Process for extracting pure fractions of lactoperoxidase and lac

Details Process for extracting pure fractions of lactoperoxidase and lac Process for extracting pure fractions of lactoperoxidase and lac

1990-05-24

1992-09-22

Robinson, Douglas W.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

530366, 530416, 435815, C12N 908, C07K 322, C07K 1300, A23J 120

Patent

active

051496470

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a process for extracting pure fractions of lactoperoxidase and lactoferrin from milk serum. By milk serum is meant both skim milk and whey.
In cheese-making, a large amount of whey is obtained as a by-product. Whey has a dry solids content of about 6%, which is composed approximately as follows:


______________________________________ % by weight ______________________________________ Lactose 4.6 Protein 0.6 thereof Lactoperoxidase 0.0020 Lactoferrin 0.0030 Fat 0.05 (after separation) Salts 0.7 Dry solids content about 6.0 ______________________________________
The protein fraction which constitutes about 10-12% of the dry solids content is composed of a number of different protein components The biggest are .beta.-lactoglobulin, .alpha.-lactalbumin and bovine serum-albumin. Also a number of bioactive components belong to the protein fraction, for example immunoglobulins, lactoperoxidase, lactoferrin and lysozyme.
Both lactoperoxidase and lactoferrin have antimicrobial properties. There is a great interest in extracting natural antimicrobial substances to be used in new contexts in food technology and in the chemico-technical and medical fields.
There are low contents of these substances in skim milk and whey (also in the original milk). Lactoperoxidase and lactoferrin are present in contents of 15-50 mg/litre, depending on the lactation state of the cow. Large quantities of whey (milk) must thus be filtered to facilitate extration of kilogram amounts of these bioactive components.
The process engineering conditions for isolating lactoperoxidase and lactoferrin, respectively, from milk/whey are based on the fact that the isoelectric point (pI) for these two proteins is about 9.5, while the main part of the whey proteins have isoelectric points around 5.1-5.4 and the casein at about 4.6. A fundamentally suitable process for separation of lactoperoxidase and lactoferrin is therefore to contact the milk/whey with a cation exchanger at a pH of <6 for selective adsorption, and use is here made of the positive net charge of lactoperoxidase and lactoferrin, which distinguishes from that of other milk proteins which have a negative charge at this pH.
The traditional way of isolating lactoperoxidase and lactoferrin in small amounts for the purpose of research is to use the precipitation technique and ion exchange chromatography, frequently combined with gel filtration, see Morrison, M., Hamilton, H-B., Stotz, E., J. Biol. Chem. 228:767 (1957); and Morrison, M., Hultquist, P-E., J. Biol. Chem. 238-2847 (1963). These methods are not suited for preparing large amounts of the bioactive components at issue in an economically defensible manner.
U.S. Pat. No. 4,436,658 (Pevrosuset) discloses adsorption of lactoferrin from casein-free milk serum (whey) by means of a silica column. The pH of the milk serum is adjusted to 7.7-8.2 before adsorption on the column. Immunoglobulins, lactoferrin and lactoperoxidase adhere to the column. After the adsorption phase, elution with a diluted saline solution at a pH of <4 takes place. No selective elution of the adsorbed proteins is obtained, particularly not regarding lactoperoxidase. A column holding about 5 g of silica compound can treat 1 litre of whey. This prior art process must be regarded as unsuitable for application on an industrial scale.
Zagulski et al. in Prace in Materialy Zootechniczne 20, (1979), p. 87-103 describes a batchwise method of obtaining lactoferrin, in which use is made of a weak cation exchanger which is mixed with milk. After equilibration, the ion exchanger is applied to a column for elution of the adsorbed proteins with a saline solution. The method thus is based on a batchwise process, and a further purification must be carried out in a second ion exchange step to obtain a high purity of the lactoferrin.
A similar process is described in BE patent specification 901,672 (J. P. Prieels and R. Peipper, Oleofina S.A.). Here, use is made of an ion exchanger based on calcium alginate, in which the i

REFERENCES:
patent: 3896241 (1975-07-01), Malaspina et al.
patent: 4436658 (1984-03-01), Peyrouset et al.
patent: 4791193 (1988-12-01), Okonogi et al.
patent: 4946944 (1990-08-01), Frankinet et al.
Chemical abstracts, vol. 105, 1986, abstract No. 41350u, J Chromatogr., 1986, 358(2), 429-433 (Eng).
Buzila et al, "The Simultaneous Preparation of the Active Components From Human Milk," Rev. roum. Biochim., vol. 21, Iss. 2, pp. 81-91 (1984).
Boyer (1986) "Modern Experimental Biochemistry" pp. 49-53.

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