Identification of lactic acid bacteria involved in the spoilage of pasteurized “foie gras” products

The spoiling microflora of a re-packaged French “foie gras” product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile “foie gras” samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of “foie gras” products and will be useful to design new quality protocols and extend the shelf-life of these products.     

Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10 °C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.     

Biocontrol as a strategy to reduce the impact of ochratoxin A and Aspergillus section Nigri in grapes

The efficacy of two strains of Kluyveromyces thermotolerans in preventing the growth and ochratoxin A (OTA) accumulation of ochratoxigenic fungi both “in vitro” and “in situ” was evaluated. The data from this study showed that both yeast strains were able to control Aspergillus carbonarius and A. niger aggregate species growth and ochratoxin A accumulation. The inhibitory effects were dependent on the ochratoxigenic species, yeast strains, a_w and temperature evaluated and their interactions. Over all conditions assayed, ochratoxin A accumulation was reduced from 3% to 100% and the growth rate from 11% to 82.5%, depending on conditions. These results are promising for future development of a bio-pesticide.     

Biodiversity of Saccharomyces cerevisiae populations in Malbec vineyards from the "Zona Alta del Río Mendoza" region in Argentina

The “Zona Alta del Río Mendoza” (ZARM) is the major Malbec grape viticulture region of Argentina. The aim of the present study was to explore Saccharomyces cerevisiae biodiversity in ZARM vineyards. Interdelta PCR and RFLP mtDNA molecular markers were applied to differentiate S. cerevisiae strains. The presence of commercial strains on ZARM vineyards was also assessed. Our results reveal a highly diverse, but genetically closely related, S. cerevisiae population (containing more than 190 molecular patterns among 590 S. cerevisiae isolates). According to the S. cerevisiae strain diversity found in vineyards, they were classified as vineyards with high and low polymorphic S. cerevisiae populations. Six vineyards showed a high polymorphic population, with more than 20 different S. cerevisiae molecular patterns. S. cerevisiae populations in these vineyards were diverse and irregularly distributed, with different strains in each vineyard site. Low polymorphic S. cerevisiae population vineyards displayed very low yeast diversity, with only 9 to 10 different S. cerevisiae strains and presence of two commercial strains widely distributed. Population diversity estimators were calculated to determine the population structure of S. cerevisiae in the ZARM vineyards. The obtained values support the hypothesis that the eight sampled subpopulations come indeed from a larger population.     

The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml⁻¹ after10 days and 400 AU m1⁻¹ after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml⁻¹ were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.     

Functional properties of Lactobacillus strains isolated from kimchi

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) from kimchi, a traditional Korean fermented vegetable product generally consumed raw as a side-dish with practically every meal.Twelve mild acid producing facultatively heterofermentative Lactobacillus strains were selected for their potential as starter cultures for fermentation of kimchi, and evaluated for their functional properties. Eleven strains were identified as Lactobacillus sakei and one as Lactobacillus plantarum. The strains identified as L. sakei differed in some physiological features; of particular interest was the fact that 9 of these strains produced L(+) lactic acid from glucose in presence of acetate.All strains were able to survive gastrointestinal conditions simulating stomach and duodenum passage. In addition, they showed higher adherence to HT-29 cells than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. These strains also showed antimicrobial activity against a number of food-borne pathogens. Their ability to lower cholesterol was demonstrated by BSH (bile salt hydrolytic) activity, and cholesterol assimilation tests in vitro. The results suggest the probiotic potential of these strains for use in kimchi fermentation.     

Chemotype and genotype chemometrical evaluation applied to authentication and traceability of “Tonda Gentile Trilobata” hazelnuts from Piedmont (Italy)

Italian hazelnuts “Tonda Gentile Trilobata” cv are recognised as high quality products with good sensory characteristics and interesting technological properties. An approach was employed to identify the “Tonda Gentile Trilobata” hazelnuts from Piedmont (Italy), differentiating them from other hazelnuts belonging to a different cultivar and/or different geographic origin, on the basis of their chemical and genetic characteristics. Proximate composition, fatty acid profile, polyphenols content and fingerprint, antioxidant activity, and protein patterns were determined. Concerning the genetic analysis, RAPD (Random Amplified Polymorphism DNA) technique was employed to detect polymorphisms amongst different hazelnut samples. Principal Component Analysis (PCA) on the chemical and genomic data sets permitted to identify the “Tonda Gentile Trilobata” cv. The analysis of chemotype allowed differentiating the Chilean sample from the other “Tonda Gentile Trilobata” hazelnuts, whilst no genetic differences were observed amongst these samples. On the other hand, genomic data confirmed that RAPD is able to discriminate amongst cultivars.     

Inhibition by Lactobacillus sakei of other species in the flora of vacuum packaged raw meats during prolonged storage

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm⁻² of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.     

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.     

Lactic acid bacteria population dynamics during minced beef storage under aerobic or modified atmosphere packaging conditions

A total of 266 lactic acid bacteria (LAB) have been isolated from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂–30% O₂–30% N₂ in the presence MAP (+) and absence MAP (−) of oregano essential oil. Sequencing of their 16S rRNA gene along with presence of the katA gene demonstrated dominance of the LAB microbiota by Leuconostoc spp. during aerobic storage at 5, 10 and 15 °C, as well as during MAP (−) and MAP (+) storage at 10 and 15 °C; Lactobacillus sakei prevailed during aerobic storage at 0 °C, as well as at MAP (−) and MAP (+) storage at 0 and 5 °C. The sporadic presence of other species such as Leuconostoc mesenteroides, Weisella viridescens, Lactobacillus casei and Lactobacillus curvatus has also been determined. Pulsed-Field Gel Electrophoresis of high molecular weight genomic DNA revealed the dynamics of the isolated LAB strains. Prevalence of Leuconostoc spp. was attributed to one strain only. On the other hand, packaging conditions affected Lb. sakei strain spoilage dynamics.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze,chill, oxidative,acid and heat stresses

The application of multilocus sequence typing (MLST) for studying Campylobacter jejuni diversity reveals that MLST clonal complex (CC) 21 and CC-45 occupies significant proportion in the diverse population of C. jejuni. These two complexes are ecologically abundant and represent an interesting subpopulation for studying C. jejuni survival under different stress conditions. In the present study we characterize and compare 19 C. jejuni strains assigned to CC-21 and CC-45, isolated from chicken meat, based on laboratory stress models maintained in Muller-Hinton broth. Model conditions were mimicking freeze, chill, oxidative, acid and heat stresses. Results show that survival patterns varied between the strains. C. jejuni strains of CC-21 survived significantly better than C. jejuni strains of CC-45 under heat (P value = 0.022) and chill (P value = 0.001) stress models. On the other hand, C. jejuni strains of CC-45 showed significantly better survival compared to C. jejuni strains of CC-21 in response to oxidative (P value = 0.003) and freeze (P value = 0.021) stress models. C. jejuni strains assigned to the founder ST-45 showed significantly better survival (P value = 0.017) under heat stress model compared to their ancestral sequence types. However, an association between survival fitness and the diversification of a clonal group cannot be demonstrated directly from the obtained results. In conclusion, findings of the present study show that genotypic variations of C. jejuni might play a role in enabling certain lineages to be selected when encountering adverse and stressful environments. In future stress response studies, it is recommended to consider the effect of genotypic diversity among C. jejuni strains as that might bias the experimental findings.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Rheological characteristics of healthy sugar substituted spreadable strawberry product

Full replacement of sucrose with healthier sugars such as fructose and/or isomaltulose is possible in spreadable strawberry products. These products formulated with different sugars (sucrose, isomaltulose, sucrose–glucose, and fructose–isomaltulose) were rheologically analysed. Static tests characterized them as Herschel–Bulkley fluids. The values of the consistency index (k) and yield stress (τ₀ were influenced by the type of sugar, the elaboration method and the pectin levels; while the fluidity index (n) was not affected by the different sugars, but by the elaboration method and the levels of pectin. The dynamic tests permitted classification of some of the products as weak gels. The strength of the network “A” increased with the pectin level, while the “coordination number” “z” did not show a clear trend depending on the different process variables.     

Effect of selected autochthonous starter cultures on processing and quality characteristics of Greek fermented sausages

Selected autochthonous starter (SAS) cultures (i.e. Lactobacillus sakei 8416, Lactobacillus sakei 4413, and L. sakei 8426, L. plantarum 7423 and L. curvatus 8427) were used as starter cultures in addition to a control treatment in the production of fermented sausages. The SAS cultures had a rapid growth and dominated the fortuitous population of LAB during the whole fermentation and ripening process improving the sensory attributes in comparison to control. Apart from the treatment produced with L. sakei 8416, all other SAS cultures prevented the lipid oxidation to values lower than 1 mg malonaldehyde/kg. The Micrococcaceae count and the redness of the sausages was not affected by the smoking and the acidification during the fermentation in the treatments produced with L. sakei 8416 and L. sakei 4413. The treatment of L. sakei 4413 had the lowest (*P < 0.05) content of all biogenic amines. In comparison to the control, the reduction of tyramine was 13%, tryptamine 55%, cadaverine 60% and putrescine 72%. Sausages produced with SAS cultures L. sakei 4413 and L. sakei 8416 had the highest scores for all sensory attributes. The results indicated that the SAS culture of L. sakei 4413 is the best autochthonous starter culture for fermented sausages.     

Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO “Fava Santorinis” (Lathyrus clymenum) adulterants

Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of “Fava Santorinis” are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO “Fava Santorinis”. Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-“Fava Santorinis” in “Fava Santorinis” commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO “Fava Santorinis” and to quantitatively detect adulterations in “Fava Santorinis” with other relative commercial “Fava” food products.     

A “successful allele” at Campylobacter jejuni contingency locus Cj0170 regulates motility; “successful alleles” at locus Cj0045 are strongly associated with mouse colonization

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific “successful alleles” at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a “successful” Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the “unsuccessful” Cj0045 11G allele experienced phase variation to “successful” 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the “successful” 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.