Yeasts associated with the worker caste of the leaf-cutting ant Atta cephalotes under experimental conditions in Colombia.

Yeasts isolated from the worker caste of the Colombian leaf-cutting ant, Atta cephalotes (Hymenoptera: Myrmicinae) were cultured and identified by molecular methods. Abundant, persistent, and omnipresent species were classified as "prevalent". Experimental data were compared with information gathered from published reports on the yeast species composition in other leaf-cutting ant species. Diversity analysis was conducted using diversity values (q0, q1, and q2) to compare the richness and abundance of yeasts present in different leaf-cutting ant species. Clustering analysis was carried out to assess the similarity of yeast community according to ant species. The yeast species composition was highly variable among the ant species. A. laevigata and A. capiguara showed the highest degree of similarity and differed from the group composed by A. cephalotes, A. sexdens, A. sexdens rubropilosa, and A. texana. The isolation of dominant yeasts in different ant castes within the different compartments of a colony strongly suggests that the identified microorganisms are not transient but are native to the soil surrounding ant colonies and the substrates used by the ants to grow their fungal cultivars. It is apparent that the ant-fungus mutualism does not operate in an environment devoid of other microbes, but rather that the association must be seen within the context of a background of other microorganisms, particularly the dominant yeasts.

Impact of Saccharomyces cerevisiae and non-Saccharomyces yeasts to improve traditional sparkling wines production.

In this study the effect of a co-inoculum of S. cerevisiae (F6789) with Torulaspora delbrueckii (TB1) or Starmerella bacillaris (SB48) on the oenological and aroma characteristics of sparkling wines obtained with the Champenoise method was investigated. The autolytic outcome and the sensory profile of sparkling wines were also evaluated. The secondary fermentations were completed by all mixed and single starter cultures with the only exception of those guided by Starm.bacillaris. Sparkling wines produced with S. cerevisiae F6789+Starm.bacillaris SB48 showed the highest amounts of glycerol (6.51 g/L). The best autolytic potential was observed in sparkling wines produced with +Starm.bacillaris (81.98 mg leucin/L) and S. cerevisiae+T. delbrueckii (79.03 mg leucin/L). The lowest value was observed for sparkling wines obtained with S. cerevisiae F6789 (53.96 mg leucin/L). Sparkling wines showed different aroma and sensory profiles. Esters were mainly present in sparkling wines obtained with S. cerevisiae F6789 (88.09 mg/L) followed by those obtained with S. cerevisiae+T. delbrueckii (87.20 mg/L), S. cerevisiae +Starm.bacillaris (81.93 mg/L). The content of esters decreased over time, and that might be related to the adsorption on lees and chemical hydrolysis. The highest concentrations of higher alcohols were found in sparkling wines produced with S. cerevisiae+T. delbrueckii (27.50 mg/L). Sparkling wines obtained with S. cerevisiae +Starm.bacillaris were well differentiated from the others due to their high score for the descriptor for spicy, bread crust, freshness and floral. Tailored strains with different autolytic potential might represent an interesting strategy to improve traditional sparkling wine production and favour their differentiation.

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry.

Brettanomyces bruxellensis is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify B. bruxellensis contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1-2 h) of B. bruxellensis in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting B. bruxellensis in the presence of other yeast species (Saccharomyces cerevisiae and Pichia spp.) and at the concentrations that produce natural contaminations (up to 105 cells/mL), as well as at lower concentrations (103-102 cells/mL). Wine samples naturally contaminated by B. bruxellensis or inoculated with four different strains of B. bruxellensis species together with Saccharomyces cerevisiae and Pichia spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of B. bruxellensis in simple and complex mixtures. Therefore, this technique has great potential for the detection of B. bruxellensis and could allow preventive actions to reduce wine spoilage.

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.

The life and times of yeasts in traditional food fermentations.

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations.

Chemical-nutritional quality and oxidative stability of milk and dairy products obtained from Friesian cows fed with a dietary supplementation of dried grape pomace.

BACKGROUND: The aim of the study was to evaluate the nutritional properties of milk and cheese obtained from Friesian cows fed with a diet supplemented with dried grape pomace, a by-product of the oenological industry, which is very rich in polyphenols. This approach is inspired by the increasing interest in foods containing functional ingredients that may have beneficial effects on human health. During the testing period, analyses of the chemical and nutritional properties of milk and dairy products derived from it were performed; particular attention was given to the effect of cheese ripening on the oxidative stability and fermentation process, evaluating respectively the presence of malondialdehyde and γ-aminobutyric acid. RESULTS: Dietary enrichment with grape pomace did not affect the milk composition but induced modifications in the fatty acid profiles in both milk and cheese with an increase in concentration of linoleic acid, trans-vaccenic acid, rumenic acid and total n-6 fatty acids. Moreover, after 30 days of cheese ripening, an increased oxidative stability and an increased concentration of γ-aminobutyric acid were found. CONCLUSIONS: Our results indicated a general improvement in nutritional parameters of milk and related cheese obtained from Friesian cows that received the feeding enrichment with dried grape pomace. Further analysis should be performed to improve knowledge of the chemical and microbiological mechanisms at the source of these findings. © 2019 Society of Chemical Industry.

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.

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.

Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation.

Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics, such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study, 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time, novel primer sets were developed to determine the expression of the genes ATG1, ATG17 and ATG29. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains and were independent of the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production. Copyright © 2016 John Wiley & Sons, Ltd.

Influence of pig rennet on fatty acid composition, volatile molecule profile, texture and sensory properties of Pecorino di Farindola cheese.

BACKGROUND: Pig rennet is traditionally used in Pecorino di Farindola cheese. In this study, different Pecorino cheeses obtained using calf, kid and pig rennets were compared in terms of fatty acids, volatile molecule profile, texture and sensory properties during ripening. RESULTS: The rennet type influenced the fatty acid composition of cheeses, though palmitic, myristic and oleic acids were always predominant. The analysis of volatiles by SPME-GC/MS showed that Pecorino from calf rennet, at the end of ripening, was the least 'evolved' in terms of volatile profile. SPME-GC/MS analysis revealed that cheeses from calf rennet showed the slowest accumulation of free fatty acids over ripening time. Volatile data permitted the differentiation of cheese samples ripened from 30 to 180 days according to the rennet used. Texture analysis differentiated cheeses made with pig and calf rennet from those made with kid rennet, which were less hard and more elastic than the former. Also sensory analysis differentiated cheese samples on the basis of rennet type, and cheeses made with pig rennet showed the lowest elasticity, bitter taste and fruity and hay flavour intensities. CONCLUSION: Pig rennet is fundamental to determine the quality parameters of Pecorino di Farindola cheese and could be used to impart peculiar quality features to ewe's milk cheeses.

Biodiversity study of wine yeasts belonging to the "terroir" of Montepulciano d'Abruzzo "Colline Teramane" revealed Saccharomyces cerevisiae strains exhibiting atypical and unique 5.8S-ITS restriction patterns.

The Montepulciano d'Abruzzo "Colline Teramane" premium wine DOCG is produced in the Teramo province (Abruzzo, Italy). This region has a great tradition in winemaking and the wine is produced by a spontaneous fermentation so it could represent a reservoir of wine natural yeasts with important oenological features. The aim of this study was to characterize the yeast community of this wine grape growing region in order to create a Saccharomyces cerevisiae bank, providing data on oenological properties for potential industrial applications. A total of 430 yeasts were isolated at the end of spontaneous fermentation. PCR-RFLP was applied for the identification at the species level and underlined that 14 strains exhibited unusual and characteristic restriction patterns different from those typical of the species S. cerevisiae. This difference was due to the insertion of base C at a position 138 in the ITS1 region that determined an additional cleavage site for the enzyme HaeIII. This insertion could be associated to the fermentative performance and associated to the relationship existing between yeasts and a viticulture region or 'terroir'.

Impact of microbial cultures on proteolysis and release of bioactive peptides in fermented milk.

This study aimed at evaluating co-cultures of selected microorganisms for their proteolytic activity and capability to produce fermented milk enriched with ACE-inhibitory (ACEI) peptides. Selected yeasts (Torulaspora delbruekii KL66A, Galactomyces geotrichum KL20B, Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26A) and lactic acid bacteria strains (Lactobacillus plantarum LAT03, Lb. plantarum KLAT01 and the not virulent Enterococcus faecalis KE06) were screened as single cultures for their capacity of releasing ACEI peptides without producing bitter taste. Three strains cultures (yeast, Lb. plantarum and E. faecalis) were performed to evaluate the combined impact on microbial growth, lactic acid production, citric acid consumption, proteolysis, ACEI activity, and bitter taste after 36 h of fermentation at 28 °C. While G. geotrichum KL20B showed a strong stimulating effect on Lb. plantarum strains and the production of peptides with ACEI activity, the presence of T. delbruekii KL26A in the cultures was deleterious both to ACEI activity and product taste. The most effective combination was P. kudriavzevii KL84A, Lb. plantarum LAT3, E. faecalis KL06, which showed the highest ACEI activity (IC50 = 30.63 ± 1.11 µg ml(-1)) and gave no bitter taste for 7 days at 6 °C. Our results highlight the importance of choosing the strains combination carefully, to obtain a high yield of ACEI activity without bitter taste.

Dual Role of Yeasts and Filamentous Fungi in Fermented Sausages.

This contribution aims to review the presence and the potential double role-positive or beneficial and negative or harmful-of fungi in fermented sausages as well as their use as starter cultures. Traditionally, studies have been focused on lactic acid bacteria; however, over the years, interest in the study of fungi has increased. The important contribution of yeasts and filamentous fungi to the quality and safety of fermented sausages has emerged from reviewing the literature regarding these fermented products. In conclusion, this review contributes to the existing literature by considering the double role of filamentous fungi and yeasts, the global fermented sausage market size, the role and use of starters, and the starters mainly present in the worldwide market, as well as the main factors to take into account to optimize production. Finally, some suggestions for future broadening of the sector are discussed.

Unlocking the potential of Kluyveromyces marxianus in the definition of aroma composition of cheeses.

Introduction: The cheese microbiota is very complex and is made up of technologically-relevant, spoilage, opportunistic and pathogenic microorganisms. Among them lactic acid bacteria and yeasts are the main ones. One of the most interesting dairy yeasts is Kluyveromyces marxianus because of its technological properties including the ability to produce aroma compounds. Methods: This study investigated the contribution of Kluyveromyces marxianus to the gross composition and aroma profile of cow cheeses. Experimental cheeses were prepared by inoculating a co-culture of K. marxianus FM09 and a commercial strain of Lacticaseibacillus casei and compared with cheeses obtained with only L. casei. The gross composition was determined by a FoodScan™ 2 Dairy Analyser, and free amino acids were evaluated at 507 nm after reaction with Cd-ninhydrin. The volatile organic compounds were extracted by head-space solid phase micro-extraction and analyzed by gas chromatography-mass spectrometry coupled with odor activity values. qRT-PCR was applied to determine the expression of genes involved in esters synthesis and degradation. Results: The inoculation of K. marxianus induced an increase of pH and a reduction of protein content of cheeses, in agreement with the stronger proteolysis detected in these cheeses. K. marxianus influenced the content of aroma compounds both quantitatively and qualitatively. In particular, an increase of higher alcohols, esters and organic acids was observed. Moreover, 12 compounds were detected only in cheeses obtained with the co-culture. These differences were in agreement with the odor activity values (OAV). In fact, only 11 compounds showed OAV > 1 in cheeses obtained with the commercial strain, and 24 in those obtained with the co-culture. The qPCR analysis revealed an over expression of ATF1, EAT1, and IAH1 genes. Conclusion: Kluyveromyces marxianus could act as an important auxiliary starter for cheese production through the development and diversification of compounds related to flavor in short-aged cow cheeses.

Sniffing the wine differences: The role of Starmerella bacillaris biofilm-detached cells.

This study investigated the impact of 10 strains of Starmerella bacillaris, co-inoculated as planktonic or biofilm-detached cells with Saccharomyces cerevisiae, on the volatilome of a red wine. The wines produced with St. bacillaris biofilm-detached cells exhibited a greater concentration of glycerol and a lower quantity of ethanol than the other wines. Furthermore, these wines exhibited elevated levels of higher alcohols, organic acids, esters, terpenes, and norisoprenoids. Based on the odor activity value and relative odor contribution, isoamyl acetate, ethyl octanoate, ethyl isobutanoate, and methyl decanoate were the main aroma components of wines made with planktonic cells. The main compounds characterizing the wines obtained with biofilm-detached cells were: phenethyl alcohol, ß-damascenone, citronellol, ß-ionone, and nerol. The sensory analysis revealed that the wines produced with biofilm-detached cells had higher scores for mouth-feel, spicy, floral, and raspberry notes than the others. The present study provides evidence that St. bacillaris biofilm-detached cells released specific volatile compounds in red wines.

Wine Barrel Biofilm as a Source of Yeasts with Non-Conventional Properties.

This study investigated the main microbial groups characterizing the interior surface of oak barrels from different years (1890, 1895, 1920, 1975, 2008) used in the production of vino cotto. The yeasts were characterized for the following properties: γ-aminobutyric acid (GABA) production, antioxidant activity, air-liquid interfacial biofilm formation, and anthocyanin adsorption capacity. Community-level physiological profile analysis revealed that the microbial communities inside the barrels used the tested carbon sources in different manners. The following yeast species were identified: Millerozyma farinosa, Zygosaccharomyces bisporus, Wickerhamiella versatilis, Zygosaccharomyces bailii, Starmerella lactis-condensi, and Zygosaccharomyces rouxii. All the strains were able to produce GABA, and S. lactis-condensi, Z. bisporus and Z. rouxii were the highest producers (more than 600 mg/L). The Z. rouxii and Z. bailii strains showed the highest antioxidant activity. Only seven strains out of ten M. farinosa formed air-liquid interfacial biofilm. None of the M. farinosa strains adsorbed anthocyanins on their cell wall. The other strains adsorbed anthocyanins in a strain-dependent way, and the highest adsorption was observed for the W. versatilis strains. The yeasts isolated in this study could be used to increase the functional properties and the quality of fermented foods and beverages.

High content of biogenic amines in Pecorino cheeses.

Pecorino refers to Italian cheeses made exclusively from raw or pasteurized ewes' milk, characterized by a high content of fat matter and it is mainly produced in the Middle and South of Italy by traditional procedures. The autochthonous microbiota plays an important role in the organoleptic traits of Pecorino cheese and it can influence biogenic amines (BA) content. The aim of this study was to characterize from microbiological and chemical point of view 12 randomly purchased commercial cheeses produced in Abruzzo region. Moreover, the BA content and the bacteria showing a decarboxylating activity were detected. For this purpose, a real-time quantitative PCR (qPCR) was applied to evaluate histamine and tyramine-producers. The samples were well differentiated for microbial groups composition, such as aerobic mesophilic bacteria, Enterobacteriaceae, coagulase-negative staphylococci, yeasts, enterococci, mesophilic and thermophilic lactobacilli. Pathogens such as Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 were absent in all samples. In most samples the content of BA resulted to be high, with prevalence of histamine and tyramine. In particular, total BA content reached 5861 mg/kg in Pecorino di Fossa cheese. The qPCR method resulted to be very useful to understand the role of autochthonous Pecorino cheese microbiota on BA accumulation in many different products. In fact, since the ability of microorganisms to decarboxylate aminoacids is highly variable being in most cases strain-specific, the detection of bacteria possessing this activity is important to estimate the risk of BA cheese content.

Contribution of Starmerella bacillaris and Oak Chips to Trebbiano d'Abruzzo Wine Volatile and Sensory Diversity.

In this study, six fermentation trials were carried out: co-inoculation and sequential inoculation of Saccharomyces cerevisiae and Starmerella bacillaris in the presence and absence of oak chips. Moreover, Starm. bacillaris strain was attached to the oak chips and co-inoculated or sequentially inoculated with S. cerevisiae. Wines fermented with Starm. bacillaris adhered to oak chips showed a higher concentration of glycerol (more than 6 g/L) than the others (about 5 g/L). These wines also showed a higher content of polyphenols (more than 300 g/L) than the others (about 200 g/L). The addition of oak chips induced an increase of yellow color (b* value of about 3). Oak-treated wines were characterized by a higher concentration of higher alcohols, esters and terpenes. Aldehydes, phenols and lactones were detected only in these wines, independently from the inoculation strategy. Significant differences (p < 0.05) were also observed in the sensory profiles. The fruity, toasty, astringency, and vanilla sensations were perceived as more intense in wines treated with oak chips. The white flower descriptor showed a higher score in wines fermented without chips. Oak surface-adhered Starm. bacillaris cells could be a good strategy to improve the volatile and sensory profile of Trebbiano d'Abruzzo wines.

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations.

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.

Microbiological and chemical profiles of naturally fermented table olives and brines from different Italian cultivars.

Six naturally fermented (Greek-style) table olives of cultivars Itrana, Peranzana, Cellina di Nardò, Nocellara del Belice and Bella di Cerignola, as well as their corresponding brines, were studied by a combined strategy consisting of chemical, microbiological and molecular analyses. In particular, organic acids, sugars, polyphenols, fatty acids, biogenic amines and cultivable microbiota were detected by standard methods. Moreover, tyramine and histamine producing bacteria were evaluated by an original approach consisting of Reverse-Transcription (RT)-qPCR. At the end of the fermentation process, mesophilic lactobacilli and yeasts in brine represented the dominating biota, ranging from 6.25 to 7.84 log CFU/ml and from 6.5 to 7.56 log CFU/ml, respectively. Enterobacteriaceae and pathogens were undetectable in all the samples. In general, table olive preparations differed in chemical composition. In particular, C16:0 and C18:2c9,12 concentrations ranged from 9.9 to 18.8 % and from 5.4 to 15.4 % of total fatty acids, respectively. The main fatty acid detected was C18:1c9 while CLAc9, t11 was present only in traces. Polyphenol concentrations greatly differentiated the final product, depending on the cultivar. A low quantity of biogenic amines was found in some samples and biogenic amines producing bacteria were rapidly detectable by RT-qPCR.