Milk samples

Fresh raw camel milk samples were collected from 20 healthy Dromedary camel females (Camelus dromedarius) ranging between 2 and 12 months into lactation from the south of Tunisia (region of Gabes), while fresh bovine milk was obtained from a local breed in the region of Sfax (Tunisia).

Bovine and camel whey preparation

Bovine and camel cheese-wheys were extracted from skimmed milk after enzymatic coagulation at 37 °C for 1–2 h in the presence of microbial rennet enzyme with amounts of 0.35 and 1.4 mL enzyme/L bovine and camel milk, respectively, followed by centrifugation at 3000 × g for 20 min at 20 °C using Thermo Scientific Heraeus Megafuge Centrifuge, Germany (Lajnaf et al., Reference Lajnaf, Trigui, Samet-Bali, Attia and Ayadi2020).

Filtration of whey

Bovine and camel wheys were filtered by dialysis against deionised water (Milli-Q system, Millipore, USA) at (sample/water, 1:100) for 24 h at 4 °C using dialysis tubing cellulose membrane (12 kDa MWCO, Sigma–Aldrich) and with continuous stirring (Virgen-Ortíz et al., Reference Virgen-Ortíz, Ibarra-Junquera, Osuna-Castro, Escalante-Minakata, Mancilla-Margalli and Ornelas-Paz2012). The distilled water was replaced after the first hour.

Physicochemical analysis

Physico-chemical composition of camel and bovine whey before and after filtration was systematically determined according to the AOAC Official Method (AOAC, 1984). Specific conductivity of whey was determined using the method of conductivity measurements of the EXPERT-002 conductometer (Khramtsov et al., Reference Khramtsov, Blinov, Blinova and Serov2017).

The demineralization rate (DR), based on conductivity analysis was then calculated by considering the specific conductivity detected in whey samples at the beginning (C_i in mS/cm) and the end (C_f in mS/cm) of the demineralization process according to Equation (1) (Beaulieu et al., Reference Beaulieu, Perreault, Mikhaylin and Bazinet2020):

(1)\begin{equation}{\text{DR}} = \left( {1 - \frac{{Cf}}{{Ci}}} \right) \times 100\end{equation}

The mineral reduction rate based on the conductivity measurement was then calculated with the same equation as for the DR (Equation (1)), considering the ash content (%) at the beginning and the end (C_i and C_f, respectively) of the demineralization process (Beaulieu et al., Reference Beaulieu, Perreault, Mikhaylin and Bazinet2020).

All analytical determinations of chemical analysis were performed in triplicate (n = 3). Data were expressed as mean ± standard deviation.

Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis

RP-HPLC (Agilent 1260 Infinity quaternary LC, Germany) was used to separate and identify the main proteins from camel and bovine whey- and milk-derived proteins before and after filtration using the method of Lajnaf et al. (Reference Lajnaf, Attia and Ayadi2022). A C18 column (Zorbax Eclipse Plus C18, 250 mm length × 4.6 mm, particle size 5 μm, Packing Lot #: B14292) was used for whey protein separation. The RP-HPLC analysis was performed using a Shimadzu SPD6A-UV detector measuring the optical density. Quantitative estimation of the main camel and bovine whey proteins was performed by calculating the peak area of each protein.

Chemical composition of camel and bovine wheys

The physicochemical composition of camel and bovine whey was determined in this study, as shown in Table 1. Camel whey has lower pH values and higher acidity compared to bovine whey. The reason behind difference in pH and acidity of the whey is the difference between camel and cow milk. Indeed, previous researches noted that pH of camel milk is similar to that of sheep milk, but significantly lower than bovine milk (Sawaya et al., Reference Sawaya, Khalil, Al‐Shalhat and Al‐Mohammad1984). Furthermore, pH values and acidity of wheys are mainly attributed to other number of factors including the lactation stage, colostrum, diseases, etc. (Huma et al., Reference Huma, Pasha, Sarwar, Ahmad and Shah2015). However, pH values significantly increased while acidity decreased after membrane dialysis regardless of the whey origin, which is explained by the dilution with deionized water upon filtration. Total solids content of both wheys didn't vary much from each other (∼5.9%), in agreement with the results of the study by Zouari et al. (Reference Zouari, Briard-Bion, Gaucheron, Schuck, Gaiani, Triki, Attia and Ayadi2020), carried out with acid and sweet wheys from camel and cow's milk. Similarly, both camel and bovine whey exhibited the same ash contents (∼0.82%) in accordance with the values of Wangoh et al. (Reference Wangoh, Farah and Puhan1998). However, these values were significantly reduced after dialysis filtration, confirming the demineralization of whey after dialysis filtration with mineral reduction rate of 25.3% and 55.2% for bovine and camel whey, respectively (Table 1). In the case of proteins, a significant difference was observed, with higher protein concentrations in camel whey than in bovine whey. The protein concentrations in sweet bovine and camel whey samples were 2.47 ± 0.06 and 4.79 ± 0.11 g/L of whey, respectively. These values are lower than those reported by (Hailu et al., Reference Hailu, Hansen, Seifu, Eshetu, Ipsen and Kappeler2016) and Zouari et al. (Reference Zouari, Briard-Bion, Gaucheron, Schuck, Gaiani, Triki, Attia and Ayadi2020). No significant differences in the lactose concentration were observed between bovine and camel wheys (∼ 45 g/L), in accordance with the finding of Bouhaddaoui et al. (Reference Bouhaddaoui, Chabir, Errachidi, El Ghadraoui, El Khalfi, Benjelloun and Soukri2019), who reported that fat contents ranged from 45 to 56 g/L in camel whey with an average of 49.8 g/L and 42 g/L for camel and bovine milk, respectively. However, dialysis filtration of whey reduced effectively lactose contents in both wheys with the reduction rate of 93.7% and 91.6% for bovine and camel wheys, respectively. Therefore, dialysis filtration proved to be a valuable tool for the reduction of lactose content in whey regardless of the milk origin. Finally, Table 1 also shows the dependence of the specific conductivity of cheese-whey on the origin of milk and the degree of its demineralization. Indeed, the highest value obtained for the camel whey with specific conductivity was 34.35 ± 0.21 mS/cm, followed by the bovine whey with 26.4 ± 0.01 mS/cm and the lowest value for the cheese-whey with conductivity of 2.5 ± 0.04 mS/cm and 4.18 ± 0.01 mS/cm for bovine and camel wheys, respectively. In the whey compartment, the decrease in conductivity was higher for bovine whey than camel whey, which corresponded to DR levels of 87.8% versus 84.2% for bovine and camel wheys, respectively. These findings are in agreement with those of Khramtsov et al. (Reference Khramtsov, Blinov, Blinova and Serov2017) who reported that the demineralization causes significant decrease to the specific conductivity of whey. These authors noted that the process of demineralization of whey was completed when the specific conductivity value reached approximately 1–1.5 mS/cm. Meanwhile, the demineralization process of acid wheys in the study of Beaulieu et al. (Reference Beaulieu, Perreault, Mikhaylin and Bazinet2020) resulted in DR values that ranged between 20.32% and 77.23% depending on membrane configuration, experimental conditions and raw materials.

Table 1. Chemical composition of camel and bovine whey as affected by dialysis filtration

^a-d samples represented with different letters are significantly different from each other (p < 0.05). error bars show the standard deviations of mean values of physicochemical characteristics (ph value, acidity, conductivity, total solids, ash, proteins and lactose).

RP-HPLC profile

Fig. 1A and B show that RP-HPLC chromatograms of bovine and camel milk protein fractions, respectively. The characterized protein fractions which are derived from bovine and camel wheys are skimmed milk, native whey and filtrated whey. For bovine milk, five major peaks with retention times (RT): 17.8 min, 22.4 min, 23.9 min, 26.1 min and 27.8 min) were detected and identified as κ-casein (∼4.4%), α-casein (∼20.5%), β-casein (∼59%), α-La (∼1.7%) and β-Lg (∼14.4%). Meanwhile, six major protein peaks were identified in camel milk (Fig. 1B). These peaks corresponded to κ-casein (∼1.4%), α-casein (∼27.1%), α-La (∼14.7%), protein fractions (F1 and F2) (∼2.3 and 1.6%) and β-casein (∼52.9%) with RT of 16.3 min, 18.3 min, 20.2 min, 22.2 min, 23.3 min and 25.3 min, respectively. Thus, chromatograms showed that β-casein is the main protein of the colloidal fraction of bovine and camel milk representing more than the half of total bovine and camel proteins in accordance with the results of Kappeler et al. (Reference Kappeler, Farah and Puhan1998). Camel milk also presented ahigher content of α-casein and lower amounts of κ-casein when compared to bovine milk proteins in agreement with the findings of Lajnaf et al. (Reference Lajnaf, Attia and Ayadi2022) and Omar et al. (Reference Omar, Harbourne and Oruna-concha2016). As expected, no peak corresponding to β-Lg was detected in camel milk and whey in agreement with previous researches (Ereifej et al., Reference Ereifej, Alu'datt, Alkhalidy, Alli and Rababah2011). Meanwhile, β-Lg is the main protein of bovine whey as shown in chromatograms (Fig. 1A) followed by α-La representing ∼ 64.9% and ∼ 32.6% of the total whey proteins, respectively. It is also possible to observe a third peak of protein fraction in bovine whey with an RT of 18.3 min, which is identified as caseinomacropeptide (CMP). Camel α-La was found to be the main protein in the camel whey accounting for 62 % of the total camel and whey. Fig. 1B, shows three other main peaks in camel whey with RT of 14.18 and 23.5 min camel whey, suggested to be identified as the CMP (∼8.8%), traces of caseins (∼13.7%), and camel serum albumin-lactoferrin (∼15.5%) in agreement with Ereifej et al. (Reference Ereifej, Alu'datt, Alkhalidy, Alli and Rababah2011)

Figure 1. RP-HPLC chromatograms recorded at 220 nm for bovine and camel milk and whey protein fractions (chromatograms A and B, respectively). Abbreviations are: β-CN, β-casein; α-CN, α-casein; CN, caseins; α-la, α-lactalbumin; β-lg, β-lactoglobulin; F, protein fraction; CMP, caseinomacropeptide.

Chromatograms showed that dialysis filtration didn't affect significantly the composition of bovine and camel whey as they show all peaks previously observed in native whey. However, the proportions of proteins have changed upon filtration. For instance, the β-Lg contents were significantly reduced to 55.8% of total whey proteins, while the percentage of α-La raised to 64.3% in agreement with the findings of Huma et al. (Reference Huma, Pasha, Sarwar, Ahmad and Shah2015). This could be of great interest to food industry, as the level of β-Lg, the main allergen in milk has been significantly reduced.