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of Buckwheat Malt
Blaise Patricia Nic Phiarais 1,2, Hilde Henny Wijngaard 1,2 and Elke Karin Arendt 1,3
tralia). In every trial steeping, germination and kilning of 24 h. Freeze-drying (AMSCO, Finn Aqua, Lyvovac
conditions were kept constant: steeping (12 h /10°C), ger- GT2, Germany) was carried out at <45°C and <0.01 mbar
mination (96 h /15°C) and kilning (48 h /40°C). For all on open petri dishes.
enzyme analysis, rootlets were removed.
Moisture content of malts
Analytical procedures After drying for 24 h at 50°C, EBC-method 4.2 was
Analytical procedures were carried out in duplicate (n followed 10.
= 2) and the means of all results were calculated. All con- Friability
centrations were based on dry weight, unless mentioned Friability was determined by following EBC-method
otherwise. 4.15 10.
Freeze-drying ␣-Amylase activity
Throughout the kilning period, samples were collected To measure ␣-amylase activity the ICC standard
and immediately frozen in a –80°C freezer for a minimum method 303 18 was followed using a Megazyme enzyme
kit (Megazyme, Bray, Ireland). One unit of ␣-amylase Analyzer (SCABA) (Tecator AB, Sweden) according to
activity is defined as the amount of enzyme required to EBC-method 9.2.2. Wort viscosity was measured using a
release 1 µmol of -nitrophenol from non-reducing-end falling ball viscometer at 20°C (Haake, Germany) accord-
blocked -nitrophenol maltoheptaoside (BPNPG7) in 1 ing to EBC-method 8.4. Colour was determined in accor-
min under the defined assay conditions as mentioned in dance with EBC-method 8.5. Fermentability was deter-
the assay procedure. mined as described in EBC-method 8.6.1. The mash was
filtered according to EBC-method 4.5.1, through filter
Total and soluble -amylase activity paper (Schleicher & Schnell, Germany) into graduated
-Amylase activities of unmalted and malted buck- cylinders. The time taken for each wort sample to filter
wheat were determined using the method described by the was recorded.
-amylase Megazyme enzyme kit. One unit of -amylase
activity is defined as the amount of enzyme required to RESULTS AND DISCUSSION
release 1 µmol of -nitrophenol from -nitrophenyl-␣-D-
maltopentaose (PNPG5) in 1 min. Moisture content
The effect of time on moisture content during buck-
-Glucanase activity wheat kilning is outlined in Fig. 1. The temperature dur-
-Glucanase activities of unmalted and malted buck- ing kilning was kept constant at 40°C. In the first hour,
wheat were determined using the method described in the the moisture content of buckwheat decreased from 43.9%
-glucanase Megazyme enzyme kit 18. to 43.1%. During the free drying stage of kilning 7, the
moisture content decreased from 43.1% to 19.2% in buck-
Protease activity wheat malt after 6 h of kilning. As the intermediate stage
The protease activity level was measured according to of kilning begins, the rate of drying begins to slow down
the method of Brijs et al.8. due to the physically or chemically bound nature of the
residual moisture, which restricts evaporation (Fig. 2). In
Mashing buckwheat, similar results were obtained to those of bar-
ley malt kilning 32 where the rate of drying and the mois-
Malted samples were mashed according to the EBC- ture content decreased from 19.3% to 10.5% after 12 h of
method 4.5.110. kilning. The final stage of barley malt kilning is character-
ised by the removal of firmly bound water in the grain.
Wort analysis The water content is reduced from 10% to 5%. This stage
EBC 10-methods 3.3.2 and 4.3.2 were used to determine is referred to as the bound water stage. To achieve the
the TN level in flour of unmalted and malted samples, removal of the bound water in barley malt, the ‘air-on’
respectively. EBC-method 8.9.2 was used to determine temperature is increased to 65–75°C 7. Results showed
TSN in wort. A nitrogen analyzer (LECO type FP-528; that in buckwheat malt the rate of drying slowed and the
LECO, St. Joseph, MI, USA) was employed for nitrogen moisture content decreased from 10.5% to a final mois-
determination. FAN in congress wort was measured ac- ture content of 5% after 48 h of kilning at 40°C.
cording to method of EBC 8.10. Extract (%) of resulting Wijngaard et al.39 found that barley, which has larger
wort was measured using a Servo-Chem Automatic Beer grains than buckwheat, takes up moisture more slowly
than buckwheat; this is most likely due to the covering phase of malt kilning may in part account for the increase
layers present in the barley kernel, which may limit water in total -amylase activity 11,22,30. Over the following 41 h
uptake during steeping 14, therefore it was expected that total -amylase activity decreased from 28.1 units g–1 to
buckwheat would dry faster than barley during kilning. 26.4 units g–1. One reason is that when germinated grain is
However Schuster and Grünewald 32 found that the mois- kilned, a fraction of -amylase activity is inactivated due
ture content in barley was reduced from approximately to the thermolability of the enzyme, even at these low
43% to 11% barley in the first 9 hours of kilning at 40°C. temperatures 11. In contrast, barley produces no extra -
In contrast, the moisture content in buckwheat was re- amylase during kilning, and Wijngaard et al.39 found a
duced from 43.9% to approximately 15.5% under the significant decrease in -amylase activity during malting.
same conditions. One reason for this may be due to the Table I shows the difference in total -amylase activ-
uptake of more free water because of the presence of extra ity determined between unmalted and malted buckwheat.
outer layers or husks around the barley kernel 31. This wa- This represents an additional total -amylase activity pro-
ter is not bound to endosperm components so therefore duced during germination. This confirms the findings of
water is removed more easily by the kilning process. Wijngaard et al.39. Unmalted and malted buckwheat were
found to contain a total -amylase activity level of 5.3
Total -amylase activity units g–1 and 24.7 units g–1 respectively, whereas malted
-Amylase is a heat labile enzyme 23 present in un- barley contained a total activity of 514 units g–1. The -
malted barley in a bound form (linked via disulphide amylase content of buckwheat malt is low compared to
bridges), a free form and a latent form 11. During malting that of barley malt and is better compared to sorghum
proteolytic enzymes cleave the disulphide bridges, solu- malt 1.
bilising the bound -amylase 16. In order to determine the Fig. 4 depicts the relative enzymatic activity of total
amount of total and soluble -amylase activity, cysteine and soluble -amylase during the kilning stage of malting
was used to free the bound enzyme. Fig. 3 shows results buckwheat. Table II underlines the relative enzymatic ac-
for total, soluble and insoluble -amylase levels through- tivity of -amylase at the end of germination and at the
out the kilning process. The determined total and soluble end of kilning. Results show approximately 90% more
-amylase activity levels of the unmalted and malted total -amylase activity than in green malt. In contrast,
buckwheat are highlighted in Table I. The insoluble levels approximately 40% less -amylase was present at the end
were calculated by subtracting the soluble levels from the of barley kilning 21. Therefore it can be concluded that
total levels. Temperature and duration of kilning were buckwheat -amylase is tolerant to 40°C for 48 h.
found to influence amylase activity in sorghum malts 27.
Fig. 3 shows that in the first 7 h of kilning, total -amy- Soluble and insoluble -amylase activity
lase activity level in buckwheat malt increased from 23.1 Results show, that in the first 8 h of kilning, soluble
units g–1 to 28.1 units g–1. This confirms the findings of -amylase activity level increased from 13.8 units g–1 to
Okungbowa et al.30, where it was noted that when kilning 24.6 units g–1 (Fig. 3). One reason is that the total -amy-
sorghum at lower temperatures, i.e. 40°C, the enzyme lase activity may increase as a result of enzymatic devel-
denaturing phase is avoided and increased enzyme devel- opment and proteolytic activation, which leads to an in-
opment is observed. In addition, possible proteolytic acti- crease in soluble -amylase activity. Over the period of
vation of -amylase zymogens during the enzymatic 16 h, soluble -amylase activity decreased from 23.2
units g–1 to 20.2 units g–1. This supports the findings of show over the subsequent 40 h of kilning, ␣-amylase ac-
Evans et al.11, where the soluble form is less resistant to tivity decreased from 40.3 units g–1 to 21.4 units g–1. This
heat denaturation and thus leads to a decrease in enzy- supports the finding that, after 10 h of drying, enzymatic
matic activity. Our results show over the final 24 h of kiln- activity in sorghum kilning was found to decrease due to
ing, there is an increase of activity from 20.2 units g–1 to heat denaturation 36. This is referred to as the enzyme-
24.6 units g–1 which correlates with increasing protease inactivating phase of the kilning process 25. Unlike barley
activity levels where proteolytic enzymes cleave the disul- amylases, where ␣-amylase is more thermostable than -
phide bridges, making the bound -amylase more soluble amylase 21, ␣ and -amylase in buckwheat were found to
and resulting in an increase in soluble -amylase activ- have similar thermostability. They both show an increase
ity 16. There is also some evidence to suggest that by kiln- in inactivation after 7–8 h of kilning due to heat denatura-
ing at lower moisture contents, towards the end of kiln- tion.
ing, enzymes become more resistant to heat 9. As soluble Like unmalted barley, unmalted buckwheat contains
-amylase activity increases, insoluble -amylase activity very little ␣-amylase activity. The production of ␣-amy-
decreases accordingly. lase is induced during germination 39. The ␣-amylase ac-
Table I depicts soluble -amylase activity in unmalted, tivity level of malted buckwheat is shown in Table I. The
green and malted buckwheat. Soluble -amylase activity buckwheat malt was found to have a final activity level of
is the active -amylase activity of the grain, therefore it is 19.9 units g–1 which is much lower when compared to
a more important parameter than total -amylase activ- malted barley, which was found to have a value of 105.9
ity 39. The results of this study reveal that soluble -amy- units g–1. Sorghum ␣-amylase activity was also higher
lase activity increased from 4.8 units g–1 to 21.3 units g–1. than in buckwheat 39. ␣- and -Amylase behave similarly
Although some of the bound -amylase is released during in buckwheat kilning, therefore when optimising the kiln-
germination either by a disulfide reductase or by a proteo- ing conditions, they should be considered together.
lytic enzyme(s)33, the results of this study reveal that most Fig. 4 depicts the relative enzymatic activity of ␣-amy-
of the -amylase activity is released during kilning. The lase during the kilning stage of malting buckwheat. En-
soluble -amylase activity at the end of germination was zymes are associated with high molecular weight pro-
found to be 13.8 units g–1. Therefore optimising the kiln- teins 21. As a result of heating during kilning, the structures
ing conditions seems to enhance soluble -amylase activi- of proteins are changed to some extent and they become
ties in malted buckwheat. denatured. Table II highlights the relative enzymatic activ-
ity of ␣-amylase at the end of germination and at the end
␣-Amylase activity
Fig. 5 outlines the changes in ␣-amylase activity dur-
Table II. Relative enzymatic activities of buckwheat malt kilned at 40°C
ing the kilning stage of buckwheat. Results show that ␣- for 48 h.
amylase activity increased from 35.6 units g–1 at the end
Buckwheat Buckwheat malt
of germination to 40.3 units g–1 after the first 8 h of kiln- Parameter green malt kilned at 40°C
ing, confirming the findings of Uriyo and Eigel 36, where
Alpha-amylase activity 100 60.2
results indicated sorghum ␣-amylase was stable during Beta-amylase (total) activity 100 191.1
drying times of 5–10 h at low temperatures. The increase Beta-amylase (soluble) activity 100 178.5
in enzymatic activity could be attributed to continued ger- Protease activity 100 119.5
mination during drying at low temperatures 5. Our results Beta-glucanase activity 100 33.9
Fig. 6. Means and standard deviations of -glucanase activity (U kg–1) in buckwheat against kilning time (h).
of kilning. Results show that at the end of kilning there is Georg-Kraemer et al.15, where results indicate barley -
approximately 40% less ␣-amylase activity than in the glucanase is highly susceptible to thermal inactivation
green malt. In contrast, approximately 15% more ␣-amy- depending on the grain variety. Over the final 43 h of kiln-
lase activity was present at the end of barley kilning 19. ing, buckwheat -glucanase activity level decreased from
Our results show that buckwheat ␣-amylase is more heat- 53 units kg–1 to 19.2 units kg–1. This compares to results
labile than barley ␣-amylase. found by Uriyo and Eigel 36, where it was noted that -
glucanase activity in sorghum decreased after 5 h of dry-
-Glucanase activity ing, possibly due to the thermolability of the enzyme 4.
There are three -glucanases, endo--1,3:1,4 gluca- The determined -glucanase levels of the unmalted,
nases, (which hydrolyze -1-4 links adjacent to -1-3 green and malted buckwheat are shown in Table I. Al-
links), endo--1-3 glucanase and exo--1-4 glucanase. though -glucanase activity (with an activity level of 56.7
These enzymes together hydrolyze -glucans to mainly units kg–1 present at the end of germination) decreased
cellobiose or laminarobiose 23. Fig. 6 outlines the changes during drying, part of the initial activity present in the
in -glucanase activity during the kilning stage of buck- green malt (mean 17.9 units kg–1 ) was retained in the fin-
wheat. The results in this study show a decrease in -glu- ished malt. This activity level is much lower when com-
canase activity from 56.7 units kg–1 to 48.8 units kg–1 over pared to malted barley, which was found to have a value
the first 4 h of kilning. This confirms the findings of of 537.3 units kg–1. A possible explanation for this may be
due to the observation that buckwheat contains no high Lewis and Young 23, exo-peptidases tolerate the heat of
molecular weight -glucan 39, therefore buckwheat has kilning because they are heat stable and persist in the en-
little use for the enzyme -glucanase. However the small dosperm after kilning.
amount of -glucanase present may also be used to hydro- Results indicate, that over the following 21 h (7–28 h)
lyze hemicellulose that may be present in buckwheat. The of kilning, a decrease in a proteolytic activity level of 6.5
method used to determine -glucanase activity does not mg of leucine h–1 g–1 to 5.2 mg of leucine h–1 g–1 was ob-
distinguish between -glucanase and cellulase activity. served, which may again be due to the inactivation of the
Analysis of the relative enzymatic activity of -gluca- heat labile endo-peptidases 23. Over the following 2 h (28–
nase activity at the end of germination and again at the 30 h) an increase in the proteolytic activity level of 5.2 mg
end of kilning clearly demonstrated that there is approxi- of leucine h–1 g–1 to 6.4 mg of leucine h–1 g–1 was observed.
mately 65% less -glucanase activity present in the final This increase may be due to the further release of amylo-
buckwheat malt than in the green malt (Table II). This lytic and proteolytic enzymes when kilning at such a low
correlates well with data on barley malt 21. temperature 36. Over the final 18 h (30–48 h), a decrease in
proteolytic activity of 6.4 mg of leucine h–1 g–1 to 5.8 mg
Protease activity of leucine h–1 g–1 was observed. This correlates to the find-
A variety of endo- and exo-proteases have been identi- ings of Dickson and Shands 9, where results indicate little
fied in barley green malt 19,29. In this study, the protease or no further decrease was observed towards the end of
activity level was measured with haemoglobin as a sub- barley malt kilning.
strate, which gives an indication of the total proteolytic Unmalted buckwheat contained a protease activity level
activity level of the grains. Fig. 7 highlights the changes of 3.7 mg of leucine h–1 g–1 (Table I), this compares fa-
in protease activity during the kilning of buckwheat. Re- vourably to the results found by Wijngaard et al.39. Pro-
sults show a decrease from a proteolytic activity level of tease activity was found to increase when buckwheat was
4.8 mg of leucine h–1 g–1 to 4.5 mg of leucine h–1 g–1 over malted. Kilned buckwheat was found to contain a proteo-
the first 3 h of kilning. This correlates with the findings of lytic activity level of 5.5 mg of leucine h–1 g–1. Most of the
Dickson and Shands 9, where a reduction in proteolytic protease was synthesised during germination 39 while the
activity in barley malt was observed in the first few hours remainder of protease was activated during kilning. The
of drying at 45°C. This reduction may be due to protease buckwheat green malt was found to have a proteolytic ac-
(endo-peptidase) enzymes being inactivated during kiln- tivity level of 4.8 mg of leucine h–1 g–1. Barley malt con-
ing, since malt endo-peptidases are relatively heat labile tains an activity level of 9.3 mg of leucine h–1 g–1 which is
and easily inactivated during kilning 23. An increase from a almost twice the level found in buckwheat malt. Approxi-
proteolytic activity level of 4.5 mg of leucine h–1 g–1 to 6.5 mately 19% more protease was found in final malt than in
mg of leucine h–1 g–1 over the following 4 h (3–7 h) of green malt (Table II). This correlates well with similar
buckwheat kilning was observed. This increase can be results for barley malt 21. Buckwheat is a friable grain be-
explained by the beginning of proteolysis initiated by a cause its endosperm only contains 35% protein, whereas
slight temperature rise within the grain bed. According to 80–90% of the protein is embedded in the endosperm of
Taylor and Boyd 35, proteolysis occurs optimally at 43°C the barley endosperm. Therefore protease activity is prob-
to 50°C in sorghum malts. Alternatively, the increase may ably not as important in buckwheat malt as it is in barley
be due to the presence of exo-peptidases. According to malt.
ERRATUM
A correction was made in this paper on November 30, 2005. In the
last paragraph on page 294, the first sentence referred incorrectly to
Fig. 5. The text was changed to refer to Fig. 4 (“Fig. 4 depicts the
relative enzymatic activity of ␣-amylase during the kilning stage of
malting buckwheat”).