physical and chemical properties of sweet juice produced ... (2004) once reported that most enzymes...
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WSN 87 (2017) 125-135 EISSN 2392-2192
Physical and chemical properties of sweet juice produced from hydrolysed acha (Digitaria exilis
Stapf) starch using crude amylase from germinated maize
Abdulquadri O. Alaka*, Rahman Akinoso
Department of Food Technology, University of Ibadan, Ibadan, Oyo State, Nigeria
*E-mail address: [email protected]
ABSTRACT
This study investigated the possibility of producing juice by hydrolyzing acha starch using
crude amylase from germinated maize. Planted maize was monitored for seven days to determine
germinative capacity and amylase activity of the seed. Extracted acha starch was hydrolysed using wet
homogenised maize seedlings in an incubator at 62-68 °C for 6-8 hours. Colour, pH, titratable acidity
(TTA), °Brix and sensory attribute of the juice were determined using standard method. A commercial
malt beverage was used as reference. The maize sample had a mean capacity of 95% while its
maximum amylase activity occurred at day 4. The pH, TTA, and °Brix of the juice were obtained as
4.5, 0.85 and 10 respectively. Colour values for L*, a*, and b* were 37.64, 7.6, and 15.3 respectively.
Sensory analysis showed no significant difference between the juice and reference sample in terms of
taste and colour. However, significant differences were observed in aroma and overall acceptability.
Hence, malt beverages can be produced using this method but requires certain additives to impart
prevalent aroma associated with malt beverages
Keywords: Maize amylase, acha, starch, germinative capacity, juice
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1. INTRODUCTION
Starch as a polysaccharide characterised with a bland taste can often be split quite easily
into its constituent monosaccharides, rich in sweetness. This process of splitting is known as
hydrolysis and can be achieved either through the use of acid or enzymatic actions. Enzymes
are complex protein molecules synthesised by living cells, acting as biocatalysts for several
reactions in living organisms. They are specific in actions as far as their substrates are
concerned. The notable enzymes employed in the starch processing industries for the
hydrolysis of starch into simple sugar constituents are enzyme amylases (Akpan et al. 1999:
77-79). Chaplin (2004) once reported that most enzymes used in the commercial production
of sweeteners come from microbes. For instance, commercial enzymes used for the industrial
hydrolysis of starch are produced by Bacillus amyloliquefaciens and by B. licheniformis.
However, the most significant limitations involve difficulties in isolating, purifying the
enzymes and the cost factor. Opportunely, the growth of maize or other cereals during
germination are regulated by certain amylases.
The use of these amylases (from germinating maize seedlings) regarded as crude
enzymes is cheap and feasible in starch hydrolysis. Production of maltose syrup from cassava
starch using amylase enzymes from rice seedlings in Indonesia has been reported (Ameko et
al. 2015).
Photo 1. Digitaria exilis Stapf
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Similarly, the use of maize seedlings as source of amylase in the processing of cassava
starch into maltose is a tradition in Vietnam (Quynh and Cecil 1996).
Acha is a cereal grain indigenous to West African countries. Usually, it is grown in
semi-arid regions which could not sustain the growth of most cereals. The 1000-kernel weight
of acha determined by Jideani and Akingbala (1993: 369-371) showed that acha is one of the
smallest cereal grains that exist. Hence, it is regarded as “a small seed with big promise”
capable of providing food early in the season when other crops are still immature for harvest.
Acha is a very nutritious food material, consumed whole, milled into flour and locally
processed into a variety of preparations such as gruels, porridges and beverages in some
African regions (Jideani and Akingbala 1993: 369-371). In recent times, acha (Digitaria exilis
Stapf) (Photo 1) and iburu (D. iburua Stapf) have gained attention in research and
development.
Studies on the functional and physicochemical properties of native and chemically
modified acha starch are not uncommon (Olu-Owolabi et al. 2014: 222-230). Yet, literature is
sparse on the production of sweet juice (i.e hydrolysate) from acha starch using crude maize
amylaze. Hence, this study investigated the possibility of producing juice by hydrolysing acha
starch using crude amylase from germinated maize [25-40].
2. RESULT / EXPERIMENTAL
Materials
Acha grain was sourced from a local market in Ilorin, Kwara State, Nigeria. Two
different samples of maize (BR9928, and DZE-MPR) were procured from the seed bank,
Institute of Agricultural Research and Training (I. A. R & T) Ibadan, Nigeria.
Starch Isolation from acha (Digitaria exilis) grains
Extraction of acha starch was done according to the method of Musa et al. (2008: 56-
66) but with very slight modification. A different blender (HR 1721 Philips Comfort, Japan)
was used. This same blender, was used to grind the dried starch lumps into fine powder. The
starch obtained was stored in a cool dry place prior to usage.
Germination of maize seedlings
High quality maize sample was germinated, in accordance with FAO corporate
document repository (n.d.) with slight modifications. Maize seeds were first soaked in a
plastic bowl for 24 hours. The seeds, still in the plastic bowl, were kept in a cool place and
wetted at least twice a day until the shoots were around 2 – 3 mm long. At this stage, the
germinated seeds were set out under shade on a plastic tray. The seedlings were watered twice
a day. After a week, the seedlings were uncovered, turned over and thoroughly washed before
use. The required quantity was pulped with a blender and used directly.
Germinative capacity test for maize
Twenty seeds per sample in duplicates were placed on moistened cotton wool in Petri
dishes (20 seeds per dish, d = 10 mm), placed in a dark environment at 22 °C for 7 days. The
percentages of germinated and un-germinated kernels were calculated. Only maize sample
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with germinative capacities > 90 % was used. This method of determination is related to that
of Hudec and Muchova (2008: 138-146).
Enzymatic hydrolysis of acha starch into juice
This was done according to the method of Quynh and Cecil (1996) and Ramachandran
et al. (2014: 1216-1219).
Determination of maximum amylase activity in maize crude enzyme extract
This was done according to the method of Ameko et al. (2013: 52-60) with minor
alterations. A 100 ml portion of 1.0 % (w/v) gelatinised acha starch solution was put in a 200
mL conical flask and to it was added 5 g of maize crude enzyme extract. The mixture was
stirred thoroughly and incubated at room temperature for 24hours, and then filtered. The time
taken by the filtrate to decolourise standard solution of acidified potassium permanganate
solution was determined. This decolorisation test took place for 7 days.
Yield of sweet juice produced
The yield of produced juice was determined using a graduated measuring cylinder. The
percent yield of sweet juice was then calculated using equation 1.
............. (1)
pH and °Brix
The pH of produced juice was determined using a pH meter (Jenway 3330, England)
and °Brix was measured using a hand-held refractometer (HANNA RB 32, Germany).
Total Titratable Acidity
The titratable acidity was determined titrimetrically.
Colour
Colour of the juice sample was determined using a Konica Minolta Chromameter (CR-
410, Japan). The colour values were expressed as L* (whiteness/darkness), a* (redness/
greenness) and b* (yellowness/blueness). The variation of colours were characterised by the
total colour difference (ΔE), which was calculated from the Hunter L*, a*, b* values using
equation 2 (Chutintrasri and Noomhorm 2007: 300-306).
Δ𝐸 = √ (Δ𝐿2 + Δ𝑎2
+ Δ𝑏2) …………… (2)
Sensory evaluation
A sensory evaluation in terms of taste, aroma, appearance and overall acceptability of
the product was done using 20 panellists. The sample were rated using five-point scale
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ranging from “5-like extremely” to “1-dislike extremely”. A commercial drink was used as
reference sample.
Data Analysis
All data were analysed using Microsoft Excel and SPSS17. Paired sample test and
correlation were done at 5% level of significance.
Starch yield
Starch yield was 60% and contained moisture content (3.50%), carbohydrate (86.54%),
crude protein (2.10%), crude fat (3.75%), crude fibre (3.41%) and ash (0.70%). The yield
(60%) was greater than 50.60% reported by Olu-Owolabi et al. (2014: 222-230). Conversely,
this yield was lesser when compared with the report of 68% percentage yield of acha starch
obtained by Cruz et al. (2011: 175). In general, the variation in starch content may be
attributed to the variety, maturity stage, different climatic and agronomic conditions (Rahman
et al. 1999: 91-97).
Germinative capacity test for maize samples
As presented in Figure 1, the mean germinative capacities for the two varieties BR9928
and DMR-ESR were 85% and 95% respectively. Consequently, only the DMR-ESR was used
as the cereal enzyme source.
Figure 1. Mean germinative capacities of the two maize varieties.
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Stage of maximum amylase activity
Figure 2. Amylase activity in germinating maize seedlings measured in terms of
decolorisation per day.
This was because a germinative capacity of at least 90.0% and preferably 95.0% and
above was required for the cereal grains to be accepted for use as an enzyme source for
hydrolyzing starch to maltose (Quynh and Cecil 1996).
The level of amylase enzyme activity was measured in terms of the concentration of
glucose in hydrolyzed solution (SAPS 2013). Thus, the time taken by the glucose to
decolourise acidified KMnO4 solution is directly proportional to the activity of the amylase
used to hydrolyse the starch. In this study, the maximum amylase activity was observed in the
germinating maize seedlings on day 4, as shown in Figure 2, during a 7- day germination
period.
This result was comparable with the reports of Ameko et al. (2013: 52-60) in which the
maximum amylase activity in Obaatanpa maize was observed on day 4 – 5 of 11 days
germination period. On the other hand, this result does not totally agree with the findings of
Subbarao et al. (1998: 657-666) in which, the time course of amylase activity in the
endosperm of maize seedlings was studied.
Physico-chemical properties of produced juice
The enzymatic hydrolysis of starch basically covers three steps; gelatinization, during
which the cooked starch granules transforms into a viscous suspension; liquefaction, which
involves partial hydrolysis and loss in viscosity; and saccharification, involving the
production of glucose and maltose through further hydrolysis (Souza and Magalhaes 2010:
850-861). During liquefaction, alpha amylases convert the gelatinised starch into
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maltodextrins, mainly composed of oligosaccharides and dextrins (Maps Enzymes 2010).
Through further hydrolysis, the maltodextrins are converted into maltose and glucose units by
beta amylases (Ameko et al. 2013: 52-60) & [25-40].
The juice physico-chemical properties are presented in Table 1. Its pH value of 4.5 is
higher than that of maltose produced from hydrolysis of cassava starch using Proso millet
(Ameko et al. 2015). In contrast, this value is lower than the values of 5.5 - 6.5 reported by
Dziedzoave et al. (2004). However, it is very close to range of 4.6-5.30 when compared with
the report of Ameko et al., (2013: 52-60).
Also, the total titratable acidity value of 0.85% of the produced juice was comparable to
the range of 0.45-1.60% obtained by Wireko-Manu et al., (2010) in the hydrolysis of sweet
potato into non-alcoholic beverage using maize malt. Degree brix value of 10 obtained was
comparable to the range of 12-13 and 3.2-12.4 given in the reports of Wireko-Manu et al.
(2010: 180-183) and Akonor et al. (2014: 20-26).
Table 1. Physicochemical properties of produced juice
Colour
Colour is an essential quality attribute that affects consumers’ view and preference of a
food. In the measurement of food sample colour, the L, a, and b colour space scale is a more
useful visual uniform scale. The L*, a*, b*, ΔC, ΔE and hue angle parameters as obtained for
the produced juice are shown in Table 2. The definition includes L*(whiteness/darkness),
a*(redness/greenness), b*(yellowness/blueness), ΔE (mean colour difference), ΔC (colour
intensity).
The standard values for L*, a*, and b* were 93.6, -0.82, and 3.68 respectively. The
measured L* value of 37.64 ±0.45 tends towards darkness indicating the dark golden brown
colour of the produced juice. In addition, the brown colour of the samples also had hues of red
Parameter Indicator
pH 4.5
Degree Brix (°) 10
Taste Strong malty
Colour Golden brown
Reducing sugar Test Positive
TTA (%) 0.85
Volume (ml) 117
Percent Yield 211.8
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and yellow, which are exhibited in the positive a* and b* values respectively. Likewise, the
lesser positive a* values when compared with positive b* values indicate a drift towards a
yellow hue in the juice sample. This may be due to the intrinsic yellow colour of the maize
variety (DMR-ESR) used.
The values of ΔC, ΔE and hue angle were calculated from L*, a*, and b* values as the
formulae suggests. However, it is noteworthy that the ΔE value obtained for the produced
juice is comparable to the range of ΔE values reported by Akonor et al. (2014: 20-26) which
were between 49.69 ± 0.25 and 55.08 ± 0.05. By and large, colour formation in malt occurs
during polymerization (Food-Info.net 2012).
Sensory attributes
In terms of taste and colour, there was no significant difference between the juice and
the reference sample (t = -2.032, df = 19, p = 0.056 > 0.05) and (t = -0.203, df = 19, p = 0.84
> 0.05). As for the aroma and overall acceptability, there was a significant difference between
the juice and the reference sample (t = -3.771, df = 19, p = 0.001 < 0.05) and (t =-3.036, df =
19, p = 0.007 < 0.05) respectively.
The significant differences observed on aroma and overall acceptability may be due to
the presence of other ingredients in the reference sample which was a malt beverage. On the
other hand, the insignificant difference between the two samples, in terms of taste and colour,
may be attributed to the overall effect of maltose as the major sugar molecule presents in the
two samples.
In addition, a fairly strong negative correlation was observed in the taste of the juice and
the control sample. On the other hand, a very weak negative correlation was noticed in the
colour, aroma and overall acceptability of the juice and the reference sample.
3. CONCLUSIONS
This study reinforced the postulate that “any starch source can be enzymatically
hydrolysed using plant crude enzymes” (Quynh and Cecil 1996). Additionally, as rated by
potential consumers, use of crude maize amylase in the hydrolysis of acha starch produced a
juice with qualities comparable with existing malt beverages or drinks. Finally, economic
value can be added to underutilised acha grains through processing into malted sweet juice.
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