CN108653059A - A kind of probiotics fermention prepares method and the application of the fermentation of seaweed liquid in cosmetics of fermentation of seaweed liquid - Google Patents
A kind of probiotics fermention prepares method and the application of the fermentation of seaweed liquid in cosmetics of fermentation of seaweed liquid Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9711—Phaeophycota or Phaeophyta [brown algae], e.g. Fucus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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Abstract
The present invention provides method and the application of the fermentation of seaweed liquid in preparing cosmetics that a kind of probiotics fermention prepares fermentation of seaweed liquid, belong to microbial fermentation seaweed technical field, the described method comprises the following steps：1) seawood meal that granularity is 1~100 μm is obtained after seaweed ultramicro grinding；2) seawood meal is mixed with fermentation of bacillus liquid, carries out the first fermentation and obtains the first fermentation of seaweed liquid；3) the inoculation yeast bacterium into the first fermentation of seaweed liquid carries out the second fermentation and obtains the second fermentation of seaweed liquid；4) inoculating lactic acid bacterium into the second fermentation of seaweed liquid carries out third fermentation and obtains third fermentation of seaweed liquid；5) the third fermentation of seaweed liquid is placed in 8~12 DEG C of 15~60d of after-ripening, be separated by solid-liquid separation, liquid phase component obtains fermentation of seaweed liquid.The method of the invention is safe, pollution-free, skin effect is good, stability is high.
Description
Technical field
The invention belongs to microbial fermentation seaweed technical fields more particularly to a kind of probiotics fermention to prepare fermentation of seaweed liquid
Method and the application of the fermentation of seaweed liquid in preparing cosmetics.
Background technology
Seaweed species are various, resourceful, are important marine resources.In China, seaweed is mainly based on kelp.Kelp
Also known as kelp, synthetic fibre cloth not only contain abundant proteins,vitamins,and minerals, also contain frequently as food and Chinese medicine material
As alginic acid, fucoidin, laminaran, brown alga fiber, mannitol, laminine, high unsaturated lipid boat acid, fucoxanthine and
The abundant physiological activator such as phytosterin compound.However, the exploitation of China kelp focuses primarily upon kelp food, sea at present
The extraction of the industrial chemicals such as algae (band) fertilizer and iodine, alginic acid, mannitol belongs to the shallow processing of seaweed, with low content of technology, production
Product added value is low, does not still carry out deep processing, the active material in high-efficiency comprehensive utilization seaweed to seaweed.
Seaweed has many advantages, such as that growing environment is pure, vitality is strong, active component content is high, American-European countries have become by
The main function raw material for the skin care field being widely recognized as, numerous line skin care brands develop seaweed element class skin care
Product, however the extraction of seaweed liquid at present is mostly based on acid, alkaline process, not only recovery rate is low for this method, it is often more important that soda acid makes
A series of the problem of with dirty useless processing are brought, environmental pollution destroys serious while improving cost.
Invention content
In view of this, the purpose of the present invention is to provide a kind of safe, free of contamination probiotics fermentions to prepare seaweed
The method of zymotic fluid and the application of the fermentation of seaweed liquid in preparing cosmetics, the fermentation of seaweed liquid that the method prepares are lived
Property ingredient type and content it is high, skin effect is good, stability is high.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：A kind of probiotics fermention preparation seaweed hair
The method of zymotic fluid, includes the following steps：1) seawood meal that granularity is 1~100 μm is obtained after seaweed ultramicro grinding；2) by the sea
Algae powder is mixed with fermentation of bacillus liquid, carries out first 24~48h of fermentation, and the first seaweed hair is obtained after removing bacillus thalline
Zymotic fluid；3) the inoculation yeast bacterium into the first fermentation of seaweed liquid carries out second 48~72h of fermentation, obtains the second fermentation of seaweed
Liquid；4) inoculating lactic acid bacterium into the second fermentation of seaweed liquid carries out third 48~72h of fermentation, obtains third fermentation of seaweed liquid；
5) the third fermentation of seaweed liquid is placed in 8~12 DEG C of 15~60d of after-ripening, be separated by solid-liquid separation, liquid phase component obtains fermentation of seaweed liquid.
Preferably, include impregnating and drying step before ultramicro grinding when seaweed raw material described in step 1) is dry seaweed
Suddenly；The time of the immersion 10~20 times that water is dry seaweed quality, the immersion is 2~5h.
Preferably, the temperature dried described in step 1) is 45~70 DEG C；Water content≤5% of seaweed after the drying.
Preferably, in step 2) bacillus be bacillus subtilis, bacillus pumilus, bacillus amyloliquefaciens,
It is one or more in clothing bacillus, bafillus natto and bacillus coagulans；Work in the fermentation of bacillus liquid
Bacterium number is 0.5~9.5 × 109cfu/mL。
Preferably, the quality of seawood meal is the 6~10% of fermentation of bacillus liquid quality in step 2).
Preferably, the saccharomycete described in step 3) is cloth Laplace saccharomycete, S. cervisiae, utilizing flavoring yeast, Wei Shi
Yeast, Hansenula anomala, schizosaccharomyces pombe, glutinous rhodotorula, pichia farinose, Candida glycerolgenesis and Candida utilis
It is one or more in yeast.
Preferably, the lactic acid bacteria described in step 4) is lactobacillus plantarum, Lactobacillus casei, streptococcus thermophilus, grignard breast
Bacillus, Bifidobacterium, lactobacillus acidophilus and one or more of curl lactobacillus；
Preferably, the lactic acid bacteria replaces with calf staphylococcus and/or staphylococcus xylosus.
Preferably, the inoculum concentration of the saccharomycete described in step 3) and the lactic acid bacteria described in step 4) be independently 4~
6% (volume).
The present invention also provides the fermentation of seaweed liquid that the method prepares, brown alga acid content in the fermentation of seaweed liquid
It is the 1.2~1.8% of seaweed raw material dry weight, the content of fucoidin sulfuric ester is the 0.6~1.0% of seaweed raw material dry weight.
The present invention also provides application of the fermentation of seaweed liquid in preparing cosmetics.
Beneficial effects of the present invention：The method that probiotics fermention provided by the invention prepares fermentation of seaweed liquid, passes through gemma
Bacillus, saccharomycete and lactic acid bacteria are fermented seawood meal acquisition successively, and the bacterial strain used that ferments is food-grade probiotics, safer,
The use of chemical reagent in chemical acid alkali method is avoided using the method for microbial fermentation, reduces pollution.The method of the invention
Active constituent type and content are high in the fermentation of seaweed liquid prepared, and the probiotics that the present invention uses has remarkable brown alga hair
Ferment ability makes active constituent in seaweed completely retain and fully discharge, while the probiotics strain itself will produce paddy Guang
Sweet peptide, gamma amino butyric acid isoreactivity ingredient increase active constituent type and content in tunning.The method of the invention system
The standby fermentation of seaweed liquid obtained has effects that good Skin Cell reparation and regeneration, and stability is high, is recorded according to embodiment,
Have by fibroblast proliferation experiment, cell scratch experiment and collagen synthesis experiment display, the fermentation of seaweed liquid
Significant ability of cell proliferation, remarkable cell repair ability and promotion collagen synthesis ability；It is tested by preservation challenge
Acceleration for stabilization challenge experiment with raw material confirms that the fermentation of seaweed liquid is with good stability.
Description of the drawings
Fig. 1 is that fermentation of seaweed liquid cut challenges experimental result picture in embodiment 4；
Fig. 2 is the fibroblastic growth curve of addition 200 times of fermentation of seaweed liquid of dilution；
Fig. 3 is a procollagen type protease reverse transcription result of the cell mixed with fermentation of seaweed liquid；
Fig. 4 is three procollagen type protease reverse transcription results of the cell mixed with fermentation of seaweed liquid.
Specific implementation mode
The present invention provides a kind of methods that probiotics fermention prepares fermentation of seaweed liquid, include the following steps：1) seaweed passes through
The seawood meal that granularity is 1~100 μm is obtained after ultramicro grinding；2) seawood meal is mixed with fermentation of bacillus liquid, is carried out
First 24~48h of fermentation, the first fermentation of seaweed liquid is obtained after removing bacillus thalline；3) into the first fermentation of seaweed liquid
Inoculation yeast bacterium carries out second 48~72h of fermentation, obtains the second fermentation of seaweed liquid；4) it is connect into the second fermentation of seaweed liquid
Kind lactic acid bacteria carries out third 48~72h of fermentation, obtains third fermentation of seaweed liquid；5) the third fermentation of seaweed liquid is placed in 8~
12 DEG C of 15~60d of after-ripening are separated by solid-liquid separation, and liquid phase component is fermentation of seaweed liquid.
The present invention does not limit the source of the seaweed, using this field routine seaweed.In the present invention, described
Seaweed is preferably kelp.In the present invention, the kelp is preferably dry kelp, and the present invention does not limit the source of the dry kelp
It is fixed, using the dry kelp of conventional commercial.In the present invention, by the dry kelp soaking；The immersion is preferably pure with water
Water purification, the amount of immersion water are preferably 10~20 times of dry kelp quality, more preferably 14~16 times, most preferably
15 times.The temperature of heretofore described immersion is preferably 20~30 DEG C, more preferably 22~28 DEG C；The time of the immersion
Preferably 2~5h, more preferably 3~4h.The purpose of heretofore described immersion is that dry kelp is made fully to be soaked.The present invention
It after the immersion, is preferably rinsed repeatedly with pure water 2~5 times, the time rinsed every time is preferably 10~15min；Institute
The purpose for stating flushing is to wash off the silt and salinity on kelp surface.
The present invention impregnated after kelp after, by after the immersion kelp dry, in the present invention, the drying
Temperature is preferably 45~70 DEG C, and more preferably 50~65 DEG C, the present invention is not particularly limited the time of the drying, to dry
The water content of rear kelp is done to determine；After the drying water content of kelp it is preferred≤5%.The present invention uses lower temperature
Kelp is dried, it is therefore intended that low temperature drying will not destroy the type, content and structure of contained active constituent in brown alga.
The present invention carries out it ultramicro grinding and obtains seawood meal after the kelp is dried.Heretofore described Ultramicro-powder
The broken Ultra-Micro Grinding Equipment using this field routine, in specific implementation process of the present invention, the ultramicro grinding is using north
Capital is started same and development in science and technology Co., Ltd the broken machine of KC-701 plant ultra-micropowders and is carried out.
In the present invention, the time of the ultramicro grinding is preferably 10~20min, more preferably 12~18min；Institute
The granularity for stating ultramicro grinding is preferably 1~100 μm, more preferably 10~50 μm.
The present invention preferably sterilizes to seawood meal after obtaining seawood meal；The sterilizing is preferably gone out using irradiation
Bacterium, irradiation sterilization entrust Shandong Quan Gang radiation developments in science and technology Co., Ltd to carry out；The metering of the irradiation sterilization is preferably
The time of 1000~2500 kilorads, the irradiation sterilization is preferably 1.5~2.5h, more preferably 2h.
The present invention mixes the seawood meal with fermentation of bacillus liquid after obtaining seawood meal, carries out the first fermentation 24
~48h obtains the first fermentation of seaweed liquid after removing bacillus thalline.The bacillus is bacillus subtilis in the present invention
One in bacterium, bacillus pumilus, bacillus amyloliquefaciens, bacillus licheniformis, bafillus natto and bacillus coagulans
Kind is a variety of；Heretofore described bacillus is probiotics, and the present invention does not limit the source of the bacillus, adopts
With the conventional above-mentioned bacillus in this field.Viable count in heretofore described fermentation of bacillus liquid is preferably 0.5
~9.5 × 109Cfu/mL, more preferably 1.0~6.0 × 109cfu/mL。
In the present invention, the fermentation of bacillus liquid is prepared by the following：Bacillus described in activation culture, so
The bacillus after the activation is inoculated into fermentation medium afterwards and carries out fermented and cultured acquisition fermentation of bacillus liquid.
In the present invention, the culture medium of the activation culture is preferably broth bouillon, the temperature of the activation culture
Preferably 35~38 DEG C, more preferably 37 DEG C；The time of the activation culture is preferably 20~28h, more preferably
22~26h, most preferably for 24 hours；The rotating speed of the activation culture is preferably 180~220rpm, more preferably 200rpm.
Bacillus after activation is inoculated into fermentation medium after the activation culture and carries out fermentation training by the present invention
It supports, in the present invention, the condition of the fermented and cultured is consistent with activation culture described in above-mentioned technical proposal, the fermentation medium
Preferably include the following components'mass percentage：1.5~2.5% glucose, 1.0~2.0% peptones, 0.04~0.06%
Sodium chloride, 0.04~0.06% beef extract and the water of surplus；More preferably include 12% glucose, 1.5% peptone,
0.05% sodium chloride, 0.05% beef extract and the water of surplus.
The present invention mixes the seawood meal with fermentation of bacillus liquid after obtaining fermentation of bacillus liquid, carries out
First fermentation.In the present invention, the quality of the seawood meal is preferably the 4~6% of fermentation of bacillus liquid quality, more preferably
It is 5%.In the present invention, the temperature of first fermentation is preferably 35~38 DEG C, more preferably 37 DEG C；First fermentation
Rotating speed be preferably 180~220rpm, more preferably 200rpm；The time of first fermentation is preferably 24~48h, more preferably
For 28~44h.During first fermentation, the seawood meal is dropped with a variety of enzymes that bacillus growth generates
Solution, the viscosity of fermented feed liquid gradually increase.The present invention described first after fermentation, to fermented feed liquid be separated by solid-liquid separation with
Bacillus thalline is removed, the first fermentation of seaweed liquid is obtained.The method of the separation of solid and liquid preferably centrifuges in the present invention,
The rotating speed of the centrifugation is preferably 5000~7000rpm, more preferably 5500~6500rpm；The time of the centrifugation is excellent
Choosing is 8~12min, more preferably 10min.
The present invention is after obtaining the first fermentation of seaweed liquid, the inoculation yeast bacterium into the first fermentation of seaweed liquid, carries out the
Two 48~72h of fermentation, obtain the second fermentation of seaweed liquid.In the present invention, the saccharomycete be preferably cloth Laplace saccharomycete,
S. cervisiae, utilizing flavoring yeast, Wei Shi yeast, Hansenula anomala, schizosaccharomyces pombe, glutinous rhodotorula, powdery finish red ferment
It is one or more in female, Candida glycerolgenesis and candida utili.In the present invention, the saccharomycete preferably passes through
It is inoculated with after activation culture；In the present invention, the culture medium of the saccharomycete activation culture is preferably malt extract medium,
The temperature of the activation culture is preferably 28~32 DEG C, more preferably 30 DEG C；The time of the activation culture is preferably
44~52h, more preferably 48h；The activation culture is preferably stationary culture.In the present invention, the saccharomycete connects
Kind amount is preferably 4~6% (volumes), more preferably 5% (volume).In the present invention, the temperature of second fermentation is excellent
It is selected as 28~33 DEG C, more preferably 30 DEG C；Second fermentation is preferably left to ferment；It is described second fermentation time be preferably
48~72h, more preferably 62~68h.In the present invention, in the second fermentation process, after yeast strain autolytic cleavage, it can discharge
Cellular content, the cellular content contains more amino acid, small peptide, nucleotide isoreactivity substance, to the new old of skin
Metabolism has facilitation.The present invention after fermentation, without being separated by solid-liquid separation, retains saccharomycete thalline described second.
The present invention is after obtaining the second fermentation of seaweed liquid, the inoculating lactic acid bacterium into the second fermentation of seaweed liquid, into
Row third 48~72h of fermentation, obtains third fermentation of seaweed liquid.In the present invention, the lactic acid bacteria is preferably lactobacillus plantarum, does
Lactobacillus paracasei, streptococcus thermophilus, lactobacillus gasseri, Bifidobacterium, lactobacillus acidophilus and one or more of curl lactobacillus.
In the present invention, the lactic acid bacteria is preferably inoculated with after activation culture；In the present invention, the lactic acid bacteria activation training
Foster culture medium is preferably MRS culture mediums, and the temperature of the activation culture is preferably 28~32 DEG C, more preferably 30
℃；The time of the activation culture is preferably 32~40h, more preferably 36h；The activation culture preferably stands training
It supports.The inoculum concentration of heretofore described lactic acid bacteria is preferably 4~6% (volumes), more preferably 5% (volume).In the present invention
In, the temperature of the third fermentation is preferably 28~33 DEG C, more preferably 30 DEG C；The third fermentation is preferably left to ferment；
The time of the third fermentation is preferably 48~72h, more preferably 62~68h.
In the present invention, lactic acid bacteria described in above-mentioned technical proposal also can be replaced staphylococcus, specially calf grape ball
Bacterium and/or staphylococcus xylosus.
The third fermentation of seaweed liquid is placed in 8~12 DEG C of after-ripening by the present invention after obtaining the third fermentation of seaweed liquid
15~60d is separated by solid-liquid separation, and is collected liquid phase component and is obtained fermentation of seaweed liquid.In the present invention, the temperature of the after-ripening is preferably
10 DEG C, the time of the after-ripening is preferably 20~50d；The acting as of heretofore described after-ripening makes hair by low temperature ageing
Active constituent in zymotic fluid tends towards stability.
It after the present invention is ripe in the rear, is separated by solid-liquid separation, the separation of solid and liquid includes preferably carrying out successively
Centrifugation and membrane filtration.The rotating speed of the centrifugation is preferably 9000~11000rpm in the present invention, more preferably 10000rpm；
The time of the centrifugation is preferably 5~20min, more preferably 8~15min.The present invention collects supernatant after the centrifugation
Then the supernatant is carried out membrane filtration by liquid, collect filtration group and be divided into fermentation of seaweed liquid.The membrane filtration in the present invention
Filter membrane diameter is preferably 0.2~0.3 μm, more preferably 0.22 μm.
The present invention also provides the fermentation of seaweed liquid that the method prepares, brown alga acid content in the fermentation of seaweed liquid
It is the 1.2~1.8% of seaweed raw material dry weight, preferably 1.5%, fucoidin sulfate content is the 0.6 of seaweed raw material dry weight
~1.0%, preferably 0.7%, both the above ingredient all have the function of significantly promoting cell Proliferation, and collagen is promoted to close
At can delay and resist cell ageing.
The present invention also provides application of the fermentation of seaweed liquid in preparing cosmetics.In the present invention, the sea
Algae zymotic fluid is mixed with cosmetics as raw material and other activated feedstocks and excipient.In the present invention, the fermentation of seaweed
Liquid can be directly appended in cosmetic formulations, additive amount is preferably 2%~50%, with specific reference to cosmetics without concentration
Purposes and cost determination.
In the present invention, the cosmetics are this field conventional cosmetic, including surfactant, facial treatment milk, skin cream, essence
Hua Su, facial mask.
Method and the sea of fermentation of seaweed liquid are prepared to a kind of probiotics fermention provided by the invention with reference to embodiment
Application of the algae zymotic fluid in preparing cosmetics is described in detail, but they cannot be interpreted as protecting model to the present invention
The restriction enclosed.
Embodiment 1：
1) dry kelp is impregnated with pure water, takes 1 portion of kelp and 15 parts of water；Dry kelp is rushed after fully impregnating with pure water repeatedly
It washes, washes off the silt and salinity on kelp surface；
2) the kelp ultramicro grinding powder diameter of clean kelp low temperature drying, drying is less than 50 μm；
3) fermentation bacterial strain used is activated, wherein the bacterial strain used that ferments is bacillus amyloliquefaciens, lichens bud
Spore bacillus；Cloth Laplace saccharomycete, S. cervisiae, utilizing flavoring yeast；Lactobacillus acidophilus curls lactobacillus.
4) kelp of low temperature drying is subjected to irradiation sterilization, 1000~2500 kilorads irradiate 2 hours；
5) activated bacillus amyloliquefaciens are cultivated with fermentation medium, culture medium prescription is：1L distilled water+
20g glucose+15g peptone+5g sodium chloride+0.5g beef extracts.Bacillus, which is cultivated to cell viable count, reaches 109cfu/mL
Afterwards, ultramicro grinding and the Kelp Powder of sterilizing are accessed into zymotic fluid according to 5% ratio, continues to cultivate bacillus；
6) Kelp Powder is degraded with a variety of enzymes that bacillus growth generates, and kelp and culture medium gradually become viscous
Larger fermented laminaria liquid is spent, after fermenting 24 hours, stops fermentation；
7) it after fermentation, is centrifuged 10 minutes with 6000rpm/min, removes bacillus thalline, bacterium is always connect according still further to 5%
Amount is respectively connected to cloth Laplace saccharomycete 1%, S. cervisiae 2% and 2% utilizing flavoring yeast and carries out secondary fermentation, and fermentation condition is
30 degree of stationary cultures 72 hours；
8) after saccharomycetes to make fermentation, third time fermentation is carried out according to the 5% bacterium amount access lactic acid bacteria that connects, fermentation condition is
30 degree of stationary cultures 48 hours；
9) after three kinds of bacterial strain mixed fermentation mixed fermentations, zymotic fluid is put into 10 degree of low temperature environments and carries out after-ripening turn
Change, ripening time is 15 days；
10) after after-ripening, liquid is centrifuged, collects supernatant, centrifugal condition is that 10000rpm/min centrifuges 10
Minute；
11) centrifugation supernatant is filtered with 0.22um filter membranes, gained clear liquid carries out preservation challenge experiment, obtains the fermentation
The preservation formulations of kelp supernatant are 0.6%PE9010；
Gained fermentation of seaweed liquid is subjected to cut challenge experiment, as a contrast with pure water.Test method is as follows：1. will be at fibre
Cell HSF-NIH3T3 is tieed up with 1 × 105A/ml density is inoculated in six orifice plates, and culture solution is the DMEM containing 10% fermented laminaria,
After the fusion of cell 80%, with self-control scraper in monolayer longitudinal scratch, cell wound model is caused；2. dividing at random after cut
At fermented laminaria and control group, control group culture solution is changed to serum-free DMEM, and photo is shot under inverted microscope as 0h；
3. wound forms rear 6h, 12h, 48h shoot photo.
4 be the cell culture photo for adding fermentation of seaweed liquid in Fig. 1, and 7 be the control for being not added with the seaweed liquid that ferments in Fig. 1.
As seen from Figure 1, cell was cultivated by 48 hours, it is seen that longitudinal scratch are obviously reduced, cell wound healing, have significant
Cell repair ability.And the cut of control group is without significant change.
Embodiment 2：
1) dry kelp is impregnated with pure water, takes 1 portion of kelp and 10 parts of water；Dry kelp is rushed after fully impregnating with pure water repeatedly
It washes, washes off the silt and salinity on kelp surface；
2) the kelp ultramicro grinding powder diameter of clean kelp low temperature drying, drying is less than 70um；
3) fermentation bacterial strain used is activated, wherein the bacterial strain used that ferments is bafillus natto；Glutinous rhodotorula,
Pichia farinose；Lactobacillus gasseri, Bifidobacterium；
4) kelp of low temperature drying is subjected to irradiation sterilization, 1000~2500 kilorads irradiate 2 hours；
5) activated bafillus natto is cultivated with fermentation medium, culture medium prescription is：1L distilled water+
20g glucose+15g peptone+5g sodium chloride+0.5g beef extracts.Bafillus natto culture to cell viable count reaches
109After cfu/mL, by ultramicro grinding and the Kelp Powder of sterilizing is according to 5% ratio access zymotic fluid, continues to cultivate bacillus；
6) Kelp Powder is degraded with a variety of enzymes that bacillus growth generates, and kelp and culture medium gradually become viscous
Larger fermented laminaria liquid is spent, after fermenting 36 hours, stops fermentation；
7) it after fermentation, is centrifuged 10 minutes with 6000rpm/min, removes bacillus thalline, bacterium amount is connect according still further to 5%
It connects glutinous rhodotorula and pichia farinose and carries out secondary fermentation, fermentation condition is 30 degree of stationary cultures 48 hours；
8) after saccharomycetes to make fermentation, lactobacillus gasseri, Bifidobacterium progress third time hair are accessed according to 5% bacterium amount that connects
Ferment, fermentation condition are 30 degree of stationary cultures 56 hours；
9) after three kinds of bacterial strain mixed fermentation mixed fermentations, zymotic fluid is put into 10 degree of low temperature environments and carries out after-ripening turn
Change, ripening time is 30 days；
10) after after-ripening, liquid is centrifuged, collects supernatant, centrifugal condition is that 10000rpm/min centrifuges 10
Minute；
11) centrifugation supernatant is filtered with 0.22um filter membranes, gained clear liquid carries out preservation challenge experiment, obtains the fermentation
It is 0.6%PE9010 that kelp supernatant, which obtains preservation formulations,；
Gained fermentation of seaweed liquid is diluted respectively and carries out fibroblastic proliferation experiment.Experimental procedure is as follows：By skin
Fibroblast kind is in 96 orifice plates, per hole 200ul, a concentration of 5 × 104, cultivate one day, dilute fermentation of seaweed liquid within second day
It 10 times, 50 times, 100 times, 200 times, is added in 96 orifice plates, per 100 μ l of hole, cultivates one day, third day is added CCK8 and is detected.
Fig. 2 is the fibroblastic growth curve of addition 200 times of fermented laminaria raw materials of dilution.As seen from Figure 2, fermentation of seaweed liquid has
Play the role of significantly promoting cell Proliferation.Fibroblastic proliferation is closely related with cell ageing, and therefore, fermented laminaria is former
Material has effects that potentially to resist cell ageing.
Embodiment 3：
1) dry kelp is impregnated with pure water, takes 1 portion of kelp and 17 parts of water；Dry kelp is rushed after fully impregnating with pure water repeatedly
It washes, washes off the silt and salinity on kelp surface；
2) the kelp ultramicro grinding powder diameter of clean kelp low temperature drying, drying is less than 100um；
3) fermentation bacterial strain used is activated, wherein the bacterial strain used that ferments is bacillus amyloliquefaciens；It is false to produce protein
Silk yeast；Streptococcus thermophilus, Lactobacillus casei；
4) kelp of low temperature drying is subjected to irradiation sterilization, 1000~2500 kilorads irradiate 2 hours；
5) activated bacillus amyloliquefaciens are cultivated with fermentation medium, culture medium prescription is：1L distilled water+
20g glucose+15g peptone+5g sodium chloride+0.5g beef extracts.Bacillus amyloliquefaciens culture to cell viable count reaches
109After cfu/mL, by ultramicro grinding and the Kelp Powder of sterilizing is according to 5% ratio access zymotic fluid, continues culture solution starch bud
Spore bacillus；
6) Kelp Powder is degraded with a variety of enzymes that bacillus growth generates, and kelp and culture medium gradually become viscous
Larger fermented laminaria liquid is spent, after fermenting 48 hours, stops fermentation；
7) it after fermentation, is centrifuged 10 minutes with 6000rpm/min, removes bacillus thalline, bacterium amount is connect according still further to 5%
It connects candida utili and carries out secondary fermentation, fermentation condition is 30 degree of stationary cultures 56 hours；
8) candida utili bacterium after fermentation, streptococcus thermophilus and Lactobacillus casei is accessed according to 5% bacterium amount that connects
Third time fermentation is carried out, fermentation condition is 30 degree of stationary cultures 72 hours；
9) after three kinds of bacterial strain mixed fermentation mixed fermentations, zymotic fluid is put into 10 degree of low temperature environments and carries out after-ripening turn
Change, ripening time is 45 days；
10) after after-ripening, liquid is centrifuged, collects supernatant, centrifugal condition is that 10000rpm/min centrifuges 10
Minute；
11) centrifugation supernatant is filtered with 0.22um filter membranes, gained clear liquid carries out preservation challenge experiment, obtains the seaweed
The preservation formulations of zymotic fluid are 0.6%PE9010；
Collagen synthesis is carried out to gained fermentation of seaweed liquid and promotes ability detection.Test method is：By cell kind at 2
In six orifice plates, per hole 2ml cell suspensions.3 samples mark fermented laminaria, water, blank (water+0.6%PE9010), cell respectively
500 μ l fermented laminaria raw materials are added in incubator culture one day, second day label fermented laminaria hole, water, sky are added in remaining hole
In vain, it cultivates two days, collects cell extraction RNA within the 4th day, reverse transcription is carried out after extracting RNA, with relevant with collagen synthesis
Two one procollagen type protease of key enzyme and three procollagen type protease are research object, using GADPH as internal reference, as a result as schemed
Shown in 3/4：The type and three collagen type reverse transcription expression quantity for being added into fermented laminaria raw material are significantly improved, i.e., significantly
The expression of two key enzymes of collagen synthesis in promotion, therefore, the fermentation of seaweed liquid tool that the method for the invention prepares
There is the effect of potential promotion collagen synthesis, delay skin aging.
By above-described embodiment it is found that the method that probiotics fermention provided by the invention prepares fermentation of seaweed liquid, prepares
Fermentation of seaweed liquid in active constituent type and content it is high, brown alga acid content is seaweed raw material dry weight in the fermentation of seaweed liquid
1.5%, fucoidin sulfate content is the 0.7% of seaweed raw material dry weight, and has good skin effect, and stability is high,
Have by fibroblast proliferation experiment, cell scratch experiment and collagen synthesis experiment display, the fermentation of seaweed liquid
Significant ability of cell proliferation, remarkable cell repair ability and promotion collagen synthesis ability；It is tested by preservation challenge
Acceleration for stabilization challenge experiment with raw material confirms that the fermentation of seaweed liquid is with good stability.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method that probiotics fermention prepares fermentation of seaweed liquid, includes the following steps：
1) seaweed obtains the seawood meal that granularity is 1~100 μm after ultramicro grinding；
2) seawood meal is mixed with fermentation of bacillus liquid, first 24~48h of fermentation is carried out, after removing bacillus thalline
Obtain the first fermentation of seaweed liquid；
3) the inoculation yeast bacterium into the first fermentation of seaweed liquid carries out second 48~72h of fermentation, obtains the second fermentation of seaweed
Liquid；
4) inoculating lactic acid bacterium into the second fermentation of seaweed liquid carries out third 48~72h of fermentation, obtains third fermentation of seaweed
Liquid；
5) the third fermentation of seaweed liquid is placed in 8~12 DEG C of 15~60d of after-ripening, be separated by solid-liquid separation, liquid phase component is fermentation of seaweed
Liquid.
2. according to the method described in claim 1, it is characterized in that, when the raw material of seaweed described in step 1) is dry seaweed,
Include immersion and baking step before ultramicro grinding；The quality of immersion water is 10~20 times of dry seaweed quality, institute
The time for stating immersion is 2~5h；The temperature of the drying is 45~70 DEG C；Water content≤5% of seaweed after the drying.
3. according to the method described in claim 1, it is characterized in that, bacillus is bacillus subtilis, short and small in step 2)
One kind or more in bacillus, bacillus amyloliquefaciens, bacillus licheniformis, bafillus natto and bacillus coagulans
Kind；Viable count in the fermentation of bacillus liquid is 0.5~9.5 × 109cfu/mL。
4. method according to claim 1 or 3, which is characterized in that the quality of seawood meal is sent out for bacillus in step 2)
The 6~10% of zymotic fluid quality.
5. according to the method described in claim 1, it is characterized in that, saccharomycete described in step 3) be cloth Laplace saccharomycete,
S. cervisiae, utilizing flavoring yeast, Wei Shi yeast, Hansenula anomala, schizosaccharomyces pombe, glutinous rhodotorula, powdery finish red ferment
It is one or more in female, Candida glycerolgenesis and candida utili.
6. according to the method described in claim 1, it is characterized in that, the lactic acid bacteria described in step 4) is lactobacillus plantarum, does
Lactobacillus paracasei, streptococcus thermophilus, lactobacillus gasseri, Bifidobacterium, lactobacillus acidophilus and one or more of curl lactobacillus.
7. according to the method described in claim 1, it is characterized in that, the lactic acid bacteria replaces with calf staphylococcus and/or wood
Sugared staphylococcus.
8. according to the method described in claim 1, it is characterized in that, described in saccharomycete and step 4) described in step 3)
The inoculum concentration of lactic acid bacteria is independently 4~6% (volumes).
9. the fermentation of seaweed liquid that the method described in claim 1~8 any one prepares, which is characterized in that the seaweed
Brown alga acid content is the 1.2~1.8% of seaweed raw material dry weight in zymotic fluid, and the content of fucoidin sulfuric ester is dry for seaweed raw material
The 0.6~1.0% of weight.
10. application of the fermentation of seaweed liquid in cosmetics described in claim 9.
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