Enzymatic determination of total starch and degree of starch gelatinization in various products

Journal Pre-proof Enzymatic determination of total starch and degree of starch gelatinization in various products Keshun Liu, Qian Liu PII:

S0268-005X(19)31603-0

DOI:

https://doi.org/10.1016/j.foodhyd.2019.105639

Reference:

FOOHYD 105639

To appear in:

Food Hydrocolloids

Received Date: 17 July 2019 Revised Date:

30 December 2019

Accepted Date: 31 December 2019

Please cite this article as: Liu, K., Liu, Q., Enzymatic determination of total starch and degree of starch gelatinization in various products, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/ j.foodhyd.2019.105639. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Starchy samples Reducing particle size

Grind dry samples and pass through a screen with 300 µm openings or less or blend wet samples with 3 parts of water for 30 s on a high speed

Resolubilizing gelatinized starch Mix in 60 mM NaOH for 15 min & neutralize with HCl

Solubilizing total starch

Mix with 0.5 M NaOH for 15 min & neutralize with HCl

Hydrolyzing solubilized starch enzymatically

Incubate with amyloglucosidase at 37°C for 45 min

Measuring D-glucose colorimetrically

React with glucose oxidase-peroxidase reagent

Gelatinized starch content

Total starch content

Expressing results relative to total starch (% gelatinized starch) Fig. 1. Schematic diagram showing key steps of the proposed method for measuring the degree of starch gelatinization.

1 2 3 4 5 6 7 8 9 10

Enzymatic Determination of Total Starch and Degree of Starch Gelatinization in Various Products

Keshun Liu a, * and Qian Liu b

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a. Grain Chemistry and Utilization Laboratory, National Small Grains and Potato Germplasm

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Research Unit, United States Department of Agriculture, Agricultural Research Service

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(USDA-ARS), 1691 South 2700 West, Aberdeen, Idaho 83210, United States

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b. College of Food Science, Northeast Agricultural University, 600 Changjiang Road, Xiangfang District, Harbin, Heilongjiang 150030, China

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* Corresponding author.

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E-mail addresses: [email protected] (K. Liu), [email protected] (Q. Liu).

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1

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Abstract

23 24

The degree of starch gelatinization (DSG) affects not only structural, physicochemical and

25

organoleptic properties but also susceptibility to enzymatic digestion and thus nutritional

26

values of starchy products. DSG determination has been conducted in many laboratories,

27

entailing measurements of both gelatinized and total starch. However, current enzymatic

28

methods are complex and inaccurate. For addressing the problems, this study was conducted.

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Results show that gelatinized and native starch solubilized maximally at different NaOH

30

concentrations and that proper sample pretreatments to solubilize starch were important for

31

obtaining accurate results. For gelatinized starch, optimal pretreatments entailed mixing in

32

40-80 mM NaOH solution at 150 rpm for 15-70 min. For total starch, mixing samples in 0.5

33

M NaOH for as short as 5 min or autoclaving for 60 min was optimal but boiling for 60 min

34

was not. Consequently, a new method was proposed to measure DSG, consisting of

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differential alkaline pretreatments of samples for determining gelatinized starch and total

36

starch, hydrolysis of solubilized starch by amyloglucosidase, and colorimetric measurement

37

of D-glucose released by glucose oxidase-peroxidase. Furthermore, DSG calculation was

38

significantly simplified by using absorbance ratio of gelatinized starch over total starch and

39

omitting a correction factor for limited hydrolysis of native starch. This calculation eliminates

40

the need for assessing absolute contents of gelatinized and total starch and determining the

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correction factor. The new method was validated and compared with a prior method. It

42

enabled simple and accurate analysis of gelatinized starch, total starch, and DSG in various

43

products in situ.

44 45

Keywords: degree of starch gelatinization, gelatinized starch, total starch, enzymatic method,

46

alkaline treatment

47 48

Abbreviations

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AGS, amyloglucosidase; DSG, degree of starch gelatinization; GOPOD, glucose

50

oxidase-peroxidase.

51

2

52

1. Introduction

53

Starch is a major component of cereal products. It is also widely used as a thickening,

54

stabilizing, gelling, bulking, binding, or water retaining agent for various food and feed

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products. Native starch granules are relatively insoluble and non-dispersible. When heated in

56

water, they undergo an irreversible order-disorder transition, characterized by taking up water,

57

swelling, unfolding double helices, altering crystalline regions, losing birefringence,

58

increasing solubility and developing viscosity (Schirmer, Jekle, & Becker, 2015; Tako,

59

Tamaki, Teruya, & Takeda, 2014; Wang & Copeland 2013). The process, known as starch

60

gelatinization, is necessary for disrupting the crystalline structure of native starch and making

61

it readily hydrolysable. Beside heating, chemical or extensive mechanical treatment can also

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induce starch gelatinization. The degree of starch gelatinization (DSG) affects not only

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physical, chemical and organoleptic characteristics of starchy foods or feeds, but also their

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susceptibility to enzymatic digestion and thus nutritional properties for humans or animals

65

(Parada & Aguilera, 2009, Ren et al., 2016; Wang & Copeland 2013). It is very important to

66

develop an accurate and simple laboratory method to determine DSG, which could serve as a

67

crucial index for assessing physiochemical characteristics and digestion potentials of starchy

68

products.

69 70

Over the years, many methods and techniques have been developed to monitor

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physicochemical changes during starch gelatinization and/or determine DSG in various

72

products (Baks, Ngene, van Soest, Janssen, & Boom, 2007; Biliaderis, Maurice, & Vose,

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1980; Birth & Priestley, 1973; Da Silva, Ciacco, Barberis, Solano, & Rettori, 1996; Di Paola,

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Asis, & Aldao, 2003; Liu & Han 2012; Marconi, Messia, Palleschi, & Cubadda, 2004;

75

Pinnavaia & Pizzirani, 1998; Schirmer et al. 2015; Shetty, Lineback, & Seib, 1974;

76

Varriano-Marston, Ke, Huang, & Ponte Jr., 1980). At present, differential scanning

77

colorimetry (DSC), amylose-iodine blue complex formation, and enzymatic hydrolysis are

78

the most commonly used methods (Wang, Liu, Wang, & Copeland 2017; Liu et al. 2017; Ren

79

et al. 2016). The DSC method can measure the precise gelatinization temperature and energy

80

changes during the whole process (Biliaderis et al. 1980; Schirmer et al. 2015), but it is less 3

81

suitable for quantitative measurement of DSG in multicomponent products due to

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interference by protein denaturation peaks (Zhu et al. 2016). It also requires a costly DSC

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instrument and unprocessed samples as a reference. The remaining two popular methods are

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chemical in nature. The amylose-iodine binding method is simple to use (Wootton, Weeden,

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& Munk, 1971, Birth & Priestley 1973, Liu et al. 2017) but also the least reliable (Baks et al.,

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2007) due to variations in stoichiometry of the iodine complexes with starch of different

87

origins.

88 89

The enzymatic method has been a method of choice for many laboratories where a DSC

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instrument is not readily available and where more measurement precision is needed,

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particularly for starch in a multicomponent matrix (Di Paola et al. 2003; Kainuma, 1994; Liu

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& Han, 2012; Shetty et al., 1974; Zhu et al. 2016). It is based on a principle that starch

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becomes solubilized during gelatinization and thus susceptible to enzyme attacks. DSG is

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proportional to the level of starch solubilization, which in turn is proportional to the level of

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enzymatic hydrolysis. When using amyloglucosidase (AGS) as a starch hydrolysing enzyme

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(Shetty et al. 1974; Liu & Han 2012), glucose released is measured colorimetrically. Because

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DSG is typically expressed as % gelatinized starch relative to total starch, its determination

98

requires two parallel measurements, one for gelatinized starch and other for total starch.

99

Therefore, for accurate DSG determination, the procedures for both tests must be equally

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reliable.

101 102

For measuring gelatinized starch, an enzymatic method works well with freshly made wet

103

samples, in which gelatinized starch is already fully solubilized. Yet, after certain levels of

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thermal processing, starchy foods and feeds are often cooled and/or dried for storage, during

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which some gelatinized starch retrogrades gradually into semi-crystalline aggregates that

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differ in form from native starch granules (Copeland, Blazek, Salman, & Tang, 2009; Tako et

107

al. 2014). Without proper sample treatments for full resolubilization before chemical or

108

enzymatic analysis, gelatinized starch measured may differ significantly from the actual value

109

(Liu & Han 2012). In developing methods for measuring gelatinized starch in dry products 4

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using an enzymatic method, some researchers isolated starch from test samples before

111

enzymatic analysis (Lineback & Wongsrikasem 1980, Varriano-Marston et al. 1980) while

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others ignored the need to fully resolubilize gelatinized starch in dried products by a

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pretreatment (Chiang & Johnson 1977; Xiong, Bale, & Preston, 1990). These methods could

114

be tedious and/or prone to errors. Still, many others described various ways (pretreatments) to

115

facilitate hydration/dispersion of gelatinized starch in their samples before enzymatic

116

measurements (Shetty et al. 1974; Zhu et al. 2016; Marconi et al. 2004; Kainuma 1994),

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Tanaka & Yukami 1969). Yet, since starch in most products is partially gelatinized, these

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products contain both gelatinized and native starches. A suitable pretreatment should enable

119

full resolubilization of gelatinized starch in a sample but minimal solubilization of native

120

starch contained in the same sample. However, authors in these cited methods did not

121

investigate whether their pretreatments met this requirement during method development.

122

Earlier in our laboratory, an enzymatic method was developed, after observations that mixing

123

a powder sample in water slowly for 70 min before AGS hydrolysis could measure

124

gelatinized starch of several grain flours in situ (Liu & Han 2012). Yet, even with the

125

pretreatment, dried autoclaved grain flours gave about 95% DSG, indicating that the

126

pretreatment developed early in our laboratory still could not fully resolubilize gelatinized

127

starch. An ideal pretreatment should enable an enzymatic method to measure dried fully

128

gelatinized starchy products for 100% DSG while keeping native (unheated) starch in the

129

products at the lowest possible measured values.

130 131

For total starch measurement, both chemical and thermal pretreatments have been used to

132

fully solubilize starch in a sample. Yet reported methods vary in chemical reagents (Shetty et

133

al. 1974; Hall, 2009; AACC Method 76-13, 2010; Liu & Han 2012; Zhu et al., 2016),

134

chemical concentrations (Chiang & Johnson 1977; Marconi et al., 2004; Baks et al., 2007;

135

Kainuma, 1994; Tovar et al. 1990; Liu & Han 2012), thermal treatment temperature, and

136

treatment duration and mode (Zhu et al., 2016; Xiong et al., 1990; Tanaka and Yukami 1969;

137

Ren et al., 2016; AACC Method 76-11, 2010). Since total starch measurement is equally

138

important, it is necessary to compare these treatments in a single study. Furthermore, even 5

139

after measurements of gelatinized and total starch, a great variation also exists in the

140

equations used for calculating DSG (Shetty et al., 1974, Liu & Han, 2012; Ren et al., 2016;

141

Zhu et al., 2016, Chiang & Johnson, 1977; Marconi et al., 2004). Some equations can be

142

rather complex, with a correction factor being difficult or impossible to determine.

143 144

The present study was systematically conducted to address the issues and variables with

145

measurements for both gelatinized starch and total starch, and with DSG calculation. Specific

146

objectives included 1) developing optimal sample treatments to maximally resolubilize

147

gelatinized starch but minimally solubilize native starch in a test sample, 2) comparing

148

thermal and chemical treatments for total starch measurement, and 3) simplifying DSG

149

calculation. The ultimate objective was to develop reliable and simple methods for measuring

150

both gelatinized and total starch in situ in various types of products.

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2. Materials and Methods

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2.1 Materials

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Seeds of several grain species and varieties, including corn (yellow dent), rice (medium grain,

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milled), hulled barley (Idaho Gold), hulless barley (Transit, a high beta-glucan and waxy

157

variety), hulled oat (Ajay), hulless oat (Lamont), soft wheat (Treasure), and hard wheat

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(Boundary), were kindly provided by local breeders or purchased from a local supermarket.

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Samples were cleaned and/or screened to remove foreign materials and broken kernels. Six

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dry (Cheerios, ramen noodles, rotini pasta, tortilla chips and two trout feeds) and six moist

161

(bagel, banana, cooked rice, corn tortilla, hot dog bun and steamed bread) starchy food or

162

trout feed products were purchased from local markets or received.

163 164

Amyloglucosidase (AGS, E.C. 3.2.1.3, also known as glucoamylase) from Aspergillus niger)

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was purchased from Megazyme International Ireland Ltd (Wicklow, Ireland) as a suspension

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(3260 U/mL). D-Glucose Assay Kit was also purchased from Megazyme. The kit contained

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two vials of glucose oxidase-peroxidase (GOPOD), two bottles of concentrated reagent buffer, 6

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and one bottle of D-glucose standard solution.

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2.2. The procedure for the proposed new method

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The whole procedure consisted of multiple steps (Fig. 1). They include: 1) sample particle

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size reduction, 2) alkaline treatment at a lower concentration to fully resolubilize gelatinized

173

starch but minimally solubilize native starch, 3) concurrent treatment with a higher alkaline

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concentration (0.5 M) to fully solubilize total starch, 4) hydrolysis of resolubilized and

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solubilized starch with AGS, respectively, 5) colorimetric measurement of D-glucose released

176

as absorbance at 510 nm, and 6) calculation of DSG.

177 178

2.2.1. Sample particle size reduction.

Dry samples were ground by a coffee grinder (Krups,

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Medford, MA) at repeated intervals until all particles passed through U.S. standard mesh, No.

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50 (300 µm opening dimension). Moist or wet samples were mixed with 3 parts of water (e.g.

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50 g sample per 150 mL water) and blended for 30 s on a high speed.

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2.2.2. Chemical and mechanical resolubilization of gelatinized starch.

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powder sample or 200 mg of a blended wet sample was weighed and put into a 50 mL plastic

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graduated centrifuge tube with a conical bottom and a flip cap (these features were important

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for mixing and fast pipetting later). To each tube, an octagonal magnet (5/16” x ½”, i.e. 7.9

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mm x 12.7 mm) was carefully added, followed by addition of 0.2 mL of 50% glycerol (for

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reducing sample clumping). A plastic rack holding the tubes together in the center (up to 12

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maximum) was placed on a stirrer (preferably with a digital speed control). While the

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weighed sample was stirred at 150 revolutions per min (rpm), 5 mL of 60 mM NaOH solution

191

was carefully pipetted into the bottom of each tube. During 15 min of stirring, sample tubes

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in the rack were rotated halfway through to minimize the positional effect of the stirring plate.

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At the end of stirring, 29.8 mL of 100 mM sodium acetic buffer, pH 4.75, was added to each

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tube, using a liquid dispenser. The mixture was vortexed before adding 5 mL of 60 mM HCl

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for neutralizing the NaOH added originally. The new mixture was vortexed again, with a total

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volume of 40 mL in each sample tube. The whole treatment was conducted at room 7

Twenty mg of a

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temperature.

198 199

2.2.3. Chemical and mechanical solubilization of total starch.

Concurrently, for the same

200

starchy samples, another set of tubes (each containing 20 mg of sample powder or 200 mg of

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blended wet sample) were used, following the same chemical and mechanical hydration

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procedure at room temperature as described above, except for: 1) using 5 mL of 0.5 M NaOH

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for initial treatment, and 2) adding 5 mL of 0.5 M HCl for NaOH neutralization.

204 205

2.2.4. Enzymatic hydrolysis of resolubilized or solubilized starch to D-glucose.

Following

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the two concurrent steps of 60 mM NaOH treatment to resolubilize gelatinized starch and 0.5

207

M NaOH treatment to solubilize total starch for the same samples, as described above, each

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50 mL centrifuge tube (containing 40 mL neutralized and buffered sample suspension) was

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vortexed, with the cap on, at a high speed for 10 s. Immediately, 2 mL of the sample

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suspension from each tube was pipetted into a 15 mL glass test tube, using a 5 mL pipette tip.

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This step was repeated one more time with just one sample suspension resulting from treating

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either with 60 mM or 0.5 M NaOH, for generating a sample blank for the subsequent

213

D-glucose measurement. Ten µL of the AGS stock solution (33 units) was added to each 15

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mL sample tube and vortexed, but to the sample blank tube, 10 µL water was added instead.

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Sample and sample blank tubes in a rack were incubated at 37°C in a covered water bath for

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45 min, and vortexed every 15 min for 5 s. At the end of incubation, each glass test tube was

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diluted to 10 mL volume with 50 mM phosphate buffer, pH 7.4 and vortexed for 10 s

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(carefully to avoid spillage over the top). Therefore, for each test sample, three 15-mL glass

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test tubes were needed: one for gelatinized starch, one for total starch, and one for sample

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blank.

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2.2.5. D-glucose measurement.

D-glucose content released from treated samples was

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determined by the Megazyme GOPOD assay procedure that came with the D-glucose

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measurement kit, but with modification. Chromogen reagent was prepared by diluting 50 mL

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(one bottle) of concentrated reagent buffer (1 M potassium phosphate, pH 7.4, 0.22M 8

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p-hydroxybenzoic acid and 0.4% w/v sodium azide) to 1000 mL with deionized water and

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dissolving the content of one vial of GOPOD reagent (also known as glucose determination

228

reagent) in this dilute buffer (50 mM phosphate buffer, pH 7.4). The GOPOD reagent should

229

be stored in a brown storage bottle in a refrigerator. From each sample or sample blank tube

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prepared from the previous step, 0.4 mL was transferred into a 4 mL cuvette (12.5 x 12.5 x 45

231

mm). One mL GOPOD reagent was then added to each cuvette. The reagent blank consisted

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of 0.4 mL of the 50 mM phosphate buffer (pH 7.4) and 1 mL of the GOPOD reagent.

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Cuvettes with added reactants were vortexed and incubated at 37°C for 30 min in the covered

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water bath. After color reaction, absorbance at 510 nm for each sample or sample blank was

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read against the reagent blank by a spectrophotometer (Genesys 6, Thermo Electron Corp.

236

Waltham, MA).

237 238

2.2.6. Calculation of results.

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expressed as % gelatinized starch relative to total starch and could be calculated simply and

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directly by the ratio of the two absorbances after correction for the sample blank, shown

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below:

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The degree of starch gelatinization in test samples was

DSG (% relative to total starch) = (A510G – A510B)/(A510T – A510B) x 100

(1)

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Where, A510G = Absorbance at 510 nm for gelatinized starch in a test sample, A510T =

244

Absorbance at 510 nm for total starch in the same test sample, and A510B = Absorbance of the

245

sample blank.

246 247

As shown in Equation (1), although calculation for the content of gelatinized starch and/or

248

total starch is unnecessary for DSG determination, it can be done by using the following

249

equation (as is basis):

250

Starch (%) = (A510-A510B) x F x FV/SV x 100/W x 162/180 = (A510-A510B) x 2250F

(2)

251 252

Where A510 = Absorbance at 510 nm for either gelatinized starch or total starch in a test

253

sample, A510B = Absorbance of the sample blank, F = Conversion factor from 1 unit

254

absorbance to mg D-glucose from a standard reading made with the D-glucose standard 9

255

provided in the Megazyme test kit, FV = Final volume of the solubilized and enzymatic

256

hydrolyzed sample solution (40 x10/2 =200 mL in this study), SV = Sample volume used for

257

the color reaction in the cuvette (0.4 mL in this study), W = Sample weight in mg (20 mg in

258

this study), 100/W = Factor to express starch content as % of sample mass, and 162/180 =

259

Adjustment from free D-glucose to anhydrous D-glucose as occurs in starch.

260 261

2.3. Experiments for the method development in the present study

262

Starch isolation and preparation from fully gelatinized grain flour or starch.

263

isolating starch, corn flour was first treated with 50 mM NaOH and centrifuged to remove

264

protein. The residue was then mixed with water and wet screened to remove fiber and recover

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starch. For preparing completely gelatinized flour, grains were cracked into grits, soaked

266

overnight, autoclaved for 60 min, dried and ground. For preparing completely gelatinized

267

starch, powder samples were mixed with 5 parts of water just before autoclaving. For details,

268

refer to the procedures of Liu and Han (2012).

Briefly, for

269 270

2.3.1. Optimization of sample pretreatments for measuring gelatinized starch.

In

271

developing a pretreatment to maximally resolubilize gelatinized starch while minimally

272

solubilizing native starch as well as a pretreatment to fully solubilize total starch in a sample,

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several factors were investigated for their effects on A510 (parallel to glucose content), using

274

both raw and dried autoclaved flours of several grain species and varieties as well as starch

275

isolated from corn flour. These included NaOH concentrations (0, 20, 40, 60, 80, 100, 120,

276

140, 500 and 2000 mM), mixing speed (50, 150, and 300 rpm), and mixing duration (5, 15

277

and 70 min). The chemical (NaOH) and mechanical (magnetic mixing) treatments were all

278

conducted at room temperature. After each treatment, each mixture was subjected to

279

neutralization with 2 N HCl and addition of 100 mM sodium acetic buffer, pH 4.75, followed

280

by AGS hydrolysis and measurement of glucose content, as described above.

281 282

2.3.2. Comparison of thermal and chemical pretreatments for total starch measurement.

283

Raw and dried autoclaved corn and rice flours were subjected to boiling (100°C) or 10

284

autoclaving (121°C) for 60 min after mixing 20 mg of each sample with 5 mL water in a

285

glass test tube. Boiling was carried out by putting the tubes in a beaker filled with water and

286

heating it on a hot plate, while autoclaving was carried out in an autoclave. Treated samples

287

were cooled to room temperature and mixed with 100 mM sodium acetic buffer (pH 4.75).

288

The final volume was also 40 mL. The samples were also subjected to a chemical treatment

289

which consisted of mixing 20 mg of each sample with 5 mL 0.5 M NaOH for 15 min,

290

neutralizing with 5 mL 0.5 M HCl, and adding 30 mL 100 mM sodium acetic buffer, pH 4.75.

291

After thermal or chemical treatment, each buffered or neutralized and buffered mixture was

292

subjected to starch hydrolysis by AGS and colorimetric measurement of glucose content, as

293

described above. Total starch content was calculated based on Equation (2). Sample moisture

294

was measured by drying in a forced air oven at 105°C for 4 hr.

295 296

2.3.3. Method validation and comparison.

For each corn and wheat flour, a set of six

297

mixtures of native and autoclaved flour samples, representing 0, 20, 40, 60, 80, and 100% of

298

fully gelatinized flour by mass, respectively, was made. All mixed samples were tested for

299

DSG, according to the proposed procedures described above. Furthermore, three enzymatic

300

methods with three different pretreatments were compared for their determination of

301

gelatinized starch in the six dry and six moist starchy food or feed products. They were

302

Method 1, the proposed method as described above (60 mM NaOH x 15 min pretreatment),

303

Method 2, an alternative to the proposed method by treating samples with 40 mM NaOH for

304

70 min, and the control method (Liu and Han 2012, featuring mixing samples in water for 70

305

min).

306

starch measurement and Equation 1 for DSG calculation.

All the three methods used the pretreatment of 0.5 M NaOH for 15 min for total

307 308

2.3.4. Data treatments and statistical analysis.

309

triplicated. Data were analyzed with JMP software, version 12.01 (SAS, Cary, NC, USA).

310

Analysis of variance was performed for determining the effect of the three pretreatments on

311

total starch measurement and the effect of three methods on DSG analysis. The Tukey’s

312

honestly significant difference test was conducted for pair-wise comparisons of means within 11

Each experiment was duplicated or

313

each sample group. The significance level was set at p <0.05. Error bars in all figures

314

represent standard deviations between or among repeats.

315 316

3. Results and discussion

317

3.1. Optimization of sample pretreatments for measuring gelatinized starch

318

Determination of DSG entails measurements of both gelatinized starch and total starch in a

319

sample. For accurate measurement of gelatinized starch, a major portion of the study was

320

devoted to developing optimized sample pretreatments. The experiments started with treating

321

native and dried fully gelatinized corn flour, wheat flour, corn starch, and flours of several

322

other species (barley, oat and wheat, each with two varieties) in aqueous NaOH solutions

323

with varying concentrations, mixing (magnetic stirring) time and speed. The levels of

324

resolubilization of gelatinized starch and solubilization of native starch were followed by

325

AGS hydrolysis and subsequent colorimetric measurement (as A510) of glucose released. The

326

objective was to determine the optimal combination of NaOH, mixing speed and time for

327

treating starchy samples before enzymatic assay for gelatinized starch, which should

328

maximally resolubilize gelatinized starch while minimally solubilizing native starch and thus

329

enable the enzymatic method based on AGS to measure dried fully gelatinized starchy

330

products for 100% DSG while keeping native (unheated) starch in products at the lowest

331

possible measured values.

332 333

Results show that all factors under investigation had significant effects (p<0.05) on starch

334

hydrolysis by AGS, as indicated indirectly by A510 values. NaOH concentration had the most

335

effect, followed by mixing time and mixing speed (Figs. 2-5). Raw and autoclaved samples

336

showed two distinct types of curves connecting A510 with NaOH concentrations, and thus had

337

differential responses to NaOH concentration, mixing time and speed. For a given starchy

338

sample, as the NaOH concentration increased from 0 to 500 mM and further to 2000 mM,

339

A510 increased and reached a plateau at a specific NaOH concentration. The minimum NaOH

340

concentration that led to the maximum A510 value depended mainly on heat treatment of flour

341

(or isolated starch), followed by mixing time and speed. Grain species and varieties had little 12

342

effect.

343 344

Taking corn as an example (Fig. 2), for raw corn flour, at 0 mM (i.e., pure water), A510 was

345

very low. As the NaOH concentration increased from 0 to 100 mM, A510 increased slowly.

346

The increasing rate was affected by mixing time and speed; the longer the mixing time and

347

the higher the mixing speed, the higher the increasing rate in A510 with increasing NaOH

348

concentration. Between 100 and 140 mM, there was a dramatic increase in A510 with

349

increasing NaOH concentration. The increasing rate in A510 at this NaOH concentration range

350

was also influenced significantly by mixing time and speed. Only when NaOH concentration

351

increased to 500 mM, did A510 reach a maximum value. Further increasing NaOH to 2000

352

mM did not cause additional gain in A510. Yet, for dried autoclaved (fully gelatinized) corn

353

flour, the curves of A510 vs. NaOH concentration drastically differed from those of raw flour.

354

At 0 mM NaOH, A510 was already very high, although not reaching to the maximum value

355

yet. The effect of mixing time was rather significant, the longer the mixing time, the higher

356

the A510 value. This effect was diminished with increasing mixing speed. Under most

357

combinations of mixing time and speed, pure water treatment could not fully resolubilize

358

gelatinized starch, confirming that the sample treatment developed in the previous study (Liu

359

& Han 2012), i.e., mixing dried samples in water at 50 RPM for 70 min, was insufficient in

360

maximally resolubilizing gelatinized starch for accurate DSG measurement. When NaOH

361

concentration increased from 0 to 120 mM, A510 in all tests reached a maximum value at

362

certain concentrations. The minimum NaOH concentration that led to the plateau A510 value

363

was determined by mixing time and speed. When mixing time was 15 or 70 min at 50-300

364

rpm, NaOH concentration as low as 40 mM could lead to the maximum A510 value. When

365

mixing time was shortened to 5 min, however, 100 or 120 mM NaOH was needed. By

366

increasing NaOH concentration from 120 mM all the way to 2000 mM, A510 remained

367

unchanged. Also, within this NaOH concentration range, mixing for 5, 15 or 70 min gave the

368

same maximum A510 values, indicating that at higher NaOH concentrations, mixing time had

369

no effect (Fig. 2).

370

13

371

In treating rice flour samples (raw and dried autoclaved), the effects of NaOH concentration,

372

mixing speed and duration on A510 (Fig. 3) generally followed those of corn samples (Fig. 2),

373

even though there were some minor differences between the two grains. Compared to raw

374

corn flour, pure water treatment of raw rice flour caused a higher A510 value, but with

375

increasing NaOH concentration in the range of 0 to 100 mM, the increasing rate in A510 was

376

relatively lower. Compared to dried fully gelatinized corn flour, starch resolubilization in

377

dried autoclaved rice flour was less influenced by NaOH concentration. For example, after 70

378

min mixing, even pure water could lead to full starch resolubilization (i.e., the maximum

379

A510 value).

380 381

A comparison of Fig. 4 with Fig. 2b shows some minor differences between starch and flour

382

samples with respect to their responses to duration of alkaline treatments. For raw samples, at

383

low NaOH concentrations, mixing duration had less effect for starch than flour, but for

384

autoclaved samples, mixing time had a much higher effect for starch than flour, with 5 min

385

treatment showing significantly lower A510 values than 15 min and 70 min treatments. The

386

major reason is that gelatinized starch was more prone to clumping than raw starch and

387

gelatinized flour. Therefore, longer mixing time was needed for full solubilization.

388

Regardless these minor differences, the results in Fig. 4 clearly show that dried native and

389

autoclaved starch had the same differential responses to the alkali treatment as corresponding

390

corn flour samples. Therefore, all dried starchy samples, whether they are in purified form or

391

in the original matrix, need pretreatments when using an enzymatic method for DSG assay.

392 393

Furthermore, some other grains, such as barley, oat and wheat, each with two varieties, had

394

the same patterns of differential responses to alkaline treatments between raw and autoclaved

395

samples (Fig. 5) as with corn and rice flours (Figs. 2 and 3). Changes among species and

396

varieties were limited to 1) absolute A510 values (which reflected in starch content variations

397

among grain species and varieties), 2) the NaOH concentration range where raw samples had

398

the fastest increasing rates in A510, and 3) the pattern of increasing A510 within the NaOH

399

range as affected by mixing duration. 14

400 401

In determining optimal treatments for dried starchy samples, a key strategy was to find a

402

condition (i.e., a combination of NaOH concentration, mixing time and speed) that could lead

403

to maximum resolubilization of gelatinized starch but minimum solubilization of native

404

starch. Fortunately, results in Figs. 2-5 showed that, at lower alkali concentrations,

405

resolubilization of gelatinized starch was much faster than solubilization of native starch.

406

Based on this strategy, several combinations, including 40 to 80 mM NaOH solution, 150

407

rpm mixing speed, and 15 to 70 min mixing duration, could be considered optimal in

408

pre-treating samples for the enzymatic analysis of gelatinized starch. Furthermore, because

409

similar patterns of A510 vs. NaOH concentration as affected by heat treatment, mixing speed

410

and time were observed (Figs. 2-5), these optimal sample pretreatments for measuring

411

gelatinized starch were applicable to all the grain species and varieties as well as purified

412

starch investigated in the present study. This was also true for waxy starch, since Transit

413

barley, a waxy high beta-glucan variety, also gave similar patterns (Fig. 5b).

414 415

In developing methods for measuring gelatinized starch in dry products using an enzymatic

416

method, a few previous investigators described several pretreatment methods, including

417

adding silica gel to aqueous sample mixtures (Shetty et al. 1974), mixing with water for 20

418

min (Zhu et al. 2016), mixing with water thoroughly (Marconi et al. 2004), making 10-20 up

419

and down piston movements for aqueous sample mixtures (Kainuma 1994), and

420

homogenizing in water for 10 min (Tanaka & Yukami 1969). Based on the findings of the

421

present study, the pretreatments described in these previous studies might be either

422

insufficient for fully hydrating gelatinized starch or counter-productive for minimizing

423

solubilization of native starch in the same samples.

424 425

Using an amylose-iodine method, Wootton et al. (1971) quantified gelatinized starch in

426

biscuits by first blending samples in water for 1 min. However, Birch & Priestley (1973)

427

found the pretreatment Wootton et al. (1971) used for biscuits was inapplicable to rice flour

428

due to lack of full solubilization of gelatinized starch in pure water and came up with an 15

429

improved pretreatment by dissolving rice flour in 0.2 M alkaline solution before measuring

430

gelatinized starch by the amylose-iodine method.

431

the effect of alkali concentration on the solubility of raw and gelatinized rice starch by

432

following changes in A600 of amylose-iodine complex. In contrast, the present study used the

433

enzymatic method and investigated the effect of not only alkali concentration but also mixing

434

time and speed on solubility of raw and gelatinized flours of several grains and species by

435

following changes in absorbance at 510 nm, which paralleled glucose released by AGS

436

hydrolysis of solubilized starch.

This was based on their investigation into

437 438

3.2. Comparison of thermal and chemical pretreatments for total starch measurement

439

DSG is commonly expressed as % relative to total starch. Therefore, the reliability of a

440

procedure for total starch measurement is another key element for accurate DSG

441

determination (Shetty et al., 1973; Liu & Han 2012). In the present study, we compared three

442

sample treatments for total starch measurement of raw and dried fully gelatinized corn and

443

rice flours. Results show that for all the samples, autoclaving (heating at 121°C) for 60 min

444

and mixing with 0.5 M NaOH for 15 min gave same total starch values (p <0.05) (Table 1).

445

However, boiling (heating at 100°C) samples for 60 min gave total starch values

446 Table 1. Comparison of three sample treatments for total starch measurement by the enzymatic method.

447

Sample pretreatment

Raw corn flour

Dried autoclaved corn flour

Raw rice flour

Dried autoclaved rice flour

Boiling in water (100C) for 60 min Autoclaving for 60 min Mixing in 0.5 M NaOH fir 60 min

70.38 ± 1.43 b 79.31 ± 0.61 a 80.00 ± 0.96 a

75.83 ± 0.93 b 79.04 ± 0.78 a 79.21 ± 0.51 a

77.32 ± 0.78 b 81.01 ± 0.30 a 81.21 ± 0.23 a

80.44 ± 1.04 ab 81.37 ± 0.09 a 81.32 ± 0.16 a

Total starch content at each data point, expressed as % dry matter, was a mean of triplicate tests. Column values with different letters differed significantly at P <0.05.

448 449

significantly lower than the other two treatments, except for the fully gelatinized rice flour.

450

The difference between the boiling treatment and other two treatments was larger for raw

451

flour than dried autoclaved flour. This finding was unexpected. It implies that boiling samples

452

for 60 min or less before enzymatic analysis, as reported previously (Xiong et al., 1990; Zhu 16

453

et al., 2016), may be insufficient for accurate measurement of total starch. Although both

454

autoclaving for 60 min (AACC Method 76-11, 2010) and mixing with 0.5 M NaOH for 15

455

min could fully gelatinize starch, the latter is recommended because the NaOH treatment is

456

much easier to implement than autoclaving.

457 458

Furthermore, in the present study, both 0.5 M or 2.0 M NaOH solutions were chosen as parts

459

of concentration series, because they are often used to chemically gelatinize starch for total

460

starch measurement. Since the two NaOH solutions were found to produce same A510 values

461

regardless of sample type, mixing speed and time (Figs. 2-5), for safety and other reasons, 15

462

min treatment with 0.5 M NaOH was chosen for total starch measurement and for calculating

463

DSG. The subject of DSG calculation will be further discussed in a following section.

464 465

3.3. Running controls and subtracting blank readings

466

For any quantitative analysis, it is important to run controls properly. With an enzymatic

467

method for DSG assay, controls tell how much of a total assay signal (i.e., absorbance

468

readings) is due to starch hydrolysis by AGS, how much arises from other elements (such as

469

colorants and free glucose present in a sample), and how much is contributed by GOPOD

470

reagents. By subtracting control data during calculation, any other elements towards

471

absorbance readings are effectively removed. An added advantage of running sample and

472

reagent blanks is that clarification of NaOH treated sample mixtures, AGS treated mixtures

473

and color reaction mixtures can all be omitted. Yet, several previous methods based on

474

enzyme hydrolysis for DSG assay lacked either sample blanks (Shetty et al. 1974) or both

475

reagent and sample blanks (Chiang & Johnson, 1977; Xiong et al., 1990; Zhu et al., 2016),

476

which could cause significant errors for samples containing colorants and free sugars.

477 478

For having proper controls, the method of Liu and Han (2012) specifies running two sample

479

blanks (one for gelatinized starch and one for total starch) and one reagent blank. Since the

480

difference in A510 between the two sample blanks was found insignificant, for simplicity, the

481

proposed method in the present study calls for running one sample blank for measurements of 17

482

both gelatinized starch and total starch during starch hydrolysis and one reagent blank during

483

glucose measurement. Since all color readings were made against the reagent blank, only the

484

sample blank was subtracted from sample readings during DSG calculation [Equation (1)].

485 486

3.4. Calculation for DSG

487

As stated before, DSG is commonly expressed as % gelatinized starch relative to total starch.

488

However, in calculating DSG following a chemical method, several equations have been used

489

over the years. These include 1) a content equation (dividing the content of gelatinized starch

490

by the content of total starch) with a correction factor to take consideration of limited

491

hydrolysis of native starch (Shetty et al., 1974; Liu & Han 2012); 2) the content equation

492

without the correction factor (Ren et al., 2016; Zhu et al., 2016); 3) an index equation

493

(dividing an index value for gelatinized starch by an index value for total starch) with a

494

correction factor (Chiang & Johnson, 1977), and 4) the index equation without the correction

495

factor (Marconi et al., 2004).

496 497

Two important issues come up when using a correction factor. First, the correction factor

498

needs to be determined under a defined assay condition (Shetty et al., 1974) and even under

499

the same assay condition, its value changes also with botanical origin of starch (Liu & Han

500

2012). When a sample contains starch from blended or unknown sources or when a native

501

starch is unavailable, estimation can be difficult or impossible. Second, there is always a

502

measurable amount of gelatinized starch in an unprocessed sample (such as a raw flour) due

503

to limited hydrolysis of native starch by AGS. By using a correction factor, DSG for the raw

504

sample is arbitrarily set to zero. An assumption for the arbitrary setting is that native starch in

505

a raw sample has not been subjected to any heat treatment. However, this assumption is rather

506

questionable. In measuring DSG for any products (including raw grains), the first step is to

507

reduce sample particle size by milling into flour or blending into a suspension. The process

508

not only generates a certain level of heat (which gelatinizes some starch) but also causes

509

damage to some starch granules. Both can induce a measurable amount of gelatinized starch

510

for a raw sample. Considering the above factors, for the proposed method, a correction factor 18

511

for the limited hydrolysis of native starch is not used in calculating DSG, as shown in

512

Equation (1). Therefore, when using the proposed method for assaying the DSG of real

513

products, native starch will have some DSG values (non-zero), while samples with low DSG

514

values will be measured a little higher than methods using a correction factor.

515 516

For further simplifying DSG calculation, the index equation is preferred over the content

517

equation. The idea of using an index equation for calculating DSG was originally proposed

518

by Wootton et al. (1971) who found that with the amylose-iodine binding method, it is

519

difficult to determine the starch content due to variations in stoichiometry of iodine

520

complexes formed with starches of different origins. By utilizing the ratio of the measured

521

color intensities of starch-iodine complexes formed in the same sample before and after

522

complete solubilization and expressing the result on a percent basis as a degree of

523

gelatinization, the necessity of assessing the absolute concentration of gelatinized starch, total

524

starch and the initial moisture content in a test sample is obviated. This method of calculating

525

DSG has been used by several later researchers who worked on method improvement for

526

DSG assay by either the amylose-iodine binding (Birch & Priestley, 1973) or enzymatic

527

hydrolysis method (Chiang & Johnson, 1977; Marconi et al., 2004).

528 529

Among the four methods for calculating DSG, the index equation without correction for

530

native starch is the simplest. Therefore, it is adopted for the present study. Furthermore, DSG

531

results based on the A510 index ratio should be equal to those based on the content ratio,

532

because, except for A510, all other factors in Equation 2 are cancelled out when dividing the

533

content of gelatinized starch over that of total starch. Based on Equation (1), after calculating

534

DSG by dividing A510 values at varying NaOH concentrations (except for 2 M NaOH

535

treatment) with the A510 value of the 0.5 M NaOH treatment, we can easily convert Figs. 2b

536

and 3b into Fig. 6a, b, respectively. Results show that mixing autoclaved flours in 20-500

537

mM NaOH at 150 rpm for 15-70 min gave 100% DSG values but the 5 min pretreatment did

538

not bring DSG to 100% until NaOH reached 120 mM. For raw flours, DSG increased

539

gradually with both NaOH concentration and treatment duration, when NaOH was within 0 19

540

-100 mM, but DSG increased dramatically, when NaOH concentration was within 100-500

541

mM. This observation partially validates the new method described in the present study.

542 543

3.5. Method validation and comparison

544

To follow a common way of validating a new method developed for measuring starch

545

gelatinization, we prepared a series of flour mixtures, each containing 0, 20, 40, 60 and 100%

546

fully gelatinized flour of corn or soft wheat. Results show that as the % of gelatinized flour

547

by mass in the sample mixture increased to 100%, DSG also increased to 100% (Fig. 7). A

548

straight-lined relationship was observed for both corn and wheat flour. Thus, the agreement

549

between measured values of gelatinized starch and the theoretical values was excellent.

550

Furthermore, as discussed before, since native starch, like gelatinized starch, was also

551

susceptible to AGS attack but at a limited scale, it showed some DSG, which is the Y-axis

552

intercept value in Fig. 7. Because susceptibility of native starch to AGS attack varied with

553

grain species, corn and wheat showed different intercept values on the Y-axis.

554 555

To further validate the new enzymatic method, we measured the 12 selected starchy products

556

for DSG by three methods. Results show that differences in DSG measured among the

557

methods varied with samples (Table 2). For some samples, the three methods gave similar Table 2. Comparison of two proposed methods with the control method in analyzing 12 dry and moist starchy products for DSG. Sample name

558

Moisture* % (wet basis)

Total starch* % (wet basis)

Degree of starch gelatinization (%)** Control method Method 1 Method 2 (Water, 70 min) (60mM NaOH, 15 min) (40mM NaOH, 70 min)

Dry samples Cheerios (breakfast cereal) Trout feed 1 Trout feed 2 Ramen noodles Rotini pasta Tortilla chips

6.43 ± 0.22 gh 5.55 ± 0.20 h 6.47 ± 0.05 gh 6.79 ± 0.01 g 9.89 ± 0.02 f 6.13 ± 0.31 gh

53.11 ± 1.02 b 14.88 ± 0.01 g 11.01 ± 0.28 h 50.37 ± 0.46 c 61.97 ± 0.87 a 51.32 ± 1.16 bc

84.87 ± 1.36 b 84.21 ± 2.66 b 74.54 ± 8.90 b 95.65 ± 3.43 a 19.63 ± 0.81 a 78.45 ± 1.32 c

86.33 ± 0.17 ab 90.82 ± 8.77 ab 80.51 ± 3.45 a 97.39 ± 2.21 a 20.26 ± 0.37 a 82.87 ± 3.18 b

89.43 ± 3.02 a 93.25 ± 0.27 a 81.92 ± 3.03 a 99.02 ± 0.18 a 20.98 ± 0.05 a 88.51 ± 0.34 a

Moist samples Bagel Banana Cooked rice Corn tortilla Hot dog bun Steamed bread

30.88 ± 0.13 e 72.69 ± 0.12 a 66.93 ± 0.16 b 48.07 ± 0.43 c 33.48 ± 0.44 d 47.43 ± 0.32 c

38.35 ± 0.13 de 6.71 ± 0.20 i 28.44 ± 0.09 f 36.98 ± 0.41 e 38.78 ± 0.39 de 39.63 ± 0.56 d

74.10 ± 1.33 b 3.51 ± 1.24 a 91.16 ± 0.87 a 77.76 ± 1.83 a 74.80 ± 2.55 b 78.39 ± 1.35 b

78.89 ± 3.28 ab 4.04 ± 0.61 a 91.18 ± 0.32 a 78.98 ± 3.30 a 80.89 ± 1.42 a 83.75 ± 0.54 a

81.57 ± 1.64 a 3.86 ± 0.77 a 91.46 ± 1.21 a 82.75 ± 3.01 a 80.65 ± 0.43 a 83.25 ± 1.61 a

Each data point was a mean of duplicate (dry samples) or triplicate (moist samples) tests. *Column values of moisture and total starch contents among samples having different letters differed significantly at P <0.05. **Row values of measured DSG by the three methods having different letters differed significantly at P <0.05.

20

559

values. Yet, for other samples, the Control Method gave significantly lower values (p<0.05)

560

than Methods 1 and 2. This finding further supported the notion that the water pretreatment in

561

the previous method (Liu and Han 2012) is still inadequate for fully solubilizing gelatinized

562

starch. Furthermore, for most samples, Methods 1 and 2 gave the same DSG values (p<0.05)

563

but using Method 1 could significantly shorten pretreatment and dramatically increase the

564

assay efficiency. Therefore, Method 1 was preferred and selected as the new method

565

described in Materials and Methods. Although it was unnecessary to measure moisture

566

content and calculate total starch content for DSG assay, as explained early, their inclusion in

567

Table 2 shows that the new method was applicable to products with varying degrees of heat

568

treatments and varying contents of starch and initial moisture.

569 570

3.8. Conclusion

571

Enzymatic determination of DSG has been complex and required measuring not only

572

contents of both gelatinized and total starch but also use of a correction factor for native

573

starch for sophisticated DSG calculations. Furthermore, many previous method developers

574

ignored the need for proper sample pretreatments for measuring both gelatinized starch and

575

total starch, while others failed to run proper sample controls. This study was conducted to

576

address all these problems. Consequently, a new enzymatic method was developed,

577

consisting of differential alkaline pretreatments of samples for measuring gelatinized and

578

total starch, hydrolysis of solubilized starch by amyloglucosidase, colorimetric measurement

579

of released D-glucose by glucose oxidase-peroxidase, and DSG calculation by A510 ratio of

580

gelatinized over total starch without a correction factor for native starch. The new method

581

was validated and compared with a prior method for accuracy and simplicity.

582 583

Acknowledgements

584

We express thanks to Mike Woolman of the USDA-ARS at Aberdeen, ID, for assistance in

585

conducting the experiments and collecting data.

586 587

This work was supported by the United States Federal Government appropriated fund for the 21

588

project “Integrating the Development of New Feed Ingredients and Functionality and Genetic

589

Improvement to Enhance Sustainable Production of Rainbow Trout” (No.

590

2050-21310-005-00-D), U.S. Department of Agriculture, Agricultural Research Service,

591

Washington DC, USA, and by a scholarship under the State Scholarship Fund administrated

592

by China Scholarship Council, Beijing, China.

593 594

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595

AACC (2010). “Approved Methods of Analysis,” 11th Edition, Method 76-11, Method 76-13,

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Da Silva, C. E. M., Ciacco, C. F., Barberis, G. E., Solano, W. M. R., & Rettori, C. (1996).

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structural properties of pregelatinized starch prepared by improved extrusion cooking

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Marconi, E., Messia, M. C., Palleschi, G., & Cubadda, R.

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determining gelatinized starch in processed cereal foods. Cereal Chemistry, 81, 6−9.

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Parada, J., & Aguilera, J. M. (2009). In vitro digestibility and glucemic response of potato

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Schirmer, M., Jekle, M., & Becker, T. (2015). Starch gelatinization and its complexity for analysis. Starch/Stärke, 67, 30–41 Shetty, R. M., Lineback, D. R., & Seib, P. A. (1974). Determining the degree of starch gelatinization. Cereal Chemistry, 51, 364-375. Tako, M., Tamaki, Y., Teruya, T., & Takeda, Y. (2014). The principles of starch gelatinization and retrogradation. Food and Nutrition Sciences, 5, 280-291. 23

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determine starch gelatinization in baked foods. Cereal Chemistry, 57, 242−248.

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Wang, S. J., & Copeland, L. (2013). Molecular disassembly of starch granules during

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gelatinization and its effect on starch digestibility: a review. Food & Function, 4,

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1564-1580.

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Wang, S. Liu, L. Wang, S., & Copeland L. (2017). Structural orders of wheat starch do not

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determine the in vitro enzymatic digestibility. J. Agric. Food Chem. 65, 1697−1706

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Wootton, M., Weeden, D., & Munk, N. (1971). A rapid method for the estimation of starch

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gelatinization in processed foods.

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Xiong, Y., Bale, S. J., & Preston, R. L. (1990). Improved enzymatic method to measure

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processing effect and starch availability in sorghum grain. J. Anim. Sci. 68:3861-3870.

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Zhu, L., Jones, C., Guo, Q., Lewis, L., Stark, C. R., & Alavi, S. (2016). An evaluation of total

656

starch and starch gelatinization methodologies in pelleted animal feed. Journal of Animal

657

Science, 94, 1501-1507.

658 659

24

660

Legends to Figures

661 662

Fig. 1.

Schematic diagram showing key steps of the proposed method for measuring the

663

degree of starch gelatinization.

664 665

Fig. 2.

Effect of NaOH concentration and treatment time on A510 (glucose released) from

666

raw and autoclaved corn flours at three mixing speeds: 50 (a), 150 (b), and 300 (c) rpm.

667 668

Fig. 3.

Effect of NaOH concentration and treatment time on A510 (glucose released) from

669

raw and autoclaved rice flours at three mixing speeds: 50 (a), 150 (b), and 300 (c) rpm.

670 671

Fig. 4.

Effect of NaOH concentration and treatment time at 150 rpm on A510 (glucose

672

released) from native and autoclaved starch isolated from corn.

673 674

Fig. 5. Effect of NaOH concentration and treatment time at 150 rpm on A510 (glucose released)

675

from native and heated flours of two barley varieties: Idaho gold (a) and Transit (b); two oat

676

varieties: Lamont (c) and Ajay (d); and two wheat varieties (soft and hard): Treasure (e), and

677

Boundary (f).

678 679

Fig. 6. Effect of NaOH concentration and treatment time at 150 rpm on the degree of starch

680

gelatinization of raw and autoclaved flours of corn (a) and rice (b).

681 682

Fig. 7. The relationship between the degree of starch gelatinization measured by the proposed

683

method and the percentage of fully gelatinized flour in corn or soft wheat samples.

25

Table 1. Comparison of three sample treatments for total starch measurement by the enzymatic method. Sample pretreatment

Raw corn flour

Dried autoclaved corn flour

Raw rice flour

Dried autoclaved rice flour

Boiling in water (100C) for 60 min Autoclaving for 60 min Mixing in 0.5 M NaOH fir 60 min

70.38 ± 1.43 b 79.31 ± 0.61 a 80.00 ± 0.96 a

75.83 ± 0.93 b 79.04 ± 0.78 a 79.21 ± 0.51 a

77.32 ± 0.78 b 81.01 ± 0.30 a 81.21 ± 0.23 a

80.44 ± 1.04 ab 81.37 ± 0.09 a 81.32 ± 0.16 a

Total starch content at each data point, expressed as % dry matter, was a mean of triplicate tests. Column values with different letters differed significantly at P <0.05.

Table 2. Comparison of two proposed methods with the control method in analyzing 12 dry and moist starchy products for DSG. Moisture* % (wet basis)

Total starch* % (wet basis)

Degree of starch gelatinization (%)** Control method Method 1 Method 2 (Water, 70 min) (60mM NaOH, 15 min) (40mM NaOH, 70 min)

Dry samples Cheerios (breakfast cereal) Trout feed 1 Trout feed 2 Ramen noodles Rotini pasta Tortilla chips

6.43 ± 0.22 gh 5.55 ± 0.20 h 6.47 ± 0.05 gh 6.79 ± 0.01 g 9.89 ± 0.02 f 6.13 ± 0.31 gh

53.11 ± 1.02 b 14.88 ± 0.01 g 11.01 ± 0.28 h 50.37 ± 0.46 c 61.97 ± 0.87 a 51.32 ± 1.16 bc

84.87 ± 1.36 b 84.21 ± 2.66 b 74.54 ± 8.90 b 95.65 ± 3.43 a 19.63 ± 0.81 a 78.45 ± 1.32 c

86.33 ± 0.17 ab 90.82 ± 8.77 ab 80.51 ± 3.45 a 97.39 ± 2.21 a 20.26 ± 0.37 a 82.87 ± 3.18 b

89.43 ± 3.02 a 93.25 ± 0.27 a 81.92 ± 3.03 a 99.02 ± 0.18 a 20.98 ± 0.05 a 88.51 ± 0.34 a

Moist samples Bagel Banana Cooked rice Corn tortilla Hot dog bun Steamed bread

30.88 ± 0.13 e 72.69 ± 0.12 a 66.93 ± 0.16 b 48.07 ± 0.43 c 33.48 ± 0.44 d 47.43 ± 0.32 c

38.35 ± 0.13 de 6.71 ± 0.20 i 28.44 ± 0.09 f 36.98 ± 0.41 e 38.78 ± 0.39 de 39.63 ± 0.56 d

74.10 ± 1.33 b 3.51 ± 1.24 a 91.16 ± 0.87 a 77.76 ± 1.83 a 74.80 ± 2.55 b 78.39 ± 1.35 b

78.89 ± 3.28 ab 4.04 ± 0.61 a 91.18 ± 0.32 a 78.98 ± 3.30 a 80.89 ± 1.42 a 83.75 ± 0.54 a

81.57 ± 1.64 a 3.86 ± 0.77 a 91.46 ± 1.21 a 82.75 ± 3.01 a 80.65 ± 0.43 a 83.25 ± 1.61 a

Sample name

Each data point was a mean of duplicate (dry samples) or triplicate (moist samples) tests. *Column values of moisture and total starch contents among samples having different letters differed significantly at P <0.05. **Row values of measured DSG by the three methods having different letters differed significantly at P <0.05.

Starchy samples Reducing particle size Grind dry samples and pass through a screen with 300 µm openings or less or blend wet samples with 3 parts of water for 30 s on a high speed

Resolubilizing gelatinized starch Mix in 60 mM NaOH for 15 min & neutralize with HCl

Solubilizing total starch Mix with 0.5 M NaOH for 15 min & neutralize with HCl

Hydrolyzing solubilized starch enzymatically Incubate with amyloglucosidase at 37°C for 45 min

Measuring D-glucose colorimetrically React with glucose oxidase-peroxidase reagent

Gelatinized starch content

Total starch content

Expressing results relative to total starch (% gelatinized starch) Fig. 1. Schematic diagram showing key steps of the proposed method for measuring the degree of starch gelatinization.

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A510

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mM NaOH

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mM NaOH

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Fig. 2. Effect of NaOH concentration and treatment time on A510 (glucose released) from raw and autoclaved corn flours at three mixing speeds: 50 (a), 150 (b), and 300 (c) rpm.

A510

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Heated, 70 min

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mM NaOH

Fig. 3. Effect of NaOH concentration and treatment time on A510 (glucose released) from raw and autoclaved rice flours at three mixing speeds: 50 (a), 150 (b), and 300 (c) rpm.

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A510

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NaOH concentration (mM)

Fig. 4. Effect of NaOH concentration and treatment time at 150 rpm on A510 (glucose released) from native and autoclaved starch isolated from corn.

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Raw, 5 min Raw, 15 min Raw, 70 min Heated, 5 min Heated, 15 min Heated, 70 min

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A510

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Fig. 5. Effect of NaOH concentration and treatment time at 150 rpm on A510 (glucose released) from native and heated flours of two barley varieties: Idaho gold (a) and Transit (b); two oat varieties: Lamont (c) and Ajay (d); and two wheat varieties (soft and hard): Treasure (e), and Boundary (f).

100.0

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Raw, 5 min Raw, 15 min Raw, 70 min Heated, 5 min Heated, 15 min Heated, 70 min

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Degree of starch gelatinization (%)

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NaOH concentration (mM)

Fig. 6. Effect of NaOH concentration and treatment time at 150 rpm on the degree of starch gelatinization of raw and autoclaved flours of corn (a) and rice (b).

Degree of starch gelatinization (%)

100.0

Corn 80.0

Soft Wheat

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Fully gelatinized flour (%)

Fig. 7. The relationship between the degree of starch gelatinization measured by the proposed method and the percentage of fully gelatinized flour in corn or soft wheat samples.

Highlights

• Gelatinized and native starch dissolved maximally at different NaOH concentrations. •

Measuring degree of starch gelatinization depended on optimal sample pretreatments

• Calculation is simplified by the ratio of absorbances with no correction factor • The new method is more accurate and simpler.

Conflict of interest statement

The authors declare that there was no conflict of interest or any potential financial or other interests that could be perceived to influence the outcomes of this research.

Statement of the Authors We declare that the work described in the manuscript has not been published previously (except in the form of an abstract, a published lecture or academic thesis, see 'Multiple, redundant or concurrent publication' for more information), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyrightholder.