Susanne Miescher Schwenninger

A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp. paracasei inhibits food spoilage yeasts

Screening for antimicrobial features of 197 propionibacteria and tests with several antifungal lactobacilli led to the development of three protective cultures containing Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp. paracasei strain SM20, SM29 or SM63. These cultures showed inhibitory activities (up to 5 orders of magnitude) against yeasts in dairy products such as yoghurt or cheese surface at refrigerator temperatures (6 degrees C) without an influence on the quality properties of the food. Initial cell numbers of 5 x 10(7) cells/g of propionibacteria and 1 x 10(8) cells/g of lactobacilli were the optimal concentrations to yield a total inhibition of the spoilage yeasts (Candida pulcherrima, Candida magnoliae, Candida parapsilosis and Zygosaccharomyces bailii).

Characterization of low-molecular-weight antiyeast metabolites produced by a food-protective Lactobacillus-Propionibacterium coculture.

We developed a pH-controlled batch fermentation process with separately immobilized cells of the protective coculture of Lactobacillus paracasei subsp. paracasei SM20 and Propionibacterium jensenii SM11 in supplemented whey permeate medium yielding cell-free supernatants with high antiyeast activity against Candida pulcherrima and Rhodotorula mucilaginosa. The antiyeast compounds were resistant to proteinase K and pronase E treatments and showed high heat resistance (121 degrees C for 15 min). Diafiltration (1,000-Da cutoff) revealed that the inhibitory metabolites have low molecular weights. Partial purification of active compounds was achieved by a microplate bioassay controlled procedure with solid-phase extraction (C18) followed by (i) gel filtration chromatography or (ii) semipreparative reverse-phase high-performance liquid chromatography (C18). In addition to propionic, acetic, and lactic acids, 2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, hydroxyphenyllactic acid, and succinic acid were identified by chromatography and mass spectrometry. Accurate quantifications revealed only low concentrations (up to 7 mM) of 2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, and hydroxyphenyllactic acid produced during fermentation in contrast to relatively high MICs (50 to more than 500 mM) determined at different pH values (4.0, 5.0, and 6.0). Succinic acid was present at higher concentrations (29 mM) in cell-free supernatants but with comparable high MICs (200 to more than 500 mM and pH 4.0, 5.0, and 6.0). Although none of these compounds was the main substance responsible per se for suppression of yeast growth, our study revealed a complex antiyeast mechanism with putative synergistic effects between several low-molecular-weight compounds.

Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

BackgroundSurface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M).ResultsTTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture.ConclusionsThis work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. TTGE revealed the presence of marine lactic acid bacteria that are likely related to the strong Listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in Listeria cell count.

Detection of Antifungal Properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and Molecular Typing of the Strains

Lactobacilli isolated from different food and feed samples such as raw milk, cheese, yoghurt, olives, sour dough, as well as corn and grass silage, were screened for their antifungal activities. Out of 1,424 isolates tested, 82 were shown to be inhibitory to different yeasts (Candida spp. and Zygosaccharomyces bailii) and a Penicillium sp., which were previously isolated from spoiled yoghurt and fruits. Carbohydrate fermentation patterns suggested that a substantial portion, 25%, belonged to the Lactobacillus casei group, including L. casei, L. paracasei, and L. rhamnosus. The isolates SM20 (DSM14514), SM29 (DSM14515), and SM63 (DSM14516) were classified by PCR using species-specific primers to target the corresponding type strains (L. casei, L. paracasei, and L. rhamnosus) as controls. Further molecular typing methods such as randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and sequencing analysis of the 16S rRNA gene allowed classifying strains SM20, SM29, and SM63 as L. paracasei subsp. paracasei in accordance with the new reclassification of the L. casei group proposed by Collins et al.

Facultative anaerobic halophilic and alkaliphilic bacteria isolated from a natural smear ecosystem inhibit Listeria growth in early ripening stages

In vitro and in situ anti-listerial properties of 3 strains of Facultative Anaerobic Halophilic and Alkaliphilic (FAHA) species, i.e. Alkalibacterium kapii ALK 6, Marinilactibacillus psychrotolerans ALK 9 and Facklamia tabacinasalis ALK 1, were investigated. The 3 strains were isolated from a smear ecosystem originating from a commercial Raclette type cheese and exhibiting strong anti-listerial activity in situ on cheese surface. In a first step, strains were tested in vitro for production of antimicrobial compounds against Listeria innocua 81000-1 and Listeria ivanovii HPB 28. M. psychrotolerans ALK 9 inhibited both indicator strains in spot-on-the-lawn tests while A. kapii ALK 6 showed no inhibiting effect. F. tabacinasalis ALK 1 exerted an in vitro inhibition on L. ivanovii HPB 28, but induced the formation of dense ball-shaped microcolonies of L. innocua 81000-1 in the soft agar, a typical biofilm microstructure. The extent of the biofilm zone was enhanced when F. tabacinasalis ALK 1 and M. psychrotolerans ALK 9 were tested together. In a second step, different combinations of strains were applied on Raclette cheeses ripened at pilot scale and contaminated with 50 cfu/cm(2)L. innocua at day 7. A control flora of 6 strains, isolated from ecosystem F and corresponding to species commonly found on smear cheeses, was applied on control and test cheeses. In test cheeses, we investigated the impact on Listeria growth of the addition of the 3 FAHA strains, applied as single or mixed cultures. A 1-log inhibition was obtained at day 15 on cheeses treated with FAHA strains applied either as single or mixed cultures. This 1-log inhibition was correlated with the development of FAHA species that reached their maximal count at day 15. This study suggests that the development of FAHA species in early ripening likely contributes to the initial part of the in situ inhibition exerted by the complex cheese surface ecosystem investigated.

Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation

To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the mediums glucose, fructose, and citric acid into 20.4-23.0 g/l of mannitol, 3.9-6.2 g/l acetic acid, and 8.6-10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4-18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51-95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.