Preparation method and application of yellow bud soup based on multi-bacteria synergistic fermentation

Abstract

The invention relates to the technical field of traditional Chinese medicine fermentation preparation, in particular to a preparation method and application of yellow bud soup based on multi-bacterium synergistic fermentation. According to the method, ginseng, poria cocos, rhizoma zingiberis and liquorice are subjected to differential pretreatment, and a composite functional flora containing ganoderma lucidum, thermophilic lactic acid bacteria and saccharomycopsis fibuligera is constructed; solid-state fermentation is carried out under the conditions of temperature gradient, segmented stirring, gradient ventilation and dynamic material supplementing, so that the contents of key active ingredients such as ginsenoside Rg3, pachymic acid and 6-gingerol are remarkably increased. The fermentation product can be used for preparing a preparation for warming yang, tonifying spleen, clearing damp, reducing phlegm and protecting gastrointestinal tracts. Through multi-bacterium synergistic fermentation and dynamic regulation, the content of ginsenoside Rg3 is increased by 3.2 times or more compared with that of a traditional water decoction method, the content of symbolic components such as pachymic acid and 6-gingerol is increased by 2 times or more, and the drug effect is greatly enhanced; and macromolecular components can be converted into micromolecular active substances through microbial metabolism, so that the digestion burden of gastrointestinal tracts is reduced.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine fermentation preparation technology, specifically to a method for preparing Huangya Tang (a traditional Chinese medicine preparation) based on multi-strain synergistic fermentation and its application. Background Technology

[0002] Huangya Decoction originates from a classic traditional Chinese medicine formula, composed of ginseng, poria cocos, dried ginger, and licorice. It has the effects of warming the yang and strengthening the spleen, dispelling dampness and resolving phlegm, and is widely used in treating spleen and stomach weakness, and internal retention of cold and dampness. Traditional Huangya Decoction is mostly prepared by decoction, which has problems such as low dissolution rate of effective components (e.g., fat-soluble components such as ginsenosides and pachymic acid are difficult to fully release), low bioavailability, and easy formation of precipitates and impurities, thus limiting its clinical application.

[0003] With the application of microbial fermentation technology in the field of traditional Chinese medicine, microbial transformation can disrupt the cell wall structure and degrade macromolecular components of Chinese medicine, thereby increasing the content and bioactivity of effective ingredients. In existing technologies, Chinese medicine fermentation often uses single-strain or simple mixed-strain methods, which suffers from low fermentation efficiency and insufficient transformation of functional components. Furthermore, the static control of parameters such as temperature, aeration, and nutrient supply during fermentation can easily lead to an imbalance in the synergistic effect of the microbial community, affecting the stability of product quality.

[0004] Based on this, developing a multi-strain synergistic and dynamically regulated fermentation method for preparing Huangyatang (a traditional Chinese medicine) to improve the content of effective ingredients, enhance efficacy, and ensure product stability has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the problems of low dissolution rate and insufficient bioavailability of effective components in existing Huangyatang preparation methods, as well as poor synergy of microbial strains and crude control of process parameters in traditional fermentation technologies, this invention provides a Huangyatang preparation method based on multi-strain synergistic fermentation. Through differentiated pretreatment of raw materials, synergistic effect of compound microbial communities, and dynamic gradient fermentation regulation, the effective components are efficiently converted and enriched.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing Huangya soup based on multi-strain synergistic fermentation, comprising the following steps: S1. Raw material differentiation pretreatment After cleaning and removing the fibrous roots, the ginseng was sliced ​​horizontally into pieces with a thickness of 3.0±0.2mm. After peeling the poria cocos, it was trimmed into cubes with a side length of 5.0×5.0×5.0±0.3mm. The dried ginger was pulverized and passed through a 10-mesh sieve (the mass percentage of residue on the sieve is ≤5%, and the mass percentage of fine powder passing through the sieve is ≤10%). The licorice was cleaned and removed from impurities, and then sliced ​​longitudinally into pieces with a thickness of 3.0±0.2mm. The ginseng, poria cocos, dried ginger, and licorice were mixed in a mass ratio of 3:3:1:2 to obtain the mixed raw materials.

[0007] The role of differentiated pretreatment: By controlling the form of raw materials (flakes, cubes, sieved powder), the specific surface area and pore structure of the raw materials are adjusted, providing a suitable microenvironment for microbial colonization and enzymatic hydrolysis in the subsequent fermentation process, and promoting the release and transformation of nutrients.

[0008] S2. Adjusting the substrate moisture content Add deionized water to the mixed raw materials to adjust the initial moisture content to 62%±2% (w / w), and stir at 30 rpm for 5 min to ensure uniform moisture distribution; load the mixed raw materials into a stainless steel fermentation pan, control the layer thickness to 15±2 mm, and sterilize by high pressure steam at 121℃ and 0.12 MPa for 30 min, then cool to 38±1℃ to obtain the sterilized substrate.

[0009] The role of substrate pretreatment: controlling moisture content and layer thickness can avoid local hypoxia or water oversaturation during fermentation; high-pressure sterilization can kill miscellaneous bacteria and endogenous enzymes in the raw materials and eliminate interference with the subsequent complex microbial community.

[0010] S3. Inoculate with a multifunctional microbial community A complex functional microbial community was inoculated into the sterilization matrix. This complex microbial community comprised *Ganoderma lucidum* (CICC NO: 14028), *Streptococcus thermophilus* (CICC NO: 6222), and *Saccharomyces cerevisiae* (CICC NO: 33186), with a viable count ratio of 2:1:1.5 and a total viable count ≥ 5 × 10⁻⁶. 8 CFU / mL; the inoculum size is 8% of the total raw material mass. 0.5% sterile sodium carboxymethyl cellulose is added to the compound microbial community as an adhesive to ensure uniform distribution of the microbial community in the matrix.

[0011] Synergistic effect of complex microbial communities: Ganoderma lucidum can produce cellulase, ligninase and other degradation enzymes to promote the rupture of cell walls of traditional Chinese medicine; Streptococcus thermophilus can regulate the pH of the fermentation system and inhibit the growth of miscellaneous bacteria; and Saccharomyces cerevisiae can convert glycosides to generate highly active secondary metabolites. The three work together to achieve efficient conversion of effective components.

[0012] S4. Dynamic gradient fermentation Dynamic gradient fermentation of the inoculated substrate was controlled, with the following specific parameters: Temperature control: Maintain 38±0.3℃ between 0-24h (adaptation period, promoting rapid colonization of the microbial community); maintain 36±0.3℃ between 24-72h (log phase, enhancing enzymatic hydrolysis and metabolism); maintain 34±0.3℃ between 72-144h (stationary phase, promoting the accumulation of active ingredients); use PID algorithm to adjust the heating / cooling rate with a control accuracy of ±0.3℃.

[0013] Segmented mixing: from 0 to 24 hours, mix once every 6 hours at a speed of 30 rpm for 10 minutes each time; from 24 to 72 hours, mix once every 4 hours at a speed of 40 rpm for 15 minutes each time; from 72 to 144 hours, mix once every 3 hours at a speed of 50 rpm for 20 minutes each time; mixing improves the permeability of the substrate and avoids the accumulation of local metabolites.

[0014] Gradual ventilation: ventilation rate 0.5 L / min·kg between 0-24 h; ventilation rate 0.8 L / min·kg between 24-72 h; ventilation rate 1.0 L / min·kg between 72-144 h; gradually increase ventilation rate as the metabolic intensity of the microbial community increases to meet oxygen demand.

[0015] Dynamic feeding: A complex enzymatic hydrolysate of Poria cocos and Glycyrrhiza uralensis was added in six separate applications (800 mL / 100 kg substrate in total). This hydrolysate was prepared by enzymatic hydrolysis of 1.5% cellulase and 0.5% papain at 50°C and pH 5.5 for 4 hours. Yeast extract containing Gly-Phe-Arg active peptides was added in three separate applications (400 mL / 100 kg substrate in total). The feeding amount was dynamically adjusted online to maintain a real-time C:N ratio of 3:1. A dual-fluid atomizer was used for feeding, with an atomization pressure of 0.2 MPa and a droplet diameter of 50-80 μm to ensure uniform nutrient supply.

[0016] Advantages of dynamic control: By varying the gradients of temperature, stirring, and aeration, the needs of the microbial community at different stages of growth and metabolism can be matched; dynamic feeding can maintain a stable nutrient environment, avoid substrate limitation or metabolite inhibition, and improve fermentation efficiency.

[0017] S5. Terminate fermentation Fermentation was terminated when the total sugar content in the fermentation substrate was ≤4.5 g / kg, the amino nitrogen content was ≤0.15 g / kg, and the pH was stable at 4.2±0.3. The fermentation product was then vacuum dried at 60-65℃ until the moisture content was ≤7.5% (w / w), pulverized and passed through an 80-mesh sieve to obtain the solid product of Huangyatang fermented by multiple bacteria.

[0018] This invention provides a method for preparing Huangya soup based on multi-strain synergistic fermentation and its application. It has the following beneficial effects: 1. This invention, through multi-strain synergistic fermentation and dynamic regulation, increases the content of ginsenoside Rg3 (lipid-soluble active ingredient) by more than 3.2 times compared with the traditional decoction method, and increases the content of marker components such as pachymic acid and 6-gingerol by more than 2 times, thus greatly enhancing the efficacy.

[0019] 2. This invention can convert macromolecular components into small molecule active substances (such as saponins, oligosaccharides, etc.) through microbial metabolism, thereby reducing the digestive burden on the gastrointestinal tract. Animal experiments have verified that the oral bioavailability of the product of this invention is more than 40% higher than that of traditional formulations.

[0020] 3. This invention ensures uniform product quality by using differentiated pretreatment of raw materials, optimization of microbial community ratio, and dynamic parameter control to achieve a coefficient of variation of active ingredient content ≤5% between batches and control of contamination rate of miscellaneous bacteria below 0.1%. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0022] This invention provides a method for preparing Huangya soup based on multi-strain synergistic fermentation, comprising the following steps: S1. Raw material differentiation pretreatment After cleaning and removing the fibrous roots, the ginseng is sliced ​​horizontally into pieces 3.0 mm thick. After peeling the poria cocos, it is trimmed into cubes with sides of 5.0 × 5.0 × 5.0 mm. The dried ginger is crushed and passed through a 10-mesh sieve (the mass percentage of residue on the sieve is ≤5%, and the mass percentage of fine powder passing through the sieve is ≤10%). The licorice is cleaned and then sliced ​​longitudinally into pieces 3.0 mm thick. The ginseng, poria cocos, dried ginger, and licorice are mixed in a mass ratio of 3:2:2:2 to obtain the mixed raw materials.

[0023] S2. Adjusting the substrate moisture content Add deionized water to the mixed raw materials to adjust the initial moisture content to 62% (w / w), and stir at 30 rpm for 5 min to ensure uniform moisture distribution. Place the mixed raw materials into a stainless steel fermentation pan, control the layer thickness to 15 mm, and sterilize by high pressure steam at 121℃ and 0.12 MPa for 30 min. Cool to 38℃ to obtain the sterilized substrate.

[0024] S3. Inoculate with a multifunctional microbial community A complex functional microbial community was inoculated into the sterilization substrate. The complex microbial community included Ganoderma lucidum (CICC NO: 14028), Streptococcus thermophilus (CICC NO: 6222), and Saccharomyces cerevisiae (CICC NO: 33186), with a viable count ratio of 2:1:1.5 and a total viable count ≥ 5 × 10⁻⁶. 8 CFU / mL; the inoculum size is 8% of the total raw material mass. 0.5% sterile sodium carboxymethyl cellulose is added to the compound microbial community as an adhesive to ensure uniform distribution of the microbial community in the matrix.

[0025] S4. Dynamic gradient fermentation Dynamic gradient fermentation of the inoculated substrate was controlled, with the following specific parameters: Temperature control: Maintain 38±0.3℃ between 0-24h (adaptation period, promoting rapid colonization of the microbial community); maintain 36±0.3℃ between 24-72h (log phase, enhancing enzymatic hydrolysis and metabolism); maintain 34±0.3℃ between 72-144h (stationary phase, promoting the accumulation of active ingredients); use PID algorithm to adjust the heating / cooling rate with a control accuracy of ±0.3℃.

[0026] Segmented mixing: from 0 to 24 hours, mix once every 6 hours at a speed of 30 rpm for 10 minutes each time; from 24 to 72 hours, mix once every 4 hours at a speed of 40 rpm for 15 minutes each time; from 72 to 144 hours, mix once every 3 hours at a speed of 50 rpm for 20 minutes each time; mixing improves the permeability of the substrate and avoids the accumulation of local metabolites.

[0027] Gradual ventilation: ventilation rate 0.5 L / min·kg between 0-24 h; ventilation rate 0.8 L / min·kg between 24-72 h; ventilation rate 1.0 L / min·kg between 72-144 h; gradually increase ventilation rate as the metabolic intensity of the microbial community increases to meet oxygen demand.

[0028] Dynamic feeding: A total of 800 mL / 100 kg of substrate was added in six separate applications of a Poria cocos-licorice compound enzymatic hydrolysate. The hydrolysate was prepared by enzymatic hydrolysis of 1.5% cellulase and 0.5% papain at 50℃ and pH 5.5 for 4 hours. A total of 400 mL / 100 kg of substrate was added in three separate applications of yeast extract containing Gly-Phe-Arg active peptides. The feeding amount was dynamically adjusted via online monitoring to maintain a real-time C:N ratio of 3:1. A dual-fluid atomizer was used for feeding, with an atomization pressure of 0.2 MPa and a droplet diameter of 50-80 μm to ensure uniform nutrient supply.

[0029] S5. Terminate fermentation Fermentation was terminated when the total sugar content in the fermentation substrate was ≤4.5 g / kg, the amino nitrogen content was ≤0.15 g / kg, and the pH was stable at 4.2±0.3. The fermentation product was then vacuum dried at 60-65℃ until the moisture content was ≤7.5% (w / w), pulverized and passed through an 80-mesh sieve to obtain the solid product of Huangyatang fermented by multiple bacteria.

[0030] The solid product of Huangya decoction obtained by multi-strain synergistic fermentation in this embodiment was compared with that obtained by traditional decoction process, and the contents of ginsenosides, pachymic acid, gingerol and glycyrrhizic acid in the product were detected. The results are shown in the table below: Table 1

[0031] The results show that when the raw material ratio of 3:2:2:2 is used in this embodiment, the key active ingredients (especially pachymic acid and glycyrrhizic acid) are significantly increased, proving the suitability of the method for the synergistic effect of poria cocos, dried ginger and licorice.

[0032] Example 2: This invention provides a method for preparing Huangya Tang (a traditional Chinese medicine formula) based on multi-strain synergistic fermentation. The mixing ratio of ginseng, poria cocos, dried ginger, and licorice is adjusted to 3:1:3:1, and the remaining steps are the same as in Example 1. The results are shown in the table below: Table 2

[0033] The results showed that after increasing the proportion of dried ginger, the content of 6-gingerol was further increased (by 4.13 times), demonstrating the method's ability to enhance the core efficacy of warming yang. Other components (such as pachymic acid and glycyrrhizic acid) also showed significant increases, indicating the adaptability of the formulation adjustment.

[0034] Example 3: This invention provides a method for preparing Huangya Tang (a type of herbal decoction) based on multi-strain synergistic fermentation. The original temperature gradient is changed to maintain 37±0.3℃ between 0-24h; 35±0.3℃ between 24-72h; and 33±0.3℃ between 72-144h. The remaining steps are the same as in Example 1. The results of comparing this example with Example 1 are shown in the table below: Table 3

[0035] The results showed that although lowering the initial fermentation temperature did not significantly affect the total amount of active ingredients (the decrease was <6%), it prolonged the fermentation cycle and reduced the risk of contamination by other microorganisms, making it suitable for scenarios with higher requirements for microbial control.

[0036] Example 4: This invention provides a method for preparing Huangya Tang (a traditional Chinese medicine formula) based on multi-strain synergistic fermentation. The original dynamic feeding method is adjusted as follows: Poria-Glycyrrhiza compound enzymatic hydrolysate is added in four portions (800 mL / 100 kg substrate in total); yeast extract containing Gly-Phe-Arg active peptides is added in two portions (400 mL / 100 kg substrate in total). The feeding amount is dynamically adjusted through online monitoring to maintain a real-time C:N ratio of 3:1. The remaining steps are the same as in Example 1. The results of this embodiment compared with Example 1 are shown in the table below: Table 4

[0037] The results showed that reducing the feeding frequency in this embodiment led to uneven nutrient supply, decreased efficiency of microbial synergistic metabolism, and a significant increase in the total number of miscellaneous bacteria, proving that the original feeding strategy was the optimal choice.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Huangya soup based on multi-strain synergistic fermentation, characterized in that: Includes the following steps: S1. Raw material differentiation pretreatment After cleaning and removing the fibrous roots, the ginseng is sliced ​​horizontally into pieces with a thickness of 3.0±0.2mm. After peeling the poria cocos, it is trimmed into cubes with a side length of 5.0×5.0×5.0±0.3mm. The dried ginger is crushed and passed through a 10-mesh sieve. The licorice is cleaned and removed of impurities, then sliced ​​longitudinally into pieces with a thickness of 3.0±0.2mm. The ginseng, poria cocos, dried ginger, and licorice are mixed in a mass ratio of 3:3:1:

2. S2. Adjusting the substrate moisture content Add deionized water to the mixed raw materials and adjust the initial water content to 62%±2% (w / w). Stir at 30 rpm for 5 min, pour into a stainless steel fermentation pan, with a layer thickness of 15±2 mm, and autoclave at 121℃ and 0.12 MPa for 30 min, then cool to 38±1℃. S3. Inoculate with a multifunctional microbial community The complex microbial flora includes Ganoderma lucidum, Streptococcus thermophilus, and Saccharomyces cerevisiae, with a viable count ratio of 2:1:1.5 and a total viable count ≥5×10⁻⁶. 8 CFU / mL, inoculum size is 8% of the total mass of raw materials; S4. Dynamic gradient fermentation Temperature control: Maintain 38±0.3℃ between 0-24h, 36±0.3℃ between 24-72h, and 34±0.3℃ between 72-144h; Segmented mixing: from 0 to 24 hours, mix once every 6 hours at a speed of 30 rpm for 10 minutes each time; from 24 to 72 hours, mix once every 4 hours at a speed of 40 rpm for 15 minutes each time; from 72 to 144 hours, mix once every 3 hours at a speed of 50 rpm for 20 minutes each time. Gradient ventilation: ventilation rate 0.5 L / min·kg between 0-24 h, ventilation rate 0.8 L / min·kg between 24-72 h, ventilation rate 1.0 L / min·kg between 72-144 h; Dynamic feeding: Poria cocos-licorice compound enzymatic hydrolysate was added in 6 batches, with a cumulative addition of 800 mL / 100 kg of substrate; yeast extract containing Gly-Phe-Arg active peptides was added in 3 batches, with a cumulative addition of 400 mL / 100 kg of substrate, maintaining real-time C:N=3:1; S5. Terminate fermentation Fermentation was terminated when total sugar ≤ 4.5 g / kg, amino nitrogen ≤ 0.15 g / kg, and pH stabilized at 4.2 ± 0.

3. The product was dried and pulverized to obtain solid fermentation product.

2. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S1, the dried ginger is sieved through a 10-mesh sieve, and the mass of residue on the sieve accounts for no more than 5% of the total mass of the dried ginger, while the mass of fine powder under the sieve accounts for no more than 10% of the total mass of the dried ginger.

3. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S3, the composite microbial community also includes 0.5% sterile sodium carboxymethyl cellulose, which serves as an adhesive to ensure that the composite microbial community is evenly distributed in the raw material matrix after inoculation.

4. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S3, the Ganoderma lucidum fungus is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC No:14028; the Streptococcus thermophilus is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC No:6222; and the Saccharomyces cerevisiae is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC No:33186.

5. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S4, the feeding of the Poria cocos-licorice complex enzymatic hydrolysate was carried out using a dual-fluid atomizer with an atomization pressure of 0.2 MPa and a droplet diameter controlled at 50-80 μm. The amount of yeast extract added was dynamically adjusted by monitoring the matrix nutrients online.

6. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S4, the temperature control uses a PID algorithm to adjust the heating or cooling rate, and the temperature control accuracy is ±0.3℃ throughout the process.

7. The method for preparing Huangya soup based on multi-strain synergistic fermentation according to claim 1, characterized in that: In step S4, the Poria cocos-licorice complex enzymatic hydrolysate is prepared by enzymatic hydrolysis of 1.5% cellulase and 0.5% papain at 50°C and pH 5.5 for 4 hours.

8. The multi-strain synergistic fermentation of Huangya soup prepared by the preparation method according to any one of claims 1-5, characterized in that: The Huangya decoction contains ginsenoside Rg3 with a content ≥1.80% (w / w), pamoate acid with a content ≥2.80% (w / w), 6-gingerol with a content ≥2.10% (w / w), moisture content ≤7.5% (w / w), and total bacterial count ≤500 CFU / g.

9. The application of the multi-strain synergistic fermented Huangya Decoction according to claim 8 in a warming and spleen-strengthening, dampness-removing and phlegm-resolving drug, wherein the drug uses the solid fermentation product obtained according to claim 1 as the active ingredient and exists in the form of tablets or granules, wherein the daily dose of the solid fermentation product is 100-500 mg / kg body weight.

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