Production of a Saccharifying Rice Malt for Brewing Using Different

May 19, 2014 - ABSTRACT: This study was conducted to produce rice malt suitable for beer brewing. An all-rice beer would be particularly appealing to ...
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Production of a Saccharifying Rice Malt for Brewing Using Different Rice Varieties and Malting Parameters Heidi Mayer,† Ombretta Marconi,*,‡ Gian Franco Regnicoli,‡ Giuseppe Perretti,‡ and Paolo Fantozzi† †

Italian Brewing Research Center and ‡Department of Agricultural, Food and Environmental Science, University of Perugia, Via San Costanzo, 06126 Perugia, Italy ABSTRACT: This study was conducted to produce rice malt suitable for beer brewing. An all-rice beer would be particularly appealing to individuals with celiac disease because rice does not contain gluten proteins. Furthermore, rice malt could also contribute to new beer flavors and brands. A screening of 10 rice varieties was conducted. The varieties Balilla and Centauro were found to be suitable for the production of an all-rice malt beer without the need of exogenous enzymes. They were characterized by a low diastatic power but nevertheless they saccharified well, likely due to other endogenous amylolytic enzymes such as limit dextrinase and α-glucosidase. The addition of CaCl2 and lactic acid during mashing lowered the pH value and increased saccharification. However, the Balilla variety saccharified without the need of these additives. We also show that the soluble nitrogen and free amino nitrogen content of rice malt wort can be increased by the incorporation of the acrospires and rootlets during mashing. KEYWORDS: rice varieties, rice malt, gluten-free, beer, saccharification

1. INTRODUCTION Rice is a staple food for nearly 50% of the world’s population. According to the Food and Agriculture Organization of the United Nations (FAO), global rice production was 730.2 million tons (486.9 million tons, milled basis) in 2012. In Europe it was about 3.1 million tons (1.8 million tons, milled basis). The leading European rice producer is Italy, yielding about 1.6 million tons per year (967,000 tons, milled basis), while the other main producers are Hungary, Portugal, Bulgaria, France, Greece, Romania, and Spain.1 Rice belongs to the Gramineae family. The two main species of cultivated rice are Oryza sativa (O. sativa) L. and Oryza glaberrima L., the latter of which is only cultivated in Africa. O. sativa L. indica is more common in tropical zones with higher temperatures and limited hours of daylight throughout the year, such as India, Southeast Asia, and southern China, while O. sativa L. japonica grows better in a temperate climate with lower temperatures and more hours of daylight during the summer, as in Japan, Korea, northern China, Turkey, Egypt, Europe, and North America. The caryopsis of O. sativa L. indica generally has an elongated shape whereas the caryopsis of O. sativa L. japonica is more rounded.2 Rice does not contain gluten-like proteins, so it is particularly suitable for consumption by individuals with celiac disease.3,4 Thus, rice could be a useful raw material for the production of a gluten-free beer-like beverage. But there is less information about rice malt for brewing an all-rice beer in a traditional way, saccharifying completely the wort without the addition of exogenous enzymes. In today’s beer brewing industry rice is used primarily as an adjunct in combination with barley malt.5,6 Only several previous studies5,7−13 showed that rice malt could be a possible raw material in brewing, but none of the studies achieved the complete saccharification of the rice malt wort, apparently because of the rice malt’s lower enzyme content and high gelatinization temperatures. However, these studies © 2014 American Chemical Society

adhered to malt quality attributes derived from official methods for analyzing barley and wheat malt such as Analytica EBC or MEBAK which are suitable for the evaluation of barley malt, but less suitable for rice malt. Indeed Taylor and colleagues5 suggested that supplementation with exogenous cell wall-degrading enzymes, namely, (1− 3,1−4)-β-D-glucanase, would benefit brewing because rice malt contains low amounts of these enzymes compared to barley malt. Also Dziedzoave and colleagues14 confirmed this. But degradation or removal of the enzyme-impermeable cell walls in the rice malt endosperm, an important step during the malting and mashing process, because it exposes starch granules to amylolytic enzymes, is different from that of barley malt. The cell walls of the endosperm of rice, a monocotyledon like barley, are more similar to the cell walls of dicotyledon plants and contain mainly pectin and xyloglucan, while in other cereals such as wheat, barley, oat, and rye the two primary cell wall components are mixed linked (1−3,1−4)-β-D-glucan and arabinoxylan.5,15 So the addition of (1−3,1−4)-β-D-glucanase, which were found in rice malt only in a negligible amount, is not necessary, but other measures are required. According to Bunzel and Steinhart,15 the pectin might simply be washed out at higher temperatures. Another difference between rice malt and barley malt is the expression of amylolytic enzymes. In barley malt, an important attribute for the activity of starchdegrading enzymes is the diastatic power, which measures the combined activity of α- and β-amylases, and for this reason it is always taken into account in the evaluation of malt. Rice malt, however, shows lower diastatic power values,11 and for this it seems less suitable for brewing. But rice malt contains other amylotytic enzymes which can act synergistically with α- and βReceived: December 10, 2013 Accepted: May 18, 2014 Published: May 19, 2014 5369

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amylases.5,16,17 This could mean that the required diastatic power for sufficient starch degradation in rice malt could be lower than in barley malt. One of these amylolytic enzymes is limit dextrinase, a debranching enzyme which catalyzes the hydrolysis of the α-1,6-glucosidic linkages in starch and related oligosaccharides and exhibits activity levels in rice several-fold higher than in barley malt.6,13 Another enzyme, α-glucosidase, shows high activity in rice malt and an optimum temperature of 55 °C, while in barley malt its activity is low and the optimum temperature is 35−40 °C. Above 50 °C in barley malt the enzyme is inactivated very rapidly.18 Evidently the same enzyme can have different features in different cereals. But the suitability of rice malt for brewing depends not only on its enzymatic activity but also on an adequate gelatinization of the starch in a temperature range where the amylolytic enzymes are still active; otherwise the amylolytic enzymes cannot degrade the starch to fermentable sugars and dextrins. This gelatinization temperature is quite high for unmalted cereals, in the case of barley 70−80 °C, but it can be lowered, according to Narziß and Back,4 up to 20 °C in the presence of enzymes. It depends on the malting conditions. So the high gelatinization temperature of rice (65−85 °C) should also be lowered after maltation. But there is no knowledge about the real optimal temperature range of the amylolytic enzymes of rice malt, which can differ from that of barley malt. Moreover Keßler18 notes that the starch granules are already attacked by enzymes during germination, and therefore the starch can be also hydrolyzed below the gelatinization temperature. Therefore, the main objective was to verify the complete saccharification of the rice malt starch during mashing with the iodine test, because in this case gelatinization occurred and enough endogenous malt enzymes were present. And, given that the overall enzymatic activity of the rice malt depends on the malting conditions,3,9,12 a key aspect of the study was to find the optimal malting parameters.

Figure 1. Improved malting conditions (steeping/germination C). (kilning program 1) and the other for 21 h at 50 °C, then 3 h at 72 °C, and finally 3 h at 82 °C (kilning program 2). An improved kilning program was tested with the green malt produced in trial steeping/germination C (kilning program 3) (Figure 1). 2.5. Malt Quality. 2.5.1. Rice Malt Analysis. Rice malt quality was assessed in duplicate using the following Analytica-EBC methods:20 moisture content (%), EBC method 4.2; extract yield (% DM), iodine test/saccharification rate time (min), and filtration rate, EBC method 4.5.1, modified EBC method 4.5.1 (changing the temperature rests to 30 min at 45 °C, 30 min at 64 °C, and 30 min at 74 °C); colored malt extract, EBC method 5.5; total nitrogen content (% DM), EBC method 4.3.1; soluble nitrogen content (mg/L), soluble nitrogen content (% DM) as a portion of total nitrogen content (% DM), and Kolbach index, EBC method 4.9.1; free amino nitrogen content (FAN; mg/L), EBC method 4.10; viscosity (mPa s at 20 °C and 8.6 °P (from the Plato scale)), EBC method 4.8; apparent final attenuation (%), EBC method 4.11.1; diastatic power (Windisch−Kolbach units (WK)), EBC method 4.12; pH, EBC method 8.17; malt color, EBC method 4.7.1. The activity of α-amylase, β-amylase, and limit dextrinase of malted rice was determined using the Megazyme Assay Kits. The dimethyl sulfide (DMS) concentration was determined in the static headspace using a gas chromatograph equipped with a mass spectrometer coupled with an autosampler after sample preparation. DMS precursors were calculated as the difference between total and free DMS.22 Aspartic acid, glutamic acid, serine, threonine, arginine, histamine, methionine, valine, leucine, isoleucine, lysine, glycine, alanine, tyrosine, and phenylalanine were determined in rice malt worts by highperformance liquid chromatography (HPLC) quantifying the fluorescence of the orthophtaldialdehyde (OPA)/mercaptoethanol derivatives.23,24

2. EXPERIMENTAL SECTION 2.1. Rice Samples. Ten paddy-rice varieties of O. sativa L. cultivated in Italy were examined. Eight belong to the subspecies japonica, Centauro, Creso, Selenio, Karnak, Arborio, Vialone Nano, Crono, and Balilla (respective amylose contents by percent: 16.3, 18.0, 18.1, 20.9, 17.3, 22.8, 16.4, 18.5),19 and two to the subspecies indica, Sirio and Libero (respective amylose contents: 26.5 and 23.0).19 Furthermore, three different samples of the Centauro variety were studied that differed in harvest year and origin. 2.2. Rice Analysis. The following quality attributes of the paddyrice grains were determined in duplicate using the Analytica-EBC methods:20 grain moisture (%), EBC method 3.2; thousand kernel weight (TKW, g), EBC method 3.4; total protein content (% dry matter (DM)), EBC method 3.3.1 (total N × 5.95); the germinative energy and water sensitivity, modified EBC method 3.6.2 (germination temperature, 28 °C). 2.3. Micromalting. An automatic micromalting system from Custom Laboratory Products (Keith, Scotland), containing a steep and germination unit separate from the kilning unit, was used as previously described.21 2.4. Malting Procedure. 2.4.1. Steeping and Germination. Three malting trials were performed. The first trial was performed by steeping the rice samples continuously in water at 28 °C for 47 h (steeping/germination A); the second, at 22 °C for 73 h (steeping/ germination B). The third trial (steeping/germination C) was done under improved conditions (Figure 1). The total steeping and germination time for all trials was 8 days. 2.4.2. Kilning. Two kilning programs were tested with the green malt produced in trial steeping/germination B, one for 21 h at 50 °C 5370

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Table 1. Rice Grain Quality Attributesa variety

subspecies

crop year

origin

moisture (%)

TKW (g)

germinative energy (%)

water sensitivity (%)

total protein (g/(100 g of DM))

Creso Selenio Karnak Arborio Vialone nano Centauro 1 Sirio Crono Libero Centauro 2 Centauro 3 Balilla

japonica japonica japonica japonica japonica japonica indica japonica indica japonica japonica japonica

2010 2010 2010 2010 2010 2010 2010 2010 2010 2011 2011 2011

Sardinia North Italy North Italy North Italy North Italy Sardinia Sardinia Sardinia Sardinia Sardinia North Italy North Italy

12.6a 13.6b 11.9c 13.2d 11e 12.6a 12.8f 14g 13.5bh 14.1g 13.4h 13i

26.6a 21.2bc 31.2d 36.3e 29.5f 23.3g 20.2bh 21.8c 19.1g 21.3bc 25.4a 21.7c

93a 90a 77b 87a 63c 92a 96a 90a 94a 90a 93a 90a

91ad 90ad 67b 69b 54c 91ad 93d 87ad 96d 81a 92d 90ad

8.51a 6.31b 7.32c 9.52d 9.40d 8.87e 8.63a 9.16f 9.58d 8.51a 7.62g 7.44g

a

n = 2 repetition; TKW = thousand kernel weight; DM = dry matter. Values in the same column followed by a different letter are statistically different (p ≤ 0.05).

Table 2. Optimization of the Malting Conditionsa steeping/germination B and kilning 1b

steeping/germination B and kilning 2c

steeping/germination C and kilning 3d

rice malt

diastatic power (WK)

moisture (%)

diastatic power (WK)

moisture (%)

diastatic power (WK)

moisture (%)

Creso Centauro 1 Sirio Libero Crono

78a 42a 118a n.t. n.t.

6.7a′ 6.1a′ 6.7a′ n.t. n.t.

56b 34b 89b 69 5

4.3b′ 4.3b′ 4.4b′ 4.6 4.5

n.t. 42a n.t. n.t. n.t.

n.t. 4.6c′ n.t. n.t. n.t.

n = 2 repetitions; n.t. = not tested. Values in the same row followed by a different letter are statistically different (p ≤ 0.05). bSteeping/germination B and kilning 1: 22 °C for 73 h (steeping)/22 °C for 120 h (germination) and 21 h at 50 °C (kilning1). cSteeping/germination B and kilning 2: 22 °C for 73 h (steeping)/22 °C for 120 h (germination) and 21 h at 50 °C, 3 h at 72 °C, and 3 h at 82 °C (kilning2). dSteeping/germination C and kilning 3: see Figure 1. a

The carbohydrate concentration in rice malt worts was determined by HPLC coupled with evaporative light scattering detector (ELSD) as reported by Floridi et al.25 2.5.2. Mashing Tests To Improve Saccharification and FAN Content. The following mashing tests were utilized: 1, EBC method 4.5.1; 2, EBC method 4.5.1 with the addition of 10 mL of an aqueous solution of CaCl2·2H2O at a concentration of 22 g/L; 3, rice malt with acrospires and rootlets applying the EBC method 4.5.1 with the addition of 10 mL of an aqueous solution of CaCl2·2H2O at a concentration of 22 g/L; 4, EBC method 4.5.1 with modified temperature rests (30 min at 45 °C, 30 min at 64 °C, and 30 min at 74 °C); 5, EBC method 4.5.1 with modified temperature rests and the addition of 10 mL of an aqueous solution of CaCl2·2H2O at a concentration of 22 g/L; 6, EBC method 4.5.1 with modified temperature rests and the addition of 0.144 mL of lactic acid at a concentration of 80% (v/v). 2.6. Statistical Analysis. Statistical analysis was performed using SigmaPlot Software (version 11.0; Systat Software, Inc., San Jose, CA, USA). Comparisons of the different matrices were made by one-way repeated measures analysis of variance, and the results were further analyzed using the Holm-Sidak test and the Tukey test.

fermentation, so the Selenio sample was excluded from malting because of its very low protein content of 6.3%. Of all rice varieties, Karnak, Arborio, and Vialone Nano showed higher TKW values, but they were lower than typical TKW values of barley (38−40 g).28 The germinative energy gives information about the proportion of grains that will germinate under the conditions of a specified test,29 and the corresponding EBC method (method 3.6.2) requires a germination temperature of 20 °C. In this study the germination temperature was set at 28 °C which is more appropriate for rice. The resulting values for the rice varieties Crono, Creso, Selenio, Centauro 1, Sirio, Libero, Centauro 2, Centauro 3, and Balilla were below 96%, which is a minimum for commercial brewing malt, but enough to try a pilot malting. They showed no water sensitivity. All of the samples with a good high germinative energy germinated on the third day. This indicates that kernel processing should be homogeneous during malting.28 On the other hand, the varieties Karnak, Arborio and Vialone Nano have an inadequate germinative energy (77, 87, and 63%, respectively) and were water sensitive (67, 69, and 54%, respectively). Because these last values were likely due to the presence of mold, these samples were excluded from malting. Three steeping/germination trials were done. Steeping/ germination A and steeping/germination B were characterized by long and unusual steeping times, in order to favor the water uptake and solubilize any inhibitors for germination, the hull features of rice and barley being different. The steeping/ germination A was performed with the varieties Creso, Centauro 1, and Sirio by steeping the rice continuously in water at 28 °C for 47 h to achieve 42−44% water uptake after

3. RESULTS AND DISCUSSION The rice grain quality attributes of the different samples are reported in Table 1. The moisture content of the rice samples (11.0−14.1%) permitted their storage at low temperatures without pretreatment. The total protein content was in a range of 7.3−9.6% (w/w) and were higher in comparison to the values found in the literature for rice (5.8−7.7% (w/w));26 however they were still low compared to brewing barley (10.5− 11.5% (w/w)).27 The protein content in brewing is very important for foam stability, taste, and yeast nutrition during 5371

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Table 3. Quality Attributes of Centauro 1 Rice Malt Wort Obtained with Congress Masha fine extract (% DM) saccharification time (min) pH color (EBC-U) total nitrogen (g/(100 g of DM)) soluble nitrogen (mg/L at 8.6 °P) Kolbach index (%) viscosity (mPa s at 8.6 °P) free amino nitrogen (mg/L at 8.6 °P) fermentability (%)

mashing test 1b

mashing test 2c

mashing test 3d

72.3a >60′ 5.96a 2.0a 1.34a 450a 27.4a n.t. 146a n.t.

72.7a 50 5.61a 1.7a 1.34a 482ab 29.5a 1.40a 148a 73.2a

71.7a 50 5.76b 1.8a 1.43b 506b 28.5a 1.48b 166b 71.2b

n = 2 repetitions; DM = dry matter; n.t. = not tested. Values in the same row followed by a different letter are statistically different (p ≤ 0.05). Mashing test 1: A-EBC method 4.5.1. cMashing test 2: A-EBC method 4.5.1 with the addition of CaCl2 aqueous solution. dMashing test 3: A-EBC method 4.5.1 with the addition of CaCl2 aqueous solution, acrospires and rootlets. a b

To increase the water absorption, another steeping and germination trial (steeping/germination C) was performed only with Centauro 1 by alternating steeping and drying periods every 8 h at 25 °C, and then decreasing the germination temperature to 20 °C (Figure 1). The water uptake of Centauro 1 was 42%. After 8 days of steeping and germination the malt did not show any evidence of mold and had a good smell and appearance. The kilning program was improved to better protect the enzymes of the green malt during kilning (kilning program 3). Rice’s hull is likely why paddy-rice absorbs water slowly and it dries also slowly for this reason; therefore the withering time was extended to 37.5 h to bring the water content to 10−12% before a higher temperature is used, because the enzymes tolerate dry heat better than wet heat.27 The first drying step at 45 °C was introduced in order to extend the time for cytolysis.29 Lastly a moderate final temperature of 70 °C for 6 h was selected. The diastatic power of the resulting Centauro 1 malt was 42 WK, which was equal to the value obtained with the kilning program 1, and also a moisture content of 60 30 5.95a″ 2.7a″ 450a″ 34a″ 1.62a″ 131a″ 85.2a″ 74.7a′ >60 25 5.45b′ 1.8a′ 541c′ 36b′ 1.44b′ 141b′ 75.0a′ 73.7a′ >60 25 5.63b′ 1.8a′ 468b′ 32a′ 1.40b′ 125a′ 70.3a′

mashing test 5

73.4a′ >60 n.s. 5.99a′ 2.0a′ 425a′ 29a′ 1.53a′ 116a′ 68.9a′

mashing test 4

73.5a >60 20 5.38c 2.2a 569c 35a 1.47ab 146b 80.6b

mashing test 6

72.8a >60 20 5.58b 2.1a 499b 31a 1.41b 134a 77.1ab

mashing test 5

73.3a >60 n.s. 5.98a 2.3a 453a 28a 1.55a 126a 74.6a

a n = 2 technological repetitions; n.s. = no saccharification; EBC-U = European brewing convention unit; DM = dry matter. Values in the same row followed by a different letter are statistically different (p ≤ 0.05). bMashing test 4: EBC method 4.5.1. with modified temperature rests (30 min at 45 °C, 30 min at 64 °C, and 30 min at 74 °C). cMashing test 5: EBC method 4.5.1 with modified temperature rests and the addition of an aqueous solution of CaCl2. dMashing test 6: EBC method 4.5.1 with modified temperature rests and the addition of lactic acid aqueous solution.

76.6ab″ >60 20 5.62b″ 2.4b″ 470b″ 36a″ 1.49b″ 135a″ 84.4a″

mashing test 5c mashing test 6

d c

Centauro 3

b d

Centauro 2

c

fine extract (% DM) filtration time (min) saccharification time (min) pH color (EBC-U) soluble nitrogen (mg/L at 8.6 °P) Kolbach index (%) viscosity (mPa s at 8.6 °P) free amino nitrogen (mg/L at 8.6 °P) fermentability (%)

°C (mashing test 4). In mashing test 5 this test was performed with the aqueous solution of CaCl2 while in mashing test 6 the CaCl2 was replaced with lactic acid to lower the pH of the mash and to determine if the improved saccharification was due to a lower pH or the presence of calcium or chloride ions. The results are shown in Table 4. Centauro 2 and Centauro 3 saccharified completely without the use of exogenous enzymes in 20 min after reaching 74 °C with the addition of an aqueous solution of CaCl2 as well as with the addition of lactic acid. Therefore, the improved saccharification in tests 5 and 6 was probably due to the lower pH value. The very low value of the diastatic power could not be decisive for assessing good saccharification, but there should be other important amylolytic enzymes in rice malt such as limit dextrinase,14 pullulanase,17

mashing test 4

Figure 2. Mashing Programs.

b

Table 4. Quality Attributes of Centauro 2, Centauro 3, and Balilla Rice Malt Wort Obtained with Modified Congress Masha

mashing test 4

b

Balilla

mashing test 6d

All FAN values were surprisingly high compared to barley malt wort in relation to the soluble nitrogen content. Normally in a good brewing malt the FAN content is around 22% of the soluble nitrogen.4 In this case it was 32, 31, and 33% respectively for mashing tests 1, 2, and 3. This could be an overstimation that depends on the method for FAN determination, which is a color reaction with ninhydrin. The test is a cumulative method for the estimation of amino acids, ammonia, and the terminal α-amino groups of the peptides and proteins,24 where the individual amino acids give different color intensities,31 and for this the comparison between barley malt and rice malt could be less significant. There was no difference in the FAN values of mashing test 1 and mashing test 2, but in mashing test 3 the FAN value was increased. The fine extracts were nearly the same for the three trials and showed very good results considering the fact that the hull accounts for up to 20% of the kernel’s mass and contributes less to the extract.1 The optimized malting program was repeated with Centauro 2 and Centauro 3, but surprisingly, the results of mashing test 2 and Analytica EBC method 5.5 revealed an incomplete saccharification (data not shown). The color of the iodine test remained light blue instead of yellow. The reason for that could be a slightly higher gelatinization temperature of Centauro 2 and Centauro 3, because the values of the diastatic power of all three rice malts were nearly the same: 42, 44, and 31 WK respectively for Centauro 1, Centauro 2, and Centauro 3. According to Narziß and Back,6 the gelatinization temperature depends on the variety and cultivation area and particularly on the climate during the crop year. Therefore, the final congress temperature of 70 °C did not permit a complete gelatinization, and a new mashing program tailored specifically for rice malt was developed. The conditions of this new standardized mashing program correspond to the typical EBC Congress Mash but with different temperature rests (Figure 2): 30 min at 45 °C, 30 min at 64 °C and 30 min at 74

77.2b″ >60 20 5.51b″ 2.5b″ 518c″ 40a″ 1.51b″ 142a″ 87.9a″

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36.54a′ 0.54a′ 15.76a′ 4.58a′ 8.24a′ 5.31a′ 6.29a′ 44.81c 0.37c 38.96c 5.90c 6.11c 1.00c 0.34c 34.64b 0.45b 24.32b 3.88b 8.61b 4.17b 8.99b

a

28.94a 0.22a 17.54a 7.74a 7.44a 3.14a 8.08a glucose sucrose maltose maltriose maltotetraose maltopentaose maltohexaose

n = 2 technological repetitions. Values in the same row followed by a different letter are statistically different (p ≤ 0.05). bMashing test 4: EBC method 4.5.1. with modified temperature rests (30 min at 45 °C, 30 min at 64 °C, and 30 min at 74 °C). cMashing test 5: EBC method 4.5.1 with modified temperature rests and the addition of an aqueous solution of CaCl2. dMashing test 6: EBC method 4.5.1 with modified temperature rests and the addition of lactic acid aqueous solution.

33.21a″ 0.12b″ 34.74a″ 4.59a″ 3.00b″ 0.01b″ 0.01b″ 32.76a″ 0.14b″ 30.57a″ 4.46a″ 6.57a″ 2.20a″ 2.71a″

0.48a″ 0.58a″

30.15a″ 0.19a″ 30.59a″ 4.65a″ 6.53a″ 2.49a″ 3.44a″ 34.63a′ 0.19b′ 32.64b′ 4.69a′ 8.08a′ 2.25b′ 3.23b′ 34.05a′ 0.19b′ 32.10b′ 4.61a′ 7.94a′ 2.21b′ 3.17b′

mashing test 5

0.45a′ 0.61a′ 0.82c 0.36b 0.56a fructose

Centauro 2

Table 6. Sugar Profile of Rice Malt Wortsa

c

mashing test 6

d

mashing test 4

b

Centauro 3

c

could also imply a greater protein enzyme endowment. For every sample the fine extract of mashing tests 4, 5, and 6 was nearly the same; meanwhile the viscosity decreased with the addition of CaCl2 and lactic acid, but for all tests it was in an acceptable range. The optimized malting and mashing trials were repeated again using the variety Balilla which has similar characteristics (subspecies japonica, 16−18% amylose content) as Centauro. This variety gave even better results than Centauro (Table 4) in spite of the low diastatic power of 34 WK. The saccharification occurred also without the addition of CaCl2 or lactic acid after 30 min. The fermentability, extract content, FAN, soluble nitrogen, and Kolbach Index were all higher than those of Centauro 2 and 3. The α- and β-amylases as well as limit-dextrinase content of the rice malts Centauro 2 and Balilla were analyzed (Table 5). The α- and β-amylase values are very low and correspond to the very low diastatic power; meanwhile the results of the limitdextrinase activity was 6940 and 6129 U/(malt kg) for Centauro 3 and Balilla, respectively, which is much higher than in barley malt (200−400 U/(malt kg));5 so limit dextrinase is certainly one of the supplemental enzymes that contribute to the degradation of the rice malt starch in conjunction with α- and β-amylases. Table 6 shows the sugar profiles of the laboratory rice malt worts. All of the rice malt worts from the different mashing programs produced a wide range of sugar spectra which are characterized by the greater amount of glucose than maltose highlighting the different trend with respect to barley malt wort and could be due to the presence of different enzymes. The data also differed from the results obtained by Agu et al. in 20128 perhaps of a different malting and mashing process. Table 7 reports the amino acid profile of the rice malt worts. Histidine and arginine are the amino acids present in major amount. The amino acid profile of the samples did not change a lot between the different mashing programs and varieties. The total amino acid concentration of the rice malt worts is in the range of a barley malt Congress wort28 even if the total nitrogen content is lower than a barley malt wort. This confirmed the high FAN values. The DMS

0.46a′

mashing test 6

n = 2 repetitions.

mashing test 5

6129 ± 25 48.3 ± 0.1 8.4 ± 0.2

mashing test 4

Balilla

6940 ± 21 47.4 ± 1.1 3.8 ± 0.3

b

a

Centauro 2

d

Table 5. Starch Degrading Enzymes in Rice Malt as Mean and Standard Deviationa limit dextrinase (U/(malt kg)) α-amylase (Ceralpha (U/g)) β-amylase (Betamyl-3 (U/g))

mashing test 5c mashing test 4

b

Balilla

mashing test 6d

and α-glucosidase18 that significantly contribute to the degradation of the rice malt starch. The filtration time of the laboratory worts last more than an hour despite rice wort’s low viscosity. The cause may be the cooling of the wort after the laboratory mashing process to 20 °C where the pectin begins to form a gel, and this could hinder the filtration; however this should not be a problem in beer production where the filtration is carried out at 78 °C. Soluble protein increased in both samples from mashing test 4 to mashing test 5 to mashing test 6. There was also a positive correlation with the FAN values. Compared to Centauro 3, Centauro 2 displayed higher rice malt quality values probably due to its higher total protein content in the crude rice (Tables 1and 5) that could provide more soluble nitrogen and FAN but

0.59a″

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c

5375

191a 21a 88a 41b 86b 55a 85c 80a 28a

190a 24a 87a 43a 83a

55a 97a 78a 31a

1119a

total

1324c

65b 105c 97b 38b

206b 29b 101b 47c 118c

51a 67c 62c 177c 49b 113c

mashing test 6

d

1131a′

56a′ 93a′ 79a′ 44a′

184a′ 42a′ 87a′ 41a′ 81a′

50a′ 54a′ 61a′ 134a′ 38a′ 89a′

mashing test 4

b

c

1134a′

62b′ 101b′ 82b′ 31b′

180b′ 42a′ 89b′ 45b′ 93b′

42b′ 41b′ 57b′ 142b′ 34b′ 93b′

mashing test 5

Centauro 3

1082b′

53c′ 86c′ 78c′ 38c′

175c′ 25b′ 82c′ 38c′ 88c′

41b′ 50c′ 56b′ 137c′ 36b′ 100c′

mashing test 6

d

1175a″

67a″ 96a″ 86a″ 40a″

185a″ 25a″ 94a″ 51a″ 100a″

50a″ 46a″ 65a″ 136a″ 39a″ 96a″

mashing test 4

b

1135b″

66a″ 91b″ 86a″ 37b″

177b″ 26a″ 90b″ 48b″ 100a″

45b″ 43b″ 59b″ 136a″ 35b″ 97a″

mashing test 5c

Balilla

1209c″

67a″ 99a″ 91b″ 42c″

176b″ 47b″ 95a″ 49b″ 110b″

46b″ 46a″ 57c″ 151b″ 39a″ 94b″

mashing test 6d

a

n = 2 technological repetitions. Values in the same row followed by a different letter are statistically different (p ≤ 0.05). bMashing test 4: EBC method 4.5.1. with modified temperature rests (30 min at 45 °C, 30 min at 64 °C and 30 min at 74 °C). cMashing test 5: EBC method 4.5.1 with modified temperature rests and the addition of an aqueous solution of CaCl2. dMashing test 6: EBC method 4.5.1 with modified temperature rests and the addition of lactic acid aqueous solution.

1107b

47b 49b 55b 148b 38a 96b

52a 54a 57a 140a 39a 90a

mashing test 5

Centauro 2

group A aspartic acid glutamic acid serine arginine treonine lysine group B histidine methionine valine leucine isoleucine group C alanine tyrosine phenylalanine glycine

mashing test 4

b

Table 7. Amino Acid Profiles of Rice Malt Worts (mg/L)a

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(3) Ceppi, E. L. M.; Brenna, O. V. Brewing with rice malt-Aglutenfree alternative. J. Inst. Brew 2010, 116, 275−279. (4) Narziß, L.; Back, W. Die Bierbrauerei: Band 2: Die Technologie der Würzebereitung; John Wiley & Sons: Weinheim, Germany, 2012. (5) Taylor, J. R. N.; Dlamini, B. C.; Kruger, J. 125th Anniversary Review: The science of the tropical cereals sorghum, maize and rice in relation to lager beer brewing. J. Inst. Brew. 2013, 119, 1−14. (6) Narziß, L.; Back, W. Die Bierbrauerei: Band 1: Die Technologie der Malzbereitung; John Wiley & Sons: Weinheim, Germany, 2012. (7) Adebowale, A. A.; Sanni, S. A.; Karim, O. R.; Ojoawo, J. A. Malting characteristics of Ofada rice: Chemical end sensory qualities of malt from Ofada rice grains. Int. Food Res. J. 2010, 17, 83−88. (8) Agu, R.; Yukihiro, C.; Goodfellow, V.; MacKinlay, J.; Brosnan, J.; Bringhurst, T.; Jack, F.; Macdonald Harrison, B.; Pearson, S.; Bryce, J. H. Effect of germination temperatures on proteolysis of the gluten-free rice and buckwheat during malting and mashing. J. Agric. Food Chem. 2012, 60, 10147−10154. (9) Ceppi, E. L. M.; Brenna, O. V. Experimental studies to obtain rice malt. J. Agric. Food Chem. 2010, 58, 7701−7707. (10) Kongkaew, A.; Usansa, U.; Wanapu, C. Optimisation of wort production from rice malt using enzymes and barley malt. Afr. J. Biotechnol. 2012, 11, 9941−9949. (11) Owusu-Mensah, E.; Oduro, I.; Sarfo, K. J. Steeping: A way of improving the malting of rice grain. J. Food Biochem. 2011, 35, 80−91. (12) Usansa, U.; Sampong, N.; Wanapu, C.; Boonkerd, N.; Teaumroong, N. The influences of steeping duration and temperature on the α- and β-amylase activities of six thai rice malt cultivars (Oryza sativa L. indica). J. Inst. Brew. 2009, 115, 140−147. (13) Usansa, U.; Burberg, F.; Geiger, E.; Black, W.; Chokchai, W.; Arendt, E. K.; Kreisz, S.; Boonkerd, N.; Teamuroong, N.; Zarnkow, M. Optimization of malting conditions for two black rice varieties, black non-waxy rice and black waxy rice (Oryza sativa L. indica). J. Inst. Brew. 2011, 117, 39−46. (14) Dziedzoave, N. T.; Graffham, A. J.; Westby, A.; Komlaga, G. Comparative assessment of amylolytic and cellulolytic enzyme activity of malts prepared from tropical cereals. Food Control 2010, 21, 1349− 1353. (15) Bunzel, M.; Steinhart, H. Ballaststoffe aus Pflanzenzellwänden. Ernaehr.-Umsch. 2003, 50 (12), 469−475. (16) Nakai, H.; Ito, T.; Hayashi, M.; Kamiya, K.; Yamamoto, T.; Matsubara, K.; Kim, Y. M.; Jintanart, W.; Okuyama, M.; Mori, H.; Chiba, S.; Sano, Y.; Kimura, A. Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare). Biochemistry 2007, 89, 49−62. (17) Yamasaki, Y.; Nakashima, S.; Konno, H. Pullulanase from rice endosperm. Acta Biochim. Polym. 2008, 3, 507−510. (18) Keßler, M.; Zarnkow, M.; Kreisz, S.; Back, W. Gelatinisation properties of different cereals and pseudocereals. Monatsschr. Brauwiss. 2005, Sep./Oct., 82−88. (19) Ente Nazionale Risi, http://www.enterisi.it (accessed September 2013). (20) European Brewery Convention. Analytica-EBC, 5th ed.; Fachverlag Hans Carl: Nürnberg, Germany, 2007. (21) Mayer, H.; Marconi, O.; Perretti, G.; Sensidoni, M.; Fantozzi, P. Investigation on suitability of hulled wheats for malting and brewing. J. Am. Soc. Brew. Chem. 2011, 69, 116−120. (22) Stafisso, A.; Marconi, O.; Perretti, G.; Fantozzi, P. Determination of dimethyl sulphide in brewery samples by headspace gas chromatography mass spectrometry (HS-GC/MS). Ital. J. Food Sci. 2011, 23, 19−27. (23) Lookhart, G. L.; Jones, B. High performance liquid chromatography analysis of amino acids at the picomole level. Cereal Chem. 1985, 62, 97−102. (24) Marconi, O.; Mayer, H.; Chiacchieroni, F.; Ricci, E.; Perretti, G.; Fantozzi, P. The influence of glumes on malting and brewing of hulled wheats. J. Am. Soc. Brew. Chem. 2013, 71, 41−48. (25) Floridi, S.; Miniati, E.; Montanari, L.; Fantozzi, P. Carbohydrate determination in wort and beer by HPLC-ELSD. Monatsschr. Brauwiss. 2001, 9/10, 209−215.

values (free, total, and precursor) for Centauro 2 and Balilla were determined (Table 8). They were higher than the Table 8. Dimethyl Sulfide (Total, Free, and Precursor) Of Rice Malts (mg/kg)a

a

rice malt

total DMS

free DMS

DMS-P

Centauro 2 Balilla

17.15 ± 0.74 12.70 ± 0.69

3.66 ± 0.01 2.52 ± 0.07

13.50 ± 0.74 10.18 ± 0.70

n = 2 repetitions.

accepted values for a commercial barley malt (5−6 mg/kg) and could be due to the high germination temperatures and the relatively low curing temperature.32 In conclusion, a malting program to produce rice malt for brewing was developed and 10 rice varieties were screened. Probably due to the resistant hull of paddy-rice, five steeping steps were necessary to achieve at least 42% of steeping-out moisture, and a long-time kilning program was developed with a particular drying step at 45 °C for 12 h to impair the degradation of the cell walls. Another 12 h at 50 °C and 13.5 h at 55 °C dried the malt enough to protect the enzymes from the following heat (6 h at 70 °C). But this curing temperature and time were not enough to eliminate the high DMS values found in the rice malt. It was shown that the amount of the endogenous rice malt enzymes is high enough to saccharify the rice malt starch and that their optimal temperature and pH conditions are different from those of barley malt. So the conventional Congress mash (mashing test 1) was slightly changed. With different temperature rests and the addition of CaCl2 and lactic acid a complete saccharification of the rice malt starch was achieved. Balilla and Centauro of the subspecies japonica are the most suitable varieties for the production of an all-rice malt beer. The addition of lactic acid also yielded higher soluble nitrogen and FAN contents and enhanced fermentability. In the case of the Balilla rice malt, the saccharification occurred also without the addition of CaCl2 and lactic acid. The soluble nitrogen and FAN values of rice malt wort can be increased by incorporating the acrospires and rootlets during mashing. Work is in progress for brewing an all-rice beer with the malt obtained from this study.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 075 585 7926. E-mail: [email protected]. Funding

We thank the Italian Ministry of Agricultural, Food and Forestry Policies for financial support of the project CERSUOM. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We are grateful to Dr. Simona Floridi for the analytical support. REFERENCES

(1) FAO. Rice Market Monitor, Vol. XVI (Issue No. 1), Food and Agriculture Organization of the United Nations: Rome, Italy, 2013; pp 2 and 14, www.fao.org/docrep/017/aq144e/aq144e.pdf (accessed September 2013). (2) Italian breeds online. http://www.agraria.org/ coltivazionierbacee/riso.htm (accessed September 2013). 5376

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(26) Bienvenido O. J. Rice in human nutrition; Food and Agriculture Organization of the United Nations: Rome, Italy, 1993; Chapter 3, http://www.fao.org/docrep/t0567e/T0567E08. htm#Grossnutrientcomposition (accessed September 2013). (27) Kunze, W. Raw materials. Technology Brewing and Malting; VLB: Berlin, 2004; pp 32−49. (28) Mitteleuropäische Brautechnische Analysenkommission. Brautechnische Analysenmethoden Rohstoffe; MEBAK: Freising-Weihenstephan, Germany, 2006. (29) Briggs, D. E. The selection and purchase of grain. Malts and malting; Blackie Academic & Professional: London, 1998; p 265. (30) Montanari, L.; Mayer, H.; Marconi, O.; Fantozzi, P. Minerals in Beer. In Beer in Health and Disease Prevention; Preedy, V. R., Ed.; Elsevier: Amsterdam, 2009; Chapter 34, pp 359−365. (31) Friedman, M. Applications of the ninhydrin reaction for analysis of aminoacids, peptides, and proteins to agricultural and biomedical sciences. J. Agric. Food Chem. 2004, 52, 385−406. (32) Anness, B. J.; Bamforth, C. W. DimethylsulphideA Review. J. Inst. Brew. 1982, 88, 244−252.

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