FI61516C - EXHAUST FOUNDATION FOR BETA-AMYLAS AND SAW SURFACES - Google Patents

Description

61 51 6

Method for extracting β-amylase from cereal grains. - For the purpose of extraction of ft-amylated urine.

The invention relates to a process for the preparation of a pure crude extract of β-amylase from cereal grains, in particular barley grains.

β-amylase is a starch hydrolyzing enzyme that forms in and is stored in grains during the growth phase. The best studied and known is barley (3-amylase because of its great importance in the brewing industry. (DE Briggs "Barley", Chapman & Hall, London, 1978 Cook "Barley and Malt", Academic Pres, London, 1862, JRA Pollock "Brewing Science. ”Academic Press, London, 1979).

There are two types of β-amylase preparations on the market, cereal and bacterial. The difference between them is e.g. composition of starch hydrolysis products. Bacterial (3-amylase-catalyzed hydrolysis) produces 15-30% maltotriose and 50-60% maltose, while cereal (i-amylases) mainly produce maltose.

The major uses of ft amylase are found in the brewing and starch industries. If enzyme with a good enzyme density of 2 6151 6 is not available for mashing, the deficiency can be remedied by adding e.g. / 3-amylase. The use of enzyme supplements is essential when using unmalted grain for mash. When hydrolysis of starch with cereal β-amylase is performed, sweet, maltose-rich products are obtained which are suitable e.g. as a raw material for beer and for use in the manufacture of confectionery. In the confectionery industry, such a maltose-rich syrup can replace the proportion of sucrose because of the high sweetness of the syrup itself.

The above-mentioned literature, its references and DE-AS 1 792 362 disclose a number of different ways of isolating β-amylase from grains and parts thereof. What all methods have in common is that the grain is initially ground or ground or that the starting material is a mechanically separated part of the grain, such as a bran. From these starting materials, the?

Since the shell layers of the grain are semipermeable and because (3-amylase is located relatively close to the shell, in the subaleuron layer of the grain, it was considered possible to extract 5-amylase from the whole grain, since Surprisingly, it was found that 60-80% of the total amount of β-amylase can be extracted from the grain by the process according to the invention, so that the extraction is carried out on whole or partially hulled grains with a weight of 0-20% of the shell material removed at an extraction temperature of 20%. 55 ° C and an extraction time of more than 5 hours.

The amount, purity and extraction time of β-amylase extracted from the grains can be controlled by changing the temperature, which affects the permeability and diffusion rate of the grain membranes. The temperature is preferably 20-55 ° C.

Another control means is the partial removal of the outer husk layers of the grain, e.g. from the husking of rice, by a known technique, whereby the permeability of the membranes of the grain becomes desired. The preferred result is obtained when about 10% of the total weight of the grain, preferably barley, is removed as peeling material so that the endosperm remains intact.

In this method, the outermost layers of the grain endosperm act as a kind of ultrafilter, completely preventing insoluble impurities from entering the soaking water and also substantially limiting the amount of soluble compounds. The extract thus obtained (containing 3-amylase) is easy to concentrate, for example, by ultrafiltration.

It is preferred to use a reducing agent (e.g. SC> 2) in the extraction to release i-amylase from other proteins, a method known in the literature. The invention is further illustrated by the following examples.

Example 1 100 g of paddy barley were soaked in 300 ml of 0.5% Na metabisulphite at 30 ° C. Dissolved (i-amylase activity was monitored as a function of time (Tables). Activity assays were performed by the modified Bendelow method (Bendelow, WM 1963, Journal of the Institute of Brewing, no. 69, pp. 467-472). The relative unit U (powdered the specific activity of barley was denoted as 100 U / g protein.) All assays were performed on filtered samples, and the amount of 3-amylase that dissolves from ground barley under 18 hours is denoted as 100% activity.

Ground barley: Soaking time (s) __2 4 6 7 12 16 18

Activity% 27 49 71 89 96 99 100

Spesif.akt. (U / g prot.) 100

Akt. (U / g dry matter in soaking water) _30

Barley without husk Soaking time (s) __10 20 30 40 50 60 70

Activity% 8 20 39 51 69 76 78

Spesif.akt. (U / g prot.) 152

Akt. (u / g dry matter in soaking water) _52 4 61516

Example 2

The experiment was carried out in the same manner as in Example 1, but the grains were peeled so that 10% of the total weight was removed as a peel fraction.

Soaking time (s) __2 5 10 15 20 25 26

Activity% 7 20 48 65 73 7Q 80

Spesif.akt. (U / g prot.) 156

Active (U / g dry matter in soaking water) _60

Example 3

The grains were peeled so that 20% of the total weight was removed as a peel fraction. In other respects, the experiment was performed as in Example 1.

Soaking time (s) ___2 5 10 15 18 20 21

Activity% 12 31 57 72 75 76 77

Spesif.akt. (U / g prot.) 135

Active (U / g dry matter in soaking water) _44

Example 4

To study the effect of temperature on the dissolution rate of β-amylase and the purity of the solution, experiments corresponding to Example 1 were performed at 25 ° C, 40 ° C and 50 ° C. Barley with a degree of peeling of 10% was used in these experiments.

Soaking time (s) _2 5 10 20 22 23 24 26

Activity% 25 ° C 4 14 35 70 76 79 82 83 40 ° C 7 19 41 69 75 78 78 74 _50 ° C_11 22 47 70 75 74 70_

The following table shows the dissolution rate,% activity, specific activity and activity per g of dry matter obtained at different temperatures.

61 51 6 5 _Peeling rate 10% ground barley

Soaking temperature (° C) 50 40 25 25

Soaking time (s) 22 23 26 19

Activity% 75 78 83 100

Specific Act (U / g prot.) 183 178 156 98

Akt. (U / g dry matter in soaking water) 74 78 65_29_

Thus, it can be seen from Examples 1-4 that - whole grains (i-amylase are extracted much purer than from flour, although the extraction time is longer (e.g. 1)) - by peeling and / or raising the temperature, the extraction time can be shortened while the extractable O-amylase is still purer when compared to the purity of {3-amylase extracted from flour (eg 2, 3 and 4) - at best 75-83% of the amount of 3-amylase extractable from flour is obtained from whole grains, in which case the purity of β-amylase is at least 156/98 - 183 / 98 i.e. 1.59 to 1.87 times the protein and 65/29 to 78/29 or 2.24 to 2.69 times the total dry matter (e.g. 4) (further purification of i-amylase can be performed by any known technique such as spray drying, ultrafiltration, precipitation etc. Since the purity of the crude extract using the presented method is higher, these operations are correspondingly facilitated.