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Meat Science 92 (2012) 538–547 Contents lists available at SciVerse ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci A new, simple method for the production of meat-curing pigment under optimised conditions using response surface methodology Nafiseh Soltanizadeh ⁎, Mahdi Kadivar Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan 84156, Iran article info abstract Article history: The production of cured meat pigment using nitrite and ascorbate in acidic conditions was evaluated. HCl, Received 22 August 2011 ascorbate and nitrite concentrations were optimised at three levels using the response surface method Received in revised form 15 March 2012 (RSM). The effects of process variables on the nitrosoheme yield, the wavelength of maximum absorbance Accepted 22 May 2012 (λmax), and L*, a* and b* values were evaluated. The response surface equations indicate that variables exerted a significant effect on all dependent factors. The optimum combinations for the reaction were Keywords: HCl=−0.8, ascorbate=0.46 and nitrite=1.00 as coded values for conversion of 1 mM hemin to Nitrosoheme Yield nitrosoheme, by which a pigment yield of 100%, which was similar to the predicted value of 99.5%, was fi λ Absorbance obtained. Likewise, the other parameters were not signi cantly different from predicted values as the max, Optimisation L*, a* and b* values were 558 nm, 47.03, 45.17 and 17.20, respectively. The structure of the pigment was iden- FTIR tified using FTIR and ESI/MS. ESI/MS © 2012 Elsevier Ltd. All rights reserved. 1. Introduction 2007). The N-Nitrosamines in meat products, are compounds that could cause carcinogenicity. These compounds (e.g., N-nitrosopyrrolidine and Current meat-curing practice, which is founded upon the ancient N-nitrosodimethylamine) are formed, albeit in the parts-per-billion art of preserving meat with salt, employs the addition of nitrite range, by the reaction of nitrite with the amines or amino acids that are (and in certain products, nitrate) along with salt, sugar, reducing present in foods. In addition, the existence of residual nitrite in cured agents, and phosphates to meat (Rubin, Diosady, O'Boyle, Kassam, & meat increases the body's total nitrite load, which in turn may lead to Shahidi, 1992). Nitrite has beneficial effects on meat products and po- an increased likelihood of nitrosamine formation within the human di- tentially detrimental effects on human health. The role of nitrite in gestive tract (Rubin et al., 1992). However all health implications being cured meat is four-fold: i) it provides the characteristic pink-red associated with nitrite/nitrate consumption are tenuous and suggestive cured-meat colour to the lean tissue; ii) it inhibits the growth of a at present and no known case of human cancer has been shown to result number of bacteria that cause food poisoning or spoilage; iii) it con- from exposure to N-nitroso compound and all datas are according to indi- tributes to the distinctive flavour of cured meats; and iv) it retards rect observations. It should be mentioned that inspite of concerns about oxidative rancidity in processed meat products, principally through consumption of cured meat products, recent studies demonstrate that ni- a process of metal chelation. tritehavebenefitialeffectsonhealthanduponitsingestionandmixture Despite these beneficial effects on cured-meatproducts,therewere with gastric acid, is a potent bacteriostatic and/or bactericidal agent for deep concerns about the use of nitrite and nitrate as food additives be- gastrointestinal, oral, and skin pathogenic bacteria (Archer, 2002). The cause both are potentially toxic for humans. The lethal oral doses for potential role in hypoxic vasodilation and protective action against ische- humans have been established in the range of 80–800 mg of nitrate and mia are other physiological and pharmacological properties of nitrite that 33–250 mg of nitrite/kg body weight (Honikel, 2008). Over time, nitrite has been recently considered (Butler & Feelisch, 2008; Parthasarathy & has been suspected of playing a role in the development of cancer, methe- Bryan, 2012) but due to the potential health hazard associated with the moglobinemia in infants, and even reproductive toxicities such as birth use of sodium nitrite, extensive studies have been conducted to find defects (Archer, 2002). Recently, epidemiological relationships were methods to reduce nitrite in cured meat products. The chance of finding shown between cancer incidence and intake of processed meats a single compound which duplicates all functions of nitrite is very slight. (Demeyer, Honikel, & De Smet, 2008; Faramawi, Johnson, Fry, Sall, & Yi, Therefore, the development of a multifunctional system, including the synthetic cooked cured meat pigment have been considered (Shahidi, Rubin, Diosady, & Wood, 1985). Cured meat pigment is ordinarily devel- oped by the reaction of nitrite with the natural meat pigment myoglobin ⁎ Corresponding author. Tel.: +98 311 3913382; fax: +98 311 3912254. to form dinitrosyl ferrochrome (DNFH). The pigment, which gives meat E-mail address: [email protected] (N. Soltanizadeh). its characteristic cured-meat colour, is formed from the meat pigment 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2012.05.024 N. Soltanizadeh, M. Kadivar / Meat Science 92 (2012) 538–547 539 myoglobin, which consists of an iron porphyrin complex, the heme group, Table 1 attached to the protein globin. In the presence of nitrite, the bright red Coded settings for the process parameters for nitrosoheme synthesis, according to a central composite design. nitrosomyoglobin is formed, in which the H2Ointheaxialpositionon the heme iron is replaced by nitric oxide (NO). The NO is formed from ni- Independnt variables Symbols Range and levels trite by the natural reducing activity of the muscle tissue, which is accel- −1.68 −1 0 1 1.68 erated by the addition of reductants such as ascorbic acid. In heat- HCl concentration (%) X 0.32 1 2 3 3.68 processed cured meat, the globin has been split off to a heat-stable pink 1 Ascorbic acid concentration (mM) X2 65.91 100 150 200 234.08 pigment, nitrosyl hemochromogen (Pegg & Shahidi, 2000). The synthetic Nitrite concentration (mM) X3 15.91 50 100 150 184.08 production of this pigment has been the subject of some researches (Shahidi & Pegg, 1991, 1995a, 1995b; Shahidi & Pegg, 1992; Shahidi et al., 1985). In a series of studies, curing colour was prepared from red blood cells and nitric oxide (Shahidi & Pegg, 1991, 1995a, 1995b; Shahidi & Pegg, 1992; Shahidi et al., 1985). The pigment 2.4. Yield could be prepared in a direct, one-step process or by an indirect method through a hemin intermediate. Hemin was dissolved in a di- The yield of nitrosoheme pigment was determined according to lute sodium carbonate solution and added to a mixture containing Hornsey (1956). Briefly, the absorption of preformed cooked cured sodium tripolyphosphate, sodium ascorbate, and sodium acetate. pigment in 80% acetone solution was read in 540 nm. With multiply- Nitric oxide was then introduced into the solution to produce the ing the absorbance in 652 (Molecular weight of hemin) and dividing mM nitrosoheme, which then was precipitated out by lowering the pH to 11.3 (E 540), the concentration of nitrosoheme was obtained as (Pegg, Shahidi, & Fox, 1997). ppm. Yield was calculated by division of nitrosoheme concentration A new, rapid and easy procedure for the production of cooked to initial concentration of hemin. cured-meat colour was developed in this study. The influence of ni- trite, ascorbic acid levels, and HCl concentration on pigment forma- tion was evaluated in the model system of hemin using response 2.5. Colour of preformed pigments surface methodology, and after comparison of results with those of others under actual curing conditions and model systems, the The lightness/darkness (L* value), red/green (+/−a* value), and optimum conditions for in vitro production of nitrosoheme were yellow/blue (+/−b* value) of the nitrosoheme pigments were deter- established. mined according to Yam and Papadakis (2004). In this procedure, 2 ml of diluted pigment was poured into a glass container with an in- ternal diameter of 2 mm. The colour of the samples was measured 2. Material and methods using an apparatus constructed in the Department of Food Science, Isfahan University of Technology, Iran. It consists of a chamber with 2.1. Material a trapezoidal cross section that is equipped with two D65 (daylight) lamps for illumination of the samples. An 8.1-mega-pixel camera All chemicals and solvents used in this study were analytical grade (Samsung L830, South Korea) was used to record the images. Samples commercial products. Sodium nitrite, chloridric acid (37%) and ace- were placed in the centre of chamber. The images were captured after tone (pro analysis grade, 99.8%) were purchased from the Merck adjustment of the lens and focusing of the camera. Colour measure- Chemical Company, Germany. L-ascorbic acid sodium salt was ments for each sample were made at 5 different locations using obtained from Alfa Aesar Chemical Company, Germany. Hemin adobe Photoshop CS version 8.0 to determine colour coordinates, (98% pure, HPLC grade) was purchased from the Fluka Company, i.e., the L*, a* and b* values. For standardization of colour, the images Switzerland. were also recorded from the Ral colour standard. 2.2. Preparation of nitrosoheme Table 2 The nitrosoheme pigment was prepared from hemin and nitrite. Experimental design used in the RSM studies and the responses. Bovine hemin (6.52 mg) was dissolved in 1.8 ml of a 0.1 N NaOH so- Exp. Coded level of variables Yield λmax L* a* b* lution. This solution was diluted with 8 ml of acetone, and then no. (%) (nm) value value value 0.2 ml of concentrated hydrochloric acid was added, giving a solu- X1 X2 X3 tion of acid hematin in 80% acetone.

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