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.

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.

Massive Survey on Bacterial-Bacteriophages Biodiversity and Quality of Natural Whey Starter Cultures in Trentingrana Cheese Production.

This study focused on the microbial and bacteriophages identification and characterization in cheese-production facilities that use natural whey starter (NWS) cultures for Trentingrana production. Bacterial and phage screening was carried out on cooked not acidified whey and NWS samples isolated from six dairy factories, for 4 consecutive days in four different months. By means of a combined approach, using plate counts, bacterial isolation, and metataxonomic analysis Lactobacillus helveticus was found occurring as the dominant species in NWS cultures and Levilactobacillus brevis as codominant in the cheese factories where the temperature of NWS production was mainly lower than 40°C, suggesting that the variability in the parameters of the NWS culture preparation could differently modulate the bacterial species in NWS cultures. Using turbidity test approach on 303 bacterial isolates from the NWS cultures, 120 distinct phages were identified. L. helveticus phage contamination of NWS cultures was revealed in most of the analyzed samples, but despite the great recovery of bacteriophage contamination cases, the microbial quality of NWS cultures was high. Our results support the presence of natural bacteriophage resistance mechanisms in L. helveticus. The use of NWS cultures probably creates an ideal environment for the proliferation of different L. helveticus strains balanced with their phages without a clear dominance. It is evident, from this study, that the presence of a high biodiversity of NWS bacterial strains is relevant to avoid phages dominance in NWS cultures and consequently to keep a good acidification ability.

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.

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.

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.

Leuconostoc strains isolated from dairy products: Response against food stress conditions.

A systematic study about the intrinsic resistance of 29 strains (26 autochthonous and 3 commercial ones), belonging to Leuconostoc genus, against diverse stress factors (thermal, acidic, alkaline, osmotic and oxidative) commonly present at industrial or conservation processes were evaluated. Exhaustive result processing was made by applying one-way ANOVA, Student's test (t), multivariate analysis by Principal Component Analysis (PCA) and Matrix Hierarchical Cluster Analysis. In addition, heat adaptation on 4 strains carefully selected based on previous data analysis was assayed. The strains revealed wide diversity of resistance to stress factors and, in general, a clear relationship between resistance and Leuconostoc species was established. In this sense, the highest resistance was shown by Leuconostoc lactis followed by Leuconostoc mesenteroides strains, while Leuconostoc pseudomesenteroides and Leuconostoc citreum strains revealed the lowest resistance to the stress factors applied. Heat adaptation improved thermal cell survival and resulted in a cross-resistance against the acidic factor. However, all adapted cells showed diminished their oxidative resistance. According to our knowledge, this is the first study regarding response of Leuconostoc strains against technological stress factors and could establish the basis for the selection of "more robust" strains and propose the possibility of improving their performance during industrial processes.

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.

Characterization of the genome of the dairy Lactobacillus helveticus bacteriophage {Phi}AQ113.

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.

Selection of Lactobacillus plantarum strains to use as starters in fermented table olives: Oleuropeinase activity and phage sensitivity.

Fermented table olives (Olea europaea L.) are largely diffused in the Mediterranean area. Olives are picked at different stages of maturity and after harvesting, processed to eliminate the characteristic bitterness caused by the presence of the oleuropein glucoside and to become suitable for human consumption. The spontaneous fermentation of table olives mainly depends on lactic acid bacteria (LAB), and in particular on Lactobacillus plantarum which plays an important role in the degradation of oleuropein. The hydrolysis of oleuropein is attributed to the ß-glucosidase and esterase activities of the indigenous LAB microflora. This study investigated the potential of L. plantarum strains isolated from dairy products and olives to be used as starters for fermented table olives. Forty-nine strains were typed by RAPD-PCR and investigated for the presence of the ß-glucosidase (bglH) gene. The full sequence of the bglH gene was carried out. All the 49 L. plantarum strains were also tested for phage resistance. A total of six strains were selected on the basis of genotypic polymorphism, bglH gene sequence analysis, and phage resistance profile. These strains were further characterized to assess the acidifying capability, the growth at different temperatures, the tolerance to different NaCl concentrations, and the oleuropeinolytic activity. Although further characterizations are required, especially concerning the influence on sensory properties, L. plantarum proved to have the potential to be used as a debittering and fermentative agent in starter culture for fermented table olives.

Cholesterol removal capability of lactic acid bacteria and related cell membrane fatty acid modifications.

Milk and dairy products play an important role in a healthy diet because of their high nutritional value, even if they represent a source of lipids and cholesterol. Nowadays, some commercially hypocholesterolemic products are available, which contain lactic acid bacteria (LAB). Therefore, the aims of this study were to test and compare the cholesterol removal abilities of different LAB species and to investigate the capacity of the cholesterol to change the cellular fatty acid composition of microorganisms. Fifty-eight strains of dairy LAB were studied for their ability to remove cholesterol during 24 h of growth. Two of them, L. plantarum 885 and L. acidophilus LA-5®, showed the higher reduction capability. For these strains, the cellular fatty acid composition was studied. They showed a different behaviour, which appeared related to the needs of the cells to maintain the characteristics of membrane fluidity, but was dependent upon their original fatty acid composition. Further studies are required to better characterise the LAB strains to be used to develop fermented dairy products with reduced cholesterol content or be able to induce hypocholesterolemic effects. It will also be interesting to investigate the possible modifications of the cell membrane caused by cholesterol and its possible involvement in cell metabolism.

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus.

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥6log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.

Molecular analysis of relapses or reinfections of Clostridium difficile-associated diarrhea.

Recurrence is a major complication of Clostridium difficile-associated diarrhea and occurs in 15 to 20% of patients after discontinuation of therapy. Strains from 53 patients with Clostridium difficile recurrences were fingerprinted by PCR ribotyping. Reinfection with a different strain occurred in 15 out of 53 patients (28,3%), while 38 patients relapsed. These data suggest the need to perform molecular typing for implementation of infection control procedures and for a more appropriate therapeutic strategy.