Method for prolonging fresh-keeping period and improving taste of craft beer by using egg white lysozyme

Abstract

The invention relates to the technical field of beer brewing, and discloses a method for prolonging the refreshing time and improving the taste of craft beer by using egg white lysozyme, and the method comprises the following steps: carrying out wort preparation and main fermentation; adding an egg white lysozyme solution prepared by purification into the wine liquid in a stage from the end of main fermentation to the front of filling, and controlling the final concentration to be 15-90mg / L; the wine liquid is subjected to cold storage and after-ripening under the condition of 0-5 DEG C, lysozyme is used for cracking gram-positive putrefying bacteria, and biological preservation is completed under the non-thermal sterilization condition; and filtering and filling after cold storage is finished. According to the method, the specific bacteriostatic action of the high-activity lysozyme is utilized, thermal sterilization and chemical preservatives are replaced by a biological enzyme method, infectious microbes are effectively inhibited, the quality guarantee period is prolonged, meanwhile, damage of high temperature to flavor substances is avoided, the fresh taste and pure flavor of the craft beer are reserved, and the quality guarantee period of the craft beer is prolonged. The problem that fresh keeping and flavor of craft beer are difficult to consider at the same time is effectively solved.

Description

Technical Field

[0001] This invention relates to the field of beer brewing technology, specifically to a method for improving the shelf life and taste of craft beer using egg white lysozyme. Background Technology

[0002] Currently, the craft beer industry faces a dilemma between maintaining product freshness and controlling biostability. Craft beer is rich in amino acids, carbohydrates, and vitamins, a nutrient-rich matrix that easily becomes a breeding ground for spoilage microorganisms, especially Gram-positive bacteria such as lactic acid bacteria and Pediococcus. Once contamination occurs, these bacteria metabolize substances such as diacetyl and lactic acid, leading to cloudy, sour, and off-flavored beer. To extend shelf life, high-temperature pasteurization or the addition of chemical preservatives such as potassium sorbate are commonly used in production. However, heat treatment inevitably accelerates the oxidation and volatilization of flavor compounds, damaging the fresh taste characteristics of the beer; while the use of chemical preservatives runs counter to the current consumer market's pursuit of clean labels and natural, additive-free products.

[0003] For biological preservation techniques, egg white lysozyme has a relatively mature application system in winemaking. As a glycoside hydrolase, egg white lysozyme can specifically act on the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of bacterial cell walls. In winemaking, by adding purified lysozyme, winemakers can precisely control or delay the malolactic fermentation process. This technology utilizes the substrate specificity of the enzyme to directionally lyse and inhibit the growth of lactic acid bacteria in the wine without interfering with yeast alcoholic fermentation, thereby achieving regulation of the fermentation rhythm and management of microbial risks.

[0004] However, the successful experience of the wine system cannot be directly replicated in the complex matrix of craft beer. The beer environment contains unique hop resins and has high requirements for foam stability, which is drastically different from the high-tannin, foam-free system of wine. Lysozyme, as a positively charged protein, readily binds non-specifically to negatively charged foam proteins or polyphenols in beer. This binding not only leads to the loss of enzyme activity but also risks causing non-biological turbidity or damaging foam persistence—a factor not considered in wine applications. Furthermore, the environmental tolerance mechanisms of beer spoilage bacteria differ from those of wine strains. Existing brewing processes lack research on the kinetic parameters of lysozyme during low-temperature cold storage. Simply applying existing technologies makes it difficult to determine the effective enzyme concentration and timing of addition without damaging flavor compounds. The industry urgently needs to establish a biopreservation solution adapted to the beer wort system that balances antibacterial efficacy with sensory quality protection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for improving the shelf life and taste of craft beer using egg white lysozyme. This method solves the problem that existing craft beer preservation technologies struggle to effectively inhibit spoilage bacteria and extend shelf life while avoiding heat damage and flavor degradation caused by chemical additives, thus balancing biological stability and fresh taste.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for improving the shelf life and taste of craft beer using egg white lysozyme, comprising the following steps: The wort is prepared according to the craft beer brewing process, brewing yeast is added, and primary fermentation is carried out under controlled temperature. After the main fermentation is completed and before bottling, egg white lysozyme solution is added to the fermentation liquid or sake liquid to control the final concentration of egg white lysozyme in the beer system to be 15-90 mg / L. The wine with added egg white lysozyme is cold-stored and matured at 0-5℃. The egg white lysozyme is used to lyse the cell walls of Gram-positive spoilage bacteria at low temperature, thus completing biological preservation without heat sterilization. After the cold storage and aging process is completed, the wine is filtered and bottled.

[0007] By adopting the above technical solution, and using egg white lysozyme to replace traditional heat sterilization and chemical preservatives, combined with low-temperature cold storage technology, a dual effect of biological preservation and flavor retention is achieved. Its mechanism of action and effects are as follows: First, the bio-enzymatic cell wall disruption and sterilization mechanism. Egg white lysozyme is a hydrolytic enzyme that specifically targets the cell walls of microorganisms. In the beer fermentation and storage environment, the main spoilage bacteria are Gram-positive bacteria such as lactic acid bacteria. Egg white lysozyme specifically hydrolyzes the β-1,4-glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the peptidoglycan backbone of bacterial cell walls. This hydrolysis disrupts the integrity of the cell wall, causing the bacterial cells to absorb water, swell, and eventually lyse under the osmotic pressure of the beer liquid, leading to their death. Since brewer's yeast is a fungus, its cell wall is mainly composed of glucan and mannan, and does not contain peptidoglycan structures. Therefore, lysozyme has no inhibitory effect on brewer's yeast and does not affect normal fermentation or post-ripening metabolism.

[0008] Secondly, the flavor preservation mechanism is achieved without heat treatment. Conventional pasteurization is usually carried out at temperatures above 60°C. High temperatures accelerate the oxidation and volatilization of alcohols, esters, and hop terpenes in beer, and induce Maillard reactions to produce aging precursors such as furfural, resulting in a cooked flavor. This invention utilizes the characteristic of lysozyme, which retains enzymatic activity at low temperatures of 0-5°C, combined with the necessary cold storage maturation period in beer production, to continuously kill potential contaminants through prolonged low-temperature contact. The entire process avoids heat shock, thus fully preserving the hop aroma and malt flavor.

[0009] Third, the mechanism for enhancing the purity of the taste. Compared to chemical preservatives such as potassium sorbate, egg white lysozyme, as a natural protein, does not alter the pH buffering system of beer or introduce metallic ion astringency or chemical off-flavors when added at a concentration of 15-90 mg / L. Furthermore, lysozyme has high substrate specificity and does not non-specifically bind to polyphenols, bitter acids, or other flavor compounds in beer, thus ensuring the purity of the beer.

[0010] Preferably, the timing for adding egg white lysozyme solution to the fermentation broth or sake broth is any one of the following: when the primary fermentation is completed, diacetyl reduction is finished, and the brewing yeast begins to settle, it is added to the fermentation tank; during the process of transporting the fermentation broth from the fermentation tank to the cold storage tank, it is added online; after the fermentation broth enters the cold storage tank and before the start of the cold storage period, it is added to the cold storage tank.

[0011] By adopting the above technical solution, the enzyme addition point is controlled after the main fermentation is completed. On the one hand, this avoids the enzyme liquid being carried away by the violent gas degassing during the main fermentation or the resulting foam causing enzyme protein denaturation. On the other hand, at this time, the yeast has settled and the wine is relatively clear, which is conducive to the even dispersion of lysozyme and contact with trace amounts of contaminating bacteria, thereby improving the antibacterial efficiency.

[0012] Preferably, the method for preparing the egg white lysozyme solution is as follows: select egg white lysozyme with an enzyme activity of 16100-19000 U / mg, dissolve or dilute it with sterile water or sterile beer to be treated, and prepare a homogeneous egg white lysozyme solution.

[0013] By adopting the above technical solution, the required antibacterial efficacy can be achieved at a lower mass concentration using highly active lysozyme preparations, reducing the total amount of exogenous protein introduced and lowering the risk of causing non-biological turbidity in beer.

[0014] Preferably, the filtration uses diatomaceous earth filtration, plate and frame filtration, or cross-flow membrane filtration to trap bacterial fragments and turbid proteins; the method of using egg white lysozyme to improve the shelf life and taste of craft beer is kept in a non-heat-treated state throughout the post-processing and bottling process, thus preserving the volatile flavor compounds of the beer.

[0015] By adopting the above technical solution, physical filtration not only removes suspended yeast and condensed solids, but also removes bacterial cell fragments broken down by lysozyme, further clarifying the wine and improving its sensory stability.

[0016] Preferably, the egg white lysozyme is prepared using an integrated ion exchange and ultrafiltration method. The preparation process of the egg white lysozyme includes: taking fresh egg white, diluting it with water and filtering it to obtain a pretreated solution; mixing the pretreated solution with a carboxymethyl cellulose cation exchange resin for adsorption; washing the carboxymethyl cellulose cation exchange resin after adsorption; eluting it with ammonium sulfate solution and collecting the eluent; pumping the eluent into an ultrafiltration system equipped with a membrane module with a molecular weight cutoff of 10,000 Daltons for desalting and concentration; and drying it to obtain the egg white lysozyme.

[0017] By adopting the above technical solution, the characteristic that egg white lysozyme carries a positive charge at near-neutral pH (isoelectric point pI of about 10.7), while other major egg white proteins (such as ovalbumin and ovomucoid) carry a negative charge, is specifically adsorbed by carboxymethyl cellulose cation exchange resin.

[0018] The specific separation process is as follows: Electrostatic adsorption: Under pH 6.0-7.0 conditions, positively charged lysozyme binds to negatively charged resin groups, causing impurities and proteins to flow out. Ion exchange elution: A high concentration of ammonium sulfate solution provides competitive cations, which displace and elute lysozyme from the resin; Ultrafiltration purification: Taking advantage of the fact that lysozyme's molecular weight (approximately 14.3 kDa) is greater than that of inorganic salts such as ammonium sulfate and close to the membrane's molecular weight cutoff, salts are removed and proteins are concentrated using a 10,000 Dalton ultrafiltration membrane. The lysozyme prepared by this integrated process has high purity and high specific activity, and removes impurities in egg white that can cause beer turbidity or off-flavors (such as ovomucoid).

[0019] Preferably, in the adsorption, the volume of the carboxymethyl cellulose cation exchange resin accounts for 30-35% of the egg white liquid volume, the adsorption temperature is 20-30℃, the adsorption time is 6.0-7.0 hours, and the pH of the system is maintained at 6.0-7.0. In the elution, the mass concentration of the ammonium sulfate solution is 9.0-10.0%. In the desalting and concentration, the pH of the feed solution is adjusted to 6.0-6.5, and the transmembrane pressure is controlled at 0.15-0.25 MPa.

[0020] By adopting the above technical solution, the thermodynamic and kinetic parameters of resin adsorption and elution are limited, ensuring that the adsorption capacity of lysozyme is maximized and not easily deactivated, while ensuring elution efficiency, and obtaining enzyme preparations with high yield and high activity.

[0021] Preferably, the egg white lysozyme is prepared by direct ultrafiltration separation. The preparation process of the egg white lysozyme includes: taking fresh egg white and diluting it with a sodium chloride solution of concentration of 0.05-0.2 mol / L at a volume ratio of 1:(0.8-1.5), adjusting the pH to 6.0-7.0, and centrifuging to remove the precipitate; introducing the supernatant into a circulation system equipped with an ultrafiltration membrane with a molecular weight cutoff of 10000 Daltons, and performing tangential flow filtration under the condition that the feed solution temperature is below 10°C; continuously concentrating to 1 / 3-1 / 6 of the original volume, and washing with water to remove salt, to obtain the egg white lysozyme in liquid form. The transmembrane pressure of the tangential flow filtration is controlled at 0.15-0.25 MPa, and the enzyme activity of the obtained liquid form of egg white lysozyme is 17000-19000 U / mg.

[0022] By employing the above technical solution, lysozyme is prepared using direct ultrafiltration. Tangential flow filtration technology avoids phase transitions and the introduction of large amounts of chemical reagents. The addition of sodium chloride solution disrupts the viscous interactions between egg white proteins, reducing the viscosity of the feed solution and increasing membrane flux. The low-temperature and low-pressure operating environment protects the enzyme's three-dimensional structure from damage by shear forces or thermal effects, resulting in a highly active liquid enzyme preparation that retains its native conformation. This preparation is suitable for direct addition to beer, exhibiting superior solubility and dispersibility.

[0023] This invention provides a method for improving the shelf life and taste of craft beer using egg white lysozyme. It has the following beneficial effects: 1. This invention utilizes the biological characteristics of egg white lysozyme's specific hydrolysis of bacterial cell wall peptidoglycan, combined with low-temperature cold storage technology, to precisely inhibit Gram-positive spoilage bacteria such as lactic acid bacteria commonly found in craft beer. Since lysozyme has no inhibitory effect on brewing yeast, this method reduces the risk of contamination by other microorganisms in the finished beer without interfering with normal post-fermentation metabolism, effectively extending the product's biological shelf life.

[0024] 2. This invention employs a bio-enzymatic preservation technology to replace the traditional high-temperature pasteurization process, avoiding the impact of heat treatment on the beer throughout the process. This prevents the volatilization of hop aromas, oxidation of malt flavors, and the development of stale or cooked flavors caused by high temperatures. Compared to the addition of chemical preservatives such as potassium sorbate, egg white lysozyme, as a natural protein, does not introduce chemical off-flavors or metallic astringent tastes. While ensuring food safety, it preserves the fresh, pure, and original flavor and taste of craft beer to the greatest extent possible.

[0025] 3. The method of this invention has good process compatibility, seamlessly integrating the enzyme addition step into existing fermentation or cold storage processes without altering the original saccharification, fermentation, and filtration procedures, and without requiring investment in large-scale equipment such as heat sterilization tunnels. This solution can be implemented using existing fermentation tanks or sake tanks, is simple to operate, and is easily applied directly to craft beer production lines. Attached Figure Description

[0026] Figure 1 This is a schematic diagram showing the number of Escherichia coli under the action of different concentrations of lysozyme according to the present invention; Figure 2 This diagram illustrates the effects of different concentrations of lysozyme of the present invention on Escherichia coli. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Preparation Examples 1-4: Preparation Example 1: This preparation example provides a high-purity egg white lysozyme extracted using an integrated ion exchange and ultrafiltration method, comprising the following steps: Take fresh egg whites and dilute them with an equal volume of sterile distilled water. Filter the solution through four layers of gauze to remove egg white residue and insoluble impurities to obtain pretreated egg white solution. The pretreated egg white solution was mixed with carboxymethyl cellulose cation exchange resin, with the resin volume accounting for 30% of the egg white solution volume. Adsorption was carried out at 20℃ and 60 rpm for 6.0 hours, during which a low concentration of acid or alkali solution was used to maintain the pH of the system at 6.0. After adsorption, the resin was filtered out and washed with a small amount of deionized water. Then, it was eluted with a 9.0% ammonium sulfate solution and the eluent rich in lysozyme was collected. The eluent was pumped into an ultrafiltration system equipped with a polyethersulfone membrane with a molecular weight cutoff of 10,000 Daltons. The pH of the feed solution was adjusted to 6.0, and the transmembrane pressure was controlled at 0.15 MPa for desalting and concentration until the conductivity of the permeate no longer decreased significantly. The retentate was collected and freeze-dried to obtain egg white lysozyme powder with an enzyme activity of 16,150 U / mg.

[0029] Preparation Example 2: This preparation example provides an egg white lysozyme extracted using a direct ultrafiltration separation method, comprising the following steps: Take fresh egg whites and dilute them with a 0.2 mol / L sodium chloride solution at a ratio of 1:0.8 (v / v). Adjust the pH of the mixture to 6.0 with citric acid solution, stir well, and then centrifuge to remove the precipitate. The diluted egg white solution was introduced into a circulation system containing a polyethersulfone hollow fiber ultrafiltration membrane module with a molecular weight cutoff of 10,000 Dalton. Start the circulation pump, control the transmembrane pressure to 0.25MPa by adjusting the opening of the feed valve and the concentration valve, and at the same time turn on the cooling circulation system to keep the feed temperature below 10℃. Use the tangential flow filtration mode to allow small molecule impurities to pass through the membrane pores and be discharged, while lysozyme is retained. The solution was continuously filtered and concentrated to 1 / 6 of its original volume. After washing and desalting with water, the concentrate was collected. The enzyme activity was tested and found to be 18900 U / mg. It can be used directly as a liquid enzyme preparation or further freeze-dried.

[0030] Preparation Example 3: This preparation example provides an egg white lysozyme extracted using a direct ultrafiltration separation method, comprising the following steps: Take fresh egg whites and dilute them with a 0.05 mol / L sodium chloride solution at a ratio of 1:1.5 (v / v). Adjust the pH of the mixture to 7.0 and centrifuge to remove the precipitate. The supernatant is introduced into a circulation system equipped with an ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons, and tangential flow filtration is performed under the condition that the feed temperature is below 10°C. The transmembrane pressure was controlled at 0.15 MPa, and the mixture was continuously concentrated to 1 / 3 of its original volume. Water was added for washing, filtration, and desalting to obtain a liquid form of egg white lysozyme preparation. The enzyme activity of the obtained liquid enzyme preparation was found to be 17100 U / mg.

[0031] Preparation Example 4: This preparation example provides an egg white lysozyme extracted using an integrated ion exchange and ultrafiltration method, comprising the following steps: Dilute fresh egg whites with an equal volume of sterile water and filter. The treated egg white liquid was mixed with carboxymethyl cellulose cation exchange resin, with the resin volume accounting for 35% of the egg white liquid volume. Adsorption was carried out at 30℃ with stirring for 7.0 hours, and the pH of the system was maintained at 7.0. After adsorption, the resin containing lysozyme was eluted with a 10.0% ammonium sulfate solution, and the eluent was collected. The eluent was desalted and concentrated by passing it through an ultrafiltration membrane system with a molecular weight cutoff of 10,000 Daltons under conditions of transmembrane pressure of 0.25 MPa and feed solution pH of 6.5, ultimately yielding lyophilized egg white lysozyme powder with an enzyme activity of 16,300 U / mg.

[0032] General process for wort preparation: In the embodiments of the present invention, unless otherwise specified, the preparation of wort according to the craft beer brewing process or the conventional craft beer mashing process are carried out according to the following steps: Select high-quality barley malt (or blend with special malt) and grind it, controlling the degree of grinding to break the husk but not crush it; mix the ground malt with brewing water at a ratio of 1:3-1:4, and carry out constant temperature saccharification in a mashing tank at 62-68℃ for 60-90 minutes until the iodine test is negative (the reaction is complete); after saccharification, filter and wash the mash in a filter tank, and collect the clear wort; boil the wort for 60-90 minutes, add bitter hops at the beginning of boiling, and add aroma hops 5-15 minutes before the end of boiling; after boiling, let it stand in a vortex settling tank for 20-30 minutes to remove hot coagulations, and then quickly cool it to the appropriate fermentation inoculation temperature through a plate heat exchanger.

[0033] Examples 1-4: Example 1: This embodiment provides a method for improving the shelf life and taste of craft beer using egg white lysozyme, specifically including the following steps: Following the conventional craft beer mashing process, crushed malt is mixed with water for mashing, filtered to obtain wort, boiled and hops are added, and after vortexing to separate the hot coagulated material, it is quickly cooled to the fermentation temperature. The cooled wort was inoculated with activated brewing yeast and primary fermentation was carried out under controlled temperature. When the primary fermentation was basically completed, the residual sugar content tended to stabilize and the yeast began to settle, the liquid egg white lysozyme obtained in Preparation Example 3 (enzyme activity 17100 U / mg) was selected. Liquid egg white lysozyme was added directly or diluted with a small amount of sterile water to the fermentation tank under stirring conditions, so that the final concentration of lysozyme in the wine was 15 mg / L. After the addition is complete, the beer is transferred to a cold storage tank for post-maturation at a low temperature. Finally, it is filtered through diatomaceous earth or membrane filtration to remove residual yeast and macromolecular substances, and then bottled and capped to obtain the finished craft beer.

[0034] Example 2: This embodiment provides a method for improving the shelf life and taste of craft beer using egg white lysozyme, specifically including the following steps: Prepare wort according to standard procedures and inoculate with brewer's yeast for fermentation; Monitor the fermentation process. When the main fermentation is finished and the diacetyl reduction is basically complete, prepare to add the biological preservative. Weigh an appropriate amount of the egg white lysozyme (enzyme activity 16150 U / mg) obtained in Preparation Example 1, and dissolve it in a small amount of sterile beer to activate it; The lysozyme solution is slowly injected into the fermentation tank through the addition port, and the enzyme is evenly distributed in the beer using a circulation pump or stirring device, controlling the final concentration of lysozyme in the beer to be 45 mg / L. The wine was then cooled to 0-4℃ for cold storage and maturation, during which time the lysozyme continued to inhibit Gram-positive spoilage bacteria. After the post-fermentation process, the wine is centrifuged and finely filtered before being aseptically bottled.

[0035] Example 3: This embodiment provides a method for improving the shelf life and taste of craft beer using egg white lysozyme, specifically including the following steps: The wort is prepared by malt saccharification, boiling and adding hops and cooling, and then inoculated with yeast for constant temperature primary fermentation. Before entering the post-ripening stage after the main fermentation period, the egg white lysozyme lyophilized powder (enzyme activity 16300 U / mg) obtained in Example 4 was dissolved in sterile water. Add the dissolved enzyme solution to the fermentation tank and adjust the amount added so that the final concentration of lysozyme in the beer system reaches 90 mg / L. After being mixed evenly, the original process parameters are maintained and the mixture is cooled and stored. At this high concentration, the lysozyme strongly inhibits potentially resistant bacteria. After cold storage, the beer is clarified through a cross-flow membrane filtration system, which also retains some unreacted enzyme proteins, before being aseptically bottled.

[0036] Example 4: This embodiment provides a method for improving the shelf life and taste of craft beer using egg white lysozyme, specifically including the following steps: After the wort preparation and primary fermentation process are completed, the fermentation liquid is transported to a cold storage tank (sake tank) through pipelines after the primary fermentation is completed. During or immediately after transport, add the concentrated egg white lysozyme solution obtained in Preparation Example 2 (enzyme activity 18900 U / mg, diluted for use) to the cold storage tank. The lysozyme used is a food-grade preparation, which is pre-diluted with low-temperature sterile water, and the amount added is controlled to make the final concentration of lysozyme in the beer in the can 45mg / L; In a cold storage environment of 0-5℃, the low temperature and long cycle conditions allow lysozyme to fully contact and lyse residual lactic acid bacteria and other bacteria. After the cold storage period, the beer is filtered through a plate and frame filter press to remove bacterial fragments and cloudy proteins before being packaged and shipped.

[0037] Comparative Examples 1-5: Comparative Example 1: Compared with Example 2, the difference is that no egg white lysozyme was added as a blank control, and the rest of the steps were the same.

[0038] Comparative Example 2: Compared with Example 2, the difference is that the addition of 45 mg / L egg white lysozyme was replaced with the addition of 200 mg / L potassium sorbate, and the rest of the steps are the same.

[0039] Comparative Example 3: Compared with Example 2, the difference is that no lysozyme is added after the main fermentation and during the cold storage stage, and the finished beer is pasteurized (at a constant temperature of 60°C for 20 minutes) after bottling. All other steps are the same.

[0040] Comparative Example 4: Compared with Example 2, the difference is that the egg white lysozyme is added at the wort boiling stage (added at the same time as hops), and is not added after the main fermentation and during the cold storage stage. The other steps are the same.

[0041] Comparative Example 5: Compared with Example 2, the difference is that the amount of egg white lysozyme added is reduced, and the final concentration is controlled at 5 mg / L. All other steps are the same.

[0042] Test Example 1-3: Test Example 1: Inhibitory effect of different concentrations of egg white lysozyme on model bacteria in beer matrix This test case aims to verify the bioactivity of the egg white lysozyme used in this invention in the complex matrix of craft beer, and to determine its dose-response relationship in inhibiting bacterial growth.

[0043] Experimental description: Prepare 8 sets of sterile test tubes, and add 12 mL of sterile filtered craft beer to each set as a substrate.

[0044] The preserved Escherichia coli were inoculated into liquid culture medium and cultured with shaking at 30°C and 120 rpm for 12-24 hours to prepare a bacterial suspension with uniform concentration.

[0045] The sample addition was carried out according to the experimental group design: Group 1 was added with an equal volume of sterile TE buffer as a blank control; Group 2 was added with 200 μL of bacterial suspension as a positive control; Groups 3 to 8 were added with 200 μL of bacterial suspension, and then pre-prepared egg white lysozyme solution was added respectively, so that the final concentration of enzyme in the system was 15 mg / L, 30 mg / L, 45 mg / L, 60 mg / L, 75 mg / L and 90 mg / L respectively.

[0046] Each group of test tubes was placed in a 30℃ constant temperature incubator and incubated for 24 hours.

[0047] After the culture was completed, the culture medium of each group was aspirated and serially diluted with sterile physiological saline. An appropriate amount of the diluted solution was spread on nutrient agar plates and incubated at 37°C for 24 hours. After that, the colony count (CFU / mL) was performed, and the lysis rate was calculated based on the data of the control group.

[0048] Experimental data: Table 1. Statistical analysis of the inhibitory effects of different concentrations of egg white lysozyme on Escherichia coli.

[0049] Note: "-" indicates that it is not applicable.

[0050] Conclusion: Based on the data in Table 1 and Figure 1 , Figure 2 The trends shown lead to the following conclusions: First, in the control group (group 2) without added lysozyme, the number of E. coli reached 2511.8 cfu / mL, confirming that the beer matrix itself has the conditions to support bacterial growth. When egg white lysozyme was introduced into the system, the number of bacteria showed a more significant decreasing trend.

[0051] Secondly, the data showed a clear dose-dependent effect. At a low concentration of 15 mg / L (Group 3), the number of E. coli decreased to 973.7 CFU / mL, and the lysis rate reached 61.23%, indicating that the enzyme molecules had begun to disrupt the bacterial cell wall structure at a low concentration. As the enzyme concentration increased, the antibacterial effect continued to strengthen. When the concentration reached 45 mg / L (Group 5), the lysis rate increased to 89.61%, and the colony count decreased to 261.0 CFU / mL; at 60 mg / L (Group 6), the lysis rate exceeded 94.99%; and at a high concentration of 90 mg / L (Group 8), the lysis rate reached 99.65%, and the colony count was only 8.7 CFU / mL, which basically achieved the elimination of the tested bacteria.

[0052] From a mechanistic perspective, egg white lysozyme hydrolyzes the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of the bacterial cell wall, disrupting the mechanical strength of the cell wall and causing bacterial lysis and death due to osmotic pressure differences. It is noteworthy that *E. coli* is a Gram-negative bacterium, and its cell wall contains a lipopolysaccharide layer, making it generally less sensitive to lysozyme than Gram-positive bacteria. Our experimental data show that this lysozyme still exhibits high lysis efficiency (up to 99.65%) against Gram-negative *E. coli* in a beer environment, indicating that it will have a stronger and faster killing effect on common Gram-positive spoilage bacteria in beer that lack outer membrane protection and have a higher content of peptidoglycan in their cell walls (such as *Lactobacillus* and *Pediococcus*).

[0053] In summary, the experimental data confirm that the 15-90 mg / L addition range set in this invention is scientifically reasonable. Within this concentration range, the egg white lysozyme maintains high biological activity in the beer matrix and can effectively control the risk of microbial contamination.

[0054] Test Example 2: Microbial Stability (Shelf Life) Test During Actual Brewing Process This test case aims to simulate the storage conditions of finished beer during its shelf life. By comparing the microbial indicators and physicochemical stability of different treatment groups under accelerated aging conditions, it verifies the ability of the process of this invention to control the main spoilage bacteria in beer.

[0055] Experimental description: Twelve bottles were randomly selected from each group of finished beer produced in Examples 1-4 and Comparative Examples 1-5, marked, and stored in a 25°C constant temperature incubator in the dark to simulate room temperature shelf storage and accelerate the aging process.

[0056] The sampling time points were set as storage day 0, day 30, day 60 and day 90. At each time point, one bottle of sample was randomly selected from each group for destructive testing.

[0057] Open the bottle cap in a sterile laminar flow hood, aseptically aspirate the wine sample, filter it using a 0.45μm pore size filter membrane, attach the filter membrane containing the retained microorganisms to an MRS agar plate (with added actinomycete ketone to inhibit yeast growth), place it in an anaerobic culture tank, and incubate at 28℃ for 72 hours. The colony count (CFU / mL) is mainly performed on anaerobic / facultative anaerobic putrefactive bacteria such as lactobacillus and Pediococcus.

[0058] Take another portion of the degassed beer sample and inject it into the cuvette of the EBC turbidimeter. Measure the turbidity value of the beer (EBC units) and record the value to assess the non-biological and biological turbidity.

[0059] Experimental data: Table 2. Statistics on changes in total anaerobic bacteria count and turbidity of each beer group during storage at 25℃

[0060] Conclusion: According to the data in Table 2, the microbial stability and apparent turbidity of the samples in each group showed significant differences during storage.

[0061] Comparative Example 1 (blank control) showed anaerobic bacterial growth (42 CFU / mL) after 30 days of storage, and the colony count surged to 3450 CFU / mL by day 90, with turbidity also increasing from the initial 0.45 EBC to 12.65 EBC. This indicates that craft beer without added antimicrobial agents is susceptible to the proliferation of residual lactic acid bacteria or Pediococcus at room temperature, leading to biological turbidity and spoilage.

[0062] The data trend of Comparative Example 4 (added during boiling) was highly consistent with that of Comparative Example 1, with the colony count reaching 2890 CFU / mL and the turbidity increasing to 9.84 EBC on day 90. This result confirms that adding egg white lysozyme during the wort boiling stage (usually at temperatures >95℃) destroys the spatial structure of the enzyme protein due to high temperature, leading to the inactivation of its active site and preventing it from lysing bacterial cell walls during subsequent storage.

[0063] Comparative Example 5 (low concentration of 5 mg / L) showed some inhibitory effect on microbial growth in the early stage of storage (0-30 days), but the colony count rebounded to 128 CFU / mL after 60 days and reached 340 CFU / mL after 90 days. This indicates that the enzyme concentration was insufficient to completely eliminate or continuously suppress potentially resistant strains, posing a risk of failure during long shelf life.

[0064] In contrast, Examples 1-4 all exhibited excellent biostability. Examples 2, 3, and 4 maintained a total anaerobic bacterial count consistently below the detection limit (<1 CFU / mL) and stable turbidity (fluctuation range <0.05 EBC) throughout the 90-day testing period. Their preservative effect was comparable to Comparative Example 2, which used a chemical preservative, and Comparative Example 3, which used heat sterilization. Although Example 1 had a lower addition level (15 mg / L), only single-digit colonies (8 CFU / mL) were detected after 90 days, and the turbidity increased slightly to 0.62 EBC, still within the acceptable range for commercial sterility.

[0065] Furthermore, comparing the data from Example 2 and Comparative Example 3 reveals that although their antibacterial effects are comparable, the initial turbidity of Comparative Example 3 (pasteurization) (0.62 EBC) is higher than that of Example 2 (0.46 EBC). This is due to the denaturation and precipitation of some heat-sensitive proteins caused by the thermal effect of pasteurization. The biocold sterilization technology employed in this invention utilizes the mechanism of lysozyme specifically hydrolyzing bacterial peptidoglycans to achieve preservation without introducing heat load, thereby better maintaining the clarity of the beer. In summary, the 15-90 mg / L egg white lysozyme addition process determined in this invention can effectively ensure the biosafety of craft beer during its shelf life.

[0066] Test Example 3: Sensory Quality and Flavor Evaluation Test This test case aims to evaluate the differences in sensory dimensions of craft beer treated with different preservation methods, and focuses on verifying the effect of the method of the present invention on preserving the original flavor, taste purity and freshness of beer while extending the shelf life.

[0067] Experimental description: Sample preparation: Craft beers prepared in Example 2 (with 45 mg / L egg white lysozyme), Comparative Example 2 (with 200 mg / L potassium sorbate), and Comparative Example 3 (pasteurized after bottling) were selected as test samples. All samples were fermented from the same batch of wort and stored at 20°C in the dark for 7 days before evaluation to ensure stability.

[0068] Tasting Panel Formation: A sensory evaluation panel consisting of 10 individuals holding national or corporate-level craft beer taster qualifications will be invited. Smoking, drinking alcohol, and consuming spicy or irritating foods are prohibited for one hour prior to the tasting.

[0069] Blind Tasting Procedure: A double-blind method was used for testing. The three wine samples were poured into clean, odorless, dedicated tulip-shaped tasting glasses, approximately 100 mL per glass, with the wine temperature maintained between 8-10°C. Each sample glass was labeled only with a randomly generated 3-digit code and no other markings.

[0070] Scoring Criteria: Wine tasters score the following four dimensions (0-10 points, with higher scores indicating better quality) using the QDA (Quantitative Descriptive Analysis) method: Aroma: Evaluate the intensity and typicality of hop aromas (such as citrus and resin) and malt aromas.

[0071] Purity: Assess whether the taste is pure and whether there are any off-flavors (such as chemical taste, astringency, or diacetyl taste).

[0072] Freshness: Assess whether the wine has a lively feel and whether there are any signs of oxidation, cardboard, or cooked taste.

[0073] Overall Preference Rating: A comprehensive score based on the overall flavor balance.

[0074] Data processing: Collect the original scores from 10 sommeliers, remove the highest and lowest scores, and take the average value, keeping one decimal place.

[0075] Experimental data: Table 3. Sensory scores of craft beer under different preservation methods

[0076] Conclusion: According to the data in Table 3, the three treatment methods showed significant differences in their impact on the sensory quality of beer, confirming that the choice of process plays a decisive role in the flavor of the final product.

[0077] Example 2 achieved the highest score across all evaluation dimensions (overall preference score 9.5), indicating that the egg white lysozyme preservation technology used in this invention can maximally restore the authentic flavor of craft beer. Mechanistically, lysozyme, as a highly substrate-specific enzyme, hydrolyzes only the peptidoglycan structure of bacterial cell walls, without reacting with flavor compounds such as esters, phenols, and terpenes in the beer. Therefore, the sample in Example 2 exhibits a rich hop aroma, prominent malt flavor, and a high degree of purity and freshness in its taste.

[0078] In contrast, Comparative Example 3 (pasteurized) scored significantly lower in aroma and freshness (6.8 and 7.1, respectively). This is because the heat treatment during pasteurization (60°C for 20 minutes) accelerated the volatilization and degradation of heat-sensitive flavor compounds (such as myrcene and linalool in hops) and induced the formation of late-stage Maillard reaction products, resulting in a noticeable cooked or aging flavor in the beer and ruining the fresh and crisp characteristics that craft beer should have. Although its purity score was high (9.1), indicating that heat sterilization effectively suppressed unwanted microorganisms, the loss of flavor came at a high cost.

[0079] The main defect of Comparative Example 2 (potassium sorbate) lies in the purity dimension, which scored only 7.2. As an organic acid salt chemical preservative, potassium sorbate, when reaching an effective antibacterial concentration (200 mg / L), will change the ionic strength and pH buffering system of the beer, giving it an unpleasant roughness and a slight chemical off-flavor, and masking the delicate flavors of malt and hops.

[0080] In summary, using egg white lysozyme for biological cold preservation avoids the flavor damage caused by heat sterilization and overcomes the off-flavor problems introduced by chemical preservatives. It is the best technical solution to improve the taste and quality of craft beer while ensuring biological stability.

Claims

1. A method for improving the shelf life and taste of craft beer using egg white lysozyme, characterized in that, Includes the following steps: The wort is prepared according to the craft beer brewing process, brewing yeast is added, and primary fermentation is carried out under controlled temperature. After the main fermentation is completed and before bottling, egg white lysozyme solution is added to the fermentation liquid or sake liquid to control the final concentration of egg white lysozyme in the beer system to be 15-90 mg / L. The wine with added egg white lysozyme is cold-stored and matured at 0-5℃. The egg white lysozyme is used to lyse the cell walls of Gram-positive spoilage bacteria at low temperature, thus completing biological preservation without heat sterilization. After the cold storage and aging process is completed, the wine is filtered and bottled.

2. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 1, characterized in that, The timing for adding egg white lysozyme solution to the fermentation broth or sake broth is any one of the following: When the primary fermentation is complete, diacetyl reduction is finished, and the brewing yeast begins to settle, it is added to the fermentation tank; The fermentation broth is added online during the process of transporting the fermentation broth from the fermenter to the cold storage tank; The fermentation broth is added to the cold storage tank after it enters the cold storage tank and before the start of the cold storage period.

3. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 1, characterized in that, The method for preparing the egg white lysozyme solution is as follows: Select egg white lysozyme with an enzyme activity of 16100-19000 U / mg, and dissolve or dilute it with sterile water or sterile beer to be treated to prepare a homogeneous egg white lysozyme solution.

4. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 1, characterized in that, The filtration process employs diatomaceous earth filtration, plate and frame filtration, or cross-flow membrane filtration to trap bacterial fragments and turbid proteins. The method of using egg white lysozyme to improve the shelf life and taste of craft beer maintains a non-heat-treated state throughout the post-processing and bottling process, thus preserving the volatile flavor compounds of the beer.

5. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 1, characterized in that, The egg white lysozyme was prepared using an integrated ion exchange and ultrafiltration method. The preparation process of the egg white lysozyme includes: Fresh egg whites were diluted with water and filtered to obtain a pretreatment solution, which was then mixed with carboxymethyl cellulose cation exchange resin for adsorption. After the adsorption process is complete, the carboxymethyl cellulose cation exchange resin is washed, eluted with ammonium sulfate solution, and the eluent is collected. The eluent was pumped into an ultrafiltration system equipped with a membrane module with a molecular weight cutoff of 10,000 Daltons for desalting and concentration, and then dried to obtain the egg white lysozyme.

6. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 5, characterized in that, In the adsorption process, the volume of the carboxymethyl cellulose cation exchange resin accounts for 30-35% of the volume of the egg white liquid, the adsorption temperature is 20-30℃, the adsorption time is 6.0-7.0 hours, and the pH of the system is maintained at 6.0-7.

0.

7. The method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 5, characterized in that, In the elution, the mass concentration of the ammonium sulfate solution is 9.0-10.0%.

8. A method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 5, characterized in that, In the desalination and concentration process, the pH of the feed solution is adjusted to 6.0-6.5, and the transmembrane pressure is controlled at 0.15-0.25 MPa.

9. A method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 1, characterized in that, The egg white lysozyme was prepared by direct ultrafiltration separation method, and the preparation process of the egg white lysozyme includes: Take fresh egg whites and dilute them with a sodium chloride solution of concentration of 0.05-0.2 mol / L at a volume ratio of 1:(0.8-1.5), adjust the pH to 6.0-7.0 and centrifuge to remove the precipitate; The supernatant is introduced into a circulation system equipped with an ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons, and tangential flow filtration is performed under the condition that the feed temperature is below 10°C. The mixture was continuously concentrated to 1 / 3 to 1 / 6 of its original volume, and then washed, filtered, and desalted with water to obtain the egg white lysozyme in liquid form.

10. A method for improving the shelf life and taste of craft beer using egg white lysozyme according to claim 9, characterized in that, The tangential flow filtration controls the transmembrane pressure to be 0.15-0.25 MPa, and the resulting liquid form of the egg white lysozyme has an enzyme activity of 17000-19000 U / mg.

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