Enzyme complex and use thereof

Abstract

The invention relates to an enzyme complex solution comprising an alpha-galactosidase and a beta-endo-mannanase, wherein the alpha-galactosidase and the beta-endo-mannanase retain at least 80% of their maximal activity at mutual exclusion ranges of temperatures and / or ranges of pH.

Description

[0001] Enzyme complex and use thereof

[0002] The invention relates to an enzyme complex comprising an alpha-galactosidase and a beta- endo-mannanase for hydrolysing a galactomannan. The invention also relates to a process for obtaining a modified galactomannan using the said enzyme complex, the modified galactomannan and a composition comprising the said modified galactomannan.

[0003] Galactomannans are polysaccharides consisting of a mannose backbone with galactose side groups, more specifically, a (1-4)-linked beta-D-mannopyranose backbone with branchpoints from their 6-positions linked to alpha-D-galactose, ( / .e. 1-6-linked alpha-D-galactopyranose). The main source of galactomannans is the endosperm of certain leguminous seeds such as fenugreek, guar, carob, tara, cassia obtusifolia and the like. In particular, the polysaccharide contained in guar seeds consists of a main chain of mannose units linked together by 1 -4-|3- glycosidic linkages from which single galactose units branch by means of 1-6-a-glycosidic linkages. The ratio of galactose units to mannose units can vary from one source to another. In the case of the polysaccharide contained in guar seeds the ratio is about 1 :2.

[0004] Galactomannans are soluble in water to give high-viscosity solutions. This property makes them particularly useful in various sectors such as hydrocarbons research and production, textile printing, food, paper, and pharmaceutics.

[0005] Forage legume seed galactomannans possess a fully galactose substituted mannose backbone that offers the advantage of facile and reproducible construction of galactomannan structure conformations (via enzymatic modification or on some occasions via fermentation) able to provide specific technofunctional and health promoting aspects.

[0006] However, the main legume seed sources for galactomannans, e.g. guar, tara and locust bean gums, have a high dependence of climatological, altitudinal, and geographical restrictions. Consequently, they are subject to market volatility and are projected to undergo loss of supply because of global warming.

[0007] The hereby invention aims at providing an alternative sustainable solution based on an ecologically resilient bioresource.

[0008] DESCRIPTION OF THE INVENTION

[0009] To this aim, the invention relates to an enzyme complex solution comprising an alphagalactosidase and a beta-endo-mannanase, wherein the alpha-galactosidase and the beta- endo-mannanase retain at least 80% of their maximal activity at mutual exclusion ranges of temperatures and / or ranges of pH.

[0010] The enzyme complex of the invention advantageously comprises two types of enzymes able to modify a galactomannan, namely an alpha-galactosidase and a beta-endo-mannanase, both enzymes working at excluding ranges of temperatures and / or pH. Consequently, the enzyme complex of the invention allows to produce modified galactomannan with specific controlled galactose side content and mannose backbone length. Depending on the modified galactomannan to produce, only one enzyme or both enzymes sequentially can be activated. Because of the very specific controlled modifications, the enzyme complex of the invention allows producing modified galactomannan from sustainable source mimicking the properties of the currently used galactomannans from non-sustainable sources. Moreover, the inventors surprisingly discovered that modified galactomannan from a unique starting galactomannan allows to mimic different of these non-sustainable galactomannans depending on the modifications applied with the enzyme complex of the invention. The production of modified galactomannan thanks to the enzyme complex of the invention are therefore real alternatives to non-sustainable galactomannans.

[0011] In one embodiment of the invention, the ranges of pH and / or temperature at which one enzyme of the couple retains at least 80% of its maximal activity correspond to at most 20% of maximal activity of the other.

[0012] The invention also relates to a process for obtaining a modified galactomannan by reducing the galactose content and / or the mannose chain length of a starting galactomannan with the enzyme complex solution as above defined.

[0013] In one embodiment of the invention, wherein the starting galactomannan is alfalfa galactomannan or fenugreek galactomannan.

[0014] In another embodiment, wherein the modified galactomannan has a mannose over galactose content ratio from 2 to 5.

[0015] In another embodiment, the process comprises the following steps: a) providing a hydrated preparation of a starting galactomannan, b) adding the enzyme complex solution to the hydrated preparation, c) depleting the galactose unit content of the starting galactomannan with the alphagalactosidase of the enzyme complex solution and / or reducing the chain length mannan of the starting galactomannan with the beta-endo-mannanase of the enzyme complex solution, d) inactivating the enzyme complex solution, and e) obtaining a modified galactomannan, wherein the galactose depleting step and the chain length mannan reducing step are conducted at mutual exclusion ranges of temperatures and / or ranges of pH. The invention also relates to a use of the enzyme complex solution as above defined for modifying a galactomannan and obtaining a modified galactomannan.

[0016] The invention further relates to a modified galactomannan obtained with the process as above defined or the use as above defined. Notably, the modified galactomannan has a mannose over galactose content ratio from 2 to 5.

[0017] The invention finally relates to a gum solution comprising the modified galactomannan as above defined.

[0018] In one embodiment, the gum solution has a complex shear modulus from 5 to 20 Pa at 1 wt%..

[0019] In another embodiment, the gum solution further comprises at least one additional polysaccharide.

[0020] Particularly, the additional polysaccharide is selected in the group consisting of a second galactomannan, a xanthan, a kappa-carrageenan, sucrose and combinations thereof.

[0021] Notably, the gum solution has an elastic modulus G’ from 4 to 25 Pa.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Enzyme complex

[0024] As a first object, the invention relates to an enzyme complex solution comprising an alphagalactosidase and a beta-endo-mannanase, wherein the alpha-galactosidase and the beta- endo-mannanase retain at least 80% of their maximal activity at mutual exclusion ranges of temperatures and / or ranges of pH.

[0025] Alpha-galactosidase or “a-galactosidase” is an enzyme that can specifically hydrolyse the 1- 6-a-glycosidic bond formed between mannose and galactose in a galactomannan molecule. Accordingly, the alpha-galactosidase acts on the galactose side content.

[0026] Beta-endo-mannanase or “P-endo-mannanase” or “P-mannanase” is an enzyme that specifically degrades p-1 ,4-glycosidic bonds on a galactomannan backbone to degrade the macromolecular polysaccharide into smaller molecules. Accordingly, the beta-endo- mannanase acts on the mannose backbone length.

[0027] By “retain at least 80% of its maximal activity”, it is meant in the invention 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% of maximal activity. The maximal activity of an enzyme corresponds to the best reaction number per time said enzyme can achieve. Maximal activity of an enzyme requires both optimal temperature and pH conditions. Determining the maximal activity of an enzyme can be done by different routine belonging to the general knowledge of the skilled person. In particular, this can be achieved by spectrophotometric, fluorometric and chromatographic methods (Practical Enzymology, Bisswanger, 2011).

[0028] Each enzyme in the couple alpha-galactosidase and beta-endo-mannanase in the enzyme complex of the invention works at excluded ranges of temperatures and / or ranges of pH, i.e. has at different maximal activity conditions. In particular, the optimal temperature and / or pH conditions of one enzyme of the couple correspond to poor temperature and / or pH conditions for the other. Accordingly, a simple change of pH and / or of temperature allows to inactivate one enzyme of the couple and activate the other to produce the desired modifications on a starting galactomannan substrate, as detailed below.

[0029] Particularly, the ranges of pH and / or temperature at which one enzyme of the couple retains at least 80% of its maximal activity correspond to at most 20% of maximal activity of the other.

[0030] By “at most 20% of maximal activity”, it is meant in the invention 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % and 0% of the maximal activity.

[0031] In one embodiment of the invention, the alpha-galactosidase and the beta-endo-mannanase respectively retains at least 80% of their maximal activity at a temperature from 25°C to 95°C and / or at a pH from 2 to 12. Especially, the alpha-galactosidase may be chosen to retains at least 80% of its maximal activity in a part of a range of temperature from 25°C to 60°C and / or in a part of a range of pH from 3.5 to 8.5. The beta-endo-mannanase retains at least 80% of its maximal activity in a part of a range of temperature from 25°C to 95°C, and in a part of a range of pH from 3 to 9.

[0032] Especially, the alpha-galactosidase may retain at least 80% of its maximal activity at a temperature from 20°C to 50°C, and preferably at a pH from 5 to 7 and the beta-endo- mannanase may retain at least 80% of its maximal activity at a temperature from 55°C to 90°C, and preferably at a pH from 3.5 to 4.5.

[0033] Alternatively, the alpha-galactosidase may retain at least 80% of its maximal activity at a temperature from 50°C to 65°C, and preferably at a pH from 3 to 5 and the beta-endo- mannanase may retain at least 80% of its maximal activity at a temperature from 25°C to 45°C, and preferably at a pH from 5.5 to 8.

[0034] The alpha-galactosidase and the beta-endo-mannanase may each originate from any source. They may originate from the same source or diverse sources. The skilled person in the art knows how to select both enzymes of the couple from the different available sources to respect the exclusion condition activity in terms of pH and / or temperature. In particular, the alphagalactosidase and the beta-endo-mannanase may respectively originate from legume plant seeds, coffee beans, Cucumis melo, fungi, yeast ( / paston s)bacteria or Helix pomatia. Legume plant seeds source comprises guar, alfalfa, clover, locust bean, and cassia. Fungi source comprises Aspergillus niger, Aspergillus oryzae and Penicillium simplissimum. Bacteria source comprises E. coli, Xanthomonas manihotis, Clostridium cellulolyticum, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacillus halodurans, Cellvibrio mixtus and Thermus brockianus, Bifidobacterium longum, Thermotoga maritima, Zobellia galactanivorans

[0035] In particular the alpha-galactosidase may originate from an organism chosen in the group consisting of Aspergillus niger, Aspergillus oryzae and Penicillium simplissimum, E. coli, Xanthomonas manihotis, Clostridium cellulolyticum, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacillus halodurans, Cellvibrio mixtus and Thermus brockianus, Bifidobacterium longum, Thermotoga maritima, Zobellia galactanivorans, Bacillus circulans, Clostridium perfringens, Streptococcus pneumonia and Native pseudomonas sp. Keratanase.

[0036] In particular, the beta-endo-mannanase may originate from an organism chosen in the group consisting of Thermotoga maritima, Clostridium thermocellum, Bacillus sp., Clostridium cellulolyticum, Podospora anserina, Thermobifida fusca, Thermotoga petrophila, Paenibacillus polymyxa, Cellvibrio japonicus, Bacteroides thetaiotaomicron, Streptomyces sp., Bacillus subtilis, Clostridium cellulovorans, Alicyclobacillus acidocaldarius, Ruminococcus champanellensis, Streptomyces coelicolor, Ruminococcus flavefaciens, Helix pomatia, Cellvibrio mixtus, Pyrococcus furiosus, Cellulomonas fimia and Pichia pastoris.

[0037] Process for modifying galactomannan

[0038] As a second object, the invention relates to a process for obtaining a modified galactomannan by reducing the galactose content and / or the mannose chain length of a starting galactomannan with the enzyme complex solution as above defined.

[0039] The starting galactomannan may be a native galactomannan ( / .e. directly purified from its natural source) or a modified galactomannan. The modified galactomannan as a starting galactomannan may be previously modified by the process described therein or any other process.

[0040] The starting galactomannan may have a low to a high content of mannose over galactose. In one embodiment of the invention, the starting galactomannan has a mannose over galactose ratio (M / G) from 0.9 to 1.5, in particular from 0.9 to 1.3, particularly from 0.95 to 1.1 , notably 0.95 to 1 .05, especially 1. The M / G of the starting material inevitably determines the obtainable modified galactomannan. Because of this, a starting galactomannan with a M / G around 1 is preferred, allowing to the obtained the widest kinds of modified galactomannan and thus allowing the production of modified galactomannan that can mimic the properties of the currently used non sustainable galactomannan. Nevertheless, thanks to the controlled modifications obtained with the enzyme complex of the invention, using a starting galactomannan with a low (0.9) or high (1.5) M / G allows to obtain a wide kind of modified galactomannan.

[0041] In one embodiment, the starting galactomannan originates from a sustainable source. Especially, the starting galactomannan originates from Fabaceae seeds and especially from alfalfa, red clover, white clover or fenugreek seeds. Alfalfa and fenugreek galactomannans, in addition to originates from a sustainable source, have a M / G around 1 , allowing production of vastly different kind of modified galactomannan.

[0042] In one embodiment of the invention, the modified galactomannan has a M / G from 3 to 5. Indeed, the inventors unexpectedly discovered that this kind of modified galactomannan can mimic the properties of different non sustainable galactomannans, such as guar gum or locust bean gum, tara gum and cassia gum in terms of viscoelastic behaviour, cold set gelling and cryogelation behaviour.. Especially, the modified galactomannan has a M / G from 3.5 to 5, particularly from 4 to 5.

[0043] In another embodiment of the invention, the process of the invention comprises the following steps: a) providing a hydrated preparation of a starting galactomannan, b) adding the enzyme complex solution to the hydrated preparation, c) depleting the galactose unit content of the starting galactomannan with the alphagalactosidase of the enzyme complex solution and / or reducing the chain length mannan of the starting galactomannan with the beta-endo-mannanase of the enzyme complex solution, d) inactivating the enzyme complex solution, and e) obtaining a modified galactomannan.

[0044] Because of the use of the enzyme complex solution of the invention, the galactose unit depletion - step and the mannose chain length reducing step are conducted at mutual exclusion ranges of temperatures and / or ranges of pH.

[0045] Step a)

[0046] The composition of the hydrated preparation of the starting galactomannan may be adjusted as function of the desired modified galactomannan.

[0047] In particular, the hydrated preparation may comprise from 0.1 to 70 wt % of the starting galactomannan by weight of the preparation. Particularly 0.1 to 50 wt %, notably 0.1 to 10 wt %, especially 0.1 to 5 wt %, in particular 0.1 to 2 wt %, particularly 0.2 to 1.5 wt %, notably 0.5 to 1% of the starting galactomannan by weight of the preparation.

[0048] Particularly, the hydrated preparation is devoid of crosslinking agent, like a boric acid, or thickening agent.

[0049] According to a preferred embodiment of the invention, the enzyme complex is added to the hydrated solution at conditions solely the alpha-galactosidase or the beta-beta-endo- mannanase is working. Alternatively, the enzyme complex is added to the hydrated solution at conditions both enzymes of the couple are net working, to control the beginning of step c). In particular, the enzyme complex comprises from 0.01 to 1000 ll / g of each enzyme of the couple relative to the galactomannan content. Particularly from 0.01 to 500 ll / g, in particular from 0.01 to 300 ll / g, especially from 0.01 to 200 ll / g, notably from 10 to 200 ll / g, particularly from 50 to 200 ll / g of each enzyme of the couple relative to the galactomannan content. For example, 80 ll / g of the alpha-galactosidase enzyme relative to the galactomannan content.

[0050] Step c) corresponds to the activation of solely one enzyme of the couple, or each enzyme of the couple successively, depending on the desired modifications on the starting galactomannan, by setting the adapted pH and temperature. By “successively”, it is meant in the invention that the temperature and / or pH are modified during step c) to activate one enzyme at the time, but different cycles of activation may be conducted for each enzyme.

[0051] In particular, step c) is conducted at a temperature from 20 to 90°C and / or at a pH from 2 to 12, corresponding to the retention of 80% of the maximal activity of both enzymes of the couple. Of course, each enzyme of the couple has its maximal activity that ranges in the previous range of temperature and / or pH in an exclusion manner.

[0052] In particular, the galactose reducing step and the chain length mannan reducing step in step c) are respectively conducted from 0.5h to 24h. The time of operation of the galactose reducing step will impact the M / G of the modified galactomannan. A longer time of operation corresponds to a higher M / G. Besides a longer time of operation of the chain length mannan reducing step will reduce the backbone length of the galactomannan. Correct equilibrium between those steps allows obtaining the desired modified galactomannan. Particularly, the galactose reducing step and the chain length mannan reducing step in step c) are respectively conducted from 1 h to 15h, especially from 3h to 10h, notably from 5h to 9h. For example, the galactose reducing step is conducted for 8h. In one embodiment of the invention, the starting galactomannan is an alfalfa seed galactomannan, the alpha-galactosidase in the enzyme complex is at an amount of 80 ll / g relative to the starting galactomannan and wherein step c) consists in reducing the galactose content of the starting galactomannan with the alpha-galactosidase of the enzyme complex solution and is conducted for 8h at a temperature of 40°C and a pH of 4.5. These conditions allow to obtain a modified galactomannan with good viscoelastic behaviour (as good as native locust bean and tara galactomannans) and cryogelation capacity (as good as native locust bean). Hence a unique modified galactomannan with the process of the invention is able to mimic several sought properties of several commercial galactomannans.

[0053] Step c) may also comprise the set of conditions for the activation of the other(s) enzyme(s) comprised in the enzyme complex, when present(s). This or these activations may be conducted before the activation of the enzymes of the couple, after or in-between.

[0054] The inactivating step is designed to alter activity of all enzymes of the enzyme complex and prevent any further activity.

[0055] In that context, the inactivating step may carry out at a temperature from 70°C to 140°C, at a pH from 1 to 14, during 5 minutes to 60 minutes. Of course, those conditions will depend on the enzymes presents in the enzyme complex and are designed to not alter the modified galactomannan. In particular, the temperature may range from 90°C to 121°C, and is especially 121°C. pH may be for example from 6 to 9 and especially 7. Time of operation may be from 10 to 30 minutes and especially 20 minutes.

[0056] Step e)

[0057] During step e), the modified galactomannan is recovered.

[0058] Notably, the modified galactomannan is purified by the following steps:

[0059] - solvent precipitation, and

[0060] - centrifugation.

[0061] In particular, the solvent precipitation is conducted in ethanol, acetone or isopropanol.

[0062] Particularly, a gum pellet of the modified galactomannan is obtained in a subsequent step by redispersion in water and dialysis for 24 to 72h.

[0063] As a third object, the invention relates to the use of the above defined enzyme complex solution for modifying a galactomannan and obtaining a modified galactomannan. The features above recited in relation to the second object of the invention apply mutatis mutandis to the third object of the invention. As a fourth object, the invention relates to a process for obtaining a modified galactomannan by reducing the galactose content with an alpha-galactosidase. The feature above recited regarding the alpha-galactosidase and the operation of the latter apply mutatis mutandis to this object of the invention.

[0064] In particular, the process comprises the following steps: a) providing a hydrated preparation of a starting galactomannan, b) adding an alpha-galactosidase to the hydrated preparation, c) reducing the galactose content of the starting galactomannan with the alphagalactosidase of the enzyme complex, d) inactivating the alpha-galactosidase, and e) obtaining a modified galactomannan.

[0065] Modified galactomannan

[0066] As a fifth object, the invention relates to a modified galactomannan, obtained with the process of the second or fourth object of invention or with the use of the third object of invention.

[0067] Particularly, the modified galactomannan has a M / G from 2 to 5.

[0068] Particularly, step b) of the process comprises adding 80 ll / g of the alpha-galactosidase, especially of guar galactosidase. Notably, the galactose reducing step of step c) laps for 8 hours at 40°C and pH 4.5.

[0069] Gum solution

[0070] As a sixth object, the invention finally relates to a gum solution comprising the modified galactomannan as above defined.

[0071] In one embodiment of the invention, the modified galactomannan is a modified alfalfa galactomannan or a modified fenugreek galactomannan, notably a modified alfalfa galactomannan.

[0072] In another embodiment of the invention, the gum solution comprises from 0.1 to 5 wt % of the modified galactomannan based on the total weight of the gum solution, especially from 0.2 to 2 wt %, in particular from 0.5 to 1 wt %.

[0073] Particularly, the gum solution has a complex shear modulus G* from 5 to 20 Pa, especially at a frequency of 1 Hz and at a temperature of 25°C with a strain of 0.5%.

[0074] In one embodiment of the invention, the gum solution further comprises at least one additional polysaccharide.

[0075] In one embodiment of the invention, the at least one additional polysaccharide is selected in the group consisting of a second galactomannan, , a xanthan, a kappa-carrageenan, sucrose and combinations thereof. The second galactomannan may be a modified galactomannan or a native galactomannan, and is especially locust bean galactomannan, particularly a native locust bean galactomannan. The second modified galactomannan is notably obtained with the process as above defined.

[0076] Particularly, the gum solution has an elastic modulus G’ from 4 to 25 Pa, especially at a frequency of 1 Hz and at a temperature of 4°C with a strain of 0.5%.

[0077] The ratio between each of the modified galactomannan and the at least one additional polysaccharide in the gum solution may be from 0.01 to 100, in particular from 0.02 to 50 and is especially 1.

[0078] In one embodiment, the gum solution comprises an alfalfa modified galactomannan with a native locust bean galactomannan.

[0079] In another embodiment of the invention, the gum solution comprises an alfalfa modified galactomannan with a xanthan.

[0080] In another embodiment of the invention, the gum solution comprises an alfalfa modified galactomannan with a kappa-carrageenan.

[0081] In another embodiment of the invention, the gum solution comprises an alfalfa modified galactomannan with sucrose.

[0082] BRIEF DESCRIPTION OF THE FIGURES

[0083] Figure 1 is a diagram representing changes in viscoelastic behaviour of a gum solution (0.5 wt %) comprising an alfalfa galactomannan modified with a process of the invention using 80 units per gram (U / g) of a guar alpha-galactosidase (mAAG) at 40°C and pH 4.5, as influenced by the time of incubation. The x-axis represents the incubation time in hours, and the y-axis represents the complex viscosity in mPa of the obtained modified alfalfa galactomannan (Grey bars: before a cryogenic processing; black bars: after a cryogenic processing).

[0084] Figure 2 represents molecular properties at 25°C of a gum solution (0.15-1 wt %) comprising a starting alfalfa galactomannan (Figures 2A and 2C) and a mAAG gum solution comprising a modified alfalfa galactomannan (Figure 2B and 2D) with a process of the invention using 80U / g of guar alpha-galactosidase for 8 hours. In Figures 2A and 2B, the reduced viscosity and inherent viscosity (in dL.g-1) are represented as function of the concentration of the galactomannan (in g.100g-1of solution), wherein the straight lines represent the Huggins’s plot, and the dashed-lines represent the Kraemer’s plot. In Figure 2A, Huggins’s equation was determined as 71.645x + 9.1566 with a R2of 0.9941 and the Kraemer equation was determined as at 2.6632x + 9.4552 with a R2of 0.8188. In Figure 2B, Huggins’s equation was determined as 89.366x + 10.225 with a R2of 0.973 and the Kraemer equation was determined as 8.4435x + 10.575 with a R2of 0.4985. In Figures 2C and 2D, the log of the specific viscosity is represented as function of the log of concentration of the galactomannan, wherein the straight line represent the Huggins’s plot and the dashed lines represent the Kraemer’s plot. In Figure 2C, Huggins’s equation was determined as 4.421x + 2.6393 with a R2of 0.9991 and the Kraemer equation was determined as 2.3318x + 3.5247 with a R2of 0.9998. In Figure 2D, Huggins’s equation was determined as 4.421x + 2.6393 with a R2of 0.9991 and the Kraemer equation was determined as 2.3318x + 3.5247 with a R2of 0.9998.

[0085] Figure 3 is a graph representing the viscoelastic behaviour and steady state flow behaviour of a mAAG gum solution with a mAAG solution (triangle curve) and locust bean gum solution (square curve). X-axis represents the Frequency (in Hz), and the y-axis represents the complex shear modulus G* (in Pa).

[0086] Figure 4 is a graph representing the viscoelastic behaviour of a mAAG gum solution with the mAAG of Figure 3 (solid square symbols: elastic modulus G’ ; void square symbols: elastic modulus G”), locust bean gum solution (solid diamond symbols: elastic modulus G’ ; void diamond symbols: elastic modulus G”) and tara gum solution (solid circle symbols: elastic modulus G’ ; void circle symbols: elastic modulus G”), each at 1 wt % of the total weight of the respective solution, 0.5% strain and 25°C. X-axis represents the Frequency (Hz), and the y- axis represents the elastic moduli G’ and G” (in Pa).

[0087] Figure 5 illustrates the cold gel-setting a mAAG gum solution with the mAAG of Figure 2, a kappa-carrageenan gum and a mixture solution thereof, each at 1 wt % of the total weight of the respective solution, 0.5% strain and 4°C. Dotted line indicates an ideal mixture (1 :1) behaviour. Y-axis represents the elastic modulus G’ (in Pa).

[0088] Figure 6 illustrates the cold gel-setting synergism of a mAAG gum solution with the mAAG of Figure 2, a xanthan gum solution and a mixture solution thereof, each at 0.5 wt % of the total weight of the respective solution, 0.5% strain and 4°C. Dotted line indicates an ideal mixture (1 :1) behaviour. Y-axis represents the elastic modulus G’ (in Pa).

[0089] Figure 7 is an histogram representing the cold gel-setting synergism of a mAAG gum solution, a locust bean gum solution and a mixture solution thereof. Dotted line indicates an ideal mixture (1 :1) behaviour. Y-axis represents the elastic modulus G’ (in Pa).

[0090] Figure 8 represents the cryogelation behaviour of a mAAG gum solution with the mAAG of Figure 2 (square symbols) and a native alfalfa galactomannan solution (triangle symbols), with 0.5% strain, 1 Hz of frequency for 5 freeze-thaw cycles. The mAAG gum solution comprises 1 wt % of modified galactomannan, and the native alfalfa galactomannan solution comprises 2 wt % of native galactomannan. X-axis represents the number of freeze thaw cycles, and the y-axis represents the normalised elastic modulus G’ from the starting value. Starting value for the mAAG gum solution was 14.2 Pa and the starting value for the native alfalfa galactomannan solution was 52.70 Pa.

[0091] Figure 9 represents cryogelation behaviour of a mAAG gum solution with the mAAG of Figure 2 (square symbols), a tara gum solution (circle symbols) and a locust bean gum solution (diamond symbols), each at 1 wt % of the total weight of the respective solution, with 0.5% strain and 1 Hz of frequency for 5 freeze-thaw cycles. X-axis represents the number of freeze thaw cycles, and the y-axis represents the elastic modulus G’ (in Pa).

[0092] Figure 10 represents cryogelation behaviour of a mAAG gum solution with the mAAG of Figure 2 (square symbols), a xanthan gum solution (diamond symbols) and a mixture thereof (circle symbols), at 0.5 wt % of the total weight of the respective solution, with 0.5% strain and frequency of 1 Hz for 5 freeze-thaw cycles. X-axis represents the number of the successive freeze thaw cycles, and the y-axis represents the elastic modulus G’ (in Pa).

[0093] Figure 11 represents cryogelation behaviour of a mAAG gum solution with the mAAG of Figure 2 (square curve) and a mixture solution of the latter with sucrose (diamond curve), at 0.5% strain and frequency of 1 Hz for 5 freeze-thaw cycles. The mAAG gum solution comprised 0.5 wt % of modified alfalfa galactomannan. The mixture comprised 0.5 wt % of modified alfalfa galactomannan and 50 wt % of sucrose. X-axis represents the number of freeze thaw cycles, and the y-axis represents the elastic modulus G’ (in Pa).

[0094] Figure 12 represents the cold-water solubility of a modified alfalfa galactomannan solution as compared to a native alfalfa galactomannan solution and a locust bean gum solution, at 1 wt % of the total weight of the respective solution and 25°C. X-axis represents the mAAG solubility in cold water (25°C) (in %).

[0095] Figure 13 represents the evolution of the normalised complex viscosity of galactomannooligosaccharides (GMO) produced by modification of a native AAG solution by an enzyme complex as function of the time of incubation (min).

[0096] EXAMPLES

[0097] Material and Method

[0098] Seed gums

[0099] Medicago sativa L. Seeds were prepared as detailed in document W02022029103 to obtained native alfalfa seed gum (AAG). Locust bean gum was obtained from Sigma-Aldrich, LBG from Ceratonia siliqua seeds, CAS: 9000-40-2.

[0100] Tara gum was obtained from Sosa Ingredients from the seeds of Caesalpinia spinosa seeds. kappa-carrageenan wad obtained from Sigma Aldrich CAS: 11114-20-8 xanthan gum from Xanthomonas campestris was obtained from Sigma Aldrich CAS: 11138- 66-2.

[0101] Enzymatic blend

[0102] An enzymatic blend was prepared by isolating an a-galactosidase from guar (Cyamopsis tetragonoloba L.) seeds with a residual p-endo-mannanase (<1%) as detailed in McCleary (1988). Two p-endo-mannanases from microbial sources were obtained from Creative Enzymes.

[0103] Example 1 : Changes in the viscoelastic behaviour of modified AAG (mAAG) as influenced by the duration of the enzymatic process

[0104] An amount of 80 ll / g of the enzymatic blend was added into a native alfalfa seed gum solution at 0.5 % wt and allowed to incubate for 10 h at 40°C and pH = 4.5. Samplings on 1h time intervals following heat inactivation of the enzyme at 90°C for 30min were carried out. The modified AAG (mAAG) was precipitated with 3 volumes of EtOH centrifuged at 4500g for 5min and the precipitate was washed twice with EtOH 70% v / v and vacuum dried. The obtained dried mAAG samples were reconstituted into MilliQ water (0.5% wt.) and characterised fortheir dynamic rheological properties (i.e. frequency sweeps at 1% strain). To evaluate their cryogel forming capacity, the mAAG solutions (0.5% wt.) were subjected into a single freeze thaw event by transferring them in a freezer at -20°C for 24 h and melting them in a cold room at 4°C for 2h. The obtained cryogels were characterised as before mentioned. A time dependent increase in the complex viscosity of the mAAG cold-set gels and cryogels was attained reaching their maximum strength at 8-9h of enzymatic treatment (Figure 1).

[0105] Example 2: Molecular properties of the native and modified AAG mAGG was obtained by adding an amount of 80 ll / g of the enzymatic blend into a native alfalfa seed gum solution at 0.5 % wt and allowed to incubate for 8h at 40°C and pH = 4.5. The native AAG as well as the obtained mAAG were characterised for their steady state flow behaviour (shear rate 0.01 - 1000 s1) and capillary flow behaviour to determine their molecular properties i.e., intrinsic viscosity [q] and coil overlap concentration [c*]. As illustrated in Figure 2, the intrinsic viscosity of the mAAG was increased by 12% (i.e. from 9.30 to 10.40 dL / g) and the c* was reduced from 0.31 to 0.3% wt. These results demonstrate an unforeseen increase in the intrinsic viscosity and critical coil overlap concentration indicating an improvement in the thickening power of the modified alfalfa galactomannan.

[0106] Example 3: Viscoelastic behaviour and steady state flow behaviour of modified AAG and LBG (purified form)

[0107] Locust bean gum (LBG) was dissolved in MilliQ water at 80°C. The obtained LBG solution was precipitated with 3 volumes of absolute EtOH, centrifuged at 4000g for 10 min and the obtained pellet was diafiltrated (12kDa membrane against MilliQ water) for 24h, and freeze-dried. The obtained purified LBG (purLBG) was dispersed into MilliQ at 80°C under magnetic stirring for 1h to obtain a purLBG solution 1 % wt. A mAAG solution 1 % wt. was obtained by using an amount of 80 U / g of the enzymatic blend added into a native alfalfa seed gum solution at 1 % wt and allowed to incubate for 8h at 40°C and pH = 4.5. The mAAG solution was then prepared in the same manner as purLBG. Both biopolymer solutions were characterised for their dynamic rheological properties (0.1-10Hz, strain 0.5%, 20°C) as shown in Figure 3. A mirroring behaviour (i.e. almost complete overlapping of the dynamic rheological spectra) in the stiffness of the purLBG and mAAG solutions was observed suggesting that mAAG8h can be employed as 100% replacer of purLBG in terms of its cold-set gelling performance.

[0108] Example 4: Viscoelastic behaviour of modified AAG, LBG and tara gum solutions

[0109] Aqueous solutions (1 % wt) of two purified commercial galactomannans i.e. tara and locust bean gum and mAAG were prepared as described in Example 3. The individual galactomannan solutions were characterised by means of dynamic oscillatory rheology as shown in Figure 4. All systems exhibited a viscous dominant viscoelastic behaviour with the mAAG analogues to exert the highest elastic modulus values over the viscous dominant region of the spectra. Purified commercial galactomannans exhibited similar viscoelastic properties.

[0110] Example 5: Cold gel-setting synergism of mAAG with kappa-carrageenan mAAG was obtained by using An amount of 80 U / g of the enzymatic blend added into a native alfalfa seed gum solution at 1 % wt and allowed to incubate for 8h at 40°C and pH = 4.5. Individual and binary (1 :1) aqueous solutions of mAAG and kappa-carrageenan (1% wt.) were prepared by dispersing under magnetic stirring the biopolymer into cold water (25°C), heating at 80°C for 30min under constant stirring (water was added to balance the total solids contents due to evaporation) and the hot solution was transferred to a sandblasted plate-plate geometry at 80°C and sealed with paraffin oil. The biopolymer solutions were cooled at 2°C / min to 4°C, aged for 1 h at 4°C and characterised for their elasticity. A strong cold-set gelling synergism between mAAG and kappa carrageenan was observed (Figure 5) as compared to the individual biopolymer solutions and their ideal binary mixture (G’ was estimated to18.9 and 207 Pa, respectively).

[0111] Example 6: Cold gel-setting synergism of mAAG with xanthan gum mAAG was obtained as described in Example 2. Individual and binary (1 :1) aqueous solutions of mAAG and xanthan (0.5% wt.) were prepared by dispersing under magnetic stirring the biopolymer into cold water (25°C) under constant stirring for 60min. The obtained biopolymer solutions were transferred to a sandblasted plate-plate geometry at 80°C and sealed with paraffin oil. The biopolymer solutions were cooled at 2°C / min to 4°C, aged for 1 h at 4°C and characterised for their elasticity (Figure 6). A high cold-set gelling synergism between mAAG and xanthan gum was detected as compared to the individual biopolymer solutions and their ideal binary mixture (G’ was 5.8 and 25.9 Pa, respectively).

[0112] Example 7: Cold gel-setting synergism of mAAG with locust bean gum mAAG solution and purLBG solution were prepared as described in Example 3. Individual and binary (1 :1) aqueous solutions of mAAG and pureLBG were prepared by dispersing under magnetic stirring the biopolymers into cold water (25°C) and heating at 80°C under constant stirring for 30min. The obtained biopolymer solutions were transferred to a sandblasted plateplate geometry at 80°C and sealed with paraffin oil. The biopolymer solutions were cooled at 2°C / min to 4°C, aged for 1 h at 4°C and characterised for their elasticity (Figure 7). A good cold-set gelling synergism between mAAG8h and purLBG was detected as compared to the individual biopolymer solutions and their ideal binary mixture (G’ was 11.8 and 19.5 Pa, respectively).

[0113] Example 8: Cryogelation behaviour of native and modified AAG mAAG was obtained as describe in Example 5. Individual aqueous solutions of native AAG (2wt%) and mAAG (1% wt.) were prepared by dispersing the gums to cold MilliQ water (25°C) under magnetic stirring and heating to 80°C for 30min under constant stirring. For a The solutions were cooled to ambient temperature (25°C), divided equally in 5mL Eppendorf tubes and transferred into a freezer (-20°C) for 1 h. The frozen biopolymer solutions transferred into a chilling chamber at 4°C and allowed to melt completely (that corresponds to one cycle). The biopolymer solutions were subjected in five freeze-thaw cycles. The viscoelastic behaviour of the cryogels obtained at the end of each cycle was determined by means of frequency sweeps (0.1 - 100Hz, strain 0.5%) at 5°C. According to the normalised elastic modulus G’ plot (Figure 8), both native AAG and mAAG were able to undergo cryogelation. Initial elastic modulus values: G0= 14.2 Pa for mAAG and G0=52.70Pa for the native AAG. However, mAAG was able to provide 8.4 times stiffer cryogels than native AAG i.e. , 8.4 and 2.4 times stiffer cryogels than their initial (non-cryogenically processed) galactomannan solutions, employing only the half biopolymer content (i.e., 1% vs 2% wt., respectively).

[0114] Example 9: Cryogelation behaviour of modified AAG, tara and LBG mAAG was obtained as describe in Example 5. Individual aqueous solutions (1% wt.) of two purified galactomannans (i.e. locust bean gum and tara gum) and mAAG were prepared by dispersing the gums to cold MilliQ water (25°C) under magnetic stirring and heating to 80°C for 30min under constant stirring. The solutions were cooled to ambient temperature (25°C), divided equally in 5mL Eppendorf tubes and transferred into a freezer (-20°C) for 1h. The frozen biopolymer solutions transferred into a chilling chamber at 4°C and allowed to melt completely for 1h (one cycle). The biopolymer solutions were subjected in five freeze-thaw cycles. The viscoelastic behaviour of the cryogels obtained at the end of each cycle was determined by means of frequency sweeps (0.1 - 100Hz, strain 0.5%) at 5°C and results are presented at Figure 9. Tara gum did not confer any significant cryogel formation (i.e. G’ increased from 11.2 to 19.3 Pa at the end of the fifth freeze-thaw cycle). In the case of the purified LBG, a steep cryogel forming response to the number of the freeze thaw cycles was observed (i.e. G’ increased from 11.9 to 110.2 Pa within the first two cycles), followed by a gradual decline of the cryogel stiffness at the end of the 4th freeze-thaw cycle. On the other hand, mAAG exerted a sustained cryogel forming capacity with the stiffness of the cryogels to increase throughout the entire freeze - thawing processing employed (G’ increased from 13.6 to 126.2 Pa at the end of the 5th cycle).

[0115] Example 10: Cryogelation enhancement between modified AAG and xanthan gum mAAG was obtained as describe in Example 2. Individual aqueous solutions (0.5% wt.) of commercial xanthan gum and mAAG as well as their binary mixture (1:1, total biopolymer content 0.5% wt.) were prepared by dispersing the gums into cold MilliQ water (25°C) under magnetic stirring and heating to 80°C for 30min under constant stirring. The single biopolymer solutions as well as their binary (1 :1) mixture were cooled to ambient temperature (25°C), divided equally in 5mL Eppendorf tubes and transferred into a freezer (-20°C) for 1h. The cryogenically processed biopolymer solutions were transferred into a chilling chamber at 4°C and allowed to melt completely for 1h (that corresponds to one freeze thaw cycle). The biopolymer solutions were subjected in five freeze-thaw cycles and results are presented at Figure 10. The viscoelastic behaviour of the cryogels obtained at the end of each cycle was determined by means of frequency sweeps (0.1 - 100Hz, strain 0.5%) at 5°C. A cryogelation enhancing interaction between xanthan gum and mAAG8h (increase in the G’ ranged from 2 to 280 %) was observed.

[0116] Example 11 : Cryogelation enhancement with sucrose of modified AAG mAAG was obtained as describe in Example 5. Aqueous solution of mAAG (1% wt.) was prepared by dispersing the gums into cold MilliQ water (25°C) under magnetic stirring and heating to 80°C for 30min under constant stirring. The solutions were cooled to ambient temperature (25°C), divided equally in 5mL Eppendorf tubes and transferred into a freezer (-20°C) for 1 h. The frozen biopolymer solutions transferred into a chilling chamber at 4°C and allowed to melt completely (that corresponds to one cycle). The biopolymer solutions were subjected in five successive freeze-thaw cycles and results are represented at Figure 11. The viscoelastic behaviour of the cryogels obtained at the end of each cycle was determined by means of frequency sweeps (0.1 - 100Hz, strain 0.5%) at 5°C. According to the elastic modulus G’ plot (Figure 11), mAAG exerts a strong cryogel formation synergism with sucrose (G’ was in average 2.5 times higher in the presence of sucrose as compared to the sucrose- free counterparts, and sucrose alone has negligible impact on elastic modulus development (i.e. G’ ~ 0)).

[0117] Example 12: Cold water solubility of modified AAG as compared to LBG mAAG was obtained as describe in Example 2. purified LBG was obtained as described in Example 3. Individual aqueous solutions of purified LBG, native AAG and mAAG were prepared by dispersing the biopolymers into cold water at 25°C under magnetic stirring. The solutions were kept under stirring overnight and then, centrifuged at 4000g for 30min. The supernatants were collected and characterised for their galactomannan content (mg / mL) by means of the phenol-sulfuric acid method as detailed in (Masuko et al., 2005). The cold water solubility of the gums was determined according to the formula: CWS (%) = [galactomannan total solids added in the solution - galactomannan total solids in the supernatant] / [galactomannan total solids added in the solution] and results are represented at Figure 12. As represented, the solubility of purLBG, native AAG and mAAG was 11.4%, 99.5% and 71.2%, respectively. The ability of galactomannans to dissolve in cold water (i.e. at 20- 25°C) is steeply reduced when M / G>2 (i.e., tara, LBG and cassia galactomannans). These results show that despite the mAAG technofunctional affinity to LBG (i.e. M / G ~4, similar thickening, gelling and cryogelling performance) it is substantially (unexpectedly) higher soluble in cold water (i.e. 71%) than LBG (i.e. 11%).

[0118] Example 13: Production of low and intermediate-to-high viscosity soluble fibres (galactomannooligosaccharides - GMO)

[0119] An alpha-galactosidase (Bifidobacterium bifiduni) and two beta-endo-mannanases (1 : Podospora anserina and 2: Clostridium thermocellum) were mixed with native AAG solutions (1% wt) at 30 ll / g and 7.5U / g, respectively. The native AAG solutions were incubated for 5h at: GM01: 30°C, pH=6.5, using beta-endo-mannanases 1 (working conditions excluding endo-beta-mannanase),

[0120] GM02: 60°C, pH=4.5, using beta-endo-mannanases 1 (working conditions excluding the alpha -galactosidase) and - GM03: 65°C, pH=7.5, using beta-endo-mannanases 2 (working conditions excluding the alpha -galactosidase).

[0121] The complex viscosity was in situ monitored in double gap geometry (strain: 0.5%, frequency 1Hz). As illustrated in Figure 13, three different classes of partially hydrolysed products: low DH - high viscosity (GMO1), medium DH - intermediate viscosity (GMO2), and high DH - low viscosity (GMO3) were obtained. The products can be employed as soluble fibre for personalised nutrition applications e.g. dysphagia formulations, prebiotics / synbiotics etc.

Claims

CLAIMS1. An enzyme complex solution comprising an alpha-galactosidase and a beta-endo- mannanase, wherein the alpha-galactosidase and the beta-endo-mannanase retain at least 80% of their maximal activity at mutual exclusion ranges of temperatures and / or ranges of pH.

2. The enzyme complex solution according to claim 1 , wherein the ranges of pH and / or temperature at which one enzyme from the alpha-galactosidase and the beta-endo- mannanase retains at least 80% of its maximal activity correspond to at most 20% of maximal activity of the other.

3. A process for obtaining a modified galactomannan by reducing the galactose content and / or the mannose chain length of a starting galactomannan with the enzyme complex solution of claim 1 or 2.

4. The process according to claim 3, wherein the starting galactomannan is an alfalfa galactomannan or a fenugreek galactomannan.

5. The process according to claim 3 or 4, wherein the modified galactomannan has a mannose over galactose content ratio from 2 to 5.

6. The process according to any of claims 3 to 5, comprising the following steps: a) providing a hydrated preparation of a starting galactomannan, b) adding the enzyme complex solution to the hydrated preparation, c) depleting the galactose unit content of the starting galactomannan with the alphagalactosidase of the enzyme complex solution and / or reducing the chain length mannan of the starting galactomannan with the beta-endo-mannanase of the enzyme complex solution, d) inactivating the enzyme complex solution, and e) obtaining a modified galactomannan, wherein the galactose depleting step and the chain length mannan reducing step are conducted at mutual exclusion ranges of temperatures and / or ranges of pH.

7. Use of the enzyme complex solution according to claim 1 or 2 for modifying a galactomannan and obtaining a modified galactomannan.

8. A modified galactomannan obtained with the process according to any of claims 3 to 6 or the use of claim 7, preferably having a mannose over galactose content ratio from 3 to 5.

9. A gum solution comprising the modified galactomannan according to claim 9.

10. The gum solution of claim 10, having a complex shear modulus G from 5 to 20 Pa.

11. The gum solution of claim 9 or 10, further comprising at least one additional polysaccharide selected in the group consisting in a second galactomannan, xanthan, kappa-carrageenan, sucrose and combinations thereof.

12. The gum solution of any of claims 9 to 11, having an elastic modulus G’ from 4 to 25 Pa.

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