In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk

Accepted Manuscript In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: a comparative study with fermented bovine milk Mutamed Ayyash, Amna K. Al-Nuaimi, Suheir Al-Mahadin, Shao-Quan Liu PII: DOI: Reference:

S0308-8146(17)31131-7 http://dx.doi.org/10.1016/j.foodchem.2017.06.149 FOCH 21371

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

4 April 2017 27 May 2017 28 June 2017

Please cite this article as: Ayyash, M., Al-Nuaimi, A.K., Al-Mahadin, S., Liu, S-Q., In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: a comparative study with fermented bovine milk, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.06.149

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In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: a comparative study with fermented bovine milk

Mutamed Ayyasha*, Amna K. Al-Nuaimia, Suheir Al-Mahadina, Shao-Quan Liub

a

Food Science Department, College of Food and Agriculture, United Arab Emirates University

(UAEU), PO Box 1555, Al Ain, UAE b

Food Science and Technology Programme, Department of Chemistry, National University of

Singapore, S14 Level 5, Science Drive 2, 117542, Singapore

*

Corresponding author:

Dr. Mutamed Ayyash Food Science Department College of Food & Agriculture United Arab Emirates University (UAEU) T: +971 3 713 4552 F: +971 3 767 5336 E: [email protected]

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ABSTRACT This study aimed to investigate in-vitro the health-promoting benefits (anticancer activity, ⍺amylase and ⍺-glucosidase inhibition, angiotensin-converting-enzyme (ACE)-inhibition, antioxidant and proteolytic activity) of camel milk fermented with indigenous probiotic strains of Lactobacillus spp., compared with fermented bovine milk. The three camel milk probiotic strains Lb. reuteri-KX881777, Lb. plantarum-KX881772, Lb. plantarum-KX881779 and a control strain Lb. plantarum DSM2468 were employed to ferment camel and bovine milks separately. The proteolytic and antioxidant activity of water soluble extracts (WSEs) from all fermented camel milks were higher than those of fermented bovine milk. α-Amylase inhibition of WSEs were > 34% in both milk types fermented with all strains during storage periods, except the WSE of camel milk fermented by Lp.K772. The highest ACE-inhibition of the WSE from camel milk fermented by Lr.K777 was > 80%. The proliferations of Caco-2, MCF-7 and HELA cells were more inhibited when treated with the WSE of fermented camel milk.

Keywords Camel milk; fermented; probiotics; ACE-inhibition; anticancer

Chemical compounds studied in this article o-Phthalaldehyde (PubChem CID: 4807); p-nitrophenyl ⍺-D-glucopyranoside (PubChem CID: 102764); 1,1-diphenyl-2-picrylhydrazyl DPPH (PubChem CID: 2735032); hippuryl-histidyl-leucine (PubChem CID: 94418);

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1. Introduction Camels are found in Africa and Asia, and are kept mostly by nomads and tribes living in the desert regions. Australia has half a million wild camels, mostly in the Northern Territory. Globally, there are two common species of camels: one-humped Arabian camels, or dromedaries, (Camelus dromedarius) – the camels of the plains; and two-humped Bactrian camels (Camelus bactrianus) – the camels of the mountains (Fukuda, 2013). Camels are raised for milk, meat, fibre (wool and hair), transport and other work; their dung is used as fuel (Elagamy, 2006). A FAO workshop (2008) estimated that global camel milk production is approximately more than 5.3 million tons (Al haj & Al Kanhal, 2010). Several products made from camel milk, including pasteurized camel milk, ice-cream, cheese, camel milk powder, latte coffee, and camel milk soap, have been developed and sold in many countries (Al haj & Al Kanhal, 2010). In the arid rural communities of Asia and Africa, camel milk has been traditionally used for many years as a biomedicine to treat several health issues such as asthma, oedema, and diabetes (Khalesi, Salami, Moslehishad, Winterburn, & Moosavi-Movahedi, 2017). Camel milk is high in vitamin C and niacin, as well as richer in Cu and Fe than bovine milk (Elagamy, 2006). Several studies have shown the nutritional benefits of camel milk, including antihypertensive, hypoglycaemic, hypoallergenic, and hypocholesterolaemic effects (Ibrahim & Khalifa, 2015; Mostafa, Abd El-Hamed, & Almetwaly, 2013; Sayed, Ahmed, & Sayed, 2013), although a few limitations have been reported by Mihic, Rainkie, Wilby, and Pawluk (2016). Camel milk contains well-balanced nutrients and biological components for fermented milk production. Moreover, camel milk is similar to human milk in terms of lack of β-lactoglobulin and that it contains ⍺-lactalbumin (Khalesi et al., 2017). The in-vitro health benefits of fermented camel milk have also been reported (El-Salam & ElShibiny, 2013). The angiotensin-converting enzyme (ACE) inhibition and antioxidant activities of fermented camel milk have been highlighted in two studies (Alhaj et al., 2017; Shori, 2013). However, few attempts have been made to compare the health benefits of fermented camel milk

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with other fermented mammalian milks, particularly fermented bovine milk (Moslehishad et al., 2013; Mymensingh, 2007; Shori, 2013) with a focus on ACE-inhibition, antioxidant and proteolytic activity. Shori and Baba (2014) examined the in-vitro antidiabetic and antioxidant activity of fermented camel milk supplemented with garlic extract. The authors employed traditional lactic acid bacteria (LAB) such as Lactobacillus bulgaricus and Streptococcus thermophilus to ferment camel milk. To the best of our knowledge, none of the above studies provided a comprehensive in-vitro investigation into the health promoting benefits (antiproliferation, antidiabetic, ACE-inhibition, antioxidant and proteolytic activity) of camel milk fermented with probiotic LAB isolated from camel milk. The current study attempted to highlight the impact of some chemical and biochemical variations between fermented camel and bovine milks with regard to potential health benefits. Nine LAB were previously isolated from camel milk and identified with having robust probiotic characteristics (Abushelaibi, Al-Mahadin, El-Tarabily, Shah, & Ayyash, 2017). Out of nine LAB isolates, Lb. reuteri KX881777, Lb. plantarum KX881772 and Lb. plantarum KX881779 were found to be most promising and hence, were used in the current study. Therefore, the objective of this study was to investigate in-vitro the health-promoting effects, namely antiproliferation activity against three cancer cell lines, ⍺-amylase and ⍺-glucosidase inhibitions, ACE-inhibition, antioxidant and proteolytic activity, of camel milk fermented with three indigenous probiotic strains of Lactobacillus spp., compared with fermented bovine milk during 21 days of storage at 4C. 2. Materials and methods 2.1. Culture propagation Lb. plantarum DSM2648 (Lp.DSM) was purchased from Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and employed as a control probiotic (Anderson, Cookson, McNabb, Kelly, & Roy, 2010). The three probiotic strains Lb. reuteri KX881777 (Lp.K777), Lb. plantarum KX881772 (Lp.K772), Lb.

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plantarum KX881779 (Lp.K779) and Lb. plantarum (Lp.DSM) were stored in de Man, Rogosa, and Sharpe (MRS) broth (Oxoid, Hampshire, UK) with 50% glycerol at −80°C. For culture activation, a 100-μL aliquot of each culture was individually transferred into MRS broth and incubated at 37°C for 24 h. A weekly culture transfer was carried out to maintain the bacterial activity. For all cultures, two successive culture transfers were carried out in MRS broth, and a third transfer was in sterilized reconstituted skim bovine or camel milk (RSM 10%, w/v) and incubated at 37°C for 24 h. 2.2. Milk fermentation Skim camel milk from one-humped Camelus dromedaries was purchased from a local supplier with a total soluble solids content of ~10.3%, protein content of 3.7%, lactose content of 4.1% and fat content of 0.47% (wet basis). Reconstituted skim bovine milk was prepared with a total soluble solids content of 10% w/v, protein content 3.4%, lactose content 4.5% and fat content 0.5%. Both milk types were pasteurized at 90C for 10 min, followed by cooling to 37C. Pasteurized milk was inoculated with 1% ( v/v) of each strain separately (~ 9.0 log10 CFU/ml = the cell count of the inoculated milk), followed by mixing for 1 min. Inoculated milk was fermented at 37C for 24 h. Afterwards, fermented milk was cooled to 4C in ice-bath, then stored at 4C for 21 days. Fermented milk was sampled at 0, 7, 14 and 21 day of storage. Milk fermentation was performed in triplicate. 2.3. Bacterial enumeration Bacterial population in fermented milk was enumerated according to Sah, Vasiljevic, McKechnie, and Donkor (2014). An aliquot (1 ml) of blended fermented milk sample was subjected to appropriate serial dilutions using 0.1% (w/v) peptone, and bacterial populations were counted using MRS agar (Oxoid). Inoculated plates in duplicate were incubated anaerobically at 37°C for 48 h using an anaerobic jar system (Don Whitley Scientific Limited, West Yorkshire, UK). 2.4. Water soluble extract (WSE) For each fermented milk sample, pH was adjusted by 1.0 M HCl or 1.0 M NaOH to pH 4.6, followed by centrifugation at 10000 x g for 15 min at 4C. The supernatants were filtered through 5

an 0.45-µm syringe filter (Mixed Cellulose Esters, EMD Millipore Corporation, MA, USA) and stored at -20C for further analysis. 2.5. Proteolytic activity assay by o-phthalaldehyde (OPA) Prior to the OPA assay, stored WSEs were vortexed for 1 min followed by centrifugation at 10000 x g for 5 min. OPA of WSE was determined as described by Sah et al. (2014). Briefly, the OPA reagent was prepared daily by combining 25 ml of sodium tetraborate buffer (100 mM; pH 9.3), 2.5 ml of sodium dodecyl sulphate (20%, w/w), 40 mg of OPA (dissolved in 1 ml of methanol), and 100 µl of β-mercaptoethanol and diluting to 50 ml with water. An aliquot (150 µl) of the WSE sample was added to a test tube containing 1.5 ml of the OPA reagent and mixed gently for 5 s. The absorbance was measured after exactly 2 min at 340 nm using a UV-spectrophotometer (JENWAY 6300, Staffordshire, UK). Proteolysis results were presented as absorbance at 340 nm. 2.6. α-Amylase inhibition assay Prior to the assay, stored WSEs were prepared as described above. The ⍺-amylase inhibition assay was carried out according to the method described by Kim, Wang, and Rhee (2004) with minor modifications. Briefly, 100 µl of ⍺-amylase from human salivary (1.0 unit/ml, Sigma, St. Louis, MO, USA) was premixed with 100 µl of a WSE. After pre-incubation at 37°C for 5 min, 250 µl of 1% starch was added as a substrate in phosphate buffer (pH 6.8) to start the reaction. The reaction was performed at 37°C for 5 min and terminated by the addition of 200 µl of DNS reagent (1% 3,5dinitrosalicylic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The reaction mixture was heated for 15 min at 100°C and diluted with 2 ml of distilled water in an ice bath. ⍺-Amylase activity was determined by measuring absorbance at 540 nm. 2.7. α-Glucosidase inhibition assay Prior to the assay, stored WSEs were prepared as described above. ⍺-Glucosidase inhibition assay was carried out according to the method detailed elsewhere (Kim et al., 2004) with some modifications. ⍺-Glucosidase (1 unit/ml, Sigma) was dissolved in 100 µl of 0.1 M potassium phosphate buffer (pH 6.8) and mixed with 50 µl of an WSE. After pre-incubation at 37°C for 10

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min, 50 µl of 5 mM p-nitrophenyl ⍺-D-glucopyranoside (pNPG) was added as the substrate. The enzymatic reaction was performed at 37°C for 30 min and stopped by the addition of 1 ml of 0.1 M Na2CO3. ⍺-Glucosidase activity was determined by measuring the release of p-nitrophenol from pNPG at 400 nm. A solution without the WSE sample was used as a control. A solution without the substrate was used as a blank. The inhibition percentage was calculated as follows:

2.8. Antioxidant activity Prior to the antioxidant activity assays, stored WSEs were prepared as described above. 2.8.1. Radical scavenging rate by DPPH assay The determination of radical scavenging activity by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was performed according to Elfahri, Vasiljevic, Yeager, and Donkor (2016). Briefly, 800 μl of the DPPH reagent (0.1 mM DPPH dissolved in 95% methanol) was added to 200 μl of an WSE in glass test tubes. The samples were shaken vigorously and incubated in the dark at room temperature for 30 min. Methanol was used as a blank. The absorbance of the incubated samples was measured at 517 nm. The percentage of radical scavenging activity was expressed as scavenging rate %:

2.8.2. Radical scavenging rate by ABTS assay Radical scavenging rate by the 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS•+) method was determined according to the procedure of Sah et al. (2014). A stock solution of ABTS was prepared by mixing stock solutions of 7.4 mM ABTS aqueous solution and 2.6 mM potassium persulphate aqueous solution in equal quantities (molar ratio = 1:0.35) and allowing them to react for 12 h in the dark at room temperature. A fresh ABTS reagent was prepared by mixing 1 ml of ABTS•+ solution with 50–60 ml of the buffered methanol to obtain an absorbance of 0.70±0.02 at 734 nm after equilibration at 30 °C. Twenty microliters of a properly diluted WSE in double

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distilled (dd) water were added to 2 ml of an ABTS reagent and incubated at 30°C for 6 min. The absorbance of the mixture was measured at 734 nm. Similarly, 20 µl of dd-water was used instead of the sample for the blank. Radical scavenging activity was calculated as follows:

2.9. ACE-inhibition ACE-inhibition activity of WSEs was assayed according to the method detailed elsewhere (Ayyash & Shah, 2011). Briefly, a 5 ml of WSEs was freeze-dried and then dissolved in 1 ml of Tris buffer (50 mM, pH 8.3) containing 300 mM NaCl. An ACE enzyme (from rabbit lung) and hippurylhistidyl-leucine (HHL) were purchased from Sigma-Aldrich and prepared in Tris buffer. The assay consisted of 100 μl of 3.0 mM HHL, 100 μl of 1.25-mU/ml ACE enzyme, and 100 μl of a dissolved WSE sample. The mixture was placed in a glass tube and then incubated for 30 min at 37°C in a water bath without mixing and then for an additional 30 min after mixing. Glacial acetic acid (200 μl) was added to stop the ACE enzyme activity. The mixture was kept at −20°C to be analyzed using HPLC. The resulting hippuric acid (HA) from the previous reaction was determined using HPLC. An external standard curve was prepared to quantify the hippuric acid in assay samples. An aliquot (20 μl) of the mixture was injected into the HPLC system from ThermoFisher Scientific (Waltham, MA USA). The system was fitted with a reverse-phase column (C18, 250-mm length × 4.6-mm diameter, 5-μm diameter of the HPLC column particles inside with a guard column (C18 4 × 3.0 mm). The separation was conducted at room temperature (~22°C) at a flow rate of 0.8 ml/min. The mobile phase was an isocratic system consisting of 12.5% (v/v) acetonitrile (Sigma) in Milli-Q water, and the pH was adjusted to pH 3.0 using glacial acetic acid (Sigma). The detection device was a UV-Vis detector set at 228 nm. The control reaction mixture contained 100 μl of buffer instead of the assay sample; the control was expected to liberate the maximum amount of hippuric acid from the substrate due to uninhibited ACE activity. The percentage inhibition of enzyme activity was calculated as follows:

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2.10. Anticancer activity Prior to the assay, WSEs were filtered through Macrosep® Advance Spin Filter 3 kDa (Pall Corporation, NY, USA). Filtrates were assayed against Caco-2 and MCF-7 carcinoma cell lines according to the method detailed by Elfahri et al. (2016). Cell lines used for the cell cytotoxicity assay included colon cancer cell line (Caco-2, provided by Dr. Carine Platat, Nutrition & Health Department, UAEU); breast cancer cell line MCF-7 (Faculty of Medicine and Health Sciences, UAEU). Cells were grown in a humidified incubator (37°C and 5% CO2) in Dulbecco’s modified Eagle’s medium (Gibco ®, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated bovine serum (FBS) and 1% penicillin and streptomycin (Invitrogen). Cell lines were initially passaged and counted in a haemocytometer prior to seeding. Cells were seeded at 1 x 103 concentration in a 96-well plate and incubated in an appropriate medium overnight. For the treatment, 25 µl of filtered WSEs was added to each well and incubated for 72 hours at 37°C and 5% CO2. Afterwards, 20 µl of a pre-warmed Abcam Cell Cytotoxicity Assay Kit (ab112118, Cambridge, MA, USA) was added and incubated at 37°C for a minimum of 5 hours. Each sample was assayed in triplicate. The absorbance was measured at 570 nm and 605 nm according to the Kit’s manufacturer’s protocol. The ratio of OD570/OD605 nm was used to determine cell viability in each well. The proliferative inhibition was calculated as follows:

where Rsample is the absorbance ratio of OD570/OD605 in the presence of the WSE. Rctrl is the absorbance ratio of OD570/OD605 in the absence of the WSE (vehicle control). Ro is the averaged background (non-cell control) absorbance ratio of OD570/OD605. 2.11. Statistical analysis All fermentation experiments (2 milk types x 4 probiotics x 4 storage time) were conducted in triplicate. Each sample was assayed in duplicate unless otherwise mentioned. Two-way ANOVA

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was carried out to investigate the effect of milk type, probiotic strain and storage period on fermented milk parameters (p < 0.05). Mean comparisons were performed using Tukey’s test (p < 0.05). Pearson’s test was carried out to find any correlations between parameters for the same milk and the same probiotic bacterium. Correlation coefficients are presented in supplementary Table S1 and S2 for fermented bovine and camel milks, respectively. All statistical analyses were carried out using Minitab 17.0 software (Minitab Inc., PA, USA). 3. Results and Discussion 3.1. Bacterial population, pH and TA The bacterial populations (log10 CFU/ml), pH values and TA (%) of fermented bovine and camel milk during 21 days of storage at 4C are presented in Table 1. In both milk types, the bacterial populations of all strains were maintained at > 8.0 logs during the storage period. The Lp.DSM population in bovine milk was higher (p < 0.05) compared with camel milk. However, no significant changes occurred in bacterial populations of Lr.K777, Lp.K77 and Lp.K772 in bovine milk compared with camel milk. In bovine milk, the changes in bacterial populations during the storage period were insignificant (p > 0.05), except Lp.DSM which dropped during storage (p < 0.05). Table 1 shows that pH values after 24 h (Day 0) dropped from ~ 6.5 to 4.3, 5.2, 5.1, and 5.4 in fermented bovine milk by Lp.DSM, Lr.K777, Lp.K772 and Lp.K779, respectively. The pH values of camel milk fermented by Lr.K77, Lp.K779 and Lp.K772 were lower (p < 0.05) compared with bovine milk. In general, pH declined slightly during 21 days of storage for both milk types (Table 1). In bovine milk, TA% of Lp.DSM was higher (p < 0.05) than other bacterial strains during the storage period (Table 1). Camel milk fermented by Lr.K777, Lp.K772 and Lp.K779 had higher (p < 0.05) TA% compared with bovine milk. According to FAO/WHO (2002) definition of probiotics, a high probiotic population in fermented milk especially at time of consumption is regarded as necessary. Our results are in accordance with the FAO/WHO (2002) guidelines for probiotics. The present results also agree with findings by

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Moslehishad et al. (2013) who reported that bacterial population maintained > 8.0 logs in camel and bovine milks fermented by Lb. rhamnosus PTCC 1637. Varga, Süle, and Nagy (2014) also reported that the population of Bifidobacterium animalis sustained > 6.0 logs during storage for fermented camel and cow milks. The relatively lower bacterial population of Lp.DSM in fermented camel milk may be attributed to the lesser ability of Lp.DSM to overcome the antimicrobial components in fermented camel milk. Elagamy, Ruppanner, Ismail, Champagne, and Assaf (1996) found that the antimicrobial quantities in camel milk were higher than bovine milk. We postulate that Lr.K777, Lp.K772 and Lp.K779 were more compatible with camel milk than Lp.DSM. This was likely due to the fact that Lr.K777, Lp.K772 and Lp.K779 were isolated from camel milk (Abushelaibi et al., 2017). The current pH and TA% results had negative (inverse) correlations for each fermented milk (Table S1). The lower pH and higher TA% of both fermented milks by Lp.DSM could be attributed to the efficiency of Lp.DSM to ferment lactose into lactic acid. Lactose fermentation by Lp.DSM was via homofermentation (a known feature of Lb. plantarum). Homofermentation produces more lactic acid than heterofermentation (Marshall & Tamime, 1997). Although camel milk is known to have more natural antimicrobial substances than bovine milk (Elagamy et al., 1996), Lr.K777, Lp.K772 and Lp.K779 had a further capacity to produce more organic acids (higher TA%) in fermented camel than bovine milk. The TA% results agree with the finding of Monteagudo-Mera et al. (2011) who reported a significant difference in acidity between fermented ewe’s and cow’s milk after 6 h of fermentation using the same strain. Our results also agree with the report of Abu-Tarboush (1996) who showed clear differences in pH between fermented camel and cow milks. Mymensingh (2007) found a significant difference in acidity of Dahi yogurt prepared from cow, goat and buffalo milks, and fermented by the same strain. However, these studies did not provide sufficient interpretations for their findings. On the other hand, Shori and Baba (2014) and Gomes et al. (2013) reported insignificant differences in acidity between fermented cow and camel or ewe milks, respectively. We postulate that the higher TA% in fermented camel milk may be attributed to the

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adaptation of Lr.K777, Lp.K772 and Lp.K779 strains to camel milk because these strains were isolated from camel milk. 3.2. Proteolysis assessment by OPA Figure 1 displays the proteolytic patterns of fermented bovine and camel milks during 21 day of storage at 4C. Interestingly, camel milk fermented by all strains had greater (p < 0.05) OPA readings than bovine milk during the storage period. Lr.K777 and Lp.K779 exhibited higher OPA absorbance than Lp.DSM and Lp.K772 in fermented camel milk. The proteolytic activities of all strains were significantly (p < 0.05) lower in bovine milk compared with camel milk. In general, proteolytic activity in both fermented milk types increased significantly (p < 0.05) with prolonged storage (Fig. 1). OPA correlated positively with TA% in camel milk fermented by Lp.DSM (r = 0.832), Lr.K777 (r = 0.532), Lp.K772 (r = 0.438) and Lp.K779 (r = 0.500) (Table S2). Pearson’s test showed relatively weak positive correlations between OPA and TA% in fermented bovine milk (Table S1). Proteolytic activity is one tentative indicator of health-promoting benefits that could be claimed in fermented product. OPA assesses free amino acids and small peptides in fermented products (McSweeney & Fox, 1997). One major group of bioactive compounds in fermented dairy products are oligopeptides (Park, 2009). Consequently, camel milk fermented by Lr.K777 and Lp.K779 could have enhanced bioactive functionality due to high OPA results compared with other strains. Several proteolytic enzymes (proteinases, peptidases and aminopeptidases) are produced by Lactobacillus spp. (Park, 2009). The higher OPA in fermented camel than bovine milk (Fig. 1) may be attributed to the susceptibility of camel milk caseins to proteolytic enzymes produced by the Lactobacillus spp. strains used. Our results agree with those of Shori and Baba (2014) who reported higher OPA values in fermented plain (without garlic extract)-camel milk than plain-cow’s milk. Abu-Tarboush (1996) also reported higher proteolytic activity in camel milk fermented by Streptococcus thermophilus and Lb. bulguricus than fermented bovine milk. The comparatively strong positive correlations between OPA and TA% in fermented camel milk support the

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assumption that proteolytic activity and lactose metabolism by all strains increased in camel milk compared with bovine milk. 3.3. Antioxidant activity determined by ABTS and DPPH (%) The scavenging rates of fermented bovine and camel milks by the four Lactobacillus spp. strains are presented in Figure 2A for ABTS and Figure 2B for DPPH. In general, the antioxidant in fermented bovine milk assessed by ABTS were markedly lower (p < 0.05) than in camel milk. The ABTS scavenging rate in fermented bovine ranged from 20% to 30%, whereas the rate in fermented camel milk ranged from > 30% up to 70% (Fig. 2A). Camel milk fermented by Lr.K777 and Lp.K779 possessed the highest ABTS rates compared with Lp.DSM and Lp.K772.

Fig. 2A exhibits that antioxidant activity measured by ABTS assay in both fermented milks increased (p < 0.05) during storage. The scavenging rate by DPPH assay in all fermented bovine milks increased (p < 0.05) during 14 days of storage, followed by significant decline at the end of the storage period (Fig. 2B). In contrast, antioxidant activity determined by DPPH assay continued rising in fermented camel milk during 21 days of storage. Again, camel milk fermented by Lr.K777 and Lp.K779 possessed the highest DPPH rates (Fig. 2B). The antioxidant activity determined by DPHH assay positively correlated with ABTS, OPA, and TA% in fermented camel milk by Lr.K777, Lp.K779 and Lp.K772 (Table S2). Different trends in antioxidant activity of camel and bovine milks could be attributed to the different mechanisms of action for the two assays used (ABTS and DPPH).

Bioactive compounds in foods especially fermented dairy products play a crucial role in reducing the impact of reactive oxygen species such as superoxide ( ●O2‾, ●OOH), hydroxyl (●OH), and peroxyl (ROO●) radicals formed by oxidatively stressed cells (Benbrook, 2005). These bioactive compounds especially protein-derived peptides possess the ability to donate electrons to neutralize free radicals. Moreover, the presence of several amino acid residues in the peptide chains can

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enhance antioxidant properties (Aluko, 2012). Bioactive peptides, as antioxidant agents, in fermented milks prevent enzymatic and non-enzymatic peroxidation of essential fatty acids (ElSalam & El-Shibiny, 2013). The higher antioxidant activity in fermented camel milk than bovine milk may be attributed to the higher proteolysis rate (high OPA, Fig. 1) and the nature of bioactive peptides in fermented camel milk. Similarly, Moslehishad et al. (2013) reported high antioxidant activity determined by ABTS assay in camel milk than bovine milk fermented by Lb. rhamnosus PTCC 1637. Shori (2013) also reported similar antioxidant activity by DPPH assay in fermented camel compared with bovine milk. 3.4. α-Amylase and α-glucosidase inhibition The inhibition results of α-amylase and α-glucosidase of bovine and camel milks fermented by Lp.DSM, Lr.K777, Lp.K779 and Lp.K772 are illustrated in Figure 3A and 3B, respectively. In general, the α-amylase inhibitions were > 34% in both milk types fermented by all strains during all storage, except camel milk fermented by Lp.K772 (Fig. 3A). The inhibition of α-amylase increased (p < 0.05) in both fermented milks with prolonged storage, except Lp.K779 in bovine milk. Comparatively, the percentages of α-amylase inhibitions in camel milk fermented by Lp.K772 were lower (p < 0.05) than bovine milk (Fig. 3A). The inhibitions of α-glucosidase in both milks fermented by Lp.K772 were higher (p < 0.05) than other counterparts in the same milk type (Fig. 3B). α-Glucosidase inhibitions increased (p < 0.05) during storage. Pearson’s test showed that αamylase and α-glucosidase inhibitions strongly correlated in bovine milk fermented by all strains except Lp.K779 (Table S1). The correlation between α-amylase and α-glucosidase inhibitions was weak in camel milk fermented by Lp.K772 and Lp.K779. α-Amylase and α-glucosidase inhibitions in bovine milk had a weak coefficient of correlation r < 0.400 with OPA, but Lp.K772 had r > 0.550 (Table S1). The inhibition of ⍺-amylase and ⍺-glucosidase activity can be considered as an effective approach to controlling diabetes via diminishing carbohydrate hydrolysis (Donkor, Stojanovska, Ginn, Ashton, & Vasiljevic, 2012). The general inhibition of both ⍺-amylase and ⍺-glucosidase enzymes

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could be attributed to bioactive peptides, particularly smaller ones (Gomes da Cruz, Buriti, Batista de Souza, Fonseca Faria, & Isay Saad, 2009), produced as a result of proteolytic enzymes secreted by the probiotic strains used. Our results contradict with findings by Shori and Baba (2014) who reported that fermented plain-camel milk had higher α-amylase and α-glucosidase inhibitions than fermented plain-cow’s milk. We assume that this contradiction may be attributed to differences in the bioactive peptides released in fermented milk between the current study and that of Shori and Baba (2014). The differences in LAB species and strains could lead to variations in proteolytic activity qualitatively and quantitatively (El-Salam & El-Shibiny, 2013). Although Lp.K772 had relatively low OPA (Fig. 1), the marked α-glucosidase inhibitions in both milk types fermented by Lp.K772 may be attributed to the nature of bioactive peptides released in milk fermented by Lp.K772. Moslehishad et al. (2013) reported that the peptide functionality varied according to the peptide fractions. The authors demonstrated that peptide fractions < 3 kDa had a higher bioactivity than fractions of 3 to 5 kDa. The moderate correlation between OPA and the inhibitions of α-amylase and α-glucosidase supports the weak proteolytic activity and lactose metabolisms (low OPA and TA%) during storage. 3.5. ACE-inhibition activity Figure 4 displays that ACE-inhibitions in camel milk fermented by Lr.K777, Lp.K779 and Lp.K772 were significantly (p < 0.05) higher than bovine milk during all storage periods. The highest ACEinhibition in camel milk fermented by Lr.K777 was > 80%, whereas the ACE-inhibition in bovine milk did not exceed 50% after 21 days of storage (Fig. 4). Lp.DSM had markedly lower (p < 0.05) ACE-inhibition in camel milk than bovine milk. The increases in ACE-inhibition activity in all fermented milk types during prolonged storage were significant (p < 0.05). In general, ACEinhibition exhibited positive correlations with proteolytic activity (OPA) in all fermented milk types, except Lp.DSM (Table S1 and S2). ACE-inhibition is an in-vitro indicator for antihypertensive properties of fermented dairy products (Gobbetti, Minervini, & Rizzello, 2004). The higher ACE-inhibition in camel milk may be due to

15

high proteolytic activity (OPA, Fig. 1). This supports our assumption that camel milk casein proteins may be more susceptible to hydrolysis by proteolytic enzymes produced by the present strains except Lp.DSM. Moreover, the peptides’ nature in camel milk fermented by Lr.K777, Lp.K779 and Lp.K772 may contribute to the higher ACE-inhibition than bovine milk. The higher proline content in camel’s caseins may explain the high ACE-inhibition in fermented camel milk (El-Salam & El-Shibiny, 2013). These results are in accordance with those reported by (Moslehishad et al., 2013) who reported greater ACE-inhibition in camel milk than bovine milk fermented by Lb. rhamnosus. The ACE-inhibitions of camel milk fermented by Lr.K777, Lp.K779 and Lp.K772 were greater than those reported by Alhaj et al. (2017) who fermented camel milk by Lb. acidophilus and Streptococcus thermophiles or Lb. helveticus. Notably, the ACE-inhibition activity in camel milk fermented by Lr.K777 correlated well with DPPH (r = 0.774), ABTS (r = 0.719), α-amylase (r = 0.669) and α-glucosidase (r = 0.791). This correlation suggests that the peptides released by the proteolytic action of Lp.K777 may possess multi-functional bioactivity (Harnedy & FitzGerald, 2012). 3.6. Anticancer activity The proliferation inhibition, as an anticancer indicator, of WSEs (< 3 kDa) of fermented bovine and camel milks against Caco-2, MCF-7 and HELA carcinoma cell lines are presented in Table 2. The proliferations of Caco-2, MCF-7 and HELA cells were more inhibited (p < 0.05) when treated with WSEs from camel milk compared with bovine milk fermented by all present strains except Lp.DSM. The Lp.DSM showed lower proliferation inhibition than counterparts in fermented camel milk. However, the proliferation inhibitions of camel milks fermented by Lr.K777, Lp.K779 and Lp.K772 differed insignificantly. Table 2 shows that the percentages of proliferation inhibitions of WSEs in both milk types fermented by Lr.K777, Lp.K779 and Lp.K772 were in the following order: HELA > MCF-7 > Caco-2 cell lines. In camel milk fermented by Lp.K779 and Lp.K772, Pearson’s test exhibited a positive correlation between OPA and proliferation inhibition of Caco-2 (r = 0.780 and 0.831),

16

MCF-7 (r = 0.706 and 0.811) and HELA (r = 0.657 and 0.808). The correlations between proliferation inhibitions of three cell lines and ACE-inhibition in fermented camel milk ranged from r = 0.500 to 0.700 (Table S2). The antiproliferative effects of these peptides were greater on cervical cancer cells (HELA) and breast cancer cells (MCF-7) than colon cancer cells (Caco-2).

The antiproliferation activity of milk peptides have been reported by several workers (Park, 2009). Several hypotheses have been proposed to explain the mechanism(s) of the antiproliferative activity of milk peptides. Competition between the peptides and cancer growth factors for cancer cellmembrane receptors is one of these hypotheses. Another hypothesis is that the released peptides have specific cytotoxicity on cancer cells which induces apoptosis (Pessione & Cirrincione, 2016; Picot et al., 2006). Therefore, the high antiproliferation activity of camel milk fermented Lr.K777, Lp.K779 and Lp.K772 may be attributed to the greater competition capability of peptides derived from fermented camel milk compared with those from fermented bovine milk. The specific cytotoxicity of the fermented camel milk peptides to induce apoptosis may also contribute to the interpretation. Several researchers have reported antiproliferation activity of camel milk but, to the best of our knowledge, without fermentation or comparisons with bovine milk (Habib, Ibrahim, SchneiderStock, & Hassan, 2013; Korashy, Maayah, Abd-Allah, El-Kadi, & Alhaider, 2012; Magjeed, 2005). The positive correlation between proliferation inhibition and ACE-inhibition suggests that peptides derived from fermented camel milk have multifunctional bioactivity. 4. Conclusion This study showed that health-promoting benefits particularly antioxidant, ACE-inhibition and antiproliferative activity of WSEs from fermented camel milk were markedly higher than those from fermented bovine milk. Moreover, the WSE from camel milk fermented by indigenous Lactobacillus spp (Lr.K777, Lp.K779 and Lp.K772) exhibited greater potential health benefits than a non-indigenous strain (Lp.DSM). The fermented camel milk requires more investigations to

17

identify proteolytic pathways of the indigenous lactic acid bacteria isolated from camel milk and to characterize the bioactive peptides derived from fermented camel milk. The characteristics of Lactobacillus spp. isolated from camel milk make them promising starter cultures to the dairy food industry. Acknowledgement Authors are thankful to Dr. Jaleel for handling antiproliferation assay. Authors acknowledge the financial support by United Arab Emirates University (UAEU) via Start-up project G00001216. References Abu-Tarboush, H. (1996). Comparison of associative growth and proteolytic activity of yogurt starters in whole milk from camels and cows. Journal of Dairy Science, 79(3), 366-371. Abushelaibi, A., Al-Mahadin, S., El-Tarabily, K., Shah, N. P., & Ayyash, M. (2017). Characterization of potential probiotic lactic acid bacteria isolated from camel milk. LWTFood Science and Technology, 79, 316-325. doi:10.1016/j.lwt.2017.01.041 Al haj, O. A., & Al Kanhal, H. A. (2010). Compositional, technological and nutritional aspects of dromedary camel milk. International Dairy Journal, 20(12), 811-821. doi:10.1016/j.idairyj.2010.04.003 Alhaj, O. A., Metwalli, A. A., Ismail, E. A., Ali, H. S., Al‐Khalifa, A. S., & Kanekanian, A. D. (2017). Angiotensin converting enzyme‐inhibitory activity and antimicrobial effect of fermented camel milk (Camelus dromedarius). International Journal of Dairy Technology. Aluko, R. E. (2012). Functional foods and nutraceuticals. New York, USA: Springer New York. Anderson, R. C., Cookson, A. L., McNabb, W. C., Kelly, W. J., & Roy, N. C. (2010). Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function. FEMS Microbiology Letters, 309(2), 184-192. doi:10.1111/j.1574-6968.2010.02038.x Ayyash, M. M., & Shah, N. P. (2011). Proteolysis of low-moisture Mozzarella cheese as affected by substitution of NaCl with KCl. Journal of Dairy Science, 94(8), 3769-3777. Benbrook, C. M. (2005). Elevating antioxidant levels in food through organic farming and food processing. Organic Center, State of Science Review, January. Donkor, O. N., Stojanovska, L., Ginn, P., Ashton, J., & Vasiljevic, T. (2012). Germinated grains sources of bioactive compounds. Food Chemistry, 135(3), 950-959. doi:10.1016/j.foodchem.2012.05.058 El-Salam, M. H. A., & El-Shibiny, S. (2013). Bioactive peptides of buffalo, camel, goat, sheep, mare, and yak milks and milk products. Food Reviews International, 29(1), 1-23. doi:10.1080/87559129.2012.692137 Elagamy, E. (2006). Camel milk. In Y. W. Park & G. F. Haenlein (Eds.), Handbook of Milk of Nonbovine Mammals. Ames, Iowa, USA: Blackwell Publishing Professional. page #? Elagamy, E., Ruppanner, R., Ismail, A., Champagne, C., & Assaf, R. (1996). Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel's milk. International Dairy Journal, 6(2), 129-145. Elfahri, K. R., Vasiljevic, T., Yeager, T., & Donkor, O. N. (2016). Anti-colon cancer and antioxidant activities of bovine skim milk fermented by selected Lactobacillus helveticus strains. Journal of Dairy Science, 99(1), 31-40. doi:10.3168/jds.2015-10160 FAO/WHO. (2002). FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food. Geneva, swiztherland: World health organization. .

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Fukuda, K. (2013). Camel milk. In Y. W. Park & F. H. George (Eds.), Milk and Dairy Products in Human Nutrition: Production, Composition and Health (pp. 172-199). Chichester, UK: John Wiley & Sons. Gobbetti, M., Minervini, F., & Rizzello, C. G. (2004). Angiotensin i-converting-enzyme-inhibitory and antimicrobial bioactive peptides. International Journal of Dairy Technology, 57(2-3), 173-188. Gomes da Cruz, A., Buriti, F. C. A., Batista de Souza, C. H., Fonseca Faria, J. A., & Isay Saad, S. M. (2009). Probiotic cheese: Health benefits, technological and stability aspects. Trends in Food Science and Technology, 20(8), 344-354. doi:10.1016/j.tifs.2009.05.001 Gomes, J. J. L., Duarte, A. M., Batista, A. S. M., de Figueiredo, R. M. F., de Sousa, E. P., de Souza, E. L., & do Egypto, R. d. C. R. (2013). Physicochemical and sensory properties of fermented dairy beverages made with goat's milk, cow's milk and a mixture of the two milks. LWT-Food Science and Technology, 54(1), 18-24. Habib, H. M., Ibrahim, W. H., Schneider-Stock, R., & Hassan, H. M. (2013). Camel milk lactoferrin reduces the proliferation of colorectal cancer cells and exerts antioxidant and DNA damage inhibitory activities. Food Chemistry, 141(1), 148-152. Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods, 4(1), 6-24. Ibrahim, A. H., & Khalifa, S. A. (2015). Effect of freeze-drying on camel's milk nutritional properties. International Food Research Journal, 22(4), 1438-1445. Khalesi, M., Salami, M., Moslehishad, M., Winterburn, J., & Moosavi-Movahedi, A. A. (2017). Biomolecular content of camel milk: A traditional superfood towards future healthcare industry. Trends in Food Science and Technology, 62, 49-58. doi:10.1016/j.tifs.2017.02.004 Kim, Y. M., Wang, M. H., & Rhee, H. I. (2004). A novel α-glucosidase inhibitor from pine bark. Carbohydrate Research, 339(3), 715-717. doi:10.1016/j.carres.2003.11.005 Korashy, H. M., Maayah, Z. H., Abd-Allah, A. R., El-Kadi, A. O., & Alhaider, A. A. (2012). Camel milk triggers apoptotic signaling pathways in human hepatoma HEPG2 and breast cancer MCF7 cell lines through transcriptional mechanism. BioMed Research International, 2012. Magjeed, N. A. (2005). Corrective effect of milk camel on some cancer biomarkers in blood of rats intoxicated with aflatoxin b1. Journal of the Saudi Chemical society, 9, 253-263. Marshall, V., & Tamime, A. (1997). Physiology and biochemistry of fermented milks. In Microbiology and Biochemistry of Cheese and Fermented Milk (pp. 153-192): Springer. place ? McSweeney, P., & Fox, P. (1997). Chemical methods for the characterization of proteolysis in cheese during ripening. Le Lait, 77(1), 41-76. Mihic, T., Rainkie, D., Wilby, K. J., & Pawluk, S. A. (2016). The therapeutic effects of camel milk: A systematic review of animal and human trials. Journal of Evidence-Based Complementary and Alternative Medicine, 21(4), NP110-NP126. doi:10.1177/2156587216658846 Monteagudo-Mera, A., Caro, I., Rodríguez-Aparicio, L., Rúa, J., Ferrero, M., & García-Armesto, M. (2011). Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products. Journal of Food Protection, 74(8), 1379-1386. Moslehishad, M., Ehsani, M. R., Salami, M., Mirdamadi, S., Ezzatpanah, H., Naslaji, A. N., & Moosavi-Movahedi, A. A. (2013). The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus ptcc 1637. International Dairy Journal, 29(2), 82-87. doi:10.1016/j.idairyj.2012.10.015 Mostafa, T. H., Abd El-Hamed, A. A., & Almetwaly, H. A. (2013). Effect of some nutritional treatments on productive performance of she-camels. Journal of Camel Practice and Research, 20(2), 217-228. Mymensingh, B. (2007). A comparative study on the quality of dahi (yoghurt) prepared from cow, goat and buffalo milk. International Journal of Dairy Science, 2(3), 260-267.

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Park, Y. W. (2009). Bioactive Components in Milk and Dairy products. editor? publisher? place? page #? Pessione, E., & Cirrincione, S. (2016). Bioactive molecules released in food by lactic acid bacteria: Encrypted peptides and biogenic amines. Frontiers in Microbiology, 7(JUN). doi:10.3389/fmicb.2016.00876. vol and page #? Picot, L., Bordenave, S., Didelot, S., Fruitier-Arnaudin, I., Sannier, F., Thorkelsson, G., . . . Piot, J. (2006). Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochemistry, 41(5), 1217-1222. Sah, B. N. P., Vasiljevic, T., McKechnie, S., & Donkor, O. N. (2014). Effect of probiotics on antioxidant and antimutagenic activities of crude peptide extract from yogurt. Food Chemistry, 156, 264-270. doi:10.1016/j.foodchem.2014.01.105 Sayed, R. G., Ahmed, A. A.-H., & Sayed, M. (2013). Nutritional value and sanitary evaluation of raw camel's milk. Emirates Journal of Food and Agriculture, 26(4), 317-326. doi:10.9755/ejfa.v26i4.16158 Shori, A. B. (2013). Antioxidant activity and viability of lactic acid bacteria in soybean-yogurt made from cow and camel milk. Journal of Taibah University for Science, 7(4), 202-208. Shori, A. B., & Baba, A. S. (2014). Comparative antioxidant activity, proteolysis and in vitro αamylase and α-glucosidase inhibition of Allium sativum-yogurts made from cow and camel milk. Journal of Saudi Chemical Society, 18(5), 456-463. doi:10.1016/j.jscs.2011.09.014 Varga, L., Süle, J., & Nagy, P. (2014). Short communication: Viability of culture organisms in honey-enriched acidophilus-bifidus-thermophilus (abt)-type fermented camel milk. Journal of Dairy Science, 97(11), 6814-6818. doi:10.3168/jds.2014-8300

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Figure Captions Figure 1: Proteolytic activity of fermented camel and bovine milks measured by OPA at 340 nm.

Figure 2: Antioxidant activity determined by ABTS (A) and DPPH (B) of fermented camel and bovine milks. Values are the mean ± standard deviation of n=6.

Figure 3: ⍺-Amylase (A) and ⍺-glucosidase (B) inhibition (%) of fermented camel and bovine milks. Values are the mean ± standard deviation of n=6.

Figure 4: ACE-inhibition of fermented camel and bovine milks. Values are the mean ± standard deviation of n=6.

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Table 1: Bacterial population, pH values and titrable acidity of fermented camel and bovine milks Bovine milk Storage Lp.DSM Lr.K777 Lp.K772 Lp.K779 Lp.DSM (day) Log10 CFU/ml

Camel milk Lr.K777 Lp.K772

Lp.K779

0 7 14 21

9.7±0.11Aa1 9.8±0.02Aba 9.6±0.04Ba 9.5±0.03Ba

9.1±0.02Ab 9.1±0.05Ab 9.1±0.03Ab 9.2±0.05Ab

9.2±0.03Ab 9.3±0.02Ab 9.2±0.06Ab 9.1±0.02Ab

9.2±0.04Ab 9.2±0.03Aa 8.5±0.06Ac 9.1±0.07Aab 8.7±0.08Ac 8.8±0.04Bb 8.5±0.05Ac 8.8±0.05Bb pH values

9.2±0.03Aa 9.4±0.03Aa 9.4±0.03Aa 9.3±0.05Aba

9.0±0.05Aa 8.9±0.07Ab 8.8±0.01Ab 8.8±0.10Ab

9.0±0.11Aa 8.4±0.09Bb 8.8±0.04Bb 8.3±0.05Bb

0 7 14 21

4.3±0.01Ad 4.3±0.01Ac 4.2±0.01Bc 4.2±0.01Bc

5.2±0.02Bb 5.4±0.01Aa 5.4±0.01Ab 5.1±0.01Cb

5.1±0.01Dc 5.3±0.01Bb 5.4±0.01Ab 5.2±0.01Ca

5.4±0.02Aa 5.4±0.01Aa 5.3±0.01Bc 5.1±0.04Cab

4.6±0.01Ac 4.6±0.01Ab 4.5±0.01Bc 4.3±0.02Cc

5.0±0.00Ab 4.9±0.02Ba 4.7±0.00Bb 4.8±0.01Ba

4.9±0.01Aa 4.8±0.01Ba 4.8±0.02Ba 4.6±0.01Cb

3.3±0.04Aa 3.2±0.03Aa 3.4±0.02Aa 3.4±0.09Aa

1.8±0.06Bc 1.9±0.03Bd 1.8±0.04Bd 2.2±0.08Ad

4.4±0.01Ad 4.3±0.02Bc 4.0±0.01Cd 3.9±0.02Dd

Titratable acidity (TA %) 0 3.8±0.04Aa 2.2±0.06ABb 1.9±0.08Bc 1.7±0.03Bc 3.4±0.09Ba 2.7±0.05Bb 7 3.8±0.04Aa 2.0±0.06Bb 2.3±0.10Ab 1.7±0.06Bc 3.9±0.06Aa 2.6±0.02Bb 14 3.8±0.03Aa 2.1±0.05ABb 2.5±0.04Ab 1.6±0.01Bc 3.8±0.03Aa 3.1±0.07Ab 21 3.8±0.05Aa 2.3±0.05Ab 2.5±0.03Ab 2.0±0.03Ac 4.0±0.04Aa 3.0±0.09Ab 1 Values are the mean ± standard deviation of n=6. a-d Mean values in same row, in the same milk, with different lowercase superscripts differ significantly (p < 0.05). A-D Mean values in the same column with different uppercase superscripts differ significantly (p < 0.05).

22

Table 2: Proliferation inhibition (%) of fermented camel and bovine milks against three cancer cell lines Bovine milk Camel milk Lr.K777 Lp.K772 Lp.K779 Lp.DSM Lr.K777 Lp.K772 Storage (days) Lp.DSM

Lp.K779

Caco-2 0 7 14 21

Aa1

38.8±0.2 38.7±0.2Aa 38.7±0.3Ab 39.4±1.4Aab

Cc

36.6±0.3 36.0±0.2Cc 41.6±0.2Ba 40.8±0.1Aa

Ab

37.6±0.2 38.1±0.1Ab 37.8±0.3Ac 37.7±0.5Ab

Ab

37.8±0.5 37.6±0.1Ab 37.9±0.2Abc 37.3±0.3Ab

37.5±0.3BCa 37.2±0.2Ca 39.5±0.8ABb 39.8±0.3Ac

37.5±0.8Ba 37.2±0.6Ba 45.1±0.1Aa 45.6±0.5Ab

36.9±0.6Ba 36.5±0.1Ba 45.2±0.2Aa 44.8±0.2Aa

37.0±0.1Ca 37.2±0.3Ca 44.9±0.2Ba 45.8±0.2Aa

42.6±1.3Ba 41.7±0.2Bb 57.5±4.4Aa 55.4±2.9Aa

42.7±1.4Ba 44.5±0.5Ba 57.1±4.3Aa 56.1±1.9Aa

41.6±1.3Ba 42.0±1.2Bb 58.9±2.1Aa 56.2±0.3Aa

44.4±0.8Ba 44.2±0.3Bb 65.0±0.3Aa 65.4±0.8Aa

44.1±0.6Ca 44.6±0.5Cb 67.5±0.4Ba 66.6±0.7Aa

MCF-7 0 7 14 21

44.9±0.3Aa 40.8±1.7Aa 43.5±1.2Ab 44.8±0.6Ab

40.3±0.6Cb 43.3±1.0BCa 46.4±1.0ABab 49.8±0.6Aab

45.7±0.7Ba 42.4±1.7Ba 45.5±1.0Bab 51.9±2.1Aa

44.7±0.7Aa 41.4±3.2Aa 47.4±0.7Aa 46.9±2.3Aab

41.5±1.3Ba 40.7±0.4Bb 46.7±1.7Ab 47.9±0.4Ab HELA

0 44.2±0.1Ab 43.7±0.1Cb 44.7±1.3BCab 50.4±3.6ABa 42.1±0.9Ba 47.2±3.1Ca 7 53.7±3.7Aa 43.2±0.3Cb 43.4±0.3Cb 46.2±0.5Bb 42.7±0.3Bc 52.9±0.5BCa 14 50.1±1.1Ab 57.7±0.4Aa 49.8±0.8ABab 58.2±0.7Aa 51.9±0.8Ac 63.6±1.1Aba 21 48.3±2.7Ab 56.1±0.3Ba 53.6±2.1Aa 54.6±1.0Aab 53.2±0.3Ac 60.0±1.4Ab 1 Values are the mean ± standard deviation of n=9. a-c Mean values in same row, in the same milk, with different lowercase superscripts differ significantly (p < 0.05). A-C Mean values in the same column with different uppercase superscripts differ significantly (p < 0.05).

23

0d

7d

14d

21d

3.0

Absorbance 340 nm

2.5 2.0 1.5 1.0

0.5 0.0 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

Camel

Figure 1.

24

A

0d

7d

14d

21d

90 80

ABTS antioxidant (%)

70

60 50 40 30 20 10 0 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

B

Camel

0d

60

7d

14d

21d

DPPH antioxidant(%)

50 40 30 20

10 0 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

Camel

Figure 2.

25

A

0d

7d

14d

21d

Amylase inhibition (%)

60 50 40 30 20 10 0 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

B

Camel

0d

7d

14d

21d

70

Glucosiade inhibition (%)

60 50 40 30 20

10 0

Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

Camel

Figure 3.

26

0d

7d

14d

21d

100 90

ACE-Inhibirion (%)

80 70 60

50 40 30

20 10 0 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Lp.DSM Lr.K777 Lp.K779 Lp.K772 Bovine

Camel

Figure 4.

27

Highlights  Fermented camel milk possesses significant health benefits  ACE-inhibition of fermented camel milk was greater than bovine milk  Antiproliferation of fermented camel milk was markedly higher than fermented bovine  Functionality of fermented camel milk value needs more investigation.

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