Method for increasing the amino acid content of animal feed products

Abstract

Disclosed are compositions and methods for increasing the amino acid content of animal feed byproducts from dry mill ethanol production facilities by reducing the amount of residual reducing sugars that can participate in, for example, the Maillard reaction.

Description

Technical Field

[0001] Disclosed are compositions and methods for increasing the amino acid content of animal feed byproducts from ethanol production facilities by reducing the amount of residual reducing sugars that can participate in Maillard and other reactions.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is a U.S. national phase application of International Application No. PCT / US2020 / 036444, filed on June 5, 2020, entitled “METHODS FORIMPROVING THE AMINO ACID CONTENT OF ANIMAL FEED PRODUCTS,” under 35 U.S.C. §371. This application claims priority to U.S. Provisional Patent Application No. 62 / 857,624, filed on June 5, 2019, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0004] 56 pounds of bushels of corn processed in a dry mill ethanol plant traditionally produce about 2.8 gallons of ethanol and 17.5 pounds of animal feed. The animal feed is in the form of distiller's dried grains (DDGS) with solubilizers and represents the starch-depleted portion of corn plus yeast organisms for fermentation. Based on weight, DDGS is more nutritious for animals than unprocessed corn because DDGS is richer in protein and fat. In addition to DDGS, dry mill ethanol plants also have the ability to produce other protein-rich corn byproducts for animal feed applications.

[0005] Unfortunately, not all amino acids withstand the ethanol production process equally well. In particular, lysine is significantly degraded by the Maillard reaction during the DDGS and corn byproduct drying steps. Lysine is an essential amino acid for animals, so if lysine cannot be provided by DDGS or other corn byproducts, it must be supplemented. In fact, a significant cost of animal feed is the free amino acid lysine.

[0006] There is a need to reduce the amount of lysine degradation in the preparation of DDGS or other corn co-products to reduce the need for subsequent lysine supplementation. Summary of the invention

[0007] The compositions and methods of the present invention are directed to increasing the amino acid content of byproducts from ethanol production facilities by reducing the amount of residual reducing sugars that can participate in, for example, the Maillard reaction. These compositions and methods are particularly valuable for preparing animal feed products. Aspects and embodiments of these compositions and methods are summarized in the following, respectively numbered paragraphs:

[0008] 1. In one aspect, a method for reducing the degradation of amino acids in a dry post-fermentation product from a dry mill ethanol production facility is provided, the method comprising contacting a wet post-fermentation product comprising residual reducing sugars and amino acids with an enzyme capable of oxidizing the residual sugars, thereby reducing the degradation of the amino acids via the Maillard reaction during a subsequent heating step for producing a heated post-fermentation product.

[0009] 2. In some embodiments of the method according to claim 1, the product after wet fermentation is fermentation broth.

[0010] 3. In some embodiments of the method of claim 1, the wet fermentation product is whole wine grains.

[0011] 4. In some embodiments of the method of claim 1, the wet fermentation product is thin stillage.

[0012] 5. In some embodiments of the method of any one of claims 1-4, the heated post-fermentation product is dried distiller's grains, dried distiller's grains with solubles, concentrated distiller's grains solubles, or other protein-containing by-products.

[0013] 6. In some embodiments of the method of any one of claims 1-5, free amino acids are present in the starch-containing substrate used to produce the mash.

[0014] 7. In some embodiments of the method of claim 6, the substrate comprises corn.

[0015] 8. In some embodiments of the method of any one of claims 1-6, the free amino acids are produced by a fermenting organism used to produce the fermentation broth.

[0016] 9. In some embodiments of the method of claim 8, the fermenting organism is yeast.

[0017] 10. In some embodiments of the method of any one of claims 1-9, the amino acid is lysine.

[0018] 11. In some embodiments of the method of any one of claims 1-10, the enzyme capable of oxidizing the residual sugar is glucose oxidase and / or hexose oxidase.

[0019] 12. In some embodiments of the method of any one of claims 1-11, the reducing sugar comprises glucose.

[0020] 13. In some embodiments, the method of any one of claims 1-12 further comprises contacting the post-distillation product comprising residual sugars and amino acids with catalase.

[0021] 14. In some embodiments of the method of claim 13, the amino acids are lysine and methionine.

[0022] These and other aspects and embodiments of the compositions and methods will be apparent from the description and drawings. DETAILED DESCRIPTION

[0023] 1. Definitions and Abbreviations

[0024] Before describing various aspects and embodiments of the compositions and methods of the present invention, the following definitions and abbreviations are described.

[0025] In light of this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "a dosage" includes reference to one or more dosages and equivalents thereof known to those of ordinary skill in the art, and so forth.

[0026] This document is organized into several sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for the different sections of this disclosure should not be interpreted as limiting.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the sake of clarity, the following terms are defined as follows.

[0028] As used herein, "whole stillage" is the byproduct of a dry-grind ethanol production facility after distillation.

[0029] As used herein, "thin stillage" is the liquid portion of whole stillage after separation of the solid matter.

[0030] As used herein, "distillers grains (DG)" is the solid / slurry component of whole distillers grains.

[0031] As used herein, "distillers dried grains (DDG)" is DG that has been dried.

[0032] As used herein, "distillers dried grains with solubles (DDGS)" is DG that has been dried with concentrated thin grains for added nutritional value.

[0033] As used herein, a "wet" by-product of distillation contains at least 20% water by weight.

[0034] As used herein, the "dry" by-product of distillation contains less than 20% water by weight.

[0035] As used herein, the term "amino acid" refers to both free amino acids and amino acid residues of synthetic proteins.

[0036] As used herein, the term "amino acid degradation" refers to the chemical alteration of amino acids through the Maillard reaction (optionally in combination with other reactions).

[0037] As used herein, the term "contacting" refers to bringing components into physical proximity in order to, for example, facilitate a chemical reaction.

[0038] As used herein, the term "about" refers to ±15% of a reference value.

[0039] Unless otherwise stated, the following abbreviations / acronyms have the following meanings: [will be used as required]

[0040] EC Enzyme Commission

[0041] ℃ Celsius

[0042] H2O water

[0043] dH2O or DI deionized water

[0044] dIH2O Deionized water, Milli-Q filtered

[0045] g or gm gram

[0046] μg microgram

[0047] mg milligram

[0048] kg kilogram

[0049] μL and μl microliter

[0050] mL and ml

[0051] M Moore

[0052] mM millimole

[0053] μM micromolar

[0054] U Unit

[0055] min

[0056] rpm revolutions per minute

[0057] hr hour

[0058] CAZy Carbohydrate Active Enzyme Database

[0059] HOx Hexose oxidase

[0060] GOx Glucose Oxidase

[0061] DG Distillers Lees

[0062] DDG Dried Distillers Grains

[0063] DDGS Distillers Dried Grains with Dissolved Matter

[0064] 2. Degradation of amino acids in animal feed products

[0065] The Maillard reaction (Maillard, LC (1912). Compt. Rend. 154:66) is a chemical reaction between amino acids and reducing sugars that occurs at temperatures between about 140° C. and 165° C. The reaction is well known for giving brown foods their distinctive flavor, including, for example, grilled steaks, fried dumplings, cookies, bread, toasted marshmallows, etc. The reaction destroys amino acids, either in the form of free amino acids or amino acid residues in proteins.

[0066] A valuable, and sometimes overlooked, byproduct of dry-milled ethanol fermentation is animal feed, usually in the form of distillers dried grains (DDG) or, more commonly, distillers dried grains with solutes (DDGS). For simplicity, unless the context indicates otherwise, these products are collectively referred to as "DDGS," which is the most common product. Such animal feed products are in many ways more nutritious than the original feedstock for ethanol production because the animal feed products are depleted of carbohydrates but enriched with amino acids from both the feedstock and the fermenting organism (i.e., the ethanolagen).

[0067] The specific amino acid composition of DDGS or other corn byproducts is important for the quality of animal feed. Some amino acids are much more important than other amino acids. For example, lysine is an essential amino acid for most farm animals and, if lysine cannot be provided by DDGS, it must be supplemented to maximize feed conversion. Synthetic lysine is expensive and represents a significant cost for animal feed. Methods for increasing the content of lysine in DDGS or other corn byproducts are highly desirable and should allow ethanol producers to extract greater value from their products.

[0068] The compositions and methods of the present invention involve adding enzymes to post-wet fermentation products from ethanol production facilities to oxidize residual reducing sugars, particularly residual glucose, in order to prevent amino acids, particularly lysine, from being lost due to the Maillard reaction during the subsequent drying step. Variants of the compositions and methods of the present invention include further use of catalase to reduce oxidation of methionine, providing added value to DDGS or other corn by-products.

[0069] 3. Residual reducing sugars in byproducts after oxidative fermentation

[0070] Reducing sugars are sugars that can act as reducing agents due to having free aldehyde groups (in the case of aldoses) or ketone groups (in the case of ketoses). All naturally occurring monosaccharides as well as some disaccharides, oligosaccharides and polysaccharides are reducing sugars. Examples of reducing sugars include glucose, lactose, maltose and cellobiose, but do not include sucrose or trehalose. The most common reducing sugar in the post-wet fermentation product from an ethanol production facility (whether from a starch-based substrate or from a cellulose-based substrate) is glucose, although there are also small amounts of other reducing sugars.

[0071] The enzymes used in the compositions and methods of the present invention oxidize residual reducing sugars, thereby reducing the destruction of amino acids in subsequent drying steps, such as by the Maillard reaction. Exemplary enzymes include hexose oxidase (HOx; EC 1.1.3.5), which has broad specificity for hexoses, and glucose oxidase (GOx; EC 1.1.3.4), which is specific for glucose. HOx can be used as SUREBAKE TM 800 (DuPont). GOx is available under the trade name GO, S 758 and GOL (DuPont), MONO TM 10.000BG (Novozymes), Go Pure (DSM) and GLOXY TM GOx from Aspergillus niger is also available from Sigma-Aldrich.

[0072] In application, HOx or GOx is preferably added at about 0.0625% by volume / dry weight fermentation residue to produce 0.01% to 1.25% by mass HOx or GOx / dry weight fermentation residue. The amount of HOx or GOx required can be determined by incrementally adjusting the amount used and measuring the amount of lysine recovered.

[0073] 4. Further addition of catalase to reduce methionine oxidation

[0074] Although the main advantage of the compositions and methods of the present invention is the reduction of lysine destruction by, for example, the Maillard reaction, a potential disadvantage is the loss of methionine due to oxidation. This disadvantage can be avoided by further including catalase in the product after wet distillation. Catalase (EC 1.11.1.6) destroys the free hydrogen peroxide produced by oxidation of reducing sugars into products that no longer participate in the Maillard reaction, thereby eliminating the unwanted chain effect caused by the addition of enzymes such as glucose oxidase or hexose oxidase.

[0075] Catalase is sold under the trade name CA 800L, CAT、OXY T400 (DuPont) and 25 (Novozymes) for sale.

[0076] In applications, catalase is added at about 1:1 mass % relative to HOx or GOx. As with HOx or GOx, the required amount can be determined by incrementally adjusting the amount used and in this case measuring the amount of methionine recovered.

[0077] 5. When to add enzymes to fermentation byproducts

[0078] According to the compositions and methods of the present invention, the above-mentioned one or more enzymes are contacted with the product after wet distillation, such as fermentation end liquid, whole grains or thin grains. In some cases, the enzyme is added to the whole grains and the final product is DDG or DDGS. In other cases, the enzyme is added to the thin grains, which is then combined with DDG to produce DDGS. Although these represent the most practical methods for implementing the compositions and methods of the present invention, other methods exist and are included in this specification, including applying the enzyme to partially dried DDG / DDGS or other corn byproducts.

[0079] 6. Dry grinding variants

[0080] There are many variations of dry mill ethanol production, including cold cooking, or no cooking, processes involving liquefaction at or below gelling temperature, simultaneous saccharification and fermentation, fractionation, etc. While such variations may alter the residual amounts of reducing sugars and amino acids in the product after wet distillation, they are not expected to be incompatible with the compositions and methods of the present invention.

[0081] 7. Compatible starch substrate

[0082] The starch substrate used to produce the wet fermentation product is not limited to corn, but can also include, for example, tubers, roots, stems, beans, cereals or whole grains. More specifically, the starch substrate can be obtained from corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes.

[0083] 8. Additional enzymes

[0084] The compositions and methods of the present invention involving oxidoreductases that catalyze the oxidation of glucose are by no means intended to exclude the use of other enzymes. In fact, it should be understood that enzymes that hydrolyze starch, such as alpha-amylases and glucoamylases, remain important for the dry milling process that produces the wet fermentation post-product. Additional enzymes that are advantageously included in the dry milling process include, but are not limited to, phytases, proteases, pullulanases, beta-amylases, isoamylases, alpha-glucosidases, cellulases, xylanases, other hemicellulases, beta-glucosidases, transferases, pectinases, lipases, cutinases, esterases, mannanases, oxidoreductases, different alpha-amylases, or combinations thereof.

[0085] 9. Fermentation organisms

[0086] The compositions and methods of the present invention do not require specific fermentation organisms. Typical fermentation organisms are yeast. Yeast is a unicellular eukaryotic microorganism classified as a member of the fungi kingdom, and includes organisms from the Ascomycota and Basidiomycota. Yeast that can be used for alcohol production includes but is not limited to Saccharomyces species, including Saccharomyces cerevisiae and Kluyveromyces, Lachancea and Schizosaccharomyces species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high ethanol production, fast growth rate, etc. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylase or alpha-amylase.

[0087] All references cited herein are hereby incorporated by reference in their entirety for all purposes.To further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative rather than limiting.

[0088] Examples

[0089] Example 1: Post-fermentation treatment using HOx and HOx + catalase

[0090] Liquefact (corn mash slurry) was prepared by adding 600 ppm urea, 0.124 SAPU / g ds acid fungal protease, 0.33 GAU / g ds variant Trichoderma reesei glucoamylase, and 1.46 SSCU / g ds Aspergillus kawachii alpha-amylase, adjusted to pH 4.8 with sulfuric acid. Simultaneous saccharification and fermentation were performed using standard procedures and conventional yeast.

[0091] At the end of SSF (66 hours), HOx alone, or HOx and catalase (HOx+catalase) were added to the flasks in duplicate, while two control flasks had no enzyme added. HOx (pre-commercial sample) was added at 1% w / w relative to the wet weight of corn liquefact (i.e., 10 mg HOx / g) and catalase was added at 0.075% v / w ( CA) (i.e., 75 μL catalase / g). After enzyme addition, the flasks were placed back in the incubator under the same SSF conditions (32°C, shaking at 200 rpm) and allowed to incubate for another 4 hours. After 4 hours, the fermentation broth from the duplicate flasks was collected in an 800 mL beaker and placed in a shaking water bath at 90°C to evaporate the ethanol. The fermentation broth was incubated for approximately 3-5 hours, or until no significant ethanol was detected by HPLC. Some water was also lost during this process. The resulting material (i.e., whole vinasse) was spun down at 6,000 rpm for 10 min. Both the supernatant (i.e., thin vinasse) and the precipitate (i.e., wet cake) were collected. The wet cake was dried at 37°C until a dry solids content of approximately 34% was reached. The thin vinasse was weighed into a 600 mL beaker and placed in a 97°C shaking water bath to concentrate the contents approximately 4.8 times (by weight) to produce a slurry. To make DDGS samples, wet cake and corresponding slurry were combined in a 2 to 1 mass ratio (as received weight) and mixed thoroughly. DDGS was spread onto metal trays and dried in a 99°C oven for approximately 3 hours (with occasional mixing) to >90% dry solids content.

[0092] For testing of bioavailable amino acids, samples of DDGS were incubated with pepsin and pancreatin based on previously reported methods (Qiao Y (2001) Routine techniques for monitoring the nutritional value of animal meals, Doctoral thesis at North Carolina State University). Briefly, 0.33 g of DDGS was added to a 20 mL scintillation vial along with 3.33 mL of 0.05 M citrate buffer (pH 2) and approximately 0.012 g of pepsin (from porcine gastric mucosa) at ≥ 400 units / mg protein. The mixture was incubated at 38° C. for approximately 24 hours with shaking at 200 rpm. After this time, 5 mL of phosphate buffer (0.2 M, pH 11.5, with 0.025% w / w sodium azide) and approximately 0.023 g of pancreatin (from porcine pancreas, 4×UXP specification) were added to each vial. The vials were returned to the 38°C incubator shaking at 200 rpm for approximately 66 hours. After this time, samples were removed from each vial, spun down through a 0.2 μM filter and analyzed by HPLC for free amino acids.

[0093] The results shown in Table 1 compare the measured bioavailable amino acid concentrations from HOx and HOx + catalase treated DDGS with control DDGS (not treated with enzymes). The HOx treated samples showed a 10% increase in bioavailable lysine, while the HOx + catalase samples showed a 25% increase in bioavailable lysine. The HOx treated samples showed a significant loss of methionine compared to the control, which may be due to the production of hydrogen peroxide caused by HOx. The inclusion of catalase prevented this loss, resulting in a 17% increase in bioavailable methionine compared to the control.

[0094] Table 1. Bioavailable amino acids in enzyme-treated DDGS

[0095]

[0096]

[0097] Example 2: Post-fermentation treatment using GOx and GOx+catalase

[0098] Whole grains from a dry mill ethanol plant were collected and treated with GOx or GOx + catalase. Specifically, GOx ( GO), and catalase ( CAT). After enzyme addition, flasks containing whole grains (200 g) and added glucose (final concentration of about 0.46 g / L) were placed in an incubator at 32°C, shaking at 200 rpm, and incubated for 3 hours. A control flask without enzyme addition (final concentration of glucose was about 0.46 g / L) was also included in the test. After 3 hours, whole grains samples were collected and spun down at 6,000 rpm for 10 minutes. The supernatant (thin grains) and precipitate (wet cake) were collected. The wet cake was dried at 37°C to a dry solids content of about 40%. The thin grains were weighed into an 800 mL beaker and placed in a 98°C shaking water bath to concentrate the contents by about 4.5 times (by weight) to produce a slurry. To make DDGS samples, the corresponding wet cake and slurry were combined in a 2 to 1 mass ratio (by original weight) and mixed thoroughly. They were spread onto a metal tray and dried in a 105°C oven for approximately 3 hours (with occasional mixing) until dry (>90% dry solids).

[0099] To test for bioavailable amino acids, samples of DDGS were incubated with pepsin and pancreatin, and the resulting products were analyzed by HPLC, as described in Example 1. The results shown in Table 3 compare the measured amino acid concentrations (bioavailable amino acids) of DDGS from GOx and GOx+catalase treatments with control DDGS. Thus, for strictly dried DDGS, a 7% increase in bioavailable lysine was observed with GOx treatment, while a 16% increase in available lysine was observed with GOx+catalase treatment. In addition, many other amino acids were shown to increase with the addition of both enzymes, including but not limited to methionine and arginine. For samples with only GOx, a significant reduction in bioavailable methionine was observed compared to the control, which may be due to the production of hydrogen peroxide caused by GOx. Adding catalase can convert this hydrogen peroxide into oxygen, thereby avoiding the destruction of methionine.

[0100] Table 2. Bioavailable amino acids in enzyme treated samples compared to controls

[0101]

[0102] Example 3: HOx+catalase or GOx+catalase with different drying conditions

[0103] Whole grains from an industrial-scale dry-milling ethanol plant were collected and treated with HOx+catalase or GOx+catalase. Specifically, HOx was added at 1% w / w (pre-commercial sample), GOx was added at 0.25% v / w ( HPL5000), and catalase ( CA). The control did not include enzyme. After the enzyme was added, the flask containing 200 g of whole grains was placed in an incubator at 32°C, shaking at 200 rpm, and allowed to incubate for another 4 hours. After 4 hours, the whole grains sample was collected and spun down at 6,000 rpm for 10 minutes. The supernatant (thin grains) and precipitate (wet cake) were collected. The wet cake was dried at 37°C until a dry solids content of about 40% was reached. The thin grains were weighed into an 800 mL beaker and placed in a 97°C shaking water bath to concentrate the contents by about 4.8 times (by weight), producing a slurry.

[0104] To make the DDGS samples, the wet cake and the corresponding slurry were combined in a 2 to 1 mass ratio and mixed thoroughly. The DDGS were spread onto metal trays and dried overnight in a 99°C oven (with occasional mixing) until >95% dry solids. These DDGS were designated "strictly dried DDGS". In addition, "mildly dried DDGS" samples were produced by combining the wet cake and slurry, respectively, in a 2 to 1 mass ratio (as is) and drying in a 99°C oven for 2-3 hours (with occasional mixing) until dry (>90% dry solids).

[0105] For testing of bioavailable amino acids, samples of DDGS were incubated with pepsin and pancreatin, and the resulting products were analyzed by HPLC, as described in Example 1. The results shown in Table 2 compare the measured amino acid concentrations (bioavailable amino acids) of DDGS from HOx+catalase and GOx+catalase treatments with control (unenzyme treated) DDGS for strict and mild drying samples. For strict drying DDGS, a 20% increase in bioavailable lysine was observed with HOx+catalase treatment, while a 33% increase in bioavailable lysine was observed with GOx+catalase treatment. In addition, with the addition of both enzymes, increases were shown in many other amino acids, including, but not limited to, methionine, threonine, and proline. For mild drying DDGS, a 20% increase in bioavailable lysine was observed with HOx+catalase treatment, while a 23% increase in bioavailable lysine was observed with Gox+catalase treatment. Similarly, increases were shown in many other amino acids, including, but not limited to, glycine and glutamic acid.

[0106] Table 3. Bioavailable amino acids in enzyme-treated DDGS with different drying conditions

[0107]

[0108]

Claims

1. A method for reducing amino acid degradation in a dry fermentation product from a dry mill ethanol production facility, the method comprising contacting a wet fermentation product comprising residual reducing sugars and amino acids with an enzyme capable of oxidizing the residual sugars, thereby reducing degradation of the amino acids via the Maillard reaction during a subsequent heating step for producing a heated fermentation product; The method further comprises contacting the wet fermentation product comprising residual sugars and amino acids with catalase, in, The amino acids are lysine and methionine.

2. The method of claim 1, wherein: The product after wet fermentation is fermentation liquid.

3. The method of claim 1, wherein: The product after wet fermentation is whole wine grains.

4. The method of claim 1, wherein: The product after the wet fermentation is thin vinasse.

5. The method according to any one of claims 1 to 4, wherein: The heated fermentation product is dried distiller's grains, dried distiller's grains with dissolved matter, concentrated distiller's grains soluble matter or other protein-containing by-products.

6. The method according to any one of claims 1 to 4, wherein: Amino acids are present in the starch-containing substrate used to produce the mash.

7. The method of claim 6, wherein: The substrate comprises corn.

8. The method according to any one of claims 1 to 4, wherein: The amino acids are produced by the fermenting organism used to produce the fermentation broth.

9. The method of claim 8, wherein: The fermenting organism is yeast.

10. The method according to any one of claims 1 to 4, wherein: The amino acid is lysine.

11. The method according to any one of claims 1 to 4, wherein: The enzyme capable of oxidizing the residual sugar is glucose oxidase and / or hexose oxidase.

12. The method according to any one of claims 1 to 4, wherein: The reducing sugar comprises glucose.

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