Influence of PDO Ragusano cheese biofilm microbiota on flavour compounds formation.

The objectives of the present study were to characterize the biofilm microbiota of 11 different farms (from A to K), producing PDO Ragusano cheese, and to investigate on its ability to generate volatile organic compounds (VOCs) in milk samples inoculated with biofilm and incubated under Ragusano cheese making conditions. The biofilms were subjected to plate counting and PCR/T/DGGE analysis and the VOCs generated in incubated milk samples were evaluated through SmartNose, GC/O, and GC/MS. Streptococcus thermophilus was the dominant species both in biofilms and in incubated milks. Lactobacillus, Lactococcus, Enterococcus and Leuconostoc were also identified. Low levels of Pseudomonas spp. and yeasts counts were detected, whereas coliforms, Listeria monocytogenes and Salmonella spp., were never found. SmartNose and GC/O analyses were able to differentiate incubated milk samples on the basis of the odour compounds, highlighting that samples E and F overlapped and sample C was clearly separated from the others. These results complied with those acquired by GC/MS analysis, that detected in total 20 VOCs. Principal component analysis showed positive correlations (r > 0.6; P < 0.05) between some lactic acid bacteria (LAB) and VOCs: such as Enterococcus hirae with alcohols, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii with aldehydes, and Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus hilgardii with ketones. This work demonstrates that biofilm represents an excellent source of LAB biodiversity, which contribute to generate VOCs during the production of PDO Ragusano cheese.

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.

Molecular Traceability Approach to Assess the Geographical Origin of Commercial Extra Virgin Olive Oil.

Extra virgin olive oil (EVOO) is a precious and healthy ingredient of Mediterranean cuisine. Due to its high nutritional value, the interest of consumers in the composition of EVOO is constantly increasing, making it a product particularly exposed to fraud. Therefore, there is a need to properly valorize high-quality EVOO and protect it from fraudulent manipulations to safeguard consumer choices. In our study, we used a straightforward and easy method to assess the molecular traceability of 28 commercial EVOO samples based on the use of SSR molecular markers. A lack of correspondence between the declared origin of the samples and the actual origin of the detected varieties was observed, suggesting possible adulteration. This result was supported by the identification of private alleles based on a large collection of national and international olive varieties and the search for them in the molecular profile of the analyzed samples. We demonstrated that the proposed method is a rapid and straightforward approach for identifying the composition of an oil sample and verifying the correspondence between the origin of olives declared on the label and that of the actual detected varieties, allowing the detection of possible adulterations.

Microbial screening of animal skin bags used in traditional cheesemaking.

Bouhezza is a traditional Algerian cheese produced and ripened in goatskin bags called Djeld. The aim of this study was to characterize the microbial ecosystem from Djeld (fresh and dried Djeld for making Bouhezza cheese) and the changes introduced by Lben microflora during its preparation and to identify its role in cheesemaking and its safety. Two replicates of fresh and dried skin bags (FS and DS) were sampled and analyzed before and after contact with Lben. The microbiological results showed no pathogens. Skins observed before the addition of Lben were less populated 2.86 and 3.20 log CFU cm-2 than skins examined after the addition of Lben (approximately 6.0 log CFU cm-2), suggesting a potential role of Lben in releasing some microorganisms into the skin during its time in the Djeld. However, an increase in mesophilic lactic acid bacteria and yeasts was observed in Lben after different periods of interaction with the skin. PCR-TTGE revealed the predominance of lactic acid bacteria (Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Staphylococcus equorum subsp. linens, Lactococcus cremoris, Streptococcus thermophilus) and a few high-GC-content bacteria (Lacticaseibacillus paracasei, Brevibacterium casei). Transfer of several microbial species was observed between the goatskin bag biofilm and Lben during the overnight interaction. Bands corresponding to Lacticaseibacillus paracasei, Brevibacterium casei, and Lactobacillus delbrueckii subsp. lactis were detected in the fresh skin profile and in Lben after contact with the fresh skin. Lacticaseibacillus paracasei was found in dried skin and Lben after contact with dry skin. Lactobacillus helveticus and Enterococcus faecalis appeared in the Lben profile and persisted in Lben and the biofilm-covered dry skin after interaction. These results demonstrate an exchange of specific microbial populations between goatskin bag biofilm and Lben during the traditional preparation method, suggesting that the diversity of goatskin biofilm contributes to the microbial diversity of Lben used in the production of Bouhezza cheese.

Influence of dietary cardoon meal on volatile compounds and flavour in lamb meat.

Cardoon meal is a by-product retained after oil extraction from the seeds of cultivated Cynara cardunculus var. altilis that has been proposed as a valuable resource for animal feeding. The study aimed to assess the influence of its dietary inclusion on volatile profile and flavour of meat and kidney fat from lambs. Fifteen Sarda × Comisana male lambs were randomly divided in two groups and fed for 75 days with a concentrate-based diet containing 15% cardoon meal (CMD, n = 7) or dehydrated alfalfa (CON, n = 8). Cardoon meal inclusion reduced the "barnyard/animal" odour perceived by a trained panel in kidney fat, which could be associated with the aromatic compound p-cresol (4-methylphenol), detected only in CON diet. Considering the other aroma volatiles regarded as the main contributors for the characteristic lamb flavour, both diets were characterized by moderate to low levels of 4-methyloctanoic acid, skatole and indole while 4-methylnonanoic acid and 4-ethyloctanoic acid were not detected.

Survival of potential probiotic lactobacilli used as adjunct cultures on Pecorino Siciliano cheese ripening and passage through the gastrointestinal tract of healthy volunteers.

In the present study, two lactobacilli strains, Lactobacillus rhamnosus H25 and Lactobacillus paracasei N24, used as adjunct cultures, were evaluated for their heat resistance both with and without prior heat adaptation and for their survival, at industrial scale, during the production and ripening of the Pecorino Siciliano cheese. In addition, the viability and persistence of the lactobacilli strains after passage through the gastrointestinal tract of healthy volunteers were evaluated by using rep-PCR analysis of viable cells. Both strains exhibited good heat resistance and survival throughout cheese production and ripening, and positively influenced the physico-chemical, the microbiological and the sensorial characteristics of the final product. In addition, the molecular typing of the lactobacilli isolates, retrieved from fecal samples of healthy volunteers during and after 15 days of the experimental cheese administration, revealed a high survival of the strains, highlighting their persistence during passage into the GI tract. In conclusion, this study proposes the two adjunct cultures as potential probiotic candidate deliverable by cheese.

The Microfloras and Sensory Profiles of Selected Protected Designation of Origin Italian Cheeses.

Approximately 39 Italian cheeses carry protected designation of origin (PDO) status. These cheeses differ in their manufacturing technology and the microbial flora which comprise the finished products. The evolution of lactic microflora in cheeses with PDO status is of particular interest because the biochemical activities of these organisms participate in cheesemaking and may play an acknowledged role in the development of organoleptic characteristics during ripening. Nonstarter lactic acid bacteria (NSLAB) constitute complex microbial associations that are characterized by the occurrence of various species and many biotypes as a result of a number of selective conditions persisting during the manufacturing process and different ecological niches. The evolution of different species during ripening of Fiore Sardo showed that, when present, Lactobacillus paracasei persists and dominates the microflora of the cheese in the last period of ripening, suggesting that this species, more resistant to the constraints of the mature cheese, could be involved in proteolysis and in other enzymatic processes occurring during cheese ripening. In contrast, the stretching step typical of pasta filata cheese, such as Ragusano, induced a simplification of the raw milk profiles, allowing the persistence only of some predominant species, such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. lactis, Lactococcus lactis, and Streptococcus macedonicus, after the stretching step. Lactobacillus plantarum and L. paracasei were isolated from ripened Castelmagno PDO cheese samples with the highest frequencies. These species, generally absent in the milk, occur in dairy ecosystems and dominate the bacterial flora of many ripened semihard cheeses. In PDO long-ripened Italian cheese such as Parmigiano Reggiano, the NSLAB population is mainly formed by L. paracasei, Lactobacillus rhamnosus, and Pediococcus acidilactici. Lactobacillus helveticus, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus were also detected. Continued insight into the microbial populations of traditional Italian cheeses will allow continued production of characteristic, high-quality cheeses which have been enjoyed for many centuries.

Uncommonly thorough hydrolysis of peptides during ripening of Ragusano cheese revealed by tandem mass spectrometry.

Ragusano is a pasta filata cheese produced from raw milk in Sicily. The proteolysis was extensively analyzed after stretching (day 0), at 4 and 7 months of ripening through soluble nitrogen, urea-PAGE, and peptide identification by tandem mass spectrometry. After stretching, 123 peptides were identified: 72 arising from ß-casein, 34 from α(s1)-casein, and 17 from α(s2)-casein. The main protein splitting corresponded to the action of plasmin, chymosin, cathepsin D, cell envelope proteinase, and peptidase activities of lactic acid bacteria. Unlike other types of cheeses, <10% residual ß- and α(s)-caseins remained intact at 7 months, indicating original network organization based on large casein fragments. The number of identified soluble peptides also dramatically decreased after 4 and 7 months of ripening, to 47 and 25, respectively. Among them, bioactive peptides were found, that is, mineral carrier, antihypertensive, and immunomodulating peptides and phosphopeptides.

Tina wooden vat biofilm: a safe and highly efficient lactic acid bacteria delivering system in PDO Ragusano cheese making.

In the Sicilian PDO Ragusano cheese making, raw milk is placed in a wooden vat called a Tina. As no starter is added, lactic acid is produced by milk flora and flora released from the Tina biofilm. The aim of this work was to assess the safety and efficiency of this natural inoculation system. From 15 Tinas' biofilms, bacteria total counts varied from 10(3) to 10(6) CFU/cm(2), with the predominance of thermophilic lactic acid bacteria. Low counts of yeasts and moulds were found in a few Tinas. Salmonella, Listeria monocytogenes, Escherichia coli O157:H7 were totally absent, as assessed by conventional plating and the Bax detection system after enrichment, highlighting the safety of the system. From four Tinas out of the 15, micropieces of wood were observed by confocal and scanning electron microscopy. The biofilm entrapped in a matrix covered almost entirely the surface of the wood. Polysaccharides were detected in the four Tinas. In three of the latter, cocci were predominant in the ecosystem whereas in the other one, cocci, bacilli, yeasts and moulds were observed. Fifty litres of microfiltrated milk (<10 CFU/mL) were poured in the four Tinas for 10 min of contact. Enumeration of lactic acid bacteria, yeasts and enterococci were performed in the milk after contact. Depending on the Tina, from 5.10(4) to 10(6) CFU/mL of Streptococcus thermophilus were released into the milk, and from 10(4) to 10(5) CFU/mL of thermophilic lactobacilli. Spontaneous acidification after contact confirmed the high efficiency of biofilm lactic acid bacteria delivery.