International Journal of Biological Macromolecules 65 (2014) 215–221
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International Journal of Biological Macromolecules
j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac
dentification and characterization of hydrocolloid fromordia myxa leaf
ahid Samavati a,∗, Mohammad Lorestanib, Sajjad Joolazadehc
Department of Food Science and Engineering, Faculty of Agricultural Engineering and Technology, University of Tehran, IranDepartment of Food Science & Technology, Islamic Azad University, Ayatollah Amoli Branch, Amol, IranDepartment of Food Science & Technology, Ramin Agricultural & Natural Resources University, Ahvaz, Iran
r t i c l e i n f o
rticle history:eceived 28 December 2013eceived in revised form 16 January 2014ccepted 19 January 2014vailable online 25 January 2014
a b s t r a c t
Hot water extraction technique was employed to extract the hydrocolloid from Cordia myxa leaf (PCM).The optimal conditions for extraction of PCM were determined using response surface methodology. ABox–Behnken design (BBD) was applied to evaluate the effects of three independent variables (extractiontime (X1: 1–4 h), extraction temperature (X2: 55–95 ◦C), and water to raw material ratio (X3: 5–30 ml/g)
eywords:ydrocolloid
dentificationox–Behnken
on the extraction yield of PCM. The content of moisture, water-soluble and water-insoluble ash, crudeprotein and total phenol were determined in the extracted hydrocolloid by standard methods. The max-imum hydrocolloid extraction yield (9.501 ± 0.15%) was achieved by using extraction time of 4.94 h,extraction temperature of 94.91 ◦C and water to raw material ratio of 21.74 ml/g. The contents of moisture,crude protein, water-soluble and water-insoluble ash and total phenol were 21.63 ± 0.94%, 14.27 ± 0.55%,
.19 m
odeling 3.07 ± 0.16% and 2.61 ± 0
. Introduction
Cordia myxa fruit is popularly used for the treatment of chestnd urinary infections, and as an anthelminthic, diuretic, astrin-ent, demulcent and expectorant agent [1]. Moreover, it has beeneported that leaf extracts of certain species of Cordia such as. myxa, C.francisci, and C. serratifolia have significant analgesic,nti-inflammatory, and antiarthritic activities in rats [2]. The anti-nflammatory properties of the C. myxa fruit preparation in thereatment of experimental colitis have been demonstrated by Alwadi et al. [3].
In the developed countries, plant-derived materials are nowegarded as either versatile functional ingredients or as biologicallyctive components [4,5]. These plant materials found broad appli-ations in the areas of foods (e.g. thickener, gelling agent, emulsifier,oating, fat substitute) and pharmaceuticals (e.g. radical scavenginggent, diet supplement) [6–8].
There are some interesting research reports about utilization ofydrocolloids obtained from plant leaves although plant leaves areenerally not source of hydrocolloids [9,10].
Hydrocolloid from plants has the advantage over those from
nimals because of their friendly image toward consumers. A lotf work has been done to study the chemical composition andunctionality of these hydrocolloids. However, there is still place in
the hydrocolloid market for new sources of plant hydrocolloids tomeet the demand for ingredients with more specific functionalityin foods [10].
It has been reported that many plant hydrocolloids have strongantioxidant abilities and should be paid more attention to exploringthem as novel potential antioxidants [11–13]. RSM is a collectionof statistical and mathematical techniques useful for developing,improving and optimizing process. The main advantage of RSM isthe reduced number of experimental trials needed to evaluate mul-tiple parameters and their interactions [14–17]. The application ofstatistical experimental design techniques in bioprocess develop-ment and optimization can result in enhanced product yields, closerconformance of the process output or response to target require-ments and reduced process variability, development time and cost[18].
This methodology can be used in developing suitable treatmenttechnology considering the effects of operational conditions on theremoval process or to determine a region that satisfies the operat-ing specifications [20,21].
Box–Behnken design (BBD) is an independent, rotatablequadratic design with no embedded factorial or fractional facto-rial points where the variable combinations are at the midpoints ofthe edges of the variable space and at the center [19]. The objectiveof this research were: (1) to investigate the significant variables
(extraction time, extraction temperature and ratio of water to rawmaterial) and further to optimize the process for extraction ofhydrocolloid from C. myxa leaf (PCM) using RSM and (2) to evaluatethe Antioxidant activity of extracted hydrocolloid (PCM).
Table 1Independent variables and their levels used in the response surface design.
Independent variables Factor level
−1 0 1
Extraction time (h) 1 2.5 4
TR
16 V. Samavati et al. / International Journal o
. Materials and methods
.1. Materials
The C. myxa leaf was obtained from Mollasani (Iran), thenashed with tap water, rinsed with deionized water, and then air-ried at ambient temperature (25 ◦C). To inactivate the enzymesaturally present, the leaves were heated in a hot air oven at the0 ◦C for 90 min. All other chemicals and solvents used were ofnalytical grade.
.2. Methods
.2.1. Extraction of PCMThe extraction of hydrocolloid from C. myxa leaf was performed
sing a method modified from that by Sun et al. [22]. The C. myxaeaf (2000 g) were ground in a blender to obtain a fine powder andhen were extracted for three times with 80% EtOH at 60 ◦C and 2 hach time to defat and remove some colored materials, oligosac-harides, and some small molecule materials under reflux in thepparatus, Soxhlet’s. The pretreated samples were separated fromhe organic solvent by centrifugation (3000 × g for 10 min). Eachried pretreated sample (20 g) was extracted by water in a designedxtraction temperature (55–100 ◦C), extraction time (1–6 h), andater to the raw material ratio (5–30). The water extraction solu-
ions were separated from insoluble residue through the nylonloth (pore diameter: 38 m), concentrated and then precipitated byhe addition of ethanol to a final concentration of 80% (v/v) to obtain. myxa leaf hydrocolloid (PCM). The precipitate was air-dried athe 50 ◦C until its weight was constant, and then was weightedith a balance (BS2202S, Germany). The percentage hydrocolloid
Extraction temperature (◦C) 55 75 95Water to raw material ratio (ml/g) 5 17.5 30
The applied design was integrated to determine a reason-able relationship between three independent variables and eachresponse and to find the optimum level of the independent vari-ables resulting in the desirable objectives (Table 2).
For statistical calculations, the relation between the coded val-ues and actual values are described as the following equation:
Xi = Ai − A0
�A(2)
where Xi is a coded value of the variable; Ai the actual value ofvariable; A0 the actual value of the Ai at the center point; and �Athe step change of variable.
A design matrix comprising of 17 experimental runs was con-structed. The non-linear computer-generated quadratic model isgiven as:
Y =∑
ˇo +4∑
i=1
ˇiXi +4∑
i=1
ˇiiX2i +
4∑
i<j=2
ˇijXiXj (3)
where Y is the response variable, ˇ0, ˇi, ˇii, ˇij are the regres-sion coefficients of variables for intercept, linear, quadratic andinteraction terms respectively, and Xi, Xj are the independent vari-ables (i–j). The coefficients of the second polynomial model andthe responses obtained from each set of experimental design weresubjected to multiple nonlinear regressions using software Design-Expert 8.
2.2.3. Antioxidant activity of PCM2.2.3.1. Assay of hydroxyl radicals scavenging activity. The hydroxylradicals scavenging activity of PCM was measured using a pre-vious method with modification [22,24]. In brief, the reactionmixture contained 1 ml of brilliant green (0.435 mM), 1.0 ml ofEDTA–ferrous ion solution (9 mM), 1.0 ml of H2O2 (8.8 mM) anddifferent volumes of the PCM solution (1 mg/ml). The final reaction
volume was made up to 4 ml with distilled water. After incuba-tion at room temperature for 20 min, the absorbance of the mixturewas measured at 624 nm against a blank (distilled water instead ofthe PCM solution). The same procedure was repeated with ascorbic
cid, as a positive control. The hydroxyl radical-scavenging activityas expressed as follows:
cavenging ability (%) = A0 − A1
A0× 100% (4)
A0 and A1 are the absorbance of control (without sample) andample, respectively.
.2.3.2. Superoxide radical scavenging activity. According to Mar-inez et al. [25] each 3 ml reaction mixture contained 50 mM sodiumhosphate buffer (pH 7.8), 13 mM methionine, various concentra-ions (100–400 �g/ml) of PCM, 100 �M EDTA, 75 �M nitro-blueetrazolium (NBT) and 2 �M riboflavin. Reaction started by illu-
inating sample with light and the absorbance was measured at60 nm after 10 min of illumination. Identical tubes with the reac-ion mixture were kept in the dark and served as blank. butylatedydroxyanisole (BHA) was used as a positive control. The degree ofcavenging was calculated by the following equation:
cavenging activity (%) = A − B
A× 100 (5)
here A was the absorbance of the control and B was thebsorbance in the presence of sample.
.2.4. Chemical characterization of PCM
.2.4.1. Determination of moisture content. Moisture content ofCM was measured according to Amin et al. [26]. Approximately
g of the air-dried gum was weighed in the crucible and it waslaced in the oven for 6 h at 1001 ◦C. After drying, the dried sampleas then covered and cooled in a desiccator. Later, the crucible waseighed without cover. The moisture content was calculated using
he following equations:
ry content (%) = weight of dried sampleinitial sample weight
.2.4.2. Determination of total ash and water soluble ash contents.he total ash content was determined according to AOAC Officialethod 923.03 [27]. The total ash content was calculated using this
.2.4.3. Measurement of crude protein content. Protein contentas measured according to the method of Bradford [28]. BSA
0.01–1 mg/ml) was used to produce standard calibration curve.otal protein content of the extracts was expressed as g of BSAquivalents/100 g of dry hydrocolloid.
. Results and discussion
.1. Extraction yield
.1.1. Effect of different times on extraction yield of PCMThe extraction yield of PCM affected by different extraction
imes (1–6 h) was seen in Fig. 1a, when other two factors (extractionemperature and water to raw material ratio) were fixed at 75 ◦C,
nd 17.5. The results showed that the extraction yield of PCM hadn obvious increase within the extraction time (1–4 h). The extrac-ion yields of the PCM significantly increased from 5.7% to 7.8% asime of extraction increased from 1 to 4, and then the extraction
Fig. 1. Effects of different (a) times, (b) temperatures and (c) water to raw materialrations on the extraction yield of PCM (%).
yield of PCM no longer obviously changed, when the extractiontime increasing. It was reported that a long extraction time favorsthe production of hydrocolloid [29–33]. This might be due to thetime requirement of the exposure of the hydrocolloid to the releasemedium where the liquid penetrated into the dried raw material,dissolved the hydrocolloid and subsequently diffused out from theraw material.
3.1.2. Effect of different temperatures on extraction yield of PCMTo study the effect of different temperatures on extraction yield
of PCM, extraction process was carried out using the differentextraction temperatures of 55–100 ◦C (Fig. 1b). The extraction time,and the ratio of water to the raw material were fixed at 2.5 h, and17.5, respectively. The extraction yield of hydrocolloid had beenincreasing when the extraction temperature increased from 50 to95 ◦C, and then there was no increase when extraction temperaturecontinued to rise. The extraction yield increased with increasing
the extraction temperature due to the increase of the hydrocolloidsolubility [34]. Similar results have been reported of other authorsin extraction of hydrocolloids [35,36].
218 V. Samavati et al. / International Journal of Biological Macromolecules 65 (2014) 215–221
Table 3Analysis of variance for the fitted models.
Source Degree of freedom Coefficient Sum of square Mean square F-value P-value
.1.3. Effect of different ratios of water to raw material onxtraction yield of PCM
The extraction yield (%) of PCM extracted by different ratio ofater to raw material from 5 to 30 was shown in Fig. 1c. The extrac-
ion time and extraction temperature were fixed at 2.5 h, and 75 ◦Cespectively. The extraction yields of the hydrocolloid significantlyncreased from 5.1% to 7.7% (w/w) as the ratio of water to raw mate-ial increased from 5 to 30, due to the increase of the driving forceor the mass transfer of the hydrocolloid [37]. However, when theatio continued to increase, the extraction yields no longer changed.imilar results have been reported in other researches [33,38].
According to the single-parameter study, extraction time of–4 h, extraction temperature of 55–95 ◦C, and the ratio of water toaw material of 5–30 were adopted for RSM experiments.
.2. Data analysis and evaluation of the model
The effects of three independent variables – extraction time1–4 h) (X1), extraction temperature (55–95 ◦C) (X2) and watero raw material ration (ml/g) (X3) – on the dependent variableextraction yield, Y (%w/w)) were considered using RSM, and theirnteractive relationship was studied. Analysis of variance (ANOVA)
as performed to investigate the adequacy of the suggested modelsnd identify the significant factors. The independent and dependentariables were fitted by the second-order polynomial equation tohe experimental data. Table 3 gives the statistical significance, theinear and quadratic equations and the interaction of effects. Bypplying multiple regression analysis on the experimental data,he response variable (extraction yield of PCM (%w/w)) and thendependent variables were related by the following second-orderolynomial equations:
The R2 and adj-R2 values were 0.994 and 0.987, respectively(Table 3). A high R2 indicates that the variation could be accountedfor by the data satisfactorily fitting the model.
The CV values were found to be 2.241 for yield extraction ofPCM. Since CV is a measure expressing the standard deviation asa percentage of the mean, smaller values of CV give better repro-ducibility. In general, a CV higher than 10 indicates that variation inthe mean value is high and does not satisfactorily develop an ade-quate response model [23]. These values showed a good agreementbetween the experimental and the predicted values. The low PRESS2.243 value suggests for the adequacy of the fitted quadratic modelsfor predictive applications (Table 3). Adequate precision measuresthe signal-to noise ratio. A ratio greater than 4 is desirable [23]. Forthe proposed models, this value was 39.960, a very good signal-to-noise ratio. All these statistical parameters show the reliability ofthe models.
The regression coefficient values of Eq. (4) were listed in Table 4.The P-values were used as a tool to check the significance of eachcoefficient, which in turn may indicate the pattern of the interac-tions between the variables. The smaller was the value of P, themore significant was the corresponding coefficient.
It can be seen from this table that the linear coefficients (X1, X2,X3), a quadratic term coefficient (X2
1, X23 ) and mutual interaction
coefficients (X1X3, X1X2, X1X3) were significant, with very small Pvalues (P < 0.05). The other term coefficient (X2
2) was not significant(P > 0.05).
3.3. Optimization of extraction conditions of PCM
Response surfaces were plotted using design expert software(version 8.0) to study the effects of parameters and their interac-tions on extraction yield of PCM. The results of extraction yield ofPCM affected by extraction temperature, time, number of extrac-tion and the ratio of water to raw material are presented inFigs. 2 and 3. These types of plots show effects of two factors on
the response at a time and the other factor was kept at level zero.
The 3D response surface plot and the contour plot in Figs. 2aand 3a, which give the extraction yield of PCM as a function ofextraction temperature and extraction time at fixed water to raw
V. Samavati et al. / International Journal of Biological Macromolecules 65 (2014) 215–221 219
Fig. 2. Response surface (3D) showing the effect of the extraction time (X1), extrac-tion temperature (X2), and water to raw material ratio (X3), on the extraction yieldof PCM (%).
Fig. 3. Contour plots showing the effect of the extraction time (X1), extraction tem-
perature (X2), and water to raw material ratio (X3), on the extraction yield of PCM(%).
material ratio 17.5 mg/l, indicated that the extraction yield of PCMincreased rapidly with the increasing of the extraction time from 1to 4 h, and the extraction yield of PCM was found to increase withthe increase of extraction temperature from 55 to 95 ◦C.
In Figs. 2b and 3b, when the 3D response surface plot and thecontour plot were developed for the extraction yield of PCM withvarying extraction time and water to raw material ratio at fixedextraction temperature 75 ◦C. The yield of PCM was increasingevidently as the increasing of extraction time and water to raw
material ratio. It can be seen that maximum recovery of PCM canbe achieved when extraction time and water to raw material ratiowere around 4 h and 30 ml/g, respectively.
220 V. Samavati et al. / International Journal of Biological Macromolecules 65 (2014) 215–221
Table 5Predicted and experimental values of the response at optimum conditions.
Optimum condition Extraction yield of PCM (%)
◦ ater to raw material ratio (ml/g) Experimental Predicted
01 ± 0.15 9.368 ± 0.24 27.41
rtotta
3
(acrcrs9tm
3
3
atoCiaPcPhs
Fc
0
10
20
30
40
50
60
70
80
90
100
0 40 80 120 160 200 240 280
Supe
roxi
de ra
dica
l sca
veng
ing
ac�v
ity
(%)
Con centra�o n of HCM (ppm )
BHA
HCM
Extraction time (h) Extraction temperature ( C) W
89.83 3.87 9.5
Figs. 2c and 3c show the effect of temperature and water toaw material ratio on the extraction yield of PCM at a fixed extrac-ion time of 2.5 h. At a definite extraction temperature, the yieldf extraction increased slightly with the increase of the extrac-ion time, and nearly reached a peak at the highest extraction timeested. The highest extraction yield occurred at extraction temper-ture 95 ◦C and water to raw material ratio 30 ml/g.
.4. Verification of predictive model
The optimal condition obtained using Box–Behnken designBBD) was as follows: extraction time, 3.75 h, extraction temper-ture, 94.98 ◦C and water to raw material ratio, 26.54 ml/g. Toompare the predicted result (9.501%) with the practical value, theechecking experiment was performed using this deduced optimalondition. The mean value of 9.368 ± 0.24% (n = 4), obtained fromeal experiments, demonstrated the validity of the RSM model,ince there were no significant (P > 0.05) differences between.501% and 9.368 ± 0.25% (Table 5). The strong correlation betweenhe real and the predicted results confirmed that the response
odel was adequate to reflect the expected optimization.
.5. Radical scavenging activity
.5.1. Scavenging activity of PCM on hydroxyl radicalAmong the reactive oxygen species, hydroxyl radical is the most
ctive free radical that attacks all the biological molecules by set-ing off free radical chain reactions [39]. Fig. 4 shows an analysisf the hydroxyl radical-scavenging activities of PCM with vitamin
as positive control. This shows PCM exhibited scavenging activ-ty toward hydroxyl radicals in a concentration-dependent mannernd the scavenging effect increased based on the concentration ofCM. As shown in Fig. 4, PCM scavenging effect of hydroxyl radi-als increased by increasing the concentration of PCM up to 280PM. PCM was found to have a higher scavenging effect (90%) on
ydroxyl radicals than vitamin C (83%), suggesting that PCM hastronger scavenging activity on hydroxyl radicals.
0
20
40
60
80
100
0 40 80 12 0 16 0 20 0 24 0 28 0
OH
radi
cal s
cave
ngin
g ac
�vity
(%)
Con centra�o n of HCM (ppm )
Ascorbic acid
HCM
ig. 4. Hydroxyl radical-scavenging activity of PCM and vitamin C at different con-entrations. Data are mean ± SD for three measurements.
Fig. 5. Superoxide radical-scavenging activity of PCM and BHA at different concen-trations. Data are mean ± SD for three measurements.
3.5.2. Superoxide radical scavenging activity of PCMSuperoxide radical is known to be very harmful to cellular com-
ponents and plays a major role in the formation of other reactiveoxygen species such as hydroxyl radical, hydrogen peroxide andsinglet oxygen in living system [40]. Regarding data presentedin Fig. 5, the scavenging activity of PCM increased with increas-ing concentration up to a certain level (210 ppm) and reached aplateau. PCM was found to have a lower scavenging effect (49%)on superoxide radicals than BHA (91%). The results suggested thatthe hydrocolloid exhibited scavenging effect on superoxide anionradical generation that could help prevent or ameliorate oxidativedamage. Scavenging effect of hydrocolloid on superoxide radicalshave been reported for various plant hydrocolloids [40–42].
3.6. Chemical composition of PCM
The PCM had 21.63 ± 0.94% of moisture content, 14.27 ± 0.55% oftotal ash content and 3.07 ± 0.16% of crude protein. It was reportedthat the ash content of arabic, guar and xanthan gum were 1.2%,11.9% and 1.5%, respectively [43]. This shows that the ash contentof PCM was higher compared to the commercial gum. The resultsshowed that only 0.32% of the ash was water soluble. The crudeprotein content of PCM is similar to previously reported results forprotein content of some other hydrocolloids like agar, carrageenan,tragacanth and Karaya gum [44].
4. Conclusion
The results that the aqueous extraction condition significantly(P < 0.05) influenced the extraction yield of hydrocolloid from C.myxa leaf. In the present work, the extraction time and temperatureexhibited the most and least significant (P < 0.05) impact on theextraction yield of hydrocolloid from C. myxa leaf, respectively. Thiswork revealed that the maximum hydrocolloid extraction yield wasachieved by using extraction time 4.94 h, extraction temperature
94.91 ◦C and water to raw material ratio of 21.74 (w/w). Radicalscavenging tests showed that the hydrocolloid extracted from C.myxa leaf has strong radical scavenging activities.
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V. Samavati et al. / International Journal o
cknowledgement
The author wishes to thank the University of Tehran for financialnd scientific supports.
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