Ragusano Cheese Research Articles

Feeding Cows with Olive Cake Enriched in Polyphenols Improves the Sustainability and Enhances the Nutritional and Organoleptic Features of Fresh Caciocavallo Cheese.

In this study, Caciocavallo, a typical cheese produced in Sicily Island (Italy), was obtained from the milk of dairy cows fed with and without enriched olive cake (ECO and CTR, respectively) in order to evaluate nutritional, microbiological, volatile, and sensory differences in cheeses. ECO cheese showed greater (p < 0.05) MUFA and PUFA and polyphenols content and lower SFA content than CTR cheese. Microbiological analyses revealed the absence of Salmonella, Listeria monocytogenes, Escherichia coli, and E. coli O157, and no significant differences in the viable counts of the remaining microbial groups analyzed, between samples. Thermophilic lactococci were more prevalent in ECO cheese. The implementation of a culture-independent method, such as PCR-DGGE analyses, revealed the presence of a more diverse microbial population in both cheeses. Regarding the volatile compounds profile, long-chain free fatty acids were more abundant in the ECO cheese, resulting in a healthier free fatty acid profile. This study also showed that, especially for their appearance and taste, consumers mostly appreciated the ECO cheese. The results show that using enriched olive cake could enhance the sustainability and the quality of Ragusano cheese, improving not only the health of its consumers but also positively influencing tastes and acceptability.

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.

Variability of volatile profiles in milk from the PDO Ragusano cheese production zone

Native pasture has been shown to have a strong influence on the sensory characteristics of dairy products. However, few studies have been carried out on milk flavor profiles. The aim of the present study was to evaluate the influence of cows’ pasture-based feeding on milk volatile profile. Four Sicilian dairy farms, with three different breeds and located in the Ragusano PDO cheese production area, were selected and bulk milk samples were collected during three seasons (two pasture seasons and one non-pasture season). Pasture was available in spring and autumn time but not in summer because of the climatic conditions. A total of 72 milk samples were collected. All 72 samples were analyzed by SMart Nose®; and taking into account SMart Nose results, only 48 of spring and summer milk samples were selected for gas chromatography/olfactometry (GC/O) coupled with gas chromatography/mass spectrometry (GC/MS) systems. By applying a principal component analysis on standardized means value, results by SMart Nose showed that if native pasture was present in cows’ diet, milk samples were similar in volatile fingerprint (spring and autumn time) but it strongly differed when cows’ diet lacked native pasture (summer time). However, spring milk samples showed a higher variability than milk samples collected in autumn. Using GC/O, 179 odor-active compounds were detected in milk extracts; 87% of which were chemically identified by GC/MS. Milk samples collected in spring presented 26% more compounds than non-pasture milk samples collected in summer, thus implying a pasture effect on milk aroma profile.

Effect of brine composition and brining temperature on cheese physical properties in Ragusano cheese

Composition and physical properties of cheeses are influenced by temperature, salt, and calcium concentration of brine. This work aimed to examine conditions of brine under which the cheese matrix contracts or expands in absence of restrictions imposed by surface rind development during overnight block formation. Three experimental 4-kg blocks of Ragusano cheese were produced at 3 different stretching temperatures (70, 80, and 90°C) and cut into pieces weighing approximately 40 to 50g. One piece from each was chemically analyzed at time 0. All other pieces were measured for weight and volume and placed in plastic bags containing 300mL of different brine solutions (2% NaCl with 0.1% Ca; 10% NaCl with 0, 0.1, 0.2, or 0.4% Ca; 18% NaCl with 0.1% Ca; and 26% NaCl with 0.1% Ca) at 3 different temperatures (4, 12, and 20°C). After 24h of brining, the cheeses were analyzed for weight, volume, chemical, and microstructural changes. Salt concentration in brine significantly influenced composition, weight, and volume of the cheeses after brining. Salt concentration was inversely related to cheese volume and weight. Changes in weight caused by altering the brining temperature were sufficient to reach statistical significance, and statistically significant volume changes were induced by brining temperature and its interaction with salt content. The highest volume increase (30%) occurred in the cheese stored in the 2% NaCl brine at the coldest temperature, whereas the greatest volume decrease was recorded in cheeses brined in the 26% NaCl brine. Composition was not affected by brining temperature. Calcium concentration did influence weight, volume, and composition, except on a fat-on-dry-basis. When cheeses were brined without added calcium, cheese volume and weight increased at all temperatures. At high calcium levels (0.4%), syneresis occurred and volume decreased, especially at 20°C (−16.5%). Microstructural investigation with porosity measurement confirmed weight and volume changes.

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 β-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 β- 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.

Segmentation of structural features in cheese micrographs using pixel statistics

Description of microscopic features is often accomplished by qualitative inspection of micrographs. However, assessing the differences responsible for the variation of rheological properties requires taking measurements of microscopic features. A statistically-founded algorithm is proposed for automatic identification of cheese micro-features in Scanning electron microscopy (SEM) imagery for quantitative analysis. Twenty SEM images of Ragusano cheese were recorded and manually labelled using a different colour coding for each of three features: whey pockets, fat globules, and protein matrix. The marked micrographs were used to train a statistical model. In turn, that model was used to mark the looked-for features on new images of the same type. Namely, pixel probabilities of belonging to the three classes above mentioned were computed, using pixel neighbourhoods at different scales. Each pixel was set within the most probable class. The proposed method could recognize the microstructure of Ragusano cheese in SEM images with higher accuracy (on average, 78.0% correct pixels) than other popular algorithms in the literature (e.g., Maximum Response 8 73.7% correct pixels). Being completely automatic in the operative phase, this algorithm allows to batch-process lots of images with no human intervention. It can also be used for any other application, since it can handle any number of phases.

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.

Variability of Bacterial Biofilms of the “Tina” Wood Vats Used in the Ragusano Cheese-Making Process

Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.

Interaction of Brine Concentration, Brine Temperature, and Presalting on Salt Penetration in Ragusano Cheese

Thirty-one 3.6-kg blocks of Ragusano cheese were made on each of 6 different days (in different weeks) starting with a different batch of milk on each day. On d 1, 3, and 5, the cheeses were not presalted and on d 2, 4, and 6, all cheeses were presalted (PS). One of the 31 blocks of cheese was selected at random for analysis before brine salting (i.e., on d 0). The remaining 30 blocks were randomly divided into 2 groups of 15 blocks each; one group was placed in 18% brine (18%B) and the other group was placed in saturated brine (SB). For the 15 blocks within each of the 2 brine concentrations (BC), 5 blocks were placed in a brine tank at 12° C, 5 at 15° C, and 5 at 18° C, and submerged for 24 d. The research objective was to determine the combined impacts (i.e., interactions) of PS the curd before stretching, BC (SB vs. 18%B), and brine temperature (BT; 12, 15, and 18° C) on salt uptake, moisture content, and yield of Ragusano cheese. Although BC, BT, and PS each had their own separate impacts on salt uptake, there was little interaction of these effects on salt uptake when they were used in combination. The PS most quickly delivered salt to the interior of the cheese and was the most effective approach to salting for controlling early gas formation. There were strong separate impacts of BC, BT, and PS on cheese moisture content, moisture loss, and net weight loss, with BC having the largest separate impact on these parameters. Reducing BT reduced salt content and increased moisture, but the effects were small. The more important effect of reduced BT was to reduce growth of gas forming bacteria. The 18%B produced higher moisture, and less moisture and weight loss than SB. The effect of interactions of BC, BT, and PS on moisture loss and net weight loss were small. To achieve the maximum benefit from the various approaches to salting for controlling early gas formation in Ragusano cheese, PS combined with slightly lower BT (i.e., 15° C instead of 18° C) should be used. Although using 18%B instead of SB did increase salt uptake, the point at which improved salt uptake occurred due to use of 18%B did not provide benefit in prevention of early gas formation, as reported separately. However, use of 18%B instead of SB provided a 9.98% increase in cheese yield due to reduced moisture loss during brining; this would be very attractive to cheese makers. The increase in yield needs to be balanced against the risk of growth of undesirable bacteria in the 18%B and the creation of another cheese quality defect.

Composition, Microstructure, and Surface Barrier Layer Development During Brine Salting

The goal of this study was to characterize the changes in chemical composition, porosity, and structure that occur at the surface of a block of brine-salted cheese and their relationship to the rate at which salt is taken up from the brine. To create a difference in composition, salt uptake, and barrier layer properties, identical blocks of Ragusano cheese were placed in saturated and 18% salt brine at 18°C for 12 d. The overall moisture content and porosity decreased, whereas salt and salt in moisture content increased near the surface of blocks of brine-salted Ragusano cheese for all treatments. The general appearance of the microstructure of the surface of the blocks of brine-salted cheese was much more compact than the microstructure 1mm inside the block at both brine concentrations. Large differences in porosity of the barrier layer were produced by brine-salting cheese in 18% vs. saturated brine, with cheese in saturated brine having much lower porosity at the surface and taking up much less salt during brining. The macro network of water channels within the microstructure of the cheese was less open near the surface of the block for cheese in both saturated and 18% brine after 4 d. However, no large differences in the size of the macro channels in the cheese structure due to the difference in brine concentration were observed by scanning electron microscopy. It is possible that the shrinkage of the much smaller pore structure within the casein matrix of the cheese is more important and will become more limiting to the rate of salt diffusion. Further microstructure work at higher resolution is needed to answer this question. The calculated decrease in porosity at the exterior 1-mm portion of the block was 50.8 and 29.2% for cheeses that had been in saturated vs. 18% brine for 12 d, respectively. The difference in brine concentration had a very large impact on the salt in moisture content of the cheese. The exterior of the cheese in 18% brine reached a salt in moisture content almost identical to that of the brine very quickly (17.3% at 4 d), whereas the salt in moisture content at the surface of the cheese block in saturated brine was only 11.9% at 4 d. There appears to be some critical concentration of salt in brine above which there is a large negative impact on salt uptake due to the creation of a barrier layer at the surface of the block of cheese.

Evaluation of Bitterness in Ragusano Cheese

The appearance of undesirable bitter taste in Ragusano cheese was investigated by comparing the composition of 9 bitter cheeses with that of 9 reference cheeses of good quality by means of chemical, electrophoretic, and chromatographic analyses. Rates of proteolysis were significantly affected in cheeses of different quality. Primary proteolysis, as measured by pH 4.6-soluble N, was significantly greater in bitter cheeses compared with reference samples. Urea-PAGE profiles showed an almost complete breakdown of caseins in bitter cheeses and the further degradation of primary peptides into smaller compounds not detectable by this technique. Cheeses with defects had significantly lower levels of secondary proteolysis as reflected by the percentage of pH 4.6-soluble N soluble in 12% trichloroacetic acid and the amounts of total free amino acids. Peptides separated by reversed phase-HPLC revealed that the large and significant differences in peptide profiles of the soluble fractions between bitter and reference cheeses were mainly due to a much higher proportion of hydrophobic peptides in the former. The occurrence of bitterness in Ragusano cheese was therefore attributable to unbalanced levels of proteolysis and peptidolysis. Extensive degradation of caseins and primary peptides by activities of proteases produced large amounts of small- and medium-sized hydrophobic peptides that were not adequately removed by peptidases of microflora and therefore accumulated in cheese potentially contributing to its bitter taste. The presence of these compounds in bitter cheeses was related to high salt-in-moisture and low moisture contents that limited the enzymatic activities of microflora important in secondary proteolysis. Combining salt-in-moisture and the ratio of hydrophobic-to-hydrophilic soluble peptides resulted in the best logistic partial least squares regression model predicting cheese quality. Although bitterness is known to be rarely encountered in cheese at salt-in-moisture levels >5.0, all of the bitter cheeses analyzed in this study had salt-in-moisture levels much greater than this value. According to the logistic model, a risk of bitterness development may exist for cheeses with a midrange (5 to 10%) salt-in-moisture content but with an inadequate level of secondary proteolysis.

Influence of Brine Concentration, Brine Temperature, and Presalting on Early Gas Defects in Raw Milk Pasta Filata Cheese

Thirty-one 3.8-kg blocks of Ragusano cheese were made on each of 6 d starting with a different batch of raw milk on each day. On d 1, 3, and 5, cheeses were not presalted and on d 2, 4, and 6, all cheeses were presalted. Before brine salting, one of the 31 blocks of cheese was selected at random for analysis (i.e., at d 0). The remaining 30 blocks were randomly divided into 2 batches of 15 blocks each, one group was placed in 18% brine, and the other group was placed in saturated brine. For the 15 blocks within each of the 2 brine concentrations, 5 blocks each were placed in brine tanks at 12, 15, and 18°C. Cheese blocks were sampled immediately before brine salting (d 0) and after 1, 4, 8, 16, and 24 d of brine salting. Presalting the curd with 2% added salt before stretching reduced the coliform count in the cheese by 1.41 log and resulted in a major reduction in early gas formation. Across all treatments in the present study, the average reduction in gas formation due to presalting was 75%. Reducing brine temperature had the second largest impact on reducing gas production, but did not reduce the coliform count in the cheese. Reducing brine temperature from 18 to 12°C made a larger reduction in early gas formation in cheeses that were not presalted (from 6.8 to 1.8% gas holes, respectively) than in cheeses that were presalted (from 1.9 to 0.5% gas holes, respectively). To achieve the same absolute level of gas production in the nonpre-salted cheese as was achieved in presalted cheese in combination with 18°C brine, the brine temperature for the nonpresalted cheese had to be lowered from 18 to 12°C. Reducing brine concentration, although effective at increasing the rate of salt penetration into the block, did not have any impact on coliform count and had minimal impact on reducing gas production. The condition where reducing brine concentration was able to make a reduction in gas production was for cheeses that were not presalted and brined at 18°C. Presalting is a very simple and practical approach to reducing the problem of early gas formation in combination with strategies to improve milk quality and cheese making conditions. Further work is needed to understand the impact of different levels of presalting on death of coliforms and gas production in the cheese.

Chemometric Analysis of Proteolysis During Ripening of Ragusano Cheese

Chemometric modeling of peptide and free amino acid data was used to study proteolysis in Protected Denomination of Origin Ragusano cheese. Twelve cheeses ripened 3 to 7 mo were selected from local farmers and were analyzed in 4 layers: rind, external, middle, and internal. Proteolysis was significantly affected by cheese layer and age. Significant increases in nitrogen soluble in pH 4.6 acetate buffer and 12% trichloroacetic acid were found from rind to core and throughout ripening. Patterns of proteolysis by urea-PAGE showed that rind-to-core and age-related gradients of moisture and salt contents influenced coagulant and plasmin activities, as reflected in varying rates of hydrolysis of the caseins. Analysis of significant intercorrelations among chemical parameters revealed that moisture, more than salt content, had the largest single influence on rates of proteolysis. Lower levels of 70% ethanol-insoluble peptides coupled to higher levels of 70% ethanol-soluble peptides were found by reversed phase-HPLC in the innermost cheese layers and as the cheeses aged. Non-significant increases of individual free amino acids were found with cheese age and layer. Total free amino acids ranged from 14.3 mg/g (6.2% of total protein) at 3 mo to 22.0 mg/g (8.4% of total protein) after 7 mo. Glutamic acid had the largest concentration in all samples at each time and, jointly with lysine and leucine, accounted for 48% of total free amino acids. Principal components analysis and hierarchical cluster analysis of the data from reversed phase-HPLC chromatograms and free amino acids analysis showed that the peptide profiles were more useful in differentiating Ragusano cheese by age and farm origin than the amino acid data. Combining free amino acid and peptide data resulted in the best partial least squares regression model (R2 = 0.976; Q2 = 0.952) predicting cheese age, even though the peptide data alone led to a similarly precise prediction (R2 = 0.961; Q2 = 0.923). The most important predictors of age were soluble and insoluble peptides with medium hydrophobicity. The combined peptide data set also resulted in a 100% correct classification by partial least squares discriminant analysis of cheeses according to age and farm origin. Hydrophobic peptides were again discriminatory for distinguishing among sample classes in both cases.

Lipolysis and Proteolysis in Ragusano Cheese During Brine Salting at Different Temperatures

The influence of temperature (12, 15, 18, 21, and 24°C) of saturated brine on lipolysis and proteolysis in 3.8-kg blocks of Ragusano cheese during 24 d of brining was determined. Twenty-six 3.8-kg blocks were made on each day. The cheese making was replicated on 3 different days. All blocks were labeled and weighed prior to brining. One block was sampled and analyzed prior to brine salting. Five blocks were placed into each of 5 different brine tanks at different temperatures. One block was removed from each brine tank after 1, 4, 8, 16, and 24 d of brining, weighed, sampled, and analyzed. Both proteolysis and lipolysis in Ragusano cheese increased with increasing brine temperature (from 12 to 24°C), with the impact of brine temperature on proteolysis and lipolysis becoming progressively larger. Proteolysis was highest in the interior of the blocks where salt in moisture content was lowest and temperature had more impact on proteolysis in the interior position of the block than the exterior position. However, the opposite was true for lipolysis. The total free fatty acid content was higher and temperature had more impact on lipolysis at the exterior position of the block where salt in moisture was the highest. This effect of increased salt concentration on lipolysis was confirmed with direct salted cheeses in a small follow-up experiment. Lipolysis increased with increasing salt in the moisture content of the direct salted cheeses. It is likely that migration of water-soluble FFA from the brine into the cheese and from the interior portion of the cheese to the exterior portion of the cheese also contributed to a higher level of FFA at the exterior portion of the blocks. As brine temperature increased the profile of individual free fatty acids released from triglycerides changed, with the proportion of short-chain free fatty acids increasing with increasing brine temperature. This effect was largest at high salt in moisture content.

Composition and aroma compounds of Ragusano cheese: native pasture and total mixed rations.

Raw milk from 13 cows fed TMR supplemented with native pasture and from 13 cows fed only TMR on one farm was collected separately 4 times with an interval of 15 d between collections. Two blocks (14 kg each) of cheese were made from each milk. The objective was to determine the influence of consumption of native plants in Sicilian pastures on the aroma compounds present in Ragusano cheese. Milk from cows that consumed native pasture plants produced cheeses with more odor-active compounds. In 4-mo-old cheese made from milk of pasture-fed cows, 27 odor-active compounds were identified, whereas only 13 were detected in cheese made from milk of total mixed ration-fed cows. The pasture cheeses were much more rich in odor-active aldehyde, ester, and terpenoid compounds than cheeses from cows fed only total mixed ration. A total of 8 unique aroma-active compounds (i.e., not reported in other cheeses evaluated by gas chromatography olfactory) were detected in Ragusano cheese made from milk from cows consuming native Sicilian pasture plants. These compounds were 2 aldehydes ([E,E]-2,4-octadienal and dodecanal), 2 esters (geranyl acetate and [E]-methyl jasmonate), 1 sulfur compound (methionol), and 3 terpenoid compounds (1-carvone, L(-) carvone, and citronellol). Geranyl acetate and (E)-methyl jasmonate were particularly interesting because these compounds are released from fresh plants as they are being damaged and are part of a possible plant defense mechanism against damage from insects. Most of the odor-active compounds that were unique in Ragusano cheese from pasture-fed cows appeared to be compounds created by oxidation processes in the plants that may have occurred during foraging and ingestion by the cow. Some odor-active compounds were consistently present in pasture cheeses that were not detected in the total mixed ration cheeses or in the 14 species of pasture plants analyzed. Either these compounds were present in other plants not analyzed, created in the rumen or in cheese after the pasture-plant material had been consumed, or the compounds were lost in the method of sample extraction used for the plant analysis (i.e., steam distillation) versus the solid-phase microextraction method used for the cheeses. This research has demonstrated clearly that some unique odor-active compounds found in pasture plants can be transferred to the cheese.

Influence of the Temperature of Salt Brine on Salt Uptake by Ragusano Cheese

The influence of temperature (12, 15, 18, 21, and 24°C) of saturated brine on salt uptake by 3.8-kg experimental blocks of Ragusano cheese during 24 d of brining was determined. Twenty-six 3.8-kg blocks were made on each of three different days. All blocks were labeled and weighed prior to brining. One block was sampled and analyzed prior to brine salting. Five blocks were placed into each of five different brine tanks at different temperatures. One block was removed from each brine tank after 1, 4, 8, 16, and 24 d of brining, weighed, sampled, and analyzed for salt and moisture content. The weight loss by blocks of cheese after 24 d of brining was higher, with increasing brine temperature, and represented the net effect of moisture loss and salt uptake. The total salt uptake and moisture loss increased with increasing brine temperature. Salt penetrates into cheese through the moisture phase within the pore structure of the cheese. Porosity of the cheese structure and viscosity of the water phase within the pores influenced the rate and extent of salt penetration during 24 d of brining. In a previous study, it was determined that salt uptake at 18°C was faster in 18% brine than in saturated brine due to higher moisture and porosity of the exterior portion of the cheese. In the present study, moisture loss occurred from all cheeses at all temperatures and most of the loss was from the exterior portion of the block during the first 4 d of brining. This loss in moisture would be expected to decrease porosity of the exterior portion and act as a barrier to salt penetration. The moisture loss increased with increasing brine temperature. If this decrease in porosity was the only factor influencing salt uptake, then it would be expected that the cheeses at higher brine temperature would have had lower salt content. However, the opposite was true. Brine temperature must have also impacted the viscosity of the aqueous phase of the cheese. Cheese in lower temperature brine would be expected to have higher viscosity of the aqueous phase and slower salt uptake, even though the cheese at lower brine temperature should have had a more porous structure (favoring faster uptake) than cheese at higher brine temperature. Therefore, changing brine concentration has a greater impact on cheese porosity, while changing brine temperature has a larger impact on viscosity of the aqueous phase of the cheese within the pores in the cheese.

Influence of Presalting and Brine Concentration on Salt Uptake by Ragusano Cheese

The impact of presalting and nonsaturated brine on salt uptake by Ragusano cheese was determined. The study included four treatments: 1) the traditional method using no presalting and saturated brine, 2) presalting and saturated brine, 3) no presalting and 18% brine for 8 d followed by 16 d in saturated brine, and 4) presalting and 18% brine for 8 d followed by 16 d in saturated brine. Cheese blocks were weighed and sampled before brine salting (time 0) and after 1, 4, 8, 16, and 24 d of brining for each treatment. Presalting delivered 60% of the normal level of salt in the center of the block prior to brine salting without decreasing the rate of uptake of salt from either saturated or 18% brine. Use of 18% salt brine for the first 8 d of 24 d of brine salting increased the rate of salt uptake, compared with 24 d in saturated brine. The increased rate of salt uptake with 18% brine compared with saturated brine was related to the impact of salt brine on the moisture content and porosity of the cheese near the surface of the block. Brine with higher salt content causes a rapid loss of moisture from cheese near the surface of the block. Moisture loss causes shrinkage of the cheese structure and decreases porosity, which impedes moisture movement out and salt movement into the block. The use of 18% salt brine for the first 8 d delayed the moisture loss and cheese shrinkage at the exterior of the block and allowed more salt penetration.

Selection of forage species by dairy cattle on complex Sicilian pasture

This study examined the selective foraging behaviour of dairy cattle on very complex pastures as a part of a larger study on the influence of diet upon cheese flavour [Selective grazing on Sicilian pasture by cattle and effects on Ragusano cheese, Ph.D. dissertation, Cornell University, Ithaca, NY, 2003]. Sicilian pastures can contain over 100 plant species belonging to over 25 families, although not all of these species may occur in a single pasture. A grazing study was conducted on a farm with a group of 13 lactating Friesian cows. Milk was used to make cheeses that were evaluated in a separate study. Pasture composition and selection of plant species by cows were studied in the years 1999 and 2000. Qualities of mixed selected forage were uniformly very high and did not vary greatly over season and years. While pasture composition varied between years, animal selection was highly correlated with species incidence ( r=0.83). Animal choices tend to follow occurrence. A few species remained uneaten including Carduus (thistle) and Biscutella. The dominant species consumed belonged to the plant families Asteraceae (Compositae), Cruciferae, Malvaceae and Fabaceae. This information was used in a larger study to relate pasture species to flavour of cheese.

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Composition of Ragusano Cheese During Aging

Ragusano cheese is a brine-salted pasta filata cheese. Composition changes during 12 mo of aging were determined. Historically, Ragusano cheese has been aged in caves at 14 to 16°C with about 80 to 90% relative humidity. Cheeses (n = 132) included in our study of block-to-block variation were produced by 20 farmhouse cheese makers in the Hyblean plain region of the Province of Ragusa in Sicily. Mean initial cheese block weight was about 14kg. The freshly formed blocks of cheese before brine salting contained about 45.35% moisture, 25.3% protein, and 25.4% fat, with a pH of 5.25. As result of the brining and aging process, a natural rind forms. After 12 mo of aging, the cheese contained about 33.6% moisture, 29.2% protein, 30.0% fat, and 4.4% salt with a pH of 5.54, but block-to-block variation was large. Both soluble nitrogen content and free fatty acid (FFA) content increased with age. The pH 4.6 acetate buffer and 12% TCA-soluble nitrogen as a percentage of total nitrogen were 16 and 10.7%, respectively, whereas the FFA content was about 643 mg/100g of cheese at 180 d. Five blocks of cheese were selected at 180 d for a study of variation within block. Composition variation within block was large; the center had higher moisture and lower salt in moisture content than did the outside. Composition variation within blocks favored more proteolysis and softer texture in the center.