Modeling of Growth and Organic Acid Kinetics and Evolution of the Protein Profile and Amino Acid Content during Lactiplantibacillus plantarum ITM21B Fermentation in Liquid Sourdough.

The application of mathematical modeling to study and characterize lactic acid bacterial strains with pro-technological and functional features has gained attention in recent years to solve the problems relevant to the variabilities of the fermentation processes of sourdough. Since the key factors contributing to the sourdough quality are relevant to the starter strain growth and its metabolic activity, in this study, the cardinal growth parameters for pH, temperature (T), water activity (aw), and undissociated lactic acid of the sourdough strain Lactiplantibacillus plantarum ITM21B, were determined. The strain growth, pH, organic acids (lactic, acetic, phenyllactic, and hydroxy-phenyllactic), total free amino acids, and proteins were monitored during fermentation of a liquid sourdough based on wheat flour and gluten (Bio21B) after changing the starting T, pH, and inoculum load. Results demonstrated that the different fermentation conditions affected the strain growth and metabolite pattern. The organic acid production and growth performance were modeled in Bio21B, and the resulting predictive model allowed us to simulate in silico the strain performances in liquid sourdough under different scenarios. This mathematical predictive approach can be useful to optimize the fermentation conditions needed to obtain the suitable nutritional and technological characteristics of the L. plantarum ITM21B liquid sourdough.

Characterization of Dextran Produced by the Food-Related Strain Weissella cibaria C43-11 and of the Relevant Dextransucrase Gene.

A metabolic feature of lactic acid bacteria (LAB) is the production of exopolysaccharides (EPSs), which have technological and functional properties of interest to the food sector. The present study focused on the characterization of the Weissella cibaria strain C43-11, a high EPS producer in the presence of sucrose, in comparison with a low-producing strain (C2-32), and on possible genetic regulatory elements responsible for the modulation of dextransucrase (dsr) genes expression. NMR analysis of the polymeric material produced by the C43-11 strain indicated the presence of dextran consisting mainly of a linear scaffold formed by α-(1-6) glycosidic linkages and a smaller amounts of branches derived from α-(1-2), α-(1-3), and α-(1-4) linkages. Molecular analysis of the dsr genes and the putative transcriptional promoters of the two strains showed differences in their regulatory regions. Such variations may have a role in the modulation of dsr expression levels in the presence of sucrose. The strong upregulation of the dsr gene in the C43-11 strain resulted in a high accumulation of EPS. This is the first report showing differences in the regulatory elements of the dsr gene in W. cibaria and indicates a new perspective of investigation to identify the regulatory mechanism of EPS production.

A Predictive Growth Model for Pro-technological and Probiotic Lacticaseibacillus paracasei Strains Fermenting White Cabbage.

Bacterial strains belonging to Lacticaseibacillus paracasei species are generally used as starters in food fermentations and/or as probiotics. In the current study, the growth cardinal parameters of four L. paracasei strains (IMPC2.1, IMPC4.1, P40 and P101), isolated from table olives or human source, were determined. Strains were grown in liquid medium and incubated at several temperatures (10 values from 5.5°C-40°C) and pH (15 values from 3.2 to 9.1) along the growth range. The cardinal temperature model was used to describe temperature effects on the maximum specific growth rate of L. paracasei whereas new equations were developed for the effect of pH. The estimated Tmin values ranged between -0.97°C and 1.95°C and were lower than 0°C for strains IMPC4.1 and P101. Strain P40 was able to grow in the most restricted range of temperature (from 1.95°C to 37.46°C), while strain IMPC4.1 was estimated to survive at extreme conditions showing the lowest pHmin . Maximum specific growth rates of L. paracasei IMPC2.1 in white cabbage (Brassica oleracea var. capitata) were used to calculate the correction factor (Cf ) defined as the bias between the bacterial maximum specific growth rate in broth and in the food matrix. A simple bi-linear model was also developed for the effect of temperature on the maximum population density reached in white cabbage. This information was further used to simulate the growth of L. paracasei strains in cabbage and predict the time to reach the targeted probiotic level (7 log10 CFU/g) using in silico simulations. This study demonstrates the potential of the predictive microbiology to predict the growth of beneficial and pro-technological strains in foods in order to optimize the fermentative process.

Effect of Amaranth and Quinoa Flours on Exopolysaccharide Production and Protein Profile of Liquid Sourdough Fermented by Weissella cibaria and Lactobacillus plantarum.

Exopolysaccharides (EPSs) are known for their positive contribute to the technological properties of many foods, including bakery products. These molecules can be obtained performing piloted fermentation with lactic acid bacteria (LAB). In order to select strains able to produce EPS, a screening test in agar medium containing sucrose, fructose or glucose as carbohydrate source was performed on 21 LAB strains. Results allowed to select 8 Weissella cibaria, 2 Weissella confusa, and 2 Leuconostoc spp. strains as EPS producers only in the presence of sucrose. A further screening in liquid medium enriched with sucrose (10%) (mMRS_S) indicated the W. cibaria strain C43-11 as the higher EPS producer. The selected strain was used to develop liquid sourdoughs (LSs) with dough yield (DY) 500, fermented for 15 h and based on wheat flour and wheat gluten or pseudocereals (quinoa or amaranth) in ratio 1:1, in the presence or not of sucrose at 3% (w/w, LS weight), in comparison to Lactobacillus plantarum ITM21B, a strain not producing EPS in mMRS_S. Results indicated that the use of pseudocereals favored the EPS production. Formulations were optimized by modifying DY (500 or 250), sucrose concentration (3 or 6%) and flour ratio. LSs were characterized for the content of organic acids (lactic, acetic, phenyllactic, OH-phenyllactic), pH, TTA, EPS, viscosity, total protein degradation and protein pattern. The highest EPS production (20.79 g/kg) and viscosity (1168 mPa s) were obtained in LS (DY 250, sucrose 6%) based on quinoa flour and started with C43-11 strain. The LS was characterized by the presence of phenyllactic and OH-phenyllactic acids, protein degradation by 51.7% and proteins in the range 14-80 kDa. In these conditions, also strain ITM21B was able to produce EPS at level of 4.61 g/kg and to degrade proteins by 53.8% in LS based on wheat and quinoa flours (1:1) (DY250 and sucrose 3%). Therefore, results demonstrated that the use of selected conditions (flour type, DY, sucrose) can stimulate specific attributes of strains making them suitable for production of short fermented (15 h) LSs which can be used as bread improvers.

Microbiological and physicochemical parameters for predicting quality of fat and low-fat raw ground beef during refrigerated aerobic storage.

The aim of the current study was to identify quality indicators of fat (14.50 ± 0.75%) and low-fat (4.79 ± 0.63%) raw ground beef by monitoring changes in physicochemical and microbiological parameters during aerobic refrigerated storage, such as water-holding capacity, pH, thiols, carbonyl compounds, thiobarbituric acid reactive substances (TBARS), metmyoglobin, deoxymyoglobin, oxymyoglobin color indices, pseudomonads, Brochothrix thermosphacta, and total viable counts. Meat packaged in air-permeable polyethylene plastic film was stored under controlled isothermal conditions (0, 5, 10, and 15 °C). A population level of pseudomonads equal to 7.0 ± 0.5 log10 colony forming units (CFU)/g was considered as the potential spoilage level. Using principal component analysis, samples were distinguished on the basis of their microbial load. A significant positive correlation between microbial population and carbonyls, metmyoglobin, TBARS, water-holding capacity, and a negative correlation with thiols and color parameters (L* , chroma) were observed. Two different approaches were followed to estimate the quality status of samples: (i) the partial least square (PLS) regression with R2 of 0.93 and root mean square error prediction of 0.44 for pseudomonads, using the above physicochemical characteristics as the dominant input variables, which allowed prediction of the microbiological status of ground beef regardless of time-temperature storage profile and fat content, and (ii) a square-root-type model (adjusted R2 of 0.952) that satisfactorily predicted the growth of spoilage pseudomonads under isothermal and dynamic conditions, regardless of the above physicochemical changes. The above results suggest that depending on the available input data, the two modeling approaches can accurately (and complementarily) assess quality of aerobically stored ground beef. PRACTICAL APPLICATION: Changes in appearance and quality of fat and low-fat raw ground beef are associated with physicochemical alteration and/or microbial growth. The study provides two different modeling approaches that can be integrated in an intelligent interface of the refrigerator having specific colorimetric and/or temperature sensors, to evaluate in a convenience way the quality of stored meat thus reducing domestic waste: the partial least square model was based on physicochemical parameters (particularly chroma, metmyoglobin, and thiobarbituric acid reactive substances), while the square root model was based on the time-temperature conditions during storage.

Thermoplastic starch and bioactive chitosan sub-microparticle biocomposites: Antifungal and chemico-physical properties of the films.

In this study, chitosan (C) tripolyphosphate (T) sub-micro particles containing ungeremine (CTUn), an alkaloid particularly active against Penicilliumroqueforti, a fungus responsible of the bakery products deterioration, were prepared through external gelation crosslinking process. The particles were included in a thermoplastic starch based polymer Mater-Bi (MBi), and MBi/CTUn bioactive biocomposites were obtained. The films showed bioactivity against P. roqueforti. In particular, the bioassays were performed on films with different concentration of CTUn and at different pH values. CTUn particles influenced MBi crystallization (DSC analysis) and promoted thermal degradation of MBi starch component (TGA). Morphological analysis confirmed even distribution of sub-micro particles into the polymeric matrix. Water permeability slightly increased, as expected, whereas oxygen permeability decreased. Tensile tests showed CTUN sub-microparticles improved rigidity and tensile strength of the films at the expense of ductility. Finally, MBi/CTUn biocomposites evidenced interesting performances potentially exploitablein bioactive bakery based food packaging materials.

Lactobacillus plantarum ITM21B fermentation product and chickpea flour enhance the nutritional profile of salt reduced bakery products.

The study aimed at improving the nutritional profile of yeast leavened salt reduced sliced bread and puccia type bread fortified with a wheat-based Lactobacillus plantarum ITM21B fermentation product (Bio21B). The protein content of bread made under laboratory conditions was increased by using: (i) chickpea flour (CF) (15% wt/wt flour) and Bio21B or (ii) the Bio21B containing a fungal protease to favour the gluten hydrolysis. Products showed increased protein and total amino acid content and improved protein digestibility. Moreover, the formula significantly affected the protein pattern of breads which, according to the results of the microfluidic two-dimensional electrophoresis (µ2DE) protein pattern, were discriminated as observed by the PCA plot. The use of CF was validated at industrial pilot plant producing salt reduced sliced bread and puccia type bread. The resulting products showed improved nutritional profile and a sensory quality comparable to the company's products containing salt.

Effect of pH and TPP concentration on chemico-physical properties, release kinetics and antifungal activity of Chitosan-TPP-Ungeremine microbeads.

In this study, chitosan based microbeads containing Ungeremine, an antimicrobial alkaloid particularly active against Penicillium roqueforti, a filamentous fungus responsible of the bakery products deterioration, were prepared by external gelation by using sodium tripolyphosphate (TPP) as crosslinking agent. The stability of the beads, as well as the loading efficiency of the bioactive molecule, were assessed at different pH and TPP concentrations resulting particularly enhanced at low pH. All the microbeads evidenced antimicrobial activity against Penicillium roqueforti. The release kinetics of Ungeremine was tailored by opportunely modulating pH and TPP concentrations. Morphological analysis evidenced the improvement of the structural crosslinking density of microbeads including Ungeremine and spectroscopic analysis emphasized the active participation of Ungeremine to the crosslinking process occurring between chitosan and TPP. Finally, thermogravimetric analysis confirmed the increasing of free volume in three-dimensional networks and their liability to thermal degradation.

Pectin-honey coating as novel dehydrating bioactive agent for cut fruit: Enhancement of the functional properties of coated dried fruits.

In this paper, a novel and sustainable process for the fruit dehydration was described. Specifically, edible coatings based on pectin and honey were prepared and used as dehydrating and antimicrobial agents of cut fruit samples, in this way promoting the fruit preservation from irreversible deteriorative processes. Pectin-honey coating was tested on apple, cantaloupe melon, mango and pineapple. The analysis were performed also on uncoated dehydrated fruits (control). The coated fruit evidenced enhanced dehydration percentage, enriched polyphenol and vitamin C contents, improved antioxidant activity and volatile molecules profile. Moreover, the antimicrobial activity against Pseudomonas and Escherichia coli was assessed. Finally, morphological analysis performed on fruit fractured surface, highlighted the formation of a non-sticky and homogeneous thin layer. These outcomes suggested that the novel fruit dehydration process, performed by using pectin-honey coating, was able to both preserve the safety and quality of dehydrated fruits, and enhance their authenticity and naturalness.

Formulation of yeast-leavened bread with reduced salt content by using a Lactobacillus plantarum fermentation product.

A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). l-Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (γ-l-glutamyl-l-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized.

Improvement of the antifungal activity of lactic acid bacteria by addition to the growth medium of phenylpyruvic acid, a precursor of phenyllactic acid.

The aim of the current study was to improve the antifungal activity of eight lactic acid bacterial (LAB) strains by the addition of phenylpyruvic acid (PPA), a precursor of the antifungal compound phenyllactic acid (PLA), to a defined growth medium (DM). The effect of PPA addition on the LABs antifungal activity related to the production of organic acids (PLA, d-lactic, l-lactic, acetic, citric, formic and 4-hydroxy-phenyllactic acids) and of other phenylpyruvic-derived molecules, was investigated. In the presence of PPA the inhibitory activity (expressed as growth inhibition percentage) against fungal bread contaminants Aspergillus niger and Penicillium roqueforti significantly increased and was, even if not completely, associated to PLA increase (from a mean value of 0.44 to 0.93 mM). While the inhibitory activity against Endomyces fibuliger was mainly correlated to the low pH and to lactic, acetic and p-OH-PLA acids. When the PCA analysis based on data of growth inhibition percentage and organic acid concentrations was performed, strains grown in DM+PPA separated from those grown in DM and the most active strains Lactobacillus plantarum 21B, Lactobacillus fermentum 18B and Lactobacillus brevis 18F grouped together. The antifungal activity resulted to be strain-related, based on a different mechanism of action for filamentous fungi and the yeast and was not exclusively associated to the increase of PLA. Therefore, a further investigation on the unique unidentified peak in HPLC-UV chromatograms, was performed by LC-MS/MS analysis. Actually, full scan mass spectra (negative ion mode) recorded at the retention time of the unknown compound, showed a main peak of m/z 291.0 which was consistent with the nominal mass of the molecular ion [M-H](-) of polyporic acid, a PPA derivative whose antifungal activity has been previously reported (Brewer et al., 1977). In conclusion, the addition of PPA to the growth medium contributed to improve the antifungal activity of LAB strains and resulted in the production of the polyporic acid, here ascertained in LAB strains.

Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients.

Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.

Bioprotection of ready-to-eat probiotic artichokes processed with Lactobacillus paracasei LMGP22043 against foodborne pathogens.

The survival of 3 pathogens Listeria monocytogenes ATCC19115, Salmonella enterica subsp. enterica ATCC13311, and Escherichia coli ATCC8739 was evaluated over time in ready-to-eat (RTE) artichoke products processed or not with the probiotic strain Lactobacillus paracasei LMGP22043. Both probiotic and standard products (final pH about 4.0; aw = 0.98) dressed with oil and packaged in modified atmosphere were inoculated with pathogens at a level of about 3 log CFU/g and stored at 4 ºC for 45 d. Pathogens decreased in the probiotic product in 2 descent phases, without shoulder and/or tailing as observed by fitting the models available in the GInaFit software to the experimental data. S. enterica subsp. enterica was completely inactivated after 14 and 28 d in probiotic and standard products, respectively; E. coli was inhibited in the probiotic food at day 4 (count