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.     

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.     

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.     

Milk nutrition and childhood epilepsy: An ex vivo study on cytokines and oxidative stress in response to milk protein fractions

We present a pilot study on the effects of milk protein fractions [α_S1-casein (CN), α_S2-CN, κ-CN, β-CN, and a mix of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG)] from different animal species (bovine, ovine, and caprine) on pro- and anti-inflammatory cytokines and oxidative status in cultured peripheral blood mononuclear cells from children with generalized epilepsy. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from blood of 10 children with generalized epilepsy (5 males; mean age 33.6 ± 5.4 mo) and 10 controls (5 males; mean age 35.6 ± 6.8 mo). Children with epilepsy were grouped according to cytokine levels as follows: children with epilepsy having low levels of cytokines not different from those of control children (LL-EC); children with epilepsy having cytokine levels at least 5-fold higher (medium levels) than those of control children (ML-EC); and children with epilepsy having cytokine levels at least 10-fold higher (high levels) than those of control children (HL-EC). The production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC incubated with α_S1-CN, α_S2-CN, κ-CN, β-CN, and a mix of α-LA and β-LG from bovine, caprine, and ovine milks. The levels of reactive oxygen and nitrogen species (ROS/RNS) and catalase activity were assessed in cultured supernatant. In the HL-EC group, β-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with α_S2-CN from ovine milk and the mix of β-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1β was higher for bovine and ovine α_S2-CN fractions and lower for caprine and ovine β-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with α_S2-CN from bovine and ovine milk than from caprine milk. The cytokine IL-10 did not differ among milking species. The highest levels of ROS/RNS were found after incubation of PBMC with the β-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with β-CN isolated from bovine and caprine milk and with α_S1-CN from ovine milk.     

Proteolysis in reduced sodium Feta cheese made by partial substitution of NaCl by KCl

Feta cheeses (five trials) of different sodium content were made, using ewes’ milk, from split lots of curd by varying the salting procedure, i.e. dry salting with NaCl (control) or mixtures of NaCl/KCl (3:1 or 1:1, w/w basis) and filling the cans with brine made with NaCl or the above NaCl/KCl mixtures, respectively, in order to study the influence of the partial substitution of NaCl by KCl on the proteolysis during cheese ripening. The extent and characteristics of proteolysis in the cheeses were monitored during aging by using Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, phosphotungstic acid-soluble nitrogen), the cadmium–ninhydrin method for the determination of total free amino acids (FAA), urea–polyacrylamide gel electrophoresis of cheese proteins followed by densitometric analysis of the α_s1- and β-casein fractions, reverse-phase HPLC analysis of the water-soluble extracts of cheeses, and ion-exchange HPLC analysis of FAA. The results showed that proteolysis was similar in control and experimental cheeses at all sampling ages, indicating that the partial substitution of NaCl by KCl in the manufacture of Feta cheese had no significant effect on the extent and characteristics of proteolysis during cheese aging.     

Robust PCR‐based method for quantification of bovine milk in cheeses made from caprine and ovine milk

To prevent fraud and enhance quality assurance, credible analysis of dairy products is crucial. A common problem is the addition of cheaper bovine milk to caprine and/or ovine dairy products and when not declared addition of bovine milk constitutes fraud. The aim was to develop a rapid, robust and sensitive method for the identification of adulteration of caprine and/or ovine cheeses with bovine milk. New quantitative real‐time polymerase (qPCR) assays were designed for the specific determination of bovine DNA (Cow1) and bovine, caprine and ovine DNA (BoCaOv). These were applied to 17 samples of caprine cheese and 24 of ovine cheese. Results showed that 17% (7/41) of these cheeses contained >5% bovine milk. As bovine milk was not declared as an ingredient in any of the samples, this represents adulteration. Other cheeses that contained detectable bovine milk at ≤5% (22%; 5/41) might pose a health risk to people allergic to bovine milk.     

Changes during ripening in chemical composition,proteolysis, volatile composition and texture in Kashar cheese made using raw bovine,ovine or caprine milk

Kashar cheeses were manufactured from pure ovine (OV), bovine (BV) and caprine (CP) milk, and the chemical composition, cheese yield, proteolysis, hardness, meltability and volatile composition were studied during 90 days. Gross chemical composition, cheese yield and level of proteolysis were higher in OV cheeses than those of BV or CP cheeses. Glu, Val, Leu, Phe and Lys were the most abundant free amino acids (FAA) in the samples, and the concentrations of individual FAA were at the highest levels in OV cheeses with following BV and CP cheeses. Urea‐PAGE patterns and RP‐HPLC peptide profiles of the BV cheeses were completely different from the small ruminants’ milk cheeses (OV or CP). Higher and lower hardness and meltability values were observed in CP cheeses, respectively. OV cheeses resulted in higher levels of the major volatile compounds. In conclusion, the Kashar cheese made using OV milk can be recommended due to high meltability, proteolysis and volatiles.     

Microbiological,chemical, textural and sensory characteristics of Hispánico cheese manufactured using frozen ovine milk curds scalded at different temperatures

Hispánico cheese is a semi-hard variety, manufactured in Spain from a mixture of pasteurized bovine and ovine milk. To study one strategy for overcoming a seasonal shortage of ovine milk in summer and autumn, curds made from ovine milk, scalded at 32, 35 or 38 °C, were pressed for 30 min and frozen at ?24 °C for 4 months. After thawing, they were added to fresh bovine milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized bovine and ovine milk in the same (80:20) proportion. No significant effect of the addition of frozen ovine milk curd or scalding temperature was found for lactic acid bacteria counts, dry matter content, hydrophilic and hydrophobic peptides, 45 out of 65 volatile compounds, texture, and sensory characteristics throughout a 60-day ripening period. Differences between cheeses, of low magnitude and little practical significance, were found for pH value, aminopeptidase activity, proteolysis, free amino acids, free fatty acids, and the remaining 20 volatile compounds. Thus, the addition of frozen ovine milk curd to fresh bovine milk curd does not alter the main physicochemical and sensory characteristics of Hispánico cheese.     

Effect of partial or total substitution of bovine for caprine milk on the compositional,volatile, non-volatile and sensory characteristics of semi-hard cheeses

Semi-hard cheeses were manufactured from caprine milk and bovine milk mixed in ratios of 100:0; 75:25; 50:50; 25:75; 0:100 (caprine:bovine) and ripened at 12 °C for 150 d. Substitution of up to 75% bovine for caprine milks had no significant effect on levels of cheese protein, moisture-in-non-fat substances, salt, pH, whiteness, total or individual free amino acids and substitution of up to 50% had no significant effect on levels of primary proteolysis and free fatty acid and fruity flavours. Increased bovine milk content resulted in decreased levels of butanoic to octanoic acids, ethyl and methyl esters of acetic to octanoic acids and acyclic, mono and bicyclic terpenes and waxy/goaty flavours to a degree dependent on the proportion of each milk type. Partial substitution of caprine with bovine milks resulted in cheeses with chemical and sensorial characteristics that may provide an available alternative to consumers desiring caprine cheeses.     

Some properties of urfa cheese (a traditional white-brined Turkish cheese) produced from bovine and ovine milks

In this study, the basic composition and ripening profile of traditional urfa cheese made from ovine and bovine milks were investigated. While cheese made from ovine milk had higher total solids, fat-in-dry matter and total nitrogen, the titratable acidity, salt-in-dry matter, pH, total mesophilic colony count and total yeasts and moulds counts were found to be close to each other. During storage, whilst the total solids content of cheese produced from ovine milk gradually decreased, the variation in the total solids content of cheese made from bovine milk was found to be insignificant. The salt penetration into the cheeses was rapid during the first two weeks of ripening, and it continued to diffuse into the samples throughout storage. Proteolysis developed faster in the cheese made from ovine milk than in cheese of bovine milk. The former sample had higher water soluble nitrogen, nonprotein nitrogen, phosphotungustic acid soluble nitrogen, Proteose-peptone nitrogen and tyrosine levels throughout storage, and the ripening index was higher as well.     

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.     

EVALUATION OF pH CHANGE KINETICS DURING DIFFERENT STAGES OF KASHAR CHEESE PRODUCTION FROM BOVINE, OVINE AND CAPRINE MILK

The objective of this study was to determine the pH change kinetics during Kashar cheese production from bovine, ovine and caprine milk. Kinetics of pH change were determined during milk ripening, cooking/holding and pressing/fermentation phases of Kashar cheese. The pH decreased logarithmically, nonlinearly, with time in the milk ripening period and was reduced linearly with time in the cooking/holding and pressing/fermentation stages. The time of pH decrease in cow's, ewe's and goat's milk was significantly different, except cooking/holding time, for three stages. There was no difference between bovine and caprine milk in cooking/holding time; however, ovine milk was significantly different from the other two kinds of milk. The shortest and the longest overall times were determined for caprine and ovine milk cheeses, respectively. The rate of change in milk pH of the three species was significantly different for three stages. The rate followed the pattern caprine  >  bovine  >  ovine milk.

PRACTICAL APPLICATIONS

Optimum acid development is essential to creating the desired cheese mass. For each major cheese type, lactic acid must develop in the appropriate time, usually not too rapidly or too slowly, and in a specific concentration. The progress of acidification is monitored by pH change in the industrial Kashar cheese production. The final pH of cheese is determined not only by the amount of lactic acid but also by the buffering capacity (BC) of the milk and curd. However, the BC is different between milk species. This study evaluates and compares the pH change kinetics during various stages of Kashar cheese making using cow's, ewe's and goat's milk. This work may help to compare milk and cheese curds made from bovine, ovine and caprine milk.     

Effects of milk high pressure homogenization on biogenic amine accumulation during ripening of ovine and bovine Italian cheeses

The aim of this work was to evaluate the biogenic amine (BA) content during the ripening of both bovine and ovine cheeses obtained using milk subjected to a homogenization treatment at 100 MPa before cheese-making. The data obtained were compared with those from cheeses produced by the same milks without any treatment or thermized. The results showed that both microbial ecology and BA concentrations of cheeses during ripening were significantly influenced by the type of milk used for cheese-making and by the treatment applied to the raw materials. In particular, the microbial counts found in Caciotta indicated that the high pressure homogenization (HPH) of milk significantly reduced the presence of the yeasts, Micrococcaceae and lactobacilli at the end of ripening. On the other hand, the HPH treatment of milk favoured the proliferation of yeasts in ovine cheese. Moreover, the ovine cheeses were characterized by a remarkably higher accumulation of BA than bovine cheeses. However, the HPH treatment of milk was able to drastically reduce the biogenic amine concentrations in both cheese typologies at the end of ripening.     

Effects of genetic type,stage of lactation,and ripening time on Caciocavallo cheese proteolysis

The objective of this experiment was to evaluate the effects of genetic type, stage of lactation, and ripening time on proteolysis in Caciocavallo cheese. One hundred twenty Caciocavallo cheeses made from the milk of 2 breeds, Italian Brown and Italian Holstein and characterized by different stages of lactation were obtained and ripened for 1, 30, 60, 90, and 150 d. Cheese proteolysis was investigated by ripening index (ratio of water-soluble N at pH 4.6 to total protein, %) and by the study of degradation of the protein fractions (α_S1-, β-, and para-κ-casein), which was determined by densitometric analysis of isoelectric focusing results. The statistical analysis showed a significant effect of the studied factors. Ripening index was higher in Italian Brown Caciocavallo cheese and in cheeses made with early lactation milk, whereas casein solubilization was greater in the first 2 mo of ripening. Isoelectric focusing analysis of cheese samples during ripening showed extensive hydrolysis of caseins. In particular, the protein fraction that underwent major degradation by proteolytic enzymes was α_S1-casein, followed by β-casein, whereas para-κ-casein was less degraded. Italian Brown cheese showed a lower residual quantity of β- and para-κ-casein, whereas Italian Holstein cheese showed a lower residual quantity of α_S1-casein. In addition, significant interactions of both first and second order were found on both ripening index and degradation of protein fractions. This study demonstrated that the analyzed factors influenced proteolysis of Caciocavallo cheese, which forms the basis of new knowledge that could lead to the production of a pasta filata cheese with specific characteristics.     

Effect of partial substitution of caprine milk for ovine milk,dry salting and cured scalding on lipolysis in Urfa cheeses

The objectives of this study were to determine the effect of partial substitution of caprine for ovine milk, dry salting and curd scalding on the free fatty acid (FFA) level of Urfa cheeses. At the end of storage, lauric and linoleic acids were lower in cheese made from milk where up to the 30% partial substitution of caprine milk for ovine milk was made. Dry salted cheeses had higher butyric, lauric, linoleic and linolenic acid levels, and lower palmitic acid levels than their scalded counterpart. A relative increase in short‐chain FFAs occurred during ripening. Palmitic and oleic acids were the most abundant FFAs in fresh and ripened Urfa cheeses.     

How milk type,coagulant, salting procedure and ripening time affect the profile of free amino acids in Picante da Beira Baixa cheese

The concentration of total free amino acids (FAA) in Picante cheese increased with ripening time irrespective of the particular protocol used for manufacture (ie ratio of caprine to ovine milks, animal or plant rennet and number of salting steps). The experimental cheeses manufactured with 20% (v/v) caprine milk, coagulated with animal rennet and salted only once exhibited the highest content of total FAA by 120 days of ripening. All four manufacture parameters were statistically significant on the 0.5% level of significance in terms of total concentration of FAA. The dominating free amino acids present in the various experimental cheeses throughout the ripening period were valine, leucine and phenylalanine, each one representing more than 10% (w/w) of the total concentration of FAA. All four manufacture parameters were, in general, statistically significant with respect to the content of every single FAA, with particular emphasis on salting and ripening time. © 1999 Society of Chemical Industry     

The effect of ovine and bovine milk on the textural properties of Lighvan cheese during ripening

The effect of milk origin on the physicochemical characteristics, microstructure and texture of Lighvan cheese was investigated over a 90‐day ripening period. Besides fat, other physicochemical properties of Lighvan cheese were affected by milk type. The moisture content of Lighvan cheese decreased when half or all the ovine milk was substituted with bovine milk. The Lighvan cheese's microstructural properties and porosity were affected by type of milk and ripening time. Compaction of cheeses manufactured from ovine and mixed ovine and bovine milk is similar, and more than that of bovine Lighvan cheese. Ovine Lighvan cheese is harder and less brittle than bovine and mixed bovine and ovine.     

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.     

SDS-PAGE of Proteins in Goat Milk Cheeses Ripened under Different Conditions

Proteolytic characteristics of five varieties of commercial goat milk cheeses and a cow milk Cheddar aged under different conditions were evaluated by SDS-PAGE and an advanced densitometry system. All fresh goat cheeses had distinctively lower intensities of α_S1-casein (CN) bands than those of cow milk Cheddar, whereas intensities of β-CN were much greater in the goat cheeses. The PAGE patterns clearly displayed α_S2-CN in all goat cheese, but it was negligible in cow milk Cheddar. The greater protein degradation in hard goat cheeses than cow milk Cheddar at 4°C and 22°C strongly correlated with water-soluble nitrogen compound concentrations and densitometric values of corresponding cheeses.