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Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA).In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition. © 2012 Elsevier B.V.

Microbial components and visual quality were determined on 1158 consumer units of ready-to-eat salads from several processors, two per each of 579 process lots, with residual shelf-life varying around a mode of five days, collected over 19 months in the years 2006-2008 from retail stores of two Italian cities close to a major producing and processing area. The salads were mainly baby leaf of single species (lettuce, arugula, spinach, lamb's lettuce), with approximately 10% of the lots made up by mixes of 2-4 species. One unit per lot was analyzed on the day of collection and the other at the consume-by date. No Salmonella or Listeria monocytogenes was found (detection limit: presence in 25 g). Escherichia coli was detected in 27% of the lots (detection limit: 5 cfu/g), with probability of occurrence and counts highest in Autumn and for lettuce and arugula. Average visual quality was higher and other components of the microbial load were lower in Winter and Spring compared to Summer and Autumn (-0.6 log cfu/g of total aerobic counts, -1.3 log cfu/g of coliforms, -0.6 log cfu/g of yeasts and moulds). Lactic acid bacteria were detected more frequently in Spring and Summer (up to 50% of the lots). The rate of increase of microbial populations during shelf life was not affected by the level of initial contamination. At the consume-by date total aerobic count exceeded 7.2 log cfu/g for 50% of the lots and 7.7 log cfu/g for 25%. Salads from the biggest processor and retailer showed slightly higher visual quality scores, lower odds of E. coli occurrence and lower microbial loads. Visual quality scores showed significant negative relationships with the levels of lactic acid bacteria, coliforms and total viable counts.

The effect of tryptone, yeast extract, Tween 80 and initial pH on the production of enterocin 1146 and lactocin D, two bacteriocins produced by lactic acid bacteria, was studied in a basal buffered medium (tryptone-yeast extract-tween, TYT) using factorial experiments and empirical modelling. Production of enterocin 1146 was affected by pH, yeast extract and Tween 80 and to a lesser degree, by the initial pH of the medium. On the basis of the predictions of the models developed, three TYT media (TYT10, TYT11 and TYT30) were designed to maximize bacteriocin production while minimizing the amount of peptides in the medium. Growth and bacteriocin production by Enterococcus faecium DPC 1146 (enterocin 1146), Lactococcus lactis subsp. lactis biovar diacetylactis DPC 3286 (lactocin D) and Lact. lactis subsp. cremoris LMG2130 (lactococcin A) was compared in TYT media and seven other culture media (Elliker lactic broth, M17, M17 dialysate, MRS, tryptose phosphate, tryptone yeast extract broth, yeast glucose Lemco broth). Bacteriocin production in TYT media was comparable with that in M17 and MRS, which had a higher peptide content. TYT30 allowed good production of enterocin 1146 and lactocin D while TYT11 proved acceptable for all the strains tested.

Streptococcus thermophilus is an important dairy starter used for the manufacture of fermented milks and cheeses. As for many other lactic acid bacteria, its survival during propagation and preservation of starter cultures and its performance in dairy fermentations are significantly affected by its ability to cope with many environmental stresses. This, in turn, is dependent on the expression of a several genes, which is often controlled by negative response regulators, and has complex relationships with the control of metabolism. In this paper we describe the effect of growth phase, adaptation and inactivation of genes for stress response regulators (hrcA, ctsR and rr01) on the growth, acid production and stress resistance and intracellular protein patterns in the yoghurt strain S. thermophilus Sfi39. Inactivation of stress response regulators significantly affected growth, acid production in milk and stress tolerance. Although mutants showed an increased resistance during the exponential phase compared to the wild type strain, the effect of the mutations was complex and in some cases mutants were unable to mount an acid- or heat-shock response after exposure to mild heat and acid stresses. Significant differences in intracellular protein patterns analyzed by SDS-PAGE and 2-DE/mass spectrometry were found as a function of growth phase, adaptation and mutation, but a detailed transcriptomic and proteomic study would be needed to provide a mechanistic interpretation of the effect of the mutations and to identify a strategy for the construction of food-grade mutants with improved growth and performances.

Fumaric acid production by Rhizopus arrhizus from potato flour was studied at different initial substrate concentrations (S), C/N ratios and fermentation times (t f) in a composite design experiment. By using response surface methodology and canonical analysis, the experimental values of fumaric acid and mycelial biomass yields and productivity were fitted to the only statistically significant factors with mean percentage errors of 11, 26 and 13%, respectively. Owing to the limited statistical significance of the C/N ratio, it was possible to determine the values of S (25–50 g/l of glucose equivalent) and t f(44–100 h) associated with fumaric acid yields ranging from 60 to 75% and productivities varying from 6 to 8.4 g/l per day. Since such results were in quite good agreement with previous experiments carried out on other starchy materials, the above operating conditions might be used to minimize fumaric acid production costs as a function of the feed-stock used.

Aim: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. Methods and Results: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD–PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. Conclusions: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. Significance and Impact of the Study: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.

Forty-one strains of lactic acid bacteria (LAB) isolated from Cornetto di Matera sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process. Urease, peptidase, phytase, phosphatase and beta-glucosidase activities were measured spectrophotometrically using synthetic substrates. Proteolytic activities were examined in model doughs, using neutral and acidified sterile doughs as controls. All strains had low urease, glutamyl aminopeptidase and iminopeptidase activities, whereas differences within species were observed for the other enzymatic activities. Leuconostoc mesenteroides and Lactobacillus curvatus strains generally showed high aminopeptidase, X-prolyl dipeptidyl aminopeptidase, beta-glucosidase and phytase activities, while the enzymatic activities of Lactobacillus plantarum, Lactobacillus pentosus and Weissella cibaria varied between strains. In order to classify the strains on the basis of similar enzymatic profiles, a hierarchical cluster analysis was carried out. Several strains of L. plantarum, L. curvatus and Leuc. mesenteroides showed an interesting combination of proteolytic, peptidase, beta-glucosidase and phytase activities, suggesting their possible usefulness as a mixed starter culture in bread-making processes.

Conventional multivariate statistical techniques (hierarchical cluster analysis, linear discriminant analysis) and unsupervised (Kohonen Self Organizing Map) and supervised (Bayesian network) artificial neural networks were compared for as tools for the classification and identification of 352 SDS-PAGE patterns of whole cell proteins of lactic acid bacteria belonging to 22 species of the genera Lactobacillus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus including 47 reference strains. Electrophoretic data were pre-treated using the logistic weighting function described by Piraino et al. [Piraino, P., Ricciardi, A., Lanorte, M. T., Malkhazova, I., Parente, E., 2002. A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol. Lett. 24, 1477-1482]. Hierarchical cluster analysis provided a satisfactory classification of the patterns but was unable to discriminate some species (Leuconostoc, Lb. sakei/Lb. curvatus, Lb. acidophilus/Lb. helveticus, Lb. plantarum/Lb. paraplantarum, Lc. lactis/Lc. raffinolactis). A 7 × 7 Kohonen self-organizing map (KSOM), trained with the patterns of the reference strains, provided a satisfactory classification of the patterns and was able to discriminate more species than hierarchical cluster analysis. The map was used in predictive mode to identify unknown strains and provided results which in 85.5% of cases matched the classification obtained by hierarchical cluster analysis. Two supervised tools, linear discriminant analysis and a 23:5:2 Bayesian network were proven to be highly effective in the discrimination of SDS-PAGE patterns of Lc. lactis from those of other species. We conclude that data reduction by logistic weighting coupled to traditional multivariate statistical analysis or artificial neural networks provide an effective tool for the classification and identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins. © 2005 Elsevier B.V. All rights reserved.

The dynamic gassing-out method was used to assess the effect of stirrer speed, air flow rate and alginate concentration on the overall volumetric oxygen mass transfer coefficient (K(L)a) in a bench-top stirred fermenter equipped with two Rushton-type turbines. As the alginate concentration in the medium was increased from 0 to 2% (w/v), the liquid film time constant (tau(F)) increased from approx. 30 to 300% of the electrode time constant (tau(E)), clearly showing that the liquid-probe diffusion film affected the oxygen-electrode response. By accounting for delayed probe response, liquid film time constant and gas residence time, the corrected values of K(L)a were empirically correlated with the above independent variables as converted into gassed power input and aeration number per unit liquid volume and the effective liquid/water shear viscosity ratio. Despite an average error of 19%, such a correlation might be useful to adjust the oxygen transfer in bench-top stirred reactors during viscous aerobic fermentations.

Rapid and reliable identification of Staphylococcus xylosus was achieved by species-specific PCR assays. Two sets of primers, targeting on xylulokinase (xylB) and 60 kDa heat-shock protein (hsp60) genes of S. xylosus, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 27 reference strains of the DSM collection, representing 23 different species of the Staphylococcus genus and 3 species of the Kocuria genus. Moreover, 90 wild strains isolated from different fermented dry sausages were included in the analysis. By using primers xylB-F and xylB-R the expected PCR fragment was obtained only when DNA from S. xylosus was used. By contrast, amplification performed by using primers xylHs-F and xylHs-R produced a single PCR fragment, of the expected length, when DNA from S. xylosus, S. haemolyticus, S. intermedius and S. kloosii were used as template. Nevertheless, AluI digestion of the xylHs-F/xylHs-R PCR fragment allowed a clear differentiation of these 4 species. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. xylosus strains.

In order to study the composition of the lactic acid bacteria (LAB) community of sourdoughs used for the manufacture of Altamura bread, a traditional durum wheat bread produced in Apulia (Southern Italy), 111 strains of LAB were isolated and characterized. The phenotypic characterization of the isolates, carried out using a set of 29 tests, allowed the identification of 15 clusters at the 80% similarity level by hierarchical cluster analysis. Of the isolates, 88% were identified as facultatively heterofermentative LAB (Lactobacillus plantarum, Lb. paracasei, Lb. casei) and 12% as heterofermentative LAB (Lb. brevis, Leuconostoc mesenteroides). SDS-PAGE profiles of whole cell proteins of 68 strains confirmed the identification. Both the diversity and structure of the lactic microflora for sourdoughs for Altamura bread varied among samples.

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus , Enterococcus faecium , and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25–36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus , E. faecium , and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.

Leucocin F10 is a bacteriocin produced by a strain of Leuconostoc carnosum isolated from fermented meat. It is sensitive to proteolytic enzymes and alpha-amylase, heat resistant up to 121 degrees C for 10 min at pH 3.0 and is inhibitory to Listeria spp., Leuconostoc spp., Enterococcus faecalis and Lactobacillus sake. Bactericidal activity against L. innocua in potassium phosphate buffer was strongly dependent of the growth phase of the indicator cells. A rapid sub-lethal damage, followed by slow death was observed for log-phase cells of L. innocua. The bacteriocin induced a rapid release of K+ from energized L. innocua cells. Leucocin F10 production occurred at temperatures between 4 and 30 degrees C and followed a primary metabolite kinetics in batch fermentations at controlled pH. Maximum bacteriocin activity was obtained at pH 6.0.

The production of enterocin 1146, a bacteriocin from Enterococcus faecium DPC1146, was studied during batch fermentation at pH 5, 5.5, 6 and 6.5. The bacteriocin was produced throughout the growth of the micro-organism, showing primary metabolite kinetics. Bacteriocin production stopped at the end of growth and was followed by a decrease in activity due primarily to adsorption on the cells of the producer. The optimal pH for enterocin 1146 production was 5.5, because of higher bacteriocin yield per unit of biomass and slower adsorption/degradation, while optimal pH for growth was between 6.0 and 6.5.

Gluconic acid production from corn starch hydrolysates by immobilised mycelia of Aspergillus niger was studied in a laboratory-scale stirred fermentor at different concentrations of glucose (S0) and dissolved oxygen (DO) in the culture broth. Its evolution was simulated quite well by applying the same unstructured model set up in previous experiments using stirred and airlift fermentors. In particular, increasing S0 in the range 70–160 g/l, although uninfluential upon the yield coefficient, resulted in an exponential decrease in the gluconic acid formation rate constant. Nevertheless, the greater the oxygen transfer rate used in the fermentor, the smaller the inhibitor effect of the higher concentrations of glucose on gluconate productivity became. This was achieved by enriching the inlet air with pure oxygen so as to maintain the DO level above 75% saturation throughout the fermentation.

The composition of 16 natural whey cultures from 3 different Mozzarella chee se plants was investigated. They consisted mainly of lactic acid bacteria, coliform bacteria and yeasts. Micrococci, butyric and propionic acid bacteria only occurred occasionally. Lactobacillus lactis was the most common species of Lactobacillus while Streptococcus lactis and Str. thermophilus were the most common species of Streptococcus. Enteropathogenic Escherichia coli were always present. Different species of Leuconostoc and yeasts belonging to the genera Candida, Kluyveromyces, Debaryomyces and Brettanomyces were also isolated. Acidifying and proteolytic capacity of the strains showed that these activities were widely affected by tempe rature and type of milk (cow or waterbuffalo milk). Streptococci were the most active acid pro du cers at the cheese vat temperature (37 oC).

Enterococcus faecium DPC 1146 produces a bacteriocin, enterocin 1146, which is inhibitory to Listeria monocytogenes . Enterocin 1146 was produced in GM17 and in milk. The bacteriocin was partially purified by ammonium sulfate precipitation. Its molecular weight, estimated by SDS-PAGE, was 3.0 kDa. It could be stored at -20°C without loss of activity, but pH had a marked effect on enterocin 1146, which was more stable at both high (up to 120°C) and low temperatures (4°C) at pH 5 than at pH 7 and 9. The sensitivity of 57 strains belonging to 35 different species was studied using a critical dilution assay. L. monocytogenes and L. innocua were most sensitive; enterocin 1146 had a bactericidal effect on Listeria . Starter and nonstarter lactic acid bacteria (except Lactobacillus sake )were insensitive or relatively resistant to the bacteriocin. Genetic determinants for bacteriocin production and immunity do not appear to be plasmid borne.

Continuous whey fermentation by Kluyveromyces fragilis was studied in a 30-dm3 stirred fermenter as a function of the dilution rate (D) and feed concentration of lactose (So). By using the concept of material and energy balances, it was possible not only to check the consistency of the experimental steady-state responses, but also to develop an unstructured model based on a Monod-type kinetic equation for cell growth and two independent linear equations for the consumption rates of lactose and oxygen. Moreover, a typical Lineweaver—Burk plot enabled the yeast specific growth rate to be described as that of an enzyme-catalysed reaction in the presence of an unknown noncompetitive inhibitor (that was identified with a reference intermediate compound produced by the microorganism itself under partially anaerobic conditions and expressed in terms of ethanol equivalent). In this way, the experimental cell concentrations were reconstructed with a mean standard error of about 10%, thus confirming the capability of this model to provide a reliable basis for further scale-up of this fermentation process.

Aims Lactobacillus plantarum is a lactic acid bacterium involved in the production of many fermented foods. Recently, several studies have demonstrated that aerobic or respiratory metabolism in this species leads to improved technological and stress response properties. Methods and Results We investigated respiratory growth, metabolite production and stress resistance of Lact. plantarum C17 during batch, fed-batch and chemostat cultivations under respiratory conditions. Sixty mutants were selected for their ability to tolerate oxidative stress using H2O2 and menadione as selective agents and further screened for their capability to growth under anaerobic, respiratory and oxidative stress conditions. Dilution rate clearly affected the physiological state of cells and, generally, slow-growing cultures had improved survival to stresses, catalase production and oxygen uptake. Most mutants were more competitive in terms of biomass production and ROS degradation compared with wild-type strain (wt) C17 and two of these (C17-m19 and C17-m58) were selected for further experiments. Conclusions This work confirms that, in Lact. plantarum, respiration and low growth rates confer physiological and metabolic advantages compared with anaerobic cultivation. Significance and Impact of the Study Our strategy of natural selection successfully provides a rapid and inexpensive screening for a large number of strains and represents a food-grade approach of practical relevance in the production of starter and probiotic cultures.

Strains of Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus , and Streptococcus thermophilus varied in their ability to grow in whey permeate-based media. Whey permeate alone was a poor medium with a relatively low buffering capacity, which supported the growth of L. delbrueckii ssp. bulgaricus and S. thermophilus only. External pH control and the addition of .1% nonfat dry milk solids and .5 to 1% yeast extract improved the growth and activity of L. delbrueckii ssp. bulgaricus, L. helveticus , and S. thermophilus and made whey permeate-based media comparable with 11% nonfat dry milk as a medium for thermophilic lactic acid starters. Internal pH control, using a phosphate buffer, did not significantly improve the growth of the strains used when compared with the unbuffered medium and was inhibitory to L. helveticus 880.