Aurora Meucci

Lactococcus hircilactis sp. nov. and Lactococcus laudensis sp. nov., isolated from milk.

Two strains of lactic acid bacteria, designated 117(T) and 4195(T), were isolated from goat milk in Valtellina, Italy and from cow milk in Valle Trompia, Italy, respectively, and characterized taxonomically by a polyphasic approach. The strains were Gram-stain-positive, coccoid, non-spore-forming and catalase-negative bacteria. Morphological, physiological and phylogenetic data indicated that these isolates belonged to the genus Lactococcus. Strain 117(T) was closely related to Lactococcus fujiensis, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae and Lactococcus taiwanensis, showing 93-94% and 82-89% 16S rRNA and rpoB gene sequence similarities, respectively. Strain 4195(T) was closely related to Lactococcus chungangensis, Lactococcus raffinolactis, Lactococcus plantarum and Lactococcus piscium, showing 92-98% and 86-99% 16S rRNA and rpoB gene sequence similarities, respectively. Based on this evidence and the data obtained in the present study, the milk isolates represent two novel species of the genus Lactococcus, for which the names Lactococcushircilactis sp. nov., and Lactococcuslaudensis sp. nov. are proposed. The respective type strains are 117(T) ( = LMG 28352(T) = DSM 28960(T)) and 4195(T )( = LMG 28353(T) = DSM 28961(T)).

Lactose, galactose and glucose determination in naturally “lactose free” hard cheese: HPAEC-PAD method validation

A chromatographic method by HPAEC-PAD was developed and in-house validated for the quantification of low sugar levels in hard cheese, specifically Grana Padano PDO cheese. Particular attention was paid to the extraction procedure, due to residual microbial and enzymatic activities. Specificity in detection and linearity were verified. Recoveries ranged from 93% for lactose to 98% for glucose and galactose. The obtained LOD and LOQ values were, respectively, 0.25 and 0.41mg/100g for lactose, 0.14 and 0.27mg/100g for galactose, and 0.16 and 0.26mg/100g for glucose. The method was applied to 59 samples of Grana Padano PDO cheese: galactose showed the highest concentration and variability among the samples (1.36±0.89), compared to both lactose (0.45±0.12) and glucose (0.46±0.13). Considering the very low levels of sugars detected, authentic PDO Grana Padano could be safely included in the diet of people suffering from lactose intolerance.

Isolation and preliminary characterization of Lactobacillus plantarum bacteriophages from table olive fermentation

Nine table olive fermentation brines were analysed to demonstrate the presence of lytic bacteriophages active against Lactobacillus plantarum strains. Five brines, out of the nine studied, were added to selected strains of L. plantarum as starter cultures, while the other four brines were left to ferment spontaneously. Turbidity tests with different host strains inoculated in growth medium with added filter-sterilized brines demonstrated the presence of phages able to lyse the sensitive strains. Phages were isolated from fermented brines and their presence was confirmed by scanning electron microscopy. A subsequent phage characterization based on host range, restriction analysis and protein profiles was performed. This study reported for the first time the isolation and characterization of L. plantarum phages from table olive fermentation. This demonstrated presence of lytic phages active against L. plantarum could represent a serious obstacle in the regular table olive fermentation process. For these reasons, the search for phage-resistant strains, to use as starter cultures, could be important to counteract the development of fermentation problems.

Applicability of Lactococcus hircilactis and Lactococcus laudensis as dairy cultures

The aim of this study was to evaluate whether Lactococcus hircilactis and Lactococcus laudensis can be used as starter cultures. To this end, the two lactococci were characterized for traits of technological and functional interest. Tests in milk included growth at 20, 25, 30, and 37 °C, flavor production, antioxidant (AO) activity, folate and exopolysaccharide (EPS) production. At 30 °C, which resulted the best growth temperature for both strains, Lc. hircilactis and Lc. laudensis lowered the pH of the milk to 4.8 and 5.5, respectively, after 24 h of incubation. Sugar and organic acid composition indicated a higher lactose utilization, coupled with a higher lactate accumulation, by Lc. hircilactis, while galactose was completely consumed by both species. Both strains showed a Cit- phenotype after growth in a selective medium containing citrate as the sole carbon source. Nevertheless, a small amount of citrate was used by both lactococci when grown in milk. The two strains were characterized by a different flavor production, showed high AO activity, and produced small amounts of EPS (~30 mg/L). Lactococcus laudensis showed a weak proteolytic activity while Lc. hircilactis was able to accumulate folate at levels four times higher than uninoculated milk. When the two lactococci were tested as starter cultures in small-scale cheesemaking trials, cheeses resulted of satisfying quality and contained amounts of ethanol, acetic acid, diacetyl and acetoin higher than controls, obtained using a commercial culture. The application of Lc. hircilactis and Lc. laudensis as aromatic cultures in cheesemaking is proposed.

Biodiversity of Lactobacillus helveticus bacteriophages isolated from cheese whey starters

Twenty-one Lactobacillus helveticus bacteriophages, 18 isolated from different cheese whey starters and three from CNRZ collection, were phenotypically and genetically characterised. A biodiversity between phages was evidenced both by host range and molecular (RAPD-PCR) typing. A more detailed characterisation of six phages showed similar structural protein profiles and a relevant genetic biodiversity, as shown by restriction enzyme analysis of total DNA. Latent period, burst time and burst size data evidenced that phages were active and virulent. Overall, data highlighted the biodiversity of Lb. helveticus phages isolated from cheese whey starters, which were confirmed to be one of the most common phage contamination source in dairy factories. More research is required to further unravel the ecological role of Lb. helveticus phages and to evaluate their impact on the dairy fermentation processes where whey starter cultures are used.