Influence of pH on retention of camel chymosin in curd

Retained coagulant in cheese initiates casein breakdown and influences cheese structure and flavour formation. This study investigated the influence of milk pH on retention of camel chymosin in curd and compared it with bovine chymosin. Milk at five different pH levels was coagulated with same coagulant activity of each chymosin and centrifuged. Chymosin activity in whey was determined using the synthetic peptide Pro-Thr-Glu-Phe-(NO₂-Phe)-Arg-Leu as substrate and HPLC analysis of the resulting product. Camel chymosin had 2.7 times lower activity in milk than bovine chymosin at the same coagulation activity. The retention of camel chymosin in curd was rather constant at ∼20% between pH 6.65 and 6.00, while it increased almost linear from 2 to 21% for bovine chymosin. The lower pH dependence for retention of camel chymosin than of bovine chymosin may be explained by a lower negative charge of the camel chymosin molecule.     

Yak milk whey protein denaturation and casein micelle disaggregation/aggregation at different pH and temperature

At the natural pH of yak milk (pH 6.6), a low level (<30%) of κ-casein (κ-CN) was found in the serum phase after heating at 95 °C for 30 min, indicating that as much as 70% of the β-lactoglobulin (β-Lg) and κ-CN complexes is associated with the micelle colloidal particles. The β-Lg and κ-CN levels increased from 13.2% and 2.6% at pH 6.0 to 35.2% and 60.1% at pH 7.0, respectively, when yak milk was heated at 95 °C for 30 min. At pH 6.0–6.4, the denatured whey proteins were associated with the caseins in the colloidal phase, resulting in milk gelation upon heating. The distribution of β-Lg and κ-CN complexes increased in the serum phase, demonstrated by the increasing levels of both β-Lg and κ-CN with increasing pH; at high pH (6.6–7.0), large proportions of β-Lg and α-lactalbumin were lost, presumably forming complexes in the colloidal phase.     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

Structure and shelf stability of milk protein gels created by pressure-assisted enzymatic gelation

In this work, pressure-assisted enzymatic gelation was applied to milk proteins, with the goal of enhancing the structure and stability of pressure-created milk protein gels. High-pressure processing (HPP) at 600 MPa, 3 min, and 5°C was applied to milk protein concentrate (MPC) samples of 12.5% protein concentration, both in the absence and in the presence of calf chymosin [up to 60 IMCU (international milk-clotting units)/kg of milk] or camel chymosin (up to 45 IMCU/kg of milk). Gel hardness, water-holding capacity, and degree of proteolysis were used to assess network strength and shelf stability. The processing trials and all measurements were conducted in triplicate. Statistical analyses of the data were performed by ANOVA, at a 95% confidence level. After HPP treatment, we observed significant structural changes for all samples. Pressurization of MPC, with or without chymosin addition, led to extensive protein aggregation and network formation. The strength of HPP-created milk protein gels without chymosin addition, as measured by the elastic modulus (G′), had a value of 2,242 Pa. The value of G′ increased with increasing chymosin concentration, reaching as high as 4,800 Pa for samples with 45 IMCU/kg of camel chymosin. During 4 wk of refrigerated storage, the HPP and chymosin MPC gels maintained higher gel hardness and better structural stability compared with HPP only (no chymosin) MPC gels. The water-holding capacity of the gels without chymosin remained at 100% during 28 d of refrigerated storage. The HPP and chymosin MPC gels had a lower water-holding capacity (91–94%) than the HPP-only counterparts, but their water-holding capacity did not decrease during storage. Overall, these findings demonstrate that controlled, fast structural modification of high-concentration protein systems can be obtained by HPP-assisted enzymatic treatment, and the created gels have a strong, stable network. This study provides insights into the possibility of using HPP for the development of milk-protein-based products with novel structures and textures and long refrigerated shelf life, along with the built-in safety imparted by the HPP treatment.     

Release of angiotensin converting enzyme-inhibitory peptides during in vitro gastro-intestinal digestion of camel milk

Angiotensin-converting enzyme (ACE)-inhibitory peptides released from camel milk after simulated gastro-intestinal digestion were identified. The hydrolysis degree increased during digestion. The highest ACE-inhibitory activity was found in the post-pancreatic <3 kDa fraction. Peptides responsible for the biological activity were isolated by reversed-phase high-performance liquid chromatography and identified by mass spectrometry. Among the identified sequences, 17 were identical to known bioactive peptides with ACE-inhibitory activity. Based on previous structure–activity relationship studies, the sequence of some peptides allowed us to anticipate the presence of biological activities. The anti-hypertensive tripeptide isoleucine-proline-proline (IPP) was identified and quantified in digested camel milk. The amount of released IPP was 2.56 ± 0.15 mg L⁻¹ of milk. For the first time, we showed that IPP is released during the gastro-intestinal digestion of camel milk κ-casein. This research provides the basis to increase the exploitation of the health benefits of camel milk.     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and β-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.     

The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological quality

The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentrations ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly regarding their sensory properties and had an acceptable microbiological quality. This study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal.     

Cholesterol reduction in camel hump fat using β-cyclodextrin

β-Cyclodextrin (β-CD) was used for removing cholesterol from fatty tissues such as lard, milk fat. This investigation was carried out to study optimum conditions of different factors (β-CD concentration, stirring temperature, stirring time, stirring rate and ratio of hump camel fat to water for reduction of cholesterol in hump camel fat by application of β-CD and resulting changes in cholesterol reducing camel hump fat). β-CD concentration at 1–10% provided about 88.89–95.3% removal of cholesterol when mixed at 40°C for 91 h. Among other factors, mixing time (0.5–2 h) did not significantly affect cholesterol reduction. Removal was enhanced with increasing stirring rate to 800 rpm and centrifugal forces up to 5,000×g (95.5%) but decreased thereafter. The obtained results showed that the optimum conditions for the process were addition of 7% β-CD, 40°C mixing temperature, 1 h of mixing time, the ratio of camel hump fat to water 1:1, and stirring rate 800 rpm and centrifugation at 5,000×g. The acid value of β-CD treated camel hump fat was significantly decreased due to removal of free fatty acid. The peroxide value of β-CD treated camel hump fat was slightly increased. No significant differences were noted in the fatty acids composition between treated with β-CD and untreated camel hump fat.     

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks β-lactoglobulin and consists of high concentration of α-lactalbumin (2.01 ± 0.02 mg mL⁻¹), lactoferrin (1.74 ± 0.06 mg mL⁻¹) and serum albumin (0.46 ± 0.01 mg mL⁻¹). Among caseins, the concentration of β-casein (12.78 ± 0.92 mg mL⁻¹) was found the highest followed by α-casein (2.89 ± 0.29 mg mL⁻¹) while κ-casein represented only minor amount (1.67 ± 0.01 mg mL⁻¹). These results were in agreement with sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.     

Synthesis of extracellular lipase by a strain ofPseudomonas fluorescensisolated from raw camel milk

Ten strains of psychrotrophic bacteria were isolated from raw camel milk and were arranged according to their lipase production. Lipolytic activities ranged between 0.26 to 3.43 meq of palmetic acid 100 g^-;1of bovine milk fat h^-;1.Pseudomonas fluorescensRM₄was the most active strain. This bacterium could grow and secrete lipase over a wide range of temperatures. The optimum temperature for growth was 37°C, and the maximum lipase production was at 25°C. Growth was maximal after 96 h of incubation at 25°C, and lipase activity was maximal at 72 h post-inoculation at 25°C (during the late logarithmic phase). Shaking of the cultures (100 rpm) led to an increase in both growth and enzyme activities. Pseudomonas fluorescensRM₄was able to grow and secrete lipase over a pH range of 5.5-;8.50. Synthesis of the enzyme appeared to be inducible because no enzyme was detected in the absence of organic nitrogen. Supplementation of the basal medium with milk proteins enhanced lipase production. Tryptophan and lysine induced enzyme synthesis most effectively. Addition of 4 g l^-;1of glucose to broth stimulated both growth and production of lipase. Beyond this level of supplementation, lipase activity was considerably depressed.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Influence of NaCl and CaCl2 on Cold-Set Gelation of Heat-denatured Whey Protein

Heat‐denatured whey‐protein isolate (HD‐WPI) solutions were prepared by heating a 10 wt% WPI solution (pH 7) to 80 °C for 10 min and then cooling it back to 30 °C. Cold‐set gelation was initiated by adding either NaCl (0 to 400 mM) or CaCl₂ (0 to 15 mM). Both salts increased the turbidity and rigidity of the HD‐WPI solutions. Gelation rate and final gel strength increased with salt concentration and were greater for CaCl₂ than NaCl at the same concentration because the former is more effective at screening electrostatic interactions and can form salt bridges.     

Partial renneting of pasteurised bovine milk: Casein micelle size,heat and storage stability

The effects of partial renneting at low temperature on the casein micelle (CM) size and the storage stability of milk were investigated. Low chymosin concentrations (≤ 0.03 IMCU mL^− 1) was applied to pasteurised skim milk at 4 °C and enzyme activity was terminated by thermal application at 60 °C/3 min and 85 °C/30 min, referred to as low heat (LHT) and high heat (HHT) treatment milk, respectively. The addition of rennet with concentrations of 0.01, 0.02 and 0.03 IMCU mL^− 1 for 15 min resulted in κ-casein hydrolysis of 10, 20 and 25%, respectively. Moreover, mean CM size of milk was reduced by up to 10 nm. For LHT milk, the renneted micelles appeared to be stable for up to 17 days, especially in response to the application of 0.01 IMCU mL^− 1 and at a storage temperature of 4 °C. Severe heating at 85 °C/30 min to inactivate the enzyme caused an increase in CM size.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Manufacture of dry‐ and brine‐salted soft camel cheeses for the camel dairy industry

Two experiments were conducted in a camel cheese study to (i) compare camel cheese to bovine cheese made from bovine milk standardised to simulate camel milk, and (ii) describe the technology for manufacture of dry (SCC‐D) and brine‐salted soft camel cheese (SCC‐B). Comparable cheese yield (camel: 7.4 ± 0.15, cow: 7.3 ± 0.55 kg/100 kg of milk) and levels of dry matter loss in whey were observed. Clotting time was 234 s for both cheeses which were made using thermophillic starters. Cheese yield was 9.31 ± 0.64 kg/100 kg with 425.6 ± 38.2 g/kg cheese dry matter for SCC‐D and 8.22 ± 0.90 kg/100 kg with 469 ± 73.8 g/kg dry matter for SCC‐B.     

Proteomic study on the stability of proteins in bovine,camel, and caprine milk sera after processing

Milk proteins have been shown to be very sensitive to processing. This study aims to investigate the changes of the bovine, camel, and caprine milk proteins after freezing, pasteurization (62 °C, 30 min), and spray drying by proteomic techniques, filter-aided sample preparation (FASP) and dimethyl labeling followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 129, 125, and 74 proteins were quantified in bovine, camel, and caprine milk sera, respectively. The milk serum protein content decreased significantly after freezing, pasteurization, or spray drying, which can be ascribed to the removal of protein aggregates by the pH adjustment and ultracentrifugation during sample preparation. Some of the immune-related proteins were heat-sensitive, such as lactoferrin (LTF), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactadherin (MFGE8), with losses of approximately 25% to 85% after pasteurization and 85% to 95% after spray drying. α-Lactalbumin (LALBA), osteopontin (SPP1), and whey acidic protein (WAP) were relatively heat stable with losses of 10% to 50% after pasteurization and 25% to 85% after spray drying. The increase of some individual proteins in concentration after freezing may be caused by the proteins originating from damaged milk fat globules and somatic cells. GLYCAM1 decreased significantly after pasteurization in bovine and camel milk but this protein is relatively stable in caprine milk. In conclusion, milk proteins changed differently in concentration after different processing steps, as well as among different species.     

Purification and characterisation of milk‐clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa

Ovine pepsin was isolated and assessed for its milk‐clotting properties and caseinolytic activity in comparison with commercial chymosin. Ovine pepsin showed similar responses to variations in pH, temperature and CaCl₂ concentration of milk compared with chymosin, although its pH sensitivity was higher. SDS‐PAGE electrophoretic analysis of the casein fractions treated with ovine pepsin showed that alpha‐casein was more susceptible to proteolysis than beta‐casein, in contrast to chymosin. Curd‐firming properties of skim milk gels obtained with ovine pepsin and chymosin were evaluated by Gelograph under the same conditions. Curd produced using ovine pepsin was less firm than that made with chymosin.     

Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins

Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7–5.3) and pepsin concentration (0.110–2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ～6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ～73% to ～33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.     

Proteolysis and storage stability of UHT milk produced in Turkey

The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 °C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 °C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 °C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 °C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 °C showed quite small number of peaks after 180 d of storage. Sterilization at 150 °C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness.