A method for preparing Rehmannia glutinosa through Bacillus pre-fermentation coupled with nine steaming and nine sun-drying processes and yeast secondary fermentation.

Abstract

This invention discloses a method for preparing Rehmannia glutinosa using Bacillus pre-fermentation coupled with nine-step steaming and drying and secondary fermentation by yeast, belonging to the field of processing technology for medicinal and edible herbs. Using raw Rehmannia glutinosa as raw material, after washing and peeling, it is steamed, cooled, and inoculated with Bacillus for solid-state pre-fermentation. After drying, it undergoes nine-step steaming and drying to obtain an intermediate. The surface is then perforated and inoculated with yeast for secondary fermentation to obtain prepared Rehmannia glutinosa. This invention, through the synergistic effect of two-step fermentation and traditional processing, results in an average reduction of over 15% in monosaccharide content, over 30% in 5-hydroxymethylfurfural content, and an average increase of over 20% in Rehmannia glutinosa polysaccharide retention rate. It significantly reduces the sweetness and bitterness, making it suitable for the large-scale production of medicinal and edible health foods, nourishing snacks, and functional beverages.

Description

Technical Field

[0001] This invention belongs to the field of processing technology of medicinal and edible herbs, specifically involving a method for preparing Rehmannia glutinosa using microbial fermentation coupled with traditional processing, as well as its products and applications. Background Technology

[0002] Prepared Rehmannia glutinosa is made from raw Rehmannia glutinosa through nine steaming and nine sun-drying processes. It is rich in active ingredients such as Rehmannia glutinosa polysaccharides and iridoid glycosides, and has the effects of nourishing yin and blood, replenishing essence and marrow. It is the core raw material of more than a thousand kinds of traditional Chinese medicines, such as Liuwei Dihuang Wan. Since Rehmannia glutinosa was included in the list of medicines and foods of the same origin, its application scenarios have expanded from traditional Chinese medicines to health foods, nourishing snacks, functional beverages and other fields, and market demand continues to grow.

[0003] Despite the broad market prospects of Rehmannia glutinosa, the current process and products still have several key pain points: (1) The traditional nine-steaming and nine-drying process relies on manual experience, has a long processing cycle, poor batch-to-batch quality consistency, and is difficult to meet the needs of large-scale production; (2) Industrial steaming is mostly a one-time high-temperature and high-pressure process, and the degradation of bitter precursors such as iridoid glycosides is not sufficient, making it difficult to eliminate the inherent greasy nature of Rehmannia glutinosa, which can easily cause gastrointestinal discomfort and limit its daily consumption scenarios; (3) Some products add exogenous sweeteners such as honey and sucrose to improve the taste, which increases the sugar burden, goes against the trend of low-sugar healthy consumption, and cannot solve the greasy problem from the root; (4) The large molecular active ingredients such as Rehmannia glutinosa polysaccharides are difficult to be absorbed by the human body, have low bioavailability, and have poor component stability during high-temperature processing. Taking Rehmannia glutinosa polysaccharides as the test object, the activity retention rate of some commercial products within 6 months of storage is less than 60%.

[0004] Currently, industry solutions to the aforementioned pain points mainly focus on two categories: process optimization and auxiliary material improvement. However, both have significant limitations. In terms of process optimization, while introducing temperature control equipment and standardized steaming parameters (such as 100℃ and 2h) can improve component conversion efficiency, it still falls under the limitations of a single steaming mode and cannot completely degrade bitter precursors or reduce greasiness. In terms of auxiliary material improvement, while adding honey and rice wine can harmonize the taste, the exogenous sugar increases health risks, and rice wine residue and fluctuations in auxiliary material usage can easily lead to batch-to-batch quality differences. Although single-strain fermentation (such as Lactobacillus plantarum and yeast) can achieve partial component degradation, it can only solve a single pain point (such as only improving bioavailability or only improving part of the taste) and cannot simultaneously solve multiple problems such as bitterness, greasiness, and low bioavailability. Furthermore, it lacks deep integration with the traditional nine-steaming and nine-drying processing method and cannot take into account both the ancient medicinal properties and the controllability of modern technology.

[0005] To address the shortcomings of existing technologies, this invention innovatively proposes a coupled process of "Bacillus pre-fermentation - nine-times steaming and drying - secondary fermentation by yeast," achieving a systematic solution to the core pain points of Rehmannia glutinosa: Bacillus pre-fermentation can directionally degrade bitter precursors such as iridoid glycosides and break down large-molecule polysaccharides into small-molecule soluble sugars, while loosening the tissue structure of raw Rehmannia glutinosa, solving the problems of heavy bitterness, strong greasiness, and uneven subsequent steaming; nine-times steaming and drying retain the medicinal property transformation advantages of traditional processing methods, ensuring the core efficacy of Rehmannia glutinosa; secondary fermentation by yeast further optimizes the flavor profile, improves the bioavailability of active ingredients such as Rehmannia glutinosa polysaccharides, and reduces the content of monosaccharides and 5-hydroxymethylfurfural, eliminating the need for exogenous sweeteners and aligning with the low-sugar trend. Furthermore, by precisely controlling the process parameters of each step, process defects such as differences in internal and external moisture content during drying are avoided, improving product stability and providing a standardized, quality-enhancing, and efficiency-improving new solution for the traditional Rehmannia glutinosa processing technology. Summary of the Invention

[0006] This invention aims to address the core pain points in existing Rehmannia glutinosa preparation processes, such as the long cycle of traditional nine-steaming and nine-drying, poor batch consistency, heavy bitterness and greasiness from industrial steaming, the addition of exogenous sweeteners not conforming to the low-sugar trend, low bioavailability of active ingredients, and uneven drying moisture. It provides a method for preparing Rehmannia glutinosa with reduced bitterness and greasiness by coupling Bacillus pre-fermentation, nine-steaming and nine-drying, and secondary fermentation by yeast. This method enables large-scale and standardized production of Rehmannia glutinosa, preserving the medicinal properties of traditional processing methods while meeting the needs of modern health consumption.

[0007] To achieve the above-mentioned objectives, this invention provides a method for preparing Rehmannia glutinosa through Bacillus pre-fermentation coupled with nine steaming and nine sun-drying processes and secondary fermentation by yeast, comprising the following steps: S1. Raw material pretreatment: Select raw Rehmannia glutinosa that is free from mold, rot, damage, impurities, and insect infestation as raw material. First, use a rotating brush roller in conjunction with a spray system to remove the mud, sand, fibrous roots, and impurities attached to the surface of the raw Rehmannia glutinosa. Then, rinse it repeatedly with running water 2-3 times until there is no visible mud or sand residue on the surface. After rinsing, use a mechanical peeling method to remove the outer skin of the raw Rehmannia glutinosa to obtain clean raw Rehmannia glutinosa raw material, which is then placed in a clean container for later use.

[0008] S2, Bacillus solid-state pre-fermentation: S2.1 Steaming and softening: The raw Rehmannia glutinosa pretreated in step S1 is steamed; S2.2 Cooling and Inoculation: After steaming, cool the food and inoculate it with Bacillus. S2.3 Solid-state fermentation: Solid-state fermentation is carried out to loosen the tissue structure of Rehmannia glutinosa and degrade its fiber; S2.4 Post-fermentation treatment: Dry after fermentation.

[0009] S3, Nine-steaming and nine-drying process: S3.1 First steaming and drying: The pre-fermented and dried product is steamed and then sun-dried or dried. S3.2 Repeated steaming and drying: Repeat the above steaming, drying or baking operations, and complete a total of 9 steaming and drying operations to obtain the intermediate product of Rehmannia glutinosa.

[0010] S4, Secondary fermentation by yeast: S4.1 Intermediate processing: The surface of the prepared Rehmannia glutinosa intermediate is scratched. S4.2 Inoculation and Fermentation: Inoculate with yeast for secondary fermentation to obtain Rehmannia glutinosa.

[0011] Preferably, in step S2.1, the steaming and softening temperature is 100±5℃ and the steaming time is 30 to 50 minutes; in step S2.2, after steaming, the mixture is cooled to room temperature, sterile water is added at a material-to-liquid ratio of 1:1 (g / mL), mixed well, and then inoculated with Bacillus.

[0012] Preferably, in step S2.2, the Bacillus is Bacillus subtilis, and the inoculation amount is 8-12% (based on the mass of Rehmannia glutinosa raw material).

[0013] Preferably, in step S2.3, the conditions for solid-state fermentation are: fermentation temperature 35-39℃, relative humidity 70-85%, and fermentation time 8-12 days.

[0014] Preferably, in step S2.4, the drying temperature is 50-60°C, and the product is dried to constant weight. The standard for constant weight is that the difference between two consecutive weighings is ≤0.5%.

[0015] Preferably, in step S3, the steaming is performed under normal pressure, and each steaming time is 3 to 5 hours. Each drying is carried out by sun-drying or drying at 50 to 60°C until the moisture content is 15 to 20% (on a wet basis).

[0016] Preferably, in step S4.1, the surface piercing treatment involves using a sterile needle-like tool to uniformly pierce the surface of the prepared Rehmannia glutinosa intermediate, with a piercing depth of 2-5 mm and a piercing spacing of 0.5-1.5 cm.

[0017] Preferably, in step S4.2, the yeast is food-grade brewing yeast, and the inoculation amount is 4-7% (based on the mass of Rehmannia glutinosa intermediate); the conditions for the secondary fermentation are: fermentation temperature 26-30℃, relative humidity 70-85%, and fermentation time 3-5 days.

[0018] This invention also protects the prepared Rehmannia glutinosa obtained by the above preparation method, which, compared with the traditional nine-steamed and nine-dried prepared Rehmannia glutinosa, has an average reduction of more than 15% in monosaccharide content, an average reduction of more than 30% in 5-hydroxymethylfurfural content, an average increase of more than 20% in the retention rate of Rehmannia glutinosa polysaccharides, and a significant reduction in sweetness and bitterness.

[0019] This invention also protects the above-described preparation method or the use of the above-described Rehmannia glutinosa in the preparation of food and medicine homologous health foods, nourishing snacks or functional beverages.

[0020] Using the above technical solution, the specific functions of each main raw material in this invention are as follows: Raw Rehmannia root: The core raw material of this invention is the dried tuberous root of Rehmannia glutinosa Libosch., a plant of the Scrophulariaceae family. After processing, it is transformed into prepared Rehmannia root, which is rich in active ingredients such as Rehmannia polysaccharides and iridoid glycosides. It is the material basis for achieving the effects of nourishing yin and blood, and replenishing essence and marrow.

[0021] Bacillus subtilis: a food-grade safe strain that secretes a complex enzyme system including glycosidases, cellulases, and hemicellulases during solid-state fermentation. Glycosidases directionally degrade bitter precursors such as iridoid glycosides, reducing bitterness at its source; cellulases and hemicellulases break down components of the Rehmannia glutinosa cell wall, loosening the dense tissue structure, improving the uniformity of subsequent steaming, and reducing the degradation of active ingredients; simultaneously, they preliminarily break down some large-molecule Rehmannia glutinosa polysaccharides into small-molecule soluble sugars, providing substrates for yeast fermentation.

[0022] Food-grade brewing yeast (Saccharomyces cerevisiae): a safe edible microorganism that metabolizes and consumes some of the monosaccharides in the intermediate of Rehmannia glutinosa during secondary fermentation, reducing sweetness and greasiness; it degrades harmful components produced by high-temperature processing such as 5-hydroxymethylfurfural, improving product safety; and the small amount of flavor substances produced during fermentation can optimize the flavor profile of Rehmannia glutinosa and improve overall palatability.

[0023] Sterile water: Adjusts the moisture content of the pre-fermentation system to provide a suitable moisture environment for the growth, reproduction, and enzyme secretion of Bacillus subtilis, ensuring the smooth progress of solid-state fermentation.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly optimizes product taste and flavor This invention utilizes a complex enzyme system, including glycosidases and cellulases, produced through pre-fermentation with Bacillus subtilis to target and degrade bitter precursors such as iridoid glycosides, thus reducing bitterness at its source. A secondary fermentation process with yeast then metabolizes and consumes monosaccharides, resulting in a monosaccharide content that is more than 15% lower than that of traditionally steamed and sun-dried Rehmannia glutinosa. This two-step fermentation process works synergistically, effectively reducing the bitterness and greasiness of Rehmannia glutinosa without the need for exogenous sweeteners, thereby improving the product's palatability.

[0025] (2) Improve the retention rate of effective ingredients and reduce the content of harmful ingredients. Bacillus pre-fermentation loosens the tissue structure of raw Rehmannia glutinosa, improving the uniformity of subsequent nine-steaming and nine-drying processes and reducing polysaccharide loss caused by localized high temperatures. Secondary yeast fermentation promotes the conversion of macromolecular Rehmannia glutinosa polysaccharides into smaller soluble sugars through metabolic regulation. High-performance liquid chromatography (HPLC) analysis shows that, compared with traditionally prepared Rehmannia glutinosa prepared through nine steaming and nine drying processes, the polysaccharide retention rate of the prepared Rehmannia glutinosa is increased by more than 20%, the 5-hydroxymethylfurfural content is reduced by more than 30%, and the bioavailability of the core active ingredients is significantly improved.

[0026] (3) Achieve process standardization and large-scale production This invention clearly defines the core process parameters for Bacillus pre-fermentation (inoculation amount 8-12%, temperature 35-39℃, relative humidity 70-85%), nine-steaming and nine-drying (steaming for 3-5 hours, moisture content 15-20%), and yeast secondary fermentation (inoculation amount 4-7%, temperature 26-30℃, relative humidity 70-85%), achieving standardized control of the entire process, avoiding batch differences caused by reliance on manual experience in traditional processes, and adapting to industrial-scale production.

[0027] (4) Improve product stability and reduce overall production costs This invention uses food-grade Bacillus subtilis and Saccharomyces cerevisiae, combined with a process of drying at 50-60℃ to constant weight (with a mass difference of ≤0.5% between two consecutive weighings), to ensure consistent moisture content inside and outside the product, avoid the risk of mold growth caused by moisture differences, and improve storage stability. At the same time, the two-step fermentation process reduces dependence on exogenous auxiliary materials such as honey and sucrose, eliminating the need to add exogenous auxiliary materials and directly reducing raw material costs. Furthermore, the fermentation pretreatment makes the material texture uniform, improving the uniformity and component stability of subsequent processing, thereby optimizing overall production efficiency.

[0028] (5) Achieve synergistic effect of "two-step fermentation + nine-steaming and nine-drying" and break through the limitations of existing technology. In existing technologies, single-strain fermentation can only solve a single problem, and single process optimization cannot completely solve the multiple technical challenges of Rehmannia glutinosa. This invention innovatively couples Bacillus pre-fermentation, yeast secondary fermentation, and the traditional nine-steaming and nine-drying processing method to form a synergistic effect: pre-fermentation directionally degrades bitter precursors, breaks down large-molecule polysaccharides, and loosens the tissue structure, reducing the degradation of active ingredients caused by localized overheating, providing homogenized raw materials for the nine-steaming and nine-drying process; the nine-steaming and nine-drying process completes the transformation of medicinal properties, preserving the ancient medicinal properties of Rehmannia glutinosa in nourishing yin and replenishing blood, and benefiting essence and marrow; secondary fermentation further optimizes flavor and improves bioavailability. This synergistic system achieves systemic technical effects that cannot be achieved by single fermentation or single process optimization, distinguishing it from existing technical solutions and highlighting the inventiveness of this invention.

[0029] (6) Expand the application scenarios of food and medicine homology products The prepared Rehmannia glutinosa prepared by this invention has good palatability, high safety and clear efficacy. It can be widely used in the large-scale production of food and medicine homology health foods, nourishing snacks and functional beverages, effectively expanding the application scenarios of prepared Rehmannia glutinosa and enhancing the market competitiveness of products. Detailed Implementation

[0030] To further understand the present invention, specific embodiments are described in detail below. These embodiments are for illustrative purposes only and do not constitute any limitation on the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Example 1

[0031] S1. Raw material pretreatment: Select raw Rehmannia glutinosa that is free from mold, rot, damage, impurities, and insect infestation as raw materials. First, use a rotating brush roller in conjunction with a spray system to remove surface mud, fibrous roots, and impurities. Then, rinse repeatedly with running water three times until there is no visible mud or sand residue on the surface. Use a mechanical peeling method to remove the outer skin to obtain clean raw Rehmannia glutinosa raw materials, and place them in a clean container for later use.

[0032] S2, Bacillus solid-state pre-fermentation: S2.1 Steaming and softening: Place the pretreated raw Rehmannia glutinosa in an atmospheric pressure steaming device and steam at 100℃ for 40 minutes; S2.2 Cooling and Inoculation: After steaming, allow the mixture to cool naturally to 25℃. Add sterile water at a material-to-liquid ratio of 1:1 (g / mL), mix well, and then inoculate with 10% (based on the mass of Rehmannia glutinosa) at a concentration of 1×10⁻⁶. 8 The Bacillus subtilis bacterial suspension at cfu / mL was thoroughly mixed using a sterile stirrer; S2.3 Solid-state fermentation: Place in a sterile fermentation device and ferment in a solid-state environment at 37°C and 80% relative humidity for 10 days; S2.4 Post-fermentation treatment: After fermentation, dry at 55℃ to constant weight (the difference between two consecutive weighings should be ≤0.5%), and then take it out for use.

[0033] S3, Nine-steaming and nine-drying process: S3.1 First steaming and drying: Place the pre-fermented and dried product in an atmospheric pressure steaming device and steam at 100℃ for 4 hours; after taking it out, let it dry naturally until the moisture content is 18% (on a wet basis) to complete the first steaming and drying. S3.2 Repeated steaming and drying: Following the parameters in step S3.1, repeat the steaming and drying operations, and complete a total of 9 steaming and drying operations to obtain the intermediate product of Rehmannia glutinosa.

[0034] S4, Secondary fermentation by yeast: S4.1 Intermediate processing: Use a sterile needle-like tool to evenly puncture the prepared Rehmannia root intermediate (puncture depth 3mm, puncture spacing 1.0cm). S4.2 Inoculation and Fermentation: Inoculate with 5% (by intermediate mass) of food-grade brewing yeast, place in a sterile fermentation device, and ferment for 4 days at 28℃ and 80% relative humidity. After fermentation, Rehmannia glutinosa is obtained. Example 2

[0035] This embodiment is basically the same as Embodiment 1, except for some process parameters: S2.1 Steaming and softening: Steaming temperature 105℃, steaming time 30 minutes; S2.2 Cooling inoculation: The inoculation amount of Bacillus subtilis is 8%; S2.3 Solid-state fermentation: fermentation temperature 35℃, relative humidity 70%, fermentation time 8 days; S2.4 Post-fermentation treatment: Drying temperature is 50℃; S3.1 First steaming and drying: Steaming time is 3 hours, and drying is carried out until the moisture content is 15% (on a wet basis). S4.1 Intermediate body treatment: piercing depth 2mm, hole spacing 0.5cm; S4.2 Inoculation and Fermentation: The amount of brewing yeast inoculated is 4%, the fermentation temperature is 26℃, the relative humidity is 70%, and the fermentation time is 3 days.

[0036] The remaining steps and parameters are the same as in Example 1, and the prepared Rehmannia glutinosa product is obtained. Example 3

[0037] This embodiment is basically the same as Embodiment 1, except for some process parameters: S2.1 Steaming and softening: Steaming temperature 95℃, steaming time 50 minutes; S2.2 Cooling inoculation: The inoculation amount of Bacillus subtilis is 12%; S2.3 Solid-state fermentation: fermentation temperature 39℃, relative humidity 85%, fermentation time 12 days; S2.4 Post-fermentation treatment: Drying temperature is 60℃; S3.1 First steaming and drying: Steaming time is 5 hours, and drying is carried out until the moisture content is 20% (on a wet basis). S4.1 Intermediate body treatment: piercing depth 5mm, hole spacing 1.5cm; S4.2 Inoculation and Fermentation: The amount of brewing yeast inoculated is 7%, the fermentation temperature is 30℃, the relative humidity is 85%, and the fermentation time is 5 days.

[0038] The remaining steps and parameters are the same as in Example 1, and the prepared Rehmannia glutinosa product is obtained.

[0039] Comparative Example 1 (Traditional Nine-Steaming and Nine-Drying Process) Select the same batch of raw Rehmannia glutinosa as in Example 1 and process it according to the traditional nine-steaming and nine-drying process: each time, place the raw Rehmannia glutinosa in a normal pressure steaming equipment and steam it at 100°C for 4 hours; after taking it out, naturally dry it until the moisture content is 18% (on a wet basis); repeat the above operation to complete a total of 9 steaming and drying cycles to obtain the traditional nine-steaming and nine-drying processed Rehmannia glutinosa product.

[0040] Comparative Example 2 (Single Bacillus fermentation + nine steaming and nine sun-drying, no secondary fermentation with yeast) This comparative example is basically the same as Example 1, except that the secondary fermentation step of yeast in S4 is omitted, and the prepared Rehmannia glutinosa product is obtained directly after S3 is completed.

[0041] Comparative Example 3 (Single yeast fermentation + nine steaming and nine sun-drying, no Bacillus pre-fermentation) This comparative example is basically the same as Example 1, except that the S2 Bacillus solid-state pre-fermentation step is omitted, and after S1 is completed, the S3 nine-steaming and nine-drying treatment is carried out directly. After S3 is completed, the S4 yeast secondary fermentation is carried out.

[0042] Product performance testing and result comparison The prepared Rehmannia glutinosa products of Examples 1, 2 and 3 and Comparative Examples 1, 2 and 3 were tested for the following indicators. Each test was repeated 3 times and the average value was taken.

[0043] (1) Determination of monosaccharide content The monosaccharide content in the samples was determined by high-performance liquid chromatography (HPLC). Detection conditions: amino column (4.6 × 250 mm, 5 μm), mobile phase acetonitrile-water (75:25, v / v), flow rate 1.0 mL / min, column temperature 30℃, injection volume 10 μL. The detection results are shown in Table 1. Table 1 Results of Monosaccharide Content Detection

[0044] The results showed that the monosaccharide content of Examples 1-3 was 3.98%~4.52%, a decrease of 9.6%~20.4% compared to Comparative Example 1 (5.00%). Specifically, the reduction rates of Examples 1 and 3 reached 15.4% and 20.4% respectively, both exceeding 15%, consistent with the technical feature of "monosaccharide content reduced by more than 15%" in claim 8. The monosaccharide reduction rate of Comparative Example 2 (omitting secondary fermentation) was only 3.8%, and the monosaccharide content of Comparative Example 3 (omitting pre-fermentation) (5.19%) was higher than that of Comparative Example 1 (5.00%). The above comparisons indicate that single-step fermentation alone cannot effectively reduce monosaccharide content; only the synergistic effect of two-step fermentation can achieve the monosaccharide reduction effect described in claim 8.

[0045] (2) Determination of 5-hydroxymethylfurfural (5-HMF) content The content of 5-HMF in the sample was determined by high performance liquid chromatography (HPLC). Detection conditions: C18 column (4.6 × 250 mm, 5 μm), mobile phase: methanol-water (10:90, v / v), flow rate: 1.0 mL / min, column temperature: 30℃, detection wavelength: 284 nm, injection volume: 10 μL. The detection results are shown in Table 2 below. Table 2 Results of 5-HMF Content Detection

[0046] The results showed that the 5-HMF content in Examples 1-3 was 17.2~20.1 mg / kg, a reduction of 26.6%~37.2% compared to Comparative Example 1 (27.4 mg / kg). Specifically, the reduction rates in Examples 1 and 3 both exceeded 30%, consistent with the technical feature of "reducing 5-hydroxymethylfurfural content by more than 30%" in claim 8. The reduction rate in Comparative Example 2 (omitting secondary fermentation) was only 7.7%, and the reduction rate in Comparative Example 3 (omitting pre-fermentation) was only 2.2%, both significantly lower than the Example groups. The above comparison demonstrates that the three-step coupled process (pre-fermentation-nine-steaming-nine-drying-secondary fermentation) described in claim 1 has a synergistic effect on reducing the harmful component 5-HMF, an effect that cannot be achieved by single-step fermentation.

[0047] (3) Determination of Rehmannia polysaccharide retention rate The content of Rehmannia glutinosa polysaccharides in the samples was determined using the phenol-sulfuric acid method. Detection conditions: glucose was used as a reference standard, and absorbance was measured at a wavelength of 490 nm. The detection results are shown in Table 3 below: Table 3. Results of Rehmannia glutinosa polysaccharide content detection

[0048] The results showed that the polysaccharide content of Examples 1-3 was 40.8~43.2 mg / g, which was 16.6%~23.4% higher than that of Comparative Example 1 (35.0 mg / g). The improvement rates of Examples 1 and 3 both exceeded 20%, consistent with the technical feature of "increasing the retention rate of Rehmannia glutinosa polysaccharides by more than 20%" in claim 8. The improvement rate of Comparative Example 2 (omitting secondary fermentation) was only 7.1%, and that of Comparative Example 3 (omitting pre-fermentation) was only 3.4%, both significantly lower than the Example groups. The above comparison demonstrates that the three-step coupling process described in claim 1 has a synergistic effect on protecting Rehmannia glutinosa polysaccharides, an effect that cannot be achieved by single-step fermentation.

[0049] (4) Sensory evaluation A panel of 10 professionals with experience in food sensory evaluation was invited to conduct sensory evaluations of the prepared Rehmannia glutinosa products from Examples 1-3 and Comparative Examples 1-3. Evaluation indicators included bitterness, sweetness, richness, and overall palatability, using a 9-point scoring system (1 being the worst and 9 the best). The sensory evaluation results are shown in Table 4 below: Table 4 Sensory evaluation results

[0050] The results showed that Examples 1-3 scored significantly higher than Comparative Examples 1-3 in terms of bitterness, sweetness, richness, and overall palatability. Specifically, Example 3 achieved an overall palatability score of 8.1, an increase of 4.1 points compared to Comparative Example 1 (4.0). While Comparative Examples 2 and 3 scored slightly higher than Comparative Example 1, they were still significantly lower than the Example 1 groups. These comparisons indicate that single-step fermentation has limited effect on improving taste; only the synergistic effect of the two-step fermentation described in claim 1 can achieve a significant improvement in taste and flavor, consistent with the technical feature of "significantly reduced sweetness and bitterness" in claim 8.

[0051] (5) Storage stability test The prepared Rehmannia glutinosa products from Example 1 and Comparative Example 1 were sealed and stored for 6 months at room temperature (25±2℃) and relative humidity (60±5%). Samples were taken at 0, 3, and 6 months to determine the Rehmannia glutinosa polysaccharide content and water activity. The storage stability results are shown in Table 5 below. Table 5 Storage stability test results

[0052] The results showed that after 6 months of storage, the polysaccharide retention rate of Example 1 was 92.2%, while that of Comparative Example 1 was 80.0%, with the former being 12.2 percentage points higher. The water activity of Example 1 was 0.57, while that of Comparative Example 1 was 0.75, with the former being significantly lower than the latter. These data indicate that the "drying to constant weight at 50-60℃" step in S2.4 of the process described in claim 1 ensures consistent moisture content inside and outside the product. Combined with the two-step fermentation process, this reduces the need for exogenous additives and effectively improves the product's storage stability.

[0053] Final Results Discussion The comparative test results of Examples 1, 2, and 3 above with Comparative Examples 1, 2, and 3 show that: 1. Taste and flavor This invention utilizes the synergistic effect of Bacillus pre-fermentation and yeast secondary fermentation to reduce monosaccharide content by 9.6% to 20.4% (average 15.1%) compared to traditional processes. Sensory evaluation indicators show significant superiority over the comparative examples. Comparative Examples 2 (omitting secondary fermentation) and 3 (omitting pre-fermentation) exhibited significantly inferior monosaccharide reduction effects compared to the example group, demonstrating that the two-step fermentation process described in claim 1 is indispensable, and only through synergy can the technical effects of reducing monosaccharides by more than 15% and significantly improving taste described in claim 8 be achieved.

[0054] 2. Efficacy ingredients and safety The prepared Rehmannia glutinosa of this invention exhibits a 16.6%–23.4% (average 20.5%) higher polysaccharide retention rate and a 26.6%–37.2% (average 32.1%) lower 5-HMF content compared to traditional processes. Comparative Examples 2 and 3 show significantly worse performance than the Example Group in all aspects, demonstrating that the three-step coupling process described in claim 1 can achieve the technical effects of claim 8, namely, a polysaccharide retention rate increase of over 20% and a 5-HMF content reduction of over 30%.

[0055] 3. In terms of process synergy Comparative Examples 2 (omitting secondary fermentation) and 3 (omitting pre-fermentation) showed significantly worse performance in all indicators compared to Examples 1-3, demonstrating that the three-step process of "Bacillus pre-fermentation - nine-times steaming and drying - yeast secondary fermentation" described in claim 1 has a synergistic effect: pre-fermentation loosens the tissue structure, providing homogenized raw materials for the nine-times steaming and drying process and reducing component loss caused by localized high temperatures; secondary fermentation metabolizes and consumes monosaccharides, reduces 5-HMF, optimizes flavor, and improves safety. Single fermentation or single process optimization cannot achieve the above-mentioned systematic technical effects.

[0056] 4. Storage stability The prepared Rehmannia glutinosa of this invention achieved a polysaccharide retention rate of 92.2% during a 6-month storage period, significantly better than the 80.0% of the traditional process, and also exhibited lower water activity (0.57 vs 0.75). This effect corresponds to the "drying to constant weight at 50~60℃" process described in claim 3 and the moisture content control described in claim 4, ensuring consistent moisture content inside and outside the product and effectively improving storage stability.

[0057] Conclusion: In summary, the method for preparing Rehmannia glutinosa by pre-fermentation of Bacillus coupled with nine steaming and nine sun-drying processes and secondary fermentation of yeast provided by this invention achieves the technical effects described in claim 8, namely, a reduction of monosaccharide content of more than 15%, a reduction of 5-HMF content of more than 30%, and an increase in the retention rate of Rehmannia glutinosa polysaccharides of more than 20%, through the synergistic effect of two-step fermentation and traditional processing. It systematically solves the core technical pain points of traditional Rehmannia glutinosa, such as bitterness, greasiness, and low bioavailability, and has significant beneficial effects, making it suitable for industrial-scale production.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing Rehmannia glutinosa through Bacillus pre-fermentation coupled with nine steaming and nine sun-drying processes and secondary fermentation by yeast, characterized in that: Includes the following steps: S1. Raw material pretreatment: Take raw Rehmannia glutinosa, wash and peel it, and set it aside; S2, Bacillus solid-state pre-fermentation; S3, Nine-steaming and nine-drying treatment; S4, secondary fermentation by yeast; Step S2 includes the following sub-steps: S2.1 Steaming and softening: The raw Rehmannia glutinosa pretreated in step S1 is steamed; S2.2 Cooling and Inoculation: After steaming, cool the food and inoculate it with Bacillus. S2.3 Solid-state fermentation: Solid-state fermentation is carried out to loosen the tissue structure of Rehmannia glutinosa and degrade its fiber; S2.4 Post-fermentation treatment: Dry after fermentation; Step S3 includes the following sub-steps: S3.1 First steaming and drying: The pre-fermented and dried product is steamed and then sun-dried or dried. S3.2 Repeated steaming and drying: Repeat the above steaming, drying or baking operations, and complete a total of 9 steaming and drying operations to obtain the intermediate product of prepared Rehmannia glutinosa; Step S4 includes the following sub-steps: S4.1 Intermediate processing: The surface of the prepared Rehmannia glutinosa intermediate is scratched. S4.2 Inoculation and Fermentation: Inoculate with yeast for secondary fermentation to obtain Rehmannia glutinosa.

2. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S2.1, the steaming and softening temperature is 100±5℃ and the steaming time is 30 to 50 minutes; in step S2.2, after steaming, the mixture is cooled to room temperature, sterile water is added at a material-to-liquid ratio of 1:1 (g / mL), mixed well, and then inoculated with Bacillus.

3. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S2.2, the Bacillus is Bacillus subtilis, and the inoculation amount is 8-12% (based on the mass of raw Rehmannia glutinosa).

4. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S2.3, the conditions for solid-state fermentation are: fermentation temperature 35-39℃, relative humidity 70-85%, and fermentation time 8-12 days.

5. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S2.4, the drying temperature is 50-60℃, and the product is dried to constant weight. The standard for constant weight is that the difference between two consecutive weighings is ≤0.5%.

6. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S3, the steaming is carried out under normal pressure, and each steaming time is 3 to 5 hours. Each time, the product is sun-dried or dried at 50 to 60°C until the moisture content is 15 to 20% (on a wet basis).

7. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S4.1, the surface piercing treatment involves using a sterile needle-like tool to uniformly pierce the surface of the prepared Rehmannia glutinosa intermediate, with a piercing depth of 2-5 mm and a pier spacing of 0.5-1.5 cm.

8. The method for preparing Rehmannia glutinosa according to claim 1, characterized in that: In step S4.2, the yeast is food-grade brewing yeast, and the inoculation amount is 4-7% (based on the mass of Rehmannia glutinosa intermediate); the conditions for the secondary fermentation are: fermentation temperature 26-30℃, fermentation humidity 70-85%, and fermentation time 3-5 days.

9. The prepared Rehmannia root obtained by the method of any one of claims 1 to 8, characterized in that: High-performance liquid chromatography (HPLC) was used to detect the effects of traditionally steamed and sun-dried Rehmannia glutinosa: the content of monosaccharides was reduced by an average of more than 15%, the content of 5-hydroxymethylfurfural was reduced by an average of more than 30%, the retention rate of Rehmannia glutinosa polysaccharides was increased by an average of more than 20%, and the sweetness and bitterness were significantly reduced.

10. The method for preparing Rehmannia glutinosa according to any one of claims 1 to 8, or the use of Rehmannia glutinosa according to claim 9 in the preparation of food and medicine homologous to health foods, nourishing snacks, or functional beverages.

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