A method for brewing a dark beer and its use

Abstract

The application discloses a brewing method of dark beer and application thereof, and comprises the following steps: raw material collocation, wort preparation and beer fermentation; the wort preparation comprises the following steps: saccharification, wort boiling process control, whirl deposition and cooling; the wort boiling process control comprises the following steps: the saccharified wort is slowly heated to the initial boiling stage, the steam pressure is controlled to be 2.4-2.6 Bar, and the heating time is 20-25 min; the wort boiling process control pH is 5.0-5.3, the steam pressure is 2.0-2.2 Bar, the boiling time is 60 min, and the boiling evaporation rate is 8%; the application reduces the generation amount of acrylamide in the wort preparation process by controlling the key process parameters of the wort preparation process, especially the boiling process; secondly, the key indexes of malt in the raw material formula are limited, more amidases are secreted by the yeast, the beer fermentation step preferably uses beer yeast with high deaminase activity, efficient deamination conversion of free acrylamide in the wort is realized, and the content of acrylamide in the beer is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of beer brewing technology, and particularly relates to a method for brewing dark beer and its application. Background Technology

[0002] During food heat processing, the Maillard reaction not only imparts unique color, aroma, and flavor to food, but may also generate a series of harmful or unpleasantly odorous substances, such as DDMP, HMF, acrylamide, and furfural. These substances have been widely detected in various foods, including honey, vegetables, fruits, condiments, baked bread, and beverages. In recent years, the formation mechanisms and control strategies of these associated hazards in heat-processed foods have become a research hotspot in the field of food safety both domestically and internationally.

[0003] Since acrylamide was first discovered in food in 2002, its potential toxicity has made it a major concern for the international food industry. Therefore, effectively reducing acrylamide formation in food processing and applications has become a key focus for manufacturers. Studies have shown that the Maillard reaction is the main pathway for acrylamide formation, with the key substances being the precursor asparagine (Asn) and intermediates produced through multiple steps such as decarboxylation and deamination, generating many intermediate products such as Schiff base and 3-aminopropionamide. Currently, among various acrylamide control methods, L-asparaginase can degrade the acrylamide precursor asparagine into aspartic acid and ammonia, effectively inhibiting acrylamide formation in food (up to 90%), while maintaining the nutritional properties, color, and taste of the food. Commercially available asparaginase preparations include Prevent ASeTM from DSM in the Netherlands, derived from Aspergillus niger, with optimal pH and temperature of pH 4-5 and 50 ℃, respectively; and Acrylaway from Novozymes in Denmark, derived from Aspergillus oryzae, with optimal pH and temperature of pH 7 and 37 ℃, respectively.

[0004] In beer brewing, the use of specialty malts can enhance the typicality and fullness of beer flavor. For example, caramelized malt can impart a prominent caramelized flavor to beer. However, during the roasting process of caramelized malt, acrylamide is generated through the Maillard reaction, leading to a significant increase in acrylamide content in beers brewed with a high proportion of caramelized malt. Conventional control methods, such as adding asparaginase during wort mashing, are unlikely to significantly reduce acrylamide levels in the beer brewing system. This may be related to the complexity of the brewing process and the environment in which the enzymes function. Therefore, how to reduce the content of potentially harmful substances while ensuring beer flavor through process optimization and innovation has become an important issue for the beer brewing industry. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is the difficulty in effectively reducing the formation of acrylamide in the existing beer brewing system, and a brewing method for dark beer and its application are proposed.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a method for brewing dark beer, including a raw material matching step, a wort preparation step, and a beer fermentation step; The wort preparation steps include saccharification, wort boiling process control, vortex sedimentation, and cooling. The wort boiling process control includes: The mashed wort is heated to the initial boiling stage at a slow temperature, with a steam pressure of 2.4-2.6 Bar and a heating time of 20-25 minutes. During the wort boiling process, the pH is controlled at 5.0-5.3, the steam pressure at 2.0-2.2 Bar, and the boiling time is 60 minutes, with a boiling evaporation rate of 8%. Hops or hop products are added during the boiling process.

[0007] As a preferred choice, hops or hop products are selected from Qingdao Dahua, Zha Yixianghua, etc.

[0008] Preferably, in the raw material blending step, the mass percentage of roasted malt in the raw materials is controlled to be 20-30%.

[0009] Preferably, in the raw material formulation step, the raw materials also include barley malt, with the aN of the barley malt controlled at ≥160mg / 100g and the saccharification power ≥300WK.

[0010] Preferably, in the raw material preparation step, the raw materials also include rye malt and auxiliary materials, and the mass ratio of barley malt, roasted malt, rye malt and auxiliary materials is 50:(20-30):(0-5):(20-30). The caramel malt has a color intensity of 90-150 EBC, and the rye malt has a color intensity of 900-1500 EBC.

[0011] Preferably, the auxiliary ingredients are rice, syrup or starch.

[0012] Preferably, the saccharification step includes: first, feeding the material at 40-50 ℃, then keeping it at the same temperature for 30-60 minutes; then raising the temperature to 63-68 ℃ and keeping it at that temperature for 60-90 minutes; then rapidly raising the temperature to 76-78 ℃, finally transferring it to a filter tank, filtering, and then going through processes such as wort boiling, vortex sedimentation and wort cooling to obtain saccharified cold wort; Preferably, in the saccharification step, the aN of the prepared saccharified cold wort is controlled to be ≥200 mg / L, and the degree of fermentation is 65-68%.

[0013] The vortex sedimentation time is controlled to be completed within 15 minutes.

[0014] As a preferred option, the beer fermentation process includes a primary fermentation step, a reduction step, and a cooling and cold storage step. The temperature of the primary fermentation step is controlled at 9.5-10℃, and the fermentation time is 96-120h. The reduction temperature for the reduction step is 12-13℃, and the reduction time is 15 days. The cooling and cold storage step involves controlling the cold storage temperature at -1 to 0°C for 7 days.

[0015] Preferably, in the beer fermentation step, brewer's yeast with an amidase activity ≥230u / L is used to ferment the wort.

[0016] A second aspect of the present invention provides the application of the above-described brewing method in reducing the acrylamide content in dark beer.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a brewing method for dark beer. By adjusting key process parameters in the wort preparation process, particularly through innovation in the wort boiling process, the method further reduces acrylamide, a byproduct of the Maillard reaction, during wort preparation, resulting in a reduction of acrylamide formation in cold wort by more than 40%. Secondly, through innovations in processes such as optimizing the malt formulation, key indicators in wort preparation, further optimizing and controlling the secretion of acrylamide enzymes in the saccharified wort, and selecting superior yeast varieties, the method achieves highly efficient conversion of acrylamide in the wort during fermentation, increasing the conversion efficiency to over 85% and significantly reducing the content of the potentially harmful acrylamide. Furthermore, the resulting beer possesses a strong caramel aroma, a rich and harmonious flavor, and a pure and clean taste, satisfying consumers' pursuit and demand for high-quality beer. Attached Figure Description

[0018] Figure 1 This is a comparison chart showing the changes in acrylamide during the brewing process; Figure 2 A graph showing the difference in acrylamide formation caused by different heating methods during wort boiling; Figure 3 To construct a standardized Pareto plot of effects based on the results of the Box-Behnken central composite design experiment; Figure 4 Figure showing the effect of aN in stout wort on the activity of yeast-secreting amidase; Figure 5The graph shows the effect of the sugar composition (degree of fermentation) of dark beer wort on the activity of yeast-secreted amidase. Figure 6 Figure 1 shows the effect of combined treatments of aN and fermentation degree on the activity of yeast-secreting amidase in stout wort. Figure 7 A comparative chart showing the ability of different brewer's yeast strains to secrete amidase. Detailed Implementation

[0019] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0020] This invention provides a method for brewing dark beer, including a raw material matching step, a wort preparation step, and a beer fermentation step; The wort preparation steps include saccharification, wort boiling process control, vortex sedimentation, and cooling. The wort boiling process control includes: The mashed wort is heated to the initial boiling stage at a slow temperature, with a steam pressure of 2.4-2.6 Bar and a heating time of 20-25 minutes. During the wort boiling process, the pH is controlled at 5.0-5.3, the steam pressure at 2.0-2.2 Bar, and the boiling time is 60 minutes, with a boiling evaporation rate of 8%. Hops or hop products are added during the boiling process.

[0021] The aforementioned wort boiling process includes two stages: the filtered wort heating to initial boiling and the full wort boiling. By controlling the process parameters of the wort boiling process, especially the steam pressure and flow rate during the wort heating stage, the formation of acrylamide during the heating process is controlled. Simultaneously, based on DOE experiments, key process parameters affecting acrylamide, such as wort pH, boiling time, and boiling temperature, are optimized to further control acrylamide formation during wort boiling. These two stages reduce acrylamide formation by 40%, effectively reducing the amount of acrylamide generated during wort preparation.

[0022] In a preferred embodiment, the hops or hop products are selected from Qingdao Dahua, Zha Yixianghua, etc.

[0023] In a preferred embodiment, in the raw material blending step, the mass percentage of roasted malt in the raw materials is controlled to be 20-30%.

[0024] In a preferred embodiment, the raw material formulation step further includes rye malt and adjuvants, and the mass ratio of barley malt, roasted malt, rye malt and adjuvants is 50:(20-30):(0-5):(20-30). The caramel malt has a color intensity of 90-150 EBC, and the rye malt has a color intensity of 900-1500 EBC.

[0025] In a preferred embodiment, the excipient is rice, syrup, or starch.

[0026] In a preferred embodiment, the raw material formulation step further includes barley malt, wherein the aN of the barley malt is controlled at ≥160mg / 100g and the saccharification power is ≥300WK.

[0027] It should be noted that among brewing ingredients, caramelized malt is the main source of acrylamide in wort, followed by rye malt, while acrylamide was not detected in base malt and adjuncts. Therefore, while beer using a high proportion of caramelized malt imparts a stronger caramelized flavor, it also significantly increases the acrylamide content. This invention, through process optimization and innovation, achieves an acrylamide reduction rate of over 85% in the wort during fermentation, significantly reducing the acrylamide content.

[0028] The aforementioned technical solution defines key control indicators for barley malt, controlling indicators such as aN and saccharification power in the base malt. By combining it with caramel malt and other raw materials, it achieves control over the aN index for yeast-secreted amidase and the carbon and nitrogen sources for fermentable sugars, meeting the needs of yeast with high amidase activity. The control levels of aN and fermentable sugars in wort are key factors in stimulating yeast to secrete amidase and improving acrylamide reduction. Malt protein dissolution mainly occurs during malting, while the protein rest process during saccharification accounts for only about 20% of protein breakdown. Adjusting the saccharification process has limited effect on aN growth, thus limiting the contribution of caramel malt, rye malt, and adjuvants to the final saccharified cold wort aN. Therefore, controlling the protein solubility and saccharification power of the base malt during raw material combination, combined with the efficient conversion of protein and starch breakdown during saccharification, is a crucial step in obtaining the required aN and fermentable sugar components.

[0029] In a preferred embodiment, the saccharification step includes: first, feeding the material at 40-50°C, then holding it at the same temperature for 30-60 minutes; then raising the temperature to 63-68°C and holding it for 60-90 minutes; then rapidly raising the temperature to 76-78°C, finally transferring it to a filter tank, filtering, and then undergoing wort boiling, vortex sedimentation, and wort cooling processes to obtain saccharified cold wort; The vortex sedimentation time is controlled to be completed within 15 minutes.

[0030] In a preferred embodiment, during the saccharification step, the aN of the prepared saccharified cold wort is controlled to be ≥200 mg / L, and the degree of fermentation is 65-68%.

[0031] Many microorganisms secrete amidases in the prior art, but there are no reports of brewer's yeast secreting this enzyme. The inventors specifically detected the amidase activity of brewer's yeast using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), confirming that brewer's yeast has the ability to secrete amidase. During fermentation, the conversion of acrylamide is achieved through the amidase secreted by brewer's yeast, and many factors affect the secretion of yeast amidase. Through wort aN and fermentable sugar gradient experiments, the supply of nitrogen and carbon sources during fermentation is a key factor in yeast amidase secretion. Among these, wort aN is the main factor affecting the activity of yeast amidase secretion, followed by the fermentable sugar components in the wort.

[0032] In a preferred embodiment, the beer fermentation step includes a primary fermentation step, a reduction step, and a cooling and cold storage step; The temperature of the primary fermentation step is controlled at 9.5-10℃, and the fermentation time is 96-120h. The reduction temperature for the reduction step is 12-13℃, and the reduction time is 15 days. The cooling and cold storage steps described above involve controlling the cooling and cold storage temperature at -1 to 0℃, and the cold storage time is 7 days.

[0033] In a preferred embodiment, during the beer fermentation step, a beer yeast variety with high acrylamide conversion efficiency is selected for fermentation, and the selected beer yeast secretes amidase activity ≥230u / L.

[0034] In a preferred embodiment, the brewer's yeast used is W-34 / 70 or S-23, purchased from Fermentis.

[0035] The aforementioned technical scope limits the activity of amidase secretion in brewer's yeast because there are significant differences in the ability of different strains to secrete amidase. Therefore, it is necessary to select yeast varieties with high acrylamide conversion rates.

[0036] A second aspect of the present invention provides the application of the above-described brewing method in reducing the acrylamide content in dark beer.

[0037] The research approach of this invention is as follows: 1. Investigation into the sources of acrylamide content in raw materials: First, the applicant systematically compared the acrylamide content of various raw materials used in dark beer production. The results showed that acrylamide was not detected in any of the base malts, including Canadian malt (8 batches), Australian malt (6 batches), wheat malt (2 batches), French malt (1 batch), and domestic malt (3 batches) (below the detection limit of 10 μg / kg). Adjuncts such as rice, syrup, and starch were also not detected (below the detection limit of 10 μg / kg). However, acrylamide was detected in all four batches of caramel malt and rye malt tested. The results are shown in Table 1. It can be seen that acrylamide in dark beer ingredients mainly comes from dark malt; the higher the proportion, the greater the contribution to acrylamide levels. Caramel malt accounts for approximately 25% of the total, which is the main reason for the high acrylamide content in dark beer.

[0038] Table 1 Comparison of Acrylamide Content in Different Raw Materials

[0039] 2. Investigate the changes of acrylamide during the brewing process: After identifying the source of the raw materials, the applicant systematically studied the dynamic changes of acrylamide throughout the brewing process: Depend on Figure 1 The data shows that acrylamide content increased by 31% during the heating stage, 14.4% during the boiling stage, and 21% during the vortex stage. Overall, the data indicates a continuous and significant cumulative increase in acrylamide content from mixed wort to cold wort before fermentation. The heating stage is the key period for early rapid growth, while the boiling and vortex stages further exacerbate its formation and accumulation. This result clearly reveals that the high-temperature stages in wort preparation, namely heating, boiling, and vortex precipitation, are the main stages for acrylamide formation.

[0040] 3. Clarify the influence of the high-temperature stage process on acrylamide formation and the key control points. Based on the understanding that acrylamide is dynamically generated during the brewing process, the applicant focused on key processes, revealed the influence of major process parameters through systematic experiments, and explored control methods.

[0041] (1) Effect of heating rate on acrylamide formation Further investigation into the heating rate during the boiling process revealed that it has a significant impact on the amount of acrylamide produced. like Figure 2 As shown: During the heating phase: using a rapid heating process, the acrylamide content increases by as much as 45%; while using a slow heating process, the increase is only 16.8%.

[0042] Throughout the boiling process (including heating): Under the rapid heating process, the total increase in acrylamide reached 108%; while under the slow heating process, the total increase decreased to 62%.

[0043] The results show that controlling and reducing the heating rate is an effective means to suppress the large-scale generation of acrylamide during the boiling process (especially in the early stage).

[0044] (2) Study on the effect of boiling process on the formation of acrylamide To investigate the effects of boiling process parameters on acrylamide formation, the applicant conducted a small-scale simulation experiment using a Box-Behnken central composite design with three factors and three levels. Temperature, time, and pH were used as independent variables, and the acrylamide content in the wort was used as the response value. The factor and level design is shown in Table 2.

[0045] Table 2 Box Factors and Levels of Behnken Central Composite Design

[0046] Table 3 Results of the Box-Behnken central composite design experiment

[0047] Based on the results of the Box-Behnken central composite design experiment, a predictive model was established to support precise regulation, and a standardized Pareto diagram of the effects was constructed, such as... Figure 3 .like Figure 3 As shown, through statistical analysis of the experimental results, the order of influence of each factor on the acrylamide formation rate was clarified as follows: pH > boiling time > temperature.

[0048] Specifically, higher pH values ​​and longer boiling times promote acrylamide formation. The key control points in the boiling process are wort pH and boiling time.

[0049] (3) Exploration of key regulatory factors in the fermentation process Given that the fermentation stage is a critical step in acrylamide reduction, the applicant investigated the effects of key wort components on the yeast's ability to secrete acrylamide enzyme, and conducted wort aN and fermentable sugar gradient experiments: Table 4. Experimental Design of Wort aN and Fermentable Sugar Gradient

[0050] By adding different amounts of neutral protease (Neutrase) to adjust the aN level in the wort, wort with aN concentrations of 183, 203, and 226 mg / L were obtained; by adding different amounts of enzyme to adjust the proportion of fermentable sugars, wort with fermentable sugar proportions of 65%, 67%, and 68% were obtained.

[0051] Combination experiment: Treatment with 150 μL Neutrase + 20 μL Attenuzyme Core yielded wort with aN of 203 mg / L and fermentable sugars of 68%.

[0052] Experimental results: like Figure 4-6 As shown, both wort aN and sugar components significantly induced yeast to secrete amidase. Compared with no enzyme added, increasing wort aN to 203 mg / L increased amidase activity by 99%; increasing the proportion of fermentable sugar by 2% increased amidase activity by approximately 30%; further increasing aN and sugar components to over 220% and 68% respectively did not significantly increase the secreted enzyme activity; the induction effect of aN was significantly better than that of sugar components (99% vs. 30%). Through the wort aN and fermentable sugar gradient experiments, the supply of nitrogen and carbon sources during fermentation is the key factor in yeast amidase secretion. It was determined that wort aN is the main factor affecting the activity of yeast deaminase secretion, followed by the fermentable sugar components in the wort.

[0053] By controlling indicators such as aN and saccharification power in basic malt, and by using it in combination with caramel malt and other raw materials, the carbon and nitrogen sources for yeast-secreted amidase and fermentable sugars can be controlled, thus meeting the needs of yeast with high amidase activity.

[0054] This study elucidates the detoxification mechanism of yeast-secreted amidases, identifying key factors in the induction of amidase production by brewer's yeast using wort aN. Combining the mechanisms of acrylamide formation during saccharification and yeast transformation during fermentation, it determines control strategies for managing the formation of Maillard reaction intermediates during the high-temperature boiling stage and for increasing acrylamide degradation conversion rates through fermentation-enhanced yeast amidase activity and metabolism, providing theoretical support for regulation. The study also identifies the influence of wort boiling process parameters on acrylamide formation and key process control points, clarifying the order of influence of control measures on acrylamide production in brewing: fermentation > boiling > raw materials.

[0055] To more clearly and in detail introduce the brewing method for dark beer and its application provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0056] In the following embodiments and comparative examples of the present invention, the caramelized malt used was purchased from Shandong Haiyue Malt Co., Ltd. The brewer's yeast used in the examples was W-34 / 70 and S-23, purchased from Fermentis. The brewer's yeast TT-1 used in the comparative example was purchased from the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19839.

[0057] The above comparison of the ability of different brewer's yeasts to secrete amidase is as follows: Figure 7 As shown.

[0058] Example 1 Raw material formula (mass ratio): Barley malt: Roasted malt: Black malt: Rice = 50:25:5:20; Key indicators for barley malt include: aN 162mg / 100g, and saccharification power 350WK. Caramel malt color: 100 EBC, Rye malt color: 1200 EBC; Brewing method: (1) Raw material crushing: Barley malt, caramel malt, rye malt and rice auxiliary material are crushed by a two-roll mill to obtain crushed raw materials; (2) The crushed rice auxiliary material is gelatinized separately in a gelatinization pot. The mash after the rice gelatinization is completed according to the standard process is mixed with the mash after the barley malt protein rests, and then saccharification is carried out. (3) Saccharification process: The crushed malt is first fed at 50°C, and then kept at the same temperature for 50 minutes (including feeding time); then the temperature is raised to 65°C and kept for 75 minutes; then the temperature is rapidly raised to 76°C. (4) Wheat filtration: The mash after saccharification is transferred into a filter tank, filtered, and then subjected to wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort. The aN of the obtained saccharified cold wort is 208 mg / L. (5) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is increased slowly. The steam pressure is controlled at 2.5 Bar and the heating time is 22.5 min. During the wort boiling process, the pH is controlled at 5.2, the steam pressure is controlled at 2.1 Bar, the boiling time is 60 min, and the boiling evaporation rate is 8%. 1% wt of Tsingtao florets is added during the boiling process. (6) Fermentation: The wort after boiling is swirled and settled, with the time controlled within 15 minutes; after thin-plate cooling, it is introduced into the fermentation tank with online oxygenation, controlling the dissolved oxygen of the wort to 10-12 mg / L, and yeast strain W-34 / 70 is introduced at the same time, with the yeast count controlled at 22 million / ml in the tank. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.05 mg / L, it is further cooled and stored at -1 to 0℃ for 7 days.

[0059] Example 2 Raw material formula: barley malt: caramel malt: syrup = 50:20:30; among which, the key indicators of barley malt are: α-N 160mg / 100g, saccharification power 350WK; Caramel malt color: 100 EBC, Caramel malt color: 1200 EBC; Brewing method: (1) Raw material crushing: Barley malt and caramel malt are crushed by a two-roll mill to obtain crushed malt; (2) Saccharification process: The crushed malt is first fed at 45°C, and then kept at the same temperature for 50 minutes (including feeding time); then the temperature is raised to 65°C and kept for 60 minutes; then the temperature is rapidly raised to 76°C; (3) Wheat filtration: The mash after saccharification is transferred into a filter tank, filtered, and then subjected to wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort. The aN of the saccharified cold wort is 220 mg / L. (4) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is slowly increased. The steam pressure is controlled at 2.5 Bar and the heating time is 22 min. During the wort boiling process, the pH is controlled at 5.0, the steam pressure is controlled at 2.0 Bar, the boiling time is 60 min, and the boiling evaporation rate is 8%. 1% wt of Qingdao Dahua is added during the boiling process. (5) Fermentation: The wort after boiling is swirl-sedimented and the time is controlled within 15 minutes; after thin plate cooling, it is introduced into the fermentation tank with online oxygenation, and the dissolved oxygen of the wort is controlled at 10 mg / L. At the same time, yeast W-34 / 70 is added, and the yeast concentration in the tank is controlled at 21 million / ml. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.06 mg / L, it is further cooled and stored at -1 to 0℃ for 7 days.

[0060] Comparative Example 1 Raw material formula: barley malt: caramel malt: rye malt: rice = 50:25:5:20; among which, the key indicators of barley malt are: aN 165mg / 100g, saccharification power 330WK; Caramel malt color: 100 EBC, Rye malt color: 1200 EBC; Brewing method: (1) Raw material crushing: Barley malt, roasted malt, rye malt and rice auxiliary material are crushed by a two-roll mill to obtain crushed raw materials; (2) The crushed rice auxiliary material is gelatinized separately in a gelatinization pot. The mash after the rice gelatinization is completed according to the standard process is mixed with the mash after the barley malt protein rests, and then saccharification is carried out.

[0061] (3) Saccharification process: The crushed malt is first fed at 50°C, and then kept at the same temperature for 50 minutes (including feeding time); then the temperature is raised to 65°C and kept for 75 minutes; then the temperature is rapidly raised to 76°C. (4) Wheat filtration: The mash after saccharification is transferred into a filter tank, filtered, and then subjected to wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort. The aN of the saccharified cold wort is 210 mg / L. (5) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is slowly increased. The steam pressure is controlled at 2.6 Bar and the heating time is 25 min. During the wort boiling process, the pH is controlled at 5.0, the steam pressure is controlled at 2.0 Bar, the boiling time is 75 min, and the boiling evaporation rate is 8%. 1% wt of Qingdao Dahua is added during the boiling process. (6) Fermentation: After boiling, the wort is vortexed and settled, and the vortexing time is controlled within 15 minutes. After thin plate cooling, oxygen is introduced into the fermentation tank online, and the dissolved oxygen of the wort is controlled at 10 mg / L. At the same time, yeast W-34 / 70 is added, and the yeast concentration in the tank is controlled at 23 million / ml. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.06 mg / L, the temperature is further lowered and stored in cold storage at -1 to 0℃ for 7 days.

[0062] Comparative Example 2 Raw material formula: barley malt: caramel malt: rye malt: rice = 50:25:5:20; among which, the key indicators of barley malt are: aN 140mg / 100g, saccharification power 280WK; Caramel malt color: 100 EBC, Rye malt color: 1200 EBC; Brewing method: (1) Raw material crushing: Barley malt, roasted malt, rye malt and rice auxiliary material are crushed by a two-roll mill to obtain crushed raw materials; (2) The crushed rice auxiliary material is gelatinized separately in a gelatinization pot. The mash after the rice gelatinization is completed according to the standard process is mixed with the mash after the barley malt protein rests, and then saccharification is carried out.

[0063] (3) Saccharification process: The crushed malt is first fed at 50°C, and then kept at the same temperature for 50 minutes (including feeding time); then the temperature is raised to 65°C and kept for 75 minutes; then the temperature is rapidly raised to 76°C. (4) Wheat filtration: The mash after saccharification is transferred into a filter tank, filtered, and then subjected to wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort. The aN of the saccharified cold wort is 160 mg / L. (5) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is slowly increased. The steam pressure is controlled at 2.5 Bar and the heating time is 23 min. During the wort boiling process, the pH is controlled at 5.0, the steam pressure is controlled at 2.0 Bar, the boiling time is 60 min, and the boiling evaporation rate is 8%. 1% wt of Qingdao Dahua is added during the boiling process. (6) Fermentation: The wort after boiling is swirl-sedimented and the time is controlled within 15 minutes; after thin plate cooling, it is introduced into the fermentation tank with online oxygenation, and the dissolved oxygen of the wort is controlled at 10 mg / L. At the same time, yeast W-34 / 70 is added, and the yeast concentration in the tank is controlled at 23 million / ml. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.06 mg / L, it is further cooled and stored at -1 to 0℃ for 7 days.

[0064] Comparative Example 3 Raw material formula: barley malt: caramel malt: rye malt: rice = 50:25:5:20; among which, the key indicators of barley malt are: α-N 160mg / 100g, saccharification power 350WK; Caramel malt color: 100 EBC, Rye malt color: 1200 EBC; Brewing method: (1) Raw material crushing: Barley malt, roasted malt, rye malt and rice auxiliary material are crushed by a two-roll mill to obtain crushed raw materials; (2) The crushed rice auxiliary material is gelatinized separately in a gelatinization pot. The mash after the rice gelatinization is completed according to the standard process is mixed with the mash after the barley malt protein rests, and then saccharification is carried out.

[0065] (3) Saccharification process: The crushed malt is first fed at 50°C, and then kept at the same temperature for 50 minutes (including feeding time); then the temperature is raised to 65°C and kept for 75 minutes; then the temperature is rapidly raised to 76°C. (4) Wheat filtration: The mash after saccharification is transferred into a filter tank, filtered, and then subjected to wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort. The aN of the saccharified cold wort is 210 mg / L. (5) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is slowly increased. The steam pressure is controlled at 2.5 Bar and the heating time is 23 min. During the wort boiling process, the pH is controlled at 5.0, the steam pressure is controlled at 2.0 Bar, the boiling time is 60 min, and the boiling evaporation rate is 8%. Hops or hop products are added during the boiling process. (6) Fermentation: The wort after boiling is swirl-sedimented and the time is controlled within 15 minutes. After being cooled by thin plate, it is oxygenated online and introduced into the fermentation tank. The dissolved oxygen in the wort is controlled at 10 mg / L. At the same time, beer yeast TT-1 is added. The yeast content in the tank is controlled at 23 million / ml. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.06 mg / L, it is further cooled and stored at -1 to 0℃ for 7 days.

[0066] Comparative Example 4 Raw material formula (mass ratio): Barley malt: Roasted malt: Black malt: Rice = 50:25:5:20; Key indicators for barley malt include: α-N 140mg / 100g, and saccharification power 280WK. Caramel malt color: 100 EBC, Rye malt color: 1500 EBC; Brewing method: (1) Raw material crushing: Barley malt, roasted malt, rye malt and rice auxiliary material are crushed by a two-roll mill to obtain crushed raw materials; (2) The crushed rice auxiliary material is gelatinized separately in a gelatinization pot. The mash after the rice gelatinization is completed according to the standard process is mixed with the mash after the barley malt protein rests, and then saccharification is carried out. (3) Saccharification process: The crushed malt is first fed at 45°C, and then kept at the same temperature for 60 minutes (including feeding time); then the temperature is raised to 65°C and kept for 75 minutes; then the temperature is rapidly raised to 78°C. (3) Wheat filtration: The mash after saccharification is transferred into a filter tank and filtered to obtain saccharified wort. The aN of the saccharified wort is 158 mg / L. (4) Boiling the wort: The saccharified wort is heated to the initial boiling stage and the temperature is slowly increased. The steam pressure is controlled at 3.5 Bar and the heating time is 14.5 min. During the wort boiling process, the pH is controlled at 5.5, the steam pressure is 3.0 Bar, the boiling time is 75 min, and the boiling evaporation rate is 12%. Hops or hop products are added during the boiling process. (5) Fermentation: The wort after boiling is vortexed and settled, with the time controlled within 15 minutes; after thin plate cooling, it is introduced into the fermentation tank with online oxygenation, and the dissolved oxygen of the wort is controlled at 10-13 mg / L. At the same time, beer yeast TT-1 is added, and the yeast concentration in the tank is controlled at 21 million cells / ml. The yeast amide activity is controlled at above 200 u / L. The main fermentation temperature is 9.5±0.5℃. When the sugar content drops to 5.0°P, the temperature is raised to the reduction temperature of 12-13℃. When the diacetyl drops to 0.05 mg / L, it is further cooled and stored at -1 to 0℃ for 7 days.

[0067] Test case According to the test method specified in the national standard GB 5009.204-2014 "National Food Safety Standard - Determination of Acrylamide in Food", the acrylamide content in the mixed wort (uncooked), mixed wort (boiled), cold wort cooled after boiling, and finished beer of Examples 1-2 and Comparative Examples 1-4 was determined. The results are shown in Table 1.

[0068] Table 5 Acrylamide content (μg / L) in wort and beer at different stages under different treatments

[0069] As shown in Table 5, in Example 1, the acrylamide content decreased significantly from the mixed wort (unboiled) to the finished beer. The acrylamide formation rate after boiling was calculated to be (29.8-18.4) / 18.4=61.96%, and the reduction rate during fermentation was (29.8-3.0) / 29.8=89.9%. Comparative Example 1 (boiling for 75 min) and Example 1 (boiling for 60 min) show that, under the same formulation and strain conditions, extending the boiling time resulted in higher acrylamide content during the boiling and cold wort stages. The conversion rate during the boiling process was (35.6-17.0) / 17.0=109.41%, significantly higher than that in Example 1, indicating that prolonged boiling promoted acrylamide formation. The main difference between Comparative Example 2 and Example 1 is that malt with a lower aN content was used. Comparing the finished beer data of the two, it can be found that the acrylamide reduction rate of Comparative Example 2 during the fermentation stage is (28.5-12.1) / 28.5=57.54%, which is significantly lower than that of Example 1. This indicates that the aN content of wort is a key factor affecting the activity of yeast deaminase, and its reduction will significantly weaken the yeast's ability to metabolize and reduce acrylamide during fermentation. The main difference between Comparative Example 3 and Example 1 is that different yeast strains TT-1 were used. Comparing the finished beer data of the two, it can be calculated that the acrylamide reduction rate of Comparative Example 3 during fermentation was 81.38%, which was lower than that of Example 1 (89.9%). This indicates that the acrylamide conversion ability of yeast strain TT-1 is lower than that of strain W-34 / 70. The results of Comparative Example 4 indicate that when three unfavorable conditions—extending the boiling time, using low aN, and selecting yeast strains with weak conversion ability—exist simultaneously, a cumulative effect occurs, leading to a significant and progressively increasing acrylamide content throughout the entire process from wort preparation to finished beer. This highlights the importance of synergistic control of boiling time, raw material selection, and strain screening in the process for systematically reducing acrylamide content in beer.

Claims

1. A method for brewing dark beer, characterized in that, This includes the steps of ingredient matching, wort preparation, and beer fermentation. The wort preparation steps include saccharification, wort boiling process control, vortex sedimentation, and cooling. The wort boiling process control includes: The mashed wort is heated to the initial boiling stage at a slow temperature, with a steam pressure of 2.4-2.6 Bar and a heating time of 20-25 minutes. During the wort boiling process, the pH is controlled at 5.0-5.3, the steam pressure at 2.0-2.2 Bar, and the boiling time is 60 minutes, with a boiling evaporation rate of 8%. Hops or hop products are added during the boiling process.

2. The brewing method according to claim 1, characterized in that, In the raw material blending step, the mass percentage of roasted malt in the raw materials is controlled to be 20-30%.

3. The brewing method according to claim 2, characterized in that, In the raw material preparation step, the aN of the prepared saccharified wort is controlled to be ≥200mg / L, and the degree of fermentation is 65-68%.

4. The brewing method according to claim 3, characterized in that, In the raw material formulation step, the raw materials also include barley malt, with the aN of the barley malt controlled at ≥160mg / 100g and the saccharification power ≥300WK.

5. The brewing method according to claim 4, characterized in that, In the raw material preparation step, the raw materials also include rye malt and auxiliary materials, and the mass ratio of barley malt, roasted malt, rye malt and auxiliary materials is 50:(20-30):(0-5):(20-30). The caramel malt has a color intensity of 90-150 EBC, and the rye malt has a color intensity of 900-1500 EBC.

6. The brewing method according to claim 5, characterized in that, The auxiliary ingredients are rice, syrup or starch.

7. The brewing method according to claim 1, characterized in that, The saccharification process includes: first, feeding the material at 40-50 ℃, then holding it at the same temperature for 30-60 minutes; then raising the temperature to 63-68 ℃ and holding it for 60-90 minutes; then rapidly raising the temperature to 76-78 ℃, and finally transferring it to a filter tank for filtration, followed by wort boiling, vortex sedimentation and wort cooling processes to obtain saccharified cold wort; The vortex sedimentation time is controlled to be completed within 15 minutes.

8. The brewing method according to claim 1, characterized in that, The beer fermentation process includes the primary fermentation step, the reduction step, and the cooling and cold storage step. The temperature of the primary fermentation step is controlled at 9.5-10℃, and the fermentation time is 96-120h. The reduction temperature for the reduction step is 12-13℃, and the reduction time is 15 days. The cooling and cold storage step involves controlling the cooling and cold storage temperature between -1 and 0°C, and the cold storage time is 7 days.

9. The brewing method according to claim 8, characterized in that, In the beer fermentation step, brewer's yeast with an amidase activity ≥230u / L is used to ferment the wort.

10. The use of the brewing method according to any one of claims 1-9 in reducing the acrylamide content in dark beer.

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