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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World Scientific News 87 (2017) 125-135

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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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( Received 08 September 2017; accepted 02 October 2017 )

physical and chemical properties of sweet juice produced ... (2004) once reported that most enzymes...

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