Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.     

Effect of different ventilation regimens on ewes' milk and Canestrato Pugliese cheese quality in summer

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4.6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.     

Microbiological and biochemical properties of canestrato pugliese hard cheese supplemented with bifidobacteria

Canestrato Pugliese cheeses from ewe milk were produced according to a traditional protocol and by adding 7.0 log10 cfu of fresh cells per gram of Bifidobacterium bifidum Bb02, Bifidobacterium longum Bb46, or both species. The traditional technology was modified slightly to favor the survival of probiotic microorganisms. After 56 d of ripening, the survival of B. bifidum Bb02 and B. longum Bb46 was 6.0 and 5.0 log10 cfu/g, respectively. After 19 d cheeses contained ca. 7.0 log10 cfu/g of bifidobacteria. Compared to traditional cheese, the addition of bifidobacteria seemed to support the growth and survival of mesophilic lactobacilli and Streptococcus thermophilus, used as starter, during ripening. No significant differences were observed in the main chemical composition, and only a slightly higher concentration of acetic acid was found in cheeses with bifidobacteria added. On the contrary, alpha- and beta-galactosidase activities were markedly more pronounced in the presence of bifidobacteria, especially with B. bifidum Bb02. In contrast with traditional cheese, the lactose was completely hydrolyzed in cheeses made with bifidobacteria. Urea-PAGE electrophoresis of the pH 4.6-soluble and pH 4.6-insoluble N fractions did not show appreciable variations. Only the reversed-phase-HPLC analysis of the pH 4.6-soluble N showed a slightly more complex profile in the presence of bifidobacteria. This finding was in agreement with the higher value of the pH 4.6-soluble N/total N ratio and with the more pronounced amino-, imino-, and dipeptidase activities found in all the cheeses with the bifidobacteria added, especially B. bifidum Bb02. No differences were found in the free amino acid and free fatty acid contents. The amino acids glutamic acid, valine, proline, leucine, and lysine and the fatty acids butyric, caproic, capric, and oleic acids were found at the highest concentrations. The sensory evaluation did not show significant differences, and Canestrato Pugliese cheeses were characterized by small and uniformly distributed eyes, were pale yellow, had an elastic consistency and a Pecorino-like smell, were very salty, and tended to be moderately piquant.     

Effects of dietary protein level on ewe milk yield and nitrogen utilization, and on air quality under different ventilation rates

The experiment, which lasted 53 d, was conducted during the winter (February and March) of 2004 and used 48 Comisana ewes in mid lactation. A 2 x 2 factorial design was used, with ewes receiving two levels of dietary crude protein (CP) (moderate, 16% CP v. low, 13% CP) in the dry matter (DM) and being exposed to two ventilation rates (moderate, 47 m3/h v. low, 23.5 m3/h per ewe) for each dietary treatment. Air concentrations of NH3 and of microorganisms were measured twice weekly. Milk yield was recorded daily. Individual milk samples were analysed weekly for composition and fortnightly for bacteriological characteristics. After the last milk sampling (day 49 of the study period), four animals from each group were placed in a metabolism box and their individual faeces and urine were collected for three consecutive days. Amounts of urine and faeces excreted, and urinary and faecal N outputs were measured. The 16% CP diet resulted in a lower milk casein content and a higher milk urea concentration than the 13% CP diet, as well as in a reduced gross efficiency of utilization of dietary N, a greater amount of N excreted and a higher total coliform concentration in milk. The moderate ventilation rate resulted in higher yields of milk, irrespective of CP content. Significant interactions of CP level x ventilation rate were found for the amounts of urine, of total water and of faecal N, and for mesophilic concentration in milk, the highest values being displayed by the ewes fed the 16% CP diet and exposed to the low ventilation rate. The moderate dietary CP level and low ventilation rate had a deleterious effect on air concentrations of microorganisms and ammonia. Results suggested that a reduction of dietary CP level from 16 to 13% of DM had no detrimental effect on ewe milk yield in mid lactation and could even improve some of its nutritional and hygienic characteristics. Our findings also indicate that the choice of a proper ventilation rate is critical for high efficiency of production in the lactating ewe, especially in intensively managed flocks receiving diets high in CP.     

Triacylglycerol composition of protected designation of origin cheeses during ripening. Authenticity of milk fat

Triacylglycerol (TAG) composition by carbon number in 2 protected designation of origin cheeses, Mahón (cheese from cow milk) and Manchego (cheese from ewe milk) that were manufactured by 3 different producers was analyzed during cheese ripening using gas chromatography with a short capillary column. The TAG composition at different times during cheese ripening was also analyzed in cheeses from different batches produced at the same plant. Lipolysis levels in the Mahón and Manchego cheeses during ripening were low; free fatty acid values ranged from 2,500 to 4,000 ppm at the end of ripening. The TAG composition did not change significantly during ripening. The TAG values obtained from each cheese sample were substituted into the multiple regression equations that have been proposed to detect foreign fats in milk fat. The values obtained using the equations for bovine (proposed by the European Union) and ovine milk (proposed by our laboratory) were within the normal range. Accordingly, these equations can be considered useful for detecting foreign fat in these cheeses during the ripening period contemplated during this study.     

Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot whey

Canestrato Pugliese cheeses were produced from raw ewes' milk (R and R(II) cheeses), pasteurized ewes' milk (P cheese) and by heating the curd in hot whey according to a traditional protocol (T cheese). R(II) differed from R cheese mainly by having been produced from raw milk with a higher number of somatic cells, 950.000 vs. 750.000 ml(-1), respectively. Compared to P and T cheeses, R and R(II) cheeses had a higher concentration (one or two orders of magnitude) of cheese-related bacteria such as adventitious mesophilic lactobacilli, enterococci and staphylococci. At the end of ripening, all cheeses contained less than 1.0 log cfu g(-1) of total and fecal coliforms, and Escherichia coli and Staphylococcus aureus were not detected. As shown by phenotypic identification and RAPD-PCR, R cheese contained the largest number of mesophilic lactobacilli species and the greatest diversity of strains within the Lactobacillus plantarum species. Primary proteolysis did not differ appreciably among the cheeses. On the contrary, both urea-PAGE and the RP-HPLC analyses of the water-soluble N fractions showed the more complex profiles in cheeses produced by raw milks. R and R(II) cheeses had the highest values of water-soluble N/total N (ca. 30%) and the highest concentration of total free amino acids (ca. 40 mg g(-1) which approached or exceeded those reported for Italian cheeses with very high level of proteolysis during ripening. The main differences between R-R(II) and P-T cheeses were the concentrations of aspartic acid, proline, alanine, isoleucine, histidine and lysine. The water-soluble extracts of R and R(II) cheeses contained levels of amino-, imino- and di-peptidase activities, which were about twice those found in P and T cheeses. Cheeses differed slightly in the concentration of total free fatty acids that ranged between 1673 and 1651 mg kg(-1) in R and R(II) cheeses, and 1397 and 1334 mg kg(-1) in P and T cheeses. Butyric, caproic, capric, palmitic, oleic and linoleic acids were found at the highest concentrations.     

Ripening of Camembert-type cheese made from caprine milk using calf rennet or kid rennet as coagulant

Camembert-type cheese was made from caprine milk using either calf rennet or kid 'Grandine' rennet as coagulant. The pH of all cheeses increased throughout ripening and levels of pH 4.6-soluble nitrogen increased from 8.1 to 18.2% of total nitrogen (TN) and from 6.9 to 20% TN for the cheeses made using calf rennet and kid rennet, respectively. Degradation of β-casein, measured by urea–polyacrylamide gel electrophoresis, and total and free amino acids were greater in the cheese made using kid rennet. Production of peptides, analysed by high performance liquid chromatography (HPLC), was slightly more extensive in the Camembert-type cheese made using calf rennet as coagulant. In general, a higher degree of proteolysis was found in Camembert-type cheese made from caprine milk using kid rennet than in cheese made using calf rennet as coagulant.     

Proteolysis and microstructure of Piacentinu Ennese cheese made using different farm technologies

The aim of this study was to provide the biochemical and structural characterization of Piacentinu Ennese cheese and to evaluate the impact of different farm technologies on cheese proteolysis and microstructure. Fifteen cheeses were manufactured according to traditional technology, i.e., from raw milk and farmhouse rennet in the absence of starter culture. Pasteurized milk, commercial rennet, and starter were used for production of 20 nontraditional cheeses. Proteolysis in Piacentinu Ennese cheese was monitored during a 2- to 10-mo ripening time. Low rates of overall proteolysis were observed in cheese, as percentages of total N soluble at pH 4.6 and in 12% trichloroacetic acid were about 11.40 and 8.10%, respectively, after 10 mo of age. Patterns of primary proteolysis by urea-PAGE showed that alpha(s)-caseins were degraded to a larger extent than were beta-caseins, although a considerable amount of both caseins was still intact after 10 mo. Reversed phase-HPLC analysis of the cheese peptide fractions showed a slow decrease in the levels of hydrophobic peptides coupled to increasing levels of hydrophilic compounds as the cheese aged. The structural characteristics of Piacentinu Ennese cheese were evaluated by scanning electron microscopy after 2, 4, and 6 mo of age. The micrographs showed a sponge-like structural network with a well-distributed system of empty spaces, originally occupied by whey and fat. The microstructure changed during cheese ripening to become more compact with cavities of smaller size. Farm technology significantly affected cheese proteolysis and microstructure. Nontraditional cheeses had higher levels of pH 4.6-soluble N and showed a larger hydrolysis of alpha(s)-casein fractions by urea-PAGE analysis than did traditional cheeses. Large differences between cheese-types also concerned the patterns of secondary proteolysis. Nontraditional cheeses had higher levels of 12% trichloroacetic acid-soluble N and showed larger proportions of free amino acids and hydrophilic peptides in the HPLC profiles of the corresponding 70% ethanol-soluble N fraction than traditional cheeses. Nontraditional cheeses also had a more open structure with a coarser and less continuous appearance than did traditional cheeses. A large amount of variability in cheese proteolysis and structure within nontraditional treatment reflected farm-dependent changes in manufacturing conditions related to the use of various types of rennet and starter.     

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: α_S1-casokinin (e.g., sheep α_S1-casein (CN) f22-30 of Pecorino Romano and cow α_S1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep α_S1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow κ-CN f106-115 of Canestrato Pugliese and Crescenza); and β-casomorphin-11 (e.g., goat β-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 μg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.     

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: proteolysis during ripening

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.     

Proteolysis in ultra-filtered and conventional Iranian white cheese during ripening

Conventional and ultra-filtered (UF) Iranian white cheeses were made with almost identical gross chemical composition and the extent and characteristics of proteolysis were studied during ripening. UF cheeses exhibited a lower rate of development of pH 4.6-soluble nitrogen than conventional cheeses. The rates of degradation of α_s1-casein and particularly β-casein were lower in UF cheeses than in conventional cheeses. Plasmin activity was lower in UF cheeses than that in conventional cheese, whereas coagulant activity was higher in the former. Noticeable qualitative and quantitative differences were observed in reverse-phase high performance liquid chromatography (RP-HPLC) peptide profiles between UF and conventional white cheeses and chemometric analysis of peak height data distributed the cheeses into two separate groups. The levels of free amino acids in UF cheeses were lower than in conventional cheeses. Lower peptide degradation and production of amino acids suggested slower ripening, which may have been associated with the weak aroma development characteristic of UF cheeses.     

Effects of somatic cell count and stage of lactation on the plasmin activity and cheese-making properties of ewe milk

The experiment was conducted from March to July 2002 using 5 intensively managed flocks of Southern Italy. In each flock, 2 groups of 50 ewes were created. The groups were designated LSCC (low somatic cell count [SCC]) when their milk SCC was lower than 500,000/mL and HSCC (high SCC) when their milk SCC was higher than 1,000,000/mL. Bulk milk and whey samples were analyzed for fat, total protein, lactose, casein, and whey protein contents. Renneting properties of milk were also determined. Moisture, NaCl, and nitrogen fractions were determined in fresh cheese curds. In addition, plasmin (PL) and plasminogen (PG) activities in milk and cheese were monitored. The proteolytic activity of plasmin by urea-polyacrylamide gel electrophoresis and the white blood cell (WBC) differentials were determined. The HSCC resulted in higher pH values in milk and in higher moisture and lower fat contents in fresh cheese curds. Moreover, a lower recovery of fat and whey proteins was obtained from the HSCC than from the LSCC raw milk. The crude protein and casein contents were higher in the HSCC than in the LSCC curds during early and midlactation; an opposite trend was observed in late lactation. Plasmin and PG activities underwent more marked fluctuations in the LSCC than in the HSCC curds through lactation. The results of this experiment demonstrate that the PL activity in ewe milk is markedly influenced by the SCC, although SCC is not the only parameter for predicting PL and PG evolution in ewe milk. The LSCC milk resulted in a higher proteolytic potential of Canestrato pugliese cheese curds.     

Proteomic study of proteolysis during ripening of Cheddar cheese made from milk over a lactation cycle

Milk for cheese production in Ireland is predominantly produced by pasture-fed spring-calving herds. Consequently, there are marked seasonal changes in milk composition, which arise from the interactive lactational, dietary and environmental factors. In this study, Cheddar cheese was manufactured on a laboratory scale from milk taken from a spring calving herd, over a 9-month lactation cycle between early April and early December. Plasmin activity of 6-months-old Cheddar cheese samples generally decreased over ripening time. One-dimensional urea-polyacrylamide gel electrophoresis (PAGE) of cheese samples taken after 6 months of ripening showed an extensive hydrolysis of caseins, with the fastest hydrolysis of α(s1)-caseins in cheeses made in August. A proteomic comparison between cheeses produced from milk taken in April, August and December showed a reduction in levels of β-casein and appearance of additional products, corresponding to low molecular weight hydrolysis products of the caseins. This study has demonstrated that a seasonal milk supply causes compositional differences in Cheddar cheese, and that proteomic tools are helpful in understanding the impact of those differences.     

Effect of high-pressure treatment of ewe raw milk curd at 200 and 300 MPa on characteristics of Hispánico cheese

Hispánico cheese is a semihard variety made from a mixture of cow and ewe milks. Production of ewe milk declines in summer and autumn. To surmount the seasonal shortage of ewe milk and prevent the inactivation of milk enzymes by pasteurization, curd made in spring from ewe raw milk was pressurized at 200 and 300 MPa and stored frozen for 4 mo. Thawed ewe milk curds were added to fresh curd made from pasteurized cow milk for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same proportions as those used for experimental cheeses. Experimental cheeses exhibited lower dry matter content, higher aminopeptidase activity and total free amino acid concentration, and higher levels of acetic and propionic acids, aldehydes, alcohols, and esters compared with control cheese. In contrast, the concentration of total free fatty acids and ketones and the levels of textural parameters were significantly higher in control cheese. The use of ewe raw milk curd pressurized at 200 and 300 MPa, stored frozen and thawed for Hispánico cheese manufacture, was generally beneficial for cheese characteristics and increased cheese yield because of the lower dry matter content of experimental cheeses.     

Microbiological changes throughout ripening of goat cheese made from raw,pasteurized and high-pressure-treated milk

The bacteriological quality during ripening of raw (RA), pasteurized (PA; 72°C, 15 s) and pressure-treated (PR; 500 MPa, 20°C, 15 min) goat milk assessed by enumeration of total bacteria, psychrotrophic bacteria, Enterobacteriaceae, lactobacilli, enterococci, Micrococcaceae and lactococci was evaluated. The high pressure treatment applied was as efficient as pasteurization in reducing the bacterial population of milk. Experimental cheeses were made from RA, PA and PR milks to study the microbial population during ripening. Lactobacilli and lactococci were the predominant microbiota present during ripening in all the cheeses. There were no differences in numbers of starter bacteria during ripening. However, lactobacilli counts for RA milk cheese were significantly higher than for PA and PR cheeses in all the ripening stages studied. Micrococcaceae and enterococci remained at a secondary level, and no differences were observed between cheeses at the end of ripening. On the other hand, the number of Enterobacteriaceae decreased during ripening, but faster in PR milk cheese than in PA and RA milk cheeses. The results of this study suggest that goat cheese made from PR milk had similar microbiological characteristics to PA milk cheeses.     

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style)

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log₁₀ cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log₁₀ cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log₁₀ cfu/g after whey drainage and during ripening and storage decreased by 2.9 log₁₀ cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.     

Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese

Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 μmol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8°C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 μmol/L of pepstatin showed complete inhibition of hydrolysis of α_S1-casein (CN) at Phe₂₃-Phe₂₄ at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact α_S1-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of α_S1-CN at Phe₂₃-Phe₂₄ is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.     

Interlaboratory validation of two multiplex quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk proportions in cheese

Milk products like yogurt, flavoured milk-drinks, curd and cheese may be composed of milk different from cow, namely of ruminant species like sheep and goat. Such products experience an increasing demand in Europe and are recognised as healthy and naturally finished specialities. To verify declared milk compositions in these dairy products, two different quantitative multiplex PCR systems have been evaluated in a comparison test with eleven participating laboratories employing two unknown, traditionally manufactured cheeses with different degrees of ripening to determine milk fractions from cow, ewe and goat. Precision and accuracy was investigated by calibration to dilutions of DNA mixtures and to homologous matrix-adapted reference cheeses, respectively. As expected, independent of the particular method, best inter- and intra-laboratory accuracy has been achieved through the use of homologous reference cheese standards. Furthermore, it has been shown that cheese ripening and the concomitant DNA degradation exert an inverse effect on the method’s sensitivity and performance characteristics. Additionally, a broad market survey of different milk products demonstrated its applicability as an efficient analytical tool for food control laboratories to challenge the authenticity of milk and its products from small ruminants.