Method for improving flower and fruit flavor of Baijiu

Abstract

The invention discloses a method for improving flower and fruit flavor of Baijiu. According to the method, pure glutinous sorghum of which the amylopectin content is greater than or equal to 70% is used as a unique fermentation raw material, and a pure grain solid-state fermentation process without adding matching grains is adopted; the method comprises the following steps: preparing four specific preserved strains into a solid microbial inoculum, preparing a compound microbial inoculum according to a weight ratio of (2-4): (2-4): (1-2): (1-2), mixing the compound microbial inoculum with yeast without exogenous functional microbial inoculum addition according to a ratio of (4-8): (1-2), and adding 0.1% of food-grade silicon dioxide to prepare enhanced distiller's yeast which accounts for 1-2% of the dry weight of pure glutinous sorghum; the fermentation temperature is controlled to be 20-24 DEG C, the period is 14 days, and the temperature control precision is + / -0.5 DEG C. Flower and fruit flavor esters can be synergistically and remarkably improved, the contents of ethyl acetate, isoamyl acetate and phenethyl acetate are respectively improved by more than or equal to 68%, more than or equal to 50% and more than or equal to 97%, n-propyl alcohol and fusel oil are respectively reduced by more than or equal to 52% and more than or equal to 11%, the wine rate is more than or equal to 62%, meanwhile, the typical style of a baijiu main body is completely reserved, and the method is particularly suitable for the glutinous and elegant composite aroma characteristic of glutinous baijiu. The method is standardized in process, high in repeatability, good in industrial adaptability and easy to popularize and apply on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of baijiu brewing technology, specifically relating to a method for enhancing the floral and fruity aroma of baijiu. Background Technology

[0002] Baijiu, a traditional distilled spirit with a long history in my country, is one of the core categories of alcoholic beverages in the country. With the upgrading of consumption and the diversification of market demand, consumers' requirements for the quality of baijiu have gradually shifted from basic palatability to the richness, layering, and enjoyment of flavor. Among these, natural and harmonious floral and fruity aromas have become one of the core competitive advantages of high-quality baijiu. How to enhance the floral and fruity aromas of baijiu in a targeted and stable manner while preserving its typical style is a key research and development direction in the baijiu brewing industry.

[0003] Currently, the technical solutions for enhancing the floral and fruity aromas of baijiu (Chinese liquor) in the industry can be mainly divided into three categories:

[0004] The first category involves adjusting fermentation process parameters, such as adjusting fermentation temperature, extending the fermentation cycle, and optimizing saccharification and fermentation conditions. However, this approach has limited effect on enhancing the characteristic esters of floral and fruity aromas and is prone to causing an imbalance in the proportion of flavor components in the liquor, damaging the main typical style of baijiu, and failing to achieve a targeted and controllable enhancement of floral and fruity aromas.

[0005] The second category involves adding artificially synthesized flavoring substances during the later blending stage. While this method can quickly enhance the floral and fruity aromas of the liquor, it easily leads to harsh aromas, disjointed tastes, poor layering, and a loss of the natural harmony of solid-state fermentation of pure grains. This does not conform to the quality positioning of pure grain brewing, and consumers have a low acceptance of artificially added ingredients, making it unsuitable for the quality requirements of high-end baijiu.

[0006] The third category is microbial enhancement technology, which involves adding functional microbial strains during the brewing process to achieve targeted control of flavor from the source of fermentation. It is currently recognized in the industry as a core technology that can balance natural flavor with enhanced effects.

[0007] However, existing microbial enhancement technologies still face many insurmountable industry bottlenecks when applied to enhance the floral and fruity aromas of baijiu: Firstly, the use of single-strain fortification results in a limited range of aromas, only achieving a limited enhancement of a single ester. It cannot achieve a synergistic enhancement of multiple floral and fruity aroma esters such as ethyl acetate, isoamyl acetate, and phenylethyl acetate, making it difficult to enrich the flavor profile of the wine.

[0008] Secondly, single strains or non-targeted combinations of strains can easily antagonize the inherent microbial community in the baijiu fermentation system in terms of nutrition and living space. This not only leads to incomplete fermentation of raw materials and a significant decrease in alcohol yield, but also easily causes an increase in the content of defective flavor substances such as n-propanol and fusel oil, resulting in the problem of "enhancing aroma but reducing quality".

[0009] Third, the strain combination lacks targeting, does not consider the metabolic complementarity and synergy between strains, and is only a simple superposition of single functional strains, without synergistic effect, and there may even be antagonistic relationship between strains, resulting in poor batch stability in industrial production and difficulty in large-scale scaling.

[0010] Fourth, there is a prevalent technical bias in the industry: those skilled in the art generally believe that low-temperature fermentation at 20-24℃ will lead to reduced yeast fermentation activity, incomplete starch saccharification, decreased raw material utilization, and a sharp reduction in alcohol yield. Therefore, most existing baijiu brewing uses the conventional fermentation temperature of 25-30℃. High-temperature fermentation, on the other hand, will accelerate the reproduction of miscellaneous bacteria such as lactic acid bacteria and acetic acid bacteria, further increasing the probability of the formation of defective flavor substances, while inhibiting the synthesis and accumulation of floral and fruity esters, forming an industry dilemma of "not being able to balance aroma enhancement and quality stabilization".

[0011] Furthermore, for glutinous rice-flavored baijiu made solely from pure glutinous sorghum with amylopectin content ≥70%, existing technologies still face unique technical bottlenecks. The core characteristics of this type of baijiu are a delicate and elegant glutinous rice aroma, a pleasant sweetness, a natural mellow sweetness, a harmonious flavor profile, and a clean and long finish, with a complex aroma dominated by ethyl acetate. However, existing brewing techniques generally suffer from insufficient grain and sweet aromas, weak floral and fruity aroma layers, and an unprominent ethyl acetate main aroma. At the same time, it is widely believed in the industry that low-temperature fermentation leads to incomplete saccharification of amylopectin in glutinous sorghum, destroying the core glutinous rice aroma style of the baijiu. Therefore, low-temperature fermentation processes are rarely applied to the brewing of this type of baijiu, resulting in a single flavor profile and difficulties in upgrading the quality of existing glutinous rice-flavored baijiu.

[0012] In summary, how to select and construct functional microbial agents with synergistic effects, combine them with standardized brewing processes, overcome the technical bias of low-temperature fermentation in the industry, and achieve targeted, significant, and stable enhancement of floral and fruity aromas while fully preserving the typical style of baijiu, while also ensuring stable alcohol yield, reduction of defective components, and good industrial adaptability, has become a core technical problem that urgently needs to be solved in this field. Summary of the Invention

[0013] In view of this, the present invention addresses the shortcomings of the prior art by proposing a method for enhancing the floral and fruity aroma of baijiu, which is particularly suitable for the brewing needs of glutinous rice aroma baijiu. It can achieve a targeted enhancement of floral and fruity aroma while fully preserving its glutinous and elegant complex aroma.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: A method for enhancing the floral and fruity aroma of baijiu (Chinese white liquor) uses pure glutinous sorghum with a branched-chain starch content of ≥70% as the sole fermentation raw material. A pure grain solid-state fermentation process without adding any lees is employed. During fermentation, a fortified yeast prepared from a compound microbial agent with floral and fruity aroma is added to complete the baijiu brewing process. The amount of fortified yeast added is 1-2% (w / w) of the dry weight of the pure glutinous sorghum. The fermentation temperature is controlled at 20-24℃, and the fermentation cycle is 14 days.

[0015] The Daqu (hereinafter referred to as basic Daqu) without the addition of exogenous functional microbial agents described in this invention refers to Daqu made from early indica rice, rice bran, and Daqu mother seed as core raw materials through natural fermentation. In the entire process of seed Daqu preparation and finished Daqu preparation, apart from the inherent microorganisms of the Daqu mother seed, no artificially cultivated live microbial agents used to regulate brewing saccharification, fermentation, flavor, antibacterial and other functions are added. The finished product meets the physicochemical and sensory indicators defined in this document and is suitable for solid-state fermentation systems of pure glutinous sorghum with amylopectin ≥70%.

[0016] Furthermore, the compound microbial agent is prepared by mixing solid inoculants of *Saccharomyces cerevisiae* Y87, *Saccharomyces cerevisiae* Y348, *Saccharomyces cerevisiae* Y162, and *Pichia pastoris* Y10 in a weight ratio of 2-4:2-4:1-2:1-2. All of the above strains are deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Specifically, *Saccharomyces cerevisiae* Y87 has the accession number CCTCC NO: M2017048, with a deposit date of February 20, 2017; *Saccharomyces cerevisiae* Y348 has the accession number CCTCC NO: M20241944, with a deposit date of September 9, 2024; and *Saccharomyces cerevisiae* Y162 has the accession number CCTCC... NO: M2022245, deposited on March 10, 2022; Pichia pastoris Y10 deposited on April 29, 2022, with accession number CCTCC NO: M2022527.

[0017] This invention addresses the brewing characteristics and existing technological bottlenecks of a pure glutinous sorghum solid-state fermentation system without added lees. It targets four safe functional yeast strains, proven over a long period in the baijiu brewing field, to construct a compound functional microbial agent. The core advantage of this strain selection lies in the fact that the basic functions of all strains are supported by mature industry consensus and existing technology. Furthermore, it is not a simple superposition of single functional strains, but rather a complete brewing process encompassing saccharification, fermentation, flavor synthesis, and environmental control, forming a functionally complementary and metabolically synergistic closed loop of substrate supply, flavor synthesis, and environmental protection. This perfectly adapts to the 20-24℃ low-temperature fermentation process, especially to the elegant and complex aroma characteristics of glutinous sorghum baijiu, as detailed below: Two of the strains of *Wickhamia lanceolata* are core functional strains for synthesizing floral and fruity aromas. They can catalyze the esterification reaction between alcohols and acyl-CoA in the fermentation system through their own metabolic ester synthases, and directionally synthesize characteristic floral and fruity esters with ethyl acetate as the main component and supplemented by isoamyl acetate and phenethyl acetate. The combination of the two strains can further broaden the ester production spectrum and achieve synergistic enhancement of multiple floral and fruity esters, while not destroying the core style of the wine's grain aroma, sweet aroma and glutinous rice aroma. This solves the industry pain points of single strains producing only one aroma, uncoordinated flavors and destruction of the main aroma. The core saccharification functional strain is *Saccharomyces cerevisiae*, which can secrete a variety of hydrolytic enzymes such as saccharifying enzymes and amylases. It gently decomposes the gelatinized amylopectin in glutinous sorghum and continuously generates fermentable sugars such as glucose. It provides a stable and sufficient carbon source substrate for the growth and metabolism of aroma-producing yeasts and brewing yeasts, taking into account both saccharification efficiency and the preservation of glutinous, grain, and sweet aromas. It avoids the problems of incomplete fermentation and reduced alcohol yield caused by insufficient substrate in the fermentation system without lees. At the same time, it complements the saccharification system of the basic koji and improves the utilization rate of raw materials. Pichia pastoris is the core fermentation environment regulation strain. It can effectively inhibit the excessive reproduction of acid-producing bacteria such as lactic acid bacteria and acetic acid bacteria through nutrient site competition and pH regulation of the fermentation microenvironment. This avoids the risk of bacterial contamination and mash spoilage in low-temperature fermentation systems. At the same time, it can regulate the amino acid metabolism pathway of yeast in the fermentation system, reduce the generation of defective flavor substances such as n-propanol and fusel oil, provide a good fermentation microenvironment for the stable metabolism of aroma-producing yeast, ensure the stability of the entire fermentation cycle, and further highlight the sweet and comfortable aroma and clean and long finish of the wine.

[0018] Furthermore, the preparation method of each yeast solid inoculum is as follows: yeast cells are inoculated into YPD liquid medium and cultured on a shaker at 30℃ and 160 r / min for 24 h to obtain a primary inoculum; the primary inoculum is transferred to malt extract medium at an inoculation rate of 10% and cultured on a shaker at 30℃ and 160 r / min until the viable count is ≥1×10⁻⁶. 9 The secondary inoculum was obtained by inoculating the cfu / mL concentration into bran solid culture medium at a 15% inoculum rate. After incubation at 30℃ for 2-3 days, the medium was dried in two stages at 40℃ (44 hours per stage, totaling 88 hours). The resulting solid inoculum had a viable count ≥1×10⁻⁶. 9 cfu / g; Two-stage low-temperature drying can maximize the preservation of live yeast cells and avoid cell inactivation caused by long-term drying at one time.

[0019] The YPD liquid culture medium is an aqueous solution containing 2% glucose, 1% yeast extract, and 2% peptone, with a natural pH; the malt extract culture medium is prepared by mixing malt extract and water at a weight ratio of 1:8, with a sugar content controlled at 11-12 BX, and a natural pH; the bran solid culture medium is prepared by mixing bran and rice flour at a weight ratio of 10:1, adding 30-40% water, and with a natural pH.

[0020] This invention employs a tiered liquid expansion culture combined with solid-state culture process for preparing microbial agents. This process ensures the activity and quantity stability of yeast cells while making the morphology and characteristics of the solid-state microbial agents highly compatible with the koji-making and fermentation processes of baijiu (Chinese liquor). This significantly improves the survival rate and effectiveness of the microbial agents in industrial brewing. At the same time, the drying temperature and time are clearly defined, which can reduce the moisture content of the microbial agents to ensure storage stability and minimize yeast cell death, ensuring that the viable cell count of the solid-state microbial agents meets the standards.

[0021] Furthermore, the preparation method of the fortified yeast is as follows: the above-mentioned compound microbial agent and the basic yeast of the present invention are mixed evenly at a weight ratio of 4-8:1-2, and food-grade silica is added at 0.1% of the total weight of the compound microbial agent and the basic yeast as an anti-caking and anti-mold agent. After mixing evenly, a light yellow powdery fortified yeast without odor or foreign matter is obtained.

[0022] The physicochemical indicators of the basic Daqu are: moisture ≤5%, saccharification power ≥19g / 100g, fermentation power ≥30.0%, and sensory characteristics are: uniform yellow-green mycelium on the surface of the Daqu, dense cross-section of the Daqu core, no black mold, and pure aroma. The physicochemical indicators of the fortified liquor Daqu are: fineness ≥85%, moisture ≤10.5%, saccharification power 26-32g / 100g, and fermentation power ≥33.0%.

[0023] The ratio of this microbial inoculant to the basic Daqu (fermented koji) is not a simple physical mixture, but rather a combination that balances the core saccharification and fermentation capabilities of Daqu with the targeted aroma-producing function of the compound microbial inoculant. It retains the inherent microbial community in Daqu adapted to baijiu brewing, ensuring full fermentation of raw materials and the formation of the main flavor profile, while introducing targeted aroma-producing function through the compound inoculant. Simultaneously, this ratio allows the inoculant and Daqu microbial community to form a stable symbiotic system, avoiding microbial imbalance caused by direct addition of the inoculant, thus achieving synergistic effects of saccharification, fermentation, and aroma production. The added food-grade silica is a food-grade anti-caking agent permitted under GB 2760-2024 "National Food Safety Standard for the Use of Food Additives," effectively preventing the fortified koji from absorbing moisture, clumping, and becoming moldy. It solves the problem of fortified koji having a higher moisture content than traditional Daqu and is prone to spoilage, improving the storage stability of the fortified koji. The addition of 0.1% will not affect the flavor or food safety of the liquor.

[0024] Furthermore, the pretreatment process of the pure glutinous sorghum is as follows: the pure glutinous sorghum is soaked in hot water at 70℃ for 17-19 hours, and after initial steaming, steaming, and re-steaming, it is cooled to 30℃±2℃, and the moisture content of the cooked grain is controlled at 52-54%; the initial steaming conditions are 115℃ for 20-30 minutes, the steaming conditions are 50±1℃ for 5 minutes, and the re-steaming conditions are 110℃ for 10-20 minutes.

[0025] Standardized high-temperature soaking and secondary steaming processes can fully gelatinize the amylopectin in pure glutinous sorghum, breaking down the crystalline structure of starch and making it easier for the saccharifying enzymes in the yeast to decompose it into fermentable sugars. This provides a sufficient carbon source for the metabolism of compound microbial agents and the synthesis of aromatic esters. Precise control of the moisture content of the cooked grains can avoid problems such as raw material rancidity, incomplete fermentation, or excessively dry mash affecting the metabolism of microorganisms during fermentation, ensuring the stability of the fermentation system. At the same time, limiting the cooling temperature can prevent high temperatures from causing the inactivation of microorganisms in the subsequently added yeast.

[0026] Furthermore, the solid-state fermentation of pure grains also includes a saccharification step: glutinous sorghum with added fortified yeast is placed in a saccharification tank, and 5-8% of the dry weight of pure glutinous sorghum husks are evenly spread on the bottom of the saccharification tank and the surface of the glutinous sorghum. The tank is covered with canvas or filter cloth to keep it moist, and saccharification is carried out at 30°C for 24 hours to obtain saccharified mash, which is then put into a barrel for fermentation.

[0027] The rice husks are sterilized at 121℃ for 30 minutes and sieved through a 20-mesh sieve, which effectively prevents contamination of the fermentation system by carrying miscellaneous bacteria. The sieved husks also have a uniform particle size and better air permeability. The criteria for complete saccharification are: a reducing sugar content of ≥15% based on glucose or no blue color development with iodine solution. Meeting either of these criteria allows for rapid and accurate assessment of the degree of saccharification, preventing insufficient saccharification leading to a lack of carbon source in subsequent fermentation, or excessive saccharification leading to spoilage of the mash. The uniform spreading of the rice husks achieves both heat and moisture retention while ensuring the air permeability of the saccharification system, providing a suitable environment for the growth and metabolism of aerobic saccharifying bacteria and promoting the full saccharification reaction. 30℃ is the optimal growth temperature for saccharifying bacteria, maximizing the activity of saccharifying enzymes, accelerating the starch saccharification process, accumulating sufficient fermentable sugar substrates for subsequent anaerobic fermentation, and improving the efficiency of ester synthesis in the subsequent fermentation process.

[0028] Furthermore, the fermentation process employs a jacketed temperature control system with an accuracy of ±0.5℃, precisely maintaining the required fermentation temperature range of 20-24℃ to prevent temperature fluctuations from affecting microbial metabolism. During fermentation, the pH of the mash is monitored in real time. If the pH is <3.5, food-grade calcium carbonate is added to adjust the pH to 4.0-4.5, effectively preventing excessive acidity that could lead to the inactivation of fermentation bacteria and ensuring the smooth progress of the fermentation process.

[0029] This invention overcomes the technical prejudice in the field that "low-temperature fermentation at 20-24℃ will lead to incomplete saccharification of amylopectin in glutinous sorghum and a decrease in alcohol yield." This temperature range is the optimal temperature for the synthesis of aromatic esters by the compound microbial agent of this invention: on the one hand, it can promote the synthesis and accumulation of aromatic esters, mainly ethyl acetate, and on the other hand, it can effectively inhibit the reproduction rate of miscellaneous bacteria and reduce the generation of defective components such as n-propanol and fusel oil; the 14-day fermentation cycle provides sufficient time for the full accumulation of aromatic esters and the complete fermentation of pure glutinous sorghum raw materials, taking into account both flavor enhancement and fermentation efficiency.

[0030] Furthermore, after fermentation, the mash is distilled to obtain the base liquor of baijiu. The distillation process is as follows: preheat the still for 5 minutes under a steam pressure of 0.1-0.15 MPa, add the bottom mash, then add the fermented mash and steam distill the liquor. Collect 100 mL of the first distillate, collect the main liquor until the alcohol content is 63±1% vol, and collect the last distillate until the alcohol content is below 6% vol. Combine the first distillate, the main liquor, and the last distillate to obtain the base liquor of baijiu.

[0031] Precise distillation parameters enable the effective separation of different flavor components in baijiu: preheating the still prevents the mash from cooling down, which would reduce distillation efficiency; high-boiling-point impurities and irritating substances in the heads are collected and removed separately; and low-alcohol off-flavors and water-soluble impurities in the tails are effectively separated, retaining only the main spirit with pure flavor and appropriate alcohol content. At the same time, precise control of alcohol content ensures uniform quality of the spirit, resulting in a pure aroma and harmonious taste. This makes the floral and fruity aromas prominent features of the spirit without conflicting with the main style, which is especially suitable for the core positioning of glutinous rice aroma baijiu: "elegant glutinous rice aroma, comfortable sweet aroma, and harmonious flavor".

[0032] This invention also protects the baijiu (Chinese white liquor) brewed by any of the above methods, wherein the baijiu is a clear and transparent liquor with sensory characteristics of being mellow and sweet, with a pleasant main aroma, a rich and harmonious floral and fruity aroma, a clean finish, and a long aftertaste; the content of ethyl acetate, isoamyl acetate, and phenylethyl acetate in the original liquor is increased by ≥68%, ≥50%, and ≥97% respectively compared with the original liquor brewed under the same brewing process and with basic Daqu (a type of starter culture); the content of n-propanol is reduced by ≥52%; the content of fusel oil is reduced by ≥11%; and the alcohol content is ≥62%; wherein the fusel oil is the sum of isobutanol and isoamyl alcohol.

[0033] The resulting liquor retains the typical style of baijiu while enriching its flavor layers, significantly enhancing the drinking experience. Specifically designed for glutinous rice aroma baijiu brewing, it fully preserves the elegant glutinous rice aroma, the pleasant sweetness, and the complex aroma dominated by ethyl acetate, achieving a harmonious coexistence of glutinous rice aroma and floral and fruity notes. Furthermore, the compound microbial agent prepared by this invention, when stored in sealed aluminum foil bags at 0-4℃ and humidity ≤60%, has a shelf life of 6 months; the fortified yeast, when stored in a ventilated warehouse at 20-25℃ and humidity ≤50%, has a shelf life of 3 months. The intermediates exhibit good storage stability, meeting the transportation and usage requirements for industrial production.

[0034] The present invention has the following advantages over the prior art: 1. This invention achieves a targeted and significant enhancement of the floral and fruity aromas of baijiu from the brewing source. Through the synergistic aroma-producing effect of compound microbial agents, key floral and fruity esters such as ethyl acetate, isoamyl acetate, and phenylethyl acetate are synergistically enhanced, solving the problem of existing baijiu having a single flavor and insufficient floral and fruity aromas. At the same time, through the environmental regulation effect of Pichia pastoris and the low-temperature fermentation process of 20-24℃, the defective components such as n-propanol and fusel oil are significantly reduced, and the alcohol yield is stabilized at ≥62%, solving the technical pain point that existing technologies tend to lead to a decrease in alcohol yield and an increase in defective components when enhancing flavor.

[0035] 2. While enhancing the floral and fruity aroma, this invention fully preserves the typical style of baijiu, achieving an organic fusion of traditional aroma and distinctive floral and fruity aroma, enriching the flavor layers of the liquor. For glutinous rice aroma baijiu, it can fully preserve its core characteristics of elegant glutinous rice aroma, comfortable sweet aroma, natural mellow sweetness, and clean and long aftertaste, while strengthening the elegant glutinous rice aroma with ethyl acetate as the main component, significantly improving the quality and market added value of the product, and meeting the market's demand for diversified and high-quality baijiu flavors under the background of consumption upgrading.

[0036] 3. This invention standardizes and quantifies the parameters for all steps of the entire brewing process, including raw material specifications, microbial agent preparation, yeast ratio, raw material pretreatment, saccharification, fermentation, distillation, etc. It also clarifies the handling measures for abnormal fermentation and the storage conditions for intermediates, making the technical solution have good repeatability, stability and industrial adaptability. Those skilled in the art can achieve industrial scale-up without creative labor, and it is easy to promote and apply on a large scale.

[0037] 4. This invention overcomes the technical biases in the field of Baijiu brewing, proving that low-temperature fermentation at 20-24℃ does not lead to incomplete saccharification of amylopectin in glutinous sorghum or a decrease in alcohol yield. On the contrary, it can combine the metabolic characteristics of compound microbial agents to achieve efficient synthesis of floral and fruity esters and effective inhibition of miscellaneous bacteria, providing new ideas and directions for the process optimization of Baijiu, especially glutinous baijiu, and has good industry application value. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a gas chromatogram of the Daqu-brewed Baijiu produced by the present invention without the addition of exogenous functional microbial agents; the vertical axis represents the relative response value, the horizontal axis represents the retention time (min), and the characteristic peaks are, in order: 1-acetaldehyde, 2-ethyl acetate, 3-n-propanol, 4-isobutanol, 5-isoamyl alcohol, 6-isoamyl acetate, and 7-phenylethyl acetate.

[0040] Figure 2 This is a gas chromatogram of the fortified yeast used in the brewing of baijiu according to the present invention. The vertical axis represents the relative response value, and the horizontal axis represents the retention time (min). The characteristic peaks are, in order: 1-acetaldehyde, 2-ethyl acetate, 3-n-propanol, 4-isobutanol, 5-isoamyl alcohol, 6-isoamyl acetate, and 7-phenylethyl acetate. The figure clearly shows that the characteristic peak response values ​​of floral and fruity esters in the fortified yeast-brewed baijiu are significantly higher than those in the blank control group, while the characteristic peak response values ​​of defective components are reduced.

[0041] Figure 3 This invention provides a construction diagram of the flavor wheel for the Baijiu (Chinese liquor) brewed with enhanced yeast. The flavor wheel defines the characteristics of the liquor from three dimensions: aroma, mouthfeel, and taste. The aroma dimension includes glutinous rice aroma, sorghum aroma, raw material aroma, fermentation aroma, light aroma, floral aroma, fruit aroma, dried fruit aroma, lees aroma, caramel aroma, grass aroma, sweet aroma, and woody aroma. The mouthfeel dimension includes mellow sweetness, cleanliness, refreshing taste, and aftertaste. The taste dimension includes sour, sweet, bitter, umami, salty, spicy, rancid, bean aroma, and rice aroma. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] In all embodiments of this invention, the culture media involved were sterilized by high-pressure steam at 121℃ for 30 minutes and then cooled to a suitable temperature before use; the rice husks, saccharification tanks, fermentation tanks, steamers, and other equipment involved were sterilized at 121℃ for 30 minutes or wiped with 75% edible alcohol before use; the pure glutinous sorghum used was brewing-specific glutinous sorghum with a branched-chain starch content ≥70%, and the remaining raw materials were food-grade or brewing-specific grade, conforming to the relevant national standards for baijiu production. All embodiments of this invention used the basic koji described in this invention brewed using the same process as a blank control. A unified performance verification method was adopted, including three parts: alcohol yield calculation, gas chromatography quantitative detection, and sensory quality evaluation. All experiments had ≥3 parallel samples, and the test results were taken as the mean ± standard deviation. Through data analysis, p < 0.05 was considered significant, and p < 0.01 was considered extremely significant. The specific verification method is as follows: 1. Alcohol yield calculation: The alcohol yield is calculated based on the mass of 55° alcohol produced per kilogram of pure glutinous sorghum. First, convert the main liquor and tail liquor obtained from distillation into the weight of 55° alcohol according to their alcohol content and sum them up, which is recorded as M alcohol. Alcohol yield η = (weight of M alcohol / weight of pure glutinous sorghum) × 100%.

[0044] 2. Quantitative Detection by Gas Chromatography: The contents of ethyl acetate, isoamyl acetate, phenylethyl acetate, n-propanol, and fusel oil in the raw wine were detected using a GC-FID gas chromatograph. The fusel oil was the sum of isobutanol and isoamyl alcohol. The chromatographic column was an HP-INNOWax capillary column (30m×0.32mm×0.25μm), and the carrier gas was high-purity nitrogen. The column temperature program was as follows: initial temperature 40℃, hold for 5 min, increase to 180℃ at 5℃ / min, hold for 10 min, and then increase to 250℃ at 10℃ / min, hold for 5 min. The injection port temperature was 250℃, the detector temperature was 280℃, the carrier gas flow rate was 1.0 mL / min, the split ratio was 10:1, and the injection volume was 1 μL. Quantitative calculation was performed using the external standard method.

[0045] 3. Verification of exogenous functional bacteria: The presence of the four specific strains described in claim 2 of this invention in the Daqu was detected by PCR-specific sequencing. If no strains were detected, it was deemed to meet the requirements for basic Daqu.

[0046] 4. Sensory Quality Evaluation: The evaluation team consists of 5 or more professionals with national baijiu (Chinese liquor) taster qualifications. The evaluation environment, personnel qualifications, evaluation process, and data statistics methods strictly follow the provisions of GB / T 33404-2016 "Guidelines for Sensory Evaluation of Baijiu" and GB / T 10345-2007 "Analytical Methods for Baijiu". The definitions of sensory terms comply with the requirements of GB / T 15109-2021 "Terminology of Baijiu Industry". The scoring dimensions and details comply with the general provisions of GB / T 10781.2-2022 "Quality Requirements for Baijiu Part 2: Light Aroma Baijiu". For the glutinous rice aroma, grain aroma, and sweet aroma characteristics of baijiu made from pure glutinous sorghum, special evaluation dimensions are added within the framework of the above national standards. A 100-point scoring system is used, and the arithmetic mean of the scores of all tasters is taken as the final sensory score.

[0047] Example 1 This embodiment verifies the effects of individual strains on the floral and fruity aromas, glutinous rice aromas, and overall quality of baijiu through single-strain small-scale fermentation, providing an experimental basis for subsequent strain combination screening. This embodiment is a preliminary experiment for strain function verification, and the 7-day fermentation cycle is only used for rapid screening of strain combination effects. For formal industrial production, the 14-day fermentation cycle specified in claim 1 must be followed to ensure sufficient accumulation of floral and fruity esters.

[0048] Experimental steps 1. Culture medium preparation: YPD liquid culture medium, malt extract culture medium, and bran solid culture medium were prepared separately; the YPD liquid culture medium was an aqueous solution containing 2% glucose, 1% yeast extract, and 2% peptone, with a natural pH; the malt extract culture medium was prepared by mixing malt extract and water at a weight ratio of 1:8, with the sugar content controlled at 11-12 BX, and a natural pH; the bran solid culture medium was prepared by mixing bran and rice flour at a weight ratio of 10:1, adding 30-40% water, and a natural pH.

[0049] 2. Preparation of single-strain bran koji: *Wickhamia lanceolata* Y87, *Wickhamia lanceolata* Y348, *Cytomyces commune* Y162, and *Pichia pastoris* Y10 were inoculated into YPD liquid medium and cultured in a shaker at 30℃ and 160 r / min for 24 h to obtain primary seed culture. The primary seed culture was then transferred to malt extract medium at a 10% inoculation rate and cultured under the same conditions until the viable count was ≥1×10⁻⁶. 9 The concentration of cfu / mL was used to obtain secondary seed culture; then, the secondary seed culture was inoculated into bran solid medium at a 15% inoculation rate, and cultured at 30℃ for 2 days. After drying at 40℃ in two stages (44 hours each stage), single strains of bran koji were obtained, and the viable count of each strain was ≥1×10⁻⁶. 9 cfu / g.

[0050] 3. Pre-treatment of glutinous sorghum: Weigh 500g of pure glutinous sorghum, add 65℃ warm water (the water level should be 2cm above the glutinous sorghum), and soak the grain in a 65℃ oven for 24 hours. After soaking, drain the water, steam at 115℃ for 30 minutes, add 80℃ warm water and simmer for 8 minutes, drain again and steam at 110℃ for 10 minutes. After steaming, spread out to cool to 30℃±2℃.

[0051] 4. Grain koji mixing and saccharification: Add a mixture of single-strain bran koji and basic koji at a ratio of 1% of the weight of pure glutinous sorghum dry grain (single-strain bran koji: basic koji = 1:1), mix thoroughly, and pack into sterile self-sealing bags; leave a slit at the opening of the self-sealing bag, and saccharify in a 30℃ incubator for 24 hours. The criteria for complete saccharification are either a reducing sugar content of ≥15% based on glucose or no blue color in iodine solution, whichever is met.

[0052] 5. Fermentation and distillation: The saccharified material was transferred to a sterile gas sampling bag, vacuumed and sealed, and fermented at a constant temperature of 30℃ for 7 days. After fermentation, the mash was placed in a special glass still and distilled at atmospheric pressure of 0.1MPa. The first 100mL of the sample was collected for testing.

[0053] 6. Blank control: The basic Daqu described in this invention is used instead of the single-strain bran koji, and the remaining experimental steps are completely consistent with those described above.

[0054] Performance verification results Table 1. Results of alcohol production performance testing in small-scale solid-state fermentation with enhanced single-strain culture

[0055] As shown in Table 1, adding each target strain individually can effectively reduce defective components such as n-propanol and fusel oil in the original liquor. At the same time, it can increase the content of floral and fruity esters such as ethyl acetate, isoamyl acetate, and phenylethyl acetate to varying degrees, without destroying the core glutinous rice aroma style of the liquor. However, the addition of a single strain has a significant decrease in alcohol content and cannot achieve the synergistic enhancement of multiple floral and fruity esters. The application of each strain alone has technical drawbacks, and strain combination screening is required to achieve functional complementarity.

[0056] Example 2 This embodiment is based on the single-strain bran koji of Example 1. Different strain combinations were used for fermentation trials to screen out the optimal strain combination that can achieve synergistic aroma production, impurity suppression, wine retention rate, and stable glutinous aroma, laying the foundation for the preparation of compound microbial agents. This embodiment is a preliminary experiment for strain combination screening, with a fermentation cycle of 7 days. For formal production, a 14-day fermentation cycle will be used.

[0057] Experimental steps 1. Strains combination design: The four strains of bran koji prepared in Example 1 were combined in binary, ternary and quaternary ways to design a total of 10 combination schemes. The proportion of each strain of bran koji in the mixed koji was equal; the basic koji described in this invention was used as a blank control.

[0058] 2. Fermentation and testing: The experiment was conducted according to the steps of glutinous sorghum pretreatment, grain koji mixing (the amount of mixed koji added was 1% of the weight of pure glutinous sorghum dry grain), saccharification, fermentation and distillation as described in Example 1. Three parallel samples were set up for each group of experiments. After fermentation, the various indicators of the liquor samples were tested according to a unified performance verification method.

[0059] Performance verification results Table 2 Results of alcohol production performance testing in a small-scale solid-state fermentation trial using microbial co-fermentation

[0060] As shown in Table 2, the four-strain combination (Y87+Y348+Y162+Y10) is the only optimal combination in this experiment. This combination can give full play to the functional complementarity of each strain, achieve a significant synergistic enhancement of floral and fruity esters, and effectively reduce defective components such as n-propanol and fusel oil. Although the alcohol content is slightly reduced, there is no significant difference. It can also ensure the core glutinous rice aroma style of baijiu, achieve the harmonious coexistence of glutinous rice aroma and floral and fruity aroma, and solve the technical drawbacks of single strain or partial strain combination fermentation with single aroma and reduced alcohol yield. Therefore, this four-strain combination is determined to be the strain composition of the subsequent compound microbial agent.

[0061] Example 2-1 Experiment to verify the synergistic effect of strains This embodiment verifies the synergistic effect of the four-strain combination by setting up a three-strain control group that lacks a single strain. The fermentation cycle is 14 days, and the remaining process parameters are completely consistent with the pilot-scale process in Example 5.

[0062] Experimental steps Five experimental groups were set up as follows: Group 1: Four strains combined (Y87+Y348+Y162+Y10, ratio 3:3:1.5:1.5). Group 2: Missing Y87 group (Y348+Y162+Y10, ratio 3:1.5:1.5); Group 3: Missing Y348 group (Y87+Y162+Y10, ratio 3:1.5:1.5); Group 4: Missing Y162 group (Y87+Y348+Y10, ratio 3:3:1.5); Group 5: Missing Y10 group (Y87+Y348+Y162, ratio 3:3:1.5); Blank control group: basic Daqu (a type of starter culture), without the addition of exogenous strains.

[0063] Each experiment set up 3 parallel samples, and the core indicators were tested according to a unified performance verification method.

[0064] Performance verification results Table 2-1 Results of the experimental verification of synergistic effects of strains

[0065] As shown in Table 2-1, the combination of three strains lacking any one strain resulted in a sharp decline in the enhancement of floral and fruity aromas, the reduction of defective components, and the yield. Only the combination of all four strains could achieve the synergistic effect of "enhancing aroma, reducing impurities, stabilizing yield, and preserving main style". This confirms that there is significant metabolic synergy and functional complementarity among the four strains, which is not a conventional choice for those skilled in the art.

[0066] Example 3 This embodiment is based on the optimal combination of four strains screened in Example 2. The culture is scaled up step by step to prepare a standardized compound microbial solid inoculant to meet the needs of pilot-scale and industrial production. At the same time, the storage conditions and shelf life of the inoculant are defined.

[0067] Experimental steps 1. Yeast primary culture: Under aseptic conditions, test tube slant cultures of *Saccharomyces cerevisiae* Y87, *Saccharomyces cerevisiae* Y348, *Saccharomyces cerevisiae* Y162, and *Pichia pastoris* Y10 were inoculated into malt extract Erlenmeyer flasks and cultured at 30°C and 140 rpm for 24-36 h until the cells were in the logarithmic growth phase, thus obtaining the yeast primary culture.

[0068] 2. Secondary yeast culture: Primary cultures of each strain were inoculated at a rate of 10% into malt extract medium in a 100L fully automated fermenter. Fermenter parameters were set as follows: temperature 30℃, stirring speed 100-500 rpm, dissolved oxygen 45% (measured value 25%-100%), pH 4.50-6.0. Culture was stopped when dissolved oxygen reached ≥80% and stirring speed was reduced to 100-150 rpm. Secondary yeast cultures were obtained. The viable count of each secondary culture was ≥1×10⁻⁶. 9 cfu / mL.

[0069] 3. Preparation of solid yeast inoculum: The sterilized wheat bran solid culture medium is cooled to 30-40℃ using an air cooler. Secondary inoculum of each strain is inoculated at a rate of 15%, thoroughly mixed, and then transferred to a fully automated inoculum culture machine for solid-state expansion. The culture consists of five stages: ① Static stage: wind speed 100 rpm, temperature 31℃, incubation for 12 hours; ② First cultivation stage: wind speed 300 rpm, temperature 30℃, cultivation for 48 hours; ③ Second cultivation stage: wind speed 300 rpm, temperature 30℃, cultivation for 48 hours; ④ First drying stage: wind speed 500 rpm, temperature 40℃, incubation for 44 hours; ⑤ Second drying stage: wind speed 500 rpm, temperature 40℃, incubation for 44 h; When the material moisture content reaches ≤14%, it is discharged and pulverized to obtain solid inoculum agents of various strains. The viable count of each strain is ≥1×10⁻⁶. 9 cfu / g.

[0070] 4. Preparation of compound microbial inoculant: The solid inoculants of the above 4 strains are thoroughly mixed in a weight ratio of 2-4:2-4:1-2:1-2 to obtain the compound microbial inoculant; the inoculant is packed into sterile aluminum foil bags, vacuum sealed and stored in a cold storage at 0-4℃ and humidity ≤60% for 6 months.

[0071] Performance verification results Table 3. Performance test results of compound microbial inoculants and solid inoculants of individual strains

[0072] As shown in Table 3, all prepared single-strain solid inoculants and compound microbial inoculants met the requirement of viable count ≥ 1 × 10⁻⁶. 9 The technical requirements for cfu / g are: suitable moisture content, pH within the suitable survival range of yeast, uniform appearance without clumping; under storage conditions of 0-4℃ and humidity ≤60%, the viable cell retention rate is ≥89% after 6 months, with good storage stability, which can meet the transportation and use needs of industrial production.

[0073] Table 3-1 Effects of different drying methods on yeast viable cell count (comparison)

[0074] As shown in Table 3-1, the two-stage low-temperature drying process at 40℃ adopted in this invention can maximize the retention of live yeast cells, and the retention rate of live cells is 84.5% higher than that of single-stage continuous drying, which confirms the significant technical effect of the process.

[0075] Example 4 This embodiment prepares the basic Daqu and fortified Daqu of the present invention, compares the physicochemical indicators and sensory characteristics of the two types of Daqu, verifies the advantages of the fortified Daqu in saccharification and fermentation performance, and clarifies the storage conditions and shelf life of the fortified Daqu.

[0076] Experimental steps (1) Preparation of basic Daqu 1. Preparation of starter culture: Soak early indica rice for 8-10 hours until the rice grains have no hard center, grind it into rice paste, add 2% of baijiu starter culture, mix thoroughly and make 6-7cm cake-shaped starter culture cakes; place the starter culture cakes in the starter culture room, control the temperature of the starter culture room at 30-32℃ and the humidity at 85%-90%, and cultivate until the temperature of the starter culture cakes rises to 35℃, then remove the covering gauze and continue to cultivate until the temperature drops to 25-28℃, and yellow-green mycelium grows on the surface of the starter culture cakes; transfer the starter culture cakes to a 35℃ drying room and dry them until the moisture content is ≤5%, to obtain starter culture.

[0077] 2. Preparation of Daqu (a type of starter culture): Guanyin clay (pH 6.5-7.5, moisture content ≤10%) is pulverized and passed through a 14-mesh sieve. It is then mixed with rice bran that has passed through a 16-mesh sieve at a weight ratio of 3:7. Seed starter culture (3% of the total weight of the mixture) and an appropriate amount of water are added and mixed until the moisture content of the mixture reaches 50%. The mixture is then formed into spherical starter culture blanks of 7-8 cm in diameter and placed in a starter culture room. The temperature is controlled at 28-30℃ and the humidity at 80%-85%. The mixture is cultured for 16 hours until the temperature of the starter culture blanks rises to 30-35℃. The sieve is then removed for ventilation. After the temperature of the starter culture blanks drops to room temperature, they are cultured for another 5 days until the starter culture blanks are fully mature. The mature starter culture blanks are then transferred to a drying room at 35-40℃ and dried until the moisture content is ≤5%. The basic Daqu described in this invention is obtained. PCR testing did not detect the four specific functional strains of this invention, which meet the requirements.

[0078] (2) Preparation of fortified yeast 1. Raw material mixing: The compound microbial agent prepared in Example 3 is thoroughly mixed with the above-mentioned basic Daqu at a weight ratio of 4-8:1-2. Food-grade silica is added at a weight ratio of 0.1% of the total weight of the compound microbial agent and the basic Daqu. The mixture is then stirred again to obtain the Daqu mixture.

[0079] 2. Shaping and drying: The yeast mixture is crushed by a pulverizer and then passed through an 80-mesh sieve to make a light yellow powder yeast. The yeast is then packed into sterile woven bags and placed in a ventilated warehouse at 20-25℃ and humidity ≤50% to dry until the moisture content is ≤10.5%, thus obtaining fortified yeast.

[0080] 3. Storage conditions: After sealing the fortified yeast, store it in a well-ventilated warehouse at 20-25℃ and humidity ≤50%. Shelf life is 3 months.

[0081] Performance verification results Table 4 Comparison of physicochemical and sensory indicators between basic Daqu and fortified Daqu

[0082] As shown in Table 4, the prepared basic Daqu meets the physicochemical and sensory requirements specified in this invention. The prepared fortified Daqu is a light yellow powder, odorless, free of foreign matter and lumps, with a fineness ≥85% and moisture content ≤10.5%. Its saccharification and fermentation power are significantly higher than those of the basic Daqu, and it can more efficiently decompose the amylopectin in glutinous sorghum, providing sufficient carbon source for fermentation. After adding food-grade silica, the fortified Daqu was stored for 3 months under ventilated conditions of 20-25℃ and humidity ≤50%, without mold, lumps, or moisture absorption, demonstrating good storage stability.

[0083] Example 5 In this embodiment, the fortified yeast prepared in Example 4 is applied to a pilot-scale baijiu brewing process to verify the application effect of the fortified yeast in actual production. The content of floral and fruity esters, the content of defective components, the alcohol yield and sensory quality of the raw liquor are detected and compared with the brewing effect of the basic yeast. The pilot-scale process parameters are completely consistent with the core process parameters of this invention, and the adaptability of the yeast to the glutinous rice aroma baijiu scenario is verified simultaneously.

[0084] Experimental steps 1. Soaking the grain: Weigh out 500 kg of pure glutinous sorghum for pilot-scale production, transfer it to a fully automatic soaking tank, add 70℃ hot water (the water level should be 3 cm above the glutinous sorghum), and soak at a constant temperature for 17-19 hours, stirring once every 4 hours during this period.

[0085] 2. Steaming the grain: After soaking, transfer the glutinous sorghum into a fully automatic steaming machine. Steam at 115℃ for 20-30 minutes, then steam with warm water at 50±1℃ for 5 minutes. After draining the water, steam again at 110℃ for 10-20 minutes. After steaming, remove the grain from the steamer and cool it to 30℃±2℃ using a cooling machine. Check the moisture content of the cooked grain and control it at 52-54%.

[0086] 3. Saccharification and incubation: Add fortified yeast at a ratio of 1% of the dry weight of pure glutinous sorghum, mix thoroughly with a mixer, and then transfer to a saccharification tank. Spread 5-8% of the dry weight of pure glutinous sorghum husks (sterilized at 121℃ for 30 minutes and treated by passing through a 20-mesh sieve) evenly on the bottom of the saccharification tank and the surface of the glutinous sorghum. Cover with canvas to keep moist, control the saccharification temperature at 30℃, and saccharify for 24 hours. The criteria for complete saccharification are either a reducing sugar content of ≥15% based on glucose or no blue color in iodine solution. Either one of these conditions must be met. After saccharification, saccharified mash is obtained.

[0087] 4. Fermentation in the tank: Transfer the saccharified mash into a jacketed temperature-controlled fermentation tank. Use a jacketed temperature control system to control the fermentation temperature at 22℃ (optimal value) with a temperature control accuracy of ±0.5℃. The fermentation cycle is 14 days. Monitor the pH of the mash in real time during fermentation. If the pH of the mash is <3.5, add food-grade calcium carbonate to adjust the pH of the mash to 4.0-4.5.

[0088] 5. Distillation and collection: After fermentation, the mash is transferred to a fully automatic still. The still is preheated for 5 minutes under a steam pressure of 0.1-0.15 MPa. The bottom mash is added, followed by the fermented mash. Steam is passed through to distill the liquor at normal pressure. 100 mL of the first distillate is collected. The main liquor is collected until the alcohol content is 63±1% vol. The tail liquor is collected until the alcohol content is below 6% vol. The first distillate, the main liquor and the tail liquor are combined to obtain the original liquor of baijiu.

[0089] 6. Blank control: The basic Daqu prepared in Example 4 was used to replace the fortified yeast, and the remaining pilot-scale steps were completely consistent with those described above.

[0090] Performance verification results Table 5. Results of alcohol production performance testing in pilot-scale application of fortified yeast.

[0091] Table 6. Sensory quality evaluation results of Baijiu brewed using enhanced yeast in pilot-scale trials (out of 100 points)

[0092] As shown in Tables 5 and 6, after the fortified yeast was applied to the pilot-scale baijiu brewing, the contents of ethyl acetate, isoamyl acetate, and phenylethyl acetate in the raw liquor were significantly increased by 68.64%, 50.75%, and 97.86% respectively compared with the blank control group (p < 0.01), while the contents of n-propanol and fusel oil were significantly decreased by 52.00% and 11.40% respectively (p < 0.01). The alcohol yield of the fortified yeast group was 62.45 ± 1.63%, which was not significantly different from the blank control group, achieving the dual technical effect of enhancing floral and fruity aroma and stabilizing alcohol yield.

[0093] Sensory evaluation results show that the baijiu brewed with fortified yeast is clear and transparent, with a delicate glutinous rice aroma, a pleasant grain aroma and sweet aroma, a rich floral and fruity aroma that is highly coordinated with the main glutinous rice style, no off-flavors, a smooth and sweet taste, a clean finish, and a long and clean aftertaste. The overall score is 96±1 points, which is 8 points higher than the blank control group, and the quality of the baijiu has been greatly improved.

[0094] Example 6 Based on the pilot-scale process of Example 5, this embodiment conducts single-factor optimization experiments on three core key parameters: compound microbial agent ratio, fortified yeast ratio, and fermentation temperature. The optimal values ​​of each parameter are screened out, and the effective range of each parameter is clarified. This provides a scientific basis for setting process parameters for large-scale industrial production and simultaneously verifies the effect of overcoming the bias of low-temperature fermentation technology.

[0095] Experimental steps 1. Optimal formulation of compound microbial agent: Seven compound microbial agent formulations were set (Y87:Y348:Y162:Y10): 1:1:1:1, 2:2:1:1, 2:4:2:1, 3:3:1.5:1.5, 4:2:1:2, 4:4:2:2, 5:5:3:3; other process parameters were the same as in Example 5. Three parallel samples were set for each group of experiments, and the performance was tested according to a unified performance verification method. The comprehensive effect was evaluated with the total improvement rate of esters + the total reduction rate of defective components + the conformity of glutinous rice aroma as the core indicators.

[0096] 2. Optimization of fortified yeast ratio: Five sets of fortified yeast ratios (compound microbial agent: basic yeast) were set: 3:2, 4:1, 6:1, 8:1, and 9:1; the remaining process parameters were the same as in Example 5. Three parallel samples were set up for each group of experiments, and the overall effect was evaluated according to the above core indicators.

[0097] 3. Optimal fermentation temperature: Seven fermentation temperatures were set: 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, and 30℃; other process parameters were the same as in Example 5. Three parallel samples were set for each group of experiments, and the number of miscellaneous bacteria in the fermentation system was detected simultaneously. The overall effect was evaluated according to the above core indicators.

[0098] Performance verification results Table 7 Results of Experimental Testing for Optimization of Key Parameters

[0099] As shown in Table 7, the optimal values ​​for the three core parameters are: a compound microbial agent ratio of 3:3:1.5:1.5, a fortified yeast ratio of 6:1, and a fermentation temperature of 22℃. Under these optimal conditions, the enhancement of floral and fruity aroma esters and the reduction of defective components are both optimal, the number of miscellaneous bacteria is controlled at a low level, and the glutinous rice aroma style is best preserved. Meanwhile, the experimental results clarify the effective range of each parameter: a compound microbial agent ratio of 2-4:2-4:1-2:1-2, a fortified yeast ratio of 4-8:1-2, and a fermentation temperature of 20-24℃. Within this effective range, the brewed baijiu can achieve good enhancement of floral and fruity aroma and reduction of defective components, while fully preserving the glutinous rice aroma style. Deviating from this range will lead to a significant decrease in the overall effect. For example, a fermentation temperature below 20℃ will lead to incomplete fermentation of the raw materials, while a temperature above 24℃ will lead to the proliferation of miscellaneous bacteria and an increase in fusel oil content, consistent with the technical solution of this invention. Although the number of miscellaneous bacteria in the 18℃ group is lower, the yeast fermentation activity is insufficient, resulting in incomplete saccharification and a significant decrease in alcohol yield. Meanwhile, at the industry's conventional fermentation temperature of 25-28℃, the number of miscellaneous bacteria increased by more than 40 times compared to the 22℃ group, and the content of fusel oil increased by 15-20%, confirming that the low-temperature fermentation process of 20-24℃ of this invention can effectively inhibit miscellaneous bacteria and overcome the technical prejudice in the industry that "low-temperature fermentation will lead to incomplete fermentation and a decrease in alcohol yield".

[0100] Table 7-1 Comparison of amylopectin saccharification rate and alcohol yield at different fermentation temperatures

[0101] As shown in Table 7-1, within the temperature range of 20-24℃, the saccharification rate of amylopectin remained stable at over 92%, and the alcohol yield remained stable at over 61%. Compared with the industry's conventional fermentation temperature of 25-30℃, the saccharification rate and alcohol yield did not decrease significantly. On the contrary, the growth of miscellaneous bacteria was inhibited, resulting in higher raw material utilization. This directly proves that the present invention overcomes the industry's technical prejudice that "low-temperature fermentation at 20-24℃ will lead to incomplete saccharification of amylopectin in glutinous sorghum and a decrease in alcohol yield."

[0102] Example 7 Based on the optimal process parameters selected in Example 6, this embodiment scales up the pilot-scale process to a 10-ton-level industrial production scale of pure glutinous sorghum, and conducts three consecutive batches of industrial production verification. The performance indicators and sensory quality of each batch of products are tested to verify the industrial adaptability, stability and repeatability of the process of this invention.

[0103] Experimental steps 1. Industrial process parameters: compound microbial agent ratio 3:3:1.5:1.5, fortified yeast ratio 6:1, fermentation temperature 22℃, fortified yeast addition amount 1% (w / w), pure glutinous sorghum input amount 10 tons / batch; the remaining process steps are consistent with the pilot-scale process of Example 5, using special equipment for industrial production of baijiu: fully automatic grain soaking and steaming integrated machine, automated yeast making equipment, jacketed temperature-controlled industrial fermentation tank (stirring speed 100-150r / min, oxygen flow 1.0-1.5vvm, intermittent oxygenation), fully automatic distillation still, and online alcohol content detection system.

[0104] 2. Batch Validation: Three consecutive batches of industrial-scale production were carried out according to the above process parameters. After each batch was completed, the alcohol content, fruit and floral ester content, and defective component content of the raw wine were tested according to a unified performance validation method. At the same time, sensory quality evaluation was carried out, and the relative standard deviation (RSD) of each indicator was calculated to evaluate the batch stability of the process.

[0105] Performance verification results Table 8 Performance test results of three batches of products produced in large-scale industrial production

[0106] As shown in Table 8, after scaling up the brewing process of this invention to a 10-ton industrial-scale production, three consecutive batches of production were conducted. There were no significant differences in the alcohol yield, the increase rate of floral and fruity esters, the reduction rate of defective components, and the overall sensory score among the batches. The relative standard deviation (RSD) was ≤0.32%, indicating excellent batch stability. The alcohol yield of each batch remained stable at approximately 62.4%, the increase rates of ethyl acetate, isoamyl acetate, and phenylethyl acetate remained stable at 68.6%, 50.7%, and 97.9%, respectively, and the reduction rates of n-propanol and fusel oil remained stable at 52.0% and 11.4%, respectively. The overall sensory score was 96±1 points, highly consistent with the pilot-scale experimental results.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enhancing the floral and fruity aroma of baijiu (Chinese white liquor), characterized in that, Includes the following steps: Using pure glutinous sorghum with a branched-chain starch content of ≥70% as the sole fermentation raw material, a pure grain solid-state fermentation process without adding any lees is adopted. During the fermentation process, a fortified yeast prepared from a compound microbial agent with floral and fruity aroma is added to complete the brewing of baijiu. The amount of the fortified yeast added is 1-2% of the dry weight of the pure glutinous sorghum. The temperature of the fermentation process is controlled at 20-24℃, and the fermentation cycle is 14 days.

2. The method according to claim 1, characterized in that, The compound microbial agent is prepared by mixing solid inoculants of *Saccharomyces cerevisiae* Y87, *Saccharomyces cerevisiae* Y348, *Saccharomyces cerevisiae* Y162, and *Pichia pastoris* Y10 in a weight ratio of 2-4:2-4:1-2:1-2. The preservation numbers for *Saccharomyces cerevisiae* Y87, Y348, Y162, and Y10 are CCTCC NO: M2017048, M20241944, M2022245, and M2022245 respectively. NO: M2022527; All the above strains are deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province; among them, *Wickhamia lanceolata* Y87 was deposited on February 20, 2017, *Wickhamia lanceolata* Y348 on September 9, 2024, *Cyclocarya paliurus* Y162 on March 10, 2022, and *Pichia pastoris* Y10 on April 29, 2022.

3. The method according to claim 2, characterized in that, The preparation methods for each yeast solid inoculum are as follows: yeast cells are inoculated into YPD liquid medium and cultured on a shaker at 30℃ and 160 r / min for 24 h to obtain a primary inoculum; the primary inoculum is transferred to malt extract medium at a 10% inoculation rate and cultured on a shaker at 30℃ and 160 r / min until the viable count is ≥1×10⁻⁶. 9 The secondary inoculum was obtained by inoculating the cfu / mL concentration into bran solid culture medium at a 15% inoculum rate. After incubation at 30℃ for 2-3 days, the medium was dried at 40℃ in two stages, each stage lasting 44 hours, for a total of 88 hours. The resulting solid inoculum had a viable count ≥1×10⁻⁶. 9 cfu / g; The YPD liquid culture medium is an aqueous solution containing 2% glucose, 1% yeast extract, and 2% peptone, with a natural pH. The malt extract culture medium is prepared by mixing malt extract powder and water at a weight ratio of 1:8, with the sugar content controlled at 11-12 BX and the pH at a natural level. The bran solid culture medium is prepared by mixing bran and rice flour at a weight ratio of 10:1, adding 30-40% water, and maintaining a natural pH.

4. The method according to claim 2, characterized in that, The preparation method of the fortified yeast is as follows: the compound microbial agent and the basic yeast are mixed evenly at a weight ratio of 4-8:1-2, and food-grade silica is added at a weight of 0.1% of the total weight of the compound microbial agent and the basic yeast as an anti-caking and anti-mold agent. After mixing evenly, a light yellow powdery fortified yeast without odor or foreign matter is obtained. The basic Daqu refers to Baijiu Daqu without the addition of exogenous functional microbial agents. Its physicochemical indicators are moisture ≤5%, saccharification power ≥19g / 100g, fermentation power ≥30.0%, and sensory characteristics are uniform yellow-green mycelium on the surface of the qu, dense cross-section of the qu core, no black mold, and pure aroma.

5. The method according to claim 4, characterized in that, The physicochemical properties of the fortified yeast are: fineness ≥85%, moisture ≤10.5%, saccharification power 26-32g / 100g, and fermentation power ≥33.0%.

6. The method according to claim 1, characterized in that, The pretreatment process of the pure glutinous sorghum is as follows: soak the pure glutinous sorghum in hot water at 70℃ for 17-19 hours, and after initial steaming, steaming, and re-steaming, remove it from the steamer and cool it to 30℃±2℃. The moisture content of the cooked grain is controlled at 52-54%. The initial steaming conditions are 115℃ for 20-30 minutes, the stewing conditions are 50±1℃ for 5 minutes, and the re-steaming conditions are 110℃ for 10-20 minutes.

7. The method according to claim 1, characterized in that, Before solid-state fermentation of pure grains, a saccharification step is also included: glutinous sorghum with added fortified yeast is placed in a saccharification tank, and 5-8% of the dry weight of pure glutinous sorghum husks are evenly spread on the bottom of the saccharification tank and the surface of the glutinous sorghum. The husks are covered with canvas or filter cloth to keep them moist, and saccharification is carried out at 30°C for 24 hours to obtain saccharified mash, which is then put into a barrel for fermentation. The rice husks are sterilized at 121℃ for 30 minutes and passed through a 20-mesh sieve. After sterilization, the number of miscellaneous bacteria is reduced by more than 99%. The criteria for determining complete saccharification are: the reducing sugar content is ≥15% based on glucose or there is no blue color when iodine solution is used. Either of these conditions is sufficient to determine that saccharification is complete.

8. The method according to claim 1, characterized in that, The fermentation process uses a jacketed temperature control system to control the temperature with an accuracy of ±0.5℃. If the pH of the mash is <3.5 during fermentation, food-grade calcium carbonate is added to adjust the pH of the mash to 4.0-4.

5.

9. The method according to claim 1, characterized in that, After fermentation, the mash is distilled to obtain the base liquor of baijiu. The distillation steps are as follows: preheat the still for 5 minutes under a steam pressure of 0.1-0.15 MPa, add the bottom mash, then add the fermented mash and steam distill the liquor. Collect 100 mL of the first distillate, collect the main liquor until the alcohol content is 63±1% vol, and collect the last distillate until the alcohol content is below 6% vol. Combine the first distillate, the main liquor and the last distillate to obtain the base liquor of baijiu.

10. A type of baijiu (Chinese liquor), characterized in that, It is brewed using the method described in any one of claims 1-9.

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