NMR-Based Metabolomic Study on Phaseolus vulgaris Flour Fermented by Lactic Acid Bacteria and Yeasts

Abstract

In recent years, fermented foods have attracted increasing attention due to their important role in the human diet, since they supply beneficial health effects, providing important sources of nutrients. In this respect, a comprehensive characterization of the metabolite content in fermented foods is required to achieve a complete vision of physiological, microbiological, and functional traits. In the present preliminary study, the NMR-based metabolomic approach combined with chemometrics has been applied, for the first time, to investigate the metabolite content of Phaseolus vulgaris flour fermented by different lactic acid bacteria (LAB) and yeasts. A differentiation of microorganisms (LAB and yeasts), LAB metabolism (homo- and heterofermentative hexose fermentation), LAB genus (Lactobacillus, Leuconostoc, and Pediococcus), and novel genera (Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus) was achieved. Moreover, our findings showed an increase of free amino acids and bioactive molecules, such as GABA, and a degradation of anti-nutritional compounds, such as raffinose and stachyose, confirming the beneficial effects of fermentation processes and the potential use of fermented flours in the production of healthy baking foods. Finally, among all microorganisms considered, the Lactiplantibacillus plantarum species was found to be the most effective in fermenting bean flour, as a larger amount of free amino acids were assessed in their analysis, denoting more intensive proteolytic activity.

Keywords: NMR; Phaseolus vulgaris; chemometrics; common bean; fermentation; lactic acid bacteria; legumes; metabolomics.

Figure 1

Score (a) and loading (b) plots of PCA performed considering all samples and the complete ¹H NMR spectra. In (a), orange pentagons, black circles, and light blue diamonds represent CTRL, LAB-fermented, and yeast-fermented samples, respectively; in (b) black triangles represent the loadings of the variables of X matrix (the numbers refer to the initial ppm values of the buckets). 3PCs, R²X = 95.6%, Q²cum = 61.1%.

Figure 2

Score (a) and loading (b) plots of PCA performed, excluding CTRL, LBC01_ss, and WS01_ss samples and considering the complete ¹H NMR spectra. In (a), black circles and light blue diamonds represent LAB-fermented and yeast-fermented samples, respectively; in (b) black triangles represent the loadings of the variables of X matrix (the numbers refer to the initial ppm values of the buckets). 2PCs, R²X = 95.1%, Q²cum = 70.7%.

Figure 3

Score plot (a) and S-plot (b) of OPLS-DA performed according to LAB metabolism, considering the complete ¹H NMR spectra. In (a), burgundy diamonds and green circles represent hetero- and homofermentative LAB-fermented samples, respectively; in (b) black triangles represent the weight of the variables of X matrix (the numbers refer to the initial ppm values of the buckets). One predictive and one orthogonal component, R²X = 79.7%, R²Y = 94.8%, Q²cum = 82.3%. Permutation tests are reported in Figure S2.

Figure 4

Score (a) and weight (b) plots of PLS-DA performed according to LAB genera in the early taxonomy, considering the complete ¹H NMR spectra. In (a), green circles and blue diamonds represent the Lactobacillus genus and the Leuconostoc and Pediococcus genera, respectively; in (b) black triangles represent the weight of the variables of X matrix (the numbers refer to the initial ppm values of the buckets) while red circles represent the weight of the variables of Y matrix. 2LCs, R²X = 71%, R²Y = 93.5%, Q²cum = 63.3%. Permutation tests are reported in Figure S3.

Figure 5

Score (a) and loading (b) plots of PCA performed on LAB-fermented flours considering only genera in the latest taxonomy and the complete ¹H NMR spectra. In (a), orange circles, blue diamonds, and green pentagons represent Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus samples, respectively; in (b) black triangles represent the loadings of the variables of X matrix (the numbers refer to the initial ppm values of the buckets). 3PCs, R²X = 97.6%, Q²cum = 74.7%.

Figure 6

Quantification of the relative content (integral values normalized to the total spectral area) of metabolites of interest for nutritional purposes in small- (ss) and large-scale (ls) fermented bean flours (with the exclusion of LBC01_ss and WS01_ss samples) compared to control (CTRL). (a) GABA, (b) RFO, (c) Valine, (d) Leucine, (e) Methionine, and (f) Phenylalanine trends. The dotted line indicates the integral value, for each metabolite, in the CTRL sample.