Maria Elisabetta Fadda

Yeasts associated with Sardinian ewe's dairy products

In the present work, the occurrence of yeasts in different types of typical Sardinian ewes cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.

Characterization of yeast population and molecular fingerprinting of Candida zeylanoides isolated from goat's milk collected in Sardinia

The occurrence of yeast microflora in raw goats milk collected from 62 dairy farms located in different areas of Sardinia was evaluated. Candida zeylanoides was the most frequently occurring species followed by different Basidiomycetous species. In the strains isolated some biochemical characteristics of technological interest were investigated and a predominance of lipolytic yeast species was found. We employed a simple method of DNA extraction that in a minimal time and with low-cost provided a high quality of DNA for RAPD analysis of 32 isolates of C. zeylanoides. The primers M13 and CDU were used and at 40% of similarity, two distinct clusters were observed. The presence of C. krissii species was supposed but further molecular studies are needed to exclude the presence of an as-yet-undescribed species.

Yeast populations in Sardinian feta cheese

In this study, the yeast populations in feta cheese from two different Sardinian dairies were examined. Samples of good quality feta (32) and samples of feta with a slimy surface defect (10) were examined from Dairy A. Similar, samples of good quality feta (23), feta with slimy surface defects (14) and samples with swelling defects (6) were examined from Dairy B. Kluyveromyces lactis was the dominating species in feta from Dairy A (95.2% of samples) followed by Debaryomyces hansenii (76.2%), Dekkera anomala (28.6%) and Dek. bruxellensis (19%). D. hansenii was dominant in samples from Dairy B (93%), followed by K. lactis (23.3%), Geotrichum candidum (23.3%) and Dek. anomala (18.6%). No significant difference was observed between the occurrence of yeast species in feta of good quality and in feta with slimy surface defects, thus confirming that slimy production is not associated with yeast contaminations. The swelling of samples observed in Dairy B seems to be caused by Dek. anomala. In fact, this strong fermenting species was present in all swelled samples in numbers exceeding 10(6) CFU g(-1), while it was isolated in very low concentration in only 5.4% of good samples.

Preliminary Evaluation of Probiotic Properties of Lactobacillus Strains Isolated from Sardinian Dairy Products

Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong β-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

Antilisterial activity of nisin-like bacteriocin producing Lactococcus lactis subsp. lactis isolated from traditional sardinian dairy products.

With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes.

Looking for Candida nivariensis and C. bracarensis among a large Italian collection of C. glabrata isolates: results of the FIMUA working group

Two recently described pathogenic Candida species, C. nivariensis and C. bracarensis, share many phenotypic characteristics with C. glabrata and are easily misidentified as such. The aim of this study was to determine the occurrence of these cryptic species in Italy. One thousand yeast isolates collected in 14 Italian regions and identified as C. glabrata by phenotypic and biochemical methods were included in this study: 928 were screened on CHROMagar and 72 were analysed by a multiplex PCR. None of these cryptic species was identified despite the nationwide distribution and the variety of biological origin of the isolates.

Fungi allergic pathologies: correlation between allergological tests and presence of fungi in the upper respiratory tract

In order to clarify the aetiological role of fungi in determining allergic diseases, we evaluated the correlation between the positive skin test response to fungal allergens and the presence of the same species in the upper respiratory tract in 193 patients with suspected allergic disease to fungi. The results of our study have shown that the isolation and identification of the fungal flora from the patient’s respiratory tract, in addition to the medical history, the symptomatology, and the diagnostic in vivo and in vitro, can be helpful to identify the species responsible for provoking the allergic manifestations, and can be recommended either as a supplement to the diagnosis or as a guidance in elimination treatment.

Application of Autochthonous Lactobacillus Strains as Biopreservatives to Control Fungal Spoilage in Caciotta Cheese

Fungal spoilage is one of the main causes of economic losses worldwide in the food industry. In the last years, consumers demands for preservative-free processed foods have increased as a result of growing awareness about the health hazards associated with chemicals. Lactic acid bacteria have been extensively studied for their antibacterial and antifungal potential in order to be used as biopreservatives. The first objective of this study was to investigate in vitro the antifungal activity of autochthonous Lactobacillus strains against moulds commonly associated with cheese spoilage. Then, the Lactobacillus strains with the highest inhibitory effect and broadest spectrum were tested in single or mixed cultures against Penicillium chrysogenum ATCC 9179 and Aspergillus flavus ATCC 46283 on miniature Caciotta cheese produced at laboratory scale to evaluate in situ their ability to prevent mould growth and to determine their impact on cheese organoleptic properties and starter culture activity. The growth of the starter lactococcal population exhibited similar trend and values during ripening, suggesting that the addition of lactobacilli did not influence its growth and survival. Inhibition of P. chrysogenum inoculated in the milk was determined in cheeses produced with single or mixed Lactobacillus adjuncts as compared to cheeses without adjunct. The mixed adjunct cultures resulted in more effective, significantly reducing mould counts of more than 2 log units at the end of ripening. The application of the adjunct cultures resulted in a delay in mycelial growth of P. chrysogenum and A. flavus inoculated on the cheese surface as well. Finally, we found no significant differences among samples for the sensory parameters evaluated that received similar ratings. Our results indicate that the selected Lactobacillus strains may have a potential effect in controlling mould contamination on cheeses. Further studies are currently being carried out to identify the molecules responsible for the antifungal activity.

Evaluation of biocidal activity of Evolyse, a disinfectant based on hydrogen peroxide and silver nitrate

The in vitro biocidal activity of a novel disinfectant, Evolyse, against a wide spectrum of microorganisms, including bacteria, spores and fungi was analysed. The antimicrobial efficacy of disinfectant delivered by a nebulising device was also evaluated under practical conditions, in different hospital environments by microbiological monitoring of air and surfaces, before and after disinfection treatment. The disinfectant exhibited bactericidal activity against all bacterial strains tested after 5 min contact at room temperature. Sporicidal activity against B. cereus ATCC 11178 spores was obtained after 45 min contact at room temperature. The disinfectant also showed fungicidal activity against A. flavus ATCC 46283 and an A. niger DBS strain after 15 min contact at room temperature and against C. albicans ATCC 10231, S. cerevisiae ATCC 2601 and P. chrysogenum ATCC 9179 after 30 min contact time. Overall, disinfection treatment with Evolyse was able to reduce up to 70% of the microbial growth of air and surfaces of the different hospital environments analysed. The results of this study showed the good biocidal activity of Evolyse and offer promising perspective for the use of this product in hospital environments.