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Process for obtaining growth factor (tgf-beta and igf-1), lactoperoxidase and immunoglobulins preparations from milk products having low mutual cross-contamination

A technology for growth factors and dairy products, applied in the direction of milk immunoglobulins, transforming growth factors, insulin-like growth factors, etc., can solve the problems of low HAP column life, low yield, less effective, and less economical feasibility.

Inactive Publication Date: 2004-12-15
CAMPINA BV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While appropriate fractions of growth factors can be obtained, this method has some disadvantages
One of these disadvantages is the relatively low lifetime and yield per cycle of the HAP column, which makes the method less economically viable
In addition, the HAP column binds lactoperoxidase, making the method less effective since the major part of the protein in this fraction consists of lactoperoxidase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Isolation of IGF-1, TGF-β and lactoperoxidase (LP) from milk (tert-butyl)

[0080] A 10 cm diameter ion exchange chromatography (IEC) column was packed with 1 L of strong cation exchanger (SP Sepharose Big Beads, Pharmacia).

[0081] The column was pretreated with phosphate buffer (pH 6.5, 0.025M phosphate). The fat fraction of the milk was removed by centrifugation, and 360 L of the resulting skim milk was passed through the column at room temperature at a flow rate of 100 BVH (column bed volume / hour). The column was washed with 5L of 0.10M NaCI, pH 6.5 solution.

[0082] Then by using:

[0083] a) 5L 0.25M NaCl solution, pH 6.5

[0084] b) 5L 1.00M NaCl solution, pH 6.5

[0085] Elution column to separate the adsorbed protein.

[0086] Fraction a) mainly contains lactoperoxidase and is rich in IGF-1 and TGF-β.

[0087] Part b) is rich in angiogenin and lactoferrin. According to the results, part a) contained 800 mg LP / g protein, 30 μg IGF-1 / g protein...

Embodiment 2

[0093] Example 2: Isolation of IGF-1, TGF-β and LP (tert-butyl) from milk with different HIC elution conditions

[0094] Fractions bound to the t-butyl column can also be separated using other elution conditions.

[0095] Under the same conditions as described in Example 1, the IEC eluate [Example 1, part a)] was loaded onto a tert-butyl column, and the tert-butyl was eluted with a buffer containing 0.25M NaCl / 25mM phosphate, pH 5.0. Butyl column. The growth factor-enriched fraction was then eluted with a linear gradient of 3.75 L of 0-40% ethanol in 0.2M ammonium acetate buffer pH 5.0. The yield of growth factors in this step was slightly lower, but the specific activity of the IGF-1 enriched fraction was much higher than that obtained using the conditions described in Example 1, ie 1250 μg / g protein. The TGF-[beta] level in the IGF fraction was 9 [mu]g / g protein.

[0096] The obtained TGF-β-enriched fraction had a specific activity of the same order as in Example 1.

Embodiment 3

[0097] Example 3: Isolation of IGF-1, TGF-β and LP(tert-butyl) from milk with different HIC loading and elution conditions

[0098] Fraction a) obtained by elution of IEC in Example 1 was divided into two fractions:

[0099] One portion was adjusted to pH 4.0 and the other to pH 6.0.

[0100] pH 4.0:

[0101] The pH 4.0 fraction was loaded onto a column containing 0.75L Macro-Prep tert-butyl at 15BVH. The pH of the wash and elution buffer was also 4.0. The column was eluted with 3 L of 0.025 M acetate pH 4.0 containing 0.25 M NaCl and 3 L of 0.1 M ammonium acetate pH 4.0. The adsorbed protein was then fractionated by eluting the column with a linear gradient of 12 L of 0-40% isopropanol in 0.1 M ammonium acetate buffer pH 4.0. Lactoperoxidase was mainly present in the unbound and washed fractions. During the linear gradient, IGF-1-enriched and TGF-β-enriched fractions were obtained. Both fractions were obtained in good yields and had high specific activities, i.e. the IG...

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Abstract

The present invention relates to a process for extracting beneficial compounds, in particular growth factors, such as TGF beta and IGF-1 from milk. In this process a hydrophobic interaction chromatography step is included. A resin having a butyl group, or a phenyl group as the ligand is used as hydrophobic interaction resin. The resin can be eluted with a salt gradient which, when the ligand is a phenyl group, contains substantially no alcohol, and thus resulting in fractions enriched in the desired growth factors. These fractions can be separated further by means of a hydroxyapatite column.

Description

technical field [0001] The present invention relates to a method for obtaining a fraction containing beneficial compounds from dairy products (milk or whey). In particular, according to the invention, a fraction enriched in growth factor compounds, such as transforming growth factor beta (TGF-beta) or insulin-like growth factor 1 (IGF-1 ), is obtained. Background technique [0002] Dairy products have been known for some time to contain growth factors with beneficial activity. These growth factors are found in very low concentrations in dairy products, which is why they are sometimes called micronutrients. They may be characterized by their isoelectric points, which are relatively higher than other milk proteins, as well as by their molecular weight. The present invention is particularly concerned with the growth factors TGF-beta and IGF-1. [0003] TGF-β is a multifunctional protein found in all mammalian tissues. Five forms of TGF-β are currently known, β1-β5. It is i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23J1/20A23L11/00C07K14/495C07K14/65C07K16/04C12N9/08
CPCA23J1/20C07K14/495C07K14/65C12N9/0065
Inventor 马里纳斯·赫拉尔杜斯·科内利斯·基维茨凯瑟琳娜·玛丽亚·加拉玛安多尔·威廉·约瑟夫·亨德里克斯
Owner CAMPINA BV
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