Phenotypic and Safety Assessment of the Cheese Strain Lactiplantibacillus plantarum LL441, and Sequence Analysis of its Complete Genome and Plasmidome.

This work describes the phenotypic typing and complete genome analysis of LL441, a dairy Lactiplantibacillus plantarum strain. LL441 utilized a large range of carbohydrates and showed strong activity of some carbohydrate-degrading enzymes. The strain grew slowly in milk and produced acids and ketones along with other volatile compounds. The genome of LL441 included eight circular molecules, the bacterial chromosome, and seven plasmids (pLL441-1 through pLL441-7), ranging in size from 8.7 to 53.3 kbp. Genome analysis revealed vast arrays of genes involved in carbohydrate utilization and flavor formation in milk, as well as genes providing acid and bile resistance. No genes coding for virulence traits or pathogenicity factors were detected. Chromosome and plasmids were packed with insertion sequence (IS) elements. Plasmids were also abundant in genes encoding heavy metal resistance traits and plasmid maintenance functions. Technologically relevant phenotypes linked to plasmids, such as the production of plantaricin C (pLL441-1), lactose utilization (pLL441-2), and bacteriophage resistance (pLL441-4), were also identified. The absence of acquired antibiotic resistance and of phenotypes and genes of concern suggests L. plantarum LL441 be safe. The strain might therefore have a use as a starter or starter component in dairy and other food fermentations or as a probiotic.

Advantages and disadvantages of non-starter lactic acid bacteria from traditional fermented foods: Potential use as starters or probiotics.

Traditional fermented foods are a significant source of starter and/or non-starter lactic acid bacteria (nsLAB). Moreover, these microorganisms are also known for their role as probiotics. The potential of nsLAB is huge; however, there are still challenges to be overcome with respect to characterization and application. In the present review, the most important steps that autochthonous lactic acid bacteria isolated from fermented foods need to overcome, to qualify as novel starter cultures, or as probiotics, in food technology and biotechnology, are considered. These different characterization steps include precise identification, detection of health-promoting properties, and safety evaluation. Each of these features is strain specific and needs to be accurately determined. This review highlights the advantages and disadvantages of nsLAB, isolated from traditional fermented foods, discussing safety aspects and sensory impact.

Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.

Lactic acid bacteria (LAB) found in milk can be responsible for organoleptic defects in cheese. To identify sources of LAB that could potentially develop during cheese making, we evaluated their prevalence and abundance in milk according to the type of forage used in dairy cow feeding. Forages and bulk tank milk were sampled 3 times on 24 farms using either hay alone (control), or grass or legume silage supplemented with corn silage or not. Both types of silage were either non-inoculated or inoculated with commercial preparations containing at least a Lactobacillus buchneri strain along with Lactobacillus casei, Lactobacillus plantarum, Enterococcus faecium, or Pediococcus pentosaceus. Our results indicate that LAB viable counts in milk samples (2.56 log cfu/mL) did not differ according to the type of forage used. A total of 1,239 LAB were isolated and identified by partial 16S rRNA gene sequencing. Although inoculation increased lactobacilli abundance in grass silage by 35%, we did not observe an effect on the LAB profile of milk. Indeed, we found no significant difference in milk LAB prevalence and abundance according to the type of forage (P > 0.05). Moreover, isolates belonging to the L. buchneri group were rarely found in bulk tank milk (3 out of 481 isolates). Random amplified polymorphic DNA typing of 406 LAB isolates revealed the plausible transfer of some strains from silage to milk (~6%). Thus, forage is only a minor contributor to LAB contamination of milk.

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production.

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality.

Microbial communities involved in Kasar cheese ripening.

The microbiota of non-starter lactic acid bacteria (NSLAB) and their concomitant community dynamics during cheese ripening were investigated for traditional Turkish Kasar cheeses made from raw cows' milk. Five batches of 15 Kasar cheeses produced in different dairy plants located in Kars were analysed during their whole ripening phase up to 180 days. Lactobacilli and lactococci were determined as the prevailing microbial groups. The molecular classification and identification of 594 LAB isolates during Kasar cheese ripening were performed through (GTG)5-PCR fingerprinting of their genomic DNA followed by verification of the (GTG)5-PCR clusters obtained after numerical analysis through 16S rRNA gene sequencing of representative isolates. Lactobacillus casei (247 isolates, 41.6%), Lactobacillus plantarum (77 isolates, 13.0%), and Pediococcus acidilactici (58 isolates, 9.8%) were the prevailing NSLAB species in all Kasar cheeses of the different dairy plants investigated throughout cheese ripening. The data of the present study contribute to the inventory of unique cheese varieties to enable the prevention of losses of microbial biodiversity and the selection of starter cultures for controlled cheese manufacturing.

Tailoring the probiotic potential of non-starter Lactobacillus strains from ripened Parmigiano Reggiano cheese by in vitro screening and principal component analysis.

Non-starter lactic acid bacteria (NSLAB) inhabiting fermented food have been recently revised as source of probiotic strains. Here, we in vitro assessed the potential probiotic aptitude of a de-replicated set of NSLAB previously isolated from long ripened Parmigiano Reggiano cheeses (22 Lactobacillus rhamnosus, 18 Lactobacillus paracasei, 3 Lactobacillus casei, 2 Lactobacillus harbinensis, and 2 Lactobacillus fermentum). Most strains showed moderate to good resistance to biological barriers, including bile salts, lysozyme, and simulated gastric and pancreatic juices. Antimicrobial susceptibility tests against seven antimicrobials belonging to different categories showed that most strains are susceptible towards all the antibiotics, with the exception of vancomycin and streptomycin. The strains lost the streptomycin resistance when assayed on agar medium containing 0.5% bile salts, suggesting that detergent-like properties of cholic acids increase membrane permeability and mediate streptomycin susceptibility. No isolate showed bile salt hydrolase (BSH) activities, supporting that bile salts resistance and BSH activity are unpaired traits. Finally, NSLAB strains had moderate to high auto-aggregative and hydrophobic phenotypes, whereas two subsets of 22 and 8 strains co-aggregated with Escherichia coli and Salmonella enterica s. typhimurium, respectively. A multivariate analysis was effective to segregate one L. casei and two L. rhamnosus strains showing physiological characteristics compatible with probiotic properties.

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese.

Introduction: Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. Methods: To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. Results and discussion: The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from ß-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from ß-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for ß-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.

The microbial and metabolite composition of Gouda cheese made from pasteurized milk is determined by the processing chain.

Gouda cheeses of different production batches and ripening times often differ in metabolite composition, which may be due to the starter culture mixture applied or the growth of non-starter lactic acid bacteria (NSLAB) upon maturation. Therefore, a single Gouda cheese production batch was systematically investigated from the thermized milk to the mature cheeses, ripened for up to 100 weeks, to identify the main bacterial species and metabolites and their dynamics during the whole production and ripening. As this seemed to be starter culture strain- and NSLAB-dependent, it requested a detailed, longitudinal, and quantitative investigation. Hereto, microbial colony enumeration, high-throughput full-length 16S rRNA gene sequencing, and a metabolomic approach were combined. Culture-dependently, Lactococcus lactis was the most abundant species from its addition as part of the starter culture up to the first two months of cheese ripening. Afterward, the NSLAB Lacticaseibacillus paracasei became the main species during ripening. The milk was a possible inoculation source for the latter species, despite pasteurization. Culture-independently, the starter LAB Lactococcus cremoris and Lc. lactis were the most abundant species in the cheese core throughout the whole fermentation and ripening phases up to 100 weeks. The cheese rind from 40 until 100 weeks of ripening was characterized by a high relative abundance of the NSLAB Tetragenococcus halophilus and Loigolactobacillus rennini, which both came from the brine. These species were linked with the production of the biogenic amines cadaverine and putrescine. The most abundant volatile organic compound was acetoin, an indicator of citrate and lactose fermentation during the production day, whereas the concentrations of free amino acids were an indicator of the ripening time.

Associations between the Bacterial Composition of Farm Bulk Milk and the Microbiota in the Resulting Swedish Long-Ripened Cheese.

The maturation of a traditional Swedish long-ripened cheese has shown increasing variation in recent years and the ripening time is now generally longer than in the past. While the cheese is reliant on non-starter lactic acid bacteria for the development of its characteristic flavour, we hypothesised that the observed changes could be due to variations in the microbiota composition and number of bacteria in the raw milk used for production of the cheese. To evaluate associations between microbiota in the raw milk and the resulting cheese, three clusters of commercial farms were created to increase variation in the microbiota of dairy silo milk used for cheese production. Cheese production was performed in three periods over one year. Within each period, milk from the three farm clusters was collected separately and transported to the cheese production facility. Following pasteurisation, the milk was processed into the granular-eyed cheese and matured at a dedicated cheese-ripening facility. For each cheese batch, farm bulk and dairy silo milk samples, a starter culture, early process samples and cheese samples from different stages of maturation (7-20 months) were collected and their microbiota characterised using 16S rRNA amplicon sequencing. The microbiota in the farm bulk milk differed significantly between periods and clusters. Differences in microbiota in dairy silo milk were observed between periods, but not between farm clusters, while the cheese microbiota differed between periods and clusters. The top 13 amplicon sequence variants were dominant in early process samples and the resulting cheese, making up at least 93.3% of the relative abundance (RA). Lactococcus was the dominant genus in the early process samples and, together with Leuconostoc, also dominated in the cheese samples. Contradicting expectations, the RA of the aroma-producing genus Lactobacillus was low in cheese during ripening and there was an unexpected dominance of starter lactic acid bacteria even at the later stages of cheese ripening. To identify factors behind the recent variations in ripening time of this cheese, future studies should address the effects of process variables and the dairy environment.

The Controversial Nature of Some Non-Starter Lactic Acid Bacteria Actively Participating in Cheese Ripening.

This mini review deals with some controversial non-starter lactic acid bacteria (NSLAB) species known to be both human and animal pathogens but also health-promoting and probiotic. The focus is on Lactococcus garvieae, two Streptococcus species (S. uberis and S. parauberis), four Weissella species (W. hellenica, W. confusa, W. paramesenteroides, and W. cibaria), and Mammalicoccus sciuri, which worldwide, are often found within the microbiotas of different kinds of cheese, mainly traditional artisanal cheeses made from raw milk and/or relying on environmental bacteria for their ripening. Based on literature data, the virulence and health-promoting effects of these bacteria are examined, and some of the mechanisms of these actions are reviewed. Additionally, their possible roles in cheese ripening are also discussed. The analysis of the literature data available so far showed that, in general, the pathogenic and the beneficial strains, despite belonging to the same species, show somewhat different genetic constitutions. Yet, when the safety of a given strain is assessed, genomic analysis on its own is not enough, and a polyphasic approach including additional physiological and functional tests is needed.

Impact of NSLAB on Kazakh cheese flavor.

Xinjiang is a multiethnic region of China. Traditionally, most ethnic minorities are known to produce and consume cheese. Nomadic people have been reported to use lactic acid bacteria (LAB) for decades to produce fermented dairy products as part of a balanced diet. Non-starter LAB (NSLAB) contribute to different degrees of ripening, depending on the cheese variety. In the present study, we screened three types of NSLAB with good proteolysis and autolytic abilities from traditional Kazakh cheese: Pediococcus acidilactici R3-5, Staphylococcus epidermidis R4-2, and Lactobacillus rhamnosus R9-6. A control (no NSLAB) was also included, resulting in four distinct types of cheese samples. We used gas chromatography-mass spectrometry and the electronic nose system to identify volatile compounds and analyze the effect of NSLAB on cheese flavor at the ripening stage. The physicochemical indicators changed significantly during the ripening of Kazakh cheese. Compared with the control, the protein content, free fatty acid content, pH, flavor compounds, and odor profiles of the test cheeses were significantly different. The major chemical differences among cheeses were the synthesis of some key volatile components (ethyl caprylate, ethyl caprate, myristyl carbonate, capric acid, caprylic acid, nonanal, and benzyl alcohol). NSLAB can be used as an adjunct starter to make Kazakh cheese and the use of NSLAB affected the cheese flavor quality positively.

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.

Diversity of non-starter lactic acid bacteria in autochthonous dairy products from Western Balkan Countries - Technological and probiotic properties.

The aim of this review was to summarize the data regarding diversity of non-starter lactic acid bacteria (NSLAB) isolated from various artisanal dairy products manufactured in Western Balkan Countries. The dairy products examined were manufactured from raw cow's, sheep's or goat's milk or mixed milk, in the traditional way without the addition of commercial starter cultures. Dairy products such as white brined cheese, fresh cheese, hard cheese, yogurt, sour cream and kajmak were sampled in the households of Serbia, Croatia, Slovenia, Bosnia and Herzegovina, Montenegro, and North Macedonia. It has been established that the diversity of lactic acid bacteria (LAB) from raw milk artisanal dairy products is extensive. In the reviewed literature, 28 LAB species and a large number of strains belonging to the Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc and Weissella genera were isolated from various dairy products. Over 3000 LAB strains were obtained and characterized for their technological and probiotic properties including: acidification and coagulation of milk, production of aromatic compounds, proteolytic activity, bacteriocins production and competitive exclusion of pathogens, production of exopolysaccharides, aggregation ability and immunomodulatory effect. Results show that many of the isolated NSLAB strains had one, two or more of the properties mentioned. The data presented emphasize the importance of artisanal products as a valuable source of NSLAB with unique technological and probiotic features important both as a base for scientific research as well as for designing novel starter cultures for functional dairy food.

Addition of selected starter/non-starter lactic acid bacterial inoculums to stabilise PDO Pecorino Siciliano cheese production.

The present study was carried out to produce Protected Denomination of Origin (PDO) Pecorino Siciliano cheese with a multi-species lactic acid bacteria (LAB) culture, composed of starter and non-starter strains in order to reduce the microbiological variability of the products derived without LAB inoculums. To this end, cheese samples produced in six factories located in five provinces (Agrigento, Catania, Enna, Palermo and Trapani) of Sicily, and previously characterised for physicochemical, microbiological and sensory aspects, have been investigated in this work for bacterial microbiome, fatty acid (FA) composition as well as volatile organic compound (VOC) profiles. Analysis of the cheese microbiomes indicated that streptococci (30.62-77.18% relative abundance) and lactobacilli (on average 25.90% relative abundance) dominated the bacterial communities of control cheeses, produced without exogenous inoculums, whereas the cheeses produced with the selected multi-strain culture saw the dominance of lactococci (in the range 6.49-14.92% relative abundance), streptococci and lactobacilli. After the addition of the selected mixed culture, Shannon index increased in all cheeses, but only the cheeses produced with the selected LAB mixed culture in the factory 2 showed Gini-Simpson diversity index (0.79) closer to the reference value (0.94) for a perfect even community. FA composition, mainly represented by saturated FA (on average 69.60% and 69.39% in control cheeses and experimental cheeses, respectively), was not affected by adding LAB culture. The presence of polyunsaturated FA ranged between 7.93 and 8.03% of FA. VOC profiles were different only for the content of butanoic acid, registered for the experimental cheeses at higher concentrations (on average 662.54 mg/kg) than control cheeses (barely 11.96 mg/kg). This study validated addition of the ad hoc starter/non-starter culture for PDO Pecorino cheese production.

Cultivable non-starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti-hypertensive peptides.

The impact of salt and fat intake on human health drives the consumer's attention towards dairy food with reduced salt and fat contents. How changes in salt and fat content modulate dairy LAB population and the associated proteolytic activities have been poorly studied. Here, non-starter LAB populations from 12 Parmigiano Reggiano (PR) cheeses (12-month ripened), clustered in low salt and fat content (LL-PR) and high salt and fat content (HH-PR) groups, were investigated and identified at specie-level with molecular assays. Lactobacillus rhamnosus was dominant in HH-PR samples, whereas Lactobacillus paracasei in LL-PR samples. (GTG)5 rep-PCR analysis discriminated 11 and 12 biotypes for L. rhamnosus and L. paracasei isolates, respectively. Screening for proteolytic activity identified L. rhamnosus strains more proteolytic than L. paracasei, and, within L. rhamnosus species, HH-PR strains were generally more proteolytic than LL-PR strains. Two L. rhamnosus representatives, namely strain 0503 from LL-PR and strain 2006 from HH-PR, were functionally characterized in cow milk fermentation assay. HH-PR strain 2006 overcame LL-PR strain 0503 in acidification performance, leading to a fermented milk with higher angiotensin I-converting enzyme inhibitory and antioxidant activities. L. rhamnosus 2006 was more prone to release VPP, while L. rhamnosus 0503 released higher amount of IPP. This study provides evidences that salt/fat content affects NSLAB cultivable fraction and the associated proteolytic ability resulting in a complex occurrence of bioactive peptides featuring health-promoting properties.

Use of autochthonous mesophilic lactic acid bacteria as starter cultures for making Pecorino Crotonese cheese: Effect on compositional, microbiological and biochemical attributes.

The use of selected autochthonous mesophilic lactic acid bacteria as starter cultures was investigated according to the traditional protocol for making Pecorino Crotonose (PC). Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C (Autochthonous Starter, AS1) and Leuc. mesenteroides subsp. mesenteroides 2A, and L. casei 25D and 16A (AS2) were isolated and identified from aged ewes' milk PC cheeses, selected based on several enzymatic activities, and used as starter cultures. As shown by the in vitro kinetic of acidification, selected starter cultures had suitable capabilities to acidify. The manufacture of PC cheeses was carried out at an industrial plant scale. A control cheese (CC) was also made, using commercial starters consisting of mesophilic and thermophilic species. Ripening lasted 105 days at 10 °C. A poly-phasic approach was used to compare cheeses during manufacture and ripening, mainly based on pyrosequencing of the 16S rRNA targeting DNA, proteolysis and volatile component analyses. Compared to CC, both autochthonous starter cultures slightly affected the gross chemical composition of PC cheese. The cell density of thermophilic starters of CC progressively decreased throughout ripening. Plate count and RAPD-PCR showed that the cell number of autochthonous lactobacilli cultures of PC cheeses, made with AS1 and AS2, was almost constant throughout ripening and abundantly higher than that observed in CC. As shown by culture-independent analysis, the OTUs found during ripening varied depending on the manufacture with or without autochthonous starter cultures. The major chemical differences among cheeses were the concentration of free amino acids and the synthesis of some key volatile components (e.g., 2-methyl-1-propanol, 2-methyl-1-butanol, isobutyric, isovaleric, and isocaproic acids). Compared to CC, the use of AS1 positively affected the overall cheese quality.

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 µg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods.

New developments in the study of the microbiota of raw-milk, long-ripened cheeses by molecular methods: the case of Grana Padano and Parmigiano Reggiano.

Microorganisms are an essential component of cheeses and play important roles during both cheese manufacture and ripening. Both starter and secondary flora modify the physical and chemical properties of cheese, contributing and reacting to changes that occur during the manufacture and ripening of cheese. As the composition of microbial population changes under the influence of continuous shifts in environmental conditions and microorganisms interactions during manufacturing and ripening, the characteristics of a given cheese depend also on microflora dynamics. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. In the past, research in this area was dependent on classical microbiological techniques. However, culture-dependent methods are time-consuming and approaches that include a culturing step can lead to inaccuracies due to species present in low numbers or simply uncultivable. Therefore, they cannot be used as a unique tool to monitor community dynamics. For these reasons approaches to cheese microbiology had to change dramatically. To address this, in recent years the focus on the use of culture-independent methods based on the direct analysis of DNA (or RNA) has rapidly increased. Application of such techniques to the study of cheese microbiology represents a rapid, sound, reliable, and effective way for the detection and identification of the microorganisms present in dairy products, leading to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality. In this article, an overview on the recent advances in the use of molecular methods for thorough analysis of microbial communities in cheeses is given. Furthermore, applications of culture-independent approaches to study the microbiology of two important raw-milk, long-ripened cheeses such as Grana Padano and Parmigiano Reggiano, are presented.

Survey on the community and dynamics of lactic acid bacteria in Grana Padano cheese.

Grana Padano (GP) is a Protected Designation of Origin cheese made with raw milk and natural whey culture (NWC) that is characterised by a long ripening period. In this study, six GP productions were considered in order to evaluate the trend of microbial dynamics and compare lactic acid bacteria (LAB) population levels in cheeses during the entire cheese-making process. To reach this goal, for each GP production, samples of vat raw milk, NWC and cheeses at 48h, 2, 6, 9 and 13 months were subjected to plate counts and direct counts by fluorescence microscopy, as well as amplicon length heterogeneity-PCR (LH-PCR). Statistical analysis was applied to the results and ecological indices were estimated. It was demonstrated that the LAB able to grow in the cheese-environment conditions could arise from both raw milk and NWC. Starter lactobacilli (SLAB) from NWC were the main species present during acidification, and non-starter LAB (NSLAB), mainly from milk but also from NWC, were able to grow after brining and they dominated during ripening. The peak areas of LH-PCR profiles were used to determine ecological indices during manufacture and ripening. Among cheese ecosystems with different ageing times, diversity, Evenness and Richness were different, with highest bacterial growth and diversity occurring in cheese ripening at 2 months. At this time point, which seemed to be a crucial moment for GP microbial evolution, cell lysis of both SLAB and NSLAB was also observed. Sampling modality and statistical analysis gave greater significance to the results used to describe the microbiological characteristics of a cheese recognised worldwide.