Bamboo-based fermented wine and preparation method thereof

Abstract

The invention provides bamboo-based fermented wine and a preparation method thereof. The preparation method comprises the following steps: a wet grinding step: performing wet grinding on a crushed product obtained by crushing and sieving a bamboo material to obtain bamboo-based slurry; an enzymolysis and liquefaction step: carrying out enzymolysis and liquefaction on the bamboo-based slurry by utilizing an enzyme preparation to obtain liquefied slurry; a fermentation step: in the presence of a carbon source and water, fermenting the liquefied slurry by using a composite strain to obtain a fermentation product; and a distillation step: carrying out distillation treatment on the fermentation product to obtain distillate and bamboo-based vinasse. In the preparation method of the bamboo-based fermented wine, the specific surface area is greatly increased in the wet grinding step, the process conditions are mild, strong acid and strong alkali are not used, and the problems of chemical pollution and wastewater treatment are solved; and in the enzymolysis and liquefaction treatment, hemicellulose is efficiently degraded and plant tissues are disintegrated by using an enzyme preparation under a mild condition, the solid bamboo material is converted into liquefied slurry rich in monosaccharide, and the utilization rate of raw materials is extremely high.

Description

Technical Field

[0001] This invention relates to a bamboo-based fermented wine and its preparation method, belonging to the field of resource utilization of bamboo materials. Background Technology

[0002] Bamboo, as an important lignocellulose biomass, has significant advantages such as a short growth cycle and abundant resources. However, the cellulose and hemicellulose it contains are tightly wrapped by a complex lignin structure, making it difficult for traditional microorganisms to directly and efficiently utilize them. This is a key technical challenge restricting the high-value utilization of bamboo-based materials.

[0003] Traditional methods for preparing bamboo wine or alcoholic beverages containing bamboo materials mainly fall into two categories: One common method is physical extraction, where base liquor obtained from grain fermentation is injected into growing bamboo nodes, allowing it to grow and associate within the bamboo cavity for 15 to 60 days to acquire bamboo flavor. This method utilizes the microenvironment of the bamboo cavity for the penetration and association of flavor substances (such as bamboo polysaccharides and flavonoids), without the bamboo itself participating in the biochemical reaction. The other method is auxiliary material blending, where bamboo powder is used as an auxiliary material in grain fermentation, mixed with grain in a certain ratio for solid-state or liquid-state fermentation and distillation. The role of bamboo powder is to regulate the fermentation microenvironment and provide some flavor precursors, but the core carbon and energy sources remain grain starch. However, the main focus of existing technologies is on flavor preservation or using bamboo materials as auxiliary raw materials, with low efficiency in the direct conversion and utilization of the abundant lignocellulose in bamboo-based materials. A large amount of bamboo cellulose resources are not fully utilized, resulting in resource waste.

[0004] Therefore, there is an urgent need to develop an efficient bioconversion process that uses bamboo-based materials as the main raw material to break through the limitations of traditional brewing technology in the utilization of cellulose, and to maximize the conversion of hemicellulose and cellulose in bamboo materials into fermentable sugars, so as to realize the full utilization and high-value output of bamboo-based resources. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] To address the technical problems existing in the prior art, this invention first provides a method for preparing bamboo-based fermented wine. This invention elevates bamboo from a supporting role to a primary one, using bamboo biomass as the carbon source for alcohol production. The method employs physical loosening, enzymatic hydrolysis, and then microbial fermentation and distillation to prepare a bamboo-based fermented wine. This invention transforms waste distiller's grains into raw materials rich in functional microorganisms and unused fibers. Through the cycle of distillation and fermentation, it achieves full utilization of bamboo resources, greatly improving raw material utilization and process economy.

[0007] The method for preparing bamboo-based fermented wine of the present invention realizes the bio-conversion of bamboo-based materials as the main fermentation raw material to produce wine, and solves the technical problem that traditional processes can only rely on external sugar sources and cannot effectively utilize the cellulose of bamboo materials.

[0008] This invention also provides a bamboo-based fermented liquor. The total ester and total acid content of this bamboo-based fermented liquor meet the national standards for strong-aroma baijiu in China, proving that this process can stably biosynthesize rich flavor substances and produce a harmonious body. The presence of amino acids such as proline effectively increases the mellowness and aftertaste of the liquor, making the taste richer. The bamboo-based fermented liquor of this invention has a high tyrosine content, a unique biomarker of bamboo raw materials, with a content far exceeding that of traditional grain-based baijiu, giving the product a highly distinctive flavor base.

[0009] Solution for solving the problem

[0010] This invention provides a method for preparing bamboo-based fermented wine, which includes the following steps:

[0011] Wet grinding steps: The pulverized bamboo material is crushed and sieved, and then wet-ground to obtain bamboo-based slurry;

[0012] Enzymatic hydrolysis and liquefaction step: The bamboo-based pulp is enzymatically hydrolyzed and liquefied using an enzyme preparation to obtain liquefied pulp;

[0013] Fermentation steps: In the presence of carbon source and water, the liquefied slurry is fermented using a compound microbial strain to obtain fermentation products;

[0014] Distillation step: The fermentation product is distilled to obtain distillate and bamboo-based lees.

[0015] According to the preparation method of the present invention, in the wet grinding step, the sieve size is 1-10 mesh;

[0016] The mass ratio of the pulverized product to water is 1:4-8, and the wet grinding time is 10-60 min.

[0017] According to the preparation method of the present invention, in the enzymatic hydrolysis and liquefaction step, the enzyme preparation includes xylanase and pectinase; preferably, based on the dry weight of the bamboo material as 100%, the amount of xylanase added is 0.1-1.5%, and the amount of pectinase added is 0.05-0.5%.

[0018] According to the preparation method of the present invention, in the enzymatic hydrolysis and liquefaction step, the pH value of enzymatic hydrolysis and liquefaction is 4.5-5.0, the temperature of enzymatic hydrolysis and liquefaction is 45-55℃, and the time of enzymatic hydrolysis and liquefaction is 4-12 hours.

[0019] Preferably, the enzymatic hydrolysis and liquefaction step further includes an inactivation treatment after the enzymatic hydrolysis and liquefaction is completed, wherein the inactivation treatment is performed at a temperature of 80-100°C and for a time of 10-20 minutes.

[0020] According to the preparation method of the present invention, in the fermentation step, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is 1-30%, and the amount of water added is 5-100%.

[0021] Preferably, the carbon source includes carbohydrates.

[0022] According to the preparation method of the present invention, the composite microbial strain includes Clostridium species, Saccharomyces cerevisiae, and lactic acid bacteria;

[0023] Preferably, the Clostridium species include one or more of Clostridium thermophilum, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficile; the Saccharomyces cerevisiae includes one or more of Pichia kudrica, Pichia kudrica, and Wickhamia spp.

[0024] Preferably, based on the mass of the liquefied slurry, the amount of Clostridium species is 0.02-0.2%; the amount of Saccharomyces cerevisiae is 0.01-0.1%; and the amount of Lactic acid bacteria is 0.001-0.02%.

[0025] According to the preparation method of the present invention, the fermentation step and the distillation step are performed 2-6 times;

[0026] Preferably, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is reduced by 0.5-3% per fermentation.

[0027] According to the preparation method of the present invention, the fermentation time for a single fermentation is 37-67 days;

[0028] Preferably, the fermentation includes aerobic fermentation and anaerobic fermentation; the aerobic fermentation time is 5-9 days, and the anaerobic fermentation time is not less than 30 days.

[0029] According to the preparation method of the present invention, the distillation includes liquid-solid mixed distillation; preferably, the distillation power is 700-900W when the distillate begins to flow out.

[0030] The present invention also provides a bamboo-based fermented wine, wherein the bamboo-based fermented wine is prepared by the preparation method described in the present invention;

[0031] Preferably, the bamboo-based fermented wine comprises amino acids, esters, and alcohol.

[0032] The effects of the invention

[0033] In the preparation method of bamboo-based fermented wine of the present invention, the wet grinding step greatly increases the specific surface area, the process conditions are mild, strong acids and alkalis are not used, and there is no chemical pollution or wastewater treatment problem; then, the enzymatic hydrolysis and liquefaction treatment uses enzymes to efficiently degrade hemicellulose and break down plant tissues under mild conditions, transforming solid bamboo material into a liquefied slurry rich in monosaccharides, resulting in extremely high raw material utilization. The batch processing time is short, making it suitable for large-scale industrial production.

[0034] The bamboo-based fermented liquor prepared by the method of this invention meets the national standards for Chinese strong-aroma baijiu in terms of total ester and total acid content. The preparation method of this invention can stably biosynthesize rich flavor substances, resulting in a harmonious liquor body. Due to the presence of amino acids such as proline in the bamboo-based fermented liquor, the body's mellowness and aftertaste are effectively increased, making the taste more complex.

[0035] The bamboo-based fermented liquor of this invention has a high tyrosine content, far exceeding that of traditional grain-based liquors, giving the product a highly distinctive flavor base. This demonstrates that the wet grinding and enzymatic hydrolysis process described in this invention can efficiently release the deep nutrients in bamboo materials. The bamboo-based fermented liquor of this invention does not contain harmful substances such as heavy metals, methanol, and cyanide. The flavor and taste of the bamboo-based fermented liquor of this invention originate entirely from the biological fermentation of bamboo materials, rather than from the later addition of any artificial sweeteners; it is a purely natural brewed product, free of cyclamate, sucralose, etc. Attached Figure Description

[0036] Figure 1 The total soluble sugar content of Examples 1-3 and Comparative Examples 1-3 before fermentation is shown.

[0037] Figure 2 The residual reducing sugar content before distillation after fermentation in Examples 1-3 and Comparative Examples 1-3 is shown.

[0038] Figure 3 The LC / MS total ion chromatograms of Examples 1-3 are shown. Detailed Implementation

[0039] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0040] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0041] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0042] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0043] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0044] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0045] <First Aspect>

[0046] A first aspect of the present invention provides a method for preparing bamboo-based fermented wine, comprising the following steps:

[0047] Wet grinding steps: The pulverized bamboo material is crushed and sieved, and then wet-ground to obtain bamboo-based slurry;

[0048] Enzymatic hydrolysis and liquefaction step: The bamboo-based pulp is enzymatically hydrolyzed and liquefied using an enzyme preparation to obtain liquefied pulp;

[0049] Fermentation steps: In the presence of carbon source and water, the liquefied slurry is fermented using a compound microbial strain to obtain fermentation products;

[0050] Distillation step: The fermentation product is distilled to obtain distillate and bamboo-based lees.

[0051] In the preparation method of bamboo-based fermented wine of the present invention, the wet grinding step greatly increases the specific surface area, the process conditions are mild, strong acids and alkalis are not used, and there is no chemical pollution or wastewater treatment problem; then, the enzymatic hydrolysis and liquefaction treatment uses a complex enzyme to efficiently degrade hemicellulose and break down plant tissues under mild conditions, transforming solid bamboo material into a liquefied slurry rich in monosaccharides, resulting in extremely high raw material utilization. The batch processing time is short, making it suitable for large-scale industrial production.

[0052] In this invention, the bamboo material is not specifically limited, and may include various bamboo species such as *Phyllostachys edulis*, *Phyllostachys pubescens*, *Phyllostachys liangshanensis*, *Phyllostachys nanmu*, *Phyllostachys pubescens*, *Phyllostachys nigra ... The bamboo species include: silver bamboo, human-faced bamboo, moso bamboo, flowery moso bamboo, flowery bamboo, purple bamboo, spotted bamboo, tortoise-shell bamboo, light bamboo, rigid bamboo, bitter bamboo, golden bamboo, tortoise-patterned bamboo, silver chain bamboo, jade-edged bamboo, tea stalk bamboo, arrow bamboo, bubble bamboo, Luohan bamboo, seedling bamboo, goose feather bamboo, thorny bamboo, yellow bamboo, Chinese bamboo, black bamboo, stone bamboo, lucky bamboo, red bamboo, red-shelled bamboo, early garden bamboo, large bamboo, water bamboo, bitter bamboo, yellow-stalked black-breasted chicken bamboo, melon bamboo, *Bambusa* species, *Bambusa* species, large green hedge bamboo, *Bambusa* species, and *Bambusa* species. Specifically, the preferred bamboo material in this invention is moso bamboo.

[0053] Wet grinding steps

[0054] This invention involves wet grinding of the pulverized and sieved bamboo material to obtain bamboo-based slurry. The wet grinding process of this invention significantly increases the specific surface area, operates under mild conditions, does not use strong acids or alkalis, and avoids chemical pollution and wastewater treatment problems.

[0055] This invention opens up structural channels through wet grinding. Wet grinding is not just "crushing", but rather using strong mechanical shearing force in a high-concentration water medium to achieve the dissociation, fibrillation and specific surface area increase of wood fibers.

[0056] The present invention does not impose any particular limitation on the pulverization process; specifically, a hammer mill can be used for pulverization followed by sieving. The sieving size is also not particularly limited, as long as it meets the requirements for subsequent enzymatic hydrolysis and liquefaction. Specifically, the sieving size is 1-10 mesh.

[0057] After sieving, wet grinding is performed. Specifically, water can be added to the sieved bamboo material, and then wet grinding is carried out using a colloid mill to form a uniform bamboo-based slurry. In the wet grinding process, the role of water is crucial. It can prevent overheating and changes in material properties caused by dry grinding, and it can also penetrate into the fiber interior, working with mechanical force to break down the macroscopic and microscopic structure of the fiber, thus opening physical channels for the large-scale, deep adhesion and action of subsequent enzyme molecules.

[0058] In some specific implementations, the mass ratio of the pulverized product to water is 1:4-8, and the wet grinding time is 10-60 min.

[0059] The present invention does not specifically limit the water used; it can be drinking water, distilled water, etc.

[0060] Enzymatic hydrolysis liquefaction steps

[0061] This invention utilizes an enzyme preparation to enzymatically hydrolyze and liquefy the bamboo-based pulp, obtaining a liquefied pulp. The enzymatic hydrolysis and liquefaction process uses enzymes to efficiently degrade hemicellulose and break down plant tissues under mild conditions, transforming solid bamboo material into a liquefied pulp rich in monosaccharides, resulting in extremely high raw material utilization.

[0062] This invention employs enzyme preparations to precisely break down lignin. The purpose of the enzymatic liquefaction step is not to completely saccharify cellulose, but rather to remove the hemicellulose and pectin matrix surrounding the cellulose. Without affecting the cellulose crystallization zone, the enzymatic liquefaction step further loosens the lignin-carbohydrate complex structure, exposing more of the internal cellulose and laying the foundation for subsequent fermentation.

[0063] This invention combines wet grinding with compound enzymatic hydrolysis, which can efficiently open the lignocellulose barrier of bamboo materials and efficiently degrade the (originally solid) hemicellulose and cellulose in bamboo materials into soluble sugars. This high concentration of soluble sugars provides a solid material basis for subsequent multi-strain fermentation and high alcohol yield. Under the promotion of high soluble sugars, Clostridium can efficiently degrade cellulose and hemicellulose and continuously release fermentable sugars in bamboo materials, providing a basis for subsequent fermentation.

[0064] In some specific embodiments, the enzyme preparation in the enzymatic hydrolysis and liquefaction step includes xylanase and pectinase; by using xylanase and pectinase, hemicellulose and pectin matrix wrapped around the outer layer of cellulose can be precisely removed.

[0065] Preferably, based on the dry weight of the bamboo material (100%), the amount of xylanase added is 0.1-1.5%, and the amount of pectinase added is 0.05-0.5%. When the amount of xylanase added is 0.1-1.5% and the amount of pectinase added is 0.05-0.5%, the effects of xylanase and pectinase can be exerted more effectively.

[0066] In some specific implementation schemes, in order to maximize the effect of the enzyme preparation, the pH value of the enzymatic hydrolysis liquefaction step is 4.5-5.0, the temperature of the enzymatic hydrolysis liquefaction is 45-55℃, and the time of the enzymatic hydrolysis liquefaction is 4-12 hours.

[0067] Furthermore, the enzymatic hydrolysis-liquefaction step also includes an inactivation treatment after the enzymatic hydrolysis-liquefaction is completed. The inactivation treatment is performed at a temperature of 80-100℃ for 10-20 minutes. This inactivation treatment facilitates the subsequent fermentation steps.

[0068] Fermentation steps

[0069] In this invention, liquefied slurry is fermented using a composite microbial strain in the presence of a carbon source and water to obtain fermentation products. This invention achieves the relay and synergy of metabolic pathways by constructing a highly efficient microbial ecological community through the use of a composite microbial strain.

[0070] In some specific implementations, the compound microbial strain includes Clostridium species, Saccharomyces cerevisiae, and lactic acid bacteria; specifically, the Clostridium species include one or more combinations of Clostridium thermocellulose, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficile. The Saccharomyces cerevisiae includes one or more combinations of Pichia kudrica, Pichia tree yeast, and Wickham's yeast aberrant. Both the Clostridium species and Saccharomyces cerevisiae can be purchased from Hangzhou Kenong Agricultural Technology Co., Ltd., and the lactic acid bacteria can be purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.

[0071] Clostridium species can degrade cellulose and / or hemicellulose, secreting cellulose granules to continuously degrade cellulose and hemicellulose in liquefied slurry, continuously releasing fermentable sugars. Clostridium species can be activated by oxygenation in the early stages of aerobic fermentation and can dominate cellulose degradation in anaerobic fermentation. Saccharomyces cerevisiae can perform alcoholic fermentation. Saccharomyces cerevisiae can efficiently convert fermentable sugars released by Clostridium and other pathways into ethanol. Lactic acid bacteria can regulate the fermentation system. In this invention, lactic acid bacteria can metabolize some sugars to produce acid, regulating the pH of the fermentation system and inhibiting other microorganisms; by degrading some complex substances, they provide growth factors for other microorganisms; in addition, the metabolites of lactic acid bacteria (such as lactic acid) may impart a special flavor to the wine.

[0072] Preferably, in order to make the compound microbial strains work more effectively, the amount of Clostridium species used is 0.02-0.2% based on the mass of the liquefied slurry; the amount of Saccharomyces cerevisiae is 0.01-0.1%; and the amount of Lactic acid bacteria is 0.001-0.02%.

[0073] In some specific implementations, in the fermentation step, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is 1-30%, and the amount of water added is 5-100%.

[0074] In the presence of a carbon source, Clostridium species can more effectively release cellulose to efficiently decompose the cellulose in bamboo materials, producing fermentable monosaccharides. In the early stages of fermentation, the presence of a carbon source allows the inoculated complex strains to rapidly multiply and reach the required dominant microbial density and biomass for fermentation. Once the carbon source is depleted, the Clostridium in the fermentation system, lacking readily available carbon sources, begins to efficiently express and release cellulose. Cellulose is a highly efficient multi-enzyme complex that can attach to bamboo-based materials and efficiently hydrolyze the complex structure of lignocellulose (cellulose and hemicellulose), degrading it into fermentable monosaccharides (such as glucose and xylose) that can be continuously utilized by Saccharomyces cerevisiae, thus achieving efficient biotransformation of the carbon source in bamboo materials.

[0075] Furthermore, in this invention, the carbon source includes sugars. Specifically, the carbon source can be fermentable sugars such as granulated sugar, sucrose, and glucose.

[0076] Furthermore, this invention utilizes multiple fermentation processes to fully utilize the carbon source of bamboo materials. Specifically, the fermentation steps can be performed 2-6 times, preferably 3-5 times. Through multiple fermentation steps, fermentable materials can be extracted in stages. Through multiple rounds of fermentation, functional microorganisms are screened and enriched, and the fermentation system matures and stabilizes, gradually converting cellulose / hemicellulose of varying accessibility and crystallinity in bamboo into alcohol. A compound microbial strain needs to be added during each fermentation process.

[0077] Preferably, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is reduced by 0.5-3% for each fermentation, and no additional carbon source is needed for the last fermentation step.

[0078] In some specific implementation schemes, the fermentation time for a single fermentation is 37-67 days; the specific fermentation time can be determined according to the room temperature. If the room temperature is too high, for example, above 20°C, the fermentation time is short, and if the room temperature is low, for example, below 20°C, the fermentation time is long.

[0079] Furthermore, the fermentation of the present invention can be carried out in a fermentation tank, which can be made of stainless steel, clay, plastic, etc.

[0080] In some specific implementation schemes, the fermentation includes aerobic fermentation and anaerobic fermentation; the aerobic fermentation time is 5-9 days, and the anaerobic fermentation time is not less than 30 days.

[0081] For aerobic fermentation, the fermenter is initially kept in a natural or microaerobic state, and stirred 2-4 times during the aerobic fermentation process, each stirring lasting 20-40 minutes. The purpose of stirring is to briefly introduce air, allowing the yeast to rapidly multiply through respiration and perform biological deoxygenation, thereby creating a deep anaerobic environment for Clostridium fibrinolyticum. After the aerobic fermentation process is complete, anaerobic fermentation begins. Specifically, this can be achieved by stopping the oxygen supply and sealing the fermenter to establish an anaerobic environment. This relay method of first aerobic enzyme production and then anaerobic fermentation maximizes the synergistic effect of different microorganisms. The temperature for anaerobic fermentation can be 20-30℃.

[0082] Distillation steps

[0083] Finally, the fermentation product is distilled to obtain distillate and bamboo-based lees. The bamboo-based lees can then be used in further fermentation and distillation steps.

[0084] In some specific implementations, the distillation includes liquid-solid mixed distillation; specifically, a still can be used for liquid-solid mixed distillation. During the initial distillation, the power can be increased to reach the required temperature as quickly as possible; once the distillate begins to flow out, the distillation power is adjusted to 700-900W. During the distillation process, the temperature of the liquor at the condenser outlet is monitored and maintained at 15-25°C.

[0085] Finally, the alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in segments. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the fermented wine was obtained.

[0086] <Second aspect>

[0087] A second aspect of the present invention provides a bamboo-based fermented wine, wherein the bamboo-based fermented wine is prepared by the preparation method described in the first aspect of the present invention.

[0088] In some specific implementations, the bamboo-based fermented wine includes amino acids, esters, and alcohol.

[0089] The bamboo-based fermented liquor prepared by the method of this invention meets the national standards for Chinese strong-aroma baijiu in terms of total ester and total acid content. The preparation method of this invention can stably biosynthesize rich flavor substances, resulting in a harmonious liquor body. Due to the presence of amino acids such as proline in the bamboo-based fermented liquor, the body's mellowness and aftertaste are effectively increased, making the taste more complex.

[0090] The bamboo-based fermented liquor of this invention has a high tyrosine content, far exceeding that of traditional grain-based liquors, giving the product a highly distinctive flavor base. This demonstrates that the wet grinding and enzymatic hydrolysis process described in this invention can efficiently release the deep nutrients in bamboo materials. The bamboo-based fermented liquor of this invention does not contain harmful substances such as heavy metals, methanol, and cyanide. The flavor and taste of the bamboo-based fermented liquor of this invention originate entirely from the biological fermentation of bamboo materials, rather than from the later addition of any artificial sweeteners; it is a purely natural brewed product, free of cyclamate, sucralose, etc.

[0091] Example

[0092] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0093] Example 1

[0094] Wet grinding process: Take 5 kg (dry weight) of one-year-old moso bamboo, grind it using a high-speed pulverizer, and pass it through a 5-mesh sieve to obtain the pulverized product. Mix the pulverized product with 30 kg of water and then feed it into a colloid mill for wet grinding for 30 minutes to form a bamboo-based slurry.

[0095] Enzymatic hydrolysis and liquefaction: Bamboo-based pulp was added to an enzymatic hydrolysis tank, the pH was adjusted to 4.8, and the temperature was raised to 50°C. Then, 40g of xylanase and 10g of pectinase were added, and the mixture was kept warm and stirred for 8 hours. The temperature was then raised to 90°C and maintained for 15 minutes for inactivation treatment, followed by cooling to 30°C to obtain liquefied pulp.

[0096] Primary fermentation: Approximately 35 kg of liquefied slurry was added to the fermentation tank, along with 5 kg of sucrose and 10 kg of water. 30 g of Clostridium cellulose, 15 g of Saccharomyces cerevisiae (a mixture of Pichia pastoris, Pichia tree, and Wickham's yeast in a 1:2:1 ratio), and 2.5 g of lactic acid bacteria were weighed and activated with 750 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with a plastic film and anaerobic fermentation was carried out at 30-35°C for a total fermentation period of 45 days, yielding the first batch of fermentation products.

[0097] Single distillation: The first batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on and set to 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40 mL / min. During distillation, the distillate temperature was monitored and maintained at approximately 20°C. The alcohol content of the distillate was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the first batch of fractionated fermented wine was obtained. The first batch of bamboo-based mash was collected and cooled.

[0098] Secondary fermentation: The first batch of bamboo-based distiller's grains (14 kg wet weight, approximately 70% moisture content) was reintroduced into the fermentation tank, along with 3.5 kg of sucrose and approximately 21 kg of water. 23 g of Clostridium fibrinolyticum, 11.5 g of brewing yeast (a mass ratio of Pichia kudrifolia, Pichia tree, and Wickham's abnormal yeast of 1:2:1), and 2 g of lactic acid bacteria were weighed and activated in 500 g of 35°C sterile saline for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 30-35°C for a total fermentation period of 45 days, yielding the second batch of fermentation products.

[0099] Secondary distillation: The second batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on and set to 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40 mL / min. During distillation, the distillate temperature was monitored and maintained at approximately 20°C. The alcohol content of the distillate was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the second batch of fractionated fermented wine was obtained. The second batch of bamboo-based mash was collected and cooled.

[0100] Three-stage fermentation process: The second batch of bamboo-based distiller's grains (wet weight 13.3 kg, moisture content approximately 70%) was reintroduced into the fermentation tank, along with 2 kg of sucrose and approximately 14.6 kg of water. 18 g of Clostridium cellulose, 9 g of brewing yeast (Pichia pastoris, Pichia tree, and Wickham's abnormal yeast in a 1:2:1 mass ratio), and 1.5 g of lactic acid bacteria were weighed and activated with 400 g of 35°C sterile saline for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 35-40°C for a total fermentation period of 30 days, yielding the third batch of fermentation products.

[0101] Triple distillation: The third batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on and set to 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40 mL / min. During distillation, the distillate temperature was monitored and maintained at approximately 20°C. The alcohol content of the distillate was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the third batch of fractionated fermented wine was obtained. The third batch of bamboo-based mash was collected and cooled.

[0102] The fourth fermentation process: The third batch of bamboo-based distiller's grains (wet weight 12.6 kg, moisture content approximately 70%) was reintroduced into the fermentation tank, along with 0.5 kg of sucrose and approximately 8.3 kg of water. Then, 13 g of Clostridium cellulose, 6.5 g of brewing yeast (a mass ratio of Pichia pastoris, Pichia tree, and Wickham's abnormal yeast in 1:2:1), and 1 g of lactic acid bacteria were weighed and activated with 300 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 35-40°C for a total fermentation period of 30 days, yielding the fourth batch of fermentation products.

[0103] Quadruple distillation: The fourth batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on and set to 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40 mL / min. During distillation, the distillate temperature was monitored and maintained at 20℃. The alcohol content of the distillate was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the fourth batch of fractionated fermented wine was obtained. The fourth batch of bamboo-based mash was collected and cooled.

[0104] Fifth fermentation process: The fourth batch of bamboo-based distiller's grains (wet weight 11.8 kg, moisture content approximately 70%) was reintroduced into the fermentation tank, along with approximately 5.9 kg of water. 10.5 g of Clostridium fibrinolyticum, 5 g of brewing yeast (a mass ratio of Pichia kudrifolia, Pichia tree, and Wickham's abnormal yeast in 1:2:1), and 1 g of lactic acid bacteria were weighed and activated with 250 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 25-30°C for a total fermentation period of 50 days, yielding the fifth batch of fermentation products.

[0105] Five-stage distillation: The fifth batch of fermentation product underwent liquid-solid mixed distillation. The fermentation product was loaded into a stainless steel still with a filling coefficient controlled at 70%. The heating device was turned on and set to 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40 mL / min. During distillation, the distillate temperature was monitored and maintained at approximately 20°C. The alcohol content of the distillate was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the fifth batch of fractionated fermented wine was obtained.

[0106] Blending process: The collected 1-5 batches of fermented wine are blended to obtain bamboo-based fermented wine with the target alcohol content of 53% vol.

[0107] Example 2

[0108] Wet grinding process: Take 5 kg (dry weight) of one-year-old moso bamboo, grind it using a hammer mill, and pass it through a 10-mesh sieve. Add 20 kg of distilled water, mix, and then feed it into a colloid mill for wet grinding for 30 minutes to form bamboo-based slurry.

[0109] Enzymatic hydrolysis and liquefaction: Bamboo-based pulp was added to an enzymatic hydrolysis tank, the pH was adjusted to 5.0, and the temperature was raised to 55°C. Then, 75g of xylanase and 25g of pectinase were added, and the mixture was kept warm and stirred for 8 hours to obtain liquefied pulp. The temperature was then raised to 90°C and maintained for 15 minutes for inactivation treatment, followed by cooling to 25°C to obtain liquefied pulp.

[0110] Primary fermentation: Approximately 25 kg of liquefied slurry was added to the fermentation tank, followed by 2.5 kg of glucose and 22.5 kg of water. Then, 50 g of Clostridium cellulose, 20 g of Saccharomyces cerevisiae (a mixture of Pichia pastoris, Pichia tree, and Wickham's yeast in a 1:2:1 ratio), and 5 g of lactic acid bacteria were weighed and activated with 1000 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was added to the fermentation tank, and manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 35-40°C. The total fermentation period was 37 days, yielding the first batch of fermentation products.

[0111] Single distillation: The first batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in stages. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the first batch of staged fermented liquor was obtained. The first batch of bamboo-based mash was collected and cooled.

[0112] Secondary fermentation: The first batch of bamboo-based distiller's grains (wet weight 13.6 kg, moisture content approximately 70%) was added back into the fermentation tank. Then, 1 kg of glucose and approximately 19.3 kg of water were added. Next, 34 g of Clostridium fibrinolyticum, 13.5 g of brewing yeast (a mass ratio of *Pichia pastoris*, *Pichia treeii*, and *Wickham's abnormal yeast* in 1:2:1), and 3.5 g of lactic acid bacteria were weighed and activated with 750 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 35-40°C for a total fermentation period of 37 days, yielding the second batch of fermentation products.

[0113] Secondary distillation: The second batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in stages. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the second batch of staged fermented liquor was obtained. The second batch of bamboo-based mash was collected and cooled.

[0114] Three-stage fermentation process: The second batch of bamboo-based distiller's grains (13 kg wet weight, 70% moisture content) was reintroduced into the fermentation tank. Approximately 13 kg of water was then added. Next, 26 g of Clostridium celluloseophilum, 10.5 g of brewing yeast (a mass ratio of *Pichia pastoris*, *Pichia treeii*, and *Wickham's abnormal yeast* in 1:2:1), and 2.5 g of lactic acid bacteria were weighed and activated with 600 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was then added to the fermentation tank. Manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 25-30°C for a total fermentation period of 45 days, yielding the third batch of fermentation products.

[0115] Triple distillation: The third batch of fermentation product underwent liquid-solid mixed distillation. The fermentation product was loaded into a stainless steel still with a filling factor controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at approximately 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the third batch of fractionated fermented liquor was obtained.

[0116] Blending process: The collected 1-3 batches of fermented wine are blended according to the target alcohol content to obtain bamboo-based fermented wine with a target alcohol content of 53% vol.

[0117] Example 3

[0118] Wet grinding process: Take 5 kg (dry weight) of one-year-old moso bamboo, grind it using a hammer mill, and pass it through a 1-mesh sieve. Add 40 kg of water, mix, and then feed it into a colloid mill for wet grinding for 30 minutes to form bamboo-based slurry.

[0119] Enzymatic hydrolysis and liquefaction: Bamboo-based pulp was added to an enzymatic hydrolysis tank, the pH was adjusted to 4.5, and the temperature was raised to 45°C. Then, 5g of xylanase and 2.5g of pectinase were added. The mixture was kept at this temperature and stirred for 8 hours. Then, the temperature was raised to 90°C and maintained for 15 minutes for inactivation treatment, followed by cooling to 25°C to obtain liquefied pulp.

[0120] Primary fermentation: Approximately 45 kg of liquefied slurry was added to the fermentation tank, followed by 1.5 kg of white sugar and 3.5 kg of water. Then, 10 g of Clostridium cellulose, 5 g of Saccharomyces cerevisiae (a mixture of Pichia pastoris, Pichia tree yeast, and Wickham's yeast in a 1:2:1 ratio), and 0.5 g of lactic acid bacteria were weighed and activated with 300 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was added to the fermentation tank, and manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 25°C. The total fermentation period was 67 days, yielding the first batch of fermentation products.

[0121] Single distillation: The first batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in stages. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the first batch of staged fermented liquor was obtained. The first batch of bamboo-based mash was collected and cooled.

[0122] Secondary fermentation: The first batch of bamboo-based distiller's grains (wet weight 14.5 kg, moisture content 70%) was added back into the fermentation tank, followed by 1 kg of glucose and approximately 25.6 kg of water. Then, 8.5 g of Clostridium fibrinolyticum, 4 g of brewing yeast (Pichia pastoris, Pichia tree, and Wickham's abnormal yeast in a 1:2:1 ratio), and 0.5 g of lactic acid bacteria were weighed and activated with 200 g of sterile physiological saline at 35°C for 30 minutes to prepare a compound bacterial suspension. This suspension was added to the fermentation tank, and manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 25°C for a total fermentation period of 67 days, yielding the second batch of fermentation products.

[0123] Secondary distillation: The second batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still, with the liquid-to-liquid ratio controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at approximately 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in stages. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the second batch of staged fermented liquor was obtained. The second batch of bamboo-based mash was collected and cooled.

[0124] Three-stage fermentation process: The second batch of bamboo-based distiller's grains (14 kg wet weight, 70% moisture content) was added back into the fermentation tank. Approximately 18.3 kg of water was then added. Next, 6.5 g of Clostridium fibrinolyticum, 3.5 g of brewing yeast (a mass ratio of Pichia kudrifolia, Pichia tree, and Wickham's abnormal yeast of 1:2:1), and 0.3 g of lactic acid bacteria were weighed and activated with 150 g of sterile saline for 30 minutes to prepare a compound bacterial suspension. This suspension was added to the fermentation tank, and manual stirring was performed once at the time of feeding, on day 3, and on day 6, each time for 30 minutes. Starting from day 7, the fermentation tank was sealed with plastic film and anaerobic fermentation was carried out at 25°C. The total fermentation period was 67 days, yielding the third batch of fermentation products.

[0125] Triple distillation: The third batch of fermentation products underwent liquid-solid mixed distillation. The fermentation products were loaded into a stainless steel still with a filling factor controlled at 70%. The heating device was turned on at 3500W for rapid heating. Once the distillate began to flow out, the heating power was reduced to 800W to maintain a stable simmer. The distillate flow rate was controlled at approximately 40mL / min. During distillation, the temperature of the liquor at the condenser outlet was monitored and maintained at 20℃. The alcohol content was monitored in real time using an alcohol meter, and the distillate was collected in fractions. When the real-time alcohol content of the distillate dropped below 3% vol, distillation was stopped, and the third batch of fractionated fermented liquor was obtained.

[0126] Blending process: The collected 1-3 batches of fermented wine are blended to obtain bamboo-based fermented wine with the target alcohol content of 53% vol.

[0127] Comparative Example 1

[0128] The only difference between Comparative Example 1 and Example 1 is that: without enzymatic hydrolysis and liquefaction, the bamboo-based pulp was directly adjusted to pH 4.8 in a tank, heated to 50°C and stirred for 8 hours, and then subjected to high-temperature inactivation. The remaining steps and the amount of materials used were exactly the same as in Example 1, and five batches of fermented wine were finally obtained.

[0129] Comparative Example 2

[0130] The only difference between Comparative Example 2 and Example 1 is that Clostridium cellulose was not added, and only the same amount of brewing yeast and lactic acid bacteria as in Example 1 were used. The remaining steps and the amount of ingredients were exactly the same as in Example 1, and five batches of fermented wine were finally obtained.

[0131] Comparative Example 3

[0132] The only difference between Comparative Example 3 and Example 1 is that wet grinding is not performed, while the remaining steps and the amount of materials used are exactly the same as in Example 1, resulting in five batches of fermented wine.

[0133] Performance testing

[0134] 1. Detection of total soluble sugars

[0135] According to GB / T 36056-2018 "Analytical Methods for Forestry Biomass Raw Materials - Determination of Soluble Sugars", the liquefied pulps of Examples 1-3 and Comparative Examples 2-3, as well as the bamboo-based pulp of Comparative Example 1, were tested, and the results are as follows: Figure 1 As shown.

[0136] The soluble sugar content of Examples 1-3 is relatively high. It can be seen from the comparison of the soluble sugar content of Examples 1-3 that the saccharification efficiency can be effectively improved by optimizing the enzymatic hydrolysis process and the particle size.

[0137] Comparative Example 1 showed the lowest total soluble sugar content, almost zero. Therefore, soluble sugars can be released from bamboo materials through an enzymatic liquefaction step.

[0138] Although the sugar content of Comparative Example 3 was higher than that of Comparative Example 1, it was much lower than that of Example 1. This demonstrates that breaking down the dense structure of lignocellulose through wet milling can increase the specific surface area, allowing the enzyme preparation to effectively contact the interior of the substrate, thus significantly improving saccharification efficiency.

[0139] 2. Reducing sugar content test

[0140] The last batches of fermented wine in Examples 1-3 and Comparative Examples 1-3 were tested according to GB 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugars in Food". The results are as follows: Figure 2 As shown.

[0141] In fermentation, the reducing sugar content is a standard for measuring whether fermentation is complete. The lower the reducing sugar content, the higher the conversion rate of sugar to ethanol, and the more efficient the fermentation process. This reducing sugar refers to the residual reducing sugar in the fermented beverage.

[0142] Depend on Figure 2 It can be seen that the reducing sugar content of Examples 1-3 is significantly lower than that of Comparative Examples 1-3, proving that the present invention can completely consume the sugar released from bamboo materials and the exogenous carbon supplementation.

[0143] Comparative Example 2 had the highest residual sugar content. Although it had almost the same initial total sugar as Example 1 before fermentation, it had the highest reducing sugar content at the fermentation endpoint. This may be because without the use of Clostridium cellulose, the brewing yeast in the system could not effectively utilize the pentose sugars, such as xylose, produced by the hydrolysis of bamboo hemicellulose, leading to the accumulation of reducing sugars. Example 1, on the other hand, introduced Clostridium cellulose, achieving synergistic utilization of hexoses and pentoses, thus resulting in the lowest reducing sugar content and the highest raw material utilization rate. Furthermore, Comparative Examples 3 and 1 also had relatively high reducing sugar contents, indicating that the absence of wet grinding or enzymatic liquefaction steps also leads to incomplete fermentation and low efficiency.

[0144] Examples 1-3 of this invention, through efficient pretreatment, provide microorganisms with abundant and easily usable sugars from bamboo material in the early stages of fermentation. This allows the microbial community to quickly establish a dominant population and maintain extremely high metabolic activity. Consequently, at the end of fermentation, all sugars from various sources, including sugars released from the bamboo material and added carbon, are consumed more thoroughly, ultimately achieving the dual advantages of lower reducing sugars and higher alcohol yield.

[0145] 3. Alcohol content test

[0146] According to GB 5009.225-2023 "National Food Safety Standard - Determination of Ethanol Concentration in Wine and Edible Alcohol", the last batch of fermented wine in Examples 1-3 and Comparative Examples 1-3 was tested, and the results are shown in Table 1; the alcohol content of each batch of fermented wine in Example 1 was also calculated, and the results are shown in Table 2.

[0147] Table 1

[0148]

[0149] Table 2

[0150]

[0151] As shown in Table 1, after the fifth batch of fermentation without external carbon supplementation, Example 1 (batch 5) was still able to distill 25 mL of high-proof (over 60% vol) heads and 160 mL of medium-proof (40-60% vol) core spirits. Example 2 (batch 3) and Example 3 (batch 3) produced 125 mL and 90 mL of medium-proof core spirits, respectively, proving that the pretreated bamboo material itself can serve as the core sugar source for continuous fermentation, supporting the continuous metabolism and alcohol production of microorganisms.

[0152] After stopping carbon supplementation, Comparative Example 2 could not distill a head of >60% vol. Its first few batches relied entirely on external carbon supplementation; once carbon supplementation ceased, fermentation stopped because the cellulose sugar source from the bamboo material could not be utilized. The 40-60% vol core yield of Comparative Example 1 was only 37.5% of that of Example 1, further demonstrating the crucial role of enzymatic hydrolysis in the release of sugars from the bamboo material. Even after stopping external carbon supplementation, the microorganisms in Examples 1-3 were still able to continuously utilize the sugars produced by the degradation of the bamboo material for fermentation and alcohol production.

[0153] As shown in Table 2, although the total amount of alcohol produced by fermentation decreased in a gradient from 5 kg in the first batch to 0 kg in the fifth batch, consistent with the law of conservation of mass, it is noteworthy that even under the oligotrophic conditions of the fifth batch (without any added sucrose), the system still produced approximately 610 mL of distillate. This demonstrates that the Clostridium cellulophilum in the fermentation system did not die off due to the lack of readily available carbon sources in the later stages. Instead, it was induced to secrete highly active cellulase systems, successfully degrading the unusable cell wall components in the bamboo material into fermentable sugars, thus maintaining the continuous fermentation process.

[0154] The high yields in batches 1-3 of Example 1 followed by low yields in batches 4-5 reflect the succession pattern of the microbial community. Sufficient sucrose in the early stages promoted the rapid proliferation and biomass accumulation of *Saccharomyces cerevisiae* and *Clostridium cellulose*. As the sucrose was depleted, the large *Clostridium* population began to degrade and attack the cellulose in the bamboo material. This strategy of "using sugar to nourish bacteria and using bacteria to treat bamboo" significantly improved the utilization rate of raw materials. Observation of the proportions of each fraction showed that as fermentation progressed, the proportion of high-proof liquor (>60% vol) gradually decreased, while the proportion of low-proof liquor (<40% vol) relatively increased. Although the later low-proof fractions had low ethanol content, they were enriched with a large number of phenolic, ester, and terpene secondary metabolites derived from the degradation of lignin and hemicellulose in the bamboo material. Therefore, the method of this invention not only recovered ethanol but, more importantly, collected the unique bamboo aroma and aging aroma substances of bamboo-based fermentation, providing a rich variety of base liquors for blending the final product.

[0155] 4. Fermented wine and component testing

[0156] The fermented wine of Example 1 was tested according to the test items in Table 3 below and the test methods described below. The results are shown in Table 3 below.

[0157] Table 3

[0158]

[0159] As shown in Table 3, the total ester and total acid content of the bamboo-based fermented liquor of this invention both meet the national standards for strong-aroma baijiu in China, proving that this process can stably biosynthesize rich flavor substances and achieve a harmonious liquor body. The presence of amino acids such as proline in the bamboo-based fermented liquor of this invention effectively increases the mellowness and aftertaste of the liquor, making the taste richer; among them, the tyrosine content is as high as 0.71g / 100g, which is a unique biomarker of bamboo material, and its content far exceeds that of traditional grain-based baijiu, giving the product a highly distinctive flavor base, proving that the wet grinding and enzymatic hydrolysis process described in this invention can efficiently release the deep nutrients in bamboo. The absence of methanol and cyanide in the bamboo-based fermented liquor of this invention proves that the raw material processing and fermentation process of this invention are safe and controllable. The absence of cyclamate and sucralose in the bamboo-based fermented liquor of this invention proves that the flavor and taste of this product originate entirely from the bio-fermentation of bamboo material raw materials, rather than from the later addition of any artificial sweeteners, making it a purely natural brewed product. No heavy metal lead was detected in the bamboo-based fermented wine of this invention, proving that the distillation process of this invention is properly controlled and has high purification efficiency, ensuring the clarity and purity of the wine.

[0160] 6. Gas chromatography-mass spectrometry detection

[0161] Gas chromatography-mass spectrometry (GC-MS) was used to analyze 1 μL of each of the bamboo-based fermented wines with a target alcohol content of 53% vol from Examples 1-3. The results are shown in Table 4. Figure 3 As shown.

[0162] Table 4

[0163]

[0164] As shown in Table 4 and Figure 3As shown, Examples 1 and 2 exhibited the highest peak densities, while Example 3 showed the lowest. The highest baseline signal-to-noise ratio indicates the richest concentration and variety of volatile flavor compounds in the wine. Examples 1 and 2 were inoculated with sufficient microorganisms, which received ample carbon sources, resulting in vigorous metabolism and the accumulation of numerous primary flavor metabolites in the early stages. This enabled them to more efficiently convert the degradation products of bamboo materials into various alcohols, esters, acids, and other flavor precursors. In contrast, Example 3, due to its lower inoculation amount and insufficient fermentation intensity, resulted in a smaller overall amount of flavor compounds, making its chromatogram appear thinner. Example 1 displayed a series of high-intensity, sharp characteristic peaks around 29 min, 33 min, and 38 min. These are typically high-boiling-point substances such as long-chain fatty acid esters, phenolic derivatives, and sesquiterpenes, which are key sources of the "aged feel," "richness," and unique "bamboo aroma" of bamboo-based fermented wine. The top 15 metabolites affecting the flavor of bamboo-based fermented wine in Examples 1-3 are ranked by their original signal intensity (abundance), as shown in Table 3. Isoamyl alcohol, ethyl decanoate, and ethyl octanoate are the three main components shared by the fermented bamboo-based wine, forming the basic fruit and aroma framework. However, Example 1 adds more unique and complex components beyond these basic components. The contents of diethyl succinate and butyl ethyl succinate are significantly higher than in other examples. These diesters typically have elegant fruit and aging aromas, indicating that the esterification reaction in Example 1 was more thorough. 2-Methylnonane is a unique substance detected in Example 1. 2-Methylnonane is a branched alkane compound and one of the characteristic components of plant waxes, giving the wine a unique oily feel and aging style. It can be seen that by adjusting parameters such as the amount of enzyme added, fermentation cycle, and amount of microbial strain added, the flavor fingerprint spectrum of the wine can be controlled.

[0165] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0166] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing bamboo-based fermented wine, characterized in that, Includes the following steps: Wet grinding steps: The pulverized bamboo material is crushed and sieved, and then wet-ground to obtain bamboo-based slurry; Enzymatic hydrolysis and liquefaction step: The bamboo-based pulp is enzymatically hydrolyzed and liquefied using an enzyme preparation to obtain liquefied pulp; Fermentation steps: In the presence of carbon source and water, the liquefied slurry is fermented using a compound microbial strain to obtain fermentation products; Distillation step: The fermentation product is distilled to obtain distillate and bamboo-based lees.

2. The preparation method according to claim 1, characterized in that, In the wet grinding step, the sieve size is 1-10 mesh; The mass ratio of the pulverized product to water is 1:4-8, and the wet grinding time is 10-60 min.

3. The preparation method according to claim 1 or 2, characterized in that, In the enzymatic hydrolysis and liquefaction step, the enzyme preparation includes xylanase and pectinase; preferably, based on the dry weight of the bamboo material as 100%, the amount of xylanase added is 0.1-1.5%, and the amount of pectinase added is 0.05-0.5%.

4. The preparation method according to any one of claims 1-3, characterized in that, In the enzymatic hydrolysis and liquefaction step, the pH value of enzymatic hydrolysis and liquefaction is 4.5-5.0, the temperature of enzymatic hydrolysis and liquefaction is 45-55℃, and the time of enzymatic hydrolysis and liquefaction is 4-12 hours. Preferably, the enzymatic hydrolysis and liquefaction step further includes an inactivation treatment after the enzymatic hydrolysis and liquefaction is completed, wherein the inactivation treatment is performed at a temperature of 80-100°C and for a time of 10-20 minutes.

5. The preparation method according to any one of claims 1-4, characterized in that, In the fermentation step, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is 1-30%, and the amount of water added is 5-100%. Preferably, the carbon source includes carbohydrates.

6. The preparation method according to any one of claims 1-5, characterized in that, The compound microbial strain includes Clostridium species, Saccharomyces cerevisiae, and lactic acid bacteria; Preferably, the Clostridium species include one or more of Clostridium thermophilum, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficile; the Saccharomyces cerevisiae includes one or more of Pichia kudrica, Pichia kudrica, and Wickhamia spp. Preferably, based on the mass of the liquefied slurry, the amount of Clostridium species is 0.02-0.2%; the amount of Saccharomyces cerevisiae is 0.01-0.1%; and the amount of Lactic acid bacteria is 0.001-0.02%.

7. The preparation method according to any one of claims 1-6, characterized in that, The fermentation and distillation steps are performed 2-6 times. Preferably, based on the mass of the liquefied slurry as 100%, the amount of carbon source added is reduced by 0.5-3% per fermentation.

8. The preparation method according to claim 7, characterized in that, The fermentation time for a single cycle is 37-67 days; Preferably, the fermentation includes aerobic fermentation and anaerobic fermentation; the aerobic fermentation time is 5-9 days, and the anaerobic fermentation time is not less than 30 days.

9. The preparation method according to any one of claims 1-8, characterized in that, The distillation includes liquid-solid mixed distillation; preferably, the distillation power is 700-900W when the distillate begins to flow out.

10. A bamboo-based fermented wine, characterized in that, The bamboo-based fermented wine is prepared by the preparation method according to any one of claims 1-9; Preferably, the bamboo-based fermented wine comprises amino acids, esters, and alcohol.

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