CA1142104A - Preparation of a low calorie beer - Google Patents

Abstract

ABSTRACT
Low calorie beer is prepared by introducing into the brewing process a debranching enzyme obtained from rice, a traditional brewing material. The debranching enzyme re-duces the calorie content of the beer by cleaving 1-6 link-ages of unfermentable limit dextrins to form alpha 1,4 dex-trins which can be converted by grain enzymes to sugars that can be fermented by brewer's yeast. She enzyme may be in-troduced into the brewing process by adding enzyme extracted from rice to the mash; or by adding the debranching enzyme previously isolated from rice to the wort prior to fermenta-tion or preferably by adding the debranching enzyme to wort in the fermentor. 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.

Description

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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 alchohol a substrate made of a mixture of ferment-able carbohydrates. The wort carbohydrates involved whichcan be fermented by brewer's yeast are primarily maltose, glucose, maltotriose and traces of sucrose and fructose.
They are obtained by allowin~ malt enzymes (alpha and beta amylase) to transform starch molecules from malt and other adjuncts into the fermentable sugars outlined above. This is done during the mashing operation. Following mashing the soluble materials are extracted during lau-tering, 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 fer-ment. 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 inabil-ity of malt amylases -to hydrolyze the alpha 1-6 linkages of ,_i, ~Zl(14 the starch. When the wort described below is fermented a product is obtained which contains approximately 110 calor-ies per 12 oz. bottle when packaged at 3.3g/100 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 superatten-uated products made were produced by a process which con-sisted of adding an external enzyme in ~he 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 difficultv 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 mater-ials, 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, pullulanases cleave the alpha 1,6 linkages of pullulan (an alpha 1,6 polymer of maltotriose isolated from a mold cell wall) to yield maltotriose. Pullulanases are specific for ~ 2~63~
alpha l,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 ~y 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 addi-tional 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 carbohy-drates. The rice adjunct used in this fashion has no en~yme 3ll.~*Z1~4 activity, all of it having been inactivated in the cereal cooker. The traditional process, therefore, does nok 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 improve-ment 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 dex-trins which are converted by 1,4 carbohydrases to ferment-able sugars which are ~ermented by the yeast to alcohol.
The enzyme can be introduced at various stages. In a pre-ferred 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 poly-saccharides into fermentable sugars which are then converted to ethanol by the yeast.
In another embodiment of the method, the debranch-ing enzyme extracted from the rice is added to the mash tohelp cleave the 1-6 linkages of the limit dextrins which otherwise would be formed. The natural malt enzymes hydrol-~14;~
yze the 1-4 linkages thus producing higher levels of fer-mentable 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 pullul-anase 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 0-60C. The preferred conditions are to slurry polished rice in 0.1 M potassium phosphate buffer--0.2 M NaCl, pH 6.0 at about 50C for about 3 hr.
The pullulanase-containing supernatant from the extraction may be further purified by~ acidification of the crude extract; and (2) precipitation of the rice enzyme with (NH4)2S04. 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.lM 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 fer-mentation an enzyme source, either extract or rice, con-taining from about 100 units to about 300 units of pullul-anase 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 pull-ulanase 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 well 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 nor-mally present in the product of the fermentation and the extent of caloric reduction desired. Normally, -the debranch-Zl~
ing enæyme will be present in an amoun-t 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 gluco-amylase will be present in about 2 units to about 10 units of glucoamylase 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 (l). Pullulanase activ-ity was determined by hydrolysis of 0.5% w/v pullulan at pH
5.0 and 50C. Amylase activity was determined by the hydrol-ysis of 0.5% w/v Linter soluble starch at pH 5.0 and 50C.
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 l mg reducing sugar (as maltose)/minute. Specific activities are expressed as units/mg protein.
~ lucoamylase activity was determined by a modi-fication of the method of Pazur (3), using maltose as 1~9LZ~

substrate, at pH 5.0 and 25C. 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 den-sitometer. When the beers were judged to be end-fermented, refractive indices were obtained on a Zeiss immersion re-fractometer. 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 calcu-lated at 3.3 g~100 ethanol, as described by Helbert (7).
Carbohydrate profiles were obtained by high-pressure liquid chromatography on Bio Rad Q 15S 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-lP-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. Fi~e-hundred grams seed grade LaBelle rice were blended in 0.1 M potassium phosphate buffer--0.2 M NaCl, pH
6.0, using a Waring blender. The blended grain was trans-ferred to a vessel in a bath maintained at 50C 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 ~421~4 removed by centrifugation, and the pH of the supernatant was readjusted to 6Ø
The extract may be purified and concentrated by (NH4)2S04 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 centri-fugation. The precipitate was dissolved in and diafiltered vs. the extraction buffer. Table 1 summarizes the extrac-tion of whole rice detailed above.
Example 2. Localization of pullulanase withinthe rice kernel. LaBelle rice from Example 1 was pearled and the following fractions isolated: (13 husks; (2) brown or dehusked rice; (3) rice bran; and (4) polished white rice. Each fraction was extracted and clarified as de-scribed in Example 1 for whole rice. Analysis of these extracts, summarized in Table 2, revealed that the great majority of pullulanase activity was localized in the endo-sperm (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 pollshed rice. Two 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 50C.
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 6Ø
For long-term storage, it was desirable to obtain the preparation as a salt-free powder. This was accomplished _g_

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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. ~he results of this extrac-tion are summarized in Table 3.
Example 4. ~H optimum of rice pullulanase. The p~ 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 ad~usted 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 pullulan-ase prepared as described in Example 3 was made to a final concentration of 2 mg/ml in 0.1 M acetate buffer, pH 5Ø
Aliquots of this mixture were incubated in the temperature range 40-70C for times ranging from 10-60 min. Aliquots of the incubates were withdrawn, cooled, and subjected to the standard pullulanase assay. The results illustrated in Fig.

3 show that the enzyme was rapidly inactivated at tempera-tures in excess of 40C. Complete inactivation occurred at 60C after 10 min.
(B) Presence of substrate. Rice pullulanase prepared as described in Example 3 was made to a concen-11~2104 tration of 1 mg/ml in 0.1 M acetate pH 5Ø Ali~uots 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 tempera-ture (ranging from 40C-70C), l-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 is is ap-parent that the enzyme is stable in the presence of sub-strate up to 60C for 30 min. This is in marked contrast to the temperature stability of the enzyme alone as described in Example 5-A.
Examples 6-15 will illustrate the application of rice debranching enzyme (pullulanase) to the brewing process.
Examples 7-12 will illustrate the use of the enzyme in com-bination with various alpha 1,4 carbohydrases with ferment-ing beer, while Examples 13 and 14 will illustrate its use prior to fermentation. In all cases/ the wort used was mashed as an all-malt wort and was adjusted to about 12 to about 15P 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 concentra-tion of 1 x 107 cells/ml and fermented at 15C.
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 5Ø The slurry was stirred for 2 hr. at 50C and the supernatant recovered as described for rice crude extract (Example 1).
The enzyme was further purified by adding (NH~)2SO4 to a final concentration of 40 g/100 ml. The precipitate was harvested by centrifugation and resuspended in 0.1 M acetate buffer pH 5Ø The suspension was clari-fied by diafiltration vs. the same buffer, concentrated and stored at 4C.
(B)_ Preparation of malt beta-amylase. Malt diastase prepared as in Example 6-A contains both alpha- and beta-amylase, with alpha~am~lase in greatest concentration.
Malt alpha-amylase can be seiectively 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 35C for 2 hr. The solution was clarified by centrifuga-tion, and the pH of the supernatant was readjusted to 5Ø
(C) Preparation of soybean 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 55C for 1 hr. The solution was clarified by centrifugation followed by filtration using a filter aid.
The supernatant was diluted 4-fold, diafiltered vs. H2O, and concentrated to the original volume. The concentrate was stored at 4C.
(D) Isolation of wheat diastase. Wheat diastase was isolated from pearled hard winter wheat ground as de-scribed in Example 6-B for soybean diastase. The powder (50 g) was doughed into 100 ml 0.1 M phosphate buffer--0.1 M
NaCl, pH 6.0, and the suspension was stirred for 3 hr at 50C. 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 NaC1 and then water.

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Table 4 summarizes the amylase activity of the grain amylases described in Example 6.
The glucoamylase used in the experiments described below was Novo 150 obtained ~rom Novo Industries, Wi.lton, Connecticut.
Example 7. Superattenuation of fermenting beer using rice pullulanase-malt diastase. The wort formulated as described above was fermented: (l) with no enzyme addi-tion (Beer #l) to establish the atten~ation limit; ~2) with the addition of glucoamylase (Beer #2) to establish the superattenuation limit; and (3) with the addition of rice pullulanase and malt diastase (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 15C. Table S summarizes the enzymes used as well as their concentration in the fermenting beer in U/1.
Table 6 lists the properties of the end-fermented beers described above. The enzyme-free control contained 0.5-0.6 g/100 less alcohol than did either Beer #2 or #3 which were superattenuated with glucoamylase and rice pull-ulanase/malt diastase, respectively. When packaged at 3.3%
ethanol the real extract in Beer #3 was reduced by about 1.0 g/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/12 oz as opposed to 108 cal/12 oz for Beer #l.
The carbohydrate profiles summarized in Table 7 show that Beers #2 and #3 have nearly identical carbohydrate compositions at end-fermentation and that in both Beers #2 and #3 the nonfermentable sugars ~greater than DP-3) are 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 and soybean diastase were added according to the schedule listed in Table 5. The beer was fermented as described in Example 7.
The end-fermented beer (Beer #4, Table 6) super-attenuated to about the same degree as the glucoamylase control (Beer #2), yielding a beer of 93.4 cal/12 oz when packaged at 3.3 g/100 ethanol. The carbohydrate profile is given in Table 7 (after 12 days of fermentation) and shows 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 and wheat diastase were added as shown in Table 5. The beer was fermented at 15C as described in Example 7.
Reference to Table 6 shows that this beer (Beer #5) superattenuated to the same level as the glucoamylase control (Beer #2). When packaged at an alcohol concentra-tion of 3.3 g/100 ethanol, the beer would contain 92.5 cal/12 oz. The carbohydrate composition shown in Table 7 shows that the nonfermentable fraction was nearly eq~al 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 and malt beta-amylase (Example 6-B) were added as described in Table 5 and the beer fer-mented at 15C in the normal manner.
The results in Table 6 show that this beer (#6) end-fermented in 8 days, which was faster than the gluco-amylase control ~Beer #2) or any of the beers formulated with rice pullulanase in conjunction with the other grain diastases. Again, the carbohydrate composition was similar to that of the glucoamylase control as shown in Table 7. A
beta-amylase appears to be superior to any of the diastases (containing both alpha- and beta-amylase) used in the previ-ous 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, Table 6) contained 5.2 g rice flour and 0.12 g malt flour/liter, while the other (Beer #8) contained twice as much of each flour. The worts were fermented as described above. The grain additions to Beer #7 were calculated to yield 15.4 units pullulanase per liter and 140 units of malt diastase per liter based on extraction as illustrated in Examples 3 and 6-A, respec-tively.
The data presented for Beers #7 ~nd #8 in Table 6 shows that both beers attenuated to the same level as the gluco-amylase control (Beer #~) and the beers illustrated in Examples 7-11 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 and glucoamylase were added as described in Table 8. From the results in Table 9, it can readily be seen that rice pullulanase significantly shortened the fermentation time over th~ glucoamylase control even at reduced glucoamylase (Beers #11 and 12) or pullulan-ase concentration (Beer #13).
Example 13. Conversion of all-malt wort prlor to fermentation with rice pullulanase-malt beta-amylase. An 1~21(~4 all-malt wort was obtained following kettle-boil. Three wort samples were converted with malt beta-amylase in con-junction with decreasing concentrations of rice pullulanase.
Another sample of the wort was converted using rice pullul-anase in conjunction with glucoamylase.
In all cases the procedure was the same. The worts were equilibrated at 60C with stirring in a water bath. The enzyme concentrations were adjusted to the levels shown in Table lOA. 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 al'owed 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 alcohol and real extract are summarized in Table 11. The beers were superattenuated relative to the no-enzyme control (Beer #1, Table 6~. When packaged at 3.3 g/100 ethanol, the caloric content of these beers would be about 98 calories, some 10 calories less than Beer #1 for-mulated with no enzyme addition.
; Example 14. Addition of grain amylases to pre-converted 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 14A and 14B. Both beers were repitched and received the enzymes listed in Table lOB. Fermentation was then continued at 15~C.

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Beers #15A and #16A were not repitched. Instead,the enzymes were injected directly into the fermenting beers as described in Table lOB.
The results of the secondary fermentation are listed in Table ll. Addition of the alpha 1,4 carbohydrase, malt beta-amylase, did not significantly reduce the specific gravity (Beer #14A), suggesting that most of the nonferment-able 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), superattenuated the beers to the same level as the gluco-amylase control (Beer #2) or the beers described in Examples 7-12.
Example 15. Addition of rice pullulanase to w~rt ~t ~a~h 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 46C. Then, 129.6 g pale malt were doughed in, and the mash was subjected to the following mash cycle^ (1~ 46C for 30 min; and (2) 60C for 120 min.
The brew was mashed-off at 77C 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 E~ample 11.
This beer end-fermented to a final specific gravity of 1.0019 ~0.49P) as opposed to 1.0029 (0.75P) for Beer #1, the no enzyme control cited in Example 7. Thus, incorporation of rice pullulanase in the mash reduced the attenuation limit by 0.26P

REFERENCES

l. Miller, G . 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 Refracto-metrically" 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. ~acGregor. J. Inst. Brew.
71, 408, 1965.

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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, Star-bonnet, 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 advantag-eously 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 (10)

1. In a method of producing a superattenuated beer by fermenting brewers malt wort with yeast, the improvement which comprises adding to the wort a rice pullulanase in an amount of at least about 2 units of pullulanase activity per liter of wort which is effective in hydrolyzing the alpha 1,6 linkages of wort limit dextrins to form alpha 1,4 dextrins, and adding an alpha 1,4 carbohydrase in an amount of at least about 20 units of amylase activity per liter of wort to convert the alpha 1,4 dextrins to fermentable sugars which are fermented by the yeast in the wort to alcohol thereby reducing the residual limit dextrins in the real extract to obtain a low calorie super-attenuated beer which contains a greater proportion of alcohol to real extract and fewer calories per unit of volume when packaged at constant alcohol than a control beer prepared by the same process without the added pullulanase.

2. The method of claim 1 in which the rice pullulanase is added to the wort before fermentation.

3. The method of claim 1 in which the rice pullulanase is added to the wort during fermentation.

4. The method of claim 1 in which the rice pullulanase is added to the wort by adding the pullulanase to the mash from which the wort is obtained.

5. The method of claim 1 in which the rice pullulanase is added to the wort by adding rice containing pullulanase.

6. The method of claim 1 in which the rice pullulanase is added to the wort as a purified enzyme extracted from rice.

7. The method of claim 1 in which the alpha 1,4 carbohydrase is glucoamylase.

8. The method of claim 1 in which the rice pullulanase is added in amounts of about 15 units of pullulanase activity per liter of wort.

9. The method of claim 1 in which the alpha 1,4 carbohydrase is added in an amount of about 100 to about 140 units of amylase activity per liter of wort.

10. The method of claim 1 in which the pullulanase and the alpha 1,4 carbohydrase are added simultaneously.