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Milk clotting aspartic protease enzyme composition

A technology of aspartic acid and protease, applied in the direction of enzyme, peptidase, hydrolase, etc., can solve the problem that milk coagulase is not clearly mentioned

Active Publication Date: 2016-03-16
CHR HANSEN AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As examples of proteins of interest bovine globulin, bovine serum albumin and lysozyme are mentioned, but no milk coagulation enzymes (e.g. chymosin) are explicitly mentioned

Method used

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  • Milk clotting aspartic protease enzyme composition
  • Milk clotting aspartic protease enzyme composition
  • Milk clotting aspartic protease enzyme composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0269] Embodiment 1: the mensuration of curd specific activity

[0270] 4.1 Determination of coagulation activity

[0271] Milk-clotting activity was determined using the REMCAT method, a standard method developed by the International Dairy Federation (IDF method).

[0272] Coagulation activity is determined by the time required for visible flocculation to occur on a standard milk substrate prepared from reduced heat, low fat milk powder using 0.5 g per liter of sodium chloride solution (pH ≈ 6.5). The clotting times of the milk coagulating enzyme samples were compared to a reference standard with known milk clotting activity and based on IDF standard 110B having the same enzyme composition as the samples. Samples and reference standards are measured under the same chemical and physical conditions. Variant samples were adjusted to approximately 3 IMCU / ml using 84 mM acetic acid pH 5.5 buffer. Thereafter, 200 μl of enzyme were added to 10 ml of pre-heated milk (32°C) place...

Embodiment 2

[0284] Example 2: Add PEG or Brij-35 to the elution buffer

[0285] Bovine chymosin or camelid chymosin was expressed recombinantly in Aspergillus niger (approximately as described in WO02 / 36752A2).

[0286] The enzyme was purified by a solid phase extraction method using a benzylamine ligand covalently bound to agarose (similar to that described in WO 01 / 58924A2).

[0287] 96-well filter plates equipped with 25 μm PE filters and 2 ml pore volume were packed with Fastline 1300 from Upfront Chromatography, Denmark.

[0288] The wells were filled with resin to obtain a bed height of 6-8 mm in all wells. The resin in all wells was equilibrated with 5 ml of 20 mM sodium malonate pH 5.7.

[0289] The supernatant from the culture was adjusted by mixing 3ml supernatant with 0.5ml 2M sodium malonate pH 5.7. 3.5 ml samples were then filtered through an 8 μm filter to remove particles and loaded into 96 individual wells of the plate. After loading, the resin was washed with 5 ml o...

Embodiment 3

[0304] Example 3: Addition of PEG to formulation (after purification)

[0305] A liquid clotting aspartic protease composition is obtained comprising clotting aspartic protease at a strength of about 1100 IMCU / g.

[0306] Compositions (ie 3 different compositions) were obtained for bovine chymosin, camelid chymosin and mucor pepsin.

[0307] PEG8000 was added to each composition to reach a level of 0.015% w / w (150 ppm w / w).

[0308] The control composition is the composition without adding PEG8000.

[0309] The compositions were stored at 5°C and 37°C for 6 months and periodically analyzed for curdling activity.

[0310] The results confirmed that after 6 months storage of the tested liquid milk-clotting aspartic protease composition ， Addition of PEG increases long-term storage stability—— That is, these compositions have higher IMCU / g activity.

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Abstract

A liquid or dried granulated milk clotting aspartic protease enzyme composition and process for isolating a milk clotting aspartic protease enzyme of interest.

Description

technical field [0001] The present invention relates to a liquid or dry granular milk-clotting aspartic acid protease composition and a method for isolating the target milk-clotting aspartic acid protease. Background technique [0002] The enzymatic coagulation of milk by milk coagulating enzymes such as rennet and pepsin is one of the most important processes in cheese making. Enzymatic milk coagulation is a two-stage process: the first stage, during which proteolytic enzymes, chymosin, or pepsin attacks κ-casein, resulting in a metastable state of the casein micellar structure, and the second stage, during which At this stage, the milk then coagulates and a curd forms. [0003] Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1) (milk coagulating enzymes of the mammalian stomach) are aspartic proteases belonging to a large class of peptidases. [0004] Commercially relevant milk coagulating enzyme products are usually liquid compositions and a number of different methods are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
CPCC12N9/6478C12N9/6481C12N9/6483A23C19/032C12Y304/23004
Inventor 马丁·伦德J·雅各布森约翰尼斯·马腾·范丹尼布林克
Owner CHR HANSEN AS
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