Combined Application of High-Throughput Sequencing and Metabolomics Reveals Metabolically Active Microorganisms During Panxian Ham Processing

Abstract

Panxian ham, a traditional Chinese dry-cured ham, is protected by national geographical indication. Similar to other fermented foods, the microbial population of dry-cured ham is pivotal to taste and flavor formation. This study aimed to establish the relationship between microorganisms and metabolites during the spontaneous fermentation of Panxian ham. Multivariate analysis based on metabolomics data revealed that continuous metabolic changes occurred during the entire fermentation process, with the most significant changes occurring in the initial stage of ripening. Thirty-one significantly different metabolites (SDMs) were identified as discriminant factor, and pathway analysis suggested that these metabolites were involved in 30 pathways, including alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; and arginine and proline metabolism. Microbial community analysis using the Illumina MiSeq platform indicated that the bacterial community was more complex than the fungal community, and their succession regulation differed during processing. At the genus level, 11 bacteria and five fungi were identified as core microbes, of which Staphylococcus was the dominant bacteria and Debaryomyces and Aspergillus were the dominant fungi. Further, statistical redundancy analysis (RDA) indicated that Staphylococcus, Debaryomyces, and Chromohalobacter promoted the production of amino and fatty acids; Cobetia and Aspergillus were associated with sugar metabolism, and Kushneria, Penicillium, and Yamadazyma were closely related with organic acids. These findings provide fundamental knowledge regarding the metabolically active microorganisms in Panxian ham, helping industrial processors to develop effective strategies for standardizing quality parameters.

Keywords: Panxian ham; high-throughput sequencing; metabolically active microorganism; metabolomics; non-volatile metabolites.

FIGURE 1

Principal component analysis (PCA) (A) and orthogonal partial least squares discriminant analysis (OPLS-DA) (B) score plots derived from the GC-TOF-MS data from Panxian ham and Quality Control (QC) samples. A–F indicate the traditional spontaneous fermentation process stage of Panxian ham: (A) raw ham; (B) post-salting; (C) post-resting; (D) initial stage of ripening; (E) middle stage of ripening; (F) final stage of ripening. (QC) represents the quality control sample.

FIGURE 2

The integrated metabolic pathway shows the dynamics of significantly different metabolites (SDMs) during Panxian ham processing. Beneath each metabolite, the color gradient and their values indicate the log2 (fold change) with respect to raw ham: red and blue represent up and downregulation of metabolites, respectively. Glycine, serine, and threonine metabolism, alanine, aspartate, and glutamate metabolism, and arginine and proline metabolism are shown in the blue, yellow, and green areas, respectively.

FIGURE 3

Effects of spontaneous fermentation on microbial diversity and community structure of Panxian ham. (A) OTU number of bacteria and fungi at different processing stages, values are presented as mean ± standard error (n = 5), ^a–e different letters represent significant differences (p < 0.05). (B) rarefaction curves of bacteria and fungi for each sample. (C,D) NMDS and PCoA score plots of bacteria and fungi. A–F indicate the traditional spontaneous fermentation process stage of Panxian ham: (A) raw ham; (B) post-salting; (C) post-resting; (D) initial stage of ripening; (E) middle stage of ripening; (F) final stage of ripening.

FIGURE 4

Relative abundance of bacteria (A) and fungi (B) at the phylum level and bacteria (C) and fungi (D) at the genus level in samples of Panxian ham. A–F indicate the traditional spontaneous fermentation process stage of Panxian ham: (A) raw ham; (B) post-salting; (C) post-resting; (D) initial stage of ripening; (E) middle stage of ripening; (F) final stage of ripening.

FIGURE 5

Redundancy analysis (RDA) of Panxian ham samples, core microbial genera, and significantly different metabolites (SDMs) during Panxian ham processing. (A) Correlation between samples, core bacteria genera, and SDMs. (B) Correlation between samples, core fungi genera, and SDMs. A–F indicate the traditional spontaneous fermentation process stage of Panxian ham: (A) raw ham; (B) post-salting; (C) post-resting; (D) initial stage of ripening; (E) middle stage of ripening; (F) final stage of ripening. The core bacterial and fungal genera were consistent with Figures 4C,D; while SDMs are represented by M1–M31. M1, valine; M2, Isoleucine; M3, proline; M4, glycine; M5, serine; M6, L-allothreonine; M7, aspartic acid; M8, methionine; M9, pyroglutamate; M10, glutamic acid; M11, phenylalanine; M12, ornithine; M13, lysine; M14, tyrosine; M15, alanine; M16, myristic acid; M17, palmitoleic acid; M18, heptadecanoic acid; M19, arachidonic acid; M20, arachidic acid; M21, stearic acid; M22, succinic acid; M23, malic acid; M24, creatine; M25, glycerol; M26, myo-inositol; M27, fructose; M28, glucose; M29, hypoxanthine; M30, inosine; M31, creatinine.