CN108653059B - Method for preparing seaweed fermentation liquor by probiotic fermentation and application of seaweed fermentation liquor in cosmetics - Google Patents
Method for preparing seaweed fermentation liquor by probiotic fermentation and application of seaweed fermentation liquor in cosmetics Download PDFInfo
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Abstract
The invention provides a method for preparing seaweed fermentation liquor by probiotic fermentation and application of the seaweed fermentation liquor in preparation of cosmetics, belonging to the technical field of microbial fermentation of seaweed, and the method comprises the following steps: 1) carrying out superfine grinding on seaweed to obtain seaweed powder with the granularity of 1-100 mu m; 2) mixing the seaweed powder with a bacillus fermentation broth, and performing first fermentation to obtain a first seaweed fermentation broth; 3) inoculating saccharomycetes into the first seaweed fermentation liquor, and performing second fermentation to obtain second seaweed fermentation liquor; 4) inoculating lactic acid bacteria into the second seaweed fermentation liquid, and performing third fermentation to obtain a third seaweed fermentation liquid; 5) and (3) after-ripening the third seaweed fermentation broth for 15-60 days at the temperature of 8-12 ℃, carrying out solid-liquid separation, and obtaining the seaweed fermentation broth from liquid-phase components. The method disclosed by the invention is high in safety, pollution-free, good in skin care effect and high in stability.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation of seaweed, and particularly relates to a method for preparing seaweed fermentation liquor by probiotic fermentation and application of the seaweed fermentation liquor in preparation of cosmetics.
Background
The seaweed has various varieties and rich resources, and is an important marine resource. In China, kelp is the main seaweed. Kelp, also known as kelp and woven fabric, is often used as a raw material of food and traditional Chinese medicine, not only contains abundant proteins, vitamins and mineral substances, but also contains abundant physiologically active substances such as alginic acid, fucoidan, brown algae starch, brown algae fiber, mannitol, laminine, highly unsaturated lipo-furan acid, fucoxanthin, sterol compounds and the like. However, at present, the development of kelp in China mainly focuses on the extraction of kelp food, seaweed (seaweed) fertilizer, iodine, alginic acid, mannitol and other chemical raw materials, and the kelp food, the seaweed (seaweed) fertilizer, the iodine, the alginic acid, the mannitol and other chemical raw materials belong to the shallow processing of the seaweed, the technical content is low, the product added value is low, the seaweed is not deeply processed, and the active substances in the seaweed are efficiently and comprehensively utilized.
The seaweed has the advantages of pure growth environment, strong vitality, high content of active ingredients and the like, is a main functional raw material in the widely accepted skin care product field in European and American countries, and seaweed element skin care products are developed by a plurality of first-line skin care brands, however, the extraction of seaweed liquid is mostly based on an acid and alkali method at present, the method has low extraction rate, more importantly, the problem of treatment of a series of sewage and waste is brought by the use of acid and alkali, the cost is improved, and the environmental pollution is seriously damaged.
Disclosure of Invention
In view of the above, the invention aims to provide a method for preparing a seaweed fermentation broth through probiotic fermentation with high safety and no pollution and an application of the seaweed fermentation broth in preparation of cosmetics.
In order to achieve the above object, the present invention provides the following technical solutions: a method for preparing seaweed fermentation liquor by probiotic fermentation comprises the following steps: 1) carrying out superfine grinding on seaweed to obtain seaweed powder with the granularity of 1-100 mu m; 2) mixing the seaweed powder with the bacillus fermentation liquor, performing first fermentation for 24-48 h, and removing bacillus thallus to obtain first seaweed fermentation liquor; 3) inoculating saccharomycetes into the first seaweed fermentation liquor, and performing second fermentation for 48-72 hours to obtain second seaweed fermentation liquor; 4) inoculating lactic acid bacteria into the second seaweed fermentation liquid, and performing third fermentation for 48-72 hours to obtain a third seaweed fermentation liquid; 5) and (3) after-ripening the third seaweed fermentation broth for 15-60 days at the temperature of 8-12 ℃, carrying out solid-liquid separation, and obtaining the seaweed fermentation broth from liquid-phase components.
Preferably, when the seaweed raw material in the step 1) is dry seaweed, the steps of soaking and drying are included before superfine grinding; the water for soaking is 10-20 times of the dry seaweed in mass, and the soaking time is 2-5 hours.
Preferably, the drying temperature in the step 1) is 45-70 ℃; the water content of the dried seaweed is less than or equal to 5 percent.
Preferably, the bacillus in the step 2) is one or more of bacillus subtilis, bacillus pumilus, bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto and bacillus coagulans; the number of viable bacteria in the bacillus fermentation liquor is 0.5-9.5 multiplied by 109cfu/mL。
Preferably, the mass of the seaweed powder in the step 2) is 6-10% of that of the bacillus fermentation liquor.
Preferably, the yeast in the step 3) is one or more of saccharomyces boulardii, saccharomyces cerevisiae, aroma-enhancing yeast, saccharomyces williamsii, hansenula anomala, schizosaccharomyces pombe, rhodotorula glutinis, pichia pastoris powder, candida glycerinogenes and candida utilis.
Preferably, the lactic acid bacteria in step 4) are one or more of lactobacillus plantarum, lactobacillus casei, streptococcus thermophilus, lactobacillus gasseri, bifidobacterium, lactobacillus acidophilus and lactobacillus huckii;
preferably, the lactic acid bacteria are replaced by staphylococcus calf and/or staphylococcus xylosus.
Preferably, the inoculation amount of the yeast in the step 3) and the inoculation amount of the lactic acid bacteria in the step 4) are independently 4-6% (volume).
The invention also provides the seaweed fermentation liquor prepared by the method, wherein the alginic acid content in the seaweed fermentation liquor is 1.2-1.8% of the dry weight of the seaweed raw material, and the fucoidan sulfate content is 0.6-1.0% of the dry weight of the seaweed raw material.
The invention also provides application of the seaweed fermentation liquid in preparing cosmetics.
The invention has the beneficial effects that: the method for preparing the seaweed fermentation liquid by fermenting the probiotics is obtained by sequentially fermenting the seaweed powder by the bacillus, the microzyme and the lactic acid bacteria, the strains used for fermentation are all food-grade probiotics, the safety is higher, the use of chemical reagents in a chemical acid-base method is avoided by adopting a microbial fermentation method, and the pollution is reduced. The seaweed fermentation liquor prepared by the method has high active ingredient types and contents, the probiotics used by the method has excellent brown seaweed fermentation capacity, so that the active ingredients in the seaweed are completely reserved and fully released, and meanwhile, the probiotic strains can generate active ingredients such as glutathione, gamma-aminobutyric acid and the like, so that the types and contents of the active ingredients in the fermentation product are increased. The seaweed fermentation liquid prepared by the method has good skin cell repairing and regenerating effects and high stability, and the fibroblast proliferation experiment, the cell scratch experiment and the collagen synthesis experiment show that the seaweed fermentation liquid has obvious cell proliferation capacity, excellent cell repairing capacity and collagen synthesis promoting capacity according to the records of the embodiment; the seaweed fermentation liquid is proved to have good stability through a preservative challenge experiment and an accelerated stability challenge experiment of raw materials.
Drawings
FIG. 1 is a graph showing the scratch challenge experiment results of the seaweed fermentation broth in example 4;
FIG. 2 is a graph showing the growth of fibroblasts with 200-fold dilution of the fermentation broth of seaweed;
FIG. 3 shows the reverse transcription results of procollagenase type I of cells mixed with fermentation broth of seaweeds;
FIG. 4 shows the reverse transcription results of the type III procollagenase of cells mixed with the fermentation broth of seaweeds.
Detailed Description
The invention provides a method for preparing seaweed fermentation liquor by probiotic fermentation, which comprises the following steps: 1) carrying out superfine grinding on seaweed to obtain seaweed powder with the granularity of 1-100 mu m; 2) mixing the seaweed powder with the bacillus fermentation liquor, performing first fermentation for 24-48 h, and removing bacillus thallus to obtain first seaweed fermentation liquor; 3) inoculating saccharomycetes into the first seaweed fermentation liquor, and performing second fermentation for 48-72 hours to obtain second seaweed fermentation liquor; 4) inoculating lactic acid bacteria into the second seaweed fermentation liquid, and performing third fermentation for 48-72 hours to obtain a third seaweed fermentation liquid; 5) and (3) after-ripening the third seaweed fermentation broth for 15-60 days at the temperature of 8-12 ℃, and performing solid-liquid separation to obtain a liquid phase component which is the seaweed fermentation broth.
The source of the seaweed is not limited in the invention, and the seaweed is conventional in the field. In the present invention, the seaweed is preferably kelp. In the present invention, the kelp is preferably dried kelp, and the source of the dried kelp is not limited in the present invention, and conventional commercially available dried kelp may be used. In the present invention, the dried kelp is soaked; the soaking water is preferably purified water, and the amount of the soaking water is preferably 10-20 times, more preferably 14-16 times, and most preferably 15 times of the mass of the dried kelp. The soaking temperature is preferably 20-30 ℃, and more preferably 22-28 ℃; the soaking time is preferably 2-5 hours, and more preferably 3-4 hours. The purpose of the soaking in the invention is to fully soak the dried kelp. After the soaking, preferably, the washing is repeatedly carried out for 2-5 times by using purified water, and the time for washing each time is preferably 10-15 min; the purpose of washing is to wash away silt and salinity on the surface of the kelp.
After the soaked kelp is obtained, drying the soaked kelp, wherein the drying temperature is preferably 45-70 ℃, more preferably 50-65 ℃, the drying time is not particularly limited and is determined by the water content of the dried kelp; the water content of the dried kelp is preferably less than or equal to 5%. The invention adopts lower temperature to dry the kelp, and aims to prevent the types, contents and structures of active ingredients contained in the brown algae from being damaged by low-temperature drying.
According to the invention, after the kelp is dried, the kelp is subjected to superfine grinding to obtain the seaweed powder. The ultrafine grinding is carried out by adopting conventional ultrafine grinding equipment in the field, and in the specific implementation process of the invention, the ultrafine grinding is carried out by adopting a KC-701 plant ultrafine grinder of Beijing Kekagaogong and science and technology development Limited company.
In the invention, the time for superfine grinding is preferably 10-20 min, and more preferably 12-18 min; the superfine grinding particle size is preferably 1-100 μm, and more preferably 10-50 μm.
According to the invention, after the seaweed meal is obtained, the seaweed meal is preferably sterilized; the sterilization is preferably performed by irradiation sterilization, and the irradiation sterilization is performed by Shandong spring harbor radiation science and technology development company Limited; the dosage of the irradiation sterilization is preferably 1000-2500 kilorads, and the time of the irradiation sterilization is preferably 1.5-2.5 h, and more preferably 2 h.
After the seaweed powder is obtained, the seaweed powder is mixed with the bacillus fermentation liquid, the first fermentation is carried out for 24-48 h, and the bacillus thallus is removed to obtain the first seaweed fermentation liquid. In the invention, the bacillus is one or more of bacillus subtilis, bacillus pumilus, bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto and bacillus coagulans; the bacillus is probiotics, the source of the bacillus is not limited, and the bacillus can be prepared by adopting the conventional bacillus in the field. The number of viable bacteria in the bacillus fermentation liquor is preferably 0.5-9.5 multiplied by 109cfu/mL, more preferably 1.0 to 6.0X 109cfu/mL。
In the invention, the bacillus fermentation liquor is obtained by the following method: activating and culturing the bacillus, and then inoculating the activated bacillus into a fermentation culture medium for fermentation culture to obtain bacillus fermentation liquor.
In the invention, the culture medium of the activation culture is preferably a broth culture medium, and the temperature of the activation culture is preferably 35-38 ℃, and more preferably 37 ℃; the activation culture time is preferably 20-28 h, more preferably 22-26 h, and most preferably 24 h; the rotation speed of the activation culture is preferably 180-220 rpm, and more preferably 200 rpm.
According to the invention, after the activation culture, the activated bacillus is inoculated into a fermentation medium for fermentation culture, and in the invention, the conditions of the fermentation culture are consistent with those of the activation culture in the technical scheme, and the fermentation medium preferably comprises the following components in percentage by mass: 1.5-2.5% of glucose, 1.0-2.0% of peptone, 0.04-0.06% of sodium chloride, 0.04-0.06% of beef extract and the balance of water; more preferably, the composition comprises 12% of glucose, 1.5% of peptone, 0.05% of sodium chloride, 0.05% of beef extract and the balance of water.
After the bacillus fermentation broth is obtained, the seaweed powder is mixed with the bacillus fermentation broth for the first fermentation. In the present invention, the amount of the seaweed powder is preferably 4 to 6% by mass, more preferably 5% by mass, based on the mass of the bacillus fermentation broth. In the invention, the temperature of the first fermentation is preferably 35-38 ℃, and more preferably 37 ℃; the rotating speed of the first fermentation is preferably 180-220 rpm, and more preferably 200 rpm; the time of the first fermentation is preferably 24-48 h, and more preferably 28-44 h. During the first fermentation period, the seaweed meal is degraded along with various enzymes generated by the growth of bacillus, and the viscosity of fermentation liquid is gradually increased. After the first fermentation is finished, performing solid-liquid separation on the fermentation liquid to remove bacillus thallus to obtain a first seaweed fermentation liquid. The solid-liquid separation method is preferably centrifugation, and the rotation speed of the centrifugation is preferably 5000-7000 rpm, more preferably 5500-6500 rpm; the time for centrifugation is preferably 8-12 min, and more preferably 10 min.
After obtaining a first seaweed fermentation broth, inoculating saccharomycetes into the first seaweed fermentation broth, and carrying out second fermentation for 48-72 hours to obtain a second seaweed fermentation broth. In the invention, the yeast is preferably one or more of saccharomyces boulardii, saccharomyces cerevisiae, aroma-enhancing yeast, saccharomyces willianus, hansenula anomala, schizosaccharomyces pombe, rhodotorula glutinis, pichia pastoris powder, candida glycerinogenes and candida utilis. In the invention, the yeast is preferably inoculated after being subjected to activated culture; in the invention, the culture medium for yeast activation culture is preferably a wort culture medium, and the temperature for activation culture is preferably 28-32 ℃, and more preferably 30 ℃; the activation culture time is preferably 44-52 hours, and more preferably 48 hours; the activation culture is preferably a static culture. In the present invention, the amount of the yeast to be inoculated is preferably 4 to 6% by volume, more preferably 5% by volume. In the invention, the temperature of the second fermentation is preferably 28-33 ℃, and more preferably 30 ℃; the second fermentation is preferably a standing fermentation; the time of the second fermentation is preferably 48-72 hours, and more preferably 62-68 hours. In the invention, in the second fermentation process, the yeast strain can release cell contents after autolysis and cracking, and the cell contents contain more active substances such as amino acids, small peptides, nucleotides and the like, thereby having the effect of promoting the metabolism of the skin. In the invention, after the second fermentation is finished, solid-liquid separation is not carried out, and the saccharomycete thalli are reserved.
After the second seaweed fermentation broth is obtained, inoculating lactic acid bacteria into the second seaweed fermentation broth, and performing third fermentation for 48-72 hours to obtain a third seaweed fermentation broth. In the invention, the lactobacillus is preferably one or more of lactobacillus plantarum, lactobacillus casei, streptococcus thermophilus, lactobacillus gasseri, bifidobacterium, lactobacillus acidophilus and lactobacillus helveticus. In the present invention, the lactic acid bacteria are preferably inoculated after being subjected to activation culture; in the invention, the culture medium for the activation culture of the lactic acid bacteria is preferably an MRS culture medium, and the temperature for the activation culture is preferably 28-32 ℃, and more preferably 30 ℃; the activation culture time is preferably 32-40 h, and more preferably 36 h; the activation culture is preferably a static culture. The amount of the lactic acid bacterium to be inoculated is preferably 4 to 6% by volume, more preferably 5% by volume. In the invention, the temperature of the third fermentation is preferably 28-33 ℃, and more preferably 30 ℃; the third fermentation is preferably static fermentation; the time of the third fermentation is preferably 48-72 hours, and more preferably 62-68 hours.
In the invention, the lactic acid bacteria can be replaced by staphylococcus, specifically staphylococcus calf and/or staphylococcus xylosus.
After the third seaweed fermentation liquid is obtained, the third seaweed fermentation liquid is subjected to after-ripening for 15-60 days at the temperature of 8-12 ℃, solid-liquid separation is carried out, and liquid-phase components are collected to obtain the seaweed fermentation liquid. In the invention, the after-ripening temperature is preferably 10 ℃, and the after-ripening time is preferably 20-50 d; the effect of the after-ripening in the present invention is to stabilize the active ingredients in the fermentation broth by low temperature after-ripening transformation.
In the present invention, after the completion of the after-ripening, solid-liquid separation is carried out, and the solid-liquid separation preferably includes centrifugation and membrane filtration which are carried out in this order. The rotating speed of the centrifugation is preferably 9000-11000 rpm, and more preferably 10000 rpm; the time for centrifugation is preferably 5-20 min, and more preferably 8-15 min. The invention collects the supernatant after the centrifugation, then carries out membrane filtration on the supernatant, and collects the filtered component as the seaweed fermentation liquor. The diameter of the membrane filtration in the present invention is preferably 0.2 to 0.3. mu.m, and more preferably 0.22. mu.m.
The invention also provides the seaweed fermentation liquor prepared by the method, the alginic acid content in the seaweed fermentation liquor is 1.2-1.8% of the dry weight of the seaweed raw material, the alginic acid content is preferably 1.5%, the fucoidan sulfate content is 0.6-1.0% of the dry weight of the seaweed raw material, the fucoidan sulfate content is preferably 0.7%, and the two components have obvious effects of promoting cell proliferation, promoting collagen synthesis and delaying and resisting cell aging.
The invention also provides application of the seaweed fermentation liquid in preparing cosmetics. In the invention, the seaweed fermentation liquor is used as a raw material to be mixed with other active raw materials and excipients to prepare the cosmetics. In the invention, the seaweed fermentation liquid does not need to be concentrated, and can be directly added into a cosmetic formula, and the addition amount is preferably 2-50%, and is specifically determined according to the application and the cost of the cosmetic.
In the present invention, the cosmetic is a cosmetic conventional in the art, and includes skin lotion, skin cream, essence, and facial mask.
The method for preparing seaweed fermentation broth by fermenting probiotics and the application of the seaweed fermentation broth in preparing cosmetics provided by the invention are described in detail below with reference to the examples, but the invention is not to be construed as being limited by the scope of the invention.
Example 1:
1) soaking dried kelp in purified water, and taking 1 part of kelp and 15 parts of water; after fully soaking the dried kelp, repeatedly washing the kelp by using purified water to remove silt and salt on the surface of the kelp;
2) drying the clean kelp at low temperature, wherein the particle size of the dried kelp superfine crushed powder is less than 50 mu m;
3) activating the strains used for fermentation, wherein the strains used for fermentation are bacillus amyloliquefaciens and bacillus licheniformis; saccharomyces boulardii, saccharomyces cerevisiae, and aroma-enhancing saccharomyces; lactobacillus acidophilus, Lactobacillus crispatus.
4) Performing irradiation sterilization on the kelp dried at low temperature, and irradiating for 2 hours at 1000-2500 kilorads;
5) culturing the activated bacillus amyloliquefaciens by using a fermentation culture medium, wherein the formula of the culture medium is as follows: 1L of distilled water +20g of glucose +15g of peptone +5g of sodium chloride +0.5g of beef extract. Culturing the bacillus till the viable count of the cells reaches 109After cfu/mL, inoculating the ultrafine-crushed and sterilized kelp powder into the fermentation liquor according to the proportion of 5%, and continuously culturing the bacillus;
6) degrading kelp powder along with various enzymes generated by the growth of bacillus, gradually changing the kelp and a culture medium into a fermentation kelp liquid with higher viscosity, and stopping fermentation after fermenting for 24 hours;
7) after fermentation, centrifuging at 6000rpm/min for 10 minutes to remove bacillus thallus, respectively inoculating 1% of Saccharomyces boulardii, 2% of Saccharomyces cerevisiae and 2% of flavor-enhancing yeast according to 5% of total inoculation amount to perform secondary fermentation, and performing static culture at 30 ℃ for 72 hours;
8) after yeast fermentation is finished, inoculating lactobacillus according to 5% of inoculation amount for third fermentation, wherein the fermentation condition is 30 ℃ for static culture for 48 hours;
9) after the mixed fermentation of the three strains is finished, putting the fermentation liquor into a low-temperature environment of 10 ℃ for after-ripening conversion, wherein the after-ripening time is 15 days;
10) after the after-ripening is finished, centrifuging the liquid, collecting supernatant, and centrifuging for 10 minutes under the centrifugation condition of 10000 rpm/min;
11) filtering the centrifugal supernatant with a 0.22um filter membrane, and performing an antiseptic challenge experiment on the obtained clear liquid to obtain an antiseptic formula of the fermented kelp supernatant, wherein the antiseptic formula is 0.6% PE 9010;
the obtained seaweed fermentation broth was subjected to a scratch challenge experiment with pure water as a control. The test method is as follows: is to getFibroblast HSF-NIH3T3 at 1X 105Inoculating the cells/ml in a six-hole plate at a density, wherein a culture solution is DMEM containing 10% of fermented kelp, and after 80% of cells are fused, longitudinally scratching a single cell layer by using a self-made scraper to form a cell wound model; randomly dividing the kelp into a fermented kelp group and a control group after scratching, replacing the culture solution of the control group with serum-free DMEM, and taking a picture under an inverted microscope as 0 h; ③ taking pictures 6h, 12h and 48h after the wound is formed.
FIG. 1 shows a photograph of cell culture with addition of seaweed fermentation broth at 4, and FIG. 1 shows a control without addition of seaweed fermentation broth at 7. As can be seen from fig. 1, the cells cultured for 48 hours showed a significant reduction in longitudinal scratches, and the wound of the cells healed with a significant cell repair capacity. Whereas the scratch of the control group did not change significantly.
Example 2:
1) soaking dried kelp in purified water, and taking 1 part of kelp and 10 parts of water; after fully soaking the dried kelp, repeatedly washing the kelp by using purified water to remove silt and salt on the surface of the kelp;
2) drying the clean kelp at low temperature, wherein the particle size of the dried kelp superfine crushed powder is less than 70 um;
3) activating a strain used for fermentation, wherein the strain used for fermentation is bacillus natto; rhodotorula glutinis, Pichia farinosa; lactobacillus gasseri, bifidobacterium;
4) performing irradiation sterilization on the kelp dried at low temperature, and irradiating for 2 hours at 1000-2500 kilorads;
5) culturing the activated bacillus natto by using a fermentation medium, wherein the formula of the culture medium is as follows: 1L of distilled water +20g of glucose +15g of peptone +5g of sodium chloride +0.5g of beef extract. Culturing Bacillus natto until viable count of cells reaches 109After cfu/mL, inoculating the ultrafine-crushed and sterilized kelp powder into the fermentation liquor according to the proportion of 5%, and continuously culturing the bacillus;
6) degrading kelp powder with various enzymes generated by growth of bacillus, gradually changing kelp and a culture medium into a fermentation kelp liquid with higher viscosity, and stopping fermentation after fermenting for 36 hours;
7) after fermentation is finished, centrifuging at 6000rpm/min for 10 minutes, removing bacillus thalli, inoculating rhodotorula glutinis and pichia pastoris powder according to 5 percent of inoculation amount for secondary fermentation, and performing static culture for 48 hours at the fermentation condition of 30 ℃;
8) after yeast fermentation is finished, inoculating lactobacillus gasseri and bifidobacterium according to the inoculation amount of 5 percent to perform third fermentation, wherein the fermentation condition is 30 ℃ static culture for 56 hours;
9) after the mixed fermentation of the three strains is finished, putting the fermentation liquor into a low-temperature environment of 10 ℃ for after-ripening conversion, wherein the after-ripening time is 30 days;
10) after the after-ripening is finished, centrifuging the liquid, collecting supernatant, and centrifuging for 10 minutes under the centrifugation condition of 10000 rpm/min;
11) filtering the supernatant with 0.22um filter membrane, and performing antiseptic challenge experiment on the obtained clear liquid to obtain fermented kelp supernatant with antiseptic formula of 0.6% PE 9010;
the obtained seaweed fermentation liquid is respectively diluted to carry out proliferation experiments of fibroblasts. The experimental procedure was as follows: skin fibroblasts were seeded in 96-well plates at a concentration of 5X 10 at 200ul per well4Culturing for one day, diluting the seaweed fermentation liquor by 10 times, 50 times, 100 times and 200 times the seaweed fermentation liquor the next day, adding the diluted seaweed fermentation liquor into a 96-well plate, adding each hole by 100 mu l, culturing for one day, and adding CCK8 for detection the third day. FIG. 2 is a graph showing the growth of fibroblasts with 200-fold dilution of the fermented kelp material. As can be seen from FIG. 2, the fermentation broth of seaweed has a significant effect of promoting cell proliferation. The proliferation of fibroblasts and cellular senescence are closely related, and therefore, the fermented kelp material has a potential efficacy against cellular senescence.
Example 3:
1) soaking dried kelp in purified water, and taking 1 part of kelp and 17 parts of water; after fully soaking the dried kelp, repeatedly washing the kelp by using purified water to remove silt and salt on the surface of the kelp;
2) drying the clean kelp at low temperature, wherein the particle size of the dried kelp superfine crushed powder is less than 100 um;
3) activating a strain used for fermentation, wherein the strain used for fermentation is bacillus amyloliquefaciens; candida utilis; streptococcus thermophilus, lactobacillus casei;
4) performing irradiation sterilization on the kelp dried at low temperature, and irradiating for 2 hours at 1000-2500 kilorads;
5) culturing the activated bacillus amyloliquefaciens by using a fermentation culture medium, wherein the formula of the culture medium is as follows: 1L of distilled water +20g of glucose +15g of peptone +5g of sodium chloride +0.5g of beef extract. Culturing the bacillus amyloliquefaciens till the number of viable cells reaches 109After cfu/mL, inoculating the ultrafine-crushed and sterilized kelp powder into the fermentation liquor according to the proportion of 5%, and continuously culturing the bacillus amyloliquefaciens;
6) degrading kelp powder along with various enzymes generated by the growth of bacillus, gradually changing the kelp and a culture medium into a fermentation kelp liquid with higher viscosity, and stopping fermentation after fermenting for 48 hours;
7) after fermentation is finished, centrifuging at 6000rpm/min for 10 minutes, removing bacillus thallus, inoculating candida utilis according to the inoculation amount of 5 percent, and performing secondary fermentation under the condition of 30-DEG standing culture for 56 hours;
8) after the fermentation of the candida utilis is finished, inoculating streptococcus thermophilus and lactobacillus casei according to the inoculation quantity of 5% to perform third fermentation, wherein the fermentation condition is 30 ℃ for static culture for 72 hours;
9) after the mixed fermentation of the three strains is finished, putting the fermentation liquor into a low-temperature environment of 10 ℃ for after-ripening conversion, wherein the after-ripening time is 45 days;
10) after the after-ripening is finished, centrifuging the liquid, collecting supernatant, and centrifuging for 10 minutes under the centrifugation condition of 10000 rpm/min;
11) filtering the centrifugal supernatant with a 0.22um filter membrane, and performing an antiseptic challenge experiment on the obtained clear liquid to obtain an antiseptic formula of the seaweed fermentation liquid, wherein the antiseptic formula is 0.6% PE 9010;
and (3) detecting the collagen synthesis promoting capacity of the obtained seaweed fermentation liquor. The test method comprises the following steps: cells were seeded in 2 six-well plates with 2ml of cell suspension per well. The 3 samples are respectively marked with fermented kelp, water and blank (water + 0.6% PE9010), cells are cultured in an incubator for one day, 500 mu l of fermented kelp raw materials are added into holes marked with the fermented kelp on the next day, water and blank are added into the rest holes, the cells are cultured for two days, the cells are collected on the fourth day to extract RNA, the RNA is extracted and then subjected to reverse transcription, two key enzymes, namely type I procollagen protease and type III procollagen protease, related to collagen synthesis are taken as research objects, GADPH is taken as an internal reference, and the result is shown in figure 3/4: the reverse transcription expression quantity of the type I collagen and the type III collagen added into the fermented kelp raw material is obviously improved, namely the expression of two key enzymes for synthesizing the inner collagen is obviously promoted, so the seaweed fermentation liquid prepared by the method has the potential effects of promoting the synthesis of the collagen and delaying the skin aging.
According to the embodiment, the method for preparing the seaweed fermentation liquid by probiotic fermentation has the advantages that the prepared seaweed fermentation liquid is high in active ingredient type and content, the alginic acid content in the seaweed fermentation liquid is 1.5% of the dry weight of the seaweed raw material, the fucoidin sulfate content is 0.7% of the dry weight of the seaweed raw material, the seaweed fermentation liquid has a good skin care effect and high stability, and the seaweed fermentation liquid has obvious cell proliferation capacity, excellent cell repair capacity and collagen synthesis capacity as shown by a fibroblast proliferation experiment, a cell scratch experiment and a collagen synthesis experiment; the seaweed fermentation liquid is proved to have good stability through a preservative challenge experiment and an accelerated stability challenge experiment of raw materials.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for preparing seaweed fermentation liquor applied to cosmetics by probiotic fermentation comprises the following steps:
1) carrying out superfine grinding on seaweed to obtain seaweed powder with the granularity of 1-100 mu m;
2) mixing the seaweed powder with the bacillus fermentation liquor, performing first fermentation for 24-48 h, and removing bacillus thallus to obtain first seaweed fermentation liquor; the number of viable bacteria in the bacillus fermentation liquor is 0.5-9.5 multiplied by 109cfu/mL；
3) Inoculating saccharomycetes into the first seaweed fermentation liquor, and performing second fermentation for 48-72 hours to obtain second seaweed fermentation liquor;
4) inoculating lactic acid bacteria into the second seaweed fermentation liquid, and performing third fermentation for 48-72 hours to obtain a third seaweed fermentation liquid;
5) after-ripening the third seaweed fermentation broth for 15-60 days at 8-12 ℃, and carrying out solid-liquid separation to obtain a liquid phase component which is the seaweed fermentation broth;
when the raw material of the seaweed in the step 1) is dry seaweed, the steps of soaking and drying are included before superfine grinding; the mass of the soaking water is 10-20 times of that of the dried seaweed, and the soaking time is 2-5 hours; the drying temperature is 45-70 ℃; the water content of the dried seaweed is less than or equal to 5 percent;
the mass of the seaweed powder in the step 2) is 6-10% of that of the bacillus fermentation liquor.
2. The method of claim 1, wherein the bacillus in step 2) is one or more of bacillus subtilis, bacillus pumilus, bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto, and bacillus coagulans.
3. The method of claim 1, wherein the yeast in step 3) is one or more of saccharomyces boulardii, saccharomyces cerevisiae, saccharomyces fragrans, saccharomyces willianus, hansenula anomala, schizosaccharomyces pombe, rhodotorula glutinis, pichia farinosa, candida glycerinogenes, and candida utilis.
4. The method according to claim 1, wherein the lactic acid bacteria in step 4) is one or more of Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus gasseri, Bifidobacterium, Lactobacillus acidophilus and Lactobacillus crudus.
5. The method according to claim 1, wherein the lactic acid bacteria are replaced by staphylococcus calf and/or staphylococcus xylosus.
6. The method according to claim 1, wherein the amount of the yeast in step 3) and the lactic acid bacteria in step 4) are independently 4-6% by volume.
7. The seaweed fermentation broth obtained by the method according to any one of claims 1 to 6, wherein the content of alginic acid in the seaweed fermentation broth is 1.2 to 1.8% of the dry weight of the seaweed raw material, and the content of fucoidan sulfate is 0.6 to 1.0% of the dry weight of the seaweed raw material.
8. The use of the algal broth of claim 7 in cosmetics wherein the algal broth achieves skin care by proliferating cells, repairing cells and promoting cellular collagen synthesis.
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