Vol. 86, No. 2, 2009 133
NOTE
Determining Corn Germ and Pericarp Residual Starch by Acid Hydrolysis
Bernardo C. Vidal, Jr.,1 Kent D. Rausch,1 M. E. Tumbleson,1 and Vijay Singh1,2
Cereal Chem. 86(2):133–135
Corn (Zea mays) is the primary feedstock for fuel ethanol pro- duction in the United States. In 2007, ≈58 million metric tons were processed by the dry-grind industry to produce ethanol and distillers dried grains with solubles (DDGS), a coproduct used as animal diet ingredient. A limited market depressed the price of DDGS, which could be eased if coproducts were diversified (Rausch and Belyea 2006). Fractionation processes (modified dry-grind processes, as they are referred to when adjunct to etha- nol production) have been developed to produce higher value coproducts such as corn germ and fiber (Singh et al 2005). Re- moving germ and fiber could increase fermentation throughput by concentrating starch in the fermentation mash (Singh et al 2005).
However, ethanol yields typically are reduced due to starch lost to germ and fiber fractions during fractionation. Maximizing starch recovery from these fractions is important for process economics.
A common way to measure the effectiveness of fractionation is by determining the residual starch in fractionated germ and fiber.
The enzymatic method is the established way of measuring total starch in cereal products (McCleary et al 1994). The procedure consisted of incubating finely milled samples with thermostable α-amylase at high temperatures to liquefy granular starch before digestion with glucoamylase and quantifying glucose by colori- metric assay (e.g., glucose oxidase-peroxidase-chromogen assay).
As a tool for measuring residual starch in samples that have gone through enzymatic processes (e.g., starch hydrolysis and fermen- tation), a method using another enzyme (thermostable α-amylase) does not appear ideal. For example, such a method would be unlikely to be capable of hydrolyzing resistant starch (McCleary et al 2002), which, expectedly, could grow in proportion to the total residual starch as the extent of enzyme reaction increases.
To a lesser degree, methods relying on acid hydrolysis have been used to determine total starch in plant tissues (Pirt and Whe- lan 1951; Ebell 1969). One potential disadvantage of assays based on acid hydrolysis is overestimation of starch content, apparently due to breakdown of nonstarch carbohydrates (Rose et al 1991;
Chow and Landhäusser 2004). In contrast, McCleary et al (1994) reported that acid hydrolysis gave comparable and sometimes lower starch concentrations in whole cereal products when com- pared with the enzymatic method. The applicability of acid hy- drolysis to residual starch analysis in fractionated grain components has not been the subject of any published study. It would be use- ful to examine whether such a method could accurately estimate starch contents in these samples (i.e., corn germ and fiber).
The use of an assay based on acid hydrolysis to measure starch in corn germ and pericarp fiber was evaluated. The assay, referred to as HCl assay, was adopted with modification from a procedure developed by Ebell (1969), using a more dilute HCl concentration and higher temperature. The use of more dilute HCl and higher temperature (thus allowing for shorter incubation time) could
minimize nonstarch degradation. The results of this assay on frac- tionated germ and fiber samples were compared with the enzy- matic method (Approved Method 76-13, AACC International 2000) using the Total Starch Kit (Megazyme, Bray, Co. Wicklow, Ireland), which shall be referred to as MZYM assay.
MATERIALS AND METHODS Germ and Fiber Fractionation
Using two fractionation processes, corn germ and pericarp fiber were obtained from yellow dent corn grown in 2007 at the Agri- cultural and Biological Engineering Research Farm (University of Illinois at Urbana-Champaign). The dry degerm defiber (3D) process is a dry-fractionation method based on the dry-mill proc- ess that produces flaking grits and smaller endosperm fractions (Murthy et al 2006). The enzymatic milling (E-Mill) process is a wet-fractionation method that uses enzymes to separate germ and pericarp fiber by gravity difference (Singh et al 2005). The modi- fied E-Mill procedure in Wang et al (2005) was followed except enzymes used were NS50086 (granular starch hydrolyzing en- zymes, 0.05% v/w slurry) and NS50045 (neutral pH protease, 0.05% v/w slurry) obtained from Novozymes (Franklinton, NC).
Germ and fiber samples obtained were assayed whole or ground using a coffee grinder until >90% of the sample mass passed through a 600-μm screen.
Destarching and Endosperm Flour Spiking
To evaluate the HCl assay over a wider range of residual starch content, and to examine whether it gives consistent response to increments in starch content, destarched samples were spiked with endosperm flour. To provide destarched germ and fiber, 3D samples were first mixed into slurries consisting of 25% germ and 14% fiber (w/w wet basis) and liquefied (82°C, 1 hr, pH 6.0) us- ing 0.025% v/w α-amylase (Liquozyme SC, Novozyme, Frank- linton, NC). Liquefied samples were incubated (60°C, 20 hr, pH 4.2) with 0.05% v/w and 0.025% v/w glucoamylase (Spirizyme Fuel, Novozyme, Franklinton, NC) for germ and fiber samples, respectively. After discarding the supernatant, remaining germ and fiber were washed with 50% ethanol and copious amounts of distilled water. After overnight drying in a 49°C oven and grind- ing, the destarched samples were assayed for starch content by both HCl and MZYM assays. To increase starch content of germ and fiber samples, endosperm flour (83% w/w starch as deter- mined by both assays) from 3D endosperm fractions was added.
Endosperm flour fraction was increased incrementally to 33% of total weight (wb), equivalent to an upper limit of 30% w/w starch content.
HCl Assay
Fiber or germ samples (≈1 g) in triplicate were placed in 100- mL autoclavable glass bottles (GL45, VWR International, West Chester, PA) containing 50 mL of HCl (0.4N) and autoclaved for 1 hr at 127°C (Napco model 9000D, Thermo Fisher Scientific, Waltham, MA). After cooling to 25°C, a 10-mL aliquot was added with 1.1 mL of Na2CO3 (2M ) to bring to pH 7. Neutralized sam- ples were centrifuged for 10 min at 1,500 × g; supernatant was
1 Agricultural and Biological Eng., University of Illinois at Urbana-Champaign, Urbana, IL 61801.
2 Corresponding author. Phone: 217-333-9510. Fax: 217-244-0323. E-mail: vsingh
@illinois.edu
doi:10.1094 / CCHEM-86-2-0133
© 2009 AACC International, Inc.
134 CEREAL CHEMISTRY
diluted with distilled water to reduce the glucose concentration to
<1 g/L. Glucose was measured using the glucose oxidase- peroxidase or GOPOD colorimetric method (Megazmye). Starch was calculated from the determined glucose concentration and corrected for glucose recovery. The latter was determined from a known glucose solution that was run along with the samples to account for glucose lost during the acid reaction (≈8% glucose lost). Starch content was reported as % dry basis after accounting for the moisture content determined by the oven method (Ap- proved Method 44-19, AACC International 2000). Six repeat as- says (in duplicates) on control starch with 98.8% purity (soluble starch, Fisher Chemical, Fairlawn, NJ) gave a result of 98.2 ± 3.4% w/w, or a coefficient of variation of 3.4%.
RESULTS AND DISCUSSIONS
To examine the effect of sample grinding on the precision of the HCl assay, three repeat assays were made on ground and whole samples (in triplicates) and the standard deviations among triplicates were averaged over the three assays (shown as error bars in Fig. 1). Assay precision, as measured by this pooled stan- dard deviation (SD), improved when samples were ground, espe- cially 3D samples wherein SD decreased by a factor of 10 in fiber and 6.5 in germ. The decrease in SD with grinding was smaller for E-Mill samples (2.8× and 5.1× reduction for fiber and germ, respectively). Both ground and whole germ samples (from E-Mill
and 3D) exhibited larger SD in starch content when compared with fiber samples. Overall, the sample grinding did not appear to increase starch content as determined by HCl assay; however, it had the positive result of homogenizing samples to attain greater assay precision (smaller SD among replicate samples). Subse- quently, all starch content analyses were done on ground samples.
Starch contents of E-Mill samples measured by HCl and MZYM assays were not different (Table I). In 3D samples, starch contents were >15% higher by HCl assay than by MZYM assay.
The MZYM assay was repeated on all samples using DMSO to dissolve starch before liquefaction with α-amylase but the results did not change (results not shown).
To evaluate the assay on a wider range of starch contents, spiked samples were prepared by adding known amounts of en- dosperm flour to destarched 3D germ and fiber samples. Starch contents of destarched samples, based on HCl and MZYM assays, were 10.0 and 7.4% w/w, respectively, for germ, and 4.0 and 2.8% w/w, respectively, for fiber. Enzymes did not digest starch completely during destarching as the assays revealed, partly be- cause samples were used without grinding. Enzymes were unable to access starch in the germ interior or were limited by mass transfer rates because of the smaller surface area. The observed bias (higher starch content by HCl assay) in 3D samples also per- sisted even after destarching. Starch contents determined by HCl assay in the spiked samples are depicted in Fig. 2A and B. Also depicted are expected starch contents (calculated based on the starch contents of destarched fiber and germ and added endo- sperm), represented by two lines owing to the two assays. Starch contents of spiked samples followed the expected line for HCl assay, showing consistent response of HCl assay to sample spik- ing. Because starch content and bias in the destarched fiber were less than half their values in destarched germ, the bias in the spiked fiber samples diminished with increasing endosperm frac- tion (decreasing percentage weight of fiber).
As inferred from Fig. 2, the observed bias in 3D samples was not dependent on starch content. One factor that could result in underestimation of starch content by the MZYM assay would be the presence of resistant starch (McCleary et al 1994). As noted earlier, DMSO treatment did not affect the outcome of the MZYM assay. Methods relying on DMSO treatment have under- estimated the amount of resistant starch present in some samples (McCleary et al 2002).
An improved alternative proposed for resistant starch determina- tion involves incubation with pancreatic α-amylase under condi- tions that mimic in vivo starch hydrolysis. Applying this method (resistant starch assay kit, Megazyme) on germ samples yielded resistant starch contents that were below detection limits (in both E-Mill and 3D). Nonresistant starch (effectively the total starch) was determined to be 17.3 ± 0.4% w/w for E-Mill germ and 33.5
TABLE I
Starch Contents of Germ and Fiber Samples (After Grinding) Obtained from E-Mill (EM) and Dry Degerm Defiber (3D) as Determined by HCl and MZYM assays
HCl Assay (% w/w)a MZYM Assay (% w/w)b Assay Comparison
Samples Meanc SEd Meanc SEd Δ (% w/w)e P Valuef
Fiber
EM 18.5 0.5 18.2 0.2 0.4 0.376
3D 29.9 0.4 25.4 1.0 4.4 0.006
Germ
EM 16.6 1.0 15.7 0.5 0.9 0.329
3D 31.8 1.1 26.5 0.3 5.3 0.008
a HCl assay, an acid hydrolysis based starch assay according to Ebell (1969) with modifications.
b MZYM assay, enzymatic starch assay (Approved Method 76-13, AACC International 2000) using Megazyme Total Starch Kit.
c Means of three measurements by HCl assay and two measurements by MZYM assay.
d Standard errors of means.
e Absolute difference between mean results of the two assays (HCl – MZYM), also referred to as bias in the text.
f P value for the difference between means obtained by t-test (two-tailed, α = 0.05).
Fig. 1. Starch content of germ and pericarp fiber, with and without grinding, obtained from E-Mill and dry degerm defiber (3D) as determined by HCl assay. Mean values (from three determinations) are shown alongside data points. Error bars represent ±1 pooled standard deviation of triplicate samples (averaged over three determinations).
Vol. 86, No. 2, 2009 135
± 1.3% w/w for 3D germ. These values were not significantly different from the starch content determined by HCl assay (Table I), indicating the likelihood that the bias was due to the MZYM assay’s underestimation of the residual starch content in these samples.
The resistant starch assay, unlike the MZYM assay, involved enzymatic hydrolysis at a temperature (37°C) below the gelatini- zation temperature (60°C). This was similar to the enzyme treat- ment used for the E-Mill process (incubation at 48°C), which may explain why E-Mill samples did not exhibit discrepancy in starch content assayed by HCl and MZYM assays. Hydrothermal pre- treatment (at temperatures below gelatinization) has facilitated enzymatic starch hydrolysis (Jacobs and Delcour 1998). In con- trast, high temperature cooking and mashing have produced cross- linking in the endosperm protein matrix, creating weblike struc- tures around starch granules that could hinder enzymatic degrada- tion (Ezeogu et al 2008; Zhao et al 2008). The destarching procedure (liquefaction at 82°C) and the DMSO treatment (incu- bation at 100°C) exemplified this high temperature treatment,
which might explain why they have perpetuated the observed underestimation by the MZYM assay.
CONCLUSIONS
The HCl assay was satisfactory for determining starch content of fractionated corn germ and fiber. Sample grinding improved the assay precision, even though grind size might not be critical for complete hydrolysis of the samples. The assay was not af- fected by starch recalcitrance to enzyme attack, especially in sam- ples undergoing high temperature treatment. The assay would be useful for evaluating the effectiveness of corn fractionation to maximize the recovery of starch in modified dry-grind processes.
ACKNOWLEDGMENTS
We thank Sidney Knight for conducting some of the assays during a summer laboratory internship.
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[Received November 12, 2008. Accepted January 31, 2009.]
Fig. 2. Starch contents of destarched germ (A) and pericarp fiber (B) as determined by HCl assay after incremental addition (spiking) with endo- sperm flour. Error bars are ±1 standard deviation of sample duplicates.
HCl expected refers to calculated starch contents based on HCl assay of starch in unspiked destarched samples; MZYM expected refers to calcu- lated starch contents based on the MZYM assay of starch in unspiked destarched samples.