Diafiltration

Abstract

To provide methods to isolate native protein having functionality and solubility from potato side streams, in a viable commercial process.SOLUTION: The present invention provides a method to isolate native tuber protein using a pretreatment of tuber processing water and diafiltration for salt solution. This sequence of steps has the advantage that protein is stabilized during diafiltration, increasing process efficiency and protein quality and yield.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a method for isolating native tuber proteins using diafiltration. [Background technology]

[0002] Vegetarians and vegans are increasingly aware of the environmental impact of meat-based foods, which has led to a growing demand for traditional food analogs. However, in various aspects, plant-based proteins still cannot compete with animal-derived products. One reason is that plant-based proteins often have to be isolated and processed before being prepared into food.

[0003] Potato protein is widely available because potatoes are processed extensively to obtain starch and a variety of potato products. Potato protein has an amino acid composition that makes it ideal for use in human food applications. However, the isolation of potato protein of sufficient quality is a tedious process.

[0004] Potato proteins are traditionally isolated from a side stream of starch production, which is prepared by crushing or grinding whole potatoes followed by separation of the starch. The resulting stream contains potato proteins, which can be isolated by a variety of methods to obtain native or coagulated proteins. Coagulated proteins can be obtained by traditional methods but suffer from the drawback of lacking functionality and solubility. Therefore, in many food applications, native proteins are more desirable.

[0005] However, isolated native potato proteins often suffer from off-tastes and excessive color, which make their use in food difficult. The best results have been obtained using absorption or chromatography, such as expanded bed adsorption, membrane adsorption or ion exchange chromatography, but these methods are expensive and laborious, especially on an industrial scale, because they require a series of pretreatments and must be operated at high concentrations to reach acceptable efficiencies.

[0006] Other methods for isolating native proteins have also been applied. Various membrane methods, such as ultrafiltration and diafiltration, have been applied in various settings. However, it remains a challenge to isolate proteins of sufficient quality using these methods alone, as the proteins are often not sufficiently pure. In addition, membrane methods suffer from membrane clogging, which precludes large-scale applications. During diafiltration, proteins tend to aggregate and precipitate, which precludes the use of diafiltration in a commercially viable manner. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2017 / 146568 [Patent Document 2] WO2016 / 036243 [Patent Document 3] WO2008 / 056977 [Patent Document 4] WO2008 / 069651 [Patent Document 5] WO2016 / 036243 A1 [Non-patent literature]

[0008] [Non-Patent Document 1] Laus MC, Klip G. & Giuseppin MLF (2016) Food Anal. Methods 10(4) “Improved Extraction and Sample Cleanup of Tri-glycoalkaloids α-Solanine and α-Chaconine in Non-denatured Potato Protein Isolates” [Non-patent document 2] ISO17294-2:2016 Summary of the Invention [Problem to be solved by the invention]

[0009] A more versatile method for isolating proteins from potato side streams would allow for greater accessibility to potato proteins and thus greater access to functionally ideal plant-based proteins, thereby increasing the sustainability of the food supply. The present invention provides an optimized method for the isolation of native potato proteins based on diafiltration that can be carried out on a large scale. [Means for solving the problem]

[0010] The present invention provides a method for isolating a native tuber protein isolate, comprising the steps of: a) processing at least one tuber to obtain a tuber processing water containing native tuber protein; b) the tuber treatment water; ba) concentrated, and / or bb) dilution, and / or bc) pH adjustment, and / or bd) condensation, and / or be) heat treatment, and / or bf) Solid content removal a step of subjecting the sample to a pretreatment comprising one or more of the steps This pretreatment resulted in a 2–20 mS·cm -1providing a pretreated tuber treatment water having a conductivity of c) the pretreated tuber treatment water is diluted with at least 5 mS·cm -1 diafiltration using a 5 to 300 kDa membrane against a salt solution having a conductivity of Including, This results in the tuber protein isolate being obtained as the retentate of the diafiltration.

[0011] An advantage of the present method is that native tuber proteins can be isolated on a large scale from a variety of process streams, which can be achieved with high efficiency, low protein loss, a small environmental impact, low cost and relatively few waste streams, resulting in proteins with high solubility, high purity and intact functional properties. [Brief explanation of the drawings]

[0012] [Figure 1] Solubility of potato proteins at various pH and conductivities. [Figure 2] Solubility of potato proteins at pH 6 and 7 and at different conductivities when exposed to mechanical stress. [Figure 3] Flux-concentration plot during the first and second diafiltration of Example 6. [Figure 4] Solubility of total potato isolates at various conductivities. [Figure 5] Solubility of total potato isolates at various conductivities. [Figure 6]Total protein isolate (MF-PFJ) containing all protein fractions present in the tuber. L protein standard, lane 1: Example 4 Experiment 9 MF-PFJ, lane 2: Example 4 Experiment 10 MF-PFJ, lane 3: Example 4 Experiment 11 MF-PFJ, lane 4: Example 5 MF-PFJ, lane 5: Example 5 Final product (retentate of DF), lane 6: Example 4 Experiment 9 Final product (dried), lane 7: Example 4 Experiment 10 Final product (dried), lane 8: Example 4 Experiment 11 Final product (dried). DETAILED DESCRIPTION OF THE INVENTION

[0013] method The present method is directed to the isolation of native tuber proteins. Tubers in this context include structures that may also be called roots. Tubers naturally contain protein, and preferred types of tubers are also rich in starch, such as the types of tubers used for starch isolation.

[0014] Preferably, tubers in this regard comprise potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (including Manihot esculenta, syn. M. utilissima, also known as manioc, mandioca or yuca, and including M. palmata, syn. M. dulcis, also known as yuca dulce), yam (Dioscorea spp.) and / or taro (Colocasia esculenta). More preferably, the tubers comprise potato, sweet potato, cassava or yam, even more preferably, the tubers comprise potato, sweet potato or cassava, even more preferably, the tubers comprise potato or sweet potato, and most preferably, the tubers comprise potato (Solanum tuberosum).

[0015] Preferred tuber proteins include potato protein, sweet potato protein, cassava protein, yam protein, and / or taro protein. Potato protein is preferred. Potato is the tuber from the potato (Solanum tuberosum) plant, which exists in many varieties. The present method of protein isolation can be carried out with any potato variety, including varieties intended for the starch industry (starch potatoes) and varieties intended for human consumption (consumable potatoes).

[0016] All tuber varieties contain native tuber proteins, which can be divided into three classes: (i) the patatin family, highly homologous acidic 43 kDa glycoproteins (40-50 wt.% of potato proteins), (ii) basic 5-25 kDa protease inhibitors (30-40 wt.% of potato proteins), and (iii) other proteins, mostly of high molecular weight (10-20 wt.% of potato proteins).

[0017] A protease inhibitor as defined herein is a root or tuber protein, preferably a potato protein, which in its native form is capable of inhibiting the protease activity of a protease. It is common knowledge that the root or tuber protein is considered to be a protease inhibitor. In the present invention, protease inhibitor refers to a root or tuber protein fraction in which at least 80 wt.%, preferably at least 85 wt.%, more preferably at least 90 wt.% of the total protein has a molecular weight of at most 35 kDa as determined by SDS-page.

[0018] Patatin, as defined herein, is a root or tuber protein, preferably a potato protein, which is an acidic glycoprotein that functions as a storage protein in tubers. In the root and tuber processing industry, it is generally known which root or tuber proteins are considered patatin. Patatin, in the context of the present invention, refers to a root or tuber protein fraction in which at least 80 wt.%, preferably at least 85 wt.%, more preferably at least 90 wt.% of the total protein has a molecular weight of more than 35 kDa as determined by SDS-page.

[0019] SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a commonly known technique for determining the molecular weight of proteins.

[0020] The present method is directed to obtaining any native tuber protein isolate. In one embodiment, the native tuber protein isolate is a native protease inhibitor isolate. In another embodiment, the native tuber protein isolate is a native patatin isolate. In these embodiments, the tuber process water may be subjected to a step of removal of a specific potato protein prior to the diafiltration step. This can be achieved by adsorption chromatography, by selective precipitation, or by any other method known to separate one protein fraction from another. During such treatment, the protein fraction remaining in solution may then be subjected to a diafiltration step as defined herein.

[0021] In a highly preferred embodiment, the tuber protein isolate is an isolate containing native protease inhibitors and native patatin, and in a further highly preferred embodiment, the tuber protein isolate is a native whole tuber protein isolate.

[0022] Total isolate, as used herein, refers to a protein isolate that includes protease inhibitors and patatin, as well as any other proteins present in the tuber under consideration. Thus, a total unmodified tuber protein isolate can be defined as an isolate that includes all tuber proteins in their unmodified form.

[0023] The protein isolate obtained by this method is a native protein isolate. "Native" in this context means that the isolation of the protein from the tuber is achieved without significantly affecting the protein. Thus, a native protein is not significantly altered or denatured, i.e., the amino acid sequence, three-dimensional structure, and functional properties (e.g., solubility and / or emulsifying properties) remain essentially the same as the protein occurring in the tuber.

[0024] The nativeness of a protein can be tested by solubilization experiments. Non-native proteins are significantly less soluble in water than native proteins. Protein solubility can be determined by dispersing the protein in water, dividing the resulting liquid into two fractions, centrifuging one fraction at 800 g for 5 minutes to create a pellet of undissolved material, and recovering the supernatant. Solubility is determined by measuring the protein content in the supernatant and in the untreated solution and expressing the protein content of the supernatant as a percentage of the protein content in the untreated solution. A convenient method for determining protein content is by measuring absorbance at 280 nm via a Sprint Rapid Protein Analyser (CEM). In the present invention, a protein is considered native if its solubility is at least 55%, preferably at least 65%, more preferably at least 75%, even more preferably at least 85% or even at least 90%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98%.

[0025] Isolation in the present invention means obtaining the protein either as a (clean) solution or as a protein powder. The powder can be obtained from the solution by drying it. Optionally, a concentration step is carried out prior to drying, for example by reverse osmosis, ultrafiltration or freeze concentration. Isolation in this context means that the tuber protein is maintained in a solubilized form until the tuber protein isolate is dried to obtain native tuber protein powder. Therefore, isolation preferably does not include a step of protein precipitation, for example with alginate, resulting in a precipitated protein fraction, and a subsequent step of resolubilization of the precipitated protein, for example by dissolving the precipitated protein fraction after isolation in an aqueous solvent to obtain native tuber protein. Protein precipitation and subsequent resolubilization may result in slight denaturation, and therefore precipitated and resolubilized protein is not a protein isolate according to the present invention.

[0026] The present invention provides a method for producing a native tuber protein isolate having a pH of at least 5 mS·cm -1 It is disclosed that the native tuber protein isolate can be obtained by diafiltration (DF) against a salt solution having a conductivity of 0.05 kDa. Diafiltration is a method of removing low molecular weight compounds by diluting the retentate while removing the filtrate using a diafiltration membrane, which is characterized by a molecular weight cut-off (MWCO). A MWCO value of 10 kDa means that the membrane can retain 90% of the molecules having a molecular weight of 10 kDa from the feed solution. The native tuber protein isolate is obtained as the retentate of the diafiltration. Using diafiltration, salts present in the pretreated tuber processing water can be removed, but are replaced by salts in the salt solution.

[0027] The diafiltration membrane (DF membrane) is a membrane used in diafiltration in the present invention. Preferably, the DF membrane has a MWCO of 3 to 500 kDa, preferably 5 to 300 kDa, more preferably 5 to 200 kDa, for example, preferably 30 to 200 kDa, more preferably 40 to 120 kDa, and even more preferably 50 to 100 kDa. In one embodiment, the MWCO can be 3 to 50 kDa, preferably 5 to 25 kDa, for example, 5 to 15 kDa or 15 to 25 kDa. In another embodiment, the MWCO can be 50 to 200 kDa, preferably 50 to 150 kDa.

[0028] Preferred DF membranes are polysulfone (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), regenerated cellulose, and polypropylene (PP) membranes, preferably PES or PS membranes. Preferred DF membranes are anisotropic DF membranes. DF membranes can be implemented as tubular, spiral wound, hollow fiber, flat plate, or cross-rotation induced shear modification units. Highly preferred DF membranes are tubular DF ​​membranes. Each of these membranes can have the MWCO defined above.

[0029] In a preferred embodiment, diafiltration is carried out as a continuous (cross-flow) process. The operating flux is, for example, from 3 to 300 l (h m 2 ) -1 Between 5 and 200 l·(h·m 2 ) -1 between 5 and 100 l·(h·m 2 ) -1 Between 6 and 70 l·(h·m 2 ) -1 Between 6 and 30 l·(h·m 2 ) -1 Between 7 and 30 l·(h·m 2 ) -1 Between 9 and 20 l·(h·m 2 ) -1 It can be between.

[0030] It has been found that the major protein fractions in tuber proteins, and in particular potato proteins, are oppositely charged at many pH values. Patatin has a pI of 4.8 to 5.2, while protease inhibitors have pIs of 5.8 up to 9. Even pH values ​​optimized for solubility cannot prevent aggregation, precipitation, and clogging, especially during diafiltration. It has been found that the conductivity of the solution has a significant effect on protein solubility, and that low solubility can be compensated for by increasing the conductivity. This is particularly important during diafiltration.

[0031] It has been found that for any solution containing a native protein isolate as defined herein, a relatively high conductivity is essential throughout the isolation process, whereas the salt solution in which diafiltration is carried out should have a conductivity of at least 5 mS cm -1 The feed solution must have a conductivity of 2-20 mS cm -1 The electrical conductivity must be

[0032] During diafiltration, proteins experience high mechanical stress in the vicinity of the membrane. The flow pattern during diafiltration forces various protein molecules together, which leads to forced aggregation and precipitation. Furthermore, proteins can interact with the membrane. The mechanical stress experienced by proteins during diafiltration, and any membrane interactions, thus result in increased membrane clogging, which hinders industrial diafiltration of protein solutions.

[0033] At least 5 mS·cm -1It has been found that a salt solution with a conductivity of at least 5 mS cm stabilizes proteins during the mechanical stresses that occur with diafiltration, thereby maintaining and even enhancing protein solubility under mechanical stress. This improves flux stability, increases DF operation time, and minimizes protein loss. Therefore, it is essential that diafiltration be performed on a salt solution. This is important to maintain protein stability in solution during diafiltration. The term "salt solution," as used herein, refers to a solution that has a conductivity of at least 5 mS cm. -1 is defined as a solution containing a salt having a conductivity of

[0034] The conductivity of the salt solution must be at least 5 mS cm to maintain good solubility of all tuber proteins during diafiltration. -1 , preferably at least 8 mS cm -1 , more preferably at least 15 mS cm -1 However, to avoid the addition of excess salt during diafiltration, the conductivity should preferably be less than 100 mS cm -1 Less than 50 mS·cm -1 Less than 20 mS·cm -1 less than, and even more preferably 18 mS·cm -1 is less than.

[0035] The conductivity of the solution to be diafiltered (diafiltration feed solution or feed) is between 2 and 20 mS cm -1 , preferably 5 to 18 mS cm -1 , more preferably 8 to 14 mS·cm -1 It is further essential that the pH of the diafiltration feed solution is less than 4.0 or greater than 5.5, more preferably between 5.5 and 12, and even more preferably between 5.5 and 7.0. This ensures that the protein does not precipitate before diafiltration.

[0036] In a further preferred embodiment, the diafiltration is preferably performed at a concentration of 5 to 20 mS·cm -1 , preferably 5 to 18 mS cm -1 , more preferably 8 to 15 mS·cm -1 , and even more preferably 9 to 14 mS·cm -1 , e.g., 9–11 or 10–13 mS cm -1 The method is carried out on a salt solution having a conductivity as defined above.

[0037] The salt solution preferably comprises a chloride salt, such as NaCl, KCl, and / or CaCl, preferably NaCl or KCl. The salt solution may preferably comprise KCl. Alternatively, the salt solution may preferably comprise NaCl. Further alternatively, the salt solution comprises a mixture of NaCl and KCl.

[0038] Preferably, the salt is NaCl. In the case of NaCl, the salt concentration in the salt solution can be 0.1 to 5 wt.%, preferably 0.2 to 2 wt.%.

[0039] Those skilled in the art can use their common general knowledge to convert the requested conductivity to a mass-based concentration (or molar concentration), with or without other solutes present. For example, the conductivity of a 0.33 wt.% NaCl solution is 5.3 mS / cm.

[0040] In a highly preferred embodiment, the salt solution does not contain heavy metal salts, such as cadmium, mercury, lead or arsenic salts. In a further preferred embodiment, the salt solution further contains NH4HCO3, which increases the flux.

[0041] In further preferred embodiments, the salt solution may have a pH of less than 4.0 or greater than 5.5, more preferably between 5.5 and 12, more preferably between 5.5 and 8.0, even more preferably between 6.0 and 8.0, for example between 5.5 and 7.0 or between 6.0 and 7.0. In preferred embodiments, this pH is maintained throughout diafiltration. In other preferred embodiments, the salt solution used for the advanced stages of diafiltration has a higher pH, for example between 8.0 and 12.0, preferably between 9.0 and 11.0, to further increase membrane flux.

[0042] Diafiltration is preferably carried out at a dilution ratio of (feed:salt solution) in the range of 5:1 to 1:10, preferably 1:1 to 1:10, preferably 1:1 to 1:5, more preferably 1:1 to 1:4. The retentate of this DF may be subjected to a second, third or further stage of DF.

[0043] These conditions result in a diafiltration retentate containing clean, native tuber protein, which as a percentage of dry matter, is at least 75 wt.%, preferably at least 80 wt.%, more preferably at least 85 wt.%, even more preferably at least 90 wt.% native tuber protein, preferably at most 1.0 wt.%, more preferably at most 0.5 wt.%, more preferably at most 0.1 wt.% total glucose, fructose and sucrose, preferably at most 1.0 wt.%, more preferably at most 0.5 wt.%, even more preferably at most 0.1 wt.% total tuber free amino acids, more preferably at most 1.0 wt.%, more preferably at most 0.5 wt.%, even more preferably at most 0.1 wt.% total glucose, fructose and sucrose, Preferably, the diafiltration retentate contains at most 0.1 wt.% tuber free amino acids, preferably at most 10 mg / kg, more preferably at most 5 mg / kg sulfites, preferably at most 200 mg / kg, more preferably at most 100 mg / kg, more preferably at most 50 mg / kg, even more preferably at most 25 mg / kg glycoalkaloids, preferably at most 5 mg / kg heavy metals selected from the group consisting of cadmium, mercury, lead, and arsenic, and / or preferably at most 10 wt.%, more preferably at most 5 wt.% chloride salts. Preferably, the ash content is less than 5 wt.%, more preferably less than 3 wt.%, more preferably less than 1 wt.%. Even more preferably, the potassium content is less than 4 wt.%, preferably less than 2 wt.%, more preferably less than 1 wt.%. In a highly preferred embodiment, the retentate of the diafiltration complies with the ranges of all these parameters combined. All amounts are expressed as percentages of dry matter.

[0044] In a preferred embodiment, diafiltration is carried out against a salt solution throughout all diafiltration stages. In a further preferred embodiment, particularly when the salt solution is applied at a relatively high conductivity within the ranges specified herein, diafiltration against a salt solution can be continued with a stage of diafiltration against water at a low conductivity or against normal water to remove the salt and isolate essentially salt-free native tuber protein.

[0045] Preferably, the conductivity of the solution to be diafiltered (diafiltration feed solution or feed) remains within the ranges specified herein. Preferably, in this embodiment, the pH remains the same throughout all diafiltration stages. Thus, membrane clogging can be balanced with the need to remove salts after diafiltration. In this way, a tuber protein isolate with a low salt content compared to the dry matter is obtained.

[0046] Alternatively, the retentate from diafiltration can optionally be subjected to an ultrafiltration (UF) step. This results in the concentration of the diafiltration retentate while simultaneously removing at least a portion of the salt added during the DF step. Preferably, the conductivity remains more or less constant during UF. In this way, a concentrated tuber protein isolate having a low salt content relative to dry matter is obtained. Preferably, the concentrated tuber protein isolate obtained from ultrafiltration complies with all the parameters described above for the diafiltration retentate, but in addition has a salt content, expressed as ash, of less than 5 wt.%, preferably less than 3 wt.%, even more preferably less than 1 wt.%. Even more preferably, the potassium content is less than 4 wt.%, preferably less than 2 wt.%, more preferably less than 1 wt.%.

[0047] Ultrafiltration can be performed using the same or a different setup as that used for diafiltration. Thus, the membrane can have a MWCO of 3 to 500 kDa, preferably 5 to 300 kDa, more preferably 5 to 200 kDa, preferably 30 to 200 kDa, more preferably 40 to 120 kDa, and even more preferably 50 to 100 kDa. In one embodiment, the MWCO, independent of the membrane used for diafiltration, can be 3 to 50 kDa, preferably 5 to 25 kDa, for example 5 to 15 kDa or 15 to 25 kDa, or 50 to 200 kDa, preferably 50 to 150 kDa.

[0048] Preferred UF membranes, also independent of the membrane used for diafiltration, are polysulfone (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), regenerated cellulose, and polypropylene (PP), preferably PES or PS membranes. Preferred UF membranes are anisotropic UF membranes. UF membranes can be implemented as tubular, spiral wound, hollow fiber, flat plate, or cross-rotation induced shear modification units. Highly preferred membranes are tubular UF membranes.

[0049] The flux may also be the same or different from that obtained in diafiltration, but is generally as described above under diafiltration. UF is preferably carried out to obtain a concentrated tuber protein isolate having a total dissolved solids of 0.5 to 25°Bx, preferably 5 to 22°Bx, more preferably 10 to 18°Bx, even more preferably 12 to 17°Bx, and even more preferably 14 to 16°Bx. In further preferred embodiments, the total dissolved solids may be up to 30°Bx, up to 40°Bx, or up to 50°Bx.

[0050] In a preferred embodiment, the same setup used for diafiltration is also used for ultrafiltration, so the membranes, flux, and other process and equipment parameters are preferably the same, which increases the operational efficiency of the process.

[0051] The native tuber protein isolate can then be dried to obtain a native tuber protein powder. Drying can be carried out by any method known in the art, preferably by spray drying or freeze drying. Optionally, the native tuber protein isolate is subjected to a further concentration step, preferably by reverse osmosis, evaporation or freeze concentration, before drying. How this is achieved is described elsewhere and is generally known.

[0052] It is highly preferred that the tuber protein isolate be adjusted to a pH of 5.5 to 7.0, preferably 6.0 to 7.0, prior to drying, as this improves the stability of the native tuber protein powder, which facilitates storage.

[0053] It is further preferred to adjust the pH of the tuber protein isolate, in particular the concentrated aqueous tuber protein isolate, to above 2.5, preferably above 2.75, in order to stabilize the viscosity of the protein solution and to avoid gelling of the solution during storage before drying. Preferably, such pH values ​​are used for tuber protein isolates containing tuber protease inhibitors.

[0054] Additionally, the pH of the concentrated tuber protein isolate may be adjusted to less than 4.0, preferably less than 3.5, more preferably less than 3.0, again to stabilize the viscosity of the protein solution during storage before drying. Preferably, such pH values ​​are used for tuber protein isolates containing tuber patatin.

[0055] In order to efficiently carry out the present diafiltration method, it is important that a relatively clean diafiltration feed solution containing native tuber proteins as defined herein is used. The clean diafiltration feed solution of the present invention is obtained by following steps a and b.

[0056] In step a of the method, at least one tuber is processed to obtain an aqueous liquid comprising tuber proteins, which may be referred to as tuber process water. This processing may include, for example, pulping, grinding, sanding, crushing, pressing or cutting the tubers, and optionally mixing with water to obtain said tuber process water comprising undenatured tuber proteins.

[0057] The aqueous liquid may comprise starch, and preferably the aqueous liquid is subjected to a step of starch removal, for example as known in the art, such as decanting, cyclone separation or filtration, to obtain tuber process water containing unmodified tuber proteins. In this embodiment, the tuber process water is preferably a by-product from the starch industry, for example potato fruit juice (PFJ) obtained after starch isolation in the potato industry.

[0058] In another embodiment, tubers can be processed by cutting, preferably from potatoes, to form shapes that are the basis for processed tuber products such as chips and fries. When such cutting is carried out in the presence of water, it results in a tuber processing water that contains undenatured tuber proteins.

[0059] In one such embodiment, the tubers may be treated with a jet of water to cut the tubers. In another embodiment, the tubers may be treated with a blade, for example in the presence of water. The water resulting from such a cutting method contains intact tuber proteins, which is a further preferred type of tuber treatment water for the purposes of step a).

[0060] In step b, the tuber processing water is subjected to at least one pretreatment step, such as concentration, dilution, pH adjustment, coagulation, solids removal, and / or heat treatment, resulting in pretreated tuber processing water containing intact proteins. These steps can be performed in any order. Solids removal refers to the removal of a small number of insoluble particles from the solution. These insoluble particles include lipid (aggregates), insoluble proteins, remaining cell wall fragments, starch granules or fragments thereof, microorganisms, and soil particles. Pretreatment is important to ensure that the tuber processing water can be efficiently processed with little or no protein degradation or denaturation, to prevent clogging of filters and membranes, and the formation of films and scales on the surfaces of processing equipment, and to ensure high processing stability and efficiency.

[0061] Concentration of tuber process water can be achieved by any method known in the art for removing excess water. Preferred methods are those that can be operated at relatively low temperatures, for example, 40°C or less, preferably 35°C or less, more preferably 30°C or less, and even more preferably 25°C or less. More preferably, pre-concentration can occur in a high-speed process. Preferred methods for concentrating tuber process water are ultrafiltration, reverse osmosis, and freeze concentration, preferably ultrafiltration. These methods are known in the art.

[0062] In one embodiment, concentration is achieved through freeze concentration, which can be performed as described in WO2017 / 146568 or by other methods known in the art.

[0063] In another embodiment, concentration is achieved through reverse osmosis. Reverse osmosis can be performed using RO membranes, which are known in the art and have no detectable pores. RO membranes separate solutes based on their different solubilities in the membrane material, as is well known in the art. The RO flux can generally be the same as the flow rate in the DF (or UF) flux, e.g., 2 to 50, preferably 5 to 30, and more preferably 10 to 25 l·(h·m) 2 ) -1 is the flux of

[0064] In a further highly preferred embodiment, the pre-concentration is achieved through ultrafiltration. Ultrafiltration has the advantage that it can be operated at high fluxes while at the same time being cost-effective. The operating fluxes are, for example, from 3 to 150 l (h m 2 ) -1 Between 5 and 50 l·(h·m 2 ) -1 Between 7 and 30 l·(h·m 2 ) -1 Between 9 and 20 l·(h·m 2 ) -1 In a preferred embodiment, ultrafiltration is carried out as a continuous (cross-flow) process.

[0065] Preferred membranes for use in the pretreatment of ultrafiltration are polysulfone (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), regenerated cellulose, and polypropylene (PP), preferably PES and PS membranes. Preferred membranes have a molecular weight cut-off (MWCO) of 3 to 500 kDa, preferably 5 to 300 kDa.

[0066] For ultrafiltration of tuber process water having a relatively low amount of suspended solids (e.g. juice having a low amount of cellular debris and / or which has already undergone a solids removal step), the membrane preferably has a MWCO of 3 to 100 kDa, for example 5 to 50 kDa, more preferably 5 to 20 kDa.

[0067] For ultrafiltration of tuber process water with a relatively high amount of suspended solids (e.g., juice with a high amount of cellular debris and which has not yet been subjected to a solids removal step), preferred membranes have a MWCO of 20 to 300 kDa, preferably 50 to 150 kDa.

[0068] Further conditions for the pretreatment ultrafiltration process may be the same as those defined above for diafiltration. In a preferred embodiment, the pretreatment ultrafiltration is carried out using the same setup as the diafiltration step. That is, the optional ultrafiltration step is preferably carried out using a membrane of 5 to 300 kDa, preferably 30 to 200 kDa, more preferably 40 to 120 kDa, and even more preferably 50 to 100 kDa. The phrase "optional ultrafiltration step" is to be interpreted as meaning that, if an ultrafiltration step is present, the ultrafiltration is preferably carried out using the membrane type described above. In other words, if present, the ultrafiltration is carried out using the membrane type described above.

[0069] Dilution of the tuber process water can be achieved by any method known in the art to achieve dilution. Thus, the tuber process water may be diluted with water (tap or demineralized), a buffer, or an acid or base solution. In some embodiments, dilution can be achieved through diafiltration as a pretreatment step, using the methodology and setup described above.

[0070] The pretreatment may include one or more pH adjustments. pH adjustments can be achieved by the addition of suitable acids or bases known in the art. Suitable acids or bases may include, for example, hydrochloric acid, citric acid, acetic acid, formic acid, phosphoric acid, sulfuric acid, and lactic acid, and suitable bases are, for example, sodium or potassium hydroxide, ammonium chloride, sodium or potassium carbonate, calcium and magnesium oxides and hydroxides.

[0071] pH adjustment can serve a variety of purposes. It can be used to change the conductivity of a solution and also to affect the solubility of proteins. In the present invention, pH adjustment should not result in complete protein denaturation, as in, for example, acid coagulation of proteins. However, pH adjustment of tuber processing water may result in partial precipitation of proteins or precipitation of other components of the tuber processing water, which can then be removed by a solids removal step.

[0072] For example, a pH adjustment to 4.0-5.5 can be used to precipitate at least a portion of the patatin fraction, particularly at high concentrations, e.g., 5-20 wt.% protein in the tuber process water, to obtain a tuber process water containing a higher relative amount of native protease inhibitors. The precipitated protein can then be removed during the solids removal step defined elsewhere. This increases the relative amount of native protease inhibitors in the native tuber protein isolate.

[0073] Coagulation can be achieved by adding a suitable flocculant, such as Ca(OH), cationic or anionic polyacrylamide, chitosan, or carrageenan, as known in the art. Methods such as those described in WO 2016 / 036243 can also be used. Following coagulation, a solids removal step is preferably carried out, for example, by decanting, filtration, centrifugation, cyclone separation, or microfiltration.

[0074] Heat treatment can also be applied as a pretreatment, provided that the heat treatment does not result in complete protein coagulation. For example, heat treatment at 40 to 55°C for 1 to 120 minutes can remove most of the patatin, which can then be removed by a solids removal step. It is also known that protease inhibitors from tubers have higher thermal stability than patatin, and that heating can cause partial or complete denaturation of patatin. Therefore, a heating step can be performed in combination with a solids removal step to obtain, for example, tuber treatment water enriched in native protease inhibitors. For example, heat treatment at 60 to 80°C, preferably 70 to 73°C, can be used to precipitate at least a portion of the patatin fraction, followed by a solids removal step to isolate native tuber protein enriched in native protease inhibitors.

[0075] Solids removal may be performed in the present invention in addition to, and preferably subsequent to, another pretreatment step described above, but may also be performed as the only pretreatment step. Solids removal, as defined herein, may be performed at another point in the process. Preferably, however, solids removal is performed during pretreatment. Pretreatment preferably includes a step of solids removal.

[0076] Solids removal in the present invention is preferably a process of filtration, centrifugation, cyclone separation, decanting, nanofiltration or microfiltration, most preferably microfiltration, which can be carried out by methods known in the art.

[0077] Microfiltration (MF) is a highly preferred pretreatment in any of the present embodiments, especially in those in which solids removal is the only pretreatment step. Microfiltration can be performed to achieve separation of particulates from a liquid. Microfiltration can be performed with a variety of membranes, such as polysulfone, polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), and polypropylene (PP), as well as with ceramic membranes, such as zirconium oxide, titanium oxide, or aluminum oxide membranes. MF is preferably performed across membranes having pore sizes of 0.1 to 10 μm, preferably 0.2 to 4 μm, and more preferably 0.3 to 1.5 μm.

[0078] The MF can be operated either at constant pressure or at constant flow. The pressure can vary between 1.5 bar and a maximum of 5 bar. The flux can vary from 0 to 350 l (h m 2 ) -1 Between 45 and 350 l·(h·m 2 ) -1 The microfiltration results in a tuber treatment water having an absorbance of the microfiltered liquid at 620 nm of preferably less than 0.2, more preferably less than 0.1, compared to a demineralized water blank.

[0079] In highly preferred embodiments, pretreatment comprises a step of microfiltration. In other highly preferred embodiments, pretreatment comprises a step of ultrafiltration. In highly preferred embodiments, pretreatment comprises or consists solely of microfiltration. In even more highly preferred embodiments, pretreatment comprises or consists of ultrafiltration followed by microfiltration, or microfiltration followed by ultrafiltration.

[0080] Pretreatment results in relatively clean tuber process water, which becomes the diafiltration feed solution. Pretreatment preferably results in a diafiltration feed solution having an absorbance at 620 nm of less than 0.2, more preferably less than 0.1, compared to a demineralized water blank. Pretreatment further preferably results in a diafiltration feed solution having a conductivity and pH as defined above. The total dissolved solids of the diafiltration feed solution are preferably 2-10°Bx, more preferably 3-8°Bx, for example 4-6°Bx. The total suspended solids are less than 0.05 vol.%, preferably less than 0.25 vol.%, more preferably less than 0.01 vol.%. Most preferably, suspended solids are essentially absent. The total suspended solids are measured at this point by centrifuging a sample and determining the vol.% of the precipitate relative to the supernatant after centrifugation.

[0081] In a further highly preferred embodiment, the method includes a glycoalkaloid removal step to obtain a tuber protein isolate containing up to 200 mg / kg of glycoalkaloids. Glycoalkaloids in this context are glycosylated alkaloids, defined as the sum of solanine and chaconine derivatives. This amount can also be referred to as total glycoalkaloid content (TGA) and can be determined according to the method of Laus et al., Laus MC, Klip G. & Giuseppin MLF (2016) Food Anal. Methods 10(4) "Improved Extraction and Sample Cleanup of Tri-glycoalkaloids α-Solanine and α-Chaconine in Non-Denatured Potato Protein Isolates." Glycoalkaloids are known to be toxic to humans, and for that reason, their presence in tuber protein isolates should be limited.

[0082] Glycoalkaloid removal is known per se and can be achieved by chromatography, acid extraction, enzymatic conversion, fermentation, or adsorption onto activated carbon, hydrophobic resins, or various types of clays. Exemplary techniques are described in WO 2008 / 056977 and WO 2008 / 069651. Preferably, glycoalkaloids are removed by adsorption, for example, by passing the glycoalkaloid-containing process stream through a column containing a suitable adsorbent, such as activated carbon, hydrophobic resins, or various types of clays. This can be done at any point in the process, but is preferably carried out as part of pretreatment step b or after step c.

[0083] In a further preferred embodiment, the method includes an initial step in which at least one tuber is peeled prior to processing. The method thus subjects peeled tubers to processing. This has the advantage that the resulting tuber processing water is much cleaner and therefore requires less pre-treatment prior to the diafiltration method. In addition, peeling the tubers results in a different protein composition, so that the resulting native tuber protein is richer in the amino acids aspartic acid and aspartine, glutamic acid and glutamine, tyrosine, proline, and arginine.

[0084] In a highly preferred embodiment, the first diafiltration or ultrafiltration provides a permeate containing tuber free amino acids. Therefore, the permeate from the first diafiltration or ultrafiltration step is not discarded as waste but is processed separately to obtain tuber free amino acids. Such processing preferably includes a step of drying, preferably by spray drying and / or freeze drying, to obtain tuber free amino acid powder. Drying may be performed after a step of concentration, such as by ultrafiltration, reverse osmosis and / or freeze concentration, as described elsewhere, before drying.

[0085] The process is preferably operated on an industrial scale. As such, the process is preferably operated to yield at least 5 kg of protein per hour, more preferably at least 25 kg of protein per hour, even more preferably at least 50 kg of protein per hour, potentially up to several tonnes per hour. In a preferred embodiment, the process is carried out at a rate of, for example, 10 to 750 m 3 / hr, preferably 50 to 450 m 3 / hr, preferably 80-300m 3 / hr. Preferably, therefore, the process is one in which the entire process is operated continuously (as opposed to batchwise), for example in a "continuous feed and bleed configuration" known to those skilled in the art.

[0086] In embodiments where the tuber processing water has a high protein concentration, such as greater than 1 wt.%, preferably greater than 1.5 wt.%, the method for treating the tuber processing water preferably comprises a microfiltration pretreatment followed by diafiltration as described above. Preferably, the method further comprises a coagulation step either before or after the microfiltration step. In a further preferred embodiment, the tuber processing water is further subjected to an ultrafiltration step prior to the diafiltration step. In this embodiment, the method comprises a first ultrafiltration step followed by diafiltration. In a further embodiment, the retentate of the diafiltration may be concentrated by ultrafiltration before drying. The first and second ultrafiltration steps can be subject to the same general method conditions as described above, but need not be identical method steps.

[0087] An optimized process for the isolation of native tuber proteins from tuber processing water containing a high protein concentration comprises or consists of the steps of microfiltration, flocculation and diafiltration, or the steps of flocculation, microfiltration and diafiltration, or the steps of flocculation, microfiltration, ultrafiltration and diafiltration, or the steps of microfiltration, ultrafiltration and diafiltration. In a highly preferred embodiment, the sequence of steps consists of the steps of microfiltration, ultrafiltration and diafiltration. Alternatively, the sequence of steps comprises or consists of the steps of flocculation, microfiltration, ultrafiltration and diafiltration, or the steps of flocculation, ultrafiltration and diafiltration, each of which optionally follows ultrafiltration.

[0088] All listed steps can be carried out according to the parameters described elsewhere herein. In these embodiments, the coagulation step is preferably followed by a solids removal step, preferably centrifugation. All embodiments are preferably complemented at any point in the process with a glycoalkaloid removal step. Each of these methods may include a drying step, which is optionally preceded by a concentration step, preferably ultrafiltration.

[0089] In embodiments where the tuber processing water has a low protein concentration, such as less than 1.5 wt.%, preferably less than 1 wt.%, the method preferably comprises a pretreatment step comprising ultrafiltration followed by diafiltration as described above. Preferably, this sequence of steps is preceded by a step of microfiltration. More preferably, the retentate of the diafiltration is then ultrafiltered. An optimized method for the isolation of native tuber proteins from tuber processing water containing low protein concentrations consists of the subsequent steps of microfiltration, ultrafiltration, diafiltration, and optionally ultrafiltration and / or drying, as defined herein, complemented at any point in the method by a step of glycoalkaloid removal.

[0090] Optionally, the retentate of the diafiltration or the concentrated solution obtained by ultrafiltration of the retentate of the diafiltration can be subjected to a fractionation step, for example by adsorption or chromatography. These methods are known to separate the native tuber proteins into a protease inhibitor fraction, a patatin fraction or a total protein fraction. Therefore, such methods can be applied to further purify the total tuber protein isolate or to obtain a protease inhibitor isolate or a patatin isolate.

[0091] However, in a preferred embodiment, protein isolation is achieved without a step of protein adsorption. The method preferably does not include a step of protein absorption onto an absorbent, where the protein (defined as a protein for which it has a higher affinity than other components of the treated fluid) is adsorbed. The method preferably does not include a step of protein absorption-elution or protein chromatography, such as expanded bed adsorption (EBA), membrane adsorption, or chromatography.

[0092] Furthermore, the method preferably also does not include a denaturing step. In a highly preferred embodiment, the method is carried out so as to keep the temperature of the tuber processing water below 40°C during pre-treatment, diafiltration, and any other steps before drying. This helps to achieve viscosity stability of the protein solution in time and also avoids protein denaturation. In addition, the method preferably does not include any further denaturing steps, nor does it include any high-shear steps, such as micronization steps.

[0093] Proteins isolated using this method The present method has several advantages over known methods for the isolation of native tuber protein isolates: the protein is cleaner, less denatured and less denatured (more native), and has improved functional properties. In addition, it has no off-tastes or harsh mouthfeel.

[0094] The protein isolated using this method is a clean, native protein with a high protein content, which, as a percentage of dry matter, comprises at least 75 wt.% native tuber protein, preferably at least 80 wt.%, more preferably at least 85 wt.%, and even more preferably at least 90 wt.% native tuber protein.

[0095] The ability of the isolated protein powder to dissolve in demineralized water is a measure of the degree of denaturation; denatured protein powder cannot dissolve in water, whereas native protein powder can. Native tuber protein isolated using the present method (and subsequently dried) can be essentially completely soluble in demineralized water, meaning that at least 55%, preferably at least 65%, more preferably at least 75%, even more preferably at least 85%, or even at least 90% of the isolated protein can be re-dissolved in demineralized water.

[0096] The functional properties, including solubility, are comparable to those of the protein naturally occurring inside the tuber. In addition, the emulsifying properties are not affected.

[0097] In addition, the protein contains a maximum of 1.0 wt.% of the total amount of glucose, fructose and sucrose, a maximum of 1 wt.% of tuber free amino acids, a maximum of 10 mg / kg, preferably a maximum of 5 mg / kg of sulfites, a maximum of 200 mg / kg, preferably a maximum of 100 mg / kg, more preferably a maximum of 50 mg / kg, even more preferably a maximum of 25 mg / kg of glycoalkaloids, a maximum of 5 mg / kg of heavy metals selected from the group consisting of cadmium, mercury, lead and arsenic, and a maximum of 10 wt.%, preferably a maximum of 5 wt.% of chloride salts. Preferably, the ash content is less than 5 wt.%, preferably less than 3 wt.%, more preferably less than 1 wt.%.

[0098] A low sugar content and a low free amino acid content are important because sugars such as glucose, fructose and sucrose are reducing sugars that can react with free amino acids to form pyrazines, which contribute significantly to off-flavors.

[0099] Preferably, the sugar content is less than 1.0 wt.%, preferably less than 0.5 wt.%, more preferably less than 0.1 wt.%, more preferably less than 0.05 wt.%, based on the dry weight of the composition. Even more preferably, the isolate contains at most 1 wt.% tuber free amino acids, more preferably at most 0.5 wt.%, even more preferably at most 0.1 wt.%, even more preferably at most 0.05 wt.% free amino acids based on the dry weight of the composition.

[0100] The method advantageously reduces the amount of both sugars and free amino acids. Diafiltration against a salt solution further provides conditions under which proteins are stabilized and therefore not or hardly altered by the forced mechanical interactions during diafiltration. This minimizes the formation of additional free amino acids.

[0101] The present method also has the effect of minimizing the presence of sulfites in the resulting tuber protein isolate. Sulfites destabilize tuber protein solutions and are conventionally added to tuber processing water during starch processing to prevent oxidation of the tuber processing water and therefore color formation. The present method effectively removes sulfites, thus leading to improved viscosity stability.

[0102] In a preferred embodiment, the method includes the removal of salts from the tuber protein isolate, in particular the removal of salts that originate from the tuber and / or that have been added during the diafiltration step. This can be achieved as described above, resulting in a protein with a low ash content, a low potassium content, and also a low amount of heavy metals.

[0103] For purposes of clarity and precise description, features are described herein as part of the same embodiment or as separate embodiments, but it will be appreciated that the scope of the invention may include embodiments having all or any combination of the described features. The invention will now be illustrated by the following non-limiting examples. [Example]

[0104] Glycoalkaloids (total glycoalkaloids or TGA) were determined essentially according to the method of Laus and coworkers, Laus MC, Klip G. & Giuseppin MLF (2016) Food Anal. Methods 10(4) "Improved Extraction and Sample Cleanup of Tri-glycoalkaloids α-Solanine and α-Chaconine in Non-denatured Potato Protein Isolates."

[0105] Briefly, samples were dissolved or diluted in 5% acetic acid solution containing 20 mM heptanesulfonic acid sodium salt (VWR 152783K) for at least 2 h. Insoluble material was removed by centrifugation at 9000 g at ambient temperature (Heraeus Multifuge 1 SR, rotor 75002006), and the supernatant was directly filtered through a GHP Acrodisc 13 mm Syringe Filter equipped with a 0.45 μm GHP membrane into a 1.5 mL HPLC vial (VWR 548-0004) and stoppered with an aluminum 11 mm rubber / butyl / TEF cap (VWR 548-0010). Samples were automatically loaded onto an SPE column (Oasis HLB prospect-2 / Symbiosis cartridge 2.0 × 10 mm, 30 μm particle size) via a Robotlon online SPE system (Separations). Glycoalkaloids were separated on a Hypersil ODS C18 (250 mm × 4.6 mm, 5 μm) column using 50% acetonitrile / phosphate buffer, pH 7.6. Analytes were detected using a Smartline UV detector 2520 (Knauer) and quantified using a calibration curve prepared from purified glycoalkaloids (α-solanine, Carl Roth 4192,1, and α-chaconine, Carl Roth 2826,1).

[0106] Metals were determined by Inductive-Coupled Plasma Mass Spectrometry (ICP-MS) according to ISO 17294-2:2016.

[0107] The elemental composition was determined via X-ray fluorescence (XRF) for all elements with atomic numbers above that of sodium via a Rigaku CG ED-XRF (Rigaku Co., Ltd.).

[0108] Ash was determined by incinerating the samples at 550°C and weighing the residue.

[0109] Sugar determination was performed with the Megazyme SuFrG kit according to the manufacturer's instructions.

[0110] The amount of total suspended solids (TSS) was measured at 620 nm for juice with a dry matter content of 4.5 wt.%. It can be determined by measuring absorbance.

[0111] The amount of dissolved solids can be determined by measuring it in a PAL Alpha manual digital refractometer (AT 3840, Atago).

[0112] The absorbance at 620 nm is determined by diluting the sample to 5.0°Bx (corresponding to 4.5 wt.% dry matter) and centrifuging at 14,000 rpm for 10 minutes in an Eppendorf centrifuge to remove insoluble material. Samples whose brix value is less than 5 are centrifuged neat. A 1 mL aliquot of the supernatant of each sample is introduced into a cuvette and placed in a BioRad SmartSpec Plus spectrophotometer. The absorbance is read in duplicate at 620 nm against a blank of demineralized water.

[0113] Conductivity can be determined at room temperature with a HI 98312 conductivity meter (Hanna Nindustries, The Netherlands) as is known in the art. Conductivity can also be calculated and determined based on various concentrations of solute, if appropriate, as is known in the art.

[0114] Protein concentration can be determined by the Kjeldahl method. The nitrogen number is then converted to protein content by multiplying by 6.25.

[0115] True protein content was determined using a CEM Sprint rapid protein analyzer. The method is based on the interaction of a negatively charged dye (iTAG) with the positively charged amino acids lysine, arginine, and histidine present in proteins under acidic conditions. The hydrophobic nature of the dye causes proteins to precipitate, and the loss of dye adsorption at 480 nm is translated into protein content using a calibration curve prepared from a Kjeldahl analysis performed on thoroughly washed protein preparations. Nitrogenous compounds such as single amino acids and small peptides do not precipitate in the dye solution and consequently do not affect the protein content measurement.

[0116] Moisture content is reported as loss on drying and is determined by placing an aluminum sample pan (VWR 611-9000) containing between 1 and 10 g of sample in a HG83 Halogen Oven (Mettler Toledo) set at 100°C for 10 minutes.

[0117] In this experiment, the following salt solutions were frequently used during diafiltration: 0.5 wt.% NaCl (conductivity 8.2 mS / cm), 0.5 wt.% KCl (conductivity 8.2 mS / m), 0.33 wt.% NaCl (conductivity 5.3 mS / cm).

[0118] Protein composition by Experion Protein composition was determined using an Experion Pro260 automated electrophoresis station (Bio-Rad, USA). First, the reagents were equilibrated to room temperature and briefly stirred, then spun down in a centrifuge at 10,000 × g for 5 minutes. After this, gel and gel staining solution were prepared. The former did not require mixing of the reagents, while the latter was made by mixing 20 μL of staining reagent with 520 μL of gel reagent. Both solutions were then stirred and spun down at 10,000 × g for 5 minutes in an Eppendorf cup equipped with a 0.2 μm filter. Sample buffer was then prepared by mixing 30 μL of sample buffer with 1 μL of β-mercaptoethanol. Sample and ladder were then prepared by mixing 4 μL of sample / ladder with 2 μL of sample buffer, followed by stirring and centrifugation at 10,000 × g for 5 minutes. The sample and ladder were subsequently diluted with 84 μL of ultrapure water. Prime the Experion tip with 12 μL of gel staining solution by running program B3 in the priming station. Then, pipette 12 μL of gel staining solution and gel solution into the chip wells. Pipette 6 μL of ladder into the ladder wells in the Experion tip. Then, load 6 μL of each sample into sample wells 1-10. Finally, load the Experion tip into the Experion Pro260 and run the Protein 260 assay to begin the analysis. After the analysis, wash the electrodes using a cleaning tip.

[0119] Example 1 The effect of salt concentration on the tendency of proteins to aggregate and precipitate was assessed using model solutions at various pHs and conductivities, including purified protease inhibitors (obtained by chromatography and extensive dialyzed) and purified patatin.

[0120] Proteins were dissolved in 30 mM potassium citrate buffer at a total protein concentration of 1 wt.% with a 1:1 mass ratio of purified protease inhibitor to purified patatin. The pH was adjusted as needed with HCl (1 M) or NaOH (1 M). The conductivity was adjusted to 2, 12, or 50 mS cm through the addition of potassium chloride. -1 The temperature was set at 0°C. The samples were incubated at ambient temperature for 1 hour and centrifuged at 14,000 rpm in an Eppendorf centrifuge for 10 minutes to remove precipitated proteins. The supernatant was diluted 1 in 25 in 100 mM NaOH solution. Protein concentrations were read at 280 nm in a BioRad Smartspec Plus spectrophotometer against a 100 mM NaOH blank and expressed as the percentage of soluble protein. The results show a strong pH dependence of potato protein solubility as a function of pH at low conductivities, while at higher conductivities the pH effect is reduced or eliminated (see Figure 1).

[0121] Example 2 The effect of salt concentration on the tendency of proteins to aggregate and precipitate under mechanical stress was evaluated at various pHs and conductivities using model solutions containing purified protease inhibitors (obtained by chromatography and extensive dialyzed) and purified patatin in a 1:1 molar ratio.

[0122] Proteins were dissolved in 10 mM citrate buffer at 2 wt.% and 6 wt.% total protein concentrations. The buffer was diluted with NaOH or HCl solution to set the pH to 6.0 or 7.0. Solid KCl was added to the resulting solution to adjust the conductivity to 2, 5, 12, and 50 mS cm. -1 The solution was centrifuged at 10,000 x g for 5 minutes to remove insoluble protein and then set to a final protein concentration of 1.5 wt.% and 4 wt.% (calibrated to the PI fraction). Protein solubility was determined separately for each fraction and then averaged to reflect the natural variability in potato juice.

[0123] The final protein solution was incubated at room temperature for 1 hour under vigorous stirring with a mechanical stirrer to mimic the mechanical force occurring during diafiltration. The protein mixture was then centrifuged at 10,000 × g for 5 minutes, and the protein concentration in the supernatant was determined. Protein loss due to aggregation was calculated by the difference between the starting protein concentration and the final protein concentration. The results are shown in Figure 2.

[0124] The results show that total potato proteins are highly soluble at elevated conductivities, even under mechanical stress. Conductivities of at least 5 mS cm -1 , preferably at least 8 mS cm -1 To avoid the addition of excess salt during diafiltration, the conductivity should preferably be 20 mS cm -1 Less than 18 mS·cm -1 The preferred conductivity is 8 to 15 mS cm -1 , preferably 9 to 14 mS cm -1 is.

[0125] Example 3 Potato process water, obtained from potato tubers after removal of starch and fiber (potato process water), was used as a feedstock for the production of unmodified total protein isolates on a pilot scale. The potato process water was subjected to a pretreatment of solids removal ((a) and (b)) or to a pretreatment of coagulation followed by solids removal (c). In all cases, the treatment was carried out at a rate of 100 to 250 l h -1 This was generated in the flow between (a) Potato process water was centrifuged in a conventional continuous stack disc centrifuge. Further removal of particles was achieved using dead end filtration with diatomaceous earth as a filter aid. The centrifuged potato process water was stored in a tank awaiting further processing. (b) Potato treatment water is passed through a ceramic membrane with a pore size of 0.8 micrometers and at a temperature of approximately 23 ± 2°C and a flow rate of 100 l (h m 2 ) -1The microfiltered potato processing water was stored in a tank awaiting further processing. (c) Potato process water was pretreated with a mixture of cationic and anionic flocculants as described in WO2016 / 036243 A1. Solids were separated from the potato process water in a disc stack centrifuge. Sludge was discarded at the bottom of the centrifuge, while the centrifuge supernatant was used as feed for diafiltration. Samples of the flocculated potato process water were taken according to the modified sludge volume index (SVI), as an indication of the quality of flocculation. The supernatant was also tested for absorbance at 620 nm. The flocculated potato process water was stored in a tank awaiting further processing.

[0126] The characteristics of these pretreatment steps are shown in Table 1.

[0127] [Table 1]

[0128] The thus pretreated potato juice was optionally subjected to TGA removal. TGA removal was carried out in a column packed with granular activated carbon (C-GRAN, Norit). The activated carbon column was pre-soaked in demineralized water for 24 hours before TGA removal was carried out. The pretreated potato juice was passed through the column using a contact time of 2 hours.

[0129] In all cases, pretreatment further consisted of a step of ultrafiltration preceded by diafiltration. The potato process water was ultrafiltered using a spiral wound membrane with a molecular weight cut-off (MWCO) of 5 kDa to obtain a concentrated potato protein solution with a dissolved solids content between 9.9 and 21.8°Bx.

[0130] [Table 2]

[0131] The retentate from ultrafiltration was subjected to further processing, including diafiltration against a salt solution with 0.33 wt.% or 0.66 wt.% NaCl in different ratios of feed to salt solution (1:3 to 1:4). This resulted in a potato protein isolate solution. The pH remained at 6.3±0.3 during the ultrafiltration and diafiltration steps. The final concentrate was then subjected to spray drying (SD, T in 175℃, T out The samples were dried by heating at 75°C or by freeze-drying. The conditions are outlined in Table 2.

[0132] Potato protein isolates obtained from ultrafiltration and diafiltration against salt solutions were characterized for conductivity and concentration of dissolved solids (°Bx). The dried product was analyzed for protein content (Kjeldhal), moisture content, protease inhibitor and TGA content, and relative amounts of sulfites and heavy metals.

[0133] Table 2 shows the effect of different process parameters on the quality of the protein isolate. All the different pretreatments can be successfully applied before diafiltration, but do not have a significant effect on the final quality of either the final concentrated aqueous protein isolate or the dried product, provided that the diafiltration is performed on a salt solution. A TGA removal step is necessary to obtain potato protein with the required low TGA level.

[0134] Run 5 shows that when the potato process water is diafiltered at a higher dilution factor (1:4), the conductivity of the protein isolate is lower than in the run where a dilution factor of 1:3 was applied. After drying, the product from Run 5 also has the highest protein content.

[0135] When the salt concentration was increased from 0.33% to 0.66%, as was done in Experiment 6, the conductivity of the protein isolate was higher. However, the TGA content of the dried protein isolate from Experiment 6 was lower than that obtained in the other experiments. The table also shows that the protein isolate could be dried by various methods, such as spray drying (Experiments 1-6) or freeze drying (Experiment 7). In all cases, products with high protein contents ranging from 79% up to 85.5% were obtained.

[0136] Example 4 Potato tuber process water (potato process water), obtained from potato tubers after removal of starch and fiber, was used as a raw material for the production of whole unmodified potato protein isolate at a pilot scale. The potato process water was filtered using a ceramic membrane with a pore size of 0.8 micrometers and at a temperature of approximately 23 ± 2 °C and 100 L (h m 2 ) -1 The microfiltered potato process water (MF-PFJ) was stored in a tank awaiting further processing.

[0137] In all cases, the microfiltered PFJ (4.2-5.0°Bx and 12 mS / cm) was subjected to ultrafiltration using polyethersulfone membranes with a molecular weight cut-off (MWCO) of 5 kDa or 50 kDa, resulting in a retentate with a dissolved solids content between 10.5 and 28.2°Bx and a conductivity ranging between 10 and 16 mS / cm.

[0138] The retentate of the ultrafiltered PFJ was subsequently subjected to TGA removal. Optionally, the retentate was diluted with soft water before TGA removal to obtain a protein solution having a conductivity between 10.5 and 12.9°Bx and a dissolved solids content between 6 and 13 mS / cm. TGA removal was performed at room temperature and a pH of 6 to 6.5 using four columns packed with hydrophobic resin. After TGA removal, a protein solution with a conductivity between 8 and 13 mS / cm was obtained, which was optionally concentrated to a solids content ranging between 12 and 18°Bx.

[0139] The retentate from the ultrafiltration was diluted with the feed (V PJ ) vs. salt solution (V DF The final concentrate was subjected to further processing, including multiple diafiltration steps at different ratios of 0.01 to 0.1 (1:1 to 1:2). This resulted in a potato protein isolate solution. The final concentrate was then subjected to spray drying (SD, T in 175℃, T out Each diafiltration step was performed using a protein solution volume V PJ Diafiltration volume V DF This requires dilution with HCl at a ratio within the ranges indicated, and concentration of the diluted protein solution back to the original volume by ultrafiltration. An overview of the conditions is shown in Table 3.

[0140] Potato protein isolates obtained from ultrafiltration and diafiltration against salt solutions were characterized for dissolved solids concentration (°Bx). The final products were analyzed for protein content, moisture content, protease inhibitor and TGA content, and relative amounts of sulfites and heavy metals.

[0141] Table 3 shows the effect of various process parameters on the quality of the protein isolate. All the various pretreatments can be successfully applied before diafiltration. Furthermore, various diafiltration conditions were tested, showing that good quality protein isolates can be obtained using various salt solutions.

[0142] Experiments 8 and 9 show that diafiltration at a salt concentration of 0.5% NaCl and at protein solution to diafiltrate ratios of 1:1 or 1:2 and 1:1, respectively, can be used to obtain potato protein isolates of good quality (84-86%). Figure 6 shows that a total protein isolate is obtained containing all protein fractions also present in the starting material.

[0143] In experiment 10, the microfiltered and ultrafiltered PFJ was subjected to five separate diafiltration steps (two before TGA removal and three after TGA removal) using 0.5% KCl. These conditions result in protein isolates of similar quality compared to when NaCl is used as the salt during diafiltration. See also Figure 6.

[0144] Furthermore, Experiment 11 demonstrates that the quality of native protein isolates can be significantly improved when UF and DF membranes with a MWCO of 50 kDa instead of 5 kDa are used. Such conditions result in protein isolates with a true protein content of 94% compared to approximately 84% when using membranes with a MWCO of 5 kDa (see Experiments 8 and 9). Figure 6 shows that a total protein isolate is obtained containing all protein fractions also present in the starting material (MF-PFJ).

[0145] [Table 3]

[0146] Example 5 Potato tuber process water (potato process water), obtained from potato tubers after removal of starch and fiber, was used as a raw material for the production of unmodified potato protein protease inhibitor isolates on a pilot scale. The potato process water was filtered using a ceramic membrane with a pore size of 0.8 micrometers and at a temperature of approximately 23 ± 2 °C and 100 L (h m 2 ) -1 The microfiltered potato process water (MF-PFJ) was stored in a tank awaiting further processing.

[0147] The microfiltered PFJ was subjected to ultrafiltration using a polyethersulfone membrane with a molecular weight cut-off (MWCO) of 5 kDa to obtain a potato protein solution with a dissolved solids content of 19.2 °Bx.

[0148] The pH of the ultrafiltered PFJ was then set to 3.0 by dropwise addition of 1 M HCl solution while stirring, forming a slurry due to precipitation of the patatin fraction of PFJ. The slurry was centrifuged at 4200 RPM for 10 minutes and decanted. The supernatant was subjected to TGA removal at a pH of approximately 3.5 at room temperature using four columns packed with hydrophobic resin, followed by concentration. The concentrate was diafiltered against a 6 kDa membrane three times using a pH 3.5 citrate buffer, followed by three times using a 0.5% KCl solution (a 5:3 ratio of concentrate to diafiltrate) to obtain a protein solution with a pH of 5.3°Bx and 90% protease inhibitor content (see also Figure 6).

[0149] The results are summarized in Table 4. It shows that pH adjustment before diafiltration confers good quality to the protein isolate, reflected in a true protein content of 81.4%. It further demonstrates that this method makes it possible to obtain a native potato protein isolate enriched in the potato protease inhibitor fraction.

[0150] [Table 4]

[0151] Example 6 The microfiltered PFJ (300 L) was subjected to ultrafiltration using a 5 kDa membrane to obtain a concentrated protein solution (35 L, 22.7°Bx). The concentrated protein solution was subjected to diafiltration by adding 50 L of water (concentrate to diafiltrate ratio 7:10). The conductivity decreased from 12 mS / cm (before adding water) to 6.2 mS / cm (after adding water). The diafiltered solution was concentrated again (35 L) and subjected to another 50 L of water, resulting in a further decrease in conductivity to 3.5 mS / cm. Upon addition of water, a white precipitate formed and the solution turned from clear and orange to white and milky, indicating protein precipitation. NaCl (250 g) was added, again resulting in a clear orange solution and an increase in conductivity to 8 mS / cm.

[0152] The flux concentration plot is shown in Figure 3. From this plot, it can be seen that the flux decreased by 50% upon addition of the second volume of water. This decrease in flux indicates clogging of the membrane. The flux recovered upon addition of NaCl, indicating that the protein had resolubilized.

[0153] These results clearly demonstrate that it is essential to maintain the conductivity of the protein solution above at least 5 mS / cm, preferably above at least 8 mS / cm, to avoid protein precipitation, which leads to both protein loss and membrane clogging and fouling. This can be achieved by monitoring the conductivity of the pretreated tuber process water and / or by monitoring the conductivity of the salt solution.

[0154] Example 7 Tuber process water (sweet potato and cassava process water) obtained from sweet potato and peeled cassava tubers after starch and fiber removal was used as the raw material for the production of intact total protein isolate. The process water was subjected to a pre-treatment step of ultrafiltration solids removal using an Amicon M-2000 ultrafiltration cell equipped with a 10 kDa MWCO membrane.

[0155] The ultrafiltered sweet potato juice was then diafiltered against a 10 kDa MWCO membrane using 0.5% NaCl solution (concentrate to diafiltrate ratio 1:5). The concentrate was subjected to two further diafiltration steps using 0.5% NaCl solution (concentrate to diafiltrate ratio 1:2 and 1:1) to obtain a protein solution.

[0156] The peeled, ultrafiltered cassava juice was subjected to diafiltration using 0.5% NaCl solution against a 10 kDa MWCO membrane (recentrate to diafiltrate ratio 1:1.5) to obtain a protein solution.

[0157] The chemical composition of the treated tuber juice is shown in the table and compared with that of the untreated juice.

[0158] [Table 5]

[0159] The results show that the method successfully yields native tuber juice with a high content of native protein. In both cases, a significant increase in the true protein content was observed, while the presence of contaminants such as sugars and free amino acids was substantially reduced.

[0160] Example 8 Total potato protein containing protease inhibitors and patatin was used to test the solubility of proteins at natural pH (6-6.3) and at various conductivities. Potato proteins were obtained by microfiltration of potato juice, ultrafiltration to 10% of the original volume, and diafiltration using the protocol of Example 4, Experiment 8, without spray drying and with pure water, until the retentate had a conductivity of 8 mS / cm.

[0161] The resulting aqueous potato protein was diluted in aqueous solutions of NaCl at conductivities of 3.7, 5.3, 6.7, 9.2 and 14 mS / cm to obtain solutions with a protein concentration of 1.3 wt.% and with purified water to produce potato protein solutions at a conductivity of 1.3 mS / cm and a protein concentration of 1.3 wt.%.

[0162] The solution was then left stirring for 1 hour and then centrifuged. The protein concentration in the supernatant was determined by Sprint and compared with the original protein concentration. The results are shown in Figure 4.

[0163] This experiment shows that protein precipitation in aqueous mixtures containing protease inhibitors and patatin is unacceptable if the conductivity falls below 5 mS / cm. Conductivity during diafiltration must always be maintained above 5 mS / cm, more preferably above 8 mS / cm.

[0164] Example 9 The protein isolate described in Example 8 was subjected to diafiltration using solutions against NaCl solutions having conductivities of 1.34, 3.65, 4.72, 5.25, 6.72, 8.72, 9.2, 12, 14 and 49.5 mS / cm.

[0165] The results are shown in Figure 5. Diafiltration against salt solutions with a conductivity below 5 mS / cm results in unacceptable precipitation. Increasing the conductivity to above 5 mS / cm, preferably above 8 mS / cm, allows for convenient protein isolation, resulting in an improved protein product.

Claims

1. 1. A native tuber protein isolate for use in human food applications, comprising: As a percentage of dry matter, it contains at least 75 wt.% undenatured tuber protein, a maximum of 1 wt.% total of glucose, fructose and sucrose, a maximum of 1 wt.% tuber free amino acids, a maximum of 10 mg / kg sulfites, a maximum of 200 mg / kg glycoalkaloids, a maximum of 5 mg / kg heavy metals selected from the group consisting of cadmium, mercury, lead and arsenic, and a maximum of 5% metal salts; - the native tuber protein isolate is a native tuber protein isolate containing native protease inhibitors and native patatin, - the native tuber protein isolate is a) processing at least one tuber to obtain a tuber processing water containing native tuber protein; b) subjecting the tuber treatment water to the following: ba) concentrated, and / or bb) dilution, and / or bc) pH adjustment, and / or bd) condensation, and / or be) solids removal, and / or bf) Heat treatment a step of subjecting the sample to a pretreatment comprising one or more of the steps the pretreatment results in a pretreated tuber treatment water containing undenatured tuber proteins and having a conductivity of 2 to 20 mS cm -1 ; c) diafiltration of the pretreated tuber treatment water using a membrane with a molecular weight cut-off of 3 to 500 kDa against a salt solution having a conductivity of at least 5 mS·cm −1 , wherein the native tuber proteins are retained in the retentate of the diafiltration; and - Undenatured tuber protein isolate, wherein the tuber is potato (Solanum tuberosum).

2. 10. The native tuber protein isolate of claim 1, containing up to 5 mg / kg of sulfites.

3. 3. The native tuber protein isolate of claim 1 or 2, containing up to 100 mg / kg of glycoalkaloids.

4. 4. A native tuber protein isolate according to any one of claims 1 to 3, containing up to 50 mg / kg of glycoalkaloids.

5. 5. The native tuber protein isolate according to any one of claims 1 to 4, containing up to 25 mg / kg of glycoalkaloids.

6. 6. The native tuber protein isolate according to any one of claims 1 to 5, wherein the pH is between 5.5 and 7.

0.

7. 1. A method for producing a native tuber protein isolate for use in human food applications, comprising: a) processing at least one tuber to obtain a tuber processing water containing native tuber protein; b) subjecting the tuber treatment water to the following: ba) concentrated, and / or bb) dilution, and / or bc) pH adjustment, and / or bd) condensation, and / or be) solids removal, and / or bf) Heat treatment a step of subjecting the sample to a pretreatment comprising one or more of the steps The pretreatment results in a soluble fiber containing undenatured tuber proteins with a concentration of 2 to 20 mS cm -1 to provide a pretreated tuber treatment water having a conductivity of c) adding the pretreated tuber treatment water to at least 5 mS cm -1 diafiltration using a membrane with a molecular weight cut-off of 3 to 500 kDa against a salt solution having a conductivity of 0.05 to 0.15 kDa, wherein the native tuber proteins are retained in the retentate of the diafiltration; Including, 1. A method according to claim 1, wherein the native tuber protein isolate comprises, as a percentage of dry matter, at least 75 wt.% native tuber protein, up to 1 wt.% total amount of glucose, fructose and sucrose, up to 1 wt.% tuber free amino acids, up to 10 mg / kg sulfites, up to 200 mg / kg glycoalkaloids, up to 5 mg / kg heavy metals selected from the group consisting of cadmium, mercury, lead and arsenic, and up to 5% metal salts.

8. 8. The method of claim 7, wherein the native tuber protein isolate is a native tuber protein isolate containing native protease inhibitors and native patatin.

9. 9. The method of claim 7 or 8, wherein the native tuber protein isolate is a native total tuber protein isolate, defined as an isolate containing all tuber proteins in their native form.

10. 10. The method according to any one of claims 7 to 9, further comprising the step of removing glycoalkaloids to obtain a tuber protein isolate containing up to 200 mg / kg of glycoalkaloids.

11. 11. The method of claim 10, wherein the step of removing glycoalkaloids is carried out as part of step b or after step c.

12. 12. The method according to any one of claims 7 to 11, wherein the processing to obtain tuber processing water comprises pulping, grinding, sanding, crushing, pressing or cutting of the tubers, with or without mixing with water.

13. 13. The method according to claim 12, wherein the processing to obtain tuber processing water further comprises a step of removing starch.

14. 14. The method of claim 13, wherein the starch removal step comprises decanting, centrifuging, cycloning, or filtration.

15. 15. The method of any one of claims 7 to 14, wherein the at least one tuber is peeled before processing.

16. 16. The method of any one of claims 7 to 15, wherein the solids removal comprises the steps of filtration, centrifugation, cyclone separation, decanting and / or microfiltration.

17. 17. The method according to any one of claims 7 to 16, wherein the pretreatment comprises a step of concentration selected from ultrafiltration, reverse osmosis and / or freeze concentration.

18. 18. The method of any one of claims 7 to 17, wherein the diafiltration step is preceded by an ultrafiltration step.

19. 18. The method of claim 17, wherein the pretreatment comprises, in any order, the steps of concentration by ultrafiltration and solids removal by microfiltration.

20. 20. The method of any one of claims 7 to 19, wherein diafiltration is carried out using a membrane with a molecular weight cut-off of 5 to 300 kDa.

21. 21. The method of any one of claims 7 to 20, wherein the diafiltration is carried out on a salt solution containing chloride.

22. The salt solution is NaCl, KCl or CaCl 2 22. The method of claim 21, comprising:

23. The conductivity of the salt solution is 5 to 20 mS cm -1 22. The method of any one of claims 7 to 21, wherein

24. 24. The method of any one of claims 7 to 23, wherein the pH during diafiltration is less than 4.0 or greater than 5.

5.

25. 25. The method according to any one of claims 7 to 24, wherein the retentate of the diafiltration is subjected to a step of ultrafiltration to obtain a concentrated tuber protein isolate.

26. 20. The method according to any one of claims 17 to 19, wherein the ultrafiltration step is carried out using a membrane with a molecular weight cut-off of 5 to 300 kDa.

27. 27. The method according to any one of claims 7 to 26, wherein the first diafiltration or ultrafiltration results in a permeate containing tuber free amino acids.

28. 28. The method according to any one of claims 7 to 27, wherein the pH of the tuber protein isolate is adjusted to above 2.

5.

29. 29. The method according to any one of claims 7 to 28, wherein the pH of the tuber protein isolate is adjusted to below 3.

5.

30. 30. A method according to any one of claims 7 to 29, wherein the tuber protein isolate is subsequently dried to obtain a native tuber protein powder.

31. 31. The method of claim 30, wherein the drying is carried out by freeze-drying or spray-drying.

32. 32. A method according to claim 30 or 31, wherein prior to said drying, the tuber protein isolate is subjected to a further step of concentration via reverse osmosis, evaporation or freeze concentration.

33. 33. The method of any one of claims 7 to 32, operated to yield at least 5 kg of protein per hour.

34. 34. The method of any one of claims 7 to 33, operated to yield at least 5 kg of protein per hour.

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