WO2005003759A1

WO2005003759A1 - A method for analysis of a beverage

Info

Publication number: WO2005003759A1
Application number: PCT/SG2004/000195
Authority: WO
Inventors: Nikolai Kocherginsky; Iouri Kostetski; Alexej I. Smirnov
Original assignee: National University Of Singapore
Priority date: 2003-07-03
Filing date: 2004-07-03
Publication date: 2005-01-13

Abstract

A method for determining one or more parameters of a beverage comprising one or more reducing agents. The method comprising the step of introducing a spin probe to a sample of the beverage, the spin probe capable of reacting with the one or more reducing agents. A plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents is obtained. The plurality of obtained EPR spectra of the sample is used to determine one or more parameters of the sample.

Description

A Method for Analysis of a Beverage

Technical Field The present invention generally relates to a method for determining one or more parameters of a beverage such as beer. More particularly, the present invention relates to an EPR based method for determining one or more kinetic parameters of a beverage that may be used to analyse the amount of one or more reducing agents in the beer and to predict beer shelf-life.

Background Oxidation reactions are a major cause for beer loosing its flavour with time. Antioxidants (reducing agents), which prevent or retard these reactions, are therefore crucial in extending the lifetime of a beer and enhancing flavour stability. Antioxidants are compounds that limit the damage caused by reactive oxidising species such as oxygen-containing free radicals by removing the reactive oxygen species. Antioxidants may be added to the beer extraneously, but many brewers prefer a natural source by utilising the antioxidants inherent within the brewing raw materials. The use of high antioxidant malts guard against oxidative damage of beer in the brewhouse and ensures long shelf life for the beverage.

One known method to determine the flavour stability of beer uses spin traps and detection of spin adducts with electron paramagnetic resonance (EPR) A disadvantage of this method is that the rate of beer deterioration through a free radical mediated process is relatively slow at typical refrigeration temperatures. For example, at 0°C a detectable (by EPR) concentration of spin- adducts appears only after 5 days. It has also been observed, that at elevated temperatures and by exposing the beer to molecular oxygen, such as for example, by equilibrating the beverage with air, the kinetics of spin-adduct formation has a lag period which is related to the content of antioxidants in beer. The value of this lag period is equal to the time when the main antioxidants have been used up. This lag period can be used for determination of beer flavor stability and prediction of beer shelf life. For example, a spin trap molecule such as PBN (N-tert-butyl- -phenylnitrone) can be added to beer, and the beer is then subjected to an accelerated oxidative stress carried out at 60°C in the presence of air. Initially, the PBN molecule is not paramagnetic, but upon reacting with intermediate and short-lived free radicals it forms stable spin-adducts which give rise to characteristic EPR spectra. The lag period of spin-adduct formation observed by such a method correlates with the beer age and it has been found to be a useful indicator of beer shelf life.

The main disadvantage of the previous method of analysis is the long time required for the observation of the lag period. For example, when beer is fresh the lag period can be as long as several hours. The fresher the beer and the more stable its flavor, the longer the lag time and thus, the time required for the analysis. In addition to this, the method requires different samples of beer to be taken at different time intervals to measure the kinetics of spin-adducts produced because beer has to be incubated with air to accelerate kinetics of oxidation. Another disadvantage of the method is that the lag period is not an accurate indicator of the remaining antioxidant pool. One more disadvantage of the method is that antioxidants in the beer are reacting with active forms of oxygen but during the storage they can also be regenerated in reactions with different reducing agents present in beer. The pool of these reducing agents (called ^""reducing power of beer") is not determined by the spin trap method. Another disadvantage of the spin trap method is that the beer is studied under conditions at elevated temperature of of about 60 °C and in the presence of molecular oxygen, which is atypical for beer storage. These conditions are artificial to that of beer and may lead to inaccurate results.

There is a need to provide a method for determining one or more parameters of a beverage that overcomes or at least ameliorates one or more of the disadvantages described above.

Summary of invention According to a first aspect of the invention, there is provided a method for determining one or more parameters of a beverage comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beverage, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra of the sample to determine one or more parameters of the sample.

In one embodiment, the plurality of obtained EPR spectra may be used to determine one or more kinetic parameters of the beverage sample. The one or more kinetic parameters may be used to determine the amount of reducing agent present in the beverage. According to a second aspect of the invention, there is provided a method for determining one or more parameters of beer comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a period of time as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra to determine one or more parameters of the sample.

In one embodiment, the plurality of obtained EPR spectra may be used to determine one or more kinetic parameters of the beer sample. The one or more kinetic parameters may be used to determine the amount of reducing agent present in the beer. According to a third aspect of the invention, there is provided a method for determining the concentration of one or more reducing agents in beer, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents; using the plurality of obtained EPR spectra to determine one or more kinetic parameters of the sample; and determining the concentration of the one or more reducing agents in the beer from the one or more kinetic parameters . According to a fourth aspect of the invention, there is provided a method for determining one or more characteristics of a sample of beer comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a period of time as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra to determine one or more kinetic parameters of the sample; comparing the one or more kinetic parameters to one or more standards for such parameters to determine a characteristic of the sample.

According to a fifth aspect of the invention, there is provided a analytical system for determining one or more parameters of a beverage comprising one or more reducing agents, the analytical system comprising: a receptacle for containing a spin probe and a sample of the beverage, the spin probe capable of reacting with the one or more reducing agents; an electron paramagnetic resonance apparatus for obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents; and a processor capable of using the plurality of obtained EPR spectra of the sample to determine one or more parameters of the sample. Definitions The following words and terms used herein shall have the meaning indicated:

The term ^λ spin probe' is to be interpreted broadly to include a nitroxide molecule with a stable NO^* paramagnetic group which is capable of attachment to a part of another molecular entity and which may be revealed by electron paramagnetic resonance spectroscopy. The term reducing agent' is to be interpreted broadly to include atoms, ions, molecules or any combination thereof that are capable of donating electrons in a reaction. The term reducing agent' may also have the same meaning as "antioxidant" .

Disclosure of embodiments

The method may comprise the step of measuring the intensity of the EPR spectrums over the time period.

The method may further comprise the step of determining the concentration of the one or more reducing agents in the beverage from the one or more kinetic parameters . The period of time that the EPR spectrums are obtained may be selected from the group consisting of: 30 to 10 minutes; 1 minute to 8 minutes; 2 minutes to 5 minutes; 3 minutes to 4 minutes; and 40 seconds to 4 minutes .

The temperature at which the spin probe reacts with the reducing agents may be at a temperature within the range selected from the group consisting of: 1°C to 99 °C;

5°C to 80°C; 10°C to 70°C; 15°C to 60°C; 18°C to 50°C;

18°C to 40°C; 18°C to 30°C; 18°C to 25°C; 18°C to 22°C.

The reaction between the spin probe and the one or more reducing agents may be conducted in an inert atmosphere. The inert atmosphere may be obtained by providing an atmosphere of nitrogen surrounding the reactants . The beverage may be a fermented beverage or a non- fermented beverage. The fermented beverage may include beer, wine, and sake. The non-fermented beverage may include fruit juice. The beverage may also be a semifinished beverage such as brewing mashes and worts.

The spin probe may be a nitroxide. The nitroxides may be selected from the group consisting of: piperidine nitroxides, oxazolidine nitroxides, pyrroline nitroxides, pyrrolidine nitroxides, imidazolidine nitroxides, and imidazoline nitroxides.

The piperidine nitroxides spin probes may be selected from the group consisting of: 2,2,6,6- Tetramethylpiperidine-1-oxyl, 4-Hydroxy-2, 2,6,6- tetramethylpiperidine-1-oxyl, 4-0x0-2.2 , 6,6 tetramethylpiperidine-1-oxyl, 4-Isothiocyanato-2 , 2 , 6,6 tetramethylpiperidine-1-oxyl, 4-Benzyl-2 , 2 ,6,6 tetramethylpiperidine-1-oxyl, 4-Phospho-2 , 2 ,6,6 tetramethylpiperidine-1-oxyl, 4-Carboxy-2 2 6,6 tetramethylpiperidine-1-oxyl, 4-Sulfate-2 2. 6,6 tetramethylpiperidine-1-oxyl, 4-Amino-2 2. 6,6 tetramethylpiperidine-1-oxyl, 4-Methylamino-2, 2 , 6,6 tetramethylpiperidine-1-oxyl, 4-Dimethylamino-2_/ 2 , 6,6 tetramethylpiperidine-1-oxyl, 4- (N,N-Dimethyl-N- (3 sulfopropyl ) ) ammonium-2 ,2,6,6 tetramethylpiperidine-1 oxyl, 4- (N,N-Dimethyl-N- (2-hydroxyethyl) ) ammonium 2,2,6, 6-tetramethylpiperidine-l-oxyl, 4

Trimethylammonium-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, 4- (N,N-Dimethyl-N-nonyl) ammonium-2, 2,6,6- tetramethylpiperidme-1-oxyl, 4- (N, N-Dimethyl-N decyl) ammonium-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4 (N, N-Dimethy1-N-tridecyl) ammonium-2, 2, 6, 6- tetramethylpiperidine-1-oxyl, 4- (N, N-Dimethyl-N' tetradecyl) ammonium-2 ,2,6, 6-tetramethylpiperidine-l-oxyl, 4- (N,N-Dimethyl-N-pentadecyl) ammonium-2, 2,6,6- tetramethylpiperidine-1-oxyl, 4- (N, -Dimethyl-N' hexadecyl) ammonium-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4-Octanoyloxy-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, 4- Dodecanoyloxy-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4-

Hexadecanoyloxy-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4- Octadecanoyloxy-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4- Dodecanoylamino-2, 2,6, 6-tetramethylpiperidine-l-oxyl, 4- Octadecanoylamino-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, 4-Acetylamino-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, 4-

(Iodoacetyl) amino-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, 4- (2-iodoacetamido) -2,2,6, 6-tetramethylpiperidine-l-oxyl, 4- (Bromoacetyl) amino-2, 2, 6, 6-tetramethylpiperidine-l- oxyl, 4- (2-bromoacetamido) -2,2, 6, 6-tetramethylpiperidine 1-oxyl, 4- (Chloroacetyl) amino-2, 2, 6, 6- tetramethylpiperidine-1-oxyl, 4- (2-chloroacetamido) - 2,2,6, 6-tetramethylpiperidine-l-oxyl, 4- (3-

Carboxypropylamido) -2,2, 6, 6-tetramethylpiperidine-l-oxyl, 4-Maleimido-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, N- (2,2,6, 6-tetramethylpiperidine-l-oxyl-4-yl)maleimide, 4- ( (N,N-Dimethyl) -2-aminoethyl) amino-2, 2,6,6- tetramethylpiperidine-1-oxyl, N,N-dimethyl-N' -(2,2,6,6- tetramethylpiperidine-l-oxyl-4-yl) -1, 2 diaminoethane bis (2,2,6, 6-Tetramethylpiperidine-l-oxyl-4-yl) succinate, N,N' -bis (2,2,6, 6-Tetramethylpiperidine-l-oxyl-4-yl) -1, 2- cis-diaminocyclopropane, N,N' -bis (2,2,6,6-

Tetramethylpiperidine-l-oxyl-4-yl) -1, 2-trans- diaminocyclopropane, and 2, 2, 6, 6-TetramethyIpiperidine-l- hydroxy.

The oxazolidine nitroxides spin probes may be selected from the group consisting of: 2- (2- Carboxyethyl) -2-tetradecyl-4, 4-dimethyloxazolidine-3- oxyl, 2- (3-Carfooxypropyl) -2-undecyl-4, 4- dimethyloxazolidine-3-oxyl, 2- ( 6-Carboxyhexyl) -2-octyl- 4, 4-dimethyloxazolidine-3-oxyl, 2- (3-Carboxypropyl) -2- tridecyL-4, 4-dimethyl oxazolidine-3-oxyl, 2- (4- Carboxybutyl) -2-dodecyl-4, 4-dimethyloxazolidine-3-oxyl, 2- ( 5-Carboxypentyl) -2-undecyl-4 , 4-dimethyloxazolidine-3- oxyl, 2- (7-Carboxyheptyl) -2-nonyI-4, 4- dimethyloxazolidine-3-oxyl, 2- (8-Carboxyoctyl) -2-octyl- 4, 4-dimethyloxazolidine-3-oxyl, 2- (10-Carboxydecyl) -2- hexyl-4 , 4-dimethyloxazolidine-3-oxyl, 2- (14-Carboxy tetradecyl) -2-ethyl-4, 4-dimethyloxazolidine-3-oxyl, 2-(4- Methoxy-4-oxobutyl) -2-undecyl-4, 4-dimethyioxazolidine-3- oxyl, 2- (11-Methoxy-ll-oxoundecyl) -2-hexyI-4, 4- dimethyloxazolidine-3-oxyl, 2- (4-Methoxy-4-oxobutyl) -2- tridecyl-4, 4-dimethyloxazolidine-3-oxyl, 2- (2- Carboxyethyl) -2, 4, 4-trimethyloxazolidine-3-oxyl, 2,4,4- Trimethyl-2-ethyIoxazolidine-3-oxyl, 2, 4, 4-Trimethyl-2- dodecyloxazolidine-3-oxyl, 4, 4-Dimethyl-2-hexyl-2- heptyIoxazolidine-3-oxyl, and Spiro [cyclohexane-1, 2 ' - (4 ', 4 ' -dimethyl oxazolidine-3 ' -oxyl) ] .

The pyrroline nitroxides spin probes may be selected from the group consisting of: 2,2,5,5- Tetramethylpyrroline-1-oxyl, 3-Carboxy-2, 2,5,5- tetramethylpyrroline-1-oxyl, 2,2,5,5 tetramethylpyrroline-l-oxyl-3-carboxylic acid, 3- Hydroxymethyl-2, 2,5, 5-tetramethylpyrroline-l-oxyl, 3- Methoxyoxomethyl-2, 2, 5, 5-tetramethylpyiroline-l-oxyl, 3- Carbamoyl-2, 2,5, 5-tetramethylpyrroline-l-oxyl, 3- (N,N- Dimethyl-N-octadecylamino) methyl-2 ,2,5, 5-tetramethyl pyrroline-1-oxyl, N, N-dimethyl-N-octadecyl-N- (3- methylene-2, 2,5, 5-tetramethylpyrroline-l-oxyl) ammonium, 3- (Octadecylphosphorylmethyl) -2,2,5,5- tetramethylpyrroline-1-oxyl, and octadecylphosphoryl-3- methylene-2, 2,5, 5-tetramethylpyrroline-l-oxyl .

The pyrrolidine nitroxides spin probes may be selected from the group consisting of: 3-Carboxy-2, 2, 5, 5- tetramethyipyrrolidine-1-oxyl, 2,2,5,5 tetramethylpyrrolidine-l-oxyl-3-carboxylic acid, 3- Carbamoyl-2, 2,5, 5-tetramethylpyrrolidine-l-oxyl, 3- (Isothiocianatomethyl) -2,2,5, 5-tetramethyl pyrrolidine-1- oxyl, 3-Amino-2, 2, 5, 5-tetramethylpyrrolidine-l-oxyl, 3- (2, 3-Dihydroxy-l-hydroxymethyl) propylamide-2, 2,5,5- tetramethylpyrrolidine-1-oxyl 12- (2, 2,5, 5-

Tetramethylpyirolidine-l-oxyl-3-carbonylamino) dodecanoic acid 1, 2, 3-Propanetriol-tris- [12- (2,2,5,5- tetramethylpyrrolidine-l-oxyl-3- carbonylamino) dodecanoate] , 2,5, 5-Trimethyl-2- ethylpyrrolidine-1-oxyl, 2,2,5, 5-Tetramethylpyrrolidine- l-oxyl-3-maleimide, 2-Carboxymethyl-2, 5, 5- trimethylpyrrolidine-l-oxyl, 2-Carboxymethyl-2-tridecyl- 5, 5-dimethylpynolidine-l-oxyl, 2, 5, 5-Trimethyl-2- ethylpyrrolidine-1-oxyl, 2,5, 5-Trimethyl-2- propylpyrrolidine-1-oxyl, 2, 5, 5-Trimethyl-2- butylpyrrolidine-1-oxyl, 2, 5, 5-Trimethyl-2- hexylpyrrolidine-1-oxyl, and 2,2,3,3,5, 5-Hexamethyl-l- pyrrolidine-1-oxyl .

The imidazolidine nitroxides spin probes may be selected from the group consisting of: 5, 5-Dimethoxy- 2, 2, 3, 4, 4-pentamethylimidazolidine-l-oxyl, and 5,5- Diphenyl-2,2,3,4, 4-pentamethylimidazolidine-l-oxyl .

The imidazoline nitroxides spin probes may be selected from the group consisting of: 4-Amino-2, 2, 5, 5- tetramethyl-3-imidazoline-l-oxyl, 5-5-Dimethoxy-2, 2,4- trimethyl-3-imidazoline-l-oxyl, and 2, 2, 5, 5-Tetramethyl- 4-phenyl-3-imidazoline-3-oxide-l-oxyl .

Another spin probes that can be used may be di-tert- butylnitroxide . In one embodiment, the nitroxide spin probes mentioned above may be perdeuterated nitroxide spin probes by comprising one or deuterium atoms that are substituted for the hydrogen atoms. The parameters may be kinetic parameters. The kinetic parameters may be the second order rate constant of reaction (fe) between the spin probe and the reducing agent within the beer sample and effective concentration of reducing agent or a pseudo first order rate constant (ki) •

In one embodiment, an excess amount of spin probes are added to a sample of the beverage relative to an amount of reducing agent in the beverage.

In one embodiment, the total decrease of nitroxide content is measured, which is a measure of the concentration of the reducing agents (b) in the beer. The kinetics of reaction between the spin probe and the reducing agents can be described based on either the second order rate constant k_r or the effective concentration of reducing agents (b) .

It should be realised that the measured rate constant is an effective rate constant of reaction as there could be several different reducing agents within a sample of beer, each of the reducing agents reacting with the spin probe at different rates.

In another embodiment, a small amount of a spin probe is provided in a sample of the beverage relative to an amount of reducing agent. The rate of reaction in this case is pseudo-first order with respect to the spin probe and is characterized by only one parameter, effective rate constant ki, which is equal to the product k_b.

The concentration a of spin probe that may be added to the sample of the beverage may be in the range selected from the group consisting of: 20μM to 400μM; 30μM to 300μM; 40μM to 250μM; 50μM to 220μM; 50μM to lOOμM; 60μM to 210μM; 70μM to 200μM; 80μM to 150μM; and 90μM to 130μM. In one embodiment, an excess amount of reducing agent is provided in a sample of the beverage relative to an amount of spin probe. The rate of reaction may be pseudo-first order (k'_r2) with respect to the spin probes.

Brief Description Of Drawings The accompanying drawings which are incorporated into and constitute a part of the description of the invention, illustrate disclosed embodiments and serve to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are for purposes of illustration only, and not as a definition of the limits of the invention.

FIG. 1 is a diagram showing the chemical structure and the corresponding EPR spectra of nitroxides in a sample of beer over the magnetic field range 3202 to 3252 Gauss: (a) - TEMPO (2, 2, 6, 6-Tetramethylpiperidine 1- oxyl); (b) - TEMPOL (4-Hydroxy-TEMPO) ; and (c) Perdeuterated - TEMPONE or PDT (4-Oxo-TEMPO-dl6) .

FIG. 2 is a graph presenting the kinetics of reduction of PDT showing actual experimental data and an exponential fit to the data.

FIG. 3 is a graph presenting the effective pseudo- first order rate constant of TEMPO reduction as a function of L-cysteine concentration in a sample of beer.

Fig. 3A is a graph presenting the data of Fig. 3 in inverse coordinates. FIG. 4 is a graph presenting the kinetics of TEMPO reduction in a sample of beer: (1 - o) - filtrated beer, Molecular Weight Cut-off (MWCO) of 30,000; (2 .#) beer tEllman' s reagent; (3 - Δ) beer + pepsin + Ellman's reagent; (4 - 1E|) boiled beer; (5 - ) control beer; (6 - ffl) beer+L-cysteine (5 x 10^"2M) ; and (7 - S) beer + pepsin.

FIG. 5 is TEMPOL based reducing power vs length of Miller Genuine Draft™ beer storage at elevated (32°C) temperature .

Detailed Description

Determination of the EPR spectra The receptacle may be for containing a spin probe and a sample of the beverage may be provided on an electron paramagnetic resonance apparatus.

The electron paramagnetic resonance apparatus may be any commercially available X-band (9-10 GHz) EPR spectrometer. Exemplary spectrometers available commercially include: an X-band VARIAN™ E-112 century series spectrometer from Varian Inc. of Palo Alto,

California, United States of America; BRUKER ELEXSYS™ E500 spectrometer, EMS series, or e-scan series of Bruker

BioSpin Gmbh, from Rheinstetten/Karlsruhe, Germany; Model

8400 X-band Electron Spin Resonance spectrometer from

Resonance Instruments, Inc., of Scokie, Illinois, United

States of America. An exemplary EPR spectrometer that could be used is disclosed in United States Patent

No. 3,931,569, which is incorporated in its entirety herein by reference. The spectra can be recorded with a microwave power in the range of 0.5 mW to 5 mW, a modulation amplitude in the range of 0.01 to 5 G, and at modulation frequency from 1 to 200 KHz.

Determination of the reducing agent concentration It has been found that the intensity of the nitroxide spin probes decrease over time. In one exemplary embodiment, the second-order rate constant k of reaction between the spin probe and the reducing agents within the beverage and the concentration b of reducing agent within the sample are determined from the following equation:

1 , b(a - x) , -In— ^L = kt a — b a(b — x)

where a is the initial concentration of the spin probe and b is the initial concentration of the reducing agent in beer, k is the second order rate constant, and x is the decrease of the spin probe concentration at time t.

A plot of the EPR spectra intensity over known time t can be used to determine the parameters b and k. Spin probe is added and its concentration a is known. To minimize possible error of a as the result of addition of the probe, the data from the EPR spectra can be plotted using a commercially available software package and all three parameters a, b, and k can be determined from the software package. A processor such as a personal computer may be used that is programmed with software to determine the reaction rate constant or the concentration of the reducing agent from the EPR spectrometer. An exemplary software package that can be used is SigmaPlot™ from SPSS Science of Chicago, Illinois, United States of America.

In the examples 1-11, it should be noted that all beers were "fresh" in that they were opened just before taking a sample and were not more than 2 weeks old from their date of production.

Example 1 A sample of bottled Miller Genuine Draft™ beer from Miller Brewing Company, Milwaukee, Wisconsin, United States of America was obtained.

Nitroxide TEMPOL (4-hydroxy-2, 2, 6, 6- tetramethylpiperidine-1-oxyl) was obtained from Sigma- Aldrich Corporation of Milwaukee, Wisconsin, United States of America and was added to the sample of beer from aqueous stock solution to a final concentration of 100 μM in beer. The sample was placed inside a gas- permeable Teflon (polytetrafluoroethylene) capillary (PTFE, 0.81 mm I.D., 0.86 mm O.D.) from Zeus Industrial Products of New Jersey, United States of America, and the ends of the capillary (length 3 cm) were closed. The capillary was then inserted into a quartz tube and the oxygen content in the beer was decreased by^' a flux of nitrogen around the gas-permeable capillary in this tube. The reaction between the TEMPOL and the reducing agents within the beer was allowed to proceed at room temperature and gas re-equilibration of a solution inside such a capillary occurred with a time constant of about 3-4 min.

EPR spectra were taken with Varian (Palo Alto, CA) Century Series E-112 X-band (8.8-9.5 GHz) operating at a microwave power of 0.5 - 2 mW, a modulation amplitude of 1 Gauss, and a modulation frequency of 100 KHz. The EPR spectra of TEMPOL in beer consisted of three spectral components due to isotropic hyperfine splitting on the nitrogen spin 1=1 (for ¹⁴N) as shown in graph (b) of Figure 1. The amplitude of these components was almost equal indicating that the nitroxide molecules tumbled freely in solution. Deoxygenation of the beer inside the gas-permeable capillary resulted in line-narrowing effects that were caused by a decrease in spin exchange interactions between TEMPOL and the dissolved molecular oxygen. Upon completing deoxygenation of the beer sample containing the spin probe, EPR spectra of the central (m.ι=0) spectral component was recorded every minute. It was observed that the peak-to-peak amplitude of the observed spectral line decreased over time until it reached a certain level. The decrease occurred without any addition of exogenous substances due to reaction between the TEMPOL and reducing agents present in beer. The intensity of sequential EPR spectra provides a qualitative measure of chemical reactions that remove paramagnetic spin probes from the beer.

It was observed that the kinetics of the reaction between the TEMPOL and the reducing agents could be explained by a mechanism according to which there is just one reducing agent irreversibly reducing the nitroxide. Kinetics of the nitroxide reaction in beer could be described as irreversible process, which is a first order reaction with respect to both TEMPOL and a reducing agent . Using the method and the equation described above, the rate constant in this example was found to be 123 M^"1 min^-1. The concentration of the reducing agent was found to be 60+1 μM.

Comparative Example 2 The experiment of the example 1 was repeated but with TEMPOL concentration at 10 μM. The kinetics was pseudo-first order with respect to TEMPOL and the EPR spectrum decayed to an undetectable level with time. It was determined that in this spin probe EPR experiment that it was not possible to determine the absolute content of the reducing agent in beer because the initial concentration of the spin probe was too low.

Comparative Example 3 The experimental procedure of example 1 was repeated but with TEMPOL concentration at 2 mM. In this experiment the intensity of the EPR signal decreased over the time but only marginally, i.e., the magnitude of decrease was much smaller than the initial intensity of the EPR signal. It was found that such a small decrease in EPR intensity led to large errors in kinetic parameters' determination.

It will be appreciated that comparative examples 2 and 3 show that too high concentration or too low concentration of TEMPOL will affect instrument sensitivity limits

Example 4 A sample was obtained by opening a bottle of Miller Genuine Draft beer was opened. Nitroxide perdeuterated TEMPONE (PDT) was added to beer from an aqueous stock solution to final concentration in beer 100 μM. Perdeuterated nitroxide, PDT, was used to improve EPR signal-to-noise ratio during registration of the spectra because of a substantial line narrowing due to substituting hydrogens by deuteriums in the spin probe molecule. The reaction was monitored for almost 10 hrs .

The kinetics eventually practically stopped, as there was no reducing agent left at the end as shown in

Figure 2. The kinetics of this reaction can be described as an irreversible reaction, first order with respect to both PDT and just one reagent in beer

The kinetic model describes the data exceptionally well as shown by Fig. 2.

The rate constant of the reaction with PDT was 54.8 M^"1min^"1, which was 2.25 times lower than with TEMPOL in example 1 and which can be explained by an isotope effect.

The concentration of the reducing agent was found to be 61.7+0.2 μM and was essentially the same as in the example 1. This indicates an excellent reproducibility of the nitroxide reduction method.

Example 5 The method is able to detect the presence of additional reducing agents in beer. Samples of bottled Tiger™ beer from Asia Pacific Breweries, Singapore were obtained. 10^_1 M L-Cysteine in ethanol stock solution from Sigma-Aldrich Corporation of Milwaukee, Wisconsin, United States of America was added into beer so that five concentrations of L-Cysteine in beer were obtained as follows: was 0.125xl0^"2 M, 0.25xl0^~2 M, 0.5 xlO^-2 M, 0.75 xlO^"2 M, lxlO^"2 M. Then TEMPO (2,2,6,6- tetramethylpiperidin-1-oxyl) was added from an aqueous stock solution (1 mM) to concentration in beer of 100 μM. Spin probe reduction kinetics were measured from EPR spectra using the procedure of the Example 1. The spectrometer used in this example was a Bruker Elexsys Series E500 CW-EPR X-band (9-10 GHz) from Bruker BioSpin Gmbh, of Rheinstetten/Karlsruhe, Germany, operating at a microwave power of 5 mW, a modulation amplitude of 1 Gauss, and a modulation frequency of 100 KHz. To simplify the analysis of TEMPO EPR spectra, the peak-to-peak amplitude of the EPR signal was used instead of the signal double-integrated intensity as the signal line width became constant after the initial 2-3 minutes. In the kinetics measurements the EPR peak of the central (m.ι=0) spectral component was recorded every minute.

Although in this example, another compound - cysteine - was present in the reaction mixture, the kinetics of chemical reaction of spin probe reduction still could be described as an apparent first order with respect to both the nitroxide and a reducing agent in beer. In this series of experiments the concentration of L-cysteine was varied (ffrom 10^~3 M to 10^~2 M ) and the kinetics of the nitroxide reaction in beer were analyzed. It was found that the rate of TEMPO reduction in anaerobic conditions increased with an increase in cysteine concentration. Specifically, it was found that in the presence of 5 x 10^~2 M cysteine, the initial rate of reduction was twice as high as that in a control beer (i.e. without cysteine) . Initially, the rate increased with the concentration of cysteine, but then reached a constant value as shown by Figure 3. Fig. 3A shows this data in inverse coordinates. The results demonstrates that the reaction has a catalytic mechanism.

Example 6 Ascorbic acid (Vitamin C) often is used as antioxidant in food and beverages. Addition of ascorbic acid in beer also increases the rate of nitroxide reduction, which is proportional to the ascorbic acid concentration .

The rate constant of TEMPONE reduction by ascorbic acid in aqueous solutions at room temperature was about 450 M^_1 rnin^"1.

Example 7 Fresh bottled Tiger™ beer was opened. A sample of beer was filtrated for 12 minutes at 10,000 rpm with a Centrifugal Filter Unit MWCO 30K, from Whatman PLC, of Maidstone Kent, United Kingdom.

The procedure and the EPR spectrometer of Example 6 were used utilised. The reaction rate constant was found to be 3.5-fold lower than for TEMPO in the fresh beer. The kinetics is shown in Figure 4 (l-o) . This example demonstrates that the ultrafiltration procedure removes some of the reducing compounds from beer and that the method is sensitive to these changes in the composition of reducing compounds in beer.

Example 8 A fresh sample of Tiger™ beer was initially purged with nitrogen for 2 minutes, heated to a boiling temperature, and then kept at this boiling temperature for 10 min in a closed, sealed flask that was purged with nitrogen.. After this treatment, the beer became cloudy. A sample was drawn from the boiled beer, cooled down to room temperature, and the experimental procedures of Example 1 was repeated, but with a Bruker spectrometer and the spin probe was TEMPO at 100 μMas in Example 6 (with L-cysteine) and 8 (with centrifugal filter)

It was determined that the rate constant was less than half of what was observed for the control sample of untreated fresh beer. Boiling of beer is expected to denature soluble proteins, which then would aggregate giving beer a cloudy appearance; these aggregates would eventually precipitate. Upon denaturing of soluble proteins, the amount of reactive S-H groups of cysteine residues is reduced and their accessibility to other solvent molecules is also reduced through the aggregation process and formation of S-S bonds. Kinetics is shown in Figure 4 (4) .

Example 9 A sample of fresh bottled Tiger™ beer was obtained. The solution was purged with nitrogen for 2 minutes and then incubated with pepsin (20 mg/ml of solid pepsin powder in fresh beer) for 30 min in a closed, sealed flask that was purged with nitrogen at a temperature of 37°C. After the treatment, the TEMPO nitroxide reduction experiment was conducted following the procedure of the Example 1.

The initial rate of nitroxide reduction was 5 times faster than that for TEMPO in fresh beer. It was also observed that the decrease between the initial and final concentration of the spin probe concentration in this case was much more than observed for the untreated beer. The overall reaction kinetics of EPR signal is shown in Figure 4 (7 - U) ) . This example demonstrates that the spin probe nitroxide reaction is sensitive to the protein hydrolysis in beer.

Example 10 A sample of fresh bottled Tiger™ beer was obtained. A sample of the beer was treated with a SH-specific Ellman' s (DTNB, 5, 5 ' -dithiobis- (2-nitrobenzoic acid)) in the following way.

8 mg of a solid Ellman' s reagent were dissolved in 0.2 ml of ethanol and added to 10 ml of fresh beer. The Ellman' s reagent is not soluble in pure water at pH 4 - 5 but is soluble in ethanol and ethanol/water mixtures. When it was added to beer in concentrations up to 10^~3 M it did not change the rate of nitroxide reduction. Nevertheless, when the pH was increased to 7.5 units by addition of NaOH, this reagent became soluble in a beer and Ellman solution and gave the solution a bright yellow color. The pH of Ellman+beer solution was adjusted by adding NaOH, i.e. Ellman was added first into beer and NaOH was added later to solution. The beer was treated with Ellman' s reagent in this way at room temperature for 1.5 hrs to allow Ellman' s reagent to react with accessible SH groups. Then TEMPO was added and the kinetics of EPR signal decay was measured using the procedure of the Example 1. The resulting curve is shown in Figure 4 (2 - .#) . The initial rate in this experiment was about one-third of what was observed for fresh beer.

This example demonstrates that the method is sensitive to chemical modification of SH- groups of proteins in beer.

Example 11 A sample of fresh bottled Tiger™ beer was obtained. A sample of beer was treated with pepsin as in Example 9. The solution was purged with nitrogen for 2 minutes and then incubated with pepsin (20 mg/ml of solid pepsin powder in fresh beer) for 30 min in a closed flask at T=37°C. After the pepsin treatment, Ellman' s reagent was added to the solutions using procedures of the Example 10. Then TEMPO was added and the kinetics of TEMPO EPR signal decay was measured using the same procedure. The initial overall rate of nitroxide reduction was only slightly higher than that after the Ellman' s treatment of beer without pepsin (Example ) , indicating that the Ellman' s reagent is effective in blocking essentially all the cysteine residues. The overall kinetics of reaction is shown in Figure 4 (3-Δ) .

Overall, examples 8-11 demonstrate that TEMPO can be reduced by SH-groups bound to proteins. The rate of chemical reduction of nitroxides is increased when these groups become more accessible to nitroxides after the proteolysis. The rate is decreased when the SH-groups are blocked by Ellman' s reagent.

Example 12 The spectrometer and procedure from example 1 was repeated with a sample of Miller Genuine Draft™ beer.

In this example, the effect of accelerated aging of beer in closed bottles was investigated. Beer bottles were opened after 4, 8, 15, 22, 28, 56, 84 days of storage at 32 °C. Each time TEMPOL was added to beer to a final concentration of 100 μM. Measurements of TEMPOL EPR signal decay were conducted using the protocol and EPR instrumentation of the Example 1. From these experiments, the effective concentration of the beer reducing agents reacting with the nitroxide was determined using the procedures of Example 1.

The effective concentration of the reducing agent reacting with nitroxides in beer can be used to characterize the beer reducing power. It can be seen from Fig. 5 that the concentration increased and then decreased upon the storage at the elevated temperature. After 3 - 4 weeks, the reducing power decreased by 10 - 15% as the result of beer aging.

Determination of beer flavour and shelf-life It will be appreciated that by using the spin probes disclosed herein, it has been possible to determine the reaction effective rate constant of the beer as reducing agents within the beer react with the spin probe (s). The reaction rate constant is an indicator of the concentration of the reducing agent in beer and therefore also serves as an indicator of beer shelf life. It will be understood, that the reaction rate obtained in the disclosed method and system will serve as an indicator shelf life of the beer. The effective reaction rate constant that is obtained will be dependent on the process conditions used to produce the beer and will vary between brewhouses and from brewing batches. It will be appreciated however, a standard reaction rate range or reducing concentration range of a particular beer can be empirically determined to infer the shelf- life of the beer as it is produced by the method disclosed herein.

It will also be appreciated, that a panel of tasting experts could empirically correlate the flavor stability of the beer from the reaction rate constant or the reducing agent concentration. In this way, the reaction rate constant obtained by the disclosed method could be used to enable the empirical determination of the projected shelf life of the beer and/or the flavour stability of the beer.

It should also _.be understood that it is possible to compare the obtained reaction rate constant with to a reaction rate constant of a standard to determine a characteristic of the sample. It is also possible to compare the obtained reducing agent concentration with the reducing agent concentration of a standard to determine a characteristic of the sample. The characteristic may be for example, an indicated shelf- life. For example, if the comparison indicates that there is an excess reducing agent in a sample compared to a standard, the results may indicate that the beer will have a longer shelf-life than the standard. Likewise, if the comparison indicates that there is less reducing agent in a sample compared to a standard, the results may indicate that the beer will have a shorter shelf-life than the standard.

Applications The developed method is simple and fast and can be used to characterize properties of beer and other beverages. Though the above examples were dealing mainly with beer, the present invention is not limited to the above- mentioned examples. It can be used to characterize transparent and, more importantly, non-optically transparent liquids, such as intermediate and semifinished products, which is impossible to assess with optical tests.

The time necessary to conduct the experiments is relatively short and may only take 3-5 minutes for fresh beer. The method described here is more convenient than spin trapping methods, especially in industrial applications .

Specifically, for fresh beer, for which the content of antioxidants is high, the spin probe method produces results in a short time, while the spin trap method based on measuring the lag time requires a longer measuring time (up to 1 hr or even longer if the amount of antioxidants is especially high) . Another advantage of the disclosed method is that the beer is studied under conditions at room temperatures

(ie about 15-28 °C) which is the temperature range at which beer is stored on the shelf. Accordingly, it is not necessary to test the beer at elevated temperatures of about 60 °C as in other methods. Furthermore, there is no need to conduct the analysis in the presence of oxygen, which is atypical for beer in-storage. It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims .

Claims

Claims 1. A method for determining one or more parameters of a beverage comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beverage, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra of the sample to determine one or more parameters of the sample.

2. A method as claimed in claim 1, wherein the one or more parameters is a kinetic parameter.

3. A methods as claimed in claim 2, further comprising determining the amount of reducing agent present in the beverage from the kinetic parameter.

4. A method as claimed in claim 1, the method further comprising measuring at least two amplitude peaks of a signal of an EPR spectra during the time period.

5. A method as claimed in claim 1, wherein the obtaining occurs for a time period selected from the group consisting of: 40 seconds to 10 minutes; 1 minute to 8 minutes; 2 minutes to 5 minutes; 3 minutes to 4 minutes; and 40 seconds to 4 minutes.

6. A method as claimed in claim 1, further comprising providing an inert atmosphere for the reaction between the spin probe and the one or more reducing agents.

7. A method as claimed in claim 1, comprising providing the beverage selected from the group consisting of: beer, wine, sake, fruit juice, brewing mashes and worts .

8. A method as claimed in claim 1, comprising providing the spin probe as a nitroxide spin probe.

9. A method as claimed in claim 8, comprising selecting the nitroxide spin probe from the group consisting of: piperidine nitroxides, oxazolidine nitroxides, pyrroline nitroxides, pyrrolidine nitroxides, imidazolidine nitroxides, and imidazoline nitroxides.

10. A method as claimed in claim 8, wherein the nitroxide spin probes are perdeuterated nitroxide spin probes.

11. A method as claimed in claim 2, wherein the kinetic parameter is the second order rate constant of reaction between the spin probe and the reducing agent.

12. A method as claimed in claim 1, further comprising adding an excess amount of spin probe to a sample of the beverage relative to the amount of reducing agent in the beverage.

13. A method as claimed in claim 1, further comprising adding a concentration spin probe to the sample of the beverage in the range selected from the group consisting of: 20μM to 400μM; 30μM to 300μM; 40μM to 250μM; 50μM to 220μM; 50μM to lOOμM; 60μM to 210μM; 70μM to 200μM; 80μM to 150μM; and 90μM to 130μM.

14. A method as claimed in claim 2, wherein the kinetic parameter is the pseudo-first order rate constant of reaction with respect to the spin probes.

15. A method as claimed in claim 2, wherein the kinetic parameter is the pseudo-first order rate constant of reaction with respect to the reducing agent.

16. An analytical system for determining one or more parameters of a beverage comprising one or more reducing agents, the analytical system comprising: a receptacle for containing a spin probe and a sample of the beverage, the spin probe capable of reacting with the one or more reducing agents; an electron paramagnetic resonance apparatus for obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period as the spin probe reacts with the one or more reducing agents; and a processor capable of using the plurality of obtained EPR spectra of the sample to determine one or more parameters of the sample.

17. An analytical system as claimed in claim 16, wherein the spin probe is a nitroxide spin probe selected from the group consisting of: piperidine nitroxides, oxazolidine nitroxides, pyrroline nitroxides, pyrrolidine nitroxides, imidazolidine nitroxides, and imidazoline nitroxides .

18. A method for determining one or more parameters of beer comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a period of time as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra to determine one or more parameters of the sample.

19. A method for determining the concentration of one or more reducing agents in beer, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a time period^' as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra to determine one or more kinetic parameters of the sample; determining the concentration of the one or more reducing agents in the beer from the one or more kinetic parameters .

20. A method for determining one or more characteristics of a sample of beer comprising one or more reducing agents, the method comprising the steps of: introducing a spin probe to a sample of the beer, the spin probe capable of reacting with the one or more reducing agents; obtaining a plurality of electron paramagnetic resonance (EPR) spectra of the sample over a period of time as the spin probe reacts with the one or more reducing agents; and using the plurality of obtained EPR spectra to determine one or more kinetic parameters of the sample; comparing the one or more kinetic parameters to one or more standards for such parameters to determine a characteristic of the sample.