Discovering the Influence of Microorganisms on Wine Color.

Flavor, composition and quality of wine are influenced by microorganisms present on the grapevine surface which are transferred to the must during vinification. The microbiota is highly variable with a prevalence of non-Saccharomyces yeasts, whereas Saccharomyces cerevisiae is present at low number. For wine production an essential step is the fermentation carried out by different starter cultures of S. cerevisiae alone or in mixed fermentation with non-Saccharomyces species that produce wines with significant differences in chemical composition. During vinification wine color can be influenced by yeasts interacting with anthocyanin. Yeasts can influence wine phenolic composition in different manners: direct interactions-cell wall adsorption or enzyme activities-and/or indirectly-production of primary and secondary metabolites and fermentation products. Some of these characteristics are heritable trait in yeast and/or can be strain dependent. For this reason, the stability, aroma, and color of wines depend on strain/strains used during must fermentation. Saccharomyces cerevisiae or non-Saccharomyces can produce metabolites reacting with anthocyanins and favor the formation of vitisin A and B type pyranoanthocyanins, contributing to color stability. In addition, yeasts affect the intensity and tonality of wine color by the action of ß-glycosidase on anthocyanins or anthocyanidase enzymes or by the pigments adsorption on the yeast cell wall. These activities are strain dependent and are characterized by a great inter-species variability. Therefore, they should be considered a target for yeast strain selection and considered during the development of tailored mixed fermentations to improve wine production. In addition, some lactic acid bacteria seem to influence the color of red wines affecting anthocyanins' profile. In fact, the increase of the pH or the ability to degrade pyruvic acid and acetaldehyde, as well as anthocyanin adsorption by bacterial cells are responsible for color loss during malolactic fermentation. Lactic acid bacteria show different adsorption capacity probably because of the variable composition of the cell walls. The aim of this review is to offer a critical overview of the roles played by wine microorganisms in the definition of intensity and tonality of wines' color.

Promoting Candida zemplinina adhesion on oak chips: A strategy to enhance esters and glycerol content of Montepulciano d'Abruzzo organic wines.

In this study cell surface hydrophobicity and the ability to adhere on abiotic surfaces (polystyrene plates, stainless steel and oak chips) of 10 Candida zemplinina strains were assessed. Moreover, the impact of C. zemplinina cells adhered on oak surface on fermentation kinetics and volatile profile of Montepulciano d'Abruzzo organic wines was evaluated. All strains showed a hydrophobic nature with a certain affinity for the apolar solvents tested (hexadecane and decane). In agreement with this data strains were able to adhere on abiotic surfaces in a strain dependent way. On polystyrene plates all strains mainly grew as planktonic cells. On stainless steel surfaces sessile cells ranged from 2.6 Log CFU/mL (SB2) to 4.1 Log CFU/mL (SB8), while on oak chips were about 2 log higher ranging from 4.3 Log CFU/mL (SB8) to 6.1 Log CFU/mL (SB10). Candida zemplinina sessile state resulted in an increase of glycerol (from 6.98 g/L to 11.92 g/L) and esters amount (from 55.47 g/L to 91.5 mg/L), and a reduction of ethanol content (from 14.13% to 9.12% v/v). As for esters, methyl vanillate, ethyl isobutyrate, and ethyl isovalerate were present only when C. zemplinina was adhered on oak chips. This study revealed that changes of concentrations in esters and glycerol content reflected the fermentation bioactivity of yeast cells attached on oak chips. Surface-adhered behaviours should be considered in the improvement of strategies for the development of high-quality organic wines and eventually obtain novel wine styles.

Correlation between IRC7 gene expression and 4-mercapto-4-methylpentan-2-one production in Saccharomyces cerevisiae strains.

Volatile thiols are not present in must but are synthesized and released by wine yeasts during alcoholic fermentation. In this study, autochthonous and commercial Saccharomyces cerevisiae strains were characterized for the expression of the main genes involved in thiols metabolism and their production in wine. New primer sets were developed on the basis of the S288c genome to evaluate the expression of Cys3, Cys4, MET17 and IRC7 genes. Obtained data revealed the occurrence of some thiols, for example, 4-mercapto-4-methylpentan-2-one (4-MMP) and 3-mercaptohexan-1-ol (3-MH) in Pecorino white wine. All genes were upregulated, but only for IRC7 was found a correlation with 4-MMP release: strains with the highest production showed the highest transcription level. IRC7 gene could be proposed as target for the selection of S. cerevisiae strains to increase thiols content in wine.

Accumulation γ-Aminobutyric Acid and Biogenic Amines in a Traditional Raw Milk Ewe's Cheese.

The influence of calf (R1), kid (R2) and pig (R3) rennets on microbiota, biogenic amines (BAs) and γ-aminobutyric acid (GABA) accumulation in raw milk ewe's cheeses was evaluated. Cheeses were investigated at different ripening times for their microbial composition, free amino acids (FAAs), BAs and GABA content. Moreover, the expression of tyrosine (tdc) and histidine (hdc) decarboxylases genes was evaluated by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Microbial counts showed similar values in all samples. Pig rennet were cheeses were characterized by higher proteolysis and the highest values of BAs. The BAs detected were putrescine, cadaverine and tyramine, while histamine was absent. qRT-PCR confirmed this data, in fact hdc gene was not upregulated, while tdc gene expression increased over time in agreement with the increasing content of tyramine and the highest fold changes were detected in R3 cheeses. GABA showed the highest concentration in R2 cheeses reaching a value of 672 mg/kg. These results showed that the accumulation of BAs and GABA in Pecorino di Farindola is influenced by ripening time and type of coagulant. Further studies are required to develop starter cultures to reduce BAs content and improve health characteristics of raw milk ewe's cheeses.

Cell Wall Surface Properties of Kluyveromyces marxianus Strains From Dairy-Products.

Thirty-three Kluyveromyces marxianus strains were tested for the ability to form biofilm and mat structures in YPD and whey and for cell surface hydrophobicity. To identify genes potentially involved in adhesion properties, a RT-qPCR analysis was performed. Eight strains were able to adhere on polystyrene plates in both media and formed a mature mat structure. These strains showed a different level of hydrophobicity ranging from 55 to 66% in YPD and from 69 to 81% in whey. Four K. marxianus orthologs genes (FLO11, STE12, TPK3, and WSC4), known from studies in other yeast to be involved in biofilm formation, have been studied. FLO11 and STE12 showed the highest fold changes in all conditions tested and especially in whey: 15.05 and 11.21, respectively. TPK3 was upregulated only in a strain, and WSC4 in 3 strains. In YPD, fold changes were lower than in whey with STE12 and FLO11 genes showing the highest fold changes. In mat structures FLO11 and STE12 fold changes ranged from 3.6-1.3 to 2-1.17, respectively. Further studies are necessary to better understand the role of these genes in K. marxianus adhesion ability.

Different genetic responses to oenological conditions between a flocculent wine yeast and its FLO5 deleted strain: Insights from the transcriptome.

During winemaking Saccharomyces cerevisiae strains are exposed continuously to environmental changes and this microorganism responds modifying its transcriptional profile. Yeast flocculation is considered a social trait that allows the cells to escape from hostile conditions by sedimentation. This behaviour is based on the self-interaction of flocculins, proteins encoded by FLO family genes. These are considered responsible of the facultative-helping type cooperation and were designed as green-beard genes. In order to understand the role of flocculation to stress response, the genome wide expression analysis of a wine flocculent S. cerevisiae F6789A strain and its FLO5 deleted strain (F6789A-Δflo5) were determined, using DNA microarray technology. Results highlighted that F6789A strain showed a shorter lag phase in winemaking condition. The comparison of transcriptomic profiles underlined that, while F6789A-Δflo5 strain seemed engaged in the re-organization of the cell wall and in finding different adhesion ways, F6789A strain presented an up-regulation of genes involved in stress response and higher alcohol production.

Influence of Iodine Feeding on Microbiological and Physico-Chemical Characteristics and Biogenic Amines Content in a Raw Ewes' Milk Cheese.

Iodine is an essential trace element involved in the regulation of thyroid metabolism and antioxidant status in humans and animals. The aim of this study was to evaluate the effect of ewes’ dietary iodine supplementation on biogenic amines content as well as microbiological and physico-chemical characteristics in a raw milk cheese at different ripening times (milk, curd, and 2, 7, 15, 30, 60, and 90 days). Two cheese-making trials were carried out using milk from ewes fed with unifeed (Cheese A) or with the same concentrate enriched with iodine (Cheese B). The results indicated that the counts of principal microbial groups and physico-chemical characteristics were quite similar in both cheeses at day 90. Cheese B was characterized by a higher content of biogenic amines and propionic acid. Propionic bacteria were found in both cheeses mainly in Trial B in agreement with the higher content of propionic acid detected.

Impact of Saccharomyces cerevisiae strains on traditional sparkling wines production.

Sparkling wine fermentation is a challenge for yeasts due to the hostile conditions. A phenotype sought in starters is flocculation, because it reduces riddling time. For this reason, six flocculent Saccharomyces cerevisiae wine strains with different flocculation degree and autolytic activity and two commercial strains were tested for traditional sparkling wine production in a winery. Yeast viability, free aminoacids and high molecular weight nitrogen release and physico-chemical composition of sparkling wines were evaluated. Moreover, strains were tested for their aromatic potential. Obtained data revealed that flocculent yeasts presented oenological performances (in terms of fermentation rate, maximum pressure reached, free aminoacids - AAN and high molecular weight nitrogen - HMWN release) similar to the commercial strains. All considered strains were able to complete fermentation and viable cells of all strains were detected in all sparkling wines produced even after 6 months. F6789 and F10471 strains showed slow fermentation kinetics reaching the maximum of pressure at 180 days. Regarding nitrogen compounds release, FI strain was characterized by the highest amount of AAN and HMWN released, followed by F6789. Strains showed a considerable diversification in terms of number and amount of aroma molecules produced and sparkling wines obtained with autochthonous flocculent strains presented a higher amount of alcohols and esters already after 3 months. Further studies are necessary to select starter strains to improve traditional sparkling wines production.

Determination of Lipophilic Marine Biotoxins in Mussels Harvested from the Adriatic Sea by LC-MS/MS.

Lipophilic marine biotoxins include okadaic acid, pectenotoxin, yessotoxin and azaspiracid groups. The consumption of contaminated molluscs can lead to acute food poisoning syndromes depending on the exposure level. Regulatory limits have been set by Regulation (European Community, 2004a) No 853/2004 and LC-MS/MS is used as the official analytical method according to Regulation (European Community, 2011) No 15/2011. In this study specimens of mussels (Mytilus galloprovincialis) were collected along the coasts of the central Adriatic Sea during the years 2015-2017 and analyzed by the European harmonized Standard Operating Procedure. The method was validated for linearity, specificity, repeatability and reproducibility and it revealed able to be used for the detection of the lipophilic marine biotoxins. Levels of okadaic acid, pectenotoxin, yessotoxin and its analogs were detected at different concentrations in 148 (37%) out of a total of 400 samples, always below the maximum limits, except for 11 (4.3%) of them that were non-compliant because they exceeded the regulatory limit. Moreover, some samples were exposed to a multi-toxin mixture with regards to okadaic acid, yessotoxin and 1-Homo yessotoxin. Following these results, the aquaculture farms from which the non-compliant samples derived were closed until the analytical data of two consecutive samplings returned favorable. Besides the potential risk of consumption of mussels contaminated by lipophilic marine biotoxins, these marine organisms can be considered as bio-indicators of the contamination status of the marine ecosystem.

Development of a rapid method for the detection of Yersinia enterocolitica serotype O:8 from food.

In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y. enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat and dairy products (n = 132). The method was validated by ISO 16140:2003 and compared with the official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food samples (n = 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The results from this study demonstrated that the developed method was rapid and cheap, specific and sensitive for the screening of the pathogen in food.

Intraspecies polymorphisms of Kluyveromyces marxianus strains from Yaghnob valley.

In this study, 29 strains of Kluyveromyces marxianus with peculiar genetic and phenotypic traits previously isolated from a fermented goat milk of Yaghnob valley were investigated for chromosome length polymorphism (CLP) by PFGE, adhesion properties and carbon usage by Biolog analysis. Obtained data showed that strains differed in terms of number and size of chromosome bands. The number of bands ranged from 5 to 7, suggesting a probable genome size from 1.4 to 2.6 Mb. Strains showed a certain level of cell surface hydrophobicity ranging from 32% to 77.7%. Strains were also tested for their ability to form a biofilm on polystyrene plates: planktonic cells ranged from 6.3 cfu/mL to 7.95 cfu/mL, while sessile from 7.11 cfu/mL to 8.6 cfu/mL. The strains able to adhere to polystyrene plates were also able to form a mature MAT. Biolog analysis revealed that almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, ß-methyl D-gucoside and xylose; 5 strains were able to grow on cellobiose and 3 were able to catabolise α-ketobutyric. The obtained data highlighted a number of interesting features underlying the peculiar capacities of these strains for industrial applications.

Development and implementation of multilocus sequence typing to study the diversity of the yeast Kluyveromyces marxianus in Italian cheeses.

The yeast Kluyveromyces marxianus possesses advantageous traits like rapid growth, GRAS (generally regarded as safe) status and thermotolerance that make it very suitable for diverse biotechnological applications. Although physiological studies demonstrate wide phenotypic variation within the species, there is only limited information available on the genetic diversity of K. marxianus. The aim of this work was to develop a multilocus sequence typing (MLST) method for K. marxianus to improve strain classification and selection. Analysis of housekeeping genes in a number of sequenced strains led to the selection of five genes, IPP1, TFC1, GPH1, GSY2 and SGA1, with sufficient polymorphic sites to allow MLST analysis. These loci were sequenced in an additional 76 strains and used to develop the MLST. This revealed wide diversity in the species and separation of the culture collection and wild strains into multiple distinct clades. Two subsets of strains that shared sources of origin were subjected to MLST and split decomposition analysis. The latter revealed evidence of recombination, indicating that this yeast undergoes mating in the wild. A public access web-based portal was established to allow expansion of the database and application of MLST to additional K. marxianus strains. This will aid understanding of the genetic diversity of the yeast and facilitate biotechnological exploitation.

FLO5 gene controls flocculation phenotype and adhesive properties in a Saccharomyces cerevisiae sparkling wine strain.

Flocculation is an important feature for yeast survival in adverse conditions. The natural diversity of flocculating genes in Saccharomyces cerevisiae can also be exploited in several biotechnological applications. Flocculation is mainly regulated by the expression of genes belonging to the FLO family. These genes have a similar function, but their specific contribution to flocculation ability is still unclear. In this study, the distribution of FLO1, FLO5 and FLO8 genes in four S. cerevisiae wine strains was investigated. Subsequently, both FLO1 and FLO5 genes were separately deleted in a flocculent S. cerevisiae wine strain. After gene disruption, flocculation ability and agar adhesion were evaluated. FLO1 and FLO5 genes inheritance was also monitored. All strains presented different lengths for FLO1 and FLO5 genes. Results confirm that in S. cerevisiae strain F6789, the FLO5 gene drives flocculation and influences adhesive properties. Flocculation ability monitoring after a cross with a non-flocculent strain revealed that FLO5 is the gene responsible for flocculation development.

Histamine Food Poisoning.

The consumption of food containing high amounts of histamine and other biogenic amines can cause food poisoning with different symptoms linked to the individual sensitivity and the detoxification activity. Histamine is the only biogenic amine with regulatory limits set by the European Commission in fish and fishery products, because it can lead to a fatal outcome. However, also fermented foods can be involved in outbreaks and sporadic cases of intoxication. The factors affecting the presence of histamine in food are variable and product specific including the availability of the precursor amino acid, the presence of microorganisms producing decarboxylases, and the conditions allowing their growth and enzyme production. Generally, the good quality of raw material and hygienic practices during food processing as well as the use of histidine decarboxylase-negative starter cultures can minimize the occurrence of histamine. Further studies are necessary to estimate the human exposure and the relationship between the total amount of the biogenic amines ingested with food and health effects.

Technological Factors Affecting Biogenic Amine Content in Foods: A Review.

Biogenic amines (BAs) are molecules, which can be present in foods and, due to their toxicity, can cause adverse effects on the consumers. BAs are generally produced by microbial decarboxylation of amino acids in food products. The most significant BAs occurring in foods are histamine, tyramine, putrescine, cadaverine, tryptamine, 2-phenylethylamine, spermine, spermidine, and agmatine. The importance of preventing the excessive accumulation of BAs in foods is related to their impact on human health and food quality. Quality criteria in connection with the presence of BAs in food and food products are necessary from a toxicological point of view. This is particularly important in fermented foods in which the massive microbial proliferation required for obtaining specific products is often relater with BAs accumulation. In this review, up-to-date information and recent discoveries about technological factors affecting BA content in foods are reviewed. Specifically, BA forming-microorganism and decarboxylation activity, genetic and metabolic organization of decarboxylases, risk associated to BAs (histamine, tyramine toxicity, and other BAs), environmental factors influencing BA formation (temperature, salt concentration, and pH). In addition, the technological factors for controlling BA production (use of starter culture, technological additives, effects of packaging, other non-thermal treatments, metabolizing BA by microorganisms, effects of pressure treatments on BA formation and antimicrobial substances) are addressed.

Marine Biotoxins: Occurrence, Toxicity, Regulatory Limits and Reference Methods.

Harmful algal blooms are natural phenomena caused by the massive growth of phytoplankton that may contain highly toxic chemicals, the so-called marine biotoxins causing illness and even death to both aquatic organisms and humans. Their occurrence has been increased in frequency and severity, suggesting a worldwide public health risk. Marine biotoxins can accumulate in bivalve molluscs and regulatory limits have been set for some classes according to European Union legislation. These compounds can be distinguished in water- and fat-soluble molecules. The first group involves those of Paralytic Shellfish Poisoning and Amnesic Shellfish Poisoning, whereas the toxins soluble in fat can cause Diarrheic Shellfish Poisoning and Neurotoxic Shellfish Poisoning. Due to the lack of long-term toxicity studies, establishing tolerable daily intakes for any of these marine biotoxins was not possible, but an acute reference dose can be considered more appropriate, because these molecules show an acute toxicity. Dietary exposure assessment is linked both to the levels of marine biotoxins present in bivalve molluscs and the portion that could be eaten by consumers. Symptoms may vary from a severe gastrointestinal intoxication with diarrhea, nausea, vomiting, and abdominal cramps to neurological disorders such as ataxia, dizziness, partial paralysis, and respiratory distress. The official method for the detection of marine biotoxins is the mouse bioassay (MBA) showing some limits due to ethical restrictions and insufficient specificity. For this reason, the liquid chromatography-mass spectrometry method has replaced MBA as the reference technique. However, the monitoring of algal blooms producing marine biotoxins should be regularly assessed in order to obtain more reliable, accurate estimates of bloom toxicity and their potential impacts.