Spray drying and ultrasonication processing of camel whey protein concentrate: Characterization and impact on bioactive properties

The production of whey protein concentrates (WPCs) from camel milk whey represents an effective approach to valorize this processing by-product. These concentrates harbor active ingredients with significant bioactive properties. Camel WPCs were spray-dried (SD) at inlet temperature of 170, 185 and 200°C, or Ultra-sonicated (US) for 5, 10 and 15 min, then freeze-dried to obtain fine powder. The impact of both treatments on protein degradation was studied by sodium dodecyl sulfate-PAGE and reverse-phase ultraperformance liquid chromatography (RP-UPLC) techniques. Significantly enhanced protein degradation was observed after US treatment when compared with SD. Both SD and US treatments slightly enhanced the WPCs samples’ anti-oxidant activities. The US exposure for 15 min exhibited highest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (12.12 mmol TE/g). Moreover, US treatment for 10 min exhibited the highest in vitro anti-diabetic properties (α-amylase and α-glucosidase in - hibition), and dipeptidyl-peptidase-IV inhibitory activity among all samples. In addition, the ultrasonication for 10 min and SD at 170°C showed the lowest IC 50 values for in vitro anti-hypercholesterolemic activities in terms of pancreatic lipase and cholesteryl esterase inhibition. Conclusively, these green techniques can be adapted in the preservation and processing of camel milk whey into active ingredients with high bioactive properties.


INTRODUCTION
According to the Food and Agriculture Organization of the United Nations (FAOSTAT, 2022), raw camel milk ranks as the third most produced commodity in the United Arab Emirates (UAE), with a production of 79,434.4tonnes in 2022.The UAE camel dairy market size reached 222.4 million USD in the year 2022, and by 2028, it is expected to reach 343.4 Million USD (IMRAC, 2022).Pasteurized camel milk is consumed in many forms such as fresh or flavored milk, powdered milk, drinking yogurt (laban), cheese and ghee etc.The production of fresh, soft, and semi-hard varieties of cheese has increased which led to increased whey production as a by-product (Jafar et al., 2018).The major components of raw whey are lactose, proteins, minerals, and water.
Camel whey proteins (CWP) apart from being rich in essential amino acids are also known to have other potential therapeutic properties including antioxidant, antidiabetic, anti-obesity, and anti-hypertensive properties (El-Agamy et al., 2009;Al Haj & Al Kanhal, 2010;Ibrahim et al., 2018;Kamal et al., 2018;Mudgil et al., 2019).The health benefits of CWP are the attribute of proteins it contains, such as α-lactalbumin (α-LA), serum albumin, lactoferrin, lactoperoxidase, lysozyme, and immunoglobulins.(Ho et al., 2019;Maqsood et al., 2019).Due to the distinctive chemical composition of camel whey, camel milk is considered to have striking similarity with human milk in lacking β-lactoglobulin (β-LG) and richness of α-LA (Izadi et al., 2019).β-LG is the protein that is responsible for causing cow milk protein allergy (CMPA).Thus, camel milk is a suitable alternative for people who have allergenicity to bovine milk (El-Hatmi et al., 2007;El-Agamy et al., 2009;Hailu et al., 2016;Benabdelkamel et al., 2017;Ho et al., 2019;Izadi et al., 2019).These properties broaden the scope of camel milk and its derived products, facilitating the development of specialty milk-based products that cater to the growing demand for healthy food among the general population, children, and individuals seeking alternatives to bovine milk.Therefore, producing camel WPC powder will be an efficient strategy to add value and effectively valorize camel milk by-product in diverse food and nutraceutical applications.High amounts of bioactive proteins, which are important for human nutrition and health, are found in the CWPs (Benabdelkamel et al., 2017;Al Haj & Al Kanhal, 2010).The protein composition of Camel milk differs from bovine counterpart which might affect the bioactive properties after gastric and intestinal digestion (Maqsood et al., 2019).
Spray-drying (SD) is a common method employed for the production of stable whey protein powders with longer shelf-life (Habtegebriel, Wawire et al., 2018;Zouari, Schuck et al., 2020).Nevertheless, production of camel milk powders using SD is still at an early stage of research and development (Habtegebriel, Wawire et al., 2018).SD has become the commercial, rapid, and costefficient method of drying for various liquid products including whey protein powder (Habtegebriel, Wawire et al., 2018;Teijeiro et al., 2018).In the SD process, it is important to preserve the physicochemical, techno-functional, and bioactive properties of the dried food sample by using moderate drying conditions (Zouari, Schuck et al., 2020).The main advantage of the SD process is that it can produce a dry powder with suitable properties such as specific moisture content, uniform shape, and size distribution (Teijeiro et al., 2018).The main limitation of the SD method is that it might lead to losses in the techno-functional and bioactive properties of the protein because of the denaturation and aggregation caused by the dehydration that occurs during spray-drying (Carter et al., 2018).The effect of spray-drying on the bioactive properties of camel whey protein powder has not been explored till date and such experimentation deserves investigation.
Apart from spray drying, ultrasonication (US) has been recently applied to dairy proteins to initiate modification in the structural conformation, thus enhancing the functionality and retaining bioactive properties.Although not enough research is available on the effect of ultrasonication on the bioactive properties of treated camel whey protein concentrate (CWPC).However, sonication has been reported to improve the antioxidant and ACE inhibition activity of the bovine WPs (Leong et al., 2018;Munir et al., 2020).The use of ultrasonic technology needs to be controlled to achieve higher yields of WPs from native protein and act as a precursor of bioactive peptides (Prabhuzantye et al., 2019).Paniwnyk (2017) reported that ultrasonication maintains more bioactive peptides in foods during processing as well as storage.In addition, Gammoh et al. (2020) reported that important functional and biological characteristics of camel milk WPs can be enhanced by sonicating the samples.Hence, US treatment of milk protein as well as the whey proteins produced by hydrolysis or digestion of milk protein with ultrasound pretreatment has potential to improve its bioactive properties, while maintaining the other physical, chemical, and nutritional characteristics.
Therefore, the major objective of this study was to investigate the effect of non-thermal ultrasound treatment and spray-drying on the bioactive properties of CWPC.Non-thermal techniques have been adopted instead of the conventional processing methods to maintain the integrity of the heat sensitive bioactive compounds and consequently improve the bioactive properties of processed foods (Ahmadi et al., 2017).There is no comparative study conducted so far on bioactive properties of CWPC as affected by SD and US.Therefore, this current study determined the influence of SD and US on the bioactive properties of SD and US CWPC.

MATERIALS AND METHODS
Camel milk (raw) was collected from a local farm located in Al Ain, UAE.All the chemicals, enzymes and reagents used were of analytical grade purchased from Sigma Aldrich (USA).

Preparation of camel whey protein
Milk samples were processed within 2 h upon their arrival.Camel milks were skimmed 2 times through centrifugation at 4200 × g, for 15 min at 10°C, and the resulting skimmed camel milk was subjected to acid precipitation by adjusting the pH at 4.0 using HCl 6 M, and then stored overnight at 4°C. Isolation of whey proteins from the skimmed camel milk was done following the method described by Jafar et al. (2018) with some modifications.Milk samples were centrifuged twice at 4200 × g and 10538 × g, for 15 min at 4°C to separate the whey from caseins and remove all casein particles.The whey proteins were frozen at −18°C until further processing and analysis.

Production of camel whey protein concentrate (CWPC) using spray drying
The obtained camel whey protein samples were spraydried using a spray dryer (Armfield, UK.) at 3 different inlet temperatures of 170, 185 and 200°C to produce camel whey protein concentrate (CWPC).The outlet temperature was set at 70°C with a solid feed rate of 20%, relative humidity around 8.5%, drying air flow rate of 7.5 m3/min, and atomization pressure of 0.52 MPa that was maintained constant throughout the drying experiment.For the control sample, camel whey protein samples were freeze-dried at −80°C using a Telstar Freeze-dryer (Terrassa, Spain) at 0.01 mbar to produce CWPC.The CWPC samples were stored at −20°C in Mylar bags (TF-4000, ImpakCrop., Central City, SD; ~1 kg per bag) until further analysis.The freeze-dried and spray-dried CWP powder were produced in 3 batches.

Ultrasonication processing of CWPC
The CWPC samples were ultrasonicated following the technique outlined by Ahmadi et al. (2017) with few modifications.CWPC samples (10 g) were dissolved in deionized water (100 mL), then the mixture was ultrasonicator (bath-type EGS5HD, EngeSolutions, São Paulo-SP, Brazil) at 20 kHz for 0, 5, 10 and 15 min at 300 W. The temperature of the mixture was kept below 30°C by adding ice in the water bath.Subsequently, the ultrasonicated CWPC samples were freeze-dried to obtain fine powders, which were kept at −20°C until further analysis.

SDS-PAGE.
The identification of the protein fractions generated from treated and control (freeze-dried) CWPC samples was carried out by employing the methodology of Maqsood et al. (2019), using SDS-PAGE in a Mini-PRO-TEAN-3 Electrophoresis Unit (Bio-Rad Laboratories, Inc., USA).Phosphoprotein Molecular Weight Standards (Catalog number: P33350) were used as protein marker.Protein samples were incubated at 100°C for 3 min with a 1:1 ratio of sample buffer (12.5 mL 1.5 M Tris pH = 8, 10 mL glycerol, 0.5 g SDS, 0.25 mL 2-mercaptoethanol, and 0.5% bromophenol blue solution).Subsequently, samples were loaded into the SDS-PAGE gel.The Gel Doc + Gel documentation system (Bio-Rad, CA, USA) under visible light was used for image visualization.
Ferric reducing/antioxidant power (FRAP Assay).The methodology outlined by Al-Shamsi et al. ( 2018) was followed to study reducing ability of control (freezedried), spray-dried and ultrasonicated CWPC.The FRAP assay values were computed based on a Trolox standard curve (10-60 mM) and articulated as Trolox equivalent (mM TE/g protein).

Inhibition of α-Amylase
The α-amylase inhibition of control (freeze-dried) and treated samples was studied by employing the methodology previously described by Mostafa et al., (2022b) with slight modifications.In a 96 well microplate, CWPC samples (25 µL) were mixed with 50 µL of 5 mM p-nitrophenyl-α-D-maltohexaoside (pNPM) and 5 mg/mL porcine pancreatic AA enzyme (50 µL).The mixture volume was completed to 250 µL using 0.02 M sodium phosphate buffer (pH 6.9), and the resulting mixture was afterward kept at 37°C for 90 min.A control sample was prepared without CWPC samples considered as a 100% enzyme activity, and the absorbance was recorded at 405 nm using a microplate reader (Epoch 2, BioTek, VT, USA).Moreover, a blank sample was also prepared to remove the background absorbance of the CWPC samples, and acarbose was used to serve as a positive inhibition control.Triplicate measurements were carried out and α-amylase inhibition (%) was calculated by using following equation: The required concentration of CWPC samples to inhibit 50% (IC 50 ) of α-amylase activity was determined by plotting the percent inhibition as a function of the test com-

Inhibition of α-Glucosidase
The α-glucosidase inhibition of control (freeze-dried) and treated CWPC was estimated by using the method described by Kamal et al. (2018), and evaluation each sample was carried out in triplicate.
To calculate the inhibition percentage of α-glucosidase the following equation was used: The required concentration of CWPC samples to inhibit 50% (IC 50 ) of α-glucosidase activity was enumerated by plotting the percent inhibition as a function of the test compound concentration, and the IC 50 values were expressed as µg protein /mL.
Inhibition of DPP-IV The method described by Mudgil et al. (2018) was outlined for DPP-IV inhibitory activity estimation of control (freeze-dried) and treated CWPC samples.The DPP-IV inhibition rate was calculated as: The required concentration of CWPC samples to inhibit 50% (IC 50 ) of DPP-IV activity was determined by plotting the percentage inhibition as a function of the test compound concentration, and the IC 50 values were expressed as µg protein /mL.

Pancreatic lipase and cholesteryl esterase inhibition
The pancreatic lipase (PL) and cholesteryl esterase (CE) inhibition activities were evaluated for control (freezedried), spray-dried and ultrasonicated CWPC samples as previously reported by Mudgil et al. (2022).For estimation of PL inhibitory activity, CWPC samples (50 µL) were mixed with sodium phosphate buffer solution containing PL (20 µL) and p-nitrophenyl butyrate (25 µL).Furthermore, the final volume was made up to 150 µL using sodium phosphate buffer followed by incubation at 37°C for 30 min.Subsequently, the absorbance was read at 405 nm in a microplate reader (Epoch 2,BioTek).The enzymes inhibition was computed using the following equation:

Statistical analysis
The SD and US treatments of CWP were carried out in 3 batches and the experimental analyses were conducted in triplicate (n = 3).All data were subjected to Analysis of Variance (ANOVA) by using IBM Statistical Package for the Social Sciences (SPSS 24.0) software (SPSS INC., Chicago, IL, USA, 2002).The differences between means were evaluated by Duncan's Multiple Range Test at 95% confidence level (P < 0.05).Principal component analysis (PCA) was used to correlate the experimental values of antioxidant and bioactive properties of CWPC samples using Minitab software (version 21.4, 2023 Minitab LLC.). 1 demonstrated that the most abundant proteins present in CWPC samples were lactoferrin, serum albumin and α-LA with electrophoretic mobility bands corresponding to 87, 67.5, and 14 kDa, respectively.The protein bands observed in this study are in line with the findings reported by Maqsood et al., (2019), in which the SDS-PAGE analysis of skimmed camel whey milk from 4 different camel breeds has produced electrophoretic mobility of lactoferrin, serum albumin and α-LA corresponding to 87, 66, 6.5 kDa respectively.Moreover, a previous study conducted by Lajnaf et al. (2018) on CWP has reported that the protein bands for lactoferrin, serum albumin and α-LA correspond to 87, 66, 14 kDa.The protein profiles of SD samples and US-treated samples showed differences in terms of the protein bands, in which the US samples had more protein degradation and fainter bands when compared with the control and SD samples of CWPC.US treatment induced smear of the major protein bands, α-LA, serum albumin and lactoferrin.In this study, US of CWPC samples have resulted in extensive protein degradation, which led to more faint bands obtained in case of sonicated samples when compared with control and spray-dried samples.This observed degradation of camel whey proteins is attributed to the acoustic cavitation caused by US waves, which generated mechanical stress and turbulence that disrupted intermolecular hydrogen and hydrophobic bonds (Khatkar et al., 2018).Notably, the controlled temperature during US treatment in this study (section 2.3) indicated that thermal effects of US treatment did not contribute to the observed protein degradation.Previous studies have also reported that ultrasound treatment of proteins has a significant effect on breaking down the protein and changing its secondary structure (Stathopulos et al., 2004;Xu et al., 2008;Liu et al., 2018).However, other studies reported that there was no difference between US-treated WP samples and untreated samples (Hu et al., 2013;Wang & Arntfield, 2016;de Figueiredo Furtado et al., 2017;Higuera-Barraza et al., 2017;Meng et al., 2021;Zhang et al., 2021).Furthermore, fainter bands for US-5 and US-10 were observed (Figure 1) when compared with US-15.Shorter US treatment times disordered protein structures into smaller and irregular fragments.However, longer US exposure durations led to smaller particles along with an increased number of free SH groups, which could react with themselves or be oxidized, consequently forming larger aggregates and hence, leading to comparatively darker bands in SDS-PAGE analysis (Rahman &Lamsal, 2021).Among the most abundant proteins (lactoferrin, serum albumin and α-LA) present in CWPC from control, US, and SD samples, the lactoferrin exhibited the highest electrophoretic mobility (87 kDa).It could be stated that CWPC treated with US showed more structural degradation compared with control and SD samples.Furthermore, no pronounced changes were noted in the electrophoresis observations among spraydried samples (SD-170, SD-185 and SD-200).Oldfield et al., (2005) also reported a minimal impact of inlet/ outlet air temperature (160/89°C -200/101°C) on whey proteins denaturation.They suggested that the whey sample enters the drying chamber, where it encounters the hot inlet air, subsequently water evaporates quickly from the whey droplet surface.Moreover, evaporation rapidly cooled the whey droplet to a temperature above its respective wet bulb temperature, also cooling the surrounding hot air.Thus, it is unlikely that the high inlet air temperature conditions (160-200°C) would cause significant whey protein degradation.Furthermore, when the whey droplet dehydrates and drops to the bottom of the spray dryer, its temperature will approach that of the outlet air temperature.

SDS-PAGE characterization of CWPC. Protein changes as indicated by SDS-PAGE inferred from Figure
Reverse phase ultra performance liquid chromatography (RP-ULPC) of CWPC.The changes occurred in camel whey protein fraction followed by US and spraydrying were analyzed using RP-UPLC (Figure 2a and 2b).
In general, β-LG was unidentified in all CWPC samples analyzed by RP-UPLC as well as by SDS-PAGE.Analysis of sonicated CWPC samples by RP-UPLC confirmed that higher degradation of proteins was observed when compared with the control samples, which was also confirmed from the results obtained by SDS-PAGE.The US treatment of CWPC completely hydrolyzed major whey protein α-LA and generated smaller peptides similar to the findings reported by Jafar et al., (2018).The complete hydrolysis of sonicated samples was mainly observed with peaks having retention time around 48 and 64 min respectively, those peaks may either completely disappear or remain with very low intensity (Figure 2a).In this study, the main whey protein present in CWPC samples were eluted at later retention times when compared with previous studies conducted by Anandharamakrishnan et al., (2010) 15-20 min.The noticeable increase in the retention time of CWP eluted in this study could be due to an increase in the hydrophobic character of whey proteins (Alizadeh &Aliakbarlu, 2020).
Regarding the effect of spray-drying on the CWPC, the obtained results of RP-UPLC have confirmed that there was slight difference between the whey protein profile of spray-dried samples and the control (Figure 2b).Previous studies have also confirmed that retention time and intensity of the main whey proteins present in SD samples were similar to the control samples (Anandharamakrishnan et al., 2010;Zouari, Briard-Bion et al., 2020).On the contrary, a study conducted by Perusko et al., (2021) stated that spray drying of protein samples has resulted in an increment of the retention time of main proteins detected up to 3.5 min when compared with control samples.

Bioactive properties of US and SD treated CWPC.
In vitro antioxidant properties of US and SD treated CWPC The antioxidant potential of CWPC was studied by using 3 In vitro methods namely, FRAP assay, ABTS and DPPH radical scavenging activities.The ABTS reagent lacks biological relevance and DPPH has limitation to the determination of hydrophilic antioxidants while FRAP assay has slower kinetics, reaction time dependency and limited determination of lipophilic antioxidants concerns (Sadowska-Bartosz & Bartosz, 2022).Therefore, multiple antioxidant activity methods were opted to rule out the limitations of individual methods and have widely accepted information about antioxidant behavior of the CWPC.

ABTS and DPPH radical scavenging activities
The radical scavenging activity of CWPC samples was determined using ABTS and DPPH radical scavenging assays (Table 1).No statistically significant effects were observed on the ABTS and DPPH radical scavenging activities of CWPC samples when exposed with different SD inlet temperatures and ultrasonication (P > 0.05).However, when the sonication time was increased to 15 min the ABTS values exhibited a pronounced increase (12.12 mmol TE/g).While during SD, the increase in temperature showed a declining trend, however, the change was insignificant.The antioxidant activity of proteins is due to amino acid residues, aromatic amino acid, and sulfur-containing amino acids because of their capability to donate protons to reactive free radicals.Hence, this increment in antioxidant activity followed by US treatment could be due to the exposure of antioxidant amino acids caused by conformational alteration and unfolding of the whey proteins (Meng et al., 2021).Our findings are in line with the observations of Wang et al., (2019), they reported that increment in the SD inlet temperature from 140°C up to 200°C to dry skimmed soybean milk negatively impacted the ABTS and DPPH radical scavenging activities.
The samples treated with US for 15 min and 5 min showed the highest ABTS radical scavenging activity and DPPH radical scavenging activity, respectively among all the samples with values of 12.12 and 6.86 mmol TE/g, respectively.Both ABTS and DPPH radical scavenging activities obtained for US-15 and US-5 samples were significantly higher (p-value <0.05) than control samples with values of 9.87 mmol TE/g and 6.26 mmol TE/g, respectively.Interestingly, Gammoh et al., (2020) studied DPPH radical scavenging activity of CWP after exposure with US for a longer period (45 min at 400 W) and observed 30% enhanced scavenging activity compared with the non-sonicated sample.Furthermore, upon sonicating bovine whey protein isolate (WPI) for 60 min, Liu et al., (2019) observed enhanced ABTS radical scavenging activity with scavenging percentage of around 50%, which was significantly higher when compared with non-sonicated samples.While for DPPH radical scavenging activity, the scavenging strength initially increased by increasing the US treatment time up to 30 min, then ahead of 30 min the scavenging strength started to decline.This led to the notion that exposure duration of whey proteins with US impacted the hydrolysis of native structure of the protein and subsequently, owing to exposure of bioactive functional groups, the antioxidant potential of proteins enhanced.However, in accordance with aforementioned studies and our findings, a general trend cannot be concluded for antioxidant potential of CWPC based on exposure duration of US treatment.
Spray drying of samples using different inlet temperatures did not have any significant effect on the ABTS and DPPH radical scavenging activities of CWPC with all the samples having values very close to the control samples (Table 1).In line with the findings of this current study, a study conducted by Wang et al., (2019) reported that by increasing the SD inlet temperature from 140°C up to 200°C to dry skimmed soybean milk have negatively impacted the ABTS and DPPH radical scavenging activities, with samples dried at 140°C exhibiting the highest radical scavenging activities.However, SD of camel milk samples at inlet temperatures between 210 to 250°C significantly improved their ABTS radical scavenging activity up to 16% compared with freeze-dried samples (Perusko et al., 2021).Coefficient of PCA was negative (−30%) for DPPH radical scavenging in PC1 (which explains major data variation) suggesting insignificant reduction DPPH scavenging activity for SD samples, US-10 and US-15 while it was positive (49.9%) in PC2 (explaining only 23% data) explained the significant enhancement of DPPH scavenging after US-5.

Ferric reducing antioxidant power (FRAP Assay)
The reducing power of CWPC samples was also measured using FRAP assay and the reducing potential of CWPC is presented in Table 1.Significant reduction in ferric reducing power of CWPC samples was observed after both US and SD treatments.However, only US-10 was found to have FRAP value comparable to the control sample (P < 0.05).The decline in bioactive properties of US treated samples could be due to the loss of some active compounds by excessive heating (Naczk& Shahidi, 2006).
The spray drying was conducted at 170, 185 and 200°C.Significant increment was observed in FRAP value when the SD temperature increased from 170°C.However, the FRAP values of all SD treated CWPC samples were significantly lower than control CWPC.This reduction observed in FRAP activity can be linked to loss of some bioactive compounds during spray-drying (Khanji et al., 2018).It is in accordance with the study conducted by Arranz et al., (2019) on whey protein-based beverage which was thermally treated to produce pasteurized, UHT, and SD whey protein-based beverage samples.The results showed that the SD samples had the lowest FRAP activity with value around 5 µmol TE/100mL, whereas pasteurized and UHT samples had FRAP values around 8 and 14.5 µmol TE/100mL, respectively.

In vitro anti-diabetic propertiesof US and SD treated CWPC
α-Amylase and α-Glucosidase Inhibition Globally, the prevalence of diabetes among adults (aged 20-79) was estimated at 10.5% in 2021, affecting 536.6 million individuals.This number is expected to increase to 783.2 million by 2045, representing a rise to 12.2% of the population in this age group (Sun et. al, 2022).Therefore, it is crucial to explore antidiabetic potential of foods or food processing by-products.The antidiabetic property of CWPC was analyzed by studying the inhibitory effect against metabolic enzymatic markers; α-amylase and α-glucosidase that are essential in regulating blood glucose levels (Figure 3a and 3b).The peptides generated after degradation can show inhibitory activity with hydrophobic interactions by binding the active site of the enzymes.It was found that SD and US treatment of CWPC samples significantly improved the inhibitory strength of CWPC samples against both antidiabetic markers, with US treatment being more effective in enhancing the inhibitory activity.The highest inhibitory strength against α-amylase and α-glucosidase was recorded for US-10 samples with IC 50 values of 81.18 and 130.10 µg/mL, followed by SD-185 with α-Amy-IC 50 value of 136.86 µg/mL.Moreover, increasing the US treatment time above 10 min had a negative effect on the inhibitory activity against α-amylase and α-glucosidase as an increment of 93% and 49.5% was observed in the IC 50 value, respectively of US-15 samples was noticed when compared with US-10.This is concordant with the exposure time impact of US against α-amylase and α-glucosidase observed by Fadimu et al., (2022).They investigated the effect of US treatment (5 and 10 min) of alcalase derived lupin protein hydrolysates on inhibitory activity against α-amylase and α-glucosidase and reported significant enhancement in the inhibition of both markers after US hydrolysis than control.However, sonicated hydrolysates for 10 min exhibited the higher α-amylase and α-glucosidase inhibitory strength when compared with samples exposed for 5 min.
Similarly, increasing the inlet SD temperature above 185°C significantly decreased the inhibitory activity of CWPC on α-amylase with SD-200 samples having the highest IC 50 value of 196.25 µg/mL among SD samples (Figure 3a).Furthermore, lowering inlet temperature (170°C) resulted in significantly reduced inhibitory activity against α-amylase, suggesting 185°C as optimum SD inlet temperature for enhanced inhibitory activity against α-amylase.In addition, SD of skimmed soybean milk at different inlet temperatures (140, 160, 180, and 200°C) have significantly improved the inhibitory potential of samples against α-amylase and α-glucosidase with inlet temperature of 200°C showed the highest inhibitory activity with IC 50 of 0.29 and 0.19 mg/g, respectively.On increasing the inlet temperatures, the inhibitory activities of SD samples increased against α-amylase and α-glucosidase (Wang et al., 2019).These results are in accordance with the results obtained in the present study that showed higher SD inlet temperature led to more inhibitory activities against α-amylase.Differences in IC 50 values observed after treatment with US and SD, and the differences among treatment variants are likely stem from differing degrees of protein degradation.This, in turn, leads to peptides and amino acids with varying hydrophobicity, impacting their ability to bind to the active sites of α-amylase and α-glucosidase.The differential degradation is evident by the SDS-PAGE (Figure 1) and RP-UPLC (Figures 2a and 2b) observations.

DPP-IV Inhibition
The inhibitory potential of US and SD CWPC samples against DPP-IV was explored to determine the antidiabetic potential of the CWPC samples and the results are presented in CWPC samples significantly enhanced the CWPC inhibitory potential against DPP-IV.However, US treated samples showed significantly pronounced inhibitory activity compared with SD and untreated samples (Figure 4).The highest inhibitory strength against DPP-IV was recorded by US-10 samples with IC 50 values of 67.92 µg/ mL, followed by US-15 and US-5 samples with DPP-IV-IC 50 values of 107.73 and 129.50 µg/mL, respectively.However, among the SD samples, increasing the inlet SD temperature slightly affected the inhibitory activity of CWPC against DPP-IV with SD-170 samples having the lowest IC 50 values of 169.58 µg/mL, followed by SD-185 and SD-200 with IC 50 values of 173.01 and 183.34µg/mL, respectively (Figure 4).Significant decline in DPP-IV inhibition could be noted when the SD inlet temperature increased from 170°C to 200°C.Peptides with DPP-IV inhibitory activity often display specific features, including hydrophobic amino acids and a proline residue located at the second position from the N terminus (Li-Chen et al., 2012).Enhanced but varied DPP-IV inhibitory activity after treatment with US and SD suggested that the hydrophobicity and conformation of amino acids were differentially affected by the hydrolysis conditions.Previously, CWP hydrolysates generated using pepsin, trypsin, and chymotrypsin have been reported to display significantly higher inhibitory activity against DPP-IV with inhibition percentages of 96%, 84%, and 89%, respectively which were significantly higher compared with control sample (17%).These findings confirmed that CWP have potent antidiabetic effects after gastric and intestinal digestion upon consumption (Kamal et al., 2018).Furthermore, Lacroix & Li-Chan., (2013), Mudgil et al., (2018), andNongonierma et al., (2017) determined the DPP-IV inhibitory activity of camel and bovine milk protein hydrolysates generated using alcalase, bromelain, papain, pepsin, and trypsin enzymes and reported that produced protein hydrolysates showed improved DPP-IV inhibitory activity compared with unhydrolyzed camel and bovine milk samples.Moreover, a study done by Akan (2021) explored the antidiabetic activity of camel whey protein hydrolysates (CWPH) after static simulated gastrointestinal digestion of CWP using pepsin and pancreatin enzymes for 2h at 37°C and reported an inhibitory strength of 20.8% against DPP-IV.

Pancreatic Lipase (PL) and Cholesteryl Esterase (CE)
Inhibition Production of bioactive peptides has gained extensive interest due to its ability to lower cholesterol concentration in human plasma, hence decreasing the risk of cardiac diseases.Specifically, the ability of the bioactive compounds to inhibit pancreatic lipase (PL) might help to control the absorption of fat in the small intestine consequently leading to reduction in human body weight (Ikeda et al., 2002;Birari & Bhutani, 2007;Singh et al., 2017;Jafar et al., 2018).Moreover, when cholesteryl esterase (CE) is inhibited, a reduction in the bioavailability of cholesterol produced from cholesterol esters is observed, which controls cholesterol absorption into micelles, and prevents free cholesterol transportation into enterocyte (Heidrich et al., 2004;Ngamukote et al., 2011).The in vitro anti-hypercholesterolemic potential of SD and US treated CWPC samples against PL and CE was determined and obtained results are presented in Figure 5a and 5b.In general, US treatment significantly reduced PL and CE inhibitory IC 50 value of CWPC samples from 120.89 and 138.55 µg/mL (control samples) to 72.83 and 79.12 µg/mL (US-10 sample), respectively.It was observed that sonicating CWPC sample for more than 10 min had a significant negative effect on the inhibitory activity of CWPC against PL and CE, however, US-15 samples exhibited improved inhibitory activity than the control samples with IC 50 values of 104.34 and 117.04 µg/mL respectively (Figure 5a and 5b).Hence, it could be concluded that the optimum US treatment time could be 10 min for CWPC at 20 kHz and 300W, since longer treatment period had a negative impact on PL and CE inhibition.enhancing the PL inhibiting activity of CWPC samples when compared with the control samples (Figure 5).Nevertheless, SD-170 samples with IC 50 values of 130.34 µg/mL exhibited significantly pronounced CE inhibiting activity when compared with control and SD treatments at elevated temperatures (185, and 200°C).A study done by Mudgil et al., (2018) reported a significant improvement in PL inhibitory properties of whole camel milk proteins after hydrolysis using alcalase, bromelain and papain.Moreover, gastric and pancreatic enzymes derived CWP hydrolysates exhibited higher PL and CE inhibitory strength when compared with unhydrolyzed samples (Jafar et al., 2018).Studies on PL inhibitory potential of CWPC are scanty and this study highlighted the PL and CE inhibitory potential of CWPC and explored the effect of SD and US treatment on enhancing the anti-hypercholesterolemic activity of CWPC.These findings confirmed that US treatment of CWPC up to 10 min has significantly improved the CWPC inhibitory activities against PL and CE.Furthermore, US treatment was found significantly more efficient in improving the anti-hypercholesterolemic activity of CWPC than SD.
Principal component analysis The differences and similarities between the CWPC samples after the ultrasonication and spray drying treatments at different levels were evaluated by PCA (Figure 6).The average values of bioactive properties i.e ABTS, antioxidan potential in terms of DPPH radical scavenging activity and FRAP, α-amylase and α-glucosidase inhibition, DPP-IV Inhibition, pancreatic lipase and cholesteryl esterase Inhibition were analyzed.Principal components 1 (PC1), 2 (PC2), and 3 (PC3) all exhibited eigenvalues exceeding 1.Specifically, the eigenvalues for PC1, PC2, and PC3 were 4.33, 1.86, and 1.306, respectively, corresponding to 54.2%, 23.3%, and 16.3% of the total variance observed among the aforementioned bioactive properties of untreated, US-treated, and SD-treated CWPC samples.Since, PC1 and PC2 cumulatively explained 77.5% of the total variation therefore, the major contributors are discussed in terms of score plot and biplot (Figure 6a  and 6b).In PC1, the contribution of ABTS, FRAP, amylase, glucosidase, DPP-IV, lipase and CE inhibition was positive while DPPH scavenging negatively contributed to PC1.However, ABTS and glucosidase inhibition contributed negatively to PC2.The score plot of PC1 and PC2 described the overall variation among CWPC samples (Figure 6a).In the score plot, CWPC samples closer to each other depicted similar bioactive properties.The CWPC samples were distributed throughout the quadrants demonstrating large variability in their bioactive traits due to the difference in treatments and conditions.Furthermore, larger variation could be observed among different levels of US treatment when compared with SD variant (Figure 6a).The biplot of PC1 vs. PC2 showed that the maximum bioactive attributes were associated with PC1 (Figure 6b).Lipase, CE and glucosidase inhibition have greater influence on PC1 while DPPH is a high value variable of PC2.The closer loading vectors of CE and lipase inhibition activity in the biplot suggested their strong positive correlation.Based on this correlation, it could be suggested that in the CWPC, the CE and lipase inhibitory peptides could have similar amino acid residues in their peptide structure.Similarly, FRAP, amylase inhibition, and DPP-IV inhibition displayed positive correlations, suggesting that the presence of homologous amino acid residues in the hydrolyzed peptide structures contributed to these functionalities.Conclusively, the enzymes inhibitory activities exhibited higher influence on PC1 while anti-oxidative properties have smaller contribution.

CONCLUSION
Current study explored for the first time an efficient drying method and green technique that can be utilized to produce WPCs from camel milk with enhanced bioactive properties.This study reported the production of SD and US CWPC powders with improved bioactive properties.The RP-UPLC analysis confirmed that β-LG was not identified in all CWPC samples and that sonicated CWPC samples exhibited high DH that was in line with the results obtained by SDS-PAGE.For the bioactive properties, SD and US treatment slightly improved the antioxidant activity of CWPCs.Moreover, both treatments significantly enhanced the inhibitory activity of CWPC samples against α-amylase, α-glucosidase, and DPP-IV with US treatment being more effective in enhancing the inhibitory activity than SD.The anti-hypercholesterolemic activity of CWPC samples was significantly enhanced by US treatment with the IC 50 of CWPC samples to inhibit PL and CE reduced from IC 50 of 120.mL (control samples) to 72.83 and 79.12 µg/mL (US-10 sample), respectively.The produced CWPCs could be incorporated in food products as an active ingredient to boost the food products' antioxidant, anti-diabetic, and anti-hypercholesterolemic activities.
Al-Thaibani et al.: Spray drying and ultrasonication… Al-Thaibani et al.: Spray drying and ultrasonication… pound concentration, and the IC 50 values were expressed as µg protein/mL.

Figure 6 .
Figure 6.The principal component analysis (PCA) score plot (a) and biplot (b) of different bioactive properties of camel whey protein concentrate samples processed with spray-drying or ultrasonication.