Article Cite This: J. Agric. Food Chem. 2018, 66, 1033−1038
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Novel Method to Quantify β‑Glucan in Processed Foods: Sodium Hypochlorite Extracting and Enzymatic Digesting (SEED) Assay Masahiro Ide,*,†,‡ Masato Okumura,† Keiko Koizumi,† Momochika Kumagai,† Izumi Yoshida,† Mikihiko Yoshida,† Takashi Mishima,† and Munetomo Nakamura† †
Japan Food Research Laboratories, Osaka 567-0085, Japan Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan
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J. Agric. Food Chem. 2018.66:1033-1038. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/25/19. For personal use only.
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ABSTRACT: Some β-glucans have attracted attention due to their functionality as an immunostimulant and have been used in processed foods. However, accurately measuring the β-glucan content of processed foods using existing methods is difficult. We demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay, in which β-glucan is extracted using sodium hypochlorite, dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into β-glucan fragments using Westase which is an enzyme having β-1,6- and β-1,3 glucanase activity. The β-glucan fragments are further digested into glucose using exo-1,3-β-D-glucanase and β-glucosidase. We measured β-glucan comprising β-1,3-, -1,6-, and -1,(3),4bonds in various polysaccharide reagents and processed foods using our novel method. The SEED assay was able to quantify βglucan with good reproducibility, and the recovery rate was >90% for food containing β-glucan. Therefore, the SEED assay is capable of accurately measuring the β-glucan content of processed foods. KEYWORDS: β-glucan, sodium hypochlorite, Westase, exo-1,3-β-glucanase, β-glucosidase, processed food
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processed foods because it is not β-glucan specific. However, there are several methods involving converting the β-glucan in a sample to glucose by enzymatic digestion.12−14 Compared with the above two methods, the enzymatic method has high selectivity owing to its utilization of β-glucan-specific enzymes, and therefore, it is possible for it to be applied to processed foods. However, previously reported methods only work for a limited range of materials. For example, the AOAC 995.16 method is limited to β-glucan in barley and oats, and the type of enzyme is also limited to β-1,(3),4-glucan.12 In 2010, the glucan enzymatic method (GEM) was reported as a very useful method intended for measuring yeast-derived β-glucan.13 In this method, cellulose is excluded from the measurement target, and the hydrolysis of yeast-derived β-1,3-glucan is efficiently performed using yeast cell-wall lytic enzymes, exo-1,3-β-Dglucanase and β-glucosidase.15 However, these methods are not originally intended for application to processed foods. In addition, these methods are not suitable for physically hard materials such as Ganoderma lucidum, easily swelling materials, and complicated matrices.16−18 It is difficult to hydrolyze all the β-glucan contained in these samples to glucose by methods that only use enzymes. Additionally, glucose is measured after enzymatic degradation in this method, and therefore, it is necessary to remove any glucose originally present in the sample. There are no existing methods that can comprehensively quantify multiple types of noncellulosic β-glucan in various
INTRODUCTION β-glucans are homopolysaccharides composed of β-glycosidic bonds of only glucose. β-glucans having a specific structure are of great interest in the fields of nutritional science, pharmaceutical science, and medicine due to their activity as an immunostimulant.1 Composed only of glucose, β-glucans exhibit diversity in structure and functionality due to differences in the coupling positions or branches in the glucose chains, and are found in various species such as yeast, fungi, lichens, algae, and cereals. For example, lentinan is a Lentinula edodes-derived β-glucan comprising β-1,3−1,6-glycosidic bonds for which antitumor effects have been reported.2,3 In addition, Curdlan derived from microorganisms and paramylon derived from Euglena gracillis comprise β-1,3-glycosidic bonds, and their immunological activity has been reported.4,5 Most of these physiological activities were reported for noncellulosic βglucans.6−8 In recent years, with growing interest in health foods, noncellulosic β-glucan is used not only as a food additive, but is also frequently added to materials to impart functionality to processed foods. Therefore, foods on the market need adequate quality control, including accurate content management. So far, several methods to measure β-glucan content have been reported, such as quantitative nuclear magnetic resonance (NMR) spectroscopy, colorimetric assay, and enzymatic assay. The quantitative NMR method is used to estimate the structure and content from the 1H NMR signal of C1 of the glucose chain.9,10 However, this method is only suitable for purified βglucan products, and it is difficult to quantify the sample when present in complicated matrices. The colorimetric assay can quantify β-glucan with triple-helix structures by reaction with Congo red.11 However, this method is also unsuitable for © 2018 American Chemical Society
Received: Revised: Accepted: Published: 1033
November 1, 2017 December 29, 2017 January 2, 2018 January 2, 2018 DOI: 10.1021/acs.jafc.7b05044 J. Agric. Food Chem. 2018, 66, 1033−1038
Article
Journal of Agricultural and Food Chemistry processed foods. Therefore, we have developed a novel method suitable for this purpose. We adopted the enzymatic digestion method that is the most specific. To achieve our goal, it is necessary to complete three tasks to measure noncellulosic βglucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in processed foods. The first task is to find an approach to extract β-glucan in hard samples, the second task is to exclude interfering substances that affect the measurements, and the last task is to make sure that all β-glucan converted into the measuring target is digested to glucose. On the basis of these considerations, the following process was devised. First, we attempted to remove starches and small saccharides such as free glucose in the sample using pancreatin enzyme and ethanol, as in the analysis method for dietary fiber.19,20 Conveniently, the lipase and protease contained in pancreatin can decompose not only fragments of starch due to amylase but also some of the fats and proteins in the sample. Subsequently, to reduce the strength of the sample, a weak sodium hypochlorite solution was added to the precipitate.21,22 βglucan was then extracted using dimethyl sulfoxide (DMSO) and 5 mol/L sodium hydroxide.23 The treatment with 5 mol/L sodium hydroxide needs to be done quickly to avoid nonspecific degradation of the polysaccharides. After such extraction processes, the samples were digested by Westase, which has β-1,3-glucanase activity and β-1,6-glucanase activity.24 Finally, fragmented β-glucan was decomposed into glucose by using exo-β-1,3-glucanase and β-glucosidase. A glucose oxidase/peroxidase (GOPOD) test was performed on the enzyme-treated solution. Our method involved making a hard sample brittle, extracting polysaccharides while excluding small sugar molecules, and enhancing the resolution of β-1,6-glycosidic bond chains. We named this method the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay. We measured noncellulosic β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in polysaccharides, fungi, cereals, and processed foods using this novel method. Moreover, the effectiveness of this method was verified through comparison with the existing targeted enzyme methods such as the GEM assay and the AOAC 995.16 method.
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Figure 1. Schematic of the SEED assay.
accurately weighed into 50 mL polypropylene conical tubes. One mL of 0.1 mol/L PBS pH 7.0 was added into each tube, and it was then heated in a boiling water bath for 10 min. After cooling to room temperature, 1 mL of 0.1 g/mL pancreatin solution was added to the tubes. The weighed pancreatin from porcine pancreas was suspended in 0.1 mol/L PBS pH7.0, and the supernatant obtained by centrifugation at 3500g for 10 min was used as the pancreatin solution. The tubes were incubated in a shaking water bath at 37 °C for 16 h with 200 shaking strokes/min for solubilization and fragmentation of the starch into small sugars. After incubation, 4.0 mL of sodium hypochlorite and 4.0 mL of 0.1 mol/L aqueous sodium hydroxide were added into the tubes, which were stirred vigorously on a vortex mixer and sonicated for 2 min. After keeping the tubes at 4 °C for 90 min, 40 mL of ethanol were added into the tubes, which were mixed vigorously on a vortex mixer and allowed to stand at 4 °C for 2 h. Then the tubes were centrifuged at 3500g for 10 min, and the supernatants were removed using an aspirator. Five mL of DMSO were added to the precipitates, followed by incubation in a boiling water bath for 2 min and then sonication for approximately 1 min; this process was repeated three times. Following this process, 25 mL of ethanol was added into the tubes, which was then mixed vigorously on a vortex mixer and allowed to stand at 4 °C for at least an hour. The tubes were then centrifuged at 3500g for 10 min, and the supernatants were removed using an aspirator. The precipitates were solubilized with 5.0 mL of 5 mol/L sodium hydroxide. After dissolution, 25 mL of ethanol was added into the tubes followed by vigorous mixing on a vortex mixer. The tubes were then immediately centrifuged at 3500g for 10 min, and the supernatants were removed using an aspirator. Into the tubes were added 600 μL of 1.2 mol/L aqueous sodium acetate buffer (pH 3.8), 400 μL of 1 mol/L aqueous hydrochloric acid, and 4.0 mL of Milli-Q water. These tubes, after adding 30 mL of ethanol again, were centrifuged to remove the supernatant and were dried at 60 °C in an incubator. 500 μL of 1 mol/L aqueous sodium hydroxide and 1 mL of 1.2 mol/L aqueous sodium acetate buffer (pH 3.8) were added to the dried precipitates, which were then dissolved using a vortex mixer. To the sample solutions was added 2.5 mL of 1% (w/v) Westase solution prepared with 0.1 mol/L sodium acetate, and the tubes were incubated in a shaking water bath at 37 °C for 24 h with 200 shaking strokes/min. Then, 600 μL of 1 mol/L aqueous hydrochloric acid and 20 units exo-1,3-β-glucanase/4 units β-glucosidase were added to the sample solutions, and the tubes were incubated in a shaking water bath at 40 °C for 24 h with 200 shaking strokes/min. Glucose in the enzyme digestion solution was measured by a GOPOD test and converted to β-glucan. Laminarin and Barley β-Glucan Recovery Test. A laminarin and a barley β-glucan were used as samples. Samples were weighed at 1, 2, 5, 10, 20, 40, 100, and 200 mg, β glucan values were measured using SEED Assay, and the sampled amount and the β glucan content were plotted.
MATERIALS AND METHODS
Materials. Sodium hypochlorite, 0.1 mol/L phosphate buffer saline (PBS) pH 7.0, sodium hydroxide, potassium hydroxide, acetic acid, hydrochloric acid, sulfuric acid, glucose, cellulose, wheat starch, and Curdlan were purchased from Wako Pure Chemical Ind. (Osaka, Japan). 300 Units exo-1,3-β-glucanase/60 Units β-glucosidase mixture, carob galactomannan (low viscosity), konjac glucomannan (high viscosity), glucose oxidase/peroxidase (GOPOD) reagent, GOPOD reagent buffer concentrate, and β-glucan assay kit (mixed linkage) were purchased from Megazyme International Ireland, Ltd. (Wicklow, Ireland). Westase was purchased from Takara Bio Inc. (Shiga, Japan). Pancreatin from porcine pancreas, barley β-glucan, and Lyticase were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Pinefiber as indigestible dextrin, sweet potato fries, and Ganoderma lucidum were purchased from commercial sources. Laminarin and DMSO were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Ethanol (99.5%) was purchased from Kishida Chemical (Osaka, Japan). In addition, 5% (w/w) Laminarin was added to commercially available sweet potato fries, which was used as a processed food sample with known β-glucan concentration. The polysaccharide product used for the test was sufficiently dried and used. SEED Assay. Figure 1 shows a schematic diagram of the SEED assay. Before beginning the assay, the sample was homogenized as much as possible using a blender. 20−200 mg of samples were 1034
DOI: 10.1021/acs.jafc.7b05044 J. Agric. Food Chem. 2018, 66, 1033−1038
Journal of Agricultural and Food Chemistry
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Glucan Enzymatic Method (GEM) Assay. The GEM assay was performed in accordance with the previously reported method.9 Briefly, to the samples were added 1.6 mL of 1.2 mol/L aqueous sodium acetate (pH 3.8) and 600 μL of 10 KU/mL lyticase solution prepared by 0.01 mol/L Tris, 0.001 mol/L EDTA, and 0.02 mol/L sodium chloride after being dissolved in 400 μL of 2 mol/L aqueous potassium hydroxide for 20 min on ice. After incubation at 50 °C for 18 h, 130 μL of solutions were collected from each sample, and 650 μL of 12 U/mL exo-1,3-β-D-glucanase/2.4 U/mL β-glucosidase mixture prepared by 0.2 mol/L aqueous sodium acetate (pH 5.0) were added to the collected solutions. These solutions were incubated in a water bath at 40 °C for 60 min. Then the tubes were centrifuged, and their glucose contents were measured using the GOPOD test kit. AOAC 995.16 Method. This method for quantifying β-glucan in barley and oats was performed in accordance with the previously reported method.12 Briefly, samples of 100 mg were accurately weighed into glass test tubes directly. 0.2 mL of 50% (v/v) ethanol was added into the tubes, which were stirred on a vortex mixer until the samples were wet. Four mL of 0.02 mol/L sodium phosphate buffer were then added into the tubes and the contents in the tubes were mixed vigorously on a vortex mixer to disperse the samples. The tubes were immediately placed in a boiling water bath for 1 min and mixed vigorously on a vortex mixer. The tubes were then returned to a boiling water bath for an additional 2 min and mixed vigorously on a vortex mixer. After that, the tubes were placed in a water bath at 50 °C for 5 min. 0.2 mL of lichenase solution was added into the tubes, where were then mixed on a vortex mixer and incubated at 50 °C for 60 min. The contents in the tubes were vigorously stirred on a vortex mixer four times during incubation. 5.0 mL of 0.2 mol/L aqueous sodium acetate buffer (pH 4.0) was added into the tubes and mixed on a vortex mixer. The tubes were equilibrated to room temperature and then centrifuged at 1000g for 10 min. 0.1 mL of each supernatant was accurately transferred to the bottom of each of three new test tubes. Only two tubes were treated with 0.1 mL of β-glucosidase solution prepared with 0.05 mol/L aqueous sodium acetate buffer (pH 4.0). As a blank sample, to the nontreated tube was added 0.05 mol/L aqueous sodium acetate buffer (pH 4.0). All test tubes were incubated in a water bath at 50 °C for 10 min. After incubation, glucose was measured by a GOPOD test and converted to β-glucan. Acid Hydrolysis Assay. Five mL of 72% sulfuric acid were added to 100 mg of polysaccharide materials, which were then incubated at 20 °C for 3 h. After that, 65 mL of Milli-Q water were added to the samples, which were then treated in a hot water bath at 90 °C for 2 h. The sample solutions were neutralized with 10% NaOH and then diluted to 250 mL in a volumetric flask using Milli-Q water. All of the polysaccharide samples and oat flour were subjected to acid hydrolysis, and glucose was measured using a GOPOD test. GOPOD Test. The GOPOD test was performed according to the manufacturer’s protocol. Briefly, 50 mL of GOPOD reagent buffer concentrate were diluted to 1 L with Milli-Q water. Then, the entire contents of a vial containing a freeze-dried glucose oxidase/peroxidase mixture were added to 1 L of GOPOD reagent buffer. 160 μL of GOPOD reagent were added to 40 μL of the sample solution on a 96well plate and incubated at 37 °C for 20 min. The absorbance of the solution at 510 nm was measured using a ultraviolet visible light spectrophotometer (SpectraMax M2e microplate reader, Molecular devices, Sunnyvale, CA). The value obtained by multiplying the result for the glucose by 0.9 to remove the molecular weight of water was taken as the result of β-glucan in all samples. HPLC. The sample concentrations in the enzyme digested solutions of pustulan in each SEED assay and GEM assay were equally diluted and used as HPLC samples. HPLC was carried out on an amino column with a pulsed amperometric detector. Statistical Analysis. All results were expressed as mean value. Statistical analysis was performed using GraphPad Prism 5 from GraphPad Software, Inc. (La Jolla, CA). In comparing the results between groups, unpaired two-tailed t test was performed. A p-value
