Composition and dynamics of microbiota in different dairy ecosystems

Il formaggio è un cibo biologicamente e biochimicamente dinamico contenente microorganismi sia intenzionalmente aggiunti come colture starter sia presenti come contaminanti avventizi di natura non-starter. La composizione della popolazione microbica si modifica sotto l’influenza di continui cambiamenti delle condizioni ambientali e delle interazioni tra i microorganismi durante la produzione e la stagionatura. Nel processo produttivo del formaggio, la selezione di parametri tecnologici può influenzare o anche indurre vari processi biochimici necessari per l’ottenimento di questo alimento. Le popolazioni microbiche presenti nel formaggio sono complesse e la loro crescita e attività rappresentano le fasi più importanti ma meno controllabili del processo di caseificazione. Lo scopo della presente tesi di dottorato è lo studio della diversità microbica e delle dinamiche delle colture starter, non-starter e dei microrganismi contaminanti coinvolti nei processi di produzione e stagionatura del formaggio. A tale scopo, diversi ecosistemi, dal latte crudo al formaggio, e la crosta sono stati studiati al fine di comprendere il ruolo specifico svolto dai vari microorganismi in ciascuna fase del processo di produzione e correlare la presenza di alcune specie microbiche con lo sviluppo di aromi e strutture desiderate del prodotto. Nello specifico, gli ecosistemi microbici di sieroinnesti naturali, formaggi duri italiani (Grana Padano e Parmigiano Reggiano), e della crosta naturale di un formaggio erborinato stagionato, sono stati studiati per tutto il processo di produzione e stagionatura mediante tecniche culture-dependent e culture-independent. Attraverso lo studio della microflora di sieroinnesti naturali e formaggi a partire dai primi giorni di produzione, è stato possibile descrivere l’attività fermentativa dei batteri lattici starter (SLAB) che determinano il processo di stagionatura del formaggio. L’acidità titolabile del sieroinnesto non è risultata essere in relazione né con la quantità di cellule (totali e coltivabili) né con il contributo specifico delle differenti specie. Elevate concentrazioni di acido lattico e aminoacidi liberi sono state trovate in formaggi con maggiori quantità della specie Lactobacillus helveticus e con alte densità di batteri termofili coltivabili. La presenza di galattosio residuo è risultata associata ad alti contenuti della specie Streptococcus thermophilus. Inoltre, la composizione in termini di biotipi dei sieroinnesti è apparsa molto più importante che la composizione in termini di specie nell’assicurare le buone performance dei sieroinnesti stessi. Una correlazione diretta tra la composizione delle specie SLAB e l’efficacia di acidificazione nei sieroinnesti non è stata trovata. Al contrario, nella matrice formaggio, l’attività fermentativa degli SLAB è sembrata essere specie-dipendente. Negli ecosistemi dei formaggi duri, sia gli SLAB che i LAB non-starter (NSLAB) sono apparsi contribuire all’acidificazione e alla maturazione. Tuttavia, gli SLAB, ed in particolare L. helveticus, sono risultati essere principalmente soggetti alla lisi che si verifica a 2 mesi di stagionatura. I NSLAB sono stati in grado di crescere dopo la salatura, diventando la maggioranza dei microorganismi durante la stagionatura. I NSLAB potrebbero provenire sia dal latte crudo che dal sieroinnesto naturale ed il loro contributo allo sviluppo delle caratteristiche del formaggio è ancora sconosciuto. Inoltre, la presenza di L. helveticus e Lactobacillus delbrueckii subsp. lactis in stato non coltivabile, fino a 13 mesi di stagionatura, suggerisce che queste specie potrebbero svolgere un ruolo diverso ma ancora sconosciuto nella stagionatura del formaggio. La microflora della superficie di un formaggio erborinato durante lo sviluppo naturale della crosta ha mostrato una diversità microbica composta da 14 generi di batteri (Enterococcus; Lactococcus; Leuconostoc; Macrococcus; Staphylococcus; Klebsiella; Brevibacterium; Corynebacterium; Brachybacterium;, Nocardiopsis;, Cobetia; Psychrobacter; Halomonas; Haererehalobacter), due generi di lieviti (Candida; Debaryomyces) e un genere di fungo filamentoso (Penicillium). Sono state osservate elevate e comparabili densità di batteri e lieviti vitali. E’ stata osservata inoltre, un’evoluzione batterica durante la formazione della crosta e nessun genere è rimasto costantemente presente per tutta la durata della stagionatura. Staphylococcus si è dimostrato il genere dominante durante le fasi precoci e successivamente è stato sostituito da Brevibacterium alla fine della stagionatura. Sfruttando esperimenti di interazione, sono stati osservati effetti di inibizione e stimolazione tra le specie; queste interazioni potrebbero spiegare come alcuni microorganismi contribuiscano alla formazione della comunità microbica. Candida catenulata e Debaryomyces hansenii hanno incrementato la crescita di Staphylococcus equorum mentre C. catenulata ha inibito la crescita di D. hansenii. Tuttavia, studi approfonditi dovranno essere svolti per valutare se le specie batteriche, di lievito e muffe sopracitate possano essere utili e svolgere un ruolo nello sviluppo dell’aroma e della struttura di altre varietà di formaggi che presentino simile crosta naturale. In conclusione, grazie ad approcci complementari culture-dependent e culture-independent, è stato possibile identificare quali microrganismi fossero principalmente coinvolti in ciascuna delle matrici casearie studiate, e definire l’importanza della loro presenza che, se in giusto equilibrio, può favorire l’ottenimento delle diverse caratteristiche peculiari di ciascun prodotto.Cheese is a biologically and biochemically dynamic food containing microorganisms both deliberately added as starters and non-starter adventitious contaminants. The composition of microbial population changes under the influence of continuous shifts in environmental conditions and interactions occurring among microorganisms during manufacturing and ripening. In cheese manufacturing, the selection of technological parameters can influence and even induce several biochemical processes needed for this product. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. The aim of the present thesis is the study of microbial diversity and dynamics of starter, non-starter and adventitious microorganisms involved in the cheese manufacturing and ripening processes. Thus, different ecosystems from raw milk to cheese and rind were investigated in order to comprehend the specific role played by microorganisms in each cheese making phase and to correlate the occurrence of certain microbial species with desired flavors and textures. In particular, the microbial ecosystems in natural whey starters, hard Italian cheeses (i.e. Grana Padano and Parmigiano Reggiano), and natural rind of mold-ripened cheese (i.e. blue cheese) were investigated throughout cheese manufacturing and ripening by culture-dependent and culture-independent techniques. By studying the microbiota in natural whey starters and cheese starting from the first days of manufacture, it was possible to describe the fermentative activity of starter lactic acid bacteria (SLAB) determining the ripening progress of the cheese. Whey starter titratable acidity did not seem to be related to the cell amount (total and cultivable cells) nor to the different species contribution. High concentrations of lactic acid and free aminoacids were found in cheeses with higher levels of Lactobacillus helveticus species and cultivable thermophilic bacterial densities. The presence of residual galactose was associated with higher contents of Streptococcus thermophilus species. In addition, the biotype composition of whey starters seemed to be far more important than the species composition in ensuring their good performances. A direct correlation between the composition of SLAB species and the acidifying efficacy in whey starters was not found. Contrarily, in the cheese matrix, the SLAB fermentative activity seemed to be species-dependent. In hard cheese ecosystems, both SLAB and non-starter LAB (NSLAB) seemed to contribute to acidification and ripening. However, SLAB, and in particular L. helveticus, resulted to be the species mainly subjected to the lysis occurring at 2 months of ripening. NSLAB were able to grow after brining, and became more relevant during ripening. NSLAB could arise both from the raw milk and the natural whey starter but their contribution to the development of cheese characteristics is still unknown. Furthermore, the presence of L. helveticus and Lactobacillus delbrueckii subsp. lactis in a non-cultivable state, up to 13 months of ripening, suggests that these species could play a different but still unknown role in cheese ripening. The microbiota on the surface of a blue cheese during the natural rind development showed microbial diversity comprising fourteen genera of bacteria (Enterococcus; Lactococcus; Leuconostoc; Macrococcus; Staphylococcus; Klebsiella; Brevibacterium; Corynebacterium; Brachybacterium;, Nocardiopsis;, Cobetia; Psychrobacter; Halomonas; Haererehalobacter), two yeasts genera (Candida; Debaryomyces) and one filamentous fungal genus (Penicillium). High and comparable densities of viable bacteria and yeasts were observed. Bacterial succession was observed during rind formation and no genus remained constant throughout ripening. The Staphylococcus genus dominated the early stages and then Brevibacterium the later stages. By using interaction experiments, inhibition and stimulation were observed among several species; these interactions could explain how some microorganisms contribute to community formation. Candida catenulata and Debaryomyces hansenii enhanced the growth of Staphylococcus equorum while C. catenulata inhibited D. hansenii growth. However, thorough studies need to be performed in order to evaluate whether the above mentioned bacteria, yeasts and molds can be beneficial and play a role in flavor and texture development of other cheese varieties with similar natural rinds. Overall, thanks to culture-dependent and culture-independent complementary approaches it was possible to identify which microorganisms were mainly involved in each dairy matrix and to address the importance of their presence that, if balanced, can help obtaining the distinctive features of each product

Izdvajanje i identifikacija mikroflore iz kravljeg kolostruma tradicionalnim i molekularnim metodama

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated.Kolostrum je prvo mlijeko, koje proizvodi mliječna žlijezda tijekom ranog postpartalnog razdoblja. Osim što sadrži hranjiva, antimikrobne tvari i hormone rasta, kravlji kolostrum sadržava dragocjenu mikrofloru, uključujući sojeve rodova Lactobacillus i Bifidobacterium što se koriste u proizvodnji probiotičke hrane. Svrha je ovoga rada bila identificirati i kvantificirati mikrofloru u uzorcima kolostruma dvaju Holstein mlječnih krava, uzetih tijekom prva tri dana postpartalnog razdoblja. Mikroorganizmi su određeni sekvenciranjem 16S rRNK gena i LH-PCR metodom kojom se ekstrahira ukupna DNK. U sintetičkoj je podlozi izolirano dvadeset i devet sojeva, identificiranih pomoću sekvencija 16S rRNK gena, te su izdvojena dva potencijalno probiotička soja (Lactobacillus casei i Bifidobacterium pseudolongum). Uzorci su kolostruma analizirani LH-PCR metodom kako bi se utvrdio DNK profil mikroflore, te je utvrđeno da u uzorcima prevladava vrsta Lactobacillus casei. Probiotička svojstva ove bakterije za proizvodnju fermentiranih i probiotičkih proizvoda tek treba istražiti

Izdvajanje i identifikacija mikroflore iz kravljeg kolostruma tradicionalnim i molekularnim metodama

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated.Kolostrum je prvo mlijeko, koje proizvodi mliječna žlijezda tijekom ranog postpartalnog razdoblja. Osim što sadrži hranjiva, antimikrobne tvari i hormone rasta, kravlji kolostrum sadržava dragocjenu mikrofloru, uključujući sojeve rodova Lactobacillus i Bifidobacterium što se koriste u proizvodnji probiotičke hrane. Svrha je ovoga rada bila identificirati i kvantificirati mikrofloru u uzorcima kolostruma dvaju Holstein mlječnih krava, uzetih tijekom prva tri dana postpartalnog razdoblja. Mikroorganizmi su određeni sekvenciranjem 16S rRNK gena i LH-PCR metodom kojom se ekstrahira ukupna DNK. U sintetičkoj je podlozi izolirano dvadeset i devet sojeva, identificiranih pomoću sekvencija 16S rRNK gena, te su izdvojena dva potencijalno probiotička soja (Lactobacillus casei i Bifidobacterium pseudolongum). Uzorci su kolostruma analizirani LH-PCR metodom kako bi se utvrdio DNK profil mikroflore, te je utvrđeno da u uzorcima prevladava vrsta Lactobacillus casei. Probiotička svojstva ove bakterije za proizvodnju fermentiranih i probiotičkih proizvoda tek treba istražiti

EuReCa ONE—27 Nations, ONE Europe, ONE Registry A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe

AbstractIntroductionThe aim of the EuReCa ONE study was to determine the incidence, process, and outcome for out of hospital cardiac arrest (OHCA) throughout Europe.MethodsThis was an international, prospective, multi-centre one-month study. Patients who suffered an OHCA during October 2014 who were attended and/or treated by an Emergency Medical Service (EMS) were eligible for inclusion in the study. Data were extracted from national, regional or local registries.ResultsData on 10,682 confirmed OHCAs from 248 regions in 27 countries, covering an estimated population of 174 million. In 7146 (66%) cases, CPR was started by a bystander or by the EMS. The incidence of CPR attempts ranged from 19.0 to 104.0 per 100,000 population per year. 1735 had ROSC on arrival at hospital (25.2%), Overall, 662/6414 (10.3%) in all cases with CPR attempted survived for at least 30 days or to hospital discharge.ConclusionThe results of EuReCa ONE highlight that OHCA is still a major public health problem accounting for a substantial number of deaths in Europe.EuReCa ONE very clearly demonstrates marked differences in the processes for data collection and reported outcomes following OHCA all over Europe. Using these data and analyses, different countries, regions, systems, and concepts can benchmark themselves and may learn from each other to further improve survival following one of our major health care events

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

Traditional detection methods available for microbial analysis of food are time consuming and not sufficiently sensitive to meet food industries requirements as rapidity, on-site applicability, and cost-effectiveness. Among the more recent rapid methods for detection of microorganisms in food, adenosine triphosphate (ATP) bioluminescence is very suitable for online monitoring of bacterial contamination in food and beverages due to no need for a culturing step or large equipment to fulfill the measurement, rapidity and sensitivity. The availability of sensitive luminometers as well as many commercial ATP-bioluminescent kits allowed the development of various applications in industrial microbiology for rapid in situ determinations. This review summarizes the scientific literature available to date on the use of microbial ATP to determine the microbial load for different beverages and/or foodstuff

Comparison of natural whey starters for Grana Padanocheese using sunray plots

Twenty-one natural whey starters, collected from dairy factories located in six provinces of the Grana Padano production area, were characterized. Basic techniques, such as acidity evaluation and microbial plate count, together with more complex methods such as the Live/Dead® BacLight™ bacterial viability kit, have been used. Seven parameters, including pH, Soxhlet Henkel degrees, microbial plate count in Man Rogosa Sharpe medium and Whey Agar medium, together with count of total, viable and non-viable cells, have been adopted to produce sunray plots. One plot for each natural whey starter sample was obtained by characterizing the status of the microbial culture and compared with three natural whey starters prepared in the laboratory. In this way, a sunray trace is suggested to define the traits of a good natural whey starter. Another multivariate technique, principal component analysis, was applied, and it should be possible to conclude that, for this particular dataset composed of 24 objects and 7 variables, PCA allowed the highlighting of the good and bad samples, while sunray plots, even if remaining only a descriptive and explorative analysis, allowed the better visualizing of the differences among all the samples

Whey starter for Grana Padano Cheese: effect of technological parameters on viability and composition of the microbial community

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4°C for 24 h (cooling treatment), or at −20°C for 24 h (freezing treatment) to evaluate the possibility of conservation, or at 54°C for 1 h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH) reverse transcription (RT)- PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4°C). However, after the freezing (−20°C) and heating (54°C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter pro- duction. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at −20°C and yeast extract addition cannot be suggested as technological innovations

Recovery and Identification of Bovine Colostrum Microflora Using Traditional and Molecular Approaches

Colostrum is the first milk produced by the mammary gland during the early postpartum period. In addition to nutrients, antimicrobial and growth factors, bovine colostrum contains valuable microflora, including members of the genera Lactobacillus and Bifidobacterium, which have been used widely in probiotic food. The aim of this work is to identify and quantify the microflora of colostrum samples from two Holstein dairy cows on the first three postpartum days through culture-dependent 16S rRNA gene sequencing and length heterogeneity polymerase chain reaction (LH-PCR) techniques, using an adapted method to extract the total DNA. Twenty-nine strains isolated in synthetic media were identified by their 16S rRNA gene sequences, revealing two potential probiotic strains (Lactobacillus casei and Bifidobacterium pseudolongum). Colostrum samples were subjected to LH-PCR analysis to obtain fingerprint profiles of the microflora. Lactobacillus casei was the main species present in the samples. The probiotic potential of these bacteria for use in fermented and functional foods remains to be evaluated