Lactobacillus gasseri JM1 acidified beer and preparation method thereof

Abstract

The present invention discloses a Lactobacillus gasseri JM1 acidified beer and a preparation method thereof, belonging to the field of microbial technology. The preparation method comprises the following steps: S01, weighing malt, crushing it, and saccharifying it at 67°C for 60 minutes at a mass ratio of malt to water of 1:4. After saccharification, filtering the wort and washing the slag with 76-78°C slag water to obtain the slag to be acidified; S02, after washing the slag, cooling the slag to 37°C, and stirring at 3.5×10 6 The acidified beer of the present invention exhibits the fastest pH decrease rate during acidification and the highest bacterial count of Lactobacillus gasseri JM1, shortens the wort acidification time, and enhances the overall fruity and floral aroma of the beer.

Description

Technical Field

[0001] The invention relates to Lactobacillus gasseri JM1 acidified beer and a preparation method thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Beer is one of the most consumed beverages in the world, and sensory flavor is a key characteristic of beer. Different raw materials and brewing techniques contribute to varying beer flavors, and a balanced flavor profile is crucial for optimal consumer experience. The wort acidification process imparts a unique flavor profile to beer, which is particularly pronounced in sour beers. Compared to "normal beer," this results in a lower pH and an increased intensity of corresponding sensory attributes (such as acidic taste).

[0003] Wort acidification is a key step in sour beer production. Acidification methods fall into two distinct categories: non-biological and biological acidification. These methods can create distinct flavor profiles, further diversifying beer flavors. Non-biologically acidified beer, also known as chemically acidified beer, is acidified by adding food-grade organic acids such as lactic acid, fresh fruit juice, or lemon juice. While non-biological acidification is suitable for large-scale production due to its speed, simplicity, and low cost, it lacks the ability to maintain changes over time due to the presence of metabolically active microorganisms, potentially resulting in a loss of flavor and mouthfeel. Biological acidification is achieved through the growth and metabolic activity of lactic acid bacteria, such as Lactobacillus plantarum or Lactobacillus amyloliquefaciens. Lactobacilli are Gram-positive, rod-shaped bacteria that produce lactic acid as the primary metabolite of carbohydrate metabolism. Their metabolism is classified as either obligate homofermentative, meaning they convert hexoses almost exclusively into lactic acid, or obligate or facultative heterofermentative, converting hexoses into lactic acid along with CO2 and ethanol or acetic acid. Lactobacilli are also important contributors to the production of many food products through mixed fermentation. While traditional beer fermentation is typically limited to a single yeast, sour beer is produced through polymicrobial fermentation. Controlled mixed fermentation is one of the mainstays of beer fermentation, allowing for the timely isolation of different microorganisms. Compared to traditional methods, pure culture fermentation using Lactobacilli, combined with careful application of processing steps, offers an effective alternative for promoting the production of sour beer. Furthermore, the addition of lactic acid bacteria improves the overall flavor intensity and flavor profile of beer. Beers produced with Lactobacillus plantarum exhibit enhanced fruity and dried fruit aroma intensity, while beers with Lactobacillus brevis exhibit higher overall flavor intensity, sourness, and astringency. The higher sourness intensity in beer is directly related to the increased content of organic acids, such as lactic acid, caused by the involvement of acid-producing bacteria during fermentation, which in turn leads to a lower pH. Thus, while biological acidification can provide a rich flavor profile, chemical and microbial pathways may be responsible for flavor development within the beer matrix.

[0004] Due to the complexity of the beer brewing process and the entire supply chain, assessing the importance of each pathway is challenging. It's well known that acidification significantly impacts beer flavor, but the extent and extent of its impact on key processes is less clear. Furthermore, existing acidification processes suffer from slow acidification rates and prolonged acidification times. This study examines the impact of bioacidification with Lactobacillus gasseri JM1 on beer sensory quality and investigates a rapid acidification process using Lactobacillus gasseri JM1 to improve production efficiency. Summary of the Invention

[0005] The present invention aims to provide a Lactobacillus gasseri JM1 acidified beer. The acidified beer has the fastest pH drop rate during acidification and the highest bacterial count of Lactobacillus gasseri JM1, thereby shortening the acidification time of wort. At the same time, the acidified beer increases the concentrations of lactic acid, citric acid and malic acid, reduces the concentration of succinic acid, and overall enhances the fruity and floral aromas of the beer.

[0006] At the same time, the purpose of the present invention is to provide a preparation method of Lactobacillus gasseri JM1 acidified beer, which can give the beer a unique flavor.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for preparing Lactobacillus gasseri JM1 acidified beer comprises the following steps:

[0009] S01, weighing malt, crushing it, and saccharifying it at 67°C for 60 minutes at a malt to water ratio of 1:4. After saccharification, filtering the wort, and washing the slag with 76-78°C slag water to obtain the acidified wort, wherein the mass percentage of malt raw material in the acidified wort is 22.5%;

[0010] S02, after washing the slop, cool the wort to be acidified to 37℃ and 6 CFU / mL (6.54logCFU / mL) was inoculated with galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1 and acidified to pH 3.66±0.03 to obtain the acidified sample AG of galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1;

[0011] S03, after the acidification of galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1, the sample AG was heated for 60 min and 0.10% w / v Saaz hop particles were added during the boiling process. 50% of Saaz hop particles were added 20 min after the initial boiling and 5 min before the end of boiling respectively. After boiling, the sample was spun out of the vortex, allowed to stand, filtered, cooled to 18 °C, and then heated to 3.5 × 10 6CFU / mL (6.54log CFU / mL) was inoculated with Saccharomyces cerevisiae S-33, and 0.1-0.5 mg / L zinc sulfate, 50-150 mg / L magnesium sulfate and 100-200 mg / L potassium dihydrogen phosphate were added at the same time. The culture was fermented at 18°C ​​for 14 days to obtain galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1. After acidification, beer AGT-S was fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate.

[0012] Preferably, in S01, the mass ratio of barley to wheat in the malt is 1:1.

[0013] Preferably, in S02, the encapsulation process of galacto-oligosaccharide encapsulating Lactobacillus gasseri JM1 is:

[0014] The probiotic protective agent is 5-15% w / v galacto-oligosaccharide, 10-20% w / v skim milk powder, 2-6% w / v glycerol, and the remainder is water, to obtain a mixture solution of the probiotic protective agent;

[0015] A mixture solution of Lactobacillus gasseri JM1 and a probiotic protective agent was treated in a mass ratio of 1:2 to prepare a probiotic emulsion preparation; the probiotic emulsion preparation was mixed with a 1.5% sodium alginate solution in a volume ratio of 1:15 to obtain a mixed solution; the mixed solution was dropped into a 1% CaCl2 solution, and the mixture was filtered after standing and rinsed with sterile saline to prepare alginate microcapsules encapsulating probiotics, namely, oligosaccharide-encapsulated Lactobacillus gasseri JM1.

[0016] Preferably, in SO2, the average rate of pH decrease is 0.243±0.003h -1 The viable count of Lactobacillus gasseri JM1 was 7.95±0.01log CFU / mL.

[0017] Preferably, in S03, the mixture is allowed to stand for 10 minutes.

[0018] The Lactobacillus gasseri JM1 acidified beer is obtained by the preparation method of the present invention.

[0019] A Lactobacillus gasseri JM1 acidified beer having high concentrations of lactic acid, citric acid and malic acid and low concentration of succinic acid.

[0020] Specifically, the specific data of organic acids are as follows:

[0021]

[0022] A Lactobacillus gasseri JM1 acidified beer has a specific gravity (SG) of 1.013±0.000, a pH of 3.86±0.01, an alcohol content (ABV) of 4.80±0.02% v / v, and a viable cell count of Saccharomyces cerevisiae S-33 of 7.51±0.02 log CFU / mL.

[0023] The present invention has the following beneficial effects:

[0024] During the acidification process of oligogalactose encapsulated Lactobacillus gasseri JM1 at 37°C, the number of Lactobacillus gasseri JM1 increased significantly, from 6.54 log CFU / mL (3.5×10 6 The results showed that the pH of the wort encapsulated by galacto-oligosaccharide increased to 7.95±0.01log CFU / mL. Within 12 hours, the initial wort pH value of 5.85 dropped to 3.66. During this process, the galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1 produced organic acids by metabolizing sugars. When acidification was performed using Lactobacillus gasseri encapsulated with oligofructose, oligogalactose, and inulin, with the acidification endpoint of 3.65±0.02, it was found that the pH drop rate was the fastest during acidification with galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1, and the bacterial count of Lactobacillus gasseri JM1 was the highest, shortening the wort acidification time.

[0025] In the present invention, galacto-oligosaccharide encapsulation showed the effect of promoting the growth and metabolism of Lactobacillus gasseri JM1. The residual substrate after acidification and the addition of supplements (zinc sulfate, magnesium sulfate and potassium dihydrogen phosphate) promoted the subsequent growth of Saccharomyces cerevisiae S-33. At the same time, in the acidified beer, AGT-S had the lowest sugar content and SG, and the highest ABV, indicating that after acidification with Lactobacillus gasseri JM1 encapsulated by galacto-oligosaccharides, fermentation with Saccharomyces cerevisiae S-33 and the addition of supplements, by promoting the increase in the number of viable cells of Saccharomyces cerevisiae S-33, the substances in the fermentation matrix were fully utilized.

[0026] In the present invention, acidification of Lactobacillus gasseri can increase the concentrations of lactic acid, citric acid and malic acid, and reduce the concentration of succinic acid, and the effect is most significant in galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1.

[0027] In the present invention, the acidified beer has increased levels of ethyl lactate, ethyl nonanoate, ethyl butyrate, ethyl phenylacetate, ethyl isovalerate, and ethyl valerate. Ethyl lactate and ethyl phenylacetate have a sweet and fruity aroma; ethyl nonanoate and ethyl butyrate have a fruity aroma; ethyl isovalerate has a distinct fruity aroma, similar to that of apple or pineapple; and ethyl valerate has a fruity aroma, similar to that of peach. The increase is greatest in AGT-S, enhancing the overall fruity and floral aroma of the beer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The organic acid content of different samples, including (a) lactic acid content; (b) citric acid content; (c) malic acid content; (d) succinic acid content. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings.

[0030] In the present invention, Lactobacillus gasseri JM1 was isolated from healthy infant feces and deposited at the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University. The 16S rDNA sequence was determined for bacterial identification and uploaded to the National Center for Biotechnology Information (NCBI). Lactobacillus gasseri JM1 is referred to as JM1.

[0031] Lactobacillus gasseri JM1, isolated from healthy infant feces, was recorded in Sun Linlin. Study on the immunomodulatory effect of a strain of Lactobacillus gasseri and its regulatory pathway[D]. Northeast Agricultural University.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] 1. Beer Preparation

[0035] Weigh 22.5% malt (barley:wheat = 1:1) and grind it into granules using a malt grinder. Saccharify at 67°C for 60 minutes at a malt:water ratio of 1:4 (kg:kg). After saccharification, filter the wort and wash the lees with 77°C lees water.

[0036] Specifically, the 22.5% malt content determines the amount of malt wash water added later. In this example, 2.25kg of barley and 2.25kg of wheat were added, for a total of 4.5kg of malt, representing 22.5%. The target total was 20kg (approximately 20L of beer). Using a 1:4 malt:water ratio, 18L of water was added for saccharification. After saccharification, the 20L volume was replenished with malt wash water.

[0037] Lactobacillus gasseri JM1 acidification group: After washing, the temperature was quickly lowered to 37℃ and 3.5×10 6 CFU / mL was inoculated with Lactobacillus gasseri JM1 and acidified to pH 3.66±0.01 to obtain the acidified sample of Lactobacillus gasseri JM1 (A).

[0038] Fructo-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidification group: After washing, the temperature was quickly cooled to 37℃ and 3.5×10 6 CFU / mL was inoculated with oligofructose-encapsulated Lactobacillus gasseri JM1 and acidified to pH 3.67±0.02 to obtain the acidified sample (AF) of oligofructose-encapsulated Lactobacillus gasseri JM1.

[0039] Encapsulation process of oligofructose encapsulated Lactobacillus gasseri JM1:

[0040] The probiotics protective agent is 10% w / v oligofructose, 15% w / v skim milk powder, 5% w / v glycerol, and the remainder is water. After mixing, a mixture solution of the probiotics protective agent is obtained.

[0041] A mixture of Lactobacillus gasseri JM1 and a probiotic protective agent (mass ratio of 1:2) was treated to produce a probiotic emulsion preparation. This probiotic emulsion preparation was then mixed with a 1.5% sodium alginate solution (volume ratio of 1:15) to obtain a mixed solution. The mixture was then added dropwise to a 1% CaCl2 solution, allowed to stand, filtered, and rinsed with sterile saline to produce alginate microcapsules encapsulating the probiotics. This increased the acid resistance of the microcapsules, promoting the maintenance of probiotic activity. The synergistic effect of the prebiotics and probiotics also promoted the proliferation of the probiotics.

[0042] Preparation of 1.5% sodium alginate solution: Dissolve 1.5% (w / v) sodium alginate in distilled water.

[0043] Preparation of 1% CaCl2 solution: Dissolve 1% (w / v) calcium chloride in distilled water.

[0044] Galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidification group: After washing, the temperature was quickly cooled to 37℃ and 3.5×10 6 The galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1 was inoculated with CFU / mL and acidified to pH 3.66±0.03 to obtain the acidified sample (AG) of galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1.

[0045] Encapsulation process of galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1:

[0046] The probiotic protective agent is 10% w / v galacto-oligosaccharide, 15% w / v skim milk powder, 5% w / v glycerol, and the rest is water.

[0047] A mixture of Lactobacillus gasseri JM1 and a probiotic protective agent (mass ratio of 1:2) was treated to produce a probiotic emulsion preparation. This probiotic emulsion preparation was then mixed with a 1.5% sodium alginate solution (volume ratio of 1:15) to obtain a mixed solution. The mixture was then added dropwise to a 1% CaCl2 solution, allowed to stand, filtered, and rinsed with sterile saline to produce alginate microcapsules encapsulating the probiotics. This increased the acid resistance of the microcapsules, promoting the maintenance of probiotic activity. The synergistic effect of the prebiotics and probiotics also promoted the proliferation of the probiotics.

[0048] Inulin encapsulated Lactobacillus gasseri JM1 acidification group: After washing, the temperature was quickly cooled to 37℃ and 3.5×10 6 CFU / mL was inoculated with inulin-encapsulated Lactobacillus gasseri JM1 and acidified to pH 3.65±0.02 to obtain the acidified sample (AI) of inulin-encapsulated Lactobacillus gasseri JM1.

[0049] Encapsulation process of inulin encapsulated Lactobacillus gasseri JM1:

[0050] The probiotic protective agent is 10% w / v inulin, 15% w / v skim milk powder, 5% w / v glycerol, and the rest is water.

[0051] A mixture of Lactobacillus gasseri JM1 and a probiotic protective agent (mass ratio of 1:2) was treated to produce a probiotic emulsion preparation. This probiotic emulsion preparation was then mixed with a 1.5% sodium alginate solution (volume ratio of 1:15) to obtain a mixed solution. The mixture was then added dropwise to a 1% CaCl2 solution, allowed to stand, filtered, and rinsed with sterile saline to produce alginate microcapsules encapsulating the probiotics. This increased the acid resistance of the microcapsules, promoting the maintenance of probiotic activity. The synergistic effect of the prebiotics and probiotics also promoted the proliferation of the probiotics.

[0052] At the same time, the acidification group of Lactobacillus gasseri JM1 with oligosaccharide alone and without oligosaccharide encapsulation was: after washing, the temperature was quickly cooled to 37℃, and 3.5×10 6 CFU / mL was inoculated with Lactobacillus gasseri JM1 and a probiotic protective agent was added at the same time. The mass ratio of Lactobacillus gasseri JM1 to the probiotic protective agent was 1:2. The probiotic protective agent was 10% w / v galacto-oligosaccharide, 15% w / v skim milk powder, 5% w / v glycerol, and the rest was water. The solution was acidified to a pH of 3.65±0.01 to obtain acidified samples of Lactobacillus gasseri JM1 with or without galacto-oligosaccharide encapsulation (A+G).

[0053] The unacidified group was heated directly for 60 minutes. During the boiling process, 0.10% (w / v) Saaz hop pellets were added, with 50% added 20 minutes after the initial boil and 5 minutes before the end of the boil. After boiling, the mixture was spun at high speed to create a vortex, allowed to stand for 10 minutes, filtered, and cooled to 18°C ​​to obtain the boiled sample (S).

[0054] Saaz hop pellets are a commercially available product and are cylindrical Saaz hop pellets.

[0055] The acidification process was studied on the boiled sample (S), the acidified sample of Lactobacillus gasseri JM1 (A), the acidified sample of Lactobacillus gasseri JM1 encapsulated with oligofructose (AF), the acidified sample of Lactobacillus gasseri JM1 encapsulated with oligofructose (AG), the acidified sample of Lactobacillus gasseri JM1 encapsulated with oligofructose (AI), and the acidified sample of Lactobacillus gasseri JM1 with or without oligofructose encapsulation (A+G).

[0056] The boiled samples of the non-acidified group (S) were 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33 and fermented at 18°C ​​for 14 days to obtain Saccharomyces cerevisiae S-33 fermented beer (ST).

[0057] After acidification with Lactobacillus gasseri JM1, the sample (A) was heated for 60 minutes and 0.10% (w / v) Saaz hop pellets were added during the boiling process. 50% Saaz hop pellets were added 20 minutes after the initial boiling and 5 minutes before the end of the boiling. After boiling, the sample was spun at high speed to create a vortex, allowed to stand for 10 minutes, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated into Saccharomyces cerevisiae S-33 and fermented at 18°C ​​for 14 days to obtain Lactobacillus gasseri JM1. After acidification, beer was fermented with Saccharomyces cerevisiae S-33 (AT).

[0058] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated into Saccharomyces cerevisiae S-33 and fermented at 18 °C for 14 days to obtain galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1. After acidification, beer was fermented by Saccharomyces cerevisiae S-33 (AGT).

[0059] After acidification with Lactobacillus gasseri JM1, the sample (A) was heated for 60 minutes and 0.10% (w / v) Saaz hop pellets were added during the boiling process. 50% Saaz hop pellets were added 20 minutes after the initial boiling and 5 minutes before the end of the boiling. After boiling, the sample was spun at high speed to create a vortex, allowed to stand for 10 minutes, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate, 100 mg / L magnesium sulfate, and 150 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain Lactobacillus gasseri JM1. After acidification, beer was fermented with Saccharomyces cerevisiae S-33, zinc sulfate, magnesium sulfate, and potassium dihydrogen phosphate (AT-S).

[0060] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate, 100 mg / L magnesium sulfate and 150 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18 °C for 14 days to obtain galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1. After acidification, beer was fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate (AGT-S).

[0061] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.7 mg / L zinc sulfate (excess), 100 mg / L magnesium sulfate and 150 mg / L potassium dihydrogen phosphate were added at the same time, and fermented at 18°C ​​for 14 days to obtain AGT-S1.

[0062] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate, 200 mg / L magnesium sulfate (excess) and 150 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain AGT-S2.

[0063] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate, 100 mg / L magnesium sulfate and 300 mg / L potassium dihydrogen phosphate (excess) were added at the same time, and fermented at 18°C ​​for 14 days to obtain AGT-S3.

[0064] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 100 mg / L magnesium sulfate and 150 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain AGT-S4.

[0065] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate and 150 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain AGT-S5.

[0066] The sample (AG) was heated for 60 min after encapsulating Lactobacillus gasseri JM1 with oligosaccharides. During the boiling process, 0.10% (w / v) Saaz hop pellets were added. 50% Saaz hop pellets were added 20 min after the initial boiling and 5 min before the end of the boiling. After boiling, the sample was spun out at high speed, allowed to stand for 10 min, filtered, cooled to 18°C, and then heated to 3.5×10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.3 mg / L zinc sulfate and 100 mg / L magnesium sulfate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain AGT-S6.

[0067] Fermentation processes were studied for beer fermented with Saccharomyces cerevisiae S-33 (ST), acidified with Lactobacillus gasseri JM1, fermented with Saccharomyces cerevisiae S-33 (AT), acidified with Lactobacillus gasseri JM1 encapsulated with galacto-oligosaccharides, fermented with Saccharomyces cerevisiae S-33 (AGT), acidified with Lactobacillus gasseri JM1, fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate (AT-S), acidified with Lactobacillus gasseri JM1 encapsulated with galacto-oligosaccharides, and fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate (AGT-S), AGT-S1, AGT-S2, AGT-S3, AGT-S4, AGT-S5, and AGT-S6. Zinc in zinc sulfate (ZnSO4) is a crucial component of the yeast enzyme system and helps improve yeast fermentation activity and alcohol tolerance. The magnesium in magnesium sulfate (MgSO4) is a vital element for cell walls and yeast metabolism, promoting yeast growth and metabolic processes, improving fermentation efficiency and alcohol production. The phosphorus and potassium in potassium dihydrogen phosphate (KH2PO4) are crucial nutrients for yeast growth and metabolism, contributing to cell membrane stability and yeast reproduction while also increasing the buffering capacity of the fermentation broth.

[0068] 2. Physical and chemical indicators

[0069] The pH value was measured using a pH meter; the Brix content was determined using a refractometer; the specific gravity (SG) was determined using a hydrometer; and the samples were counted for viable bacteria using the gradient dilution method and the spreading method. Yeast was counted using potato dextrose agar (PDA) after incubation at 28°C for 72 h, and lactic acid bacteria were counted using MRS agar after incubation at 37°C for 48 h.

[0070] 3. Organic acid content

[0071] The analysis was performed using a 1260 Infinity II HPLC (Agilent, Santa Clara, CA, USA). The samples were first centrifuged and filtered through a 0.22 μm microfilter. 18 Isocratic elution was performed on a chromatographic column (150 × 4.6 mm, Agilent, Santa Clara, CA, USA) with a mobile phase of 0.1% phosphoric acid / methanol (95:5 v / v) at a flow rate of 1.0 mL / min and a column temperature of 40° C. Samples were detected using a UV absorption detector at a wavelength of 210 nm.

[0072] 4. Determination of volatile substances

[0073] Samples (1 mL) were transferred to 20 mL headspace vials, and 10 μL of a 10 mg / L deuterated n-hexanol-d13 solution was added to each sample. A solid-phase microextraction (SPME) fiber coated with divinylbenzene / carboxyl / polydimethylsiloxane (DVB / CAR / PDMS, 50 / 30 μm × 1 cm) was incubated at 60°C for 30 min. In the gas chromatograph injector, the SPME fiber was desorbed at 250°C for 5 min. Following the injection step, the fiber was placed in the sample chamber at 270°C for 10 min. Saturated n-alkanes (10 μL) were transferred to 20 mL headspace vials for incubation, extraction, and injection.

[0074] 5. Results and Discussion

[0075] 5.1 Physical and chemical indicators

[0076] During the acidification of Lactobacillus gasseri JM1 at 37°C, the number of Lactobacillus gasseri JM1 increased significantly from 6.54 log CFU / mL (3.5×10 6 The results show that the pH value of Lactobacillus gasseri JM1 increased from 5.85 to 3.66 in 12 hours, and the Brix value decreased by 1.05. In this process, Lactobacillus gasseri JM1 produces organic acid by metabolizing sugar. When Lactobacillus gasseri encapsulated with oligofructose, oligogalactose and inulin was acidified, the pH value decreased at 3.65 ± 0.02, and the pH value decreased the fastest when encapsulating Lactobacillus gasseri JM1 with oligofructose, oligogalactose and inulin, and the bacterial count of Lactobacillus gasseri JM1 was the highest, which shortened the acidification time of wort.

[0077] When equal amounts of galacto-oligosaccharide and Lactobacillus gasseri JM1 without galacto-oligosaccharide encapsulation were added for acidification, the acid reduction rate and viable bacterial count of the samples were lower than those of Lactobacillus gasseri JM1 encapsulated with galacto-oligosaccharide. The specific results are shown in Table 1 below.

[0078] Table 1 Physical and chemical indicators of different acidified samples

[0079]

[0080] Note: S: sample after boiling;

[0081] A: acidified sample of Lactobacillus gasseri JM1;

[0082] AF: acidified sample of oligofructose-encapsulated Lactobacillus gasseri JM1;

[0083] AG: galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidified sample;

[0084] AI: acidified sample of inulin-encapsulated Lactobacillus gasseri JM1;

[0085] A+G: Acidified samples with the addition of galacto-oligosaccharide alone and without galacto-oligosaccharide encapsulation of Lactobacillus gasseri JM1.

[0086] Subsequently, yeast S. cerevisiae S-33 was inoculated to enter the yeast fermentation stage. After acidification, the yeast S-33 count was significantly lower than that of the ST control, indicating that the yeast count in the acidified beer increased less significantly compared to the unacidified control. This is related to the metabolism of some wort substrate components by Lactobacillus gasseri JM1 during the acidification process, resulting in reduced growth of S. cerevisiae S-33 due to substrate acidification. Furthermore, the lower pH of the fermentation substrate after acidification inhibits yeast growth.

[0087] In the final fermentation product, the SG (Specific Gravity) of ST is lower than that of AT, while the ABV (Alcohol by Volume) of ST is higher than that of AT. The viable Saccharomyces cerevisiae S-33 count in ST is higher than that in AT. This indicates that the viable Saccharomyces cerevisiae S-33 count has a significant impact on the ABV and SG of beer. Saccharomyces cerevisiae is a key bacterial species in the beer production process. Increased Saccharomyces cerevisiae counts result in higher alcohol content. Furthermore, supplements (zinc sulfate, magnesium sulfate, and potassium dihydrogen phosphate) were added to the Saccharomyces cerevisiae S-33 fermentation stage to enhance its activity and metabolism. In samples that had undergone the acidification process, Saccharomyces cerevisiae S-33 and supplements were added before fermentation. The study found that the addition of supplements significantly increased the viable count of Saccharomyces cerevisiae S-33 in AT-S and AGT-S beers compared to AT and AGT (p < 0.05), indicating that supplementation promoted yeast growth, with the most significant increase in AGT-S. This was related to the fact that the acidification endpoint was reached quickly after encapsulation of Lactobacillus gasseri JM1 with galacto-oligosaccharides. Galacto-oligosaccharides promoted the growth and metabolism of Lactobacillus gasseri JM1, and the residual substrate after acidification and the addition of supplements promoted the subsequent growth of Saccharomyces cerevisiae S-33. Furthermore, the acidified beer AGT-S had the lowest sugar content and SG, and the highest ABV. This suggests that after acidification with Lactobacillus gasseri JM1 encapsulated with galacto-oligosaccharides, fermentation with Saccharomyces cerevisiae S-33, and the addition of supplements, the active count of Saccharomyces cerevisiae S-33 was increased, leading to the full utilization of the fermentation substrate.

[0088] Compared to AGT-S, the bacterial count of S. cerevisiae S-33 decreased to varying degrees when zinc sulfate, magnesium sulfate, and potassium dihydrogen phosphate were deficient or excessive (AGT-S1 to AGT-S6). This also reduced the ABV, which in turn reduced the proliferation and metabolism of S. cerevisiae and the alcoholic flavor of the beer. See Table 2 for the detailed structure.

[0089] Table 2 Physical and chemical indicators of beer samples with different fermentation treatments

[0090]

[0091] Note: ST: Saccharomyces cerevisiae S-33 fermented beer;

[0092] AT: beer fermented by Saccharomyces cerevisiae S-33 after acidification by Lactobacillus gasseri JM1;

[0093] AGT: After galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidified, beer was fermented by Saccharomyces cerevisiae S-33; AT-S: After Lactobacillus gasseri JM1 acidified, beer was fermented by Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate;

[0094] AGT-S: Galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidified and fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate;

[0095] AGT-S1: After galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 and acidified, beer was fermented with Saccharomyces cerevisiae S-33 + excess zinc sulfate + magnesium sulfate + potassium dihydrogen phosphate;

[0096] AGT-S2: After galacto-oligosaccharide encapsulation and acidification of Lactobacillus gasseri JM1, beer was fermented with Saccharomyces cerevisiae S-33, zinc sulfate, excess magnesium sulfate, and potassium dihydrogen phosphate;

[0097] AGT-S3: After galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 acidified, beer was fermented by Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate + excess potassium dihydrogen phosphate.

[0098] AGT-S4: After galacto-oligosaccharide encapsulation and acidification of Lactobacillus gasseri JM1, beer was fermented with Saccharomyces cerevisiae S-33, magnesium sulfate, and potassium dihydrogen phosphate;

[0099] AGT-S5: After galacto-oligosaccharide encapsulation and acidification of Lactobacillus gasseri JM1, beer was fermented with Saccharomyces cerevisiae S-33, zinc sulfate, and potassium dihydrogen phosphate;

[0100] AGT-S6: Galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1 was acidified and then fermented with Saccharomyces cerevisiae S-33 + zinc sulfate + magnesium sulfate.

[0101] 5.2 Organic acid content

[0102] The acidification process is related to the production of organic acids. Lactic acid is the main organic acid released by Lactobacillus gasseri JM1. The lactic acid content in the acidified wort (A) increased significantly (p < 0.05), while no lactic acid was detected in the wort that was not acidified by Lactobacillus gasseri JM1 (S), indicating that acidification by Lactobacillus gasseri JM1 promoted the massive production of lactic acid. Furthermore, in the prebiotic-encapsulated Lactobacillus gasseri JM1, the lactic acid content increased most significantly after acidification by galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1. At the same time, compared to the unacidified sample, the contents of citric acid and malic acid in the wort (A) acidified by Lactobacillus gasseri JM1 increased significantly, and the increase was most significant after acidification by galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1 (p < 0.05), indicating that galacto-oligosaccharides promote acidification by Lactobacillus gasseri JM1. In addition, after acidification of Lactobacillus gasseri JM1 (A, compared with S), the succinic acid content decreased significantly (p<0.05). Acidification of Lactobacillus gasseri JM1 can reduce the content of succinic acid, and the reduction is most significant after acidification of Lactobacillus gasseri JM1 encapsulated with oligosaccharides (p<0.05). The common sensory characteristic of organic acids is sourness, but the sourness of different organic acids is also different. The sourness of citric acid is relatively fresh and refreshing, and the sourness of malic acid is refreshing, slow in flavor, and slightly bitter; succinic acid has a special unpleasant taste that causes astringency in the mouth and throat. Previous studies have emphasized the effect of succinic acid in alcoholic fermentation products on gastric acid. Succinic acid is a potential stimulant of gastric acid secretion and can be used as a key indicator of food drinkability. In summary, this study shows that acidification of Lactobacillus gasseri can increase the concentrations of lactic acid, citric acid, and malic acid, and reduce the concentration of succinic acid, and it is most significant in Lactobacillus gasseri JM1 encapsulated with oligosaccharides. The results are as follows Figure 1 shown.

[0103] 5.3 Volatile flavor compounds

[0104] Esters and higher alcohols are the primary flavor compounds in beer. Alcohols are one of the main byproducts of beer fermentation and are crucial components of beer flavor. An appropriate composition and content of higher alcohols not only enhances the beer's rich aroma but also enhances its balance and body. Acidified beer exhibits increased levels of 2,3-butanediol, lauryl alcohol, and 3-hexen-1-ol. 2,3-Butanediol exhibits sweet, fruity, and floral aromas; lauryl alcohol has a fresh, aromatic character; and 3-hexen-1-ol exhibits notes of orange and flowers, similar to isoamyl alcohol, and rich grass. Esters are the primary flavor compounds in beer. Although present in relatively small amounts, they significantly contribute to beer's flavor, often imparting floral and fruity notes and adding complexity and character to the beer. Compared to the ST group, acidified beer exhibited increased levels of ethyl lactate, ethyl nonanoate, ethyl butyrate, ethyl phenylacetate, ethyl isovalerate, and ethyl valerate. Ethyl lactate and ethyl phenylacetate have sweet and fruity aromas; ethyl nonanoate and ethyl butyrate have fruity aromas; ethyl isovalerate has a distinct fruity aroma, similar to apple or pineapple; and ethyl valerate has a fruity aroma, similar to peach. The increase in AGT-S was greatest, enhancing the overall fruity and floral aromas of the beer. See Table 3 below for detailed results.

[0105] Table 3 Peak areas of main volatile substances in each beer

[0106]

[0107]

[0108] Note: For example, 1.95E+08 represents 1.95×10 8 .

[0109] Example 2

[0110] The difference between this embodiment and embodiment 1 is that:

[0111] Zinc sulfate (ZnSO4) is added at a rate of 0.1 mg / L. Zinc is a vital component of the yeast enzyme system and helps improve yeast fermentation activity and alcohol tolerance. Magnesium sulfate (MgSO4) is added at a rate of 50 mg / L. Magnesium is a key element in the cell wall and yeast metabolism, promoting yeast growth and metabolic processes, improving fermentation efficiency and alcohol production. Potassium dihydrogen phosphate (KH2PO4) is added at a rate of 100 mg / L.

[0112] Encapsulation process of probiotic Lactobacillus gasseri JM1:

[0113] The probiotic protective agent is 5% w / v oligosaccharide (fructooligosaccharide / galacto-oligosaccharide / inulin), 10% w / v skim milk powder, 2% w / v glycerol, and the rest is water.

[0114] A mixture of probiotic bacteria (i.e., Lactobacillus gasseri JM1) and a protective agent (mass ratio of 1:2) was treated to produce a probiotic emulsion preparation. This probiotic emulsion preparation was then mixed with a 1.5% sodium alginate solution (volume ratio of 1:15) to obtain a mixed solution. The mixture was then added dropwise to a 1% CaCl2 solution, allowed to stand, filtered, and rinsed with sterile saline to produce alginate microcapsules encapsulating the probiotics. This increased the acid resistance of the microcapsules, promoting the maintenance of probiotic activity. The synergistic effect of the prebiotics and probiotics also promoted the proliferation of the probiotics.

[0115] Example 3

[0116] The difference between this embodiment and embodiment 1 is that:

[0117] The addition amount of zinc sulfate (ZnSO4) is 0.5mg / L. Zinc is an important component of the yeast enzyme system and helps improve the fermentation activity and alcohol tolerance of yeast. The addition amount of magnesium sulfate (MgSO4) is 150mg / L. Magnesium is an important element of cell wall and yeast metabolism. It can promote the growth and metabolism of yeast, improve fermentation efficiency and alcohol production. The addition amount of potassium dihydrogen phosphate (KH2PO4) is 200mg / L.

[0118] Encapsulation process of probiotic Lactobacillus gasseri JM1:

[0119] The probiotic protective agent is 15% w / v oligosaccharide (fructooligosaccharide / galacto-oligosaccharide / inulin), 20% w / v skim milk powder, 6% w / v glycerol, and the rest is water.

[0120] A mixture of probiotic bacteria (i.e., Lactobacillus gasseri JM1) and a protective agent (mass ratio of 1:2) was treated to produce a probiotic emulsion preparation. This probiotic emulsion preparation was then mixed with a 1.5% sodium alginate solution (volume ratio of 1:15) to obtain a mixed solution. The mixture was then added dropwise to a 1% CaCl2 solution, allowed to stand, filtered, and rinsed with sterile saline to produce alginate microcapsules encapsulating the probiotics. This increased the acid resistance of the microcapsules, promoting the maintenance of probiotic activity. The synergistic effect of the prebiotics and probiotics also promoted the proliferation of the probiotics.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Lactobacillus gasseri JM1 acidified beer, characterized in that: The following steps are involved: S01, weighing malt, crushing it, and saccharifying it at 67°C for 60 min at a mass ratio of malt to water of 1:

4. After saccharification, filtering the wort, and washing the slag with 76-78°C slag water to obtain the malt wort to be acidified, wherein the mass percentage of malt raw material in the malt wort is 22.5%; S02, after washing the slop, cool the wort to be acidified to 37℃ and 6 CFU / mL was inoculated with galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1 and acidified to pH 3.66±0.03 to obtain the acidified sample AG of galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1; S03, after the acidification of galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1, the sample AG was heated for 60 min and 0.10% w / v Saaz hop particles were added during the boiling process. 50% of Saaz hop particles were added 20 min after the initial boiling and 5 min before the end of boiling respectively. After boiling, the vortex was removed, the sample was allowed to stand, filtered, cooled to 18 °C, and then heated to 3.5 × 10 6 CFU / mL was inoculated with Saccharomyces cerevisiae S-33, and 0.1-0.5 mg / L zinc sulfate, 50-150 mg / L magnesium sulfate, and 100-200 mg / L potassium dihydrogen phosphate were added at the same time. The mixture was fermented at 18°C ​​for 14 days to obtain galacto-oligosaccharide-encapsulated Lactobacillus gasseri JM1. After acidification, beer AGT-S was fermented with Saccharomyces cerevisiae S-33, zinc sulfate, magnesium sulfate, and potassium dihydrogen phosphate. In S02, the encapsulation process of galacto-oligosaccharide encapsulating Lactobacillus gasseri JM1 is as follows: The probiotic protective agent comprises 5-15% w / v galacto-oligosaccharide, 10-20% w / v skim milk powder, 2-6% w / v glycerol, and the remainder water, to obtain a mixture solution of the probiotic protective agent; A mixture solution of Lactobacillus gasseri JM1 and a probiotic protective agent was treated in a mass ratio of 1:2 to prepare a probiotic emulsion preparation; the probiotic emulsion preparation was mixed with a 1.5% sodium alginate solution in a volume ratio of 1:15 to obtain a mixed solution; the mixed solution was dropped into a 1% CaCl2 solution, and the mixture was filtered after standing and rinsed with sterile saline to prepare alginate microcapsules encapsulating probiotics, namely, oligosaccharide-encapsulated Lactobacillus gasseri JM1.

2. The preparation method according to claim 1, characterized in that In S01, the mass ratio of barley and wheat in malt is 1:

1.

3. The preparation method according to claim 1, characterized in that In S02, the average rate of pH decrease was 0.243±0.003h -1 In the sample AG after acidification of galacto-oligosaccharide encapsulated Lactobacillus gasseri JM1, the viable count of Lactobacillus gasseri JM1 was 7.95±0.01 log CFU / mL.

4. The preparation method according to claim 1, characterized in that S03, and let it stand for 10 minutes.

5. The Lactobacillus gasseri JM1 acidified beer obtained according to the preparation method according to any one of claims 1 to 4.

6. A Lactobacillus gasseri JM1 acidified beer according to claim 5, characterized in that: The specific gravity (SG) was 1.013 ± 0.000; the pH was 3.86 ± 0.01; the alcohol content (ABV) was 4.80 ± 0.02% v / v; and the viable count of Saccharomyces cerevisiae S-33 was 7.51 ± 0.02 log CFU / mL.

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