A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Effect of Pod Storage and Drying Temperature on Fermentation Dynamics and Final Bean Quality of Cacao Nacional in Ecuador.

The impact of pod storage (PS) and two drying temperatures of fermented cocoa beans was investigated in Ecuador. Therefore, four variations were simultaneously carried out three times at two locations, independently: 0, 3, and 5 days of PS, dried at 60 °C and 0 days of PS, dried at 80 °C. Pod weight during storage, pulp content, pH, temperature, microbial counts, total free amino acids, protein profiles, sugars, organic acids, cut-test, fermentation index, and sensory profiles were analyzed. Minor differences in fermentation dynamics and bean quality were found between variations with and without PS. A rather accelerated fermentation with pod-stored beans was observed (e.g., faster color change, slightly lower pH in cotyledon after 48 h), along with a significantly higher maximal temperature during 24-42 h (43.1 ± 3.2 °C compared to 39.2 ± 2.0 °C without PS). More well-fermented beans were reached with PS (52.3 ± 22.6%) than without (62.7 ± 9.2%). Differences during fermentation were observed between the locations (e.g., pH, acids, sugars), but sensory evaluation indicated that the impact of location was mitigated with PS. Drying at 80 °C showed no adverse effects, as evidenced by the results of the cut-test and fermentation index. However, sensory evaluations revealed significant differences between 80 °C and 60 °C, with the former exhibiting more bitter and astringent cocoa liquor.

Growth Control of Listeria monocytogenes in Raw Sausage via Bacteriocin-Producing Leuconostoc carnosum DH25.

The current study addresses the critical issue of Listeria monocytogenes growth in raw sausage/meat products leading to human infections, most commonly listeriosis, which is known for its high fatality rate. This research focuses on the isolation, identification, and screening of lactic acid bacteria from various meat and fish products in Switzerland. In total, 274 lactic acid bacteria strains were isolated from 30 different products and were screened for their ability to inhibit Listeria monocytogenes growth, with 51 isolates demonstrating anti-Listeria activity at 8 °C, 15 °C, 25 °C, and 37 °C. Further experiments, using a meat model and a raw sausage challenge test, demonstrated that Leuconostoc carnosum DH25 significantly inhibited Listeria monocytogenes growth during the ripening and storage of the tested meat/sausage. This inhibitory effect was found to be attributed to the bacteriocins produced by Leuconostoc carnosum DH25 rather than factors like pH or water activity. The stability of the anti-Listeria substances was examined, revealing their resistance to temperature and pH changes, making Leuconostoc carnosum DH25 a promising protective culture for raw sausages. The genome sequencing of this strain confirms its safety, with no antibiotic resistance genes or virulence factors detected, and reveals the presence of the structural genes for the production of the bacteriocin LeucocinB-Ta11a. This study underscores the potential of LAB strains and their bacteriocins as effective tools for enhancing food safety and preventing Listeria monocytogenes growth in meat products, offering valuable insights into biocontrol strategies in the food industry.

Variations in Ecuadorian Cocoa Fermentation and Drying at Two Locations: Implications for Quality and Sensory.

In Ecuador, various processes are applied during cocoa post-harvesting. This study, therefore, explored fermentation parameters across two locations with 2-7 independent runs, focusing on temperature, microbial counts, pH during fermentation and drying, and their impact on cocoa bean quality. Factors including fermentation devices (jute bags, plastic bags, and wooden boxes), pre-drying, turning during fermentation, fermentation duration, and drying temperature were investigated. Fermenting in plastic bags without pre-drying or turning and fermenting in jute bags for only 40 ± 2.0 h yielded low maximal fermentation temperatures Tmax (31.1 ± 0.4 °C and 37.6 ± 1.8 °C), leading to bitter, astringent, woody, and earthy cocoa liquor. Longer fermentation (63 ± 6 h) in wooden boxes with turning (Wt) and in jute bags with pre-drying and turning (Jpt) achieved the highest Tmax of 46.5 ± 2.0 °C, and a more acidic cocoa liquor, particularly in Wt (both locations) and Jpt (location E). Therefore, it is recommended to ferment for a minimum duration from day 1 to 4 (63 ± 6 h), whether using plastic bags (with mandatory pre-drying) or jute bags (with or without pre-drying or turning). Furthermore, this study underscores the risks associated with excessively high drying temperatures (up to 95.2 ± 13.7 °C) and specific dryer types, which can falsify cut-tests and introduce unwanted burnt-roasted off-flavors in the cocoa liquor.

Monitoring of cocoa post-harvest process practices on a small-farm level at five locations in Ecuador.

Cocoa post-harvest practices were monitored on a small-farm scale (ca. 50 kg fresh beans) at five intermediaries from four provinces in Ecuador: (A) in Manabí, (B) and (E) in Los Ríos, (C) in Cotopaxi, (D) in Guayas. Temperature, pH (pulp, cotyledon), cell counts (yeasts, lactic acid bacteria, acetic acid bacteria) were recorded daily, and cut-tests and sensory descriptive analysis evaluated end quality. An overall inconsistency and variability in processing were observed with different fermentation devices (jute/plastic bags, wooden boxes), pre-drying, turning during fermentation, fermentation duration, and different drying processes (temperatures, direct/indirect). Key parameters (maximum temperature, pH cotyledon development) revealed a significant impact of the fermentation device on the post-harvest process and, therefore, on the fermentation development. 67-74 h in jute bags without turning was sufficient to reach well-fermented cocoa beans without moldy off-flavors, whereas 133 h in plastic bags without turning resulted in 3 ± 1% moldy beans and cocoa liquor with moldy off-flavor. Drying at high temperatures (80 ± 10 °C) with direct heat contact resulted in beans roasted to burnt off-flavor. Conclusively, the whole post-harvest process was crucial for well-fermented beans without off-flavor. Plastic bags seemed unsuitable, while jute bags could be an alternative to wooden boxes.

MALDI-TOF MS profiling and exopolysaccharide production properties of lactic acid bacteria from Kunu-zaki - A cereal-based Nigerian fermented beverage.

MALDI-TOF MS is a technique for high-throughput characterization of foodborne microbiota, however, its application for studying African traditional fermented foods is limited. A total of 164 out of 220 lactic acid bacterial (LAB) isolates from Kunu-zaki were identified using MALDI-TOF MS, with 100% identity of representative strains compared to 16S rRNA gene sequencing. MALDI-TOF MS profiling combined with 16S rRNA gene sequencing revealed a total of 15 LAB species in Kunu-zaki, where the most predominant species were Lactiplantibacillus plantarum (40.46%), Weissella confusa (27.27%), and Pediococcus pentosaceus (15.00%). Phenotypic screening of all isolates revealed strains of W. confusa (57), Lactiplantibacillus sp. (9), Companilactobacillus musae (1), Ligilactobacillus saerimneri (1) and Leuconostoc citreum (1) that are capable of producing dextran and/or fructan. Dextransucrase genes were detected in all EPS-producing strains by PCR. Weissella confusa YKDIA1 and YKDIA4 produced 11.93 and 11.70 g/L dextran from millet-sorghum flour hydrolysate-sucrose, respectively. Kunu-zaki produced using W. confusa YKDIA1 had high water holding capacity (100%) and viscosity ranging from 49.46-139.24 mPas. In this study, MALDI-TOF MS adequately revealed the LAB species composition in Kunu-zaki in a high-throughput strategy and further, the dominant occurrence of EPS-producing LAB strains and their potentials to influence the rheological properties of Kunu-zaki were demonstrated.

Multiple Techno-Functional Characteristics of Leuconostoc and Their Potential in Sourdough Fermentations.

In this study, the potential of Leuconostoc as non-conventional sourdough starter cultures was investigated. A screening for antifungal activities of 99 lactic acid bacteria (LAB) strains revealed high suppression of bakery-relevant moulds in nine strains of Leuconostoc with activities against Penicillium sp., Aspergillus sp., and Cladosporium sp. Mannitol production was determined in 49 Leuconostoc strains with >30 g/L mannitol in fructose (50 g/L)-enriched MRS. Further, exopolysaccharides (EPS) production was qualitatively determined on sucrose (40 g/L)-enriched MRS agar and revealed 59 EPS positive Leuconostoc strains that harboured dextransucrase genes, as confirmed by PCR. Four multifunctional Lc. citreum strains (DCM49, DCM65, MA079, and MA113) were finally applied in lab-scale sourdough fermentations (30 °C, 24 h). Lc. citreum was confirmed by MALDI-TOF MS up to 9 log CFU/g and pH dropped to 4.0 and TTA increased to 12.4. Antifungal compounds such as acetic acid, phenyllactic and hydroxyphenyllactic acids were determined up to 1.7 mg/g, 2.1 µg/g, and 1.3 µg/g, respectively, mannitol up to 8.6 mg/g, and EPS up to 0.62 g/100 g. Due to the observed multifunctionalities and the competitiveness in the natural flour microbiota present in sourdoughs, non-conventional LAB genera such as Leuconostoc seem promising for application in sourdough-based bakery products.

Enzymatic and microbial conversions to achieve sugar reduction in bread.

A standard level of sugar addition to bread is 2% (flour base) but sweet baked goods including hamburger buns, hot dog buns and some sandwich bread contain more than 10% sucrose. This study aimed to provide an integrated assessment of different strategies for sugar-reduced bread by using isomaltooligosaccharides (IMO) as bulk sweetening agent, polysaccharide hydrolases to generate sugars from flour polysaccharides, and sourdough. Trained panel sensory analyses of the intensity of sour and sweet tastes were compared to the concentration of organic acids and the sugar concentration of bread. Sourdough fermentation reduced the sweet taste intensity of bread produced with 9% sucrose. This effect was more pronounced with Leuconostoc mesenteroides, which converts fructose to mannitol with concomitant production of acetate. Addition of up to 20% sourdough fermented with Weissella cibaria 10 M, which does not produce mannitol and less acetate when compared to L. mesenteroides, did not substantially reduce the sweet taste intensity. Bread produced with 9% IMO tasted less sweet than bread prepared with 9% sucrose but partial replacement of sucrose with IMO maintained the sweet taste intensity. Addition of 4.5% IMO in combination with W. cibaria sourdough, amyloglucosidase and the fructosidase FruA enabled production of bread with 50% reduced sucrose addition while maintaining the sweet taste intensity. In conclusion, the single use of a sweet bulking agent, of amyloglucosidase or fructanases or the use of sourdough alone, did not maintain the sweet taste intensity of sugar-reduced bread, however, a combination of the three approaches allowed a reduction of sucrose addition without reducing the sweet taste intensity.

Influence of two anti-fungal Lactobacillus fermentum-Saccharomyces cerevisiae co-cultures on cocoa bean fermentation and final bean quality.

The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.

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 medium's 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.

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.

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

BACKGROUND: Surface 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). RESULTS: TTGE 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 x 10(2) 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 (10(5) CFU cm(-2) on cheeses smeared with a defined surface culture. CONCLUSIONS: This 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.