Interrelationships among physical and chemical traits of cheese: Explanatory latent factors and clustering of 37 categories of cheeses.

Cheese presents extensive variability in physical, chemical, and sensory characteristics according to the variety of processing methods and conditions used to create it. Relationships between the many characteristics of cheeses are known for single cheese types or by comparing a few of them, but not for a large number of cheese types. This case study used the properties recorded on 1,050 different cheeses from 107 producers grouped into 37 categories to analyze and quantify the interrelationships among the chemical and physical properties of many cheese types. The 15 cheese traits considered were ripening length, weight, firmness, adhesiveness, 6 different chemical characteristics, and 5 different color traits. As the 105 correlations between the 15 cheese traits were highly variable, a multivariate analysis was carried out. Four latent explanatory factors were extracted, representing 86% of the covariance matrix: the first factor (38% of covariance) was named Solids because it is mainly linked positively to fat, protein, water-soluble nitrogen, ash, firmness, adhesiveness, and ripening length, and negatively to moisture and lightness; the second factor (24%) was named Hue because it is linked positively to redness/blueness, yellowness/greenness, and chroma, and negatively to hue; the third factor (17%) was named Size because it is linked positively to weight, ripening length, firmness, and protein; and the fourth factor (7%) was named Basicity because it is linked positively to pH. The 37 cheese categories were grouped into 8 clusters and described using the latent factors: the Grana Padano cluster (characterized mainly by high Size scores); hard mountain cheeses (mainly high Solids scores); very soft cheeses (low Solids scores); blue cheeses (high Basicity scores), yellowish cheeses (high Hue scores), and 3 other clusters (soft cheeses, pasta filata and treated rind, and firm mountain cheeses) according to specific combinations of intermediate latent factors and cheese traits. In this case study, the high variability and interdependence of 15 major cheese traits can be substantially explained by only 4 latent factors, allowing us to identify and characterize 8 cheese type clusters.

Major Causes of Variation of External Appearance, Chemical Composition, Texture, and Color Traits of 37 Categories of Cheeses.

Cheeses are produced by many different procedures, giving rise to many types differing in ripening time, size, shape, chemical composition, color, texture, and sensory properties. As the first step in a large project, our aim was to characterize and quantify the major sources of variation in cheese characteristics by sampling 1050 different cheeses manufactured by over 100 producers and grouped into 37 categories (16 with protected designation of origin, 4 traditional cheese categories, 3 pasta filata cheese categories, 5 flavored cheese categories, 2 goat milk categories, and 7 other categories ranging from very fresh to very hard cheeses). We obtained 17 traits from each cheese (shape, height, diameter, weight, moisture, fat, protein, water soluble nitrogen, ash, pH, 5 color traits, firmness, and adhesiveness). The main groups of cheese categories were characterized and are discussed in terms of the effects of the prevalent area of origin/feeding system, species of lactating females, main cheese-making technologies, and additives used. The results will allow us to proceed with the further steps, which will address the interrelationships among the different traits characterizing cheeses, detailed analyses of the nutrients affecting human health and sensorial fingerprinting.

Assessment of the microbiological origin of blowing defects in Grana Padano Protected Designation of Origin cheese.

Recognized worldwide for its history, flavor, and high nutritional quality, Grana Padano (GP) is one of the most traditional Italian raw-milk, hard-cooked, long-ripened cheese. Throughout GP manufacturing, some well-known and undesired bacterial species, such as clostridia, can proliferate and lead to spoilage defects that mischaracterize the final product; however, little is known about the development of late-blowing defects in hard cheese samples without clostridia. Therefore, in this study we aimed to use metataxonomic analysis to identify bacterial taxa associated with the development of late-blowing defect in GP samples. Furthermore, the presence of several heterofermentative lactobacilli species in defective zones were verified by primer-specific PCR assay. Considering α- and ß-diversity analyses, no statistically significant differences were detected between cheese samples with and without blowing defect. Following taxonomic assignment, Lactobacillus and Streptococcus were the dominant genera, whereas clostridia-related taxa were not detected in any of the 20 analyzed samples. Using EdgeR, the genera Propionibacterium and Acinetobacter were found to be prevalently more abundant in samples categorized as having "big regular holes." In samples with "small regular holes," multiplex PCR amplification revealed differences in terms of Lactobacillus population composition, mainly obligate homofermentative lactobacilli, between defective and non-defective zones of the same cheese wheel. This study demonstrated that GP samples with blowing defects not caused by clostridial development share similar biodiversity indices with GP collected from control zones, but an imbalance of obligate homofermentative lactobacilli was noticed between samples, which requires further analysis to better comprehend the exact mechanism involved in this process.

Thermal resistance and high-performance microwave decontamination assessment of Bacillus endospores isolated from food-grade herbal extracts.

Generally, endospore contamination can occur from different sources during product manufacturing in many industries and therefore lower its quality by affecting physicochemical properties and shelf-life. Bacterial endospores can germinate inside the product and produce several enzymes, which can cause several undesirable changes. This study assessed the spores thermal resistance and applied a microwave decontamination technique toward herbal extracts (Tilia tomentosa and Centella asiatica) containing ethanol or glycerol. Based on 16S rRNA analysis, the detected contaminant endospores belonged to different Bacillus species, namely B. subtilis, B. zhangzhouensis, and B. pumilus. The thermal resistance assessment using inoculated endospores in the actual products revealed B. pumilus T2 as the most resistant endospore to the heat treatments tested in both T. tomentosa and C. asiatica extracts. Finally, a high-performance microwave technique was used to decontaminate T. tomentosa extract against the mixture of Bacillus spores. Results from the microwave technique indicate that the increase of temperature from 100°C to 105°C not only decontaminated the product but also could dramatically decrease the effective thermal treatment time (10 times), which can benefit the product quality. The results provided in this study considerably contribute to improving an original decontamination method for products containing glycerol and ethanol with the most negligible effect on product quality.

The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination.

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota.

Analysis of Process Factors of Dry Fermented Salami to Control Listeria Monocytogenes.

Challenge tests are a clear opportunity for manufacturers interested in the evaluation of their management system with the aim to reduce the spread of foodborne pathogens. This is a main concern especially in ready-to-eat food in relation to the risk associated with Listeria monocytogenes. For small and medium-scale food industry the manufacturing practices and products formulation are characterised by a wider variability and poor repeatability. The use of ad hoc challenge test and the comparison among different processing systems are strongly required. This paper reports a preliminary comparison among different challenge tests (n=12) commissioned by three manufacturers of raw-fermented salami during a period of three years (2013-2016). The challenge tests were designed to evaluate the growth potential (δ) of L. monocytogenes during the whole processing period of the salami. The doughs were prepared according to different formulations: the simplest formulation was represented by the use of salt, potassium nitrate, black pepper and starter cultures, while the most composited formulations also included the use of sugars and ascorbic acid in addition to nitrite salt. All the processing steps were conducted within an experimental laboratory dedicated for the processing of meat. After stuffing, the salami were dried and ripened under temperature and relative humidity control. The sugar inclusion can be considered as a protective factor, while the drying step at high temperature (above 20°C) was associated with higher δ values (δ>0.5 log10 cfu/g). The addition of starter cultures, and the subsequent acidification highlighted the importance of pH as the parameter able to affect the L. monocytogenes growth.

Genome comparison and physiological characterization of eight Streptococcus thermophilus strains isolated from Italian dairy products.

Eight Streptococcus thermophilus strains of dairy origin isolated in Italy were chosen to investigate autochthonous bacterial diversity in this important technological species. In the present study a comparative analysis of all the 17 S. thermophilus genomes publicly available was performed to identify the core and the variable genes, which vary among strains from 196 to 265. Additionally, correlation between the isolation site and the genetic distance was investigated at genomic level. Results highlight that the phylogenetic reconstruction differs from the geographical strain distribution. Moreover, strain M17PTZA496 has a genome of 2.15 Mbp, notably larger than that of the others, determined by lateral gene transfer (including phage-mediated incorporation) and duplication events. Important technological characters, such as growth kinetics, bacteriocin production, acidification kinetics and surface adhesion capability were studied in all the Italian strains. Results indicate a wide range of variability in adhesion properties that significantly clustered strains into four groups. Genomic differences among strains in relation to these characters were identified but a clear correlation between genotype and phenotype was not always found since most of the genomic modifications arise from single nucleotide polymorphisms. This research represents a step forward in the identification of strains-specific functions in Streptococcus thermophilus and it has also the potential to provide valuable information to predict strain specific behaviors in industrial processes.

Isolation and characterisation of lactic acid bacteria from donkey milk.

During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

Outlining a selection procedure for Saccharomyces cerevisiae isolated from grape marc to improve fermentation process and distillate quality.

Nowadays grape marc represents one of the main by-product of winemaking. Many South Europe countries valorize this ligno-cellulosic waste through fermentation and distillation for industrial alcoholic beverage production. The storage of marcs is a crucial phase in the distillation process, due to the physicochemical transformations ascribed to microbial activity. Among the methods adopted by distillers to improve the quality of spirits, the use of selected yeasts has not been explored so far, therefore in this work we evaluated the selection criteria of Saccharomyces cerevisiae strains for grape marc fermentation. The proposed selection procedure included three steps: characterization of phenotypical traits, evaluation of selected strains on pasteurised grape marc at lab-scale (100 g) and pilot-scale fermentation (350 kg). This selection process was applied on 104 strains isolated from grape marcs of different origins and technological treatment. Among physiological traits, ß-glucosidase activity level as quality trait seems to be only partially involved in increasing varietal flavour. More effective in describing yeast impact on distillate quality is the ratio higher alcohols/esters that indicates strain ability to increase positive flavours. Finally, evaluating grape marc as source of selected yeasts, industrial treatment rather than varietal origin seems to shape strain technological and quality traits.

Exploring the use of Saccharomyces cerevisiae commercial strain and Saccharomycodes ludwigii natural isolate for grape marc fermentation to improve sensory properties of spirits.

In Mediterranean countries the most diffuse practice to obtain the valorization of grape marc, the main by-product from winemaking, is the production of spirits. During this process, marc storage for sugar fermentation represents a crucial step, since side-fermentations leading to off-flavours production can very easily occur. In this study we evaluated the effect of the addition of two yeast strains, inoculated separately at the beginning of the storage period, into marcs from two Italian grape varieties with the aim to control the development of autochthonous microbiota and to improve spirit quality. The presence of the inoculated strains was monitored by means of PCR-based approaches. A commercial Saccharomyces cerevisiae strain, chosen as this species is notably the best ethanol producer, showed excellent ability to dominate the autochthonous microflora and to reduce off-flavours as demonstrated by chemical analysis and sensory evaluation. A Saccharomycodes ludwigii strain, chosen for increasing varietal compounds thus enhancing spirit aroma, showed a level of implantation not sufficient to assure a clear beneficial effect on quality. The implantation level of this strain was affected by S. cerevisiae competition since the highest level was found in grape marc with lower sugar content, where indigenous S. cerevisiae were less persistent.

Biodiversity and characterization of indigenous coagulase-negative staphylococci isolated from raw milk and cheese of North Italy.

The aim of the current study was to detect coagulase-negative staphylococci (CNS) in raw milk and cheeses produced in North Italy, and to analyze isolates for their biodiversity, safety aspects and technological properties. Molecular identification methods revealed a high biodiversity among isolates and assigned them to 17 species. The most recovered species were Staphylococcus equorum (12%), Staphylococcus lentus (12%), Staphylococcus simulans (12%), Staphylococcus sciuri (10%), and Staphylococcus xylosus (9%). The presence of ten transferable antibiotic resistance (AR) genes was verified by PCR and 19% of isolates were positive, with tet(K) being the most frequent gene (10%); interestingly, no strain carried multiple AR genes. Twenty-four isolates displayed hemolytic activity; tyrosine decarboxylase gene (tdcA) was found in two isolates, while histidine decarboxylase gene (hdcA) and enterotoxin genes (se) were not detected. Isolates were further characterized for the presence of some relevant technological properties; 16% of isolates displayed proteolytic activity and 39% lipolytic activity, while no one of the isolates was found to exhibit antimicrobial activity against Staphylococcus aureus. This study provided evidence of a low occurrence of safety hazards in CNS isolated from dairy products.

Identification of microbiota present on the surface of Taleggio cheese using PCR-DGGE and RAPD-PCR.

UNLABELLED: Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. PRACTICAL APPLICATION: Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.

A longitudinal study on thermophilic Campylobacter spp. in commercial turkey flocks in northern Italy: occurrence and genetic diversity.

Poultry are recognized as a main reservoir of thermophilic campylobacters, but few studies have been carried out on commercial meat turkeys. This study was aimed at assessing the occurrence of thermophilic Campylobacter spp., their genetic diversity, and the trend of the infection during the whole production cycle of three turkey flocks from different farms in Northern Italy. Flocks were monitored from the time of housing 1-day-old poults to slaughter time by collecting samples (meconium and cloacal swabs) at weekly intervals up to the recovery of Campylobacter spp. and then twice a month. A conventional culture method and a multiplex PCR assay were used for Campylobacter detection and identification. A subset of isolates was genetically characterized by random amplified polymorphic DNA-PCR (RAPD-PCR) and flagellin gene A short variable region (flaA-SVR) sequencing. Although at different times, all flocks became colonized by Campylobacter jejuni or Campylobacter coli (or both) that persisted throughout the entire production cycle. Overall, nine RAPD types and 14 flaA-SVR types were detected with differences in their distribution among flocks and sampling times. Moreover, changes in the Campylobacter genotypes colonizing turkeys were observed over time within each flock. These findings suggest that Italian commercial turkeys might be widely colonized by different genotypes of C. jejuni and C. coli and also suggest that differences in the distribution and epidemiologic dynamics of these microorganisms might occur among flocks.

Yeast population dynamics during pilot-scale storage of grape marcs for the production of Grappa, a traditional Italian alcoholic beverage.

The composition and population dynamics of the yeast microflora of grape marcs were investigated during a pilot scale fermentation study using two white grape varieties, namely Moscato and Prosecco, from two distinct areas of the Veneto Region. Yeast counts were made at the beginning, after 4 and after 15 days of marc storage under anaerobic conditions. Seventy isolates from each sampling time were identified to species by RAPD-PCR analysis and subsequent ITS region sequencing. A good biodiversity of yeasts occurred in both marcs at the beginning of fermentation, with high presence of Hanseniaspora opuntiae, but without detectable presence of Saccharomyces strains, which instead became the dominant yeast after just 4 days of fermentation, remaining at that level until the end of fermentation. Colonization of Moscato marc by S. cerevisiae resulted better, in relation to its higher sugar content. Characterization of S. cerevisiae isolates by mitochondrial DNA restriction analysis revealed the presence of 66 different strains in the marc from the Moscato grapes, without the occurrence of a clearly dominant strain, while in the marc from the Prosecco grapes only 23 different profiles were scored, with a dominant strain that accounted for 62.7% of the Saccharomyces population after 4 days of fermentation.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Strains from Italian Dairy Products.

Staphylococcus aureus is a known major cause of foodborne illnesses, and milk and dairy products are often contaminated by enterotoxigenic strains of this bacterium. In the present study, 122 S. aureus isolates collected from different dairy products were characterised by phenotypic properties, by the distribution of genes encoding staphylococcal enterotoxins (sea, sec, sed, seg, seh, sei, sej, and sel) and by randomly amplified polymorphic DNA PCR (RAPD-PCR). Moreover, strain resistance to vancomycin and methicillin (oxacillin) was studied. The differences in the RAPD-PCR profiles obtained with the primers M13 and AP4 revealed the presence of a great genetic heterogeneity among the different S. aureus strains. Using the primer AP4 and M13, eight groups were distinguished by RAPD-PCR cluster analysis, although, except in few cases, it was not possible to correlate the isolates of different animal species (cow or ovine) with the presence of se genes. None of the isolates showed resistance to vancomycin or methicillin.

Occurrence of Streptococcus macedonicus in Italian cheeses.

A new approach for the detection and enumeration of Streptococcus macedonicus in cheese was developed. The method which is based on a first screening of cheeses by a PCR assay specific for S. macedonicus followed by plating positive samples on a differential medium (SM medium) was applied to 51 samples derived from PDO and traditional Italian cheeses. Streptococcus macedonicus was found in 16 of the 51 samples examined in the present work. With the exclusion of an Asiago cheese sample in which very high numbers of S. macedonicus (7.13 log CFU g(-1)) were found, the counts of S. macedonicus in SM medium ranged from 2.48 to 4.70 log CFU g(-1). In the same cheeses, total streptococci enumerated onto M17 agar were found at higher concentrations with values up to 7.88 log CFU g(-1). The system developed was particularly useful for the differential count of S. macedonicus in cheese and allowed to evaluate the occurrence of this species within the complex microbial lactic acid bacteria (LAB) population, which is typical of traditional cheeses. Results showed that in the examined cheeses S. macedonicus cannot be considered as a dominant LAB species.

A polyphasic approach to highlight genotypic and phenotypic diversities of Lactobacillus helveticus strains isolated from dairy starter cultures and cheeses.

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided stran-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities: the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309.

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity.

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.