Antonello Paparella

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must.

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.

Characterization of the Enterobacteriaceae isolated from an artisanal Italian ewe's cheese (Pecorino Abruzzese)

Aims:  To evaluate some physiological characteristics of the Enterobacteriaceae isolated from Pecorino cheese.

Comparison of the compositional, microbiological, biochemical and volatile profile characteristics of three Italian PDO fermented sausages.

Three Italian PDO fermented sausages, Varzi, Brianza and Piacentino, were compared for compositional, microbiological, biochemical and volatile profile characteristics. Mean values for the gross composition varied especially due to moisture, fat, total protein and nitrate concentration which reflected differences in the ingredients and some technological parameters. Cell numbers of the major microbial groups were almost similar among sausages. The major differences were found for Brochothrix thermosphacta, enterococci and moulds. Apart from their use as starters, Lactobacillus sakei and Lactobacillus curvatus were the dominant lactic acid bacteria and, as well as Staphylococcus xylosus, dominated the population of coagulase-negative staphylococci. Sausages differed for the hydrolysis of myofibrillar proteins and secondary proteolysis. Varzi, the sausage subjected to prolonged fermentation at 23-25°C for 10 days before ripening, showed the highest degree of secondary proteolysis. Varzi and Brianza, the two fermented sausages manufactured by using microbial starters, showed the highest concentration and similar profiles of free amino acids. The peptidase activities contained in the aqueous extracts agreed with the above findings. A total of 52 volatile components, mainly alcohols, aldehydes and terpenes, were identified by solid-phase micro-extraction coupled with gas chromatography-mass spectrometry analysis. The volatile profiles of the three Italian PDO fermented sausages differed in part and, except for terpenes, the highest levels of the other chemical classes were found in Varzi and Brianza sausages. The composition of free fatty acids of the three Italian PDO sausages was rather similar. Monounsaturated fatty acids (MUFA) were found at the highest relative percentage followed by saturated (SFA) and polyunsaturated (PUFA) fatty acids. Oleic, palmitic, linoleic and stearic were the main free fatty acids found in all fermented sausages.

Use of Staphylococcus xylosus as a starter culture in dried sausages: effect on the biogenic amine content.

The main objective of this work was to investigate if the use of Staphylococcus xylosus S81 as a starter culture in sausage production can influence the amount of histamine during ripening, and the concentration of the other most important biogenic amines, by influencing the activity of the microbial amino oxidases as well as the aminoacid decarboxylases. The results confirm that the biogenic amines presence in foods is the consequence of a complex equilibrium between the composition of the medium and the enzymatic activities of the microbial population. In addition, the results suggest that the presence and relative activity of amino oxidases should be considered as an important characteristic in the selection of starter cultures used in the production of fermented foods.

Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects.

The presence of enterococci in Pecorino Abruzzese cheese during ripening was evaluated. Counts were high, especially in fully ripened summer batches. Seventy strains were isolated and identified based on phenotypical and genotypical features as Enterococcus faecium (48.5%), Enterococcus faecalis (40%), and Enterococcus durans (11.5%), with the first species predominant in spring batches and the second predominant in summer batches. High biodiversity was revealed by random amplification of polymorphic DNA and a PCR assay, suggesting the presence of autochthonous strains. E. faecium isolates were the most resistant to the tested antibiotics, especially to erythromycin, chloramphenicol, and penicillin, but all strains were susceptible to vancomycin, as confirmed by the absence of vanA and vanB genes. The presence of some virulence determinants was investigated, revealing the diffusion of aggregation substance (asal) and gelatinase (gelE) genes in 37.5% of E. faecalis strains. However, none of the isolates produced gelatinase in vitro, suggesting the presence of silent genes. The virulence genes were absent in E. durans. Among E. faecium strains, only Lab 41/1 possessed gelE and asal, whose presence previously has been reported only in E. faecalis. Decarboxylating activity was revealed for phenylalanine (27% of the strains) and tyrosine (96%) but not histidine. The presence of a tyrosine decarboxylase-encoding gene was observed for all strains. A comparison of these results with those of previous studies of clinical and food isolates indicates that enterococci from Pecorino Abruzzese cheese have low pathogenic potential.

Yeasts from Colombian Kumis as source of peptides with Angiotensin I converting enzyme (ACE) inhibitory activity in milk

This study investigated the possibility of using yeast strains in fermented milks to obtain products with high Angiotensin I-converting enzyme (ACE) inhibitory activity and low bitter taste. Ninety-three yeast strains isolated from Colombian Kumis in different geographic regions were molecularly identified, and their milk fermentation performances were determined. Molecular identification evidenced that Galactomyces geotrichum, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis, were the dominant species. Eighteen out of 93 strains produced fermented milk with ACE-inhibitory (ACEI) activity values ranging from 8.69 to 88.19%. Digestion of fermented milk samples by pepsin and pancreatin demonstrated an increase in ACEI activity, with C. lusitaniae KL4A as the best producer of ACEI peptides. Moreover, sensory analysis of the products containing the major ACE-inhibitory activity pointed out that P. kudriavzevii KL84A and Kluyveromyces marxianus KL26A could be selected as potential adjunct starter cultures in Kumis, since they made a considerable contribution to the ACE inhibitory activity and produced fermented milk without bitter taste. In this study we observed that Colombian Kumis can be an excellent vehicle for the isolation of yeasts with a potential to enhance bioactive peptides produced during milk fermentation.

Combination of multiplex PCR and PCR-denaturing gradient gel electrophoresis for monitoring common sourdough-associated Lactobacillus species.

ABSTRACT A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs.

Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage

The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content. Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.

Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population

Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main objective of this research was to studied the enterococcal population, present in 13 kumis samples traditionally manufactured, for their role as beneficial organisms or opportunistic pathogens. The molecular identification of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in enterococci, were detected in 30 isolates, while 42 were free of virulence determinants. Skim milk media were fermented by all the different isolates and further tested for proteolysis (free NH(3) groups), Angiotensin-I Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE inhibitory activity, with E. faecalis KE09 as the best producer (IC50 = 14.25 μg ml(-1)). Moreover, the strains showed a very low tyrosine decarboxylase activity and did not produce histamine during 48 h fermentation in milk. This study underlines the that Colombian kumis is a good source of not virulent enterococci able to produce fermented milks with ACE-inhibitory activity.

Traditional Fermented Foods and Beverages from a Microbiological and Nutritional Perspective: The Colombian Heritage

Fermentation has been used for preserving foods for centuries prior to the invention of pasteurization and sterilization, and every culture has a variety of fermented products as part of its diet. This paper reviews the diversity of fermented foods and beverages from Latin America; these fermented products are produced by traditional methods that exploit mixed cultures of various nonpathogenic microorganisms. Fermented foods covered in this review include maize, cassava, palm sap, sugar cane juice, cocoa, and milk. We explore the history of some Colombian fermented foods and beverages, which are today part of the tradition of some ethnic groups, and evaluate their technology, microbiology, the presence of some nutritional factors, and safety concerns. To the best of our knowledge, this is the 1st systematic review on Colombian fermented beverages and foods, and we believe that it may contribute to valorize these products that are still part of the Latin America tradition.