WO1996025483A1

WO1996025483A1 - Process for producing beer

Info

Publication number: WO1996025483A1
Application number: PCT/JP1996/000346
Authority: WO
Inventors: Yuji Shibano; Hideko Yomo; Takehiro Matsumoto; Hirofumi Koda; Yoshihide Suwa; Teruo Amachi; Haruyo Hatanaka; Sakayu Shimizu
Original assignee: Suntory Limited
Priority date: 1995-02-17
Filing date: 1996-02-16
Publication date: 1996-08-22

Abstract

A process for producing beer with a reduced purine compound content, which comprises using the wort with the purine nucleoside content thereof reduced by the decomposition of the nucleoside into purine bases by nucleoside phosphorylase or nucleosidase, or which comprises decomposing the purine nucleoside into purine bases during fermentation and metabolizing the bases with yeast.

Description

Description Beer production method Field of the invention

The present invention relates to a method for producing beer with a reduced concentration of a purine compound. More specifically, the present invention relates to a method for producing beer, which comprises decomposing a purine nucleoside in wort by applying an enzyme to a purine base that yeast can assimilate. Background art

In recent years, with the westernization and hypernutrition of dietary habits, an increase in blood uric acid level has been observed, and there is a concern that gout onset through asymptomatic hyperuricemia may increase. Purine bases, prinnucleotides, prinnucleotides, and high-molecular-weight nucleic acids in the diet are digested and absorbed in the digestive tract, and decomposed to uric acid by the prin-degradation system in the liver. Epidemiological findings on hyperuricemia and gout caused by consumption of diets high in purine compounds have been compiled, and it is most important to reduce the amount of purine compounds to be consumed. It is a preventive measure for illness or gout.

Meat, milt, fish eggs, liver, etc. are mentioned as high-purine-compound diets, but alcoholic beverages, especially beer, have a very high purine-compound content. In fact, Kaneko (Kaneko Kiyoko; Japan Clinical, 49: 1108—115, (1991)) compared the content of purine compounds in various alcoholic beverages and found that beer, sake, wine, and other brews were It reports that it contains a higher amount of purine compounds than distilled spirits such as whiskey and shochu, and that beer has a particularly high purine compound content among brewed sakes. Fujimori et al. Reported that beer contained 50-70 mg of total purine compounds. It has been reported that Z1 is contained (Shin Fujimori et al., Uric acid, Vol. 9, No. 2, pp. 128-133).

This value was obtained by hydrolyzing a phenol compound in beer to a pyridine base with perchloric acid and measuring it by high performance liquid chromatography. However, compared to liver, it is 1/100 to 1/100, and the intake is large, so the risk of hyperuricemia and gout is greater than that of distilled spirits. Tof 1 er and Woodings (0. B. Toiler and TL Woodings; Med. J. Aust. 2, 479-481, (1981)) track the beer population by beer intake for 13 years. A survey was conducted and noted that there was a positive correlation between beer intake and gout frequency. Thus, in epidemiological studies, beer is now regarded as an alcoholic beverage with a high risk of hyperuricemia and gout. Disclosure of the invention

Accordingly, the present invention provides a method for producing beer with a reduced content of a purine compound.

The inventors of the present invention have obtained the knowledge of decomposing prin nucleosides in wort into prin base to produce beer with a reduced content of a brin compound, and have further studied. As a result, the present invention has been completed.

That is, the present invention provides nucleosides by causing nucleoside 'phosphorylase or nucleosidase to act on wort to decompose prin nucleoside contained in wort into purine bases. Provided is a method for producing beer, which is characterized by producing nucleoside-decomposed wort and producing using the nucleoside-decomposed wort. The present invention further provides a method for producing wort, which comprises causing nucleoside 'phosphorylase or nucleoside to act on wort. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the fate of a purine compound in a Ί0 L scale beer test brew, where the reference shows the purine nucleoside and the 〇 shows the purine base. This is a graph showing the amount of each purine compound when Reoside's phosphorylase was added.

FIG. 3 shows the activity when inosin was used as a substrate and reacted at pH 5.0 at each temperature for 10 minutes.

Fig. 4 shows the results of incubating at 70 ° C for each hour at each pH using inosine as a substrate, adding the same volume of a 10 mM inosine solution, and incubating at 70 ° C for 10 minutes. This shows the activity when the reaction is performed.

Figure 5 shows that after incubation at 70 ° C for 50 hours in 50% sodium acetate buffer pH5.0, the same volume of lOmM inosin solution was added, and the mixture was incubated at 70 ° C for 10 hours. It shows the activity when reacted for 1 minute.

Figure 6 shows that each sample was incubated at 70 ° C in 50mM sodium acetate buffer pH 5.0 at 70 ° C for each time, and the same amount of 10mM inosin solution was added. Shows the activity when reacted for 10 minutes.

FIG. 7 shows the substrate specificity for inosine, adenosine, and guanosine at 60 ° C. and 70 ° C. at pH 5.0 by reducing sugar determination.

FIG. 8 shows the activity when inosine was used as a substrate at 60 ° C. after incubation at 100 ° C. for each hour.

Fig. 9 shows the time-temperature curves of the charging pot and the charging tank in the wort production process. FIG. 10 shows the results of analyzing the purified form of wort produced with the addition of the enzyme and wort produced without the addition of the enzyme.

FIGS. 11A and 11B show the fate of the purified form during fermentation using the nucleotide-decomposed wort produced in Example 4 and ordinary wort. Specific description

The present inventors have measured the amounts of prin nucleotides, prin nucleotides, prin bases and high molecular nucleic acids in beer, and provided a method for producing beer in which the concentration of these prin compounds was reduced. As a result of various studies for this purpose, the present invention has been completed. Purine base is a generic term for derivatives in which various parts of purine (9H-imidazo [4,5-d] pyrimidine) are substituted, and includes adenine, guanine, and xanthine. Prin nucleoside is a generic name for glycoside compounds in which a princ base and a reducing group of a sugar are N-glycosidically bonded, and includes adenosine, guanosine, and inosine.

Prinnucleotide is a generic name for compounds in which the sugar moiety of prinnucleotide forms an ester with phosphoric acid, and includes adenylic acid, guanylic acid, inosinic acid, and the like.

The term “purine compound” is a generic term for compounds having a skeleton such as the above-mentioned princ base, princnucleotide, and princnucleotide. The present inventors have clarified the following points by measuring the amount of each purine compound in beer and in the production process thereof.

1) The purine compounds in various commercial beers vary from 40 to 10 Omg / l, but the purine nucleotides are 2 to 25 times as large as the purine base. That is, most of the purine compounds in beer exist as purine chloride.

2) During the fermentation process, the purine base in the wort is absorbed by the yeast. I almost apologize and almost disappear.

Based on the above analysis results, the present inventors have conducted intensive studies in order to solve this problem, and as a result, have invented a method for producing beer in which the content of a purine compound is reduced.

In other words, ordinary yeast cannot absorb princnucleoside, but can absorb and metabolize princ base. Therefore, in the present invention, an enzyme is used in wort to decompose princ nucleotides contained in the wort into princ bases, thereby producing nucleoside-decomposed wort. The use of decomposed wort makes it possible to reduce the content of the purine compound in beer. The nucleoside-digested wort is obtained by degrading a part or all of the princ nucleoside in the wort into pryne base by an enzyme. Can be adjusted by the amount of enzyme to be added, the reaction time, the reaction temperature, etc., but preferably, all of the prinnucleoside is decomposed into prinbase, Wort without nucleosides is preferred.

This enzyme can act in (1) wort production, (2) wort production and before fermentation, or (3) fermentation. In order for the enzyme to work during the wort production process, the enzyme can be added at the beginning of wort production (saccharification) or at an appropriate time during wort production. In order for the enzyme to work before the fermentation process, the enzyme may be added to the wort in the wort production process and left for a predetermined time before the start of fermentation, but the most desirable of these is during the wort production process. This is an addition before boiling. This is because boiling can deactivate enzymes, so there is no possibility of bringing active enzymes into beer products and affecting the quality in any way. In order for the enzyme to work during the fermentation process, the enzyme is added at the start of fermentation or during fermentation. However, the printer produced by the action of the enzyme Since the base must be metabolized by yeast, the enzyme is preferably added at the beginning of the fermentation or at the beginning of the fermentation period.

Since wort is acidic throughout the manufacturing process (PH 5.0-5, 5) and the temperature of the manufacturing process is 50-80 ° C, enzymes that can react at high temperatures in the acidic range are preferred. .

The enzyme for this purpose may be a malt-derived enzyme or an enzyme of another origin, such as nucleoside 'phosphophorase or nucleoside enzyme. it can. As an enzyme source other than malt, for example, nucleoside'phosphorylase (EC 2.4.2.1) derived from calf spleen or bacteria can be used.

The nucleosidase used in the present invention refers to a substance that degrades prin nucleode into a prim base and ribose, and particularly has an optimum pH of 4.5 to 6.5 and an optimum temperature of 50 ° C to 80 °. Those in C are desirable, and there are no particular restrictions on the type of inhibitor, Km value, molecular weight, etc., which represent enzymes and other general properties.

Nucleosidase is usually produced by culturing a microbial strain capable of producing nucleosidase. Microbial strains include, for example, the genus Ochrobactrum, the genus Streptococcus, the genus Pediococcus, and the genus Leuconostoc. Genus (Leuconostoc), genus Lactobacillus, genus Escheri chia, genus Citrobacter, genus Serratia, and Alcaligenes (Al calogenes) i genes), genus Flavobacterium, genus Bacillus 厲, genus Corynebacteriuni, staphylococcus Genus (Staphylococcus), genus Arthrobacter, genus Comamonas, genus Pseudomonas, genus Kluyveromyces, genus Saccharomyc es), genus Debaryomyces, genus Pichia, genus Hansenula, genus Sporobol omyces, genus Sporidiobolus, genus Aspergillus Aspergillus) genus, Penicillium (Penici 11 ium) genus and the like are used, but the strain is not particularly limited, and newly added to soil, lactic acid bacteria, rancid food, animal organs and excrement A strain isolated from E. coli can be used as long as it has the ability to produce nucleosidase. In addition, these strains were subjected to ultraviolet irradiation or mutagen treatment to artificially induce mutations and artificially required gene fragments required for the expression of the nucleosidase activity. However, other transformants incorporating the same can also be used in the method of the present invention.

Specific examples of strains capable of producing nucleosidase include Ochrobactrum anthropi (FER BP-5377), Streptococcus citrus Robots (Streptococcus ci trovorum), Pediococcus pentosaceus (IF0 3182), Leuconostoc 'dextranicum (Leuconostoc d ex tran i cum), rack Lactobacillus pi an tarum (Lactobacillus pi an tarum) (Lactobacillus arabinosu s) (IF0 3070), Lactobaci 1 lus p lan tarum ) (Lactobacillus cucumer is) (IF0 3074), Escherichia coli B biotin less, Sitrono Inverter (Ci troba cter freundi i) (IF ♦ Serratia marcescens IFO 3736, Alcaligenes faecal is, Flabotobacterium meningosepticum (DSM2) ), Nocicles cereus (Baci 11 us cereus), Corynebacterium-ku "(Corynebac terium gl u tami cum) (ATCC13060), Staphylococcus aureus (IFO 3060), Earth bacterium (Arthrobacter ureafaciens) Arthrobacter globiformis) (IFO 12140), Comamonas testosteroni (Pseudomonas dacunhae) (IF0140) 12048), Pseudomonas put i da, Bacillus Bacillus aneurinolyticus, Bacillus turieniens (IFO 3951), Ms. Marxianus (Kluyveromyces marxianus) (Saccharomyces maxianus) (IF0 0277), Deno Rio Myces * (Debaryomyces pse udopo 1 ymorphus) (Pichia pseudopol ymorpha) (IF0 1026), Pichia capsulata (Hansenula capsulata) (Hansenula capsulata) IF0 0721), Sporobolomyces salmon i co 1 or (I F0 1038), Sporidiobulus salmon i co 1 or Sporobos; Sporobolomyces odorus) (lF0 1035), Aspergillus niger (IF0 4416), Penicillium spinulosum (Penicillium spinulosum) ) (IAM 7047), Aspergillus awamori (IF0 4033), Aspergillus oryzae (lAM 2630), Aspergillus flavus (lF58) ), Aspergi 1 lus terreus (I F0 5445), Aspergi 1 lus sojae (IF0 4386), Aspergilus' parasites (Aspergi 1 lus parasiticus) (IFO 4082), Penicillium sp ( Penicillium sp.), Etc. Kutrum * Ochrobactrum anthrop i (FERM BP-5377), Aspergilus niger (IFO 4416), Aspergillus oryzae (IAM 2630), Aspergillus Rus flavus (IFO 5839) and Aspergillus terreus (XIFO 5445).

Of the above strains, those with the ATCC number are available from the ATCC, and those with the IF0 number are the Fermentation Research Institute (2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi). More available.

The newly isolated bacterial strain Ochrobactrum anthropi in the present invention has the following mycological properties.

(a) Morphological properties

① Cell morphology: Bacillus

② Presence or absence of cell polymorphism: None

③ Mobility: Yes

の Spore presence: None

(b) Cultural properties

① Gravy agar plate culture (cultured at 30 ° C for 2 days):

The shape of the colony is a perfect circle with a convex ridge. The surface is smooth and shiny.

(c) Physiological properties

① Gram staining: negative

② Nitrate reduction: Negative

③ Denitrification reaction: Negative

④ Indole formation: negative

酸 Production of acid from glucose: negative

ゥゥ Rease: positive ⑦ Chitochrome: positive

カタ Catalase: positive

酸 Acid production from Kinloth: positive

最適 Optimal growth temperature: 28-37 ° C

色素 Dye formation in King's B medium: negative

⑫ 0— F test (sugar: by glucose) oxidation ⑬ Sugar assimilation:

(1) Glucose: +

(2) Fara Vinose: +

(3) Mannose: +

(4) Mannitol: one

(5) N-acetylglucosamine: +

(6) Maltese: +

(7) Dalconic acid: one

(8) Cabric acid: +

(9) Adipic acid: one

(10) Lingoic acid: +

(11) Cuenoic acid: +

(12) Funylacetic acid: one

(d) Other properties

① Esculin hydrolysis: Negative

② Garbage: Negative

③ Gelatin hydrolysis: Negative

④ Arginine dihidrorase: negative

生 Growth on MacConkey medium: positive

⑥Polymixin sensitivity: Negative

⑦ Use of alanine: positive グリ Glycine use: negative

Physiological and biochemical properties were investigated using API20NE (BI0 MERIEUX S.A.). Furthermore, based on these results, a search for species names was performed using the Analytical Profile Index for API20NE. The presence or absence of nitrate reduction and glycine utilization was described by Holmes et al. (International Journal of System atic Bacteriology, Vol. 38, No. 4, 1988, p. 406-416) was different, and it was identified as Ochrobactrum anthropi according to the Index.

This strain was identified as Ochrobactrum anth ropi. This strain was deposited internationally under the Budapest Treaty on January 26, 1996, as FERMBP — 53777, at the Research Institute of Biotechnology, Industrial Technology Institute of Japan.

In order to produce nucleosidase by culturing these microorganism strains, continuous or intermittent cultivation can be carried out by usual stationary culture, shaking culture, aeration stirring culture or solid culture. The nucleosidase after culturing is usually contained in microbial cells, and the cells are obtained by ordinary enzyme purification means to obtain a crude enzyme or a purified enzyme having a higher specific activity than live cells. It can be used in the method of the present invention. When nucleosidase is secreted into the culture solution, the culture solution can be used as it is. Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Test Example 1. Measurement of purine compounds in beer

Purine compounds in beer were analyzed by EP-10 type high performance liquid chromatograph (manufactured by EIC OM). The column is GS320-H (7.6 mm DX 250 mm L, manufactured by Asahipak) and the moving bed The analysis was performed at 10 mM sodium phosphate (PH 5.0), at a flow rate of 0 m1 / min, and at a temperature of 30 ° C. After removing the solid content by membrane filtration of the sample, 101 was injected, and the amount of the pyridine compound was quantified by the absorbance at 260 nm.

In advance, three known purine bases (adenine, guanine, and xanthine) and three purine nucleosides (adenosine, guanosine, and inosine) were analyzed as reference samples. Then, the retention time and peak area of each print compound are determined. The identity of each purine compound in the actual sample was determined from the retention time of each peak, and the concentration was determined from a calibration curve obtained from the peak area of a reference sample of known concentration. Note that the three types of purine nucleotides (adenylic acid, guanylic acid, and inosinic acid) are eluted quickly under the conditions of this analysis, and do not interfere with the analysis of the above six types of purine compounds. .

Table 1 shows the analysis results for seven types of commercially available beer and two types of wine. The total amount of prin, expressed as the sum of plin base and prin nucleotide converted to the amount of plin base, fluctuated depending on the type of beer, and was in the range of 40 to 1OOmgZl. In addition, most of the purine compounds existed as nucleotides.

In wine, which is the same brew as beer, red wine is as high as 60 m / 1, but white wine is as low as 20 mg / l.

table 1

(mg / 1) f 1, J

PX7J ゾギギ ✓ ✓ ✓ PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX PX Amount of dough in beer Beer A 18.4 37.1 19.5 4.2 1.9 8.0 14.1 39.0 53.1 Beer B 15.5 32.1 15.2 4.9 7.8 4.7 17.4 32.6 50.0 Beer C 7.8 34.2 19.1 5.5 3.1 8.8 17.4 31.8 49.2 Beer D 8.1 36.9 20.5 6.6 3.9 7.8 18.3 34.2 52.5 E 5.0 35.4 19.4 6.7 3.8 8.6 19.1 31.2 50.3 Beer F 6.6 32.2 17.2 6.6 5.3 nd 11.9 29.2 41.1 Beer G 16.2 136.4 40.8 4.0 ndnd 4.0 101.7 105.7 Red wine H 0.5 1.3 12.6 34.3 20.0 nd 54.3 7.4 61.7 White wine I 0.3 7.0 9.8 ndnd 14.0 14.0 8.9 22.9

When the prinnucleotides in beer and wort were analyzed by ion-exchange ram, prinnucleotide was not present in the beer or wort. In addition, beer and wort were acid-hydrolyzed and analyzed, and the total amount of purine compounds did not change from that before acid hydrolysis.Therefore, high-molecular-weight nucleic acids such as RNA and DNA were present in beer and wort. It is thought that it does not exist.

Test Example 2—Displacement of purine compounds during the beer production process

The fate of the purine compound during the beer production process was investigated in a test brew at the 70 L scale. The initial extract concentration is 12.5%, the input yeast amount is 5 g wet weight I, the fermentation temperature is 11.5 ° C, and when the extract reaches 2.5% (260 hours after the start of fermentation) The temperature was reduced to 0 ° C, except for the yeast that settled from the bottom of the fermentation tank, and storage was continued for another 300 hours.

The results are shown in Figure 1, where the sum of adenosine, guanosine, and inosine is shown as purine nucleotides, and the sum of adenine, guanine, and xanthine is shown as purine bases. Indicated. Purine base is present in wort at about 7 OmgZl, but decreases to 1 OmgZl at the beginning of fermentation and does not increase during the subsequent fermentation and storage periods. On the other hand, prin nucleoside was present in the wort in a large amount of about 9 O mg Zl, and did not change during the fermentation period or the storage period.

This result indicates that the purin base contained in ordinary wort is hardly absorbed or metabolized by the yeast during the yeast growth phase at the beginning of fermentation.

Example 1. Nucleo-leoside ♦ Degradation to purine base by phosphorylase

Extract concentration 12.5% wort, Nucleoside 'fosphorylase from calf spleen (Boehringer) to 7 units / ml Was added and reacted at 30 ° C. for 3 hours. For the wort to which the enzyme was added and the wort which was kept at 30 ° C. for 3 hours without adding the enzyme as a control, the amount of the purine compound was measured by the method described in Test Example 1.

As shown in Fig. 2, the wort with the enzyme reduced the adenosine, guanosine, and inosine purine nucleotides compared to the wort without the enzyme, and correspondingly reduced the wort. Adenine, guanine and xanthine had increased purine bases. In addition, the decomposition rate of each nucleoside was about 60%.

This result indicates that nucleoside phosphorylase from calf spleen is effective in degrading prin nucleoside in wort. According to the results of Test Example 2, purine base is absorbed and metabolized by the yeast during the yeast growth phase in the early stage of fermentation, so that nucleoside's phosphorylase decomposes purine base. Purine base obtained in this way is also absorbed and metabolized by yeast during fermentation, and if beer is produced using wort treated with this enzyme, the purine nucleotide content in beer can be reduced to about 60%. % Can be reduced.

In addition, the beer shown in Test Example 2 contains 90 mg of purine chloride and 10 ml of purine base, so that wort should be treated with this enzyme. As a result, the content of prinnucleoside in beer can be reduced to about half.

Example 2. Screening of nucleosidase which can be used in wort production process

Nucleosidase was screened from mold, bacteria and yeast by the following procedure.

Each strain was cultured and collected in the following medium and culture conditions. The cells were suspended in 20 mM Tris-HCl buffer, pH 7.4, and then disrupted by ultrasonic waves (for mold, mortar with sea sand). Unnecessary substances were removed by heart to obtain a crude enzyme solution. The crude enzyme solution was reacted at 60 ° C and 70 ° C in 100 mM sodium acetate buffer pH 4.5 in the presence of 5 mM inosine, adenosine, or guanosine substrate. I went. The solution was spotted on a thin layer of silica gel, and thin layer chromatography was performed using a developing solution of normal butanol: acetic acid: water = 3: 1: 1 to obtain a thin layer. From the size of the spots of the reoside and the corresponding purine base, the degree of degradation of the nucleoside was examined. Table 2 shows the results for bacteria with high activity.

Medium composition and culture conditions

1) Mold

Malt extract 5% 28 ° C, 3-5 days

Yeast extract 0.3% shaking culture

pH 5.5

2) Bacteria (excluding lactic acid bacteria)

Tripton 0.5% 28 ° C, 2-4 days

Yeast extract 0.5% shaking culture

Glucose 0.1%

0.1% dicalcium phosphate

pH7.0

3) Lactic acid bacteria

Peptone 1% 28 ° C, 4-7 days

Meat extract 1% static culture

Yeast extract 0.5%

Glucose 2%

Sun 80 0.1%

Ammonium citrate 0.2%

Sodium acetate 0.5% Magnesium sulfate heptahydrate 0.01% Manganese chloride tetrahydrate 0.005%

Dicalcium phosphate 0.2%

ρΗ6

4) Yeast

Yeast extract 0.2% 28 ° C, 2-4 days Peptone 0.5% Shaking culture Glucose 5%

0.4% dicalcium phosphate

Monolithium phosphate 0.2%

Magnesium sulfate heptahydrate 0.02%

ΡΗ6.0

Table 2

◎> 70%, △> 50%, Δ> 20%, X 10% Example 3 Properties of Nucleosidase

Of the bacteria obtained above, the bacterium Ochrobactrum anthropi was treated at 70 ° C with inosine, adenosin, and guanosine at 60 ° C. Produces highly active nucleosidase that can degrade inosine and guanosine. The crude enzyme solution of Ochrobactrum anthropi was dialyzed against 50 mM MES buffer-PH6.0 to remove low molecules in the crude enzyme solution. The properties of reosidase were investigated in detail.

Activity measurement method using inosine

Appropriate buffer (25 mM sodium acetate pH 4.5-5.0, Tris-HCl buffer pH 7.0-9.5, MES buffer pH 5.5) -6.5), Incubate the 5 mM inosin solution 1951 in advance at each temperature, add the enzyme solution 51, and start the reaction. After 10 minutes, add 200 jul of 0.2 hydrochloric acid solution to stop the reaction, and remove the denatured protein by centrifugation. The supernatant 1001 was mixed with 0.1N sodium hydroxide solution 1001, 1M Tris-HCl buffer pH 8.0501, and water 345u1, and the absorbance at 293nm was measured. Troll. Xanthine oxidase 51 (5 mU) is further added and mixed. When the increase in absorbance at 293 nm stops, the absorbance is measured. The unit of activity U for nucleosidase is expressed as the amount of enzyme that hydrolyzes 1 / mole of nucleoside per minute, and the activity (U / ml) of nucleosidase in enzyme solution is 3.33X Calculated as ΔΑ293. In addition, room temperature A293 shows the value obtained by subtracting the absorbance of the control from the absorbance of xanthine oxidase added. Activity measurement method by reducing sugar determination

Incubate a 25 mM sodium acetate buffer, pH 5.0, 5 mM nucleoside solution 1951 at each temperature in advance and add the enzyme solution 5i1 to start the reaction. 10 minutes later Add 200 1 of 0.1N zinc sulfate solution

1 Θ Stop the reaction by adding 200N 0.1N sodium hydroxide solution, and centrifuge to remove the protein. Supernatant 200 u 1 and 4% sodium carbonate, 1.6% glycine, 0.045% copper sulfate pentahydrate 300 1 and 0.04% 2,9-dimethyl-1,10-phena Mix 0.9ml of slurolin and 2001 of water and incubate at 95 ° C for 8 minutes. Measure the absorbance at 450 nm. Perform the same treatment using a ribose solution with a known concentration, create a calibration curve from the absorbance at 450 nm, and calculate the ribose concentration of the enzyme reaction mixture. The activity (シ / ml) of nucleosidase in the enzyme solution is determined at ribose concentration (M) x 0.004.

(1) Optimum temperature

Optimal temperature 50 ° C ~ 80 ° C. Figure 3 shows the activity when inosine was used as a substrate and reacted at pH 5.0 for 10 minutes at each temperature. The activity is highest at 60 ° C and drops to about 80% at 70 ° C, but does not change at 80 ° C.

(2) Optimum pH

Optimum pH pH 4.5-6.5. Figure 4 shows the activity of each reaction at 70 ° C for 10 minutes at each pH using inosine as a substrate. There are peaks at pH5.0 and pH6.0.

(3) Heat resistance

After incubation at 70 ° C in 50mM sodium acetate buffer pH5.0 for each hour, the same volume of 10mM inosin solution was added and reacted at 70 ° C for 10 minutes. The activity at that time is shown in FIG. Figure 6 shows the activity when the reaction was performed at 40 ° C for 10 minutes. When the reaction is performed at 70 ° C with a heat treatment for 10 minutes, the activity decreases to about 70%, but hardly decreases thereafter. When the reaction is carried out at 40 ° C by heat treatment for 10 minutes, the activity decreases to about 25%, but hardly decreases thereafter.

Considering the results of (1)-(3), Ochrobactrum anthropi has at least two optimal temperatures, Expected to have nucleosidases with different pH and thermostability

(4) Substrate specificity

The substrate specificity for inosine, adenosine, and guanosine at 60 ° C and 70 ° C at pH 5.0 was determined by quantification of reducing sugars. Figure 7 shows this. The loss of adenosine activity at 70 ° C means that there are at least two types of nucleosidases that can degrade adenosine and those that cannot degrade adenosine. Is expected from the substrate specificity.

(5) Heat deactivation

To determine whether the enzyme was inactivated by boiling wort, the enzyme was incubated at 100 ° C for each hour, and then the activity was measured at 60 ° C using inosine as a substrate. Figure 8 shows the results. It is completely inactive at 100 ° C for 10 minutes. The activity of degrading inosine at 60 ° C is possessed by both nucleosidase that can degrade adenosine and nucleosidase that cannot degrade adenosine. Easily deactivated.

Based on the above results, this enzyme has sufficient activity around the wort pH of 5.0, and adenosine decomposes at 60 ° C, while inosine and guanosine decompose even at higher 80 ° C I knew I was able to do it. In addition, the active enzyme does not directly change the quality of the product beer after the boiling process in a normal beer production process because it is completely deactivated by boiling.

Example 4 Production of nucleoside-decomposed wort

Figure 9 shows the time-temperature curve of the charging pot and the charging tank in the wort production process. Table 3 shows the proportions of the ingredients in the pot and tank. In this manufacturing process, heat treatment, salting out, ion exchange Chromatography and gel chromatography Nucleoside of Ochrobactrum anthropi partially purified by first-class chromatography, about 20, OOOu (substrate: a) Nosin, reaction temperature: 60 ° C) was added together with the crushed malt to produce wort. Figure 10 shows the results of the analysis of the purified form of wort produced with the addition of the enzyme and wort produced without the addition of the enzyme. Purine nucleosides were not detected in the wort to which the enzyme had been added, and the amount of purine base was increased. Hereinafter, this wort is referred to as nucleotide-decomposed wort.

Table 3

Example 5. Beer brewing using nucleotide-decomposed wort

Beer brewing was performed using the nucleotide-decomposed wort produced in Example 4 and ordinary wort. Beer yeast was suspended in 2 liters of wort at a wet weight of 10.5 g and fermented at 12 ° C for 8 days. After that, it was stored at 4 ° C for 5 days. Figure 11 shows the fate of the purified form during fermentation. Adenine was completely utilized during the fermentation time. The amount of guanine in the fermentation liquor decreases with time and becomes undetectable at the end of the fermentation.

Guanine, equivalent to about 90 M after subtraction, was assimilated by the yeast. Therefore, a total of 260 fiW of whey in the wort disappears during the fermentation process. Beer brewed using wort produced without enzyme treatment has a total of 500 プ M beer, and beer brewed using enzyme-treated wort. Since the total size of the pudding was 180 M, we were able to brew beer with a reduced total of 320 β Μ pudding. In fermentation, both yeast growth and consumption of extract were almost the same as those using normal wort, and there was no significant difference in taste. Industrial applicability

In the present invention, the enzyme of the present invention is allowed to act on wort in a beer production process to decompose prin nucleoside among the prin compounds derived from the raw materials into a prin base, thereby obtaining prin nucleoside. Wort in which beer is substantially absent, the beer is produced from the wort, and the purine base is metabolized to yeast in a fermentation step, thereby obtaining a content of a purine compound in beer. It is possible to obtain a beer with reduced emissions. The beer obtained by the method of the present invention can reduce the risk of disease such as hyperuricemia or gout.

The enzyme of the present invention can be made to act on wort in the beer production process without changing the production process. In particular, when the enzyme of the present invention is made to act in the wort production process, ordinary wort can be produced. The temperature and ρΗ in the juice production process (saccharification) are within the optimal range of the enzyme, so it works effectively, and the enzyme can also act on the wort within the time required for the wort production process. In addition, the enzyme can be deactivated in the wort boiling step immediately after the wort production step, which is one of the bead production steps, so that the enzyme can be made to act more easily and can be carried out economically. In addition, when an enzyme is allowed to act on wort immediately before or during fermentation, if there is a production step in which heat treatment is performed after the fermentation step, the enzyme can also be deactivated by that step. Furthermore, the obtained beer does not show a great difference in terms of taste from ordinary product beer, and it is possible to obtain a beer that is preferable as a palatable product. References to deposited microorganisms in Rule 13bis and depository communication Research Institute of Biotechnology, Industrial Technology Institute, Ministry of Commerce and Industry Address: 1-3-1, Tsukuba-Higashi, Ibaraki, Japan Home Deposit Number and Date of Deposit

FERM BP-5377 January 26, 1996

Claims

Scope of the claim 1. A process for producing beer, which comprises causing nucleoside phosphorylase and Z or nucleosidase to act on wort.

2. Nucleoside 'phosphorylase and / or nucleosides are allowed to act on the wort to decompose the nucleosides contained in the wort into purine bases. A method for producing beer, comprising using the nucleoside-decomposed wort obtained by the above method.

3. The method according to claim 2, wherein the nucleoside-decomposed wort does not contain prinnucleoside.

4. The nucleosidase is a genus Ochrobactrum, a genus Streptococcus, a genus Pediococcus, a genus Leuconostoc, Lactobacillus genus, Escherichia genus, Citrobacter genus, Serratia genus, Alcaligenes Genus, genus Flavobacteri urn, genus Bacillus, genus Corynebacter ium, genus Staphylococcus, earthlo Genus Arthrobacter, genus Comamonas, genus Pseudomonas, Kluyy veromyces, Saccharomyces ¾, τ Debaryomyces, Pichia (P genus i chia, genus Hansenula, genus Sporobolomyces, genus Sporidi obo 1 us, genus Aspergillus, and Penicici 11 The method according to any one of claims 1 to 3, wherein the method is a microorganism-derived nucleosidase having a nucleosidase-producing ability selected from the group consisting of the genus ium).

5. The optimum pH range of nucleoside's phosphorylase or nucleoside is 4.5 to 6.5 and the optimum temperature range is 50 ° C to 80 ° C. The method according to any one of the preceding items 4.

6. The method according to any one of claims 1 to 5, wherein a nucleoside 'phosphorylase and a nucleoside or a nucleoside are added to the wort before or during the fermentation. The described method.

7. A claim according to any one of claims 1 to 5, wherein the wort is subjected to nucleoside 'phosphorylase and / or nucleosidase during the wort production process. the method of.

8. A process for producing wort, which comprises reacting wort with nucleoside 'phosphorylase and Z or nucleonidase.

9. Nucleoside's phosphorylase and Z or nucleosidase are allowed to act on the wort to decompose the nucleosides contained in the wort into purine bases. A method for producing wort to obtain decomposed wort.

10. The method according to claim 9, wherein the nucleoside-decomposed wort does not contain bleach nucleoside.

11. The nucleoside is a genus of Ochrobactrum, a genus of Streptococcus, a genus of Pediococcus, or a genus of Leuconos. toe), Lactobacillus, Escherichia, Ci trobacter, Serratia, Alcal igenes Genus, genus Flavobacterium, genus Bacillus, genus Corynebacterium, genus Staphylococcus, earth locust Genus (Arthrobacter), genus Comamonas, genus Pseudomonas, genus Kluyveromyces, Genus Saccharomyces, genus Debaryomyces, genus Pichia, genus Hansenula (Hansenu 1a), genus Sporobol omyces, genus Sporidi obonoles 10. A nucleosidase derived from a microorganism having a nucleosidase-producing ability selected from the group consisting of the genus Spor idi obol us), the genus Aspergillus, and the genus Penicillium. Any of 10

The method of paragraph 1.

12. The method according to any one of claims 8 to 11, wherein the optimum pH range of nucleoside 'phosphorylase or nucleosidase is 4.5 to 6.5 and the optimum temperature range is 50 ° C to 80 ° C. The method of paragraph 1.

13. Any one of claims 8 to 12, characterized in that wort is subjected to nucleoside 'phosphorylase and / or nucleosidase during the wort production process. The method described in the section.