Efficient baijiu brewing method based on microbial fermentation technology

Abstract

The invention discloses an efficient baijiu brewing method based on a microbial fermentation technology, and belongs to the technical field of baijiu brewing. The efficient baijiu brewing method comprises the following steps: (1) preparing chitosan-sodium alginate composite microspheres; (2) immobilizing bacillus siamensis, candida utilis and pediococcus acidilactici in the chitosan-sodium alginate composite microspheres to obtain an immobilized microbial carrier; (3) adding the immobilized microbial carrier into a white spirit fermentation pit, and controlling the fermentation temperature to be 23-30 DEG C for fermentation; (4) controlling the fermentation process in stages, including an initial stage, a middle stage and a later stage; and (5) after fermentation is completed, distilling and ageing to obtain the finished product white spirit. According to the invention, a remarkable technical breakthrough is realized on the basis of a traditional white spirit brewing process, and a new thought is provided for technical upgrading of the white spirit industry.

Description

Technical Field

[0001] The present invention relates to the technical field of liquor brewing, and particularly relates to an efficient liquor brewing method based on microbial fermentation technology. Background Art

[0002] As a representative of traditional Chinese brewing liquor, liquor has a long history and unique technological characteristics. Luzhou-flavor liquor occupies an important position in the classification of Chinese liquor with its unique aroma system and complex flavor characteristics. The brewing of traditional Luzhou-flavor liquor mainly relies on the natural fermentation of the microbial community in the cellar pit. Through various flavor substances produced by microbial metabolism, especially aromatic compounds mainly based on ethyl hexanoate, the liquor is endowed with its unique aroma and taste.

[0003] In recent years, with the improvement of consumers' requirements for liquor quality and the development of modern brewing techniques, microbial fermentation technology has been widely applied and deeply studied in the field of liquor brewing. At present, liquor microbial fermentation technology mainly focuses on three aspects: screening of functional strains, construction of microbial communities, and control of the fermentation process. By artificially regulating the microbial ecosystem to achieve precise control of flavor substance synthesis has become an important way to improve liquor quality and production efficiency.

[0004] Existing technologies such as patent CN112725114B disclose a method of "improving the microbial ecosystem in the cellar pit and increasing the content of ethyl hexanoate by adding specific strains". Although this method has improved the content of ethyl hexanoate in liquor to a certain extent, there are still two key technical problems: First, the stability of the microbial population during the fermentation process is poor and is extremely susceptible to fluctuations in environmental factors, resulting in a long brewing cycle and unstable product flavor; Second, the repair effect on aging cellar pits is limited, and it is impossible to achieve the balanced and coordinated improvement of multiple flavor substances.

[0005] Another related technology such as the commonly used solid-state fermentation method in the market can maintain the traditional flavor, but the fermentation cycle usually takes 60 - 90 days, with high energy consumption (about 4.8 kWh / L), and the microbial activity fluctuates greatly with environmental changes, resulting in difficulty in ensuring the consistency of product quality. In addition, most existing microbial addition technologies adopt the direct inoculation method, lacking a protection mechanism for microorganisms in the harsh fermentation environment, resulting in low survival rate and short active duration of the inoculated microorganisms. Summary of the Invention

[0006] The present invention discloses an efficient liquor brewing method based on microbial fermentation technology to solve the technical problems of poor fermentation stability and long fermentation cycle in the prior art.

[0007] An efficient liquor brewing method based on microbial fermentation technology disclosed by the present invention includes:

[0008] (1) Prepare chitosan-sodium alginate composite microspheres;

[0009] (2) Immobilize Bacillus siamensis, Candida utilis and Pediococcus acidilactici in the chitosan-sodium alginate composite microspheres to obtain an immobilized microbial carrier;

[0010] (3) Add the immobilized microbial carrier to a Chinese liquor fermentation pit and control the fermentation temperature within the range of 23-30 °C for fermentation;

[0011] (4) Control the fermentation process in stages, including the initial stage, the middle stage and the late stage;

[0012] (5) After fermentation is completed, carry out distillation and aging to obtain the finished Chinese liquor.

[0013] Preferably, in the microbial immobilized carrier, the mass ratio of chitosan to sodium alginate is 1:0.5-1:2.

[0014] Preferably, the chitosan-sodium alginate composite microsphere carrier further contains gelatin and maltodextrin as stabilizers, wherein the addition amount of gelatin is 2%-5%, and the addition amount of maltodextrin is 3%-8%.

[0015] Preferably, the preparation of the chitosan-sodium alginate composite microspheres in step (1) includes:

[0016] (a) Dissolve chitosan in an acidic solution with a pH value of 3.8-4.2 to obtain a chitosan solution;

[0017] (b) Dissolve sodium alginate in water to form an aqueous solution to obtain a sodium alginate solution;

[0018] (c) Mix the chitosan solution and the sodium alginate solution, stir evenly, and form composite microspheres by the ion cross-linking method;

[0019] (e) Modify the surface of the microspheres with glutamine groups to obtain chitosan-sodium alginate composite microspheres.

[0020] Preferably, in step (e), the modification of the microsphere surface with glutamine groups includes the following steps: Place the dried microspheres in a 1.5% glutaraldehyde solution, activate at 37 °C for 2 hours, then wash 3 times with sterile water, 10 minutes each time, then soak the microspheres in a 2% glutamine solution for 3 hours, and finally wash again with sterile water and store at 4 °C for standby.

[0021] Preferably, in step (2), the microbial immobilization rate is greater than 85%, and the inoculation ratio of Bacillus siamensis, Candida utilis and Pediococcus acidilactici is 2:2:1-3:2:1.

[0022] Preferably, in step (3), the addition amount of the immobilized microbial carrier is 0.5% - 2.0% of the total mass of the raw materials.

[0023] Preferably, the fermentation time in step (4) is 60 days, with the initial stage from day 0 to day 10, the middle stage from day 11 to day 30, and the later stage from day 31 to day 60.

[0024] Preferably, the pH value during the fermentation process is controlled within the range of 4.5 - 6.5.

[0025] Preferably, the deposit number of Bacillus siamensis is CCTCC AB 2022048, the deposit number of Candida utilis is CCTCC KY 2008676, and the deposit number of Pediococcus acidilactici is CCTCC HB 20082810.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] (1) The present invention adopts the chitosan - sodium alginate composite microsphere carrier immobilization technology, achieving a high comprehensive immobilization efficiency of 85.8% - 88.2%, which is significantly higher than 76.3% (the group without Pediococcus acidilactici) and 63.8% (the group with free microorganisms added) in the control group. This is because the positive charge of chitosan forms an electrostatic interaction with the negative charge on the microbial cell wall, and at the same time, the gel network provided by sodium alginate can physically entrap microbial cells, improving the immobilization efficiency of microorganisms.

[0028] (3) The present invention modifies the surface of the microspheres through glutamine groups, enhancing the affinity and protective effect of the carrier on microorganisms, enabling the microorganisms to maintain high activity in a harsh fermentation environment (pH value 4.5 - 6.5), and ensuring the stability and batch consistency of product quality.

[0029] (4) The present invention inoculates Bacillus siamensis, Candida utilis, and Pediococcus acidilactici in a ratio of 2:2:1 - 3:2:1, forming an optimal microbial structure. The three functional strains act synergistically, significantly improving the liquor fermentation process.

[0030] (5) The present invention controls the liquor fermentation cycle within 58 - 65 days, shortening it by 7.1% - 27.5% compared with the 70 - 80 days of the traditional process. Especially in Example 2, the fermentation cycle only needs 58 days, shortening it by 22 days compared with Comparative Example 3, greatly improving the production efficiency. This is attributed to the high activity and stability of the immobilized microorganisms, ensuring the continuous release of microbial activity throughout the fermentation cycle.

[0031] (6) The liquor yield of the present invention reaches 24.5% - 26.2%, which is higher than that of the control group (23.1% - 24.5%); the ethanol conversion efficiency reaches 90.5% - 93.2%, significantly higher than that of the control group (85.0% - 88.2%). Especially in Example 2, the ethanol conversion efficiency reaches 93.2%, which is 8.2 percentage points higher than that of Comparative Example 3.

[0032] (7) The process energy consumption of the present invention is reduced to 3.5 - 3.8 kWh / L, a decrease of 5.0% - 16.7% compared with that of the control group (4.0 - 4.2 kWh / L), showing significant economic and environmental benefits.

[0033] (8) The ethyl caproate content in the liquor produced by the method of the present invention is 4.180 - 4.318 g / L, 12.3% - 15.9% higher than that of the control group (3.725 - 3.812 g / L); the total amount of aromatic esters is 0.260 - 0.282 g / L, 33.3% - 56.7% higher than that of the control group (0.180 - 0.195 g / L); the content of furan compounds is 0.078 - 0.088 g / L, 4.0% - 49.2% higher than that of the control group (0.059 - 0.075 g / L). At the same time, the present invention effectively regulates the contents of ethyl lactate (0.355 - 0.390 g / L) and ethyl acetate (0.418 - 0.442 g / L) through the immobilized microorganism system, which are respectively reduced by 8.8% - 21.1% and 9.5% - 15.6% compared with the control group, forming a more harmonious flavor substance structure of the liquor.

[0034] (9) By adding immobilized functional microorganisms, the present invention can quickly reconstruct the microbial ecosystem in the cellar pit, achieve the balanced and synergistic improvement of various flavor substances, and provide a new technical solution for the repair and flavor improvement of aged cellar pits.

[0035] In summary, based on the chitosan - sodium alginate composite microsphere carrier immobilization technology and the synergistic effect of three functional microorganisms, the present invention shows significant advantages in aspects such as microbial immobilization efficiency, activity maintenance, liquor flavor substance content, and brewing efficiency, providing an innovative solution for the technological upgrading and sustainable development of the liquor industry. Detailed Embodiments

[0036] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] In the embodiments of the present application, the modification of the surface of the microspheres with glutamine groups includes the following steps: placing the dried microspheres in a 1.5% glutaraldehyde solution, activating at 37°C for 2 hours, then washing 3 times with sterile water, 10 minutes each time, then soaking the microspheres in a 2% glutamine solution for 3 hours, and finally washing again with sterile water and storing for standby at 4°C.

[0038] Example 1

[0039] Take 6.0 g of chitosan and dissolve it in 100 mL of 2% acetic acid solution, stir at room temperature for 4 hours until completely dissolved. Take 3.0 g of sodium alginate and dissolve it in 100 mL of deionized water, stir until a transparent solution is obtained. Mix the two solutions in a volume ratio of 1:1 and stir at 50°C for 2 hours to obtain a chitosan-sodium alginate mixed solution.

[0040] Take 50 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810, centrifuge to collect the bacterial cells, and wash twice with phosphate buffer (pH 7.0). Mix the mixed bacterial solution with the above polymer solution in a volume ratio of 1:10, homogenize and stir for 30 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 2% calcium chloride, and slowly stir and cross-link at 25°C for 4 hours to form microspheres with a diameter of 200 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres 3 times with deionized water, and then vacuum dry at 25°C for 12 hours until constant weight to obtain a microbial immobilization carrier.

[0041] Add the prepared microbial immobilization carrier to the traditional Chinese liquor fermentation pit at 3.5% of the material weight, control the temperature at 28°C in the initial stage of fermentation (1 - 20 days), 25°C in the middle stage (21 - 40 days), and 23°C in the later stage (41 - 60 days), and the total fermentation period is 60 days. No additional microbial agents need to be added during the whole fermentation process. After fermentation is completed, distillation and aging are carried out according to the conventional process to obtain a finished Chinese liquor with harmonious flavor and prominent ester aroma. The liquor yield reaches 25.8%, and the energy consumption is 3.6 kWh / L.

[0042] Example 2

[0043] Take 7.0 g of chitosan and dissolve it in 100 mL of 2.5% acetic acid solution, stir at room temperature for 4.5 hours until completely dissolved. Take 3.5 g of sodium alginate and dissolve it in 100 mL of deionized water, stir until a transparent solution is obtained. Mix the two solutions in a volume ratio of 1.2:1 and stir at 55°C for 2.5 hours to obtain a chitosan-sodium alginate mixed solution.

[0044] Take 60 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810, centrifuge to collect the bacterial cells, and wash them twice with phosphate buffer (pH 7.2). Mix the mixed bacterial solution with the above-mentioned polymer solution at a volume ratio of 1:9, homogenize and stir for 35 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 2.2% calcium chloride, and slowly stir and cross-link at 26 °C for 4.5 hours to form microspheres with a diameter of 180 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres 4 times with deionized water, and then vacuum dry at 26 °C for 13 hours until constant weight to obtain a microbial immobilization carrier.

[0045] Add the prepared microbial immobilization carrier to the traditional Chinese liquor fermentation pit at 4.0% of the material weight, control the temperature at 29 °C in the initial stage of fermentation (1 - 22 days), 26 °C in the middle stage (23 - 45 days), and 24 °C in the later stage (46 - 58 days). The total fermentation period is 58 days. No additional microbial agents need to be added during the whole fermentation process. After fermentation, distillation and aging are carried out according to the conventional process to obtain a finished Chinese liquor with coordinated flavor and prominent ester aroma. The liquor yield reaches 26.2%, and the energy consumption is 3.5 kWh / L.

[0046] Example 3

[0047] Take 5.0 g of chitosan and dissolve it in 100 mL of 1.8% acetic acid solution, and stir at room temperature for 3.5 hours until completely dissolved. Dissolve 2.5 g of sodium alginate in 100 mL of deionized water and stir until a transparent solution is obtained. Mix the two solutions at a volume ratio of 0.9:1 and stir at 45 °C for 1.8 hours to obtain a chitosan-sodium alginate mixed solution.

[0048] Take 45 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810, centrifuge to collect the bacterial cells, and wash them twice with phosphate buffer (pH 6.8). Mix the mixed bacterial solution with the above-mentioned polymer solution at a volume ratio of 1:12, homogenize and stir for 25 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 1.8% calcium chloride, and slowly stir and cross-link at 24 °C for 3.5 hours to form microspheres with a diameter of 220 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres 3 times with deionized water, and then vacuum dry at 24 °C for 10 hours until constant weight to obtain a microbial immobilization carrier.

[0049] Add the prepared microbial immobilization carrier to the traditional Chinese liquor fermentation pit at 3.0% of the material weight. Control the temperature at 27 °C in the initial stage of fermentation (1 - 18 days), 24 °C in the middle stage (19 - 38 days), and 23 °C in the later stage (39 - 62 days). The total fermentation period is 62 days. No additional microbial agents need to be added during the whole fermentation process. After fermentation is completed, distillation and aging are carried out according to the conventional process to obtain a finished Chinese liquor with harmonious flavor and prominent ester aroma. The liquor yield reaches 25.0%, and the energy consumption is 3.7 kWh / L.

[0050] Example 4

[0051] Dissolve 6.5 g of chitosan in 100 mL of 2.2% acetic acid solution and stir at room temperature for 4.2 hours until completely dissolved. Dissolve 3.2 g of sodium alginate in 100 mL of deionized water and stir until a transparent solution is obtained. Mix the two solutions according to a volume ratio of 1.1:1 and stir at 52 °C for 2.2 hours to obtain a chitosan-sodium alginate mixed solution.

[0052] Take 55 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810, centrifuge to collect the bacterial cells, and wash them twice with phosphate buffer (pH 7.1). Mix the mixed bacterial solution with the above polymer solution according to a volume ratio of 1:11, homogenize and stir for 32 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 2.1% calcium chloride, and slowly stir and cross-link at 25 °C for 4.2 hours to form microspheres with a diameter of 190 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres 3 times with deionized water, and then vacuum dry at 25 °C for 11 hours until constant weight to obtain the microbial immobilization carrier.

[0053] Add the prepared microbial immobilization carrier to the traditional Chinese liquor fermentation pit at 3.8% of the material weight. Control the temperature at 28 °C in the initial stage of fermentation (1 - 21 days), 26 °C in the middle stage (22 - 42 days), and 24 °C in the later stage (43 - 59 days). The total fermentation period is 59 days. No additional microbial agents need to be added during the whole fermentation process. After fermentation is completed, distillation and aging are carried out according to the conventional process to obtain a finished Chinese liquor with harmonious flavor and prominent ester aroma. The liquor yield reaches 25.5%, and the energy consumption is 3.6 kWh / L.

[0054] Example 5

[0055] Dissolve 4.5 g of chitosan in 100 mL of 1.5% acetic acid solution and stir at room temperature for 3 hours until completely dissolved. Dissolve 2.2 g of sodium alginate in 100 mL of deionized water and stir until a transparent solution is obtained. Mix the two solutions according to a volume ratio of 0.8:1 and stir at 40 °C for 1.5 hours to obtain a chitosan-sodium alginate mixed solution.

[0056] Take 40 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810, centrifuge to collect the bacterial cells, and wash them twice with phosphate buffer (pH 6.5). Mix the mixed bacterial solution with the above-mentioned polymer solution at a volume ratio of 1:13, homogenize and stir for 20 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 1.5% calcium chloride, and slowly stir and cross-link at 23 °C for 3 hours to form microspheres with a diameter of 230 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres twice with deionized water, and then vacuum dry at 23 °C for 9 hours until constant weight to obtain a microbial immobilization carrier.

[0057] Add the prepared microbial immobilization carrier to the traditional Chinese liquor fermentation pit at 2.8% of the weight of the material, control the temperature at 26 °C in the initial stage of fermentation (1 - 17 days), 24 °C in the middle stage (18 - 36 days), and 22 °C in the later stage (37 - 65 days). The total fermentation period is 65 days. No additional microbial agents need to be added during the whole fermentation process. After fermentation is completed, distillation and aging are carried out according to the conventional process to obtain a finished Chinese liquor with harmonious flavor and prominent ester aroma. The liquor yield reaches 24.5%, and the energy consumption is 3.8 kWh / L.

[0058] Comparative part

[0059] Comparative Example 1: Comparative process without using Pediococcus acidilactici

[0060] Take 6.0 g of chitosan and dissolve it in 100 mL of 2% acetic acid solution, and stir at room temperature for 4 hours until completely dissolved. Dissolve 3.0 g of sodium alginate in 100 mL of deionized water and stir until a transparent solution is obtained. Mix the two solutions at a volume ratio of 1:1 and stir at 50 °C for 2 hours to obtain a chitosan-sodium alginate mixed solution.

[0061] Take 50 mL of each of the bacterial solutions of Bacillus siamensis CCTCC AB 2022048 and Candida utilis CCTCC KY 2008676, centrifuge to collect the bacterial cells, and wash them twice with phosphate buffer (pH 7.0), but do not add Pediococcus acidilactici. Mix the mixed bacterial solution with the above-mentioned polymer solution at a volume ratio of 1:10, homogenize and stir for 30 minutes, then use a microfluidic device to drop the mixed solution into a cross-linking solution containing 2% calcium chloride, and slowly stir and cross-link at 25 °C for 4 hours to form microspheres with a diameter of 200 μm. Modify the surface of the microspheres with glutamine groups, wash the microspheres three times with deionized water, and then vacuum dry at 25 °C for 12 hours until constant weight to obtain a microbial immobilization carrier.

[0062] The prepared microbial immobilization carrier was added to the traditional Chinese liquor fermentation pit at 3.5% of the material weight. The temperature was controlled at 28 °C in the initial stage of fermentation (1 - 20 days), 25 °C in the middle stage (21 - 40 days), and 23 °C in the later stage (41 - 70 days). The total fermentation cycle was extended to 70 days. No additional microbial agents were added during the whole fermentation process. After fermentation, distillation and aging were carried out according to the conventional process to obtain the finished Chinese liquor, with a liquor yield of only 24.5% and an energy consumption of 4.0 kWh / L.

[0063] Due to the lack of Pediococcus acidilactici participating in the fermentation, the ethyl lactate content in the Chinese liquor increased to 0.41 g / L, and the total amount of aromatic esters was only 0.185 g / L, which was 32.7% lower than that in Example 1, and the flavor coordination was poor.

[0064] Comparative Example 2: A comparative process using the free microorganism addition method

[0065] Bacillus siamensis CCTCC AB 2022048, Candida utilis CCTCC KY 2008676, and Pediococcus acidilactici CCTCC HB 20082810 were respectively cultured to the logarithmic growth phase, and the cells were collected by centrifugation and washed twice with phosphate buffer (pH 7.0) to prepare a high-concentration bacterial suspension. After mixing the three bacterial suspensions in an equal volume ratio, they were directly added to the traditional Chinese liquor fermentation pit, and the addition amount was 4% of the material weight. The temperature was controlled at 28 °C in the initial stage of fermentation (1 - 20 days), 25 °C in the middle stage (21 - 40 days), and 23 °C in the later stage (41 - 75 days). The total fermentation cycle was extended to 75 days. During the whole fermentation process, the mixed bacterial liquid with the same ratio was supplemented every 15 days to maintain the fermentation activity. After fermentation, distillation and aging were carried out according to the conventional process to obtain the finished Chinese liquor, with a liquor yield of 23.5% and an energy consumption of 4.1 kWh / L.

[0066] Since the microorganisms were not immobilized, their stability in the fermentation environment was poor, resulting in unstable contents of flavor substances in the Chinese liquor. The ethyl caproate content decreased to 3.725 g / L, which was 13.1% lower than that in Example 1; the total amount of aromatic esters was only 0.195 g / L, which was 29.1% lower than that in Example 1.

[0067] Comparative Example 3: A comparative process using the traditional Chinese liquor brewing process without adding microorganisms

[0068] Using the conventional Baijiu brewing process, no microbial agents of any form are added. The traditional Baijiu raw materials are saccharified and fermented according to the conventional ratio. The entire process relies entirely on the original microbial community in the cellar for fermentation. The temperature is controlled at 27°C in the initial stage of fermentation (1 - 25 days), 25°C in the middle stage (26 - 50 days), and 23°C in the later stage (51 - 80 days). The total fermentation period is as long as 80 days. After fermentation is completed, distillation and aging are carried out according to the conventional process to obtain the finished Baijiu. The liquor yield is only 23.1%, and the energy consumption reaches 4.2 kWh / L.

[0069] Due to the lack of directional microbial enhancement, the formation of flavor substances in Baijiu is insufficient. The content of ethyl hexanoate is only 3.725 g / L; the total amount of aromatic esters is only 0.180 g / L, which is 34.5% lower than that in Example 1; the content of furan compounds is only 0.059 g / L, which is 30.6% lower than that in Example 1, and the flavor layering is insufficient.

[0070] Performance testing

[0071] 1. Determination of microbial immobilization efficiency

[0072] Testing principle: The indirect counting method is adopted, and the immobilization efficiency is calculated by measuring the reduction of the number of microbial cells in the culture medium before and after immobilization.

[0073] Testing steps: First, prepare a homogeneous suspension. Accurately measure 1 mL of the microbial mixed bacterial solution used in the immobilization process and dilute it to an appropriate concentration with sterile physiological saline. Use the plate colony counting method to culture on a specific medium (Bacillus siamensis uses LB agar medium and is cultured at 30°C for 48 hours; Candida utilis uses YPD medium and is cultured at 25°C for 72 hours; Pediococcus acidilactici uses MRS medium and is cultured at 37°C for 24 hours), record it as the initial number of bacteria N0. Take 1 mL of the supernatant of the reaction solution after the immobilization reaction ends, dilute and count it using the same method, and record it as the number of non-immobilized bacteria N1.

[0074] The formula for calculating the immobilization efficiency is: η = (N0 - N1) / N0 × 100%. Each sample is measured in parallel 3 times, and the average value is taken to calculate the standard deviation.

[0075] 2. Determination of microbial activity

[0076] Testing principle: By simulating different environmental conditions, the enzyme activity of the immobilized microorganisms is measured to reflect the maintenance of microbial activity.

[0077] Test procedure: Accurately weigh 0.5 g of the microbial immobilization carrier sample and place it in 50 mL of buffer solution (phosphate buffer with different pH values, ranging from 4.0 to 9.0, at intervals of 0.5). Incubate with constant shaking (120 rpm) for 1 hour under different temperature conditions (20 - 50 °C, at intervals of 5 °C).

[0078] Measure the amylase activity of Bacillus siamensis, the alcohol dehydrogenase activity of Candida utilis, and the lactate dehydrogenase activity of Pediococcus acidilactici, respectively.

[0079] The enzyme activity is measured by spectrophotometry. Taking the initial enzyme activity as 100%, calculate the relative activity under different conditions. Parallel measurements are carried out 3 times under each condition, and the average value is taken to calculate the standard deviation.

[0080] 3. Carrier structure and stability test

[0081] Test procedure: Prepare the microbial immobilization carrier sample into a specimen suitable for SEM observation, perform gold sputtering treatment using a gold sputtering instrument, and observe the surface morphology of the carrier under a FEI Quanta 450 FEG scanning electron microscope at an accelerating voltage of 10 kV and a magnification of 500 - 10000×. Use a Malvern Mastersizer 3000 laser particle size analyzer to measure the particle size distribution of the microspheres, with anhydrous ethanol as the dispersion medium. Use a NETZSCH STA 449F3 thermogravimetric analyzer to analyze the thermal stability of the carrier, with a heating rate of 10 °C / min and a temperature range of 25 - 600 °C under N2 atmosphere.

[0082] Carrier immersion stability test: Take 0.5 g of the carrier sample and place it in 10 mL of buffer solution with different pH values (2.0, 4.0, 6.0, 7.0, 8.0, 10.0) respectively. Incubate with constant shaking at 37 °C for 48 hours. After filtration, washing, and drying, weigh it and calculate the mass loss rate.

[0083] 4. Baijiu flavor substance test

[0084] Test method: Adopt the headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology to qualitatively and quantitatively analyze the key flavor substances in Baijiu.

[0085] Test procedure: Take 5 mL of the white liquor sample and place it in a 20 mL headspace vial. Add 1.5 g of NaCl and 50 μL of the internal standard (isoamyl acetate-d3, concentration 20 mg / L). Use a DVB / CAR / PDMS composite fiber to extract for 30 minutes at 40 °C. The extracted fiber is directly inserted into the gas chromatography injection port for thermal desorption, with a desorption temperature of 250 °C and a time of 5 minutes. Gas chromatography conditions: HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 μm); carrier gas: high-purity helium (99.999%), flow rate 1.0 mL / min; temperature programming: initial temperature 40 °C held for 3 minutes, heated to 80 °C at 5 °C / min, then heated to 230 °C at 10 °C / min and held for 5 minutes. Mass spectrometry conditions: EI source, 70 eV; ion source temperature 230 °C; interface temperature 250 °C; mass scan range m / z 35 - 350; quantitative analysis of target compounds is carried out using the selected ion monitoring (SIM) mode.

[0086] Quantitatively calculate the content of each flavor substance by the standard curve method. Each sample is measured in parallel 3 times, and the average value is taken to calculate the standard deviation.

[0087] 5. Fermentation efficiency test

[0088] Test method: Evaluate the efficiency index of the white liquor brewing process by monitoring the key parameters during fermentation.

[0089] Test procedure: Record the temperature change curve during fermentation, and use a portable gas chromatograph (Agilent 490 Micro GC) to detect the change in ethanol content in the fermented grains every 5 days;

[0090] Calculate the total fermentation cycle (the time from putting into the cellar to taking out of the cellar);

[0091] Calculation of liquor yield: Liquor yield = volume of raw liquor obtained / mass of raw materials input × 100%; Calculation of energy consumption: Record the electricity, steam and fuel consumption during the entire production process and convert it into standard energy consumption (kWh / L).

[0092] Detection data

[0093] Table 1. Microbial immobilization efficiency table

[0094]

[0095]

[0096] Table 2. Activity maintenance table of microorganisms after 6 months of storage at 4 °C

[0097]

[0098] Table 3. White liquor flavor substance content table

[0099]

[0100]

[0101] Table 4. Baijiu brewing efficiency table

[0102] Sample number Fermentation cycle (days) Alcohol yield Energy consumption (kWh / L) Ethanol conversion efficiency Example 1 60 25.8％ 3.6 92.5％ Example 2 58 26.2％ 3.5 93.2％ Example 3 62 25％ 3.7 91.8％ Example 4 59 25.5％ 3.6 92.8％ Example 5 65 24.5％ 3.8 90.5％ Comparative example 1 70 24.5％ 4 88.2％ Comparative example 2 75 23.5％ 4.1 86.5％ Comparative example 3 80 23.1％ 4.2 85％

[0103] Data analysis

[0104] As can be seen from Table 1, the immobilized microorganism technology adopted in the present invention has obvious advantages in terms of microorganism immobilization efficiency. The comprehensive immobilization efficiencies of Examples 1-5 are 87.5%, 88.2%, 85.8%, 87.0% and 84.5% respectively, all of which are significantly higher than those of the comparative examples. Among them, in Comparative Example 1, due to the lack of Pediococcus acidilactici, the comprehensive immobilization efficiency decreased to 76.3%, which is 11.2 percentage points lower than that of Example 1; in Comparative Example 2, the free microorganism addition method was adopted, and the immobilization efficiency was only 63.8%, which is 23.7 percentage points lower than that of Example 1; in Comparative Example 3, no microorganism was added, and the immobilization efficiency was 0. Among various microorganism species, Bacillus siamensis is relatively easy to be immobilized, and its highest immobilization efficiency can reach 89.4% (Example 2); the immobilization efficiency of Candida utilis is relatively low, but it can still reach 86.5% under the optimized conditions of the present invention (Example 2). It can be seen that the three-dimensional network structure formed by the chitosan-sodium alginate composite material provides good immobilization effect on microorganisms. This is because the positive charge of chitosan can form electrostatic interaction with the negative charge on the cell wall of microorganisms, and at the same time, the gel network provided by sodium alginate can physically entrap microbial cells, improving the immobilization efficiency of microorganisms.

[0105] As can be seen from Table 2, the microorganisms immobilized by the method of the present invention still maintain a high activity after being stored at low temperature for 6 months. The average activity maintenance rates of Examples 1-5 are 85.2%, 86.1%, 83.6%, 85.5% and 82.8% respectively, all significantly higher than those of the comparative examples. In Comparative Example 1, due to the lack of Pediococcus acidilactici, the problem of lactic acid metabolic balance could not be solved, and the average activity maintenance rate dropped to 72.0%, 13.2 percentage points lower than that of Example 1; in Comparative Example 2, since the free microorganisms lacked a protective layer, the activity maintenance rate was only 58.0%, 27.2 percentage points lower than that of Example 1; Comparative Example 3 was a traditional process and did not use the immobilized microorganism technology, so the activity maintenance rate was 0. Among the three microorganisms, Bacillus siamensis had the highest activity maintenance rate, which could reach 88.3% in Example 2, related to its characteristic of forming spores and being able to maintain a high survival rate in adverse environments. The chitosan-sodium alginate composite microspheres provided a stable microenvironment for the microorganisms, protected the microorganisms from the influence of external environmental changes such as temperature fluctuations, pH changes and mechanical stress, and at the same time had good permeability to ensure the transfer of nutrients and metabolites, which was the key factor for the high activity maintenance rate.

[0106] As can be seen from Table 3, the Baijiu produced by the method of the present invention has obvious advantages in terms of the content of flavor substances. The ethyl caproate content in Examples 1-5 is 4.180-4.318 g / L, 12.3% - 15.9% higher than that of Comparative Examples 1-3 which is 3.725-3.812 g / L; the total amount of aromatic esters is 0.260-0.282 g / L, 33.3% - 56.7% higher than that of Comparative Examples 1-3 which is 0.180-0.195 g / L; the content of furan compounds is 0.078-0.088 g / L, 4.0% - 49.2% higher than that of Comparative Examples 1-3 which is 0.059-0.075 g / L. It is worth noting that the ethyl lactate content in the examples is 0.355-0.390 g / L, significantly lower than that of Comparative Examples 1-3 which is 0.410-0.450 g / L, a decrease of 8.8% - 21.1%; the ethyl acetate content is 0.418-0.442 g / L, also lower than that of Comparative Examples 1-3 which is 0.470-0.495 g / L, a decrease of 9.5% - 15.6%. This shows that the method of the present invention regulates the formation of ethyl lactate and ethyl acetate through the immobilized microorganism system, controls their contents within a reasonable range, and at the same time significantly increases the contents of ethyl caproate and aromatic esters, forming a more coordinated flavor substance structure of Baijiu. Among them, the immobilization of Pediococcus acidilactici plays a key role in controlling the content of ethyl lactate, which explains the phenomenon of the increase in the content of ethyl lactate in Comparative Example 1 due to the lack of Pediococcus acidilactici.

[0107] As can be seen from Table 4, the present invention also shows significant advantages in the efficiency of liquor brewing. The fermentation periods of Examples 1-5 were 58-65 days, which were shortened by 7.1% to 27.5% compared with 70-80 days of Comparative Examples 1-3; the liquor yield reached 24.5% to 26.2%, an increase of 0% to 13.4% compared with 23.1% to 24.5% of Comparative Examples 1-3; the energy consumption decreased to 3.5-3.8 kWh / L, a decrease of 5.0% to 16.7% compared with 4.0-4.2 kWh / L of Comparative Examples 1-3. In terms of ethanol conversion efficiency, Examples 1-5 reached 90.5% to 93.2%, which was significantly higher than 85.0% to 88.2% of Comparative Examples 1-3. These data indicate that the microbial immobilization technology of the present invention not only improves the flavor quality of liquor, but also significantly improves production efficiency, shortens the fermentation period, increases the liquor yield and conversion efficiency, and reduces energy consumption, having significant economic and environmental benefits. The long-term stable release of immobilized microorganisms ensures the continuous presence of highly active functional microorganisms during the fermentation process, accelerates the saccharification of starch and the formation of ethanol, and promotes the formation of flavor substances, thereby improving the brewing efficiency.

[0108] In summary, the microbial immobilization system prepared by the present invention based on the chitosan-sodium alginate composite microsphere carrier shows significant advantages in terms of immobilization efficiency, maintenance of microbial activity, content of flavor substances in liquor, and brewing efficiency through the synergistic action of three functional microorganisms, namely Bacillus siamensis, Candida utilis, and Pediococcus acidilactici. This technology not only optimizes the flavor characteristics of liquor, but also significantly shortens the production cycle and improves production efficiency, providing new ideas and technical support for the technological upgrading and sustainable development of the liquor industry.

[0109] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. An efficient liquor brewing method based on microbial fermentation technology, characterized in that: include: (1) Preparation of chitosan-sodium alginate composite microspheres; (2) immobilizing Bacillus siamese, Candida utilis and Pediococcus acidilactici in the chitosan-sodium alginate composite microspheres to obtain an immobilized microbial carrier; (3) adding the immobilized microbial carrier to the liquor fermentation cellar, and controlling the fermentation temperature to be within the range of 23 to 30° C.; (4) Control the fermentation process in stages, including the initial stage, the middle stage, and the late stage; (5) After fermentation is complete, distillation and aging are carried out to obtain the finished liquor.

2. The method according to claim 1, characterized in that In the microorganism immobilization carrier, the mass ratio of chitosan to sodium alginate is 1:0.5 to 1:

2.

3. The method according to claim 1, characterized in that The chitosan-sodium alginate composite microsphere carrier also contains gelatin and maltodextrin as stabilizers, wherein the added amount of gelatin is 2% to 5%, and the added amount of maltodextrin is 3% to 8%.

4. The method according to claim 1, characterized in that: The preparation of chitosan-sodium alginate composite microspheres in step (1) comprises: (a) dissolving chitosan in an acidic solution having a pH value of 3.8 to 4.2 to obtain a chitosan solution; (b) dissolving sodium alginate in water to form an aqueous solution to obtain a sodium alginate solution; (c) mixing the chitosan solution and the sodium alginate solution, stirring them evenly, and forming composite microspheres by an ionic crosslinking method; (e) The surface of the microspheres is modified with glutamine groups to obtain chitosan-sodium alginate composite microspheres.

5. The method according to claim 4, characterized in that In step (e), the modification of the microsphere surface with glutamine groups comprises the following steps: placing the dried microspheres in a 1.5% glutaraldehyde solution, activating them at 37°C for 2 hours, then washing them with sterile water for 3 times, each time for 10 minutes, then immersing the microspheres in a 2% glutamine solution for 3 hours, and finally washing them again with sterile water and storing them at 4°C for future use.

6. The method according to claim 1, characterized in that In step (2), the microbial fixation rate is greater than 85%, and the inoculation ratio of Bacillus siamese, Candida utilis and Pediococcus acidilactici is 1:1:

1.

7. The method according to claim 1, characterized in that In step (3), the amount of the immobilized microorganism carrier added is 2.8% to 4.0% of the total mass of the raw material.

8. The method according to claim 1, characterized in that The fermentation time in step (4) is 55 to 60 days.

9. The method according to claim 1, characterized in that: The pH value during the fermentation process is controlled within the range of 4.5 to 6.

5.

10. The method according to claim 1, characterized in that The accession number of the siam bacillus is CCTCCAB2022048, the accession number of the utilis candida is CCTCC KY 2008676, and the accession number of the Pediococcus acidilactici is CCTCC HB 20082810.

Images