A method for purifying microecology of live bacteria in a second time in a craft beer bottle

Abstract

The application discloses a method for purifying microecology of two times of live bacteria in a craft beer bottle, and relates to the technical field of craft beer brewing. The method comprises the following steps: yeast strain propagation, malt crushing, primary saccharification, in-tank first-time brewing, second-time base wine preparation, aseptic filling, in-bottle second-time brewing and low-temperature storage. In the first-time stage, the temperature is increased by 2-3 DEG C when the sugar content is 4.2-4.5 DEG P to promote diacetyl reduction, and the temperature is decreased by 4 DEG C when the sugar content is lower than 0.8-1.2 DEG P to stand and settle to remove yeast; the second-time wort addition amount is 4-10%, and the carbon dioxide is replaced by aseptic nitrogen gas; the in-bottle second-time brewing is first subjected to aerobic fermentation at 12-20 DEG C to consume dissolved oxygen, and then subjected to anaerobic reduction of diacetyl by increasing the temperature by 2-3 DEG C on the basis of the temperature; and the low-temperature storage is carried out at 0 DEG C for 5-7 days. The method can reduce wine loss, prolong the product shelf life to 18 months and reduce production cost.

Description

Technical Field

[0001] This invention belongs to the field of craft beer brewing technology, specifically relating to a method for secondary purification of the microecological environment of live bacteria inside craft beer bottles. Background Technology

[0002] Bottled craft beer refers to bottled beer products containing yeast. It has a cloudy appearance, fresh taste, and is characterized by aroma, carbonation, mellowness, sweetness, and fullness. Because no adjuncts are added, the live bacteria in the bottle remain fresh, and its quality is superior to pasteurized beer, draft beer, and ordinary fresh beer.

[0003] In existing technologies, craft breweries often adopt a simplified version of the technology used in large breweries. This involves processing the fermented liquid, brewed using a single-tank fermentation process, through heat sterilization and other treatments to obtain the final product. This technology has drawbacks such as the inactivation of yeast in the bottle due to heat, resulting in a less fresh product, significant beer loss during processing, and high production costs.

[0004] To address the aforementioned issues, there are existing technologies for mass production of craft beer via in-bottle secondary fermentation. However, these technologies still suffer from shortcomings such as short shelf life of live bacteria, high risk of near-expiration, and high beer spoilage. Therefore, there is an urgent need to further optimize process conditions to improve product quality and reduce production costs. Summary of the Invention

[0005] The purpose of this invention is to provide a method for purifying the microbial ecosystem of live bacteria in the secondary fermentation of craft beer bottles, in order to solve the problems of short shelf life, high risk of near expiration, high beer loss and high production cost of live bacteria in the secondary fermentation of craft beer in bottles, as well as insufficient diacetyl reduction in the first fermentation stage, poor purification effect of the base beer in the first fermentation stage, and product aging caused by residual dissolved oxygen in the secondary fermentation stage.

[0006] The present invention adopts the following technical solution: This solution provides a method for purifying the microbial ecosystem of secondary viable bacteria inside craft beer bottles, including the following steps: S1. Yeast strain expansion culture to obtain an inoculum containing live yeast cells; S2, malt powder; S3. Preparation of wort through a single saccharification process; S4. In-tank fermentation: The inoculum is inoculated into the wort for primary fermentation, which proceeds sequentially through the aerobic fermentation stage, the high-foaming fermentation stage, the diacetyl reduction stage, and the primary base liquor stage. During the diacetyl reduction stage, when the sugar content of the fermentation liquid drops to 4.2–4.5°P, the fermentation temperature is increased by 2–3°C. During the primary base liquor stage, when the sugar content of the fermentation liquid is lower than 0.8–1.2°P, the fermentation temperature is decreased by 4°C. After standing for 48–72 hours, the yeast and coagulated matter at the bottom of the tank are drained to obtain the primary base liquor. S5. Preparation of second fermented base liquor: Add second wort to the first fermented base liquor. The amount of second wort added is 4-10% of the volume of the first fermented base liquor, preferably 4-8%. Inflate with sterile nitrogen for 10-20 minutes to replace part of the carbon dioxide. Let stand at 0.01 MPa pressure for 12-24 hours and then drain the condensate at the bottom of the tank to obtain the second fermented base liquor. S6. Aseptic filling: The second-stage base spirit is filled into beer containers and sealed under sterile nitrogen protection throughout the process; S7. Second fermentation in bottle: The bottled beer is allowed to stand at 12-20°C for 36-48 hours, with the bottom fermentation yeast preferably at 12-14°C and the top fermentation yeast preferably at 18-20°C, so that the yeast in the bottle can carry out aerobic fermentation to consume dissolved oxygen; then the temperature is increased by 2-3°C from the above-mentioned second fermentation temperature in the bottle, and anaerobic reduction of diacetyl in the bottle is carried out for 72-90 hours. S8. Low-temperature aging: Place the beer in the bottle after the second fermentation in a 0℃ environment for 5 to 7 days.

[0007] Furthermore, the inoculum in S1 contains more than 100 million live yeast cells per milliliter.

[0008] Furthermore, the diameter of the coarse particles after malt grinding in S2 is 0.4–0.8 mm, and the mass ratio of coarse to fine particles is 2–5:1.

[0009] Furthermore, the S3 saccharification process sequentially includes low-temperature enzymatic hydrolysis, medium-temperature enzymatic hydrolysis, and high-temperature backwash enzymatic hydrolysis. The low-temperature enzymatic hydrolysis temperature is 50–55°C, with a settling time of 20–40 minutes; the medium-temperature enzymatic hydrolysis temperature is 60–65°C, with a settling time of 50–80 minutes; and the high-temperature backwash enzymatic hydrolysis temperature is 75–80°C. After saccharification, the wort is filtered and boiled, resulting in a wort concentration of 10–12°P. The boiling process includes pre-cooking and the addition of hops. The pre-cooking temperature is 100–110°C, with bittering hops added after 5–10 minutes of boiling, followed by boiling for another 50–60 minutes, then adding aroma hops, and boiling for another 5–10 minutes.

[0010] Furthermore, in S4, the inoculation amount is 5‰ of the volume of the inoculation liquid, and the pressure inside the tank during inoculation is 0.01 MPa; the temperature of the aerobic fermentation stage is 8-18℃, of which the bottom fermentation yeast is preferably 8-10℃ and the top fermentation yeast is preferably 16-18℃, and the fermentation lasts for 3-4 days; the temperature of the high-foaming fermentation stage is the same as that of the aerobic fermentation stage, and the fermentation lasts for 3-4 days.

[0011] Furthermore, the secondary wort in S5 is pure wort without added hops, and it is not oxygenated during the cooling process. After adding the secondary wort, expanded yeast culture is also introduced, and sterile nitrogen is used to backflush and stir from the bottom of the tank to ensure even mixing.

[0012] Furthermore, in S6, sterile nitrogen is used to pressurize and transport the second-stage base liquor to the aseptic filling production line. The empty bottles are sterilized and then aseptically filled, with the entire filling process completely isolated from air.

[0013] Furthermore, after the second fermentation in the S7 bottle, the dissolved oxygen content in the beer is less than 0.04 mg per liter, and the diacetyl content is less than 0.09 mg per liter.

[0014] Furthermore, in S8, the beer is transferred to a 0°C cellar. Timing begins when the temperature of the beer in the container drops to 0°C. During cellaring, the container is stored upside down to ensure that the yeast metabolites are fully mixed. After cellaring is completed, samples are taken for testing, and the beer is removed from the cellar only if it passes the test.

[0015] The present invention also provides a craft beer prepared using the above method.

[0016] The beneficial effects achieved by adopting the above solution are as follows: 1. By triggering temperature control based on changes in the sugar content of the fermentation broth during the first fermentation diacetyl reduction stage, the yeast's ability to reduce diacetyl is enhanced, allowing the diacetyl produced in the first fermentation to be fully reduced in the first fermentation stage. This reduces the diacetyl flavor content, eliminates the stale taste, produces more 2,3-butanediol, and improves the antioxidant and preservative effects of the craft brewing microecology, which is beneficial for extending the product's shelf life.

[0017] 2. By triggering cooling and allowing the fermentation liquid to settle during the first fermentation stage of the base liquor based on changes in sugar content, the residual yeast in the first fermentation base liquor is separated to the maximum extent without the use of external forces such as centrifuges, filters, and precipitants. This accelerates the sedimentation and removal of dead yeast and solidified matter, resulting in better original ecological purification of the first fermentation base liquor in the tank. This is beneficial for reducing sediment in the second fermentation product in the bottle, and making the taste more fragrant, gritty, mellow, and sweet.

[0018] 3. By controlling the amount of secondary wort added to a low level of 4-10% (preferably 4-8%), the carbon dioxide content and pressure index generated during secondary fermentation in the bottle are reduced, thereby reducing the stringent requirements on the pressure resistance parameters of the packaging container. Low-cost packaging containers can be selected, and the cost of packaging materials is significantly reduced.

[0019] 4. By introducing sterile nitrogen after secondary saccharification to replace some of the carbon dioxide and remove other off-flavors, and allowing the fermenter to stand under slightly positive pressure to enhance the secondary discharge of condensate from the bottom of the fermenter, the final product is cleaner and of better quality.

[0020] 5. By controlling the temperature in stages during the second fermentation in the bottle, the yeast is first allowed to undergo aerobic fermentation at a suitable temperature to consume the oxygen in the residual air at the bottle opening during bottling, reducing the dissolved oxygen to an extremely low level; then the temperature is increased to anaerobically reduce diacetyl, reducing the diacetyl content to below the flavor threshold, thus providing preservation conditions and effectively preventing the reaction of residual dissolved oxygen with amino acids to produce an aging flavor, thereby improving the freshness of the craft beer in the bottle.

[0021] 6. Through low-temperature aging, the containers are stored upside down and fully exchanged for cold. The yeast metabolites are fully mixed and recombined under low-temperature conditions. The live yeast forms a suspended complex with amino acids, alcohols, minerals, etc. The microecology inside the bottle gradually ages and stabilizes, and the secondary fermentation of live bacteria in the bottle matures. Attached Figure Description

[0022] Figure 1 This is a flowchart of the secondary fermentation process for the microecological purification of live bacteria inside craft beer bottles according to the present invention. Detailed Implementation

[0023] The technical solutions of 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.

[0024] Example 1: Bottled Live Culture Craft Lager Brewing This experiment was a pilot-scale test using 1000L of saccharification and 1000L of fermentation as feed batches. The steps are as follows: I. Process Steps: such as Figure 1 As shown, the process is as follows: fermentation of liquid yeast culture → malt crushing → primary saccharification to prepare malt wort → in-tank fermentation and primary fermentation of base wine → secondary saccharification and secondary fermentation of base wine → aseptic bottling → in-bottle secondary fermentation → cellar storage → packaging and warehousing.

[0025] II. Process conditions and parameters: 1. Propagation of bottom-fermented liquid yeast strain: A suspension-type bottom-fermented yeast strain was selected. Following the strain propagation process, the slant culture was aseptically inoculated into pre-sterilized 10ml original wort 12°P test tubes in a sterile operating table in a sterile room. Three test tubes were inoculated simultaneously, and the tubes were placed in a shaker incubator set to 25°C for 12–24 hours for activation. Propagation was then carried out sequentially to 100ml Erlenmeyer flasks, 1000ml Erlenmeyer flasks, and 10L cartridge tanks. Finally, the 10L cartridge tank with the most vigorous fermentation was inoculated into a 100L incubator containing hop-added 12°P wort. The incubator was set to 25°C for 24–48 hours. Successful propagation was indicated by a microscopic examination showing more than 100 million healthy cells per milliliter of liquid culture.

[0026] 2. Malt Crushing: 200 kg of brewing barley malt is moistened with 50℃ warm water to increase the malt moisture content by 0.7-1.5%, and then fed into the hopper for crushing. Dry crushing technology is used, adjusting the roller gap to 0.5-1.0 mm. The malt husk is broken but not crushed, and the malt grains are crushed to a coarse particle diameter of approximately 0.5 mm, with a coarse-to-fine particle ratio of approximately 4:1. The malt is simultaneously fed into the mashing pot, and feeding is completed in approximately 30 minutes.

[0027] 3. Preparation of wort through single-stage saccharification: (1) Saccharification: ① Feeding and low-temperature enzymatic hydrolysis: The saccharification pot is filled with brewing hot water in advance, prepared according to the ratio of feed to water by weight of 1:4, and the water temperature is 53℃. After feeding, the saccharification mash is kept at 53℃ for 30 minutes; ② Medium-temperature enzymatic hydrolysis: the temperature is raised to 65℃ and kept for 60 minutes; ③ High-temperature mash diversion enzymatic hydrolysis: after the temperature is raised to 78℃, all the saccharification mash is transferred to the wort filter tank.

[0028] (2) Filtration: After circulating filtration, first wort filtration, second wort filtration and third wort filtration, the concentration of mixed wort was measured to be 11°P.

[0029] (3) Boiling: ① Pre-boil at normal pressure with a boiling temperature of about 105℃; ② Add hops: After boiling for 5 minutes, add 800g of William Meat bitter hops, continue boiling and maintain boiling for 60 minutes, then add 700g of Cascade aromatic hops, maintain boiling for 5 minutes, then stop heating and let stand for 10 minutes. The mature wort concentration was measured to be 12.1°P, and the hot wort volume was 1080L.

[0030] (4) Vortex sedimentation: The mature hot wort is transported to the vortex sedimentation tank, circulated internally for 15 minutes, and then left to stand for 20 minutes.

[0031] (5) Cooling into the tank: The hot wort is cooled to 9°C by a two-stage plate cooler and sterile oxygen is added simultaneously. The cold wort is then transported to a fermentation tank with a volume of 1040L.

[0032] 4. In-tank fermentation of the first and second base spirits: (1) Inoculation: Before the cold wort is put into the fermentation tank, add 50L of pre-cultured active liquid yeast at an inoculation rate of 5‰ and mix with 1090L of cold wort. The pressure of the fermentation tank is set to 0.01MPa, and the dissolved oxygen concentration of the cold wort in the tank is measured to be 14ppm.

[0033] (2) Primary fermentation: Aerobic fermentation: Set the temperature inside the fermenter to 9°C. On the 4th day after the yeast is added to the fermenter, the number of yeast cells is measured and the culture is expanded to ≥15 million per milliliter. The sugar reduction curve is 0.5~0.7°P.

[0034] High-foaming fermentation: On days 5 to 8 after entering the tank, the sugar reduction curve is 1.0 to 1.5°P, the sugar content drops to 4.5°P, the diacetyl content is greater than 0.17 mg per liter, and the yeast count drops to about 10 million per milliliter.

[0035] Diacetyl reduction: When the sugar content dropped to 4.5°P, the pressure was kept constant at 0.01 MPa, and the temperature inside the fermenter was set to 12°C and maintained for 5 days. The sugar content gradually decreased, and on the fifth day, the sugar content was measured at 1.8°P with no change. The diacetyl content dropped to 0.11 mg per liter.

[0036] First-stage base liquor: When the sugar content of the fermentation liquid in the tank is below 1°P, the temperature inside the fermentation tank is set to decrease to 8°C, automatically decreasing by 0.5°C per hour, maintaining a pressure of 0.01MPa inside the tank, and left to stand for 3 days. The condensate is discharged daily to obtain the first-stage base liquor.

[0037] 5. Secondary saccharification and second fermentation of base spirits: (1) Secondary saccharification: Secondary saccharification begins on the third day of the initial stabilization period of the base wine. 100L of mature hot wort at 12°P is prepared, boiled at an evaporation intensity of 8.5%, and cooled to 8°C before being added to the initial fermentation tank of the base wine.

[0038] (2) Mixed inoculation: Take the same fresh liquid yeast strain for bottom fermentation and inoculate it into the first fermentation tank of base wine. Use sterile nitrogen to backflush from the bottom material outlet of the tank for 15 minutes to mix thoroughly and replace part of the carbon dioxide in the tank. Maintain the pressure in the tank at 0.01 MPa, let it stand for 12-24 hours, and drain the condensate at the bottom of the tank to obtain 1140L of second fermentation base wine.

[0039] 6. Aseptic Filling: The secondary fermented base liquor is pressurized with sterile nitrogen and transported to the aseptic filling production line. The entire process is protected by sterile nitrogen. After cleaning, the tin cans are aseptically filled. The liquid level valve is adjusted so that the base liquor in the bottle exceeds the standard capacity of 630ml. The bottles are then sealed, rinsed, and air-dried. Online monitoring of the sealing quality and weight is conducted to remove substandard products. A total of 1702 bottles of semi-finished base liquor were obtained, with a filling loss of 2.74%.

[0040] 7. Secondary Fermentation in Bottles: The qualified product containers are transferred to a craft beer reconstitution chamber, where the temperature is controlled at 12℃ and the mixture is left to stand for 48 hours. During this time, the yeast in the bottles undergoes autophagy of dissolved oxygen, secondary aerobic fermentation, and yeast cell division. The chamber temperature is then increased by 2℃, and the time is set when the temperature of the canned product reaches 14℃. This process continues for 72 hours, during which the yeast in the bottles undergoes autophagy of diacetyl. If the diacetyl content is measured to be <0.09 mg / L, the secondary reconstitution is complete.

[0041] 8. Cellaring: Transfer the semi-finished products from the second fermentation process to the cellar. The temperature inside the cellar is 0℃. The time is set when the temperature of the canned wine drops to 0℃. The wine is stored in the cellar for 7 days. After passing the quality inspection of 4 bottles, the wine is removed from the cellar.

[0042] 9. Packaging and Warehousing: The appearance and weight are inspected on the packaging line, the barcode is scanned and the bottles are packed into boxes of 630ml x 6 bottles each, and qualified products are put into the warehouse.

[0043] III. Quality Inspection: Sensory, physicochemical, and hygiene indicators were tested according to GB / T4927 standards. Sensory inspection revealed a golden-yellow body with uniform suspension and no sediment; the foam was white and fine, clinging to the glass for more than 130 seconds; and the flavor was aromatic, mellow, and sweet. Physicochemical testing showed an alcohol content ≥4.2% vol, original wort concentration 12.1°P, carbon dioxide content ≥0.50%, dissolved oxygen <0.04 mg / L, diacetyl content <0.09 mg / L, and a shelf life of 18 months at room temperature. Hygiene indicators showed an active yeast count of 1 x 10^7 cells / mL, and no pathogenic bacteria were detected. Conclusion: The beer meets the GB / T4927 premium grade standard for specialty beers.

[0044] Example 2: Bottled Live Culture Craft Ale Brewing This experiment was a pilot-scale test using 1000L of saccharification and 1000L of fermentation as feed batches. The experimental steps were the same as in Example 1, but the process conditions and parameters differed as follows: 1. Propagation of liquid yeast strains for top fermentation: Use suspension-type top fermentation yeast strains in test tubes, and use the same propagation conditions and parameters as Lager.

[0045] 2. Malt grinding: 120 kg of beer barley malt, 70 kg of wheat malt, and 5 kg of caramel malt with a color intensity of 50 EBC. Only the barley malt is moistened with 50°C warm water; the wheat malt and caramel malt are kept dry. The barley malt grinding process is the same as for lager, and the average particle size of the wheat malt and caramel malt is less than 0.2 mm.

[0046] 3. Preparation of wort through primary saccharification: Boil and add 700g of bitter chrysanthemum and 1000g of fragrant chrysanthemum. Cool the wort to a temperature of 18℃ and pour it into a tank with a capacity of 1060L.

[0047] 4. In-tank fermentation of the first and second base spirits: For aerobic fermentation, the fermentation tank temperature is set at 18°C. On the third day after fermentation, the yeast count is checked and increased to ≥15 million cells per milliliter. For high-foam fermentation, on days 4-6 after fermentation, the sugar content is checked and measured to have decreased to 4.5°P, with diacetyl content greater than 0.18 mg / L. When the diacetyl reducing sugar content decreases to 4.5°P, the fermentation tank temperature is raised to 20°C and maintained for 4 days, until the diacetyl content decreases to 0.12 mg / L. For the second base spirit, the fermentation tank temperature is lowered to 16°C.

[0048] 5. Secondary saccharification and preparation of base wine: 100L of mature hot wort from secondary saccharification is cooled to 16℃ and added to the primary base wine fermentation tank.

[0049] 6. Aseptic filling: 630ml cans, actually yielded 1686 bottles of semi-finished base wine, with a 2.9% loss during filling.

[0050] 7. Second aging in the bottle: The container is transferred to the Shangfa craft brewing reduction room and left to stand at 20°C for 24 hours. The temperature is then increased by 2°C. When the temperature of the sample reaches 22°C, the time is started. The reduction in the bottle lasts for 48 hours. The diacetyl content is tested to be <0.1mg per liter.

[0051] 8. Cellar storage: The temperature inside the cellar is 0℃, and the cellar storage is maintained for 5 days.

[0052] III. Quality Inspection: Sensory inspection revealed an orange-yellow, uniformly turbid beer with no sediment; fine, white foam that clings to the glass for over 150 seconds; and a smooth, rich, and mellow taste with a sweet aftertaste. Physicochemical testing showed an alcohol content ≥4.2% vol, original wort concentration 12.0°P, carbon dioxide content ≥0.45%, dissolved oxygen <0.04 mg / L, diacetyl content <0.09 mg / L, and a shelf life of 18 months at room temperature. Hygiene indicators showed an active yeast count of 1 x 10^7 cells / mL, and no pathogenic bacteria were detected. Conclusion: The beer meets the GB / T4927 Special Grade Beer Standard.

[0053] Example 3: In-bottle live culture craft brewing of IPA varietal This experiment was a scale-up pilot-scale test using 2000L saccharification and 2000L fermentation batches as the feed batches. The steps were the same as in Example 2, but the process conditions and parameters differed as follows: 2. Malt grinding: 240 kg of beer barley malt, 140 kg of wheat malt, 10 kg of caramel malt with a color intensity of 100 EBC, and 20 kg of dark caramel malt with a color intensity of 400 EBC. The dark caramel malt is ground and added to the filter tank during filtration. 800 L of washer water and 2400 L of mixed wort are also used.

[0054] 3. First mashing: Boil and add 1200g of bitters hops and 2500g of aroma hops. Obtain 2180L of mature hot wort, then cool and transfer to tanks to obtain 2100L of wort.

[0055] 4. First fermentation in tank: Inoculate 100L of liquid yeast, ferment 2200L of mixed liquid in fermentation tank, and obtain 2100L of base wine for the first fermentation.

[0056] 5. Secondary saccharification: Prepare 200L of secondary wort to obtain 180L of mature cold wort and 2270L of second fermentation base spirit.

[0057] 6. Aseptic filling: Using a 1L tinplate can aseptic filling machine, 2202 bottles of base wine semi-finished product were actually filled, with a wine loss of 2.9% during filling and pipeline.

[0058] 7. Secondary evaporation in the bottle: After completing the secondary reduction, the diacetyl content was detected to be <0.1 mg per liter.

[0059] III. Quality Inspection: Sensory inspection revealed a deep red color with uniform suspension and no sediment; the foam was white and delicate, clinging to the glass for over 160 seconds; the flavor was fresh, with a mature floral aroma, a mellow and sweet aftertaste. Physicochemical testing showed an alcohol content ≥4.2% vol, original wort concentration 12.2°P, carbon dioxide content ≥0.46%, dissolved oxygen <0.04 mg / L, diacetyl content <0.1 mg / L, and a shelf life of 18 months at room temperature. Hygiene indicators showed an active yeast count of 1 x 10^7 cells / mL, and no pathogenic bacteria were detected. Conclusion: This beer meets the GB / T4927 Special Grade Beer Standard.

[0060] Example 4: Brewing of Stout with live bacteria in a bottle This experiment was conducted on a scaled-up pilot production line with a feed batch of 2000L saccharification and 4000L fermentation. The steps were the same as in Example 3, but the process conditions and parameters differed as follows: 2. Malt Crushing: 240 kg of beer barley malt, 140 kg of wheat malt, 10 kg of caramel malt with a color intensity of 100 EBC, 10 kg of dark caramel malt with a color intensity of 400 EBC, and 30 kg of rye malt. The crusher has a capacity of 1 ton per hour and can complete the feeding within 30 minutes.

[0061] 3. First saccharification: Two batches of feed are completed within 24 hours, with a total of 4200L of cold wort added to the tank.

[0062] 4. First fermentation in tank: 100L of liquid yeast is inoculated at once and mixed with 2200L of the first batch of cold wort. The second batch of cold wort is added to the tank 4-6 hours later, with a total of 4300L of the mixture added to the tank, resulting in 4200L of base spirit for the first fermentation.

[0063] 5. Secondary saccharification: 400L of mature cold wort with secondary sugars is obtained, and 4560L of base spirit is obtained after secondary fermentation.

[0064] 6. Aseptic filling: Using a 1L tinplate can aseptic filling machine, 4423 bottles of base wine semi-finished product were filled, with a wine loss of 2.96% during filling and pipeline.

[0065] III. Quality Inspection: Sensory Inspection: The beer has a coffee-black, uniform suspension with no sediment; the foam is white and delicate, clinging to the glass for more than 170 seconds; the taste is coffee aroma with a hint of milkiness, a mellow and sweet finish. Physicochemical Testing: Alcohol content ≥ 4.2% vol, original wort concentration 12.0°P, carbon dioxide content ≥ 0.50%, dissolved oxygen < 0.04 mg / L, diacetyl content < 0.1 mg / L, shelf life 18 months at room temperature. Hygiene Indicators: Active yeast count 1 x 10^7 cells / mL, no pathogenic bacteria detected. Conclusion: Meets the GB / T4927 Special Grade Beer Standard.

[0066] Table 1 Comparison of Key Process Parameters for Each Variety Table 2 Comparison of Inspection Results for Various Varieties Comparative Example 1 The difference from Example 1 is that the temperature during the diacetyl reduction stage is not increased to 12°C, but maintained at the original temperature of 9°C; the temperature inside the tank during the first fermentation of the base spirit is not decreased to 8°C, but maintained at the original temperature. Everything else is the same as in Example 1.

[0067] In Comparative Example 1, when the diacetyl reduction stage was maintained at 9°C, the diacetyl content in the first-fermented base spirit was approximately 0.1 mg / L. This increased the pressure on the yeast reduction process during the subsequent secondary fermentation, necessitating an increase in the amount of wort added and a longer reduction time, yet diacetyl content remained poorly controlled. When the temperature was not lowered during the first-fermentation stage, visually, the remaining dead, shocked, and weakened yeast cells did not precipitate completely, with yeast counts exceeding 1.2 x 10^7 cells per milliliter. This resulted in excessive turbidity of the spirit, leading to weakened carbonation and poor taste in the final product, along with increased sediment and unpleasant sensory characteristics.

[0068] Comparative Example 2 The difference from Example 1 is that: 120L of mature hot wort at 12°P was prepared by secondary saccharification, the boiling evaporation intensity was 8.5%, and it was cooled to 8°C before being added to the primary fermentation tank. Everything else is the same as in Example 1.

[0069] In Comparative Example 2, the increased amount of wort during secondary saccharification led to a rise in the temperature of the base spirit during secondary fermentation, causing fermentation to begin in the fermentation tank. The residual yeast did not settle completely during secondary fermentation, resulting in excessive foam that severely affected aseptic bottling, impacting the effects of secondary fermentation and secondary reduction, and resulting in poor quality of the secondary fermentation.

[0070] Comparative Example 3 The difference from Example 1 is that: after the secondary fermentation in the bottle was carried out at a temperature of 12°C for 48 hours, the temperature of the incubator was maintained at 12°C for 72 hours of in-bottle reduction without raising the temperature. Everything else is the same as in Example 1.

[0071] Table 3 Comparison of effects between comparative examples and Example 1 In Comparative Example 3, when the greenhouse temperature is not raised, the yeast activity is very low under low temperature conditions. Its own aerobic fission consumes dissolved oxygen slowly, the second fermentation time is long, it occupies the second fermentation greenhouse, it cannot effectively reduce diacetyl, and it may be surpassed by the reproduction rate of other bacteria, or be rancid by aerobic bacteria, resulting in failure of the second fermentation and loss.

[0072] In summary, all four batches of pilot-scale experiments passed, with a beer loss rate not exceeding 3%, which is more than 70% lower than that of pasteurized and flash-pasteurized craft beer. Ultra-low dissolved oxygen was <0.04 mg / L, and diacetyl was controlled below 0.1 mg / L for all batches. Bottled craft beer produced using the bottled live bacteria room-temperature preservation technology of this invention passed follow-up tests within its shelf life. The product maintains freshness and quality under room-temperature conditions with live bacteria in the bottle, exhibiting stable quality and meeting the enterprise's internal control standards for bottled live bacteria craft beer and the national standard GB / T4927. The above embodiments are preferred embodiments of this invention, but the embodiments of this invention are not limited to the above embodiments.

[0073] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0074] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for secondary fermentation of live bacteria microecological purification in craft beer bottles, characterized in that, Includes the following steps: S1. Yeast strain expansion culture to obtain an inoculum containing live yeast cells; S2, malt powder; S3. Preparation of wort through a single saccharification process; S4. In-tank fermentation: The inoculum is inoculated into the wort for primary fermentation, which proceeds sequentially through an aerobic fermentation stage, a high-foaming fermentation stage, a diacetyl reduction stage, and a primary base liquor stage. During the diacetyl reduction stage, when the sugar content of the fermentation liquid drops to 4.2–4.5°P, the fermentation temperature is increased by 2–3°C compared to the high-foaming fermentation stage. During the primary base liquor stage, when the sugar content of the fermentation liquid is lower than 0.8–1.2°P, the fermentation temperature is decreased by 4°C. After standing for 48–72 hours, the yeast and coagulated matter at the bottom of the tank are discharged to obtain the primary base liquor. S5. Preparation of second fermented base liquor: Add second wort to the first fermented base liquor. The amount of second wort added is 4-10% of the volume of the first fermented base liquor. Inflate with sterile nitrogen for 10-20 minutes to replace part of the carbon dioxide. Let stand at 0.01 MPa pressure for 12-24 hours and then drain the condensate at the bottom of the tank to obtain the second fermented base liquor. S6. Aseptic filling: The second-stage base spirit is filled into beer containers and sealed under sterile nitrogen protection throughout the process; S7. Second fermentation in the bottle: After bottling, the beer is allowed to stand at 12-20°C for 36-48 hours to allow the yeast in the bottle to undergo aerobic fermentation to consume dissolved oxygen; then the temperature is increased by 2-3°C from the above-mentioned second fermentation temperature in the bottle, and anaerobic reduction of diacetyl in the bottle is carried out for 72-90 hours. S8. Low-temperature aging: Place the beer in the bottle after the second fermentation in a 0℃ environment for 5 to 7 days.

2. The method for secondary viable microbial purification of craft beer bottles according to claim 1, characterized in that, In S1, the inoculum contains more than 100 million live yeast cells per milliliter.

3. The method for secondary purification of craft beer bottles using live microbial microecology according to claim 1, characterized in that, In S2, the diameter of the coarse particles after malt crushing is 0.4-0.8 mm, and the mass ratio of coarse to fine particles is 2-5:

1.

4. The method for secondary viable microbial purification of craft beer bottles according to claim 1, characterized in that, In S3, the first saccharification process sequentially includes low-temperature enzymatic hydrolysis, medium-temperature enzymatic hydrolysis, and high-temperature backwash enzymatic hydrolysis. The low-temperature enzymatic hydrolysis temperature is 50–55°C, and the mixture is allowed to stand for 20–40 minutes. The medium-temperature enzymatic hydrolysis temperature is 60–65°C, and the mixture is allowed to stand for 50–80 minutes. The high-temperature backwash enzymatic hydrolysis temperature is 75–80°C. After saccharification, the wort is filtered and boiled, and the wort concentration after filtration is 10–12°P. The boiling process includes pre-cooking and the addition of hops. The pre-cooking temperature is 100–110°C. After boiling for 5–10 minutes, bitter hops are added, and after boiling for another 50–60 minutes, aroma hops are added, and the mixture is boiled for another 5–10 minutes.

5. The method for secondary purification of craft beer bottles using live bacteria microecological processes according to claim 1, characterized in that, In S4, the inoculation amount is 5‰ of the volume of the inoculation liquid, and the pressure inside the tank during inoculation is 0.01 MPa; the temperature of the aerobic fermentation stage is 8-18℃, and the fermentation time is 3-4 days; the temperature of the high-foaming fermentation stage is 8-18℃, and the fermentation time is 3-4 days.

6. The method for secondary purification of craft beer bottles using live microbial microecology according to claim 1, characterized in that, In S5, the secondary wort is pure wort without added hops, and the secondary wort is not oxygenated during the cooling process; after adding the secondary wort, expanded yeast culture is also inoculated, and sterile nitrogen is used to backflush and stir from the bottom of the tank to make it evenly mixed.

7. The method for secondary purification of craft beer bottles using live bacteria microecological processes according to claim 1, characterized in that, In S6, the base liquor from the second fermentation is transported to the aseptic bottling production line under sterile nitrogen pressure. The empty bottles are sterilized and then aseptically filled, with the entire bottling process completely isolated from air.

8. The method for secondary viable microbial purification of craft beer bottles according to claim 1, characterized in that, In S7, after the second fermentation in the bottle, the dissolved oxygen content in the beer is less than 0.04 mg / L and the diacetyl content is less than 0.09 mg / L.

9. The method for secondary evaporation of live bacteria microecological purification in craft beer bottles according to claim 1, characterized in that, In S8, the beer is transferred to a 0℃ cellar. The timing begins when the temperature of the beer in the container drops to 0℃. During the cellaring period, the container is stored upside down to ensure that the yeast metabolites are fully mixed. After the cellaring is completed, samples are taken for testing, and the beer is removed from the cellar if it passes the test.

10. A craft beer, characterized in that, It is prepared using the secondary viable microbial purification method for craft beer bottles as described in any one of claims 1 to 9.

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