DE2209530B2 - Process for the production of an aqueous extract, in particular of wort - Google Patents

Description

Recently there are processes for the production of sorghum and tapioca.

Seasoning has been suggested which has a use as a starch dissolving enzyme being a commercially available one

Avoid malt or prefer to minimize bacterial α-amylase, which z. B. from the

Bacillus subtilis, Bacillus megaterium, Bacillus cereus, work being thinned. The wort is preferred Bacillus amyloliquefaciens or Bacillus polymyxa filtered without prior cooling,

can be won. Instead, however, in another application example, the

-starch-liquefying enzyme z. B. also ground material or grist from a mixture that is started by malt

-will create. 5 made from unmalted grain and malt. The malt is

If the starch liquefaction is carried out before the protein, an aqueous preparation dei a-amylase is used

carried out, the temperature of the and proteinase is sprayed, and the resulting MaIz-

Proteolysis is advantageous at 60 to 70 ⁰ C and preferably enzyme preparation is the raw or cooked food

about 62 ° C, and the pH value advantageously between 5 grains at a temperature of 60 to 70 ⁰ C, preferably

and 6 and preferably at about 5.5. io wise a temperature of 65 ° C, added. Here

If an aqueous slurry, in particular one made from, is the preferred lower temperature limit of 60 ° C

malted barley, at the same time as the starch liquefies - lower than in the case of unmalted grain

the or digesting enzyme and the proteolytide the preferred lower temperature limit 63 ° C

If the enzyme is treated, then the treatment is given. The procedure is then described in above

preferably at a temperature of 63 to 75 ° C, e.g. B. 15 described manner continued

5ei65 ° to 70 ⁰ C, and preferably at approximately 65 C transit In another application example, the

leads, the pH-wide between 5.5 and 8.5, z. B. proteolytic enzyme a normal brewing malt or

»Between 6.5 and 8.5, and preferably around 7.5. a mash of malt and unmalted grain

A standard meal made from raw grain or raw set and the seasoning in one, not commercially available

tetwide and malt would normally use 0.5% her- ao enzymes, conventional other way

conventional proteolytic enzyme and 0.5% "-Amy". Since the proteolytic enzyme during the

each with standard activity and a total temperature found for mashing

jLStunden period followed at 55 ° C and of a region (of 55 to 75 ⁰ C, with optimum value at 65 C)

4-hour period at 65 to 70 ⁰ C need to be ielativ stable, the malt protein is through the

To liquefy starch and to dissolve protein. With the action of the proteolytic enzyme it can still be released Method according to the invention, however, in which made. Unexpectedly, the additional MaIz

At the same time, the thermostable proteolytic enzyme protein that has been made solvable is normal

and «-amylase used at the same levels of activity in the proportions to be expected in the wort

are only 4 hours of digestion at 65 to high and low molecular weight, so that

70 ⁰ C is necessary to achieve the same results. 30 Protein-carbohydrate ratio in the wort increased

2-hen will. As a result, the mash can be used for reconstitution

According to an example of performing or setting the ratio or equilibrium of Applying the method of the invention turns protein into carbohydrate to the value required for a Raw grain or a mixture of raw grain and malt special brewing process is most desirable,

Rohkom Or an IVLlSvlluiig au »imjuhuiu uiiu ινιαίί. spvuvuvs umuTviiu.iivu —— ■ ·

ground dry or wet and mixed with additional unmalted grain at a temperature of 35

below about 55 ° C with water to a smooth, not enriched, that a larger proportion of un-

Processed gelatinizing paste, after which the Tem- malted carbohydrate substance is used as usual

temperature is increased to 65 ° C. If also malt types with a low

exercise «-amylase and thermostable proteinase add-on soluble protein content used in brewing

under about 33 ^ mil of water tu a giaucii, uiviii i ^ iwiviι vtviuvu, - ~ w ν. « _β

gelatinizing paste processed, after which the Tem- malted carbohydrate substance is used as usual, temperature is increased to 65 ° C. If also malt types with a low

Usual α-amylase and thermostable proteinase increases the soluble protein content used in brewing and allows digestion until an adequate 4 »must be achieved, e.g. B. Poor quality barley for starch liquefaction and conversion of protein 5-.u malt purposes is used, the addition of soluble nitrogen-containing compounds has occurred. the thermostable proteinase during mashing for necessary, the temperature then raised to 70 a releasably making additional protein and up to 75 ⁰ C to the liquefaction of the starch, the protein-carbohydrate ratio and brings to the complete the protein solution. Is the desired level.

content of fermentable sugar is now sufficient. In the following examples, certain

so the temperature is lowered to 60 to 65 ° C and units for expressing the enzyme activity combine saccharification enzyme that turns fermentable sugar. These units are defined as follows: can dissolve from dextrins, added and the dige-, _v _. . .

ration continued. 50 1st Xn units

The saccharification enzyme can be a commercially available one. A proteinase preparation becomes an activity

Be an enzyme or e.g. B. assumed in the form of ground of 36 Xn units per gram if / 9-amylase added to malt. Commercially available 5 ml of a 2 g / l concentration of an enzyme in one-0-amylase can e.g. B. obtained from soy. effect on 10 ml of an aqueous "Hammerstein" Other saccharification enzymes, e.g. B. Amyloglu- 55 casein solution of 2 percent by weight in a phosphosidase can be used. phate-buffered pH value of 6.5 and a temperature

The saccharification enzyme is preferably used at 35 ° C, a total of 4 milligrams, a temperature of 55 to 60 ° C and a pH of 4.5 to 6 in 4% trichloric acid solution in 30 minutes. If, however, amylogluclear nitrogen is used to generate sidase, it may be appropriate to outside 6 ° - _v _. . .

this pH range to work. ^L - * inside

After the enzyme treatment, the resulting A bacterial amylase preparation has an active

Wort eg filtered and / or centrifuged and an intensity of 1X unit per gram, if 25OMilliII finally fed directly to normal processing to the gram of the preparation to 1 g dry weight of Farina production of beer. Another possibility 65 strength in a total volume of 55 ml at 30 ⁰ C is z. B. is to filter the wort and to a and a pH of 6.0 (phosphate buffer) to evaporate syrup in such a way that stored and later react to BiI- that dextrinization in 15 minutes is achieved by a wort for a normal subsequent Ver - is. "Dextrinization" is to be understood when the

by adding 1 ml of the above reaction solution Experiment B - Proteinase T to 5 ml of 0.0007 N iodine solution obtained color a

Standard color corresponds. This color can be applied to a watery pulp made from ground barley

a »Lovibonde color knife disc No. 4/29, a temperature of 65 ° C was 0.5% proteinase T

A. S. B. C standard, for amylase in malt visually 5 (36 Xn units / g) and 0.5% nervanase («-amylase

are checked or preferably added to a Spktro 9 X units / g), both enzyme

phqiometer with 1 cm glass cells at one wavelength rebalanced to the original barley weight

can be read from 6175A against distilled water. The digestion was at 65 ^° C. for 4 hours

when the optical density is 0.800. held, and the resulting wort was filtered.

ίο After adjusting the wort to a specific gravity of 1.040

3.Tyn units resulted in the following analysis:

One Tyn unit is the amount of enzyme activity, extract yield 73.1%

the reaction with 200 milligrams of Hammerstein wort mock steaming 79.0%

Casein in a total volume of 15 ml, buffered 15 wort total nitrogen 88.7 mg / 100 ml

with phosphate to a pH value of 6.5 and with a wort a-amino nitrogen 20.1 mg / 100 ml

Temperature of 35 ° C, proteinase activity time q 5 for a reaction period

a microgram tyro product of 30 minutes per minute - ^ - · 36 · 4 = 0.72

^{sine equivalent of 10} ° in 4% trichloroacetic acid

Solution generated soluble total nitrogen. so

When comparing the results from the experi-

1 ν τ ™, ιιϊϊιτ ™ "" j 1 ν "" Anτ "" ment A and B shows that the spices are mutually exclusive

1 K Tyn = 1000 Tyn and 1 Xn = 5860 Tyn. _sehf _ _similar ^ _{J (} f _but ^^ _{the proteinase} acti.

4. Tys units of the vity time product in experiment B about half of the

as stated for experiment A. It follows that

A Tys unit is the amount of enzyme activity obtained by using the thermostable proteinase

that when responding with 200 milligrams of Hammerstein- versus the method using

Casein in a total volume of 15 ml, buffered with a normal proteinase, either the plant

with phosphate to a pH value of 6.5 and doubled or the proteinase activity by when it is used

temperature of 35 ° C, can be reduced by half over a reaction period of 30. In a similar way

30 minutes per minute a microgram of tyrosine is the α-amylase activity time product

Equivalent of nitrogen containing aromatic substances - according to Experiment B half of that after Ex-

compounds produced in 4% trichloroacetic acid experiment A.

acid solution are soluble, as by measuring the opti- Example II

see density in 1 cm Siüka cells against a reagent using heat-stable proteinase

sample at a wavelength of 2750 A intended to increase the wort nitrogen content

1 K Tys Tys ^ = 1000 ^{s WUR <^ e eme} malt wort by two hours

Mashing 1 part by weight of ground malt with For the 1 I * ° 'parts by weight of water at a temperature of

65 ° C. The wort was caught by filtering to compare 100% fermentation. For comparison purposes, a similar one was made using a conventional mash. However, 0.5% Proteinase T was added to bacterial proteinase and a thermostable (46 Xn units / g) based on the weight of the bacterial Proteinase _4S malt at the start of mashing. The Wür experiment A - normal bacterial proteinase _zen from both methods were analyzed and the

noted the following results: A watery pulp made from ground barley

0.5% proteinase 36Xn (36 Xn units / g - of A. B. M. Industrial Products Limited) and 0.5% Nervanase ("-amylase 9 X units / g - from A. B. M. Industrial Products Limited placed on the market), both enzyme weights referring to the original barley weight. Digestion was carried out at 55 ° C for 4 hours (proteolytic level) and held at 65 ° C (amylolytic level) for 4 hours. The resulting Wort was filtered and after setting resulted in a;:? a weight (gravity) of 1.040 the following analysis:

Extract yield 73.6% Scheindämpfuög of the wort .. 78.1% Total nitrogen of the wort .. 82.6 mg / 100 ml ^From these results it can be seen that the addition

«-Amino nitrogen of the put of proteinase T to the mash the degree of

Wort 19.4 mg / 100 ml ⁶ S nitrogen solubilization while maintaining the Proteinase activity time - _{0 5} total nitrogen spectrum!) Of the wort increased. Like him-

product —— · 36 · ξ = 1.44 visible, is the apparent attenuation in the control approach

100 'lower.

Normal mash mash + Proteinase T. (Control) yield 78.5 79.1 Apparent attenuation,% 81.9 82.3 55 total nitrogen, 78.4 90.1 mg / 100 ml Lundin fraction A,% 24.6 25.1 Lundin fraction B,% 10.2 10.8 Lundin fraction C,% 65.4 64.1 6a «-amino nitrogen, 20.1 23.6 mg / 100 ml

Example III

Fermentation scheme for seasonings prepared with thermostable proteinase

A control wort and a thermostable proteinase wort were prepared using the procedure outlined in Example II. A 22 ml fraction of each wort was taken and added to this x ⁵ g of pressed yeast. The fermentation rate, as 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 the prepared using the thermostable proteinase Wort fermented at a greater rate and to a greater extent than the control wort.

Example IV

Use of thermostable proteolytic enzymes for digesting mixed grist or grist

from barley and malt Experiment A - Conventional proteinase

An aqueous slurry of 50% ground barley and 50% ground malt was given 1.0% pioteinase 36 Xn (36 Xn units / g) plus 1.0% Nervanase (α-amylase 9 X units / g), added, both Enzymes are related to barley weight. The digestion was held at 65 ° C for 2 hours The resulting wort is then filtered and adjusted to a specific gravity of 1.040 °. The analysis of the wort was like follows:

Extract yield 76.9%

Wort steaming 81.7%

Wort total nitrogen 70.6 mg / 100 ml

Wort-a-amino nitrogen ... 13.7 mg / 100 ml

Experiment B - Thermostable Proteinase

An aqueous slurry of 50% ground barley in 50% ground malt was given 1.0% proteinase T 36 (36 Xn units / g) plus 1.0% Nervanase (α-amylase 9 X units / g) added, whereby both enzymes relate to barley weight. The digestion was 2 hours at one temperature kept at 65 ° C, the resulting wort then filtered and adjusted to a density of 1.040. the Analysis of the wort was as follows:

Lyric enzyme caused the protein in the meal to be broken down more completely.

Example V

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

ίο Experiment A - Conventional Bacterial Proteinase

The barley was wet-ground and made into a pulp with water at a temperature of 53 ° C. It an a-amylase (Nervanase 10 X) and a pro-

teolytic enzyme (Proteinase 36 Xn) each with a concentration of 1 percent by weight, based on the barley weight, added. The mash was kept at a temperature of 53 ° C. for 30 minutes (proteolytic level), and the temperature was

ao then raised to 72 ⁰ C and held for 40 minutes this state (stand amylolytic). The mash was then cooled down to 62 ° C and 5% ground malt was added. After 1 hour the temperature was increased to 75 ° C. 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 wort analysis was as follows:

Extract yield 73.4%

Wort steaming ... 73.8%

Total nitrogen 113.6 mg / 100 ml

α-amino nitrogen 19.9 mg / 100 ml

Proteinase activity time- ._.

product illl. 36. _ = 1.05

100 60

Experiment B - Thermostable Proteinase

The barley was wet-ground and made into a pulp with water. An a-amylase (Nervanase 10X) was added at a concentration of 1.0% (based on the weight of barley) thermostable proteinase (proteinase T 36) in a concentration of 0.5% (based on the barley weight) added The temperature was quickly increased to 65 ° C. This temperature became 60 minutes held. The mash temperature was then increased to 72 ° C, held for 10 minutes and then to 62 ° C cooled down The ground malt was added and after 1 hour the temperature was increased to 75 ° C The wort was then obtained by filtering. The entire process time was again 175 minutes. The wort analysis was as follows:

Extract yield

Wort steaming ...
Wort total nitrogen ...
Wort-a-amino nitrogen - -

77.3% 82.9%

81.6 mg / 100 ml 20.4 mg / 100 ml

Replacing the normal proteinase enzyme with an activity equivalent of thermostable proteo-

Extract yield _T .. 74.7%

Wort steaming 72.9%

Total nitrogen 168 mg / 100 ml

«-Amino nitrogen 23.9 mg / 100 ml

Proteinase activity time- , _c _.

product M.36.175 _{= 052;}

100 1 60

509547/61

10

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

B e i s ρ i e 1 VI

Comparison of common beers made from wort,

prepared using a conventional and a thermostable proteinase cooled down and the other mash, which was added to the thermostable enzyme, was cooled down to 62 ⁰ C. A proteolytic enzyme concentration equivalent to 1% Proteinase 36 Xn (based on barley weight) was added to each mash. Digestions of 6 hours were then allowed, after which each sample was tested for soluble total nitrogen (TSNj) and α-amino nitrogen-ia-amino N- content. The results are summarized in the following table:

601 wort were prepared using the procedures outlined in Example I, Experiments A and B, and the wort was each boiled with hops to obtain a tartness value of 40 ppm isohumulones. The wort was 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 by the yeast ^ao Proteinase 36 Xn and analyzed.

Table of the degree of protein solubilization in gelled barley when using proteolytic Enzymes

Enzymes 6h

Proteinase T 36

TSN ₂

47

55

«-Amino N 7.9 11.9

Beer analysis

Normal proteinase

The values are as mgNi / 100ml of digestion 35 expressed. Thermostable These results show the improved releasability

Proteinase

Original weight or
Extract content 1.040 1.041 Final weights or
Extract content 1.007 1.0Oi Total nitrogen,
mg / 100 ml 46.8 53.9 Color ⁰ EBC 11.0 12.5 PH 4.0 3.9 Foam retention Σ sec 110 108 Isohumulones p. p. m. 28.5 28.0 Taste rating 6th 8th

The figures for the final extract content are again remarkable. Unexpectedly it was The beer obtained from the use of thermostable proteinase tastes significantly better than that of the beer brewed according to normal procedures. The difference was noticeable in that the severe bitter aftertaste often found in beers brewed with normal proteinase was absent.

making protein using the thermostable enzyme, regardless of the fact that both Enzymes at the same activity levels and under opti · paint pH and temperature conditions appli found.

The system has further improved by using the thermostable enzyme and digesting with a! higher temperature than biologically stable ge

shows. So fell z. B. after 6 hours digestion at 55 ° 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 heiab was set.

Example VIII Characterization of proteinase T

(Daiwa Kaiseis Thermoase or Thermolysin)

description

♦ 5 Proteionase T is a proteolytic enzyme that aui the Bacillus thermoproteolytikus Rokko and excellent heat stability and sub has strate specificity.

ExampleVn Comparison between thermostable proteolytic

Enzymes and a conventional proteinase in the breakdown of the protein in pre-gelled grain

5o

55

6o

Enzymatic properties

(a) Molecular weight: 37,500 (approximate).

(b) Optimal pH standard Tys at 35 ° C: PH: 5.5 6.5 8.5 10.2 K Tys: 10.0 20.0 21.2 4.8

(c) Optimal Temperature - Standard Tys at pH 6.5

K Tys

Two individual 50 g samples of finely ground barley were mashed with 150 ml of water and 1% (based on barley weight) Nervanase 1OX (starch dissolving α-ainylase). The temperature was raised for 1 hour fast at 70 ⁰ C followed by 1 hour at 80 ⁰ C. The mash, the normal proteinase (Proteinase 36 Xn) was added, was heated to 55 ° C 10.0 20.0 48.9

60.2 81.2

66.0

(d) Temperature stability - enzyme solution heated with (g) activator: pH 5.5 over a period of 30 minutes before calcium.

Testing at pH 6.5 to 35 ° C:

Heating temperature rest activity 55 ° C 100% 60 ° C 100% 65 ° C 100% 70 ° C 98% 75 ⁰ C 94% 80 ° C 49% 85 ⁰ C 0 (e) Ratio - = 33. Tys (f) inhibitors: Phosphate, E. D. T. A.

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

(a) Ma t s über ar a, H., Si nger, A., S asa ki, R., ίο and Jukes, T. H., Biochem. Biphys. Res. Commun., 21, 242 (1965).

(b) E d do, S., J. Fermentation Tech., 40, 346 (1962),

(c) Ohta, Y., Ogura, Y., and Wada, A., J.Biol, Chem., 241, 5919 (1966).

(d) Biotechnology No. 2/1970.

and Bioengineering, Vol. 12

Claims (2)

and instead use commercial α-amylase and 7 patent claims: normal commercial proteolytic enzymes for breaking down protein and starch in raw; m or 1. Use a method for producing an aqueous ex-cooked grain, namely at high temperatures, especially of wort, from an aqueous 5 to about 50 ° C industrially (e.g. by fermentation) in substance (e.g. barley), in which starch has been produced by treating the pulp with a starch-liquefying form suitable for use in industrial processes. The enzymes ah such enzyme, e.g. B. commercially available bacterial α-Amy can, for. B. in the form of a solution or in Verbinlase, liquefied or dissolved and protein with io dung with a solid diluent used to dissolve a proteolytic enzyme, stick. Substance-containing compounds is converted, The treatment with α-amylase and proteinase characterized by a treatment is often followed by a treatment with a saccharifying agent with a thermostable proteolytic, from the enzyme to soluble carbohydrate - either through Thermoproteolytikus-Rokko-Bacillus obtained 15 use of / 3-amylase (as with malt or Sqja ( Enzyme. or an amyloglucosidase enzyme or both, depending 2. The method according to claim 1, characterized in accordance with the required degree of saccharification - draws that the treatment at a temperature to saccharify fermentable sugars. of above about 55 ° C, preferably a temperature. The method according to the invention is in contrast temperature between 60 and 75 ° C. 30 characterized by treatment with a thermostable proteolytic, from the Thermoproteolytic Rokko Bacillus recovered enzyme. A treatment with such a thermostable proteolytic enzyme yields compared to hei-35 conventional proteolytic enzymes surprisingly The invention relates to a method for wise higher solubility of protein. Furthermore, production of an aqueous extract, in particular the process with higher temperatures durchgevon wort, can be made from an aqueous pulp of starch and leads, so that simultaneously with the thermoprotein-containing vegetable matter (e.g. barley), the stable profiolytic enzyme and a starch agent -Starch by treating the pulp with a starch- 30 liquid enzyme starch liquefied and protein to liquefied enzyme, eg commercially available bacteria- soluble, nitrogen-containing compounds converted rial α-amylase, liquefied or dissolved and pro- can be. The treatment is advantageous in the case of tein with a proteolytic enzyme to be soluble at a temperature of about 55 ^° C., preferably nitrogen-containing compounds. After between 60 and 75 ° C, the extracts obtained in such a process can be used in half a working range for starch liquefaction: for example, in the production of industrial chemicals, enzymes and above the normal working range can be used vegetable raw materials suitable for conventional proteolytic enzymes, e.g. B. Spirit is present. In this way, the digestion period can actually be used as a wart for brewing purposes, which is why in comparison to previously proposed methods, the following explanations focus on starch liquefaction and protein conversion in the area of application. different temperatures are carried out, With normal wort production, malt will be shortened. or a mixture of malt and unmalted grain A thermostable proteolytic, made from thermite mashed in hot water and then gefil- moproteolytikus Rokko Bacillus obtained enzyme tert. The resulting filtrate contains soluble charcoal- 45 is Proteinase T (A. B. M. Industrial Products Limited, hydrate obtained from dissolved starch and soluble stick- Woodley, Cheshire, England) originally from Substances containing substances including proteins and Daiwa Kasei K.K., Osaka, Japan, under the names Breakdown products. The filtrate is called "Thermoase" or "Thermolysin" suitable fermentation agent for yeast in manufacture. of alcohol. Most of the nitrogenous substances 50 in one by the method according to the invention in the wort come from the malt, the previously produced wort becomes their apparent steaming Malt process has been won. The soluble heights and the subsequent fermentation of the wort Carbohydrate results from the action of the amylo-relieved. Also the taste of one has changed lytic malt enzymes on the starch in the malt and such wort beer brewed are quite unexpected Starch in the unmalted grain. Hence, 55 wears any wise compared to a sample that is conventional unmalted Liches proteolytic enzyme used in the process was used when Grain mainly to the carbohydrate content in the better exposed. Furthermore, through the seasoning at. If a satisfactory fermentation is to be carried out and proteolysis at a higher temperature is possible, so it is essential to reduce the risk of infection in the wort to a minimum Set balance between carbohydrate and 6a. Maintain protein. Since unmalted carbohydrates are examples of starchy and protein-rich plant However, it is cheaper than malt, most of the substances take are primarily barley, wheat, corn, rice Breweries a maximum of carbohydrate-protein grains and other grains, but also roots, sponge ratio in the case of one of the quality of a product or mushroom materials and by-products of measured Spice. 65 fahien to which grain was subjected, as well as