RU2817795C2 - Vertical flow process for bioconversion of biomass comprising enzymes - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: described is a method for the production of a solid product selected from a solid feed ingredient, or a solid food ingredient, or a solid food additive, involving production of a biomass substrate, including carbohydrates and a protein substance originating from soya bean seeds, rape seeds or mixtures thereof, wherein at least 20 wt.% of said biomass include carbohydrates and a protein substance originating from soya bean seeds, rape seeds or mixtures thereof, mixing said substrate with an enzyme preparation or a combination of enzyme preparations, and adding water in an amount which provides the initial mixture for incubation, having water content of 30 wt.% to 70 wt.% and ratio of wet bulk density to dry bulk density of 0.60 to 1.45, incubating said initial mixture for incubation for 1–48 hours at temperature of 20–70 °C and extracting said end products from the incubated mixture, wherein the incubation step is performed as a continuous process in a uniform plug flow in a vertical reactor for incubation without mixing with devices for inlet of said mixture and additives and devices for outlet of said solid transformation product, and biomass movement is mediated by gravitational force.

EFFECT: invention enables to obtain bioproducts with a high protein content and/or a modified sugar profile, which include a reduced amount of indigestible carbohydrates and/or anti-nutritional factors.

24 cl, 3 dwg, 5 tbl, 9 ex

Description

Field of technology to which the invention relates

The present invention relates to a solid substrate bioconversion process for obtaining a valuable solid substrate transformation product, wherein the bioconversion is carried out using one or more suitable enzyme preparations through a continuous plug flow process in an unstirred vertical reactor where the movement is mediated by gravitational force.

Prerequisites for creating the invention

There is a need for bioproducts that can primarily be used as food or feed or as ingredients in food or feed. The main elements in such products are proteins, fats and carbohydrates. Suitable biomass for such products are oilseed crops such as oilseeds, grains and legumes. Cereals have a protein content, relative to dry matter, of up to 15%, such as wheat, and legumes have a protein content of up to 40%, such as soybeans. Soybeans are primarily an industrial crop grown for their oil and protein. It has a relatively low oil content in the beans, but soybeans are one of the major sources of edible oil.

The nutritional quality of protein, measured by its chemical evaluation as a composition of essential amino acids, and the palatability of protein products are important and mandatory parameters for nutritional purposes in food and feed and food additives. Applications of protein products in pharmaceutical and cosmetic products may sometimes also require high palatability and/or specific functional properties.

A common problem associated especially with beans and legume fruits and seeds, which include carbohydrates and protein matter, as sources of bioproducts (in particular protein products), is the content of indigestible oligosaccharides such as stachyose and raffinose, which cause flatulence and diarrhea when fermented in colon.

The approximate average composition of example soybeans, measured on an anhydrous basis, is 40% protein; 20% fat, mainly triglycerides and some phospholipids; 35% carbohydrates in the form of soluble oligosaccharides (sucrose, raffinose, stachyose, verbascose) and insoluble fiber, and 5% ash, including minerals, particularly potassium, calcium and magnesium.

Proteins from beans, seeds, grains and herbs, including soy proteins such as trypsin inhibitors, allergens and lectins, are known as anti-nutrients. They exhibit specific physiological effects. Trypsin inhibitors impair protein digestion by inactivating trypsin and are considered detrimental to the nutritional value of soybeans and are thought to be responsible for poor growth in chickens. The soy allergen is β-conglycinin, which causes inflammation and intestinal dysfunction.

An object of the present invention is a method for producing a biomass substrate transformation product through a vertical plug flow bioconversion process performed in the presence of one or more suitable enzymes.

Another object is a method that can be carried out in a large reactor of simple design and thereby at low cost.

Another object is an efficient method for the bioconversion of biomass, in particular soybeans or rapeseeds or mixtures thereof, in order to produce bioproducts with desired properties, such as high protein content and/or a modified sugar profile and/or improved nutritional value and/ or reduced anti-nutritional factors and/or improved palatability and/or enhanced organoleptic properties and/or improved functional properties.

The final object of the invention is an improved method for the production of bioproducts comprising reduced amounts of indigestible carbohydrates and/or anti-nutritional factors.

These goals are achieved by the method of the present invention.

The essence of the invention

Accordingly, in one aspect, the present invention relates to a method for producing a solid substrate transformation product, comprising the following steps:

- obtaining a biomass substrate comprising carbohydrates and proteinaceous matter originating from soybeans, rapeseeds or mixtures thereof, optionally in an additional mixture with carbohydrates and proteinaceous matter originating from fava beans, peas, sunflower seeds, lupine, cereals and/or grasses;

- mixing said substrate with an enzyme preparation or combination of enzyme preparations, and adding water in an amount that provides an initial incubation mixture having a water content of from 30% to 70%, and a ratio of wet bulk density to dry bulk density in the resulting mixture from 0, 60 to 1.45;

- incubating said initial incubation mixture for 0.15-240 hours at a temperature of 20-70°C and removing the wet solid transformation product from the incubated mixture;

further comprising that the incubation step is performed as a continuous plug flow process in a vertical unstirred incubation reactor with means for inlet of said mixture and additives and means for outlet of said solid transformation product.

The method of the present invention for processing biomass uses gravitational force to move/move the biomass during bioconversion. Although the use of gravity for transport is generally simple, careful selection of reaction conditions for a particular purpose is required, as is the case with the slug flow process of the present invention.

Typically, when the water content increases, the incubation mixture tends to become compacted by reducing void volume, so that transfer behavior is negatively affected. When a certain amount of water is reached, the mixture is compacted to the point where transport by gravity stops. Material may adhere to the reactor walls and the uniform slug flow is disrupted, resulting in fluctuating residence times of the biomass.

The solution to the problem of transfer of incubation mixture by gravitational force according to the present invention would involve the use of an incubation reactor as defined in the claims, wherein the flow of material can be maintained so high and uniform that slug flow conditions are achieved and maintained. The flow rate is controlled by inlet and outlet fixtures and by the dimensions (width to height ratio) of the reactor.

In addition, the solution according to the present invention must balance the water content of the incubation mixture such that the action of water on the surface of the particles is sufficient for the reaction process. This is achieved by maintaining the ratio of wet bulk density to dry bulk density of the substrate at a low level and within certain limits, as defined in paragraph 1.

More specifically, the present inventors have discovered that the desired uniformity of the process can be achieved by using an initial incubation mixture having a water content of 30 wt% to 70 wt% and a wet bulk density to dry bulk density ratio of 0.60 to 1.45 . In combination with the above, the vertical design for the slug flow process can ensure uniform slug flow and guarantee the same processing time of the incubation mixture. Moreover, the method of the present invention is carried out without stirring. If the water content exceeds about 70 wt.%, the biomass cannot retain water and the incubation mixture becomes a slurry having an aqueous phase and a solid phase. The two phases do not flow at the same flow rates and a uniform plug flow will not result and the incubation mixture may stick to the walls of the incubator. A water content greater than about 70% by weight will result in a wet to dry bulk density ratio greater than 1.45, which is the upper limit of the invention.

The vertical design is less expensive in terms of investment than the horizontal design due to its greater throughput in a single production line. It also requires less maintenance due to fewer mechanical movements. The use of a non-stirred reactor also contributes to lower operating costs.

The method of the present invention is effective in particular if the biomass substrate is pre-treated before it is mixed with the enzyme preparation or combination of enzyme preparations, since the pre-treatment improves the access of the enzymes to the components in the biomass that are to be transformed. Pre-treatment is usually carried out by chemical or physical treatment, for example, by crushing, grinding, flattening, heat treatment, pressure treatment, ultrasonic treatment, hydrothermal treatment or acid or alkali treatment.

The method according to the invention can be used to obtain a solid substrate transformation product, which is a transformation product of protein matter and/or carbohydrates originating from said biomass.

Such solid transformation products can be used, for example, in a processed food product or as an ingredient in a food or feed product or as an ingredient in a cosmetic or pharmaceutical product or dietary supplement. The solid transformation product of the substrate can be included, for example, in a food product, feed, cosmetic or pharmaceutical product or nutritional supplement, up to a content of from 1 wt.% to 99 wt.% solid transformation product.

Definitions

In the context of the present invention, the following terms are intended to include the following unless otherwise defined in the description.

The terms "about", "about", "approximately" or "~" are intended to indicate, for example, the commonly expressed variability of a measurement in a technique, which may be of the order of, for example, +/- 1, 2, 5, 10 , 20 or even 50%.

The term "including" should be interpreted as specifying the presence of specified part(s), step(s), feature(s), composition(s), chemical(s) or component(s), but does not exclude the presence of one or more additional parts, steps, features, compositions, chemicals or components. For example, a composition comprising a chemical compound may thus include other chemical compounds, etc.

Slug flow process

In this type of continuous process, the reaction mixture flows through, for example, a tubular or polyhedral reactor with limited back mixing. The flow is a laminar flow, in which the composition of the reaction mixture varies along the axial direction of the reactor, or a uniform mass flow.

Biomass

Includes biological material both produced by photosynthesis and material that can be used as raw material in industrial production. In this context, the term "biomass" refers to plant material in the form of seeds, grains, legumes, grasses, such as beans and peas, etc., and mixtures thereof, in particular legume fruits and seeds. In addition, biomass including beans is particularly useful due to its protein content and composition.

The biomass substrate can be destroyed by pre-treatment, such as chemical or physical pre-treatment, for example by crushing, grinding, flattening, heat treatment, pressure treatment, ultrasonic treatment, hydrothermal treatment or acid or alkali treatment.

Bioconversion

It is the process of incubating enzymes on a substrate for a specific purpose, such as incubating a protease on a protein to produce peptides or individual amino acids.

Solid product of substrate transformation

In the context of the present invention, the term "solid substrate transformation product" refers to the product resulting from incubation of a selected biomass with an enzyme preparation or combination of enzyme preparations that can convert the substrate material into the desired product, and optionally auxiliary processing.

Bulk Density

Bulk density is a parameter important for the physical behavior of biomass, which is in the form of powder, granules, etc. The parameter is defined as the ratio of mass to volume and can be measured, for example, in g/ml. It is not an inherent property, but may vary with processing and can be used as an indicator of structural changes. The density of a material is determined by placing a fixed volume of material in a measuring cup and determining the mass, or by determining the mass of a measured volume of material. This test can determine the following properties.

Bulk density (also known as bulk density) = mass/volume in the original dry state, in g/ml or kg/ ^m3 .

Wet bulk density (also known as total density) = the ratio of the total mass (M _s +M _l ) to its total volume;

M _s = mass of solids, and M _l = mass of liquids.

Thus, in the context of the present invention, "dry bulk density" is the measured bulk density of the biomass without the addition of water, namely bulk density/bulk density. "Wet bulk density" is the bulk density measured after adding a specified amount of water.

Typically, bulk density is measured in accordance with International Standards ISO 697 and ISO 60, but due to the nature of the substances this is not acceptable in the present context. The individual method used is described in the examples.

Oligosaccharides and polysaccharides

An oligosaccharide is a saccharide polymer containing at least two simple sugar components. Polysaccharides are polymers of saccharides containing several simple sugar components, also known as complex carbohydrates. Examples include storage polysaccharides such as starch and structural polysaccharides such as cellulose.

Carbohydrates

Includes mono-, di-, oligo- and polysaccharides.

Protein materials

Include organic compounds with a significant protein content of amino acids arranged in one or more chains. With a chain length of up to approximately 50 amino acids, the compound is called a peptide; at higher molecular weights, the organic compound is called a polypeptide or protein.

Fats

Includes esters of fatty acids and glycerol. One glycerol molecule can be esterified with one, two, or three fatty acid molecules to form a monoglyceride, diglyceride, or triglyceride, respectively. Typically, fats consist primarily of triglycerides and small amounts of lecithins, sterols, etc. If a fat is a liquid at room temperature, it is usually called an oil. For oils, fats and related products in this context, reference is made to "Physical and Chemical Characteristics of Oils, Fats and Waxes", AOCS, 1996, and to "Lipid Glossary 2", F.D. Gunstone, The Oily Press, 2004.

Glycerides

Includes mono-, di- and triglycerides.

Enzymes

Enzyme(s) are a very large class of protein substances with the ability to act as catalysts. Typically, and as recommended by the Commission on Enzyme Nomenclature, they are divided into six classes.

Typical examples in the context of the invention may include, but are not limited to, protease(s), peptidase(s), phytase(s), carbohydrase(s), lipase(s), amylase(s), glucosidase(s), amyloglucosidase(s). ), galactosidase(s), decarboxidase(s), glucanase(s), pectinase(s), cellulase(s), hemicellulase(s), phospholipase(s), transferase(s) and oxidoreductase(s).

Auxiliary agents for processing

1. Plant components and organic substances for processing

Some of the functional properties that are important in the present context are antioxidant antibacterial action, wetting properties and stimulation of enzymatic activity.

The list of plant components for modulating soluble carbohydrates is vast, but the most important are the following: rosemary, thyme, oregano, flavonoids, phenolic acids, saponins and α- and β-acids from dried hop cones, such as α-lupulinic acid.

In addition, organic acids such as sorbic, propionic, lactic, citric and ascorbic acid and their salts for pH adjustment, preservation and complexation properties are part of this group of processing aids.

2. Inorganic processing auxiliaries

Includes inorganic compositions that can protect against the attack of bacteria during processing, for example, sodium bisulfite, etc.; anti-caking agents and agents that improve the flow of the final product, for example, potassium aluminum silicate, etc.

Inorganic acids are included, such as hydrochloric acid or sulfuric acid.

Processed foods

Includes dairy products, processed meats, sweets, desserts, ice cream desserts, canned goods, freeze-dried foods, dressings, soups, prepared foods, breads, cakes, etc.

Processed feed

Includes ready-to-eat feed for animals such as piglets, calves, poultry, fur-bearing animals, sheep, cats, dogs, fish and crustaceans, etc.

Pharmaceutical products

Includes products typically in tablet or granular form containing one or more biologically active ingredients intended to treat and/or alleviate symptoms of a disease or condition. In addition, pharmaceutical products include pharmaceutically acceptable excipients and/or carriers. The biosolids disclosed herein are very suitable for use as a pharmaceutically acceptable ingredient in a tablet or granule.

Cosmetic products

Includes products designed for personal care as well as appearance enhancements such as conditioners and bath products.

Brief description of drawings

Figure 1 shows in lane 1 the marker, in lane 2 the protease-treated sample from example 2, in lane 3 the reference sample for both examples 2 and 3, in lane 4 the protease-treated sample from example 3.

Figure 2 shows the composition of soluble sugars and oligosaccharides. Lane 1 - sample treated with carbohydrase, lane 2 - standard (soybean flour).

Figure 3 shows thin layer chromatography showing soluble sugars and oligosaccharides. Lane 1 - sample treated with carbohydrase, lane 2 - standard.

Detailed Description of the Invention

In a first embodiment of the method according to the invention, at least 20% by weight of biomass, for example at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, or at least 90% by weight is a proteinaceous substance derived from optionally defatted soybeans. Soybeans can also be shelled.

In a second embodiment of the method according to the invention, at least 20% by weight of biomass, for example at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight or at least 90% by weight is a proteinaceous substance derived from optionally defatted rapeseed.

In a third embodiment of the method according to the invention, the biomass comprises a protein substance derived from optionally defatted soybean flakes, in an amount of from 5 wt.% to 95 wt.%, mixed with a protein substance derived from optionally defatted rapeseed, in an amount from 95 wt. % to 5% by weight, optionally further mixed with proteinaceous matter derived from fava beans, peas, sunflower seeds and/or cereals, in amounts bringing the total amount of proteinaceous matter to 100% by weight.

In any of the embodiments of the invention, the biomass comprising proteinaceous matter may also include oligosaccharides and/or polysaccharides, and/or also include oils and fats, for example from oilseeds.

In any of the embodiments of the invention, the solid transformation product of the substrate may be a transformation product of proteinaceous matter and/or carbohydrates originating from said biomass, such as a transformation product of legumes such as soybeans, peas, lupine, sunflower seeds and/or cereals such as wheat or corn, or from oilseeds, such as rapeseed.

In any of the embodiments of the invention, the substrate or substrate, when mixed with the enzyme preparation or combination of enzyme preparations and water, cannot include any live baker's yeast, and/or it cannot include any live yeast selected from strains of Saccharomyces cerevisiae , including yeast , grown for the manufacture of beer, and yeast grown for distillate, and baker's yeast and yeast grown for winemaking, or does not include any live yeast; and/or in particular it may not include baker's yeast when the enzyme is α-galactosidase.

In any of the embodiments of the invention, the solid transformation product of the substrate may be a transformation product of a protein substance and/or carbohydrates originating from said biomass.

In any of the embodiments of the invention, the enzyme preparation or mixture of enzyme preparations includes one or more enzymes selected from proteases, peptidases, phytases, carbohydrases, such as α-galactosidase, amylases, amyloglucosidases, cellulases, pectinases and hemicellulases, such as xylanases, mannases or glucanases , and lipases and oxidoreductases.

In any of the embodiments of the invention, the dry matter ratio of said biomass substrate to said enzyme preparation or said combination of enzyme preparations may be from 2:1 to 100,000,000:1, such as 1000:1, 10000:1, 50000:1, 100000:1. 500000:1, 1000000:1, 5000000:1, 10000000:1, 50000000:1 or 100000000:1. One skilled in the art will appreciate that the ratio can be selected depending on parameters such as process conditions, enzyme activity, and the desired product, and one skilled in the art will be able to optimize the ratio according to these parameters.

In any embodiment of the invention, water can be added to the substrate in an amount that provides the substrate with a wet bulk density to dry bulk density ratio of from about 0.65 to 1.45, for example 0.70, 0.75, 0.80, 0. 85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25, 1.30 or 1.35.

In any of the embodiments of the invention, at least 40 wt.% biomass, for example, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.% or at least at least 90% by weight may comprise proteinaceous matter derived from optionally defatted rapeseed, while water may be added to the substrate in an amount to provide a wet bulk density to dry bulk density ratio of from about 0.65 to about 1.10, such as 0.75, 0.80, 0.85, 0.90, 0.95, 1.00 or 1.05.

In any of the embodiments of the invention, one or more processing aids selected from enzymes, botanicals, and organic and inorganic processing aids may be added to the substrate before or during incubation.

In any of the embodiments of the invention, the α-galactosidase preparation may be added to the biomass substrate and/or to the initial incubation mixture in an amount of from 0.05 to 50 units of α-galactosidase per g of dry matter of the biomass substrate, for example, from 0.5 to 25 units of α-galactosidase per g of dry matter of the biomass substrate, for example, from 1 to 10, from 2 to 8, from 3 to 6 or from 4 to 5 units of α-galactosidase per g of dry matter of the biomass substrate.

In any of the embodiments of the invention, the fill level of said incubation reactor may remain constant. This will result in an even flow.

In any of the embodiments of the invention, the incubation can be performed under anaerobic conditions.

In any of the embodiments of the invention, the water content of the incubation mixture can be from 35 wt.% to 70 wt.%, for example, 40 wt.%, 45 wt.%, 50 wt.%, 55 wt.%, 60 wt.% or 65 wt.%. Thus, the water content of the initial mixture does not exceed 70 wt.%, and it can vary, for example, from 40% to 65%, from 45% to 60%, from 48% to 52% or from 50% to 55%, for example, be 49, 50, 51, 52, 53 or 54%.

In any of the embodiments of the invention, the mixture can be incubated for 1-240 hours at 20-70°C. One skilled in the art will know how to optimize reaction time and reaction temperature in connection with other reaction conditions such as the choice of enzyme(s). Thus, the temperature can vary, for example, within the range of 20-65°C, 25-60°C, 30-55°C, 35-50°C or 40-45°C; and the reaction time can be selected, for example, from 1 to 180 hours, such as 2 to 150 hours, 3 to 120 hours, 5 to 90 hours, 8 to 72 hours, or 12 to 48 hours, at any of the temperatures mentioned herein intervals.

In any of the embodiments of the invention, the solid substrate transformation product can be dried, optionally followed by grinding.

In any of the embodiments of the invention, the substrate mixture can be incubated for a time and at a temperature sufficient to inactivate the enzyme(s), any anti-nutritional factors and any processing aids, if used in part or in whole and if desired.

In any of the embodiments of the invention, the vertical reactor for incubation without stirring may be closed.

In any of the embodiments of the invention, the unstirred incubation reactor may be of the vertical, elongated cylindrical or polyhedron type. The advantage of using this type is that it is compact, and since the reactor is unstirred, there are no operating and maintenance costs for stirring equipment.

In any of the embodiments of the invention, the area at the top of said incubation reactor without stirring may be smaller than at the bottom, ie, the reactor has a conical shape. The advantage of this is that the sliding effect is increased, so that biomass with reduced fluidity can be used.

In any embodiment of the invention, the unstirred incubation reactor may have insulation mats or a thermal dimple jacket and provisions for controlling the temperature in the incubation reactor.

The solid transformation product of the substrate of the invention can be dried to a water content of no more than 15 wt%, 13 wt%, 10 wt%, 6 wt%, 4 wt% or 2 wt% and is optionally in crushed form .

The solid product of the invention may be a transformation product of protein matter, oligosaccharides and/or carbohydrates originating from said biomass. The solid transformation product may have reduced levels of antinutritive factors such as trypsin inhibitors, antigens, flatulence-inducing oligosaccharides such as stachyose and raffinose; phytic acid and lectin.

The solid product of the invention may comprise at least 40 wt.% proteinaceous matter, based on the dry matter weight, derived from soybeans.

The solid product of the invention may comprise at least 40 wt.% proteinaceous matter based on the dry weight of rapeseed.

The solid product of the invention may comprise proteins in an amount of 30-65% by weight of the dry matter originating from soybean, rapeseed or sunflower plant parts or mixtures thereof.

The solid product of the invention may include raffinose, stachyose and verbacose in a total amount of 3% by weight or less, such as 2% or less, 1% or less, 0.5% or less, or 0.4% or less.

Finally, the invention relates to a food product, feed, cosmetic or pharmaceutical product or dietary supplement containing from 1 wt.% to 99 wt.% solid transformation product obtained according to the invention.

EXAMPLES

DENSITY RATIO

Example 1

Wet bulk density/dry bulk density ratio for preferred substrates based on different biomass

1.1. Biomass used in the procedure

Soybeans

The soy used is defatted soybean meal (SBM).

Corn

The corn used is whole corn ground in a hammer mill with a 3.5mm sieve.

Wheat

The wheat used is whole wheat, ground in a hammer mill with a 3.5 mm sieve.

Sunflower

The sunflower used is sunflower meal (SSM).

Rapeseed seeds

The rapeseed used is rapeseed meal (RSM).

Fava beans

The beans used are whole fava beans.

Pea protein

The pea protein used is pea protein concentrate.

1.2. Description of the procedure

Biomass and water in the quantities indicated in the table below are mixed in a closed container for ten minutes, followed by five minutes of equilibration.

The material is then discharged into a 500 ml measuring cup and its mass is determined by weighing the cup and subtracting the tare weight of the cup.

Bulk density is calculated as mass/initial volume in kg/m ³ .

The dry bulk density used is the measured bulk density of the biomass without the addition of water.

Wet bulk density is the bulk density of biomass with added water.

The ratio is calculated as wet bulk density divided by dry bulk density.

The moisture content in biomass is determined by drying to constant weight.

After adding water, the moisture content in the mixture is determined by calculation.

1.3. results

Results for 100% soy and 80% soy mixtures are shown in the table below.

Soybeans Corn Wheat Sunflower Rapeseed seeds Fava beans Peas Water in g Humidity in % Bulk Density
kg/m ³ Attitude 1000 g 0 10.9 665 - 1000 g 100 19.0 638 0.96 1000 g 250 28.7 500 0.75 1000 g 450 38.6 476 0.72 1000 g 750 49.1 470 0.71 1000 g 900 53.1 572 0.86 1000 g 1100 57.6 655 0.98 1000 g 1400 62.9 715 1.07 1000 g 1900 69.3 889 1.34 800 g 200 g 0 11.4 703 - 800 g 200 g 450 38.9 617 0.88 800 g 200 g 900 53.4 634 0.90 800 g 200 g 1900 69.4 1008 1.43 800 g 200 g 0 11.7 694 - 800 g 200 g 450 39.1 580 0.84 800 g 200 g 900 53.5 623 0.90 800 g 200 g 1900 69.5 960 1.38 800 g 200 g 0 10.4 683 - 800 g 200 g 450 38.2 554 0.81 800 g 200 g 900 52.9 598 0.88 800 g 200 g 1900 69.1 926 1.36 800 g 200 g 0 11.3 711 - 800 g 200 g 100 19.4 576 0.81 800 g 200 g 250 29.0 514 0.72 800 g 200 g 450 38.8 483 0.68 800 g 200 g 750 49.3 490 0.69 800 g 200 g 900 53.3 597 0.84 800 g 200 g 1100 57.8 528 0.74 800 g 200 g 1900 69.4 908 1.28 800 g 200 g 0 11.1 691 - 800 g 200 g 450 38.7 569 0.82 800 g 200 g 900 53.2 605 0.88 800 g 200 g 1900 69.3 941 1.36 800 g 200 g 0 11.2 703 - 800 g 200 g 450 38.7 488 0.69 800 g 200 g 900 53.2 728 1.04 800 g 200 g 1900 69.4 964 1.37

Results for mixtures of 60% and 40% soybeans with corn, sunflower and rapeseed, as well as for 100% rapeseed, are shown in the table below.

Soybeans Corn Sunflower Rapeseed seeds Water Humidity in % Bulk density kg/m ³ Attitude 600 g 400 g 0 g 11.8 703 - 600 g 400 g 250 g 29.5 651 0.93 600 g 400 g 450 g 39.2 626 0.89 600 g 400 g 750 g 49.6 631 0.90 600 g 400 g 900 g 53.6 666 0.95 600 g 400 g 1100 g 58.0 723 1.03 600 g 400 g 1400 g 63.3 796 1.13 600 g 400 g 0 g 10.0 644 - 600 g 400 g 100 g 18.2 530 0.82 600 g 400 g 250 g 28.0 435 0.68 600 g 400 g 450 g 37.9 433 0.67 600 g 400 g 750 g 48.6 436 0.68 600 g 400 g 900 g 52.6 480 0.75 600 g 400 g 1100 g 57.1 449 0.70 600 g 400 g 1400 g 62.5 616 0.96 600 g 400 g 0 g 11.7 643 - 600 g 400 g 100 g 19.7 560 0.82 600 g 400 g 250 g 29.4 502 0.78 600 g 400 g 450 g 39.1 503 0.78 600 g 400 g 750 g 49.5 492 0.77 600 g 400 g 900 g 53.5 516 0.80 600 g 400 g 1100 g 57.9 545 0.85 600 g 400 g 1400 g 63.2 655 1.02 400 g 600 g 0 g 12.3 718 - 400 g 600 g 250 g 29.9 636 0.89 400 g 600 g 450 g 39.5 638 0.89 400 g 600 g 750 g 49.9 666 0.93 400 g 600 g 900 g 53.8 721 1.00 400 g 600 g 1100 g 58.2 802 1.12 400 g 600 g 1400 g 63.5 988 1.38 400 g 600 g 0 g 9.5 654 - 400 g 600 g 100 g 17.7 535 0.82 400 g 600 g 250 g 27.6 422 0.65 400 g 600 g 450 g 37.6 487 0.74 400 g 600 g 750 g 48.3 491 0.75 400 g 600 g 900 g 52.4 512 0.78 400 g 600 g 1100 g 56.9 585 0.89 400 g 600 g 1400 g 62.3 612 0.94 400 g 600 g 0 g 12.1 658 - 400 g 600 g 100 g 20.1 556 0.84 400 g 600 g 250 g 29.7 471 0.72 400 g 600 g 450 g 39.4 458 0.70 400 g 600 g 750 g 49.8 486 0.74 400 g 600 g 900 g 53.7 486 0.74 400 g 600 g 1100 g 58.1 531 0.81 400 g 600 g 1400 g 63.4 605 0.92 0 g 1000 g 0 g 12.9 616 - 0 g 1000 g 100 g 20.8 484 0.79 0 g 1000 g 250 g 30.3 438 0.71 0 g 1000 g 450 g 39.9 457 0.74 0 g 1000 g 750 g 50.2 507 0.82 0 g 1000 g 900 g 54.1 535 0.87 0 g 1000 g 1100 g 58.5 585 0.95 0 g 1000 g 1400 g 63.7 688 1.12

BIOCONVERSION ON A PILOT SCALE

Materials and methods

Materials

Biomasses : soybean meal (SBM), soy flakes, rapeseed meal (RSM) and sunflower meal (SSM).

Water : regular tap water.

Enzymes

Protease: papain from Enzybel; Ronozyme Pro Act from DSM; acid protease from Suntaq International Ltd;

α-galactosidase: from Bio-Cat (12500 U/g);

phytase: Natufos from BASF.

Other carbohydrases: Viscozyme L from Novozymes, Ronozyme VP from Novozymes.

Example 2

Bioconversion with SBN and RSM protease (50:50 ratio)

2.1. Incubator

The laboratory incubator used is an insulated cylindrical elongated stainless steel tube with a working volume of 2 m ³ and an inlet and outlet. In addition, the incubator is equipped with a temperature probe at the inlet as well as at the outlet.

2.2. Method

A laboratory vertical reactor with a total volume of 2.0 m ³ is continuously fed with 250 kg/h of soybean meal (SBM), 250 kg/h of rapeseed meal (RSM), 967 kg/h of water at 25°C, 0.5 kg /hour of protease (papain from Enzybel) and 0.5 kg/hour of protease (ronozyme ProAct from DSM). The wet bulk density/dry bulk density ratio of the incubation mixture is 1.10.

The output quantity is adjusted to maintain a constant reactor fill level, and the fill level is adjusted to obtain a total processing time of 1.0 hour. Immediately after leaving the vertical reactor, the product is subjected to heat treatment at 99°C for 15 minutes, followed by air drying.

2.3. Product Test Procedure

The enzyme-treated product and its untreated reference, a 50:50 mixture of SBM and RSM, were analyzed by SDS-PAGE for soluble peptides using the following method.

Suspend 5.0 g of the product in water, adjust the pH to 8.5, and make up with water to a total mass of 50.0 g. The suspension is heated at 90°C for 15 minutes, followed by centrifugation at 3000 RCF for 15 minutes. The supernatant is mixed 1+5 with Laemmli sample buffer+50 mM DTT (Laemmli, 1970) and heated at 90°C for 15 min. Load 15 µl of each sample onto a TGX Any kD gel and run the test following the manufacturer's instructions. The gel is stained with colloidal Coomassie (Kang et al., 2002).

Links

Kang, D., Gho, Y. S., Suh, M. and Kang, C., Bull. Korean Chem. Soc., 2002, Vol. 23, No. 11, pp. 1511-1512.

Laemmli UK, Nature, 1970, Vol. 227, pp. 680-685.

2.4. results

The SDS-PAGE results are shown in Figure 1, lanes 2 and 3.

It is clear from SDS-PAGE that most of the various protein bands in the crude SBM/RSM mixture (lane 3) are hydrolyzed to peptides by treatment with proteases in the vertical reactor used according to the invention (lane 2).

Example 3

Bioconversion with SBN and RSM protease (50:50 ratio)

3.1. Incubator

The laboratory incubator used is an insulated cylindrical elongated stainless steel tube with a working volume of 2 m ³ and an inlet and outlet. In addition, the incubator is equipped with a temperature probe at the inlet as well as at the outlet.

3.2. Method

A laboratory vertical reactor with a total volume of 2.0 m ³ is continuously fed with 15.6 kg/hour of soybean flour (SBM), 15.6 kg/hour of rapeseed meal, 24 kg/hour of water at 40°C, 0.03 kg /hour protease (acid protease from Suntaq International Ltd.) and 0.88 kg/hour H ₂ SO ₄ . The wet bulk density/dry bulk density ratio of the incubation mixture is 0.75.

The outlet quantity is adjusted to maintain a constant reactor fill level, and the fill level is adjusted to obtain a total processing time of 16 hours. Immediately after leaving the vertical reactor, the product is subjected to heat treatment at 99°C for 15 minutes, followed by air drying.

3.3. Product Test Procedure

The enzyme-treated product and its untreated reference, a 50:50 mixture of SBM and RSM, were analyzed by SDS-PAGE for soluble peptides using the following method.

Suspend 5.0 g of the product in water, adjust the pH to 8.5, and make up with water to a total mass of 50.0 g. The suspension is heated at 90°C for 15 minutes, followed by centrifugation at 3000 RCF for 15 minutes. The supernatant is mixed 1+5 with Laemmli sample buffer+50 mM DTT (Laemmli, 1970) and heated at 90°C for 15 min. Load 15 µl of each sample onto a TGX Any kD gel and run the test following the manufacturer's instructions. The gel is stained with colloidal Coomassie (Kang et al., 2002).

Links

Kang, D., Gho, Y. S., Suh, M. and Kang, C., Bull. Korean Chem. Soc., 2002, Vol. 23, No. 11, pp. 1511-1512.

Laemmli UK, Nature, 1970, Vol. 227, pp. 680-685.

3.4. results

SDS-PAGE results are shown in Figure 1, lanes 3 and 4.

It is clear from SDS-PAGE that most of the various protein bands in the untreated SBM/RSM mixture (lane 3) were hydrolyzed to peptides by 16 h of protease treatment in a vertical reactor (lane 4).

Example 4

Bioconversion with α-galactosidase of soy flakes

Biomass includes polysaccharides and proteins from beans.

4.1. Incubator

The incubator used is an insulated cylindrical elongated stainless steel tube with an internal diameter of 1.55 m and a total height of 4.75 m. At the top there is a set of three rotating blade type level monitors to regulate the intake and distribution system to a level of 4.25 m This gives the incubator an effective working volume of 8 m ³ . In addition, the incubator is equipped with a temperature probe at the inlet as well as at the outlet.

4.2. Method

A mixture of dehulled defatted and solvent-free soybean flakes, sulfuric acid, α-galactosidase and water at 60°C is prepared continuously in quantities to achieve a mixture dry matter content of 50 wt.%, pH 4.7 and α-galactosidase concentration 1.1 kg/ton soybeans. The wet bulk density/dry bulk density ratio of the incubation mixture is 0.73.

The incubator is filled with the incubation mixture to a suitable extent per hour. After 16 hours, the incubator is loaded to operating level and the outlet fixtures are set to speed to maintain the fill level constant.

An aliquot volume is approx. 30 liters are taken after 18 hours of testing and incubated at 100°C with fresh steam for 25 minutes.

The wet solid biomass transformation product is then spray dried under vacuum and ground.

General incubation parameters are as follows:

incubation time 16 hours,

inlet temperature 45°C,

outlet temperature 45°C.

4.3. results

The solid product of biomass transformation has a total crude protein content (N×6.25) of 52.6 wt.% and a water content of 5.6 wt.%, corresponding to 55.5% protein in dry matter. In addition, the content of stachyose and raffinose in the dry solid transformation product was significantly reduced, as shown in Table 1.

Table 1 Soy flakes Product Dry matter protein 56% +/- 1% 55.5% Stachyose+raffinose 5-6% None

The solid transformation product is highly nutritious and palatable and is therefore suitable as an ingredient in a range of food and feed products or dietary supplements. In addition, it can be used as an excipient in pharmaceutical products and cosmetic products such as bath products.

Example 5

Bioconversion with soy flake phytase

This biomass includes polysaccharides and proteins from beans.

5.1. Incubator

The incubator used is an insulated cylindrical elongated stainless steel tube with an internal diameter of 1.55 m and a total height of 4.75 m. At the top there is a set of three rotating blade type level monitors to regulate the intake and distribution system to a level of 4.25 m This gives the incubator an effective working volume of 8 m ³ . In addition, the incubator is equipped with a temperature probe at the inlet as well as at the outlet.

5.2. Method

A mixture of dehulled defatted and solvent-free soybean flakes, heat-stable phytase and water at 95°C is produced continuously in quantities to achieve a mixture dry matter content of 46 wt.% and a phytase concentration of 250 g/ton soybean.

The wet bulk density/dry bulk density ratio of the incubation mixture is 0.87.

The incubator is filled with incubation mixture at a rate of 750 liters per hour. After 12 hours, the incubator is loaded to operating level and the outlet fixtures are set to speed to maintain the fill level constant.

An aliquot volume is approx. 30 liters are taken after 14 hours of testing and incubated at 100°C with fresh steam for 25 minutes.

The wet solid biomass transformation product is then dried and ground.

General incubation parameters are as follows:

incubation time 12 hours,

inlet temperature 67°C,

outlet temperature 66°C.

5.3. results

The solid biomass transformation product has a total crude protein content (N×6.25) of 51.0 wt.% and a water content of 8.0 wt.%, corresponding to 55.4% protein in dry matter. In addition, the content of phosphorus-binding phytic acid (anti-nutrient) in the dry solid transformation product is significantly reduced, as shown in Table 2.

table 2 Soy flakes Product Dry matter protein 55% +/- 1% 55.4% Phosphorus binding phytic acid 0.42% 0.08%

The solid transformation product is highly nutritious and palatable and is therefore suitable as an ingredient in a range of food and feed products or dietary supplements. In addition, it can be used as an excipient in pharmaceutical products and cosmetic products such as bath products.

Example 6

Bioconversion with soybean meal carbohydrase (SBM)

This biomass includes polysaccharides and proteins from beans.

6.1. Incubator

The incubator is an industrial vertical reactor with a total volume of 96 m ³ .

6.2. Method

The inlet uses a constant amount of ingredients of 1800 kg/h soya flour, 2800 kg/h water at 60°C, 6.5 kg/h viscozyme L from Novozymes and 2.8 kg/h Depol 679 from Biocatalysts. The wet bulk density/dry bulk density ratio of the incubation mixture is 1.1.

The outlet quantity is adjusted to maintain the fill level constant, and the fill level is adjusted to obtain a total processing time of 16 hours. Immediately after leaving the vertical reactor, the product is heated at 99°C for 15 minutes, followed by air drying.

6.3. Product Test Procedure

The composition of soluble sugars and oligosaccharides was analyzed by thin layer chromatography, extracting an aqueous suspension of 10% DM for 30 min, followed by centrifugation for 10 min at 3000 g and applying the supernatant to TLC plates with silica gel 60 (Merck). The various components are quantified by comparison with standards of known concentration (Chaplan and Kennedy, 1986).

Links

Chaplan MF and Kennedy JF. Carbohydrate analysis - practical approach; I RL Press, Oxford, 1986.

6.4. results

The thin layer chromatography results are shown in Figure 2. It is apparent that carbohydrase treatment (lane 1) releases some soluble carbohydrates, as visualized by the additional spot between sucrose and raffinose on the thin layer chromatography, as well as the overall blurring of the band compared to the standard (lane 2).

Example 7

Bioconversion with carbohydrase of soybean meal (SBM), rapeseed meal (RSM) and wheat

This biomass includes polysaccharides and proteins from beans.

7.1. Incubator

The incubator is a laboratory plastic container.

7.2. Method

Mix 60 g soy flour, 45 g rapeseed meal, 45 g crushed wheat, 180 g water, 3.0 g H ₂ SO ₄ , 0.525 g viscozyme L from Novozymes, 0.225 g Depol 679 from Biocatalysts, 0.30 g BAN 480 L from Novozymes and 0.30 g AMG 300 L from Novozymes and left to incubate at 37°C for 16 hours. The wet bulk density/dry bulk density ratio of the incubation mixture is 0.90.

After incubation, the product is heated at 100°C for 15 minutes, followed by air drying and grinding.

7.3. Product Test Procedure

The enzyme-treated product and its untreated comparison sample - mixed 60 g soybean meal + 45 g rapeseed meal + 45 g cracked wheat - were analyzed for soluble/insoluble non-starch polysaccharides (NSP) using the method of Englyst et al., 1994. The carbohydrate content of the aqueous extract was analyzed by the phenol-sulfuric acid method, and the composition of soluble sugars and oligosaccharides was analyzed by thin layer chromatography, extracting the aqueous suspension with 10% DM for 30 min, followed by centrifugation for 10 min at 3000 g and applying the supernatant to TLC plates with silica gel 60 (Merck). The various components are quantified by comparison with standards of known concentration (Chaplan and Kennedy, 1986).

Links

Chaplan MF and Kennedy JF. Carbohydrate analysis - practical approach; I RL Press, Oxford, 1986.

Englyst HN, Quigley ME. and Hudson GJ; Analyst, 1994, Vol. 119, pp. 1497-1509.

7.4. results

The results for soluble and insoluble NSP, as well as the content of soluble carbohydrates are given in Table 3. The thin layer chromatography results are shown in Figure 3.

It is apparent that carbohydrase treatment releases some soluble carbohydrates, as visualized by a blurred spot on thin layer chromatography, and that, compared to the reference, carbohydrase treatment alters the amounts of soluble carbohydrates and NSPs present in the product.

Table 3. Soluble/insoluble NSP content as well as soluble carbohydrate content of the enzyme treated product and its untreated comparison sample, respectively The product's name Fraction Total g/100 g SD Example 7, product Soluble NSPs 4.0 0.1 Insoluble NSP 8.5 0.1 All NSPs 12.5 0.3 Soluble carbohydrates 28.7 4.8 Example 7, comparison sample Soluble NSPs 2.8 0.2 Insoluble NSP 12.0 0.2 All NSPs 14.8 0.5 Soluble carbohydrates 15.0 4.2

Example 8

Bioconversion with carbohydrase of soybean meal (SBM) and sunflower meal (RSM)

This biomass includes polysaccharides and proteins from beans.

8.1. Incubator

The incubator is a laboratory vertical reactor with a total volume of 2.0 m ³ .

8.2. Method

At the inlet, a constant amount of ingredients is used: 18.8 kg/h soybean flour, 12.5 kg/h sunflower meal, 30 kg/h water at 40°C, 0.03 kg/h carbohydrase (Ronozyme VP from Novozymes) and 0.88 kg/hour H ₂ SO ₄ . The wet bulk density/dry bulk density ratio of the incubation mixture is 0.80.

The outlet quantity is adjusted to maintain the reactor fill level constant, and the fill level is adjusted to obtain a total processing time of 16 hours. Immediately after leaving the vertical reactor, the product is heated at 99°C for 15 minutes, followed by air drying.

8.3. Product Test Procedure

The enzyme-treated product and its untreated comparison sample - 90 g soybean meal + 60 g sunflower meal - were analyzed for soluble/insoluble non-starch polysaccharides (NSP) using the method of Englyst et al., 1994.

References : Englyst HN, Quigley ME. and Hudson GJ; Analyst, 1994, Vol. 119, pp. 1497-1509.

8.4. results

The results of the NSP analysis are shown in Table 4. It is clear that the treatment changes the amounts of NSP fractions.

Table 4. Soluble/insoluble NSP content as well as soluble carbohydrate content of the enzyme treated product and its untreated comparison sample, respectively The product's name Fraction Total g/100 g SD Example 8, product Soluble NSPs 3.8 1.4 Insoluble NSP 12.6 1.4 All NSPs 16.3 0.2 Example 8, comparison sample Soluble NSPs 3.4 1.0 Insoluble NSP 14.8 1.0 All NSPs 18.2 0.1

Example 9

Bioconversion on a large scale

Incubator

The reactor used is a vertical cylinder with an effective height of 7.3 m and a diameter of 4.3 m.

At the top of the vertical reactor, the incoming mixture is loaded almost into the center of the reactor. To ensure uniform distribution, the mixture entering the inlet is distributed around the perimeter of the reactor using a scraper knife or a planer rod.

The product is recovered at the bottom of the reactor, achieving a constant residence time for any particle scatter at the top of the reactor.

Checking the uniformity of slug flow

The reactor inlets and outlets are adjusted to achieve an expected residence time of 12 hours. To ensure constant distribution time, an inert indicator substance is added to the feed mixture. The feed mixture used in the experiment has a natural iron content of about 143 mg/kg dry matter (= initial concentration); Therefore, iron sulfate (FeSO ₄ ) is used as an indicator at a concentration of 1167 mg FeSO ₄ /kg of dry matter of the loaded mixture, which is equal to a total iron content of 572 mg Fe/kg of total dry matter. At time 0 hours, FeSO ₄ is added to the feed mixture fed into the reactor for 60 minutes. Samples are taken every 20 minutes, dried and analyzed for iron content and a FeSO ₄ -enriched product is found to exit the reactor 12-13 hours after FeSO ₄ is added to the inlet mixture and a maximum Fe concentration of 355 mg/kg is found 12.5 hours after the start of the experiment.

Claims (30)

1. A method for producing a solid product selected from a solid feed ingredient, or a solid food ingredient, or a solid food additive, where the method includes the following steps: - obtaining a biomass substrate comprising carbohydrates and proteinaceous matter originating from soybean seeds, rapeseeds or mixtures thereof, wherein at least 20 wt.% of said biomass includes carbohydrates and proteinaceous matter originating from soybean seeds, rapeseed seeds or mixtures thereof ; - mixing said substrate with an enzyme preparation or combination of enzyme preparations and adding water in an amount that provides an initial incubation mixture having a water content of from 30 wt.% to 70 wt.% and a ratio of wet bulk density to dry bulk density of from 0.60 up to 1.45; - incubating said initial incubation mixture for 1-48 hours at a temperature of 20-70°C, and - extracting from the incubated mixture the specified solid feed ingredient, or solid food ingredient, or solid food additive obtained as a result of incubation of a protein substance and/or carbohydrates originating from the specified biomass, with the specified enzyme preparation or combination of enzyme preparations; wherein the incubation stage is performed as a continuous process in a uniform a plug flow in a vertical reactor for incubation without stirring, with means for inlet of said mixture and additives and means for outlet of said solid transformation product, and where the movement of biomass is mediated by gravitational force. 2. The method according to claim 1, wherein said biomass is further mixed with carbohydrates and protein matter originating from fava bean seeds, pea seeds, sunflower seeds, lupine seeds and/or cereal seeds. 3. The method according to claim 1 or 2, further comprising pre-treating said biomass substrate prior to its mixing with said enzyme preparation or said combination of enzyme preparations, such as chemical or physical pre-treatment, for example by crushing, grinding, flattening, heat treatment, pressure treatment, ultrasonic treatment, hydrothermal treatment or acid or alkali treatment. 4. Method according to any one of paragraphs. 1-3, wherein at least 30 wt.% of said biomass, for example at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least at least 80% by weight or at least 90% by weight of said biomass comprises carbohydrates and protein matter derived from optionally defatted and/or optionally dehulled soybeans, optionally defatted rapeseed or mixtures thereof. 5. The method according to claim 4, where the specified biomass includes carbohydrates and protein matter originating from defatted and/or hulled soybeans, defatted rapeseeds or mixtures thereof. 6. The method according to any of the preceding claims, wherein said biomass includes oligosaccharides and/or polysaccharides. 7. The method according to claim 6, where said biomass additionally includes oils and fats, for example, from the seeds of oilseeds. 8. A method according to any one of the preceding claims, wherein said biomass substrate or said initial incubation mixture does not include any live yeast. 9. The method according to any of the preceding claims, wherein the solid feed ingredient, or solid food ingredient, or solid food additive is obtained by incubating a protein substance and/or carbohydrates derived from soybean seeds, rapeseeds or mixtures thereof, in a further mixture with material originating from beans and/or cereals and/or oilseeds. 10. The method according to any of the preceding claims, wherein the solid feed ingredient, or solid food ingredient, or solid food additive is obtained by incubating a protein substance and carbohydrates originating from soybean seeds, rapeseeds or mixtures thereof, in a further mixture with the material , originating from peas, lupine, sunflower, wheat, corn or rapeseed. 11. A method according to any one of the preceding claims, wherein said enzyme preparation or combination of enzyme preparations comprises one or more enzymes selected from proteases, peptidases, phytases, carbohydrates, lipases and oxidoreductases. 12. The method according to any of the preceding paragraphs, wherein said enzyme preparation or combination of enzyme preparations comprises one or more carbohydrases selected from α-galactosidase, amylase, amyloglucosidase, pectinase, cellulase and hemicellulase, for example xylanase, mannase or glucanase. 13. The method according to any of the preceding claims, wherein the dry matter ratio of said biomass substrate to said enzyme preparation or said combination of enzyme preparations is from 2:1 to 100000000:1, such as 1000:1, 10000:1, 50000:1, 100000 :1. 500000:1, 1000000:1, 5000000:1, 10000000:1, 50000000:1 or 100000000:1. 14. A method as claimed in any one of the preceding claims, wherein said initial incubation mixture is incubated for 1-36 hours, 2-32 hours, 3-28 hours, 5-24 hours, 8-20 hours or 12-16 hours. 15. The method of any one of the preceding claims, wherein water is added to said biomass substrate in an amount that provides an initial incubation mixture having a wet bulk density to dry bulk density ratio of from 0.65 to 1.40, such as 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25, 1.30 or 1.35. 16. A method according to any one of the preceding claims, wherein the water content of said initial incubation mixture is from 35% by weight to 70% by weight, such as 40%, 45%, 50%, 55%, 60% or 65%. 17. A method according to any one of the preceding claims, wherein one or more processing auxiliaries selected from plant components and organic or inorganic processing agents are added to said biomass substrate and/or to said initial incubation mixture. 18. The method according to claim 17, wherein α-galactosidase is added to said biomass substrate and/or to said initial incubation mixture. 19. The method according to any of the preceding claims, wherein the α-galactosidase preparation is added to the biomass substrate and/or to the initial incubation mixture in an amount of from 0.05 to 50 units of α-galactosidase per g of dry matter of the biomass substrate, such as from 0. 5 to 25 units of α-galactosidase per g dry matter of biomass substrate, for example 1 to 10, 2 to 8, 3 to 6 or 4 to 5 units of α-galactosidase per g dry matter of biomass substrate. 20. The method according to any one of the preceding claims, further including that the vertical reactor for incubation without stirring is closed. 21. A method according to any one of the preceding claims, wherein said reactor for incubation without stirring is vertical, elongated cylindrical or polyhedron type. 22. A method according to any of the preceding claims, wherein the area of the upper part of said incubation reactor without stirring is less than the area of the lower part, i.e. the reactor has the shape of a cone. 23. A method according to any one of the preceding claims, wherein said unstirred incubation reactor has insulation with mats or a cooling jacket and means for regulating the temperature in the reactor. 24. A method according to any one of the preceding claims, wherein the fill level of said incubation reactor is maintained constant.

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