Low-Level Clostridial Spores' Milk to Limit the Onset of Late Blowing Defect in Lysozyme-Free, Grana-Type Cheese.

The growth of clostridial spores during ripening leads to late blowing (LB), which is the main cause of spoilage in Grana Padano Protected Designation of Origin (PDO) cheese and other hard, long-ripened cheeses such as Provolone, Comté, and similar cheeses. This study aimed to verify the cause-effect relationship between the level of clostridial butyric spores (BCS) in milk and the onset of the LB defect. To this end, experimental Grana-type cheeses were produced without lysozyme, using bulk milk with different average BCS content. The vat milk from the so-called "virtuous" farms (L1) contained average levels of BCS of 1.93 ± 0.61 log most probable number (MPN) L-1, while the vat milk from farms with the highest load of spores (L2), were in the order of 2.99 ± 0.69 log MPN L-1. Cheeses after seven months of ripening evidenced a strong connection between BCS level in vat milk and the occurrence of LB defect. In L2 cheeses, which showed an average BCS content of 3.53 ± 1.44 log MPN g-1 (range 1.36-5.04 log MPN g-1), significantly higher than that found in L1 cheeses (p < 0.01), the defect of LB was always present, with Clostridium tyrobutyricum as the only clostridial species identified by species-specific PCR from MPN-positive samples. The L1 cheeses produced in the cold season (C-L1) were free of defects whereas those produced in the warm season (W-L1) showed textural defects, such as slits and cracks, rather than irregular eyes. A further analysis of the data, considering the subset of the cheesemaking trials (W-L1 and W-L2), carried out in the warm season, confirmed the presence of a climate effect that, often in addition to the BCS load in the respective bulk milks (L1 vs. L2), may contribute to explain the significant differences in the chemical composition and some technological parameters between the two series of cheeses. Metagenomic analysis showed that it is not the overall structure of the microbial community that differentiates L1 from L2 cheeses but rather the relative distribution of the species between them. The results of our trials on experimental cheeses suggest that a low-level BCS in vat milk (<200 L-1) could prevent, or limit, the onset of LB in Grana-type and similar cheeses produced without lysozyme.

The Reduction of Salt in Different Cheese Categories: Recent Advances and Future Challenges.

Public awareness about excessive sodium intake and nutrition claims related to salt content entail the need for food industries to carefully reconsider the composition and processing of high sodium foods. Although in some products the reformulation with alternative ingredients is commonly practiced, in cheese the reduction of salt is still a challenging task, as sodium chloride exerts multiple and fundamental functions. Salt favors the drainage of the residual whey, enhances the taste and the aroma profile, regulates the texture, the final pH, the water activity, and affects the microbial growth. Ultimately, salt content modulates the activity of starter and non-starter lactic acid bacteria (NSLAB) during cheese manufacturing and ripening, influencing the shelf-life. Any modification of the salting procedure, either by reducing the level of sodium chloride content or by replacing it with other salting agents, may affect the delicate equilibrium within the above-mentioned parameters, leading to changes in cheese quality. The decrease of Na content may be differently approached according to cheese type and technology (e.g., soft, semi-hard, hard, and mold-ripened cheeses). Accordingly, targeted strategies could be put in place to maintain the overall quality and safety of different cheeses categories.

The Microbiota of Grana Padano Cheese. A Review.

Grana Padano (GP) is the most appreciated and marketed cheese with Protected Designation of Origin in the world. The use of raw milk, the addition of undefined cultures (defined as 'sieroinnesto naturale'), the peculiar manufacturing proces, and the long ripening make the cheese microbiota play a decisive role in defining the quality and the organoleptic properties of the product. The knowledge on the microbial diversity associated with GP has been the subject, in recent years, of several studies aimed at understanding its composition and characteristics in order, on the one hand, to improve its technological performances and, on the other hand, to indirectly enhance the nutritional quality of the product. This review aims to briefly illustrate the main available knowledge on the composition and properties of the GP microbiota, inferred from dozens of studies carried out by both classical microbiology techniques and metagenomic analysis. The paper will essentially, but not exclusively, be focused on the lactic acid bacteria (LAB) derived from starter (SLAB) and the non-starter bacteria, both lactic (NSLAB) and non-lactic, of milk origin.

Bacterial Community of Grana Padano PDO Cheese and Generical Hard Cheeses: DNA Metabarcoding and DNA Metafingerprinting Analysis to Assess Similarities and Differences.

The microbiota of Protected Designation of Origin (PDO) cheeses plays an essential role in defining their quality and typicity and could be applied to protect these products from counterfeiting. To study the possible role of cheese microbiota in distinguishing Grana Padano (GP) cheese from generical hard cheeses (HC), the microbial structure of 119 GP cheese samples was studied by DNA metabarcoding and DNA metafingerprinting and compared with 49 samples of generical hard cheeses taken from retail. DNA metabarcoding highlighted the presence, as dominant taxa, of Lacticaseibacillus rhamnosus, Lactobacillus helveticus, Streptococcus thermophilus, Limosilactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus spp., and Lactococcus spp. in both GP cheese and HC. Differential multivariate statistical analysis of metataxonomic and metafingerprinting data highlighted significant differences in the Shannon index, bacterial composition, and species abundance within both dominant and subdominant taxa between the two cheese groups. A supervised Neural Network (NN) classification tool, trained by metagenotypic data, was implemented, allowing to correctly classify GP cheese and HC samples. Further implementation and validation to increase the robustness and improve the predictive capacity of the NN classifier will be needed. Nonetheless, the proposed tool opens interesting perspectives in helping protection and valorization of GP and other PDO cheeses.

Design of an autochthonous starter culture using strains isolated from traditional Matsoni.

Artisanal products support the conservation of the indigenous biodiversity of food microbiomes, although they do not always comply to quality and hygienic requirements for the dairy industry. This study describes the development of an autochthonous starter culture to produce Matsoni, a traditional Georgian fermented milk. To this end, strains of lactic acid bacteria isolated from artisanal Matsoni samples were used to design a starter formulation reproducing the dominant microbial diversity, also preserving quality characteristics and ensuring the safety of the product. As a result, strains that represent the acidifying portion of the starter (Lactobacillus delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus) were combined in different ratios and strain combinations, together with cultures of Lactobacillus rhamnosus that were chosen for their potential beneficial traits. The strain association acting better in milk cultures at laboratory scale was selected as starter culture for the production of Matsoni in pilot-scale industrial trials.

Functional characterization and immunomodulatory properties of Lactobacillus helveticus strains isolated from Italian hard cheeses.

Lactobacillus helveticus carries many properties such as the ability to survive gastrointestinal transit, modulate the host immune response, accumulate biopeptides in milk, and adhere to the epithelial cells that could contribute to improving host health. In this study, the applicability as functional cultures of four L. helveticus strains isolated from Italian hard cheeses was investigated. A preliminary strain characterization showed that the ability to produce folate was generally low while antioxidant, proteolytic, peptidase, and ß-galactosidase activities resulted high, although very variable, between strains. When stimulated moDCs were incubated in the presence of live cells, a dose-dependent release of both the pro-inflammatory cytokine IL-12p70 and the anti-inflammatory cytokine IL-10, was shown for all the four strains. In the presence of cell-free culture supernatants (postbiotics), a dose-dependent, decrease of IL-12p70 and an increase of IL-10 was generally observed. The immunomodulatory effect took place also in Caciotta-like cheese made with strains SIM12 and SIS16 as bifunctional (i.e., immunomodulant and acidifying) starter cultures, thus confirming tests in culture media. Given that the growth of bacteria in the cheese was not necessary (they were killed by pasteurization), the results indicated that some constituents of non-viable bacteria had immunomodulatory properties. This study adds additional evidence for the positive role of L. helveticus on human health and suggests cheese as a suitable food for delivering candidate strains and modulating their anti-inflammatory properties.

Evaluation of bacterial communities of Grana Padano cheese by DNA metabarcoding and DNA fingerprinting analysis.

The composition of the bacterial community of Grana Padano (GP) cheese was evaluated by an amplicon-based metagenomic approach (DNA metabarcoding) and RAPD-PCR fingerprinting. One hundred eighteen cheeses, which included 118 dairies located in the production area of GP, were collected. Two hundred fifty-four OTUs were detected, of which 82 were further discriminated between dominant (32 OTUs; > 1% total reads) and subdominant (50 OTUs; between 0.1% and 1% total reads) taxa. Lactobacillus (L.) delbrueckii, Lacticaseibacillus (Lact.) rhamnosus, Lact. casei, Limosilactobacillus fermentum, Lactococcus (Lc.) raffinolactis, L. helveticus, Streptococcus thermophilus, and Lc. lactis were the major dominant taxa ('core microbiota'). The origin of samples significantly impacted on both richness, evenness, and the relative abundance of bacterial species, with peculiar pattern distribution among the five GP production regions. A differential analysis allowed to find bacterial species significantly associated with specific region pairings. The analysis of pattern similarity among RAPD-PCR profiles highlighted the presence of a 'core' community banding pattern present in all the GP samples, which was strictly associated with the core microbiota highlighted by DNA metabarcoding. A trend to group samples according to the five production regions was also observed. This study widened our knowledge on the bacterial composition and ecology of Grana Padano cheese.

Characterization and pre-industrial validation of Streptococcus thermophilus strains to be used as starter cultures for Crescenza, an Italian soft cheese.

The aim of this work was to search for new candidate strains to be included in a culture for Crescenza, a rindless soft cheese, today produced mainly at industrial level using selected starter cultures composed of S. thermophilus. Performance testing was applied to 29 pre-selected strains and a scoring approach was developed to identify the most suitable candidates to be employed in Crescenza cheesemaking. Eight S. thermophilus strains fulfilling most of the desired properties (e.g., high phage resistance, fast acidification rate, no growth below 20 °C, NaCl sensibility, no post acidification at 4 °C) were selected. These strains were grouped in pairs to design different starter culture formulations, which were preliminary tested for the production of Crescenza cheeses at laboratory scale. Two couples of binary cultures (designed Phage rotation 1 and Phage rotation 2) were finally designed and used as starters in pilot scale cheesemaking. The combinations, especially those designed in Phage rotation 1, appeared to be suitable for Crescenza production and showed mutual similarity in terms of strain characteristics, technological performance, and cheese quality. The selection method and ranking approach presented in this work may be adapted to other species of LAB showing traits of industrial interest.

Application of Recombined Milk to Produce Crescenza-Type Cheese in Laboratory-Scale Cheesemaking: Implications on Technology and Sensory Properties.

This work evaluated the effect of recombined skimmed milk (RM), mixed in different ratios (40, 60, and 100%) with fresh cow milk, on the processing technology and quality of Crescenza, an industrial soft cheese of the Italian dairy tradition. Crescenza-type cheeses were produced at a laboratory scale, following the industrial process. Control cheese consisted of Crescenza-type cheese produced with 100% whole fresh milk. Compared to control cheese, the substitution of fresh milk with 60-100% of RM deteriorated the coagulation properties and led to a higher moisture retention, whereas, with 40% of RM, the differences were not statistically significant. Cheeses produced with any concentration of RM, although of acceptable quality, differed significantly in terms of sensory properties from control cheese. The addition of colloidal calcium phosphate, or CaCl2 together with a reduction in the size of the curd at cutting, minimized the differences in composition and sensory properties between cheeses produced with 40% RM and control cheese. This study suggested the applicability of 40% RM to obtain Crescenza-type cheese with suitable quality characteristics. The type of product, the technology, the quality, and quantity of the powders are all key factors to be taken into account for a successful application.

Extracellular and intracellular DNA for bacterial profiling of long-ripened cheeses.

A novel approach was developed to extract the extracellular DNA (eDNA), i.e. the free DNA outside the microbial cell, compared to the intracellular DNA (iDNA). The two DNA fractions were investigated in seven long-ripened cheeses. Among different buffer solutions tested, EDTA 0.5 M at pH 8 enabled a mild homogenization of cheese samples and the highest eDNA recovery. The extraction protocol was tested on single strains of lactic acid bacteria characterizing many Italian long-ripened cheeses, such as Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus rhamnosus. The method resulted suitable for eDNA extraction because it minimized cell-lysis, avoiding the leakage of iDNA from the cells. The yields of eDNA, ranging from 0.01 to 0.36 µg g-1 cheese, were generally higher than the iDNA, indicating that autolytic phenomena prevailed over intact cells after 8-12 months of ripening. In four of the seven cheeses, the same LAB species were detected in the eDNA and iDNA fractions by length-heterogeneity PCR, while in the remaining three samples, a higher number of species was highlighted in the eDNA compared to the corresponding iDNA. The sequential extraction of eDNA and iDNA can be applied to obtain additional information on the composition of the bacterial community in long-aged cheeses.

The impact of sodium chloride reduction on Grana-type cheese production and quality.

With the aim to reduce the Na content, hard cheeses manufactured using the same technology as for Grana cheese (Grana-type) were salted using three brines containing different amounts of KCl (K-brines) and compared with control cheeses, salted with marine NaCl. A lower weight loss was observed in cheeses salted with K-brines (K-cheeses), whereas the yield and dry matter did not differ significantly between K-cheeses and controls. After 3 months of ripening (T3), the distribution of the Na cations (Na) was centripetal, with a higher Na concentration in the outer (0-3 cm of depth) layer, whereas the K cations (K) seemed to diffuse into the cheese more rapidly and homogeneously. Starting from the 6th month (T6), the distribution of both Na and K was stabilized through the different cheese layers. The use of the brine with the highest concentration of potassium (53.8% K) enabled us to successfully halve the Na content compared to the controls whereas, with the other brines, the reduction of Na was below 30%. At the end of ripening (T9), all the cheeses were without defects and the partial substitution of Na with K did not impact on the chemical composition, microbiological characteristics and ripening process. The sensory evaluation did not show any difference between K-salted and control cheeses in discriminant analysis.

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.

Folates biosynthesis by Streptococcus thermophilus during growth in milk.

The ability of folate-producer strains of Streptococcus thermophilus to accumulate folates and the expression of two target genes (folK and folP), involved in the folate biosynthesis, were studied during milk fermentation. An over-expression of folK took place only in the early phase of growth, whereas folP was mainly expressed in the mid log-phase of growth and declined thereafter. The accumulation of total folates, which was quantified by a microbiological assay, was strain-dependent. Two major forms of folates, i.e. tetrahydrofolate (THF) and 5-methyl-tetrahydrofolate (5-Met-THF), were identified and quantified by HPLC. With respect to the level accumulated by a weak folate producer (St 383), used as calibrator in the expression experiments and as control in folate quantification in milk, the strains St 563 and St 399 produced 5-Met-THF in amounts significantly higher than THF. The possibility of using selected folate-producer S. thermophilus strains as functional cultures for a bio-fortification of dairy products is discussed.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain.

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems.

Depletion of tyrosyl-DNA phosphodiesterase 1α (MtTdp1α) affects transposon expression in Medicago truncatula.

The role of plant tyrosyl-DNA phosphodiesterase 1α in genome stability is studied using a Medicago truncatula MtTdp1α-depleted line. Lack of MtTdp1α results in a 39% reduction of methylated cytosines as compared to control. RNA-Seq analyses revealed that 11 DNA transposons and 22 retrotransposons were differentially expressed in the Tdp1α-2a line. Among them all, DNA transposons (MuDR, hAT, DNA3-11_Mad) and seven retrotransposons (LTR (Long Terminal Repeat)/Gipsy, LTR/Copia, LTR and NonLTR/L1) were down-regulated, while the 15 retrotransposons were up-regulated. Results suggest that the occurrence of stress-responsive cis-elements as well as changes in the methylation pattern at the LTR promoters might be responsible for the enhanced retrotransposon transcription.

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)).

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.

Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants.

KEY MESSAGE: Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress. Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.2 mM CuCl2. The DNA diffusion assay revealed a significant reduction in the percentage of necrosis caused by copper in the aerial parts of the Tdp2α-13c and Tdp2α-28 plants while neutral single cell gel electrophoresis highlighted a significant decrease in double strand breaks (DSBs), compared to CTRL. In the copper-treated Tdp2α-13c and Tdp2α-28 lines there was up-regulation (up to 4.0-fold) of genes encoding the α and ß isoforms of Tyrosyl-DNA phosphodiesterase 1, indicating the requirement for Tdp1 function in the response to heavy metals. As for DSB sensing, the MtMRE11, MtRAD50 and MtNBS1 genes were also significantly up-regulated (up to 2.3-fold) in the MtTdp2α-overexpressing plants grown under physiological conditions, compared to CTRL line, and then further stimulated in response to copper. The basal antioxidant machinery was always activated in all the tested lines, as indicated by the concomitant up-regulation of MtcytSOD and MtcpSOD genes (cytosolic and chloroplastic Superoxide Dismutase), and MtMT2 (type 2 metallothionein) gene. The role of MtTdp2α gene in enhancing the plant response to genotoxic injury under heavy metal stress is discussed.