Process for producing a lactoferrin-containing product

Abstract

The invention relates to a process for producing a lactoferrin-containing product by cation exchange using milk with a lactoferrin concentration of at least 4.0 per gram protein. The invention also relates to a lactoferrin-containing product containing at least 96 wt% lactoferrin and less than 0.60 wt% immunoglobulins (IgG+IgA+IgM), based on total protein.

Description

[0001] PROCESS FOR PRODUCING A LACTOFERRIN-CONTAINING PRODUCT

[0002] The present invention relates to a process for producing a lactoferrin-containing product and a lactoferrin-containing product with a high lactoferrin content and a high purity.

[0003] Lactoferrin is a protein that is present in both human milk and cow’s milk. Although it is present in relatively small amounts, it plays a vital role in regulating iron metabolism, immune functions, and embryonic development. In addition, it has anti-microbial activity against a range of pathogens.

[0004] In view of these important functions, lactoferrin is added to infant formula, (dairy-based) nutraceuticals, and oral care products.

[0005] Lactoferrin can be isolated from bovine milk. The main proteins present in bovine milk are caseins and whey proteins. The main whey proteins are beta-lactoglobulin, alphalactalbumin, and bovine serum albumin. Other whey proteins include immunoglobulins (IgG, IgA, IgM), lactoperoxidase, angiogenin (RNase 5), RNase 4, and lactoferrin. In the winter season, the average mature bovine milk has a total protein content (determined as total nitrogen content according to the Kjedahl method times 6.38) of about 36.5 g / L and a lactoferrin content of about 135 mg / L, resulting in a concentration of about 3.6 mg per gram total protein. During summer, the lactoferrin content is generally lower, around 3.2 mg per gram total protein.

[0006] Lactoferrin can be isolated from (un)pasteurized skim milk or whey using chromatography. Types of chromatography that have been applied are ion exchange chromatography, hydrophobic interaction chromatography, metal chelation chromatography, and affinity chromatography. Commercial production is generally performed by cation exchange chromatography at about neutral pH. Lactoferrin binds to the resin and is subsequently eluted by an increasing salt buffer. The salts can subsequently be removed from the lactoferrin product by conventional techniques, like membrane filtration, ion-exchange, and / or electrodialysis.

[0007] The object of an isolation or purification method is generally the provision of the target compounds in a purity as high as possible. The main impurities in isolated lactoferrin are lactoperoxidase, angiogenin (RNase 5), and RNase 4. Another important impurity is immunoglobulin (Ig), and in particular IgM. The total Ig content - defined as the total content of IgG, IgM, and IgA - in conventional lactoferrin products is generally about 0.76 wt%, based on total protein. The IgM content in conventional lactoferrin products is generally about 0.40 wt% IgM, based on total protein. Immunoglobulins may tend to aggregate during application of the lactoferrin. This could potentially negatively affect the product properties and the further processing of the lactoferrin or products containing said lactoferrin.

[0008] It is therefore an object of the present invention to provide a high purity lactoferrin product with a relatively low immunoglobulin content

[0009] A further compound that is conventionally found in purified lactoferrin is vitamin B12. The concentration of this vitamin is much lower than that of immunoglobulins; it generally varies in the range 0-5 mg per kg dry matter.

[0010] Vitamin B12 exists in different forms: cyanocobalamin, methylcobalamin, hydroxocobalamin and adenosylcobalamin.

[0011] The synthetic form of vitamin B12 is cyanocobalamin. This form, wherein a cyanide anion is bound to the cobalt ion of vitamin B12, is easy to crystallize and purify. The cyanide group is removed in the human body. It is this synthetic form of vitamin B12 that is currently added to infant formula in order to reach the legally required vitamin B12 levels.

[0012] Milk of all mammals, including cows and humans, contains the natural forms of vitamin B12: methylcobalamin, hydroxocobalamin, and adenosylcobalamin. A major part of the vitamin B12 content in milk is bound to transcobalamin (TC) - also called the vitamin B12 binding protein - which protects the vitamin B12 from acid degradation in the stomach.

[0013] The metabolic fate of natural and the synthetic forms of vitamin B12 in the human body is not the same. The natural forms are present in human milk and are therefore preferred in products mimicking human milk, such as infant formula. One way of introducing these natural forms into such products is by applying a lactoferrin product with a high vitamin B12 content.

[0014] It is therefore a further object of the present invention to provide a high purity lactoferrin product with a relatively high vitamin B12 content. It has now been found that these objects can be met by the process of the present invention, which relates to the isolation of lactoferrin from milk or whey with a high lactoferrin content. Surprisingly, it has been found that by starting from a milk source with a high lactoferrin content, a purer lactoferrin product could be obtained. Furthermore, it appeared that this product contained a relatively high vitamin B12 content, despite its conventional concentration in the starting milk.

[0015] This process is not only able to provide lactoferrin with a higher purity, it also allows for a higher lactoferrin yield.

[0016] The present invention also relates to lactoferrin products with such improved purity. More in particular, it relates to a lactoferrin-containing product comprising at least 96 wt% lactoferrin and less than 0.60 wt% immunoglobulins (IgG+lgA+IgM), based on total protein. Preferably, the IgM concentration in the lactoferrin-containing product is less than 0.30 wt%. The vitamin B12 content of the lactoferrin-containing product is preferably in the range 1 .5-5.0 mg. This vitamin B12 content refers to the total content of natural vitamin B12, i.e. the total content of methylcobalamin, hydroxocobalamin, and adenosylcobalamin.

[0017] The lactoferrin content of milk, based on total protein, and the lactoferrin content of the lactoferrin-containing product, based on dry weight, are determined by reversed phase HPLC on samples that are purified using a heparin cleanup protocol. In liquid samples, this can be done according to GB 1903.17-2016, issued by the Standards Administration of China (SAC); for liquid samples, this can be done according to Y. Zhang Y, et al., J AO AC I nt. 100 (2017) 133-138.

[0018] The total protein content in milk can be determined by either the well-known standard Kjeldahl method, with a nitrogen to protein conversion factor of 6.38, or by infrared spectroscopy using Milkoscan™ (compliant with ISO 9622:2013). Since the first method is used to calibrate the latter, both methods will result in the same values for the same milk sample.

[0019] The immunoglobulin content, defined as the content of IgG, IgA, plus IgM, and the IgM content of the lactoferrin-containing product according to the invention is determined using the ELISA quantitation set as described by R.L. Valk-Weeber, T. Eshuis-de Ruiter, L. Dijkhuizen, and S.S. van Leeuwen, International Dairy Journal, Volume 110, November 2020, 104814. The vitamin B12 content in milk and in the lactoferrin-containing product can be determined by extracting samples at 100°C using an acetate buffer with potassium cyanide in presence of an internal vitamin B12 standard, purifying and concentrating the extract with an immunoaffinity column, and determining the vitamin B12 content with UPLC-MS / MS, in accordance with GB 5413.14-2010 (issued by the Standards Administration of China) and ISO 20634:2015.

[0020] The lactoferrin content of the lactoferrin-containing product according to the invention is at least 96 wt%, preferably at least 96.5 wt%, more preferably at least 97 wt%, even more preferably at least 97.5 wt%, and most preferably at least 98 wt%, based on dry weight.

[0021] Should the lactoferrin-containing product be heat treated, e.g. pasteurized, this heat treatment is preferably conducted such that the majority of the lactoferrin remains in native state and the denaturation is minimized. Therefore, it is preferred that at least 80 wt%, more preferably at least 85 wt%, even more preferably at least 90 wt%, and most more preferably at least 95 wt% of the lactoferrin-containing product consists of native lactoferrin; all based on total protein.

[0022] This native lactoferrin content can be determined by the Mono S method described by J. Billakanti et al., Int. Dairy J. 99 (2019) 104546.

[0023] The immunoglobulin content of the lactoferrin-containing product according to the invention is less than 0.60 wt%, preferably less than 0.50 wt%, even more preferably less than 0.40 wt%, and most preferably less than 0.30 wt%, based on total protein.

[0024] The IgM content of the lactoferrin-containing product according to the invention is less than 0.30 wt%, preferably less than 0.25 wt%, and most preferably less than 0.20 wt%, based on total protein.

[0025] The vitamin B12 content of the lactoferrin-containing product according to the invention is preferably in the range 1.5-5.0 mg per kg dry matter, more preferably 2.0-4.0 mg per kg dry matter, and most preferably 2.0-3.0 mg per kg dry matter.

[0026] The present invention further relates to a process for obtaining a lactoferrin product, said process comprising the steps of:

[0027] - passing milk or whey through at least one column comprising a bed of cation exchange resin, thereby selectively binding lactoferrin on said resin, and - eluting the lactoferrin from the resin by passing a salt solution with increasing concentration over said column(s), wherein the milk has a lactoferrin concentration of at least 4.0 mg per gram protein, preferably at least 4.5 mg per gram protein, more preferably at least 5.0 mg per gram protein, even more preferably at least 5.5 mg per gram protein, and most preferably at least 6.0 mg per gram protein, and said whey has been obtained from milk having a lactoferrin concentration in the range of at least 4.0 mg per gram protein, preferably at least 4.5 mg per gram protein, more preferably at least 5.0 mg per gram protein, even more preferably at least 5.5 mg per gram protein, and most preferably at least 6.0 mg per gram protein.

[0028] The milk that is passed over the column, or that is used to make the whey that is passed over the column, is mature milk of healthy cows.

[0029] Mature milk is defined as milk obtained at least 4 days after calving. Milk up to 3 days after calving is defined as colostrum. Although colostrum is much higher in lactoferrin than mature milk, it is not an option to use colostrum instead of or in admixture with mature bovine milk. First of all, the composition of colostrum (e.g. its high concentration of whey proteins) is such that it negatively affects various industrial dairy processing steps: it tends to precipitate on the surface of heat exchangers and evaporators, causing problems in their cleaning and maintenance. In addition, the produced quantities of colostrum are only a fraction of that of normal milk and colostrum is normally not collected from farms. So, using colostrum to adjust lactoferrin levels would cause a significant price increase.

[0030] Milk from healthy cows is defined as milk with a somatic cell count (SSC) below 200,000 cells / mL, preferably below 150,000 cells / mL, and most preferably below 100,000 cells / mL as determined by microscopy according to ISO 13366-1 :1977 [Milk - Enumeration of somatic cells - Part 1 : Microscopic method (Reference method); International Organization for Standardization (ISO), Geneva, Switzerland], Milk with such low cell count implies that the cows were not suffering from mastitis. Mastitis results in an increase in lactoferrin in the milk; but the milk has low quality.

[0031] There are different ways to obtain such mature milk from healthy cows with a lactoferrin content of at least 4.0 mg per gram protein. According to one embodiment, the milk is obtained by collecting milk from cows over 200 days, preferably over 250 days, and most preferably over 300 days in lactation.

[0032] According to another embodiment, the milk is obtained by collecting milk from cows with a parity of at least 3, preferably at least 4, more preferably at least 5.

[0033] According to a further embodiment, the milk is obtained by collecting milk from cows which genetically produce milk with a relatively high lactoferrin concentration.

[0034] According to a further embodiment, the milk is obtained by collecting milk from cows that have been milked only once a day.

[0035] According to a further embodiment, the milk is obtained by collecting milk from cows that yield less than 25 kg of milk per day.

[0036] It is also possible to apply a blend of (i) milk with a high lactoferrin content, such as milk collected from specifically selected cows according to the above embodiments, with (ii) milk with a regular lactoferrin content, provided that the lactoferrin content of the blend is of at least 4.0 mg per gram protein, preferably at least 4.5 mg per gram protein, more preferably at least 5.0 mg per gram protein, even more preferably at least 5.5 mg per gram protein, and most preferably at least 6.0 mg per gram protein.

[0037] Before isolating the lactoferrin from the milk as defined above, it is preferably skimmed to remove fat. Skimming of the milk can be performed by conventional techniques.

[0038] Alternatively, the milk is used to create whey. This whey can be obtained by cheese making (resulting in cheese whey), by acidification to obtain acid casein and so-called acid whey, and by microfiltration t obtain so-called ideal whey.

[0039] In order to ensure the nativity of the lactoferrin, the milk and whey are preferably not heat treated, e.g. pasteurized, prior to isolation of the lactoferrin.

[0040] The milk of whey can be clarified in order to remove microbes, particulate matter, and / or remaining fat before submission to the column. This can be done by, for instance, microfiltration with a ceramic membrane with a pore size in the range 0.5-2.0 micrometer. The temperature during this step is preferably in the range 5-18°C or 50- 55°C; the trans membrane pressure is preferably in the range 0.1 -2.5 bar.

[0041] The lactoferrin is isolated from the milk or whey by cation exchange chromatography. The isoelectric point ( I EP) of lactoferrin is about 8.7, while that of casein is around 4.6 and that of most other whey proteins around 5.1 -5.4. This means that lactoferrin is positively charged in raw milk (pH around 6.5-7.0), cheese whey (pH 5.9-6.6), and acid whey (pH 4.3-4.6), while most protein impurities are negatively charged. Lactoferrin will therefore bind to the cation exchange resin, while most other proteins will flow through the column. Exceptions are, e.g., lactoperoxidase (IEP around 9.5), angiogenin (RNase 5), RNase 4, and Vitamin B12 binding protein (IEP around 8.7).

[0042] In view of the similarity of the lEPs of lactoferrin and Vitamin B12 binding protein, it is theorized that the vitamin B12 present in the purified lactoferrin product of the present invention is mainly bound to the binding protein, thereby enabling better absorption by the body.

[0043] There is no need to adjust the pH of the milk or whey or to add salts to the milk or whey prior to their submission to the cation exchange resin. However, if so desired, salts and pH buffers may be added.

[0044] Suitable cation exchange resins for use in the process according to the present invention are strong cation exchange resins, preferably having strongly acidic sulfonic acid-based functional groups such as sulfopropyl or methyl sulfonate, or weak cation exchangers like caboxymethyl groups. Strong cation exchange resins are preferred, as they give full protein binding over the whole pH range. Even more preferred are strong cation exchange resins in the sodium form (i.e. sodium as counter ion).

[0045] The cation exchange resin can be a gel-type or a microporous resin. Macroporous resins are preferred since they have a higher protein binding capacity.

[0046] The resin bead size is preferably in the range 100-300 micrometer, since this allows high liquid flow without too much pressure on the resin.

[0047] An example of a suitable cation exchange resin is SP sepharose big beads.

[0048] Examples of suitable columns are axial and radial flow columns.

[0049] The cation exchange resin can be present in a packed or a settled bed, either compressed or uncompressed.

[0050] Although the milk and whey used in the process of the present invention contains a relatively large amount of lactoferrin, the absolute lactoferrin concentration in a litre of milk is still in the order of about 120 ppm, meaning that large volumes of milk have to be treated in order to obtain significant lactoferrin yields. Therefore, a high flow through the column is desired. The flow through the column is preferably in the range 3-150 bed volumes / hr, more preferably 45-120 bed volumes / hr, and most preferably 60-100 bed volumes / hr. The column is preferably loaded with 150-250, more preferably 160- 200 bed volumes of milk or whey,

[0051] The milk entering the column is preferably at a temperature in the range 50-55°C or 7- 25°C, more preferably 50-55°C.

[0052] The lactoferrin is eluted by flowing an eluent with an increasing salt concentration over the column.

[0053] The salt can be selected from, e.g, NaCI, KCI, or CaC , and is preferably NaCI.

[0054] The salt concentration may increase from 0.1 M at the start up to 2.0 M at the end of the process, preferably from 0.2 M to 1.5 M, and most preferably from 0.3 M to 1 M. Lactoperoxidase will elude at salt concentrations of 0.1 -0.4 M; lactoferrin will elude at salt concentrations of 0.5-2.0M.

[0055] The eluent preferably flows through the column with a rate of 5-20, more preferably 5- 15 bed volumes per hour.

[0056] The isolated lactoferrin can be de-salted and / or concentrated by conventional techniques, such as ultrafiltration, and / or electrodialysis. Ultrafiltration is preferred. The isolated lactoferrin can be pasteurized either before or after de-salting.

[0057] The lactoferrin can finally be dried, e.g. freeze-dried or spray-dried.

[0058] EXAMPLE

[0059] Mature milk was collected at farm level from cows that had a parity of at least 3 and were at least 250 days in lactation.

[0060] This milk had a lactoferrin concentration of 8.3 mg / g protein and is herein referred as Lf-enriched milk.

[0061] A conventional milk from a very large pool of cows not selected on parity or lactation was used as reference. This milk had a lactoferrin concentration of 3.4 mg / g protein and is herein referred as Reference Milk.

[0062] Blends were made by mixing

[0063] - 20 wt% of the Lf-enriched milk with 80 wt% of the Reference milk (20% enriched milk)

[0064] - 50 wt% of the Lf-enriched milk with 50 wt% of the Reference milk (50% enriched milk)

[0065] Lactoferrin was isolated from these (blends of) raw whole milk as follows:

[0066] A column with 1 liter of Sepharose big beads in the sodium form was cleaned with 1 M NaCI to ensure all impurities were removed.

[0067] Around 200 bed volumes of milk (blend) were loaded on the column.

[0068] The lactoferrin was eluted using two steps of salt elution, with the first step of < 0.5 M concentration to remove impurities such as lactoperoxidase, and the second step of > 0.5 M to elute Lactoferrin.

[0069] After elution, the eluate was analyzed for lactoferrin content and purity of this lactoferrin. The lactoferrin yield, purity, and Ig content of the resulting eluate is displayed in the following Table:

Claims

CLAIMS1 . Process for producing a lactoferrin-containing product comprising the steps of:- passing milk or whey through at least one column comprising a bed of cation exchange resin, thereby selectively binding lactoferrin on said resin, and- eluting the lactoferrin from the resin by passing a salt solution with increasing concentration over said column(s), wherein the milk has a lactoferrin concentration of at least 4.0 mg per gram protein and said whey has been obtained from milk having a lactoferrin concentration in at least 4.0 mg per gram protein.

2. The process according to claim 1 wherein the milk has a lactoferrin concentration of at least 4.5 mg per gram protein, preferably at least 5.0 mg per gram protein, more preferably at least 5.5 mg per gram protein, and most preferably at least 6.0 mg per gram protein.

3. The process according to claim 1 or 2 wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram protein comprises milk that has been obtained by collecting milk from cows over 200 days, preferably over 250 days, and most preferably over 300 days in lactation.

4. The process according to any one of claims 1 -3 wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram protein comprises milk that has been obtained by collecting milk from cows with a parity of at least 3 preferably at least 4, more preferably at least 5.

5. The process according to any one of claims 1 -4 wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram protein comprises milk that has been obtained by collecting milk from cows which genetically produce milk with a relatively high lactoferrin concentration.

6. The process according to any one of claims 1 -5 wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram protein comprises milk thathas been obtained by collecting milk from cows that have been milked only once a day.

7. The process according to any one of claims 1 -6 wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram protein comprises milk that has been obtained by collecting milk from cows that produce less than 25 kg milk per day.

8. Lactoferrin-containing product containing at least 96 wt% lactoferrin and less than 0.60 wt% immunoglobulins (IgG+lgA+IgM), based on total protein.

9. Lactoferrin-containing product according to claim 8 containing less than 0.50 wt%, preferably less than 0.40 wt%, and most preferably less than 0.30 wt% immunoglobulins (IgG+lgA+IgM), based on total protein.

10. Lactoferrin-containing product according to claim 8 or 9 containing less than 0.30 wt%, preferably less than 0.25 wt%, and most preferably less than 0.20 wt% IgM, based on total protein.

11. Lactoferrin-containing product according to any one of claims 8-10 comprising at least 80 wt%, preferably at least 85 wt%, even more preferably at least 90 wt%, and most more preferably at least 95 wt% native lactoferrin, based on total protein.

12. Lactoferrin-containing product according to any one of claims 8-11 comprising 1 .5- 5.0 mg, preferably 2.0-4.0 mg, more preferably 2.0-3.0 mg vitamin B12 per kg dry matter.

13. Lactoferrin-containing product according to any one of claims 8-12 containing at least 96.5 wt%, preferably at least 97 wt% lactoferrin, based on dry matter.

14. Lactoferrin-containing product according to any one of claims 8-13 obtainable by the process of any one of claims 1-7.

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