Inactive wheat protein and its production method

Abstract

The present invention relates to an inactive wheat protein, preferably wheat albumin, characterized by its high emulsifying activity, high foaming power and its low bitterness. The present invention also relates to a process allowing the industrial production of this inactive wheat protein, preferably wheat albumin. Finally, the present invention relates to industrial uses, including food, feed, cosmetic and pharmaceutical applications.

Description

Technical Field

[0001] The present invention relates to an inactive wheat protein, preferably wheat albumin, characterized by its high emulsifying activity, high foaming power and its low bitterness. The present invention also relates to a process allowing the industrial production of this inactive wheat protein, preferably wheat albumin. Finally, the present invention relates to industrial uses, including food, feed, cosmetic and pharmaceutical applications. Background Art

[0002] As is known, wheat is a seed that allows the industrial production of starch, lipids and proteins for the food, feed, cosmetic and pharmaceutical industries. The main components sought are wheat starch and wheat gluten. The two most commonly used processes in the production of starch and gluten from wheat are the so-called Halle or fermentation process and the Martin's process.

[0003] In the fermentation process, the mashed grains are subjected to a relatively long period of natural fermentation, thereby changing the properties of the wheat gluten so that they no longer seriously interfere with the washing out of the starch. The fermented liquid is then stirred with water in a finely perforated container, thereby producing a suspension of starch in water, which escapes through the perforated walls of the container. The decay processes that occur during the fermentation make this method extremely annoying to operate. In addition, this process does not allow the recovery of the wheat gluten in commercial form.

[0004] In the Martin process, wheat flour is made into a dough with water and stirred and kneaded by grooved rollers that repeatedly roll and turn the dough over a fixed bed with sieve surfaces on both sides, while the starch is washed out by water jets. The operation of this process is extremely cumbersome and has the additional disadvantage that a relatively large proportion of inferior starch is produced that is strongly contaminated with finely dispersed gluten, which cannot be separated by conventional purification methods.

[0005] In all wheat starch extraction processes, proteins are separated into two main streams: wheat gluten and wheat albumin. Wheat gluten is separated from the beginning because it is insoluble. Wheat albumin is concentrated in the soluble fraction. All processes produce a liquid fraction called wheat solubles. Such a typical by-product of a wheat milling refinery usually contains 30% residual starch, 4%-7% ash, and 15%-35% soluble proteins. These proteins are mainly wheat albumin.

[0006] Wheat solubles are mainly used in the feed and fermentation industries. In the food sector, some companies have successfully purified wheat albumin in order to sell it on the human food market. For example, Nisshin Pharma disclosed a method for obtaining a purified fraction of wheat albumin in JP2009000017686. The method includes aqueous extraction of whole wheat flour, boiling and ultrafiltration to precipitate contaminating compounds and other proteins with low temperature stability. The purified fraction can be eaten directly or in soup (see "Shelf Life of Drink and Powdered Soup Containing Wheat Albumin" by Miyazaki et al.). Hassan also studied the valorization of wheat albumin in 2010. In his work, it was concluded that wheat albumin attracted attention due to its functional properties. Unfortunately, the foaming power (also known as whipping properties) can only reach 1.68 ml foam / ml solution (or, if expressed as a percentage, a foam volume of 168%), and this value is achieved at a very alkaline pH of about 9-10, which can be detrimental to the protein (see Table 10 of Hassan 2010). As the industry seeks more foaming proteins, the functional properties of wheat albumin need to be improved to make it exhibit more foam.

[0007] Another major disadvantage of wheat albumin is that it has a very strong bitter effect when consumed. Due to this extreme bitterness, the food industry is not an application area. When food companies add such wheat solubles containing albumin, they have to add compounds in their formulations that are intended to cover / mask the bitter taste. US9259454 from Kao Corporation teaches, for example, a solid composition comprising wheat albumin and maltitol, sorbitol, lactitol, or any combination thereof, intended to provide a food formulation based on wheat albumin without a bitter off-flavor.

[0008] Finally, wheat solubles also have very low emulsifying activity, which avoids some special uses in the food industry, like calf milk replacer or ice cream industry. If a person skilled in the art can remove the bitter taste by adding polyols, he cannot obtain a highly emulsifying albumin-enriched fraction.

[0009] In summary, there is still a need for a purified and enriched fraction of wheat albumin with low bitter off-taste without the addition of extraneous compounds and with high emulsifying power and high foaming power (also known as whipping properties). Summary of the invention

[0010] A first object of the present invention is an inactive wheat protein, preferably wheat albumin, more preferably native wheat albumin, characterized by an emulsifying activity higher than 500 g oil / g protein, preferably higher than 600 g oil / g protein, and more preferably higher than 1000 g oil / g protein (see Test A below).

[0011] In preferred embodiments, the proteins of the invention have a low bitter off-taste when consumed by a human panel. Such bitterness can also be assessed by methods known in the art including an electronic tongue.

[0012] In another preferred embodiment, the protein of the present invention also has a whipping property (also called foaming power) higher than 250%, preferably higher than 300%.

[0013] In a more preferred embodiment, the protein of the present invention in the above embodiments has a protein enrichment expressed as N×6.25 based on dry matter of higher than 80%, preferably higher than 90%.

[0014] A second object of the present invention is a method allowing the production of the above protein of the invention, comprising the following steps:

[0015] 1. Starting from wheat seeds, extract the outer fiber, wheat germ, starch and wheat gluten and obtain a fraction called wheat solubles,

[0016] 2. Microfiltration of wheat solubles to obtain microfiltration permeate,

[0017] 3. applying cation chromatography to the microfiltration permeate so as to produce a fraction enriched in wheat albumin,

[0018] 4. Ultrafiltration is performed on the above wheat albumin-enriched fractions together or separately to obtain more albumin-enriched retentate;

[0019] 5. Concentrating and / or drying the albumin-enriched retentate described above.

[0020] In a preferred embodiment, the method of the present invention is characterized in that the cation chromatography of step 3 comprises the following steps:

[0021] a. Feed the microfiltration permeate onto a cationic resin to bind the protein

[0022] b. Feeding different eluents onto the resin to release and separate the proteins bound to the resin

[0023] In a more preferred embodiment, the method of the present invention is characterized in that the different eluents of step a are continuous:

[0024] a) 20 mM malic acid and 3 mM sodium carbonate solution at pH 6.2

[0025] b) 20 mM malic acid, 3 mM sodium carbonate and 0.5 M NaCl solution at pH 6.2

[0026] c) 30 mM sodium carbonate and 0.45 M NaCl solution at pH 12

[0027] In a preferred embodiment, the concentration in step 5 is performed using reverse osmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The wheat soluble matter purification process of the present invention is shown

[0029] Figure 2 Represents a chromatogram showing PN1, PN2 and PN3 peaks DETAILED DESCRIPTION

[0030] In the present invention, "inactive wheat protein" is understood to be a protein obtained from wheat seeds that does not include wheat gluten. Wheat gluten, which is composed of gliadin and glutenin, can be easily characterized by its viscoelastic properties. The "active" property can be better understood by reading "Codex Standard for Wheat Protein Products Including Wheat Gluten [International Food Codex Standard for Wheat Protein Products Including Wheat Gluten] (Codex Stan 163-1987, Revised Edition 1-2001)", which summarizes for us that "active wheat gluten is characterized by its high viscoelastic properties when hydrated". The characteristic of deactivated wheat gluten is that its viscoelastic properties when hydrated are reduced due to denaturation. For "inactive wheat protein", those skilled in the art will understand all wheat proteins including albumin but not including all wheat gluten.

[0031] According to the invention, "wheat" is understood to be a grass (cereal, which is a staple food worldwide) widely cultivated for its seeds. Many species of wheat together constitute the genus Triticum; the most widely cultivated is common wheat (T. aestivum).

[0032] According to the present invention, "protein" is understood to be a large biological molecule or macromolecule composed of one or more long chains of amino acid residues. Such proteins can be natural or modified, including hydrolysis or chemical modification.

[0033] In the present invention, "emulsion" is understood to be a mixture of two or more liquids that are generally immiscible (cannot be mixed or blended). Emulsions are part of a more general class of two-phase material systems called colloids. Although the terms colloid and emulsion are sometimes used interchangeably, when both phases (dispersed and continuous) are liquids, emulsions should be used. In an emulsion, one liquid (the dispersed phase) is dispersed in another liquid (the continuous phase). Examples of emulsions include salad dressings, homogenized milk, mayonnaise, and some cutting fluids used for metalworking.

[0034] In the present invention, "emulsifying activity" is understood to be the maximum amount of oil that can be emulsified by one gram of protein with water.

[0035] In the present invention, "albumin" is understood to be a family of globular proteins. All proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and undergo thermal denaturation.

[0036] In the present invention, "bitter" is understood to be the most sensitive taste and is considered by many to be very unpleasant or unpleasant, but is sometimes desired and intentionally added with the aid of various bittering agents. Common bitter foods and beverages include coffee, unsweetened cocoa, yerba mate, bitter melon, olives, citrus peels, many plants in the cruciferous family, dandelion greens, wild chicory, and broadleaf chicory. The ethanol in alcoholic beverages tastes bitter,

[30] as do other bitter ingredients found in some alcoholic beverages, including hops in beer and citrus in bitters. Quinine is also known for its bitter taste and is found in tonic water.

[0037] As mentioned above, a first object of the present invention is an inactive wheat protein, preferably wheat albumin, more preferably native wheat albumin, characterized by an emulsifying activity higher than 500 g oil / g protein, preferably higher than 600 g oil / g protein, more preferably higher than 1000 g oil / g protein (see Test A below).

[0038] As exemplified below, wheat solubles produced by the wheat refining industry are concentrated in wheat albumin. Native wheat albumin means albumin in its native state that has not been hydrolyzed. Albumin is well known for its properties including foaming, transparency and emulsifying ability. Unfortunately, wheat solubles (even if albumin is concentrated) have low emulsifying power or activity. Industrial applications including food, feed, cosmetics and pharmaceuticals are unable to use these albumin sources as functional compounds in their products.

[0039] Emulsifying activity is defined as above. Those skilled in the art are aware of various protocols that can help measure this emulsifying activity. Emulsifying properties can generally be described by two factors: emulsifying activity (EA) and emulsion stability (ES). (Boye et al. 2010a). Emulsifying activity (EA) can be defined by the maximum amount of oil that can be dispersed in an aqueous solution containing a known amount of emulsifier before destroying or transforming the emulsion phase (Sherman, 1995).

[0040] On the basis of the prior art, the applicant of the present invention has developed Test A described below, which allows to quantify the emulsification properties easily, quickly and definitively:

[0041] 1. Introduce 0.2 g of protein sample into 20 ml of water;

[0042] 2. Homogenization was performed with the aid of Ultraturax IKA T25 at 9500 rpm within 30 seconds;

[0043] 3. Add 20 ml corn oil under the same conditions as step 2 above while homogenizing;

[0044] 4. Centrifuge at 3100 g for 5 minutes.

[0045] a. In case the emulsion was good (the emulsion was not broken or inverted), a new experiment was performed after increasing the amount of water and oil by 50%.

[0046] b. In case of bad emulsion (emulsion breakdown or inversion), new experiments were performed after reducing the amount of water and oil by 50%.

[0047] 5. Repeat this process to determine the maximum amount of oil that can be emulsified (called Qmax, in ml).

[0048] 6. Emulsification capacity = (Qmax / 0.2)*100

[0049] Such emulsification capacity analysis can be performed at different pH levels in order to assess its effectiveness.

[0050] In a preferred embodiment, when the above protein of the invention is consumed by a human panel, it has a low bitter off-taste. Such bitterness can also be assessed by methods known in the art including electronic tongue.

[0051] The assessment of bitterness can be easily and accurately performed with the help of a trained human panel. The preferred protocol for this assessment is described below:

[0052] Six panelists trained to detect bitterness were selected. The tasting base was a 5% suspension in water (Evian), then manually homogenized and pasteurized at 70°C / 30min. The panel tasted each product in the same order and shared their feelings about the bitterness experience. The descriptor "bitter" was specifically tracked.

[0053] Bitterness can also be assessed with the aid of an electronic tongue, such as the SA402B electronic tongue produced and sold by Insent. A preferred approach for such an assessment is described below:

[0054] 1. Weigh about 4 g of sample into a 250.0 mL volumetric flask;

[0055] 2. Add 100 mL of purified water to the sample and mix on a mechanical shaker for about 60 minutes;

[0056] 3. Centrifuge the suspension at about 3000 rpm for about 10 min;

[0057] 4. The supernatant was transferred to 2 sampling cups for analysis with the electronic tongue, which involved inserting the electronic tongue into the supernatant and waiting for stable measurements (bitterness, but also umami, astringency and richness).

[0058] In another preferred embodiment, the protein of the present invention also has a whipping property (also called foaming power) higher than 250%, preferably higher than 300%.

[0059] The whipping properties (also called foaming power) can be analyzed according to the following procedure (referred to as Test B in this specification):

[0060] √ Disperse 1g of sample in 40ml of distilled water (hereinafter, the volume is referred to as Vi);

[0061] √Use Ultraturax IKA T25 to stir at 8000rpm for 30s,

[0062] √Use Ultraturax IKA T25 to stir for the second time at 9500rpm for 8min,

[0063] √ Transfer to a 100ml graduated test tube

[0064] √Measure the foam volume (hereinafter referred to as Vf) and liquid volume immediately and after 90 minutes;

[0065] √ Whipping characteristics =Vf (foam volume) / Vi (volume before stirring).

[0066] The whipping properties can also be expressed as a percentage:

[0067] Whipping characteristics=(Vf (foam volume) / Vi (volume before stirring))*100

[0068] In a more preferred embodiment, the protein of the invention presented in the above two examples has an enrichment higher than 80%, preferably higher than 90%, expressed as N×6.25 based on dry matter.

[0069] The enrichment of albumin can be measured by well known protocols known to those skilled in the art, including electrophoresis and nitrogen dosing. The most preferred protocol involves the Kjærdahl assay protocol.

[0070] A second object of the invention is a method allowing the production of a protein according to the invention, comprising the following steps:

[0071] 1. Starting with wheat seeds, extract the outer fiber, wheat germ, starch, and wheat gluten to obtain a fraction called wheat solubles

[0072] 2. Microfiltration of the wheat soluble fraction to obtain microfiltration permeate

[0073] 3. applying cation chromatography to the microfiltration permeate so as to produce a fraction enriched in wheat albumin;

[0074] 4. subjecting the wheat albumin-enriched fractions to an ultrafiltration step together or separately to obtain a more albumin-enriched retentate;

[0075] 5. Concentrating and / or drying the albumin-enriched retentate described above.

[0076] For step 1, well known protocols can be applied to obtain wheat solubles.

[0077] As an example of a method suitable for recovering wheat solubles, wheat grains are soaked in warm water and ground with a roller mill. During grinding, the bran and germ are separated by air flow and sieving, so that wheat flour is obtained. This flour is then mixed with 2 parts of water and stirred for 10 minutes to allow glutenin and gliadin to form a network and produce viscoelastic gluten. The flour slurry is then centrifuged. Starch, fiber and gluten paste are deposited. The remaining upper layer of clear water, which includes salt, sugars and wheat albumin, is obtained, which is called "wheat solubles".

[0078] For example, wheat solubles are obtained from a separate stream of wheat "B" starch produced by starch separation during the milling of wet wheat starch. B starch or "second starch" is a starch that consists essentially of a large portion of small starch granules or damaged granules. In addition to this B starch, wheat solubles are also known to contain non-negligible amounts of high molecular weight proteins that can constitute a nitrogen source for the microorganisms of interest.

[0079] For step 2, microfiltration removes residual macromolecules and helps to obtain filtered and clarified wheat solubles in the permeate. A ceramic microfiltration membrane with a pore size of 0.1um and a permeability gradient (GP) is preferred. The microfiltration (MF) permeate is introduced into the next step, and the retentate (rich in protein and oil) after washing out (diafiltration) can be used for high-protein feed production or recycling. Microfiltration is preferably carried out in a 3-sequential stacking system, in which each next step is fed by the retentate of the previous step, and the permeates are collected together from all 3 stacks. Diafiltration of the final retentate is not carried out, but diafiltration of the final retentate may be carried out in the 3rd or additional stack.

[0080] Step 3 includes chromatographic separation of specific albumin-enriched fractions. Preferably, cation exchange chromatography is used. Preferably, macroporous beads of hydrophilic copolymers of glycidyl methacrylate and propyl sulfonic acid groups are used as cation exchangers. The resin is pre-conditioned by a low ionic strength acid (HCl or lactic acid, or malic acid) solution of low pH (2.5). The pH of the microfiltration permeate is adjusted to 3.8-4.0 by acid (HCl or lactic acid, or malic acid), and passed through the resin until protein absorption is saturated (this can be detected when protein leaks out at the resin output port). The unbound complexes mainly containing fibers in the so-called raffinate stream can be directed to soluble fiber recovery, or only recycled back to the original stream starch process water (possibly after optional nanofiltration or ultrafiltration). As pH and ionic strength gradients increase (using NaOH and NaCl solutions buffered by malic acid or carbonate), 3 to 6 independent peaks, preferably 3 peaks, are recovered from the resin to bind proteins.

[0081] In a preferred embodiment, the method of the present invention is characterized in that the cation chromatography of step 3 comprises the following steps:

[0082] c. Feed the microfiltration permeate onto a cationic resin to bind the protein

[0083] d. Feeding different eluents onto the resin to release and separate the proteins bound to the resin

[0084] In a more preferred embodiment, the method of the present invention is characterized in that the different eluents of step a are continuous:

[0085] i) 20 mM malic acid and 3 mM sodium carbonate solution at pH 6.2

[0086] ii) 20 mM malic acid, 3 mM sodium carbonate and 0.5 M NaCl solution at pH 6.2

[0087] iii) 30 mM sodium carbonate and 0.45 M NaCl solution at pH 12

[0088] For further processing, the peaks can be transferred or mixed separately into so-called PN1, PN2 and PN3 fractions, in particular depending on their palatability. Three separate protein fractions are collected with the following characteristics:

[0089] PN1: pH 6.1-6.3; conductivity 3-3.5mS; low bitterness

[0090] PN2: pH 6.1-6.4; conductivity 45-50mS; low bitterness;

[0091] PN3: pH 12.0-12.2; conductivity 45-50mS; high bitterness

[0092] In step 4, the protein fraction is concentrated by ultrafiltration (UF) with a PES membrane in a tangential flow filtration (TFF) system. Different molecular weight cut-offs from 1 to 6 kDa can be used. In order to increase the protein content, at the end of the ultrafiltration, the PN2 fraction is diafiltered with reverse osmosis quality water. The PN3 fraction before UF requires additional pH adjustment (as low as 5.5-6) ​​and is clarified by a centrifuge. The UF permeate or non-permeate can be recycled as PW (optionally, after reverse osmosis filtration) to starch production. After this step, a purified protein fraction of 9%-10% dry matter, even preferably 10%-20% dry matter, even more preferably 20%-30% dry matter can be obtained.

[0093] Step 5 aims at removing the water. Possible solutions are ultrafiltration, short residence time evaporation or forward osmosis. In the case of forward osmosis, the osmoticum is re-concentrated by reverse osmosis or evaporation. The filtrate or condensate is reused in starch processing.

[0094] Step 6 aims at drying the protein. A spray dryer is preferred. Usually, the product is non-sticky and dries easily. The protein isolate is obtained in the form of a light, cream-colored dry powder with a dry matter content of about 95% and a protein content of 85%-97%. Microagglomerated products may require better commercial properties, which requires special drying schemes like a spray / fluidized bed combination.

[0095] In a more preferred embodiment, the concentration of step 5 is performed by reverse osmosis.

[0096] The present invention will be better understood through the following examples.

[0097] Examples

[0098] Example 1: Production of wheat solubles by conventional methods in the prior art

[0099] Wheat kernels are tempered for 8 hours in water heated to 28°C. Water is removed and ground by a roller mill. After grinding, the bran and germ are separated by a combination of sieving at 1400 microns, 530 microns and finally 300 microns to obtain wheat flour with about 14% moisture and 0.8% ash (dry basis). The flour obtained is mixed with 2 parts of water and stirred for 10 minutes so that glutenin and gliadin form a network to produce viscoelastic gluten. The flour slurry is then centrifuged at 3-10x1000G-force for 5 minutes. Starch, fiber and gluten paste are deposited. The remaining upper layer of clear water including salt, sugars and wheat albumin is obtained and is called "wheat solubles". The average composition of such wheat solubles is summarized in Table 1 below:

[0100] analyze unit value Dry matter ％ 5.5-7.5 Total Protein %Dry Matter 19-22 Soluble protein % of total protein 47-51 Soluble Fiber %Dry Matter 14-17 carbohydrate %Dry Matter 33-35 Lipids (crude fat) %Dry Matter 2-3 Organic acids (lactic acid, acetic acid, etc.) %Dry Matter 15-25

[0101] Table 1: Average composition of wheat solubles

[0102] Example 2: Production of wheat albumin extract with no bitterness and high emulsifying activity

[0103] This example involves 1000 kg of wheat solubles containing 20% ​​protein on a dry matter basis. The wheat solubles are fed into a membrane graded microfiltration unit, more precisely a PALL ceramic microfiltration membrane with a pore size of 0.1 um with a permeability gradient (GP). The microfiltration permeate will be fed to the next step, while the retentate after washing out (diafiltration) is discarded. A 3-sequential stacking system is used, in which each next step is fed by the retentate of the previous step and the permeates are collected together from all 3 stacks. Diafiltration of the final retentate is not performed, but it may be performed in the 3rd or additional stack. Filtration is terminated after the VCF (volume concentration factor) reaches 5. The main parameters of microfiltration (MF) are summarized in the following Table 2:

[0104] Temperature, °C 45-60 Inlet pressure, bar 2.5 Cross-molded, bar 1 Recirculation speed, m3 / h 33 Linear velocity of retentate, m / s 5 Average flow, l / h / m2 50

[0105] Table 2: Main parameters of microfiltration

[0106] This step produces 722 kg of clarified permeate containing 6.5 kg of protein. The permeate is then purified by chromatography (more precisely, cation exchange chromatography). Macroporous beads of hydrophilic copolymers of glycidyl methacrylate and propyl sulfonic acid groups are used as cation exchangers. The beads are pre-conditioned by a low ionic strength acid (HCl or lactic acid, or malic acid) solution at a low pH (2.5) and loaded into a standard exchange column. The pH of the clarified permeate is adjusted to 3.8-4.0 with an acid (HCl or lactic acid, or malic acid) and the clarified permeate is fed to the resin at a linear speed of 2 cm / min and 20°C. The first unbound stream equivalent to 840 kg with 2.3% dry matter, also referred to as raffinate, is discarded. As the pH and ionic strength are gradually increased, the bound protein is recovered from the resin so that three different peaks are produced. In order to achieve this separation with 3 different peaks, 3 eluents are continuously fed to the resin as described in Table 3.

[0107]

[0108] Table 3: Description of the three eluents fed continuously onto the resin.

[0109] Three distinct peaks were detected using a UV detector placed at the output of the resin scanning at 280 nm (see Figure 2 ). Peak protein fractions with the following characteristics summarized in Table 4 were collected.

[0110]

[0111] Table 4: Peak protein fraction characteristics

[0112] This step produces 442 kg of PN1 fraction containing 2.21 kg of protein, 195 kg of PN2 fraction containing 7.80 kg of protein, and 149 kg of PN3 fraction containing 4.47 kg of protein. Then, PN1, PN2 and PN3 are purified separately by ultrafiltration. The PN3 fraction before ultrafiltration requires additional pH adjustment (down to 5.5-6) ​​and is clarified by a centrifuge. PES membranes in tangential flow filtration (TFF) systems are preferred, with a molecular weight cutoff of 6 kDa. In order to increase the protein content, at the end of ultrafiltration, the PNx (PNx refers to PN1, PN2 and PN3 fractions) fractions are diafiltered with reverse osmosis quality water. The UF permeate or non-permeate can be recycled as PW to starch production (optionally, after RO filtration). After this step, a PNx protein fraction with a protein enrichment of 9%-10% DS and more than 90% expressed as N×6.25 can be obtained. The main ultrafiltration parameters are summarized in Table 5 below:

[0113] MWCO, kDa 3-6 Temperature, °C 6-10 or 55-60 Inlet pressure, bar 2 Cross-molded, bar 1.5 Recirculation speed, m3 / h Depends on system Linear velocity of retentate, m / s 3 Average flow, l / h / m2 3 or 10 (depending on temperature)

[0114] Table 5: Main ultrafiltration parameters.

[0115] The concentration of all PNx protein fractions was too low in dry matter to be spray dried. The dry matter was concentrated to 20% by an ultrafiltration membrane filtration process, where the permeate was discarded and the retentate was retained, thereby concentrating the wheat albumin. The main parameters of ultrafiltration (UF) are summarized in Table 6 below:

[0116] MWCO, kDa 5 Temperature, °C 6-10 or 55-60 Inlet pressure, bar 2 Cross-molded, bar 1.5 Recirculation speed, l / min 3 Linear velocity of retentate, m / s 3 Average flow, l / h / m2 3 or 14 (depending on temperature)

[0117] Table 6: Main parameters of ultrafiltration

[0118] Finally, all concentrated PNx protein fractions were dried in a Fujisaki spray dryer. The protein isolate was obtained in the form of a light, cream-colored dry powder with a dry matter content of about 95% and a protein content of 85%-97%. The parameters of the currently used spray dryer are summarized in Table 7 below:

[0119] Temperature (inlet), ℃ 190 Temperature (out), ℃ 70

[0120] Table 7: Spray dryer parameters

[0121] Example 3: Comparison between the present invention and the prior art

[0122]

[0123] Table 8: Emulsifying activity, whipping properties / foaming power and foaming stability of wheat solubles and PN1, 2 and 3 fractions at different pH.

[0124] As described in Table 8, the PN2 and PN3 fractions could emulsify more than 500 ml oil / g sample. Furthermore, only the PN2 fraction allowed emulsification of about 1000 ml oil / g protein.

[0125] On the other hand, it can be seen that, thanks to the process of the invention, the PN2 and PN3 fractions present whipping properties higher than 300%, allowing the production of large amounts of foam with the same amount of protein. Finally, it can be seen that the PN2 fraction allows the production of very stable foams.

[0126] Example 4: Sensory comparison by a sensory panel

[0127] product:

[0128] Pea isolate, NUTRALYS S85F batch W122K

[0129] Hydrolyzed wheat gluten, NUTRALYS W batch E5166

[0130] Whey Protein Isolate WPI 894

[0131] Milk casein salt

[0132] · Microfiltration clarified permeate, feed for chromatography in the present invention

[0133] PN1 peak obtained in Example 2

[0134] PN2 peak obtained in Example 2

[0135] PN3 peak obtained in Example 2

[0136] Six panelists trained to detect bitter taste participated in the trial.

[0137] The tasting base was a 5% suspension in water (Evian), homogenized manually and pasteurized at 70°C / 30 min.

[0138] The panel tasted each product in the same order and shared their feelings about the bitterness

[0139] turn out

[0140] NUTRALYS S85F: Strong pea / raw smell and taste, bitter, astringent, chickpea soup

[0141] Nutralys W: Strong smell and taste: raw, rice, fermented (like Chinese green tea), very bitter

[0142] ·WPI 894: Milk, boiled eggs, sandy, dry

[0143] Milk casein salt: milk, washing water, pungent, sour and bitter

[0144] · Microfiltration clarified permeate, feed for chromatography in the present invention: bitter

[0145] PN2 and PN1 peak fractions: The smell is fairly neutral, a bit fruity, like ice tea. Not bitter / astringent.

[0146] PN3 fraction: very bitter, even more bitter than the feed

[0147] In summary, the process of the present invention can produce wheat protein isolates (PN2) without bitterness, without the need for polyol formulation as taught in the prior art, and can also produce very bitter wheat protein isolates (PN3). If desired, very bitter fractions can be used in some industrial applications.

[0148] Example 5: Antioxidant capacity of the inactive wheat protein of the present invention

[0149] This example aims to evaluate the antioxidant capacity of PN2 sample from Example 4 relative to common protein samples on HaCaT human keratinocyte model using LUCS technology.

[0150] The LUCS method is based on the generation of cellular radical species following the addition of light-inducible fluorescent nucleic acid biosensors to the culture medium. The effect of applying light in the presence of the cellular biosensors triggers the generation of singlet oxygen, which in turn leads to the generation of ROS (reactive oxygen species) in a biochemical cascade associated with an increase in released fluorescence.

[0151] LUCS analysis measures the ability of antioxidant compounds to remove ROS in living cells and neutralize oxidative stress. The effect is measured by the delay in the kinetic evolution of fluorescence emission. The method has been standardized on a high-throughput 96-well plate to allow reliable statistical analysis (EP 2235505, US 20110008783 and Derick S. et al. Sci Rep. [Science Report] 2017, 7 (1): 18069).

[0152] Before the experiment, the protein samples were digested according to the in vitro gastrointestinal (GI) protocol. First, 5 g of the PN2 sample from Example 4 was dissolved in 95 mL of ultrapure water and 250 μL of 0.3 M CaCl 2 , and incubated at 37 °C under magnetic stirring for 1 h. Then, 80 mL of ultrapure water, 15 mL of HCl (1 M), and 50 μL of CaCl 2The gastric juice made of (0.3M) is added to the protein solution. Before adding pepsin, pH is adjusted to 2 with HCl (4M). Pepsin (from porcine gastric mucosa EC3.4.23.1, CAS 9001-75-6, index number Sigma P7000) of 0.5g amount is added to the solution, which is kept for 2 hours under magnetic agitation at 37 ℃. pH is measured every 30min, and is adjusted to 2 (if necessary). Then, the intestinal juice made of 175mL phosphate buffered saline (PBS) solution (pH 7.4), 35mL ultrapure water, 400 μL CaCl (0.3M) and 4.5mL NaOH (1M) is added to the GI system, and pH is adjusted to 6.8 (if necessary) with NaOH (4M). A 1 g amount of pancreatin (EC 232-468-9 from porcine pancreas, CAS 8049-47-6, index number Sigma P7545) was added to the solution and the whole system was kept at 37°C under magnetic stirring for 2 hours. The pH was measured every 30 min and adjusted to 6.8-7 (if necessary). Once the in vitro GI digestion was complete, the solution was heated at 90°C for 10 min to inactivate the enzyme. The sample was centrifuged at 10 000 g for 10 min and the supernatant was stored at -20°C.

[0153] A volume of 10 ml of DMEM (Dulbecco's Modified Eagle Medium) cell culture medium without FCS (fetal calf serum) was added to 500 mg of each sample (whey protein, egg white and PN2 sample from Example 4). The solution (50 mg / ml w / v) was vortexed and then centrifuged (8700 rpm, 10 min). The supernatant was aliquoted and stored at -20°C until the day of the experiment. The pH of the samples was 8, which is compatible with AOP analysis. Before the experiment, the samples were then warmed to 90°C for 10 min to inactivate the digestive enzymes. Studies were performed on human HaCaT cells. Cells were seeded in 96-well plates at a density of 75 000 cells / well in DMEM medium supplemented with fetal calf serum (FCS) and incubated at 37°C / 5% CO 2 Then, the samples (8 concentrations obtained by serial log2 dilution) were incubated at 37°C / 5% CO 2 The cells were incubated for 4 hours at 4 °C. The experiments were performed in DMEM medium without FCS. Two independent experiments were performed on three replicate wells.

[0154] After 4 hours of incubation, cells were treated with the fluorescent biosensor within 1 hour and fluorescence (RFU at 535 nm) was measured following repeated 480 nm LED applications (20 iterations) across the 96-well plate. Kinetic curves were recorded.

[0155] The antioxidant cell index (AOP index) was calculated from the normalized kinetic curve according to the following formula:

[0156] AOP index (%) = 100–100 (0∫ 20 RFU sample / 0∫ 20 RFU control)

[0157] The dose-response curve obtained by compiling the AOP index according to the logarithm (10) of the sample concentration was subjected to a sigmoid fit according to the following formula:

[0158] AOP index = minimum AOP index + (maximum AOP index - minimum AOP index) / (1 + 10 (Log (EC50-SC) * HS))

[0159] Where SC = sample concentration and HS = Hill slope. EC50 (50% efficacy concentration), EC10 and EC90 were then estimated where possible.

[0160] Table 9 below shows the results:

[0161]

[0162] Table 9: Comparison of EC50 of different samples

[0163] The results clearly showed that the PN2 sample protein had strong antioxidant activity compared to prior art proteins from common animals.

Claims

1. A method which facilitates the production of an inactive wheat protein having an emulsifying activity of 1000 mL oil / g protein at pH 6-6.5 and / or at pH 8-8.5, It is characterized in that The method comprises the following steps: (1) Starting from wheat seeds, the outer fiber, wheat germ, starch and wheat gluten are extracted and a fraction called wheat solubles is obtained, (2) microfiltration of the wheat solubles to obtain a microfiltration permeate, (3) applying cation chromatography to the microfiltration permeate so as to produce a fraction enriched in wheat albumin having an emulsifying activity of 1000 mL oil / g protein at pH 6-6.5 and / or at pH 8-8.5, (4) ultrafiltration of the wheat albumin-enriched fraction to obtain a retentate enriched with more albumin; (5) concentrating and / or drying the albumin-enriched retentate described above, The cation chromatography of step (3) comprises the following steps: a. feeding the microfiltration permeate onto a cationic resin to bind the protein; b. feeding different eluents onto the resin to release and separate the proteins bound to the resin, The macroporous beads of hydrophilic copolymer of glycidyl methacrylate and propyl sulfonic acid groups are used as cationic resin. Wherein the different eluents of step b are continuous: i) 20 mM malic acid, 3 mM sodium carbonate solution and NaOH at pH 6.2-6.4; ii) 20 mM malic acid, 2.5 mM sodium carbonate, 0.5 M NaCl solution and NaOH at pH 6.2-6.4; iii) 30 mM sodium carbonate, 0.45 M NaCl solution and NaOH at pH 12; Wherein, the emulsifying activity is quantified according to Test A described below: (1) Introduce 0.2 g of protein sample into 20 mL of water; (2) homogenization at 9500 rpm within 30 seconds using Ultraturax IKA T25; (3) adding 20 mL of corn oil under the same conditions as in step (2) above while homogenizing; (4) Centrifugation at 3100 g for 5 minutes; a. In the case of a good emulsion, a new experiment was performed after increasing the amount of water and oil by 50%, wherein the good emulsion means that the emulsion was not destroyed or transformed; b. In the case of a poor emulsion, a new experiment was performed after reducing the amount of water and oil by 50%, wherein the poor emulsion refers to emulsion destruction or conversion; The maximum amount of oil that can be emulsified is determined repeatedly in this way, and the maximum amount of oil is called Qmax, in mL; Emulsifying capacity = (Qmax / 0.2) × 100.

2. The method according to claim 1, in, The concentration step was performed using ultrafiltration.

3. The method according to claim 1 or 2, wherein the wheat protein is wheat albumin.

4. Inactive wheat protein having an emulsifying activity of 1000 mL oil / g protein at pH 6-6.5 and / or at pH 8-8.5 obtainable by the process according to any one of claims 1 to 3.

5. The inactive wheat protein according to claim 4, wherein the wheat protein is wheat albumin.

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