Metabolomics Analysis for Nitrite Degradation by the Metabolites of Limosilactobacillus fermentum RC4

Abstract

Nitrite (NIT), a commonly used food additive, especially in pickled and cured vegetables and meat products, might cause acute and chronic diseases. Fermentation with lactic acid bacteria (LAB) is an effective method for degrading NIT and improving the flavor of pickled and cured foods. In this study, Limosilactobacillus fermentum (L. fermentum) RC4 with a high NIT degradation ability was found to degrade NIT in a new manner when compared with reported enzymatic and acid degradation, namely, metabolite degradation during fermentation in MRS broth, which shows a synergistic effect with acid to increase NIT degradation. Liquid chromatography-mass spectrometry analysis identified 134 significantly different metabolites, of which 11 metabolites of L. fermentum RC4, namely, γ-aminobutyric acid (GABA), isocitric acid, D-glucose, 3-methylthiopropionic acid (MTP), N-formyl-L-methionine, dimethyl sulfone (MSM), D-ribose, mesaconate, trans-aconitic acid, L-lysine, and carnosine, showed significant NIT degradation effects compared with the control group (MRS broth). Verification experiments showed that adding the above 11 metabolites to 100 mg/L NIT and incubating for 24 h resulted in NIT degradation rates of 5.07%, 4.41%, 6.08%, 16.93%, 5.28%, 2.41%, 0.93%, 18.93%, 12.25%, 6.42%, and 3.21%, respectively. Among these, three metabolites, namely, mesaconate, MTP, and trans-aconitic acid, showed efficient NIT degradation abilities that might be related to the degradation mechanism involving decarboxylation reactions. This is the first systematic study of NIT degradation by LAB, resulting in the identification of a new metabolite degradation pathway and three efficient NIT degradation metabolites.

Keywords: LCMS; lactic acid bacteria; metabolites; nitrite degradation.

Figure 1

The growth curve and nitrite degradation curves for L. fermentum RC4.

Figure 2

Acid production curves of L. fermentum RC4 (a) Nitrite degradation curves of L. fermentum RC4 metabolites and MRS culture broth; (b) Nitrite degradation curves in different pH; (c) Nitrite degradation curve of L. fermentum RC4 metabolites in synergy with acid (d).

Figure 3

Metabolite full identification chemical structure subclass classification chart of L. fermentum RC4.

Figure 4

The z-score plot of the L. fermentum RC4 differential metabolites (a,b). Note—D: Experimental group; M: MRS broth group. The red and green dots represent the fermentation broth group and culture broth group, respectively, with four replicates in each group, and a greater distance between groups indicates the greater difference in relative content. As shown in Figure 4, the two groups were well aggregated within the group, and the difference between the groups was obvious.

Figure 4

The z-score plot of the L. fermentum RC4 differential metabolites (a,b). Note—D: Experimental group; M: MRS broth group. The red and green dots represent the fermentation broth group and culture broth group, respectively, with four replicates in each group, and a greater distance between groups indicates the greater difference in relative content. As shown in Figure 4, the two groups were well aggregated within the group, and the difference between the groups was obvious.

Figure 5

A heat map of L. fermentum RC4 differential metabolites (a,b). Note—D: Experimental group; M: MRS broth group. The magnitude of the relative content in the graph is shown by the different colors: red represents high sample content, blue represents low sample content, and the darker the color, the higher or lower the relative content. The columns represent sample groups and the rows represent metabolites. Both fermentation broth and culture broth groups were divided into four replicates: D1, D2, D3, and D4, and M1, M2, M3, and M4, respectively. Two metabolite groups were clustered within the group, and the metabolic patterns or metabolic pathways were distinct between the groups.

Figure 5

A heat map of L. fermentum RC4 differential metabolites (a,b). Note—D: Experimental group; M: MRS broth group. The magnitude of the relative content in the graph is shown by the different colors: red represents high sample content, blue represents low sample content, and the darker the color, the higher or lower the relative content. The columns represent sample groups and the rows represent metabolites. Both fermentation broth and culture broth groups were divided into four replicates: D1, D2, D3, and D4, and M1, M2, M3, and M4, respectively. Two metabolite groups were clustered within the group, and the metabolic patterns or metabolic pathways were distinct between the groups.

Figure 6

A histogram of nitrite degradation by target metabolites in 24 h. Note: A-C represent that there are significant differences between metabolites with different letters, p ≤ 0.05.