Method for producing lactoferrin-containing products

Abstract

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

Description

[0001] The present invention relates to a method for producing a lactoferrin-containing product and a lactoferrin-containing product having a high content of lactoferrin and high purity.

[0002] Lactoferrin is a protein present in both human milk and cow's milk. Although the amount of lactoferrin present is relatively small, it plays a crucial role in regulating iron metabolism, immune function, and embryonic development. In addition, lactoferrin has antimicrobial activity against a range of pathogens.

[0003] Given these important functions, lactoferrin is added to infant formula, (dairy-based) nutritional products, and oral care products.

[0004] Lactoferrin can be isolated from cow's milk. The main proteins present in cow's milk are casein and whey proteins. The main whey proteins are β-lactoglobulin, α-lactalbumin, and bovine serum albumin. Other whey proteins include immunoglobulins (IgG, IgA, IgM), lactoperoxidase, angiogenin (RNase 5), RNase 4, and lactoferrin. In winter, on average, mature cow's milk has a total protein content of about 36.5 g / L (determined as the total nitrogen content multiplied by 6.38 according to the Kjeldahl method) and a lactoferrin content of about 135 mg / L, resulting in a concentration of about 3.6 mg / g of total protein. In summer, the lactoferrin content is typically lower, about 3.2 mg / g of total protein.

[0005] Lactoferrin can be isolated from (un)pasteurized skim milk or whey using chromatography. The types of chromatography that have been applied are ion exchange chromatography, hydrophobic interaction chromatography, metal chelate chromatography, and affinity chromatography. Commercial production is typically carried out by cation exchange chromatography at about neutral pH. Lactoferrin binds to the resin and is subsequently eluted by increasing the salt buffer. These salts can then be removed from the lactoferrin product by conventional techniques such as membrane filtration, ion exchange, and / or electrodialysis.

[0006] The aim of separation or purification methods is usually to provide the target compound with the highest possible purity. The main impurities in isolated lactoferrin are lactoperoxidase, angiogenin (RNase 5), and RNase 4.

[0007] Another important impurity is immunoglobulin (Ig), and particularly IgM. The total Ig content (defined as the total content of IgG, IgM, and IgA) in conventional lactoferrin products is typically about 0.76 wt% based on total protein. The IgM content in conventional lactoferrin products is typically about 0.40 wt% IgM based on total protein. During the application of lactoferrin, immunoglobulins may tend to aggregate. This may potentially have a negative impact on product properties and on the further processing of lactoferrin or products containing said lactoferrin.

[0008] Accordingly, an object of the present invention is to provide a high-purity lactoferrin product having a relatively low content of immunoglobulins.

[0009] Another compound commonly found in purified lactoferrin is vitamin B12.

[0010] The concentration of this vitamin is much lower than that of immunoglobulins; it usually varies in the range of 0 - 5 mg / kg dry matter.

[0011] Vitamin B12 exists in different forms: cyanocobalamin, methylcobalamin, hydroxocobalamin, and adenosylcobalamin.

[0012] The synthetic form of vitamin B12 is cyanocobalamin. This form (wherein the 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. Currently, this synthetic form of vitamin B12 is added to infant formula to achieve the legally required vitamin B12 level.

[0013] The milk of all mammals (including cows and humans) contains natural forms of vitamin B12: methylcobalamin, hydroxocobalamin, and adenosylcobalamin. The major part of the vitamin B12 content in milk is bound to transcobalamin (TC) (also known as vitamin B12-binding protein), thus protecting vitamin B12 from acid degradation in the stomach.

[0014] The metabolic fates of natural and synthetic forms of vitamin B12 in the human body are different. Since natural forms of vitamin B12 are present in human milk, they are preferred in products that mimic human milk, such as infant formula. One way to introduce these natural forms of vitamin B12 into such products is through the application of lactoferrin products having a high content of vitamin B12.

[0015] Accordingly, another object of the present invention is to provide a high-purity lactoferrin product having a relatively high content of vitamin B12.

[0016] It has now been found that these objects can be achieved by the method of the present invention, which involves separating lactoferrin from milk or whey having a high content of lactoferrin. Surprisingly, it has been found that starting from a milk source having a high content of lactoferrin, a purer lactoferrin product can be obtained. In addition, although the vitamin B12 in the starting milk is at a conventional concentration, the product appears to contain a relatively high content of vitamin B12.

[0017] This method is not only capable of providing lactoferrin with higher purity, but also allows for a higher lactoferrin yield.

[0018] The present invention also relates to lactoferrin products having such improved purity. More particularly, the present invention relates to a lactoferrin-containing product comprising at least 96 wt% lactoferrin based on total protein and less than 0.60 wt% immunoglobulins (IgG + IgA + IgM). 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 of 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.

[0019] The lactoferrin content based on total protein in milk and the lactoferrin content based on dry weight in the lactoferrin-containing product were determined by reverse-phase HPLC for samples purified using a heparin clearance protocol. In liquid samples, this can be carried out according to GB 1903.17 - 2016 issued by the Standards Administration of China (SAC); for liquid samples, this can be carried out according to Y. Zhang Y et al., JAOAC Int. [Journal of AOAC International] 100 (2017) 133 - 138.

[0020] The total protein content in milk can be determined by the well-known standard Kjeldahl method (where the nitrogen to protein conversion factor is 6.38) or by infrared spectroscopy using Milkoscan TM in accordance with ISO 9622:2013. Since the first method is used to calibrate the latter, for the same milk sample, both methods will give the same value.

[0021] The immunoglobulin content (defined as the content of IgG, IgA plus IgM) and the IgM content of the lactoferrin-containing product according to the present invention were determined using an ELISA quantification device as described below: R.L. Valk-Weebera, T. Eshuis-de Ruiter, L. Dijkhuizen, and S.S. van Leeuwen, International Dairy Journal, Volume 110, November 2020, 104814.

[0022] The vitamin B12 content in milk and the lactoferrin-containing product can be determined by: extracting the sample at 100 °C using an acetate buffer containing potassium cyanide in the presence of a vitamin B12 internal standard, purifying and concentrating the extract using an immunoaffinity column, and determining the vitamin B12 content by UPLC-MS / MS according to GB 5413.14 - 2010 (issued by the Standards Administration of China) and ISO 20634:2015.

[0023] The lactoferrin content of the lactoferrin product according to the present 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.

[0024] If the lactoferrin product is heat-treated (e.g., pasteurized), this heat treatment is preferably carried out such that most of the lactoferrin remains in the native state and denaturation is minimized. Thus, preferably at least 80 wt%, more preferably at least 85 wt%, even more preferably at least 90 wt%, and most preferably at least 95 wt% of the lactoferrin product consists of native lactoferrin; the data are all based on total protein.

[0025] This native lactoferrin content can be determined by the Mono S method as described below: J. Billakanti et al., Int. Dairy J. 99 (2019) 104546.

[0026] Based on total protein, the immunoglobulin content of the lactoferrin product according to the present 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%.

[0027] Based on total protein, the IgM content of the lactoferrin product according to the present invention is less than 0.30 wt%, preferably less than 0.25 wt%, and most preferably less than 0.20 wt%.

[0028] The vitamin B12 content of the lactoferrin product according to the present invention is preferably in the range of 1.5 - 5.0 mg / kg dry matter, more preferably in the range of 2.0 - 4.0 mg / kg dry matter, and most preferably in the range of 2.0 - 3.0 mg / kg dry matter.

[0029] The present invention further relates to a method for obtaining a lactoferrin product, the method comprising the following steps:

[0030] - passing milk or whey through at least one column comprising a bed of cation exchange resin, thereby selectively binding lactoferrin to the resin, and

[0031] - eluting the lactoferrin from the resin by passing a salt solution of increasing concentration through the one or more columns,

[0032] wherein the lactoferrin concentration of the milk is at least 4.0 mg per gram of protein, preferably at least 4.5 mg per gram of protein, more preferably at least 5.0 mg per gram of protein, even more preferably at least 5.5 mg per gram of protein, and most preferably at least 6.0 mg per gram of protein,

[0033] and the whey is obtained from milk having a lactoferrin concentration in the range of at least 4.0 mg per gram of protein, preferably at least 4.5 mg per gram of protein, more preferably at least 5.0 mg per gram of protein, even more preferably at least 5.5 mg per gram of protein, and most preferably at least 6.0 mg per gram of protein.

[0034] The milk passing through the column, or the milk used for manufacturing the whey passing through the column, is mature milk from healthy cows.

[0035] Mature milk is defined as milk obtained at least 4 days after calving. Milk within the first 3 days after calving is defined as colostrum. Although the lactoferrin in colostrum is much higher than that in mature milk, it is not possible to choose to use colostrum instead of or mixed with mature cow milk. First, the composition of colostrum (such as its high concentration of whey protein) will have a negative impact on various industrial dairy processing steps: it tends to precipitate on the surfaces of heat exchangers and evaporators, causing problems in their cleaning and maintenance. In addition, the yield of colostrum is only a small fraction of normal milk, and colostrum is usually not collected from farms. Therefore, using colostrum to adjust the lactoferrin level will result in a significant price increase.

[0036] Milk from healthy cows is defined as milk having a somatic cell count (SSC) of less than 200,000 cells / mL, preferably less than 150,000 cells / mL, and most preferably less than 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 a low cell count means that the cows do not have mastitis. Mastitis causes an increase in lactoferrin in milk; however, the milk quality is poor.

[0037] There are different ways to obtain such mature milk with a lactoferrin content of at least 4.0 mg per gram of protein from healthy cows.

[0038] According to one embodiment, the milk is obtained by collecting milk from dairy cows with a lactation period of more than 200 days, preferably more than 250 days, and most preferably more than 300 days.

[0039] According to another embodiment, the milk is obtained by collecting milk from dairy cows with a parity of at least 3, preferably at least 4, more preferably at least 5.

[0040] According to another embodiment, the milk is obtained by collecting milk from dairy cows that genetically produce milk with a relatively high lactoferrin concentration.

[0041] According to another embodiment, the milk is obtained by collecting milk from dairy cows that are milked only once a day.

[0042] According to another embodiment, the milk is obtained by collecting milk from dairy cows that produce less than 25 kg of milk per day.

[0043] It is also possible to apply a blend of (i) milk with a high content of lactoferrin (such as milk collected from specially selected dairy cows according to the above embodiments) and (ii) milk with a conventional content of lactoferrin, provided that the lactoferrin content of the blend is at least 4.0 mg / g of protein, preferably at least 4.5 mg / g of protein, more preferably at least 5.0 mg / g of protein, even more preferably at least 5.5 mg / g of protein, and most preferably at least 6.0 mg / g of protein.

[0044] Before separating lactoferrin from the milk as defined above, it is preferably defatted to remove fat. Defatting of the milk can be carried out by conventional techniques.

[0045] Alternatively, the milk is used to produce whey. Such whey can be obtained by: cheesemaking (producing cheese whey), acidification to obtain acid casein and so-called acid whey, and microfiltration to obtain so-called ideal whey.

[0046] To ensure the naturalness of lactoferrin, the milk and whey are preferably not heat-treated (e.g., pasteurized) before separating lactoferrin.

[0047] The milk of the whey can be clarified before being fed to the column in order to remove microorganisms, particulate matter, and / or residual fat. This can be done, for example, by microfiltration with a ceramic membrane having a pore size range of 0.5 - 2.0 microns. The temperature during this step is preferably in the range of 5°C - 18°C or 50°C - 55°C; the transmembrane pressure is preferably in the range of 0.1 - 2.5 bar.

[0048] Lactoferrin is separated from the milk or whey by cation exchange chromatography.

[0049] The isoelectric point (IEP) of lactoferrin is approximately 8.7, while that of casein is approximately 4.6, and that of most other whey proteins is approximately 5.1 - 5.4. This means that lactoferrin is positively charged in raw milk (pH approximately 6.5 - 7.0), cheese whey (pH 5.9 - 6.6), and acidic whey (pH 4.3 - 4.6), while most protein impurities are negatively charged. Thus, lactoferrin will bind to the cation exchange resin, while most other proteins will flow through the column. Exceptions are, for example, lactoperoxidase (IEP approximately 9.5), angiogenin (RNase 5), RNase 4, and vitamin B12 binding protein (IEP approximately 8.7).

[0050] Given the similarity in the IEP of lactoferrin and vitamin B12 binding protein, it is speculated that the vitamin B12 present in the purified lactoferrin product of the present invention is mainly bound to the binding protein, enabling better absorption by the body.

[0051] Before delivering milk or whey to the cation exchange resin, it is not necessary to adjust the pH of the milk or whey or add salt to the milk or whey. However, if desired, salt and a pH buffer can be added.

[0052] Suitable cation exchange resins for use in the method according to the present invention are strong cation exchange resins, preferably having a strong acidic sulfonic acid group functional group (such as sulfopropyl or methyl sulfonate), or weak cation exchange agents (such as carboxymethyl groups). Strong cation exchange resins are preferred because they provide complete protein binding over the entire pH range. Even more preferred are strong cation exchange resins in the sodium form (i.e., sodium as the counter ion).

[0053] The cation exchange resin can be a gel type or a microporous resin. Macroporous resins are preferred because they have a higher protein binding capacity.

[0054] The resin bead size is preferably in the range of 100 - 300 microns, as this allows for high liquid flow rates without imposing excessive pressure on the resin.

[0055] An example of a suitable cation exchange resin is SP Sepharose Big Beads.

[0056] Examples of suitable columns are axial flow columns and radial flow columns.

[0057] The cation exchange resin can be present in a compressed or uncompressed packed bed or a sedimentation bed.

[0058] Although the milk and whey used in the method of the present invention contain a relatively large amount of lactoferrin, the absolute lactoferrin concentration in one liter of milk is still on the order of about 120 ppm, which means that a large amount of milk must be processed to obtain a significant lactoferrin yield. Therefore, a high flow rate through the column is desired. The flow rate through the column is preferably in the range of 3 - 150 bed volumes per hour, more preferably 45 - 120 bed volumes per hour, and most preferably 60 - 100 bed volumes per hour. The column is preferably loaded with 150 - 250, more preferably 160 - 200 bed volumes of milk or whey.

[0059] The temperature range of the milk entering the column is preferably 50°C - 55°C or 7°C - 25°C, more preferably 50°C - 55°C.

[0060] Lactoferrin is eluted by flowing an eluent with increasing salt concentration through the column.

[0061] The salt can be selected from, for example, NaCl, KCl or CaCl2, and is preferably NaCl.

[0062] The salt concentration can be increased from 0.1 M at the start to 2.0 M at the end of the method, preferably from 0.2 M to 1.5 M, and most preferably from 0.3 M to 1 M. Lactoperoxidase will be eluted at a salt concentration of 0.1 - 0.4 M; lactoferrin will be eluted at a salt concentration of 0.5 - 2.0 M.

[0063] The eluent preferably flows through the column at a rate of 5 - 20, more preferably 5 - 15 bed volumes per hour.

[0064] The separated lactoferrin can be desalted and / or concentrated by conventional techniques such as ultrafiltration and / or electrodialysis. Ultrafiltration is preferred.

[0065] The separated lactoferrin can be pasteurized before or after desalting.

[0066] Finally, the lactoferrin can be dried, for example by freeze-drying or spray-drying.

[0067] Examples

[0068] Mature milk was collected at the farm level from cows with at least 3 parities and a lactation period of at least 250 days.

[0069] This milk has a lactoferrin concentration of 8.3 mg / g protein and is referred to herein as Lf-rich milk.

[0070] Conventional milk from a large number of cows not selected by parity or lactation period was used as a reference.

[0071] This milk has a lactoferrin concentration of 3.4 mg / g protein and is referred to herein as reference milk.

[0072] The blend is prepared by mixing the following:

[0073] - 20 wt% of Lf-rich milk with 80 wt% of reference milk (20% milk-rich)

[0074] - 50 wt% of Lf-rich milk with 50 wt% of reference milk (50% milk-rich)

[0075] Lactoferrin is separated from these raw whole milks (the blends) as follows:

[0076] The column with 1 liter of agarose beads in sodium form is cleaned with 1 M NaCl to ensure removal of all impurities.

[0077] Approximately 200 bed volumes of milk (the blend) are loaded onto the column.

[0078] Lactoferrin is eluted using a two-step salt elution, where the first step concentration < 0.5 M to remove impurities (such as lactoperoxidase), and the second step concentration > 0.5 M to elute lactoferrin.

[0079] After elution, the lactoferrin content and the purity of this lactoferrin in the eluate are analyzed. The lactoferrin yield, purity, and Ig content of the resulting eluate are shown in the following table:

[0080]

Claims

1. A method for producing a lactoferrin-containing product, the method comprising the following steps: - passing milk or whey through at least one column comprising a bed of cation exchange resin, thereby selectively binding lactoferrin to the resin, and - eluting the lactoferrin from the resin by passing a salt solution of increasing concentration through the one or more columns, wherein the lactoferrin concentration of the milk is at least 4.0 mg per gram of protein, and the whey is obtained from milk having a lactoferrin concentration of at least 4.0 mg per gram of protein.

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

3. The method according to claim 1 or 2, wherein the milk having a lactoferrin concentration of at least 4.0 mg per gram of protein comprises milk obtained by collecting milk from dairy cows with a lactation period of more than 200 days, preferably more than 250 days, and most preferably more than 300 days.

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

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

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

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

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

9. The lactoferrin-containing product according to claim 8, wherein the lactoferrin-containing product contains less than 0.50 wt%, preferably less than 0.40 wt%, and most preferably less than 0.30 wt% immunoglobulins (IgG + IgA + IgM) based on total protein.

10. The lactoferrin-containing product according to claim 8 or 9, wherein the lactoferrin-containing product contains 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. The lactoferrin-containing product according to any one of claims 8-10, wherein the lactoferrin-containing product comprises at least 80 wt%, preferably at least 85 wt%, even more preferably at least 90 wt%, and most preferably at least 95 wt% native lactoferrin based on total protein.

12. The lactoferrin-containing product according to any one of claims 8-11, wherein the lactoferrin-containing product comprises 1.5 mg - 5.0 mg, preferably 2.0 mg - 4.0 mg, more preferably 2.0 mg - 3.0 mg of vitamin B12 per kg of dry matter.

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

14. The lactoferrin-containing product according to any one of claims 8-13, wherein the lactoferrin-containing product can be obtained by the method according to any one of claims 1-7.

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