CN110267968B - Purification method of whey protein - Google Patents
Purification method of whey protein Download PDFInfo
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- CN110267968B CN110267968B CN201880012175.0A CN201880012175A CN110267968B CN 110267968 B CN110267968 B CN 110267968B CN 201880012175 A CN201880012175 A CN 201880012175A CN 110267968 B CN110267968 B CN 110267968B
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
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- 108060003951 Immunoglobulin Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
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- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
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- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- 239000008101 lactose Substances 0.000 description 1
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- 239000002244 precipitate Substances 0.000 description 1
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- 238000001742 protein purification Methods 0.000 description 1
- 239000012508 resin bead Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C21/00—Whey; Whey preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Pediatric Medicine (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Dairy Products (AREA)
Abstract
The present invention describes a method for purifying alpha-lactalbumin from whey using a single chromatographic step. The method comprises adjusting the pH of the whey and loading the whey onto a cation exchanger for an extended time/volume, after which high purity alpha-lactalbumin can be eluted from the column. The present method uses only a single chromatographic step to obtain a high purity alpha-lactalbumin eluate and allows further processing of the alpha-lactalbumin depleted whey, for example by adjusting the pH back to the target set point, drying and recovery as a whey product or further processing.
Description
Technical Field
A method of purifying whey protein and alpha-lactalbumin is described. The present method involves separating and purifying alpha-lactalbumin from whey (including different whey types, such as sweet whey, acid whey or ideal whey) during the use of a single chromatographic step to produce high purity alpha-lactalbumin or an alpha-lactalbumin-enriched whey protein isolate wherein the nutritional and functional properties of the protein are retained allowing use such as ingredients in various food products, infant formulas, adult nutritional formulas, sports formulas, medical formulas enteral formulas and other specialty nutritional, health and medical products. In addition, the present method allows further processing of alpha-lactalbumin depleted whey to extract other whey proteins and produce other whey protein products.
Background
Whey is a liquid component of milk and is typically obtained during the manufacture of cheese or casein as a result of separation and clarification from the curd or casein. Whey is composed of water, lactose, salts, residual fat and several proteins. Whey proteins possess interesting nutritional, functional, physiological and pharmaceutical properties and are divided into two main groups: a globulin fraction comprising mainly beta-lactoglobulin and immunoglobulins; and an albumin moiety comprising alpha-lactalbumin and serum albumin.
Alpha-lactalbumin is a globular protein consisting of 123 amino acids, found in both human and bovine milk, for which it has 74% conserved amino acid sequence homology. The alpha-lactalbumin appears as an acidic single chain protein with molecular weights corresponding to 14070 Da (in human milk) and 14178 Da (in cow milk). In humans, alpha-lactalbumin is the dominant whey protein, and constitutes approximately 75% of the total whey protein and 40% of the total protein weight. However, in cattle, alpha-lactalbumin is the second important protein in whey in number, constituting 25% of the total whey protein and 5% of the total protein weight, which is replaced by the predominant protein beta-lactoglobulin present in bovine whey, which is believed to be absent from human milk REF.
Whey proteins have different uses, such as ingredients in various food products, infant formulas, adult nutritional formulas, sports formulas, medical formulas, enteral formulas, and other professional nutritional, health, and pharmaceutical products. Among whey proteins, α -lactalbumin has been shown to exhibit a number of important nutritional and functional characteristics due to its amino acid content and functional properties (Marshall, 2004, k. Lisak Jakopovic et al, 2015).
As there are a variety of commercial applications and products that have used alpha-lactalbumin, and new human health and nutrition applications are being produced, there are several processes for purification of whey protein and alpha-lactalbumin, including ultrafiltration, thermal precipitation, and ion exchange methods.
Ultrafiltration processes use membranes that allow separation of proteins and molecules based on their physical size, allowing proteins and molecules up to a certain size to pass through into the exudate. These methods are effective in achieving separation in the absence of similarly sized proteins or molecules in the feed. However, in order to separate larger and smaller molecules, multiple steps are required. Such a process is described in U.S. patent No. 5,008,376 (bottom) and further described in U.S. patent No. 6,613,377 (Chao Wu), which further employs pH adjustment to below 4 to further increase the effectiveness of the process. US 6,613,377 B2 describes a whey treatment process for the specific enrichment of alpha-lactalbumin. The whey protein product is acidified to a pH below 4, concentrated and then the desired alpha-lactalbumin is precipitated to form a low calcium product.
Thermal precipitation is another method and involves adjusting the pH and heating the whey to denature and precipitate the protein of interest. U.S. Pat. No. 5,455,331 (Pearce) describes the use of this method to produce an alpha-lactalbumin product.
Ion exchange methods involve contacting whey with an anion or cation exchanger to selectively retain the protein fraction. Such a process is described in U.S. patent No. 5,077,067 (Thibault), which selectively removes lactoglobulin from whey; and US 5,756,680 (Sepragen), which applies a continuous process of separating different proteins in whey to other methods, including El-Sayed & Chase 2009 and 2010, which describes a two-step chromatographic process to produce alpha-lactalbumin and beta-lactoglobulin at room temperature to purify alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) from whey concentrate.
The above-described method requires a plurality of steps, a long treatment time, a plurality of buffers or chemical treatments, causes denaturation of proteins, and cannot provide a product with high purity and high yield. There is therefore a need for an improved process which allows improved handling of whey protein purification, especially in the production of alpha-lactalbumin.
Brief description of the invention
The present invention provides a novel method for purifying alpha-lactalbumin (ALA). The present invention describes a method for purifying alpha-lactalbumin to high purity from whey in a single chromatographic step. The process involves adjusting the pH of the whey to a pH of 3.5-4 (which can be achieved with a variety of acids) and adjusting the temperature to-50 ℃, for example 42-55 ℃. The whey is then loaded onto a cation exchanger for an extended time/volume, after which the high purity alpha-lactalbumin can be eluted from the column.
The novel aspects of the process lie in both the simplicity of the process and the manner in which the separation is achieved. The inventors have determined that both alpha-lactalbumin and beta-lactoglobulin bind to a cation exchanger under the pH conditions described. However, due to the difference in charge and conformation under this condition, α -lactalbumin is slightly inhibited by mass transport limitations, for which β -lactoglobulin is not experienced. This effect means that the cation exchange medium must allow equal access to the available binding sites by physical or kinetic limitations.
The inventors have found that the use of large-sized resin bead cation exchangers facilitates an equal proximity to the cation exchanger at defined contact or retention times. However, it is estimated that other cation exchanger media and forms may facilitate the same capabilities, including but not limited to chromatographic membranes, open beds, and the like.
Another aspect of the invention (facilitating the replacement of bound beta-lactoglobulin) is by extending the time/volume of whey loaded onto the cation exchanger. The combined effect of these allows separation by a single step and enables the displacement effect that occurs as the alpha-lactalbumin displaces the bound beta-lactoglobulin from the cation exchanger.
The method of the invention uses only a single chromatographic step, using only a simple low cost buffer to obtain high purity alpha-lactalbumin eluate with a purity of >80% (range 70-95%) in 75% (range 65-85%). Furthermore, the alpha-lactalbumin depleted whey may be further processed, for example by adjusting the pH back to the target set point, dried and recovered as a whey product or otherwise further processed.
There are several advantages to using the method of the present invention, some of which are listed below:
processing is simple-a single chromatographic step-also means less buffer/less program requirements and shorter processing time (although processing time is somewhat longer).
With simple buffers-providing pH and conductivity conditions are almost met by any acid/base and salts can be used so a lower cost more economical option can be used.
High purity provides a high value product with more versatility, which can be used for more applications, and high yield maximum recovery means less losses in the process, maximum return on investment and maximum material utilization from the feed.
Whey may also be used-requiring additional processing but basically the process takes a low cost product stream and extracts a higher cost product stream, making the lower cost stream still useful.
Accordingly, the present invention relates to a method for purifying whey protein, said method comprising the steps of:
-providing a cation exchanger allowing both alpha-lactalbumin and beta-lactoglobulin to equally access the available binding sites, as well as a complete displacement effect;
-acidifying the whey fraction;
-balancing the cation exchanger;
-loading the whey fraction onto the cation exchanger;
-washing the cation exchanger; and
-eluting whey protein from the cation exchanger.
Preferably, the cation exchanger comprises chromatographic beads having an average bead size diameter of 130-300 μm, preferably about 200 μm. In one embodiment, the cation exchanger is packed in a column operated with radial flow. The chromatographic beads may be of natural or synthetic origin, preferably made of polysaccharides such as cellulose or agarose.
In the process of the invention, the whey is acidified to a pH of 3.5-4.0, preferably pH 3.7. Equilibration and washing can be performed with 0.05-0.2% HAc or just water (but with a larger volume) or weaker acid (but also working with a buffer liquid system, such as citric acid + Na2HPO 4). Preferably, the loading is carried out at a linear speed of 200-1000 cm/and the process is preferably operated at a temperature of 42-55 ℃, preferably about 50 ℃.
The elution is preferably a 1 step elution with 30-200mM, preferably 75mM-125mM NaOH or KOH. The eluate is preferably re-titrated from pH 12 to pH 4.5-8. This is to restore the whey to its pH prior to acid titration, requiring that the remaining whey protein remaining in the flow-through is still used.
The column can be washed in place (CIP) with 1M NaOH/KOH at a high flow rate >800cm/h for >3 hours at 50-60 ℃. The range is 100-800. The initial flow rate is slow to avoid overpressure and a high flow rate is later required to wash away dirt, most likely minerals that were previously covered by protein.
The eluate obtained by the method of the present invention comprises alpha-lactalbumin having a purity of 70-95% (preferably 85-95%) and a yield of more than 65-85% (preferably 75-85%). Other whey proteins, such as beta-lactoglobulin, may be collected in the flow-through.
Brief Description of Drawings
FIG. 1 is a schematic representation of a one-step purification process of the present invention.
FIG. 2 is a chromatogram according to experiment 1 in the experimental section, showing UV absorbance at 280nm during the process of extracting alpha-lactalbumin from pH adjusted whey. It can be observed that the point at which the beta-lactoglobulin starts to be replaced by alpha-lactalbumin is in the range of 400L, at which point the level of beta in the flow-through exceeds the level at which the feed is started.
FIG. 3 is a chromatogram showing ion exchange analysis of the elution pool at 220nm as described in experiment 3 below.
FIG. 4 is a chromatogram showing ion exchange analysis of the elution pool at 280nm as described in experiment 3 below.
Detailed Description
The invention will now be described in more detail in connection with a few non-limiting examples and the accompanying drawings.
An overview of the process of the present invention is depicted in fig. 1.
The described invention relates to a method for treating whey to obtain purified alpha-lactalbumin or enriched alpha-lactalbumin whey protein isolate or fraction. The present invention uses a cation exchanger that promotes equal access to the binding sites for alpha-lactalbumin and beta-lactoglobulin. In the example given, this is achieved by using a large bead cation exchange resin. However, other forms of chromatographic media may be used that allow both alpha-lactalbumin and beta-lactoglobulin to equally access the available binding sites. The cation exchange resin is suitably packed into a column or suitable container. Radial flow columns have been used in the examples provided to allow for high flow rates and reduced processing times. The cation exchanger is prepared for operation by using an appropriate equilibration buffer. In the examples 0.1% acetic acid was used.
Whey, ideal whey, acid whey, whey concentrate or whey isolate (including all whey protein products) is then adjusted to a pH of 3.5-4, with an optimal pH of 3.7. This pH adjustment can be achieved using a variety of acids. Acetic acid, citric acid, phosphoric acid, and HCl are suitable and compatible with the processing environment. This pH adjustment may be achieved in several ways by in-line dilution or by adjustment in a balancing tank or other methods. The amount of acid required to achieve this adjustment can be readily determined by one skilled in the art by titration with the intended feed. At this pH, the proteins present in whey undergo various changes due to the influence of pH, which interfere with the tertiary and quaternary structural elements of the protein structure, causing some proteins to change in function, denature and aggregate. Under these conditions, alpha-lactalbumin appears in the form of a melt sphere (Laureto et al, 2002, r sener and Redfield, 2009).
The pH adjusted whey protein product is then loaded onto a cation exchanger. In the example provided, this is done using a large bead size cation exchange chromatography resin packed in a radial flow column. This may be done by using a positive displacement pump or other means, as long as a consistent residence contact time is maintained. The flow rate required in the examples is the flow rate required to achieve a contact time of 3-6 minutes, preferably about 4 minutes. This is a critical element and depends on the nature of the cation exchanger used. In other embodiments using other cation exchanger forms, different requirements may exist. The time or volume of the loading phase is also a critical aspect of the invention, as this aspect of the process allows the alpha-lactalbumin to displace the bound beta-lactoglobulin and any other bound proteins. Fig. 2 shows that the process of substitution occurs gradually during the loading phase. The inventors have recognised that a longer loading stage will result in a higher purity of alpha-lactalbumin and a shorter loading stage will provide an alpha-lactalbumin enriched whey protein isolate.
Table 1 below shows that as β -lactoglobulin is displaced from the column by α -lactalbumin, the amount of β -lactoglobulin in the flow-through increases during operation and exceeds the amount in the starting material. The alpha-lactalbumin present in the flow-through also increases during operation, however, the conventional penetration profile is not reflected. Analysis of the composite sample of flow-through was analyzed using the method described in experiment 2. The table shows the amount of alpha-lactalbumin relative to beta-lactoglobulin by sample analysis area versus initial feed whey area. Because some UV absorbance values were greater than 2AU, the area was calculated from ml×mau. It can be seen that both alpha-lactalbumin and beta-lactoglobulin initially bind to the ion exchanger, and then after a period of loading, the alpha-lactalbumin displaces the bound beta-lactoglobulin with a substitution or displacement effect. This is seen because the level of beta-lactoglobulin in the flow-through exceeds the level of the initial feed.
TABLE 1
The alpha-lactalbumin level in the whey flow-through may be measured by analysis using analytical anion exchange or other suitable methods. However, after process optimization, the time or volume set point may be used for simplified process operation.
After the loading phase, a wash of any unbound material in the column is performed. This is the standard operation of any chromatographic method. In the example given, 0.1% acetic acid is used depending on the equilibration stage. However, there are a variety of other suitable buffers, including water.
Thereafter, elution of the bound alpha-lactalbumin was performed. The elution of bound alpha-lactalbumin can be replaced by using an elution buffer comprising a suitable level of free cations or a suitable level of conductivity or by changing the pH (with salts, such as NaCL or citrate or PO 4 ) Is achieved by changing the charge of the bound protein. In the example provided, this is achieved by using a KOH buffer of 22 mSc and pH 11. However, there are a variety of other suitable buffers, including NaOH, etc., the key aspect is conductivity>10 mSc and pH>5. However, the use of elution buffers under these lower constraints will elute the product much slower and will require higher levels of elution buffer, which affects the economics of the process and any downstream processing steps.
After elution, the eluate containing high purity alpha-lactalbumin may be pH adjusted back to 4-7 for further processing.
Experimental part
Experiment 1: extraction of alpha-lactalbumin in radial flow column using SP sepharose macrobeads
This experiment was performed using SP agarose macrobeads (GE Healthcare) packed in a 21L radial flow column. The column was equilibrated with 0.1% acetic acid and the pH adjustment of the whey was performed by adding 8.5% phosphoric acid by volume of 5% whey. The pH adjusted whey was then loaded onto the column at 5.25L/min for a residence time of about 4 min for a total of 105 column volumes. After loading the column, a washing step before elution was performed with 0.1% acetic acid, followed by elution of the binding material with 125mM NaOH. It can be seen on the above chromatogram that the point where beta starts to be displaced by alpha is at 400L, where the level of beta in the flow-through exceeds the level of the initial feed.
Experiment 2: analysis of flow-through-alpha-lactalbumin depleted whey
Samples of the flow-through from experiment 1 were collected intermittently and analyzed according to the following.
The results of this experiment (table 3) show how the amount of beta-lactoglobulin in the flow-through increases and exceeds the amount in the starting material during operation because the beta-lactoglobulin is displaced from the column by alpha-lactalbumin. The alpha-lactalbumin present in the flow-through also increases during operation, however, the conventional penetration profile is not reflected. In this AEX test, there is sufficient resolution to distinguish between two forms of α -lactalbumin, the fused (resolution at 11.5-12.5 mL) and the native (resolution at 14.7 mL). Furthermore, beta-lactoglobulin is also distinguished into two different forms.
Table 2 below shows the purity of the library of α -lactalbumin eluates as measured by UV absorbance at 220nm and 280nm using the techniques described in experiment 2.
Table 3 below shows the yield of the materials evaluated using the technique described in experiment 2, measured as UV absorbance of 220 nm.
Experiment 3: the eluent library purity was analyzed by analytical ion exchange.
The purity of the samples of the composite eluent library resulting from experiment 1 was analyzed.
The data shown in fig. 3 and 4 were collected in the above manner.
Reference to the literature
Claims (13)
1. A method of purifying whey protein comprising the steps of:
-providing a cation exchanger packed in a radial flow column, the cation exchanger comprising chromatographic beads having an average bead size diameter of 130-300 μm, the cation exchanger allowing equal access to both alpha-lactalbumin and beta-lactoglobulin to the available binding sites, and a complete displacement effect;
-acidifying the whey fraction;
-equilibrating the cation exchanger with 0.05-0.2% hac;
-loading the whey fraction onto the cation exchanger at a linear velocity of 200-1000 cm/h;
-washing the cation exchanger with 0.05-0.2% hac; and
-eluting whey protein from said cation exchanger with 30-200mM NaOH or KOH.
2. The method according to claim 1, wherein the cation exchanger comprises chromatographic beads having an average bead size diameter of 200 μm.
3. A process according to claim 1 or 2, wherein the whey is acidified to a pH of 3.5-4.0.
4. A process according to claim 3, wherein the whey is acidified to pH 3.7.
5. A process according to claim 3, wherein the acidification is with H 3 PO 4 HCl, HAc or citric acid.
6. A process according to claim 1 or 2, wherein the process is run at a temperature of 42-55 ℃.
7. A process according to claim 1 or 2, wherein the process is run at a temperature of 50 ℃.
8. The method according to claim 1 or 2, wherein the elution is a 1 step elution with 75mM to 125mM NaOH or KOH.
9. The method according to claim 1 or 2, wherein the eluate is re-titrated from pH 12 to a pH of 4.5-8.
10. The process according to claim 1 or 2, wherein the column is washed in place (CIP) with 1M NaOH/KOH at a high flow rate >800cm/h for >3 hours at 50-60 ℃.
11. The method according to claim 1 or 2, wherein the eluent comprises alpha-lactalbumin in a purity of 70-95% and a yield of 65-85%.
12. A method according to claim 1 or 2, wherein the other whey proteins are collected in a flow-through.
13. A method according to claim 1 or 2, wherein the β -lactoglobulin is collected in a flow-through.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999015024A1 (en) * | 1997-09-22 | 1999-04-01 | Sepragen Corporation | Sequential separation of whey proteins and formulations thereof |
| US6096870A (en) * | 1994-01-05 | 2000-08-01 | Sepragen Corporation | Sequential separation of whey |
| WO2005110112A1 (en) * | 2004-05-07 | 2005-11-24 | Wisconsin Alumni Research Foundation | Methods and compositions involving whey protein isolates |
| WO2010037736A1 (en) * | 2008-09-30 | 2010-04-08 | Upfront Chromatography A/S | A METHOD FOR PROVIDING A β-LACTOGLOBULIN PRODUCT AND AN α-ENRICHED WHEY PROTEIN ISOLATE |
| CN102286096A (en) * | 2011-07-08 | 2011-12-21 | 上海交通大学 | Scale purification method for lactalbumin |
| CN104513305A (en) * | 2013-09-27 | 2015-04-15 | 北京济福霖生物技术有限公司 | Purification method for whey protein |
-
2017
- 2017-02-16 GB GBGB1702499.3A patent/GB201702499D0/en not_active Ceased
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2018
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- 2018-02-13 CN CN201880012175.0A patent/CN110267968B/en active Active
- 2018-02-13 CN CN202311332100.XA patent/CN117402229A/en active Pending
- 2018-02-13 EP EP18705372.3A patent/EP3583114A1/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6096870A (en) * | 1994-01-05 | 2000-08-01 | Sepragen Corporation | Sequential separation of whey |
| WO1999015024A1 (en) * | 1997-09-22 | 1999-04-01 | Sepragen Corporation | Sequential separation of whey proteins and formulations thereof |
| WO2005110112A1 (en) * | 2004-05-07 | 2005-11-24 | Wisconsin Alumni Research Foundation | Methods and compositions involving whey protein isolates |
| WO2010037736A1 (en) * | 2008-09-30 | 2010-04-08 | Upfront Chromatography A/S | A METHOD FOR PROVIDING A β-LACTOGLOBULIN PRODUCT AND AN α-ENRICHED WHEY PROTEIN ISOLATE |
| CN102286096A (en) * | 2011-07-08 | 2011-12-21 | 上海交通大学 | Scale purification method for lactalbumin |
| CN104513305A (en) * | 2013-09-27 | 2015-04-15 | 北京济福霖生物技术有限公司 | Purification method for whey protein |
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| GB201702499D0 (en) | 2017-04-05 |
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| WO2018149809A1 (en) | 2018-08-23 |
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