DEG C It), 300L thermus thermophilus enrichment liquids are transferred in this fermentation tank immediately, ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred It mixes, pH value is adjusted to 7.8 ± 0.2,150rpm, 65 DEG C with lye and cultivates 18h or so.

In some preferred modes, the formula of culture solution is：Here the formula of culture liquid is：20L culture solutions Proportioning be 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, 2.4g potassium dihydrogen phosphates, 40g ammonium sulfate, 1g anhydrous ferric chlorides, Control is adjusted in 5g sodium chloride, pH value acid or alkali, and water supplies 20L.Different amounts of culture solution contracts according to ratio Small or amplification.

On the other hand, Thermophilic Bacteria and the tunning of yeast co-fermentation culture are making skin anti-inflammatory or skin moisture-keeping Or skin is dispelled the purposes on yellow reagent, wherein the Thermophilic Bacteria and yeast co-fermentation method include：First yeast is carried out Low temperature, the lower carry out of acidity is primary or multiple liquid increases bacterium and ferments, it is allowed to be in exponential phase of growth, then allows the temperature of zymotic fluid Spend from low temperature become high temperature after 0 day or 1 day after, addition thermus thermophilus carries out secondary fermentation, and the condition of fermentation is alkalinity Condition.

Preferably, the fermentation process is aforementioned any fermentation process.

In some preferred modes, wherein the method for thermus thermophilus and saccharomycete combined fermentation, this method include such as Lower step：(1) S. cervisiae under low temperature, acid condition is first subjected to one or many fermentations, increasing bacterium is carried out to saccharomycete； (2) etc. saccharomycete are in the exponential phase later stage and again increase temperature, are inoculated with thermus thermophilus and carry out the under alkaline condition Secondary fermentation obtains tunning.

In some preferred modes, wherein in step (1), at least two or more Zengjing Granule is carried out to yeast, To allow yeast to be in exponential phase；In step (2), before being inoculated with thermus thermophilus into Yeast Cultivation liquid, dwell to thermophilic Hot bacterium carries out primary or multiple Zengjing Granule.

In some preferred modes, wherein the bacterial strain of the saccharomyces cerevisiae is CICC1596, and it is micro- to be purchased from Chinese industrial Biological inoculum preservation administrative center, preserving number CICC1596；Institute is purchased from American Type Culture using thermus thermophilus HB27 and receives Collection center (ATCC), preserving number are BAA-163.

In some preferred modes, wherein Low- temperature culture is carried out to yeast, the temperature is 20 DEG C -30 DEG C；It is right Yeast carries out acid culture, and the pH value is 4.0-5.0；Saccharomycete be in 0-1 days exponential phase later stage start increase temperature Degree；Wherein, 55 DEG C -70 DEG C are increased the temperature to.

In some preferred modes, wherein increase temperature start after 0 day be inoculated with Thermophilic Bacteria, while inoculation after 1 Its pH value for starting to adjust culture is alkalinity, pH value 7.0-8.5.In some preferred modes, wherein the anti-inflammatory Include the effect of the reduction to inflammatory factor, the inflammatory factor includes IL-1 α, IL-6, IL-8 or TNF-α.

In some preferred modes, wherein the reagent is skin care formulation, can be in the form of any type In the presence of can be solution, aqua, suspension, facial mask, lotion, creme, paste, gel, dry powder, wet-milling, one kind in spray.

On the other hand, Thermophilic Bacteria and the tunning of saccharomycete co-fermentation culture are making anti-inflammatory or skin moisture-keeping examination Purposes in agent, wherein the Thermophilic Bacteria and yeast co-fermentation method include：

Wherein, it is as follows to increase bacterium method for S. cervisiae：

(3) the 3L strains after cultivating step (2) are inoculated into the first order seed equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) In 50L tanks, ventilatory capacity maintains 5m3/h/50L, 200rpm, 30 DEG C of culture 10h or so；

(4) 30L bacterium solutions are transferred in the secondary seed 500L tanks equipped with 300L.Ventilatory capacity maintains 5m3/h/50L, 200rpm, 30 DEG C of culture 10h or so；

Wherein, it is as follows to increase bacterium method for thermus thermophilus：Thermus thermophilus HB27 frozen stock solutions 2mL is taken to connect on superclean bench Enter in the 2.5L triangular flasks equipped with 2L culture solutions (pH is 7.8 ± 0.2), ventilatory capacity maintains 5m3/h/50L, 150rpm, 65 DEG C 12h or so is cultivated, after thalline is in exponential phase, 30L bacterium solutions are inoculated into the 500L two level 500L seeds of 300L culture solutions In tank；

Step 2:It is as follows to the one time fermentation method of yeast：

It is as follows to the one time fermentation method of yeast：By the yeast in the secondary seed tank of 300L (after step (4)) Bacteria culture fluid is transferred in the 5000L fermentation tanks equipped with 2400L culture solutions (pH is 4.8 ± 0.2), and ventilatory capacity maintains 5m3/h/ 50L, 200rpm, 30 DEG C of culture 14h or so；

Step 3:Secondary fermentation

One time fermentation culture-liquid temp is promoted from 30 DEG C to after 65 DEG C in 0-1 days, by 300L thermus thermophilus enrichment liquids It being transferred in this fermentation tank, ventilatory capacity maintains 5m3/h/50L, 150rpm stirrings, is adjusted pH value to 7.8 ± 0.2 with lye, 150rpm, 65 DEG C of culture 18h or so；

Wherein, the formula of culture solution is：The proportioning of 20L culture solutions be 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, Control is adjusted in 2.4g potassium dihydrogen phosphates, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid or alkali, remaining For water.

Preferably, wherein the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese industrial Microbiological Culture Collection Administrative center, preserving number CICC1596；Institute is purchased from American Type Culture Collection center (ATCC) using thermus thermophilus HB27, Preserving number is BAA-163.

In some preferred modes, the reagent is the purposes in cosmetic agent, such as skin care reagent.

On the other hand, a kind of reagent of body of the present invention, the reagent can reduce the percutaneous moisture stream of skin, which includes By the mixture for the tunning that the method for the present invention is obtained.

In some preferred modes, the purposes or reagent, Thermophilic Bacteria are that thermus thermophilus HB27 is purchased from the U.S. Type Culture Collection (ATCC), preserving number are BAA-163.

In some preferred modes, the yeast is that CICC1596 is purchased from Chinese industrial Microbiological Culture Collection management Center, it is CICC 1596 that bacterial strain, which preserves number,.

In other preferred modes, reagent here can also include other compositions other than including tunning, Such as the ingredient of other cosmetic agents, such as moisturizer, emulsifier, light stabilizer, thickener, solubilizer, preservative, antioxygen Any one or a few mixed in agent, sun-screening agent, pH adjusting agent, penetrating agent, liposome, skin-nourishing component, essence, pigment It closes, passes through some external and human trial surfaces of the mixture of the tunning to the present invention, tunning phase of the invention There is significant improvement result to individual yeast tunning and Thermophilic Bacteria tunning and the mixed reagent of fermentation, this Seem to illustrate, the co-incubation of two kinds of bacterium has mutual promoting action, and the active content or activity of active material are all significantly Raising.

In some preferred modes, the combining form that affiliated cosmetic agent can be following carries out limited herein It enumerates, these forms can be the forms such as facial mask, toner, lotion, face cream.

In some modes, the formula of the facial mask, for some following modes：

One：In parts by weight, following components is included at least：

0.05~1 part of Sodium Hyaluronate；

1~5 part of tremella polysaccharides；

0.01~3 part of ursin；

0.1~20 part of thermus thermophilus and saccharomycete combined fermentation product；

70~90 parts of deionized water.

Two：In parts by weight, following components is included at least：

0.1~3.0 part of ursin；

0.05~1 part of polyglutamic acid sodium；

5~20 parts of Aloe Vera Gel；

0.1~20 part of Co-Q10；

1~10 part of sodium gluconate；

1~10 part of thermus thermophilus and saccharomycete combined fermentation product；

30~50 parts of deionized water.

Can be the formula of toner in some modes：

Example one：In parts by weight, following components is included at least：

1~10 part of water soluble humectants；

2~10 parts of Radix et Caulis Opuntiae Dillenii extract；

0.1~1 part of disodium ethylene diamine tetraacetate；

50~90 parts of deionized water.

Example two：In parts by weight, following components is included at least：

0.1~5 part of water soluble humectants；

0.1~5 part of thermus thermophilus and saccharomycete combined fermentation product；

1~10 part of pH adjusting agent；

50~80 parts of deionized water.

Can be the form of lotion, such as following formula in some modes：Example one：In parts by weight, it includes at least Following components：

1~5 part of white oil；

0.1~10 part of vaseline；

1~5 part of stearic acid；

0.1~5 part of polyoxyethylene laurel ether；

0.1~0.8 part of ethylparaben；

3~10 parts of thermus thermophilus and saccharomycete combined fermentation product；

3~80 parts of deionized water.

Can be the form of face cream, such as following formula in some modes：

Example one：In parts by weight, following components is included at least：

0.01~1 part of hyaluronic acid；

1~10 part of glycerin monostearate；

0.1~10 part of lanolin；

1~20 part of olive oil；

1~3 part of antioxidant；

0.1~0.5 part of natural essence；

1~70 part of deionized water.

On the other hand, present inventors have unexpectedly found that, when allow yeast and thermus thermophilus co-incubation when, essence change Become thermus thermophilus and generate the yield of B family vitamin, but promote the generation of some active materials, these active materials can To improve the activity of B family vitamin.So the present invention provides yeast and generates B family vitamin activity as promotion thermus thermophilus The upper new application of substance.So the present invention provides a kind of purposes, the S. cervisiae that bacterial strain is CICC1596 is as raising The thermus thermophilus of HB27 bacterial strains generates the purposes on the active material fermentation Enhanced agents for promoting B cluster microorganisms to enhance.

Advantageous effect

Vitro Experimental Results show：Thermus thermophilus and S. cervisiae combined fermentation product are sent out with single thermus thermophilus Ferment product can improve skin fibroblasts 20% compared to promoting the ability that skin fibroblasts increase stronger in 3% concentration More growth rate.

The anti-inflammatory effect of thermus thermophilus and the more single thermus thermophilus tunning of saccharomycete combined fermentation product is stronger, 10% thermus thermophilus and saccharomycete combined fermentation product, inflammation is added under the stimulation of inflammatory effects in skin keratinocytes The secretion of factor IL-1 α, IL-6, IL-8 and TNF-α significantly reduce, suitable with the dexamethasone anti-inflammatory effects of 100ppm, still Than dexamethasone milder, anti-inflammatory effects are substantially better than 10% single thermus thermophilus tunning or single yeast hair Ferment.

Human clinical trial the result shows that：Essence containing 1% thermus thermophilus and saccharomycete combined fermentation product, After 7 days, the percutaneous water loss for reducing skin has remarkable result, illustrates the thermus thermophilus and saccharomycete combination Tunning has the function of repairing and strengthening skin barrier function.Contain 10% thermus thermophilus and saccharomycete combined fermentation The Essence of product can significantly reduce the yellowing of skin after 7 days, illustrate the thermus thermophilus and saccharomycete combined fermentation Product has the improvement colour of skin, removes dark yellow effect.

Description of the drawings

Fig. 1 is that the tunning of examples of implementation 1 using the present invention dispels to facial skin yellow comparison diagram, wherein Fig. 1's The left side is face's effect before use, and the right is face effect of the tunning of examples of implementation 1 after 7 days.On the contrary, examples of implementation 2-6 is but without material change.

Specific implementation mode

The present invention illustrates how the present invention realizes by specific embodiment, these explanations are only to this hair The explanation that bright marrow carries out, cannot do any restrictions to the present invention.Specific protection domain is subject to claim.

Examples of implementation 1：Thermus thermophilus and saccharomycete combined fermentation method (1)

Material：

Culture solution is prepared and ingredient：The proportioning of 20L culture solutions be 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, 2.4g potassium dihydrogen phosphates, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid or alkali (hydrochloric acid or sodium hydroxide Solution) control is adjusted, water supplies 20L, and different amounts of culture solution is zoomed in or out according to ratio.The present invention does not have There is spy is standby to indicate, the culture solution that all examples of implementation are all cultivated using the culture medium prescription matched above as bacterium.Certainly, in reality It in the industrial production of border, can be matched according to different requirements, these formulas are all culture yeasts and/or thermus thermophilus Regular convention formula.

Saccharomyces cerevisiae is that CICC1596 is purchased from Chinese industrial Microbiological Culture Collection administrative center, preserving number CICC1596 (commercially available acquisition).

Thermus thermophilus HB27 is purchased from American Type Culture Collection center (ATCC), and preserving number is BAA-163 (commercially available It obtains).

1. strain expands culture

Step 1：

1.1 S. cervisiaes increase bacterium：

(3) the 3L strains after cultivating step (2) are inoculated into the first order seed equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) In 50L tanks, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.

(4) 30L bacterium solutions are transferred in the secondary seed 500L tanks equipped with 300L.Ventilatory capacity maintains 5m³/h/50L, 200rpm, 30 DEG C of culture 10h or so.

1.2 thermus thermophilus increase bacterium：

Take thermus thermophilus HB27 frozen stock solutions 2mL accesses equipped with 2L culture solutions on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flasks in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thalline is in logarithmic growth After phase, 30L bacterium solutions are inoculated into the 500L two level 500L seeding tanks of 300L culture solutions.

Here the formula of culture liquid is：The proportioning of 20L culture solutions is 60g peptones, 4g brewer's worts, 7g anhydrous slufuric acids Control, water is adjusted in magnesium, 2.4g potassium dihydrogen phosphates, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid or alkali Supply 20L.Different amounts of culture solution is zoomed in or out according to ratio.

2. step 2:

One time fermentation

Yeast bacteria culture fluid in the secondary seed tank of 300L (after step (4)) is transferred to and is trained equipped with 2400L In the 5000L fermentation tanks of nutrient solution (pH is 4.8 ± 0.2), ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 14h is left It is right.

The formula of culture solution is at this time：Here the formula of culture liquid is：The proportioning of 20L culture solutions be 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, 2.4g potassium dihydrogen phosphates, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid Or control is adjusted in alkali, water supplies 20L.Different amounts of culture solution is zoomed in or out according to ratio.

3. step 3:

Secondary fermentation

One time fermentation (step 2) culture-liquid temp is promoted from 30 DEG C to after 65 DEG C, waits 65 DEG C of arrival immediately that 300L is thermophilic Hot Thermus enrichment liquid is transferred in this fermentation tank, and ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred, with lye by pH value It adjusts to 7.8 ± 0.2,150rpm, 65 DEG C and cultivates 18h or so.

4. isolating and purifying

Bactofugation：To the culture solution of the secondary fermentation Jing Guo step 3, by plate-frame filtering, thalline is intercepted at filtering On film (0.22um), fermented supernatant fluid is obtained.

Purification：Activated carbon adsorption impurity therein is added in fermented supernatant fluid, dispelling abnormal flavor recycles multilayer absorbent gauze And diatomite filtering breeze (adding proportion of activated carbon is 6%, and activated carbon is bought in global charcoal industry).

Filtering：The zymotic fluid of purification is filtered by the filter membrane of 0.22um, obtains thermus thermophilus and saccharomycete group Close tunning.

Examples of implementation 2：Individual yeast-leavened method；

The specific implementation mode of individual yeast tunning is as follows：

Culture solution is prepared：The proportioning of 20L culture solutions is 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, 2.4g phosphoric acid Control is adjusted in potassium dihydrogen, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid or alkali, and water supplies 20L, Different amounts of culture solution is zoomed in or out according to ratio.

1. strain expands culture

2. fermentation

Yeast bacteria culture fluid in the secondary seed tank of 300L is transferred to equipped with 2700L culture solutions (pH is 4.8 ± 0.2) 5000L fermentation tanks in, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 14h or so.

3. isolating and purifying

Bactofugation：To the culture solution to ferment by step 2, by plate-frame filtering, thalline is intercepted at filter membrane On (0.22um), fermented supernatant fluid is obtained.

Filtering：The zymotic fluid of purification is filtered by the filter membrane of 0.22um, obtains big individually saccharomycetes to make fermentation production Object.

Examples of implementation 3：The fermentation process of individual Thermophilic Bacteria

The specific implementation mode of individual thermus thermophilus tunning is as follows：

1. strain expands culture

2. fermentation

300L thermus thermophilus enrichment liquids are transferred in the fermentation tank containing 2700L culture solutions, ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred, and pH value adjusts to 7.8 ± 0.2,150rpm, 65 DEG C and cultivates 18h or so.

3. isolating and purifying

Filtering：The zymotic fluid of purification is filtered by the filter membrane of 0.22um, obtains individual thermus thermophilus fermentation Product.

Examples of implementation 4：The reagent that the tunning mixing of examples of implementation 2 and 3 obtains

The tunning of Example 2 and 3 carries out 1 according to weight:The complex reagent that 1 ratio mixes.

Examples of implementation 5：Yeast and Thermophilic Bacteria co-incubation method (2)

The specific implementation mode of thermus thermophilus and saccharomycete combined fermentation product is as follows：

1. strain expands culture

Saccharomycete increases bacterium：

Take mono- rings of saccharomyces cerevisiae CICC1596 on test tube slant, access that 50mL culture solutions are housed on superclean bench In the 250mL triangular flasks of (pH is 4.8 ± 0.2), 200rpm, 30 DEG C of culture 10h or so, thalline is in exponential phase；Inoculation Into the 2.5L triangular flasks equipped with 2L (pH be 4.8 ± 0.2), inoculum concentration is that 10% (volume ratio, here inoculation are in logarithmic growth 200 milliliters of phase), 200rpm, 30 DEG C of culture 10h or so；3L strains are inoculated into equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) Level-one 50L seeding tanks in, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.30L bacterium solutions are shifted Into the two level 500L seeding tanks equipped with 300L culture solutions.Ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h is left It is right.

Thermus thermophilus increases bacterium：

Take thermus thermophilus HB27 frozen stock solutions 2mL accesses equipped with 2L culture solutions on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flasks in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thalline is in logarithmic growth After phase, 30L bacterium solutions is inoculated into the 500L two level 500L seeding tanks of 300L culture solutions and is cultivated, ventilatory capacity maintains 5m³/ H/50L, 150rpm, 65 DEG C of culture 12h or so.

2. fermentation

By the thermus thermophilus culture solution transfer in the saccharomycete and 300L secondary seed tanks in the secondary seed tank of 300L Into the 5000L fermentation tanks equipped with 2400L culture solutions (pH is 4.8 ± 0.2), ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 14h or so.Broth temperature is promoted to 65 DEG C again, ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred, and are used PH value is adjusted to 7.8 ± 0.2,150rpm, 65 DEG C and cultivates 18h or so by lye.

3. isolating and purifying

Examples of implementation 6：Yeast and Thermophilic Bacteria co-incubation method (3)

1. strain expands culture

Saccharomycete increases bacterium：

Take mono- rings of saccharomyces cerevisiae CICC1596 on test tube slant, access that 50mL culture solutions are housed on superclean bench In the 250mL triangular flasks of (pH is 4.8 ± 0.2), 200rpm, 30 DEG C of culture 10h or so, thalline is in exponential phase, inoculation Into the 2.5L triangular flasks equipped with 2L (pH is 4.8 ± 0.2), inoculum concentration is 10% (volume ratio), 200rpm, 30 DEG C of culture 10h 3L strains are inoculated into the level-one 50L seeding tanks equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) by left and right, and ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.30L bacterium solutions are transferred in the two level 500L seeding tanks equipped with 300L.It is logical Tolerance maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.

Thermus thermophilus increases bacterium：

Take thermus thermophilus HB27 frozen stock solutions 2mL accesses equipped with 2L culture solutions on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flasks in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thalline is in logarithmic growth After phase, 30L bacterium solutions are inoculated into the 500L two level 500L seeding tanks of 300L culture solutions.Broth temperature is promoted to 65 again DEG C, ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred, and are adjusted pH value to 7.8 ± 0.2,150rpm, 65 DEG C training with lye Support 18h or so.

2. fermentation

Simultaneously by the thermus thermophilus culture solution in the saccharomycete and 300L secondary seed tanks in the secondary seed tank of 300L It is transferred in the 5000L fermentation tanks equipped with 2400L culture solutions (pH is 7.8 ± 0.2), ventilatory capacity maintains 5m³/h/50L, 150rpm, 65 DEG C of culture 18h or so.Broth temperature is reduced to 30 DEG C, ventilatory capacity maintains 5m³/ h/50L, 200rpm are stirred It mixes, pH value is adjusted to 4.8 ± 0.2,200rpm, 30 DEG C with acid solution and cultivates 14h or so.

3. isolating and purifying

Bactofugation：To the culture solution by step secondary fermentation, by plate-frame filtering, thalline is intercepted at filter membrane On (0.22um), fermented supernatant fluid is obtained.

Examples of implementation 7：Thermus thermophilus and saccharomycete combined fermentation method (4)

With examples of implementation 1 the difference is that：In secondary fermentation, by one time fermentation (step 2) culture-liquid temp from 30 DEG C It is promoted to after 65 DEG C, behind wait 65 DEG C of arrival 1 day, 300L thermus thermophilus enrichment liquids is transferred in this fermentation tank, ventilatory capacity Maintain 5m³/ h/50L, 150rpm are stirred, and are adjusted pH value to 7.8 ± 0.2,150rpm, 65 DEG C with lye and are cultivated 18h or so.

Examples of implementation 8：Thermus thermophilus and saccharomycete combined fermentation method (5)

With examples of implementation 1 the difference is that：In secondary fermentation, by one time fermentation (step 2) culture-liquid temp from 30 DEG C It is promoted to after 65 DEG C, behind wait 65 DEG C of arrival 2 days, 300L thermus thermophilus enrichment liquids is transferred in this fermentation tank, ventilatory capacity Maintain 5m³/ h/50L, 150rpm are stirred, and are adjusted pH value to 7.8 ± 0.2,150rpm, 65 DEG C with lye and are cultivated 18h or so.

Examples of implementation 9：Thermus thermophilus and saccharomycete combined fermentation method (6)

With examples of implementation 1 the difference is that：In secondary fermentation, by one time fermentation (step 2) culture-liquid temp from 30 DEG C It is promoted to after 75 DEG C, behind wait 75 DEG C of arrival 3 days, 300L thermus thermophilus enrichment liquids is transferred in this fermentation tank, ventilatory capacity Maintain 5m³/ h/50L, 150rpm are stirred, and are adjusted pH value to 7.8 ± 0.2,150rpm, 65 DEG C with lye and are cultivated 18h or so.

Examples of implementation 10：Cytotoxicity experiment

Logarithmic phase cell (human skin fibroblasts and application on human skin horn cell) is collected, individual cells are made with culture solution Suspension, adjusting final concentration of cells are 40000cell/mL, and 100uL cell suspensions are added in 96 orifice plates, and 5%CO2,37 DEG C are incubated, After cell is adherent, detectable substance (cell culture fluid of each tunning of the 1-9 containing examples of implementation) is added, culture is arrived to 80% After 90% fusion, adds MTT solution 20uL. to continue to be incubated 3h per hole, terminate culture.Culture medium in hole carefully is sucked, is added per hole 100uL DMSO vibrate 10min, crystal are made fully to melt.Each hole absorbance value is measured with microplate reader, selects 490nm wavelength, Using 630nm as reference wavelength.Each processing is repeated 5 times.

The high glycoform DMEM cell culture fluids of each tunning of the 1-9 containing examples of implementation are added, cell culture is containing 3% The high glycoform DMEM cell culture fluids of tunning, 3% examples of implementation 1 (thermus thermophilus and saccharomycete combined fermentation product) Human skin fibroblasts growth rate is 131%, is improved compared with 3% examples of implementation 3 (single thermus thermophilus tunning) 20%, referring specifically to following table.

The application on human skin Keratiocyte growth of 10% examples of implementation 1 (thermus thermophilus and saccharomycete combined fermentation product) is added Rate is 101%, and the application on human skin Keratiocyte growth rate that 100ppm dexamethasone is added is 89%.10% examples of implementation 1 are (thermophilic Thermus and saccharomycete combined fermentation product) it is more mild compared with 100ppm dexamethasone.As a result such as following table.

Table 1：The tunning that each examples of implementation obtain is to cytotoxicity experiment result.

By variance analysis, examples of implementation 1 and 7 and examples of implementation, the difference of 2-6,8-9 are respectively P<0.01, reach pole Significant difference, and without significant difference between examples of implementation 2-6,8-9.This explanation, the side of 1 combination culture of the embodiment of the present invention Method can remarkably promote human skin fibroblasts growth rate, and remarkably promote effect for the also pole of application on human skin horn cell, And the effect of mixed culture is best, and the immixture effect of examples of implementation 1 or examples of implementation 7 is best.Also illustrate simultaneously, When mixed culture, mixed opportunity is also very important, and carries out mixing immediately after increasing temperature or 1 day laggard Row mixing, effect is best, but the effect more than mixing in 2 days drastically declines fibroblastic effect.Similar effect Influence for the growth rate of the application on human skin horn cell of cell is similar.

Examples of implementation 8：Anti-inflammatory effect is tested

Logarithmic phase application on human skin horn cell is collected, individual cells suspension is made with culture solution, adjusting final concentration of cells is 100uL cell suspensions are added in 96 orifice plates by 40000cell/mL, and 5%CO2,37 DEG C are incubated for 24 hours, and culture solution is sucked out, is washed with PBS Once, it is sucked out, adds the PBS of 20uL, carry out UVB irradiations, dose of radiation control is 60mJ/cm², change detectable substance (implementation Each tunning of example 1-9 is diluted, a concentration of 10% each tunning with high glycoform DMEM cell culture fluids), training After supporting for 24 hours, culture solution supernatant is collected.The content of inflammatory factor (IL-1a, IL-6, IL-8, TNF-a) is detected (according to finished product reagent The specification of box is detected).Experiment is repeated 5 times.

The result shows that：The high glycoform containing 10% thermus thermophilus and saccharomycete combined fermentation product (examples of implementation 1) is added DMEM cell culture fluids, Inflammatory Factors Contents IL-1 α are 110.12pg/mL, IL-6 16.23pg/mL, IL-8 are 1300.20pg/mL and TNF-α are 42.25pg/mL, and secretory volume obviously subtracts compared with 10% single thermus thermophilus tunning Few, anti-inflammatory effects are apparent (by analysis, significantly reducing Inflammatory Factors Contents), the dexamethasone anti-inflammatory effects phase with 100ppm When.

Table 2：Experimental result of the tunning that each examples of implementation obtain to inflammatory factor.

This explanation, the activated product that cultural method of the invention obtains can significantly reduce the generation of inflammatory factor, compare Individually fermentation or fermenting mixture, by variance analysis, examples of implementation 1,7 and other processing all reach pole significant difference (tool Body experimental data is omited).This explanation, yeast and thermus thermophilus co-incubation, are once cultivated using yeast, are then carried out again secondary Mixed culture can have the active material of two kinds of bacterium facilitation, and activity also significantly improves.On the contrary, individually culture And individually cultivate and then mix and the mixed culture of other manner, such effect can not be obtained.This may It is because containing B family vitamin in thermus thermophilus, and the main effect of B family vitamin may be exactly to play skin anti-inflammatory, this Seem to illustrate, after two kinds of different bacterium co-incubations, the activity of B family vitamin in thermus thermophilus, Huo Zheqi can be improved His unknown substance obtains the activity of bigger by B family vitamin.

Meanwhile the present invention has carried out the measurement of conventional method to the B family vitamin in 9 processing simultaneously, finds each place The B family vitamin of every 100 grams of tunning in reason does not change significantly, this seems to illustrate, combined fermentation is for one A little factors for allowing B family vitamin to have greater activity generate, to improve the activity of B family vitamin, to better Antiinflammation.And the mechanism that these specific active factors generate, need further to be studied.

Examples of implementation 9：Skin barrier recovery test

Keratoderma have good barrier function, but due to influenced by extraneous factors such as temperature, wind, sunlight and by Damage, it is critically important to carry out barrier reparation in time.This experiment uses TEWL (Trans epidermal Water Loss, TEWL) And as index, TEWL is lower, and display skin barrier repair ability is higher.Wherein, test equipment is：German CK companies skin water Part is lost in (TEWL) test probe TM300；Test condition is：Subject is 22 DEG C ± 1 DEG C in temperature, and humidity is 50% ± 5% Room in sit quietly 20min.

Experimental method：

Select 20 volunteers, the age between 22~55 years old, no skin disease or once to some drugs, cosmetics or one A little chemical substances have highly sensitive situation.Sooner or later continuous half face using control group and contains 1% examples of implementation 1-9 fermentation productions twice (main component of Essence has the Essence of object：Deionized water, Tween-80, propylene glycol, preservative and 1% the present invention The tunning of examples of implementation 1-9, wherein the solvent that 1% tunning uses configures for PBS as solvent, PH= 7.1) it, is tested using test TEWL before and after product 7 days, and calculates the change rate of TEWL, the calculation of the change rate of TEWL is： [value of the TEWL of (value of TEWL of the value of the TEWL after 7 days before -7 days)/before 7 days] * 100%；The change rate of TEWL is lower, right The barrier action of skin will be better (each examples of implementation carry out 5 repetitions).As a result 3 be see the table below.

Table 3：The skin barrier recovery test result for the tunning that each examples of implementation obtain

Examples of implementation	Content	The change rate (%) of TEWL
			1	1%	-12.63
2	1%	-6.62
			3	1%	-5.86
4	1%	-6.65
			5	1%	-8.75
6	1%	-7.86
			7	1%	-13.05
8	1%	-6.05
			9	1%	-5.86
Control	0%	-1.80

It is analyzed by significant difference, examples of implementation 1,7 reach pole with examples of implementation 2-6,8-9 for barrier repairing effect Significant difference (summary of specific experiment data), this explanation, the co-cultivation obtained by the method culture of the embodiment of the present invention 1 are produced Object can have significant repair function to keratoderma, may improve the active material for repairing skin keratin confluent monolayer cells The active either yield or activity of yield or other confactors.

Examples of implementation 10：Skin is dispelled yellow test

CIEXYZ systems are converted to CIEL*a*b* color spaces by mathematical method.The space by a brightness (L) and Two color (a, b) axis compositions.Brightness is a kind of scale indicating gray scale, its value is between 0-100,0 expression black, 100 Indicate white.A* is from red to the color saturation between green, and variation range is+60 to -60, and positive value indicates red strong The variation of degree.B* indicates yellow to the color saturation between blue, and variation range is+60 to -60, and positive value indicates that yellow is strong The variation of degree.The color of skin is also to be indicated with L*a*b*, and the lower explanation of b* values is dispelled, and yellow effect is more apparent, and △ b* are smaller, dispel Yellow effect is also better.

Experimental method：

20 volunteers are selected, the age, continuous half face using control group and contained twice sooner or later between 22~55 years old (main component of Essence has the Essence of 10% examples of implementation 1-9 tunnings：Deionized water, Tween-80, propylene glycol, Preservative), continuous use 7 days (repeating experiment 5 times).It takes pictures using before product, using carrying out VISIA after product 7 days, every group of meter Calculate average △ b* values.

Wherein, test equipment is：LAB testers：Spectrophotometer CM2600d (Minolta, Osaka, day This), subject is dried after thoroughly cleaning face with toilet paper before testing, and is 22 DEG C ± 1 DEG C in temperature, humidity is 50% ± 5% Room in sit quietly 20min.

Table 4：Skin is dispelled yellow test and comparison test result

By our variance analyses, the reagent of examples of implementation 1 and the reagent of examples of implementation 7 and other each processing all exist Extremely significant difference illustrates the co-incubation of the present invention, to being conducive to remove aging and pigment deposition active material increase, In addition, it is special, have the suitable addition time important co-incubation.And it is not deposited between examples of implementation 2-6,8-9 At significant difference (Fig. 1).

We in human skin cell it was found that, with the increase of raw material (the examples of implementation 1 or 7) additive amount and Cell viability increases (concentration 1.25%-12%), therefore the raw material improves the proliferation updating ability of Skin Cell.On the contrary, right In same examples of implementation 2-6, the addition of the same concentration of the raw material of 8-9 does not increase the vigor of cell, some, such as The cell viability that examples of implementation 8-9 is reduced instead.Rational explain may be in this way：The normal update cycle of skin is 3-4 weeks, The metabolism of skin epidermis can be accelerated by accelerating the proliferation update of cell, to remove aging and pigment deposition angle Matter so that skin appearance has the effect of Huang of dispelling.It the reason of as skin cell proliferation updating ability is promoted, should through analysis test Containing the cytotrophies ingredient such as a large amount of polysaccharide, polyphenol, amino acid in raw material, these nutritional ingredients contribute to cell proliferation and Update improves cell viability.This further instruction, it is aobvious for the opportunity of the mixing of two kinds of bacterium in co-incubation fermentation process Must be especially important, the selection on suitable opportunity contributes to the production of some benefit actives, and it is harmful may to obtain some on the contrary Substance generation or generation for the harmful substance of certain symptoms.

Examples of implementation 11：The anti-inflammatory of skin preparation is tested

Anti-inflammatory effective percentage is tested：Choose 3X160=480 ages 15~35 years old whelk (acne rank be 3-4 Grade) patient tests, wherein and each group of man, each 80 of female, the tunning obtained to embodiment 1,8 are tested,.

Application method is：After face cleaning, the tunning of embodiment 1,8 is used (to be configured with sterile water twice sooner or later daily The skin preparation of 15% tunning), wherein sterile water as a contrast, continuous use 10 days, affected part is rubescent for observation, swelling, Skin lesion degree, being subject to expert estimation, (for 0-4 point to acne classification of severity, 0- is without acne, 1- mild acnes, blackhead It is dispersed in and multiple, the inflammatory skin lesion of being dispersed in property；The medium acnes of 2-, shallow warts, inflammatory skin lesion number is more, is only limitted to Face；3- severes add the inflammatory skin lesion of deep-seated；4- severes, tumour easily form scar) it is assessed.

Specific data are as follows：The tunning of examples of implementation 1 can significantly reduce the grade of acne, and wherein women has 75 3 or 4 grades of variations before using are the 0-1 grades after using 10 days, wherein there is 50 women to become 0 grade；For male patient, There are 68 4 grades of 1 grades become after using before using.For those without significant change, it may be possible to caused by other reasons Acne does not have effect.And use same method, from the point of view of the anti-inflammatory effects of examples of implementation 2-6 tunnings, using preceding and After use, acne rank does not have significant change, and using the patient of examples of implementation 3 and 4, women only only has 6 to be become from grade 3 Grade 1, male only has 4 becomes grade 1 from class 4.From the point of view of using the anti-inflammatory effects of 8 tunning of examples of implementation, With certain anti-inflammatory effects, but effect is not obvious, and only there was only 7 in 80 female patients becomes grade from grade 3 1,80 male patient only has 6 becomes grade 1 from class 4.By human experimentation the result shows that embodiment 1 compares comparative example 2-6,8-9 suppression acne anti-inflammatory effects are more preferable, and effect is more obvious, is mutually confirmed with the anti-inflammatory effects in examples of implementation 8；And sterile water Also it does not change significantly.

The terms and expressions mode for being used herein to description method is not unique constant, and we do not have any meaning Figure excludes any mutually convertible expression way of the description present invention or feature using these terms and expressions modes, we Accept a variety of different expression ways in the range of the present invention states.It is therefore believed that although of the invention herein With various concrete schemes and arbitrary feature description clearly demonstrate that come, but change design disclosed herein table Those experienced professional technique personages, and the statement one that these changes will be subsidiary with the present invention are also sought help from up to mode It causes.Article, patent, content and the useful electronization referred to herein and citation of patent application and all other document What information was bound together, it is necessary to be referred to as a complete content, delivering one part of any of which will be special Indicate this point.Applicant has the information and material of these any and whole articles, patent, patent application or other documents Material is merged into the right for the part that the application is disclosed as patent specification.

Claims

1. Thermophilic Bacteria and the tunning of saccharomycete co-fermentation culture are dispelled yellow reagent making anti-inflammatory, skin moisture-keeping or skin On purposes, wherein the Thermophilic Bacteria and yeast co-fermentation method include：Low temperature first is carried out to yeast, under acid condition Liquid it is primary or repeatedly increase bacterium fermentation, allow it be in exponential phase of growth, the temperature of zymotic fluid then allowed to become height from low temperature Wen Hou, addition thermus thermophilus carry out secondary fermentation, and the condition of fermentation is alkaline condition.

2. purposes according to claim 1, wherein the method for thermus thermophilus and saccharomycete combined fermentation, this method packet Include following steps：(1) S. cervisiae under low temperature, acid condition is first subjected to one or many fermentations, saccharomycete is carried out Increase bacterium；(2) etc. saccharomycete are in the exponential phase later stage and again increase temperature, be inoculated with thermus thermophilus and under alkaline condition into Second of fermentation of row, obtains tunning.

3. purposes according to claim 2, wherein in step (1), the increasing bacterium that at least two or more are carried out to yeast is trained It supports, to allow yeast to be in exponential phase；In step (2), before being inoculated with thermus thermophilus into Yeast Cultivation liquid, to thermophilic Thermus carries out primary or multiple Zengjing Granule.

4. purposes according to claim 3, wherein the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese work Industry Microbiological Culture Collection administrative center, preserving number CICC1596；Institute is purchased from US mode bacterium using thermus thermophilus HB27 Kind collection center (ATCC), preserving number is BAA-163.

5. purposes according to claim 3, wherein carry out Low- temperature culture to yeast, the temperature is 20 DEG C -30 DEG C； Acid culture is carried out to yeast, the pH value is 4.0-5.0；Saccharomycete is in 0-1 days exponential phase later stage and starts to increase Temperature；Wherein, 55 DEG C -70 DEG C are increased the temperature to.

6. purposes according to claim 3, wherein increase temperature start after 0 day be inoculated with Thermophilic Bacteria, while inoculation after The pH value for starting for 1 day to adjust culture is alkalinity, pH value 7.0-8.5.

7. purposes according to claim 3, wherein the anti-inflammatory includes the effect of the reduction to inflammatory factor, described Inflammatory factor include IL-1 α, IL-6, IL-8 or TNF-α.

8. purposes according to claim 1, wherein the reagent is skin care formulation, can be with any type shape State exists, and can be one in solution, aqua, suspension, facial mask, lotion, creme, paste, gel, dry powder, wet-milling, spray Kind.

9. the purposes of Thermophilic Bacteria and the tunning of saccharomycete co-fermentation culture on making anti-inflammatory or skin moisture-keeping reagent, Wherein, the Thermophilic Bacteria and yeast co-fermentation method include：

Wherein, it is as follows to increase bacterium method for S. cervisiae：

(1) take mono- rings of saccharomyces cerevisiae CICC1596 on test tube slant, access that 50mL culture solutions (pH is housed on superclean bench Be 4.8 ± 0.2) 250mL triangular flasks in, 200rpm (per minute turn 200 turns), 30 DEG C of culture 10h or so, thalline is in logarithm Growth period；

(2) thalline in exponential phase is inoculated into the 2.5L triangular flasks equipped with 2L (pH is 4.8 ± 0.2), inoculum concentration For 10% (percentage by volume, the Yeast Cultivation liquid in exponential phase of 200 milliliters of steps (1)), 200rpm, 30 DEG C of cultures 10h or so；

(3) the 3L strains after cultivating step (2) are inoculated into the first order seed 50L equipped with 30L zymotic fluids (pH is 4.8 ± 0.2) In tank, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so；

(4) 30L bacterium solutions are transferred in the secondary seed 500L tanks equipped with 300L.Ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so；

Wherein, it is as follows to increase bacterium method for thermus thermophilus：Thermus thermophilus HB27 frozen stock solutions 2mL access dresses are taken on superclean bench In the 2.5L triangular flasks for having 2L culture solutions (pH is 7.8 ± 0.2), ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture After thalline is in exponential phase, 30L bacterium solutions are inoculated into the 500L two level 500L seeding tanks of 300L culture solutions by 12h or so；

Step 2:It is as follows to the one time fermentation method of yeast：

It is as follows to the one time fermentation method of yeast：By the saccharomycete training in the secondary seed tank of 300L (after step (4)) Nutrient solution is transferred in the 5000L fermentation tanks equipped with 2400L culture solutions (pH is 4.8 ± 0.2), and ventilatory capacity maintains 5m³/h/50L, 200rpm, 30 DEG C of culture 14h or so；

Step 3:Secondary fermentation

One time fermentation culture-liquid temp is promoted from 30 DEG C to after 65 DEG C in 0-1 days, by the transfer of 300L thermus thermophilus enrichment liquids Into this fermentation tank, ventilatory capacity maintains 5m³/ h/50L, 150rpm are stirred, and are adjusted pH value to 7.8 ± 0.2 with lye, 150rpm, 65 DEG C of culture 18h or so；

Wherein, the formula of culture solution is：The proportioning of 20L culture solutions is 60g peptones, 4g brewer's worts, 7g anhydrous magnesium sulfates, 2.4g Control is adjusted in potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chlorides, 5g sodium chloride, pH value acid or alkali, remaining is water.

10. purposes according to claim 9, wherein the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese work Industry Microbiological Culture Collection administrative center, preserving number CICC1596；Institute is purchased from US mode bacterium using thermus thermophilus HB27 Kind collection center (ATCC), preserving number is BAA-163.