VERTICAL PLUG-FLOW PROCESS FOR BIO-CONVERSION OF BIOMASS INVOLVING ENZYMES

Abstract

The invention relates to a method for producing a solid transformation product of a substrate comprising the following steps: ⋅preparing a substrate of biomass comprising carbohydrates and proteinaceous matter that originates from soya bean, rape seed, or mixtures thereof, optionally in further 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 a combination of enzyme preparations and adding water in an amount which provides an initial incubation mixture having a water content from 30 to 70% by weight, and a ratio of wet bulk density to dry bulk density from 0.60 to 1.45 in the resulting mixture; ⋅incubating said initial incubation mixture for 0.15-72 hours at a temperature of 20-70° C.; and thereafter recovering wet solid transformation product from the incubated mixture; further comprising that the incubating step is performed as a continuous plug-flow process in a vertical, non-agitated incubation tank with inlet means for said mixture and additives and outlet means for said solid transformation product.

Claims

1-25. (canceled)

26. A method for producing a solid transformation product of a biomass substrate, wherein the solid transformation product is a product of the transformation of one or more of proteinaceous matter and carbohydrates originating from a biomass substrate, comprising: (a) preparing a substrate of a biomass comprising carbohydrates and proteinaceous matter that originate from soya bean seed, rape seed, or mixtures thereof, wherein at least 20% by weight of said biomass comprises carbohydrates and proteinaceous matter originating from soya bean seeds, rape seeds, or mixtures thereof, optionally in further mixture with carbohydrates and proteinaceous matter originating from one or more of seeds of fava beans, seeds of peas, sunflower seeds, seeds of lupine, cereals, and grasses; (b) mixing said substrate with an enzyme preparation or a combination of enzyme preparations and adding water in an amount which provides an initial incubation mixture having a water content from 30% to 70% by weight, and a ratio of wet bulk density to dry bulk density from 0.60 to 1.45; (c) incubating said initial incubation mixture for 0.15-240 hours at a temperature of 20-70° C.; and (d) recovering solid transformation product from the incubated mixture; wherein the incubating step is performed as a continuous plug-flow process in a vertical, non-agitated incubation tank with an inlet for said mixture and additives and an outlet for said solid transformation product.

27. A method according to claim 26, further comprising pre-treating said substrate before mixing with said enzyme preparation or said combination of enzyme preparations by one or more selected from disintegration, milling, flaking, heat treatment, pressure treatment, ultrasonic treatment, hydrothermal treatment, or acid or alkaline treatment.

28. A method according to claim 26, wherein at least 30% by weight of said biomass comprises carbohydrates and proteinaceous matter originating from one or more of optionally defatted and optionally dehulled soya bean seeds, optionally defatted rape seeds, and mixtures thereof.

29. A method according to claim 28, wherein said weight of said biomass comprising carbohydrates and proteinaceous matter originating from one or more of optionally defatted and optionally dehulled soya bean seeds, optionally defatted rape seeds, and mixtures thereof is selected from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% by weight of said biomass.

30. A method according to claim 26, wherein said biomass comprises one or more of oligosaccharides and polysaccharides, and optionally further comprises oils and fats.

31. A method according to claim 26, wherein said substrate and said initial incubation mixture do not comprise any live yeast.

32. A method according to claim 26, wherein said substrate and said initial incubation mixture do not comprise any live yeast of Saccharomyces cerevisiae strains.

33. A method according to claim 26, wherein said solid transformation product is a product of the transformation of proteinaceous matter, or of the transformation of carbohydrates, or of the transformation of proteinaceous matter and carbohydrates originating from seeds of soya, pea, lupine, or sunflower, or from wheat, maize, or rape seed.

34. A method according to claim 26, wherein said enzyme preparation or combination of enzyme preparations comprises one or more enzymes selected from proteases, peptidases, phytases, carbohydrases, lipase, and oxidoreductase.

35. A method according to claim 26, wherein said enzyme preparation or combination of enzyme preparations comprises one or more carbohydrases selected from α-galactosidase, amylase, amyloglucosidase, pectinase, cellulase, and hemi-cellulases.

36. A method according to claim 26, wherein the dry matter ratio of said substrate to said enzyme preparation or said combination of enzyme preparations is from 2:1 to 100,000,000:1.

37. A method according to claim 26, wherein said initial incubation mixture is incubated for 1 to 180 hours.

38. A method according to claim 26, wherein water is added to said substrate in an amount which provides an initial incubation mixture having a ratio of wet bulk density to dry bulk density from 0.65 to 1.40.

39. A method according to claim 26, wherein water is added to said substrate of biomass in an amount which provides an initial incubation mixture having a ratio of wet bulk density to dry bulk density selected from 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25, 1.30, and 1.35.

40. A method according to claim 26, wherein the water content in said initial incubation mixture is from 35% to 70% by weight.

41. A method according to claim 26, wherein the water content in said initial incubation mixture is selected from 40%, 45%, 50%, 55%, 60%, and 65%.

42. A method according to claim 26, further comprising adding one or more processing aids selected from plant components and organic and inorganic processing agents to one or more of said substrate and said initial incubation mixture.

43. A method according to claim 42, wherein α-galactosidase is added to one or more of said substrate and said initial incubation mixture.

44. A method according to claim 26, comprising adding an α-galactosidase preparation to one or more of the substrate and the initial incubation mixture in an amount of from 0.05 to 50 α-galactosidase units per g dry matter of the substrate.

45. A method according to claim 26, wherein the vertical, non-agitated incubation tank is closed.

46. A method according to claim 26, wherein the non-agitated incubation tank is of a vertical, oblong cylindrical or polyhedral type.

47. A method according to claim 26, wherein the area in the upper part of said non-agitated incubation tank is less than the area in the lower part.

48. A method according to claim 26, where said non-agitated incubation tank has insulating matting or a thermal dimple jacket.

49. A method according to claim 26, wherein the filling degree of said incubation tank is kept constant.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 shows: in lane 1: Marker; Lane 2: Protease treated sample from example 2; Lane 3: Reference sample for both examples 2 and 3; Lane 4: Protease treated sample from example 3.

[0074] FIG. 2 shows composition of soluble sugars and oligosaccharides. Lane 1: Carbohydrase-treated sample; Lane 2: Reference (soya bean meal).

[0075] FIG. 3 shows thin layer chromatography showing soluble sugars and oligosaccharides. Lane 1: carbohydrase-treated product; lane 2: reference.

DETAILED DESCRIPTION OF THE INVENTION

[0076] In a first embodiment of the method of the invention at least 20% by weight of the biomass, such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by weight, comprises proteinaceous matter originating from optionally defatted soya. The soya may also be dehulled.

[0077] In a second embodiment of the method of the invention at least 20% by weight of the biomass, such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by weight, comprises proteinaceous matter originating from optionally defatted rape seeds.

[0078] In a third embodiment of the method of the invention the biomass comprises proteinaceous matter originating from defatted soya flakes in an amount of from 5% to 95% by weight in mixture with proteinaceous matter originating from optionally defatted rape seed in an amount of from 95% to 5% by weight optionally in further mixture with proteinaceous matter originating from fava beans, peas, sunflower seeds and/or cereals in amounts to make up a total amount of the proteinaceous matter of 100% by weight.

[0079] In any of the embodiments of the invention the biomass comprising proteinaceous matter may further comprise oligosaccharides, and/or polysaccharides, and/or further comprises oils and fats, e.g. from seeds of oil bearing plants.

[0080] In any of the embodiments of the invention the solid transformation product of the substrate may be a product of the transformation of proteinaceous matter and/or carbohydrates originating from said biomass, such as a transformation product of pulses, such as soya, pea, lupine, sunflower, and/or cereals, such as wheat, or maize, or from seeds of oil bearing plants, e.g. rape seed.

[0081] In any of the embodiments of the invention the substrate, or the substrate after mixing with an enzyme preparation or a combination of enzyme preparations and water, may not comprise any live baker's yeast, and/or it may not comprise any live yeast selected among Saccharomyces cerevisiae strains, including spent brewer's yeast and spent distiller's yeast and baker's yeast and spent yeast from wine production, or does not comprise any live yeast; and/or in particular it may not comprise baker's yeast when the enzyme is α-galactosidase.

[0082] In any of the embodiments of the invention the solid transformation product of the substrate may be a product of the transformation of proteinaceous matter and/or carbohydrates originating from said biomass.

[0083] In any of the embodiments of the invention the enzyme preparation or mixture of enzyme preparations comprises one or more enzymes selected from proteases, peptidases, phytases, carbohydrases, such as α-galactosidase, amylase, amyloglucosidase, cellulase, pectinase, and hemi-cellulases, e.g. xylanase, mannanase, or glucanase; and lipase, and oxidoreductase.

[0084] In any of the embodiments of the invention the dry matter ratio of said substrate of biomass to said enzyme preparation, or said combination of enzyme preparations, may be from 2:1 to 100,000,000:1, such as 1,000:1, 10,000:1, 50,000:1, 100,000:1. 500,000:1, 1,000,000:1, 5,000,000:1, 10,000,000:1, 50,000,000:1, or 100,000,000:1. The skilled person will appreciate that the ratio can be selected in dependence of parameters, such as process conditions, the activity of the enzyme, and the desired product, and the skilled person will be able to optimise the ratio in accordance with these parameters.

[0085] In any of the embodiments of the invention water may be added to the substrate in an amount to provide a ratio of wet bulk density to dry bulk density from about 0.60 to 1.45 in the substrate, such as from about 0.65 to about 1.40, e.g. 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.

[0086] In any of the embodiments of the invention at least 40% by weight of the biomass, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight, may comprise proteinaceous matter originating from optionally defatted rape seeds, whereas water may be added to the substrate in an amount to provide a ratio of wet bulk density to dry bulk density 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.

[0087] In any of the embodiments of the invention one or more processing aids selected from enzymes, plant components, and organic and inorganic processing agents may be added to the substrate before or during incubation.

[0088] In any of the embodiments of the invention an α-galactosidase preparation may be added to the substrate of biomass and/or to the initial incubation mixture in an amount of from 0.05 to 50 α-galactosidase units pr. g. dry matter of substrate of biomass, such as from 0.5 to 25 α-galactosidase units pr. g. dry matter of substrate of biomass, e.g. from 1 to 10, from 2 to 8, from 3 to 6, or from 4 to 5 α-galactosidase units pr. g. dry matter of substrate of biomass.

[0089] In any of the embodiments of the invention the filling degree of said incubation tank may be kept constant. This will result in a uniform flow.

[0090] In any of the embodiments of the invention the incubation can be carried out under anaerobic conditions.

[0091] In any of the embodiments of the invention the water content in the incubation mixture may be from 35% to 70% by weight, such as 40%, 45%, 50%, 55%, 60%, or 65% by weight. Thus, the water content in the initial mixture does not exceed 70% by weight and it may vary from e.g. from 40% to 65%, from 45% to 60%, from 48% to 52%, or 50% to 55%, such as 49, 50, 51, 52, 53, or 54%.

[0092] In any of the embodiments of the invention the mixture may be incubated for 1-240 hours at 20-70° C. The skilled person will know how to optimise the reaction time and the reaction temperature in view of the other reaction conditions, such as the selection of enzyme(s). Thus, the temperature may vary as e.g. 20-65° C., 25-60° C., 30-55° C., 35-50° C., or 40-45° C.; and the reaction time may be selected as e.g. 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 each and every one of the here mentioned temperature intervals.

[0093] In any of the embodiments of the invention the solid transformation product of the substrate may by dried, optionally followed by milling.

[0094] In any of the embodiments of the invention the substrate mixture may be incubated at a time and a temperature sufficient to inactivate the enzyme(s), any anti-nutritional factors, and any processing aids, and if used partly or totally, and if desired.

[0095] In any of the embodiments of the invention the vertical, non-agitated incubation tank may be closed.

[0096] In any of the embodiments of the invention the non-agitated incubation tank can be of a vertical, oblong cylindrical or polyhedral type. The advantage of using this type is that it is space-saving and as it is non-agitated the operating costs and maintenance costs for mixing equipment are avoided.

[0097] In any of the embodiments of the invention the area in the upper part of said non-agitated incubation tank may be less than the area in the lower part i.e. the tank is of conical shape. The advantage of this is that the slip effect is increased so that biomasses with a reduced flowability can be used.

[0098] In any of the embodiments of the invention the non-agitated incubation tank may have insulating matting or a thermal dimple jacket and means to control the temperature inside the incubation tank.

[0099] The solid transformation product of the substrate of the invention may be dried to a water content of not more than 15%, 13%, 10%, 6%, 4%, or 2% by weight and optionally be in milled form.

[0100] The solid product of the invention can be a product of the transformation of proteinaceous matter, oligosaccharides, and/or polysaccharides originating from said biomass. The solid transformation product may have reduced content of anti-nutritional factors, such as trypsin inhibitors, antigens, flatulence-producing oligosaccharides, e.g. stachyose and raffinose; phytic acid, and lectin.

[0101] The solid product of the invention may comprise at least 40% proteinaceous matter by weight of dry matter originating from soya.

[0102] The solid product of the invention may comprise at least 40% proteinaceous matter by weight of dry matter originating from rape seed.

[0103] The solid product of the invention may comprise proteins in an amount of 30-65% by weight on dry matter basis originating from plant parts of soya, rape seed, or sun flower, or mixtures thereof.

[0104] The solid product of the invention may comprise a total amount of raffinose, stachyose, and verbascose of 3% by weight or less, such as 2% or less, 1% or less, 0.5% or less, or 0.4% or less.

[0105] Finally, the invention provides a food, feed, cosmetic or pharmaceutical product or a nutritional supplement containing from 1% to 99% by weight of a solid transformation product provided according to the invention.

EXAMPLES

[0106] Density Ratio

Example 1

[0107] Ratio of Wet Bulk Density/Dry Bulk Density for Preferred Substrates Based on Various Biomasses

[0108] 1.1 Biomasses Used in the Procedure:

[0109] Soya

[0110] The soya used was defatted Soya Bean Meal (SBM).

[0111] Maize

[0112] The maize used was whole maize, ground on a hammer mill through a 3.5 mm sieve.

[0113] Wheat

[0114] The wheat used was whole wheat, ground on a hammer mill through a 3.5 mm sieve.

[0115] Sunflower

[0116] The sunflower used was defatted Sunflower Seed Meal (SSM).

[0117] Rapeseed

[0118] The rapeseed used was defatted Rape Seed Meal (RSM).

[0119] Fava Beans

[0120] The beans used were whole fava beans.

[0121] Pea Protein

[0122] The pea protein used was a pea protein concentrate.

[0123] 1.2 Description of the Procedure:

[0124] The amount(s) of biomass and water tabulated in the following was mixed for ten minutes followed by fifty minutes of equilibration in a closed container.

[0125] After this the material was poured into a measuring cup of 500 mL and its mass determined by weighing the cup and subtracting the tare of the cup.

[0126] The bulk density was calculated as mass/untapped volume in kg/m.sup.3.

[0127] The dry bulk density used was the measured bulk density of the biomass without addition of water.

[0128] The wet bulk density was the bulk density of the biomass with added water.

[0129] The ratio was calculated as wet bulk density divided by the dry bulk density.

[0130] The moisture content of the biomasses was determined by drying to constant weight.

[0131] After addition of water the moisture in the mixture was determined by calculation.

[0132] 1.3 Results:

[0133] The results for 100% soya and 80% mixtures with soya are tabulated in the following:

TABLE-US-00001 Bulk Fava Moisture Density Soya Maize Wheat Sunflower Rapeseed bean Pea Water In % kg/m.sup.3 Ratio 1000 g 0 g 10.9 665 — 1000 g 100 g 19.0 638 0.96 1000 g 250 g 28.7 500 0.75 1000 g 450 g 38.6 476 0.72 1000 g 750 g 49.1 470 0.71 1000 g 900 g 53.1 572 0.86 1000 g 1100 g 57.6 655 0.98 1000 g 1400 g 62.9 715 1.07 1000 g 1900 g 69.3 889 1.34 800 g 200 g 0 g 11.4 703 — 800 g 200 g 450 g 38.9 617 0.88 800 g 200 g 900 g 53.4 634 0.90 800 g 200 g 1900 g 69.4 1008 1.43 800 g 200 g 0 g 11.7 694 — 800 g 200 g 450 g 39.1 580 0.84 800 g 200 g 900 g 53.5 623 0.90 800 g 200 g 1900 g 69.5 960 1.38 800 g 200 g 0 g 10.4 683 — 800 g 200 g 450 g 38.2 554 0.81 800 g 200 g 900 g 52.9 598 0.88 800 g 200 g 1900 g 69.1 926 1.36 800 g 200 g 0 g 11.3 711 — 800 g 200 g 100 g 19.4 576 0.81 800 g 200 g 250 g 29.0 514 0.72 800 g 200 g 450 g 38.8 483 0.68 800 g 200 g 750 g 49.3 490 0.69 800 g 200 g 900 g 53.3 597 0.84 800 g 200 g 1100 g 57.8 528 0.74 800 g 200 g 1900 g 69.4 908 1.28 800 g 200 g 0 g 11.1 691 — 800 g 200 g 450 g 38.7 569 0.82 800 g 200 g 900 g 53.2 605 0.88 800 g 200 g 1900 g 69.3 941 1.36 800 g 200 g 0 g 11.2 703 — 800 g 200 g 450 g 38.7 488 0.69 800 g 200 g 900 g 53.2 728 1.04 800 g 200 g 1900 g 69.4 964 1.37

[0134] The results for 60% and 40% of soya mixtures with maize, sunflower and rapeseed as well as 100% rapeseed are tabulated in the following:

TABLE-US-00002 Bulk Moisture Density Soya Maize Sunflower Rapeseed Water In % kg/m.sup.3 Ratio 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

[0135] Pilot-Scale Bio-Conversion

[0136] Materials and Methods:

[0137] Materials

[0138] Biomasses:

[0139] Soya Bean Meal (SBM), Soya Flakes, Rape Seed Meal (RSM) and Sunflower Seed Meal (SSM).

[0140] Water:

[0141] Normal tap water

[0142] Enzymes:

[0143] Protease: papain from Enzybel; Ronozyme Pro Act from DSM; Acid protease from Suntaq International Ltd;

[0144] α-galactosidase: from Bio-Cat (12,500 U/g);

[0145] Phytase: Natuphos from BASF

[0146] Other carbohydrases: Viscozyme L from Novozymes, Ronozyme VP from Novozymes

Example 2

[0147] Bio-Conversion with Protease on SBM and RSM (50:50 Ratio)

[0148] 2.1 Incubator:

[0149] The pilot incubator used was an insulated, cylindrical oblong stainless-steel tube with an effective operating volume of 2 m.sup.3 and in- and outlets. Furthermore, the incubator was equipped with a temperature probe at the inlet as well as at the outlet.

[0150] 2.2 Method:

[0151] In a pilot scale vertical reactor with a total volume of 2.0 m.sup.2, a continuous inlet amount of 250 kg/h soya bean meal (SBM), 250 kg/h rapeseed meal (RSM), 967 kg/h water at 25° C., 0.5 kg/h protease (papain from Enzybel), and 0.5 kg/h protease (Ronozyme ProAct from DSM) was applied. The ratio wet bulk density/dry bulk density of the incubation mixture was 1.10.

[0152] The outlet amount was adjusted to keep a constant level of filling inside the reactor, and the level of filling was set to yield a total processing time of 1.0 hour. Immediately after leaving the vertical reactor, the product was heat treated at 99° C. for 15 min followed by air drying.

[0153] 2.3 Test Procedure for Product:

[0154] The enzyme-treated product and its untreated reference, a 50:50 mixture of SBM and RSM, were analysed for soluble peptides by SDS-PAGE using the following method:

[0155] 5.0 g of product was suspended in water, pH adjusted to 8.5, and adjusted with water to a total weight of 50.0 g. The suspension was heated to 90° C. for 15 min followed by centrifugation at 3000 RCF for 15 min. The supernatant was mixed 1+5 with Laemmli sample buffer+50 mM DTT (Laemmli, 1970) and heated to 90° C. for 15 min. 15 μL of each sample was loaded onto a TGX Any kD gel and run following the manufacturer's instructions. The gel was stained with colloid coomassie (Kang et al., 2002).

REFERENCES

[0156] Kang D, Gho Y S, Suh M, and Kang C, Bull. Korean Chem. Soc., 2002, Vol. 23, No. 11, pp. 1511-1512. [0157] Laemmli U K, Nature, 1970, Vol. 227, pp. 680-685.

[0158] 2.4 Results:

[0159] Results of the SDS-PAGE are shown in FIG. 1, lanes 2 and 3.

[0160] It is clear from the SDS-PAGE that most of the distinct protein bands in the untreated SBM/RSM mixture (lane 3) have been hydrolysed to peptides by the protease treatment in the vertical reactor used according to the invention (lane 2).

Example 3

[0161] Bio-Conversion with Protease on SBM and RSM (50:50 Ratio)

[0162] 3.1 Incubator:

[0163] The pilot incubator used was an insulated, cylindrical oblong stainless-steel tube with an effective operating volume of 2 m.sup.3 and in- and outlets. Furthermore, the incubator was equipped with a temperature probe at the inlet as well as at the outlet.

[0164] 3.2 Method:

[0165] In a pilot scale vertical reactor with a total volume of 2.0 m.sup.2, a continuous inlet amount of 15.6 kg/h soya bean meal (SBM), 15.6 kg/h rapeseed meal (RSM), 24 kg/h water at 40° C., 0.03 kg/h protease (Acid protease from Suntaq International Ltd.), and 0.88 kg/h H.sub.2SO.sub.4 was applied. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.75.

[0166] The outlet amount was adjusted to keep a constant level of filling inside the reactor, and the level of filling was set to yield a total processing time of 16 hour. Immediately after leaving the vertical reactor, the product was heat treated at 99° C. for 15 min followed by air drying.

[0167] 3.3 Test Procedure for Product:

[0168] The enzyme-treated product and its untreated reference, a 50:50 mixture of SBM and RSM, were analysed for soluble peptides by SDS-PAGE using the following method:

[0169] 5.0 g of product was suspended in water, pH adjusted to 8.5, and adjusted with water to a total weight of 50.0 g. The suspension was heated to 90° C. for 15 min followed by centrifugation at 3000 RCF for 15 min. The supernatant was mixed 1+5 with Laemmli sample buffer+50 mM DTT (Laemmli, 1970) and heated to 90° C. for 15 min. 15 μL of each sample was loaded onto a TGX Any kD gel and run following the manufacturer's instructions. The gel was stained with colloid coomassie (Kang et al., 2002).

REFERENCES

[0170] Kang D, Gho Y S, Suh M, and Kang C, Bull. Korean Chem. Soc., 2002, Vol. 23, No. 11, pp. 1511-1512. [0171] Laemmli U K, Nature, 1970, Vol. 227, pp. 680-685.

[0172] 3.4 Results:

[0173] Results of the SDS-PAGE are shown in FIG. 1, lanes 3 and 4.

[0174] It is clear from the SDS-PAGE that almost all of the distinct protein bands in the untreated SBM/RSM mixture (lane 3) have been hydrolysed to peptides by the 16 hour protease treatment in the vertical reactor (lane 4).

Example 4

[0175] Bio-Conversion with α-Galactosidase on Soya Flakes

[0176] This biomass comprises polysaccharides and proteins from pulses

[0177] 4.1 Incubator

[0178] The incubator used was an insulated, cylindrical oblong stainless-steel tube with an internal diameter of 1.55 m and a total height of 4.75 m. In the upper part, there was an array of three rotating paddle type level monitors to regulate the inlet and distribution system to a level at 4.25 m. This gives the incubator an effective operating volume of 8 m.sup.3. Furthermore, the incubator was equipped with a temperature probe at the inlet as well as at the outlet.

[0179] 4.2 Method

[0180] A mixture of dehulled, defatted and desolventised soya flakes, sulfuric acid, α-galactosidase, and water at 60° C. was prepared continuously in amounts to reach a dry matter content of 50% by weight in the mixture, a pH of 4.7, and an alpha-galactosidase concentration of 1.1 kg/ton soya. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.73.

[0181] The incubator was filled with incubation mixture at a suitable rate per hour. After 16 hours the incubator was filled to operating level and the outlet means were set at a rate to keep the level of filling constant.

[0182] An aliquot volume of approx. 30 liters was taken after 18 hours of the test run and incubated at 100° C. with live steam for 25 min.

[0183] Subsequently, the wet solid transformation product of the biomass was flash dried and milled.

[0184] The overall incubation parameters were the following:

[0185] Incubation time—16 hours

[0186] Temperature inlet—45° C.

[0187] Temperature outlet—45° C.

[0188] 4.3 Results:

[0189] The solid transformation product of the biomass had a total crude protein (N×6.25) content of 52.6% and a water content of 5.6% by weight, which corresponds to a protein of dry matter of 55.5%. Furthermore, stachyose and raffinose in the dried, solid transformation product were significantly reduced as shown in table 1:

TABLE-US-00003 TABLE 1 Soya flakes Product Protein of dry matter 56% +/− 1% 55.5% Stachyose + raffinose 5-6% Absent

[0190] The solid transformation product is highly nutritious and palatable and thus suitable as an ingredient in a number of food and feed products or nutritional supplements. Furthermore, it can be used as an excipient in pharma products and in cosmetics e.g. bath formulations.

Example 5

[0191] Bio-Conversion with Phytase on Soya Flakes

[0192] This biomass comprises polysaccharides and proteins from pulses

[0193] 5.1 Incubator

[0194] The incubator used was an insulated, cylindrical oblong stainless-steel tube with an internal diameter of 1.55 m and a total height of 4.75 m. In the upper part, there was an array of three rotating paddle type level monitors to regulate the inlet and distribution system to a level at 4.25 m. This gives the incubator an effective operating volume of 8 m.sup.3. Furthermore, the incubator was equipped with a temperature probe at the inlet as well as at the outlet.

[0195] 5.2 Method

[0196] A mixture of dehulled, defatted and desolventised soya flakes, thermostable phytase, and water at 95° C. was prepared continuously in amounts to reach a dry matter content of 46% by weight in the mixture, and a phytase concentration of 250 g/ton soya.

[0197] The ratio wet bulk density/dry bulk density of the incubation mixture was 0.87.

[0198] The incubator was filled with incubation mixture at the rate of 750 liters per hour. After 12 hours the incubator was filled to operating level and the outlet means were set at a rate to keep the level of filling constant.

[0199] An aliquot volume of approx. 30 liters was taken after 14 hours of the test run and incubated at 100° C. with live steam for 25 min.

[0200] Subsequently, the wet solid transformation product of the biomass was flash dried and milled.

[0201] The overall incubation parameters were the following:

[0202] Incubation time—12 hours

[0203] Temperature inlet—67° C.

[0204] Temperature outlet—66° C.

[0205] 5.3: Results

[0206] The solid transformation product of the biomass had a total crude protein (N×6.25) content of 51.0% and a water content of 8.0% by weight, which corresponds to a protein of dry matter of 55.4%. Furthermore, phytic acid bound phosphor (an anti-nutrient) in the dried, solid transformation product were significantly reduced as shown in table 2:

TABLE-US-00004 TABLE 2 Soya flakes Product Protein of dry matter 55% +/− 1% 55.4% Phytic acid bound phosphor 0.4% 0.08%

[0207] The solid transformation product is highly nutritious and palatable and thus suitable as an ingredient in a number of food and feed products or nutritional supplements. Furthermore, it can be used as an excipient in pharma products and in cosmetics e.g. bath formulations.

Example 6

[0208] Bio-Conversion with Carbohydrase on Soya Bean Meal (SBM)

[0209] This biomass comprises polysaccharides and proteins from pulses

[0210] 6.1 Incubator

[0211] The incubator was a production scale vertical reactor with a total volume of 96 m.sup.2.

[0212] 6.2 Method

[0213] A continuous inlet amount of 1800 kg/h soya bean meal, 2800 kg/h water at 60° C., 6.5 kg/h Viscozyme L from Novozymes, and 2.8 kg/h Depol 679 from Biocatalysts was applied. The ratio wet bulk density/dry bulk density of the incubation mixture was 1.1.

[0214] The outlet amount was adjusted to keep a constant level of filling inside the reactor, and the level of filling was set to yield a total processing time of 16 hours. Immediately after leaving the vertical reactor, the product was heat treated at 99° C. for 15 min followed by air drying.

[0215] 6.3 Test Procedure for Product

[0216] Composition of soluble sugars and oligosaccharides was analysed by thin layer chromatography by extracting a watery suspension slurry of 10% DM for 30 min followed by centrifugation for 10 min at 3,000×g and applying the supernatant onto TLC silica gel 60 plates (Merck). The different components were quantified by comparison to standards of known concentration (Chaplan and Kennedy, 1986).

REFERENCES

[0217] Chaplan M F and Kennedy J F. Carbohydrate analysis—practical approach; I RL Press, Oxford, 1986

[0218] 6.4 Results

[0219] Results of the thin layer chromatography are shown in FIG. 2. It is evident that the carbohydrase treatment (lane 1) liberates some soluble carbohydrates, visualised by the additional spot between sucrose and raffinose on the thin layer chromatography as well as a general smear throughout the lane compared to the reference (lane 2).

Example 7

[0220] Bio-Conversion with Carbohydrase on Soya Bean Meal (SBM), Rapeseed Meal (RSM) and Wheat

[0221] This biomass comprises polysaccharides and proteins from pulses.

[0222] 7.1 Incubator

[0223] The incubator was a laboratory scale plastic container.

[0224] 7.2 Method

[0225] An amount of 60 g soya bean meal, 45 g rapeseed meal, 45 g ground wheat, 180 g water, 3.0 g H.sub.2SO.sub.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 was mixed and left to incubate at 37° C. for 16 hours. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.90.

[0226] Following incubation, the product was heat treated at 100° C. for 15 min followed by air drying and milling.

[0227] 7.3 Test Procedure for Product

[0228] The enzyme-treated product and its untreated reference, 60 g soya bean meal+45 g rapeseed meal+45 g ground wheat, milled, were analysed for soluble/insoluble non-starch polysaccharides (NSP) using the method of Englyst et al. 1994. Content of carbohydrate content in watery extract was analysed by the phenol sulphuric acid method and composition of soluble sugars and oligosaccharides was analysed by thin layer chromatography by extracting a watery suspension slurry of 10% DM for 30 min followed by centrifugation for 10 min at 3,000×g and applying the supernatant onto TLC silica gel 60 plates (Merck). The different components were quantified by comparison to standards of known concentration (Chaplan and Kennedy, 1986).

REFERENCES

[0229] Chaplan M F and Kennedy J F. Carbohydrate analysis—practical approach; I RL Press, Oxford, 1986 [0230] Englyst H N, Quigley M E, and Hudson G J; Analyst, 1994, Vol 119, pp. 1497-1509.

[0231] 7.4 Results

[0232] Results of the soluble and insoluble NSP as well as the contents of soluble carbohydrates are shown in table 3. Results of the thin layer chromatography are shown in FIG. 3.

[0233] It is evident that the carbohydrase treatment liberates some soluble carbohydrates, visualised by a smear on the thin layer chromatography, and that the carbohydrase treatment changes the amounts of soluble carbohydrates and NSP present in the product compared to the reference.

TABLE-US-00005 TABLE 3 Contents of soluble/insoluble NSP as well as contents of soluble carbohydrates of enzyme-treated product and its untreated reference, respectively. Total Product Name Fraction g/100 g SD Example 7, product Soluble NSP 4.0 0.1 Insoluble NSP 8.5 0.1 Total NSP 12.5 0.3 Soluble carbohydrates 28.7 4.8 Example 7, reference Soluble NSP 2.8 0.2 Insoluble NSP 12.0 0.2 Total NSP 14.8 0.5 Soluble carbohydrates 15.0 4.2

Example 8

[0234] Bio-Conversion with Carbohydrase on Soya Bean Meal (SBM) and Sunflower

[0235] This biomass comprises polysaccharides and proteins from pulses

[0236] 8.1 Incubator

[0237] The incubator was a pilot scale vertical reactor with a total volume of 2.0 m.sup.2.

[0238] 8.2 Method

[0239] A continuous inlet amount of 18.8 kg/h soya bean meal, 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/h H.sub.2SO.sub.4 was applied. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.80.

[0240] The outlet amount was adjusted to keep a constant level of filling inside the reactor, and the level of filling was set to yield a total processing time of 16 hour. Immediately after leaving the vertical reactor, the product was heat treated at 99° C. for 15 min followed by air drying.

[0241] 8.3 Test Procedure for Product

[0242] The enzyme-treated product and its untreated reference, 90 g soya bean meal+60 g sunflower meal, were analysed for soluble/insoluble non-starch polysaccharides (NSP) using the method of Englyst et al. 1994.

REFERENCES

[0243] Englyst H N, Quigley M E, and Hudson G J; Analyst, 1994, Vol 119, pp. 1497-1509.

[0244] 8.4 Results

[0245] The results of the NSP analysis are shown in table 4. It is evident that the processing changes the amounts of the NSP fractions.

TABLE-US-00006 TABLE 4 Contents of soluble/insoluble NSP as well as contents of soluble carbohydrates of enzyme-treated product and its untreated reference, respectively. Total Product Name Fraction g/100 g SD Example 8, product Soluble NSP 3.8 1.4 Insoluble NSP 12.6 1.4 Total NSP 16.3 0.2 Example 8, reference Soluble NSP 3.4 1.0 Insoluble NSP 14.8 1.0 Total NSP 18.2 0.1

Example 9

[0246] Large Scale Bioconversion

[0247] Incubator:

[0248] The reactor used was a vertical cylinder with an effective height of 7.3 m and a diameter of 4.3 m.

[0249] In the top of the vertical reactor, the feed mixture falls on position near the centre of the reactor. For even distribution, a scraper blade or level arm distributes the inlet feed mixture over the perimeter of reactor.

[0250] In the bottom of the reactor, the product was extracted by means to achieve a uniform residence time for any particle spread on the top of the reactor.

[0251] Testing Uniform Plug Flow

[0252] The inlet and outlet means of the reactor were adjusted to achieve an expected residence time of 12 hours. For proving the uniform distribution time, an inert tracer substance was added to the feed mixture. The feed mixture used in the experiment had a natural content of iron of around 143 mg/kg dry matter (=off-set concentration); therefore, iron sulphate (FeSO.sub.4) was used as a tracer in a concentration of 1167 mg FeSO.sub.4/kg feed mixture dry matter equal to a total iron content of 572 mg Fe/kg total dry matter. At time 0 hours, FeSO.sub.4 was added to the feed mixture dosed to the reactor for a period of 60 minutes. Samples were drawn every 20 minutes, dried, and analysed for content of iron, and it was found that the FeSO.sub.4 enriched product leaves the reactor 12-13 hours after dosing FeSO.sub.4 to the inlet feed mixture, and a maximum concentration of 355 mg/kg Fe was found at 12.5 hours after start.