Means and methods for hydrolyzing carbohydrates present in raw materials containing carbohydrates

Abstract

The present invention relates to a method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, to a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, to a method for simultaneously liquifying and saccharifying starch, a method for simultaneously liquifying, saccharifying and fermenting starch, and to a method for producing a biomass, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes on a medium comprising complex carbohydrates as a major carbon source.

Description

[0001] Means and methods for hydrolyzing carbohydrates present in raw materials containing carbohydrates

[0002] Field of the invention

[0003] The present invention relates to a method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, to a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, to a method for simultaneously liquifying and saccharifying starch, a method for simultaneously liquifying, saccharifying and fermenting starch, and to a method for producing a biomass, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes on a medium comprising complex carbohydrates as a major carbon source.

[0004] Background of the invention

[0005] Fermentation is a metabolic process by which substances containing carbon are modified by the action of enzymes from organisms. Many microorganisms, such as yeast and bacteria, produce different compounds during fermentation that have industrial and / or commercial use such as organic acids, alcohols, polymers and proteins. Moreover, many food products deemed usual in the human diet (like bread, cheese, soy sauce, chocolate...) result from fermentation.

[0006] Carbohydrates are a major substrate for microbial fermentation, with glucose being easily consumed by many species of bacteria, yeast and fungi. However, other carbon sources, namely disaccharides and longer C chain sugars, must be enzymatically processed to release the fermentable sugars (monosaccharides) that the microbial cells can further metabolize at industrially relevant rates.

[0007] These cells can produce enzymes that are used to chemically modify the nutrient sources so that they can be effectively used, but different microorganisms produce different enzymes with different kinetic properties, making them better adapted to grow in certain substrates and fermenting conditions than in others. Furthermore, using complex media such as sidestreams from other industries can be an effective way to reduce costs of production and promote circularity and environmental practices. However, the use of these substrates can also bring further challenges since the carbohydrates present in these sidestreams can be of very different nature (in opposition to a media with a single defined carbon source) and more challenging to process. This poses a challenge for the industrial production of certain products through fermentation of microorganisms that are not able to readily metabolize these substrates at rates deemed economically feasible for the industry. Solutions to facilitate the consumption of more complex substrates can carry extra costs, such as adding commercial enzymes for preprocessing the substrate, or high energy consumption to achieve the optimal temperature for enzyme activity.

[0008] An example of such a complex substrate is starch, one of the dominant biomass carbon sources on earth that is constituted by glucose molecules linked by glycosidic bonds and is available in several sidestreams. For it to be used as a fermentation substrate, it must be processed which typically involves an initial gelatinization at high temperatures to release the molecules of starch, followed by liquefaction through enzymatic degradation. The maltodextrines that result from this step are further processed into dextrose during saccharification, after which microbial fermentation starts with the consumption of the monosaccharide. Traditionally, these steps are performed sequentially at high temperature and specific pH range, requiring enzymes active at those specific conditions.

[0009] Different solutions have been procured to decrease the energy and overall production cost of the starch processing industry. Namely, US10351836B2 discloses alpha-amylase variants that have an improved stability at low pH and / or high temperatures, in particular at low calcium concentrations, and that can be used to liquefy the starch containing material prior to fermentation. However, the following processes of saccharification and fermentation still require extra enzymes and a fermenting microorganism. Document US20210040523A1 discloses isolated polypeptides with glucoamylase activity that can be added to saccharify starch-containing materials at low pH, and that can be used during simultaneous saccharification and fermentation (SSF). Also in this case, despite the increased efficiency in the process of sugar hydrolysis there is the need to add a fermenting microorganism or use an engineered strain that can ferment and produce the enzyme. The use of enzymes that are active at low temperatures has also been proposed, like the one disclosed in CN 104962503B, regarding a Bacillus sp ZE01 capable of producing an amylase that is active at low temperature. The same principle concerns the use of Raw- starch digesting amylases (RSDA) that can hydrolyze raw starch at lower temperatures, like the one reported by Xu Q-S et al, 2016 or Ubi DS et al, 2023. However, despite the advances proposed by the above-mentioned inventions, their application has different drawbacks depending on the case, such as slow catalysis, incomplete degradation of the starch molecule, inhibition by glucose and maltose or the need to add another enzyme or fermenting organism.

[0010] Another relevant fermentation feedstock is sucrose, a disaccharide composed of sucrose and glucose subunits. A sucrose containing media can comprise not only solutions with defined components and pure sucrose, but also sidestreams from other industries like juice or molasses from sugar cane, sugar beet or any other sucrose-containing plant material. While many microorganisms are capable of hydrolyzing sucrose into glucose and fructose by the action of enzymes such as invertases and levansucrases, this hydrolysis is very often incomplete and / or it can take several hours (Khattab SM 2016, Bersaneti GT et al, 2017). This fact implies that a part of the energy source is wasted in the process and / or that there is an increase in the time needed for the fermentation to occur. A strategy to improve this process is disclosed by EP2652122A2 about a method to improve the capacity of a biocatalyst to transport and metabolize sucrose.

[0011] Summary of the invention

[0012] Accordingly, the present invention is based, at least in part, on a surprising discovery that certain bacteria are capable of secreting enzymes that can hydrolyse complex carbohydrates to fermentable carbohydrates at rates deemed novel for the field. It has been further established by the present inventors that the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented, which also may be referred to as a composition comprising carbohydrate-hydrolyzing enzymes secreted by the bacteria secreting carbohydrate-hydrolyzing enzymes, and in particular in the case of Vibrio spp and Priestia spp bacteria, that such composition / such medium is particularly useful in hydrolyzing carbohydrates present in certain raw materials, for example a fast and complete hydrolysis of sucrose present in molasses or in the processes known as simultaneous starch liquefaction, saccharification and fermentation. Such a process has significant advantages in comparison to the processes of prior art, which require a cascade of biotechnological use of different enzymes, and cycling / changing of the reaction conditions, such as pH or temperature, for each step catalyzed by a different enzyme. In contrast, according to the method of the invention, different hydrolysis steps, as required in starch liquefaction and saccharification, can be performed simultaneously. Moreover, the rate of hydrolysis of complex sugars to fermentable carbohydrates is higher, which is reflected in a faster and more complete consumption of the carbohydrates. Thus, the medium according to the present invention, and the methods of the present invention using the same, are particularly advantageous as they allow robust and fast hydrolysis of carbohydrates.

[0013] The present invention is summarized in the following embodiments.

[0014] In a first embodiment, the present invention relates to a method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, the method comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented.

[0015] Brief description of figures

[0016] The invention is illustrated by using the following figures. These are however not meant to be considered limiting and merely serve illustrative purposes.

[0017] Fig. 1 shows a graph presenting bacterial growth (increase in relation to population at the beginning of the fermentation) and starch consumption over time for Priestia flexa during fermentation.

[0018] Fig. 2 shows a graph presenting the variation in concentration of starch derived carbohydrates in relation to initial concentration during fermentation with a medium with starch as a carbon source (see Fig. 1 for reference) for Priestia flexa.

[0019] Fig. 3 shows alpha-amylase activity of the fermentation broth obtained in the fermentation of Priestia flexa strain with a medium with starch as a carbon source (see Fig. 1 for reference).

[0020] Fig. 4 shows the concentration of unhydrolyzed starch over time in a sample of corn starch in contact with the fermentation broth obtained in the fermentation of Priestia flexa strain on corn starch

[0021] Fig. 5 shows change in concentration of sucrose, glucose and fructose over time when the supernatant and / or fermentation broth from a fermentation of Vibrio natriegens using molasses is in contact with a sucrose solution Fig. 6 shows concentration of sucrose, glucose and fructose over time when the supernatant and / or fermentation broth from a fermentation of Priestia flexa using molasses is in contact with a sucrose solution

[0022] Fig. 7 presents the SDS page gel of the fermentation broth obtained in the fermentation of Priestia flexa in molasses.

[0023] Detailed description of the invention

[0024] As mentioned before, in one embodiment, the present invention relates to a method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented.

[0025] As understood herein, carbohydrates refer collectively to polysaccharides and monosaccharides. Polysaccharides are herein preferably understood as molecules comprising more than 1 monosaccharide molecules, connected to each other through glycosidic bond. In other words, polysaccharides may be understood as comprising more than 1 monosaccharide moiety. Monosaccharide is preferably defined as a simple sugar, that is a compound of a linear and unbranched carbon skeleton with one carbonyl functional group and one hydroxyl functional group on each of the remaining carbon atoms. For certain carbon atoms, hydroxy group may be absent. Accordingly, a monosaccharide is a compound of formula H-(CHX)n-(C=0)-(CHX)m-H, wherein n+m+1 is preferably selected from 3, 4, 5, 6 and 7, wherein each X is independently H or -OH, provided that at least two instances ofX are OH and not more than 2 instances of X are H, preferably wherein not more than 1 instance of X is H, more preferably wherein each X is - OH. As it is further apparent to the skilled person monosaccharides may exist in a circular form, by formation of cyclic hemiacetals, referred to as pyranoses (when 6-membered ring is present) or furanoses (when 5-membered ring is present). Preferably, the monosaccharide is a hexose or a pentose. A hexose is a monosaccharide as defined hereinabove wherein n+ +1 = 6, and preferably wherein each instance of X is -OH. A pentose is a monosaccharide as defined hereinabove, wherein n+m+-1 = 5 and preferably wherein each instance of X is -OH). The skilled person recognizes that monosaccharides may also include compounds as defined herein, which have been further oxidized, for example uronic acids. As understood herein, hydrolyzing the carbohydrates refers to any reaction wherein a glycosidic bond in a polysaccharide compound reacts with water to yield smaller carbohydrate molecules, in particular to yield monosaccharide molecules. As encompassed by the present invention, said hydrolysis is in particular hydrolysis catalyzed by enzymes, which are described herein in detail.

[0026] As referred to herein, the medium in which the bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented or, in other words, the medium in which the bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented comprises hydrolytic enzymes of bacterial origin, produced by said bacteria upon their fermentation. As apparent to the skilled person, the medium is preferably suitable for use in bacterial culture, and in particular in cultivation / fermentation of bacteria secreting carbohydrate-hydrolyzing enzymes.

[0027] The medium is not particularly limited, and the skilled person is capable of formulating a medium that is suitable for use in bacterial culture. Such a medium, suitable for bacterial culture, in particular liquid bacterial culture, as apparent to the skilled person, necessarily includes a carbon source, water, salts and trace elements, and a source of amino acids and nitrogen. Carbon source may be for example glucose. However, other carbon-containing molecules, such as glycerol, can also be used as a carbon source. A source of amino acid and nitrogen may for example be yeast extract, beef extract, or peptone.

[0028] Preferably, before subjecting to use according to the present invention, bacterial cells and bacterial cell debris (understood to constitute insoluble remaining of dead bacterial cells) are removed from the medium. This can be achieved by any biomass separation or biomass harvesting methods apparent to the skilled person, for example by centrifugation and / or filtration. However, the present invention also encompasses the embodiments wherein the bacterial cells (which may be viable or not viable) are still present in the medium. Furthermore, it is apparent to the skilled person that the medium of the present invention may have undergone further processing steps, in particular concentration and / or heat inactivation (which is to be conducted so that the remaining bacterial cells in the medium are heat- inactivated while the activity of hydrolytic enzymes is not, or substantially not, affected).

[0029] Accordingly, the medium may comprise cells and / or cell debris. However, the medium may also be free, or substantially free, of cells and / or cell debris. Said hydrolytic enzymes are enzymes capable of catalyzing the reaction of carbohydrate hydrolysis, as defined herein. Preferably, said carbohydrate-hydrolyzing enzymes are selected from invertase, levansucrase, levanase, pullulanase (other that produces levan), amylase (such as alpha-amylase, betaamylase, or gamma-amylase), cellulase and hemicellulase.

[0030] As understood herein, invertase is an enzyme that hydrolyzes (i.e., catalyzes the reaction of hydrolysis) sucrose into glucose and fructose. Invertase may also be referred to as -d- fructofuranosidefructohydrolase, 0-fructofuranosidase, sucrase, or saccharase. As apparent to the skilled person, invertase can be classified as EC 3.2.1.26.

[0031] As understood herein, levansucrase is an enzyme that catalyzes the reaction involving hydrolysis of sucrose and incorporation of the resulting fructose into a polysaccharide, according to formula: sucrose + (2, 6-beta-D-fructosyl)nglucose + (2,6-beta-D-fructosyl)n+i

[0032] Thus, accordingly, levansucrase can be also described as glycosyltransferase with sucrase activity. Other systematic names that can be used to describe this enzyme are sucrose:2,6-beta-D-fructan 6-beta-D- fructosyltransferase. Other names in common use include sucrose 6-fructosyltransferase, beta-2, 6- fructosyltransferase, and beta-2, 6-fructan:D-glucose 1-fructosyltransferase. As apparent to the skilled person, levansucrase can be classified as EC 2.4.1.10.

[0033] As understood herein, levanase is an enzyme that hydrolyzes levan into levan-type fructooligosaccharides (L-FOSs) being fructose its predominant product. It belongs to the class of Levan-degrading enzymes, and as apparent to the skilled person, invertase can be classified as EC 3.2.1.65.

[0034] As understood herein, pullulanase is an enzyme that hydrolyses glycosidic bonds within pullulan. Type I pullulanases specifically attack a-1 ,6 linkages, while type II pullulanases are also able to hydrolyse a-1 ,4 linkages. As understood herein, and as apparent to the skilled person, pullulan is a polysaccharide wherein three glucose units in maltotriose are connected by an a-1,4 glycosidic bond, whereas consecutive maltotriose units are connected to each other by an a-1 ,6 glycosidic bond. As it is apparent to the skilled person, pullulanase can be classified as EC 3.2.1.41. As understood herein, amylase is an enzyme that catalyzes the hydrolysis of starch into simpler sugars. All amylases are glycoside hydrolases that act on an a-1 ,4 glycosidic bond. The a-amylases, also referred to as 1 ,4-a-D-glucan glucanohydrolase or glycogenase, are calcium metalloenzymes that act at random locations along the starch chain. The a-amylases, as apparent to the skilled person, can be classified as EC 3.2.1.1. 0-Amylase, also referred to as 1 ,4-a-D-glucan maltohydrolase, glycogenase, or saccharogen amylase, catalyzes the hydrolysis of the second a-1 ,4 glycosidic bond, cleaving off two glucose units, i.e. a maltose disaccharide, at a time. As apparent to the skilled person, 0-amylase can be classified as EC 3.2.1.2. y-Amylase, as understood herein, also referred to as Glucan 1 ,4-a-glucosidase; amyloglucosidase; exo-1,4-a-glucosidase; glucoamylase; lysosomal a-glucosidase; or 1 ,4-a-D-glucan glucohydrolase) cleaves a(1— 6) glycosidic linkages, as well as the last a-1 ,4 glycosidic bond at the nonreducing end of amylose and amylopectin, yielding glucose. As it is apparent to the skilled person, y- amylase can be classified as EC 3.2.1.3.

[0035] As understood herein, cellulase is any of several enzymes that catalyzes cel lulolysis, the decomposition of cellulose and other polysaccharides. They promote the endohydrolysis of (1 — ->4)-fJ-D-glucosidic linkages, releasing monosaccharides such as 0-glucose or shorter polysaccharides and oligosaccharides. There are several types of cellulases, depending on the type of reaction they catalyze. Endocellulases randomly cleave internal bonds at amorphous sites. As it is apparent to the skilled person, endocellulases can be classified as EC 3.2.1.4. Exocellulases or cellobiohydrolases attack the ends of cellulose chains and cleave two to four glucose units from the end in a sequential maner. As it is apparent to the skilled person, exocellulases can be classified as EC 3.2.1.91. - glucosidades hydrolyse the cellubiose (a disacharide) into two individual glucose molecules. As it is apparent to the skilled person, 0- glucosidades can be classified as EC 3.2.1 .21 .

[0036] As understood herein, hemicellulase is any of several enzymes that specifically degrade hemicellulose, by hydrolyzing the glycosidic bonds within it. There are different hemicelluloses that are modified by different hemicellulases such as xylanases and mannanases. As it is apparent to the skilled person, xylanases can be classified as EC 3.2.1.8. and mannanases can be classified as EC 3.2.1.78.

[0037] As understood herein, starch is a polysaccharide composition comprising (or substantially consisting of) amylose and amylopectin. Amylose is a linear polysaccharide made of a-D-glucose units, bonded to each other through a(1— >4) glycosidic bonds. Amylose makes up typically 20 to 30 w / w% of starch weight. Amylopectin is a branched polysaccharide made of a-D-glucose units, wherein the glucose units are linked in a linear way with a(1— >4) glycosidic bonds. Branching usually occurs at intervals of 25 residues. At the places of origin of a side chain, the branching that takes place bears an a(1— >6) glycosidic bond. This results in a water-soluble molecule that can be quickly degraded as it has many end points onto which enzymes can attach. Further a(1 — >3)linkages have also been reported to be present in case of amylopectin.

[0038] As understood herein, bacteria capable of secreting carbohydrate-hydrolyzing enzymes, which may also be referred to as bacteria secreting carbohydrate-hydrolyzing enzymes, may be bacteria selected from the bacterial strain Bacillus spp or bacteria selected from the bacterial strain Priestia spp. It is to be understood that several organisms are described as belonging to both these genera, or have been reclassified from one to another genera. For example, Bacillus flexus is presently referred to as Priestia flexa. Similarly, Bacillus megaterium, an organism very important from the biotechnological point of view due to its ability of producing vitamin B12, is more recently referred to as Priestia megaterium. Thus, in one embodiment, the bacterial strain may be Bacillus spp. strain. In one embodiment, the bacterial strain may be Priestia spp. strain. Accordingly, one exemplary and particularly preferred strain of such bacterium is Bacillus flexus, which may also be referred to as Priestia flexa.

[0039] However, other Bacillus spp can also be used in the method of the present invention. Accordingly, another suitable Bacillus include Bacillus stearothermophilus and Bacillus subtilis. Further suitable bacteria capable of secreting carbohydrate-hydrolyzing enzymes, which may also be referred to as bacteria secreting carbohydrate-hydrolyzing enzymes, may be selected from Vibrio spp (such as Vibrio natriegens) and Geobacillus spp (such as Geobacillus LC300) bacteria.

[0040] Alternatively, the bacteria capable of secreting carbohydrate-hydrolyzing enzymes may also be characterized as follows. Accordingly, the bacteria capable of secreting carbohydrate-hydrolyzing enzymes are characterized by a growth rate of at least 0.4 h1. Preferably, fast-growing bacterial strains, for which the growth rate in the process, also referred to as the growth rate, as defined herein, is >0.85 h-1, are used within the method of the present invention. Preferably, the bacteria, or the bacterial strain, used in step (a) is / are characterized by a growth rate in process of at least 0.4 h1, preferably at least 0.85 h1, more preferably at least 1.0 h1. Also encompassed by the present invention is an embodiment, wherein said bacteria / said bacterial strain are / is characterized by a growth rate in process of at least 2 h-1, at least 3 h1, or of at least 4 h1. The growth rate as understood herein, unless stated otherwise, is the growth rate in the process, as defined herein. As it is apparent to the skilled person, the growth rate in the process may depend on the growth medium, and be different for minimal medium and rich medium, as understood to the skilled person.

[0041] The growth rate in the process is defined herein as the growth rate achievable under the fermentation conditions. I n other words, for a microorganism to be suitable for the use within the method of the present invention it must be possible to cultivate it with the growth rate as defined herein in the fermentation conditions, in particular on a specific growth medium. It is particularly preferred that the growth rate meant herein refers to the growth on the medium as used in, and as referred to in step a) of the method of the present invention, preferably before further processing steps such as concentration and / or heat inactivation. The growth rate in the process as defined herein refers to a growth rate of a particular microorganism, herein particular bacterium, unless indicated otherwise. In the case of co-cultivation of more than one microorganism, the growth rate may refer to a combined growth rate of all the microorganism in the culture, and describe the increase in combined biomass over time.

[0042] As apparent to the skilled person, the growth rate in the process is preferably determined by plotting the In (natural logarithm) of biomass amount as a function of time and using a linear regression to calculate the slope in the linear range that corresponds to the exponential growth phase.

[0043] The growth rate in the process may also be referred to as p [IT1].

[0044] Suitable examples of such bacteria include Bacillus spp., Priestia spp., Vibrio spp., and Geobacillus spp. strains. Thus preferably, in the method of the present invention the bacteria secreting carbohydratehydrolyzing enzymes are selected from Vibrio spp, in particular Vibrio natriegens, Geobacillus spp, in particular Geobacillus LC300, Priestia spp, in particular Priestia flexa and Bacillus spp, in particular Bacillus subtilis. Preferably, the at least one microorganism strain is selected from Vibrio spp, in particular Vibrio natriegens. Further suitable examples of Geobacillus spp. may include Geobacillus uralicus, and Geobacilus stearothermophilus. Further suitable examples of Priestia spp. may include Priestia megaterium. Further suitable examples of Bacillus spp may include Bacillus coagulans and Bacillus stearothermophilus.

[0045] In a particularly preferred embodiment of the present invention, the bacteria capable of secreting carbohydrate hydrolysing enzymes (i.e., the bacteria secreting carbohydrate hydrolysing enzymes) are selected from Priestia flexa, Vibrio natriegens and Geobacilus stearothermophilus. Preferably, the bacteria capable of secreting carbohydrate hydrolysing enzymes (i.e., the bacteria secreting carbohydrate hydrolysing enzymes) are selected from Priestia flexa, and Vibrio natriegens. Accordingly, said bacteria may be Priestia flexa. Alternatively, said bacteria may be Vibrio natriegens. Alternatively, said bacteria may be Geobacilus stearothermophilus.

[0046] Thus, as surprisingly discovered by the present inventors, medium in which a fast-growing microbe was grown, as defined in the foregoing, is characterized by hydrolytic activity against carbohydrates and upon being contacted with a raw material containing carbohydrates, can be useful in hydrolysing carbohydrates contained in said raw materials.

[0047] As understood herein, fermentation may refer to any type of microbial cultivation in or on a medium. In particular, the term fermentation includes both aerobic and anaerobic fermentation. As known to the skilled person, the fermentation may be performed as a batch process, fed-batch process or (semi)continuous process. The conducting of fermentation is a routine operation to the skilled person. Particular fermentation types, which are of importance to the method of the present invention, are described in the following.

[0048] The batch processes are characterized by lack of inflow of material into the fermentation vessel. In a batch process, all nutrients are provided at the beginning of the cultivation, without adding any more in the subsequent bioprocess. During the entire bioprocess, no additional nutrients are added with the exception of gases, acids and bases. In certain embodiments, an antifoaming agent may also be added. The bioprocess then lasts until one of the nutrients required for growth becomes limiting. This strategy is suitable for rapid experiments such as strain characterization or the optimization of nutrient medium. The disadvantage of this convenient method is that the biomass and product yields are limited. Since the carbon source and / or oxygen transfer are usually the limiting factor, the microorganisms are not in the exponential growth phase for a long time. After the end of a bioprocess run in batch mode, the biomass and / or medium, in particular only the biomass or the medium is harvested and appropriately processed to obtain the desired product. From the bioreactor point of view, the process is repeatedly interrupted by cleaning and, when needed, sterilization steps, and the biomass is only produced in stages.

[0049] In the fed batch process, substrate, nutrients and other substances may be added (preferably, in a form of a concentrated solution) into the fermentation vessel, to extend the possible culture time or increase the yield, among others. The advantage of feeding during cultivation is that it allows to achieve higher product quantities overall. Under specific growth conditions, the microorganisms and / or cells constantly double and therefore follow an exponential growth curve. Therefore, in certain embodiments the feed rate may be increased exponentially as well. Generally, the substrate is pumped from the supply bottle (or a feed tank) into the culture vessel, for example through a silicone tube (or a sterilizable piping). The user can either manually set the feed at any time (linear, exponential, pulse-wise), or add nutrients when specific conditions are met, such as when a certain biomass concentration is reached or when a nutrient is depleted. The fed-batch process offers a wide range of control strategies and is also suitable for highly specialized applications. However, it may increase the processing time and potentially leads to inhibition through the accumulation of toxic by-products. At high cell density, limitation through limited oxygen transfer from the gas phase may also occur.

[0050] Preferably, in the method of the present invention the fermentation is operated as a continuous process. After a batch growth phase, an equilibrium is established with respect to a particular component (also called steady state). Under these conditions, as much fresh culture medium is added, as it is removed (chemostat). These bioprocesses are referred to as continuous cultures and are particularly suitable when an excess of nutrients would result in inhibition due to, e.g., acid or ethanol build up or excessive heating. Other advantages of this method include reduced product inhibition and an improved space-time yield. When medium is removed, cells are harvested, which is why the inflow and outflow rates must be less than the doubling time of the microorganisms. Alternatively, the cells can be retained in a wide variety of ways (for example, in a spin filter), which is called perfusion. In a continuous process, the space-time yield of the bioreactor can be even further improved compared to that of a fed-batch process. However, the long cultivation period also increases the risk of contamination and long-term changes in the cultures. The three most common types of continuous culture are chemostat (The rate of addition of a single growthlimiting substrate controls cell multiplication), turbidostat (an indirect measurement of cell numbers - turbidity or optical density - which needs an additional sensor but is driven by real-time feedback, controls addition and removal of liquid), and perfusion (this type of continuous bioprocessing mode is based on either retaining the cells in the bioreactor or recycling the cells back to the bioreactor; fresh medium is provided and cell-free supernatant gets removed at the same rate).

[0051] In other words, the present invention requires the use of the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented. The medium can be as described herein, and as exemplified in the present application. As explained herein, the medium used in the method of the present invention is modified by fermentation of said bacteria in that said bacteria secrete the carbohydrate-isolating enzymes into the medium. The skilled person is capable of incubating the bacterial cells in the medium, i.e., fermenting the bacterial cells in the medium, until desired, required or suitable concentration thereof is achieved. To this end, this can be done for example by monitoring the activity of the enzymes in medium during the growth. Once the maximum activity (or desired activity) is obtained, the fermentation of said bacteria may be terminated, for example by inactivating or separating the bacteria. However, the bacteria may also be left in the medium, as also encompassed by the present invention in an alternative embodiment.

[0052] As apparent to the skilled person, the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented may also be referred to as a composition comprising carbohydrate- hydrolyzing enzymes secreted by the bacteria secreting carbohydrate-hydrolyzing enzymes.

[0053] The present invention is based, at least in part, on a surprising discovery of the present inventors that the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented, which also may be referred to as a composition comprising carbohydrate-hydrolyzing enzymes secreted by the bacteria secreting carbohydrate-hydrolyzing enzymes, and in particular in the case Vibrio spp and Bacillus spp bacteria, or in the case of Vibrio spp and Priestia spp bacteria, that such composition / such medium is particularly useful in hydrolyzing carbohydrates present in certain raw material, for example in the processes known as starch liquefaction and saccharification.

[0054] As understood herein, the raw material containing carbohydrates is not particularly limited and any such raw material that is apparent to the skilled person can be used. It is preferred that the raw material containing carbohydrates, as referred to herein, is a solid material. However, the present invention also encompasses embodiments, when liquid raw material containing carbohydrates is used, for examples molasses.

[0055] The raw material containing carbohydrates may comprise starch. Specific hydrolytic enzymes that hydrolyze starch are disclosed herein. Thus, in one embodiment, the method of the present invention for hydrolyzing carbohydrates present in raw materials containing carbohydrates may be a method for hydrolyzing starch. In other words, the method of the present invention for hydrolyzing carbohydrates present in raw materials containing carbohydrates may be a method for simultaneously liquifying and saccharifying starch. As understood herein, the liquefying (or liquefaction) of starch is a hydrolysis process that converts starch into dextrins, i.e. lower molecular weight polysaccharides that are made of D-glucose units linked by a- (1— >4) and / or a-(1 — >6) glycosidic bonds. In the process of liquefaction of starch, the highly viscous gelatinized starch granules are dispersed, through their partial enzymatic hydrolysis. Further in the process maltose and glucose may be produced. As obtained products are significantly more water soluble than starch, the process is referred to as liquefaction. As understood herein, saccharification is a process that follows liquefaction of starch, wherein the remaining polysaccharides are further hydrolysed into maltose and glucose. The combined process of liquefaction and saccharification of starch is known to the skilled person to be catalyzed by amylase enzymes, and optionally also pullanase enzymes, as defined herein.

[0056] Thus, in one embodiment of the present invention, the present invention relates to a method for simultaneously liquifying and saccharifying starch, the method comprising the steps of the method for hydrolyzing carbohydrates present in raw materials containing carbohydrates (herein, raw materials containing starch)

[0057] Encompassed by the present invention is further an embodiment, wherein said raw material containing carbohydrates may comprise sucrose. The sucrose-hydrolyzing and sucrose utilizing enzymes are disclosed and discussed herein.

[0058] As encompassed by the present invention, it is preferred that the raw material containing carbohydrates, which also may be referred to as carbohydrate raw material, is selected from sugarcane, sugarbeet, soybean and fababean molasses and / or juice, permeates from the dairy industry, potato peels, potato pulp, beet pulp, pea starch, wheat starch, grey starch, cassava starch, peels from different fruits such as (but not limited to) oranges and apples, stillage, brewers spent grain, corn fermented protein, corn gluten feed, corn gluten meal, corn protein concentrate, distiller's dried grains with solubles (DDGS), and soy protein meal. More preferably, the raw material containing carbohydrates, which also may be referred to as carbohydrate raw material, is selected from sugarcane, sugarbeet, soybean and fababean molasses and / or juice, permeates from the dairy industry, potato peels, potato pulp, pea starch, wheat starch, grey starch, cassava starch, peels from different fruits such as (but not limited to) oranges and apples, stillage, brewers spent grain, corn fermented protein, corn gluten feed, corn gluten meal, corn protein concentrate, distiller's dried grains with solubles (DDGS), and soy protein meal. As understood herein, said raw material containing carbohydrates may include wastes and side-streams from other industries where the original raw material, as any one listed hereinabove, has been used, for example such as wheat waste, or any waste product from the process of making beer, such as brewers spent grain. It is further to be understood that raw material containing carbohydrates may refer to more than one material, for example a mixture of two or three materials, as defined hereinabove.

[0059] It is further envisaged in the method of the present invention that the raw material containing carbohydrate, as referred to herein, is a lignocellulosic material. As understood herein, the lignocellulosic material comprises cellulose, preferably the lignocellulosic material comprises lignin, hemicellulose and cellulose.

[0060] As understood herein, cellulose is a polysaccharide consisting of a linear chain of several hundred to many thousands of (1 — >4) linked D-glucose units.

[0061] As encompassed by the present invention, the lignocellulosic material is selected from spent grain, cereal brans, cotton, cotton seed husks, bagasse, cocoa shells, cocoa, cocoa pods, cotton and oil press cakes from sunflower, peanut, hazelnut, palm oil, olive, shells and husks from nuts, grass and leaves waste, wood chips, wood pulp, paper, crop straws (including but not limited to rice, wheat, corn, cotton) coffee grounds, coffee husks, coffee silverskin, byproducts from the soy industry like soybean pulp (“okara”) and rapeseed.

[0062] As understood herein, the raw material containing carbohydrate may also be referred to as material containing carbohydrate, carbohydrate-containing material, composition comprising carbohydrate(s) and carbohydrate composition. The reference is herein made to a raw material to define that the material, or the composition, to be used in the method of the present invention, is in no way the final industrial product, but requires further processing to be used in industry. This definition is however not meant to be particularly limiting and any carbohydrate composition can be used in the method of the present invention. It is further noted that while preferably the raw material is an unprocessed material, potentially sourced directly from agriculture or another industrial process, potentially as a byproduct or a side stream, it is not excluded that said material is pre-processed in certain ways. In particular, the raw material, or the carbohydrate composition, or the like, as used herein, can undergo (i.e. has undergone, before application in the method of the present invention), pretreatment according to the method selected from washing, solvent-extraction, solvent-swelling, comminution, milling, steam pretreatment, explosive steam pretreatment, dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolvent pretreatment, biological pretreatment, ammonia percolation, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, and gamma irradiation.

[0063] As preferably understood herein, the method of the present invention includes the step of b) separating and isolating the hydrolyzed carbohydrates from the medium. As understood herein, step b) can be performed by any separation method known to the skilled person, which may include filtration, precipitation, crystallization and chromatography-based methods. In one exemplary embodiment, the water is removed from the medium, for example by evaporation, and the composition comprising solid carbohydrates is isolated. Accordingly, the steps of separating and isolating said hydrolyzed carbohydrates are not meant to be particularly limited, insofar the purity or the content of said hydrolyzed carbohydrates in the medium is increased.

[0064] However, as discussed herein, the present invention also refers to a method not including the step b), wherein the obtained composition comprising hydrolyzed carbohydrates may be used directly.

[0065] As required in step a) of the method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, said raw material is to be contacted with the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented. Any form of contacting, as referred to herein, and as compatible with the biotechnological application, is considered encompassed in the method of the present invention. Accordingly, the material containing carbohydrates may be suspended or dissolved in a solution, preferably an aqueous solution, and said medium in which bacteria secreting carbohydrate- hydrolyzing enzymes have been fermented can be mixed therewith or added thereto, and incubated, optionally under stirring or agitation. Alternatively, said medium in which bacteria secreting carbohydrate- hydrolyzing enzymes have been fermented can be directly contacted with the raw material containing carbohydrates. As further apparent herein, the medium can also be used while the bacteria secreting carbohydrate-hydrolyzing enzymes are still present in the medium or are still being fermented in said medium.

[0066] In one embodiment, the present invention relates to a medium in which bacteria secreting carbohydrate- hydrolyzing enzymes have been fermented. The medium is obtainable according to the process, as described herein. As understood herein, the medium may or may not include the bacterial cells. Further the present invention also encompasses the medium as referred to herein, which has further undergone certain further processing steps, for example involving inactivation of the bacteria, separation of the bacteria or concentration / dilution steps.

[0067] Preferably, the medium of the present invention comprises the carbohydrate-hydrolyzing enzyme selected from invertase, levansucrase, levanase, pullulanase (other that produces levan), amylase (such as alphaamylase, beta-amylase, or gamma-amylase), cellulase and hemicellulase. Said enzymes are as defined herein.

[0068] In one embodiment, the present invention relates to a composition comprising hydrolyzed carbohydrates, obtainable in the method for hydrolyzing carbohydrates present in raw materials containing carbohydrates of the present invention. Said composition preferably comprises fermentable sugars (in particular monosaccharides, such as glucose) and is highly applicable in the production of microbial culture media, as well as the production of biomass based on said microbial culture media.

[0069] In one embodiment, the present invention relates to a method for producing a biomass, the method comprising the steps of:

[0070] (i) providing a fermentation medium comprising the composition comprising hydrolyzed carbohydrates of the present invention;

[0071] (ii) cultivating at least one microorganism on the medium provided in step (i), and

[0072] (iii) harvesting the biomass.

[0073] The skilled person is capable of formulating a medium suitable for culturing the microorganism of choice. It is in particular apparent to the skilled person that the medium requires the carbon source, which is an essential element for the microbial, in particular bacterial growth. Thus, preferably, the medium provided in step (i) comprises the composition comprising hydrolyzed carbohydrates of the present invention as a major carbon source. As referred to herein, the major carbon source refers to a situation wherein said composition would constitute at least 50% w / w, preferably at least 60% w / w, more preferably at least 70% w / w, even more preferably at least 80%w / w, most preferably at least 90% w / w of the carbon content in the medium.

[0074] In one embodiment, the medium to be provided in step (i) may also be generated in situ, by contacting the carbohydrate-containing material with bacteria (preferably cultured, growing bacteria) secreting carbohydrate-hydrolyzing enzymes. It is to be understood that in such a setup, culturing of said bacteria would happen consecutively with culturing of the microorganism in step (ii), or the bacteria and said microorganism could also be co-cultured.

[0075] The skilled person is capable of performing the cultivation step (ii) of the method of the present invention, which may also be referred to fermentation of at least one microorganism on the medium provided in step (i). In particular, the conditions of the cultivation, including the medium and its pH, the temperature, aeration, duration of the culture, among others, can be selected by the skilled person. Furthermore, different ways of performing the fermentation, including the batch mode, fed batch mode and continuous fermentation, are described herein. Accordingly, as understood herein, the step (ii) can be performed as described herein for the fermentation.

[0076] Step (ii) of the method of the present invention, is not particularly limited and any microorganism, such as bacterium, algae, yeast or fungus, can be cultured. In one embodiment, the microorganism to be cultured in step (ii) is a plurality of co-cultured organisms, in particular two organisms to be co-cultured. In one embodiment, the organism to be cultured in step (ii) is bacterium secreting carbohydrate-hydrolyzing enzymes, as defined herein.

[0077] In step (iii) of the method of the present invention, the obtained biomass is harvested according to the methods known well to the skilled person.

[0078] Accordingly, in one embodiment, the present invention relates to the biomass, obtainable in the method of the present invention. In one embodiment, the present invention relates to a single cell protein comprising the biomass of the present invention. It is preferably to be understood that single cell protein consists substantially of non-viable cells of said biomass.

[0079] In one embodiment, the present invention relates to a method for producing a biomass, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes on a medium comprising complex carbohydrates as a major carbon source.

[0080] In another embodiment, the present invention relates to a method for producing biomass, the method comprising the step of preparing the growth medium, wherein said preparing of the growth medium comprises contacting the raw material containing carbohydrate with a medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented. It is to be understood that the medium in which the bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented is as defined herein. In particular said medium comprises hydrolytic enzymes of bacterial origin, produced by said bacteria upon their fermentation. Furthermore, it is to be understood that said medium may or may not include the bacterial cells, as described herein. It is further to be understood that the method comprises the step of cultivating the microorganism, as defined herein, on the growth medium, as defined herein.

[0081] According to the present invention, and as surprisingly demonstrated by the present inventors, certain bacteria are capable of secreting carbohydrate-hydrolyzing enzymes and can accordingly grow (be grown) on medium that includes complex carbohydrates. Accordingly, said bacteria can process the complex carbohydrates to obtain simple fermentable sugars, which are readily consumed in the process of fermentation. The cultivation of said cells is preferably as described herein in the context of bacterial fermentation / cultivation.

[0082] In one embodiment, the method of the present invention comprises a step of cocultivation of said bacteria capable of secreting carbohydrate-hydrolyzing enzymes with another microorganism. Said microorganism is not particularly limited and any microorganism, such as bacterium, algae, yeast or fungus, can be cocultured according to the method of the present invention.

[0083] In a particular embodiment, the present invention relates to a method for simultaneously liquifying, saccharifying and fermenting starch, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes, as described herein, on a medium comprising starch as a major carbon source.

[0084] In a particular embodiment, the present invention relates to a method of fermenting raw materials such as brewers spent grain, corn fermented protein, corn gluten feed, corn gluten meal, corn protein concentrate, distiller's dried grains with solubles (DDGS), soy protein meal, the method comprising the step of contacting the raw material containing carbohydrate with a medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented. As understood herein, fermenting refers to growing a biomass on said raw materials (either directly or indirectly). Thus, it is further to be understood that the method comprises the step of cultivating the microorganism, as defined herein, on the products obtained upon said step of contacting raw materials with said bacteria. The raw material containing carbohydrate, and the medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented, is as described herein. In particular said medium comprises hydrolytic enzymes of bacterial origin, produced by said bacteria upon their fermentation. Furthermore, it is to be understood that said medium may or may not include the bacterial cells, as described herein.

[0085] In a further embodiment, the present invention relates to the use of the biomass of the present invention or the single cell protein of the present invention, as described herein, in the production of animal feed, pet food, nutraceuticals, food products or flavor enhancers.

[0086] Animal feed is herein defined as a food product intended for nutrition of preferably domestic animals, especially of livestock and aquaculture. It can, for example, be manufactured in the solid form, e.g., in the form of pellets, or in any other suitable form for feeding animals.

[0087] Petfood as defined herein is intended for small domestic animals, including dogs, cats, mice, guinea pigs, domestic birds, aquarium fish. However, this list is not meant to be in any way limiting.

[0088] Nutraceutical, which also may be referred to as functional food as understood herein is defined as any food that goes beyond simple nutrition and has at least one specific targeted action to improve the health and / or well-being of the host and / or prevent pathological states in the host.

[0089] In a further embodiment, the present invention relates to the use of the composition comprising hydrolyzed carbohydrates in the production of sweeteners.

[0090] As referred to herein, the major carbon source refers to a situation wherein said starch would constitute at least 50% w / w, preferably at least 60% w / w, more preferably at least 70% w / w, even more preferably at least 80%w / w, most preferably at least 90% w / w of the carbon content in the medium.

[0091] Further embodiments of the invention are illustrated / disclosed in the following numbered items.

[0092] 1. A method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, the method comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented.

[0093] 2. The method of item 1 , further comprising the step of b) separating and isolating the hydrolyzed carbohydrates from the medium. The method of item 1 or 2, wherein the raw material containing carbohydrate comprises starch. The method of item 1 to 2, wherein the raw material comprising carbohydrate raw material comprises sucrose. The method of item 1 or 2, wherein the raw material containing carbohydrate comprises lignocellulosic material The method of any one of items 1 to 3, wherein hydrolyzing the carbohydrates present in a carbohydrate raw material comprises hydrolysis of starch. The method of item 6, wherein the method is a method for simultaneous liquefaction and saccharification of starch, preferably wherein the method is performed at a pH of 6.5 to 8.5 and at a temperature from 30 to 60 °C. The method of any one of items 1 to 7, wherein the raw material containing carbohydrate is selected from sugarcane, sugar beet, soybean and faba bean molasses and / or juice, permeates from the dairy industry, potato peels, potato pulp, pea starch, wheat starch, grey starch, cassava starch, peels from different fruits such as (but not limited to) oranges and apple, stillage, brewers spent grain, corn fermented protein, corn gluten feed, corn gluten meal, corn protein concentrate, distiller's dried grains with solubles (DDGS), and soy protein meal. The method of any one of items 1 to 8, wherein the bacteria capable of secreting carbohydratehydrolyzing enzymes are selected from Priestia spp (such as Priestia flexa), Bacillus spp (such as Bacillus subtilis), Vibrio spp (such as Vibrio natriegens) and Geobacillus spp (such as Geobacillus LC300) bacteria The method of any one of items 1 to 9, wherein the carbohydrate-hydrolyzing enzyme is selected from invertase, levansucrase, levanase, pullulanase, amylases, cellulase and hemicellulase. A medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, as described in any one of items 1 to 10. 12. A composition comprising hydrolyzed carbohydrates, obtainable in the method for hydrolyzing carbohydrates present in raw materials containing carbohydrates of any one of items 1 to 10.

[0094] 13. A method for producing a biomass, the method comprising the steps of:

[0095] (i) providing a fermentation medium comprising the composition comprising hydrolyzed carbohydrates of item 11;

[0096] (ii) cultivating at least one microorganism on the medium provided in step (i), and

[0097] (iii) harvesting the biomass.

[0098] 14. A method for producing a biomass, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes on a medium comprising complex carbohydrates as a major carbon source.

[0099] 15. A method for producing biomass from raw materials containing carbohydrates, the method comprising the step of contacting the raw material containing carbohydrate with a medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented.

[0100] 16. The method of item 15, wherein the raw material containing carbohydrate is selected from ethanol production side streams, such as corn gluten feed, corn gluten meal, corn protein concentrate, dried distillers grains with soluble (DDGS), and products derived from soybean processing such as soybean meal.

[0101] 17. The method of items 15 and 16, wherein the carbohydrates contained in raw materials containing carbohydrates are selected from starch, sucrose and lignocellulosic material, or a mixture of the same.

[0102] 18. A biomass, obtainable in the method according to any one of items 13 to 17.

[0103] 19. A method for simultaneously liquifying, saccharifying and fermenting starch, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes, as described in any one of items 1 to 10, on a medium comprising starch as a major carbon source. 20. Use of the biomass of item 18 in the production of animal feed, pet food, fermentation medium or supplement, food products, nutraceuticals, pharmaceuticals or flavour enhancers.

[0104] 21. Use of the composition of item 12 in the production of sweeteners.

[0105] The invention will be illustrated with the use of the following examples. These are not meant to be considered limiting the scope of protection in any way which is directed in the claims, and serve merely an illustrative purpose.

[0106] Examples

[0107] Example 1. Medium for culture with a fermenting organism of the current invention.

[0108] The fermenting microorganism of the present invention was pre-cultured and cultured in a minimal medium with glucose at a concentration between 2 and 5 g / L and yeast extract at a concentration of 1 g / L. The minimal medium for preculture and culture contains the elements shown in Table 1 at a concentration of at least the one defined as “minimum value” and at a maximum of the one defined as “maximum value”. The elements are in a form which enables bioavailability of the element, such as, but not limited to mineral, ionic or inorganic. An example medium is composed by 2 g / L of glucose, 1 g / L of yeast extract, 15g / L of NaCI, 0.097g / L of FeCI3-6H20, 0.026 g / L of MnCI2-4H20, 0.00043 g / L of CuCI2-2H20, 0.0006 g / L of CoCI2- 6H2O, 0.0006 g / L of Na2MoO4-2H2O, 0.5 g / L of MgSO4-7H2O, 1.6 g / L of KH2PO4, 6.25 g / L of NH4CI, 0.015g / L of CaCI2-2H2O, 0.0017 g / L of ZnCI2, 1 .25 g / L of (NH4)2SO4, 8.5 g / L of Na2HPO4The skilled person is capable of formulating a medium in which the elements will be present, as provided in the following. For the culture step it is added a carbon source at a concentration of 20g / L. It is to be understood that provided concentrations refers to final concentrations in the medium. The medium components are sterilized either by autoclaving (carbon source and yeast extract 121°C 20 min) or filtration.

[0109] Table 1 . Element composition of medium for preculture and culture Example 2: Fermentation of a fermenting organism of the current invention

[0110] This protocol is for the cultivation of a fermenting organism of the present invention in a medium with a carbon source rich in starch (refers to Fig. 1 and Fig. 2) or in sucrose using bench scale bioreactors

[0111] Preculture of fermenting organism of the present invention

[0112] 1. Prepare 50 ml of the preculture medium as described hereinabove (Example 1) and inoculate with the strain from a cryovial in a shake flask.

[0113] 2. Place the shake flasks in an incubator set at 37°C and 275 rpm and inoculate bioreactor when an OD600 value between 1.5 and 2 is reached.

[0114] Bioreactor batch fermentation

[0115] 1. Prepare the culture medium as described in Example 1 in a bioreactor.

[0116] 2. When the desired OD is reached, the preculture medium is inoculated in the bioreactor and operated at a temperature between 30°C and 40°C, pH between 6.8 and 7.6 with control using 25% w / w NH3 (aq) and stirring of 1800 rpm.

[0117] Example 3: Method to quantify OD600

[0118] An aliquote from the preculture or culture media is analysed in the spectrophotometer with a light source of 600 nm.

[0119] Example 4: Method to quantify dextrin content in samples using HPLC (Refers to Figure 2)

[0120] This protocol is used to quantify dextrines (glucose polymers) containing 1 to 7 glucose units, as well as arabinose and xylose. Fermentation broth samples are filtered with 0.2 m filters and diluted, if necessary, before adding it to HPLC vials. Samples are quantified with a HPLC setup (Shimadzu, Japan) equipped with an Aminex HPX-42A (4%) column (7.8 x 300 mm) at 85 °C using a mobile solvent composed of milliQ water, at a flowrate of 0.5 mL / min using an RID detector.

[0121] Example 5: Method to quantify alpha-amylase activity in fermentation broth (refers to Figure 3) Xanthan gum (Sigma Life Sciences) was dissolved at 3 mg / mL in Britton-Robison buffer. The solution was heated at in a water bath at 60°C for 30 minutes and let to cool down at room temperature. CaCb was added to achieve concentration of 5mM. AZCL-amylose 2mg / mL solution was added to the xanthan gum solution, and vortexed.

[0122] The testing samples were prepared by spinning the fermenting broth for 20 minutes at 4°C, 4754 ref. 10 pl of the supernatant with the secreted enzymes were pipetted to a flat bottom polystryrene 96-wells plate and 190 pl of AZCL-amylose solution were added to the plate (Greiner ref 651101). The plate was incubated in a plate reader at 37°C while shaking and absorption was measured at 600nm every 3 minutes.

[0123] Example 6: Method for quantifying starch liquefaction (refers to Figure 4)

[0124] Starch stock solution (60 g / L) was autoclaved and mixed with 5 ml of supernatant from fermenting broth prepared as described in Example 5. 3mL of 0.1 M potassium phosphate buffer pH 7.2 was added and the mix was incubated at room temperature. 1 mL samples were taken and centrifuged for 10 min at 12500 rpm. The supernatant was discarded and pellets were dried at 60°C until stable mass was registered.

[0125] Example 7: Method to quantify sugar content in samples using HPLC (Refers to Figure 5 and Figure 6)

[0126] This protocol is used to quantify sugars such as sucrose, glucose, and fructose in the samples of fermentation broth. Fermentation broth samples are filtered with 0.2 pm filters and diluted, if necessary, before adding it to HPLC vials. Samples are quantified with a HPLC setup (Shimadzu, Japan) equipped with an Agilent Metacarb 67H (8%) column (6.5 x 300 mm) at 40 °C using a mobile solvent composed of 2.5 mM H2SO4, at a flowrate of 0.8 mL / min using an RID detector. Standards used for the quantification were analytical grade.

[0127] Example 8: Method to quantify sugar concentration over time in sucrose sample in contact with fermentation broth (Refers to Figure 5 and Figure 6)

[0128] This protocol is used to measure the concentration of different sugars when fermentation broth is in contact with a carbohydrate.

[0129] A known amount of the fermentation broth and / or the supernatant is put into contact with a known amount of sucrose solution of known concentration at room temperature, and samples are collected at specific time points. Sugar concentrations are measured through HPLC as described in Example 7.

[0130] Example 9: Method for determining molecular mass of enzymes in the fermentation broth (refers to Fig. 7)

[0131] SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed using a 12.5% acrylamide separating gel, Samples of fermentation medium centrifuged at 130000 rpm for about 4 min and to 10pl of the sample were added 5 pL of SDS-sample buffer (4x). Samples were incubated for 5 minutes at 95°C in a thermoshaker (600 rpm). 10 pl of the samples were loaded into the gel, which was run at 190 V for 50 minutes. Protein gels were stained with Coomassies Brilliant Blue R-250 and destained using a destaining solution.

Claims

CLAIMS1. A method for hydrolyzing carbohydrates present in raw materials containing carbohydrates, the method comprising the step a) of contacting the raw material containing carbohydrate with a medium in which bacteria secreting carbohydrate-hydrolyzing enzymes have been fermented.

2. The method of claim 1 , further comprising the step of b) separating and isolating the hydrolyzed carbohydrates from the medium.

3. The method of claim 1 or 2, wherein the raw material containing carbohydrate comprises starch.

4. The method of claim 1 or 2, wherein the raw material comprising carbohydrate raw material comprises sucrose.

5. The method of claim 1 or 2, wherein the raw material containing carbohydrate comprises lignocellulosic material.

6. The method of any one of claims 1 to 3, wherein the raw material containing carbohydrate comprises starch and wherein hydrolyzing the carbohydrates present in a carbohydrate raw material comprises hydrolysis of starch.

7. The method of claim 6, wherein the method is a method for simultaneous liquefaction and saccharification of starch, preferably wherein the method is performed at a pH of 6.5 to 8.5 and at a temperature from 30 to 60 °C.

8. The method of any one of claims 1 to 7, wherein the raw material containing carbohydrate is selected from sugarcane, sugar beet, soybean and faba bean molasses and / or juice, permeatesfrom the dairy industry, potato peels, potato pulp, beet pulp, pea starch, wheat starch, grey starch, cassava starch, peels from different fruits such as (but not limited to) oranges and apple, stillage, brewers spent grain, corn fermented protein, corn gluten feed, corn gluten meal, corn protein concentrate, distiller's dried grains with solubles (DDGS), and soy protein meal.

9. The method of any one of claims 1 to 8, wherein the bacteria capable of secreting carbohydratehydrolyzing enzymes are selected from Priestia spp (such as Priestia flexa), Bacillus spp (such as Bacillus subtilis), Vibrio spp (such as Vibrio natriegens) and Geobacillus spp (such as Geobacillus LC300) bacteria.

10. The method of any one of claims 1 to 8, wherein the bacteria capable of secreting carbohydratehydrolyzing enzymes are selected from Priestia flexa, Vibrio natriegens and Geobacilus stearothermophilus.

11. The method of claim 10, wherein the bacteria capable of secreting carbohydrate-hydrolyzing enzymes are selected from Priestia flexa, and Vibrio natriegens.

12. The method of claim 10, wherein the bacteria capable of secreting carbohydrate-hydrolyzing enzymes are Geobacilus stearothermophilus.

13. The method of any one of claims 1 to 12, wherein the carbohydrate-hydrolyzing enzyme is selected from invertase, levansucrase, levanase, pullulanase, amylase, cellulase and hemicellulase.

14. A medium in which bacteria capable of secreting carbohydrate-hydrolyzing enzymes have been fermented, as described in any one of claims 1 to 13.

15. A composition comprising hydrolyzed carbohydrates, obtainable in the method for hydrolyzing carbohydrates present in raw materials containing carbohydrates of any one of claims 1 to 13.

16. A method for producing a biomass, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes on a medium comprising complex carbohydrates as a major carbon source.

17. A biomass, obtainable in the method according to claim 16.

18. A method for simultaneously liquifying, saccharifying and fermenting starch, the method comprising the step of cultivating bacteria secreting carbohydrate-hydrolyzing enzymes, as described in any one of claims 1 to 13, on a medium comprising starch as a major carbon source.

19. Use of the biomass of claim 17 in the production of animal feed, pet food, fermentation medium or supplement, food products, nutraceuticals, pharmaceuticals or flavour enhancers.

20. Use of the composition of claim 15 in the production of sweeteners.