Novel Method to Quantify Î²-Glucan in Processed Foods: Sodium

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A novel method to quantify #-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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05044 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Journal of Agricultural and Food Chemistry

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A novel method to quantify β-glucan in processed foods:

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Sodium hypochlorite Extracting and Enzymatic Digesting

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(SEED) assay

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Masahiro Ide1,2*, Masato Okumura1, Keiko Koizumi1, Momochika Kumagai1, Izumi

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Yoshida1, Mikihiko Yoshida1, Takashi Mishima1, and Munetomo Nakamura1.

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1

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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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AUTHOR INFORMATION

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Corresponding Author

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* Phone: +81(0)72 641 8958; fax: +81(0)72 641 8969; E-mail: [email protected]

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1 Environment ACS Paragon Plus


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ABSTRACT: Some of β-glucans has attracted attention due to its functionality as an

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immunostimulant and has been used in processed foods. However, accurately measuring

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the β-glucan content of processed foods using existing methods is difficult. We

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demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic

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Digesting (SEED) assay, in which β-glucan is extracted using sodium hypochlorite,

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dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into β-glucan

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fragments using Westase which is an enzyme having β-1,6- and β-1,3 glucanase activity.

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The β-glucan fragments are further digested into glucose using exo-1,3-β-D-glucanase

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and β-glucosidase. We measured β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds

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in various polysaccharide reagents and processed foods using our novel method. The

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SEED assay was able to quantify β-glucan with good reproducibility, and the recovery

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rate was >90% for food containing β-glucan. Therefore, the SEED assay is capable of

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accurately measuring the β-glucan content of processed foods.

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KEYWORDS: β-glucan; sodium hypochlorite; Westase; exo-1,3-β-glucanase; β-

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glucosidase; processed food

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INTRODUCTION

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The β-glucan is homo-polysaccharides composed of β-glycosidic bonds of only glucose.

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Some of β-glucans having a specific structure is of great interest in the fields of

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nutritional science, pharmaceutical science, and medicine due to its activity as an

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immunostimulant.1 β-glucan is a homopolysaccharide comprising only glucose. It

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exhibits diversity in its structure and functionality due to differences in coupling

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positions or branches in the glucose chains, and it is found in various species such as

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yeast, fungi, lichens, algae, and cereals. For example, lentinan is a Lentinula edodes-

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derived β-glucan comprising β-1,3-1,6-glycosidic bonds for which anti-tumor effects

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have been reported.2,3 In addition, curdlan derived from microorganisms and paramylon

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derived from Euglena gracillis comprise β-1,3-glycosidic bonds, and their

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immunological activity has been reported.4,5 Most of these physiological activities were

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reported for non-cellulosic β-glucans.6-8 In recent years, with growing interest in health

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foods, non-cellulosic β-glucan is not only used as a food additive, it is also frequently

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added to materials in order to impart functionality to processed foods. Therefore, foods

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on the market need adequate quality control, including accurate content management.

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So far, several methods to measure β-glucan content have been reported, such as

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quantitative nuclear magnetic resonance (NMR) spectroscopy, colorimetric assay, and

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enzymatic assay. The quantitative NMR method is used to estimate the structure and

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content from the 1H-NMR signal of C1 of the glucose chain.9, 10 However, this method

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is only suitable for purified β-glucan products and it is difficult to quantify the sample

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when present in complicated matrices. The colorimetric assay can quantify β-glucan

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with triple-helix structures by reaction with Congo red.11 However, this method is also



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unsuitable for processed foods because it is not β-glucan specific. On the other hand,

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there are several methods involving converting the β-glucan in a sample to glucose by

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enzymatic digestion.12-14 Compared with the above two methods, the enzymatic method

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has high selectivity owing to its utilization of β-glucan-specific enzymes, and therefore,

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it is possible for it to be applied to processed foods. However, previously reported

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methods only work for a limited range of materials. For example, the AOAC 995.16

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method is limited to β-glucan in barley and oats, and the type of enzyme is also limited

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to β-1,(3),4-glucan.12 In 2010, the glucan enzymatic method (GEM) was reported as a

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very useful method intended for measuring yeast-derived β-glucan.13 In this method,

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cellulose is excluded from the measurement target, and the hydrolysis of yeast-derived

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β-1,3-glucan is efficiently performed using yeast cell-wall lytic enzymes, exo-1,3-β-D-

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glucanase and β-glucosidase.15 However, these methods are not originally intended for

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application to processed foods. In addition, these methods are not suitable for physically

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hard materials such as Ganoderma lucidum, easily swelling materials, and complicated

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matrices.16-18 It is difficult to hydrolyze all β-glucan contained in these samples to

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glucose by methods that only use enzymes. Additionally, glucose is measured after

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enzymatic degradation in this method, and therefore, it is necessary to remove any

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glucose originally present in the sample.

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There are no existing methods that can comprehensively quantify multiple types of

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non-cellulosic β-glucan in various processed foods. Therefore, we have developed a

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novel method suitable for this purpose. We adopted the enzymatic digestion method that

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is the most specific. To achieve our goal, it is necessary to complete three tasks to

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measure non-cellulosic β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in


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processed foods. The first task is to find an approach to extract β-glucan in hard samples,

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the second task is to exclude interfering substances that affect the measurements, and

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the last task is to make sure that all β-glucan converted into the measuring target is

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digested to glucose. Based on these considerations, the following process was devised.

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First, we attempted to remove starches and small saccharides such as free glucose in

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the sample using pancreatin enzyme and ethanol, as in the analysis method for dietary

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fiber.19, 20 Conveniently, the lipase and protease contained in pancreatin can decompose

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not only fragments of starch due to amylase but also some of the fats and proteins in the

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sample. Subsequently, to reduce the strength of the sample, a weak sodium hypochlorite

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solution was added to the precipitate.21, 22 β-glucan was then extracted using dimethyl

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sulfoxide (DMSO) and 5 mol/L sodium hydroxide.23 The treatment with 5 mol/L

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sodium hydroxide needs to be done quickly to avoid non-specific degradation of the

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polysaccharides. After such extraction processes, the samples were digested by

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Westase, which has β-1,3-glucanase activity and β-1,6-glucanase activity.24 Finally,

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fragmented β-glucan was decomposed into glucose by using exo β-1,3-glucanase and β-

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glucosidase. A glucose oxidase/peroxidase (GOPOD) test was performed on the

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enzyme-treated solution.

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Our method involved making a hard sample brittle, extracting polysaccharides while

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excluding small sugar molecules, and enhancing the resolution of β-1,6-glycosidic bond

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chains. We named this method the Sodium hypochlorite Extracting and Enzymatic

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Digesting (SEED) assay. We measured non-cellulosic β-glucan comprising β-1,3-, -1,6-

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, and -1,(3),4- bonds in polysaccharides, fungi, cereals, and processed foods using this

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novel method. Moreover, the effectiveness of this method was verified through



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comparison with the existing targeted enzyme methods such as the GEM assay and the

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AOAC 995.16 method.

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MATERIALS AND METHODS

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Materials

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Sodium hypochlorite, 0.1 mol/L phosphate buffer saline (PBS) pH 7.0, sodium

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hydroxide, potassium hydroxide, acetic acid, hydrochloric acid, sulfuric acid, glucose,

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cellulose, wheat starch, and curdlan were purchased from Wako Pure Chemical Ind.

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(Osaka, Japan). 300 Units exo-1,3-β-glucanase/60 Units β-glucosidase mixture, carob

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galactomannan (low viscosity), konjac glucomannan (high viscosity), glucose oxidase/

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peroxidase (GOPOD) reagent, GOPOD reagent buffer concentrate, and β-glucan assay

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kit (mixed linkage) were purchased from Megazyme International Ireland Ltd.

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(Wicklow, Ireland). Westase was purchased from Takara Bio Inc. (Shiga, Japan).

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Pancreatin from porcine pancreas, barley β-glucan, and Lyticase were purchased from

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Sigma-Aldrich Japan (Tokyo, Japan). Pinefiber as indigestible dextrin, sweet potato

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fries, and Ganoderma lucidum were purchased from commercial sources. Laminarin and

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DMSO were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Ethanol (99.5%) was

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purchased from Kishida Chemical (Osaka, Japan). In addition, 5% (w/w) Laminarin was

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added to commercially available sweet potato fries, which was used as a processed food

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sample with known β-glucan concentration. The polysaccharide product used for the

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test was sufficiently dried and used.

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SEED assay


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Figure 1 shows a schematic diagram of the SEED assay. Before beginning the assay, the

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sample was homogenized as much as possible using a blender. 20–200 mg of samples

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were accurately weighed into 50 mL polypropylene conical tubes. 1 mL of 0.1 mol/L

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PBS pH 7.0 was added into each tube, and it was then heated in a boiling water bath for

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10 min. After cooling to room temperature, The tubes were added the 1 mL of 0.1 g/mL

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pancreatin solution. The weighed pancreatin from porcine pancreas was suspended in

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0.1 mol/L PBS pH7.0, and the supernatant obtained by centrifugation at 3500g for 10

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min was using as pancreatin solution. The tubes were incubated in a shaking water bath

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at 37°C for 16 h with 200 shaking strokes/min for solubilization and fragmentation of

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starch to small sugars. After incubation, 4.0 mL of sodium hypochlorite and 4.0 mL of

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0.1 mol/L aqueous sodium hydroxide were added into the tubes, which were stirred

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vigorously on a vortex mixer and sonicated for 2 minutes. After keeping the tubes at

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4°C for 90 min, 40 mL of ethanol were added into the tubes, which were mixed

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vigorously on a vortex mixer and allowed to stand at 4°C for 2 h. Then the tubes were

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centrifuged at 3500g for 10 min and the supernatants were removed using an aspirator.

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5 mL of DMSO were added to the precipitates, followed by incubation in a boiling

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water bath for 2 min and then sonication for approximately 1 min; this process was

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repeated three times. Following this process, 25 mL of ethanol were added into the

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tubes, which were then mixed vigorously on a vortex mixer and allowed to stand at 4°C

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for at least an hour. The tubes were then centrifuged at 3500g for 10 min and the

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supernatants were removed using an aspirator. The precipitates were solubilized with

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5.0 mL of 5 mol/L sodium hydroxide. After dissolution, 25 mL of ethanol were added

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into the tubes followed by vigorous mixing on a vortex mixer. The tubes were then



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immediately centrifuged at 3500g for 10 min and the supernatants were removed using

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an aspirator. Into the tubes were added 600 µL of 1.2 mol/L aqueous sodium acetate

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buffer (pH 3.8), 400 µL of 1 mol/L aqueous hydrochloric acid, and 4.0 mL of Milli-Q

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water. These tubes, after adding 30 mL of ethanol again, were centrifuged to remove the

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supernatant and were dried at 60°C in an incubator. 500 µL of 1 mol/L aqueous sodium

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hydroxide and 1 mL of 1.2 mol/L aqueous sodium acetate buffer (pH 3.8) were added to

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the dried precipitates, which were then dissolved using a vortex mixer. To the sample

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solutions were added 2.5 mL of 1% (w/v) Westase solution prepared with 0.1 mol/L

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sodium acetate, and the tubes were incubated in a shaking water bath at 37°C for 24 h

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with 200 shaking strokes/min. Then, 600 µL of 1 mol/L aqueous hydrochloric acid and

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20 units exo-1,3-β-glucanase/4 units β-glucosidase were added to the sample solutions,

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and the tubes were incubated in a shaking water bath at 40°C for 24 h with 200 shaking

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strokes/min. Glucose in the enzyme digestion solution was measured by a GOPOD test

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and converted to β-glucan.

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Laminarin and barley β-glucan recovery test

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A laminarin and a barley β-glucan were used as samples. Samples were weighed at 1, 2,

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5, 10, 20, 40, 100 and 200 mg, β glucan values were measured using SEED Assay, and

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the sampled amount and the β glucan content were plotted.

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Glucan Enzymatic Method (GEM) assay

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The GEM assay was performed in accordance with the previously reported method9.

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Briefly, to the samples were added 1.6 mL of 1.2 mol/L aqueous sodium acetate (pH


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3.8) and 600 µL of 10 KU/mL lyticase solution prepared by 0.01 mol/L Tris, 0.001

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mol/L EDTA, and 0.02 mol/L sodium chloride after being dissolved in 400 µL of 2

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mol/L aqueous potassium hydroxide for 20 min on ice. After incubation at 50 °C for 18

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h, 130 µL of solutions were collected from each sample, and 650 µL of 12 U/mL exo-

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1,3-β-D-glucanase/2.4 U/mL β-glucosidase mixture prepared by 0.2 mol/L aqueous

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sodium acetate (pH 5.0) were added to the collected solutions. These solutions were

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incubated in a water bath at 40 °C for 60 min. Then, the tubes were centrifuged, and

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their glucose contents were measured using the GOPOD test kit.

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AOAC 995.16 method

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This method for quantifying β-glucan in barley and oats was performed in accordance with

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the previously reported method12. Briefly, samples of 100 mg were accurately weighed into

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glass test tubes directly. 0.2 mL of 50% (v/v) ethanol was added into the tubes, which were

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stirred on a vortex mixer until the samples were wet. 4 mL of 0.02 mol/L sodium phosphate

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buffer were then added into the tubes and the contents in the tubes were mixed vigorously

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on a vortex mixer to disperse the samples. The tubes were immediately placed in a boiling

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water bath for 1 min and mixed vigorously on a vortex mixer. The tubes were then returned

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to a boiling water bath for an additional 2 min and mixed vigorously on a vortex mixer.

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After that, the tubes were placed in a water bath at 50 °C for 5 min. 0.2 mL of lichenase

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solution was added into the tubes, where were then mixed on a vortex mixer and incubated

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at 50 °C for 60 min. The contents in the tubes were vigorously stirred on a vortex mixer

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four times during incubation. 5.0 mL of 0.2 mol/L aqueous sodium acetate buffer (pH 4.0)



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was added into the tubes and mixed on a vortex mixer. The tubes were equilibrated to room

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temperature and then centrifuged at 1000 g for 10 min. 0.1 mL of each supernatant was

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accurately transferred to the bottom of each of three new test tubes. Only two tubes were

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treated with 0.1 mL of β-glucosidase solution prepared with 0.05 mol/L aqueous sodium

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acetate buffer (pH 4.0). As a blank sample, to the non-treated tube was added 0.05 mol/L

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aqueous sodium acetate buffer (pH 4.0). All test tubes were incubated in a water bath at 50

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°C for 10 min. After incubation, Glucose was measured by a GOPOD test and converted to

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β-glucan.

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Acid hydrolysis assay

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5 mL of 72% sulfuric acid were added to 100 mg of polysaccharide materials, which

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were then incubated at 20 °C for 3 h. After that, 65 mL of Milli-Q water were added to

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the samples, which were then treated in a hot water bath at 90 °C for 2 h. The sample

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solutions were neutralized with 10% NaOH and then diluted to 250 mL in a volumetric

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flask using Milli-Q water. All of the polysaccharide samples and oat flour were

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subjected to acid hydrolysis and glucose was measured using a GOPOD test.

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GOPOD test

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The GOPOD test was performed according to the manufacturer’s protocol. Briefly, 50

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mL of GOPOD reagent buffer concentrate were diluted to 1 L with Milli-Q water. Then,

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the entire contents of a vial containing a freeze-dried glucose oxidase/peroxidase

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mixture were added to 1 L of GOPOD reagent buffer. 160 µL of GOPOD reagent were

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added to 40 µL of the sample solution on a 96-well plate and incubated at 37 °C for 20


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min. The absorbance of the solution at 510 nm was measured using a ultraviolet visible

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light spectrophotometer

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Sunnyvale, CA). The value obtained by multiplying the result for the glucose by 0.9 to

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remove the molecular weight of water was taken as the result of β-glucan in all samples.

(SpectraMax M2e microplate reader, Molecular devices,

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HPLC

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The sample concentrations in the enzyme digested solutions of pustulan in each SEED

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assay and GEM assay were equally diluted and used as HPLC samples. HPLC was

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carried out on an amino column with a pulsed amperometric detector.

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Statistical analysis

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All results were expressed as mean value. Statistical analysis was performed using

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GraphPad Prism 5 from GraphPad Software, Inc. (La Jolla, CA). In comparing the

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results between groups, unpaired two-tailed t-test was performed. A p-value < 0.05 was

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considered was deemed significant.

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RESULTS AND DISCUSSION

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First, in order to ascertain how much β-glucan can be degraded by the enzyme used in

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the SEED assay, a laminarin and a barley β-glucan recovery test was conducted. The

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recovery rates were more than 90% for both laminarin and barley β-glucan (Figure 2A

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and 2B). A linear approximation for amounts of β-glucans in the range 1–200 mg

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maintained a slope corresponding to recovery rates of more than 90% for both samples.



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This result shows that the SEED assay was not affected by the difference between β-

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1,3-1,6- and β-1,(3),4- bonds.

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Subsequently, a performance test of the SEED assay was carried out by inspection for

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variability calculation by iterative analysis of the processed food and measurement at

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low concentration sample of 1% (w/v) (Table 1a). The RSD was 1.64 %, and it was

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confirmed that this method is effective even for concentration of 1% (w/v) β-glucan

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sample. Furthermore, the limits of detection and quantification were calculated from the

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result of a 1% (w/v) laminarin solution. In the 1% (w/v) laminarin solution, the standard

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deviation predicted by a Horwitz correction type is a desirable 1–2% of the intermediate

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precision, and the target standard deviation is 0.020. The limits of detection and

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quantification were calculated to be 0.08% and 0.16%, respectively. Similarly, a 0.5%

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laminarin solution was examined, and the recovery rate obtained was more than 90%

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(Supporting Information, Table 1). Thus, β-glucan could be quantified with 0.1% (w/w

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or w/v) digits using this method in the processed food. In the nutritional analysis,

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0.1%(w/w or w/v) digits are used to display common nutrients such as carbohydrates for

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processed foods. The sensitivity of SEED assay is comparable to the methods for other

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nutrients. Moreover, considering the effects of the matrix components in processed food

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samples, the actual lower limit of quantification needs to be set higher than 0.16%,

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which is a result of the cellulose present in the matrix.

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In order to check the inhibition of free sugar, laminarin at a weight ratio of 5% was

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added to glucose. Subsequent SEED assay revealed that the quantitative value was not

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affected at all (Supporting Information, Table 2). This result shows that even foods with

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large amounts of free sugar do not affect the measurements.


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To test samples containing lipids, proteins, and carbohydrates in the sample matrix,

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we selected commercially available sweet potato fries containing many saccharides such

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as cellulose, starch, and free sugar. The sweet potato fries were measured by the SEED

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assay, and the β-glucan level was less than 0.2% (Supporting Information, Table 3). In

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addition, a sample in which 5% (w/w) of the total weight was replaced with laminarin

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was used as the processed food sample for testing (Table 1b). In the SEED assay, the

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recovery rate between 95% and 100 % was obtained comparing with the added amount,

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whereas in the GEM assay, which is not applicable for processed foods, the β-glucan

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value was higher than the added amount (Supporting Information, Table 4).

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Table 2 shows the measured values of β-glucan from polysaccharides, edible

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mushrooms, and yeast-processed food samples. The structural selectivity of the SEED

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assay was investigated based on the results of polysaccharide samples. Indigestible

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dextrin gave a low value in the SEED assay. Because indigestible dextrin composing of

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α-1,4-, α-1,6- glycosidic bonds was not digested by the enzyme used in this assay, it did

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not contribute to the measured value. Additionally, the indigestible dextrin of low

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molecular weight was removed during the small sugar removal process of the method.

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Therefore, we considered that polydextrose is measuring by the SEED assay does not

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depart from the definition of β-glucan in our manuscript. In glucomannan, there was no

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significant difference between the two methods, but the results by the SEED assay

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tended to be lower comparing with the results by GEM assay. In galactomannan, there

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was a significant difference between the results of both methods. These results

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suggested that they were tended to suppress the overvaluation of β-glucan causing by

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the non-β-glucan polysaccharide such as glucomannan and galactomannan in the SEED



288

assay comparing with the GEM assay. Regarding β-glucan material, a difference in the

289

result was not found for curdlan, which is a straight chain of β-1,3-glycoside bonds.

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Similarly, the result of laminarin which main chain is β-1,3- glycosidic bonds were no

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significant difference, but the results using the SEED assay tended to be high than using

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the GEM assay. On the other hand, for pustulan containing β-1,6-glycosidic bonds, a

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high recovery rate was obtained by the SEED assay compared with the GEM assay. The

294

difference in these results is considered to be due to the difference in the enzyme

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activity; lyticase was used in the GEM assay, and Westase, which exhibits β-1,6-

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glucanase activity, was used in the SEED assay. Mushrooms such as Lentinula edodes

297

are typical foods that often contain β-1,6-glucan. Therefore, β-1,6-glucanase activity

298

was indispensable in constructing a general-purpose β-glucan assay for processed foods.

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In addition, it was investigated whether all the sugars contained in enzyme treatment

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solution of pustulan were glucose. The chromatograph of pustulan was obtained using

301

an electrochemical detector and a column for oligosaccharide analysis in the enzyme-

302

treated solution in the SEED assay, in which only the peak of glucose was confirmed,

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and the recovery rate of the collected pustulan was 96.6% (for reference, the recovery

304

rate was 97% by a GOPOD test kit). On the other hand, in the enzyme-treated solution

305

of GEM, also only the peak of glucose was confirmed, but the glucose concentration

306

was very low (Supporting Information, Figure 1). As a result, since lyticase used in

307

GEM does not have β-1,6-glucanase activity, it is possible that pustulan was not

308

fragmented and remained as a relatively long chain. The result of the yeast-processed

309

food, which is one representative example of β-glucan processed foods, also suggested a

310

difference in the enzyme activity between Westase used in the SEED assay and lyticase


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used in the GEM assay. As for the yeast cell wall having a β-1,3-1,6-glycosidic chain, it

312

is thought that the result was higher in the SEED assay due to the β-1,6-glucanase

313

activity of Westase.

314

Ganoderma lucidum was chosen as a hard sample that is difficult to extract. The

315

lyophilized Sarcomyxa serotine was used as a mushroom sample, which swells upon

316

adding a buffer. In the GEM assay, the hard sample could not be solubilized after

317

lyticase digestion, whereas in the SEED assay, the sample could be solubilized before

318

the enzymatic digestion (Supporting Information, Figure 2). The result of the SEED

319

assay was higher than that of GEM. In addition, in the GEM assay, it was observed that

320

Ganoderma lucidum and Sarcomyxa serotine were swollen by the buffer, whereas in the

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SEED assay, the extraction process worked well even if the mushroom sample was

322

swollen by the buffer.

323

In addition to lichens, fungi, and yeast, β-glucan derived from cereals was also

324

investigated (Table 3). Cellulose and wheat-derived starch are the most typical

325

polysaccharides contained in many cereals and processed foods. Although starch and

326

cellulose may be interfering substances in a conventional β-glucan assay, it was

327

observed that they caused very low measured values in the SEED assay as well as in the

328

AOAC method. The result of sweet potato fries, which contain abundant starch and

329

cellulose, also showed a similar trend (Supporting Information, Table 4).

330

Next, we examined β-glucan derived from oats and barley (Table 3). Also, we did

331

unpaired two-tailed t-test in the result of wheat starch, cellulose, oats and barley β-

332

glucan, there was no significant difference in β-glucan value in SEED assay and AOAC

333

method. The SEED assay showed no difference in the results of oat flour compared with



334

the AOAC method and thus can be considered suitable. For barley-derived β-glucan, the

335

result of the SEED assay was close to the total glucose level determined by the acid

336

hydrolysis assay compared with result of the AOAC method. There is no significant

337

difference, but the result of the AOAC method tends to be smaller than the result of the

338

SEED assay. As one possible reason, it is thought that the lichenase and β-glucosidase

339

used in the AOAC method could not completely decompose purified barley-derived

340

mixed-linkage glucan. Considering the results of β-glucan by AOAC and total glucose

341

by acid hydrolysis, the SEED assay was able to measure β-1,(3),4-glucan without

342

overvalue by other polysaccharides, cellulose and starch in the cereals.

343

This study has shown that the SEED assay is applicable to many sample types such as

344

purified β-glucans, hard samples, water-absorbing samples, and processed foods. In this

345

novel assay method, the target is glucose derived from β-1,3, β-1,6, and β-1,(3),4 bonds,

346

and compared with the previous method. On the other hand, not only indigestible

347

dextrin but also polydextrose composing of β-1,4-, β-1,6- glycosidic bonds are often

348

added to health food as dietary fiber. In the quantitation of β-glucan in processed foods

349

including polydextrose, the glucan degree of polymerization included in ethanol

350

precipitation in the SEED assay is measured and it is considered that only β-1,3- and β-

351

1,6-glycosidic bonds are digested. Therefore, this method can be applied to more

352

broadly sample types and it can be said that the SEED assay is convenient for

353

quantifying the total amount of β-glucan with a specific structure in processed foods.

354

Prior to this study, no method existed for directly measuring processed foods to

355

control the β-glucan content. The SEED assay developed here can directly evaluate the


Page 16 of 27

Page 17 of 27


356

β-glucan content of a product, and it may become an effective quality control method

357

for β-glucan-containing foods in the health food market.

358 359

ABBREVIATIONS

360

EDTA,

361

chromatography; SD, standard deviation; RSD, relative standard deviation.

ethylenediaminetetraacetic

acid;

HPLC,

high-performance

liquid

362 363

ACKNOWLEDGMENTS

364

The authors would like to thank Mr. Masatoshi Watai and Dr. Kazuhiro Fujita at the

365

Japan Food Research Laboratories for their useful comments and constructive advice.

366 367 368

ASSOCIATED CONTENT

369

Supporting Information

370

The supporting Information is available free of charge on the ACS Publications website

371

at DOI:

372

HPLC chromatograms of pustulan, photographs of Ganoderma lucidum in the SEED

373

assay and GEM assay, β-glucan recovery rate in 0.5% aqueous laminarin in the SEED

374

assay, β-glucan value in 5% laminarin/glucose in the SEED assay, β-glucan value in

375

sweet potato fries in the SEED assay, and β-glucan value in 5% laminarin/sweet potato

376

fries in the GEM assay.

377



378 379

REFERENCES

380

(1) Akramiene, D.; Kondrotas, A.; Didziapetriene, J.; Kevelaitis, E. Effects of beta-

381

glucans on the immune system. Medicina (Kaunas, Lithuania) 2007, 43, 597–606.

382

(2) Chihara G.; Hamuro J.; Maeda, Y. Y.; Arai, Y.; Fukuoka, F. Fractionation and

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purification of the polysaccharides with marked antitumor activity, especially lentinan,

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from Lentinus edodes (Berk.) Sing. (an edible mushroom). Cancer Res. 1970, 30, 2776–

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2781.

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(3) Maeda, Y. Y.; Hamuro, J.; Chihara, G. The mechanisms of action of anti-tumour

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polysaccharides. I. The effects of antilymphocyte serum on the anti-tumour activity of

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lentinan. Int. J. Cancer. 1971, 8, 41–46.

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(4) Marchessault, R. H.; Yves, D. Fine structure of (1→3)-β-D-glucans: curdlan and

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paramylon. Carbohydr. Res. 1979, 75, 231–242.

391

(5) Kondo, Y.; Atsusi, K.; Hiroshi, H.; Nozoe, S.; Takeuchi, M. Cytokine-related

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immunopotentiating activities of Paramylon, a β-(1→3)-D-glucan from Euglena gracilis.

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J. Pharmacobio-dyn. 1992, 15, 617–621.

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(6) Tani, M.; Tanimura, H.; Yamaue, H.; Tsunoda, T.; Iwahashi, M.; Noguchi, K.;

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Tamai, M.; Hotta, T.; Mizobata, S. Augmentation of lymphokine-activated killer cell

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activity by lentinan. Anticancer Res. 1993, 13, 1773–6.

397

(7) Jiezhong, C.; Robert, S. Medicinal importance of fungal β-(1→3),(1→6)-glucans.

398

Mycol Res. 2007, 111, 635–652.


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(8) Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kuge, T.; Kitamura, S. Macromolecular

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cyclization of (1→6)-branched-(1→3)-β-D-glucans observed after denaturation–

401

renaturation of the triple-helical structure. Biopolymers 1993, 33, 193–198.

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(9) Ciucanu, I. Per-O-methylation reaction for structural analysis of carbohydrates by

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mass spectrometry. Anal. Chim. Acta. 2006, 576, 147–155.

404

(10) Lowman.; D. W.; David L. W. A proton nuclear magnetic resonance method for

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the quantitative analysis on a dry weight basis of (1→ 3)-β-D-glucans in a complex,

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solvent-wet matrix. J. Agric. Food Chem. 2001, 49, 4188–4191.

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(11) Nitschke, J.; Modick, H.; Busch, E.; Von Rekowski, R. W.; Altenbach, H. J.;

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Mölleken, H. A new colorimetric method to quantify β-1,3-1,6-glucans in comparison

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with total β-1,3-glucans in edible mushrooms. Food Chem. 2011, 127, 791–796.

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(12) Mccleary, B. V.; Mugford, D. C. Determination of β-glucan in barley and oats by

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streamlined enzymatic method: Summary of collaborative study. J. AOAC. Int. 1997,

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80.3: 580–583.

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(13) Michael E. D.; Rosmarie D.; Natalie A. E.; Ryan R. L.; Andrew S. M.; Paul M. W.

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Enzymatic method to measure β-1,3-β-1,6-glucan content in extracts and formulated

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products (GEM Assay). J. Agric. Food Chem. 2010, 58, 10305–10308.

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(14) Zygmunt, LC.; Paisley, SD. Enzymatic method for determination of (1-->3)(1--

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>4)-beta-D-glucans in grains and cereals: collaborative study. J. AOAC. Int. 1993, 76,

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1069–1082.

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(15) Scott J. H.; Schekman R. Lyticase: Endoglucanase and Protease Activities That Act

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Together in Yeast Cell Lysis. J. Bacteriol. 1980, 142, 414–23.



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products. J. AOAC. Int. 2016, 99, 364–373.

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(17) Liu, Y.; Zhang, J.; Tang, Q.; Yang, Y.; Guo, Q.; Wang, Q.; Wu, D.; Cui, S. W.

424

Physicochemical characterization of a high molecular weight bioactive β-d-glucan from

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the fruiting bodies of Ganoderma lucidum. Carbohydr. Polym. 2014, 101, 968–974.

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(18) Freimund, S.; Janett, S.; Arrigoni, E.; Amado, R. Optimised quantification method

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for yeast derived (1,3)-β-D-glucan and R-D-mannan. Eur. Food Res. Technol. 2005,

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insoluble and soluble dietary fiber. J. Agric. Food Chem. 1983, 31, 476–482.

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(20) Prosky, L.; Asp, N. G.; Furda, I.; DeVries, J. W.; Schweizer, T. F.; Harland, B. F.

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Determination of total dietary fiber in foods and food products: collaborative study. J.

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Assoc. Off. Anal. Chem. 1984, 68, 677–679.

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(21) Miura, N. N.; Miura, T.; Ohno, N.; Adachi, Y.; Watanabe, M.; Tamura, H.;

435

Tanaka, S.; Yadomae, T. Gradual solubilization of Candida cell wall β-glucan by

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oxidative degradation in mice. FEMS Immunol. Med. Microbiol. 1998, 21, 123–129.

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(22) Miura, N. N.; Adachi, Y.; Yadomae, T.; Tamura, H.; Tanaka, S.; Ohno, N.

438

Structure and biological activities of beta-glucans from yeast and mycelial forms of

439

Candida albicans. Microbiol Immunol. 2003, 47, 173–82.

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(24) Hirokawa, Y.; Fujiwara, S.; Suzuki, M.; Akiyama, T.; Sakamoto, M.; Kobayashi,

444

S.; Tsuzuki, M. Structural and physiological studies on the storage β-polyglucan of

445

haptophyte Pleurochrysis haptonemofera. Planta 2008, 227, 589–599.

446 447 448

Notes

449

The authors declare no competing financial interest.



Figure and Table Captions

Figure 1. Schematic of the SEED assay.

Figure 2. Recovery rates of laminarin and barley-derived β-glucan in the SEED assay. 1, 2, 5, 10, 20, 40, 100, and 200 mg of (A) laminarin and (B) barley-derived β-glucan were accurately weighed, and the results of the SEED assay were plotted against the amount collected. The blank was subtracted, and the results followed a linear approximation formula passing through the origin.

Table 1a. Analysis method validation results based on the measurement of a lowconcentration β-glucan sample using the SEED assay.

Table 1b. β-glucan measurement precision and accuracy when using the SEED assay for a processed food.

Table 2. Comparison of results obtained by SEED and GEM assays for various types of sample.

Table 3. Comparison of typical results for glucan in cereals obtained using various methods.


Page 22 of 27

Page 23 of 27


Figures and Tables Figure 1.

Figure 2. A

200 175

y = 0.9247x R² = 0.999

β-glucan equivalent (mg)

plns

150 125 100 75 50 25

B

0 0

25

50

75

100 125 150 175 200

Laminarin (mg)



Page 24 of 27

B 200 B β-glucan equivalent (mg)

175

y = 0.9767x R² = 0.999

150 125

plns100 75 50 25 0 0

25

50

75 100 125 150 175 200

Barley β-glucan (mg)

Table 1a. Sample

% β-glucan

S.D.

%RSD

%Recovery

LOD%

LOQ%

1% laminarin solution

0.99

0.016

1.64

95.4

0.08

0.16

The results are representative of eight independent experiments. The recovery rate was calculated from the measured value assuming that the amount of β-glucan added was 100%. SD, standard deviation; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantification.


Page 25 of 27


Table 1b. Sample

% β-glucan

S.D.

%RSD

%Recovery

4.77

0.16

3.35

95.4

5% laminarin/sweet potato fries

The results are representative of nine independent experiments. The recovery rate was calculated from the measured value assuming that the amount of β-glucan added was 100%. SD, standard deviation; RSD, relative standard deviation. Table 2.

GEM assay

SEED assay

% total glucose

Sample % β-glucan

S.D.

% β-glucan

S.D.

Indigestible dextrin

0.45

0.28

4.68

0.60

105

Glucomannan

0.45

0.21

0.94

0.29

37.4

Galactomannan

0.08

0.02

0.61

0.22

2.00

Curdlan

93.2

3.39

91.2

2.18

102

Laminarin

89.6

1.18

86.3

3.24

95.9

Pustulan

93.3

4.04

8.28

0.32

99.3

Yeast-processed food

8.29

0.06

7.69

0.07

-

Ganoderma lucidum

30.0

1.05

24.5

1.79

-

Sarcomyxa serotine

19.6

0.39

16.6

2.56

-

The results are representative of three independent experiments. % total glucose was measured by a GOPOD test in an acid hydrolysis assay. SD, standard deviation.



Page 26 of 27

Table 3. SEED assay

GEM assay

AOAC 995.16 method

Sample

% total glucose % β-glucan

S.D.

% β-glucan

S.D.

% β-glucan

S.D.

Wheat starch

0.15

0.09

1.32

0.11

0.44

0.33

101

Cellulose

0.16

0.09

2.52

1.19

0.03

0.01

99.9

Oats flour

8.13

0.14

8.24

0.16

8.18

0.14

62.1

Barley β-glucan

92.4

5.08

90.9

6.08

83.5

1.55

96.5

The results are representative of more than three independent experiments. Barley and oats were selected as representative grains. Wheat starch and cellulose were selected as representative of the matrices of cereals. % total glucose was measured by a GOPOD test in an acid hydrolysis assay. SD, standard deviation.


Page 27 of 27


Table of Contents/Abstract Graphics


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