Suitability of a probiotic Lactobacillus paracasei strain as a starter culture in olive fermentation and development of the innovative patented product “probiotic table olives”

Probiotic bacteria are generally available for consumers as concentrated preparations or incorporated in milk-based foods. Due to an increased interest of the market for probiotic foods as well as to meet a demand of industry for innovation, a new kind of probiotic food has been developed using table olives as a carrier. Green table olives, produced according to the Spanish-style, are obtained by a fermentation which can be carried out by spontaneous microflora, even if the use of starter cultures is desirable to obtain a more controlled process. In this regard, the selected strain Lactobacillus paracasei IMPC 2.1 of human origin was used in the dual role of starter and probiotic culture, and here we describe the different aspects which have been evaluated and solved to utilize that strain for the development of a new table olive-based probiotic food. These aspects include selection of the strain on the basis of its probiotic properties, molecular characterization, compatibility with the carrier food, and efficacy as starter. The final product meets commercial and functional requirements throughout its shelf-life

Production of a yeast-free focaccia with reduced salt content using a selected Leuconostoc citreum strain and seawater

Abstract A biotechnological protocol to produce a focaccia (a typical Italian flat bread) without bakers' yeast addition and with reduced salt was developed, to meet the current needs of the consumer. Based on its leavening capability, the Leuconostoc citreum strain C2.27 was selected to be used as a starter instead of the baker's yeast and inoculated in a liquid sourdough (type-II) for the production of the "yeast-free" focaccia. The addition of different NaCl concentrations and the replacement of the salt with food grade seawater were evaluated, and the capability of the selected strain to affect technological, nutritional and sensory features of the focaccia investigated. A significant improvement of the nutritional characteristics of the focaccia was observed compared to the control (leavened with bakers' yeast and added with NaCl 1.5 g/100 g) using 0.7 g/100 g of salt in the form of NaCl or seawater. Besides the reduced Na content (66% lower than the control), focaccia with seawater also showed a higher content of Ca2+ and Mg2+ (ca. 36% and 53%, respectively), and the lowest predicted glycemic index compared to the other experimental focaccia

Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml(−1) was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of ≤10 mg ml(−1). Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml(−1) in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs

Lactobacillus brevis-based bioingredient inhibits Aspergillus niger growth on pan bread

Bread shelf life is generally compromised by fungi mainly belonging to Aspergillus and Penicillium genera, which colonise the surface of the product within few days from the production. The aim of this study was to select a Lactobacillus brevis-based bioingredient (LbBio) able to inhibit the growth of Aspergillus niger ITEM5132 on pan bread in order to prolong its shelf life. Four LbBio formulations, obtained by growing a selected L. brevis strain in a flour-based medium containing different carbon sources or acid precursors (fructose, LbBio1; fructose and maltose, LbBio2; α-chetoglutaric acid, LbBio3; short-chain fructooligosaccharides, LbBio4), were evaluated for their content of organic acids (lactic, acetic, propionic, phenyllactic, 4-hydroxy-phenyllactic, valeric, isovaleric acids). The LbBio formulations were applied in yeast-leavened bread during bread-making trials and the resulting products were inoculated after baking with A. niger spore’s suspension and the fungal growth was monitored during storage (25°C for 6 days). The formulation showing the highest inhibitory activity was separated by ultra-filtration method, and whole and fractions obtained were evaluated for their in vitro activity. The fraction showing the highest activity was further separated by gel-filtration and the resulting products were investigated for their protein content and in vitro inhibition. The results from the bread-making trials performed using different formulations of LbBio showed a delay in fungal growth (1 day) respect to the bread not containing the bioingredient (control) or including calcium propionate (0.3% w/w). The formulation LbBio2, prepared with fructose and maltose 1% (w/vol), contained the highest amount of total organic acids, including phenyllactic and hydroxyl-phenyllactic acids, and reduced the visual spoilage of bread. This formulation was separated by ultra-filtration and fractions containing metabolites with molecular weight higher than 30 kDa showed high inhibitory effect in the in vitro assay. In particular, the microfluidic analysis highlighted the presence of a protein with a molecular weight of 56 kDa only in the active fraction. Further studies have to be done in order to identify the protein involved in the antifungal activity

Lactobacillus brevis-based bioingredient inhibits Aspergillus niger growth on pan bread

Bread shelf life is generally compromised by fungi mainly belonging to Aspergillus and Penicillium genera, which colonise the surface of the product within few days from the production. The aim of this study was to select a Lactobacillus brevis-based bioingredient (LbBio) able to inhibit the growth of Aspergillus niger ITEM5132 on pan bread in order to prolong its shelf life. Four LbBio formulations, obtained by growing a selected L. brevis strain in a flour-based medium containing different carbon sources or acid precursors (fructose, LbBio1; fructose and maltose, LbBio2; α-chetoglutaric acid, LbBio3; short-chain fructooligosaccharides, LbBio4), were evaluated for their content of organic acids (lactic, acetic, propionic, phenyllactic, 4-hydroxy-phenyllactic, valeric, isovaleric acids). The LbBio formulations were applied in yeast-leavened bread during bread-making trials and the resulting products were inoculated after baking with A. niger spore’s suspension and the fungal growth was monitored during storage (25°C for 6 days). The formulation showing the highest inhibitory activity was separated by ultra-filtration method, and whole and fractions obtained were evaluated for their in vitro activity. The fraction showing the highest activity was further separated by gel-filtration and the resulting products were investigated for their protein content and in vitro inhibition. The results from the bread-making trials performed using different formulations of LbBio showed a delay in fungal growth (1 day) respect to the bread not containing the bioingredient (control) or including calcium propionate (0.3% w/w). The formulation LbBio2, prepared with fructose and maltose 1% (w/vol), contained the highest amount of total organic acids, including phenyllactic and hydroxyl-phenyllactic acids, and reduced the visual spoilage of bread. This formulation was separated by ultra-filtration and fractions containing metabolites with molecular weight higher than 30 kDa showed high inhibitory effect in the in vitro assay. In particular, the microfluidic analysis highlighted the presence of a protein with a molecular weight of 56 kDa only in the active fraction. Further studies have to be done in order to identify the protein involved in the antifungal activity

Reduction of Olive Knot Disease by a Bacteriocin from Pseudomonas syringae pv. ciccaronei

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10(5) and 10(8) CFU ml(−1), respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml(−1) at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml(−1)) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites

Purification and Characterization of Novel Antifungal Compounds from the Sourdough Lactobacillus plantarum Strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(−1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(−1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(−1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B