Antioxidant Activity of Barley and Malt: Relationship with Phenolic Content

Lebensm.-Wiss. u.-Technol., 29, 238–244 (1996)

Antioxidant Activity of Barley and Malt: Relationship with Phenolic Content M.-N. Maillard*, M.-H. Soum, P. Boivin and C. Berset M.-N. Maillard, M.-H. Soum, C. Berset: Laboratoire de Chimie des Substances Naturelles, Departement ´ Science de l’Aliment, E.N.S.I.A. 1, avenue des Olympiades, F-91 305 Massy (France) P. Boivin: Institut Fran¸cais de la Brasserie et de la Malterie, Pole ˆ technologique de Brabois, 7, rue du bois-de-la-champelle B.P. 267, F-54 512 Vandoeuvre Cedex (France) (Received March 29, 1995; accepted July 3, 1995)

One way to naturally inhibit the oxidative deterioration of beer would involve protecting the antioxidants present in barley (mainly polyphenols) during the malting process. Depending on the variety, the antioxidant activity of barley is not negligible. Its relationship with the content of the three main phenolic groups (flavan-3-ols, hydroxycinnamic derivatives and flavonols), determined by a new ultraviolet spectrophotometric method, is proposed. It increases during malting probably not only by the modification or releasing of phenolic compounds, but also by the formation of new antioxidants, such as Maillard reaction products.

©1996 Academic Press Limited

Introduction The brewing industry is concerned with the stability of its final products. Under long storage conditions, beer develops colloidal haze and off-flavours. Among phenomena leading to such problems is the oxidation of linoleic acid to yield trans 2-nonenal, which gives beer a cardboard flavour (‘papery’) and has a flavour threshold of as little as 1.10–10 g/g (1). Germinated barley can contain up to 45 mg/g of its dry weight as lipids, with linoleic acid as the main constituent (50–60%). During malting, a significant decrease in the lipid content can be observed, indicating rapid degradation. The free fatty acids generated during lipolysis can be dioxygenated by autoxidation and by the action of lipoxygenase yielding highly reactive hydroperoxides. These hydroperoxides can enzymatically produce carbonyl compounds including trans 2-nonenal (2). Two ways may be used to naturally inhibit the oxidative deterioration of beer by optimizing the malting procedure: the protection of antioxidants present in barley (mainly polyphenols) and the promotion of new antioxidants produced in situ. In order to understand better the role of the phenolic compounds in the antioxidant power of barley and malt, a new spectrophotometric method has been developed. Compared to the existing colorimetric methods such as the Folin–Ciocalteu method, this UVmeasurement not only allows evaluation of the total phenolic content, but also the content of the three main *To whom correspondence should be addressed.

0023-6438/96/030238 + 07$18.00/0

groups of phenolic compounds. It was applied to different varieties of barley and their corresponding malts. We attempted to establish a relationship between the level and nature of phenolic compounds and the antioxidative activity of the malts, at each step of malting.

Materials and Methods Plant material Malts from barley (Hordeum vulgare) varieties Baraka, Magie, Plaisant, Triumph and Volga harvest 1991 and Triumph harvests 1992 and 1993 were prepared in a micro-malthouse (2 kg) using standard malting conditions. After steeping and germination, the kilning procedure occurred in five successive steps of heating: 50°C for 8 h, 64°C for 10 h, 80°C for 4 h, 85°C for 4 h and 90°C for 2 h. For the variety Triumph harvests 1992 and 1993, the malt was sampled at each step of the kilning.

Extraction of the antioxidants from barley and malt Two grams of barley (or malt) were finely ground in an analysis blender IKA A10. The sample was extracted four times (for periods of 20 min) with 20 mL of methanol (HPLC grade, Carlo Erba, Milano, Italy). It was then filtered through Whatman No.1 filter paper. The filtrate (80 mL) was evaporated to dryness at 35°C under vacuum. The residue was dissolved either in 10 mL of water and then purified for the analysis of ©1996 Academic Press Limited

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phenolic compounds by UV-measurement, or in 5 mL of butan-1-ol (Aldrich, St Quentin Fallavier, France) for the determination of the antioxidant activity.

Enrichment in phenolic compounds Ten millilitres of distilled water were added to the extraction residue. After modification of the ionic force by 10 mL of 400 g/L ammonium sulfate and acidification to pH 3 by 2 mL of 200 g/L metaphosphoric acid, the aqueous phase was extracted three times by 10 mL of ethyl acetate (Pestinorm grade, Prolabo, Paris, France) into a separating funnel. The ethyl acetate phase was then evaporated to dryness at 40°C under vacuum. The residue was dissolved in 5 mL of methanol and analysed by ultraviolet spectrophotometry.

Determination of total phenolic compounds by the Folin–Ciocalteu method Total phenolic compounds were measured with Folin– Ciocalteu reagent using gallic acid as a standard (3,4). Five millilitres of Folin–Ciocalteu reagent (diluted tenfold in distilled water), 2 mL of 200 g/L sodium bicarbonate and 2 mL of distilled water were added to 1 mL of the raw methanolic extract (filtrate) of barley or malt, already diluted tenfold in distilled water. After 1 h at 20°C, the absorbance at 755 nm was read. Results are expressed in gallic acid equivalents.

UV-measurement of phenolic compounds The purified methanolic extracts were analysed by ultraviolet spectrophotometry, using a Uvikon 810 Kontron spectrophotometer. Extinction coefficients at 280, 320 and 370 nm were determined from three pure compounds: DL-catechin, chlorogenic acid and quercetin (Extrasynthese, ` Genay, France) and a system of three equations was established in order to measure each of them in a mixture (see Results and Discussion). The concentrations of flavan-3-ols, hydroxycinnamic derivatives and flavonols were expressed in mg/g of dry matter, in DL-catechin, chlorogenic acid and quercetin equivalents, respectively.

methanol and then dissolved in butan-1-ol), we determined the effective quantity (EQ) required to double the half-life time of the control. EQ was expressed in gram of dry matter of barley or malt per mL of medium. The lower the EQ value, the stronger was the antioxidant. To make these values easier to understand, 1/EQ, proportional to the antioxidant activity, was used.

Results and Discussion Antioxidant activity of barley and malt Antioxidant activity of barley is not negligible (Fig. 1), even though it is significantly lower than that of a pure powerful antioxidant such as butylated hydroxytoluene (BHT) or gallic acid (6). The variety of barley has a significant influence on antioxidant efficiency. 1/EQ varies from 16 for Baraka barley (1991) to 37 for Plaisant barley (1991). Slight differences are observed for Triumph barley between 1991, 1992 and 1993. Presence of phenolic compounds in barley has often been reported. Main phenolic compounds already identified in barley and malt are ( + )-catechin, procyanidins B3 and C2, prodelphinidin B3 (7–12), p-coumaric acid, ferulic acid and vanillic acid (13). Taking into account their tendency to polymerize, causing colloidal haze, polyphenols are generally considered as undesirable compounds, and are, therefore, often removed by addition of polyvinyl polypyrolidone (PVPP) before bottling. Nevertheless, some of these phenolic compounds have strong antioxidant activity, as shown in Table 1, where the values of 1/EQ for several commercial standards are reported. Among the standards tested, quercetin and ( + )-catechin show higher antioxidant activity than BHT (1/EQ = 35 L/g) (6). The molar activities of (–)-epicatechin and flavonol glycosides are nearly identical, while those of hydroxycinnamic acids are slightly inferior. These compounds are classified as free radical terminators which interfere with lipid oxidation by rapid donation of a hydrogen

16

Baraka 91 Volga 91

24 22.5

Triumph 91 Variety

Antioxidant activity determination The antioxidant activity was measured according to the method of Cuvelier et al. (5) based on the accelerated autoxidation of methyl linoleate (Fluka, St Quentin Fallavier, France) in anhydrous dodecane (Aldrich), under strong oxidizing conditions (110°C, intensive oxygenation), for several hours. Gas chromatography was used to monitor the degradation of methyl linoleate from the reaction medium (initial concentration = 40 g/L). Antioxidant activity was assessed by the percentage increase in the half-life time of methyl linoleate without any antioxidant. This activity varied according to the antioxidant and its concentration. To compare the activity of barley and malt extracts (extracted by

30 18 29 21

Triumph 92

25.5 22 22 22

Triumph 93 Magie 91

45 37

Plaisant 91

43

0

10 20 30 40 –3 (1/EQ) × 10 (dodecane L/g dry matter)

50

Fig. 1 Antioxidant activity of different varieties of barley and malt. h = 1/EQ barley; C = 1/EQ malt. Bars indicate standard deviations calculated from three values; numbers 91, 92 and 93 indicate harvest years

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Table 1 Antioxidant activity of phenolic standards. Values represent means (¯x) and standard deviations (sx) calculated from two determinations 1/EQ (dodecane L/g phenolic compound) Phenolic group

Phenolic standard

Flavan-3-ols

(+)-catechin (–)-epicatechin p-coumaric acid ferulic acid quercetin isoquercitrin rutin

Hydroxycinnamic acids Flavonols

x¯

sx

x¯

59.2 15.3 8.4 14.3 125.0 13.4 8.0

2.4 1.0 0.6 0.6 5.1 0.2 0.0

17168 4466 1378 2697 37750 6217 4880

1/EQ × 10–3 (dodecane L/g dry matter)

atom to lipid radicals (14). Phenolic antioxidants are excellent hydrogen or electron donors. In addition, their radical intermediates are relatively stable due to resonance delocalization and lack of suitable sites for attack by molecular oxygen. Except for the variety Triumph harvest 1993, malts were found to have a higher antioxidant activity than their corresponding barley (Fig. 1), but the increase of 1/EQ varied greatly from one variety to another: it was 16% for Plaisant 1991 and more than 100% for Magie 1991. The evolution of the antioxidant activity, followed during malting for the variety Triumph harvests 1992 and 1993, is reported in Fig. 2. For Triumph 1992, the antioxidant activity of the germinated barley slightly decreases until 50°C, and then increases up to 80°C. A similar result was found with Triumph 1993, except for a very low value for germinated barley. This result may be due to bad extraction of the antioxidant compounds, because of the particularly high moisture of this sample (0.45 g/g). This problem could have been solved by freezing and drying the material before extraction, but it has not been done. If this value is discarded, a good parallelism of the two curves can be seen. The increase in antioxidant activity between barley and final malt may be explained in two different ways: (i) A release of phenolic compounds bound to cellular structures and better extraction (15). Besides the 30

20

10

0

Barley Germinated 50°C barley Kilning step

64°C

1/EQ (dodecane L/mol phenolic compound)

80°C

Fig. 2 Evolution of the antioxidant activity during malting of Triumph barley harvests 1992 and 1993. s = 1/EQ 92 (mL/g); d = 1/EQ 93 (mL/g). Bars indicate standard deviations calculated from two determinations

sx 696 292 98 113 1540 93 0.0

synthesis of amylases, proteases and β-glucanases causing polymer degradation (16), other hydrolytic enzymes can lead to the release of bound phenolic compounds, mainly the phenolic acids associated to lignin and arabinoxylans (17). Moreover, kilning leads to more friable tissues and probably allows better extraction of phenolic acids, mostly present in the outer layers of the grain. (ii) Appearance of Maillard reaction products during kilning. It has been shown that beers naturally contain products resulting from the thermal breakdown of carbohydrates or from the non-enzymic browning reaction (Amadori compounds, enediols, enaminols, enediamines, reductones and melanoidins) (1). In fact, germination releases reducing sugars and amino acids. During the first steps of kilning, the water at the surface layers of the grain is removed (the humidity falls from 0.45 g/g to 0.12 g/g) and the first intermediates of Maillard reaction can be produced (18). Then, high temperatures and low humidity contents achieve the extension of the Maillard reaction. Caramelization of sugars can also occur during the last steps of kilning, catalysed by the low concentration of organic acids (18). The effect of barley and malt extracts on the lipoxygenase activity was followed and showed a decrease in activity during kilning (19). This inhibition of the enzyme can be explained by an increase in free phenolic compounds, since Maillard reaction products do not exert any effect on the lipoxygenase (ForgetRichard F., personal communication (1994)). In order to verify the assertion concerning the role of phenolic compounds, two experiments were developed. First, a rapid evaluation of the level of phenolic compounds in the extracts of barley and malt was done. Then, the evolution of phenolic compounds concentration was followed during kilning for malt Triumph 1992 and 1993.

Rapid spectrophotometric evaluation of phenolic compounds In order to estimate the levels of total phenols and the main groups of phenols in barley and malt, a UVspectrophotometric method was developed. A similar principle has already been used by Billot (20) and Amiot et al. (21) for pear and olive, respectively.

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The methanolic extracts show an absorption band at 280 nm and a shoulder at 320 nm (Fig. 3). Analysis by thin layer chromatography shows the presence of three main phenolic groups: flavan-3-ols (i.e. catechin), hydroxycinnamic derivatives (i.e. chlorogenic acid) and flavonols (i.e. quercetin). The determination of the extinction coefficients (Table 2) of the standards determined by reporting their absorbance at 280, 320 and 370 nm as a function of their concentration expressed in mol/L, permitted the following equations to be established: A370 = 63.40×[Q]

Eqn [1]

A320 = (23.60×[Q]) + (40.70×[ACQ])

Eqn [2]

Absorbance (arbitrary units)

280

320

250

300 350 Wavelength (nm)

400

Fig. 3 Ultraviolet spectra of a purified methanolic barley extract, and of phenolic standards. — = barley; — . — = DLcatechin; – – – – = chlorogenic acid; .... = quercetin

A280 = (23.20×[Q]) + (21.90×[ACQ]) + (12.15×[C]) Eqn [3] where Q = quercetin, ACQ = chlorogenic acid and C = catechin: concentrations are expressed in g/L; Aλ = absorbance at the suitable wavelength. The resolution of this system of equations allows evaluation of the level of each phenolic group in barley and malt. Results are expressed in quercetin, chlorogenic acid and catechin equivalents, respectively. [Q] = 15.8×10–3×A370

Eqn [4]

[ACQ] = 24.6×10 ×A320–9.2×10 ×A370 –3

–3

Eqn [5]

[C] = 82.3×10–3×A280–44.3×10–3×A320 –13.6×10–3×A370 Eqn [6] with concentrations expressed in g/L. The concentration of total phenolic compounds is determined by adding up the three concentrations, [Q],[ACQ] and [C]. The recovery yield of these three groups of phenolic compounds, determined on a mixture of the three standards submitted to the enrichment procedure, is always higher than 80%: respectively 81, 85 and 87% for catechin, chlorogenic acid and quercetin. The concentrations of flavonols, hydroxycinnamic derivatives and flavan-3-ols will be discussed, taking into account these recovery yields. In order to verify the validity of this method, the level of total phenolic compounds in several varieties of barley was also determinated by the reference colorimetric method of Folin–Ciocalteu. Differences observed by these two methods for the studied barleys spread over 5 to 15%, except for Plaisant barley (Table 3). This could be considered as acceptable because of the poor specificity of the Folin–Ciocalteu reagent. The UV-measurement can be used for the determination of total phenolic compound content in barley and will be preferred, because it also allows the concentrations of the three main groups of phenolic compounds in barley to be determined.

Table 2 Extinction factors of DL-catechin, chlorogenic acid and quercetin at 280, 320 and 370 nm in methanol Extinction coefficients (L/(mol.cm)) Phenolic compound DL-catechin chlorogenic acid quercetin

280 nm

320 nm

370 nm

3523 8103 7006

0 15059 7127

0 0 19147

Phenolic compounds in barley Phenolic compounds in the studied barley samples are mainly flavan-3-ols ( > 85%) (Table 4). Hydroxycinnamic derivatives represent about 10% and flavonols less than 5% of total phenolic compounds of the purified methanolic extracts. The variety of barley does

Table 3 Determination of total phenolic content in barley: comparison between the UV-measurement and the Folin–Ciocalteu method. The coefficient of variation between the two methods, expressed in %, is calculated by dividing the standard deviation by the average of the concentrations of total phenolics obtained by the two methods Total phenolic compounds (mg/g dry matter) Variety of barley Baraka 91 Volga 91 Triumph 91 Magie 91 Plaisant 91

UV-measurement

Folin–Ciocalteu

Coefficient of variation (%) UV-measurement/ Folin–Ciocalteu

1.22 1.16 1.04 1.39 1.87

1.04 1.01 0.93 1.02 1.14

8.0 6.9 5.6 15.3 24.2

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Table 4 Phenolic composition and antioxidant activity of different varieties of barley. Values represent means (¯x) and standard deviations (sx) calculated from two determinations; numbers 92 and 93 indicate harvest years Flavan-3-ols (mg/g dry matter) Variety Baraka 91 Volga 91 Triumph 91 Triumph 92 Triumph 93 Magie 91 Plaisant 91

Hydroxycinnamic derivatives (mg/g dry matter)

Flavonols (mg/g dry matter)

x¯

sx

x¯

sx

x¯

sx

Total phenolics (mg/g dry matter)

1.04 1.07 0.95 1.12 0.89 1.25 1.68

0.15 0.02 0.00 0.03 0.06 0.00 0.08

0.15 0.08 0.08 0.11 0.08 0.12 0.16

0.04 0.00 0.00 0.00 0.00 0.00 0.01

0.03 0.01 0.01 0.02 0.01 0.02 0.03

0.00 0.00 0.00 0.00 0.00 0.00 0.00

1.22 1.16 1.04 1.25 0.98 1.39 1.87

not influence the proportion of each phenolic group, but only their overall contents, which may vary from 1 to 1.9 mg/g of dry matter. The analysis of seven different varieties of barley did not allow a clear correlation between the level of phenolic compounds and the antioxidant activity to be demonstrated because the differences between the samples were not significant. However in the case of Plaisant barley, a great increase in total phenolics also led to an important increase of the 1/EQ value. Thus, even if phenolic compounds are not the only components responsible for the antioxidant activity of barley, they could play a major role.

Phenolic compounds in malt Contents of phenolic compounds in malt are higher than in barley (Table 5), but proportions of the different groups are nearly identical. These results strengthen the hypothesis that better extraction of flavonoids and phenolic acids (13, 17) is possible after kilning. No correlation between the antioxidant activity and phenolic content is found for the five studied malts. On one hand, for Baraka and Magie varieties, the increase of 1/EQ value cannot be explained by the slight increase of phenolic content (Table 5), confirming that phenolic compounds are not the only constituents responsible for the antioxidant activity in malts. On the other hand, Triumph 1992 and 1993 showed higher level

1/EQ (dodecane L/g dry matter) 16 × 10–3 18 × 10–3 22.5 × 10–3 21 × 10–3 22 × 10–3 22 × 10–3 37 × 10–3

of phenolics without a high increase of 1/EQ. In order to better understand these surprising results, the evolution of each group of phenolic compounds during malting has been followed (Table 6) for the variety Triumph harvests 1992 and 1993. For Triumph 1992, an important increase in the amount of phenolic compounds is observed during germination (50%) while the antioxidant activity only increases by 5%. Bound phenolic compounds, released during this step, either seem to present low antioxidant activity or they are inhibited by other constituents of the extracts. Another hypothesis could be that part of the molecules is unstable in the operating conditions of the antioxidant test at 110°C. The decrease in activity and phenolic contents at 50°C can probably be explained by a poor extraction due to a high humidity and also by bad milling of the grains. At 80°C, both an increase in the antioxidant activity and the phenolic contents is observed. This increase can be due to a release of compounds, mainly flavan-3-ols ( > 86% of total phenolic compounds). It should also be noticed that Maillard reaction products or intermediates can absorb at 280 and 320 nm, and even if they will mainly be recovered in the residual aqueous phase, some of them will probably go into the ethyl acetate phase, altering the quantification. For Triumph 1993, except during the germination period, the antioxidant activity progressively increases without a concomitant important rise of the phenolic compounds. We can assume that as early as 64°C,

Table 5 Phenolic composition and antioxidant activity of different varieties of malt. Values represent means (¯x) and standard deviations (sx) calculated from two determinations; numbers 92 and 93 indicate harvest years. The percentage increase between malts and barleys is noted in parentheses Flavan-3-ols (mg/g dry matter) Variety Baraka 91

Hydroxycinnamic derivatives (mg/g dry matter)

x¯

sx

x¯

sx

x¯

sx

1.15

0.00

0.14

0.00

0.03

0.00

(+10%) Triumph 92

1.84

(–7%) 0.16

(+64%) Triumph 93

1.09

0.04 (+22%)

Magie 91

1.32

0.16 (+6%)

Plaisant 91

Flavonols (mg/g dry matter)

1.99

0.37 (+18%)

0.20

(+0%) 0.01

0.03

0.00

(+82%)

(+50%)

0.17 0.00 (+112%) 0.16 0.01 (+33%) 0.26 0.06 (+62%)

0.04 0.00 (+300%) 0.03 0.00 (+50%) 0.05 0.01 (+67%)

Total phenolics (mg/g dry matter)

1/EQ (dodecane L/g dry matter)

1.32 (+8%) 2.07 (+66%) 1.30 (+33%) 1.51 (+9%) 2.30 (+23%)

30 × 10–3 (+88%) 25.5 x 10–3 (+21%) 22 × 10–3 (+0%) 45 × 10–3 (+105%) 43 × 10–3 (+16%)

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Table 6 Evolution of the antioxidant activity and of the phenolic composition of Triumph barley harvests 1992 and 1993 during malting. Values represent means (¯x) and standard deviations (sx) calculated from two determinations 1/EQ (× 10–3 dodecane L/g dry matter)

Flavan-3-ols (mg/g dry matter)

Hydroxycinnamic (mg/g dry matter)

Flavonols derivatives (mg/g dry matter)

sx

x¯

sx

x¯

sx

x¯

sx

Total phenolics (mg/g dry matter)

21 22 19 21 25.5

0.38 1.58 0.80 0.64 0.36

1.12 1.63 1.36 1.45 1.84

0.03 0.04 0.00 0.15 0.17

0.12 0.22 0.14 0.18 0.20

0.00 0.00 0.00 0.04 0.01

0.02 0.03 0.02 0.03 0.03

0.00 0.00 0.00 0.00 0.00

1.26 1.88 1.52 1.66 2.07

22 10 15.5 19 22

0.50 1.06 0.52 0.51 1.45

0.89 0.65 1.22 0.92 1.09

0.06 0.03 0.12 0.00 0.04

0.08 0.09 0.19 0.14 0.17

0.00 0.00 0.01 0.00 0.00

0.01 0.02 0.02 0.03 0.04

0.00 0.00 0.00 0.00 0.00

0.98 0.76 1.43 1.09 1.30

Kilning step

x¯

Triumph 1992 Barley Germinated barley 50˚C 64˚C 80˚C Triumph 1993 Barley Germinated barley 50˚C 64˚C 80˚C

Maillard reaction and caramelization products are formed.

Conclusion This work could contribute to the selection of barley varieties, knowing their antioxidant efficiency and their phenolic composition. The choice of a variety must take into account two parameters: the antioxidant power of barley and its increase during malting. Antioxidant activity of barley seems to be somewhat related to the total phenolic content, particularly to the content of flavan-3-ols. Thus, it could be more judicious to choose a variety of barley showing a polyphenolic profile with the most antioxidative compounds. However, the importance of the increase of 1/EQ value during malting must be considered. Moreover, compounds other than polyphenols could participate in the antioxidant activity of malt, including Maillard reaction and caramelization products. At the moment, flavonoids involved in the formation of unacceptable colloidal haze are eliminated during the last steps of beer production. Thus, it is necessary to find a compromise between the antioxidant activity of polyphenols and their participation in beer colloidal haze. Indeed, it would be interesting to know exactly the effect of PVPP, used before bottling, on the polyphenols having the highest antioxidant activity and therefore protecting beer against oxidation.

Acknowledgements The authors would like to thank the Ministere ` de l’Enseignement Superieur ´ et de la Recherche, the Ministere ` de l’Agriculture et de la Peche ˆ (France), the

Chambre Syndicale de la Malterie Fran¸caise and the Brasseurs de France for financial support.

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