Histologic and histomorphometric analysis of bone regeneration with bovine grafting material after 24 months of healing. A case report

Anorganic bovine bone mineral matrix (ABBMM) has been reported to have osteoconductive properties and no inflammatory or adverse responses when used as grafting material in sinus augmentation procedures. However, controversy remains in regard to degradation rate of ABBMM. The aim of this study was to histologically and histomorphometrically evaluate the degradation of ABBMM in human bone samples obtained in one patient 24 months after sinus augmentation. Materials and Methods: The histologic and histomorphometric analysis was performed by means of light microscopy in three specimens harvested from the same patient, Results: After 24 months the tissue pattern appeared to be composed of residual particles, some in close contact with the newly formed bone, others separated by translucent areas and osteoid tissues. Newly-formed bone presented different levels of maturation and numerous osteocytes, with greater numbers in bone closer to the grafted particles (27.3% vs. 11.2%, p < 0.05). The histomorphometric analysis showed mean values of 40.84% newly-formed bone, 33.58% residual graft material, 23.84% marrow spaces, and 1.69% osteoid tissue, Conclusions: Even though ABBMM underwent considerable resorption, a great amount of residual grafting material was still present after two years of healing following sinus augmentation. This study confirms that the bovine grafts can be classified as long-term degradation materials

Discovering microbiota and volatile compounds of surströmming, the traditional Swedish sour herring

none13noIn this study, the microbiota of ready-to-eat surströmming from three Swedish producers were studied using a combined approach. The pH values of the samples ranged between 6.67±0.01 and 6.98±0.01, whereas their aw values were between 0.911±0.001 and 0.940±0.001. The acetic acid concentration was between 0.289±0.009 g/100 g and 0.556±0.036 g/100 g. Very low concentrations of lactic acid were measured. Viable counting revealed the presence of mesophilic aerobes, mesophilic lactobacilli and lactococci as well as halophilic lactobacilli and lactococci, coagulase-negative staphylococci, halophilic aerobes and anaerobes. Negligible counts for Enterobacteriaceae, Pseudomonadaceae and total eumycetes were observed, whereas no sulfite-reducing anaerobes were detected. Listeria monocytogenes and Salmonella spp. were absent in all samples. Multiplex real-time PCR revealed the absence of the bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP) genes, which encode botulinic toxins, in all the samples analyzed. Metagenomic sequencing revealed the presence of a core microbiota dominated by Halanaerobium praevalens, Alkalibacterium gilvum, Carnobacterium, Tetragenococcus halophilus, Clostridiisalibacter, and Porphyromonadaceae. Psychrobacter celer, Ruminococcaceae, Marinilactibacillus psychrotolerans, Streptococcus infantis and Salinivibrio costicola were detected as minority OTUs. GC-MS analysis of volatile components revealed the massive presence of trimethylamine and sulfur compounds. Moreover, 1,2,4-trithiolane, phenols, ketones, aldehydes, alcohols, esters and long chain aliphatic hydrocarbons were also detected. The data obtained allowed pro-technological bacteria, which are well-adapted to saline environments, to be discovered for the first time. Further analyses are needed to better clarify the extent of the contribution of either the microbiota or autolytic enzymes of the fish flesh in the aroma definition.restrictedLuca Belleggia, Lucia Aquilanti, Ilario Ferrocino, Vesna Milanović, Cristiana Garofalo, Francesca Clementi, Luca Cocolin, Massimo Mozzon, Roberta Foligni, M. Naceur Haouet, Stefania Scuota, Marisa Framboas, Andrea OsimaniBelleggia, Luca; Aquilanti, Lucia; Ferrocino, Ilario; Milanovic, Vesna; Garofalo, Cristiana; Clementi, Francesca; Cocolin, Luca; Mozzon, Massimo; Foligni, Roberta; Naceur Haouet, M.; Scuota, Stefania; Framboas, Marisa; Osimani, Andre

Exploitation of Tenebrio molitor larvae as biological factories for human probiotics, an exploratory study

The exploitation of yellow mealworm (Tenebrio molitor) larvae for the bioaugmentation of probiotic Bacillus clausii strains was evaluated during a 7-day rearing period. qPCR was applied to evaluate the persistence and growth of B. clausii in the rearing substrate and larvae (washed and non-washed). Moreover, the effect of freeze-drying of larvae on B. clausii viability was evaluated. The results demonstrated the suitability of yellow mealworm as biological factories for the multiplication of B. clausii through a simple and inexpensive procedure, in view of the further application of larvae as foods and food ingredients. In more detail, an increase in the load of B. clausii was observed during the 7-day rearing of larvae fed wheat middlings spiked with 1 Log cells g−1. Further research is needed to evaluate the most suitable technologies and processing parameters for obtaining yellow mealworm-based ingredients with a stable and active population of probiotic B. clausii

The Microbial Diversity of Non-Korean Kimchi as Revealed by Viable Counting and Metataxonomic Sequencing

Kimchi is recognized worldwide as the flagship food of Korea. To date, most of the currently available microbiological studies on kimchi deal with Korean manufactures. Moreover, there is a lack of knowledge on the occurrence of eumycetes in kimchi. Given these premises, the present study was aimed at investigating the bacterial and fungal dynamics occurring during the natural fermentation of an artisan non-Korean kimchi manufacture. Lactic acid bacteria were dominant, while Enterobacteriaceae, Pseudomonadaceae, and yeasts progressively decreased during fermentation. Erwinia spp., Pseudomonas veronii, Pseudomonas viridiflava, Rahnella aquatilis, and Sphingomonas spp. were detected during the first 15 days of fermentation, whereas the last fermentation phase was dominated by Leuconostoc kimchi, together withWeissella soli. For the mycobiota at the beginning of the fermentation process, Rhizoplaca and Pichia orientalis were the dominant Operational Taxonomic Units (OTUs) in batch 1, whereas in batch 2 Protomyces inundatus prevailed. In the last stage of fermentation, Saccharomyces cerevisiae, Candida sake, Penicillium, and Malassezia were the most abundant taxa in both analyzed batches. The knowledge gained in the present study represents a step forward in the description of the microbial dynamics of kimchi produced outside the region of origin using local ingredients. It will also serve as a starting point for further isolation of kimchi-adapted microorganisms to be assayed as potential starters for the manufacturing of novel vegetable preserves with high quality and functional traits

New insights into the microbiota of European typical fermented foods of animal origin

La pratica della fermentazione è stata sviluppata empiricamente da varie civiltà antiche con lo scopo di preservare gli alimenti ed esaltare le loro caratteristiche nutrizionali e sensoriali. Ad oggi, gli alimenti fermentati caratterizzano profondamente le abitudini alimentari e il folclore di numerosi paesi del mondo. La presente tesi di dottorato ha lo scopo di esplorare il microbiota e il suo impatto sui tratti microbiologici e fisico-chimici di alimenti fermentati di origine animale tipici europei. Sono stati presi in considerazione prodotti caratterizzati da una fermentazione spontanea, tra cui alimenti a base di pesce (l'aringa acida svedese Surströmming, lo squalo fermentato islandese Hákarl, i salami di pesce fermentati del Sud Italia) e a base di carne (il Ciauscolo e i salami di fegato fermentati, entrambi derivanti dalla regione Marche d'Italia, i salami Cacholeira e Painho de Porto Preto, entrambi derivanti dal Portogallo). A tale scopo, è stato adottato un approccio combinato di differenti tecniche analitiche. Inizialmente, sono stati utilizzati metodi coltura-dipendenti, tra cui l'uso di terreni selettivi di crescita e l'identificazione genotipica di isolati. Tra i metodi coltura-indipendenti, sono stati utilizzati la qPCR con specifici primer di interesse microbiologico, e il NGS di regioni conservate di batteri ed eumiceti. Inoltre, sono state condotte misurazioni fisico-chimiche di parametri chiave che influenzano la crescita microbica. I risultati hanno confermato il maggior contributo dei batteri lattici durante il processo fermentativo sia di prodotti a base di pesce che di carne. Nonostante ci fossero variazioni tra le popolazioni di batteri lattici degli alimenti a base di carne (ad esempio, Leuconostoc carnosum, Dellaglioa algida) e di pesce (ad esempio, Tetragenococcus halophilus, Lactiplantibacillus plantarum), è stata evidenziata la costante presenza di Latilactobacillus sakei in entrambe le matrici. I dati raccolti hanno mostrato anche popolazioni microbiche minori regolarmente identificate negli alimenti fermentati di origine animale, ossia gli stafilococchi coagulasi-negativi (sono state identificate le specie Staphylococcus succinus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus xylosus, Staphylococcus sciuri, Staphylococcus saprophyticus, e Staphylococcus caprae) e i lieviti (Debaryomyces hansenii era prevalente). Sono state condotte analisi per verificare la presenza di geni coinvolti nella produzione di istamina, così come la sua quantificazione, nel Surströmming, nell'Hákarl e nei salami fermentati di pesce. In più, è stato dato un primo sguardo alla presenza di geni coinvolti nella resistenza alle tetracicline nei campioni di Surströmming. La determinazione di istamina e di geni relativi sia alla produzione di istamina che alla resistenza alle tetracicline hanno dimostrato la necessità per un monitoraggio costante e ulteriori investigazioni, inclusa l'applicazione di microrganismi selezionati in base all'assenza di tali determinanti estrinseci. Per ottenere specifiche colture aggiunte o starter in grado di assicurare sicurezza e qualità di prodotti fermentati sia a base di carne che di pesce, sono state isolate specie di batteri lattici, tra cui L. sakei e Latilactobacillus curvatus. La caratterizzazione di questi isolati ha permesso di collezionare alcuni candidati con caratteristiche pro-tecnologiche. La presente tesi di dottorato getta le fondamenta di fonti di biodiversità non ancora esplorate. Le informazioni ottenute forniscono un terreno solido per studi futuri mirati a comprendere le dinamiche di fermentazione e ottimizzare i processi di produzione delle prelibatezze tipiche analizzate.Fermentation was empirically developed by several ancient societies with the aim of preserving foods and enhancing their nutritional and sensory attributes. To date, fermented foods deeply characterize the eating habits and folklore of numerous countries around the world. The present Ph.D. thesis is aimed to explore the microbiota and its impact on microbiological and physico-chemical traits of European traditional fermented foods of animal origin. Spontaneously fermented fish-based products of Northern European countries (Swedish sour herring Surströmming, Icelandic fermented shark Hákarl, fermented fish sausage of Southern Italy) and meat-based products of Mediterranean countries (Ciauscolo salami and fermented liver sausage, both handcrafted in the Marche Region of Italy, Cacholeira blood sausage and Painho de Porco Preto sausage, both manufactured in Portugal) were considered. To reach this goal, a combined approach of different analytical techniques was applied. First, culture-dependent methods, including the use of growth selective media and genotypic identification of selected isolates, were used. Among culture-independent methods, qPCR with specific primers of microbiological interest, and NGS of bacteria and eumycete conserved regions were adopted. In addition, physico-chemical measurements on key parameters affecting microbial growth were carried out. The major contribution of lactic acid bacteria during the fermentative process of both fish-based and meat-based products was clearly confirmed. Although there was variation between lactic acid bacteria populations of meat-based (i.e., Leuconostoc carnosum, Dellaglioa algida) and fish-based products (i.e., Tetragenococcus halophilus, Lactiplantibacillus plantarum), the regular detection of Latilactobacillus sakei in both the food matrices was highlighted. The data collected allowed to notice also minor microbial populations constantly occurring in typical fermented foods of animal origin, namely coagulase-negative staphylococci (the species Staphylococcus succinus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus xylosus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae were identified) and yeasts (Debaryomyces hansenii was prevalent). The occurrence of genes involved in histamine production as well as its quantification in Surströmming, Hákarl and fermented fish sausage were assessed. In addition, a first sight on the occurrence of genes involved in tetracycline resistance in Surströmming samples was given. The detection of histamine and genes related both to histamine production and tetracycline resistance suggested the need for continuous monitoring and further investigations, including the application of microorganisms selected for the absence of such extrinsic determinants. In order to obtain specific adjunct or starter cultures for safety and quality assurance of both fish-based and meat-based products, lactic acid bacteria species, namely L. sakei e Latilactobacillus curvatus, were isolated from fermented fish sausage and Ciauscolo salami samples. The characterization of these isolates allowed to collect few candidates with potential pro-technological features. The present Ph.D. thesis allowed to build the first foundations of biodiversity sources not yet explored. The collected information provides solid ground for future studies aimed to comprehend fermentation dynamics and optimize production processes of typical delicacies analyzed

Fermented fish and fermented fish-based products, an ever-growing source of microbial diversity: a literature review

Fermented fish and fermented fish-based products are part of the diet of many countries all over the world. Their popularity is not only due to the unique flavor, the distinct texture, and the good nutritional quality, but also to the easiness of the production process, that is commonly based on empirical traditional methods. Fish fermentation techniques ususally rely on the combination of some key steps, including salting, addition of spices or additives, and maintenance of anaerobic conditions, thus selecting for the multiplication of some protechnological microorganisms. The objective of the present review was to provide an overview of the current knowledge of the microbial communities occurring in fermented fish and fish-based products. Specific information was collected from scientific publications published from 2000 to 2022 with the aim of generating a comprehensive database. The production of fermented fish and fish-based foods was mostly localized in West African countries, Northern European countries, and Southeast Asian countries. Based on the available literature, the microbial composition of fermented fish and fish-based products was delineated by using viable counting combined with identification of isolates, and culture-independent techniques. The data obtained from viable counting highlighted the occurrence of microbial groups usually associated with food fermentation, namely lactic acid bacteria, staphylococci, Bacillus spp., and yeasts. The identification of isolates combined with cultureindependent methods showed that the fermentative process of fish-based products was generally guided by lactobacilli (Lactiplantibacillus plantarum, Latilactobacillus sakei, and Latilactobacillus curvatus) or Tetragenococcus spp. depending on the salt concentration. Among lactic acid bacteria populations, Lactococcus spp., Pediococcus spp., Leuconostoc spp., Weissella spp., Enterococcus spp., Streptococcus spp., and Vagococcus spp. were frequently identified. Staphylococcus spp. and Bacillus spp. confirmed a great adaptation to fermented fish-based products. Other noteworthy bacterial taxa included Micrococcus spp., Pseudomonas spp., Psychrobacter spp., Halanaerobium spp., and Halomonas spp. Among human pathogenic bacteria, the occurrence of Clostridium spp. and Vibrio spp. was documented. As for yeast populations, the predominance of Candida spp., Debaryomyces spp., and Saccharomyces spp. was evidenced. The present literature review could serve as comprehensive database for the scientific community, and as a reference for the food industry in order to formulate tailored starter or adjunctive cultures for product improvement

Prevalence of histidine decarboxylase genes of Gram-positive bacteria in surströmming as revealed by qPCR

Histamine is a degradation product of the bacterial decarboxylation of the amino acid histidine; such activity is determined by histidine decarboxylase encoded by a gene cluster, carried by some Gram-positive bacteria, that includes the hdcA gene. In this study, the presence of the hdcA gene in ready-to-eat surströmming samples collected from three producers based in Sweden was directly assessed via qPCR analysis for the very first time. Samples from producer A showed hdcA average gene abundance of 6.67±0.13 Log cells/gene copies g-1; in samples from producer B the average value attested at 5.56±0.06 Log cells/gene copies g-1, whereas for samples of producer C hdcA average gene abundance attested at 5.30±0.08 Log cells/gene copies g-1. ANOVA showed a significantly higher average hdcA gene copy number in samples from producer A, whereas no significant differences were seen between average values of hdcA gene copy numbers detected in samples from producer B and C. The hdcA gene copies detected in the present study could give an estimation of the load of potential histamine-producing bacteria in surströmming

Microbial Dynamics of a Specialty Italian Raw Ewe’s Milk Cheese Curdled with Extracts from Spontaneous and Cultivated Onopordum tauricumWilld

Milk coagulants prepared by maceration of flowers harvested from both spontaneous and cultivated Onopordum tauricum Willd. and a commercially available coagulant obtained from Cynara cardunculus L. (control) were assayed for small-scale manufacturing of Caciofiore, an Italian specialty raw ewe’s milk cheese produced in a family run dairy farm located in the Marche region (Central Italy). The microbiota of the three thistle-based milk coagulants and their effect on the microbial dynamics of raw milk cheeses during fermentation and maturation (from day 0 up until day 60) were investigated through a combined approach based on viable counting and Illumina DNA sequencing. In both the control and experimental cheeses, despite the slight differences emerged depending on the coagulant used, Lactococcus lactis and Debaryomyces hansenii were the prevalent species among bacteria and fungi, respectively. Moreover, raw ewe’s milk was the main factor affecting the evolution of both the bacterial and fungal microbiota in all cheeses. The overall similarities between control and experimental cheeses herein analyzed supports the exploitation of Onopordum tauricum Willd. as an alternative milk coagulating agent for production of Caciofiore and, more in general, raw ewe’s milk cheeses

Survey on the willingness to consume insect-based foods [Indagine sulla propensione al consumo di alimenti a base di insetti edibili]

RIASSUNTO Negli ulti mi anni l’interesse negli insetti edibili come fonte di proteine è aumentato, sebbene vi sia ancora scetti cismo da parte dei consumatori. Il presente lavoro ha valutato la propensione al consumo di insetti o alimenti a base di insetti di 222 potenziali consumatori italiani. A tale scopo sono stati uti lizzati insetti edibili reperibili in commercio (cavallett e, camole della farina e grilli) e prodotti da forno sperimentali (pane, waffl e e muffin) con insetti o loro trasformati . Gli insetti commerciali hanno incuriosito l’80% degli intervistati. Il 25% degli intervistati si è dichiarato favorevole all’acquisto degli alimenti contenenti insetti edibili interi. Dai dati è emersa una prevalente neofobia alimentare nelle femmine, che hanno espresso una sensazione di disgusto nel 27% dei casi. Il 45% degli intervistati ha evidenziata una sorprendente propensione al consumo di alimenti contenenti farine di insett o fra i quali il pane con grillo polverizzato che ha ott enuto il 29% delle preferenze. ABSTRACT In recent years interest in the adoption of edible insects as an alternati ve source of protein has increased, although in Europe consumers still react with disgust and rejection. Familiarity with insect-based products could increase the likelihood that they will be used as meat substitutes.The present work aims to analyze the propensity to consumption of insects of 222 potenti al Italian consumers. For this purpose, edible insects available on the market and experimental preparations of bakery products containing insects or processed ones were used. The results showed a prevalent food neophobia in females, which expressed a feeling of disgust in 27% of cases. Furthermore, a surprising propensity (45%) to the consumption of foods containing insect flours was highlighted. These results could be useful for the formulation of insect-based products to be introduced to the market

Profiling of autochthonous microbiota and characterization of the dominant lactic acid bacteria occurring in fermented fish sausages

The use of fish flesh to produce fermented sausages is uncommon, especially in European countries where fermented sausages are mainly obtained using mammalian meat. In the present study, the microbiota naturally occurring in novel fermented fish sausages, handcrafted using marine fish species caught in the Mediterranean Sea, was studied. To this end, fish sausages were subjected to physico-chemical analyses (including histamine quantification). Microbiological traits of sausages were studied via viable counting and metataxonomic analysis. Sausages were also subjected to the detection of genes encoding histidine decarboxylase of both Gram-positive (hdcA) and -negative (hdc) bacteria. The results of histamine quantification showed different contents among fish sausage samples. Moreover, the presence of the hdcA gene was below the detection limit in all the samples, whereas the hdc gene was detected only in samples from batch 2, characterized by high levels of Enterobacteriaceae. In the analysed samples, viable lactic acid bacteria, coagulase-negative staphylococci, and eumycetes were detected. Bacterial composition displayed the highest frequency of Latilactobacillus sakei, whereas eumycetic composition displayed the highest frequency of Kurtzmaniella zeylanoides. In order to select potential adjunct cultures for product improvement, 60 lactic acid bacteria (22 isolates of L. sakei and 38 of Latilactobacillus curvatus) were isolated from sausage samples and characterized for: i) the presence of the hdcA gene; ii) the production of exopolysaccharides (EPS); iii) the presence of genes involved in the production of EPS; iv) the production of bacteriocins against Listeria innocua. None of the isolates tested positive for the presence of the hdcA gene. Moreover, 39 out of 60 isolates showed the formation of mucoid colonies, thus attesting the production of EPS. Interestingly, 56 out of 60 isolates were positive for the gene epsD/E, whereas 37 out of 60 isolates were positive for the gene epsA, all these genes encoding the production of heteropolysaccharides. Of note, the EPS production capability and the absence of hdcA gene could represent a starting point for future selection of the isolates as autochthonous adjunct cultures to improve texture, sensory traits and safety of the fermented fish sausages under study. None of the L. sakei or L. curvatus isolates exerted a bactericidal effect against L. innocua