George E. Inglett,'2 Devin J. Rose,' David C. Stevenson," Diejun Chen,' and Atanu Biswas4. ABSTRACT .... DOGS was obtained from MGP Ingredients (Atchison, KS), .... tracts of barley, rice, oat, wheat, buckwheat, and millet using the.
eXtra* Total Phenolics and Antioxidant Activity of Water and Ethanolic Extracts from Distillers Dried Grains with Solubles With or Without Microwave Irradiation George E. Inglett,' 2 Devin J. Rose,' David C. Stevenson," Diejun Chen,' and Atanu Biswas4 ABSTRACT
Cereal Chem. 86(6);661-664
The purpose of this study was to determine the efficacy of extracting phenolic compounds with antioxidant activity from distillers' dried grains with solubles (DOGS) with water. 50% aqueous ethanol, and absolute ethanol, using microwave irradiation or a water bath at various temperatures. DDGS was extracted for IS min with each solvent while heating at 23, 50, 100. and 150°C by microwave irradiation or in a water bath at 23, 50, and 100°C. Phenolic content of extracts increased with increasing temperature to a maximum of 12.02 mg/g in DDGS extracts that were microwave irradiated in water or with 50% aqueous ethanol at 150°C.
Antioxidant activity range was l.49-.53 imol of Trolox equivaients/g of DOGS. Highest antioxidant activities were obtained from 50% aqueous ethanol extracts at all temperatures, and water extracts that were heated at 1011) and 150°C. These data indicate that DOGS extracts with high phenolic content and antioxidant activity can be obtained from DDGS, particularly with the use of water or 50% ethanol and high temperature (100 or 1500 C). This may be valuable to ethanol manufacturers, livestock producers, and food and nutraccutical companies.
Currently, corn starch accounts for z98% of the feedstock used for ethanol production in the United States (Hoffman et al 2007), and demand for ethanol pushed production of corn to a record level in 2007 (Baker and Lutman 2008). The residues remaining after fermentation of starch to ethanol are dried and-referred to as distillers' dried grains (DOG) or distillers' dried grains with solubles (DOGS), depending on whether or not the soluble fraction was blended with the insoluble fraction. As the bio-fuel industry continues to expand, the supplies of DDG or DOGS will increase proportionately. The composition of DOGS can vary widely depending on production parameters and fermentation conditions but they contain 50% carbohydrate (mostlydietary fiber), 30% protein, and 12% oil (San Buenaventura et at 1987; Singh et at 2002); the remainder being ash, phenolic compounds, and other minor components. DOGS are commonly used as animal feed and fertilizer (Boydston et al 2008; Swiatkiewicz and Koreleski 2008). However, for the ethanol industry to succeed, it will be critical to develop new, value-added uses for its byproducts that promote bio-fuel profitability (Hoffman et at 2007). It has been well accepted that antioxidants such as phenolics inhibit lipid peroxidation in food products and improve food quality (Velasco and Dobarganes 2002). Antioxidants may also improve redox status in biological systems and reduce risk of health problems such as cancer, diabetes, cardiovascular disease, and neurodegeneration (Srinivasan et al 2007). Residues remaining
after fermentation of other grain's are high in these beneficial phenolic acids (Mussatto et al 2006). Because corn is also an abundant source of phenolic acids (Adorn Liu 2002), DDGS may exhibit important market value for its phenolic content and antioxidant activity. Phenolic content and antioxidant activity of extracts from agricultural products may be affected by processing conditions. For example, ethanol was the best extraction solvent for solubilizing phenolic compounds from corn tassels when tested against water, methanol, acetone. hexane, chloroform, butano!, petroleum ether, and niethylene chloride (Mohsen and Ammar 2009). Dry-heat treatments up to 160°C significantly increased total extracted phenolics and antioxidant activity of mango seeds (Soong and Barlow 2004). Because DOGS represents a potentially valuable source of phenolic acids with antioxidant activity, studies are needed to determine conditions that yield high extraction of these compounds. Therefore, in this study, we investigated the effectiveness of extracting phenolic compounds with antioxidant activity from DOGS with water, 50 97o aqueous ethanol, or absolute ethanol, using microwave irradiation or a water bath at various temperatures.
*The e-Xtra logo stands for "electronic extra" and indicaics that Fig. 3 appears in color online. Cereal Products & Food Science Research Unit. National Center for Agricultural Utilization Research. USDA, ARS. 1815 N. University Street, Peoria. IL 51604. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product. and the use or the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. 2 Corresponding author. Phone; 309-681-6363. Fax; 309-681-6685. E-mail address: George.lnglett(lars.usda.gov 3 Present address; National Starch. 10 Finderne Ave. Mail Stop 2130. Bridgewater, NJ 08807. Plant Polymers Research Unit. National Center for Agricultural Utilization Research, USDA. ARS. ISIS N. University Street, Peoria, 1L51604. doi;lO.1o94/CCHEM'86-6-0661 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. AACC International, Inc., 2009.
MATERIALS AND METHODS DDGS Source and Experimental Design DOGS was obtained from MGP Ingredients (Atchison, KS), ground using a Fritsch rotor speed-mill (Idar-Oberstein, Germany), and sieved through a 1-mm screen (No. 8 mesh). DOGS was then suspended in water, 50% aqueous ethanol, or absolute ethanol. While other solvents have been used to extract phenolic compounds from cereal grains (Sun and He 2005; Mohsen and Ammar 2009). a focus of this study was to produce extracts that could be utilized by food manufacturers and nutraeeutical companies to improve nutrition of human and animal diets. Water and ethanol are readily available at plants that produce DOGS, and may be food-grade; therefore. these solvents were used in this study. Slurries were then extracted at 23, 50, 100, or 150°C using microwave irradiation, or using a water bath as the heat source at 23, 50. and 100°C. A temperature of 150°C was not used in the water bath due to limitation of conditions. The extracts obtained were centrifuged and analyzed for total phenolics, antioxidant activity, and color. The effect of extraction temperature, solvent Vol. 86, No. 6, 2009 661
type, and heating method were then compared to determine which conditions resulted in the highest level of total phenolics and antioxidant activity, accompanied by a moderate degree of color. Extraction with Microwave Irradiation Ground DDGS (1 g, in triplicate) was suspended in 50 mL of the desired solvent in a 100-mL perfluoroalkoxy, Teflon reactor vessel containing a stir bar. Vessels were then sealed with special lids equipped with a temperature sensor and placed in an advanced microwave system (Ethos 1600, Milestone, Monroe, CT) which was controlled and monitored by EasyWave software (v.3.5.4.1). During treatment, the vessels were stirred magnetically at 320 rpm, and microwave power was continually varied such that each vessel took 5 min to reach the set temperature, and then the set temperature was maintained for 15 mm. Set temperatures were 50, 100, and 150°C. For an extraction temperature of 23°C (room temperature), the extraction vessels were placed in the microwave without heating. The average microwave wattage required to reach and maintain set temperature is shown in Table I. After the microwave irradiation, vessels were allowed to cool before samples were transferred to test tubes and centrifuged at 1,000 x g for 10 mm. The supernatant was used directly for analysis of total phenolics and antioxidant activity. TABLE I Average Microwave Wattage Required to Reach and Maintain Set Temperature Set Temp Come-up Time Treatment Time (°C) (0-5 mm) (5-20 mm)
Solvent
Water
23 50 100 150 23 50 100 150 23 50 100 150
50% Aqueous ethanol
Absolute ethanol
na
na
86 210 346
24 76 116
na
na
79 196 234
28 73 146
na
na
50 126 160
22 58 136
Average wattage required to reach set temperature (come-up time) and to maintain set temperature (treatment time) during extraction of distillers' dried grains with water, 50% aqueous ethanol, or absolute ethanol using microwave irradiation at three temperatures. At 23°C no microwave power was required; na, not applicable.
Extraction with Water Bath Heating DDGS was ground and suspended in solvent as described above and then heated in water bath at 23, 50, or 100°C for 15 min in triplicate. Tubes were removed from water bath and centrifuged at 1,000 x g for 10 min and analyzed for total phenolics and antioxidant activity. Phenolic Content and Antioxidant Activity of DDGS Extracts The phenolic contents of the DDGS extracts were analyzed by the Folin-Ciocalteau colorimetric method (Singleton et al 1999; Waterhouse 2001). Briefly, to 100 tL of extract, 7.9 mL of deionized water and 0.5 ml. of Folin-Ciocalteau reagent (F-9252, Sigma Aldrich, St. Louis, MO) were added, mixed on a vortex mixer; 1.5 mL of 1 .85M sodium carbonate was added after 8 mm. Absorbance of samples was measured at 765 nm after 2 hr and gallic acid was used as an external standard. Antioxidant activity was determined using the 1,1-diphenyl-2picryl-hydrazyl (DPPH) method as described by Sensoy et al (2006). Results were expressed as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalents. Data Analysis Data were analyzed using statistical software (v.8, SAS Institute, Cary, NC) using a mixed model analysis of variance with Tukey's test to determine significant differences between treatment combinations. Significance was defined as P < 0.05. RESULTS Effect of Extraction Temperature on DDGS Extracts Phenolic content. Extraction temperature had a clear influence on the phenolic content of DDGS extracts. In general, higher temperature resulted in more solubilized phenolic compounds. Significant increases in phenolic compounds for the microwave irradiated extracts were found at 50-100°C and 100-150°C for all solvent types (Fig. IA). In the water bath heated extracts, significant increases in phenolic content were observed for increasing temperature intervals in all cases, except when comparing water extracts at 23 and 50°C (Fig. 113). DDGS extraction at 150°C using either water or 50% aqueous ethanol resulted in the highest degree of solubilized phenolic compounds. Antioxidant activity. The effect of extraction temperature on antioxidant activity was not as clear as the effect on total phenolics. Extraction temperature did not affect the antioxidant activity of 50% aqueous ethanol extracts; at all temperatures these extracts
15
10 0
a a
12 0
a 0 I0
C
C a.
0 a-
C -o 0 C 23
50
100
150
23
50
100
Temperature (CC) Fig. 1. Phenolic content of distillers dried grains with solubles (DDGS) extracted with water (black bars). 50% aqueous ethanol (gray bars), or absolute ethanol (white bars) at various temperatures with microwave irradiation (A) or water bath (B). Bars labeled with different letters are significantly different (P -o CU ci) 0 - -c
oc
23 50 100 150 23 50 1300 23 3 00L5O
100
23 ,fl 100
23 50 100
Fig. 3. l:\LrdCls from distilici'. dried 9idifl' with soluble, DIX1Si under different conditions. Numbers on test tubes indicate extraction temperattire ( 'C).
significantly higher than the corresponding extract that had been heated in a water bath. Microwave irradiation also resulted in significantly higher antioxidant activities in absolute ethanol extracts that were heated to 50 and 100°C compared to heating in a water bath. Color The color of DDGS extracts was noticeably affected by heating method (Fig. 3). At all comparable temperatures, microwave irradiation resulted in darker extracts than those heated in a water bath.
DISCUSSION Because DDGS represents a potentially valuable source of phenolic compounds with antioxidant activity, we tested effects of heating and solvent type on these parameters in DDGS extracts. DDGS was extracted with water, 50% aqueous ethanol, or absolute ethanol, assisted by heating using microwave irradiation (23, 50, 100, and 150°C) or a water bath (23, 50, and 100°C). Total phenolic content of methanol and aqueous methanolic extracts of barley, rice, oat, wheat, buckwheat, and millet using the Folin-Ciocalteau phenol reagent have a range of 0.50-10.5 mg of gallic acid equivalents/g (Sensoy et al 2006; Stratil et a! 2007). Absolute ethanolic extracts from DDGS were 1.49-9.25 mg of gallic acid equivalents/g (Fig. li). Therefore, ethanol may be used as a suitable extraction solvent for producing DDGS extracts with a phenolic content that is typical of other grains. Water and aqueous ethanol were also suitable solvents for extracting phenolic compounds from DDGS. Indeed, at all temperatures tested, these solvents outperformed absolute ethanol with respect to total phenolic content, extracting a maximum of 12.02 mg/g of DDGS using 50% aqueous ethanol and an extraction temperature of 150°C. Water and ethanol have an advantage over methanol as an extraction solvent if the extract is to be for food use, as these so!vents may be food-grade. It is surprising that such high levels of phenolics could be extracted from DDGS. In corn, phenolic acids are mainly present in the bound form, esterified to cell wall structural components such as hernicellulose (Saulnier and Thibault 1999). Therefore, extracts from corn are usually very low in phenolic compounds (Adorn and Liu 2002). While the yeasts used in fermentation do not grow on phenolic compounds (Baranowski 1980), they do produce enzymes that free bound phenolic acids (Goodey 1982). This may explain why some of the DDGS extracts were so high in phenolic compounds. In accordance with Hinneburg and Neubert (2005), higher extraction temperatures resulted in greater solubilization of phenolic compounds from DDGS (Fig. 1). The high temperatures used in this study may have been able to liberate some of the phenolic compounds that remained bound after fermentation. Alternatively, the color of the extracts became darker as extraction temperature increased (Fig. 3), and heat-induced formation of brown pigments may have contributed to the enhanced phenolic content (Makris and Rossiter 2000). The major phenolic compounds present in corn and other cereal grains include the cinnamic acid derivatives: p-coumaric, caffeic, ferulic, and sinapic acids, with ferulic being the most abundant in corn (Adorn and Liu 2002; Mattila et al 2005). These compounds exhibit antioxidant activity that may help prevent disease (Slavin 2003) so antioxidant activity was tested in each DDGS extract. Water, 50% aqueous ethanol, or absolute ethanol produced extracts with antioxidant activities of 1.49-6.52 f.Imol of Trolox equivalents/g of DDGS (Fig. 2). Sensoy et al (2006) reported antioxidant activities of 1.97 and 1.68 timol of Trolox equivalents/g in 50% aqueous methanolic extracts from corn meal and whole wheat, respectively, while Fernandez-Orozco et al (2007) reported an antioxidant activity of 2.54 .tmo1 of Trolox equivalents/g in 80% methanolic extracts from soybean. Therefore, extraction of DDGS under certain conditions can produce extracts Vol86. No. 6, 2009 663
that are 2-3x higher than reported for other cereal and legume products. Fifty percent aqueous ethanol at all temperatures tested, or water at high temperature (i.e.. IOU or 150°C), was consistent with these outcomes. The extracts in absolute ethanol had significantly lower antioxidant activities compared to water and 50% aqueous ethanol, although these values were still in the range of extracts from other products that are considered good sources of antioxidants (Stratil et at 2007). Because p-counianc, caffcic, ferulic, and sinapic acids are the most abundant phenolic compounds in corn, and they also exhibit considerable antioxidant activity, it would be expected that extracts with high phenolic content would be accompanied by a proportional increase in antioxidant activity. Surprisingly, this was not necessarily the case (Figs. I and 2). Liazid et al (2007) showed that phenolic compounds with fewer substituents on aromatic rings have greater stability during heat treatment. Furthermore, they showed that hydroxylated phenolic acids were more labile than those that were methylated. Because the hydroxyl groups play such an important role in the antioxidant activity of phenolic compounds, it may be that high temperatures partially destroy the antioxidant capacity of hydroxylated phenolic acids, or that high temperatures extract a greater degree of phenolic compounds from DDGS that lack substituents that possess antioxidant activity. We hypothesized that the method of heating (microwave irradiation or water bath) may affect the extraction of phenolic compounds or the antioxidant activity of DDGS. No significant differences were observed between heating methods with respect to phenolic content (Fig. 1). However, some significant differences between microwave heating and water bath heating with respect to antioxidant activity were apparent (Fig. 2). fit cases, microwave irradiation was higher than water bath heating. It is possible that the differences may have been caused by better mixing technology employed in the microwave system compared with the water bath and not necessarily due to heating mechanism. The microwave system was equipped with magnetic stirring capabilities, thus samples could be continually stirred, whereas during water bath heating, the tubes were only vortex-mixed every 5 mm. Because such moderate differences were discovered between heating methods, in the future, heating using water may he more feasible as it is more conducive to large-scale operation and requires less specialized equipment compared to microwave irradiation. CONCLUSIONS This is the first report to show that DDGS represent a significant source of phenolic compounds with antioxidant activities that may be as much as 2-3x higher than extracts from other common grains or legumes. Overall, 50% aqueous ethanol extracted the most phenolic compounds with antioxidant activity, followed closely by water, and then absolute ethanol. In general, higher extraction temperatures resulted in higher phenolic content of extracts, but not necessarily higher antioxidant activity. The use of DDGS extracts in the food. nutraceutical, and farm industries will depend on further characterization of the phenolic acids in DDGS and optimization of extraction conditions. The findings from this study will provide a solid foundation for these future studies. ACKNOWLEDGMENTS We wish to thank Janet Berfield for microwave assistance and Debra Palmquist for help on statistical analysis. LITERATURE CITED Adorn. K. K., and Liu, R. H. 2002. Antioxidant activity of grains. J. Agric. Food Chem. 50:6182-6187. Baker, A.. and Lutman. H. 2008. Peed year in review (domestic): Record demand drives U.S. feed grain prices higher in 2007/08. Available online at: http://usda.mannlib.cornell.edu/usda/currentiFDS-yearboolciFDS 664 CEREAL CHEMISTRY
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[Received November 17, 2008. Accepted August 17, 2009.1
