Food Chemistry 135 (2012) 950959

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Germinated grains Sources of bioactive compounds

O.N. Donkor a,, L. Stojanovska a, P. Ginn b, J. Ashton b, T. Vasiljevic a
a b

School of Biomedical and Health Sciences, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, Vic 8001, Australia Sanitarium Development and Innovation, P.O. Box 40, Cooranbong, NSW 2265, Australia

a r t i c l e

i n f o

a b s t r a c t
Germination of seven selected commercially important grains was studied to establish its effects on the nutritional and chemical composition. The changes in the concentration of the nutrients, bioactive compounds and the inhibitory effect of extracts on a-glucosidase and a-amylase activities were investigated. These were measured through proximate analysis, inhibition assays and HPLC. Germinated sorghum and rye extracts inhibited (p < 0.05) a-glucosidase activity, whereas barley and sorghum extracts exhibited higher inhibitory activities against a-amylase. Germinated grains contained substantial amounts of total phenolics with rye having signicantly higher content compared with the non-germinated grains. Radical scavenging activities of the phenolic extracts were between 13% and 73% for non-germinated and 14% and 53% for germinated. Inositol phosphate (InsP) 4, 5 and 6 were noted in all the grains, but InsP 6 was signicantly lower in concentration. This study indicates the potential of germinated barley, sorghum and rye for the development of effective physiologically bioactive compounds for the reduction of the risk of diabetic agents and colon cancer. Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.

Article history: Received 22 March 2012 Received in revised form 18 April 2012 Accepted 14 May 2012 Available online 23 May 2012 Keywords: Germination Phenolic compound Antioxidant a-Glucosidase a-Amylase

1. Introduction A number of epidemiological studies have shown that regular consumption of whole grains reduces risks of various types of chronic diseases, such as cardiovascular disease (Anderson, Hanna, Peng, & Kryscio, 2000), type 2 diabetes (Liu et al., 2000; Meyer et al., 2000), some cancers (Kasum, Jacobs, Nicodemus, & Folsom, 2002; Nicodemus, Jacobs, & Folsom, 2001) and reduced mortality (Jacobs, Meyer, & Solvoll, 2001). Whole grains are rich sources of bre, vitamins, minerals, and phytochemicals, including phenolics, carotenoids, vitamin E, lignans, b-glucan, inulin, resistant starch, sterols, and phytates. Bioactive phytochemicals, found in signicant amounts in fruits, vegetables and whole grains, may provide desirable health benets beyond basic nutrition to reduce the risk of chronic diseases (Liu, 2004; Slavin, 2000). Recent evidence suggests that the complex mixture of bioactives in whole grain foods may be more healthful than individual isolated components (Liu, 2004). Wheat, rice, and corn are the major important grains in the human diet. The minor grains include oats, barley, rye, triticale, sorghum, millet, and buckwheat. Notably buckwheat is referred to as a pseudocereal related to sorrels, knotweeds, and rhubarb

Corresponding author. Tel.: +61 3 9919 8059; fax: +61 3 9919 8284.
E-mail address: Osaana.Donkor@vu.edu.au (O.N. Donkor).

(Chlopicka, Pasko, Gorinstein, Jedryas, & Zagrodzki, 2011). Whole grains contain unique phytochemical complex that complement those in fruits and vegetables when consumed together. The various classes of phenolic compounds in grains include phenolic acids, anthocyanidins, quinones, avonols, chalcones, avones, avanones, and amino phenolic compounds (Lloyd, Siebenmorgen, & Beers, 2000). However, germinated cereals/pseudocereal or sprouts are believed to have a greater nutritive and physiological value than cereal and pseudocereal grains and their products (Price, 1988; Prodanov, Sierra, & Vidal-Valverde, 1997; Rozan, Kuo, & Lambein, 1999, 2000). Numerous studies have shown that the nutritional and chemical prole is altered following germination of cereal grains such as wheat, rice, barley, oats and rye (Price, 1988). The most signicant change occurs in starch, which is broken to simpler sugars by amylases. The degree of the changes seen in starch depends on various germination conditions, such as temperature, humidity, culturing media, steeping (soaking) and the length of germination (Koehler, Hartmann, Wieser, & Rychlik, 2007). This means direct comparison is difcult, and optimum conditions will need to be dened for individual cereals. At the same time with synthesis of novel compounds, the concentrations of some nutrient inhibitors may decrease. For example, phytic acid is hydrolysed by increased phytase activity, decreasing phytate concentration subsequently leads to releasing phosphate, inositol and minerals (Bohn, Meyer, & Rasmussen, 2008). In germinated our, protein, bre, fat and energy increased signicantly as did anti-nutrients (tannins, phytates and trypsin) and total amino acid content increased, while mineral

0308-8146/$ - see front matter Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.058

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alterations included signicant increases (Fe, Se) and decreases (Ca, K, Mg, Na, P) (Ljarotimi & Esho, 2009). Polyphenols found in wheat sprouts may be of benet associated with a particular group of population as they appear to improve glucose metabolism (Hanhineva et al., 2010) and counteract inammation and oxidative stress associated with obesity (Detopoulou et al., 2010). Other examples of plant derived bioactives are c-aminobutyric acid (GABA), inositol, and phytic acid (IP6) (Nagaoka, 2005). GABA acts as an inhibitory neurotransmitter in the brain and spinal cord of mammals and shows a series of functions, such as regulation of blood pressure and heart rate, and alleviation of pain and anxiety (Mody, Dekoninck, Otis, & Soltesz, 1994). Studies show that GABA is also a strong secretagogue of insulin from the pancreas, and effectively prevents diabetes (Adeghate & Ponery, 2002; Hagiwara, Seki, & Ariga, 2004). Furthermore, it appears that an inverse relation between dietary intake of IP6 and cancer rates in some countries exist (Shamsuddin & Ullah, 1989). Administration of exogenous IP6 in vivo animal models has been negatively correlated with the development of several types of cancer (Estensen & Wattenberg, 1993; Shamsuddin & Ullah, 1989; Vucenik, Shamsuddin, & Zhang, 1998). Arabinoxylan (AX) is a major bre component in many cereal grains. The nutritional values of other bre components in grains, most notably arabinoxylans, have not been investigated to the same extent as those of b-glucans. However, recent studies revealed positive effects of water soluble maize, wheat and rye arabinoxylans on caecal fermentation, production of short-chain fatty acids, reduction of serum cholesterol and improved adsorption of calcium and magnesium (Hopkins et al., 2003; Lopez et al., 1999). It has been shown to decrease ghrelin, serum insulin and glucose response in 11 subjects with impaired glucose tolerance following 6 weeks supplementation of 15 g in a randomised crossover trial (Garcia et al., 2007). Five weeks of arabinoxylan supplementation, signicantly reduced fasting plasma glucose and postprandial glucose in diabetic subjects compared with a control diet (Lu, Walker, Muir, & ODea, 2004). It is anticipated, therefore, that the health benets of grains will stimulate interest among food producers and consumers in using them for food purposes. To date in the public domain, common grains and their germinated counterparts have not been assessed in Australia, thus the main aim of this project was to establish the effects of germination on the synthesis of selected physiologically important compounds in selected commercially important grain varieties.

Grains. Wheat, oats, barley, rye, sorghum, buckwheat and brown rice (hulled), were kindly supplied by Sanitarium (Cooranbong, NSW, Australia). 2.2. Germination process Germination of grains was conducted according to the method described by Yang, Basu, and Ooraikul (2001). Briey, grain samples were submerged in 1.25% sodium hypochlorite (NaClO) (seed:water ratio of 1:5, w/v) at room temperature for 30 min to disinfect any contaminating micro-organisms on the grain surface prior to germination process. This was followed by constant washing under tap water, at approximately 10 C, for 2030 min to thoroughly rinse off residual NaClO. The disinfected grains were then steeped in tap water for 24 h at 16.5 C. For sprouting, each sample of grains (approx. 20 g) was placed into aluminium foil pan with draining holes at the bottom to drain excess moisture. Germination was accomplished in incubators at 98% RH and 16.5 C for 5 days in the dark. The humidity was maintained at 98% to facilitate reduction in the drying of the sprouts by periodic watering. The grains were wrapped with sterile cheesecloth to maintain the moisture. The total germination length included the time for steeping and the time for sprouting. The grains were sprinkled with tap water for 15 min at 12 h intervals and aerated by hand once every 24 h in order to (1) prevent rootlets matting, (2) ensure that on average all grains were treated as nearly evenly as possible, (3) break up hot spots and (4) reduce its bulk density and ensure that the grain bed was saturated with water vapour. After 5 days the grains were harvested, carefully washed with distilled water (3 800 ml) to remove the testas and stored at 80 C prior to freeze drying. Subsequently, the dried grains were ground using mortar and pestle into ne powder and stored at 20 C before analysis. The analyses included vitamin C, phenolic compounds and antioxidants. 2.3. Proximate analysis of germinated and non-germinated grains Moisture, crude protein, fat, crude bre, carbohydrates and total ash were determined using Association of Ofcial Analytical Chemists (AOAC) (2002). The crude fat was determined using Soxhlet extraction method of Association of Analytical Chemists (AOAC) (1984). The crude protein content of the rice samples was determined using the Micro Kjeldahl method of AOAC (1984). Crude bre was determined in the sample using the standard methods of analysis of the AOAC (1984). The total percentage carbohydrate content was determined by the difference method as described by Edeogu, Ezeonu, Okaka, Ekuma, and EIom (2007). The moisture content was determined by drying overnight in a convection oven at 90 C as described by the method of the standard ofcial methods of analysis (AOAC, 1984). The ash content was carried out according to Method 25.092 (AOAC, 1984). 2.4. Extraction of phenolic compounds Freeze dried germinated/control grains (1 g) were weighed and ground into a ne powder using mortar and pestle. The samples were mixed with 80% ethanol (EtOH) and extracted for 10 min followed by vortexing (Mt19 Auto Vortex mixer, Chiltern Scientic, Biolab Ltd., Auckland, New Zealand) for further 15 min and supernatant was collected after centrifugation (Sorvall RT7 centrifuge, DuPont, Newtown, CT, USA) at 4000g for 30 min. Extraction was repeated three times and supernatants were pooled together. The extract was concentrated by removal of ethanol using rotary evaporator (EYELA, Tokyo Rikakikai Co. Ltd., Japan) and the sample was freeze dried (Dynavac freeze drier, FD300, Dynavac Eng. Pty. Ltd., Melbourne, Australia). The freeze dried sample was re-sus-

2. Materials and methods 2.1. Chemicals and reagents Methanol, acetonitrile (CH3CN) and phosphoric acid were HPLCgrade and purchased from Merck (Darmstadt, Germany). Folin Ciocalteau reagent and highly puried polyphenols as (+)-catechin (C), ()-epicatechin (EC), epigallocatechin-3-gallate (EGCG), ()epigallocatechin (EGC), ellagic acid (AE), quercetin (Q), luteolin (L) and gallic acid (AG), Sodium phytate (dodecasodium salt hydrate), phytic acid (40% w/w solution in water), phytic acid (50% in water) and tetrabutylammonium hydroxide (TBAOH, 40% w/w solution in water), a-amylase from porcine pancreas (A3176) were purchased from SigmaAldrich (St. Louis, MO, USA). Other used chemicals and solvents were of the highest analytical grade available. The Total Starch Assay Kit and the Mixed-Linkage b-glucan Assay Kit were from Megazyme (Bray, Ireland). Sodium hypochlorite, ethanol, methanol, sodium carbonate, silica-based anion exchange columns (SAX, size 3cc) were purchased from Varian (Harbor City, CA). Cation exchange resin AG 50W-X4 50100 mesh H+ was purchased from Bio-Rad (Richmond, CA).

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pended in methanol to a nal volume of 10 ml, ltered and stored at 80 C until further analysis. 2.5. Inhibition assay for a-glucosidase activity Inhibition of a-glucosidase assay was performed as described previously (Kim, Hyun, & Kim, 2011) with some modications. aGlucosidase (1 U/ml) was dissolved in 100 ll of 0.1 M potassium phosphate buffer (pH 6.8) and mixed with 50 ll of sample extract. After pre-incubation at 37 C for 10 min, 5 mM p-nitrophenol from p-nitrophenyl a-D-glucopyranoside (pNPG) (50 ll) was added as substrate. The enzymatic reaction was allowed to proceed at 37 C for 30 min and was stopped by the addition of 1 ml of 0.1 M Na2CO3. a-Glucosidase activity was determined by measuring the release of p-nitrophenol from pNPG with a Pharmacia UV spectrophotometer at 400 nm. Solution without sample was used as a control. Solution without substrate was used as a blank. The experiment was performed in triplicate:

methanol extract of each grain sample was placed in separate tubes and 0.5 ml of FolinCiocalteu reagent was added to each tube and vortexed for 1 min. After 3 min of incubation, 10 ml of sodium carbonate solution (75 g/l) and 5 ml distilled water were added, mixed and incubated for 1 h at room temperature in the dark. Absorbance of the resulting solutions were measured at 750 nm using a Pharmacia UV spectrophotometer (LKB NOVASPEC II, LKB Biochrom, St. Albans, UK). The results were expressed in ferulic acid equivalents. 2.9. Determination of individual polyphenols Quantication of polyphenols in grain extracts were performed using HPLC as described by Amici et al. (2008). A Varian HPLC system (Varian Analytical Instruments, Walnut Creek, CA, USA) equipped with a C18 Phenomenex Luna column (2.5 mm porosity, 250 4 mm, with an UltraSep ES RP18 pre-column) was used with a three-step linear gradient for the separation of all polyphenols in the samples. The mobile phase consisted of: (A) 0.3% phosphoric acid and (B) 100% CH3CN with the following gradient: from 10% to 20% of B in 45 min, from 20% to 60% of B in 20 min then 60% to 90% of B in 20 min (ow rate 0.7 ml/min, temperature 20 C). The elution pattern was monitored with a Varian UVVIS detector at 220 nm. The methanol extracts were ltered through 0.45 lm membrane lter (Schleicher & Schuell GmbH, Dassel, Germany) injected in a 10 ll loop. Each marker was identied by comparison with the retention time of the reference standard and by internal standard. Quantitative analysis was performed by calibration curves using the reference standards of ferulic acid, gallic acid, catechin, epicatechin, epigallocatechin, epigallocatechingallate, luteolin and p-coumaric acid (SigmaAldrich) and the linearity was investigated in the range of 05 mg at ve increasing concentrations. Even levels of concentration tested were performed in triplicate. Intra-day analyses of the same solution containing all phenolic compounds tested were used to validate the precision of the chromatographic system. 2.10. Determination of arabinoxylan in grains Arabinoxylan (AX) content in the ground germinated/non-germinated grains (0.25 g) was determined after hydrolysis with 4 N triuoroacetic acid at 120 C for 4 h as described by Holgun-Acua et al. (2008) with modications. The reaction was stopped on ice and the extracts were evaporated to dryness using a Savant Speedvac concentrator (model SC110, Savant Instruments Inc., Farmingdale, NY, USA) at 40 C, and rinsed twice with 300 ll of water. The evaporated extract was solubilised in 2 ml of water. Samples were ltered through 0.45 lm membrane lters and analysed using a Varian HPLC equipped with a Star Chromatography Workstation system control software version 5.50 and a refractive index detector (Varian Star 9040, St. Helens, Australia). Separation of arabinoxylan was achieved by a Supelcogel Pb column (300 7.8 mm; Supelco Inc., Bellefont, PA) eluted with water (ltered 0.2 lm lter, Whatman) at a ow rate of 0.6 ml/min and a column temperature of 80 C. Arabinoxylan content was estimated from the sum of xylose + arabinose. 2.11. Extraction of GABA from grains Free amino acids were extracted using the procedure of Oh and Choi (2001) with modications. Approximately 0.25 g of ground samples were placed in eppendorf tube containing 1 ml of 70% ethanol. The mixture was vortexed for 1 min and centrifuged at 12,000g at 4 C for 10 min. The supernatant was collected and the above step was repeated. The supernatants were pooled to-

  Abssample Absblank 100 Inhibition % 1 Abscontrol

where Abssample represents the absorbance of the experimental sample, Absblank represents the absorbance of the blank, and Abscontrol represents the absorbance of the control. 2.6. Inhibition assay for a-amylase activity The inhibition assay for a-amylase activity was performed by the method described previously (Kim, Jeong, Wang, Lee, & Rhee, 2005) with some modications. Briey, 100 ll a-amylase from porcine pancreas (A3176, 23 units/mg solid, Sigma) was premixed with 100 ll of methanol extract and after pre-incubation at 37 C for 5 min, 250 ll of 1% starch was added as substrate in phosphate buffer solution (pH 6.8) to start the reaction. The reaction was carried out at 37 C for 5 min and terminated by the addition of 200 ll DNS reagent (1% 3,5-dinitrosalicylic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The reaction mixture was heated for 15 min at 100 C and diluted with 2 ml distilled water on an ice bath. a-Amylase activity was determined by measuring absorbance at 540 nm using a Pharmacia UV spectrophotometer. 2.7. DPPH free radical scavenging activity of grain extracts The free radical scavenging activity of different grain methanol extracts was evaluated according to the method described by Bouaziz, Fki, Jemai, Ayadi, and Sayadi (2008) with some modications. Briey, 3.9 ml of DPPH solution (0.075 mM in methanol) was added to 0.1 ml of properly diluted grain extracts followed by 30 min incubation in dark and absorbance was read at 515 nm using a Pharmacia UV spectrophotometer. A standard curve was prepared at different concentrations (10, 25, 50, 75 and 100 lg/ml) of ascorbic acid (SigmaAldrich). The reduction in the absorbance of DPPH solution at different concentrations of ascorbic acid over a period of 30 min was measured and plotted. The DPPH radical scavenging activities of grain extracts were expressed in ascorbic acid equivalent antioxidant capacity. The antioxidant activity was calculated as percent inhibition caused by the hydrogen donor activity of each sample:

  absorbance of sample 100 Inhibition % 1 absorbance of blank

2.8. Determination of total phenol content in grain extracts Total phenol content (TPC) of the grain extracts was determined according to Angioloni and Collar (2012). Briey, 0.5 ml of the

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gether. The volume of obtained crude extract was adjusted to 2 ml and derivatized using 2-hydroxynaphthaldehyde (HN).

2.13. Statistical analysis Values are expressed as mean values and standard deviation of results obtained from 3 separate experiments, subsequently subsampled once giving a number of independent observations of 6. One-way Analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons, was used to assess differences among the grains. Statistical tests were performed with Sigma-Stat 3.1 software (SPSS, Chicago, IL). A value of p < 0.05 was considered signicant.

2.11.1. Derivatization of samples and GABA standards The crude extract samples (1 ml) was added to 0.6 ml of borax buffer pH 8 and 1 ml of HN (0.3% w/v in methanol) as described by Khuhawar and Rajper (2003). The solution was heated in a water bath at 80 C for 10 min and was allowed to cool, and then passed through 0.45 lm membrane lter for analysis by HPLC. The steps were then repeated for the GABA standards. A standard curve was constructed through analysis by HPLC from ve standard solutions of GABA (25, 50, 75 and 100 lg/ml of GABA). 2.11.2. GABA content determination by HPLC The presence of GABA in the samples was determined following the method described by Khuhawar and Rajper (2003) with some modications. A Varian HPLC system equipped with a C18 Phenomenex Luna column (5 lm 100A 250 4.6 mm with C18 4 3.0 mm pre-column) and a linear gradient was used to analyse GABA in the samples. The mobile phase consisted of: (A) 0.1% formic acid (HCOOH) and (B) 100% CH3CN with the following gradient: from 30% to 40% of B in 5 min, from 40% to 55% of B in 510 min then 55% to 30% of B in 1025 min with an injection volume of 10 ll (ow rate 1 ml/min, temperature 20 C). The elution pattern was monitored with a UVVIS detector at 330 nm.

3. Results and discussion 3.1. Proximate composition The proximate composition of seven grains assessed in this study is shown in Table 1. Generally, ash, protein, fat and starch contents signicantly (p < 0.05) depended on the grain variety and the treatment. As expected the main proportion of these grains was composed of carbohydrates followed by the crude protein (Edeogu et al., 2007). The moisture content (not shown) was similar to that reported for germinated and non-germinated grains while fat and ash levels were lower (Obatolu, 2002). Proximate and nutrient analysis of grains and vegetables plays a crucial role in assessing their nutritional signicance. As various grains are used as food along with their health benets, evaluating their nutritional signicance can help to understand the worth of these grains (Pandey, Abidi, Singh, & Singh, 2006), which was also emphasised by WHO (1993). 3.2. Inhibition of a-glucosidase and a-amylase activity The methanolic extracts from 7 germinated/non-germinated grains were tested for a-glucosidase inhibitory activity (Fig. 1). Extract from germinated sorghum signicantly (p < 0.05) inhibited aglucosidase activity followed by germinated rye. On the other hand barley, wheat, buckwheat, brown rice and oats showed low inhibitory effects. A similar pattern of inhibition was observed in the non-germinated samples. Degradation of starch by a-amylase was inhibited signicantly (p < 0.05) by all the germinated grains in comparison to the non-germinated samples (Fig. 2). Germinated barley and sorghum were the most potent inhibitors of a-amylase activity. Kim et al. (2011) reported similar ndings with sorghum. The inhibition of a-amylase activity, together with a-glucosidase is considered to be an effective strategy for the control of diabetes by diminishing the absorption of glucose (Hara & Honda, 1990). Our investigation indicated that grains, particularly the germinated form, may be effective inhibitors of glucosidase, with inhibitory activity against porcine pancreatic a-amylase. Although the inhibitory effect of the grain extracts has been established in vitro, these ndings indicate that the extracts may potentially suppress the increase in post-prandial glucose caused by starch hydrolysis. 3.3. Radical scavenging activity The inuence of germination on TPC of 7 germinated and nongerminated grains were examined and quantitative measurements performed with the FolinCiocalteau reagent. All germinated grains contained substantial amounts of total phenols with rye having signicantly (p < 0.05) higher content compared with the non-germinated grains (Figs. 3a and 3b). Yang et al. (2001) also showed increased phenolic content during and after germination of wheat which was in line with our study. However the TPC in non-germinated buckwheat appeared signicantly (p < 0.05) high-

2.12. Determination of inositol phosphates content Freeze-dried, ground grains samples (0.5 g) were mechanical agitation with 2.5 ml 0.5 M HCl for 2 h at 20 C (Frontela, GarcaAlonso, Ros, & Martnez, 2008; Lehrfeld, 1994) with some modications. The mixture was centrifuged at 12,000g for 10 min and the supernatant decanted. Two and a half millilitres of 0.5 M HCl was added to the pellet, the steps were repeated and the supernatant pooled together. The resulting solution was ltered through 0.22 lm membrane lter. The inositol phosphates were separated from the ltrate and concentrated by ion-exchange procedure as follows: the aliquot from the extract (2.5 ml) was diluted with water (1/10) and placed onto an anion exchange (SAX) column (500 mg; Supelco, Bellefonte, PA, USA). The column was prewashed with 2 ml of 0.05 M HCl, and the inositol phosphates were eluted with 2 ml of 2 M HC1 (trace element grade). The eluate was evaporated to dryness with a Savant Speedvac concentrator at 40 C. The residue was dissolved in 0.5 ml vacuum-ltered buffer solution prepared by adding 1.6 ml of TBAOH, 0.2 ml of 5 M sulphuric acid, and 0.1 ml of formic acid (91%) to 100 ml of a methanolwater solution (51.5%) sonicated using ultrasonic bath (Unisonics Pty. Ltd., Sydney, Australia) for 6 min The solution was centrifuged at 12,000g for 5 min to remove any suspended material prior to injection into the HPLC. Twenty microlitres of sample was injected into a Varian HPLC system equipped with a refractive index detector (ERC-7515A, ERMA CR Inc., Kawaguchi city, Japan), Alltech C18 (5 lm 250 4.6 mm) reversed phase analytical column and a guard column (adsorbosphere C18 (5 lm 150 4.6 mm). The isocratic mobile phase was prepared by mixing 430 ml Milli-Q water with 20 ml tetrabutylammonium hydroxide pH adjusted to 2.65 with 85% phosphoric acid. Methanol (570 ml) and 20 ll of phytic acid were added to the resulting solution and the pH was adjusted to 4.3 to achieve an optimal separation of the inositol phosphates. The injection volume was 20 ll and the ow rate was 0.6 ml/min. Identication of the inositol phosphate was achieved by comparison with the external standard (sodium phytate). Commercial 40% phytic acid (SigmaAldrich) was used for calibration.

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Table 1 Proximate analysis of germinated and non-germinated grains. Sample Ash, g/100 g DM Non-germ Buckwheat Barley Oat Wheat Sorghum Rye Brown rice 1.89 0.09 2.16 0.06 1.77 0.04 1.85 0.05 1.84 0.04 2.08 0.06 1.53 0.14 Germ 1.91 0.08 2.33 0.10 1.56 0.19 2.18 0.04 1.81 0.04 3.04 0.10 1.37 0.04 Protein, g/100 g DM Non-germ 12.88 0.57 15.69 0.16 10.59 0.08 11.56 0.10 10.57 0.09 16.34 0.37 7.81 0.32 Germ 16.93 0.09 19.57 0.61 13.14 0.25 13.69 0.01 12.55 0.25 26.38 0.66 9.52 0.11 Fat, g/100 g DM Non-germ 4.10 0.19 2.37 0.22 6.81 0.21 1.95 0.14 4.11 0.13 1.61 0.24 3.16 0.16 Germ 1.93 0.12 3.01 0.69 8.20 0.15 1.81 0.22 3.53 0.16 3.52 0.18 1.95 0.01 Starch, g/100 g DM Non-germ 46.88 0.82 28.23 0.19 40.61 1.95 41.90 0.99 33.65 0.33 30.43 0.90 54.71 0.36 Germ 62.60 1.47 45.22 3.10 46.57 2.53 41.12 1.32 47.72 0.41 30.07 2.41 59.69 0.92

Non-germ non-germinated; Germ germinated; DM dry matter.

Fig. 1. Effect of methanol extracts from germinated and non-germinated grain varieties on a-glucosidase activity. Data shown represent mean standard deviation, p < 0.05.

Fig. 2. Inhibitory effects of methanol extracts from germinated and non-germinated grain varieties on a-amylase activity. Data shown represent mean standard deviation, p < 0.05.

er than in the germinated form whereas TPC in oats and brown rice were similar in both forms (Figs. 3a and 3b). Bleaching of DPPH induced by germinated and non-germinated grain extracts was also measured to test for their free radical scavenging activity (Figs. 3a and 3b). DPPH is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. When DPPH is reduced to yellow-coloured diphenyl picryl hydrazine by antioxidants, hydrogen atom-donating activity is measured by providing an index of the free radical quenching capacity (Hong, Kang, Kang, & Cho, 2001). The decrease in absorbance of DPPH radical caused by antioxidants results in the

scavenging of the radical by hydrogen donation. Radical scavenging activities of the phenolic extracts were between 13% and 73% for non-germinated and 14% and 53% for germinated grains. Even though all the grains showed substantial inhibitory activity, buckwheat and rye in the germinated form appeared to have exhibited more than 50% radical scavenging activity. The non-germinated buckwheat also showed the highest inhibition. A similar observation was previously made by Oki et al. (2002). Grains that contained higher levels of phenolic compounds also displayed higher scavenging activity (Figs. 3a and 3b). The TPC and DPPH results indicate that TPC is positively correlated with antioxidant func-

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Fig. 3a. Radical scavenging capacity against DPPH in comparison to total polyphenolic content of non-germinated grain varieties. Data shown represent mean standard deviation, p < 0.05.

Fig. 3b. Radical scavenging capacity against DPPH in comparison to total polyphenolic content of germinated grain varieties. Data shown represent mean standard deviation, p < 0.05.

tionality (Figs. 3a and 3b) whereby antioxidant capacity increases with TPC. Similar results were reported previously (Chung & Woo, 2001). The results reported in our study provide a sharp indication that the mixture of powerful antioxidant compounds found in several grain tissues may be a potent protection for human against the pathologies linked to oxidative stress. In this context it is important to stress the signicance of natural sources that not only supply a series of natural antioxidant compounds, but supply them in natural ratios between themselves (Calzuola, Gianfranceschi, & Marsili, 2006). The nutritional and physiological relevance of grain sprouts in terms of bioactive compounds are of importance in strengthening antioxidant defence. 3.4. Analysis of polyphenols Polyphenol identication was determined and quantied using RP-HPLC analysis comparing the retention time of reference standards (Table 2). The reference standard mixture and the extract elution proles are shown in [Fig. 4 (Chromatograph A and B)]. Comparing the retention times of the peaks obtained from the selected germinated grains and control hydroalcholic extracts with the peaks obtained from the standard mixture, it was possible to

identify eight components: gallic acid (GA), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechingallate (EGCG), ferulic acid (FA), p-coumaric (PC), and luteolin (L) (Table 2). The differences observed between the elution times related to the extract and the standard mixture could be due to the matrix effect. This effect depends upon variations of ionisation efciency in the presence of coeluting substances and causes analytes to elute at different retention times, considering the same polyphenolic compound dissolved either in a specic buffer or in a whole grain extract. The amounts of each identied polyphenol were determined by RP-HPLC analysis using standard peak area values obtained by serial dilutions of the polyphenols standard mixture. Data reported in Table 2 show the polyphenol concentrations present in the selected germinated grains and non-germinated methanolic extract with high catechin content and ferulic acid and luteolin were detected in all grain extracts. Amici et al. (2008) similarly reported high catechins content, particularly catechin, epicatechin, epigallocatechin and epigallocatechingallate in the extracts. Others have reported four polyphenol catechins in green tea (C, EGC, EC and EGCG). These have been reported to have potent antioxidant capabilities, and are being investigated for their ability to prevent cancer and heart disease (Basu, 1999). In exper-

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Table 2 HPLC quantication of polyphenolic compounds in germinated and non-germinated grains. Sample Barley Barley-G Buckwheat Buckwheat-G Oat Oat-G Rye Rye-G Sorghum Sorghum-G Wheat Wheat-G Brown rice Brown rice-G Gallic acid (lg/ml) 4.70 0.23c 2.90 0.53cd 0.73 0.08cd ND ND ND 2.26 0.04cd 22.61 4.32a ND 16.59 2.96b ND 0.77 0.34cd ND ND Epigallocatechin (lg/ml) 10.94 2.70bcd 36.59 11.96a 14.04 2.72b 1.41 0.64cde ND ND 0.88 0.01cde 11.22 2.70bc ND 19.24 2.42b ND 0.59 0.10de ND 1.26 1.27cde Catechin (lg/ml) 20.81 0.75ab 18.94 3.15bc 26.80 3.10a 13.53 6.21cd ND ND 4.02 0.28e 12.82 1.95cd 0.35 0.01e 18.34 0.43bc 6.95 3.48de 0.16 0.16e ND 1.22 1.22e Epicatechin (lg/ml) 1.51 0.96d 2.71 0.77d 38.30 2.74a 9.45 1.78c 0.15 0.15d 1.25 1.26d 5.37 1.00cd 21.05 6.23b 3.29 0.25d 11.03 0.03c ND 11.07 2.36c 1.22 0.06d ND Epigallocatechingallate (lg/ml) 4.67 0.70a 1.73 0.62cd 4.03 1.51ab 3.08 1.04abc 0.11 0.11d ND 0.14 0.07d 0.27 0.28d 0.22 0.04d 1.82 1.03cd 2.50 0.10bc 1.70 0.74cd 0.30 0.02d ND P-coumaric acid (lg/ml) 2.43 0.19bc 0.68 0.68c 13.39 3.20a 6.63 2.62b ND ND 0.74 0.11c 3.49 0.87bc 16.77 4.36a 5.95 1.13b ND 1.82 0.60bc ND ND Ferulic acid (lg/ml) 12.53 6.20cd 14.03 1.29cd 29.34 2.81b 4.45 0.56def 3.72 2.38def 1.04 0.80ef 14.82 1.08cd 13.12 2.98cd 12.03 0.42cde 10.73 0.72cdef 20.05 8.74bc 52.94 7.38a 1.13 0.17ef 0.72 0.73f Luteolin (lg/ml) 159.41 28.87a 12.29 6.53b 17.35 1.37b 3.96 1.66b 2.93 1.42b 2.15 0.16b 3.71 1.05b 4.03 1.59b 9.03 1.84b 3.85 1.08b 0.40 0.40b 5.31 0.19b 3.66 1.11b 2.87 2.87b

ND not detected; G denotes germinated variety. The values are expressed as means of 6 independent observations standard deviation. ae Means in the same column with different small letter superscripts are signicantly different.

Fig. 4. HPLC analysis. Chromatogram A: elution prole of a standard mixture of polyphenols gallic acid (GA), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin-3-gallate (EGCG), p-coumaric (PC), ferulic acid (FA) and luteolin (L). Chromatogram B: elution prole of a germinated sorghum hydroalcolic extract. Chromatographic analysis was performed on a Varian HPLC system equipped with a C18 Phenomenex Luna column and a guard column with a three-step linear gradient of solvent A 0.3% phosphoric acid and solvent B 100% CH3CN. The elution pattern was monitored with a Varian UVVIS detector at 220 nm.

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Fig. 5. Content of arabinoxylan in germinated and non-germinated grain varieties. Each bar represents the mean of 6 independent observations with error bars showing the standard deviation, p < 0.05. DM dry matter.

Fig. 6. Content of c-amino butyric acid (GABA) in germinated and non-germinated grain varieties. Each bar represents the mean of 6 independent observations with error bars showing the standard deviation, p < 0.05.

imental models, catechins show a wide range of protective effects, including cardioprotective, chemoprotective, and anitmicrobial properties (Scalbert, Manarch, Morand, Remesy, & Jimenez, 2005). 3.5. Arabinoxylan content The chromatographic quantication of individually released monosaccharides after degradation of water-soluble polysaccharides, showed that the actual amount of AX (arabinose + xylose) can be analysed (Fig. 5). After 5 days of germination, the total AX content in barley, oats and rye grains decreased, while AX content in buckwheat, sorghum wheat and brown rice increased compared with AX content in non-germinated grains (Fig. 5). The total AX content varied from 0.11 to 2.58 g/100 g of grain. The different types of grains have on average measurable AX levels, for barley, oats and rye, non-germinated grain samples evidently are richer in AX, where as AX differences are noticeable between the different types of germinated grains. Arabinoxylan is a major component of hemicelluloses in insoluble and soluble bres and is found in many

Table 3 Concentrations of inostiol phosphates in germinated and non-germinated grains. Sample Barley Barley-G Buckwheat Buckwheat-G Oat Oat-G Rye Rye-G Sorghum Sorghum-G Wheat Wheat-G Brown rice Brown rice-G IP4 (g/100 g DM) 0.69 0.09a 0.94 0.30a 0.63 0.03a 0.61 0.08a 2.39 1.88b 1.16 0.58c 1.50 0.79c 1.20 1.07c 0.92 0.73a 0.60 0.81a 0.68 0.08a 1.27 0.67c 0.58 0.52a 0.33 0.18a IP5 (g/100 g DM) 0.05 0.09a 0.35 0.31a ND 0.05 0.09a 1.66 1.48b 0.11 0.2a 0.45 0.79a 0.85 1.13a 0.39 0.51a 0.06 0.10a 0.05 0.09a 0.18 0.31a ND ND IP6 (g/100 g DM) 0.09 0.08a 0.14 0.01a 0.04 0.08a ND 1.69 1.58b ND 0.63 0.98a 0.79 1.25a 0.04 0.08a 0.04 0.08a ND 0.04 0.08a 0.05 0.08a ND

ND not detected; G denotes germinated variety; DM dry matter. The values are expressed as means of 6 independent observations standard deviation. ac Means in the same column with different small letter superscripts are signicantly different.

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cereal grains. Studies have revealed positive effects of water soluble maize, wheat and rye arabinoxylans on caecal fermentation, production of short-chain fatty acids, reduction of serum cholesterol and improved adsorption of calcium and magnesium (Hopkins et al., 2003; Lopez et al., 1999). Furthermore, AX bre has been reported to improve metabolic control in people with type 2 diabetes (Lu et al., 2004). It is anticipated, therefore, that the health benets of oats, barley, sorghum, buckwheat and brown rice will stimulate interest among food producers and consumers in using these grains for food purposes. 3.6. GABA contents in grains HPLC analysis showed that each sample of germinated/non-germinated grain studied contained GABA (Fig. 6). The study also observed that the amino acid content in all the grains increased during the germination process. The analysis further showed that the GABA content in barley and rye were signicantly (p < 0.05) higher than that of the other examined grains. Oh (2003) reported signicant increases in the concentration of GABA in brown rice during germination. c-Aminobutyric acid is a nonprotein amino acid that is primarily produced from the c-decarboxylation of Lglutamic acid that is catalysed by the enzyme glutamate decarboxylase (Park et al., 1999). GABA functions in animals as a major inhibitory neurotransmitter (Mody et al., 1994). There has been an increased interest in utilisation of GABA as a bioactive plant component. Several lines of evidence suggest that plant extract containing high levels of GABA are effective in blood pressure regulation (Nakagawa & Onota, 1996) and in the recovery of alcohol related symptoms (Oh & Cha, 2001). This shows that there is potential for producing GABA-enriched breakfast cereals with barley and rye. 3.7. Inositol phosphates The qualitative and quantitative analyses of inositol phosphates were also carried out on germinated and non-germinated grains (Table 3). In these materials, the presence of inositol phosphate (InsP) 4, 5 and 6 were noted with InsP 6 showing signicantly (p < 0.05) higher concentration in comparison to InsP 4 and 5. These results are in accordance with Frontela et al. (2008) and conrm that cereal whole grains are a rich source of inositol hexaphosphate (Lasztity, 1998). It has been reported that InsP6 in the diet has the potential to reduce the risk of colon cancer (Shamsuddin, 1995). Moreover, among its lower phosphorylated form InsP4 has been noted to have the ability to chelate the Fe ion in vitro (Zielinski, Kozowska, & Lewczuk, 2001). It has been reported that, germination causes considerable degradation of InsP6 (Honke, Kozlowska, Vidal-Valverde, Frias, & Gorecki, 1998). This was in accordance with our study, which showed in general a reduction, although not signicant (p > 0.05) in concentration of InsP6 in the germinated buckwheat, oats, brown rice, rye and sorghum. InsP6 and its lower phosphorylated forms (InsP15) are important biologically active component of plant origin in human nutrition. Recent interest in inositol phosphates are due to their antioxidant functions as a result of their ability to chelate divalent cations. InsP6 inhibits hydroxyl radical (OH) production by chelating iron in the presence of oxygen (O2) or superoxide anion radicals O 2 or any reducing agent; it can also maintain the redox potential of iron by accelerating both the reduction of Fe3+ by ascorbic acid (AH2) and oxidation of Fe2+ by O2. Therefore, InsP6 could reduce the active oxygen species-mediated cell injury via its antioxidant function (Frontela et al., 2008). On the grounds of obtained results, it can be suggested that cereal whole grains are a good source of inositol hexaphosphate.

4. Conclusions Grains contain several compounds that could be benecial to health in addition to their nutritional benets. Among these compounds, arabinoxylans are a signicant source of bre, while phenolic compounds provide a strong antioxidant activity. Moreover, upon germination the concentrations of these antioxidants increase. This research showed that it may be possible to develop innovative, low cost process for increasing and diversifying utilisation of common grain varieties. These modied (germinated) grains, with increased levels of bioactive compounds, may have capacity to serve as preventative strategies in combating burning health issues among the obese and diabetics, as well as the potential to reduce the risk of colon cancer. Acknowledgements This research was funded by Victoria University out of cycle research grant and Sanitarium Development and Innovation. The authors thank Sanitarium, Cooranbong, NSW, Australia for providing the miscellaneous grain samples. The authors also acknowledge the contribution of Mr. Mamilla Ravikumar and Mrs. Kristina Nelson as the research assistants for this project. References
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