ISOLATION AND CHARACTERIZATION OF DISTILLER GRAINS DERIVED FROM STARCH HYDROLYSIS OF AGRICULTURAL PRODUCTS

This application claims priority to US provisional application number 60/992,847, filed December 6, 2007, the contents of which are incorporated by reference in its entirety. BACKGROUND

In view of the current demand and shortage of crude oil, alternative sources of energy such as ethanol are gaining significant interest. One source of ethanol is agricultural products. In particular, agricultural products having a high starch content can be processed to produce ethanol. The general process for ethanol production involves fermenting and distilling the starch present in the agricultural product. Although ethanol production is important, the agricultural product also contains other valuable compounds. Distillers grain is a by-product of the process. Distillers grain is generally used as feedstuff for livestock and domestic pets. Thus, the use of the distiller grain is an efficient use of the agricultural product when used in combination with ethanol production. The nutrients present in agricultural products can be temperature sensitive. For example, certain compounds present in the agricultural product can decompose at elevated temperatures. This is an important consideration due to the fact that current techniques for hydrolysing starch to produce ethanol involve high temperature techniques. For example, the "jet cooking" method involves the direct injection of steam to grain mash under high pressure. The near instantaneous temperature increase from 107 to 120 ⁰ C results in the efficient interruption of enzyme-resistant starch structures, which can constitute up to 30% of the total starch in some small grain crops. As a result, starch hydrolysis efficiencies and subsequent ethanol yields are typically higher by several percent. In addition to the stringent conditions during hydrolysis that can decompose valuable compounds, this process involves the use of expensive and intricate devices.

Thus, what is needed is a methodology for hydrolysing starch present in agricultural products for producing high yields of ethanol that does not decompose or reduce the amount of valuable components present in the agricultural product.

SUMMARY

Described herein are methods for isolating and characterizing distiller grains derived from starch hydrolysis that contain high amounts of nutrients and other valuable compounds. The methods for producing the distiller grains generally involve reduced temperatures, which helps minimize the decomposition of nutrients and other valuable compounds present in the distiller grain. The advantages of the materials, methods, and articles described herein will be set forth-in part in the description which follows, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF FIGURES

The accompanying Figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. Figure 1 shows a flow diagram of an exemplary process described herein.

Figure 2 shows the phenolics content in grains and DDGS derived from several agricultural products using the methods described herein.

DETAILED DESCRIPTION

Before the present materials, articles, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

Throughout this specification, unless the context requires otherwise, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a fatty acid" includes a single fatty acid or mixtures of two or more fatty acids. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

Described herein are methods for isolating and characterizing distiller grains derived from starch hydrolysis that contain high amounts of nutrients and other valuable compounds. In one aspect, the starch hydrolysis process comprises (a) contacting an agricultural product with an enzyme for a sufficient time and temperature to hydrolyse starch present in the product, wherein ethanol and distiller grain is produced; (b) isolating the ethanol; (c) isolating the distiller grain; and (d) characterizing the distiller grain for nutritional components.

Figure 1 provides one embodiment of the methods described herein. The first step involves milling or grinding the agricultural product. Examples of agricultural products include, but are not limited to, corn, wheat, milo, oat, barley, rice, rye, sorghum, potato, whey, sugar beets, taro, cassava, fruits, fruit juices, and sugar cane. The agricultural products useful herein can be hulled or dehulled. In one aspect, the agricultural product can be an industrial grade or brewery grade of grain. In this aspect, the industrial grade grain is used to produce industrial grade ethanol and the brewery grade grain is used to produce brewery grade ethanol, respectively.

Upon selecting the agricultural product of choice, the agricultural product can be milled or ground using techniques known in the art (e.g., hammer mills or roller mills) (step 1 in Figure 1). The particle size can vary depending upon the selection of the agricultural product and the reaction conditions such as the selection of the enzyme. In one aspect, the particle size is 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 38

mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, 55 mm, 56 mm, 57 mm, 58 mm, 59 mm, 60 mm, 61 mm, 62 mm, 63 mm, 64 mm, 65 mm, 66 mm, 67 mm, 68 mm, 69 mm, 70 mm, 71 mm, 72 mm, 73 mm, 74 mm, 75 mm, 76 mm, 78 mm, 79 mm, 80 mm, 81 mm, 82 mm, 83 mm, 84 mm, 85 mm, 86 mm, 87 mm, 88 mm, 89 mm, 90 mm, 91 mm, 92 mm, 93 mm, 94 mm, 95 mm, 96 mm, 98 mm 99 mm, and 100 mm, where any particle size can form a lower or upper endpoint for a particle size range. The milled or ground agricultural product can then mixed with water to make a mash that ready for further processing.

In certain aspects, it may be desirable to contact the mash with one or more liquefaction enzymes in order to facilitate processing (step 3 in Figure 1). For example, amylase (e.g., alpha amylases), starch liquefying enzymes (e.g., enzymes sold under the tradename SPEZYME ^® manufactured by Genencor), starch saccharifying enzymes (e.g., enzymes sold under the tradename DISTILLASE manufactured by Genencor), cellulose/hemicellulose hydrolyzing enzymes (e.g., enzymes sold under the tradename OPTIMASH™ manufactured by Genencor), or any combination thereof can be added to the mash. The selection of these enzymes can vary depending upon the agricultural product selected. Not wishing to be bound by theory, the liquefaction enzyme decreases the viscosity of the mash by breaking the starch at α-(l,4)-glucosidic bonds to produce smaller dextrin chains. In certain aspects, the mash is not subjected to extreme heat (e.g., greater than 90 ⁰ C) when the mash is exposed to the liquefaction enzyme. An example of this is jet cooking, which includes very high gravity jet cooking (hereinafter VHG jet cooking). During jet cooking, the mash undergoes direct steam injections at temperatures of 100 to 120 ⁰ C. Although this methodology helps reduce the viscosity of the mash when used in combination with a liquefaction enzyme, this approach has considerable economic drawbacks. First, the process uses a large amount of energy to produce ethanol. In addition, the applied heat can damage temperature sensitive proteins, sterols, tocopherols, tocotrienols, phenolic s, fatty acids, and other nutrients within the distiller grains and thin stillage. Therefore, not exposing the mash to extreme heat can minimize degradation of the proteins, sterols, tocopherols, tocotrienols, phenolics, fatty acids, and other nutrients within the distiller grains and thin

stillage. The Examples below demonstrate the adverse effects of jet cooking with respect to the degradation of nutrients in the distiller grains. Although it not always desirable to use jet cooking and other heat treatments, in certain aspects it may be desirable. Therefore, the present invention also contemplates the use of jet cooking where appropriate. In certain aspects, the mash is sterilized preferably with a chemical sterilizing agent.

Such an agent includes, but is not limited to, diethyl pyrocarbonate. Chemical sterilization is particularly important when producing industrial ethanol; however, when producing various forms of ethanol for consumption (e.g., beer), chemical sterilization is unneeded and in some aspects discouraged. When a liquefaction enzyme is used, the mash is then transferred to a saccharification reactor. The conditions of the saccharification reactor can vary. In one aspect, the temperature in the reactor is from room temperature to 70 ⁰ C, from 30 ⁰ C to 60 ⁰ C, or from 50 ⁰ C to 60 ⁰ C. In another aspect, the pH is from 2 to 5, 3 to 4, or 3.5 to 4. In a further aspect, the mash is in the saccharification reactor from 20 minutes to one hour, or from 30 minutes to 40 minutes.

The mash is then generally fed from the saccharification reactor to a fermentation reactor, where sugars are converted to ethanol and carbon dioxide. Alternatively, saccharification and fermentation can be performed in the same reactor simultaneously, which is depicted in Figure 1 (steps 4 and 5). Yeast and other compounds useful in fermentation are added to the mash in the fermentor. The addition of nitrogen sources such as, for example, urea, can be added to facilitate the release of starch from the protein matrix. In one aspect, the enzyme useful for fermentation is a protein hydrolyzing enzyme sold under the tradename FERMGEN™ manufactured by Genencor, which is a protease.

It is at this stage a starch hydrolyzing enzyme is added (step 4 of Figure 1). The starch hydrolyzing enzyme is an enzyme that facilitates the cleavage of glucosidic bonds with the addition of water molecules and further facilitates fermentation. In one aspect, the enzyme is a granular starch hydrolyzing enzyme (GSHE), which is an enzyme having glucoamylase activity and having the ability to hydrolyze starch in granular form. Granular starch hydrolyzing enzymes useful herein, include, but are not limited to, those disclosed in U.S. Patent No. 7,303,899, which are incorporated by reference in their entirety. In one

aspect, the granular starch hydrolyzing enzyme is produced by Trichoderma strain (e.g., T. reesei strain) transformed with a heterologous polynucleotide encoding the granular starch hydrolyzing enzyme. In another aspect, the starch hydrolyzing enzyme is STARGEN™ 001 manufactured by Genencor. Other examples of starch hydrolyzing enzymes useful herein can be found in Table 1.

During fermentation, the carbon dioxide can be removed using techniques known in the art. The resulting liquid can be fed to a distillation system composed of distillation columns and a stripping column. The ethanol stream can be directed to a molecular sieve where remaining water is removed using adsorption technology. The yield of ethanol production can be evaluated by techniques known in the art such as, for example, HPLC and GC (see the Examples).

The whole stillage (i.e., the resulting solids after distillation) can be withdrawn from the bottom of the distillation system and centrifuged to produce distillers wet grains (DWG) and thin stillage (liquids). Using techniques known in the art (e.g., evaporation, freeze drying (steps 7 and 8 of Figure I)), the thin stillage (liquid) can be concentrated to form distillers solubles, which can be added back to and combined with a distillers grains process stream and dried. This combined product is referred to herein as distillers dried grains with solubles (DDGS). Alternatively, the distillers wet grains (DWG) can be dried to remove moisture and produce distillers dry grains (DDG). However, when drying either the DDGS or DDG, it is preferable to avoid the use of excessive heat. If high temperatures are used, sterols, tocopherols, tocotrienols, phenolics, fatty acids, and other nutritional components present in the distiller grain can be degraded. Thus, in one aspect, the distiller grains are not subjected to heat having a temperature greater than 80 ⁰ C. In other aspects, the distiller grains are not subjected to heat greater than 85 ⁰ C, greater than 90 ⁰ C, greater than 95 ⁰ C, or greater than 100 ⁰ C. For example, the distiller grain is not subjected to spray drying, which is typically performed at 100 ⁰ C to 120 ⁰ C.

The methods described herein produce distiller grains (DWG, DDG, and DDSG) having increased amounts of nutrients and compounds. As shown below, the high amounts of sterols, tocopherols, tocotrienols, phenolics, and fatty acids are present in the distiller grains. This feature has not been recognized and provides a source of nutrients and other

compounds that can be subsequently isolated and used in supplements in the health industry. In one aspect, the nutrients and other valuable compounds can be separated from the distiller grains in the fermentor after fermentation is completed. For example, organic solvents or supercritical fluids can be used to extract nutrients from the distillers grains. By isolating higher concentrations of these components, the overall process of ethanol production from agricultural products is much more efficient with respect to the use of the agricultural products. Indeed, more nutrients and other valuable components of the agricultural product are available for use as opposed to being wasted due to decomposition or some other mechanism. In certain aspects such as industrial applications, distiller wet grains (DWG) can be extracted continuously during ethanol production. Thus, the distillers grains are never dried using industrial techniques and, thus, any nutritional components present in the grains are preserved.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the materials, articles, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ⁰ C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions. The experimental procedures and data presented below have been accepted for publication by the journal Applied & Environmental Microbiology (American Society for Microbiology).

Abbreviations. DDGS, dried distiller's grain with solubles; DEPC, diethyl pyrocarbonate; HPLC, high-pressure liquid chromatography; GC, gas chromatography; SSF, simultaneous saccharification and fermentation; VHG, very high gravity fermentation (fermentation that

contains 27 grams or more of solids/100 grams of mash); DM, dry matter; GE, gross energy. Grains involved in this study. Three 2-row spring barley varieties, Bold, Xena, and Fibar, were used in this study. Bold was grown in 2006 at the Kaun Seed Farm in Red Deer, AB. Xena feed barley was grown in 2006 and developed at the Crop Development Centre at the University of Saskatchewan (Saskatoon, SK, Canada). Fibar barley was a hull-less variety developed at the Crop Development Centre at the University of Saskatchewan. Pioneer Hi- Bred corn was supplied by Pioneer Hybrid Ltd. (Chatham, ON, Canada), whereas CPS wheat was provided by Jim Greilach (Alberta Agriculture and Food, Barrhead, AB, Canada). Grain samples were ground in a Jacobson-Carter Day Cutler-Hammer mill (using a 1.98 mm sieve) or in a Retsch mill model ZM 100 (using a 0.5 mm sieve). Ground grains were stored in airtight plastic bags at room temperature.

Enzymes, reagents, and chemicals. STARGEN 001 (an enzyme cocktail containing Aspergillus kawachi α-amylase expressed in Trichoderma reesei and a glucoamylase that work synergistically to hydrolyze granular starch to glucose), Optimash TBG (viscosity reducing) and Fermgen (protease) enzymes were provided by Genencor International (Palo Alto, CA, USA). Viscozyme Barley (viscosity reducing), Viscozyme Wheat (viscosity reducing), Liquozyme SC (α-amylase), and Spirizyme® Fuel (glucoamylase) enzymes were obtained from Novozyme (Bagavaerd, Denmark). SuperStart Yeast was provided by Ethanol Technology (Milwaukee, WI, USA). Urea was purchased from Fisher Scientific. Traditional Stargen 001 treatment at 48 ⁰ C and 20% solids

The ground grains (0.5 mm) were mixed with water (1.5 kg grain/2.78 kg water) to obtain a mash with 35% solids. Using 12M hydrochloric acid, the mash was adjusted to pH 4.0 for the viscosity and raw starch hydrolysis (RSH) treatments. The mash was heated to 53 to 55 ⁰ C with frequent stirring. Optimash TBG (80 μl/kg of grain) was added to the mash at 53 to 55 ⁰ C for 1 hour with frequent stirring. A pasturalant, diethyl pyrocarbonate (DEPC), was then added (450 μl/4.28 kg of mash). The mash was then transferred using aseptic techniques to a sterile container and kept at 4.0 ⁰ C for 72 hours. The mash (1.71 kg) was then transferred into sterile fermenters using a specially designed transfer system (tube-to-tube lock fittings) and the temperature increased to 48 ⁰ C at 500 RPM. Once the temperature target was reached, Stargen 001 (2.8 ml/kg of grain) was added to each fermentor for 1 hour.

After the initial treatment with Stargen 001, the temperature was reduced to 30 ⁰ C at 200-300 RPM for the rest of the fermentation. Water and urea were added to the mash in each fermenter to produce 20% solids w/w and 16 mM urea. The pH was adjusted to 4.0. Yeast was prepared using 200 ml of sterile filtered water and 40 g of yeast. The yeast was hydrated with water and then incubated at 30 ⁰ C for 30 min with shaking at 200 RPM. The yeast mixture was then introduced to the fermentors. Samples were taken at various points throughout the fermentation process. The initial inoculum of the yeast at the beginning of the fermentation was approximately 2x10 CFU/ml.

Each sample was analyzed by gas chromatography (GC) to determine ethanol yield whereas high performance liquid chromatograph (HPLC) was performed to determine the sugar contents of the samples.

Stargen 001 treatment at 55 ⁰ C with the addition of urea and protease at 20% solids

The addition of urea as a nitrogen source for yeast and Fermgen enzyme as a protease treatment to release starch from a protein matrix was also tested in conjunction with a slightly higher Stargen 001 treatment temperature of 55 ⁰ C. The temperature was raised to help prevent contamination of Lactobacillus sp. while maintaining the enzyme activity. The ground grains (0.5 mm) were mixed with water (1.5 kg grain/2.78 kg water) to obtain a mash with 35% solids. Due to the high viscosity of Fibar barley, additional water was added to the mash which reduced its % solids to 27.53. Using hydrochloric acid, the mash was adjusted to pH 4.0 for the viscosity and RSH treatments. Mash was heated to 53-55 ⁰ C with frequent stirring. Optimash TBG (80 μl/kg of grain) and Fermgen (940 μl/kg of grain) were added to the mash at 53-55 ⁰ C for 1 hour with frequent stirring. DEPC was then added (900 μl/4.28 kg of mash). The mash was then transferred using aseptic technique to a sterile container and kept at 4.O ⁰ C for 72 hours. The mash (1.71 kg) was then transferred into sterile fermentors and the temperature increased to 55 ⁰ C at 500 RPM. The fermentors containing Fibar mash received 2.17 kg instead of 1.71 kg and enzyme amounts were adjusted to compensate for the additional water. Once the temperature target was reached, Stargen 001 (1.646 ml) was added to each fermentor for 1 hour. After Stargen 001 treatment, the temperature was reduced to 30 ⁰ C at 200 RPM 200-300 RPM for the rest of the fermentation. Urea (16 mM) and the remaining amount of water were added to the mash to produce 20% solids. Due to an

increase in pH by urea the mash was again adjusted to pH 4.0. The yeast mixture was prepared as described previously and then introduced to the fermentors. The initial inoculum of the yeast at the beginning of the fermentation was approximately 2x10 CFU/ml.

Samples were taken at various points throughout the fermentation process. As previously mentioned, each sample was analyzed by GC and HPLC.

Star gen 001 treatment at 55 ⁰ C with the addition of urea and protease at 30% solids

The ground grains (0.5 mm) were mixed with water (1.60 kg grain/2.40 kg water) to obtain a mash with 40% solids. Using hydrochloric acid, the mash was adjusted to pH 4.0 for the viscosity and RSH treatments. Mash was heated to 53-55 ⁰ C with frequent stirring. Optimash TBG (80 μl/kg of grain) and Fermgen (940 μl/kg of grain) was added to the mash at 53-55 ⁰ C for 1 hour with frequent stirring. Viscozyme Barley (300 μl/kg of grain) at pH 5.0 was used in the case of Fibar barley to help reduce the high viscosity of the mash. Viscozyme Barley was able to reduce the viscosity of Fibar due to its ability to hydrolyze β- glucans. DEPC was then added (900 μl/4.28 kg of mash). 1.875 kg of the mash was then transferred using aseptic technique to a sterile container and kept at 4.0 ⁰ C for 72 hours. The mash was then transferred into sterile fermentors and the temperature increased to 55 ⁰ C at 500 RPM. Once the temperature target was reached, Stargen 001 (1.646 ml) was added to each fermentor for 1 hour. After the treatment with Stargen 001, the temperature was reduced to 30 ⁰ C at 200 RPM for the rest of the fermentation. Urea (16 mM) and the remaining amount of water were added to the mash in each fermenter to produce 30% solids. Due to an increase in pH by urea, the mash was again adjusted to pH 4.0. The yeast mixture was prepared and then introduced to the fermentors with an initial inoculum of approximately 2x10 CFU/ml. Samples were taken at various points throughout the fermentation process. Each sample was analyzed by GC and HPLC as mentioned above. Jet cooking preparation of the mash and fermentation at 30% solids

The ground grains (1.99 mm) were mixed with water (5.25 kg grain/10.50 kg water) to obtain a mash with 35% solids. Using hydrochloric acid (12M), the mash was adjusted to pH 4.8 for the viscosity and protease treatments. Mash was heated to 53-55 ⁰ C with frequent stirring in a groen kettle model TS/9. Viscozyme Wheat (for viscosity reduction, 300 μl/kg

of grain) and Fermgen (940 μl/kg of grain) was added to the mash at 53-55 ⁰ C for 1 hour with frequent stirring. Viscozyme Barley (for viscosity reduction, 300 μl/kg of grain) at pH 4.8 was used in the case of Fibar barley to help reduce the high viscosity of the mash. At the end of the treatment with Viscozyme and Fermgen, the pH was changed to 5.25 using 5N NaOH. One fourth of the dose of liquozyme SC (21 μl/kg of grain) was added to the mash and the temperature was changed from 55 ⁰ C to 85 ⁰ C. Liquozyme treatment was done for 30 min. The first liquefaction step was carried out to further reduce the viscosity of the mash (through hydrolyzing α-(l,4)-glucosidic bonds of the starch) in order to avoid jet-cooker plugging. The mass of the mash was determined at the end of the Liquozyme treatment and water was added if necessary to compensate for any loss in the mass by evaporation. During the VHG jet cooking (Very High Gravity), the mash was heated to 110 to 120 ⁰ C by direct injection of high pressure (50 psig) clean steam at approximate rate of 150 lb/h. The mash was passed through a jet cooker five times where the mash was heated to 110-120 ⁰ C by the injection of high-pressure steam into the mash. After jet-cooking, the mass of the mash was determined and adjusted by the addition of water if necessary. The jet-cooked mash was transferred again to a groen kettle (model TS/9) adjusted to 85 ⁰ C and a % dose of the liquozyme SC (63 μl/kg of grain) was added to the mash and kept for 90 min to completely liquefy the starch. DEPC was then added (105 μl/kg of mash) and the mash was transferred using aseptic technique to a sterile container and kept at 4.0 ⁰ C for 72 hours. For fermentation, the mash was transferred into sterile fermentors and the temperature was adjusted to 30 ⁰ C at 200 to 300 RPM. Urea (16 mM) and the remaining amount of water were added to the mash in each fermenter to produce 30% solids in the fermenters. Due to an increase in pH by urea, the mash was again adjusted to pH 4.0. Once the temperature target was reached, Spirizyme® Fuel (600 μl/kg of grain) was added to each fermentor for 15 min as a presaccharification step. The yeast mixture was prepared and then introduced to the fermentors with an initial inoculum of approximately 2x10 CFU/ml after finishing the spirizyme treatment. Samples were taken at various points throughout the fermentation process. Each sample was analyzed by GC and HPLC as mentioned above.

Preparation of DDGS samples

All DDGS samples were prepared in two phases: 1) RotoVap evaporation of the liquid phase (ethanol and water) at 72 ⁰ C under vacuum with constant mixing. 2) Freeze drying at - 6O ⁰ C at -0.4 bar for 72 h. This method of preparation helps keep all unique chemical and nutritional properties intact.

Analysis of the Nutritional Characteristics of DDGS.

The impact of grain source, type of fermentation, and percentage of solids on nutritional characteristics of DDGS is described in Table 2. The DDGS samples were analyzed for initial feed value variables such as in vitro energy digestibility, crude protein and crude fiber content. DDGS samples can vary widely in nutritional value. In vitro energy digestibility was used as an initial indicator of energy digestibility in swine. The focus on characterizing energy is because this is the most important cost component in feed formulation for livestock.

A 3-step in vitro energy digestibility technique was used. The barley samples were finely ground to pass through a 1-mm mesh size screen in a Retsch mill (model ZMl, Brinkman Instruments, Rexdale, ON, Canada). A ground sample (1 ± 0.1 mg) was weighed into a 125-mL conical flask. Phosphate buffer (25 ml, 0.1 N, pH 6) solution was added to the flask and stirred using a small magnetic rod. After adding 10 mL of 0.2 N HCl solution to the flask, the pH of the solution was adjusted to 2 using 1 N HCl or 1 N NaOH solutions. 1 mL of freshly prepared pepsin (P-7000, Sigma, St. Louis, MO, USA; 800-2500 units/mg protein, from porcine gastric mucosa) and 0.5 mL chloramphenicol solutions (0.5 g/100 mL ethanol) were added to the flask and then incubated in a water bath at 39 ⁰ C for 6 h. After the incubation, 10 mL of 0.2 N phosphate buffer (pH 6.8) and 5 mL of 0.6 N NaOH solutions were added to the flask, and the pH of the solution was adjusted to 6.8 with 1 N HCl or 1 N NaOH solutions. Thereafter, 3 mL of freshly prepared pancreatin (P-1750, Sigma, St. Louis, MO, USA; activity equivalent to 8 x USP specification, from porcine pancreas) solution was added to the flask. The flask was incubated in a water bath at 39 ⁰ C for 18 h. 20 mL of freshly prepared cellulase solution (C-9422, Sigma, St. Louis, MO, USA; 3 to 10 units/ mg solid, from Trichoderma viridae) was added, and the flask was incubated at 39 ⁰ C for 24 h. The enzymatic digestion was terminated by addition of 5 mL 20% sulpho-salicylic acid, and the

flask was kept at room temp for 30 min to facilitate precipitation of undigested soluble proteins. The undigested residues were then collected in a filtration unit using porcelain filtration funnel lined with pre-weighed filter paper (Whatman no. 54). The residues along with the filter paper were dried at 8O ⁰ C overnight. In vitro dry matter (DM) digestibility was calculated by deducting the residue DM from the sample DM following by division by the sample DM.

A. Analysis of Sterols in DDGS and Grains

1. Preparation of Sample

(a) Raw sample (0.3 g ) (DDGS or Grains) was weighed. (b) To this, 0.05 mg of dihydrocholesterol internal standard and 5 ml 2 N alcoholic KOH was added.

(c) The mixture was saponified at 70 ⁰ C for 1 h and allowed to stand overnight at room temperature.

(d) Deionized water (5 ml) and cyclohexane (15 mL) was added. (e) The mixture was centrifuged at l,000rpm for 10 min.

(f) The cyclohexane fraction was transferred into a glass tube and washed three times with deionized water until the pH is neutral

(g) Dry under N ₂

(h) Dissolve in 0.25 ml pyridine and 0.25 ml BSTFA containing 1 % TMCS (i) Heat at 50 ⁰ C for 30 min and allowed to stand overnight at room temperature (j)

Dry under N ₂

(k) Dissolve in 0.4 ml hexane (1) Transfer the solution into GC vial

2. GC Analysis Conditions

GC Column: J&W Scientific DB-5 capillary column (30m x 0.25 mm ID, 0.25 μm film,

Agilient Technologies)

Inlet temperature: 280 ⁵ C Detector temperature: FID 280 ⁵ C Head pressure: 25 PSI Injection volume: Splitless, 1.0 μl Column temperature: Start 70 ⁵ C, hold 0.5 min; 70 ⁵ C ~ 250 ⁵ C, 20 ^Q C/ min; 250 ⁵ C ~ 280 ⁵ C, 15 ^Q C/ min; 280 ^Q C, hold 17min.

Run time: 28.5 min 3. Calculation

Peak identification was achieved by comparing the retention of authentic sterols in a mixture containing campesterol, stigmasterol, sitosterol and brassicasterol. For quantitative analysis, stigmasterol standard was used to calculate the relative response factor (RRF) for all the sterol. Results are shown in Table 3.

B. Analysis of Fatty Acids in DDGS and Grains 1. Preparation of Sample (a) Weigh 50mg raw sample (DDGS or Grains)

(b) Add 50μl chloroform

(c) Leave it over night (Dark)

(d) Add 2ml methanolic HCl (3N)

(e) Heat at 50 ⁰ C for 20 Min (f) Cool to room temp.

(g) Add lOOμl H ₂ O, 5ml Hexane and 100 μl internal standard (Methyl C17:0)

(h) Draw off hexane layer into second tube containing a pinch of anhydrous sodium sulfate

(i) Centrifuge it at 2,500rpm for 5Min

(j) Take out ImI into GC Vial. 2. GC Analysis

Conditions

GC Column: Supeclo SP-2560 capillary column(100m x 0.25 mmID, 0.2 μm film thickness)

Inlet temperature: The initial injector temperature of 50 ⁵ C was maintained for 0.2 min and then ramped to 230 ⁵ C at 150 ⁵ C. Detector temperature: FID 230 ⁵ C Head pressure: 40 PSI Injection volume: Cool-on column

Column temperature: Start 45 ⁵ C, hold 4.0 min; 45 ⁵ C ~ 175 ⁵ C, 13 ⁵ C/ min; 175 ⁵ C, hold 27 min; 175 ⁵ C ~ 215 ⁵ C, , 4.0 ⁵ C/ min; 215 ⁵ C, hold 29.0 min Run time: 80.0 min 3. Calculation

Peak identification was achieved by comparing the retention of authentic fatty acids in a mixture containing 48 species fatty acids(Standard 463, Nu-Chek Pucp Inc., Elysion, MN S6028). All species of fatty acids were quantified using an internal standard (Methyl C17:0) with the same response factor of 1.0. Results are shown in Table 4.

C. Analysis Total Phenolics Content in DDGS and Grains:

1. Preparation of Extracts from DDGS and Grains

(a) Weigh 50 mg sample (DDGS or Grains)

(b) Add 2ml 80% methanol in water (V/V) (c) Sonicate for 1 hour under nitrogen at room temperature

(d) Centrifuge at 4,000 rpm for 15Min

(e) Take out 0.2 ml DDGS extract or ImI

(f) Extract grains and react with Folin-Ciocalteu's phenol reagent.

2. Determination of Total Phenolics Content (a) Place sample extract into 10-ml volumetric flask

(b) Mix with 2.0 ml Folin-Ciocalteu's phenol reagent(10%, v/v, in water) and react for 5 min

(c) Add 2.0 ml sodium carbonate (10%, v/v, in water)

(d) Add deionized water and the final volume was made up to 10 ml (e) Mix and allowed to stand 1 h at room temperature

(f) Measure absorbance at 750 nm and the total phenolic content was expressed as gallic acid equivalents (GAE) in mg per g material using the method as described in Zhao et al, J. Agric. Food Chem. (2006) 54, pp. 7277-7286. The results are shown in Figure 2.

D. Analysis Tocols Content in DDGS and Grains 1. Preparation of Extracts from DDGS and Grains

(a) Weigh 2.0 g sample (DDGS or Grains)

(b) Add 2 ml KOH (600 g/L), 2 ml ethanol(95%), 2 ml NaCl(IO g/L), and 5 ml ethanolic BHT(30 g/L)

(c) Saponify at 70 ⁰ C for 45 min under nitrogen and mixed every 5 min during saponification (d) Cool in an ice bath and add 15 ml NaCl (10%)

(e) Extract suspension twice with a 15 ml portion of n-hexane/ethyl acetate (9: 1 v/v) and collect the organic layer

(f) Evaporate under vacuum at 35 ⁰ C

(g) Dissolve 2 ml isopropyl alcohol (1%) in n-hexane and transfer 1 ml solution into HPLC vial.

2. HPLC Analysis

(a) HPLC Separation Technique Parameter

Column: Supelco LC-Diol column ( 25cm x 4.6 cm, 5 μm, Supelco, Inc. Bellefonate, PA) Mobile Phase: 0.6% isopropanol in n-hexane Column Flow: 1.00 ml/min;

Run time: 30 min

Column Temperature: Room temperature Injection Volume: 35 μl

Detector: Fluorescence detector, EX 300 nm, EM 330 nm (b) Preparation of Calibration Curve

A calibration curve prepared for external standards of α-, β-, γ-, and δ-tocopherols (2.5-50μg/ml) is used for identification and quantification purposes. The linear regression coefficient for each tocopherol standard cure has at least two nines. All standards need to be done in duplicate. (c) Calculation

For sample, each α-, β-, γ-, and δ-tocopherols peak identification was achieved by comparing the retention time of authentic tocols in a mixture containing α-, β-, γ-, and δ- tocopherols standard solution and quantified using calibration curve respectively. Each α-, β- , γ-, and δ-tocotrienols peak identification is achieved by comparing the peak position according to chromatograph (2). α-tocotrienol is quantified by α-tocopherol calibration curve, β- and γ-tocotrienols is quantified by γ -tocopherol calibration curve respectively, and δ-tocotrienols is quantified by δ-tocopherol calibration curve. Results are shown in Table 5.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the materials, methods, and articles described herein. Other aspects of the materials, methods, and articles described herein will be apparent from consideration of the specification and practice of the materials, methods, and articles disclosed herein. It is intended that the specification and examples be considered as exemplary.