Traditional Chinese medicine compound fermented wine as well as preparation method and application thereof

Abstract

The invention aims to break through the limitation of the traditional Chinese medicine wine preparation technology, realize efficient release and functional conversion of active ingredients through a microorganism-traditional Chinese medicine synergistic system, and solve the problems of low ingredient dissolution rate and single function; directional fermentation is used for replacing high-concentration ethanol soaking, so that dissolution of toxic components is reduced, and the safety risk of a traditional process is overcome; through strain metabolism regulation and control, the health-care value and sensory acceptability of the product are synchronously improved, and a'flavor-function 'double-excellent system is constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of food fermentation, and specifically relates to a Chinese medicine composite fermented wine prepared by synergistic fermentation of composite bacteria using a specific Chinese medicine composition as raw material, a preparation method thereof, and application thereof in health food. Background Art

[0002] As a traditional health-preserving beverage, Chinese medicinal liquor has a history of thousands of years. According to the "White Paper on Chinese Infused Liquor," approximately 87% of the currently available Chinese medicinal liquors still use the baijiu infusion method (such as Guogong liquor and ginseng liquor). This technical limitation is mainly reflected in:

[0003] 1. Low dissolution efficiency of ingredients

[0004] The cell walls of medicinal materials (especially those with high lignification, such as Astragalus and Poria) hinder the release of active ingredients. Studies have shown that after immersion in 50% vol ethanol for 30 days, the dissolution rate of astragaloside IV is only 12.3±1.8% (HPLC determination, see "Chinese Patent Medicine" 2021, 43(5): 1125-1130).

[0005] Fat-soluble components (such as wolfberry carotenoids) have insufficient solubility in low-alcohol soaking solutions, and their bioavailability is reduced by 40-60% (Food Chemistry 2020, 316:126235).

[0006] 2. Functional component destruction and toxicity risks

[0007] Long-term high-concentration ethanol immersion causes degradation of heat-sensitive substances (such as total flavonoids in hawthorn), with a loss rate of 35-48% after 30 days of immersion (Journal of Ethnopharmacology, 2019, 241:111956).

[0008] The toxic alkaloids in some medicinal materials (such as aconite and Chuanwu) continue to dissolve during the soaking process, posing a safety risk (China Journal of Traditional Chinese Medicine 2020, 45(7): 1693-1698).

[0009] 3. Unutilized Microbial Metabolic Potential

[0010] Traditional processes rely on passive dissolution and fail to generate new active substances through microbial transformation. For example, glycyrrhizic acid cannot be directly absorbed by the human body, but microorganisms can convert it into glycyrrhetinic acid, which is more active (Biotechnology Advances 2018, 36(1):268-280). Polysaccharides are difficult to be directly utilized by the intestines, but low molecular weight functional oligosaccharides can be produced after enzymatic hydrolysis.

[0011] In recent years, some research methods have adopted the fermentation method instead of the soaking method (such as CN112175776A, CN113234572A), but the following technical bottlenecks still exist:

[0012] 1. Single-function bacterial strain. The process uses only Saccharomyces cerevisiae for fermentation, resulting in incomplete decomposition of macromolecules such as cellulose and pectin (medicinal material utilization rate <50%). The metabolite spectrum is narrow (only ethanol and a small amount of esters are produced), lacking functional components such as GABA and organic acids. A few solutions incorporate fungi such as Aspergillus oryzae (e.g., CN114456999A), but their protease enzymes are poorly compatible with traditional Chinese medicine ingredients, and the content of bitter amino acids (e.g., leucine) in the fermentation broth increases by 22-35%, affecting palatability.

[0013] 2. Insufficient scientific compatibility. The selection of medicinal ingredients often follows ancient texts (e.g., Shiquan Dabu Tang), without formula design tailored to modern metabolic syndromes (e.g., insulin resistance and intestinal flora disturbances). Over 70% of the formulas contain warming tonic herbs like ginseng and deer antler, which can easily induce dryness and heat reactions. The formulas lack ingredients like hawthorn and poria, which have both flavor-regulating and metabolic-stimulating properties and are considered "medicinal and edible."

[0014] 3. Extensive process control. The fermentation phases are unclear, and the aerobic / anaerobic transition is not precisely controlled. This results in: the Monascus esterification phase overlaps with the yeast ethanol fermentation phase, inhibiting enzyme activity (a 40-60% decrease); and premature introduction of lactic acid bacteria causes a drop in pH, inhibiting yeast proliferation (a 50-70% reduction in biomass).

[0015] Therefore, for the preparation of Chinese herbal wine, establishing a bacteria-drug adaptation system (screening exclusive bacteria species that can efficiently decompose the cell walls of Chinese medicine), realizing metabolic pathway regulation (directional synthesis of health factors such as GABA and short-chain fatty acids), and developing flavor modification technology (reducing the bitterness of Chinese medicine through microbial co-fermentation) have become current industry needs and innovation directions. Summary of the Invention

[0016] The present invention aims to break through the limitations of traditional Chinese medicine wine preparation technology, realize the efficient release and functional transformation of active ingredients through the synergistic system of microorganisms and traditional Chinese medicine, solve the problems of low ingredient dissolution rate and single function; use directed fermentation to replace high-concentration ethanol soaking to reduce the dissolution of toxic ingredients and overcome the safety risks of traditional processes; through the metabolic regulation of bacterial strains, simultaneously improve the health value and sensory acceptance of the product, and construct a "flavor-function" dual-excellence system.

[0017] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0018] The first aspect of the present invention provides a Chinese medicine composite fermented wine, which is prepared by fermenting the following Chinese medicine raw materials in the following mass percentages through composite strains in stages: 15-30% astragalus, 10-20% wolfberry, 8-15% poria, 5-12% hawthorn, 3-8% licorice, and 5-10% longan meat.

[0019] Furthermore, the composite bacterial strain includes Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum, wherein the number of Monascus spores is 1×10 6 -5×10 6 CFU / g, the ratio of viable bacteria count of Saccharomyces cerevisiae to Lactobacillus plantarum is (1-3):1.

[0020] Furthermore, the Monascus purpureus is a strain with a preservation number of CGMCC NO.7603, the Lactobacillus plantarum is a strain with a preservation number of CGMCC NO.1.12934, and the Saccharomyces cerevisiae is a strain with a preservation number of CCTCCNO:M2018669.

[0021] Another aspect of the present invention provides a method for preparing a traditional Chinese medicine compound fermented wine, comprising the following steps:

[0022] (1) The Chinese medicine raw materials are crushed to 60-100 mesh and steam sterilized;

[0023] (2) Solid-state fermentation: Monascus is inoculated into the sterilized raw materials and fermented at 28-32°C and 70-80% humidity for 3-7 days;

[0024] (3) Liquid fermentation: Add 5-10 times the mass of water to the solid fermentation product, inoculate with Saccharomyces cerevisiae and Lactobacillus plantarum, and ferment in a closed container at 25-30°C for 10-15 days;

[0025] (4) Aging and clarification: After filtration, add 0.05-0.2% chitosan for clarification and age at 40-55°C for 20-40 days.

[0026] Furthermore, the inoculation amount of Monascus in step (2) is 2-4wt%, and the material is turned over every 8-12 hours during the fermentation period; the inoculation amount of Saccharomyces cerevisiae in step (3) is 2×10 7 -5×10 7 CFU / mL.

[0027] Furthermore, step (3) further includes adding 2-5 wt% of a longan extract when the pH of the fermentation liquid drops to 3.8-4.2, and cooling the fermentation liquid to 20-25° C. and maintaining the temperature for 24-48 hours. The longan extract contains natural reducing sugars (e.g., fructose and glucose, with a content of about 12-15%), which can provide a readily usable carbon source for microorganisms in the middle and late stages of fermentation (when the pH drops to 4.0). The longan extract contains organic acids (e.g., malic acid and citric acid) and minerals (e.g., potassium and calcium), which form a natural buffer system to stabilize the pH within the range of 3.8-4.2.

[0028] Furthermore, the content of gamma-aminobutyric acid (GABA) in the final product is ≥50 mg / L, the content of astragalus polysaccharide is ≥10 mg / L, and the volume fraction of ethanol is 8-12% vol.

[0029] Another aspect of the present invention provides a use of a traditional Chinese medicine compound fermented wine in the preparation of a health food having antioxidant and intestinal flora regulating functions.

[0030] Furthermore, the dosage form of the health food includes liquid beverage, freeze-dried powder or capsule preparation.

[0031] The core innovation of this invention lies in the "one match, two bacteria, three stages" technical system, in which:

[0032] "One pairing" refers to the precise design of Chinese medicine compatibility: the Chinese medicine components of the present invention are reconstructed using monarch, minister, assistant and envoy ingredients. The monarch ingredients are astragalus (tonifying qi and strengthening the exterior) and hawthorn (promoting blood circulation and reducing fat), which have good efficacy against metabolic syndrome; the minister ingredients are tuckahoe (strengthening the spleen and removing dampness) and wolfberry (nourishing the liver and improving eyesight), which can regulate water metabolism and oxidative stress; the assistant ingredients are licorice (adjusting and detoxifying) and longan meat (soothing and flavoring), which can reduce the toxicity of fermentation by-products. In addition, the present invention designs an efficient dissolution-conversion synergistic system for the above-mentioned important ingredients, in which high-starch medicinal materials (tuckahoe and longan meat) can serve as microbial carbon sources to promote enzyme secretion; and low-molecular-weight glycosides (such as glycyrrhizic acid) are designed as fermentation conversion precursors.

[0033] The two bacteria refer to the fact that the present invention adopts a combination of functionally complementary bacterial species, and utilizes the bacterial community interaction mechanism to obtain a "metabolic relay" mechanism of cross-kingdom bacterial species. The cross-kingdom combination of fungi (Monascus) → yeast (fungus) → lactic acid bacteria (bacteria) forms a three-level metabolic chain of decomposition → transformation → functionalization. Among them, Monascus can decompose medicinal macromolecules and release precursors such as polysaccharides and flavonoid glycosides; yeast uses monosaccharides to generate ethanol and synthesize ester flavor substances; lactic acid bacteria uses free amino acids (such as glutamic acid) to synthesize functional factors such as GABA. For the first time, cross-kingdom synergy between fungi and bacteria has been achieved in the fermentation of traditional Chinese medicine, breaking through the metabolic limitations of traditional microorganisms of the same type (such as yeast + Aspergillus).

[0034] For strain screening, Monascus purpureus prioritizes edible, safe strains that do not produce citrinin, such as the Monascus GL-1 strain with a preservation number of CGMCC NO.7603, which has a high color value. Plant Lactobacillus screens strains with high gadB gene expression, such as the plant Lactobacillus with a preservation number of CGMCC NO.1.12934, whose glutamate decarboxylase (GAD) activity is ≥85U / mg. Saccharomyces cerevisiae selectively breeds strains with overexpressed ATF1 genes, such as the strain with a preservation number of CCTCC NO:M2018669, whose ester synthesis capacity is 3 times higher than that of ordinary strains. The present invention constructs a dedicated strain library with safety and functionality as core indicators.

[0035] The third stage refers to a fermentation process with time and space separation, and its process steps include the following stages:

[0036] 1. Solid-state stage (Monascus-dominated): Select a Monascus strain that produces a highly active complex enzyme system. During this stage, Monascus secretes extracellular enzymes, achieving a cell wall rupture rate of ≥80% and a starch conversion rate of 92±3%. This breaks through the traditional passive "immersion and dissolution" model and achieves efficient cell wall rupture through active microbial decomposition.

[0037] 2. Liquid Phase (Yeast + Lactic Acid Bacteria Collaboration): Ethanol-tolerant Saccharomyces cerevisiae and GABA-producing Lactobacillus plantarum are introduced to create a dual-barrier system of "ethanol-acidity." The yeast rapidly produces ethanol (8-12% vol), inhibiting contaminants while promoting the production of ester flavor compounds. The lactic acid bacteria metabolize and produce lactic acid (final concentration 1.2-1.8 g / L), precisely regulating the pH to 3.8-4.2 and creating a stable window for flavonoids. During this phase, microbial interactions achieve the dual goals of environmental self-purification and functional ingredient protection. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific embodiments. The specific embodiments are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and intent of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0039] The reagents and raw materials used in the present invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained from commercial sources. Experimental methods not specifying specific conditions in the present invention are generally carried out under conventional conditions or conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not be construed to limit the scope of the present invention in any way.

[0040] Example 1: Preparation of the Chinese medicinal composite fermented wine of the present invention.

[0041] 1. Raw material preparation and processing.

[0042] The Chinese medicine composition is as follows: 15-30% of astragalus, 10-20% of wolfberry, 8-15% of poria, 5-12% of hawthorn, 3-8% of liquorice, and 5-10% of longan meat (all percentages are by mass).

[0043] Pretreatment: The medicinal materials were crushed to 80 mesh and sterilized by steam at 121℃ for 15 min.

[0044] 2. Solid-state enzyme fermentation.

[0045] Species: Monascus CGMCC NO.7603;

[0046] Inoculum size: 3% (w / w, spore count 3×10 6 CFU / g);

[0047] Conditions: temperature 30°C, humidity 75%, oxygen flow 0.3vvm, fermentation for 5 days, turning every 12 hours;

[0048] Key indicators: amylase activity 720±25U / g, Poria β-glucan molecular weight from 2.5×10 5 Da is reduced to 8.1×10 3 Da (SEC-MALLS detection).

[0049] 3. Liquid collaborative fermentation.

[0050] Bacterial species: Lactobacillus plantarum CGMCC NO.1.12934, Saccharomyces cerevisiae CCTCC NO:M2018669;

[0051] Inoculation volume: 5×10 Saccharomyces cerevisiae 7 CFU / mL, Lactobacillus plantarum 2.5×10 7 CFU / mL;

[0052] Conditions: Fermentation was carried out in a closed chamber at 28°C for 12 days. When the pH dropped to 4.0, 3 wt% longan extract was added.

[0053] Dynamic monitoring: ethanol concentration reached 11.2% vol on the 8th day (GC detection), and GABA content reached 68.5 mg / L on the 12th day (HPLC-FLD detection).

[0054] 4. Post-processing

[0055] Clarification: add 0.1% chitosan, turbidity ≤ 5NTU;

[0056] Aging: Gradual cooling from 50℃ to 30℃, maintaining for 30 days.

[0057] Comparative Example 1: Traditional immersion method.

[0058] Process: Soak in 50% vol liquor for 30 days.

[0059] Comparative Example 2: Single bacteria fermentation.

[0060] Process: Only Monascus and Saccharomyces cerevisiae are used, without adding Lactobacillus plantarum.

[0061] Table 1: Comparison of active ingredient dissolution and functional indicators.

[0062]

[0063]

[0064] It can be seen that the present invention uses Monascus to produce enzymes in a targeted manner, so that the dissolution amount of astragalus polysaccharide reaches 15.2 mg / mL, which is 300% higher than the traditional soaking method; the high content of total flavonoids in hawthorn in Example 1 is due to the efficient release of flavonoid glycosides wrapped in the hawthorn cell wall by the cellulase of Monascus, while Comparative Example 1 only dissolves a small part of free flavonoids due to the polarity limitation of ethanol, and Comparative Example 2 does not completely hydrolyze flavonoid glycosides due to the lack of an acidic environment for lactic acid bacteria; Example 1 converts glycyrrhizic acid into glycyrrhetinic acid through the β-glucosidase of Monascus, while Comparative Example 2 only dissolves unconverted glycyrrhizic acid, and Comparative Example 2 has an insufficient conversion rate due to the lack of acidic protease; Lactobacillus plantarum metabolizes and synthesizes GABA (68.5 mg / L), filling the functional gap of traditional medicinal wine. In addition, the synergistic fermentation makes the antioxidant activity (DPPH clearance rate 89.7%) significantly higher than that of the traditional process.

[0065] Table 2: Comparison of process efficiency and stability.

[0066]

[0067] It can be seen that Example 1 reduces the liquid fermentation time by pre-production of enzymes by Monascus (enzyme reserve) and dynamic pH control (feeding trigger); the traditional soaking method relies on high-concentration ethanol preservation (50% vol), while the present invention achieves antibacterial effect through a lactic acid-ethanol double barrier (pH 3.8-4.2 + 11.2 vol% ethanol), with lower ethanol concentration and improved safety; the composite clarification process (chitosan + gradient cooling) adopted in the example reduces precipitation, and the polysaccharide retention rate is significantly higher than that of the traditional process comparative example 1.

[0068] Table 3: Comparison of sensory qualities and flavor substances.

[0069] Aroma and bitterness: Blind test and scoring by a 10-person professional sensory evaluation panel (referring to GB / T 15038-2006).

[0070] Ethyl acetate: headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0071] Turbidity: turbidimeter (HACH2100AN, ISO7027 standard).

[0072]

[0073] It can be seen that the overexpression of ATF1 gene in Saccharomyces cerevisiae increases the content of ester substances, which can mask the bitter taste of traditional Chinese medicine; the acid protease of Monascus purpurogenus can selectively decompose bitter amino acids such as leucine and isoleucine, and the content is detected by HPLC.

[0074] Table 4: Comparison of functional verification experiments.

[0075] SOD activity: WST-1 method (kit, Beyotime S0101).

[0076] Intestinal Lactobacillus proliferation: qPCR quantification (primers: Lacto-F / R, refer to the "Technical Specifications for Intestinal Flora Detection").

[0077] ATL reduction rate: fully automatic biochemical analyzer (Roche Cobas 8000).

[0078] Blood pressure monitoring: non-invasive blood pressure monitor (Softron BP-98A), SHR rat model.

[0079]

[0080]

[0081] It can be seen that the produced astragalus polysaccharides (scavenging free radicals) + monascus pigments + flavonoids (chelating metal ions) form a complex antioxidant network; the short-chain fatty acids (acetic acid, propionic acid) produced by Lactobacillus plantarum metabolism promote the colonization of probiotics; GABA lowers blood pressure by inhibiting angiotensin-converting enzyme (ACE), and flavonoids inhibit lipid peroxidation in hepatocytes.

[0082] Table 5: Comparison of synergistic effects of bacterial strains.

[0083] Cell wall rupture rate: Scanning electron microscopy (SEM) image analysis (ImageJ software was used to calculate the cell wall rupture area).

[0084] GABA production: HPLC-FLD (mobile phase: 0.1% formic acid water-acetonitrile, excitation wavelength 338 nm).

[0085] AI-2 signaling molecule: q-PCR was used to detect the expression of the luxS gene (primers: luxS-F / R).

[0086]

[0087] It can be seen that the cellulase activity of Monascus is significantly higher than that of Aspergillus niger, so the cell wall rupture rate of Example 1 is higher than that of Comparative Example 2; Lactobacillus plantarum carries a highly active gadB gene with GABA specificity, which other bacteria do not have; the AI-1 signal molecule coordinates the metabolic rhythm of yeast and lactic acid bacteria to avoid resource competition.

[0088] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A Chinese medicine composite fermented wine, which is prepared by fermenting the following Chinese medicine raw materials by composite strains in stages in the following mass percentages: 15-30% astragalus, 10-20% wolfberry, 8-15% poria, 5-12% hawthorn, 3-8% liquorice, and 5-10% longan meat.

2. The Chinese medicinal compound fermented wine according to claim 1, characterized in that: The composite bacterial species includes Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum, wherein the number of Monascus purpureus spores is 1×10 6 -5×10 6 CFU / g, the ratio of viable bacteria count of Saccharomyces cerevisiae to Lactobacillus plantarum is (1-3):

1.

3. A Chinese medicinal compound fermented wine according to claim 1 or 2, characterized in that: The Monascus purpureus is a strain with a preservation number of CGMCC NO.7603, the Lactobacillus plantarum is a strain with a preservation number of CGMCC NO.1.12934, and the Saccharomyces cerevisiae is a strain with a preservation number of CCTCC NO:M2018669.

4. The method for preparing a traditional Chinese medicine composite fermented wine according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) The Chinese medicine raw materials are crushed to 60-100 mesh and steam sterilized; (2) Solid-state fermentation: Monascus is inoculated into the sterilized raw materials and fermented at 28-32°C and 70-80% humidity for 3-7 days; (3) Liquid fermentation: Add 5-10 times the mass of water to the solid fermentation product, inoculate with Saccharomyces cerevisiae and Lactobacillus plantarum, and ferment in a closed container at 25-30°C for 10-15 days; (4) Aging and clarification: After filtration, add 0.05-0.2% chitosan for clarification and age at 40-55°C for 20-40 days.

5. The method for preparing a traditional Chinese medicine compound fermented wine according to claim 4, characterized in that: The inoculation amount of Monascus in step (2) is 2-4wt%, and the material is turned over every 8-12 hours during the fermentation period; the inoculation amount of Saccharomyces cerevisiae in step (3) is 2×10 7 -5×10 7 CFU / mL.

6. The method for preparing a traditional Chinese medicine compound fermented wine according to claim 4, characterized in that: Step (3) further comprises adding 2-5 wt% of longan pulp extract when the pH of the fermentation liquid drops to 3.8-4.2, and cooling the fermentation liquid to 20-25° C. and maintaining the temperature for 24-48 hours.

7. The method for preparing a traditional Chinese medicine compound fermented wine according to any one of claims 4 to 6, characterized in that: The content of gamma-aminobutyric acid (GABA) in the final product is greater than or equal to 50 mg / L, the content of astragalus polysaccharide is greater than or equal to 10 mg / L, and the volume fraction of ethanol is 8-12% vol.

8. Use of the traditional Chinese medicine compound fermented wine according to any one of claims 1 to 7 in the preparation of health food with antioxidant and intestinal flora regulating functions.

9. Use of a Chinese medicinal compound fermented wine according to claim 8 in preparing a health food having antioxidant and intestinal flora regulating functions, characterized in that: The dosage form of the health food includes liquid beverage, freeze-dried powder or capsule preparation.

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