Formación y control de comunidades polimicrobianas sobre la epidermis de las aceitunas de mesa: bases para un nuevo alimento multifuncional

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biotecnología de Productos AlimentariosClave Programa: DBICódigo Línea: 10La presente Tesis doctoral está centrada en el estudio de la biodiversidad de BAL que se encuentran exclusivamente en los biofilms de aceitunas de mesa, para la selección como cultivos iniciadores de las cepas que presentan las mejores características tecnológicas y probióticas (cultivos multifuncionales) y su validación y posterior respuesta en fermentaciones reales de aceitunas de mesa, determinando su perfil de producción de volátiles, como marcadores de sus características sensoriales y presencia durante la fermentación. Para este fin, se ha seguido un enfoque multidisciplinar, utilizando técnicas de microbiología clásica, microbiología predictiva, microscopia electrónica de barrido, biología molecular, metagenómica, análisis bioinformático (Bionumerix y QIIME), química analítica (HLPC y GC-MS), análisis de datos composicional, análisis multivariante y finalmente estudios en modelos animales con el fin de validar el potencial probiótico de los microorganismos seleccionados.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Main challenges expected from the impact of climate change on microbial biodiversity of table olives: Current status and trends

Climate change is a global emergency that is affecting agriculture in Mediterranean countries, notably the production and the characteristics of the final products. This is the case of olive cultivars, a source of olive oil and table olives. Table olives are the most important fermented vegetables in the Mediterranean area, whose world production exceeds 3 million tons/year. Lactic acid bacteria and yeast are the main microorganisms responsible for the fermentation of this product. The microbial diversity and population dynamics during the fermentation process are influenced by several factors, such as the content of sugars and phenols, all of which together influence the quality and safety of the table olives. The composition of fruits is in turn influenced by environmental conditions, such as rainfall, temperature, radiation, and the concentration of minerals in the soil, among others. In this review, we discuss the effect of climate change on the microbial diversity of table olives, with special emphasis on Spanish and Portuguese cultivars. The alterations expected to occur in climate change scenario(s) include changes in the microbial populations, their succession, diversity, and growth kinetics, which may impact the safety and quality of the table olives. Mitigation and adaptation measures are proposed to safeguard the authenticity and sensorial features of this valuable fermented food while ensuring food safety requirements.TOBE project (RTI2018-100883-B-I00, MCIU/AEI/FEDER,UE; PAIDI2020-00162info:eu-repo/semantics/publishedVersio

Lactic Acid Bacteria and Yeast Inocula Modulate the Volatile Profile of Spanish-Style Green Table Olive Fermentations

In this work, Manzanilla Spanish-style green table olive fermentations were inoculated with Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, the yeast Wickerhanomyces anomalus Y12 and a mixed culture of all them. After fermentation (65 days), their volatile profiles in brines were determined by gas chromatography-mass spectrometry analysis. A total of 131 volatile compounds were found, but only 71 showed statistical differences between at least, two fermentation processes. The major chemical groups were alcohols (32), ketones (14), aldehydes (nine), and volatile phenols (nine). Results showed that inoculation with Lactobacillus strains, especially L. pentosus Lp13, reduced the formation of volatile compounds. On the contrary, inoculation with W. anomalus Y12 increased their concentrations with respect to the spontaneous process, mainly of 1-butanol, 2-phenylethyl acetate, ethanol, and 2-methyl-1-butanol. Furthermore, biplot and biclustering analyses segregated fermentations inoculated with Lp13 and Y12 from the rest of the processes. The use of sequential lactic acid bacteria and yeasts inocula, or their mixture, in Spanish-style green table olive fermentation could be advisable practice for producing differentiated and high-quality products with improved aromatic profile.Gobierno de España-OliFilm-AGL-2013-48300-

Tracking microbial diversity and hygienic-sanitary status during processing of farmed rainbow trout (Oncorhynchus mykiss)

Aquaculture is becoming a strategic sector for many national economies to supply the increasing demand for fish from consumers. Fish culture conditions and processing operations can lead to an increase in microbial contamination of farmed fish that may shorten the shelf-life of fish products and byproducts, and ready-to-eat fishery products. The objective of this study was to evaluate the hygienic-sanitary status of water, environment, and processing of fresh-farmed rainbow trout (Oncorhynchus mykiss) fillets produced in a local fish farm in Andalusia, Spain. To achieve this, a longitudinal study was carried out by collecting environmental (air and food-contact surfaces), water from fish ponds, and rainbow trout samples. Thereby, seven sampling visits were performed between February 2021 and July 2022, where foodborne pathogens and spoilage microorganisms, together with physicochemical parameters, were analysed in the collected samples. Further, microbial identification of microbiota was achieved through a culture-dependent technique using blast analysis of 16S RNA gene sequencing. The results showed that Listeria monocytogenes and Salmonella were not detected in the analysed samples. Regarding the hygienic-sanitary status of the fish farm, the slaughtering bath, the eviscerating machine and the outlet water from fish ponds presented the highest counts of coliforms, Enterobacteriaceae, and Aerobic Mesophilic Bacteria. Staphylococcus aureus and sulphite-reducing Clostridium were identified in the conveyor belts, fish flesh, and viscera. The 16S RNA identification confirmed the presence of viable spoilage bacteria such as Citrobacter gillenii, Macrococcus caseolyticus, Hafnia paralvei, Lactococcus lactis, Lactococcus cremoris, Klebsiella, Escherichia coli, Morganella morganii, and Shewanella. Three of these genera (Citrobacter, Hafnia, and Pseudomonas) were present in all types of samples analysed. The results evidenced potential transmission of microbial contamination from contaminated packaging belts and boxes, evisceration and filleting machines to flesh and viscera samples, thus the establishment of control measures should be implemented in fish farm facilities to extend the shelf-life of farmed fishery products

Understanding the transcriptomic response of Lactiplantibacillus pentosus LPG1 during Spanish-style green table olive fermentations

Lactiplantibacillus pentosus (Lbp. pentosus) is a species of lactic acid bacteria with a great relevance during the table olive fermentation process, with ability to form non-pathogenic biofilms on olive epidermis. The objective of this work is to deepen into the genetic mechanisms of adaptation of Lpb. pentosus LPG1 during Spanish-style green table olive fermentations, as well as to obtain a better understanding of the mechanisms of adherence of this species to the fruit surface. For this purpose, we have carried out a transcriptomic analysis of the differential gene expression of this bacterium during 60 days of fermentation in both brine and biofilms ecosystems. In brines, it was noticed that a total of 235 genes from Lpb. pentosus LPG1 were differentially expressed during course of fermentation and grouped into 9 clusters according to time-course analysis. Transport and metabolism of carbohydrates and amino acids, energy production, lactic acid and exopolysaccharide synthesis genes increased their expression in the planktonic cells during course of fermentation. On the other hand, expression of genes associated to stress response, bacteriocin synthesis and membrane protein decreased. A total of 127 genes showed significant differential expression between Lpb. pentosus LPG1 planktonic (brine) and sessile (biofilms) cells at the end of fermentation process (60 days). Among the 64 upregulated genes in biofilms, we found genes involved in adhesion (strA), exopolysaccharide production (ywqD, ywqE, and wbnH), cell shape and elongation (MreB), and well as prophage excision. Deeping into the genetic bases of beneficial biofilm formation by Lpb. pentosus strains with probiotic potential will help to turn this fermented vegetable into a carrier of beneficial microorganisms to the final consumers

RT-PCR-DGGE analysis to elucidate the dominant bacterial species of industrial Spanish-style green table olive fermentations

This paper describes the dominant bacterial species metabolically active through the industrial production of Spanish-style Manzanilla and Gordal olives. For this purpose, samples (brines and fruits) obtained at 0, 15, and 90 fermentation days were analyzed by a culture-independent approach to determine viable cells by reverse transcription of RNA and further PCR-DGGE analysis, detecting at least 7 different species. Vibrio vulnificus, Lactobacillus plantarum group, and Lactobacillus parafarraginis were present in samples from both cultivars; Lactobacillus sanfranciscensis and Halolactobacillus halophilus were detected only in Gordal samples, while Staphylococcus sp. was exclusively found at the onset of Manzanilla fermentations. Physicochemical data showed a typical fermentation profile while scanning electron microscopy confirmed the in situ biofilm formation on the olive epidermis. Different Bacillus, Staphylococcus, and Enterococcus species, not detected during the fermentation process, were also found in the solid marine salt used by the industry for preparation of brines. Elucidation of these non-lactic acid bacteria species role during fermentation is then an appealingly challenge, particularly regarding safety issues.The research has received funding from the Spanish Government (Project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es). AB-C and FA-L wish to express thanks to the Spanish Government for their pre-doctoral fellowship and postdoctoral research contract (Ramón y Cajal), respectively.Peer Reviewe

Microbiological and Physicochemical Changes in Natural Green Heat-Shocked Aloreña de Málaga Table Olives

11 Páginas; 5 Tablas; 6 FigurasPreserving the highly appreciated natural freshness of Aloreña de Málaga table olives and preventing their progressive darkening during processing is a major challenge. In this work, heat-shocked (60°C, 5 min) fruits were processed according to the three denominations referred to in the Protected Designation of Origen (cured, fresh green, and traditional) and their characteristics compared with those that followed the habitual industrial process (controls). The results revealed that the effects of the heat treatment on the evolution of pH, titratable acidity, salt, sugar, organic acid, ethanol content, texture, and color of fruits as well as on microbial populations (yeasts and lactic acid bacteria) were slight in the case of the fresh green and cured presentations. However, the differences between heat-shocked and its control were remarkable in the traditional process. Notably, the heat treatment favored lactic acid fermentation, retention of the green appearance of the fruits, stability during packaging, and led to the highest sensory evaluation. The metagenomic analysis carried out at the end of the fermentation revealed the presence in all samples of three genera (Lactobacillus, Pediococcus, and Celerinatantimonas) which encompassed most of the sequences. The number of Lactobacillus sequences was statistically higher (p ≥ 0.05) in the case of traditional heat-shocked fruits than in its control.The research leading to these results has received funding from Junta de Andalucía Government through the PrediAlo project (AGR-7755: www.predialo.science.com.es) and FEDER European funds. FA-L wishes to express thanks to the Spanish government for his RyC postdoctoral research contract while VR-G would like to thank ceiA3, Spanish Government, Bank of Santander, IG-CSIC and ‘Aloreña de Málaga’ Olive Manufacturing Association for her predoctoral fellowship. Authors express their gratitude to Dr. Borja Sánchez (IPLA-CSIC) for his invaluable help in the metagenomic analysis.Peer reviewe

Biodiversity and Multifunctional Features of Lactic Acid Bacteria Isolated From Table Olive Biofilms

In the present study, a total of 554 lactic acid bacteria (LAB) isolates were obtained from the olive surface of Manzanilla, Gordal, and Aloreña cultivars processed as green Spanish-style or directly brined (natural) olives. The isolates obtained from industrial processes were genotyped by rep-PCR with primer GTG5, collecting a total of 79 different genotypes. The α-biodiversity indexes showed that the LAB diversity was higher in the biofilms on the fruits which followed the Spanish-style process than in those just brined. Sixteen genotypes had a frequency higher >1% and were identified, by multiplex PCR recA gene and 16S gene sequencing, as belonging to Lactobacillus pentosus (n = 13) and Lactobacillus plantarum (n = 3) species. A multivariate analysis based on a dataset with 89,744 cells, including technological (resistance to salt and pH, production of lactic acid, auto and co-aggregation with yeast species, β-glucosidase and esterase activities), and potential probiotic characteristics (survival to gastric and pancreatic digestions, resistance to antibiotics, inhibition of pathogens, presence of bsh genes, cholesterol removal, hemolytic, α-glucosidase, β-galactosidase, and phytase activities) showed that the 16 genotypes could be grouped into 3 great phenotypes. Thus, the genotype biodiversity in table olive biofilms was limited but, at phenotype level, it was even lower since L. pentosus predominated clearly (80.15% isolates). L. pentosus Lp13 was the genotype with the most promising characteristics for its use as a multifunctional starter, with this strain being and ubiquitous microorganism present in both natural and lye-treated olive fermentations

Reduction of the Bitter Taste in Packaged Natural Black Manzanilla Olives by Zinc Chloride

The work assays the use of various concentrations of ZnCl2 (0.0–0.1%, w/v) in packaged natural black Manzanilla table olives. The transformations were followed for 4 months. The presence of Zn modified the leaching of total sugars (sucrose, glucose, fructose, and mannitol) into the brine, which decreased as the ZnCl2 content increased. Over the study, sucrose and glucose were exhausted while fructose, although consumed, left some final residues and the use of mannitol was limited. Titratable acidity was always gradually formed causing the subsequent pH decrease, which stabilized at ≈3.5. Acetic and mainly lactic acid were also formed during the assay, reaching the highest level of lactic acid in the 0.050% ZnCl2 treatment, followed by the Control. The acids were formed by lactic acid bacteria (LAB) (Lactobacillus pentosus, 39%, and Lactobacillus plantarum, 61%). However, the most outstanding Zn effect was found on the olive sensory characteristics: its presence markedly reduced the bitter notes, increased the overall appreciation, and the treatment containing 0.075% ZnCl2 had the highest scores in hardness, crunchiness, and overall appreciation. Therefore, the addition of ZnCl2 into packaged natural table olives may lead to healthy products with desirable sensory characteristics which, in turn, could promote consumption

Acceleration of the DNA methylation clock among lynch syndrome‑associated mutation carriers

The research leading to these results has received funding from "la Caixa" Foundation (Ref: CAIXA2017/1) for library preparation, sequencing, and employment of research personnel, from The Fundacion Progreso y Salud, Junta de Andalucia, Spain and from DPI2017-84439-R of MINECO, Madrid and FEDER for sequencing and employment of research personnel. Finally, grant ref. A-BIO-470-UGR20 from University of Granada and FEDER has funded article processing charges (APC) and sample processing expenses.Background: DNA methylation (DNAm) age metrics have been widely accepted as an epigenetic biomarker for biological aging and disease. The purpose of this study is to assess whether or not individuals carrying Lynch Syndromeassociated mutations are affected in their rate of biological aging, as measured by the epigenetic clock. Methods: Genome-wide bisulfite DNA sequencing data were generated using DNA from CD4 + T-cells obtained from peripheral blood using 27 patient samples from Lynch syndrome families. Horvath’s DNAm age model based on penalized linear regression was applied to estimate DNAm age from patient samples with distinct clinical and genetic characteristics to investigate cancer mutation-related aging effects. Results: Both Lynch mutation carriers and controls exhibited high variability in their estimated DNAm age, but regression analysis showed steeper slope for the Lynch mutation carriers. Remarkably, six Lynch Syndrome-associated mutation carriers showed a strong correlation to the control group, and two sisters carrying Lynch Syndrome-associated mutations, with no significant difference in lifestyle and similar chronological age, were assigned very different DNAm age. Conclusions: Future studies will be required to explore, in larger patient populations, whether specific epigenetic age acceleration is predictive of time-to-cancer development, treatment response, and survival. Epigenetic clock DNAm metrics may be affected by the presence of cancer mutations in the germline, and thus show promise of potential clinical utility for stratified surveillance strategies based on the relative risk for imminent emergence of tumor lesions in otherwise healthy Lynch Syndrome-associated mutation carriers.La Caixa Foundation CAIXA2017/1Junta de AndaluciaSpanish Government DPI2017-84439-REuropean CommissionUniversity of Granada A-BIO-470-UGR2