Classifications

• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
  • C01B25/00—Phosphorus; Compounds thereof
  • C01B25/16—Oxyacids of phosphorus; Salts thereof
  • C01B25/26—Phosphates
  • C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
  • C01B25/00—Phosphorus; Compounds thereof
  • C01B25/16—Oxyacids of phosphorus; Salts thereof
  • C01B25/26—Phosphates
  • C01B25/45—Phosphates containing plural metal, or metal and ammonium
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

• the present invention relates to a method for the simultaneous production of inositol and inorganic phosphate, particularly by hydrolysing phytate in co-products of bioethanol production, including wheat yeast concentrate, corn concentrate, and sorghum concentrate. These co-products are typically employed as animal feed, which makes the present method well-suited to reduce phytate levels in animal feed and thereby avoid phosphate pollution.
• Ethanol can be produced by processing starch containing biomass such as wheat, corn, and sorghum.
• the ethanol production process results in several important co-products, including for example wheat yeast concentrate, corn concentrate and sorghum concentrate, and dried distillers grain and solubles (DDGS).
• DDGS dried distillers grain and solubles
• phosphate (P) in phytate form cannot be digested by nonruminant animals such as poultry and swine, which results in significant amounts of phytate P in their excreta.
• Phytates can strongly bind to divalent minerals and proteins, preventing their assimilation by the digestive system. In view thereof, phytates are also known as antinutrients.
• the phytate P-rich manure can cause P pollution in soil and surface water.
• inorganic P supplements are also added to the feed, resulting in a further increased P content in the animal excreta and worsening of potential P pollution.
• Noureddini 2010, Biosource Technology 101 ; 9106-9113
• phytates are hydrolysed to produce inorganic phosphate and myo-inositol.
• the process of Noureddini is costly, complex and requires multiple steps.
• the present invention provides a method for simultaneously producing inositol and inorganic phosphate, wherein the method preferably is an integrated part of an ethanol production process.
• the method uses a co-product of the ethanol production process, i.e. a stream containing phytate, phytic acid, and/or phytin.
• a co-product of the ethanol production process i.e. a stream containing phytate, phytic acid, and/or phytin.
• this can be wheat yeast concentrate, corn concentrate or sorghum concentrate, depending on the raw material used in the ethanol production.
• the method then uses a hydrolysis step to convert the phytate, phytic acid, and/or phytin to inositol and inorganic phosphate.
• the present inventors found that the method is drastically improved by employing a separation step, preferably by means of a nanofiltration element, which separates inorganic phosphate from the reaction mixture during the hydrolysis step, and recirculates the partly hydrolysed phytate (i.e. a mixture of inositol phosphates, e.g. IP5, IP4, IP3, IP2, and/or IP1) back to the reaction mixture.
• a separation step preferably by means of a nanofiltration element, which separates inorganic phosphate from the reaction mixture during the hydrolysis step, and recirculates the partly hydrolysed phytate (i.e. a mixture of inositol phosphates, e.g. IP5, IP4, IP3, IP2, and/or IP1) back to the reaction mixture.
• the present method produces valuable products in high concentrations, namely inositol and inorganic phosphate, and at the same time greatly contributes to the prevention of P pollution by lowering phytate P content in animal feed.
• an enzymatic hydrolysis step can be improved by using a synergetic combination of phytase(s) and/or acidic phosphatase(s), which can optionally be immobilized.
• the present method does not require to perform a partial hydrolysis and subsequently a complete hydrolysis under different reaction conditions. Instead, the present method allows to perform the complete hydrolysis in a single step. Additionally, it was found that inositol can be isolated very efficiently by means of anti-solvent crystallization also in combination with chromatography.
• the present method can be performed in a continuous stirred tank reactor (CSTR) containing immobilized phytase and immobilized acid phosphatase, or in a continuous flow reactor set-up (CFR) comprising two column reactors with (1) immobilized phytase and (2) immobilized acid phosphatase linked in series.
• CSTR continuous stirred tank reactor
• CFR continuous flow reactor set-up
• the effluent of the CSTR or CFR system, respectively can be fed to a nanofiltration element (see Example 5) to separate the released free inorganic phosphate from the inositol and inositol phosphates.
• inorganic phosphate-free solution enriched in inositol and inositol phosphates is recycled to the enzyme reactors, to complete the hydrolysis.
• inositol can be isolated from the enriched solution by anti-solvent crystallization.
• the present invention relates to a method for producing inositol and inorganic phosphate, comprising the steps of:
• step (c) preferably separating inorganic phosphate from the composition provided in step (b) to provide a composition enriched in inositol phosphate, i.e. a mixture of IP5, IP4, IP3, IP2, and/or IP1 , (and/or inositol) and a composition comprising inorganic phosphate;
• step (d) hydrolysing the inositol phosphate obtained in step (c) to provide a composition comprising inositol and inorganic phosphate.
• Steps (b), (c), and/or (d) are not necessary performed in this order.
• steps (b) and (d) simultaneously and/or in the same reactor, although it is also possible to perform said steps in separate reactors.
• the step of separating the inorganic phosphate can thus be performed during at least part of the process of converting the phytate, phytic acid and/or phytin to IP5, IP4, IP3, IP2, IP1 respectively, and eventually to inositol, e.g. during at least 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 90, or 100% of the time applied for the hydrolysis reaction.
• Inositol (or cyclohexane-1 ,2,3,4,5,6-hexol) is a compound with formula C6H12O6 or (-CHOH- )e, a derivative of cyclohexane with six hydroxyl groups:
• Inositol exists in nine possible stereoisomers, of which cis-1 ,2,3,5-trans-4,6- cyclohexanehexol, or myo-inositol (also referred to as meso-inositol or i-inositol), is the most widely occurring form in nature. Inositol is a sugar alcohol with about half the sweetness of sucrose.
• Myo-inositol is the structural basis for a number of secondary messengers in eukaryotic cells, i.e. various inositol phosphates.
• inositol serves as an important component of the structural lipids phosphatidylinositol (PI) and its various phosphates, the
• PIP phosphatidylinositol phosphate
• Inositol can be used for diverse applications, including
• a nutritional supplement e.g. in (baby) food or animal feed including fish feed;
• inositol has been proposed as selective serotonin reuptake inhibitor (SSRI);
• inositol is thought to be essential for bone formation, osteogenesis and bone mineral density;
• step (b) and step (d) can be performed in a single reactor or single set of reactors, preferably by recycling the composition enriched in inositol phosphate as provided in step (c) to said reactor or set of reactors.
• step (b) and (d) can be performed as a single step, which is an embodiment of the above-described method and which comprises the steps of:
• step (a) the method starts with providing a composition comprising phytate, phytic acid, and/or phytin.
• the composition provided in step (a) or (a’) preferably comprises at most 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 , 0.5 wt.% other constituents next to water and the phytate, phytic acid, and/or phytin, with respect to the total weight of the composition.
• IP6 inositol hexakisphosphate
• IP6 inositol polyphosphate
• (1 R,2S,3r,4R,5S,6s)-cyclohexane-1 ,2,3,4,5,6-hexa-yl hexakis[dihydrogen (phosphate)], or phytate when in salt form, can be characterized as a saturated cyclic acid, and is the principal storage form of phosphate in many plant tissues, especially bran and seeds. It is typically found in cereals and grains.
• Phytin (CeHeCaeMgeC ⁇ Pe) is the calcium magnesium salt of phytic acid as it occurs naturally in plants.
• the composition comprising phytate, phytic acid, and/or phytin according to the present disclosure may be derived from wheat, corn, and/or sorghum.
• the composition refers to wheat yeast concentrate, corn concentrate (or corn steep water), and/or sorghum concentrate, which can be processed to Distillers Dried Grains with Solubles (DDGS) for use as animal feed.
• DDGS Distillers Dried Grains with Solubles
• the present method allows to produce animal feed with reduced levels of organic P, i.e. comprising at most 5000, 4000, 3000, 2500, 2000, 1500, 1000, 800, 600, 400, 200, 100, or 50 mg total phosphate per kg of the animal feed.
• Total phosphate can for example be determined by a photometric dry-ashing procedure based on the standard method as described by Pulliainen and Wallin (1994 J. AOAC Int. 77, 1557-1561). Briefly, measurements can be based on a colorimetric method wherein the colour of the treated sample reflects the concentration of P, and wherein the samples are first ashed to remove organic (C, H, O) materials. Hydrochloric acid is added to the remaining inorganic ash residue to dissolve P, and the resulting solution is used for the colour reaction based on the formation of a blue complex between phosphate and sodium molybdate, in the presence of ascorbic acid as the reducing agent. The blue colour of the complex is directly proportional to the total P.
• Wheat yeast concentrate is a co-product from bioethanol production wherein starch from grain is converted into ethanol by means of fermentation. After the ethanol has been separated, a brown-coloured protein-rich substance remains, i.e. wheat yeast concentrate. The biggest part of the wheat yeast concentrate thus obtained is commonly used as animal feed. From 800 kiloton of wheat comes 250 kiloton of wheat malt concentrate. Similarly, corn concentrate or sorghum concentrate can be obtained.
• step (a) of the method further involves purifying and/or concentrating the provided composition and/or removing constituents other than phytate, phytic acid and/or phytin from the provided composition, preferably by
• centrifugation step preferably at 2000-8000 rpm;
• precipitating the phytate, phytic acid and/or phytin for example by hydroxide precipitation, i.e. through adding calcium hydroxide (or magnesium hydroxide) to the provided composition, subsequently dissolving the obtained precipitate, and optionally disposing the supernatant.
• hydroxide precipitation i.e. through adding calcium hydroxide (or magnesium hydroxide) to the provided composition, subsequently dissolving the obtained precipitate, and optionally disposing the supernatant.
• this can be achieved by first adjusting the pH to a range of pH 2-9, or 3-8, 3-7, or pH 4-5. Then, the composition can be mixed or agitated, and subsequently centrifuged, preferably at 2000-8000 rpm for a period of e.g. 1-30 min, or 1-10 min. Then, the supernatant can be removed, and the precipitate can be dissolved by adding e.g. HCI.
• a hydrolysis step is performed, preferably in a single (set of) containers, more preferably in a continuous stirred tank reactor, or in a continuous flow reactor which may comprise two column reactors.
• the container or reactor(s) is preferably set up to allow hydrolysis of phytic acid and/or inositol phosphate to inositol. Accordingly, the reaction mixture is preferably under conditions allowing such hydrolysis.
• Hydrolysis of the phytate, phytic acid, and/or phytin refers to the hydrolytic cleavage of at least one, two, three, four, five, or six phosphate group(s) of the phytate, phytic acid, and/or phytin in order to obtain the different inositol phosphates being (myo-)inositol pentakis-, tetrakis-, tris-, bis- and monophosphate, i.e. IP5, IP4, IP3, IP2, and IP1 respectively, and eventually inositol. Additionally, inorganic phosphate is obtained, which refers to the anion P0 4 3 , and which can form a salt together with for example hydrogen or calcium, magnesium, sodium, kalium, or ammonium.
• the concentration of inositol and inorganic phosphate in the reaction mixture will increase, while the concentration of phytic acid and/or inositol phosphates will decrease.
• the method does not provide for further hydrolysis steps apart from step (b) and/or (d) (or b’).
• the method according to the present disclosure may comprise additional steps, for example - step (e) (or c’) of separating the inorganic phosphate from the composition provided in step (d) (or b’) to provide a composition comprising inositol and a composition comprising inorganic phosphate.
• This step thus lowers the concentration of inorganic phosphate of the starting composition, i.e. the composition provided in step (d) (or b’), such that a composition comprising inositol is provided which contains at most 1000, 800, 600, 400, 200, 100, 50,
• inorganic phosphate content can be determined as described by Noureddini et al (2009, Bioresour. Technol. 100 731-736).
• a separation step is performed to separate and/or remove inorganic phosphate from the reaction mixture, e.g. by (nano)filtration, ion exchange chromatography, size exclusion chromatography, electrodialysis.
• a particular advantage thereof is that a composition enriched in inositol is provided, with reduced inorganic phosphate content so as to significantly reduce product inhibition during the hydrolysis reaction.
• enriched means that the proportion of inositol relative to inorganic phosphate is increased relative to the starting composition.
• inorganic phosphate is isolated.
• the method according to the present disclosure preferably does not involve further separation steps such as by column chromatography.
• the separation step according to the present method lowers the concentration of inorganic phosphate of the starting composition, i.e. the composition provided in step (d), such that a composition comprising inositol and/or inositol phosphate is provided which contains at most 1000, 800, 600, 400, 200, 100, 50, 40, 30, 20, 10, 5, or 1 mg inorganic phosphate (free phosphate) per kg of the composition.
• the separation step in step (c) (or b’) and/or step (e) is performed by membrane filtration, preferably by a nanofiltration element, more preferably a nanofiltration element with a pore dimension of at most 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, or between 1-10, 1-15 nanometer.
• a DOW Filmtec NF270-400 nanofiltration element at 1-200, or 20-120, or 50-90, 65-75 psi may be used, during e.g. 0.5- 600 min, preferably 1-100 min or 20-40 min, at a temperature of 0-100, preferably 1-50, or 5-35 degrees Celsius.
• the nanofiltration element will produce a retentate comprising phytate, inositol and/or inositol phosphate which can be recycled to the reaction mixture, while the permeate will comprise inorganic phosphate.
• composition provided in step (b) and/or the enriched composition provided in step (c) according to the method of the present disclosure may further comprise inositol, phytate, phytic acid, and/or phytin (as well as inorganic phosphate).
• the method according to the present disclosure may further comprise step (f) (or c’ or d’) of precipitating the inositol from the composition comprising inositol provided in step (d) or step (e) (or b’), preferably by anti-solvent crystallisation, by selectively removing solvent from the reaction mixture (i.e. water) and/or adding anti-solvent to the reaction mixture, thereby allowing the precipitation of inositol.
• the anti-solvent crystallisation is performed as described in WO2012128624.
• the process for the crystallisation of the water-soluble inositol from the reaction mixture comprises the following steps: 1) providing, preferably in a crystallisation vessel, the water-soluble inositol in a mixture of water and a solvent in which the inositol has a lower solubility than in water; 2) preferably, passing vapour phase of the mixture (preferably at elevated temperature such as between 30-100, or 40-80 or 50-70 degrees Celsius) through a sorption zone containing a water vapour sorbent to selectively adsorb water from the vapour phase to obtain a vapour phase depleted in water and enriched in the solvent and water-saturated water vapour sorbent; 3) enriching the mixture in the crystallisation vessel in solvent by recycling at least part of the vapour phase depleted in water and enriched in the solvent to the crystallisation vessel or withdrawing vapour phase depleted in water from the process
• inositol produced according to the present disclosure typically will be myo-inositol.
• the hydrolysis in step (b) (or b’) and/or step (d) is acid hydrolysis (or chemical proton hydrolysis).
• Acid hydrolysis can be seen as a process in which a protic acid is used to catalyse the cleavage of a chemical bond via a nucleophilic substitution reaction, typically with the addition of the elements of water (H2O).
• the pH of the reaction mixture is preferably adjusted to pH 1-8, or 2-6, more preferably 3-5 or 3.5-4.5 and/or the temperature of the reaction mixture is adjusted to 50-200, or 75-175 or 125-175 or 130-170 degrees Celsius, preferably for a period of 0.5-80, or 1-80, 1-50, 10-40, 20-30 hours, and preferably subsequent cooling to a temperature of 10-100, or 15-40 or 15-30 degrees Celsius, at a pH in the preferred range of pH 3-10, or 4-9, or 6-8.
• the hydrolysis in step (b) (or b’) and/or step (d) is enzymatic hydrolysis, which allows for very efficient conversion of the phytate, phytic acid and/or phytin into inositol.
• enzymatic hydrolysis is that it does require heating and cooling steps, nor adjustment of the pH, while high concentrations of inositol can be obtained.
• the enzymatic hydrolysis can be performed by using at least one phytase and/or at least one acidic phosphatase, preferably the combination and/or in a single container/reactor.
• the phytase may be also be provided in a container/reactor different from the container/reactor where the acidic phosphatase is provided, preferably wherein the effluent of the phyase containing reactor is the input for the acidic phosphatse containing reactor.
• the separation step e.g. the nanofiltration element may be provided in between, or after these reactors.
• hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol can be achieved by enzymatic catalysis using an enzyme preparation rich in phytase, that can catalyze the partial hydrolysis of phytate to myo-inositol 2-monophosphate (IP1), and/or an acidic phosphatase that can hydrolyse the myo-inositol 2-monophosphate further to release myo-inositol and inorganic phosphate.
• IP1 myo-inositol 2-monophosphate
• phytate hydrolysis by phytases occurs in a step-wise process, with the release of myo-inositol pentakis-, tetrakis-, tris-, bis- and monoposphates, depending on the type and the substrate specificity of the enzymes used.
• Plant, fungal and bacterial phytases can be used. Most suited is a phytase with broad substrate specificity, like phytase derived from Aspergillus fumigatus, Emericella nidulans and/or Myceliophthora thermophila, that can readily hydrolyse phytate and catalyse the intermediate hydrolysis of myo-inositol phosphates and accumulate myo-inositol 2- monophopshate as major product, and/or a phytase with high specificity for phytate, such as a phytase from Aspergillus niger and Escherichia coli.
• a phytase with broad substrate specificity like phytase derived from Aspergillus fumigatus, Emericella nidulans and/or Myceliophthora thermophila, that can readily hydrolyse phytate and catalyse the intermediate hydrolysis
• an acidic phosphatase from A. niger can be used and is particularly preferred.
• a phytase and acid phosphatase can act in synergy to hydrolyse phytate in order to produce myo-inositol and inorganic phosphate, preferably at conditions of pH and temperature that favour the activity of both enzymes, e.g. pH of at least 1 , 2, or at least 2.5 and/or below pH 9,8, 7 or 6 and/or temperature of 20-80 degrees Celsius, or 30-70, or 40-60, 45-55 °C.
• the at least one phytase can be a fungal or bacterial phytase, particularly an phytase from Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, Aspergillus niger and/or Escherichia coli; and/or the at least one acidic phosphatase can be a fungal or bacterial acidic phosphatase, particularly an acidic phosphatase from Aspergillus niger.
• a combination of enzymes is used which provide synergistic conversion reactions.
• the following combinations of at least one phytase and at least one acidic phosphatase provide for a synergistic conversion efficiency:
• Said synergistic conversion reactions preferably are performed in one container/reactor at conditions of pH and temperature that favour the activity of both enzymes, e.g. a pH of within 3 pH units, preferably within 2 pH units, more preferably within 1 pH unit, of the pH optimum of both enzymes, and at a temperature of within 10 °C, preferably within 5 °C, more preferably within 2 °C, of the temperature optimum of both enzymes.
• pH and temperature that favour the activity of both enzymes
• Aspergillus niger phytase and Aspergillus niger acid phosphatase is between 2.5-6.5 pH and 30-55 °C, preferably about pH 4.5 and 40 °C;
• Aspergillus fumigatus phytase and Aspergillus niger acid phosphatase is between 4- 6 pH and 40-55 °C, preferably about pH 5 and 45°C;
• Myceliophthora thermophila phytase and Aspergillus niger acid phosphatase is between 3-6.5 pH and 30-55°C, preferably about pH 4.5 and 40°C;
• Escherichia coli phytase and Aspergillus niger acid phosphatase is between 2.5-6.5 pH and 30-55 °C, preferably about pH 5.5 and 50°C.
• the at least one phytase and/or at least one acidic phosphatase as used according to the present disclosure can be immobilized e.g. to a solid support, or to solid particles of at least 0.1 , 0,5, 1 , 2, 4, 6, 8, 10, 15 mm, or at most 15, 20,
• phytate hydrolysis can thus be performed in e.g. a continuous stirred tank reactor (CSTR) containing (immobilized) phytase and (immobilized) acid phosphatase, or in a continuous flow reactor set-up preferably comprising two column reactors with (1) (immobilized) phytase and (2) (immobilized) acid phosphatase, preferably linked in series (CFR).
• CSTR continuous stirred tank reactor
• CFR system preferably comprises two column reactors with (1) (immobilized) phytase and (2) (immobilized) acid phosphatase, preferably linked in series (CFR).
• the effluent of the CSTR or CFR system respectively, can be fed to a nanofiltration element (see Example 5) to separate the released free inorganic phosphate from the reaction mixture comprising inositol and inositol phosphates (and phytate).
• the inorganic phosphate- free solution enriched in inositol and inositol phosphates can be recycled to the enzyme reactor(s), to complete the hydrolysis. At the end, inositol can be separated from the reaction mixture.
• FIG. 1 relates to a preferred embodiment of the present disclosure.
• the figure shows a Continuous Stirred Tank Reactor (CSTR), wherein phytate (1) is introduced and hydrolysis is carried out with immobilized enzymes, using an immobilized phytase (A) and an immobilized acid phosphatase (B).
• the effluent (2) of the CSTR is fed to a nanofiltration element (C) to separate the released free phosphate (3) from the inositol and inositol phosphates.
• the phosphate-free solution enriched in inositol and inositol phosphates (4) is recycled to the CSTR, to complete the hydrolysis.
• inositol is separated from the enriched solution by anti-solvent crystallisation (not shown).
• Example 3 0.5 g calcium hydroxide was suspended in 20 ml water and slowly added to the supernatant of Example 1 which was stirred during the addition of the calcium hydroxide. After 1 hour at room temperature the treated supernatant was centrifuged during 5 minutes at 4000 rpm. The clear supernatant contained 1.04 g/L total phosphate and 0.14 g/L free phosphate. By calculation it was concluded that 67% of the total phosphate and 71% of the free phosphate was precipitated. It was concluded that the precipitated phosphate was calcium phosphate and the bound phosphate was calcium phytate.
• Example 3 Example 3
• Example 2 The precipitate of Example 2 was taken and a 1 Molar solution of HCI was added until the pH was 4.2 for more than 5 minutes. A clear solution containing phosphoric acid and phytin resulted.
• crystallisation tank After 24 hours the volume has decreased from about 1250 ml to 550 ml, and crystals of myo- inositol could be collected by sieving with a yield of 52 gram (yield 69.3%) after washing the precipitate with 96% ethanol and drying the crystals at room temperature. After analysis, the crystals were identified as myo-inositol while less than 1 % (w/w) of phosphate could be detected.
• Hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol can be achieved by enzymatic catalysis using an enzyme preparation rich in phytase, that catalyses the partial hydrolysis of phytate to myo-inositol 2-monophosphate (IP1), and an acidic phosphatase that hydrolyses further the myo-inositol 2-monophosphate to release myo-inositol and phosphate.
• IP1 myo-inositol 2-monophosphate
• Phytate hydrolysis by phytases occurs in a step-wise process, with the release of myo-inositol pentakis-, tetrakis-, tris-, bis- and monoposphates, depending on the type and the substrate specificity of the enzymes used. Plant, fungal and bacterial phytases can be used.
• phytases with broad substrate specificity like phytases from Aspergillus fumigatus, Emericella nidulans and Myceliophthora thermophila, that readily hydrolyse phytate and the intermediate hydrolysis myo-inositol phosphates and accumulate myo-inositol 2-monophopshate as major product, and phytases with high specificity for phytate, such as phytases from Aspergillus niger and Escherichia coli.
• an acidic phosphatase from A. niger can be used.
• Any combination of a phytase and acid phosphatase can act in synergy, in one reaction vessel, to hydrolyse phytate to produce myo-inositol and phosphate, at conditions of pH and temperature that favour the activity of both enzymes, (e.g. pH higher than 2.5 and below pH 6 and temperature around 40 and 50 °C).
• Suitable enzymes mixtures acting in synergy for the total hydrolysis of phytate to myo-inositol are: (1) A. niger phytase and A. niger acid phosphatase, (2) A. fumigatus phytase and A. niger acid phosphatase, (3) M. thermophila phytase and A.
• niger acid phosphatase (4) E. coli phytase and A. niger acid phosphatase.
• Hydrolysis of phytate can be carried in solution, with soluble enzymes, in a membrane reactor to separate continuously the phosphate released from the reaction mixture containing the inositol phosphates and free inositol, in order to prevent enzyme inhibition by phosphate.
• the inositol rich solution is separated and inositol can be isolated from the solution by antisolvent crystallisation as described in example 6, while the phopshate is precipitated from the phosphate-rich fraction using known procedures.
• phytate hydrolysis can be carried out with immobilized enzymes, using an immobilized phytase and an immobilized acid phosphatase, as described above.
• the enzymes can be immobilized by adsorption, by encapsulation, by cross-linking as cross- linked enzyme aggregates, by covalent binding to an inert solid support or by attachment on magnetic active particles.
• Phytate hydrolysis can be performed in a continuous stirred thank reactor (CSTR) containing the immobilized phytase and acid phosphatase, or in a continuous flow reactor set-up consisting of two column rectors with (1) immobilized phytase and (2) immobilized acid phosphatase linked in series (CFR).
• CSTR continuous stirred thank reactor
• the effluent of the CSTR and CFR systems, respectively, is fed to a nanofiltration element (see Example 5) to separate the released free phosphate from the inositol and inositol phosphates.
• the phosphate-free solution enriched in inositol and inositol phosphates is recycled to the enzyme reactors, to complete the hydrolysis.
• inositol is separated from the enriched solution by anti- solvent crystallisation.
• the Table below shows the estimated end concentrations of inorganic phosphate and myo inositol in the reaction mixture as obtained by the method according to the present disclosure.
• Table 1 Estimated concentration in Pi and myo-inositol as obtained by the method according to the present disclosure upon processing wheat yeast concentrate.

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