High Prevalence of Antibiotic-Resistant Escherichia coli Isolates from Retail Poultry Products in Spain.

The prevalence of Escherichia coli was analysed in poultry products from different Spanish retailers and determined its antibiotic resistance capability by phenotypic (ampicillin, amoxicillin, chloramphenicol, gentamicin, imipenem, cefotaxime, tetracycline, ciprofloxacin, trimethoprim, and colistin) and genotypic assays. A total of 30 samples (hindquarters or livers) were collected from supermarkets and butchers. Enterobacteriaceae counts ranged between 3.2 and 6.5 log colony-forming units (CFU)/g, and the highest values were found in livers and in samples from supermarkets. E. coli was detected in 83% of the samples tested, and the highest prevalence was observed in livers (100%) and supermarkets (91%). Regarding the antibiotic sensitivity test, 100% of the E. coli showed resistance to at least one antibiotic. The highest resistance rates were detected for colistin (87%) and gentamicin (79%), while only two antibiotics (chloramphenicol and cefotaxime) showed a resistance lower than 10%. Furthermore, the resistance genes of tetracycline and beta-lactams were analysed by multiplex PCR, revealing that tet(A) and blaTEM were the majority genes, respectively.

Free and Immobilised ß-Glucosidases in Oenology: Biotechnological Characterisation and Its Effect on Enhancement of Wine Aroma.

In grapes, monoterpenes and norisoprenoids are in the form of non-volatile compounds, flavourless glycosides which could enhance the aroma of wines after its hydrolysis using ß- glucosidases enzymes. It is known that the use of immobilised enzymes offers advantages such as reusability and easy recuperation. In this study, a commercial ß-glucosidase was immobilised by absorption in sodium alginate. Biotechnological characteristics and terpen hydrolysis (hydrolysis aroma precursors) in muscat wines were studied after treatment with both free and immobilised commercial ß- glucosidase with two different concentrations. It was revealed that both forms shared an optimal pH (4.5) and a maximum temperature (64°C), even an increment on the activity between 40and 60°C. A similar Km value has been determined while Vmax from the immobilised enzyme was higher than the free (3.35 and 2.52 µmol min-1 mg-1, respectively). Additionally, the immobilised enzyme showed a better hydrolytic activity during 24 h, and its reusability has been proven. Regarding enzymatic hydrolysis in grape must, the best results were observed for the highest concentration of free ß-glucosidase although glucose release was also determined for the immobilised enzyme along the days. In contrast, maximum activity was reached by the immobilised ß-glucosidase in less time but in no case equalled the free ones. Finally, volatile compound liberation in wines treated with free or immobilised enzymes was analysed using HRGC-MS. Liberation for both enzymes and the greatest concentrations of some volatiles were detected when a double dose of the free ß-glucosidase was used. Nevertheless, the wines treated with the immobilised ß-glucosidase showed a high concentration of some volatile compounds such as nerol or geraniol.

Potential Probiotic Strains of Saccharomyces and Non-Saccharomyces: Functional and Biotechnological Characteristics.

Due to the evident demand for probiotic microorganisms, a growing number of scientific studies have involved the preliminary selection of new strains, but deeper studies for knowing specific functional and biotechnological properties are needed. In the present work, twenty yeasts (Saccharomyces and non-Saccharomyces) with potential probiotic characteristics, selected in previous works, were evaluated. The following assays were realized: adhesion to Caco-2/TC7 cells, prebiotic metabolisms, assimilation of cholesterol, enzymatic and antioxidant activity, and antifungal resistance. In addition, the effect of ultrasonic treatment was evaluated for attenuating the cultures before their possible incorporation into a food or supplement. In all of the cases, the unique commercial probiotic yeast (S. boulardii CNM I-745) was used as positive control. Results show different capabilities depending on the property studied. In general, no Saccharomyces yeasts were better in the adhesion to Caco cells, prebiotic metabolism, and presented higher variability of enzymatic activities. The ones related to cholesterol assimilation and antioxidant capability did not show a marked trend, and with respect to the attenuation process, the Saccharomyces yeasts were more resistant. For selecting the potential probiotic yeasts with better balance among all characteristics, a principal component analysis (PCA) was carried out. The most promising yeasts for use as health-promoting probiotics are Hanseniaspora osmophila 1056 and 1094, Lachancea thermotolerans 1039, and S. cerevisiae 3 and 146.

Potential probiotic and food protection role of wild yeasts isolated from pistachio fruits (Pistacia vera).

BACKGROUND: The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties. RESULTS: Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of < 22 h. Additionally, some strains showed better kinetic parameters than Saccharomyces boulardii (positive control). Complementary tests were done to determine their auto-aggregation capacity, cell surface hydrophobicity, behaviour in a sequential simulated digestion, biofilm formation capability and carbon source assimilation. Finally, 67% and 13% of the studied yeasts showed biocontrol and antioxidant activities, respectively. CONCLUSIONS: Diutina rugosa 14 followed by Diutina rugosa 8 were the best wild yeast from Pistacia vera as potential probiotic and in carbon source utilization. However, Hanseniaspora guilliermondii 6 and Aureobasidium proteae 5 could be used to improve food or feed product preservation because of their notable biocontrol and antioxidant capabilities. © 2020 Society of Chemical Industry.

Saccharomyces cerevisiae and Hanseniaspora osmophila strains as yeast active cultures for potential probiotic applications.

This work allowed the evaluation of the gastrointestinal resistance of five yeasts (Saccharomyces and non-Saccharomyces) in order to assess some biotechnological characteristics linked to the potential probiotics, using a dynamic gastrointestinal simulator (simgi®). The best results obtained were for strains Saccharomyces cerevisiae 3 and Hanseniaspora osmophila 1056. Having optimised the method, the yeasts were subsequently lyophilised, and the one that showed the least loss of viability, S. cerevisiae 3, was used in a freeze-dried form to obtain a new functional food. On the other hand, some characteristics of the product were compared with those of probiotic supplements and other commercial probiotic foods. The obtained functional product showed better parameters than the rest of the samples containing yeasts which, together with the great acceptance shown after the consumer tests, means that it can be presented as a possible commercial functional product.

Disposable amperometric immunosensor for Saccharomyces cerevisiae based on carboxylated graphene oxide-modified electrodes.

A sensitive and disposable amperometric immunosensor for Saccharomyces cerevisiae was constructed by using carbon screen-printed electrodes modified with propionic acid-functionalized graphene oxide as transduction element. The affinity-based biosensing interface was assembled by covalent immobilization of a specific polyclonal antibody on the carboxylate-enriched electrode surface via a water-soluble carbodiimide/N-hydroxysuccinimide coupling approach. A concanavalin A-peroxidase conjugate was further used as signaling element. The immunosensor allowed the amperometric detection of the yeast in buffer solution and white wine samples in the range of 10-107 CFU/mL. This electroanalytical device also exhibited low detection limit and high selectivity, reproducibility, and storage stability. The immunosensor was successfully validated in spiked white wine samples.

Selection of potential non-Sacharomyces probiotic yeasts from food origin by a step-by-step approach.

Due to healthcare is increasing in nowadays, the use of the commercial probiotics is in progress and each day they are more demanded. The challenge of this study is to identify yeast species for using as probiotic organisms. Thus, the research applied a step-by-step approach, to study the probiotic potential of non-Saccharomyces yeast strains. The 215 yeasts were isolated from different environments such as wineries, oil mills, brines cheeses, fermented vegetables and distilleries in previous works and were identified to strain level by RAPD-PCR technique resulting 108 different strains. A general screening was carried out to know the probiotic capability of the yeasts, following the next steps: study of the ability to resist and grow of the yeasts when they exposed to simulated in vitro digestion conditions and influence of time, temperature, pH and the presence of enzymes on the kinetic growth parameters (lag phase (λ), generation time (G), maximum OD (ODmax) and the specific growth rate constant (µmax)). The results made possible the selection of the 23% of the strains and they were assayed for knowing their capability of self-aggregation and hydrophobicity. Biofilm formation capacity and viability after simulated sequential salivary-gastric-intestinal digestion were then studied for the 10 best strains. Statistical analyses were applied in each step to make the selection. The final results showed that two yeasts, H. osmophila and P. kudriavzevii, were the most promising strains.

Yeast Identification During Fermentation of Turkish Gemlik Olives.

Naturally fermented black table olives of the Gemlik variety are one of the most consumed fermented products in Turkey. The objective of this work was to identify yeast strains isolated during their natural fermentation by using Restriction Fragments Lengths Polymorphism-Polimerase Chain Reaction (RFLP-PCR) and DNA sequencing methods. The study also focused on determining the effect of regional differences on yeast microflora of naturally fermented Gemlik olives. A total of 47 yeast strains belonging to 12 different species which had been previously isolated from the natural brine of Akhisar and Iznik-Gemlik cv. olives were characterized by molecular methods. Forty-two of the tested strains could be identified by RFLP-PCR to species level. These yeast species were determined as Candida mycetangi, Candida hellenica, Candida membranaefaciens, Candida famata, Candida pelliculosa, Saccharomyces cerevisiae, and Zygosaccharomyces mrakii. Five strains were identified by DNA sequencing. These strains belonged to three different species: Aureobasidium pullulans, Kloeckera apiculate, and Cryptococcus saitoi. The most frequent species were C. famata and C. pelliculosa in both regions. PRACTICAL APPLICATION: This work studies the yeasts from Turkish table olives which could prove to be of importance to the food industry in that area. On the other hand, it compares identification by molecular and classical biochemical methods and offers an idea about the differences between the ecosystems of Gemlik olives in the Akhisar (AO) and Iznik (IO) regions. The study could be useful in characterizing a very important product and, in this way, could help to promote its marketing.

Disposable electrochemical immunosensor for Brettanomyces bruxellensis based on nanogold-reduced graphene oxide hybrid nanomaterial.

The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10-106 CFU/mL and 102-106 CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability. Graphical abstract A novel disposable electrochemical immunosensor for the detection of the red wine spoilage yeast B. bruxellensis.

Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste.

The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. Castilla-La Mancha produces around three million tons of waste coming from olive oil and wine industries, both of which have a pivotal role in the economy of this region. For this reason, this study reports on the exploitation of grape skins and olive pomaces for the production of lignocellulosic enzymes, which are able to deconstruct the agroindustrial waste and, therefore, reuse them in future industrial processes. To this end, solid-state fermentation was carried out using two local fungal strains (Aspergillus niger-113 N and Aspergillus fumigatus-3). In some trials, a wheat supplementation with a 1:1 ratio was used to improve the growth conditions, and the particle size of the substrates was altered through milling. Separate fermentations were run and collected after 2, 4, 6, 8, 10 and 15 days to monitor enzymatic activity (xylanase, cellulase, ß-glucosidase, pectinase). The highest values were recorded after 10 and 15 days of fermentation. The use of A. niger on unmilled grape skin yielded the best outcomes (47.05 U xylanase/g by-product). The multi-enzymatic extracts obtained were purified, freeze dried, and immobilized on chitosan by adsorption to assess the possible advantages provided by the different techniques.

Fungi isolated from olive ecosystems and screening of their potential biotechnological use.

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for ß-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for ß-glucosidase, CMCase and lipase); Rhizopus oryzae (for ß-glucosidase and lipase); Rhizomucor variabilis (for ß-glucosidase, CMCase and polygalacturonase); Mucor fragilis (ß-glucosidase, CMCase and lipase); Galactomyces geotrichum (for ß-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, ß-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and ß-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.

Extracellular beta-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology.

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Yeast biodiversity from oleic ecosystems: study of their biotechnological properties.

The aim of this study was to know the yeast biodiversity from fresh olive (Olea europaea L.) fruits, olive paste (crush olives) and olive pomace (solid waste) from Arbequina and Cornicabra varieties. Yeasts were isolated from fruits randomly harvested at various olive groves in the region of Castilla La Mancha (Spain). Olive paste and pomace, a byproduct of the processing of this raw material, were also collected in sterile flasks from different oil mills. Molecular identification methodology used included comparison of polymerase chain reaction (PCR) amplicons of their 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2 followed by restriction pattern analysis (RFLP). For some species, sequence analysis of the 5.8S rDNA gene was necessary. The results were compared to sequences held in public databases (BLAST). These techniques allowed to identify fourteen different species of yeasts, belonging to seven different genera (Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida, Torulaspora) from the 108 yeast isolates. Species diversity was thus considerable: Pichia caribbica, Zygosaccharomyces fermentati (Lachancea fermentati) and Pichia holstii (Nakazawaea holstii) were the most commonly isolated species, followed by Pichia mississippiensis, Lachancea sp., Kluyveromyces thermotolerans and Saccharomyces rosinii. The biotechnological properties of these isolates, was also studied. For this purpose, the activity of various enzymes (beta-glucosidase, beta-glucanase, carboxymethylcellulase, polygalacturonase, peroxidase and lipase) was evaluated. It was important that none of species showed lipase activity, a few had cellulase and polygalacturonase activities and the majority of them presented beta-glucanase, beta-glucosidase and peroxidase activities.

Relationship between Debaryomyces pseudopolymorphus enzymatic extracts and release of terpenes in wine.

As a result of the interest that exists in the liberation of aromas in young wines, we obtained some different enzymatic extracts (purified extract, P; lyophilized purified extract, LP; immobilized purified extract, IP; and immobilized lyophilized purified extract, ILP) with beta-glucosidase activity from Debaryomyces pseudopolymorphus, which excreted the enzyme in the growth medium. The extracts were added to natural glycosides isolated from different grape varieties. The results were compared with the effect of seven commercial enzyme preparations (CEP), obtained from molds used in wine making. It was shown that some yeast extracts had effects similar to those of the CEP, and the next step was to use them on wine samples elaborated in the laboratory. The effect was studied at 9 and 16 days of contact, quantifying both the precursors that were retained and the liberated terpenes. The results were compared with a control wine (without any extract) and with wine treated with a commercial enzyme preparation specially indicated for the liberation of aromas. It was observed that the enzymatic extracts from Db. pseudopolymorphus hydrolyzed the precursors in wine and that they could compete with the commercial preparations since the liberation was produced in even less time.