The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation.

The ability of coagulase-negative staphylococci (CNS) to use alternative energy sources in meat may partially explain their occurrence in fermented meats. Of 61 CNS strains tested, all metabolized adenosine and inosine in a meat simulation medium (MSM). The ability to catabolize arginine via the arginine deiminase (ADI) pathway varied between strains. All tested strains of Staphylococcus carnosus and Staphylococcus epidermidis possessed an arcA gene and showed ADI activity, whereas other species, such as Staphylococcus equorum and Staphylococcus succinus, did not. Arginine catabolic mobile elements (ACME), as in the positive control S. epidermidis ATCC 12228, were uncommon and only found in Staphylococcus xylosus 3PA6 (sausage isolate) and Staphylococcus chromogenes G222 (teat apex isolate). Monoculture experiments were performed in MSM with S. carnosus 833 and SS3-4, S. xylosus G211, and S. epidermidis ATCC 12228 and 2S7-4. At all pH values tested (5.3, 5.8, and 6.5), the strains of S. carnosus catabolized arginine faster than the strains of S. xylosus and S. epidermidis. Only at pH 6.5 could a low ADI activity be found for S. xylosus G211. Increased ADI activity occurred in the case of the ACME-positive S. epidermidis ATCC 12228, when compared to the ACME-negative S. epidermidis 2S7-4.

Community dynamics of coagulase-negative staphylococci during spontaneous artisan-type meat fermentations differ between smoking and moulding treatments.

Coagulase-negative staphylococci (CNS) that are naturally present in the raw meat batter of fermented sausages or that originate from the addition of a starter culture play a role in flavour development. A wide species diversity of CNS can be present in fermented meats, but it is not fully clear yet how specific process parameters select for specific CNS by affecting their community dynamics. Therefore, the influence of smoking and moulding treatments on the CNS community dynamics in spontaneously fermented, artisan-type sausages was investigated. During the fermentation stage, the meat batter was in all cases dominated by Staphylococcus saprophyticus, in addition to Lactobacillus sakei as governing lactic acid bacterium. Following fermentation, the bacterial communities were not perturbed by the smoking treatment, since both L. sakei and S. saprophyticus remained dominant throughout the ripening stage and prevailed in the end-products. Yet, when fermentation was followed by a moulding step with Penicillium nalgiovense, a shift of the CNS communities towards dominance by Staphyloccocus equorum was seen, despite a similar evolution of L. sakei. This effect was possibly due to a pH rise caused by the mould, a hypothesis which was reinforced by the finding that the isolated strain S. equorum DBX-S-17 was more sensitive to low pH than the isolated strain S. saprophyticus DFL-S-12 during growth experiments in brain heart infusion (BHI). Differences in CNS communities may affect sausage flavour, due to intraspecies variations in metabolic conversions of, for instance, amino acids. The fact that 3-methyl-butanal was only found in the moulded sausage, which was dominated by S. equorum, may be related to the finding that the isolated strain of this species was able to produce this compound in BHI medium, whereas the isolated strain of S. saprophyticus was not.

Application' and validation of autochthonous lactic acid bacteria starter cultures for controlled leek fermentations and their influence on the antioxidant properties of leek.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important outdoor vegetables, mainly cultivated for its white shaft. Fermentation of leek offers opportunities in view of biomass valorization and product diversification. This study deals with the implementation and validation of starter cultures to perform controlled leek fermentations and to ensure a high quality of the end-products. Therefore, a thorough study of the fermentation microbiology and the influence of three starter culture strains (Lactobacillus plantarum IMDO 788, Lactobacillus sakei IMDO 1358, and Leuconostoc mesenteroides IMDO 1347) on the metabolite kinetics of leek fermentation and antioxidant properties of leek was performed. Overall, the application of lactic acid bacteria starter cultures resulted in a fast prevalence of the species involved, coupled to an accelerated acidification. Of the three starter cultures tested, the mixed starter culture of L. plantarum IMDO 788 and L. mesenteroides IMDO 1347 was most promising, as its application resulted in fermented leek of good microbiological quality and in a more extensive carbohydrate consumption, whereby diverse end-metabolites were produced. However, high residual fructose concentrations allowed yeast outgrowth, resulting in increased ethanol and glycerol concentrations, and indicated the lack of a prevailing strictly heterofermentative LAB species. The antioxidant capacity of fermented leek samples, as measured with the oxygen radical absorbance capacity assay, increased when starter cultures were used, whereas with regard to 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, only leek fermented with L. sakei IMDO 1358 scored higher than spontaneously fermented leek. The total phenolic content was not influenced by the use of starter cultures, while the S-alk(en)yl-L-cysteine sulfoxides content decreased strongly. A preliminary sensory analysis revealed that the spontaneously fermented leek and the one obtained with the mixed starter culture were preferred by consumers, emphasizing again the importance of microbial successions in vegetable fermentations.

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

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.

Leuconostoc durionis sp. nov., a heterofermenter with no detectable gas production from glucose.

Three lactic acid bacterial (LAB) strains obtained from a Malaysian acid-fermented condiment, tempoyak (made from pulp of the durian fruit), showed analogous but distinct patterns after screening by SDS-PAGE of whole-cell proteins and comparison with profiles of all recognized LAB species. 16S rRNA gene sequencing of one representative strain showed that the taxon belongs phylogenetically to the genus Leuconostoc, with its nearest neighbour being Leuconostoc fructosum (98 % sequence similarity). Biochemical characteristics and DNA-DNA hybridization experiments demonstrated that the strains differ from Leuconostoc fructosum and represent a single, novel Leuconostoc species for which the name Leuconostoc durionis sp. nov. is proposed. The type strain is LMG 22556(T) (= LAB 1679(T) = D-24(T) = CCUG 49949(T)).

Detection of cleavage of a prenisin-mimicking decapeptide by Lactococcus lactis subsp. lactis endoproteinase activity.

As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate.