A Strategy for the Recovery of Raw Ewe's Milk Microbiodiversity to Develop Natural Starter Cultures for Traditional Foods.

Commercial starter cultures, composed of high concentrations of a few species/strains of lactic acid bacteria (LAB), selected based on their strong technological aptitudes, have been developed to easily and safely carry out food fermentations. Frequently applied to industrial productions, selected starter LAB easily become the dominant microbiota of products, causing a dramatic decrease in biodiversity. On the contrary, natural starter cultures, which usually characterize the most typical and Protected Designation of Origin (PDO) food products, are constituted by a multitude and an indefinite number of LAB species and strains, both starter and nonstarter, thus contributing to preserving microbial biodiversity. However, their use is not risk-free since, if obtained without heat treatment application, natural cultures can contain, together with useful, also spoilage microorganisms or pathogens that could be allowed to multiply during fermentation. In the present study, an innovative method for the production of a natural starter culture directly from raw ewe's milk, inhibiting the growth of spoilage and potentially pathogenic bacteria without applying any heat treatment, was described. The culture developed show a good degree of microbial biodiversity and could be applied to both artisanal and industrial scales, guaranteeing safety, quality constancy, technological performance reproducibility, preserving biodiversity and peculiar sensory characteristics, usually linked to traditional products, while overcoming the problems associated with the daily propagation of natural cultures.

Biodiversity and Safety Assessment of Half-Century Preserved Natural Starter Cultures for Pecorino Romano PDO Cheese.

The use of biodiverse autochthonous natural starter cultures to produce typical and PDO cheeses contributes to establishing a link between products and territory of production, which commercial starters, constituted by few species and strains, are not able to. The purpose of this work was the assessment of biodiversity, at strain level, and safety of natural scotta-innesto cultures whose use is mandatory for the Pecorino Romano PDO cheese manufacturing, according to its product specification. The biodiversity of three scotta-innesto, collected in the 1960s and preserved in lyophilised form, was assessed by molecular biotyping using both PFGE and (GTG)5 rep-PCR profiling on 209 isolates belonging to Streptococcus thermophilus (30), Lactobacillus delbrueckii subsp. lactis (72), Enterococcus faecium (87), and Limosilactobacillus reuteri (20), revealing high biodiversity, at the strain level, in the cultures. The cultures' safety was proved through a new approach assessing phenotypic and molecular antibiotic resistance of the cultures in toto, instead of single strains, while the safety of Enterococcus faecium isolates was investigated according to EFSA guidelines. The use of natural biodiverse cultures for the production of microbial starters for typical and PDO cheeses, such as Pecorino Romano, could be an opportunity for recovering the cheese microbiota biodiversity lost during years of commercial starters use.

Optimization of scotta as growth medium to preserve biodiversity and maximise bacterial cells concentration of natural starter cultures for Pecorino Romano PDO cheese.

Preservation of cheese microbiota biodiversity is central to the sensory quality of traditional and PDO cheeses. Lyophilized commercial selected starters, being advantageous in terms of cells concentration, are supplanting natural cultures causing important loss of microbial biodiversity in the dairy environment. Biodiversity could be recovered using natural starter cultures, however their cells concentration after propagation is lower than the commercial ones. Two autochthonous and biodiverse starter cultures (MixA and MixB) coming from scotta (residual whey from Ricotta cheese manufacture), collected in the 1960 s from Pecorino Romano PDO cheese manufactures, were revitalized in reconstituted commercial powder scotta. The aim of this study was the propagation of the microbial starter mixes increasing their bacterial concentration in the pellet, reducing nonessential scotta components by a fast and not-expensive method, without changing the microbial community balance. The behaviour of each mix inoculated in scotta was compared to that in half-concentrated, clarified, and half-concentrated-clarified scotta. Higher cells concentration in the pellets from the modified scotta was obtained, without changing technological performances and microbial fingerprint. The pellets obtained were reinoculated in commercial scotta for the preparation of the scotta-innesto (the typical starter for Pecorino Romano), and no differences were observed among the treatments after incubation. The reduction of nonessential scotta's components could help the reproduction of natural starter cultures preserving their properties.

Do Best-Selected Strains Perform Table Olive Fermentation Better than Undefined Biodiverse Starters? A Comparative Study.

Twenty-seven Lactobacillus pentosus strains, and the undefined starter for table olives from which they were isolated, were characterised for their technological properties: tolerance to low temperature, high salt concentration, alkaline pH, and olive leaf extract; acidifying ability; oleuropein degradation; hydrogen peroxide and lactic acid production. Two strains with appropriate technological properties were selected. Then, table olive fermentation in vats, with the original starter, the selected strains, and without starter (spontaneous fermentation) were compared. Starters affected some texture profile parameters. The undefined culture resulted in the most effective Enterobacteriaceae reduction, acidification and olive debittering, while the selected strains batch showed the lowest antioxidant activity. Our results show that the best candidate strains cannot guarantee better fermentation performance than the undefined biodiverse mix from which they originate.

Shelf Life Evaluation of Ricotta Fresca Sheep Cheese in Modified Atmosphere Packaging.

Ricotta fresca cheese is the product of Sardinian dairy industry most exposed to microbial post-process contamination. Due to its technological characteristics, intrinsic parameters, pH (6.10-6.80) and water activity (0.974-0.991), it represents an excellent substrate for the growth of spoilage and pathogenic microorganisms, which are usually resident in cheese-making plants environments. Generally, ricotta fresca has a shelf life of 5-7 days. For this reason, at industrial level, modified atmosphere packaging (MAP) is used to extend the durability of the product. However, few investigations have been conducted to validate the use of MAP in ricotta fresca. The aim of this work is to evaluate the shelf life of ricotta fresca under MAP. A total of 108 samples were collected from three Sardinian industrial cheese-making plants and analysed within 24 h after packaging and after 7, 14 and 21 days of refrigerated storage. Aerobic mesophilic bacteria, mesophilic and thermophilic cocci and lactobacilli, Enterobacteriaceae and E. coli, L. monocytogenes, Pseudomonas spp, Bacillus cereus, yeasts and moulds, and the chemical-physical parameters and composition of the product were determined. At the end of the shelf life, Pseudomonas spp. and Enterobacteriaceae reached high concentrations, 5 to 7 and 3 to 6 log10 colony forming unit g-1, respectively. The presence of environmental contaminants indicates that the use of MAP without the appropriate implementation of prerequisite programmes is not sufficient to extend the durability of ricotta fresca. Gas mixture and packaging material should be selected only on the basis of scientific evidence of their effectiveness.

Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods.

The aim of this study was to evaluate the susceptibility of 197 isolates of Lactobacillus paracasei, isolated from Italian fermented products coming from different geographical areas, to tetracycline and erythromycin, two antimicrobials widely used in clinical and animal therapy. Isolation media were supplemented with antibiotics according to the microbiological breakpoints (BPs) defined by European Food Safety Authority (EFSA). Isolates were identified at the species level and were typed by rep-PCR using the (GTG)(5) primer. A total of 121 genotypically different strains were detected and their phenotypic antimicrobial resistance to tetracycline and erythromycin was determined as the minimum inhibitory concentration (MIC) using the broth microdilution method. The presence of the genes ermB, ermC and tetL, tetM, tetS, tetW, in the phenotypically resistant isolates was investigated by PCR. Tetracycline induction of tetM expression on representative resistant strains, grown in medium either lacking or containing the antibiotic, was also analyzed by RT-PCR. Among the 121 tested strains, 77.7% were susceptible to tetracycline (MICor=1024 microg/ml) (Erm(R)). The tetM and ermB genes were the most frequently detected in the Tet(R) and/or Erm(R) strains. The tetM expression was induced by antibiotic addition to the growth medium. Our study confirmed that L. paracasei is quite sensitive to tetracycline and erythromycin, but the high level of resistance of Erm(R) strains suggested that acquired resistance took place. Further investigations are required to analyze whether the genes identified in L. paracasei isolates might be horizontally transferred to other species. Since "commensal" bacteria, which L. paracasei belongs to, may play an active role in the spreading of antibiotic resistance, a series of measures inspired from a principle of precaution should be taken before they are used as commercial starters or probiotic cultures in food products, complemented by a more prudent use of antibiotics in agriculture, veterinary, and human medicine.