Botrytis adeninivorans BA0616 and application of botrytis adeninivorans BA0616 in preparation of low-purine food

Abstract

The invention discloses a botrytis adeninivorans BA0616 strain and application thereof in low-purine food, and relates to the technical field of microorganisms. The preservation number of the adeninivorous botrytis cinerea BA0616 disclosed by the invention is GDMCC (China General Microbiological Culture Collection Center) NO: 65287. The adeninivorous botrytis cinerea can take a purine substance as a carbon source, has a very strong metabolic capability on the purine substance, and can be applied to fermentation of low-purine food. The adeninivorous botrytis cinerea is applied to beer production, and the low-purine beer brewed through co-fermentation of the adeninivorous botrytis cinerea and beer yeast is rich in foam, fine and smooth in taste and rich in purine content on the premise that the usage amount of barley malt is not reduced and exogenous additives such as adsorbents, strong acids, strong bases and enzymes are not additionally added. And the purine content in the final beer is only 8-15mg / L, which is only 1 / 5-1 / 10 of that of common beer, so that the beer is healthier to drink.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more particularly to a strain of adenine-feeding budding botrytis cinerea BA0616 and application thereof in preparing low-purine food. Background Art

[0002] Among alcoholic beverages, the purine content of fermented wines such as sake, beer and wine is much higher than that of distilled wines such as Japanese shochu and whiskey, among which beer has the highest purine content (40-100 mg / L). Many epidemiological studies have shown that there is a high dependence between a high-purine diet and the formation of gout. Since 1980, the scale of beer production in my country has continued to expand. In 2002, it surpassed the United States for the first time to become the world's largest producer and consumer. The beer consumer group has gradually expanded and is showing a trend of younger age, and the incidence of gout is also increasing year by year. Therefore, reducing the intake of purine substances is considered to be the best way to prevent hyperuricemia and gout.

[0003] At present, researchers at home and abroad have conducted some research in the field of reducing beer purine, mainly by reducing the amount of barley malt used (application number 201210012067); adding acid, alkali (application number 201310600728, 2017102912509) and enzyme preparations (application number 201510575000) in the saccharification stage; adding activated carbon and zeolite (application number 2005100946709) and other adsorbents to the body of the beer. Although the purine substances in beer can be reduced to varying degrees, the too low proportion of malt used does not meet the requirements of beer brewing (that is, malt is the main raw material, the malt content is ≥50%), and there are chemical or physical additives in the brewing process.

[0004] Therefore, safe and efficient brewing of low-purine beer is a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides adenine-eating budding grape spore yeast (Blastobotrysadeninivorans) BA0616, and its application in brewing low-purine food. The strain can efficiently utilize purine substances, and it is co-fermented with brewer's yeast, without reducing the amount of malt used or requiring additional additives, and the brewed beer has low purine content and unchanged taste and flavor.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Blastobotrys adeninivorans, wherein the deposit number of the Blastobotrys adeninivorans is GDMCC NO:65287.

[0008] Another object of the present invention is to provide a microbial agent comprising the above-mentioned adenine-feeding budding Botrytis cinerea.

[0009] Another object of the present invention is to provide the use of the above-mentioned adenine-eating budding Botrytis cinerea or the above-mentioned microbial agent in the preparation of low-purine brewed food.

[0010] Another object of the present invention is to provide a low-purine brewed beer, using the above-mentioned adenine-eating budding Botrytis cinerea or the above-mentioned microbial agent as a fermentation agent to prepare brewed food.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned low-purine brewed beer, which is obtained by co-fermenting the above-mentioned adenine-eating budding grape spore yeast or the above-mentioned microbial agent with beer yeast.

[0012] Preferably, the preparation method specifically comprises the following steps:

[0013] (1) Wort saccharification:

[0014] The barley malt was ground using a standard mill and saccharified at a material-water ratio of 1:4. Saccharification process: 48°C for 30 minutes, then heated to 65°C for 60 minutes, then heated to 72°C for 10 minutes, and finally heated to 78°C for 10 minutes, with a heating rate of 1°C / min. After saccharification, filter and adjust the wort concentration to 10°P.

[0015] (2) Wort boiling:

[0016] 10°P wort and 10°P syrup were mixed in a volume ratio of 1:1, hops were added in an amount of 0.2 g / L, boiled for 60 min, vortexed and precipitated, and cooled;

[0017] (3) Fermentation:

[0018] Adenine-eating budding yeast and brewer's yeast were inoculated into the wort at a ratio of 1:50, with an inoculation volume of 1×10 5 CFU / mL, 5×10 6 CFU / mL, the inoculation temperature is 18±0.2℃, the temperature during the main fermentation is maintained at 20±0.2℃, sterile air is filled to make the dissolved oxygen reach 8-10mg / L, when diacetyl ≤0.05mg / L, the main fermentation ends, the temperature is lowered to 0-4℃, the yeast mud is separated, and the wine enters the post-ripening stage;

[0019] (4) Filtration: After 14 days of post-aging storage, the beer is filtered to obtain the finished beer.

[0020] Beneficial effects: The adenine-eating budding grape spore yeast provided by the present invention can use purine substances as carbon sources, has a strong metabolic ability for purine substances, and can be used for the fermentation of low-purine foods. The adenine-eating grape spore yeast is applied to beer production. Under the premise of not reducing the use amount of barley malt and not adding extra adsorbents, strong acids, strong bases, enzymes and other exogenous additives, the low-purine beer brewed by co-fermenting the adenine-eating grape spore yeast with beer yeast has rich foam and delicate taste. The purine content in the final beer is only 8-15 mg / L, which is only 1 / 5-1 / 10 of that of ordinary beer, and it is healthier to drink. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 The accompanying drawing is a schematic diagram of the colony of adenine-feeding budding Botrytis cinerea GDMCC 65287 provided by the present invention.

[0023] Figure 2 The accompanying drawing is a phylogenetic tree of the adenine-feeding budding Botrytis cinerea GDMCC 65287 of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 Isolation, screening and identification of strains

[0026] 1. Isolation of strains

[0027] Sample preparation: 5 g of ripe Pu'er tea (Yunnan Pu'er Tea Factory) was ground in a pre-sterilized mortar, added to 100 mL of sterile distilled water and mixed, and vortexed for 20 min. After the bacterial solution was fully mixed, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5Use a pipette to take 0.2 mL of the diluted bacterial suspension and spread it on YPD plate culture medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder). Spread 3 plates of each culture medium for each sample and each dilution, and place them in a constant temperature incubator at 30°C for culture.

[0028] After the colonies grow, they are separated and purified according to their growth characteristics. Pick out a single colony on the plate medium, use an inoculation needle to streak on the YPD medium, and repeat the inoculation and separation 2 to 3 times until a pure strain is obtained, and then culture it in a constant temperature incubator.

[0029] 2. Determination of the ability of strains to degrade purine substances

[0030] Preparation of purine-reducing strain screening medium: barley malt was ground using a standard grinder and saccharified at a material-water ratio of 1:4. Saccharification process: keep warm at 48℃ for 30min, heat to 65℃ for 60min, then heat to 72℃ for 10min, and finally heat to 78℃ for 10min, with a heating rate of 1℃ / min. After saccharification, filter, adjust the wort concentration to 10°P, add 0.2g / L hops, boil for 60min, filter, and cool.

[0031] Each triangular flask (150 mL) was filled with 30 mL of 10°P wort medium. The isolated candidate bacteria were inoculated into the wort medium alone, placed in a shaker at 30°C and 200 rpm, and cultured for 12 hours. After the end, the supernatant was collected by centrifugation at 8000 rpm for 5 minutes, filtered with a 0.22 μm filter membrane, and the degradation rates of adenine, guanine, hypoxanthine, xanthine, adenosine, guanosine and hypoxanthine were determined according to the method of Li et al. (Li H, Liu F, Hao J, et al. Determination of purines in beer by HPLC using a simple and rapid sample pretreatment [J]. Journal of the American Society of Brewing Chemists, 2015, 73 (2): 137-142.) (see Table 1).

[0032] Total purines = adenine + guanine + hypoxanthine + xanthine + adenine + guanosine + hypoxanthine

[0033] Total purine degradation rate = total purine content degraded / total purine content before degradation

[0034] Table 1

[0035]

[0036] 3. Identification of strains

[0037] Extraction of strain DNA: The purified BA0616 strain was used to extract genomic DNA using a fungal DNA extraction kit (Shanghai Sangon Biotechnology Co., Ltd.) to obtain template DNA for later use.

[0038] PCR amplification and sequencing: PCR amplification primers: ITS1 (5-TCCGTAGGTGAACCTGCGG-3, SEQ ID NO.2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3, SEQ ID NO.3). PCR system (25μL): template DNA 0.5μL, mix enzyme 12.5μL, ITS 10.5μL, ITS 10.5μL, sterile water 11μL. PCR amplification conditions: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 35s, 72℃ extension for 1min, 35 cycles, and finally 72C extension for 8min.

[0039] The PCR product was sent to Shanghai Biotechnology Co., Ltd. for sequence determination. The sequencing results were compared with BLAST software on NCBI (SEQ ID NO. 1), and the isolated strain BA0616 was determined to be Blastobotrys adeninivorans.

[0040] , SEQ ID NO.1.

[0041] The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 21, 2024, with the deposit number GDMCC No: 65287, classified as Blastobotrys adeninivorans, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0042] Example 2 Strain fermentation of beer

[0043] 1. Raw material ratio: barley malt 100%.

[0044] 2. Saccharification:

[0045] The barley malt was ground using a standard mill and saccharified at a material-water ratio of 1:4. Saccharification process: 48°C for 30 minutes, heated to 65°C for 60 minutes, then heated to 72°C for 10 minutes, and finally heated to 78°C for 10 minutes, with a heating rate of 1°C / min. After saccharification, filter, adjust the wort concentration to 10°P, add 0.2g / L hops, boil for 60 minutes, swirl and settle, and cool.

[0046] 3. Fermentation:

[0047] Adenine-eating budding yeast BA0616 and commercial beer yeast Mangrive Jacks M29 were inoculated into the wort at a ratio of 1:50, with an inoculum of 1×10 5 CFU / mL, 5×10 6 CFU / mL. One in the control group was inoculated with brewer's yeast Mangrive Jacks M29 at a volume of 5×10 6 CFU / mL. The inoculation temperature is 18±0.2℃, and the temperature during the main fermentation is maintained at 20±0.2℃. Sterile air is filled to make the dissolved oxygen reach 8-10mg / L. When diacetyl ≤0.05mg / L, the main fermentation ends. Cool down to 0-4℃, separate the yeast mud, and the wine enters the post-ripening stage.

[0048] 4. Filtration: After 14 days of post-ripening storage, filter to obtain the finished beer.

[0049] Example 3 Strain fermentation of beer

[0050] 1. Raw material ratio: barley malt 50%, 10°P syrup 50%.

[0051] 2. Saccharification:

[0052] The barley malt was ground using a standard mill and saccharified at a material-water ratio of 1:4. Saccharification process: 48°C for 30 minutes, heated to 65°C for 60 minutes, then heated to 72°C for 10 minutes, and finally heated to 78°C for 10 minutes, with a heating rate of 1°C / min. After saccharification, filter and adjust the wort concentration to 10°P. 10°P wort and 10°P syrup were mixed in a ratio of 1:1, and the hops were added in an amount of 0.2g / L. Boiled for 60 minutes, vortexed and cooled.

[0053] 3. Fermentation:

[0054] Adenine-eating budding yeast BA0616 and brewer's yeast Mangrive Jacks M29 were inoculated into the wort at a ratio of 1:50, with an inoculum of 1×10 5 CFU / mL, 5×10 6 CFU / mL, and two mice in the control group were inoculated with brewer's yeast Mangrive Jacks M29 at an inoculation volume of 5×10 6 CFU / mL. The inoculation temperature is 18±0.2℃, and the temperature during the main fermentation is maintained at 20±0.2℃. Sterile air is filled to make the dissolved oxygen reach 8-10mg / L. When diacetyl ≤0.05mg / L, the main fermentation ends. Cool down to 0-4℃, separate the yeast mud, and the wine enters the post-ripening stage.

[0055] 4. Filtration: After 14 days of post-ripening storage, filter to obtain the finished beer.

[0056] Example 4 Effect of strains on purine degradation

[0057] The purine content of the beer brewed in Example 2 and Example 3 was determined by referring to the method of Li et al. (Li H, Liu F, Hao J, et al. Determination of purines in beer by HPLC using a simple and rapid sample pretreatment [J]. Journal of the American Society of Brewing Chemists, 2015, 73 (2): 137-142.). The results are shown in Table 2.

[0058] Table 2

[0059]

[0060] It can be seen that the present invention uses adenine-eating budding grape spore yeast BA0616 to brew beer, and the purine content of the beer is 14.7 mg / L and 9.4 mg / L, respectively, which are significantly reduced compared with the control group (the reduction rate is 81.6% and 80.8%, respectively). The purine content of domestic commercial beer is between 36.80 mg / L and 150.42 mg / L, with an average value of 74.89 mg / L (Li et al. 2015). The finished beer prepared by the adenine-eating grape spore yeast BA0616 of the present invention has a purine content of only 1 / 5-1 / 10 of that of ordinary beer, and is healthier to drink.

[0061] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0062] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of adenine-feeding budding yeast (Blastobotrys adeninivorans) BA0616, characterized in that: The deposit number of the adenine-feeding budding botrytis cinerea is GDMCC NO:65287.

2. A microbial agent, characterized in that: The invention comprises the adenine-feeding budding Botrytis cinerea of ​​claim 1.

3. Use of the adenine-eating budding Botrytis cinerea of ​​claim 1 or the microbial agent of claim 2 in the preparation of low-purine brewed food.

4. A low-purine brewed beer, characterized in that: The brewed food is prepared by using the adenine-eating budding botrytis cinerea described in claim 1 or the microbial agent described in claim 2 as a fermentation agent.

5. The method for preparing low-purine brewed beer according to claim 4, characterized in that: The product is obtained by co-fermenting the adenine-eating grape spore yeast of claim 1 or the microbial agent of claim 2 with brewer's yeast.

6. The method for preparing low-purine brewed beer according to claim 5, characterized in that: The preparation method specifically comprises the following steps: (1) Wort saccharification: The barley malt was ground using a standard mill and saccharified at a material-water ratio of 1:4; saccharification process: 48°C for 30 min, then heated to 65°C for 60 min, then heated to 72°C for 10 min, and finally heated to 78°C for 10 min, with a heating rate of 1°C / min; after saccharification, the wort was filtered and the wort concentration was adjusted to 10°P; (2) Wort boiling: 10°P wort and 10°P syrup were mixed in a volume ratio of 1:1, hops were added in an amount of 0.2 g / L, boiled for 60 min, vortexed and precipitated, and cooled; (3) Fermentation: Adenine-eating budding yeast and brewer's yeast were inoculated into the wort at a ratio of 1:50, with an inoculation volume of 1×10 5 CFU / mL, 5×10 6 CFU / mL, the inoculation temperature is 18±0.2℃, the temperature during the main fermentation is maintained at 20±0.2℃, sterile air is filled to make the dissolved oxygen reach 8-10mg / L, when diacetyl ≤0.05mg / L, the main fermentation ends, the temperature is lowered to 0-4℃, the yeast mud is separated, and the wine enters the post-ripening stage; (4) Filtration: After 14 days of post-aging storage, the beer is filtered to obtain the finished beer.

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