Insect frass in stored cereal products as a potential source of Lactobacillus sanfranciscensis for sourdough ecosystem.

AIM: Insect frass samples were collected from Drosophila melanogaster, Plodia interpunctella, Rhyzopertha dominica, Sitophilus granarius, Sitophilus oryzae, Sitophilus zeamais, Tribolium confusum and Tribolium castaneum to elucidate if they can be the origin of Type I sourdough micro-organisms (Lactobacillus sanfranciscensis and Candida milleri). METHODS AND RESULTS: Selective enrichments were carried out to isolate lactic acid bacteria (LBA) and yeast. A metagenetic analysis, targeted on bacterial 16S rRNA gene and fungal ITS region, was performed by using Illumina MiSeq protocol. In cultivation conditions, Lactococcus garvieae and Rhodotorula mucilaginosa were the most frequently species among LAB and yeasts respectively. The Next Generation Sequencing approach revealed that Enterobacteriaceae, Pseudomonadacae and Bacillaceae were the dominating taxa, accounting for 61% of the bacterial community. Lactobacillus genus showed a relative abundance of only 0·36%, but L. sanfranciscensis proved to be the species most frequent between lactobacilli and predominant in faecal samples of T. castaneum and T. confusum larvae. The core fungal microbiota was constituted by Saccharomycetales, Pleosporaceae and Nectriaceae that attained the 51% of recognized OTUs. While the most abundant yeast genus was Candida (17·1%), sequences belonging to C. milleri were not found. CONCLUSIONS: Frass released by the insects of stored cereal products can be the natural reservoir of L. sanfranciscensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Insect dejections are potential sources of novel strains for controlled bakery productions.

Characterization of malolactic bacteria isolated from Aosta Valley wines and evidence of psychrotrophy in some strains.

AIM: A survey on indigenous malolactic bacteria populations isolated from wines produced in 13 different wineries of Aosta Valley, the highest vine-growing area in Europe, was carried out in order to characterize the dominant strains in spontaneous malolactic fermentation (MLF) and to reveal the appearance of psychrotrophic ones. METHODS AND RESULTS: Fifty-four isolates were identify by ITS rDNA region analysis and partial sequencing of 16S rDNA gene: 80% were ascribed to Oenococcus oeni while the remaining 20% were attributed to Pediococcus damnosus species. The genetic diversity of 43 O. oeni isolates was investigated through PFGE analysis after ApaI and SfiI restriction: 28 different pulso-types were discriminated at a level of similarity of 90%. In general, the MLF was led by more than one strain simultaneously. No connection between genotype and grape variety or vine-training system or geographical site of isolation was observed. The histidine decarboxylase gene was not found in any isolate. Pediococci proved to be more resistant than oenococci to lysozyme. Three O. oeni strains (2A1, 6A2 and 11A4) were able to develop at 10°C in Petit Rouge wine. CONCLUSIONS: Psychrotrophy is a phenotypic trait present in O. oeni species and it may be possible to select strains for the management of MLF in cold climate territories where this biologic transformation is very difficult to control. SIGNIFICANCE AND IMPACT OF THE STUDY: The natural emergence of strains able to perform the MLF at 10°C in wine is a new finding, interesting because it confirms the ecological ability of O. oeni species to adapt itself to environmental conditions by strain phenotype variations. It can be also a starting point for more sustainable oenological practices, since it would be alternative to the conditioning systems of the tanks or of the wineries where they are costly in terms of investment and energy consumption.

Development of a Type I gluten-free sourdough.

UNLABELLED: The aim of this study was the setting up of a gluten-free sourdough from selected lactobacilli and yeasts isolated from a traditional wheat-based Type I sourdough. A gluten-free matrix was inoculated with Lactobacillus sanfranciscensis and Candida humilis, fermented to pH 4·0, and constantly propagated for ten times. A stable association between micro-organisms was observed from the second refreshment with mean values of 9·08 ± 0·25 log CFU g(-1) for lactobacilli and 7·81 ± 0·07 log CFU g(-1) for yeasts. In order to have a good workability of the dough, a 230 BU consistency was considered. Rheofermentographic indices remained constant over the ten refreshments, showing an average value of 23·2 mm dough height in about 7·5 h. The CO2 production and retention volumes reached average values of 1430 and 1238 ml respectively. The microbiological and technological data obtained highlighted that a GF sourdough was effectively developed. SIGNIFICANCE AND IMPACT OF THE STUDY: Type I sourdough has a long tradition as a leavening agent of baked goods as its use results in an improved texture, flavour, taste and extended shelf-life of the final products. In this study a Type I gluten-free sourdough was obtained. After few refreshments in controlled conditions, the sourdough presented a stable association between Lactobacillus sanfranciscensis and Candida humilis, constant fermentation times and technological properties (in terms of dough consistency, dough maximum height, CO2 production and retention). The results showed that the gluten-free sourdough developed in this study can improve the overall quality of gluten-free baked products.

Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment.

UNLABELLED: Cell suspensions of four Dekkera bruxellensis strains (CBS 2499, CBS 2797, CBS 4459 and CBS 4601) were subjected to heat treatment in deionized water at four different temperatures (55·0, 57·5, 60·0 and 62·5°C) to investigate their thermal resistance. The decimal reduction times at a specific temperature were calculated from the resulting inactivation curves: the D-values at 55·0°C ranged from 63 to 79·4 s, at 57·5°C from 39·6 to 46·1 s, at 60·0°C from 19·5 to 20·7 s, at 62·5°C from 10·2 to 13·7 s. The z-values were between 9·2 and 10·2°C, confirming that heat resistance is a strain-dependent character. A protocol for the sanitization of 225 l casks by immersion in hot water was set up and applied to contaminated 3-year-old barrels. The heat penetration through the staves was evaluated for each investigated temperature by positioning a thermal probe at 8 mm deep. A treatment at 60°C for an exposure time of 19 min allowed to eliminate the yeast populations up to a log count reduction of 8. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces/Dekkera bruxellensis is the main yeast involved in red wine spoilage that occurs during ageing in barrel, generating considerable economic losses. Current sanitization protocols, performed using different chemicals, are ineffective due to the porous nature of the wood. The thermal inactivation of D. bruxellensis cells by hot water treatment proves to be efficacious and easy to perform, provided that the holding time at the killing temperature takes into account the filling time of the vessel and the time for the heat penetration into the wood structure.

Short communication: Characterization of Shiga toxin 2-carrying bacteriophages induced from Shiga-toxigenic Escherichia coli isolated from Italian dairy products.

Forty samples of raw milk cheese and 25 samples of raw milk itself were subjected to enrichment culture for Shiga-toxigenic Escherichia coli (STEC), and a single Shiga toxin 2- (Stx(2)) positive strain was obtained from one of the cheese samples. Thus, aged cheeses in which the curd is subsequently heat treated (48°C) cannot be presumed to be STEC free. Infective Stx(2) bacteriophages were induced from 3 STEC strains isolated elsewhere from raw milk and 1 STEC strain from aged cheese sourced in Italy. Data on E. coli host range, morphology, genome size, and genetic variation determined by restriction fragment length polymorphism and multi-locus genotyping are presented. Although all 4 bacteriophages were found to be short-tailed Podoviridae, they exhibited considerable variation in both genome size and content. This extended to the Stx(2) genes themselves, whose sequences contained several point mutations, but these did not translate to amino acid substitutions.

Inactivation of wine spoilage yeasts Dekkera bruxellensis using low electric current treatment (LEC).

AIMS: The objective of this study was to investigate the inactivation of a selected yeast Dekkera bruxellensis strain 4481 in red wine by application of low electric current treatment (LEC). METHODS AND RESULTS: LEC (200 mA) was applied for 60 days to a red wine, Montepulciano d'Abruzzo, in an alternative strategy to the SO(2) addition during wine storage. The LEC effect on both cell activity and microflora viability was assessed. LEC decreased significantly the survival viable cells and increased the death rate of D. bruxellensis strain 4481 yeast. A final comparison was made of the main physico-chemical parameters of the wine after the different treatments. The study suggests the importance of an appropriate LEC treatment which limits wine deterioration in terms of off-flavours synthesis. CONCLUSIONS: The results demonstrate that the growth of undesirable Dekkera can be inhibited by low voltage treatment; LEC was shown to be useful to prevent wine spoilage and has the potential of being a concrete alternative method for controlling wine spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing the growth of undesirable Dekkera yeasts, through the effective use of LEC in the winemaking process.

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines.

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l(-1)), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing.

AIMS: To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau-PCR and amplified fragment length polymorphism (AFLP). METHODS AND RESULTS: Eighty-one isolates from bovine and caprine dairy products (n = 53) and from diseased rainbow trouts and other fishes (n = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84.4% to 97.5% and discriminatory powers from 0.798 to 0.986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau-PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes. CONCLUSION: L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau-PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.

Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments.

AIMS: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. METHODS AND RESULTS: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. CONCLUSIONS: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.

Isolation and characterization of a virulent Lactobacillus sanfranciscensis bacteriophage and its impact on microbial population in sourdough.

Thirty-five sourdough samples used for sweet and salted Italian baked products were checked for the presence of a virus active on Lactobacillus sanfranciscensis species. One phage, named EV3, was isolated and its phenotypic and genotypic features were investigated. It belonged to the Siphoviridae family (morphotype B1); its life cycle at 25 degrees C lasted 3 h with a burst size of about 30 viral particles per infected cell. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed one major structural protein of 35 kDa and four minor proteins. The genome, approximately 32 kb long, was a double-stranded linear DNA molecule with a pac-type system. Phage spreading into sourdough did not adversely affect acidification and volume increase of the dough neither lactobacilli counts; the propagation of viral particles was shown to be hindered. This is the first report of the isolation of a L. sanfranciscensis phage.

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region.

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.

Comparative study of nine Lactobacillus fermentum bacteriophages.

AIMS: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization. METHODS AND RESULTS: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations. CONCLUSION: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. SIGNIFICANCE AND IMPACT OF THE STUDY: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation.

Bactericidal activity of chlorine dioxide against Escherichia coli in water and on hard surfaces.

The efficacy of chlorine dioxide as a disinfectant was evaluated against cells of Escherichia coli ATCC 11229 in aqueous suspension and adhering to the surfaces of stainless steel AISI 304 and PVC. The concentrations tested ranged from 0.7 to 14 mg/liter; the exposure times investigated were 30 s and 1, 2, 4, and 8 min. When the bacteria were suspended in water with 1.4 mg/liter of chlorine dioxide, a 10(5)-fold reduction of the initial viable count occurred within 30 s; when cells were attached to the steel surface, the same rate of inactivation took place only after 6 min with 7 mg/liter or 4 min with 14 mg/liter of chlorine dioxide. A 5-log reduction was not obtained when organisms were adhered to polyvinyl chloride (PVC). Scanning electron microscope micrographs of contaminated surfaces revealed that the PVC was very rough with pores much larger in diameter than the cells. Time values determining a 90% reduction of the E. coli population (90% killing time) were calculated for each concentration of disinfectant tested in suspension and on the steel surface. If the same experimental conditions were strictly adopted, linear functions of the log of bacterial inactivation could be plotted (log 90% killing time versus log concentration of disinfectant). This work showed that results obtained with suspension tests could not be used to estimate disinfection of hard surfaces.