DE2209530A1 - - Google Patents

Description

PATENT Attorney E.

DiPL-INQ. DR. IUR. DIPL.-ING. VOLKER BUSSE DIETRICH BUSSE

45 Osnabrück »February 28th, 1972

MOSERSTRASSE 2O / 24 D B / Ka

2 ono con

A.B.M. INDUSTRIAL PRODUCTS LIMITED Newark on Trent, Nottinghamshire, England

Process for the production of brew wort

The invention relates to a method for producing brewed wort.

In the normal production of wort, malt or a mixture of malt and unmalted grain is mashed with hot water and then filtered. The resulting piltrat contains soluble carbohydrates, which are made up of dissolved starch and soluble nitrogenous substances including proteins and their breakdown products. The Piltrat is a suitable fermentation agent for yeast in the production of Alcohol. Most of the nitrogenous substances in eggs The wort comes from the Mals, which was previously obtained by aera Malsverfahren. The soluble carbohydrate results from the action of the amylolytic malt enzymes on the starch in the malt and the starch in the unmalted grain "

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Therefore, any unmalted grain used in the process contributes primarily ζμ of the carbohydrate content in the wort.

If a satisfactory fermentation is to take place, it is essential to have the right balance between the wort Maintain carbohydrate and protein. However, since unmalted carbohydrate is cheaper than malt, most breweries use it a maximum carbohydrate / protein ratio with a seasoning appropriate to the quality of a product.

Recently, methods for producing wort have been proposed that avoid or minimize the use of malt and instead use commercial ones • A / amylase and commercial proteolytic enzymes to break down of protein and starch in raw or cooked grains.

A commercial enzyme is one that is industrially (e.g., by fermentation) in for use in industrial processes has been produced in a suitable shape. The enzyme as such can be used, for example, in the form of a solution or in conjunction with a solid Diluents can be used.

Treatment with et-amylase and proteinase is often followed by treatment with a saccharifying enzyme to convert soluble carbohydrates ^{- either by using Ζ * 1} amylase (as in malt or soy) or an amyloglucosidase enzyme, or both, depending

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according to the required degree of saccharification - to fermentable To saccharify sugars.

The object of the invention is now to create an improved method for producing wort.

It was found that seasoning can be prepared in an advantageous V / else using a thermostable, commercially available bacterial proseolytischen enzyme at a temperature v / ground for example, the 50 to 55 ⁰ C amount terminate above of eg normal working range for conventional proteolytic enzymes is located.

It has surprisingly been found that the thermostable proteolytic enzyme in comparison to conventional proteolytic enzyme creates higher solubility of protein.

Furthermore, by simultaneous treatment with the thermostable proteolytic enzyme and the starch-liquefying enzyme in the same Temperature starch liquefies and protein is converted into soluble, nitrogenous compounds, with the Temperature within a working range for the starch-liquefying enzyme and above the normal working range for conventional proteolytic enzymes. Thus, compared to previously proposed

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drive where starch liquefaction and protein conversion carried out at different temperatures can be shortened.

The thermostable proteolytic enzyme has an optimal activity at a temperature of eg 65 to 75 ° C, for example about 70 ⁰ C.

Examples of thermostable proteolytic enzymes include:

Proteinase T (ABM Industrial Products Limited, Woodley, Cheshire, England), originally developed by Daiwa Kasei K.K., Osaka, Japan under the names "Thermoase" or "Therraolysin" ,

Astra STl (Lake Medel AB, Sweden) Astra 1048 (Lake Medel AB, Sweden) Alcalase (Novo Industri A / S, Denmark) Bioprase (Nagase & Co. Ltd., Osaka, Japan) D66 / 72 (ABM Industrial Products Limited, England).

Proteinase T is preferred.

The inventive method for the production of wort from an aqueous pulp of starch and protein-containing plant material, in which starch is liquefied by treating the pulp with a starch-liquefying enzyme and protein to be dissolved

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Union, nitrogen-containing compounds is implemented, now provides a treatment at a temperature of about 55 ⁰ C with a thermostable commercial proteolytic enzyme.

The invention also provides a simultaneous treatment of the pulp with a starch-liquefying enzyme and a thermostable enzyme commercial proteolytic enzyme at a temperature that is within a working range for the starch liquefying Enzyme and is above the normal working range for conventional proteolytic enzymes.

It has been found that the apparent attenuation of a wort produced by the method according to the invention increased and the subsequent fermentation of the wort is facilitated. Also, the taste of from such a seasoning has brewed beer quite unexpectedly compared to a sample using conventional proteolytic enzyme as better exposed. Furthermore, by carrying out the proteolysis at a higher temperature, there is a possible risk of infection reduced to a minimum.

Preferably, the proteolytic '-ic the enzyme ^ Ba ^ i thermoproteolytikus Rokko, £, B. of the aforementioned PPofceinaso won.

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- O -

In a modified method according to the invention, instead of carrying out the starch liquefaction at the same time and protein conversion essentially liquefies the starch completed before the Prottinum enclosure.

Examples of plant substances containing starch and protein except Grains are roots, sponge or fungus materials, and by-products of processes to which grain has been subjected.

Other examples of suitable materials are both sorghum, Tapioca and rice, as well as wheat, barley and corn.

The invention further relates to a wort produced by a method according to the invention, a method for brewing of beer in which the wort according to the invention is fermented by yeast and a beer produced in this way. Also creates the invention relates to a method for producing a seasoning syrup in which the seasoning according to the invention is concentrated, and relates to a concentrated seasoning syrup produced by such a process.

A commercially available one is preferably used as the starch-dissolving enzyme bacterial c6 amylase derived, for example, from the following Sources can be obtained:

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Bacillus subtilis Bazillusmegaterium Bazilluscereus Bazillus amyloliquefaciens Bacillus Polymyxa

Instead, the starch-liquefying enzyme can also be created using malt, for example.

The treatment with proteolytic enzyme is preferably carried out at a temperature of 60 to 75 ⁰ C.

If starch liquefaction is carried out before protein conversion, for example, the temperature of the proteolysis is advantageously 60 to 70 ° C. and preferably about 62 ° C., and the pH is advantageously between 5 and 6 and preferably about 5.5.

The invention also provides a method for producing wort from an aqueous mash of unmalted barley, in which starch is first liquefied by treating the mash with ^ -amylase and then the mash at a temperature of 60 to 70 ⁰ C with a thermostable, commercially available proteolytic, Enzyme obtained from the Bacillus thermoproteolytic Rokko.

If the slurry simultaneously treated with the strengthening liquefying ends or -aufschließendem enzyme, and the proteolytic enzyme, the treatment is preferably at a temperature of 63 ° to 75 ° C, for example at 65 to 70 ° C, and preferably at about 65 ⁰ C

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carried out, the pH value between 5> 5 and 8.5, e.g. between 6.5 and 8.5> and preferably about 7 »5.

The invention further comprises a process for the preparation of wort from an aqueous slurry of ungemalzter barley, liquefied at the strength and at the same temperature protein is converted to soluble stick--containing compounds by passing the slurry with simultaneously at a temperature of 65 to 7O ⁰ C. ^ -Amylässe and a thermostable commercially available proteo-Iytic, from the Bacillus thermoproteolyticus Rokko gwonnenen enzyme is treated.

A standard meal from raw grain or raw grain and malt would usually encode 0,52 conventional proteolytic enzyme and 0.5% "t-Amyläse require standard type respectively, and followed a 4-hour period at 55 ⁰ C and a 4 hour period at 65 to 70 ⁰ C need to liquefy starch and to dissolve protein. In the method according to the invention, however, be used simultaneously for the same type heights a thermostable proteolytic enzyme and £ ° -Amyläse in which, only 4 hours digestion at 65 to 70 ° C _(necessary to achieve the same results.

In a method embodying the invention Example Rohkorn or a mixture of Rohkorn and malt is dry or wet

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milled and processed at a temperature below about 55 ° C with water to a smooth, non-gelatinizing paste, whereupon the temperature is raised to 65 ⁰ C. Both commercially available ^ -amylase and commercially available thermostable proteinase are then added and digestion is made possible until adequate starch liquefaction and conversion of protein into soluble nitrogen-containing compounds has taken place. If necessary, the temperature is then raised to 70 to 75 ° C to complete the liquefaction of the starch and the protein solution. If the content of fermentable sugar now sufficient, the temperature is lowered to 60 to 65 ⁰ C and can solve xtrinen a saccharifying enzyme, the fermentable sugar from D ^e added and forge constitutes digestion.

The saccharification enzyme can be a commercially available enzyme or e.g. consist of P-amylase added in the form of ground Mals. Commercially available F-amylase can be obtained from soy, for example will. Other saccharification enzymes, e.g. amyloglucosidase, can be used.

The saccharification enzyme is preferably used at a temperature of 55 to 60 ^° C. and a pH of 4.5 to δ. However, if amyloglycosidase is used, it can be useful to work »outside this pH range.

After the enzyme treatment, the resulting wort is

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filtered and / or centrifuged and then fed directly to normal processing for producing beer. One Another possibility is, for example, to filter the wort and evaporate it into a syrup, which is stored and later for formation a wort is diluted for normal subsequent processing. The wort is preferably filtered without prior cooling.

In a further example of the method according to the invention, the initial grist consists of a mixture of unmalted grain and malt. The malt is first treated with an aqueous preparation of the - sprayed Amyläse and proteinase and the resulting malt / enzyme preparation is added to the raw or cooked cereal at a temperature of 60 to 7O ⁰ C, preferably a temperature of 65 ⁰ C was added - ^ /. Here, the preferred lower temperature limit of 6O ⁰ C lower than in the case of ungemalztem cereals, in which the preferred lower temperature limit is 63 ⁰ C. The process is then continued in the manner described above.

In another example of the method according to the invention proteolytic enzyme is added to normal brewing malt or a mash of malt and unmalted grain and the wort is added to it manufactured in a different conventional manner not using commercial enzymes. As the proteolytic enzyme during of the entire + mashing temperature range (from 55 to ♦ to

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75 ⁰ C, with the optimum value at 65 ⁰ C) is relatively stable, the malt protein is made widely soluble by the action of the proteolytic enzyme. Unexpectedly, the additional malt protein that has been made soluble has the normal level of high and low molecular weight proportions expected in the wort, so that the protein / carbohydrate ratio in the wort is increased. Accordingly, in order to restore the protein to carbohydrate ratio or balance to the level most desirable for a particular brewing process, the mash can be fortified with additional unmalted grain such that a greater than normal amount of unmalted carbohydrate material is used.

If, in addition, malts with a low content of soluble protein have to be used in brewing, e.g. barley worse Quality for malt purposes is used, the addition of a thermostable proteinase during mashing leads to a solution of additional protein and brings the protein / carbohydrate ratio to the desired level.

In the following examples, certain units are used to express the enzyme activity. These units will be defined as follows:

1. Xn units

For a proteinase preparation, an activity of 36 Xn single

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units per gram assumed if 5 ml. of a 2 gr / ltr. Concentration of an enzyme in action on 10 ml. Of an aqueous "Hammerstein" casein solution of 2 parts by weight at a phosphate-buffered pH of 6.5 and a temperature of 35 ° C, a total of ¹ l milligrams of Ίϊ trichloric acid solution Generate soluble nitrogen in 30 minutes.

2. X units

A bacterial ^ -Amylasepräparat has an activity of lX-Einheitpro grams when 250 milligrams of the preparation to 1 gr dry weight Parinastärke in a total volume of 55 m at 30 ⁰ C and a pH of 6.0 (phosphate buffer) to react in such a way that Dextrinization is achieved in 15 minutes. "Dextrinization" means when the color obtained by adding 1 ml of the above reaction solution to 5 ml of 0.0007 N iodine solution corresponds to a standard color. This color can ^{be checked visually using a II} Lovibond "color knife disc No. ^ / 29» ASBC standard for amylase in malt or, preferably, read off on a spletrophotometer with 1 cm glass cells at a wavelength of 6175 A ⁰ against distilled water, when the optical density is 0.800.

3. Tyn units

One Tyn unit is the amount of enzyme activity that occurs at Reaction with 200 milligrams of Hammerstein casein in a Qe

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total volume of 15 ml buffered with phosphate to a pH value of 6.5 and at a temperature of 35 ° C, over a reaction period of 30 minutes per minute, a microgram tyrosine equivalent of total nitrogen soluble in ^% trichloroacetic acid solution generated.

1 K Tyn = 1000 Tyn and 1 Xn = 5860 Tyn

4. Tys units

A Tys unit is the amount of enzyme activity that is produced when reacting with 200 milligrams of Hammerstein casein in a total volume of 15 ml, buffered with phosphate to pH 6.5 and at a temperature of 35 ° 0, over a reaction period of 30 minutes per minute produces a microgram of tyrosine equivalent of flavoring nitrogen compounds that are soluble in 4? -trichloroacetic acid solution, as by measuring the optical density in learning silica cells against a reagent sample is determined at a wavelength of 275 0A °.

1 K Tys κ 1000 Tys
EXAMPLE I

Comparison of 100 ^ -Ger Digestion Using a Conventional Bacterial Proteinase and a thermostable bacterial proteinase.

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Experiment A - Normal bacterial proteinase

A watery pulp made from ground barley was 0.5? Proteinase 36Xn (36 Xn units / gr. - marketed by ABM Industrial Products Limited) and 0.5? Nervanase (// - Amylase 9X units / gr. - marketed by ABM Industrial Products Limited) added, both enzyme weights referring to the original barley weight. The digestion was held at 55 ° C. (proteolytic level) for 4 hours and at 65 ° C. (amylolytic level) for 4 hours. The resulting wort was filtered and, after adjusting to a gravity of 1.040, gave the following analysis:

Extract yield 73.6?

Sham steaming of the wort 78.1?

Total nitrogen of the wort 82.6 mg / 100 ml

* t Aminos ticks off the wort 19.4 mg / 100 ml

Proteinase activity time product = O ₁ S χ 36 χ 8 = 1.44

Ϊ00

Experiment B - Proteinase T

An aqueous pulp from ground barley with a temperature of 65 ⁰ C was 0.5? Proteinase T (36 Xn units / gr.) And 0.5? Nervanase («UAmylase 9X units / gr.) Added, both enzyme weights referring to the original barley weight. The digestion was held at 65 ^° C. for 4 hours and the resulting

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The wort that had stood was filtered. After adjusting the wort on a specific weight of 1.040 resulted in the following analysis:

Extract yield 73.1 %

Wort steaming 79, OJi

Wort total nitrogen 88.7 mg / 100 ml

Wort- ⁰ ^ amino nitrogen 20.1 mg / 100 ml

Porteinase activity time product = OJj χ 36 χ 4 = 0.72

100

A comparison of the results from experiments A and B shows that the seasonings are very similar to one another. However the proteinase activity time product in experiment B is about half that given in experiment A. As a result, results that by using the thermostable proteinase as compared to the method using a normal Proteinase can either double the plant utilization or reduce proteinase activity by half. In a similar way Thus the «t-amylase activity time product is correspondingly Experiment B half that of experiment A.

EXAMPLE II

Use of heat-resistant proteinase to increase the wort nitrogen content .

It has a malt wort by mashing two hours by a

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Part by weight of ground malt with seven parts by weight of water at a temperature of 65 ⁰ C prepares. The wort was collected by filtering. A similar mash was made for comparison purposes. However, Q.5% proteinase T (46 Xn units / gr.) Was added based on the weight of the malt at the start of mashing. The condiments from both procedures were analyzed and the following results noted:

yield

Apparent attenuation %
Total nitrogen mg ^
Lundin fraction A %
Lundin fraction B %
Lundin fraction C %

Normal mash
(Control) Mash +
Proteinase T 78.5 79.1 81.9 82.3 100 ml 78.4 90.1 24.6 25.1 10.2 10.8 65.4 64.1 100ml 20.1 23.6

From these results it can be seen that the addition of Proteinase T to the mash increases the level of nitrogen solubilization while maintaining the total nitrogen spectrum of the wort. As can be seen, the apparent attenuation is lower in the control approach.

EXAMPLE III 100? Barley fermentation using by Bi oprase

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An aqueous slurry of ground barley with a temperature of 65 ⁰ C were 0,1883t Bioprase PNlO (100 Xn units / gr.) And 0.5 £ Nervonase (^ 9X-amylase units / gr.) Was added, both enzyme weights to refer to the original barley weight. The digestion was held for 4 hours at 65 ⁰ C and the resulting wort then filtered. The wort was adjusted to a specific gravity of 1.040 and analyzed as follows:

Ex tr act yield 74.0 %

Wort-mock steaming $ 77.8

Wort total nitrogen 80.2 mg / 100 ml

Wort-o ^ amino nitrogen 17.4 mg / 100 ml

Ptoteinase activity time product; 0.188 χ 100 χ 1 = 0.75

100

EXAMPLE IV 100% barley fermentation using D66 / 72

An aqueous slurry of ground barley with a temperature of 65 ⁰ C, O, 25Z, D66 / 72 (72 Xn units / gr) zuz üglich-05? Nervanase (06-amylase 9X units / gr) added, both enzyme weights referring to the original barley weight. The digestion was held at 05 ° C. for 4 hours and the resulting wort was then filtered. The wort was adjusted to a specific gravity or extract content of 1.040 and analyzed as follows;

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Extract yield 72.82 Wort steaming 77.9 * Wort total nitrogen 78.4 mg / 100 ml Wort - «v amino nitrogen 11.9 mg / 100 ml Proteinase activity time product: 0.25 72 4: 0.72

100

Example V

Fermentation scheme for seasonings prepared with thermostable proteinase

A control wort and a thermostable proteinase wort became using the procedure given in Example II. A fraction of 22 ml. Of each wort was taken and this one 0.5 gr pressed yeast added. The rate of permeation determined by the decrease in specific gravity was measured.

0 hours 12 hours 24 hours 36 hours 48 hours

Normal wort 1.040 1.035 1.021 1.009 1.007 (control)

thermostable 1.040 1.032 1.014 1.006 1.006 proteinase wort

From these results it can be seen that using the thermostable proteinase prepared wort at a greater rate and fermented to a greater extent than the control wort.

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- 19 EXAMPLE VI

Use of thermostable proteolytic enzymes for digesting mixed grist or grist from barley and malt

Experiment A - Conventional Proteinase

A watery pulp from 50? ground barley and 50? ground malt was 1.0? Proteinase 36Xn (36 Xn units / gr) plus 1.0? Nervanase (6-amylase 9X units / gr), added, whereby both enzymes are related to barley weight. The digestion was held for two hours at 65 C, the resulting wort then filtered and adjusted to a specific weight of 1.0M0 °. The analysis of the wort was as follows:

Extract yield 76.9?

Wort steaming 81.7?

Wort total nitrogen '70.6 mg / 100 ml

Wort- ^ amino nitrogen 13.7 mg / 100 ml

Experiment B - thermostable proteinase

A watery pulp from 50? ground barley and 50? ground malt was 1.0? Proteinase T 36 (36 Xn units / gr) plus 1.0? Nervanase (oL-amylase 9X units / gr) added, whereby

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both enzymes relate to barley weight. The digestion was held at a temperature of 65 ^° C. for two hours, and the resulting wort was then filtered and adjusted to a specific gravity of 1.0 ^ 0. The analysis of the wort was as follows:

Extract yield 77.3Z

Wort steaming 82.9 *

Wort total nitrogen 8l, 6 mg / 100 ml

Wort- o ^ amino nitrogen 20, ¹ J mg / loo ml

Replacing the normal proteinase enzyme with an activity equivalent thermostable proteolytic enzyme has resulted in a more complete breakdown of the protein in the meal.

EXAMPLE VII

Comparison between a wet-milled digestion of 95 * barley and 5ί malt using a conventional proteinase and a thermostable proteinase.

Experiment A - Conventional Bacterial Proteinase.

The barley was wet-milled and processed at a temperature of 53 ⁰ C with water to a pulp. There were eine06-Amyläse (Nervanase 10X) and a proteolytic enzyme (proteinase 36Xn) was added in each case based with a concentration of 1 wt. J weight on the device. The mash was left on for 30 minutes

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Maintained temperature of 53 ⁰ C (proteolytic level) and the temperature was then increased to 72 ° C and held at this level for 40 minutes (amylolytic level). The mash was then cooled down to 62 C and 53 »ground malt was added. After one hour the temperature was increased to 75 °. This temperature was held for 15 minutes and then the wort was collected by filtration. The total process time during which the enzymes reacted with the grain, including the times to reach the various temperatures, was 175 minutes. The Wür analysis was as follows:

Extract yield 73.4%

Wort steaming 73.8 *

Total nitrogen 113.6 mg / 100 ml

ot amino nitrogen 19.9 mg / 100 ml

Proteinase activity time product 1.0 36 175 = 1.05

100 60

Experiment B - thermostable proteinase

The barley was wet-ground and made into a pulp with water. A 06 amylase (Nervanase 10X) was added at a concentration of 1.0% (based on barley weight). A thermostable + (proteinase T 36) was also added in a concentration of 0.5% (in relation to the barley weight). The temperature was quickly increased to 65 ^° C. This temperature was held for 60 minutes. The mash temperature was then increased to 72 ⁰ C, ♦ proteinase

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Held for 10 minutes and then cooled down to 62 ° C. That ground malt was added and after an hour the Temperature increased to 75 ° C. The wort was then obtained by piling. The total process time was again 175 minutes. The wort analysis was as follows:

Extract yield 7 ^, 7% Wort steaming 12.9% Total nitrogen 168 mg / 100 ml

oL-amino nitrogen 23.9 mg / 100 ml

Proteinase activity time product : 0.5 x 36 χ 175 ^{s 0.525}

100 1 TO

A comparison between experiments A and B shows "that that Proteinase activity time product has been decreased and protein digestion has been improved by the use of the thermostable enzyme.

EXAMPLE VIII

Comparison of conventional beers made from worts that have been prepared using a conventional and a thermostable proteinase.

60 l of wort was prepared using the methods described in Example I, Experiments A and B, given in the procedure, and the wort was each prepared to obtain a tartness value of ΊΟ p.p.m. Isohumulone with

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Boiled hops. The worts were ^{cooled down to 16 °} C. and pressed yeast was added to the wort in a ratio of 3.5 g / l. The respective brew was then allowed to ferment for a total of 5 days and the beer was filtered off from the yeast and analyzed.

Beer analysis

Normal thermostable proteinase proteinase

Original specific gravity or extract content 1.040 1.040

Final specific gravity or extract content 1.007 1.006

Total nitrogen mg / 100 ml 46.8 53.9

Color ⁰ EBC "11.0 12.5

pH 4.0 3.9

Foam retention ■ £. lake. 110 108

Isohumulone p.p.m. 28.5 28.0

Taste rating 6 8

The figures for the final extract content are again worth noting. Unexpectedly, the taste of the was out of use thermostable proteinase-derived beer is significantly better than that of beer brewed by normal methods. The difference was noticeable in that it lacked the severe bitter aftertaste that is often found in brewed with normal proteinase Beers occurs.

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EXAMPLE IX

Comparison between thermostable proteolytic enzymes and a conventional proteinase in the breakdown of the protein in pre-gelled cereals ■

Four individual 50 g samples of finely ground barley were mixed with 150 ml of water and 1? (in relation to the barley weight) Nervanase 1OX (starch dissolving «c-amylase) mashed. The temperature was raised for one hour fast at 70 °, followed by one hour at 80 ⁰ C. The slurry which was added (36Xn proteinase) the normal proteinase was cooled down to 55 ⁰ C and the other mashing, where thermostable enzymes were added, were cooled down to 62 ° C. A proteolytic enzyme concentration equivalent to 1% proteinase 3öXn (in relation to the barley weight) was added to all mashes. Digestions were then allowed for 6 hours, after which each sample was tested for total _{soluble nitrogen (TSN 2} ) and amino nitrogen (<£ amino N) content. The results are summarized in the following table:

Table of the degree of protein solubilization in gelled barley using different droteolytic enzymes

Enzymes 6 hours

Ji Amino N

7.9 10.7

8.8 11.9

TSN ₂ Proteinase 36 Xn 47 Bioprase PN 10 58 A.B.M.I.P. D66 / 72 51 Proteinase T 36 55

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- 25 The values are expressed as mgN./100 ml of digestion.

These results show the improved solubilization of protein when using the thermostable enzymes, regardless of the fact that all enzymes have the same activity levels and found application under optimal pH and temperature conditions. This also results in the superior mode of action of Proteinase T compared to the other thermostable enzymes.

Further, by using a thermostable enzyme and digesting it at a higher temperature, the system has been shown to be more biologically stable. Thus, after 6 hours of digestion, for example, dropped at 55 ⁰ C the pH of the reaction mixture of 5 »8 to 4.9, whereas at 62 ⁰ C the pH-value of 5.8 to 5.6 was reduced.

EXAMPLE X

Characterization of Proteinase T (Daiwa Kaisei ¹ S Thermoase or Thermolysin) ; ,

description

Proteionase T is a proteolytic enzyme obtained from the Bacillus thermoproteolytikus Rokko and has excellent thermal stability and has substrate specificity.

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Enzymatic properties Molecular weight: 37 _: 25 ⁰ C 2209530 .500 (approximate) 10.0 ⁵ C Residual activity (a) Optimal pH standard 35 ° C 1 Tys at 35 ⁽ 20.0 , 2 100t - 26 - (b) PH: 5.5 6.5 55 ° C 8.5 10 48.9 ,8th 1002 K Tys: 10.0 20.0 60 ⁰ C 21.2 4 60.2 at pH 6.5. 100% Optimal temperature - 7O ⁰ C Standard Tys 81.2 98% (C) K 8O ⁰ C Tys 66.0 94% Temperature stability - • Enzyme solution 49% O heated at pH 5.5 over a period of 30 minutes before testing at pH 6.5 to 35 ^° C. (d) Heating temperature 55 ° C 60 ° C 65 ° C 70 ⁰ C 75 ° C 80 ⁰ C 85 ° C Ratio Ty_n = 33
. Tys (β)

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(f) Inhibitors: Phospaht, E.D.T.A.

(g) Activator: calcium

For further identification, express reference is made to the publications listed below, the content of which is the subject matter to form the description.

(a) Matsubara, H., Singer, A., Sasaki, R., and Jukes, T.H., Biochem.Biphys. Res. Commun. 2JL ^ 242 (1965)

(b) Endo, S., J. Fermentation Tech., JjO 3M6 (1962)

(c) Ohta, Y., Ogura, Y. and Wada, A., J. Biol. Chem., 5919 (1966)

(d) Biotechnology and Bioengineering Volume 12 No. 2/1970.

EXAMPLE XI Characterization of Astra STl description

Astra STl is a thermostable proteolytic enzyme, obtained from Streptomycesgriseus.

Enzymatic properties

(a) Optimal pH - Standard Tys at 35 ° C pH 5.5 6.5 8.5 10.0 K Tys 9.0 20.0 35.0 28.6

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- 28 - 2209530 at pH 6.5 C. Tys
, 5 t 35 ⁰ C • (b) Optimal Temperature - Standard Tys K
"HM
9 , 0 temperature
25 ° C 20th , 0 35 ° C 45 , 4 50 ⁰ C 34 , 2 6O ⁰ C 36 , 6 70 ° C 27 , 0 8O ⁰ C 23 , 6 85 ⁰ C 9 90 ⁰ C (c) Ratio Ty_n = 30
Tys (d) Inhibitors: phosphate, E.D.T.A. EXAMPLE XI] Marking of Astra 1048 Astra 1048 is a thermostable proteolytic bacterial description Enzyme. Enzymatic properties (a) Optimal pH - Standard Tys at pH 5.5 6.5 8.5 10.2 K Tys 10.0 20.0 35.8 23.6

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Optimal temperature - i 2209530 - 29 - Residual activity ;, Ξ.Ο.Τ.Α. temperature Standard Tys at pH 6.5 ,. (b) 25 ° K Tys 45 * 35 ° 9.0 38 * 45 ° 20.0 20 * 50 ° 43.8 0 55 ° 56.2 65 ° 64.6 70 ° 113.6 75 ° 115.0 80 ° 126.0 Temperature stability - 55.0 Enzyme solution heated over at pH 5.5 (c) a period of 30 minutes before testing at pH 6.5 to 35 ^° C. Heating temperature 55 ⁰ C 6O ⁰ C 65 ⁰ C 70 ° C 75 ° C Ratio Ty_n = 49.4
Tys (d) Inhibitors: phosphate (e) Activator: calcium (f)

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EXAMPLE XIII

Characterization of Alcalase, Bioprase and D66 / 72 description

Alcalase and 066/72 are thermostable proteolytic enzymes obtained from the bacillus subtilis. Bioprase is a thermostable proteolytic enzyme that is obtained from the Bacillus alieniformis. The other distinctive features of the three enzymes are essentially the same.

Enzymatic properties

(a) Molecular weight: 27,000-30,000

(b) Optimal pH - Standard Tys at 35 ° C pH: 5.5 6; 5 8.5 10.2 K Tys: 7.0 20.0 31.8 44.0

(c) Optimal temperature - standard Tys at pH 6.5

K Tys 10.0 20, o 93.0

144.6 118.4

(d) Temperature stability - Enzyme solution heated, at pH 5> 5

temperature C. 25 ° C. 35 ° C. 55 ° C. 65 ° C. 70 °

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- 31 for a period of 30 minutes before testing at pH 6.5

35 ⁰ C Heating temperature Residual activity 55 ⁰ C 92 * 60 ° C 77% 65 ⁰ C 7456 70 ° C 26 * (e) Ratio Τχη = 58
Ty s (f) Activator: calcium

209838/0121

Claims (9)

Patent claims: 1. A method for producing wort from an aqueous pulp of starch and protein-containing vegetable matter (e.g. barley), in which starch is liquefied or dissolved by treating the pulp with an etark-liquefying enzyme, e.g. commercial bacterial ^ -amylase, and protein with a proteolytic enzyme to soluble, nitrogen-containing compounds is converted, characterized by a treatment at a temperature above about 55 ° C (preferably between 60 and 75 ⁰ C) hen-handelsüblic with a thermostable proteolytic enzyme. 2. The method according to claim 1, characterized in that a proteolytic obtained from the Thermoproteolytikus Rokko Bacillus Enzyme is used. 3. The method according to claim 1, characterized in that a product obtained from Streptomycesgriseus proteolytic enzyme is used. 4. The method according to claim 1, characterized in that a proteolytic enzyme obtained from the subtilis or licheniformis bacillus is used. 5. The method according to any one of claims 1 to ² I, characterized in that by simultaneously treating the pulp with the starch-liquefying enzyme and the proteolytic enzyme starch 209838/0121 dissolved or liquefied and at the same temperature · protein is converted to soluble, nitrogen-containing compounds. 6. The method according to any one of claims 1 to 4, characterized in that the starch liquefaction is essentially completed before the protein conversion. 7. The method according to any one of claims 1 to 5, characterized in that that as a starch liquefying enzyme, a commercial enzyme and also malt is used with a aqueous preparation of the commercially available starch-liquefying enzyme and the proteolytic enzyme is sprayed. 8. The method according to any one of claims 1 to 6, characterized that the watery pulp from a normal brewing mash consists, which comprises malt with the addition of commercially available, proteolytic enzyme, the malt being the starch-liquefying Enzyme forms. 9. The method according to claim 8, characterized in - that the watery pulp is enriched with additional unmalted grain will. 0 9 8 3 3/0121 ORIGINAL INSPECTED