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Department of Nutrition and Health Sciences, College of Medicine and Health Sciences, United Arab Emirates University (UAEU), Al Ain, PO Box 15551, UAE
Key Laboratory of Agro-Product Quality and Safety, Institute of Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China, 100081
The objective of this study was to assess protein degradation and biological activities of the water-soluble extract (WSE) and the 10 kDa permeable and nonpermeable fractions of in vitro digesta of low-fat Akawi cheese made from blends (100:0, 85:15, or 70:30) of bovine milk and camel milk and ripened for 28 d. Biological activities, such as antioxidant activities, amylase and glucosidase inhibition, angiotensin-converting enzyme inhibition, and antiproliferative of the WSE, and the 10 kDa permeable and nonpermeable fraction of the digesta were assessed. To identify the nature of the bioaccessible compounds, untargeted metabolomic analysis was carried out by ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Higher o-phthaldialdehyde absorbances were observed in cheeses made of bovine-camel milk blends compared with cheese from bovine milk only. The WSE from these blends also exhibited higher angiotensin-converting enzyme inhibitory effects and higher antiproliferative effects than from bovine milk. The results from this study suggest that the use of blends of camel milk and bovine milk can modulate biological activities of low-fat Akawi cheese.
). African, Gulf Cooperation Council, and Central Asian countries are the major locations of camel species (dromedary and bactrian). Camel milk is widely available in these countries, with estimated production exceeding 150 million L/yr worldwide. Unlike goat and sheep milk, camel milk is available throughout the year due to the long lactation period of she-camels (
Microbiological quality and somatic cell count in bulk milk of dromedary camels (Camelus dromedarius): Descriptive statistics, correlations, and factors of variation.
In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.
). Nonetheless, making cheese and set-yogurt from camel milk is very challenging and has thus limited uptake at an industrial level. Acid-induced or rennet-induced coagulation produces weak and fragile coagulum (
). As a result, producing cheese and set-yogurt from camel milk alone has not yet become successful. Therefore, the concept of utilizing camel milk has changed to incorporating it in other dairy products as an ingredient or mixing it with milk from other species, such as bovine, ovine, caprine, or buffalo milk (
Study of the soft cheese composition produced from a mixture of sheep and camel milk at different storage periods using the enzyme trypsin and calcium chloride.
), or buffalo milk to make soft white cheese. However, the focus in these studies was largely on physicochemical properties, whereas biological functionality of these cheeses has not been investigated.
Akawi cheese is a rennet-coagulated white brined cheese, which is appreciated in North African and Middle Eastern countries as a table cheese and for making sweets called Kunafah (
The effect of NaCl substitution with KCl on Akawi cheese: Chemical composition, proteolysis, angiotensin-converting enzyme-inhibitory activity, probiotic survival, texture profile, and sensory properties.
). This study focused on the biological activities of the bioaccessible fraction of low-fat Akawi cheese after in vitro digestion using the INFOGEST2.0 model (
). Assessing the bioaccessible fraction is important to ensure that any claimed nutritional benefits are attributed to compounds that can be absorbed by the human intestine (
). The study used in vitro assays to assess the antioxidant (DPPH and ABTS) activity, α-amylase and α-glucosidase inhibition, angiotensin-converting enzyme (ACE)-inhibition, antiproliferative activity (colon and breast cell lines), as well as proteolytic activities of the bioaccessible (10 kDa permeable) fraction of low-fat Akawi cheese (LFAC) made from blends of bovine and camel milk. The abovementioned biological activities were also assessed for the pH4.6-water-soluble extract (WSE; before digestion) and 10 kDa-nonpermeable fractions of the digesta, for comparison purposes. To identify the nature of the bioaccessible compounds, untargeted metabolomic analysis was carried out using ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-QTOF). The physicochemical parameters of the cheese, such as texture profile, rheological properties, and microstructure will be presented elsewhere.
MATERIALS AND METHODS
Low-Fat Akawi Cheese Making
Low-fat pasteurized camel (2.7% protein, 1.0% fat, and 4.2% carbohydrates) and bovine milk (3.2% protein, 1.0% fat, and 4.6% carbohydrates) were purchased from a local market. As preliminary work, camel milk was blended with bovine milk at various ratios from 0 to 50% followed by renneting using camel-origin chymosin FAR-M (Chr. Hansen Holding A/S). Acceptable curds were obtained when the proportion of camel milk in the mixture did not exceed 30%. Therefore, 3 bovine-camel milk blends were prepared: bovine only (BM), camel milk 15% (CM15%), and camel milk 30% (CM30%). The milk was tempered at 38°C for 30 min in a 13-L stainless steel cheese vat (Pignat). The CH-1 starter culture consisting of Streptococcus thermophilus and Lactobacillus bulgaricus (Chr. Hansen) was added (0.5 g/vat). After 60 min incubation at 42°C with the starter culture, the camel chymosin FAR-M (Chr. Hansen) was mixed for 2 min and subsequently allowed to form the curd. After 45 min, the formed curd was cut into 1-cm3 cubes and stirred for 20 min at 40°C. The whey was drained and the curd was transferred to cheesecloth to complete the draining. The curd was molded into ~300-g blocks using cheesecloth and pressed at 35 g/cm2 for 90 min. Afterward, the cheese blocks were brined in cold (4°C) 7% NaCl solution (wt/vol, 1:3) and kept overnight at 4°C. Next day, the brined cheese blocks were dried with cheesecloth and vacuum-packaged (oxygen transfer rate 0.0094 mL/cm2 per 24 h at 22.7°C, moisture vapor transfer 1.4 mg/cm2 per 24 h at 37.7°C). The vacuum-packaged cheese blocks were stored at 4°C for 28 d and samples were taken for analysis on d 0 and d 28. The whole cheesemaking procedure was repeated twice (2 experimental units) and sampled in triplicates (3 blocks). Cheeses had 59.7 to 60.1 g/100 g moisture, 23.3 to 24.1 g/100 g protein, 9.1 to 9.6 g/100 g fat, and 3.3 to 3.9 g/100 g ash content.
Lactic Acid Bacteria Enumeration
An aliquot of grated cheese (10 g) was taken from the shredded cheese block, placed in a stomacher bag, and blended with 90 mL of sterile distilled water using a Stomacher-400 laboratory blender (Seward Medical). Appropriate serial dilutions were made using 0.1% (wt/vol) peptone, and lactic acid bacteria (LAB) populations were counted using MRS agar (Neogen). Inoculated plates were incubated anaerobically in duplicate at 37°C for 48 h using anaerobic incubator with CO2 5.0% (Binder GmbH).
pH Measurement and pH4.6 Water-Soluble Extract
Thirty grams of the grated cheese was homogenized with 30 mL of demineralized water for 2 min at 15,000 rpm using the Ultra Turrax Homogenizer T25 (Ika). The pH was recorded using digital-calibrated pH meter. Subsequently, an additional 30 mL of demineralized water was added to the slurry and homogenized for 1 min. The pH of this slurry was decreased to 4.6 using 1.0 N HCl followed by centrifugation at 5,000 × g at 4°C for 15 min. The supernatant (WSE) and precipitate were separately stored at −20°C for further analysis.
In Vitro Digestion (Bioaccessible and Excreta Fractions)
The in vitro digestion protocol developed by INFOGEST group was employed for this study (
). Briefly, 3-g grated cheese samples were subjected to in vitro oral (amylase 75 U/mL, salivary fluid SSF, 0.3 M CaCl2, 2 min), gastric (pepsin 2,000 U/mL, gastric lipase RGE 60 U/mL, gastric juice SGF pH 3.0, 0.3 M CaCl2, 120 min), and intestinal (pancreatin 100 U/mL, bile 10 mmol/L, duodenal juice SIF pH 7.0, 0.3 M CaCl2, 120 min) digestion. At the start of the intestinal phase of the in vitro digestion, a dialysis membrane (10 kDa MWCO, Thermofisher) filled with 25 mL of 0.5 M NaHCO3 solution (
) was immersed in the digesta. The fraction inside the dialysis membrane (10 kDa permeable) was described as bioaccessible, whereas the fraction remaining outside the dialysis membrane (10 kDa-nonpermeable) was described as excreta. Both were stored separately at −20°C for further analysis. Excreta was centrifuged at 14,000 × g for 10 min at 4°C before assays.
Proteolysis Assessment
The SDS-PAGE of the precipitates resulted from the pH4.6-WSE preparation was performed according to (
The determination of radical scavenging activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) radical (ABTS•+) were assayed according to
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.
Exploration of the molecular interactions between angiotensin-I-converting enzyme (ACE) and the inhibitory peptides derived from hazelnut (Corylus heterophylla Fisch.).
. The ACE-inhibition activity (%) was calculated as
where Ab is the absorbance without the addition of sample solution (buffer solution added instead of sample), Aa is the absorbance in the presence of ACE and the sample solution, and Ac is the absorbance of the blank (HCl was added before the addition of ACE).
Antiproliferative Activity
The antiproliferative effect was assayed against colon (Caco-2) and breast (MCF-7) carcinoma cell lines according to method detailed by
. Cells were grown in a humidified incubator (37°C and 5% CO2) in Dulbecco's modified Eagle's medium (Gibco, Invitrogen) supplemented with 10% heat-inactivated bovine serum and 1% penicillin and streptomycin (Invitrogen). Cell lines were initially passaged and counted in a hemocytometer before seeding. Cells were seeded at 1 × 103 concentration in a 96-well plate and incubated in an appropriate medium overnight. For the treatment, 25 µL of filtered WSE, excreta, or bioaccessible was added to each well and incubated for 72 h at 37°C and 5% CO2. Afterward, 20 µL of a pre-warmed Abcam Cell Cytotoxicity Assay Kit (ab112118) was added and incubated at 37°C for at least 5 h. Each sample was assayed in triplicate. The absorbance was measured at 570 nm and 605 nm according to the manufacturer's protocol. The ratio of optical density (OD)570/OD605 nm was used to measure 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, and Rctrl is the absorbance ratio of OD570/OD605 in the absence of the WSE (vehicle control). Ro is the averaged background (noncell control) absorbance ratio of OD570/OD605.
Untargeted Metabolomic Analysis
The bioaccessible fractions of the 3 experimental cheeses at d 28 were analyzed by UPLC-QTOF. All chromatographic separations were performed using an UPLC system (Waters). An ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) was used for the reversed phase separation. The column oven was maintained at 40°C. The flow rate was 0.4 mL/min and the mobile phase consisted of solvent A (water + 0.1% formic acid) and solvent B (methanol + 0.1% formic acid). Gradient elution conditions were set as follows: 0 to 1 min, 1% phase B; 1 to 3 min, 1 to 15% phase B; 3 to 6 min, 15 to 50% phase B; 6 to 9 min, 50 to 95% phase B, 9 to 10 min, 95% phase B; 10.1 to 12 min, 99% phase B. The injection volume for each sample was 5 μL. A high-resolution tandem mass spectrometer Xevo G2 XS QTOF (Waters) was used to detect metabolites eluted form the column. The Q-TOF was operated in both positive and negative ion modes. For the positive ion mode, the capillary and sampling cone voltages were set at 3.0 kV and 40.0 V, respectively. For the negative ion mode, the capillary and sampling cone voltages were set at 2.0 kV and 40.0 V, respectively. The mass spectrometry data were acquired in Centroid MSE mode. The TOF mass range was from 50 to 1200 Da and the scan time was 0.2 s. For the MS/MS detection, all precursors were fragmented using 20 to 40 eV, and the scan time was 0.2 s. During the acquisition, the LE signal was acquired every 3 s to calibrate the mass accuracy. The commercial software Progenesis QI (Waters) and self-developed metabolomics analysis process were used to conduct in-depth analysis of mass spectrometry data, wherein metabolite identification was based on Human Metabolome Database (http://www.hmdb.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/). Principal component analysis (PCA) and unsupervised clustering analysis (heatmap) were performed using the metabolomics R software package metaX developed by BGI. The mass difference between observed and the database value was less than 10 ppm. All analyses were performed by BGI Company.
Statistical Analysis
Cheesemaking was carried out in duplicate. Each experimental unit was subsampled in triplicate. Thus, result values are mean ± standard deviation. One-way ANOVA was performed to investigate the effect of the blended ratio at same storage period and to investigate the effect of storage at same blended ratio on the measured parameters (P < 0.05). Means comparisons at the same storage time were performed using Tukey's test (P < 0.05). The PCA was performed to assess the structure correlation of the variables (loadings) and observations (scores) to visualize the relationship between different cheeses. The statistical analyses were carried out using XLSTAT software (Addinsoft).
RESULTS AND DISCUSSION
Bacterial Enumeration and pH
The LAB counts and pH values of the cheese sample are presented in Figure 1A, B. Small differences (P > 0.05) between the 3 cheeses were apparent in relation to LAB counts (Figure 1A). The viable numbers of LAB in fresh cheeses (at d 0) were higher in BM and CM15% cheeses compared with CM30% cheese (P > 0.05). At d 28, the LAB viable numbers had decreased (P < 0.05) by less than 1 log10 cfu/g in BM and CM15% cheeses, whereas it remained the same in CM30% cheese. Nevertheless, the numbers of LAB remained >5.5 log10 cfu/g in all cheeses during cold storage (Figure 1A).
Figure 1(A) Lactic acid bacteria enumeration (log10 cfu/g) and (B) pH values of the experimental low-fat Akawi cheese during storage. Bars are mean ± SD. Bars with different lowercase letters (a, b) at the same cheese type differed significantly (P < 0.05). BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%.
Significant reductions in the pH of all cheeses were apparent by end of the storage period (P < 0.05). On d 0, the highest pH value was observed in CM30% cheese (pH 6.5) compared with both BM and CM15% cheeses (pH 6.2). By the end of storage period (at d 28), decreases in the pH were seen for all 3 cheeses. The pH of BM and CM15% decreased from 6.2 to 5.9, whereas the highest reduction was observed in CM30% cheese (from 6.5 to 5.9; P < 0.05). This suggests that lactose fermentation to lactic acid and other organic acids continued as a result of continued starter culture activity in the cheeses during the storage. At d 28, CM15% and CM30% had lower (P < 0.05) values compared with BM. This may be attributed to the greater fermentation rate of lactose in cheeses made from blended milk compared with bovine only.
have reported differences in pH of the soft white cheese made with different starter cultures, but no storage data were reported. In agreement with our results,
have reported lower pH values in fresh panela cheeses made with mixed milk (goat and cow) compared with cow milk only.
Proteolysis Assessment
The SDS-PAGE profiles display changes in the protein bands of the pH 4.6-water insoluble fraction of different cheeses at d 0 and after 28 d of storage at 4°C (Figure 2A), which shows similar proteolytic patterns between the experimental cheeses at d 0. At d 28, slight reductions in the protein band intensities (β-casein, α-casein, and κ-casein) for all of the cheeses. The proteolytic patterns were distinct in CM30% after 28 d of storage. However, band intensities decreased slightly at d 28 and new bands appeared in the range of 15 kDa to 10 kDa especially in CM30% cheese (Figure 2A). This implies that slight degradation occurred in all cheeses during storage at 4°C. This finding can be explained by the proteolytic activity of chymosin residues in cheese and possibly plasmin on the caseins (
). Additionally, starter cultures produce enzymes including proteinases, peptidases, and aminopeptidases that may have a role in the formation of intermediate and small peptides resulting from the primary hydrolysis by chymosin residues (
Figure 2(A) SDS-PAGE of pH 4.6-water-soluble extract (WSE) and (B) o-phthaldialdehyde (OPA) absorbances (340 nm) of the experimental low-fat Akawi cheese during storage. Bars are mean ± SD. Bars with different lowercase letters (a, b) at the same storage time and type differed significantly (P < 0.05). BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%; MR = molecular ladder.
). The values of OPA in WSE, excreta, and bioaccessible fractions from different cheeses at d 0 and after 28 d of storage at 4°C are presented in Figure 2B. At d 0, the bioaccessible fraction showed the highest OPA absorbances (0.62–0.63) compared with WSE (0.45–0.59) and excreta (0.56–0.56; Figure 2B). This implies that the in vitro digestion enhanced the proteolytic rate. As can be seen from Figure 2B, the OPA absorbances in WSE from BM were lower (P < 0.05) compared with CM15% and CM30% at d 0 and 28. Our results agree with the concept that camel milk caseins are more susceptible to proteolytic enzymes compared with bovine caseins (
). On the other hand, the variations between the experimental cheeses became insignificant (P > 0.05) after the in vitro digestion (excreta and bioaccessible). This suggests that variations between the experimental cheeses in OPA absorbances before in vitro digestion did not have a significant effect on the postdigestion fractions (excreta and bioaccessible). The OPA absorbances in WSE from CM30% and CM15% cheeses differed insignificantly (P > 0.05). The higher OPA values observed in cheeses made from mixed milk reflected the changes in SDS-PAGE protein profile (Figure 2A). The OPA absorbances of WSE increased (P < 0.05) with prolonged storage for all cheeses (Figure 2B). This may be attributed to the proteolytic activity of the hydrolytic enzymes produced by the starter culture (
Proteolytic activity is an important indicator of the presence of bioactive compounds, particularly small peptides and free amino acids, which may possess potential health benefits in fermented dairy products such as cheese (
; Egger et al., 2020). Gastrointestinal enzymes (pepsin, trypsin, and chymotrypsin) have a significant effect on the hydrolysis of the milk proteins. The positions of pepsin action on milk protein are different from those of the intestinal chymotrypsin and trypsin (
). Pepsin is known by its unspecific proteolytic activities to bonds having Trp, Tyr, Leu, or Val residues; accordingly pepsin initiates hydrolysis of the milk's proteins. Subsequently, trypsin and chymotrypsin continue the main hydrolytic action on the milk protein (Huppertz and Chia, 2020). This could explain why the differences between the experimental cheeses diminished after the in vitro digestion. It also could explain the higher OPA absorbances after in vitro digestion (Figure 2B).
Antioxidant Activities
The results of radical scavenging activity as determined by ABTS and DPPH assays of the WSE, excreta, and bioaccessible fractions from the different cheeses at d 0 and d 28 of storage are shown in Figure 3. At d 0 and d 28, the highest ABTS values were observed in the bioaccessible fraction, followed by excreta from all cheeses, whereas the lowest was observed in the WSE (Figure 3A). The same trend was apparent in DPPH test (Figure 3B). The ABTS increased by the end of the storage period (after 28) for all cheeses (Figure 3A and 3B). The bioaccessible fraction from CM30% cheese followed by CM15% cheese exhibited higher ABTS rates compared with those of BM cheese at d 0 and d 28 (P < 0.05). Similarly,
have reported that ABTS increased gradually during ripening period of cow and buffalo milk Cheddar cheeses.
Figure 3Antioxidant activities by (A) ABTS % and (B) DPPH % of the experimental low-fat Akawi cheese during storage. Bars are mean ± SD. Bars with different lowercase letters (a–c) at the same storage time and type differed significantly (P < 0.05). BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%; WSE = water-soluble extract.
The higher ABTS and DPPH values of bioaccessible and excreta compared with WSE (Figure 3A, B) may be attributed to the greater number of bioactive compounds with low molecular weight resulted after the in vitro digestion (Figure 3A). These bioactive compounds possess greater antioxidant activities than those in WSE (
). This implies that the in vitro digestion could improve the antioxidant activities of the fermented camel and bovine milk. These results agree with the proteolysis assessment in Figure 2, Figure 3. Interestingly, the differences in antioxidant activities between the storage times (d 0 and 28) diminished (P > 0.05) after the in vitro digestion. This suggests that antioxidant benefits could be attained regardless of the cheese storage time.
The ABTS test exhibited higher scavenging values compared with DPPH test which could be due to the difference in the nature of these tests (
In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.
). Bioactive compounds have a vital role in reducing the effect of reactive oxygen species, for instance peroxyl (ROO•), superoxide (O2•-), prehydroxyl (HOO•), and hydroxyl (HO•) radicals in foods generally, and in fermented dairy products as well (
). Peptides produced from αs-casein have been reported to possess free radical scavenging activities as well as the ability to inhibit lipid peroxidation both enzymatic and nonenzymatic (
The inhibition of α-glucosidase and α-amylase is recognized as an effective approach for controlling diabetes through decreasing carbohydrate hydrolysis (
). The inhibitory effects of the WSE, excreta, and bioaccessible fractions from all cheeses on α-amylase and α-glucosidase activity at d 0 and 28 of storage are presented in Figure 4. At d 28, the WSE from CM30% cheese followed by CM15% cheese had higher (P < 0.05) α-amylase (>50%) and α-glucosidase (>20%) inhibitory rates compared with those of BM cheese (Figure 4A, B). These results agree with the OPA results observed in the 2 cheeses made from mixed milk suggesting that adding camel milk enhanced the α-amylase and α-glucosidase inhibitions in agreement with previous results
In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.
. These observations may be due to 2 reasons: (1) camel milk's caseins more susceptible to the hydrolytic enzymes (residual chymosin and LAB enzymes) that producing bioactive peptides, and (2) the nature of the bioactive compounds (especially bioactive peptides) that possess higher biological activity than those derived from bovine milk.
Figure 4α-Amylase (A) and α-glucosidase (B) inhibitions of the experimental low-fat Akawi cheese during storage. Bars are mean ± SD. Bars with different lowercase letters (a–c) at the same storage time and type differed significantly (P < 0.05). BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%; WSE = water-soluble extract.
As can be seen from Figure 4A and B, the differences in the α-amylase and α-glucosidase inhibitions of the bioaccessible fractions from all experimental cheeses diminished after the in vitro digestion. This suggests that the strong hydrolytic actions, due to the digestive enzymes, released a comparable level of bioactive compounds possessing similar α-amylase and α-glucosidase inhibitions. These results agree with the OPA and antioxidant results in Figure 3, Figure 4. Moreover, Figure 4A and B demonstrates that the α-glucosidase and α-amylase inhibitory activities of the bioaccessible fractions were not influenced (P > 0.05) by storage time. This suggests that the same α-amylase and α-glucosidase inhibitions could be achieved at any time of storage. The α-glucosidase inhibitions trends of the excreta were similar to WSE. Further investigations are necessary to fully understand these observations.
ACE Inhibition and Antiproliferative Activity
Among bioactive peptides, the ACE-inhibitory effects of the dairy product are the most extensively investigated. Their biochemical characteristics, physiological activities, and mechanisms of action are well documented (
). In the present study, the results for ACE-inhibitory activity (%) and antiproliferative activities of WSE, excreta, and the bioaccessible fraction from all cheeses at d 0 and after 28 d of storage are presented in Figure 5A–C. At d 1 and 28, WSE from CM15% and CM30% cheese exhibited higher (P < 0.05) ACE-inhibitory activity than BM cheese (Figure 5A). These results are in line with the higher OPA values for WSE observed in (Figure 2B) for cheeses made from mixed milk (CM15% and CM30%). This implies that camel milk compounds, especially the released peptides, enhanced the ACE-inhibitions of the cheeses made from blended milk. This could be due to the vulnerability of camel milk caseins to the proteolytic enzymes (chymosin residues and LAB) as reported by
In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.
. Interestingly, the ACE-inhibitions of the excreta and bioaccessible fractions were slightly lower (P > 0.05) than WSE (Figure 5A). This suggests that the in vitro digestion had minimal effect on the bioactive compounds released in the WSE. This may be due to resistance of these bioactive compounds toward the digestive enzymes (gastric and intestine). Our results agree with
who reported an increase in ACE-inhibitions of the bioaccessible compounds in the dialyzed fraction.
Figure 5ACE-inhibition (A) and antiproliferative activities against Caco-2 (B) and MCF-7 (C) of the experimental low-fat Akawi cheese during storage. Bars are mean ± SD. Bars with different lowercase letters (a–c) at the same storage time and type differed significantly (P < 0.05). BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%; WSE = water-soluble extract.
Figure 6B and C display the antiproliferative activities of the WSE, excreta and bioaccessible fractions against breast cancer (MCF-7) and colon cancer (Caco-2) cell lines, respectively. At d 0 and 28, the highest antiproliferative activity against MCF-7 and Caco-2 cell lines were observed in bioaccessible from all cheeses, whereas the lowest was detected with WSE (Figure 5B, C). This suggests the digestive system enhanced (P < 0.05) the antiproliferative activities of the experimental cheeses. Moreover, the antiproliferative effect progressed (P < 0.05) by the end of the storage period (after 28 d) for all cheeses (Figure 6B, C).
Figure 6Principal component analysis (PCA) of the variables (loading plot, A) and observations (score plot, B). AMY = amylase; GLU = glucosidase; CACO = Caco-2 cell line; MCF = MCF-7 cell line; W = water-soluble extract pH 4.6 (WSE) fraction; Ex = excreta fraction; B = bioaccessible fraction; BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%.
For cell lines MCF-7 and Caco-2, bioaccessible from CM30% and CM15% cheeses revealed higher antiproliferative rates compared with those of BM cheese at d 0 and d 28, demonstrating that the antiproliferative activity of the cheese made from mixed milk increased during storage. This could be due to the nature of bioactive compounds, especially small peptides, derived from camel milk especially caseins. Our results are in accordance with
In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.
who found that antiproliferative activities against MCF-7 and Caco-2 cell lines were significantly superior in fermented camel milk compared with fermented bovine milk. Several hypotheses have been proposed to explain the mechanism(s) of the antiproliferative activities of milk peptides. Competition between the peptides and cancer growth factors for cancer cell-membrane receptors is one of these hypotheses. Another hypothesis claims that the released peptides have specific cytotoxicity on cancer cells which induces apoptosis (
Principal component analysis was performed to explore relations between the antioxidant (DPPH and ABTS) activity, amylase and glucosidase inhibition, ACE-inhibition, antiproliferative (Caco-2 and MCF-7) activity, proteolysis (DH% and OPA), and fermentation (LAB count and pH). Two principal components (PC) explained about 64.3% of the total variance in the data (PC1 36.1% and PC2 28.2%). The loading plot (Figure 6A) indicate positive correlations between antioxidant activity, proteolysis, amylase and glucosidase inhibition, and antiproliferative activities, suggesting that the compounds behind these parameters could share similar characteristics and structures. The score plot (Figure 6B) presented clear variations between the experimental cheeses (BM, CM15%, and CM30%) and also between the 2 storage times (D0 and D28). At d 0, the cheeses made of mixed milk (CM15% and CM30%) were distinguished from the BM cheese but this situation noticeably changed after 28 d of storage and the CM15% cheeses became more closer to BM cheese and distinguished from the CM30%. This suggests that adding camel milk up to 30% resulted a cheese with distinct characteristics compared with BM only.
Untargeted Metabolomic Analysis
To the best of our knowledge, this the first attempt to perform untargeted metabolomics for Akawi cheese made from bovine milk only and from blended milk (bovine and camel). The following metabolites classes were annotated in the bioaccessible portions of 3 cheeses at d 28 of storage: lipids and lipid-like molecules (47145), fatty acyls (9989), glycerophospholipids (8725), glycerolipids (4202), phenylpropanoids and polyketides (4106), sterol lipids (3954), polyketides (2760), organoheterocyclic compounds (2634), benzenoids (2590), organic acids and derivatives (2567), prenol lipids (1685), organic oxygen compounds (1475), sphingolipids (697), organooxygen compounds (405), alkaloids and derivatives (218), nucleosides, nucleotides, and analogs (188), organic nitrogen compounds (165), lignans, neolignans and related compounds (75), hydrocarbons (63), organosulfur compounds (45), organohalogen compounds (34), organonitrogen compounds (30), hydrocarbon derivatives (25), organic compounds (22), glycolipids (22), homogeneous nonmetal compounds (11), organometallic compounds (10), homogeneous metal compounds (2), organic 1,3-dipolar compounds (2), organic polymers (2), organic salts (2), and unknown (15174). Thus, the bioaccessible fractions from the 3 cheeses host a very large collection of bioactive components many of which are biochemically related.
have reported similar metabolites classes in protected designation of origin (PDO) Grana Padano cheeses.
The unsupervised PCA analysis exhibited clear variations between the Akawi cheeses made of CM15% and CM30% versus the BM only (Figure 7A). The first PC, explaining 73% of the variation in the metabolites data, separates the bioaccessible fractions of the bovine cheese from those of cheeses containing 15 and 30% camel milk. This suggests that the addition of CM at these ratios influenced the metabolites released in the bioaccessible fraction qualitatively. The second PC, explaining 27% of the variance in the data, separates the bioaccessible fraction of the cheese containing 30% CM from the BM only and the CM15% cheeses. This implies that increasing the camel milk percentage affected the quantity of some metabolites in the bioaccessible portions.
Figure 7Unsupervised principal component (PC) analysis (A) and heatmaps (B) of the untargeted metabolomic analysis of the bioaccessible fractions from the 3 experimental cheeses. BM = bovine milk; CM 15% = camel milk 15%; CM 30% = camel milk 30%.
The unsupervised hierarchical cluster analysis represented in the heatmap (Figure 7B) revealed distinct clustering of the bioaccessible fractions of the 3 cheeses. The cheeses made of blended milk (CM15% and CM30%) were clustered together and separated from the BM cheese in agreement with the PCA analysis (Figure 7A). The detected metabolites in the experimental cheeses clustered into 4 main groups (G1, G2, G3, and G4; Figure 7B). Metabolites of G1 presented in higher concentrations in CM15% and CM30% cheeses compared with BM. The metabolites of G3 exhibited lower concentrations in BM cheese compared with CM15% and CM30%. The KEGG database revealed the top 20 metabolic pathways (Supplemental Figure S1;
). The type and counts of the top 20 KEGG in the bioaccessible portions were similar. However, the order of the metabolic pathways changed when camel milk was added (CM15% and CM30%).
CONCLUSIONS
The addition of camel milk to produce LFAC slightly enhanced the ABTS, DPPH, α-amylase, α-glucosidase, ACE inhibition, and antiproliferative activities, especially before in vitro digestion (WSE). Nonetheless, the digestion conditions generally increased the activities of the measured parameters but diminished the difference between the experimental cheeses. In general, storage time had a minor effect on the biological activities of the fractions after digestion. The stability of the biological activities after the gastrointestinal condition requires further investigation.
ACKNOWLEDGMENTS
We are grateful to United Arab Emirates University for funding this project. We thank Arachchige Ranasinghe for support with the SDS-PAGE. We are grateful to United Arab Emirates University (UAEU) for funding this project. The author contributions include M. Ayyash: conceptualization, formal analysis, writing of the original draft, and supervision; A. Abdalla: writing of original draft; M. Alameri, M. Affan Baig, and J. Kizhakkayil: investigation; T. Huppertz, A. Kamal-Eldin, and G. Chen: writing review and editing. The authors have not stated any conflicts of interest.
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