The effects of camel chymosin and Withania coagulans extract on camel and bovine milk cheeses

Withania coagulans (W. coagulans) extract and camel chymosin have aspartic protease capable of coagulating milk for cheese production. This study investigated the quality of camel and bovine milk cheeses coagulated using Withania extracts, came chymosin, and their mixture in two experiments. In Experiment (1), a factorial design with four factors (W. coagulans, camel chymosin, incubation time, and incubation temperature) was performed. The effect of these factors on cheese’s yield and hardness were assessed. An enzyme concentration corresponding to a 36 µg/L of milk of W. coagulans, 50 IMCU/L of camel chymosin, holding time of 4 h, and incubation temperature of 60 °C provided the optimal textural hardness for both camel and bovine milk cheeses. Seven treatments were analyzed in experiment (2) were analyzed for physicochemical properties, yield, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGEitation). The results showed that pure Withania extract exhibited the lower coagulating effect resulting in cheeses with low yield, hardness, fat, protein, and total solids. The SDS-PAGE electropherograms of camel cheese showed several low molecular weight bands as compared to bovine cheese. This phenomenon is due to excessive proteolysis in camel cheese, which we believed is caused by the presence of endogenous enzymes.

Experimental design. The first experiment was performed using a central composite rotatable design, with varying combinations of W. coagulans concentration (7,21,36,50, and 65 µg/1000 mL milk), camel chymosin concentration (10,30,50,70, and 90 IMCU/1000 mL milk), incubation time, (1, 2, 4, 6 and 8 h), and incubation temperature (40,50,60,70 and 80 °C), which were independent variables; then, the response variables (cheese yield and hardness) were measured (Table 1). In the second experiment, which was performed in triplicate, three sets of cheeses were made from each treatment using pure Withania extract, pure camel chymosin, or their mixture, as shown in Table 2. In this experiment, several other traits were measured in addition to yield and hardness, including cheese color, titratable acidity/pH, protein, fat content, and SDS-PAGE electrophoretic profiles of both camel and bovine cheese, whey, and milk.
Enzyme extraction. Enzymes were extracted from W. coagulans following a previously described method 34 .
W. coagulans berries were washed and dried in a cool place and then ground. The powder (10 g) was mixed with 100 mL of 1% saline solution for 24 h at 4 °C with agitation. The mixture was centrifuged at 9000×g at 4 °C for 30 min. The supernatant was filtered through Whatman paper No. 1 30 . The protein content in W coagulans crude extract was determined following Bradford method 37 using bovine serum albumin (BSA) as a standard. The absorbance of the supernatant was measured at 595 using a UV/visible spectrophotometer (Pharmacia Biothch ultrospect 3000, Cambridge, England). A freshly crude extract was used in making the cheeses.
Cheese preparation. One litter of raw camel or bovine milk was processed into cheeses, in each of three repetitions per treatment, using camel chymosin or W. coagulans, or the mixture of the two coagulans. The milk was heated to 63 °C for 30 min and calcium chloride (3%) was added based on earlier reports 12 . The milk's temperature was brought down to 43 °C and it was inoculated with 3% (w⁄v) of an active thermophilic yogurt starter culture Yoflex Express ® 1.0 (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus). The incubation continued for 60 min until the milk's pH was lowered to 6.2, and then the camel chymosin (CHY-MAX ® M), W. coagulans extract, or the mixture was added to the milk and stirred thoroughly 38 . The enzyme concentrations, incubation time, and incubation temperature in Experiment (1) are given in Table 1. While in Experiment (2) after the addition of (3%) starter culture and 50 IMCU/L of milk camel chymosin or 36 µg/L of milk or the mixture. The milk was incubated at room temperature (25 °C) for four hours until the pH reached 4.8, and the firm curd was observed, and then the curd was placed in cheesecloth to drain for 8 h 16,22,39 . Cheese yield and physicochemical properties. Fresh cheese prepared from 1 L of milk per trial was weighed, after 8 h of draining, using Metis digital weighing scale (Dubai, UAE). The cheese yield was calculated as the percentage of the weight of the fresh cheese as follows (Yield = kg of fresh cheese × 100/mL of processed milk) 40 . The pH of the samples from both experiments was determined using a digital pH meter (Starter3100; Ohaus, New Jersey, USA), and the titratable acidity was determined in triplicate using the standard method ISO/ TS 11869:2012 (IDF/RM 150:2012) 22 . The seven treatments of camel and bovine cheeses and wheys in experiment (2) were evaluated for their contents of fat, protein, and total solid using a Near-Infrared Multipurpose Analyzer (Bruker Optik GmbH, Ettlingen, Germany). All the samples were tested on the same day, with each sample analyzed in triplicate. The texture profile analysis (TPA) of the camel and bovine cheeses from both experiments was analyzed for textural hardness using a CT III texture analyzer equipped with a 4.5-kg load cell (Brook-field, Middleboro, Massachusetts, USA). TPA was performed with a compression test of the cheese in a 40-mL cup using a 25-mm-diameter perplex cylindrical probe (TA11/1000) with a test speed of 2 mm/s and target distance of 5 mm 22 . The hardness pattern (force-time) was analyzed in triplicate. The color characteristics of camel and bovine cheeses in experiment (2)  www.nature.com/scientificreports/ each value being the average of four measurements. This color system comprises a lightness component (L*), a* component for green (− a) to red (+ a), and a b* component from blue (− b) to yellow (+ b). Fourier-transform infrared (FTIR) spectroscopy of the cheese samples from experiment (2) was performed using an ATR-FTIR Spectrometer (Nicole™ 1S50 FTIR; Thermo Fisher Scientific, Massachusetts, USA) at mid-infrared wavelengths. The infrared spectrum was recorded between 400 and 4000 cm −1 at a resolution of 4 cm −1 . To improve the signalto-noise ratio, 124 scans were used per spectrum. Three spectra were taken from each sample.  www.nature.com/scientificreports/ SDS-PAGE of cheeses and whey proteins. Proteolytic activity of the seven treatments of camel and bovine cheese, whey, and milk samples from camel and bovine in Experiment (2) were analyzed using SDS-PAGE. Cheese samples were prepared using previously reported methods 21, 41 . The fresh cheese samples (0.6 g) were dissolved in 25 mL of 8 M urea and whey samples (0.6 g) were dissolved in 8.3 mL of 8 M urea. The samples were homogenized for 2 min using T 25 digital Ultra-Turrax (IKA-Werke GmbH and Co. KG, Staufen, Germany). For the complete solubilization of caseins, the sample and urea mixtures were incubated in a temperature-controlled water bath at 37 °C for 2 h. The cheese and urea mixture was defatted by centrifugation at 9150×g at 4 °C for 35 min. The solution was filtered through Whatman no. 1 filter paper (pore size, 11 µm). 10 µL of the filtered sample was added to 30 µL of 4 × Lamelli buffer solution containing 50 mM Dithiothreitol (added freshly). The sample and sample buffer mixture were heated in a temperature-controlled water bath for 5 min at 90 °C. From this mix, 6 µL was loaded on the hand-casted polyacrylamide gels. Electrophoresis was performed at 200 V using a power supply from PowerPac™ Basic Power supply (Bio-Rad Laboratories Inc., Hercules, California, USA). The running buffer (pH = 8.3) used was a 10 × Tris/Glycine/Sodium dodecyl sulphate Buffer (25 mM Tris, 192 mM glycine, and 0.1% (w/v) sodium dodecyl sulphate. Gels with 1 mm thickness were prepared using the gel hand casting accessories provided with the Bio-Rad Mini-PROTEAN Tetra cell (Bio-Rad Laboratories Inc., Hercules, California, USA). A 12% resolving gel and 4% stacking gel were prepared. To prepare a quantity of 15 mL of 12% resolving gel solution the following were added: 6 mL 30% acrylamide/Bis Solution 29:1, 3.75 mL 1.5 M Tris HCl (pH 8.8), 150 μL of SDS solution 10% (w/v), 5.03 mL deionized water, 75 μL of 10% APS (ammonium persulphate), 7.5 μL N, N, N′, N′-Tetramethylethylenediamine. To prepare a quantity of 15 mL of 4% stacking gel solution the following were added: 1.98 mL 30% acrylamide/Bis Solution (29:1, v/v), 3.78 mL 0.5 M Tris HCl (pH 6.8), 150 μL SDS solution 10% (w/v), 9 mL deionized water, 75 μL 10% APS, 15 μL TEMED. The gels were kept for one hour in a solution of 40% ethanol and 10% acetic acid for fixation of the protein bands. Gels were stained for 20 h using the QC colloidal Coomassie stain. The gels were de-stained for three hours by changing the distilled water three times. Gels image acquisition and densitometry were performed by Gel Doc™ XR+ and Chemidoc™ XRS+ Imaging Systems (Bio-Rad Laboratories Inc., Hercules, California, USA).
For imaging the gels, a UV/White light conversion screen was used. The instrument was operated by the Image lab software (Bio-Rad Laboratories Inc., Hercules, California, USA). The software was used to determine the protein bands' molecular weights, integrate the peaks, and determine their relative densities. (1). The values of four independent factors (W. coagulans extract concentration, camel chymosin concentration, incubation time, and incubation temperature) and their response variable are shown in (Table 1). The design consisted of 31 experiments including 7 central point repetitions that would account for the error in the model. This experiment was designed using Minitab ® 19 (Connecticut, USA). The model design was fitted to each response using the following equation:

Statistical analysis. A full factorial central composite design was used in experiment
where y is the response, β 0 is a constant, β i is the linear coefficient, β ii is the quadratic coefficient, and β ij is the interaction coefficient. X i and X j are two independent variables. In this experiment, raw data from freshly extracted cheese was analysed.
In the second experiment, the physicochemical, and yield from freshly extracted cheese were analyzed in triplicate, and the mean values were used in the calculations. The statistical analysis was performed using IBM SPSS (SPSS Inc., Chicago, Illinois, USA). Data were analyzed using a one-way analysis of variance. The results were presented as the mean values of triplicate and their standard deviations. Mean values were compared using the least significant difference test, and p ≤ 0.05 was considered to represent statistical significance.

Results and discussion
Effects of camel chymosin and Withania coagulans on the yield and hardness of camel and bovine cheeses. Table 1 presents the experimental design for the independent variables (W. coagulans concentration, camel chymosin concentration, incubation time, and incubation temperature) and the results of two associated response variables (cheese yield and hardness) in experiment (1). Among the different combinations, crude extracts of W. coagulans (36 µg/L milk), camel chymosin (50 IMUC/L milk), coagulation time (4 h), and incubation temperature (60 °C) were the optimum conditions providing the lower fresh yield (Fig. 1) and the highest hardness (Fig. 2) for both camel and bovine milk cheeses. The negative correlation between the fresh yield and the hardness of both camel and bovine cheeses (p < 0.001) as shown in Fig. 3 is consistent with our previous observations and is explained by the increased retention of moisture in soft cheeses 22 . As the concentrations of added enzyme and the incubation temperature and time exceeded the optimum concentrations, the cheese started to become softer in agreement with literature 27 . This decrease in hardness may be explained by increased proteolysis of the casein proteins 22,43,44 . Table 3 presents mathematical models that indicate the significance of the independent variables and their interactions in affecting the yield and hardness of camel and bovine cheeses in experiment (1). All models significantly (p < 0.05) suggest that the independent variables (concentration of W. coagulans and camel chymosin, and incubation time and temperature) collectively have similar effects on the yield and hardness of both camel and bovine cheese although the magnitude of the effect is different. However, higher yield and lower hardness are noticed more in camel cheeses than bovine cheeses 42 . The results also show that chymosin interactions are www.nature.com/scientificreports/ more important than those of Withania in terms of affecting cheese yield and hardness. The significant terms in the models were the constants related to the milks and the quadratic terms C*C, TP*TP, and TM*TM, all contributing negatively to the hardness of camel and bovine cheeses. These models suggest that the independent variables affected the camel and bovine milk cheeses fresh yield and hardness in similar ways but the magnitude of the effects was different due to differences between the two milks.

Camel Cheeses
Camel chymosin www.nature.com/scientificreports/ The second experiment, based on seven treatments Table 2, was performed by combining different concentrations of chymosin and W. coagulans extracts at fixed incubation temperature (60 °C) and incubation time (4 h) that were chosen based on the results of Experiment 1. Table 4 presents results of the yield, hardness, and color of camel and bovine milk cheeses prepared using W. coagulans extracts, pure chymosin, and their mixtures. Cheese made from pure W. coagulans alone has the lowest cheese yield and hardness and is more yellow compared to the other cheeses. This yellow color of the Withania-treated cheeses may be the result of the presence of some water-soluble compounds in the berries 31   www.nature.com/scientificreports/ the W. coagulans extract could be associated with poor coagulating properties of its protease compared with chymosin. The mixture of low camel chymosin and low Withania extract produced cheeses of higher hardness and total solids from both camel and bovine milks (Fig. 4). In agreement with the results of Experiment 1, the camel chymosin was more crucial for the cheese yield and hardness than the Withania extract. However, as the concentration of either enzyme increased, the cheeses became soft and fragile with high moisture content due to excessive hydrolysis of caseins 22,43,44,45,46 . Thus, the results from the two experiments showed that unripened cheeses produced from camel milk have higher moisture contents and lower hardness compared to bovine cheeses in agreement with our previous findings 22 . This difference was suggested to result from low κ-casein content in camel milk compared to bovine milk 5,47 . However, we have suggested that the high percentage of β-casein might also contribute to the soft and smooth nature of camel cheeses 41 . Moreover, the SDS-PAGE electropherograms (see below) suggest that some endogenous enzymes in camel milk might contribute to the softness of camel cheeses. Table 5 presents the results of pH/acidity, fat, protein, and total solids in camel and bovine cheeses and whey. The camel milk cheeses were generally more acidic than bovine milk cheese. Withaniatreated cheeses possessed higher acidity and lower pH in both camel and bovine cheeses and whey products. The , and (c) cheese yield and cheese hardness for camel (red) and bovine (blue) cheeses. All correlations are significant (p < 0.001). The experimental design is presented in www.nature.com/scientificreports/ high pH in the chymosin-treated cheeses may be associated with rapid coagulation of milk after the addition of chymosin, which triggers rapid casein cleaving and quick rearrangement of the caseins 48 . This results in larger pore spaces in the casein microstructure 22 that enhance water drainage leading to increased pH due to lower activities of lactic acid bacteria in the dry gels 49 . In this study, we added calcium chloride (3%) to both kinds of milk as usually done during the preparation of bovine cheeses but it was reported that there is no observed improvement by adding calcium chloride with camel chymosin 50 . Table 5 shows significant differences between the different treatments with respect to the pH and acidity of the different cheeses. The importance of electrolyte balance for enzyme activities and casein coagulation during cheese-making is not well understood and deserves further investigations. The equilibria involving minerals (mainly calcium and magnesium but also sodium) and anions (such as phosphate, citrate, and acetate) are important determinants of casein micelle stability, pH, and www.nature.com/scientificreports/ enzyme activity 30 . Moreover, the high acidity and low pH of the camel compared with the bovine cheeses may be explained by an increased degree of proteolysis in camel milk cheeses, because proteolytic activities may produce peptides with acidifying effect (see below). Cheeses made from a mixture of chymosin and W. coagulans had the highest total solids, protein, and fat. Furthermore, the results also show fat, protein, and total solid contents were significantly higher in the bovine milk cheeses than in the camel milk cheeses (Fig. 4), which is in agreement with previous findings 21, 51 . The lower protein, fat, and total solid contents of camel cheeses and the higher total solid in whey shown in Table 5 may be associated with the softer nature of these cheeses. Another factor could be due to a lower concentration of κ-casein in camel milk compared to bovine milk (3.3% vs. 13%). The proportions of αs1-:αs2-:β-:κ-caseins in camel milk were 2.6:0.4:6.7:0.3, compared with 4:1:4:1 in bovine milk 52 . κ-Casein is known to enhance the coagulation properties of milk, leading to a denser casein matrix, which reduces the loss of fat and protein to the whey [53][54][55] . The κ-casein concentration and its relative proportion to αS1-and β-casein concentrations are usually low in poorly and non-coagulating bovine milk 56 . Determination of the exact contribution of the different caseins in camel milk to the texture of camel milk cheese remains a challenge. Figure 6 presents the spectra of cheeses at various wavelengths, ranging from 400 to 4000 cm −1 . The spectra showed in both camel and bovine cheeses were similar to what was reported by previous researchers [57][58][59] .

SDS-PAGE results on the proteolysis of camel and bovine milk cheeses. The SDS-PAGE elec-
tropherograms showing differences in the protein and peptide profiles of camel and bovine cheeses and whey are presented in Fig. 7. It can be observed that camel cheeses show several low molecular weight bands compared to bovine cheeses suggesting that excessive proteolysis of caseins has occurred presumably catalyzed by endogenous enzymes such as plasmin in camel milk 60,61 . The proteolysis of β-CN by the natural milk proteases (plasmin) was successfully found in milk samples analysis before 62 . Thus, the high proportion of β-casein and possibly more active proteolytic activity in camel milk may lead to an increased level of proteolytic products. It was reported that high levels of β-casein affect milk coagulation causing softness of cheeses 63 . We have observed similar behavior in camel milk fermented by the lactic acid bacteria used to make yogurt (results not shown). Some of the low molecular weight peptides from camel milk cheese seem to migrate into the whey fraction explaining the low total solid content in camel milk cheeses and casein bands seen in the SDS-PAGE whey results. Correlations for camel (red) and bovine (blue) cheeses between (a) lightness and yellowness (CM cheeses are lighter in color and less yellow than BM cheeses; p < 0.001) and between (b) acidity and pH (CM cheeses have higher acidity and lower pH than BM cheeses; p < 0.001). All correlations are significant (p < 0.001).
Chymosin yields cheeses that are less yellow, lighter in color, less acidic, and with higher pH than those yielded with Withania treatment (p < 0.001). The codes are presented in Table 2 (Experiment 2).

Conclusions
This study investigated the effect of different combinations of W. coagulans extract and camel chymosin on the yield, hardness, and total solids (protein, fat, and other solids) contents of cheeses prepared from camel and bovine kinds of milk. The results revealed that too high concentrations of the enzymes resulted in the production of soft cheeses. W. coagulans extract protease alone is not sufficient to produce good quality cheese especially camel milk cheese but a mixture of W. coagulans and camel chymosin produced better quality camel and bovine www.nature.com/scientificreports/ milk cheeses than chymosin alone. SDS-PAGE showed camel cheeses to more hydrolysis products compared to bovine cheeses suggesting possible participation of endogenous enzymes in camel milk. Further studies are needed to identify the enzyme(s) responsible for proteolytic activity in camel milk and their contribution to milk coagulation and cheese softness.