Effect of blends of camel chymosin and microbial rennet (Rhizomucor miehei) on chemical composition, proteolysis and residual coagulant activity in Iranian Ultrafiltered White cheese.

Iranian Ultrafiltered White cheese was produced by using different blends of coagulants (100:0, 75:25, 50:50, 25:75 and 0:100; Rhizomucor miehei and camel chymosin, respectively) and ripened for 90 days. The effect of different combinations of these coagulants on chemical composition, proteolysis and residual coagulant activity of the cheeses were studied. The results showed that pH, fat-in-dry matter, salt-in-dry matter and protein contents of the cheeses were significantly influenced by type and concentration of the coagulants. The difference between proteolytic activities of the two coagulants resulted in different levels of proteolysis in the cheeses. A direct relationship was determined between using higher concentrations of R. miehei and increasing the hydrolysis of αs1-casein in the cheeses, during ripening. The residual coagulant activity was influenced by the type and concentration of the coagulant as well. In conclusion, R. miehei provided a higher level of proteolysis and residual coagulant activity compared with camel chymosin.

Investigating the properties of high-pressure-treated, reduced-sodium, low-moisture, part-skim Mozzarella cheese during refrigerated storage.

We proposed that the performance and sensory properties of reduced-Na, low-moisture, part-skim (LMPS) Mozzarella cheese could be extended by the application of high hydrostatic pressure (HHP) to cheese postmanufacture and thereby decrease microbial and enzymatic activity. Fermentation-produced camel chymosin was also used as a coagulant to help reduce proteolysis during storage. Average composition of the LMPS Mozzarella cheeses was 48.6 ± 0.6% moisture, 22.5 ± 0.4% fat, 24.5 ± 0.6% protein, and 1.0 ± 0.1% NaCl. Blocks of cheeses were divided into 3 groups randomly after manufacture and stored at approximately 4°C for 20 wk. The control group was not HHP treated. Two weeks after manufacture, 2 groups of cheese samples were treated with HHP at 500 or 600 MPa for 3 min and then returned to storage at approximately 4°C. Analysis was performed during 20 wk of storage after cheese manufacture. Texture profile analysis (TPA) and dynamic low-amplitude oscillatory rheology were used to monitor cheese functionality. Quantitative descriptive analysis was conducted with 9 trained panelists using a 15-point scale to evaluate texture and flavor attributes of unmelted cheese as well as cheeses melted on pizzas. Pressure treatments at 500 and 600 MPa resulted in approximately 1 and 2 log reduction in the numbers of starter culture, respectively, compared with the control when measured 1 d after HHP treatment. Starter numbers continued to decrease in all cheeses over the 20 wk of storage, but the decrease was larger in the HHP-treated cheeses. Even though the initial numbers of nonstarter lactic acid bacteria were the same in all cheeses, the numbers of these bacteria increased faster in the control cheeses. High-pressure treatment of LMPS Mozzarella cheese resulted in an initial (1 d after HHP treatment) increase in pH, but by 2 wk after HHP treatment there was no statistical difference in pH values between control and HHP-treated samples. Immediately after treatment, HHP-treated cheeses exhibited significantly lower TPA and sensory (unmelted) hardness. However, by 14 wk after pressure treatment, the 600-MPa HHP-treated cheese had significantly higher TPA compared with control or 500-MPa HHP-treated cheeses. Sensory panels also indicated that by 14 wk after HHP treatment, the 600-MPa treated samples were significantly firmer than the control or 500-MPa treated cheeses. Compared with control cheese, cheeses treated at 600 or 500 MPa exhibited lower water-soluble nitrogen values at 6 and 10 wk after pressure treatment, respectively. By 10 wk after pressure treatment, the levels of intact αS1-casein were significantly higher in all HHP-treated cheeses compared with the control. Pizza sensory panels indicated that 600-MPa treated cheese was significantly chewier and exhibited lower blister quantity and higher strand thickness compared with control cheeses. High hydrostatic pressure treatment of low-Na, LMPS Mozzarella cheese could result in the extension of its desired baking characteristics when the cheese is stored at refrigerated temperature.

Expression and characterization of camel chymosin in Pichia pastoris.

Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45-50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese.

Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses.

In this work, we studied the influence of the type of coagulant enzyme and the curd scalding temperature on the proteolysis and residual coagulant and plasmin activities of a cooked cheese, Reggianito, in the interest of reducing ripening time. A two-factor experimental design was applied in two levels: type of coagulant enzyme, bovine chymosin or camel chymosin, and curd scalding temperature, 50 or 56 °C. The experimental treatments were applied in Reggianito cheese making experiments, and the samples were ripened for 90 d at 12 °C. Scalding temperature influenced residual coagulant activity; the cheeses cooked at 50 °C had significantly higher activity than those treated at 56 °C. In contrast, scalding temperature did not modify plasmin activity. Proteolysis was primarily affected by curd cooking temperature because chymosin-mediated hydrolysis of αs1 casein was slower in cheeses treated at 56 °C. Additionally, the nitrogen content in the cheese soluble fractions was consistently lower in the cheeses scalded at 56 °C than those cooked at 50 °C. A significant influence of the type of coagulant enzyme was observed, especially in the nitrogen fractions and peptide profiles, which demonstrated that camel chymosin was slightly less proteolytic; however, these differences were lower than those caused by the scalding temperature.

Effect of camel chymosin on the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese.

The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer ripening time than cheeses made with BCC, indicating that use of CC could help to extend the performance shelf-life of LMPS Mozzarella.

Using recombinant camel chymosin to make white soft cheese from camel milk.

Bovine derived chymosin in rennet cannot coagulate camel milk (CAM). The study aimed at producing cheese curd from CAM using a recombinant camel chymosin. Pasteurized CAM was prepared for curdling using a recombinant camel chymosin (50 IMCU/ kg). CAM (pH 6.65) contained 2.83% Fat, 3.34% proteins and 9.11% non-fat solids. Physicochemical properties of soft cheese from cultured CAM were 51.89%, 15.62%, 20.21% and 2.47% for moisture, protein, fat and ash, respectively. Cultured CAM afforded higher cheese yield (8.75%) than non-cultured CAM (3.34%). CAM cheese whey had 48.94% and 76.80% of the fat and proteins, respectively, of their corresponding concentrations in CAM. The study is the first report on soft cheeses from CAM (cultured and non-cultured) using a recombinant camel chymosin, with reference to whey constituents. CAM cheese and whey could be added-value products.

Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris.

Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45-50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications.

Effect of Oryctolagus cuniculus (rabbit) rennet on the texture, rheology, and sensory properties of white cheese.

Calf rennet has long been used in cheese-making. Because of calf rennet shortage and high cost, novel proteases were needed to meet industry's increasing enzyme demand. Recombinant chymosins and camel chymosin were started to be used in the industry. There is no study in the literature subjecting use of rabbit rennet in cheese production. Chemical, rheological, and sensorial characteristics of white cheese made with rabbit rennet were investigated in this study. Quality characteristics of rabbit rennet cheese (RC) were compared to cheeses produced with commercial calf (CC) and camel chymosins (CLC). RC and CLC exhibited higher hardness and dynamic moduli values throughout the storage as compared to CC. Although moisture levels of cheese samples were similar at day 60, CC had much lower hardness and dynamic moduli values than CLC and RC. While the appearance and structure were better for CLC, the highest odor and taste scores were obtained by RC during 60 days of storage. The results of this investigation proposed that rabbit rennet could be a suitable milk coagulant for white cheese production. Our results showed that rabbit rennet has comparable cheese-making performance with camel chymosin and could be a good alternative for calf chymosin.