Probiotic GR-5 and its application in producing low-purine beer

Abstract

The present invention provides a probiotic strain and a method for producing low-purine beer fermented by the strain, characterized in that: (1) the strain is isolated from local fermented slurry in Gansu; (2) the degradation ability of the candidate bacteria for purine substances is determined; (3) the fermentation ability of the candidate bacteria for beer is tested; (4) the strain is identified and its properties are detected; (5) the preparation and improvement of low-purine probiotic beer. The beer produced by this method has a unique taste while retaining the beer flavor, and its purine content is lower than that of commercially available beer and is rich in Pediococcus acidilactici GR-5 that can degrade purine substances, thereby reducing the high purine risk of beer drinking.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics and beer preparation, and more particularly to a probiotic and an application thereof in producing low-purine beer. Background Art

[0002] The rapid development of today's society has led to a continuous improvement in people's living standards. Long-term excessive intake of nutrients may cause various metabolic diseases, such as high blood pressure, fatty liver, obesity and other "rich diseases". As one of the important metabolites of the human body, purine is a normal part of the body, but the accumulation of its end product uric acid may cause gout. The continuous intake of high-purine foods will affect purine metabolism and greatly increase the risk of gout. Beer is a recognized high-purine beverage and one of the low-alcohol beverages commonly consumed by the public. Therefore, low-purine beer is a common demand of the market and consumers.

[0003] Beer is a low-alcohol beverage made from barley malt, hops, and water, fermented with yeast. It is rich in nutrients and is known as "liquid bread". Its unique flavor comes from malt and hops, and the specific fermentation strains and fermentation processes also have a great impact on it. The relative purine content of beer itself is not high, but continuous and large-scale drinking as a daily beverage and the alcohol it contains will affect the body's purine metabolism, and the accumulation of the final product uric acid may cause clinical symptoms - gout.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. Traditional fermented foods such as yogurt, soy sauce, and vinegar contain a wide range of microorganisms. Probiotics extracted from these natural fermentation products have both probiotic effects and safety. The colonization of probiotics in the intestine can build a probiotic barrier to protect the intestine, and can also improve the intestinal environment through its own metabolism, reduce the oxidative stress of the human body, and inhibit the proliferation of harmful bacteria to a certain extent. With the continuous development of the craft beer industry, the types of beer are becoming more and more diverse. In life, fresh (draft) beer containing live yeast can be purchased, and its unique flavor and fresh taste are quite popular. In addition to yeast, some lactic acid bacteria can also grow normally in beer fermentation liquid, which also provides a realistic basis for using beer as a carrier to deliver probiotics. Through controllable and limited biological acidification, probiotics can be introduced without significantly affecting the original flavor of beer, so that the purpose of supplementing probiotics can be achieved when drinking.

[0005] The purine content of the novel GR-5-rich beer produced by the present invention is lower than that of commercially available beer. After being ingested by the human body, the rich probiotics contained in the beer can assist in degrading food-borne purines, thereby achieving a probiotic effect while satisfying people's demand for beer. Summary of the invention

[0006] In continuation of the above, the present invention aims to provide a method for producing low-purine probiotic beer, aiming to solve the problem of high purine content and homogeneous taste of existing commercially available beer. By adding a probiotic-assisted fermentation process to the traditional beer production process, low-purine probiotic beer is obtained while retaining the characteristic flavor of beer, meeting consumers' dual needs for beer and health.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A probiotic strain, the probiotic strain is Pediococcus acidilactici GR-5, which was deposited on September 16, 2022 in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No.25729.

[0009] 2. A method for separating probiotics that can efficiently degrade purine substances, comprising the following steps:

[0010] (1) To isolate microorganisms from Gansu Jiangshui, Qinyi brand fresh Jiangshui was selected for gradient dilution and evenly spread on MRS culture medium. The obtained single colonies were picked out and inoculated into MRS liquid culture medium for expansion.

[0011] (2) Gram staining of strains: the candidate bacteria were initially stained with crystal violet and then washed with water, mordanted with iodine solution and then washed with water, decolorized with 95% ethanol, restained with safranin solution, washed with water, dried, and observed with an oil microscope to screen out negative strains and yeast.

[0012] (3) The ability of the strain to degrade pure purine compounds in vitro was determined by using HPLC to measure the content of hypoxanthine, xanthine, guanine, and adenine after treatment for a unit time to evaluate the degradation effect;

[0013] (4) Identification of strains: 16S rRNA gene sequencing was used to identify the phylogenetic characteristics of the isolates. Scanning electron microscopy was used to determine the morphological characteristics of strain GR-5.

[0014] 3. A method for screening the most suitable candidate strain for low-purine probiotic beer, comprising the following steps:

[0015] (1) Weigh the dry barley and food-grade saccharifying enzyme from the raw materials of the low-purine probiotic beer, crush and sieve the dry barley. Add water to the dry material components of each raw material to obtain a first mixed solution; the amount of 70°C drinking water added is 2.5 times the weight of the dry matter.

[0016] (2) Saccharifying the first mixed solution to obtain a second mixed solution: maintaining the temperature of the first mixed solution between 55-65° C., stirring it thoroughly to make it evenly mixed, maintaining it for 60 minutes, and testing whether the saccharification is complete with an iodine-potassium iodide solution. After the saccharification is complete, heating the wort to boiling, maintaining the boiling state for 10 minutes, and filtering the obtained mixture with eight layers of medical gauze to obtain a second mixed solution.

[0017] (3) After filtering the mixture 2 with gauze, add 60°C drinking water in a 1:1 ratio of dry matter mass to the residue, mix thoroughly for 10 min, filter again to collect the filtrate, repeat the operation once, collect all the filtrate and mix to obtain the mixture 3; mix the mixture 2 and the mixture 3, adjust the Brix value to 10, and obtain the mixture 4.

[0018] (4) The fourth mixed solution is sterilized at high temperature to remove most of the precipitate, yeast is added to the mixed solution for primary fermentation to obtain original beer crude liquid, and probiotics are added to the beer crude liquid for secondary fermentation to obtain probiotic beer crude liquid.

[0019] (5) Add the diluted sterilized hop extract to the obtained probiotic beer crude solution, with the final concentration of hop isomers being 2‰.

[0020] (6) Purine content determination: HPLC was used to detect the free purine and total purine content.

[0021] 4. A method for preparing low-purine probiotic beer from scratch, comprising the following steps:

[0022] (1) Weigh the dry barley and food-grade saccharifying enzyme from the raw materials of the low-purine probiotic beer, crush and sieve the dry barley. Add water to the dry material components of each raw material to obtain a first mixed solution; the amount of 70°C drinking water added is 2.5 times the weight of the dry matter.

[0023] (2) Saccharifying the first mixed solution to obtain a second mixed solution: maintaining the temperature of the first mixed solution between 55-65° C., stirring the first mixed solution to make it evenly mixed, maintaining the temperature for 60 minutes, and testing whether the saccharification is complete with an iodine-potassium iodide solution. After the saccharification is complete, heating the wort to boiling, maintaining the boiling state for 10 minutes, and filtering the obtained mixture with eight layers of medical gauze to obtain a filtered mixed solution II.

[0024] (3) After the mixture 2 was filtered through gauze, the residue was added with 60°C drinking water in a 1:1 ratio of dry matter mass, and the mixture was fully mixed for 10 minutes. The filtrate was collected again by filtration, and the operation was repeated once. All the filtrates were collected and mixed to obtain the mixture 3. The mixture 2 and the mixture 3 were mixed and the Brix value was adjusted to 10 to obtain the mixture 4.

[0025] (4) The fourth mixed solution is sterilized at high temperature to remove most of the precipitate, and yeast is added to the mixture for primary fermentation to obtain the original beer crude solution, and probiotics are added to the beer crude solution for secondary fermentation to obtain the probiotic beer crude solution. Specific implementation plan: The fourth mixed solution is sterilized at high temperature, the supernatant is transferred to a fermentation bottle, the turbid precipitate is discarded, and 1% volume of yeast seed solution is added to make the final yeast concentration reach 1*10 5 cfu / mL, and the first anaerobic fermentation was carried out at 20°C. During the fermentation, the sugar content and pH changes were continuously monitored. The initial fermentation was considered to be completed when the sugar content change was less than 0.1 for two consecutive days. GR-5 seed liquid was added to make the final concentration reach 1*10 6 cfu / ml, and fermented at 37°C for one day, then the temperature was lowered to 20°C and maintained for one day to obtain a crude probiotic beer solution, of which a portion of the beer was not added with Pediococcus acidilactici GR-5 as a control.

[0026] (5) Add the diluted sterilized hop extract to the obtained probiotic beer crude liquid, and the final concentration of hop isomers is 2‰. Add 25°C saturated baking soda to the obtained probiotic beer crude liquid to adjust the palatability. The pH of the beer before addition is 3.49, and the pH of the beer after addition is 4.1.

[0027] (6) The finished low-purine probiotic beer was mixed and divided into sterile glass bottles and stored at 4°C, 6°C, and 10°C respectively. During this period, the concentration of GR-5 and yeast were measured. When the concentration dropped to 1*10 8 The content is considered expired if the content is below cfu / mL.

[0028] (7) Quality determination: determination of pH, sugar content, strain concentration, and purine content.

[0029] 5. A simple method for preparing low-purine probiotic beer comprises the following steps:

[0030] (1) Sterilize commercially available wort at 110°C for 20 min, filter out the precipitate, and adjust the sugar content to 10°Bx.

[0031] (2) Yeast was added for primary fermentation to obtain the original beer crude liquid, and probiotics were added to the beer crude liquid for secondary fermentation to obtain the probiotic beer crude liquid. Specific implementation plan: The commercially available malt juice was sterilized at 110°C for 20 minutes, the supernatant was transferred to the fermentation bottle, the turbid precipitate was discarded, and 1% yeast seed liquid was added to make the final yeast concentration reach 1*10 5 cfu / mL, and the first anaerobic fermentation was carried out at 20°C. The sugar content was continuously monitored during the fermentation. When the sugar content changed less than 0.1 for two consecutive days, the initial fermentation was considered to be over. The seed liquid of Pediococcus acidilactici GR-5 was added to make the final concentration reach 1*10 6cfu / mL, after fermentation at 37°C for one day, the temperature was reduced to 20°C and maintained for one day to obtain a probiotic beer crude liquid, of which a portion of the beer was not added with GR-5 as a control. The yeast seed liquid preparation method: after the malt juice was sterilized, the preserved beer yeast was inoculated and cultured at 37°C for 1 day. The GR-5 seed liquid preparation method: after the malt juice was sterilized, the preserved GR-5 was inoculated and cultured at 37°C for 1 day.

[0032] (3) Add the diluted sterilized hop extract to the obtained probiotic beer crude liquid, and the final concentration of hop isomers is 1.5‰. Add 25°C saturated baking soda to the obtained probiotic beer crude liquid, and the pH of the wine body before addition is 3.49, and the pH of the wine body after addition is 4.1. The finished low-purine probiotic beer is mixed and divided into sterile glass bottles and stored at 4°C.

[0033] (4) Comparative analysis with commercially available beer: collect common beers on the market, conduct sensory evaluation and comparison with the beer of the present invention, and measure the purine content of the collected beers.

[0034] 6. A method for producing low-purine probiotic beer, wherein the content of free purine substances adenine, guanine, hypoxanthine, xanthine, inosine and guanosine in the beer is less than 40 mg / L. The method comprises the following steps:

[0035] (1) Wash and dry the dry stored barley and crush it into a small particle powder mixture, and pass it through a 2.5 mm sieve to remove large un-crushed grains. The higher the degree of barley crushing, the higher the saccharification efficiency and the greater the difficulty of filtering. When using it, comprehensive considerations should be made and appropriate adjustments should be made based on the final sugar content.

[0036] (2) Add food-grade saccharifying enzyme at a ratio of 1:200, add drinking water at a weight ratio of 1:2.5 at 70°C, maintain the temperature at 55-60°C for 60 minutes, and complete saccharification in one step; the saccharifying enzyme is preferably a food-grade saccharifying enzyme produced by Shandong Longkote Enzyme Preparation Co., Ltd. The ratio of drinking water for saccharification to barley flour is 2.5:1, and the temperature of the mixture after mixing is about 60°C. The sugar content will still be adjusted later. Complete saccharification means that the iodine test does not change color.

[0037] (3) Heat and boil, filter out the residue, use 60℃ drinking water to heat extract again for 10min, filter and mix with the former, remove the precipitate after heating, adjust the initial sugar content to 10°Bx; use gauze filtration, let stand at high temperature and take the supernatant for fermentation. Sterile wort may contain a certain amount of precipitate, most of which is protein, which can be used by yeast during fermentation.

[0038] (4) Sterilized wort was inoculated with yeast to a final concentration of 1*10 5cfu / mL, fermented at 20℃, the first fermentation ended after 5-6 days of constant sugar content, constant sugar content means that the sugar content change is less than 0.1 within two days. The yeast used is commercially available brewer's yeast (Saccharomyces cerevisiae). However, it is necessary to ensure the sterilization effect, avoid contamination with miscellaneous bacteria, and pay attention to timely exhaust during fermentation.

[0039] (5) After the yeast fermentation cycle in step (4) is completed, the highly efficient purine-degrading probiotic GR-5 screened from the slurry is inoculated and the final concentration is adjusted to 1*10 6 cfu / mL, and fermented at 37°C for 1 day. Probiotic GR-5 is the strain described in Example 1, with Fig.12 and Fig.13 The lactic acid fermentation bacteria JL-3 involved is the probiotic bacteria deposited in the applicant's aforementioned patent application CN202111503162.3 with CCTCC number CCTCC NO: M2021902.

[0040] (6) After one day of fermentation in step (5), the temperature was lowered back to 20° C. and fermentation was continued for another day to stabilize the fermentation system.

[0041] (7) Add 2‰ of boiled hop extract and baking soda solution to the obtained bacteria-containing beer in proportion after step (6) to adjust the pH to the pH of the beer before the probiotics are added (it can be adjusted after the fermentation is completed or continuously added during the fermentation). The hop extract is a pure natural 10:1 hop powder of the brand "Zhizhijia". 2‰ refers to the original unconcentrated ratio of hops. The baking soda used in the example is "Xuehou" edible baking soda produced by Inner Mongolia Etuoke Banner Shuangxin Chemical Co., Ltd.

[0042] (8) The finished product after seasoning should be packaged in sterile containers and is recommended to be stored in a low temperature environment to extend the shelf life. The low temperature environment should be below 10°C.

[0043] The outstanding advantage of the present invention is that: through the above technical scheme, compared with the existing beer making technology, the present invention adopts pure barley as the main raw material, uses a two-stage brewing process, and uses hop extract and baking soda as auxiliary seasoning. The obtained beer has a high fermentation degree, good sensory effect, and an overall purine content lower than that of commercially available beer. It also contains abundant lactic acid bacteria that can degrade purine and has a unique flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The colony morphology (left) and Gram staining results (right) of GR-5.

[0045] Among them, the GR-5 colonies on the MRS solid culture medium showed a standard oval shape, with a colony size of 0.5-1.0 mm, milky white, round protrusions, neat edges, glossy, and no transparent circles. Gram staining indicated that GR-5 was a Gram-positive bacterium.

[0046] Figure 2 The peak diagrams of 6 purine substances under the chromatographic conditions of the embodiment are shown in FIG.

[0047] Among them, the standard concentration of each purine is 0.5 mg / L, and the separated peaks represent different purine substances. The larger the peak area, the higher the content of this substance. From front to back, the characteristic peaks are adenine (3.861s), guanine (4.128s), hypoxanthine (4.793s), xanthine (5.021s), inosine (5.742s), and guanosine (5.992s). The peak areas of multiple concentrations are used for curve fitting to obtain the working curve.

[0048] Figure 3 The evolutionary tree (a) and electron microscopic morphology (b) of GR-5.

[0049] Among them, GR-5 belongs to the genus Pediococcus and Pediococcus acidilactici on the evolutionary tree. The electron microscope image shows that the GR-5 bacteria are short rod-shaped, and multiple single bacteria are stacked in a pile shape.

[0050] Figure 4 This is the chromatogram of beer sample.

[0051] The above figure is the chromatogram of the fermentation group sample without GR-5. Figure 2 The peak times of the substances in the figure are 3.859s, 4.009s, 4.733s, 4.947s, 5.675s, and 5.985s. The figure below is the chromatogram of the fermentation group sample with GR-5 added. The peak times of the substances in this figure are 3.824s, 3.996s, 4.732s, 4.945s, not detected, and 5.927s, respectively. Figure 2 The elution time of pure purine chromatogram is slightly different, but the characteristic peaks can still be identified.

[0052] Figure 5 The sugar content of beer changes during fermentation and storage.

[0053] Sugar content refers to the proportion of solids dissolved in a solution, usually expressed as grams of solids dissolved in 100g of solution. In beer fermentation, the degree of fermentation can be measured by the change in sugar content. Usually, fermentation is considered complete when the final sugar content drops to half of the initial sugar content and the sugar content is stable. This figure shows that the addition of GR-5 does not significantly affect the overall process of beer yeast fermenting wort, and maintains a similar trend during storage.

[0054] Figure 6 The pH of beer changes during fermentation and storage.

[0055] Among them, pH refers to the concentration of hydrogen ions in the solution, which is better understood as the acidity of beer, which is used to measure the quality and acidification of beer. From this figure, it can be seen that the addition of GR-5 significantly reduces the pH of beer and can be maintained for a long time, and it still shows a slowly declining trend during storage.

[0056] Figure 7 is the change of GR-5 concentration during storage.

[0057] Among them, different legend shapes represent different storage temperatures. The concentration of GR-5 can reflect the relative activity of probiotics. It can be seen that with the addition of GR-5 during fermentation, the number of live bacteria increased rapidly and remained stable during storage. During low-temperature storage, the number of live bacteria of GR-5 gradually decreased. On the 14th day, the number of live bacteria of beer GR-5 stored at various temperatures dropped to about 1*108cfu / mL, which still retained a relatively high amount of live bacteria and could provide live probiotics when drinking.

[0058] Figure 8 The change in yeast concentration during storage.

[0059] The different shapes in the legend represent different storage temperatures. The concentration of yeast can reflect the relative activity of yeast. It can be seen that the number of yeast at each temperature during low-temperature storage is 1*10 7 ~1*10 8 The cfu / mL showed an oscillating change, indicating that the system after fermentation and storage can be maintained for a long time and the yeast can still survive stably.

[0060] Fig. 9 To study the effect of adding GR-5 on purine substances in beer.

[0061] The height of each column indicates the specific content of each substance in the sample. It can be seen that compared with the CK group, the environmental hypoxanthine and inosine guanosine content of the CK+GR-5 group added with GR-5 for co-fermentation was significantly reduced, and the same trend was seen in the total purine content.

[0062] Fig.10 To investigate the effect of adding GR-5 on the total purine in beer.

[0063] The height of each column represents the total content of purine substances in the sample. Specifically, the addition of GR-5 significantly reduced the content of total purine and showed significant degradation efficiency for nucleosides.

[0064] Fig.11For packaging beer during storage.

[0065] Among them, it can be observed that there is milky white precipitation at the bottom of the packaged beer, and the beer body is yellowish brown, which is a cloudy beer.

[0066] Fig.12 This is a characteristic analysis of GR-5.

[0067] The small figures show the salt and acid resistance curves of GR-5 (a), bile salt resistance (b), harmful bacteria inhibition ability (transparent circle method, c) and probiotic cell adsorption ability (d).

[0068] Fig.13 Analysis of the purine degradation ability of GR-5.

[0069] The sub-figures show the degradation ability of GR-5 for various purines (a), inosine (b), guanosine (c), and adenosine (d). DETAILED DESCRIPTION

[0070] In order to make the purpose, technical solution and advantages of the invention clearer, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present invention shown in the accompanying drawings and described according to the accompanying drawings are merely exemplary, and the present invention is not limited to these embodiments. It should also be noted that in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only structures and / or processing steps closely related to the solution according to the present invention are shown in the accompanying drawings, and other details that are not very relevant are omitted.

[0071] Example 1

[0072] This embodiment provides a probiotic strain, which is Pediococcus acidilactici GR-5, which was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on September 16, 2022, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No. 25729.

[0073] Example 2

[0074] This embodiment provides a method for separating probiotics that can efficiently degrade purine substances, comprising the following steps:

[0075] 1. Isolation of microorganisms from Gansu Jiangshui

[0076] Fresh pulp water (Qin Yi in November 2021 / January 2022, Meng Gu in July 2021, and Qin Yaya in November 2021) was diluted 10 times, 100 times, 1000 times, and 10000 times with sterile water and evenly spread on MRS solid medium (10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 2.0 g diammonium hydrogen citrate [(NH4)H2C6H5O7], 20.0 g glucose (C6H 12 O6·H2O), 1.0mL Tween 80, 5.0g sodium acetate (CH3COONa·3H2O), 2.0g potassium dihydrogen phosphate (K2HPO4·3H2O), 0.58g magnesium sulfate (MgSO4·7H2O), 0.25g manganese sulfate (MnSO4·H2O), 15.0g agar, 1000mL distilled water, the above components were stirred evenly and then sterilized by high pressure at 121℃ for 15min, cooled and poured into plates), cultured at 37℃ for 48h, and the single colony obtained ( Figure 1 Left) The cells were picked out and inoculated into MRS liquid medium.

[0077] 2. Gram staining identification of strains

[0078] Perform Gram staining on the candidate bacteria obtained in 1 ( Figure 1 Right) (Take 100 μL of the culture medium and fix it on a slide with an alcohol lamp, initially stain it with crystal violet and then wash it with water, then add iodine solution for mordant staining and then wash it with water and decolorize it with 95% ethanol, then add safranin solution for restaining and wash it with water and dry it, then observe it with an oil objective lens) and observe the morphology under a light microscope, and screen out negative strains and yeast.

[0079] 3. Determination of the strain's ability to degrade pure purine compounds in vitro.

[0080] The candidate bacteria screened in Example 2 were inoculated into MRS liquid culture medium and cultured at 37°C for 48 h. After that, the OD was adjusted with sterile water. 600 To 0.7, after the dilution is completed, 2mL of each candidate single bacterial suspension is drawn into a centrifuge tube, centrifuged at 8000rpm for 10min, the supernatant is discarded and washed twice with sterile water, and the centrifugation operation is repeated to obtain a centrifuge tube containing a strain precipitate. Use analytically pure adenine, guanine, hypoxanthine, and xanthine to prepare a mother solution with a purine concentration of 1g / L (add 1mol / L NaOH to aid dissolution), and dilute 20 times to obtain a purine solution with a purine concentration of 50mg / L. Add 2mL of the prepared 50mg / L purine solution to the centrifuge tube containing the strain precipitate, shake and mix well, incubate at 37°C for 1h, centrifuge at 8000rpm for 5min after the incubation, and filter the supernatant with a 0.22μm filter membrane and measure it on the machine. The clearance rate = degraded purine content / purine content before degradation.

[0081] Purine content determination method: HPLC Agilent 1260, C18 column (Dim.mm 20*4.6, SN: 70744669), aqueous phase: 997mL ultrapure water + 1.5mL glacial acetic acid + 1.5mL tetrabutylammonium hydroxide, organic phase: methanol, aqueous phase: organic phase = 9:1, isocratic elution, VWD detector λ = 254nm, sample volume 10μL. The characteristic spectrum of the standard under the above chromatographic conditions is as follows Figure 2 shown.

[0082] Table 1. Purine degradation rate of strains screened from pulp water

[0083]

[0084] 4. Identification of strains

[0085] 16S rRNA gene sequencing was used to identify the phylogenetic characteristics of the isolates. 16S rRNA was amplified using bacterial universal specific primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTAC CTTGTTACGACTT-3'). The 20 mL PCR reaction system contained the following: 1 μL template, 0.5 μL primer 27F, 0.5 μL primer 1492R, 0.2 μL Taq DNA polymerase (Takara Bio, Japan), 2 μL reaction buffer, 1.65 μL dNTPs and 13.2 μl ddH2O. The amplified product was sent to BGI MiSeq for sequencing (BGI, China), and the 16S rRNA sequence (submitted on 2022-11-15 23:55:36, GenBank OP829805) was compared with the sequences of other related organisms in NCBI in Clustal X. Phylogenetic trees were constructed by the neighbor-joining method in MEGA software (version 5.1). Figure 3 a), the alignment result is Pediococcus, Pediococcus acidilactici. The bootstrap values ​​obtained by 1000 repetitions are expressed as percentages at the nodes.

[0086] The morphological characteristics of strain GR-5 were determined using a scanning electron microscope (SEM). After growing strain GR-5 in LB medium for 12 hours, it was washed three times with phosphate buffered saline buffer (PBS, pH maintained at 7.2). After centrifugation, the bacterial precipitate was gently blown up with 2.5% glutaraldehyde and placed at 4°C for fixation overnight. After that, the GR-5 strain precipitate was dehydrated in a gradient of 10%-100% ethanol series concentrations, and each gradient dehydration took about 15 minutes. The final dehydrated bacterial liquid was dropped onto a coverslip with a diameter of 1 cm and placed in a vacuum freeze dryer for freeze drying overnight. After gold spraying, the colonies on the coverslip were imaged and photographed using S-3400N SEM (Hitachi, Japan) ( Figure 3 b), the bacteria are stacked in the shape of short rods.

[0087] Example 3

[0088] This embodiment provides a method for screening the most suitable candidate strain for low-purine probiotic beer, comprising the following steps:

[0089] Taking 5L of the low-purine beer as an example, the raw materials of the low-purine probiotic beer include the following weight components: 1.2kg of dry barley, 6g of food-grade saccharifying enzyme, and 1g of hop extract (10:1). The preparation method of the low-purine probiotic beer is as follows:

[0090] 1. Weigh the dry barley and food-grade saccharifying enzyme from the raw materials of the low-purine probiotic beer, crush and sieve the dry barley. Add water to the dry material components of each raw material to obtain a first mixed solution; the amount of 70°C drinking water added is 2.5 times the weight of the dry matter.

[0091] 2. The first mixed solution is saccharified to obtain a second mixed solution: the temperature of the first mixed solution is maintained between 55-65°C, and it is stirred thoroughly to mix evenly. After maintaining for 60 minutes, iodine-potassium iodide solution is used to test whether saccharification is complete (saccharification is considered complete if the original color of the iodine-potassium iodide solution does not change after the addition). After saccharification is complete, the wort is heated to boiling, and the boiling state is maintained for 10 minutes. The obtained mixture is filtered through eight layers of medical gauze to obtain a filtered mixed solution II.

[0092] 3. After filtering the mixture 2 with gauze, add 60℃ drinking water with a dry matter mass ratio of 1:1, mix thoroughly for 10 minutes, filter again to collect the filtrate, repeat the operation once, collect all the filtrates and mix to obtain the mixture 3. Mix the mixture 2 and the mixture 3, adjust the Brix to 10, and obtain the mixture 4.

[0093] 4. The fourth mixed solution is sterilized at high temperature to remove most of the precipitate, and yeast is added for primary fermentation to obtain the original beer crude liquid, and probiotics are added to the beer crude liquid for secondary fermentation to obtain the probiotic beer crude liquid. Specific implementation plan: The fourth mixed solution is sterilized at high temperature (110°C, 20min), the supernatant is transferred to the fermentation bottle, the turbid precipitate is discarded, and 1% yeast seed liquid is added (seed liquid preparation method: after the malt juice is sterilized, the preserved beer yeast is added and cultured at 37°C for 1 day) to make the final yeast concentration reach 1*10 5 cfu / mL, the first fermentation was carried out at 20℃, and the sugar content was continuously monitored during the fermentation. The initial fermentation was considered to be over when the sugar content changed less than 0.1 for two consecutive days; the seed solution of the candidate bacteria was inoculated (seed solution preparation method: the preserved candidate bacteria were inoculated after the malt juice was sterilized and cultured at 37℃ for 1 day) to make the final concentration reach 1*10 6 cfu / ml, after fermentation at 37°C for one day, the temperature was lowered to 20°C and maintained for one day to obtain the probiotic beer crude liquid, of which a part of the beer was not added with the alternative bacteria as the control (CK).

[0094] 5. Add the diluted sterilized hop extract (1 g dissolved in 30 mL of drinking water, 6 mL added per liter) to the obtained probiotic beer crude liquid, with a final hop isomer concentration of 2‰.

[0095] 6. Purine content determination

[0096] Purine content: HPLC Agilent 1260, C18 column (Dim.mm 20*4.6, SN: 70744669), aqueous phase: 997 mL ultrapure water + 1.5 mL glacial acetic acid + 1.5 mL tetrabutylammonium hydroxide, organic phase: methanol, aqueous phase: organic phase = 9:1, isocratic elution, VWD detector λ = 254 nm, sample volume is 10 μL.

[0097] Preparation of standard solution: Weigh 0.025g of each purine and transfer it to a 50mL volumetric flask. Add a small amount of ultrapure water and then use NaOH to dissolve it. After filtering the prepared purine solution through a sterile filter membrane, store it in a sterile centrifuge tube and store it at 4°C. If a white precipitate appears, it needs to be re-prepared.

[0098] Here, purine in beer is divided into two types: one is free purine that exists directly in the form of purine. This part of purine is directly dissolved in beer and can be directly detected; the other is purine substances that exist in microorganisms or insoluble substances in the form of purine compounds. This part of purine needs to be acid-lyzed before it can be detected. The sum of the two types of purine is called the total purine content of beer. For the convenience of description, the legends involving the total purine content in the following text are marked with "total".

[0099] Method for determination of free purine: ultrasonically degas beer for 20 minutes, centrifuge at 4000rpm for 2 minutes, take the supernatant and filter it through a 0.22μm water filter membrane before loading directly. Method for determination of total purine: ultrasonically degas beer for 20 minutes, mix well and take 5mL into a 25mL stoppered colorimetric tube, add 0.25mL 70% perchloric acid, boil in water bath for 1h, filter quickly in ice bath, adjust pH to neutral with KOH solution, filter with qualitative filter paper, use a volumetric flask to dilute to 25mL with ultrapure water, filter through a 0.22μm water filter membrane before loading on the machine for determination.

[0100] Figure 4 To show the effect of adding GR-5 to the co-fermentation of beer samples, the spectrum of beer without adding the screening bacteria is as follows Figure 4 The spectrum of beer with the addition of the candidate bacteria GR-5 is shown in Figure 4 Under the condition of 4.7min, hypoxanthine and 5.9min appeared in the best degradation effect. The addition of Pediococcus acidilactici GR-5 can significantly reduce the content of total purine substances, especially hypoxanthine. The degradation efficiency of free nucleoside substances can reach 93%.

[0101] Table 2. Purine content in beer with different selected bacteria (mg / L)

[0102]

[0103] Example 4

[0104] This embodiment provides a method for preparing low-purine probiotic beer from scratch, comprising the following steps:

[0105] Taking 5L of the low-purine beer as an example, the raw materials of the low-purine probiotic beer include the following components by weight: 1.2kg of dry stored barley, 6g of food-grade saccharifying enzyme, 1g of hop extract (1:10), and 100mL of saturated baking soda solution (25°C) (using baking soda can produce more CO2 and improve palatability, and Na2CO3 can be used to adjust the pH in actual application).

[0106] The preparation method of the low-purine probiotic beer is as follows:

[0107] 1. Weigh the dry barley and food-grade saccharifying enzyme from the raw materials of the low-purine probiotic beer, crush and sieve the dry barley. Add water to the dry material components of each raw material to obtain a first mixed solution; the amount of 70°C drinking water added is 2.5 times the weight of the dry matter.

[0108] 2. The first mixed solution is saccharified to obtain a second mixed solution: the temperature of the first mixed solution is maintained between 55-65°C, and it is stirred thoroughly to mix evenly. After maintaining for 60 minutes, iodine-potassium iodide solution is used to test whether saccharification is complete (saccharification is considered complete if the original color of the iodine-potassium iodide solution does not change after the addition). After saccharification is complete, the wort is heated to boiling, and the boiling state is maintained for 10 minutes. The obtained mixture is filtered through eight layers of medical gauze to obtain a filtered mixed solution II.

[0109] 3. After filtering the mixture 2 with gauze, add 60℃ drinking water with a dry matter mass ratio of 1:1, mix thoroughly for 10 minutes, filter again to collect the filtrate, repeat the operation once, collect all the filtrates and mix to obtain the mixture 3. Mix the mixture 2 and the mixture 3, adjust the Brix to 10, and obtain the mixture 4.

[0110] 4. The fourth mixed solution is sterilized at high temperature to remove most of the precipitate, and yeast is added for primary fermentation to obtain the original beer crude liquid, and probiotics are added to the beer crude liquid for secondary fermentation to obtain the probiotic beer crude liquid. Specific implementation plan: The fourth mixed solution is sterilized at high temperature (110°C, 20min), the supernatant is transferred to the fermentation bottle, the turbid precipitate is discarded, and 1% yeast seed liquid is added (seed liquid preparation method: after the malt juice is sterilized, the preserved beer yeast is added and cultured at 37°C for 1 day) to make the final yeast concentration reach 1*10 5 cfu / mL, the first fermentation was carried out at 20°C, and the sugar content was continuously monitored during the fermentation (e.g. Figure 5 ) and pH (such as Figure 6 ) changes, and the initial fermentation is considered to be completed when the sugar content changes less than 0.1 for two consecutive days (such as Figure 5 ); inoculate GR-5 seed solution (seed solution preparation method: inoculate the preserved lactic acid bacteria GR-5 after malt juice sterilization, and culture at 37°C for 1 day) to make the final concentration reach 1*10 6 cfu / ml, fermented at 37°C for one day, then the temperature was lowered to 20°C and maintained for one day to obtain the probiotic beer crude liquid, of which a portion of the beer was not added with Pediococcus acidilactici GR-5 as the control (CK).

[0111] To the obtained probiotic beer crude liquid, add the diluted sterilized hop extract (1 g dissolved in 30 mL of drinking water, 6 mL added per liter), with a final hop isomer concentration of 2‰. Add saturated baking soda (25°C) to the obtained probiotic beer crude liquid. The pH of the wine before addition is 3.49, and the pH of the wine after addition is 4.1.

[0112] The finished low-purine probiotic beer was mixed and packaged into sterile glass bottles and stored at 4°C, 6°C, and 10°C respectively. During this period, the GR-5 concentration was measured ( Figure 7 ) and yeast concentration ( Figure 8 ), when its concentration drops to 1*108 When the concentration is below cfu / mL, it is considered expired.

[0113] 5. Quality determination method:

[0114] pH: An electronic pH meter (Alalis pH 400 Basic Acidity Meter) was used.

[0115] Sugar content: Handheld refractometer.

[0116] Strain concentration: GR-5 (selective plate method, diluted and coated on MRS solid medium plate, the concentration of natamycin in the medium is 150ppm), brewer's yeast (microscopic counting method, diluted 100 times and counted under a microscope using a hemocytometer)

[0117] Purine content: HPLC Agilent 1260, C18 column (Dim.mm 20*4.6, SN: 70744669), aqueous phase: 997 mL ultrapure water + 1.5 mL glacial acetic acid + 1.5 mL tetrabutylammonium hydroxide, organic phase: methanol, aqueous phase: organic phase = 9:1, isocratic elution, VWD detector λ = 254 nm, sample volume is 10 μL.

[0118] Preparation of standard solution: Weigh 0.025g of each purine and transfer it to a 50mL volumetric flask. Add a small amount of ultrapure water and then use NaOH to dissolve it. After filtering the prepared purine solution through a sterile filter membrane, store it in a sterile centrifuge tube and store it at 4°C. If a white precipitate appears, it needs to be re-prepared.

[0119] Method for determination of free purine: ultrasonically degas beer for 20 minutes, centrifuge at 4000 rpm for 2 minutes, take the supernatant and filter it through a 0.22 μm water filter membrane before loading the sample directly.

[0120] Total purine determination method: ultrasonically degas beer for 20 minutes, mix well, take 5 mL into a 25 mL stoppered colorimetric tube, add 0.25 mL 70% perchloric acid, boil in water bath for 1 hour, quickly filter in ice bath, adjust pH to neutral with KOH solution, filter with qualitative filter paper, use a volumetric flask to make up to 25 mL with ultrapure water, and determine after passing through a 0.22μm water filter membrane.

[0121] Changes in beer sugar content during fermentation Figure 5 , pH changes as Figure 6 At the end of fermentation, the sugar content dropped to half of that at the beginning of fermentation and remained stable. The addition of GR-5 promoted the acidification of beer. After 14 days of storage, the number of viable bacteria in GR-5 remained at 1*10 7 cfu / ml or more ( Figure 7 ).

[0122] Table 3. Peak time of each purine standard under the above chromatographic conditions (s)

[0123]

[0124] When the concentration of purine substances is between 0.5-50 mg / L, a linear fit is performed according to the concentration of the standard sample and the corresponding peak area. 2 >0.9996.

[0125] The contents of various purine substances were converted according to the standard curve. The results are as follows: Fig. 9 and Fig.10 Compared with the CK group, the environmental hypoxanthine and inosine guanosine contents of the CK+GR-5 group co-fermented with GR-5 were significantly reduced, and the same trend was found in the total purine content; specifically, the addition of GR-5 significantly reduced the total purine content and showed significant degradation efficiency for nucleoside substances.

[0126] Example 5

[0127] The simple preparation method of the low-purine probiotic beer of the present invention comprises the following steps:

[0128] Taking 5L of the low-purine probiotic improved beer as an example, the finished malt juice of a local brewery (purchased from Lanzhou Red Star Brewery on December 7, 2021 and stored in an ultra-low temperature refrigerator at -80°C) was used for fermentation, and the remaining components were 0.75g of hop extract (1:10) and 100mL of saturated baking soda solution (25°C) (the use of baking soda can produce more CO2, and NA2CO3 can be used to adjust the pH in actual applications).

[0129] The preparation method of the low-purine probiotic beer is as follows:

[0130] 1. The wort obtained from a local brewery was sterilized (110°C, 20 min), the precipitate was filtered out, and the sugar content was adjusted to 10°Bx.

[0131] 2. Inoculate yeast for primary fermentation to obtain original beer crude liquid, add probiotics to the beer crude liquid for secondary fermentation to obtain probiotic beer crude liquid. Specific implementation plan: sterilize the fourth mixed liquid at high temperature (110°C, 20min), transfer the supernatant to the fermentation bottle, discard the turbid precipitate, inoculate 1% yeast seed liquid (seed liquid preparation method: inoculate the preserved beer yeast after the malt juice is sterilized, and culture at 37°C for 1 day) to make the final yeast concentration reach 1*10 5cfu / mL, the first fermentation was carried out at 20℃, and the sugar content was continuously monitored during the fermentation. The initial fermentation was considered to be completed when the sugar content changed less than 0.1 for two consecutive days; the seed liquid of Pediococcus acidilactici GR-5 was inoculated (seed liquid preparation method: after the malt juice was sterilized, it was inoculated with preserved GR-5 and cultured at 37℃ for 1 day) to make the final concentration reach 1*10 6 cfu / mL, fermented at 37°C for one day, then the temperature was lowered to 20°C and maintained for one day to obtain the crude probiotic beer solution, of which a portion of the beer was not added with GR-5 as a control (CK1).

[0132] 3. Add the diluted sterilized hop extract (0.75 g dissolved in 30 mL of drinking water, 6 mL per liter) to the obtained probiotic beer crude liquid, the final concentration of hop isomers is 1.5‰, add saturated baking soda (25°C) to the obtained probiotic beer crude liquid, the pH of the wine before addition is 3.49, and the pH of the wine after addition is 4.1. The finished low-purine probiotic beer is mixed and divided into sterile glass bottles and stored at 4°C ( Fig.11 ).

[0133] 4. Comparative analysis with commercially available beer

[0134] Common beers on the market were collected, and sensory evaluation was compared with the beer of the present invention, and the purine content of the collected beers was measured.

[0135] (1) The purine detection method is the same as above, and the chromatographic conditions are as follows:

[0136] Chromatograph: Agilent 1260; chromatographic column: C18 chromatographic column; aqueous phase: 997 mL ultrapure water + 1.5 mL glacial acetic acid + 1.5 mL tetrabutylammonium hydroxide; organic phase: methanol, aqueous phase: organic phase = 9:1; isocratic elution, flow rate 1 mL / min; injection volume 10 μL; column temperature 25°C; VWD detector λ = 254 nm, sample volume 10 μL.

[0137] (2) Preparation of standard samples: Accurately weigh 0.025 g each of inosine, guanosine, adenine, guanine, xanthine, and hypoxanthine, transfer to a 25 mL volumetric flask, add ultrapure water to dissolve, and use a small amount of 10 mol / L NaOH to assist dissolution to obtain a standard stock solution with a standard concentration of 500 mg / L.

[0138] (3) Determination of purine in beer: Take 20 mL of beer, degas by ultrasonication for 20 min, centrifuge at 4000 rpm for 2 min, and filter the supernatant through a 0.22 μm filter for testing.

[0139] (4) Determination of total purine content in beer: ultrasonically degas beer for 20 min, mix well, take 5 mL into a 25 mL stoppered colorimetric tube, add 0.25 mL 70% perchloric acid, boil in water bath for 1 h, quickly filter in ice bath, adjust pH to neutral with KOH solution, filter with qualitative filter paper, dilute to 25 mL in a volumetric flask with ultrapure water, and filter through a 0.22 μm water filter membrane before determination.

[0140] The results are shown in the following table. In the low-purine probiotic beer obtained by the preparation method provided by the example of the present invention, the purine content of the beer fermented with Pediococcus acidilactici GR-5 in Example 4 is 37.8999 mg / L, and the purine content of the beer without GR-5 is 100.961 mg / L. The purine content of the beer fermented with GR-5 in Example 5 is 35.0662 mg / L, and the purine content of the beer without GR-5 is 61.5207 mg / L. The free purine content of commercial beer is 75.420 mg / L. It can be seen that the beer prepared by the present invention has a low purine content.

[0141] Table 4. Comparison of purine content in various examples (mg / L)

[0142]

[0143] Example 6

[0144] This embodiment provides a method for making low-purine probiotic beer, comprising the following steps:

[0145] (1) Wash, dry and crush the dry stored barley into a small particle powder mixture, and pass it through a 2.5 mm sieve to remove large uncrushed grains.

[0146] (2) adding food-grade saccharifying enzyme at a ratio of 1:200, adding drinking water at 70°C at a weight ratio of 1:2.5, and completing saccharification in one step at 60°C;

[0147] (3) Heat and boil, filter out the residue, and heat-extract the residue again with 60°C drinking water for 10 min. Filter and mix with the previous one, remove the precipitate after heating, and adjust the initial sugar content to 10°Bx;

[0148] (4) Sterilized wort was inoculated with yeast to a final concentration of 1*10 5 cfu / mL, fermented at 20°C, and the first fermentation ended after 5-6 days of constant sugar content (the sugar content changed less than 0.1 within two days).

[0149] (5) After the yeast fermentation cycle in step (4) is completed, the highly efficient purine-degrading probiotic GR-5 screened from the slurry is inoculated and the final concentration is adjusted to 1*10 6 cfu / mL, fermented at 37°C for 1 day;

[0150] (6) After one day of fermentation in step (5), the temperature was lowered back to 20° C. and fermentation was continued for another day to stabilize the fermentation system.

[0151] (7) Add 2‰ boiled hop extract and baking soda solution to the obtained bacteria-containing beer in step (6) to adjust the pH to the pH of the beer before the probiotics are added (the pH can be adjusted after the fermentation is completed or continuously added during the fermentation).

[0152] (8) The finished product after seasoning should be stored in a low temperature environment after packaging to extend the shelf life.

[0153] The crusher used in step (1) is not limited to any model, but the higher the degree of barley crushing, the higher the saccharification efficiency and the greater the difficulty of filtering, which should be comprehensively considered when using it.

[0154] The saccharifying enzyme used in step (2) is a food-grade saccharifying enzyme produced by Shandong Longkote Enzyme Preparation Co., Ltd.

[0155] The ratio of drinking water to barley flour for saccharification in step (2) is 2.5:1. After mixing, the temperature of the mixture is about 60° C. The sugar content will still be adjusted later. Complete saccharification means that the iodine test does not change color.

[0156] The filtering method adopted in step (3) is gauze filtering, and the supernatant is taken for fermentation after standing at high temperature.

[0157] The sterile wort obtained in step (3) may contain a certain amount of precipitate, most of which is protein. This part of the precipitate can be used by yeast during fermentation.

[0158] The yeast used in step (4) is brewer's yeast (Saccharomyces cerevisiae).

[0159] The fermentation device used in step (4) is not restricted in form, but must ensure the sterilization effect, avoid contamination by miscellaneous bacteria, and pay attention to timely exhaust during fermentation.

[0160] The probiotics used in step (5) are highly efficient purine-degrading strains of Pediococcus acidilactici GR-5 obtained by the Environmental Microbiology Laboratory of Lanzhou University from the natural fermented food "slurry". The probiotics used in step (5) are GR-5, and the properties of GR-5 are shown in the attached figure ( Figure 12-13 The JL-3 (lactic acid fermentation bacteria) mentioned in the figure is a probiotic bacteria deposited in CCTCC with the patent number CCTCC NO: M2021902, which has been applied for by our unit in patent CN202111503162.3).

[0161] The seed liquid used in step (4) and step (5) should be the same as malt extract for expansion.

[0162] The hop extract used in step (7) is 2‰, which refers to the unconcentrated proportion of the original hops. The baking soda used in the example is the "After Snow" edible baking soda produced by Inner Mongolia Etuoke Banner Shuangxin Chemical Co., Ltd.

[0163] The low temperature environment mentioned in step (8) should be below 10°C.

[0164] The low-purine probiotic beer refers to beer whose free purine content (adenine, guanine, hypoxanthine, xanthine, inosine, guanosine) is less than 40 mg / L.

[0165] The above is only a specific implementation of the present application. 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 application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A probiotic strain, the probiotic strain is Pediococcus acidilactici GR-5, which was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on September 16, 2022, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No. 25729.

2. A method for preparing low-purine probiotic beer from scratch, characterized in that The method comprises the following steps: (1) Weighing the dry barley and food-grade saccharifying enzyme from the raw materials of the low-purine probiotic beer, crushing and sieving the dry barley; adding water to the dry material components of each raw material to obtain a first mixed solution; adding 70° C. drinking water in an amount of 2.5 times the weight of the dry matter; (2) saccharifying the first mixed solution to obtain a second mixed solution: maintaining the temperature of the first mixed solution between 55° C. and 65° C., stirring the first mixed solution to make it uniformly mixed, maintaining the temperature for 60 minutes, and testing whether the saccharification is complete with an iodine-potassium iodide solution. After the saccharification is complete, heating the wort to boiling, maintaining the boiling state for 10 minutes, and filtering the obtained mixture with eight layers of medical gauze to obtain a second mixed solution; (3) adding 60°C drinking water in a 1:1 ratio of dry matter mass to the residue after filtering with gauze, mixing thoroughly for 10 minutes, filtering again to collect the filtrate, repeating the operation once, collecting all the filtrates and mixing to obtain a mixed solution 3; mixing the mixed solution 2 and the mixed solution 3, adjusting the Brix to 10, to obtain a mixed solution 4; (4) sterilizing the fourth mixed solution at high temperature to remove most of the precipitate, adding yeast for an anaerobic fermentation to obtain an original beer crude liquid, and adding probiotics to the beer crude liquid for a secondary fermentation to obtain a probiotic beer crude liquid; Specific implementation plan: sterilize the fourth mixed solution at high temperature, transfer the supernatant to the fermentation bottle, discard the turbid precipitate, add 1% volume of yeast seed solution to make the final yeast concentration reach 1*10 5 cfu / mL, and the first anaerobic fermentation was carried out at 20°C. During the fermentation, the sugar content and pH changes were continuously monitored. The initial fermentation was considered to be completed when the sugar content change was less than 0.1 for two consecutive days. GR-5 seed liquid was added to make the final concentration reach 1*10 6 cfu / ml, fermented at 37°C for one day, then the temperature was lowered to 20°C for one day to obtain a crude probiotic beer solution, of which a portion of the beer was not added with Pediococcus acidilactici GR-5 as a control; (5) adding the diluted sterilized hop extract to the obtained probiotic beer crude liquid, the final concentration of hop isomers is 2‰, adding 25°C saturated baking soda to the obtained probiotic beer crude liquid to adjust the palatability, the pH of the beer before addition is 3.49, and the pH of the beer after addition is 4.1; (6) The finished low-purine probiotic beer was mixed and packaged into sterile glass bottles and stored at 4°C, 6°C, and 10°C. During the storage, the GR-5 concentration and yeast concentration were measured. When the concentration dropped to 1*10 8 cfu / mL or less is considered expired; (7) Quality determination: determination of pH, sugar content, strain concentration, and purine content.

3. A simple method for preparing low-purine probiotic beer, characterized in that The method comprises the following steps: (1) Sterilize the commercially available wort at 110°C for 20 min, filter out the precipitate, and adjust the sugar content to 10°Bx; (2) inoculating yeast for primary anaerobic fermentation to obtain original beer crude liquid, and adding probiotics to the beer crude liquid for secondary fermentation to obtain probiotic beer crude liquid; Specific implementation plan: sterilize the commercially available wort at 110°C for 20 min, transfer the supernatant to a fermentation bottle, discard the turbid precipitate, add 1% yeast seed liquid, and make the final yeast concentration reach 1*10 5 cfu / mL, and the first anaerobic fermentation was carried out at 20°C. The sugar content was continuously monitored during the fermentation. When the sugar content changed less than 0.1 for two consecutive days, the initial fermentation was considered to be over. The seed liquid of Pediococcus acidilactici GR-5 was added to make the final concentration reach 1*10 6 cfu / mL, fermented at 37°C for one day, then the temperature was reduced to 20°C and maintained for one day to obtain a probiotic beer crude liquid, wherein a portion of the beer was not added with GR-5 as a control; the yeast seed liquid preparation method: after the malt juice was sterilized, the preserved beer yeast was inoculated and aerobically cultured at 37°C for one day; the GR-5 seed liquid preparation method: after the malt juice was sterilized, the preserved GR-5 was inoculated and aerobically cultured at 37°C for one day; (3) adding the diluted sterilized hop extract to the obtained probiotic beer crude liquid, the final concentration of hop isomers is 1.5‰, adding 25°C saturated baking soda to the obtained probiotic beer crude liquid, the pH of the wine before addition is 3.49, and the pH of the wine after addition is 4.1; the finished low-purine probiotic beer is mixed and divided into sterile glass bottles and stored at 4°C; (4) Comparative analysis with commercially available beer: collect common beers on the market, conduct sensory evaluation and comparison with the beer of the present invention, and measure the purine content of the collected beers.

4. A method for preparing low-purine probiotic beer, characterized in that The low-purine probiotic beer refers to beer in which the content of free purine substances such as adenine, guanine, hypoxanthine, xanthine, inosine and guanosine is less than 40 mg / L; the production method adopts a probiotic strain according to claim 1 and comprises the following steps: (1) Wash, dry and crush the dry stored barley into a small particle powder mixture, and pass it through a 2.5 mm sieve to remove large un-crushed grains; the higher the degree of barley crushing, the higher the saccharification efficiency and the greater the difficulty of filtering. When using it, comprehensive considerations should be made and appropriate adjustments should be made based on the final sugar content; (2) Add edible grade saccharifying enzyme at a ratio of 1:200, add 70℃ weight ratio of 1: 2.5 drinking water, maintaining the temperature between 55-60°C and completing saccharification in one step for 60 minutes; the saccharifying enzyme is preferably a food-grade saccharifying enzyme produced by Shandong Longkote Enzyme Preparation Co., Ltd.; the ratio of drinking water for saccharification to barley flour is 2.5:1, and the temperature of the mixture after mixing is about 60°C. The sugar content will still be adjusted later. Complete saccharification means that the iodine test does not change color; (3) heating and boiling, filtering out the residue, and re-extracting the residue with 60°C drinking water for 10 min, filtering and mixing with the former, removing the precipitate after heating, and adjusting the initial sugar content to 10°Bx; using gauze filtering, standing at high temperature, and taking the supernatant for fermentation; sterile malt juice may contain a certain amount of precipitate, most of which is protein, and this part of the precipitate can be used by yeast during fermentation; (4) Sterilized wort was inoculated with yeast to a final concentration of 1*10 5 cfu / mL, fermented at 20℃, and the first fermentation ended after 5-6 days of constant sugar content. Constant sugar content means that the sugar content changes less than 0.1 within two days. The yeast used is commercially available beer yeast Saccharomyces cerevisiae. However, the sterilization effect must be ensured to avoid contamination with miscellaneous bacteria, and timely exhaust should be paid attention to during fermentation. (5) After the yeast fermentation cycle in step (4) is completed, the highly efficient purine-degrading probiotic GR-5 screened from the slurry is inoculated and the final concentration is adjusted to 1*10 6 cfu / mL, fermented at 37°C for 1 day; (6) After one day of fermentation in step (5), the temperature was lowered back to 20° C. and fermentation was continued for another day to stabilize the fermentation system; (7) After the fermentation in step (6) is completed, or during the fermentation period, 2‰ of boiled hop extract and 25°C baking soda solution are continuously added to adjust the pH to the pH of the beer before the probiotics are added; the brand of hop extract is pure natural 10:1 hop powder from Plant Extract Home, and 2‰ refers to the original unconcentrated ratio of hops; (8) The finished product after seasoning should be packaged in sterile containers and is recommended to be stored in a low temperature environment to extend the shelf life. The low temperature environment should be below 10°C.

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