Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1999-08-26
2002-09-17
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
Reexamination Certificate
active
06451552
ABSTRACT:
The present invention relates to a method for the selective degradation of milk proteins, and in particular to a method for the selective hydrolysis of casein and/or casein/caseinate in the presence of at least one further protein constituent other than casein/caseinate.
The invention more in particular relates to such a method wherein the further protein constituent is a milk protein (constituent) other than casein, in particular a whey protein (constituent), and/or to such a method wherein the casein/caseinate and preferably also the at least one further protein constituent are essentially in solution, in particular essentially in an aqueous solution.
According to the method of the invention, the casein/caseinate is specifically degraded in the presence of the at least one further protein constituent, said further protein constituent essentially remaining intact in the process. This allows the casein hydrolysis fragments to be separated, in a further separation step, from the one or more further protein constituents which have essentially remained intact.
The invention is based on the surprising finding that certain proteinases exhibit a very high specificity with respect to casein in the presence of other proteins, in particular in the presence of other milk proteins such as whey proteins. The invention is further based on the use of these specific proteinases in the hydrolysis of casein-containing protein preparations, in particular casein preparations or (aqueous) solutions of milk proteins, such as milk.
The method of the invention can be used, in a first embodiment, for the preparation of a casein hydrolysate which has been stripped of one or more immunogenic protein components other than casein. In a second embodiment, the invention can be used for preparing a milk protein preparation, in particular a whey protein preparation which is essentially stripped of casein/caseinate, starting from a casein-containing milk protein preparation such as milk, whey or a solution of milk or whey proteins. These whey protein preparations stripped of casein likewise have beneficial properties from an immunological point of view.
The invention will be further discussed with reference to these specific, non-limiting embodiments “A” and “B”, which are schematically shown in FIG.
6
. Both embodiments comprise the step of selectively degrading casein in the presence of at least one further proteinaceous component, and separating the hydrolysed fragments from the non-degraded proteins, but differ in the starting material used, the product desired, as well as the intended use and required (immunological) properties of said product, as will become clear hereinbelow.
EP-A-0 250 501 describes a method for producing a whey protein hydrolysate, comprising the steps of:
a) removing casein from the whey protein starting material;
b) hydrolysis of the casein-free protein material obtained in (a) with at least one protease;
c) ultrafiltration of the hydrolysate of (b) using a membrane with a “cut-off” value of no more than 20,000 Dalton;
and optionally further processing of the filtate thus obtained.
In step a), the casein is removed from the whey protein starting material by either physical separation such as precipitation or filtration; or enzymatically by the use of the neutral metalloprotease of
Bacillus subtilis
(Neutrase®, Novo Industrie A/S), which is said to have “a high activity against casein but no or only a weak activity against both native as well as heat-denatured whey proteins”.
The enzymatic hydrolysis is carried out at a pH of 4-9, preferably pH 7.5, and a temperature of 30-60° C., preferably 50° C., until “completion”, which is said to be a degree of hydrolysis (DH) of about 4%, based upon total protein. The casein fragments are then removed by means of centrifugation or ultrafiltration using—when the whey protein starting material has not been heat-treated—a membrane with a “cut-off” value of no more than 20,000.
Because the molecular weight of the different caseins (19.5-25 kDa) are in the same range as the molecular weights of the native whey proteins &agr;-lactalbumin (14.2 kDa) and &bgr;-lactoglobulin (18.2 kDa (monomer)) it is doubtful whether the use of a 20 kDa membrane will lead to the separation of the hydrolysed caseins from the whey proteins.
The “casein-free” whey proteins thus obtained are then further hydrolysed using enzymes such as Alkalase® 2.4 L and Rhozyme P41®, and then ultrafiltrated. According to this disclosure, by selectively removing the casein before the whey proteins are hydrolysed, the final whey protein hydrolysate will have a less bitter taste.
However, the casein depleted whey protein preparation obtained after said step a) is only an intermediate product, and not the end product. Accordingly, no immunological determination of the allergenic properties of this intermediate product is given. In the method of EP-A-0 250 501, this is also not essential, as the proteins obtained are hydrolysed further, after which any still remaining antigenic fragments can be removed by ultrafiltration. However, from the low degree of hydrolysis (4%) in step a), it is expected that at least some antigenic casein fragments will be present in said intermediate product.
EP-A-0 384 303 describes a method for hydrolysing proteins, especially milk proteins and whey proteins, in which a combination of a proteinase preparation from Aspergillus sp. and a bacterial aminopeptidase is used. The use of this combination provides an end product with a less bitter taste, at a higher degree of hydrolysis: with said combination, the “bitter point” is reached at a DH of 4.4%, whereas the bitter point is said to be reached at 1.2% when only the proteinase is used.
EP-A-0 610 411 discloses a method for obtaining a casein hydrolysate, in which a casein or caseinate is suspended in an aqueous medium and hydrolyzised to a DH of 15-35%, preferably 22-28%, using a combination of proteases from the following three groups:
1) one or more neutral endoproteases of a Bacillus (such as Neutrase®);
2) one or more basic endoproteases of Bacillus (such as Alcalase®, Esperase® and Savinase®) and
3) an endoprotease of Aspergillus (such as Novozym® 515).
According to this reference, the use of such a combination of proteases, and hydrolysis to a DH of 15-35% (compared to 4.4% such as disclosed in EP-A-0 250 501 as well as for instance EP-A-0 384 303) provides an improved casein hydrolysate. Nevertheless, no immunological determination of the antigenic properties of the hydrolysate thus obtained is disclosed. Also, the enzymes used are obtained from
Bacillus subtilis
, which is not a food grade microorganism.
EP-A-0 631 731 describes a method for producing a partial hydrolysate of milk protein by enzymatic hydrolysis of a mixture of whey protein and casein to a DH between 4 and 10%. Although a reduction in antigenicity of 80% or more was obtained, according to the examples the resulting hydrolysates still had some residual antigenicity as determined by an ELISA.
EP-A-0 421 309 describes a method for preparing a whey protein hydrolysate free of allergenics by pepsin prehydrolysis, followed by trypsin-chymotwpsin hydrolysis in the presence of a cathionic serine endoprotease of type elastase 2.
Derwent Abstracts AN-96-471202 and AN-94-337356 (corresponding to JP-08238059-A and JP-06261691-A) disclose a method for producing a low-allergenic milk protein preparation, in which alpha-casein is selectively decomposed using a fungus derived protease, in particular from the geni Mucor and Cladosporium.
Derwent Abstract AN-95-307065 (corresponding to JP027203844 A) discloses a method for preparing an emulsified whey protein hydrolysate with good thermo-stability and up to 0.0001 residual antigen in ELISA, obtained by hydrolysing a solution of milk whey protein with a mixture of endotype proteases from
Bacillus subtilis
, trypsin and papain.
EP-A-0 601 802 describes a method for removing allergenic compounds from proteinaceous compositions, in which the protein in said composition is decomposed with proteolytic enz
Alting Aart Cornelis
Van Beresteijn Emmerentia Catharine Henriëtte
Nederlands Instituut voor Zuivelonderzoek
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
Weber Jon P.
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