Food Sci. Biotechnol. 23(6): 2075-2082 (2014)
DOI 10.1007/s10068-014-0282-2
Antioxidant Activities and Functional Properties of Tea Seed Protein Hydrolysates (Camellia oleifera Abel.) Influenced by the Degree of Enzymatic Hydrolysis
Xu Li, Shian Shen, Junlin Deng, Tian Li, and Chunbang Ding
Received April 22, 2014; revised June 24, 2014; accepted June 26, 2014; published online December 31, 2014
© KoSFoST and Springer 2014
Abstract Antioxidant activities and functional properties of tea seed protein hydrolysates (TSPH) prepared using alcalase with different (10, 20, 30 and 40%) values of the degree of hydrolysis (DH) were investigated. The effect of hydrolysis time on antioxidant activity was also investigated. As the hydrolysis time was extended, the DPPH radical scavenging activity increased and finally reached a plateau, the copper chelating capacity decreased, and the superoxide radical scavenging and iron chelating activities increased initially, then subsequently slowed. The solubility, foaming properties, and emulsification prope
rties of TSPH were affected by pH and DH. As the DH value increased, the DPPH radical scavenging activity and the reducing power increased and the copper chelating capacity decreased. TSPH at 20 and 30% DH values exhibited higher superoxide radical scavenging and stronger iron chelating activities respectively, than TSPH at other DH values. The DH value of TSPH affected the antioxidant activity and functional properties.
Keywords: protein hydrolysate, antioxidant activity, functional property, hydrolysis, Camellia oleifera Introduction
Camellia oleifera Abel is an evergreen oilseed tree crop that is widely distributed in south and central China. The seed is used for extraction of camellia oil, which is rich in unsaturated fatty acids and is used extensively as a cooking oil and as an adjuvant medicine for burn injuries and stomachache. The oil has strong antioxidant compounds that resist degenerative pathologies (1,2). However, a large amount of tea seed cake residue containing 14-20% of crude protein is discarded in oil industry (3). This byproduct can be used as an animal feed, fuel, or organic fertilizer. The amino acid profile of tea seed protein indicates that the product can be a source of high nutritional value plant protein (4).
reaction between pvp and aminoFunctional properties of proteins are important in food applications. Most native proteins do not show d
esirable functional properties. Thus, protein modification for property improvement, especially protein solubility, needs to be addressed (5). Enzymatic hydrolysis is an attractive tool to modify proteins due to mild reaction conditions and ease of control (6). Enzymatic hydrolysis disrupts the 3-dimensional protein structure, reduces the molecular weight (Mw), and consequently alters the functional properties (7). Enzymatic hydrolysis has been applied successfully for improvement of functional properties by cleaving proteins into peptides of desired sizes, charge, and surface properties (5). These peptides exert different physiological functions after they are released by enzymatic hydrolysis, but they are inactive within the parent protein (8). Hydrolysates have excellent solubility values at high degrees of hydrolysis (DH). However, excessive hydrolysis has a negative effect on functional properties, including foaming and emulsification (9,10).
Apart from functional properties, protein hydrolysates from food resources, such as fish (11), blood hemoglobin (12), rice endosperm (13), and rapeseed (14), have been shown to possess antioxidant activities. Enzymatic hydrolysis
Xu Li, Shian Shen, Junlin Deng, Chunbang Ding ( )
College of Life Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, China
Tel: +86-0835-*******; Fax: +86-0835-*******
E-mail: dcb@sicau.edu
Tian Li
College of Agronomy, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
RESEARCH ARTICLE
2076Li et al.
affects the molecular size and hydrophobicity, and polar and ionizable groups of protein hydrolysates (9).
The main aim of this study was to investigate antioxidant activities and functional properties of tea seed protein hydrolysates (TSPH) using different DH values with alcalase. Additionally, the effect of the hydrolysis time on antioxidant activities and metal chelating activities was also investigated.
Materials and Methods
Materials Camellia seed cake was provided by Times Biotech Co., Ltd. (Y a’an, Sichuan, China). Alcalase, pyrocatechol violet, 2,4,6-trinitrobenzen sulfonic acid (TNBS), and Sephadex G-50 were purchased from Kayon Biological Technology Co., Ltd. (Shanghai, China). DPPH and ferrozine were purchased from Sigma-Aldrich Chemical Co. (St. Louis. MO, USA). All other chemicals and regents used were of analytical grade. Preparation of TSPH Tea seed protein (TSP) was extracted by suspension of tea seed flour in distilled water (1:10, w/v). The pH value was adjusted and maintained at pH 10.0 using 1 M NaOH with agitation by homogenizer (FSH-2; Puguang Physical Optics Instrument Co., Ltd., Changzhou, China) for 1h at 40o C, followed by centrifugation using centrifugal (Thermo Fisher Scientific,Waltham, MA, USA) at 5,000×g for 20min at room temperature. The supernatant pH was adjusted to 4.0-4.5 using 1M HCl, followed by centrifugation at 10,000×g for 20min. The precipitate was collected and vacuum-dried using vacuum-drying oven (DZF; Puguang Physical Optics Instrument Co., Ltd., Changzhou, China) at 40o C. Alcalase was dissolved in distilled water, then added to a slurry of the resulting precipitate with the enzyme at a substrate ratio (E/S) of 2:100. The resulting slurry was incubated at 55o C for 12h with a constant pH value of 8.5 maintained using addition of 1M NaOH. At the end of hydrolysis, the resulting hydrolysate was adjusted to pH 7.0 by using 1M HCl and heated in boiling water for 10min to inactivate the enzyme. The next step was centrifugation by using centrifugal at 10,000×g for 20min. The supernatant was vacuum-dried by using vacuum-dried oven, then stored at 20o C for further use.
Determination of the DH value The DH value was determined using the method of Benjakul and Morrissey (15) based on a reaction between the α-amino group of the sample and TNBS. The α-amino groups were obtained using complete sample acid hydrolysis with 6 M HCl at 110o C for 24 h.Antioxidant activities
DPPH radical scavenging activity: The DPPH radical scavenging activity of TSPH was determined as described by Wu et al. (16) with some modification. An amount of 0.5mL of sample solution of different concentrations from 0.15-2.5mg/mL was added to 4.5mL of DPPH (0.15mM in 95% ethanol). The mixture was mixed vigorously using vortex mixer and incubated for 30min in the dark at room temperature. The absorbance of the resulting mixture was measured by applying UV-spectrophotometer (UV-1750; Shimadzu, Kyoto, Japan) at 517nm. A blank was prepared in the same manner, except that distilled water was used instead of a sample. The DPPH radical scavenging activity was defined as:
DPPH radical scavenging effect (%)
=×100
where A0 is the absorbance of a blank, A s is the absorbance of a sample, and A h is the absorbance o
f a sample without addition of the DPPH solution.
Superoxide radical scavenging activity: The superoxide radical scavenging activity was measured based on monitoring inhibition of pyrogallol auto-oxidation according to the method of Zhang et al. (17). An amount of 0.1mL of sample was dissolved in the Tris-HCl-EDTA buffer (0.1 M, pH 8.0). After 10min at room temperature, 0.1mL of 3mM pyrogallol was added to initiate a reaction. The absorbance of the mixture was measured by using UV-spectrophotometer at 325nm. The superoxide radical scavenging activity was determined as the percent inhibition of pyrogallol autooxidation, calculated using sample absorbance values in the presence and absence of pyrogallol.
Determination of the reducing power: The reducing power of TSPH was determined as described by Oyaizu (18) with minor modification. A series of TSPH solutions at concentrations of 0.15-2.5mg/mL were prepared, then 1mL of each concentration was mixed with 2.5mL of 0.2M phosphate buffer (pH 6.6) and 2.5mL of 1% potassium ferricyanide. The mixtures were mixed vigorously by using vortex mixer, then incubated at 50o C for 20min after which 2.5mL of 10% trichloroacetic acid was added to the mixtures, followed by centrifugation for 10min at 3,000×g. The supernatant (2.5mL) was diluted using 2.5mL of distilled water and 0.5mL of 0.1% ferric chloride. The absorbance of the resulting solution was measured by using UV-spectrophotometer at 700nm. An increase in the absorbance of th
e mixture indicated an increase in the reducing power.
A0A s A h
–
()
–
A0
----------------------------
Antioxidant and Functional Properties of Peptides 2077
Metal chelating activities
Iron chelating capacity : The Fe 2+-chelating activity was determined as described by Decker et al. (19). TSPH solutions at concentrations of 0.15-2.5mg/mL were prepared,then 3.7mL of distilled water and 0.1mL of 2 mM FeCl 2were added to 1mL of each solution. Ferrozine (0.2mL,5mM) was then adde
d, and the mixture was allowed to react for 20min at room temperature. The absorbance was measured by using UV-spectrophotometer at 562nm. A blank was prepared in the same manner except that distilled water was used instead of a sample. The iron chelating activity was calculated as:
Chelating activity (%)=×100
where A 0 is the absorbance of a blank and A s is the absorbance of a sample.
Copper chelating activity : The Cu 2+-chelating activity was determined as described by Saiga et al. (20) with slight modification. A 4mM pyrocatechol violet solution and 0.1mg/mL of a CuSO 4 solution were prepared using sodium acetate buffer (pH 6.0, 50mM), then 2mL of a CuSO 4solution was added to 1mL of sample with a series of concentrations (0.3-5.0mg/mL), followed by stirring by using vortex mixer. Subsequently, 0.5mL of a pyrocatechol violet solution was added, mixed completely by using vortex mixer, and then allowed to react for 20min at room temperature. The absorbance was measured by using UV-spectrophotometer at 632nm. The copper chelating capacity was calculated as described above for iron.
Functional properties
Solubility : The solubility was tested at different pH values following the method of Castellani et al. (21) with minor modification. A sample was dispersed in distilled water and the pH of the mixture was adjusted using either 1M HCl or 1M NaOH. After stirring though applying a shaker table for 30min at room temperature, mixtures were centrifuged by using centrifugal at 8,000×g for 15min.The supernatant was collected and the protein content was determined using the method of Lowry et al. (22).
Foaming property : The foaming ability and the foam stability of TSPH were determined according to the method of Shahidi et al. (23). Samples were dispersed in distilled water of pH values at a range of 3-11 to obtain a concentration of 5mg/mL, then 20mL of the solutions was in a 50mL graduated bottle, and homogenized at a speed of 10,000rpm for 1min using a homogenizer. The total volume was read though graduated bottle prior and after 30min, of homogenization. The foaming ability was expressed as foam expansion at 0min, while the foam stability was expressed as foam expansion after 30min.The foam expansion was calculated as (24):
Foam expansion (%)=×100
Foam stability (%)=×100
where A =the volume after homogenization (mL) and B =the volume before homogenization (mL).
Emulsification property : The emulsion activity index (EAI)and the emulsion stability index (ESI) were determined according to the method of Pearce and Kinsella (25) using samples of 5mg/mL prepared with distilled water and pH adjusted to values of ranges of 3-11 using either 1M HCl or 1M NaOH. Resulting samples (6mL) and oil (2mL) were homogenized by homogenizer at a speed of 10,000rpm for 1min and resulting emulsions were pipetted out at 0 min and again after 10min, then diluted using 0.1% SDS to 100× and completely mixed though using vortex mixer.The absorbance was measured immediately by aping UV-spectrophotometer at 0min (A 0) and after 10 min (A 10) at 500nm, and values were used to calculate EAI and the ESI values as:
EAI (m 2/g)=ESI (min)=where A 0=the absorbance at time zero, A 10=the absorbance at 10min; ϕ=the oil volume fraction, and C=the protein concentration in the aqueous phase (g/m 3).
Amino acid composition analysis Samples of TSPH were hydrolysed using 6M HCl for 24h at 110o C in sealed tubes. The amino acid composition, except for tryptophan,was measured using an automatic amino acid analyser (L-8800; Hitachi, Tokyo, Japan). The TSPH and TSP amino acid compositions were reported as g/100 g of protein.Size exclusion chromatography TSPH peptide fractions were sepa
rated using a Sephadex G-50 column. Samples were dispersed in distilled water to obtain a concentration of 20mg/mL, then 2mL of the resulting suspension was fractioned using a Sephadex G-50 colunm (1.0×60cm) and eluted with distilled water. Each 4 mL of separated peptide fractions was collected at a flow rate of 80mL/h, and the absorbance values of separated peptide fractions were monitored by using UV-spectrophotometer at 280nm.Statistical analysis All tests were peformed at least in triplicate and data were expressed as mean±standard deviation (SD). Data were subjected to statistical analysis using the Origin Pro 8.0 statistics program (OriginLab
A 0A s
–A 0
--------------A 0min B
–B ---------------------A 30min B
–A 0min B
–-----------------------2 2.303A ×()100
×ϕC
-----------------------------------------A 010×A 0A 10
–-----------------
2078Li et al. Corporation, Northampton, MA, USA), and statistical
differences were defined as p<0.05 using Tukey’s test.
Results and Discussion
Enzymatic hydrolysis TSP was subjected to hydrolysis
using alcalase. The hydrolysis process was monitored
based on DH values. The rate of hydrolysis initially
progressed rapidly for 1.5h, then subsequently slowed
(Fig. 1A), similar to typical hydrolysis curves (9,26). DH
values of 10% (10.9±0.5%), 20% (19.3±0.9%), 30%
(30.7±0.8%), and 40% (39.3±1.5%) were observed from
the hydrolysis curve (Fig. 1A). The hydrolysis reaction
continued after the hydrolysis rate slowed after 1.5h to
obtain a 30% DH value at 4h and a 40% value at 12h.
Extension of the hydrolysis time resulted in more small
size peptides.
Effect of hydrolysis time on antioxidant and metal
chelating acticvities The effect of hydrolysis time on the
radical scavenging and metal chelating activities was
investigated in order to determine the relationship of these
activities to the DH. The DPPH and superoxide radical
scavenging activities both increased with the hydrolysis
time for 120min (Fig. 1B). Thereafter, the DPPH radical
scavenging activity was steady after reaching a plateau
while the superoxide radical scavenging activity decreased
after reaching the highest value. The relationship between
the metal chelating activities and hydrolysis time was complex. The copper chelating activity decreased with hydrolysis time while the iron chelating activity showed an initial rise followed by a subsequent decline. The iron chelating activity was steady at approximately 42% during the hydrolysis time period of 0.5-1.5h, subsequently increased to the highest point (67.5%) at 6h, then declined linearly to the end of the hydrolysis process with an activity of just 20.8%. Hydrolysis time apparently affected both antioxidant activity and metal chelating capacity of TSPH.
Enzymatic hydrolysis exposes the inner peptide domains of proteins where active sites are likely found. However, excessive hydrolysis resulting in cleavage of proteins into small peptides breaks the structure of the active peptide, causing a decrease in bioactivity (27). The progress of hydrolysis affect
ed peptide bioactivity by DH and enzyme used, and hydrolysis time changes the profile of the hydrolysate, resulting in variation of hydrolysate functions (9,12).
Antioxidant activities
DPPH radical scavenging activity: DPPH, a stable free radical, has been widely used to determine the radical scavenging activities of different antioxidants with an absorbance maximum at 517nm in ethanol. TSPH exhibited a dose-dependent increase in the DPPH radical scavenging activity (Fig. 2A). At 2.5mg/mL, the TSPH activities at DH values of 10, 20, 30, and 40% were 50.9, 59.7, 64.4, and 67.7%, respectively. A higher degree of protein cleavage caused higher DPPH radical scavenging activities of hydrolysates. An increase in the DH value causing increased DPPH radical scavenging of hydrolysates has also been reported for porcine plasma protein (12), for porcine collagen protein (28), and for yellow stripe trevalley protein (9). In peanut protein hydrolysate, further hydrolysis had no effect on the enzyme activity (26).
Superoxide radical scavenging activity: The superoxide anion radical is the most common free radical generated in organisms and can be converted to more destructive free radical forms, including hydrogen peroxide and the hydroxyl radical, via dismutation and other reactions. The superoxide anion
radical and derivatives can damage DNA, proteins, and cell membranes. Therefore, identification of antioxidants that can scavenge the superoxide radical is important. The antioxidant activity of TSPH based on monitoring of the superoxide anion radical scavenging
activity is shown in Fig. 2B for different sample Fig. 1. An enzymatic hydrolysis curve (A) prepared using alcalase, and the effect of hydrolysis time on the radical scavenging and metal chelating activities (B).
Antioxidant and Functional Properties of Peptides 2079
concentrations. TSPH exhibited a strong superoxide radical scavenging activity and showed an activity increase with an increased sample concentration. The highest activity of 82.7% at a concentration of 2.5mg/mL was observed for TSPH with a 20% DH value. TSPH with a 10% DH value exhibited the lowest activity of 48.3%. However, higher hydrolysis (30 and 40% DH) values did not result in any increase in the radical scavenging activity. TSPH effectively inhibited auto-oxidation of pyrogallol, indicating that TSPH probably contains substances that retard the oxidation reaction induced by the superoxide anion radical.
Reducing power : The reducing power of TSPH at different
DH values with different sample concentrations is shown in Fig. 2C. The reducing power of TSPH increased with an increasing DH value, similar to the DPPH radical scavenging activity. The highest reducing power of TSPH was observed at a 40% DH value, while the lowest activity was exhibited at a DH value of 10%. Excessive hydrolysis probably generated small peptides that contributed to scavenging of the radical to retard the oxidation reaction. An increase in the reducing power with an increase in the DH value has also been reported for hydrolysates using alcalase from porcine plasma (12) and protease N from mackerel (16).Metal chelating capacities : Transition metal ions catalyze generation of reactive oxygen species that cause cell damage in organisms by acceleration of auto-oxi
dation and breakdown of volatile compounds in food storage (20). Therefore,chelation of transition metal ions by antioxidants or antioxidant peptides can retard the oxidation reaction. The metal chelating capacities of TSPH were affected by the DH value and the sample concentration (Fig. 3). The iron chelating capacity of TSPH exhibited a dose-dependent increase (Fig. 3A). The highest capacity of 78.5% at 2.5mg/mL was observed for TSPH with a 30% DH value,while the lowest capacity was found for at a 20% DH value
with the capacity of 61.7%. However, further hydrolysis
Fig. 2. The antioxidant activities of TSPH at different DH values with different sample concentrations, including DPPH (A), the superoxide radical scavenging activity (B), and the reducing power (C). V alues reported represent mean±SD (n
=3).
Fig. 3. The metal chelating activity of TSPH at different DH values with different sample concentrations. Iron (A), and copper (B) chelating activities. V alues reported represent mean±SD (n =3).
2080Li et al. (40% DH) did not increase the iron chelating capacity. An
appropriate degree of protein cleavage produced hydrolysates
with strong iron chelating capacities.
Copper chelating capacities of TSPH decreased as the
DH value increasing (Fig. 3B). And at 5.0mg/mL, copper
chelating capacities of TSPH with DH, from 10 to 40%,
showed a decrease from 74.8 to 47.5%. Higher DH value
means more small peptides generated (9). Copper chelating
activities of TSPH at DH values 30% were weaker than
iron chelating activities at the same DH values (Fig. 3),
indicated that binding of copper was more sensitive to the
size of the peptide than binding of iron. These results were
in disagreement with results of a study of porcine plasma
protein hydrolysates in which the copper chelating and iron
chelating activities increased with an increase in the DH
value, and the copper chelating activity was higher than the
iron chelating activity (12).
Functional properties
Protein solubility: The solubility of TSPH at different DH values in a pH range of 2-12 is shown in Fig. 4. All hydrolysates exhibited an increase in the solubility of TSPH as the DH increasing, especially at pH 4. Hydrolysates had excellent solubility values at high DH values. The lowest TSPH solubility value was observed at pH 4, probably due to the least repulsive force between proteins at isoelectric points. Solubility values of all samples were good, especially at pH7, except at the isoelectric points. Generally, excessive hydrolysis of proteins produced more small peptides that produced more polar residues with an ability to form hydrogen bonds and increase the solubility (29). TSPH at DH values 30% was observed to have high solubility values about 90% under both neutral and alkaline conditions.
Foaming properties: The foam expansion ability (FA) and the foam stability (FS) of TSPH are shown in Fig. 5A and 5B at different DH and different pH values. At pH 5 and 7, TSPH with a 30% DH value showed the highest value of FA, following by 20% DH, 10% DH, and 40% DH. FS values of TSPH at 10 and 20% DH values were stronger than at other pH values. The FA and FS values of TSPH were poor at pH 9. TSPH with a 40% DH value showed the lowest FA and FS values at all values of pH, suggesting that excessive hydrolysis has a negative effect on the foaming properties. Hydrolysates with higher DH values contained smaller peptides that were able to incorporate more air into solution than larger peptides, and to increase the foam capacity, but lacked sufficient strength to produce stable foam (10). An appropriate degree of hydrolysis presumably produces proteins that can incorporate more air into a solution and produce greater foam strength. In this study, TSPH with a 30% DH value exhibited good FA and FS values.Emulsification properties: The EAI and ESI values of TSPH are shown in Fig. 5C and 5D at different pH values. The EAI value of TSPH increased with an increase in the pH value. The emulsifying activity of all preparations was independent of the DH value. The emulsifying activity was highest at pH 9 due to changes in the surface charges of peptides with changing pH values. Excessive hydrolysis commonly produces more small peptides that decrease EAI values due to a low efficiency in reduction of interfacial tension (9,10). However, in this study, additiona
l hydrolysis from DH values of 20 to 40% resulted in no significant (p>0.1) decrease in the EAI value, compared with TSPH of 20% DH. The behavior of the ESI values of TSPH was influenced by pH. At pH 3, the ESI value of TSPH decreased with an increase in the DH value. At pH 5, 7, and 9, TSPH at a 40% DH value exhibited the strongest emulsifying stability of all preparations. Additional hydrolysis (DH≥20%) did not decrease the emulsifying activity, and the emulsifying stability was improved under neutral and alkaline conditions.
Amino acid profile and size exclusion chromatography TSP peptides with 40% DH values were subjected to amino acid composition analysis to evaluate the effect of the amino acid profile on the antioxidant activity. Amino acid profiles of TSP peptides are shown in Table 1. The antioxidant activity is influenced by the amino acid peptide composition (8). The amino acid profile of TSP peptides was nonsignificant difference from the profile of TSP (p>0.1), and TSP and TSP peptides was rich of acidic amino acids such as Asp and Glu. Je et al. (30) reported that peptides containing acidic amino acids exhibited high degrees of antioxidant activity due to acidic amino acid residues.
The Mw of TSPH with a 40% DH value is shown in
Fig. 6, based on size exclusion chromatography using a Fig. 4. The protein solubility of TSPH at different DH values with different pH values. V alues reported represent mean±SD (n=3).
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