Spectral characterisation of red pigment in Italian-type dry-cured ham. Increasing lipophilicity during processing and maturation

Spectroscopic studies of Parma ham during processing revealed a gradual transformation of muscle myoglobin, initiated by salting and continuing during ageing. Electron spin resonance spectra did, however, conclusively show that the pigment in dry-cured Parma ham at no stage is a nitrosyl complex of ferrous myoglobin as found in brine-cured ham and Spanish Serrano hams. Both near-infra red reflectance spectra of sliced ham and UV/visible absorption spectra of extract of hams, obtained with aqueous buffer or acetone, showed the presence of different red pigments at varying processing stages for both solvents. Especially, the pigment extracted with aqueous buffer exhibited unique spectral features different from those of well-known myoglobin derivatives. At the end of processing, the pigment(s) becomes less water extractable, while the fraction of red pigment(s) extractable with acetone/water (75%/25%) increases throughout the processing time up to full maturation at 18 months. The chemical identity of the 6th ligand of myoglobin could not be conclusively established, but possible candidates are discussed. The partition of the pigment(s) between pentane and acetone/water showed a strong preference for pentane, suggesting that only the heme moiety is present in the acetone/water extract, and that Parma ham pigment is gradually transformed from a myoglobin derivative into a non-protein heme complex, which was found to be thermally stable in acetone/water solution     

巴马火腿红色色素的研究进展

综述了巴马火腿红色色素研究的最新进展。巴马火腿在腌制时不添加硝酸盐或亚硝酸盐 ,经过长期的加工过程 ,巴马火腿肉具有稳定的红色。巴马火腿的红色色素与添加硝酸盐或亚硝酸盐腌制的普通火腿的红色色素具有不同的吸收光谱、颜色稳定性和抗氧化性。根据巴马火腿红色色素的吸收光谱、荧光光谱、高压液相色谱和高分辨率电喷雾电离质谱 ,证实了巴马火腿的红色色素是锌 原卟啉IX。     

Changes in Zn-porphyrin and proteinous pigments in italian dry-cured ham during processing and maturation

Substitution of iron with zinc in myoglobin during maturation of Parma ham to yield zinc porphyrin extractable by 75% vol/vol acetone/water solution and detectable by fluorescence spectroscopy, was found to occur concomitant with protein modification in myoglobin. The content of zinc porphyrin increases throughout the whole processing and maturation of Parma ham, from I(fl) 0.1±0.06 for green ham to I(fl) 84.4±48.8 for fully matured Parma ham. In an aqueous extract of Parma ham with pH 6.0 protein alteration in myoglobin, as detected by size-exclusion chromatography, is initiated during the resting period following salting and seems to precede formation of zinc porphyrin. During maturation the results indicate that the modified myoglobin could undergo polymerization, and it is suggested that initial protein denaturation or degradation facilitates substitution of iron with zinc. The pigment polymerization may be a result of non-covalent protein association to zinc porphyrin in denatured or partly degraded myoglobin.     

A Zn-porphyrin complex contributes to bright red color in Parma ham

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.     

Proteolysis involvement in zinc–protoporphyrin IX formation during Parma ham maturation

During maturation of Parma ham a continuing increase in concentration of glycerol, choline, and free amino acids, as followed for 15 months by high resolution ¹H NMR of ham extracts, indicates activity of lipolytic and proteolytic enzymes. Proteolysis was shown to be mediated by endogenous cathepsins with detectable activity even at the latest stage of maturation. The concentration of red proteinous pigments containing zinc–protoporphyrin-IX (Zn–PP), as measured by fluorescence spectroscopy, increased throughout the whole maturation period concomitant with formation of low molecular weight peptides. EPR spectroscopy showed that the concentration of the high-spin iron(III) species metmyoglobin present at early stages of production, decreased during maturation to form a EPR-silent pigment suggested to be labile, partly proteolyzed iron(II) myoglobin gradually loosing iron forming non-heme colloidal ferric hydroxide. Proteolytic degradation of myoglobin and precipitation of inorganic iron(III) in the meat matrix are essential for the metal exchange to form Zn–PP and non-heme iron(III) species.     

Observation of the distribution of Zn protoporphyrin IX (ZPP) in Parma ham by using purple LED and image analysis

We investigated the distribution of Zn protoporphyrin IX (ZPP) in Parma ham by using purple LED light and image analysis in order to elucidate the mechanism of ZPP formation. Autofluorescence spectra of Parma ham revealed that ZPP was present in both lean meat and fat, while red emission other than that of ZPP was hardly detected. Although ZPP was found to be distributed widely in Parma ham, it was more abundant in intermuscular fat and subcutaneous fat than in lean meat. The intensity of red emission was weak in muscles that were exposed during the processing. ZPP in both lean meat and subcutaneous fat tended to be more abundant in the inner region than in the outer region. It was thought that ZPP is transferred from lean meat to fat tissue during the processing, resulting in the small amount of ZPP in the lean meat adjacent to subcutaneous fat. Our results led to a completely new hypothesis that ZPP is formed in lean meat and transferred to fat tissue.     

Thermal and photochemical degradation of myoglobin pigments in relation to colour stability of sliced dry-cured Parma ham and sliced dry-cured ham produced with nitrite salt

Lipophilic and hydrophilic extracts of the red pigments from Parma ham and nitrosylated pigment of dry-cured ham produced with nitrite salt were prepared with acetone/water (75/25 v/v %) solution and aqueous phosphate buffer, respectively. The spectral characteristics differed for both the lipophilic and the hydrophilic Parma ham pigment compared with the dry-cured ham produced with nitrite salt. The red lipophilic pigment(s) extractable from Parma ham was(were) found to be very stable towards thermal degradation in acetone/water (75/25 v/v %) solution for temperatures up to 70 °C in contrast to the lipophilic pigment(s) extractable from dry-cured ham produced with nitrite salt, which was(were) found to have an energy of activation of 99 kJ/mol for thermal degradation. In contrast, quantum yields for photodegradation of the lipophilic ham pigments exposed to 366 nm (420 nm) monochromatic light were larger for Parma ham than for nitrite-cured ham [1.6×10^–5 (6.9×10^–6) versus 1.6×10^–6 (2×10^–6) mol einstein^–1] as determined for acetone/water (75/25 v/v %) solution. In agreement with these findings for the extracted lipophilic pigments, sliced Parma ham showed better colour stability than sliced dry-cured ham produced with nitrite salt, when stored in the dark at low oxygen concentration, in contrast to a faster initial discolouration for Parma ham when exposed to light, as shown for chilled storage for 35 days under retail conditions for the two products each packed at two oxygen levels (0.4 and 21%).     

Direct demonstration of the presence of zinc in the acetone-extractable red pigment from Parma ham

We studied the presence of zinc in the red pigment extracted from Parma ham by scanning electron microscopy/energy dispersive X-ray microanalysis. The large peak of about 8.6keV and the small peaks at about 1.1keV and 9.5keV were derived from K(α), L(α1) and K(β) X-ray of zinc, respectively. Thus, the results suggested that zinc existed in the red pigment extracted from Parma ham. In contrast, the K(α) X-ray peak of iron (6.4keV) was not detected and the K(α) X-ray peak of magnesium (1.3keV) was hardly detected. These results revealed that the red pigment contained not iron but zinc. In addition to the results of mass analysis in previous studies, the red pigment was demonstrated to be zinc protoporphyrin IX.     

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

Zn-porphyrin (Zn-pp) was quantified by fluorescence spectroscopy in the cured and dry cured meat products: Parma ham, Iberian ham, dry-cured ham with added nitrite, cooked ham with added nitrite, raw ham meat, raw bacon and Karree-Speck. The highest amount 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 completely the formation of Zn-pp in meat products. A positive correlation between both Zn content and Fe content and the logarithmic transformed Zn-pp content (measured as fluorescence intensity I_fl) was found for the different cured and dry cured meat products, with correlation coefficients of 0.79 (p < 0.001) and 0.71 (p < 0.01), respectively. Log I_fl correlates best with the Zn content, indicating that the formation of Zn-pp is proportional to the Zn content. A model system with vacuum packed pork in brine with different added levels of sodium 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%. Addition of nitrite or Zn(II) in the curing brine was found to inhibit formation of Zn-pp confirming the observations from the various cured meat products. It is suggested that a chloride anion assisted dissociation of iron from myoglobin could be rate-determining for Zn-pp formation in meat products.     

Quantification of zinc-porphyrin in dry-cured ham products by spectroscopic methods Comparison of absorption, fluorescence and X-ray fluorescence spectroscopy

Zinc–protoporphyrin (Zn–pp), which has been identified as the major pigment in certain dry-cured meat products, was extracted with acetone/water (75%) and isolated from the following meat products: Parma ham, Iberian ham and dry-cured ham with added nitrite. The quantification of Zn–pp by electron absorption, fluorescence and X-ray fluorescence (XRF) spectroscopy was compared (concentration range used [Zn–pp] = 0.8–9.7 μM). All three hams were found to contain Zn–pp, and the results show no significant difference among the content of Zn–pp quantified by fluorescence, absorbance and X-ray fluorescence spectroscopy for Parma ham and Iberian ham. All three methods can be used for quantification of Zn–pp in acetone/water extracts of different ham types if the content is higher than 1.0 ppm. For dry-cured ham with added nitrite, XRF was not applicable due to the low content of Zn–pp (<0.1 ppm). In addition, XRF spectroscopy provides further information regarding other trace elements and can therefore be advantageous in this aspect. This study also focused on XRF determination of Fe in the extracts and as no detectable Fe was found in the three types of ham extracts investigated (limit of detection; Fe ? 1.8 ppm), it allows the conclusion that iron containing pigments, e.g., heme, do not contribute to the noticeable red colour observed in some of the extracts.     

Colour stability and vitamin E content of fresh and processed pork

The effect of increasing muscular content of vitamin E on colour intensity and stability of pork (both fresh and processed) was studied by feeding manipulation. Fresh pork was represented by loin chops and processed pork was represented by a dry cured fermented sausage (salame Milano) and raw cured ham (Parma ham). Dietary vitamin E was increased by feeding the animals with sunflower oil and α-tocopheryl acetate at 100 and 200 ppm. Chops were packed in oxygen-permeable film and in a protective atmosphere (80% O₂ and 20% CO₂) whereas salame Milano and Parma ham were packed under vacuum and in a protective atmosphere (85% N₂ and 15% CO₂). No differences in colour intensity or type were observed in relation to vitamin E content. Colour stability tests suggested, on the other hand, that higher vitamin E contents could be associated with slower myoglobin oxidation rates in fresh chops packed in a protective atmosphere. No effect of vitamin E on colour stability was observed in salame, probably because of the soft fat induced by sunflower oil. Significant differences among dietary groups were also not observed in Parma ham.     

Mass spectrometric evidence for a zinc-porphyrin complex as the red pigment in dry-cured Iberian and Parma ham

Extracts containing red pigment complexes from the two types of dry-cured hams, Italian Parma and Spanish Iberian ham, were obtained using water and acetone as extraction solvents followed by a crude purification with C18 column filtration. The purified extracts were then analyzed spectroscopically by recording absorption and fluorescence spectra (λ(ex)=420nm), which both indicate the presence of chemically identical red chromophores with properties similar to a complex of transition metals and protoporphyrin IX. Electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode confirms the presence of identical chemical compounds. ESI-MS in the negative ion mode detects a cluster of seven isotopologue ions (that of m/z 623.2 as the most intense) with a pattern matching that of a Zn protoporphyrin IX complex. Based on mass spectral data it is concluded that a Zn-porphyrin complex constitutes a major chromophore in dry-cured Iberian ham as well as in Parma ham.     

射频等离子体活性水处理对火腿发色的影响

探讨射频等离子体活性水（radio frequency plasma activated water,RF-PAW）处理对肌红蛋白颜色及火腿发色的影响,对比亚硝酸钠溶液与RF-PAW的发色效果以及这2 种发色剂用于火腿发色后亚硝酸盐的残留量。结果表明,等离子体处理6 min以内得到的PAW,不会对高铁肌红蛋白（metmyoglobin,metMb）的颜色产生显著影响,会使氧合肌红蛋白逐渐氧化成metMb,颜色由鲜红色变为红棕色;肉眼观察发现,采用RF-PAW、亚硝酸盐溶液（nitrite,NI）作为亚硝酸盐来源来腌制火腿,都能使新鲜猪肉发色,且RF-PAW腌制的火腿发色效果更好,RF-PAW中的活性物质并未对火腿发色造成不良影响;色差分析结果显示,PAW腌制出的火腿有更高的a*、C*值以及更低的b*值,色素含量测定显示,PAW处理的火腿具有更高的亚硝化肌红蛋白色素百分比（43.52%）,说明PAW腌制比NI腌制形成更多的亚硝化肌红蛋白色素,使火腿的红色更深;通过亚硝酸盐残留测定发现,NI腌制的火腿亚硝酸盐残留量为54.45 mg/kg,而PAW腌制的火腿亚硝酸盐残留量为52.79 mg/kg,均小于国标限量值70 mg/kg。该研究结果为肉品低温等离子体保鲜与腌制技术的工程化应用提供理论基础和科学指导。     

熟食肉鱼制品氧化安全性评价

对市售火腿肠、肉片、肉松、腊肠和鱼片5类19种熟食肉鱼制品的氧化产物丙二醛(MDA)和亚硝酸盐(NO2)这2种致癌物的含量进行了测定和分析。结果表明,只有1种鸡肉卷火腿肠的NO2残留量(36mg/kg)超标,其余熟食制品的NO2含量均在允许安全范围。猪肉和牛肉火腿肠的MDA值均低于05mg/kg,未出现任何异味,表现出很高的安全性,但是脂肪和蛋白质含量普遍偏低,影响了口感和应有的品质。鸡肉火腿肠的MDA值在05～10mg/kg范围内,出现氧化异味。肉片、鱼片、肉松及腊肠制品的MDA值在抽样时多数都超过10mg/kg,氧化安全期与标签保质期相差42～177d,对这类熟食肉鱼制品在标签保质期内所存在的氧化安全隐患,应引起足够的重视。     

Studies on the antioxidative activity of red pigments in Italian-type dry-cured ham

Aqueous phosphate buffer extracts and acetone/water extracts of pigments from Parma ham were assessed as antioxidants by (1) electron spin resonance spectroscopy using a spin probing technique to evaluate their efficiencies as scavengers of free radicals, and (2) by electrochemical measurement of oxygen depletion rate in an aqueous methyl linoleate emulsion to evaluate their efficiencies as chain-breaking antioxidant, and using both methods, compared with the effect of apomyoglobin and nitrosylmyoglobin. Aqueous phosphate extracts and acetone/water extracts of Parma ham pigment both scavenged a semi-stable nitroxide radical (Fremy's salt), and both extracts reduced the rate of oxygen consumption for lipid peroxidation (initiated by metmyoglobin) very efficiently. For apomyoglobin no antioxidative capacity was observed, and the heme moiety of the pigment(s) of Parma ham were concluded to have antioxidative properties. The more lipophilic pigment, as extracted by acetone/water, had the most significant effect, and its ability to inhibit lipid oxidation was further tested in a model food system based on cooked pork. The lipid oxidation was increasingly inhibited by increasing additions from 0.12 ppm to 0.24 ppm Parma ham pigment, and the pigment protected -tocopherol against degradation in a concentration dependent manner.     

Contribution of pigment content, myoglobin forms and internal reflectance to the colour of pork loin and ham from pure breed pigs

The colour of loin, M. longissimus dorsi (LD), and ham, M. biceps femoris (BF), from pure breed Hampshire, Swedish Landrace and Swedish Yorkshire pigs was studied. The contribution of the pigment content, the myoglobin forms deoxymyoglobin (Mb), oxymyoglobin (MbO) and metmyoglobin (MetMb) and the internal reflectance to the colour of pork of normal meat quality was evaluated using partial least squares regression (PLS). The colour of LD and BF from the Hampshire breed was more red and yellow and more saturated than the colour of the same muscles from the Swedish Landrace and the Swedish Yorkshire breeds. Furthermore, BF from Hampshire was darker than BF from the other two breeds. These differences in colour were related to the lower pH in Hampshire, resulting in more blooming and in higher internal reflectance, and to the higher pigment content. The colour of BF was darker and more red than the colour of LD within each breed. No colour difference was found between gilts and castrates within each breed. Most of the variation (86-90%) in lightness (L* value), redness (a* value) and yellowness (b* value), chroma (saturation) and hue angle of pork of normal meat quality was explained by the pigment content, myoglobin forms and internal reflectance. The L* value, a* value, chroma and hue angle were influenced by both the pigment content and by the myoglobin forms to almost the same extent, while the internal reflectance was of no significance to these colour parameters. The b* value was influenced most by the myoglobin forms, less by the internal reflectance and almost not at all by the pigment content.     

Establishment of a model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX is formed in nitrite-free dry-cured ham

The aim of this study was to establish a model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX (ZPP) is formed in Parma ham. The established model consisted of myoglobin, meat and antibiotics, and incubation under anaerobic conditions resulted in a greater yield of ZPP. Formation of ZPP was observed even in the presence of various antiseptics. The amount of ZPP formed increased as the period of incubation increased. ZPP formation was inhibited by heating meat homogenate depending on the heating temperature. Our results show that anaerobic conditions are suitable for the formation of ZPP in meat products without nitrate or nitrite and that endogenous enzymes as well as microorganisms may be involved in ZPP formation.     

无硝干腌火腿中红色素形成机制研究进展

干腌火腿经过长时间腌制发酵成熟,富含生物活性物质,具有良好的感官特性。在传统干腌火腿加工过程中常添加硝盐（硝酸盐或亚硝酸盐）以获得稳定的色泽,然而,由亚硝酸盐与仲胺反应生成的亚硝胺具有致癌风险,已引起人们的广泛关注。研究发现,国外的著名干腌火腿如帕尔玛火腿,在腌制过程中不添加硝盐,其红色的主要贡献者被证实为Zn-原卟啉IX。本文综述了添加硝盐与未添加硝盐干腌火腿中红色素的研究进展,重点探讨Zn-原卟啉IX在无硝干腌火腿加工过程中的形成机制,以期为干腌火腿加工过程的色泽调控提供科学理论依据。     

Novel Synthesis of Cooked Cured-Meat Pigment

Cooked cured-meat pigment was synthesized from bovine blood and a nitrosating agent in aqueous solutions at elevated temperatures. Effects of reductant(s) and base in the reaction mixture were investigated on the yield and purity of the pigment. The best yield of pigment was 95% with purity >98% in most cases. Absorption characteristics of the pigment were identical to those of pigments extracted from a nitrite-cured ham or from hemin-nitric oxide synthesis. Color imparted to comminuted pork by the pre-formed pigment, upon cooking, and its color stability thereafter, were indistinguishable from that of nitrite-cured meat, yet depended on the myoglobin content.     

Extraction properties and absorption spectra of dry cured hams made with and without nitrate

The spectral and extraction properties of nitrate-free dried hams (Parma hams) were studied as a function of maturing time and muscle type (semimembranosus and biceps femoris muscles). To understand the red colour of the processed meat, a three-step extraction using water, acetone and tetrahydrofuran (THF) as solvents was employed and the extracts from each step were submitted to spectrophotometric (visible) analysis while the corresponding residues underwent instrumental (CIE L* a* b*) colour evaluation. Results showed that the drier the meat the more the pigment is soluble in organic solvents, THF proving a major extractant in matured hams, regardless of muscle type. When Parma hams were compared with hams made using nitrate, the former displayed more intense (P<0.05) absorption in the red region in the aqueous solution, lower intensities in acetone and higher values in THF solution. Major differences in spectral characteristics were found between water and organic extracts of Parma hams, with both acetone and THF solutions exhibiting a doublet at 544–584 nm, and water extracts displaying two distorted peaks at 551 and 595 nm. It is concluded that dry curing of meat without nitrate yields a lipophylic stable red pigment whose hydrophobicity increases with ageing.