Effect of Temperature and Germination Time on Quality Attributes of Teff

May 22, 2017 - and Germination Time on Quality Attributes of Teff [Eragrostis tef. (zucc.) Trotter] ... brewing.13−15 Authors concluded that the opt...
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Gluten-free sources of fermentable extract: effect of temperature and germination time on quality attributes of teff [Eragrostis tef (zucc.) trotter] malt and wort. Lidia Di Ghionno, Eung Gwan Lee, Ombretta Marconi, Christopher J. Rice, Valeria Sileoni, and Giuseppe Perretti J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Journal of Agricultural and Food Chemistry

Gluten-free sources of fermentable extract: effect of temperature and germination time on quality attributes of teff [Eragrostis tef (zucc.) trotter] malt and wort.

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Lidia Di Ghionno1, Eung Gwan Lee3, Ombretta Marconi2, Christopher J. Rice3, Valeria Sileoni1*, Giuseppe Perretti2

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

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University of Perugia, Italian Brewing Research Centre Perugia, Italy

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Campden BRI, Nutfield, United Kingdom

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* Corresponding author

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e-mail: [email protected]

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Tel: +39 075 585 7926 - Fax: +39 075 585 7946

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

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via San Costanzo s.n.c.

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06126 Perugia,

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Italy 1 ACS Paragon Plus Environment

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Abstract

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This study was conducted to evaluate the behavior of a white teff variety called Witkop during

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malting by using different parameters (germination temperature and duration) and to identify the

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best malting program. Samples were evaluated for standard quality malt and wort attributes, pasting

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characteristics, β-glucan and arabinoxylan content and sugar profile. It was concluded that malting

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teff at 24°C for 6 days produced acceptable malt in terms of quality attributes and sugar profile for

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brewing. The main attributes were: 80.4% extract, 80.9 % fermentability, 1.53 mPa·s viscosity, 7.4

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EBC-U colour, 129 mg/L FAN and 72.1 g/L of total fermentable sugars. Statistical analysis showed

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that pasting characteristics of teff malt were negatively-correlated with some malt quality attributes,

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such as extract and fermentability. Witkop teff appeared to be a promising raw material for malting

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and brewing. However, the small grain size may lead to difficulties in handling malting process and

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a bespoke brewhouse plant should be developed for the production at industrial scale.

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Keywords

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Arabioxylans, Beta-glucans, Eragrostis tef, Gluten-free beer, Malt quality, Rapid Visco Analysis 2 ACS Paragon Plus Environment

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1. Introduction

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Worldwide, barley is the main raw material used in the malt and beer production. Among other

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characteristics, its suitability for malting and brewing is mainly due to the high content of starch, as

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well as the presence of hulls, which work as a filtering aid during brewing, and the high content of

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hydrolytic enzymes produced during germination, which ensure an high fermentable extract yield.1

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However, because of its content of hordeins,2 barley is not suitable for people with coeliac disease,

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which is estimated to affect up to 1% of the global population.3 Hordeins, as well as wheat gliadins

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and rye secalins, represent the alcohol-soluble fraction of gluten responsible for the immunological

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reactions in coeliac patients. Together, these compounds are termed prolamins, the ingestion of

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which, in genetically predisposed individuals, could lead to intestinal villous atrophy,

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malabsorption of nutrients resulting in loss of weight or failure to thrive in infants, and to a high

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risk of gastrointestinal cancer.3 In addition, other gluten-related diseases, such as gluten ataxia,

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dermatitis herpetiformis, wheat allergy and gluten sensitivity, affect up to 6% of the global

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population.4 At the present, no pharmacological treatments are available and all patients are

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required to adhere to a strict lifelong gluten-free diet. As a consequence, an increasing number of

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studies have been focused on the evaluation of naturally gluten-free cereals (rice, corn, millet,

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sorghum, teff) and pseudocereals (quinoa, buckwheat and amaranth) in order to replace gluten

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containing raw materials in cereal-based food and beverages, including beer.5–8 Among these

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grains, rice, sorghum, millet and buckwheat, have been most widely studied for malting and

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brewing purposes, with promising results.7–11 In contrast, only very little published material is

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available on teff [Eragrostis tef (Zucc.) Trotter] malt and its use in the production of gluten-free

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beer.12–16

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Teff, is a small cereal native to Ethiopia where is considered as staple food, used to make injera

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flatbread, porridge or the home-brewed alcoholic drink tella.17 Teff is a cereal belonging to the

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poaceae family of grasses, which does not contain gluten, is rich in starch (73%), protein (11 -

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12%), minerals and is well-balanced in all essential amino acids.18–20 For these reasons teff 3 ACS Paragon Plus Environment

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represents an alternative source of fermentable extract, potentially suitable for the production of

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malt and gluten-free beer. However, the diameter of the grain is less than 1 mm (less than 1% the

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size of a barley grain19), which makes it a challenging raw material for the production of malt and

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beer. Recent studies investigated the suitability of different teff varieties, for the production of an

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acceptable malt for brewing.13–15 Authors concluded that the optimal malting conditions were 4

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days of germination with 48 % degree of steeping, and a set temperature of 24 °C for steeping and

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germination. The optimal kilning conditions were found to be 18 h at 30 °C+1h at 60 °C+3 or 5 h at

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65 °C. According to the authors, Quncho teff variety, cultivated in Ethiopia, showed the highest

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enzyme activity, malt quality attributes and concentration of fermentable sugar when compared

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with Ivory, Brown, Dessie and Sirgynia varieties grown in North America. However, since the

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Ethiopian government in 2006 has implemented an export ban on grain teff and flour in an effort to

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reduce domestic prices (a ban partially-lifted specifically for teff flour in 2015),21 there is a need to

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evaluate the physicochemical characteristics and the suitability for malting and brewing purpose of

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other teff varieties cultivated outside Ethiopia. Moreover, considering that different varieties can

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give a different response to the malting process, the specific optimal technological parameters need

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to be defined. In fact, nowadays some countries such as Canada, China, India, Netherlands, South

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Africa, Uganda, Cameroon, United Kingdom and the United States have begun to produce and

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export teff because of its high rusticity and adaptability to a wide range of environments.22

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The present study was focused on the evaluation of the effect of temperature and germination time

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on malt quality attributes enzymatic-activity and sugar profile of Witktop teff variety cultivated in

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South Africa, never investigated before, in order to identify the optimal combination of these two

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parameters for malting process. Since arabinoxylan and beta-glucan content are known to be

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important, quality-limiting attributes of brewers’ malts, their total content have also been evaluated.

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In addition, Rapid Visco Analyser (RVA) profile of the Witkop teff variety was analysed and

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reported in order to correlate the pasting and gelatinization properties with the quality attributes.

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The main goal of this study was to look into the malting performance of this emerging cereal, and in 4 ACS Paragon Plus Environment

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particular into a variety available outside Ethiopia (i.e. Witkop teff), in response to the global

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increasing demand for teff as part of the growing gluten-free market.

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2. Materials and methods

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2.1 Grain teff

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Witkop teff variety, cultivated in South Africa in 2014, was purchased from Millets Place BV

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company (Gasselte, Netherlands). The sample was analyzed in duplicate following the Analytica-

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EBC23 and Mitteleuropäische Brautechnische Analysenkommision (MEBAK) methods.24

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germinative capacity (%) by EBC method 3.5.2, germinative energy and water sensitivity (%) by

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EBC method 3.6.2, grain moisture (%) by EBC method 3.2, total nitrogen (% dm) and total protein

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(total nitrogen × 6.25) by EBC method 3.3.1, fatty substances (% dm) by EBC method 6.10.

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Furthermore, for the rate of water uptake 50 g of sample was steeped at two different temperatures

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(22 °C and 24 °C). Samples from the steeping phase were taken over time (at 15, 30, 60, 120, 180,

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300 and 360 min), dried with blotting paper to remove surface moisture, quickly milled with a

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mortar and analyzed with moisture analyzer (Sartorius) for determining the moisture content.

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2.2. Micromalting procedure

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Samples of teff grains were washed a potable water to remove dust and possible microorganisms

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from the surface of the grains. Micromalting trials were carried out in 400 g batches in a nylon bag

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in the micromalting plant at Campden BRI (UK) at two different temperatures (22°C and 24°C) and

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five different times of germination (2, 3, 4, 5 and 6 days) in duplicate. In all trials, the steeping

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procedure was performed as follows: 3 hour wet, 2 hour air-rest followed by 2 hour wet, in order to

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reach the steeping out moisture of 48 %, which, according to Zarnkow et al.,13 is the optimal

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moisture content for steeped teff grain. Kilning was carried out for all trials with a slightly modified

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program proposed by Gebremariam et al.:15 20 hours at 30°C, 2 hours at 60°C and 3 hours at 65°C.

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The malts were shaken manually within the nylon bags to remove rootlets. Malt loss was about 20%

The

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for all experiments performed, that is perfectly in line with the typical values reported for barley

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malt.25

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2.3. Malted grain teff analysis

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2.3.1. Quality attributes

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Each sample was analyzed in duplicate following standard Analytica-EBC23 methods. The extract

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yield (% dm) and filtration rate by EBC method 4.5.1, total nitrogen (TN) content (% dm) by EBC

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method 4.3.1, soluble nitrogen (SN) content (% dm) and Kolbach index (SN/TN ratio) by EBC

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method 4.9.1, free amino nitrogen content (FAN; mg/L) by EBC method 4.10, viscosity (mPa s at

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20 °C and 8.6 °P (from the Plato scale)) by EBC method 4.8, fermentability (%) by EBC method

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4.11.1, diastatic power (Windisch−Kolbach units (WK)) by EBC method 4.12, pH by EBC method

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8.17, malt color (EBC units) by EBC method 4.7.1.

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2.3.2. Starch-degrading enzyme activity

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The activity of α-amylase, β-amylase and limit-dextrinase of malted teff was determined using the

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Megazyme Assay Kits (Megazyme International Ireland)26–28.

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2.3.3. Sugar profile

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The concentrations of fermentable sugars (g/L) in malted grain teff congress worts were determined

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by HPLC coupled with evaporative light scattering detector (ELSD) as previously reported by

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Floridi et al.29

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2.3.4. Total β-glucan and total arabinoxylan content

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The β-glucan were analyzed using a commercial assay kit (Megazyme International Ireland),

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following the Analytica-EBC26 methods 3.11.1 and 4.16.1., The total arabinoxylan was determined

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by improving the methods of Bell30 and Englyst et al.31 In fact, both the extraction procedures

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described in the reference methods, namely a treatment with sulfuric acid for Bell30 and with an

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enzyme solution (α-amylase + pullulanase) for 16 h at 40 °C for Englyst et al.31, were made more

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efficient by the separate use of the different enzymes at their optimal temperatures, allowing an

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important time reduction. This procedure includes the extraction of the arabinoxylan from the 6 ACS Paragon Plus Environment

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sample and various purification steps to obtain arabinoxylan as pure as possible as follows: (A) For

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the solubilization of arabinoxylan, acetate buffer (pH 5.2, 10 mL) were added to 500 mg of milled

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sample. (B) The enzymatic degradation of the interferences was performed by adding α-amylase

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(Megazyme 3000 U/mL, 200 µL) while stirring at 80°C for 15 min, by subsequent addition of

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amyloglucosidase (Megazyme 3260 U/mL, 23 µL), stirring at 60°C for 15 min, and by subsequent

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addition of NaOH 1M until the solution reached pH 7. Finally pancreatin (4 mg) and lichenase (20

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µL) were added and the solution was stirred at 40°C for 1 hour. (C) Precipitation of the purified

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pellet was performed by the addition of pure ethanol (30 mL), with the test tubes left on ice for 30

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min, and then centrifuged (4000 rpm, 10 min) and the supernatant fraction discarded. Subsequently,

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the pellet was washed by the addition of ethanol (85 % v/v, 10 mL) and stirring by vortexing,

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followed by the addition of ethanol (85% v/v, 30 mL) and mixing by inversion, centrifuging at 4000

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rpm for 10 min and discarding the supernatant fraction. Acetone (20 mL) was added, mixed by

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inversion and centrifuged at 4000 rpm for 10 min. After discarding the supernatant fraction, the

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uncapped test tube was left in a heated bath at 80 °C for 15 min to allow the solvent evaporation.

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(D) Acid hydrolysis of the pellet was carried out by adding sulfuric acid (12 M, 5 mL) and heating

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at 35°C for 1 hour, then deionized water (25 mL) was added and boiled for 30 min before the

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sample was cooled and diluted 1:3 (0.5 mL of samples with 1.5 mL of deionized water). For the

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arabinoxylan quantification through a spectrophotometric measure, 10 mL of a pentosans-specific

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reagent (phloroglucinol) was added to the sample, which was boiled for 22 min, cooled in ice for 10

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min and left at 20 °C for 5 min. Finally, the absorbance versus blank reagent was read at 510 and

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subtracted to absorbance at 552 nm. In fact, the absorbance of interfering sugars differs very little at

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these two wavelengths but is considerable for the pentosans. Accordingly, the pentosans may be

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estimated, even when an excess of interfering sugars is present, by subtracting the absorbance at

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510 nm from that at 552 nm30. Arabinoxylan concentration was calculated by mean of a calibration

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curve. A control sample was prepared with 10 mg of standard arabinoxylan HV (Megazyme)

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treated in the same way as the sample. For the blank solution, 2 mL of deionized water were used

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for the reaction, with phloroglucinol used in place of the diluted sample.

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2.3.5. Rapid Visco Analysis (RVA)

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A RVA was carried out using a Rapid Visco Analyser (Newport Scientific Pty. Ltd.) in accordance

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with the MEBAK method 2.7. A known amount of sample (5 g), adjusted for moisture, was slurried

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with a known volume of water (total weight 30 g) and then processed in the RVA analyser using the

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following standard test profile: starting temperature of 50 °C held for 1 min, raised to 95 °C over

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3.7 min, held for 2.5 min, cooled to 50 °C in 3.8 min and held for 2 min. To ensure the dispersion of

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the grist, stirring was performed, initially at 960 rpm, 10 s before being decreased to 160 rpm for

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the remainder of the analysis. The RVA measurements were as follows: peak viscosity (PV) –

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maximum viscosity developed during heating; Peak time (Pt) – time at which the peak viscosity

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occurred; Trough (T) – lowest viscosity after cooling started; Breakdown (BD) – peak viscosity

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minus trough; Final viscosity (FV) – viscosity at the end of the test; Setback (SB) – final viscosity

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minus trough; Pasting temperature (PT) – temperature at which the viscosity increases by 25 mPa·s

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within a 20 s period, as defined by the manufacturer (approx. gelatinization temperature).

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2.4. Statistical analyses

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The micromalting trials and each analysis were all performed in duplicate. Thus, the data were

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reported as the mean of four values ± standard deviations. The statistical analyses were performed

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(Statgraphics Centurion XVI version 16.1.11, StatPoint Technologies, Inc., Warrenton, VA, USA)

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on the data collected. The significant variations between the different samples were discriminated

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using a one-way Analysis Of Variance (ANOVA, p