PROCESS FOR THE EXTRACTION OF BETA-AMYLASE FROM GRAINS

Abstract: A process for the extraction of β-amylase from grains, especially from barley. The extraction is effected by using whole or partially hulled grains, some of the hull layer being removed with the grain remaining intact or whole.

Description: Process for the extraction of (3-amylase from grains The present ivention relates to a process for the preparation of pure raw extract of ss-amylase from grains, especially from grains of barley. ss-amylase is a starch-hydrolyzing anzyme developed in grain during the growth period and stored therein. The best-known and studied is ss-amylase of barley for its major importance in brewing industrv. (D.E. Briggs "Barley", Chapman & Hall, London, 1978 Cook "Barley and Malt11, Academic Press, London, 1862, J.R.A. Pollock "Brewing Science Academic Press, London, 1979). There are two types of ss-amylase preparation on the market, those based on grain and bacteria. A difference between them is e.g. tine composition of starch hydrolysis products. A hydrolysis catalyzed by bacteria-based 5-amylases produces 15 - 30 % of maltotriose and DO - 60 % of maltose, while grain-based ss-amylases produce vainly maltose. Therefore, a starch hydrolyzed by the grain-based ss-amylase has increased sweetness and ability to serve as a nutrient of yeast. The most important applicatios of ss-amylase are found in brewing and starch industries. If there is not available a malt with sufficient entymatic activities for mashing, the defect can be overcome by the addition of e.g. ss-amylase. Enzyme additions are necessary when using unmalted grain for mashing. Effecting the hydrolysis of starch with a grain-based ss-amylase will result in sweet, maltose-rich products which can be employed e.g. as a raw material of beer and in the production of sweets. In confectioney industry, it is possible to substitute such maltose-rich syrup for some of the saccharose, since the syrup is inherently very sweet indeed. The above-cited literature, its references and publication DE-AS 1 792 362 disclose a plurality of methods for the separation of 0 -amylase from grains and components thereof. A common feature of all these methods is that the grain is initially ground or crushed or that the starting material comprises some mechanically separated component of grain, such as bran. From these starting materials ss-amylase is extracted with water, a buffer solution etc., whereby the obtained raw extrate contains, together with ss-amylase, a lot of other soluble grain ingredients which leads to a very arduous purification and concentration of enzyme. Since the hull layers of a grain are semi-permeable and since ss-amylase is found relatively near the hull, in the subaleurone layer of a grain, studies were initiated to find out whether it is possible to extract ss-amylase from a whole grain as it was presuree that, with the grain's own membranes acting as a kind of filter, it would be possible to obtain already at "the raw extract stage" a preparation of substantially improved purity compared to these extracted from flour or grit. It was unexpectedly discoverer tat 60 - 80 % of the total amount of (3-amylase can be extracte rc- z grain by the process of the invention in such a manner that the extraction is effected from whole or partially hulled grain, the weight of removed hull material being 0 - 20 % of the weight of a grain, the extraction temperature being 20-55 C, and the extraction period exceeding 5 hours. The amount, purity and extraction period of the ss-amylase to be extracted from grain can be controlled by- chanciinc-- t;- temperature which effects on the permeability and rate of diffusion of the gain membranes, Temperature is preferably within the rane of fro. 20 to 550C. Another means of control is partial removal of the outermost hull layers of a grain by applying e.g. the technique known from rice hulling, thus obtaining the desired permeability for the grain membranes. A preferred result is achieved by removing circa 10 % of the total weight of a grain, preferably barley, as a hull material in such a manner that endosperm remains intact. In this process, the outermost layers of the grain endosperm serve as a kind of ultrafilter preventing entirely the passage of insoluble impurities into the leaching water and also substantially restricting the amount of soluble compounds. The obtained 0 -amylase containing extract can be readily concentrated e.g. by ultrafiltration. It is preferable to use in the extraction some reducing agent (e.g. SO2) for liberating ss-amylase from other pro.ei.s, this process being known in the literature. The invention will now be illustrated in more detail by means of the following examples. Example 1 100 g of unhulled barley was leached in 300 ml of 0,5 i Na metabisulphite at 300C. Activity of leached ss-amylase was observe; as a function of time (tables). Activity determinations were carried out by modified Bendelow method (Bendelow, W.M. Journal of Institute of Brewing, No. 69, p. 467-473). The unit applied was a relative unit U (specific activity of around barley was rarked as 100 U/g of protein). All determina' sns were effected from filtered samples. A 100 % activity was meant by the amount of ss-amylase which in corresponding conditions dissolves from groung barley in 18 hours. Ground barley: Leaching time (h) 2 4 6 7 12 16 18 Activity% 27 49 71 89 96 99 100 Specific activity (U/g protein) 100 Activitv (U/g drv matter in leachinq water) Unhulled barley Leaching time (h) 10 20 30 40 50 60 70 Activity % 8 20 39 51 69 76 78 Specific activity (U/g protein) 152 Activitv (U/q drv matter in leaching water) 52 Example 2 The test was effected the same way as in example 1 except that the grains were hulled so that 10 % of total weight was removed as hull fraction, Leaching time (h) 2 5 10 15 20 25 Activity z 7 20 48 65 73 7C 80 Specific activity (U/g protein) Activitv (U/q drv matter in leaching water) Example 3 Grains were hulled so that 20 8 of total weight was removed as hull fraction. Otherwise the test was effected the same way as in example 1. Leaching tine (h) 2 5 10 15 1R 20 21 Activity % 12 31 57 72 75 76 Specific activity (U/g protein) 13r Activitv (U/q drv matter in leachinq water) 44 Example 4 For studying the effect of temperature on the dissolving rate of ss -amylase and on the purity of the solution, the test corresponding to that of example 1 were run at temperatures 250C, 400C and 500C, respectively. Material used in these tests was barley with hulling degree of 10 %. Leaching time (h) 2 5 10 20 - 22 23 24 26 Activity % 250C 4 14 35 70 76 79 82 3 40 C 7 19 41 69 75 78 78 74 50 C 11 22 47 70 75 76 70 The following table illustrates the dissolving rate, activity %, specific activity and activity per g dry matter. Hulling degree ground 10% barley Leaching temperature ( C) 50 40 25 25 Leaching time (h) 22 23 26 19 Activity % 75 78 83 100 Specific activity (U/g protein) 183 1 8 156 OP Activitv (U/q dry matter in leachinq water) 74 78 65 65 20 Thus, it can be noted from examples 1-4 that - Puritv of p-amylase extracted from whole grain is considerahly improved compared to that extracted from flour, although the extraction time is longer (example 1) - by hulling the grains and/or by elevating temperature the extraction time can be shortened and the extracting /2-amylase is still of improved purity as compared to the purity of ss-amylase extracted from flour (examples 2, 3 and t) - in the most favourable conditions it is possible to extract from whole grains 75 - 83 8 of that amount of p-amylase which is extractable from flour. In this case the.purity of pss-amylase is at least 156/98 - 183/98 or 1.59 - 1.87 times higher with respect to protein and 65/29 - 78/29 or 2.24 - 2.6 time higher with respect to total dry matter (example 4). Further purification of ss-amylase can be effected bv applying any conventional technique, such as spray drying, ultrafiltration, precipitation etc. Since the purity of raw extract can be substantially improved by the present process, the above procedures will be facilitated accordingly.

Claims: CLAIM A process for the preparation of pure raw extract of p-amylase from grains, particularly from grains of barley, c h a r a c t e r i z e d in that the extraction is effected from whole or partially hulled grains, the weight of the removed hull material being 0 - 20 % of the weight of a grain, the extraction temperature being 20 - 550C, and the extraction time exceeding 5 hours.

Inventors: LEHMUSSAARI, ANTTI, MANSIKKAMAKI, AINO, Mansikkamäki, Aino
Applicants: SUOMEN NESTESOKERI OY
Application year: 1982
Main classification: C12N9
IPC codes: C12N9