GB2056484A - Preparation of low calorie beer - Google Patents

Abstract

Low calorie beer is prepared by introducing into the brewing process a rice pullulanase, which reduces the calorie content of the beer by cleaving 1-6 linkages of unfermentable limit dextrins to form alpha 1,4 dextrins which can be converted by grain enzymes to sugars that can be fermented by brewer's yeast. The enzyme may be introduced into the brewing process by adding enzyme extracted from rice to the mash; by adding the debranching enzyme previously isolated from rice to the wort prior to fermentation or preferably by adding the debranching enzyme to wort in the fermentor, or by adding rice to the wort. The debranching enzyme may be obtained from commercially polished dry milled rice. Methods of isolating the enzyme and a storage stable form of the enzyme are disclosed, wherein rice is treated with an aqueous buffer at pH 6 and 0 to 60 DEG C, e.g. in 0.1M potassium sorbate-0.2M NaCl at 50 DEG C for 3 hours; the extract being dialyzed against a solution of a sublimable salt e.g. NH4CO3 and freeze-dried.

Description

SPECIFICATION Preparation of low calorie beer The present invention relates generally to a method of preparing a beer. More particularly, it relates to a method of preparing a low calorie beer which comprises introducing an enzyme extracted from rice, a traditional brewing source, into the brewing process. It also relates to a method of extracting the enzyme from whole or polished rice and a storage stable form of the enzyme.

In the production of beer, yeast is used to ferment into ethyl alcohol a substrate made of a mixture of fermentabie carbohydrates. The wort carbohydrates involved which can be fermented by brewer's yeast are primarily maltose, glucose, maltotriose and traces of sucrose and fructose.

They are obtained by allowing malt enzymes (alpha and beta amylase) to transform starch molecules from malt and other adjuncts into the fermentabie sugars outlined above. This is done during the mashing operation. Foliowing mashing the soluble materials are extracted during lautering, leaving behind the spent grain. A clear liquid (wort) is obtained which is transferred to a brew kettle and boiled for a period of time (kettle boil) to inactivate all malt enzymes. Hops are usually added at kettle boil after which the wort is cooled, aerated, pitched with yeast and allowed to ferment. Wort compositions vary depending on the bill of materials, mash cycle employed, etc.

However, a typical wort is made up of approximately 65 to 80% fermentable carbohydrates of the type mentioned before and about 20 to 35% nonfermentable carbohydrates.

After fermentation a beverage is obtained which usually contains from 3 to 5% alcohol with approximately equal amounts of residual dextrin forming the bulk of dissolved solids, commonly referred to as real extract. This residue remains because of the inability of malt amylases to hydrolyze the alpha 1-6 linkages of the starch.

When the wort described below is fermented a product is obtained which contains approximately 110 calories per 1 2 oz bottle when packaged at 3.3g/1 00 ethanol.

In the production of low calorie, superattenuated beers, an attempt is made to obtain a higher proportion of alcohol and a much lower amount of residual dextrin. This results in a beer which has a lower specific gravity at end fermentation than normally obtained. The first superattenuated products made were produced by a process which consisted of adding an external enzyme in the fermenter. That particular enzyme, a glucoamylase, has the capability of hydrolyzing both alpha 1-4 and alpha 1-6 linkages of the starch and is usually obtained from the mold Aspergillus niger. The use of glucoamylase is not without certain disadvantages. They are the following: (a) The enzyme has some difficulty hydrolyzing the alpha 1-6 linkages. It is much more efficient at hydrolyzing alpha 1-4 linkages, and, (b) The enzyme may be considered to be exogenous to the brewing process.That is, it is not present nor is it isolated from traditional brewing materials, e.g., malt, rice, corn, or yeast.

Another approach which has been suggested consists of using an alpha 1-6 carbohydrase or pullulanase combined with a beta amylase of microbiological origin.

There are three basic classes of starch debranching enzymes. They are the glucoamylases, the isoamylases, and the pullulanases. The distinctions between these classes are well known to those skilled in the art.

Basically, puilulanases cleave the alpha 1,6 linkages of pullulan (an alpha 1,6 polymer of maltotriose isolafed from a mold cell wall) to yield maltotriose. Pullulanases are specific for alpha 1,6 linkages and can debranch the wort limit dextrins producing alpha 1,4 polysaccharides which can be converted by various alpha 1,4 carbohydrases to sugars which are fermentable by brewer's yeast.

Attempts have been made in the past to isolate a debranching enzyme from sources germane to beer production, such as malt. The so-called "R enzyme" has been reported in the literature. It seems, however, that to date a good efficient way of isolating the "R enzyme" has not been found.

It is the general object of the present invention to disclose the discovery that a well known traditional brewing material can be used as a source for a debranching enzyme to produce a superattenuated beer.

The present invention broadly comprises using rice as the source of debranching enzyme in the preparation of a low calorie beer. It also includes a method of isolating the debranching enzyme from rice.

Rice has traditionally been used in the brewing industry. Usually it is used as an adjunct, or an additional source of carbohydrates, like corn grits or corn syrup. The rice used for this purpose is usually a food grade rice, that is rice which has been put through the conventional drying process and subsequently dry milled. Brewers generally use the broken kernels from the polishing operation. The traditional process consists of using rice in the cereal cooker. Usually some malt is added, together with enough water so that some conversion of rice starch is obtained in the cooker. This mixture is cooked for a period of time and added to the mash where malt enzymes convert the starch from the malt and the rice into fermentable carbohydrates. The rice adjunct used in this fashion has no enzyme activity, all of it having been inactivated in the cereal cooker.The traditional process, therefore, does not use rice as a source of enzyme.

We have discovered that the use of a debranching enzyme which naturally occurs in rice provides good results when used in the brewing process to prepare a low calorie beer.

The debranching enzyme from rice originates in a traditional brewing ingredient, and it appears to be more effective than glucoamylase in reducing the highly branched high molecular weight dextrin fraction.

The method of the present invention is an improvement in the method of producing a superattenuated beer by fermenting brewers wort with yeast which comprises adding a rice pullalanase in an amount effective to reduce the amount of residual dextrins in the real extract by cleaving the alpha 1,6 linkages of limit dextrins to form alpha 1,4 dextrins which are converted by 1,4 carbohydrases to fermentable sugars which are fermented by the yeast to alcohol. The enzyme can be introduced at various stages. In a preferred embodiment, either rice or the debranching enzyme extracted from the rice is added to the wort which contains grain amylase from a suitable source, i.e., malt at the fermentor.The debranching enzyme from the rice hydrolyzes the residual 1-6 linkages of the limit dextrins and the grain amylase cleaves the resulting linear alpha 1-4 polysaccharides into fermentable sugars which are then converted to ethanol by the yeast.

In another embodiment of the method, the debranching enzyme extracted from the rice is added to the mash to help cleave the 1-6 linkages of the limit dextrins which otherwise would be formed. The natural malt enzymes hydrolyze the 1-4 linkages thus producing higher levels of fermentable sugars.

Beer of palatable quality can be produced by each of the above-described procedures. In each instance, the end product has been found to contain a greater proportion of alcohol to real extract and fewer calories per unit of volume when packaged- at constant alcohol than a control beer produced with no enzyme addition.

The enzyme which has been found to be useful in the preparation of a low calorie or superattenuated beer is a starch debranching enzyme which naturally occurs in rice. The enzyme of the present invention is classed as a pullulanase because it hydrolyzes the alpha 1,6 linkages of the diagnostic substrate pullulan.

In the extraction method of the present invention the enzyme is extracted from whole or commercially polished rice with an aqueous buffer system having a pH of about 6 at temperatures ranging from 0--600C. The preferred conditions are to slurry polished rice in 0.1 M potassium phosphate buffer -0.2 M NaCI, pH 6.0 at about 500C for about 3 hr.

The pullulanase-containing supernatant from the extraction may be further purified by (1) acidification of the crude extract; and (2) precipitation of the rice enzyme with (NH4)2SO4. These procedures will be illustrated in the examples below.

The enzyme may be stored in a liquid form, or as a freeze-dried or spray dried powder. The freeze dried powder is obtained by diafiltering the enzyme containing buffered pH 6 extract against 0.1 M ammonium bicarbonate solution.

Ammonium bicarbonate is a volatile salt which sublimes on freeze drying to yield a salt free enzyme powder Other sublimable salts which do not interfere with the process or adversely affect the enzymatic activity also may be used if desired.

The amount of rice or extracted enzyme to be 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 it is added and the brewing conditions, e.g., pH, temperature, and time. Generally, the amount of rice or extract to be added will be an amount effective to reduce the amount of residual limit dextrins in the real extract by about 30 to about 80%. Normally, for preconversion of the dextrins prior to fermentation an enzyme source, either extract or rice, containing from about 100 units to about 300 units of pullulanase activity per liter will be added to the wort or about 300 units to about 700 units per liter to the mash.Smaller amounts containing about 2 units to about 75 units of pullulanase activity per liter are effective when added to the wort in the fermenter. Larger amounts than those normally used can be used if desired or needed. Obviously some testing may be required to determine the precise amounts to be used. However, such testing and determination are weli within the skill of those skilled in brewing art.

In one embodiment of the method, the debranching enzyme is added to the fermentor with a carbohydrase, such as a grain diastase. This combination is required because the rice debranching enzyme cleaves the highly branched alpha 1,6 limit dextrins, and the added carbohydrase cleaves the resulting alpha 1,4 dextrins into sugars that can be used by the yeast.

The effective amounts of each enzyme to be added will depend upon the content of limit dextrins normally present in the product of the fermentation and the extent of caloric reduction desired. Normally, the debranching enzyme will be present in an amount of about 2 units to about 60 units of pullulanase activity per liter of wort and the carbohydrase or grain diastase will be present in an amount ranging from about 20 units per to about 140 units of amylase activity per liter of wort.

In still another embodiment, the addition of the rice debranching enzyme to the fermentor is accompanied by the addition of a glucoamylase such as that derived from Aspergillus niger which is active vs. both alpha 1,6 and alpha 1,4 linkages.

The introduction of the combination of these two enzymes at the fermentation stage significantly reduces the fermentation time normally required to prepare a superattenuated beer, e.g., from 12 to 7 days. Although both of the enzymes possess debranching activity the rice enzyme is more potent than glucoamylase and as a result the fermentation time is reduced. The concentration of the rice enzyme in such a mixture may be lower than that normally employed, e.g., 2 4 units of pullulanase and the glucoamylase will be present in about 2 units to about 10 units of glocoamylase activity per liter.

The following analytical procedures were used in the examples described below. Protein was determined by the Lowry method as modified by Miller (1). Pullulanase activity was determined by hydrolysis of 0.5% w/v pullulan at pH 5.0 and 500 C. Amylase activity was determined by the hydrolysis of 0.5% w/v Linter soluble starch at pH 5.0 and 500C. The appearance of reducing sugars was monitored by the dinitrosalicylic acid method of Bernfield (2). A unit of activity in both assays was defined as the appearance of 1 mg reducing sugar (as maltose)/minute. Specific activities are expressed as units/mg protein.

Glucoamylase activity was determined by a modification of the method of Pazur (3), using maltose as substrate, at pH 5.0 and 250C. The appearance of glucose was monitored using the coupled glucose oxidase-peroxidase reaction with o-dansidine as the indicator dye (3). A unit of activity was defined as the hydrolysis of 1 micromole maltose/minute under these conditions.

Fermentations were monitored by the decrease in specific gravity using the Mettler DMA-45 calculating densitometer. When the beers were judged to be end-fermented, refractive indices were obtained on a Zeiss immersion refractometer. These measurements were used to calculate the alcohol (4,5,6) and real extract (5,6) of the beers. The caloric content of a standard 12 oz container was calculated at 3.3 g/1 00 ethanol, as described by Helbert (7).

Carbohydrate profiles were obtained by highpressure liquid chromatography on Bio Rad Q 1 5S 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-1 P-10 cartridge (m.w.

cutoff = 10,000) (Amicon Corporation, Lexington, Mass.).

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

EXAMPLE 1 Isolation of Pullulanase from Whole Rice Five-hundred grams ssed grade LaBelle rice were blended in 0.1 M potassium phosphate buffer - 0.2 M NaCI, pH 6.0, using a Waring blender. The blended grain was transferred to a vessel in a bath maintained at 500C and stirred under 2 liters buffer for 3 hr. The spent grain was removed by filtration through cheesecloth and the filtrate clarified by centrifugation.

Further clarification may be achieved by reducing the pH of the extract to 5.0. The resulting precipitate was removed by centrifugation, and the pH of the supernatant was readjusted to 6.0.

The extract may be purified and concentrated by (NA4)2SO4 fractionation. This salt was added to the pH adjusted supernatant at the rate of 40 g solid (NH4)2SO4 per 100 ml solution. The suspension was stirred for 1 hr at room temperature, and the precipitate was removed by centrifugation. The precipitate was dissolved in and diafiltered vs. the extraction buffer.

EXAMPLE 2 Localization of Pullulanase within the Rice Kernel LaBelle rice from Example 1 was pearled and the following fractions isolated: (1) husks; (2) brown or dehusked 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 these extracts revealed that the great majority of pullulanase activity was localized in the endosperm (polished rice). In addition, this preparation had a much greater specific activity than preparations obtained from either whole or brown rice. Thus, polished rice is the preferred enzyme source.

EXAMPLE 3 Extraction of Pullulanase from Polished Rice Twa kilograms of commercially polished rice were ground to .02 inch in a barley mill. The ground rice was doughed into 4 liters of pH 6 extraction buffer, and the suspension was stirred for 3 hours at 500C.

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

For long-term storage, it was desirable to obtain the preparation as a salt-free powder. This was accomplished by diafiltering the supernatant from the pH adjustment step vs. 0.1 M NH4HCO3.

This salt was chosen since: (1) the preparation requires salt to remain in solution; and (2) NH4HCO3 sublimes and is removed by subsequent freeze drying.

After diafiltration vs. 4 volumes of 0.1 M NH4HCO3, the retentate was freeze dried.

EXAMPLE 4 pH Optimum of Rice Pullulanase The pH optimum range was determined on rice pullulanase isolated by the procedure outlined in Example 3. The following buffer systems were used: (1) pH 4.0--5.5 -- 0.1 M acetic acid adjusted to the appropriate pH with NaOH; (2) pH 6-7 0.1 M KH2PO4 adjusted to the appropriate pH with NaOH. Stock pullulan (10% w/v in H2O) 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 is obtained in the pH range of 5-6.5.

EXAMPLE 5 Temperature Optimum of Rice Pullulanase (A) In the absence of substrate Rice pullulanase prepared as described in Example 3 was made to a final concentration of 2 mg/ml in 0.1 M acetate buffer, pH 5.0. Aliquots of this mixture were incubated in the temperature range 40-700C for times ranging from 10-60 min. Aliquots of the incubates were withdrawn, cooled, and subjected to the standard pullulanase assay. The results indicated that the enzyme was rapidly inactivated at temperatures in excess of 4000. Complete inactivation occurred at 600C after 10 min.

(B) Presence of substrate Rice pullulanase prepared as described in Example 3 was made to a concentration of 1 mg/ml in 0.1 M acetate pH 5.0. Aliquots of this preparation (sufficient to yield a final concentration of 0.2 mg/ml incubate) were delivered into tubes containing pullulan and 0.1 M acetate buffer, pH 5.0 which had been equilibrated to the desired temperature. At each temperature (ranging from 400C--700C), 1-ml aliquots were withdrawn after 10,20, and 30 minute incubations and inactivated by delivering them into the dinitrosalicylic acid solution used for color development. The reducing sugars were determined in the standard manner.

From the results of these experiments it is apparent that the enzyme is stable in the presence of substrate up to 600C for 30 min. This is in marked contrast to the temperature stability of the enzyme alone as described in Example 5-A.

Examples 6-1 5 will illustrate the application of rice debranching enzyme (pullulanase) to the brewing process. Examples 7-12 will illustrate the use of the enzyme in combination with various alpha 1,4 carbohydrases with fermenting beer, while Examples 13 and 14 will iI!ustrate its use prior to fermentation. In all cases, the wort used was mashed as an all-malt wort and was adjusted to about 120 to about 1 50P with a commercial converted corn-derived syrup, prior to fermentation. In the examples which follow the original gravity was constant. The worts were pitched with a stock brewing culture of S. uvarum to a final concentration of 1 x 107 cells/ml and fermented at 1 50C.

EXAMPLE 6 Preparation of Grain Diastases for Use with Rice Pullulanase (A) Malt diastase High-gib distiller's malt was ground in a standard barley mill. The powder (150 g) was doughed into 1.5 liters 0.1 M acetate buffer, pH 5.0. The slurry was stirred for 2 hr at 500C and the supernatant recovered as described for rice crude extract (Example 1).

The enzyme was further purified by adding (NH4)2SO4 to a final concentration of 40 g/1 00 ml.

The precipitate was harvested by centrifugation and resuspended in 0.1 M acetate buffer pH 5.0.

The suspension was clarified by diafiltration vs.

the same buffer, concentrated and stored at 40C.

(B) Preparation ofmalt beta-amylase Malt diastase prepared as in Example 6-A contains both alpha- and beta-amylase, with alpha-amylase in greatest concentration. Malt alpha-amylase can be selectively inactivated at acid pH (9). The pH of a portion of the malt diastase, prepared as described in Example 6-A, was adjusted to 3.6 and incubated at 350C for 2 hr. The solution was clarified by centrifugation, and the pH of the supernatant was readjusted to 5.0.

(C) Preparation ofsoybean diastase Whole soybeans were ground in a Wiley mill using a 20 mesh screen. Ten gm of powder were stirred in 100 ml .01 M acetate buffer, pH 5.2 at 550C for 1 hr. The solution was clarified by centrifugation followed by filtration using a filter aid. The supernatant was diluted 4-fold, diafiltered vs. H20, and concentrated to the original volume.

The concentrate was stored at 40C.

(D) Isolation of wheat diastase Wheat diastase was isolated from pearled hard winter wheat ground as described in Example 6-B for soybean diastase. The powder (50 g) was doughed into 100 ml 0.1 M phosphate buffer-0.1 M NaCI, pH 6.0, and the suspension was stirred for 3 hr at 500C. The suspension was clarified as described in Example 1 for the rice enzyme.

The supernatant was dialyzed vs. .02 M phosphate buffer - 0.2 M NaCI and then water.

The glucoamylase used in the experiments described below was Novo 150 obtained from Novo Industries, Wilton, Connecticut.

EXAMPLE 7 Superattenuation of Fermenting Beer Using Rice Pullulanase-Malt Diastase The wort formulated as described above was fermented: (1) with no enzyme addition (Beer #1) to establish the attenuation limit; (2) with the addition of glucoamylase (8.1 U/I Beer #2) to establish the superattenuation limit; and (3) with the addition of rice pullulanase (15.3 U/I) and malt diastase (140 U/I Beer #3). In all cases, the worts were pitched and aerated as described above after which the appropriate enzymes were added. The beers were fermented at 150C.

The enzyme-free control contained 0.5-0.6 9/100 less alcohol than did either Beer #2 or#3 which were superattenuated with glucoamylase and rice pullulanase/malt diastase, respectively. When packaged at 3.3% ethanol the real extract in Beer #3 was reduced by about 1.0 9/100 over that in Beer #1 and was nearly identical to that obtained when glucoamylase was used (Beer #2). At this alcohol concentration Beers #2 and #3 would contain 92-93 cal/1 2 oz as opposed to 108 cal/1 2 oz for Beer #1.

The carbohydrate profiles demonstrated that Beers #2 and #3 had nearly identical carbohydrate compositions at end-fermentation and that in both Beers #2 and #3 the nonfermentable sugars (greater than DP-3) were substantially reduced over that obtained in Beer #1.

EXAMPLE 8 Superattenuation of Fermenting Beer Using Rice Pullulanase-Soybean Diastase The wort was aerated and pitched as in Example 7. Rice pullulanase (15.3 U/I) and soybean diastase (140 U/I) were added. The beer was fermented as described in Example 7.

The end-fermented beer containing 1 5.3 U/I of the rice pullulanase and 140 U/I of soybean diastase (Beer #4) superattenuated to about the same degree as the glucoamylase control (Beer #2), yielding a beer of 93.4 cay/12 oz when packaged at 3.3 g/1 00 ethanol. The carbohydrate profile after 12 days of fermentation showed that the nonfermentable fraction was nearly identical to that of the glucoamylase control (Beer #2).

EXAMPLE 9 Superattenuation of Fermenting Beer Using Rice Pullulanase-Wheat Diastase The wort was aerated and pitched as described in Example 7. Rice pullulanase (15.3 U/I) and wheat diastase (140 U/I) were added as shown.

The beer was fermented at 1 50C as described in Example 7.

The beer (Beer #5) superattenuated to the same level as the glucoamylase control (Beer #2).

When packaged at an alcohol concentration of 3.3 g/1 00 ethanol, the beer would contain 92.5 cay/12 oz. The carbohydrate composition indicated that the nonfermentable fraction was nearly equal to that of Beer #2.

EXAMPLE 10 Superattenuation.of Fermenting Beer with Rice Pullulanase-Malt Beta-Amylase The wort was aerated and pitched as described in Example 7. Rice pullulanase (15.3 U/I) and malt beta-amylase (140 U/I) were added and the beer fermented at 1 50C in the normal manner.

The results indicated that this beer (#6) endfermented in 8 days, which was faster than the glucoamylase control (Beer #2) or any of the beers formulated with rice pul!ulanase in conjunction with the other grain diastases. Again, the carbohydrate composition was similar to that of the glucoamylase control. A beta-amylase appears to be superior to any of the diastases (containing both alpha and beta-amylase) used in the previous examples.

EXAMPLE 11 Superattenuation of Fermenting Beer with Rice and Malt Flours Polished #4 brewer's rice and high gib distiller's malt were ground to 20 mesh in a Wiley mill. They were added to the wort aerated, and pitched as described above. One wort (Beer #7) contained 5.2 g rice flour and 0.12 g malt flour/liter, while the other (Beer #8) coritained twice as much of each flour. The worts were fermented as described above. The grain additions to Beer S7 were calculated to yield 1 5.4 units pullulanase per liter and 140 units of malt diastase per liter based on extraction as illustrated in Examples 3 and 6-A, respectively.

The data obtained for Beers #7 and #8 indicated that both beers attenuated to the same level as the glucoamylase control (Beer #2) and the beers illustrated in Examples 7-11 1 in which enzyme extracts were employed.

EXAMPLE 12 Rice Pullulanase Used with Glucoamylase to Shorten Fermentation Time The wort was used to set up 5 separate fermentations. All the worts were aerated and pitched after which rice pullulanase (2.0 U/I; 4.0 U/I or 8.1 U/I) and glucoamylase (3.8 U/I or 1 5.3 U/I) were added. The results demonstrated that the addition of rice pullulanase significantly shortened the fermentation time over the glucoamylase control even at reduced glucoamyiase (Beers #1 1 and 12) or pullulanase concentration (Beer #13).

EXAMPLE 13 Conversion of All-Malt Wort Prior to Fermentation with Rice Pulluianase-Malt Beta-Amylase An all-malt wort was obtained following kettle boil. Three wort samples were converted with malt beta-amylase (1450 U/I) in conjunction with decreasing concentrations of rice pullulanase (150 U/I; 75 U/I and 38 U/I). Another sample of the wort was converted using rice pullulanase in conjunction with glucoamylase.

In all cases the procedure was the same. The worts were equilibrated at 600C with stirring in a water bath. Incubation was allowed to continue for 30 minutes after which they were delivered into a flask contained in a vigorously boiling water bath. They were allowed to remain there for 2 hr to inactivate the enzymes. The worts were then cooled, and the resulting trub was removed by centrifugation.

The malt to syrup adjunct ratio was adjusted to the same level as the wort described in Examples 7-12. The worts were then pitched, aerated, and fermented as described in Example 7.

The beers were superattenuated relative to the no-enzyme control (Beer #1). When packaged at 3.3 g/1 00 ethanol, the caloric content of these beers would be about 98 calories, some 10 calories less than Beer #1 formulated with no enzyme addition.

EXAMPLE 14 Addition of Grain Amylases to Preconverted Beers Since the beers cited in Example 13 did not superattenuate to the same level as the beers cited in Examples 7-12, various enzymes were added to see if the attenuation limit could be decreased. The yeast was removed from Beer #14 by centrifugation, and the clarified beer was split into two equal portions designated 1 4A and 1 4B.

Both beers were repitched. To Beer #1 4A was added 1 40 U/I of malt beta-amylase and to Beer #1 4B was added 1 5.3 U/I of the rice pullulanase and 140 U/I of malt beta-amylase. Fermentation was then continued at 1 50C.

Beers #1 5A and #1 6A were not repitched.

Instead, the enzymes were injected directly into the fermenting beers. To Beer #1 5A was added 1 5.3 U/I of rice pullulanase and to Beer #1 6A was added 15.3 U/I of rice pullulanase and 140 U/I of malt beta-amylase.

The addition of the alpha 1,4 carbohydrase, malt beta-amylase, did not significantly reduce the specific gravity (Beer #1 4A), suggesting that most of the nonfermentable sugars contained alpha 1,6 linkages. In contrast, malt beta-amylase in conjunction with rice pullulanase (Beers #14B and #1 6A), or rice pullulanase alone (Beer #1 5A), superattenuated the beers to the same level as the glucoamylase control (Beer #2) or the beers described in Examples 7-12.

EXAMPLE 1 5 Addition of Rice Pullulanase to Wort at Mash-in Rice pullulanase, prepared as described in Example 3, was added to 400 ml foundation water to a final concentration of 520 U/1 at 460C. Then, 129.6 g pale malt were doughed in, and the mash was subjected to the following mash cycle; (-1) 460C for 30 min; and (2) 600C for 120 min.

The brew was mashed-off at 770C after which the spent grain was removed by filtration through cheesecloth. The first wort was clarified by centrifugation, diluted, and placed in a boiling water bath for 2 hr. All subsequent steps were as described in Example 11.

This beer end-fermented to a final specific gravity of 1.0019 (0.490 P) as opposed to 1.0029 (0.750 P) for Beer #1 ,the no enzyme control cited in Example 7. Thus, incorporation of rice pullulanase in the mash reduced the attenuation limit by 0.260P.

REFERENCES 1. Miller, G. Anal. Chem. 31,964, 1959.

2. Bernfield, P. Advances in Enzymology XII (Nord, F., ed.) 379, Intersciences Publishers, New York, 1951.

3. Pzur, 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, 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.

The rice which may be used as the source of the enzyme of the present invention is food-grade rice which has been treated at conditions mild enough to preserve the enzymatic activity. Either seed rice or polished dry milled rice may be used.

The enzyme may be extracted from a wide variety of rice, including LaBelle, LeBonnet, Nato, Starbonnet, or Brazos. However, commercially polished dry milled brewer's rice is preferred.

If the enzyme is extracted from rice prior to use in this process, the spent rice from which the enzyme has been extracted can be utilized as a starch source in mashing or for adjunct syrup formulation to make the use of the rice enzyme more economical.

Although the use of the rice debranching enzyme which has been described is its use in preparing a low calorie or superattenuated beer, it might possibly have other applications. For example, a mixture of the rice debranching enzyme and a grain diastase may be advantageously used to prepare a starch conversion product having a high maltose content.

Because of its natural origin, the debranching enzyme from rice would no doubt be approved for use in food products without too much difficulty.

It will be apparent to those skilled in the art that a number of modifications and changes can be made without the departing from the spirit and scope of the present invention. Therefore, it is intended that the scope of the invention be limited only by the claims which follow.

Claims (19)

1. A method of producing a superattenuated beer by fermenting brewers wort with yeast which comprises adding a rice pullulanase in an amount effective to reduce the amount of residual dextrins in the real extract by cleaving the alpha 1,6 linkages of limit dextrins to form alpha 1,4 dextrins which are converted by 1,4 carbohydrases to fermentable sugars which are fermented by the yeast to alcohol.

2. The method according to Claim 1 in which the rice pullulanase is added in an effective amount to the wort.

3. The method according to Claim 2 in which the rice pullulanase is added to the wort before fermentation.

4. The method according to Claim 2 in which the rice pullulanase is added to the wort during fermentation.

5. The method according to any one of claims 2 to 4 in which the rice pullulanase is added to the wort by adding rice containing the pullulanase to the wort.

6. The method according to any one of claims 2 to 4 in which the rice pullulanase is added to the wort as a purified enzyme extracted from rice.

7. The method according to any one of claims 2 to 6 in which the amount of rice pullulanase added is about 15 to about 60 units of pullulanase per liter of wort.

8. The method according to any one of claims 2 to 6 in which the rice pullulanase is added in an amount of about 2 units to about 60 units of pullulanase activity per liter of wort.

9. The method according to any one of claims 1 to 8 in which the rice pullulanase is added in an amount effective to reduce the amount of residual dextrins in the real extract by about 30% to about 80%.

10. The method according to any one of claims 1 to 9 in which a 1,4 carbohydrase is added to the wort to convert the alpha 1,4 dextrins formed by the rice pullulanase to fermentable sugars which can be fermented by the yeast to alcohol.

11. A method according to Claim 1 and substantially as herein described.

12. A method of extracting a pullulanase from rice which contains enzymatic activity, which comprises treating the rice with an aqueous buffer of about pH 6 at a temperature of about 0 to about 600C. to obtain an extract containing the pullulanase.

13. The method according to Claim 12 in which whole rice is extracted.

14. The method according to Claim 12 in which commercially polished rice is extracted.

1 5. The method according to Claim 12 in which either whole or commercially polished rice is extracted at pH 6.0 in 0.1 M potassium phosphate - 0.2 M NaCI at 500C for 3 hours.

1 6. The method according to any one of claims 12 to 1 5 in which the pullulanase-containing extract is dialyzed against a solution of a sublimable salt and freeze dried to yield a salt-free enzyme powder which is storage stable.

17. The method according to Claim 16 in which the sublimable salt is NH4HCO3.

18. A method according to Claim 12 and substantially as herein described.

19. A pullulanase obtained from rice by the method of any one of claims 12 to 18.