A method for preparing Polygonatum wine through co-fermentation of compound microorganisms

Abstract

This invention discloses a method for preparing Polygonatum wine through co-fermentation of a compound microbial strain. Firstly, this invention provides a compound microbial fermentation agent composed of *Lactobacillus plantarum*, *Bacillus lactis*, and *Saccharomyces cerevisiae* in a mass ratio of 2–4:1–2:50–100. This compound microbial fermentation agent, through the synergistic effect of the compound microorganisms, effectively balances and improves the original bitterness of Polygonatum, significantly improving the overall quality of the Polygonatum wine; it accelerates the fermentation process, significantly shortens the fermentation cycle, and improves production efficiency, thereby reducing production costs. This invention further provides a method for preparing Polygonatum wine based on this compound microbial fermentation agent. This method exhibits significant effects in improving taste, increasing fermentation efficiency, enhancing product stability, reducing adverse metabolites, and improving nutritional and health benefits, thus meeting consumers' pursuit of high-quality Polygonatum health drinks.

Description

Technical Field

[0001] This invention belongs to the field of fermented wine technology, specifically, it relates to a method for preparing Polygonatum wine through co-fermentation of compound microorganisms. Background Technology

[0002] Polygonatum, or Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua, is a traditional Chinese medicine used for both medicinal and culinary purposes. It contains various beneficial components such as polysaccharides, saponins, flavonoids, alkaloids, lignin, and anthraquinones, as well as essential amino acids and trace elements. Polygonatum possesses numerous health benefits, including tonifying the kidneys and replenishing essence, nourishing yin and moistening dryness, delaying aging, combating fatigue, providing antioxidant effects, enhancing immune function, lowering blood sugar, and lowering blood lipids. It has significant therapeutic effects on diseases such as diabetes, coronary heart disease, and hypertension.

[0003] Based on the health benefits of Polygonatum, it is frequently used to make various health-promoting wines, known as "Polygonatum wine." The main production methods for Polygonatum wine are soaking or extracting Polygonatum extract and then blending. However, these two methods often fail to remove the bitterness of the herb, resulting in a relatively poor taste and flavor. Especially when Polygonatum is soaked or blended with other herbs to prepare compound Polygonatum wine, the herbal flavor becomes even stronger, resulting in a generally unpleasant taste, a darker color, and poor visual appeal. Furthermore, Polygonatum wine prepared by soaking or blending methods also exhibits poor stability. Because the large molecular components in Polygonatum (such as polysaccharides, proteins, and polypeptides) have low solubility and poor stability in alcohol, sedimentation easily occurs after a period of time, significantly affecting the sensory quality of the Polygonatum wine.

[0004] Traditional yeast fermentation can also be used to prepare Polygonatum wine. This method can improve the taste and color of the wine to a certain extent, but it also has some shortcomings. Specifically: (1) Long fermentation cycle: Traditional yeast fermentation requires a certain amount of time, and the temperature, humidity and other conditions during this period are strictly controlled. Otherwise, it is easy to cause fermentation failure or off-flavors. (2) Not rich enough taste: If only traditional yeast is used for fermentation, the chemical components in the fermentation products may not be rich enough, thus affecting the layering of flavor. (3) Poor quality stability: The number and types of microorganisms in traditional yeast vary, and it is impossible to guarantee that the number and types of microorganisms in each batch of yeast are exactly the same. At the same time, the metabolic activities of microorganisms during fermentation are complex and variable, making it difficult to accurately control the fermentation process of traditional yeast, thus affecting the quality stability of the final Polygonatum wine. (4) May produce adverse metabolites: Some adverse metabolites, such as methanol, may be produced during the fermentation process of traditional yeast. This is because traditional yeast is composed of a mixture of various microorganisms, including yeast, mold and other bacteria. Among them, mold, such as Aspergillus niger, can decompose the pectin in the raw materials to produce methanol. However, high concentrations of methanol and other harmful metabolites may have negative effects on human health.

[0005] There are also methods on the market that use a single strain of microorganisms, such as Saccharomyces cerevisiae, to ferment and prepare Polygonatum wine. However, this method often results in a Polygonatum wine with a relatively simple flavor profile, low fermentation efficiency, and the quality of the final product is easily affected by environmental changes.

[0006] In summary, current Huangjing (Polygonatum sibiricum) wine products and their brewing processes still have some shortcomings, which seriously affect their sensory evaluation and overall quality. Therefore, improving the taste, stability, and safety of Huangjing wine are issues that still need to be addressed in its fermentation process. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a compound microbial fermentation agent.

[0008] The second objective of this invention is to provide a method for preparing Polygonatum wine through co-fermentation of compound microorganisms.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] The present invention first provides a compound microbial fermentation agent, which is composed of Lactobacillus plantarum, Bacillus lactis and Saccharomyces cerevisiae in a mass ratio of 2-4:1-2:50-100.

[0011] This invention innovatively employs three microbial strains—Lactobacillus plantarum, Bacillus lactis, and Saccharomyces cerevisiae—as a compound fermentation agent, using a specific ratio (2-4:1-2:50-100) for co-fermentation to prepare Polygonatum wine. Through the synergistic effect of the compound microbial strains, the multi-strain co-fermentation technology effectively balances and improves the original bitterness of Polygonatum, significantly enhancing the overall quality of the wine. The Polygonatum wine produced using this compound fermentation agent has a refreshing taste, a balanced sweet and sour flavor, and a fragrant aroma, while preserving the original delicate aroma of Polygonatum. It boasts a rich and layered flavor profile, a harmonious blend of sweet and sour, and a lingering finish. The wine is clear and transparent with a pale yellow color. Its taste, aroma, and color all reach a high standard. In contrast, Polygonatum wine prepared using traditional yeast and one or two of the aforementioned microbial strains, with the same amount of fermentation agent, suffers from inferior taste and flavor. Furthermore, the synergistic effect of multiple microbial strains accelerates the fermentation process, significantly shortens the fermentation cycle, improves production efficiency, and thus reduces production costs. In particular, the strong resistance of Bacillus lactis to adverse external factors further promotes the rapid progress of the fermentation process. The compound microbial fermentation technology optimizes microbial metabolic pathways, reduces the formation of harmful metabolites (such as methanol), improves the safety of Polygonatum wine, and reduces potential risks to human health.

[0012] Furthermore, the mass ratio of Lactobacillus plantarum, Bacillus lactis, and Saccharomyces cerevisiae is 2-3:1-1.5:50-75.

[0013] Furthermore, the viable count of the *Lactobacillus plantarum* is 10. 10 ~10 12 CFU / g, the viability counts of Bacillus lactis and Saccharomyces cerevisiae were 10. 9 ~10 11 CFU / g.

[0014] Preferably, the viable count of the *Lactobacillus plantarum* is 10. 11 CFU / g, viable count of Bacillus lactis is 10. 10 CFU / g, viable count of Saccharomyces cerevisiae is 10. 10 CFU / g.

[0015] Furthermore, the *Lactobacillus plantarum* is *Lactobacillus plantarum* LP45, the *Bacillus lactis* is *Bacillus lactis* DU-106, and the *Saccharomyces cerevisiae* is *Angel Wine Saccharomyces RV171*.

[0016] This invention provides the application of any of the above-described compound microbial fermentation agents in the preparation of Polygonatum wine.

[0017] This invention provides a method for preparing Polygonatum wine through co-fermentation of compound microorganisms, comprising the following steps:

[0018] S1. Activate any of the above-mentioned compound microbial fermentation agents to obtain a compound microbial solution;

[0019] S2. Saccharify the Polygonatum slices, then mix them with distilled water and compound bacterial liquid, seal and ferment at 25-35℃ for 5-10 days to obtain fermented wine.

[0020] S3. The fermented wine is then subjected to a series of processes to remove impurities, including filtration and sterilization, to remove macromolecular compounds with a molecular weight greater than 10,000, thus obtaining Polygonatum wine.

[0021] This invention achieves precise control over the fermentation process through scientifically and rationally controlled microbial strain ratios and fermentation conditions (fermentation time, temperature, and filtration sterilization). This reduces the complexity and variability of microbial metabolic activities during fermentation, significantly improving the quality stability of the Polygonatum wine and ensuring that the product maintains stable quality over a relatively long period (120 days). Simultaneously, a multi-stage filtration process removes large-molecule polysaccharides, proteins, and other impurities from the wine, ensuring its clarity, transparency, and stability, and preventing secondary sedimentation after prolonged storage.

[0022] Further, the activation in step S1 involves adding the compound bacterial fermentation agent to a mixed solution of physiological saline and sugar water.

[0023] Preferably, the volume ratio of physiological saline to sugar solution in the mixed solution is 1:1.

[0024] Preferably, the sugar syrup is 4-6 wt% sugar syrup.

[0025] Preferably, the sugar syrup is 5% wt% sugar syrup.

[0026] Furthermore, the activation conditions described in step S1 are activation at 25–35°C for 25–35 minutes.

[0027] Preferably, the activation conditions in step S1 are activation at 30°C for 30 minutes.

[0028] Furthermore, the Polygonatum slices mentioned in step S2 are used after soaking, washing, and decocting.

[0029] Furthermore, in step S2, the mass ratio of Polygonatum tablets, sugar, distilled water, and compound bacterial solution is 1:10-20:35-70:1.

[0030] Preferably, the mass ratio of Polygonatum sibiricum tablets, sugar, distilled water and compound bacterial solution in step S2 is 1:10-15:35-52.5:1.

[0031] Furthermore, the fermentation conditions described in step S2 are 25–35°C and a fermentation time of 5–10 days.

[0032] Furthermore, the method for removing impurities described in step S3 involves using a coarse fiber filter bag for initial coarse filtration to remove large particulate impurities; secondly, using a plate and frame filter to further refine the filtration effect; and finally, using a diatomaceous earth filter to deeply remove tiny suspended solids.

[0033] Furthermore, the filtration sterilization described in step S3 involves sequentially using microporous membranes with diameters of 0.4–0.5 μm and 0.1–0.2 μm.

[0034] Preferably, the filtration and sterilization is performed by sequentially using microporous membranes of 0.45μm and 0.15μm.

[0035] Furthermore, the removal of compounds with a molecular weight greater than 10,000 in step S3 is performed by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10,000.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The compound microbial fermentation agent of the present invention is composed of a specific ratio of Lactobacillus plantarum, Bacillus lactis, and Saccharomyces cerevisiae. This compound microbial fermentation agent can efficiently transform and fully utilize the complex metabolic components in Polygonatum sibiricum, and through the interaction between the microorganisms, produce a series of small molecule flavor substances during the metabolic process. These substances can greatly enrich the taste and flavor characteristics of Polygonatum sibiricum wine. It can effectively balance and improve the original bitterness of Polygonatum sibiricum, and significantly improve the overall quality of Polygonatum sibiricum wine. The Polygonatum sibiricum wine made with the compound microbial fermentation agent has a refreshing taste, a moderate sweet and sour taste, and a fragrant mellow aroma. At the same time, it retains the original fresh aroma of Polygonatum sibiricum, with a rich taste, a harmonious blend of mellow, sweet and sour, and a long aftertaste. The wine is clear and transparent with a light yellow color. Its taste, aroma, and color have all reached a high level. Furthermore, this compound microbial fermentation agent can also significantly shorten the fermentation cycle, effectively improve the stability of the product, optimize the microbial metabolic pathway, and reduce the generation of potentially harmful metabolites such as methanol, thereby improving the safety of Polygonatum sibiricum wine and reducing potential risks to human health.

[0038] (2) The method for preparing Polygonatum wine by co-fermentation of compound microorganisms in this invention achieves precise control of the fermentation process through scientific and reasonable microbial strain ratio and fermentation condition control. This reduces the complexity and variability of microbial metabolic activities during fermentation, significantly improves the quality stability of Polygonatum wine, and enables the product to maintain stable quality over a relatively long period (120 days). At the same time, by using plate and frame filters, diatomaceous earth filters, microfiltration, ultrafiltration and other equipment and technologies, impurities such as macromolecular polysaccharides and proteins in the wine are removed, ensuring the clarity and stability of the wine and avoiding secondary sedimentation after prolonged storage. Attached Figure Description

[0039] Figure 1The methanol detection results are for the Polygonatum wine of Examples 1-3 and Comparative Example 1; where A is the sample of Example 1, B is the sample of Example 2, C is the sample of Example 3, and D is the sample of Comparative Example 1.

[0040] Figure 2 Sensory evaluation of Polygonatum wine during storage ( (n=10).

[0041] Figure 3 The results show the alcohol content of the Polygonatum wine during storage.

[0042] Figure 4 The results show the total acidity determination during the storage of Polygonatum wine.

[0043] Figure 5 The results show the color difference value (ΔE) measured during the storage of Polygonatum wine.

[0044] Figure 6 The results show the light transmittance of Polygonatum wine during storage. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] The traditional yeast was purchased from "Liangshi-Plant Sweet Wine Yeast" produced by Hubei Quzixiang Food Co., Ltd.

[0048] Lactobacillus plantarum LP45 (100 billion CFU / g) was purchased from Hebei Yiran Biotechnology Co., Ltd.; Saccharomyces cerevisiae was Angel Yeast RV171 (10 billion CFU / g) purchased from Angel Yeast Co., Ltd.; Bacillus lactis DU-106 (10 10 The CFU / g was provided by the College of Food Science and Technology, South China Agricultural University.

[0049] Example 1

[0050] (1) Raw material processing:

[0051] Weigh 200g of Polygonatum rhizome slices, soak and rinse them in distilled water for several minutes to thoroughly remove impurities from the surface. Then, add distilled water again to submerge the Polygonatum rhizome, decoct for 3-5 minutes, remove the slices, and place them in a fermentation tank for later use.

[0052] (2) Saccharification:

[0053] Boil 1000mL of distilled water, add 2000g of granulated sugar, and stir until the sugar is completely dissolved. Then, transfer the dissolved sugar water into the prepared fermentation tank.

[0054] (3) Selection and activation of strains:

[0055] The selected bacterial strains were *Lactobacillus plantarum*, *Bacillus lactis* DU-106, and *Saccharomyces cerevisiae*. Appropriate amounts of physiological saline and an equal volume of 5% sugar solution were prepared separately, mixed well, and sterilized in an autoclave at 121°C for 15 minutes. Then, 0.1 g of *Lactobacillus plantarum*, 0.05 g of *Bacillus lactis* DU-106, and 2.5 g of *Saccharomyces cerevisiae* were added to each 100 mL of solution. Finally, activation was performed at 30°C for 30 minutes to obtain the activated compound bacterial strain.

[0056] (4) Inoculation and fermentation:

[0057] Add another 6000mL of distilled water to the fermenter and wait for the solution to cool to room temperature. Then, add the 200g of activated compound microbial solution from step (3) (i.e., the amount of compound microbial solution added is equal to the amount of Polygonatum sibiricum medicinal material added) to the fermenter and stir evenly. After that, seal the fermenter and maintain the temperature at 25℃ for 5 days of fermentation.

[0058] (5) Filtration and sterilization:

[0059] After fermentation, the wine is coarsely filtered through a coarse fiber filter bag to remove the Polygonatum rhizome and larger impurities. Next, a plate and frame filter press is used to remove larger suspended solids and impurities, reducing the workload for subsequent filtrations. Then, a diatomaceous earth filter is used to further remove fine particles and suspended solids, including some plant fibers and colloids. Afterward, the wine is filtered sequentially using 0.45μm and 0.15μm microporous membranes to achieve sterility. Finally, an ultrafiltration membrane with a molecular weight cutoff of 10,000 is used to remove large molecular compounds, preventing the precipitation of large molecules such as Polygonatum rhizome polysaccharides and proteins, which would otherwise form flocculent precipitates, thus ensuring the clarity and stability of the wine.

[0060] (6) Filling:

[0061] The filtered Polygonatum fermented wine is transferred into bottles for bottling.

[0062] Example 2

[0063] (1) Raw material processing:

[0064] Weigh 200g of Polygonatum rhizome slices, soak and rinse them in distilled water for several minutes to thoroughly remove impurities from the surface. Then, add distilled water again to submerge the Polygonatum rhizome, decoct for 3-5 minutes, remove the slices, and place them in a fermentation tank for later use.

[0065] (2) Saccharification:

[0066] Boil 1500mL of distilled water, add 3000g of granulated sugar, and stir until the sugar is completely dissolved. Then, transfer the dissolved sugar water into the prepared fermentation tank.

[0067] (3) Selection and activation of strains:

[0068] The selected bacterial strains were *Lactobacillus plantarum*, *Bacillus lactis* DU-106, and *Saccharomyces cerevisiae*. Appropriate amounts of physiological saline and an equal volume of 5% sugar solution were prepared separately and sterilized in an autoclave at 121°C for 15 minutes. Then, 0.15 g of *Lactobacillus plantarum*, 0.075 g of *Bacillus lactis* DU-106, and 3.75 g of *Saccharomyces cerevisiae* were added to each 100 mL of solution. Finally, the mixture was activated at 30°C for 30 minutes to obtain the activated compound bacterial strain.

[0069] (4) Inoculation and fermentation:

[0070] Add another 9000mL of distilled water to the fermentation tank and wait for the solution to cool to room temperature. Then, add the 200g of activated compound microbial solution from step (3) (i.e., the amount of compound microbial solution added is equal to the amount of Polygonatum sibiricum medicinal material added) to the fermentation tank and stir evenly. After that, seal the fermentation tank and maintain the temperature at 30℃ for 7 days of fermentation.

[0071] (5) Filtration and sterilization:

[0072] After fermentation, the wine is coarsely filtered through a coarse fiber filter bag to remove the Polygonatum rhizome and larger impurities. Next, a plate and frame filter press is used to remove larger suspended solids and impurities, reducing the workload for subsequent filtrations. Then, a diatomaceous earth filter is used to further remove fine particles and suspended solids, including some plant fibers and colloids. Afterward, the wine is filtered sequentially using 0.45μm and 0.15μm microporous membranes to achieve sterility. Finally, an ultrafiltration membrane with a molecular weight cutoff of 10,000 is used to remove large molecular compounds, preventing the precipitation of large molecules such as Polygonatum rhizome polysaccharides and proteins, which would otherwise form flocculent precipitates, thus ensuring the clarity and stability of the wine.

[0073] (6) Filling:

[0074] The filtered Polygonatum fermented wine is transferred into bottles for bottling.

[0075] Example 3

[0076] (1) Raw material processing:

[0077] Weigh 200g of Polygonatum rhizome slices, soak and rinse them in distilled water for several minutes to thoroughly remove impurities from the surface. Then, add distilled water again to submerge the Polygonatum rhizome, decoct for 3-5 minutes, remove the slices, and place them in a fermentation tank for later use.

[0078] (2) Saccharification:

[0079] Boil 2000mL of distilled water, add 4000g of granulated sugar, and stir until the sugar is completely dissolved. Then, transfer the dissolved sugar water into the prepared fermentation tank.

[0080] (3) Selection and activation of strains:

[0081] The selected bacterial strains were *Lactobacillus plantarum*, *Bacillus lactis* DU-106, and *Saccharomyces cerevisiae*. Appropriate amounts of physiological saline and an equal volume of 5% sugar solution were prepared separately and sterilized in an autoclave at 121°C for 15 minutes. Then, 0.2 g of *Lactobacillus plantarum*, 0.1 g of *Bacillus lactis* DU-106, and 5.0 g of *Saccharomyces cerevisiae* were added to each 100 mL of solution. Finally, activation was performed at 30°C for 30 minutes to obtain the activated compound bacterial strain.

[0082] (4) Inoculation and fermentation:

[0083] Add 12,000 mL of distilled water to the fermenter and wait for the solution to cool to room temperature. Then, add the 200 g of activated compound microbial solution from step (3) (i.e., the amount of compound microbial solution added is equal to the amount of Polygonatum sibiricum medicinal material added) to the fermenter and stir evenly. After that, seal the fermenter and maintain the temperature at 35°C for 10 days of fermentation.

[0084] (5) Filtration and sterilization:

[0085] After fermentation, the wine is coarsely filtered through a coarse fiber filter bag to remove the Polygonatum rhizome and larger impurities. Next, a plate and frame filter press is used to remove larger suspended solids and impurities, reducing the workload for subsequent filtrations. Then, a diatomaceous earth filter is used to further remove fine particles and suspended solids, including some plant fibers and colloids. Afterward, the wine is filtered sequentially using 0.45μm and 0.15μm microporous membranes to achieve sterility. Finally, an ultrafiltration membrane with a molecular weight cutoff of 10,000 is used to remove large molecular compounds, preventing the precipitation of large molecules such as Polygonatum rhizome polysaccharides and proteins, which would otherwise form flocculent precipitates, thus ensuring the clarity and stability of the wine.

[0086] (6) Filling:

[0087] The filtered Polygonatum fermented wine is transferred into bottles for bottling.

[0088] Comparative Example 1

[0089] This comparative example does not use specific fermentation strains, but instead uses traditional yeast for natural fermentation. Specifically, in the selection and activation of the strains, *Lactobacillus plantarum*, *Bacillus lactis* DU-106, and *Saccharomyces cerevisiae* used in Example 1 were replaced with an equal mass of traditional yeast for fermentation. Except for the above strain replacement, all other fermentation steps and production conditions are the same as in Example 1.

[0090] Comparative Example 2

[0091] This comparative example uses a single-strain fermentation, specifically using only *Lactobacillus plantarum*. In the selection and activation of the strain, the three strains mixed in proportion in Example 1 were replaced with an equal mass of plant yeast for fermentation. Apart from this, all fermentation steps and production conditions were the same as in Example 1.

[0092] Comparative Example 3

[0093] This comparative example uses a single-strain fermentation, specifically using only *Bacillus lactis* DU-106. In the selection and activation of the strain, the three strains mixed in proportion in Example 1 were replaced with an equal mass of *Bacillus lactis* DU-106 for fermentation. Apart from this, all fermentation steps and production conditions were the same as in Example 1.

[0094] Comparative Example 4

[0095] This comparative example uses a single-strain fermentation, specifically using only *Saccharomyces cerevisiae*. In the selection and activation of the strain, the three strains mixed in proportion in Example 1 were replaced with an equal mass of *Saccharomyces cerevisiae*. Apart from this, all fermentation steps and production conditions are the same as in Example 1.

[0096] Comparative Example 5

[0097] This comparative example uses dual-strain fermentation, specifically selecting *Lactobacillus plantarum* and *Bacillus lactis* DU-106. During the strain selection and activation stage, the three strains mixed in proportion in Example 1 were replaced with equal masses of *Lactobacillus plantarum* and *Bacillus lactis* DU-106, maintaining the same proportions as in Example 1. Except for the strain adjustment, all fermentation steps and production conditions were the same as in Example 1.

[0098] Comparative Example 6

[0099] This comparative example uses a dual-strain fermentation, specifically selecting *Bacillus lactis* DU-106 and *Saccharomyces cerevisiae*. During the strain selection and activation stage, the three strains mixed in proportion in Example 1 were replaced with equal masses of *Bacillus lactis* DU-106 and *Saccharomyces cerevisiae*, while maintaining the same proportions as in Example 1. Except for the strain adjustment, all fermentation steps and production conditions were the same as in Example 1.

[0100] Comparative Example 7

[0101] This comparative example uses dual-strain fermentation, specifically selecting *Lactobacillus plantarum* and *Saccharomyces cerevisiae*. During the strain selection and activation stage, the three strains mixed in proportion in Example 1 were replaced with equal masses of *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, while maintaining the same proportions as in Example 1. Except for the strain adjustment, all fermentation steps and production conditions are the same as in Example 1.

[0102] Comparative Example 8

[0103] This comparative example used three strains for fermentation, but unlike Example 1, the mixing ratio of these three strains (Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae) was 1:1:1, while the total mass remained consistent with Example 1. During the selection and activation stage of the strains, they were mixed in a 1:1:1 ratio. Apart from this, all fermentation steps and production conditions were the same as in Example 1.

[0104] Comparative Example 9

[0105] This comparative example used three strains for fermentation, but unlike Example 1, the mixing ratio of these three strains (Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae) was 1:10:50, while the total mass remained consistent with Example 1. During the selection and activation stage of the strains, they were mixed at a ratio of 1:10:50. Apart from this, all fermentation steps and production conditions were the same as in Example 1.

[0106] Comparative Example 10

[0107] This comparative example used three strains for fermentation, but unlike Example 1, the mixing ratio of these three strains (Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae) was 50:10:1, while the total mass remained consistent with Example 1. During the selection and activation stage of the strains, the strains were mixed at a ratio of 50:10:1. Apart from this, all fermentation steps and production conditions were the same as in Example 1.

[0108] Comparative Example 11

[0109] The filtration and sterilization method in this comparative example differs from that in Example 1. In the filtration step, the fermented wine is coarsely filtered through a coarse fiber filter bag, then larger suspended solids and impurities are removed using a plate and frame filter press, and finally, a diatomaceous earth filter is used to further remove small particles and suspended solids, including some plant fibers and colloids. However, this comparative example does not use microporous membranes or ultrafiltration membranes for deep filtration. Apart from this, the remaining steps and production conditions are the same as in Example 1.

[0110] Comparative Example 12

[0111] The filtration and sterilization method in this comparative example differs from that in Example 1. In the filtration step, the fermented wine is coarsely filtered through a coarse fiber filter bag, then a plate and frame filter press is used to remove larger suspended solids and impurities, followed by a diatomaceous earth filter to further remove fine particles and suspended solids, including some plant fibers and colloids. It is then filtered sequentially through 0.45μm and 0.15μm microporous membranes. However, ultrafiltration is not used for deep filtration in this comparative example. Apart from this, the remaining steps and production conditions are the same as in Example 1.

[0112] Test Example 1

[0113] Sensory evaluation of Polygonatum wine

[0114] Sensory evaluations were conducted on the Polygonatum wines of Examples 1-3 and Comparative Examples 1-12 in terms of aroma, taste, color, and clarity. The specific sensory evaluation criteria are shown in Table 1. The evaluation scores are shown in Table 2.

[0115] 1. Experimental Objective

[0116] Through systematic sensory evaluation, the Polygonatum wines of Examples 1-3 and Comparative Examples 1-12 were systematically evaluated in terms of aroma, taste, color, and clarity, so as to objectively compare the quality of different Polygonatum wines.

[0117] 2. Experimental materials and personnel

[0118] Experimental materials: Polygonatum wine samples from Examples 1-3 and Comparative Examples 1-12, clean and odorless tasting glasses, light source for observing clarity, and transparent glass cups.

[0119] Personnel: 10 trained sensory evaluators with keen sense of smell, taste and sight, and familiar with the sensory evaluation process and standards for alcoholic beverages.

[0120] 3. Experimental Procedure

[0121] 3.1 Preparation Phase:

[0122] Ensure the evaluation environment is clean and tidy, free from odors, and that the temperature and humidity are controlled within a suitable range (room temperature 20-25℃, humidity 50%-60%).

[0123] All samples of Polygonatum wine were numbered and randomly placed to ensure that the evaluators were unaware of the samples' origin.

[0124] Prepare tasting cups, a light source, and a clear glass.

[0125] The evaluators first tasted a glass of pure water to remove any lingering taste from their mouths.

[0126] Pour the Polygonatum wine sample into tasting glasses and transparent glasses respectively, ensuring that the amount of sample in each glass is consistent.

[0127] 3.2 Sensory evaluation process:

[0128] Aroma evaluation: The evaluator lightly smells the sample and records its aroma characteristics (such as intensity, purity, and presence of off-odors). Then, after a period of time (30 seconds), the evaluator smells the sample again to assess the aroma persistence and scores it according to the standards in Table 1.

[0129] Taste evaluation: Evaluators take small sips of the sample, paying attention to the balance and harmony of its sweetness, acidity, bitterness, astringency, and other flavors, as well as whether the aftertaste is long-lasting, and score it according to the standards in Table 1.

[0130] Color evaluation: Under a uniform light source, observe the color of each Polygonatum wine sample, record its depth, transparency and uniformity, and score according to the standards in Table 1.

[0131] Clarity evaluation: Place the sample in a transparent glass and observe its clarity, whether there are any suspended or sedimentary substances, and score it according to the standards in Table 1.

[0132] 3.3 Precautions:

[0133] Before the evaluation, evaluators should avoid eating spicy or irritating foods and maintain oral hygiene.

[0134] During the evaluation process, rinse your mouth with water after tasting each sample to avoid residual taste affecting subsequent evaluations.

[0135] Evaluators should conduct independent evaluations to avoid interference from each other.

[0136] For each evaluation indicator, each evaluator needs to repeat the evaluation three times to improve the accuracy of the results.

[0137] 4. Data Recording and Analysis

[0138] Data recording: Collect scores from 10 evaluators on each sample in four aspects: aroma, taste, color, and clarity.

[0139] Results Analysis: The sum of the scores for each sample across all evaluation indicators was calculated as the final comprehensive score for that sample. A higher comprehensive score indicates better overall quality of the Polygonatum wine.

[0140] Table 1. Sensory Evaluation Criteria for Polygonatum Wine

[0141]

[0142]

[0143] Table 2. Sensory evaluation results of Polygonatum wines from Examples 1-3 and Comparative Examples 1-12 ( n=10)

[0144]

[0145]

[0146] Sensory comparisons were performed between Comparative Examples 1-12 and Example 1. The results of the sensory comparisons are as follows:

[0147] Compared to Example 1, the Polygonatum wine prepared in Comparative Example 1 differs significantly in quality. Firstly, the wine prepared in Comparative Example 1 has a distinctly bitter taste characteristic of Polygonatum, and its fermentation period needs to be significantly extended. Specifically, when the alcohol content of the Polygonatum wines prepared in Example 1 and Comparative Example 1 was measured, after 5 days of fermentation, the alcohol content of the wine obtained in Comparative Example 1 was still relatively low, requiring approximately 15 days to reach an alcohol content level similar to that of Example 1 (approximately 8.7°). This indicates that the synergistic effect of multiple microbial strains in this invention can accelerate the fermentation process and significantly shorten the fermentation period. Furthermore, due to the complexity and uncertainty of the natural fermentation environment, competition and interaction among multiple microorganisms may lead to the generation of undesirable metabolites.

[0148] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 2 had no alcoholic taste, only a sour taste. This is because the main metabolite of Lactobacillus plantarum during fermentation is lactic acid rather than alcohol. This indicates that using Lactobacillus plantarum alone is not suitable for fermenting and preparing Polygonatum wine with a typical alcoholic taste, but is more likely to yield a sour product similar to "Polygonatum vinegar".

[0149] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 3 had no alcoholic taste, only a sour taste. This is because Bacillus lactis DU-106, similar to Lactobacillus plantarum, produces lactic acid rather than alcohol as its main metabolite during fermentation. This indicates that using Bacillus lactis DU-106 alone is not suitable for fermenting and preparing Polygonatum wine with a typical alcoholic taste, but is more likely to yield a sour product similar to "Polygonatum vinegar".

[0150] Compared to Example 1, the Polygonatum wine prepared in Comparative Example 4 was inferior in both taste and aroma. Specifically, due to the use of a single strain of microorganisms for fermentation, the Polygonatum wine had a rather monotonous taste, lacking the sweet and sour, smooth balance found in Example 1. Furthermore, the bitterness of Polygonatum was more pronounced, affecting the overall drinking experience. This indicates that the synergistic effect of multiple microorganisms during the fermentation process of Polygonatum wine is crucial for improving the product's taste and aroma.

[0151] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 5 exhibited significant differences in flavor. Specifically, this product lacked the aroma of traditional Polygonatum wine, instead possessing a strong, prominent sour taste. This phenomenon can be attributed to the fact that the main metabolic products of *Lactobacillus plantarum* and *Bacillus lactis* DU-106 during fermentation are lactic acid, rather than alcohol. Therefore, using only these two strains for fermentation is unlikely to yield a Polygonatum wine with a typical alcoholic flavor, and is more likely to result in a beverage with a strong sour taste.

[0152] Compared with Example 1, the Polygonatum wine prepared in Comparative Example 6 showed certain differences in flavor. Specifically, the Polygonatum wine had a milder acidity and a higher sweetness. At the same time, its flavor complexity and layering were insufficient, failing to fully demonstrate the rich and harmonious taste of the Polygonatum wine in Example 1.

[0153] Compared to Example 1, the Polygonatum wine prepared in Comparative Example 7 exhibited some changes in flavor. Specifically, the wine was milder in taste and acidity, while being sweeter. However, this flavor adjustment also resulted in a lack of complexity and depth in the flavor profile, failing to fully showcase the rich and harmonious taste of the Polygonatum wine from Example 1.

[0154] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 8 exhibited a significant change in flavor. This product displayed sharp acidity and high sweetness, but with extremely low alcohol content, resulting in a taste that leaned more towards a tangy and refreshing flavor than the mellow aroma of traditional Polygonatum wine. Overall, its flavor profile was closer to that of a fermented vinegar beverage with a distinctly tangy and refreshing taste, rather than "Polygonatum wine" in the traditional sense.

[0155] Compared with Example 1, the Polygonatum wine prepared in Comparative Example 9 tasted more acidic and not sweet enough. This reflects a possible incoordination in the adjustment of the strain ratio, resulting in the failure to achieve an ideal balance between acidity and sweetness.

[0156] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 10 exhibited a significant imbalance in flavor. The product displayed a pronounced sourness that almost masked the mellow aroma of the wine, resulting in an overall taste that was too extreme and failed to achieve the proper flavor balance expected of Polygonatum wine.

[0157] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 11 had a highly consistent taste, aroma, and color. However, in terms of clarity, the "Polygonatum wine" in Comparative Example 11 appeared less transparent and slightly cloudy. After being left at room temperature for several days, obvious flocculent precipitate formed in the wine. In contrast, the Polygonatum wine prepared in Example 1 did not show any obvious precipitate even after being left at room temperature for three months. This indicates that the filtration and sterilization method, especially the microfiltration and ultrafiltration steps, affects the clarity and stability of the Polygonatum wine.

[0158] Compared to Example 1, the "Polygonatum wine" prepared in Comparative Example 12 had a highly consistent taste, aroma, and color. Immediately after preparation, its clarity was also not significantly different from that of Example 1. However, after being left at room temperature for 3 months, a small amount of flocculent precipitate appeared in the Polygonatum wine of Comparative Example 12, while the Polygonatum wine prepared in Example 1 remained clear without any obvious precipitate. This phenomenon indicates that the filtration and sterilization method, especially the ultrafiltration step, has a significant impact on the long-term clarity and stability of Polygonatum wine.

[0159] Through systematic sensory evaluation analysis, it can be clearly observed from Table 2 that the Polygonatum wines of Examples 1-3 exhibit superior performance in multiple aspects such as aroma, taste, color and clarity. Their average comprehensive scores are significantly higher than those of Comparative Examples 1-12, and this difference is statistically significant. This fully demonstrates that the Polygonatum wines of Examples 1-3 are of better quality and have a superior production process.

[0160] This significant advantage can be attributed to the innovative use of three microbial strains—Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae—in a specific ratio (2–4:1–2:50–100) during the production of Polygonatum wine in Examples 1–10. In contrast, the Polygonatum wines in Comparative Examples 1–10 showed significant differences in the use or ratio of microbial strains. Some did not use the above-mentioned combination of microbial strains (e.g., Comparative Example 1), some only used some of the microbial strains (e.g., Comparative Examples 2–7), and some used all three microbial strains but in an inappropriate ratio (e.g., Comparative Examples 8–10).

[0161] The Polygonatum wines prepared in Examples 1-3 exhibit a richer and more harmonious flavor and taste compared to the Polygonatum wine prepared in the comparative example. Since flavor and taste reflect the overall chemical composition of the wine, this indicates that the Polygonatum wines prepared in Examples 1-3 have a richer and more harmonious chemical composition. The synergistic effect of the three microbial strains—Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae—promoted the efficient transformation and utilization of complex metabolites in Polygonatum. Therefore, it is clear that the precise ratio and synergistic fermentation mechanism of Lactobacillus plantarum, Bacillus lactis DU-106, and Saccharomyces cerevisiae play a crucial role in improving the overall quality of Polygonatum wine. The absence or imbalance of any microbial component will adversely affect the fermentation process, thereby weakening the quality of the final product.

[0162] Furthermore, Examples 1-3 featured meticulously designed filtration and sterilization processes, employing a multi-stage filtration method: coarse fiber filter bag filtration, plate and frame filter filtration, diatomaceous earth filter filtration, microporous membrane filtration, and ultrafiltration with a molecular weight cutoff of 10,000. This ensured the clarity and transparency of the wine. Even after being stored at room temperature for three months, no sediment was observed. Comparative Examples 11 and 12, however, simplified their filtration methods. Comparative Example 11 omitted the microporous membrane filtration and ultrafiltration stages, while Comparative Example 12 omitted the ultrafiltration stage altogether. This resulted in significantly lower sensory evaluation scores for clarity compared to Examples 1-3. This comparison clearly demonstrates the crucial role of microporous membrane filtration and ultrafiltration with a molecular weight cutoff of 10,000 in improving the clarity of the Polygonatum wine.

[0163] In summary, Examples 1-3 significantly improved the quality of Polygonatum wine through precise strain ratios, synergistic fermentation mechanisms, and multi-level filtration and sterilization processes.

[0164] Test Example 2

[0165] Methanol determination in Examples 1-3 and Comparative Example 1.

[0166] The main component of alcoholic beverages is ethanol. However, due to simplified brewing methods, low-quality raw materials, inadequate brewing techniques, or improper practices such as the illegal addition of industrial methanol, the methanol content in some alcoholic beverages may be abnormally high. During metabolism in the body, methanol is converted into formaldehyde and formic acid. Both of these substances are far more toxic than methanol itself. Excessive intake can rapidly lead to retinal damage, and in severe cases, even blindness or death.

[0167] Methanol formation is influenced by various factors, including the brewing process, the types of raw materials used, and fermentation temperature. Specifically, the selection of inferior alcohol and raw materials, as well as failure to follow standardized production procedures, are significant causes of increased methanol content. Incomplete fermentation or improper brewing techniques can lead to methanol levels exceeding safe standards. Controlling brewing conditions, such as temperature and fermentation conditions, and using high-quality raw materials are crucial for reducing methanol formation.

[0168] 1. Experimental objective:

[0169] The purpose of this invention is to evaluate whether the compound microbial fermentation technology can optimize the metabolic pathway of microorganisms, effectively reduce the generation of harmful metabolites (methanol), and thus improve the safety of Polygonatum wine.

[0170] 2. Detection method:

[0171] A methanol detection kit was used. The kit was manufactured by Shenyang Kaicheng Technology Co., Ltd.

[0172] 3. Detection principle:

[0173] During the testing process, methanol in the wine sample is oxidized to formaldehyde under the action of specific oxidizing and decolorizing agents. Subsequently, the formaldehyde reacts chemically with the color developer in the reagent kit to generate a purple quinone pigment. The intensity of this reaction product's color is directly proportional to the methanol content in the wine sample; that is, the darker the color, the higher the methanol content. By comparing the color intensity with a standard color scale, the methanol content in the wine sample can be roughly determined, and it can be used to judge whether it exceeds the national safety standard.

[0174] 4. Testing steps:

[0175] (1) Take 100 mL of the Polygonatum wine sample to be tested and place it in the distillation flask of a rotary evaporator. Set the water bath temperature to 80℃ and the cooling circulation pump temperature to -20℃. Do not start the vacuum pump during this process, and keep the gas pressure inside the rotary evaporator at atmospheric pressure. Continue distilling until no more liquid flows into the receiving flask, and collect the obtained distillate. Then, add an appropriate amount of distilled water to the collected distillate to make up to 100 mL, which will be used as the sample to be tested later. This step aims to distill out the alcohols in the Polygonatum wine and remove as much as possible pigments, sugars, and other substances that may cause interference.

[0176] (2) Use a pipette to take 0.1 ml of the above sample to be tested and inject it into the test tube. Then, add distilled water to the test tube until the liquid level reaches the 1.0 ml mark (i.e., the 1.0 mark on the test tube), and immediately put on the cap and gently shake to mix thoroughly.

[0177] (3) In the mixed test tube, add 2 drops of Test Solution A (oxidizing agent). Subsequently, shake the test tube for about 1 minute and then let it stand for 10 minutes to allow the reaction to proceed fully.

[0178] (4) After 10 minutes, add 1 drop of Test Solution B (decolorizing agent) to the test tube. After covering the lid again, shake it up and down several times to ensure the solution is fully mixed. Then, wait for the solution to decolorize on its own. If the solution has not completely decolorized within 10 minutes, 1 more drop of the decolorizing agent can be added.

[0179] (5) After the solution has completely decolorized to light yellow or colorless, add 3 drops of Test Solution C (color-developing agent) to the test tube. Cover the lid again and gently shake to mix. Then, let it stand for 30 minutes (this time point is crucial, and the result must be observed at the 30 - minute mark). Finally, observe the color change of the solution. If the solution shows a purple color, it indicates that the sample contains a relatively large amount of methanol; and the darker the color, the higher the methanol concentration. By comparing with the colorimetric card, the methanol content in the wine sample can be roughly judged.

[0180] 5. Evaluation Criteria:

[0181] According to the relevant regulations of GB 2757 - 2012 "National Food Safety Standard - Distilled Wines and Their Blended Wines", for wines brewed from non - grain cereal raw materials, the methanol content ≤ 2.0 g / L.

[0182] 6. Experimental Results

[0183] The methanol detection results of Examples 1 - 3 and Comparative Example 1 are as Figure 1 shown. Figure 1 Visually presented the colors of the final test sample solutions of the polygonatum wines in Examples 1 - 3, which are significantly lighter than the color of the final test sample solution of Comparative Example 1. Through precise comparison with the colorimetric card provided with the methanol test tube, it can be confirmed that the methanol content in the polygonatum wines in Examples 1 - 3 is all lower than 0.1 g / L, while the methanol content of the polygonatum wine in Comparative Example 1 falls within the range of 0.1 - 0.2 g / L. Although it meets the limit standard for the methanol content of fermented wines, its methanol content is significantly higher compared to the polygonatum wine prepared in Example 1. This result indicates that the composite strain fermentation technology of the present invention can inhibit the formation of the undesirable metabolite methanol by precisely regulating the metabolic pathways of microorganisms.

[0184] It should be noted that the methanol content in the polygonatum wines in Examples 1 - 3 is much lower than the limit (i.e., not exceeding 2.0 g / L) specified by the national food safety standard GB2757 - 2012. This result proves that the polygonatum wines brewed using the technology of the present invention not only meet the national safety standards in terms of methanol control but also demonstrate extremely high safety, providing a healthier choice for consumers.

[0185] In summary, the compound microbial fermentation technology involved in this invention has significant effects in reducing the methanol content in Polygonatum wine, providing a solid technical foundation and guarantee for improving the overall quality and safety of Polygonatum wine.

[0186] Test Example 3

[0187] Stability evaluation of Polygonatum wine during storage:

[0188] The Polygonatum wines of Examples 1-3 were stored at room temperature for 0, 10, 30, 60, 90 and 120 days, respectively. A comprehensive sensory evaluation and tests of alcohol content, total acidity, color and clarity were conducted to assess the stability of the Polygonatum wines during storage.

[0189] 1. Sensory evaluation of Polygonatum wine during storage

[0190] The methods, procedures, and evaluation criteria for the sensory evaluation of Polygonatum wine all followed the methods and procedures specified in Test Example 1. The evaluation results are shown below. Figure 2 Sensory evaluation scores can comprehensively and intuitively reflect the changes in Polygonatum wine during storage. Figure 2 The sensory scores of the Polygonatum wines in Examples 1-3 are clearly shown to vary with different storage days. The results indicate that the sensory scores of the Polygonatum wines did not fluctuate significantly with the gradual increase in storage days, suggesting that the flavor and quality of the Polygonatum wines maintained good stability during storage at room temperature for up to 120 days.

[0191] 2. Alcohol content determination during the storage of Polygonatum wine

[0192] According to the alcohol content determination method in GB5009.225-2023 National Food Safety Standard for Determination of Ethanol Concentration in Wine and Edible Alcohol, the alcohol meter method was used to determine the alcohol content of the Polygonatum wine in Examples 1 to 3.

[0193] principle:

[0194] Non-volatile substances in the sample were removed by distillation. The volume fraction of alcohol was measured by an alcohol meter. The temperature was corrected by referring to the conversion table between alcohol meter temperature and ethanol concentration (alcohol content) at 20℃, and the ethanol concentration (alcohol content) of the sample at 20℃ was obtained.

[0195] Instruments and equipment:

[0196] (1) Precision alcohol meter: graduation value is 0.1% vol.

[0197] (2) All-glass stills: 500mL, 1000mL.

[0198] (3) Graduated cylinders: 100mL, 200mL, 500mL, 1000mL.

[0199] (4) Thermometer: scale division value is 0.1℃.

[0200] Analysis steps:

[0201] (1) Sample preparation

[0202] After heating a clean, dry 100mL volumetric flask and sample to 20℃, accurately measure 100mL of sample into a 500mL distillation flask. Rinse the volumetric flask three times with 50mL of water, and combine the washings with the 500mL distillation flask. Add a few boiling stones, connect a serpentine condenser, and use the original volumetric flask used for sampling as a receiver (with an ice bath). Turn on the cooling water (cooling water temperature below 15℃) and slowly heat and distill, collecting the distillate. When near the mark, remove the volumetric flask, stopper it, and incubate it in a 20℃ water bath for 30 minutes. Then add water (20℃) to the mark, mix well, and set aside.

[0203] (2) Determination of sample solution

[0204] Pour the sample solution into a clean, dry graduated cylinder and let it stand for several minutes until the air bubbles disappear. Then, place a clean, dry alcohol meter in the cylinder and press it gently, ensuring it does not touch the cylinder wall. Simultaneously, insert a thermometer and allow it to equilibrate for about 5 minutes. Observe horizontally and read the scale reading at the point tangent to the meniscus, while recording the temperature.

[0205] (3) Presentation of analysis results

[0206] Based on the measured alcohol meter reading and temperature, refer to Appendix B of GB5009.225-2023 National Food Safety Standard for Determination of Ethanol Concentration in Wines and Edible Alcohol to convert the alcohol content of the sample at 20℃, expressed as a volume fraction "%vol". The result is expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, rounded to one decimal place. The absolute difference between two independent determinations obtained under repeatability conditions should not exceed 0.5%vol. See results below. Figure 3 .

[0207] Alcohol content is one of the core indicators for evaluating the quality of wines, and its stability is particularly important during the storage process. If the alcohol content can remain constant during storage, this is not only an important sign of the wine's maturity and excellent quality, but also a key indicator of its outstanding aging potential.

[0208] Figure 3The changes in alcohol content of the Polygonatum wines from Examples 1-3 after storage at room temperature for different number of days are visually demonstrated. The figures clearly show that the alcohol content of the Polygonatum wines did not fluctuate significantly with prolonged storage time. This fully demonstrates that the alcohol content of the Polygonatum wines from Examples 1-3 remained highly stable and at a relatively constant level during the 120 days of storage at room temperature.

[0209] 3. Determination of total acidity during the storage of Polygonatum wine

[0210] The test was conducted according to the acid-base indicator titration method in the "National Food Safety Standard GB / T12456-2021 Determination of Total Acid in Food".

[0211] principle:

[0212] Based on the principle of acid-base neutralization, the acid in the test solution is titrated with an alkaline solution, and the titration endpoint is determined using phenolphthalein as an indicator. The total acid content in the food is calculated based on the amount of alkaline solution consumed.

[0213] Reagents and preparation:

[0214] Phenolphthalein indicator solution (10g / L): Weigh 1g of phenolphthalein, dissolve it in ethanol (95%), and dilute it to 100mL with ethanol (95%).

[0215] Sodium hydroxide standard titration solution (0.1 mol / L): Prepared and standardized according to the requirements of GB / T 5009.1.

[0216] Analysis steps:

[0217] Pipette 25.0 mL of the Polygonatum odoratum wine sample into a 250 mL volumetric flask, dilute to the mark with carbon dioxide-free water, and mix well. Filter through rapid filter paper, collect the filtrate, and use it as the test solution for determination.

[0218] Pipette 25 mL of the test solution into a 250 mL Erlenmeyer flask. Add 2–4 drops (10 g / L) of phenolphthalein indicator solution and titrate with 0.1 mol / L sodium hydroxide standard titration solution until a faint pink color persists for 30 seconds. Record the volume of 0.1 mol / L sodium hydroxide standard titration solution consumed. Simultaneously, perform a blank test using the same volume of carbon dioxide-free water instead of the test solution, and record the volume of sodium hydroxide standard titration solution consumed.

[0219] Result calculation:

[0220] The total acid content in the sample is calculated according to formula (1).

[0221]

[0222] In the formula:

[0223] X — The total acid content in the sample, expressed in grams per liter (g / L);

[0224] c — Concentration of the sodium hydroxide standard titration solution, in moles per liter (mol / L);

[0225] V1—The volume of sodium hydroxide standard titration solution consumed during the titration of the test solution, in milliliters (mL);

[0226] V2—The volume of sodium hydroxide standard titration solution consumed in the blank test, in milliliters (mL);

[0227] K – Conversion factor for acids. Polygonatum wine mainly contains lactic acid, and the conversion factor for lactic acid is 0.090.

[0228] F – the dilution factor of the test solution, which is 10 in this formula;

[0229] m — the volume of the sample taken, in milliliters (mL);

[0230] 1000 — Conversion factor.

[0231] The calculation results are expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions, and the results are shown in [see figure]. Figure 4 .

[0232] During the storage of Polygonatum sibiricum wine, the stability of total acidity plays a crucial role in maintaining its unique varietal characteristics and superior quality. The flavor and taste of Polygonatum sibiricum wine, as a direct reflection of its core quality, are deeply influenced by total acidity, a vital component of the wine. Total acidity not only enriches the flavor profile but also profoundly shapes its unique taste experience. Therefore, maintaining a stable total acidity level during storage is essential for the effective preservation of the wine's flavor and taste. Furthermore, an appropriate level of total acidity effectively inhibits the activity of potentially harmful microorganisms in the wine, thereby extending its shelf life and ensuring its safety and quality. Conversely, insufficient total acidity may provide a breeding ground for microorganisms, threatening the wine's flavor, taste, and even safety. Therefore, the stability of total acidity is of great importance for the long-term preservation of Polygonatum sibiricum wine.

[0233] from Figure 4 It can be seen that during the storage of the Polygonatum wine in Examples 1-3 at room temperature for up to 120 days, the total acid content did not fluctuate significantly, but remained at a relatively constant level.

[0234] 4. Color evaluation during the storage of Polygonatum wine

[0235] Based on the fact that a colorimeter can simulate the process of human eyes observing color, by accurately measuring and quantifying subtle differences in color, the color stability of the Polygonatum wine in Examples 1-3 during storage can be analyzed.

[0236] By periodically measuring the color difference values ​​of Polygonatum wine stored for different periods, it is possible to observe how the color of Polygonatum wine changes as the storage time increases.

[0237] The colorimeter measures the L, a, and b values ​​of a standard sample (newly brewed Polygonatum wine, i.e., Polygonatum wine aged for 0 days) and the sample to be tested (Polygonatum wine aged for different times), and calculates the color difference (ΔE) between the two, as shown in Formula 2. In the formula, L represents brightness, with a larger value indicating greater brightness; a represents red-green, with a larger positive value indicating deeper red and a larger negative value indicating deeper green; b represents yellow-blue, with a larger positive value indicating deeper yellow and a larger negative value indicating deeper blue. A smaller ΔE value indicates minimal color change and good stability; conversely, a larger ΔE value indicates significant color change and poor stability.

[0238]

[0239] in:

[0240] △L——The difference between two consecutive measurements of L

[0241] △a——The difference between the two measurements of a

[0242] △b — the difference between the two measurements of b

[0243] The stability of color during the storage of Polygonatum wine is of immeasurable importance to its quality. The consistency of color directly reflects the stability of the wine's quality. If the color remains unchanged or undergoes only minor changes during storage, it indicates that most components in the wine have maintained good stability, avoiding significant oxidation and degradation processes, thus ensuring the overall good and stable quality of the Polygonatum wine.

[0244] There is a close relationship between color and flavor. A stable color often indicates that the loss of flavor components in the wine is minimal, and the unique medicinal and wine aroma of Polygonatum can be well preserved. Conversely, if the color changes significantly, such as darkening or fading, this is often a sign of damage to flavor substances, which may be accompanied by the development of unpleasant flavors, thus affecting the overall flavor of Polygonatum wine.

[0245] Figure 5 The figures specifically illustrate the color difference between the Polygonatum wines of Examples 1-3 and their original state after storage at room temperature for different numbers of days. The ΔE value in the figures directly reflects the degree of color difference; a larger ΔE value indicates a more significant color difference. (Observation) Figure 5It can be seen that the color difference value of the Polygonatum wine did not fluctuate significantly as the storage time increased, which fully proves that the color of the Polygonatum wine maintained a high degree of stability during the 120 days of storage at room temperature.

[0246] 5. Evaluation of Clarity During the Storage of Polygonatum Wine

[0247] Based on light transmittance, a physical indicator that measures the ability of light to penetrate a medium, the dynamic changes in the transparency of a liquid can be directly observed, thereby effectively assessing whether the Polygonatum wines of Examples 1-3 maintained a clear and stable state during storage.

[0248] Transmittance (T) represents the ability of light to pass through a medium; it is the percentage of luminous flux passing through a transparent or translucent medium relative to the incident luminous flux. When parallel monochromatic light passes through a uniform, non-scattering medium, part of the light is absorbed, part passes through the medium, and part is reflected by the medium's surface. Transmittance T is equal to the ratio of the intensity of the transmitted light (It) to the intensity of the incident light (I0), i.e., T = It / I0. A higher transmittance indicates a stronger ability of the medium to transmit light, and thus higher transparency.

[0249] The clarity of Polygonatum wine is closely related to its transparency. During storage, if Polygonatum wine becomes cloudy or develops sediment, its transparency will decrease, and its light transmittance will also decrease. Therefore, the light transmittance of Polygonatum wine can be periodically measured using a spectrophotometer to intuitively grasp the trend of its transparency changes and further evaluate the stability of its clarity.

[0250] Considering the testing range of the spectrophotometer and relevant research, 680 nm was selected as the wavelength for measuring the clarity of Polygonatum odoratum wine. A suitable sample of Polygonatum odoratum wine was taken, and its transmittance was measured at 680 nm. By comparing the transmittance data of Polygonatum odoratum wine stored for different periods, changes in its clarity can be directly assessed. Generally, a higher transmittance value indicates better clarity of the Polygonatum odoratum wine; conversely, a lower transmittance indicates a decrease in clarity. Results are shown below. Figure 6 .

[0251] Figure 6 The transmittance of the Polygonatum wines of Examples 1-3 at room temperature varies with the number of storage days. The figures clearly show that the transmittance of the Polygonatum wines did not fluctuate significantly with prolonged storage time, demonstrating that the clarity of the Polygonatum wines of Examples 1-3 remained highly stable during 120 days of storage at room temperature.

[0252] In summary, the Polygonatum wines of Examples 1-3, after being stored at room temperature for up to 120 days, exhibited excellent stability in various aspects, including sensory evaluation, alcohol content, total acid content, color, and clarity. Their flavor and quality changed very little during storage; alcohol content and total acid content remained constant, color remained stable without significant change, and clarity was also well maintained. These results demonstrate that the Polygonatum wines of Examples 1-3 possess excellent storage characteristics under room temperature storage conditions, maintaining good quality stability for 120 days.

Claims

1. A method for preparing a compound microbial strain co-fermentation of Polygonatum sibiricum wine, characterized in that, Includes the following steps: S1. Activate the compound microbial fermentation agent to obtain the compound microbial liquid; S2. Soak, wash, and decoct the Polygonatum slices before use. Stir sugar and distilled water until the sugar is completely dissolved to obtain sugar water. Then mix the sugar water with the Polygonatum slices, distilled water, and compound bacterial liquid. Seal and ferment at 25-35℃ for 5-10 days to obtain fermented wine. S3. Remove impurities from the fermented wine in sequence, filter and sterilize it, and remove macromolecular compounds with a molecular weight greater than 10,000 to obtain Polygonatum wine. The compound microbial fermentation agent consists of *Lactobacillus plantarum* LP45, *Bacillus lactis* DU-106, and *Saccharomyces cerevisiae* RV171 in a mass ratio of 2–3:1–1.5:50–75, with *Lactobacillus plantarum* LP45 having a viable count of 10-1. 10 ~10 12 The CFU / g count of Bacillus lactis DU-106 and Saccharomyces cerevisiae RV171 was 10. 9 ~10 11 CFU / g; The mass ratio of Polygonatum tablets, sugar, distilled water and compound bacterial solution in step S2 is 1:10-20:35-70:

1.

2. The preparation method according to claim 1, characterized in that, The activation described in step S1 is performed using a mixed solution of physiological saline and sugar water.

3. The preparation method according to claim 2, characterized in that, The sugar solution mentioned in step S1 is a 4-6 wt% sugar solution.

4. The preparation method according to claim 1, characterized in that, The activation conditions described in step S1 are: activation at 25–35°C for 25–35 minutes.

5. The preparation method according to claim 1, characterized in that, The mass ratio of Polygonatum sibiricum tablets, sugar, distilled water and compound bacterial solution in step S2 is 1:10-15:35-52.5:

1.

6. The preparation method according to claim 1, characterized in that, The filtration sterilization described in step S3 involves sequentially using microporous membranes with a diameter of 0.4–0.5 μm and 0.1–0.2 μm.

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