The role of myoglobin degradation in the formation of zinc protoporphyrin IX in the longissimus lumborum of pork

https://doi.org/10.1016/j.lwt.2017.06.047Get rights and content

Highlights

  • Within 72 h incubation the meat extract fluorescence rose due to Fe-Zn replacement.

  • The first 24 h of incubation are rate determining for Fe-Zn replacement (p < 0.001).

  • The changes in myoglobin concentration were clearly non-significant (p = 0.4–0.9).

  • Myoglobin protein degradation is not a necessity for Zn integration in heme.

Abstract

Investigations on the post mortal formation of fluorescent zinc protoporphyrin (ZnPP) IX in pork meat are currently in focus of meat science research. The role of myoglobin degradation in this context appears to be one of the most diversely discussed issues. To address this question meat-extracts of longissimus lumborum (LL) muscle (0.8 mg/mL) were incubated at 30 °C for up to 72 h and investigated by HPSEC-UV-fluorescence, SDS-PAGE and MALDI-TOF-MS. Between 0 and 72 h of incubation the fluorescence intensity (λex./em. = 420/590 nm) of the meat-extracts rose significantly (p < 0.001) from 10.9 ± 0.8 to 34.8 ± 0.3 (rel. units) while the staining intensity of the SDS-PAGE of myoglobin non-significantly (p > 0.4) changed from 6.2 ± 0.5 × 105 to 5.0 ± 0.3 × 105 (rel. units). The results indicate that ZnPP is formed by a Fe(II)-Zn(II)-substitution in myoglobin heme where an accompanying myoglobin degradation is not necessarily obligatory.

Introduction

The luminescent metalloporphyrin ZnPP is a by-product of the heme biosynthesis, which is present in almost all forms of life (Heinemann, Jahn, & Jahn, 2008). Due to its color and its specific fluorescence at the excitation and emission wavelengths λex. = 420 nm and λem. = 590 nm (Camadro et al., 1984, Wakamatsu et al., 2004b) ZnPP has technologically become a subject of interest, as its physical features have been discussed in terms of their potential benefits during meat processing: ZnPP has been proven as the main chromophore in Parma ham giving the product its characteristic red color (Adamsen et al., 2006, Moller et al., 2007, Wakamatsu et al., 2004a). Parma ham traditionally is cured without the addition of nitrite salts, which are typically used to achieve a stable red color in meat, but also represent a source of concern due their role in the formation of carcinogenic N-nitrosamines (Pegg & Shahidi, 2008, p. 259). In this context ZnPP might represent a nitrite free alternative for the reddening of meat and meat products (Tuan Thanh, Ishigaki, Kataoka, & Taketani, 2011). On the other hand the accumulation of ZnPP during the storage of the longissimus lumborum (LL) of pork has been attested by Schneider et al. (2008), so that ZnPP has been discussed as a potential indicator in the evaluation of LL quality.

Nevertheless, the biochemical reaction pathway leading to the formation of ZnPP in the aforementioned kinds of pork meat (Parma ham and LL) still has to be clarified. There are three assumptions on the post mortem formation of ZnPP, which have been discussed using the example of Parma ham:

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    i) a non-enzymatic chelation of Zn(II) by protoporphyrin IX (PP) (Becker, Westermann, Hansson, & Skibsted, 2012)

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    ii) an enzyme-induced substitution of Fe(II) from heme with Zn(II) by endogenous ferrochelatase (FECH) (Becker et al., 2012, Benedini et al., 2008, Parolari et al., 2009, Wakamatsu et al., 2007),

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    iii) a bacteria-induced enzymatic reaction (Morita et al., 1996, Wakamatsu et al., 2004a, Wakamatsu et al., 2004b),

whereby the possibility of an enzymatic and the non-enzymatic pathway, respectively, is assumed to be affected by the storage temperature (Parolari, Aguzzoni, & Toscani, 2016). Myoglobin is supposed to be the heme-donor for the ZnPP formation (Wakamatsu et al., 2004a, Wakamatsu et al., 2004b). Using the example of Parma ham Grossi, do Nascimento, Cardoso, and Skibsted (2014) have reported, that the degradation of myoglobin is essential for the formation of ZnPP, as the degraded myoglobin would allow a Fe(II)-Zn(II)-transmetallation in myoglobin heme. However, the increase of ZnPP concentrations in LL within the storage at room temperature (RT) for 72 h has been attested in our recent research works taking the view that the accumulation of ZnPP has not been associated with a degradation of myoglobin in LL. Thus the aim of this study was to find out, whether myoglobin degradation, as it had been described in the case of Parma ham, could also be observed in LL samples in spite of the very short storage time as compared to Parma ham (>9 month).

To pursue this question LL extracts were incubated at RT for up to 72 h and analysed by various analytical techniques: a HPSEC-UV-fluorescence setup was used in order to separate the myoglobin from most of other meat-inherent proteins and to determine the occurrence of ZnPP fluorescence in the myoglobin during the incubation of the extracts. For the determination of a probable myoglobin degradation during the storage of the LL extracts the myoglobin-containing protein bands of the LL extracts were collected after HPSEC by a fraction collector and investigated qualitatively and semi-quantitatively by MALDI-TOF-MS and SDS-PAGE, respectively: MALDI-TOF-MS spectra were supposed to enable the detection of lower molecular myoglobin fragments, which might indicate a myoglobin degradation during the storage of the LL extracts. SDS-PAGE analyses were supposed to enable the detection of a probable decrease of the myoglobin concentration during the storage of the LL extracts.

The present research study delivers new insights into the chemical nature of the ZnPP formation during the storage of LL, in which the fluorophore is accumulated at RT within a few days of storage.

Section snippets

Preparation and incubation of the water-soluble LL proteins

LL of pork was purchased from a local butcher (Potsdam, Germany) one day after slaughter and minced with a hand mixer. Thirty two g of that meat homogenate were weighed into a beaker and diluted with 40 mL of a pH 7.4, 4 °C phosphate buffer (10 mmol/L Na2HPO4, 1.8 mmol/L NaH2PO4, 9 g/L NaCl, 0.2 g/L NaN3, all from Carl Roth, Karlsruhe, Germany). Protein extraction was performed on ice by treating the meat homogenate with an Ultra Turax (IKA GmbH, Staufen, Germany) at 12.000 rpm for 1 min.

Results and discussion

The chromatogram of the HPSEC analysis showed the separation of the LL extract into four protein bands of high intensity in the time-ranges of tR = 12–14 min, 19–22 min, 24–27 min, 29–31 min and into five protein bands of low intensity in the time-ranges of tR = 22–24 min, 28–29 min, 32–34 min, 34–36 min, 42–44 min (Fig. 1). The retention time of the myoglobin standard was tR = 23.5 min and therefore the myoglobin in the LL extract was expected in the protein band in the time range tR

Conclusion

The comprehension of the biochemical mechanism leading to the formation of ZnPP in stored porcine muscles still remains a challenge in the research field of the post mortem biochemistry of meat. Though the results of this study indicate, that Mb heme most probably is the primary substrate for the Fe(II)-Zn(II)-substitution forming ZnPP and that the ZnPP formation takes place at an early stage of meat incubation before Mb degradation begins, the biomolecular formulation for this reaction pathway

Acknowledgements

This study was supported partially by a promotion fellowship from the Avicenna foundation from the Federal Ministry of Education and Research of Germany.

References (23)

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