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.

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.

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.

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.

A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters.

A Qualified Presumption of Safety (QPS) approach was applied to dominant lactic acid bacteria (LAB) associated with Grana Padano cheese whey starters. Thirty-two strains belonging to Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum, and representing the overall genotypic LAB diversity associated with 24 previously collected whey starters [Rossetti, L., Fornasari, M.E., Gatti, M., Lazzi, C., Neviani, E., Giraffa, G., 2008. Grana Padano cheese whey starters: microbial composition and strain distribution. International Journal of Food Microbiology 127, 168-171], were analyzed. All L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus isolates were susceptible to four (i.e. vancomycin, gentamicin, tetracycline, and erythromycin) of the clinically most relevant antibiotics. One L. fermentum strain displayed phenotypic resistance to tetracycline (Tet(R)), with MIC of 32 microg/ml, and gentamycin (Gm(R)), with MIC of 32 microg/ml. PCR was applied to this strain to test the presence of genes tet(L), tet(M), tet(S), and aac(6')-aph(2')-Ia, which are involved in horizontal transfer of Tet(R) and Gm(R), respectively but no detectable amplification products were observed. According to QPS criteria, we conclude that Grana cheese whey starters do not present particular safety concerns.

Grana Padano cheese whey starters: microbial composition and strain distribution.

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization.

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.

Detection and identification of Lactobacillus helveticus bacteriophages by PCR.

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.

Cultivability of Streptococcus thermophilus in Grana Padano cheese whey starters.

The application of a culture-independent approach, that of reverse transcriptase-length heterogeneity-PCR coupled with epifluorescence microscopy, allowed us to observe that Streptococcus thermophilus is metabolically active, but only partially cultivable in Grana Padano cheese whey starters. A short preincubation of the starters in sterile skimmed whey was followed by cultivation in sterile skimmed whey-enriched M17. This procedure restored the cultivability of S. thermophilus and enabled us to detect S. thermophilus at ranges (10(7)-10(8) CFU mL(-1)) which have rarely been reported in these cultures. The use of cheese whey as a cultivation-revitalization substrate can be useful to obtain an unbiased picture of the microbial composition of whey starters for Grana Padano cheese, thus avoiding an underestimation of S. thermophilus in these cultures.

Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations.

A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.

Heterogeneity of putative surface layer proteins in Lactobacillus helveticus.

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

Monitoring of the bacterial composition of dairy starter cultures by RAPD-PCR.

Randomly amplified polymorphic DNA (RAPD)-PCR was used to verify the species composition of commercial dairy starters and to detect possible shifts in strain composition of these cultures. After RAPD-PCR analysis, not all the strains isolated in the years 2001 and 2002 fell within the same dendrogram cluster of the strains isolated in the year 2000 and used as reference strains. Changes in composition of the microbial population and/or voluntary immission of new biotypes with respect to the original strain formulation had occurred in the starters. The microbial composition of modern dairy starters represents a key point because the complex relationships among microorganisms can easily be altered. Little variations in the microbial composition could have unexpected effects on cheese quality.

Comparison of cultural methods for the identification and molecular investigation of yeasts from sourdoughs for Italian sweet baked products.

Twenty-five yeast strains isolated from sourdough samples for Panettone, Pandoro and Cornetto brioche manufactured by eight different bakeries in northern Italy were characterised. Classification was performed by the simplified identification method (SIM), Kurtzman and Fell's identification protocol, the API system from bioMérieux (France) and the MicroLog system from Biolog (USA). Genetic diversity was investigated by randomly amplified polymorphic DNA fingerprinting and mitochondrial-DNA restriction enzyme analysis. Sequences of the internal transcribed spacers between 18S and 26S rDNA genes were analysed. Candida humilis was the predominant species (56% of isolates), whereas the remaining strains (44%) were related to the Saccharomyces cerevisiae sensu stricto group. Identification systems based on phenotypic analysis proved to be unreliable to identify yeasts from sourdough. Either RAPD-PCR or mtDNA restriction analysis showed to be suitable for the identification of species, but could not be used to differentiate among the isolates at the strain level. Sequencing of the ITS region permitted a consistent classification of the sourdough yeasts.