Zn porphyrin formation in cured meat products_effect of added salt and nitrite.pdf

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Zn-porphyrin formation in cured meat products: Effect of addedsalt and nitrite

Christina E. Adamsen, Jens K.S. Mller, Kristoffer Laursen, Karsten Olsen, Leif H. Skibsted *

Food Chemistry, Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 4th. DK-1958 Frederiksberg C, Denmark

Received 26 April 2005; received in revised form 16 September 2005; accepted 29 September 2005

Abstract

Zn-porphyrin (Zn-pp) was quantified by fluorescence spectroscopy in the cured and dry cured meat products: Parma ham, Iberianham, dry-cured ham with added nitrite, cooked ham with added nitrite, raw ham meat, raw bacon and Karree-Speck. The highestamount of Zn-pp was found in dry-cured Parma ham and Iberian ham, while the use of nitrite as curing agent was found to inhibit com-pletely the formation of Zn-pp in meat products. A positive correlation between both Zn content and Fe content and the logarithmictransformed Zn-pp content (measured as fluorescence intensity Ifl) was found for the different cured and dry cured meat products, withcorrelation coefficients of 0.79 (p< 0.001) and 0.71 (p< 0.01), respectively. LogIflcorrelates best with the Zn content, indicating that theformation of Zn-pp is proportional to the Zn content. A model system with vacuum packed pork in brine with different added levels ofsodium chloride with or without nitrite and Zn acetate was investigated in order to further elucidate the mechanism of Zn-pp formation.Zn-pp increased with time (up to 42 days investigated) in non-cured meat and for meat cured solely with NaCl lower than 9%. Additionof nitrite or Zn(II) in the curing brine was found to inhibit formation of Zn-pp confirming the observations from the various cured meatproducts. It is suggested that a chloride anion assisted dissociation of iron from myoglobin could be rate-determining for Zn-pp forma-

tion in meat products. 2005 Elsevier Ltd. All rights reserved.

Keywords: Dry cured meat products; Colour; Zn-protoporphyrin; Nitrite content; Salt content

1. Introduction

Visual appearance is of vital importance for the qualityof most food products, and especially the colour affectsconsumers when they evaluate freshness and quality ofmeat and meat products. The colour of meat products is

determined by a combination of different factors includingmoisture and fat content, but more important is the chem-ical form and concentration of the hemoproteins, especiallythat of myoglobin (Mb) (Fox, 1966; Lawrie, 1991; Led-ward, 1992). Mb is affected by processing parametersincluding heat treatment and the use of nitrite/nitrate inmeat curing is of particularly interest together with thepacking method used for the product, because compounds

formed by reduction of nitrite or nitrate react with Mbforming the pink-coloured pigment, nitrosylmyoglobin(MbFe(II)NO) (Fox, 1966; Mller & Skibsted, 2002). Inproducts with added nitrite or nitrate the complexMbFe(II)NO is the main contributor to the characteristiccolour, e.g. in dry-cured ham, brine-cured meat products

and cooked cured ham. Cured meats and meat productswithout nitrite/nitrate addition will normally attain a dullbrown colour in raw products or a grey colour in heatedproducts, which influences consumer acceptance nega-tively. Also safety aspects of adding nitrite/nitrate as aningredient to meat have been discussed over the past dec-ades (Cassens, 1990), and topics such as microbiologicalsafety versus human health concerns have to be consideredand analyzed.

The traditional dry-cured ham from Northern Italy,Parma ham, is made from pork legs and only sodium

0309-1740/$ - see front matter 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.meatsci.2005.09.017

* Corresponding author. Tel.: +45 35283221; fax: +45 35283344.E-mail address: [email protected](L.H. Skibsted).

www.elsevier.com/locate/meatsci

Meat Science 72 (2006) 672679

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chloride in the form of sea salt from the Mediterranean Seais added, and notably the product obtains a stable red col-our without any addition of nitrite or nitrate (Parolari,1996). Theories and hypotheses regarding the chemical nat-ure and identity of the Parma ham pigment are numerousincluding suggestions for the reaction pathway for their

formation. Virgili et al. (1999), thus, suggested that low-molecular weight compounds containing electron donatingatoms, formed during maturation of Parma ham, in partic-ular basic peptides or amino acids resulting from an exten-sive proteolysis, may play a role as Fe ligands in Mb.Morita, Niu, Sakata, and Nagata (1996) claimed that theMb derivative present in matured Parma ham was a nitro-sylated Mb formed by microbial activity in the ham duringthe prolonged processing. Other studies showed that thecolour or the change of colour in meat and meat productsmay depend on microbial growth (Kalchayanand, Ray,Field, & Johnson, 1989; Arihara et al., 1993). However, itseems highly speculative that the pigment in Parma ham

is formed solely by microbial action, since microorganismsare mainly found at the surface of the meat during the pro-cess while a uniform colour is developed throughout thewhole ham. The colour of Parma ham has also been sus-pected to be due to contamination with nitrate from thesea salt, but the total amount of nitrate from the salt hasbeen found negligible (Sakata, 2000). Mller, Adamsen,and Skibsted (2003)have by electron spin resonance spec-troscopy shown that the Parma ham pigment is differentfrom MbFe(II)NO and is not a nitric oxide complex.

Morita et al. (1996) found that the pigment of Parmaham is easily extracted with 75% acetone/water solution

and that it is a different Mb derivative not known fromother meat and meat products. Recently, both Mlleret al. (2003) and Parolari et al. (2003) further showed thatboth a lipophilic (75% acetone) and a hydrophilic pigment(phosphate buffer pH = 6.00) can be extracted from Parmaham through processing. Both pigments exhibit uniqueUVVis spectral features different from those of otherwell-characterized Mb derivatives, and during processingthe lipophilic pigment becomes predominant.

The lipophilic pigment extracted from Parma ham hasmore recently been identified as Zn-protoporphyrin IX(Zn-pp), believed to originate from Mb in which Fe hasbeen substituted by Zn and the heme separated from thenative heme-protein (Wakamatsu, Nishimura, & Hattori,2004a). Another study from Wakamatsu, Okui, Ikeda,Nishimura, and Hattori (2004b) showed, using a modelsystem, that anaerobic conditions favour formation ofZn-pp, and that endogenous enzymes as well as microor-ganisms may be involved in the formation of Zn-pp. Threepossible substitution patterns may accordingly be sug-gested: (i) a non-enzymatic reaction in which Zn(II) substi-tutes Fe(II) under anaerobic condition with concomitantdissociation of the heme; (ii) a bacterial enzymatic reactionwhere bacterial growths naturally degrade the meat pro-teins including the pigment, or (iii) an enzymatic reaction

where an endogenous ferrochelatase interchanges the two

metals. More knowledge about the chemical nature andmechanisms of formation of the stable red pigment inParma ham is of general interest, since such knowledge pre-sents future prospects for manufacturing cured meat prod-ucts with a desirable and stable red colour without the useof nitrite/nitrate.

The present work is part of a project investigating theformation of stable red pigments in Parma ham, with theaim of using such information for designing processes formanufacturing other meat products without nitrite ornitrate. The objective of the current study was to determinethe Zn-pp content in different types of meat products pro-duced with variations in curing and processing technology,in order to obtain more information about how the pro-cessing parameters affect the formation of Zn-pp. More-over, a model system with vacuum packed pork cured atdifferent levels of sodium chloride with and without nitriteand added zinc acetate was established in order to investi-gate how varying combinations of sodium chloride, nitrite

and Zn affect the formation of Zn-pp. The sodium nitriteconcentration used for the brine was as high as 1% (w/w)which notably is far beyond the legal permission. However,the aim was solely to investigate the possible mechanism ofnitrite on formation of Zn-pp.

2. Materials and methods

2.1. Chemicals

Sulphuric acid (9597%), hydrochloric acid (37%),sodium chloride, sodium nitrite, silver nitrate, nitric acid

(65%), ammonium iron(III) sulphate, potassium thiocya-nate and hydrogen peroxide (30%) were all of analyticalgrade and obtained from Merck (Darmstadt, Germany).The Fe standard (1 mg/l, Titrisol) and the Zn standard(1 mg/l, Titrisol) were obtained from Merck (Darmstadt,Germany). Acetone and methanol were obtained fromLab-Scan (Dublin, Ireland). MES (Z-[N-morpho-lino]ethanesulfonic acid), zinc acetate dihydrate and ribo-flavin were all obtained from Sigma-Aldrich ChemieGmbH (Steinheim, Germany). Certified reference material,bovine muscle CRM148, was obtained from The Institutefor Reference Materials and Measurements (Geel, Bel-gium). All other chemicals were of analytical grade andused without further purification. Water was purifiedthrough a Millipore Q-Plus unit (Millipore, Bedford,Mass., USA).

2.2. Cured meat products

Seven different types of cured meat products wereincluded in this study. Fully matured Parma ham (12months manufacturing time) was obtained from a localproducer in Parma through Stazione Sperimentale perIIndustria delle Conserve Alimentari, Parma, Italy. Fullymatured Iberian ham (30 months manufacturing time)

was obtained from Senorio de Montanera S.L. Criadores

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de Cerdo Iberico Asociados, Badajoz, Spain. Dry-curedham with added nitrite (3 months manufacturing time)and cooked, nitrite-cured ham was obtained from TulipInternational (Viby J., Danmark). Raw meat from theham region of the pig and raw bacon salted with nitriteas standard Danish products were obtained locally. A spe-

cial dry-cured product, Karree-Speck (3 months manufac-turing time), locally produced in Austria was obtainedfrom a local shop in Zillertal, Austria. From all seven typesof meat products a slice, without visible fat, selected as typ-ical for the product was finely chopped and used for furtheranalysis.

2.3. Meat model system

For the meat model systems Semimembranosus (SM)muscles were used. The meat was obtained from The Dan-ish Meat Trade College through the Danish Meat ResearchInstitute, Roskilde, Denmark. The muscles were from 6 dif-

ferent animals (total 12 SM muscles) originating from thesame producer. The animals had been treated equallyunder production, during the slaughtering process andafter slaughter. The muscles were removed from the animaljust after slaughter and immediately packed in vacuumbags and stored at 2 C until used. All the animals had apH value post-mortem between 5.5 and 5.7.

The model experiment was carried out as two separateexperiments. The first experiment included 5 different con-dition and the second experiment included 4 different con-dition, and all 9 curing condition are described in Table 1.

For each experiment, 6 SM muscles were cut in to cubes

of approximately 2 2 2 cm and mixed. The meat wasthen divided into the curing-groups, as described above.The curing was carried out as brine salting. Equal quantityof meat and brine were added to a 13 20 cm pouches oflaminated packaging material consisting of 30 lm polyam-ide (PA)/100 lm polyethylene (PE) (SFK Meat system,Denmark) with an oxygen transmission rate of 21 cm3 m2 d1 bar1 (at 23 C and 75% RH), a carbon diox-ide transmission rate of 97 cm3 m2 d1 bar1 (at23 C and 75% RH), and a watervapour transmission rate

of 0.8 g cm3 d1 (at 23 C and 85% RH) and werevacuum packed (Electronic VacMit, Duggendorf, Ger-many). The vacuum packed meat in brine was stored at5 C in darkness in a refrigerator for two days. After cur-ing, the meat pieces were drained for of excess brine andvacuum packed in 30 lm PA/100 lm PE laminated bags

(as described above). The meat was then stored at 5 C indarkness in a refrigerator for 42 days. On day 0, 7, 14and 42, randomly selected samples (n= 3) of each meatsample were analysed.

2.4. Chemical analysis

2.4.1. Water content analysis

Two grams of finely chopped meat were placed on analuminium tray (with known weight) and the precise weightwas noted. The samples were dried in a thermo cabinet(WTB Binder GmbH, Tuttingen, Germany) at 104 C in4 h. Immediately after drying the samples were placed in

a desicator and after 15 min, the samples were weightedand the water content was calculated.

2.4.2. Salt content

The chloride was quantified as gram/100 g meat in themeat sample. Ten gram of homogenized meat sample wastransfer into a 500 ml conical flask and 200 ml of boilingmilli-Q-water was added. The sample was mixed andcooled to room temperature. The meat solutions were fil-tered through an S&S filter 5892 (Scheicher & SchuellGmbH, Germany). Ten milliliters of the filtrate was mixedwith 10 ml a 0.1 M silver nitrate solution, 1.5 ml of a 65%

nitric acid and 0.5 ml ammonium iron(III) sulfate solution(5 g ammonium iron(III) sulfate dissolved in a 10% (w/w)nitric acid solution). The solution was boiled for 5 minand cooled to room temperature, filtrated through a S&Sfilter 5892 and titrated with 0.05 M potassium thiocyanatesolution.

2.4.3. Water activity

Water activity was measured using a CX-2 water activityanalyzer (AQUA LAB, Pullman W.A., USA). All sampleswere chopped with a knife very fine and the analysis wasperformed in triplicate.

2.4.4. Nitrite content

The nitrite content was measured by an NOx

biosensor(Unisense A/S, Arhus, Denmark) connected to a bio-chamber with nitrite reductase activity. The biosensorwas calibrated by two solutions, one containing 1.0 mMsodium nitrite with 1% NaCl and another 0.1 mM sodiumnitrite with 1% NaCl both dissolved in a 50 mM MESbuffer (pH = 5.5). The nitrite contents in the meat sampleswere measured in meat slurries. The meat slurries wereprepared by homogenization of the different ham or meatsamples with a 50 mM MES-buffer (pH = 5.5), thus thefinal meat slurry had a salt content of approximately 1%.

Each sample was measured in triplicate.

Table 1

The sodium chloride content (w/w), sodium nitrite content (w/w) and zincacetate content (w/w) of the brine for the nine meat curing systems

Number Sodium chloride(w/w)

Sodium nitrite(w/w)

Zn-acetate(w/w)

1 Controla b 2 15% 3 25% 4 15% 1% 5 25% 1% 6 15% 0.6%7 25% 0.6%8 15% 1% 0.6%9 25% 1% 0.6%

a Meat without curing.

b No addition.

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2.4.5. Colour measurements

The colour of the cured meat pieces was measured witha Color-guide 45/0 (BYK-Gardner GmbH, Geretsried,Germany) using the L*, a*, b* coordinates (CIE L*a*b*colour system). Red colour was expressed as the a*-value;the higher the a*-value the more red was the sample.

Approximately 10 g of each meat type was chopped veryfine using a mincing machine (KRUPS, Speedy Pro plus,Groupe SEB, RCS Lyon, France) and placed betweentwo glass Petri-dishes, and the colour was measured thoughthe glass on three randomly selected locations of thespread-out mince. For the different ham types the colourwas measured once. In the experiment with meat curingthe colour was measured on day 0, 7, 14 and 42.

2.4.6. Isolation of Zn-pp from ham

Zn-pp from the ham/meat was isolated as described byWakamatsu et al. (2004a)with minor modification. Afterhomogenizing the meat in distilled water, the homogenate

was centrifuged at 3000 rpm for 10 min at 4 C and filtered.

2.4.7. Fluorescence-spectroscopy

All measurements of fluorescence emission intensities(Ifl) and spectra were made using an Aminco Bowman ser-ies 2 luminescence spectrometer (SLM-Aminco, Urbana,IL, USA). All spectra were recorded at 25C. Sampleswere excited at 420 nm and the emission spectra were mea-sured from 500 to 700 nm with 1 nm intervals and theintensity of the emission maximum at 588590 nm wasmonitored relative to a riboflavin standard (1 mg/ml in75% acetone/water-solution equals to 1). The index for

fluorescence emission intensities was calculated from:

Ifl flsample ex420 nm;em589 nm

flriboflavin 1 mg=ml ex420 nm;em589 nmM1dry matter 1

whereMdry matteris the dry matter content of the sample ingram.

Only solvents stored in glass containers were used andthroughout the analytical procedure, only glassware wasused. Fluorescence was measured in quartz cuvettes withteflon stoppers. For the seven different ham types the fluo-rescence emission intensities were measured three times foreach ham types. In the curing experiment the fluorescenceemission intensities were measured on day 0, 7, 14 and42. All measurements were carried out three times on eachmeat sample.

2.4.8. Total Fe and Zn content

The total Fe and Zn content was quantified using atomicabsorption spectrophotometry (AAS) based on the methodof the Danish Standardization Board (Dansk Standard 259,Copenhagen, Denmark 1982). Samples (0.500 0.004 g) offreeze-dried meat from the seven different types of meatproducts (Parma ham, Iberian ham, dry-cured ham withnitrite, raw bacon with nitrite, raw ham meat, Kaaree-Speck with nitrite, and cooked ham with nitrite) and a

freeze-dried SM sample from 6 different animals were

weighed into vessels (Teflon) (special for heating in amicrowave oven, CEA Advanced composite vessel assem-blies, CEM Innovators in microwave technology, CEMMatthews, USA), and 7 ml of concentrated nitric acidwas added. The mixture was heated in a microwave oven(MES-81 D, CEM Matthews, USA) for 25 min (5 min at

full micro-wave power, 10 min at 75% of full micro-wavepower and 10 min at full micro-wave power). After heat-ing, 5 drops of H2O2 (30%) were added to each vesseland the vessels were left at room temperature overnight.The samples were quantitatively transferred to 10 ml volu-metric flasks with Milli-Q water. After 1:10 dilution thesolution was analysed for iron using AAS (Atomic Absorp-tion Spectrometer 3300, Perkin-Elmer with an Iron-lamp,Intensitron lamp, Perkin-Elmer, Wellesley, MA 02481-4078, USA) using a standard curve based on an iron stan-dard (Titrisol). These solutions were analysed for zinc usingan AAS (Atomic Absorption Spectrometer 3300, with aZinc-lamp, Intensitron lamp, Perkin-Elmer, Wellesley,

MA 02481-4078, USA) using a standard curve based ona zinc standard (Titrisol). The AAS method was controlledwith a standard certified reference material (Bovine muscle,Commission of the European communities, Communitybureau of reference BCR, Reference material no. 184).The presented values are means of duplicate measurements.

2.5. Data analysis

Average values of the Fe content, Zn content, wateractivity, water content, salt content and the initial colourand Ifl were compared by Students t-test and the correla-

tions between pairs of variables were calculated as Pear-sons correlation coefficients. The formation of Zn-ppmeasured as Ifl in the meat curing experiment were ana-lyzed by a two-way analysis of variance, including as mainfactors curing method and storage time. All the statisticalanalyses were done in the Analyst application within theSAS system statistical software 8.02 (SAS Institute, Cary,NC, USA).

3. Results and discussion

Zinc porphyrin has now been demonstrated to be themain pigment in Parma ham and, in order to obtain moreknowledge about how processing parameters affect the for-mation of Zn-pp in meat products, Parma ham, Iberianham, dry-cured ham with nitrite, raw bacon with nitrite,Kaaree-Speck with nitrite, cooked ham with nitrite andraw ham meat were compared with respect to their Zn-ppcontent. The results obtained for Fe content, Zn content,water activity, water content, salt content, the initial colourand the fluorescence intensityIflof the extract are shown inTable 2. Due to large variation in origin and processing,the seven ham/meat types were rather different in composi-tion and initial colour (a*-value). Especially, Fe and Zncontents were found to be higher for Parma ham and

Iberian ham compared to other meat products. For the



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initial colour, it was observed that the addition of nitriteleads to higher a*-values (more red colour) as expected,since nitrite is known to contribute to the colour formationin cured meat products (Mller & Skibsted, 2002).

Fig. 1 shows fluorescence spectra of the red pigmentextracted with 75% acetone/water solution as describedby Wakamatsu et al. (2004a) from dry-cured ham pro-duced without nitrite/nitrate (Fig. 1A), and of extractsfrom the five other meat products cured with nitrite andof extract from raw ham (Fig. 1B). The fluorescence spectraobtained using excitation at 420 nm of extracts from theseven different types of ham/meat, all show two emissionmaxima, a strong band at 588590 nm and a weak bandat 640642 nm (Fig. 1), respectively, and the spectra clearlydiffer in intensity. The fluorescence spectra from all seventypes of ham/meat show the characteristic spectrum ofZn-pp (the fluorescence emission maximum of Zn-pp is

583589 nm) comparable with the findings for extractsfrom Parma ham and with the fluorescence spectrum of

purified Zn-pp as reported by Wakamatsu et al. (2004a).Mean values of the fluorescence emission intensities at589 nm, Ifl, are reported in Table 2, and the intensitiesclearly differ. The highest Ifl is observed for Parma ham

and Iberian ham, which are significantly higher (p< 0.001)than dry-cured ham produced with nitrite, raw bacon pro-duced with nitrite, raw ham meat, Karree-speck andcooked ham produced with nitrite (Table 2).

The possible relationship between Ifl of the meat prod-ucts extracts and the physiochemical parameters measuredfor the different ham/meat products (Fe, Zn, salt and watercontent; plus aw, and initial colour) has been assessed byanalysis of Pearsons correlations. The fluorescence intensi-ties are logarithmically transformed (log Ifl) prior to corre-lation analysis in order to obtain a normal distribution. ThePearsons correlation coefficients (Table 3) show that bothZn (p< 0.001) and Fe (p< 0.01) content are positively cor-

related to logIfl, with values of 0.79 and 0.71, respectively.Fig. 2shows scatter plots for log Iflas a function of the Zncontent and Fe content. It can be observed that the log Iflcorrelates much better with the Zn content compared toFe content indicating that the formation of Zn-pp is loga-rithmically proportional to the Zn content in the rangeinvestigated. In contrast, a negative correlation betweenlog Ifl and either aw (p< 0.05) or water content (p< 0.05)can be observed (r= 0.56 and 0.61, respectively), in

Table 2Content of Fe (lg/g dry matter), Zn (lg/g dry matter), water activity, water content (g water/100 g meat), salt content (g NaCl/ 100 g meat), initial colour(a*value) and fluorescence intensity Ifl(kex= 420 nm, kem= 590 nm, relative to a 1 mg/ml riboflavin standard) of Zn-porphyrin in the extract of differentham types, produced by varying process (dry-cured, brine-cured, cooked and raw) and with or without nitrite added

Type of ham Fe (Fe lg/g dry matter)

Zn (Zn lg/g dry matter)

aw Water (g water/100 g meat)

Salt (g NaCl/100 g meat)

Colour(*a-value)

Ifl

Dry-cured Parma ham 35.4 1.6 107.2 2.9 0.915 0.001 57.5 1.2 4.1 0.2 5.1 0.04 39 7

Dry-cured Iberian ham 50.8 8.4 110.6 6.6 0.872 0.001 44.0 0.5 5.9 0.2 9.8 0.5 25 4Dry-cured ham with nitrite 28.3 2.1 92.5 0.1 0.901 0.001 58.0 0.8 7.7 0.1 13.4 0.7 0.51 0.05Raw bacon with nitrite 30.2 1.6 110.6 8.4 0.947 0.002 69.1 0.05 3.8 0.4 7.4 0.2 0.25 0.02Raw ham meat 27.1 1.6 72.2 1.4 0.994 0.003 73.3 0.8 ND 9.3 0.4 0.13 0.02Karree-Speck with nitrite 29.2 1.3 83.2 4.2 0.896 0.004 54.1 2.5 6.1 0.1 12.2 0.03 0.06 0.04Cooked ham with nitrite 27.5 1.0 68.3 2.9 0.967 0.001 74.2 0.6 3.8 0.2 9.9 0.4 0.005 0.002

ND = non detectable.Values are the means standard deviations.

500 550 600 650 700

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400A

Fluorescen

ceIntensity(EX420nm)

B

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Fig. 1. Fluorescence spectra of red pigment extracted with 75% acetone/water solution from Parma ham (full line), Iberian ham (broken line) (A),dry-cured ham with nitrite (dotted line), raw bacon with nitrite (dot-dashline), raw ham meat (dot-dot-dash line), Kaaree-Speck with nitrite (full line)and cooked ham with nitrite (broken line) (B). All spectra were recorded at25 C. Samples were excitated at 420 nm and the emission spectra were

measured from 500 to 700 nm.

Table 3

Correlation coefficients of composition Fe (lg/g dry matter), Zn (lg/g drymatter), water activity, water content (g water/100 g meat), salt content (gNaCl/100 g meat) and initial colour (a*value) and logIflfor the 7 differentham types (seeTable 2)

Variable Fe Zn aw Watercontent

Saltcontent

Colour

Zn 0.58*

aw 0.61* 0.60*

Water content 0.49 0.54 0.94***

Salt content 0.26 0.36 0.86*** 0.75**

Colour 0.58* 0.39 0.66** 0.70** 0.42Log Ifl 0.71

** 0.79*** 0.56* 0.61* 0.19 0.65*

* Significant p < 0.05.** Significant p < 0.01.

*** Significant p < 0.001.



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agreement with the observed higher values for Ifl in dry-cured products (higher concentration of Zn-pp) comparedto other types of meat/meat products. A positive correla-tion between log Ifl and surface colour measured as a*-value(p< 0.05; r= 0.65) was found, which is in agreement withthe recent suggestion that Zn-pp is the principal pigmentin dry-cured ham produced without nitrite or nitrate (Wak-amatsu et al., 2004a). However, no significant correlationbetween log Ifland the salt content was found, despite the

fact that the salt content is much higher for dry-cured meatproducts compared to brine-cured bacon and ham.A clear correlation between aw, water content, and salt

content was observed (Table 3), which is expected due tothe interrelationship between these parameters. Also a neg-

ative correlation between surface colour and aw (p< 0.01)or water content (p< 0.01) (r= 0.56 and 0.70, respec-tively) was found confirming that meat products withreduced water content and low awcontain a higher concen-tration of pigments. A weaker correlation (p< 0.05) wasobserved between Fe, Zn and aw, and between Fe content

and surface colour (p< 0.05), indicating also that Fe con-tent, possibly through a higher pigment concentration,has an effect on the observed colour of the meat/meat prod-ucts, although the relationship is less clear.

The total processing time seems to have a positive effecton the formation of the Zn-pp. The dry-cured hams, Parmaham and Iberian ham (manufacturing time > 12 months)both have much higher Ifl than all other types of meatproducts considered, which in general all have muchshorter processing times


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colour (Table 4) of meat cured with nitrite was more red asevidenced by higher a*-values as expected from the reac-tion of nitrite with meat products.

Formation of Zn-pp was followed by fluorescence spec-troscopy, and as may be seen from Fig. 3, fluorescenceintensity Ifl (kex= 420 nm, kem= 590 nm, relative to a

1 mg/ml riboflavin standard) for extracts of the meatexposed to the nine different curing conditions at 5 C for42 days varied considerably. Ifl was measured at days 0,7, 14 and 42 and as further seen from Fig. 3A, Zn-pp (Ifl)was formed and the amount increased with time for rawmeat (control), meat cured in 15% (w/w) brine and meatcured in 25% (w/w) brine. The concentration of Zn-ppreached a maximum after 14 day of storage followed by adecrease in the Zn-pp content for raw meat (control), meatcured in 15% (w/w) brine and meat cured in 25% (w/w)brine. The formation of Zn-pp was more pronounced forraw meat (control) and meat cured in 15% (w/w) brine.For meat cured in 25% (w/w) brine, the formation of

Zn-pp was significantly lower compared with raw meat(control) and meat cured in 15% (w/w) brine. For meatcured with nitrite and 15% (w/w) salt brine or nitrite and25% (w/w) brine, zinc acetate and 15% (w/w) brine or25% (w/w) brine, nitrite and zinc acetate and 15% (w/w)brine or 25% (w/w) brine, no formation of Zn-pp wasobserved through the curing period.

The change in red colour measured as a*-value was alsofollowed on days 0, 7, 14 and 42, but no correlation betweenthe formation of Zn-pp and the change in the red colour wasobtained (data not shown). Only the well known effect onred colour from curing with nitrite was observed.

The observation that Zn(II), added as zinc acetate, had

no effect on the formation of Zn-pp was remarkable, sinceit suggests that the displacement of Fe from Mb is not abimolecular substitution process. The rate determining stepmay rather be a dissociation of Fe(II) from Mb under non-oxidizing condition followed by a fast binding of Zn (II).

In conclusion, our results show that Zn-pp is presentnot only in Parma ham, but also in other meat productsalthough in a lower concentration. In addition, our exper-iments have demonstrated that the use of nitrite as a cur-ing ingredient inhibits the formation of Zn-pp. Thisinhibition was demonstrated for different cured and drycured meat products and the effect was confirmed in ameat model system, although the content of nitrite in

the model system was far beyond what is legally permittedand much higher than in real meat products. It seems,however, not possible to identify the fundamental mecha-nism behind formation of Zn-pp in meat products. Threepossible mechanisms for the metal substation are: (i) anon-enzymatic enzymatic reaction driven by binding ofiron in the high chloride meat matrix, (ii) a bacterial enzy-matic reaction or (iii) an endogenous enzymatic reaction.A more detailed understanding of the chemistry and themechanism behind the substitution between Fe and Znwill, however, have to await a kinetic study of salt-drivendissociation of Fe from myoglobin and the reaction

between nitrite and myoglobin, which seems to inhibitthe dissociation of Fe from myoglobin.

Acknowledgements

The present study was partly funded by the ResearchSchool for Organic Agriculture and Food Systems (SOAR)and the Norma and Frode S. Jacobsens Fond, Copenhagen,Denmark. We would like to thank Dr. Giovanni Parolari,Stazione Sperimentale per lIndustria delle Conserve Ali-mentari, Parma, Italy, for the kind donation of Parmaham samples, Dr. Ana Isabel Andres Nieto, the Universityof Extremadura, Spain, for the arranging the contact to theproducer of Iberian ham, Puro Iberico Artesana, S.L., Bad-ajoz, Spain and Bent Olesen, Tulip, Vejle, Denmark, for thekind donation of nitrite based dry-cured ham samples.

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0 20 30 40 50

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0 20 30 40 5010 0 20 30 40 5010

Time (Day)

Fig. 3. Changes in fluorescence intensityIfl (kex= 420 nm, kem= 590 nm,relative to a 1 mg/ml riboflavin standard) for Zn-pp in extract of: (A)Meat without curing (control) (square), meat cured with 15% (w/w) brine(circle) and meat cured with 25% (w/w) brine (triangle). (B) Meat curedwith 15% (w/w) brine plus 1% (w/w) sodium nitrite (circle) and meat curedwith 25% (w/w) brine plus 1% (w/w) sodium nitrite (triangle). (C) Meatcured with 15% (w/w) brine plus 0.6% (w/w) zinc acetate ( open circle) andmeat cured with 25% (w/w) brine plus 0.6% (w/w) zinc acetate ( opentriangle). (D) Meat cured with 15% (w/w) brine plus 0.6% (w/w) zincacetate and 1%(w/w) sodium nitrite (open circle) and meat cured with 25%(w/w) brine plus 0.6% (w/w) zinc acetate and 1% (w/w) sodium nitrite(open triangle) during curing. Values are means of three independent

measurements standard deviations, indicated by bars.



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