ASTM E3417 - 25a 测定固体玉米、玉米-高粱混合物和高粱生物质样品中纤维素半纤维素衍生的葡聚糖和半乳聚糖含量的标准试验方法

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Designation: E3417 −25a

Standard Test Method for

Determination of Cellulose/Hemicellulose-Derived Glucan

and Galactan Content in Solid Corn, Corn-Sorghum Blended

and Sorghum Biomass Samples

This standard is issued under the ﬁxed designation E3417; the number immediately following the designation indicates the year of

original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A

superscript epsilon (´) indicates an editorial change since the last revision or reapproval.

INTRODUCTION

The U.S. biofuel industry generates ~15 billion gal of bioethanol annually. Most of this bioethanol

is produced using processes that convert the starch content of corn biomass to glucose through

enzymatic degradation and subsequently to ethanol via yeast fermentation. Starch (a branched

polymer of α-(1-4)-D-glucose units) is the source of the majority of the glucose content present in corn

and sorghum. A tiny proportion of bioethanol is generated using processes that convert cellulosic

feedstock to glucose before yeast fermentation. The emergence of in-situ corn kernel ﬁber (CKF)

conversion processes that allow the simultaneous conversion of starch and cellulosic content in

biomass presents an excellent opportunity for the sector. In order to correctly apportion the ethanol

produced during such a fermentation as either starch-derived or cellulosic, starch content and

cellulosic content must be measured in the biomass material before and after fermentation. While

starch measurement methodology has been well described in literature, the analytical methodology

required to measure cellulosic content in corn biomass was not published until recently by Sluiter et

al.

The procedure outlined herein seeks to address a number of biases that have been identiﬁed and

acknowledged by the authors of that work.

1. Scope

1.1 This procedure can be used to quantify the cellulose/

hemicellulose-derived glucan and galactan (CHDGG) content

in corn biomass samples, corn-sorghum blended biomass

samples and sorghum biomass samples, that also contain

varying levels of starch-derived and yeast-derived glucan. The

method has been shown to provide accurate values for samples

with cellulose content up to 40 % w/w.

1.2 Units—The values stated in SI units are to be regarded

as the standard. No other units of measurement are included in

this standard.

1.3 This standard does not purport to address all of the

safety concerns, if any, associated with its use. It is the

responsibility of the user of this standard to establish appro-

priate safety, health, and environmental practices and deter-

mine the applicability of regulatory limitations prior to use.

1.4 This international standard was developed in accor-

dance with internationally recognized principles on standard-

ization established in the Decision on Principles for the

Development of International Standards, Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade (TBT) Committee.

2. Referenced Documents

2.1 ASTM Standards:

E1705 Terminology Relating to Bioenergy and Industrial

Chemicals from Biomass

E1757 Practice for Preparation of Biomass for Composi-

tional Analysis

This test method is under the jurisdiction of ASTM Committee E48 on

Bioenergy and Industrial Chemicals from Biomass and is the direct responsibility of

Subcommittee E48.05 on Biomass Conversion.

Current edition approved Nov. 1, 2025. Published December 2025. Originally

approved in 2024. Last previous edition approved in 2025 as E3417 – 25. DOI:

10.1520/E3417-25A.

Sluiter, J. B. et al., “Direct Determination of Cellulosic Glucan Content in

Starch-Containing Samples,” Cellulose, 2021, https://doi.org/10.1007/s10570-020-

03652-2.

See supporting data document — Determination of Cellulosic Carbohydrate

Content in Corn Biomass Samples. Available from the ASTM Committee E48

website, https://mcsdocs.astm.org/committee-documents/

E3417%20Supporting%20Document.pdf.

For referenced ASTM standards, visit the ASTM website, www.astm.org, or

contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM

Standards volume information, refer to the standard’s Document Summary page on

the ASTM website.

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the

Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

E3181 Practice for Determination of the Converted Fraction

of Starch and Cellulosic Content From a Fuel Ethanol

Production Facility

2.2 Other Documents:

Supporting information document – Determination of cellu-

losic carbohydrate content in corn and corn-sorghum

blended biomass samples

3. Terminology

3.1 Deﬁnitions of terms:

3.1.1 For deﬁnitions of terms used in this standard, refer to

Terminology E1705.

NOTE 1—The user is advised that the deﬁnitions used by various

industries, marketers, regulatory bodies, and other ASTM Committees can

differ from those speciﬁc to this speciﬁcation. When using this standard,

it is critical to use terminology from Committee E48.

3.1.2 biomass, n—substance wholly comprised of living or

recently living (non-fossil) material.

3.1.2.1 Discussion—This method is suitable for corn

biomass, which is deﬁned as the biomass from large kernals set

in rows on a cob from a cereal plant, sorghum biomass deﬁned

as the biomass from the grains obtained from the bushy

ﬂowerhead of a sorghum plant, and corn-sorghum blended

biomass samples. Note that sorghum is also commonly referred

to as milo and both terms can be used interchangeably.

3.1.3 calibration veriﬁcation standard, CVS, n—standards

used in determining the quality of the calibration curve as well

as the quality of the standard reagents used in preparing the

calibration standards.

3.1.3.1 Discussion—The concentration of the CVS should

be set in the middle of the chosen calibration range and is used

to ensure the calibration remains accurate throughout the

entirety of the sample run. Additional CVS concentrations at

the lower or higher end of the calibration curve can also be run

to ensure accuracy of a sample’s respective concentrations.

3.1.4 cellulose, n—a crystalline, straight chain glucan with

β-(1→4) linkages.

3.1.5 cellulose/hemicellulose-derived glucan and galactan,

n—carbohydrate content present in a sample comprising glucan

and galactan, but speciﬁcally does not include glucan from

starch or yeast.

3.1.5.1 Discussion—For the purpose of this test method,

cellulose/hemicellulose-derived glucan and galactan is abbre-

viated as CHDGG.

3.1.6 cellulosic content, n—as deﬁned by EPA RFS

documentation, the sum of cellulose, hemicellulose, and lignin

in cellulosic feedstock.

3.1.7 cellulosic pellet, n—solid fraction of sample remain-

ing on quartz ﬁlter after precipitation and ﬁltration following

starch and yeast glucan removal.

3.1.8 galactose, n—six-carbon monosaccharide common in

biomass.

3.1.9 glucose, n—six-carbon monosaccharide common in

biomass.

3.1.10 glycogen, n—high molecular weight branched ho-

mopolymer found in yeast consisting of linear α-(1,4)-glucosyl

chains with α-(1,6)-branch points.

3.1.11 hemicellulose, n—a polymeric, branched carbohy-

drate with mixed C5 and C6 monomeric sugars, typically with

a xylose or mannose backbone.

3.1.12 starch, n—a polysaccharide consisting of glucose

monomers joined in α-(1,4)-linkages; the simplest form of

starch is the linear polymer amylose, while amylopectin is the

branched form.

3.1.13 yeast degrading cocktail, n—an enzymatic reagent

for degradation of yeast glucan.

3.1.13.1 Discussion—The reagent should contain the neces-

sary suite of hydrolytic activities to effect yeast glucan removal

under the conditions speciﬁed in this test method, but should

not exhibit contaminating activities that would degrade cellu-

losic or hemicellulosic derived glucan and galactan.

3.1.14 yeast glucan, n—any glucan from a yeast source.

3.1.14.1 Discussion—Includes compounds such as yeast

β-glucan; a branched polymer of (1,3/1,6)-β-D-glucose units,

glycogen, and trehalose; an α-(1,1)-linked glucosyl disaccha-

ride.

3.2 Abbreviations:

3.2.1 CF—Converted Fraction

3.2.2 CHDGG—Cellulose/Hemicellulose-Derived Glucan

and Galactan

3.2.3 CVS—Calibration Veriﬁcation Standard

3.2.4 SRS—Sugar Recovery Standard

3.2.5 YDC—Yeast Degrading Cocktail

4. Signiﬁcance and Use

4.1 The procedure, in brief, consists of solubilization of

biomass using cold caustic extraction, an enzymatic removal of

starch and yeast glucan followed by precipitation of hemicel-

lulosic material, and acid hydrolysis of the residual carbohy-

drate pellet before measurement of glucose and galactose

derived from cellulose and hemicellulose.

4.2 The starch removal procedure is identical to that em-

ployed in the NREL assay

which was itself adapted from the

Megazyme-published starch analysis procedure: RTS-NaOH,

2019.

5,6

4.3 The cellulosic pellet hydrolysis and monosaccharide

measurements are based on those described in the NREL

laboratory analytical procedure “Determination of Structural

Carbohydrates and Lignin in Biomass,” NREL/TP-5100-42618

and “Determination of Cellulosic Glucan Content in Starch

Containing Feedstocks,” NREL/TP-2800-76724.

McCleary, B. V., Charmier, L. M. J., and McKie, V. A., Megazyme, “Measure-

ment of Starch: Critical Evaluation of Current Methodology,” Starch, 1800146, No.

71, 2018, pp. 1-13, DOI: 10.1002/star.201800146.

The Megazyme starch assay procedure referenced in this test method can be

found at:

https://www.megazyme.com/documents/Booklet/K-TSTA-100A_DATA.pdf.

All NREL biomass compositional analysis laboratory procedures referenced in

this test method can be found at: https://www.nrel.gov/bioenergy/biomass-

compositional-analysis.html.

E3417 − 25a

4.4 This test method references Practices E1757 and E3181.

4.5 This test method is intended for use in the measurement

of the fermentable portion of cellulosic content in pre- and

post-fermentation corn, sorghum, and corn-sorghum blended

biomass speciﬁcally for the apportionment of a percentage of

the total ethanol produced in a fermentation as cellulosic.

5. Apparatus

5.1 Analytical balance with precision to 0.1 mg.

5.2 Water bath capable of maintaining 50 °C 62 °C, 40 °C

62 °C, and 30 °C 62 °C.

5.3 HPLC system equipped with a refractive index detector,

column oven capable of reaching 85 °C, and the following

column: Concise Separations CarboSep CHO782, Lead Form

column (or equivalent) with ionic form H

/CO

deashing

guard column.

NOTE 2—The deashing guard column should be replaced every ~50

injections to protect the analytical column.

5.4 pH meter, accurate to 0.01 pH unit.

5.5 Vortex mixer, variable speed, capable of reaching

3000 rpm.

5.6 Magnetic stir plate, capable of stirring at 600 rpm

minimum.

5.7 Filtration apparatus, accommodates 47 mm diameter

ﬁlter.

5.8 Vacuum pump, for ﬁltration.

5.9 Autoclave, capable of reaching and maintaining 121 °C

for 60 min on liquids cycle.

5.10 Freeze dryer/lyophilizer, drying oven or vacuum oven.

6. Reagents and Materials

6.1 Purity of Reagents—Reagent grade chemicals shall be

used in all tests. Unless otherwise indicated, it is intended that

all reagents conform to the speciﬁcations of the Committee on

Analytical Reagents of the American Chemical Society where

such speciﬁcations are available.

Other grades may be used,

provided it is ﬁrst ascertained that the reagent is of sufficiently

high purity to permit its use without lessening the accuracy of

the determination.

6.1.1 D-(+)-glucose—≥99.5 % (GC).

6.1.2 An aliquot of high purity D-(+)-glucose—≥99.5 %

(GC) from a different source (manufacturer or lot), to be used

to prepare CVS.

6.1.3 D-(+)-galactose—≥99.5 % (GC).

6.1.4 An aliquot of high purity D-(+)-galactose—≥99.5 %

(GC) from a different source (manufacturer or lot) to be used to

prepare CVS.

6.1.5 Sodium hydroxide pellets (for solution preparation) or

solution—ACS grade.

6.1.6 Water—HPLC grade, 0.2 µm ﬁltered (or deionized

18 MΩ⁄cm ﬁltered water).

6.1.7 Sulfuric acid—72 % w/w (speciﬁc gravity 1.6338 at

20 °C).

6.1.8 Glacial acetic acid—ACS grade.

6.1.9 Thermostable α-amylase

—(3000 U ⁄mL; one unit of

α-amylase activity is deﬁned as the amount of enzyme required

to release one µmole of 4-nitrophenol from blocked

4-nitrophenyl-maltoheptaoside per minute (in the presence of

excess α-glucosidase) at pH 6.5 and 40 °C).

6.1.10 Amyloglucosidase

—(3260 U ⁄mL; one unit of amy-

loglucosidase activity is deﬁned as the amount of enzyme

required to release one µmole of D-glucose reducing-sugar

equivalents per minute from soluble starch at pH 4.5 and

40 °C).

6.1.11 Yeast-degrading enzyme cocktail (YDC)—

>800 U ⁄mL; one unit of yeast degradation activity is deﬁned as

the amount of enzyme required to release one µmole of

D-glucose reducing-sugar equivalents per minute from soluble

yeast beta glucan (USP 1048288) at pH 5 and 40 °C.

6.1.12 Calcium carbonate—ACS reagent grade.

6.1.13 Calcium chloride dihydrate—ACS reagent grade.

6.1.14 Ethanol or denatured ethanol (for example, Indus-

trial methylated spirits, ~95 % v/v ethanol denatured with

methanol, or similar)—99 % v/v. This solution is used neat or

diluted in deionised water to prepare 95 % v/v or 85 % v/v

solutions as needed.

6.2 Materials:

6.2.1 Positive displacement automatic pipettors, capable of

multi-dispensing with corresponding compatible tips.

6.2.2 Pressure tubes, Of sufficient capacity (for example,

120 mL), glass, with screw on polytetraﬂuoroethylene (PTFE)

caps and O-ring seals or glass vessels (that is, 250 mL

Erlenmeyer ﬂask, beaker, or serum bottle), autoclave safe.

Pressure vessels shall be pressure rated for at least 10.5 kg ⁄cm

at 120 to prevent potential rupture during the autoclave step.

6.2.3 PTFE or glass stir rods, in appropriate size for dilute

acid hydrolysis vessel.

6.2.4 Magnetic stir bars, 10 mm by 6.4 mm or similar.

6.2.5 Nylon or polypropylene sample racks, to accommo-

date tubes with outer diameters (OD) of 30 mm and 16 mm.

6.2.6 Metal sample racks, to accommodate pressure tubes

for acid hydrolysis.

6.2.7 Conical polypropylene tube, approximately 50 mL

capacity, with a tightly ﬁtting screw cap.

6.2.8 Quartz ﬁber ﬁlters, 47 mm, efficiency rating: >99 %

retention dispersed oil particulate (DOP) at 3 µm.

ACS Reagent Chemicals, Speciﬁcations and Procedures for Reagents and

Standard-Grade Reference Materials, American Chemical Society, Washington,

DC. For suggestions on the testing of reagents not listed by the American Chemical

Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,

U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-

copeial Convention, Inc. (USPC), Rockville, MD.

Note that both starch degrading enzymes must be essentially devoid of

cellulose or hemicellulose degrading activities. E-BSTAA and E-AMGDF available

from Neogen, 620 Lesher Place, Lansing, MI 48912, have been shown to be suitable

for this application. Equivalent enzymes from an alternative supplier with the same

or better purity speciﬁcation can also be used.

The sole source of supply of the YDC reagent known to the committee at this

time is Neogen, 620 Lesher Place, Lansing, MI 48912. If you are aware of

alternative suppliers, please provide this information to ASTM International

Headquarters. Your comments will receive careful consideration at a meeting of the

responsible technical committee, which you may attend. Appendix X1 provides

additional information on this reagent.

E3417 − 25a

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标签： #标准试验 #纤维 #试验方法

摘要：

了解 ASTM E3417-25a 标准试验方法，该标准专门用于测定固体玉米、玉米-高粱混合物以及高粱生物质样品中由纤维素和半纤维素衍生的葡聚糖与半乳聚糖含量。本方法为生物质原料的糖类组分定量提供可靠依据，适用于生物燃料、生物基产品及可再生资源研究领域。通过规范化的水解与检测流程，该标准有助于评估生物质转化效率、优化原料筛选及工艺开发。无论是科研机构、检测实验室还是工业生产企业，均可依据此标准实现对生物质中关键多糖成分的精准分析，从而推动生物质能产业的标准化与高效利用。

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ASTM E3417 - 25a 测定固体玉米、玉米-高粱混合物和高粱生物质样品中纤维素半纤维素衍生的葡聚糖和半乳聚糖含量的标准试验方法

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