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Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group ( P < 0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari’s volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota. [ABSTRACT FROM AUTHOR]

Parmigiano Reggiano (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value. Long ripening times allow for extensive proteolysis of milk proteins to yield a number of peptides, some of which have potential healthy bioactive properties. This study aimed to: i) determine the peptide profile of PR cheese subjected to simulated gastrointestinal transit; ii) evaluate in vitro whether the peptides could support growth of beneficial microbial groups of the gut microbiota. PR samples were subjected to in vitro digestion, simulating oral, gastric, and duodenal transit. Liquid chromatography coupled with tandem mass spectrometry revealed that digestion caused the disappearance of the serum proteins and most of the original peptides, while 71 new peptides were found, all ranging from 2 to 24 residues. The digests were given as sole nitrogen source to pure cultures of Bifidobacterium (27 strains) and Lactobacillus (30 strains), and to bioreactor batch cultures of human gut microbiota. Most of bifidobacteria and lactobacilli grew more abundantly on PR digests than on the control peptone, and exhibited strain- or species-specific peptide preferences, as evidenced by principal component analysis. Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptide consumption and the number of peptides consumed. For bifidobacteria, peptide preferences were very diverse, but a core of 10 peptides with 4 or 5 residues were consumed by all the strains. Lactobacilli behaved more homogenously and consumed nearly only the same 6 peptides, mostly dipeptides. The peptide preferences of the different groups of bifidobacteria and lactobacilli could not be ascribed to features such as the length of the peptide or the abundance of residues with peculiar properties (hydrophobicity, polarity, charge) and likely depend on specific proteases and/or peptide transporters preferentially recognizing specific sequence motifs. The cultures of human colonic microbiota confirmed that PR digest promoted the growth of commensal bifidobacteria. This study demonstrated that peptides derived from simulated gastrointestinal digestion of PR supported the growth of most lactobacilli and bifidobacteria. [ABSTRACT FROM AUTHOR]

Artisanal Minas cheese is produced in Minas Gerais state, Brazil and its varieties are named according to their geographical origin (Serro, Canastra, Serra do Salitre, Araxá and Campo das Vertentes). The cheese is produced with raw cow's milk and the whey from the previous cheese production (“pingo”). The high economic and cultural importance of artisanal cheese in Brazil justifies the efforts to ensure its safety, quality and provenance. This study aimed to characterize the microbial diversity composition, and geographical distribution of artisanal Minas cheese, focusing on the characterization of its autochthonous lactic acid bacteria (LAB) microbiota. Artisanal Minas cheese varieties from Serro, Canastra, Serra do Salitre, Araxá and Campo das Vertentes were analyzed by culture-dependent (culturing and LAB sequencing) and -independent (repetitive extragenic palindromic-PCR (rep-PCR) and length heterogeneity-PCR, LH-PCR) methods to characterize the microbiota. The microbial counts were variable between cheese samples, and some samples presented high number of coagulase positive bacteria and coliforms that may be associated with hygienic issues. In all samples was observed a prevalence of LAB. 16S rRNA sequencing and rep-PCR of the LAB strains identified four genus ( Lactobacillus , Lactococcus , Enterococcus and Weissella ), ten species and more than one strain per species. Lactobacillus was the most prevalent genera in all the cheeses. LH-PCR revealed a further six genera and ten species that were not identified by culturing, highlighting the importance of combining both culture-dependent and -independent methods to fully characterize microbiota diversity. Principal component analysis of the LH-PCR data and cluster analysis of rep-PCR data revealed that the artisanal Minas cheese microbiota was influenced not only by their geographical origin but also by the cheese farm. The lack of standardization in the milking and cheese manufacturing procedures between artisanal cheese farms could explain the microbial diversity. [ABSTRACT FROM AUTHOR]

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads < 5.0 Log cfu g − 1 . Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. [ABSTRACT FROM AUTHOR]

In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13 months of ripening in two dairies. The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2 months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria ( Lactobacillus helveticus and Lactobacillus delbrueckii ) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus / Lactobacillus casei and Pediococcus acidilactici . On the other hand, the cheese from dairy 1 that ripened for 2 months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13 months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus / L. casei ) growth during ripening. [ABSTRACT FROM AUTHOR]

Commission Regulation (EC) No. 2073/2005 established, as a food safety criterion for tolerable levels of Listeria monocytogenes in ready-to-eat foods which do not support the growth of the pathogen or with shelf life below 5 days, a maximum of 100 cfu g −1 . Blue-veined cheeses are among these foods because their rinds can be contaminated, and the pathogen can be transferred to the paste during slicing. The aim of this research was to investigate whether cutting procedures could be responsible for cheese paste contamination. Considering that the Commission Regulation limit is allowed when the pathogen does not grow during the shelf life, we also wanted to verify whether, in the case of positive dragging, L. monocytogenes was able to grow on cut slices beyond the limit imposed, thereby becoming a risk for consumers during storage at 4 °C. Gorgonzola cheese was chosen for this investigation. The cutting simulation on artificially inoculated wheel rinds indicated that greater rind contamination corresponded to a higher percentage of contaminated paste samples. The growth of L. monocytogenes transferred to cut slices was variable relative to the physicochemical characteristics of the cheese, to the contamination level and to the time of storage. In particular, the sweet typology was able to support the growth of L. monocytogenes in the shelf life conditions considered and the quick overcoming of the limit imposed by food safety criteria would not ensure the safety for consumption. [ABSTRACT FROM AUTHOR]

The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: β-naphthyl amide derivatives of l-anomers of leucine, lysine, proline, glycine-proline, and phenilalanine-proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile. [ABSTRACT FROM AUTHOR]

Abstract: Blue-veined cheeses may allow Listeria monocytogenes survival and multiplication due to the biochemical characteristics of the cheese and the growth characteristics of the pathogen. Because of the availability of a considerable number of samples, we wanted this study to take a large view of the microbiota and of the incidence and genetic diversity of L. monocytogenes in blue-veined cheese rinds. Moreover, we wanted to determine if the pathogen present on the rinds at the end of ripening represented a risk to the consumer if the cheese should exceed the domestic storage limit imposed by Commission Regulation (EC) No 2073/2005 for food safety criteria. The rind microflora showed a high level of heterogeneity, and the incidence of L. monocytogenes (never found in the paste samples) was 55%, with an increased presence and concentration associated with a longer ripening time. Amplified Fragment Length Polymorphism (AFLP) was used to characterise L. monocytogenes isolated from blue-veined cheese rinds and highlighted the heterogeneity among the strains, demonstrating its suitability for studying the biodiversity of the pathogen in this environment. L. monocytogenes was able to grow during the shelf life of the cheese and was influenced by the refrigeration temperatures and the physicochemical characteristics of the cheese. [Copyright &y& Elsevier]

Physical properties, microbiological quality and volatile compounds of sous-vide-processed carrots and Brussels sprouts were analysed after 1, 5 and 10 days of refrigerated storage. Similar analyses were performed on raw and steamed carrots and Brussels sprout for comparison. Sous-vide carrots were firmer than steamed and firmness increased during storage. Similar values of colour indices were obtained for both steamed and sous-vide carrots, which also showed an increase of redness and colour saturation during storage. Aerobic and anaerobic counts of raw carrots were significantly reduced both by steaming and sous-vide procedures at values lower than 1 log cfu/g; these values were maintained until the end of storage. Among volatiles, terpenes were better preserved in sous-vide than steamed carrots, but all volatile fractions were significantly affected during storage. Steamed Brussels sprouts were softer and greener than sous-vide-processed ones, but softening and loss of green of the latter increased under vacuum storage. Microbiological counts of raw products were more efficiently reduced by sous-vide procedures than steaming. Higher amounts of nitriles and terpenes were shown in sous-vide vegetables in comparison to steamed, whereas thiocyanates and isothiocyanates occurred in lower quantities. Volatiles were not affected during 5 days of storage. [ABSTRACT FROM AUTHOR]

The achievement of a sufficient acidity level by starters at early stages of manufacture is essential for a good quality Parmigiano Reggiano (PR) cheese. In this work, the chemical parameters and microbial characteristics of 18 PR cheeses during the moulding phase were analysed to determine how the microbial growth takes place conditioning acidification performances. Based on the mean values of lactic acid content, two groups of cheeses were observed. High numbers of culturable thermophilic bacteria and in particular Lactobacillus helveticus able to metabolise galactose but also grow faster in the cheese appeared to be essential conditions to achieve adequate lactic acid content. On the other hand, low acidification and residual galactose, as observed in the second group, may be associated with a lower amount of metabolically active L. helveticus and higher relevance of Streptococcus thermophilus contents in the cheese. While culturable thermophilic bacteria were fundamental for higher lactic acid production, non-culturable cells did not seem to contribute to the acidification. [ABSTRACT FROM AUTHOR]

Bacterial growth media represent a high cost in industrial applications, and for this reason, it is economically important to find less expensive supplements to replace the traditional ones. In the present work, peptide hydrolysates obtained from poultry meat and bone residues (functional animal protein [FAP]) and from feathers (functional feather protein [FFP]) were studied to determine their ability for the production of microbial biomass with improved viability. The results obtained were compared with those obtained with other supplement nutritive compounds used in fermentation growth media. The molecular composition of the hydrolysates in terms of total and soluble nitrogen, molecular weight distribution, total and free amino acids, was determined. The growth and cellular state of Bifidobacterium and Lactobacillus strains were studied by turbidimetric measurements and direct count by fluorescence microscopy. Overall, this study suggested that by-products from poultry industry provide a good alternative to substitute expensive supplements for growth of Lactobacillus and Bifidobacterium with a high level of viability [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to evaluate the potential of target tRNAAla–23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNAAla–23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNAAla–23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods – P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) – for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNAAla–23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNAAla–23S rDNA has high potential in bacterial identification and tRNAAla–23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products. [Copyright &y& Elsevier]

Abstract: Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFR-tyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC50 values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC50, while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR B max and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM. [Copyright &y& Elsevier]

Abstract: CXCR4 and CXCR7 chemokine receptors, and their ligands CXCL11 and CXCL12, have been often involved in tumor cell proliferation and survival. We report the expression pattern of these ligand/receptor pairs in 22 human meningiomas. High CXCR7 and CXCL12 expression was associated with high-proliferative tumors. CXCR7 levels were correlated to the content of both ligands, suggesting a possible autocrine regulation. CXCR4 and CXCL12 were homogeneously expressed within tumor cells, while CXCR7 was mainly detected in tumor endothelial cells and CXCL11 in pericytes. Our results highlight the preferential CXCR7 and CXCL12 expression within more aggressive tumors and the possible role of CXCR7 in meningioma vascularization. [Copyright &y& Elsevier]

Abstract: A partial picture of the typical microflora of PDO Parmigiano Reggiano cheese was achieved by studying the cultivability of lactic acid bacteria associated with its manufacturing and ripening. A comprehensive sampling design allowed for the analysis of the cheese microflora during its production over 20 months of ripening. An innovative cheese agar medium (CAM) was prepared after testing 18 formulations all based on grated Parmigiano Reggiano ripened cheese. During cheese manufacturing and ripening, different samples were sampled and their microflora was recovered using CAM in comparison with other traditional media. Colonies which formed units from the different agar media tested were picked and isolated; the phylogenetic positions of 154 isolated strains were studied at level of species by 16S-rRNA gene sequencing. CAM seems to be able to recover the minority population coming from milk and whey starter, hardly estimable, during the first hours of production, on traditional media. [Copyright &y& Elsevier]

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD 133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotimb and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relation- ship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect. [ABSTRACT FROM AUTHOR]

Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (104–105 cfu g−1) and then decreased to 102 at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45°C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese. [Copyright &y& Elsevier]

• Natural and synthetic parazoanthines were identified as CXCR4 antagonists. • Parazoanthine-B was the most potent compound with an IC 50 of 9.3 nM. • An optimized pharmacophoric model was obtained from parazoanthine scaffold. CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer metastasis and inflammatory diseases. Starting from our previously-developed pharmacophoric model, we applied a combined computational and experimental approach that led to the identification of the hydantoin alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae , as novel CXCR4 antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B was identified as the most potent CXCR4 antagonist with an IC 50 value of 9.3 nM, even though all the investigated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency and efficacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure–activity relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening. [ABSTRACT FROM AUTHOR]

In recent years, increasing consumer's demand for even cleaner label, functional, safe and high quality products has led to searching for new antimicrobial agents of natural origin that can improve quality and safety with reducing the impact on the product composition. In this scenario the use of algae extracts in food formulations as antimicrobial agents is taking more and more interest. In particular, the antimicrobial potential of Arthrospira platensis has already been tested in vitro , using the agar well diffusion method or the broth dilution method, that can provide a quantitative estimation of minimum inhibitory concentration (MIC). Even if several methods are already in use to test the MIC and minimum bactericidal concentration (MBC) of antimicrobials, the aim of the present research was to propose impedance measurement as a valid method for the in vitro evaluation of MIC and MBC of a natural antimicrobial extract of A. platensis. To this purpose, six different concentrations of A. platensis extract (0.1%, 0.15%, 0.2%, 0.25%, 0.3% and 0.5% v/v) were tested on Pseudomonas fluorescens and Serratia liquefaciens , two species commonly involved in food spoilage, and Listeria innocua , as representative of the human food borne pathogenic species Listeria monocytogenes. The results obtained confirmed the in vitro antimicrobial potential of A. platensis extract, but also highlighted how MIC and MBC could be different depending on both the concentration of antimicrobial and the tested strain. Furthermore, the proposed method allowed the identification of MIC and MBC values in a new way never used before for this purpose. • Impedometric method for the evaluation of MIC and MBC values. • A. platensis extract activity against pathogen and spoilage bacteria. • MIC and MBC defined by two impedometric parameter. • MIC and MBC varying depending on the strains and antimicrobic concentration. [ABSTRACT FROM AUTHOR]

• Wild Lb. paracasei enhances flavour of pasteurized cow milk Caciotta cheese. • Experimental production of test and control Caciotta cheeses. • QRT-PCR quantification of starter and adjunct culture in curd and cheeses. • Aromatic profile, colour, texture and sensorial perception significantly different. Caciotta is the name used to define a type of Italian semi-hard cheese Caciotta-type cheese. Due to the short ripening time, pasteurization is necessary to eliminate the potential pathogenic bacteria, which may be present in raw milk, causing also the reduction of ripened cheese flavor. The purpose of this research was to evaluate the effect of a selected wild Lactobacillus paracasei strain experimentally used as adjunct culture to enhance the flavour formation in a short-ripened caciotta-type cheese. An integrated polyphasic approach was used to compare the experimental and control Caciotta produced in a company located in Emilia Romagna region (Italy). It was demonstrated how the L. paracasei 4341 was able to develop in curd and cheese interacting with the acidifying commercial starter. The main acidifying starter species, were differently affected by the presence of the adjunct culture. Streptococcus thermophilus shown comparable behavior in all cheese-making step of control and experimental Caciotta, while Lactobacillus delbrueckii subsp bulgaricus, growth was slowed down by the presence of the adjunct culture during the whole ripening time. The higher amount of volatile compounds and organic acids due to the adjunct L. paracasei 4341 lead to a clear differentiation of the experimental Caciotta respect to the control, in terms of aromatic profile, color, texture and sensorial perception. [ABSTRACT FROM AUTHOR]

Thermophilic and mesophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus , Lactobacillus delbrueckii subsp. bulgaricus , Lactobacillus helveticus, and Lactococcus lactis , play a crucial role in the technological and sensory quality of Mozzarella cheese. In this study, the safety (genes encoding virulence factors and antibiotic resistance) and acidifying activity of autochthonous S. thermophilus cultures were evaluated in order to choose the most suitable strain for industrial application. The safe and good acidifying culture was tested in two buffalo Mozzarella cheese batches: Mozzarella cheeses produced with autochthonous culture (SJRP107) and commercial culture (STM5). The cultivable LAB was evaluated by culture-dependent method (plate counting) and the quantification of S. thermophilus cultures (commercial and autochthonous) were evaluated by culture-independent method RealT-qPCR (real-time quantitative polymerase chain reaction). The texture, physicochemical and proteolytic properties of the Mozzarella cheeses were similar for both batches. The nonstarter LAB count was higher during manufacture than in the storage, and the RealT-qPCR indicated the presence of S. thermophilus culture until the end of storage. S. thermophilus SJRP107 presented high potential for safety application in the production of Mozzarella cheese. Furthermore, considering the culture characteristics and their relationship with product quality, further studies could be helpful to determine their effect on the sensory characteristics of the cheese. • Mozzarella cheese produced with autochthonous or commercial cultures were compared. • Texture, chemical and proteolytic properties were similar for both cultures. • St. thermophilus SJRP107 proved to be safe and stable all over the storage time. • St. thermophilus SJRP107 showed high technological aptitude for industrial use. [ABSTRACT FROM AUTHOR]

• qPCR and metataxonomics were applied to describe the dynamics of NWS microbiota. • Species present in the whey respond differently to cheese-making technology. • The microbiota of NWS, regardless of initial composition, is shaped by technology. Natural starter cultures are undefined multiple-strains culture communities of mostly thermophilic lactic acid bacteria (LAB), in association with minor amounts of mesophilic bacteria, which structure could be affected by small changes in the parameters of the cheese/whey-making process. This study aims to investigate the complex microbiota of natural whey starter (NWS) used in Parmigiano Reggiano (PR) cheese-making, focusing on both the absolute and relative abundance of bacterial species and on the modification of the bacterial community under environmental and technological pressures. To reach this purpose a combined approach, using quantitative PCR (qPCR) and High-Throughput Sequencing (HTS), was used to investigate the bacterial dynamics of 91 whey samples collected during different steps of PR cheese-making, in one dairy, through two different lines of production, one Conventional and one Organic, over a 10 weeks period. Our results highlighted that NWS used for the production of PR cheese is a dynamic microbial community, which adapts to the different technological parameters encountered in the cheese/NWS manufacturing process, while retaining a high level of resilience of the thermophilic LAB species mainly involved in the steps of curd acidification and the early maturation process. Differences were also observed in bacterial species diversity between samples from Conventional and Organic line but, in conclusion, NWS resulted to be shaped by technological treatments, regardless of its initial different composition. [ABSTRACT FROM AUTHOR]

Bacterial production of exopolysaccharides (EPS) is of increasing interest near food manufacturers, biotechnology industries and nutritionists because of their different roles. Several analytical methods are available for recovery, quantification and characterization of EPS from lactic acid bacteria (LAB) in food. However, direct screening method for production of EPS is still based on the visual observation of filamentous texture of the colonies developed on supplemented solid growth media. To overcome weaknesses of many currently used screening methods, we propose adopting impedance microbiology to evaluate the EPS production from LAB in milk. In this work we have proven that the peculiar shape of capacitance curve of Lactobacillus delbrueckii subsp. bulgaricus 2214, measured in milk by means of a BacTrac 4300® system, is due to production of EPS. Besides the pH measurement, the amounts of EPS evaluated after 0, 8, 13 and 55 h of incubation in milk, were in agreement with the evaluation of gene expression and confirmed by the observations by confocal laser scanning microscopy and by transmission electron microscopy. With the aim to verify the applicability of the proposed method, the drop entity of the capacitance curve (ΔE%) of 22 EPS-producing (EPS+) LAB strains and one negative (EPS-) control was evaluated both in broth medium and in milk. The positive ΔE% value found for all of the strains cultivated in the clear broth medium allowed to confirm the EPS production, simply observing a strain-dependent amount of EPS on surface of the measurement electrodes of the device. When the same EPS+ strains were cultivated in milk, the obtained ΔE% values showed that only a few of them were able to produce EPS in this environment, supporting their diversified ability to utilize lactose for this purpose. Results obtained by this multidisciplinary study demonstrate that impedance microbiology represents a suitable method to overcome the limits of the most commonly used methods to screen LAB for EPS production in milk. Moreover, these results also open a door to the application to other food and beverages, in which the EPS produced in situ could be of great interest for food industry. • We hypothesized descending shape of conductance curve was due to EPS production. • Multidisciplinary study was used to prove our hypothesis. • The positive value of ΔE% is a measure of LAB EPS production. • This method could overcome the lacks of the common used screening methods in food. [ABSTRACT FROM AUTHOR]

The microbial ecology fundamentals of raw milk and long-ripened cheeses consist of a complex interaction between starter lactic acid bacteria (SLAB) and non-starter LAB (NSLAB). Although NSLAB aromatic properties are paramount, other phenotypic traits need to be considered for their use as adjunct cultures, such as the capability to endure technological parameters encountered during cheesemaking. The present study focused on the isolation and characterization of NSLAB from spontaneously fermented raw cow's milk coming from 20 dairies that produce Grana Padano PDO cheese. From 122 isolates, the screening process selected the 10 most diverse strains belonging to Lacticaseibacillus spp. to be phenotypically characterized. The strains were tested for their growth performance in milk in combination with the application of technological stresses, for their ability to produce volatile compounds after their growth in milk, and for their ability to use different nutrient sources and resist chemicals. The complex characterization qualified the strains 5959&#95;Lbparacasei and 5296&#95;Lbparacasei as the best candidates to be used as adjunct strains in the production of raw milk and long-ripened cheeses, provided that antibiotic resistance is measured before their employment. Other strains with interesting aromatic capabilities but lower heat resistance were 5293&#95;Lbparacasei, 5649&#95;Lbparacasei and 5780&#95;Lbparacasei, which could be candidates as adjunct strains for uncooked cheese production.

The present study investigated some physico-chemical and microbiological traits of 20-month ripened hard cheeses produced from low-temperature high-speed centrifuged raw milk that developed a structural defect consisting of eyes or slits in the paste. Cheeses obtained using the same process and that did not develop the defect were used as controls. The colour, texture, moisture, water activity, proton molecular mobility, microstructure, extent of proteolysis, and viable microorganisms have been evaluated in all the cheese samples, and the significant differences between the defective and non-defective cheeses have been critically discussed. At a microstructural level, the defects caused fat coalescence and an unevenly organised protein matrix with small cracks in the proximity of the openings. The different fat organisation was correlated to a different transverse relaxation time of 1 H population relaxing at higher times. The textural and colour features were not different from those of the control cheeses and were comparable with those reported in the literature for other long-ripened hard cheeses. On the other hand, the defective cheeses showed a higher moisture level and lower lactobacilli and total mesophilic bacteria concentrations, but the microbial origin of the defect remains an open hypothesis that deserves further investigation.

In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures' preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bettera, Dreier, Schmidt, Gatti, Berthoud and Bachmann.)

Lactic Acid Bacteria (LAB) exert a fundamental activity in cheese production, as starter LAB in curd acidification, or non-starter LAB (NSLAB) during ripening, in particular in flavor formation. NSLAB originate from the farm and dairy environment, becoming natural contaminants of raw milk where they are present in very low concentrations. Afterward, throughout the different cheesemaking processes, they withstand chemical and physical stresses becoming dominant in ripened cheeses. However, despite a great body of knowledge is available in the literature about NSLAB effect on cheese ripening, the investigations regarding their presence and abundance in raw milk are still poor. With the aim to answer the initial question: "which and how many LAB are present in cow raw milk used for cheese production?," this review has been divided in two main parts. The first one gives an overview of LAB presence in the complex microbiota of raw milk through the meta-analysis of recent taxonomic studies. In the second part, we present a collection of data about LAB quantification in raw milk by culture-dependent analysis, retrieved through a systematic review. Essentially, the revision of data obtained by plate counts on selective agar media showed an average higher concentration of coccoid LAB than lactobacilli, which was found to be consistent with meta-taxonomic analysis. The advantages of the impedometric technique applied to the quantification of LAB in raw milk were also briefly discussed with a focus on the statistical significance of the obtainable data. Furthermore, this approach was also found to be more accurate in highlighting that microorganisms other than LAB are the major component of raw milk. Nevertheless, the variability of the results observed in the studies based on the same counting methodology, highlights that different sampling methods, as well as the "history" of milk before analysis, are variables of great importance that need to be considered in raw milk analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bettera, Levante, Bancalari, Bottari and Gatti.)

Exopolysaccharides (EPS) are complex molecules produced by some microorganisms and used in foods as texturizers and stabilizers, their properties depending on their chemical structure. In this work, three different lactic acid bacteria (LAB), were tested for their ability to produce EPS, by using five different mono- and disaccharides as their sole carbon source. The growth and acidifying ability were analysed, the EPSs were quantified by the official method AOAC 991.43, and their chemical structure was investigated. The amount of EPS varied from 0.71 g/L to 2.38 g/L, and maltose was the best sugar for EPS production by Lacticaseibacillus paracasei 2333. Lacticaseibacillus rhamnosus 1019 produced the highest amount when fed with lactose, whereas the EPS amount of Lactobacillus bulgaricus 1932 was not significantly different depending on the sugar type. The EPS chains consisted of fructose, galactose, glucose, mannose, ribose, glucosamine, galactosamine, and in some cases rhamnose in different proportions, depending on the strain and carbon source. The molecular weight of EPS ranged from <10 KDa to >500 KDa and was again highly dependent on the strain and the sugar used, suggesting the possibility of growing different strains under different conditions to obtain EPS with different potential applications in the food system.

Time of ripening has a strong impact on shaping the valuable and recognizable characteristics of long-ripened types of cheese such as Parmigiano Reggiano (PR) due to the interrelationship between microbiota and proteolysis that occurs during ripening. The derived peptide profile is linked to cheese quality and represents the canvas for enzymes upon digestion, which could be responsible for the release of potentially bioactive peptides (BPs). In this study, we aimed at investigating the presence of BP in 72 PR cheese samples of different ripening times, from curd to 24 months of ripening, produced in six different dairies, and following their fate after simulated gastrointestinal digestion. A small number of peptide sequences sharing 100% similarity with known antimicrobial, antioxidant, and ACE-inhibitor sequences were found in PR cheeses, while a higher number of potential BPs were found after their simulated gastrointestinal digestion, in different amounts according to ripening time. Taking advantage of the complex organization of the sampling plan, we were able to follow the fate of peptides considered quality drivers during cheese ripening to their release as functional compounds upon digestion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Castellone, Prandi, Bancalari, Tedeschi, Gatti and Bottari.)

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E% max -E% 40h ) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lactic acid bacteria (LAB) are involved in producing a considerable number of fermented products consumed worldwide. Many of those LAB fermented foods are recognized as beneficial for human health due to probiotic LAB or their metabolites produced during food fermentation or after food digestion. In this review, we aim to gather and discuss available information on the health-related effects of LAB-fermented foods. In particular, we focused on the most widely consumed LAB-fermented foods such as yoghurt, kefir, cheese, and plant-based products such as sauerkrauts and kimchi.

This study aims at proposing the use of Arthrospira platensis, commonly known as Spirulina, extract as a non-invasive method to attenuate the growth rate of non-starter adjunct cultures, thus preventing the over-acidification that may occur during cheese manufacturing. A preliminary screening using four different concentrations (0.20%, 0.30%, 0.50%, and 0.70%) of A. platensis extract and four starter and three non-starter lactic acid bacteria strains was performed by impedometric analysis. This allowed us to select one starter and one non-starter strain to be used in the in vitro simulation of a co-culture in milk with the best antimicrobial concentration (0.3%). The growth dynamics of the two selected strains, starter Lactococcus lactis 1426 and non-sarter Lacticaseibacillus rhamnosus 1473, co-cultured for 120 h was monitored by three different approaches: (i) plate counting on M17, for the enumeration of lactococci, and MRS for lactobacilli; (ii) fluorescence microscopic counting of viable and non-viable coccoid Lactococcus lactis 1426 and rod-shaped Lacticaseibacillus rhamnosus 1473 cells; (iii) the overall estimation of co-culture growth behavior by impedometric parameters Lag, Rate, and yEnd. All the data obtained from the in vitro simulation were in agreement, revealing that a slowdown of non-starter growth occurred, while the starter strain was not affected, or slightly stimulated, from the antimicrobial presence. In particular, the growth of Lb. rhamnosus 1473 was delayed without adversely compromise the cells' integrity, connected with metabolic functions, showing a great potential for use in cheese production.

Background: Biologically appropriate raw food (BARF) diet is becoming more and more popular among pet owners in Europe. However, there are documented microbiological risks associated with raw feeding, and this study aimed to determine the presence of human pathogens in commercially frozen BARF products sold in Italy., Methods: Salmonella species, Escherichia coli O157:H7, Listeria monocytogenes and Campylobacter species were identified. The general microbiological quality of BARF products and hygiene were also evaluated. Sample size was limited and therefore the study may not be representative of a larger sample., Results: None of the tested samples showed total bacterial count (TBC) higher than the limit set to consider a sample unacceptable. However, 14 out of 21 samples showed TBC higher than the limit set to consider a sample marginally acceptable. A high percentage of samples were contaminated by the aforementioned pathogens, highlighting the need for pet owners to be aware of the risks of this feeding strategy both to themselves and to their pets., Conclusions: Considering that BARF diet meals can be prepared at home using the hands, as well as tools and spaces that could be shared, guidelines on safer handling of these pet food products should be recommended by veterinarians and nutritionists., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Cheese microbiota contribute significantly to the final characteristics of cheeses due to the growth and interaction between cheese microorganisms during processing and ripening. For raw milk cheeses, such as Parmigiano Reggiano (PR), the microbiota derive from the raw milk itself, the dairy environment, and the starter. The process of cheese making and time of ripening shape this complex ecosystem through the selection of different species and biotypes that will drive the quality of the final product by performing functions of their metabolism such as proteolysis. The diversity in the final peptide and amino acid composition of the cheese is thus mostly linked to the diversity of this microbiota. The purpose of this study was to get more insight into the factors affecting PR cheese diversity and, more specifically, to evaluate whether the composition of the bacterial community of cheeses along with the specific peptide composition are more affected by the ripening times or by the cheese making process. To this end, the microbiota and the peptide fractions of 69 cheese samples (from curd to cheese ripened 24 months) were analyzed during 6 complete PR production cycles, which were performed in six different dairies located in the PR production area. The relation among microbial dynamics, peptide evolution, and ripening times were investigated in this unique and tightly controlled production and sampling set up. The study of microbial and peptide moieties in products from different dairies - from curd to at least 12 months, the earliest time from which the cheese can be sold, and up to a maximum of 24 months of ripening - highlighted the presence of differences between samples coming from different dairies, probably due to small differences in the cheese making process. Besides these differences, however, ripening time had by far the greatest impact on microbial dynamics and, consequently, on peptide composition., (Copyright © 2020 Bottari, Levante, Bancalari, Sforza, Bottesini, Prandi, De Filippis, Ercolini, Nocetti and Gatti.)

Lactobacillus helveticus is a thermophilic lactic acid bacterium that is widely employed as a starter culture for manufacturing several Swiss and Italian hard-cooked cheeses. The sequencing of L. helveticus Lh 23, which consists of 2,100,230 bp with a GC content of 36.5%, reveals industrially useful traits and interesting metabolic pathways., (Copyright © 2020 Bertani et al.)

Arthrospira platensis, commercially known as Spirulina, is a fresh-water cyanobacterium that has been gaining increasing attention in recent years due to its high biological and nutritional value. For this reason, it has been employed in several food applications, to obtain or enhance functional and technological properties of cheese, yogurt, bread, cookies or pasta. The aim of this work was to evaluate the potential boosting effect of two different concentrations (0.25% and 0.50% w / v ) of A. platensis on the fermentation capability of several starter lactic acid bacteria (LAB) strains, 1 probiotic and 4 commercial mix culture. These strains were used to ferment three different substrates and their fermentation behaviors were evaluated by impedance analyses together with rheological and color measurements. In tryptic soy broth (TSB), the A. platensis boosting effect was significantly higher if compared to yeast extract for all the starter LAB strains except for Lb. casei , which was equally stimulated. Different results were found when the same LAB strains were cultivated in SSM. The most evident boosting effect was found for S. thermophilus and Lb. casei. LAB growth was promoted by A. platensis , confirming that it could be a useful tool in the production of novel functional fermented dairy foods. The potential boosting effect was evaluated on four commercial mix cultures used to produce milk and soy fermented beverages. It was demonstrated that the booster effect took place, but it was variable and dependent not only on the mix culture used, but also on the substrate and A. platensis concentration. Also, rheological and color modifications were found to be dependent on these factors.

Plant derived beverages have recently gained consumers' interest, particularly due to their intrinsic functional properties. They can also act as non-dairy carriers for probiotics and prebiotics, meeting the needs of lactose allergic/intolerant people and vegans. Direct fermentation of fruit and vegetables juices by probiotic lactic acid bacteria could be a tool to increase safety, shelf-life, nutrients bioavailability and to improve sensorial features of plant derived juices. This study aims to screen wild Lactobacillus casei -group strains isolated from dairy matrices for probiotic features, such as acid and bile salts resistance, and test them for the potentiality to ferment celery and orange juices. Strains' ability to produce exopolysaccharides (EPS) in situ is also checked. These evaluations were performed for the first time in fruit and vegetables matrices by means of an impedometric analysis, recently shown to be a suitable and rapid method to measure microorganisms' growth, acidification performances and EPS production. This study allowed the selection of three potentially probiotic L. casei -group wild strains able to ferment fruit and vegetable juices and also producing EPS. These strains with three-in-one abilities could be used to produce new functional fermented plant derived juices.

Despite the fact that strains belonging to Weissella species have not yet been approved for use as starter culture, recent toxicological studies open new perspectives on their potential employment. The aim of this study was to evaluate the ability of a wild Weissella minor ( W 4451) strain to modify milk viscosity compared to Lactobacillus delbrueckii subsp. bulgaricus , which is commonly used for this purpose in dairy products. To reach this goal, milk viscosity has been evaluated by means of two very different instruments: one rotational viscometer and the Ford cup. Moreover, water holding capacity was evaluated. W 4451, previously isolated from sourdough, was able to acidify milk, to produce polysaccharides in situ and thus improve milk viscosity. The ability of W 4451 to produce at the same time lactic acid and high amounts of polysaccharides makes it a good candidate to improve the composition of starters for dairy products. Ford cup turned out to be a simple method to measure fermented milk viscosity by small- or medium-sized dairies., (Copyright © 2020 Bancalari, Alinovi, Bottari, Caligiani, Mucchetti and Gatti.)

Autochthonous lactic acid bacteria (LAB) play a key role in the development of cheese flavor. As the pasteurization treatment on raw milk causes the elimination of LAB, secondary starter cultures are used in cheese manufacture to obtain cheeses with improved and standardized flavors. In this work, strains of the L. casei group isolated from traditional Italian cheeses were screened for their phenotypic features of technological interest for use as secondary starters. Their milk acidifying performance and the production of volatile compounds when grown in milk were evaluated. Simultaneously, the acetoin metabolic pathway presence was screened in the strains and assessed for its transcriptional activation. The results showed that the analyzed strains, despite belonging to taxonomically-related species, vary greatly according to the measured phenotypes. Four strains among the fourteen screened could be potentially used as adjunct cultures for cheese-making processes. The strain that showed the highest production of acetoin upregulated the aspartate pathway. An increased knowledge of volatile compounds' production and acidifying properties of LAB strains isolated from traditional dairy products might guide the selection of strains for industrial applications.

Members of the Lactobacillus casei group, including species classified currently as L. casei , L. paracasei , and L. rhamnosus , are among the most frequently found species in raw milk, hard cooked, long-ripened cheeses. Starting from very low numbers in raw milk, they become dominant in the cheese during ripening, selected by physical and chemical changes produced by cheese making and ripening. Their presence at different stages of cheese making and ripening is crucial in defining product features. For these reasons, the scientific community has been more and more interested in studying these "tiny but mighty microbes" and their implications during cheese making and ripening. The present paper reviews the current literature on the effect of L. casei in cheeses, with particular reference to the case of Parmigiano Reggiano and Grana Padano, two of the most famous PDO (Protected Designation of Origin) Italian cheeses. Recent advances regarding the selection of new wild strains able to persist until the end of ripening and carrying out slow but crucial activities resulting in specific aromatic features, are also presented.

Background: Fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) are an heterogeneous group of compounds that can be poorly digested and may have a range of effects on gastrointestinal processes. FODMAPs are found in a wide variety of foods, including bread. FODMAPs' intake is associated with the onset of symptoms of irritable bowel syndrome (IBS). On the other hand, some FODMAPs contribute to the healthy maintenance of intestinal microbiota. Volume increase of bread dough commonly relies on the use of two biological leavening agents, sourdough and baker's yeast and, in some cases, a combination of both. Scope and Approach: The main objective of this review is to discuss the association between FODMAPs and IBS, beneficial effects of FODMAPs on healthy subjects and potential impact of biological leavening agents on FODMAPs content of bread. Key Findings and Conclusion: Given that yeasts and lactic acid bacteria, the dominant microorganisms in sourdough, may degrade FODMAPs, it would be possible to modulate the FODMAPs concentration in bread, thus positively affecting consumers' health.

Lactobacillus helveticus is a lactic acid bacterium widely used in cheese-making and for the production of bioactive peptides from milk proteins. Here, we describe the draft genome sequence and annotation of L. helveticus strain Lh 12 isolated from natural whey starter used in the production of Grana Padano cheese., (Copyright © 2018 Bertani et al.)

Pectin oligosaccharides (POS) have been indicated as a new class of potential prebiotic compounds, which can be produced from pectin-rich food byproducts. In the present study, different technological means of POS production were explored to produce tailor-made POS mixtures starting from sugar beet pulp. The overall POS production process consisted of two steps: the extraction of pectin and the hydrolysis of pectin to tailored POS by combined hydrolysis/fractionation approaches. Different extraction as well as hydrolysis and fractionation methodologies were applied. The obtained POS were characterized for their total galacturonic acid content and, at a deeper level, using a HILIC-ESI/MS methodology, for the POS structure and composition. The composition of POS fractions was studied as a function of the technology used to obtain them. Finally, the potential prebiotic properties of the POS mixtures obtained were thoroughly explored by several in vitro experiments aimed at detecting lactic acid bacteria (LAB) stimulation by POS fractions. Several fractions were very efficient in stimulation, in a species-dependent manner. The overall best fractions were in general those rich in arabinans having a low degree of polymerization, obtained from the enzymatic extraction of biomass and subsequent fractionation with low-medium molecular weight cut-off. Quite interestingly, no POS fraction was able to stimulate pathogenic E. coli strains. The data reported here clearly indicate the possibility to obtain diverse fractions with different prebiotic properties starting from the same biomass, and outline clear potential for POS obtained from sugar beet pulp with the appropriate technology to act as prebiotic compounds.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM), the most common primary brain tumor in adults, is an aggressive, fast-growing and highly vascularized tumor, characterized by extensive invasiveness and local recurrence. In GBM and other malignancies, cancer stem cells (CSCs) are believed to drive invasive tumor growth and recurrence, being responsible for radio- and chemo-therapy resistance. Mesenchymal stem cells (MSCs) are multipotent progenitors that exhibit tropism for tumor microenvironment mediated by cytokines, chemokines and growth factors. Initial studies proposed that MSCs might exert inhibitory effects on tumor development, although, to date, contrasting evidence has been provided. Different studies reported either MSC anti-tumor activity or their support to tumor growth. Here, we examined the effects of umbilical cord (UC)-MSCs on in vitro GBM-derived CSC growth, by direct cell-to-cell interaction or indirect modulation, via the release of soluble factors. We demonstrate that UC-MSCs and CSCs exhibit reciprocal tropism when co-cultured as 3D spheroids and their direct cell interaction reduces the proliferation of both cell types. Contrasting effects were obtained by UC-MSC released factors: CSCs, cultured in the presence of conditioned medium (CM) collected from UC-MSCs, increased proliferation rate through transient ERK1/2 and Akt phosphorylation/activation. Analysis of the profile of the cytokines released by UC-MSCs in the CM revealed a strong production of molecules involved in inflammation, angiogenesis, cell migration and proliferation, such as IL-8, GRO, ENA-78 and IL-6. Since CXC chemokine receptor 2 (CXCR2), a receptor shared by several of these ligands, is expressed in GBM CSCs, we evaluated its involvement in CSC proliferation induced by UC-MSC-CM. Using the CXCR2 antagonist SB225002, we observed a partial but statistically significant inhibition of CSC proliferation and migration induced by the UC-MSC-released cytokines. Conversely, CXCR2 blockade did not reduce the reciprocal tropism between CSCs and UC-MSCs grown as spheroids. In conclusion, we show that direct (cell-to-cell contact) or indirect (via the release of soluble factors) interactions between GBM CSCs and UC-MSCs in co-culture produce divergent effects on cell growth, invasion and migration, with the former mainly causing an inhibitory response and the latter a stimulatory one, involving a paracrine activation of CXCR2.

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.