The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494

Abstract

The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.

Fig. 1.

Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of inosine (mMRS4 medium) (A and B) or 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of adenosine (mMRS5 medium) (C and D) with free pH, starting at an initial pH of 5.5. (A) Course of lactic acid (×), acetic acid (▲), formic acid (■), inosine (○), and hypoxanthine (◆) and pH profile (–). (C) Course of lactic acid (×), acetic acid (▲), formic acid (■), adenosine (○), and adenine (◆) and pH profile (–). (B and D) Course of arginine (□), citrulline (Δ), and ornithine (⋄).

Fig. 2.

(A to F) Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of inosine (mMRS4 medium) at different constant pH values: (A and B) pH 6.5, (C and D) pH 5.5, and (E and F) pH 5.0. (A, C, and E) Course of lactic acid (×), acetic acid (▲), formic acid (■), inosine (○), and hypoxanthine (◆). (B, D, and F) Course of arginine (□), citrulline (Δ), and ornithine (⋄). Solid lines are according to the model; symbols represent the experimental data.

Fig. 3.

(A to F) Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of adenosine (mMRS5 medium) at different constant pH values: (A and B) pH 6.5, (C and D) pH 5.5, and (E, F) pH 5.0. (A, C, and E) Course of lactic acid (×), acetic acid (▲), formic acid (■), adenosine (○), and adenine (◆). (B, D, and F) Course of arginine (□), citrulline (Δ), and ornithine (⋄). Solid lines are according to the model; symbols represent the experimental data.

Fig. 4.

Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of ribose (mMRS6 medium) at constant pH 6.5. (A) Course of lactic acid (×), acetic acid (▲), and ribose (○). (B) Course of arginine (□), citrulline (Δ), and ornithine (⋄). Solid lines are according to the model; symbols represent the experimental data.

Fig. 5.

Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine, 3 g liter⁻¹ of inosine, and 1 g liter⁻¹ of glucose (mMRS7 medium) (A and B) or 3 g liter⁻¹ of arginine, 3 g liter⁻¹ of adenosine, and 1 g liter⁻¹ of glucose (mMRS8 medium) (C and D) at constant pH 5.5. (A) Course of glucose (+), lactic acid (×), acetic acid (▲), formic acid (■), inosine (○), and hypoxanthine (◆). (C) Course of glucose (+), lactic acid (×), acetic acid (▲), formic acid (■), adenosine (○), and adenine (◆). (B and D) Course of arginine (□), citrulline (Δ), and ornithine (⋄). Solid lines are according to the model; symbols represent the experimental data.

Fig. 6.

Growth (in log [CFU ml⁻¹]) of Lactobacillus sakei CTC 494 (●) in a modified MRS medium containing 3 g liter⁻¹ of arginine and 3 g liter⁻¹ of inosine (mMRS4 medium) at constant pH 6.5 under anaerobic conditions. (A) Course of lactic acid (×), acetic acid (▲), formic acid (■), inosine (○), and hypoxanthine (◆). (B) Course of arginine (□), citrulline (Δ), ornithine (⋄), and carbon dioxide (–).

Fig. 7.

Metabolic scheme representing the steps involved in the catabolism of inosine and adenosine in Lactobacillus sakei CTC 494 based on the metabolic end products measured during the present study in combination with genomic information of L. sakei 23 K (2). Step 1, nucleoside transporter; 2, nucleoside phosphorylase; 3, nucleoside hydrolase; 4, phosphoketolase; and 5, pyruvate formate lyase.