WO2018231565A1

WO2018231565A1 - Green biomass modification

Info

Publication number: WO2018231565A1
Application number: PCT/US2018/035807
Authority: WO
Inventors: Jens Frisbaek Sorensen; Lone Vendel NIELSEN
Original assignee: Dupont Nutrition Biosciences Aps; E.I. Du Pont De Nemours And Company
Priority date: 2017-06-15
Filing date: 2018-06-04
Publication date: 2018-12-20
Also published as: WO2018231565A9

Abstract

Disclosed is an enzymatic treatment of green biomass to improve the quality and/or the quantity of protein isolated from green biomass compared to protein obtained from green biomass that has not been enzymatically treated.

Description

GREEN BIOMASS MODIFICATION

FIELD

The field relates to the modification of green biomass to improve the efficiency and sustainability of monogastric livestock production systems.

BACKGROUND

The demand for high quality protein for animals and humans is increasing as a consequence of global population growth. Typically, animal feed mainly has been based on energy from cereals and protein from legumes. Green biomass holds the potential for production of high quality protein for feed and food as alternative to soy protein.

Today green biomass is used for grazing or stored as hay/silage for ruminant animals.

Ruminants do not need high quality soluble proteins and monogastric animals cannot digest the fiber.

It would be desirable to extract the soluble protein from green biomass and use the fiber for ruminants.

Green biomass also has the potential, not only as a food or feed ingredient, but as well as a medical or nutritional product. One challenge being faced is devising a way to extract protein from green biomass in a way that makes it competitive with respect to the protein obtained from soybeans in terms of cost and quality. Thus, due to the high fiber content of green biomass, there is a need to develop a way to process it to decrease the amount of antinutritional factors that may be present while improving not only the nutritional quality but also the purity and/or quantity of protein obtained.

SUMMARY

In one embodiment, there is described an enzymatic treatment of green biomass comprising a water content of at least 60% to improve purity and/or yield of protein isolate as compared to green biomass that has not been enzymatically treated.

In a second embodiment, the enzymatic treatment comprises at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase.

In a third embodiment, the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase. In a fourth embodiment, the purity and/or the quantity of the protein isolate obtained from the green biomass is increased at least 5% when compared to protein isolate obtained from green biomass that has not been enzymatically treated.

In a fifth embodiment, the green biomass can be any plant material comprising at least 60% water.

In a sixth embodiment, there is described a protein isolate derived from enzymatically- treated green biomass comprising a water content of at least 60% wherein said protein has improved purity and/or yield as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated and further wherein said protein can be used in feed or food.

In a seventh embodiment, the enzyme treatment comprises at least one enzyme selected from the group consisting of at least xylanase, at least one glucanase and at least one pectinase

In an eighth embodiment, the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

In a ninth embodiment, the enzyme treatment increases the purity and/or quantity of protein isolate obtained by at least 5% when compared to purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

In a tenth embodiment, the green biomass can be any plant material comprising at least 60% water.

In an eleventh embodiment, there is described feed or feedstuff for a monogastric animal comprising the protein isolate described herein.

In a twelfth embodiment, there is described a method for improving the yield and/or purity of protein isolate obtained from enzymatically-treated green biomass, said method comprising:

a) contacting green biomass comprising at least 60% water with at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase; and

b) obtaining protein isolate having improved quantity and/or purity as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated. In a thirteenth embodiment, the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

In a fourteenth embodiment, the purity and/or quantity of protein isolate obtained is increased at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

In a fifteenth embodiment, the green biomass can be any plant material comprising at least 60% water.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow diagram of laboratory-scale processing of green biomass.

Figure 2 presents results for mono-saccharide composition of protein

isolates/concentrates from laboratory-scale processing of ryegrass with enzyme mix 1,2, 4 or no enzymes (blank) obtained by HPLC (LOQ is 5000ppm). Despite large variation in data it can be concluded that mix 2 and 4 perform better than mix 1.

DETAILED DESCRIPTION

All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

The articles "a", "an", and "the" preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a", "an", and "the" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term "comprising" means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term

"comprising" is intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of . Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of. Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be construed as including ranges "1 to 4", "1 to 3", "1- 2", "1-2 & 4-5", "1-3 & 5", and the like.

As used herein in connection with a numerical value, the term "about" refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context. For instance, the phrase a "pH value of about 6" refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.

It is intended that every maximum numerical limitation given throughout this

Specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this Specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this

Specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The term "green biomass" as used herein means any plant material having a water content greater than 60% and, preferably, greater than 70%. Such plant material can include perennial forage grasses and legumes, forage grasses such as ryegrass, clover such as red and white clover, lucerne, immature cereals and the like.

The terms "animal" and "subject" are used interchangeably herein. An animal includes all non-ruminant (including humans) and ruminant animals. In a particular embodiment, the animal is a non-ruminant animal, such as a horse and a mono-gastric animal. Examples of mono- gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns.

A "feed" and a "food," respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by a non- human animal and a human being, respectively.

As used herein, the term "food" is used in a broad sense and covers food and food products for humans as well as food for non-human animals (i.e. a feed).

The term "feed" is used with reference to products that are fed to animals in the rearing of livestock. The terms "feed" and "animal feed" are used interchangeably. The terms "peptides", "proteins" and "polypeptides are used interchangeably herein and refer to a polymer of amino acids joined together by peptide bonds. A "protein" or

"polypeptide" comprises a polymeric sequence of amino acid residues. The single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

The terms "derived from" and "obtained from" refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question.

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations used herein to identify specific amino acids can be found in Table 1.

Table 1. One and Three Letter Amino Acid Abbreviations

Three-Letter One-Letter

Amino Acid Abbreviation Abbreviation

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Thermostable serine acid Asp D

Cysteine Cys C

Glutamine Gin Q Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Tip W

Tyrosine Tyr Y

Valine Val V

Any amino acid or as defined herein Xaa X

Figure 1 depicts a flow scheme for processing of green biomass. Typically, the green biomass can be put through a screw press from which (a) pulp containing fiber and insoluble protein is obtained for use in cattle feed, and (b) juice is obtained from which protein is precipitated. This precipitation can be done in several ways, such as, under acidic conditions (pH is less than 7) or by using heat treatment (temperature in the range of 60°C to 140°C, preferably, around 80°C). The protein-containing fraction (i.e., the protein isolate or concentrate) contains soluble protein that can be used in feed for monogastric animals. During the processing of the green biomass, some NSPs are co-extracted which may induce anti -nutritional effects in monogastric animals. NSPs of grass and clover changes with maturity of plant and includes pectin (5-35%), cellulose (10-60%), hemicellulose (10-40%, incl. mixed linkage beta-glucan, xyloglucan, (arabinoxylan and arabinogalactan) and lignin (5-20%).

The green biomass should have a water content of at least 60%, preferably 70% or even

80%). Any plant material having a water content greater than 60% and, preferably, greater than 70%) or even more preferably, greater than 80%> can be used as a source of green biomass. There can be mentioned plant material such as perennial forage grasses and legumes, forage grasses such as ryegrass, clover such as red and white clover, lucerne, immature cereals, beets, whole crop and the like. Green biomasses such as red clover and ryegrass present crops with a water content of at least 70% and a protein content of 3-4%.

Certain cultivated grasses and grassland legumes may have protein contents up to 30%> of dry matter. Typically, these grasses are utilized by ruminants (multi -gastric animals). On the other hand, monogastric animals such as pigs and poultry can only use grass on a limited basis due to the high fiber content.

In one embodiment, there is described enzymatic treatment of green biomass comprising a water content of at least 60%> to improve purity and/or yield of protein isolate as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated. It has been found that the purity and/or quantity of protein extracted from green biomass can be improved by treating the green biomass with at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase, at least one pectinase and at least one protease. Preferably, the enzymatic treatment of the green biomass comprises an enzyme cocktail or mixture comprising at least one xylanase, at least one glucanase and at least one pectinase. The improvement in purity and/or quantity of protein isolated from

enzymatically-treated green biomass is increased as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated. It is believed that this enzyme treatment is capable of decreasing the amount of non-starch polysaccharides (NSP) that are present in the protein isolate/concentrate.

The enzyme treatment may be applied to the green biomass after harvesting. The enzyme(s) will than act in the processing of the green biomass and be inactivated during protein precipitation. Alternatively, the enzyme(s) may be added to the protein-containing juice before any protein is precipitated.

Xylanase (EC 3.2.1.8) is the name given to a class of enzymes which catalyze the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans one of the major components of plant cell walls. Xylanases are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect, seeds, etc. but the principal source is filamentous fungi.

Glucanases are enzymes that break down a glucan, a polysaccharide made of glucose sub-units. As they perform hydrolysis of the glucosidic bond, they are hydrolases. Glucanases occurs in two forms: alpha and beta. Beta-glucanase enzymes (EC 3.2.1.4) digests fiber. It helps in the breakdown of plant walls.

Pectinase or pectinases refers to a class of enzymes that can break down pectin. Pectins consist of a complex set of polysaccharides. Several distinct polysaccharides have been identified and characterized within the pectic group. Homogalacturonans are linear chains of a- (l-4)-linked D-galacturonic acid. Substituted galacturonans are characterized by the presence of saccharide appendant residues (such as D-xylose or D-apiose in the respective cases of xylogalacturonan and apiogalacturonan) branching from a backbone of D-galacturonic acid residues. Rhamnogalacturonan I pectins (RG-I) contain a backbone of the repeating disaccharide: 4-a-D-galacturonic acid-(l,2)-a-L-rhamnose. From many of the rhamnose residues, sidechains of various neutral sugars branch off. The neutral sugars are mainly D-galactose, L-arabinose and D- xylose, with the types and proportions of neutral sugars varying with the origin of pectin.

Another structural type of pectin is rhamnogalacturonan II (RG-II), which is a less frequent, complex, highly branched polysaccharide. Rhamnogalacturonan II is classified by some authors within the group of substituted galacturonans since the rhamnogalacturonan II backbone is made exclusively of D-galacturonic acid units.

Isolated pectin may have a molecular weight of typically 60,000-130,000 g/mol, varying with origin and extraction conditions.

Pectins, also known as pectic polysaccharides, contain two different defined regions. The "smooth" region consists of an alpha- 1,4-linked galacturonic acid residues. The "hairy" region consist of galacturonic acid residues in the backbone, interrupted by alpha- 1,2-linked rhamnose residues, to which long arabinan and galactan chains can be attached at 04 position. The enzymes hydrolyzing these polysaccharides are pectin lyases (E.C. 4.2.2.10), pectate lyase (E.C. 4.2.2.2), polygalacturonases (E.C. 3.2.1.15 and E.C. 3.2.1.67) and finally rhamnogalacturonan hydrolases and rhamnogalacturonan lyases.

Once the green biomass has been enzymatically treated it can then be processed to extract the protein using any standard means known to those skilled in the art. For example, the enzymatically-treated green biomass can be mixed and comminuted and then subjected to at least one screw press to produce a fiber fraction and a liquid protein-containing fraction. The fiber fraction may be used as feed for ruminants or a raw material for ethanol production.

Protein can be precipitated from the liquid protein-containing fraction using pH or heat treatment. The precipitated protein can be separated from the liquid using any standard means such as a centrifuge. This precipitated protein can be referred to as protein isolate or protein concentrate which may be used as feed for monogastric animals.

Once the protein has been extracted, the remaining liquid fraction contains very low residual organic matter, mainly, readily soluble carbohydrates, some salts and non-protein nitrogen.

Thus, the three main fractions produced are a fiber fraction, a protein isolate or concentrate fraction and a liquid fraction containing sugar, salts and non-protein nitrogen.

The composition of essential amino acids in protein extracted from green biomass is expected to be very beneficial in improving the efficiency and sustainability of monogastric livestock production systems. For example, the content of sulfur-containing amino acids is higher than for soya.

It is believed that a highly purified protein fraction from green biomass may be of sufficient quality and nutritional value to be used in human food.

Thus, in another aspect, there is described protein isolate obtained from enzymatically- treated green biomass comprising a water content of at least 60% wherein said protein has improved purity and/or quantity as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated and further wherein said protein can be used in feed or food.

As was noted above, the enzymatically-treatment of the green biomass comprises at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase. Preferably, the enzymatic treatment is an enzyme cocktail or mixture comprising at least one xylanase, at least one glucanase and at least one pectinase. The protein isolate obtained from enzymatically-treated green biomass may have a purity and/or quantity increase of at least 5% when compared to the purity and/or quantity of protein obtained from green biomass that has not been enzymatically treated.

In still another aspect, such protein may be used in food or feed. Preferably, it may be used in feed for monogastric animals.

The terms "animal feed composition," "feed", "feedstuff and "fodder" are used interchangeably and can comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grains with Solubles (DDGS) (particularly corn based Distillers

Dried Grains with Solubles (cDDGS)), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; and/or e) minerals and vitamins.

Preferably, the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof. A feed additive composition may be admixed with a compound feed, a compound feed component or to a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder.

The term "fodder" as used herein means any food which is provided to an animal (rather than the animal having to forage for it themselves). Fodder encompasses plants that have been cut. Furthermore, fodder includes silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from: corn (maize), alfalfa (Lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsike clover, red clover, subterranean clover, white clover, fescue, brome, millet, oats, sorghum, soybeans, trees (pollard tree shoots for tree-hay), wheat, and legumes.

The term "compound feed" means a commercial feed in the form of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be blended from various raw materials and additives. These blends are formulated according to the specific requirements of the target animal. Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a part of the ration (protein, energy) or supplements that only provide additional micronutrients, such as minerals and vitamins.

The main ingredients used in compound feed are the feed grains, which include corn, wheat, canola meal, rapeseed meal, lupin, soybeans, sorghum, oats, and barley.

Suitably a premix as referred to herein may be a composition composed of

microingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.

Feedstuff may comprise or consists of corn, DDGS (such as cDDGS), wheat, wheat bran or any combination thereof.

In another aspect, the feed component may be corn, DDGS (e.g. cDDGS), wheat, wheat bran or a combination thereof. In one embodiment, the feedstuff comprises or consists of corn, DDGS (such as cDDGS) or a combination thereof.

A feedstuff described herein may contain at least 30%, at least 40%, at least 50% or at least 60% by weight corn and soybean meal or corn and full fat soy, or wheat meal or sunflower meal.

For example, a feedstuff may contain between about 5 to about 40% corn DDGS. For poultry, the feedstuff on average may contain between about 7 to 15% corn DDGS. For swine (pigs), the feedstuff may contain on average 5 to 40% corn DDGS. It may also contain corn as a single grain, in which case the feedstuff may comprise between about 35% to about 80% corn.

In feedstuffs comprising mixed grains, e.g. comprising corn and wheat for example, the feedstuff may comprise at least 10% corn.

In addition, or in the alternative, a feedstuff also may comprise at least one high fibre feed material and/or at least one by-product of the at least one high fibre feed material to provide a high fibre feedstuff. Examples of high fibre feed materials include: wheat, barley, rye, oats, by products from cereals, such as corn gluten meal, corn gluten feed, wet-cake, Distillers Dried Grains (DDG), Distillers Dried Grains with Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and pulp obtained from green biomass as described in Figure 1. Some protein sources may also be regarded as high fibre: protein obtained from sources such as sunflower, lupin, fava beans and cotton. In one aspect, the feedstuff as described herein comprises at least one high fibre material and/or at least one byproduct of the at least one high fibre feed material selected from the group consisting of

Distillers Dried Grains with Solubles (DDGS), particularly cDDGS, wet-cake, Distillers Dried Grains (DDG), particularly cDDG, wheat bran, and wheat for example. In one embodiment, the feedstuff may comprise at least one high fibre material and/or at least one by-product of the at least one high fibre feed material selected from the group consisting of Distillers Dried Grains with Solubles (DDGS), particularly cDDGS, wheat bran, and wheat for example.

The feed may be one or more of the following: a compound feed and premix, including pellets, nuts; a crop or crop residue: corn, soybeans, sorghum, oats, barley copra, straw, chaff, sugar beet waste; fish meal; meat and bone meal; molasses; oil cake and press cake;

oligosaccharides; conserved forage plants: silage; seaweed; seeds and grains, either whole or prepared by crushing, milling etc.; sprouted grains and legumes; yeast extract.

The term "feed" as used herein encompasses in some embodiments pet food. A pet food is plant or animal material intended for consumption by pets, such as dog food or cat food. Pet food, such as dog and cat food, may be either in a dry form, such as kibble for dogs, or wet canned form. Cat food may contain the amino acid taurine.

Animal feed can also include a fish food. A fish food normally contains macro nutrients, trace elements and vitamins necessary to keep captive fish in good health. Fish food may be in the form of a flake, pellet or tablet. Pelleted forms, some of which sink rapidly, are often used for larger fish or bottom feeding species. Some fish foods also contain additives, such as beta carotene or sex hormones, to artificially enhance the color of ornamental fish.

In still another aspect, animal feed encompasses bird food. Bird food includes food that is used both in birdfeeders and to feed pet birds. Typically, bird food comprises of a variety of seeds, but may also encompass suet (beef or mutton fat).

It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared.

Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins. In some embodiments, the feedstuff is a corn soybean meal mix. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting, in particular by suitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), and swine (all age categories), , horses, sheep, a pet (for example dogs, cats) or fish (for example gastric fish, freshwater fish such as salmon, cod, trout and carp, e.g. koi carp, marine fish such as sea bass, and crustaceans such as shrimp, mussels and scallops). In still another aspect, there is described a method for improving the purity and/or quantity of protein isolate obtained from enzymatically-treated green biomass, said method comprising:

b) obtaining protein isolate having improved purity and/or quantity as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

Preferably, the enzyme treatment is an enzyme cocktail or mixture comprising at least one xylanase, at least one glucanase and at least one pectinase. The purity and/or quantity increase of the protein isolate obtained by this method is at least 5% when compared to purity of protein isolate obtained from green biomass that has not been enzymatically treated.

Also described here is a method for improving the yield and/or purity of protein isolate obtained from enzymatically-treated green biomass, said method comprising:

a) contacting green biomass comprising at least 60% water with at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase; and b) obtaining protein isolate having improved yield and/or purity as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

The enzyme treatment is as described above.

The purity and/or quantity of protein isolate obtained is at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

Furthermore, the green biomass can be any plant material comprising at least 60% water. Non-limiting examples disclosed herein include: 1. Enzymatic treatment of green biomass comprising a water content of at least 60% to improve purity and/or quantity of protein isolate as compared to purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

2. The enzymatic treatment of green biomass of embodiment 1 wherein the enzyme treatment comprises at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase.

3. The enzymatically-treated green biomass of embodiment 1 wherein the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

4. The enzymatically-treated green biomass of embodiment 1 wherein the purity and/or the quantity of the protein isolate obtained is increased at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

5. The enzymatically-treated green biomass of embodiment 1 wherein the green biomass is any plant material comprising at least 60% water.

6. Protein isolate derived from enzymatically-treated green biomass comprising a water content of at least 60% wherein said protein isolate has improved purity and/or yield as compared to protein isolate from green biomass that has not been enzymatically treated and further wherein said protein can be used in feed or food.

7. The protein isolate derived from enzymatically-treated green biomass of embodiment 6 wherein the enzyme treatment comprises at least one enzyme selected from the group consisting of at least xylanase, at least one glucanase and at least one pectinase 8. The protein isolate derived from enzymatically-treated green biomass of embodiment 6 wherein the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

9. The protein isolate derived from enzymatically-treated green biomass of embodiment 6 wherein the enzyme treatment increases the purity and/or quantity of protein obtained at least

5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

10. The protein isolate derived from enzymatically-treated green biomass of claim 6 wherein the green biomass is any plant material comprising at least 60% water.

11. Feed or feedstuff for a monogastric animal comprising the protein isolate of embodiment 6.

12. A method for improving the yield and/or purity of protein isolate obtained from enzymatically-treated green biomass, said method comprising:

b) obtaining protein isolate having improved yield and/or purity as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

13. The method of embodiment 12 where the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

14. The method of embodiment 12 or 13 wherein the purity and/or quantity of protein isolate obtained is increased at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

15. The method of embodiment 12 wherein green biomass is any plant material comprising at least 60% water.

EXAMPLES

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE

HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used with this disclosure.

The disclosure is further defined in the following Examples. It should be understood that the Examples, while indicating certain embodiments, is given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain essential

characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt to various uses and conditions. Example 1.

Production and Analysis of Protein Obtained from Enzymatically-Treated Green Biomass as compared to Protein Obtained from Green Biomass without Enzymatically Treating a. Laboratory scale processing - simulation of industrial conditions for processing green biomass

300 g of freshly cut ryegrass was dispersed at the bottom of a plastic container and 5 ml of enzyme cocktail (mix 1, mix2, mix3 and mix4 as described in Table 2 below) were distributed with a spray as shown in Figure 1. A timer was set at 30 min and started and the first 5 min was used for cutting grass in smaller pieces with scissors. The grass was then passed through a table top screw-press to make juice. The juice was poured into 50 ml tubes, balanced and left standing at room temperature (RT) for 30 minutes. The tubes then were put into a 90°C water bath for 12 minutes to allow the enzymatically-treated sample of green biomass to reach >80°C for 1.5 min. The samples were then quickly cooled to RT using an ice water bath. To separate precipitated protein from supernatant the tubes were centrifuged 10 min at 950 g (2800 rpm) and 4°C, followed by decanting of supernatant, freezing of pellet and freeze drying over night before pulverization. Pellets were analyzed as described below. b) Enzymes used in processing

Based on a literature review of ryegrass and clover NSP composition four mixtures of carbohydrases were chosen for this experiment. The four enzyme mixtures or cocktails used are set forth in Table 2 below. Most of these enzymes are or will be commercially available from Danisco US, Inc. Genencor Division. LAMINEX® BG2 is an enzyme complex hydrolyzing beta-glucans and non-starch polysaccharides like arabinoxylans.

LAMINEX® 750 is an enzyme complex hydrolyzing beta-glucans and non-starch

polysaccharides like arabinoxylans.

LAMINEX® MaxFlow 4G, contain beta-glucanases and xylanases.

POWERMill™ enzymes are used in wheat processing. PowerMill™ 8500 is an uninhibited bacterial xylanase which is produced by fermentation with a selected strain of Bacillus subtilis. MULITFECT® BGL is a Genencor glycosidase having cellulase-like activity.

MULITFECT® Pectinase FE is a concentrated liquid pectinase complex containing pectinase, cellulase and hemicellulose activities. It is derived from a selected strain of Aspergillus niger. Genencor xylanase NGX is an endo-1,4- beta-xylanase.

Treatment mixtures and abbreviations used in

Enzyme class Activity product names figures

LAM IN EX^® MaxFlow Glucanase/ 1750 BGL U/g

LM F4G

4G Xylanase 14261 XBU/g mixl 220000 DAN xyl

Powermill 8500 BS3 Xylanase

u/g

Multifect Pectinase

MpectFE Pectinase 154 U/g

FE

LAM IN EX^® BG2 LBG2 Glucanase 5555 BGL U/g

Genencor xylanase 51219 DAN xyl

NGX Xylanase

mix2 NGX u/g

Multifect Pectinase

MpectFE Pectinase 154 U/g

FE

LAM IN EX^® 750 L750 Glucanase 887 BGL U/g

LAM IN EX^® Glucanase/ 1750 BGL U/g

LM F4G

mix3 MaxFlow 4G xylanase 14261 XBU/g

Genencor xylanase 51219 DAN xyl

NGX Xylanase

NGX u/g

Multifect BGL MBGL Glucanase 2863 BGL U/g

Genencor xylanase 51219 DAN xyl

NGX Xylanase

mix4 NGX u/g

Multifect Pectinase

MpectFE Pectinase 154 U/g

FE

Table 2. Information on enzymes and mixtures used in processing of green biomasses c. Unit definitions

(i) Beta- glucanase

One BGL U is the amount of enzyme that releases 2.4 μπιοΐ of reducing sugar equivalents (as glucose by the DNS reducing sugar method) from barley glucan per min at pH 5.0 and 50°C.

(ii) Xylanase

One DAN xyl U is the amount of enzyme that releases 0.48 μπιοΐ of reducing sugar (as xylose by the DNS reducing sugar method) from wheat arabino xylan per minute at pH 4.2 and 50°C. (iii) Pectinase

The activity of pectinase, U/g, is determined by a relative viscometric determination of pectolytic activity in apple juice against a reference standard of known activity. d) Assays (i) Measurement of protein in pellet (Kjeldahl)

Pellet samples of 1 g each were analyzed by Eurofins, Denmark for determination of total nitrogen (N) by Kjeldahl method, protein = total Nx6.25. Reference method MKL 6.4:2003 mod. (titrimetry).

(ii) Measurement of monosaccharides in pellet (HPLC)

Samples were weighed out in quadruplicate (20- 40 mg). Two samples were added to 0.25 mL

12 M H2S04 and reacted for 30 min at 35 C. After this pre-treatment, 2.75 mL water was added to make the final H2S04 concentration 1M. The other two samples had 0.25 mL of 12M H2S04 and 2.75 mL of water added but were not pre-treated. All four test tubes were reacted at 100° C for 6 hours in a boiling water bath. Samples were transferred to 20 mL measuring flasks which were filled to the mark with water. Samples were further diluted lOx before analysis. Samples were analyzed on a Thermo ISC3000 BioLC (or ISC5000). Columns used were a CarboPac PA1 column, 2mm*250 mm. Flow= 0.25 mL/min. temperature: ambient. Eluents: A: water, B 600 mM NaOH.

Gradient:

Calculations based on area under curves were done using external standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, glucuronic acid and galacturonic acid) in the

chromatographic software Chromeleon. The sum of all measured sugars was taken as total sugars measured. e) Protein and sugar content

Protein and sugar in concentrates were determined as described above. f) Results and Discussion

Enzyme mixtures 1, 2 and 4 were selected for enzymatically-treating green biomass under lab-scale conditions with heat precipitation as described above in simulating the conditions assumed to apply to industrial processing of green biomass with enzymes.

Beta-glucanases were dosed at 2500 U, xylanases at 100 U and pectinase at 50 mg (7.7 U) per 300g whole plant. Large variation in data on both total protein and mono-saccharide composition of pellets exist, however, sugar analysis confirms that mix 2 and 4 appear to be better than mix 1 and mix 3 and protein to sugar ratio of protein precipitates also confirm this (Table 3). Data for mix 3 was comparable to the data for mix 1 and, thus, was not included. Furthermore, it is clear that the enzyme pre-processing has a significant positive effect versus not applying enzymes (blank).

Table 3. Values of protein determined by Kjeldahl method, total sugar calculated as sum of

monosaccharides measured by H PLC and the ratio of protein to sugar are listed for ryegrass isolates or concentrates obtained after laboratory-scale processing with enzymes or without (blank), st.dev. are shown in parentheses. Surprisingly, it was found that enzymatically treating biomass improved the purity and/or the quantity of protein isolated from green biomass compared to the purity and/or quantity of protein isolate obtained from green biomass that had not been enzymatically treated.

The best enzyme candidate combination tested in the present example appears to be mix 4 which comprised Multifect BGL, Genencor xylanase NGX and Multifect Pectinase FE, i.e., at least one glucanase, at least one xylanase, and at least one pectinase. This enzyme cocktail or mixture increased the protein to sugar ratio 28% by increasing the protein concentration and decreasing the carbohydrate concentration in the obtained protein fraction, i.e., the protein isolate.

Claims

CLAIMS What is claimed is:

1. Enzymatic treatment of green biomass comprising a water content of at least 60% to improve purity and/or quantity of protein isolate as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

2. The enzymatic treatment of green biomass of claim 1 wherein the enzyme treatment comprises at least one enzyme selected from the group consisting of at least one xylanase, at least one glucanase and at least one pectinase.

3. The enzymatically-treated green biomass of claim 1 wherein the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

4. The enzymatically-treated green biomass of claim 1 wherein the purity and/or the quantity of the protein isolate obtained is increased at least 5% when compared to the purity and/or the quantity of protein isolate obtained from green biomass that has not been

enzymatically treated.

5. The enzymatically-treated green biomass of claim 1 wherein the green biomass is any plant material comprising at least 60% water.

6. Protein isolate derived from enzymatically-treated green biomass comprising a water content of at least 60% wherein said protein has improved purity and/or yield as compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated and further wherein said protein can be used in feed or food.

7. The protein isolate derived from enzymatically-treated green biomass of claim 6 wherein the enzyme treatment comprises at least one enzyme selected from the group consisting of at least xylanase, at least one glucanase and at least one pectinase

8. The protein isolate derived from enzymatically-treated green biomass of claim 6 wherein the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

9. The protein isolate derived from enzymatically-treated green biomass of claim 6 wherein the enzyme treatment increases the purity and/or quantity of protein obtained at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

11. Feed or feedstuff for a monogastric animal comprising the protein of claim 6.

b) obtaining protein isolate having improved purity and/or quantity as compared to purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

13. The method of claim 12 where the enzyme treatment comprises at least one xylanase, at least one glucanase and at least one pectinase.

14. The method of claim 12 or 13 wherein the purity and/or quantity of protein isolate obtained is at least 5% when compared to the purity and/or quantity of protein isolate obtained from green biomass that has not been enzymatically treated.

15. The method of claim 12 wherein green biomass is any plant material comprising at least 60% water.