The Chemical Essence And Formation Mechanism Of Red Pigment In Nuodeng Ham

Nuodeng ham is a representative traditional meat product from Yunnan Province,favoured by consumers for its bright color and unique flavor.Color is one of the most important quality evaluation indices of dry-cured ham,and the discoloration of Nuodeng ham sections is the main factor hindering the development of the ham industry.It is of practical significance to explore the chemical essence of the red pigment in Nuodeng ham and clarify its formation mechanism for solving the problem of the discoloration of Nuodeng ham.Based on this,in this study,Nuodeng hams processed for two years were selected for the first time to characterize the chemical structure of the red pigment by ultraviolet spectroscopy（UV-Vis）,fluorescence spectrum,ultra-high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）,fourier transform infrared（FTIR）and nuclear magnetic resonance（NMR）techniques.The 36 hind legs of Nuodeng black pigs were further selected and randomly divided into two groups of 18 in each group and processed with 100%Na Cl（control group）and a compound curing agent（Na Cl+KCl+Na NO₂,treatment group）,respectively.Six hams were selected as test samples from the control and treatment groups during the processing of Nuodeng ham（3,6and 9 months）.The differences of bacterial community diversity,small molecule metabolites and lipid compound were analyzed using high-throughput sequencing and ultra-high performance liquid chromatography-high-resolution mass spectrometry（UHPLC-QE-MS）techniques to explore their intrinsic links with the red pigment of Nuodeng ham during processing.The aim is to clarify the formation mechanism of red pigment in Nuodeng ham and to provide a scientific theoretical basis for solving the problem of discoloration in Nuodeng ham sections.The main results were as follows:1.The red pigment in Nuodeng ham was extracted by acetone aqueous solution（75%,V/V）and purified by a C₁₈ solid-phase extraction column.The chemical structure of red pigment from Nuodeng ham was characterized by UV-Vis,fluorescence spectroscopy,UPLC-MS/MS,FTIR and NMR.The results showed that there was a strong absorption peak at 416 nm and two weakly symmetrical absorption peaks at 546 nm and 584 nm in UV-Vis,which was consistent with the characteristics of metalloporphyrins.The red pigment in Nuodeng ham was excited at 420 nm,with a strong fluorescence emission peak at 590 nm and a weak fluorescence emission peak at 644 nm,which highly coincided with the Zn-protoporphyrin（Zn PP）standard,indicating that the metal ions in the porphyrin ring were Zn²⁺.The Zn PP was detected in both positive ion（m/z 625.1779[M+H]⁺）and negative ion（m/z 623.1614[M-H]^-）modes of UPLC-MS/MS,and the stretching vibrations of characteristic functional groups on porphyrin rings such as-CH₃,-CH2-,C=O,-C-OH in carboxyl group,C-H on olefin and porphyrin ring C=N were found in FTIR.In addition,the ¹H-NMR results of red pigment in Nuodeng ham were consistent with the structure of the target compound Zn PP.Therefore,the chemical essence of red pigment in Nuodeng ham was identified as Zn PP.2.The color change of Nuodeng ham during processing was analyzed by a colorimeter combined with sensory evaluation.The results of instrumental analysis showed that with the processing time,the brightness（L^*）decreased,the yellowness（b^*）increased,the redness（a^*）decreased first and then increased.The treatment group showed higher a^*,b^*and color saturation（C）,and the C value was significantly different from the control group at the end of the 9th month of processing（P<0.05）.The results of sensory evaluation showed that the red intensity increased with the processing time,while the purple and brown intensities decreased.There was a significant difference between the treatment group and the control group at the end of the 9th month of processing（P<0.05）,and the treatment group showed higher red intensity and lower purple intensity.3.During the processing of Nuodeng ham,the p H gradually increased while the Aw and moisture content gradually decreased.The chloride content tended to increase and was significantly lower in the treatment group than that in the control group at the end of the6th and 9th month of processing（P<0.05）.The nitrite content in the treatment group was significantly higher than that in the control group（P<0.05）,and the nitrite content of both groups gradually decreased with the processing time.The protein content remained basically unchanged,while the proteolysis index increased significantly with the extension of processing time（P<0.05）,and the treatment group was higher than the control group.The total Fe content was higher in the treatment group,while the total Zn content was lower,and the total Zn content was about two times that of the total Fe content.With the processing time,the content of Zn PP increased significantly（P<0.05）,while the content of heme decreased significantly（P<0.05）,and the contents of Zn PP and heme in the treatment group were significantly lower than those in the control group（P<0.05）.The Pearson’s correlation analysis showed that the Zn PP content was significantly positively correlated with chloride content and proteolysis index（P<0.05）,and had an extremely significant negative correlation with nitrite content and total Fe content（P<0.01）.4.The bacterial community diversity in Nuodeng ham was analyzed by high-throughput sequencing technology.The results showed that the bacterial community of Nuodeng ham was significantly different during processing,while the control group and treatment group had similar bacterial community compositions.At the phylum level,the dominant bacteria phyla were Proteobacteria and Firmicutes,which were inhibited by Actinobacteriota during processing.At the genus level,the relative abundance of Staphylococcus gradually increased with the processing time,which was the dominant genus in the processing of Nuodeng ham,and its relative abundance was reduced by the compound curing agent.Correlation analysis showed that Delftia and Acinetobacter were significantly positively correlated with Zn PP content（P<0.05）,and Staphylococcus,Clostridium＿sensu＿stricto＿1 and unclassified＿f＿＿Enterobacteriaceae showed an extremely significant positive correlation with Zn PP content（P<0.01）.5.Using UHPLC-QE-MS technology,292 and 308 small molecule metabolites were detected from the Nuodeng ham in positive and negative ion modes,respectively.A total of 47 differential metabolites were screened by the PLS-DA model based on VIP≥1.5 and P<0.05,mainly including amino acids,peptides,and analogues,lipids and lipid-like molecules,and organic acids and derivatives.Correlation analysis showed that 15differential metabolites,such as 11,13-Dihydrotaraxinic acid glucosyl ester and sonchuionoside C had an extremely significant positive correlation with the Zn PP content（P<0.01）,and N（6）-（Octanoyl）lysine and 3alpha,7alpha-Dihydroxycopro-stanic acid were significantly positively correlated with the Zn PP content（P<0.05）.These seventeen differential metabolites were mainly amino acids,peptides,and analogues and lipids and lipid-like molecules,indicating that the degradation of some proteins and lipids during the processing of Nuodeng ham contributed to the formation of Zn PP.6.1002 and 470 lipid compounds,including glycerophospholipids（GP）,glycerolipids（GL）,sphingolipids（SP）,fatty acyls（FA）and sterolipids（ST）,were detected from the Nuodeng ham by UHPLC-QE-MS in positive and negative ion mode,respectively.Among them,a total of 50 differential lipids（VIP≥1.5,P<0.05 and the top 25）were screened from positive and negative ion modes,mainly including glycerolipids and glycerophospholipids.Correlation analysis showed that 12 differential lipids,such as Cer（d18:1/18:2+2O）and TG（19:0/12:4/18:2）showed an extremely significant positive correlation with the Zn PP content（P<0.01）,and TG（18:0/17:1/18:1）was significantly positively correlated with the Zn PP content（P<0.05）.These 13 differential lipids were mainly glycerolipids and glycerophospholipids.The results showed that the decomposition of some glycerolipids and glycerophospholipids during the processing of Nuodeng ham contributed to the formation of Zn PP.7.Pearson’s correlation analysis was performed on the top 20 bacterial genera with differential metabolites and differential lipid compounds.It was found that a strongly positive correlation between the bacteria of Staphylococcus,Delftia and Acinetobacter with the seventeen differential metabolites that had a significantly positive correlation with Zn PP formation.The bacteria of Staphylococcus and Delftia also had a strongly positive correlation with the thirteen differential lipids that had a significantly positive correlation with Zn PP formation.The results demonstrated that Staphylococcus,Delftia and Acinetobacter may play an important role in the formation of ZnPP.