Giorgia Perpetuini

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.

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.

Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods

Biogeography is the descriptive and explanatory study of spatial patterns and processes involved in the distribution of biodiversity. Without biogeography, it would be difficult to study the diversity of microorganisms because there would be no way to visualize patterns in variation. Saccharomyces cerevisiae, “the wine yeast,” is the most important species involved in alcoholic fermentation, and in vineyard ecosystems, it follows the principle of “everything is everywhere.” Agricultural practices such as farming (organic versus conventional) and floor management systems have selected different populations within this species that are phylogenetically distinct. In fact, recent ecological and geographic studies highlighted that unique strains are associated with particular grape varieties in specific geographical locations. These studies also highlighted that significant diversity and regional character, or ‘terroir,’ have been introduced into the winemaking process via this association. This diversity of wild strains preserves typicity, the high quality, and the unique flavor of wines. Recently, different molecular methods were developed to study population dynamics of S. cerevisiae strains in both vineyards and wineries. In this review, we will provide an update on the current molecular methods used to reveal the geographical distribution of S. cerevisiae wine yeast.

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

Characterization of specialized flocculent yeasts to improve sparkling wine fermentation

Flocculent wine yeasts were characterized for the expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes, growth kinetics and physicochemical properties of the cell surface during a 6‐month sparkling wine fermentation period.

Chromosome arrangement, differentiation of growth kinetics and volatile molecule profiles in Kluyveromyces marxianus strains from Italian cheeses

Thirty-nine strains of Kluyveromyces marxianus from Pecorino di Farindola cheese in comparison with 3 strains from Parmigiano Reggiano cheese, 1 from fermented milk, 3 from cow whey and two type strains K. marxianus CBS 834(T) and Kluyveromyces lactis CBS 683(T) were tested for genetic and metabolic characteristics. Intraspecific diversity of chromosome arrangements was evaluated by pulsed field gel electrophoresis (PFGE) analysis. Among K. marxianus strains chromosome polymorphisms were evident with 11 patterns that differed in size and number of the chromosomal bands. The number of the bands varied from 4 to 7 with sizes ranging from about 1.0 to 2.7 Mb. Twelve strains were selected for determining their growth capacity and volatile compound production in two wheys (raw cheese whey and ricotta cheese whey) under limited oxygen availability. The growth kinetics highlighted four different biotypes and the influence of whey composition on K. marxianus development. The main volatile compounds detected after the growth were alcohols, acids, esters, ketones and aldehydes. Ethanol was the most abundant in both wheys. Aldehydes and other minor compounds were produced only when the strains were inoculated in ricotta cheese whey, while esters, butanoic, decanoic and octanoic acids were qualitatively and quantitatively more present in raw cheese whey. This study highlights a great genetic and metabolic biodiversity within Pecorino di Farindola K. marxianus strains and it could be exploited to improve the knowledge of this yeast for biotechnological uses.

Long-term impact of farm management and crops on soil microorganisms assessed by combined DGGE and PLFA analyses

In the present study, long-term organic and conventional managements were compared at the experimental field of Monsampolo del Tronto (Marche region, Italy) with the aim of investigating soil chemical fertility and microbial community structure. A polyphasic approach, combining soil fertility indicators with microbiological analyses (plate counts, PCR-denaturing gradient gel electrophoresis [DGGE] and phospholipid fatty acid analysis [PLFA]) was applied. Organic matter, N as well as some important macro and micronutrients (K, P, Mg, Mn, Cu, and Zn) for crop growth, were more available under organic management. Bacterial counts were higher in organic management. A significant influence of management system and management x crop interaction was observed for total mesophilic bacteria, nitrogen fixing bacteria and actinobacteria. Interestingly, cultivable fungi were not detected in all analyzed samples. PLFA biomass was higher in the organic and Gram positive bacteria dominated the microbial community in both systems. Even if fungal biomass was higher in organic management, fungal PCR-DGGE fingerprinting revealed that the two systems were very similar in terms of fungal species suggesting that 10 years were not enough to establish a new dynamic equilibrium among ecosystem components. A better knowledge of soil biota and in particular of fungal community structure will be useful for the development of sustainable management strategies.

Biogenic Amines: Toxicology and Health Effect

Biogenic amines (BAs) are organic bases that play several physiological roles in humans, acting as local hormones and neurotransmitters modulating smooth muscle cell contraction, vasodilatation, increased vascular permeability and mucus secretion, tachycardia, alterations of blood pressure, arrhythmias, gastric acid secretion, and nociception in nerve fibers. However, when ingested in high concentrations, they can cause a range of toxicological effects such as migraine, headaches, gastric and intestinal problems, and pseudoallergic responses, mainly due to the toxic action of histamine and tyramine. In particular, histamine toxicity seems enhanced by the presence of other amines such as cadaverine, putrescine, and tyramine, which act as potentiators of its toxicity. Moreover, some BAs (spermidine, spermine, tyramine, putrescine, and cadaverine), when subjected to heat, can give rise to the formation of secondary amines, and in the presence of nitrites, these can generate nitrosamines, chemical agents considered to possess major carcinogenic properties. Under normal conditions, the human body is able to detoxify BAs ingested from foods by acetylation and oxidation by monoamine oxidase (MAO) and diamine oxidase (DAO) and specific amine methyltransferases. However, this detoxification system can be negatively influenced by some food components (other amines, alcohol and its metabolite acetaldehyde, phenols, etc.), drugs acting as inhibitors of MAO and DAO and tobacco.

Food borne bacterial models for detection of benzo[a]pyrene-DNA adducts formation using RAPD-PCR

Random amplified polymorphic DNA (RAPD) PCR is a feasible method to evaluate genotoxin‐induced DNA damage and mutations. In this study, Lactobacillus plantarum ATCC 14917T, Enterococcus faecium DSMZ 20477T, Escherichia coli PQ37 and Saccharomyces cerevisiae S441 were screened for DNA genetic alterations by DNA fingerprinting using M13 and LA1 primers after treatment with three compounds forming covalent adducts with DNA [benzo[a]pyrenediol epoxide (BPDE), methyl methanesulfonate and 1,2,3,4‐diepoxybutane (DEB)]. M13 RAPD fingerprinting revealed that the total number of bands decreased in all treated DNA compared to control samples and generally the lost bands were characterized by high molecular weight. Some extra bands were detected for L. plantarum and E. faecium, while in E. coli and S. cerevisiae DNAs BPDE and DEB treatments did not result in new extra bands. Besides qualitatively analysis, cluster analysis based on Unweighted Pair‐Group Method with Average algorithm was performed to compare DNA fingerprints before and after treatments. This analysis confirmed the absence of significant differences between negative controls and treated DNA in S. cerevisiae and E. coli however the disappearance of some bands can be detected. The data indicate that this approach can be used for DNA damage detection and mutations induced by genotoxic compounds and highlighted the possible use of L. plantarum and E. faecium M13 based fingerprinting as reference for hazard identification in risk assessment.