No abstract

No abstract

The application of a culture-independent approach, that of reverse transcriptase-length heterogeneity-PCR coupled with epifluorescence microscopy, allowed us to observe that Streptococcus thermophilus is metabolically active, but only partially cultivable in Grana Padano cheese whey starters. A short preincubation of the starters in sterile skimmed whey was followed by cultivation in sterile skimmed whey-enriched M17. This procedure restored the cultivability of S. thermophilus and enabled us to detect S. thermophilus at ranges (10(7)-10(8) CFU mL(-1)) which have rarely been reported in these cultures. The use of cheese whey as a cultivation-revitalization substrate can be useful to obtain an unbiased picture of the microbial composition of whey starters for Grana Padano cheese, thus avoiding an underestimation of S. thermophilus in these cultures.

Significance and Impact of the Study: There is increased interest in using donkey's milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey's milk provide protection from microbial infections and distinguish donkey's milk from the milks of other mammals. However, the microbiota in donkey's milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey's milk. AbstractThe diversity of lactic acid bacteria (LAB) species in donkey's milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heattreated at 63°C for 10 min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey's milk have been discussed below.

Some species of Leuconostoc are very important for fermented dairy products, as they contribute to the organoleptic characteristics of butter and cream, and also contribute to the formation of openings in some soft, semi-hard (Edam and Gouda cheeses), many artisanal or in blue-veined cheeses, such as Roquefort. In this study, 14 Leuconostoc strains isolated from cheese and cheese-related products were characterized by genotypic and phenotypic methods, and their technological performance assessed for their potential use as dairy adjunct starters. Phenotypic characterization allowed these strains to be classified to genus level, and genotypic studies (RAPD-PCR and 16S rRNA gene sequencing) identified them to species/ subspecies level. Five Leuconostoc strains grew well and acidified milk, and most of them grew even at 8°C. They showed moderate resistance to heat treatments (30 min at 63°C) and grew well in the presence of 3% and 4% NaCl, and were significantly inhibited at pH ≤ 5. All strains showed resistance against the bacteriophages tested. In general, the antibacterial properties observed were slight and due to acid production, with the exception of Leuconostoc citreum MB1, which strongly inhibited Listeria monocytogenes ATCC 15313 by the production of a bacteriocin-like compound. All Leuconostoc strains studied were susceptible to gentamicin, tetracycline, erythromycin and ampicillin. Some strains also showed interesting technological and antimicrobial properties, thus being potentially appropriate as adjunct starters in fermented dairy products. This study highlights that adventitious lactic acid bacteria can be a great source of novel strains with interesting technological features that could be used for fermented dairy foods.

Lactic acid bacteria (LAB) counts, PepX activity towards H‐Phe‐Pro‐βNA, and aminopeptidase activity towards H‐Arg‐βNA.HCl, H‐Lys‐βNA, H‐Leu‐βNA, H‐Pro‐βNA, H‐Glu‐βNA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo‐peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo‐peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid beta-naphthylamide (betaNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Artificial manipulation of sink and source was carried out in several bread wheat varieties in order to study the variations in the pattern of storage products accumulation in the grain. In a first experiment, partial ablation of the laminae did not cause any significant variation in the yield components, while total elimination of the laminae resulted in a decreased fertility of the spikelets and in a lower kernel weight. The protein content of grain turned out to be a function of the amount of vegetative organs left in the different treatments, and ranged from 11.1 % in leafless plants to 16.5% in the control. The halving of the spikes led to a 14% increase in the size of the kernels, while the protein content per spike showed a 20 % drop in comparison to the control. Reduction of sink affected nitrogen accumulation to a lesser extent than carbohydrates storage in the grain. Reduction of sink induced remarkable increases in the protein fractions: gliadins +59%, glutelins +44%, insoluble residue +30%, non‐proteic nitrogen +28%, albumins and globulins + 16%. The increase of GPC or of specific solubility classes did not influence significantly the quality of the flour evaluated by the Zeleny and Pelshenke indices. The presented data suggest some caution in adopting kernel size and GPC as main selection criteria in early generations. It seems more convenient to select for a prolonged grain filling period and for a higher biomass without increasing plant height.