Inhibitory Effect of Lactiplantibacillusplantarum and Lactococcus lactis Autochtonous Strains against Listeria monocytogenes in a Laboratory Cheese Model.

In the present study, six Lactococcus lactis and seven Lactiplantibacillus plantarum strains isolated from artisanal Sardinian dairy products were evaluated for their efficacy in controlling the growth of Listeria monocytogenes during the storage of miniature fresh cheese manufactured on a laboratory scale to exploit their possible use as biopreservatives. The strains were tested for antimicrobial activity and some technological characteristics before using them in miniature fresh cheese to evaluate their in situ antilisterial effect. Our results showed that five strains (L. lactis 16FS16-9/20234-11FS16 and Lpb. plantarum 1/14537-4A/20045) could be considered suitable candidates for use as protective cultures in fresh cheese manufacture since they significantly lowered the pathogen counts by 3-4 log units compared to the control; however, all strains tested were capable of decreasing L. monocytogenes numbers. Our results suggest that the single and combined action of the acidifying power and the production of bacteriocin of these strains was capable of controlling and/or reducing the growth of L. monocytogenes. Considering their technological characteristics, they might be used as starter/adjunct cultures to increase the safety of the products, perhaps in association with other antimicrobial hurdles.

Influence of Autochthonous Putative Probiotic Cultures on Microbiota, Lipid Components and Metabolome of Caciotta Cheese.

The present study was undertaken to produce probiotic Caciotta cheeses from pasteurized ewes' milk by using different combinations of autochthonous microbial cultures, containing putative probiotic strains, and evaluate their influence on gross composition, lipid components, sensory properties and microbiological and metabolite profiles of the cheeses throughout ripening process. A control cheese was produced using commercial starter cultures. The hydrophilic molecular pools (mainly composed by amino acids, organic acids, and carbohydrates) were characterized by means of 1H NMR spectroscopy, while the cholesterol, α-tocopherol and fatty acid composition by HPLC-DAD/ELSD techniques. Conventional culturing and a PCR-DGGE approach using total cheese DNA extracts were used to analyze cheese microbiota and monitor the presence and viability of starters and probiotic strains. Our findings showed no marked differences for gross composition, total lipids, total cholesterol, and fatty acid levels among all cheeses during ripening. Differently, the multivariate statistical analysis of NMR data highlighted significant variations in the cheese' profiles both in terms of maturation time and strains combination. The use of autochthonous cultures and adjunct probiotic strains did not adversely affect acceptability of the cheeses. Higher levels of lactobacilli (viability of 108-109 cfu/g of cheese) were detected in cheeses made with the addition of probiotic autochthonous strains with respect to control cheese during the whole ripening period, suggesting the adequacy of Caciotta cheese as a carrier for probiotic bacteria delivery.

Microbiota of Sardinian Goat's Milk and Preliminary Characterization of Prevalent LAB Species for Starter or Adjunct Cultures Development.

This work was performed to study the microbiota of raw goat's milk (67 samples) collected in different areas of Sardinia, in order to select autochthonous lactic acid bacteria (LAB) strains for use in goat cheese manufacturing. Total mesophilic bacteria ranged between 105 and 107 cfu/mL; mean counts of Enterobacteriaceae did not exceed 4 log cfu/mL whereas those of E. coli and coagulase-positive staphylococci were lower than 1.5 and 2 log ufc /ml, respectively. Neither Salmonella spp. nor Listeria monocytogenes were recovered. The numbers of total LAB were in the range from 104 to 107 cfu/mL and mean yeasts counts varied between 103 and 105 cfu/mL. The most frequently isolated LAB species were Lactococcus lactis subsp. lactis and Lactobacillus paracasei. The presence of Enterococcus faecium was also noteworthy. The in vitro study of some functional characteristics related to technological properties of the strains belonging to these species allowed to point out some strains possessing good potential for use as adjunct or starter cultures in the production of cheese.

Application of Autochthonous Lactobacillus Strains as Biopreservatives to Control Fungal Spoilage in Caciotta Cheese.

Fungal spoilage is one of the main causes of economic losses worldwide in the food industry. In the last years, consumer's demands for preservative-free processed foods have increased as a result of growing awareness about the health hazards associated with chemicals. Lactic acid bacteria have been extensively studied for their antibacterial and antifungal potential in order to be used as biopreservatives. The first objective of this study was to investigate in vitro the antifungal activity of autochthonous Lactobacillus strains against moulds commonly associated with cheese spoilage. Then, the Lactobacillus strains with the highest inhibitory effect and broadest spectrum were tested in single or mixed cultures against Penicillium chrysogenum ATCC 9179 and Aspergillus flavus ATCC 46283 on miniature Caciotta cheese produced at laboratory scale to evaluate in situ their ability to prevent mould growth and to determine their impact on cheese organoleptic properties and starter culture activity. The growth of the starter lactococcal population exhibited similar trend and values during ripening, suggesting that the addition of lactobacilli did not influence its growth and survival. Inhibition of P. chrysogenum inoculated in the milk was determined in cheeses produced with single or mixed Lactobacillus adjuncts as compared to cheeses without adjunct. The mixed adjunct cultures resulted in more effective, significantly reducing mould counts of more than 2 log units at the end of ripening. The application of the adjunct cultures resulted in a delay in mycelial growth of P. chrysogenum and A. flavus inoculated on the cheese surface as well. Finally, we found no significant differences among samples for the sensory parameters evaluated that received similar ratings. Our results indicate that the selected Lactobacillus strains may have a potential effect in controlling mould contamination on cheeses. Further studies are currently being carried out to identify the molecules responsible for the antifungal activity.

Preliminary evaluation of probiotic properties of Lactobacillus strains isolated from Sardinian dairy products.

Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong ß-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

Antilisterial activity of nisin-like bacteriocin-producing Lactococcus lactis subsp. lactis isolated from traditional Sardinian dairy products.

With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes.

Mitochondrial alterations and autofluorescent conversion of Candida albicans induced by histatins.

The mechanism of the candidacidal activity of histatins 3 and 5 (Hst) is still a matter of debate. Previous studies have indicated that Hst induce cell permeabilization, generation of reactive oxygen species (ROS) by mitochondria, inhibition of the respiratory chain, and energy-dependent cytotoxic release of ATP. On the other hand, the multiplicity of effects and the apparent contrast between experimental data continue to render the mechanism of Hst-induced killing of C. albicans unclear. In this investigation, using fluorescent probes (the potential-sensitive mitochondrial probe tetramethylrhodamine methyl ester perchlorate, TMRM; the ROS-sensitive probe dihydrofluorescein diacetate, DHF; the membrane-impermeant probe, calcein) and autofluorescence data we observed that Hst induce ROS generation by mitochondria undergoing a high energy swelling condition, accompanied by oxidation of cytosolic NAD(P)H and mitochondrial flavoproteins. ROS generation and swelling, attributable to an inhibition of the respiratory chain and to impairment of the K/H-exchanger, were followed by mitochondrial depolarization. Mitochondrial changes were accompanied by massive calcein influx, indicative of cell permeabilization, and prominent alterations of the cell size, shape, and optical density. The loss of proliferative activity was correlated, on a single cell basis, to the acquisition of a lipofuscin-like autofluorescence.