Luca Settanni

Non-starter lactic acid bacteria used to improve cheese quality and provide health benefits.

Non-starter lactic acid bacteria (NSLAB) dominate cheese microbiota during ripening. They tolerate the hostile environment well and strongly influence the biochemistry of curd maturation, contributing to the development of the final characteristics of cheese. Several NSLAB are selected on the basis of their health benefits (enhancement of intestinal probiosis, production of bioactive peptides, generation of gamma-aminobutyric acid and inactivation of antigenotoxins) and are employed in cheese-making. This review describes the ecology of NSLAB, and focuses on their application as adjunct cultures, in order to drive the ripening process and promote health advantages. The scopes of future directions of research are summarised.

Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding

Thirty-five isolates from pig feces were identified as Lactobacillus reuteri (12 strains), Lactobacillus mucosae (7), Lactobacillus plantarum (6), Lactobacillus kitasatonis (3), Lactobacillus rossiae (2), Lactobacillus ultunensis (2), Lactobacillus crispatus (2), and Lactobacillus intestinalis (1) by partial sequence analysis of the 16S rRNA. All isolates were detected at 8-9 log CFU g(-1). Preliminarily, strains were selected based on resistance to heat treatments (ca. 70 degrees C for 10 s). The decrease in viability for some L. reuteri, L. mucosae, L. plantarum, L. kitasatonis, and L. rossiae strains was lower than 1 log cycle. Selected strains were further characterized for acid and bile salt resistance, and antibacterial activity. Except for L. kitasatonis, tolerance to simulated gastric and intestinal conditions was enhanced for all strains by addition of reconstituted skimmed milk. Antibacterial activity was found against Gram-positive and -negative potential pathogens. L. reuteri 8.1, 3S7, 6.2, and 1.2, L. mucosae 1.1R, L. plantarum 4.1, and L. rossiae 4.4 were freeze-dried and mixed (1%, w/w) into pig feed before pelleting. After pelleting, pig feed contained 10-9 log CFU kg(-1) of lactobacilli. L. plantarum 4.1, and L. reuteri 3S7 were selected based on their bile salt resistance, pH tolerance, antimicrobial activity and heat resistance. The findings in this study provide a strong basis for exploring the potential of porcine lactobacilli isolates to be used in pelleted feeding as probiotic additives.

Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a traditional Caciocavallo type cheese

The present work was undertaken to evaluate the influence of the wooden dairy plant equipment on the microbiological characteristics of curd to be transformed into Caciocavallo Palermitano cheese. Traditional raw milk productions were performed concomitantly with standard cheese making trials carried out in stainless steel vat inoculated with a commercial starter. Milk from two different farms (A and B) was separately processed. The wooden vat was found to be a reservoir of lactic acid bacteria (LAB), while unwanted (spoilage and/or pathogenic) microorganisms were not hosted or were present at very low levels. All microbial groups were numerically different in bulk milks, showing higher levels for the farm B. LAB, especially thermophilic cocci, dominated the whole cheese making process of all productions. Undesired microorganisms decreased in number or disappeared during transformation, particularly after curd stretching. LAB were isolated from the wooden vat surface and from all dairy samples, subjected to phenotypic and genetic characterization and identification. Streptococcus thermophilus was the species found at the highest concentration in all samples analyzed and it also dominated the microbial community of the wooden vat. Fourteen other LAB species belonging to six genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Weissella) were also detected. All S. thermophilus isolates were genetically differentiated and a consortium of four strains persisted during the whole traditional production process. As confirmed by pH and the total acidity after the acidification step, indigenous S. thermophilus strains acted as a mixed starter culture.

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

ABSTRACT A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.

Diversity and technological potential of lactic acid bacteria of wheat flours

Lactic acid bacteria (LAB) were analysed from wheat flours used in traditional bread making throughout Sicily (southern Italy). Plate counts, carried out in three different media commonly used to detect food and sourdough LAB, revealed a maximal LAB concentration of approximately 4.75 Log CFU g(-1). Colonies representing various morphological appearances were isolated and differentiated based on phenotypic characteristics and genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR. Fifty unique strains were identified. Analysis by 16S rRNA gene sequencing grouped the strains into 11 LAB species, which belonged to six genera: Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella. Weissella cibaria, Lactobacillus plantarum, Leuconostoc pseudomesenteroides and Leuconostoc citreum were the most prevalent species. The strains were not geographically related. Denaturing gradient gel electrophoresis (DGGE) analysis of total DNA of flour was used to provide a more complete understanding of the LAB population; it confirmed the presence of species identified with the culture-dependent approach, but did not reveal the presence of any additional LAB species. Finally, the technological characteristics (acidifying capacity, antimicrobial production, proteolytic activity, organic acid, and volatile organic compound generation) of the 50 LAB strains were investigated. Eleven strains were selected for future in situ applications.

Yeast microbiota associated with spontaneous sourdough fermentations in the production of traditional wheat sourdough breads of the Abruzzo region (Italy)

The aims of this study were to describe the yeast community of 20 sourdoughs collected from central Italy and to characterize the sourdoughs based on chemical properties. A polyphasic approach consisting of traditional culture-based tests (spore-forming and physiological tests) and molecular techniques (PCR-RFLP, RAPD-PCR, PCR-DGGE) and chemical analysis (total acidity, acids, and sugar contents), was utilized to describe the yeast population and to investigate the chemical composition of the doughs. PCR-RFLP analysis identified 85% of the isolates as Saccharomycescerevisiae, with the other dominant species being Candidamilleri (11%), Candidakrusei (2.5%), and Torulaspora delbrueckii (1%). RAPD-PCR analysis, performed with primers M13 and LA1, highlighted intraspecific polymorphism among the S. cerevisiae strains. The diversity of the sourdoughs from the Abruzzo region is reflected in the chemical composition, yeast species, and strain polymorphism. Our approach using a combination of phenotypic and genotypic methods identified the yeast species in the 20 sourdough samples and provided a complete overview of the yeast populations found in sourdoughs from the Abruzzo region.

Yeast ecology of vineyards within Marsala wine area (western Sicily) in two consecutive vintages and selection of autochthonous Saccharomyces cerevisiae strains.

In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards showed some differences in species composition and concentration levels among 2008 and 2009 vintages. Due to the enological relevance, all Saccharomyces cerevisiae isolates were differentiated applying two genotypic tools (interdelta analysis and microsatellite multiplex PCR of polymorphic microsatellite loci) that recognized 51 strains. Based on the low production of H(2)S, acetic acid and foam, ethanol resistance, growth in presence of high concentrations of potassium metabisulphite (KMBS) and CuSO(4) and at low temperatures, 14 strains were selected and used as starter to ferment grape must at 13 °C and 17 °C in presence of 100 mg/L of KMBS. Three strains (CS160, CS165 and CS182) showed optimal technological aptitudes.

Application of a novel polyphasic approach to study the lactobacilli composition of sourdoughs from the Abruzzo region (central Italy).

Aims:  To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach.

Dissemination of wine-related yeasts by migratory birds.

The present work was undertaken to evaluate the contribution of migratory birds in the environmental dissemination of yeasts. Four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points in Sicily, were analysed during spring and autumnal bird migration and 349 birds (belonging to 10 families) were ringed and analysed for yeast presence. A total of 125 yeasts were isolated and identified by a multiple genotypic approach, consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 18 species, including the technological relevant Saccharomyces cerevisiae which were characterized at strain level applying three techniques (interdelta analysis, minisatellite analysis based on the separate amplification of three genes and microsatellite multiplex PCR of polymorphic microsatellite loci). The evaluation of the persistence of living S. cerevisiae in birds for about 12 h from ingestion of inoculated feed allowed the conclusion that yeasts with technological potential are disseminated during migration.

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.