189

Scientia Agricola
http://dx.doi.org/10.1590/0103-9016-2015-0290

Review
Sulfites in beer: reviewing regulation, analysis and role
Luis F. Guido*

University of Porto/Faculty of Science – Dept. of Chemistry ABSTRACT: Beer is an extremely complex mixture of more than 3,000 different compounds in
and Biochemistry − REQUIMTE/LAQV, R. do Campo Alegre, an aqueous environment. Thus, it is perhaps not surprising that the maintenance of beer quality
687 − 4169-007 − Porto – Portugal. throughout its lifetime has been a considerable challenge for brewers. Whilst it is inevitable that
*Corresponding author <[email protected]> chemical changes will occur in beer with the passage of time, it is the formation of flavor-active
components which is of immediate concern to an overview of beer shelf life stability. Sulfur diox-
Edited by: Paulo Cesar Sentelhas ide has long been recognized by brewers as the most important factor in delaying flavor staling,
and prolonging the shelf life of beer. However, nowadays, sulfur dioxide and sulfites are consid-
ered allergens and concerns about the safety of their use as food additives have been on the
increase. The present review is structured into three main parts. Firstly, the chemical properties
of sulfur dioxide are presented, along with the toxic effects and maximum legal levels permitted
according to U.S. and EU legislation. As the accurate determination of the free, bound and total
sulfur dioxide in beer is essential to ensuring regulatory compliance, several methods have been
developed for analyzing sulfur dioxide in beer. Thus, secondly, various types of methods are
reported and compared with the officially recommended ones. Finally, the crucial role of sulfite
in the control of flavor instability of beer is discussed in light of the current data. Two courses of
action have been proposed, which are elucidated in detail relating firstly to the fact that sulfite
inhibits beer oxidation during storage by acting as an antioxidant and, secondly, sulfite reacts
Received July 17, 2015 with the carbonyl staling compounds in beer, and thereby masks stale flavors.
Accepted November 04, 2015 Keywords: beer flavour, sulfur dioxide, legislation toxic effects, antioxidants

Introduction ic studies revealed that undissociated sulfurous acid,

H2SO3, may be assumed to be a short-lived intermediate
1. Chemical properties of sulfur dioxide, safety in the acidification of sulfites, but does not exist in de-
concerns and regulations tectable amounts in aqueous solutions of sulfur dioxide
Sulfur dioxide (SO2) is a colorless gas at room tem- (Senning, 1982). At the usual pH of beers (3.8 – 4.4),
perature, with a pungent odor which is readily soluble in most of the SO2 is present as the bisulfite (or hydrogen
water (3927 cm3 SO2 in 100 g H2O at 20 ºC). Maximum sulfite) anion, as illustrated in Figure 1.
permitted atmospheric concentration for humans is 5 Sulfur dioxide, sulfurous acid and sulfites can
ppm but many green plants suffer severe distress from function as mild oxidizing agents; however, reactions in
concentrations as low as 1-2 ppm (Earnshaw, 1999). A which these compounds react as reducing agents (and
number of molecular and physical properties of sulfur are oxidized to the sulfate ion, SO42-) are more numerous
dioxide are listed in Table 1. and more important (Earnshaw, 1999), and a compre-
Sulfur dioxide and sulfur (IV) oxoanions in solu- hensive monograph on the chemistry of sulfur dioxide in
tion undergo pH-dependent equilibration reactions be- foods has been published (Wedzicha, 1984). In the pres-
tween sulfur dioxide, sulfurous acid (H2SO3), bisulfite ent review, the terms sulfur dioxide and sulfite will be
ion (HSO3-), and sulfite ion (SO32-): used interchangeably to represent all forms of SO2.
Sulfites occur naturally in a number of foods and
K1 K2 beverages as a result of fermentation, such as occurs in
beer and wine. As a food additive, sulfites have been
SO2n H2O  HSO3− (aq) + H3O+(aq)  SO32 − (aq) + 2 H3O+(aq)
used since 1664 and have been approved for use in the
United States since the 1800s (Lester, 1995). With such
Sulfur dioxide dissolves fairly readily in water a history of widespread application, sulfites have been
to give a rather complicated reaction mixture, strongly generally regarded as safe (GRAS) by the FD; however,
dependent on concentration, temperature and pH (Sen- it is suspected that a low percentage of the population is
ning, 1982). Therefore, the concentration of each of the sensitive to sulfites. The manifestations of sulfite sensi-
different forms is a function of the pH medium and is tivity include a large array of dermatological, pulmonary,
determined by the equilibrium constants K1 (1.6 × 10−2 gastrointestinal, and cardiovascular symptoms. Asthmat-
mol dm−3) and K2 (1.0 × 10−7 mol dm−3). At a pH lower ics, for example, who are steroid-dependent or have a
than 2 most of the SO2 is in a loosely hydrated undissoci- great degree of airway hyperreactivity may be at an in-
ated form, due to its high water solubility. Spectroscop- creased risk of having a reaction to a sulfite-containing

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Guido Sulfites in beer: a review

Table 1 − Selected molecular and physical properties of SO2 (adapted from Greenwood and Earnshow, 1999).
Property Value Property Value
MP /ºC -75.5 Electrical conductivity κ /ohm−1 cm−1 < 10−8
BP /ºC -10.0 Dielectric constant ε (0º) 15.4
Critical temperature / ªC 157.5 Dipole moment m /D 1.62
Critical pressure / atm 77.7 Angle O-S-O 119º
Density (-10º) / g cm−3 1.46 Distance r(S-O) / pm 143.1
Viscosity η (0 ºC) / centipoise 0.403 DHfo (g) / kJ mol−1 -296.9

duce beers with a sulfite content well below 10 mg L−1,

the level at which declaration of sulfites has been man-
dated for the labelling of alcoholic beverages by both US
and EU legislation. The statement “Contains sulfites” or
“Contains (a) sulfiting agent(s)” or a statement identify-
ing the specific sulfiting agent where sulfur dioxide or a
sulfiting agent is detected at a level of 10 or more parts
per million (ppm), measured as total sulfur dioxide, is
mandatory in the USA (GPO, 2000). The 2000/13/EC Di-
rective from the European Parliament and Council on
the labelling of foodstuffs (EC, 2000) requires sulfur di-
oxide and/ or sulfites to be labeled when present in beer
above 10 mg L−1 (calculated in terms of the total SO2).
For products where a list of ingredients is not required
(e.g. alcoholic beverages with > 1.2 % by volume of al-
cohol) the presence of the allergen must still be shown
Figure 1 − The effect of pH on the equilibria of SO2 species in on the label e.g. ‘contains sulfur dioxide’. In Canada,
aqueous solution (adapted from Ilett, 1995). the new food allergen labelling regulations require that
sulfites at a level of 10 ppm or more must be declared
when present in alcoholic beverages. Beer, ale, stout,
food (Lester, 1995). As a result, sulfiting agents are not porter and malt liquor are exempt from the requirement
considered GRAS for use in meats, i.e. foods recognized to declare food allergen, gluten or added sulfites unless
as a major source of vitamin B1 (thiamine), or “fruits or a list of ingredients is shown on the product's label in
vegetables intended to be served raw to consumers or to a statement entitled ‘Contains’. Any list of ingredients
be presented to consumers as fresh” (FDA, 1988). that is voluntarily provided must be complete, and de-
The European Commission’s Scientific Committee clare all priority allergens and gluten sources, and added
on Food (SCF) put forward in 1994 an acceptable daily sulfites when present at 10 ppm or more [B.01.010.2,
intake (ADI) of 0.7 mg kg−1 body weight/day for sulfur B.01.010.3, Food and Drug Regulations] (Government
dioxide and sulfur dioxide equivalents (SCF, 1994). The of Canada, 2015). Brazil’s regulatory agency (ANVISA,
ADI is the amount of food additive, expressed on a body 2011) issued a resolution (Resolution RDC Nº 65 of 29
weight basis, which can be ingested daily over a lifetime November 2011) establishing a maximum legal limit of
without appreciable health risk. Toxicity was in part at- 0.005 g / 100 g or 0.005 g / 100 mL to earn a free of total
tributed to the well-known destructive action of sulfite sulfur dioxide in beer classification (Brazil, 2011).
on thiamine. It has been suggested that the ingestion of
SO2 in a beverage may effectively reduce the level of thi- 2. Sulfur dioxide analysis in beer
amine in the rest of the diet (Cremer and Hotzel, 1970). Potential problems with the usage of sulfiting
Human studies over short periods showed that 400 mg agents in foods and the investigations revealing wide
d−1 produced no effect on thiamine excretion. Sulfites and increased utilization of these compounds in food-
have also a certain irritant action on the skin, but the stuffs have raised questions as to the suitability of avail-
risks involved in handling these compounds are appar- able methodologies for determining sulfites in food and
ently rather small (Senning, 1982). beverages. Many methods are currently in use, varying
The use of sulfites in food products became an is- in specificity and sensitivity, applicable to specific matri-
sue of concern to both consumer and regulatory agen- ces but lacking a suitable data-base for comparison with
cies when it was found that certain sensitive individuals each other.
exhibited adverse reactions to sulfite residues in foods. Currently, many different procedures are being
Further investigations into this potential health hazard used by the industry to determine the sulfite content of
led agencies to promulgate regulations concerning the their products including various versions of the classi-
usage and labelling of sulfiting agents. Brewers can pro- cal Monier-Williams method, colorimetric procedures

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Guido Sulfites in beer: a review

such as the p-rosaniline and to a limited extent, flow p-rosaniline to give a colored product which is measured
injection analysis, pulse polarography and ion chroma- using a spectrophotometer. Several applications of FIA
tography. The Monier-Williams method has been the towards the determination of sulfite in wine by spectro-
reference method for many years, but it has drawbacks photometric detection have been recently reported (Ma-
at the 10 mg L−1 level. Minor procedural changes made quieira et al., 1993; Decnop-Weever and Kraak, 1997;
to the Monier-Williams method by the Food and Drug Richter et al., 1993; Gonçalves et al., 2010).
Administration (FDA) improved accuracy at the 10 ppm A chronopotentiometric method for the on-line
level used in the labelling of sulfite content in commer- determination of total sulfur dioxide in beer has been
cial products (Bubnis, 1988). The Monier-Williams tech- reported (Dvořák et al., 2006). Free and bound sulfites
nique, adopted by the Institute of Brewing, involves the were converted to sulfite anions on alkalising the sample
removal of SO2 from acidified beer in a stream of carbon solutions. On acidifying the solution, the sulfur dioxide
dioxide and nitrogen at 100 ºC. The gas is absorbed in released was separated on-line through a semiperme-
hydrogen peroxide and the sulfuric acid formed titrated able membrane and transported by an electrolyte into
against NaOH. Minor modifications made by the FDA the measuring cell and measured by stripping chrono-
include reduction in concentration of the titrant by a potentiometry. The results corresponded well with those
factor of 10, the replacement of the pyrogallol trap by obtained with the alternative EBC methods, with the ad-
a GLC-type oxygen scrubber and elimination of the hot vantage of being much faster and simpler.
condenser step (Fazio, 1988; Fazio and Warner, 1990). A An enzymatic assay employing sulfite reductase
coulometric adaptation of the Monier-Williams method is currently recommended by the EBC (Method 9.25.2,
yielded significant correlation with the p-rosaniline ref- EBC, 1987). It is based on the oxidation of NADH by the
erence method, permitting a reduced sample size while hydrogen peroxide formed during the oxidation of sulfite
improving precision and significantly reducing analysis by the action of sulfite oxidase. Changes in absorbance
time (Zeller, 1988). of NADH at 340 nm are used for determining the con-
The colorimetric method using rosaniline hydro- centration of total sulfite.
chloride was adopted by the American Society of Brew- Since the advent of flame photometric detection
ing Chemists (ASBC). This method proved to be quite (FPD), the use of various gas chromatographic methods
acceptable until the last few years when questions about has been reported (Moreno and Vega, 1989) using GC to
the carcinogenicity of the p-rosaniline reagent were accurately quantify the amount of SO2 in beer. A method
raised. This colorimetric analysis is based on the reac- for the routine determination of both free and total sul-
tion between SO2, p-rosaniline and formaldehyde. After fur dioxide residues in malt and total SO2 in beer using
reaction with p-rosaniline and development of the color the technique of headspace GC was developed by Mu-
for 30 minutes, the absorbance of the p-rosaniline-sulfite nar and co-authors (Munar et al., 1994). They showed
complex is measured at 550 nm. This method has been that both the colorimetric and the headspace methods
widely used as it is precise and allows for measurement are comparable. The use of a highly sensitive and se-
of free and total SO2. Another colorimetric method uses lective chemiluminescence sulfur detector was reported
DTNB, which is 5.5-dithiobis (2-nitrobenzoic acid), as (Osborne, 1992; Burmeister et al., 1992). The sulfur che-
the color reagent after a distillation step. It is a method miluminescence detector was evaluated as a replace-
for total SO2 determination in beer recommended by ment for the FPD as it was found to have better selectiv-
the EBC. Sulfur dioxide is distilled from acidified 25 mL ity, sensitivity, and linearity. It was not susceptible to
samples into a buffered DTNB solution, with a nitrogen hydrocarbon-quenching effects shown by the FPD detec-
carrier gas, and absorbance is measured at 415 nm. A tor. These methods have the additional advantage over
simpler version of the DTNB method has been recently the colorimetric methods for dark beers.
applied to beer samples with 97 % of sulfite recovery (Li Ion exclusion chromatography with electrochemi-
and Zhao, 2006). cal detection was proven to be a lot simpler and quicker
During the past few years, flow injection analysis with the results compared more favourably with the
(FIA) methods for determining sulfites in beer and other Monier-Williams technique and to correlate well with
beverages have been widely developed. Most flow injec- the p-rosaniline technique (Kim, 1990; Wygant et al.,
tion analyses are adaptations of other existing methods, 1997). This method showed good reproducibility at lev-
mainly spectrophotometric methods. Application of FIA els close to 0.5 mg L−1 and an analysis time of approxi-
systems for both the malachite green (Atanassov et al., mately half an hour. Wagner and McGarrity used ion
2000; Bendtsen and Jorgensen, 1994) and p-rosaniline exclusion chromatography with pulsed amperometric
(Fernandes et al., 1998) methods are known. The sam- detection to avoid the loss of detector sensitivity that oc-
ple is injected into the carrier stream which contains curs over time when direct amperometry is used (Wag-
NaOH for on-line release of bound sulfites. Sulfuric acid ner and McGarrity, 1991; Wagner and McGarrity, 1992).
is introduced to lower the pH and convert all existing A rapid and sensitive polarographic method was
equilibrium products into the sulfur dioxide form which also presented for determining sulfiting agents in foods
then diffuses across a gas permeable membrane. The and beverages (Stonys, 1987; Holak and Patel, 1987). This
sulfur dioxide is then reacted with malachite green or method is based on the modified Monier-Williams distil-

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Guido Sulfites in beer: a review

lation followed by polarographic detection by differen- Table 2 − Uses of sulfur dioxide in the brewing industry (adapted
tial pulse polarography or square wave voltammetry and from Ilett, 1995).
is specific for total SO2. A method involving voltammet- Stage of beer production Use of sulfur dioxide
ric determination of free and total sulfur dioxide in beer
germinating reduce malting losses, enhanced yields of
was reported by Almeida et al., (2003). Voltammetric de-
grain extracts
terminations are in agreement with those obtained by
the p-rosaniline reference method, with the advantage of malt kilning control the formation of nitrosamines, bleach
excluding the use of toxic reagents and providing more malt, increase soluble nitrogen
accurate and precise results. kilning of hops bleaching agent, preservative
More recently, a method was developed for the storage of syrups preservative
quantification of sulfite in beer based on derivatization storage of finings preservative
with the maleimide-derived probe ThioGlo I followed fermentation vessels antimicrobial agent
by separation of fluorescent adducts by reversed-phase, proteolytic enzymes preservative
high-performance liquid chromatography and fluores- beer additive antioxidant, preservative
cence detection. ThioGlo I was found to have generated
fluorescent adducts with both bound and free sulfites,
providing a quantification of total sulfite content in beer.
The limit of quantification of sulfite was 0.6 mg L−1 and 4. Antioxidant activity of sulfur dioxide in beer
the method can be used for quantification of sulfite in Antioxidants can be broadly defined as com-
highly colored beers (Abrahamsson et al., 2012). pounds that inhibit an oxidative reaction. They can
Albeit numerous methods for determining sulfur act by decreasing molecular oxygen levels, scavenging
dioxide in beer have been reported in the literature, the chain-initiating and chain-propagating free radicals, che-
brewing industry is still searching for the ideal procedure. lating metals, or decomposing peroxides (Halliwell et al.,
It should be fast, simple, inexpensive, allow for measure- 1987). They are, therefore, thought to have a significant
ment of free or/and total SO2 and exclude the use of toxic effect in malting and brewing as inhibitors of oxidative
reagents. Most of these conditions, or at least a number of damage. In their general reactions, sulfites and hydrogen
them, are met in the aforementioned methods. sulfites are moderatetely strong reducing agents, and to-
gether with antioxidant properties, yield either dithion-
3. Uses of sulfur dioxide in the brewing industry ate (S2O62-) or sulfate (SO42-) upon reaction. This reducing
Sulfites in various forms have been added to foods (antioxidant) behavior under all pH conditions may be
for centuries. The usage of sulfiting agents or S (IV) com- seen in the following redox potentials (Senning, 1982):
pounds to foods has become widespread because of their
varied functional applications such as bleaching agents, S2O62 − + 4 H + + 2e−  2H2SO3 E =
0.564V
antimicrobials, oxygen scavengers, reducing agents and
SO42- + 4 H++2e-  H2SO3+H2O E  = 0.119V
enzyme inhibitors. Sulfur dioxide and several forms of
sulfites, which generate sulfur dioxide when used in food,
can be used as sulfiting agents. These include sulfur di- Beer flavor stability is influenced by a multiplicity
oxide (E220), potassium bisulfite (potassium hydrogen of factors; some of them are certainly disputed, but there
sulfite) (E228), potassium metabisulfite (E224), sodium is no question of the crucial role of oxygen in packaged
bisulfite (sodium hydrogen sulfite) (E222), sodium metabi- beer. It has been repeatedly demonstrated that high air
sulfite (E223), sodium sulfite (E221), calcium sulfite (E226) levels in the final package greatly reduce shelf life (Back
and calcium hydrogen sulfite (E227) after the Nº 95/2/EC et al., 1999; Bamforth, 2000). Therefore, modern fillers
(EC, 1995) Directive. As all the above can convert to sul- are designed to achieve very low O2 levels and several
fur dioxide, sulfites are measured and expressed as sulfur measures have been adopted in order to keep ingress of
dioxide. Sulfur dioxide found in beer is due not only to oxygen into the packaged beer as low as possible. Vari-
the addition of sulfiting agents (exogenous SO2) but is also ous antioxidants may be used in beer (depending on the
derived from the yeast metabolism or as a component of legislation), with the most prominent of these being sul-
finings or primings (endogenous SO2). Yeast has the ability fur dioxide and ascorbic acid (Bamforth, 1999). These
to produce sulfur dioxide, from the reduction of sulfate agents can only protect against new oxidation occurring
in water and grist material. SO2 levels will be increased in beer, and do not rectify any damage which has oc-
if the sulfate supply to the yeast is increased, wort clar- curred upstream. Sulfur dioxide is the more effective,
ity is increased, wort oxygenation and pitching rate are either in the role of carbonyl binder (discussed below) or
lowered and fermentation temperature is reduced (Ilett, radical scavenger.
1995). The sources of sulfur dioxide in beer were compre- The production of reactive oxygen species (ROS)
hensively reviewed by Ilett, (1995). Table 2 summarizes is promoted by light and certain enzyme systems. Of
the uses of sulfur dioxide in the brewing industry. The particular importance are transition metal ions, such
different roles of sulfur dioxide on beer flavor stability as iron and copper, which are effective in stimulating
will be emphasised and discussed below. the formation and multiple-interconversions of radicals

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Guido Sulfites in beer: a review

from oxygen (Bamforth, 2001). The deleterious effect of concluded that aldehyde-bisulfite adducts have radical
ROS has led most brewers to make efforts to eliminate scavenging activity and protect free radical chain reac-
these substances from their processes and products. The tions during beer storage, leading to stability of beer
reactivity of oxygen and its activation by acquisition of quality (Kaneda et al., 1994). Additionally, it was shown
electrons was described by Bamforth et al.,(1993) and by Kaneda and co-authors that the lower the dissolved
Hughes,(2000). As oxygen passes successively through oxygen in pitching wort, the higher the pitching rate of
superoxide (O2·-), peroxide (O22-) and hydroxyl (HO·) it yeast. Clearer pitching wort leads to higher sulfite con-
becomes increasingly reactive. The hydroxyl radical is tent level, inhibition of CL production, and better flavor
an immensely reactive species generated in wort and stability of the resulting beers (Kaneda et al., 1992). Le-
beer, which reacts instantly with a great many types of rmusieau and co-authors confirmed in laboratory-scale
molecule. Superoxide, on the other hand, is less reactive. experiments that SO2 can reduce both lipid autooxida-
At beer pHs, the majority of the superoxide will be in a tion and the nonenal potential rise while the wort is boil-
perhydroxyl form (HO2·). Hydroxyl and perhydroxyl are ing (Lermusieau et al., 1999).
capable of reacting with unsaturated fatty acids, such as Much of the SO2 added to beers is rapidly bound up
linoleic acid, and thereby set in motion a chain reaction with components other than carbonyls, with an addition
that leads to stale flavor development (Bamforth, 2001). of 20 ppm revealing only approximately 12 ppm of mea-
The 1-hydroxyethyl radical, which can be formed surable total SO2. The majority of this is lost through ag-
by reaction between hydroxyl radical and ethanol, was ing, especially in the presence of high levels of air (Bush-
recently found to be, quantitatively, the most important nell et al., 2003). Recent sensory and chemical evidence
radical in beer, detectable by electron spin resonance from Bushnell and co-authors support the argument that
(ESR). Oxidation processes were characterised in beer sulfites exert their protective effect as antioxidants rather
involving reaction of the 1-hydroxyethyl radical in an than as agents that bind carbonyls as adducts.
important reaction with oxygen, converting it into hy-
droperoxyl radicals (Andersen and Skibsted, 1998). A 5. Formation of adducts with carbonyl compounds
number of potential antioxidants have been evaluated Sulfur dioxide reacts with a wide range of food
for their effect on the formation of radicals in beer us- components. It forms adducts by reversible action with
ing the ESR lag phase method. Sulfite was found to be aldehydes and ketones (including reducing sugars, acet-
the only compound that was able to delay the formation aldehyde, quinones, and ketoacids), with anthocyanins,
of radicals, whereas phenolic compounds such as phe- and with cysteine residues in proteins. In most foods and
nolic acids, catechin, epicatechin, and proanthocyanidin beverages, adducts with carbonyl compounds, the hy-
dimers had no effect on the formation of radicals. It was droxysulfonates (Figure 2), comprise most of the bound
suggested that antioxidants must be able to either scav- sulfite, and this equilibrium reaction has been studied
enge peroxides or trap metal ions in order to be effective in detail. In the range pH 1 to 8 the hydroxysulfonates
in beer. The effectiveness of sulfite was suggested to be predominate, while at higher pH values dissociation oc-
a consequence of its two-electron nonradical producing curs (Adachi et al., 1979). It is also believed that sulfite
reaction with peroxides (Andersen et al., 2000). Addi- stabilizes intermediates of the Maillard reaction by form-
tionally, the concept of Endogenous Antioxidant (EA) ing adducts. For example, glyceraldehyde forms stable
value, which is the time taken before an ESR signal is hydroxysulfonate adduct, which could contribute to the
developed in an aging test, showed a clear relationship mechanism of the inhibition of Maillard browning by
between the EA value and sulfite level in beer (Uchida sulfite species (Keller et al., 1999). Dissociation constants
and Ono, 2000). More recently, Karabín and co-authors of carbonyl bisulfite compounds at pH 3.5 have been
have shown that one of the reasons for the decline in reported by Azevedo et al., (2007). They revealed that
the endogenous antioxidative potential of beer during aliphatic aldehydes form adducts with S (IV), whereas
storage is a significant decrease in SO2 content, probably ketones, cyclic aldehydes, and trans-alkenes interact
caused by chain oxidation reactions catalysed by transi- weakly and are found predominantly in the free form.
tion metal ions, particularly ferric ions (Karabin et al., Range of dissociation constants of bisulfite compounds
2014). are 1.09 × 10−7 for formaldehyde, 2.06 × 10−6 for acetal-
It is postulated that the deterioration rates of beers dehyde, 3.45 × 10−6 for hexanal, 2.52 × 10−6 for octanal
might be assessed from the chemiluminescence (CL) and 1.33 × 10−4 for hexan-2-one. Determination of the
producing patterns in fresh beer before storage. The
presence of sulfite in beer depressed CL production dur-
ing its storage indicating that there is some contribution
of sulfite to flavor stability due to its inhibitory effect on
radical reactions (Kaneda et al., 1994). Formaldehyde-
and acetaldehyde-bisulfite adducts inhibited the chemi-
luminescence production of beer and Cypridina lucifern
analog dependent luminescence in beer, indicating that Figure 2 − Formation of α-hydroxysulfonates by the addition reaction
they inhibit free radical reactions. Therefore, it could be of sulfite to the carbonyl group.

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Guido Sulfites in beer: a review

predicted sulfite binding power was carried out by Lea The protective effect of sulfite against beer stal-
and co-authors. They found equilibrium constants of 1.6 ing is not a passive event. During fermentation, the
× 10−4 for pyruvate, 1.8 × 10−2 for galacturonic acid and bisulfite excreted by yeast can potentially bind to the
6.4 × 10−1 for glucose (Lea et al., 2000). The equilibrium carbonyl compounds present in the medium, and pre-
constants remain fairly constant between pH 2 to 6, a vent them from being reduced by yeast into the corre-
pH range that encompasses all beers. At pH > 7 dis- sponding alcohols. These adducts might pass into beer,
sociation of adducts is favored to give the free carbonyl. where they progressively break down to free the car-
Beer flavor instability is caused by the formation of bonyls which render the beer stale. Thus, brewers have
volatile, long chain, unsaturated carbonyls with low fla- contradictory opinions on the efficiency of naturally
vor thresholds and unpleasant flavors. Long chain unsatu- produced bisulfite by yeast as compared to that added
rated aldehydes, such as E-2-nonenal which contributes to the beer before bottling (Dufour et al., 1999). Partial
a cardboard-like flavor, are prime contributors (Huige, or full elimination of MET10 gene activity in brewer's
2009). Sulfite, produced by yeast during fermentation or yeast resulted in increased sulfite accumulation. Beer
added before bottling, readily form adducts with carbonyl produced with such yeasts was quite satisfactory and
compounds, rendering them nonvolatile and flavour-in- showed increased flavor stability (Hansen and Kielland-
active (Nyborg et al., 1999). As a result, SO2 plays a role Brandt, 1996).
in masking the stale flavors that develop in beer during Acetaldehyde has a far greater capacity for binding
storage. Appearance of the cardboard flavor in aged beer bisulfite than have the staling aldehydes and its pres-
is strongly retarded when such beers are supplemented ence interferes with the ability of agents to bind staling
with sulfite. The papery, cardboard stage of beer staling substances (Bamforth, 2000). Studies on the behavior
was ascribed to a decrease in bisulfite concentration and of sulfites during fermentation and storage of beer re-
subsequent bisulfite transfer from unsaturated aldehyde vealed that acetaldehyde-bisulfite adduct and free sul-
bisulfite addition complexes (Barker et al., 1983). Nyborg fite produced during fermentation are oxidized by free
and co-authors demonstrated that flavor-active E-2-none- radical reactions during beer storage and inhibit the
nal has disappeared from the beer upon addition of sul- oxidation of the other beer components (Kaneda et al.,
fite, suggesting the formation of flavor-inactive adducts 1996a; Kaneda et al., 1996b). These observations most
(Nyborg et al., 1999). They confirmed the adduct forma- likely explain the increase obtained for the ratio of acet-
tion between E-2-nonenal and sulfite in aqueous solution aldehyde and SO2 during extended storage of beer. Us-
at beer pH indirectly by 1H NMR spectroscopy and direct- ing a voltammetric-based approach, the formation of an
ly by liquid chromatography coupled to mass spectrom- acetaldehyde-sulfite adduct was followed, and showed
etry (LC-MS). Dufour and co-authors have showed, using that the reaction is rapid and equilibrium is reached af-
the unsaturated aldehyde E-2-butenal to model the behav- ter 30 minutes (Guido et al., 2003).
iour of E-2-nonenal, that the mechanism of the addition The complexity of carbonyl-bisulfite adducts for-
reaction proceeds by a two-step process, to initially give mation is enhanced if the release mechanism of the
a carbonyl adduct and ultimately yield a disulfonate as carbonyl compounds from their adducts with amino
the thermodynamic product, as illustrated in the Figure 3 acids and proteins of the wort is considered. Within
(Dufour et al., 1999). A reversible binding occurs between the pH range of wort, a Schiff base formation is pro-
the carbonyl functional group and bisulfite, whereas the posed between carbonyl compounds and either free
addition of bisulfite to the double bound of unsaturated amino acids or proteins (Noel et al., 1999; Lermusieau
aldehydes was shown to be irreversible. The stability of et al., 1999). 1H-NMR evidence of model systems sug-
such adducts does not support the generally accepted gest that N-adducts of alkenals, Michael adducts and
mechanism for the release of unsaturated aldehydes from Schiff bases, are relatively unstable particularly in the
non-volatile species. presence of bisulfite (Dufour et al., 2000). Formation of
carbonyl-adducts and their subsequent release appears
to interact in a complicated and dynamic way. Various
linked equilibria, including those involving sulfite, are
responsible for the release of carbonyl compounds and,
thus, for the stale character typical of aged beers.

6. The dualistic mode of action of sulfites

In terms of the contribution of sulfite to control
flavor instability of beer, a dualistic mode of action has
been proposed (Figure 4). Firstly, sulfite inhibits beer ox-
idation during storage by acting as an antioxidant. Sulfite
has radical scavenging activity and avoids the free radi-
cal chain reactions during beer storage, and contributes
Figure 3 − Equilibria of E-2-butenal based on the 1H-NMR kinetic data to the stability of beer quality. Secondly, sulfite can re-
proposed by Dufour et al., (1999). act with the carbonyl staling compounds in beer, which

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 195

Guido Sulfites in beer: a review

Andersen, M.L.; Outtrup, H.; Skibsted, L.H. 2000. Potential

antioxidants in beer assessed by ESR spin trapping. Journal of
Agricultural and Food Chemistry 48: 3106-3111.
Andersen, M.L.; Skibsted, L.H. 1998. Electron spin resonance spin
trapping identification of radicals formed during aerobic forced
aging of beer. Journal of Agricultural and Food Chemistry 46:
1272-1275.
Atanassov, G.; Lima, R.C.; Mesquita, R.B.R.; Rangel, A.O.S.S.;
Tóth, I.V. 2000. Spectrophotometric determination of carbon
dioxide and sulphur dioxide in wines by flow injection.
Figure 4 − The dualistic mode of action of sulfites to control the Analusis 28: 77-82.
flavor instability of beer. Azevedo, L.C.; Reis, M.M.; Motta, L.F.; Rocha, G.O.; Silva, L.A.;
Andrade, J.B. 2007. Evaluation of the formation and stability
of hydroxyalkylsulfonic acids in wines. Journal of Agricultural
masks stale flavors. Adducts formed are non-volatile and Food Chemistry 55: 8670-8680.
and, therefore, have much higher flavor thresholds than Back, W.; Forster, C.; Krottenthaler, M.; Lehmann, J.; Sacher,
free carbonyls. B.; Thum, B. 1999. New research findings on improving taste
The most effective role of SO2 in delaying flavor stability. Brauwelt International 5: 394-405.
staling of beer, beit acting as an antioxidant or mask- Bamforth, C.W. 1999. The science and understanding of the
ing stale flavors, is still an open question. Sulfur dioxide flavour stability of beer: a critical assessment. Brauwelt
is progressively lost from beer in a first order reaction. International 2: 98-110.
The constant rate for the reaction increased as the tem- Bamforth, C.W. 2000. Making Sense of Flavor Change in Beer.
perature increased in the range of 0 to 40 ºC and was MBAA Technical Quarterly 37: 165-171.
dependent to some extent, on the initial SO2 concentra- Bamforth, C.W. 2001. Oxido-reduction processes and active forms
tion. Storage of beer at 0 ºC considerably reduced the of oxygen in aqueous systems. Cerevisia 26: 149-154.
rate of SO2 loss, but did not prevent it (Ilett and Simpson, Bamforth, C.W.; Muller, R.E.; Walker, M.D. 1993. Oxygen and
1995). Lowering the temperature is, thus, the main mea- oxygen radicals in malting and brewing: a review. Journal of
sure that must be adopted by the brewer in an attempt the American Society of Brewing Chemists 51: 79-81.
to minimize the loss of SO2 and to successfully preserve Barker, R.L.; Gracey, D.E.F.; Irwin, A.J.; Pipasts, P.; Leiska, E.
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Journal of the Institute of Brewing 89: 411-415.
Acknowledgements Bendtsen, A.B.; Jorgensen, S.S. 1994. Determination of total and
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The author is grateful to the Foundation for Sci- Journal of AOAC International 77: 948-951.
ence and Technology Portugal (FCT) for his Sabbatical Bubnis, B. 1988. Methods of determining sulfites in beer and
Leave Grant (SFRH/BSAB/1272/2012). This study also other beverages. Brewers' Digest 63: 20-21.
received financial support from the European Union Burmeister, M.S.; Drummond, C.J.; Pfisterer, E.A.; Hysert, D.W.
(FEDER funds) under the framework of QREN through 1992. Measurement of volatile sulfur compounds in beer
Project NORTE-07-0124-FEDER-000069. using gas chromatography with a sulfur chemiluminescence
detector. Journal of the American Society of Brewing Chemists
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