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Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.

) as a Raw Material for Gluten-free Beverages and Beers


Martin Zarnkow1,3,*, Matthias Keler2, Werner Back1, Elke K. Arendt3 and Martina Gastl1
ABSTRACT

INTRODUCTION
Celiac disease (CD) (also known as non-tropical sprue, gluten-sensitive enteropathy, celiac sprue, idiopathic steatorrhea, primary malabsorption, Gee-Herter disease, gluten-induced enteropathy, adult celiac disease) is an intolerance to the gliadin fraction of wheat and the prolamins of rye (secalins), barley (hordeins) and possibly oats (avidins)29 and affects approximately 1% of the world population11. Gluten consumption by celiacs excites an inflammation of the small intestine leading to the malabsorption of several important nutrients including iron, folic acid, calcium and fat-soluble vitamins47,50. The symptoms of CD can develop at any age and include severe symptoms of malabsorption such as steatorrhea, abdominal discomfort, weight loss, tiredness, anaemia and severe diarrhoea47,50. The only way to treat CD is the total lifelong avoidance of gluten consumption. The total avoidance of gluten and gluten-related proteins leads to a recovery of the mucosa. A review presented by Kasarda suggests that species of cereals which are less closely related to wheat such as sorghum, millet, rice, and maize as well as pseudo-cereals such as buckwheat, amaranth and quinoa, are safe for the consumption of persons suffering from CD20. One of the products not recommended for celiac patients is beer made from barley; therefore, it is essential to develop beer from grains which are recognized as gluten-free. Most studies dealing with the production of gluten-free malt have been focused on sorghum13 and buckwheat33,34,4246. Proso millet (Panicum miliaceum L.) is one of the oldest crops known to mankind. It was cultivated in the Neolithic period (80002000 BC) in China17. It is reported, that the Chinese farmers cultivated a waxy and a regular species of proso millet during the Second Chinese Dynasty23. Discoveries made in the middle and eastern parts of Europe point to a cultivation of proso millet in this area in the Age of the Band Ceramic (about 5000 BC)22. Due to the cultivation of potatoes and an expansion of potato acreage, proso millet became increasingly less important7. At the beginning of the 20th century, proso millet almost disappeared as a field crop in the western part of Europe14. Today however, proso millet plays an important role in northwest China, Kazakhstan, as well as the central and
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J. Inst. Brew. 116(2), 141150, 2010 Proso millet is a gluten-free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH-adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The -amylase enzyme showed highest activity at a temperature of 60C and at pH 5.0, whereas the -amylase activity was optimum at 40C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control. Key words: Gluten, malted proso millet, mashing, Panicum miliaceum L.

1 Lehrstuhl

fr Brau- und Getrnketechnologie (formerly Lehrstuhl fr Technologie der Brauerei I), Technische Universitt Mnchen Weihenstephan, Weihenstephaner Steig 20, 85354 Freising, Germany. 2 Saatenunion GmbH, Eisenstrae 12, 30916 Isernhagen, Germany. 3 Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, College Road, Cork, Ireland. * Corresponding author. E-mail: Martin.Zarnkow@wzw.tum.de
Publication no. G-2010-0709-1060 2010 The Institute of Brewing & Distilling

southern states of India, Eastern Europe, USA and Australia. Proso millet is the third most important millet, after pearl millet (Pennisetum glaucum) and foxtail millet (Setaria italica). The production of millets has increased slightly from 29 million tons in the 1980s up to 33.6 to 37.3 million tons from 2001 to 2005.53 Proso millet is also referred to as common millet, hog millet, broom corn, yellow hog, hershey, and white millet3. It is an annual cereal grain producing bright green leaves and small seeds. The proso millet plant is considered a short-day plant, growing to 30 to 100 cm tall with few tillers and an adventitious root system. It is considered a self-pollinated crop, but natural cross-pollination may exceed 10%2. The seeds are generally oval in shape and about 3 mm long and 2 mm wide and normally smaller than pearl millet (Pennisetum glaucum)2. Proso millet is well adapted to many soil and climatic conditions. Being a short-season crop (6075 days) with a low water requirement, it grows further north (up to 54 N latitude) than the other millets and also adapts well to plateau conditions and high elevations26. Proso millet can be cultivated up to altitudes of 3,500 m3. The annual precipitation average should be less than 600 mm and the average daytime temperature during vegetation should be above 17C16. Furthermore, the plant requires only small amounts of nitrogen fertilizer and crop rotation is not completely necessary. Moreover, it is quite resistant to plant diseases18. Proso millet belongs to the order of Poales, to the family of Gramineae, to the subfamily of Panicoideae and therein to the tribe of Paniceae, which includes 80 genera36. Proso millet is classified in five subspecies depending on the type of panicle (patetissimum, effusum, contractum, ovatum and compactum)37. Within these subspecies, proso millet is divided into varieties, whose distinctive characteristics are the colour of the grain, the type of husk and the appearance of anthocyanin dye in the peduncle-spikes and husks24. There are numerous studies about malting and brewing with sorghum, pearl and finger millet9, but so far only a few about proso millet51,52. The objective of this study was to find optimal mashing conditions, with a special emphasis on the amylolytic enzyme activity of P. miliaceum malt. Size exclusion chromatography was used to fractionate and extract the amylolytic enzymes. These different enzyme fractions were added to enzyme-inactive proso millet worts with various mashing temperatures and

pH. Enzyme optima were determined by comparing the resulting carbohydrate spectra of the resulting worts.

MATERIALS AND METHODS


Barley starch As purified proso millet starch was not commercially available, barley starch (Altia/Finland) was used as the substrate for the preliminary mashing trials. The starch was suspended in bi-distilled water at a concentration of 10% (w/w). Proso millet malt Proso millet (cv. brown wild form) was grown and harvested near Erding/Bavaria in 2004. The raw material for malting had a moisture content of 10.4% (m/m) and a protein content of 11.4% (w/w) (calculated as N 6.25). Germination capacity was determined to be 98%. Proso millet was malted by applying the optimised malting procedure.51 Briefly, the proso millet was steeped to a moisture level of 44%, followed by five days of germination at 22C. The characteristics of the proso millet malt (measured in the Congress wort) are shown in Table I and compared to the corresponding mash produced from barley malt. Analytical procedures Analytical procedures were carried out in duplicate (n = 2), and the means of all results were calculated. All concentrations are based on dry weight unless stated otherwise. All analyses were carried out according to the standard methods of the Mitteleuropische Brau- und Analysenkommision (MEBAK)1, and Internationale Gesellschaft fr Getreidewissenschaft und -Technologie, International Association for Cereal Science and Technology (ICC)19. Extract. The malt extract was determined by using an Anton Paar Alcolyzer (Anton Paar, Graz, Austria) following the MEBAK method 4.1.4.2.21. Apparent attenuation limit. Apparent attenuation limit (AAL) was determined following MEBAK method 4.1.4.101. Gelatinisation temperature. Gelatinisation temperature (GT) was measured with a Rapid Viscoanalyser RVA Super 4 (Newport Scientific, Warriewood, Australia) fol-

Table I. Malt quality attributes of proso millet malt compared to barley malt.32 Attributes Extract Viscosity (8.6%) Colour Protein Soluble N Kolbach index FAN -Amylase activity -Amylase activity Limit dextrinase activity AAL Gelatinisation temperature
a Dry

Unit %, d.m.a mPa s EBC % mg/100 g % mg/100 g U/g U/g U/kg % C

Proso millet malt (congress wort) 64.1 1.529 5.2 11.0 786 39.5 219 124 107 2073 74.3 67.1

Barley malt (congress wort)32 7685 1.511.63 2.08.0 9.011.5 600900 3545 85237 84410 225627 0800 7484 5866

matter.

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lowing MEBAK method 2.71. The weight-in was 7.5 g malt (5% moisture) and 15 g dist. H2O. -Amylase activity. The -amylase activity was determined using the ICC standard method 30312 and a Megazyme enzyme kit (Megazyme, Wicklow, Ireland); 1.0 g of proso millet sample was used in the test instead of 0.5 g, as recommended in the standard method. The Ceralpha procedure for the assay of -amylase, employs as substrate, the defined oligosaccharide non-reducingend blocked p-nitrophenyl maltoheptaoside in the presence of excess levels of a thermostable -glucosidase, which has no action on the native substrate due to the presence of the blocking group. On hydrolysis of the oligosaccharide by endo-acting -amylase, the excess quantities of -glucosidase present in the mixture give an instantaneous and quantitative hydrolysis of the p-nitrophenyl maltosaccharide fragment to glucose and free pnitrophenol. -Amylase activity. The -amylase activity was determined using Megazyme test kits (Megazyme, Wicklow, Ireland)1,27,38. The reagent consists of a mixture of p-nitrophenyl--D-maltopentaoside (PNPG5) and p-nitrophenyl-D-maltohexaoside (PNPG6). These substrates are rapidly hydrolysed by -amylase, but are only slowly cleaved by cereal -amylase, which requires a longer stretch of 1,4-linked D-glucosyl residues to satisfy the substrate sub-site binding requirements. Limit dextrinase activity. Limit dextrinase activity was determined using a Megazyme test kit (LDZ 7/98; Megazyme, Wicklow, Ireland). The substrate is an azurine-crosslinked-pullulan, which is hydrolysed by limit dextrinase and pullulanase, but is resistant to attack by other commonly occurring amylolytic enzymes such as amylase, -amylase and amyloglucosidase. The substrate is slowly hydrolysed by an Aspergillus niger -amylase. This enzyme can act at the -1,4-maltotetraosyl structural units in pullulan. The substrate is prepared by dyeing and crosslinking a pullulan polysaccharide to produce a material which hydrates in water, but is water insoluble. Hydrolysis by limit dextrinase produces water soluble dyed fragments and the rate of release of these (increase in absorbance at 590 nm) can be related directly to enzyme activity. Glucose, fructose, sucrose, maltose, maltotriose (fermentable carbohydrates). Fermentable carbohydrates were determined using high-performance anionexchange chromatography (HPAEC) with a pulsed amperometric detection system (PAD). The equipment used was supplied by Dionex Cooperation, Sunnyvale, USA21.

Deionised water (resistivity = 18.2 Mcm) was used for both the method and for sample preparation. Samples were boiled for 5 sec to inactivate all amylolytic enzymes. The 2-deoxy-D-glucose (Sigma, St. Louis, USA), cellobiose (Sigma, St. Louis, USA) and deionised water were added for a final dilution of the sample of 1:1,000. The 2deoxy-D-glucose was used as the internal standard for glucose, fructose and sucrose. Cellobiose was used as the internal standard for maltose and maltotriose. Aliquots of 2.5 L of sample were injected and separated using a CarboPac PA 10 guard column (25 2) as well as a CarboPac PA 10 analytical column (100 2). The oven compartment was set at 30C and the flow rate was set at 0.25 mL/min. The gradient used is shown in Table II. Eluent A consisted of 250 mM NaOH prepared by using 50% (w/w) sodium hydroxide (Baker, Netherlands). Eluent B was deionised water. Both eluents were kept under pressure using helium gas. The potentials for the PAD system are shown in Table III. The coefficient of variation was 4.2% for glucose, 3.1% for fructose, 1.7% for sucrose, 0.3% for maltose and 0.9% for maltotriose. Kolbach index (ratio S/T). The effects of malting conditions on proteolytic activities in proso millet were determined by calculating the Kolbach index (MEBAK method 4.1.4.5.3)1. -Amino-nitrogen. Determination of -amino-nitrogen, also referred to as free amino nitrogen (FAN), was based on MEBAK method 4.1.4.5.51 using a Skalar working station (Skalar, Breda, Netherlands). Viscosity. Wort viscosity was measured at 20C using a falling ball viscosimeter AMVn-Automated Micro Viscometer (Anton Paar, Graz, Austria) according to MEBAK method 4.1.4.4.11. Wort colour. Wort colour was determined according to MEBAK method 4.1.4.2.8.11 using a Cadas 200 Spectral Photometer (Dr. Lange, Dsseldorf, Germany). Experimental procedure A cold water extract of proso millet malt was produced by adding 5 g of fine grist into 20 mL of phosphate buffer (5 M, pH 7). The suspension was centrifuged at 3,500 rpm at 4C. Approximately 10 to 15 mL of the supernatant was filtered through a membrane filter with a pore size of 0.45 m. A volume of 3 mL of the filtrate was then injected into a Superdex 200 prep grade 26/60 column (Fa. Amersham Biosciences) to perform size exclusion chromatography (SEC) at a flow rate of 2.2 mL/minute. The chosen setting should enable the different amylolytic enzymes to only occur in certain fractions, so that cross-contamination is avoided. For verification purposes, each fraction was analysed for its enzyme activity. Enzyme kits (Megazyme, Wicklow, Ireland) were used to determine the enzymatic activity of the fractions obtained by SEC. Fig. 1 shows the extinctions of each fraction in comparison to the blank samples.

Table II. Gradient for carbohydrate separation. Time (min) Eluent A (%) Eluent B (%) 10.0 9 91 0.0 9 91 17.9 9 91 18.5 80 20 55.0 80 20 55.1 9 91 75.0 9 91

Table III. Settings of the pulsed amperometric detection system (PAD). Time (min) Potential (V) Integration 0.00 0.1 0.20 0.1 begin 0.40 0.1 end 0.41 2.0 0.42 2.0 0.43 0.6 0.44 0.1 0.50 0.1

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Fractions were considered as pure, when no other amylolytical enzyme activity occurred. Fractions 7 and 8 were used as limit dextrinase, 10 and 11 for -amylase and 13 and 14 for -amylase source. In preliminary trials, enzymes were added into a barley-starch solution (10% w/w). For further trials, the cold proso millet malt wort extracts were prepared following the procedure mentioned above with the difference that the suspension was boiled for 5 min afterwards to inactivate endogenous malt enzymes and pregelatinisation. The blank carbohydrate profile was 0.15 g/L glucose, 0.70 g/L maltose, 0.10 g/L maltotriose, 0.01 g/L fructose and 0.09 g/L sucrose. By extracting with 70% ethanol solution, Becker and Lorenz studied the saccharide composition of eight proso millet varieties. In their research they could only find monosaccharides traces of glucose, fructose and galactose (data were not shown). Sucrose prevailed with a fraction of 0.50.9% of dry weight, values which are comparable to other cereal grains.4,5 Neither maltose nor maltotriose have been detected in proso millet4. Aliquots of 1 mL cold wort extract and 0.5 mL of the fractions containing enzymes were poured together and incubated in a Thermomixer (Thermomixer 5436, Eppendorf-Nethler-Hinz, Hamburg) for 30 min at temperatures of 30, 40, 45, 50, 60, 65, 70 and 75C respectively with a pH of 5.4. Experimental procedures were carried out in duplicate (n = 2), and the means of all results were calculated.

RESULTS AND DISCUSSION


-Amylase -Amylase hydrolyses starch, glycogen, and other 1,4-glucans. The cleavage occurs from inside of the molecules. Amylose is split into oligosaccharides of 67 glucose units, whereas amylopectin is cleaved nonspecifically, leaving out the 1,6--branching points6. Ungermi-

nated, mature proso millet seeds have a very low -amylase activity. In papers by Parvathy and Sadasivam36 as well as Zarnkow et al.51, an increase in -amylase activity has been reported during germination. The -amylase activity in the proso millet malt measured by Zarnkow et al.51 was much lower than the enzyme activity normally found in barley malt. The enzyme -amylase plays a vital role during the mashing process. It is one of the essential enzymes responsible for the degradation of starch into fermentable carbohydrates. All enzymes have specific temperature optima, which need to be considered when mashing regimens are designed. However, there is no published literature available on the optimal temperature and pH profile of the -amylase derived from proso millet malt. The amylase was isolated from proso millet malt using size exclusion chromatography and subjected to an isothermal mashing regimen in a temperature range of 30 to 75C. Since proso millet starch was not commercially available, barley starch was used as the substrate for the enzyme. The impact of the enzyme on starch degradation was measured by using the carbohydrate profile determined by HPLC. The impact on the carbohydrate profile from proso millet malt -amylase is shown in Fig. 2. It can be seen from Fig. 2 that temperature at 50C has a significant impact on the carbohydrate profile. The temperature had very little influence on the maltotriose released. Also, the glucose, fructose and sucrose content were not influenced by temperature to the same extent as maltose. Based on the data presented in Fig. 2, the temperature optimum for proso millet malt -amylase activity in starch was 50C, which was only slightly different from barley, which was 55C28. In a second trial, the impact of pH on the -amylase activity of malt was determined using different pHs and temperatures. In this trial, only maltose and maltotriose are shown, since these were the main sugars released by this particular enzyme in these trials (Fig. 3).

Fig. 1. The -amylase, -amylase and limit dextrinase activities of different fractions obtained with size exclusion chromatography.
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The results (Fig. 3) show that the highest amount of maltose and maltotriose was obtained when the mixture of the -amylase containing SEC-fraction and cold wort extract from proso millet malt was mashed isothermally at 60C and at a pH of 5.0 (Fig. 3). The -amylase activity was clearly influenced by the pH value with pH 5.0 being optimal. However, at a temperature of 70C, the -amylase activity was strongly reduced, independent of the pH value. Based on these data, the optimal conditions for the highest -amylase activity should be set at a temperature of 60C and a pH of 5, which is much lower than that for barley malt30.

-Amylase The -amylases are exo-hydrolases, releasing maltose from the non-reducing end of -1,4-linked poly- and oligo-glucans until the first -1,6-branching point along the substrate molecule is reached54. Yamasaki isolated amylase from germinating proso millet seeds47. The molecular weight (Mr) of the enzyme was estimated to be 58,000. The enzyme activity significantly increased during days one and four of germination. In accordance with the increased enzyme activity, the rootlet length was markedly enhanced. The enzyme hydrolysed malto-oligo-

Fig. 2. The carbohydrate patterns obtained by isothermal mashing of barley starch solution at different temperatures using the SEC-fraction containing proso millet -amylase.

Fig. 3. The maltose and maltotriose patterns obtained by isothermal mashing of pH adjusted cold water extract from proso millet malt at different temperatures using the SEC-fraction containing proso millet -amylase.
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Fig. 4. The carbohydrate patterns obtained by isothermal mashing of barley starch solution at different temperatures using the SEC-fraction containing proso millet -amylase.

saccharides more readily, as their degree of polymerization increased, this being strongest for the malto-oligosaccharides larger than 13 glucose residues and very low for maltotriose. For example, amylopectin and soluble starch were hydrolysed three times faster than maltoheptose. The optimum pH of the enzyme was found to be 5.56.0 which is 0.51.0 higher than Skovron and Lorenz hypothesised39. It was stable in a range of pH 3.59.0. The optimum temperature was found to be 55C47. However, the differences found between the results of the different authors could be due to the use of different cultivars, which has been reported by Skovron and Lorenz39. In the current study, -amylase was extracted from proso millet using size exclusion. The same experimental setup was used as described earlier for -amylase. The results of the carbohydrate profile analysis are shown in Fig. 4. A comparison of Fig. 2 and Fig. 4 reveals a similar carbohydrate pattern for both -amylase and -amylase. The SEC-fraction revealed high amounts of maltose as seen for -amylase (Fig. 2). A consistent decrease in the carbohydrate between 30 and 50C was evident. At 55C, maltose content was increasing again followed by another decrease with a minimum of maltose and maltotriose at 75C. The slight ascent at 55C agrees with the observations made by Yamasaki.47 Nevertheless, concerning these data, the entire temperature range from 30 to 55C is highly activating for -amylase. However, from these data it is difficult to produce an explanation for the high maltose amounts at 30C. One possibility would be the existence of a second -amylase (which might be also the case for -amylase). The impact of pH in combination with temperature, on the starch breakdown caused by -amylase, is shown in Fig. 5. In the pH-adjusted cold water extract from proso millet wort, a non-significant maximum amount of maltose was reached at a temperature of 40C and pH 5.3. At this pH
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level, the activity decreased just slightly up to a temperature of 60C. Further elevation of the temperature to 70C inhibited the generation of maltose considerably. The quantity of maltotriose generated during these mashing trials was neither affected by temperature nor the pH, since maltotriose is not a primary product of -amylase starch degradation. Limit dextrinase Limit dextrinase plays an important role during the early stage of germination in terms of respiration of the seed as its activity in liberating glucose from starch. As discussed in chapter 6, it is assumed that the Megazyme limit dextrinase kit is probably able to measure some type of -glucosidase activity. Furthermore, it is remarkable that in the literature nothing could be found concerning limit dextrinase and proso millet, but many publications were available concerning -glucosidase and proso millet. The -glucosidase activity had already doubled after 24 hours of germination (soaking) whereas -amylase activity was still in a lag period48. Some authors35,41 claim that -glucosidase is part of the non-phosphorolytic pathway for the breakdown of starch by hydrolyzing the oligosaccharides produced by - and -amylases. However, Sun and Henson reported, that barley seed -glucosidase can initiate the breakdown of raw starch granules and that this degradation is independent of the presence of -amylase40. Using fractionation and preparative gel electrophoresis, Yamasaki et al. isolated two types of -glucosidase (I and II) from proso millet seeds50. The optimum pH of the two -glucosidases was found to be 3.5 and they were stable in a pH-range of 3.56.0 (I) and 3.55.5 (II). The temperature optimum of the two -glucosidases was found to be 60C. The two enzymes hydrolysed malto-oligosaccharides at a similar rate as maltose. The -glucosidase (II) hydrolysed native starch from millet seeds poorly. The glucosidase (I) showed a reduced activity by incubation with Hg2+, Zn2+ and Cu2+ of 20% and more, whereas -

Fig. 5. The maltose and maltotriose patterns obtained by isothermal mashing of pH adjusted cold water extract from proso millet malt at different temperatures using the SEC-fraction containing proso millet -amylase.

Fig. 6. The carbohydrate patterns obtained by isothermal mashing of barley starch solution at different temperatures using the SEC-fraction containing proso millet limit dextrinase.

glucosidase (II) activity was not inhibited by Zn2+ and Cu2+. Yamasaki et al. also reported that the activity doubled after the proso millet seeds had been soaked in water for 24 h48. Thus, the conformation of the enzyme protein may have changed to show higher enzyme activity than that in intact seeds. The -glucosidases showed a higher affinity for polysaccharides than for maltose, even at considerably low concentrations compared with maltose. The Michaelis-Menten constant, Km value, for maltose is lower than those of -glucosidases from other plants, such as rice seed10, buckwheat8, sugar beet25,49, and barley15. The Km value for malto-oligosaccharides decreased with an increase of the substrates molecular weight. The glucosidase may preferably hydrolyse oligosaccharides

liberated from starch by -amylase to glucose without the preceding action of -amylase during the germination of millet seeds, although it has been suggested that -glucosidase needs the preceding action of - and -amylases to play a role during the germination of plants35,41. In these trials, the limit dextrinase was isolated in the same way as the - and -amylase and the impact of limit dextrinase on barley starch was determined using isothermal mashing procedures at different temperatures and constant pH. The results of these measurements are shown in Fig. 6. Isothermal mashing of the mixture of barley starch solution and SEC-fraction containing proso millet limit dextrinase showed maximum amounts of maltose at temVOL. 116, NO. 2, 2010 147

Fig. 7. The maltose and maltotriose patterns obtained by isothermal mashing of pH adjusted cold water extract from proso millet malt at different temperatures using the SEC-fraction containing proso millet limit dextrinase.

Fig. 8. The mashing program with optimal temperatures and pH adjusting with respect to amylase, -amylase and limit dextrinase of proso millet malt.

peratures of 40 and 50C. Nevertheless, amounts for all other sugars generated during the various mashing procedures stayed constant and were not affected by temperature. A progressive inactivation of limit dextrinase appeared at temperatures above 60C. When isothermal mashing of the SEC-fraction containing proso millet limit dextrinase was performed in cold wort extract of proso millet malt, highest values for maltose could be reached again at temperatures of 40 and 50C with a single peak at 50C and pH 5.3 (Fig. 7). Again, the influence of pH was clearly noticeable. Comparable to barley, an incipient inactivation of limit dextrinase has already occurred at 60C.31 Mashing procedure On the basis of the above results, it was possible to create an optimised mashing regime (Fig. 8). A modified decoction mashing regimen was designed in which 40%
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of the total mash was removed and heated for fully proso millet starch gelatinisation to one degree over gelatinisation temperature (68C). The gelatinisation temperature of proso millet malt was 67.1C in these trials. The remaining 60% of the original mash was mashed-in at 22C in order to achieve 40C in the combined mash so that the breakdown of the starch by the malt-based amylolytic enzymes was possible. After 10 min the two parts of the mash were combined and mashed for a further 10 min at 40C. At this point the pH of the mash was adjusted to 5.3 using lactic acid to improve -amylase activity. After a 20 min rest, the mash was heated to 50C at a heating rate of 1C/min (general heating rate) and kept constant for 20 min. To optimise the limit dextrinase activity, the mashing temperature was then increased to 60C and the pH was adjusted to pH 5.0, to take the optimal -amylase activity into consideration. After 30 min, the temperature was raised to 70C in order to maintain enzyme activity as

Table IV. Proso millet wort attributes compared to barley malt wort (ale type Klsch Bier1). Attributes Original gravity (GG %) AAL (%) pH FAN (mg/100 mL) BU (EBC) TBI Iodine test Viscosity (mPa s) Zinc (mg/L) Proso millet wort 12.88 79.1 5.59 37.7 38 79 0.266 1.649 1.8 Barley malt wort (ale type Klsch Bier1) 1114 8085 5.35.6 1430 3054 9.659.1 <0.45 1.72.2 0.011.08

Further clarification of enzyme activity during mashing with proso millet malt is needed. More precisely, work needs to be done to examine the effect of calcium ions on -amylase activity, as well as the formation of high amounts of sugar by -amylase and -amylases in barley starch solution at very low temperatures. However, this research revealed that it is possible to produce proso millet malt wort that is comparable to barley malt wort. It represents another important step on the way to producing a gluten-free beer from proso millet, which has been until now unknown in brewing technology.
REFERENCES 1. Anger, H.-M., Brautechnische Analysenmethoden Band Rohstoffe. Selbstverlag der MEBAK: Freising, 2006. 2. Baltensperger, D. D., Progress with proso, pearl and other millets. ASHS Press: Alexandria, VA, 2002, pp. 100-103. 3. Baltensperger, D. D., Foxtail and proso millet. ASHS Press: Alexandria, VA, 1996, pp. 182-190. 4. Becker, R. and Lorenz, K., Saccharides in proso and foxtail millets. J. Food Sci., 1978, 43(5), 1412-1414. 5. Becker, R., Lorenz, K. and Saunders, R. M., Saccharides of maturing triticale, wheat and rye. J. Agric. Food Chem., 1977, 25, 1115. 6. Belitz, H. D., Grosch, W. and Schieberle, P., Lehrbuch der Lebensmittelchemie. 5th ed., Springer-Verlag: Berlin, 2001. 7. Bckler, W., Relikte unter den Kulturpflanzen. Zeitschrift fr Agrargeschichte und Agrarsoziologie, 1954, 2(1), 22-40. 8. Chiba, S., Kanaya, K., Hiromi, K. and Shimomura, T., Substrate specificity and subsite affinities of buckwheat -glucosidase. Agric. Biol. Chem., 1979, 43, 237-242. 9. Daiber, K. H. and Taylor, J. R. N., Opaque Beers. American Association of Cereal Chemists: St. Paul, USA, 1995, p. 307. 10. Eksittikul, T., Svendsby, O., Yazmaguchi, H., Iizuka, M. and Minamimura, N., Thai rice seed -glucosidase and its specificity. Biosci. Biotechnol. Biochem., 1993, 57, 319-321. 11. Fasano, A. and Catassi, C., Current approaches to diagnosis and treatment of Celiac disease: an evolving spectrum. Gastroenterology, 2001, 120, 636-651. 12. Hberli, G., ICC-Standards Standardmethoden der Internationalen Gesellschaft fr Getreidewissenschaft und Technologie, Vienna, 2004. 13. Hallgren, L., Lager beers from sorghum. In: Sorghum and Millets: Chemistry and Technology, D. A. V. Dendy, Ed., AACC: St. Paul, USA, 1995, pp. 293-294. 14. Hanelt, P., Zur Geschichte des Anbaus von Buchweizen und Rispenhirse in der Lausitz. Abh. Ber. Naturkundemuseums Grlitz, 1981, 55, 1-13. 15. Henson, C. A. and Sun, Z. Barley seed -glucosidases: their characteristics and roles in starch degradation. In: Enzymatic Degradation of Insoluble Carbohydrates. J. N. Saddler and M. H. Penner, Eds., American Chemical Society Symposium series 618: Washington, DC, 1995, pp. 51-58. 16. Hoffmann-Bahnsen, R., Alte Kulturpflanze neu entdeckt - Perspektiven und Mglichkeiten der Wiederinkulturnahme von Rispenhirse (Panicum miliaceum) im kologischen Landbau, Onfarm-Erhaltung genetischer Ressourcen von Getreide und lpflanzen, Kern-Verbund, 2004, pp. 91-100. 17. Hoffmann-Bahnsen, R. and Plessow, J., Alte Kulturpflanzen neu entdeckt, Rispenhirse (Panicum miliaceum) eine ideale Sommerung fr den kologischen Landbau., Mitteilungen der Gesellschaft fr Pflanzenbauwissenschaften - 46. Jahrestagung, Gieen. D. Kauter, A. Kmpf, W. Claupein, and W. Diepenbrock, Eds., Verlag Gnter Heimbach: Stuttgart, 2003, pp. 31-33. 18. Humphrys, C., Raps und Hirse im Biologischen Anbau. Last acessed June 2010 at http://www.agroscope.admin.ch/aktuell/ 02720/02722/02771/02772/index.html?lang=de#sprungmarke0_ 13 (24.07.2006).

well as diminish wort viscosity during the lautering process. Lautering was accomplished in a standard lauter tun. Resulting wort characteristics are shown in Table IV and compared to a Klsch-beer type barley malt wort. The latter was chosen due to the fact that in Germany, fermentations with other than barley malt have to be top-fermented5. The wort achieved by applying the optimised mashing regimen on proso millet malt clearly showed that its quality was within the range of published data for worts produced from barley malt, even though the zinc content and thiobarbituric acid index (TBI) were quite distinct. Mainly the latter (TBI) could be regarded as critical, as high values of TBI usually correlate with a negative influence on flavour stability in the final beer. On the contrary, a high zinc content can be seen as positive and may correlate to proso millet malt being an adjunct having zinc-donating properties.

CONCLUSIONS
The gluten-free cereal proso millet (Panicum miliaceum L.) is suitable as a raw material for the manufacture of foods and beverages for people suffering from celiac disease. The object of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity (- and -amylases and limit dextrinase) during mashing was investigated. Size exclusion chromatography was used to isolate the different enzymes. These enzymes were added into a pH-adjusted, cold water extract of proso millet malt and isothermal mashing procedures were applied. Optimum temperature and optimal pH for these amylolytic enzyme activities was determined. The -amylase showed its highest activity in proso millet malt wort at a temperature of 60C and at pH of 5.0, whereas -amylase had its optimum at 40C and pH 5.3. The limit dextrinase activity reached its maximum at 50C and at pH 5.3. In the subsequent mashingregimen, the mash was separated and 40% was kept for 10 min at 68C to achieve gelatinisation. In the next step the mash was combined. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the adapted proso millet mash were comparable to barley wort, which acted as a control.

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19. ICC. ICC-Standards Standardmethoden der Internationalen Gesellschaft fr Getreidewissenschaft und Technologie: Wien, 2004, pp. 1-5. 20. Kasarda, D. D., Grains in relation to celiac disease. Cereal Foods World, 2001, 46, 209-210. 21. Keler, M., Zarnkow, M., Kreisz, S. and Back, W., Gelatinisation properties of different cereals and pseudocereals. Monatsschr. Brauwiss., 2005, (9/10), 82-88. 22. Knrzer, K. H., Pflanzenspuren, Archobotanik im Rheinland: Agrarlandschaft und Nutzpflanzen im Wandel der Zeiten. Rheinland Verlag: Kln, 1999. 23. Kolb, R. T., Landwirtschaft im Alten China. Systemata Mundi Institut zur Erforschung fremder Denksysteme und Organisationsformen: Berlin, 1992, Vol. 1. 24. Lysov, V. N., Proso. Kolos: Leningrad, 1968. 25. Matsui, H., Chiba, S. and Shimomura, T., Substrate specificity of an -glucosidase in sugar beet seed. Agric. Biol. Chem., 1978, 42, 1855-1860. 26. Matz, S. A., Millet, wild rice, adlay, and rice grass. Avi Press: Westport, CT, 1986, pp. 225-229. 27. McCleary, B. V. and Codd, R., Measurement of -amylase in cereal flours and commercial enzyme preparations. J. Cereal Sci., 1989, 9(1), 17-33. 28. Muralikrishna, G. and Nirmala, M., Cereal -amylases-an overview. Carbohydrate Polymers, 2005, 60(2), 163-173. 29. Murray, J. A., The widening spectrum of celiac disease. Am. J. Clin. Nutr., 1999, 69(3), 354-365. 30. Narzi, L., Die Technologie der Malzbereitung. Enke Verlag: Stuttgart, 1999, pp. 152-154. 31. Narzi, L., Die Technologie der Malzbereitung. Enke Verlag: Stuttgart, 1999, pp. 402-417. 32. Narziss, L., Back, W., Die Technologie der Wrzebereitung. Wiley-VCH Verlag: Weinheim, 2009, p. 627. 33. Nic Phiarais, B. P., Wijngaard, H. H. and Arendt, E. K., The impact of kilning on enzymatic activity of buckwheat malt. J. Inst. Brew., 2005, 111(3), 290-298. 34. Nic Phiarais, B. P., Wijngaard, H. H. and Arendt, E. K., Kilning conditions for the optimisation of enzyme levels in buckwheat. J. Am. Soc. Brew. Chem., 2005, 64(4), 187-194. 35. Nomura, T., Kono, Y. and Akazawa, T., Enzymic mechanism of starch breakdown in germinating rice seeds. II. Scutellum as the site of sucrose synthesis. Plant Physiol., 1969, 44, 765-769. 36. Parvathy, K. and Sadasivam, S., Comparison of amylase activity and carbohydrate profile in germinating seeds of Setaria italica, Echinochloa frumenatcea, and Panicum miliaceum. Cereal Chemistry, 1982, 59, 543-544. 37. Prosvirkina, A. G., Agrometeorologitscheskije usslovija i produktivnost prosa. Gidrometeoisdat: Leningrad, 1987. 38. Santos, M. M. M. and Riis, P., Optimized McCleary method for measurement of total beta-amylase in barley and its applicability. J. Inst. Brew., 1996, 102(4), 271-275. 39. Skovron, J. and Lorenz, K., Enzymatic activities in proso millets. Cereal Chem., 1979, 56, 559-562.

40. Sun, Z. and Henson, C. A., Degradation of native starch granules by barley -glucosidases. Plant Physiol., 1990, 94, 320327. 41. Swain, R. R. and Dekker, E. E., Seed germination studies II. Pathways for degradation in germinating pea seedlings. Biochim. Biophys. Acta, 1966, 122, 87-100. 42. Wijngaard, H. H. and Arendt, E. K., Buckwheat - a review. Cereal Chem., 2006, 83, 391-401. 43. Wijngaard, H. H. and Arendt, E. K., Optimisation of a mashing program for 100% malted buckwheat. J. Inst. Brew., 2006, 112(1), 57-65. 44. Wijngaard, H. H., Nic Phiarais, B. P., Ulmer, H. M., Goode, D. L. and Arendt, E. K., Gluten-free beverages based on buckwheat. Proceedings of the European Brewery Convention Congress, Prague, IRL Press: Oxford, 2005, pp. 101/1-101/11. 45. Wijngaard, H. H., Ulmer, H. M. and Arendt, E. K., The effect of germination temperature on malt quality of buckwheat. J. Am. Soc. Brew. Chem., 2005, 63(1), 31-36. 46. Wijngaard, H. H., Ulmer, H. M. and Arendt, E. K., The effect of germination temperature on malt quality of buckwheat. J. Am. Soc. Brew. Chem., 2005, 63(1), 31-36. 47. Yamasaki, Y., -Amylase in germinating millet seeds. Phytochemistry, 2003, 64(5), 935-939. 48. Yamasaki, Y., Fujimoto, M., Kariya, J. and Konno, H., Purification and characterization of an -glucosidase from germinating millet seeds. Phytochemistry, 2005, 66(8), 851-857. 49. Yamasaki, Y. and Konno, H., Purification and properties of glucosidase from suspension-cultured sugar-beet cells. Phytochemistry 1991, 30, 2861-2863. 50. Yamasaki, Y., Konno, H. and Masima, H., Purification and properties of -glucosidase from millet seeds. Phytochemistry, 1996, 41(3), 703-705. 51. Zarnkow, M., Keler, M., Burberg, F., Back, W., Arendt, E. K. and Kreisz, S., The use of response surface methodology to optimise malting conditions of proso millet (Panicum miliaceum L.) as a raw material for gluten-free foods. J. Inst. Brew., 2007, 113(3), 280-292. 52. Zarnkow, M., Keler, M., Burberg, F., Kreisz, S. and Back, W., Gluten free beer from malted cereals and pseudocereals. Proceedings of the European Brewery Convention Congress, Prague, IRL Press: Oxford, 2005, pp 104/1-104/8. 53. Zarnkow, M., Mauch, A., Burberg, F., Back, W., Arendt, E. K., Kreisz, S. and Gastl, M., Proso millet (Panicum miliaceum L.) a sustainable raw material for the malting and brewing process: A review. Brew. Sci., 2009, 62(7/8), 119-140. 54. Ziegler, P., Cereal -amylases. J. Cereal Sci., 1999, 29(3), 195204. 55. Zipfel, W. and Rathke, K.-D., Lebensmittelrecht. C. H. Beck: Mnchen, 2008.

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