Botrytis cinerea BA0616 and its application in the preparation of low-purine foods

Abstract

This invention discloses a strain of *Botrytis cinerea* BA0616 and its application in low-purine foods, relating to the field of microbial technology. The *Botrytis cinerea* BA0616 strain of this invention has the accession number GDMCC NO: 65287. This *Botrytis cinerea* strain can use purines as a carbon source and has a strong metabolic capacity for purines, making it suitable for fermentation in low-purine foods. Applying *Botrytis cinerea* to beer production, without reducing the amount of barley malt used or adding adsorbents, strong acids, strong alkalis, or exogenous additives such as enzymes, the resulting low-purine beer, co-fermented with brewer's yeast, has rich foam and a delicate taste. The final purine content of the beer is only 8-15 mg / L, only 1 / 5-1 / 10 of that of ordinary beer, making it healthier to drink.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of adenine-producing Botrytis cinerea BA0616 and its application in the preparation of low-purine foods. Background Technology

[0002] Among alcoholic beverages, fermented wines such as sake, beer, and wine have a much higher purine content than distilled spirits such as Japanese shochu and whiskey, with beer having the highest purine content (40-100 mg / L). Numerous epidemiological studies have shown a strong correlation between high-purine diets and the development of gout. Therefore, reducing purine intake is considered the best way to prevent hyperuricemia and gout.

[0003] Currently, researchers both domestically and internationally have conducted some studies on reducing purines in beer, mainly through methods such as reducing the amount of barley malt used (application number 201210012067); adding acids, alkalis (application numbers 201310600728, 2017102912509) and enzyme preparations (application number 201510575000) during the mashing stage; and adding adsorbents such as activated carbon and zeolite (application number 2005100946709) to the beer. Although these methods can reduce purines in beer to varying degrees, excessively low malt usage does not meet beer brewing requirements (i.e., malt as the main raw material, malt content ≥ 50%), and the brewing process involves chemical or physical additives.

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

[0005] In view of this, the present invention provides *Blastobotrys deninivorans* BA0616, and its application in brewing low-purine foods. This strain can efficiently utilize purine substances, and its co-fermentation with brewer's yeast does not require reducing the amount of malt or additional additives, resulting in beer with low purine content and unchanged taste and flavor.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A strain of *Blastobotrys adeninivorans*, with accession number GDMCC NO: 65287.

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

[0009] Another object of the present invention is to provide the application of the above-mentioned adenine-producing Botrytis cinerea or the above-mentioned microbial agent in the preparation of low-purine brewed foods.

[0010] Another object of the present invention is to provide a low-purine brewed beer, which uses the above-mentioned adenine-producing Botrytis cinerea or the above-mentioned microbial inoculum as a fermenting 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, wherein the beer is obtained by co-fermentation with the above-mentioned adenine-producing Botrytis cinerea or the above-mentioned microbial inoculum.

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

[0013] (1) Wort saccharification:

[0014] Barley malt was ground using a standard grinder and saccharified at a feed-to-water ratio of 1:4. The saccharification process consisted of: holding at 48℃ for 30 minutes, increasing to 65℃ and holding for 60 minutes, then increasing to 72℃ and holding for 10 minutes, and finally increasing to 78℃ and holding for 10 minutes, with a heating rate of 1℃ / min. After saccharification, the mixture was filtered, and the wort concentration was adjusted to 10°P.

[0015] (2) Boil the wort:

[0016] Mix 10°P wort and 10°P syrup in a 1:1 volume ratio, add 0.2 g / L of hops, boil for 60 minutes, swirl to settle, and cool.

[0017] (3) Fermentation:

[0018] Botrytis cinerea and brewer's yeast were inoculated into the wort at a ratio of 1:50, with an inoculation amount of 1×10⁻⁶. 5 CFU / mL, 5×10 6 CFU / mL, inoculation temperature is 18±0.2℃, temperature is maintained at 20±0.2℃ during the main fermentation, sterile air is introduced to make 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-maturation storage, the beer is filtered to obtain the finished product.

[0020] Beneficial Effects: The *Botrytis cinerea* budding yeast provided by this invention can use purines as a carbon source and has a strong metabolic capacity for purines, making it suitable for fermentation of low-purine foods. Applying *Botrytis cinerea* budding yeast to beer production, without reducing the amount of barley malt used or adding adsorbents, strong acids, strong alkalis, or exogenous additives such as enzymes, results in a low-purine beer with rich foam and a delicate taste. The final purine content of the beer is only 8-15 mg / L, only 1 / 5-1 / 10 of that of ordinary beer, making it healthier to drink. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure is a schematic diagram of the colony of *Botrytis cinerea* GDMCC 65287 provided by this invention.

[0023] Figure 2 The attached figure is a phylogenetic tree of the adenine-producing Botrytis cinerea GDMCC 65287 of this invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Isolation, Screening and Identification of Strains

[0026] 1. Isolation of strains

[0027] Sample preparation: Grind 5g of ripe Pu-erh tea (from Yunnan Pu-erh Tea Factory) in a pre-sterilized mortar, add 100mL of sterile distilled water and mix well. Vortex for 20 minutes to ensure thorough mixing of the bacterial solution, then prepare 10... -1 10 -2 10 -3 10 -4 10 -5Diluted bacterial suspensions were prepared by pipetting 0.2 mL of each suspension onto YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder). Three plates were prepared for each sample, each dilution, and each culture medium. The plates were then incubated at 30°C.

[0028] After colonies have grown, isolate and purify them based on their growth characteristics. Pick out a single colony from the plate medium and streak it onto YPD medium using an inoculation needle. Repeat the inoculation and isolation process 2-3 times until a pure strain is obtained, and then incubate it in a constant temperature incubator.

[0029] 2. Determination of the strain's ability to degrade purines

[0030] Preparation of the screening medium for purine-lowering strains: Barley malt was ground using a standard grinder and saccharified at a material-to-water ratio of 1:4. The saccharification process was as follows: 48℃ for 30 min, then heated to 65℃ for 60 min, then heated to 72℃ for 10 min, and finally heated to 78℃ for 10 min, with a heating rate of 1℃ / min. After saccharification, the mixture was filtered, the wort concentration was adjusted to 10°P, the hop addition was 0.2 g / L, and the mixture was boiled for 60 min, filtered, and cooled.

[0031] Each Erlenmeyer flask (150 mL) contained 30 mL of 10°P wort medium. The isolated candidate bacteria were individually inoculated into the wort medium and placed in a shaker at 30°C and 200 rpm for 12 h. After the incubation, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the degradation rates of adenine, guanine, hypoxanthine, xanthine, adenine nucleoside, guanine nucleoside, and hypoxanthine nucleoside 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 nucleoside + guanine nucleoside + hypoxanthine nucleoside

[0033] Total purine degradation rate = Total purine content after degradation / Total purine content before degradation

[0034] Table 1

[0035]

[0036] 3. Identification of strains

[0037] DNA extraction from the strain: Genomic DNA was extracted from the purified BA0616 strain using a fungal DNA extraction kit (Shanghai Sangon Biotech 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 (25 μL): template DNA 0.5 μL, mix enzyme 12.5 μL, ITS 10.5 μL, ITS 10.5 μL, sterile water 11 μL. PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 35 s, 72℃ extension for 1 min, 35 cycles, final extension at 72℃ for 8 min.

[0039] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the BLAST sequence on NCBI using SEQ ID NO.1, which confirmed that the isolated strain BA0616 was *Blastobotrys adeninivorans*.

[0040] , SEQ ID NO.1.

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

[0042] Example 2: Fermented Beer by Strains

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

[0044] 2. Saccharification:

[0045] Barley malt was ground using a standard grinder and saccharified at a feed-to-water ratio of 1:4. The saccharification process was as follows: 48℃ for 30 minutes, then heated to 65℃ for 60 minutes, then heated to 72℃ for 10 minutes, and finally heated to 78℃ for 10 minutes, with a heating rate of 1℃ / min. After saccharification, the mixture was filtered, the wort concentration was adjusted to 10°P, hops were added at 0.2 g / L, the mixture was boiled for 60 minutes, swirl to settle, and then cooled.

[0046] 3. Fermentation:

[0047] Botrytis cinerea BA0616 and Mangrive Jacks M29, a commercial brewer's yeast, were inoculated into the wort at a ratio of 1:50, with inoculation amounts of 1×10⁶ spores each. 5 CFU / mL, 5×10 6 CFU / mL. One control group was inoculated with Mangrive Jacks M29 brewer's yeast at a density of 5 × 10⁻⁶. 6 CFU / mL. The inoculation temperature was 18±0.2℃, and the temperature was maintained at 20±0.2℃ during the primary fermentation. Sterile air was introduced to achieve dissolved oxygen levels of 8–10 mg / L. Primary fermentation ended when diacetyl ≤0.05 mg / L. The temperature was then lowered to 0–4℃, the yeast sludge was separated, and the wine entered the post-fermentation stage.

[0048] 4. Filtration: After 14 days of post-maturation storage, the beer is filtered to obtain the finished product.

[0049] Example 3: Fermented Beer with Strains

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

[0051] 2. Saccharification:

[0052] Barley malt was ground using a standard grinder and saccharified at a feed-to-water ratio of 1:4. The saccharification process was as follows: 48℃ for 30 minutes, then heated to 65℃ for 60 minutes, then heated to 72℃ for 10 minutes, and finally heated to 78℃ for 10 minutes, with a heating rate of 1℃ / min. After saccharification, the mixture was filtered, and the wort concentration was adjusted to 10°P. The 10°P wort was mixed with 10°P syrup in a 1:1 ratio, and hops were added at a rate of 0.2 g / L. The mixture was boiled for 60 minutes, swirl to settle, and then cooled.

[0053] 3. Fermentation:

[0054] Botrytis cinerea BA0616 and Mangrive Jacks M29 were inoculated into the wort at a ratio of 1:50, with inoculation amounts of 1×10⁶ spores each. 5 CFU / mL, 5×10 6 CFU / mL, the control group consisted of 2 mice inoculated with Mangrive Jacks M29 brewer's yeast at a dose of 5 × 10⁻⁶. 6 CFU / mL. The inoculation temperature was 18±0.2℃, and the temperature was maintained at 20±0.2℃ during the primary fermentation. Sterile air was introduced to achieve dissolved oxygen levels of 8–10 mg / L. Primary fermentation ended when diacetyl ≤0.05 mg / L. The temperature was then lowered to 0–4℃, the yeast sludge was separated, and the wine entered the post-fermentation stage.

[0055] 4. Filtration: After 14 days of post-maturation storage, the beer is filtered to obtain the finished product.

[0056] Example 4: Purine Degradation Effect of Strains

[0057] The purine content of the beers brewed in Examples 2 and 3 was 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.). The results are shown in Table 2.

[0058] Table 2

[0059]

[0060] It is evident that the beer brewing method using *Botrytis cinerea* BA0616 of this invention resulted in beer purine contents of 14.7 mg / L and 9.4 mg / L, respectively, which were significantly lower than the control group (reduction rates of 81.6% and 80.8%, respectively). The purine content of commercially available beers in China ranges from 36.80 mg / L to 150.42 mg / L, with an average of 74.89 mg / L (Li et al. 2015). The beer prepared using *Botrytis cinerea* BA0616 of this invention has a purine content that is only 1 / 5 to 1 / 10 of that of ordinary beer, making it healthier to drink.

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

[0062] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of adenine-producing Botrytis cinerea ( Blastobotrys adeninivorans BA0616, characterized in that, The accession number of the adenine-producing Botrytis cinerea BA0616 is GDMCC NO: 65287.

2. A microbial inoculant, characterized in that, Includes the adenine-producing Botrytis cinerea BA0616 as described in claim 1.

3. The use of the adenine-producing Botrytis cinerea BA0616 of claim 1 or the microbial agent of claim 2 in the preparation of low-purine brewed beer.

4. A method for preparing low-purine brewed beer, characterized in that, The product is obtained by co-fermentation of *Botrytis cinerea* BA0616 (as described in claim 1) and *Saccharomyces cerevisiae* (as described in claim 2) with *Saccharomyces cerevisiae*.

5. The method for preparing low-purine brewed beer according to claim 4, characterized in that, The preparation method specifically includes the following steps: (1) Wort saccharification: The barley malt was ground using a standard grinder and saccharified with a material-to-water ratio of 1:

4. The saccharification process was as follows: 48℃ for 30 minutes, then heated to 65℃ for 60 minutes, then heated to 72℃ for 10 minutes, and finally heated to 78℃ for 10 minutes, with a heating rate of 1℃ / min. After saccharification, the mixture was filtered and the wort concentration was adjusted to 10ºP. (2) Boil the wort: Mix 10ºP wort and 10ºP syrup at a volume ratio of 1:1, add 0.2 g / L of hops, boil for 60 min, swirl to settle, and cool. (3) Fermentation: Botrytis cinerea BA0616 and brewer's yeast were inoculated into the wort at a ratio of 1:50, with inoculation amounts of 1×10⁶ spores each. 5 CFU / mL, 5×10 6 CFU / mL, inoculation temperature is 18±0.2℃, temperature is maintained at 20±0.2℃ during the main fermentation, sterile air is introduced to make dissolved oxygen reach 8~10mg / L, when diacetyl ≤0.05mg / L, the main fermentation ends, the temperature is lowered to 0~4℃, the yeast sludge is separated, and the wine enters the post-ripening stage; (4) Filtration: After 14 days of post-maturation storage, the beer is filtered to obtain the finished product.

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