Post-treatment method of microbial fermentation product

Abstract

The invention belongs to the field of cosmetic raw materials, and particularly discloses a post-treatment method of a microbial fermentation product for cosmetics. The method a comprises the following steps: adding activated carbon into fermentation filtrate or supernate obtained by removing thalli after microbial fermentation, stirring, and filtering or centrifuging to remove the activated carbon; a method b comprises the following steps: filling activated carbon into a filtering device, carrying out microbial fermentation, removing thalli, and filtering the obtained fermentation filtrate or supernatant by using the filtering device; the microorganisms are yeast and lactic acid bacteria. The activated carbon is one or a mixture of wood activated carbon, coconut shell activated carbon and coal activated carbon, the particle size of the activated carbon is 10-500 meshes, and the use amount of the activated carbon is 1-5 wt% of the fermentation filtrate or supernate. According to the decolorized and deodorized fermentation product obtained by the post-treatment method, the fermentation taste, sour and odor taste and soy sauce taste can be obviously removed, so that the decolorized and deodorized fermentation product can be used for preparing cosmetics or foods, and the dosage of the fermentation product in the product is increased.

Description

Technical Field

[0001] The invention relates to the field of cosmetic raw materials, and specifically discloses a post-processing method for microbial fermentation products used in cosmetics. Background Art

[0002] Fermentation products produced through microbial fermentation processes have a wide range of applications, from food processing, pharmaceutical manufacturing to biochemical engineering and cosmetics industries. Especially in the field of cosmetics, fermentation products are widely used due to their multiple functions.

[0003] The main advantages of fermentation products are as follows: 1. Antioxidation. Fermentation products contain a variety of antioxidants such as glutathione and superoxide dismutase, which can effectively inhibit the generation of free radicals. 2. Moisturizing. They are rich in amino acids and polysaccharides, which can enhance the skin's bonding ability. 3. Whitening. Fermentation products contain ingredients such as niacinamide and tyrosine, which can reduce the production of melanin.

[0004] However, in addition to nutrients and functional substances, fermentation products generally contain a variety of odor substances such as acids, alcohols, aldehydes, phenols, sulfides, etc. Among them, acids represented by butyric acid and its derivatives and sulfides produced in some fermentation processes will produce unpleasant odors such as saliva, sweat, foot odor, rotten eggs, etc. In addition, fermentation products are mostly yellow to brownish yellow, and when used in certain cosmetics, lighter colors may be required to meet the appearance requirements.

[0005] Due to different bacterial strains and fermentation processes, the unpleasant odors of the fermentation liquid also have different types. In addition to the common fermentation smell, there are also sour smells, soy sauce smells, etc.

[0006] In the existing cosmetics containing fermented products, users often report that there will be sour and smelly odors such as saliva. This is not because the product is spoiled, but because the fermented products cannot remove these odors.

[0007] The usual solution is to reduce the content of fermentation products in the product, or add flavors and fragrances to cover up the unpleasant smell. Reducing the content of fermentation products will result in less active substances in them, affecting the skin care effect; using flavors and fragrances may not completely cover up the unpleasant smell, or it may be difficult to reconcile with the smell of flavors and fragrances; at the same time, too high a concentration of flavors and fragrances will also affect the user experience.

[0008] These disadvantages limit the application of fermentation products in cosmetics, and it is even more difficult to use them in cosmetic oral preparations, foods and other products. Therefore, if the fermentation filtrate is to be more widely used, certain post-treatment is required to solve the above-mentioned defects.

[0009] The existing post-processing process for fermentation products used in cosmetics only performs simple sterilization and filtration. The fermentation liquid after this treatment still retains unpleasant odor and pigments, which affects the user experience of the fermented raw materials in cosmetic formulas. In the subsequent formula development, further color adjustment or fragrance masking is required, which increases the difficulty of cosmetic formula development and affects the application of fermentation products.

[0010] Although activated carbon is used to decolorize and deodorize the fermentation liquid in the existing process, in fact, this method cannot completely remove the sour smell and soy sauce smell in the fermentation product. The content of these odor substances is low but they are easy to remain and difficult to remove.

[0011] CN115554978A discloses a composite material for post-treatment of fermentation liquid and a post-treatment method for fermentation liquid. Activated carbon and filter aids (pearlite powder, diatomaceous earth, etc.) are dispersed in water respectively, and a composite filter layer is formed on the filter cloth of the filter press after pressure filtration. Fermentation liquid can be decolorized and deodorized by filtering through the composite layer, and the recovery rate of target protein in the fermentation liquid can be improved.

[0012] It can be seen that in the prior art, activated carbon treatment mainly solves the problem of removing fermentation odor. For sour and soy sauce odors, conventional activated carbon adsorption cannot solve them well.

[0013] Therefore, it is necessary to improve the existing method and decolorize and deodorize the fermentation product to obtain a fermentation product with no bad odor and light color, so that it can be used as a cosmetic raw material or directly used as a cosmetic; and, by decolorizing and deodorizing, the fermentation product can also be used as food or food additives. Summary of the invention

[0014] The present invention aims to provide a post-treatment method for microbial fermentation products to effectively remove the unpleasant odor therein.

[0015] A post-treatment method for microbial fermentation products, for removing bad odor and reducing color, is selected from method a or b:

[0016] Method a: After microbial fermentation, activated carbon is added to the fermentation filtrate or supernatant obtained by removing the bacterial cells, and the activated carbon is removed by filtering after stirring;

[0017] Method b: The activated carbon is loaded into a filtering device, and the fermentation filtrate or supernatant obtained by removing the bacterial cells after microbial fermentation is filtered using the filtering device.

[0018] The activated carbon particle size is 10-500 mesh, preferably 100-500 mesh, and more preferably 200-500 mesh.

[0019] The activated carbon is any one of wood activated carbon, coconut shell activated carbon and coal-made activated carbon, or any mixture thereof.

[0020] The amount of activated carbon used is 1wt%-5wt% of the fermentation filtrate or supernatant, preferably 1wt%-2.5wt%, more preferably 1wt%-2wt%.

[0021] The wood activated carbon is made from trees, such as firewood, sawdust or wood blocks, etc. It is preferably any one of pine activated carbon and fir activated carbon or any mixture thereof.

[0022] Preferably, the activated carbon is a mixture of coconut shell activated carbon and coal-made activated carbon; or a mixture of wood activated carbon and coconut shell activated carbon.

[0023] In a preferred embodiment of the present invention, the activated carbon is a mixture of wood activated carbon and coconut shell activated carbon, and the mass ratio thereof is 1:0.5-10, preferably 1:0.6-6, and more preferably 1:2-5. In one embodiment of the present invention, the mass ratio thereof is 1:4.

[0024] In a preferred embodiment of the present invention, the activated carbon is a mixture of coal-based activated carbon and coconut shell activated carbon, with a mass ratio of 1:1-10, more preferably 1:5-10; in an embodiment of the present invention, the mass ratio is 1:9.

[0025] The microorganism is yeast, fungus or bacteria; preferably Saccharomyces cerevisiae, Pichia pastoris, Bacillus, Escherichia coli, Bifidobacterium, Pediococcus, Lactobacillus, Lactococcus, Streptococcus or Aspergillus. In a preferred embodiment of the present invention, it is Saccharomyces cerevisiae or Lactobacillus paracasei.

[0026] The decolorized and deodorized fermentation product obtained by the above-mentioned post-treatment method can significantly remove the fermentation smell, sour smell and soy sauce smell, especially the sour smell and soy sauce smell, so that it can be used to prepare cosmetics, increase the amount of fermentation products in cosmetics, or directly use the filtrate obtained by post-treatment of the fermentation filtrate as lotion.

[0027] The content of the fermentation product in the cosmetics may be 0.001 wt%-10 wt% based on dry matter.

[0028] The amount of the active carbon in cosmetics is 0.05wt%-100wt% based on the filtrate after removing the activated carbon.

[0029] The decolorized and deodorized fermentation product obtained by the above post-treatment method can be used to prepare cosmetics, external skin preparations, oral beauty preparations, foods or food additives.

[0030] A cosmetic or skin external preparation contains the decolorized and deodorized fermentation product obtained by the above post-treatment method.

[0031] Preferably, the preservative in the cosmetic or skin topical preparation is a mixture of benzoic acid and polyol. The benzoic acid is one of benzoic acid, benzoate and benzoate, or a combination thereof. The polyol is one of 1,3-propylene glycol, 1,2-pentanediol, 1,2-butanediol, 1,2-hexanediol, caprylyl glycol, decanediol, or any mixture thereof. The mass ratio of benzoic acid to polyol is 1:4-200.

[0032] A cosmetic oral preparation, food or food additive contains the decolorized and deodorized fermentation product obtained by the above post-treatment method.

[0033] After the above-mentioned post-treatment, the fermented liquid after decolorization and deodorization still retains the original skin care or health care effects, for example, promoting the proliferation of basal cells, increasing the thickness of the epidermis, reducing inflammation caused by ultraviolet rays, and promoting skin cell autophagy.

[0034] The beneficial effect of the present invention is that after yeast fermentation and lactobacillus fermentation, the initial fermentation liquid obtained by sterilization is dark in color and strong in smell, and it can be seen from the results of physical and chemical tests and sensory evaluation that the sour and soy sauce smell is strong. If it is directly added to the cosmetic formula, it will affect the quality and stability of the product, bring difficulties to product development, and it is even more difficult to use it in food products. Through the post-treatment method of the present invention, decolorization and deodorization can be carried out in a targeted manner; after treatment, aldehydes, heterocyclics, medium and short chain carboxylic acids, alcohols, phenols and other odor substances in the fermentation liquid can be effectively removed, thereby reducing the fermentation smell, sour smell and soy sauce smell, especially being able to significantly remove the sour smell and soy sauce smell, so that the fermentation product can be used in cosmetics without adding flavors to cover up the smell; it can also meet the use requirements as food or food additives. The present invention can simplify the formula and process, and increase the amount of fermentation products as effective ingredients.

[0035] The present invention uses a compound of one or more specific activated carbons to specifically decolorize and remove odor from the fermentation product, and ultimately obtains a fermentation product with good odor, light color, and enhanced multiple functions. The fermentation product can be used as a cosmetic raw material or directly used as a cosmetic, and can also be used in cosmetic oral preparations, foods or food additives, making its use more extensive.

[0036] The method of the present invention can quickly process a large amount of fermentation liquid, and the obtained product has no unpleasant odor and the quality meets the use requirements; the amount of fermentation liquid processed in a single time can range from several kilograms to dozens of tons, meeting the needs of industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 HE staining results of the epidermal model treated with post-treated yeast fermentation products

[0038] Figure 2Results of IHC Ki67 staining of the epidermal model treated with post-treated yeast fermentation products (A: NT; B: UV irradiation; C: UV irradiation + 0.5 mg / mL decolorized and deodorized filtrate).

[0039] Figure 3 This is a picture of the HE staining results of the 3D full-thickness skin model irradiated with UBA&UVB and treated with post-treated yeast fermentation products to analyze the expression of IL-1α.

[0040] Figure 4 Effects of post-processed yeast fermentation products on IL-1α expression in the dermis and epidermis of a 3D full-thickness skin model irradiated with UBA & UVB. DETAILED DESCRIPTION

[0041] The present invention is described below by specific examples, but the protection scope of the present invention is not limited thereby. Experimental methods without specifying specific operating methods and conditions are selected according to conventional operating methods and experimental conditions, or product instructions.

[0042] Example 1 Preparation of fermentation products

[0043] The fermentation product includes, but is not limited to, fermentation broth, fermentation filtrate, supernatant, concentrate or dry powder derived from yeast, bacteria or fungi.

[0044] The following is an example of the fermentation filtrate of Saccharomyces cerevisiae or Lactobacillus paracasei:

[0045] 1.1 Yeast fermentation process:

[0046] Take a single colony of Saccharomyces cerevisiae and inoculate it into liquid PDB or YPD medium. Cultivate it on a shaker at 20-30°C and 0-400 rpm for 8-24 hours until the OD 600 The fermentation time is preferably 16-20 hours, and the OD 600 The fermentation liquid of the brewer's yeast has the effects of promoting the proliferation of basal cells, anti-inflammation and anti-ultraviolet.

[0047] The seed liquid bacteria are inoculated into the fermentation medium at an inoculation amount of 0.1%-10.0% (v / v), and the fermentation is carried out at a stirring speed of 0-500rpm, a ventilation of 0-4.0vvm, and 15-30°C for 12-96 hours. Preferably, the inoculation amount is 0.2-0.5%, and the first stage is fermented at 15-30°C for 12-24 hours, the stirring condition is 200-400rpm, and the ventilation is 0-4.0vvm; in the second stage, the fermentation medium is supplemented with 0.1wt%-5.0wt% of carbon source and 0.1wt%-2.0wt% of nitrogen source in a fed-addition manner, and the fermentation is continued at 15-30°C for 12-72 hours, the stirring speed is 0-400rpm, and the ventilation is 0.2-2.0vvm.

[0048] The nitrogen source is at least one or a combination of yeast extract (yeast extract or yeast extract powder), soybean meal, peptone or yeast peptone; the carbon source is at least one or a combination of corn syrup, molasses or glucose, sucrose and xylose.

[0049] The fermentation liquid is subjected to physical filtration, centrifugation and other methods to remove bacteria and impurities to obtain fermentation filtrate and supernatant, or is further concentrated by membrane filtration to form a concentrate, or is further formed into dry powder by spray drying, freeze drying and other methods.

[0050] 1.2 Lactic acid bacteria fermentation process:

[0051] (I) Culture medium

[0052] The seed culture medium is an MRS culture medium or a composite culture medium, preferably 0.5% to 3% glucose, 2% to 5% peptone, 2% to 5% beef extract, 0.5% to 3% yeast extract, 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% manganese sulfate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% diammonium hydrogen citrate, 0.01% to 1% sodium acetate, 0.01% to 1% potassium chloride, 0.01% to 1% sodium chloride, 0.01% to 1% ferrous sulfate, 0.01% to 1% Tween-80, and the remainder is water, the percentages are mass percentages, and the pH is 6.5.

[0053] The fermentation medium mainly comprises a carbon source, a nitrogen source, an inorganic salt and a growth factor, preferably 0.5% to 5% glucose, 1% to 5% yeast extract, 0 to 2% yeast peptone, and according to the growth requirements of the bacteria, 0.5% to 5% sodium acetate, 0.01% to 0.5% magnesium sulfate heptahydrate, 0.01% to 1% manganese sulfate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% diammonium hydrogen citrate, 0.01% to 0.5% manganese sulfate, Tween-80 1mL / L to 5mL / L are added in appropriate amounts, and the balance is water, and the percentages are mass percentages. The initial pH is 5.5-6.5.

[0054] The feed medium mainly consists of a carbon source, a nitrogen source, an inorganic salt and a growth factor, preferably a carbon source of 1% to 6% glucose or lactose or a combination thereof; a nitrogen source of 0 to 2% yeast peptone, 1% to 3% yeast extract, or a combined nitrogen source; an inorganic salt of 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% manganese sulfate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% diammonium hydrogen citrate, 0.01% to 0.5% manganese sulfate or a combination thereof; the growth factor is an amino acid or Tween-80, and the remainder is water, and the percentages are weight percentages.

[0055] (ii) Fermentation

[0056] The seed culture medium, fermentation culture medium and feed culture medium are prepared according to the growth requirements of lactic acid bacteria, sterilized at 115° C. and high temperature and pressure for 20 to 30 minutes, and cooled for later use.

[0057] A single colony is inoculated into a seed culture medium, and cultured in a shaker or fermenter at 28°C to 38°C with nitrogen aeration or static culture or low speed culture for 16-24 hours to obtain a lactic acid bacteria seed culture liquid.

[0058] The seed bacterial liquid is added to the sterilized fermentation medium at an inoculation rate of 0.1-10.0%, and the shaker or fermenter is kept at 28-38°C and 50-150rpm, and the pH is controlled at 5.5-6.5 using alkalis such as ammonia water and sodium hydroxide. After inoculation, the culture is cultured, the dissolved oxygen and pH curves are observed, and the feed is fed at a constant rate for about 6h-18h of fermentation. The total culture time is 12-48 hours, preferably 18-24 hours. The percentages are mass percentages.

[0059] The fermentation liquid is subjected to physical filtration, centrifugation and other methods to remove bacteria and impurities to obtain fermentation filtrate and supernatant, or is further concentrated by membrane filtration to form a concentrate, or is further formed into dry powder by spray drying, freeze drying and other methods.

[0060] 1.3 Preparation of sterilized fermentation filtrate

[0061] The fermentation broth is filtered by centrifugation (tubular centrifuge, 10000-13000rpm, preferably 12000rpm; disc centrifuge, 5000-6000rpm, preferably 5300rpm, flow rate 400L / h, slag discharge time 200-500s, preferably 300s; plate centrifuge, 600-800 mesh, made of nylon or polyester or polypropylene) or membrane filtration system (plate and frame filter, filter membrane material nylon / polypropylene / polyethersulfone membrane, 0.1-0.2μm, filter bag material nylon or polyester or polypropylene, 600-800 mesh, filter aid perlite or diatomaceous earth, 200-500 mesh, added in an amount of 1-5%w / w; or hollow fiber membrane filtration system, 0.1-0.2μm, filtration area 14-70m2 ) Remove the bacteria to obtain a clarified yeast fermentation product filtrate semi-finished product with a solid content of 0.1-5.0% (w / w). The purpose of this step is to remove the bacteria to ensure the adsorption efficiency of activated carbon during the decolorization and deodorization process. After the fermentation broth is sterilized and filtered, its physical parameters are measured and sensory evaluation is performed.

[0062] 1.4 Sensory evaluation method of fermentation filtrate

[0063] The sensory evaluation results of the physicochemical parameters of the initial fermentation filtrates of yeast and lactobacillus are shown in Tables 1 and 2.

[0064] Preparation stage: Select and train people with keen sense of smell and taste and no obvious cold or olfactory interference as judges. All fermentation samples are prepared at 25℃ to avoid the influence of environmental factors on the smell. Samples should be coded and tested blindly to avoid preconceived ideas of judges.

[0065] Environmental setting: It should be carried out in a quiet, well-ventilated, odor-free professional sensory evaluation room, with the temperature maintained at 20-25℃ and the humidity appropriate.

[0066] Evaluation process: Before the evaluation, the judges were asked to smell a neutral odor (such as pure water vapor) to clear their nasal memory. The judges applied an equal amount of sample on their arms or backs of their hands, and recorded their first impression and detailed description of the odor during the absorption process, including the odor type (such as fermented smell, sour smell, soy sauce smell, etc.) and the corresponding intensity (such as 1-10 points, 1 point is almost odorless, 10 points is very strong).

[0067] Recording and analysis: Use standardized forms or electronic systems to record, including scoring criteria and detailed sensory descriptions. Collect data from all reviewers, calculate average scores, and identify major odor characteristics and abnormalities. Based on the evaluation results, summarize the typical odor characteristics of fermented products, evaluate their impact on consumer acceptance, and provide a basis for product improvement or standardization.

[0068] Table 1 Initial fermentation broth parameters

[0069]

[0070]

[0071] Table 2 Sensory index of initial fermentation broth

[0072]

[0073] From the results of physical and chemical tests and sensory evaluation, it can be seen that the initial fermentation liquid of yeast fermentation and lactobacillus fermentation after sterilization is dark in color and strong in odor. If it is directly added to the cosmetic formula, it will affect the quality and stability of the product and bring difficulties to product development. Therefore, targeted decolorization and deodorization post-treatment is required. The fermentation filtrate of the above yeast and lactobacillus is used for post-treatment.

[0074] Example 2 Decolorization and deodorization of fermentation broth (method 1)

[0075] Take the yeast fermentation filtrate obtained in Example 1, add wood (pine or fir) granular activated carbon or coconut shell granular activated carbon with a particle size of 10-30 mesh to the above fermentation filtrate semi-finished product for decolorization and deodorization, the addition amount is 1% (w / w), and stir at a speed of 20 rpm for 16 hours; the temperature during the stirring process is controlled at 0-10°C.

[0076] After the stirring process is completed, the temperature is continued to be controlled at 0-10°C, and the stirring device is connected to the filtering device. The filtering device can be selected according to the total volume of the decolorized and deodorized liquid, such as a positive pressure filter (0.1-0.2μm, the membrane material is polypropylene or nylon or polyethersulfone), a plate centrifuge (speed 3000-5000rpm, the filter cloth material is nylon or polyester or polypropylene, the pore size is 600-800 mesh), a bag filter (600-800 mesh, the material is nylon or polyester) . nylon or polypropylene, filtration pressure is 2.0-6.0bar), plate and frame filter (0.1-0.2μm, filter membrane material is nylon or polypropylene or polyethersulfone; 600-800 mesh, filter cloth material is nylon or polyester or polypropylene, filtration pressure is 2.0-6.0bar), disc centrifuge (5000-6000rpm, optimal is 5300rpm, flow rate is 400L / h, slag discharge time is 200-500s, optimal is 300s). The corresponding decolorized and deodorized filtrate is obtained.

[0077] After decolorization and deodorization, the filtrate was comprehensively evaluated by physical and chemical parameters and sensory evaluation. The results are as follows:

[0078] Table 3 Physicochemical parameters of yeast fermentation filtrate after treatment by method 1

[0079]

[0080] Table 4 Sensory evaluation of yeast fermentation filtrate after treatment by method 1

[0081]

[0082] The results of sensory evaluation showed that compared with the initial fermentation filtrate, the fermentation liquid treated by method one had improved odor performance in three dimensions. Physical and chemical tests showed that the conductivity and color of the fermentation liquid had obvious improvements.

[0083] The characteristic odor substances of the fermentation filtrate before and after activated carbon treatment were analyzed and compared by headspace solid phase microextraction (HP-SPME) combined with comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS). The results are as follows:

[0084] Table 5 HP-SPME-GC×GC-MS test results of yeast fermentation filtrate after treatment by method 1

[0085]

[0086]

[0087] It can be seen from the test results that the odor substances are mainly aldehydes, acids and heterocyclic substances. The content of these substances is significantly reduced after being treated with activated carbon. Among these substances, aldehydes have odors such as nuts, sweet oranges, and oils, and heterocyclic compounds can provide products with odors such as burnt sweetness and baking. Aldehydes and heterocyclic compounds are both characteristic flavor substances that provide soy sauce flavor to the fermentation broth. Carboxylic acid substances, especially butyric acid and its homologues, often have unpleasant odors such as sweat and rot, and their content is relatively high in the initial fermentation broth, which seriously affects the odor. After treatment with activated carbon, the content of such substances is significantly reduced, and the sour smell of the fermentation broth is also significantly improved. In addition, alcohol substances are contributors to the characteristic fermentation taste of the fermentation broth. After treatment with activated carbon in method one, the content of such substances is slightly reduced, and the characteristic fermentation taste of the fermentation broth is less affected.

[0088] Example 3 Method 2

[0089] Wood (pine or fir) granular activated carbon or coconut shell granular activated carbon with a particle size of 10-30 mesh is loaded into a bag filter device, and the added amount is 1.5wt% of the yeast fermentation filtrate. The pore size of the bag filter bag is 600-800 mesh, and the material is nylon, polyester or polypropylene. The discharge port of the sterilization liquid equipment is connected to the feed port of the bag filter, and the flow rate is controlled to be 1-10kg / min, the temperature is 20-30℃, and the pressure is 2-6bar to obtain a clear decolorized and deodorized liquid.

[0090] Table 6 Physical and chemical parameters of yeast fermentation filtrate after treatment by method 2

[0091]

[0092]

[0093] Table 7 Sensory evaluation of yeast fermentation filtrate after treatment by method 2

[0094]

[0095] From the sensory evaluation results, it can be seen that after the fermentation liquid was treated by method 2, the fermentation taste did not significantly weaken, and the sour smell and soy sauce taste were significantly improved. The physical and chemical test results also showed that the color and conductivity were significantly reduced.

[0096] Using stir bar sorptive extraction (HBSE) combined with gas chromatography-mass spectrometry (GC-MS), the odor components of yeast fermentation broth before and after decolorization by method 2 were compared. The results are as follows

[0097] Table 8 HBSE-GC-MS test results of yeast fermentation filtrate after treatment by method 2

[0098]

[0099]

[0100] The test results show that the relative content of carboxylic acid substances has been significantly reduced, indicating that the sour smell of the fermentation liquid has been improved. The content of aldehydes, heterocyclics, and phenols has also been reduced. These substances are the main odor substances of soy sauce and provide the fermentation liquid with soy sauce flavor. They have also been reduced. Alcohol substances such as phenylethanol are the product of yeast fermentation and degradation of phenylalanine. It is one of the main contributors to the fermentation taste and its content has also been significantly reduced. The above test results are also reflected in the sensory evaluation.

[0101] Example 4 Method 3

[0102] Coconut shell powder activated carbon and coal-based activated carbon are added to the sterilized yeast fermentation filtrate for decolorization and deodorization. The activated carbon particle size is 200-300 mesh, the addition amount is 0.9% and 0.1% (w / w) respectively, the stirring rate is 50-500rpm, the decolorization and deodorization process temperature is controlled at 20-30℃, and the decolorization and deodorization time is 0.5-3 hours. The single processing volume is 100-200L.

[0103] After the decolorization and deodorization process is completed, the temperature of the decolorization and deodorization tank is continued to be controlled at 0-10°C, and the decolorization and deodorization equipment is connected to the filtering equipment. The filtering equipment can select a positive pressure filter (0.1-0.2μm, the membrane material is polypropylene or nylon or polyethersulfone), a plate centrifuge (600-800 mesh, the material is nylon or polyester or polypropylene), a bag filter (600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), a plate and frame filter (0.1-0.2μm filter membrane, the material is nylon or polypropylene or polyethersulfone; 600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), and a disc centrifuge (5000-6000rpm, preferably 5300rpm, flow rate 400L / h, slag discharge time 200-500s, preferably 300s).

[0104] Table 9 Physicochemical parameters of fermentation filtrate treated by method 3

[0105]

[0106] Table 10 Sensory evaluation index of fermentation filtrate after treatment by method 3

[0107]

[0108] From the results of sensory evaluation and physical and chemical testing, it can be seen that the color of the yeast and lactobacillus fermentation broth treated with a mixture of different types of activated carbon is significantly lighter, and the odor (sour smell and soy sauce smell) is significantly improved.

[0109] Therefore, for the product obtained by this method, the odor substances were detected by headspace solid phase microextraction combined with gas chromatography-mass spectrometry (HP-SPME-GC-MS). The results are as follows:

[0110] Table 11 Content of odor substances in fermentation liquid before and after treatment

[0111]

[0112]

[0113]

[0114] From the identification results of odor substances, it can be seen that compared with the yeast filtrate without decolorization and deodorization, the yeast filtrate treated by method 3 has significantly reduced the contents of aldehydes, heterocyclics and phenols, which are the main odor components of soy sauce flavor. In addition, the content of alcohol substances is also significantly reduced. In the initial fermentation broth, the content of phenylethanol is relatively high. It is the product of microbial metabolism of phenylalanine and the main representative substance of fermentation flavor. The content is significantly reduced after activated carbon treatment. In addition, the content of carboxylic acid substances, especially medium and short chain carboxylic acids, is also significantly reduced. This type of substance mainly has sour taste and odor. Compared with the yeast filtrate, the aldehyde content of the lactobacillus filtrate after decolorization and deodorization is lower, and the content of carboxylic acids such as isobutyric acid and isovaleric acid is also relatively low. The content of alcohol substances represented by phenylethanol is also lower than that of the yeast filtrate. Combined with the sensory evaluation results, the sensory aspects of both filtrates are greatly improved after decolorization. Compared with the yeast filtrate, the lactobacillus filtrate also shows a similar odor.

[0115] Example 5 Method 4

[0116] Sawdust (pine, fir or a 1:1 mixture of the two) powdered activated carbon and coconut shell powdered activated carbon are added to the above-mentioned fermentation liquid semi-finished product for decolorization and deodorization. The particle size of the two activated carbons is 300-500 mesh, and the addition amounts are 0.4% and 1.6% (w / w) respectively. The decolorization and deodorization stirring rate is 50-200rpm, the decolorization and deodorization process temperature is controlled at 20-30°C, and the decolorization and deodorization time is 0.5-3 hours.

[0117] After the decolorization and deodorization process is completed, the temperature of the decolorization and deodorization tank is controlled at 0-10°C, and the decolorization and deodorization equipment is connected to the filtering equipment. The filtering equipment can select a positive pressure filter (0.1-0.2μm, the membrane material is polypropylene or nylon or polyethersulfone), a plate centrifuge (600-800 mesh, the material is nylon or polyester or polypropylene), a bag filter (600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), a plate and frame filter (0.1-0.2μm filter membrane, the material is nylon or polypropylene or polyethersulfone; 600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), and a disc centrifuge (5000-6000rpm, preferably 5300rpm, flow rate 400L / h, slag discharge time 200-500s, preferably 300s).

[0118] Table 12 Sensory evaluation index of fermented liquid obtained by decolorization and deodorization method 4

[0119]

[0120]

[0121] Table 13 Sensory index of fermented liquid obtained by decolorization and deodorization method 4

[0122]

[0123] According to the results of physical and chemical tests and sensory evaluation, the fermentation filtrate obtained by this method is light in color and has a light odor. Therefore, the odor components were compared with those of the untreated fermentation filtrate by HPSPME-GC-MS. The results are as follows:

[0124] Table 14 Comparison of odor components of fermentation liquid before and after treatment

[0125]

[0126]

[0127] According to the above table, the treatment of method 4 has a good adsorption effect on the characteristic odor components of soy sauce flavor such as aldehydes, phenols, and heterocyclics in the yeast filtrate, and the content of carboxylic acids is also significantly reduced. It also has a good adsorption effect on alcohol substances such as phenylethanol.

[0128] 3. Detection of amino acid content in fermentation broth

[0129] Liquid chromatography-mass spectrometry (LC-MS) was used to test the active substance content of the same sterilized fermentation broth after different activated carbon treatments. The results are as follows:

[0130] Table 15 Active substance content of yeast fermentation broth after treatment with different activated carbons

[0131]

[0132]

[0133] According to the test results of the above amino acid content, after different activated carbon treatments, the change in the amino acid content of the fermentation broth is not proportional to the change in the solid content. It can be seen that the adsorption efficiency of the active substance content of the fermentation broth is lower than that of the chromogenic substance and other odor substances. In addition, the glutamic acid content of the yeast fermentation broth before and after the above activated carbon treatment is the highest, which is the main odor substance of the soy sauce flavor. The content of aspartic acid, isoleucine, valine, etc. is relatively high, and these substances are also the main contributors to the salty and fresh taste of soy sauce.

[0134] Example 6 Methods 5-8

[0135] According to the ratio in Table 16, add wood (pine, fir) powdered activated carbon, coconut shell powdered activated carbon or coal powdered activated carbon to the sterilized fermentation filtrate semi-finished product, and compound it for decolorization and deodorization. The particle size of the activated carbon is 300-500 mesh. Decolorization and deodorization process: stirring rate 50-200rpm, temperature control at 20-30℃, time 0.5-3 hours.

[0136] After the decolorization and deodorization process is completed, the temperature of the decolorization and deodorization tank is controlled at 0-10°C, and the decolorization and deodorization equipment is connected to the filtering equipment. The filtering equipment can select a positive pressure filter (0.1-0.2μm, the membrane material is polypropylene or nylon or polyethersulfone), a plate centrifuge (600-800 mesh, the material is nylon or polyester or polypropylene), a bag filter (600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), a plate and frame filter (0.1-0.2μm filter membrane, the material is nylon or polypropylene or polyethersulfone; 600-800 mesh, the material is nylon or polyester or polypropylene, and the filtering pressure is 2.0-6.0bar), and a disc centrifuge (5000-6000rpm, preferably 5300rpm, flow rate 400L / h, slag discharge time 200-500s, preferably 300s).

[0137] After decolorization and deodorization, the filtrate was evaluated for physical and chemical parameters and sensory evaluation. The results are as follows:

[0138] Table 16 Effect of different types and proportions of activated carbon on compounding results

[0139]

[0140] At the same amount of activated carbon added, only adding coal powder activated carbon (method 5) will make it difficult to remove the final sour smell of the product, and a salty smell will appear; reducing the addition of coal powder activated carbon and replacing part of it with wood powder activated carbon (method 6), the deodorization effect will be significantly improved, but the fermentation smell and soy sauce smell are still heavy.

[0141] At the same amount of activated carbon added, only adding wood powder activated carbon (method 7) can improve the soy sauce taste and fermentation taste, but the effect is still not ideal; after replacing part of the wood powder activated carbon with coconut shell powder activated carbon (method 8), the deodorization effect will be significantly improved.

[0142] The above method can process several tens of kilograms to ten tons of sterilized fermentation liquid each time, and is suitable for industrial-scale production.

[0143] Example 7

[0144] If the fermentation product is to be used as a cosmetic raw material, a certain proportion of preservatives needs to be added to the finished product. Use the fermentation stock solution of lactobacillus and yeast obtained by fermentation or the filtrate obtained in Example 4, and add preservatives thereto. The method is as follows:

[0145] Take 2 portions of the fermentation filtrate obtained in step 1.5 or the decolorized and deodorized fermentation broth obtained in Example 4 (method 3), and add 0.15-2% of aromatic alcohol preservatives such as benzyl alcohol, dichlorobenzyl alcohol, phenylethyl alcohol, phenylpropanol, phenoxyethanol or phenoxyisopropanol to one portion according to the requirements of the "Technical Specifications for Safety of Cosmetics (2015 Edition)" to obtain product A; add 2-8% of polyols to the other portion, the polyols can be one or more of 1,3-propylene glycol, 1,2-pentanediol, 1,2-butanediol, 1,2-hexanediol, caprylyl glycol, decanediol, etc., and 0.01-0.5% of benzoic acid and its salts and esters to obtain product B. The above addition amounts are mass percentages.

[0146] Taking the product obtained by obtaining the decolorized and deodorized filtrate of lactobacillus obtained in Example 4 as an example, sensory evaluation was performed on the different products obtained, and the results are as follows:

[0147] Table 17

[0148]

[0149] The sensory evaluation of product B made from the yeast decolorization and deodorization filtrate was the same as that of the decolorization and deodorization filtrate; while the soy sauce taste of product A was stronger than that of the filtrate.

[0150] Products A and B obtained from the fermentation filtrate obtained in step 1.5 have strong fermentation, sour and soy sauce smells, which result in poor user experience and cannot be used.

[0151] From the evaluation results, it can be seen that when the fermentation product remains unchanged, the use of different preservative systems will also affect the final smell of the fermentation product. Aromatic alcohols will cause the final soy sauce flavor of the product to be prominent, which is the result of the synergistic production of odor substances, while benzoic acid and its salts and esters, combined with polyols, will retain the original smell of the fermentation product to the greatest extent.

[0152] Example 8 Efficacy detection of fermentation products

[0153] 1. Anti-aging

[0154] The epidermal model was constructed using P1 generation KC and cultured for 18 days. Samples were added on the 6th day of culture. The untreated group was the NT group, and the group with 0.1 mg / mL yeast fermentation product (in terms of solid content, the same below) treated by Example 4, i.e., method 3, was the sample group. HE staining and IHC Ki67 staining were performed after the culture to explore the effect of yeast fermentation products on the 3D skin model. The staining results were photographed using a Scope A1 microscope and statistically analyzed. The results are shown in Figure 1 and Figure 2 .

[0155] Ki67 is a marker protein for the proliferation ability of human skin cells and is expressed in the basal layer. Figure 1 The results of IHC staining of the epidermal model showed that there was obvious expression in the basal layer of the epidermal model using yeast fermentation products, and the expression level was significantly higher than that of the control group, indicating that the epidermal model using 0.1 mg / mL yeast fermentation products had stronger proliferation activity. Figure 2 The quantitative analysis results showed that the relative positive rate of the sample group was 41%, and the difference was statistically significant. This shows that by observing the expression of Ki67, it can be concluded that yeast fermentation products can indeed enhance the proliferation of the basal layer after use, thereby playing an anti-aging role.

[0156] 2. Anti-inflammatory

[0157] The 3D full-thickness skin model constructed by keratinocytes and fibroblasts has functions similar to normal human skin and is an important tool for evaluating anti-inflammatory ability. The anti-inflammatory function of yeast fermentation products was evaluated using an in vitro reconstructed 3D full-thickness skin model. The total culture period was 46 days, with 28 days of dermal culture and 5 days of immersion culture. On the 21st day, 0.5 mg / mL of yeast fermentation products were added, and UVA5J&UVB0.05J irradiation was performed on the 43rd day for 3 consecutive days. A control group and a model group were set up, with 3 samples in each group. Immunohistochemistry and immunofluorescence staining were performed on the full-thickness skin model.

[0158] Hematoxylin-eosin (HE) staining was used to analyze the tissue structure of the cultured 3D full-thickness skin model. Figure 3 As shown, UBA&UVB irradiation can cause epidermal thinning. Immunofluorescence staining was used to analyze the expression of IL-1α in the 3D full-thickness skin model. The immunofluorescence analysis method was to take pictures of the control group, model group, and sample group at the same exposure and background, and then use Image-Pro Premier 9.2 to analyze the optical density of the pictures. The statistical method t-test was used, and p<0.05 was marked with "*".

[0159] IL-1α, a member of the interleukin (IL)-1 family, is a ubiquitous and key proinflammatory cytokine. IL-1α is central to the pathogenesis of many diseases characterized by organ or tissue inflammation. Figure 3 It can be seen that UV irradiation significantly increased the expression of IL-1α in the epidermis and dermis of the skin model, while the addition of 0.5 mg / mL yeast fermentation product can significantly reduce the expression of IL-1α in the dermis and epidermis caused by UV irradiation, reducing it by 6.80% and 14.16%, respectively ( Figure 4 ), indicating that yeast fermentation products have anti-inflammatory effects.

[0160] The above are preferred embodiments of the present invention, which are used to illustrate the technical concept and features of the present invention. The purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A post-treatment method for microbial fermentation products, characterized in that: Either method a or method b: Method a: After microbial fermentation, activated carbon is added to the fermentation filtrate or supernatant obtained by removing the bacterial cells, and the activated carbon is removed by filtration or centrifugation after stirring; Method b: The activated carbon is loaded into a filtering device, and the fermentation filtrate or supernatant obtained by removing the bacterial cells after microbial fermentation is filtered using the filtering device; The microorganisms are yeast, fungi and bacteria.

2. The post-processing method according to claim 1, characterized in that: The microorganism is Pichia pastoris, Bacillus, Escherichia coli, Bifidobacterium, Pediococcus, Lactobacillus, Lactococcus, Streptococcus or Aspergillus.

3. The post-processing method according to claim 1, characterized in that: The amount of activated carbon used is 1wt%-5wt% of the fermentation filtrate or supernatant.

4. The post-processing method according to claim 1, characterized in that: The activated carbon is one of wood activated carbon, coconut shell activated carbon and coal activated carbon or a mixture thereof, and its particle size is 10-500 meshes.

5. The post-processing method according to claim 1, characterized in that: The particle size of the activated carbon is 100-500 meshes.

6. The post-processing method according to claim 1 or 4, characterized in that: The activated carbon is a mixture of coconut shell activated carbon and coal-made activated carbon; or a mixture of wood activated carbon and coconut shell activated carbon.

7. The post-processing method according to claim 1, characterized in that: The activated carbon is a mixture of coconut shell activated carbon and coal-based activated carbon, with a mass ratio of 1:0.5-10; or the activated carbon is a mixture of coal-based activated carbon and coconut shell activated carbon, with a mass ratio of 1:1-10.

8. The post-processing method according to claim 1, characterized in that: The filtering conditions described in method a and method b are: filtering pressure 2.0 to 6.0 bar.

9. A microbial fermentation product, characterized in that: Obtained by the post-treatment method according to any one of claims 1 to 7.

10. The microbial fermentation product of claim 9 is used for preparing cosmetics, external skin preparations, oral beauty preparations, foods or food additives.

11. A cosmetic or skin external preparation, characterized in that: Contains the microbial fermentation product according to claim 9.

12. The cosmetic or skin external preparation according to claim 10, characterized in that: It also contains a preservative, which is a mixture of benzoic acids and polyols.

13. A food, food additive or cosmetic oral preparation, characterized in that: Contains the microbial fermentation product according to claim 9.

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