WO2008040358A1

WO2008040358A1 - A method for providing proteins and fermentation products from a plant material

Info

Publication number: WO2008040358A1
Application number: PCT/DK2007/050135
Authority: WO
Inventors: Pauli Kiel; Nicolaas M . G. Oosterhuis
Original assignee: Protanol Bv
Priority date: 2006-10-03
Filing date: 2007-10-03
Publication date: 2008-04-10

Abstract

The present invention relates to a method for providing of protein and fermentation products from a plant material. In particular, there is provided a method wherein said proteins are obtained from the liquid phase of a processed plant material. Furthermore, the invention pertains to a high K-vinasse obtainable by the method of the invention. A method of modifying compost and other organic fertilizers, comprising adding to the compost or other organic fertilizers the high K-vinasse is also disclosed in the present invention.

Description

A METHOD FOR PROVIDING PROTEINS AND FERMENTATION PRODUCTS FROM A PLANT MATERIAL

Technical field of the invention The present invention pertains in general to the provision of proteins and fermentation products from a plant material. In particular, there is provided a method wherein said proteins are obtained from the liquid phase of the plant material. Furthermore, the invention pertains to a high K-vinasse obtainable by the method of the invention.

Background of the invention

Limited recourses of fossil fuels and an increasing demand for energy in the industrial countries as well as in a growing number of fast developing countries has resulted in an increased focus on alternatives to fossil fuels as energy sources. In addition, fossil fuels contribute significantly to the amount of CO₂ released into the atmosphere, and as CO₂ is defined as a "greenhouse gas" it is believed to contribute significantly to the global warming. Consequently, the demand for alternative- and environmental friendly combustion fuels is evident.

Biogenic fuels derived from harvested plant material are combustion fuels defined as "Cθ2-neutral", as the amount of CO2 released by combustion correlates to the amount of CO2 originally taken up by the plant during biomass build-up. Moreover said biogenic fuels neither contain sulphur nor aromatic compounds.

Approved candidates to replace or at least supplement regular fossil fuels are biogenic fuels such as ethanol, buthanol and methane.

Besides from being an apparent alternative to fossil fuels, the production of biogenic fuels are used to remedy the overproduction of some agricultural plant materials, as these fuels can be produced from starch crops, sugar crops or lignocellulosic feedstock. As it is well known that such plants contain valuable proteins, the industry has developed alcohol fermentation processes where proteins contained in the remaining fermentation effluent, the so-called "distillation grain", are recovered after the fermentation process and subsequently utilised as an animal feed.

For example Gibbons et al. (1987) discloses a semi-continuous diffusion fermentation process for obtaining ethanol and cubed protein feed from cubed fodder beets. The process is performed in a special shaped fermentor wherein ethanol is continuously exiting from one end of the fermentor and the fermentation effluent is continuously recovered at the other end in form of a cubed protein feed.

However, the traditional alcohol fermentation processes endure from inefficiencies in utilizing the plant material in an optimal energy effective way, disclosing the need for an additional and more effective process, which simultaneously provides high value protein products and valuable fermentation products from a plant material. Accordingly, the process of the invention will not only provide improved process economy, but also a more sustainable utilization of agricultural plant materials.

Summary of the invention

Accordingly, the present invention pertains to a method for providing at least one protein and at least one fermentation product from a plant material, said method comprises the steps of (i) disintegrating the plant material in order to obtain a solid phase and a liquid phase, (ii) separating the liquid phase, containing dissolved compounds from the solid phase, containing fibres and other insoluble compounds, (iii) recovering at least one protein from the liquid phase obtaining an isolated protein and a supernatant, and (iv) subjecting the supernatant to a fermentation process obtaining at least one alcohol as fermentation product.

In further aspects, the invention relates to a high K-vinasse obtainable by the method of the invention and to the use of such high K vinasse as organic fertilizer. A still further aspect relates to a method of modifying compost comprising adding to the compost the high K vinasse according to the invention.

Brief description of the figures The invention may be described in the following non-limiting figures, which are presented for illustrative purposes only.

Figure 1 shows the method for providing at least one protein and at least one alcohol fermentation product, biogas and a plant based organic fertilizer from a plant material. The dashed line symbolizes optional steps, and

Figure 2 shows the foaming ability and solubility of acid precipitated proteins (native proteins) and acid + heat precipitated proteins (non-native proteins).

Detailed description of the invention

It is the aim of the present invention to provide a method for producing valuable proteins from selected plant material before subjecting the plant material to an alcohol fermentation. The inventors found that there is a large potential for recovering valuable proteins, e.g. native proteins, present in plant materials which are processed for an alcohol fermentation process, such as an ethanol fermentation process. Furthermore, the inventors found that such proteins with advantage can be isolated from the liquid of the processed plant materials, whereby the remaining liquid is subsequently subjected to a fermentation to obtain alcohols useful as biogenic fuels.

Thus, the invention is based on a combination of three main process steps, namely a first step where the disintegrated plant material is separated in a liquid phase and a solid phase as this facilitates the present process. Then, proteins are recovered for further use from the liquid phase resulting in a liquid which has a high content of fermentable, soluble carbohydrates and/or organic acids and a low amount of protein and nitrogen. After the protein removal, the remaining liquid is subjected to a fermentation, preferable an alcohol fermentation. Accordingly, a first aspect of the present invention relates to a method for providing at least one protein and at least one fermentation product from a plant material, said method comprises the steps of (i) disintegrating the plant material in order to obtain a solid phase and a liquid phase, (ii) separating the liquid phase from the solid phase, (iii) recovering at least one protein from the liquid phase obtaining an isolated protein and a supernatant, and (iv) subjecting the supernatant to a fermentation process obtaining at least one fermentation product.

Optional further steps of the invention, as described later, relate to the conversion of the remaining organic compounds in the fermentation effluent, i.e. the stillage or vinasse, from the alcohol fermentation or methane fermentation, and the solid phase of the processed plant material may be added to the alcohol fermentation effluent to produce biogas (methane). The biogas may be used internally as energy source or sold for energy purposes.

The plant material

Any kind of material from various plant species and genera may be useful in the method of the present invention. However, it is presently preferred to employ plant materials comprising carbohydrates and even more preferable a plant material comprising a high content of carbohydrates. Such carbohydrates may be soluble carbohydrates or fructans (e.g. levan and graminan) as these are readily utilized by the normally used fermentation organisms such as Saccharomyces cerevisiae. Preferred carbohydrates in the plant material useful in the present method include, but are not limited, to saccharides like sucrose, fructose, glucose, galactose, xylose, mannose and maltose.

Thus, plants or parts of plants, where the main part of the carbohydrates are contained in starch granules, like e.g. in cereals and corn, are of no particular interest in the present process. In an embodiment, the plant material originates from a plant comprising no or a limited content of starch and/or starch granules. As starch is composed of a mixture of two complex carbohydrates, namely amylose and amylopectin, starch cannot be readily utilized by the normally used fermentation organisms such as Saccharomyces cerevisiae and is therefore not of any particular interest in respect of the present invention. The standard method for utilising starch containing plant materials such as potatoes, wheat, corn etc. for ethanol production involves a pre-treatment of the plant material. Accordingly, the plant material is milled wet or dry, mixed with water, heated up to about 80⁰C and amylolytic enzymes are added. After about one hour the temperature is decreased to about 60⁰C and glycosidase enzymes are added and stored for 12 to 24 hours at that temperature. After the enzymatic treatment, the hydrolysed starch medium is cooled down to 25-30⁰C, and subsequently yeast is added and the medium is fermented to ethanol.

If the plant juice contains significant amounts of mono- and di- saccharides like in the present invention, these carbohydrates can be converted to ethanol by yeast cells substantially without any pre-treatment, including enzymatic pre-treatment. Thus, denaturation of proteins is avoided, as no heat treatment is involved. The process economy is therefore significantly improved, by using the method disclosed in the present invention.

It will be appreciated, that the plant material may be selected from a genetically modified plant of one of the above plants or a plant originally having a low content of soluble carbohydrates or containing starch and/or originally having a low content of protein (the term "originally having" relates to plant materials before being genetically modified). Useful genetically modified plants would in accordance with the present invention contain a higher amount of soluble carbohydrates or have a reduced content of starch compared to the plant from which it is derived. As used herein the expression "genetically modified plant" is used in the conventional meaning of that term and refers to a plant which by the process of transformation is made to contain DNA sequences which are not normally present in the plant, or DNA sequences which are in addition to the sequences which are normally in the plant, or DNA sequences which are normally in the plant but which are altered compared to the native sequence.

Useful plant materials may be selected from plants having no or a limited content of starch and/or starch granules, such as beet, pineapple, chicory, sugar cane, carrot, parsnip, high sugar containing fruits, such as grape and apples and combinations thereof. Plant materials such as Jerusalem artichoke are of no particular relevance to the present invention as the polysaccharide inulin contained herein cannot (like starch) be readily utilized by the normally used fermentation organisms such as Saccharomyces cerevisiae. Accordingly, plant materials containing simple carbohydrates like mono- and di-saccharides are presently preferred as such sugars may be directly converted to ethanol by the normally used fermentation organisms such as Saccharomyces cerevisiae, thus providing a higher ethanol yield.

Even though the fermentation processes of the present invention are applicable to any kind of plant material it may be useful to employ low cost plant materials (e.g. plan materials with a high carbohydrate production (high ethanol potential) per hectare) in order to reach an improved process economy. In useful embodiments, low-cost plant materials include plant species of the genera Beta vulgaris such as sugar beet, fodder beet, garden beet, white beet or red beet.

In a preferred embodiment the beet is sugar beet or fodder beet.

Other low-cost plant materials may include waste materials from the industrial processing of various kinds of fruits, vegetables and agricultural crops. For example, it was found by the present inventors that the leaves of a harvested sugar beet (sugar beet top) provided a valuable plant material for the present process. Green juice from grass, clover alfalfa, a residue from the green crop drying industry could be a cheap source for protein production using the described technology. Accordingly, any type of plant part such as stem, leaves, root, fruits, seeds, tubers and combination thereof are useful in the present invention. As the content of soluble carbohydrates and protein changes during the growing season of a plant, it will be understood, that e.g. at the beginning of the season the steam and leaves of the plants may be of interest, and later on carbohydrate containing tubers may particular be useful.

According to the present invention it is desirable to use plant materials comprising a high content of carbohydrates, as this will improve the process economy significantly. This improvement is obtained by means of e.g. optimizing the efficiency of the normally used ethanol fermentation organisms Saccharomyces cerevisiae as these organisms are able to readily utilize simple carbohydrates. If the used plant materials contained significant amounts of starch, which is composed of complex carbohydrates, the efficiency of the normally used 5 fermentation organisms Saccharomyces cerevisiae would decrease as these complex carbohydrates were first to be degraded into less complex carbohydrates, before they could be used by the fermentation organisms.

In a preferred embodiment of the present invention, the carbohydrate content in 10 the plant material is of at least 0.1% (w/w). In preferred embodiments for combined protein and alcohol production, the plant material has a carbohydrate content of at least 0.5% (w/w), e.g. at least 1% (w/w), such as at least 2% (w/w), e.g. at least 5% (w/w), such as at least 10% (w/w), e.g. at least 15% (w/w), including at least 20% (w/w), such as at least 25% (w/w). 15

In further embodiments, the plant material has a carbohydrate content in the range of 0.1%-25% (w/w), such as 0.5%-25% (w/w), e.g. in the range of 1%- 25% (w/w), including in the range of 2%-10% (w/w), such as in the range 10%- 25% (w/w), e.g. in the range of 15%-25% (w/w), such as in the range of 1%- 20 20%, e.g. in the range of 5%-20% (w/w), such as in the range of 15%-20% (w/w), including in the range of 1%-15% (w/w), such as in the range of 5%-15% (w/w).

As one of the objects of the present invention is to provide plant material derived 25 proteins, it is preferred to use plant materials with a high content of proteins. Hence, in a useful embodiment of the present invention the plant material has a protein content of at least 0.1% (w/w), e.g. least 0.2% (w/w), such as 0.5% (w/w), including least 1% (w/w), such as at least 2% (w/w), e.g. at least 3% (w/w), including at least 4 % (w/w), e.g. at least 5% (w/w). 30

In further embodiments, the plant material has a protein content in the range of 0.1% - 5% (w/w), such as in the range of 0.2% - 3% (w/w), e.g. in the range of 0.5% - 5% (w/w), such as in the range of 1% - 5% (w/w), e.g. in the range of 1.5% - 5% (w/w), such as in the range of 0.5 % - 4% (w/w), including in the 35 range of 1% - 3% (w/w) , e.g. in the range of 2%-5% (w/w), such as in the range of 2%-3% (w/w), e.g. in the range of 0.1%-2.5% (w/w), including in the range of l%-2% (w/w).

Processing the plant material In accordance with the present invention, the plant material is subjected to a mechanical process for the purpose of open up the plant cells to make the proteins available for recovery and the carbohydrates available for fermentation. Furthermore, the mechanical process or the disintegration of the plant material results in a composition comprising liquid (juice) and solid plant components, i.e. the composition comprises a liquid phase and a solid phase as defined below. As will be apparent to the skilled person the terms "processing" and "disintegration" is used herein interchangeably, as both terms pertains to ways of opening up plant cells.

The plant material can be processed by a variety of well known processes which results in an efficient opening of the plant cells. Such efficient mechanical processes include grinding, milling, hacking, squeezing, slicing, abrading, pressing, crushing, chipping and combination thereof.

The mechanical process employed depends on the plant material used. For example, a person skilled in the art would in most cases grind a beet tuber, whereas a beet top would in most cases be pressed. However, an efficient mechanical process useful in the method according to the invention, is one which efficiently enhance I) the overall surface area to mass ratio in order to enable degradation of the material into a satisfactory level, and II) cell opening thus providing an efficient release of cell juice containing carbohydrates and proteins. In a useful embodiment, the grinding process is a two-step grinding process which results in an even more efficient release of proteins and carbohydrates.

Temperature is of significant to the present invention, as high temperatures lead to denatured proteins (e.g. proteins altered in the native 3-D structure). Proteins isolated after precipitation by heat are denatured and thus only valuable as animal feed. Accordingly, such proteins cannot be used for purposes where the properties of native proteins are needed. Accordingly, denatured or partly denatured proteins, which are considered as feed grade proteins, are of lower priority or no priority to the present invention, but still a product which may be obtainable by the present invention. Denaturation of proteins involves the breaking of many of the weak linkages, or bonds (e.g. hydrogen bonds) within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state. Denatured proteins have a looser, more random structure and most are insoluble and have lost some the properties which native proteins comprise (see e.g. Example 4).

In preferred embodiments, the processing or the disintegration of the plant material is conducted at a temperature of 70°C or less, such as 60°C or less, e.g. 50°C or less, such as 45°C or less, e.g. 40°C or less, such as 40°C or less, including 30°C or less, e.g. 20°C or less, such as 10°C or less, but not less than 5°C.

All temperatures mentioned in the present application refer to temperatures relative to the local atmospheric or ambient pressure.

In useful embodiments, the temperature is in the range of 5-70°C, e.g. in the range of 15-50°C, such as 20-40°C, including in the range 30-45°C, such as in the range of 10-70°C, e.g. in the range of 5-60°C. It has been found that good results are obtainable at temperatures in the range of 20-40°C, such as in the range of 25-35°C, e.g. in the range of 30-35⁰C.

During the above described processing of the plant material, enzymes may be added in order to obtain an at least partial hydrolysis of pectin, cellulose and other carbohydrates in the plant material resulting in a processed material containing an increased amount of microbially fermentable sugars which are used in the subsequent alcohol fermentation. Besides from increasing the amount of microbially fermentable sugars, the at least partial hydrolysis of pectin may also lead to the liberation of pectin bound protein. In a useful embodiment, the enzyme is added to the processed plant material, e.g. after the processing of the material.

In preferred embodiments, the at least one enzyme added during the processing of the plant material and/or to the processed plant material (i.e. the liquid phase and the solid phase) is selected from a group consisting of cellulase, kitinase, β- fructosidase, β-glucanase, hemicellulase, xylanase, invertase, glactosidase, polygalacturonase, xylosidase and arabinosidase. In useful embodiments, two or more enzymes, such as three or more enzymes, four or more enzymes or five enzymes or more enzymes, are added to the plant material and/or processed plant material. Under some circumstances it may be useful to add the two or more enzymes together or subsequently during the processing of the plant material and/or to the processed plant material.

In useful embodiments, the enzyme is added to the plant material and/or processed plant material in a quantity of at least 1 ng per kg material dry weight, such as at least 5 ng per kg material dry weight, e.g. 10 ng per kg material dry weight, including at least 25 ng per kg material dry weight, such as at least 50 ng per kg material dry weight. The amount of the enzyme added to the plant material and/or processed plant material is an amount which results in the presence in the material of 10 to 5000 units per gram material, such as in the range of 100 to 3000 units per gram material, including in the range of 250 to 2500 units per gram material, such as in the range of 500 to 1000 units per gram material, including in the range of 750 to 1000 units per gram material. In the present context, the term "units" relates to the activity of an enzyme and is defined as μmoles of substrate reacted per minute per gram of the measured sample at fixed standard conditions.

During the above described processing of the plant material it may be useful to prevent enzymatic browning (see e.g. Example 4, 5, 6, 7 and 8). Accordingly, sulphite (K₂S₂O₅) may be added during the processing of the plant material and/or to the processed plant material and/or to the plant juice. In a preferred embodiment 0.003% w/w sulphite, including 0.004% (w/w) sulphite is added during the processing of the plant material and/or to the processed plant material and/or to the plant juice, such as 0.005% (w/w) sulphite, including 0.006% (w/w) sulphite, such as 0.007% (w/w) sulphite, including 0.008% (w/w) sulphite, such as 0.009% (w/w) sulphite, including 0.010% (w/w) sulphite, such as 0.011% (w/w) sulphite, including 0.012% (w/w) sulphite, such as 0.013% (w/w) sulphite, including 0.014% (w/w) sulphite, such as 0.015% (w/w) sulphite, including 0.016% (w/w). In a useful embodiment, the addition of sulphite is combined with the lowering the pH of the plant juice.

In preferred embodiments sulphite in the range of 0.003% (w/w) - 0.016 % (w/w), such as in the range of 0.005% (w/w) - 0.010 % (w/w), e.g. in the range of 0.009% (w/w) - 0.015 % (w/w), such as in the range of 0.004% (w/w) - 0.012 % (w/w) is added during the processing of the plant material and/or to the processed plant material and/or to the plant juice.

Separation of the liquid phase from the solid phase

Subsequently, the liquid phase of the processed plant material is separated from the solid phase of the processed plant material. By removing the solids of the plant material, such as cellulose, hemicellulose, pectin and lignin, the recovery of pure protein from the liquid phase is possible, using simple technologies as precipitation filtration and centrifugation. Another advantage is that it is possible to run continuous alcohol fermentation with recirculation of cells, when the suspended or solid material is/are removed from the liquid and therefore it will not be re-circulated together with the microorganisms (e.g. yeast) in the continuous process.

In preferred embodiments, the separation of the liquid phase from the solid phase is obtained by centrifugation, filtration or decanting or a combination thereof. Such methods are well known by persons skilled in the art.

In the present context, the expression "liquid phase" is used interchangeable with the expression "juice" and relates to the phase or fraction of the processed plant material after the solid plant material has been removed or partially removed, e.g. by a process described above. As will be apparent to the skilled person the carbohydrates are present in the liquid phase. In preferred embodiments, the liquid phase contains after the separation from the solid phase at the most 3% solids, such as at the most 5%, e.g. at the most 10%, such at the most 20% including at the most 25% solids.

The expression "solid phase" relates in the present context to the phase of the processed plant material after the original liquid or juice has been removed or partially removed, e.g. by a process described above. The solid phase consists mainly of fibres and organic polymers such as cellulose, hemicellulose, pectin and lignin. In preferred embodiments, the solid phase contains after the separation from the liquid phase at the most 20% juice, such as at the most 25%, e.g. at the 5 most 30%, such at the most 30% including at the most 30% such as at the most 40%, e.g. at the most 50%, including at the most 60% juice.

From an economical point of view it is desirable to use a plant material in the present process with a high amount of dissolved protein for subsequently to

10 obtain a liquid phase with a high content of protein. Thus, in preferred embodiments, the liquid phase has a protein content of at least 0.1% (w/w), such as at least 0.2% (w/w), e.g. 0.5% (w/w), such as at least 1%, e.g. at least 1.5%, such at least 2%, including at least 2.5% (w/w), such as at least 5% (w/w), e.g. at least 10% (w/w).

15

In further embodiments, the liquid phase has a protein content in the range of 0.1% - 10% (w/w), such as in the range of 0.5% - 10% (w/w), e.g. in the range of 5% - 10% (w/w), such as in the range of 1% - 5%, e.g. in the range of 2% - 5% (w/w), such as in the range of 4% - 5%, e.g. in the range of 0.5% - 2.5%

20 (w/w), such as in the range of 1% - 2% (w/w), e.g. in the range of 1.5% - 2% (w/w), such in the range of 0.1% - 2% (w/w), including in the range of 1%- 2.5% (w/w) protein.

In accordance with the present invention, the carbohydrates or fermentable 25 sugars in the plant material, and thus in the liquid phase of the processed plant material, are used for an alcohol fermentation. In useful embodiments, the liquid phase has a carbohydrate content of at least 0.1% (w/w), e.g. 0.5% (w/w), such as at least 1% (w/w), e.g. at least 5% (w/w), such as at least 10% (w/w), e.g. at least 15% (w/w), such at least 20% (w/w), including at least 25% (w/w). In 30 further embodiments, the liquid phase has a carbohydrate content in the range of 0.1% - 25% (w/w), e.g. in the range of 0.5 - 50% (w/w), such as in the range of 1% - 10% (w/w), e.g. in the range of 5% - 25% (w/w), such as in the range of 10% - 20% (w/w), e.g. in the range of 15% - 20% (w/w), such in the range of 5% - 20% (w/w), e.g. in the range of 5% - 15% (w/w) including in the range of 35 10%- 25% (w/w). In a useful embodiment, the separation of the liquid phase from the solid phase is conducted at a temperature of between 5°C and 70°C as it is contemplated that proteins in the processed plant material at temperatures above 70⁰C or when the 5 liquid freezes, coagulates or denaturizes which as mentioned previously is not desirable in the present context. In further embodiments, the separation of the liquid phase from the solid phase is conducted at a temperature of 70°C or less, e.g. 60°C or less, such as 50°C or less, e.g. 45°C or less, such as 40°C or less, including 30°C or less, e.g. 20°C or less, such as 10°C or less but not less than 10 5°C.

In useful embodiments, the temperature is in the range of 5-70°C, such as in the range of 15-50°C, such as 20-40°C, more typically in the range 30-45°C. It has been found that good results are obtainable at temperatures in the range of 20- 15 40°C, such as in the range of 25-35°C, e.g. at 30-35⁰C, such as 10-70°C, e.g. 5- 60°C.

As described above, the amount of protein and carbohydrate in the liquid phase depends on which parts of the plant material is used to provide the liquid. For 20 example, a liquid phase obtained from processed beet leaves or beet top comprises more protein but less carbohydrate than the beet root.

Preservation of the plant juice

To prevent growth of undesired microorganisms in the juice the pH of the liquid

25 phase or plant juice may be reduced immediately after separation of the liquid and solid phase. The pH is reduced by addition of inorganic or organic acids or by addition of a pre-culture of organic acid producing microorganisms as for example lactic acid bacteria. As mentioned previously sulphite may be added to the juice in order to prevent enzymatic browning hereof.

30

In an embodiment of the present invention the pH is lowered during preservation to the optimal pH for the subsequent protein precipitation. In this manner two steps may be combined in one. Precipitation and recovery of the protein

Following the separation, the liquid phase is subjected to a precipitation and protein recovery process in order to obtain a high-value protein.

In a preferred embodiment of the present invention the protein is a native protein.

In the present context, the expression "native protein" relates to a properly folded protein (e.g. proteins having a preserved biological function). In accordance with the invention, useful "native proteins" includes proteins which have preserved at least one of the properties selected from the group consisting of protein activity, protein solubility, gelatinizing, water absorption, oil absorption, emulsifying, foaming properties or combinations hereof (see e.g. Example 5 and 9).

Protein properties can be measured by methods known to the skilled person, thus, it will be apparent form the art that proteins can be characterized by e.g. X-ray crystallography, Nuclear Magnetic Resonance, Cryo-electron microscopy, Circular dichroism or combinations hereof. If the protein for instance is an enzyme, the activity can be measured as either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist in the art and many enzymes can be assayed in several different ways such as but not limited to initial rate expression, progress curve experiments, transient kinetics experiments and/or relaxation experiments. Enzyme assays can be split into two groups according to their sampling method: continuous assays (e.g. spectrophotometric, fluorometric, calorimetric and/or chemiluminescent), where the assay gives a continuous reading of activity, and discontinuous assays (e.g. radiometric and/or chromatographic), where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

In preferred embodiments of the present invention the amount of the native protein compared to the total amount of protein in the plant material is more than 20%, such as 30%, e.g. 40%, such as 50%, e.g. 60%, such as 70%, e.g. 80%, such as 90% including 100%. In further embodiments the amounts of native protein compared to the total amount of protein in the plant material is in the range of 20% - 100%, e.g. in the range of 40%-90%, such as in the range of 50% - 60%, e.g. in the range of 30% - 80%.

In the art other means for measuring protein solubility, gelatinizing, water absorption, oil absorption, emulsifying, and foaming properties exists and are known to the skilled person (see e.g. Example 9).

In the present invention the terms "protein" and "protein product" are used herein interchangeably. A protein product comprises besides the at least one protein one or more undefined CHO products. From the protein product the one or more proteins may be isolated my means known to the skilled addressee. Both the native protein and protein product comprising native protein, may according to the present invention be used as feed grade protein.

Proteins isolated after precipitation by heat combined with low pH are denatured but are only valuable as animal feed and thus not for purposes where the properties of native proteins are needed. Accordingly, denatured or partly denatured proteins, which are considered as feed grade proteins, are of lower priority in the present invention, but still a product obtainable by the present invention. Denaturation of proteins involves the breaking of many of the weak linkages, or bonds (e.g. hydrogen bonds) within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state. Denatured proteins have a looser, more random structure and most are insoluble and have lost some of the above mentioned native properties. In useful embodiments, the proteins are recovered by precipitation, chromatography or a combination thereof. Such precipitation includes acid precipitation or salt precipitation.

Acid precipitation may be preferred because acid precipitation may involve a cheaper method for obtaining proteins. Furthermore, the protein product obtained by acid precipitation comprises a limited amount of impurities.

In a presently preferred embodiment the proteins or the at least one protein are/is recovered by acid precipitation. In preferred embodiments the acid precipitation is performed by the addition of at least one organic or inorganic acid or combinations thereof (see e.g. Example 5). Examples of useful protein precipitating acids are CH₃CHOHCOOH, CH₃COOH, HCOOH, HCL, H₂SO₄, HNO₃, H₃PO₄ or a combination hereof.

In a useful embodiment, at least one protein precipitating acid is produced by an acid producing microorganism. Useful acid producing microorganims includes lactic acid bacteria such as Lactobacillus species, e.g. L. helveticus, L delbrueckii, L. casei, L. acidophilus, L. bulgaricus , L. plantarum, L. paracasei spp. paracasei and L. salivarius, Lactococcus species, such as Lactococcus lactis, Streptococcus species such as S. thermophilus, Leuconostoc species, Pediococcus species, Propionibacterium species, Bacillus species, such as B. stearothermophilus and Bifidobacterium species (see e.g. Example 7).

It will be understood that a useful acid precipitation process, is a process where a combination of one or more acids and one or more acid producing bacteria selected from the above defined group are used. As example addition of sulphuric acid combined with inoculation with a strain of Lactobacillus salivarius could be a useful strategy.

In preferred embodiments the pH is accustomed to a pH where proteins are precipitated. Thus, in useful embodiments pH is adjusted in the range of pH 1 to 7 such as in the range of pH 2 to 6, e.g. in the range of pH 3 to 5, such as in the range of pH 3.5 to 4.5, e.g. in the range of pH 4 to 5, including the range of pH 3 to 6 by the addition of the at least one acid and/or acid producing microorganisms.

In another preferred embodiment the protein precipitation is conducted at a temperature range of 5-70°C, e.g. in the range of 15-50°C, such as 20-40°C, more typically in the range 30-45°C. It has been found that good results are obtainable at temperatures in the range of 20-40°C, such as in the range of 25- 35°C, e.g. at 30-35⁰C, such as 10-70°C, e.g. 5-60°C.

After the protein precipitation, notwithstanding whether the protein is in its native or non-native form, the precipitated protein may be recovered or isolated from the supernatant by conventional procedures including, but not limited to, centrifugation, filtration, decanting or a combination thereof. It is however, as mentioned previously, a preferred embodiment of the present invention that the isolated protein is in its native form.

As will be apparent to the skilled addressee, the terms "recovered" and "isolated" are used herein interchangeably and relates gaining proteins from the juice.

Pre-treatment of the remaining liquid and first fermentation The terms "remaining liquid" and "supernatant" are in the present context used interchangeably, and thus relates to the de-proteinated liquid which remains after protein precipitation and centrifugation.

Following the removal of the protein from the supernatant, the remaining liquid may be concentrated by removing water. In useful embodiments, 2% (w/w) or more of the water present in the supernatant is removed, such as 5% (w/w) or more is removed, e.g. 10% (w/w) or more is removed, such as 15% (w/w) or more is removed, e.g. 25% (w/w) or more is removed.

In an embodiment of the present invention the supernatant has a carbohydrate content in the range of 2 to 30% (w/w), such as in the range of 2-25% (w/w), e.g. in the range of 2-20, such as in the range of 2-15% (w/w), e.g. in the range of 2-10, such as in the range of 2-5% (w/w), e.g. in the range of 5-30, such as in the range of 15-30% (w/w), e.g. in the range of 10-30% (w/w).

In accordance with the present invention, the supernatant may be subjected to an alcohol fermentation process such as ethanol fermentation or butanol fermentation. The microbial fermentable sugars in the supernatant can be utilized by one or more microorganisms to produce fermentation products such as ethanol or butanol. Any microorganism, such as yeast and a bacterium, capable of converting sugar to an alcohol can be used in the process according to the invention. For example, suitable yeast may be selected from the group consisting of Saccharomyces cerevisiae, Clostridium species, Pichia species such as P. stipitis, P. segobiensis, P. guillermondii, and P. naganishii, Candida species such as C. shehatae, C. tenuis, C. albicans C. tropicalis, C. maltosa and C. torresii, Hansenula species such as H. polymorpha, Pachysolen species such as P. tannophilus, Brettanomyces species such as B. naardenensis, Metschnikowia species such as M. bicuspidata and M. zobellii, Sporopachydermia quercuum and Wingea robertsii. Useful bacteria may be Zymomonas mobilis E. coli and Klebsiella oxytoca. Fungi useful in the present process may be Fusarium oxysporum, Candida guillermondii, C. millerii, C. tropicalis, C. parapsilosis, Petromyces albertensis, Debaromyces hansenii, Cellulomonas cellulans, and Corynebacterium sp.

It will be understood, that a useful ethanol-fermenting or butanol-fermenting organism can be selected from a genetically modified organism of one of the above useful organisms having, relative to the organism from which it is derived, an increased or improved ethanol-fermenting or butanol-fermenting activity.

The fermenting organism can be added to the supernatant or fermentation by any of a variety of methods known in the alcohol fermentation industry. For example, may yeast, such as S. cerevisiae, be added as a dry batch or by conditioning/propagating batch. The fermentation may be carried out in fermentation reaction vessels (fermentors) of any suitable, known type. In an embodiment, the alcohol fermentation is conducted at a temperature of about 25°C to 70⁰C or about 30⁰C to about 40⁰C, depending on the growth requirements of the organism used in the fermentation.

After the alcohol fermentation is completed or partially completed, a fermentation broth comprising an alcohol and a fermentation effluent is obtained. In preferred embodiments, the fermentation broth comprises at least 2% (w/w) alcohol, such as at least 5% (w/w) alcohol e.g. at least 10% (w/w) alcohol. When producing ethanol, it is preferred that the fermentation broth comprises at least 2% (w/w) ethanol, such as at least 5% (w/w) ethanol e.g. at least 10% (w/w) ethanol.

The alcohol, such as the ethanol and butanol, produced by the above fermentation may be isolated or recovered from the fermentation broth by a variety of known processes, such as a distillation process. Pre-treatment of solid phase

The solid phase, obtained by the above-described separation of the processed plant material, is preferable pre-treated by methods known in the art, such as impregnation with sodium hydroxide, wet oxidation or steam explosion, resulting in a partial separated material wherein the fibers are opened up and more available for enzymatic attack.

The wet oxidation process takes traditionally place in an aqueous medium in the presence of an oxidising agent which reacts oxidatively with the components pres- ent in the solid phase. Steam explosion is a thermal-mechanical-chemical process that combines the presence of heat (as steam), mechanical forces (shearing effect) and chemical action (hydrolysis). The result of the two pre-treatments is the alteration of the microfibrillar packing inside the cell wall and the rupture of the fibre, which causes an increase in the accessibility of the cellulose to hydrolytic enzymes. The optimum temperature and reaction time conditions in the two processes vary depending on the kind of material.

After the pre-treatment, the partial separated material is typically treated with enzymes to release sugars that can be fermented to ethanol. In a useful embodiment, the partial separated material is subjected to a hydrolysis selected from the group consisting of an enzyme hydrolysis, an acid hydrolysis or an alkaline hydrolysis resulting in a slurry containing fermentable sugars.

In order to increase the amount of fermentable sugars, the hydrolysed solid phase (i.e. slurry) may be added directly to the alcohol fermentation process and combined with the liquid phase after removal of the proteins or it can be separated in a new liquid phase containing the dissolved carbohydrates and a new solid phase containing fibres in suspension.

In order to liberate the fermentable sugars the enzymatic treatment as mentioned earlier in the may be used.

The new liquid phase can be added to the alcohol fermentation process, whereas the new solid phase is added to the anaerobic fermentation process, as described below. Second fermentation

As the proteins are removed before the alcohol fermentation, the remaining fermentation effluent after the recovery of the alcohol comprises a low amount of proteins. Proteins and other nitrogen containing compounds, will in a conventional, subsequent, anaerobic fermentation process be converted to ammonia and thereby have an inhibitory effect on the anaerobic fermentation process, resulting in inhibition of the methane production. Thus, by using the present alcohol fermentation effluent with a low protein and nitrogen content in the methane fermentation process, no inhibition caused by ammonia will occur.

Accordingly, in one embodiment of the present invention, the alcohol fermentation effluent is subjected to an anaerobic fermentation process employing one or more anaerobic fermenting microorganisms capable of degrading or converting substances present in said effluent to form combustible fuel or gas such as methane. In useful embodiments, this fermentation is performed using methane- producing microorganisms (methanogens) which are capable of forming methane from certain classes of organic substrates, methyl substrates or acetate under anaerobic conditions. Thus, in a preferred embodiment, the alcohol fermentation effluent, also called "stillage", is subjected to a methane fermentation process.

The anaerobic fermentation process may result in a combustible fuel or gas, such as methane, and an anaerobic fermentation effluent. This anaerobic fermentation effluent may comprise a high content of potassium and a one or more anaerobic fermenting microorganisms.

Production of a high potassium (K) vinasse (high K-vinasse)

According to the present invention, one object is to obtain a sustainable utilization of agricultural plant materials. One way of obtaining this is by employing a method re-using products that traditionally have been termed waste products. The alcohol fermentation effluent described above is such a product, as all valuable, easy convertible, organic compounds are isolated or converted to fuel alcohol or methane. After the fermentation product, e.g. ethanol and/or methane, has been extracted from the fermentation broth, a liquid/anaerobic fermentation effluent with an increased level of potassium is obtained. This liquid is traditionally concentrated and termed "vinasse" or "stillage", and these terms may be used interchangeably, when the effluent comes from the alcohol fermentation and anaerobic fermentation effluent and when the effluent comes from anaerobic fermentation (e.g. from the production of methane). The concentrated liquid is in the present context termed "high K-vinasse". In addition to the potassium, the liquid normally contain valuable organic materials, such as amino acids, that can be used in cattle feed. However, after removal of the proteins and conversion of most of the organic compounds into methane, the stillage contains mainly inorganic compounds combined with difficult convert able organic compounds and is therefore not useable as cattle feed.

The vinasse, when produced by conventional ethanol fermentation processes, contains high levels of potassium, organic matter, proteins, amino acids, calcium and a moderate amounts of nitrogen and phosphorous. However, the high K- vinasse product obtained by the present invention, is characterized by having, compared to conventional vinasse or stillage products, a low content of proteins, as the proteins have been recovered in a previous step of the present method, and a low content of convertible organic compounds, but a high content of potassium, which is of great value as fertilizer.

Accordingly, the present invention relates to the use of the high K-vinasse or stillage obtainable by the method according to the invention as an organic fertilizer. In a preferred embodiment, the high K-vinasse or stillage is used in combination with compost and other organic based fertilizers.

In a further aspect, there is provided a high K-vinasse obtainable by the method of the invention. The high K-vinasse of the present invention is further characterized in having a low protein content and/or a low content of convertible organic compounds. The high K-vinasse is a valuable fertilizer product due to the high content of potassium. A further aspect, relates to a method of modifying compost and other organic fertilizers, comprising adding to the compost the high K-vinasse or stillage product according to the invention.

In preferred embodiments of the above aspects, the high K-vinasse comprise at the least 5% potassium on a dry-matter basis, such as at the least 10%, e.g. at least 15%, such at the least 20%, including at the most 30%, such as at the least 50% potassium on a dry-matter basis.

General

Reference to prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

The reference cited in the present application, are hereby incorporated by reference in its entirety.

As will be apparent, preferred features and characteristics of one aspect of the invention may be applicable to other aspects of the invention. The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated be the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced by reference therein.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. In addition, the terms "at least one" and "one or more" is in this specification used interchangeably.

The invention will hereinafter be described by way of the following non-limiting figures and examples.

Figure 1 represents a first embodiment of the present invention and shows a schematic overview of the method providing at least one protein and at least one fermentation product from a plant material. In this case the fermentation product is fuel alcohol.

Referring to figure 1, the plant material (1) is subjected to at least one integration and separation process (2) obtaining a solid phase (3) and a liquid phase (4). In an embodiment at least one enzyme (5) selected from a group consisting of cellulase, kitinase, galactosidase, β-fructosidase, β-glucanase, hemicellulase, xylanase polygalacturonase, xylosidase and arabinosidase is added to the solid phase. Preferably the separation is conducted by centrifugation, filtration, decantation or a combination thereof.

Subsequently, at least one protein is recovered (6) from the liquid phase preferably by precipitation or chromatography or a combination thereof. Such precipitation includes acid precipitation, precipitation by acid or salt precipitation. In preferred embodiments the acid precipitation agent is selected from a group consisting of HCL, H₂SO₄, HNO₃, H₃PO₄ CH₃CHOHCOOH, CH₃COOH, HCOOH, or combinations thereof. The recovered protein (8) may be isolated (7) by centrifugation, filtration, decanting or a combination thereof creating a supernatant comprising a low protein content.

Following protein isolation the supernatant is subjected to a fermentation process (9) providing a fermentation broth comprising alcohol (11), which is isolated by distillation (10) and an fermentation effluent. In a preferred embodiment the fermentation process is an alcohol fermentation process selected from a group consisting of an ethanol fermentation and butanol fermentation. The obtained alcohol may be isolated from the fermentation broth by a distillation process. In a subsequent procedure the fermentation effluent may be either subjected to (I) an anaerobic fermentation process (12) (methan fermentation) or (II) used directly as organic fertilizer (14).

The previously isolated solid phase (3) may be subjected to an enzyme hydrolysis process (5) combining wet oxidation or steam explosion and enzymatic treatment, hence obtaining a slurry which may be added to the alcohol fermentation (9) or the biogas fermentation (12).

Figure 2 illustrate foaming ability and solubility of A. acid precipitated proteins (native proteins) and B. acid + heat precipitated proteins (non-native proteins). Vial A, show the ability of native proteins to be dissolved in mild sodium hydroxid as well as the ability of the proteins to produce foam. Vial B, shows the ability of non-native proteins to be dissolved in mild sodium hydroxid as well as the ability of the proteins to produce foam. In vial A, no or minor sediment production can be observed whereas vial B shows significant and clearly visible sediment production. The foaming ability of the proteins in vial A is increased when compared to the proteins in vial B.

Examples

Example 1

Isolation of feed grade (non-native) protein from sugar beet

Sugar beet is harvested, top and root removed and the root disintegrated in a food processor. The beet mass is separated in a liquid phase and a solid phase by pressing the disintegrated sugar beet root using a Speidel Hydropresse (90 I). The juice is immediately added sulphuric acid to pH 4 and heated up to 100⁰C in an autoclave in order to precipitate non-native protein.

After 15 minutes the juice is cooled down to 20⁰C and the protein separated on a Watmann X filter paper. The protein is harvested from the surface of the filter and dried in an own at 50⁰C. The filtrate (remaining liquid) is used as feed in a continuous ethanol process.

Example 2 Isolation of native protein from sugar beet by acid precipitation

Sugar beet is harvested, top and root removed and the root disintegrated in a food processor. The beet mass is separated in a liquid phase and a solid phase by pressing the disintegrated sugar beet root using a Speidel Hydropresse (90 I). The juice is immediately added sulphuric acid to pH 4 and stored at 20⁰C for 24 hours. The native protein was separated on a Watmann X filter paper. The protein is harvested from the surface of the filter and dried in an own at 50⁰C.

The filtrate (remaining liquid) is used as feed in a continuous ethanol process.

Example 3

Isolation of native protein from sugar beet by using acid producing organisms

Sugar beet is harvested, top and root removed and the root disintegrated in a food processor. The beet mass is separated in a liquid phase and a solid phase by pressing the disintegrated sugar beet root using a Speidel Hydropresse (90 I). The juice is immediately added a pre culture of Lactobacillus salivarius BC 1001 and heated up to 40⁰C. The pH drops to pH 3.8. The juice is cooled down to 20⁰C and stored for 24 hours at said temperature. The protein is separated on a Watmann X filter paper. The protein is harvested from the surface of the filter and dried in an own at 50⁰C.

The filtrate is used as feed in a continuous ethanol process.

Example 4

Isolation of feed grade (non-native) protein from sugar beet juice by a combination of heat and acid precipitation (pH 4.0)

Sugar beet was harvested, and the top and root were separated. Subsequently, the root was washed and disintegrated. The liquid phase (juice) of the obtained disintegrated beet mass was separated from the solid phase, the pulp, in a Juice centrifuge (Kenwood JE 810). The juice was immediately added 1 ml potassium disulphite, K₂S₂O₅ solution 4 % per litre of juice and sulphuric acid to pH 4. The juice was subsequent heated up to 100⁰C in an autoclave.

After 15 minutes in the autoclave the juice was cooled down to 20⁰C and the protein separated in a Bench top centrifuge (Jouan CR 412 with 4 x500 ml rotor). The protein was washed with acidified water, pH=4.0 with sulphuric acid and dried in a freeze drier (FetoFrig). 28.9 % of the nitrogen (N) in the juice was precipitated as protein material. The content of the non-native protein in the protein product was 46.7 %. This is a measurement for the purity of the protein product.

The remaining liquid was used as feed in a continuous ethanol process.

Due to the heat precipitation step, the proteins in the precipitated protein product were non-native proteins. As can be seen from Example 9, 72% of the proteins were native (as only 28% were able to be dissolved). Thus, such proteins are low value proteins and may be used e.g. as feed grade proteins.

Example 5

Isolation of native protein from sugar beet juice by acid precipitation (pH to 4.0)

Sugar beet was harvested, and the top and root were separated. Subsequently, the root was washed and disintegrated.

The liquid phase (juice) of the obtained disintegrated beet mass was separated from the solid phase, the pulp, in a Juice centrifuge (Kenwood JE 810). The juice was immediately added 1 ml potassium disulphite, K₂S₂O₅ solution 4 % per litre of juice and sulphuric acid to pH 4,0 and stored at room temperature (20-30⁰C) for 20 hours.

The protein was separated in a Bench top centrifuge (Jouan CR 412 with 4 x500 ml rotor) and washed three times in the centrifuge with water, acidified to pH=4,0 with sulphuric acid and dried in a freeze drier (FetoFrig). 28.8 % of the nitrogen (N) in the juice was precipitated as protein material. The content of the protein in the protein product was 47.7 %. This is a measurement for the purity of the protein product.

The remaining liquid was used as feed in a continuous ethanol process.

As can be seen from Example 9, 46% of the proteins obtained by this method were native proteins (high value proteins), as these proteins were able to be dissolved.

Example 6

Isolation of native protein from fodder beet juice by lactic acid fermentation

Fodder beet (Colosse) was harvested and the top and root separated.

Subsequently the root was washed and disintegrated. The liquid phase (1.47 kg juice) of the disintegrated beet mass (2.5 kg) was separated from the solid phase (0.93 kg pulp) in a juice centrifuge (Kenwood JE 810). The liquid yield of juice was 58.7 % when compared to the whole mass of the plant material.

The juice was immediately added 1 ml potassium disulphite, K₂S₂O₅ solution 4 % per litre of juice and Ig freeze dried, Thermophilic Lactic Culture, TH-4 from Chr. Hansen a/s. The inoculated juice was incubated at 40⁰C for 20 hours.

The protein was separated in a Bench top centrifuge (Sigma 3-18K with cooling and 12 x 50 ml rotor) and washed three times in the centrifuge with water, acidified to pH=4.0 with sulphuric acid.

The protein content in the fodder beet protein product was 34.7 %. This is a measurement for the purity of the protein product. The protein yield from juice was 0.14 g protein/kg.

The remaining liquid from the centrifugation was used as feed in a continuous ethanol process. The results thus show that the amount of protein product resembles the amount obtained in examples 4 and 5, the obtained product is however not as pure as the obtained product in Examples 4 and 5.

Example 7

Isolation of native protein from fodder beet juice by lactic acid fermentation combined with enzymatic treatment

Fodder beet (Colosse) was harvested, top and root was separated and the root was washed. Subsequently the root was disintegrated and separated in a juice centrifuge (Kenwood JE 810).

The obtained juice was immediately added 1 ml potassium disulphite, K₂S₂O₅ solution 4 % and Ig freeze dried, Thermophilic Lactic Culture, TH-4 from Chr. Hansen a/s per litre of juice. The inoculated juice was separated in four portions. Each portion was added different enzymes and incubated at 40⁰C or 20⁰C for 20 hours.

Portion 1 was incubated at 40⁰C; Portion 2 was incubated at 20⁰C;

Portion 3 was added 2 ml Viscozyme L, Novozymes per kg juice and incubated at 20 ⁰C;

Portion 4 was added 2 gram Pananzym pr kg of juice and incubated at

20⁰C.

In all cases pH dropped from about 6.2 to about 4.0. The protein yield was highest when the incubation took place at 20⁰C without addition of enzymes, whereas the quality of the enzymes measured as protein content in per cent (%) was highest in the case of combined action of lactic acid bacteria and hydrolytic enzymes. Table 1 Fodder beet (Colosse) juice added LAB, TH-4 and enzymes, incubated and centrifuged

DM= Dry matter

Example 8

Purification of native protein from fodder beet juice by centrifuqation after precipitation at different pH

Fodder beet (Colosse) was harvested top and root separated and the root was washed. Subsequently the root was disintegrated and separated in a juice centrifuge: Kenwood JE 810.

The juice was immediately added 1 ml potassium disulphite, K₂S₂O₅ solution 4 % per litre of juice and divided in three portions.

The first portion (1) was acidified to pH=4.0 with sulphuric acid and centrifuged.

The second portion (2) was centrifuged at the initial pH (6.17). The third portion (3) was derived from the supernatant from the second portion. This portion was acidified to pH=4.0 with sulphuric acid and centrifuged.

The protein was in all three cases separated in a Bench top centrifuge: Sigma 3- 18K with cooling and 12 x 50 ml rotor. The protein of the first portion was washed three times in the centrifuge with water, acidified to pH=4.0 with sulphuric acid. After centrifugation at pH 4.0 the precipitate was separated in two layers; a small grey layer and a white layer (the white layer containing a more pure protein than the grey layer). The protein purity in this fodder beet protein product was 35.8 %.

The protein purity of the precipitate of the second portion isolated at pH 6.17 was only 31.4 % (light grey layer sediment), whereas the protein purity of the third portion acidified supernatant 42.6 % and white.

The remaining liquid from the centrifugation was used as feed in a continuous ethanol process.

Table 2 Purification of native protein from fodder beet juice by centrifugation after precipitation at different pH

DM= Dry matter The results show that it is possible to purify the protein product or separate the different proteins in the protein product by isolating the proteins by centrifugation after precipitation at different pH.

Example 9

Comparison of native and non-native protein

The protein product was isolated as described in Examples 4 and 5, before 200 mg protein product was dissolved in mild sodium hydroxide, for demonstrating whether the protein was native or non-native.

The results show that the proteins precipitated at pH = 4.0, were able to be dissolved in mild sodium hydroxid (low amount of sediment product) which demonstrate that the proteins are native. The amount of dissolved protein was 0.2 - 0.108 = 0.092, corresponding to 46 % of the native protein precipitated at pH 4.0. Furthermore, as can be seen from Table 3 and Figure (A), the native protein had preserved its foaming ability.

Table 3 Comparison of native protein, precipitated at pH = 4.0 and non-native protein, precipitated at pH=4.0 and heating to 100⁰C for 15 min

The non-native protein (Figure 2,B) which was precipitated at pH=4.0 and heated up to 100⁰C for 15 min, produced when compared to the native protein, significantly more sediment demonstrating that the protein is denaturated., The amount of dissolved non-native protein was in this case 0.2 - 0.144 = 0.056, corresponding to 28 % of protein. Furthermore the non-native protein produced less foam when compared to the native protein, thus indicating a decreased protein activity. The activity which remains is probably due to dissolved proteins.

References

Gibbson, W. R., Westby, CA. and Arnold, E. 1987. Semicontinuous diffusion fermentation of fodder beets for fuel ethanol and cubed protein feed production. Biotechnology and Bioengineering, 31, pp. 696-704.

Claims

1. A method for providing at least one protein and at least one fermentation product from a plant material, said method comprises the steps of:

(i) disintegrating the plant material in order to obtain a solid phase and a liquid phase,

(ii) separating the liquid phase from the solid phase,

(iii) recovering at least one protein from the liquid phase obtaining an isolated protein and a supernatant, and

(iv) subjecting the supernatant to a fermentation process obtaining at least one fermentation product.

2. The method according to claim 1, wherein said plant material comprises a high content of carbohydrate.

3. The method according to claim 1 or 2, wherein the carbohydrate is a soluble carbohydrate or fructan.

4. The method according to claim 3, wherein the carbohydrate is selected from a group consisting of sucrose, fructose, glucose, galactose, xylosemannose and maltose.

5. The method according to any one of the preceding claims, wherein said plant material originates from a plant material comprising no or limited amount of starch and/or starch granules.

6. The method according to claim 5, wherein said plant originate from a group consisting of beet, pineapple, chicory, sugar cane, carrot and parsnip.

7. The method according to claim 6, wherein the beet is selected from a group consisting of sugar beet, fodder beet, garden beet, white beet and red beet.

8. The method according to claim 7, wherein said beet is sugar beet or fodder beet.

5 9. The method according to any one of the preceding claims, wherein said plant material comprise a plant part selected from the group consisting of stem, leaves, root, fruits, seeds, tubers and combination thereof.

10. The method according to any one of the preceding claims, wherein said plant 10 material has a carbohydrate content in the range of 0.1 to 25 % (w/w).

11. The method according to any one of the preceding claims, wherein said plant material has a protein content in the range of 0.1 to 5% (w/w).

15 12. The method according to any one of the preceding claims, wherein the processed plant material is provided by at least one mechanical process selected from a group consisting of grinding, milling, hacking, squeezing, slicing, abrading, pressing, crushing, chipping and combinations hereof.

20 13. The method according to any one of the preceding claims, wherein one or more enzymes are added during the processing of the plant material and/or to the processed plant material.

14. The method according to claim 13, wherein the one or more enzymes are 25 selected from the group consisting of cellulase, kitinase, galactosidase, β- fructosidase, β-glucanase, hemicellulase, xylanase polygalacturonase, xylosidase and arabinosidase.

15. The method according to any of the preceding claims, wherein the separation 30 of the liquid phase from the solid phase is obtained by centrifugation, filtration or decanting or a combination thereof.

16. The method according to any one of the preceding claims, wherein the pH in the liquid phase of step (ii) is lowered by the addition of at least one acid in order

35 to prevent growth of undesired microorganisms.

17. The method according to any one of the preceding claims, wherein the liquid phase has a carbohydrate content in the range of 0.1 to 25% (w/w).

5 18. The method according to any one of the preceding claims, wherein the liquid phase has a protein content in the range of 0.1 to 10% (w/w).

19. The method according to any one of the preceding claims, wherein K₂S₂O₅ is added the processing of the plant material and/or to the processed plant material

10 and/or to the plant juice.

20. The method according to any one of the preceding claims, wherein the processing of the plant material and/or the separation of the liquid phase from the solid phase and/or the recovering of the at least one protein is conducted at a

15 temperature in the range of 5°C-70°C.

21. The method according any one of the preceding claims, wherein said protein is recovered by precipitation or chromatography or a combination thereof.

20 22. The method according to claim 21, wherein the precipitation is an acid precipitation or a salt precipitation.

23. The method according to claim 21, wherein the precipitation is acid precipitation.

25

24. The method according to claim 22 or 23, wherein the acid precipitation is performed by the addition of at least one acid.

25. The method according to claim 24, wherein the at least one acid is selected

30 from a group consisting of CH₃CHOHCOOH, CH₃COOH, HCOOH, HCL, H₂SO₄, HNO₃, H₃PO₄ and combinations hereof.

26. The method according to claim 24, wherein the at least one acid is produced by an acid producing microorganism.

35

27. The method according to claim 26, wherein the acid producing microorganisms is used in combination with an acid according to claim 25.

28. The method according to any one of claims 7.1-11 , wherein pH of the liquid 5 phase is adjusted to a pH in the range of pH 1 to 7.

29. The method according to any one of the preceding claims, wherein the precipitated protein is recovered or isolated from the supernatant by means of centrifugation, filtration or decanting or a combination thereof.

10

30. The method according to any one of the preceding claims, wherein the recovered or isolated protein is in its native form.

31. The method according to any one of the preceding claims, wherein the 15 supernatant is concentrated.

32. The method according to claim 31, wherein the supernatant has a carbohydrate content in the range of 2 to 30% (w/w).

20 33. The method according to any one of the preceding claims, wherein the fermentation process is an alcohol fermentation process, preferably selected from a group consisting of ethanol fermentation and butanol fermentation.

34. The method according to claim 33, wherein the alcohol fermentation is

25 performed by one or more microorganisms selected from the group consisting of yeast and bacteria.

35. The method according to claims 33 or 34, wherein the alcohol fermentation provides a fermentation broth comprising an alcohol and an alcohol fermentation

30 effluent.

36. The method according to claim 35, wherein the fermentation broth comprises at least 2% (w/w) alcohol.

37. The method according to claim 35, wherein the fermentation broth comprises at least 2% (w/w) ethanol.

38. The method according to claims 35-37, wherein the alcohol or ethanol is 5 isolated from the fermentation broth by a distillation.

39. The method according to any one of the preceding claims, wherein the alcohol fermentation effluent is subjected to a methane fermentation process.

10 40. The method according to any of the preceding claims, wherein the solid phase of the plant material is added to the alcohol fermentation process and/or to the methane fermentation process.

41. The method according to any one of the preceding claims, wherein the solid 15 phase is subjected to a pre-treatment resulting in a partially separated material.

42. The method according to claim 41, wherein the pre-treatment comprises a wet oxidation or a steam explosion.

20 43. The method according to claims 41 or 42, wherein the partially separated material is subjected to a hydrolysis selected from the group consisting of an enzyme hydrolysis, an acid hydrolysis or an alkaline hydrolysis resulting in a slurry containing fermentable sugars.

25 44. The method according to claim 43, wherein the slurry is added to the alcohol fermentation process and/or to the methane fermentation process.

45. Use of a high K-vinasse obtainable by the method according to claims 1-44 as organic fertilizer.

30

46. The use according to claim 45, wherein the high K-vinasse is used in combination with compost and other organic based fertilizers.

47. A high K-vinasse obtainable by the method according to claims 1-44 35 comprising a high content of potassium.

48. The high K-vinasse according to claim 47 further characterized in comprising a low protein content and/or a low content of convertible organic compounds.

49. A method of modifying compost and other organic fertilizers, comprising adding to the compost or other organic fertilizers the high K-vinasse according to claim 47.