Chapter 5
Additives: Preservatives
Francisco Jiménez-Colmenero and José Blázquez Solana
Contents
5.1 Introduction ......................................................................................................................91
5.2 General Considerations Regarding Analytical Methods ................................................... 93
5.3 Sorbates, Benzoates, and p-Hydroxybenzoate Esters ........................................................ 94
5.3.1 Analytical Methods .............................................................................................. 94
5.4 Sulfites ............................................................................................................................. 97
5.4.1 Analytical Methods .............................................................................................. 97
5.5 Nitrite and Nitrate ......................................................................................................... 100
5.5.1 Analytical Methods ............................................................................................ 100
5.6 Concluding Remarks ......................................................................................................102
References ................................................................................................................................103
5.1 Introduction
Processed meats and poultry are extremely perishable products, and one of the principal agents of
their spoilage is microorganisms (bacteria, yeasts, and molds). Microorganisms cause nutritional
and sensory deterioration of meat products, producing loss of quality and limiting shelf life. Besides
the economic implications of meat spoilage (deterioration of raw materials and processed products
before they can be sold, loss of brand image, etc.), microorganisms can also be responsible for
human illness.
A variety of preservation procedures have been tried to limit the speed and extent of such
processes and their consequences. Traditionally, much use has been made of physical, chemical,
and microbial methods of preservation. Preserving processed meats by chemical means is based,
among other possibilities, on the use of additives known as preservatives. Preservatives are chemical
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Table 5.1
E No.
E-200
E-202
E-203
E-210
E-211
E-212
E-213
E-214
E-215
Preservatives Permitted in the EU for Use in Meat Products
Formula
C6H8O2
C6H7O2K
(C6H7O2)2Ca
C7H6O2
C7H5O2Na
C7H5O2K
(C7H5O2)2Ca
C9H10O3
C9H10O3Na
E-218 C8H8O3
E-219 C8H8O3Na
a
b
c
d
Name
a
Sorbic acid
Potassium sorbatea
Calcium sorbatea
Benzoic acidc
Sodium benzoatec
Potassium benzoatec
Calcium benzoatec
Ethyl-p-hydroxybenzoated
Sodium ethyl-phydroxybenzoated
Methyl-p-hydroxybenzoated
Sodium methyl-phydroxybenzoated
E No.
Formula
Name
E-220
E-221
E-222
E-223
E-224
E-226
E-227
E-228
E-249
SO2
Na2SO3
NaHSO3
Na2S2O5
K2S2O5
CaSO3
Ca(HSO3)2
KHSO3
KNO2
Sulphur dioxideb
Sodium sulfiteb
Sodium hydrogen sulfiteb
Sodium metabisulfiteb
Potassium metabisulfiteb
Calcium sulfiteb
Calcium hydrogen sulfiteb
Potassium hydrogen sulfiteb
Potassium nitrite
E-250 NaNO2
E-251 NaNO3
E-252 KNO3
Sodium nitrite
Sodium nitrate
Potassium nitrate
Abbreviation for this group: Sa.
Abbreviation for this group: SO2. An SO2 content of not more than 10 mg/kg is not considered
to be present.
Abbreviation for this group: Ba.
Abbreviation for this group: PHB.
Note: The European Food Safety Authority2 has recommended withdrawal of approval for propyl
paraben; propyl-p-hydroxybenzoate (E-216), and sodium propyl-p-hydroxybenzoate (E-217).
E number (E No.) is used to classify food additive and signifies approval of an additive by
the European Union.
Source: Directive No. 95/2/EC of the European Parliament and of the Council of February 20, 1995
on food additives other than colors and sweeteners; Directive 2006/52/EC of the European
Parliament and of the Council of 5 July amending Directive 95/2/EC on food additives other
than colors and sweeteners and Directive 94/35/EC on sweeteners for use in foodstuffs.
compounds that, when added to foods, inhibit, retard, or prevent the activity and growth of spoilage
and pathogenic microorganisms. Their chief purposes are to extend the shelf life of foodstuff s by
protecting them against deterioration caused by microorganisms, and to enhance their safety.
Control and regulation of the use of preservatives is essential both to ensure their effectiveness and
because in inappropriate amounts and conditions these additives can have adverse health effects.
The use of preservatives is therefore subject to strict legal regulation to protect consumers, who are
increasingly aware of aspects of food that affect health, most especially the presence of additives,
and among these preservatives. Legislative requirements relating to the use of preservatives in
meat products are regulated by the European Union (EU) through various European Community
Directives1,2 (Tables 5.1 and 5.2).
In view of the importance of preservatives in terms of the law and food safety, it is essential
to have accurate analytical methods. A variety of published analytical methods are available in
the literature, mostly cited in reviews relating to their application to food analysis.3–5 However,
depending on the preservative, such reviews are not generally concerned specifically with analytical
methods for processed meats and poultry. This chapter considers the published methodology
available for determining permitted preservatives designed for use in meat matrices.
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Table 5.2
93
Meat Products and Levels of Conditionally Permitted Preservatives in the EU
Meat Products
Surface treatment of dried
meat products
Jelly coating of meat products
(cooked, cured, or dried); pâté
Surface treatment of dried,
cured sausages
Fresh “longaniza” and fresh
“botifarra” sausage
Burger meat with a minimum
vegetable and cereal content
of 4%
Breakfast sausages
Meat products
Sterilized meat products (F0 > 3)
Traditional immersion-cured
meat products
Other traditionally cured meat
products
Non–heat-treated meat
products
a
䡲
Preservativea
Maximum Level (mg/kg)
Maximum
Residual
Level (mg/kg)
Sa + Ba + PHB
Quantum satis
—
Sa + PHB
1000
—
E-235
—
SO2
1 mg/dm2 surface (not present
at a depth of 5 mm)
450
—
SO2
450
—
SO2
E-249 and E-250
E-249 and E-250
E-249 and E-250
E-251 and E-252
E-249 and E-250
E-251 and E-252
E-251 and E-252
450
150
100
—
—
—
50–175
10–250
0–50
10–250
—
0–300
0–180
250–300
150
For abbreviations see Table 5.1. Sa + Ba + PHB: Sa, Ba, and PHB used separately or in combination. For these preservatives the indicated maximum use levels refer to ready-to-eat foodstuffs
prepared following manufacturers’ instructions.
Note: For E-249, E-250, E-251, and E-252, maximum use levels refer to the maximum amount that
may be added during manufacture.
Source: Directive No. 95/2/EC of the European Parliament and of the Council of February 20, 1995
on food additives other than colors and sweeteners; Directive 2006/52/EC of the European
Parliament and of the Council of 5 July amending Directive 95/2/EC on food additives other
than colors and sweeteners and Directive 94/35/EC on sweeteners for use in foodstuffs.
5.2
General Considerations Regarding Analytical Methods
There are many methods available for the analysis of preservatives in foods. Although they vary
according to the preservative, in many cases the methodologies are not specific to meat matrices;
this means that further development to adapt them to processed meat and poultry analysis is
required. In general, the choice of an analytical method must take into account the means available,
the selectivity and sensitivity necessary to achieve the required level of detection in a complex
matrix, and the possibility of high throughput analysis. Versatility and minimal requirements
for sample preparation and handling are also very useful. Several preservatives are frequently
added simultaneously, and therefore the preferred methods will be those that allow for the analysis
of several preservatives in a single operation, especially all the compounds in the same family.
Quantitative analysis may be the ultimate objective in most cases, but there are many occasions
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when it may be enough to use a qualitative method that checks for the absence of the additive
(e.g., control of raw materials or verification of labeling in consumer products).
Generally speaking, there are two distinct stages in the methods used to determine
preservatives: (a) extraction of the preservative(s), frequently followed by a cleanup procedure to
eliminate interferences; and (b) separation, identification, and quantification of the preservative(s).
Preservative extraction in meat products can be complex due to diversity of properties and of
modes of interaction between functional groups and components of food matrix and solvent
systems. A variety of different separation methodologies and detection systems have been used for
determination of preservatives.
5.3
Sorbates, Benzoates, and p-Hydroxybenzoate Esters
Sorbic acid, benzoic acid, and the methyl and ethyl esters of 4-hydroxybenzoic acid (parabens)
or their salts are organic acids widely used as preservatives, and present the antimicrobial activity
typical of undissociated acids. They act as effective antimicrobial agents (e.g., mold and yeast
growth inhibitors) in meat. They are allowed in the surface treatment and jelly coating of many
processed meats, used singly or in combination (sorbates, benzoates, and parabens in concentrations ranging from 1000 mg/kg to quantum satis), and in pâtés (sorbates and parabens used singly
or in combination), in the latter case up to a maximum level of 1000 mg/kg (Table 5.2). The
acceptable daily intake (ADI) is 25 mg/kg for sorbic acid6 and 5 mg/kg body weight for benzoic
acid.7 The European Food Safety Authority has established a full-group ADI of 10 mg/kg body
weight for the sum of methyl and ethyl-p-hydroxybenzoic acid and esters and their sodium salts.2
5.3.1 Analytical Methods
Methods that have been reported for the determination of organic preservatives in foods
include spectrophotometry, thin-layer chromatography (TLC), gas chromatography (GC), highperformance liquid chromatography (HPLC), capillary electrophoresis (CE), and others.4,5,8 Most
published studies on methodology for the determination of these preservatives have been conducted
on beverages and dairy products; there are very few references to their specific application in meat
products.
Extraction procedure. Sorbates, benzoates, and parabens show moderate reactivity and can
easily be isolated from food and beverage matrices.5 However, depending on the type of food
matrix and the determination methodology, efficient sample cleanup procedures are essential
to eliminate various interferences in the matrix (e.g., proteins, fats, and polysaccharides).
Generally speaking, solid, complex matrices (such as meat products) require more cleanup.
Specific extraction methods for analysis of these preservatives in food matrices apply some
of the following procedures: direct extraction of an acidified sample by an organic solvent,
solid-phase extraction (SPE), extraction as an ion pair, and steam distillation.5,8–11
Spectrophotometric methods. Sorbic acid, benzoic acid, and parabens present strong ultraviolet
(UV) absorption, so UV detection is the method most commonly used. However, the
sensitivity of detection differs as a result of the considerable differences between them in
maximum absorbances.5 Benzoate and sorbate determination in ground beef12 includes
extraction with water, which is mixed with HCl and petroleum ether followed by
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Table 5.3 Examples of Official and Internationally Recommended Methods
for Determination of Preservatives in Meat Products
Preservative
Methods
Matrix
Saa + Baa
Ba
Sa + Ba + sulfites
Sulfurous acid (free)
Sulfurous acid
Sulfites
Sulfurous acid (total)
Sulfite (total)
Sulfites
Sulfites (total)
Sulfite (total)
GC
TLC
Spectrophotometry
Titrimetric
Color
Color
Modified Monier-Williams
Optimized Monier-Williams
Enzymatic
DPP
FIA
Sulfites
Ion-exclusion
chromatography
Colorimetry
Xylenol
Spectrophotometric
Ion-exchange
chromatography
Food
Food
Ground beef
Meats
Food
Meats
Food
Foodstuffs
Foodstuffs
Food
Foods and
beverages
Foods and
beverages
Cured meat
Meat
Meat products
Meat products
Nitrites
Nitrates and nitrites
Nitrate and nitrite
Nitrate and nitrite
a
Applicable to
Determination
of (mg/kg)
Reference
—
—
—
—
Qualitative
Qualitative
—
≥10
—
≥10
≥5
14
15
12
16
17
18
19
20,21
22
23
24
≥10
25
—
—
—
Nitrite >40
26
27
28
29
For meanings of these abbreviations see Table 5.1.
measurement of absorbance at 250 nm for sorbates and 225 nm for benzoates (Table 5.3).
Because this type of aqueous extraction or steam distillation gives relatively poor recoveries
(sorbic acid) when applied to raw beef, Campos et al.13 described an improved procedure for
sorbic extraction based on disintegration and dispersion of raw beef with sand prior to steam
distillation and determination by UV absorption.
Thin-layer chromatographic methods. TLC and high-performance thin-layer chromatography
(HPTLC) have been used in qualitative and quantitative determination of preservatives.
Qualitative determination of benzoates and hydroxybenzoates in foods (including seafoods)
based on TLC separation was developed by Pinella et al.30 These authors also described the
quantitative determination of benzoic acid by steam distillation, extraction (ethanol) after
the TLC separation, and determination in the UV region (310–205 nm). A similar procedure
is reported as an Association of Official Analytical Chemists (AOAC) method15 for benzoic
acid determination in food (Table 5.3). Quantitative (TLC and HPTLC) methods for
determination of mixtures of benzoic acid and sorbic acid without an extraction or cleanup
step,31 or previously isolated and concentrated by SPE,32 have been applied in beverages.
Gas chromatographic methods. GC, with or without derivatization, has been widely employed
for the determination of sorbates, benzoates, and parabens in foods and beverages.4,5,33
GC methods are sensitive, specific, and accurate, but may require lengthy extraction
prior to GC analysis. Sample pretreatment prior to GC analysis involves organic solvent
extraction, precipitation of protein, liquid–liquid extraction, SPE,33,34 headspace solid-phase
microextraction,35 or stir-bar sorptive extraction.36 An AOAC GC method14 (Table 5.3) for
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determination of benzoic acid and sorbic acid in foods involves extraction with ether and
successive partitionings into aqueous NaOH and CH2Cl2, derivatization to a trimethylsilyl
ester, and flame ionization detection (FID). A GC-mass spectrometric technique has been
reported for the simultaneous determination of sorbic acid, benzoic acid, and parabens
in foods.37 A new application of pyrolytic methylation has been developed to determine
benzoic acid in soft drinks by GC without any pretreatment and using a special pyrolyzer.
Samples containing benzoic acid could be accurately determined by direct-injection GC on
the medium polar stationary phase column.38
Specific GC determination of benzoates in meat products has been reported.39,40
Simultaneous GC determination of preservatives (sorbic acid, benzoic acid, and their esters)
in fatty foods (pâté) without derivatization has been described.41 Sample pretreatment
includes solvent extraction and SPE. GC-FID for quantification and mass spectrometry has
been used in the conventional electron impact mode for identification. Sorbates, benzoates,
and parabens were simultaneously analyzed in cured meat products by GC-FID.42
High performance liquid chromatographic methods. Determination of preservatives by HPLC
offers high specificity with minimal preparation and does not require derivatization.
Numerous HPLC methods for simultaneous determination of benzoic acid, sorbic acid,
and parabens have been reported as applicable to selected foodstuffs.4,5,43 In general, the
extraction system of the applicable procedures varies according to the complexity and
composition of the foodstuffs, and may include clarification/purification, which is essential
for eliminating high–molecular-mass matrix interferences (e.g., proteins, fats, and polysaccharides). Sample pretreatment prior to HPLC analysis involves solvent extraction,
filtration, centrifugation, and SPE. Separation is done essentially by reversed-phase HPLC
with UV detection at the wavelengths of maximum absorption of the compounds. Different
eluents have been used, including phosphate buffer, methanol, tetrahydrofuran, acetate
buffer, and acetonitrile.4,43–46
Nowadays HPLC is the most common analytical procedure for the detection and
quantification of these preservatives in foods, although there are very few published
analytical methods that are specifically applicable to meat systems. Ali47 reported that
benzoic acid, sorbic acid, and parabens were extracted from meat with 70% ethanol. After
filtration, extracts were analyzed using reversed-phase liquid chromatography. An analytical
procedure has been developed for the analysis of benzoic acid; p-hydroxybenzoic acid; and
methyl-, ethyl-, propyl-, isopropyl-, and butyl esters of p-hydroxybenzoic acid by micellar
liquid chromatography in food samples, including chicken spread.48
Capillary electrophoretic methods. CE has recently been employed as an efficient tool for
preservative determination in food due to its many advantages, which include high separation
efficiency, excellent resolution, and short analysis time. Various CE methods, such as
capillary zone electrophoresis, micellar electrokinetic chromatography (MEKC), and microemulsion electrokinetic chromatography (MEEKC), have been reported for determination
of preservatives in foods. These methods have generally been used on carbohydrate-rich
matrices (soft drinks, wine, jam, soy sauce, etc.). In most cases, real samples cannot be
injected directly into CE systems, and an extraction cleanup process is necessary. Th is stage
can be even more necessary in complex matrices (protein, fat, etc.), like meat derivatives, for
which no published methods have been found.
Kuo and Hsieh9 described a CE method for the simultaneous separation of nine
preservatives, including benzoic acid, sorbic acid, p-hydroxybenzoic acid, and six alkyl esters of
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p-hydroxybenzoic acid in plum preserves, bean curd, and soy sauce. MEKC has been
successfully used to simultaneously analyze p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid ethyl ester, benzoic acid, and sorbic acid.49 Huang et al.11 used the MEEKC
method to separate parabens (methyl, ethyl, propyl, and butyl), sorbic acid, and benzoic acid
in various food products (soft drinks, soy sauces, and wines). The separation and detection of
benzoate and sorbate in soft drinks by both conventional CE and microchip electrophoresis
with capacitively coupled contactless conductivity detection has been reported.50 Capillary
electrochromatography, a hybrid separation technique that combines the features of HPLC
and CE, has been used to analyze sorbic acid, benzoic acid, and parabens in some products
(cold syrups, lotions, soy sauces, and wines).10
Enzymatic determination. A method for the determination of sorbic acid based on spectrophotometric measurement of sorbyl coenzyme A at 300 nm has been reported.51 The method
has been tested for various food matrices (wine, alcoholic and nonalcoholic beverages, fruit
preserves, and tomato ketchup).
5.4 Sulfites
Sulfur dioxide and sulfites comprise the group of compounds known collectively as sulfites
(Table 5.1). These sulfiting agents, or S(IV) oxoanion compounds, are considered relatively strong
preservatives, because of their strong antimicrobial activity. Moreover, even a small amount of
sulfite in meat imparts a bright red color. These compounds are not permitted for use in meat
in the United States. In the European Community, the maximum permitted amount of added
sulfiting agents in the various different meat products is 450 mg/kg, expressed as SO2 (Table 5.2).
Sulfites have been associated with allergic reactions and food intolerance symptoms. They are
known to degrade thiamine (vitamin B1), of which meat is a good source. The ADI for sulfite
(expressed as SO2) is 0.7 mg/kg body weight.52
When added to a food matrix, some of the sulfiting agents bind to different components of
food. The portion of sulfiting agent that does not combine with the food is called free sulfite.
Bound sulfite can be categorized as reversibly or irreversibly bound sulfite. The relative presence
of each one varies according to the reactivity of sulfur dioxide in meat systems; this in turn is
associated with factors involved with composition or with processing and storage conditions. For
instance, following incorporation of additives to meat products, there can be irreversible losses
of as much as 50%, depending on these factors.53,54 Cooking meat products also causes sulfite
reduction.55 Then again, sulfite ions may cleave disulfide bonds in meat proteins.54 The analytical
determination of sulfite, then, does not reflect the preservatives that were initially added.
5.4.1 Analytical Methods
Many analytical methods have been reported for sulfite determination in foods and beverages.
These methods include titrimetry, spectrophotometry, enzymatic analysis, chromatography, flow
injection analysis (FIA), and electroanalysis.5,56,57 However, not all of them are equally suitable
for the determination of sulfites in solid, complex protein matrices such as processed meats, where
sulfite-binding problems may arise from interaction with other food components or entrapment
within food particles.56,58
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Extraction procedure. Analytical determination requires some means of removing and recovering
the sulfur dioxide (free and reversibly bound) and then quantifying the level found. Sample
preparation and analysis should be as rapid as possible to avoid loss of labile forms of sulfite.
Numerous procedures utilize the Monier-Williams distillation process for sulfite separation
by means of distillation from a suspension or solution of the food in hot HCl. In some cases,
Monier-Williams distillation has been used as a preparatory tool for obtaining free and
bound sulfite fractions to accommodate more selective quantitation techniques, whereas in
others extraction has been used instead of distillation.56 Other procedures that do not utilize
distillation or vapor phase transfer require the conversion of sulfite to a stabilized sulfite
derivative compound to take advantage of some property that serves for quantitation.
Titrimetry (Monier-Williams) method. This procedure, derived from the classic studies of
Monier-Williams, measures free sulfite plus the reproducible portion of bound sulfite. It
is based on acid distillation followed by vapor phase transfer of the SO2, facilitated by a
carrier gas stream, to an oxide-trapping solution. The sulfur dioxide is oxidized to sulfuric
acid for quantitation by titration or determination of sulfite by precipitation with barium.
The sulfuric acid is stoichiometrically related to the sulfur dioxide distilled from the test
solution (modified Monier-Williams method)19 (Table 5.3). An optimized Monier-Williams
method 20 for the analysis of sulfites in foods has been collaboratively tested and accepted as
an official method (Table 5.3). Very few foods not treated with sulfite give a false positive
even at levels below 10 ppm; Allium and Brassica vegetables and isolated soy protein are
important exceptions to this rule. As a comparative procedure, this methodology has been
assayed in different muscle foods including beef, pork, and chicken meat products.53,55
Monier-Williams methods are the ones most commonly employed by food control laboratories for meat product analysis (Table 5.4). A method based on distilling of sample in
an acidic medium followed by iodometric titration has also been also assayed (Table 5.4),
although its use is limited to products with high levels of sulfite.56
Spectrophotometric methods. Various spectrophotometric procedures have been reported for
determination of sulfiting agents in foods. An AOAC method12 (Table 5.3) for sulfite
Table 5.4 Examples of the Analytical Methods Used for Meat Product Preservative
Determination in Evaluation of Proficiency Testing of Analytical Laboratories
(Interlaboratory Comparisons)
Sulfitea in Pork Sausage
(Number of Laboratories Participating: 75)
Analytical Methods
Modified Monier-Williams
Optimized Monier-Williams
Distillation and titration
with iodine
Spectrophotometry
IC
Others
a
b
Nitriteb in Gammon Steak
(Number of Laboratories Participating: 101)
Used by
Laboratories (%)
Analytical
Methods
Used by
Laboratories (%)
43.9
28.1
15.8
Colorimetry
IC
HPLC
52.2
21.7
18.5
8.8
1.8
1.8
FIA
Enzymatic
CE
4.4
2.2
1.1
Food Analysis Performance Assessment Scheme (FAPAS), Proficiency Test 2046. August–October
2006.
FAPAS, Proficiency Test 1547. January–February 2007.
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determination in ground beef is based on a colorimetric reaction with p-rosaniline after
reaction with mercuric extractant. Another method often used for determination of sulfur
dioxide in meat products is based on distillation/spectrophotometric analysis using 5,5′
di-thiobis-(2-nitrobenzoic acid) (DTNB). It has been used in fresh sausages,53 and comminuted pork meat.54 Determination of sulfites has been performed in commercial sausages
by direct extraction and spectrophotometric methods based on a step reaction using the
reagent DTNB.58
Diff erential pulse polarographic method. A method based on differential pulse polarography
(DPP) and applicable to the determination of sulfites (total) in foods has been proposed 23
(Table 5.3). The method, based on a collaborative study,59 was tested on a number of muscle
foods including shrimp. It measures SO2, which is purged with N2 from acidified test suspension, collected in electrolyte-trapping solution, and then determined by DPP. Modified
Monier-Williams distillation followed by DPP has also been used to determine sulfiting
agents in foods.60
Chromatographic methods. Anion exclusion chromatography has proven a useful technique
for determining sulfites in foods. An electrochemical detection system is the most commonly
used,55,61–63 although conductivity detection64,65 and direct UV detection have been also
reported.66 In the AOAC chromatographic method 25 (Table 5.3), SO2 is released by direct
alkali extraction, followed by anion exclusion chromatographic separation and electrochemical (amperometric) detection.62,67 The method was tested on different food matrices,
including a muscle food (dehydrated seafood). Improvements in amperometric detection of
sulfite in food matrices have been reported.68
Although chromatographic methods may be appropriate for use on meat products, very
little has been published in the literature in that respect. Free and total sulfite have been
determined in fresh sausages by HPLC;63 the method includes extraction of both free and
total sulfite by dissolution of the sample in a suitable solvent and determination by HPLC
(anion exclusion column) using electrochemical detection. This procedure was also used to
determine sulfite content in fresh and cooked (beef, pork, and chicken) burgers.55
Enzymatic methods. Enzymatic methods have been developed for sulfite analysis in food. In
these procedures, sulfite is usually oxidized to sulfate; this is catalyzed by sulfite oxidase to
release hydrogen peroxide, and the hydrogen peroxide is measured by linking it to the oxidation of reduced nicotinamide adenine dinucleotide (NADH) in the presence of NADH
peroxidase. Hydrogen peroxide was measured by spectrophotometry.22,56,69 Various sulfite
oxidase biosensors have also been reported70,71 (Table 5.3). Enzymatic methods have been
used to determine SO2 in muscle foods (shrimp).72
FIA. This method offers the advantages of simplicity and precision with a high analytical
sampling rate, while requiring only low-cost equipment, reducing the need for large volumes
of toxic reagents, and requiring little analysis time. Several FIA methods have been used for
sulfite determination in food and beverages,4,72,73 but few published reports can be found
dealing with their application to muscle foods, and most of these refer to shrimp.72,74 With
the support of an interlaboratory study,74 an AOAC method24 has been proposed for FIA
sulfite determination (Table 5.3) based on sulfite reaction with malachite green. A test
solution is made to react with NaOH to release aldehyde-bound sulfite; then, the test stream
is acidified to produce SO2 gas, which diff uses across a Teflon membrane in the gas diff usion
cell into a flowing stream of malachite green, which is discolored. With this procedure it
is possible to assay samples containing ingredients from liliaceae (garlic, onions, leeks) for
which the Monier-Williams reference method is not suitable.
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Capillary electrophoresis. Sulfite content in foods and beverages can be determined by CE. The
sulfite is converted to sulfur dioxide and finally to sulfate by Monier-Williams distillation.
The sulfate is then determined by CE. The results for sulfite content of seafood agree very
well with those determined by titrimetry.75
5.5 Nitrite and Nitrate
Sodium and potassium nitrates and sodium and potassium nitrites (Table 5.1) are used in meat
curing because they stabilize red meat color, inhibit some spoilage and food poisoning anaerobic
microorganisms, delay the development of oxidative rancidity, and contribute to flavor development.
Depending on the type of processed meat, processing conditions, presence of sodium ascorbate,
and other factors, the added nitrite reacts with many components in the matrix (myoglobin,
nonheme proteins, lipids), so that the analytical detection of the nitrite or nitrate content does not
reflect the preservative initially added. Analytical methods therefore usually determine the residual
nitrate/nitrite, which can reach only about 10–20% of the original nitrite amount added.76,77 To
detect bad practice and use of high nitrite levels, it is more effective to control nitrite at input.77
Nitrite levels in meat products are important because nitrite can react with secondary amines to
form nitrosamines, which are recognized as having carcinogenic effects. In the EU, potassium
and sodium nitrite and nitrate are authorized for use in different meat products, and maximum
ingoing amounts (150 mg/kg for nitrite and 300 mg/kg for nitrate) are established for all products,
as well as maximum residual levels for some of them (Table 5.2). Current regulations on use of
nitrite and nitrate in the United States vary depending on the curing method used and the product
that is cured. For comminuted products, the maximum ingoing nitrite and nitrate limits are
156 mg/kg and 1718 mg/kg, respectively. For immersion-cured and massaged or pumped products,
those limits are 200 mg/kg and 700 mg/kg, respectively. In dry-cured products nitrite is limited
to 625 ppm and nitrate to 2187 ppm.78 The ADI for nitrites, as nitrite ion, is 0.07 mg/kg body
weight, and 3.7 mg/kg for nitrate, as nitrate ion.79,80
5.5.1 Analytical Methods
Several methods have been reported for quantitative determination of nitrate and nitrite in
foods, including spectrophotometry, chromatography, electrochemical detection (potentiometry,
amperometry, polarography), CE, and others.4,5,81,82 However, not all of them are equally suitable
for use on highly heterogeneous solid matrices like processed meats and poultry.
Extraction procedure. Nitrite and nitrate determination requires an extraction stage, generally
involving dispersion in hot water. Meat products contain various compounds (ascorbic acid,
fat, protein, sodium chloride, etc.) that can interfere in nitrite and nitrate measurement, and
so a number of procedures have been tried to clean up the extracts prior to determination.
These include clarification stages (fat and protein precipitation, filtration, etc.) using different
compounds or solvents (Carrez or borax reagents, zinc sulfate or potassium ferrocyanide,
acetonitrile, and others) or pretreatment cartridges to remove sample matrix interferences.3,83
Another possibility is to separate the fat by centrifugation and rapid cooling followed by
in-line dialysis to remove protein and remaining fat. Obviously the choice of a specific
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procedure to clean up an extract prior to determination will depend on the analytical
method used.
Spectrometric methods. Of the methods available for quantitative determination of residual
nitrite in meat products, the most commonly used are based on colorimetric determination
using Griess diazotization, which involves the formation of azo dye produced by coupling
a diazonium salt with an aromatic amine or phenol. The diazo compound is formed when
nitrite (aqueous extract from meat) reacts with sulfanilamide and the coupling agent
N-(1-naphtyl) ethylenediamine-2HCl.3,4,81 The color that develops is measured spectrophotometrically (540 nm). The same reaction can be used to determine nitrate. To do this,
the relatively inert nitrate is reduced to nitrite, which can then be determined by Griess
diazotization. Nitrate can be reduced by chemical and enzymatic procedures. A variety of
agents have been investigated for chemical reduction,81 the most common arrangement
being a spongy cadmium column, which can achieve efficiencies of nitrate-to-nitrite conversion approaching 100%. Enzymatic reduction of nitrate to nitrite has been accomplished
with nicotinamide adenosine dinucleotide phosphate in the presence of the enzyme nitrate
reductase.28 Spectrophotometric methods involving the reduction of nitrate to nitrite and
subsequent colorimetric determination of nitrite with a diazo coupling reaction have been
adopted26,28 for meat products (Table 5.3). Nitrite and nitrate have also been determined in
meat products using m-xylenol27 (Table 5.3).
Other colorimetric reactions have been used to determine nitrites and nitrates in
meat products. A number of these are based on the reduction of phosphomolybdic acid
to phosphomolybdenum blue complex by sodium sulfide, which is oxidized by the addition of nitrite, causing a reduction in the intensity of the blue color and a reduction in
the absorbance measured at 814 nm;84 others are based on the catalytic effect of nitrite
on the oxidation of methyl red by bromate, and the absorbance is measured at 520 nm.85
A spectrofluorimetric method has been developed for nitrite determination in meat
systems.86
Enzymatic methods. Procedures based on enzymatic reduction coupled with spectrophotometric
detection can be used to determine nitrite and nitrate in meat samples. Nitrite is measured
enzymatically through its reaction with nitrite reductase coupled with NADH, and the
reaction is measured spectrophotometrically.87
FIA. Several FIA-based methodologies for the determination of nitrite and nitrate in meat
products have been reported. Most of the FIA methods that are used to simultaneously
determine nitrates and nitrites in meat products are based on a diazotization/coupling
reaction.88–91 There have also been other applications of FIA, based on the reduction of nitrite
and nitrate to nitric oxide followed by reaction with iron (II) and thiocyanate in an acid
medium to form FeSCNNO+ chromophore, which is measured at 460 nm;82 based on the
reaction of nitrite with safranine to form diazonium salt, which absorbs at 520 nm;92,93 based
on the catalytic effect of nitrite on the oxidation of gallocyanin by bromate and the decrease
in absorbance of the system at 530 nm;94 or based on gas phase molecular absorption UV
(205 nm) spectrophotometry.95 Spectrophotometric determination of nitrite and nitrate in
cured meat has been reported using sequential injection analysis, a feasible and mechanically
simpler alternative to FIA.96
An FIA method with flame atomic absorption spectrometry (FAAS) detection has been
used to determine nitrite and nitrate in meat products. It is based on the oxidation of nitrite
to nitrate using a manganese (IV) dioxide oxidant microcolumn, where the flow of the
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sample through the microcolumn reduces the MnO2 solid-phase reagent to Mn(II), which
is measured by FAAS.97
Chromatographic methods. Ion chromatography (IC) and HPLC methods for the detection of
nitrite and nitrate have been developed in pursuit of procedures that are faster, more accurate, and more sensitive than spectrophotometric methods.98 Derivatization protocols are
essential for GC, whereas it is relatively easy to insert the sample in most HPLC and IC
systems.81 However, in the case of meat matrices, some form of cleaning up of the extracts is
required to avoid interference, which makes such methods less attractive.
Separation techniques based on ion-exchange chromatography have been used to evaluate
residual nitrite and nitrate in various meat products using detection systems based on
conductivity,99,100 UV absorption,98,101 and bulk acoustics.102 Determination of nitrite in cured
meats by ion exclusion chromatography with electrochemical detection has been reported.103
Bianchi et al.99 reported determination of nitrates in 76 different pork meat products
using IC with a conductivity detector coupled to an anion micromembrane suppressor.
Commercial samples of ham and salami have been analyzed by IC with UV absorbance
(225 nm) detection.98 Nitrite and nitrates have been determined in muscle tissue (beef, pork,
horse, and chicken) and dry-cured meat by anion-exchange chromatography/conductivity
and mass spectrometry detection.100
IC has been used in a collaborative study to devise an alternative chromatographic method
for determining residual nitrite and nitrate in meat products.101 This method, which has
been accepted by the European Committee for Standardization29 (Table 5.3), is based on
extraction of nitrite and nitrate from the sample with hot water followed by treatment in
an aqueous solution with acetonitrile to remove any interfering substances. The nitrite and
nitrate contents of the solution are then determined by ion-exchange chromatography separation and UV detection at 205 nm.
HPLC techniques have also been reported for determination of nitrate and nitrite in processed meats.104–109 These analytical procedures vary in terms of the extraction conditions
and the need to limit interference by means of protein precipitation or sample processing
steps using reversed-phase or ion-exchange pretreatment cartridges.98 Similarly, there are
varying conditions of separation (ion-exchange or ion-pair reversed phase) and anion detection by UV absorption, conductivity, indirect photometry, fluorometry, chemiluminescence,
or electrochemical detection.109
Capillary electrophoretic methods. CE is a powerful separation technique for determination of
nitrite and nitrates.81 These methods has been used for simultaneous analysis of nitrite and
nitrite in meat products using UV detection.110,111
Electrochemical methods. Various electrochemical detection techniques based on amperometric,112,113 voltametric,114–116 or potentiometric117,118 procedures have been used in determining nitrites and nitrates in food samples. However, only a few were used in processed
meat and poultry analysis.112,114,117
5.6 Concluding Remarks
Preservatives in meat products need to be quantified to assure quality and compliance with legal regulations and to minimize the heath risk to consumers. There are numerous methods for determining
preservatives in muscle foods, but in some cases such procedures have not been assayed in highly
heterogeneous solid matrices like meat products. Because of the complexity of the additive/matrix,
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these methodologies generally require further development for use in processed meat and poultry
analysis. Generally speaking, to be suitable for use in laboratories (both official and private) for food
analysis, a procedure must meet standards of sensitivity, versatility, effectiveness, rapidity, and cost.
The number of such criteria helps to explain the diversity of analytical methodologies normally
chosen by food analysis laboratories to determine preservatives in meat products (Table 5.4).
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