High pressure homogenization increases antioxidant capacity and short-chain fatty acid yield of polysaccharide from seeds of Plantago asiatica L.

Food Chemistry 138 (2013) 2338–2345

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High pressure homogenization increases antioxidant capacity and short-chain fatty acid yield of polysaccharide from seeds of Plantago asiatica L. Jie-Lun Hu, Shao-Ping Nie ⇑, Ming-Yong Xie ⇑ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

a r t i c l e

i n f o

Article history: Received 10 August 2012 Received in revised form 19 November 2012 Accepted 5 December 2012 Available online 26 December 2012 Keywords: Plantago asiatica L. Polysaccharide High pressure homogenization Antioxidant capacity Short-chain fatty acid

a b s t r a c t Physiological properties of homogenized and non-homogenized polysaccharide from the seeds of Plantago asiatica L., including antioxidant capacity and short-chain fatty acid (SCFA) production, were compared in this study. High pressure homogenization decreased particle size of the polysaccharide, and changed the surface topography from large flake-like structure to smaller porous chips. FT-IR showed that high pressure homogenization did not alter the primary structure of the polysaccharide. However, high pressure homogenization increased antioxidant capacity of the polysaccharide, evaluated by 4 antioxidant capacity assays (hydroxyl radical-scavenging, superoxide radical-scavenging, 1,1-diphenyl-2picryl-hydrazyl radical (DPPH)-scavenging and lipid peroxidation inhibition). Additionally, the production of total SCFA, propionic acid and n-butyric acid in ceca and colons of mice significantly increased after dieting supplementation with homogenized polysaccharide. These results showed that high pressure homogenization treatment could be a promising approach for the production of value-added polysaccharides in the food industry. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Polysaccharides have been widely used in food processing and preparation as stabilizers, thickeners, texturizers or emulsifiers (Huck-Iriart, Pizones Ruiz-Henestrosa, & Candal, 2012). An emulsion is a mixture of two immiscible liquids in which one phase is dispersed through the other as small, discrete droplets. Polysaccharides as emulsifiers can assist in obtaining a fine dispersion and in maintaining the homogeneous mixture through homogenization (Lapasin & Pricl, 1995). Many studies have shown that viscosity, rheological and gelling properties of polysaccharides could be influenced by the high pressure homogenization process (Pouliot, Britten, & Latreille, 1990; Silvestri & Gabrielson, 1991). In addition, it was also reported that high pressure homogenization could reduce the particle size of polysaccharides and induce some degradation, but it did not destroy the primary structure of polysaccharides (Chen et al., 2012; Floury, Desrumaux, Axelos, & Legrand, 2002). Concerning the effects of high pressure homogenization on physiological properties of polysaccharides, Lyons and Deidra Shannon (2011) reported that the antimicrobial properties of chitosan could be improved after being treated by high pressure homogenization. However, no study has been conducted to examine the effects of high pressure homogenization on several other properties of ⇑ Corresponding authors. Tel./fax: +86 791 83969009 (M.-Y. Xie), +86 791 88304452 (S.-P. Nie). E-mail addresses: [email protected] (S.-P. Nie), [email protected] (M.-Y. Xie). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.12.016

polysaccharides, such as their antioxidant capacity or their influence on the production of total SCFA, propionic acid and n-butyric acid in ceca and colon once ingested. Antioxidant capacity of polysaccharides is considered as one of the important physiological properties, for it could help prolong the shelf life of food products (Gan & Latiff, 2011). Short-chain fatty acid production of polysaccharides is also increasingly attracting attention, because it is beneficial for human intestinal health (Huang, Chu, Dai, Yu, & Chau, 2012). Researchers showed that antioxidant activity of the polysaccharides may be influenced by the reduction of the particle size (Pérez-Jiménez et al., 2008), and the particle size of the polysaccharides (determining the surface available to bacteria) may also influence fermentation and thus affect the SCFA production (Guillon, Auffret, Robertson, Thibault, & Barry, 1998). Thus, our hypothesis is that the antioxidant activity and SCFA production of the polysaccharides may be influenced by high pressure homogenization treatment, resulting in the particle size reduction. Polysaccharides could be extracted from many kinds of herbal or plant materials. Some Plantago plants, such as Plantago afra L., Plantago psyllium L., Plantago ovata Forsk (isabgul), Plantago indica L. and Plantago major L., are often used in traditional medicine throughout the world because the soluble fibres in their seeds are able to improve some intestinal functions (Mahady, Fong, & Farnsworth, 1999; Samuelsen, 2000). Our research group has recently isolated a pure and homogeneous polysaccharide from the seeds of P. asiatica L. with a molecular weight of 1894 kDa (Yin, Nie, Zhou, Wan, & Xie, 2010). The polysaccharide was found to

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be a highly branched heteroxylan which consisted of a b-1, 4linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of b-T-linked Xylp, a-T-linked Araf, aT-linked GlcAp, b-Xylp-(1 ? 3)-a-Araf and a-Araf-(1 ? 3)-b-Xylp (Yin, Lin, Li, Wang, Cui, Nie, & Xie, 2012). In addition, our recent studies have also shown that this polysaccharide may induce maturation of murine dendrite cells, and may have antioxidant activity and promote defaecation (Huang, Xie, Yin, Nie, & Xie, 2009; Tang, Huang, Yin, Zhou, Xie, & Xie, 2007; Wu, Tian, Xie, & Li, 2007). In the study, the effect of high pressure homogenization on the polysaccharide from the seeds of P. asiatica L. was evaluated by using dynamic light scattering, environmental scanning electron microscopy and FT-IR spectroscopy. In addition, the effects of high pressure homogenization on some properties of the polysaccharide, such as antioxidant activity and SCFA production, were for the first time examined. 2. Materials and methods 2.1. Materials and reagents The seeds of P. asiatica L. were purchased from Ji’an, Jiangxi Province, China and dried in the sun before use. Highly pure SCFAs were used to prepare the standard solutions for gas chromatography (GC) determination. Acetic acid (P99.5% purity) and n-valeric acid (P99.5% purity) were obtained from Merck (Darmstadt, Germany). Propionic acid (P99.5% purity) was purchased from Janssen Chimica (Belgium), while i-butyric acid (P99.5% purity), n-butyric acid (P99% purity), i-valeric acid (P99% purity) and 4-methylvaleric acid (internal standard) (P99% purity) were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of analytical grade and purchased from Shanghai Chemicals and Reagents Co. (Shanghai, China). 2.2. Animals Kunming mice, weighing 20.0 ± 2.0 g [Grade II, Certificate Number SCXK (gan) 2006-0001], were purchased from Jiangxi College of Traditional Chinese Medicine, Jiangxi Province, China. In this study, 36 mice were used in total. Mice were randomly divided into three treatment groups and in each treatment group there were 12 mice. All animals used in this study were cared for in accordance with the Guidelines for the Care and Use of Laboratory Animals, published by the United States National Institute of Health (NIH, Publication No. 85-23, 1996), and all procedures were approved by the Animal Care Review Committee (Animal application approval number 0064257), Nanchang University, China. 2.3. Polysaccharide preparation Polysaccharide from P. asiatica L. seeds was prepared using our published method (Yin et al., 2010). Briefly, the seeds of P. asiatica L. (50 g) were defatted with 1 l of ethanol at room temperature for 24 h under stirring, and then extracted with 500 ml of doubly distilled water at 100 °C for 2 h. The residue was re-extracted. The combined aqueous extract, a gel of high viscosity, was centrifuged (4800g, 10 min), prefiltered through a cotton cloth bag and concentrated in a rotary evaporator under reduced pressure at 55 °C, to yield P. asiatica L. water extract. The filtrate was mixed with 1.5 g/l of papain and heated in water at 60 °C for 2 h. The resulting aqueous solution was extensively dialyzed against doubly distilled water for 72 h and precipitated by adding 4 volumes of anhydrous ethanol at 4 °C for 12 h. After centrifugation the precipitate was washed with anhydrous ethanol, dissolved in water and lyophilized to yield the polysaccharide (soluble fibre content >90.0%).

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2.4. High pressure homogenization treatment The polysaccharide solution (2.5 mg/ml) was dispersed in deionized water and stirred gently at room temperature to achieve complete solubilization. High pressure homogenization was performed using a M-100EH-30 microfluidizer (Microfluidics Co., Newton, USA) at 160 MPa for 5 passes. The experiment was conducted three times with different solutions of polysaccharide in order to verify the repeatability of the homogenization process. Parts of the solutions, after high pressure homogenization, were lyophilized for the Fourier transform infrared (FT-IR) and scanning microscopy analyses. 2.5. Average particle size (PS) and distribution determination The average particle size and distribution of the polysaccharide in solutions were determined by Nicomp 380/ZLS Zeta potential/ Particle sizer (PSS Nicomp, Santa Barbara, USA), based on dynamic light scattering (DLS). The solutions were diluted to a concentration of 0.5 mg/ml with deionized water, and all measurements were carried out at 25 °C (Chen et al., 2012). 2.6. Scanning microscopy analysis Polysaccharide samples were taken after freeze–drying, and samples were prepared by sticking the polysaccharide onto double-sided adhesive tape attached to a circular specimen stub. The samples were viewed using an environmental scanning electron microscope (ESEM) (Quanta 200F, FEI Deutschland GmbH, Kassel, Germany) at 30 kV voltage and 3.0 spot size. Low vacuum mode was used for the ESEM. 2.7. FT-IR spectroscopy The FT-IR spectra of polysaccharide samples were recorded on a Nicolet 5700 spectrometer (Thermo Co., Madison, USA). The dried samples were ground with KBr powder (spectroscopic grade) and pressed into pellets for spectra measurement in the frequency range of 4000–400 cm1. The data was collected and plotted as transmittance (%) in function of the wave number (cm1) and analyzed with Ominic 7.2 software. 2.8. Antioxidant activity assays 2.8.1. Hydroxyl radical-scavenging activity The hydroxyl radical-scavenging ability was measured, using a modified method of Halliwell, Gutteridge, and Aruoma (1987). A reaction mixture was prepared by adding 0.1 ml of EDTA (1 mM), 0.01 ml of FeCl3 (10 mM), 0.1 ml of H2O2 (10 mM), 0.36 ml of deoxyribose (10 mM), 1.0 ml of polysaccharide samples (0.1–10 mg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid (1 mM) in sequence. The mixture was then incubated at 37 °C for 1 h. 1.0 ml of the incubated mixture was mixed with 1.0 ml of 10% trichloroacetic acid and 1.0 ml of 0.5% thiobarbituric acid (in 25 mM NaOH containing 0.025% butylated hydroxyl anisole) to develop the pink chromogen measured at 532 nm. The hydroxyl radical-scavenging activity of the polysaccharide samples was reported as the percentage of inhibition of deoxyribose degradation and was calculated according to the following equation:

% inhibition ¼ ðA0  AtÞ=A0  100

ð1Þ

where A0 was the absorbance of the control (blank, without polysaccharide samples) and At was the absorbance in the presence of the polysaccharide samples. All of the tests were carried out in

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triplicate and IC50 values were expressed as means ± standard deviation (SD). Ascorbic acid was used as a positive control. 2.8.2. Superoxide radical-scavenging activity This activity was measured using nitro blue tetrazolium (NBT) reagent as described by Sabu and Kuttan (2002). The method is based on generation of superoxide radical O 2 by auto-oxidation of hydroxylamine hydrochloride in the presence of NBT, which gets reduced to nitrite. Nitrite, in the presence of EDTA, gives a colour that was measured at 560 nm. Test solutions of polysaccharide (0.1–10 mg/ml) were taken in a test tube. To this, a reaction mixture consisting of 1 ml of (50 mM) sodium carbonate, 0.4 ml of (24 mM) NBT and 0.2 ml of 0.1 mM EDTA solution was added to the test tube and an immediate reading was taken at 560 nm. 0.4 ml of (1 mM) of hydroxylamine hydrochloride was added to initiate the reaction. The reaction mixture was then incubated at 25 °C for 15 min and the reduction of NBT was measured at 560 nm. Decreased absorbance of the reaction mixture indicates increased superoxide anion-scavenging activity. All the samples were treated in a similar manner, absorbance was recorded and the percentage of inhibition was calculated according to the following equation:

% inhibition ¼ ðA0  AtÞ=A0  100

ð2Þ

where A0 was the absorbance of the control (blank, without polysaccharide samples) and At was the absorbance in the presence of the polysaccharide samples. All the tests were performed in triplicate and IC50 values were obtained. Ascorbic acid was used as a positive control. 2.8.3. DPPH radical-scavenging activity DPPH radical-scavenging activity of polysaccharide samples was measured according to our published method (Chen, Xie, Nie, Li, & Wang, 2008). The 0.2 mM solution of DPPH in 95% ethanol was prepared daily before UV measurements were taken. One millilitre of the polysaccharide samples of different quantities (0.1–10 mg) in water was thoroughly mixed with 2 ml of freshly prepared DPPH and 2 ml of 95% ethanol. The mixture was shaken vigorously and left to stand for 30 min in the dark. The absorbance of the supernatant obtained after centrifugation was then measured at 517 nm. The DPPH radical-scavenging ability was calculated using the following equation:

I% ¼ ½1  ðAi  Aj Þ=Ac   100%

ð3Þ

where Ac is the absorbance of DPPH solution without polysaccharide sample (2 ml DPPH + 3 ml of 95% ethanol), Ai is the absorbance of the test polysaccharide sample mixed with DPPH solution (1 ml of polysaccharide sample + 2 ml of DPPH + 2 ml of 95% ethanol) and Aj is the absorbance of the polysaccharide sample without DPPH solution (1 ml of polysaccharide sample + 4 ml of 95% ethanol). All the tests were performed in triplicate and IC50 values were obtained. Ascorbic acid was used as a positive control. 2.8.4. Inhibition of lipid peroxidation This test was conducted using the method of Zhang, Yu, Zhou, and Xiao (1996) with some modifications. Briefly, an equal volume of egg yolk was added to 0.1 M phosphate-buffered saline (PBS, pH 7.45). The mixture was stirred magnetically for 10 min and then diluted with 24 volumes of PBS. The yolk homogenate (1 ml), polysaccharide samples (0.5 ml, 0.1–10 mg/ml), PBS (1 ml) and 25 mM FeSO4 (1 ml) were mixed in a tube and shaken at 37 °C for 15 min. The reaction was stopped by the addition of trichloroacetic acid and the mixture was centrifuged. Then 1 ml of 8 g/l thiobarbituric acid solution was added to 3 ml of the supernatant. This solution was heated at 10 °C for 10 min, after which its absorbance at 532 nm was measured. The ability to inhibit lipid peroxidation was calculated as follows:

inhibitory effectð%Þ ¼ ½ðB0  BÞ=B0   100

ð4Þ

where B0 is the absorbance of the control and B is the absorbance in the presence of polysaccharide samples. All the tests were performed in triplicate and IC50 values were obtained. Ascorbic acid was used as a positive control. 2.9. Changes in SCFA production of polysaccharide treated by high pressure homogenization 2.9.1. Animal experiment design Six-week-old male Kunming mice (20.0 ± 2.0 g) were individually housed in stainless steel cages in a room with controlled temperature (25 ± 0.5 °C), relative humidity (50 ± 5%) and 12 h/12 h light/dark cycle. All mice were fed with the same amount of basal diet (Supplementary Table 1) which was prepared according to the published formula (Adachi et al., 2004; Berggren, Björck, Nyman, & Eggum, 1993), and water was provided ad libitum. Mice were randomly divided into three groups: (1) non-treated polysaccharide group: mice were given oral administration of non-treated polysaccharide at the dose of 0.1 g/kg body weight (BW); (2) homogenization-treated polysaccharide group: mice were given oral administration of homogenization-treated polysaccharide at the dose of 0.1 g/kg BW; (3) control group: mice were given distilled water of the same volume as the mean volume of the polysaccharide groups. Non-treated polysaccharide and homogenization-treated polysaccharide were dissolved in distilled water before administration. Each group had 12 mice, and each mouse was housed in one cage. All the mice were given oral administration of polysaccharide or water (control) by gavage at about 9:00 am everyday for 4 weeks. In addition, we determined the body weight of each mouse everyday before gavage and changed the volume of the polysaccharide in order to make sure the mice were given polysaccharide at the dose of 0.1 g/kg BW by gavage everyday. Throughout the experiment, the animals’ general health status and body mass were observed twice daily. At the end of the designated experimental period, the mice were sacrificed, and the liver, kidney, spleen, heart, lung, intestine, cecum and colon were excised and immediately weighed. After that, the ceca and colons were aseptically removed, immediately, and placed on an ice-cold plate, longitudinally opened and the cecal and colonic contents were collected for further use. 2.9.2. Measurement of cecal and colonic SCFA concentration A portion (0.1 g) of the sample (cecal or colonic content) was rapidly put into a round-bottomed stoppered tube in an ice-cold water bath. One millilitre of deionized water was added to the tube and mixed intermittently on a vortex-mixer for 2 min. The tube was allowed to stand in the ice-cold water bath for 20 min and then centrifuged at 4800g for 20 min at 4 °C. The supernatant was transferred to another round-bottomed stoppered tube. This process was repeated once. The supernatant was analyzed by injection into the chromatographic system. Chromatographic analysis was carried out according to our published method (Hu, Nie, Min, & Xie, 2012), using an Agilent 6890N GC system equipped with a flame ionization detector (FID) and an N10149 automatic liquid sampler (Agilent, USA). A GC column (HP-INNOWAX, 190901N-213, J & W Scientific, Agilent Technologies Inc., USA) of 30 m  0.32 mm ID coated with 0.50 lm film thickness was used. Nitrogen was supplied as the carrier gas at a flow rate of 19.0 ml/min with a split ratio of 1:10. The initial oven temperature was 100 °C and it was kept there for 0.5 min, and then raised to 180 °C by 4 °C/min. The temperatures of the FID and injection port were 240 °C. The flow rates of hydrogen and synthetic air were 30 and 300 ml/min, respectively. The injected sam-

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ple volume for GC analysis was 0.2 ll, and the running time for each analysis was 20.5 min. The independently replicated determinations were performed three times for standard solutions and each sample. Data handling was carried out with a HP ChemStation Plus software (A.09.xx, Agilent). Mean values and standard deviations were calculated from triplicate determinations. Results were expressed as means ± standard deviation. 2.10. Statistical analysis All the experiments were done in triplicate. Results were expressed as means ± standard deviation (SD). Data were evaluated by 1-way analysis of variance, using SPSS 10.0 software (Version 16.0, Chicago, United States). The difference between different groups was evaluated by SNK test. The level of significance was set at p < 0.05. 3. Results and discussion

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nization, the distribution profile of particle size shifted left and narrowed (a typical Gaussian distribution), indicating that the particle size of the polysaccharide decreased. The size of the polysaccharide decreased from 4758.7 ± 153.8 nm to 226.3 ± 4.7 nm after high pressure homogenization. In addition, as shown by ESEM, the non-treated polysaccharide was relatively tidy, with flake-like lamella (Fig. 2A). However, after high pressure homogenization, many pores appeared in the polysaccharide sample (Fig. 2B). The original flake-like structure was totally changed into smaller fragments after homogenization. Interestingly, it was found that Fig. 2A and B were similar to the microstructure of pectin, with and without high pressure homogenization, respectively (Chen et al., 2012). It was reported that the complexes could be broken into microfragments by homogenization and thus produce spherical particles with a very low diameter (Chen et al., 1989). Homogenization could result in a change in the degree of aggregation of polymer, as well as irreversible disruption of the polymer (Lagoueyte & Paquin, 1998). These results were similar to ours.

3.1. Particle size and surface topography 3.2. FT-IR spectroscopy The particle size and distribution of the polysaccharide from P. asiatica L. seeds, before and after being treated by high pressure homogenization, are shown in Fig. 1. After high pressure homoge-

The chemical structures of the non-treated and homogenization-treated polysaccharide samples were analyzed by FT-IR

Fig. 1. Particle size of polysaccharide from P. asiatica L. seeds. A: non-treated; B: homogenization-treated.

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Fig. 2. Environmental scanning electron micrograph of polysaccharide from P. asiatica L. seeds. A: non-treated; B: homogenization-treated.

spectroscopy (Fig. 3). The strong absorption at 1048 cm1 was due to stretching vibration of the pyranose ring. In the anomeric region (950–700 cm1), the spectrum exhibited characteristic absorption at 810 cm1 due to the presence of mannose. There are two types of end carbon–glucoside bonds (a and b) that can be distinguished by IR. In IR spectra, the a-type C–H bond has an absorption peak near 844 cm1, while that of the b-type C–H bond is near 891 cm1 (Chen et al., 2008). A characteristic absorption at 899 cm1, indicating the b configuration of the sugar units, was also observed, but there was no absorption near 844 cm1 corresponding to the a configuration. The absorption band at 1628 cm1 indicated the presence of uronic acid. The band at 3423 cm1 was due to hydroxyl stretching vibration of the polysaccharide. The bands at 2928 cm1 and 1403 cm1 were due to C–H stretching vibration. For non-treated and homogenizationtreated polysaccharide samples, the spectra were rather similar and there was no significant difference for characteristic absorption bands, as described above between them. These results showed that high pressure homogenization treatment had not altered the primary structure of the polysaccharide. 3.3. Antioxidant activity Extended shelf-life is a key factor for making any food commodity more profitable and commercially available for long periods of

time at the best possible quality. The producer will benefit from the longer shelf-life to develop the market over greater distances. Natural antioxidant compounds could help prolong the shelf-life of food, and might also be beneficial for human health to some extent. In this study, antioxidant activity of the polysaccharide samples was evaluated using four different assays. The IC50 values of different polysaccharide samples are summarized in Table 1. Hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in the living cells (Hochstein & Atallah, 1988). As shown in Table 1, homogenization-treated polysaccharide had much more scavenging power for the hydroxyl radical than had non-treated polysaccharide (p < 0.05). Superoxide anion plays an important role in the formation of reactive oxygen species, such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, and induces oxidative damage in lipids, protein, and DNA (Pietta, 2000). In this study, the IC50 value for superoxide radical-scavenging ability of non-treated polysaccharide (2421 ± 2 lg/ml, Table 1) was higher than that for homogenization-treated polysaccharide (2271 ± 3 lg/ml) (p < 0.05). The model of scavenging the stable DPPH radical is a widely used method for evaluating the free radical-scavenging ability of natural compounds. The effect of antioxidants on DPPH radicalscavenging was thought to be due to their hydrogen-donating ability (Chen et al., 2008). The DPPH-scavenging activity of the nontreated polysaccharide, expressed in terms of IC50, was 503 ± 8 lg/ml (Table 1), with a lower DPPH-scavenging power than that of homogenization-treated polysaccharide (384 ± 5 lg/ ml) or ascorbic acid (205 ± 3 lg/ml) (p < 0.05). Lipid peroxidation (oxidative degradation of polyunsaturated fatty acid in the cell membranes) generates a number of degradation products, such as malondialdehyde (MDA), which is found to cause cell membrane destruction and cell damage, leading to liver injury, atherosclerosis, kidney damage, ageing, and susceptibility to cancer (Rice-Evans & Burdon, 1993). The IC50 value (lg/ml), related to the lipid peroxidation inhibitory capacity assay, showed that the lipid peroxidation inhibitory capacity of non-treated polysaccharide (1703 ± 8 lg/ml) was only about 60% of the homogenization-treated polysaccharide (1076 ± 8 lg/ml, Table 1). In these four antioxidant activity assays, except for the DPPHscavenging assay, the non-treated polysaccharide and homogenization-treated polysaccharide both have good solubility and no aggregates formed. Possibly some small aggregates were formed after addition of 95% ethanol in the DPPH-scavenging assay. However, these small aggregates could be removed by centrifugation before absorbance determination; thus they did not influence the accuracy of the absorbance. The decrease of absorbance was obvious, in the presence of polysaccharides, compared to the control. In addition, the procedures for DPPH-scavenging assay have been successfully used in our previous studies (Chen et al., 2008; Yin et al., 2010). The antioxidant capacity results indicated that the antioxidant capacity of the polysaccharide from P. asiatica L. was improved after the high pressure homogenization treatment. It is reported that one of the antioxidant mechanisms of polysaccharides may be due to the hydrogen supplied from some substituent groups in the polysaccharides (such as ferulic acids), which combine with radicals and form a stable radical to terminate the radical chain reaction (Chen et al., 2008). The other possibility is that the polysaccharide itself combines with metal ions or free radicals that are necessary for the radical chain reaction, so the reaction is terminated (Yin et al., 2010). In this study, the antioxidant capacity of original polysaccharide from P. asiatica L. may be attributed to both of them. On the one hand, the polysaccharide may have strong ability to chelate metal ions or free radicals, as inferred from

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Fig. 3. Effect of high pressure homogenization on FT-IR spectra of polysaccharide from P. asiatica L. seeds.

Table 1 Half-inhibition (IC50) values of antioxidant activities of non-treated polysaccharide and homogenization-treated polysaccharide measured using hydroxyl radical-scavenging, superoxide radical-scavenging, DPPH radical-scavenging and inhibition of lipid peroxidation assays. IC50 (lg/ml)a

Sample

Non-treated polysaccharide Homogenization-treated polysaccharide Ascorbic acid c

Hydroxyl radical-scavenging

Superoxide radical-scavenging

DPPH radical-scavenging

Lipid peroxidation inhibition

3084 ± 2ab 2394 ± 3b 220 ± 3c

2421 ± 2a 2271 ± 3b 304 ± 5c

503 ± 8a 384 ± 5b 205 ± 3c

1703 ± 8a 1076 ± 8b 440 ± 3c

a The IC50 value is expressed as lg/ml and represents the concentration of sample that is required for 50% inhibition of hydroxyl radical, superoxide radical, DPPH radical, and lipid peroxidation. A lower IC50 value indicates a higher antioxidant activity. Each value in the table was obtained by calculating the average of three determinations ± standard deviation. b Mean values in the same column with different letters are significantly different (Tukey test, p < 0.05). c Ascorbic acid was used as a positive control.

Table 2 Effect of non-treated and homogenization-treated polysaccharide from seeds of P. asiatica L. on short-chain fatty acid production in mouse cecum. Control group SCFA (lg/g of cecal content) Acetic acid 2525 ± 46ac Propionic acid 1022 ± 24a Butyric acid 1386 ± 21a i-Butyric acid 117 ± 11a Valeric acid 73 ± 6a i-Valeric acid 125 ± 15a Total SCFA 5248 ± 132a

NTP groupa

HTP groupb

3026 ± 51b 1108 ± 28b 2101 ± 33b 105 ± 19a 74 ± 7a 120 ± 11a 6534 ± 144b

3043 ± 41b 1499 ± 17c 2604 ± 25c 112 ± 12a 72 ± 9a 123 ± 12a 7453 ± 152c

a

Table 3 Effect of non-treated and homogenization-treated polysaccharide from seeds of P. asiatica L. on short-chain fatty acid production in mouse colon.

SCFA (lg/g of colonic Acetic acid Propionic acid Butyric acid i-Butyric acid Valeric acid i-Valeric acid Total SCFA

Control group

NTP groupa

HTP groupb

content) 2332 ± 57ac 949 ± 39a 1167 ± 21a 171 ± 14a 62 ± 8a 146 ± 11a 4827 ± 147a

3074 ± 61b 1064 ± 27b 1509 ± 36b 164 ± 15a 58 ± 6a 144 ± 13a 6013 ± 142b

3090 ± 49b 1468 ± 40c 2206 ± 30c 160 ± 20a 60 ± 10a 146 ± 18a 7130 ± 156c

a

NTP group, non-treated polysaccharide group. HTP group, homogenization-treated polysaccharide group. c Each value is the mean ± standard deviation (n = 12); means in the same line not sharing a common letter are significantly different (p < 0.05).

NTP group, non-treated polysaccharide group. HTP group, homogenization-treated polysaccharide group. c Each value is the mean ± standard deviation (n = 12); means in the same line not sharing a common letter are significantly different (p < 0.05).

our previous research (Yin, Lin, Li, Wang, Cui, Nie, & Xie, 2012). On the other hand, there are some ferulic acids present in the molecule of the polysaccharide (Yin et al., 2012), which have been proved to be hydrogen-donors and have obvious antioxidant ability (Graf, 1992). After high pressure homogenization, the particle size of the polysaccharide was significantly reduced (Fig. 1), resulting in an increase of total particle surface area for combining with

metal ions or free radicals. Hence, the antioxidant capacity of the polysaccharide was improved after high pressure homogenization. In addition, it was reported that the viscosity of the polysaccharide may influence its chelation ability towards metal ions or free radicals, and polysaccharides with lower viscosity exhibited higher antioxidant ability due to their greater chelation capacity (Kamil, Jeon, & Shahidi, 2002). In this study, it was found that the viscosity

b

b

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of the polysaccharide decreased after high pressure homogenization treatment, which was consistent with another report (Chen et al., 2012). Thus, the higher antioxidant ability of the homogenization-treated polysaccharide may also be attributed to its lower viscosity as compared to the non-treated polysaccharide. Furthermore, the process of homogenization may lead the polysaccharide molecule being dispersed more homogeneously, which resulted in the ferulic acid present in the molecule also being dispersed more homogeneously. This could mean that the ferulic acid provides hydrogen more effectively and performs antioxidant activity better. For the above reasons, the antioxidant capacity of the polysaccharide could be improved after high pressure homogenization treatment. 3.4. SCFA production of polysaccharide 3.4.1. General health status and organ weight of mice Throughout the experimental period, no noticeable behavioral or activity changes were observed in the mice, and no treatmentrelated illness or death occurred. The growth of mice appeared normal throughout the experimental period, and no mouse experienced diarrhea or constipation. After 4 weeks, the average body weight gain was 18.6 g per mouse, and the average dietary intake was 213.5 g per mouse. There was no observable difference in the animals’ body mass and hair lustre between the polysaccharidetreated groups and control group. Supplementary Table 2 summarizes the effects of the polysaccharide administration on the weights of various mouse organs. Organ weights were not different amongst the groups, except for the cecal weight and colon weight. The cecum and colon of the polysaccharide-treated groups were all significantly heavier than those of the control group (p < 0.05). This phenomenon has generally been observed when mice were fed with soluble dietary fibre and other carbohydrates (Arjmandi, Craig, Nathani, & Reeves, 1992; Moundras, Behr, Demigné, Mazur, & Rémésy, 1994). There was no significant difference in mouse cecum and colon weight between the homogenization-treated polysaccharide group and the non-treated polysaccharide group (p > 0.05). 3.4.2. Cecal and colonic SCFA concentration of mice In mice, the cecum and colon are sites of vigorous microbial activity where polysaccharides undergo fermentation, yielding SCFA. SCFA was reported to provide more than 70% of the oxygen consumed by human cecal and colonic tissue. Acetate is oxidized by brain, heart and peripheral tissues; propionic acid affects the liver and cholesterol metabolism; butyric acid serves as an energy source for colonic epithelium, regulates epithelial and immune cell growth and apoptosis, and provides protection against colonic cancer and colitis (Pryde, Duncan, Hold, Stewart, & Flint, 2002). The SCFA concentrations in the mouse cecum and colon in different groups are presented in Tables 2 and 3. The polysaccharidetreated groups had significantly higher concentrations of total SCFA, acetic acid, propionic acid and n-butyric acid in mouse cecum and colon than had the control group (p < 0.05). It was reported that the production of propionic acid could be promoted by the fermentation of glucose, xylose and arabinose (Mortensen, Holtug, & Rasmussen, 1988). The polysaccharide from P. asiatica L was high in xylose and arabinose content with a relatively lower amount of glucose (Yin et al., 2010), so the increase of propionic acid might result from the fermentation of xylose, arabinose and glucose of the polysaccharide. It was also found that xylose tended to have a greater impact on the production of butyric acid (Salvador et al., 1993). The polysaccharide from P. asiatica L was rich in xylose (Yin et al., 2010), so the increase of butyric acid might be due to the fermentation of xylose in the polysaccharide. In addition, there were no significant differences in the concentrations

of i-butyric acid, n-valeric acid, and i-valeric acid in mouse cecum and colon amongst non-treated polysaccharide group, homogenization-treated polysaccharide group and control group, throughout the experimental period (p > 0.05). It could also be seen from Tables 2 and 3 that the total SCFA, propionic acid and n-butyric acid concentrations in mouse cecum and colon of homogenization-treated polysaccharide group were both significantly higher than that of the non-treated polysaccharide group (p < 0.05). Researchers showed that the porosity of the polysaccharide and the particle size of the polysaccharide (determining the surface available to bacteria) may influence the fermentation and thus affect the SCFA production (Guillon et al., 1998). To degrade polysaccharides, microbial glycosidases must have access to their substrates. One factor, important for the control of accessibility, is particle size. Decreasing particle size increases the external surface area and so increases the area exposed to bacteria (Gama, Teixeira, & Mota, 1994). For example, the small-particle wheat bran produced greater SCFA concentrations during in vitro fermentation than did large-particle bran (Stewart & Slavin, 2009); the concentrations of total SCFA, acetic acid, propionic acid and n-butyric acid in the cecal content generally increased in rats as the particle size of corn bran decreased (Ebihara & Nakamoto, 2001); vegetable fibre of smaller particle size results in longer transit times in human intestine and is more extensively degraded (Lampe, Slavin, Melcher, & Potter, 1992). In addition, the pores in the polysaccharides could also improve the surface availability for bacteria and the surface availability for enzymes, and thus the polysaccharides are more extensively degraded (Guillon et al., 1998). In the present study, the particle size of the polysaccharide from P. asiatica L. decreased significantly after high pressure homogenization (Fig. 1). Meanwhile, the surface topography of the polysaccharide was changed from large flake-like structure to smaller porous chips, which increased the porosity of the polysaccharide (Fig. 2). Therefore, the accessible surface area of the polysaccharide for the cecal and colonic microbiota to contact was increased, and the glucose, xylose and arabinose in the polysaccharide could be more easily utilized by the microbiota after high pressure homogenization. 4. Conclusions The effect of high pressure homogenization on the physiological properties of polysaccharide, such as antioxidant capacity and SCFA production, was for the first time investigated. It was found that the particle size of polysaccharide from the seeds of Plantago asiatica L. decreased and the surface topography of the polysaccharide changed from large flake-like structure to smaller porous chips after high pressure homogenization. The antioxidant capacity and SCFA production of the polysaccharide were improved by the homogenization treatment. Our results showed that high pressure homogenization treatment could be a promising approach for the production of value-added polysaccharides in the food industry. 5. Conflict of interest The authors declare no competing financial interest. Acknowledgements This study is financially supported by Key Program of National Natural Science Foundation of China (No. 31130041), National Key Technology R & D Program of China (2012BAD33B06), National Natural Science Foundation of China (20802032 and 21062012), Objective-Oriented Project of State Key Laboratory of Food Science and Technology (SKLF-MB-201001), Training Project of Young

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Scientists of Jiangxi Province (Stars of Jing gang) and the Open Project Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-KF-201202), are gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem. 2012.12.016. References Adachi, T., Ono, Y., Koh, K. B., Takashima, K., Tainaka, H., Matsuno, Y., et al. (2004). Long-term alteration of gene expression without morphological change in testis after neonatal exposure to genistein in mice. Toxicogenomic analysis using cDNA microarray. Food and Chemical Toxicology, 42(3), 445–452. Arjmandi, B. H., Craig, J., Nathani, S., & Reeves, R. D. (1992). Soluble dietary fiber and cholesterol influence in vivo hepatic and intestinal cholesterol biosynthesis in rats. The Journal of Nutrition, 122(7), 1559–1565. Berggren, A. M., Björck, I. M. E., Nyman, E., & Eggum, B. O. (1993). Short-chain fatty acid content and pH in caecum of rats given various sources of carbohydrates. Journal of the Science of Food and Agriculture, 63(4), 397–406. Chen, W. S., Henry, G. A., Gaud, S. M., Miller, M. S., Kaiser, J. M., Balmadeca, E. A., Morgan, R. G., Baer, C. C., Borwankar, R. P., & Hellgeth, L. C. (1989). Microfragmented ionic polysaccharide/protein complex dispersions. EP Patent 0340035. Chen, J., Liang, R. H., Liu, W., Liu, C. M., Li, T., Tu, Z. C., et al. (2012). Degradation of high-methoxyl pectin by dynamic high pressure microfluidization and its mechanism. Food Hydrocolloids, 28(1), 121–129. Chen, Y., Xie, M. Y., Nie, S. P., Li, C., & Wang, Y. X. (2008). Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chemistry, 107(1), 231–241. Ebihara, K., & Nakamoto, Y. (2001). Effect of the particle size of corn bran on the plasma cholesterol concentration, fecal output and cecal fermentation in rats. Nutrition Research, 21(12), 1509–1518. Floury, J., Desrumaux, A., Axelos, M. A. V., & Legrand, J. (2002). Degradation of methylcellulose during ultra-high pressure homogenisation. Food Hydrocolloids, 16(1), 47–53. Gama, F., Teixeira, J., & Mota, M. (1994). Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique. Biotechnology and Bioengineering, 43(5), 381–387. Gan, C. Y., & Latiff, A. A. (2011). Extraction of antioxidant pectic-polysaccharide from mangosteen (Garcinia mangostana) rind: Optimization using response surface methodology. Carbohydrate Polymers, 83(2), 600–607. Graf, E. (1992). Antioxidant potential of ferulic acid. Free Radical Biology and Medicine, 13(4), 435–448. Guillon, F., Auffret, A., Robertson, J., Thibault, J. F., & Barry, J. L. (1998). Relationships between physical characteristics of sugar-beet fibre and its fermentability by human faecal flora. Carbohydrate Polymers, 37(2), 185–197. Halliwell, B., Gutteridge, J., & Aruoma, O. I. (1987). The deoxyribose method: A simple ‘‘test-tube’’ assay for determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry, 165(1), 215–219. Hochstein, P., & Atallah, A. S. (1988). The nature of oxidants and antioxidant systems in the inhibition of mutation and cancer. Mutation Research/ Fundamental and Molecular Mechanisms of Mutagenesis, 202(2), 363–375. Hu, J.-L., Nie, S.-P., Min, F.-F., & Xie, M.-Y. (2012). Polysaccharide from seeds of Plantago asiatica L. increases short-chain fatty acid production and fecal moisture along with lowering pH in mouse colon. Journal of Agricultural and Food Chemistry. http://dx.doi.org/10.1021/jf302169u. Huang, D. F., Xie, M. Y., Yin, J. Y., Nie, S. P., & Xie, M. Y. (2009). Immunomodulatory activity of the seeds of Plantago asiatica L. Journal of Ethnopharmacology, 124(3), 493–498. Huang, Y.-L., Chu, H.-F., Dai, F.-J., Yu, T.-Y., & Chau, C.-F. (2012). Intestinal health benefits of the water-soluble carbohydrate concentrate of wild grape (Vitis thunbergii) in hamsters. Journal of Agricultural and Food Chemistry, 60(19), 4854–4858.

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Huck-Iriart, C., Pizones Ruiz-Henestrosa, V. M., Candal, R. J., & Herrera, M. L. (2012). Effect of aqueous phase composition on stability of sodium caseinate/sunflower oil emulsions. Food and Bioprocess Technology. http://dx.doi.org/10.1007/ s11947-012-0901-y. Kamil, J. Y. V. A., Jeon, Y. J., & Shahidi, F. (2002). Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus). Food Chemistry, 79(1), 69–77. Lagoueyte, N., & Paquin, P. (1998). Effects of microfluidization on the functional properties of xanthan gum. Food Hydrocolloids, 12(3), 365–371. Lampe, J. W., Slavin, J. L., Melcher, E. A., & Potter, J. D. (1992). Effects of cereal and vegetable fiber feeding on potential risk factors for colon cancer. Cancer Epidemiology Biomarkers & Prevention, 1(3), 207–211. Lapasin, R., & Pricl, S. (1995). Rheology of industrial polysaccharides: theory and applications (1st ed.). London: Blackie Academic & Professional. Lyons, Deidra Shannon. (2011). Depolymerization of chitosan by high-pressure homogenization and the effect on antimicrobial properties. Master’s Thesis. University of Tennessee. . Mahady, G. B., Fong, H. H. S., & Farnsworth, N. R. (1999). WHO monographs on selected medicinal plants (1st ed.). Geneva: World Health Organisation. Mortensen, P. B., Holtug, K., & Rasmussen, H. S. (1988). Short-chain fatty acid production from mono-and disaccharides in a fecal incubation system: Implications for colonic fermentation of dietary fiber in humans. The Journal of Nutrition, 118(3), 321. Moundras, C., Behr, S. R., Demigné, C., Mazur, A., & Rémésy, C. (1994). Fermentable polysaccharides that enhance fecal bile acid excretion lower plasma cholesterol and apolipoprotein E-rich HDL in rats. The Journal of Nutrition, 124(11), 2179. Pérez-Jiménez, J., Arranz, S., Tabernero, M., Díaz-Rubio, M. E., Serrano, J., Goñi, I., et al. (2008). Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results. Food Research International, 41(3), 274–285. Pietta, P. G. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63(7), 1035–1042. Pouliot, Y., Britten, M., & Latreille, B. (1990). Effect of high-pressure homogenization on a sterilized infant formula: Microstructure and age gelation. Food Structure, 9(1), 1–8. Pryde, S. E., Duncan, S. H., Hold, G. L., Stewart, C. S., & Flint, H. J. (2002). The microbiology of butyrate formation in the human colon. FEMS Microbiology Letters, 217(2), 133–139. Rice-Evans, C., & Burdon, R. (1993). Free radicals–lipid interaction and their pathological consequences. Progress in Lipid Research, 32, 71–110. Sabu, M., & Kuttan, R. (2002). Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. Journal of Ethnopharmacology, 81(2), 155–160. Salvador, V., Cherbut, C., Barry, J. L., Bertrand, D., Bonnet, C., & Delort-Laval, J. (1993). Sugar composition of dietary fibre and short-chain fatty acid production during in vitro fermentation by human bacteria. British Journal of Nutrition, 70(1), 189–197. Samuelsen, A. B. (2000). The traditional uses, chemical constituents and biological activities of Plantago major L. A review. Journal of Ethnopharmacology, 71(1–2), 1–21. Silvestri, S., & Gabrielson, G. (1991). Degradation of tragacanth by high shear and turbulent forces during microfluidization. International Journal of Pharmaceutics, 73(2), 163–169. Stewart, M. L., & Slavin, J. L. (2009). Particle size and fraction of wheat bran influence short-chain fatty acid production in vitro. British Journal of Nutrition, 102(10), 1404–1407. Tang, Y., Huang, D., Yin, J., Zhou, C., Xie, X., & Xie, M. (2007). Effects of Semen plantaginis polysaccharides on phenotypic and endocytosis of murine dendritic cells. Food Science, 28(10), 517–520. Wu, G., Tian, Y., Xie, M., & Li, C. (2007). Effect of Semen plantaginis polysaccharides on defecating function in constipated mice. Food Science, 28(10), 514–516. Yin, J.-Y., Nie, S.-P., Zhou, C., Wan, Y., & Xie, M.-Y. (2010). Chemical characteristics and antioxidant activities of polysaccharide purified from the seeds of Plantago asiatica L. Journal of the Science of Food and Agriculture, 90(2), 210–217. Yin, J., Lin, H., Li, J., Wang, Y., Cui, S. W., Nie, S., et al. (2012). Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L. Carbohydrate Polymers, 87(4), 2416–2424. Zhang, E. X., Yu, L. J., Zhou, Y. L., & Xiao, X. (1996). Studies on the peroxidation of polyunsaturated fatty acid from lipoprotein induced by iron and the evaluation of the anti-oxidative activity of some natural products. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao, 28(2), 218–222.