Maotai-flavor vinasse fermentation product as well as preparation method and application thereof

Abstract

The invention discloses a Maotai-flavor vinasse fermentation product as well as a preparation method and application thereof, relates to the technical field of biology, and is used for solving at least one of the problems that the Maotai-flavor vinasse is difficult to be used in the field of cosmetics due to the fact that a leaching solution of the existing Maotai-flavor vinasse is dark in color and the Maotai-flavor vinasse is heavy in smell, and sensory experience is affected. According to the method, lactobacillus plantarum X028 and / or lactobacillus paracasei X016 are / is adopted to ferment the Maotai-flavor vinasse, the obtained initial fermentation mixed solution is subjected to the steps of centrifugation, filtration, decoloration, sterilization and the like, and the final Maotai-flavor vinasse fermentation product is obtained. The application of the Maotai-flavor vinasse in the field of cosmetics can be realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a fermentation product of soy sauce-flavored distiller's grains, its preparation method, and its application. Background Technology

[0002] The term "sauce-aroma type distiller's grains" mainly refers to the residue left after nine rounds of steaming, eight rounds of fermentation, and seven rounds of distillation during the production of sauce-aroma type liquor. It primarily originates from sauce-aroma type liquor production enterprises in Guizhou Province. The main components of sauce-aroma type distiller's grains include crude starch, sugar, protein, cellulose, and ash. In addition, relevant data shows that sauce-aroma type distiller's grains contain 16 hydrolyzed amino acids, including 7 essential amino acids, with a total amino acid content of 16.8% and an essential amino acid content of 5.9%. Sauce-aroma type distiller's grains are also rich in trace elements such as iron, manganese, and zinc.

[0003] Currently, the main applications of soy sauce-flavored distiller's grains are in the food and livestock industries. They are used as raw materials for brewing soy sauce and vinegar, animal feed, and edible fungi culture media, but their high-value and specialized utilization has not been achieved. Physicochemical analysis shows that soy sauce-flavored distiller's grains are rich in nutrients and have four main characteristics: ① High solids content, averaging 45-55%; ② High organic matter content, rich in protein, cellulose, starch, etc.; ③ Rich in nitrogen, phosphorus, potassium, calcium, and various trace elements; ④ Rich in organic acid salts, such as lactate and acetate, resulting in a rich nutritional profile. Therefore, developing application technologies for soy sauce-flavored distiller's grains in the cosmetics field can better realize their high-value and specialized utilization.

[0004] However, the dark color of the fermented grains and their extracts, as well as the presence of various microorganisms such as Bacillus and yeast during the brewing process, limit their application in cosmetics. Summary of the Invention

[0005] Based on the above analysis, the present invention aims to provide a fermented product of soy sauce-flavored lees, its preparation method and application, in order to solve at least one of the following problems: the extract of existing soy sauce-flavored lees is dark in color and has a strong odor, which affects the sensory experience and makes it difficult to use soy sauce-flavored lees in the cosmetic field.

[0006] In a first aspect, the present invention provides a fermentation method for a soy sauce-flavored liquor lees fermentation product, specifically comprising the following steps:

[0007] Step 1: Mix the soy sauce-flavored distiller's grains, glucose, yeast extract powder, and water, adjust the pH value, and sterilize to obtain the fermentation culture medium;

[0008] Step 2: Inoculate the fermentation medium with Lactobacillus plantarum X028 bacterial solution and / or Lactobacillus paracasei X016 bacterial solution, introduce sterile air, ferment under positive pressure and stirring conditions to obtain the initial fermentation mixture;

[0009] Step 3: Centrifuge the initial fermentation mixture and collect the supernatant;

[0010] Step 4: Filter the supernatant from Step 3 and collect the filtrate;

[0011] Step 5: Decolorize the filtrate to obtain a decolorized filtrate;

[0012] Step 6: Sterilize the decolorized filtrate to obtain a soy sauce-flavored fermented liquor product.

[0013] Furthermore, in step 1, according to the following weight parts, the following are also included: 10-20 parts of soy sauce-flavored lees, 0.1-0.5 parts of glucose, 0.1-0.5 parts of yeast extract, and 79.6-89.8 parts of water.

[0014] Furthermore, in step 1, the pH value is adjusted to 5-6.

[0015] Furthermore, in step 2, the preservation number of Lactobacillus plantarum X028 is CGMCC No. 33256, and the preservation number of Lactobacillus paracasei X016 is CGMCC No. 33255.

[0016] Furthermore, in step 2, the viable count of the *Lactobacillus plantarum* X028 bacterial solution is 1-5 × 10⁻⁶. 8 CFU / mL, inoculum size 2-5%; viable count of Lactobacillus paracasei X016 in the bacterial culture 1-5 × 10⁻⁶ 8 CFU / mL, inoculation amount 3-5%.

[0017] Furthermore, in step 2, when both Lactobacillus plantarum X028 and Lactobacillus paracasei X016 bacterial solutions are added simultaneously, the volume ratio of Lactobacillus plantarum X028 to Lactobacillus paracasei X016 is 1-2:1.

[0018] Furthermore, in step 2, the fermentation temperature is 30-37℃ and the fermentation time is 48-72h.

[0019] Furthermore, in step 6, the sterilization temperature is 80-85℃ and the sterilization time is 15-25 minutes.

[0020] Secondly, the present invention provides a soy sauce-flavored fermented product prepared by the above method.

[0021] Thirdly, the present invention provides an application of the above-mentioned soy sauce-flavored fermented liquor products in the preparation of cosmetics.

[0022] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0023] 1. This invention uses Lactobacillus plantarum X028 and / or Lactobacillus paracasei X016 to ferment soy sauce-flavored lees. The initial fermentation mixture is centrifuged, filtered, decolorized, and sterilized to obtain the final soy sauce-flavored lees fermentation product. The soy sauce-flavored lees fermentation product obtained by this invention has good soothing and moisturizing effects, and can realize the application of soy sauce-flavored lees in the cosmetic field.

[0024] 2. In this invention, the growth of *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016 is limited under the acidic conditions of soy sauce-flavored distiller's grains. Therefore, in order to increase the number of viable bacteria of *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016, sodium hydroxide solution is used to adjust the pH value to 5-6, so that *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016 can achieve a higher proliferation rate.

[0025] 3. The method of the present invention ensures the removal of residual microorganisms in the fermented mash of soy sauce-flavored liquor, as well as microorganisms introduced during the fermentation process, through two sterilization processes in steps 1 and 6; the fermentation process adopts positive pressure conditions in the tank to minimize the introduction of microorganisms; by maintaining aseptic conditions during the fermentation process, the normal growth of Lactobacillus plantarum X028 and Lactobacillus paracasei X016 is ensured, thereby guaranteeing fermentation quality and fermentation efficiency.

[0026] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0027] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0028] Figure 1 This refers to the fermented product of soy sauce-flavored distiller's grains obtained by the method in Example 1 of this invention;

[0029] Figure 2 This is a color comparison diagram of the filtrate before and after decolorization in step 5 of the method in Embodiment 1 of the present invention. Detailed Implementation

[0030] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0031] The Lactobacillus plantarum X028 strain in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 33256 and deposit date of December 30, 2024.

[0032] The Lactobacillus paracasei X016 strain in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 33255 and deposit date of December 30, 2024.

[0033] In existing technologies, the high acidity of soy sauce-flavored fermented grains makes them unsuitable for the growth of common bacterial strains, requiring the selection of specific fermentation strains; the dark color of the extract makes it unsuitable for products requiring specific color characteristics, thus limiting its application range; and the strong odor of the grains negatively impacts sensory experience and acceptance. Therefore, existing technologies for using soy sauce-flavored fermented grains in the cosmetics industry are unsuitable.

[0034] A specific embodiment of the present invention discloses a fermentation method for a soy sauce-flavored liquor lees fermentation product, which specifically includes the following steps:

[0035] Step 1: Mix the soy sauce-flavored distiller's grains, glucose, yeast extract powder, and water, adjust the pH value, and sterilize to obtain the fermentation culture medium;

[0036] Step 2: Inoculate the fermentation medium with Lactobacillus plantarum X028 bacterial solution and / or Lactobacillus paracasei X016 bacterial solution, and introduce sterile air to maintain positive pressure for fermentation to obtain the initial fermentation mixture;

[0037] Step 3: Centrifuge the initial fermentation mixture and collect the supernatant;

[0038] Step 4: Filter the supernatant from Step 3 and collect the filtrate;

[0039] Step 5: Decolorize the filtrate to obtain a decolorized filtrate;

[0040] Step 6: Sterilize the decolorized filtrate to obtain a soy sauce-flavored fermented liquor product.

[0041] Compared with the prior art, the present invention uses Lactobacillus plantarum X028 and / or Lactobacillus paracasei X016 to ferment the fermented lees of soy sauce-flavored liquor. The initial fermentation mixture is then subjected to centrifugation, filtration, decolorization, sterilization and other steps to obtain the final fermented product of soy sauce-flavored liquor. The fermented product of soy sauce-flavored liquor obtained by the present invention has good soothing and moisturizing effects, and can realize the application of soy sauce-flavored liquor in the field of cosmetics.

[0042] The beneficial effects achieved by the above preparation method are as follows: the two-step sterilization process in steps 1 and 6 ensures the removal of residual microorganisms in the fermented mash, as well as microorganisms introduced during fermentation; the fermentation process adopts positive pressure conditions to minimize the introduction of microorganisms; through centrifugation, filtration, decolorization and other processes, the original and generated solids in the fermentation system are effectively removed, and the color of the fermentation product is improved, resulting in a fermented product of fermented mash with soothing and moisturizing effects, realizing the fermentation of fermented mash for use in the cosmetics field.

[0043] Specifically, in step 1, the following proportions by weight are used: 10-20 parts of soy sauce-flavored distiller's grains, 0.1-0.5 parts of glucose, 0.1-0.5 parts of yeast extract, and 79.6-89.8 parts of water.

[0044] Specifically, the weight parts of the soy sauce-flavored distiller's grains are, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts; the weight parts of glucose are, for example, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.5 parts; and the weight parts of yeast extract are, for example, 0.1, 0.15, or 0.20 parts. 0.25 parts, 0.30 parts, 0.35 parts, 0.40 parts, 0.45 parts, 0.5 parts, the weight parts of water, for example, 79.6 parts, 80 parts, 80.5 parts, 81 parts, 81.5 parts, 82 parts, 82.5 parts, 83 parts, 83.5 parts, 84 parts, 84.5 parts, 85 parts, 85.5 parts, 86 parts, 86.5 parts, 87 parts, 87.5 parts, 88 parts, 88.5 parts, 89 parts, 89.8 parts;

[0045] Preferably, the ingredients are: 15 parts of soy sauce-flavored distiller's grains, 0.3 parts of glucose, and 0.3 parts of yeast extract.

[0046] It should be noted that too many fermentation tanks in sauce-flavored liquor can affect the efficiency of fermentation and subsequent effects.

[0047] Specifically, in step 1, an aqueous solution of sodium hydroxide is used to adjust the pH value to 5-6, such as 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6; the preferred pH value is 5.6.

[0048] It should be noted that the advantage of using a pH range of 5-6 in this invention is that it satisfies the growth requirements of the fermentation strains while avoiding excessively high salt content in the fermentation products. Too high a pH requires the addition of more sodium hydroxide, resulting in excessively high salt content in the fermentation products, while too low a pH inhibits the growth of the fermentation strains. The purpose of this invention is to address the odor of the fermentation tanks. For fermentation to proceed, excessive acidity is detrimental; therefore, this invention adjusts the pH to 5-6.

[0049] Specifically, the mass concentration of the sodium hydroxide aqueous solution is 1%-5%, such as 1%, 2%, 3%, 4%, or 5%, preferably 3%.

[0050] Specifically, in step 1, the sterilization temperature is 115-118℃, such as 115℃, 116℃, 117℃, or 118℃, preferably 116℃; excessively high sterilization temperatures will lead to the loss of nutrients in the culture medium; the sterilization time is 15-20 minutes, such as 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes, preferably 17 minutes.

[0051] Specifically, in step 2, the positive pressure of the tank is 0.02-0.05 MPa, such as 0.02 MPa, 0.03 MPa, 0.04 MPa, or 0.05 MPa, preferably 0.03 MPa; excessive positive pressure can cause stress response and damage to enzyme activity, inhibiting cell growth.

[0052] Specifically, in step 2, the stirring speed is 80-120 rpm. For example, 80 rpm, 85 rpm, 90 rpm, 95 rpm, 100 rpm, 105 rpm, 110 rpm, 115 rpm, 120 rpm, with 90 rpm being the preferred stirring speed. Excessive stirring speed can cause physical damage to cells and produce foam, etc.

[0053] Specifically, in step 2, the fermentation temperature is 30-37℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, or 37℃, preferably 34℃; excessively high fermentation temperatures will inhibit cell growth. The fermentation time is 48-72 hours, such as 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, or 72 hours, preferably 66 hours.

[0054] Specifically, the viable count in the *Lactobacillus plantarum* X028 bacterial solution is 1-5 × 10⁻⁶. 8 CFU / mL, for example, 1×10 8 CFU / mL, 2×10 8CFU / mL, 3×10 8 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL; preferably 2×10 8 CFU / mL;

[0055] It should be noted that the viable count of *Lactobacillus plantarum* X028 selected in this invention is 1-5 × 10⁻⁶. 8 The concentration of CFU / mL should be within this range to ensure normal cell growth. Too low a concentration will result in an excessively long cell growth arrest phase, potentially leading to contamination by other microorganisms. Too high a concentration will cause cells to grow too quickly, shorten the fermentation cycle, and prematurely enter the death phase.

[0056] Specifically, the viable count in the Lactobacillus paracasei X016 bacterial culture is 1-5 × 10⁻⁶. 8 CFU / mL, for example, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 3×10 8 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL; preferably 2×10 8 CFU / mL;

[0057] When only *Lactobacillus plantarum* X028 bacterial culture is selected, the inoculum concentration is 2-5% (volume fraction), such as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%; preferably 3%. Insufficient inoculum concentration will result in an excessively long cell growth arrest phase, potentially leading to contamination by other microorganisms. Insufficient inoculum concentration will cause excessively rapid cell growth, shorten the fermentation cycle, and prematurely induce cell death.

[0058] When only Lactobacillus paracasei X016 bacterial suspension is selected, the inoculum amount is 3-5% (volume fraction), such as 3%, 3.5%, 4%, 4.5%, 5%; preferably 4%.

[0059] When two bacterial solutions are selected, the volume ratio of *Lactobacillus plantarum* X028 to *Lactobacillus paracasei* X016 is 1-2:1, such as 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1; preferably 1.6:1. When the volume ratio of *Lactobacillus plantarum* X028 to *Lactobacillus paracasei* X016 is in the range of 1-2:1, if the simultaneous growth and fermentation of the two solutions is outside the range of this application, it will result in the growth of only one bacterium, while the other bacterium cannot grow normally.

[0060] When the bacterial solution is a mixed bacterial solution, the inoculation amount is 2-5% (volume fraction), such as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%; preferably 3%.

[0061] It should be noted that the bacterial solution described in this invention is preferably a mixed bacterial solution of Lactobacillus plantarum X028 and Lactobacillus paracasei X016; using a mixed bacterial solution has the following advantages:

[0062] 1. *Lactobacillus plantarum* X028 can ferment various sugars (such as glucose, fructose, and maltose), while *Lactobacillus paracasei* X016 prefers lactose and galactose. Since the fermented mash of soy sauce-flavored liquor contains abundant nutrients such as starch, sugars, and proteins, which can serve as carbon and nitrogen sources during fermentation, using a mixed culture of *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016 allows for more efficient utilization of the mixed carbon source, reduces substrate competition, and improves fermentation efficiency.

[0063] 2. *Lactobacillus plantarum* X028 has a strong ability to produce lactic acid (homogeneous fermentation), while *Lactobacillus paracasei* X016 may produce acetic acid or other metabolites (heterogeneous fermentation). When used together, they can produce a synergistic effect, accelerating acidification and inhibiting other microorganisms.

[0064] 3. Enzyme Complementarity: Proteases (such as peptidases) secreted by *Lactobacillus plantarum* X028 can break down proteins into small peptides, which *Lactobacillus paracasei* X016 further utilizes to convert into amino acids. Therefore, fermentation using a mixed culture of *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016 can accelerate the fermentation process. Furthermore, the complementary nature of their enzyme systems can increase the peptide and amino acid content in the fermentation products.

[0065] It is worth noting that the preparation method of the Lactobacillus plantarum X028 bacterial solution is as follows: the Lactobacillus plantarum X028 seed solution is diluted to the corresponding concentration using sterile physiological saline to obtain the Lactobacillus plantarum X028 bacterial solution.

[0066] The method for preparing the Lactobacillus paracasei X016 bacterial suspension is as follows: using sterile physiological saline, the Lactobacillus paracasei X016 seed liquid is diluted to the corresponding concentration to obtain the Lactobacillus paracasei X016 bacterial suspension.

[0067] Furthermore, the method for preparing the *Lactobacillus plantarum* X028 seed culture is as follows:

[0068] S1: Take Lactobacillus plantarum X028 from the glycerol preservation tube stored at -80℃, drop it onto an MRS agar plate and streak it. Cultivate until a single colony grows to obtain the plate-revitalized and activated primary Lactobacillus plantarum X028 seed.

[0069] S2: In order to improve fermentation efficiency and the quality of fermentation products, select 5-10 fully grown and uniformly sized single colonies of this bacterium and inoculate them into MRS liquid culture medium. After static culture, obtain Lactobacillus plantarum X028 seed culture.

[0070] Specifically, in S1, the streaking process is carried out by streaking individual bacteria separately until a single colony grows, in order to ensure the stability of subsequent fermentation.

[0071] Specifically, in S1, the culture conditions for growing a single colony are 32℃ for 36-48 hours.

[0072] Specifically, in S2, the static culture conditions are: static culture at 32℃ for 18-26 hours.

[0073] The method for preparing Lactobacillus paracasei X016 seed culture in this invention is the same as the method for preparing Lactobacillus plantarum X028 seed culture, except that Lactobacillus paracasei X016 in a glycerol preservation tube stored at -80°C is taken by pipette.

[0074] Specifically, in step 3, the centrifugation process uses a tubular centrifuge at 16000-19000 r / min, such as 16000 r / min, 17000 r / min, 18000 r / min, or 19000 r / min, preferably 17000 r / min. Since the fermented mash of soy sauce-flavored liquor contains a large amount of organic matter, a large amount of solids are produced during the fermentation process. Therefore, the solids produced during the fermentation process can be effectively separated from the supernatant by using a tubular centrifuge at 16000-19000 r / min.

[0075] During the centrifugation process, each centrifugation session lasts 20-30 minutes, such as 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or 30 minutes, preferably 24 minutes. A centrifugation time of 20-30 minutes achieves the desired centrifugation effect without excessive energy consumption. The number of centrifugations is 1-3 times, such as 1 time, 2 times, or 3 times, preferably 2 times. Through the centrifugation process, the solid-liquid separation effect is achieved.

[0076] Specifically, in step 4, the filtration process uses a cellulose acetate membrane, PP membrane, or paperboard with a filter membrane pore size of 0.1-0.5μm, such as 0.1μm, 0.12μm, 0.22μm, 0.45μm, or 0.5μm; the preferred filter membrane pore size is 0.45μm; and the preferred material is PP membrane.

[0077] The filtration process is performed 1-3 times, such as 1 time, 2 times, or 3 times; preferably 2 times; through filtration, a further solid-liquid separation effect is achieved.

[0078] Specifically, in step 5, the decolorization treatment uses macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:2-4, such as 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, preferably 1:3. When the material ratio of macroporous adsorption resin to filtrate is 1:2-4, it can ensure decolorization efficiency without causing excessive energy consumption. However, this is outside the scope of the present invention, as it would result in low decolorization efficiency or increased energy consumption.

[0079] Specifically, in step 6, the sterilization temperature is 80-85℃, such as 80℃, 82℃, 83℃, 84℃, or 85℃; preferably 82℃. Excessively high temperatures will reduce the yield of effective substances such as polyphenols and weaken the antioxidant properties. The sterilization time is 15-25 minutes, such as 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, or 25 minutes; preferably 20 minutes.

[0080] Specifically, in step 6, the total bacterial count in the fermented product of the soy sauce-flavored liquor is ≤500 cfu / g, and the mold and yeast count is ≤10 cfu / g.

[0081] The fermented product of soy sauce-flavored lees prepared by the method of the present invention can effectively improve the original lees odor of soy sauce-flavored lees and contains high levels of organic acids and polyphenols. The fermented product of soy sauce-flavored lees prepared by the present invention shows significantly improved DPPH scavenging rate, ABTS free radical scavenging rate, and TNF-α inhibition rate, demonstrating good antioxidant and soothing effects. Human experimental results show that the skin hydration level is significantly higher than that of the control group. Therefore, the fermented product of soy sauce-flavored lees prepared by the method of the present invention has a good moisturizing effect.

[0082] The technical solution of the present invention will be explained below with reference to specific embodiments.

[0083] Description of raw materials, reagents, materials, and microorganisms involved in the embodiments and comparative examples of this invention:

[0084] Sauce-flavored liquor lees: The lees remaining after brewing sauce-flavored base liquor from Guizhou Maotai Distillery (Group) Circular Economy Industry Investment and Development Co., Ltd., with a pH value of 3.7.

[0085] Glucose: Purchased from Xiwang Pharmaceutical Co., Ltd., named glucose monohydrate (food grade);

[0086] Yeast extract powder: Purchased from Angel Yeast Co., Ltd., named Angel Yeast Extract Powder;

[0087] Fiber acetate film, PP film or paperboard: purchased from Shanghai Xulun Environmental Protection Technology Co., Ltd.;

[0088] Macroporous adsorption resin: purchased from Zhejiang Zhengguang Industrial Co., Ltd.

[0089] Preparation Example 1

[0090] Isolation of Lactobacillus plantarum X028 and Lactobacillus paracasei X016:

[0091] Acid-producing strains were screened from 12 samples of soy sauce-flavored liquor lees from different production areas in Guizhou using MRS+BCP (bromocresol purple) / CaCO3 screening medium.

[0092] The specific separation method is as follows: 10g of soy sauce-flavored distiller's grains sample was placed in 90g of sterile physiological saline, shaken evenly, and diluted 10g using a serial dilution method. -1 -10 -6 Take 1 mL of each gradient and spread it on an MRS+BCP / CaCO3 plate. Incubate at 32℃ for 48 h. Pick single colonies with yellow circles / clear circles around the colonies and purify them repeatedly to obtain a single strain.

[0093] Identification of Lactobacillus plantarum X028 and Lactobacillus paracasei X016:

[0094] A total of 39 acid-producing strains were identified through screening. Morphological analysis combined with 16S rDNA amplification and sequencing, followed by sequence alignment using NCBIBLAST, yielded 8 lactic acid bacteria strains.

[0095] Lactic acid bacteria were inoculated into 10% of soy sauce-flavored distiller's grains (pH adjusted to 5.5, live lactic acid bacteria count 2 × 10⁻⁶). 8 Based on the combined acid production capacity experiment and growth capacity test, two lactic acid bacteria strains capable of growing in distiller's grains were obtained: *Lactobacillus plantarum* X028 (serial number 1) and *Lactobacillus paracasei* X016 (serial number 2). The remaining strains did not grow in distiller's grains. Specific results are shown in Table 1 below.

[0096] Table 1

[0097] Serial Number strain name viable cell count (cfu / g) pH 1 Lactobacillus plantarum <![CDATA[7.9×10 8 ]]> 3.92 2 Lactobacillus paracasei <![CDATA[5.8×10 8 ]]> 4.04 3 Lactobacillus acidophilus <![CDATA[0.68×10 6 ]]> 4.28 4 Lactobacillus bulgaricus <![CDATA[0.92×10 6 ]]> 4.32 5 Lactobacillus casei <![CDATA[1.01×10 6 ]]> 5.43 6 Lactococcus lactis <![CDATA[0.76×10 6 ]]> 5.52 7 Pediococcus pentosaceus <![CDATA[0.85×10 6 ]]> 5.45 8 Digestive lactobacilli <![CDATA[0.77×10 6 ]]> 5.43

[0098] Preparation Example 2

[0099] The preparation method of Lactobacillus plantarum X028 seed culture is as follows:

[0100] S1: Take 20 μL of *Lactobacillus plantarum* X028 selected in Preparation Example 1 from a glycerol storage tube stored at -80℃ and streak it onto an MRS agar plate. To ensure the stability of subsequent fermentation, streak individual bacteria separately during the streaking process. Incubate at 32℃ for 36-48 h until a single colony grows, thus obtaining the plate-revitalized and activated primary *Lactobacillus plantarum* X028 seed.

[0101] S2: In order to improve fermentation efficiency and the quality of fermentation products, eight fully grown and uniformly sized single colonies of this bacterium were selected and inoculated into 200 mL of MRS liquid medium and cultured at 32℃ for 18-26 h to obtain Lactobacillus plantarum X028 seed culture.

[0102] Preparation Example 3

[0103] The preparation method of Lactobacillus paracasei X016 seed culture is the same as that of Lactobacillus plantarum X028 in Preparation Example 2. The difference is that Lactobacillus paracasei X016 selected in Preparation Example 1 was taken from a glycerol preservation tube stored at -80°C using a pipette.

[0104] Preparation Example 4

[0105] The preparation methods for Lactobacillus plantarum X028 bacterial suspension and Lactobacillus paracasei X016 bacterial suspension are as follows:

[0106] Lactobacillus plantarum X028 bacterial culture: The Lactobacillus plantarum X028 seed culture obtained in Preparation 2 was diluted to the appropriate concentration (1-5×10⁻⁶) using sterile 0.9% NaCl. 8 The bacterial culture of Lactobacillus plantarum X028 was obtained by mixing CFU / mL.

[0107] Lactobacillus paracasei X016 bacterial culture: The Lactobacillus paracasei seed culture obtained in Preparation Example 3 was diluted to the appropriate concentration (1-5×10⁻⁶) using sterile 0.9% NaCl. 8 The bacterial culture of Lactobacillus paracasei X016 was obtained by mixing CFU / mL.

[0108] The ratio of live bacteria count of Lactobacillus plantarum X028 to Lactobacillus paracasei X016 is 1-2:1. The preparation method is to mix 1-2 volumes of Lactobacillus plantarum X028 with 1 volume of Lactobacillus paracasei X016.

[0109] Example 1

[0110] The fermentation method of a soy sauce-flavored liquor lees fermentation product according to this embodiment includes the following steps:

[0111] Step 1: According to the weight parts, mix 15 parts of soy sauce-flavored distiller's grains, 0.3 parts of glucose, 0.3 parts of yeast extract powder, and 84.6 parts of water. Adjust the pH of the mixture with a 2% sodium hydroxide aqueous solution to 5.5. Sterilize at 115℃ for 15 minutes to obtain the fermentation culture medium.

[0112] Step 2: Inoculate the fermentation medium with 3% (v / v) of the *Lactobacillus plantarum* X028 bacterial suspension obtained in Preparation Example 4 (viable count 2 × 10⁻⁶). 8 The mixture was fermented for 60 hours at a temperature of 32°C and a stirring speed of 90 rpm (CFU / mL) with sterile air introduced.

[0113] Step 3: Centrifuge the above initial fermentation mixture using a tubular centrifuge at 16000 r / min, twice for 20 min each time, and collect the supernatant.

[0114] Step 4: Filter the supernatant from Step 3 by filtering twice with a PP membrane with a pore size of 0.45μm, and collect the filtrate.

[0115] Step 5: Pass the filtrate through a decolorization column. The decolorization material is macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:3 to obtain the decolorized filtrate.

[0116] Step 6: Sterilize the decolorized filtrate by heating at 82℃ for 20 minutes to obtain the fermented product of soy sauce aroma type.

[0117] The actual image of the fermented product of the soy sauce-flavored liquor lees obtained in this embodiment is shown below. Figure 1 As shown; in this embodiment, the color comparison diagram of the filtrate before and after decolorization in step 5 is as follows. Figure 2 As shown.

[0118] Example 2

[0119] The fermentation method of a soy sauce-flavored liquor lees fermentation product according to this embodiment includes the following steps:

[0120] Step 1: According to the weight parts, mix 20 parts of soy sauce-flavored lees, 0.2 parts of glucose, 0.2 parts of yeast extract powder, and 79.6 parts of water. Adjust the pH of the mixture with a 2% sodium hydroxide aqueous solution to 5.8. Sterilize at 116℃ for 17 minutes to obtain the fermentation culture medium.

[0121] Step 2: Inoculate the fermentation medium with 4% (v / v) of the *Lactobacillus plantarum* X028 bacterial suspension obtained in Preparation Example 4 (viable count 3 × 10⁻⁶). 8The mixture was fermented for 72 hours at a temperature of 32℃ and a stirring speed of 90 rpm (CFU / mL) with sterile air introduced. The initial fermentation mixture was obtained.

[0122] Step 3: Centrifuge the above initial fermentation mixture using a tubular centrifuge at 16000 rpm for 3 times, 25 min each time, and collect the supernatant.

[0123] Step 4: Filter the supernatant from Step 3 using a 0.45μm PP membrane three times and collect the filtrate.

[0124] Step 5: Pass the filtrate through a decolorization column. The decolorization material is macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:2 to obtain the decolorized filtrate.

[0125] Step 6: Sterilize the decolorized filtrate by heating at 85℃ for 20 minutes to obtain the fermented product of soy sauce aroma type.

[0126] Example 3

[0127] The fermentation method of a soy sauce-flavored liquor lees fermentation product according to this embodiment includes the following steps:

[0128] Step 1: According to the weight parts, mix 15 parts of soy sauce-flavored distiller's grains, 0.5 parts of glucose, 0.5 parts of yeast extract powder, and 84 parts of water. Adjust the pH of the mixture with a 3% sodium hydroxide aqueous solution to 5.5. Sterilize at 118℃ for 20 minutes to obtain the fermentation culture medium.

[0129] Step 2: Inoculate the fermentation medium with 5% (v / v) of the Lactobacillus paracasei X016 culture obtained in Preparation Example 4 (viable count 5 × 10⁻⁶). 8 The mixture was fermented for 48 hours at a temperature of 37°C and a stirring speed of 120 rpm, with sterile air introduced into the tank and a pressure of 0.03 MPa maintained. The initial fermentation mixture was obtained.

[0130] Step 3: Centrifuge the above initial fermentation mixture twice using a tubular centrifuge at 16,000 rpm for 20 minutes each time, and collect the supernatant.

[0131] Step 4: Filter the supernatant from Step 3 by filtering twice with a 0.45μm PP membrane and collect the filtrate.

[0132] Step 5: Pass the filtrate through a decolorization column. The decolorization material is macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:4 to obtain the decolorized filtrate.

[0133] Step 6: Sterilize the decolorized filtrate by heating at 82℃ for 20 minutes to obtain the fermented product of soy sauce aroma type.

[0134] Example 4

[0135] The fermentation method of a soy sauce-flavored liquor lees fermentation product according to this embodiment includes the following steps:

[0136] Step 1: According to the weight parts, mix 20 parts of soy sauce-flavored distiller's grains, 0.2 parts of glucose, 0.2 parts of yeast extract powder, and 79.6 parts of water. Adjust the pH of the mixture with a 3% sodium hydroxide aqueous solution to 5.8. Sterilize at 112℃ for 15 minutes to obtain the fermentation culture medium.

[0137] Step 2: Inoculate the fermentation medium with 4% (v / v) of the Lactobacillus paracasei X016 culture obtained in Preparation Example 4 (viable count 3 × 10⁻⁶). 8 The mixture was fermented for 72 hours at a temperature of 32℃ and a stirring speed of 90 rpm (CFU / mL) with sterile air introduced. The initial fermentation mixture was obtained.

[0138] Step 3: Centrifuge the above initial fermentation mixture twice using a tubular centrifuge at 16,000 rpm for 20 minutes each time, and collect the supernatant.

[0139] Step 4: Filter the supernatant from Step 3 by filtering twice with a 0.45μm PP membrane and collect the filtrate.

[0140] Step 5: Pass the filtrate through a decolorization column. The decolorization material is macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:3 to obtain the decolorized filtrate.

[0141] Step 6: Sterilize the decolorized filtrate by heating at 85℃ for 20 minutes to obtain the fermented product of soy sauce aroma type.

[0142] Example 5

[0143] The fermentation method of a soy sauce-flavored liquor lees fermentation product according to this embodiment includes the following steps:

[0144] Step 1: According to the weight parts, mix 15 parts of soy sauce-flavored distiller's grains, 0.5 parts of glucose, 0.5 parts of yeast extract powder, and 84 parts of water. Adjust the pH of the mixture with a 4% sodium hydroxide aqueous solution to 5.5. Sterilize at 115℃ for 15 minutes to obtain the fermentation culture medium.

[0145] Step 2: Inoculate the fermentation medium with 3% (v / v) of the *Lactobacillus plantarum* X028 bacterial suspension obtained in Example 4 (viable count 2 × 10⁻⁶). 8CFU / mL): The Lactobacillus paracasei X016 bacterial suspension obtained in Example 4 (viable count 2 × 10⁻⁶) 8 A mixed bacterial culture with a CFU / mL volume ratio of 1.6:1 was prepared by introducing sterile air, maintaining a pressure of 0.03 MPa in the tank, fermenting at 32°C, and stirring at 90 rpm for 60 hours to obtain the initial fermentation mixture.

[0146] Step 3: Centrifuge the above initial fermentation mixture twice using a tubular centrifuge at 16000 rpm for 20 minutes each time, and collect the supernatant.

[0147] Step 4: Filter the supernatant from Step 3 by filtering twice with a 0.45μm PP membrane and collect the filtrate.

[0148] Step 5: Pass the filtrate through a decolorization column. The decolorization material is macroporous adsorption resin, and the material ratio of macroporous adsorption resin to filtrate is 1:3 to obtain the decolorized filtrate.

[0149] Step 6: Sterilize the decolorized filtrate by heating at 82℃ for 20 minutes to obtain the fermented product of soy sauce aroma type.

[0150] Example 6

[0151] The fermentation method of the fermented product of soy sauce-flavored liquor lees in this embodiment is similar to that in embodiment 5, except that in step 2, the volume ratio of Lactobacillus plantarum X028 bacterial solution to Lactobacillus paracasei X016 bacterial solution is 1:1.

[0152] Example 7

[0153] The fermentation method of the fermented product of soy sauce-flavored liquor lees in this embodiment is similar to that in embodiment 5, except that in step 2, the volume ratio of Lactobacillus plantarum X028 bacterial solution to Lactobacillus paracasei X016 bacterial solution is 2:1.

[0154] Comparative Example 1

[0155] The preparation method of the fermented product of soy sauce-flavored liquor in this comparative example is similar to that in Example 1, except that step 2 is not included.

[0156] Comparative Example 2

[0157] The preparation method of the fermented product of the soy sauce-flavored liquor in this comparative example is similar to that in Example 5, except that in step 2, the volume ratio of Lactobacillus plantarum X028 bacterial solution to Lactobacillus paracasei X016 bacterial solution is 0.5:1.

[0158] Comparative Example 3

[0159] The preparation method of the fermented product of soy sauce-flavored liquor in this comparative example is similar to that in Example 1, except that in step 2, the Lactobacillus plantarum X028 bacterial solution is replaced with the Lactobacillus acidophilus solution screened in Preparation Example 1. The preparation method of the screened Lactobacillus acidophilus bacterial solution is the same as that of the Lactobacillus plantarum X028 bacterial solution.

[0160] Comparative Example 4

[0161] The preparation method of the fermented product of soy sauce-flavored liquor in this comparative example is similar to that in Example 1, except that in step 2, the Lactobacillus plantarum X028 bacterial solution is replaced with the Lactobacillus bulgaricus bacterial solution screened in Preparation Example 1. The preparation method of the screened Lactobacillus bulgaricus bacterial solution is the same as that of the Lactobacillus plantarum X028 bacterial solution.

[0162] Comparative Example 5

[0163] The preparation method of the fermented product of soy sauce-flavored liquor in this comparative example is similar to that in Example 1, except that in step 1, the pH value is adjusted to 4.0.

[0164] Experimental Example 1

[0165] The appearance, odor, solids content, organic acids, total phenols content, and total bacterial count of the fermented products of soy sauce-flavored liquor prepared in Examples 1-7 and Comparative Examples 1-5 were determined. The specific determination methods are as follows:

[0166] (1) Solid content detection: Weigh 5g of the sample to be tested (m1=5), dry it at 95℃ for 120min, take it out and put it in a desiccator, cool it to room temperature, weigh it, and record it as m2;

[0167] The solid content can be calculated using the formula: Solid content = (m2 / m1) × 100%.

[0168] (2) Organic acid content detection: Lactic acid and acetic acid are determined according to the method in GB 5009.157-2016, and acetic acid is determined according to the following method:

[0169] Accurately weigh 2.0 g (accurate to 0.001 g) of homogeneous sample into a 20 mL headspace vial.

[0170] Add 1.0 mL of internal standard working solution (containing 100 μg / mL propionic acid) and 0.5 g of sodium chloride (NaCl).

[0171] Immediately seal with an aluminum cap fitted with a PTFE / silicone gasket and vortex mix for 30 seconds.

[0172] Placed in a headspace autosampler for testing.

[0173] Headspace sampler parameters:

[0174] Heating chamber temperature: 80℃;

[0175] Metering loop temperature: 90℃;

[0176] Transmission line temperature: 100℃;

[0177] Equilibrium time: 15 min (oscillation);

[0178] Injection volume: 500 μL;

[0179] GC-MS parameters:

[0180] Column: Agilent J&W HP-INNOWax (30m × 0.25mm × 0.25μm);

[0181] Inlet temperature: 250℃, split mode, split ratio 10:1;

[0182] Carrier gas: high-purity helium (He), constant flow mode, flow rate 1.0 mL / min;

[0183] Column temperature program: 40℃ (hold for 2 min) → increase to 240℃ at 10℃ / min (hold for 5 min);

[0184] MS interface temperature: 250℃;

[0185] Ion source: EI source, 70 eV, temperature 230℃;

[0186] Quadrupole temperature: 150℃;

[0187] Data acquisition mode:

[0188] Scan mode: m / z 30~200 (for qualitative screening);

[0189] SIM mode: used for quantification (acetic acid: quantitative ion m / z 43, qualitative ion m / z 45; internal standard propionic acid: quantitative ion m / z 74).

[0190] Quantitative:

[0191] Quantification was performed using the internal standard curve method. A calibration curve was plotted against the concentration ratio by comparing the peak area ratio of acetic acid to that of the internal standard propionic acid. The acetic acid content was then calculated.

[0192] (3) Detection of polyphenol content

[0193] Accurately weigh 10 mg of gallic acid standard and dilute to 100 mL with water to obtain a gallic acid standard solution with a mass concentration of 0.1 mg / mL. Using a pipette, transfer 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 mL of each standard solution into 25 mL volumetric flasks, add ultrapure water to dilute to volume, and mix well to obtain a series of gallic acid standard solutions with mass concentrations of 0, 1, 2, 3, 4, 5, 6, and 7 μg / mL. Take 1 mL of the standard solution, add 0.65 mL of Folin-Ciocalteu reagent and mix well, then add 0.6 mL of 20% sodium carbonate solution, mix well, and heat in a water bath at 60 °C for 1 h. After cooling, obtain a series of gallic acid solutions with mass concentrations of 0, 1, 2, 3, 4, 5, 6, and 7 μg / mL. Each sample was repeated three times. Measure the absorbance of the series of solutions at 720 nm and plot a standard curve.

[0194] Take 1 mL of the sample to be tested (diluted to the corresponding concentration according to the standard curve), add 0.65 mL of mol / L Folin-Ciocalteu reagent, mix well, then add 0.6 mL of 20% sodium carbonate solution, and react in a 60℃ water bath for 1 h. Measure the absorbance of the sample solution at 720 nm. Repeat three times. Substitute the measured absorbance into the standard curve to obtain the polyphenol content.

[0195] The appearance, odor, solids content, organic acid, total phenol content, and total bacterial count of the fermented products of soy sauce-flavored liquor prepared in Examples 1-7 and Comparative Examples 1-5 are shown in Table 1.

[0196] Table 1

[0197]

[0198]

[0199] As shown in Table 1, the solid content of the fermented product of soy sauce-flavored liquor obtained by the method of this invention is 0.94-1.26%, the organic acid content is 0.78-1.18%, the polyphenol content is 44.2-73.9%, and the total bacterial count is 0. Furthermore, the mixed fermentation of *Lactobacillus plantarum* and *Lactobacillus paracasei* resulted in higher organic acid and polyphenol content than single-strain fermentation, with organic acid content ranging from 0.98-1.18% and polyphenol content from 64.2-73.9%. The fermentation product obtained by the specific preparation process involved in this invention can effectively improve the original aroma of soy sauce-flavored liquor and contains high levels of organic acids and polyphenols, providing a foundation for its excellent antioxidant and soothing effects.

[0200] Experiment Example 2

[0201] (1) DPPH scavenging experiment

[0202] According to the "Experimental Method for Scavenging Free Radicals (DPPH) in Cosmetics (T / SHRH006-2018)," the DPPH free radical scavenging rate (%) of the fermentation products prepared in Examples 1-7 and Comparative Examples 1-5 and the control group were tested respectively.

[0203] Experimental method: A 96-well plate was used, with three replicates in each group, and the system volume was 200 μL.

[0204] Sample group: Dilute the fermentation product in 100 μL of distilled water to make the final concentration of the sample in the system 10%, and add 100 μL of 0.1 mM DPPH solution to the reaction system;

[0205] Control group: Take 100 μL of distilled water and add 100 μL of 0.1 mM DPPH solution.

[0206] After the reaction system was constructed, it was shaken in the dark for 10 minutes, and the absorbance at 520 nm was measured using an ELISA reader.

[0207] The clearance rate is calculated as follows: clearance rate (%) = [(A0-Ax) / A0] × 100%.

[0208] Where A0 is the absorbance of the control group and Ax is the absorbance of the sample group.

[0209] (2) Determination of ABTS free radical scavenging ability

[0210] Preparation of ABTS stock solution (7.4 mmol / L): Weigh 38.4 mg of 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) and dissolve in 10 mL of distilled water. Preparation of K2S2O8 stock solution (2.6 mmol / L): Weigh 7 mg of K2S2O8 and dissolve in 10 mL of distilled water.

[0211] Working solution: Mix the two solutions above in a 1:1 ratio until homogeneous, and let stand at room temperature in the dark for 12 hours to obtain the ABTS working solution. Dilute the ABTS solution with anhydrous ethanol to an absorbance of 0.70 ± 0.02, and then the sample can be measured.

[0212] Sample Measurement Tube: Take 1.2 mL of sample solution, add 4.8 mL of ABTS working solution, vortex to mix, react precisely for 6 minutes, and immediately measure the absorbance at 734 nm, denoted as A. Reagent Blank Tube: Take 1.2 mL of anhydrous ethanol, add 4.8 mL of ABTS working solution, mix well, react for 6 minutes, and measure the absorbance, denoted as A0. This tube represents the absorbance at 100% free radical concentration. Sample Background Blank Tube: Take 1.2 mL of sample solution, add 4.8 mL of anhydrous ethanol, mix well, zero the sample using anhydrous ethanol as a reference, and measure the absorbance at 734 nm. This value is used to correct the sample solution's own values, denoted as A1.

[0213] The clearance rate is calculated using the following formula: [(A0-(A-A1)) / A0]×100%.

[0214] (3) TNF-α inhibition experiment

[0215] Experimental Methods: RAW264.7 macrophages were used as the research subject. A cellular inflammation model was established by stimulating cells with lipopolysaccharide (LPS), a bacterial endotoxin. Macrophages were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. 3% dilutions of the products from Examples 1-7 and Comparative Examples 1-5 were added. After 2 h, LPS (1 μg / mL) was added. Groups with only LPS and no LPS were established. Stimulation was continued for 24 h, and the supernatant was collected, centrifuged, and analyzed. The release level of the pro-inflammatory inflammatory factor TNF-α in RAW264.7 cells was analyzed using an ELISA kit.

[0216] The TNF-α inhibition rate is calculated using the following formula:

[0217] TNF-α inhibition rate (%) = [(concentration of inflammatory factors in the LPS-stimulated group - concentration of inflammatory factors in the analyte-treated group) / (concentration of inflammatory factors in the LPS-stimulated group - concentration of inflammatory factors in the unstimulated group)] × 100%.

[0218] The DPPH scavenging rate, ABTS free radical scavenging ability, and TNF-α inhibition rate of the fermented products of soy sauce-flavored liquor prepared in Examples 1-7 and Comparative Examples 1-5 of this invention are shown in Table 2.

[0219] Table 2

[0220]

[0221] As shown in Table 2, the fermentation products prepared by the method of the present invention exhibited a DPPH scavenging rate of 40.39–58.72%, an ABTS free radical scavenging rate of 15.78–27.86%, and a TNF-α inhibition rate of 29.36–43.42%. The fermentation products prepared in Examples 5-7 showed better performance than those prepared in Examples 1-4, with a DPPH scavenging rate of 50.35–58.72%, an ABTS free radical scavenging rate of 20.76–27.86%, and a TNF-α inhibition rate of 38.37–48.42%. This is attributed to the synergistic effect between *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016. Table 2 also shows that the fermentation products prepared by the method of the present invention possess good antioxidant and soothing effects.

[0222] Experimental Example 3

[0223] The skin moisture content was measured using a capacitance method skin moisture meter according to the "QB-T 4256-2011 Guidelines for Evaluation of Moisturizing Efficacy of Cosmetics". Thirty volunteers participated. For 2-3 days prior to the test, no products (cosmetics or topical medications) were used on the test areas, and the areas should not come into contact with water for 1-3 hours. Before the test, all participants were required to clean the inner forearms of both hands uniformly. Cleaning was done by wiping with a dry tissue. Measurement areas (5cm x 5cm) were marked on the inner forearms of both participants. Multiple areas could be marked on the same arm, with at least 1cm between each area. Before the formal test, participants should sit quietly in a compliant room for at least 20 minutes, without drinking water or beverages. The forearms were exposed, placed in the testing position, and kept relaxed.

[0224] The smear areas for 10% of the fermentation products from Examples 1-7 and Comparative Examples 1-5, and the blank control area (water), should be randomly distributed across the marked areas on both arms to ensure statistically balanced positions of all products and blank areas. Test samples should be applied at (2.0 ± 0.1) mg / cm³. 2 Apply the sample in a single coat using the specified amount of latex, and use a latex finger cot to evenly coat the sample in the test area, and record the actual amount of sample coated.

[0225] The skin moisture content of the product area and the control area was measured using a capacitive skin moisture meter, with each area measured at least three times in parallel. The initial values ​​of each test area (before sample use) were measured at day 0, and then the skin moisture content of the test area and the control area was measured at days 7 and 14 (Table 3).

[0226] Table 3

[0227]

[0228] Table 3 shows that the skin hydration level of the fermentation product prepared by the method of this invention was 19.82-25.27% after 7 days and 28.36-39.37% after 14 days. *Lactobacillus plantarum* X028 and *Lactobacillus paracasei* X016 significantly improved skin hydration compared to unfermented products, and the combined fermentation of the two showed the best effect. Free amino acids, lactate, and acetate in the fermentation product are important components of the stratum corneum of the skin. Free amino acids are the skin's natural moisturizing factor (NMF), and lactate and acetate can also be classified as NMF. The fermentation product of the soy sauce-flavored distillers' grains prepared by this invention exhibited a good moisturizing effect.

[0229] Experiment Example 4

[0230] Safety test

[0231] Select a qualified patch tester and use the closed patch test method. Add 200-250 mg of the test substance (fermentation product prepared in Examples 1-7) into the patch tester and apply it to the skin on both sides of the spine on the back. Observe the skin reaction at 0.5, 24 and 48 hours. Use water as a control. According to the skin grading standard in the "Cosmetic Safety Technical Specifications" (2022 edition) (Table 4), record the results (Table 5).

[0232] Table 4. Skin Grading Standards in the "Cosmetic Safety Technical Specifications" (2022 Edition)

[0233]

[0234] Table 5

[0235]

[0236] As shown in Table 5, the fermentation product of the method described in this invention has no adverse effects on the skin and has good safety performance.

[0237] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation method for a soy sauce-flavored liquor lees fermentation product, characterized in that, Specifically, the following steps are included: Step 1: Mix the soy sauce-flavored distiller's grains, glucose, yeast extract powder, and water, adjust the pH value, and sterilize to obtain the fermentation culture medium; Step 2: Inoculate the fermentation medium with Lactobacillus plantarum X028 bacterial solution and / or Lactobacillus paracasei X016 bacterial solution, introduce sterile air, ferment under positive pressure and stirring conditions to obtain the initial fermentation mixture; Step 3: Centrifuge the initial fermentation mixture and collect the supernatant; Step 4: Filter the supernatant from Step 3 and collect the filtrate; Step 5: Decolorize the filtrate to obtain a decolorized filtrate; Step 6: Sterilize the decolorized filtrate to obtain a soy sauce-flavored fermented liquor product.

2. The fermentation method for a soy sauce-flavored liquor lees fermentation product according to claim 1, characterized in that, In step 1, according to the following weight proportions: 10-20 parts of soy sauce-flavored lees, 0.1-0.5 parts of glucose, 0.1-0.5 parts of yeast extract, and 79.6-89.8 parts of water.

3. The fermentation method for a soy sauce-flavored liquor lees fermentation product according to claim 1, characterized in that, In step 1, adjust the pH value to 5-6.

4. A fermentation method for a soy sauce-flavored liquor lees fermentation product according to any one of claims 1-3, characterized in that, In step 2, the preservation number of Lactobacillus plantarum X028 is CGMCC No. 33256, and the preservation number of Lactobacillus paracasei X016 is CGMCC No. 33255.

5. A fermentation method for a soy sauce-flavored liquor lees fermentation product according to any one of claims 1-3, characterized in that, In step 2, the viable count of the *Lactobacillus plantarum* X028 bacterial solution is 1-5 × 10⁻⁶. 8 CFU / mL, inoculum size 2-5%; and / or, the viable count in the Lactobacillus paracasei X016 bacterial suspension is 1-5 × 10⁻⁶. 8 CFU / mL, inoculation amount 3-5%.

6. A fermentation method for a soy sauce-flavored liquor lees fermentation product according to any one of claims 1-3, characterized in that, In step 2, when both Lactobacillus plantarum X028 and Lactobacillus paracasei X016 bacterial solutions are added simultaneously, the volume ratio of Lactobacillus plantarum X028 to Lactobacillus paracasei X016 is 1-2:

1.

7. A fermentation method for a soy sauce-flavored liquor lees fermentation product according to any one of claims 1-3, characterized in that, In step 2, the fermentation temperature is 30-37℃ and the fermentation time is 48-72h.

8. A fermentation method for a soy sauce-flavored liquor lees fermentation product according to any one of claims 1-3, characterized in that, In step 1, the sterilization temperature is 115-118℃ and the sterilization time is 15-20 min; In step 6, the sterilization temperature is 80-85℃ and the sterilization time is 15-25 minutes.

9. A fermented product of soy sauce aroma type prepared by any one of claims 1-8.

10. The use of a soy sauce-flavored fermented product prepared by any one of claims 1-8 or the soy sauce-flavored fermented product described in claim 9 in the preparation of cosmetics.

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