The role of myoglobin degradation in the formation of zinc protoporphyrin IX in the longissimus lumborum of pork
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),
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.
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2020, Food Research InternationalCitation Excerpt :However, the higher lipolysis extent in hams with a reduced salting time had no effect on pigments content. The substitution of Fe by Zn in the heme group catalyzed by the enzyme takes place in both native and partly proteolyzed myoglobin (Khozroughi et al., 2017; Paganelli et al., 2016; Wakamatsu et al., 2019). Despite that, it seems that the formed ZnPP bound to protein can be transitioned into free ZnPP during the process of incubation (Khozroughi et al., 2019).