Nadia Innocente

The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process.

A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10(4) CFU/g.

Effect of high-pressure homogenization on droplet size distribution and rheological properties of ice cream mixes.

The effect of different homogenization pressures (15/3 MPa and 97/3 MPa) on fat globule size and distribution as well as on structure-property relationships of ice cream mixes was investigated. Dynamic light scattering, steady shear, and dynamic rheological analyses were performed on mixes with different fat contents (5 and 8%) and different aging times (4 and 20 h). The homogenization of ice cream mixes determined a change from bimodal to monomodal particle size distributions and a reduction in the mean particle diameter. Mean fat globule diameters were reduced at higher pressure, but the homogenization effect on size reduction was less marked with the highest fat content. The rheological behavior of mixes was influenced by both the dispersed and the continuous phases. Higher fat contents caused greater viscosity and dynamic moduli. The lower homogenization pressure (15/3 MPa) mainly affected the dispersed phase and resulted in a more pronounced viscosity reduction in the higher fat content mixes. High-pressure homogenization (97/3 MPa) greatly enhanced the viscoelastic properties and the apparent viscosity. Rheological results indicated that unhomogenized and 15/3 MPa homogenized mixes behaved as weak gels. The 97/3 MPa treatment led to stronger gels, perhaps as the overall result of a network rearrangement or interpenetrating network formation, and the fat globules were found to behave as interactive fillers. High-pressure homogenization determined the apparent viscosity of 5% fat to be comparable to that of 8% fat unhomogenized mix.

Proteose-peptone whey fraction as emulsifier in ice-cream preparation

In the light of the results obtained from previous studies, which demonstrated the potential emulsifying and foaming capabilities of the proteose-peptone fraction of milk, the efficacy of the functional properties of these protein components when used as ingredients in ice-cream preparation was studied. In order to compare the characteristics of ice-cream obtained from mixes containing added proteose-peptones with those of ice-cream produced without emulsifiers or with the addition of mono and diglycerides of fatty acids, analyses were conducted to evaluate the viscosity, the amount of incorporated air, the resistance to melting, as well as sensory characteristics. In every case, the ice-cream containing proteose-peptones as functional ingredients, exhibited similar if not better characteristics compared to those of the ice-cream containing classical commercial emulsifiers. As regards rheological properties, the experimental ice-cream mix with added proteose-peptones had a moderately higher consistency index compared to the commercial mix with mono and diglycerides. Finally, no effect of the proteose-peptones fraction on ice-cream flavour was observed.

Emulsifying Properties of the Total Fraction and the Hydrophobic Fraction of Bovine Milk Proteose-peptones

Since proteose-peptone is a mixture of heterogeneous proteins and peptides, its functional properties have not yet been fully understood. The present study was undertaken to elucidate the emulsifying properties of proteose-peptones and a parallel evaluation was performed of the emulsifying activity of the total fraction prepared from bulk skimmed milk by precipitation with ammonium sulphate and of the most hydrophobic fraction purified by hydrophobic interaction fast protein liquid chromatography. A turbidimetric technique was used and the absorbance value at 500 nm was then directly used as the emulsifying activity index. The results obtained confirmed the good emulsifying activity of this protein fraction of the whey and demonstrated the considerable influence of the concentration on the capacity of the proteose-peptones to stabilize a model oil-in-water emulsion. Moreover, at all the concentrations examined, the purified component 3 showed a higher emulsifying activity than the total unpurified fraction.

Monitoring dry-curing of S. Daniele ham by magnetic resonance imaging

S. Daniele hams were collected at different stages during dry-curing and submitted to magnetic resonance imaging (MRI) according to the acquisition Spin-Echo sequences T1 and T2. The intensity of the MR signals in the images of the Semimembranosus, Semitendinosus, Rectus femoris and Biceps femoris muscles of the hams was computed and expressed in grey levels. Muscles were also submitted to traditional analyses, including aw, soluble solids, sodium chloride, total and water soluble nitrogen. T1 and T2 MR signals well described the evolution of the phenomena occurring in the different muscles during dry-curing. MR signal acquired in T2 mode well correlated with traditional indicators in Semitendinosus, Rectus femoris and Biceps femoris muscles. Predictive models estimating the value of aw, moisture, salt content and proteolysis extent on the basis of the MR signal intensity were proposed.

Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica.

Characterization by solid-phase microextraction-gas chromatography of the volatile profile of protected designation of origin Montasio cheese during ripening

Montasio is a typical protected designation of origin (PDO) Italian semi-hard and semi-cooked cheese produced in northeast Italy from raw or thermized cows milk. The PDO label implies that the product has distinctive characteristics that are connected to traditional production methods. The aim of this work was to precisely characterize the volatile fraction of this Italian cheese. The volatile profile can be considered a fingerprint because the flavor of a cheese variety is the result of a specific balance between the volatile compounds produced during the ripening process. Analysis of the volatile profile of Montasio cheese was performed by solid-phase microextraction-gas chromatography. Six cheesemaking trials were performed, each in a different dairy located within the Montasio cheese production area. Cheeses were analyzed at 5 stages of ripening (60, 90, 170, 300, and 365 d). Only 11 compounds were identified and measured: 5 fatty acids, 3 alcohols, 2 ketones, and 1 ester. The limited number of volatile compounds measured in the headspace of the Montasio cheese is probably due to the specific making process of this cheese, which affects evolution of the microflora and the biochemical processes of ripening. The total volatile fraction profile progressively increased from 60 to 170 d, after which time it remained almost steady. The most important contributors were found to be ethanol, short-chain fatty acids (C(2) to C(6)), diacetyl, and ethyl hexanoate. Ethanol and short-chain fatty acids increased up to 170 d, diacetyl increased up to 300 d and then declined, and ethyl hexanoate increased until the final stage.

Automatic milking systems in the Protected Designation of Origin Montasio cheese production chain: Effects on milk and cheese quality

Montasio cheese is a typical Italian semi-hard, semi-cooked cheese produced in northeastern Italy from unpasteurized (raw or thermised) cow milk. The Protected Designation of Origin label regulations for Montasio cheese require that local milk be used from twice-daily milking. The number of farms milking with automatic milking systems (AMS) has increased rapidly in the last few years in the Montasio production area. The objective of this study was to evaluate the effects of a variation in milking frequency, associated with the adoption of an automatic milking system, on milk quality and on the specific characteristics of Montasio cheese. Fourteen farms were chosen, all located in the Montasio production area, with an average herd size of 60 (Simmental, Holstein-Friesian, and Brown Swiss breeds). In 7 experimental farms, the cows were milked 3 times per day with an AMS, whereas in the other 7 control farms, cows were milked twice daily in conventional milking parlors (CMP). The study showed that the main components, the hygienic quality, and the cheese-making features of milk were not affected by the milking system adopted. In fact, the control and experimental milks did not reveal a statistically significant difference in fat, protein, and lactose contents; in the casein index; or in the HPLC profiles of casein and whey protein fractions. Milk from farms that used an AMS always showed somatic cell counts and total bacterial counts below the legal limits imposed by European Union regulations for raw milk. Finally, bulk milk clotting characteristics (clotting time, curd firmness, and time to curd firmness of 20mm) did not differ between milk from AMS and milk from CMP. Montasio cheese was made from milk collected from the 2 groups of farms milking either with AMS or with CMP. Three different cheese-making trials were performed during the year at different times. As expected, considering the results of the milk analysis, the moisture, fat, and protein contents of the experimental and control cheeses were comparable. The milking system was not seen to significantly affect the biochemical processes associated with ripening. In fact, all cheeses showed a normal proteolysis trend and a characteristic volatile compound profile during aging. Therefore, the milking system does not appear to modify the distinctive characteristics of this cheese that remain dependent on the area and methodology of production.

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines.

Inactivation of Foodborne Bacteria Biofilms by Aqueous and Gaseous Ozone

In this study, the efficacy of treatments with ozone in water and gaseous ozone against attached cells and microbial biofilms of three foodborne species, Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes, was investigated. Biofilms formed on AISI 304 stainless steel coupons from a mixture of three strains (one reference and two wild strains) of each microbial species were subjected to three types of treatment for increasing times: (i) ozonized water (0.5 ppm) by immersion in static condition, (ii) ozonized water under flow conditions, and (iii) gaseous ozone at different concentrations (0.1–20 ppm). The Excel add-in GinaFit tool allowed to estimate the survival curves of attached cells and microbial biofilms, highlighting that, regardless of the treatment, the antimicrobial effect occurred in the first minutes of treatment, while by increasing contact times probably the residual biofilm population acquired greater resistance to ozonation. Treatment with aqueous ozone under static conditions resulted in an estimated viability reduction of 1.61–2.14 Log CFU/cm2 after 20 min, while reduction values were higher (3.26–5.23 Log CFU/cm2) for biofilms treated in dynamic conditions. S. aureus was the most sensitive species to aqueous ozone under dynamic conditions. With regard to the use of gaseous ozone, at low concentrations (up to 0.2 ppm), estimated inactivations of 2.01–2.46 Log CFU/cm2 were obtained after 60 min, while at the highest concentrations a complete inactivation (<10 CFU/cm2) of the biofilms of L. monocytogenes and the reduction of 5.51 and 4.72 Log CFU/cm2 of P. fluorescens and S. aureus respectively after 60 and 20 min were achieved. Considering the results, ozone in water form might be used in daily sanitation protocols at the end of the day or during process downtime, while gaseous ozone might be used for the treatment of confined spaces for longer times (e.g., overnight) and in the absence of personnel, to allow an eco-friendly control of microbial biofilms and consequently reduce the risk of cross-contamination in the food industry.