Patent Application
20190309240
The present disclosure relates to methods for improving the nutritional quality of fermentation by-products derived from starch-containing material, wherein the fermentation step is carried out in the presence of a first enzyme composition comprising at least one hemicellulase, and wherein the process comprises further the steps of i) subjecting the fermented mash after the fermentation to a second enzyme composition comprising a beta-1,3 glucanase and a xylanase, ii) separating the desired fermentation product by distillation.
Fermentation products, such as ethanol, are produced by first degrading starch−containing material into fermentable sugars by liquefaction and saccharification and then converting the sugars directly or indirectly into the desired fermentation product using a fermenting organism. Liquid fermentation products such as ethanol are recovered from the fermented mash (often referred to as “beer” or “beer mash”), e.g., by distillation, which separate the desired fermentation product from other liquids and/or solids. The remaining faction, referred to as “whole stillage”, is dewatered and separated into a solid and a liquid phase, e.g., by centrifugation. The solid phase is referred to as “wet cake” (or “wet grains” or “WDG”) and the liquid phase (supernatant) is referred to as “thin stillage”. Dewatered wet cake is dried to provide “Distillers Dried Grains” (DDG) used as nutrient in animal feed. Thin stillage is typically evaporated to provide condensate and syrup (or “thick stillage”) or may alternatively be recycled directly to the slurry tank as “backset”. Condensate may either be forwarded to a methanator before being discharged or may be recycled to the slurry tank. The syrup consisting mainly of limit dextrins and non-fermentable sugars may be blended into DDG or added to the wet cake before drying to produce DDGS (Distillers Dried Grain with Solubles).
It is known to commercially use the various byproducts and residues derived from the fermentation processes like the ethanol production process. Distillers residues or byproducts, as well as by-products of cereal and other food industry manufacturing, are known to have a certain value as sources of protein and energy for animal feed. Furthermore, the oil from the by-products like DDGS can be recovered as a separate by-product for use in biodiesel production or other biorenewable products are sought.
The by-products like DDG, DDGS or WDG comprises proteins, fibers, fat and unconverted starch. For example DDGS contains typically about 30% of protein. While the protein content is high the amino acid composition is not well suited for monogastric animals if used as animal feed. In general processing of DDGS, especially drying time and temperature are effecting the availability and digestibility of the amino acids, especially lysine.
Furthermore, the by-products are mainly fibrous by-products comprising Crude Fibers (CF), which are structural carbohydrates consisting of cellulose, hemicellulose and indigestible materials like lignin. The structural carbohydrates are not digestible in animal's small intestine. Fibers are characterized and analyzed by different methods and can be divided into crude fibers (CF), neutral detergent fibers (NDF) and acid detergent fibers (ADF). The proportion of cellulose and lignin in the crude fibers fraction also determines the digestibility of crude fibers or its solubility in the intestine. High cellulose and lignin concentrations mean reduced digestibility and vice versa. Hemicelluloses are capable to bind water. The soluble part of fibres the soluble non-starch-polysaccharides (NSP) cannot be digested by monogastric animals like swine and poultry, but increase viscosity, due to their capability to bind water, and are a nutritional constraint, since they can cause moist, sticky droppings and wet litter. The antinutritional effect of soluble NSP's is mainly related to the increase in digesta viscosity. The increased viscosity is slowing down the feed passage rate and hinders the intestinal uptake of nutrients and can lead to decreased feed uptake The viscosity increase a) hinders the intestinal absorption of nutrients and can result in negative effect on the consistency on faces and even symptoms of diarrhea, b) slowing down the feed passage rate and possibly to decreased feed intake. Another effect of NSP's is the so-called “Nutrient Encapsulation”. The NSP's in plant cell wall encapsulated starch, protein, oil and other nutrients within the plant cell which is an impermeable barrier preventing full utilization of the nutrients within the cell.
Furthermore the soluble NSP's are responsible for high viscosities during fermentation and are directly influencing separation and drying conditions of fermentation by-products like DDGS in the production process. The bound or encapsulated water in the product is difficult to remove and causes the use of higher drying temperatures and also longer drying time, adversely affecting the quality of temperature-sensitive products like amino acids. The availability and digestibility of essential amino acids in the by-products are lowered by high temperatures and long drying time during production. Examples for NSPs are arabinoxylans, beta-glucans, galactomannans and alpha-galactosides.
As the by-products are used in animal feed for monogastrics animals like pigs and poultry it is important that the by-products have high concentrations of protein with a good amino acid composition and high availability and low soluble fibers content.
Therefore, the two ways for an improvement of a fermentative production plant ton increase their efficiency and profitability are an improved production process and the improvement of the quality of the by-products.
In the prior art, a lot of specific processes or treatment methods are described to improve fermentative production processes.
For example, WO 2007/056321 A1 discloses a method of dewatering whole stillage comprising adding enzymes to whole stillage in the ethanol production to improve the solid-liquid separation in the process.
WO 02/38786 describes a process of ethanol production, whereby enzymes are used for thinning the liquefied whole grain mash and the thin stillage. Enzymes are applied to the liquefied mash before the fermentation starts as well as to the thin stillage after centrifugation of the whole stillage.
The US 2006/0275882 A1 describes a process for producing a fermentation product wherein the viscosity of the mash is reduced by the application of enzymes before or during the fermentation.
The US 2006/0233864 A1 describes a method for improving the nutritional quality of fibrous by-products for a food manufacturing process, wherein the fibrous by-products like DDGS are inoculated with a filamentous fungus to improve the quality of the by-product.
Some ethanol plants use milo, wheat, or barley in the fermentation process, depending on geographical location and time of the year. As a result, nutrient composition can vary among DDGS sources. Because of the near complete fermentation of starch, the remaining amino acids, fat, minerals and vitamins increase approximately three-fold in concentration compared to levels found in corn. Despite the significant increase in crude protein, the poor amino acid balance of DDGS must be addressed when formulating swine and poultry diets.
Therefore, it is an object of the present disclosure to provide improved methods for improving the nutritional quality of the by-products from fermentation processes.
The present disclosure relates to methods for the improvement of the nutritional quality of fermenation by-products or residues derived from fermented mash comprising the steps of: i) subjecting the fermented mash during the fermentation to a first enzyme composition comprising at least one hemicellulase, and subjecting the fermented mash after the fermentation to a second enzyme composition comprising a beta-1,3 glucanase and a xylanase.
In a first aspect, the present disclosure relates to methods to improve the nutritional quality of by-products or residues derived from starch-containing material in a processes for producing ethanol, wherein the process comprises a fermentation step, wherein the fermentation step is carried out in the presence of a first enzyme composition comprising at least one hemicellulase, and wherein the process comprises further the steps of i) subjecting the fermented mash after the fermentation to a second enzyme composition comprising a beta-1,3 glucanase and a xylanase, ii) separating the desired fermentation product by distillation.
In a second aspect, the present disclosure also relates methods of of producing ethanol from starch containing material, said method comprising the steps of:
The present disclosure also relates to uses of a first enzyme composition comprising a xylanase and preferably also a carboxymethylcellulase (CMCase) during the fermentation step and of a second enzyme composition comprising a beta-1,3 glucanase and a xylanase after the fermentation step for the improvement of the nutritional quality of the by-products or residues derived from fermented mash in a fermentative production process.
The object of the present invention is to provide improved fermentative production processes due to a better process ability and to provide by-products from the fermentation process with an improved nutritional quality.
One aspect of the present disclosure relates to methods for improving the the nutritional quality of by-products or residues derived from starch-containing material in a processes for producing ethanol, wherein the process comprises a fermentation step, wherein the fermentation step is carried out in the presence of a first enzyme composition comprising at least one hemicellulase, and wherein the process comprises further the steps of i) subjecting the fermented mash after the fermentation to a second enzyme composition comprising a beta-1,3 glucanase and a xylanase, ii) separating the desired fermentation product by distillation.
By-products or residues of the fermenting process includes distillers' grain, brewer's grains, dried distiller's grains, dried distiller's solubles, distiller's dried grains with solubles, WDG or/and residues of the cereal processing industry, or mixtures thereof. For example, DDGS are the dried residue remaining after the starch fraction of corn is fermented with selected yeasts and enzymes to produce ethanol and carbon dioxide. After complete fermentation, the alcohol is removed by distillation and the remaining fermentation residues are dried.
Stillage is the product which remains after the mash has been converted to sugar, fermented and distilled into ethanol. Stillage can be separated into two fractions, such as, by centrifugation or screening: (1) wet cake (solid phase) and (2) the thin stillage (supernatant). The solid fraction or distillers' wet grain (DWG) can be pressed to remove excess moisture and then dried to produce distillers' dried grains (DDG). After ethanol has been removed from the liquid fraction, the remaining liquid can be evaporated to concentrate the soluble material into condensed distillers' solubles (DS) or dried and ground to create distillers' dried solubles (DDS). DDS is often mixed with DDG to form distillers' dried grain with solubles (DDGS). DDG, DDGS, and DWG are collectively referred to as distillers' grain(s).
In one embodiment of the present disclosure the first enzyme composition is added during and the second enzyme composition is added after the fermentation in the production process but before the separation step like distillation, where the desired fermentation main product is separated from the rest of the fermented mash. The addition of the two enzyme compositions at different time points results in an improved nutritional value of the fermentation by-product like DDGS. This is a result of the combination of the treatment with an enzyme composition comprising xylanase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and subsequent treatment with an enzyme composition comprising xylanase and beta 1,3-glucanase in the fermented mash simulating the beer well tank is described. These shows, that the combination of the treatment with an enzyme composition comprising xylanase and preferably with a CMCase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and the treatment with an enzyme composition comprising xylanase and glucanase in the fermented mash is more effective and therefore more economic compared to the single treatment with the enzyme composition comprising xylanase and beta 1,3-glucanase in the fermented mash.
In one aspect of the present disclosure, the quality of by-products from a fermentative production process like DDG, DDGS or WDG can be improved with the methods according to the present disclosure by increasing the digestibility. For DDGS and other feedstuff increased in vitro dry-matter digestibility reflects improved nutritional value (Van Der Klis, J. D., Kwakernaak, C. Proving a concept: An in vitro approach. Journal of Applied (2014) Poultry Research, 23(2), pp 301-305; Weurding, R. E., Veldman, A., Veen, W. A. G., Van Der Aar, P. J., Verstegen, M. W. A. In vitro starch digestion correlates well with rate and extent of starch digestion in broiler chickens (2001) Journal of Nutrition, 131 (9), pp. 2336-2342; Gehring, C. K., Bedford, M. R., Cowieson, A. J., Dozier III, W. A. Effects of corn source on the relationship between in vitro assays and ileal nutrient digestibility (2012) Poultry Science, 91 (8), pp. 1908-1914; Valdes, E. V., Leeson, S. Measurement of metabolizable energy in poultry feeds by an in vitro system. (1992) Poultry science, 71 (9), pp. 1493-1503., Boisen, S., Fernández, J. A. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses (1997) Animal Feed Science and Technology, 68 (3-4), pp. 277-286.)
In one aspect, the present disclosure relates also to methods for improving the nutritional quality of by-products or residues derived from starch-containing material, said method comprising the steps of:
In another aspect, the present disclosure relates also to methods for improving the nutritional quality of by-products or residues derived from starch-containing material, said method comprising the steps of:
In an advantageous embodiment, the methods according to the present disclosure comprises the additional steps of:
The fermenting microorganism may be a fungal organism, such as yeast, or bacteria. Suitable bacteria may, e.g., be Zymomonas species, such as Zymomonas mobilis and Escherichia coli. Examples of filamentous fungi include strains of Penicillium species. Preferred organisms for ethanol production are yeasts, e.g., Pichia or Saccharomyces. Preferred yeasts according to the disclosure are Saccharomyces species, in particular Saccharomyces cerevisiae or baker's yeast.
Preferred organisms for ethanol production are yeasts, such as e. g. Pichia or Saccharomyces. Preferred yeast according to the disclosure is Saccharomyces species, in particular Saccharomyces cerevisiae or baker's yeast. The yeast cells may be added in amounts of 105 to 1012, preferably from 107 to 1010, especially 5×107 viable yeast count per ml of fermentation broth. During the ethanol producing phase the yeast cell count should preferably be in the range from 107 to 1010, especially around 2×108. Further guidance in respect of using yeast for fermentation can be found in, e. g., “The alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference
The feedstock for producing the fermentation product may be any starch/hemicellulose-containing material, preferably starch-containing plant material, including tubers, roots, whole grain or any combination thereof. The starch/hemicellulose-containing material may be obtained from cereals. Suitable starch/hemicellulose-containing material includes corn (maize), wheat, barley, cassava, sorghum, rye, potato, or any combination thereof. Corn is the preferred feedstock, especially when the fermentation product is ethanol. The starch-containing material may also consist of or comprise, e.g., a side stream from starch processing, e.g., C6 carbohydrate containing process streams that may not be suited for production of syrups. In particular, the starch-containing material comprises hemicelluloses like lignocellulose. A hemicellulose (also known as polyose) is any of several heteropolymers (matrix polysaccharides), such as arabinoxylans, present along with cellulose in almost all plant cell walls (see Scheller H V, Ulvskov P., Hemicelluloses. //Annu Rev Plant Biol. 2010; 61:263-89. doi: 10.1146/annurev-arplant-042809-112315). Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. These polysaccharides contain many different sugar monomers. In contrast, cellulose contains only anhydrous glucose. For instance, besides glucose, sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars, and occasionally small amounts of L-sugars as well. Xylose is in most cases the sugar monomer present in the largest amount, although in softwoods mannose can be the most abundant sugar.
Processes for producing fermentation products, such as ethanol, from a starch- or hemicellulose like lignocelluloses-containing material are well known in the art. In a preferred embodiment, the starch/lignocellulose containing material is milled cereals, preferably barley or corn, and the methods comprise a step of milling the cereals before step (a). The preparation of the starch/hemicellulose-containing material such as corn for utilization in such fermentation processes typically begins with grinding the corn in a dry-grind or wet-milling process. Grinding is also understood as milling, as is any process suitable for opening the individual grains and exposing the endosperm for further processing. Wet-milling processes involve fractionating the corn into different components where only the starch fraction enters into the fermentation process. Dry-grind processes involve grinding the whole corn kernels into meal, e.g., by hammer or roller mills, and subsequently mixing the meal with water and enzymes.
Generally two different kinds of dry-grind processes are used. The most commonly used process, often referred to as a “conventional process,” includes grinding the starch/hemicellulose-containing material and then liquefying gelatinized starch at a high temperature using typically a bacterial alpha-amylase, followed by “simultaneous saccharification and fermentation” (SSF) carried out in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as a “raw starch hydrolysis” (RSH) process, includes grinding the starch-containing material and then simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.
The term “alpha-amylase” means an alpha-1,4-glucan-4-glucanohydrolase (E.C. 3.2.1.1) that catalyzes the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
The mash may be provided by forming a slurry comprising the milled starch/hemicellulose-containing material and brewing water. The brewing water may be heated to a suitable temperature prior to being combined with the milled starch-containing material in order to achieve a mash temperature of 45-70° C., preferably of 53-66° C., more preferably of 55-60° C. The mash is typically formed in a tank known as the slurry tank.
The aqueous slurry may contain from 10-55 wt-% dry solids, preferably 25-45 wt-% dry solids, more preferably 30-40 wt-% dry solids of the starch-containing material. The slurry is heated to above the gelatinization temperature and an alpha-amylase, preferably a bacterial and/or acid fungal alpha-amylase, may be added to initiate liquefaction (thinning). The slurry may be jet-cooked to further gelatinize the slurry before being subjected to an alpha-amylase in step (a).
In the liquefaction step the gelatinized starch (downstream mash) is broken down (hydrolyzed) into maltodextrins (dextrins). To achieve starch hydrolysis a suitable enzyme, preferably an alpha-amylase, is added. Liquefaction may be carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C., preferably 80-85° C., and an alpha-amylase may be added to initiate liquefaction (thinning). Then the slurry may be jet-cooked at a temperature between 95-140° C., preferably 105-125° C., for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes. The slurry is cooled to 60-95° C. and more alpha-amylase may be added to complete the hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at a pH of 4.0 to 6.5, in particular at a pH of 4.5 to 6.
The saccharification step and the fermentation step may be performed as separate process steps or as a simultaneous saccharification and fermentation (SSF) step. The saccharification is carried out in the presence of a saccharifying enzyme, e.g., a glucoamylase, a beta-amylase or maltogenic amylase. Optionally a phytase and/or a protease is added.
Saccharification may be carried out using conditions well known in the art with a saccharifying enzyme, e.g., beta-amylase, glucoamylase or maltogenic amylase, and optionally a debranching enzyme, such as an isoamylase or a pullulanase. For instance, a full saccharification process may last up to from about 24 to about 72 hours, however, it is common to do a pre-saccharification for typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process. Saccharification is typically carried out at a temperature from 20-75° C., preferably from 40-70° C., typically around 60° C., and at a pH between 4 and 5, normally at about pH 4.5.
The most widely used process to produce a fermentation product, especially ethanol is the simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that a fermenting organism, such as a yeast, and enzyme(s), including the hemicellulase(s) and/or specific endoglucanase(s), may be added together. SSF is typically carried out at a temperature from 25-40° C., such as from 28-35° C., from 30-34° C., preferably around about 32° C.
The phrase “fermentation media” or “fermentation medium” refers to the environment in which fermentation is carried out and comprises the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism(s). The fermentation medium may comprise other nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; vitamins and minerals, or combinations thereof.
In an embodiment, fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
Product and by-Product Recovery
According to the invention the fermentation product may be any fermentation product, including alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, succinic acid, 2,5-diketo-D-gluconic acid), ketones (e.g., acetone), amino acids (e.g., glutamic acid), gases (e.g., H2 and CO2), and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline), enzymes; vitamins (e.g., riboflavin, B12, beta-carotene), and hormones. Fermentation is also commonly used in the consumable alcohol (e.g., beer and wine), dairy (e.g., in the production of yogurt and cheese), leather, and tobacco industries. In a preferred embodiment the fermentation product is a liquid, preferably an alcohol, especially ethanol.
The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation as mentioned above. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
As mentioned above, the by-product or residue is preferably a fibrous by-product selected from the group consisting of spent brewer's grains, dried distiller's grains, dried distiller's soluble, distiller's dried grains with soluble, wet grains, and mixtures thereof.
In an embodiment, the aqueous by-product (whole stillage) from the distillation process is separated into two fractions, e.g., by centrifugation: wet grain (solid phase), and thin stillage (supernatant). In another embodiment, the methods of the disclosure further comprise separation of the whole stillage produced by distillation into wet grain and thin stillage; and recycling thin stillage to the starch containing material prior to liquefaction. In one embodiment, the thin stillage is recycled to the milled whole grain slurry. The wet grain fraction may be dried, typically in a drum dryer. The dried product is referred to as distillers dried grains, and can be used as mentioned above as high quality animal feed. The thin stillage fraction may be evaporated providing two fractions (see
Further details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovering of ethanol are well known to the skilled person.
Methods for dewatering stillage and for extracting oil from a fermentation product are known in the art. These methods include decanting or otherwise separating the whole stillage into wet cake and thin stillage. See, e.g., U.S. Pat. Nos. 6,433,146, 7,601,858, and 7,608,729, and U.S. Application Publication No. 2010/0058649. Furthermore, the thin stillage can be evaporated or condensed into syrup or thick stillage from which the oil can be extracted utilizing centrifugation, filtering, heat, high temperature, increased pressure, or a combination of the same. Another way to extract oil is to lower the pH of the thin stillage or syrup. The use of surfactants to break emulsions also enhances oil extraction. Presses can also be used for dewatering.
Whole stillage typically contains about 10-15 wt-% dry solids. Whole stillage components include fiber, hull, germ, oil and protein components from the starch-containing feedstock as well as non-fermented starch.
The whole stillage is separated into solid and liquid fractions (i.e., wet cake and thin stillage containing about 35 wt-% and 7 wt-% solids, respectively). The thin stillage is often condensed by evaporation into syrup, subsequently recombined with the wet cake and further dried into DDGS for use in animal feed.
Subsequent to fermentation the fermentation product may be separated from the fermentation medium. The beer mash may be distilled to extract the desired fermentation product or the desired fermentation product from the beer mash by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping.
The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation as mentioned above. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
The saccharification and fermentation steps may be carried out either sequentially or simultaneously. The enzyme composition may be added during saccharification and/or after fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out simultaneously (SSF process).
In one embodiment of the present disclosure the first enzyme composition were added during the fermentation in the production process to the fermentation medium.
In some advantageous embodiments, the first enzyme composition is present or added during the optional pre-saccharification step, saccharification step (b), and/or fermentation step (c), or simultaneous saccharification and fermentation. In further examples, the fermentation medium is subjected before, during and/or after the fermentation process to an enzyme composition.
Further, by adding the enzymes according to the present disclosure to the fermentation medium or the beer mash before the distillation step is an advantage since the enzymes in the enzyme compositions are inactivated during the distillation.
In an advantageous embodiment, the first enzyme composition comprises a hemicellulose, in particular xylanase.
In a further embodiment, the first enzyme composition comprises a cellulose, in particular a carboxymethylcellulase (CMCase).
In a further embodiment, the first enzyme composition comprises at least a further enzyme selected from the group consisting of phytase, pectinase, cellulase, glucanase and protease.
In an advantageous embodiment, the second enzyme composition comprises a beta-1,3 glucanase and a xylanase.
In a further embodiment, the second enzyme composition comprises at least a further enzyme selected from the group consisting of phytase, pectinase, cellulase, glucanase and protease.
In an advantageous embodiment, the first enzyme composition comprises a xylanase and a carboxymethylcellulase (CMCase) and the second enzyme composition comprises a beta-1,3 glucanase and a xylanase.
Hemicellulases as used herein are enzymes capable to break down hemicellulose like lignocellulose. Any hemicellulase suitable for use in hydrolyzing hemicellulose, preferably into xylose, may be used. Preferred hemicellulases include acetylxylan esterases, endo-arabinases, exo-arabinases, arabinofuranosidases, feruloyl esterase, endo-galactanases, exo-galactanases, glucuronidases, mannanases, xylanases, and mixtures of two or more thereof. Preferably, the hemicellulase for use in the present invention is an exo- and endo-acting hemicellulase, and more preferably, the hemicellulase is an exo-acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7, preferably pH 3-7.
In one aspect, the hemicellulase(s) comprises a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, Cellic® HTec, Cellic® HTec2, Viscozyme®, Ultraflo® and Pulpzyme® HC (from Novozymes A/S), Accellerase® XY, Accellerase® XC (from Genencor Int.), Ecopulp® TX-200A (from AB Enzymes GmbH), Bakezyme® HSP 6000 (from DSM Food Specialties), Depol™ 333P, Depol™ 740L, and Depol™762P (from Biocatalysts Ltd).
In an embodiment of the present disclosure the xylanase may preferably be of microbial origin, such as of fungal origin (e.g., Aspergillus, Fusarium, Humicola, Meripilus, and Trichoderma) or from a bacterium (e.g., Bacillus). In a preferred embodiment the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus; or a strain of Humicola, preferably Humicola lanuginosa. Examples of xylanases useful in the methods of the present invention include, but are not limited to Aspergillus aculeatus xylanase (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO 2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO 2009/079210). The xylanase may preferably be an endo-1,4-beta-xylanase, more preferably an endo-1,4-beta-xylanase of GH 10 or GH 11. Examples of commercial xylanases include Shearzyme® 500L, Bio-Feed™ Wheat (from Novozymes A/S), Econase® CE (from AB Enzymes GmbH), Depol™ 676 (from Biocatalysts Ltd.) and Multifect® Xylanase, Spezyme® CP (from Genencor Int.).
Examples of exo-1,4-beta-xylosidase useful in the methods of the present invention include, but are not limited to, Trichoderma reesei beta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromyces emersonii (SwissProt accession number Q8X212), and Neurospora crassa (SwissProt accession number Q7SOW4).
Examples of suitable bacterial xylanases include xylanases derived from a strain of Bacillus, such as Bacillus subtilis, such as the one disclosed in U.S. Pat. No. 5,306,633.
Xylanase may be added in an amount effective in the range from 0.16×106-460×106 Units per ton beer mash or fermentation medium.
Mannanases as used herein are enzymes capable to break down the part of the hemicelluloses fraction consisting of mannans and heteromannans in plant walls. The mannanases as used in the present disclosure may be any mannanase either endo- and exo--1,4-mannanases but preferably exo-
-1,4-mannananse, in particular of microbial origin, in particular fungal or bacterial origin.
This may be a mannanase such as a mannanase derived from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Phanerochaete). Preferably, the mannanases act on different compositions of lignocellulosic material. Preferred mannanases for use in the present invention include endo-acting mannanases, exo-acting mannanases, and combinations thereof. Examples of commercially available mannanases suitable according to the present invention include, for example, Hemicell® (from Elanco Animal Health) and Purabrite® CP (from Genencor Int.).
The pectinase used in the present disclosure may be any pectinase, in particular of microbial origin, in particular of bacterial origin, such as a pectinase derived from a species within the genera Bacillus, Clostridium, Pseudomonas, Xanthomonas and Erwinia, or of fungal origin, such as a pectinase derived from a species within the genera Trichoderma or Aspergillus, in particular from a strain within the species Aspergillus niger and Aspergillus aculeatus. Contemplated commercially available pectinases include Pectinex® Ultra-SPL, Pectinex® Ultra Color (from Novozymes A/S), Rohapect® Classic, Rohapect® 10L (from AB Enzymes GmbH).
Pectinase may be added in an amount effective in the range from 1.4×109-23500×109 Units per ton beer mash or fermentation medium.
The proteases as used in the present disclosure may be any protease, in particular of microbial origin, in particular of fungal and bacterial origin. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium and Torlopsis. Commercial proteases include GC 106′ and Spezyme® FAN (from Genencor Int.). Suitable microbial proteases, although not acidic proteases, include the commercially available products Alcalase® and Neutrase® (both from Novozymes A/S), EX-Protin (from C. Schliessmann Kellerei-Chemie GmbH & Co. KG) and Ronozyme® ProAct (from DSM Nutritional Products).
The cellulase as used in the present disclosure may be any cellulase, in particular of microbial origin, in particular fungal or bacterial origin such as a cellulase derived from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium). Preferably, the cellulase acts on both cellulosic and lignocellulosic material. Preferred cellulases for use in the present invention include endo-acting cellulases, exo-acting celluases and cellobiases, and combinations thereof. Examples of commercially available cellulases suitable according to the present invention include, for example, Celluclast® (from Novozymes A/S), LAMINEX® and Spezyme® CP (from Genencor Int.) and Econase® CE (from AB Enzymes GmbH), Rohalase® BX (from AB Enzymes GmbH), Cellulase 13P (from Biocatalysts Ltd.).
In advantageous embodiments, the first enzyme composition comprises an endoglucanase (1,4-β-D-glucan-4-glucanohydrolase, carboxymethylcellulase or CMCase; EC 3.2.1.4). CMCase are e.g. described in Kotchoni, O. S., W. E. Gachomo, B. Omafuvbe and 0.0. Shonukan, 2006. Purification and biochemical characterization of carboxymethyl cellulase (CMCase) from a catabolite repression insensitive mutant of Bacillus pumilus. Int. J. Agric. Biol., 8: 286-292.
Determination of Carboxymethyl-cellulase (CMCase) (Endoglucanase) activity: CMCase activity may be assayed using a modified method described by Wood and Bhat (1998) with some modifications. The CMCase activity was measured by mixing 0.1 mL of enzyme solution with 0.1 mL of 1.0% (w/v) CMC in 10 mM sodium phosphate buffer, pH 7.0 at 37° C. for 60 min. The reaction was stopped by adding 1.0 mL 3,5-dinitro salicylic acid (DNS) regent. The mixture was boiled for 10 min cooled in ice and its optical density at 546 nm was determined. The CMCase activity was measure by using a calibration curve for glucose. One unit of CMCase was defined as the amount of enzyme that released 1 μmot of glucose per min.
Cellulases may be added in amounts effective in the range or from 0.03×106-16×106 Units per ton beer or fermentation medium.
Glucanase
Glucan and chitin are far more resistant to microbial degradation than cellulose, which is the major constituent of the cell wall of many yeasts and fungi-like organisms. Glucan is predominantly beta-1,3-linked with some branching via 1,6-linkage (Manners et al., Biotechnol. Bioeng, 38, p. 977, 1973), and is known to be degradable by certain beta-1,3-glucanase systems. Beta-1,3-glucanase includes the group of endo-beta-1,3-glucanases also called laminarinases (E.C. 3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc. 1992). Beta-1,6-glucanases are enzymes hydrolyzing the beta-1,6-linkage in glucan.
The beta-1,3-glucanases as used in the present disclosure may be any 1,3-glucanase, in particular of microbial origin, in particular fungal or bacterial origin such as a beta-1,3-glucanase derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Penicillium, Humicola). The beta-1,3-glucanases may preferably be an endo-1,3-beta-glucanase. The beta-1,6-glucanases as used in the present disclosure may be any 1,6-glucanase, in particular of microbial origin, in particular fungal or bacterial origin such as a beta-1,6-glucanase derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Penicillium, Humicola). The beta-1,6-glucanases may preferably be an endo-1,6-beta-glucanase.
Examples for commercial available beta-1,3-glucanases suitable according to the present invention include, for example, Rohalase® BX (from AB Enzymes GmbH), Dyadic® Beta Glucanase BP CONC (from Dyadic International (USA), Inc.) and Rapidase® Glucalees (from DSM Food Specialties). Examples for commercial available beta-1,6-glucanases suitable according to the present invention include, for example, ThermoActive™ Pustulanase Ce1136 (from Prokazyme Ltd.).
1,3-glucanases and 1,6-glucanases may be added in an amount effective in the range from 0.08×106-920×106 Units per ton beer mash or fermentation medium.
Any phytase may be used in the methods of the present disclosure. Phytases are enzymes that degrade phytates and/or phytic acid by specifically hydrolyzing the ester link between inositol and phosphorus. Phytase activity is credited with phosphorus and ion availability in many ingredients. In some embodiments, the phytase is capable of liberating at least one inorganic phosphate from an inositol hexaphosphate (e.g., phytic acid). Phytases can be grouped according to their preference for a specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated (e.g., 3-phytase (EC 3.1.3.8) or 6-phytase (EC 3.1.3.26)). An example of phytase is myo-inositol-hexakiphosphate-3-phosphohydrolase. Phytases can be obtained from microorganisms such as fungal and bacterial organisms. For example, the phytase may be obtained from filamentous fungi such as Aspergillus (e.g., A. ficuum, A. fumigatus, A. niger, and A. terreus), Cladospirum, Mucor (e.g., Mucor piriformis), Myceliop t ora (e.g., M. t ermop ila), Penicillium (e.g., P. ordei (ATCC No. 22053)), P. piceum (ATCC No. 10519), or P. brevi-compactum (ATCC No. 48944), Talaromyces (e.g., T. thermophilus), Thermomyces (WO 99/49740), and Trichoderma spp. (e.g., T. reesei). In an embodiment, the phytase is derived from Buttiauxiella spp. such as B. agrestis, B. brennerae, B. ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B. warmboldiae. In some embodiments, the phytase is a phytase disclosed in WO 2006/043178.
The inventions described and claimed herein are not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following example that should not be construed as limiting the scope of the invention.
In the present example the improved nutritional value of DDGS as result of the combination of the treatment with an enzyme composition comprising xylanase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and subsequent treatment with an enzyme composition comprising xylanase and beta 1,3-glucanase in the fermented mash simulating the beer well tank is described.
For DDGS and other feedstuff increased in vitro dry-matter digestibility reflects improved nutritional value (Van Der Klis, J. D., Kwakernaak, C. Proving a concept: An in vitro approach. Journal of Applied (2014) Poultry Research, 23(2), pp 301-305; Weurding, R. E., Veldman, A., Veen, W. A. G., Van Der Aar, P. J., Verstegen, M. W. A. In vitro starch digestion correlates well with rate and extent of starch digestion in broiler chickens (2001) Journal of Nutrition, 131 (9), pp. 2336-2342; Gehring, C. K., Bedford, M. R., Cowieson, A. J., Dozier III, W. A. Effects of corn source on the relationship between in vitro assays and ileal nutrient digestibility (2012) Poultry Science, 91 (8), pp. 1908-1914; Valdes, E. V., Leeson, S. Measurement of metabolizable energy in poultry feeds by an in vitro system. (1992) Poultry science, 71 (9), pp. 1493-1503., Boisen, S., Fernandez, J. A. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses (1997) Animal Feed Science and Technology, 68 (3-4), pp. 277-286.)
In the in vitro digestion system dry matter of DDGS (in weight-%) is digested by different digestive enzymes. The more dry matter is digested by the in vitro digestion system the more the nutritional value of DDGS has improved.
Therefore an example of increased in vitro dry matter digestibility of DDGS as a result of the combination of the treatment with an enzyme composition comprising xylanase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and the subsequent treatment with an enzyme composition comprising xylanase and beta 1,3-glucanase in the fermented mash simulating the beer well tank is described.
In one embodiment, the process of the production of DDGS from corn was performed as follows:
DNSA solution: For the DNSA solution the following compounds were used:
Substrate Xylan from beechwood (Sigma X4252)
Buffer: 100 mM sodium acetate, pH 5.0 containing 20 mM CaCl2 and 0.04 g/L Tween20
Substrate was dissolved in the buffer to a concentration of 1.5% (w/v). 1.5 g of xylan from beechwood low viscosity was suspended in 100 ml of buffer was continuously and mixed well. For dissolving the xylan suspension it is autoclaved (121° C. for 20 min, cooling slowly) together with a magnetic stir bar. For using the exact xylan concentration the xylan suspension is weighed before and after autoclaving and the volume lost by autoclaving was added with autoclaved (121° C. for 20 min) deionized water.
For the assay 1.5 mL reaction tubes (Eppendorf) were used. The enzymes were diluted in buffer. 200 μL of the 1.5% xylan substrate was pre heated for 5 minutes at 40° C. in a Thermomixer (Eppendorf). Then a 20 μL enzyme solution was added. A blank was measured replacing enzyme solution with water. Incubation was carried out exactly for 20 min at 40° C., followed by the addition 100 μL of the DNSA solution stopping the enzyme reaction and vigorously vortexing for 15 seconds. Then the sample is stored in an ice-bath.
144 μL of the stopped substrate-enzyme mix was pipetted into a well of a 96 well PCR microtiter plate (Greiner). For a xylose standard curve 99 μl of xylose standards and 45 μl DNSA reagent were added and mixed with a pipette.
The 96 well PCR plate, which was covered with sticky aluminum foil, which was sealed tightly, was incubated in a Thermal Cycler (BIO-RAD) for 10 minutes at 99° C. and 5 min for 4° C. with heated lid (temperature is calculated from the cycler). Then the 96 well PCR plate was centrifuged in a plate centrifuge at 3000 g for 5 min at room temperature (20-25° C.). 25 μl of the supernatant with room temperature (20-25° C.) was transferred into a 96 well Nunc plate (transparent, flat) into which 78 μl deionized water had been added and mixed with pipette two times. The adsorption was measured at wavelength 540 nm by a micro titer plate reader (Tecan Infinite M1000).
The activity is calculated as Units per μL or mg of enzyme product. 1 Unit is defined as the amount of formed reducing ends in μmot per minute. The enzyme activities are shown in Table 1.
Substrates: Barley beta-glucan (low viscosity) (Megazyme P-BGBL)
Buffer: 100 mM sodium acetate, pH 5.0 containing 20 mM CaCl2 and 0.4 g/L Tween20
Substrate was dissolved in the buffer to a concentration of 1.5% (w/v). 1.5 g of Barley beta-glucan low viscosity was suspended in 100 ml of buffer was continuously and mixed well. For dissolving the Barley beta-glucan suspension it is autoclaved (121° C. for 20 min, cooling slowly) together with a magnetic stir bar. For using the exact Barley beta-glucan concentration the Barley beta-glucan suspension is weighed before and after autoclaving and the volume lost by autoclaving was added with autoclaved (121° C. for 20 min) deionized water.
For the assay 1.5 mL reaction tubes (Eppendorf) were used. The enzymes were diluted in buffer. 200 μL of the 1.5% Barley beta-glucan substrate was pre heated for 5 minutes at 40° C. in a Thermomixer. Then a 20 μL enzyme solution was added. A blank was measured replacing enzyme solution with water. Incubation was carried out exactly for 20 min at 40° C., followed by the addition 100 μL of the DNSA solution stopping the enzyme reaction and vigorously vortexing for 15 second. Then the sample is stored in an ice-bath.
144 μL of the stopped substrate-enzyme mix was pipetted into a well of a 96 well PCR microtiter plate (Greiner). For a glucose standard curve 99 μl of glucose standards and 45 μl DNSA reagent were added and mixed with a pipette.
The 96 well PCR plate, which was covered with sticky aluminum foil, which was sealed tightly, was incubated in a Thermal Cycler (BIO-RAD) for 10 minutes at 99° C. and 5 min for 4° C. with heated lid (temperature 105° C. to prevent condensation). Then the 96 well PCR plate was centrifuged in a plate centrifuge at 3000 g for 5 min at room temperature (20-25° C.). 25 μl of the supernatant with room temperature (20-25° C.) was transferred into a 96 well Nunc plate (transparent, flat) into which 78 μl deionized water had been added and mixed with a pipette two times. The adsorption was measured at wavelength 540 nm by a micro titer plate reader (Tecan Infinite M1000).
The activity is calculated as Units per μL or mg of enzyme product. 1 Unit is defined as the amount of formed reducing ends in μmol per minute. The enzyme activities are shown in Table 1.
The determination of the in vitro dry matter digestibility of DDGS, which has been dried and ground to 1 mm particles, was performed based on the method published by Boisen and Fernandez (Boisen, S., Fernandez, J. A. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses (1997) Animal Feed Science and Technology, 68 (3-4), pp. 277-286.)
In this process a defined mass of an initial DDGS sample produced by enzymes added to the fermentor and after 45.75 hours to the fermented mash (beer) is treated with the digestive enzymes pepsin and pancreatin, resulting in the liquefaction of the digestible components of DDGS.
The liquefied digestible components of DDGS are separated by centrifugation from the non-digestible compounds of DDGS. The mass of the non-digestible compounds of the DDGS is determined and after subtracting it from the initial DDGS the mass of the digested compounds of DDGS are calculated.
For evaluation the effects of the combination of the treatment with the enzyme composition comprising xylanase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and the treatment with an enzyme composition comprising xylanase and beta 1,3-glucanase in the beer well tank the mass of the digested compounds of DDGS was plotted relative to the reference, i.e. without enzyme treatment during the fermentation process and without enzyme treatment of the fermented mash (beer). The reference is set to 100%.
The analysis of each setup was performed in triplicates.
About 0.6 to 0.7 g of DDGS, which was dried and ground to particle sizes of about 1 mm, was added into a 100 mL shake flasks containing a magnetic stirrer. 25 mL of 0.1 M sodium phosphate buffer pH 6.0 was added into each shake flask and stirred for 5 minutes on the magnetic stirrer to homogenize the suspension. 10 ml of 0.2 M HCl was added to the suspension, which was adjusted to obtain pH 2.0 by adding 1 M HCl or 1 M NaOH. To this suspension 1 mL of a pepsin solution containing 0.1 mg/mL pepsin (Sigma, P6887, porcine, 500 U/mg) and 0.5 mL chloramphenicol solution in concentration of 0.5 g chloramphenicol/100 ml EtOH was added. The shake flasks were closed with rubber stoppers and incubated at for 1 hour at 60 rpm at 39° C. in a shaker.
After pepsin digestion DDGS was digested by pancreatin. 10 mL 0.2 M sodium phosphate buffer pH 6.8 was added to the shake flasks, followed by adding 5 mL of a 0.6 M NaOH solution and adjustment to obtain pH 6.8 by adding 1 M HCl or 1 M NaOH. To this suspension 1 mL of a centrifuged (4000 g for 30 minutes) pancreatin solution containing 100 mg/mL pancreatin (Sigma, porcine, P1750) was added. The shake flasks were closed with rubber stoppers and incubated at for 2 hours at 60 rpm at 39° C. in a shaker.
The mass of paper filters (Whatman No. 1, 125 mm Ø, CatNo. 1001125), which were died at 90° C. over night, was determined. Then the content of each of the shake flasks was filtered through a dried and weighed filter. In order to obtain also the residual non-digestible compounds the shake flasks were washed with deionized water (about 100 mL), which was previously heated at 40° C. The obtained suspensions were also additionally poured onto the corresponding filters. Finally the filters were dried over night at 90° C. and the cooled in a desiccator before weighing them.
Absolute weight of dried (100% dry weight) DDGS matter after dry matter in vitro dry matter digestion=(Filter weight of dried filter with digested DDGS)−(Filter weight of empty dried filter) a)
% of non-digestible compounds of DDGS (based on 100% dry weight) related to initial DDGS (based on 100% dry weight)=100*Absolute weight of dried (100% dry weight) DDGS matter after in vitro dry matter digestion [g]/initial DDGS (100% dry weight) [g] b)
Definition: Digestible compounds of DDGS in %=100−% of non-digestible compounds of DDGS (based on 100% dry weight) related to initial DDGS (based on 100% dry weight) c)
d) Calculation of the mass of digestible compounds of DDGS normalized to the reference in % (Treatment after start of Fermentation (fermentor): 0 g/t xylanase; Treatment after 45.75 h (simulation of beer well): 0 g/t xylanase plus 0 g/t beta 1,3-glucanase):
100*digestible compounds of DDGS in %/digestible compounds of DDGS in %; which is designated as in vitro dry matter digestibility of DDGS [%; 100% is set to the reference DDGS sample; (Setup #1 (pooled DDGS from shake flasks #1A and #1B))].
Reference DDGS samples: DDGS made from fermented mash (beer), into which neither a xylanase (Xyl40) (0 g/t) had been added into the fermentation process in the fermentor nor the combination of xylanase (Xyl41) and beta 1,3-glucanase (Glu40) (0 g/t xylanase plus 0 g/t beta 1,3-glucanase) had been added to the fermented mash (beer) after 45.75 h of the fermentation process:
In the depicted examples this is DDGS created from set up #1, i.e. pooled DDGS from shake flasks #1A and #1B.
The results of analysis of in vitro dry matter digestibility of the DDGS sample are presented in Table 2.
Table 2 shows that the combination of the treatment with an enzyme composition xylanase (Xyl40) (140 g/t) during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and the treatment with an enzyme composition comprising xylanase (Xyl41) and beta 1,3-glucanase (Glu40) (50 g/t of both enzymes) in the fermented mash simulating the beer well tank resulted in a higher in vitro dry matter digestibility (134.4%, Setup#4) compared to single enzyme treatment in the fermentor (112.9%, Setup#3) or in the beer well (125.6%, Setup#2).
Even a 12-fold increase of the enzyme amount of xylanase (Xyl41) and beta 1,3-glucanase (Glu40) (600 g/t of both enzymes) in the fermented mash was not able to compensate the effect on DDGS digestibility (125.6%, Setup#2), which was observed by the synergistic effect of treatment in the fermentor with the enzyme composition comprising xylanase (Xyl40) and with the enzyme composition comprising xylanase (Xyl41) and beta 1,3-glucanase (Glu40) in the fermented mash (134.4%, Setup#5).
These results demonstrate, that the combination of the treatment with an enzyme composition comprising xylanase during the fermentation process from starch-containing material using the fermenting microorganism wild-type yeast (Saccharomyces cerevisiae) and the treatment with an enzyme composition comprising xylanase and glucanase in the fermented mash is more effective and therefore more economic compared to the single treatment with the enzyme composition comprising xylanase and beta 1,3-glucanase in the fermented mash.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/079453 | 11/16/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62423559 | Nov 2016 | US |
