DE3028165C2 - Process for producing a highly fermented beer - Google Patents

Description

The present invention relates to a process for producing a highly fermented beer according to the preamble of patent claim 1.

In the production of beer, yeast is used to ferment a substrate of a mixture of carbohydrates into 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 are obtained by allowing malt enzyme (alpha and beta amylase), starch molecules from malt and other additives to convert into the above-mentioned fermentable sugars. This occurs during mashing. After mashing, the soluble materials are extracted during lautering, leaving the spent grain behind. A clear liquid (wort) is obtained which is transferred to a kettle and boiled for a period of time to inactivate any malt enzymes. Hops are usually added during boiling, after which the wort is cooled, aerated and yeasted and allowed to ferment. The compositions of the wort depend on the type of materials used, the mashing process, etc. A typical wort consists of about 65-80% fermentable carbohydrates of the type mentioned above and about 20-35% non-fermentable carbohydrates. After the fermentation process, a beverage is obtained which normally contains 3-5% alcohol with about equal amounts of residual dextrin which forms the majority of the dissolved solids and is usually referred to as the true extract. This residue remains as a result of the inability of the malt amylases to hydrolyze the alpha-1,6 bonds of the starch. When the wort described above is fermented, a product is obtained which contains about 110 calories per 340 ml bottle containing 3.3 g ethanol/100 g bottled.

Attempts have been made to produce highly fermented, low-calorie beers by using a higher alcohol content and a much lower residual dextrin content. This produces a beer that has a lower specific gravity after fermentation than normal. The first highly fermented products were obtained by a process that involved adding an external enzyme to the fermenter. This special enzyme, a glucoamylase, has the ability to hydrolyze both alpha-1,4 and alpha-1,6 bonds in starch and is usually obtained from the mold Aspergillus niger.

US-PS 42 11 842 discloses an exo-pullulanase which is produced by microorganisms of the species Cladosporium residiae and can be used alone or in a mixture with other starch-degrading enzymes for the production of low-calorie beers.

Such processes for producing highly fermented beers have the disadvantage that the enzymes used in these processes are sometimes much more effective at hydrolyzing the alpha-1,4 bonds and only to a lesser extent the alpha-1,6 bonds of the dextrins. In addition, these enzymes cannot be isolated from traditional brewing substances such as malt, grain, rice or yeast, and are not found in them, so they must be considered exogenous in relation to these substances.

The present invention is based on the object of providing a process for producing a highly fermented beer which enables effective cleavage of the alpha-1,6 bonds with a pullulanase contained in traditional brewing substances.

This object is achieved according to the invention by a method having the characterizing features of patent claim 1.

In the process according to the invention, the alpha-1,6 bonds of the limit dextrins are hydrolyzed by a rice pullulanase to alpha-1,4-dextrins, which are broken down by 1,4-carbohydrases to fermentable sugars, which can then be converted into ethanol by yeast. The rice-based enzyme thus comes from a traditional brewing substance and has a greater effectiveness in hydrolyzing the highly branched dextrin fractions with a high molecular weight than known enzymes, to which the good results in the production of a highly fermented, low-calorie beer are attributed.

In addition to the state of the art, it should be noted that the literature reference ( 11 ) proposed the use of an alpha-1,6-carbohydrase or pullulanase combined with a beta-amylase of microbiological origin.

Attempts to isolate enzymes that cause hydrolysis of the alpha-1,6 bonds are also described in the literature reference ( 12 ). Attempts were made to isolate such enzymes from substances used in beer production, such as malt, but such isolation of the "R-enzyme" has not yet been successfully carried out.

In a preferred embodiment of the process according to the invention for producing a highly fermented beer, the rice pullulanase is added to the wort before or during fermentation together with the 1,4-carbohydrase. The rice pullulanase can be added in the form of polished and ground rice.

It is also possible to use a rice pullulanase extract. This involves an extraction process in which the enzyme is extracted from whole or commercially polished rice using an aqueous buffer system. The buffer system has a pH value of about 6. The temperature during extraction is between 0°C and 60°C. Preferably A slurry is prepared from the polished rice in 0.1 M potassium phosphate buffer - 0.2 MNaCl at pH 6.0 and 50°C for three hours.

• (1) Ansäuern des Rohextraktes; und
• (2) Ausfällen des Reisenzymes mit (NH₄)₂SO₄.

Diese Vorgänge werden in den nachfolgenden Beispielen beschrieben.The pullulanase-containing slurry from the extraction can be further purified by the following steps:

• (1) acidifying the crude extract; and
• (2) Precipitation of the rice enzyme with (NH ₄ ) ₂ SO ₄ .

These processes are described in the following examples.

The enzyme can be stored in liquid form or as a freeze-dried or spray-dried powder. The freeze-dried powder is obtained by diafiltering the buffered pH 6 extract containing the enzyme against 0.1 M ammonium bicarbonate solution. Ammonium bicarbonate is a volatile salt which sublimates upon freeze-drying to yield a salt-free enzyme powder. Other sublimable salts which do not interfere with the process or impair enzymatic activity may also be used if desired.

Such pullulanase isolated from enzymatically active rice can be used at various stages in beer production. For example, it is possible to add the extract extracted from rice to the wort containing grain amylase from a suitable source. The enzyme from the rice hydrolyses the remaining alpha-1,6 bonds of the limit dextrins and the grain amylase breaks down the resulting linear alpha-1,4 polysaccharides into fermentable sugars, which are then converted into ethanol by the yeast.

Similarly, to produce a highly fermented beer, the extract can be added to the mash to help break down the alpha-1,6 bonds of the limit dextrins. The natural malt enzymes hydrolyze the alpha-1,4 bonds, thus producing a higher level of fermentable sugars.

The amount of rice pullulanase or extract added in the brewing process 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 brewing process, i.e. pH, temperature and time. Typically this is an amount that will reduce the level of residual limit dextrins in the actual extract by about 30 to about 80%. To pre-convert the dextrins prior to fermentation, extract or rice containing about 100 units to about 300 units of pullulanase activity per liter is normally added to the wort, or about 300 units to about 700 units per liter of the mash. Lower amounts containing about two units to about 75 units of pullulanase activity per liter are effective when added to the wort in the fermenter. Larger quantities than those normally used may be used if desired or necessary. Some experimentation may be necessary to determine the exact quantities, but such experimentation is well within the skill of the brewer.

In one embodiment of the process, the enzyme is added to the fermenter together with a carbohydrase, such as a grain diastase. This combination is necessary because the rice enzyme breaks down the highly branched alpha-1,6 limit dextrins and the added carbohydrase breaks down the resulting alpha-1,4 dextrins into sugars that can be fermented by yeast. The effective amount of each enzyme added depends on the level of limit dextrins that is normally present. Typically, the degrading enzyme is present in an amount of from about 2 units to about 60 units of pullulanase activity per liter of wort and the carbohydrase or grain diastase is present in an amount of from about 20 units to about 140 units of amylase activity per liter of wort.

In yet another embodiment, the addition of the rice extract to the fermenter is combined with the addition of a glucoamylase, for example one derived from Aspergillus niger and active with respect to both alpha-1,6 and alpha-1,4 linkages. By introducing the combination of these two enzymes at the fermentation stage, the fermentation time normally required to produce a highly fermented beer is significantly reduced, i.e. from 12 to 7 days. Although both enzymes have degradation capabilities, the rice enzyme is more effective than glucoamylase, resulting in a reduction in fermentation time. The concentration of the rice enzyme in such a mixture may be lower than normal, i.e. 2-4 units of pullulanase, and the glucoamylase may be present in about 2 units to about 10 units of glucoamylase activity per liter.

The rice which can be used as the enzyme source in accordance with the invention is food grade rice which has been treated under sufficiently mild conditions to maintain enzymatic activity. Either seed rice or polished dry milled rice can be used. The enzyme can be extracted from a wide variety of rice varieties, such as LaBele, LeBonnet, Nato Starbonnet or Brazos. However, commercially available polished dry milled rice is preferred for brewing purposes.

In order to make the process according to the invention for producing a highly fermented beer more economical, the extracted and thus enzyme-free rice can be used as a starch source during mashing or for an additional syrup formulation.

The beer produced by the process according to the invention using a rice pullulanase or the corresponding extract obtained from enzymatically active rice has in any case a higher alcohol content and correspondingly fewer calories compared to a beer produced without the addition of such a pullulanase.

The following analytical procedures were used in the examples described below.

Protein was determined by the Lowry method modified by Miller (1). Pullulanase activity was determined by hydrolysis of 0.5% w/v pullulan at pH 5.0 and 50°C. Amylase activity was determined by hydrolysis of 0.5% w/v soluble starch at pH 5.0 and 50°C. The appearance of reduced sugars was monitored by the dinitrosalicylic acid method of Bernfield (2). One unit of activity was defined as the appearance of 1 mg reduced sugar (as maltose)/minute in both assays. Specific activities were expressed as units/mg protein.

Glucoamylase activity was determined by a modification of the method of Pazur (3.), whereby Maltose was used as substrate at pH 5.0 and 25°C. The appearance of glucose was monitored using the coupled glucose oxidase-peroxidase reaction with o-dansidin as indicator dye (3.). One unit of activity was defined as the hydrolysis of one micromole of maltose/minute under these conditions.

The fermentation processes were monitored by the decrease in specific gravity using the Mettler DMA-45 densitometer. When it was considered that the fermentation process of the beers was finished, the refractive indices were measured on a Zeiss immersion refractometer. These measurements were used to determine the alcohol content (4., 5., 6.) and the real extract (5., 6.) of the beers. The heat content was determined according to the method of Helbert (7.).

Carbohydrate profiles were obtained by high pressure liquid chromatography on Bio Rad Q 155 resin as described by the ASBC Subcommittee on brewery sugars and syrups (8.) and by Scobell et al. (9.). Unless otherwise stated, all diafiltrations were performed on an Amicon DC-2 apparatus equipped with an H-1P-10 cartridge (m.w. cutoff = 10,000).

Examples 1-5 illustrate the isolation and some properties of rice pullulanase extract.

example 1 Isolation of pullulanase from whole rice

Five hundred grams of LaBelle rice (seed grade) was blended in 0.1 M potassium phosphate buffer-0.2 M NaCl, pH 6.0 using a Waring blender. The blended grain was transferred to a jar in a bath maintained at 50°C and stirred under two liters of the buffer for three hours. The spent grain was removed by gauze filtration and the filtrate was clarified by centrifugation.

Further clarification can be achieved by lowering the pH of the extract to 5.0. The resulting precipitate was removed by centrifugation and the pH of the slurry was readjusted to 6.0.

The extract can be purified and concentrated by (NH ₄ ) ₂ SO ₄ fractionation. This salt was added to the pH-adjusted slurry at a rate of 40 g of solid (NH ₄ ) ₂ SO ₄ per 100 ml of solution. The suspension was stirred for one hour at room temperature and the precipitate was removed by centrifugation. The precipitate was dissolved in the extraction buffer and diafiltered against it.

Example 2 Determination of pullulanase within the rice kernel

• (1) Schalen;
• (2) brauner oder entschalter Reis;
• (3) Reiskleie; und
• (4) polierter weißer Reis.

Jede Fraktion wurde, wie im Beispiel 1 für ganzen Reis beschrieben, extrahiert und geklärt. Die Analyse dieser Extraktion ergab, daß der größte Anteil der Pullulanaseaktivität im Endosperm (polierter Reis) aufgefunden wurde. Hinzu kam, daß diese Zubereitung eine viel größere spezifische Aktivität besaß als die von ganzem oder braunem Reis hergestellten Präparate. Folglich ist polierter Reis die bevorzugte Enzymquelle.LaBelle rice from Example 1 was pearled and the following fractions were isolated:

• (1) shells;
• (2) brown or de-hulled rice;
• (3) rice bran; and
• (4) polished white rice.

Each fraction was extracted and clarified as described in Example 1 for whole rice. Analysis of this extraction showed that the majority of the pullulanase activity was found in the endosperm (polished rice). In addition, this preparation had a much higher specific activity than the preparations made from whole or brown rice. Consequently, polished rice is the preferred enzyme source.

Example 3 Extraction of pullulanase from polished rice

2 kg of commercial 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 for 3 hours at 50°C.

The pH of the extract was adjusted to 5.0 and the resulting slurry was clarified by centrifugation. The pH of the slurry was readjusted to 6.0.

• (1) das Präparat Salz benötigt, um in Lösung zu verbleiben; und
• (2) NH₄HCO₃ sublimiert und durch nachfolgendes Gefriertrocknen entfernt wird.

Nach der Diafiltration gegen 4 Volumeneinheiten von 0,1 M NH₄HCO₃ wurde das Retentat gefriergetrocknet.For long-term storage, it was desirable to obtain the preparation as a salt-free powder. This was done by diafiltering the suspension from the pH adjustment step against 1.0 M NH ₄ HCO ₃ . This salt was chosen because

• (1) the preparation requires salt to remain in solution; and
• (2) NH ₄ HCO ₃ is sublimated and removed by subsequent freeze-drying.

After diafiltration against 4 volumes of 0.1 M NH ₄ HCO _{3 ,} the retentate was freeze-dried.

Example 4 pH optimum of rice pullulanase

• (1) pH 4,0-5,5-0,1 M Essigsäure eingestellt auf den richtigen pH-Wert mit NaOH;
• (2) pH 6-7 0,1 M KH₂PO₄ eingestellt auf den richtigen pH-Wert mit NaOH.

Gelagertes Pullulan (10% G/V in H₂O) wurde im geeigneten Puffer auf 1% G/V verdünnt. Reispullulanase wurde dann über den pH-Bereich 4-7 unter Standardbedingungen geprüft. Die Ergebnisse zeigten an, daß eine optimale Aktivität im pH-Bereich von 5-6,5 erhalten wird.The optimal pH range was determined for rice pullulanase isolated according to the procedure described in Example 3. 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 KH ₂ PO ₄ adjusted to the correct pH with NaOH.

Stored pullulan (10% w/v in H ₂ O) was diluted to 1% w/v in the appropriate buffer. Rice pullulanase was then assayed over the pH range 4-7 under standard conditions. The results indicated that optimal activity was obtained in the pH range 5-6.5.

Example 5 Temperature optimum of rice pullulanase (A) in the absence of substrate

Rice pullulanase prepared according to Example 3 was adjusted to a final concentration of 2 mg/ml in 0.1 M acetate buffer, pH 5.0. Samples of this mixture were incubated for periods of 10-60 min at a temperature range of 40-70°C. heated. Grab samples of the heated samples were removed, cooled and subjected to the standard pullulanase test. The results indicated that the enzyme was inactivated at temperatures above 40°C. Complete inactivation occurred after 10 minutes at 60°C.

(B) in the presence of substrate

The pullulanase prepared in Example 3 was adjusted to a concentration of 1 mg/ml in 0.1 M acetate, pH 5.0. Samples of this preparation (sufficient to give a final concentration of 0.2 mg/ml incubate) were introduced into tubes containing pullulan and 0.1 M acetate buffer (pH 5.0) equilibrated to the desired temperature. At each temperature (from 40°C to 70°C), 1 ml samples were withdrawn after 10, 20 and 30 minutes of heating and inactivated by introduction into the dinitrosalicylic acid solution used for color development. The reduced sugars were determined by the standard method.

The results of these experiments show that the enzyme is stable up to 60°C for 30 minutes in the presence of substrate. This is in contrast to the temperature stability of the enzyme alone as described in Example 5(A).

Examples 6-15 below describe the use of rice pullulanase extract or rice pullulanase in conjunction with the brewing process. Examples 7-12 illustrate the use of the enzyme in conjunction with various alpha-1,4-carbohydrases in beer undergoing fermentation, while Examples 13 and 14 illustrate its use prior to fermentation. In all cases, the wort was mashed as malt-only wort and adjusted to approximately 12° to 15° P with a commercially available converted grain syrup prior to fermentation. In the following examples, the original specific gravity was constant. The worts were brought to a final concentration of 1 × 10 ⁷ cells/ml with S. uvarum and fermented at 15°C.

Example 6 Production of cereal diastases (A) Malt diastase

High-gib distiller's malt was milled in a standard barley mill. The powder (150 g) was added to 1.5 liters of 0.1 M acetate buffer (pH 5.0). The slurry was stirred for two hours at 50°C and the slurry was recovered in the manner described for the crude rice extract (Example 1).

The enzyme was further purified by adding (NH ₄ ) ₂ SO ₄ to a final concentration of 40 g/100 ml. The precipitate was collected by centrifugation and resuspended in 0.1 M acetate buffer (pH 5.0). The suspension was clarified by diafiltration against the same buffer, concentrated and stored at 4°C.

(B) Production of malt beta-amylase

The malt diastase prepared according to Example 6(A) contains both alpha- and beta-amylase, with alpha-amylase being present in the greatest concentration. Malt alpha-amylase can be selectively inactivated at acidic pH (9). The pH of a portion of the malt diastase prepared as described in Example 6(A) was adjusted to 3.6 and heated to 35°C for two hours. The solution was clarified by centrifugation and the pH of the slurry was readjusted to 4.0.

(C) Production of soybean diastase

Whole soybeans were ground in a Wiley mill using a 20 mesh sieve. 10 g of powder was stirred in 100 ml of 0.1 M acetate buffer (pH 5.2) at 55°C for 1 hour. The solution was clarified by centrifugation, after which it was filtered using a filter aid. The slurry was diluted four-fold, diafiltered against H ₂ O and concentrated to the original volume. The concentrate was stored at 4°C.

(D) Isolation of wheat diastase

Wheat diastase was isolated from pearled hard winter wheat milled as described in Example 6(B) for soybean diastase. The powder (50 g) was added to 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. It was then clarified as described in Example 1 for the rice enzyme.

The suspension was dialyzed against 0.02 M phosphate buffer-0.2 M NaCl and then water.

The glucoamylase used in the following experiments was Novo 150.

Example 7 High fermentation of beer using rice pullulanase extract malt diastase

• (1) ohne Enzymzusatz (Bier + 1), um die Gärungsgrenze festzustellen;
• (2) mit Zusatz von Glucoamylase (8,1 U/l, Bier + 2), um die Gärungsgrenze festzustellen; und
• (3) unter Zusatz von Reispullulanase (15,3 U/l) und Malzdiastase (140 U/l Bier + 3).

In allen Fällen wurden die Würzen mit Hefe versetzt und belüftet, wonach die entsprechenden Enzyme zugesetzt wurden. Die Biere wurden bei 15°C vergoren.The wort, which was mashed from malt and adjusted to about 12° P to about 15° P with grain syrup, was fermented:

• (1) without enzyme addition (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 pullulanase (15.3 U/l) and malt diastase (140 U/l beer + 3).

In all cases, the worts were mixed with yeast and aerated, after which the appropriate enzymes were added. The beers were fermented at 15°C.

The enzyme-free control beer contained 0.5-0.6 g less alcohol per 100 g of beer than Beer + 2 or Beer + 3, each of which was highly fermented with glucoamylase and rice pullulanase extract malt diastase. At 3.3% ethanol, the true extract in Beer + 3 was reduced by about 1.0 g/100 g compared to that of Beer + 1 and was almost equal to the value obtained using glucoamylase (Beer + 2). At this alcohol concentration, Beers + 2 and + 3 contain 92-93 cal/340 g, while Beer + 1 has 108 cal/340 g in comparison.

The carbohydrate profiles showed that the beers + 2 and +3 had almost identical carbohydrate compositions and that in both beers + 2 and +3 the non-fermentable sugars were considerably reduced compared to those of beer + 1.

Example 8 High fermentation of beer using rice pullulanase extract-soybean diastase

This wort was aerated and yeasted as described in Example 7. Rice pullulanase extract (15.3 U/l) and soybean diastase (140 U/l) were added. The beer was fermented as described in Example 7.

The fully fermented beer containing 5.3 U/l rice pullulanase extract and 140 U/l soybean diastase (beer + 4) was highly fermented to approximately the same level as the glucoamylase control beer (beer + 2), yielding a beer of 93.4 cal/340 g when bottled with 3.3 g/100 g ethanol. The carbohydrate profile after 12 days of fermentation showed that the non-fermentable fraction was nearly equal to that of the glucoamylase control beer (beer + 2).

Example 9 High fermentation of beer using rice pullulanase extract-wheat diastase

The wort was aerated and yeasted as described in Example 7. Rice pullulanase extract (15.3 U/l) and wheat diastase (140 U/l) were added. The beer was fermented at 15°C as described in Example 7.

This beer (Beer + 5) was highly fermented to the same level as the glucoamylase control beer (Beer + 2). When bottled at an alcohol concentration of 3.3 g/100 g ethanol, the beer contains 29.5 cal/340 g. The carbohydrate composition indicated that the non-fermentable fraction was almost the same as that of Beer + 2.

Example 10 High fermentation of beer with rice pullulanase extract malt beta-amylase

The wort was aerated and yeasted as in Example 7. Rice pullulanase extract (15.3 U/l) and malt beta-amylase (140 U/l) were added and the beer was fermented at 15°C in the usual manner.

The results indicated that this beer (+6) completed the fermentation process in 8 days, i.e. sooner than the glucoamylase control beer (beer +2) or any of the beers made with rice pullulanase extract in conjunction with the other cereal diastases. Again, the carbohydrate composition was similar to that of the glucoamylase control beer. Thus, a beta-amylase appears to be superior to the diastases used in the previous examples (which contain both alpha- and beta-amylase).

Example 11 High fermentation of beer with rice and malt flour

Polished +4 brewer's rice and high-gib distiller's malt were milled to 20 mesh in a Wiley mill. The resulting flour was added to the aerated and loaded wort as described above. One wort (Beer +7) contained 5.2 g rice flour and 0.12 g malt flour/liter, while the other wort (Beer +8) contained twice as much of each flour. The worts were fermented as described above. Grain additions to Beer +7 were calculated to give 15.4 units pullulanase per liter and 140 units malt diastase per liter based on the extraction illustrated in Examples 3 and 6(A).

The results obtained for beers +7 and +8 indicated that both beers fermented to the same level as the glucoamylase control beer (beer +2) and the beers listed in Examples 7-11 where enzyme extracts were used.

Example 12 Use of rice pullulanase in combination with glucoamylase to shorten fermentation time

Wort was used to perform five different fermentations. All worts were aerated and inoculated, after which rice pullulanase extract (2.0 U/L; 4.0 U/L or 8.1 U/L) and glucoamylase (3.8 U/L or 15.3 U/L) were added. The results indicated that the addition of rice pullulanase extract significantly reduced the fermentation time compared to the glucoamylase control beer, even at reduced glucoamylase or pullulanase concentration.

Example 13 Conversion of malt-only wort before fermentation with rice pullulanase extract malt beta-amylase

After boiling, a malt-only wort was obtained. Three samples of the wort were converted using malt beta-amylase (1450 U/L) in conjunction with decreasing concentrations of rice pullulanase extract (150 U/L; 75 U/L and 38 U/L). Another sample of the wort was converted using rice pullulanase extract in conjunction with glucoamylase.

In all cases, the procedure was the same. The worts were equilibrated in a water bath at 60°C with stirring. The worts were heated for 30 minutes, after which they were placed in a glass flask contained in a vigorously boiling water bath. The wort was left to stand for two hours to inactivate the enzymes.

The worts were then cooled and the resulting slurry was removed by centrifugation.

The malt/syrup addition ratio was adjusted to the same value as for the wort described in Examples 7-12. The worts were then inoculated, aerated and fermented as described in Example 7.

The beers were highly fermented compared to the enzyme-free comparison beer (Beer + 1). When bottled with 3.3 g/100 g ethanol, the heat content of these beers was around 98 calories, i.e. around 10 calories less than Beer + 1, which was produced without the addition of enzymes.

Example 14 Addition of grain amylase to pre-converted beers

Since the beers in Example 13 were not highly fermented to the same level as the beers described in Examples 7-12, various enzymes were added to determine if the high fermentation limit could be lowered. The yeast was removed by centrifugation and the clarified beer was divided into two equal parts designated Beer + 14A and Beer + 14B. Both beers were re-yeasted. Beer + 14A was added with 140 U/L malt beta-amylase and Beer + 14B was added with 15.3 U/L rice pullulanase and 140 U/L malt beta-amylase. The fermentation process was then continued at 15°C.

Beers +15A and +16A were not re-yeasted. Instead, the enzymes were added directly to the fermenting beers. Beer +15A was added with 15.3 U/l rice pullulanase and beer +16A was added with 15.3 U/l rice pullulanase and 140 U/l malt beta-amylase.

Addition of the alpha-1,4-carbohydrase, malt beta-amylase, did not noticeably reduce specific gravity (beer +14A) because most of the unfermentable sugars contained alpha-1,6 linkages. In contrast, malt beta-amylase in conjunction with rice pullulanase (beers +14B and +16A) or rice pullulanase alone (beer +15A) fermented the beers to the same level as glucoamylase (beer +2) or the beers described in Examples 7-12.

Example 15 Addition of rice pullulanase extract to the wort during mashing

• (1) 46°C über 30 min; und
• (2) 60°C über 120 min.

Die Maische wurde bei 77°C abgemaischt, wonach das verbrauchte Korn durch Mullfiltration entfernt wurde. Die erste Würze wurde durch Zentrifugieren geklärt, verdünnt und zwei Stunden lang in einem kochenden Wasserbad gehalten. Alle nachfolgenden Schritte wurden wie in Beispiel 11 beschrieben durchgeführt.Rice pullulanase extract prepared according to Example 3 was added to 400 ml of water to give a final concentration of 520 U/l at 46°C. Then 129.6 g of pale malt was added and the mash was subjected to the following mashing procedure:

• (1) 46°C for 30 min; and
• (2) 60°C for 120 min.

The mash was mashed at 77°C, after which the spent grain was removed by gauze filtration. The first wort was clarified by centrifugation, diluted and kept in a boiling water bath for two hours. All subsequent steps were carried out as described in Example 11.

This beer fermented to a final specific gravity of 1.0019 (0.49° P) compared to 1.0029 (0.75° P) for Beer + 1, the control beer of Example 7 produced without enzyme addition. Consequently, the incorporation of rice pullulanase into the mash reduced the fermentation limit by 0.26° P.

• 1. Miller, C., Anal. Chem. 31, 964, 1959
• 2. Bernfield, P., Advances in Enzymology XII (Nord, F., ed.) 379, Intersciences Publishers, New York, 1951
• 3. Pazur, J., Methods 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., Brewers 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, S. 104, 1978
• 9. Scobell, H., Brobst, K. und Steele, F., Cereal Chem. 54, S. 905, 1975
• 10. Greenwood, C. and A. MacGregor, J. Inst. Brew. 71, 408, 1965
• 11. MBBA Technical Quarterly, Vol. 14, 1977, S. 105
• 12. Boyer (ed.), The Enzymes, 3rd Edition, V, 1971, S. 191 ff.

In the above description, reference was made to the following publications:

• 1. Miller, C., Anal. Chem. 31, 964, 1959
• 2. Bernfield, P., Advances in Enzymology XII (Nord, F., ed.) 379, Intersciences Publishers, New York, 1951
• 3. Pazur, J., Methods 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., Brewers Digest 27, 45, 1952
• 6. Olshausen, J., Brewers Digest 27, 53, 1952
• 7. Helbert, JR, 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
• 11. MBBA Technical Quarterly, Vol. 14, 1977, p. 105
• 12. Boyer (ed.), The Enzymes, 3rd Edition, V, 1971, p. 191 ff.

Claims (4)

1. Process for producing a highly fermented beer by fermenting brewer's wort with yeast, wherein a pullulanase is added to cleave the alpha 1,6 bonds of the limit dextrins, and the alpha-1,4-dextrins formed are degraded by 1,4-carbohydrases to fermentable sugars, characterized in that a rice pullulanase is used as the pullulanase. 2. Process according to claim 1, characterized in that the rice pullulanase is added to the wort before or during fermentation together with a 1,4-carbohydrase. 3. Process according to claim 2, characterized in that the rice pullulanase is added to the wort in the form of polished and milled rice. 4. Process according to one of the preceding claims, characterized in that the rice pullulanase is used as an extract.