Maria Schirone

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.

Diversity of Candida zemplinina strains from grapes and Italian wines

The aim of this research was to genetically and technologically characterize Candida zemplinina strains isolated from different sources of enological interest. Phenotypic and genotypic subtyping, as well as enological characterization, were carried out on 36 C. zemplinina isolates collected from grapes, must and wines of different regions of Italy. RAPD-PCR fingerprinting of the isolates revealed a high genetic heterogeneity. At physiological level, yeasts were grouped into different clusters on the basis of sugar and ethanol tolerance. Common enological characteristics were examined and strains resulted to be highly fructophilic while presenting low ethanol and acetic acid production, high glycerol production, capacity to metabolize malic acid and slower fermentation kinetics when compared to Saccharomyces cerevisiae. The genetic and phenotypic intraspecies biodiversity of C. zemplinina gave useful data to understand its potential technological role in winemaking. This research represents a first step for the selection of C. zemplinina strains to be used as a starter in co-culture or in sequential inoculation with S. cerevisiae to improve the complexity and to enhance the particular characteristic of wines.

Biogenic amines in raw and processed seafood.

The presence of biogenic amines (BAs) in raw and processed seafood, associated with either time/temperature conditions or food technologies is discussed in the present paper from a safety and prevention point of view. In particular, storage temperature, handling practices, presence of microbial populations with decarboxylase activity and availability of free amino acids are considered the most important factors affecting the production of BAs in raw seafood. On the other hand, some food technological treatments such as salting, ripening, fermentation, or marination can increase the levels of BAs in processed seafood. The consumption of high amount of BAs, above all histamine, can result in food borne poisoning which is a worldwide problem. The European Regulation established as maximum limits for histamine, in fishery products from fish species associated with high histidine amounts, values ranging from 100 to 200 mg/kg, while for products which have undergone enzyme maturation treatment in brine, the aforementioned limits rise to 200 and 400 mg/kg. Preventive measures and emerging methods aiming at controlling the production of BAs are also reported for potential application in seafood industries.

Application of starter cultures to table olive fermentation: an overview on the experimental studies.

Table olives are one of the oldest fermented foods and are considered as an important component of the Mediterranean diet, since their richness in monounsaturated fats (primarily oleic acid) and phenolic compounds may function as antioxidants in the human body; in the Western world they represent one of the most popular fermented vegetables but, despite its economic significance, table olive fermentation is still craft-based and empirical. In particular, such a type of fermentation results from the competitive activities among indigenous, contaminating microorganisms, the microbial balance depending on several intrinsic (pH, water activity, diffusion of nutrients from the drupe, and level of anti-microbial compounds) and extrinsic (temperature, oxygen availability, and salt concentration) factors. At present, to reduce the risk of spoilage and to achieve a more predictable process there is an increasing interest in developing starter cultures for table olives fermentation. Anyway, the application of starter cultures in the field of table olives is quite far from reaching the diffusion as it has in other sectors of food industry (e.g., dairy products and alcoholic beverages). This review focuses on experimental researches devoted to studying starter cultures for possible application to table olive fermentation both at artisan and industrial level.

Yeast biota associated to naturally fermented table olives from different Italian cultivars

The yeast communities associated with the fermentation of six different cultivars of Italian table olives were studied. Molecular identification of a total of 117 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. The yeast population was also monitored by a culture-independent method based on PCR-DGGE analysis. This combined strategy resulted to be a powerful and reliable tool to investigate table olives yeast ecology and revealed that Saccharomyces cerevisiae was present in all the processed olives. Moreover, strains were characterized on the basis of different properties of technological interest. In particular, β-glucosidase, catalase, pectinolytic, xylanolytic, esterase and lipase activities were investigated and the ability to grow up in presence of different salt concentration (5-7.5-10-14-20% w/v) was evaluated. The majority of strains showed catalase activity and none of them expressed pectinolytic, xylanolytic, esterase or lipase activities. Six strains belonging to Pichia galeiformis and six strains of Wicheramomyces anomalus showed β-glucosidase activity. Only 10 S. cerevisiae strains were able to grow in presence of 14% NaCl. The obtained results offer valuable information on yeast population biodiversity and dynamics in naturally fermented Italian table olives and show the potential use of some yeast strains, besides lactic acid bacteria, as a part of mixed starter cultures for table olive fermentation.

Yeasts associated with Manteca

Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese.

Influence of organic viticulture on non-Saccharomyces wine yeast populations

This study evaluated the population dynamics of non-Saccharomyces biota during spontaneous fermentation of organic musts. Non-Saccharomyces yeasts were found to be present at high levels during all fermentations. A total of 543 yeast colonies were isolated, 190 from Lysine-Medium (LM) agar, 254 from Wallerstein Laboratory Nutrient (WLN) agar and 99 from YPD agar. To estimate yeast population dynamics during spontaneous fermentation a genotypic approach was applied. PCR-RFLP of the ITS1-5.8S rDNA-ITS2 region and sequence determination of the D1/D2 region of the 26S rRNA gene enabled identification of the yeast isolates at the species level. Hanseniaspora uvarum, Metschnikowia fructicola and Candida zemplinina predominated, while Issatchenkia terricola, Issatchenkia orientalis and Pichia sp. were identified with a lower frequency. Hanseniaspora uvarum, M. fructicola and C. zemplinina represented 43%, 31% and 11% of the total non-Saccharomyces population isolated, respectively. Some yeast isolates were shown to be closely related to Hanseniaspora spp. and Candida spp. on the basis of the D1/D2 sequences. Based on those results, the coexistence of different Hanseniaspora and Pichia species in grape musts was supposed, and their complete identification was achieved using additional molecular markers. Moreover, strain typing and differentiation was carried out by RAPD-PCR. High strain polymorphisms were observed in the different species. For some strains, appreciable properties were demonstrated both in vitro by the API-ZYM test and in must. In must microvinification some strains showed good fermentation performances, low production of acetic acid and a partial capability to degrade malic acid.

Histamine poisoning and control measures in fish and fishery products

Histamine poisoning is one of the most common form of intoxication caused by the ingestion of fish and fishery products. Cooking, canning, or freezing cannot reduce the levels of histamine because this compound is heat stable. All humans are susceptible to histamine and its effects can be described as intolerance or intoxication depending on the severity of the symptoms. The amount of histamine in food, the individual sensitivity, and the detoxification activity in human organism represent the main factors affecting the toxicological response in consumers. Histamine is the only biogenic amine with regulatory limits set by European Legislation, up to a maximum of 200 mg/kg in fresh fish and 400 mg/kg in fishery products treated by enzyme maturation in brine.

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.

Biogenic Amines in Italian Pecorino Cheese

The quality of distinctive artisanal cheeses is closely associated with the territory of production and its traditions. Pedoclimatic characteristics, genetic autochthonous variations, and anthropic components create an environment so specific that it would be extremely difficult to reproduce elsewhere. Pecorino cheese is included in this sector of the market and is widely diffused in Italy (∼62.000t of production in 2010). Pecorino is a common name given to indicate Italian cheeses made exclusively from pure 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 from raw or pasteurized milk. The microbiota plays a major role in the development of the organoleptic characteristics of the cheese but it can also be responsible for the accumulation of undesirable substances, such as biogenic amines (BA). Bacterial amino acid decarboxylase activity and BA content have to be investigated within the complex microbial community of raw milk cheese for different cheese technologies. The results emphasize the necessity of controlling the indigenous bacterial population responsible for high production of BA and the use of competitive adjunct cultures could be suggested. Several factors can contribute to the qualitative and quantitative profiles of BA’s in Pecorino cheese such as environmental hygienic conditions, pH, salt concentration, water activity, fat content, pasteurization of milk, decarboxylase microorganisms, starter cultures, temperature and time of ripening, storage, part of the cheese (core, edge), and the presence of cofactor (pyridoxal phosphate, availability of aminases and deaminases). In fact physico-chemical parameters seem to favor biogenic amine-positive microbiota; both of these environmental factors can easily be modulated, in order to control growth of undesirable microorganisms. Generally, the total content of BA’s in Pecorino cheeses can range from about 100–2400 mg/kg, with a prevalence of toxicologically important BA’s, tyramine and histamine. The presence of BA is becoming increasingly important to consumers and cheese-maker alike, due to the potential threats of toxicity to humans and consequent trade implications.