Non-Saccharomyces yeasts represent a very attractive alternative for the production of beers with superior sensory quality since they are able to enhance the flavour of beer. Furthermore, they can produce beers with low ethanol content due to the weak fermentative capacity of a large percentage of non-Saccharomyces species. The objective of this study was to evaluate the ability of 34 non-Saccharomyces yeast strains isolated from Madrilenian agriculture to produce a novel ale beer. The non-Saccharomyces yeast strains were screened at two scales in the laboratory. In the first screening, those with undesirable aromas were discarded and the selected strains were analysed. Thirty-three volatile compounds were analysed by GC, as well as melatonin production by HPLC, for the selected strains. Thirteen strains were then fermented at a higher scale in the laboratory for sensory evaluation. Only yeast strains of the species Schizosaccharomyces pombe and Lachancea thermotolerans were able to complete fermentation. Species such as Torulaspora delbrueckii, Metschnikowia pulcherrima, Wickerhamomyces anomalus, Hanseniaspora vineae, and Hanseniaspora guilliermondii could be used both for production of low ethanol beers and co-fermentation with a Saccharomyces yeast to improve the organoleptic characteristics of the beer. In addition, for these strains, the levels of melatonin obtained were higher than the concentrations found for Saccharomyces strains subjected to the same study conditions. The selected strains can be used in future trials to further determine their viability under different conditions and for different purposes.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving β-galactosidase (β-gal) and 6Pβ-galactosidase (6Pβ-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pβ-gal gene, the 11 GH1 family proteins, in which their 6Pβ-glucosidase (6Pβ-glc) activity was experimentally demonstrated,, were assayed for 6Pβ-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pβ-gal activity. The sequence comparison of this dual 6Pβ-gal/6Pβ-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pβ-gal/6Pβ-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pβ-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pβ-gal activity with possible relevance for lactose maldigestion management.

Microbial enzymes can be used as processing aids or additives in food and feed industries. Enzymatic detoxification of ochratoxin A (OTA) is a promising method to reduce OTA content. Here, we characterize the full-length enzyme ochratoxinase (AnOTA), an amidohydrolase from Aspergillus niger. AnOTA hydrolyzes OTA and ochratoxin B (OTB) mycotoxins efficiently and also other substrates containing phenylalanine, alanine, or leucine residues at their C-terminal position, revealing a narrow specificity profile. AnOTA lacks endopeptidase or aminoacylase activities. The structural basis of the molecular recognition by AnOTA of OTA, OTB, and a wide array of model substrates has been investigated by molecular docking simulation. AnOTA shows maximal hydrolytic activity at neutral pH and high temperature (65 °C) and retained high activity after prolonged incubation at 45 °C. The reduction of OTA levels in food products by AnOTA has been investigated using several commercial plant-based beverages. The results showed complete degradation of OTA with no detectable modification of beverage proteins. Therefore, the addition of AnOTA seems to be a useful procedure to eliminate OTA in plant-based beverages. Moreover, computational predictions of in vivo characteristics indicated that AnOTA is neither an allergenic nor antigenic protein. All characteristics found for AnOTA supported the suitability of its use for OTA detoxification in food and feed.

To increase our knowledge on the functionality of 6-phospho-β-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-β-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-β-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.