The diversity of microorganisms present in cheeses contributes to their typicity and organoleptic properties. Inhibitory strains found in these ecosystems could be used as biopreservative agents in order to replace chemicals, which are increasingly decried by consumers. Here, we isolated from cheese two yeasts named, Metschnikowia pulcherrima LMA 2038 and Trichosporon asahii LMA 810, which are active against fungi, such as Yarrowia lipolytica LMA 800, Rhodotorula mucilaginosa LMA 808, Cladosporium cladosporioides LMA 31, and Penicillium commune LMA 72, as well as against bacteria, such as Listeria innocua ATCC 33090 and Clostridium tyrobutyricum LMG 1285. When cultivated on model fresh cheese, these antagonistic yeasts significantly reduced the number of Y. lipolytica LMA 800 and R. mucilaginosa LMA 808. Preliminary data enabled us to establish a snapshot of the molecules responsible for these antifungal and antibacterial inhibitions. The molecules presumed to be present in the culture supernatant were subjected to various treatments, including heat, pH variations and enzymatic treatments. Thus, we hypothesize that different antimicrobial compounds were produced by the aforementioned antagonistic yeasts, and these molecules act in a target strain-specific manner. In summary, our findings demonstrate that cheeses possess an innumerable supply of antagonistic microbial strains, which can be exploited in the field of biopreservation to substitute chemicals and extend the shell-life of food products, including cheese.

Probiotics are live yeast or bacterial organisms that have beneficial effects on the host. Several microorganisms exhibit probiotic properties, the most common types being lactic acid bacteria, Bifidobacteria, spore-forming bacteria, and some yeast strains. Saccharomyces cerevisiae var. boulardii is the most important probiotic yeast species. However, another group of foodborne microorganisms, the so-called non-Saccharomyces yeasts (NSYs), has recently been re-evaluated and shown to have enormous potential in various fields of application, ranging from food fermentation to human and animal applications. NSYs are able to produce a range of bioactive compounds such as antimicrobials, mannoproteins, enzymes, polyunsaturated fatty acids, essential amino acids, vitamins, and β-glucans, which increases their potential applications as a new class of probiotics and/or alternatives to antibiotics in animal husbandry. In this review, we aim to highlight the potential and benefits of NSYs as probiotics and natural antimicrobials to improve animal health. Furthermore, the use of NSYs as biological alternatives to antibiotics to control foodborne pathogens in animal production is discussed.

The genus Trichosporon includes yeasts that are naturally present within the human gastrointestinal tract, on the skin, and as part of the vaginal microbiota. This genus is an opportunistic pathogen, commonly found in fungal infections affecting immunocompromised individuals. The species Trichosporon asahii (T. asahii) causes the majority of trichosporonoses and is therefore widely studied, particularly in relation to its pathogenicity and its emerging resistance to antifungal drugs used to treat the disease. However, T. asahii also has important biotechnological applications, particularly its depolluting abilities and its bioproduction of flavor compounds (e.g., terpenes, C13-Norisoprenoids, C6 compounds, methyl hexanoate, and ethyl isovalerate) and antioxidant molecules. T. asahii also produces substances that inhibit certain contaminants found in dairy products, such as Kocuria rhizophila, Clostridium tyrobutyricum, and Salmonella enterica. Paradoxically, this yeast species also has some potential probiotic applications. This review aims to discuss and provide updates on the taxonomy, pathogenicity, and biotechnological relevance of T. asahii.

We describe and discuss the intestinal mycobiota of dairy cows reared in France following variations in dietary regimes and two seasons. Two groups of 21 animals were followed over a summer and winter period, and another group of 28 animals was followed only during the same summer season. The summer diet was based on grazing supplemented with 3–5 kg/d of maize, grass silage and hay, while the winter diet consisted of 30% maize silage, 25% grass silage, 15% hay and 30% concentrate. A total of 69 DNA samples were extracted from the feces of these cows. Amplification and sequencing of the ITS2 region were used to assess mycobiota diversity. Analyses of alpha and beta diversity were performed and compared statistically. The mycobiota changed significantly from summer to winter conditions with a decrease in its diversity, richness and evenness parameters, while beta diversity analysis showed different mycobiota profiles. Of note, the Geotrichum operational taxonomic unit (OTU) was prevalent in the winter group, with a mean relative abundance (RA) of 65% of the total mycobiota. This Geotrichum OTU was also found in the summer group, but to a lesser extent (5%). In conclusion, a summer grazing diet allowed a higher fecal fungal diversity. These data show, for the first time, that a change in diet associated with seasonality plays a central role in shaping hindgut fungal diversity.

Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.

Antimicrobial resistance is a global health concern across the world and it is foreseen to swell if no actions are taken now. To help curbing this well announced crisis different strategies are announced, and these include the use of antimicrobial peptides (AMP), which are remarkable molecules known for their killing activities towards pathogenic bacteria. Bacteriocins are ribosomally synthesized AMP produced by almost all prokaryotic lineages. Bacteriocins, unlike antibiotics, offer a set of advantages in terms of cytotoxicity towards eukaryotic cells, their mode of action, cross-resistance and impact of microbiota content. Most known bacteriocins are produced by Gram-positive bacteria, and specifically by lactic acid bacteria (LAB). LAB-bacteriocins were steadily reported and characterized for their activity against genetically related Gram-positive bacteria, and seldom against Gram-negative bacteria. The aim of this study is to show that lacticaseicin 30, which is one of the bacteriocins produced by Lacticaseibacillus paracasei CNCM I-5369, is active against Gram-negative clinical strains (Salmonella enterica Enteritidis H10, S. enterica Typhimurium H97, Enterobacter cloacae H51, Escherichia coli H45, E. coli H51, E. coli H66, Klebsiella oxytoca H40, K. pneumoniae H71, K. variicola H77, K. pneumoniae H79, K. pneumoniae H79), whereas antibiotics failed. In addition, lacticaseicin 30 and colistin enabled synergistic interactions towards the aforementioned target Gram-negative clinical strains. Further, the combinations of lacticaseicin 30 and colistin prompted a drastic downregulation of mcr-1 and mcr-9 genes, which are associated with the colistin resistance phenotypes of these clinical strains. This report shows that lacticaseicin 30 is active against Gram-negative clinical strains carrying a rainbow of mcr genes, and the combination of these antimicrobials constitutes a promising therapeutic option that needs to be further exploited.

Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive Lacticaseibacillus paracasei CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including Escherichia coli strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in Escherichia coli and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive Listeria innocua. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli.

Bacteriocins produced by lactic acid bacteria (LAB-bacteriocins) may serve as alternatives for aging antibiotics. LAB-bacteriocins can be used alone, or in some cases as potentiating agents to treat bacterial infections. This approach could meet the different calls and politics, which aim to reduce the use of traditional antibiotics and develop novel therapeutic options. Considering the clinical applications of LAB-bacteriocins as a reasonable and desirable therapeutic approach, it is therefore important to assess the advances achieved in understanding their modes of action, and the resistance mechanisms developed by the producing bacteria to their own bacteriocins. Most LAB-bacteriocins act by disturbing the cytoplasmic membrane through forming pores, or by cell wall degradation. Nevertheless, some of these peptides still have unknown modes of action, especially those that are active against Gram-negative bacteria. Regarding immunity, most bacteriocin-producing strains have an immunity mechanism involving an immunity protein and a dedicated ABC transporter system. However, these immunity mechanisms vary from one bacteriocin to another.

Five open reading frames viz orf010, orf12, orf023, orf030 and orf038 coding class II bacteriocins in Lacticaseibacillus paracasei CNCM I-5369 strain previously isolated from an Algerian dairy product, were found to be expressed after 24 h of growth. The strain has also shown anti-E. coli activity in a narrow pH range between 4.5 and 5. Then, expression and purification of these bacteriocins was conducted in the heterologous host E. coli. This strategy enabled us to purify the peptide encoded by orf030 in large quantities, in contrast to other peptides that were produced but required to be released from the insoluble fraction following 4 M urea and desalting treatments. All peptides heterologously produced were characterized by MALDI TOF Mass spectrometry and successfully tested for their anti-E. coli activity. Furthermore, in silico transcriptional analysis was determined by Findterm tool and with Bagel4 software permitted to locate potential promoters and co-transcription events.

Six Trichosporon asahii strains designated ICVY021, ICVY022, ICVY023, ICVY024, ICVY025 and ICVY026 were isolated from camel milk and studied for their inhibitory activities against a large number of fungi and bacteria as target organisms. Only strain ICVY021 was able to inhibit Kocuria rhizophila CIP 53.45, through production of an extracellular heat-stable, proteinaceous antibacterial peptide, with partial amino acid sequences of PPFPK and CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q), determined using LC/MS/MS. This peptide, named oranicin P16, was thought to impede the cell-division mechanism. The anti-K. rhizophila activity was confirmed, in situ , using skim milk as a food matrix. T. asahii ICVY021 did not show any hemolytic activity or any cytotoxicity effect against eukaryotic Caco-2 cells. T. asahii ICVY021 was sensitive to azoles antifungal compounds, but not to 5-fluorocytosine and anidulafungin.

During this study, we characterized the seasonality’s impact and environmental conditions on the yeast diversity from raw camel’s milk collected in Algeria. The yeast counts were estimated to 3.55 × 102 CFU mL−1, with a maximum of 6.3 × 102 CFU mL−1. The yeasts were categorized phenotypically by API 20C AUX, MALDI-TOF and genetically by sequencing 26S rDNA and ITS1-5.8S-ITS2. The rDNA sequencing approaches revealed 12 species including unusual ones such as Trichosporon asahii, Pichia fermentans, Millerozyma farinosa, Pichia galeiformis, Candida tartarivorans and Pichia manshurica. The most dominant species were T. asahii (23%), P. fermentans (19%) and Rhodotorula mucilaginosa (14%). The high occurrence and large diversity were registered in samples collected during the autumn season, in the semi-arid and arid highlands regions with 0.66 × 103 CFU mL−1 and 0.51 × 103 CFU mL−1, respectively. Interestingly, T. asahii, R. mucilaginosa, P. fermentans, C. parapsilosis and C. zeylanoides were detected during both spring and autumn.

Tomme d'Orchies is an artisanal pressed and uncooked cheese produced and marketed in the north of France. This study aimed at showing the fungal microbiota evolution of this cheese using a metagenetic based Illumina technology targeting the ITS2 domain of 5.8S fungal rDNAs. To this end, samples were taken from the rind and the core of different cheeses, after 0, 1, 3, 7, 14 and 21days of ripening. The data underpinned the prevalence of Yarrowia lipolytica and Galactomyces geotrichum for both microbiotas. Unusual species including Clavispora lusitaniae, Kazachstania unispora and Cladosporium cladosporioides were also detected, but their origins remain to be ascertained. The metagenomic revealed also the presence of Kluyveromyces and Debaryomyces species.

The effects of yeast ICluyveromyces marxianus S-2-05, of cheese origin, were assessed on the intestine anaerobe symbiont Bacteroides thetaiotaomicron ATCC 29741 to unveil any changes in its antioxidant properties. To this end, these microorganisms were grown and incubated either separately, or co-incubated, under anaerobic atmosphere. Afterwards, the microbial cells were recovered and washed, and extracts were prepared using a sterile detergent solution to mimic the intestine detergent content. The extracts prepared from K. marxianus S-2-05 and reference strain K. marxianus MUCL 29917, grown under different conditions, were assessed for their antioxidant properties against superoxide anion and hydrogen peroxide. Extracts from both yeasts showed antioxidative effects, which were particularly important for K. marxianus S-02-5 after anaerobic incubation. Moreover, K. marxianus S-02-5 displayed a high level of activity against the aforementioned reactive oxygen species, enhancing that of B. thetaiotaomicron ATCC 29741, after the co-incubation process. Two-dimensional polyacrylamide gel electrophoresis was used to separate the proteins extracted. Superoxide dismutase, thiol peroxidase, rubrerythrin-intensively produced by B. thetaiotaomicron induced by the yeast-were identified by mass spectrometry. The antioxidative potential evidenced for K. marxianus S-02-5 is another advantage which could justify the utilization of this strain as a probiotic for countering intestinal inflammatory processes. (C) 2017 Elsevier Ltd. All rights reserved.

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains.

Kluyveromyces marxianus S-2-05 and Kluyveromyces lactis S-3-05 were recently isolated from a traditional French cheese, Tomme d'Orchies, and characterized here for their advantages using a different application perspective. First, we established their anti-Salmonella activity and downregulation of the virulence sopD gene of Salmonella enterica subsp. enterica serovar Typhimurium, mainly in the presence of K. marxianus S-2-05. In addition to their antagonism, these non-Saccharomyces yeasts were able to survive under conditions mimicking the gastrointestinal environment and to form biofilms on an abiotic device such as polystyrene. These strains also displayed highly hydrophilic cell wall surfaces properties and capacity for adhesion to intestinal Caco-2 cells, thus enhancing their potential as probiotic strains.