Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Effect of β-casein concentration in cheese milk on rennet coagulation properties,cheese composition and cheese ripening

Effects of the use of a β-casein powder to enrich cheese milk on rennet coagulation properties of milk, cheese composition and cheese ripening were investigated. Casein content of control milk was 2.5%, whereas that for the three enriched milks was adjusted with β-casein powder at 2.7%, 2.9% and 3.1%. The β-casein to α-casein ratio of these cheese milks was, respectively, 0.70, 0.79, 0.89 and 0.99. Rennet coagulation properties were related not only to casein concentration but also to the proportion of β-casein and α_s-casein presents in milks. Milk with higher concentration of β-casein had poorer coagulation properties. Cheeses could be produced by using a miniature cheese making process. Moisture, ash and calcium contents decreased, while protein content and β-casein increased in cheese as casein and β-casein concentration increased in milk. As a result, hardness was higher in enriched cheeses than in control cheese. During cheese ripening, α-casein was hydrolyzed, but the rate of degradation of α-casein decreased as protein and β-casein concentration increased in cheese. β-Casein seemed to be not hydrolyzed. The rate of decrease of hardness was also slower for enriched cheeses.     

Occurrence,origin and fate of pyroglutamyl-γ3-casein in cheese

During plasmin-catalysed proteolysis of β-casein (β-CN) in cheese, pyroglutamyl-γ₃-casein (pγ₃-CN), originates from the cyclisation of the N-terminal Glu of γ₃-CN (β-CN f108–209). This peptide progressively increased in content during ripening of Grana Padano and Parmigiano-Reggiano Protected Designation of Origin cheeses. A preliminary survey revealed pγ₃-CN in both hard and extra-hard cheese varieties; in semi-hard cheeses it was present only in mature samples. To understand the mechanisms of pγ₃-CN formation/degradation, in vitro trials (real cheese, model cheeses and buffered γ₃-CN solutions) were performed. Both the effect of cheese pH and the capacity of certain microorganisms to cyclise the N-terminal Glu residue of γ₃-CN were studied. The results suggested that pγ₃-CN formed spontaneously during cheese ripening, mainly at the acid pH characteristic of hard and extra-hard cheeses; the role of cyclase enzymes was negligible. Differences in pγ₃-CN degradation were observed due to the proteolytic activity of bacteria involved in the cheese ripening.     

Proteolysis in goat cheese made from raw,pasteurized or pressure-treated milk

Primary and secondary proteolysis of goat cheese made from raw (RA), pasteurized (PA; 72 °C, 15 s) and pressure-treated milk (PR; 500 MPa, 15 min, 20 °C) were examined by capillary electrophoresis, nitrogen fractionation and HPLC peptide profiles. PA milk cheese showed a more important hydrolysis (P<0.05) of α_s1-casein than RA milk cheese at the first stages of ripening (15 days), while PR milk cheese had a level between those seen in PA and RA milk cheeses. Degradation of β-casein was more important (P<0.05) in PA and PR than in RA milk cheeses at 15 days of ripening. However, from thereon β-casein in PR and RA milk cheeses was hydrolyzed at essentially similar rates, but at lower rates (P<0.05) than in PA milk cheeses. Pressure treatment could induce proteolysis of β-casein in a way, which is different from that produced by heat treatment. There was an increase in 4.6-soluble nitrogen (WSN) and in trichloroacetic acid (TCASN) throughout ripening in cheeses, but higher contents (P<0.05) in PA and PR milk cheeses at the end of ripening were observed. PR milk cheeses contained considerably higher content (P<0.05) of free amino acids than RA or PA milk cheeses. In general, heat and pressure treatments had no significant effect on the levels of hydrophobic and hydrophilic peptides.     

Evolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertime

Biochemical, volatile and textural profiles during manufacture and ripening were determined in samples of Castelmagno PDO cheese obtained from three different batches in the main artisan cheese plant of Castelmagno PDO production area. At the end of manufacture, samples were characterised by a pH of 6.57% and 52.4% moisture content. The HPLC analysis of organic acids and sugars showed the exhaustion of lactose content, while Urea-PAGE indicated extensive primary proteolysis of both β-casein and α_s1-casein. During ripening, cheeses were characterised by high degradation of β-casein and α_s1-casein, due to bacterial action. RP-HPLC profiles showed a high production of peptides eluted between 20 and 30 min. In total, 92 volatile compounds were identified in cheese headspace. Texture profiles showed an increase in hardness, gumminess, chewiness and adhesiveness values, as well as a decrease in cohesiveness during ripening.     

Using High-Pressure Processing for Reduction of Proteolysis and Prevention of Over-ripening of Raw Milk Cheese

High-pressure-processing (HPP) at 400 or 600 MPa was applied to cheeses made from ewe raw milk, on days 21 or 35 after manufacturing, to reduce proteolysis and prevent over-ripening. The characteristics of HPP and non-pressurized (control) cheeses were compared during ripening at 8 °C until day 60 and further storage at 4 °C until day 240. HPP and control cheeses showed similar pH values throughout ripening, but on day 240 pH values remained 0.4–0.6 units lower for HPP cheeses than for the control cheeses. Casein degradation was significantly retarded in the 600 MPa cheeses. Their α-casein concentration was 48–52 % higher on day 60 and 30–33 % higher on day 240 than in the control cheeses while β-casein concentration was 25–26 % higher on day 60 and 100–103 % higher on day 240. No significant differences in para-κ-casein concentration between cheeses were found on day 60, but on day 240, it was 22–35 % higher in the 600 MPa cheeses than in the control cheese. Hydrophilic peptides, hydrophobic peptides and total free amino acids evolved similarly in HPP and control cheeses during the 60-day ripening period. However, on day 240 hydrophilic peptides were at 34–39 % lower levels in the 600 MPa cheeses than in the control cheeses, hydrophobic peptides at 7–16 % lower levels and total free amino acids at 25–29 % lower levels. Flavour intensity scores increased at a slower rate in HPP cheeses than in the control cheese. Flavour quality declined markedly in the control cheeses during refrigerated storage while it did not vary significantly in 600 MPa cheeses.     

Study of the chemical composition,proteolysis, volatile compounds,and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese

The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and α_s-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for α_s-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.     

Study of the biochemical changes during ripening of Ahumado de Áliva cheese: a Spanish traditional variety

The changes in chemical composition, main physico-chemical parameters, classical nitrogen fractions, caseins and their degradation products, and some fat characteristics were studied during the ripening of ten batches of Ahumado de Áliva cheese, a traditional variety made in the north of Spain. The values of the different compositional and physico-chemical parameters at the end of the ripening did not differ significantly from those found in other cows' milk cheeses elaborated by similar technology. The low pH values are outstanding. The presence of residual lactose at the end of ripening is also relevant. Total soluble nitrogen and non-protein nitrogen increased very little during ripening. The evolution of the values of the different nitrogen fractions show that this cheese undergoes very little proteolysis and that the rennet is the main proteolytic agent. Using PAGE, it was possible to show that, throughout ripening, only 22% of α_s-casein and 9% of β-casein were degraded. The TBA value indicated that the fat of Ahumado de Áliva cheese does not undergo noticeable autooxidation during ripening. The acidity index of the fat also indicated that this cheese underwent little lipolysis during ripening.     

Kinetics of Casein Degradation during Ripening of Fynbo Cheese Salted with NaCl/KCl Brine

First-order kinetics with respect to the α_s1-casein concentration was used to study casein degradation during low-fat Fynbo cheese ripening. Effects of partial NaCl replacement by KCI during cheese salting were studied by statistical treatment of the casein degradation results. Four zones from cheeses at 1, 5, 10, 20, and 30 ripening days were analyzed by a polyacrylamide gel electrophoresis method. Similar kinetic parameters were obtained for a cheese salted with a NaCl/KCl brine and for a control cheese during ripening. Results were more affected by salt concentration than by salt substitution. KCl did not strongly influence kinetics of Fynbo cheese proteolysis.     

Residual clotting activity and ripening properties of vegetable rennet from Cynara cardunculus in La Serena cheese

Vegetable rennet extracted from Cynara cardunculus flowers is traditionally used in the manufacture of La Serena cheese. High levels of proteolytic enzymes of the flowers are responsible for its clotting activity and strong proteolytic action. The presence of residual coagulant in cheese and whey was measured by adding known amounts of vegetable rennet as internal standard. We found no differences between the residual coagulant activity of La Serena cheese compared with other types of cheese. The coagulant content detected at the end of four cheesemakings (vat of 830 l) in cheese and whey represented 27 and 78%, respectively, of the total amount added to milk. When measurements were carried out in 16 different cheeses, vegetable rennet appeared to be highly stable during cheese ripening. Cheese composition (moisture, pH, NaCl, fat and protein) was kept relatively constant during ripening, which seems to contribute to stability of residual activity. Electrophoretic analyses of water insoluble fractions from cheeses manufactured with vegetable rennet showed that α_s-casein was less susceptible to proteolysis than β-casein. The water soluble nitrogen/total nitrogen (WSN/TN) exhibited higher levels only during the first 30 days of ripening although non-protein nitrogen/total nitrogen (NPN/TN) ratio and amino acid nitrogen (NH₂-N) increased with ripening time.     

The influence of ripening temperature and sampling site on the proteolysis in Reggianito Argentino cheese

The effects of elevated ripening temperature and sampling site on proteolysis in Reggianito Argentino cheese were evaluated. Cheeses ripened at 12 or 18 °C and 85% relative humidity were analysed at 2, 4 and 6 months in 2 sampling zones (central and external). Samples were analysed to determine the physicochemical and proteolysis parameters through the urea-PAGE of the urea-soluble fraction, RP-HPLC analysis of the water-soluble fraction at pH 4.6, and the free amino acid analysis. Proteolysis was significantly affected by ripening temperature and sampling site. Urea-PAGE analysis showed that elevated temperature increased the degradation of α_s1- and β-casein. The degradation of α_s1-casein was larger in the central zone than in the external one, while β-casein degradation was similar in both zones. The majority peaks detected by RP-HPLC of the water-soluble fraction at pH 4.6 and free amino acids were significantly affected by ripening temperature and sampling site. Glu, His, Val, Leu, and Lys had the higher concentrations. Principal component analysis showed useful groupings when results from chromatograms were studied. In conclusion, the results obtained not only are useful to characterise the ripening of an Argentinean hard cheese, but also to evaluate the effect of an increase of ripening temperature on Reggianito Argentino cheese proteolysis.     

A Survey on the Some Chemical and Biochemical Properties of Civil Cheese,a Traditional Turkish Cheese

In this article, 15 randomly selected samples of Civil cheese, were purchased from different retail markets in the Erzurum province, Turkey and were investigated for some chemical and biochemical analyses. All cheese samples were analyzed for dry matter, fat, salt, ash, titrable acidity, total nitrogen, soluble nitrogen, ripening index, α_s-and β-casein degradation, γ-casein, and peptides. Dry matter, fat, fat in dry matter, salt, salt in dry matter, ash, and acidity values in samples analyzed were found to be as found between 31.33 and 40.12 g/100 g cheese; 1.00 and 7.00 g/100 g cheese; 2.49 and 18.98 g/100 g cheese; 0.11 and 0.34 g/100 g cheese; 0.27 and 1.04 g/100 g cheese; 1.42 and 5.14 g/100 g cheese and, 0.63 and 2.16%, respectively. TN, WSN/TN, TCA-SN/TN, and PTA-SN/ TN values, expressed as TN%, were found between 3.01 and 5.57 g/100 g cheese, 4.25 and 8.80 g/100 g cheese, 3.23 and 6.12 g/100 g cheese, 1.03, and 5.53 g/100 g cheese in Civil cheese samples analyzed, respectively. SDS-PAGE showed that both α_s-CN and β-CN ratios were not high compared with similar cheeses, and are not completely hydrolyzed in all Civil cheese samples. A broad range of values from chemical and biochemical analysis indicated that Civil cheeses collected from retail markets lacked standardization. Consequently, it was decided that Civil cheese samples do not undergo an excessive proteolysis.     

Microbiological,biochemical and compositional changes during ripening of São Jorge – a raw milk cheese from the Azores (Portugal)

The microbial, compositional and biochemical profiles of São Jorge cheese (PDO) obtained from three distinct cheese plants, throughout the ripening period were determined. Fully ripened cheeses (i.e. by 130 days) contained a total of 3.1 × 10⁷ CFU g⁻¹ mesophilic bacteria, and a decrease in moisture content, concomitantly with an increase in salt content, was observed throughout the same time frame. The pH decreased until 30 days of ripening; thereafter, a slight increase was reported, up to 5.6 by the end of ripening. Urea-PAGE results showed extensive primary proteolysis, of both β-casein and α_s1-casein − degraded at essentially similar rates; plasmin and chymosin accordingly appear to be active in the cheese curd. RP-HPLC profiles of water-soluble fractions showed minor differences between 1 and 130 day old cheeses, whereas equivalent profiles of 7% (v/v) ethanol-soluble fractions contained several peaks, indicative of a heterogeneous mixture of products of proteolysis, that evolved with time.     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.     

Application of encapsulated enzymes to accelerate cheese ripening

The suitability of gellan, κ-carrageenan and a high-melting-fat-fraction of milk fat (HMFF) to encapsulate protease enzymes (Flavourzyme) and impact in accelerating Cheddar cheese ripening were studied. The rates of enzyme entrapment were 48.2%, 55.6%, and 38.9% for gellan, κ-carrageenan and HMFF, respectively. The enzyme capsules were incorporated into milk during cheese manufacture. The moisture content of cheeses with added gum capsules was higher than control cheeses. Casein (β) degradation was monitored by High-Performance Capillary Electrophoresis. All cheeses treated with encapsulated enzyme showed higher rates of proteolysis than the control cheese throughout the ripening period. The rate of proteolysis was greater with cheeses made incorporating κ-carrageenan capsules containing protease. Cheese texture and sensory quality were not significantly influenced by the type of encapsulating material (gum or milk fat). Differences in textural and sensory quality between treated and control cheeses were consistent with release of protease enzymes from capsules.     

Effect of sodium,potassium, magnesium,and calcium salt cations on pH,proteolysis, organic acids,and microbial populations during storage of full-fat Cheddar cheese

Sodium reduction in cheese can assist in reducing overall dietary Na intake, yet saltiness is an important aspect of cheese flavor. Our objective was to evaluate the effect of partial substitution of Na with K on survival of lactic acid bacteria (LAB) and nonstarter LAB (NSLAB), pH, organic acid production, and extent of proteolysis as water-soluble nitrogen (WSN) and protein profiles using urea-PAGE, in Cheddar cheese during 9 mo of storage. Seven Cheddar cheeses with molar salt contents equivalent to 1.7% salt but with different ratios of Na, K, Ca, and Mg cations were manufactured as well as a low-salt cheese with 0.7% salt. The 1.7% salt cheeses had a mean composition of 352 g of moisture/kg, 259 g of protein/kg and 50% fat-on-dry-basis, and 17.5 g of salt/kg (measured as Cl⁻). After salting, a faster initial decrease in cheese pH occurred with low salt or K substitution and it remained lower throughout storage. No difference in intact casein levels or percentage WSN levels between the various cheeses was observed, with the percentage WSN increasing from 5% at d 1 to 25% at 9 mo. A greater decrease in intact α_s1-casein than β-casein was detected, and the ratio of α_s1-casein (f121–199) to α_s1-casein could be used as an index of ripening. Typical changes in bacteria microflora occurred during storage, with lactococci decreasing gradually and NSLAB increasing. Lowering the Na content, even with K replacement, extended the crossover time when NSLAB became dominant. The crossover time was 4.5 mo for the control cheese and was delayed to 5.2, 6.0, 6.1, and 6.2 mo for cheeses with 10, 25, 50, and 75% K substitution. Including 10% Mg or Ca, along with 40% K, further increased crossover time, whereas the longest crossover time (7.3 mo) was for low-salt cheese. By 9 mo, NSLAB levels in all cheeses had increased from initial levels of ≤10² to approximately 10⁶ cfu/g. Lactococci remained at 10⁶ cfu/g in the low-salt cheese even after 9 mo of storage. The propionic acid concentration in the cheese increased when NSLAB numbers were high. Few other trends in organic acid concentration were observed as a function of Na content.     

Evaluation of cheese authenticity and proteolysis by HPLC and urea–polyacrylamide gel electrophoresis

Chromatographic and electrophoretic methods have been established as useful tools in characterising cheese ripening and in the detection of milk adulteration. The purpose of this work was to evaluate casein proteolysis of cheeses made from bovine, ovine or mixtures of bovine and ovine milks, as well as ovine cheese authenticity, for 30 days of ripening by HPLC and urea–polyacrylamide gel electrophoresis.Complementary information was obtained by both techniques when applied to the study of casein proteolysis during 30 days of ripening of ovine milk cheeses, ovine milk cheeses with 10% and 20% of bovine milk and bovine milk cheeses, manufactured according to the traditional Terrincho technology. For ovine cheeses, α-casein was the fraction that showed the higher degradation during cheese ripening. A similar behaviour was observed for ovine milk cheese with 10% of bovine milk. The profile for ovine milk cheese with 20% of bovine milk was more similar to that obtained for bovine cheese. Concerning bovine milk cheeses, electrophoresis was the most sensitive technique for the evaluation of proteolysis in these cheeses.Ten and 20% of bovine milk could be detected in ovine milk cheeses by urea–polyacrylamide gel electrophoresis and HPLC, respectively, even after 30 days of ripening.     

Comparative biochemical evolution during ripening of bovine, ovine and caprine cheeses manufactured with extracts of flowers of Cynara cardunculus

Changes in the main physicochemical and biochemical characteristics of bovine, ovine and caprine milk cheeses manufactured with aqueous extracts of flowers of Cynara cardunculus were studied throughout ripening (0?–?68 days). At the end of ripening the pH in the centre was (mean ± ISD) 5.05±0.07, 5.15±0.21 and 4.91±0.07 for bovine, ovine and caprine milk cheeses, respectively; whereas the pH at the top of the cheese was 5.21±0.09, 5.44±0.12 and 5.12±0.07, respectively; the moisture content values of bovine, ovine and caprine milk cheeses were 37.68±2.32, 40.23±4.34 and 45.73±1.44%, respectively; the NaCl content values were 4.00±0.43, 4.00±0.56 and 4.08±0.58 (%TS); the fat content values were 41.01±5.32, 63.60±7.80 and 45.14±9.14 (%TS); the protein content values were 28.96±1.84, 22.89±2.37 and 30.02±1.22 (%TS); the water-soluble nitrogen values were 32.91±1.76, 46.44±1.86 and 48.72±1.08 (%TN); the values of nitrogen soluble in trichloroacetic acid were 10.25±1.93, 10.41±2.91 and 7.25±0.84 (%TN); the values of nitrogen soluble in phosphotungstic acid were 1.50±0.18, 1.87±0.65 and 1.38±0.32 (%TN); finally, the free amino acid content values were 21.33±5.34, 17.51±5.08 and 23.93±4.80 μM equivalents of leucine. Urea-polyacrylamide gel electrophoresis of water-insoluble fractions from cheeses indicated high degrees of proteolysis of α_s1-casein in bovine cheeses and more extensive proteolysis of the α_s-casein region in caprine than in ovine cheeses, but a moderate degree of proteolysis of β-casein in all types of cheese; electrophoregrams of the water-soluble fraction displayed increasing numbers of bands throughout ripening, an observation that is consistent with the increase in the ratio of water-soluble nitrogen to total nitrogen.     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.