DE3028165A1 - Prodn. of low-calorie beer - by fermentation in presence of rice pullulanase to reduce dextrin content - Google Patents

Abstract

Prodn is carried out by fermenting brewer's wort with yeast in the presence of rice pullulanase (I) in an amt sufficient to reduce the residual dextrin content by cleaving the dextrin alpha-1,6 linkages to form alpha-1,4 dextrins, which are converted by 1,4-carbohydrases into fermentable sugars which are fermented by the yeast to alcohol. Method of extracting (I) from rice comprises treating the rice with an aq. buffer (pH6) at 0-60 degC. (I)is more effective than glucoamylase in debranching high-molecular-wt. dextrins, allowing more rapid fermentation.

Description

Process for the production of a low-calorie beer

Generally speaking, the present invention relates to a method for making a beer. More precisely, it relates to a procedure for making a low-calorie beer using rice, a traditional one Brewing base, extracted enzyme is introduced into the brewing process. The invention further relates to a method for extracting the enzyme from whole or polished Rice as well as a memory-stable form of the enzyme.

In the manufacture of beer, yeast is used to make a substrate to ferment from a mixture of fermentable carbohydrates in ethyl alcohol. Such Carbohydrates that can be fermented by brewer's yeast are primarily maltose, Glucose, maltotriose and traces of sucrose and fructose. These substances will obtained by extracting malt enzymes (alpha and beta amylase) starch molecules from malt and other additives can be converted into the fermentable sugars mentioned above. This happens during mashing5. After mashing, the soluble materials become extracted during the lautering, leaving behind the spent grain. A clear one Liquid (wort) is obtained, which is transferred to a brewing kettle and over a Length of time is cooked (kettle boiling) to get all malt enzymes to inactivate. 8hops are normally added to the boiler, after which the wort is cooled, aerated and provided with yeast, after which it is allowed to ferment. The compositions the wort depend on the type of materials used, the mashing cycle, etc. away. A typical seasoning, however, consists of around 65-80 * fermentable carbohydrates of the aforementioned type and made from about $ 20-35 non-fermentable carbohydrates. After the fermentation process, a drink is obtained that normally contains 3 - 5 qd alcohol with approximately equal amounts of residual dextrin, which contains the bulk of the dissolved Forms solids and is usually referred to as active extract. This rest remains as a result of the inability of the malt amylases to form the alpha 1-6 bonds of the Hydrolyze starch. When the wort described above is fermented, becomes Get a product that has about 110 calories per 12 ounce bottle with 3.3 g / 100 ethanol contains bottled.

In the production of highly fermented beers with few calories was used already tried a higher percentage of alcohol and a much lower one To achieve proportion of residual dextrin. This gives a beer that will ferment during the final fermentation has a lower specific weight than in the normal case. The first highly fermented Products were obtained by a process which consisted of having an external Enzyme clogged in the fermenter. This special enzyme, a glucoamylase, possesses the ability to have both Alpha 1-4 and Alpha 1-6 bonds of strength to hydrolyze and is usually extracted from the Aspergillus niger mold. However, the use of glucoamylase has certain disadvantages. These disadvantages can be summarized as follows: (a) During hydrolysis There are some difficulties with the alpha 1-6 pinions by the enzyme. The enzyme is much more effective at hydrolyzing alpha 1-4 bonds; and (b) the enzyme can be viewed as exogenous with respect to the brewing process. In other words, it is not in the traditional brewing substances malt, rice, grain or yeast present and it is not isolated from it.

Another approach, which has already been suggested, exists by combining an alpha 1-6 carbohydrase or pullulanase with a Beta-amylase of microbiological origin is used.

There are three basic classes of starch degrading enzymes. It acts these are the glucoamylases, the isoamylases and the pullulanases. The differences between these classes are well known to those skilled in the art. Basically they split Pullulanases the alpha 1,6 bonds of pullulan (an alpha 1,6 polymer of maltotriose, which is isolated from a mold cell wall) so that maltotriose is obtained will. Pullulanases are especially suitable for alpha 1,6 bonds and can break down the wort's borderline dextrins, creating alpha 1,4-polysaccharides which are converted into sugar by various alpha 1,4-carbohydrases which can be fermented by brewer's yeast.

Attempts have been made in the past to achieve such a thing Enzyme from substances associated with beer production, such as malt, for example isolate. The so-called WR-EnzymH has been mentioned in the literature.

However, it seems to be the case to date that none an effective way to isolate the "R-enzyme" has been found.

The main aim of the present invention is to make the discovery useful to make that a well-known, traditionally used effervescent substance as a source for a starch-degrading enzyme can be used to make a highly fermented beer to manufacture.

Generally speaking, the present invention relates to use of rice as a source of the starch-degrading enzyme in the production of a beer low in calories. The invention also relates to a method for isolating of the enzyme from rice.

Rice is a raw material traditionally used in the brewing industry. Usually it is used as an additive or as an additional source of carbohydrates used, wis grain bran or grain syrup. The one used for this purpose Rice is usually food grade, i.

This is rice, which is a common drying process subjected and then dry-milled. Usually found in the brewing industry Use the broken grains from this polishing process. The traditional method consists in using rice in the grain cooker. Usually something will Malt is added along with enough water so that some conversion of the rice starch occurs is reached in the cooker. This mixture is cooked over a period of time and the Mash added, with malt enzymes turning the starch from the malt and the rice into fermentable e convert carbohydrates. The rice additive used in this way has none Enzyme activity, as this has been completely inactivated in the grain cooker. at therefore, rice is not used as an enzyme source in the traditional process.

It has now been found that by using a starch degrading Enzymes that are naturally present in rice work well in a brewing process Obtained results in relation to the production of a low calorie animal can be.

The enzyme based on rice comes from a traditional one Effervescent substance and seems to be one of greater effectiveness in terms of reducing the having highly branched high molecular weight dextrin fraction as 6lucoamylaso.

The method of the present invention is an improvement the process for the production of a high-fermentation beer by fermenting wort with yeast and involves the addition of a rice pulp alanase in one for reduction the amount of residual dextrins in the active extract by breaking down the alpha 1,6 bonds the border dextrins for the formation of alpha 1,4-dsxtrins, which are caused by 1,4-carbohydrases converted into fermentable sugars, which are fermented into alcohol by the yeast, effective amount.

The enzyme can be introduced at different stages.

In a preferred embodiment, either rice or the Enzyme extracted from the rice is added to the seasoning, the corn amylase from a suitable Source contains, i.e.

Malt at the fermenter. The enzyme from the rice hydrolyzes the rest 1-6-8 bonds of the frontier dextrins, and the cornamylase cleaves the resulting linear alpha 1-4 polysaccharides in fermentable sugars, which are then made up by the yeast converted into ethanol.

In another embodiment of the method, the Rice extracted enzyme is added to the mash to help break down the 1-6 bonds contribute to border dextrins, that would otherwise be formed. The natural malt enzymes hydrolyze the 14 bonds and thus produce a higher level of fermentable sugars.

8ier tasting quality can be obtained by any of the above Process are produced. It was found that the end product in each Fall for a larger proportion of alcohol in relation to the active extract and fewer calories per volume unit when bottling with constant alcohol content than a comparative beer, which was produced without the addition of enzymes.

In the case of the enzyme that is used in the production of a highly fermented beer has been found suitable with few calories, it is a starch-degrading one Enzyme naturally found in rice. The enzyme according to the present Invention is classified as pullulanase because it has the alpha 1-6 bonds of the diagnostic substrate pullulan hydrolyzed.

In the extraction process according to the invention, the enzyme is eliminated whole or commercially polished rice with an aqueous buffer system extracted, which has a pH of about 6 at temperatures of 0-600C.

It is preferred to use a slurry of the polished rice in 0.1 M potassium phosphate buffer-0.2 M NaCl at pH 6.0 and about 50 ob over about three hours to manufacture.

The pullulanase-containing suspension from the extraction can further purified by the following steps: (1) acidifying the crude extract; and (2) precipitating the travel enzyme with (NH4) 2SO4. These operations are discussed in the following Examples described.

The enzyme can be in liquid form or as freeze-dried or spray-dried powder can be stored. The freeze-dried powder is through Diafilter the buffered pH 6 extract containing the enzyme against 0.1 M ammonium bicarbonate solution obtain. Ammonium bicarbonate is a volatile salt that freezes when freeze drying sublimed and results in a salt-free enzyme powder. Other sublimable salts that do not interfere with the process or impair the enzymatic activity, can also be used to. Apply if diss is desired.

The amount of rice or enzyme extracted that goes into the brewing process is added depends on many factors, such as the enzymatic content of the rice, the activity of the enzyme, the stage of the brewing process at which the Addition takes place, and the conditions of the roughening process, i.e. pH, temperature and Time. Usually it will be the amount of rice or extract added act in an amount intended to reduce the amount of residual border dextrins is effective in the active extract by about 30 to about 80%. To pre-convert the Dextrins before fermentation of an enzyme source, either extract or rice, of about 100 units to about 300 units of pullulanase activity per liter containing, added to the wort, or about 300 units to about 700 units per Liters of mash. Lower amounts that are about two units to about 75 units of pullulanase activity per liter are effective when added to the wort in the Fermenter are added. Larger amounts than those normally used can apply if this should be desired or necessary. To the exact amounts some experimentation may be required to determine. The implementation of such However, attempts are well within the skill of the brewery specialist.

In one embodiment of the method, the enzyme is added to the fermenter added together with a carbohydrase, for example a grain diastasis. These Combination is required because the rice enzyme contains the highly branched alpha 1,6-frontier dextrins splits up and the added carbohydrase the resulting alpha, 4-dextrins splits into sugars, which can be further treated by yeast. The effective The amount of each enzyme to be added depends on the content of borderline dextrins, which is normally present in the product of fermentation and to the extent desired Calorie reduction. Usually the degrading enzyme is in an amount of about 2 units to about 60 units of pullulanase activity per liter of wort and the Carbohydrase or grain diastasis in an amount of about 20 units up to about 140 units of amylase activity per liter of wort.

In yet another embodiment, the addition of the rice degradation enzyme connected to the fermenter with the addition of a glucoamylase, for example one those derived from Aspergillus niger and with respect to both alpha 1,6 and Alpha 1,4 bonds are also active.

By introducing the combination of these two enzymes in the fermentation stage is the fermentation time that is normally required to produce a highly fermented beer is significantly reduced, i.e. from 12 to 7 days.

Although both enzymes have degradative abilities, the rice enzyme is more effective as glucoamylase, which results in a reduction in fermentation time. The concentration of the rice enzyme in such a mixture can be lower than in the normal case, i.e. 2-4 units of pullulanase, and the glucoamylase can be in about 2 units to about 10 units of glucoamylase activity per liter.

The following analytical procedures resulted in those described below Examples of application. Protein was obtained by the Lowry method modified by Miller (1) certainly. The pullulanase activity was determined by hydrolysis of 0.5 46 w / v pullulan determined at pH 5.0 and 500C. The amylase activity was determined by hydrolysis of 0.5 $ w / v Linter soluble starch determined at pH 5.0 and 500C. That Occurrence of reducing sugars was made by the dinitrosalicylic acid method monitored by Bernfield (2). A unit of activity was identified as a the occurrence of 1 mg reducing sugar (as maltose) / minute is defined. Specific Activities were expressed as units / mg protein.

The glucoamylase activity was determined by a modification of the procedure determined by Pazur (3), using maltose as substrate at pH 5.0 and 250C became. The appearance of glucose was determined using the coupled glucose oxidase-peroxidase reaction monitored with o-dansidine as an indicator dye (3). A unit of activity became defined as hydrolysis of one micro »« altose / minute under these conditions.

The fermentation processes were based on the decrease in the specific weight using the Mettler DMA-45 computational densitometer It was assumed that the beer fermentation process had ended, when the refractive indices were used measured on a Zeiss immersion refractometer. These measurements were used to determine the alcohol content (4,5,6) and the active extract content (5,6) of the beers.

The heat content of a standard 12 ounce container was found to be 3.3 g / 100 Ethanol determined according to the method of Helbert (7).

Hydrocarbon profiles were determined by high pressure liquid chromatography obtained on Bio Rad Q 158 resin as dated ASBC Subcommitment on brewery sugars and syrups (8) and by Scobell et al (9). Unless otherwise stated, were all diafiltrations were carried out on an Amicon M; -2 apparatus, which was connected to an H-1P-10 Cartridge (m.w.

cutoff 1,10000) (Amicon Corporation, Lexington, Mass.).

was equipped.

Examples 1-5 show the insulation and some properties illustrated by Reispullulanase.

Example 1 Isolation of Pullulanase from Whole Rice 500 g LaBelle rice (Seed quality) were in 0.1 M potassium phosphate buffer-0.2 M Nah1, pH 6.0, under Mixed using a Waring mixer. The mixed grain was placed in a jar transferred to a bath kept at So0C and under two liters of buffer three Was stirred for hours.

The spent grain was removed by gauze filtration, and that The filtrate was clarified by centrifugation.

Further clarification can be achieved by adjusting the pH of the extract is reduced to 5.0. The resulting precipitate was removed by centrifugation removed and the awakening pH was readjusted to 6.0.

The extract can be purified and concentrated by (NH4) zS04 fractionation will. This salt was the pH-sinyestelle suspension with an amount of 40 g solid (NH4) 2S04 per 100 ml of solution are added. The suspension was stirred for one hour at room temperature and the precipitation proceeded Centrifugation removed. The precipitate was dissolved in the extraction buffer and diaflitrated against this.

Example 2 Determination of pullulanase within the rice kernel LaDelle rice from Example 1 was bubbled and the following fractions were isolated: (1) Peel; (2) brown or broken rice; (3) rice bran; and (4) polished white Rice. Each fraction was extracted as described in Example 1 for whole rice and clarified. Analysis of these extracts revealed that the majority of the pullulanase activity was found in the endosperm (polished rice).

In addition, this preparation had a much greater specific activity possessed as the preparations made from whole or brown rice. Hence is polished rice is the preferred source of enzyme.

Example 3 Extraction of pullulanas. from polished. Rice 2 kg commercially available polished rice was ground to 0.02 inches in a Barley mill. The ground Rice was mixed into four liters of extraction buffer pH 6, and the suspension was stirred at 50 ° C. for 3 hours.

The pH of the extract was adjusted to 5.0, and the resulting The suspension was cleared by centrifugation. The pH of the suspension was reset to 6.0.

For long-term storage, it was desirable to use the preparation as a to obtain salt-free powder. This was done by the suspension from the pH adjustment step was diafiltered against 0.1 M NH4HCO3. This salt was chosen because (1) the preparation requires salt to dissolve remain; and (2) NH4HCO3 sublimed and removed by subsequent freeze drying will.

After diafiltration against 4 volume units of 0.1 M NH4HCO3 the retentate was freeze-dried.

Example 4 Optimum pH of Rice Pululanase The optimum pH range was determined for rice pululanase following the procedure described in Example 3 had been isolated. The following buffer systems were used: (1) pH 4.0-5.5-0.1 M acetic acid adjusted to the correct pH with NaOH; (2) pH 6-7 0.1 M KH2P04 adjusted to the correct pH with NaOH. Stored pullulan (10% w / v in H2O) was diluted to 1% w / v in the appropriate buffer. Rice pulpulanase was then tested over the pH range 4-7 under standard conditions. The results indicated that optimal activity in the pH range of 5-6.5 is obtained.

Example 5 Temperature Optimum of Rice Pululanase (A) In Absence of substrate rice pululanase, which had been prepared according to Example 3, were obtained a final concentration of 2 mg / ml in 0.1 M acetate buffer, pH 5.0, is set. Spot checks this mixture was heated to a temperature range over periods of 10-60 minutes heated from 40-70 0c. Samples of the heated samples were withdrawn, cooled and subjected to the standard pululanase test. The results indicated that the enzyme was quickly inactivated at temperatures above 40 C. Complete inactivation occurred after 10 minutes at 60 ° C.

(B) Rice pululanase prepared as in Example 3 in the presence of substrate was adjusted to a concentration of 1 mg / ml in 0.1 M acetate, pH 5.0. Spot checks of this preparation (sufficient for a final concentration of 0.2 mg / ml incubate) were placed in tubes containing pullulan and 0.1 M acetate buffer (pH 5.0) and which had been equilibrated to the desired temperature. At any temperature (from 400C to 7000) samples of 1 ml were made after heating for 10, 20 and 30 minutes stripped and inactivated by adding them to the dinitrosalicylic acid solution used for color development were introduced. The reducing sugars were according to the standard method certainly.

From the results of these experiments it can be seen that the enzyme in The presence of substrate is stable up to 60 ° C for 30 minutes. This stands in contrast to the temperature resistance of the enzyme alone, as in example 5 (A) described.

In Examples 6-15, the use of the travel enzyme is described below (Pullulanase) described in connection with the brewing process. Examples 7 - 12 illustrate the use of the enzyme in connection with various types Alpha 1,4-Garbohydrasen in beer in the fermentation process, while the examples 13 and 14 illustrate its use before the fermentation process. In all cases it was the wort is mashed as wort consisting only of malt and before the fermentation process a commercial converted corn syrup set to about 120 to about 150 P. In the following examples, the original specific weight was constant. The worts were with a brewing culture of S. Uvarum to a final concentration of 1 x 10 cells / ml and fermented at 150C.

Example 6 Preparation of grain diastases for use with rice pululanase (A) Mdzdiastase high-gib distiller's malt was ground in a Standerd Barley mill. The powder (150 g) was dissolved in 1.5 liters of 0.1 U acetate buffer (pH 5.0) singing. The slurry was stirred for two hours at 50 0C, and the Suspension was in the manner described for the raw rice extract (Example 1) won.

The enzyme was further purified by adding (NH4) 2504 to a final concentration of 40 g / 100 ml was added. The precipitate was collected by centrifugation and resuspended in 0.1 M acetate buffer (pH 5.0). The suspension was through Diafiltration cleared against the same buffer, concentrated and stored at 40C.

L8) Production of Malt Beta-Aiylase The one produced according to Example 6 (A) Malt diastase contains both alpha and beta amylase, with alpha amylase in the greatest concentration is present. Malt alpha-amylase can at acidic pH (9) can be selectively inactivated.

The pH of a portion of the malt diastase obtained as in Example 6 (A) was set to 3.6 and two hours heated to 350C for a long time.

The solution was clarified by centrifugation, and the pH of the Suspension was set back to 4.0.

(c) Production of Soybean Diastase Whole soybeans were made in a Wiley Mill using a 20 mesh screen. 10 gm of powder were made stirred in 100 ml of 0.01 M acetate buffer (pH 5.2) at 550 ° C. for one hour. The solution was clarified by centrifugation, after which using a Filter aid was filtered.

The suspension was diluted fourfold, diafiltered against H2O and focused on the original volume.

The concentrate was stored at 40C.

(D) Isolation of Wheat Diastase Wheat diastase was made from pearled isolated hard winter wheat milled as in Example 6 (B) for soybean diastase had been. The powder (50 g) was dissolved in 100 ml of 0.1 M phosphate buffer-0.1 M NaCl (pH 6.0) and the suspension was stirred at 50 ° C. for three hours. Thereafter it was clarified as described in Example 1 for the rice enzyme.

The suspension was against 0.02 M phosphate buffer-O, 2 M NaCl and then dialyzed water.

The glucoamylase used in the subsequent experiments acted it is Novo 150 (manufactured by Novo Industries, Wilton, Connecticut).

Example 7 High fermentation of beer using rice pululanase malt diastate The wort prepared in the above manner was fermented: (1) without the addition of enzymes (Beer +1) to determine the fermentation limit; (2) with the addition of glucoamylase (8.1 U / l beer +2) to determine the fermentation limit; and (3) with the addition of rice pululanase (15.3 zero) and malt diastasis (140 U / l beer +3).

In all cases the condiments were yeasted as described above added and ventilated, after which the appropriate enzymes were added. The beers were fermented at 150C.

The enzyme-free comparison beer contained 0.5-0.6 g / 100 less alcohol as beer +2 or beer +3, both with glucoamylase and rice pulpulanase / malt diastase were highly fermented. At 3.3 * ethanol, the active extract in the beer was +3 um about 1.0 g / 100 compared to that of beer +1 and almost corresponded to that of the value obtained using glucoamylase (beer +2). At this alcohol concentration the +2 and +3 beers would be 92-93 cal / 12 ounces compared to the 108 cal / 12 ounces for beer +1 included.

The carbohydrate profiles indicated that the +2 and +3 beers were nearly identical Have carbohydrate compositions in the final fermentation and that in both beers +2 and +3 the non-fermentable sugars (greater than DP-3) compared to that of beer +1 were significantly reduced.

Example 8 High fermentation of beer using rice pululanase-soybean diastase The wort was aerated as described in Example 7 and provided with yeast. Rice pulpulanase (15.3 nil) and soybean diastase (140 nil) were added. The beer was like Fermented described in Example 7.

The fully fermented beer, the 5.3 Ull Reispullulanase and 140 U / l of boybean diastase (beer +4) was about on the same Value like the glucoamylase reference beer (beer +2) fermented, what a beer of 93.4 cal / 12 ounces when bottled with 3.3 g / 100 ethanol. The carbohydrate profile after 12 days of fermentation showed that the non-fermentable fraction was close to that of the glucoamylase comparison sbiere s (beer +2) corresponded.

Example 9 High fermentation of beer using rice pululanase-wheat diastase The wort was aerated as described in Example 7 and provided with yeast. Rice pulpulanase (15.3 U / L) and wheat diastase (140 nil) were added as shown. The beer was fermented as described in Example 7 at 150C.

This beer (beer +5) was fermented at the same level as the glucoamylase comparison beer (beer +2). When bottling with an alcohol concentration of 3.3 g / 100 of ethanol the beer would contain 29.5 cal / 12 ounces. The carbohydrate composition indicated that the non-fermentable fraction was almost equal to that of beer +2.

Example 10 High fermentation of beer with rice pululanase malt beta-amylase The wort was aerated as in Example 7 and provided with yeast. Rice pulpulanase (15.3 zero) and malt beta-amylase (140 U / L) were added and the beer was made Fermented at 150C in the usual way.

The results indicated that this beer (+6) was fermenting ended in 8 days, i.e. earlier than the glucoamylase comparison beer (beer +2) or any of those with rice pululanase in conjunction with the other grain diastases produced beers. Again the carbohydrate composition was that of the glucoamylaia reference beer similar. Thus, a beta-amylase appears to be that used in the previous examples Diastases (which contain both alpha and beta amylase) to be superior.

Example 11 High fermentation of beer with irritation of paint flour Polished +4 brewing ice and high-gib distiller's malt were reduced to a grain size of 20 mesh ground in a Wiley mill.

The flour obtained became aerated as described above and loaded wort added. One seasoning (8ier +7) contained 5.2 g of rice flour and 0.12 g malt flour / liter, while the other wort (beer +8) twice as much of each Contained flour. The worts were fermented as described above. The grain additives for beer +7 were calculated in such a way that 15.4 units of pullulanase per liter and 140 units of malt diastase per liter based on the methods used in Examples 3 and 6 (A) revealed extraction.

The results obtained for beers +7 and +8 indicated that Both beers ferment to the same level as the glucoamylase comparison beer (Beer +2) and the beers listed in Examples 7-11, in which enzyme extracts Found application.

Example 12 Use of rice pululanase in conjunction with glucoamylase to shorten the fermentation time The wort was used in five different fermentation processes perform. All condiments were aerated and loaded, after which rice pululanase (2.0 Zero; 4.0 U / l or 8.1 U / l) and glucoamylase (3.8 Ull or 15.3 zero) were added became. The results indicated that by adding rice pululanase, the Fermentation time even with reduced glucoamylase (beers +11 and +12) or pullulanase concentration (Beer +13) was considerably shortened compared to the glucoamylase comparison beer.

Example 13 Conversion of wort consisting only of malt before Fermentation with rice pululanase-malt beta-amylase After the boiler boil was only one get wort consisting of malt. Three samples of the wort were made with malt beta-amylase (1450 zero) in connection with decreasing concentrations of rice pullulanase (150 Zero; 75 U / l and 38 zero). Another sample of the wort was transformed, using rice pululanase in conjunction with glucoamylase.

The procedure was the same in all cases. The condiments were at 60 0C brought into equilibrium with stirring in a water bath. The condiments were Heated for 30 minutes, after which they were placed in a glass flask that was in a violently boiling water bath was included. The worts were left in there for two hours stand for a long time to inactivate the enzymes.

After that, the condiments were cooled and the resulting turbidity turned removed by centrifugation.

The malt / syrup addition ratio was adjusted to the same value as in the case of the wort described in Examples 7-12. The condiments were then loaded, ventilated and fermented as described in Example 7.

The beers were compared to the enzyme-free comparative beer (beer +1) highly fermented, when bottled with 3.3 g / loo ethanol, the heat content would be of these beers are around 98 calories, i.e. around 10 calories less than with Beer +1 that was produced without the addition of enzymes.

Example 14 Addition of corn amylases to pre-converted beers Since the beers mentioned in example 13 do not brew to the same level as / in various enzymes were added to the beers described in Examples 7-12, to determine whether the high fermentation limit can be lowered.

The yeast was removed from beer +14 by centrifugation, and that Clarified beer was divided into two equal parts, labeled 14A and 148 became. Both beers were yeasted again. Beer + 14A became 140 U / l of malt Beta-amylase and beer +148 15.3 U / l rice pululanase and 140 U / 1 malt beta-amylase added. The fermentation process was then continued at 150C.

The levels + 15A and + 16A were not re-yeasted.

Instead, the enzymes were added directly to the fermenting beers. Beer + 15A became 15.3 U / l rice pululanase and beer + 16A became 15.3 U / l rice pululanase and 140 U / l of malt beta-amylase added.

By adding the alpha 1,4-carbohydrase, malt beta-amylase, was the specific gravity (beer + 14A) is not noticeably reduced, as most of it the non-fermentable sugar contained alpha 1,6 bonds. In contrast, ferment Malt beta-amylase in combination with rice pululanase (beers +148 and + 16A) or rice pululanase alone (beer + 15A) the beers to the same level as glucoamylase (beer +2) or in the beers described in Examples 7-12.

Example 15 Addition of rice pululanase to the wort when mashing According to Example 3 produced rice pululanase was added to 400 μl of groundwater, see above that a final concentration of 520 U / l at 460C was obtained. Then were 129.6 g light malt was introduced and the mash was subjected to the following mashing cycle: (1) 450C for 30 minutes; and (2) 600C for 120 min. The brew was brewed at 7? C mashed, after which the spent grain was removed by gauze filtration. The first seasoning was clarified by centrifugation, diluted and in a boiling for two hours Water bath held. All subsequent steps were as described in Example 11 carried out.

This beer fermented to a final specific gravity of 1.0019 (0.490 P) versus 1.0029 (0.750 P) for beer +1, the one made without the addition of enzymes Comparative beer of Example 7. Thus, by incorporating rice pululanase in the mash, the fermentation limit is reduced by 0.260 P.

In the above description, reference was made to the following publications Reference: 1. Miller, G., Anal. Chem. 31, 964, 1959 2. Bernfield, P., Advances in Enzymology XII (Nord, F., ed.) 379, Interschiences Publishers, New York, 1951 3. Pazur, J., Method in Enzymology XXVIII, Ginsberg, V. (ed.) 931, Academic Press, 1975 4. Kneen, E., (ed.)., Alcohol Determined Refractometrically "in Methods of Analysis of the American Society of Brewing Chemists, 7th revised edition, published by the Society, 1976 5. Olshausen, J., Brewern Digest 27, 45, 1952 6. Olshausen, J., Brewers Digest 27, 53, 1952 7. Helbert, J.R., J. Amer. Soc. Brew. Chem. 36, 66, 1978 8. Martinelli, L. (Chairman), ASBC Journal 35, p. 104, 1978 9. Scobell, H., Brobst, K. and Steele, F., Cereal Chem. 54, p. 905, 1975 10. Greenwood, C. and A. MacGregor, J. Inst. Brew. 71, 408, 1965 With the rice that is in according to the invention can be used as an enzyme source, it is to food grade rice that helps maintain enzymatic activity has been treated under sufficiently mild conditions. It can be either seed rice or polished dry milled rice can be used. The enzyme can come from a large variety of rice species can be extracted, e.g. LaBelle, LeBonnet, Nato, Starbonnet or Brazos. However, it is commercially available polished dry milled Rice preferred for brewing purposes.

If the enzyme is removed from the rice before use in this process extracted, the spent rice from which the enzyme was extracted can used as a starch source during mashing or for an additional syrup formulation to make the use of the travel enzyme more economical.

Although the rice enzyme, as described above, for the preparation a beer with few calories or a highly fermented beer is used, it may possibly also have other areas of application. For example, can a mixture of the rice enzyme and a grain diastasis in an advantageous manner for Manufacture of a starch conversion product that has a high maltose content, be used. Due to its natural origin, it is likely to be of use of the enzyme obtained from rice in food undoubtedly do not pose any concerns.

Claims (17)

Claims 1. A method for producing a highly fermented Beer by fermentation of brewed wort with yeast, characterized in that a rice pulpulanase in an amount to reduce the amount of residual dextrins in the active extract is added to break down the alpha 1,6 bonds of the frontier dextrins and alpha Forming 1,4-dextrins, which are converted into fermentable sugars by 1,4-carbohydrases which are fermented to alcohol by the yeast. 2. The method according to claim 1, characterized in that the s Reipullilanase is added in an effective amount to the wort. 3. The method according to claim 2, characterized in that the rice pululanase is added to the wort before fermentation. 4. The method according to claim 2, characterized in that the rice pululanase is added to the wort during fermentation. 5. The method according to claim 2, characterized in that the rice pululanase is added to the seasoning by adding rice, which contains pullulanase, to the seasoning contains. 6. The method according to claim 2, characterized in that the rice pululanase added to the wort as a purified enzyme extracted from rice. 7. The method according to claim 2, characterized in that the amount of the added rice pululanase from about 15 to about 60 units of pullulanase per liter Wort amounts. 8. The method according to claim 2, characterized in that the rice pululanase in one to reduce the amount of residual dextrins in the active extract by about 30% until about 80 qd effective amount is added. 9. The method according to claim 2, characterized in that the rice pululanase in an amount of from about 2 units to about 60 units of pullulanase activity per Liter of wort is added. 10. The method according to claim 2, characterized in that the wort a 1,4 ~ carbohydrase is added to the formed by the rice pululanase Convert alpha 1,4-dextrins into fermentable sugars that ferment into alcohol through yeast can be. 11. Method for extracting a pullulanase from rice that is enzymatically is active, characterized in that the rice with an aqueous buffer of pH 6 is treated at a temperature of about OOC to about 600C to give an extract to win, which contains the pullulanase. 12. The method according to claim 11, characterized in that the whole Rice is extracted. 13. The method according to claim 11, characterized in that in commercially available Way polished rice is extracted. 14. The method according to claim 11, characterized in that either Whole rice or commercially polished rice at pH 6.0 in 0.1 M potassium phosphate-O, 2 M NaCl is extracted at 500C for three hours. 15. The method according to claim 11, characterized in that the pullulanase-containing Extract dialyzed against a solution of a sublimable salt and freeze-dried to obtain a salt-free enzyme powder that is shelf-stable. 16. The method according to claim 11, characterized in that the sublimable Salt is NH4HC03. 17. Pullulanase, characterized in that it is obtained from rice by the The method of claim 11 has been obtained.