Meat Science 80 (2008) 744752

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

The roles of pH extraction and colloidal protein solubility in the optimization of spectrophotometric nitrite determination in meat products via response surface methodology
F. Rincn a,*, B. Martnez b, R. Prez-Olmos c, A. Berzosa d
a

Universidad de Crdoba, Departamento de Bromatologa, Campus Rabanales, Edicio C-1, 14.014 Crdoba, Spain Estacin Tecnolgica de la Carne, Instituto Tecnolgico Agrario de Castilla y Len, 37.770 Guijuelo (Salamanca), Spain c Universidad del Pas Vasco, Laboratorio de Qumica Analtica, Plaza de la Casilla 3, 48.012 Bilbao, Spain d Centro Nacional de Alimentacin, Ctra. de Pozuelo a Majada-honda, 28.220 Madrid, Spain
b

a r t i c l e

i n f o

a b s t r a c t
The inuence of four critical factors such as sample weight/borax reagent ratio (BR factor), ascorbic acid content (AR factor), neutralization with HCl 1 N (NR factor) and stirring extraction time (SET factor), was investigate in order to nd the best conditions (optimization) to develop the ofcial ISO 2.918 spectrophotometric method to determine the residual nitrite content in meat products, using the response surface methodology (RSM) as optimization tool. The factors most strongly affecting nitrite determination in meat products are BR, NR and AR, due to their respective effects on pH extraction parameters and on the amount of colloidal protein present in the sample extract. At pH 6 6, for example, the extract though appearing clear and transparent to the analyst contains a considerable amount of hydrolyzed protein, which will severely interfere with measurements, generating false-positive results. The colloidal protein present in the extract (620 mg/g, corresponding in these working conditions to an OD340 value of 60.600) will lead to the recording of nitrite values greater than those actually present in the sample. In order to avoid these drawbacks, this paper proposes that the amount of borax added (BR) varies as a function of sample weight (WS), using the ratio WS/BR = 1.11. In order to monitor the analytical method, it is further recommended that pH be adjusted to 67 (lower protein solubility) and that colloidal protein levels be P20 mg/g, as conrmed by an OD340 value of P0.600. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 2 March 2007 Received in revised form 11 March 2008 Accepted 13 March 2008

Keywords: Nitrite Meat products Response surface methodology Ofcial methods of analysis

1. Introduction In addition to effectively inhibiting the growth and toxicogenic effect of Clostridium botulinum (Roberts, 1975), nitrite is responsible for the development of typical cured-meat color and avour, and also functions as an antioxidant (MacDougall, Mottram, & Rhodes, 1975). In many countries, however, the use of nitrite has been restricted for some years (Commission of the European Communities, 1995) due to its ability, under certain conditions, to react with amines to form carcinogenic nitrosamine compounds (Walters, 1992). The need for accurate measurement of nitrite levels in foods has long been recognised. Of the many methods available for measuring this additive, the most commonly used are based on the principle of diazotization of an aromatic amine followed by reaction of the diazonium compound with a coupling reagent to form an azo dye (Sen

* Corresponding author. Tel.: +34 9 57 212008; fax: +34 9 57 212000. E-mail address: frincon@uco.es (F. Rincn). 0309-1740/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.03.016

& Donaldson, 1978), possibly because spectroscopic procedures require relatively simple and inexpensive instrumentation. The mechanism involved in the colorimetric reaction is the nitrosation of sulphanilamide to form the diazonium salt, which then couples with 1-naphthyl-ethylene diamine to form a brilliant pink azo compound; it has been reported, however, that the presence of either ascorbic acid (Nicholas & Fox, 1973) or protein (Fox, Doerr, & Lakritz, 1982) may interfere with this reaction. The technique consists in extraction of nitrite, purication using various cleanup procedures, addition of colorimetric reagents and spectrophotometric determination of color intensity. On the basis of this principle, the ISO 2918 analytical method was developed (ISO, 1975) and this was adopted as ofcial method in Spain (Presidencia de Gobierno, 1979). However, there is considerable disagreement regarding the parameters to be considered critical for the development of a standard analytical method (Rincn, Martnez, & Delgado, 2003). A number of parameters have been considered with a view to enhancing standard methods for measuring nitrite levels in food using a one-variable-at-a-time strategy (Binstok, Campos, &

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Gerschenson, 1996; Pringuez, Saude, & Hulent, 1995); drawbacks to this strategy, however, include inability to chart interactions, inability to determine optimum values, and inefciency of experimentation (Joglekar & May, 1987). To avoid these disadvantages, a previous paper (Rincn et al., 2003) showed that among the major inuencing factors (IF) are the Carrez reagent and the sample weight/borate reagent ratio. Having identied the IF, the next step must be their optimization, i.e. in order to standardize nitrite determinations, it is essential to identify the optimal set of IF values that will ensure not only efcient extraction but also the most sensitive and efcient quantication by obtaining the greatest analytical response for a given nitrite level. Validation of an analytical method through a series of experiments may be used to ascertain whether the method is really suitable for its intended purposes (Ye, Liu, Ren, & Okafo, 2000). At the same time, response surface methodology, a collection of statistical and mathematical techniques useful for developing, improving, and optimizing process (Myers and Montgomery, 1995a,b), has been used successfully to ne-tune other analytical methods (Lombardi-Boccia, Martnez, Aguzzi, & Rincn, 2002; Martinez & Rincn, 1997; Martnez, Rincn, & Ibez, 2000). The aim of this study was to identify the best combination of IFs in order to standardize nitrite measurements in meat products. 2. Materials and methods 2.1. Sample preparation Since the aim was to ascertain the best conditions in which to develop the analytical method, it was necessary to identify those analytical conditions ensuring the greatest response. However, since the sample extract contains a number of interfering substances such as protein or ascorbic acid, the highest absorbance is not the most accurate way of identifying the best IF combination and so optimize the analytical method. In such cases, it is considered advisable to add to the sample a known amount of the analyte (Davis, McKeegan, Cardos, & Vaughan, 1999). Fresh loin meat, purchased in a local market, was homogenized twice; 150 g of homogenate was then diluted in 240 ml of nitrite solution at a concentration of 50 mg/L and slurred for 10 min at room temperature. This yielded 390 g of slurry with a nitrite concentration of 30.8 ppm (mg/kg). The homogenized mixture was stored in a screw-cap glass container at room temperature until extraction, but always for less than 2 h in order to prevent detectable changes in nitrite content, following Siu and Henshall (1998). 2.2. Nitrite analysis 2.2.1. Colorimetric measurements In general, the ofcial ISO 29181975 method was used, but adding ascorbic acid, modifying pH with HCl and modifying borax addition and heat extraction times according to the experimental design shown in Table 1. In the light of the results from an earlier study (Rincn et al., 2003), Carrez reagent was not used, and the

extraction temperature was also changed from 80 C to 60 C. Absorbance (path length 1 cm) of dye solutions was measured using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). 2.2.2. Potentiometric measurements Potentiometric measurements were made as reported in a previous paper (Prez-Olmos, Yoldi, Ruiz, & Merino, 1998), using an Orion 710 A digital pH/mV meter and an Orion 9346 nitrite-selective electrode (ThermoOrion, Beverly, MA). Direct interference of ascorbic acid was eliminated by applying a cellulose acetate membrane of 100 Da molecular weight cut-off on the electrode (Badea et al., 2004). 2.2.3. Chromatographic measurements Chromatographic measurements were made following Dionex application note #112 (Dionex, 1998). The chromatograph comprised a Dionex DX-600 chromatography system equipped with a GP50 gradient pump, a AD25 UV/Visible absorbance detector, a LC 30 chromatography workstation and a IonPac AS11 4 250 mm column with AG11 guard 4 50 mm, a low-capacity, hydroxide-selective, anion-exchange column designed for the fast proling of inorganic anions in foods (Dionex, Salt Lake City, UT). The mobile phase was pumped at a rate of 1 ml/min and consisted of deionised water (95%) and 100 mM NaOH (5%) from 0 to 10 min and 100 mM NaOH (100%) from 10 to 15 min. Briey, this method enables quantication of nitrite by direct extraction from meat products followed by direct determination using anion-exchange chromatography with UV detection. 2.3. Protein measurements Protein content in the sample extract was measured using the Lowry method as modied by Bensadoun and Weinstein (1976). 2.4. pH determination pH was measured at room temperature using a Crison 501 pH meter (Crison Instruments, Barcelona). 2.5. Critical factor selection The following critical factors or inuencing factors (IF) were selected for inclusion in the experimental design: Sample weight/Borax reagent ratio (WS/BR): according to the results of an earlier paper (Rincn et al., 2003), the WS/BR ratio must be considered as an IF because the interaction between sample weight and borax reagent either enhanced or impaired nitrite extraction depending on the WS/BR ratio. This parameter was monitored using a constant sample weight (10 g of slurry) and varying borax reagent addition (BR factor). Ascorbic acid: ascorbic acid reduction of nitrite plays an important role in meat processing, inuencing both the color and the avor of cured meats (Izumi, 1992); at the same time, however,

Table 1 Levels of factors for optimizing assay conditions for nitrite determination Factors Symbol Code Borax reagent addition, ml Ascorbic acid reagent addition, ppm Neutralization, as HCl addition, ml Stirring extraction time, h X1 X2 X3 X4 Actual BR AR NR SET Levels 2 0 0 1.4 0.1 1 5 50 2 0.6 0 10 100 2.6 1.1 +1 15 150 3.2 1.6 +2 20 200 3.8 2.1

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reduction of nitrite may interfere considerably with nitrite measurements. High residual ascorbate levels have been shown to result in false-low nitrite values in cured meats when analyzed according to the ofcial AOAC method (Sen & Donaldson, 1978; Sen, Lee, & McPherson, 1979), possibly because ascorbic acid results in the formation of less azo dye than might have been expected from the amount of nitrite present (Nicholas & Fox, 1973). For that reason, ascorbic acid also was also considered an IF (AR factor). pH in the extraction process: for meat product samples, such as fermented sausages, neutralization has been recommended prior to analysis (Fiddler & Fox, 1978); however, preliminary studies (Rincn et al., 2003) show that the addition of borate to, and the elimination of Carrez reagent from, the extraction process in fact yields a neutralization of pH to 89. At the same time, correct pH ensures both nitrite stability during heating and the obtaining of a clear ltrate (Sen & Donaldson, 1978). This parameter was therefore monitored by neutralization with HCl 1 N (NR factor). Stirring extraction time: an earlier paper (Rincn et al., 2003) showed that increasing the stirring extraction time from 1 to 2 h failed to improve the amount of nitrite extracted; thus, a 1 h extraction period was recommended. However, these results were obtained when ascorbic acid was not present in the sample. In such conditions, a minimum extraction time of 2 h in a steam bath has been proposed in order to oxidize reducing compounds, such as ascorbic acid, if present in the sample (Siu & Henshall, 1998). In conclusion, stirring extraction time had to be reconsidered as a factor in this experiment, in order to ascertain the time during which nitrite detection is not impaired by ascorbic acid (SET factor). It has been reported that a higher concentration of coupling reagent solution enhances the analytical procedure by improving both recovery and variation coefcients (Binstok et al., 1996). However, this conclusion was not properly tested, due to several errors in the experimental design used by these authors: (i) the effect attributed to the double concentration of coupling reagent is in fact attributable to pH extraction by NaOH addition (differences between methods II and III), since as was demonstrated long ago (Sen & Donaldson, 1978) the absorbance of the nal solution can vary widely with pH; (ii) the positive effect of the addition of sand to the sample to improve dispersion may in fact be due to considerable nitrite contamination, since sands/gravels often contain nitrite and/or nitrate, which was not ruled out in the study (method V); and (iii) the improved method proposed (method VIII) was interpreted only on the basis of main effects, but failed to investigate secondary effects and interactions when discussing results and drawing conclusions. In fact, a simple trial shows that doubling the coupling reagent does not enhance the sensibility of method, but rather impairs it but because the slope obtained is lower (Fig. 1). For that reason, coupling reagent concentration was not considered as a factor in the present experimental design. The effect of the four factors selected (BR, AA, NR and SET) on the nitrite measurement method was investigated simultaneously. 2.6. Experimental design The nitrite extraction procedure followed a central composite rotatable (CCR) experimental design, and response surface methodology (RSM) was used to establish a relationship between factors (BR, AA, NR and SET) and responses (R1 = pH in the extraction process; R2 = pH before colorimetric reaction; R3 = protein content extracted as mg/g; R4 = protein hydrolysis at OD340; R5 = nitrite content expressed in lg/g; R6 = agreement between duplicate determinations).

0.60
AOAC (1.999) method # 973.31

0.50

0.40

A 520

0.30
y = 0.006 + 0.256 x

Binstok et al. (1.996)

0.20
y = 0.008 + 0.215 x

0.10

0.00 0.25 0.50 1.00 2.00

ppm
Fig. 1. Differences in sensitivity between AOAC (1999) and Binstok et al. (1996). Both methods use NED reagent [N-(1-naphthyl) ethylenediamine dihydrochloride], but the latter uses a double concentration.

R2 was considered because it has been shown that the nal pH must be maintained within strict limits in order to guarantee complete color development (Davis, Lefer, Anderson, Soderbeg, & Meredith, 1985), since in buffer systems the rate of the reaction is pH-dependent, with an optimum at pH 3.0 (0.2) (Hildrum, 1979). R4 was included because optical density at 340 nm (OD340) is a good index for monitoring protein hydrolysis (Fadda, Vignolo, Bru, Holgado, & Oliver, 1999), and this may be important when determining the nal pH value and the total protein extracted. R6 was obtained by the term [(a b)/(a + b)] 100, where a and b are the repeat determination values, according to Dennis, Key, Papworth, Pointer, and Massey (1990). To ensure correct application of RSM, a CCR design with equal predictability in all directions from the center was used, considering the levels for each factor shown in Table 1. The theoretical aspects and experimental implications of RSM have been described elsewhere (Khuri & Cornell, 1996). RSM is currently the most popular optimization technique, probably because of its comprehensive theory, reasonably high efciency and simplicity (Arteaga, Li-Chan, Vazquez, & Nakai, 1994). The response variables were assumed to be inuenced by the four independent variables or factors shown in Table 1 (BR, AA, NR and SET), so that: ni = f (BR, AA, NR and SET), where ni (for i = 16) is each of the responses. So the unknown function f was assumed to be approximated by a second degree polynomial equation: ni b0 b1 X 1 b2 X 2 b11 X 2 b22 X 2 b12 X 1 X 2 e 1 2 where ni is the response, b0 (center point of system), bi (coefcient of linear effects), bii (coefcient of quadratic effects) and bij (coefcient of interactive effects) are the different constant coefcients of the model, Xi are the coded independent factor-related variables and e is the error of model. In RSM work it is advisable to transform natural variables into coded variables, and these coded variables are usually dened as dimensionless with mean zero and the same spread or standard deviation (Myers and Montgomery, 1995a,b). Runs were performed in random order (trial order), because randomization helps protect the experimenter against reaching erroneous conclusions due to extraneous sources of variability (Joglekar & May, 1987; Robinson, 2000). A summary of the experimental design is shown in Table 1. Both Statistica (StatSoft, Inc., Tulsa) and Design-Expert (Stat-Ease, Inc., Minneapolis) software were used to generate designs, carry out

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regression analysis, t the response surface model to the experimental data and draw response surface gures. 3. Results and discussion Response values obtained under different extraction and determination conditions are shown in Table 2; signicant standardized effects (p 6 0.05) of each factor on each response are shown in Table 3. Both initial pH (i.e. pH before extraction, R1) and nal pH (i.e. pH before colorimetric reagent addition, R2) responses were affected by the same factors, the two being closely related (r2 = 0.99, p 6 0.05). Since very similar values were obtained for the effects of borax (X1) and HCl (X3) on these two responses (Table 3), the discussion will focus only on the results obtained for R1. For response surface designs, the perturbation plots show how the response changes as each variable deviates from the reference point, while all other variables are held constant. These plots show that addition of borax had an expected positive overall effect (linear + quadratic) on R1 (Fig. 2A, X1 and Table 3), whilst the addition of HCl had a negative linear effect on R1 (Fig. 2A, X3 and Table 3). It should be noted here that the ISO 2918 method includes borax as reagent, whereas the AOAC 973.31 method does not. However, a previous study has shown that the benecial or undesirable effect of the borax reagent depends on the sample weight/borax reagent (WS/BR) ratio (Rincn et al., 2003). The problem of nitrite stability in an acid pH, particularly a pH below 5, has long been recognized (Sen et al., 1979; Usher & Telling, 1975); in meat products, nitrite instability prompts losses and interferences which, according to Norwitz and Keliher (1986, 1987), can be dened as: (i) nitrite oxyreduction in the presence of diverse mineral or organic agents or (ii) a phenomenon of competition in the diazotization reaction between the nitrites or the reagents and a variety of organic compounds, such as amines. On this basis, and in view of the results themselves, Pringuez et al. (1995)

Table 3 Signicant (p 6 0.05) standardized effects of different factors affecting each response Effect Responses R1 X1-linear (L) X1-quadratic (Q) X2-L X2-Q X3-L X3-Q X4-L X4-Q X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4
a a

R2 9.21 2.60

R3 3.62 5.03 2.64 3.92 4.70 2.22 2.53 3.96 2.30

R4 6.49 3.42

R5 5.44 2.91

R6

8.67 2.38

3.49

3.59

4.65 2.94

3.13

3.34

2.34

R1 = pH at extraction; R2 = pH before colorimetric reagent addition; R3 = protein content as mg/g; R4 = OD340; R5 = nitrite content as lg/g; R6 = agreement between duplicate determinations.

propose that HCl should not be added at rst when the chromogenic reagent is prepared; however, the formation of azo coloring occurs only in an acid medium (Middleton, 1957) and low nitrite stability in an acid pH (Sen et al., 1979; Usher & Telling, 1975), so a compromise must be achieved in order to ensure adequate color development and acceptable nitrite stability. Nitrite salts are soluble in water, and the nitrite ion NO is the 2 conjugate base of a weak acid (nitrous acid, HNO2) with a pKa of 3.36; since meat is usually mildly acidic, only a small quantity of nitrous acid is formed. Among the spectrophotometric methods for quantitative determination of nitrite ofcially recognized by the AOAC, the method based on the reaction with N-(1-naphtyl)ethylenediamine 2HCl and sulphanilamide (AOAC, 1999) requires careful control of acid-

Table 2 Experimental design and response values obtained under different extraction conditions Trial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16c 17 18 19c 20c 21 22 23 24 25 26 27 Run 8 16 11 1 6 13 15 5 20 24 21 12 9 3 18 26c 7 19 25c 27c 10 2 14 23 4 17 22 X1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 2 0 1 0 0 0 1 1 1 0 1 2 0 X2 1 1 1 1 1 1 1 1 2 0 0 1 1 1 0 0 1 2 0 0 1 1 1 0 1 0 0 X3 1 1 1 1 1 1 1 1 0 0 2 1 1 1 0 0 1 0 0 0 1 1 1 0 1 0 2 X4 1 1 1 1 1 1 1 1 0 2 0 1 1 1 0 0 1 0 0 0 1 1 1 2 1 0 0 R1 4.06 8.27 8.19 2.58 4.85 8.58 8.05 4.33 7.34 6.97 8.44 8.02 8.50 2.50 8.51 7.39 2.57 7.11 7.35 7.45 8.19 8.18 8.43 7.46 2.48 2.13 3.65 R2 4.63 8.27 8.10 2.82 5.21 8.48 8.16 4.53 7.51 7.93 8.37 8.04 8.51 2.71 8.35 7.28 2.96 7.37 7.33 7.44 8.45 8.13 8.48 7.26 2.79 2.44 4.17 R3 32.49 19.48 17.55 34.25 6.40 20.92 18.40 5.67 5.56 18.62 20.59 20.56 20.64 34.63 18.88 9.69 32.86 11.39 9.07 10.90 19.18 20.53 18.79 14.61 32.91 35.76 31.57 R4 1.303 0.264 0.195 1.787 0.067 0.221 0.207 0.063 0.126 0.392 0.286 0.542 0.204 1.753 0.212 0.133 1.864 0.179 0.127 0.185 0.239 0.327 0.244 0.152 1.901 1.824 1.486 R5 39.75 26.04 24.80 60.93 22.55 26.17 24.58 26.64 23.29 24.80 27.50 30.60 22.72 59.86 21.39 21.82 69.07 21.90 21.28 21.26 20.77 24.14 22.85 18.65 70.24 63.60 42.92 R6 2.68 0.30 1.04 2.50 0.12 0.66 0.87 3.20 1.37 1.04 0.15 0.34 0.36 2.40 1.19 0.73 2.16 0.00 1.04 1.50 1.68 1.98 0.90 0.51 2.78 0.39 1.04

R1 = pH at extraction; R2 = pH before colorimetric reagent addition; R3 = protein content as mg/g; R4 = OD340; R5 = nitrite content as lg/g; R6 = agreement between duplicate determinations.

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X1 X4 X2

X3

-2

-1

+1

+2

Deviation from reference point

B
X3

R3

X1 X4

extract pH determination (Fig. 3A). A pH value of 8.87 yielded minimum protein solubility, although the conditions in which that pH was achieved enabled the extraction of only 20.94 lg/g of nitrite, i.e. only 68% of the nitrite present in the sample. Carrez reagents were not used, due to the undesirable effects observed in a previous study (Rincn et al., 2003); it was therefore necessary to identify optimum conditions allowing extraction of all the nitrite present and as little protein as possible. Factor HCl (X3) acted in a contrary manner to borax with regard to the linear component of the effect (Table 3), and produced a perturbation plot practically the reverse of that produced by borax (Fig. 2B, X3), suggesting that pH at extraction is the main parameter determining protein solubility; the two factors had opposing effects on R3 (Fig. 2A). A strong correlation was noted between pH at extraction (R1) and extract protein content (R3) (r2 = 0.48, p 6 0.05), and both factors interacted with the WS/BR ratio (Fig. 4). These results are readily explained by the fact that total protein solubility is affected by pH, so an increase in pH value from 5.65 to 6.21 increases sarcoplasmic protein solubility by 20% (Young, Zhang, Farouk, & Podmore, 2005); by contrast, an increase in pH prompts signicantly lower myobrillar protein solubility (Zhang, Farouk, Young, Wieliczko, & Podmore, 2005). Maximum sarcoplasmic protein solubility in meats is obtained at a pH of 7.2 (Joo, Kayffman, Kim, & Park, 1999), whilst myobrillar protein solubility increases at lower pH values, possibly due to proteolysis (Zhang et al., 2005).

R1

2
70 60 50 40 30 20

X2

Minimun 10,22 mg/g

o

-2

-1

+1

+2
X3
X3
-2 -2 -1 0 1 2 0

Deviation from reference point

C
-1

R4

X1

B
X4 X1 X2

2
20 25 15 10 15 20 30

-2

X2

+1 +2 -1 0 Deviation from reference point

30

Fig. 2. Perturbation plots showing (A) the effects of factors on R1 (pH before extraction), (B) R3 (protein content as mg/g) and (C) R4 (protein hydrolysis as OD at 340 nm) responses, respectively.

-1

40

ity (Zatar, Abu-Eid, & Eid, 1999), so borax and HCl, given their effect on pH (Table 3) must be carefully controlled. Sample extract protein content (R3) was signicantly affected by all factors except extraction time (Table 3). Addition of borax (X1) had a negative linear effect and a positive quadratic effect (Fig. 2B, X1, Table 3); this enabled identication of a point of minimum protein solubility as a function of the two factors involved in

-2 -2

-1

0 X1

Fig. 3. Surface plot showing the effect of factors X1 and X3 on protein solubility during the extraction process (A) and the synergic effect of X1 and X2 on protein solubilization for values of +1 < X1 < 1 (B).

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40 35 30

2
higher NO2 higher protein lower pH area

Protein ( g g -1

25

X3

20 15 10 5 0 1 2 3 4 5 6 7 8 9
Stationary point located W S / B R = 1.11

lower NO2 higher pH area

Fig. 4. Overlapping of weight sample and borax reagent (WS/BR) ratio on extract protein content and pH.

The presence of ascorbic acid (X2) decreased protein solubility, with a linear effect of 2.64 (Table 3). This is a positive nding for the purposes of nitrite determination, since it allows more transparent extracts to be obtained, with lower colloidal protein content and thus less interference in the analytical process; however, from X1 P 1 onwards, this factor acted synergically with borax on protein solubility (interaction effect = 2.53, Table 3, Fig. 3B). Extraction time had a positive quadratic effect on R3 (Fig. 2B, X4, Table 3), prompting a decline in colloidal protein content to 0 (1.1 h), probably due to gradual precipitation, followed by an increase to +2 (2.1 h), attributable to redissolving of the protein precipitate. A strong secondary effect was observed in the X1 X3 interaction (Table 3, Fig. 3B): a negative interaction (antagonism) was noted between X1 and X3 in the effect on R3, due to their opposing effects on the value of pH at extraction; this was to be expected given their individual effects on pH at extraction (Table 3, Fig. 1A). There was also a clear positive interaction (synergy) between X2 and X3 (effect = 2.30, Table 3), the presence of both together prompting a greater decrease in pH than that caused by the sole presence of either. These effects suggest that extract protein content (R3) and pH at extraction (R1) are inversely related (r2 = 0.61, p < 0.00, Fig. 4), so that factors affecting R1 have the reverse effect on R3 (Table 3). The degree of protein hydrolysis (R4) was strongly related to pH at extraction (R1) (r2 = 0.76, p 6 0.05) and to extract protein content (R3) (r2 = 0.81, p 6 0.05), which would explain why R4 was inuenced by these factors (X1 and X3, Table 3). In practice, this means that increased extract protein content is the result of either (1) increased protein hydrolysis due to low pH during the nitrite extraction process, so that the two responses give very similar perturbation plots (Fig. 2B and C), or (2) the existence of WS/BR ratio values of P2 (Fig. 4). It is known that pH values of <6 gave OD340 values of >0.600 (Table 2), prompting interference in nitrite measurements (Fig. 5), and ultimately the generation of false-positive results, as reported by Okemgbo, Hill, Scienms, and Matcalf (1999). In these conditions, protein content was greater than 20 mg/g, and interference from colloidal organic matter prompted a false increase in the nitrite content measured (Figs. 3A and 5). These results conrm those of an earlier study, which found that an extract protein content from 0.21 to 16.65 mg/g prompted no interference in nitrite measurements (Rincn et al., 2003). The present study established that the maximum extract protein content should be 20 mg/g; beyond this value, interference in nitrite

R5

WS / BR ratio as follow:

=>2 =2 =1 = 0.67 = 0.50

-1

pH = 6

nitrite = 30.8 ppm protein = 20 mg/g

pH = 7

-2 -2

-1

pH

0 X1

Fig. 5. Contour lines plot showing the simultaneous effect of X1 (borax reagent) and X3 (HCl reagent) on pH of extraction, protein extracted and nitrite nally quantied in the analytical assay.

measurements will lead to the detection of a higher amount of nitrite than is actually present (Fig. 5). The WS/BR ratio also proved to be a decisive factor; protein values of >20 mg/g were generally associated with a WS/BR ratio of P2 (Fig. 4). The addition of borax (X1) had a negative overall effect on the amount of nitrite detected (Table 3, Fig. 6). It has been reported that borax enhances nitrite extraction, enabling detection of higher levels (Fiddler & Fox, 1978); however, the present results suggest that the addition of borax increases extract protein content and the degree of protein hydrolysis (Table 3), leading to higher absorbance values which are wrongly measured as higher nitrite content when X1 6 0.21 (BR = 8.95 ml, Fig. 5); at the same time, borax has been found to cause nitrite losses (Rincn et al., 2003), and this is also the case when X1 P 0.21 (Fig. 5). A similar but contrary effect is produced by X3 on the nitrite level detected (R5) (Fig. 5). In conclusion, nitrite extraction can be enhanced using a given WS/BR ratio, and at the same time a pH value of between 6 and 7 (Figs. 4 and 5). This is achieved with a WS/BR ratio of 1.11 (10 g of slurry/8.95 ml BR), equivalent to 0.48 (0.50) in terms of original sample (4.3 g of sample/8.95 ml). Use of larger amounts of borax gave rise to pH values of <6, leading to considerable protein hydro-

70 60

X1

X1= -0.21 BR = 8.95 ml

X3= 0.09 NR = 2.65 ml

X3

50 40
true nitrite level (30.8 ppm)

30 20
-2 -1 0 1
X2 X4

Deviation from reference point

Fig. 6. Perturbation plot showing the effects of factors on R5 (nitrite content as lg/g). Apparently, values lower than X1 = 0.21 can yield false-positive results, while those greater than X3 = 0.09 can yield false negatives.

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lysis and solubilization, and prompting interference with nitrite measurements due to the large amount of colloidal protein present; at pH > 7 (high BR values) the WS/BR ratio is 60.50, leading to loss of nitrite (Figs. 5 and 6), simply due to nitrite destruction during extraction (Sen et al., 1979), either because at acid pH some nitrite is protonated and this proportion will play no part in the colorimetric reaction (Alonso, Etxaniz, & Martinez, 1992), or possibly due to the precipitation of protein, some of which may be bound to nitrite (Rincn et al., 2003). These results may explain why Sen et al. (1979) recovered only 47.2% of the nitrite present, when pH at extraction was <5. Sen & Donaldson (1978) suggested that samples should be alkalized, recommending that the pH be adjusted to over 6, but without specifying a particular range. More recently, Pringuez et al. (1995) have suggested a pH range of 78 for measuring nitrite in dried milk. The results obtained here would indicate that the most suitable range would be 67 (Figs. 4 and 5). Other authors have similarly suggested adjusting the pH of the sample ltrate to 6.0 0.5 (Glover & Johnson, 1973) for the spectrophotometric measurement of nitrite in meat products, or to 6.5 0.5 (Badea et al., 2004) for nitrite measurement in cured meats using a selective electrochemical method; in both cases, however, the conclusion was reached empirically, rather than being the outcome of experimental design. Finally the recommended adjustment of pH to 8 (Sen et al., 1979) reportedly provides satisfactory results for dairy products; however, the present ndings suggest that this value would be unsuitable for meat products. It is worth recalling here that the pKa of HNO2 is 3.36, so that since the pH of meats and meat products (usually 5.66.5) is greater than the pKa the NO2 concentration in cured meat is low, at between 0.1% and 1.0% (Pegg & Shahidi, 1997). This would account for the empirical conclusion of some authors that it is necessary to neutralize samples prior to nitrite measurement, particularly in the case of fermented products (Fiddler & Fox, 1978). However, the addition of borax reagent in the absence of a Carrez reagent gives a pH value of 89 (Rincn et al., 2003). As Sen & Donaldson (1978) have suggested, establishing a suitable pH at extraction appears to be essential both for nitrite stability and for obtaining transparent extracts, i.e. containing little or no colloidal protein. Similar conclusions have been reported, again empirically, with regard to the elimination of ascorbic acid interference, which has been shown to be dependent on pH (Norwitz & Keliher, 1986), leading to the suggestion that nitrite extraction be performed in 0.01 M NaOH (Norwitz & Keliher, 1987). Although in the overall conditions tested (from 0 to 200 ppm), ascorbic acid (X2) did not exert a statistically-signicant (p < 0.28) effect on nitrite measurement (Table 3), the presence of ascorbic acid did diminish the amount of nitrite detected beyond X2 < 0 (or 100 ppm of ascorbic acid, Table 1), as readily observed in the pertrubation plot (Fig. 6); similar ndings are reported by Sen and Donaldson (1978) and Sen et al. (1979), who found that high residual ascorbate levels result in false-low measurements of nitrite in cured meats when analyzed according to the ofcial AOAC method, and also by Usher and Telling (1975), who observed a nitrite-recovery rate of 68.1% in the presence of 4000 ppm of ascorbic acid. Nicholas and Fox (1973), in seeking to account for the interference prompted by high ascorbic levels, suggest that ascorbic acid results in the formation of less azo dye than would have been expected from the amount of nitrite present; however, the results obtained here suggest that ascorbic acid, whilst not exerting a signicant direct effect (p 6 0.80) on pH at extraction (Table 3, Fig. 2A, X2), did prompt a signicant decrease in extract protein content (R3) (Table 3, Fig. 2B, X2), leading to a larger amount of protein precipitate, part of which is bound to some of the nitrite present in the extract (Rincn et al., 2003). The presence of ascorbic acid triggers the nitrosation of cytochrome c (Izumi,

Cassens, & Greaser, 1989), which may be interpreted as the source of the interference attributed to ascorbic acid, due to protein precipitation during the extraction process. Subsequently, when the extract is shaken during extraction/handling, nitroso cytochrome c spontaneously generates free nitrite (Izumi, 1992); thus, some nitrite might be lost when shaking during extraction. Moreover, ascorbic acid reduces nitrous acid, resulting in the production of nitric oxide which, under normal circumstances, will be lost from the sample solution (Badea et al., 2004); additionally, the Griess reaction detects only NO (Dusse, Morais, Viera, & Carvallo, 2005). 2 The results obtained here suggest that the undesirable effect of ascorbic acid in the sample should be tackled not by the destruction of ascorbic acid during extraction (Kim & Conca, 1990; Nicholas & Fox, 1973), for example 2 h at 80 C (AOAC, 1999), which also causes nitrite loss (Rincn et al., 2003; Sen et al., 1979), but rather by acting on the parameter which is modied by the presence of ascorbic acid, i.e. extract protein content, which is strongly related to extract pH (r = 0.61, p < 0.00). These results would explain why, at pH values of 6 or above, Fox et al. (1982) empirically found an effective way of eliminating ascorbate interference. This wholly supports the pH value recommended in the present study (Fig. 6). Taken in conjunction with the results obtained here on the basis of the hypothesis to be tested, the ndings reported earlier by Norwitz and Keliher (1987) conrm the conclusion that in the presence of up to 100 ppm of ascorbic acid, nitrite recovery is satisfactory; these authors obtained acceptable nitrite recovery rates with 50 ppm of ascorbic acid. This conclusion is borne out by the ndings of Riise and Berg-Nielsen (1990), who noted that elimination of the interfering effect of ascorbic acid by addition of ascorbate oxidase was similar to the effect obtained by extraction with 0.01 M sodium hydroxide, which alkalizes the medium and thus reduces protein extraction; this is conrmed by the relationship observed in the present study between R1 and R3 (r2 = 0.48, p 6 0.05). Extraction time (X4) had no direct effect on the amount of nitrite extracted (Table 3); thus, in the experimental conditions applied here, the shortest extraction time tested (X4 2 or SET = 6 min) was sufcient to extract all the nitrite present. Similar ndings are reported by Kim and Conca (1990), who observed acceptable nitrite-recovery rates with an extraction time of 1 min, when measuring nitrite content by ion-exclusion chromatography with electrochemical detection. However, since at values of X4 > 0 (equivalent to extract times longer than 1.1 h) there is some redissolving of protein precipitate, a time of 1.1 h is to be recommended when nitrite measurement is performed by spectrophotometry, since this enables extraction of all the nitrite present whilst at the same time ensuring that extract colloidal protein content will not prompt interference. Finally, the repeatability of the method (R6) was not signicantly affected by any of the factors tested (Table 3). For comparison of both ofcial and proposed methods, Table 4 shows residual nitrite levels for ten meat products, measured by the original method ISO 2918, the same method modied in the light of the results obtained, and both potentiometric and chromatographic alternative measurements. For a nitrite level of approximately 20 mg g1 (according to the proposed method), clear differences were apparent in the residual nitrite measurements obtained: lower levels were detected by both potentiometric and chromatographic methods, while higher levels were detected using both the ofcial and the proposed ISO/DIS methods. Comparison of these two methods was conclusive: for concentrations of <10 mg g1, the ofcial method measured a mean 124% more nitrite than was actually present, while for concentrations of >20 mg g1 the mean over measurement dropped to 29%. To summarize, the four critical factors for nitrite measurement in meat products acted indirectly on the nitrite levels detected,

F. Rincn et al. / Meat Science 80 (2008) 744752 Table 4 Comparison of results obtained by both ofcial and proposed methods with those obtained with the potentiometric and the chromatographic methods Sample Analytical method Spectrophotometric Ofcial pH 1 Cooked ham 2 Cooked ham 3 Cooked ham 4 Salami 5 Chopped pork 6 Trufed chicken 7 Cured ham 8 Sausage 9 Frankfurter 10 Frankfurter 5.6 4.7 6.6 4.2 5.5 6.8 5.1 4.6 5.6 4.9 Protein (mg g1) 37 42 31 120 62 28 165 82 94 73 OD340 1.4 0.9 0.7 1.6 0.8 0.4 2.1 1.4 0.8 0.8 Nitrite (mg g1) 12.9 21.3 84.1 9.2 16.5 66.3 7.9 7.5 62.4 33.0 Proposed pH 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5
a

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Potentiometric (lg g1) OD340 0.4 0.4 0.3 0.5 0.4 0.2 0.3 0.3 0.4 0.3 Nitrite (mg g1) 5.3 5.6 65.6 4.4 6.6 46.1 6.5 5.5 48.7 27.7 9.0 12.0 55.7 8.6 9.6 39.6 7.2 7.0 38.2 25.8

Chromatographic (lg g1)

Protein (mg g1) 12 10 9 18 7 16 6 12 10 8

13.1 15.2 43.3 6.8 8.5 32.8 <5 <5 31.8 15.6

a pH in the proposed method was adjusted to 6.5 using HCl, because unlike the original method no Carrez reagents are used, due to detection of undesirable effects in a previous study (Rincn et al., 2003).

either by inuencing the sample extract pH and thus the amount of colloidal protein present in the extract, or by inuencing the amount of protein previously precipitated by the addition of borax reagent and subsequently redissolved. Acknowledgement This study was partially funded by the AGR-170 Research Group belonging to the Plan Andaluz de Investigacin (Spanish Government Andalusian Research Plan). References
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