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Endogenous Antioxidants and Antioxidant Activities of Beers
C H A P T E R
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Endogenous Antioxidants and Antioxidant Activities of Beers Haifeng Zhao College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China
significantly different contributions to the antioxidant activities of beers. • Optimization of the brewing process and screening of raw materials are considered as efficient measures to increase the antioxidant activities of beers, which is extremely important to beer flavor stability. Knowledge of the relationships between • endogenous antioxidants and antioxidant activities of beers can be used to develop appropriate technologically innovative processes to control the flavor stability or to reinforce the nutritional properties of beer.
CHAPTER POINTS • P henolic compounds, Maillard reaction products, and sulfites constitute the major endogenous antioxidants in beers, although other antioxidants such as reducing sugars, vitamins, carotenoids and chelating agents also exist in beer at very low levels. • Endogenous antioxidants in beers originate from brewing raw materials or are formed during the brewing process; all of them make significant contributions to beer flavor stability and nutritional properties due to their antioxidant and antiradical properties, and other biological effects. • The beneficial impacts of these antioxidant compounds on the stability of beer flavor have been demonstrated, but there has been a lack of consensus concerning the effectiveness of these compounds as potential antioxidants. The antioxidant or pro-oxidant activity of these compounds is determined partly by their content in beers. • The content of endogenous antioxidants and antioxidant activities of beers are beer brand and type dependent. Generally, dark or brown beers contain higher levels of antioxidants and show higher antioxidant activity than pale or lager beers, because the former are brewed from worts with higher extracts or supplemented with some toast malts. • Due to the different reaction mechanisms and antioxidant activity assays involved, various kinds of endogenous antioxidants make
Processing and Impact on Antioxidants in Beverages http://dx.doi.org/10.1016/B978-0-12-404738-9.00002-7
INTRODUCTION Beer is one of the most widely consumed alcoholic beverages in the world for its fresh taste, low calories, and nutritional value. The main raw materials of beers are water, malt, hops, and brewer’s yeast, and some beers also are brewed with some non-malted cereals, starch, or starch syrups as adjuncts. The production of beer involves extremely complicated processes and chemical and biochemical reactions. Thus, the final beer contains various compounds with antioxidant activity mainly originated from raw materials or formed during processing. The species and concentrations of these antioxidants in final beer varies, largely because brewing technology, raw materials, and yeast applied in the brewing can differ Slight changes in structure or conformation of these compounds can cause significant changes of the antioxidant activity, which alters the overall oxidative or flavor stability of beer. Nowadays, flavor stability has become
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2. ANTIOXIDANT ACTIVITIES OF BEERS
the most important factor in determining the shelf-life of packaged beer, and prolonging shelf-life by delaying flavor staling is one of the greatest challenges facing the brewer. Although the flavor stability of beer depends primarily on the oxygen content of the packaged beer, the brewing process and the raw materials used can influence the flavor stability. Therefore, attention is now increasingly shifting towards increasing the antioxidant activity of beer itself due to the fact that oxidative staling of beer is still noticeable even if the level of total packaged oxygen might be as low as 0.1 mg/l (Bamforth, 2000). Antioxidants are generally thought to play a significant role in malting, mashing and brewing due to their ability to delay or prevent oxidation reactions and oxygen free radical reactions. Some synthetic antioxidants such as sulfites, formaldehyde, or ascorbate, can be added into the brewing process to improve beer flavor stability, but the effectiveness of these compounds is in doubt (Andersen et al., 2000). In recent years, there has been a general trend toward minimizing the use of additives in beer production because of consumer demand and stiffening regulations. As a result, it is desirable to prevent oxidation by protection of endogenous antioxidants in beer and in its raw materials, or those formed during the processes of kilning, boiling, and fermentation (mainly Maillard reaction products and sulfites). These endogenous antioxidants can offer an overall antioxidant activity in the beer to increase its stability during storage. This issue – to keep or increase the antioxidant activity of beer so as to improve its flavor stability – is greatly recognized by consumers and brewers. Beer generally contains a range of antioxidants, such as phenolic compounds, Maillard reaction products (melanoidins and reductones), sulfites, thiols, non-fermentable reducing sugars, vitamins, carotenoids, and chelating agents. Among the antioxidants mentioned above, phenolic compounds, melanoidins, and sulfites are of particular interest to brewers because they are described as a ntioxidants that possess antioxidant and antiradical properties as well as other biological effects (Zhao et al., 2013). The beneficial impacts of these antioxidant compounds on the stability of beer flavor have been demonstrated, whereas there has been a lack of consensus concerning the effectiveness of these compounds as potential antioxidants. For instance, results from electron spin resonance (ESR) lag phase studies showed that there were no significant effects of polyphenols on the formation of free radicals in beer during storage or in wort during brewing, while the author’s group’s previous study showed that phenolic compounds investigated made up 55.0–88.1% of the antioxidant activity of beer (Andersen et al., 2000; Zhao et al., 2010). Moreover, although the effects of sulfites on beer flavor stability evaluated by various assays have shown that sulfites stabilize the flavor in two ways, as antioxidants and as carbonyl scavengers in
aldehyde–bisulfite adducts, while the efficiency of naturally produced bisulfites by yeast is controversial (Guido, 2005). In addition, antioxidant activity of melanoidins has been mentioned in previous studies (Papetti et al., 2006), but melanoidins or their precursors may also exhibit pro-oxidative properties as indicated by their involvement in the oxidation of alcohols to aldehydes during beer storage (Martins et al., 2001). Therefore, this overview aims to clarify the contributions of endogenous antioxidants to antioxidant activities of beers, which helps us to better understand beer flavor stability.
ENDOGENOUS ANTIOXIDANTS IN BEERS Phenolic Compounds Beer is generally considered to be one of the major sources of phenolic compounds, and the presence of these compounds contributes to colloidal, foam, flavor, color, and sensory properties of beer. Beers of various types and brands show similar phenolic profiles, but significant variations exist in the total and individual phenolics content due to various materials and brewing processes used (Table 2.1). The values of total phenolic content (TPC) in beers examined by the Folin–Ciocalteu assay usually exceeded 100 mg gallic acid equivalents (GAE)/l (Table 2.1), while the sums of individual phenolics content determined by high-performance liquid chromatography (HPLC) were in the range of 4.47 to 15.50 mg/l (Zhao et al., 2010). The considerable difference in both assays for evaluating of the content of phenolic compounds is because the Folin–Ciocalteu method is not specific for phenolic compounds and does suffer interference from other compounds, and HPLC only reflects the sum content of limited or detectable phenolic species. The phenolic compounds in beer mainly are phenolic acids and flavonoids (Zhao et al., 2010; Piazzon et al., 2010; Gorinstein et al., 2000; McMurrough et al., 1996). The main phenolic acids, flavanol and isoflavonoid, identified in beers are gallic and ferulic acids, (+)-catechin, and formononetin, respectively, but p-coumaric, caffeic, ferulic, sinapic, chlorogenic, p-hydroxybenzoic, and vanillic acids, (−)-epicatechin and their dimers, and the isoflavonoids of genistein, daidzein, and biochanin A have also been detected in beers (Table 2.1). Several research studies indicated that most dimers in beer were procyanidins B3 whilst most trimers were prodelphinidins (Callemien and Collin 2008; Callemien et al., 2008). The brewing materials of malt and hop contain various phenolic acids and flavonoids, which are partially recovered in the final beer. Callemien and Collin (2010) reviewed the phenolic compounds in malts and hop, and found that the levels of p-coumaric, caffeic,
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
TABLE 2.1 Phenolic Compounds in Beers Phenolic Acids (mg/l) Gallic Acid
Protocatechuic Acid
Vanillic Acid
Caffeic Acid
Syringic Acid
p-Coumaric Acid
Ferulic Acid
4-Hydroxyphenylacetic Acid
Sinapic Acid
Chlorogenic Acid
Total phenolic content (TPC)
Lager
1.81–10.39a
0.02–1.30a,c
0.22–2.98a,b,c
0.08–1.22a,b,c
0.06–0.99a,b
0.01–1.12a,b,c
0.51–3.1a,b,c
0.27–0.61b,c
0.20–0.84b,c
0.21–0.26c
138.0–476.2a,b,e,f,g
Ale
—
—
0.61
0.14
0.20
0.58
1.54
0.53
0.29
—
280.10–563.0b,e,h
Pilsner
—
—
0.56
0.14
0.27
0.76
1.03
0.69
0.44
—
484b
Wheat
—
—
0.67
0.19
0.35
0.26
0.34
0.52
0.55
—
270.1–366.0b,e
Abbey
—
—
0.65
0.25
0.22
0.23
0.92
0.59
0.34
—
622b
Bock
—
—
0.81
0.17
0.22
0.72
2.00
0.81
0.45
—
875b
Dealcoholized
—
—
0.45
0.14
0.16
0.40
1.40
0.32
0.26
—
125.0–366b,e
Dark
—
—
—
—
—
—
—
—
—
—
333.6–600e,f
Flavanoids
Lager
(+)-Catechin
(−)-Epicatechin
(−)-Gallocatechin
(−)-Epigallocatechin
Procyanidin Dimers B2
Procyanidin Dimers B3
Prodelphinidin B3
Genistein
Biochanin A
Daidzein
Formononetin
0.03–4.00a,i
0.02–0.73a,i
0.1d,i
0.1d,i
0.16i
0–3.6i,j
0–4.5j
0.05–1.82k
0.15–1.38k
0.08–0.65k
0.32–4.02k
Endogenous Antioxidants in Beers
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
Beer Type
aZhao
et al. (2010) et al. (2010) cNardini and Ghiselli (2004) dCallemien et al. (2008) eGorjanović et al.(2010) fLugasi (2003) gMartinez-Periñan et al. (2011) hGranato et al. (2011) ide Pascual-Teresa et al. (2000) jAndersen et al.(2000) kLapcík et al. (1998). bPiazzon
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ferulic, sinapic, and chlorogenic acids in malt reached ppm levels, while p-coumaric, caffeic, and ferulic acids in hop was more than 10 ppm in most cases. Most phenolic acids in malt or hop are esterified with starch and other polysaccharides by forming bridges or cross-links through hydrogen bonds, chelation, or covalent bonds. These phenolic acids bound with starch can be released by enzymatic hydrolysis of starch during mashing, which causes significantly higher levels of bound phenolic acids than those of free forms in raw materials and final beers. In addition, malt and hop also are an excellent source of catechins, proanthocyanidins and flavonoid oligomers. (+)-Catechin in hop dried cones or pellets can reach 2821 mg/kg (Callemien and Collin, 2010), while the major monomeric and dimeric flavan3-ol in malts identified as (+)-catechin, prodelphinidin B3 and procyanidin B3 were found to be at values of 6-16 mg/kg, 99–175 mg/kg and 9–85 mg/kg, respectively (Dvorakova et al., 2008). The phenolic compounds content in beer is beer-type dependent. Piazzon et al. (2010) reported that the content of phenolic compounds increased in the order: dealcoholized < lager< pilsner < wheat < ale < abbey < bock. Vinson et al. (2003) also observed that total polyphenols in ales/ porters/stouts beers were higher than those in lager and nonalcoholic beers. However, other research indicated
that although higher TPC was found in dark beers, its difference between dark and lager beers was not statistically different (Lugasi, 2003). The higher phenolic compounds in dark, strong beers is because these beers are brewed from wort with higher extract content, while alcohol-free beers are usually brewed with lower original wort extract and inhibition of alcohol formation. Moreover, beers with higher original gravity also generally showed higher TPC (> 180 mg GAE/l), further indicating that phenolic compounds in beer mainly originate from barley malt and hops, although the brewing process also has some impact on phenolic compound content (Zhao et al., 2010).
Melanoidins Melanoidins are macromolecular, nitrogenous, and brown-colored final products of the Maillard reactions between reducing sugars and proteins or amino acids, which are formed during malt kilning, mashing and wort boiling, and are retained at least partially up to the final beer (Kuntcheva and Obretenov, 1996; Rivero et al., 2005). Melanoidins are generally formed by cyclizations, dehydrations, retroaldolizations, rearrangements, isomerizations, and condensations of initial Maillard reaction products (Figure 2.1), but only very few structures have been identified completely (Martins et al., 2001).
+ amino compound
+ H2O
N-substituted glycosylamine
Aldose Sugar
Amadori rearrangement
Amadori rearrangement product (ARP) 1-amino-1-deoxy-2-ketose -2H O
> pH 7
> pH 7
Reductones -2H
Fission products (acetol, diacetyl,pyruvaldehyde, etc)
+2H
-3H O
pH 7
Schiff’s base of hydroxymethylfurfural (HMF) or furfural
Dehydroreductones + amino compound -CO
Strecker degradation
+H O - amino compound
-amino acid
Aldehydes + amino compound
(HMF) or furfural Aldols and N-free polymers
+ amino compound
Aldimines and Ketimes
FIGURE Melanoidins (brown nitrogenous polymers)
2.1 Maillard reaction scheme. Adapted from Martins et al., (2001).
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
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Endogenous Antioxidants in Beers
Sulfites Sulfites, one of important constituents of beer, are produced by yeast as by-products of the synthesis of sulfur-containing amino acids during fermentation and survive into the finished beer. The raw materials used in brewing may contain different amounts of sulfur dioxide but most of it disappears during wort boiling. Sulfites in beer are not only important antioxidants to prevent beer from oxidative staling, but they also act as important mask agents for stale flavor by reacting with carbonyl staling compounds in beer to form bisulfite–carbonyl adducts, which is important for beer flavor stability. The differences in brewer’s yeast strain and brewing process lead to significant variations in sulfite content of final beers. The total concentration of SO2 typically
ranged from 1 to 30 mg/l (Illet, 1995). For example, the total SO2 concentrations determined by the p-rosaniline method and the chronopotentiometric method in beers were found in the range of 1.15–37.32 mg/l and 0–4.1 mg/l, respectively (Zhao et al., 2013; Dvořák et al., 2006). Almeida et al. (2003) reported that the levels of free and total sulfur dioxide in beers determined by a proposed voltammetric methods were in the ranges of 0–3.0 mg/l and 8.1–14.3 mg/l, respectively. The production of sulfites in beer is affected by yeast strain, fermentation conditions, and wort components. Saison et al. (2009) observed that sulfite production in beers appeared to be yeast strain dependent (Figure 2.2), but their content was quite low (2.44–3.91 mg/l) and their differences were small. Moreover, lager beers contained substantially higher levels of SO2 than the ale because lager yeast strains produce more SO2 than ale strains (Bushnell et al., 2003). Over-production of sulfites by yeast can occur when the brewing yeast strain is restricted in growth during fermentation because endogenous sulfites in beer are produced from sulfates in the wort by the brewing yeast strain during synthesis of hydrogen sulfide. Thus, all conditions which promote yeast growth will reduce the amount of sulfites excreted in the medium. Guido et al. (2004) examined the effects of the physiological condition of the pitching yeast on sulfites production in beer, and fermentations with the lower yeast cell count secreted higher levels of sulfites (Figure 2.3). In recent decades, modification of characteristics of brewer’s yeast by genetic engineering has been considered to be an efficient measure to control the accumulation of sulfites (Dequin, 2001). Korch et al. (1991) suggested that the overexpression of ATP sulfurylase (MET3) and APS kinase (MET14) in brewer’s yeast could increase sulfites production (Figure 2.4). While Hansen
5
Total SO2 content (mg/l)
Melanoidins contribute to the formation of color, aroma, and flavor, and also play an important role in stabilization of the foam and influence the oxidation–reduction processes of beer due to their strong antioxidant properties and other biological effects (Rivero et al., 2005). The quantitative analysis of melanoidins in beers is very limited; a more than eight-fold difference in melanoidin content, varying from 1.64 mg/1 to 13.71 mg/1, occurred between beers which can be ascribed to the different raw materials and brewing process used during beer brewing (Zhao et al., 2013; Kuntcheva and Obretenov, 1996). The content of melanoidins in beers was influenced by the type of beers, and their content was found to be the highest in dark beer with values of 1.49 g/l, compared with to 0.61 g/l in blond beer and 0.58 g/l for alcohol-free beer (Rivero et al., 2005). The main difference between the types of beers, especially for dark and blond beers, is that they are brewed from malt with diverse grades of toasting, although they are also largely influenced by the technological brewing factors. Indeed, malts contained more melanoidins than barley, and the melanoidins isolated from malt were observed as a group of compounds with high molecular weight (> 3500 Da), brown color, and with a small percentage content of non-amino acidic nitrogen (1.12%) due to the relatively high polysaccharide and low protein content of barley grain and because of the specific reaction conditions of low water activity and lengthy roasting periods applied during the roasting process (Papetti et al., 2006). Most of the molecular weight of such polymeric compounds isolated from food did not exceed 100,000 Da, while the molecular weight of melanoidins formed in roasted barley malt of was found to be up to 200,000 Da (Faist et al., 2002). Moreover, significant differences in the distribution of the nitrogen content in different molecular weights of melanoidins isolated from barley were observed, and its content reached 30% in low-molecular-weight compounds, whereas only 3% was found in the high-molecular-weight polymers (30,000–60000 Da) (Papetti et al., 2006).
4 3 2 1 0 DS05
DS08
DS10
DS12
Yeast strain
FIGURE 2.2 Influence of fermentation with different top-fermenting yeast strains on final total SO2 content in beer. Adapted from Saison et al. (2009).
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
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2. ANTIOXIDANT ACTIVITIES OF BEERS
The total SO 2 content (mg/l)
16
12
8
4
0 1
2
Fermentation
3
4
FIGURE 2.3 The effect of yeast physiological condition on total SO2
content at the end of fermentation. Fermentation 1 and 3: higher vital pitching yeast; fermentation 2 and 4: lower vital pitching yeast. Adapted from Guido et al. (2004). SO42-
SO42MET3 APS MET14 PAPS Serine
MET16
O-Acetylserine
MET10
Homoserine MET2
SO2
MET25
O-Acetylhomoserine H2S
MET25
Homocysteine
Cysteine NHS5 Cystathionine
Methionine
FIGURE 2.4 Pathways for sulfite production in S. cerevisiae. APS, adenosine 5′-phosphosulfate; MET2, serine acetyltransferase; MET3, ATP sulfurylase; MET10, sulfite reductase subunit; MET14, APS kinase; MET16, 3′-phospho adenosine 5′-phosphosulfate (PAPS) reductase; MET25, O-acetylhomoserine or O-acetylserine sulfhydrylase; NHS5, cystathionine β-synthase. Adapted from Dequin, (2001).
and Kielland-Brandt (1996) indicated that inactivation of serine acetyltransferase (MET2) in brewer’s yeast could produce higher amounts of sulfites and hydrogen sulfide, and the inactivation of sulfite reductase
subunit (MET10) was shown to be an efficient strategy for increasing sulfites formation and decreasing hydrogen sulfide production. However, it should be noted that the SO2 level must be controlled at the end of beer production for human health and beer-quality reasons. A maximum level of total SO2 in beer at 20 mg/l was established by the European Union and other countries. The SO2 level in beer must also be controlled during storage because sulfites produced during fermentation influence the flavor staling of finished beer; a content of 8–9 mg/l of sulfites in packaged beer was considered to be the most appropriate for the flavor stability of beer (Narziss et al., 1993).
ANTIOXIDANT ACTIVITIES OF BEERS Antioxidant Activity Assays for Beers Endogenous antioxidants present in beer are complex, and their activities and mechanisms largely depend on the composition and condition of the test system. The results are quite different even when an analysis of the same sample is carried out by each method since the antioxidant activity assays each rely on different chemical backgrounds and reaction mechanisms. Thus, many researchers have stressed the need to perform more than one type of antioxidant activity measurement to evaluate the antioxidant activity of beers. In the past few years, various in vitro methods have been investigated to estimate the antioxidant activity of beers or brewing raw materials. The varieties, principles, analytical performances and characteristics of methods for antioxidant activity evaluation have been reviewed by Pisoschi and Negulescu (2011) and Badarinath et al. (2010). These methods include 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation scavenging activity (ABTS) assay, oxygen radical absorbing capacity (ORAC) assay, superoxide anion radical scavenging activity (SASA), ferric-reducing antioxidant power (FRAP) assay, total radical trapping antioxidant parameter (TRAP), lipid peroxidation inhibition capacity (LPIC) assay, cupric ion reducing antioxidant capacity (CUPRAC) assay, metal-chelating activity (MCA) assay, hydroxyl radical averting capacity (HORAC) assay, potassium ferricyanide reducing power (PFRAP) method, enhanced chemiluminescence (ECL), and ESR. The various analytical methods for antioxidant activity evaluation fall into distinct categories, which involve different reaction mechanisms, reaction conditions, and analytical techniques (Table 2.2). ORAC, LPIC, TRAP, ABTS, and SASA are classified as hydrogen atom transfer methods of monitoring competitive kinetic reactions,
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
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Antioxidant Activities of Beers
TABLE 2.2 Antioxidant Activity Assays for Beers Antioxidant Activity Assays
Type of Method
Principle of Method
End-Product Determination
ORAC
Hydrogen atom transfer
Antioxidant reaction with peroxyl radicals, induced by AAPH
Loss of fluorescence of fluorescein
LPIC
Hydrogen atom transfer
Antioxidant to inhibit peroxidation of lipids induced by a Fenton-like system
Colorimetry
TRAP
Hydrogen atom transfer
Antioxidant capacity to scavenge luminol-derived radicals, generated from AAPH decomposition
Chemiluminescence quenching
SASA
Hydrogen atom transfer
Antioxidant to scavenge superoxide anion radical formation by alkaline or enzyme
Colorimetry
ABTS
Hydrogen atom transfer
Antioxidant to scavenge an organic cation radical
Colorimetry
HORAC
Hydrogen atom transfer
Antioxidant capacity to quench OH radicals generated by a Co(II) based Fenton-like system
Loss of fluorescence of Fluorescein
CUPRAC
Electron transfer
Cu(II) reduction to Cu(I) by antioxidants
Colorimetry
FRAP
Electron transfer
Antioxidant reaction with a Fe(III) complex
Colorimetry
DPPH
Electron transfer
Antioxidant reaction with an organic radical
Colorimetry
PFRAP
Electron transfer
Potassium ferricyanide reduction by antioxidants and subsequent reaction of potassium ferrocyanide with Fe3+
Colorimetry
MCA
Other
Chelating ferrous ions with ferrozine
Colorimetry
ECL
Other
Emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength
Recording of fluorescence excitation/emission spectra
ESR
Other
Free radicals by electron spin resonance
Recording the time of occurring of free radicals
AAPH, 2,2′-azinobis (2-amidinopropane) dihydrochloride; ABTS, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation scavenging activity assay; CUPRAC, cupric ion reducing antioxidant capacity assay; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay; ECL, enhanced chemiluminescence; ESR, electron spin resonance; FRAP, ferric-reducing antioxidant power assay; HORAC, hydroxyl radical averting capacity assay; LPIC, lipid peroxidation inhibition capacity assay; MCA, metal-chelating activity assay; ORAC, oxygen radical-absorbing capacity assay; PFRAP, potassium ferricyanide reducing power method; SASA, superoxide anion radical scavenging activity; TRAP, total radical trapping antioxidant parameter
while FRAP, DPPH, CUPRAC, HORAC, and PFRAP are grouped together as the electron transfer methods of a reduction reaction, and others such as MCA, ECL, and ESR are categorized as other methods. For routine evaluation of the antioxidant activity of beers or monitoring the brewing process with respect to antioxidant activity, spectrometry analytical techniques such as DPPH, ABTS, ORAC, FRAP, TRAP, etc. should be selected due to their better sensitivity, convenience, and short assay times.
Antioxidant Activities of Beers Evaluated by Different Assays As mentioned above, beer contains numerous endogenous antioxidants, which endows beer with significant antioxidant activity. Zhao et al., (2010) observed significant antioxidant activities in lager beers, suggesting that overall antioxidant activity evaluation results for beer samples using DPPH, ABTS, SASA, FRAP, MCA assays were consistent even though these assays involved
different reaction mechanisms. Another study performed by the same group indicated that all 40 beers showed significant antioxidant activity evaluated by DPPH, ABTS, ORAC, FRAP, MCA assays, but clear discrimination among beer samples with significantly different antioxidant activity was achieved by principal component analysis (Zhao et al., 2013). The antioxidant activity of 27 beers was tested by TRAP, TEAC, DPPH, FRAP, CUPRAC, and ORAC assays, and the results pointed out that all beers displayed antioxidant activities, although the values varied a lot with the sample, method, and standard (Tafulo et al., 2010). Similarly, Piazzon et al. (2010), Granato et al. (2011), and Lugasi (2003) also found that all beers showed antioxidant activity measured with various assays, but a wide range of antioxidant activity was observed. There are significant differences in antioxidant activity among different commercial beer types. Piazzon et al. (2010) reported that antioxidant activities decreased in the order: bock > abbey > ale > wheat > pilsner > lager > dealcoholized, and the antioxidant activities of bock beers
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
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2. ANTIOXIDANT ACTIVITIES OF BEERS
were found to be about 3 times higher with respect to dealcoholized beers. In a comparative study, lager-type beers were found have lower antioxidant activity than the ale ones, and Portuguese beers showed slightly lower antioxidant activity then Belgian ones, regardless of the method or standard (Tafulo et al., 2010). Granato et al. (2011) also examined the antioxidant activity of Brazilian lager and brown ale beers using ORAC and DPPH assays: higher antioxidant activity in brown ale beers was observed. However, another study performed by Lugasi (2003) indicated that there were no significant differences in antioxidant activity between lager and dark beers. It should also be noted that the differences in antioxidant activity across beer samples and types were due to the different parameters during brewing, such as the variety of barley and hop, the malting and mashing process, and the yeast fermentation employed.
Relationships between Endogenous Antioxidants and Antioxidant Activities of Beers All endogenous antioxidants in beers may contribute to some extent to the overall antioxidant activities of beers although present in beer at very low levels. However, the real contributions of these antioxidants to antioxidant activities of beers have not been clarified yet.
Contributions of Phenolic Compounds to Antioxidant Activities of Beers Most research has suggested that phenolic compounds are the most important antioxidants in beers. In addition to acting as chelating agents of metallic catalysts, phenolic compounds in beer have another indirect role in the antioxidant capacity due to their reversible interaction with sulfite (Illet, 1995). Our previous study observed that phenolic compounds including ferulic acid, syringic acid, (+)-catechin, caffeic acid, protocatechuic acid, and (−)-epicatechin contributed 55.0–88.1% of beer antioxidant activity evaluated by different assays. McMurrough et al. (1996) also found that partial removal of the polyphenol fraction by polyvinylpolypyrrolidone treatment diminished the reducing power by 9–38%. Piazzon et al. (2010) observed a strong correlation between FRAP values and total phenolic acids content of beers and a lack of correlation between FRAP values and the content of free phenolic acids, which indicated that conjugated forms of phenolic acids in beer were responsible for the antioxidant activity of beer. Granato et al. (2011) reported that the content of flavonoids and total phenolic compounds seemed to be strongly correlated with the antioxidant activity measured by ORAC and DPPH assays. TPC in the 40 commercial beers were also found to give strong correlations with DPPH, ABTS, ORAC, MCA, and FRAP assays tested, which strongly suggested that
phenolic compounds were mainly responsible for the majority of the antioxidant activity of beer (Zhao et al., 2013). However, some phenolic compounds can also act as pro-oxidants by transferring electrons to metal ions, and the levels of ferulic acid in beer were found to determine whether it is pro-oxidant or anti-oxidant (Walters et al., 1997). Thus, some controversy concerning the relevance of polyphenolic antioxidants in beer has been reported in the literature. In an ESR lag phase study, phenolic compounds such as phenolic acids, catechin, epicatechin, and proanthocyanidin dimers had no effect on the formation of radicals, and they are neither antioxidants nor pro-oxidants (Andersen et al., 2000). A Laccase–Sonogel–Carbon biosensor method proposed by Martinez-Periñan et al. (2011) for beer stability evaluation showed that the polyphenols cannot delay the formation of free radicals in beers, and polyphenols have an indirect role in beer aging by avoiding rapid SO2 loss.
Contributions of Melanoidins to Antioxidant Activities of Beers Maillard reaction products acting as antioxidants generally are involved in various mechanisms, and include: oxygen, reactive oxygen, peroxyl, or some specific stable radicals scavengers, reducing agents, and metal chelating agents. The overall effects of Maillard and caramelization products on the oxidative stability of beer are unknown. However, traditionally, the use of colored malt is known to improve the stability of the finished beer, and it has also been shown that more-highly colored beers retain a greater reducing power during storage (Coghe et al., 2003). The final beer produced has a reducing power mainly dependent on melanoidin compounds and simple and polymerized polyphenols. Indeed, positive correlations between antioxidant activity and malt color also have been observed, which have been ascribed to the presence of Maillard components (Coghe et al., 2003; W offenden et al., 2001). Moreover, the radical-scavenging and iron-chelating ability of melanoidins from dark beer has been found to be higher compared to melanoidins from pale beers (Coghe et al., 2003; Morales et al., 2005; Morales and Jiménez-Pérez, 2004). Furthermore, Woffenden et al. (2002) reported that correlations between levels of catechin or ferrulic acid and antioxidant activity are poor, suggesting that other components, such as polyphenols, oligomers, and Maillard reaction products also contribute to the total antioxidant activity of beer. Zhao et al. (2013) also indicated significant positive correlations between melanoidins content in beers and DPPH, FRAP, as well as MCA, and their content made an 8% contribution to ORAC of beers. But Rivero et al. (2005) concluded that the hydrogen-donating ability of beers is dependent on the total polyphenol levels but not on the total melanoidin content. Hydroxyl
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
References
radical-scavenging activity of beer melanoidins was found to be unrelated to their color, suggesting that colored structures were not involved in such antioxidant effects (Morales, 2009). Although melanoidins are known to act as antioxidants, some reports have also indicated a pro-oxidant activity. Pro-oxidative activity of melanoidins has mainly been established for low-molecular-weight melanoidins p roduced from model systems (Ames, 2001). A pro-oxidative effect of stout also has been observed in studies with stout added to lager beer analyzed by ESR spectroscopy (Nøddekær and Andersen 2007). Also, beers produced with dark malts kilned at high temperatures formed high concentrations of radicals, and also had a lower oxidative stability as compared to using paler malts (Cortés et al., 2010). This might be because the pro-oxidative effect of Maillard reaction products was caused by reactions that were able to increase radical levels by other mechanisms than Fenton-catalysis since stout was found to decrease the level of radicals in a beer model system where radical formation was induced by the Fenton reaction (Nøddekær and Andersen, 2007).
Contributions of Sulfites to Antioxidant Activities of Beers Sulfites formed in beer have been considered as one of the most effective antioxidants for beer flavor stability. Two main mechanisms for sulfites to control beer stabilization have been proposed: one is to inhibit beer oxidation during storage by acting as an antioxidant, the other is to combine with several carbonyls to produce sulfite adducts in beer and so mask staling flavor since the adducts are ionized and thus non-volatile. According to Andersen et al. (2000), sulfites were the most efficient naturally occurring antioxidants in beer, while thiols and ascorbic acid were shown to be pro-oxidants. Other authors obtained similar results studying flavor stability using ESR to detect free radicals, which supported the theory that phenolic compounds did not contribute to the antioxidant capacity but SO2 did increase the lag time (Foster et al., 1999). In another study performed by Martinez-Periñan et al. (2011), only SO2 concentration showed relevant differences after the ageing process, thus, it was responsible for the antioxidant capacity of beers. Our study by stepwise regression analysis also indicated that total SO2 content contributed 5% of the antioxidant activity of beer evaluated by reducing power assay. However, no significant correlations were found between SO2 and all five antioxidant activity assays used in this study (Zhao et al. 2013). Moreover, the effects of sulfites on beer flavor stability evaluated by various assays have shown that sulfites in beer are not only one of the important antioxidants but also one of the important masking agents; however, the efficiency
23
of naturally produced bisulfite by yeast was controversial (Guido, 2005).
Acknowledgments I gratefully acknowledge the National Natural Science Foundation of China (No. 31000810), the Key Technology R&D Program of Guangdong Province (Nos. 2012A080107005 and 2011A020102001) and the Fundamental Research Funds for the Central Universities (No. 2012ZM0069) for their financial support.
References Almeida, P.J., Rodrigues, J.A., Guido, L.F., Santos, J.R., Barros, A.A., Fogg, A.G., 2003. Voltammetric determination of free and total sulfur dioxide in beer. Electroanal 15, 587–590. Ames, J.M., 2001. Melanoidins as pro- or antioxidants. Cerevisia 26, 210–216. Andersen, M.L., Outtrup, H., Skibsted, L.H., 2000. Potential antioxidants in beer assessed by ESR spin trapping. J. Agric. Food Chem. 48, 3106–3111. Badarinath, A.V., RAo, K.M., Chetty, C.M.S., Ramkanth, S., Rajan, T.V.S., Gnanaprakash, K., 2010. A Review on In-vitro Antioxidant Methods: Comparisions, Correlations and Considerations. Int. J. PharmTech Res. 2, 1276–1285. Bamforth, C.W., 2000. Beer quality: oxidation. Brewer’s Guardian 4, 31–34. Bushnell, S.E., Guinard, J.X., Bamforth, C.W., 2003. Effects of sulfur dioxide and polyvinylpolypyrrolidone on the flavor stability of beer as measured by sensory and chemical analysis. J. Am. Soc. Brew. Chem. 61, 133–141. Callemien, D., Collin, S., 2008. Use of RP-HPLC-ESI(−)-MS/MS to differentiate various proanthocyanidin isomers in lager beer extracts. J. Am. Soc. Brew. Chem. 66, 109–115. Callemien, D., Collin, S., 2010. Structure, organoleptic properties, quantification methods, and stability of phenolic compounds in beer — a review. Food Rev. Int. 26, 1–84. Callemien, D., Guyot, S., Collin, S., 2008. Use of thiolysis hyphenated to RP-HPLC-ESI(−)-MS/MS for the analysis of flavanoids of fresh lager beers. Food Chem. 110, 1012–1018. Coghe, S., Vanderhaegen, B., Pelgrims, B., Basteyns, A.V., Delvaux, F.R., 2003. Characterization of dark specialty malts: New insights in color evaluation and pro- and antioxidative activity. J. Am. Soc. Brew. Chem. 61, 125–132. Cortés, N., Kunz, T., Suárez, A.F., Hughes, P., Methner, F.J., 2010. Development and correlation between the organic radical concentration in different malt types and oxidative beer stability. J. Am. Soc. Brew. Chem. 68, 107–113. de Pascual-Teresa, S., Santos-Buelga, C., Rivas-Gonzalo, J.C., 2000. Quantitative analysis of flavan-3-ols in Spanish foodstuffs and beverages. J. Agric. Food Chem. 48, 5331–5337. Dequin, S., 2001. The potential of genetic engineering for improving brewing, wine-making and baking yeasts. Applied Microbiol. Biot. 56, 577–588. Dvořák, J., Dostálek, P., Štěrba, K., Čejka, P., Kellner, V., Čulík, J., Beinrohr, E., 2006. Determination of total sulphur dioxide in beer samples by flow-through chronopotentiometry. J. Inst. Brew. 112, 308–313. Dvorakova, M., Moreira, M.M., Dostalek, P., Skulilova, Z., Guido, L.F., Barros, A.A., 2008. Characterization of monomeric and oligomeric flavan-3-ols from barley and malt by liquid chromatography–ultraviolet detection–electrospray ionization mass spectrometry. J. C hromatogr. A. 1189, 398–405.
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Faist, V., Lindenmeier, M., Geisler, C., Erbersdobler, H.F., Hofmann, T., 2002. Influence of molecular weight fractions isolated from roasted malt on the enzyme activities of NADPH‒cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells. J. Agric. Food Chem. 50, 602–606. Foster, C., Schwieger, J., Narzib, L., Back, W., Uchida, M., Ono, M., Yanagi, K., 1999. Monatsschr. Brauwiss 5/6, 86–93. Gorinstein, S., Caspi, A., Zemser, M., Trakhtenberg, S., 2000. Comparative contents of some phenolics in beer, red and white wines. Nutr. Res. 20, 131–139. Gorjanović, S.Z., Novaković, M.M., Potkonjak, N.S., LeskošekCukalović, I., Sužnjević, D.Z., 2010. Application of a novel antioxidative assay in beer analysis and brewing process monitoring. J. Agric. Food Chem. 58, 744–751. Granato, D., Branco, G.F., Faria, J.A.F., Cruzb, A.G., 2011. Characterization of Brazilian lager and brown ale beers based on color, phenolic compounds, and antioxidant activity using chemometrics. J. Sci. Food Agric. 91, 563–571. Guido, L.F., 2005. How do sulfites help to control beer ageing. Cerevisia 30, 132–138. Guido, L.F., Rodrigues, P.G., Rodrigues, J.A., Gonçalves, C.R., Barros, A.A., 2004. The impact of the physiological condition of the pitching yeast on beer flavour stability: an industrial approach. Food Chem. 87, 187–193. Hansen, J., Kielland-Brandt, M., 1996. Inactivation of MET10 in brewer’s yeast specifically increases SO2 formation during beer production. Nature Biotechnol. 14, 1587–1589. Illet, D.R., 1995. Aspects of the analysis, role, and fate of sulphur dioxide in beer – A review. Tech. Q. Master Brew. Assoc. Am. 32, 213–221. Korch, C., Moutain, H.A., Gyllang, H., Winge, M., Brehmer, P., 1991. A mechanism for sulphite production in beer and how to increase sulphite levels by recombinant genetics. In: Brewing Convention, European (Ed.), The proceedings of the 23rd Congress of the European Brewery Convention. IRL Press, Eynsham, pp. 201–208. Kuntcheva, M.J., Obretenov, T.D., 1996. Isolation and characterization of melanoidins from beer. Z. Lebensm.Unters. F. A. 202, 238–243. Lapcík, O., Hill, M., Hampl, R., Wähälä, K., Adlercreutz, H., 1998. Identification of isoflavonoids in beer. Steroids 63, 14–20. Lugasi, A., 2003. Polyphenol content and antioxidant properties of beer. Acta. Aliment. 32, 181–192. Martinez-Periñan, E., Hernández-Artiga, M.P., Palacios-Santander, J.M., ElKaoutita, M., Naranjo-Rodriguez, I., Bellido-Milla, D., 2011. Estimation of beer stability by sulphur dioxide and polyphenol determination. evaluation of a Laccase–Sonogel–Carbon biosensor. Food Chem. 127, 234–239. Martins, S.I.F.S., Jongen, W.M.F., van Boekel, M.A.J.S., 2001. A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci. Technol. 11, 364–373. McMurrough, I., Madigan, D., Kelly, R.J., Smyth, M.R., 1996. The role of flavanoid polyphenols in beer stability. J. Am. Soc. Brew. Chem. 54, 141–148. Morales, F.J., 2009. Non-specific hydroxyl radical scavenging properties of melanoidins from beer. In: Preedy, V.R. (Ed.), Beer in health and disease prevention. Elsevier Inc., San Diego, pp. 765–774.
Morales, F.J., Fernández-Fraguas, C., Jiménez-Pérez, S., 2005. Ironbinding ability of melanoidins from food and model systems. Food Chem. 90, 821–827. Morales, F.J., Jiménez-Pérez, S., 2004. Peroxyl radical scavenging activity of melanoidins in aqueous systems. Eur. Food Res. Technol. 218, 515–520. Nardini, M., Ghiselli, A., 2004. Determination of free and bound phenolic acids in beer. Food Chem. 84, 137–143. Narziss, L., Miedaner, H., Graf, H., Eichhorn, P., Lustig, S., 1993. Technological approach to improve flavour stability. Tech. Q. Master Brew. Assoc. Am. 30, 48–53. Nøddekær, T.V., Andersen, M.L., 2007. Effects of Maillard and caramelization products on oxidative reactions in lager beer. J. Am. Soc. Brew. Chem. 65, 15–20. Papetti, A., Daglia, M., Aceti, C., Quaglia, M., Gregotti, C., Gazzani, G., 2006. Isolation of an in vitro and ex vivo antiradical melanoidin from roasted barley. J. Agric. Food Chem. 54, 1209–1216. Piazzon, A., Forte, M., Nardini, M., 2010. Characterization of phenolic content and antioxidant activity of different beer types. J. Agric. Food Chem. 58, 10677–10683. Pisoschi, A.M., Negulescu, G.P., 2011. Methods for total antioxidant activity determination: a review. Biochem. Anal. Biochem. 1, 1–10. Rivero, D., Pérez-Magariño, S., González-Sanjosé, M.L., Valls-Belles, V., Codoñer, P., Muñiz, P., 2005. Inhibition of induced DNA oxidative damage by beers: Correlation with the content of polyphenols and melanoidins. J. Agric. Food Chem. 53, 3637–3642. Saison, D., De Schutter, D.P., Overlaet-Michiels, W., Delvaux, F., Delvaux, F.R., 2009. Effect of fermentation conditions on staling indicators in beer. J. Am. Soc. Brew. Chem. 67, 222–228. Tafulo, P.A.R., Queirós, R.B., Delerue-Matos, C.M., Sales, M.G.F., 2010. Control and comparison of the antioxidant capacity of beers. Food Res. Int. 43, 1702–1709. Vinson, J.A., Mandarano, M., Hirst, M., Trevithick, J.R., Bose, P., 2003. Phenol antioxidant quantity and quality in foods: beers and the effect of two types of beer on an animal model of atherosclerosis. J. Agric. Food Chem. 51, 5528–5533. Walters, M.T., Heasman, A.P., Hughes, P.S., 1997. Comparison of (+)-catechin and ferulic acid as natural antioxidants and their impact on beer flavor stability. 1. Forced-aging. J. Am. Soc. Brew. Chem. 55, 83–89. Woffenden, H.M., Ames, J.M., Chandra, S., 2001. Relationships between antioxidant activity, color, and flavor compounds of crystal malt extracts. J. Agric. Food Chem. 49, 5524–5530. Woffenden, H.M., Ames, J.A., Chandra, S., Anese, M., Nicoli, M.C., 2002. Effect of kilning on the antioxidant and pro-oxidant activities on pale malt. J. Agric. Food Chem. 50, 4925–4933. Zhao, H., Chen, W., Lu, J., Zhao, M., 2010. Phenolic profiles and antioxidant activities of commercial beers. Food Chem. 119, 1150–1158. Zhao, H., Li, H., Sun, G., Yang, B., Zhao, M., 2013. Assessment of endogenous antioxidative compounds and antioxidant activities of lager beers. J. Sci. Food Agric. 93, 910–917.
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Endogenous Antioxidants and Antioxidant Activities of Beers Haifeng Zhao College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China
significantly different contributions to the antioxidant activities of beers. • Optimization of the brewing process and screening of raw materials are considered as efficient measures to increase the antioxidant activities of beers, which is extremely important to beer flavor stability. Knowledge of the relationships between • endogenous antioxidants and antioxidant activities of beers can be used to develop appropriate technologically innovative processes to control the flavor stability or to reinforce the nutritional properties of beer.
CHAPTER POINTS • P henolic compounds, Maillard reaction products, and sulfites constitute the major endogenous antioxidants in beers, although other antioxidants such as reducing sugars, vitamins, carotenoids and chelating agents also exist in beer at very low levels. • Endogenous antioxidants in beers originate from brewing raw materials or are formed during the brewing process; all of them make significant contributions to beer flavor stability and nutritional properties due to their antioxidant and antiradical properties, and other biological effects. • The beneficial impacts of these antioxidant compounds on the stability of beer flavor have been demonstrated, but there has been a lack of consensus concerning the effectiveness of these compounds as potential antioxidants. The antioxidant or pro-oxidant activity of these compounds is determined partly by their content in beers. • The content of endogenous antioxidants and antioxidant activities of beers are beer brand and type dependent. Generally, dark or brown beers contain higher levels of antioxidants and show higher antioxidant activity than pale or lager beers, because the former are brewed from worts with higher extracts or supplemented with some toast malts. • Due to the different reaction mechanisms and antioxidant activity assays involved, various kinds of endogenous antioxidants make
Processing and Impact on Antioxidants in Beverages http://dx.doi.org/10.1016/B978-0-12-404738-9.00002-7
INTRODUCTION Beer is one of the most widely consumed alcoholic beverages in the world for its fresh taste, low calories, and nutritional value. The main raw materials of beers are water, malt, hops, and brewer’s yeast, and some beers also are brewed with some non-malted cereals, starch, or starch syrups as adjuncts. The production of beer involves extremely complicated processes and chemical and biochemical reactions. Thus, the final beer contains various compounds with antioxidant activity mainly originated from raw materials or formed during processing. The species and concentrations of these antioxidants in final beer varies, largely because brewing technology, raw materials, and yeast applied in the brewing can differ Slight changes in structure or conformation of these compounds can cause significant changes of the antioxidant activity, which alters the overall oxidative or flavor stability of beer. Nowadays, flavor stability has become
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© 2014 Elsevier Inc. All rights reserved.
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2. ANTIOXIDANT ACTIVITIES OF BEERS
the most important factor in determining the shelf-life of packaged beer, and prolonging shelf-life by delaying flavor staling is one of the greatest challenges facing the brewer. Although the flavor stability of beer depends primarily on the oxygen content of the packaged beer, the brewing process and the raw materials used can influence the flavor stability. Therefore, attention is now increasingly shifting towards increasing the antioxidant activity of beer itself due to the fact that oxidative staling of beer is still noticeable even if the level of total packaged oxygen might be as low as 0.1 mg/l (Bamforth, 2000). Antioxidants are generally thought to play a significant role in malting, mashing and brewing due to their ability to delay or prevent oxidation reactions and oxygen free radical reactions. Some synthetic antioxidants such as sulfites, formaldehyde, or ascorbate, can be added into the brewing process to improve beer flavor stability, but the effectiveness of these compounds is in doubt (Andersen et al., 2000). In recent years, there has been a general trend toward minimizing the use of additives in beer production because of consumer demand and stiffening regulations. As a result, it is desirable to prevent oxidation by protection of endogenous antioxidants in beer and in its raw materials, or those formed during the processes of kilning, boiling, and fermentation (mainly Maillard reaction products and sulfites). These endogenous antioxidants can offer an overall antioxidant activity in the beer to increase its stability during storage. This issue – to keep or increase the antioxidant activity of beer so as to improve its flavor stability – is greatly recognized by consumers and brewers. Beer generally contains a range of antioxidants, such as phenolic compounds, Maillard reaction products (melanoidins and reductones), sulfites, thiols, non-fermentable reducing sugars, vitamins, carotenoids, and chelating agents. Among the antioxidants mentioned above, phenolic compounds, melanoidins, and sulfites are of particular interest to brewers because they are described as a ntioxidants that possess antioxidant and antiradical properties as well as other biological effects (Zhao et al., 2013). The beneficial impacts of these antioxidant compounds on the stability of beer flavor have been demonstrated, whereas there has been a lack of consensus concerning the effectiveness of these compounds as potential antioxidants. For instance, results from electron spin resonance (ESR) lag phase studies showed that there were no significant effects of polyphenols on the formation of free radicals in beer during storage or in wort during brewing, while the author’s group’s previous study showed that phenolic compounds investigated made up 55.0–88.1% of the antioxidant activity of beer (Andersen et al., 2000; Zhao et al., 2010). Moreover, although the effects of sulfites on beer flavor stability evaluated by various assays have shown that sulfites stabilize the flavor in two ways, as antioxidants and as carbonyl scavengers in
aldehyde–bisulfite adducts, while the efficiency of naturally produced bisulfites by yeast is controversial (Guido, 2005). In addition, antioxidant activity of melanoidins has been mentioned in previous studies (Papetti et al., 2006), but melanoidins or their precursors may also exhibit pro-oxidative properties as indicated by their involvement in the oxidation of alcohols to aldehydes during beer storage (Martins et al., 2001). Therefore, this overview aims to clarify the contributions of endogenous antioxidants to antioxidant activities of beers, which helps us to better understand beer flavor stability.
ENDOGENOUS ANTIOXIDANTS IN BEERS Phenolic Compounds Beer is generally considered to be one of the major sources of phenolic compounds, and the presence of these compounds contributes to colloidal, foam, flavor, color, and sensory properties of beer. Beers of various types and brands show similar phenolic profiles, but significant variations exist in the total and individual phenolics content due to various materials and brewing processes used (Table 2.1). The values of total phenolic content (TPC) in beers examined by the Folin–Ciocalteu assay usually exceeded 100 mg gallic acid equivalents (GAE)/l (Table 2.1), while the sums of individual phenolics content determined by high-performance liquid chromatography (HPLC) were in the range of 4.47 to 15.50 mg/l (Zhao et al., 2010). The considerable difference in both assays for evaluating of the content of phenolic compounds is because the Folin–Ciocalteu method is not specific for phenolic compounds and does suffer interference from other compounds, and HPLC only reflects the sum content of limited or detectable phenolic species. The phenolic compounds in beer mainly are phenolic acids and flavonoids (Zhao et al., 2010; Piazzon et al., 2010; Gorinstein et al., 2000; McMurrough et al., 1996). The main phenolic acids, flavanol and isoflavonoid, identified in beers are gallic and ferulic acids, (+)-catechin, and formononetin, respectively, but p-coumaric, caffeic, ferulic, sinapic, chlorogenic, p-hydroxybenzoic, and vanillic acids, (−)-epicatechin and their dimers, and the isoflavonoids of genistein, daidzein, and biochanin A have also been detected in beers (Table 2.1). Several research studies indicated that most dimers in beer were procyanidins B3 whilst most trimers were prodelphinidins (Callemien and Collin 2008; Callemien et al., 2008). The brewing materials of malt and hop contain various phenolic acids and flavonoids, which are partially recovered in the final beer. Callemien and Collin (2010) reviewed the phenolic compounds in malts and hop, and found that the levels of p-coumaric, caffeic,
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
TABLE 2.1 Phenolic Compounds in Beers Phenolic Acids (mg/l) Gallic Acid
Protocatechuic Acid
Vanillic Acid
Caffeic Acid
Syringic Acid
p-Coumaric Acid
Ferulic Acid
4-Hydroxyphenylacetic Acid
Sinapic Acid
Chlorogenic Acid
Total phenolic content (TPC)
Lager
1.81–10.39a
0.02–1.30a,c
0.22–2.98a,b,c
0.08–1.22a,b,c
0.06–0.99a,b
0.01–1.12a,b,c
0.51–3.1a,b,c
0.27–0.61b,c
0.20–0.84b,c
0.21–0.26c
138.0–476.2a,b,e,f,g
Ale
—
—
0.61
0.14
0.20
0.58
1.54
0.53
0.29
—
280.10–563.0b,e,h
Pilsner
—
—
0.56
0.14
0.27
0.76
1.03
0.69
0.44
—
484b
Wheat
—
—
0.67
0.19
0.35
0.26
0.34
0.52
0.55
—
270.1–366.0b,e
Abbey
—
—
0.65
0.25
0.22
0.23
0.92
0.59
0.34
—
622b
Bock
—
—
0.81
0.17
0.22
0.72
2.00
0.81
0.45
—
875b
Dealcoholized
—
—
0.45
0.14
0.16
0.40
1.40
0.32
0.26
—
125.0–366b,e
Dark
—
—
—
—
—
—
—
—
—
—
333.6–600e,f
Flavanoids
Lager
(+)-Catechin
(−)-Epicatechin
(−)-Gallocatechin
(−)-Epigallocatechin
Procyanidin Dimers B2
Procyanidin Dimers B3
Prodelphinidin B3
Genistein
Biochanin A
Daidzein
Formononetin
0.03–4.00a,i
0.02–0.73a,i
0.1d,i
0.1d,i
0.16i
0–3.6i,j
0–4.5j
0.05–1.82k
0.15–1.38k
0.08–0.65k
0.32–4.02k
Endogenous Antioxidants in Beers
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
Beer Type
aZhao
et al. (2010) et al. (2010) cNardini and Ghiselli (2004) dCallemien et al. (2008) eGorjanović et al.(2010) fLugasi (2003) gMartinez-Periñan et al. (2011) hGranato et al. (2011) ide Pascual-Teresa et al. (2000) jAndersen et al.(2000) kLapcík et al. (1998). bPiazzon
17
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2. ANTIOXIDANT ACTIVITIES OF BEERS
ferulic, sinapic, and chlorogenic acids in malt reached ppm levels, while p-coumaric, caffeic, and ferulic acids in hop was more than 10 ppm in most cases. Most phenolic acids in malt or hop are esterified with starch and other polysaccharides by forming bridges or cross-links through hydrogen bonds, chelation, or covalent bonds. These phenolic acids bound with starch can be released by enzymatic hydrolysis of starch during mashing, which causes significantly higher levels of bound phenolic acids than those of free forms in raw materials and final beers. In addition, malt and hop also are an excellent source of catechins, proanthocyanidins and flavonoid oligomers. (+)-Catechin in hop dried cones or pellets can reach 2821 mg/kg (Callemien and Collin, 2010), while the major monomeric and dimeric flavan3-ol in malts identified as (+)-catechin, prodelphinidin B3 and procyanidin B3 were found to be at values of 6-16 mg/kg, 99–175 mg/kg and 9–85 mg/kg, respectively (Dvorakova et al., 2008). The phenolic compounds content in beer is beer-type dependent. Piazzon et al. (2010) reported that the content of phenolic compounds increased in the order: dealcoholized < lager< pilsner < wheat < ale < abbey < bock. Vinson et al. (2003) also observed that total polyphenols in ales/ porters/stouts beers were higher than those in lager and nonalcoholic beers. However, other research indicated
that although higher TPC was found in dark beers, its difference between dark and lager beers was not statistically different (Lugasi, 2003). The higher phenolic compounds in dark, strong beers is because these beers are brewed from wort with higher extract content, while alcohol-free beers are usually brewed with lower original wort extract and inhibition of alcohol formation. Moreover, beers with higher original gravity also generally showed higher TPC (> 180 mg GAE/l), further indicating that phenolic compounds in beer mainly originate from barley malt and hops, although the brewing process also has some impact on phenolic compound content (Zhao et al., 2010).
Melanoidins Melanoidins are macromolecular, nitrogenous, and brown-colored final products of the Maillard reactions between reducing sugars and proteins or amino acids, which are formed during malt kilning, mashing and wort boiling, and are retained at least partially up to the final beer (Kuntcheva and Obretenov, 1996; Rivero et al., 2005). Melanoidins are generally formed by cyclizations, dehydrations, retroaldolizations, rearrangements, isomerizations, and condensations of initial Maillard reaction products (Figure 2.1), but only very few structures have been identified completely (Martins et al., 2001).
+ amino compound
+ H2O
N-substituted glycosylamine
Aldose Sugar
Amadori rearrangement
Amadori rearrangement product (ARP) 1-amino-1-deoxy-2-ketose -2H O
> pH 7
> pH 7
Reductones -2H
Fission products (acetol, diacetyl,pyruvaldehyde, etc)
+2H
-3H O
pH 7
Schiff’s base of hydroxymethylfurfural (HMF) or furfural
Dehydroreductones + amino compound -CO
Strecker degradation
+H O - amino compound
-amino acid
Aldehydes + amino compound
(HMF) or furfural Aldols and N-free polymers
+ amino compound
Aldimines and Ketimes
FIGURE Melanoidins (brown nitrogenous polymers)
2.1 Maillard reaction scheme. Adapted from Martins et al., (2001).
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
19
Endogenous Antioxidants in Beers
Sulfites Sulfites, one of important constituents of beer, are produced by yeast as by-products of the synthesis of sulfur-containing amino acids during fermentation and survive into the finished beer. The raw materials used in brewing may contain different amounts of sulfur dioxide but most of it disappears during wort boiling. Sulfites in beer are not only important antioxidants to prevent beer from oxidative staling, but they also act as important mask agents for stale flavor by reacting with carbonyl staling compounds in beer to form bisulfite–carbonyl adducts, which is important for beer flavor stability. The differences in brewer’s yeast strain and brewing process lead to significant variations in sulfite content of final beers. The total concentration of SO2 typically
ranged from 1 to 30 mg/l (Illet, 1995). For example, the total SO2 concentrations determined by the p-rosaniline method and the chronopotentiometric method in beers were found in the range of 1.15–37.32 mg/l and 0–4.1 mg/l, respectively (Zhao et al., 2013; Dvořák et al., 2006). Almeida et al. (2003) reported that the levels of free and total sulfur dioxide in beers determined by a proposed voltammetric methods were in the ranges of 0–3.0 mg/l and 8.1–14.3 mg/l, respectively. The production of sulfites in beer is affected by yeast strain, fermentation conditions, and wort components. Saison et al. (2009) observed that sulfite production in beers appeared to be yeast strain dependent (Figure 2.2), but their content was quite low (2.44–3.91 mg/l) and their differences were small. Moreover, lager beers contained substantially higher levels of SO2 than the ale because lager yeast strains produce more SO2 than ale strains (Bushnell et al., 2003). Over-production of sulfites by yeast can occur when the brewing yeast strain is restricted in growth during fermentation because endogenous sulfites in beer are produced from sulfates in the wort by the brewing yeast strain during synthesis of hydrogen sulfide. Thus, all conditions which promote yeast growth will reduce the amount of sulfites excreted in the medium. Guido et al. (2004) examined the effects of the physiological condition of the pitching yeast on sulfites production in beer, and fermentations with the lower yeast cell count secreted higher levels of sulfites (Figure 2.3). In recent decades, modification of characteristics of brewer’s yeast by genetic engineering has been considered to be an efficient measure to control the accumulation of sulfites (Dequin, 2001). Korch et al. (1991) suggested that the overexpression of ATP sulfurylase (MET3) and APS kinase (MET14) in brewer’s yeast could increase sulfites production (Figure 2.4). While Hansen
5
Total SO2 content (mg/l)
Melanoidins contribute to the formation of color, aroma, and flavor, and also play an important role in stabilization of the foam and influence the oxidation–reduction processes of beer due to their strong antioxidant properties and other biological effects (Rivero et al., 2005). The quantitative analysis of melanoidins in beers is very limited; a more than eight-fold difference in melanoidin content, varying from 1.64 mg/1 to 13.71 mg/1, occurred between beers which can be ascribed to the different raw materials and brewing process used during beer brewing (Zhao et al., 2013; Kuntcheva and Obretenov, 1996). The content of melanoidins in beers was influenced by the type of beers, and their content was found to be the highest in dark beer with values of 1.49 g/l, compared with to 0.61 g/l in blond beer and 0.58 g/l for alcohol-free beer (Rivero et al., 2005). The main difference between the types of beers, especially for dark and blond beers, is that they are brewed from malt with diverse grades of toasting, although they are also largely influenced by the technological brewing factors. Indeed, malts contained more melanoidins than barley, and the melanoidins isolated from malt were observed as a group of compounds with high molecular weight (> 3500 Da), brown color, and with a small percentage content of non-amino acidic nitrogen (1.12%) due to the relatively high polysaccharide and low protein content of barley grain and because of the specific reaction conditions of low water activity and lengthy roasting periods applied during the roasting process (Papetti et al., 2006). Most of the molecular weight of such polymeric compounds isolated from food did not exceed 100,000 Da, while the molecular weight of melanoidins formed in roasted barley malt of was found to be up to 200,000 Da (Faist et al., 2002). Moreover, significant differences in the distribution of the nitrogen content in different molecular weights of melanoidins isolated from barley were observed, and its content reached 30% in low-molecular-weight compounds, whereas only 3% was found in the high-molecular-weight polymers (30,000–60000 Da) (Papetti et al., 2006).
4 3 2 1 0 DS05
DS08
DS10
DS12
Yeast strain
FIGURE 2.2 Influence of fermentation with different top-fermenting yeast strains on final total SO2 content in beer. Adapted from Saison et al. (2009).
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
20
2. ANTIOXIDANT ACTIVITIES OF BEERS
The total SO 2 content (mg/l)
16
12
8
4
0 1
2
Fermentation
3
4
FIGURE 2.3 The effect of yeast physiological condition on total SO2
content at the end of fermentation. Fermentation 1 and 3: higher vital pitching yeast; fermentation 2 and 4: lower vital pitching yeast. Adapted from Guido et al. (2004). SO42-
SO42MET3 APS MET14 PAPS Serine
MET16
O-Acetylserine
MET10
Homoserine MET2
SO2
MET25
O-Acetylhomoserine H2S
MET25
Homocysteine
Cysteine NHS5 Cystathionine
Methionine
FIGURE 2.4 Pathways for sulfite production in S. cerevisiae. APS, adenosine 5′-phosphosulfate; MET2, serine acetyltransferase; MET3, ATP sulfurylase; MET10, sulfite reductase subunit; MET14, APS kinase; MET16, 3′-phospho adenosine 5′-phosphosulfate (PAPS) reductase; MET25, O-acetylhomoserine or O-acetylserine sulfhydrylase; NHS5, cystathionine β-synthase. Adapted from Dequin, (2001).
and Kielland-Brandt (1996) indicated that inactivation of serine acetyltransferase (MET2) in brewer’s yeast could produce higher amounts of sulfites and hydrogen sulfide, and the inactivation of sulfite reductase
subunit (MET10) was shown to be an efficient strategy for increasing sulfites formation and decreasing hydrogen sulfide production. However, it should be noted that the SO2 level must be controlled at the end of beer production for human health and beer-quality reasons. A maximum level of total SO2 in beer at 20 mg/l was established by the European Union and other countries. The SO2 level in beer must also be controlled during storage because sulfites produced during fermentation influence the flavor staling of finished beer; a content of 8–9 mg/l of sulfites in packaged beer was considered to be the most appropriate for the flavor stability of beer (Narziss et al., 1993).
ANTIOXIDANT ACTIVITIES OF BEERS Antioxidant Activity Assays for Beers Endogenous antioxidants present in beer are complex, and their activities and mechanisms largely depend on the composition and condition of the test system. The results are quite different even when an analysis of the same sample is carried out by each method since the antioxidant activity assays each rely on different chemical backgrounds and reaction mechanisms. Thus, many researchers have stressed the need to perform more than one type of antioxidant activity measurement to evaluate the antioxidant activity of beers. In the past few years, various in vitro methods have been investigated to estimate the antioxidant activity of beers or brewing raw materials. The varieties, principles, analytical performances and characteristics of methods for antioxidant activity evaluation have been reviewed by Pisoschi and Negulescu (2011) and Badarinath et al. (2010). These methods include 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation scavenging activity (ABTS) assay, oxygen radical absorbing capacity (ORAC) assay, superoxide anion radical scavenging activity (SASA), ferric-reducing antioxidant power (FRAP) assay, total radical trapping antioxidant parameter (TRAP), lipid peroxidation inhibition capacity (LPIC) assay, cupric ion reducing antioxidant capacity (CUPRAC) assay, metal-chelating activity (MCA) assay, hydroxyl radical averting capacity (HORAC) assay, potassium ferricyanide reducing power (PFRAP) method, enhanced chemiluminescence (ECL), and ESR. The various analytical methods for antioxidant activity evaluation fall into distinct categories, which involve different reaction mechanisms, reaction conditions, and analytical techniques (Table 2.2). ORAC, LPIC, TRAP, ABTS, and SASA are classified as hydrogen atom transfer methods of monitoring competitive kinetic reactions,
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
21
Antioxidant Activities of Beers
TABLE 2.2 Antioxidant Activity Assays for Beers Antioxidant Activity Assays
Type of Method
Principle of Method
End-Product Determination
ORAC
Hydrogen atom transfer
Antioxidant reaction with peroxyl radicals, induced by AAPH
Loss of fluorescence of fluorescein
LPIC
Hydrogen atom transfer
Antioxidant to inhibit peroxidation of lipids induced by a Fenton-like system
Colorimetry
TRAP
Hydrogen atom transfer
Antioxidant capacity to scavenge luminol-derived radicals, generated from AAPH decomposition
Chemiluminescence quenching
SASA
Hydrogen atom transfer
Antioxidant to scavenge superoxide anion radical formation by alkaline or enzyme
Colorimetry
ABTS
Hydrogen atom transfer
Antioxidant to scavenge an organic cation radical
Colorimetry
HORAC
Hydrogen atom transfer
Antioxidant capacity to quench OH radicals generated by a Co(II) based Fenton-like system
Loss of fluorescence of Fluorescein
CUPRAC
Electron transfer
Cu(II) reduction to Cu(I) by antioxidants
Colorimetry
FRAP
Electron transfer
Antioxidant reaction with a Fe(III) complex
Colorimetry
DPPH
Electron transfer
Antioxidant reaction with an organic radical
Colorimetry
PFRAP
Electron transfer
Potassium ferricyanide reduction by antioxidants and subsequent reaction of potassium ferrocyanide with Fe3+
Colorimetry
MCA
Other
Chelating ferrous ions with ferrozine
Colorimetry
ECL
Other
Emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength
Recording of fluorescence excitation/emission spectra
ESR
Other
Free radicals by electron spin resonance
Recording the time of occurring of free radicals
AAPH, 2,2′-azinobis (2-amidinopropane) dihydrochloride; ABTS, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation scavenging activity assay; CUPRAC, cupric ion reducing antioxidant capacity assay; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay; ECL, enhanced chemiluminescence; ESR, electron spin resonance; FRAP, ferric-reducing antioxidant power assay; HORAC, hydroxyl radical averting capacity assay; LPIC, lipid peroxidation inhibition capacity assay; MCA, metal-chelating activity assay; ORAC, oxygen radical-absorbing capacity assay; PFRAP, potassium ferricyanide reducing power method; SASA, superoxide anion radical scavenging activity; TRAP, total radical trapping antioxidant parameter
while FRAP, DPPH, CUPRAC, HORAC, and PFRAP are grouped together as the electron transfer methods of a reduction reaction, and others such as MCA, ECL, and ESR are categorized as other methods. For routine evaluation of the antioxidant activity of beers or monitoring the brewing process with respect to antioxidant activity, spectrometry analytical techniques such as DPPH, ABTS, ORAC, FRAP, TRAP, etc. should be selected due to their better sensitivity, convenience, and short assay times.
Antioxidant Activities of Beers Evaluated by Different Assays As mentioned above, beer contains numerous endogenous antioxidants, which endows beer with significant antioxidant activity. Zhao et al., (2010) observed significant antioxidant activities in lager beers, suggesting that overall antioxidant activity evaluation results for beer samples using DPPH, ABTS, SASA, FRAP, MCA assays were consistent even though these assays involved
different reaction mechanisms. Another study performed by the same group indicated that all 40 beers showed significant antioxidant activity evaluated by DPPH, ABTS, ORAC, FRAP, MCA assays, but clear discrimination among beer samples with significantly different antioxidant activity was achieved by principal component analysis (Zhao et al., 2013). The antioxidant activity of 27 beers was tested by TRAP, TEAC, DPPH, FRAP, CUPRAC, and ORAC assays, and the results pointed out that all beers displayed antioxidant activities, although the values varied a lot with the sample, method, and standard (Tafulo et al., 2010). Similarly, Piazzon et al. (2010), Granato et al. (2011), and Lugasi (2003) also found that all beers showed antioxidant activity measured with various assays, but a wide range of antioxidant activity was observed. There are significant differences in antioxidant activity among different commercial beer types. Piazzon et al. (2010) reported that antioxidant activities decreased in the order: bock > abbey > ale > wheat > pilsner > lager > dealcoholized, and the antioxidant activities of bock beers
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
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2. ANTIOXIDANT ACTIVITIES OF BEERS
were found to be about 3 times higher with respect to dealcoholized beers. In a comparative study, lager-type beers were found have lower antioxidant activity than the ale ones, and Portuguese beers showed slightly lower antioxidant activity then Belgian ones, regardless of the method or standard (Tafulo et al., 2010). Granato et al. (2011) also examined the antioxidant activity of Brazilian lager and brown ale beers using ORAC and DPPH assays: higher antioxidant activity in brown ale beers was observed. However, another study performed by Lugasi (2003) indicated that there were no significant differences in antioxidant activity between lager and dark beers. It should also be noted that the differences in antioxidant activity across beer samples and types were due to the different parameters during brewing, such as the variety of barley and hop, the malting and mashing process, and the yeast fermentation employed.
Relationships between Endogenous Antioxidants and Antioxidant Activities of Beers All endogenous antioxidants in beers may contribute to some extent to the overall antioxidant activities of beers although present in beer at very low levels. However, the real contributions of these antioxidants to antioxidant activities of beers have not been clarified yet.
Contributions of Phenolic Compounds to Antioxidant Activities of Beers Most research has suggested that phenolic compounds are the most important antioxidants in beers. In addition to acting as chelating agents of metallic catalysts, phenolic compounds in beer have another indirect role in the antioxidant capacity due to their reversible interaction with sulfite (Illet, 1995). Our previous study observed that phenolic compounds including ferulic acid, syringic acid, (+)-catechin, caffeic acid, protocatechuic acid, and (−)-epicatechin contributed 55.0–88.1% of beer antioxidant activity evaluated by different assays. McMurrough et al. (1996) also found that partial removal of the polyphenol fraction by polyvinylpolypyrrolidone treatment diminished the reducing power by 9–38%. Piazzon et al. (2010) observed a strong correlation between FRAP values and total phenolic acids content of beers and a lack of correlation between FRAP values and the content of free phenolic acids, which indicated that conjugated forms of phenolic acids in beer were responsible for the antioxidant activity of beer. Granato et al. (2011) reported that the content of flavonoids and total phenolic compounds seemed to be strongly correlated with the antioxidant activity measured by ORAC and DPPH assays. TPC in the 40 commercial beers were also found to give strong correlations with DPPH, ABTS, ORAC, MCA, and FRAP assays tested, which strongly suggested that
phenolic compounds were mainly responsible for the majority of the antioxidant activity of beer (Zhao et al., 2013). However, some phenolic compounds can also act as pro-oxidants by transferring electrons to metal ions, and the levels of ferulic acid in beer were found to determine whether it is pro-oxidant or anti-oxidant (Walters et al., 1997). Thus, some controversy concerning the relevance of polyphenolic antioxidants in beer has been reported in the literature. In an ESR lag phase study, phenolic compounds such as phenolic acids, catechin, epicatechin, and proanthocyanidin dimers had no effect on the formation of radicals, and they are neither antioxidants nor pro-oxidants (Andersen et al., 2000). A Laccase–Sonogel–Carbon biosensor method proposed by Martinez-Periñan et al. (2011) for beer stability evaluation showed that the polyphenols cannot delay the formation of free radicals in beers, and polyphenols have an indirect role in beer aging by avoiding rapid SO2 loss.
Contributions of Melanoidins to Antioxidant Activities of Beers Maillard reaction products acting as antioxidants generally are involved in various mechanisms, and include: oxygen, reactive oxygen, peroxyl, or some specific stable radicals scavengers, reducing agents, and metal chelating agents. The overall effects of Maillard and caramelization products on the oxidative stability of beer are unknown. However, traditionally, the use of colored malt is known to improve the stability of the finished beer, and it has also been shown that more-highly colored beers retain a greater reducing power during storage (Coghe et al., 2003). The final beer produced has a reducing power mainly dependent on melanoidin compounds and simple and polymerized polyphenols. Indeed, positive correlations between antioxidant activity and malt color also have been observed, which have been ascribed to the presence of Maillard components (Coghe et al., 2003; W offenden et al., 2001). Moreover, the radical-scavenging and iron-chelating ability of melanoidins from dark beer has been found to be higher compared to melanoidins from pale beers (Coghe et al., 2003; Morales et al., 2005; Morales and Jiménez-Pérez, 2004). Furthermore, Woffenden et al. (2002) reported that correlations between levels of catechin or ferrulic acid and antioxidant activity are poor, suggesting that other components, such as polyphenols, oligomers, and Maillard reaction products also contribute to the total antioxidant activity of beer. Zhao et al. (2013) also indicated significant positive correlations between melanoidins content in beers and DPPH, FRAP, as well as MCA, and their content made an 8% contribution to ORAC of beers. But Rivero et al. (2005) concluded that the hydrogen-donating ability of beers is dependent on the total polyphenol levels but not on the total melanoidin content. Hydroxyl
1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS
References
radical-scavenging activity of beer melanoidins was found to be unrelated to their color, suggesting that colored structures were not involved in such antioxidant effects (Morales, 2009). Although melanoidins are known to act as antioxidants, some reports have also indicated a pro-oxidant activity. Pro-oxidative activity of melanoidins has mainly been established for low-molecular-weight melanoidins p roduced from model systems (Ames, 2001). A pro-oxidative effect of stout also has been observed in studies with stout added to lager beer analyzed by ESR spectroscopy (Nøddekær and Andersen 2007). Also, beers produced with dark malts kilned at high temperatures formed high concentrations of radicals, and also had a lower oxidative stability as compared to using paler malts (Cortés et al., 2010). This might be because the pro-oxidative effect of Maillard reaction products was caused by reactions that were able to increase radical levels by other mechanisms than Fenton-catalysis since stout was found to decrease the level of radicals in a beer model system where radical formation was induced by the Fenton reaction (Nøddekær and Andersen, 2007).
Contributions of Sulfites to Antioxidant Activities of Beers Sulfites formed in beer have been considered as one of the most effective antioxidants for beer flavor stability. Two main mechanisms for sulfites to control beer stabilization have been proposed: one is to inhibit beer oxidation during storage by acting as an antioxidant, the other is to combine with several carbonyls to produce sulfite adducts in beer and so mask staling flavor since the adducts are ionized and thus non-volatile. According to Andersen et al. (2000), sulfites were the most efficient naturally occurring antioxidants in beer, while thiols and ascorbic acid were shown to be pro-oxidants. Other authors obtained similar results studying flavor stability using ESR to detect free radicals, which supported the theory that phenolic compounds did not contribute to the antioxidant capacity but SO2 did increase the lag time (Foster et al., 1999). In another study performed by Martinez-Periñan et al. (2011), only SO2 concentration showed relevant differences after the ageing process, thus, it was responsible for the antioxidant capacity of beers. Our study by stepwise regression analysis also indicated that total SO2 content contributed 5% of the antioxidant activity of beer evaluated by reducing power assay. However, no significant correlations were found between SO2 and all five antioxidant activity assays used in this study (Zhao et al. 2013). Moreover, the effects of sulfites on beer flavor stability evaluated by various assays have shown that sulfites in beer are not only one of the important antioxidants but also one of the important masking agents; however, the efficiency
23
of naturally produced bisulfite by yeast was controversial (Guido, 2005).
Acknowledgments I gratefully acknowledge the National Natural Science Foundation of China (No. 31000810), the Key Technology R&D Program of Guangdong Province (Nos. 2012A080107005 and 2011A020102001) and the Fundamental Research Funds for the Central Universities (No. 2012ZM0069) for their financial support.
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2. ANTIOXIDANT ACTIVITIES OF BEERS
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1. COMPOSITION AND CHARACTERIZATION OF ANTIOXIDANTS