A Simple Method for Simultaneous Quantification of Total

Article pubs.acs.org/JAFC

A Simple Method for Simultaneous Quantification of Total Arabinoxylans and Fructans in Wheat Flour Zhiqian Liu† and Simone Rochfort*,†,‡ †

Department of Primary Industries, Biosciences Research Division, Bundoora, Victoria 3083, Australia La Trobe University, Bundoora, Victoria 3086, Australia

‡

S Supporting Information *

ABSTRACT: Current methods for measuring fructan and arabinoxylan concentrations in wheat flour are time-consuming, and each type of the polymers requires a separate method. Here, we report the development of a new method that allows simultaneous determination of arabinoxylan and fructan contents in wheat flour. The new method is based on a single hydrolysis procedure for both arabinoxylans and fructans and an optimized separation technique for all monomers released. Owing to the use of milder hydrolysis conditions that afforded higher recovery of pentoses, the level of arabinoxylans in wheat flour determined by this new method is slightly higher than that measured with the widely used hydrolysis protocol. On the basis of the finding that, for a given flour sample, the total fructose concentration after hydrolysis is highly correlated with its total fructan concentration, the fructan content of a wheat flour sample can thus be estimated directly by the total fructose content. By simplifying and combining the two separate methods used for arabonoxylan and fructan analysis, this new method enables the quantification of arabinoxylans and fructans in wheat flour using a single acid hydrolysis step and a single high-performance liquid chromatography run. KEYWORDS: arabinoxylans, fructans, quantification, HPLC, wheat flour

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quantification of fructans in plant tissues,14−16 they may not be suitable for wheat flour samples that contain only a small amount of fructans.3 Recently, specific methods for determination of fructan content in wheat flour have been developed.17,18 These methods are accurate but complex and time-consuming because they involve two steps in sample treatment (extraction of fructans from flour samples using hot water followed by acid or enzymatic hydrolysis of fructans into hexoses), and two high-performance liquid chromatography (HPLC) runs are required to determine the level of total fructans.17,18 Therefore, the current methods are low throughput and not suited for processing a large number of samples. Different methods can also be found in the literature for determination of arabinoxylan content in wheat flour.19−23 As colorimetric methods cannot provide information on pentose composition and gas chromatography based methods require an additional derivatization step following hydrolysis,24,25 HPAEC is increasingly being used for arabinoxylan quantification. Similarly, the amount of arabinoxylans can be determined by quantifying the level of arabinose and xylose released after hydrolysis of the pentose polymers. A detailed method comprising optimization of the acid hydrolysis condition and HPAEC separation of released monomers was reported by Houben et al.,22 and the hydrolysis protocol has been widely used since.13,26 However, a recovery of about 85% for arabinose and about 75% for xylose was observed under these hydrolysis

INTRODUCTION Fructans are linear or branched polymers of fructose molecules, with or without a single internal or terminal glucose molecule. Fructans are present in approximately 15% of the angiosperm flora.1 Arabinoxylans, consisting of a linear xylan backbone to which mostly single arabinose units are linked as side residues, are one of the major polysaccharides in all major cereal cell walls.2 In white wheat flour, fructan content is below 3%,3 whereas arabinoxylan content ranges from 1.5% to 2.5%.4 Despite being a minor component of wheat flour, fructans and arabinoxylans are soluble dietary fiber and are claimed to have beneficial effects on human health by selectively promoting certain bacterial species of the gut flora (prebiotic effect).5−7 For this reason, fructans are increasingly being used as functional additives to foods.8 In addition to conferring health benefits when included in human diets,9 arabinoxylans play an important role in the rheological properties of dough and bread-making quality of wheat flours.10,11 Since wheat is a staple food crop, increasing its fructan and arabinoxylan levels could increase the intake of these nonstarch carbohydrates for a large number of people. As significant genotypic variation has been reported for fructan and arabinoxylan concentrations in wheat grain,3,12 developing higher fructan and arabinoxylan wheat cultivars can be envisaged.3,13 Therefore, a highthroughput yet reliable method for evaluating fructan and arabinoxylan concentrations in wheat flour samples has the potential to facilitate both genetic mapping of loci responsible for controlling fructan and arabinoxylan accumulation in grain and rapid and accurate screening for the identification of elite or improved germplasm. Although various high-performance anion-exchange chromatography (HPAEC) based methods have been reported for © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8319

March 13, 2014 July 15, 2014 July 22, 2014 July 22, 2014 dx.doi.org/10.1021/jf501198k | J. Agric. Food Chem. 2014, 62, 8319−8324

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Table 1. Fructan Content of 12 Flour Samples Determined by the 2-Step Method sample

free sucrosea (%)

total glucoseb (%)

total fructosec (%)

glucose and fructose ex sucrosed (%)

fructose ex fructane (%)

glucose ex fructanf (%)

DPg

fructanh (%)

1 2 3 4 5 6 7 8 9 10 11 12

0.32 0.33 0.41 0.40 0.29 0.44 0.32 0.42 0.36 0.37 0.33 0.31

0.52 0.53 0.47 0.57 0.46 0.65 0.46 0.55 0.34 0.38 0.35 0.37

1.75 1.63 1.20 1.63 1.33 2.28 1.25 1.45 0.80 0.96 1.04 0.98

0.17 0.17 0.22 0.21 0.15 0.23 0.17 0.22 0.19 0.19 0.17 0.16

1.58 1.46 0.98 1.42 1.18 2.05 1.08 1.23 0.61 0.77 0.87 0.82

0.36 0.36 0.25 0.36 0.30 0.42 0.29 0.33 0.15 0.18 0.18 0.20

5.5 5.1 4.9 4.9 4.9 5.9 4.8 4.7 5.0 5.2 5.8 5.0

1.78 1.67 1.14 1.64 1.36 2.27 1.26 1.44 0.70 0.88 0.96 0.94

a

Obtained from analyzing the aqueous extract. bObtained from analyzing the hydrolysate of the extract. cObtained from analyzing the hydrolysate of the extract. dCalculated from a(d = 0.526 × a). eCalculated from c and d (e = c − d). fCalculated from b and d ( f = b − d). gCalculated from e and f (g = (e/f) + 1). hCalculated from e, f, and g (h = K(e + f), where K = [180 + 162(g − 1)]/180g).

conditions (i.e., 90 min in 2 M HCl at 100 °C),22 which could lead to underestimation of arabinoxylan content. Although separate methods are available for the determination of fructans and arabinoxylans in wheat flour, to our knowledge, no attempt has been made to develop a comprehensive method that enables simultaneous quantification of both fructans and arabinoxylans in wheat flour. Here, we report on the development and validation of a new method for the quantitative analysis of fructans and arabinoxylans in wheat flour based on a direct acid hydrolysis of both types of polymers and a single HPLC run to quantify all monomers released.

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was then calculated as described by Liu et al.16 For the second method, the hydrolysis was performed by adding 1 mL of HCl of various concentrations into 2 mL screw-cap tubes containing 10 mg of a flour sample, followed by incubating the tubes in heating blocks (90 °C) for 30−120 min. The amount of fructose released from the flour sample was measured by HPAEC. HPAEC Analysis. Carbohydrate analysis was performed using an ICS-5000 HPLC system (Dionex) equipped with a CarboPac PA100 column (4 × 250 mm) and an electrochemical detector working in pulsed amperometric mode using a gold working electrode and a combined pH−Ag/AgCl reference electrode. A standard quadruplepotential waveform for carbohydrates was used for all analyses (Supporting Information, Table S-1). Two elution programs were used in this work (Supporting Information, Tables S-2 and S-3): one for the quantification of arabinose, xylose, and fructose after hydrolysis, and the other for measuring free glucose, fructose, sucrose, and raffinose content before hydrolysis.

MATERIALS AND METHODS

Wheat Flour. Wet-milled white flour samples (150 in total) were prepared from different bread wheat cultivars and/or locations throughout Australia. Chemicals. Standards of glucose, fructose, sucrose, arabinose, xylose, and raffinose were purchased from Sigma (USA). Sodium hydroxide (50% solution) and sodium acetate anhydrous used in the mobile phase were obtained from Fluka (Germany). Hydrolysis of Arabinoxylans. Arabinoxylan content in wheat flour was determined by measuring the amount of arabinose and xylose released after hydrolysis of arabinoxylans. The method of direct acid hydrolysis of wheat flour was adopted in this work. Briefly, 10 mg of flour samples was accurately weighed out into 2 mL screw-cap tubes, and 1 mL of HCl of an appropriate concentration (as detailed below) was added to each tube. The hydrolysis was accomplished by incubating the tubes in heating blocks (90 °C) for 60 min. Following hydrolysis, the sample was neutralized by adding NaOH, and the amount of arabinose and xylose released was determined by HPAEC. The amount of arabinoxylans in a sample is calculated as the sum of arabinose and xylose, multiplied by 0.88 to correct for water uptake during hydrolysis.22 Extraction and Hydrolysis of Fructans. Fructan content in wheat flour was determined by measuring the amount of fructose and glucose released after hydrolysis of the fructose polymers. Two hydrolysis methods were used in this work: (1) extraction of fructans from wheat flour by hot water, followed by acid hydrolysis of the extracted fructans; (2) direct acid hydrolysis of fructans from wheat flour. The first method was adapted from that of Verspreet at al.18 and is referred to as the 2-step method in this paper. Briefly, the extraction of fructans by hot water was performed in heating blocks (90 °C) for 60 min (step 1), followed by hydrolysis of an aliquot of the extract with 30 mM HCl in heating blocks (70 °C) for 90 min (step 2). The amount of free glucose, fructose, sucrose, and raffinose present in the aqueous extract before hydrolysis as well as that of total glucose and fructose after hydrolysis was determined by HPAEC. Fructan content

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RESULTS Correlation of the Total Fructose Content (after Hydrolysis) and the Fructan Content. Rhamnose was used as an internal standard in our preliminary analysis, and comparable results were obtained with and without the internal standard. So, no internal standards were used in subsequent experiments. By using the 2-step method, the fructan content of a flour sample can be calculated based on the amount of glucose and fructose liberated from fructan molecules. To deduce the portion of fructose and glucose derived solely from fructans, two separate analyses are needed to quantify, respectively, total fructose and glucose after hydrolysis and free glucose, fructose, sucrose, and raffinose before hydrolysis, as both sucrose and raffinose also contain glucose and fructose units. The analysis of the aqueous extract of 54 random flour samples indicates that free glucose, fructose, and raffinose are present in very small amount in wheat flour (Supporting Information, Table S-4). Consequently, only the amount of fructose and glucose derived from sucrose needs to be subtracted from the total fructose and glucose to calculate the fructan content. This implies that the concentration of fructan in a wheat flour sample can be calculated using only three values (total glucose and total fructose content after hydrolysis and free sucrose content before hydrolysis), as the amount of glucose and fructose released from sucrose hydrolysis can be directly calculated from sucrose content. 8320

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Table 1 shows the fructan content of 12 random flour samples as calculated using the three values determined by two separate HPLC analyses. A close examination of the data in Table 1 reveals that the fructan content of the 12 samples is very close to their total fructose content after hydrolysis. The strong correlation between these two data series is clearly seen in Figure S-1 (Supporting Information). This suggests that the concentration of total fructose after hydrolysis can be potentially used to estimate the content of fructans in a flour sample. This will greatly simplify the fructan analysis procedure, as the total fructose content after hydrolysis can be determined by one single HPLC run. Simplification of the 2-Step Method in Fructan Analysis. All the current methods for fructan analysis involve two steps: a hot water extraction step and a hydrolysis step.16−18 Given the fact that, in the case of wheat flour, fructan content could be estimated directly by the total fructose content after hydrolysis, the 2-step method in fructan quantification can also be simplified into one single step, that is, direct hydrolysis of fructans from wheat flour under the same hydrolysis conditions. Indeed, the total fructose contents of 24 random flour samples determined after direct hydrolysis are very close to those measured using the 2-step method (Figure 1).

Figure 2. HPAEC separation of the four monomers (A) from a standard mix and (B) from the hydrolysate of a wheat flour sample. Peak annotations: A, arabinose; G, glucose; X, xylose; F, fructose. The hydrolysis of wheat flour was performed with 1 M HCl at 90 °C for 60 min.

Optimization of Acid Hydrolysis for Arabinoxylans and Fructans. Having demonstrated that fructan and arabinoxylan monomers can be quantified by a single HPLC analysis, a unified acid hydrolysis protocol for both types of polymers is needed in order to develop a higher throughput method. The yield of arabinose, xylose, and fructose was compared when one random flour sample was subjected to various hydrolysis conditions (by varying HCl concentrations, incubation time, and temperatures). Figure 3 shows the yield of arabinose, xylose, and fructose as a function of HCl concentration and incubation time at a fixed incubation temperature (90 °C). Clearly, the hydrolysis rate of arabinoxylans and fructans, as reflected by the yield of arabinose/xylose and fructose, respectively, is influenced by both the HCl concentration and the incubation time. It is also observed that the xylan backbone of arabinoxylans is more resistant to acid hydrolysis than the arabinose branches, whereas fructans can be depolymerised readily. Lowering the incubation temperature to 70 or 80 °C significantly prolongs the time required for complete hydrolysis of arabinoxylans (results not shown). A combination of HCl concentration of 1 M, incubation time of 60 min, and incubation temperature of 90 °C appears to be the best compromise for the hydrolysis of both types of carbohydrate polymers in wheat flour. This unified hydrolysis protocol and the one-step HPAEC analysis of all monomers form our new method for simultaneous quantification of fructans and arabinoxylans in wheat flour.

Figure 1. Correlation of the total fructose content of 24 wheat flour samples, as determined by the direct hydrolysis method and by the 2step method. The direct hydrolysis was performed by incubating flour samples with 30 mM HCl at 70 °C for 90 min.

Optimization of HPLC Separation for All Fructan and Arabinoxylan Monomers. In order to develop a method allowing simultaneous quantification of fructans and arabinoxylans in wheat flour, chromatographic separation of all monomers (i.e., arabinose, xylose, glucose, and fructose) contained in these two types of carbohydrate polymers is required. A systematic evaluation of the performance of various columns and mobile phase concentrations allowed us to achieve baseline resolution of the four monomer in a standard mix as well as in a wheat flour hydrolysate, in which a large amount of glucose (derived from starch) is present (Figure 2). The optimum chromatographic conditions consist of a Carbopac PA100 ion-exchange column (Dionex) and a mobile phase containing 40 mM NaOH. 8321

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contrast, a slight degradation (or transformation) was observed for fructose (92% recovery after 60 min) (Figure 4). Comparison between the New Method and the Standard Method for Arabinoxylan Quantification. Ten randomly selected flour samples were used to compare our new method with the standard hydrolysis method in quantitative analysis of arabinoxylans. Overall, our new method produced similar results compared to the currently widely used direct hydrolysis method. However, owing to the improved recovery of arabinose and xylose, a slightly higher arabinxylan content was obtained with our new method for nearly all samples (Table 2). Table 2. Comparison of Arabinoxylan Content of 10 Flour Samples, as Determined by the New Method and by the Standard Method mean contenta (%) ± SD sample A B C D E F G H I J a

Figure 3. Yield of monomers as a function of HCl concentration and incubation time. All hydrolyses were performed at 90 °C, and all data points are the means of three replicates.

new method 2.33 2.13 2.22 2.46 2.36 2.81 2.74 2.56 1.94 2.08

± ± ± ± ± ± ± ± ± ±

0.06 0.04 0.04 0.02 0 0.02 0.01 0.03 0.02 0.05

standard method 2.24 1.99 2.15 2.40 2.20 2.68 2.59 2.42 1.77 1.96

± ± ± ± ± ± ± ± ± ±

0.07 0.08 0.04 0.04 0.10 0.07 0.10 0.08 0.10 0.05

Mean and SD values are calculated from two replicates.

Comparison between the New Method and the 2Step Method in Fructan Quantification. To confirm the reliability of the new method in fructan quantification, a further set of 24 randomly selected samples was analyzed in parallel by the new method and the standard 2-step method. Fructan contents determined by the new method were highly correlated with those determined by the standard 2-step method (Figure 5). However, the overall fructan content measured by our new method is about 95% that determined by the 2-step method. As a result, a correction factor of 1.05 should be applied when using this new method for fructan quantification in wheat flour.

Stability of Arabinose, Xylose, and Fructose under Acidic Conditions. Having found the unified hydrolysis conditions for the two types of polymers, the stability of the monomers under these hydrolysis conditions needs to be assessed, as the content of the polymers is calculated based on the concentrations of the monomers. The stability of arabinose, xylose, and fructose was assessed by measuring the recovery of standard compounds (applied at 0.01%) after incubation in 1 M HCl at 90 °C for 30−90 min. Figure 4 shows that both arabinose and xylose are stable under the hydrolysis conditions, with a recovery rate close to 100% even after 90 min. By

Figure 4. Recovery (%) of arabinose, xylose, and fructose after incubation in 1 M HCl at 90 °C. All values in this figure are the means of two replicates.

Figure 5. Correlation of fructan content of 24 random flour samples, as determined by the new method and by the standard 2-step method. 8322

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breakdown during acid hydrolysis, this method allowed accurate estimation of wheat flour fructans. However, this method involved two steps, that is, hot water extraction and acid hydrolysis, and is thus low throughput. We have found that, in the case of wheat flour, the total fructose content after hydrolysis can be used directly to estimate the content of fructans. This is an important finding for simplifying the fructan analysis procedure. The possibility of using direct hydrolysis of flour to measure the total fructose content is a key step toward a unified hydrolysis protocol for both arabinoxylans and fructans. However, the easy breakdown of fructan molecules and the relative instability of fructose under acidic conditions imply that mild conditions would be preferred for fructan hydrolysis, whereas the backbone of arabinoxylans is much harder to break. Considerable effort was made to find a three-way combination (HCl concentration, incubation time, and temperature) of hydrolysis conditions that ensure complete conversion of arabinoxylans (into arabinose and xylose) and at the same time minimum degradation of fructose liberated from fructans. Under the optimized hydrolysis conditions, the content of arabinoxylans measured is slightly higher compared to the widely used hydrolysis protocol, and the content of fructan is about 95% that measured by the 2-step method. A correction factor of 1.05 is thus needed when using this method for fructan quantification in wheat flour. The reproducibility of the new method in the quantification of both arabinoxylans and fructans was finally confirmed by replicate analyses of the same sample. In conclusion, a combined method for arabinoxylan and fructan quantification in wheat flour has been developed. This method, requiring one single sample preparation step and one single HPLC run, is simple and reliable and thus particularly suited for screening a large number of samples. Although it has been validated in wheat flour, this method should be applicable to other cereal species.

Indeed, a recovery of about 94% and 99% before and after correction, respectively, was obtained with this new method when wheat flour was spiked with commercial fructan at two different concentrations (1% and 2% fructan of the flour wheat). Reproducibility of the New Method in Arabinoxylan and Fructan Quantification. The reproducibility of the method in the quantification of fructans and arabinoxylans in wheat flour was assessed by the variation of measurement results between technical replicates taken from one single flour sample (Table 3). The content of both arabinoxylans and Table 3. Reproducibility of the Method in Arabinoxylan and Fructan Analysisa carbohydrate

rep

content (%)

mean

SD

RSD (%)

arabinoxylans

1 2 3 4 1 2 3 4

2.12 2.16 2.16 2.07 1.97 1.98 1.94 1.97

2.13

0.04

1.88

1.97

0.02

1.02

fructans

a

Arabinoxylan content is calculated as the sum of arabinose and xylose multiplied by 0.88 to correct for water uptake. Fructan content is calculated using the total fructose content (after direct hydrolysis) multiplied by 1.05. SD, standard deviation. RSD, relative standard deviation.

fructans shows very small variation between replicates, demonstrating the excellent repeatability of the method in fructan and arabinoxylan quantification.

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DISCUSSION Quantitative analysis of arabinoxylans and fructans in grains is an important step in the understanding of the genetic control mechanisms for the accumulation of these carbohydrates and in research programs aiming to develop high arabinoxylan and/or fructan germplasm. Currently, separate methods are available for the determination of total arabinoxylan and fructan contents in wheat flour. A systematic method development for arabinoxylan analysis was reported by Houben et al.,22 in which a direct hydrolysis of the flour sample was adopted, resulting in significant simplification of the sample pretreatment procedure. However, an extra step was needed in this method to convert glucose into gluconic acid to prevent its interference in xylose quantification. Nguyen et al.13 further simplified this method by a direct quantification of xylose and arabinose in the hydrolysate. We also found it unnecessary to convert or remove glucose (derived from starch) after hydrolysis, as the use of a CarboPac PA100 column together with an optimized mobile phase allowed complete separation of the four monomeric sugars. In addition, by fine-tuning the parameters used in hydrolysis (reducing the HCl concentration from 2 to 1 M and decreasing the incubation temperature from 100 to 90 °C), we have improved slightly the recovery of the pentoses while achieving complete breakdown of arabinoxylans. In the case of fructan analysis in wheat flour, an excellent method was published by Verspreet et al.,18 in which a mild acid hydrolysis (60 mM) was used to breakdown fructan polymers with no degradation of starch. By avoiding starch

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ASSOCIATED CONTENT

S Supporting Information *

Quadruple potential waveform used in HPAEC-PAD analysis of carbohydrates (Table S-1); gradient elution program used for the quantification of arabinose, xylose, and fructose after hydrolysis (Table S-2); gradient elution program used for the quantification of free glucose, fructose, sucrose, and raffinose before hydrolysis (Table S-3); content of free sugars in wheat flour (Table S-4); correlation between the total fructose content after hydrolysis and fructan content determined by the 2-step method (Figure S-1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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