Identification and biotechnological characterisation of yeast microbiota involved in spontaneous fermented wholegrain sourdoughs.

BACKGROUND: New strategies in the cereal-based industry has brought about the elaboration of new sourdoughs with better microbial stability and safety as well as nutritional value such as those based on wholegrain flours. This has led to an increasing interest in the selection of adapted yeasts for using them as new starters. Therefore, this study aimed to isolate, identify, and characterise diverse yeast strains from wholegrain spontaneous sourdoughs. RESULTS: Three wholegrain sourdoughs (wheat, rye, and oat) were fermented and monitored for 96 h. Minimum pH values ranged from 3.1 to 3.5 while maximum yeast counts were reached at 72 h. A total of 76 yeast isolates were identified by polymerase chain reaction random amplification of polymorphic DNA (PCR-RAPD) and catalogued in six different species by sequencing the internal transcribed spacer (ITS) region. The major species were Candida glabrata, Saccharomyces cerevisiae, Kazachstania unispora, and Wickerhamomyces anomalus. The studied kinetic parameters of the growth curves (λ, G, ODmax , and µmax ) and the fermentation capacity allowed to ascertain that 12 and 5 strains, respectively, were better than baker's yeast control. The fibre assimilation ability (cellulose, xylose, and ß-glucan) was observed in the 27% of the strains and only four strains showed phytase activity. CONCLUSIONS: The yeast population in the three wholegrain sourdoughs were variable along the fermentation time. Genetic identification showed that strains and species presented a different trend for each sourdough although common species were determined (e.g., W. anomalus). Candida glabrata (4T1) and Saccharomyces cerevisiae (3A6) showed, respectively, better kinetics and impedance results than the positive control, while W. anomalus (C4) was notorious in fibre assimilation and phytase degradation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development and Innovation in Cooked Ham Produced in Spain.

The production of cooked ham has been gaining popularity in recent years in Spain. In general, the production process carried out by the companies remains traditional, and different production methods are therefore being sought to innovate and improve the quality of the product. This is either through pig crossbreeding, varying additives and ingredients, improving some stages of the production process, or providing nutritional and health claims that are useful to guiding the purchasing decision of consumers. Obviously, this series of changes must be subject to Spanish and European regulations in order to be marketed inside and outside the country.

Extracellular microbial proteases with specificity for plant proteins in food fermentation.

Plant-based food products are generating a growing interest as part of the ongoing transition to a primarily plant-based diet, which makes demands to the quality, functionality, and health properties of plant proteins. Microbes used for traditional food fermentations such as lactic acid bacteria (LAB) and fungi (yeasts and molds) carry out enzymatic changes on their protein substrates by which technological and sensorial characteristics can be improved. The literature on extracellular proteases targeting plant proteins, on the other hand, is scattered with only a narrow representation of plants even for traditionally plant-based products. Therefore, this review aims to explore the current state of knowledge regarding the application potential of microbial extracellular proteases targeting plant proteins, with a focus on traditional applied food microbes. Plant proteins are targeted by proteolytic microbes of both animal and plant origins, and their proteases show a wide range of activities. Extracellular microbial proteases can hydrolyze specific protein-based allergens and even reduce the toxicity of plant proteins. Additionally, microbial assisted proteolysis can improve plant protein digestibility by increasing availability of peptides and amino acids. This catabolic process will change the organoleptic characteristics of fermented plant proteins, and the release of bioactive peptides can provide additional functionalities to the plant matrix. The proteolytic activity is determined by the microbial strain, and it can be quite substrate selective, which is why proteases may be overlooked by the prevalent use of casein as substrate in proteolytic screenings. The synergetic effects of LAB and fungal species consortia can facilitate and steer plant protein hydrolysis by which co-fermentation may increase or change the properties of plant protein hydrolysates. Microbes do not necessarily require extracellular proteases because endogenous proteases in a plant-matrix may meet the microbial amino acid requirements. However, extracellular proteases have the potential to provide central properties to diverse food-matrixes by which the full proteolytic potential of food microbes needs to be explored in order to facilitate the development of high-quality plant-based food products.

Graphene quantum dots an efficient nanomaterial for enhancing the photostability of trans-resveratrol in food samples.

Given the health-promoting properties associated with trans-resveratrol consumption and knowing its photochemical instability, as it rapidly isomerizes to the less biologically active conformer, the photoprotective role of a carbon-based nanomaterial was investigated. The resveratrol adsorption on graphene quantum dots (GQDs), synthesized from uric acid, produces a considerable inhibition in the trans- to cis-resveratrol conversion process under light-induced radiation, conferring photostability to the bioactive. The influence of different parameters affecting the adsorption efficiency and loading capacity of resveratrol on GQDs was explored. Several characterization techniques confirmed this interaction, even proving to be non-toxic at the concentration at which the maximum inhibition of isomerization occurs. This adsorption also implies an increase in the antioxidant capacity of the polyphenol. The photoprotective effect was evaluated in food samples, resulting in a considerable slowing down of isomerization. This fact confirms the potential of GQDs to be an effective vehicle of trans-resveratrol to supplement food systems.

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.

Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B1 elimination ability.

The use of microorganisms for Aflatoxin B1 elimination has been studied as a new alternative tool and it is known that cell wall carried out a critical role. For that reason, cell wall and soluble intracellular fraction of eight yeasts with AFB1 detoxification capability were analysed. The quantitative and qualitative comparative label-free proteomic allowed the identification of diverse common constituent proteins, which revealed that putative cell wall proteins entailed less than 10% of the total proteome. It was possible to characterize different enzymes linked to cell wall polysaccharides biosynthesis as well as other proteins related with the cell wall organization and regulation. Additionally, the concentration of the principal polysaccharides was determined which permitted us to observe that ß-glucans concentration was higher than mannans in most of the samples. In order to better understand the biosorption role of the cell wall against the AFB1 , an antimycotic (Caspofungin) was used to damage the cell wall structure. This assay allowed the observation of an effect on the normal growth of those yeasts with damaged cell walls that were exposed to AFB1 . This effect was not observed in yeast with intact cell walls, which may reveal a protective role of this structure against mycotoxins.

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.

Autochthonous Yeast from Pork and Game Meat Fermented Sausages for Application in Meat Protection and Aroma Developing.

The wild yeast community was studied in fermented sausages from pork and game meat (deer and wild boar) during the maturation process from different curing rooms. Although the biotechnological importance of yeasts in the maturation process of pork sausages is known, there is a lack of information for sausage maturation involving game meat. A total of 123 yeasts were isolated and, by amplifying and sequencing of the ITS region, were classified in 14 species. Debaryomyces hansenii, Kazachstania servazzii, and Wickerhamomyces anomalus were isolated in both pork and game samples. The PCR-RAPD technique differentiated between 26 and 18 strains from pork and game meat sausages, respectively. The physicochemical parameters and their relationship with the yeast community were also studied. The antioxidant and anti-lipid peroxidation capability were analyzed and the 70% and 50% of the tested strains showed these abilities, respectively. Moreover, the biocontrol capability against mycotoxigenic molds was found in 19 strains, but better results were observed in game meat yeasts. On the other hand, almost 30% of strains produce a pleasant olfactory aroma, and volatile compounds associated with the yeast pathway metabolic during the maturation process have been characterized such as esters, aldehydes, fusel alcohols, etc. This study has allowed a better understanding of the biodiversity of this type of food, as well as selecting potential yeast strains for their future use as starters.

Differential distribution and proteomic response of Saccharomyces cerevisiae and non-model yeast species to zinc.

Zinc surplus in yeast cells has been previously investigated thanks to transcriptomic studies by using traditionally Saccharomyces cerevisiae as a model. However, proteome response under zinc-replete conditions needs to be further studied in yeast. For that reason, eight yeast strains from seven different species were inoculated in zinc-depleted and zinc-replete media. The quantitative and qualitative comparative label-free proteomic analysis enabled the identification of between 2000 and 3000 proteins from each strain, and changes to the proteome ranged from 2.5% to 43.7% of identified proteins. Functional analysis (Blast2Go) has allowed the characterization of differentially abundant proteins. Common zinc-responsive proteins have been detected for the eight strains such as oxidoreductases and transferases (increased in abundance) although more of the changes detected were not shared by all the strains tested. Zinc distribution under replete conditions has been analysed in cell wall fractions, and cytoplasm plus organelles (intracellular fraction), with the latter identified to be the main zinc reservoir. Additionally, the energy dispersive spectroscopy coupled to the scanning electron microscopy technique has permitted the visualization of zinc in the whole cell. Proteomic analysis revealed that while there were some shared responses, the non-model yeast species also showed distinct proteomic profiles in zinc-replete conditions, compared to S. cerevisiae, revealing new zinc-responsive proteins in yeast.

Study of the bioremediatory capacity of wild yeasts.

Microbial detoxification has been proposed as a new alternative for removing toxins and pollutants. In this study, the biodetoxification activities of yeasts against aflatoxin B1 and zinc were evaluated by HPLC and voltammetric techniques. The strains with the best activity were also subjected to complementary assays, namely biocontrol capability and heavy-metal resistance. The results indicate that the detoxification capability is toxin- and strain-dependent and is not directly related to cell growth. Therefore, we can assume that there are some other mechanisms involved in the process, which must be studied in the future. Only 33 of the 213 strains studied were capable of removing over 50% of aflatoxin B1, Rhodotrorula mucilaginosa being the best-performing species detected. As for zinc, there were 39 strains that eliminated over 50% of the heavy metal, with Diutina rugosa showing the best results. Complementary experiments were carried out on the strains with the best detoxification activity. Biocontrol tests against mycotoxigenic moulds showed that almost 50% of strains had an inhibitory effect on growth. Additionally, 53% of the strains grew in the presence of 100 mg/L of zinc. It has been proven that yeasts can be useful tools for biodetoxification, although further experiments must be carried out in order to ascertain the mechanisms involved.

Dominant Yeast Community in Raw Sheep's Milk and Potential Transfers of Yeast Species in Relation to Farming Practices.

Yeasts are always present in any type of cheese, as well as in the factories where it is produced. However, the role of the yeast community in the cheese making process, as well as the routes of contamination used by yeast species to contaminate milk from the dairy farm environment, are not well known. The objectives of this study were to broaden the knowledge of the dominant yeast community in Manchega sheep's milk and to assess the contamination routes of the yeast species depending on the farm practices. Milk, teat surface (collected from ten ewes per farm), feed, and air (collected in milking parlours and livestock housing) samples were collected from 12 typical farms in Castilla-La Mancha, Spain with differences in farming practices, and the yeast species were identified using DNA sequencing methods. To evaluate whether certain farming practices have an effect on the distribution of species of yeast in the milk samples, a mixed model was used. The results showed that most of the dominant yeast species (mainly belonging to the genus Candida) found in milk were also found in the other samples, indicating a microbial transfer from the farm environment to the milk. Furthermore, the statistical model showed that factors influencing yeast counts in milk were the presence of yeasts in the milking parlour, the use of silage, and the frequency of acid treatment for cleaning the milking machines. In conclusion, milk contamination from the yeast species present in the dairy farm environment is related to certain farming practices such as the use of silage and the daily use of acid in the cleaning of the milking machines, which favours the presence of desirable microbiota in milk.