Subscriber access provided by Binghamton University | Libraries
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 37
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.
1 2 3
Lidia Di Ghionno1, Eung Gwan Lee3, Ombretta Marconi2, Christopher J. Rice3, Valeria Sileoni1*, Giuseppe Perretti2
4 5 6 7 8 9
1
University of Perugia, Department of Agricultural, Food and Environmental Science, Perugia, Italy
10
2
University of Perugia, Italian Brewing Research Centre Perugia, Italy
11
3
Campden BRI, Nutfield, United Kingdom
12 13 14 15 16 17 18 19 20 21 22
* Corresponding author
23
e-mail:
[email protected] 24
Tel: +39 075 585 7926 - Fax: +39 075 585 7946
25
Department of Agricultural, Food and Environmental Science - University of Perugia,
26
via San Costanzo s.n.c.
27
06126 Perugia,
28
Italy 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 37
29
Abstract
30
This study was conducted to evaluate the behavior of a white teff variety called Witkop during
31
malting by using different parameters (germination temperature and duration) and to identify the
32
best malting program. Samples were evaluated for standard quality malt and wort attributes, pasting
33
characteristics, β-glucan and arabinoxylan content and sugar profile. It was concluded that malting
34
teff at 24°C for 6 days produced acceptable malt in terms of quality attributes and sugar profile for
35
brewing. The main attributes were: 80.4% extract, 80.9 % fermentability, 1.53 mPa·s viscosity, 7.4
36
EBC-U colour, 129 mg/L FAN and 72.1 g/L of total fermentable sugars. Statistical analysis showed
37
that pasting characteristics of teff malt were negatively-correlated with some malt quality attributes,
38
such as extract and fermentability. Witkop teff appeared to be a promising raw material for malting
39
and brewing. However, the small grain size may lead to difficulties in handling malting process and
40
a bespoke brewhouse plant should be developed for the production at industrial scale.
41 42 43 44 45 46 47 48 49 50 51 52 53
Keywords
54
Arabioxylans, Beta-glucans, Eragrostis tef, Gluten-free beer, Malt quality, Rapid Visco Analysis 2 ACS Paragon Plus Environment
Page 3 of 37
Journal of Agricultural and Food Chemistry
55
1. Introduction
56
Worldwide, barley is the main raw material used in the malt and beer production. Among other
57
characteristics, its suitability for malting and brewing is mainly due to the high content of starch, as
58
well as the presence of hulls, which work as a filtering aid during brewing, and the high content of
59
hydrolytic enzymes produced during germination, which ensure an high fermentable extract yield.1
60
However, because of its content of hordeins,2 barley is not suitable for people with coeliac disease,
61
which is estimated to affect up to 1% of the global population.3 Hordeins, as well as wheat gliadins
62
and rye secalins, represent the alcohol-soluble fraction of gluten responsible for the immunological
63
reactions in coeliac patients. Together, these compounds are termed prolamins, the ingestion of
64
which, in genetically predisposed individuals, could lead to intestinal villous atrophy,
65
malabsorption of nutrients resulting in loss of weight or failure to thrive in infants, and to a high
66
risk of gastrointestinal cancer.3 In addition, other gluten-related diseases, such as gluten ataxia,
67
dermatitis herpetiformis, wheat allergy and gluten sensitivity, affect up to 6% of the global
68
population.4 At the present, no pharmacological treatments are available and all patients are
69
required to adhere to a strict lifelong gluten-free diet. As a consequence, an increasing number of
70
studies have been focused on the evaluation of naturally gluten-free cereals (rice, corn, millet,
71
sorghum, teff) and pseudocereals (quinoa, buckwheat and amaranth) in order to replace gluten
72
containing raw materials in cereal-based food and beverages, including beer.5–8 Among these
73
grains, rice, sorghum, millet and buckwheat, have been most widely studied for malting and
74
brewing purposes, with promising results.7–11 In contrast, only very little published material is
75
available on teff [Eragrostis tef (Zucc.) Trotter] malt and its use in the production of gluten-free
76
beer.12–16
77
Teff, is a small cereal native to Ethiopia where is considered as staple food, used to make injera
78
flatbread, porridge or the home-brewed alcoholic drink tella.17 Teff is a cereal belonging to the
79
poaceae family of grasses, which does not contain gluten, is rich in starch (73%), protein (11 -
80
12%), minerals and is well-balanced in all essential amino acids.18–20 For these reasons teff 3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 37
81
represents an alternative source of fermentable extract, potentially suitable for the production of
82
malt and gluten-free beer. However, the diameter of the grain is less than 1 mm (less than 1% the
83
size of a barley grain19), which makes it a challenging raw material for the production of malt and
84
beer. Recent studies investigated the suitability of different teff varieties, for the production of an
85
acceptable malt for brewing.13–15 Authors concluded that the optimal malting conditions were 4
86
days of germination with 48 % degree of steeping, and a set temperature of 24 °C for steeping and
87
germination. The optimal kilning conditions were found to be 18 h at 30 °C+1h at 60 °C+3 or 5 h at
88
65 °C. According to the authors, Quncho teff variety, cultivated in Ethiopia, showed the highest
89
enzyme activity, malt quality attributes and concentration of fermentable sugar when compared
90
with Ivory, Brown, Dessie and Sirgynia varieties grown in North America. However, since the
91
Ethiopian government in 2006 has implemented an export ban on grain teff and flour in an effort to
92
reduce domestic prices (a ban partially-lifted specifically for teff flour in 2015),21 there is a need to
93
evaluate the physicochemical characteristics and the suitability for malting and brewing purpose of
94
other teff varieties cultivated outside Ethiopia. Moreover, considering that different varieties can
95
give a different response to the malting process, the specific optimal technological parameters need
96
to be defined. In fact, nowadays some countries such as Canada, China, India, Netherlands, South
97
Africa, Uganda, Cameroon, United Kingdom and the United States have begun to produce and
98
export teff because of its high rusticity and adaptability to a wide range of environments.22
99
The present study was focused on the evaluation of the effect of temperature and germination time
100
on malt quality attributes enzymatic-activity and sugar profile of Witktop teff variety cultivated in
101
South Africa, never investigated before, in order to identify the optimal combination of these two
102
parameters for malting process. Since arabinoxylan and beta-glucan content are known to be
103
important, quality-limiting attributes of brewers’ malts, their total content have also been evaluated.
104
In addition, Rapid Visco Analyser (RVA) profile of the Witkop teff variety was analysed and
105
reported in order to correlate the pasting and gelatinization properties with the quality attributes.
106
The main goal of this study was to look into the malting performance of this emerging cereal, and in 4 ACS Paragon Plus Environment
Page 5 of 37
Journal of Agricultural and Food Chemistry
107
particular into a variety available outside Ethiopia (i.e. Witkop teff), in response to the global
108
increasing demand for teff as part of the growing gluten-free market.
109
110
2. Materials and methods
111
2.1 Grain teff
112
Witkop teff variety, cultivated in South Africa in 2014, was purchased from Millets Place BV
113
company (Gasselte, Netherlands). The sample was analyzed in duplicate following the Analytica-
114
EBC23 and Mitteleuropäische Brautechnische Analysenkommision (MEBAK) methods.24
115
germinative capacity (%) by EBC method 3.5.2, germinative energy and water sensitivity (%) by
116
EBC method 3.6.2, grain moisture (%) by EBC method 3.2, total nitrogen (% dm) and total protein
117
(total nitrogen × 6.25) by EBC method 3.3.1, fatty substances (% dm) by EBC method 6.10.
118
Furthermore, for the rate of water uptake 50 g of sample was steeped at two different temperatures
119
(22 °C and 24 °C). Samples from the steeping phase were taken over time (at 15, 30, 60, 120, 180,
120
300 and 360 min), dried with blotting paper to remove surface moisture, quickly milled with a
121
mortar and analyzed with moisture analyzer (Sartorius) for determining the moisture content.
122
2.2. Micromalting procedure
123
Samples of teff grains were washed a potable water to remove dust and possible microorganisms
124
from the surface of the grains. Micromalting trials were carried out in 400 g batches in a nylon bag
125
in the micromalting plant at Campden BRI (UK) at two different temperatures (22°C and 24°C) and
126
five different times of germination (2, 3, 4, 5 and 6 days) in duplicate. In all trials, the steeping
127
procedure was performed as follows: 3 hour wet, 2 hour air-rest followed by 2 hour wet, in order to
128
reach the steeping out moisture of 48 %, which, according to Zarnkow et al.,13 is the optimal
129
moisture content for steeped teff grain. Kilning was carried out for all trials with a slightly modified
130
program proposed by Gebremariam et al.:15 20 hours at 30°C, 2 hours at 60°C and 3 hours at 65°C.
131
The malts were shaken manually within the nylon bags to remove rootlets. Malt loss was about 20%
The
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 37
132
for all experiments performed, that is perfectly in line with the typical values reported for barley
133
malt.25
134
2.3. Malted grain teff analysis
135
2.3.1. Quality attributes
136
Each sample was analyzed in duplicate following standard Analytica-EBC23 methods. The extract
137
yield (% dm) and filtration rate by EBC method 4.5.1, total nitrogen (TN) content (% dm) by EBC
138
method 4.3.1, soluble nitrogen (SN) content (% dm) and Kolbach index (SN/TN ratio) by EBC
139
method 4.9.1, free amino nitrogen content (FAN; mg/L) by EBC method 4.10, viscosity (mPa s at
140
20 °C and 8.6 °P (from the Plato scale)) by EBC method 4.8, fermentability (%) by EBC method
141
4.11.1, diastatic power (Windisch−Kolbach units (WK)) by EBC method 4.12, pH by EBC method
142
8.17, malt color (EBC units) by EBC method 4.7.1.
143
2.3.2. Starch-degrading enzyme activity
144
The activity of α-amylase, β-amylase and limit-dextrinase of malted teff was determined using the
145
Megazyme Assay Kits (Megazyme International Ireland)26–28.
146
2.3.3. Sugar profile
147
The concentrations of fermentable sugars (g/L) in malted grain teff congress worts were determined
148
by HPLC coupled with evaporative light scattering detector (ELSD) as previously reported by
149
Floridi et al.29
150
2.3.4. Total β-glucan and total arabinoxylan content
151
The β-glucan were analyzed using a commercial assay kit (Megazyme International Ireland),
152
following the Analytica-EBC26 methods 3.11.1 and 4.16.1., The total arabinoxylan was determined
153
by improving the methods of Bell30 and Englyst et al.31 In fact, both the extraction procedures
154
described in the reference methods, namely a treatment with sulfuric acid for Bell30 and with an
155
enzyme solution (α-amylase + pullulanase) for 16 h at 40 °C for Englyst et al.31, were made more
156
efficient by the separate use of the different enzymes at their optimal temperatures, allowing an
157
important time reduction. This procedure includes the extraction of the arabinoxylan from the 6 ACS Paragon Plus Environment
Page 7 of 37
Journal of Agricultural and Food Chemistry
158
sample and various purification steps to obtain arabinoxylan as pure as possible as follows: (A) For
159
the solubilization of arabinoxylan, acetate buffer (pH 5.2, 10 mL) were added to 500 mg of milled
160
sample. (B) The enzymatic degradation of the interferences was performed by adding α-amylase
161
(Megazyme 3000 U/mL, 200 µL) while stirring at 80°C for 15 min, by subsequent addition of
162
amyloglucosidase (Megazyme 3260 U/mL, 23 µL), stirring at 60°C for 15 min, and by subsequent
163
addition of NaOH 1M until the solution reached pH 7. Finally pancreatin (4 mg) and lichenase (20
164
µL) were added and the solution was stirred at 40°C for 1 hour. (C) Precipitation of the purified
165
pellet was performed by the addition of pure ethanol (30 mL), with the test tubes left on ice for 30
166
min, and then centrifuged (4000 rpm, 10 min) and the supernatant fraction discarded. Subsequently,
167
the pellet was washed by the addition of ethanol (85 % v/v, 10 mL) and stirring by vortexing,
168
followed by the addition of ethanol (85% v/v, 30 mL) and mixing by inversion, centrifuging at 4000
169
rpm for 10 min and discarding the supernatant fraction. Acetone (20 mL) was added, mixed by
170
inversion and centrifuged at 4000 rpm for 10 min. After discarding the supernatant fraction, the
171
uncapped test tube was left in a heated bath at 80 °C for 15 min to allow the solvent evaporation.
172
(D) Acid hydrolysis of the pellet was carried out by adding sulfuric acid (12 M, 5 mL) and heating
173
at 35°C for 1 hour, then deionized water (25 mL) was added and boiled for 30 min before the
174
sample was cooled and diluted 1:3 (0.5 mL of samples with 1.5 mL of deionized water). For the
175
arabinoxylan quantification through a spectrophotometric measure, 10 mL of a pentosans-specific
176
reagent (phloroglucinol) was added to the sample, which was boiled for 22 min, cooled in ice for 10
177
min and left at 20 °C for 5 min. Finally, the absorbance versus blank reagent was read at 510 and
178
subtracted to absorbance at 552 nm. In fact, the absorbance of interfering sugars differs very little at
179
these two wavelengths but is considerable for the pentosans. Accordingly, the pentosans may be
180
estimated, even when an excess of interfering sugars is present, by subtracting the absorbance at
181
510 nm from that at 552 nm30. Arabinoxylan concentration was calculated by mean of a calibration
182
curve. A control sample was prepared with 10 mg of standard arabinoxylan HV (Megazyme)
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 37
183
treated in the same way as the sample. For the blank solution, 2 mL of deionized water were used
184
for the reaction, with phloroglucinol used in place of the diluted sample.
185
2.3.5. Rapid Visco Analysis (RVA)
186
A RVA was carried out using a Rapid Visco Analyser (Newport Scientific Pty. Ltd.) in accordance
187
with the MEBAK method 2.7. A known amount of sample (5 g), adjusted for moisture, was slurried
188
with a known volume of water (total weight 30 g) and then processed in the RVA analyser using the
189
following standard test profile: starting temperature of 50 °C held for 1 min, raised to 95 °C over
190
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
191
the grist, stirring was performed, initially at 960 rpm, 10 s before being decreased to 160 rpm for
192
the remainder of the analysis. The RVA measurements were as follows: peak viscosity (PV) –
193
maximum viscosity developed during heating; Peak time (Pt) – time at which the peak viscosity
194
occurred; Trough (T) – lowest viscosity after cooling started; Breakdown (BD) – peak viscosity
195
minus trough; Final viscosity (FV) – viscosity at the end of the test; Setback (SB) – final viscosity
196
minus trough; Pasting temperature (PT) – temperature at which the viscosity increases by 25 mPa·s
197
within a 20 s period, as defined by the manufacturer (approx. gelatinization temperature).
207
2.4. Statistical analyses
208
The micromalting trials and each analysis were all performed in duplicate. Thus, the data were
209
reported as the mean of four values ± standard deviations. The statistical analyses were performed
210
(Statgraphics Centurion XVI version 16.1.11, StatPoint Technologies, Inc., Warrenton, VA, USA)
211
on the data collected. The significant variations between the different samples were discriminated
212
using a one-way Analysis Of Variance (ANOVA, p
