Vertical plug-flow process for bio-conversion employing microorganisms

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 a live microorganism or a combination of live microorganisms, which live microorganism or mixture of live microorganisms is not live yeast, 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 1-240 hours at a temperature of 15-70° C.; and thereafter recovering wet solid transformation product from the incubation 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. 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, the method 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 a live microorganism or a combination of live microorganisms, which live microorganism or combination of live microorganisms is not, and does not comprise, live yeast, 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 1-240 hours at a temperature of 15-70° 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, and wherein transport of the biomass is mediated by gravitational force.

2. The method according to claim 1, further comprising pre-treating said substrate before mixing with said live microorganism or said combination of live microorganisms by one or more selected from disintegration, milling, flaking, heat treatment, pressure treatment, ultrasonic treatment, hydrothermal treatment, acid treatment and alkaline treatment.

3. The method according to claim 1, 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.

4. The method according to claim 3, wherein the weight of said biomass comprising carbohydrates and proteinaceous matter originating from optionally defatted and/or optionally dehulled soya bean seeds, optionally defatted rape seeds, or 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.

5. The method according to claim 1, wherein said biomass comprises one or more of oligosaccharides and polysaccharides, and optionally further comprises oils and fats.

6. The method according to claim 1, 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.

7. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is one or more microorganisms which can produce one or more organic acids from carbohydrates selected from formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid.

8. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is one or more microorganisms which can produce one or more alcohols from carbohydrates.

9. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is of a genus selected from: Lactobacillus Lactococcus Streptococcus Pediococcus Enterococcus Leuconostoc Weisella Bifidobacterium Bacillus Brevibacillus Propionibacterium Clostridium Trichoderma and Aspergillus.

10. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is selected from one or more of Lactobacillus, Pediococcus, Enterococcus, Lactococcus, Streptococcus, and Weisella strains, and wherein the initial incubation mixture is incubated at a temperature of 15-50° C.

11. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is selected from Bacillus strains, and wherein the initial incubation mixture is incubated a temperature of 20-60° C.

12. The method according to claim 1, wherein the live microorganism or combination of live microorganisms is selected from Bifidobacterium strains, and wherein the initial incubation mixture is incubated at a temperature of 20-45° C.

13. The method according to claim 1, wherein said initial incubation mixture is incubated for 2 to 180 hours.

14. The method according to claim 1, 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.

15. The method according to claim 1, 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.

16. The method according to claim 1, wherein the water content in said initial incubation mixture is from 35% to 70% by weight.

17. The method according to claim 1, wherein the water content in said initial incubation mixture is selected from 40%, 45%, 50%, 55%, 60%, and 65%.

18. The method according to claim 1, wherein said live microorganism or combination of live microorganisms is used in an amount of 10.sup.3 to 10.sup.11 CFU (colony forming units) per g of said substrate.

19. The 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 one or more of the substrate and the initial incubation mixture.

20. The method according to claim 1, further comprising adding α-galactosidase to one or more of the substrate and the initial incubation mixture.

21. The method according to claim 1, further 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.

22. The method according to claim 1, wherein the vertical, non-agitated incubation tank is closed.

23. The method according to claim 1, wherein said incubation is carried out under anaerobic conditions.

24. The method according to claim 1, wherein said non-agitated incubation tank is of a vertical, oblong cylindrical or polyhedral type.

25. The method according to claim 1, wherein the area in the upper part of said non-agitated incubation tank is less than the area in the lower part.

26. The method according to claim 1, where said non-agitated incubation tank has insulating matting or a thermal dimple jacket.

27. The method according to claim 1, wherein the filling degree of said incubation tank is kept constant.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) 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.

(2) 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.

(3) In a third embodiment of the method of the invention the biomass comprises proteinaceous matter originating from optionally defatted soya 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.

(4) 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.

(5) In any of the embodiments of the invention the solid transformation product of the substrate may be a product of the transformation of carbohydrates, in particular oligosaccharides and polysaccharides, and/or proteinaceous matter 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.

(6) In any of the embodiments of the invention the live microorganism or mixture of live microorganisms may be one or more microorganisms which can produce one or more organic compounds, such as organic acids, e.g. formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid, or alcohols, e.g. ethanol, from carbohydrates.

(7) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be one or more organic acid producing microorganism(s).

(8) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be selected from the following list of genera: Lactobacillus Lactococcus Streptococcus Pediococcus Enterococcus Leuconostoc Weisella Bifidobacterium Bacillus Brevibacillus Propionibacterium Clostridium Trichoderma Candida Aspergillus.

(9) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be selected from Lactobacillus strains, and the mixture may be incubated at a temperature of 15-50° C.

(10) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be selected from Lactobacillus, Pediococcus, Enterococcus, Lactococcus, Streptococcus, and Weisella strains, and the mixture may be incubated at a temperature of 15-50° C.

(11) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be selected from Bacillus strains, and the mixture may be incubated a temperature of 20-60° C.

(12) In any of the embodiments of the invention the live microorganism or combination of live microorganisms may be selected from Bifidobacterium strains, and the mixture may be incubated at a temperature of 20-45° C.

(13) In any of the above embodiments 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 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.

(14) In any of the above embodiments the live microorganism or combination of live microorganisms is used in an amount of 10.sup.3 to 10.sup.11 CFU (colony forming units) per g of said substrate of biomass, such as 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or 10.sup.10 CFU/g substrate of biomass. The skilled person would now how to select a suitable amount, depending on the selected process conditions, such as reactor dimension, the process time and temperature, the applied microorganism, and the transformation product to be produced.

(15) 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.

(16) 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.

(17) 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 of biomass and/or to the initial incubation mixture.

(18) 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.

(19) In any of the embodiments of the invention a processing aid selected as α-galactosidase may be added to the substrate of biomass and/or to the initial incubation mixture, e.g. an α-galactosidase preparation is 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.

(20) 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.

(21) 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%.

(22) In any of the embodiments of the invention the mixture is incubated for 1-240 hours at 15-70° C. The skilled will know how to optimise the reaction time and the reaction temperature in view of the other reaction conditions, such as the selection of microorganisms. 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. 2 to 180 hours, such as 5 to 150 hours, 7 to 120 hours, 10 to 80 hours, 20 to 60 hours, or 28 to 48 hours, at each and every one of the here mentioned temperature intervals.

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

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

(25) In any of the embodiments of the invention the non-agitated incubation tank may be closed.

(26) 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.

(27) 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.

(28) 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.

(29) The solid transformation product of the substrate provided by 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.

(30) The solid product of the invention can be a product of the transformation of proteinaceous matter and/or carbohydrates 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.

(31) The solid product of the invention may comprise at least 40% proteinaceous matter by weight of dry matter originating from soya.

(32) The solid product of the invention may comprise at least 40% proteinaceous matter by weight of dry matter originating from rape seed.

(33) 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.

(34) 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 produced according to the invention.

EXAMPLES

(35) Density Ratio

Example 1

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

(37) 1.1 Biomasses Used in the Procedure:

(38) Soya

(39) The soya used was defatted Soya Bean Meal (SBM).

(40) Maize

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

(42) Wheat

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

(44) Sunflower

(45) The sunflower used was defatted Sunflower Seed Meal (SSM).

(46) Rapeseed

(47) The rapeseed used was defatted Rape Seed Meal (RSM).

(48) Fava Beans

(49) The beans used were whole fava beans.

(50) Pea Protein

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

(52) 1.2 Description of the Procedure:

(53) 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.

(54) 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.

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

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

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

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

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

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

(61) 1.3 Results:

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

(63) TABLE-US-00001 Bulk Fava Water Moisture Density Soya Maize Wheat Sunflower Rapeseed bean Pea In g In % kg/m.sup.3 Ratio 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

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

(65) 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

Example 2

(66) Ratio of Wet Bulk Density/Dry Bulk Density for Substrates Based on Various Biomasses and Used in Experiments with Various Microorgansims

(67) The determination of bulk density was performed by pouring an amount of material (approx. 250 ml) in a 250 ml measuring cylinder and reading the volume after leveling the surface by gently shaking the cylinder. Following this, the weight of the material was determined. Dry bulk densities and wet bulk densities were done in triplicates.

(68) The results are summarised in the following table:

(69) TABLE-US-00003 Density ratio = Dry matter in wet bulk density/ Biomass % by weight dry bulk density 100% SBM 35 1.13 100% SBM 40 0.95 100% SBM 42.5 0.86 100% SBM 52 0.85 100% SBM 55 0.84 80% SBM + 20% RSM 35 1.05 80% SBM + 20% RSM 42.5 0.88 80% SBM + 20% RSM 52 0.78 60% SBM + 40% SSM 35 0.94 60% SBM + 40% SSM 42.5 0.84 60% SBM + 40% SSM 52 0.73

(70) Lab-Scale Incubation Tests of New Technology Method

(71) The following examples 3 to 9 were lab scale experiments conducted under the following conditions:

(72) Background:

(73) The background for the following lab-scale incubation tests was to imitate the conditions in the method of the present invention.

(74) Materials and Methods:

(75) Materials

(76) Biomasses: Soya Bean Meal (SBM), Rape Seed Meal (RSM) and Sunflower Seed Meal (SSM) —as described in section 1.1.

(77) Water: Normal tap water

(78) Microorganisms: The microorganism(s) used are specified for each example. For all experiments, unless indicated in the specific example, microorganisms were dosed with approximately 10.sup.8 CFU/g DM. Lactic acid bacteria and Bifidobacteria were grown in MRS broth, washed in 0.9% NaCl, and dosed to the incubation based on a relationship between OD.sub.600 and CFU/ml. The ml amount needed to dose 10.sup.8 CFU/g DM was subtracted from the total water amount stated under each example. For the Bacillus strains, most of them were dosed as dry formulated cultures, but Geobacillus denitrificans and Bacillus smithii were grown in Nutrient Broth, and washed in the same way, and dosed in the same way, as described for the Lactic acid bacteria strains.

(79) The microorganisms and their origin used in the examples are shown in the following table:

(80) TABLE-US-00004 Strain Origin Lactobacillus plantarum Pangoo Lactobacillus paracasei 5622 DSMZ Lactobacillus fermentum Bio Growing Lactobacillus acidophilus Bio Growing Lactobacillus delbruckii bulgaricus Bio Growing Lactobacillus debruckii sunkii 24966 DSMZ Lactobacillus farci minis Own isolate Lactobacillus formosensis Own isolate Lactobacillus salivarius 20554 DSMZ Bacillus coagulans Pangoo Bacillus licheniformis BioCat Bacillus subtilis BioCat Bacillus smithii 2319 DSMZ Lactococcus lactis Bio Growing Bifidobacterium animalis Bio Growing Pediococcus acidolactici Pangoo Enterococcus faecium Pangoo Enterococcus faecalis Pangoo Enterococcus durans Own isolate Weisella hellenica Own isolate Streptococcus thermophiles Bio Growing Geobacillus thermodenitrificans 466 DSMZ DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen

(81) Processing aid: α-galactosidase from Bio-Cat (12,500 U/g). The α-galactosidase was dosed in 1 ml water, which was substrated from the total addition of water stated in the table of each example.

(82) Experimental Method Used

(83) Incubation Tank:

(84) To imitate bio-conversion conditions where oxygen become non-available, bio-conversion where performed in strong plastic bags, squeezed by hand to remove air and closed tightly with a strap, still allowing CO.sub.2 to escape.

(85) Incubation:

(86) Samples were incubated at different temperatures, different water contents and at different length in time, specified for each example. The incubation was stopped by heating 100° C. for 30 min.

(87) Analytical Methods:

(88) Acid Analysis:

(89) The analysis was conducted by LUFA Oldenburg, Germany, using an aqueous digestion with membrane filtration and subsequent measurement by an ion chromatograph.

(90) Sucrose and Galactose (Sugars):

(91) The content of sucrose and galactose was determined by thin-layer chromatography.

(92) Stationary phase—Silica gel 60 (Merck 1.05553.0001)

(93) Mobile phase—120 mL n-butanol, 80 mL pyridine and 60 mL demineralized water

(94) Spots are visualized with a liquid composed of 8 g diphenylamine, 335 mL acetone 8 mL aniline and 60 mL phosphoric acid.

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

(96) pH:

(97) pH was measured in 10% DM dilutions with a HQ 411d from HACH.

(98) CFU:

(99) CFU were determined by plate spreading, using MRS agar plates for lactic acid bacteria, and Nutrient agar for the Bacillus strains.

Example 3

(100) Testing Different Production Organisms (LAB) at 20° C., at Different Dry Matter Ratios

(101) Experimental Set-Up:

(102) TABLE-US-00005 Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain % of weight CFU/g DM g mg Ml Lactobacillus 42.5 1*10.sup.8 113.6 120 122 salivarius Lactobacillus 42.5 1*10.sup.8 113.6 120 122 debruckii sunkii Lactobacillus 35 1*10.sup.8 113.6 120 172 plantarum Lactobacillus 42.5 1*10.sup.8 113.6 120 122 plantarum Lactobacillus 52 1*10.sup.8 113.6 120 79 plantarum Lactobacillus 35 1*10.sup.8 113.6 120 172 paracasei Lactobacillus 42.5 1*10.sup.8 113.6 120 122 paracasei

(103) Samples were incubated in a 20° C. thermostatic water bath.

(104) Results:

(105) After 44 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production:

(106) TABLE-US-00006 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 35 4.9 1.2 6.1 4.9 3*10.sup.10 0 0 plantarum Lactobacillus 42.5 3.7 1.3 5.0 5.2 2*10.sup.10 0 0 plantarum Lactobacillus 52 3.2 0.9 4.1 5.2 2*10.sup.10 0 0 Plantarum

(107) After 116 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production:

(108) TABLE-US-00007 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 42.5 3.4 1.0 4.4 4.9 9.5*10.sup.9  0.5 1.6 salivarius Lactobacillus 42.5 3.7 0.5 4.2 4.9 3.9*10.sup.9  0 1.6 debruckii sunkii Lactobacillus 35 7.3 1.1 8.4 4.5 2.0*10.sup.10 0 0 plantarum Lactobacillus 42.5 5.7 1.1 6.8 4.7 2.3*10.sup.10 0 0 plantarum Lactobacillus 52 5.1 1.2 6.3 4.8 2.2*10.sup.10 0 0 plantarum Lactobacillus 35 4.7 0.8 5.5 4.8 1.9*10.sup.10 6 0 paracasei Lactobacillus 42.5 3.2 0.6 3.8 4.8 1.8*10.sup.10 6 0 paracasei Part of the sugars was still bound in oligosaccharides in this experiment, even after 166 hours. The potential for acid production is thereby larger than obtained in this test.

Example 4

(109) Testing Different Production Organisms (LAB) at 30° C., at 40% DM

(110) Experimental Set-Up:

(111) TABLE-US-00008 Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain % of weight CFU/g DM G mg Ml Lactobacillus 40 1*10.sup.8 68.2 72 82 plantarum Lactococcus 40 1*10.sup.8 68.2 72 82 lactis Enterococcus 40 1*10.sup.8 68.2 72 82 faecium

(112) Samples were incubated in a 30° C. thermostatic water bath.

(113) Results:

(114) After 45 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production:

(115) TABLE-US-00009 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 40 6.2 1.1 7.3 4.6 1*10.sup.10 0 1.4 plantarum Lactococcus 40 3.7 0.9 4.6 4.8 1*10.sup.10 1.8 1.8 lactis Enterococcus 40 5.1 1.4 6.5 4.8 2*10.sup.10 0.4 1.4 faecium

(116) After 69 hours of incubation the following results were obtained, showing sugar conversion and acid production (CFU not determined):

(117) TABLE-US-00010 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 40 7.0 1.0 8.0 4.5 0 0.5 plantarum Lactococcus 40 4.3 1.2 5.5 4.6 1.8 1.2 lactis Enterococcus 40 5.8 1.3 7.1 4.6 0 0.6 faecium

Example 5

(118) Testing Different Production Organisms at 37° C., at Different Dry Matter Ratios

(119) Experimental Set-Up:

(120) TABLE-US-00011 Inoculation Exp. Dry matter level SBM α-galactosidase Water Strain No. % of weight CFU/g DM (88% DM) mg Ml Lactobacillus 1 35 1*10.sup.8 113.6 120 172 plantarum Lactobacillus 2 42.5 1*10.sup.8 113.6 120 122 plantarum Lactobacillus 3 42.5 1*10.sup.8 113.6 Not added 122 plantarum Lactobacillus 4 42.5 1*10.sup.7 68.2 72 73 plantarum Lactobacillus 5 42.5 1*10.sup.9 68.2 72 73 plantarum Lactobacillus 6 52 1*10.sup.8 113.6 120 79 plantarum Lactobacillus 7 35 1*10.sup.8 113.6 120 172 paracasei Lactobacillus 8 42.5 1*10.sup.8 113.6 120 122 paracasei Lactobacillus 9 52 1*10.sup.8 113.6 120 79 paracasei Bacillus 10 35 1*10.sup.8 68.2 72 103 coagulans Bacillus 11 42.5 1*10.sup.8 68.2 72 73 coagulans Bacillus 12 42.5 1*10.sup.8 113.6 Not added 122 coagulans Bacillus 13 42.5 1*10.sup.7 68.2 72 73 coagulans Bacillus 14 55 1*10.sup.8 68.2 72 41 coagulans Bacillus 15 35 1*10.sup.8 68.2 72 103 licheniformis Bacillus 16 42.5 1*10.sup.8 68.2 72 73 licheniformis Bacillus 17 55 1*10.sup.8 68.2 72 41 licheniformis Bacillus 18 35 1*10.sup.8 68.2 72 103 subtilis Bacillus 19 42.5 1*10.sup.8 113.6 120 122 subtilis Bacillus 20 55 1*10.sup.8 68.2 72 41 subtilis Lactobacillus 21 42.5 1*10.sup.8 68.2 72 73 fermentum Lactobacillus 22 42.5 6*10.sup.7 68.2 72 73 acidophilus Lactobacillus 23 42.5 2*10.sup.7 68.2 72 73 delbruckii bulgaricus Lactobacillus 24 42.5 6*10.sup.6 68.2 72 73 farciminis Lactobacillus 25 42.5 1*10.sup.8 113.6 120 122 formosensis Lactococcus 26 42.5 4*10.sup.7 68.2 72 73 lactis Bifidobacterium 27 42.5 1*10.sup.8 68.2 72 73 animalis Pediococcus 28 42.5 1*10.sup.8 68.2 72 73 acidolactici Enterococcus 29 42.5 1*10.sup.8 68.2 72 73 faecium Enterococcus 30 42.5 1*10.sup.8 68.2 72 73 faecalis Enterococcus 31 42.5 1*10.sup.8 113.6 120 122 durans Weisella 32 42.5 1*10.sup.8 113.6 120 122 hellenica Lactobacillus 33 42.5 1*10.sup.8 and 113.6 120 122 salivarius + 3*10.sup.7 Lactobacillus paracasei Streptococcus 34 42.5 5*10.sup.7and 113.6 120 122 thermophilus + 5*10.sup.7 Bifidobacterium animalis Pediococcus 35 42.5 5*10.sup.7and 113.6 120 122 acidolactici + 5*10.sup.7 Lactobacillus plantarum Lactobacillus 36 42.5 5*10.sup.7and 113.6 120 122 farciminis + 5*10.sup.7 Lactobacillus plantarum Lactobacillus 37 42.5 1*10.sup.8 113.6 120 122 plantarum + sucrose (5% of DM)

(121) Results:

(122) After 18.5 to 20 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production

(123) TABLE-US-00012 Inoculation Exp DM Lactic acid Acetic acid Total acid level CFU/g Sucrose Galactose Strain No. % % of DM % of DM % of DM pH DM % of DM % of DM Lactobacillus 1 35 6.1 1.2 7.3 4.6 nd 1 2 plantarum Lactobacillus 2 42.5 5.3 1.2 6.5 4.7 nd 1 2 plantarum Inoc: 10.sup.8 CFU/g Lactobacillus 4 42.5 5.5 1.2 6.7 5.0 nd 2 2 plantarum Inoc: 10.sup.7 CFU/g Lactobacillus 5 42.5 6.5 1.2 7.7 4.8 nd 1 2 plantarum Inoc: 10.sup.9 CFU/g Lactobacillus 6 52 4.7 1.1 5.8 4.8 nd 2 2 plantarum Lactobacillus 7 35 5.3 0.1 5.4 4.4 nd 2 2 paracasei Lactobacillus 8 42.5 4.5 0.1 4.6 4.5 nd 2.6 2 paracasei Bacillus 11 42.5 4.5 1.4 5.9 5.3 8*10.sup.9 2.5 2 coagulans Inoc: 10.sup.8 CFU/g Bacillus 13 42.5 3.6 1.4 5.0 5.4 7*10.sup.9 2.5 2 coagulans Inoc: 10.sup.7 CFU/g Lactobacillus 24 42.5 4.2 0.1 4.3 4.8 nd 4 3.5 farciminis (no 1) Lactococcus 26 42.5 3.0 2.0 5.0 5.0 nd 2 2 lactis Bifidobacterium 27 42.5 4.1 2.0 6.1 5.0 nd 2 2 animalis Pediococcus 28 42.5 3.7 1.5 5.2 5.1 nd 2 2 acidolactici Enterococ- 29 42.5 5.4 1.4 6.8 5.1 nd 2 2 cus faecium Lactobacillus 33 42.5 5.1 0.1 5.2 4.4 nd 2 2 salivarius + Lactobacillus paracasei Streptococcus 34 42.5 4.1 1.9 6.0 4.9 nd 2 2 thermophiles + Bifidobacterium animalis Pediococcus 35 42.5 5.4 1.2 6.6 4.7 nd 1 2 acidolactici + Lactobacillus plantarum Lactobacillus 36 42.5 6.0 0.9 6.9 4.5 nd 1 2 farciminis + Lactobacillus plantarum Lactobacillus 37 42.5 5.3 1.1 6.4 4.7 nd 6 2 plantarum + sucrose nd: not determined

(124) After 42.5 and 44 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production:

(125) TABLE-US-00013 Inoculation Exp DM Lactic acid Acetic acid Total acid level CFU/g Sucrose Galactose Strain No. % % of DM % of DM % of DM pH DM % of DM % of DM Lactobacillus 1 35 7.3 1.0 8.3 4.4 .sup. 1*10.sup.10 0 1.4 plantarum Lactobacillus 2 42.5 6.8 1.2 8.0 4.4 .sup. 1*10.sup.10 0 1.8 plantarum Inoc: 10.sup.8 CFU/g Lactobacillus 3 42.5 3.6 1.2 4.8 5.1 5*10.sup.9 0 0 plantarum (no α-gal) Lactobacillus 4 42.5 7.3 1.5 8.8 4.6 nd 0 1.1 plantarum Inoc: 10.sup.7 CFU/g Lactobacillus 5 42.5 7.9 1.1 9.0 4.6 nd 0 1.25 plantarum Inoc: 10.sup.9 CFU/g Lactobacillus 6 52 6.6 1.2 7.8 4.5 .sup. 1*10.sup.10 0 1.8 plantarum Lactobacillus 7 35 7.2 0.1 7.3 4.1 .sup. 3*10.sup.10 0 2.4 paracasei Lactobacillus 8 42.5 6.6 0.1 6.7 4.2 .sup. 2*10.sup.10 0.5 2.6 paracasei Lactobacillus 9 52 5.3 0.1 5.4 4.4 .sup. 2*10.sup.10 3 3 paracasei Bacillus 10 35 6.9 1.2 8.1 4.5 nd 0 1.3 coagulans Bacillus 11 42.5 8.2 1.3 9.5 4.6 4*10.sup.9 0.4 1 coagulans Inoc: 10.sup.8 CFU/g Bacillus 12 42.5 5.5 1.2 6.7 4.7 2*10.sup.9 0 0 coagulans (no α-gal) Bacillus 13 42.5 7.3 1.3 8.6 4.7 3*10.sup.9 0.5 1 coagulans Inoc: 10.sup.7 CFU/g Bacillus 14 55 3.7 0.8 4.5 5.1 nd 2 2 coagulans Bacillus 15 35 2.7 0.0 2.7 5.1 nd 2.5 4 licheniformis Bacillus 16 42.5 0.8 0.0 0.8 6.0 nd 2.5 4 licheniformis Bacillus 17 55 0.2 0.0 0.2 6.4 nd 2.7 3.3 licheniformis Bacillus 18 35 2.4 0.1 2.5 5.1 nd 3 4 subtilis Bacillus 19 42.5 2.5 0.9 3.4 5.3 3*10.sup.9 3.6 2.6 subtilis Bacillus 20 55 0.5 0.1 0.6 6.0 nd 5 2 subtilis Lactobacillus 21 42.5 4.6 2.1 6.7 4.9 .sup. 5*10.sup.10 1 1 fermentum Lactobacillus 22 42.5 4.2 0.2 4.4 4.7 4*10.sup.9 1 1 acidophilus Lactobacillus 23 42.5 3.7 1.7 5.4 5.2 8*10.sup.9 3.3 2.5 delbruckii bulgaricus Lactobacillus 24 42.5 7.9 0.3 8.2 4.2 5*10.sup.9 0.8 2.8 farciminis Lactobacillus 25 42.5 6.5 0.2 6.7 4.2 3*10.sup.9 0.5 2 formosensis Lactococcus 26 42.5 4.0 2.3 6.3 4.8 8*10.sup.9 0.8 1 lactis Bifidobacterium 27 42.5 4.5 2.1 6.6 4.9 6*10.sup.9 1 0.8 animalis Pediococcus 28 42.5 6.9 1.5 8.4 4.6 9*10.sup.9 0.5 0.7 acidolactici Enterococcus 29 42.5 7.6 1.5 9.1 4.6 7*10.sup.9 0.5 0.7 faecium Enterococcus 30 42.5 5.8 1.5 7.3 4.7 9*10.sup.9 0.3 0.3 faecalis Enterococcus 31 42.5 2.7 0/2 2.9 4.9 2*10.sup.9 3 2 durans Weisella 32 42.5 4.1 1.6 5.7 4.9 3*10.sup.9 1 1 hellenica Lactobacillus 33 42.5 6.2 0.1 6.3 4.2 .sup. 1*10.sup.10 1 1.9 salivarius + Lactobacillus paracasei Streptococcus 34 42.5 5.1 2.0 7.1 4.8 .sup. 8*10.sup.10 0.2 1 thermosphiles + Bifidobacterium animalis Pediococcus 35 42.5 6.9 1.2 8.1 4.4 .sup. 1*10.sup.10 0 1 acidolactici + Lactobacillus plantarum Lactobacillus 36 42.5 7.4 1.0 8.4 4.4 9*10.sup.9 0 1.4 farciminis + Lactobacillus plantarum Lactobacillus 37 42.5 6.5 1.0 7.5 4.4 .sup. 1*10.sup.10 4 2.2 plantarum + sucrose nd: not determined

(126) After 116 hours of incubation the following results were obtained, showing sugar conversion and acid production.

(127) TABLE-US-00014 Exp. DM Lactic acid Acetic acid Total acid Sucrose Galactose Strain No. % % of DM % of DM % of DM pH % of DM % of DM Bacillus 10 35 7.7 1.2 8.9 4.3 0 0 coagulans Bacillus 11 42.5 7.7 1.2 8.9 4.3 0.1 0.5 coagulans Bacillus 14 55 4.8 0.7 5.5 4.8 1.2 1.6 coagulans Bacillus 15 35 2.5 0.1 2.6 4.8 0.3 3.5 licheniformis Bacillus 16 42.5 1.7 0.1 1.8 5.5 2 3.3 licheniformis Bacillus 17 55 0.4 0.1 0.5 6.3 2.9 3.3 licheniformis Bacillus 18 35 1.6 0.1 1.7 4.9 0 3.5 subtilis Bacillus 19 42.5 1.4 0.1 1.5 5.1 1.5 3.5 subtilis Bacillus 20 55 0.8 0.2 1.0 5.9 5 3 subtilis

Example 6

(128) Testing Different Production Organisms at 44° C., at 40% DM

(129) Experimental Set-Up:

(130) TABLE-US-00015 Inoculation SBM Dry matter level 88% DM α-galactosidase Water Strain % of weight CFU/g DM g mg Ml Lactobacillus 40 1*10.sup.8 68.2 72 73 plantarum Pediococcus 40 1*10.sup.8 68.2 72 73 acidolactici Bacillus 40 1*10.sup.8 68.2 72 73 coagulans Bacillus 40 1*10.sup.8 68.2 72 73 licheniformis Bacillus subtilis 40 1*10.sup.8 68.2 72 73

(131) Samples were incubated in a 44° C. thermostatic water bath.

(132) Results:

(133) After 20 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production:

(134) TABLE-US-00016 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 40 5.1 0.2 5.3 4.8 Nd 3 2.5 plantarum Pediococcus 40 4.7 0.2 4.9 4.8 Nd 3 3 acidolactici Bacillus 40 4.4 0.1 4.5 5.0 .sup. 2*10.sup.10 2 2.5 coagulans Bacillus 40 1.1 0.0 1.1 6.0 2*10.sup.8 2.5 3.6 licheniformis Bacillus 40 0.7 0.2 0.9 6.0 1*10.sup.9 6.5 3.8 subtilis nd: not determined

(135) After 44 hours of incubation the following results were obtained, showing sugar conversion and acid production (CFU not determined):

(136) TABLE-US-00017 DM Lactic acid Acetic acid Total acid Sucrose Galactose Strain % % of DM % of DM % of DM pH % of DM % of DM Lactobacillus 40 6.8 0.3 7.1 4.4 3 2.2 plantarum Pediococcus 40 6.6 0.3 6.9 4.4 2 1.5 acidolactici Bacillus 40 7.2 0.2 7.4 4.4 0.5 1.0 coagulans Bacillus 40 1.5 0.1 1.6 5.9 1 2.9 licheniformis Bacillus 40 1.3 0.1 1.4 5.7 4.5 2.9 subtilis

Example 7

(137) Testing Different Production Organisms at 52° C., at 52% DM

(138) Experimental Set-Up:

(139) TABLE-US-00018 Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain % of weight CFU/g DM g mg Ml Bacillus smithii 42.5 1*10.sup.8 113.6 120 172 Bacillus smithii 42.5 1*10.sup.8 113.6 No addition 172 Bacillus 42.5 1*10.sup.8 113.6 120 172 licheniformis Bacillus 42.5 1*10.sup.8 113.6 No addition 172 licheniformis Bacillus 42.5 1*10.sup.8 113.6 120 172 coagulans

(140) Samples were incubated in a 52° C. thermostatic water bath.

(141) Results:

(142) After 116.5 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production

(143) TABLE-US-00019 Inoculation DM Lactic acid Acetic acid Total acid level Sucrose Galactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Bacillus smithii 42.5 3.3 0.1 3.4 5.2 1*10.sup.6 4 2 Bacillus smithii 42.5 2.2 0.1 2.3 5.3 Nd 2 0 (no α-gal) Bacillus 42.5 3.8 0 3.8 5.4 5*10.sup.7 3 2 licheniformis Bacillus 42.5 2.7 0 2.7 5.5 Nd 0.5 0 licheniformis (no α-gal) Bacillus 42.5 1.8 0.2 2.0 4.9 4*10.sup.8 1.5 1.5 coagulans nd: not determined

Example 8

(144) Testing Different Production Organisms at 60° C.

(145) Experimental Set-Up:

(146) TABLE-US-00020 Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain % of weight CFU/g DM g mg Ml Bacillus coagulans 42.5 1*10.sup.8 113.6 120 122 Bacillus smithii 42.5 1*10.sup.8 113.6 120 122 Geobacillus 35 1*10.sup.8 113.6 120 172 thermodenitrificans

(147) Samples were incubated in a 60° C. incubator.

(148) Results:

(149) After 44.5 and 116.5 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production

(150) TABLE-US-00021 Incubation Inoculation Time DM Lactic acid Acetic acid Total acids level Sucrose Galactose Strain Hours % % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Bacillus 116.5 42.5 1.3 0.2 1.5 5.3 6*10.sup.6 7 4 coagulans Bacillus 116.5 42.5 0.8 0.4 1.2 5.7 5*10.sup.6 7 4 smithii Geobacillus 44.5 35 2.0 0.2 2.2 5.2 9*10.sup.7 8 4 thermodenitrificans

Example 9

(151) Bioconversion with Alternative Biomasses

(152) Incubation at 37° C. for 42.5 to 45.5 hours

(153) TABLE-US-00022 Inoculation SBM RSM SSM Dry matter level (88% DM) (88% DM) (91% DM) α-galactosedase Water Strain % of weight CFU/g DM g g g mg Ml Lactobacillus 35 1*10.sup.8 113.6 — — 120 172 plantarum Lactobacillus 42.5 1*10.sup.8 113.6 — — 120 122 plantarum Lactobacillus 52 1*10.sup.8 113.6 — — 120 79 plantarum Lactobacillus 35 1*10.sup.8 90.1 22.8 — 120 172 plantarum Lactobacillus 42.5 1*10.sup.8 90.1 22.8 — 120 122 plantarum Lactobacillus 52 1*10.sup.8 90.1 22.8 — 120 79 plantarum Lactobacillus 35 1*10.sup.8 67.8 — 43.8 120 174 plantarum Lactobacillus 42.5 1*10.sup.8 67.8 — 43.8 120 124 plantarum Lactobacillus 52 1*10.sup.8 67.8 — 43.8 120 81 plantarum Bacillus 35 1*10.sup.8 68.2 — — 72 103 coagulans Bacillus 42.5 1*10.sup.8 113.6 — — 120 122 coagulans Bacillus 55 1*10.sup.8 68.2 — — 72 41 coagulans Bacillus 35 1*10.sup.8 90.1 22.8 — 120 172 coagulans Bacillus 42.5 1*10.sup.8 90.1 22.8 — 120 122 coagulans Bacillus 52 1*10.sup.8 90.1 22.8 — 120 79 coagulans Bacillus 35 1*10.sup.8 67.8 — 43.8 120 174 coagulans Bacillus 42.5 1*10.sup.8 67.8 — 43.8 120 124 coagulans Bacillus 52 1*10.sup.8 67.8 — 43.8 120 81 coagulans

(154) Results:

(155) After 42.5 to 45.5 hours of incubation the following results were obtained, showing growth, sugar conversion and acid production

(156) TABLE-US-00023 Inoculation DM Bio- Lactic acid Acetic acid Total acids level Sucrose Galactose Strain % mass % of DM % of DM % of DM pH CFU/g DM % of DM % of DM Lactobacillus 35 SBM 7.2 0.1 7.3 4.1 .sup. 3*10.sup.10 0 2.4 plantarum Lactobacillus 42.5 SBM 6.8 1.2 8.0 4.4 .sup. 1*10.sup.10 0 1.8 plantarum Lactobacillus 52 SBM 6.6 1.2 7.8 4.5 .sup. 1*10.sup.10 0 1.8 plantarum Lactobacillus 35 SBM/ 7.3 0.9 8.2 4.4 5*10.sup.9 0 1 plantarum RSM Lactobacillus 42.5 SBM/ 6.8 1.0 7.8 4.4 5*10.sup.9 0 1 plantarum RSM Lactobacillus 52 SBM/ 5.9 0.9 6.8 4.5 7*10.sup.9 0.8 1 plantarum RSM Lactobacillus 35 SBM/ 6.5 0.7 7.2 4.4 4*10.sup.9 0 1 plantarum SSM Lactobacillus 42.5 SBM/ 6.3 0.7 7.0 4.4 4*10.sup.9 0 1 plantarum SSM Lactobacillus 52 SBM/ 5.7 0.7 6.4 4.4 4*10.sup.9 0.8 1 plantarum SSM Bacillus 35 SBM 6.9 1.2 8.1 4.5 nd 0 1.3 coagulans Bacillus 42.5 SBM 6.5 1.3 7.8 4.5 7*10.sup.9 0 0.5 coagulans Bacillus 55 SBM 3.7 0.8 4.5 5.1 nd 2 2 coagulans Bacillus 35 SBM/ 7.2 0.9 8.1 4.4 2*10.sup.9 0 0.5 coagulans RSM Bacillus 42.5 SBM/ 6.3 1.0 7.3 4.4 3*10.sup.9 0.3 0.8 coagulans RSM Bacillus 52 SBM/ 5.6 0.9 6.5 4.5 2*10.sup.9 1 1 coagulans RSM Bacillus 35 SBM/ 6.1 0.7 6.8 4.4 2*10.sup.9 0 0 coagulans SSM Bacillus 42.5 SBM/ 5.8 0.7 6.5 4.4 5*10.sup.9 0.3 0.5 coagulans SSM Bacillus 52 SBM/ 4.7 0.7 5.4 4.6 3*10.sup.9 1 1 coagulans SSM

Example 10

(157) Pilot Scale Bioconversion

(158) Incubator:

(159) The incubator was a pilot scale vertical reactor with a total volume of 2.0 m.sup.2. The incubator was equipped with a temperature probe at the inlet as well as at the outlet.

(160) Incubation Mixture:

(161) The incubator was incubated with a preheated mixture of 250 kg soya bean meal (88% DM); 264 g α-galactosidase from Bio-Cat (12,500 U/g), dry formulation of Bacillus coagulans to reach a final inoculation level of 1*10.sup.7 cells/g DM, and 268 litre tap water. The ratio wet bulk density/dry bulk density of the incubation mixture was 0.88. This resulted in a DM of 42.5% of the incubation mixture.

(162) Test Procedure:

(163) After filling of the reactor, it was flushed with N.sub.2 gas, to get rid of O.sub.2. The biomass was incubated at 60 hours at 37° C.

(164) Results:

(165) After 60 hours a product comprising 7.5% of DM lactic acid and 1.3% of DM acetic acid was obtained.

(166) pH had dropped to 4.6.

Example 11

(167) Large Scale Bioconversion

(168) Incubator:

(169) The reactor used was a vertical cylinder with an effective height of 7.3 m and a diameter of 4.3 m.

(170) 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.

(171) 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.

(172) Testing Uniform Plug Flow

(173) 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.