Journal of the Science of Food and Agriculture J Sci Food Agric 81:17±21 (online: 2000)

Evaluation of starch analysis methods for feed

samples†
Mary B Hall,1* Jocelyn P Jennings,1 Betty A Lewis2 and James B Robertson3
1
Department of Animal Sciences, University of Florida, PO Box 110920, Gainesville, FL 32611, USA
2
Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA
3
Department of Animal Science, Morrison Hall, Cornell University, Ithaca, NY 14853, USA

Abstract: The objectives of this work were to evaluate the ef®cacies of commercial starch analyses and
of starch analysis extraction and gelatinisation procedures. In Study 1, accuracy and speci®city of
commercially available starch analyses were evaluated with six co-operating laboratories (®ve
commercial, one university). Results from 11 test samples showed three laboratories with recoveries of
puri®ed starch of 92 g kgÿ1 or less. Three and four laboratories had in¯ated values when samples
contained glucose or sucrose, respectively. Analyses appeared to have good speci®city for glucose.
Incompleteness of starch detection and interference by non-starch carbohydrates can affect
commercially available analyses. In Study 2, extraction with 80:20 ethanol/water (v/v; 80EtOH) or
90:10 ethanol/water (v/v; 90EtOH) to remove low-molecular-weight carbohydrates, and gelatinisation
with heat, alkali (KOH), 6 M urea or 8 M urea were evaluated. Extraction with 80EtOH or 90EtOH
reduced interference from non-starch carbohydrates. Gelatinisation with heat was adequate for good
recoveries of starch glucose for both control (non-extracted) and 80EtOH-extracted samples;
gelatinisation with alkali was required for 90EtOH-extracted samples. Recoveries of pure starch
samples were greatest with no extraction and heat gelatinisation. 80EtOH extraction with heat
gelatinisation appears to be an adequate preparation method when removal of low-molecular-weight
carbohydrates is desired.
# 2000 Society of Chemical Industry

Keywords: starch; feed analysis; methods

INTRODUCTION EXPERIMENTAL
Analysis of starch in feedstuffs is often accomplished Study 1. Laboratory comparison
by enzymatic methods in order to distinguish the Six feed analysis laboratories (®ve commercial, one
glucose in starch from that in other carbohydrates. university) collaborated in this study. Each laboratory
Complete hydrolysis of starch to glucose and speci®c was sent 11 samples identi®ed only by sample number
measurement of the released glucose are required to (Table 1), for starch analysis according to their usual
obtain accurate starch values. However, endogenous methods (Table 2). The samples included pure
glucose in the sample, glucose released by non- samples and feed samples. Pure samples included
amylolytic enzymes, or interference of mono- corn starch (CS; Sigma Chemical Co, St Louis, MO,
saccharides other than glucose can overestimate the USA), potato starch (isolated in our laboratory), a-D-
starch content. Pre-extraction with aqueous ethanol glucose (Glc; Sigma Chemical Co, St Louis, MO,
can reduce this interference through removal of low- USA), D-fructose (Fru; Aldrich Chemical Co, Inc,
molecular-weight carbohydrates. However, it has Milwaukee, WI, USA), cellobiose (Cb; Acros Organ-
been reported that ethanol pre-extraction of samples ics, New Jersey, USA) and confectioners' sugar (Dixie
can make starch resistant to enzymatic hydrolysis and Crystals, Savannah, GA, USA). The moist total mixed
consequently to analysis.1 Incomplete hydrolysis of ration containing citrus pulp was dried in a 55 °C
starch to glucose or incomplete assay of released forced-air oven. All feed samples were ground to pass
glucose will result in low starch values. The aims of the 1 mm screen of a Wiley mill (Arthur H Thomas,
this study were to evaluate the accuracy of starch Philadelphia, PA, USA).
analyses performed by feed analysis laboratories and Collaborating laboratories used various methods of
to evaluate the effectiveness of aqueous ethanol gelatinisation followed by enzymatic hydrolysis in
extraction and starch gelatinisation methods in starch sodium acetate buffer (Table 2). Glucose detection
analysis. methods varied. Laboratories A, B, C and D analysed

* Correspondence to: Mary B Hall, Department of Animal Sciences, University of Florida, PO Box 110920, Gainesville, FL 32611, USA
†
Florida Agricultural Experiment Station, Journal Series No R-07473
(Received 31 March 2000; accepted 20 July 2000)

# 2000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2001/$30.00 17

MB Hall et al

Table 1. Starch content of samples (g kgÿ1) Samples

Corn starch a
Corn starch a Corn starch was used for the initial evaluation of
(air dry (dry matter 90EtOH extraction for starch recovery by the four
Sample Number basis) basis) gelatinisation methods. Corn hominy was used for the
initial evaluation of extraction with 80EtOH. Corn
Soybean meal (48%) 1 Ð Ð
Citrus pulp 2 Ð Ð
hominy was used because extractions of corn starch
Distillers' grains 3 Ð Ð with 80EtOH gave low and variable starch recoveries.
Hominy feed 4 Ð Ð A visible residue of corn starch remained in the
Corn starch ‡ glucose 5 785 873 80EtOH extraction tubes and persisted through
Corn starch ‡ fructose 6 789 882 repeated rinsings and scrubbing with a rubber police-
Corn starch ‡ cellobiose 7 786 872 man. This residue was not visible with 90EtOH. In
Confectioners' sugar 8 Ð Ð addition to corn starch (CS), the potato starch,
Corn starch 9 878 1000 CS ‡ Glc, CS ‡ Fru, CS ‡ Cb and confectioners' sugar
Citrus pulp TMRb 10 Ð Ð samples from Study 1 were analysed using heat gela-
Potato starch 11 Ð Ð
tinisation of unextracted samples, and as 90EtOH-
a
Dashes denote an unknown starch content. extracted samples with heat or KOH gelatinisation.
b
A total mixed ration for dairy cattle containing approximately 25% of dry The starch values of control, 80EtOH and 90EtOH
matter as citrus pulp.
treatments were compared using the soybean meal,
citrus pulp, distillers' grains, hominy feed and citrus
TMR samples from Study 1. The gelatinisation
glucose with an automated method using glucose method used with each extraction type was Heat or
oxidase (YSI Incorporated, Yellow Springs, OH, the gelatinisation method that provided the highest
USA), in which free glucose was measured as starch value for a given extraction method in the initial
hydrogen peroxide released from the action of glucose evaluations.
oxidase on glucose. Laboratory E analysed for glucose
with an automated system with glucose oxidase± Aqueous ethanol extractions
peroxidase and phenol reagents, with glucose 80EtOH was prepared by combining 840 ml of 95%
measured as the absorbance of the sample at ethanol and 160 ml of distilled water (dH2O), and
l = 505 nm. Laboratory F measured free monosacchar- 90EtOH contained 950 ml of 95% ethanol and 50 ml
ides by a ferricyanide method after enzymatic hydro- of dH2O. Samples were extracted by continuously
lysis of the starch. Only laboratory F used sample shaking 0.2 g of air-equilibrated sample with 40 ml of
blanks, which were cold water extracts that were acid the selected ethanol/water solution at room tempera-
hydrolysed. The laboratories reported their results on ture (solution temperature 17±24 °C) for 4 h in a
air dry and dry matter (DM) bases. 25 mm  150 mm Pyrex tube with a Te¯on-lined screw
cap. Capped tubes were placed horizontally in a rack
within a mechanical shaker, with the length of the tube
Study 2. Evaluation of ethanol extraction and parallel to the motion of the shaker for maximum
gelatinisation methods agitation. Extracted samples were ®ltered under
The effectiveness of extraction and gelatinisation vacuum through 70 mm diameter Whatman GF/A
methods on starch analysis values was evaluated. glass ®bre ®lters (Fisher Scienti®c, Atlanta, GA, USA)
Samples were analysed for starch without pre-extrac- in a BuÈchner funnel. The aqueous ethanol solution
tion (control) or after extraction with 80:20 ethanol/ used for the extraction was used to rinse the residues
water (80EtOH) or 90:10 ethanol/water (90EtOH). from the tubes, and for two additional rinses (10 ml
Four gelatinisation methods were investigated: heating each) of the residue. Filters with residues were stored
(Heat), KOH, 6 M urea and 8 M urea. Control samples in 100 ml Pyrex beakers capped with aluminium foil
were heat gelatinised but without pre-extraction with until analysis. The entire ®lter with residue was used
aqueous ethanol. for the subsequent starch assay.

Table 2. Descriptions of starch assay methods used by participating laboratories

Lab Gelatinisation Enzyme Buffer Sugar detected

A Autoclave 1 h in distilled water Glucoamylase (Diazyme L-200) 0.2 M Na acetate Glucose
B Autoclave 1 h in distilled water Glucoamylase (Diazyme L-200) 0.2 M Na acetate Glucose
C Boiling water bath for 1 h in acetate Amyloglucosidase (Sigma Chemical A-7555) 0.2 M Na acetate Glucose
buffer
D Boil 1 h in distilled water Amyloglucosidase (Sigma Chemical A-7255) 0.1 M Na acetate Glucose
E Boil 10 min in distilled water Amyloglucosidase (source unidenti®ed) 0.16 M Na acetate Glucose
F 110 °C for 90 min in distilled water a-Amylase (Aspergillus oryzae, Sigma 0.08 M Na acetate Monosaccharides
Chemical A-2611)

18 J Sci Food Agric 81:17±21 (online: 2000)

 Evaluation of starch analysis methods for feed samples

Gelatinisation separation when more than two treatments were

All gelatinisations were performed with stirring in compared. Orthogonal contrasts of the gelatinisation
100 ml Pyrex beakers. Sample size was 100 mg for corn methods (`Heat ‡ KOH vs 6 M urea ‡ 8 M urea', `Heat
starch and puri®ed samples and 200 mg for feed vs KOH' and `6 M urea vs 8 M urea') were also
samples. For heat gelatinisation, 20 ml of dH2O was performed and are reported when they differed from
added to samples with stirring to moisten. A 0.1 ml the means separation test. Initial evaluations of
aliquot of heat-stable a-amylase (Termamyl 120L, gelatinisation method within extraction type were
Novo Nordisk Biochem, Franklinton, NC, USA) was analysed as a 1  4 factorial arrangement of treatments
added with stirring. The beaker was capped with with gelatinisation method, `assay run' (starch ana-
aluminium foil and heated in a 90±92 °C water bath for lyses made on the same day) and the interaction term
1 h. The sample was cooled at ambient temperature for as factors. The comparison of Heat or KOH gelatini-
15 min prior to ®ltration. sation methods on 90EtOH-extracted puri®ed
The KOH method was a modi®cation of Englyst et samples from Study 1 was examined as a 2  6 factorial
al. 2 dH2O (10 ml) was stirred with the sample. The arrangement of treatments with gelatinisation method,
beaker was then capped with aluminium foil and sample, their interaction term and run as factors. The
heated in a 90±92 °C water bath for 10 min. Termamyl comparison of extraction treatments on Study 1 feed
120L (100 ml) was added with stirring, and the beaker samples using Heat or the gelatinisation method that
was recapped and returned to the water bath for provided the highest values for a given extraction
15 min. After cooling the samples in an ice bath for method was analysed as a 4  5 factorial arrangement
20 min, 10 ml of 4 M KOH was added to each sample of treatments with extraction  gelatinisation, sample
while stirring, followed by the immediate addition of and their interaction term as factors. Signi®cance was
4 M HCl and dilute HCl to adjust the pH to 6±6.5 (pH declared at P < 0.05.
meter).
Gelatinisations with 6 and 8 M urea were performed
by the same procedure. Urea solutions were made RESULTS AND DISCUSSION
fresh with dH2O on the day of analysis. Urea solution Study 1. Laboratory comparison
(5 ml) was added to the sample and stirred. After The recovery values (starch measured divided by
5 min, 10 ml of dH2O and 0.1 ml of Termamyl 120L starch in sample dry matter) for corn starch and potato
were added with stirring. The beaker was capped with starch ranged from 881 to 1015 g kgÿ1. Laboratories
aluminium foil and held at ambient temperature for B, C and F showed consistently low recoveries for
10 min, followed by heating in a 90±92 °C water bath these samples, whereas the other laboratories were
for 30 min. After heating, the sample was cooled at within 5% of the predicted values. A variety of
ambient temperature for 5 min before ®ltering. factors can contribute to low starch recovery, with
errors in starch hydrolysis or glucose measurement, or
Starch analysis miscellaneous technical errors among them. Incom-
After gelatinisation, all samples were processed ac- plete hydrolysis of starch can arise from incomplete
cording to the method of Holm et al. 3 Samples were gelatinisation or conditions that reduce enzymatic
®ltered through glass wool with quantitative transfer activity. Enzyme ef®cacy can be reduced by incubation
into 100 ml volumetric ¯asks. Samples were brought to at temperatures or pH outside the optima for the
volume with dH2O and mixed by repeated inversion of enzyme, insuf®cient time of incubation, or insuf®cient
the stoppered ¯ask. A 1 ml aliquot of sample was enzyme relative to amount of substrate and ¯uid
transferred to a 50 ml volumetric ¯ask. Sodium acetate volume. Inaccurate quanti®cation of glucose may
buffer (0.1 M, pH  4.5, 8 ml) and 50 ml of amyloglu- occur through errors in preparation of the glucose
cosidase (Sigma A-3514 from A niger in ammonium standard, de®cits in equipment calibration, or con-
sulphate, Sigma Chemical Co, St Louis, MO, USA; sumption of glucose in standards by microbes if the
EC 3.2.1.3) were added to the ¯ask. After swirling to solutions are not prepared with preservative (eg
mix, the aluminium foil-capped ¯ask was incubated in benzoic acid) or fresh on the day of analysis.
a 60 °C water bath for 30 min. Flasks were gently Blended pure samples were selected to examine the
swirled to mix every 10 min. After removal from the speci®city of the starch analyses to measure only starch
water bath, samples were cooled to room temperature, or starch-derived glucose. Interfering carbohydrates
brought to volume with dH2O and mixed by repeated may be present in a sample, or generated through
inversion. Glucose was assayed using glucose oxidase± chemical or enzymatic action of non-amylolytic
peroxidase reagent.4 enzymes. The method used to quantify starch hydro-
lysis products, either as glucose or monosaccharides,
Statistical analysis determines the type of carbohydrate that can interfere.
Effects of extraction and gelatinisation methods on The CS ‡ Glc sample presented a situation in which an
analysed starch values were evaluated. Statistical interfering substance (glucose) was present in the
analyses were performed by least squares means unprocessed sample. Laboratories A, B and E reported
ANOVA using the general linear model of SAS.5 starch values greater than 106% of actual starch
The Student±Newman±Keuls test was used for means content for the CS ‡ Glc sample (Table 3), suggesting

J Sci Food Agric 81:17±21 (online: 2000) 19

MB Hall et al

Laboratory
Sample a A B C D E F Mean SE b
Soybean meal (48%) 36 53 35 31 22 69 41 7.0
Citrus pulp 51 50 58 0 39 139 56 18.6
Distillers' grains 81 80 96 33 72 85 75 8.9
Hominy feed 624 652 628 683 637 619 641 9.7
CS ‡ Glc 955 926 894 826 992 778 895 32.9
CS ‡ Fru 854 838 822 845 896 809 844 12.4
CS ‡ Cb 857 847 870 964 915 799 875 23.4
Confectioners' sugar 204 207 184 129 42 8 129 35.0
Corn starch 947 885 921 987 984 894 936 17.9
Citrus pulp TMR 138 126 153 121 135 167 140 7.0
Potato starch 972 913 881 1014 1015 916 952 23.1
a
Table 3. Starch analysis results from six CS ‡ Glc, corn starch ‡ glucose; CS ‡ Fru, corn starch ‡ fructose; CS ‡ Cb, corn starch ‡ cellobiose.
b
laboratories (starch g kgÿ1 sample dry matter) Standard error.

that their analysis methods did not correct for pure starch samples raise questions about the com-
endogenous glucose. Free fructose in the CS ‡ Fru pleteness of gelatinisation or hydrolysis, or the ef®cacy
sample did not appear to in¯ate the starch values for of enzymes. Evaluation of puri®ed samples of the type
any laboratory (Table 3), suggesting good speci®city included in this study allows laboratories to assess the
for glucose. Laboratory F used a cold water extract accuracy and ef®cacy of their starch method.
that was acid hydrolysed as a sample blank with each
assay. Because of this correction, interference from Study 2. Comparison of extraction and gelatinisation
endogenous glucose, fructose or sucrose was elimi- methods
nated. Starch recovery differed with extraction type and
The pure carbohydrates that required hydrolysis to gelatinisation method (Table 4). `Assay run' was not
present interference were cellobiose and sucrose. a signi®cant factor for any of the treatments. Heat,
Laboratories D and E reported starch values greater KOH and 6 M urea gelatinisation methods provided
than 104% for the CS ‡ Cb sample, suggesting equally good recoveries for unextracted corn starch,
cellobiase contamination or technical errors. Results whereas gelatinisation with 8 M urea gave a lower
of the analyses of confectioners' sugar suggest that value. The KOH and Heat gelatinisation treatments
enzyme preparations used by four laboratories con- provided higher starch values than both urea treat-
tained invertase (sucrase) activity. Confectioners' ments for 90EtOH-extracted corn starch. Further
sugar contains only 33 g kgÿ1 starch and 14 g kgÿ1 free evaluation of Heat and KOH methods on 90EtOH-
glucose, yet starch values for four laboratories ranged extracted pure samples showed a higher average starch
from 129 to 207 g kgÿ1. Again, laboratory F's water value for KOH (772 g kgÿ1) than for Heat (763 g kgÿ1;
extract/acid hydrolysis correction prevented interfer- P = 0.0001). The four gelatinisation methods did not
ence by sucrose, exemplifying the advantage of differ in their starch recoveries for 80EtOH-extracted
including appropriate sample blanks. hominy feed according to the results of the Student±
Among the feed samples tested, the standard errors Newman±Keuls test. However, the orthogonal con-
across laboratories for soybean meal, distillers' grains, trast of Heat ‡ KOH vs 6 M urea ‡ 8 M urea for
hominy feed and citrus pulp TMR were small (Table 80EtOH-extracted samples indicated that the two sets
3). In contrast, the standard error for citrus pulp was
more than twice as large. This may be due to the
presence in citrus pulp of approximately 360 g kgÿ1 of Table 4. Starch analysis results for extraction and gelatinisation methods
DM of mono- and oligosaccharides, including glucose (starch g kgÿ1 sample dry matter, mean standard error)
and sucrose, which may interfere with the starch
Gelatinisation method Control a 90EtOH b 80EtOH c
analysis.6 In addition to the analytical issues previously
discussed, variation in results across laboratories may Heat 954  4.8a 915  5.3a,x 608  0.15a
be due to errors in subsampling, or other factors. Dry KOH 945  7.0a 929  4.2a,y 609  0.08a
matter estimates for samples were similar among the 6 M urea 947  1.8a 879  13.2b 591  0.11b
8 M urea 927  6.0b 869  10.2b 580  0.18b
laboratories (data not shown).
The differences in results among laboratories a
Unextracted corn starch.
b
indicate the importance of speci®city and complete- c
Corn starch extracted with 90:10 ethanol/water (v/v) solution.
Corn hominy feed extracted with 80:20 ethanol/water (v/v) solution.
ness of recovery in starch analysis. Interference can be
a,b Comparison of all gelatinisation methods. Values in same column with
minimised through use of adequate sample blanks, different letters differ, P < 0.05.
purer enzyme reagents and assays with high speci®city x,y Comparison of Heat and KOH gelatinisation. Values in same column with
for starch glucose. Starch recoveries below 95% for different letters differ, P < 0.05.

20 J Sci Food Agric 81:17±21 (online: 2000)

 Evaluation of starch analysis methods for feed samples

Extraction method Ctrl a 90EtOH b 90EtOH b 80EtOH c

Gelatinisation method d Heat Heat KOH Heat
Corn starch 954 915 929 Ð
Potato starch 962 916 930 Ð
Corn starch ‡ glucose 946 749 836 Ð
Corn starch ‡ fructose 864 810 847 Ð
Corn starch ‡ cellobiose 830 800 827 Ð
Confectioners' sugar 53 25 31 Ð
Soybean meal (48%) 22 20 24 29
Citrus pulp 38 14 13 22
Distillers' grains 56 45 52 63
Hominy feed 667 610 649 620
Citrus pulp TMR 146 124 126 131
Treatment averagee 186a 162c 173b 173b
a
Unextracted samples.
b
Samples extracted with 90:10 ethanol/water (v/v) solution.
c
Samples extracted with 80:20 ethanol/water (v/v) solution.
d
Gelatinisation methods: Heat, heating; KOH, alkali.
e
Table 5. Comparison of extraction methods (starch Average includes soybean meal, citrus pulp, distillers' grains, hominy feed and citrus pulp TMR.
g kgÿ1 of sample dry matter) a±c Values with different letters differ, P < 0.05.

of gelatinisation methods did differ. The urea gelati- ment of released glucose, pre-extraction with aqueous
nisation methods were less effective than either Heat ethanol of samples containing substantial amounts of
or KOH, particularly with the aqueous ethanol- low-molecular-weight carbohydrates, and use of a
extracted samples. The need for KOH gelatinisation gelatinisation method appropriate to the sample. Use
to achieve the greatest starch recovery for the of 80:20 ethanol/water (v/v) as an extractant and
90EtOH-extracted samples is in agreement with gelatinisation by heating appears to be an adequate
reports that ethanol extraction makes samples more method when removal of low-molecular-weight car-
resistant to starch analysis.1 However, the 80EtOH- bohydrates is needed, whilst heat gelatinisation ap-
extracted samples did not re¯ect this resistance. pears to suf®ce for unextracted samples.
The unextracted feed samples gave higher starch
values than did the aqueous ethanol-extracted samples
(Table 5). This may be at least partially due to
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of 80EtOH-extracted samples with Heat gave similar York, p 171 (1981).
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non-starch polysaccharides in plant foods by gas±liquid chroma-
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analysis of starch. Starch/Starke 38:224 (1986).
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Cary, NC (1996).
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these errors may be accomplished by speci®c measure- hydrates. J Sci Food Agric 79:2079 (1999).

J Sci Food Agric 81:17±21 (online: 2000) 21