Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1994-09-20
1997-01-21
Achutamurthy, Ponnathapura
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530366, 530380, 530832, 530833, 435192, 426657, A23J 120
Patent
active
055960820
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for isolating the metalloprotein lactoferrin and the enzyme lactoperoxidase from milk and milk products on an industrial scale.
BACKGROUND OF THE INVENTION
Lactoferrin (LF), a metalloprotein, occurs in minor amounts in milk from mammals. It is capable of binding two molecules of iron per molecule of protein. It thus provides the young mammal with iron in assimilable form. It also functions as a bacteria-inhibiting agent for pathogenic microorganisms that are iron-dependent.
The enzyme lactoperoxidase (LP) catalyzes the breakdown of hydrogen peroxide in the presence of a hydrogen donor or an oxidizable substance. In milk, it functions as a bacteria inhibitor of pathogenic Streptococci and Salmonella in the presence of thiocyanate and peroxide, both of which are present in milk. In raw cow milk, concentrations of about 200 mg/l have been reported for LF and concentrations of about 30 mg/l for LP depending in part on the stage of lactation and breed.
LP in particular is thermolabile. Upon pasteurization, this enzyme will be inactivated in part or in whole, depending on the intensity of the heat treatment. It is therefore typically necessary to isolate these two components from raw milk. However, this may be technologically difficult because small amounts of LP and LF must be separated from large amounts of other substances, e.g., proteins, sugars and fats.
Further, it is not known whether the milk from which LP or LF has been withdrawn can still be used as "milk" in terms of the official food regulations, i.e., for human consumption and/or as raw material for making cheese and butter. It is therefore desirable to avoid using raw milk as the starting product, and instead, use a by-product such as those formed in cheese or butter making, e.g., whey or skim milk.
In cheese making, the milk is subjected only to a minor heat treatment. As a consequence, substantially all of the LP is still present in cheese whey. However, only a part of the LF, namely about 25%, remains. Nevertheless, whey is an attractive starting material. It is available in large amounts; it is cheap; and, in a manner of speaking, it is prepurified.
Reference NL-A 8201947 (U.S. Pat. No. 4,436,658) discloses a process for isolating LP and LF from cheese whey. Whey having a pH between 7.5 and 8.2 is applied to a column filled with buffered silica gel. The proteins adsorbed to the column can be eluted to obtain a LF and a LP. The purity of the preparations obtained is low, e.g., only 66%.
EP-A 0 253 395 discloses the isolation of LF alone from raw milk and whey. Milk or whey is brought in contact with a weakly-acid ion exchanger with carboxymethyl groups as active groups. Various types of exchangers were compared and characterized by a binding capacity for hemoglobin. The examples describe long contact and elution times. The experiments were performed both in columns and in batches. This implies that the process described in EP-A 0 253 395 is less suitable for large-scale industrial production. Moreover, only LF is isolated, while LP is not.
EP-A 418 704 discloses the extraction of LP and LF via affinity chromatography in a column with sulfonated polysaccharide resin. LP and LF are adsorbed and subsequently eluted and purified. In the examples described, a small liquid load is used (about 80 bed volumes per hour), yielding good separation of LP and LF.
WO-A 89/04608 discloses a process whereby LP and LF can be isolated from cheese whey on a semi-industrial scale. Here, too, the rate of throughput of 1-1.5 bed volume per minute is a limiting factor, in view of the large volumes of cheese whey that must be passed through the columns.
The above-mentioned processes are based on known chromatographic techniques for the extraction and purification of proteins from a matrix containing many attendant substances, such as carbohydrates, fats and salts. The above-mentioned techniques are used in particular on a laboratory scale and also on a semi-industrial scale.
The resin and ion exchang
REFERENCES:
patent: 4436658 (1984-03-01), Peyrouset et al.
patent: 4667018 (1987-05-01), Prieels et al.
patent: 4791193 (1988-12-01), Okonogi et al.
patent: 5087369 (1992-02-01), Tanimoto et al.
patent: 5179197 (1993-01-01), Uchida et al.
patent: 5516675 (1996-05-01), Uchida et al.
Kivits Marinus G. C.
Kussendrager Klaas D.
Verver Albert B.
Achutamurthy Ponnathapura
Campina Melkunie BV
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