Rice malting optimization for the production of top fermented gluten-

free beer
Accepted Article

Dayana Ceccaroni1,2, Ombretta Marconi1,2, Valeria Sileoni1,2*, Edward Wray3, Giuseppe

Perretti1,2

1 University of Perugia, Department of Agricultural, Food and Environmental Science,

via San Costanzo s.n.c., 06126, Perugia, Italy

2
University of Perugia, Italian Brewing Research Centre, via San Costanzo s.n.c., 06126,

Perugia, Italy

3
Campden BRI, Nutfield, Centenary Hal, Coopers Hill Road, Nutfield, Surrey, RH1

4HY, United Kingdom

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/jsfa.9440

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 Abstract

BACKGROUND: A safe method to obtain gluten free beer lead to the use of naturally

gluten free grains, such as rice, but the specific malting program for rice was long and
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requiring high amount of water, and the resulting beer showed a flat flavor profile. In this

study an optimization of malting and brewing procedure is proposed to overcome the

above mentioned issues. Different steeping conditions and kilning temperatures are

considered and a top fermented beverage from rice malt is obtained for the first time.

RESULTS: The malting procedure has been optimized assessing the use of short time

steeping alternate to long air rest to obtain sufficient moisture content in the green malt

saving water consumption. The obtained malt allowed a regular fermentation as

confirmed by the sensorial analysis, which did not reveal any off-flavours. The use of a top

fermenting yeast formed high content of higher alcohol and relatively low amount of

esters.

CONCLUSION: This study confirms the potential of rice for the production of malt and

beer. The optimized malting program allowed water saving. The production of a top

fermented rice malt beer was a successful attempt to introduce a new flavoured product

for consumption by individuals affected by celiac disease.

Keywords

rice malt, malting optimization, wheat yeast, gluten-free beer, sensory profile, volatile

compounds

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 INTRODUCTION

Rice (Oryza sativa), known as ‘Queen of cereals’, is a staple food for nearly 50% of the

world population, particularly for the Asians. According to the Food and Agriculture

Organization of the United Nations (FAO), world paddy production in 2017 by 2.1 million
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tonnes to 756.7 million tonnes (502.2 million tonnes, milled basis). More than 80% of world

rice production is destined to food, about the 4 % is used for feed and the 16 % for other

uses1. Rice kernel or the caryopsis consists of seed coat, embryo and endosperm as

prominent botanical tissues. The seed coat comprises of husk and bran tissues, which are

distinctly separate entities with respect to their physical as well as morphological nature

and chemical composition. The kernel from which the husk is separated is known as

brown rice and it contains about 5% bran2,3. The rice composition makes this cereal

particularly suitable for human nutrition. The dry matter consists of about 75 - 80 % starch,

5 – 9.1%> protein, 0.2 – 2.2 % oil and small amounts of inorganic substances4. The chemical

composition of rice, especially the high starch content, makes this cereal also almost

perfectly suitable for brewing apart from the low nitrogen content2. Beer is one of the

oldest and widespread beverage in the world. It is principally produced from barley or

wheat malt, but other cereals are used and have been investigated for the production of

malt and beer5–8. The use of alternative cereals for production of beer is challenging in

terms of different physics and technological properties and taste deviation from original

beer. The chemical composition of alternative grains is generally suboptimal for the

production of a beverage similar to a traditional beer. The investigation of rice as material

for the production of malt9–12 and beer13–15 is still weak16,17. Among the available literature,

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 the experiments on the production of 100 % rice malt beer have been conducted by using

bottom fermenting yeast15 or bottom fermenting yeast combined with a top fermenting

yeast for the bottle conditioning14 or top fermenting yeast at 12°C13, which is a temperature

normally used for bottom fermentation18. Rice shape is only slightly different from barley
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and the facilities used in barley beer production could be easily employed for processing

it. Furthermore, the rice husks could play a fundamental role in the lautering process19,20,

indeed it is already sold in craft brewing market. By the literature enzymatic content of the

rice is lower than barley malt but it is higher compared to other gluten free grains. Recent

studies demonstrated that the suitable conditions for malting and brewing allow the

complete conversion of rice starch malt in fermentable sugar 12,15. A long malting time

combined with a low kilning temperature allows the preservation of the enzymatic

content but the resulting malt and beer are pale in colour and with a flat flavour profile.

Use of rice as raw material for malt and beer production is a valid alternative for the

valorisation of this cereal through innovative products. Furthermore the proposed

products can enhance the variety and taste of gluten free food for coeliac people, which is

a still limited factor. Coeliac disease (CD) is an autoimmune disorder that is estimated to

affect up to 1% of the global population. In genetically predisposed individuals the

ingestion of alcohol-soluble fractions of gluten proteins from wheat, barley, rye and oat

(gliadins, hordeins, secalins and avidins, respectively) could lead to intestinal villous

atrophy, malabsorption of nutrients resulting in loss of weight or failure to thrive in

infants, and a high risk of gastrointestinal cancer. In addition, other gluten-related

diseases, such as gluten ataxia, dermatitis herpetiformis, wheat allergy, and gluten

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 sensitivity, affect up to 6% of the global population7. At present, no pharmacological

treatments are available, and all patients are required to adhere to a strict lifelong gluten-

free diet. Follow a gluten free diet represents a difficult challenge for the consumers and

their family and might seriously compromise the quality of life. Poor availability of gluten
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free products means that people end up losing the balance between health benefits and

social sacrifices16. Consequently, the sensory aspect is one of the main attribute considered

by individuals with celiac disease when purchasing food items21.

The aim of this work was to obtain a fermented beverage from whole rice malt for the

overall improvement of this product in terms of technological conditions, aroma and

flavour profile. For these purposes different malting conditions and kilning temperatures

were considered in order to obtain an increase in colour preserving the enzymatic content.

The optimized malting procedure was then chosen to produce rice malt in sufficient

amount to brew in a pilot plant. To improve the flavour of the rice malt new product a top

fermentation was conducted for the first time. The potential suitability of rice to obtain an

innovative products has been investigated performing chemical and sensory analysis. The

volatile profile and the flavour of the final beer were analysed to assess the obtained

attributes and to study the suitability of this new product process.

MATERIALS AND METHODS

Two Italian paddy rice (Oryza sativa L. subspecie japonica) varieties were used for the

malting optimization, Balilla harvested in 2013 and Centauro harvested in 2011.

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 Technological trials

Malting

The malting trials optimization was carried out in duplicate in an automatic micromalting
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system (Custom Laboratory Products, U.K.). Paddy rice was steeped and germinated in

rotating drums containing 520 g. The green malts were dried in controlled kilning

chamber.

The four different operating conditions shown in Fig 1 were performed.

Trials 1, 2 and 3 were carried out to observe the effect of water uptake, curing temperature

and germination time on the quality of the malt, and compared with trial 4 from a

previous study12. Four short steeping steps of 4 hours with three long air rests of 20 hours

in trials 1, 2 and 3, were compared to five longer steeping steps of 8 hours alternate to 8

hours air rest of trial 4. In the different trials the germination time was stopped at the 7th

(trials 1 and 2) or the 8th day (trial 3). The kilning program was: 12 hours at 45 °C, 12 hours

at 50 °C, 13 and half hours at 55 °C. The final curing temperature was of 70 °C for 6 hours.

In trial 2 the final curing temperature raised to 80 °C for the last three hours in order to

observe the effect of a different kilning temperature in colour development and enzymatic

content.

Brewing trial

The brewing was performed in a 100-L pilot scale brewery (Campden BRI, Nutfield,

United Kingdom) in duplicate.

Wort production
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 10 kg of malt produced as described in the optimized process (trial 3) were milled to 0.45

mm twice and mixed to 40 litres of water treated with 216 ppm CaSO4·2H2O and 276 ppm

of CaCl2·2H2O. The pH was adjusted at 5.3 with lactic acid and the mashing temperature

rests were 30 min at 45 °C, 45 min at 65 °C, 60 min at 74 °C, 10 min at 78 °C increasing the
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temperature by 1 °C per minute between the rests. The saccharification test with iodine

solution was performed every 10 minutes during the rest at 74 °C. The wort completely

saccharified after 50 minutes. The wort was than boiled for 60 min. Hop, Saaz variety

(3.7% alpha acids) was added at the beginning of boiling to achieve 19 International

Bitterness Units (IBU).

Fermentation

Pitching of the yeast, 107 cells/mL in the wort, was carried out at 15 °C. A dry, top-

fermenting yeast (Munich yeast, Lallemand, Austria), defined by the producer as wheat

beer yeast. was used. The fermentation was performed at 22 °C for 6 days and then the

temperature was cooled down to 2 °C. The beer was then added of 5 grams of sugar per

litre and bottle conditioned at 22°C for ten days.

Analysis

Chemical Analysis

The quality attributes of the paddy rice were determined in duplicate, following

Analytica-EBC methods22: moisture content (%), EBC method 3.2; thousand kernel weight

(TKW, g), EBC method 3.4; total protein content (% dry matter (d. m.)), EBC method 3.3.1

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 (total N × 5.95); the germinative energy and water sensitivity, modified EBC method 3.6.2

(germination temperature, 28 °C).

The quality attributes of the rice malt were assessed in duplicate following the Analytica-
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EBC methods22: moisture content (%), EBC method 4.2; extract yield (% DM), iodine

test/saccharification rate time (min), 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)12; 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 colour, EBC method 4.7.1. Enzymatic assay kits (Megazyme

International Ireland Ltd.) were used to determine the activity of α-amylase, β-amylase,

and limit dextrinase of malted rice. According to Megazyme, the activity of α-amylase is

expressed in Ceralpha Units, while the activity of β-amylase is reported as Betamyl-3

Units. These units are described by Megazyme as the amount of enzyme required to

release one μmole of p-nitrophenol from defined substrates in one minute under the

defined assay conditions. The activity of limit dextrinase is expressed in Units defined as

the amount of enzyme required to release one μmole of glucose reducing-sugar

equivalents per minute from pullulan under the defined assay conditions.

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 The quality attributes of the wort were assessed in duplicate following the Analytica-EBC

methods22: extract of wort (°P), EBC method 8.3; pH of wort, EBC method 8.17; color of

wort: spectrophotometric method, EBC method 8.5; attenuation limit of wort (%), EBC

method 8.6.2; total nitrogen in wort: EBC method 8.9.1; free amino nitrogen (FAN) in wort,
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EBC method 8.10.

The quality attributes of the beer were assessed in duplicate following the Analytica-EBC

methods22: Original, Real and Apparent Extract of Beer (°P), EBC method 9.4; Alcohol in

Beer by Distillation (% v/v), EBC method 9.2.1; Real Degree of Fermentation (%), EBC

method 9.5; pH of Beer (method 9.35); Colour of Beer: Spectrophotometric Method (EBC-

U), EBC method 9.6; Total Nitrogen in Beer: Kjeldahl Method (mg/L), EBC method 9.9.1;

Free Amino Nitrogen (FAN) in Beer by Spectrophotometry (mg/L), EBC method 9.10;

Foam Stability of Beer using the NIBEM-T Meter 30 s (sec), EBC method 9.42. The

apparent degree of fermentation was determined by following the Mebak method 2.8.4 15.

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 high performance liquid chromatography (HPLC) quantifying the

fluorescence of the orthophtaldialdehyde (OPA)/mercaptoethanol derivatives13.

The composition of sugars in the worts and beers from rice malt were determined by

HPLC coupled with evaporative light scattering detector (ELSD) as reported by Floridi, et

al 23.

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 Aldehydes, alcohols and esters in beer were assessed as described by De Francesco,

Turchetti, Sileoni, Marconi & Perretti in 201524, based on solid-phase micro extraction with

on-fibre derivatization. An Agilent Technologies 6850 gas chromatograph equipped with

an Agilent Technologies Mass Spectrometer 5975C (Santa Clara, CA) coupled with a
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Maestro Autosamples Gerstel Multi Purpose Sampler (Baltimore, MD) was used. The gas

chromatograph-mass spectrometer was equipped with a glass direct inlet liner (1.5 mm

inner diameter and 140 μl volume) and a DB-5MS capillary column of 60 m X 0.32 mm X 1

μm (J&W Scientific, Folsom, CA) consisting of cross linked 5% phenyl methyl siloxane. A

65-μm poly(dimethylsiloxane)/divinyl benzene (PDMS/DVB) fiber coating (Supelco,

Bellefonte, PA) was used.

Sensory analysis

A sensory evaluation of the rice beer was carried out by a trained, 12-member tasting

panel. A blind-tasting was conducted. 50 ml of sample were served anonymously in dark

glasses and under red light to minimize visual cues at a temperature between 8 and 10 °C

that, according to the Analytica EBC guidelines, is suitable for full perception of flavour,

such as detecting faults or small differences22. The overall flavour (aroma, taste and

aftertaste) of beer was considered using the glossary generated in a pre-tasting of the

samples. The intensity of each attribute was scored on a scale of 0 to 9, where 0 = absent, 5

= medium intensity and 9 = intense. Data for each attribute were collected using

Compusense@hand sensory software (Compusense Inc, Ontario, Canada). The tasting

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 results for each technological replicate were combined and average scores calculated and

plotted in spider diagrams.

Statistical analysis
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The statistical analysis was performed using SigmaPlot Software (version 12.0; Systat

Software, Inc., San Jose, CA). Different matrices originated from technological and

analytical replicates were compared by one-way analysis of variance (ANOVA), and the

results were further analysed using the Holm-Sidak test and the Tukey test.

RESULTS AND DISCUSSION

Rice analysis were performed to asses quality attributes for malting and reported in table

1. The moisture content of the rice samples was in the same range of barley suitable for

malting or slightly higher, but still fine to permit the storage without pre-treatment. The

TKW values were about the half of barley, as expected considering the different dimension

of grains. The total protein content was comparable with the values found in the literature

for rice (5.8−7.7% 12

) however they were still low compared to brewing barley (10.5 −

11.5%22). Germinative energy was good in Balilla variety and acceptable for Centauro

variety.

In the current study the optimization of malting conditions for rice malt has been

achieved. The first optimization step has been to change the steeping rest time from 8

hours in the trial 4 to 4 hours in the trials 1, 2, 3, increasing the air rest time in order to

obtain a higher water absorption. Moreover, four steeping steps were performed instead

of five. The two different steeping programs allow the green malt to reach the same

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 moisture content of about 42%, thus the optimized steeping program saves the water

consumption. In table 2 quality attributes of the malts are shown. The germination time

influenced the saccharification, which was not achieved with the short germination time

(trials 1 and 2), while a complete conversion of starch was obtained with the long
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germination time (trials 3 and 4). Evidently, 8 days of malting (steeping and germination)

are needed to reach the optimal modification of rice malt in this condition. The extract

yield significantly rose at the 8th day, reaching suitable values for brewing. Indeed the

other attributes that confirm these results are soluble nitrogen and consequently the

Kolbach index, which were significantly higher in the samples of both rice varieties malted

for the longer time. In fact, Kolbach index values higher than 35 are considered adequate

in barley malt22, because the degradation of proteins in the grains expressed by the

Kolbach index as the ratio of total and soluble nitrogen is linked to the availability of the

starch by the enzymes and it probably affects the saccharification12. Concerning the quality

attributes, it can be stated that in these conditions 8 days of malting are the required time

to obtain the optimal results. Furthermore, the two trials with 8 days of malting showed

the same quality attributes, so the trial 3, performed with 4 short steeping steps, is an

optimized condition because it allows obtaining the same quality saving the water

consumption. Concerning the kilning program the higher curing temperatures did not

involve any colour improvement, probably because the difference between 70 °C and 80

°C is too low to induce a relevant difference in colour formation. By the way a higher

temperature could damage the enzymatic content.

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 The diastatic power value was very low compared to barley malt (240 – 260 WK22). This

value could be related to the low content of α- and β-amylases. Nevertheless, the content

of limit dextrinase, between 3763 and 6587 U/(malt kg), which is much higher than in

barley malt (200−400 U/(malt kg))12, can explain the acceptable values of extract and
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fermentability, showing a similar increasing trend in the different trials. Enzymatic content

of rice malts showed then a rising trend according to the length of germination,

confirming the results of standard quality attributes about the required malting time,

supported also by the literature10. Trials with a malting time of 8 days gave the highest

enzyme content in both varieties. More in detail, α-amylase significantly increased in trial

3 and 4 and Centauro variety showed a higher content than Balilla. On the other hand, β-

amylase and limit dextrinase showed a significant increase in trials 3 and 4 only for Balilla

variety which had higher content than Centauro. The increment of 10 °C in the final

kilning temperature tested in trial 2 did not affect enzymes preservation.

The sugar profiles of the malts are shown in table 3. According to literaure12 the content of

glucose in the rice malt was in the same range than maltose differently than in barley malt.

In Centauro variety the content of fructose and glucose seemed to be affected by the time

of malting, in fact there was a significant increase in trials 3 and 4. This result can be

explained by the increase of α-amylase content. The maltopentaose showed the same trend

of fructose and glucose. As consequence, the total amount of sugars also significantly

increased according to the longer malting time. In Balilla variety only fructose showed

significantly higher content in trial 3 and 4, as consequence of the α-amylase increase.

Sucrose, maltohexaose and maltoheptaose showed instead a slight decrease. As

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 consequence, the total sugar amount did not change in the different trials. This result may

be linked with the lower amount of α-amylase in Balilla than in Centauro. The sugar

profile confirmed that the malting program long 8 days with 4 short steeping steps

resulted as the best to obtain a well modified malt saving the water consumption.
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Consequently, the Centauro rice from 2014 was malted following the program of trial 3.

The quality attributes of the obtained rice malt were coherent with the results of the

optimization study, shown in Table 2.

The study conducted on a pilot scale of 100 litres is to our knowledge the first attempt to

brew a rice malt beer in a so big scale. In previous study the filtration of rice malt during

brewing resulted normal in a 25 litres scale.

In this study, despite the chosen small size of milling (0.45 mm), the husks resulted very

resistant, and lautering happened at a temperature of 78°C without any difficulty, with a

duration of about one hour, which is a standard performance in our pilot plant for barley

malt brewing. The brewery wort (Table 4) was adjusted by boiling to an extract content of

12.1 Plato degrees. pH of wort during mashing was adjusted in order to allow protein

degradation and consequent conversion of rice malt starch12,15. The complete

saccharification was indeed ascertained after 50 minutes of the rest at 74 °C. FAN content

in wort was adequate for yeast metabolism in fermentation process. Nitrogen content was

slightly low due to the lower protein content of rice, but in line with the literature 15. Beer

fermentation proceeded regularly reaching the attenuation limit as assessed by the sugar

consumption showed in table 4.

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 Concerning the sugar profile of wort (Table 4) as expected the content of glucose was in the

same range of maltose. The proportion of each single sugar was coherent with the results of

the optimization study. The amounts were higher for most of the analysed sugar, due to the

differences in the mashing program between congress and pilot plant. During fermentation,
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fructose, glucose, and sucrose were completely consumed by the yeast. Also maltose and

maltotriose showed a significant decrease even if a low amount was still present in the beer.

The dextrins content did not significantly change.

The quality attributes of final beer are reported in table 4. The alcohol content was

appropriate considering the wort extract. pH normally decreased during fermentation.

The colour of the final beer was very clear. Concerning the foam stability, typically Nibem

values above the 200 s are considered good25. In this case, the low value of Nibem is linked

to the low nitrogen content of wort (table 4) which negatively influences the foam stability.

Anyway, the foam stability is a well-known issue for gluten-free beer. In fact, the

exogenous enzymes added for gluten reduction negatively impact foam stability because

of hordein hydrolysation16. In a previous study, the same authors found an mean decrease

of about 14 % of foam stability after enzyme addition on three different kinds of beer, with

an average value of about 230 s26. On the other hand, the use of gluten-free cereals and

pseudocereals lead to different results, with good foam stability for maize, unsatisfactory

for amaranth, acceptable for quinoa and poor for oat16. The same authors found a value of

181 s for a beer of 100 % malted teff8 and a mean value of 164 s for bottom fermented beer

with 100 % malted rice27.

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 Amino acid profile of wort and beer was analysed (Table 5). The amino acid consumption

by the yeast during fermentation is very important, as they are involved in formation of

volatile compounds and also in flavour of final beer28. The amino acid content of wort was

perfectly comparable with the values expected for a wort obtained from barley malt (1200 –
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1500 mg/l)15. During fermentation and bottle conditioning, a great consumption of amino

acids happened, with a decrease of about 97 %, and this result was correlated with volatile

profile of the final beer showed in table 6. The higher alcohols are formed during the

fermentation and contribute an alcoholic or solvent-like aroma and a warm mouthfeel. The

most prevalent higher alcohols are n-propanol and isobutanol, which may cause ‘rough’

flavour and harshness, 2-phenylethanol, which causes ‘sweet’ or ‘rose’ flavour, and the amyl

alcohols (2-methylbutanol and 3-methylbutanol), which cause ‘fruity and sweetish’ flavors29.

Higher alcohols achieve maximum concentrations during batch fermentation at a time

roughly coincident with cell growth arrest and minimum FAN concentration. Their

formation takes place from 2-oxo acids arising from carbohydrates (anabolic route) or amino

acids (catabolic route) metabolism. The final concentration of higher alcohols is therefore

determined by the uptake efficiency of the corresponding amino acid and the sugar

utilization rate30. In this study, a relevant content of higher alcohols was registered. In fact,

concentrations of higher alcohols above 100 mg/l can damage the flavour and acceptability

of the beer. This high concentration of fusel alcohols may be linked to the low FAN content

of the wort. In fact, generally, for a barley malt wort of 12 °P the FAN content should not be

less than 150 mg/L for a proper fermentation, but 200 - 250 mg/L are recommended since a

low amount of total FAN promote overproduction of fermentation by-products like higher

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 alcohols25. Anyway, only the 3-methyl butanol content was above the perception threshold.

Esters represent a large group of favour-active compounds conferring to beer a ‘fruity–

flowery’ aroma. Beer esters can be divided into acetate esters (such as ethyl acetate and

isoamyl acetate, which confer to the beer a fruity, banana and solvent-like aroma), and ethyl
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or medium-chain fatty acid esters, (ethyl butyrate, ethyl hexanoate and ethyl octanoate

which give an ‘apple’ aroma to beer). Ester formation arises from enzyme-catalysed

condensation between higher alcohol and Acyl- or Acetyl-CoA, so their presence in beer is

linked to the concentration of these two substrates30. In this study, the ester concentration

was low. In fact, even considering that the ester content depends on the beer type and the

original wort gravity, usually top fermented beers contain up to 80 mg ester/125.

Furthermore, no one of detected ester was above the perception, with the exception of

isoamyl acetate, that is a typical flavour in wheat beer style and confers a banana or pear

drops aroma. This compound was detected in rice beer in a low quantity. The sugar

composition of wort has a direct influence on the organoleptic profile of final product and

high concentration of glucose in the wort increase the production of esters28. Considering the

high concentration of glucose in the wort, the high production of higher alcohols and the

suitability of the selected yeast strain for ester production, the resulting low amount of ester

can be explained by a low availability of Acyl- or Acetyl-CoA. In fact, top fermenting strains

generally produce more esters and higher alcohols than bottom fermenting strains, because

the uptake efficiency of the amino acid depends on the cell membrane, which is a strain-

specific property in yeast29. As confirmation of this statement, the higher alcohols content

results with an increment of more than 50 % compared to a previous study conducted on

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 rice malt beer with a bottom fermenting yeast27. Furthermore, despite the obtained content

of esters can be considered low for a top fermenting yeast, it shows an increase of about 40

% compared with the same study27.

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4-vinylguaiacol compound is the main typical aroma developed by wheat yeast strain

characterizing the wheat beer style. It is formed from free phenolic acids and it gives to the

beer a cloves flavour25,18. The presence of that compound was assessed in a lower range than

expected. Aldehydes and diketones in rice beer were in a low range and below the threshold

limit assessing that the principal off flavour of beer were absent.

The aroma and taste profiles of rice beer are shown in figure 2.

On the spider plot are reported the values for the chosen attributes of the beers. The beer

sensory profile did not revealed off flavour.

The aroma (fig 2 a) is characterized by fruity/esters attributes, which presence was assessed

over 3 points. The pear drop flavour is about 2.5 point, this attribute was recognized as the

main flavour among esters and its presence is ascribed in beer to isoamyl acetate. The value

than confirm the analytical results being present over the perception threshold. Other esters,

even if present at lower concentration than the perception threshold, could have concurred

to the fruity flavour thanks to synergistic effect. Also Alcoholic/Solvent flavour assessed

over 3 points was in line with the volatile analysis of the 3- Methyl-1-butanol which was

sensibly higher than the threshold. The phenolic aroma originate from phenols compounds

such as 4-vinyl-guaiacol which value was indeed close to the perception threshold. The

caramel cereal and vanilla attributes are related to the malt raw material. In previous work

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 have been reported vanilla flavour as a potential typical attribute for rice malt, further

confirmed thus by our results15. About taste of beer (fig 2 b) values of the attributes are in

line with the aroma results for the abovementioned attributes. Bitter value is coherent with

the used amount of hop. The beer was slightly sour probably due to the relative low pH
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(3,9) and could have influenced the astringency that was about 4 points.

The obtained results clearly show that the use of wheat top fermenting yeast allowed the

enhancement of the flat flavour profile achieved in a previous study27 with an increment of

the scores of fruity/estery and alcoholic/solvent attributes. Moreover, some specific

attributes related to the yeast strain, such as pear drop and phenolic, have been identified by

the panellists. However, it is worth of consideration that in this study a bottle conditioning

technique has been used which probably affected the overall sensorial profile. In fact,

secondary bottle fermentation results in a fully saturated beer with a richer, more stable

flavour profile. During refermentation, carbon dioxide as well as ethanol increase, while

esters and higher alcohol concentrations does not change sensibly. Carbonation gives beer a

refreshing taste and creates foam31–33. These effects allowed reaching the aim of this study to

improve the sensorial profile of 100 % rice malt beer.

CONCLUSION

This study confirms the potential of rice for the production of malt and beer. The malting

procedure has been optimized assessing the effect of short time steeping alternate to a

long air rest to obtain a sufficient moisture content in the green malt saving water

consumption. Long-time malting of 8 days allowed an optimal malt modification.

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 Unfortunately, the obtained rice malt did not show any colour increase, probably due to

the low kilning temperature. On the other hand, the chosen kilning temperature allows

preserving the enzymatic content, ensuring a complete saccharification. The performance

of rice malt for brewing tested on a pilot scale showed that lautering process can be
Accepted Article

conducted normally using rice malt husk. The use of a top fermenting yeast lead to a high

content of fusel alcohols. Sensory profile revealed pear drop, fruity and solvent notes,

without significant off-flavours. The production of a top fermented rice malt beer was a

successful attempt to introduce new flavoured product for consumption by individuals

affected by celiac disease. Further study should be conducted to improve the beer colour.

REFERENCES

1 FAO. December 2017. Rice Mark Monit 20: 1–27 (2017).

2 Marconi O, Sileoni V, Ceccaroni D, Perretti G. The Use of Rice in Brewing. In:

Advances in International Rice Research. InTech, 2017, pp 75–100.

3 Mohan BH, Malleshi NG, Koseki T. Physico-chemical characteristics and non-starch

polysaccharide contents of Indica and Japonica brown rice and their malts. LWT -

Food Sci Technol 43: 784–791 (2010).

4 Briggs DE. Malts and malting. 1998.

5 De Meo B, Freeman G, Marconi O, Booer C, Perretti G, Fantozzi P. Behaviour of

malted cereals and pseudo-cereals for gluten-free beer production. J Inst Brew 117:

541–546 (2011).

This article is protected by copyright. All rights reserved.

 6 Mayer H, Brewing I, Marconi O, Science F. The Investigation of the Suitability of

Hulled Wheats. J Am Soc Brew Chem 69: 116–120 (2011).

7 Di Ghionno L, Lee EG, Marconi O, Rice CJ, Sileoni V, Perretti G. Gluten-free sources
Accepted Article
of fermentable extract: behaviour of wiktop teff variety on malting process. In:

Trends in Brewing 2016. Ghent, Belgium, 2016, p 70.

8 Di Ghionno L, Sileoni V, Marconi O, De Francesco G, Perretti G. Comparative study

on quality attributes of gluten-free beer from malted and unmalted teff [Eragrostis

tef (zucc.) trotter]. LWT - Food Sci Technol 84 (2017). doi:10.1016/j.lwt.2017.06.044.

9 Ceppi ELM, Brenna OV. Experimental studies to obtain rice malt. J Agric Food Chem

58: 7701–7707 (2010).

10 Usansa U, Burberg F, Geiger E, Back W, Wanapu C, Arendt EK et al. Optimization of

Malting Conditions for Two Black Rice Varieties, Black Non-Waxy Rice and Black

Waxy Rice (Oryza sativa L. Indica). J Inst Brew 117: 39–46 (2011).

11 Agu RC, Chiba Y, Goodfellow V, MacKinlay J, Brosnan JM, Bringhurst TA et al.

Effect of germination temperatures on proteolysis of the gluten-free grains rice and

buckwheat during malting and mashing. J Agric Food Chem 60: 10147–10154 (2012).

12 Mayer H, Marconi O, Regnicoli GF, Perretti G, Fantozzi P. Production of a

saccharifying rice malt for brewing using different rice varieties and malting

parameters. J Agric Food Chem 62: 5369–5377 (2014).

13 Zarnkow M, Kessler M, Burberg F, Kreisz S, Back W. Gluten free beer from malted

This article is protected by copyright. All rights reserved.

 cereals and pseudocereals. In: European Brewery Convention Congress. Nürnberg:

Fachverlag Hans Carl: Prague, 2005, p 104.

14 Ceppi ELM, Brenna O V. Brewing with rice malt - a gluten-free alternative. J Inst
Accepted Article
Brew 116: 275–279 (2010).

15 Mayer H, Ceccaroni D, Marconi O, Sileoni V, Perretti G, Fantozzi P. Development of

an all rice malt beer: A gluten free alternative. LWT - Food Sci Technol 67: 67–73

(2016).

16 Hager A-S, Taylor JP, Waters DM, Arendt EK. Gluten free beer – A review. Trends

Food Sci Technol 36: 44–54 (2014).

17 Phiarais BPN, Arendt EK. Malting and brewing with gluten-free cereals. In: Arendt

EK, Dal Bello F (eds). Gluten-Free Cereal Products and Beverages. Academic Press, 2008,

pp 347–372.

18 Briggs DE, Boulton CA, Brookes PA, Steven R. Brewing: science and practice.

Woodhead Publishing Limited Eds: Cambridge, UK, 2004 doi:10.1007/s11947-012-

0942-2.

19 Kumar R, Singh T. Rice Husk-Reinforced Composites: A Review. In: Khangura SS,

Singh P, Singh H, Brar GS (eds). Proceedings of the International Conference on Research

and Innovations in Mechanical Engineering: ICRIME-2013. Springer India: New Delhi,

2014, pp 395–405.

20 Marconi O, Mayer H, Chiacchieroni F, Ricci E, Perretti G, Fantozzi P. The influence

This article is protected by copyright. All rights reserved.

 of glumes on malting and brewing of hulled wheats. J Am Soc Brew Chem : 41–48

(2013).

21 Do Nascimento AB, Fiates GMR, dos Anjos A, Teixeira E. Gluten-free is not enough -
Accepted Article
perception and suggestions of celiac consumers. Int J Food Sci Nutr 65: 394–398

(2014).

22 European Brewery Convention. Analytica-EBC. 5th ed. Fachverlag ans Carl

urnberg, 2007.

23 Floridi S, Miniati E, Montanari L, Fantozzi P. Carbohydrate determination in wort

and beer by HPLC-ELSD. Monatsschrift für Brauwiss 54: 209–215.

24 De Francesco G, Turchetti B, Sileoni V, Marconi O, Perretti G. Screening of new

strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce

low-alcohol beer. J Inst Brew 121: 113–121 (2015).

25 Kunze W. Technology brewing and malting. VLB Berlin : 949 (2004).

26 Di Ghionno L, Marconi O, Sileoni V, De Francesco G, Perretti G. Brewing with prolyl

endopeptidase from Aspergillus niger : the impact of enzymatic treatment on gluten

levels, quality attributes and sensory profile. Int J Food Sci Technol 52 (2017).

doi:10.1111/ijfs.13375.

27 Mayer H, Ceccaroni D, Marconi O, Sileoni V, Perretti G, Fantozzi P. Development of

an all rice malt beer: A gluten free alternative. LWT - Food Sci Technol 67 (2016).

doi:10.1016/j.lwt.2015.11.037.

This article is protected by copyright. All rights reserved.

 28 He Y, Dong J, Yin H, Zhao Y, Chen R, Wan X et al. Wort composition and its impact

on the flavour-active higher alcohol and ester formation of beer - A review. J Inst

Brew 120: 157–163 (2014).

Accepted Article
29 Rossi S, Sileoni V, Perretti G, Marconi O. Characterization of the volatile profiles of

beer using headspace solid-phase microextraction and gas chromatography-mass

spectrometry. J Sci Food Agric 94: 919–928 (2014).

30 Pires EJ, Teixeira JA, Brányik T, Vicente AA. Yeast the soul of beer’s aroma—a

review of flavour-active esters and higher alcohols produced by the brewing yeast.

Appl Microbiol Biotechnol 98: 1937–1949 (2014).

31 Marconi O, Rossi S, Galgano F, Sileoni V, Perretti G. Influence of yeast strain,

priming solution and temperature on beer bottle conditioning. J Sci Food Agric 96:

4106–4115 (2016).

32 Vanbeneden N, Vanderputten D, Vanderhaegen B, Landschoot A Van, Vanbeneden

N, Vanderputten D et al. Influence of the Sugar Composition of the Added Extract

on the Refermentation of Beer in Bottles. J Am Soc Brew Chem 470 (2006).

doi:10.1094/ASBCJ-64-0206.

33 Dekoninck T, Mertens T, Delvaux F, Delvaux FR. Influence of Beer Characteristics

on Yeast Refermentation Performance During Bottle Conditioning of Belgian Beers. J

Am Soc Brew Chem 71: 23–34 (2013).

This article is protected by copyright. All rights reserved.

 Tables

Table 1 Rice quality attributes

Varieties Centauro Balilla

Moisture (%) 13.42 ± 0.02 13.81 ± 0.02

Accepted Article

Thousand Kernel Weight (g) 25.4 ± 0.1 22.8 ± 0.1

Germinative Energy (% at 28°C) 93 ± 1 99 ± 1

Total Protein (% d.m.) 7.62 ± 0.06 7.14 ± 0.06

n = 2 analytical replicates, d.m. = dry matter.

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 Table 2 Rice malts quality attributes and enzymes content

Centauro Balilla

Trial 1 2 3 4 1 2 3 4

Extract Yield (% d.m.) 74.5 a 74.2 a 76.4 b 75.8 b 77.3A 77.3A 77.6AB 77.8B
Accepted Article

Saccharification Time (min) > 30 a >30 25-30 25-30 > 30 >30 15-20 15-20

Colour of Malt (EBC-U) 1.7 a 1.7 a 1.8 a 1.7 a 1.4A 1.5A 1.5A 1.5A

Total Protein (% d.m.) 7.1 a 7.1 a

7.1 a 7.7 a 6.6A 6.6A 6.6A 6.6A

Kolbach Index 31.8 a 32.3 a 37.8 b 36.8 b 29.3A 30.1A 35.4B 39.3B

Total Soluble Nitrogen at 8.6 °P 459 a 466 a 540 b 547 b 378A 381A 438B 495B
(mg/L)

Fermentability (%) 66.2 a 67.4 a 71.3 a 70.1 a 81.3B 80.2A 86.2D 85C

Diastatic Power (WK) 22 a 20 a 37 b 39 b 19A 26A 62B 58B

FAN at 8.6 °P (mg/L) 117 b 104 a 134 c 131 c 90A 81A 105B 104B

Enzymes

α-amylase (Ceralpha (U/g)) 27.5a 24.0a 43.9b 40.1b 20.0A 18.5A 28.1B 28.1B

β-amylase (Betamyl-3 (U/g)) 1.8a 1.6a 2.9a 2.4a 3.1A 3.1A 7.2B 6.9B

Limit Dextrinase (U/(malt kg)) 4114a 3763a 4680a 4242a 5160A 4656A 6587B 6355B

n =2 two technological replicates; d.m.=dry matter; min = minutes; EBC-U = European Brewery Convention

Units; WK = Windisch−Kolbach units; FAN = Free Amino Nitrogen; U = units. Values in the same row

followed by different letters are statistically different; p <0.05, lowercase letters for Centauro samples and

capital letters for Balilla samples.

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 Table 3. Sugars composition (g/L) of rice malt Congress worts

Centauro Balilla
Accepted Article
Trial 1 2 3 4 1 2 3 4

Fructose 0.29 a 0.29 a 0.49 b 0.51 b 0.42 A 0.39 A 0.66 B 0.65 B

Glucose 28.77 a 29.98 a 39.48 b 37.13 b 32.28 A 30.59 A 36.11 A 33.21 A

Sucrose 0.23 a 0.24 a 0.30 a 0.26 a 0.82B 0.96 B 0.27 A 0.19 A

Maltose 16.65 a 17.42 a 19.70 a 18.07 a 29.57 A 30.7 A 32.97 A 30.39 A

Maltotriose 6.41 a 6.72 a 6.88 a 6.45 a 8.62 A 8.82 A 8.43 A 7.74 A

Maltotetraose 3.43 a 3.62 a 4.46 a 3.94 a 1.72 A 1.72 A 1.77 A 1.74 A

Maltopentaose 2.24 a 2.35 a 3.06 b 2.88 b 1.43 A 1.22 A 1.66 A 1.63 A

Maltohexaose 13.55 a 14.35 a 13.90 a 13.20 a 11.79 B 10.04AB 8.31 A 8.13 A

Maltoheptaose 0.46 b 0.34 b 0.12 a 0.13 a 0.56 B 0.58 AB 0.17 A B 0.17 A

Dextrin DP8-DP15 0.41 b 0.46 b 0.60 a 0.55 ab n.d. n.d. n.d. n.d.

Total sugars 72.44 a 75.77 a 88.99 b 83.12 ab 87.21 A 85.02 A 90.35 A 83.85 A

n=2 technological replicates. DP = Degree of Polymerization; n.d. = not detected. Values in the same row
followed by different letters are statistically different, p <0.05.

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 Table 4 Quality attributes and sugar composition of rice malt wort and final beer

Centauro Rice Malt Wort Centauro Rice Malt Beer

Extract (°P)/Original extract (°P) 12.1 ± 0.1 12.7 ± 0.1

Real Extract (°P) - 4.99 ± 0.06

Apparent Extract (°P) 3.24 ± 0.06

Accepted Article

Alcohol (% v/v) - 5.08 ± 0.10

Fermentability (%)/ RDF (%) 74.4 ± 3.3 62.2 ± 0.7

pH 5.22 ± 0.01 3.93 ± 0.01

Colour (EBC-U) 8.3 ± 0.1 5.1 ± 0.4

FAN (mg/L at 12°P) 173 ± 2 29 ± 1

Total nitrogen (mg/L at 12 °P) 741 ± 3 356 ± 9

Attenuation limit (°P) 3.3 ± 0.4 -

Foam stability (s) - 182 ± 6

Haze (U-EBC) - 45.5 ± 4.9

Sugar composition (g/L)

Fructose 0.57 ± 0.06 n.d.

Glucose 56.27 ± 1.03 n.d.

Sucrose 0.22 ± 0.07 n.d.

Maltose 43.55 ± 2.80 1.76 ± 0.13

Maltotriose 13.95 ± 1.14 2.49 ± 0.45

Maltotetraose 16.61 ± 0.24 14.59 ± 0.86

Maltopentaose 4.88 ± 0.14 6.39 ± 0.43

Maltohexaose 10.37 ± 0.53 17.13 ± 1.32

Maltoheptaose 5.56 ± 3.17 1.07 ± 0.17

n=2 technological replicates; °P = Plato degree; v/v = volume/volume; EBC-U = European Brewery
Convention Units; RDF = Real Degree of Fermentation; FAN = Free Amino Nitrogen; ; n.d. = not detected.

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 Table 5 Amino acids profile (mg/L) of rice malt wort and final beer

Wort Beer

Aspartic acid 73.9 ± 4.5 2.3 ± 0.2

Glutamic acid 104.3 ± 3.6 2.1 ± 0.1

Accepted Article

Serine 73.7 ± 2.3 2.8 ± 0.2

Histidine 95.3 ± 3.6 2.4 ± 0.2

Arginine 201.0 ± 4.0 2.8 ± 0.1

Glycine 38.8 ± 3.5 2.8 ± 0.1

Treonine 66.6 ± 1.1 12.0 ± 0.8

Alanine 93.4 ± 4.5 2.5 ± 0.3

Tirosine 113.3 ± 0.1 2.5 ± 0.2

Methionine 50.4 ± 0.9 0.5 ± 0.0

Valine 109.9 ± 0.1 0.5 ± 0.1

Phenylalanine 105.0 ± 0.8 1.1 ± 0.1

Leucine 53.5 ± 0.0 1.3 ± 0.1

Isoleucine 131.1 ± 0.5 1.1 ± 0.1

Lysine 113.2 ± 7.5 2.0 ± 0.1

n=2 technological replicates.

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 Table 6 Volatile profile of rice malt beer

Rice Beer Perception threshold18

Higher Alcohols 1-propanol 28.0 ± 1.6 600

(mg/L) 2-Methyl-1-propanol 75.5 ± 3.4 100

Accepted Article

3- Methyl-1-butanol 75.2 ± 2.1 50-65

2- Methyl-1-butanol 23.4 ± 1.2 50-70

2-Phenylethanol 49.3 ± 4.6 125

Total Higher Alcohols 251.6 ± 7.8

Esters Ethyl acetate 16.5 ± 3.2 25-30

(mg/L) Ethyl butanoate 0.10 ± 0.01

Isoamyl acetate 1.2 ± 0.4 1.2-2

Ethyl hexanoate 0.10 ± 0.01 0.2-0.23

Ethyl octanoate 0.10 ± 0.01 0.9-1.0

Total Esters 17.7 ± 3.4

Phenols (mg/L) 4 vinylguaicol 0.25 ± 0.02 0.3

Aldehydes Acetaldehyde (mg/L) 14.2 ± 1.6

(μg/L)
Furfural 49.7 ± 11.5

Methional 11.9 ± 4.8

Phenylacetaldehyde 18.7 ± 2.4

Diketones (μg/L) Diacetyl 53.4 ± 3.6

n=2 technological replicates

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Accepted Article

Figure 1 Malting programs

The malting days (from 1 to 8) are indicated in the first row, while the corresponding temperatures are
indicated in the second row. Along the horizontal bars, representing the trials, the space between two
consecutive vertical lines indicates one day and sections of different colours indicate the phase of the
process that occurs. The coloured sections are proportionated to the duration of the phase.

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Accepted Article
Fo
rP
ee
rR
ev
iew

Figure 2 Aroma (a) and Taste (b) sensorial analysis of beer

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