CN108553403B

CN108553403B - A kind of purposes of thermus thermophilus and saccharomycete combined fermentation product

Info

Publication number: CN108553403B
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: 2019-09-27
Anticipated expiration: 2038-04-20
Also published as: CN108553403A; JP6793692B2; JP2019189596A

Abstract

The invention discloses the purposes that a kind of Thermophilic Bacteria and the tunning of saccharomycete co-fermentation culture are dispelled on yellow reagent in production anti-inflammatory, skin moisture-keeping or skin, wherein, the Thermophilic Bacteria and yeast co-fermentation method includes: that the liquid first carried out to yeast under low temperature, acid condition once or repeatedly increases bacterium fermentation, it is allowed to be in exponential phase of growth, then after allowing the temperature of fermentation liquid to become high temperature from low temperature, it adds thermus thermophilus and carries out 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, in particular to thermus thermophilus and saccharomycete combination hair The method and application of ferment.

Background technique

Biofermentation raw material includes cosmetic field using more and more, nearly 2 years ferment makeup in skin care formulation 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 easier to dissolve out, and extracts whole active constituents by fermenting, can generate new nutrient, allow nutritional ingredient more Add equilibrium, make nutritional ingredient small molecule, activity is stronger, can be quickly absorbed by the skin.Itself is containing many in fermentative microorganism Enzyme and microelement 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.Natural moisturizing factor (NMF) rich in yeast cells, 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, and moisture can be absorbed 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 production 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 oxidized damage 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 cytothesis, and a part of 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.: also revealing in 2017107329367 using thermus thermophilus 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 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.

It is all that single bacterium kind carries out after being respectively completed fermentation, then carries out two kinds of fermentation liquids 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 improve existing fermentation process and product, it is desirable to obtain more active materials, and reduce Independent tunning mixes mutual effect neutralization effect.

Summary of the invention

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, can not only ferment out Type is more beneficial to enzyme, moreover it is possible to which the quantity for making every fermentoid output is more times of common single strain fermentation quantity, and enzyme is more, to people The catalysis healthcare function of body is stronger, although having had already appeared 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 resulting product of 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 Thermophilic Bacteria, which is individually cultivated, can produce some beneficial active materials, 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 mention The activity of the product of the active material of high Thermophilic Bacteria, especially some active materials significantly increases.

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

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

(2) etc. saccharomycete be in the logarithmic growth phase later period temperature is increased again after 0 day or 1 day after, be inoculated with thermophilic dwells 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, so that yeast be allowed to be in logarithmic growth phase.

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

In some preferred modes, this method further includes step (3), carries out degerming to step (2) fermentation liquid, 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, deposit number CICC1596.

In some preferred modes, thermus thermophilus HB27 used herein is purchased from American Type Culture Collection Center (ATCC), deposit 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 logarithmic growth phase later period 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.High temperature is increased to from low temperature Time generally pass through a few minutes and can reach, heat up for example, by using electrically heated method.

In some modes, increase temperature 0 day or 1 day after, after start 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- 200rpm, the number of days of fermentation are 1-3 days.

In some modes, the expansion cultural method of the yeast includes the following steps: that (1) takes on superclean bench Mono- ring of saccharomyces cerevisiae CICC1596 on test tube slant, 250mL triangle of the access equipped with 50mL culture solution (pH is 4.8 ± 0.2) In bottle, 200rpm (turns 200 turns) per minute, and 30 DEG C of culture 10h or so, thallus is in logarithmic growth phase；It (2) will be raw in logarithm Long-term thallus is inoculated into the 2.5L triangular flask equipped with 2L (pH be 4.8 ± 0.2), inoculum concentration be 10% (percentage by volume, The Yeast Cultivation liquid in logarithmic growth phase of 200 milliliters of steps (1)), 200rpm, 30 DEG C of culture 10h or so；(3) by step (2) the 3L strain after cultivating is inoculated into the first order seed 50L tank equipped with 30L fermentation liquid (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 solution is 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 being carried out to S. cervisiae and thermus thermophilus respectively；

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

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

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

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

(4) 30L bacterium solution is transferred in the secondary seed 500L tank 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 solution 2mL access equipped with 2L culture solution on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flask in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thallus is in logarithmic growth After phase, 30L bacterium solution is inoculated into the 500L second level 500L seeding tank of 300L culture solution；

Step 2: 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 fermentor equipped with 2400L culture solution (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 liquid is transferred in this fermentor 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 are as follows: the formula of culture liquid here are as follows: 20L culture solution Proportion be 60g peptone, 4g brewer's wort, 7g anhydrous magnesium sulfate, 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, Control is adjusted in 5g sodium chloride, pH value acid or alkali, and water supplies 20L.Example contracts different amounts of culture solution according to the proportion Small or amplification.

On the other hand, Thermophilic Bacteria and the tunning of yeast co-fermentation culture are in production 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 includes: first to carry out to yeast 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 fermentation liquid 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) first carrying out one or many fermentations for S. cervisiae under low temperature, acid condition, carries out increasing bacterium to saccharomycete； (2) etc. saccharomycete are in the logarithmic growth phase later period 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 logarithmic growth 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, deposit number CICC1596；Institute is purchased from American Type Culture using thermus thermophilus HB27 and receives Collection center (ATCC), deposit 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 logarithmic growth phase later period start increase temperature Degree；Wherein, 55 DEG C -70 DEG C are increased the temperature to.

In some preferred modes, wherein increase temperature 0 day after start 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 Effect including the reduction to inflammatory factor, the inflammatory factor include 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 In the presence of can be one of solution, aqua, suspension, facial mask, lotion, creme, paste, gel, dry powder, wet-milling, spray.

On the other hand, Thermophilic Bacteria and the tunning of saccharomycete co-fermentation culture are in production 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 strain after step (2) culture is inoculated into the first order seed equipped with 30L fermentation liquid (pH is 4.8 ± 0.2) In 50L tank, ventilatory capacity maintains 5m3/h/50L, 200rpm, 30 DEG C of culture 10h or so；

(4) 30L bacterium solution is transferred in the secondary seed 500L tank 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 solution 2mL being taken to connect on superclean bench Enter in the 2.5L triangular flask equipped with 2L culture solution (pH is 7.8 ± 0.2), ventilatory capacity maintains 5m3/h/50L, 150rpm, 65 DEG C 12h or so is cultivated, after thallus is in logarithmic growth phase, 30L bacterium solution is inoculated into the 500L second level 500L seed of 300L culture solution In tank；

Step 2: 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 fermentor equipped with 2400L culture solution (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 liquid It being transferred in this fermentor, ventilatory capacity maintains 5m3/h/50L, 150rpm stirring, pH value is adjusted to 7.8 ± 0.2 with lye, 150rpm, 65 DEG C of culture 18h or so；

Wherein, the formula of culture solution are as follows: the proportion of 20L culture solution be 60g peptone, 4g brewer's wort, 7g anhydrous magnesium sulfate, Control is adjusted in 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 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, deposit number CICC1596；Institute is purchased from American Type Culture Collection Center (ATCC) using thermus thermophilus HB27, Deposit 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 of method tunning obtained of the invention.

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), deposit 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 saves 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 Agent, sun-screening agent, pH adjusting agent, penetrating agent, liposome, skin-nourishing component, essence, any one or a few mixed in pigment It closes, passes through some external and human trial surfaces of the mixture to tunning of the 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 is following some modes:

One: in parts by weight, include at least following components:

0.05~1 part of Sodium Hyaluronate；

1~5 part of tremella polysaccharides；

0.01~3 part of arbutin；

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

70~90 parts of deionized water.

Two: in parts by weight, include at least following components:

0.1~3.0 part of arbutin；

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.

In some modes, it can be the form of lotion, such as following formula: example one: in parts by weight, include 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, which provides yeast as promotion thermus thermophilus, generates B family vitamin activity 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 strain generates the purposes on the active material fermentation Enhanced agents for promoting B cluster microorganism to enhance.

Beneficial effect

Vitro Experimental Results show: thermus thermophilus and S. cervisiae combined fermentation product and single thermus thermophilus are sent out Ferment product is stronger compared to the ability for promoting skin fibroblasts to increase, and can improve skin fibroblasts 20% 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: containing 1% thermus thermophilus and the Essence of 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.

Detailed description of the invention

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

Specific embodiment

The present invention is illustrated 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 scope is subject to claim.

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

Material:

Culture solution prepare and ingredient: the proportion of 20L culture solution be 60g peptone, 4g brewer's wort, 7g anhydrous magnesium sulfate, 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH value acid or alkali (hydrochloric acid or sodium hydroxide Solution) control is adjusted, water supplies 20L, and example is zoomed in or out different amounts of culture solution according to the proportion.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, deposit number CICC1596 (commercially available acquisition).

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

1. strain expands culture

Step 1:

1.1 S. cervisiaes increase bacterium:

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

(4) 30L bacterium solution is transferred in the secondary seed 500L tank 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 solution 2mL access equipped with 2L culture solution on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flask in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thallus is in logarithmic growth After phase, 30L bacterium solution is inoculated into the 500L second level 500L seeding tank of 300L culture solution.

Here the formula of culture liquid are as follows: the proportion of 20L culture solution is 60g peptone, 4g brewer's wort, 7g anhydrous slufuric acid Control, water is adjusted in magnesium, 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH value acid or alkali Supply 20L.Example is zoomed in or out different amounts of culture solution according to the proportion.

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 fermentor 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 at this time are as follows: here culture liquid formula are as follows: the proportion of 20L culture solution be 60g peptone, 4g brewer's wort, 7g anhydrous magnesium sulfate, 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH value acid Or control is adjusted in alkali, water supplies 20L.Example is zoomed in or out different amounts of culture solution according to the proportion.

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 fermentor, and ventilatory capacity maintains 5m³/ h/50L, 150rpm stirring, 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, thallus 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 active carbon is 6%, and active carbon is bought in global charcoal industry).

Filtering: the fermentation liquid 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 embodiment of individual yeast tunning is as follows:

Culture solution is prepared: the proportion of 20L culture solution is 60g peptone, 4g brewer's wort, 7g anhydrous magnesium sulfate, 2.4g phosphoric acid Control is adjusted in potassium dihydrogen, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH value acid or alkali, and water supplies 20L, Ratio is zoomed in or out different amounts of culture solution according to the proportion.

1. strain expands culture

2. fermentation

Yeast bacteria culture fluid in the secondary seed tank of 300L is transferred to equipped with 2700L culture solution (pH is 4.8 ± 0.2) 5000L fermentor 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, thallus is intercepted at filter membrane On (0.22um), fermented supernatant fluid is obtained.

Filtering: the fermentation liquid of purification is filtered by the filter membrane of 0.22um, is obtained big individually saccharomycetes to make fermentation and is produced Object.

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

The specific embodiment of individual thermus thermophilus tunning is as follows:

1. strain expands culture

2. fermentation

300L thermus thermophilus enrichment liquid is transferred in the fermentor containing 2700L culture solution, ventilatory capacity maintains 5m³/ h/50L, 150rpm stirring, pH value adjust to 7.8 ± 0.2,150rpm, 65 DEG C and cultivate 18h or so.

3. isolating and purifying

Filtering: the fermentation liquid 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 the complex reagent that 1:1 ratio mixes according to weight.

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

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

1. strain expands culture

Saccharomycete increases bacterium:

Mono- ring of saccharomyces cerevisiae CICC1596 on test tube slant is taken on superclean bench, access is equipped with 50mL culture solution In the 250mL triangular flask of (pH is 4.8 ± 0.2), 200rpm, 30 DEG C of culture 10h or so, thallus is in logarithmic growth phase；Inoculation Into the 2.5L triangular flask equipped with 2L (pH be 4.8 ± 0.2), inoculum concentration is that 10% (volume ratio, inoculation is in logarithmic growth here 200 milliliters of phase), 200rpm, 30 DEG C of culture 10h or so；3L strain is inoculated into equipped with 30L fermentation liquid (pH is 4.8 ± 0.2) Level-one 50L seeding tank in, ventilatory capacity maintains 5m³/ h/50L, 200rpm, 30 DEG C of culture 10h or so.30L bacterium solution is shifted Into the second level 500L seeding tank equipped with 300L culture solution.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 solution 2mL access equipped with 2L culture solution on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flask in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thallus is in logarithmic growth After phase, 30L bacterium solution is inoculated into the 500L second level 500L seeding tank of 300L culture solution 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 tank in the secondary seed tank of 300L Into the 5000L fermentor equipped with 2400L culture solution (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 stirring, 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:

Mono- ring of saccharomyces cerevisiae CICC1596 on test tube slant is taken on superclean bench, access is equipped with 50mL culture solution In the 250mL triangular flask of (pH is 4.8 ± 0.2), 200rpm, 30 DEG C of culture 10h or so, thallus are in logarithmic growth phase, inoculation Into the 2.5L triangular flask equipped with 2L (pH is 4.8 ± 0.2), inoculum concentration is 10% (volume ratio), 200rpm, 30 DEG C of culture 10h 3L strain is inoculated into the level-one 50L seeding tank equipped with 30L fermentation liquid (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 solution is transferred in the second level 500L seeding tank 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 solution 2mL access equipped with 2L culture solution on superclean bench (pH is 7.8 ± 0.2) 2.5L triangular flask in, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture 12h or so, thallus is in logarithmic growth After phase, 30L bacterium solution is inoculated into the 500L second level 500L seeding tank of 300L culture solution.Broth temperature is promoted to 65 again DEG C, ventilatory capacity maintains 5m³/ h/50L, 150rpm stirring 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 tank in the secondary seed tank of 300L It is transferred in the 5000L fermentor equipped with 2400L culture solution (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, thallus 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 1 day for waiting 65 DEG C of arrival, 300L thermus thermophilus enrichment liquid is transferred in this fermentor, ventilatory capacity Maintain 5m³PH value is adjusted to 7.8 ± 0.2,150rpm, 65 DEG C with lye and cultivates 18h or so by/h/50L, 150rpm stirring.

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 2 days for waiting 65 DEG C of arrival, 300L thermus thermophilus enrichment liquid is transferred in this fermentor, ventilatory capacity Maintain 5m³PH value is adjusted to 7.8 ± 0.2,150rpm, 65 DEG C with lye and cultivates 18h or so by/h/50L, 150rpm stirring.

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 3 days for waiting 75 DEG C of arrival, 300L thermus thermophilus enrichment liquid is transferred in this fermentor, ventilatory capacity Maintain 5m³PH value is adjusted to 7.8 ± 0.2,150rpm, 65 DEG C with lye and cultivates 18h or so by/h/50L, 150rpm stirring.

Examples of implementation 10: cytotoxicity experiment

It collects logarithmic phase cell (human skin fibroblasts and application on human skin horn cell), individual cells is made with culture solution Suspension, adjusting final concentration of cells are 40000cell/mL, and 100uL cell suspension is added in 96 orifice plates, and 5%CO2,37 DEG C are incubated for, It after cell is adherent, is added detectable substance (cell culture fluid of each tunning of the 1-9 containing examples of implementation), culture is arrived to 80% After 90% fusion, every hole adds MTT solution 20uL. to continue to be incubated for 3h, terminates culture.Culture medium in hole is carefully sucked, every hole is added 100uL DMSO vibrates 10min, melts crystal sufficiently.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 fluid of each tunning of the 1-9 containing examples of implementation is added, cell culture is containing 3% The high glycoform DMEM cell culture fluid 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 for the also extremely significant facilitation 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 sharply 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 experiment

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 suspension is added in 96 orifice plates by 40000cell/mL, and 5%CO2,37 DEG C are incubated for 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 irradiation, dose of radiation control is 60mJ/cm², change detectable substance (implementation Each tunning of example 1-9 is diluted with high glycoform DMEM cell culture fluid, each tunning that concentration is 10%), 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 for containing 10% thermus thermophilus and saccharomycete combined fermentation product (examples of implementation 1) is added DMEM cell culture fluid, Inflammatory Factors Contents IL-1 α is 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 obvious (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 be significantly reduced the generation of inflammatory factor, compare Individually fermentation or fermenting mixture, by variance analysis, examples of implementation 1,7 and other processing all reach extremely 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 facilitation to the active material of two kinds of bacterium, and activity also significantly improves.On the contrary, individually culture And individually cultivate and then mix and the mixed culture of other way, 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 He obtains bigger activity by B family vitamin by unknown substance.

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 The factor for allowing B family vitamin to have greater activity a bit generates, so that the activity of B family vitamin is improved, to have 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 for carrying 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 are as follows: German CK company skin water Part is lost (TEWL) test probe TM300；Test condition are as follows: 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 is produced using control group and containing 1% examples of implementation 1-9 fermentation twice Object Essence (main component of Essence has: 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 7 days front and back test TEWL of product, and calculates the change rate of TEWL, the calculation of the change rate of TEWL are as follows: [value of the TEWL of (value of the TEWL before value -7 days of the TEWL after 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 and examples of implementation 2-6,8-9 reach pole for barrier repairing effect Significant difference (summary of specific experiment data), this explanation are produced by the co-cultivation that the method culture of the embodiment of the present invention 1 obtains Object can have significant repair function to keratoderma, may improve the active material for repairing skin keratin confluent monolayer cells The active perhaps yield or activity of yield or other confactors.

Examples of implementation 10: skin is dispelled yellow test

CIEXYZ system is converted to CIEL*a*b* color space by mathematical method.The space by a brightness (L) and Two color (a, b) axis compositions.Brightness is a kind of scale for 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 yellow effect of dispelling of b* value is more obvious, and △ b* is smaller, dispels 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 10% examples of implementation 1-9 tunning Essence (main component of Essence has: deionized water, Tween-80, propylene glycol, Preservative), continuous use 7 days (repeating test 5 times).It takes pictures using before product, using progress VISIA after product 7 days, every group of meter Calculate average △ b* value.

Wherein, test equipment are as follows: LAB tester: 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 co-incubation of the invention, increases removal aging and pigment deposition active material is conducive to, 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.Reasonably explain in this way and may be: the normal update cycle of skin as 3-4 weeks, The proliferation for accelerating cell updates the metabolism that can accelerate skin epidermis, 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 facilitate cell proliferation and It updates, improves cell viability.This further instruction, it is aobvious for the mixed opportunity of two kinds of bacterium in co-incubation fermentation process Must be especially important, the selection on suitable opportunity facilitates the production of some benefit actives, may obtain some nocuousness on the contrary Substance generation or substance harmful for certain symptoms generation.

Examples of implementation 11: the anti-inflammatory test of skin preparation

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

Application method are as follows: after face cleaning, (configured twice with sterile water using the tunning of embodiment 1,8 sooner or later daily The skin preparation of 15% tunning), wherein sterile water is as control, 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 acne, blackhead It is dispersed in and multiple, the inflammatory skin lesion of being dispersed in property；The medium acne of 2-, shallow warts, inflammatory skin lesion number is more, is only limitted to Face；3- severe is deepened in the inflammatory skin lesion of property；4- severe, tumour, easily formation scar) it is assessed.

Specific data are as follows: the grade of acne can be significantly reduced in the tunning of examples of implementation 1, and wherein women has 75 3 or 4 grades of variations before using are the 0-1 grade 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 tunning, 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, which 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, which 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, mutually confirms with the anti-inflammatory effects in examples of implementation 8；And sterile water Also it does not change significantly.

It is not unique constant for being used herein to the terms and expressions mode of description method, 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 present invention statement.It is therefore believed that although of the invention herein Is clearly demonstrated that and with various concrete schemes and the description of arbitrary feature, but the table of change design disclosed herein 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, the content and 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 any one part 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 of a part that the application is disclosed as patent specification.

Claims

1. Thermophilic Bacteria and the tunning of S. cervisiae combined fermentation culture are dispelled Huang in production anti-inflammatory, skin moisture-keeping or skin Purposes on reagent, wherein the Thermophilic Bacteria and saccharomyces cerevisiae combined fermentation method include:

(1) low temperature first is carried out to saccharomyces cerevisiae, the primary or multiple enrichment liquid body fermentation under acid condition allows it to be in index Growth period；

(2) then allow the temperature of fermentation liquid from low temperature become high temperature after in 1 day, addition thermus thermophilus carry out secondary fermentation, hair The condition of ferment is alkaline condition, and the low temperature is 20 DEG C -30 DEG C；Acid culture is carried out to yeast, the acid condition PH value is 4.0-5.0；Start to increase temperature after saccharomycete is in logarithmic growth phase 1 day；Wherein, raised temperature be 55 DEG C- 70℃；

Wherein, for carrying out purification procedures after the secondary fermentation, wherein described isolates and purifies including bactofugation Step,

Wherein, the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese industrial Microbiological Culture Collection administrative center, is protected Hiding number is CICC1596；Institute is purchased from American Type Culture Collection Center (ATCC) using thermus thermophilus HB27, and deposit number is BAA-163。

2. purposes according to claim 1, wherein in step (1), the increasing above at least twice is carried out to saccharomyces cerevisiae Bacterium culture, so that yeast be allowed to be in logarithmic growth phase；In step (2), thermus thermophilus is inoculated with into saccharomyces cerevisiae culture solution Before, primary or multiple Zengjing Granule is carried out to thermus thermophilus.

3. purposes according to claim 1, wherein the pH value of the alkaline condition is 7.0-8.5.

4. purposes according to claim 1, 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.

5. purposes according to claim 1, wherein the reagent is skin care formulation, and said preparation is solution, suspends One of liquid, facial mask, lotion, creme, paste, gel, dry powder, wet-milling, spray.

6. purposes according to claim 1, wherein the reagent is skin care formulation, and said preparation is aqua.

7. Thermophilic Bacteria and the tunning of S. cervisiae combined fermentation culture are in production anti-inflammatory or skin moisture-keeping reagent Purposes, wherein the Thermophilic Bacteria and yeast combined fermentation method include:

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

(1) mono- ring of saccharomyces cerevisiae CICC1596 on test tube slant is taken on superclean bench, pH of the access equipped with 50mL is 4.8 In the 250mL triangular flask of ± 0.2 culture solution, 200rpm, 30 DEG C of culture 10h or so, thallus is in logarithmic growth phase；

(2) thallus in logarithmic growth phase is inoculated into equipped with 2L, pH is to connect in the 2.5L triangular flask of 4.8 ± 0.2 culture solutions Kind amount is 10%, 200rpm, 30 DEG C culture 10h of percentage by volume or so；

(3) the 3L strain after step (2) culture is inoculated into equipped with 30L, the first order seed 50L for the culture solution that 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 solution is transferred in the culture solution secondary seed 500L tank 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 solution 2mL access dress is taken on superclean bench There is a 2L, in the 2.5L triangular flask for the culture solution that pH is 7.8 ± 0.2, ventilatory capacity maintains 5m³/ h/50L, 150rpm, 65 DEG C of culture After thallus is in logarithmic growth phase, 30L bacterium solution is inoculated into the 500L second level 500L seeding tank of 300L culture solution by 12h or so；

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

It is as follows to the one time fermentation method of yeast: will be by the saccharomycete culture in the secondary seed tank of the 300L after step (4) Liquid is transferred to equipped with 2400L, and in the 5000L fermentor for the culture solution that pH is 4.8 ± 0.2, 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 into 1 day after 65 DEG C, 300L thermus thermophilus enrichment liquid is shifted Into this fermentor, ventilatory capacity maintains 5m³/ h/50L, 150rpm stirring are adjusted pH value to 7.8 ± 0.2 with lye, 150rpm, 65 DEG C of culture 18h or so；

Wherein, the formula of the culture solution are as follows: the proportion of 20L culture solution is 60g peptone, 4g brewer's wort, 7g anhydrous slufuric acid Control is adjusted in magnesium, 2.4g potassium dihydrogen phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH value acid or alkali, Yu Weishui；

Wherein, the bacterial strain of the saccharomyces cerevisiae is CICC1596, is purchased from Chinese industrial Microbiological Culture Collection administrative center, is protected Hiding number is CICC1596；Institute is purchased from American Type Culture Collection Center (ATCC) using thermus thermophilus HB27, and deposit number is BAA-163,

It wherein, further include carrying out purification procedures, wherein described isolates and purifies after for the secondary fermentation of the step 3 Including bactofugation step.