NON-ALCOHOLIC BEER-FLAVORED BEVERAGE

Abstract

The present invention aims to provide a beverage with increased fullness. In particular, the present invention aims to provide a non-alcoholic beer-taste beverage with increased fullness. The present invention relates to a non-alcoholic beer-taste beverage containing 2′-deoxyadenosine at a concentration of 1 ppm or more.

Claims

1. A non-alcoholic beer-taste beverage comprising 2′-deoxyadenosine at a concentration of 1 ppm or more.

2. The non-alcoholic beer-taste beverage according to claim 1, wherein the concentration of 2′-deoxyadenosine is 1 to 10 ppm.

3. The non-alcoholic beer-taste beverage according to claim 1, further containing a protein having a molecular weight of 35 to 50 kDa, wherein the protein has a concentration of 5 ppm or more.

4. The non-alcoholic beer-taste beverage according to claim 3, wherein the protein has a concentration of 30 ppm or less.

Description

EXAMPLES

[0065] Hereinafter, the present invention is specifically described with reference to examples, but the present invention is not limited to the following examples.

Purification of 2′-deoxyadenosine)

[0066] 2′-deoxyadenosine (2′DA) was purified as described below.

(1) Fractionation of Beer by HP-20

[0067] Beer (60 L) was fractionated using 10 L Diaion® HP-20 (Mitsubishi Chemical Corporation). The HP-20 was washed with ethanol three times, and then washed with 50% ethanol three times before use. The washed HP-20 was packed in a mass fractionation column and replaced by water. The degassed beer (60 L) was mixed with the same amount of distilled water, and the resulting solution was introduced into the HP-20 column using a medium pressure pump. The solution that passed through the HP-20 column was obtained as a flow-through fraction. Distilled water (40 L) was introduced using a medium pressure pump, and an eluate was obtained as a water-eluted fraction. Likewise, hydrous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was introduced at an amount of 40 L for each concentration, and eluates were obtained as a 10% ethanol eluted fraction, a 30% ethanol eluted fraction, and a 70% ethanol eluted fraction. These eluted fractions were, dried and refrigerated using an evaporator and a freeze dryer.

(2) LH-20 Fractionation of 30% Ethanol Eluted Fraction

[0068] The 30% ethanol eluted fraction among HP-20 fractions was fractionated using 1.2 kg Sephadex® LH-20. The ethanol washed LH-20 was packed in a mass fractionation column and replaced by water. Then, 17.6 g of the 30% ethanol eluted fraction (87.9 g) obtained by HP-20 fractionation was dissolved in distilled water and applied to the LH-20 column. Distilled water (13.5 L) was introduced using a medium pressure pump, and water eluted fractions 1 to 6 were obtained. Subsequently, hydrous ethanol (35% ethanol, 70% ethanol, and 100% ethanol) was introduced at an amount of 7 L for each concentration, and eluates were obtained as a 35% ethanol eluted fraction, a 70% ethanol eluted fraction, and a 100% ethanol eluted fraction. These eluted fractions were dried and refrigerated. using an evaporator and a freeze dryer.

(3) Isolation of 2′DA

[0069] An amount of 86.4 mg of the water eluted fraction 4 (0.56 g) obtained by LH-20 fractionation was eluted with 10% ethanol using in a HPLC (COSMOSIL 5C18-PAQ, 20×250 mm). Subsequently, an eluate from 10 min to 13 min was concentrated and eluted with a mixture with an ethanol/water concentration gradient (5:95.fwdarw.15:85) using an HPLC (COSMOSIL 5C18-PAQ, 20×250 mm), whereby a compound (I) (0.5 mg, tR=21 min) was obtained.

[0070] The compound (I) was identified as 2′-deoxyadenosine from analysis of MS and NMR physical data and comparison with samples.

[0071] The following analytical instruments were used. LC-MS: Q Exactive, Thermo Fisher Scientific NMR: AVANCE 400, Bruker

Purification of 40 kDa Protein

[0072] A 40 kDa protein was purified from commercially available beer (1 L) as follows.

(1) Fractionation by Cation Exchange Resin

[0073] A cation-exchange resin “SP Sepharose” (50 ml) was planed in an empty column. Beer adsorbed onto the resin. Subsequently, the resin used for adsorption was transferred to another column, washed with a 20 mM sodium acetate buffer (pH 4.5), and then eluted with 20 mM sodium acetate (pH 4.5)+0.5 M-NaCl, whereby fractions were collected. The resulting fractions were evaluated by SDS-PAGE, and fractions containing a 40 kDa protein were collected as cation-exchange resin-bound fractions.

(2) Ultrafiltration (Buffer Exchange)

[0074] The cation-exchange resin-bound fractions obtained in (1) above were added in increments of 10 mL to an ultrafiltration unit. (Amicon Ultra-15 30K, Merck KGaA) washed with water, and centrifuged at 3500 rpm and ultrafiltered, whereby a concentrate was obtained.

(3) Ammonium Sulfate Fractionation

[0075] The concentrate obtained in (2) above was dropped into a beaker charged with a 20 mM phosphate buffer (pH 7.0) and 2 M ammonium sulfate, followed by stirring. The resulting suspension was then centrifuged (2330 g, 10 min, room temperature). The supernatant was collected in a different container. The collected solution was concentrated using an ultrafiltration unit. To the concentrate was added 20 mM sodium acetate (pH 4.5), followed by centrifugation (2330 g, 10 min, room temperature) for concentration, so that a purified 40 kDa protein product (quantified by the Bradford assay (in bovine serum albumin (BSA) equivalent), 20.4 mg/mL, 2.21 mL) was obtained. The purity of the resulting purified 40 kDa protein was confirmed by SDS-PAGE.

[0076] After the 40 kDa protein was digested by an enzyme, identification of the protein was attempted by LC-MS/MS analysis.

[0077] The band around 40 kDa isolated by SDS-PAGE was sliced, followed by reduction with dithiothreitol (56° C., 1 hr) and carbamide methylation with iodoacetamide (room temperature under light-shielded conditions, 45 min). Then, a 0.01% ProteaseMAX-containing 10 ng/μL chymotrypsin solution (5 mM calcium chloride, 50. mM ammonium bicarbonate solution) (15 μL), 5 mM calcium chloride, and a 50 mM ammonium bicarbonate solution (15 μL) were added, followed by overnight incubation. Subsequently the resulting enzyme digestion solution was collected. The collected solution was solidified by drying in vacuum, which was then re-dissolved in a 0.1% formic acid solution.

[0078] The resulting product was used for LC-MS/MS analysis.

Measurement by LC-MS/MS

[0079] LC-MS/MS measurement was performed under the following conditions.

Device used: direct flow type nano LC system “Easy-nLC 100™” (Thermo Fisher Scientific)
Trap column: Acclaim PepMap® (Thermo Fisher Scientific)
Analysis column: Nano HPLC Capillary Column (Nikkyo Technos Co., Ltd.)
Liquid chromatograph mass spectrometer: Q Exactive Pius (Thermo Fisher Scientific)
Mobile phase: solvent A: 0.1% formic acid/water; solvent B: 0.1% formic acid/acetonitrile
Flow rate: 300 nL/min
Gradient: 0-40% B/0-30 min, 40-60% B/30-35 min, 60-90% B/35-37 min, 90% B/37-45 mm
Amount introduced: 10 μL
Ionization mode: EST Positive
Measurement range: MS1 (m/z 350-750)
Data dependent scan mode

(4) Analysis of Protein

[0080] The protein was identified under the following conditions.

Search software: Proteome Discoverer 2.2.0.388 (Thermo Fisher Scientific)
Species: barley (Hordeum vulgare), hop (Humulus), yeast (Saccharomyces cerevisiae)
Search conditions:
Digestive enzyme: Chymotrypsin
Precursor ion mass error range: Monoisotopic, ±10 ppm
Production mass error range: ±0.02 Da
Maximum number of missed cleavages: 5
Confidence level (Percolator): High (level with the highest confidence of the three levels of confidence)

Database: SwissProt

[0081] As a result, the 40 kDa protein was found to be barley-derived Serpin Z4 (sequence coverage: 77.2) and barley-derived Serpin Z7 (sequence coverage: 72.8%).

Sensory Evaluation of Commercially Available Non-Alcoholic Beer-Taste Beverage to Which 2′DA is Added

[0082] 2′-deoxyadenosine (2′DA) was added to a commercially available non-alcoholic beer-taste beverage for sensory evaluation of the fullness.

[0083] The non-alcoholic beer-taste beverage is a non-alcoholic beer-taste beverage containing malt in its ingredients.

[0084] Raw materials include malt, hops, carbonic acid, flavorings, acidulants, caramel color, vitamin C, bittering agents, and sweeteners. The nutritional components per 100 ml include 0% alcohol content, 0 g protein, 0 g sugar, 0 to 0.1 g dietary fiber, and about 0 mg purine.

[0085] Reference points of sensory evaluation are as follows.

[0086] Five special panelists rated in increments of 0.05 points based on the following criteria, and the points were averaged.

[0087] The criteria, for the intensity of fullness are as follows.

0 points: no fullness at all
1 point: slight fullness
2 points: definite fullness
3 points: very intense fullness

[0088] A commercially available alcoholic beer-taste beverage different from the above-described commercially available non-alcoholic beer-taste beverage (the subject of evaluation) was provided as a reference alcoholic beer-taste beverage (I), and its fullness was given 0.7 points as the reference point. Another commercially available alcoholic beer-taste beverage was also provided as a reference alcoholic beer-taste beverage (II), and its fullness was given 1.5 points as the reference point.

[0089] The fullness of the commercially available non-alcoholic beer-taste beverage as the subject of evaluation was given 0.5 points based on the fullness of the commercially available alcoholic beer-taste beverages (I) and (II).

[0090] The reference alcoholic beer-taste beverage (I) is an alcoholic beer-taste beverage in which the proportion of malt in the mixture of the ingredients is more than 0 wt % and less than 50 wt %.

[0091] The raw materials include low-malt beer, malt, hops, sugars, dietary fiber, and spirit (wheat). The nutritional components per 100 ml include, 4% alcohol content, 0 to 0.2 g protein, 0.5 to 0.8 g sugar, and about 2.0 mg purine.

[0092] The reference alcoholic beer-taste beverage (II) is an alcoholic beer-taste beverage in which the proportion malt in the mixture of the ingredients is 50 wt. % or more.

[0093] The raw materials include malt and hops. The nutritional components per 100 ml include 5.5% alcohol content, 0.4 to 0.6 g protein, 3.6 g sugar, and about 12.5 mg purine.

[0094] The procedure for the sensory evaluation is as follows.

(1) The non-alcoholic beer-taste beverage is dispensed into vials at a volume of 1/10 (v/v) of the final volume.
(2) 2′DA is weighed out at a predetermined weight and added to each vial.
(3) The vials are sonicated for 30 seconds.
(4) The vials are left to stand at room temperature for 30 minutes.
(5) The non-alcoholic beer-taste beverage is filled up to the final volume.
(6) The non-alcoholic beer-taste be is dispensed and ingested for evaluation.

Analysis of Commercially Available Non-Alcoholic Beer-Taste Beverage

[0095] The concentration of 2′DA in the commercially available non-alcoholic beer-taste beverage used for the sensory evaluation was quantitated by LC-MS.

(1) Preparation of Samples and Calibration Curves

[0096] 2′DA was diluted to the following concentrations and passed through a 0.22 μm filter before measurement. Final concentration: 0.001 ppm, 0.025 ppm, 0.050 ppm, 0.100 ppm, 0.200 ppm, 0.300 ppm, 0.500 ppm, 0.750 ppm, and 1.000 ppm

(1 ppm=1 μg/mL)

[0097] A 5% (v/v) aqueous ethanol solution was used as a diluent.

[0098] The results of sample analysis were based on values measured at a dilution factor at which the measured values would fall in the range (R.sup.2>0.99) maintaining the linearity of the calibration curve.

[0099] LC measurement conditions are as follows.

LC-MS: X500R, AB Sciex Ltd.

[0100] Separation column.: HSS T3 1.8 μm, 2.1×150 mm, Waters Eluent:
Solvent A: 0.1% formic acid/water; solvent B: formic acid/acetonitrile
Gradient: solvent A:solvent B=98:2.fwdarw.2:98 (27 min)
Amount introduced: 5 μL
Flow rate: 0.2 mL/min
Column oven: 40° C.

(MS)

[0101] Ionization mode: ESI Positive
Measurement range: MS1 (m/z 100-1000)
Data Independent Scan mode
Ion source temperature: 350° C.

(2) Preparation of Sample for Measurement from Commercially Available Non-Alcoholic Beer-Taste Beverage

[0102] A commercially available non-alcoholic beer-taste beverage was degassed by sonication, appropriately diluted after air bubbles settled, and passed through a 0.22 μm filter before measurement.

[0103] A 5% (v/v) aqueous ethanol solution was used as a diluent.

[0104] The concentration of 2′DA in the commercially available non-alcoholic beer-taste beverage was used as the control.

Example 1: Evaluation by Addition of 2′DA

[0105] 2′DA in the commercially available non-alcoholic beer-taste beverage (control) had a concentration of 0 ppm.

[0106] 2′DA was added to the non-alcoholic beer-taste beverage such that the concentration of 2′DA would be 1 ppm, 6 ppm, and 10 ppm for sensory evaluation. In addition, 2′DA was added to the commercially available non-alcoholic beer-taste beverage such that the concentration of 2′DA would be 0.1 ppm for sensory evaluation (comparison sample 1).

[0107] Table 1 shows the results of the sensory evaluation.

TABLE-US-00001 TABLE 1 Comparison Control sample 1 Sample 1 Sample 2 Sample 3 2′DA concentration (ppm) 0 0.1 1 6 10 PanelistA 0.50 0.50 0.60 0.65 0.70 PanelistB 0.50 0.50 0.55 0.60 0.65 PanelistC 0.50 0.55 0.65 0.75 0.80 PanelistD 0.50 0.50 0.55 0.65 0.70 PanelistE 0.50 0.50 0.60 0.70 0.80 Average of sensory evaluation 0.50 0.51 0.59 0.67 0.73

[0108] The results in Table 1 show that the fullness of the non-alcoholic beer-taste beverages is increased when the concentration of 2′DA is 1 ppm or more.

Example 2: Evaluation of Synergy of 2′DA an 40 kDa Protein

[0109] 2′DA was added to a commercially available non-alcoholic beer-taste beverage (control) to obtain a beverage containing a 2′DA at a concentration of 1 ppm as a reference, and a 40 kDa protein was further added to the beverage to evaluate the synergy of the 2′PA and the 40 kDa protein. The concentration of the 40 kDa protein was set to 5 ppm and 10 ppm. The 40 kDa protein was one purified above.

[0110] For comparison, an evaluation was also performed on a commercially available non-alcoholic beer-taste beverage to which only a 40 kDa protein was added and in which the concentration of the 40 kDa protein was adjusted to 5 ppm (comparison sample 2).

[0111] Table 2 shows the results of the sensory evaluation.

TABLE-US-00002 TABLE 2 Comparison Control Sample 1 Sample 2 Sample 3 sample 2 2′DA concentration (ppm) 0 1 1 1 0 40 kDa protein concentration 0 0 5 10 5 (ppm) PanelistA 0.50 0.60 0.65 0.75 0.50 PanelistB 0.50 0.55 0.55 0.70 0.50 PanelistC 0.50 0.65 0.70 0.75 0.50 PanelistD 0.50 0.55 0.70 0.80 0.50 PanelistE 0.50 0.60 0.65 0.75 0.55 Average of sensory evaluation 0.50 0.59 0.65 0.75 0.51

[0112] The results in Table 2 show that adding 2′DA to a commercially available non-alcoholic beer-taste beverage and further adding a 40 kDa protein thereto can further increase the fullness.

[0113] The results from the comparison sample 2 show that the fullness can be increased by, simply adding a 40 kDa protein. The results also show that the increment (0.15) from the control in the sensory evaluation of the sample 2 is greater than the additive effect (0.10) predictable from the combination of the 2′DA and the 40 kDa protein as determined as the sum of the increment (0.09) from the control in the sensory evaluation of the sample 1 and the increment (0.01) from the control in the sensory evaluation of the comparison sample 2. This indicates unpredictable synergy by the combination of the 2′DA and the 40 kDa protein.

INDUSTRIAL APPLICABILITY

[0114] The present invention can provide a non-alcoholic beer-taste beverage with increased fullness.