New method for preparing cationic whey protein isolate and products obtained therefrom

Abstract

The present invention relates to a novel method for preparing a cationic whey protein isolate containing high-purity lactoferrin.

Description

Technical Field

[0001] The present invention relates to a novel method for preparing high-purity lactoferrin cationic whey protein isolate. Summary of the Invention

[0002] The applicant has developed a process for obtaining a whey protein isolate having a lactoferrin purity higher than 90%; this process allows controlling the vitamin B12 (cobalamin) content in the lactoferrin isolate.

[0003] The method is characterized in that, on the one hand, a previously concentrated milk material (such as concentrated skim milk or concentrated whey) is used by membrane technology (such as reverse osmosis, nanofiltration or ultrafiltration) and, on the other hand, a strong cation exchange resin of the sulfopropyl (SP) type packed in a radial chromatography column is used for selective extraction. The eluted lactoferrin pure fraction is concentrated and desalted by ultrafiltration to obtain a cationic whey protein isolate having a lactoferrin purity of at least 90%, preferably 95%. The liquid isolate obtained is debacterialized or sterilized by microfiltration and optionally dried by spray drying or freeze drying to obtain a powdered isolate.

[0004] The method for preparing a high-purity lactoferrin cationic whey protein isolate comprises the following steps a) to f):

[0005] a) The starting material may be mammalian milk that has been pre-skimmed and concentrated by membrane technology; it may also be a mixture of mammalian milk that has been pre-skimmed and concentrated by membrane technology and skimmed (unconcentrated) milk; the mammalian milk is, for example, cow's milk or goat's milk; the starting material may also be whey from mammalian milk that has been pre-concentrated;

[0006] i. When the starting material is prepared from mammalian milk (e.g. cow's or goat's milk), it is defatted and optionally pasteurized, for example by a short-term heat treatment between 60 and 78° C. (the minimum heat treatment equivalent level is 72° C. for 15 seconds; defatting can be performed before or after pasteurization) or by microfiltration using a membrane with a porosity between 0.8 and 1.4 μm for debacterization and subsequent concentration by reverse osmosis (OI) or nanofiltration (NF) or ultrafiltration (UF); for the implementation of the method, a mixture of skim milk (unconcentrated) and skim milk previously concentrated by membrane technology as described above can also be used; the protein content (MP) concentration of the product to be treated in step b) is preferably between 40 and 72 g / L, preferably between 43 and 57 g / L; when using an OI membrane, the dry matter (MS) concentration of the pasteurized skim milk is between 110 and 200 g / L, preferably between 120 and 160 g / L;

[0007] ii. When the starting material is prepared from whey, the whey can be concentrated after the separation of the caseins by acidification or renneting, by microfiltration (with a membrane porosity of about 0.1 μm), by reverse osmosis (OI) or by nanofiltration (NF) or by ultrafiltration (UF); for the implementation of the method, a mixture of whey (unconcentrated) and whey previously concentrated by membrane techniques as described above can also be used; the protein concentration of the product to be treated in step b) is preferably between 20 and 100 g / L, preferably between 30 and 80 g / L;

[0008] It should be noted that pasteurization and microfiltration are not necessary for the process.

[0009] b) selectively extracting cationic proteins comprising the following steps:

[0010] i. The starting material (eg pre-concentrated pasteurized skim milk) is passed through a flow-through chromatography column containing a sulfopropyl SP type strong cation exchange resin, preferably with a diameter greater than 100 μm (eg SP Sepharose Big Beads from Cytiva, Sweden):

[0011] the volume of the starting material (expressed as the volume corresponding to the unconcentrated material; i.e. the volume indicated is the volume the material had before concentration) is between 40 and 500 times the resin volume (BV, bed volume), in particular between 80 and 500 times, preferably between 80 and 300 BV;

[0012] - the linear velocity of the starting materials is between 1.0 and 4.0 m / h, preferably between 2.0 and 3.0 m / h;

[0013] ii. Rinse with softened water, preferably treated with an OI membrane (permeate water):

[0014] - the volume of demineralized water is between 2 and 6 BV, preferably between 3 and 5 BV;

[0015] - demineralized water throughput rate between 3.0 and 5.0 m / h, preferably between 3.5 and 4.5 m / h;

[0016] iii. eluting the bound cationic protein with a saline solution (a demineralized aqueous solution of NaCl, preferably osmotic water) having a conductivity between 30 and 50 mS / cm:

[0017] - the volume of the saline solution is between 4 and 8 BV, preferably between 5 and 7 BV;

[0018] - the passage speed of the salt solution is between 0.3 and 2.0 m / h, preferably between 0.5 and 1.0 m / h;

[0019] iv. eluting the bound cationic protein with a saline solution (a demineralized aqueous solution of NaCl, preferably permeate) having a conductivity between 80 and 140 mS / cm, preferably between 90 and 110 mS / cm:

[0020] - the volume of the saline solution is between 3 and 6 BV, preferably between 4 and 5 BV;

[0021] - the passage speed of the salt solution is between 0.5 and 2.5 m / h, preferably between 1.0 and 2.0 m / h;

[0022] The passage through the cation exchange resin serves to bind cationic proteins present in the starting material, while allowing passage of major components of skim milk (such as lactose), minerals, acidic proteins (such as casein, β-lactoglobulin, α-lactoglobulin, serum albumin, and most immunoglobulins). The first elution step serves to selectively extract specific cationic proteins by retaining the majority of lactoferrin (the main milk cationic protein) bound to the resin. Thus, a pure bovine lactoferrin fraction elutes in the second eluent.

[0023] c) concentrating the high-purity lactoferrin cationic protein eluted in the saline solution using an ultrafiltration membrane with a cut-off threshold (MWCO) between 10 and 20 kDa;

[0024] d) desalting the high-purity lactoferrin cationic protein by diafiltration with demineralized water, preferably permeate water, using an ultrafiltration membrane with a MWCO between 10 and 20 kDa to reach an ash / protein ratio between 0.001 and 0.03, preferably between 0.003 and 0.01;

[0025] e) microfiltration of the concentrated solution of the specific cationic protein using a double-layer membrane with a cut-off threshold between 0.2 and 1.4 μm, preferably between 0.8 and 1.4 μm, to reduce the microbial load;

[0026] f) Optionally, spray drying or freeze drying the previously microfiltered concentrated solution of the specific cationic protein to obtain a powdered lactoferrin isolate.

[0027] Advantageously, the use of mammalian milk materials concentrated by UF / NF / OI membranes (e.g., pasteurized skim milk, cheese whey from pasteurized goat milk) allows for a reduction in the flow rate through the extraction column for the same amount of protein present. Due to the extended contact time with the SP type strong cation exchange resin, the extraction efficiency of cationic proteins is significantly improved.

[0028] Furthermore, due to their trapezoidal geometry, the use of radial flow columns (eg Albert Handtmann Armaturenfabrick GmbH) allows for the pressure generated by the concentrated mammalian milk material passing through the filling resin to be sustainably withstood.

[0029] This combination of concentrated milk material and runoff column is essential for performing stable and constant industrial production.

[0030] Another advantage of the process according to the invention is that it can be carried out effectively over a wide temperature range; in particular, although resin manufacturers recommend operation at temperatures between 30 and 50°C, the Applicant has successfully developed a process that is effective at low temperatures, i.e. at temperatures below 15°C, preferably below 10°C.

[0031] Therefore, the present invention relates to a cationic whey protein isolate enriched in lactoferrin obtained or obtainable by the process according to the invention, such that the protein proportion of the dry matter is greater than or equal to 90% by weight and the proportion of lactoferrin in the total protein of the isolate is greater than 90% by weight, preferably greater than 95% by weight, more preferably greater than 98% by weight.

[0032] The present invention also relates to a cationic whey protein isolate enriched in lactoferrin from milk or whey (from bovine or goat milk), obtained or obtainable by the process according to the invention, having a protein proportion of the dry matter of greater than or equal to 90% by weight, a lactoferrin proportion in the total protein of greater than 95% by weight (w / w), preferably greater than 98% by weight, and containing cobalamin in complex with transcobalamin protein in a concentration of less than or equal to 5 μg / g protein, in particular a concentration of cobalamin in complex with transcobalamin protein of between 1 and 5 μg / g protein.

[0033] The present invention also relates to a cationic whey protein isolate from milk or whey (from bovine or goat milk) enriched in lactoferrin obtained or obtainable by the process according to the invention, having a protein proportion of the dry matter greater than or equal to 90% by weight, a lactoferrin proportion in the total protein greater than 90% (w / w), preferably greater than 95% by weight, and containing cobalamin in complex with transcobalamin protein in a concentration greater than or equal to 5 μg / g, preferably greater than or equal to 8 μg / g, still more preferably greater than or equal to 10 μg / g of protein.

[0034] The isolate according to the invention can be in liquid form (without step f) or in powder form (with step f). If it is in liquid form, it has the same characteristics as the powder in terms of composition relative to the dry matter and generally contains between 5 and 25% by weight, preferably between 10 and 20% by weight, of water.

[0035] According to another object, the present invention relates to a food product for human or animal consumption, a human or animal medicament or a food supplement containing the cationic protein isolate according to the invention.

[0036] Preferably, the isolate according to the invention has a microbial load such that the aerobic mesophilic bacterial count of the isolate powder according to the invention is less than 1000, preferably less than 100 or 10, even more preferably less than 1 uFc / g, or such a count of the liquid isolate is less than 100, preferably less than 10, even more preferably less than 1 uFc / ml. The combined use of concentrated milk material and flow-through columns allows, under appropriate conditions, the stable and efficient production of two high-purity lactoferrin isolates by cation exchange chromatography:

[0037] - isolates with a lactoferrin purity >95% or 98% and a vitamin B12 content ≤5 μg / g protein or between 1 and 5 μg / g protein;

[0038] Such isolates according to the invention are of particular interest for the preparation of infant formulas (infant milk or follow-up milk) based on cow's or goat's milk.

[0039] as well as

[0040] - Lactoferrin isolates with a purity of >90% or 95% and a vitamin B12 content of ≥5 μg / g protein, preferably ≥8 μg / g protein, more preferably ≥10 μg / g protein;

[0041] The isolate may have nutritional benefits as a food supplement for vegetarians or in nutritional preparations for people with deficient vitamin B12 absorption, such as those who have undergone gastrectomy or those who receive long-term IPP (proton pump inhibitor) therapy. In fact, in addition to the benefits of lactoferrin, the isolate may provide an important source of vitamin B12 that is highly bioavailable even in the absence of intrinsic factor secreted by the stomach.

[0042] Therefore, the present invention also relates to a dietary supplement comprising an isolate enriched in vitamin B12 according to the present invention, i.e. an isolate having a lactoferrin purity of >90% or 95% and a vitamin B12 content of ≥5 μg / g protein, preferably ≥8 μg / g protein, more preferably greater than or equal to 10 μg / g protein.

[0043] The amount of the isolate according to the invention in the food supplement will be selected according to the profile of the population to be supplemented, and thus the dose of vitamin B12 to be administered, as well as the vitamin B12 content of the isolate. For example, a daily dose of 150 to 1000 mg of protein of an isolate enriched in vitamin B12 (6 μg / g protein) according to the invention can provide 0.9 to 6.0 μg of vitamin B12 in complex with transcobalamin. Similarly, a daily dose of 100 to 600 mg of protein of an isolate enriched in vitamin B12 (10 μg / g protein) according to the invention can provide 1.0 to 6.0 μg of vitamin B12 in complex with transcobalamin. Thus, even if the intestinal absorption of vitamin B12 is disturbed, this food supplement can meet the needs of each population as shown in the table below.

[0044] Nutritional reference for vitamin B12 (μg / d) according to ANSES 2016

[0045] crowd Adequate intake (μg / day) Infants under 6 months old 0.4 Infants 6 months and older 1.5 Children aged 1 to 3 years 1.5 Children aged 4 to 10 years 1.5 Teens aged 11 to 17 2.5 Men and women aged 18 and over 4 Pregnant women 4.5 breastfeeding women 5

[0046] The present invention further relates to an isolate having a lactoferrin purity of >90% or 95% and a vitamin B12 content of ≥5 μg / g protein, preferably ≥8 μg / g protein, more preferably ≥10 μg / g protein, for use in preventing and / or treating vitamin B12 deficiency, for example in patients who have undergone gastrectomy or who have been receiving long-term IPP (proton pump inhibitor) therapy.

[0047] Cobalamin (vitamin B12) is present in milk in the form of a complex with binding proteins. In bovine milk, it exists in the form of a complex with transcobalamin, a 43 kDa cationic protein (S.N. Fedosov, T.E. Petersen, E. Transcobalamin from cow milk: isolation and physico-chemical properties, Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology. 1292 (1996) 113-119). The cobalamin-transcobalamin protein complex is of great nutritional significance as it is believed to be responsible for the bioavailability of vitamin B12 (SNFedosov, Ebba Nexo, Christian W. Heegaard, Vitamin B12 and its binding proteins in milk from cow and buffalo in relation to bioavailability of B12, Journal of DairyScience. American Dairy Science Association. 102 (2019) 4891-4905).

[0048] Although the behavior of the complex during cation exchange chromatography is close to that of lactoferrin, the vitamin B12 content of the eluate obtained by the method of the invention may vary depending on the conditions used.

[0049] Furthermore, despite its nutritional significance, for certain applications (e.g. infant formula, i.e. infant formula / milk and / or second formula / milk), in certain specific cases (high incorporation doses), it may be meaningful to limit the vitamin B12 content in the composition of the pure lactoferrin fraction. In this case, the method according to the invention allows the final vitamin B12 content to be adjusted, which is of great significance.

[0050] The present invention therefore relates to a food product for human or animal consumption comprising the isolate according to the invention; in the case of infant formula (i.e. infant milk / milk and / or second milk / milk formula), preferably an isolate is used having a lactoferrin purity of >95% and a vitamin B12 content of ≤5 μg / g protein. The incorporation rate of the isolate according to the invention is 50 to 1000 mg of protein per liter of ready-to-feed formula.

[0051] The present invention also relates to non-food products such as hygiene products and cosmetics comprising the isolate according to the invention.

[0052] The invention also includes products for oral hygiene, such as toothpaste, mouthwash, chewing gum in gel or paste form, comprising the isolate according to the invention. The incorporation rate of the isolate according to the invention is 1 to 100 mg of protein per gram of product. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : Schematic representation of the method for obtaining high purity lactoferrin cationic whey protein isolate.

[0054] Figure 2 : Pressure loss generated by the runoff column and different parameters (MS and MP concentrations, MS and MP flow rates) of pasteurized skim milk passing through it (Example 4)

[0055] Figure 3 : Correlation between the pressure loss generated by the runoff column and different parameters (MS and MP flow rates) of pasteurized skim milk passing through (Example 4)

[0056] Figure 4 : CLHP PI spectrum of component 1 of Example 5 (reversed phase high performance liquid chromatography; C18 column 0.1% TFA in H2O / CH3CN gradient, detection at 280 nm).

[0057] Figure 5 : CLHP SEC spectrum (size exclusion high performance liquid chromatography; TSK G3000PWxl column, CH3CN / H2O / TFA, detection at 210 nm) of component 1 of Example 5. The molecular weight at the top indicates the retention time according to the control protein.

[0058] Figure 6 : CLHP PI spectrum of component 2 of Example 6.

[0059] Figure 7 : CLHP PI spectrum of component 3 of Example 6. DETAILED DESCRIPTION

[0060] Example 1: Small-scale assay using pasteurized skim milk (control)

[0061] 1) Skim milk with an MS of 92 g / L was pasteurized at 73° C. for 20 seconds and then cooled to 6° C. The bovine lactoferrin concentration in the pasteurized skim milk was measured by CLHP SCX (strong cation exchange high performance liquid chromatography; Propac SCX column, 20 mM phosphate buffer NaCl gradient, detection at 280 nm);

[0062] 2) Pasteurized skim milk was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose BigBeads at a linear velocity of 400 cm / h. The volume of the pasteurized skim milk was variable.

[0063] 3) After washing with 5 BV of permeate water, the bound protein was eluted with 6 BV of 10% (w / v) NaCl solution at 20°C;

[0064] 4) by CLHP PI (reverse phase high performance liquid chromatography; C18 column The bovine lactoferrin content in each eluate was measured using a 0.1% TFA / CH3CN gradient and detection at 280 nm. Thus, the amount of bovine lactoferrin in each eluate was obtained.

[0065] The measurement conditions and results are shown in Table 1:

[0066] Table 1 - Bovine lactoferrin obtained by passing 92 g / L of skim milk

[0067]

[0068] Example 2: Small-scale assay using concentrated pasteurized skim milk

[0069] 1) Bovine milk was skimmed, then pasteurized at 73°C for 20 seconds, and then cooled to 6°C. The pasteurized skim milk having a MS of 92 g / L was concentrated to a MS of 130 g / L by reverse osmosis at 6°C. The bovine lactoferrin concentration in the concentrated pasteurized skim milk was measured using CLHP SCX (Propac SCX column, 20 mM phosphate buffer NaCl gradient, detection at 280 nm);

[0070] 2) Concentrated pasteurized skim milk (MS of 130 g / L) was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a variable linear velocity; the volume of pasteurized skim milk was variable;

[0071] 3) After washing with 5 BV of permeate water, the bound protein was eluted with 5 BV of 10% (w / v) NaCl solution at 20°C;

[0072] 4) Through CLHP PI (C18 column The bovine lactoferrin content of each eluate was measured using a 0.1% TFA / CH3CN gradient and detection at 280 nm. Thus, the amount of bovine lactoferrin in each eluate was obtained.

[0073] The measurement conditions and results are shown in Table 2:

[0074] Table 2 - Bovine lactoferrin obtained by passing 130 g / L of concentrated skim milk

[0075]

[0076]

[0077] *Equivalent to the value of pasteurized non-concentrated skim milk (MS is 92 g / L).

[0078] Comparison of Tables 1 and 2 shows that using comparable binding conditions (e.g., passing the equivalent of approximately 300 BV of pasteurized skim milk and a MS flow rate of 37-39 g / cm 2 / h), the yield of bovine lactoferrin obtained with previously concentrated pasteurized skim milk was much higher (>20%).

[0079] Example 3: Small-scale assay using concentrated pasteurized skim milk

[0080] 1) Bovine milk was skimmed, then pasteurized at 73°C for 20 seconds, and then cooled to 6°C. The pasteurized skim milk having a MS of 92 g / L was concentrated to a MS of 130 g / L by reverse osmosis at 6°C. The bovine lactoferrin concentration in the concentrated pasteurized skim milk was measured using CLHP SCX (Propac SCX column, 20 mM phosphate buffer NaCl gradient, detection at 280 nm);

[0081] 2) Concentrated pasteurized skim milk (MS of 130 g / L) was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a variable linear velocity; the volume of pasteurized skim milk was variable;

[0082] 3) After rinsing with 5 BV of permeate water, the bound proteins were partially eluted with 6 BV of 2.6% (w / v) NaCl solution at 38 mS / cm at 20°C. Lactoperoxidase, ribonuclease, and other basic proteins were recovered from the eluate.

[0083] 4) Elute the remaining bound protein with 5 BV of 10% (w / v) NaCl solution at 20°C. Recover bovine lactoferrin from the eluate;

[0084] 5) Through CLHP PI (C18 column The ratio of lactoferrin in the total protein in the second eluate, ie, the relative area of ​​the bovine lactoferrin peak, was determined using a 0.1% TFA H2O / CH3CN solution gradient, detected at 280 nm.

[0085] The cobalamin (vitamin B12) content of the second eluate was also determined by the AOAC method. Thus, its total protein content was obtained.

[0086] The conditions and results of the two series of measurements are shown in Table 3:

[0087] Table 3 - Bovine lactoferrin obtained in the second eluate by passing 130 g / L of concentrated skim milk

[0088]

[0089] These results show that using appropriate conditions, two fractions of high lactoferrin purity (e.g. >95% total protein) can be obtained by cation exchange chromatography and passing concentrated pasteurized skim milk:

[0090] - fractions with a lactoferrin purity >95% and a vitamin B12 content ≤5 μg / g protein;

[0091] - Fractions with a lactoferrin purity >90% and a vitamin B12 content ≥10 μg / g protein.

[0092] Example 4: Industrial scale determination using pasteurized skim milk (control)

[0093] Although pasteurized skim milk concentrated to about 130 g / L can be passed through an axial flow column on a small scale, it is difficult to envision the passage of complex matrices (such as dairy materials, especially concentrated dairy materials) through the column in a stable and long-term manner on an industrial scale due to the large pressure loss and clogging of the filter surface.

[0094] We examined the behavior of pressure loss by passing skim milk of different MS through an industrial runoff column at different flow rates.

[0095] 1) A 260 L industrial flow-through column (Albert Handtmann Armaturenfabrick GmbH) was prepared using 280 L of SP Sepharose Big Beads food-grade resin. The column was regenerated with 10% NaCl, saturated with 1 N NaOH, and finally rinsed with permeate.

[0096] 2) The milk was skimmed and then pasteurized at 73°C for 20 seconds and then cooled to 6°C. A portion of the pasteurized skimmed milk was concentrated by reverse osmosis at 6°C. The compositions of these unconcentrated and concentrated pasteurized skimmed milks were as follows:

[0097] Table 4 - Composition of skim milk starter

[0098]

[0099]

[0100] MS: dry matter; MAT: total nitrogenous matter; MP: protein matter

[0101] 3) After the pasteurized skim milk concentration levels were prepared by in-line mixing, it was passed through the previously prepared flow-through column at a temperature of 10°C at different flow rates.

[0102] The observed compositions of skim milk, flow rates, and pressures are shown in Table 5. The pressure loss (i.e., pressure) generated by the radial column increases with increasing flow rate and mobile phase material concentration ( Figure 2 ) and has a good correlation with the flow rate of MS or MP ( Figure 3 These results show that under conventional industrial production conditions, using appropriate flow rates, this industrial-scale flow-through column allows up to 200 g / L MS or 72 g MP (or 75 g / L MAT) of concentrated pasteurized skim milk to pass through (by reverse osmosis) with acceptable pressure losses.

[0103] Table 5 - Skim milk composition, flow rate and pressure observed using an industrial runoff column

[0104]

[0105]

[0106]

[0107] Example 5: Industrial Assay for the Production of Pure Bovine Lactoferrin Whey Isolate from Concentrated Pasteurized Skim Milk

[0108] 1) The milk was defatted, then pasteurized at 73°C for 20 seconds, cooled to 6°C, and then concentrated by reverse osmosis at 6°C to a MS of 128 g / L;

[0109] 2) Make 80m 3 The concentrated pasteurized skim milk was passed through a 260 L industrial flow-through column (Albert Handtmann Armaturenfabrick GmbH) filled with 280 L SP Sepharose Big Beads food grade resin at a flow rate of 2.6 m / h;

[0110] 3) After rinsing with 5 BV of permeate water, the bound proteins were partially eluted with 6 BV of 38 mS / cm NaCl solution at 20°C. Lactoperoxidase, ribonuclease, and other basic proteins were recovered from this eluate;

[0111] 4) Elute the still bound protein with 4 BV of 10% (w / v) NaCl solution at 20°C. Allow the eluate containing bovine lactoferrin to cool and store at 6°C;

[0112] 5) Repeat steps 2-4 10 times;

[0113] 6) Concentrate 11.2 mM on an ultrafilter (organic spiral membrane with MWCO of 20 kDa) 3 The second mixed eluate was then diafiltered on a UF (MWCO of 20 kDa) with permeate water as low as 1 mS / cm and finally microfiltered on a 1.4 μm double-layer ceramic membrane ( Pall Corporation);

[0114] 7) spray drying the whey protein isolate rich in bovine lactoferrin obtained in the form of microfiltrate to obtain 40 kg of powder (component 1);

[0115] 8) Component 1 was analyzed; in particular, by CLHP PI (C18 column The ratio of lactoferrin in total protein, i.e. the relative peak area of ​​bovine lactoferrin ( Figure 5 The cobalamin (vitamin B12) content in the second eluate was also determined by the AOAC method. The analytical results are shown in Table 6.

[0116] Example 6: Industrial Assay for the Production of Pure Bovine Lactoferrin Whey Isolate from Concentrated Pasteurized Skim Milk

[0117] 1) The milk was defatted, then pasteurized at 73°C for 20 seconds, cooled to 6°C, and then concentrated by reverse osmosis at 6°C to a MS of 120 g / L;

[0118] 2) Make 60m 3 The concentrated pasteurized skim milk was passed through a 260 L flow-through industrial column (Albert Handtmann Armaturenfabrick GmbH) filled with 280 L SP Sepharose Big Beads food grade resin at a flow rate of 2.6 m / h;

[0119] 3) After rinsing with 5 BV of permeate water, the bound proteins were partially eluted with 6 BV of 36 mS / cm NaCl solution at 20°C. Lactoperoxidase, ribonuclease, and other basic proteins were recovered from this eluate;

[0120] 4) Elute the still bound protein with 4 BV of 10% (w / v) NaCl solution at 20°C. Allow the eluate containing bovine lactoferrin to cool and store at 6°C;

[0121] 5) Repeat steps 2-4 15 times;

[0122] 6) Concentrate 116.8 mM on an ultrafilter (organic spiral membrane with a cut-off threshold (MWCO) of 20 kDa) 3 The second mixed eluate was then diafiltered on a UF (MWCO of 20 kDa) with permeate water as low as 1 mS / cm and finally microfiltered on a 0.8 μm double-layer ceramic membrane ( Pall Corporation);

[0123] 7) The whey protein isolate enriched in bovine lactoferrin obtained in the form of microfiltrate is subjected to the following additional treatments to ensure the stability of this protein fraction:

[0124] i. A portion of the microfiltrate was filtered through a 0.2 μm PES membrane ( Pall) and then placed in sterile 1L bottles (component 3)

[0125] ii. The remaining microfiltrate was spray-dried to obtain 60 kg of powder (component 2).

[0126] 8) Analyze components 2 and 3; in particular, by CLHP PI (C18 column The ratio of lactoferrin in the total protein in the second eluate, i.e., the relative peak area of ​​bovine lactoferrin ( Figure 6 and Figure 7 The cobalamin (vitamin B12) content in the second eluate was also determined by the AOAC method. The analytical results are shown in Table 6.

[0127] Table 6 - Physicochemical and microbiological characteristics of ingredients 1, 2 and 3

[0128]

[0129] Example 7: Small-scale assay using concentrated whey from pasteurized skim goat milk

[0130] 1) Goat milk was defatted, then pasteurized at 74°C for 30 seconds, and then cooled to 6°C;

[0131] 2) 3000L pasteurized skim goat milk was kept at 50℃ for 30 minutes and then passed through a 0.1μm ceramic microfilter ( Pall Corporation) to obtain whey as a microfiltrate of goat milk free of fat and casein micelles;

[0132] 3) 2000 L of whey from goat milk were concentrated on an ultrafilter (organic spiral membrane with a cut-off threshold (MWCO) of 10 kDa). The composition of the resulting retentate (450 L) is given in Table 7 below:

[0133] Table 7 - Composition of concentrated whey from goat milk

[0134]

[0135] The concentrations of goat β-lactoglobulin and goat α-lactalbumin in the concentrated whey were measured by CLHP SEC (TSK G3000PWxl column, CH3CN / H2O / TFA, detection at 210 nm). The concentration of goat lactoferrin in the concentrated whey was measured by CLHP SCX (Propac SCX column, 20 mM phosphate buffer NaCl gradient, detection at 280 nm).

[0136] 4) 3 L of concentrated goat whey was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at linear velocities of 200 and 300 cm / h;

[0137] 5) After rinsing with 5 BV of permeate water, the bound proteins were partially eluted with 6 BV of 2.2% (w / v) NaCl solution at 20°C. Cationic proteins other than lactoferrin, such as lactoperoxidase, were recovered from the eluate;

[0138] 6) Elute the still bound protein with 5 BV of 10% (w / v) NaCl solution at 20°C. Recover goat lactoferrin from the eluate. The goat lactoferrin content in the eluate was measured using a 0.1% TFA in H2O / CH3CN gradient, detected at 280 nm.

[0139] As shown in Table 8, a goat lactoferrin fraction having a very high protein purity was very efficiently extracted from the concentrated whey from goat milk.

[0140] Table 8 - Goat lactoferrin obtained in the second eluate by passing concentrated whey from goat milk

[0141]

[0142] Example 8: Small-scale assay using concentrated cheese whey from pasteurized skim goat milk

[0143] 1) 240 L of cheese whey from pasteurized goat milk were concentrated on an ultrafilter (organic spiral membrane with a MWCO of 10 kDa) (at 74° C. for 30 seconds). The composition of the retentate obtained (60 L) is given in the table below:

[0144] Table 9 - Composition of concentrated cheese whey from goat milk

[0145]

[0146] The concentrations of goat β-lactoglobulin and goat α-lactalbumin in the concentrated whey were measured by CLHP SEC (TSK G3000PWxl column, CH3CN / H2O / TFA, detection at 210 nm). The concentration of goat lactoferrin in the concentrated whey was measured by CLHP SCX (Propac SCX column, 20 mM NaPB / NaCl gradient, detection at 280 nm).

[0147] 2) 3 L of concentrated goat whey was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at linear velocities of 200 and 300 cm / h;

[0148] 3) After rinsing with 6 BV of permeate water, the bound proteins were partially eluted with 6 BV of 2.2% (w / v) NaCl solution at 20°C. Cationic proteins other than lactoferrin, such as lactoperoxidase, were recovered from the eluate;

[0149] 4) Elute the still bound protein with 5 BV of 10% (w / v) NaCl solution at 20°C. Recover goat lactoferrin from the eluate. The goat lactoferrin content in the eluate was measured using a 0.1% TFA in H2O / CH3CN gradient, detected at 280 nm.

[0150] As shown in Table 10, a goat lactoferrin fraction having a high protein purity was very efficiently extracted from concentrated cheese whey from goat milk.

[0151] Table 10 - Bovine lactoferrin obtained in the second eluate by passing concentrated cheese whey from goat milk

[0152]

[0153] Example 9: Preparation and determination of infant milk powder supplemented with bovine lactoferrin (low vitamin B12 content)

[0154] 1) A cow's milk-based infant formula was prepared by a standard manufacturing method and formulated with the following: skimmed cow's milk, lactose, maltodextrin, oleic sunflower oil, anhydrous milk fat, demineralized whey, soluble protein, galacto-oligosaccharides, sunflower oil, rapeseed oil, soy lecithin, sunflower lecithin, calcium phosphate, fish oil, potassium phosphate, Mortierella alpina oil, choline bitartrate, calcium chloride, potassium citrate, magnesium chloride Sodium, Fructooligosaccharides, Vitamin C, Ferric Pyrophosphate, Calcium Carbonate, Taurine, Potassium Hydroxide, Potassium Chloride, Inositol, Nucleotides, L-Phenylalanine, Tocopherol-Rich Extract, L-Ascorbyl Palmitate, Zinc Sulfate, L-Tryptophan, Vitamin E, Potassium Iodide, L-Carnitine, Niacinamide, Sodium Selenite, Calcium Pantothenate, Copper Sulfate, Thiamine, Vitamin A, Vitamin B6, Manganese Sulfate, Folic Acid, Vitamin K, Biotin, Vitamin D, Riboflavin, Vitamin B12.

[0155] 2) Infant milk powder was mixed with ingredient 1 at a blending rate of 82 mg / 100 g.

[0156] Table 11 - Composition of infant formula incorporating ingredient 1

[0157]

[0158]

[0159]

[0160] Example 10: Determination of powdered nutritional formula milk supplemented with bovine lactoferrin (high vitamin B12 content)

[0161] 1) A cow's milk-based infant formula (food for special medical purposes, i.e., DADFMS) was prepared by standard manufacturing methods and formulated with the following: skim milk, vegetable oils (palm oil, rapeseed oil, coconut oil, sunflower oil), demineralized soluble protein, lactose, starch, locust bean flour, lecithin, calcium citrate, fish oil, Mortierella alpina oil, calcium carbonate, vitamin C, calcium phosphate, potassium citrate, sodium citrate, calcium hydroxide, choline chloride, taurine, vitamin E, inositol, ferrous sulfate, L-tryptophan, potassium chloride, calcium chloride, tocopherol-rich extract, L-ascorbyl palmitate, L-carnitine, magnesium sulfate, nucleotides, zinc sulfate, vitamin A, niacinamide, vitamin K, vitamin D, calcium pantothenate, copper sulfate, thiamine, vitamin B6, riboflavin, manganese sulfate, folic acid, potassium iodide, sodium selenite, and biotin.

[0162] 2) DADFMS infant formula was mixed with ingredient 2 at an incorporation rate of 400 mg / 100 g. By incorporating ingredient 2, an intake of 3.1 μg of vitamin B12 in complex with transcobalamin protein was achieved per 100 g of powdered formula.

[0163] Table 12 - Composition of DADFMS powder incorporated with ingredient 2

[0164]

[0165]

[0166] Example 11: Determination of powdered nutritional formula milk supplemented with bovine lactoferrin (high vitamin B12 content)

[0167] 1) A bovine milk-based liquid infant formula (food for special medical purposes, DADFMS) was prepared by standard manufacturing methods and formulated with the following: skim milk, demineralized soluble protein, vegetable oils (palm oil, palm kernel oil, rapeseed oil, sunflower oil), lactose, soy lecithin, sunflower lecithin, sodium citrate, calcium phosphate, potassium citrate, calcium chloride, calcium carbonate, vitamin C, Mortierella alpina oil, fish oil, calcium hydroxide, potassium chloride, vitamin E, choline chloride, taurine, ferrous sulfate, tocopherol-rich extract, L-ascorbyl palmitate, inositol, zinc sulfate, nucleotides, L-carnitine, niacinamide, vitamin A, magnesium sulfate, vitamin K, vitamin D, calcium pantothenate, copper sulfate, thiamine, vitamin B6, riboflavin, manganese sulfate, folic acid, potassium iodide, sodium selenite, and biotin.

[0168] 2) Liquid DADFMS infant milk was sterilized by ultra-high temperature (UHT) heat treatment and then mixed with ingredient 3 at an incorporation rate of 410 mg / 100 g (dry matter). By incorporating ingredient 3, an intake of 0.43 μg of vitamin B12 complexed with transcobalamin protein was achieved per 100 mL of the liquid formulation.

[0169] Table 13 - Composition of liquid DADFMS spiked with ingredient 3

[0170]

[0171]

[0172] Example 12: Preparation of capsule food supplement using bovine lactoferrin (high vitamin B12 content)

[0173] A capsule food supplement was prepared from a mixture of ingredient 2 (99.5% of the mixture) and colloidal silicon dioxide (0.5% of the mixture) of Example 5. Each capsule contained 200 mg of protein.

[0174] The content of vitamin B12 in complex with transcobalamin protein is 1.6 μg per capsule. The recommended daily dose for each population is as follows:

[0175] Table 14

[0176] crowd Daily dose Vitamin B12 complex forms bovine lactoferrin Children aged 4 to 10 years 1 capsule 1.6 μg 189mg Teens aged 11 to 17 2 capsules 3.2 μg 379mg aldult 3 capsules 4.8 μg 568mg Pregnant / breastfeeding women 4 capsules 5.6 μg 757mg

Claims

1. A method for preparing a cationic whey protein isolate, comprising the following steps a) to f): a) the starting material is mammalian skim milk or whey from mammalian milk, which has been previously concentrated by membrane technology so that the concentration of proteinaceous matter MP is between 40 and 72 g / L when said starting material is prepared from mammalian skim milk and between 20 and 100 g / L when said starting material is prepared from whey; b) selectively extracting cationic proteins comprising the following steps: i. passing the starting material through a flow-through chromatography column containing a sulfopropyl SP type strong cation exchange resin having a diameter greater than 100 μm: - the volume of the starting material corresponding to the unconcentrated volume is between 40 and 500 times the resin volume BV; - the linear velocity of the starting material is between 1.0 and 4.0 m / h; ii. Rinse with softened water: - the volume of demineralized water is between 2 and 6 BV; - Softened water flow rate between 3.0 and 5.0 m / h; iii. Elute the bound cationic protein with a salt solution having a conductivity between 30 and 50 mS / cm: - The volume of saline solution is between 4 and 8 BV; - the passage speed of the salt solution is between 0.3 and 2.0 m / h; iv. Elute the bound cationic protein with a saline solution having a conductivity of 80 to 140 mS / cm: - The volume of saline solution is between 3 and 6 BV; - the passage speed of the salt solution is between 0.5 and 2.5 m / h; c) concentrating the high-purity lactoferrin cationic protein eluted in the saline solution using an ultrafiltration membrane with a cut-off threshold between 10 and 20 kDa; d) desalting the high-purity lactoferrin cationic protein by diafiltration against demineralized water using an ultrafiltration membrane with a cut-off threshold between 10 and 20 kDa to achieve an ash / protein ratio between 0.001 and 0.03; e) microfiltration of concentrated solutions of specific cationic proteins using membranes with a cut-off threshold between 0.2 and 1.4 μm to reduce the microbial load; f) Optionally, spray drying or freeze drying the previously microfiltered concentrated solution of the specific cationic protein to obtain a powdered lactoferrin isolate.

2. The cationic whey protein isolate obtained by the method according to claim 1, characterized in that The proportion of protein in the dry matter is greater than or equal to 90% by weight, and the proportion of lactoferrin in the total protein is greater than 98% by weight.

3. The cationic whey protein isolate obtained from cow's milk or goat's milk according to the method of claim 1, characterized in that The proportion of lactoferrin in the total protein is greater than 95% by weight and contains cobalamin in a complexed form with transcobalamin protein at a concentration of less than or equal to 5 μg / g protein.

4. The cationic whey protein isolate obtained from cow's milk or goat's milk according to the method of claim 1, characterized in that The proportion of lactoferrin in the total protein is greater than 90% by weight and contains cobalamin in a complexed form with transcobalamin protein at a concentration greater than or equal to 5 μg / g protein.

5. The cationic whey protein isolate according to claim 4, which is used for preventing and / or treating vitamin B12 deficiency in patients who have undergone gastrectomy or who have received long-term treatment with a proton pump inhibitor IPP.

6. A food product for human or animal consumption comprising the isolate according to any one of claims 2 to 4.

7. A non-food product comprising the isolate according to any one of claims 2 to 4.

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