Vertical plug-flow process for simultaneous production of ethanol and a fermented, solid transformation product of the substrate

Abstract

The invention relates to a method for the simultaneous production of a solid transformation product of the substrate and crude ethanol comprising the following steps: •preparing a substrate from milled or flaked biomass comprising proteinaceous matter which originates from soya bean, rape seed, or mixtures thereof, optionally in further mixture with proteinaceous matter originating from fava beans, peas, sunflower seeds, lupine, cereals, and/or grasses, •mixing said substrate with live yeast in a dry matter ratio of from 1:1 to 10,000:1 and adding water in an amount which provides a ratio of wet bulk density to dry bulk density from 0.60 to 1.45 in the resulting mixture; •incubating said mixture for 1-48 hours at a temperature of about 20-60° C.; and •separating crude ethanol and wet solid transformation product from said mixture; further comprising that the incubation is performed as a continuous plug-flow process in a vertical, non-agitated, closed incubation tank with inlet means for said mixture and additives and outlet means for the solid transformation product and crude ethanol. The invention further relates to the products of this method as well as uses thereof.

Claims

1. A method for the simultaneous production of a solid transformation product of a biomass substrate and crude ethanol, comprising: preparing a substrate from a milled or flaked biomass comprising proteinaceous matter which originates from one or more of soya bean, rape seed, and mixtures thereof; mixing said substrate with live yeast in a dry matter ratio of from 1:1 to 10,000:1 and adding water in an amount which provides a water content of from 35% to 70% by weight, and a ratio of wet bulk density to dry bulk density of from 0.60 to 1.45 in the resulting mixture; incubating said mixture for 1-48 hours at a temperature of 20-60° C.; and separating crude ethanol and wet solid transformation product from said mixture; wherein said incubating is performed as a continuous plug-flow process in a vertical, non-agitated, closed incubation tank wherein transport of the biomass is mediated by gravitational force, with an inlet for said mixture and additives and an outlet for said solid transformation product and crude ethanol.

2. A method according to claim 1, wherein at least 20% by weight of said biomass comprises proteinaceous matter originating from defatted soya flakes.

3. A method according to claim 1, wherein at least 20% by weight of said biomass comprises proteinaceous matter originating from defatted rape seeds.

4. A method according to claim 1, wherein at least 60% by weight of said biomass comprises proteinaceous matter originating from defatted soya flakes.

5. A method according to claim 1, wherein at least 60% by weight of said biomass comprises proteinaceous matter originating from defatted rape seed.

6. A method according to claim 1, wherein said biomass further comprises one or more of oligosaccharides, polysaccharides, and oils and/or fats from seeds of oil-bearing plants.

7. A method according to claim 1, wherein said solid transformation product is a product of the transformation of proteinaceous matter, oligosaccharides, and/or polysaccharides originating from said biomass.

8. A method according to claim 1, wherein the dry matter ratio of biomass to yeast is from 2:1 to 5000:1.

9. A method according to claim 1, wherein water is added to said substrate in an amount to provide a ratio of wet bulk density to dry bulk density of from 0.65 to 1.40 in the substrate.

10. A method according to claim 1, wherein said yeast is selected from Saccharomyces cerevisiae strains of spent brewer's yeast, spent distiller's yeast, baker's yeast, spent yeast from wine production, and yeast strains fermenting C5 sugars.

11. A method according to claim 1, further comprising adding one or more processing aids selected from enzymes, plant components, and organic and inorganic processing agents to the substrate or mixture before or during said incubating.

12. A method according to claim 1, comprising keeping the filling degree of said closed incubation tank constant.

13. A method according to claim 1, further comprising adding α-galactosidase to the substrate or mixture before or during said incubating.

14. A method according to claim 13, wherein the α-galactosidase is added as an α-galactosidase preparation having an activity of about 5,000 α-galactosidase units/g, and is added to the substrate or mixture in an amount of from about 0.001% to about 1% by weight of the dry matter of the substrate or mixture.

15. A method according to claim 1, wherein said incubating is carried out under anaerobic conditions.

16. A method according to claim 1, wherein the water content in said mixture is from 40% to 70% by weight.

17. A method according to claim 1, further comprising drying the solid transformation product.

18. A method according to claim 17, further comprising milling the dried solid transformation product.

19. A method according to claim 1, where said non-agitated, closed incubation tank is of a vertical, oblong cylindrical, or polyhedral type.

20. A method according to claim 1, wherein said non-agitated, closed incubation tank has a conical shape wherein an upper region has a smaller diameter than a lower region.

21. A method according to claim 1, wherein said non-agitated, closed incubation tank is provided with insulating matting or a thermal dimple jacket.

22. A method according to claim 1, wherein at least 90% of crude ethanol produced is recovered.

23. A method according to claim 1, wherein the solid transformation product comprises proteins in an amount of 25-90% by weight on dry matter basis.

24. A method according to claim 23, further comprising drying the solid transformation product to a water content of not more than 15%.

25. A method according to claim 1, wherein the solid transformation product comprises at least 40% proteinaceous matter originating from soya.

26. A method according to claim 1, wherein the solid transformation product comprises at least 40% proteinaceous matter originating from rape seed.

27. A method according to claim 1, wherein the solid transformation product comprises proteins originating from plant parts of soya, rape seed, or sun flower, or mixtures thereof in an amount of from 30% to 65% by weight on dry matter basis.

28. A method according to claim 1, wherein the solid transformation product comprises a total amount of raffinose and stachyose of 3% by weight or less.

29. A method according to claim 1, wherein the solid transformation product comprises organic acids selected from succinic acid, acetic acid, and mixtures thereof in amounts of from 0.01% to 0.5% by weight.

30. A method according to claim 1, wherein the biomass further comprises proteinaceous matter originating from fava beans, peas, sunflower seeds, lupine, cereals, and/or grasses.

31. A method according to claim 1, wherein the solid transformation product comprises proteins in an amount of 25-90% by weight on dry matter basis, and glyceride in an amount of 0.05-30% by weight on dry matter basis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the temperature and pressure increase as a function of incubation time.

(2) FIG. 2 illustrates the temperature development during the test fermentation at atmospheric pressure as a function of incubation time.

(3) FIG. 3 shows the density ratios (wet bulk density/dry bulk density) vs. moisture for soya- and rape seed meal and their mixtures.

DETAILED DESCRIPTION OF THE INVENTION

(4) 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 defatted soya flakes. The soya flakes may also be dehulled.

(5) 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 defatted rape seeds.

(6) 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 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.

(7) 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.

(8) 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, oligosaccharides and/or polysaccharides 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.

(9) In any of the above embodiments the dry matter ratio of biomass to live yeast is from about 1:1 to about 10000:1, such as from about 2:1 to about 8000, e.g. 3:1, 5:1, 10:1, 15:1, 20:1, 30:1, 50:1, 70:1, 75:1, 85:1, 100:1, 200:1, 300:1, 500:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, or 7000:1.

(10) In any of the embodiments of the invention water is 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.

(11) 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 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.

(12) In any of the embodiments of the invention said yeast may be selected among Saccharomyces cerevisiae strains, including spent brewer's yeast and spent distiller's yeast and spent yeast from wine production, baker's yeast, as well as yeast strains fermenting C5 sugars.

(13) 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.

(14) In any of the embodiments of the invention the filling degree of said closed incubation tank may be kept constant. This will result in a uniform flow.

(15) In any of the embodiments of the invention α-galactosidase may be added to the substrate before or during incubation, e.g. an α-galactosidase preparation having an activity of about 5,000 α-galactosidase units pr. g. of enzyme product is added in an amount of from about 0.001% to about 1% by weight of the dry matter of the substrate, such as an amount of 0.005%, 0.01%, 0.015%, 0.02%, 0.03%, 0.05%, 0.1%, 0.5%, or 0.75% by weight.

(16) In any of the embodiments of the invention the incubation can be carried out under anaerobic conditions. The anaerobic conditions are facilitated by the present invention.

(17) 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 normally 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%.

(18) In any of the embodiments of the invention the mixture is incubated for 1-48 hours at 20-60° C., e.g. for 1-42 hours at 20-60° C. or 25-60° C. or 30-50° C. or 30-40° C., or for 2-36 hours at one of the here mentioned temperature intervals, or for 3-24 hours at one of the here mentioned temperature intervals, or for 5-18 hours at one of the here mentioned temperature intervals, or for 8-15 hours at one of the here mentioned temperature intervals, or for 10-12 hours at one of the here mentioned temperature intervals.

(19) In any of the embodiments of the invention the solid transformation product of the substrate may by dried, optionally followed by milling.

(20) In any of the embodiments of the invention the substrate mixture may be incubated at a time and a temperature sufficient to inactivate the yeast, anti-nutritional factors and the enzyme(s) if used partly or totally, and if desired. Generally, a higher temperature is applied for a shorter period, whereas a lower temperature is applied for a longer time to inactivate the live yeast, the anti-nutritional factors and the enzymes.

(21) In any of the embodiments of the invention the non-agitated, closed 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.

(22) In any of the embodiments of the invention the area in the upper part of said non-agitated, closed 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.

(23) In any of the embodiments of the invention the non-agitated, closed incubation tank may have insulating matting or a thermal dimple jacket and means to control the temperature inside the incubation tank.

(24) By the present method the ethanol can be separated from the substrate mixture by conventional methods, such as vacuum and/or injection of steam, and more than 98% by weight of the crude ethanol produced may be recovered. The yield of ethanol is dependent upon the content of carbohydrates in the incubation mixture and the conversion into fermentable sugars. Based on defatted soya it is possible to generate 4-5% by weight of ethanol, whereas on wheat approx. 20% by weight can be obtained.

(25) Thus, the invention in its second aspect provides a crude ethanol obtainable by a method according to the invention and further comprising small amounts of by-products resulting from the incubation of said biomass, e.g. other alcohols and ethers.

(26) In a first embodiment of the solid transformation product of the substrate of the invention it has been dried to a water content of not more than 15%, 13%, 10%, 6%, 4%, or 2% by weight and is optionally being in milled form.

(27) In any embodiments of the solid product of the invention it can be a product of the transformation of proteinaceous matter, oligosaccharides and/or polysaccharides originating from said biomass. The solid transformation product will have reduced content of anti-nutritional factors, such as trypsin inhibitors, antigens, flatulence-producing oligosaccharides, e.g. stachyose and raffinose; phytic acid, and lecithins.

(28) In any of the embodiments of the solid product of the invention it may comprise at least 40% proteinaceous matter by weight of dry matter originating from soya.

(29) In any of the embodiments of the solid product of the invention it may comprise at least 40% proteinaceous matter by weight of dry matter originating from rape seed.

(30) In any of the embodiments of the solid product of the invention it 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.

(31) In any of the embodiments of the solid product of the invention it 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.

(32) In any of the embodiments of the solid product of the invention is may comprise organic acids selected from succinic acid, acetic acid, and mixtures thereof in amounts of 0.01% to 0.5% by weight.

(33) Finally, the invention relates to a food, feed, cosmetic or pharmaceutical product or a nutritional supplement containing from 1% to 99% by weight of a solid transformation product according to the invention.

EXAMPLES

Example 1

(34) Comparative Incubation in an Open and a Closed Batch Tank of a Biomass Comprising Polysaccharides and Proteins from Pulses

(35) In the following the incubation at atmospheric pressure and elevated pressure of a biomass based on soya is compared.

(36) 1.1 Substrate Mixture:

(37) 10 kg of dehulled, defatted and desolventised soya flakes were mixed with 3 kg of a slurry of yeast (10% dry matter) and added water in an amount to reach a dry matter content of 51% by weight in the mixture. The ratio wet bulk density/dry bulk density of the substrate mixture was 0.832.

(38) 1.2 Incubation Tank:

(39) A 40-litre insulated autoclave equipped with a temperature- and pressure gauge probe was used as an open and closed batch tank.

(40) The tank was filled with approx. 15 kg of substrate mixture and left open for the test at atmospheric pressure, and when closed for the incubation under pressure the lid had a valve with a pressure regulator set to open at 6.0 bar.

(41) 1.3 Results:

(42) FIG. 1 illustrates the temperature and pressure increase as a function of incubation time.

(43) FIG. 2 illustrates the temperature development during the test at atmospheric pressure as a function of incubation time.

(44) The reduction of oligosaccharides after 6 hours of incubation is tabulated in the following:

(45) TABLE-US-00001 Stachyose Raffinose Parameter In % by weight In % by weight Substrate mixture 4.0 1.5 Incubation at elevated pressure 1.5 0.0 Incubation at atmospheric pressure 1.6 0.0

(46) From the results it can be deducted, that it is possible to incubate under pressure, and at the same time get a reduction of the oligosaccharide content similar to what can be obtained at atmospheric pressure by choosing at the right moisture conditions i.e. the ratio wet bulk density/dry bulk density.

Example 2

(47) Ratio of Wet Bulk Density/Dry Bulk Density for Preferred Substrates Based on Various Biomasses

(48) 2.1 Biomasses Used in the Examination:

(49) Soya

(50) The soya used was defatted soya bean meal.

(51) Maize

(52) The maize used was whole maize, ground on a hammer mill through a 3.5 mm sieve.

(53) Wheat

(54) The wheat used was whole wheat, ground on a hammer mill through a 3.5 mm sieve.

(55) Sunflower

(56) The sunflower used was defatted sunflower pellets, ground in a blender.

(57) Rapeseed

(58) The rapeseed used was defatted rapeseed meal.

(59) Fava Beans

(60) The beans used were whole fava beans, ground in a blender.

(61) Pea Protein

(62) The pea protein used was a pea protein concentrate.

(63) 2.2 Description of the Procedure:

(64) 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.

(65) 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.

(66) The bulk density was calculated as mass/untapped volume in kg/m.sup.3.

(67) The dry bulk density used was the measured bulk density of the biomass without addition of water.

(68) The wet bulk density was the bulk density of the biomass with added water.

(69) The ratio was calculated as wet bulk density divided by the dry bulk density.

(70) The moisture content of the biomasses was determined by drying to constant weight.

(71) After addition of water the moisture in the mixture was determined by calculation.

(72) 2.3 Results:

(73) The results for 100% soya and 80% mixtures with soya are tabulated in the following:

(74) TABLE-US-00002 Bulk Rape Fava Moisture Density Soya Maize Wheat Sun-flower seed 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

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

(76) TABLE-US-00003 Bulk Sun- Moisture Density Soya Maize flower 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

(77) The density ratios (wet bulk density/dry bulk density) vs. moisture for soya- and rape seed meal and their mixtures are shown graphically in FIG. 3.

Example 3

(78) Lab-scale incubation tests of prior art method vs. new technology method

(79) 3.1 Background:

(80) The background for the following lab-scale incubation tests was to imitate the conditions for the prior art method (PAM) as described in WO 2013050456 and the conditions in the method of the present invention (NTM).

(81) In the prior art method (PAM) the developed CO.sub.2 has free access to be released to the surroundings, whereas this is more limited in the new technology method (NTM) of the present invention i.e. the partial CO.sub.2 pressure is higher.

(82) 3.2 Materials and Methods:

(83) 3.2.1—Materials

(84) Biomasses: Soya Bean Meal (SBM), Rape Seed Meal (RSM) and Sunflower Seed Meal (SSM)—as described in section 2.1.

(85) Water: Normal tap water

(86) Yeast: Baker's yeast from De Danske Grfabrikker, Grenå, DK

(87) Processing aid: α-galactosidase from Bio-Cat (12,500 U/g)

(88) Each incubation mixture had a biomass content of 150 g of DM, varying amount of water, 0.4% yeast by weight of DM of biomass and 0.12% α-galactosidase by weight of DM of biomass.

(89) The compositions of the incubation mixtures are tabulated in the following:

(90) TABLE-US-00004 Yeast Moisture SBM RSM SSM (20% α- in % by 90.1% DM 87.2% DM 90.7% DM Water DM) galactosidase Biomass weight (g) (g) (g) (mL) (mL) (mg) 100% SBM 45 166.7 — — 103 3.0 182 100% SBM 60 166.7 — — 205 3.0 182 100% SBM 65 166.7 — — 259 3.0 182  80% SBM/ 45 133.3 34.4 — 102 3.0 182  20% RSM  80% SBM/ 60 133.3 34.4 — 204 3.0 182  20% RSM  60% SBM/ 45 100 — 66.2 104 3.0 182  40% SSM  60% SBM/ 60 100 — 66.2 206 3.0 182  40% SSM

(91) 3.2.2—Experimental Method Used

(92) Incubation Tank:

(93) For the PAM-method conical flasks half-filled and loosely closed with aluminium foil were used. For the NTM-method strong plastic bags, squeezed by hand to remove air and closed with a strap so that CO.sub.2 could escape were used.

(94) Incubation:

(95) All samples were incubated for 16 hours at 32° C. in a thermostated water bath. The incubation was stopped by heating to 100° C. for 30 minutes, except for the samples for ethanol analyses where the incubation was stopped by freezing.

(96) Analytical Methods:

(97) The analytical methods used are referred under the following individual results.

(98) 3.3 Results:

(99) 3.3.1—Density

(100) The determination of bulk density was performed by pouring an amount of material (250-460 mL) in a 500 mL graduated cylinder and reading the volume after levelling the surface by gently shaking (not tapping) the cylinder. Following this, the weight of the material was determined.

(101) The calculations were done as described in section 2.2.

(102) The results are tabulated in the following:

(103) TABLE-US-00005 Wet bulk Wet bulk Dry bulk density- density- Δ Wet bulk Moisture density- substrate After incubation density in % by Density substrate kg/m.sup.3 kg/m.sup.3 kg/m.sup.3 Method Biomass weight ratio kg/m.sup.3 (1) (2) (2) minus (1) PAM 100% SBM 45 0.89 654 583 568 −15 100% SBM 60 1.05 642 673 645 −28 100% SBM 65 1.29 654 843 552 −291 NTM 100% SBM 45 0.90 641 580 926 346 100% SBM 60 1.06 641 682 852 170 100% SBM 65 1.27 641 817 992 175 PAM  80% SBM/ 45 0.89 671 598 530 −68  20% RSM  80% SBM/ 60 1.05 671 701 635 −66  20% RSM NTM  80% SBM/ 45 0.90 641 580 869 289  20% RSM  80% SBM/ 60 1.11 658 732 970 238  20% RSM PAM  60% SBM/ 45 0.82 652 536 506 −30  40% SSM  60% SBM/ 60 1.06 665 703 518 −185  40% SSM NTM  60% SBM/ 45 0.84 639 535 858 323  40% SSM  60% SBM/ 60 1.06 652 692 1045 353  40% SSM

(104) Comments:

(105) From the results, it is noticed that after the incubation by the PAM-method the bulk density is reduced and with the NTM-method the density is increased.

(106) To test the influence of squeezing the bags, four different substrates were treated without incubation. The results for the four measurements were a maximum increase of 60 kg/m.sup.3 and a mean value of 33.5 kg/m.sup.3. Consequently, the density increase is an obvious fact.

(107) 3.3.2 Protein Content

(108) The crude protein content was determined according to the Dumas method, using 6.25 as conversion factor.

(109) The results are tabulated in the following:

(110) TABLE-US-00006 Protein content in the Protein trans- content in the formation Δ Protein biomass product content In % by In % by In % by Moisture weight of weight of weight of Protein content in % by DM DM DM Increase in % Method Biomass weight (1) (2) (2) minus (1) relative PAM 100% SBM 45 54.8 60.4 5.6 10.2 100% SBM 60 54.8 61.6 6.8 12.4 100% SBM 65 54.8 62.1 7.3 13.3 NTM 100% SBM 45 54.8 59.9 5.1 9.3 100% SBM 60 54.8 60.8 6.0 10.9 100% SBM 65 54.8 61.7 6.9 12.6 PAM  80% SBM/ 45 52.6 56.0 3.4 6.4  20% RSM  80% SBM/ 60 52.6 58.3 5.7 10.8  20% RSM NTM  80% SBM/ 45 52.6 56.6 4.0 7.6  20% RSM  80% SBM/ 60 52.6 57.3 4.7 8.9  20% RSM PAM  60% SBM/ 45 48.9 51.7 2.8 5.7  40% SSM  60% SBM/ 60 48.9 54.3 5.4 11.0  40% SSM NTM  60% SBM/ 45 48.9 54.1 5.2 10.6  40% SSM  60% SBM/ 60 48.9 52.0 3.1 6.3  40% SSM

(111) 3.3.3 Alcohol Content in Incubated Mixture

(112) The ethanol content was analyzed by Eurofins according to their method with internal code—RTTEF.

(113) The results are tabulated in the following:

(114) TABLE-US-00007 Ranking Moisture EtOH content in gram EtOH content in gram Within Biomass in % by per kg wet weight of per kg dry weight of biomass Method group weight incubated mixture incubated mixture group PAM 100% SBM 45 12.0 21.8 6 100% SBM 60 20.0 50.0 3 100% SBM 65 13.0 48.6 4 — NTM 100% SBM 45 18.0 32.7 5 100% SBM 60 21.0 52.6 1 100% SBM 65 18.0 51.4 2 PAM  80% SBM/ 45 19.0 34.5 3  20% RSM  80% SBM/ 60 13.0 32.5 4  20% RSM NTM  80% SBM/ 45 23.0 41.8 2  20% RSM  80% SBM/ 60 20.5 51.3 1  20% RSM PAM  60% SBM/ 45 20.0 36.4 2  40% SSM  60% SBM/ 60 13.0 32.5 4  40% SSM NTM  60% SBM/ 45 18.0 32.7 3  40% SSM  60% SBM/ 60 18.0 45.0 1  40% SSM

(115) Comments:

(116) From the ranking of the EtOH formed within biomass group, it is shown that the NTM-method results in more alcohol detected than the PAM-method does.

(117) 3.3.4 Oligosaccharides

(118) The content of stachyose and raffinose was determined by thin-layer chromatography. Stationary phase—Silica gel 60 (Merck 1.05553.0001)

(119) Mobile phase—120 mL n-butanol, 80 mL pyridine, and 60 mL demineralized water Spots are visualized with a liquid composed of 8 g diphenylamine, 335 mL acetone, 8 mL aniline, and 60 mL phosphoric acid.

(120) Sugar concentrations were determined by comparison with known standards.

(121) The results are tabulated in the following:

(122) TABLE-US-00008 Stachyose + Stachyose Raffinose Stachyose + Stachyose Raffinose Raffinose After incu- After incu- Raffinose Moisture In biomass In biomass In biomass bation bation After incubation Reduction In % by In % by In % by In % by In % by In % by In % by in % Method Biomass weight DM weight DM weight DM weight DM weight DM weight DM weight DM relative PAM 100% SBM 45 4.7 1.0 5.7 0.2 0.2 0.4 93 100% SBM 60 4.7 1.0 5.7 0 0 0 100 100% SBM 65 4.7 1.0 5.7 0 0 0 100 NTM 100% SBM 45 4.7 1.0 5.7 0.2 0.2 0.4 93 100% SBM 60 4.7 1.0 5.7 0 0 0 100 100% SBM 65 4.7 1.0 5.7 0 0 0 100 PAM  80% SBM/ 45 4.0 0.9 4.9 0.2 0.2 0.4 92  20% RSM  80% SBM/ 60 4.0 0.9 4.9 0 0 0 100  20% RSM NTM  80% SBM/ 45 4.0 0.9 4.9 0.2 0.2 0.4 92  20% RSM  80% SBM/ 60 4.0 0.9 4.9 0 0 0 100  20% SBM PAM  60% SBM/ 45 2.9 1.2 4.1 0.2 0.2 0.4 90  40% SSM  60% SBM/ 60 2.9 1.2 4.1 0 0 0 100  40% SSM NTM  60% SBM/ 45 2.9 1.2 4.1 0.2 0.2 0.4 90  40% SSM  60% SBM/ 60 2.9 1.2 4.1 0 0 0 100  40% SSM

(123) Comments:

(124) The two methods performed equally well, and at a moisture content higher than 45% the reduction of the oligosaccharides measured was 100%.

(125) 3.3.5 Organic Acids

(126) The contents of succinic- and acetic acids were analyzed by Eurofins according to their method with internal code—HEG12.

(127) The results are tabulated in the following:

(128) TABLE-US-00009 Succinic + Ranking Moisture Acetic acid Acetic acid Within in % Succinic acid in mg in mg in mg biomass Method Biomass group by weight per kg DM per kg DM per kg DM group PAM 100% SBM 45 1818 3091 4909 3 100% SBM 60 4500 1625 6125 1 100% SBM 65 1314 1114 2428 5 — NTM 100% SBM 45 600 1818 2418 6 100% SBM 60 1300 1150 2450 4 100% SBM 65 4000 1371 5371 2 PAM  80% SBM/ 45 3636 3636 7272 1  20% RSM  80% SBM/ 60 4250 1750 6000 2  20% RSM — NTM  80% SBM/ 45 1818 1437 3255 3  20% RSM  80% SBM/ 60 1925 925 2850 4  20% RSM PAM  60% SBM/ 45 5091 1527 6618 1  40% SSM  60% SBM/ 60 5000 600 5600 2  40% SSM — NTM  60% SBM/ 45 2545 636 3181 3  40% SSM  60% SBM/ 60 2500 325 2825 4  40% SSM

(129) Comments:

(130) From the ranking within biomass group, it is shown that the PAM-method produces more succinic and acetic acid than the NTM-method does.

(131) 3.4 Conclusions:

(132) On the basis of the obtained results it can be seen that the NTM-method produces a solid transformation product with slightly less protein and less succinic and acetic acid than the product based on the PAM-method. At the same time the alcohol content in the incubation mixture resulting from the NTM-method is higher.

Example 4

(133) Incubation in a Continuous Vertical Incubator of a Biomass Comprising Polysaccharides and Proteins from Pulses

(134) In the following the incubation in a vertical, closed and non-stirred incubator of a biomass based on defatted soya is illustrated.

(135) 4.1 Incubation Mixture:

(136) A mixture of dehulled, defatted and desolventised soya flakes and a slurry of yeast and water was prepared continuously in amounts to reach a dry matter content of 45% by weight in the mixture.

(137) The incubation mixture had a content of 3.5% by weight of yeast based on total dry matter. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.895.

(138) 4.2 Incubator:

(139) The pilot 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.

(140) Furthermore, the incubator was equipped with a temperature probe at the inlet as well as at the outlet.

(141) 4.3 Test Procedure:

(142) The incubator was filled with incubation mixture at the rate of 1000 litre per hour. After eight hours the incubator was filled to operating level and the outlet means were set at a rate to keep the level of filling constant.

(143) An aliquot volume of approx. 30 litres was taken after 10 hours of the test run and incubated at 100° C. with live steam for 25 min. and the surplus steam comprising crude ethanol was transferred to a cooling heat exchanger.

(144) Subsequently, the wet solid transformation product of the biomass was flash dried and milled.

(145) The overall incubation parameters were the following:

(146) Incubation time—8 hours

(147) Inlet temperature—25.2° C.

(148) Outlet temperature—32.3° C.

(149) 4.4 Results:

(150) The separated crude ethanol content corresponded to a yield of 4.7 kg per 100 kg of biomass. The solid transformation product of the biomass had a total crude protein (N×6.25) content of 58.3%. The water content of the dried product was 5.4% by weight.

(151) Furthermore, anti-nutritional factors in the dried, solid transformation product were significantly reduced:

(152) TABLE-US-00010 Solid transformation Parameter Biomass product Oligosaccharides 10.2% 0.5% Trypsin inhibitor 43,000 TIU/g 1,900 TIU/g B-conglycinin 130,000 ppm 2 ppm

(153) 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

(154) Incubation in a Continuous Vertical Incubator of a Biomass Comprising Polysaccharides and Proteins from a Mixture of Pulses and Cereals

(155) In the following the incubation in a vertical, closed and non-stirred incubator of a biomass based on a mixture of defatted soya and wheat is illustrated.

(156) 5.1 Incubation Mixture:

(157) A mixture containing 10% by weight of dry matter of crushed wheat and 90% by weight of dry matter of dehulled, defatted and desolventised soya flakes and a slurry of yeast, enzymes and water was prepared continuously in amounts to reach a dry matter content of 40% by weight in the mixture. The incubation mixture had a content of 3% by weight of yeast based on total dry matter and 0.4% by weight based on dry matter of wheat of Viscozyme Wheat, Spirizyme Fuel and Liquozyme all from Novozymes.

(158) The ratio wet bulk density/dry bulk density of the incubation mixture was 0.984.

(159) 5.2 Incubator:

(160) The incubator was as described in example 4.

(161) 5.3 Test Procedure:

(162) The test procedure was as described in example 4 except that in this process the inlet means was set to run at a volume speed of 500 liters/h.

(163) The overall incubation parameters were the following:

(164) Incubation time—16 hours

(165) Inlet temperature—25.1° C.

(166) Outlet temperature—33.1° C.

(167) 5.4 Results:

(168) The separated crude ethanol content corresponded to a yield of 8.4 kg per 100 kg of biomass. The solid transformation product of the biomass had a crude protein content (N×6.25) of 59.4% and a water content of 6.2% by weight.