Article pubs.acs.org/JAFC
Antioxidant Potential of Date (Phoenix dactylifera L.) Seed Protein Hydrolysates and Carnosine in Food and Biological Systems Priyatharini Ambigaipalan and Fereidoon Shahidi* Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada A1B 3X9 ABSTRACT: Date seed protein hydrolysates were evaluated for antioxidant activity as well as solubility and water-holding capacity in food and biological model systems. Date seed protein hydrolysates as well as carnosine exhibited >80% of solubility over a pH range of 2−12. The hydrolysates and carnosine at 0.5% (w/w) were also found to be effective in enhancing waterholding capacity and cooking yield in a fish model system, which was nearly similar to sodium tripolyphosphate (STPP; 0.3%, w/ w). Incorporation of hydrolysates (200 ppm) in fish model systems resulted in the highest inhibition (30%) of oxidation in comparison to butylated hydroxytoluene (BHT; 9%). In addition, hydrolysates and carnosine inhibited β-carotene oxidation by 75%. The hydrolysates (0.1 mg/mL) inhibited LDL cholesterol oxidation by 60%, whereas carnosine inhibited oxidation by 80% after 12 h of incubation. Additionally, hydrolysates and carnosine effectively inhibited hydroxyl (6 mg/mL) and peroxyl (0.1 mg/ mL) radical-induced DNA scission. Therefore, date seed protein hydrolysates could be used as a potential functional food ingredient for health promotion. KEYWORDS: date seed, protein hydrolysate, solubility, water-holding capacity, carnosine, LDL cholesterol, DNA scission
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INTRODUCTION Hydrolysates are defined as proteins that are chemically or biologically broken down to peptides of various sizes. Enzymatic hydrolysis of food proteins is an efficient way to recover potent bioactive peptides.1 Hydrolysis of proteins using various enzymes could improve the functional properties by influencing the molecular size, hydrophobicity, and polar groups of peptides.2 Functional properties of protein hydrolysates such as solubility, water-holding capacity, and emulsifying and foaming properties are substantially useful characteristics for many food applications.1 Apart from functionalities, several studies have shown the potential application of protein hydrolysates for the nutritional or pharmaceutical industry due to their antioxidant activity.3−6 Bioactive peptides generally contain 3−20 amino acid units,7 but in some cases this range may be extended. Lunasin, for example, is a food-derived peptide with anticancer activity, composed of 43 amino acids with a molecular weight of 5400 Da.8 Depending on the amino acid sequence, these peptides may be involved in various biological functions such as antihypertension, opioid agonists or antagonists, immunomodulatory, antithrombotic, antioxidant, anticancer, and antimicrobial activities, in addition to nutrient utilization.9,10 The reactivity of free radicals on biological molecules (lipids, proteins, and DNA) may be considered as the initiating stage for several chronic diseases. For example, oxidation of lowdensity lipoprotein (LDL) cholesterol by free radicals or metal ions is an important cause for developing atherosclerotic lesions that lead to coronary heart disease.11 Furthermore, Wettasinghe and Shahidi12 reported that oxidation of DNA and strand scission play an important role in carcinogenesis. Thus, researchers have a greater interest in developing novel antioxidative compounds from various plant and animal sources. Carnosine is a β-alanylhistidine dipeptide, present at millimolar concentrations in skeletal muscle and absorbed into © XXXX American Chemical Society
plasma, that has been shown to have antioxidative property.13−15 Thus, a well-known bioactive peptide, carnosine, was used in this study to compare the functional and antioxidative properties of date seed protein hydrolysates. In this study, date seed protein hydrolysates hydrolyzed using various enzymes (Alcalase, Flavourzyme, and Thermolysin) were compared to carnosine, an antioxidative peptide, with respect to functional (solubility and water-holding capacity) and antioxidative properties in various food (cooked comminuted fish model and β-carotene−linoleate model) and biological (human LDL and DNA) model systems.
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MATERIALS AND METHODS
Materials. Ground date (Phoenix dactylifera L.) seed sample was provided by Dr. A. S. Al-Khalifa of King Saud University, Riyadh, Saudi Arabia. Alcalase (2.4 AU/g) and Flavourzyme (1000 LAPU/g) were procured from Novozymes, Bagsvaerd, Denmark. Thermolysin (50− 100 units/mg protein) was purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). All chemicals used were obtained from Fisher Scientific Ltd. (Ottawa, ON, Canada) or Sigma-Aldrich Canada Ltd. The solvents used were of ACS grade, pesticide grade, or HPLC grade and were used without any further purification. Preparation of Protein Hydrolysate. Ground date seed sample was defatted with hexane (1:5, w/v) for 5 min. The crude protein content was determined by using micro-Kjeldhal analysis.16 The date seed protein hydrolysates were prepared using Alcalase (AL), Flavourzyme (FL), and Thermolysin (TH) by individual or sequential treatment as described in Table 1. Twenty grams of defatted date seed sample was suspended in 100 mL of water and hydrolyzed batchwise in a reaction vessel, equipped with a stirrer, thermometer, and pH electrode according to the conditions presented in Table 1. The pH (changes as a result of hydrolysis) was kept constant by the addition of Received: November 4, 2014 Revised: December 31, 2014 Accepted: January 1, 2015
A
DOI: 10.1021/jf505327b J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
fish samples were transferred into plastic bags and stored in a refrigerator at 4 °C for 14 days. Thiobarbituric acid reactive substances (TBARS) were analyzed on days 0, 7, and 14 using a modified version of the method described by Wijeratne, Abou-Zaid, and Shahidi.20 Fish samples (1 g) in centrifuge tubes were mixed with trichloroacetic acid (10%, 2.5 mL) and vortexed for 2 min at high speed. TBA reagent (0.02 M, 2.5 mL) was then added to the tube, followed by further vortexing for 30 s. Samples were centrifuged at 3000g for 10 min and the supernatants filtered through Whatman no. 3 filter paper. The tubes were covered with aluminum foil, placed in a boiling water bath for 45 min, and subsequently cooled to room temperature under running cold tap water. The absorbance of the resultant pink TBA− MDA (malondialdehyde) adduct was read at 532 nm. A standard curve was prepared using 1,1,3,3-tetramethoxypropane, a precursor of MDA (0−6 ppm). TBARS values, calculated using the standard curve, were expressed as milligrams of MDA equivalent per kilogram of sample. Antioxidant Activity in Oil-in-Water Emulsions. A β-carotene− linoleate model system was used to evaluate the antioxidant activity of date seed protein hydrolysates in an oil-in-water emulsion.1 βCarotene (10 mg) was dissolved in chloroform (10 mL), and an aliquot (1.2 mL) of it was transferred into a flask containing linoleic acid (40 mg) and Tween 40 (400 mg). A blank without β-carotene was also prepared (40 mg of linoleic acid + 400 mg of Tween 40). Chloroform was removed under a nitrogen stream; 100 mL of oxygenated distilled water was added to the flask, and the mixture was stirred vigorously for 30 min. Date seed protein hydrolysates (0.5 mg/ mL; 0.5 mL) dissolved in deionized water were mixed with 4.5 mL of the above emulsion. A control without protein hydrolysate and a mixture of blank (without β-carotene) was prepared for each sample. The absorbance at 470 nm was read immediately after the addition of the emulsion. The tubes were incubated in a shaking water bath at 50 °C, and the absorbance was read over a 105 min period at 15 min intervals. A kinetic curve was plotted against blank-corrected absorbance and time. One hundred parts per million of Trolox and carnosine were used as a positive control. Antioxidant activity of date seed protein hydrolysates in protecting β-carotene/linoleic acid oxidation was calculated using the equation
Table 1. Hydrolysis Conditions Used for Preparation of Date Seed Protein Hydrolysates sample
enzyme
enzyme/protein
temperature (°C)
pH
time (h)
AL FL TH
Alcalase Flavourzyme Thermolysin
0.3 AU/g 50 LAPU/g 50 U/mg
50 50 50
8 7 8
1 2 3
AL + FL
Alcalase + Flavourzyme
0.3 AU/g 50 LAPU/g
50
8 7
1 2
AL + TH
Alcalase + Thermolysin
0.3 AU/g 50 U/mg
50
8 8
1 3
FL + TH
Flavourzyme + Thermolysin
50 LAPU/g 50 U/mg
50
7 8
2 3
AL + FL + TH
Alcalase + Flavourzyme + Thermolysin
0.3 AU/g 50 LAPU/g 50 U/mg
50
8 7 8
1 2 3
a known amount of 4 M NaOH. The degree of hydrolysis (%) was then determined by the trinitrobenzenesulfonic acid (TNBS) method described by Alder-Nissen (1979). The date seed protein hydrolysates obtained were freeze-dried and stored at −20 °C for subsequent analyses. Solubility of Protein Hydrolysates. The solubility of date seed protein hydrolysate was determined according to the method described by Shahidi, Han, and Synoweicki17 with modifications. Freeze-dried date seed protein hydrolysate (10 mg/mL, w/v) was dispersed in distilled water, and its pH was adjusted to 2, 5, 8, and 12 by the addition of a 1 N or 6 N HCl or 1 N or 6 N NaOH solution. The contents were mixed, and the mixture was centrifuged at 7500g for 15 min. The protein contents in the supernatant were determined using the Biuret method, and the total protein content in the sample was determined by solubilizing the sample in 0.5 N NaoH. The solubility of date seed protein hydrolysates was determined using the following equation: solubility (%) =
antioxidant activity (%) = [(A 0 − A t )/(A°0 − A°t )] × 100
where A0 and At are corrected absorbance values for test samples measured at zero time and after incubation, respectively, whereas A°0 and A°t are corrected absorbance values for control at time zero and at time t after incubation, respectively. Cupric Ion-Induced Human Low-Density Lipoprotein (LDL) Peroxidation. The inhibitiory effect of cupric ion-induced human LDL peroxidation was determined according to the method described by Liyana-Pathirana and Shahidi.21 LDL (5 mg/mL) was dialyzed against 100 volumes of 10 mM phosphate buffer (pH 7.4, 0.15 M NaCl) using a dialysis tube with a molecular weight cutoff of 12−14 kDa (Fisher Scientific, Nepean, ON, Canada) at 4 °C under a nitrogen blanket in the dark for 12 h. Diluted LDL cholesterol (0.04 mg LDL/ mL) was mixed with the date seed protein hydrolysate solutions (0.1 mg/mL). Carnosine was used as a positive control. The samples were preincubated at 37 °C for 15 min. The reaction was initiated by adding a solution of cupric sulfate (50 μM), and the samples were then incubated at 37 °C for 22 h. The formation of conjugated dienes (CD) was recorded at 234 nm using a diode array spectrophotometer (Agilent, Palo Alto, CA, USA). The appropriate blanks were run for each sample by replacing LDL cholesterol and CuSO4 with distilled water for background correction. The inhibitory effect of tested samples on the formation of conjugated dienes (% inhibition CD) was then calculated using the equation
protein content in supernatant × 100 total protein content in sample
Determination of Water-Holding Capacity. The water-holding capacity of date seed protein hydrolysates was determined according to the method described by Shahidi and Synowiecki.18 A fish model system was prepared using 8.5 g of ground salmon and 1.5 g of distilled water in a preweighed 50 mL centrifuge tube. Date seed protein hydrolysates (0.5%, w/w) were added and mixed thoroughly. A control without any protein hydrolysate and positive controls sodium tripolyphosphate (0.3%, w/w) and carnosine were also prepared. The mixture was allowed to stand in a cold room for 1 h and subsequently cooked at 95 °C in a water bath for 1 h. The cooked samples were cooled under a stream of cold tap water, and the drip water was determined using a filter paper. The final weight of the cooked fish was recorded. The drip volume was calculated as the difference between the initial weight and final weight. The waterholding capacity for different date seed protein hydrolysates was expressed as decrease of drip volume against the control. Antioxidant Activity in Cooked Comminuted Fish Model System. The antioxidant activity of date seed protein hydrolysates in the cooked comminuted fish model system was determined as described by Wettasinghe and Shahidi.19 Ground salmon (40 g) was mixed with 10 g of deionized water in Mason jars. Date seed protein hydrolysate (200 ppm) was added directly to the mixture. A control without protein hydrolysate and a positive control using butylated hydroxytoluene (BHT; 200 ppm) were also prepared. Samples were cooked in a thermostated water bath at 80 °C for 40 min while stirring every 5 min with a glass rod. After cooling to room temperature, the
% inhibition CD = [(Abscontrol − Abssample )/(Abscontrol − Absnative )] × 100 Abscontrol = (A°0 − A°t ) Abssample = (A 0 − A t ) B
DOI: 10.1021/jf505327b J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry where Abscontrol is the absorbance of LDL + CuSO4 + PBS, Abssample is the absorbance of LDL + CuSO4 + sample/standard, and Absnative is the absorbance of LDL + PBS. A0 and At are absorbance values for test samples measured at zero time and at time t after incubation, respectively, whereas A°0 and A°t are corrected absorbance values for control at time zero and at time t after incubation, respectively. Inhibition of Peroxyl and Hydroxyl Radical-Induced Supercoiled DNA Strand Scission. Peroxyl and hydroxyl radical-induced supercoiled plasmid DNA strand scission inhibitory activity of date seed protein hydrolysate was determined according to the method described by Chandrasekara and Shahidi.22 Supercoiled plasmid DNA (pBR 322) (50 μg/mL) was dissolved in 10 mM phosphate buffer (PBS, pH 7.4), and date seed protein hydrolysates were dissolved in distilled water. In a 0.5 mL eppendorf tube date seed protein hydrolysate (0.1 mg/mL, 2 μL), PBS (2 μL), pBR 322 (50 μg/mL, 2 μL), and 4 μL of 7 mM 2,2′-azobis(2-methylpropanimidamide dihydrochloride (AAPH) were added to generate peroxyl radicals, whereas date seed protein hydrolysate (6 mg/mL, 2 μL), PBS (2 μL), pBR 322 (50 μg/mL, 2 μL), FeSO4 (0.5 mM, 2 μL), and H2O2 (0.5 mM, 2 μL) were added to produce hydroxyl radicals. The mixture was incubated at 37 °C for 1 h in the dark. A control with DNA alone, a positive control with carnosine (0.1 mg/mL), and a blank devoid of protein hydrolysates were also prepared. After incubation, 1 μL of the loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, and 50% glycerol) were added to the reaction mixture. The mixture (10 μL) was loaded onto 0.7% agarose gel prepared in Tris−acetic acid−EDTA (TAE) buffer (40 mM Tri−-acetate containing 1 mM EDTA, pH 8.5). SYBR safe (5 μL) was added into agarose gel solution (50 mL) as a gel stain. Electrophoresis was conducted at 80 V for 90 min using a model B1A horizontal mini gel electrophoresis system (Owl Separation Systems Inc., Portsmonth, NH, USA) and a model 300 V power supply (VWR International Inc., West Chester, PA, USA) in TAE buffer. The DNA bands were visualized under trans-illumination of UV light using the Alpha-Imager gel documentation system (Cell Biosciences, Santa Clara, CA, USA). The intensity (area %) of bands was quantified with the Chemi-Imager 4400 software (Cell Biosciences). The retention of supercoiled DNA strand (%) was calculated using the following equation:
Figure 1. Solubility of date seed protein hydrolysates and carnosine at various pH values.
solubility of silver carp hydrolysates increases with lower molecular mass protein fraction at high degrees of hydrolysis. Gbogouri, Linder, Fanni, and Parmentier24 suggested that the smaller peptides from proteins have proportionally more polar residues, which could form hydrogen bonds with water and thus increase solubility. The high solubility of date seed protein hydrolysate reveals its potential applications in formulated food systems. Water-Holding Capacity. All date seed protein hydrolysates enhanced the water-holding capacity of salmon fish and thus improved cooking yield (Figure 2). There was no
DNA retention (%) = (area of supercoiled DNA with oxidative radical and protein hydrolysate/area of supercoiled DNA in control)
Statistical Analysis. All determinations were replicated three times and mean values and standard deviations reported. One-way ANOVA was performed, and the mean separations were performed by Tukey’s HSD test (p < 0.05) using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA).
Figure 2. Decrease in drip volume (%) of date seed protein hydrolysates, date seed flour, carnosine, and STPP.
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RESULTS AND DISCUSSION Solubility of Protein Hydrolysates. The protein content of defatted date seed sample was 6.7%, on a dry weight basis, and the degree of hydrolysis ranged between 11 and 13%. Molecular weights of peptides, determined using a quadrupole orthogonal time-of-flight hybrid tandem mass spectrometer, ranged from 2062.9 ± 0.0 to 116803.8 ± 0.4 Da (unpublished data). Solubility is one of the most important properties of protein hydrolysates, which is required in many functional applications such as emulsions, foams, and gels.2,3 The solubility of date seed protein hydrolysate in the pH range of 2−12 is shown in Figure 1. All date seed protein hydrolysates as well as carnosine exhibited >80% of solubility at range of pH 2−12. Solubility of date seed hydrolysates increased to 97% with increasing pH. Hydrolysis of proteins by enzymes potentially affects the molecular size and hydrophobicity, as well as polar and ionizable groups of protein hydrolysates.23 Dong, Zeng, Wang, Liu, Zhao, and Yang4 showed that the
siginificant difference among protein hydrolysates (0.5%) and sodium tripolyphosphate (STTP; 0.3%) with respect to decrease in drip volume. However, date seed flour exhibited a low water-holding capacity (1.8%) in comparison to protein hydrolysates. Cumby, Zhong, Naczk, and Shahidi25 observed an increase water-holding capacity of ground pork meat model system for rapeseed proteins hydrolyzed using Alcalase and Flavourzyme. The authors suggested that that lower molecular weight peptides could be more effective in holding water than are larger-sized peptides due to its greater hydrophilicity. However, the type of amino acid present in the peptides could also play an important role. Decrease in drip volume percentage of carnosine (4.3%) was also lower than date seed protein hydrolysates. Das, Anjaneyulu, and Biswas26 showed that ground buffalo meat containing 1.0 and 1.5% carnosine significantly improved water-holding capacity and lowered cooking loss. Thomsen and Zeuthen27 showed that the waterholding capacity increases with increasing meat pH. Thus, the C
DOI: 10.1021/jf505327b J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry authors26 concluded that the increase in meat pH by carnosine probably accounts for the decrease in cooking loss. Our study shows that 0.5% of date seed protein hydrolysates could be used as an alternative to STPP to increase cooking yield of fish meat. Antioxidant Activity of Date Seed Protein Hydrolysates in Cooked Comminuted Fish Meat Model System. The antioxidant activity of date seed protein hydrolysates in cooked salmon was measured by the thiobarbituric acid (TBA) assay. TBA assay is a spectrophotometric detection of TBARS, namely, MDA, one of the secondary lipid peroxidation products used to measure the extent of lipid degradation. Ground salmon was treated with BHT (200 ppm) or carnosine (200 ppm) or date seed protein hydrolysates (200 ppm) before cooking. Table 2 shows the
Figure 3. Inhibition of TBARS production in fish model system containing date seed protein hydrolysates, carnosine, and BHT.
Table 2. TBARS Values of Fish Model System Containing Date Seed Protein Hydrolysates, BHT, and Carnosine Stored for 14 Days
to fish system containing AL + FL, AL + TH, and FL + TH hydrolysates (200 ppm), BHT (200 ppm) showed a lower inhibition after 7 days of storage. Thus, date seed protein hydrolysates prepared using combinations of AL + FL, AL + TH, and FL + TH could be used to control oxidation in a fish model system. Antioxidant Activity of Date Seed Protein Hydrolysates in Oil-in-Water Emulsion. A β-carotene−linoleate model system was used to evaluate the antioxidant activity of date seed protein hydrolysates in an oil-in-water emulsion. Oxidation of linoleic acid in the emulsion system gives rise to the formation of free radicals and hydroperoxides. The free radicals so formed attack the β-carotene molecule, causing it to lose its conjugation and eventually decolorize. This loss of color could be monitored spectrophotometrically at 470 nm. The presence of antioxidants can counteract the bleaching of βcarotene during the coupled oxidation of linoleic acid and βcarotene in the emulsified aqueous system. Figure 4 shows the effect of date seed protein hydrolysates, at a concentration of 0.1 mg/mL, on β-carotene bleaching in the aforementioned system. β-Carotene content in the control samples decreased to a greater extent compared to those containing date seed protein hydrolysates or trolox or carnosine, which retained high amounts of β-carotene after 105 min. Positive controls trolox (∼54−63%) and carnosine (∼58−75%) showed significantly (p < 0.05) higher inhibitory effect against β-carotene bleaching than date seed protein hydrolysates up to 60 min. The percent inhibition of β-carotene oxidation inhibition by date seed protein hydrolysates ranged between 66.9 and 73.7%. Hydrolysates prepared using a combination of FL and TH showed the highest inhibition (73.7%) followed by AL + TH ∼ FL ∼ AL > AL + FL+TH ∼ TH ∼ AL + FL. Several studies have shown that amino acids possess strong antioxidant activity in linoleic acid and methyl linoleate model systems,29 for example, tryptophan and lysine in butter fat,30 proline in sardine oil,31 methionine in vegetable oils,32 and histidine, threonine, lysine, and methionine in a sunflower oil emulsion.33 Furthermore, Aruoma, Laughton, and Halliwell34 showed the antioxidative effect for taurine, hypotaurine, carnosine, and anserine using in vivo models. However, Pratt and Hudson35 reported that some amino acids such as cysteine, histidine, and tryptophan at higher concentration may act as prooxidants. Copper bound to amino acids or peptides has been shown to have a strong catalyzing effect on the oxidation of linoleic acid.1 Amarowicz and Shahidi1 suggested that differences in the antioxidant properties of peptides might originate from their synergistic
TBARSa (mg/kg) 0 days control BHT carnosine AL FL TH AL + FL AL + TH FL + TH AL + FL + TH
4.6 3.8 4.0 4.1 4.7 3.4 3.6 3.0 2.5 4.6
± ± ± ± ± ± ± ± ± ±
0.1e 0.0cd 0.0d 0.2d 0.1e 0.2bc 0.0c 0.1b 0.2a 0.1e
7 days 5.6 5.2 4.9 5.4 5.5 5.0 3.8 4.6 3.9 4.8
± ± ± ± ± ± ± ± ± ±
0.2e 0.0de 0.0cd 0.1e 0.1e 0.1cd 0.0a 0.0b 0.1a 0.0bc
14 days 5.9 5.6 5.0 5.7 5.9 5.5 5.5 5.0 4.8 5.5
± ± ± ± ± ± ± ± ± ±
0.0 0.1 0.1 0.0 0.0 0.0 0.1 0.0 0.3 0.0
a
All data represent the mean of triplicates. Values followed by the same letter are not significantly different (P > 0.05) by Tukey’s HSD test.
TBARS values of samples throughout the storage period. The initial TBARS value of salmon (4.6 mg MDA equiv/kg) were found to be higher than that of pork model system (∼1.0 mg MDA equiv/kg) studied elsewhere.6 This could be due to the presence of polyunsaturated fatty acids in fish that could increase TBARS during cooking. The fish system containing FL + TH hydrolysate displayed significantly (p < 0.05) lower TBARS value compared to BHT upon cooking. Generally, the TBARS of cooked salmon increased as the storage time increased up to 14 days of storage (p < 0.05). Inhibition percentage of TBARS production in fish model system containing date seed protein hydrolysates or carnosine or BHT is shown in Figure 3. After 7 days of storage, fish systems with AL + FL (32%) and FL + TH (30%) hydrolysate showed the highest percentages of TBARS inhibition in comparison to BHT (7%). Carnosine exhibited a higher percentage of inhibition (12%) compared to BHT (6.6%) after 7 days of storage. Decker and Crum28 showed that the antioxidant activity of carnosine (0.5−1.5%) in cooked salted and unsalted ground pork is greater than those of the lipid-soluble free radical scavengers BHT and α-tocopherol but less than that of STPP. In another study on ground buffalo meat, carnosine at 1.0 and 1.5% significantly lowered the TBARS values as compared to control sample. The inhibition of TBARS production in the fish system containing date seed protein hydrolysate could be most likely due to its radical-scavenging activity as well as chelating activity toward Fe2+. In comparison D
DOI: 10.1021/jf505327b J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 4. (a) β-Carotene bleaching as affected by date seed protein hydrolysates, trolox, and carnosine as measured by changes in absorbance value at 450 nm; (b) antioxidant activity of date seed protein hydrolysates, trolox, and carnosine against β-carotene bleaching.
action with the emulsifier (Tween 40) used in the analysis. Furthermore, Wijeratne, Abou-Zaid, and Shahidi20 reported that hydrophobic antioxidants have higher efficiency than hydrophilic ones in preventing oxidation in oil-in-water emulsion systems by preferentially orienting themselves at the oil−water interface. Emulsifiers decrease the particle size of peptides and increase the contact surface of the phases.29 This lends support to findings of this study that could be attributable to the presence of hydrophobic amino acids in date seed protein hydrolysates. Cupric Ion-Induced Human Low-Density Lipoprotein Peroxidation. Cupric ion has been shown to be effective in initiating the oxidation of EDTA-free human LDL, as measured by the formation of conjugated dienes. The presence of both cholesteryl linoleate and cholesteryl arachidonate within the LDL core provides a rich source of lipid peroxidation substrate.36 Therefore, the greater the lipophilic property of the antioxidant, the more effective it will be to extend the LDL oxidation lag phase once induced by37 Cu2+. LDL oxidation plays a major role in atherogenesis and coronary heart disease. Date seed protein hydrolysates and carnosine at a concentration of 0.1 mg/mL rendered a protective effect against human LDL oxidation (Figure 5), and this was higher. Date seed protein hydrolysates and carnosine exhibited higher after 12 h of incubation, being 50−60 and 80.9%, respectively. There was no significant difference (p > 0.05) among date seed protein hydrolysates after 12 h of incubation. Hydrolyates prepared using AL and TH showed the highest percentage of inhibition among all enzymatic treatments. Hydrolysate
Figure 5. Inhibition against human LDL cholesterol oxidation by date seed protein hydrolysates and carnosine incubated at 37 °C for 25 h.
prepared using only TH had no inhibition at 8 h, but it increased to ∼57% after 12 h. However, percent inhibition decreased after 22 h of incubation in all date seed hydrolydates as well as in carnosine. Decker, Ivanov, Zhu, and Frei16 investigated the ability of carnosine to inhibit the oxidation of LDL in comparison to its constituent amino acid, histidine. They showed that carnosine is capable of inhibiting copperpromoted oxidation reactions by chelating copper ions. However, carnosine’s inhibitory activity against copper is shown to be less than that of histidine in LDL model, due to E
DOI: 10.1021/jf505327b J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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protein hydrolysates exhibited inhibitory effects against peroxyl and hydroxyl radical-induced DNA scission in a concentrationdependent manner (Figure 6). Although date seed protein hydrolysates exhibited a protective effect against peroxyl radical-induced DNA scission at a concentration of 0.1 mg/ mL, they were effective only at 6 mg/mL against hydroxyl radical-induced DNA scission, ranging from 13.8 to 33.0%. The positive control, carnosine, exhibited an inhibition of 30.6%. AL hydrolysate exhibited the highest inhibition of 33.0% against hydroxyl radical, which is slightly higher than that rendered by carnosine, whereas the mixture of three enzymes (AL + FL + TH) showed the lowest inhibition of 13.8%. All other enzymatic treatments exhibited no significant difference (p > 0.05) in inhibition of hydroxyl radicals (19.5−24.2%). For peroxyl radical-induced DNA scission, inhibition by the date seed protein hydrolysates ranged from 22.1 to 83.2% at a concentration of 0.1 mg/mL. All enzymatic treatments of date seed proteins exhibited significantly (p < 0.05) higher percentage of inhibition against peroxyl radical compared to that of carnosine (22.1%). Decker, Livisay, and Zhou46 observed the changes in carnosine concentrations in oxidizing skeletal muscle and suggested that carnosine may not be as effective a free radical scavenger in vivo as other antioxidants such as α-tocopherol. This seems plausible, because carnosine exhibited a lower percentage of inhibition against peroxyl radical-induced DNA scission compared to date seed protein hydrolysates. AL hydrolysate showed significantly (p < 0.05) higher inhibition percentage of both peroxyl and hydroxyl radical-induced DNA scission, whereas the mixture of three enzymes (AL + FL + TH) showed the lowest percentage of inhibition. The inhibitory effect of date seed protein hydrolysates may be due to their ability to scavenge hydroxyl and peroxyl radicals and via their chelating activity. In this experiment, it was evident that the level of inhibition against hydroxyl radical was substantially lower compared to that against peroxyl radical even at a higher concentration. A similar observation was reported by Madhujith and Shahidi47 for barley extracts. Hydroxyl radicals are short-lived and extremely reactive among all reactive oxygen species.22 In contrast, peroxyl radicals exhibit a long half-life and have a greater affinity to diffuse into cells.6 Thus, Hunag, Ou, and Prior48 suggested that the direct scavenging of the hydroxyl radical by dietary antioxidants in a biological system is unrealistic as the cellular concentration of dietary antioxidants is negligible compared with DNA and other biological molecules. Therefore, the protective effect of date seed protein hydrolysates toward hydroxyl radical could be mainly due to the chetaltion of Fe(II), whereas that of peroxyl radicals may be due to a scavenging effect. This may explain the large variations observed in inhibition efficacies observed for hydroxyl and peroxyl radicalinduced DNA scission. Regardless, this study demonstrated that date seed protein hydrolysates could serve as potential antimutagenic agents in food and biological systems. This study revealed that date seed protein hydrolysates have a good solubility over a wide range of pH (pH 2−12) and improved water-holding capacity of fish products. In addition, date seed protein hydrolysates exhibited different antioxidant activities depending on the test model systems. Thus, date seed protein hydrolysates could be used as a natural additive possessing functionalities and antioxidative properties in food systems. Furthermore, date seed protein hydrolysates inhibited human LDL cholesterol oxidation and peroxyl and hydroxyl radical-induced supercoiled plasmid DNA strand scission.
its lower ability to remove copper from the surface of the macromolecules.16 Alvarez-Parrilla, de la Rosa, Amarowicz, and Shahidi5 reported that the inhibition of LDL oxidation by polyphenols could be attributed to different mechanisms, including hydrogen donation, lipid radical scavenging, metal chelation,38 and protein binding.21 Chen and Frei39 reported that direct binding of cupric ions to LDL is important for copper reactivity because modification of histidine decreased the binding of copper to LDL and decreased copper-promoted oxidation rates. In this study, the inhibitiory effect of date seed protein hydrolysates on human LDL cholesterol oxidation could be attributed to the chelating power of protein hydrolysates for copper ions as well as scavenging of free radicals in LDL. In a previous study, Kittiphattanabawon, Benjakul, Visessanguan, and Shahidi6 showed that gelatin hydrolysate from blacktip shark at various degrees of hydrolysis showed promising inhibitiory effect on LDL cholesterol oxidation. Several studies have shown that the presence of certain amino acids in protein hydrolysates, such as histidine, tryptophan, tyrosine, phenylalanine, methionine, leucine, glycine, or proline enhances the scavenging and chelating activities of peptides.40−42 Thus, date seed protein hydrolyzed using Alcalase, Flavourzyme, and Thermolysin alone or in combination could protect human LDL cholesterol from oxidation and reduce oxidative stress in the body. Inhibition of Peroxyl and Hydroxyl Radical-Induced Supercoiled DNA Strand Scission. Free radicals generated in living cells have been shown to induce base modification and DNA strand breakage, which may subsequently lead to mutation and carcinogenesis.43 The most common radical in biological systems is the superoxide radical anion, which is produced mostly within the mitochondria of a cell.44 Superoxide alone or combination with other free radicals could damage the DNA at both the phosphate backbone and the nucleotide bases. Davies45 reported that this could lead to strand scission, sister chromatid exchange, and DNA−DNA and DNA−protein cross-links. Supercoiled DNA (form I) may be converted to a nicked open circular form (form II) and a linear form (form III) when its single strands are cleaved by free radicals.43 To assess the protective effect of date seed protein hydrolysates on hydroxyl and peroxyl radical-induced DNA scission, agarose gel electrophoresis was used, which could be used to detect the forms of DNA separately. Percentage inhibition of DNA strand scission induced by peroxyl and hydroxyl radicals is presented in Table 3. Date seed Table 3. Inhibition of Peroxyl Radical-Induced DNA Scission by Date Seed Protein Hydrolystes and Carnosinea DNA scission inhibition (%) protein hydrolysate carnosine AL FL TH AL + FL AL + TH FL + TH AL + FL + TH
hydroxyl radical 30.6 33.0 20.5 24.2 23.9 19.5 21.4 13.8
± ± ± ± ± ± ± ±
2.1c 1.4c 1.6b 1.6b 1.4b 1.9ab 1.3b 2.2a
peroxyl radical 22.1 83.2 47.8 78.5 76.8 75.2 77.4 55.2
± ± ± ± ± ± ± ±
0.4a 1.5e 1.0b 2.4de 1.3d 2.2d 1.0d 0.4c
a
All data represent the mean of triplicates. Values followed by the same letter are not significantly different (P > 0.05) by Tukey’s HSD test. F
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Figure 6. (a) Agarose gel electrophoresis of inhibition of hydroxyl radical-induced DNA scission by date seed protein hydrolystes and carnosine (6 mg/mL); (b) peroxyl radical-induced DNA scission by date seed protein hydrolystes and carnosine (0.1 mg/mL). prepared from silver carp (Hypophthalmichthys molitrix). Food Chem. 2008, 107, 1485−1493. (5) Alvarez-Parrilla, E.; de la Rosa, L. A.; Amarowicz, R.; Shahidi, F. Protective effect of fresh and processed Jalapeño and Serrano peppers against food lipid and human LDL cholesterol oxidation. Food Chem. 2012, 133, 827−834. (6) Kittiphattanabawon, P.; Benjakul, S.; Visessanguan, W.; Shahidi, F. Inhibition of angiotensin converting enzyme, human LDL cholesterol and DNA oxidation by hydrolysates from blacktip shark gelatin. LWT−Food Sci. Technol. 2013, 51, 177−182. (7) Pihlanto-Leppala, A. Bioactive peptides derived from bovine proteins: opioid and ACE-inhibitory peptides. Trends Food Sci. Technol. 2001, 11, 347−356. (8) Jeong, H. J.; Lam, Y.; de Lumen, B. O. Barley lunasin suppresses ras-induced colony formation and inhibits core histone acetylation in mammalian cells. J. Agric. Food Chem. 2002, 50, 5903−5908. (9) Clare, D. A.; Swaisgood, H. E. Bioactive milk peptides: a prospectus. J. Dairy Sci. 2000, 83, 1187−1195. (10) Elias, R. J.; Kellerby, S. S.; Decker, E. A. Antioxidant activity of proteins and peptides. Crit. Rev. Food Sci. Nutr. 2008, 48, 430−441. (11) Halliwell, B.; Zentella, A.; Gomez, E. O.; Kershenobich, D. Antioxidants and human disease: a general introduction. Nutr. Rev. 1997, 55, S44−S52. (12) Wettasinghe, M.; Shahidi, F. Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food Chem. 2000, 70, 17−26. (13) Boldyrev, A. A.; Dupin, A. M.; Bunin, A. Y.; Babizhaev, M. A.; Severin, S. E. The antioxidative properties of carnosine, a histidine containing dipeptide. Biochem. Int. 1987, 15, 1105−1113. (14) Kohen, R.; Yamamoto, Y.; Cundy, K. C.; Ames, B. Antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain. Proc. Natl. Acad. Sci. U.S.A. 1998, 85, 3175−3179. (15) Decker, E. A.; Ivanov, V.; Zhu, B. Z.; Frei, B. Inhibition of lowdensity lipoprotein oxidation by carnosine and histidine. J. Agric. Food Chem. 2001, 49, 511−516. (16) AOAC. Protein (crude) determination in animal feed: copper catalyst Kjeldahl method (984.13). Official Methods of Analysis, 15th ed.; Association of Official Analytical Chemists: Rockville, MD, USA, 1990.
Therefore, it could be a potential functional food ingredient for health promotion. However, the antioxidant effect of date seed protein hydrolysate could also be influenced by the presence of other biomolecules, including minute amounts of water-soluble phenolics in the crude material, but this effect is considered to be minimal. Therefore, purification of date seed proteins, fractionation of the resultant peptides, and their sequencing may provide further clarity.
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AUTHOR INFORMATION
Corresponding Author
*(F.S.) Phone: (709) 864-8552. Fax: (709) 864-4000. E-mail:
[email protected]. Funding
We are grateful to the Natural Science and Engineering Council (NSERC) of Canada for financial support in the form of a discovery grant to F.S. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED AL, Alcalase; FL, Flavourzyme; TH, Thermolysin REFERENCES
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