Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX

The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation–reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.     

Quantitative determination of Zn protoporphyrin IX,heme and protoporphyrin IX in Parma ham by HPLC

We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate–acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0–29.9, 27.7–47.0 and 0.4–1.1 μg/g, respectively, and total content of porphyrin was 43.7–76.6 μg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60–70% of all porphyrins.     

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.     

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.     

猪副产物中锌原卟啉形成差异分析

本研究的目的为探究猪副产物间锌原卟啉(Zinc-protoporphyrin,ZnPP)形成的差异性。以猪副产物为研究对象,分析猪副产物间ZnPP含量的差异并以铁螯合酶(Ferrochelatase,FECH)活性为指标探究产生差异性原因。采用紫外分析光谱、荧光光谱及化学模拟实验测定了猪副产物中ZnPP的含量、FECH的活性两个指标,验证了ZnPP与FECH之间的联系。UV-Vis检测结果表明在416 nm有1个强吸收峰,546 nm和584 nm处有两个弱对称吸收峰。以420 nm波长激发,红色素在590 nm处有一个强荧光发射峰,644 nm处有一个弱荧光发射峰,与ZnPP标准品的荧光光谱对比后高度重合,证明从副产物中所提取的红色素为ZnPP。猪副产物间ZnPP的含量存在显著性差异,其中猪肝中ZnPP含量在孵育72 h的条件下达到最大值,此时测得的荧光强度高达25655.44 A.U。而FECH活性测定研究结果表明猪肝中FECH的活性远优于其他副产物。利用皮尔逊(Pearson)相关性分析结果表明ZnPP与FECH之间存在极显著关系。本研究结果进一步确定了肝脏具有良好的ZnPP形成能力,从肝脏中提取的ZnPP可用于改善肉制品的颜色。本研究不仅为副产物附加值利用提供了方向,同时为ZnPP的研究提供新的思路。     

Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX

We examined the effects of exogenous myoglobin, a bivalent chelator, and nitrite on Zn protoporphyrin IX (ZPP) formation by using model systems. ZPP was formed in a model solution without addition of exogenous myoglobin. After incubation, the amount of ZPP in a model solution was increased but that of heme was not decreased compared with the amounts before incubation. Protoporphyrin IX (PPIX) instead of ZPP also accumulated in a model solution with addition of EDTA, but the amount of heme was not reduced. These results suggested that ZPP was not formed by the Fe–Zn substitution in heme but was formed by the insertion of Zn into PPIX, which was formed independently. The fact that the effects of various factors in model systems with/without addition of a bivalent chelator were similar suggested that ZPP formation was strongly affected by PPIX formation. Inhibition of PPIX formation by nitrite might be the reason for the low levels of ZPP in cured meats.     

血红素和原卟啉光敏作用的研究

研究了肌红蛋白的组成成分血红素和原卟啉的光敏能力,还分析了在不同光照强度下贮存期间血红素和原卟啉对猪肉脂肪氧化的影响情况。结果表明:原卟啉产生单线态氧的能力强于血红素,它的光敏能力强于血红素。加入原卟啉样品的脂肪氧化指标在430lux以上就与其他组出现了显著性差异(P<0.05),而加入血红素的样品组在630lux以上时才出现了显著性差异(P<0.05),且随着光强度的增加,这种差异越显著。     

Red Pigment of Parma Ham and Bacterial Influence on its Formation

The red pigment of Parma ham was compared with the myoglobin derivatives present in meat and meat products. Spectral patterns of 75% acetone extracts and electron spin resonance spectra from Parma ham differed from those of the myoglobin derivatives. Staphylococci isolated from Parma ham generated red myoglobin derivative from metmyoglobin. Model fermented sausage prepared by inoculation with the isolates developed a more desirable red color without nitrite or nitrate treatment. The red pigment of Parma ham and the model sausage appeared to be the same myoglobin derivative. The reddening of Parma ham is probably caused by the action of bacteria.     

Effects of different storage conditions on quality related porphyrin fluorescence signatures of pork slices

This study evaluated the potential of fluorescence as an indicator of pork quality by determining the effects of various conditions on fluorescence signatures (excitation at 420 nm, emission at 550-750 nm).Storage of porcine musculus longissimus dorsi in PE bags led to a clear increase in porphyrin fluorescence intensity after approx. 10 d post mortem. Modified gas atmosphere (70% O₂, 30% CO₂) inhibited the fluorescence emission of zinc protoporphyrin and protoporphyrin IX due to quenching by oxygen. Bleaching processes caused similar effects by halogen light exposure during meat storage. However, already formed signals could not be manipulated by oxygen or halogen light. Storage under vacuum reduced the quenching effects and resulted in increased fluorescence intensities. Freezing and thawing of meat samples delayed and reduced the increase in fluorescence intensity. Only minor effects could be detected at long-term frozen storage for two months.Consequently porphyrin fluorescence analysis is a potential means to indicate changes of pork quality and remaining shelf life.     

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.     

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%).     

Fluorimetric detection of protoporphyrins as an indicator for quality monitoring of fresh intact pork meat

In fresh meat production fast and non-destructive quality monitoring along the distribution chain is a key aspect to guaranteeing high quality and safe products for consumption. The applicability of fluorescence spectroscopy using protoporphyrins as indicators for meat ageing was investigated. Porcine musculus longissimus dorsi (MLD) was stored in slices over 20 days at 5 and 12 °C and measured every day with an excitation of 420 nm and an emission range of 550–750 nm. Additionally, pH, drip loss and colour were examined to assess possible correlations.The obtained spectra of the MLD showed an increase in three peaks at 592, 638 and 705 nm which could be reconstructed using the spectra of standard solutions of protoporphyrin IX (PP) and zinc protoporphyrin IX (ZnPP) or magnesium protoporphyrin (MgPP), respectively.Using principal component analysis (PCA) on the fluorescence spectral data, the meat slices stored at 5 °C showed differences in the fluorescence signal after the 10th day and 5th day when stored at 12 °C. An interrelationship between the additional analyses and the fluorescence intensities on these relevant days could not be established.In conclusion, the increase of ZnPP fluorescence due to temperature related changes of physiological meat properties is capable of serving as a quality indicator with regards to inadequate conditioning (e.g. during transportation and/or storage) of pork meat.     

巴马火腿酶解物中呈味肽的分离纯化及其结构研究

采用Sephadex G-15凝胶色谱柱分离得到巴马火腿酶解物9个组分,结合感官分析和电子舌测定得出呈味组分为G-15-P2-E2。用半制备RP-HPLC制备此组分中G-15-P2-E2-r1、G-15-P2-E2-r2,并结合串联质谱分析,得出G-15-P2-E2-r1组分可能的氨基酸序列为Leu-Ser-Glu-Arg-Tyr-Pro(LSERYP,LP6)或Asn-Gly-Lys-Glu-Thr (NGKET,NT5),G-15-P2-E2-r2组分可能的氨基酸序列为Pro-Asp-Leu-Pro-Asn-Thr(PDLPNT,PT6)经合成后电子舌测定发现Leu-Ser-Glu-Arg-Tyr-Pro的呈味特性与火腿酶解物呈味特性相近。     

Quenching of excited states of red-pigment zinc protoporphyrin IX by hemin and natural reductors in dry-cured hams

Zinc protoporphyrin IX (ZnPP), the major red pigment in hams dry-cured without nitrates/nitrites, is an efficient photosensitizer, which upon absorption of visible light forms short-lived excited singlet state (¹ZnPP*) and by intersystem crossing yields the very reactive triplet-excited state (³ZnPP*). Using nano-second laser flash photolysis and transient absorption spectroscopy NADH, ascorbic acid, hemin and dehydroascorbic acid were each found to be efficient quenchers of ³ZnPP*. The deactivation followed, in homogeneous dimethyl sulfoxide (DMSO) or DMSO:water (1:1) solutions, second-order kinetics. The rate constant for ascorbic acid and NADH for reductive quenching of ³ZnPP* was at 25 °C found to be 7.5 ± 0.1 × 10⁴ L mol⁻¹ s⁻¹ and 6.3 ± 0.1 × 10⁵ L mol⁻¹ s⁻¹, respectively. The polyphenols catechin and quercetin had no effect on ³ZnPP*. The quenching rate constant for oxidative deactivation of ³ZnPP* by dehydroascorbic acid and hemin was at 25 °C: 1.6 ± 0.1 × 10⁵ L mol⁻¹ s⁻¹ and 1.47 ± 0.1 × 10⁹ L mol⁻¹ s⁻¹, respectively. Oxidized glutathione did not act as an oxidative quencher for ³ZnPP*. After photoexcitation of ZnPP to ¹ZnPP*, fluorescence was only found to be quenched by the presence of hemin in a diffusion-controlled reaction. The efficient deactivation of ³ZnPP* and ¹ZnPP* by the metalloporphyrin (hemin) naturally present in meat may accordingly inherently protect meat proteins and lipids against ZnPP photosensitized oxidation.     

Sensory and texture properties of Italian typical dry-cured hams as related to maturation time and salt content

Maturing time and salt were fixed factors and fat was a covariate in a full factorial design study of sensory and texture properties of 36 dry cured hams. Samples were chosen to fit three ageing and salt classes. Differences (P < 0.05) in sensory scores were found between whole slices (with cover fat) and their biceps femoris (BF) counterparts, with sweet taste enhanced in whole samples and conversely unpleasant bitter taste more intense in BF portions. Increased ageing time resulted in better sensory profiles, as documented by greater scores (P < 0.05) for matured odour, matured taste and sweet taste, whereas salty taste was reduced. Decreased salt was associated with greater intensities (P < 0.05) of undesired green odour and taste. Based on chemical composition, the ham consistence appears improved by long-term dehydration, provided that protein breakdown is under control. It is concluded that for salt reduction of up to 25% to be achieved without negative side effects, extended ageing and restrained proteolysis are key factors.     

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。     

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.     

Chemical, instrumental and sensory characteristics of cooked pork ham

Instrumental, chemical and sensory parameters of cooked pork ham were evaluated. Principal component analysis was carried out on the basis of the instrumental variables related to colour and texture. The four PCs account for almost 94% of the total variance in the data set. The PCA only separated 3 hams with a^* > 10. Hardness was correlated with non-collagen muscle protein (P  0.01), gumminess (P  0.01) and ash (P  0.05). Sensory evaluated tenderness showed positive significant correlation with L^* (P  0.01). The most important colour parameter seems to be a^*, which was negatively correlated with sensory evaluated parameter colour (P  0.01). The PCA performed on all parameters (sensory, chemical and textural) discriminated two groups of hams differing in non-collagen muscle protein content and hardness.     

Evaluation of enzymatic and non-enzymatic softening in low salt cucumber fermentations

Retention of a firm, crisp fruit texture is a major consideration for pickled vegetables including pickles made from fermented cucumbers. It is known that cucumbers soften rapidly when fermented at low salt concentrations (<0.5 M) without added calcium. This study has shown that there is non-enzymatic softening in low salt fermentations because cucumbers soften even when heated sufficiently to inactivate pectinesterase and several glycosidases that can hydrolyse glycosidic linkages that are present in cell wall polysaccharides. Though pectinesterase activity declines and these glycosidases lose activity within the first week of fermentation there is generally greater loss of cucumber tissue firmness when enzymes are not inactivated by heat. While heating cucumbers prior to fermentation reduces softening during subsequent storage, a heat treatment after 2 weeks of fermentation does not reduce softening. This result suggested that the enzymatic reactions responsible for softening occur early in the fermentation process even though the softening does not become evident until later in the storage period. Despite the evidence of an enzymatic component of tissue softening in low salt cucumbers, softening could not be associated with specific enzymes.     

Cooked ham classification on the basis of brine injection level and pork breeding country

The aim of this study was to classify whole-leg cooked hams, made without polyphosphates, by linear discriminant analysis. Principal component analysis (PCA) was used for the selection of significant variables. Thirty-two variables were evaluated on 26 cooked hams prepared using different levels of brine injection and legs from pork bred in different countries (France or Denmark). Previously published data related to 20 hams were also used for classification. A chemometric model, based on ten variables, was obtained by using PCA. The variables were pH, moisture, protein, fat, NaCl, superficial wateriness, L* and a*/b* of biceps femoris muscle, modulus and elasticity index of semitendinosus muscle. Discriminant functions calculated using PCA-selected variables enable correct classification of the cooked hams according to the origin of the meat used and, when this is the same, according to the percentage of brine injected.