Use of Galactose-Fermenting Streptococcus thermophilus in the Manufacture of Swiss,Mozzarella, and Short-Method Cheddar Cheese

Galactose-fermenting (galactose-positive) strains of Streptococcus thermophilus, alone and combined with galactose-positive and galactose-negative strains of Lactobacillus bulgaricus, were used as starter cultures in the manufacture of Swiss and Mozzarella cheese and were paired with Streptococcus lactis (also galactose-positive) in short-method Cheddar cheese manufacture. Experimental Swiss cheese made with the galactose-positive Streptococcus thermophilus starter alone contained a large amount of galactose (ca. 26 to 28 µmol/g of curd) 28 h after hooping compared with control Swiss (< 2 µmol/g) made with a nongalactose fermenting strain of Streptococcus thermophilus and a galactose-positive strain of Lactobacillus bulgaricus. Mozzarella and short-method Cheddar made with only galactose-positive Streptococcus thermophilus also contained large amounts of galactose. Swiss cheese made with a galactose-positive strain of Streptococcus thermophilus and a galactose-negative strain of Lactobacillus bulgaricus had little galactose remaining after 28 h, indicating that the Lactobacillus had a stimulatory effect on galactose metabolism in Streptococcus thermophilus. These results indicate that galactose-fermenting Streptococcus thermophilus may have limited potential when used as single strain starter cultures in Swiss cheese, but may be useful when combined with galactose-positive Lactobacillus in the manufacture of Mozzarella cheese.     

Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100 kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45 °C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with k_cat/K_m of 8.1 × 10⁶ M^?1 s^?1, and the activation energy (E_a) of 72.5 kJ M^?1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn²⁺, Mn²⁺ and Co²⁺ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.     

Migration of α-tocopherol from LDPE films to corn oil and its effect on the oxidative stability

The migration of α-tocopherol (α-T) from low density polyethylene (LDPE) films, added with 20 (film A) and 40 mg g^?1 (film B) to corn oil for 12 weeks at 5, 20 and 30 °C was determined. A LDPE film added with no α-T was used as control (film C). Diffusion coefficient (D) values for the film A system were 1.4 × 10^?11, 7.1 × 10^?11 and 30.3 × 10^?11 cm² s^?1 at 5, 20 and 30 °C, respectively. Meanwhile, D values for the film B system were 1.3 × 10^?11, 9.6 × 10^?11 and 51.1 × 10^?11 cm² s^?1 at the same temperatures. The activation energy (E_a) for the diffusion of α-T was 126.5 (film A) and 105.9 kJ mol^?1 (film B). The effect of the migration of α-T on the oxidative stability of corn oil was evaluated by monitoring hexanal content by solid phase micro-extraction (SPME) and gas chromatography. The hexanal content in the oil showed that both films added with α-T resulted suitable to maintain the oxidative stability of the oil for about 16 weeks at 30 °C, compared to 12 weeks for the oil in contact with the film C.     

Manufacture of Cheddar Cheese Using Proteinase-Negative Mutants of Streptococcus cremoris

Cheddar cheese was manufactured with a proteinase-negative mutant of Streptococcus cremoris UC 73 and from a commercial lactic culture blend. Soluble nitrogen was analyzed and the cheese graded at intervals to 365 d of age. The cheese made with proteinase-negative cultures graded equal to the control cheese up to 90 d. It was best in overall texture and body and lowest in cheese flavor and flavor intensity after 90 d. It also had significantly higher soluble nitrogen throughout storage. No significant differences in yields were found.Cheddar cheese made at a constant 39°C temperature with a 2% inoculum of proteinase-negative S. cremoris UC 73 increased manufacturing time by 40 min over cheese made by conventional cooking procedures. When inoculum was increased to 4% and the constant temperature to 42°C, manufacturing time was 3.6 h, which was 1 h less than with a 2% inoculum and conventional cooking. By providing a yeast extract carry-over of .0175% from the proteinase-negative bulk culture, it would be possible to produce Cheddar cheese within a normal time frame with only .7% inoculum.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties.     

Thermal and light stabilities and antioxidant activity of carotenoids from tomatoes extracted using an ultrasound-assisted completely solvent-free method

The use of petroleum-derived solvents, particularly volatile organic compounds (VOCs), in the chemical industry has increased the contamination and residual effects of these solvents. Ionic liquids (ILs) can potentially replace VOCs, thereby reducing the risks of environmental contamination and toxicity. In this context, the objectives of this study were as follows: 1 — to obtain an ionic liquid for use in extracting carotenoids from tomatoes with ultrasound assistance; and 2 — to determine the stability and antioxidant activity of tomato carotenoid extracts. Ultrasound can also efficiently extract carotenoid compounds with ionic liquids in comparison with conventional VOC solvents (obtained from an all-trans-lycopene 7.5–8.0 μg·g^− 1 tomato sample by IL and 6.2–7.7 μg·g^− 1 by acetone). Similarly, the activation energies (E_a) in aqueous medium were obtained for the IL carotenoid extract (10.8 kJ·mol^− 1) and acetone carotenoid extract (9.4 kJ·mol^− 1). The antioxidant activities of the tomato carotenoid extract were 7.4 and 12.4 relative to α-tocopherol for the ionic liquid extract and acetone extract, respectively. The combination of chromatographic analysis and degradation kinetics provided data for positive assessment similarity of thermal and light stabilities of tomato carotenoids extracted by IL and extracted by acetone.     

Adsorption kinetics,thermodynamics and isotherm of Hg(II) from aqueous solutions using buckwheat hulls from Jiaodong of China

The adsorption kinetics and adsorption isotherms of buckwheat hulls in the region of Jiaodong, China (BHJC) for Hg(II) were investigated. Results revealed that the adsorption kinetics of BHJC for Hg(II) were well described by a pseudo second-order reaction model, and the adsorption thermodynamic parameters ΔG, ΔH and ΔS were –5.83 kJ mol^?1(35 °C), 73.1, and 256 J K^?1 mol^?1, respectively. Moreover, Langmuir, Freundlich and Redlich–Peterson isotherm models were applied to analyse the experimental data and to predict the relevant isotherm parameters. The best interpretation for the experimental data was given by the Langmuir isotherm equation, and the maximum adsorption capacity for Hg(II) is 243.90 mg/g at 35 °C. Furthermore, investigation of the adsorption selectivity showed that BHJC displayed strong affinity for mercury in the aqueous solutions and exhibited 100% selectivity for mercury in the presence of Zn(II) and Cd(II).     

Development of a device and method for the time-course estimation of low water fluxes and mean surface water activity of food products during ripening and storage

Accurate measurement of water activity (a_w) is an important goal for the food industry because a_w is a key parameter in microbial growth, biological reaction rates and physical properties. An experimental device was setup using air-product water balance to non-destructively estimate the time-course of mean a_w at the food product surface under well-controlled airflow conditions. The device is especially suited for studying the ripening of cheeses and fermented meat products, where water fluxes exchanged between products and air are very low. The validation tests performed with a_w-known model products showed that water fluxes of 10^?7 kg s^?1 can be estimated with an accuracy better than 2% over very short periods of time, and that surface a_w can be estimated with an absolute uncertainty of less than 0.01 a_w units. A handful of cheese ripening trials illustrate the potential of the method, highlighting the effects of a low air velocity and high air RH on the water exchanges occurring at a cheese surface, thus demonstrating the strong surface sensitivity to external air conditions.     

Occurrence of genetic polymorphism at the αs1-casein locus in Mediterranean water buffalo milk

A study on casein genetic polymorphism in water buffalo milk of Mediterranean breed, reared in Southern Italy, was carried out by means of immunoelectrophoretic and chromatographic techniques coupled with mass spectrometry analysis. When compared with reference α_s1-CN variant A, the results showed the occurrence of a new α_s1-casein (α_s1-CN) B genetic variant having a single silent amino acid substitution Leu¹⁷⁸(A) → Ser¹⁷⁸(B). In Mediterranean buffalo milks the allelic frequency of new α_s1-CN B was very similar to that determined in Mozzarella di bufala campana (MBC), a Protected Denomination of Origin (PDO) cheese, made exclusively from whole raw buffalo milk of this breed. The relative percentage of the two α_s1-CN variants, either in bulk milk or MBC cheese, can be used in defining the authenticity of PDO MBC cheese.     

Applicability of heat-stable deoxyribonuclease assay for assessment of staphylococcal growth and the likely presence of enterotoxin in cheese

The relationships between growth of Staphylococcus aureus and production of deoxyribonuclease and enterotoxin A in cheese were evaluated. Conditions of cheese manufacture, such as the nature of milk used (heated or raw), type of lactic starter, and degree of starter activity, influenced deoxyribonuclease production. There was a close correlation between the S. aureus population and deoxyribonuclease content (correlation .88 in Cheddar and Colby cheeses for normal or inhibited starter, and .85 in Brick cheese for normal starter). Conditions which affected deoxyribonuclease production also had a similar influence on production of enterotoxin A. Detection of the former is especially useful in cheeses which may have had a partial starter failure not detected by the usual criteria of starter activity such as the titratable acidity of whey or the final pH of cheese. While the viable S. aureus population declined during aging, both deoxyribonuclease and enterotoxin A persisted for an extended time (3 yr at 4.4 C) in cheese of normal or inhibited starter.     

Biochemical changes throughout the ripening of a traditional Spanish goat cheese variety (Babia-Laciana)

The physico-chemical characteristics, proteolysis (classical nitrogen fractions, caseins and their degradation products and free amino acids), and lipolysis (fat acidity and free fatty acids) were studied throughout the ripening of three batches of Babia-Laciana cheese, a Spanish traditional variety made from raw goats’ milk. The main compositional characteristics of this cheese at the end of the ripening are its high content of total solids (TS) (78.0±2.4 g 100 g⁻¹ of cheese) and fat (61.1±1.2 g 100 g⁻¹ of TS), the presence of residual lactose (1.6±0.8 g 100 g⁻¹ of TS) and its low content of sodium chloride (1.1±0.7 g 100 g⁻¹ of TS) and ash (2.8±0.5 g 100 g⁻¹ of TS). Its pH values (4.44±0.72) are extraordinarily low. The evolution and final values of the different nitrogen fractions show that this cheese undergoes a very slight proteolysis, a fact which was corroborated when the caseins and their degradation products were quantified: β-casein did not undergo any modification throughout ripening, while only 21% of the α_s-caseins were degraded. Free amino acids content increased by a factor of about 7 throughout ripening, resulting in a high content of γ-amino butyric acid and a low content of glutamic acid at the end of the process. Fat acidity increased very slightly, approximately 4.5 times, during ripening, reaching final values of 3.5±2.2 mg KOH g⁻¹ of fat. The total free fatty acids content showed a similar evolution to fat acidity. At the end of the ripening process, the main free fatty acid was C_18:1, followed by C₁₆ and C₁₀.     

Texture characteristics and pizza bake properties of low-fat Mozzarella cheese as influenced by pre-acidification with citric acid and use of encapsulated and ropy exopolysaccharide producing cultures

Low-fat Mozzarella cheeses containing 6% fat were made by pre-acidification of milk with citric acid to pH 6.1 and using encapsulated ropy or non-ropy exopolysaccharide (EPS) producing Streptococcus thermophilus. Moisture retention, changes in texture profile analysis (TPA), meltability and stretchability of cheese, and changes in colour, surface scorching and shred fusion were analysed after baking over 90 days (d). Control cheeses and those made from pre-acidified milk without EPS cultures had the lowest moisture content at 54.84% and 55.28%, respectively. Control cheeses were hardest and their meltability and stretchability were initially low. Hardness was reduced and the melt and stretch distances increased with time. When baked, control cheeses showed incomplete shred fusion. Pre-acidification reduced hardness and increased meltability. Capsular- and ropy-EPS were quantified at 30.42 and 30.55 mg g⁻¹ of cheese, respectively, and increased moisture retention in pre-acidified cheese to 56.67% and 56.21%, respectively. These cheeses were softer and exhibited lower springiness. Greater meltability was observed initially but became similar to control cheeses after 90 d of storage. When baked after 45 d of storage, cheeses containing EPS producing cultures showed improved shred fusion, meltability and a reduction in surface scorching.     

Routine and sensitive SPE-HPLC method for quantitative determination of pheophytin a and pyropheophytin a in olive oils

A sensitive and specific HPLC method with tandem diode array-fluorescence detection (DAD-FL) has been developed and validated for the simultaneous determination of pheophytin a (phy a) and pyropheophytin a (pyrophy a) in olive oils. Pigments were extracted with reverse phase solid phase extraction (RP-SPE) and subsequently analysed by HPLC-DAD-FL. The chromatographic analysis was carried out isocratically on ODS2 RP column using methanol–acetone (1:1 v/v) at flow-rate of 2.0 ml min⁻¹. Specificity of the method was assured by the simultaneous detection by UV–visible (410 nm) and FL (λ_Ex: 410 nm; λ_Em: 672 nm). Both compounds could be baseline separated within 7 min. The method was validated and applied in olive oil samples recently extracted as well as stored during 12 months. The limit of detection (LOD) defined at a signal-to-noise ratio of about 3 was ∼21.6 ng g⁻¹ for pyrophy a and ∼24.6 ng g⁻¹ for phy a under FL detection, and ∼148.0 ng g⁻¹ for both analytes under UV–visible detection. The calibration graphs were linear (r² > 0.9999; p < 0.01) between 0.25–14.00 ng μl⁻¹ for pyrophy a and 0.25–19.00 ng μl⁻¹ for phy a, under both fluorescence and UV–visible detection conditions. Recoveries of phy a and pyrophy a were over 94% as estimated by the standard addition method. Relative standard deviation for the intra-day and inter-day determination of phy a and pyrophy a were lower than 3.7% and 8.0%, respectively.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.     

Primary proteolysis and textural changes during ripening in Cheddar cheeses manufactured to different fat contents

The effects of varying fat content in Cheddar cheese, from 6.3 to 32.5 g 100 g⁻¹, on changes in pH, primary proteolysis and texture were monitored over a 225 d ripening period. Reduction in the fat content resulted in significant (P<0.05) increases in pH, moisture and protein contents and decreases in the concentration of moisture in the non-fat substance. The increase in pH as the fat content increased was attributed to the concomitant decrease in the lactate-to-protein ratio. Polyacrylamide gel electrophoresis showed that the concentration of intact casein decreased in all cheeses during ripening and that the rate of decrease was not affected by the fat content. However, for a given concentration of casein, α_s1-casein was degraded more slowly, and β-casein more rapidly, as the fat content was reduced. The slower degradation of α_s1-casein with decreased fat content coincided with a decrease in the ratio of residual chymosin activity to protein in the cheese. At most ripening times, reduction in the fat content resulted in significant increases in the concentration of intact casein, fracture stress, fracture strain, and cheese firmness. The effects of fat reduction on proteolysis and rheology are probably due to the interactive effects of the concomitant changes in composition.     

Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese

The effect of two commercially available adjunct cultures, LBC 80 (Lactobacillus casei subsp. rhamnosus) and CR-213 (containing Lactococcus lactis subsp. cremoris and Lc. lactis subsp. lactis) on the proteolysis in low-fat hard ewes’ milk cheese of Kefalograviera-type was investigated. Two controls, a full-fat cheese (306 g kg⁻¹ fat, 378 g kg⁻¹ moisture) and a low-fat cheese (97 g kg⁻¹ fat, 486 g kg⁻¹ moisture, made using a modified procedure), were also prepared. The effect of adjunct culture on proteolysis, as examined by polyacrylamide gel electrophoresis of cheese and water soluble cheese extracts, was marginal. The reverse-phase HPLC peptide profiles of the water soluble extracts from low-fat cheeses were similar although some quantitative differences were observed between low-fat control cheese and experimental cheeses. The fat content as reflected by the differences in peptide profiles affected the pattern of proteolysis. Proteolysis, as measured by the percentage of total nitrogen soluble in water or in 120 g L⁻¹ trichloroacetic acid, was significantly (P<0.05) affected by the addition of adjunct cultures. Furthermore, the adjunct cultures enhanced the production of low molecular mass nitrogenous compounds; the levels of total nitrogen, soluble in 50 g L⁻¹ phosphotungstic acid, and of free amino acids were significantly (P<0.05) higher in the low-fat experimental cheeses than in the low-fat control cheese.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

Microbiological,compositional, biochemical and textural characterisation of Caciocavallo Pugliese cheese during ripening

A microbiological, compositional, biochemical and textural characterisation of the pasta filata Caciocavallo Pugliese cheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g⁻¹ mesophilic bacteria and ca. log 6.0 cfu g⁻¹ presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum, Lb. gasseri, Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of α_s1-casein was more rapid than that of β-casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of Caciocavallo Pugliese cheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese.