Real-time PCR-based quantitative microbiome profiling elucidates the microbial dynamic succession in backslopping fermentation of Taiwanese pickled cabbage.

BACKGROUND: Microbiota succession determines the flavor and quality of fermented foods. Quantitative PCR-based quantitative microbiome profiling (QMP) has been applied broadly for microbial analysis from absolute abundance perspectives, transforming microbiota ratios into counts by normalizing 16S ribosomal RNA (16S rRNA) gene sequencing data with gene copies quantified by quantitative PCR. However, the application of QMP in fermented foods is still limited. RESULTS: QMP elucidated microbial succession of Taiwanese pickled cabbage. In the spontaneous first-round fermentation (FR), the 16S rRNA gene copies of total bacteria increased from 6.1 to 10 log copies mL-1. The dominant lactic acid bacteria genera were successively Lactococcus, Leuconostoc and Lactiplantibacillus. Despite the decrease in the proportion of Lactococcus during the succession, the absolute abundance of Lactococcus still increased. In the backslopping second-round fermentation (SR), the total bacteria 16S rRNA gene copies increased from 7.6 to 9.9 log copies mL-1. The addition of backslopping starter and vinegar rapidly led to a homogenous microbial community dominated by Lactiplantibacillus. The proportion of Lactiplantibacillus remained consistently around 90% during SR, whereas its absolute abundance exhibited a continuous increase. In SR without vinegar, Leuconostoc consistently dominated the fermentation. CONCLUSION: The present study highlights that compositional analysis would misinterpret microbial dynamics, whereas QMP reflected the real succession profiles and unveiled the essential role of vinegar in promoting Lactiplantibacillus dominance in backslopping fermentation of Taiwanese pickled cabbage. Quantitative microbiome profiling (QMP) was found to be a more promising approach for the detailed observation of microbiome succession in food fermentation compared to compositional analysis. © 2024 Society of Chemical Industry.

Determination of the soybean allergen Gly m 6 and its stability in food processing using liquid chromatography-tandem mass spectrometry coupled with stable-isotope dimethyl labelling.

A cost-effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable-isotope dimethyl labelling was used for the determination of Gly m 6. The validation results revealed that the recoveries and precisions obtained from five spiked levels were in the ranges of 88.8-113.0% and 8.3-22.0%, respectively. The content and stability of the major soybean allergen Gly m 6 in various food processing procedures were evaluated by the quantification results of its surrogate signature peptide. The Gly m 6 content in soybean decreased by 42% after natto fermentation, and by 31% and 35% in pasteurised soymilk and sterilised soymilk, respectively, relative to the raw soymilk. Only 19% of Gly m 6 in raw soymilk was retained in the soymilk film. This study extended the feasibility of dimethyl labelling to soy-based food samples and examined the proteolysis of Gly m 6 in natto fermentation and its thermal instability.

Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography⁻Tandem Mass Spectrometry-Based Determination for Food Allergens-Case of Kiwifruit.

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography- tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5-109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86-9.92%. The limit of quantification (LOQ) was set at 8 pg mL-1 for SPA1-H and at 8 ng mL-1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175-0.0515 mg g-1 of Act d 1 and 0.0252-0.0556 mg g-1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.