IMPROVING THE SACCHARIFICATION OF SORGHUM MASH WITH SUPPLEMENTARY ENZYMES FROM LOCAL CROPS

Transcription

1 IMPROVING THE SACCHARIFICATION OF SORGHUM MASH WITH SUPPLEMENTARY ENZYMES FROM LOCAL CROPS * Peter Abum Sarkodie 1, Daniel Agyapong 1, Solomon Dawuona 2, Abraham Janice Dwomoh 1 and Ernest Owusu-Ansah 2 1 Department of Science Education, University of Education Winneba- Ghana 2 Department of Chemistry, University of Cape Coast, Ghana *Author for Correspondence ABSTRACT Barley which is the main cereal for brewing beer is imported into most African countries by brewing companies; this comes at cost and also affects the cost of production. To address these problems and also to improve the income of African farmers, a lot of interest has been generated in the use of sorghum as substitute for barley. Brewing solely with sorghum comes with a lot of challenges due to the low level of α and β amylases in the malt. To improve the saccharification, sorghum malt was augmented with 30% rice malt, sweet potato, soya bean and barley malt as enzyme sources. Dorado, the sorghum variety that was used was malted, dried and mailed into grits. The mailed sorghum malt was mixed with the various enzyme sources and the hot water extracted (mashed) using 100% barley as the control. The saccharification rate, extracts (%), extract yield (%), limit attenuation (%) at 24 and 48 hours and ph of the worts was determined. The diastatic power ( WK) of sorghum malt, rice malt, barley malt, sweet potato and soya beans were also determined. mash converted when augmented with 30% rice and barley malt and sweet potato while that of soya beans and 100% sorghum showed partial conversion. One way ANOVA of the diastatic power, extract and the limit attenuation showed a significant difference between the samples and the control at 95% confidence level. It was thus concluded that, sorghum mash will saccharify when augmented with 30% supplementary enzymes from barley malt, rice malt and sweet potato. Key Words: Saccharification, Diastatic Power, Attenuation Limit, Supplementary Enzymes, β-amylase and INTRODUCTION The process of brewing lager beers with cereals other than barley is becoming a common practice especially in nonbarley producing countries. This process of barley malt replacement in brewing is accelerating rapidly (Taylor et al., 2013). Barley has been the basic raw material for brewing (Yonkova et al., 2007) for both barley and non-barley producing countries. Its malt is known to be rich in protein and enzymes and has a high β-glucan and pentosan level when compared to other local grains (Malomo et al., 2011). Its high diastatic power and high level of maltose producing β amylase, a key enzyme in breaking down malt starch into fermentable sugars ensuring efficient saccharification, than other cereals makes it brewers choice for beer production. Barley is a temperate cereal and does not thrive on African soil because of the unfavorable climatic condition. It is therefore imported by African brewers for brewing; however, skyrocketing prices due to strong global demand and high shipping costs have forced a re-think by most African countries to invest in other cereals as a malting substitute (Wolfgang, 2004). Nigerian government as a means of conserving its foreign exchange banned the importation of barley which forced local breweries to look at alternative indigenous cereals as a replacement for malted barley (Goode et al., 2003). It was once believed that beer could not be produced without barley. However, it has been well documented that cereals like sorghum, millet and maize have the potential to be alternative substrates for conventional beer brewing (Bubacz et al., 2013). These cereals were commonly used as adjuncts and where utilized wholly, they were used with less success relative to malted barley (Boboye, 2007). They were normally used in the production of opaque beers in tropical climates (Bubacz et al,. 2013) but since the beginning of the twentieth century there has been increasing development of European type lager beer brewing using high proportions of cereals other than barley malt (Taylor et al., 2013). Among these cereals, sorghum is now the leading contender for replacing barley malt in beer production, particularly in tropical countries where barley cultivation is unsuccessful (Raji et al., 2014). grows well in semi-arid climatic region and is the fifth largest cereal produced in the world. It is droughtresistant and indigenous to Africa. Utilization of sorghum in brewing local drinks has been noted in many areas across the world. It has been the basis of traditional African beers such as the clear beers of West Africa (dolo and pito) and the opaque beers of southern Africa (Malomo et al., 2012). like other cereals, is a good source of starch with its content at 69.5 % compared to barley that has 60.0 % (Nandwa et al., 2013) and has an approximately 71

2 75:25 amylopecting/amylose ratio (Ezeogu et al., 2005) which makes it a good carbohydrate source for efficient brewing. It is one of the cereals which is highly contending for barley replacement in brewing European lager beer and until now most of the clear beer produced in Nigeria is brewed with at least some level of sorghum present (Taylor et al., 2006). It is also a very good adjunct material because of its ability to produce a spectrum of fermentable sugar after it has undergone enzymatic conversion (Ogbeide, 2010). Nevertheless, brewing with sorghum is characterized by complexity in the malting process. has low level of β-amylases, enzyme responsible for the hydrolysis of starch into maltose. The inadequacy of β-amylase in sorghum affects its maltogenesis resulting in lower levels of fermentable extracts in brewing. Thus, when sorghum malt is used, starch may not be sufficiently hydrolyzed causing difficulties during wort filtration and beer haziness (Raji et al., 2014). Ogbonna (2011) in his literature outlined some major biochemical problems brewing with sorghum comes with such as high malting losses estimated at 10 30% as against 8 10% for barley; high gelatinisation temperatures which limited starch solubilisation/hydrolysis by the amylolytic enzymes during mashing; low extract yield/low diastatic power (DP) due to inadequate hydrolytic enzyme activities especially β-amylase, which is noted in the literature to affect saccharification of sorghum (Lyumugabe et al., 2012). Inadequate diastatic power according to Nandwa et al., (2013), results in the development of under-modified malt giving rise to poor yields of sugar extracts (maltose) and reduced starch conversion causing insufficient fermentation and consequently production of low volume of ethanol. The lower levels of maltose in malt worts make them less fermentable. Given that the production of maltose, a fermentable sugar, is an important aspect of the brewing process, mashing process for sorghum malt requires modification (Chikezie, 1999).The use of commercial enzymes (Zangue et al., 2011) when mashing with sorghum has been known in the literature to help alleviate the levels of fermentable sugars in its worts and has however become a common practice (Goode et al., 2003). The use of other cereals as adjuncts to sorghum malt during mashing process has also been noted in literature. Adjuncts from roots and tubers are quiet noted to also provide efficient saccharification of sorghum when used. Research by Etim and Etokakpan (1992) shows that, 20% (w/v) sweet potato flour substitution for sorghum malt, was able to increase the level of β amylase in sorghum wort which increased saccharification of the sorghum malt apparently because of the presence of β glucanase in sweet potato which is limited in sorghum. In addition, Onyeze et al., (2013) successfully used low cyanide cassava as an adjunct in beer production and concluded that satisfactory beer could be produced with the use of low cyanide cassava as an adjunct to sorghum. According to Lyumugabe et al., (2012), problem of lack of β amylase in sorghum malt could be solved when sorghum is brewed in association with other crops available in Africa and avoiding the use of commercial enzymes. This current review seeks to investigate the association of rice, sweet potato and soya bean as supplementary enzyme sources to augment the saccharification process of sorghum malt. MATERIALS AND METHODS Malting of rice (KRC Baika) The malting procedure was carried out as describe by Morrall et al., (1985). The variety of rice that was used is KRC-Baika, Cultivated at the Kpong Research Center of University of Ghana. 1Kg of rice sample was weighed and washed with water after which it was steeped in 4L of water and kept overnight. The steep water was then discarded and the sample was spread in a malting tray. The tray was then incubated at a temperature of 28 C for seven days. 500ml of water was sprayed on the sample every morning and evening. (Dorado): The malting procedure was carried out as described by Morrall et al., (1985) The sample (Dorado) was obtained from the Savanna Research Institute of the Council for Scientific and Industrial Research. 8Kg of sorghum sample was washed after which it was steeped in 15L of 0.2% caustic soda solution for 4 hours. The caustic soda solution was discarded after the 4 hours and the sample was washed 3 times with water. The sample was then allowed 2 hours of air rest, after which it was steeped with 15L of water for 6 hours. The steeped water was then discarded and the sample was spread in a malting tray. The sample was then incubated at a temperature of 28 C for five days. 200ml of water was sprayed on the sample every morning and evening. Malt kilning: Kilning of malted sorghum and rice samples were done at a temperature of 40 C for 24hours. The dried samples were then de-rooted after which it was milled into suitable grits size. Malt milling: The malt samples was prepared for analysis by milling to fine grind consistency with a water cooled laboratory mill IKAM20 ( IKA Labortecnik, Janke and Kunkel GmbH & Co. kg, Staufen, Germany). 72

3 Sweet Potato: White sweet potatoes was obtained from the market; these were then washed and peeled, after which they were thinly sliced into drying trays and dried at a temperature of 40 C. The dried samples were then finely milled into flour. Soya Beans: The soya beans were washed dried and were finely milled into soya powder. Experimental Design Table 1 shows the various treatments with grist percentages of main malt and their respective grist percentages of supplementary enzyme sources. Table 1: Treatments and their composition Treatment Main Malt Supplementary Enzyme Sources Percentage of A B C D E F Types Barley Key: Treatment A = 100% Barley Treatment B = 100% Treatment C = 70% + 30% sweet potato Treatment D = 70% + 30% Soya bean Treatment E = 70% + 30% Rice malt Treatment F = 70% + 30% Barley malt Grist (%) Type 0 0 Sweet potato Soya beans Rice malt Barley malt Percentage of Grist (%) Assessment of enzyme availability: The Diastatic power of the various enzymes sources, namely, sorghum malt, rice malt, barley malt, soya beans and sweet potatoes were determined to provide information on the enzyme levels available for the conversion of starch to sugars. Barley malt was used as the reference malt. Diastatic Power Procedure: Diastatic power, the measure of the joint enzymatic activity (mainly α and β amylase) in the malt was determined according to the procedure in the European Brewing Convention (EBC, 1987). The method will be used to determine the Diastatic power of rice malt, barley malt, soya beans and Sweet potato. Mashing experiments (Hot water extraction): Hot Water Extract of malt samples treatments was prepared using standard mashing procedure by Duffour et al., (1992) with some modifications. In the mashing experiment, sorghum malt was replaced with 30% of the various enzyme sources. Iodine Test, Extract Yield, Apparent Attenuation Limit, Reducing Sugars and ph of the wort was measured to assess the extent of saccharification. Procedure for hot water extraction: Milled malt, 200g was transferred into a mashing beaker and then mixed with 800m1 of distilled water at ambient temperature. The mash was then left at ambient temperature for 1 hour to sediment. 360m1 of the supernatant enzyme extract, making 40% of the total mash was decanted using a pipette and kept aside. The remaining thick mash was boiled for 30 minutes under continues stirring after which the supernatant enzyme extract was added to the cooked sediment. The mash was then transferred to a mashing bath at 72 C ± 1 C and held at this temperature for 60 minutes, during which saccharification (Iodide test ) was checked every 10 minutes using iodine starch test. At the end of the 60th minutes, the mash was cooled to room temperature and the weight adjusted to 1.2 kg with distilled water. The resulting wort was then filtered (wort separation) through a fluted filter paper Whatmann No. 3 for analysis. Wort analysis: Extract in % mass saccharose, was done using an ATC refractometer. Wort ph was measured using a Hanna ph meter, model HI9025C. Extract yield was calculated by: Extract yield %, as-is, Y 1 = E (H+ 500) / (l00-e) Extract yield % on dry matter (dm), Y 2 Y 1 x 100 / (100) Where, Y 1 = Extract content of sample, as-is in %; Y 2 = Extract content on dry matter, in %; E = Extract content of wort in g/l00g; H = Moisture content of malt in % 73

4 Attenuation Limit: The wort sample (250g) was quickly brought to boil in a conical flask. After boiling for 10 minutes, the sample was cooled to room temperature and the weight was re-adjusted to the initial 250g with distilled water. The extract was then determined after which the wort was transferred into a Schott fermentation bottle. 1.0g Brewers of yeast was then added and thoroughly mixed with l00ml of the wort. The fermentation bottle was fitted with fermentation lock filled with water and was left to ferment at ambient temperature. The fermenting wort was swirled periodically. The residual wort extract (non-fermentable sugars) after the fermentation period was then determined by measuring the wort extract periodically in the course of 72 hours. Apparent Attenuation Limit, which is the percentage of fermentable sugars in the total extract, was determined as follows: Apparent Attenuation Limit (%) = (initial extract - Residual Extract) 100 Initial Extract A flow diagram of the methodology is shown in figure 1. Figure 1: Flow diagram of methodology RESULTS AND DISCUSSIONS The results in Table 2 show the pattern of saccharification of sorghum mash at 70 C with the various enzyme extracts. Although 70% sorghum malt + 30% sweet potato, 70% sorghum malt + 30% rice malt and 70% sorghum malt + 30% barley malt all converted totally (yellow) by the 60 minute, that of 70% sorghum malt + 30% barley malt converted totally at start just as the standard (100% barley malt) with that of 70% sorghum malt + 30% sweet potato and 70% sorghum malt + 30% rice malt converting totally by the 20th minute. 100% sorghum malt was light blue by the 60th minute and confirmed the report that sorghum lacks enough α and β amylases to self convert (Palmer, 1989). 74

5 Table 2: Saccharification test of various mash combinations Treatment Saccharification (Iodine Test) Colour change (dark blue, light blue, purple, brown, yellow) Start 10mins 20mins 30mins 40mins 50mins 60mins A yellow yellow Yellow Yellow yellow yellow yellow B dark blue dark blue dark blue light blue light blue light blue light blue C brown brown yellow yellow yellow yellow yellow D purple purple purple purple brown brown brown E brown brown yellow yellow yellow yellow yellow F yellow yellow yellow yellow yellow yellow yellow Table 3: One way ANOVA examining differences in diastatic power ( WK) Sum of Squares Df Mean Square F Sign. Between Groups Within Groups Total Table 4: Games-Howell Multiple Comparisons: Diastatic power (I) Standard (J) Combinations Mean Differences (I-J) Std. Error Sign. Barley malt malt * Rice malt * Sweet potato * Soya bean * * The mean difference is significant at the 0.05 level Table 5: One way ANOVA examining differences in Extract (%) Sum of Squares Df Mean Square F Sign. Between Groups Within Groups Total Table 6: Tukey HSD Multiple Comparisons: Extract (%) (I) Standard (J) Combinations Mean Differences (I-J) Std. Error Sign. 100% Barley malt 100% sorghum malt * % sorghum+30% sp * % sorghum+30% sb * % sorghum+30% rm * % sorghum+30% bm * * The mean difference is significant at the 0.05 level Diastatic power in diastatic units of rice malt, sorghum malt, soya beans and sweet potato all showed significant difference (p < 0.05: Table 3) when compared to diastatic power of barley malt using one-way ANOVA and Games- Howell multiple comparisons (Table 4). From literature, malt with enough power to self- convert should have a diastatic power near 94 WK, which explains why 100% sorghum mash did not saccharify. Fig. 2 shows the various distributions of the diastatic power in barley malt, sorghum malt, rice malt sweet potato and soya beans. The percent extract (Fig. 3) of the various combinations of wort all showed significant differences when compared to barley wort at 95% confidence level for both One way ANOVA and Turkey HSD Multiple Comparisons ( p= 0: Table 5 & 6 respectively). These results suggest that, amount of dissolved solutes in the various combinations cannot match that of the standard which fully converted at the start of the saccharification test, but this is contrary to that of 70% sorghum malt + 30% barley malt which also fully converted at start of saccharification test, showed significant difference as compared to 100% barley malt. These results are complex and difficult to understand and explain. 75

6 Fig 2: Mean values of disatatic power of samples Fig. 3: Mean extract of various treatments Table 7: Games-Howell Multiple Comparisons: 24hrs Limit attenuation (%) (I) Standard (J) Combinations Mean Differences (I-J) Std. Error Sign. Treatment A Treatment B * Treatment C Treatment D Treatment E Treatment F * The mean difference is significant at 0.05 level Table 8: LSD Multiple Comparisons: 48 hrs Limit attenuation (I) Standard (J) Combinations Mean Differences (I-J) Std. Error Sign. Treatment A Treatment B * Treatment C Treatment D * Treatment E Treatment F * The mean difference is significant at 0.05 levels 76

7 Fig. 4: Mean Value of limit attenuations at 24 and 48 hours The results of the Games-Howell multiple comparisons for limit attenuation at 24 hours showed significant difference for 100% sorghum (p=0.007: Table 7), while that of 70% sorghum malt + 30% sweet potato, 70% sorghum malt + 30% soya beans, 70% sorghum malt + 30% rice malt and 70% sorghum malt + 30% barley malt all showed no significant difference at 95% confidence level (Table 7). For the 48 hours limit attenuation, there was no significant difference for all the combinations except that of 100% sorghum and 70% sorghum malt + 30% soya beans which showed significant difference 95% confidence level (p=0 and p=0.023 respectively (Table 8). This explains why l00% sorghum malt and 70% sorghum malt + 30% soya beans did not fully saccharify during the saccharification test (Table 2). Fig. 4 shows the mean limit attenuations of treatments at 24 and 48 hours. CONCLUSION This study showed that sorghum mash can saccharify when augmented with 30% supplementary enzyme extracts from barley, sweet potato and rice. The study further confirmed that sorghum lack the necessary amount of α and β - amylase to self convert, as the diastatic power value for sorghum was found to be WK, which is far below the least value for self conversion of mash (94 WK), and the one-way ANOVA for diastatic power showed significant difference between barley (control) and that of sorghum. Although 30% of these supplementary enzymes were able to convert the sorghum mash, the degree of saccharification, as in time of conversion is not the same as compared to the control (Table 2). It is therefore recommended that further studies have to be conducted using a different malting procedure and also, other varieties of sorghum, rice and sweet potato have to be tried to see if the outcome will be better and also, further studies must be carried out to determine the turbidity, foaming capacity of the worts and the free amino nitrogen of the malts. REFERENCES Boboye B (2007). Bacteria, biochemical changes and sensory characterization of sorghum lager beer production. Current World Environment, 2(2) Bubacz M, Philip T, McCreanor PT & Jenkins HE (2013). Engineering of Beer: Hard Work or Too Much Fun?. American Society for Engineering Education, 2013 (Accessed at: Accessed date: June 1, 2014] Chikezie IO (1999). Brewing Beer with. Journal of the Institute of Brewing, 105(1) Dufour JP, Melotte L & Srebrnik S (1992). malts for the production of a lager beer. Journal of America Society for Brewing and Chemistry, Etim MN & Etokakpan OU (1992). brewing using sweet potato enzymic flour to increase saccharification. World Journal for Microbiology and Biotechnology, 8(5) Ezeogu LI, Duodu KG & Taylor JRN (2005). Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours. Journal of Cereal Science, 42(1) Goode DL, Halbert C & Arendt EK (2003). Mashing studies with unmalted sorghum and malted barley. Journal of the Institute of Brewing,

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