Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-08-20
2003-04-29
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C426S007000, C426S020000, C426S032000, C426S049000
Reexamination Certificate
active
06555336
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method for preparing albumin hydrolysates from a starting material containing albumin in concentrated of isolated form.
BACKGROUND OF THE INVENTION
Albumin hydrolysates are albumin fragments of different lengths, which may be produced by chemical hydrolysis by means of alkali or acid or by enzymatic hydrolysis. In the chemical hydrolysis protein damages like lysinoalanine and chloropropanol may occur as a drawback. Known disadvantages of the enzymatic hydrolysis are long hydrolysis times and bitter peptides. Further, the albumin yield in relation to the albumin input is clearly smaller with enzymatic hydrolysis than with chemical hydrolysis. On the other hand, the chemical hydrolysis takes place more or less non-specifically. In weighing up all factors, the enzymatic hydrolysis is presently preferred for preparing albumin hydrolysates for the food and pharmaceutics sectors.
Prior art methods for preparing albumin hydrolysates from a starting material containing albumin in concentrated or isolated form by means of enzymatic hydrolysis generally have a similar structure, the albumin, as a rule being dissolved before or after the enzymatic hydrolysis at an alkaline pH.
It is known from U.S. Pat. No. 1,231,652, for example, to carry out an enzymatic hydrolysis at the isoelectric point of the albumin with acid proteases being active within this pH-range for reducing the viscosity of albumin dispersions. Afterwards the pH is raised, sometimes up to 11.8, by means of alkali, and the albumins are alkalinally hydrolysed for 1 to 2 hour at temperatures of 50 to 85° C. By means of this, the albumins are dissolved, and are then further cleaved by means of the protease pepsin within a high acid pH-range. It is a drawback of this known method that a strong formation of salt is associated with the frequently changing pH of the albumin containing dispersion or solution, and that the enzymatic hydrolysis is only carried out within the acid pH range, in which hydrolysis times of more than 20 hours are necessary to obtain an acceptable albumin hydrolysate yield.
From U.S. Pat. No. 4,473,589 it is known to combine an enzymatic hydrolysis with an alkaline hydrolysis. To this end, it is at first alkalinally hydrolysed under strongly alkaline conditions at a pH of over 12, and then the albumin dispersion is further liquefied by means of an alkaline protease. The temperatures of the alkaline hydrolysis over 1 to 2 hours are between 50 to 80° C., and the temperatures of the enzymatic hydrolysis are between 40 to 60° C. The time of the enzymatic hydrolysis is up to 48 hours. Like the method according to U.S. Pat. No. 1,231,652 described above, the method according to U.S. Pat. No. 5,473,589 results into considerable alkali damages of the obtained albumin hydrolysates because of the high temperatures and of the high pH applied simultaneously. This strongly limits a use of the obtained albumin hydrolysates in the food and pharmaceutics sector. Additionally, especially the long hydrolysis times in the method according to U.S. Pat. No. 4,473,589 are unsuitable for an economic preparation of albumin hydrolysates at a large scale.
A method for preparing a soya albumin hydrolysate is known from EP 0 199 981 A2. Here, a neutral protease is used at a neutral pH. To support the effect of this neutral protease, a fine dispersion of the starting material is initially prepared in that the starting material is homogenized at 60 to 100° C. over a pressure drop of 100 to 800 bars so that the starting material is present with a low particle size and thus a great engageable surface. Depending of the wanted solubility of the desired product, it is then hydrolysed for a period of 5 to 180 min. The resulting dispersion is dried after inactivation of the used enzymes without further processing. It is one of the drawbacks of this known method that a dispersion of different albumin fragments up to non-processed albumins is obtained. Further, the availability of neutral proteases is limited. Additionally, the high pressures and the simultaneous high temperatures during the homogenization lead to a strong protein denaturation so that as a result the possibilities of engagement by the protease are strongly limited which increases the hydrolysis time. All at all, the obtained albumin hydrolysates are limited to a small number of fields of application.
A method for preparing albumin hydrolysates from a starting material containing albumin in a concentrated or isolated form is known from EP 0 298 419 A2, which method is closest to the present invention of all present prior art. In this known method, vegetable albumins are hydrolysed over 10 to 60 min. chemically by acid or alkali at 60 to 180° and/or enzymatically and/or by means of reducing or oxidizing agents. The kind and the way of hydrolysis shall be selected based on the application of the albumin hydrolysis to be prepared. A clear concept for the preparation of albumin hydrolysates does appear from EP 0 298 419. Instead, all known working mechanisms are simply listed.
The preparation of functional disperse as well as of fully soluble albumin hydrolysates is described in EP 0 480 104 A1. Here, the positive effect of a high alkaline pH of 9 to 11 is emphasized. It is particularly pointed out that the toxic amino acid lysinoalanine, which is a measure of albumin damages, does not exceed a concentration of 300 ppm. The temperatures in the enzymatic hydrolyses are between 10 and 75° C., and they are not tuned to the different proteases used. The hydrolysis time is 10 to 240 min.
A special method for preparing rice albumin hydrolysates is disclosed in DE 195 02 167 C2. Here, a hydrolysis takes place in the present of adsorbents. The pH is between 8 and 10, the temperature is between 40 and 90° C., and the hydrolysis time is between 1 and 24 hours. Because of the multistage processing of the starting material using filtration by means of filter presses and processing with activated carbon, the known preparation method is very cost intensive. It is also a particular drawback that the conserved albumin hydrolysate solution can only be processed after a maturation time of 14 days and after a repeated application of activated carbon. Thus, a preparation of albumin hydrolysates at low cost is not possible.
From DE 33 06 009 C2 it is known to use enzymes on a fixed support for the hydrolysis of albumins. The main emphasis is on fungal proteases. The pH at which the hydrolysis takes place is between 6 and 9. The hydrolysis time is adjusted to values between 10 and 100 min. by means of the flow velocity along the fixed support. At the end, the high cost of coupling the enzymes to the support material, the disposal of the support material and the difficulties with a technical layout stand against a commercial application of this known method.
It is common to all of the above described prior art methods, except of EP 0 199 981 A2, that, after the hydrolysis, the enzymes are heat inactivated and the non-hydrolysed albumin parts are separated from the albumins hydrolysed from the starting material by precipitation at the isoelectric point of the non-hydrolysed albumin parts to obtain a albumin hydrolysate solution as pure as possible.
Inter alia from Ullmanns Encyclopaedia of technical Chemistry, Vol. 10, page 494 (1975) it is known that the activity of enzymes is considerably reduced as an effect of thermal, enzymatic-proteolytic and mechanical stress during a step of homogenization, particularly by high pressure disintegration.
The invention is based on the problem to provide a method for preparing albumin hydrolysates from a starting material containing albumins in a concentrated or isolated form which may by applied in a continuous and thus economic form because of a short hydrolysis time and which, with a great flexibility with regard to the starting material and the albumin hydrolysates to be prepared, results into method product with a low bitter content.
SUMMARY OF THE INVENTION
The invention discloses a method for preparing albumin hy
Tate Christopher R.
Thomas Kayden Horstemeyer & Risley
Winston Randall
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