Characterization of Enterococcal Community Isolated from an Artisan Istrian Raw Milk Cheese: Biotechnological and Safety Aspects

U ovom su radu istraženi prevalencija, biotehnološki i sigurnosni profil 588 izolata enterokoka prikupljenih iz uzoraka sirovog mlijeka te istarskog sira tijekom zrenja. Unatoč sporadičnoj i varijabilnoj prisutnosti enterokoka u mlijeku ((3,65±2,93) log CFU/mL), nakon 30 dana sazrijevanja sira broj enterokoka je usporedivo visok ((7,96±0.80)) log CFU/g), što potvrđuje da su enterokoki sastavni dio temeljne mikrobiote istarskog sira. Dominantne vrste enterokoka identificirane su kao E. faecium (53,8 %) i E. faecalis (42,4 %), dok su vrste E. durans (2,84 %) i E. casseliflavus (0,95 %) bile zastupljene u manjem broju. Na temelju analize obrazaca metoda otiska prsta (RAPD-PCR), ustanovljena je i velika varijabilnost unutar vrste te detektirano 35 sojeva (genotipova). Većina sojeva bila je specifična za određenu farmu, dok je jedna trećina genotipova detektirana u uzorcima svih šest istraživanih farmi. Ova varijabilnost sojeva odraz je razlika u tehnologiji proizvodnje istarskog sira na različitim farmama, kao što su: različita koncentracija soli, temperatura i relativna vlažnost zraka tijekom zrenja sira, te mikroklimatski ili vegetacijski uvjeti. Među sojevima iste vrste utvrđena je znatna varijabilnost u pogledu biotehnoloških karakteristika, kao i značajne razlike u sposobnosti preživljavanja u simuliranim gastrointestinalnim uvjetima. Znatan broj sojeva bio je otporan na klinički važne antibiotike, kao što su tetraciklin (43,56 %), eritromicin (35,79 %) i vankomicin (23,48 %). Analizom lančane reakcije polimerazom (PCR) nije detektirana niti jedna od genskih determinanti za otpornost na vankomicin i eritromicin; za tetraciklin je detektiran gen tetM.In this study, prevalence, biotechnological and safety profiles of 588 Enterococcus isolates isolated from raw milk and Istrian cheese during different stages of ripening were analyzed. Despite the low and variable presence of enterococci in milk ((3.65±2.93) log CFU/mL), highly comparable enterococcal populations were established after 30 days of cheese ripening ((7.96±0.80) log CFU/g), confirming Enterococcus spp. as a major part of the core microbiota of Istrian cheese. The dominant species were E. faecium (53.8 %) and E. faecalis (42.4 %), while minor groups, consisting of E. durans (2.84 %) and E. casseliflavus (0.95 %), also occurred. A pronounced intraspecies variability was noticed based on molecular fingerprinting, with 35 strains (genotypes) detected. Most of the genotypes were farm-specific with one third being shared between the farms. This genotype variability reflected particular differences of Istrian cheese production, mainly variable salt concentration, ripening temperature and air humidity as well as microclimatic or vegetation conditions. There was considerable variation between the strains of the same species regarding wide range of biotechnologically important traits as well as their ability to survive in simulated gastrointestinal conditions. A considerable number of strains were resistant to critically important antibiotics such as tetracycline (43.56 %), erythromycin (35.79 %) and vancomycin (23.48 %). Polymerase chain reaction-based detection did not identify any of the common genetic determinants for vancomycin and erythromycin resistance; for tetracycline tetM gene was detected. The presence of virulence genes including agg, efaAfs, gelE, cylM, cylB, cylA, esp, efaAfm, cob and cpd was frequently recorded, especially among E. faecalis strains

Proteolitički soj Lactococcus lactis i lipolitički soj Enterococcus durans, izolirani iz tradicionalnog sira i upotrijebljeni kao funkcionalne mesne starter kulture

Research background. As fermentation is an integral feature of both, dry sausage and cheese production, this has led to the evaluation of bacterial cultures Lactococcus lactis ssp. cremoris (LL8307) and Enterococcus durans (ED0207) originally isolated from artisanal Croatian hard type cheese to diversify the range of flavours of dry fermented sausages and to increase their microbiological safety. Both strains were chosen for their high or medium acidifying, proteolytic and/or lipolytic activity and bioprotective potential after step-by-step selection of wild isolates. Therefore, this study aims to evaluate the survival rate of selected starter cultures in wild boar meat sausages during the ripening period of 40 days at a local small-scale facility under artisanal conditions as well as their influence on sausage quality parameters. Experimental approach. Safety, biotechnological and probiotic properties of twenty-three enterococcal and lactococcal isolates of dairy origin were studied. Based on the results, two best candidates were selected and added to the meat batter during the artisanal wild boar meat sausage preparation where their survival rate, effect on physicochemical, microbiological and sensorial properties and histamine content were evaluated. Results and conclusions. As revealed by repetitive element-polymerase chain reaction (rep-PCR), native starter cultures survived up to 15 days of ripening and were either absent from (LL8307) or reduced by 80% (ED0207) in final products. The application of native starter cultures rapidly decreased pH (p<0.05), leading to the significantly lower load of E. coli, coliforms and Enterobacteriaceae in ready-to-eat sausages prepared by the addition of starter cultures (3.04-3.94 log CFU/g) than in the control (3.88-5.00 log CFU/g). Analysis of hedonic test data revealed that some of the sensory traits (odour, flavour, juiciness) of treatments with starter cultures were highly liked by the higher percentage of consumers. The results suggest that these starter cultures would represent a valuable tool to improve the homogeneity of artisanal manufacture and hygienic quality of fermented sausages and can be safely used for food application. Novelty and scientific contribution. This is the first study to explore in depth the biotechnological potential of bacterial cultures isolated from artisanal Croatian cheese as functional starter cultures for high-quality game meat sausage production.Pozadina istraživanja. S obzirom na to da se fermentacija podjednako koristi za dobivanje trajnih kobasica i tvrdih sireva, bakterijske kulture Lactococcus lactis ssp. cremoris (LL8307) i Enterococcus durans (ED0207) izolirane su iz tradicionalnih hrvatskih tvrdih sireva, te detaljno okarakterizirane radi postizanja palete okusa trajnih fermentiranih kobasica i povećanja njihove mikrobiološke sigurnosti. Oba soja su odabrana nakon postupne selekcije autohtonih izolata zbog njihove velike ili srednje velike sposobnosti zakiseljavanja, proteolitičke i/ili lipolitičke aktivnosti, te moguće biozaštitne uloge. Stoga je svrha ovog istraživanja bila procijeniti stopu preživljavanja odabranih starter kultura u kobasicama od mesa divlje svinje tijekom perioda zrenja od 40 dana pri tradicionalnim uvjetima proizvodnje, te ispitati njihov utjecaj na parametre kakvoće kobasica. Eksperimentalni pristup. Ispitana su sigurnosna, biotehnološka i probiotička svojstva dvadeset i triju izolata enterokoka i laktokoka podrijetlom iz hrvatskih tradicionalnih tvrdih sireva. Na osnovi dobivenih rezultata izabrana su dva najbolja kandidata, koja su dodana nadjevu kobasica od mesa divlje svinje, te je procijenjena stopa preživljavanja izolata, njihov utjecaj na fizikalno-kemijska, mikrobiološka i senzorska svojstva kobasica te udjel histamina. Rezultati i zaključci. Analizom ponavljajućih elemenata lančane reakcije polimerazom (rep-PCR) utvrđeno je da su autohtone starter kulture preživjele u kobasicama do 15 dana zrenja, a da ih nije bilo (soj LL8307) ili je njihov broj bio reduciran za 80 % (soj ED0207) u gotovim proizvodima. Primjena odabranih starter kultura brzo je smanjila pH-vrijednost kobasica (p<0,05) i znatno smanjila broj E. coli, koliforma i enterobakterija u gotovim proizvodima pripremljenim s dodatkom starter kultura (3,04-3,94 log CFU/g), u usporedbi s kontrolnim uzorkom (3,88-5,00 log CFU/g). Analiza rezultata hedonističkog testa otkrila je da su se neke od senzornih osobina (miris, okus, sočnost) kobasica proizvedenih s odabranim starter kulturama svidjele većem broju potrošača. Rezultati upućuju na zaključak da ove starter kulture mogu pridonijeti standardizaciji proizvodnje i poboljšanju higijenske ispravnosti tradicionalnih fermentiranih kobasica, te da se mogu sigurno primijeniti u proizvodnji hrane. Novina i znanstveni doprinos. Ovo je prva studija koja je detaljno istražila biotehnološki potencijal bakterijskih kultura izoliranih iz tradicionalnog hrvatskog sira i upotrijebljenih kao funkcionalne starter kulture u proizvodnji visokokvalitetnih kobasica od mesa divljači

N2 gas flushing alleviates the loss of bacterial diversity and inhibits psychrotrophic Pseudomonas during the cold storage of bovine raw milk

Peer reviewe

Strukturelle und funktionelle Diversität der proteolytischen Gene in einem Ackerboden

The present study aimed at investigating the indigenous bacterial community harboring genes encoding for extracellular proteases as well as investigating regulation of extracellular proteases activity in soil. For this purpose, a uniformly managed arable field under integrated management was studied at four sampling sites representing four different soil types and at three different depths. Using different culture independent techniques, it was shown that spatial, vertical and temporal effects exhibit a direct influence on abundance and structure of the proteolytic gene community as well as on the activity of extracellular proteases in soil. The high dynamic of soil bacterial proteolytic communities can be related to differences in quantity and heterogeneity of available substrate, soil texture as well as spatial isolation caused by varying water amount and connectivity of soil particles.Die vorliegende Arbeit hat sich zum Ziel gesetzt, autochthone Bakteriengemeinschaften in Agrarböden auf Gene die für Proteasen kodieren sowie die Regulation der extrazellulären Proteasen zu untersuchen. Dazu wurde ein einheitlich integriert bewirtschafteter Schlag an vier Probenahmestellen mit jeweils verschiedenen Bodentypen und in drei unterschiedlichen Tiefen untersucht. Mittels verschiedener kultivierungsunabhängiger Techniken wurde gezeigt, dass räumliche Heterogenität, vertikales Profil und jahreszeitliche Effekte sowohl auf Abundanz und Struktur von bakteriellen proteolytischen Genen, als auch auf die Aktivität extrazellulärer Proteasen in Böden einen direkten Einfluss hatten. Die hohe Dynamik proteolytischer Gemeinschaften wurde durch Faktoren wie Quantität und Heterogenität verfϋgbarer Substrate, Bodentextur sowie räumliche Isolation aufgrund schwankenden Wassergehalts und Zusammenhalts von Bodenpartikeln beeinflusst

Bacterial diversity of naturally fermented game meat sausages: Sources of new starter cultures.

Bacterial communities associated with the ripening process in artisanal wild boar and deer meat sausages were investigated by molecular barcoding using the 16S rRNA gene as a marker. A core microbiota shared by 83.54% of the samples indicated remarkable level of Lactobacillus sake/and Lactobacillus curvatus, accounting for 20.55% in initial and 70.48% in final products as well as spoilage-associated bacteria including Stenotrophomonas, Bacillus, Pseudomonas, Carnobacterium and Brochothrbc, with an average abundance 44.15% at the beginning and 13.98% at the end of the production. Of selected LAB isolates (n = 555), 43.83% were not suitable for food application due to the antibiotic resistance or the presence of the tric gene. Most of the strains designated as safe were able to grow at 25 degrees C even in the presence of 3.0 and 6.0% of NaCl or pH 4.5, but exposure to the same stressors resulted in growth reduction at 12 degrees C. Acidification and antimicrobial activity were found in 65.62% and 37.50% of strains, respectively. Most of the strains showed lipolytic and proteolytic activity, but only 9.37% were able to degrade sarcoplasmic proteins. These results give important information for the development of new starter formulation for the production of high quality game meat sausages

Microbial key players involved in P turnover differ in artificial soil mixtures depending on clay mineral composition.

Nutrient turnover in soils is strongly driven by soil properties, including clay mineral composition. One main nutrient is phosphorus (P), which is known to be easily immobilized in soil. Therefore, the specific surface characteristics of clay minerals might substantially influence P availability in soil and thus the microbial strategies for accessing P pools. We used a metagenomic approach to analyze the microbial potential to access P after 842 days of incubation in artificial soils with a clay mineral composition of either non-expandable illite (IL) or expandable montmorillonite (MT), which differ in their surface characteristics like soil surface area and surface charge. Our data indicate that microorganisms of the two soils developed different strategies to overcome P depletion, resulting in similar total P concentrations. Genes predicted to encode inorganic pyrophosphatase (ppa), exopolyphosphatase (ppx), and the pstSCAB transport system were higher in MT, suggesting effective P uptake and the use of internal poly-P stores. Genes predicted to encode enzymes involved in organic P turnover like alkaline phosphatases (phoA, phoD) and glycerophosphoryl diester phosphodiesterase were detected in both soils in comparable numbers. In addition, P-o concentrations did not differ significantly. Most identified genes were assigned to microbial lineages generally abundant in agricultural fields, but some were assigned to lineages known to include oligotrophic specialists, such as Bacillaceae and Microchaetaceae

Analytical Methods: Microbiological.

The evaluation of dairy products with reference to compliance with regulatory standards has led to an increased testing of the products for specific microbiological parameters. Milk and dairy products are generally tested for food borne pathogens in addition to spoilage bacteria that can reduce the shelf life of products and cause product spoilage. Culture techniques have traditionally been used, while several rapid techniques based on immunologic and DNA-based methodologies have also been introduced recently. It is important to learn about different microbiological analytical methods that are available and to use the appropriate methods to achieve the desired results