A lab-scale model system for cocoa bean fermentation.

Lab-scale systems modelling the spontaneous cocoa bean fermentation process are useful tools to research the influence of process parameters on the fermentation and the final bean quality. In this study in Honduras, a 1-kg lab-scale fermentation (LS-F) was compared to a 300-kg on-farm fermentation (OF-F) in a multiphasic approach, analysing microbial counts, microbial species diversity, physico-chemical parameters, and final dried bean quality. Yeast and total aerobic counts of up to 8 log CFU/g during the LS-F were comparable to the OF-F, while counts for lactic acid bacteria and acetic acid bacteria were up to 3 log CFU/g lower during the LS-F than during the OF-F. While species of the genera Hansenia, Saccharomyces, and Acetobacter dominated most of the fermentation processes, the genera dominating the drying phases were Pichia, Trichosporon, Pediococcus, and Acetobacter. Dried beans resulting from the LS-F, compared to the OF-F, were similar in contents of acetic acid, 6 times lower in lactic acid, up to 4 times higher in residual sugars, and 3-12 times higher in polyphenols. Dried beans processed at LS showed a similar flavour profile in terms of astringency, bitterness, acidity, and brown, fine, and cocoa flavours, but 2 units higher off-flavours than OF processed beans. With 81%, the share of well-fermented beans from the LS-F complied with industrial standards, whereas 7% over-fermented beans were above the threshold. Conclusively, the 5-day model fermentation and subsequent drying successfully mimicked the on-farm process, providing a high-throughput method to screen microbial strains to be used as starter cultures.

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.

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.

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.

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).