Ginseng ladder fermentation extraction process and application thereof

Abstract

This invention belongs to the field of ginseng fermented food technology, specifically relating to a ginseng step-fermentation extraction process and its application. The process includes: S1: The raw material is washed, dried, pulverized, and sterilized to obtain coarse powder; S2: Water is added for moistening, and mild enzymatic hydrolysis pretreatment is performed using cellulase and hemicellulase; S3: A compound sugar source and a compound microbial strain are added for semi-solid pre-fermentation; S4: Water is added to transfer to a liquid system and stage-switching heat treatment is performed; S5: Compound sugar source and microbial strain are added again for staged temperature-controlled secondary liquid fermentation; S6: After settling, filtration, low-temperature vacuum concentration, and refrigeration, the extract is obtained. This invention effectively improves the release efficiency of ginseng active ingredients and enhances the targeted enrichment of rare saponins through the synergy of pretreatment and step-fermentation; the product effectively reduces the raw, grassy smell and bitterness, has a harmonious flavor, and exhibits good system stability.

Description

Technical Field

[0001] This invention belongs to the field of ginseng fermented food technology, specifically relating to a ginseng step fermentation extraction process and its application. Background Technology

[0002] Ginseng is a common food and medicinal ingredient, containing ginsenosides, polysaccharides, amino acids, volatile components, and various trace elements. It has been widely used in the food, health food, and fermented product industries. In existing technologies, ginseng raw materials are typically prepared into related products through slicing, crushing, water extraction, alcohol extraction, and concentration. Alternatively, ginseng is added as a plant substrate to microbial fermentation systems such as lactic acid bacteria and yeast to improve flavor and promote the release or transformation of some components.

[0003] However, ginseng raw materials have a relatively dense structure, and the cell wall components limit the release of intracellular and bound components. Furthermore, ginseng has a pronounced bitter taste, woody aroma, or raw, grassy smell, often resulting in insufficient palatability when used directly in food processing. While existing technologies can improve the flavor of fermented ginseng products to some extent through microbial fermentation, they still generally suffer from the following shortcomings: Firstly, using only liquid fermentation can easily lead to inefficient release of ginseng soluble components due to insufficient substrate utilization; secondly, excessive heat treatment, prolonged fermentation, or simply increasing the amount of added sugar can easily lead to flavor imbalance, rapid increase in acidity, loss of aroma, or decreased system stability.

[0004] Furthermore, existing fermentation techniques for ginseng raw materials mostly focus on single-stage fermentation, single inoculation, or single-feeding models, with relatively insufficient attention paid to substrate state adjustment before fermentation, switching between fermentation stages, and the differences in the effects of sugar sources and strains at different fermentation stages. Particularly when using ginseng powder or high-solids ginseng materials as substrates, how to ensure good fermentability while avoiding excessive liquefaction due to over-pretreatment; and how to promote the release and fermentation conversion of ginseng soluble components while maintaining flavor harmony and subsequent concentration stability, remain technical issues requiring further optimization in this field. Summary of the Invention

[0005] This invention addresses the shortcomings of existing ginseng fermentation processing techniques by providing a ginseng step-fermentation extraction process and its application. This invention resolves the contradiction between the relatively dense structure of ginseng tissue, insufficient release of soluble components, and the potential for excessive softening of the material due to overly strong pretreatment, which could even affect subsequent stacking fermentation. It also resolves the contradiction between promoting fermentation conversion and maintaining the harmony of fermentation flavor and the stability of the system.

[0006] The technical effects described in this invention are achieved through the following technical solution: a ginseng step fermentation extraction process, specifically including the following steps: S1: Select food-grade ginseng raw materials, remove surface impurities, wash and drain the surface water, and dry them with hot air at 50-65℃ until the ginseng moisture content drops to 8-12%; pulverize the dried ginseng and pass it through a 20-40 mesh sieve, and pre-sterilize it to obtain ginseng coarse powder for later use. S2: Add purified water equivalent to 20-30% of the mass of the ginseng powder in step S1 to moisten the powder, let it stand for 0.5-1.5 hours to allow the water to be evenly distributed in the powder, and obtain moistened ginseng powder; on this basis, add a mild enzymatic hydrolysis system to the moistened ginseng powder for pretreatment to obtain pretreated ginseng material. S3: In the pretreated ginseng material in step S2, add 0.5-2% honey, 1-3% white sugar powder, 1-3% glucose powder and 1-3% brown sugar powder according to the mass of ginseng powder, and stir to evenly disperse the compound sugar source in the material; then inoculate with food-grade compound fermentation bacteria, mix well to obtain primary fermentation material, and then carry out fermentation treatment; S4: After the first solid-state pre-fermentation is completed, the material is transferred to a fermentation tank, and purified water equivalent to 15 to 25 times the mass of the first fermentation material is added. The mixture is stirred evenly, and then a stage switching heat treatment is performed. After the heat treatment is completed, the system is cooled to 30 to 38°C in order to carry out a second inoculation. S5: In the liquid fermentation system cooled in step S4, add 0.5-2% honey, 1-3% white sugar powder, 1-3% glucose powder and 1-3% brown sugar powder according to the weight of the original ginseng powder, and add food-grade compound fermentation bacteria again. After mixing evenly, carry out secondary liquid fermentation. S6: After the secondary liquid fermentation is completed, the fermentation broth is allowed to stand for 4–12 hours to allow larger suspended particles to settle naturally. Then, it is subjected to coarse filtration at 80–120 mesh and fine filtration at 200–400 mesh to remove insoluble residues, resulting in a clear fermentation filtrate. The obtained fermentation filtrate is then placed in a vacuum concentration device for low-temperature concentration, with the concentration temperature controlled at 50–60°C. The relative density is measured to be 1.15–1.25 at 60°C, yielding a ginseng fermentation concentrate. The ginseng fermentation concentrate is then filtered again and refrigerated at 2–8°C for 12–24 hours to obtain the ginseng fermentation extract. Preferably, in step S1, the pre-sterilization treatment parameters are 95-100℃ for 20-30 minutes; this reduces the risk of contamination of ginseng powder by miscellaneous bacteria during subsequent fermentation. Preferably, in step S2, the mild enzymatic hydrolysis system is a compound system composed of cellulase and hemicellulase in a mass ratio of 1:1 to 2. The cellulase and hemicellulase are commonly used enzyme preparations in the food industry. The cellulase activity is 10,000 to 50,000 U / g, and the hemicellulase activity is 10,000 to 50,000 U / g. The two work synergistically to the cell wall structure of ginseng tissue, promoting moderate loosening of ginseng tissue without excessive liquefaction of the material, thereby improving substrate accessibility and the release efficiency of soluble components during subsequent fermentation. Preferably, in step S2, the amount of the mild enzymatic hydrolysis system added is 0.2-0.6% of the mass of the crude ginseng powder; the enzymatic hydrolysis temperature is 40-55℃, the enzymatic hydrolysis time is 1-2 hours, and the pH is the natural pH of the material; the mild enzymatic hydrolysis pretreatment is intended to promote a moderately loose ginseng tissue structure, reduce the mass transfer resistance of subsequent fermentation, and improve the release efficiency of soluble components, without aiming at excessive liquefaction of the material; In step S2, the pretreated material should preferably be kept in a semi-solid state that allows it to be stacked and turned over. Preferably, in step S3, the food-grade compound fermentation strain is a compound system composed of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in a mass ratio of 2–4:1; wherein the viable count of *Lactobacillus plantarum* is not less than 1 × 10⁻⁶. 9 CFU / g, viable count of brewer's yeast not less than 1×10⁻⁶ 8 CFU / g; the inoculation amount of the food-grade compound fermentation strain is 2-5% of the mass of the pretreated ginseng material; among which, Lactobacillus plantarum is used to regulate the acidity of the fermentation system, inhibit miscellaneous bacteria and establish a stable fermentation environment, and Saccharomyces cerevisiae is used to improve the fermentation aroma, mitigate the original raw green smell and bitterness of ginseng, thereby jointly enhancing the flavor coordination of the fermentation system. Preferably, in step S3, the fermentation process specifically involves: placing the primary fermentation material in a sterilized container, controlling the material thickness to be 3-10 cm, and carrying out semi-solid stacking pre-fermentation at 36-40°C for 10-15 days; during the fermentation period, stirring is performed 1-2 times daily to promote uniform temperature and humidity and full contact between the microorganisms and the substrate; Preferably, in step S4, the stage switching heat treatment parameters are 95-100℃ and the holding time is 20-30 minutes. The purpose is to terminate the excessive fermentation of the previous stage, reduce the risk of interference from miscellaneous bacteria, and use the heat to further soften some ginseng tissue and release soluble components, thus creating conditions for subsequent liquid-state in-depth fermentation. Preferably, in step S5, the inoculation amount of the food-grade compound fermentation strain is 1-3% of the mass of the liquid fermentation system; Preferably, in step S5, the secondary liquid fermentation is carried out using a staged temperature control method, specifically: Early stage of enhanced fermentation: the temperature is controlled at 36–38°C for 4–7 days to promote the rapid establishment of a dominant metabolic state in the microorganisms and accelerate the release and transformation of ginseng soluble components; Later stage of coordinated fermentation: the temperature is adjusted to 30–34°C and fermentation continues for 5–10 days to slow down the rapid rise in acidity, promote flavor coordination, and improve system stability; More preferably, pH, odor, and soluble solids changes are periodically monitored during fermentation; when the pH of the fermentation broth changes by no more than 0.10 for two consecutive days, and the soluble solids content changes by no more than 0.5°Brix for two consecutive days, and the fermentation broth has no obvious abnormal rancid odor, the fermentation is considered complete. In this process, the compound sugar source and compound fermentation strain are added in two stages. The first addition occurs in the semi-solid pre-fermentation stage, specifically in S3, where the compound sugar source and compound fermentation strain are added. This is mainly used to establish a dominant fermentation microecology under semi-solid conditions, promote the adaptation of the microorganisms to the ginseng substrate and initial metabolism, and pre-regulate the original bitter flavor of ginseng, laying the foundation for subsequent liquid-state deep fermentation. The second addition occurs in the liquid-state deep fermentation stage after heat treatment, specifically in S5, where the compound sugar source and compound fermentation strain are added again. This is mainly used to rebuild and enhance fermentation activity in the liquid system, enabling the microorganisms to re-establish a dominant metabolic state after heat treatment, and further promoting the release of soluble components of ginseng, flavor coordination, and fermentation transformation. In step S6, low-temperature vacuum concentration helps to increase the solid content while reducing the adverse effects of prolonged high-temperature treatment on fermentation aroma, ginseng characteristic flavor and some heat-sensitive components. Another aspect of the present invention is to provide an application of the ginseng fermentation extract obtained by the above process in functional foods or health products.

[0007] The beneficial effects of this invention are as follows: This invention first activates crude ginseng powder by wetting it, and then further pretreats the ginseng tissue using a mild enzymatic hydrolysis system composed of cellulase and hemicellulase. This moderately loosens the ginseng tissue structure without excessive liquefaction, thereby improving substrate accessibility and the release efficiency of soluble components during subsequent fermentation. Simultaneously, the pretreated material remains in a semi-solid state that allows for stacking and turning, facilitating seamless transition to subsequent semi-solid stacking pre-fermentation. Thus, a good synergistic relationship is established between the pretreatment and subsequent fermentation steps, improving mass transfer conditions while also ensuring the adaptability of the material morphology to the fermentation operation.

[0008] This invention employs a complex sugar source system composed of honey, white sugar, glucose, and brown sugar during fermentation, combined with a complex fermentation strain consisting of *Lactobacillus plantarum* and *Saccharomyces cerevisiae*. The complex sugar source provides a continuous carbon source for the microorganisms and improves the overall flavor profile; *Lactobacillus plantarum* helps regulate system acidity, inhibits unwanted microorganisms, and establishes a more stable fermentation environment; and *Saccharomyces cerevisiae* improves the fermentation aroma and mitigates the original raw, grassy, ​​and bitter taste of ginseng. Furthermore, this invention utilizes a two-stage addition of the complex sugar source and the complex fermentation strain. Stage S3 primarily focuses on establishing the dominant microbial community, microbial adaptation, and pre-regulating the flavor of the raw materials, while Stage S5 primarily focuses on rebuilding fermentation activity in the liquid system, further releasing soluble components, and perfecting the flavor. This creates a synergistic relationship between the two additions in terms of function.

[0009] This invention employs a stepped fermentation path, consisting of a semi-solid stacked pre-fermentation followed by a liquid-state in-depth fermentation. The higher solids environment in the first stage facilitates the gradual adaptation of the microorganisms to the ginseng substrate and allows for initial flavor adjustment. The second stage, through the addition of water and the construction of a liquid system, further improves mass transfer and component dissolution conditions, enabling the previously activated ginseng substrate to continue its release and fermentation transformation. A stage-switching heat treatment step between the two stages helps to terminate over-fermentation in the first stage and reduce the risk of continuous interference from other microorganisms. It also promotes further softening of some ginseng tissues and the release of soluble components, creating more suitable system conditions for subsequent liquid-state in-depth fermentation. Combined with the staged temperature control method in the secondary liquid fermentation, this invention achieves a good balance between early fermentation efficiency and later flavor coordination and system stability. After completing the secondary liquid fermentation, the invention further undergoes post-processing steps including settling, filtration, low-temperature vacuum concentration, and refrigerated settling. Among them, settling and filtration are beneficial for removing larger particulate matter and improving the uniformity of the system. Low-temperature vacuum concentration is beneficial for increasing the solid content while reducing the adverse effects of long-term high-temperature treatment on fermentation aroma, ginseng characteristic flavor and some heat-sensitive components. Refrigerated settling is beneficial for further promoting flavor coordination and sedimentation of unstable suspended components. Attached Figure Description

[0010] Figure 1 The graph shows the pH changes of ginseng fermentation extracts from Example 1 and Comparative Examples 2, 3, and 5 under different storage conditions. Figure 2 The graph shows the changes in soluble solids in the ginseng fermentation extracts of Example 1 and Comparative Examples 2, 3, and 5 under different storage conditions. Figure 3 The graph shows the turbidity changes of ginseng fermentation extracts from Example 1 and Comparative Examples 2, 3, and 5 under different storage conditions. Figure 4 This is the HPLC chromatogram of the ginseng fermentation extract from Example 1. Detailed Implementation

[0011] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer. Example

[0012] A stepwise fermentation extraction process for ginseng specifically includes the following steps: S1: Select food-grade ginseng raw materials, remove surface impurities, wash and drain the surface moisture, and dry with hot air at 58℃ until the ginseng moisture content drops to 10%; pulverize the dried ginseng, pass it through a 30-mesh sieve, and treat it at 98℃ for 25 minutes to obtain coarse ginseng powder for later use. S2: Add purified water equivalent to 25% of the ginseng powder mass from step S1 to moisten the powder, let it stand for 1 hour to allow the moisture to be evenly distributed in the powder, and obtain moistened ginseng powder; on this basis, add a compound system of cellulase and hemicellulase in a mass ratio of 1:1.5 to the moistened ginseng powder, with cellulase activity of 30000 U / g and hemicellulase activity of 30000 U / g, and the addition amount is 0.4% of the ginseng powder mass; the enzymatic hydrolysis temperature is 48℃ and the enzymatic hydrolysis time is 1.5 hours; to obtain pretreated ginseng material; S3: In the pretreated ginseng material of step S2, add 1.2% honey, 2% white sugar powder, 2% glucose powder, and 2% brown sugar powder by weight of ginseng powder, and stir to evenly disperse the compound sugar source in the material; then inoculate with Lactobacillus plantarum and Saccharomyces cerevisiae in a 3:1 mass ratio to form a compound fermentation strain, with the viable count of Lactobacillus plantarum not less than 1×10⁻⁶. 9 CFU / g, viable count of brewer's yeast not less than 1×10⁻⁶ 8 CFU / g, the inoculation amount is 3% of the mass of the pretreated ginseng material; after mixing, the primary fermentation material is obtained, and the primary fermentation material is placed in a sterilized container, the material thickness is controlled to be 6cm, and semi-solid stacking pre-fermentation is carried out at 38℃ for 12 days; during the fermentation period, it is turned over twice a day. S4: After the first solid-state pre-fermentation is completed, the material is transferred to a fermentation tank, and purified water equivalent to 20 times the mass of the first fermentation material is added. The mixture is stirred evenly, and then a stage switching heat treatment is performed, which is kept at 98℃ for 25 minutes. After the heat treatment is completed, the system is cooled to 35℃ in order to carry out the second inoculation. S5: In the liquid fermentation system cooled in step S4, add 1.2% honey, 2% white sugar powder, 2% glucose powder and 2% brown sugar powder according to the original ginseng powder mass, and add 2% food-grade compound fermentation bacteria according to the liquid fermentation system mass again. After mixing evenly, carry out secondary liquid fermentation. In the early stage of enhanced fermentation, the temperature is controlled at 37℃ and fermentation lasts for 5 days. In the later stage of coordinated fermentation, the temperature is adjusted to 32℃ and fermentation continues for 7 days. S6: After the secondary liquid fermentation is completed, the fermentation broth is allowed to stand for 8 hours to allow larger suspended particles to settle naturally. Then, it is subjected to 100-mesh coarse filtration and 300-mesh fine filtration to remove insoluble residues and obtain a clear fermentation filtrate. The obtained fermentation filtrate is placed in a vacuum concentration device for low-temperature concentration, with the concentration temperature controlled at 55℃. The relative density is measured to be 1.2 at 60℃, and ginseng fermentation concentrate is obtained. The ginseng fermentation concentrate is filtered again and then placed at 4℃ for 18 hours to obtain ginseng fermentation extract. Example

[0013] A stepwise fermentation extraction process for ginseng specifically includes the following steps: S1: Select food-grade ginseng raw materials, remove surface impurities, wash and drain the surface moisture, and dry with hot air at 50℃ until the ginseng moisture content drops to 8%; pulverize the dried ginseng, pass it through a 20-mesh sieve, and treat it at 95℃ for 30 minutes to obtain coarse ginseng powder for later use. S2: Add purified water equivalent to 20% of the mass of the ginseng crude powder from step S1 to moisten it, let it stand for 0.5 hours to allow the moisture to be evenly distributed in the powder, and obtain moistened ginseng powder; on this basis, add a compound system of cellulase and hemicellulase in a 1:1 mass ratio to the moistened ginseng powder, with cellulase activity of 10000 U / g and hemicellulase activity of 10000 U / g, and the addition amount is 0.6% of the mass of the ginseng crude powder; the enzymatic hydrolysis temperature is 40℃ and the enzymatic hydrolysis time is 2 hours; obtain pretreated ginseng material; S3: In the pretreated ginseng material of step S2, add 0.5% honey, 1% white sugar powder, 1% glucose powder, and 1% brown sugar powder by weight of ginseng powder, and stir to evenly disperse the complex sugar source in the material; then inoculate with a compound system of Lactobacillus plantarum and Saccharomyces cerevisiae in a 2:1 mass ratio, with the viable count of Lactobacillus plantarum not less than 1×10⁻⁶. 9 CFU / g, viable count of brewer's yeast not less than 1×10⁻⁶ 8 CFU / g, the inoculation amount is 2% of the mass of the pretreated ginseng material; after mixing, the primary fermentation material is obtained, and the primary fermentation material is placed in a sterilized container, the material thickness is controlled to be 3cm, and semi-solid stacking pre-fermentation is carried out at 36℃ for 15 days; the material is turned over once a day during the fermentation period. S4: After the first solid-state pre-fermentation is completed, the material is transferred to a fermentation tank, and purified water equivalent to 15 times the mass of the first fermentation material is added. The mixture is stirred evenly, and then a stage switching heat treatment is performed, which is kept at 95°C for 30 minutes. After the heat treatment is completed, the system is cooled to 30°C in order to carry out the second inoculation. S5: In the liquid fermentation system cooled in step S4, add 0.5% honey, 1% white sugar powder, 1% glucose powder and 1% brown sugar powder according to the original ginseng powder mass, and add 1% food-grade compound fermentation bacteria according to the liquid fermentation system mass again. After mixing evenly, carry out a second liquid fermentation. In the early stage of enhanced fermentation, the temperature is controlled at 36℃ and fermentation lasts for 7 days. In the later stage of coordinated fermentation, the temperature is adjusted to 30℃ and fermentation continues for 10 days. S6: After the secondary liquid fermentation is completed, the fermentation broth is allowed to stand for 4 hours to allow larger suspended particles to settle naturally. Then, it is subjected to coarse filtration at 80 mesh and fine filtration at 200 mesh to remove insoluble residues, resulting in a clear fermentation filtrate. The obtained fermentation filtrate is placed in a vacuum concentration device for low-temperature concentration, with the concentration temperature controlled at 50℃. The relative density is measured to be 1.15 at 60℃, resulting in ginseng fermentation concentrate. The ginseng fermentation concentrate is filtered again and then refrigerated at 8℃ for 12 hours to obtain ginseng fermentation extract. Example

[0014] A stepwise fermentation extraction process for ginseng specifically includes the following steps: S1: Select food-grade ginseng raw materials, remove surface impurities, wash and drain the surface moisture, and dry with hot air at 65℃ until the ginseng moisture content drops to 12%; pulverize the dried ginseng, pass it through a 40-mesh sieve, and treat it at 100℃ for 20 minutes to obtain coarse ginseng powder for later use. S2: Add purified water equivalent to 30% of the mass of the ginseng crude powder from step S1 to moisten it, and let it stand for 1.5 hours to allow the moisture to be evenly distributed in the powder, thus obtaining moistened ginseng powder. Then, add a compound system of cellulase and hemicellulase in a 1:2 mass ratio to the moistened ginseng powder. The cellulase activity is 50,000 U / g, and the hemicellulase activity is 50,000 U / g. The addition amount is 0.2% of the mass of the ginseng crude powder. The enzymatic hydrolysis temperature is 55℃, and the enzymatic hydrolysis time is 1 hour, thus obtaining pretreated ginseng material. S3: In the pretreated ginseng material of step S2, add 2% honey, 3% white sugar powder, 3% glucose powder, and 3% brown sugar powder according to the mass of ginseng powder, and stir to evenly disperse the complex sugar source in the material; then inoculate with a compound system of Lactobacillus plantarum and Saccharomyces cerevisiae in a 4:1 mass ratio, with the viable count of Lactobacillus plantarum not less than 1×10⁻⁶. 9 CFU / g, viable count of brewer's yeast not less than 1×10⁻⁶ 8CFU / g, the inoculation amount is 5% of the mass of the pretreated ginseng material; after mixing, the primary fermentation material is obtained, and the primary fermentation material is placed in a sterilized container, the material thickness is controlled to be 10cm, and semi-solid stacking pre-fermentation is carried out at 40℃ for 10 days; during the fermentation period, it is turned over twice a day. S4: After the first solid-state pre-fermentation is completed, the material is transferred to a fermentation tank, and purified water equivalent to 25 times the mass of the first fermentation material is added. The mixture is stirred evenly, and then a stage switching heat treatment is performed, which is kept at 100℃ for 20 minutes. After the heat treatment is completed, the system is cooled to 38℃ in order to carry out the second inoculation. S5: In the liquid fermentation system cooled in step S4, add 2% honey, 3% white sugar powder, 3% glucose powder and 3% brown sugar powder according to the original ginseng powder mass, and add 3% food-grade compound fermentation bacteria according to the liquid fermentation system mass again. After mixing evenly, carry out secondary liquid fermentation. In the early stage of enhanced fermentation, the temperature is controlled at 38℃ and fermentation lasts for 4 days. In the later stage of coordinated fermentation, the temperature is adjusted to 34℃ and fermentation continues for 5 days. S6: After the secondary liquid fermentation is completed, the fermentation broth is allowed to stand for 12 hours to allow larger suspended particles to settle naturally. Then, it is subjected to coarse filtration at 120 mesh and fine filtration at 400 mesh to remove insoluble residues and obtain a clear fermentation filtrate. The obtained fermentation filtrate is placed in a vacuum concentration device for low-temperature concentration, with the concentration temperature controlled at 60℃. The relative density is measured to be 1.25 at 60℃, and ginseng fermentation concentrate is obtained. The ginseng fermentation concentrate is filtered again and then placed at 2℃ for 24 hours to obtain ginseng fermentation extract.

[0015] Comparative Example 1: Compared with Example 1, mild enzymatic pretreatment was performed without the addition of cellulase and hemicellulase, and the remaining steps were the same.

[0016] Comparative Example 2: Compared with Example 1, the semi-solid stacking pre-fermentation in step S3 was not performed. The pretreated ginseng material obtained in step S2 directly entered step S4, and the remaining steps were the same.

[0017] Comparative Example 3: Compared with Example 1, the complex sugar source and complex fermentation strain in S3 and S5 were added all at once in the initial stage of fermentation, and no further additions were made thereafter. The remaining steps were the same.

[0018] Comparative Example 4: Compared with Example 1, the food-grade compound fermentation strain was changed to use only Lactobacillus plantarum, while the other steps remained the same.

[0019] Comparative Example 5: Compared with Example 1, the secondary liquid fermentation stage did not use a staged temperature control method, but instead used a constant temperature fermentation at 36°C throughout the process, while the other steps were the same.

[0020] Performance testing: The samples tested were Examples 1-3 and Comparative Examples 1-5. Heavy metal residues were determined according to GB 5009.12 (lead), GB 5009.11 (arsenic), GB 5009.17 (mercury), and GB 5009.15 (cadmium). The test results are shown in Table 1 below. Microbial contamination was determined according to GB 4789.2 (total bacterial count), GB 4789.3 (coliform bacteria), and GB 4789.15 (mold and yeast). Physicochemical indicators were determined according to GB 5009.3 (moisture) and GB 5009.4 (ash). The test results are shown in Table 2 below.

[0021] Table 1. Results of heavy metal residue tests in ginseng fermentation extracts from Examples 1-3 and Comparative Examples 1-5

[0022] Table 2. Microbial contamination and basic physicochemical index test results of ginseng fermentation extracts from Examples 1-3 and Comparative Examples 1-5

[0023] As shown in Tables 1 and 2, the residual levels of lead, arsenic, mercury, and cadmium in each group of samples were low, indicating that the ginseng raw materials used were relatively clean and that no significant heavy metal contamination was introduced during the entire fermentation and extraction process. Furthermore, no significant abnormal microbial contamination was detected in any group of samples, indicating that the fermentation, filtration, concentration, and refrigeration steps generally ensured the hygienic quality of the product. The results of this group indicate that heavy metal residues are mainly affected by the background of the raw materials and the degree of final concentration. The differences between the examples and the comparative examples were small, indicating that the technical effect of this invention does not stem from differences in raw material safety. Microbial contamination better reflects the quality of the process. Example 1, due to its use of mild enzymatic hydrolysis, semi-solid pre-fermentation, two-stage sugar and bacteria supplementation, and staged temperature-controlled fermentation, achieved a more complete establishment of dominant microbial communities and a more stable system, resulting in the lowest total bacterial count and the lowest number of molds and yeasts. Although Examples 2 and 3 were both superior to the comparative examples, their overall control effect was slightly inferior to Example 1. Comparative Examples 2 and 3 had the weakest microecological control due to the weakened synergistic effect of stepped fermentation and staged supplementation.

[0024] Flavor Evaluation Test: Ten evaluators with experience in ginseng flavor sensory evaluation were selected and given 15 minutes of specialized training to clarify the scoring rules: bitterness (1-5 points, 5 points indicating no bitterness and a smooth taste), grassy aroma (1-5 points, 5 points indicating no grassy taste interference), ginseng fragrance (1-5 points, 5 points indicating a prominent unique ginseng fragrance), and aftertaste harmony (1-5 points, 5 points indicating a refreshing aftertaste without lingering). The test was conducted in an independent sensory chamber with no off-odors, uniform lighting, and a temperature of 25°C. The samples were ginseng fermentation extracts obtained from Examples 1-3 and Comparative Examples 1-5. After diluting with purified water at a ratio of 1:10, the evaluators observed the appearance, smelled the aroma, and tasted 5 mL of the diluted solution, holding it for 10 seconds before spitting it out. They immediately scored the four indicators (1-5 points each). There was a 3-minute interval between each two samples, and the mouth was rinsed with purified water and cleaned with salt-free soda crackers. Each evaluation consisted of no more than 5 samples and was completed in two rounds. The overall acceptability was calculated using a weighted average (formula: 0.4 × bitterness + 0.3 × raw grassiness + 0.2 × ginseng aroma + 0.1 × aftertaste harmony). All scores were taken as the average of 10 evaluators. The test results are shown in Table 3 below.

[0025] Table 3. Flavor evaluation test results of ginseng fermentation extracts from Examples 1-3 and Comparative Examples 1-5

[0026] As shown in Table 3, Example 1 achieved the highest scores in all four indicators: bitterness, grassy aroma, ginseng fragrance, and aftertaste harmony. Its overall acceptability was higher than that of Examples 2 and 3, as well as the comparative examples, indicating that this example's process achieves a better balance in terms of mitigating bitterness, reducing grassy aroma, preserving the characteristic ginseng fragrance, and harmonizing the aftertaste. In Comparative Example 1, the overall acceptability decreased significantly after the mild enzymatic hydrolysis pretreatment was removed, indicating that mild enzymatic hydrolysis improves substrate accessibility, allowing for more complete subsequent fermentation, thereby reducing bitterness and improving ginseng aroma release. In Comparative Example 2, the scores decreased after the S3 semi-solid-state stacked pre-fermentation was removed, indicating that the stepped fermentation path plays an important role in the early substrate pre-adaptation, flavor pre-adjustment, and subsequent liquid-state deep fermentation. In Comparative Example 3, the overall acceptability decreased after the complex sugar source and complex fermentation strain were added all at once, indicating that the two-stage additions are not simply repetitive but contribute to the seamless transition between the early and late fermentation stages and the formation of flavor layers. Comparative Example 4, which used only *Lactobacillus plantarum*, had the lowest scores for ginseng aroma and aftertaste harmony, as well as the lowest overall acceptability. This indicates that brewing yeast has a significant synergistic effect in improving aroma, mitigating the grassy smell, and enhancing aftertaste harmony. Comparative Example 5, which underwent constant fermentation at 36℃ throughout, showed slightly better bitterness and grassy smell compared to Comparative Examples 2 and 4, but its aftertaste harmony was still lower than Example 1, and its overall acceptability also decreased significantly. This suggests that a staged temperature control method is more conducive to balancing early fermentation efficiency and later flavor harmony.

[0027] Component release and fermentation effect test: Ginseng fermentation extract samples obtained in Examples 1-3 and Comparative Examples 1-5 were used to determine the soluble solids, total saponin content, and total polysaccharide content, respectively. Soluble solids were measured using a handheld refractometer and expressed as °Brix. Total saponin content was determined using the vanillin-glacial acetic acid colorimetric method, calculated with ginsenoside Re as the reference standard. Total polysaccharide content was determined using the phenol-sulfuric acid method, calculated with glucose as the reference standard. Each group of samples was measured in triplicate, and the results are expressed as mean ± standard deviation. The test results are shown in Table 4.

[0028] Table 4. Results of component release and fermentation effect tests of ginseng fermentation extracts from Examples 1-3 and Comparative Examples 1-5

[0029] As shown in Table 4, there are significant differences in soluble solids, total saponin content, and total polysaccharide content among Examples 1-3 and Comparative Examples 1-5. Example 1 exhibits the highest total saponin and total polysaccharide content, indicating that under these process parameters, mild enzymatic hydrolysis, semi-solid pre-fermentation, staged addition of compound sugar sources and compound fermentation strains, and staged temperature control during secondary liquid fermentation better balance ginseng component release, fermentation transformation, and retention of effective components, thus achieving the best overall component release effect. In Comparative Example 1, the removal of mild enzymatic hydrolysis pretreatment resulted in a decrease in both total saponin and total polysaccharide content, indicating that the synergistic effect of cellulase and hemicellulase promotes moderate loosening of ginseng tissue, improving substrate accessibility and component release efficiency. In Comparative Example 2, the removal of the S3 semi-solid stacked pre-fermentation resulted in the lowest total saponin and total polysaccharide content, demonstrating that the stepped fermentation pathway plays a crucial role in early substrate pre-activation, cell adaptation, and subsequent liquid-state in-depth fermentation. In Comparative Example 3, after adding the compound sugar source and compound fermentation strain all at once, the component release effect was better than that of Comparative Examples 1 and 2, but still lower than that of Example 1. This indicates that the two-stage addition method is more conducive to maintaining the metabolic connection between the early and late stages and promoting subsequent release and transformation. In Comparative Example 4, when only *Lactobacillus plantarum* was used, the total saponin and total polysaccharide contents were also lower than those of Example 1, indicating that the compound strain system has a greater advantage in component release and fermentation synergy. In Comparative Example 5, after using constant temperature fermentation throughout the process, although the total saponin and total polysaccharide contents were relatively higher among the comparative examples, they were still lower than those of Example 1. This indicates that the staged temperature control method is beneficial to balancing the fermentation efficiency in the early stage and the retention of components in the later stage. Stability test: Ginseng fermentation extract samples obtained in Example 1 and Comparative Examples 2, 3, and 5 were stored at 4℃ and 25℃ for 0, 7, 14, and 30 days, respectively. The pH value, soluble solids content, and turbidity changes of the samples were measured periodically. Each group of samples was measured in triplicate, and the results are expressed as mean ± standard deviation. The test results are as follows: Figures 1-3 As shown.

[0030] Depend on Figures 1-3 It is evident that under both storage conditions of 4℃ and 25℃, the pH value of each sample group decreased with prolonged storage time, the soluble solids content gradually decreased, and the turbidity gradually increased. Furthermore, the change was significantly greater at 25℃ than at 4℃, indicating that higher storage temperatures accelerate residual metabolism, component aggregation, and the precipitation of unstable particles within the system. Example 1 exhibited the smallest pH fluctuation, the slowest decrease in soluble solids, and the lowest turbidity increase under both storage conditions. This demonstrates that the present invention, through its synergistic design involving semi-solid pre-fermentation, two-stage supplementation of compound sugar sources and compound fermentation strains, and staged temperature control during secondary liquid fermentation, can more effectively establish a stable fermentation system and improve the homogeneity of the final product, thereby enhancing storage stability. Comparative Example 2, after omitting the S3 semi-solid stacked pre-fermentation, exhibited the most significant pH decrease, soluble solids loss, and turbidity increase under both storage conditions. This indicates that the lack of microbial adaptation and substrate pre-regulation under the high-solids environment weakens the homogeneity and stability of the subsequent liquid system, resulting in the worst stability for this group. Comparative Example 3, with all sugar source and inoculum added at once, also showed significantly worse storage stability than Example 1, demonstrating that the two-stage addition method is beneficial for maintaining the continuity of fermentation activity between the early and late stages and reducing subsequent unstable changes. Comparative Example 5, using a constant temperature of 36℃ throughout fermentation, showed better storage stability than Comparative Example 2, but still lower than Example 1. This indicates that the staged temperature control method is beneficial for the formation of flavor and system harmony in the later stages and further improves the product's stability during storage. The results of this study indicate that the storage stability of the ginseng fermentation extract obtained in this invention is due to the synergistic regulation of multiple factors. Semi-solid pre-fermentation is beneficial for the establishment of dominant microbial communities and substrate pre-activation in the early stage, reducing uneven changes in the subsequent liquid system. The two-stage addition of compound sugar source and compound fermentation strain is beneficial for maintaining a relatively stable metabolic state at different fermentation stages, avoiding imbalance between the early and late stages after a one-time addition. Staged temperature control is beneficial for balancing the efficiency of early fermentation and the coordination of the later system, thereby reducing the continuous decrease in pH, loss of soluble solids, and particle aggregation and precipitation during storage.

[0031] Spectral testing: An appropriate amount of the ginseng stepwise fermentation extract obtained in Example 1 was extracted with methanol using ultrasound. The supernatant was collected by centrifugation and filtered through a 0.22 μm microporous membrane as the test solution. Reference solutions of Rb1, Rc, 20(S)-Rg3, 20(R)-Rg3, F2, Rh2, and Compound K were prepared separately. High-performance liquid chromatography (HPLC) was used for detection. The chromatographic column was a C18 reversed-phase column, the mobile phase was acetonitrile-water, gradient elution was used, the detection wavelength was 203 nm, the column temperature was 30℃, and the injection volume was 10 μL. The corresponding characteristic peaks in the test sample were assigned according to the retention time of the reference standards. The test results are as follows: Figure 4 As shown.

[0032] Depend on Figure 4 As can be seen, in addition to the detection of some original ginsenosides such as Rb1 and Rc, the ginseng step fermentation extract obtained in Example 1 also showed the detection of transformation products such as 20(S)-Rg3, 20(R)-Rg3, F2, Rh2 and Compound K. This indicates that the process of the present invention does not simply cause the ginseng components to dissolve, but rather promotes the staged transformation of some ginsenosides under the combined action of mild enzymatic hydrolysis, semi-solid pre-fermentation and subsequent liquid-state in-depth fermentation, thereby forming a component profile with multi-level transformation characteristics. This result is consistent with the step fermentation process path of Example 1 of the present invention.

[0033] 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 ginseng step fermentation extraction process, characterized in that, Specifically, the process includes the following steps: S1: Ginseng raw material for food is washed, dried, pulverized and pre-sterilized to obtain crude ginseng powder; S2: Add purified water to the crude ginseng powder for wetting, and then add a mild enzymatic hydrolysis system for pretreatment to obtain pretreated ginseng material; S3: Add a compound sugar source consisting of honey, white sugar powder, glucose powder and brown sugar powder to the pretreated ginseng material, and inoculate it with food-grade compound fermentation bacteria, mix well and carry out a first fermentation. S4: After the first fermentation is completed, purified water is added to the fermentation material, and a stage switching heat treatment is performed. After cooling, a second inoculation is performed. S5: Add the compound sugar source and food-grade compound fermentation strain to the liquid fermentation system after step S4 for secondary liquid fermentation; S6: After the secondary liquid fermentation is completed, the fermentation liquid is allowed to stand, filtered, concentrated under reduced pressure, and then filtered again and refrigerated to obtain ginseng fermentation extract.

2. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S1, the pre-sterilization treatment parameters are 95-100℃ for 20-30 minutes.

3. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S2, the mild enzymatic hydrolysis system is a compound system composed of cellulase and hemicellulase in a mass ratio of 1:1 to 2, wherein the cellulase activity is 10,000 to 50,000 U / g and the hemicellulase activity is 10,000 to 50,000 U / g.

4. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S2, the amount of the mild enzymatic hydrolysis system added is 0.2-0.6% of the mass of the crude ginseng powder; the enzymatic hydrolysis temperature is 40-55℃, and the enzymatic hydrolysis time is 1-2 hours.

5. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S3, the food-grade compound fermentation strain is a compound system composed of Lactobacillus plantarum and Saccharomyces cerevisiae in a mass ratio of 2 to 4:1; the inoculation amount of the food-grade compound fermentation strain is 2 to 5% of the mass of the pretreated ginseng material.

6. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S3, the specific operation of the fermentation process is as follows: the primary fermentation material is placed in a sterilized container, the material thickness is controlled to be 3-10cm, and semi-solid stacking pre-fermentation is carried out at 36-40℃ for 10-15 days; the material is turned over 1-2 times a day during the fermentation period.

7. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S4, the stage switching heat treatment parameters are 95-100℃, and the holding time is 20-30 minutes.

8. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S5, the inoculation amount of the food-grade compound fermentation strain is 1 to 3% of the mass of the liquid fermentation system.

9. The ginseng step fermentation extraction process according to claim 1, characterized in that, In step S5, the secondary liquid fermentation is carried out using a staged temperature control method, specifically: the early stage of enhanced fermentation: the temperature is controlled at 36-38℃, and fermentation lasts for 4-7 days to promote the rapid establishment of a dominant metabolic state of the microorganisms and accelerate the release and transformation of ginseng soluble components; the later stage of coordinated fermentation: the temperature is adjusted to 30-34℃, and fermentation continues for 5-10 days to slow down the rapid rise in acidity, promote flavor coordination, and improve the stability of the system.

10. The application of ginseng fermentation extract prepared by the ginseng step fermentation extraction process according to any one of claims 1-9 in the preparation of functional foods or health products.

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