Fapurusi: Sugar Changes during Preparation of Burukutu Beer 79density (1 ‘4)and it is readily available within the dried fruit C.S.I.R.O., industry. Division of Horticultural Research, Owing to the simplicity of the gritometer, the cost of Glen Osmond, South Australia 5064 production and maintenance is low and operators need have and no special skills. Furthermore the few steps involved in the Merbein, procedure allow determinations to be carried out quickly. Victoria, Australia After a few days’ experience, operators in Packing Houses can Received 28 July, 1969 average 10 estimations per hour. Siro gritometers are currently being used in most dried fruit Packing Houses in Austrialia. References 1. Cruess, W. V., ‘Commercial fruit and vegetable products’, Acknowledgments p. 593 (New York: McGraw-Hill) 2. Moyer, J. C., ‘Dirt content of grapes’, Research Circular No. 6, The authors thank Miss S. Oliver and Miss J. Evans for 1966, (Geneva, N.Y.: New York State Agric. Exp. Stn) valuable technical assistance, Mr. P. Johnston for construction 3. Hawker, J. S., & Grncarevic, M., Fd Technol. A m . , 1968, 20, work, the Australian Dried Vine Fruits Research Committee 218 4. Aust. P., Appln No. 39619168 for financial support and Mr. K. M. Cellier of the C.S.I.R.O., 5. Harris, J., Grncarevic, M., & Hawker, J. S., Fd Techno[. Ausr., Division of Mathematical Statistics for statistical analyses. 1968,20, 537 SUGAR CHANGES DURING THE PREPARATION OF BURUKUTU BEER By S. I. FAPARUSI* In the study of sugar changes during preparation of Burukutu beer, samples analysed were obtained from a local brewer. A freshly prepared drink was analysed together with gari - a starch adjunct. The pattern of sugar changes during this 5-day malting stage was also followed. Twelve sugars were identified in the beverage while six sugars were shown to be present in the gari adjunct. Analyses of both germinated and ungerminated sorghum grains show that no new types of sugars resulted from the process of germina- tion. In the study of amylase activity of the malts it was shown that detectable amylase activity began from the second day of germination onwards. Because of the relatively high concentrations of malto- oligosaccharides in the drink, starch amylosis should have continued during the fermentation stage. Introduction The mixture is left to ferment for 2 days. At the end of Burukutu is an alcoholic beverage brewed from the guinea fermentation, the mixture is boiled for about 4 h. The drink corn grains (Sorghum vulgare; = S. bicolor (Moench)); in the is then left to mature for another period of 2 days. The end savannah regions of Nigeria. During the preparation, product is a suspension of some particles in a creamy liquid. sorghum grains are steeped in water overnight. The grains Similar alcoholic beverages produced from cereal grains are then poured into a basket and the water is allowed to have been described in other parts of the world, e.g. in the drain off for about 2 hours. Republic of South Africa, a kaffir beer is produced from The grains are spread out on leaves or mats in a bed of Sorghum cafrorum Beauv.lT2 In Nigeria little study has been about 3-4 in. in thickness and covered with another layer of made on any of the beverages produced from the cereals. leaves. During this malting period, the grains are watered Recently, a study of the microflora of Pito drink was started on alternate days and occasionally turned over. (Ekundayo, J. A., personal communication). Pito is a Germination, which starts within 24 h after steeping, is beverage which can be brewed from millet or corn or sorghum allowed to continue for a period of 4-5 days. The length of grains. plumule is used in judging when germination has gone far The present study was undertaken to determine the different enough. However, germination is usually complete after a types of sugars in the Burukutu beer. The changes which period of 4 days. occur in the various sugar concentrations were followed After germination the malt is spread in thin layers in the during the malting stage in order to ascertain the type and sun to dry for 1 or 2 days. The dried malt is ground into amount of sugars formed during the germination of the powder. grains. In the next stage, gari (a starchy powder produced from the tuber of cassava plant, Manihot utilissima Pohl.) is added Experimental to a mixture of the ground malt and water and stirred with a Preparation of sorghum malt stick. The resulting mixture (gari-malt powder-water) is All the samples analysed were obtained from a local roughly in the ratio 1 : 2 : 6 by vol. brewer, who bought the sorghum grains from the local markets. The grains were steeped in water overnight (about 16 h). The water was allowed to drain off from a basket for * Present address: Department of Brewing and Biochemistry, about 1 h. Heriot-Watt University, Edinburgh J. Sci. Fd Agric., 1970, Vol. 21, February 80 Fapurusi: Sugar Changes during Preparation of Burukutu Beer The grains were then spread out in a bed of up to 3 in. in After drying, one of the triplicate columns was cut off and thickness and covered with banana leaves and sacks. Each the sugars on it were located using the method described by day samples were taken out from the germinating grains and Dube & Nordin.3 Then, areas containing the spots were spread out in thin layers to dry in the sun. marked out, and corresponding areas on each of the remaining The remaining grains were watered and turned over every duplicates were removed and eluted for 3 h with 5 * O ml dis- day. The dried malt was ground in a Waring blender. tilled water. Germination was allowed to continue for five days. To 2.0 ml of an aqueous eluate was added 0.05 ml of 80% (wt./vol.) aqueous phenol reagent followed by a rapid Extraction of sugars from the samples addition of 5.0 ml of conc. HzS04. After the sample had 5.0 g of ground malt were weighed into a 100 ml conical stood at room temperature (25") for 30 min, the optical flask. The sugars were extracted with three 50 ml portions density was determined at 480 nm. The concentration of of 80% ethanol on a boiling water bath. Each portion was each sugar was calculated from the standard curve. All extracted for 1 h. Three such extractions were shown from samples were read against a blank containing distilled water the preliminary experiments, to be sufficient for complete in place of the sugar solution.'L extraction of the sugars. The solid particles in the extract were centrifuged and the Assay of amylases supernatant concentrated in vacuo at a temperature of 60" to 100 ml of 0.2% (wt./vol.) calcium acetate at pH 4.6 were a volume of about 20 ml. The proteins were precipitated used to extract the amylases from 10 g of the ground sorghum with a suspension of basic lead acetate in water. The red- malt for 3 hours.s The extract was centrifuged at 12,000 brown pigment got from the coat of the grains was also ad- rev/min for 30 min at a temperature of between - 1O and 1". + sorbed by the thick precipitate formed. After centrifuging The clear supernatant extract was assayed at 37" using a the precipitate the lead ions were removed by two successive modified method of Street & Closes as described by Street.6 bubblings of H2S gas through the solution. After the preliminary experiments it was considered essential H2S was boiled off and the volume of the extract was to dilute the extract 10 times. The extract was always diluted reduced in vacuo at a temperature of 60" to about 3 . 0 ml. with the calcium acetate solution. The total volume was made up to 5 .O ml with 20 % (by vol.) aqueous isopropanol. Results and Discussion Table I shows the sugar content of the gari adjunct. The Sugar analysis sugar content in the Burukutu beer is shown in Table 11. 0.01 ml of each sugar extract was spotted in triplicate on The sugars identified in the beverage were fructose, mannose, Whatman No. 1 chromatography paper. The chromato- galactose, glucose, galacturonic acid, sucrose, maltose, gram was developed for 72 h in 1-butanol-acetic acid-water isomaltose, raffinose, maltotriose, stachyose and malto- (4 : 1 : 1 by vol.) using a descending paper chromatographic tetraose. method. Fig. 1 shows the variation of sugars with the number of days of the germination of the sorghum grains. The pattern TABLEI of variation of the sugars during this malting stage showed that the sugars could be divided into three distinct groups: Analysis of sugars in gari those sugars which decreased in concentrations on the first Sugar Conc., %by wt. day of germination and then showed a rapid increase in con- centration on the second day until the third day after which Mannose 0.90 there was no further substantial increase in concentration, Galactose 0.50 e.g. glucose and sucrose; those sugars which showed a steady Maltose 0.32 increase in their concentrations until the second day, after Maltotriose 0.30 Maltotetraose 0-09 Raflinose 0.45 TABLEI1 Analysis of sugars in Burukutu beer *-O t The samples were analysed immediately after the 2-day maturing period Sugar Conc., %by wt. Fructose 0.20 Mannose 0-65 Glucose 0.60 Galactose 0.36 Galacturonic acid 0.48 Sucrose 0.20 Maltose 0.42 0 I 1 I Isomaltose 0.38 1 3 5 Raffinose 0.60 DAYS OF GERMINATION Maltotriose 0.56 Stachyose 0.43 FIG.1. Concentration of sugar vs. days of germination Maltotetraose 0.16 A Sucrose; 0 glucose; 0 fructose; 0 raffinose; A maltose; x maltotriosc; W isomaltose; V stachyose J. Sci. Fd Agric., 1970, Vol. 21, February Faparusi: Sugar Changes during Preparation of Burukutu Beer 81 which there was a relatively sharp rise in the concentrations but they did not increase after the third day, e.g. fructose, maltose, isomaltose and maltotriose; and those sugars which did not show any increase in their concentrations, e.g. raffinose and stachyose. Previous analyses of sorghum grains have shown the presence of a number of sugars. Von Holdt & Brand7 reported that only fructose, glucose and sucrose were present in ungerminated sorghum grains. They also showed, in addition to these three sugars, that maltose, isomaltose, maltotriose and traces of higher malto-oligosaccharides were present in germinated grains. Nordins reported the presence of stachyose in sorghum grains. The purpose of the malting stage is to effect the hydrolysis of starch to fermentable sugars. It is the view of many workers that amylases are responsible for this process of hydrolysis. There have been conflicting views as to when the activity of these enzymes becomes manifested. Novellie2 concluded that the enzymes are synthesised during the FIG.2. Graph of amyalse activity against days of germination Enzyme activity is expressed in Street-Close units germination stage. However, Dube & Nordin3 reported that the amylases are usually present in the insoluble condition in the grains even before germination begins. Here, there is would be produced.ll Because some of the sugars which evidence (Fig. 2) that detectable amylase activity started from arise from starch hydrolysis, e.g. glucose and maltose, could the second day. be used up in the fermentation process while the malto- During the steeping stage the metabolic activities of the oligosaccharides are unfermented, it is expected that these grains which were previously dormant, were resumed. Some maltosugars would remain in relatively high concentrations. of the sugars would be used in the metabolic processes. Sucrose appears to be the sugar used mainly in the fermenta- Sorghum grains germinate rapidly and hence will require high tion. This is not surprising since some of the micro- energy-level compounds. This could be responsible for the organisms which could be responsible for the fermentation of initial fall in the concentrations of some of the sugar. How- the drink usually ferment sucrose in preference to other ever, when the amylase activity resumes, more sugars will be sugars. Although these micro-organisms have not been produced than is required for metabolism; thus the concen- analysed, however, it has recently been shown that Saccharo- trations of these sugars increase. myces cerevisiae is one of the yeasts which ferment Pito drink It appears that all these sugars were already present in the (Ekundayo, J. A., personal communication). It is expected grains before germination started. Some suggestions have that this yeast would play a major role in this beverage. been made that the moulds which sometimes grow on the Also, from the sour taste of the drink, some lactobacilli may grains, produce amylases which would attack the ~ t a r c h . ~ have played a substantial r d e in the fermentation. This This initial sugar production cannot be excluded because question will be resolved when the study of the microflora Nigeria is a humid country and moulds grow readily on food which ferment Burukutu beer is completed. substances. The local brewers add gari to the mash to increase the Acknowledgment viscosity of the drink. A consumer regards a thin drink as being too dilute and is therefore considered to be of an The author wishes to thank Mme Adamo Yinusa for her co- operation in preparing the samples analysed in this study. inferior quality. Analysis of the gari powder (Table I) showed that mannose, galactose, maltose, raffinose, maltotriose and maltotetraose Biochemistry Department, were present. Thus, only galacturonic acid was not identified University of Ibadan, Nigeria free in any of the ingredients used in the preparation of the Received 24 July. 1969 drink. Galacturonic acid is usually obtained by enzymic hydrolysis of pectin in plant materials. It is expected that pectic substance in gari could have been hydrolysed by the References enzymes of the microflora which ferment the drink. 1. Schwartz, H. M., J. Sci. Fd Agric., 1956, 7, 101 From Table I1 it can be seen that there are relatively high 2. Novellie, L., J. Sci. Fd Agric., 1960, 11, 408 3. Dube, S. K., & Nordin, P., Archs Biochem. Biophys., 1961,94, concentrations of the malto-oligosaccharides, in the drink. ,*, 1Ll Since the mash was not boiled the amylases of the malt 4. Dubois, M., Giles, K. A., Hamiltom, J. K., Rebers, P. A., & remained active and a certain degree of amylosis could occur Smith, F., Analyt. Chem., 1956, 28, 350 during fermentation. Also the micro-organisms responsible 5. Street, H. V., & Close, J. R., CIinica chim. Acta, 1956, 1, 256 6. Street, H. V., ‘Methods of Enzymatic Analysis’, (Ed. Berg- for this fermentation could produce amylases which could mayer, H. U.) 1963 (New York: Academic Press) hydrolyse the starch. Novellielo reported such a starch I . Von Hodlt, M. W., & Brand, J. C., J. Sci. Fd Agric., 1960, 11, amylosis during kaffir beer fermentation. 467 During the preparation of gari the starch should have 8 . Nordin, P., Chem. Absfr., 1959,55, 8548h 9. Platt, B. S., &Webb, R. A., Proc. Nutr. Soc., 1946,4, 132 undergone some degree of gelatinisation. This would make 10. Novellie, L., J . Sci. Fd Agric., 1966, 17, 354 the breakdown of the starch rapid and thus more sugars 11. Novellie, L., & Schutte, R. J., J. Sci. Fd Agric., 1961, 12, 552 J. Sci. Fd Agric., 1970, Vol. 21, February
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