Isolation of bioactive polysaccharide from acorn and evaluation of its functional properties

International Journal of Biological Macromolecules 72 (2015) 179–184

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Isolation of bioactive polysaccharide from acorn and evaluation of its functional properties Mehrnoosh Tadayoni a,∗ , Mahmoud Sheikh-Zeinoddin b , Sabihe Soleimanian-Zad c a Department of Food Science, College of Agriculture, Isfahan University of Technology, Isfahan, Iran, Islamic Azad University Baghmalek Branch, Baghmalek, Iran b Department of Food Science, College of Agriculture, Isfahan University of Technology, Isfahan, Iran c Department of Food Science, College of Agriculture, Biotechnology Research Institute, Isfahan University of Technology, Isfahan, Iran

a r t i c l e

i n f o

Article history: Received 28 May 2014 Received in revised form 17 July 2014 Accepted 7 August 2014 Available online 23 August 2014 Keywords: Prebiotic Functional food Antioxidant activity Polysaccharide Acorn

a b s t r a c t The aim of this study was to evaluate the prebiotic potential and some functional properties of polysaccharides isolated from acorn fruit. The FTIR spectrum of isolated acorn polysaccharide (IAP) showed the typical bands corresponding to sugars and polysaccharides. The IAP was resistant to simulated acidic and enzymatic digestion even more than Inulin (In). The prebiotic activity, which was tested using IAP as a carbon source, showed significant increase in the growth and viability of Lactobacillus plantarum A7 (probiotic). Viability of Lactobacillus plantarum A7 in IAP and In supplemented media was stable even after 72 h, whereas in glucose supplemented medium, bacterial growth showed a notable decrease after 24 h. Lipid absorption capacity (LAC) and water holding capacity (WHC) of IAP were 5.44 ± 0.02 (g oil/g DM) and 4.33 ± 0.03 (g water/g DM), respectively, which were comparable to some dietary fibers and were more than In. IAP scavenged DPPH radicals by 82.24%. IAP was found to have a high scavenging ability compared to the reference prebiotic (In), giving a scavenging ability of about 20%. Therefore, due to prebiotic capability, high WHC, LAC and good antioxidant activity, IAP can be a suitable candidate for technological applications and health improving effects in functional food. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In recent years, natural plant compounds have received considerable attention from scholars because of health benefits from their relevant bioactive constituents [1]. Health improving claims and preventive or therapeutic effects of bioactive compounds correspond to polyphenols, proteins, lipids, vitamins, polysaccharides and other constituents [2]. Polysaccharides isolated from plants, mushrooms or seaweeds have emerged as an important class of bioactive natural products [1–4]. They exhibit various biological effects including antitumor, anticoagulant, antiviral, immune modulatory and anti-inflammatory, anti-diabetic, hepato-protective, hematopoietic, anti-oxidant or free radical scavenging activity and anti-lipidemic effects [5–9]. Some studies are now focused on the prebiotic potential of polysaccharides extracted from natural sources such as artichoke [10] seaweeds [11] bamboo shoot [12] edible burdock [13]. Prebiotics are defined as non-digestible ingredients that beneficially affect host’s health

∗ Corresponding author. Tel.: +98 9161720691; fax: +98 3113912254. E-mail address: [email protected] (M. Tadayoni). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.015 0141-8130/© 2014 Elsevier B.V. All rights reserved.

by selectively stimulating the growth or activity of one or a limited number of bacteria in the colon [14]. Prebiotics alone or with probiotic bacteria (symbiotic) can exert health improving effects by influencing the formation of blood glucose, increasing mineral absorption, reducing the cholesterol and serum lipid level, reducing the risk of colon cancer, artroscelrosis and immunomodulatory activities [15–20]. Additionally, due to their porous matrix structure, polysaccharides could absorb and hold oil or water in their matrix, and therefore could be valuable additives in food and drug industries because of their rheological properties like gelling and a thickening agent for stabilizing and modifying the texture of food or drug [21,22]. In addition, these properties are very important for helping to prevent or control obesity and abnormal blood lipid profiles [21,23,24]. Therefore, isolation of bioactive polysaccharides from new sources and evaluation of their functional property for technological applications or popular food formulation have become a hot research spot. Oak or acorn fruit is indigeneous to Italy, Spain, Iran, North America and India [25]. The species of oak belonged to Quercus genus and are classified into the Fagaceae family including about 200 species. Different species of oak grow in Iran, but four species of oak are found in the Zagrossian region. Quercus branti is the most famous and dominant

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species in the Zagros mountain chain in Iran, and this study was carried out on this species [25]. The use of acorn in local diet and empirical treatment of some human diseases such as diarrhea is not something new to Iranians. Due to its anti-nutritional substances, bitter constituents, wooden texture and astringent taste, acorn is scarcely used in normal diet [25]. However, its widespread availability and empirical use for medical purposes suggest some possibilities of using acorn in food and drug industries. Therefore, this study was carried out to isolate and characterize of acorn polysaccharide and evaluate its prebiotic and functional potential. 2. Material and methods 2.1. Materials Acorn fruit was obtained from a local market in Baghmalek, Iran. All chemical reagents, enzyme and media used in this study were purchased from the Merck Chemical Company (Darmstadt, Germany) and Sigma-Aldrich. Inulin (In) was obtained from Sensus, USA. 2.2. Extraction Acorn fruit was ground in a blender and then defatted with three volumes of 80% ethanol at 60 ◦ C for 8 h. Ethanol extract was then separated from defatted ground acorn through filtration, and the defatted ground acorn was air dried. The pretreated sample was extracted by hot water bath (Memmert, Germany) at 90 ◦ C for 3 h. Then, the extract containing water-soluble polysaccharide was centrifuged (Sigma k-16, Germany), to remove insoluble residues (2000 × g for 10 min, at 20 ◦ C). The solution was concentrated in a rotary evaporator (BUCHI 011, Switzerland) under reduced pressure at 60 ◦ C. The water extraction solution was separated from debris fragments (8000 rpm for 10 min, at 20 ◦ C). The water extraction solutions were precipitated by addition of three volumes of 80% ethanol at 4 ◦ C for 48 h. The precipitate was collected by centrifugation (2000 × g, 10 min, at 4 ◦ C) and washed three times with ethanol, and then the extract was distilled by a rotary evaporator to remove the residual ethanol in it and finally lyophilized in a freeze dryer (Christ alpha-1-4, Germany) [12,22,26]. The purity of crude polysaccharide is calculated by phenol-sulphuric method [22]. 2.3. FTIR spectroscopy One mg of dried polysaccharide was weighed and mixed with 100 mg of potassium bromide. The mixture was made into a pellet for FTIR measurement in the frequency range of 4000–400 cm−1 [3]. The IR specta of the polysaccharides were determined using a Fourier transform IR spectrophotometer (JASCO, Japan). 2.4. Resistance to acidic and enzymatic digestion In vitro resistance of polysaccharide to acidic and enzymatic digestion was studied based on Jain et al. [27] and was compared with Inulin (In) as a typical prebiotic reference. Simulation of gastric intestinal transit conditions was carried out using different dissolution media. Simulated gastric fluid (SGF) pH 1.2 consisting of NaCl (2 g), HCl (7 ml) and pH level was adjusted to 1.2 ± 0.5. Simulated intestinal fluid (SIF) pH 7.4 included KH2 PO4 (6.8 g), NaOH (190 ml) and ␣-amylase (2 unit/ml) (Sigma-Aldrich). Simulation of the mixture of simulated gastric and intestinal fluid (SMF) of pH 4.5 was performed by mixing SGF and SIF in a ratio of 39:61. The dissolution studies were carried out in 900 ml of dissolution medium, which was stirred at 100 rpm at 37 ◦ C [27]. A sample was taken at 1, 2, 3 h to determine the percentage of hydrolysis, which was calculated based on reducing liberated sugar and total sugar content according

to DNS (3,5-Dinitrosalicylic acid) and phenol-sulfuric acid method [28,29].

2.5. Effect of extracted polysaccharide on probiotic growth Lactobacillus plantarum A7 obtained from the microbial collection of Food Science and Technology Department, Isfahan University of Technology (Isfahan, Iran). The probiotic properties of Lactobacillus plantarum A7 were previously reported by Mirlohi et al. [30,31]. To obtain sufficient cells for each inoculum, cultivation was performed on MRS broth (Merck, Germany) at 37 ◦ C for overnight. MRS free carbohydrate supplemented with 2% isolated acorn polysaccharide (IAP), a reference prebiotic (In) and glucose (Glu), were used for the growth of probiotic. The culture was incubated at 37 ◦ C for 72 h. Samples were taken at 24, 48, 72 h for bacterial enumeration and pH. To evaluate the metabolization of the extracted polysaccharide through probiotic, the effect of its different concentrations on pH changes was studied [32].

2.6. Functional properties Water holding and lipid absorption capacity of the IAP and Inulin (In) (commercial prebiotic) were determined as reported by Carvalho et al. (2009) [23]. 30 ml of distilled water was added to the sample (1 g) in a centrifuge tube. The sample was agitated and left in room temperature. Then, the mixture was centrifuged (12,000 × g, 20 min) (Sigma k-16, Germany), The supernatant was discarded and the residue was weighed. The WRC was expressed as g of water g−1 dry sample. For LAC, samples (3 g) were added to sunflower oil (18 ml), left overnight in room temperature (25 ◦ C), centrifuged at 1500 × g for 10 min and the supernatant was disposed. LAC was expressed as grams of oil g−1 dry sample [23].

2.7. Antioxidant property The ground IAP (0.2 g) was mixed with 5 ml of methanol and was stirred vigorously using shaker incubator (200 rpm, 3 h) (IKA® KS 4000 I control, Germany), and then it was centrifuged at 3000 rpm for 20 min (Sigma k-16, Germany). The obtained supernatant was analyzed for its DPPH radical (2, 2-Diphenyl-1-picrylhydrazyl) scavenging. In the second series, IAP (0.025–0.2 g) was mixed with 5 ml of methanol and was stirred vigorously for 3 h, and then it was centrifuged at 3000 rpm for 20 min. The obtained supernatant was analyzed for its DPPH radical scavenging.

2.7.1. DPPH radical scavenging activity An aliquot of methanol extracts was measured in terms of hydrogen-donating or radical scavenging ability using the stable radical DPPH (2, 2-Diphenyl-1-picrylhydrazyl). Then, 500 ␮l of the extract was quickly added to 5 ml of a 0.1 mM methanol solution of DPPH. After incubating this solution in room temperature for 30 min, the absorbance was read using spectrophotometer (UV-VIS 2100, USA) against a blank at 517 nm. Ascorbic acid (As) (1000 ␮M) and In (40 mg/ml) were used as a positive control and reference prebiotic under the same assay conditions. The assay was carried out in triplicate [4,33]. The DPPH radical scavenging activity was calculated according to the following: Scavenging activity% =

(Absblank − Abssample ) Absblank

× 100

where Absblank is the absorbance of the control and Abssample is the absorbance of the sample.

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181

Fig. 1. FTIR spectra for IAP.

2.8. Statistical analysis

3.2. Evaluation of resistance to acidic and enzymatic digestion

All determinations were carried out three times. Mean values and standard deviations calculated and the results were analyzed using the SAS package (version 8). The least significant difference (LSD) test was used to describe means at the 1% significance level.

To evaluate prebiotic potential of IAP, its resistance to simulated acidic and enzymatic digestion was studied. This study evaluated IAP resistance to simulated digestion condition in 3 steps, and the calculated degree of hydrolysis of IAP was shown in Fig. 2. In the first step, simulated gastric fluid (SGF) in stomach at pH 1.2 ± 0.5, percentage of calculated hydrolysis was 1.27%. IAP was more resistant to SGF than commercial prebiotic (In), which underwent calculated hydrolysis of 15.58 ± 0.78% (p < 0.01). In the second step, the mixture of simulated gastric and intestinal fluid (SMF) of pH 4.5, the degree of hydrolysis was 0.14 ± 0.03% whereas the maximum hydrolysis of a positive control (In) was 1.7 ± 0.3% (p < 0.01). In the third step, (SIF) resistance to intestinal condition in addition to ␣amylase was studied. Results showed that the resistance of IAP to ␣-amylase hydrolysis is similar to that of inulin. Thus, IAP could be a useful candidate as a prebiotic substrate because approximately morethan 97% of ingested polysaccharide could reach colon to be metabolized by probiotic bacteria. This result is comparable

3.1. Isolation and identification of acorn polysaccharide structure by FTIR The crude polysaccharide was extracted from the acorn fruit contained 90% carbohydrate based on phenol-sulfuric acid method and the extraction yield was about 5% on dry basis. FTIR spectroscopy was used to study chemical structure and to identify its functional group, type of monosaccharide or glycosidic bonds involved in isolated acorn polysaccharide (IAP) structure. In this method, deformation vibrations are used as a base to recognize monosaccharide types, glycosidic bonds and functional groups [5]. The FTIR spectrum is shown in Fig. 1 for IAP. The band at 2923 cm−1 was due to the C–H stretching. The observed band at 3420 cm−1 was attributed to the hydroxyl group. The absorptions at 1081, 1023 and 1155 cm−1 are caused mainly by C–C and C–O deformation vibrations in pyranose form of sugars and C–O–C glycosidic band vibration [3,4,34]. Absorptions at 1418, 1370, 1260 and 1236 are attributed to CH stretching deformation and COH bending vibration [12,34]. Absorptions at 857 cm−1 , 931 cm−1 were typical for ␣ and ␤ configurations in pyranose form [3,12,34]. The band observed at 764 cm−1 could be attributed to ␣-D-xylose [5]. As typical indicator bands of polysaccharides in 1000–1200 cm−1 and 2923 cm−1 regions were observed in this spectrum, this spectrum could confirm that polysaccharide is a dominant component in the studied extract [3,4,12,34]. These results are consistent with other studied polysaccharides reported by other researchers [12,34,35].

a

18 16 14

Hydrlysis (%)

3. Results and discussion

12 10

IAP

8 6 4

In

b

a

b

a

a

2 0 1

2

3

Digeson Fig. 2. Degree of hydrolysis of IAP and In (Inulin) to simulated digestion condition. Step 1: SGF, Step 2: SMF. Step 3: SIF and ␣-amylase. Average of triplicate analyses. Different letters indicate significant differences in the same digestion step (p < 0.01).

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6.2

7

a

6.1

6

6

b

5.9

d

5.7

pH

pH

c

b

5.6

e

5.5 5.4 5.3

c

b

d

d

4

d

In

3

Glu

2

IAP

1

5.2

0

5.1 0

0.5

1

2

3

0

24

Fig. 3. Effects of IAP in different concentration on the growth of Lp A7. The overnight culture of this strain was diluted to a cell density of 107 cells/ml and 2% of cells were inoculated into carbohydrate-free MRS medium supplemented with 0, 0.5%, 1%, 2%, or 3% (wt/vol) IAP. This strain was cultured at 37 ◦ C for 24 h. The growth was monitored by pH value determination. Average of triplicate analyses. Different letters mean a significant difference (p < 0.01).

to other studied oligo or polysaccharides such as BSCP (Bamboo shoot crude polysaccharide), which were 99% resistant to artificial human gastric juice [12]. Gluco-oligosaccharide produced by Gluconobacter oxidants NCIMB 4943 was (98.4%) resistant. Galactooligosaccharide (commercial prebiotic) was reported to be more than 90% indigestible [12]. Pitaya oligosaccharides isolated from dragon fruit showed high acid resistance to artificial human gastric juice (96%) [28]. 3.3. Effect of acorn polysaccharide on probiotic growth To evaluate the potential of IAP to be metabolized by colon microbiota, the effect of IAP on the growth of Lactobacillus plantarum A7 (Lp A7) was studied in vitro. It was cultured in carbohydrate free MRS-medium supplemented with 2% (w/v) of In, 2% (w/v) IAP and 2% (w/v) Glu at 37 ◦ C for 72 h. During the first 72h fermentation, the pH measurement and bacterial enumeration were carried out at 24-h intervals. In addition, Lp A7 cultured in carbohydrate-free MRS medium supplemented with various concentrations (0, 0.5, 1, 2, 3) % (w/v) of IAP and pH values were reported. As shown in Fig. 3, pH decrease correlates with the concentration of IAP, which could indicate that Lp A7 was able to metabolize IAP. As shown in Fig. 4, IAP had a stimulatory effect on cell growth after 24 h (from 7 logcfu/ml to 8.29 log cfu/ml). Viability of Lp A7 in IAP and In supplemented media was stable even after 72 h, whereas in Glu supplemented medium, bacterial growth showed a notable decrease after 24 h. As defined by Huebner et al. [36], the prebiotic effect of a substrate refers to 9 In

8

IAP

7

Glu

48

72

Time(hour)

Concentraon of IAP (%)

Log cfu/mL

c

b

5

c

5.8

a a a

Fig. 5. Effect of IAP compared to In (inulin) and Glu (glucose) on the reduction of pH in the medium by Lp A7 at different cultivation times. Average of triplicate analyses. Different letters mean a significant difference (p < 0.01).

its ability to support the growth of probiotic organism relative to growth on a non-prebiotic substrate such as glucose. As shown in Fig. 5, pH decrease in Glu supplemented medium was more than IAP or In supplemented media which could be attributed to the use of diverse metabolic pathways for metabolizing substrates in each medium (Fig. 5). It seems that due to the complexity of the chemical structure of IAP and lack of simple carbon sources, bacteria could maintain their viability longer and their growth rate has not been negatively affected. On the contrary, in Glusupplemented medium, microorganisms produce more acid due to catabolic repression since more simple carbon sources are available [37]. More viability and higher population of probiotic bacteria are very important in playing their positive role in the colon. As reported by Firdus et al., a similar result was observed on crude polysaccharide extracted from Gigantochloa Levis, which resulted in a significant increase in the growth of B. animalis ATCC 1053, B. longum BB 536 and L. acidophilus ATCC 4356 as compared to the use of FOS (fructooligosaccharide) and viability of probiotics in BSCP (Bamboo shoot crude polysaccharide)-supplemented media showing a positive increase, even after 48 h [12]. As mentioned by Ramnani et al. [38], low molecular weight polysaccharides derived from agar and alginate seaweeds caused a significant increase in bifidobacterial populations after 24 h. As reported by Wichienchot et al. [28], Oligosaccharides extracted from white-flesh dragon fruit stimulated the growth of L. delbrueckii BCC13296 by increasing its number within 48 h. It is complicated to evaluate the prebiotic potential of new component and to compare it with other studied prebiotics. Due to different chemical structures of prebiotics, varied concentrations or dose–response relationships, choices of probiotic microorganisms, metabolic capability of selected probiotic or pathogenic bacteria, and types of growth media (ingredients, buffering capacity) in distinctive periods (24,48, and 72 h) affect prebiotic activity of the new compound. Therefore, in this study, prebiotic capability of IAP was compared to In in the same conditions. The results of this section and section 3.2 could be regarded as evidence to prebiotic potential of IAP compared to In (commercial prebiotic). 3.4. Functional properties

6

5 0

24

Time(hr)

48

72

Fig. 4. Microbial counts of using IAP, In (inulin) and Glu (glucose) as carbon sources. The overnight culture of Lp A7 was diluted to a cell density of 107 cells/ml, and 2% of cells was inoculated into carbohydrate-free MRS medium supplemented with 2% (w/v) IAP, In and Glu. This strain was cultured at 37 ◦ C for 72 h. The growth was monitored by measuring bacteria counts at the 24, 48, 72 h.

To evaluate the potential of IAP for technological applications and its physiological effects, the functional properties were studied. Water holding capacity (WHC) and lipid adsorption capacity (LAC) were evaluated in this study. The results of WHC and LAC of IAP and In were shown in Table 1. IAP was found to have higher WHC than In. WHC of IAP is comparable to other studied compounds including defatted rice bran, sugarcane bagasse, coconut fiber and carbohydrate of chitosan ranging from 4.42–4.98 [23,24]

M. Tadayoni et al. / International Journal of Biological Macromolecules 72 (2015) 179–184 Table 1 Hydration properties and lipid adsorption capacities of IAP. Polysaccharide

WHC (g water/g DM)

LAC (g oil/g DM)

Inulin IAP

0.68 ± 0.03b 4.33 ± 0.03a

1.62 ± 0.05b 5.44 ± 0.02a

Average of triplicate analyses. Different letters in the same column mean a significant difference (p < 0.01).

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researchers. Ingredients with high lipid absorption capacity could affect dietary lipid absorption in the gastrointestinal tract and decrease lipid absorption from ingested food. IAP, therefore, could help control blood lipid and body weight [23]. This property is also very important in helping to stabilize the texture of high fat food and emulsions [24]. Therefore, IAP with high WHC and LAC can be used as a functional ingredient to provide healthier food and to modify the texture of formulated food.

120

3.5. Antioxidant property of acorn polysaccharide

Scavenging ability (%)

100

80

60

40

20

0

In

IAP

As

Fig. 6. DPPH radical scavenging activity of IAP (40 mg/ml), In (Inulin, 40 mg/ml) and As (Ascorbic acid, 1000 ␮M).

reported by different researchers. WHC of IAP is more than dietary fiber in some plants or fruit sources such as wheat bran, apple pomace, grape fruit, lemon and orange peel reported by other researchers. It ranges from 1.62 to 2.7 [21,24]. Water holding capacity of IAP refers to its porous matrix structure and its ability to hold water in its matrix. Ingredients with high hydration property could increase viscosity, fecal volume, the frequency of depositions and they could reduce the rate of nutrient absorption. Thus, increased intake of such polysaccharides could reduce the risk of coronary heart disease, diabetes and obesity. Hydration property of acorn polysaccharide suggests some technological applications about the use of polysaccharide as functional compound to increase viscosity, prevent synersis, stabilize and modify the texture of the formulated food [21,23,24]. IAP was found to contain higher LAC than In. LAC of IAP is comparable to other studied compounds such as defatted rice bran, sugarcane bagasse, asparagus by-products and carrot dietary fiber ranging from 4.54 to 5.5 [23] reported by different researchers. LAC of IAP is more than dietary fiber in some plants or fruit sources such as wheat bran, apple pomace, grapefruit, lemon and orange peel, mango dietary fiber, peach dietary fiber and carbohydrate of chitosan ranging from 0.6 to 2.41 [21,23,24] reported by other

To evaluate the antioxidant activity of IAP, the scavenging ability of DPPH radicals was studied. The mechanism of DPPH radical scavenging assay is based on the reduction of DPPH− solution by a hydrogen-donating compound causing the formation of nonradical form DPPH-H [39]. IAP scavenged DPPH radicals by 82.24%. IAP was found to have a high scavenging ability compared to the reference prebiotic (In) giving the scavenging ability of about 20% (Fig. 6). The high DPPH radical scavenging activity of IAP indicated its effective hydrogen-donating capacity. The scavenging property of IAP correlated with its concentration and increased by increasing its concentration (Fig. 7). Numerous studies showed that some isolated compounds from herbal sources and food plants were capable antioxidants correlating with their phenolic and polysaccharide compounds [4]. In this study scavenging property of IAP is likely due to the fact that the crude polysaccharide may contain other components in addition to polysaccharides such as polyphenols or tannin [4]. It should be mentioned that the polysaccharide extraction from acorn goes through some steps in which ethanol-based extract is removed (including heating in ethanol, separation, precipitation with ethanol and finally washing with ethanol). Thus, it is expected that major part of the phenolic compound and tannin can be removed from the extract. Nevertheless, crude polysaccharide may contain tannin residue that might affect antioxidant capability of the extract [40]. Different studied polysaccharides showed different scavenging properties suggested to be related to structural characteristics of polysaccharide including monosaccharide composition, glycosidic bonds, molecular weight, configuration types or possessed carbonyl, carboxyl, amino or sulfate groups [4,6,35,39,41]. As mentioned by Yang et al. [39], soy sauce lees oligosaccharides possessed a very low DPPH radical scavenging activity. In the range of 50–500 ␮g/ml, less than 3% of DPPH radical scavenging activity was detected [39]. The carrageenan oligosaccharides at 400 ␮g/ml also exhibit a DPPH radical scavenging activity of approximately 10%. The sulphate groups in carrageenan and agaro-oligosaccharides are responsible for the radical scavenging activity [39]. The antiradical property of IAP also could act as a modulating agent for oxidative stress in the growth medium of bacteria. Therefore, it could support more viability of probiotic bacteria as shown in section 3.3 [42]. It seems that the prebiotic effect of IAP was correlated with antioxidant activity. In addition, further research on the antioxidant mechanism of IAP is worth being conducted. 4. Conclusion

Fig. 7. The scavenging effect of different concentration of IAP on DPPH radical. Average of triplicate analyses.

Typical indicator bands of polysaccharides were observed in IAP spectrum confirming that polysaccharide is a dominant component in the studied extract. IAP was more resistant to simulated digestion condition than commercial prebiotic (In) as approximately more than 97% of ingested polysaccharide could reach the colon so that it could be metabolized by probiotic bacteria. IAP had a stimulatory effect on cell growth and viability of Lp A7 in IAP, and In-supplemented media were stable even after 72 h, whereas in Glu-supplemented medium, bacterial growth showed a notable

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decrease after 24 h. LAC and WHC of IAP were 5.44 ± 0.02 (g oil/g DM), 4.33 ± 0.03 (g water/g DM), respectively, which were comparable to dietary fiber and were more than In. IAP scavenged DPPH radicals by 82.24%. IAP was found to possess high scavenging ability compared to the reference prebiotic (In) yielding only 20% scavenging ability. The scavenging property of IAP correlated with its concentration and increased with increasing concentration. Therefore, IAP, with good prebiotic capability, high WHC and LAC, and suitable antioxidant activity, can be used as a functional ingredient to produce healthier food and to modify the texture of formulated food. Acknowledgments Financial support and facilities for this work were provided by Isfahan University of Technology. I am very grateful for their services all through this study. References [1] M. Jin, K. Zhao, Q. Huang, C. Xu, P. Shang, Carbohyd. Polym. 89 (2012) 713–722. [2] Y. Zhang, S. Li, X. Wang, L. Zhang, P.C.K. Cheung, Food Hydrocolloid. 25 (2011) 196–206. [3] Z. Yu, G. Ming, W. Kaiping, C. Zhixiang, D. Liquan, L. Jingyu, Z. Fang, Fitoterapia 81 (2010) 1163–1170. [4] H. Fan, G. Mazza, X. Liao, J. Food Sci. Technol. 2 (2010) 9–17. [5] L. Yang, L. Zhang, Carbohyd. Polym. 76 (2009) 349–361. [6] G. Jiao, G. Yu, J. Zhang, H. Stephen Ewart, Drugs 9 (2011) 196–223. [7] I. Dahech, K.S. Belghith, K. Hamden, A. Feki, H. Belghith, H. Mejdoub, Int. J. Biol. Macromol. 49 (2011) 742–746. [8] W. Cao, Q. Li, X. Wang, T. Li, X. Chen, S.B. Liu, et al., Int. J. Biol. Macromol. 46 (2010) 115–120. [9] Y. Sun, J. Tang, X. Gu, D. Li, Int. J. Biol. Macromol. 36 (2005) 283–289. [10] D. Lopez-Molina, M.D. Navarro-Martinez, F. Rojas Melgarejo, A.N.P. Hiner, S. Chazarra, J.N. Rodriguez-Lopez, Phytochemistry 66 (2005) 1476–1484. [11] S. Patel, A. Goyal, Biotech. 2 (2012) 115–125. [12] A. Firdaus, M. Nurul Azmi, S. Mustafa, D. Hashim, Y. Abdul Manap, Molecules 17 (2012) 1635–1651. [13] D. Li, J.M. Kim, Z. Jin, J. Zhou, Anaerobe 14 (2008) 29–34.

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