 *Corresponding author.

Email: nganp1115002@gstudent.ctu.edu.vn , nvmuoi@ctu.edu.vn

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 1082 - 1088 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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The study of angiotensin converting enzyme isolation from crossbred rabbit

lung and enzyme storage capacity in frozen conditions

1*Nguyen, V.M., 1Tran, T.T. and 2*Vo, H.N.

1College of Agriculture, Can Tho University, Campus 2, 3/2 street, Ninh Kieu District, Can Tho City,

Vietnam.
2Department of Science and Technology of Vinh Long province, 111 Nguyen Hue street, Ward 2, Vinh

Long City, Vinh Long Province, Vietnam.

Article history:

Received: 2 January 2020

Received in revised form: 29

February 2020

Accepted: 3 March 2020

Available Online: 26 March

2020

Keywords:

Ammonium sulfate,

Angiotensin converting

enzyme (ACE),

Activity,

Crossbred rabbit lung,

Extract,

Frozen

DOI:
https://doi.org/10.26656/fr.2017.4(4).011

Abstract

The study was carried out to obtain angiotensin converting enzyme (ACE) with high

specific activity and to evaluate the ability to maintain enzyme activity in extract products

as well as in rabbit lungs using frozen condition. In the scope of the content, the study

conducted an evaluation to select the appropriate extraction solvent of four solvents

including acetone, ethanol, Tris-HCl and distilled water. Initially, the research results have

helped determine distilled water as the suitable extraction solvent and the difference is not

statistically significant compared to Tris-HCl solvent. Angiotensin converting enzyme

extract that was obtained by distilled water solvent has a specific activity of 10.35 U/g

protein. In addition, the study investigated the ability to maintain angiotensin converting

enzyme activity in rabbit lung and crude enzyme product during frozen storage (-18±2°C).

The results of the study showed that angiotensin converting enzyme activity could be

maintained for 3 months in rabbit lungs and 4 months in the crude product. Besides, the

study also used ammonium sulfate with different concentrations to conduct angiotensin

converting enzyme collection from extract product. The results of this content help

determine the use of saturated ammonium sulfate with concentrations of 50% to 60% for

the highest efficiency. The precipitation process helped obtain ACE products with a purity

of 4.22 times, the specific activity of 42.64 U/g protein and the recovery rate of ACE up to

29.37%.

1. Introduction

Enzymes play an important role in scientific research

as well as in medicine and food processing. The

technology in enzyme production has brought significant

profits to many countries, production and sales of

enzymes in the world market increased by 20-30% per

year (Murado et al., 2009). In recent years, the

technology of producing enzymes such as protease,

lipase and cellulase has been studied widely, to the point

of being easily extracted. Nowadays, angiotensin

converting enzyme (ACE, peptidyl dipeptide hydrolase,

EC 3.4.15.1) is increasingly interested in studies of

hypertension. This is an enzyme that plays an important

role in regulating blood pressure through the Renin

Angiotensin Aldosterone system (Muñoz-Durango et al.,

2016). In Vietnam, the use of this enzyme for research is

very limited due to the high cost and the difficulty of

finding on the market. Studies of ACE extract from the

lungs of mammals have also been conducted in various

studies, and in many countries around the world (Shafiee

et al., 2005; Abdulazeez and Kurfi, 2016). In 1971,

Cushman and Cheung showed that the lung tissue of

mammals (especially rabbit lungs) contained ACE with

high activity. On the other hand, rabbit lung is a

byproduct in rabbit processing with low economic value.

All of this shows the potential to use rabbit lungs as a

cheap ACE supply for research. Therefore, the study was

conducted to obtain ACE with high specific activity and

to evaluate the ability to maintain enzyme activity in

extract products as well as in rabbit lungs using frozen

condition.

2. Materials and methods

The research was conducted at the Laboratory of

Food Technology, Faculty of Agriculture, Can Tho

University. Major equipment and chemicals used in the

study include Thermo Multiskan Spectrum Reader

(USA), Cold centrifuge (HermLe Labortechnik GmbH

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Siemensstr., Type: Z232K, Germany), Micropipet (EMC

Lab), N-[3-(2-Furyl)acryloyl]-Phe-Gly-Gly (Sigma-

Aldrich), Tris-HCl (Merck), Ammonium sulfate (China).

Rabbit lungs are selected from healthy male

crossbred rabbits (New Zealand x local) were purchased

directly from the farming area in Vinh Long province,

Vietnam, with a weight range 3.5 to 4 kg. The lungs

were collected immediately after slaughter and kept cold

afterwards. After collection, lungs were washing,

separating bronchial and fat and keeping in polyamide

(PA) packaging at frozen condition (-18±2°C) before

further processing.

2.1 Research procedures

Frozen rabbit lungs were finely cut about 3 mins in

cold condition before extraction. In the study, the

investigation of ACE extraction by 4 solvents was

conducted (Experiment 1) using the ratio of lung and

solvent is 1:2 (Abdulazeez and Kurfi, 2016; Gupta et al.,

2018). All solvents were cooled to 4±2°C before

conducting the study. Acetone and ethanol were added

slowly at grinding time. If the extraction solvent was

water or Tris-HCl, the supernatant after centrifugation

(10,000 x g, 30 mins, 4°C) would be recovered, filtered

and adjusted to a fixed volume to obtain crude ACE. On

the contrary, the residue would be the main component

of the extraction process if the extraction solvent was

acetone or ethanol. The centrifugal residue after solvent

removal was dissolved in Tris-HCl and centrifuged again

to extract raw ACE. Analyze the activity indicators of

crude ACE to determine the appropriate extraction

solvent. To assess the possibility of maintaining ACE

activity in freezing conditions, frozen storage

experiments were also carried out for rabbit lungs

contained in Falcon 15 mL and crude ACE contained in

Falcon 15 mL (Experiment 2). Storage temperature was

fixed at -18±2°C and sample analysis was expected after

each month of storage. In the other hand, the study also

used cold ammonium sulfate with different

concentrations (from 30% to 70% saturated

concentration) to collection ACE (Mansurah et al., 2013)

(Experiment 3). The addition of ammonium sulfate was

carried out by magnetic stirring and the mixture was kept

cold by ice. After dissolving the salt completely, leave

the mixture in the refrigerator for 1 hr before

centrifuging to obtain a residue. The centrifugal residue

would be dialyzed by cellophane membrane in the

solvent which is a result of Experiment 1 to remove

ammonium sulfate out of ACE. The activity indicators of

the product would be analyzed as a basis for selecting the

appropriate salt concentration. The result of Experiment

1 was chosen as a fixed factor for Experiment 2 and

Experiment 3.

Experiment 1. Investigated the ACE extraction

ability of different solvents in four solvents of distilled

water, 50 mM Tris-HCl pH 8.3 containing 30 mM NaCl,

acetone and ethanol.

Experiment 2. Evaluated the ability to maintain

ACE activity in rabbit lungs and crude enzyme product

in frozen storage (-18±2°C).

Experiment 3. Investigated the efficiency to collect

ACE of ammonium sulfate with different concentrations

(from 30% to 70% saturated concentration).

2.2 Proximate analysis

The proximate analysis was determined according to

AOAC methods (AOAC, 1995). Moisture content was

determined by AOAC 925.04, protein content was

determined by the Kjeldahl method (AOAC 981.10) and

lipid content was determined by Soxhlet method (AOAC

920.39). ACE activity (U/L) was analyzed based on the

method of Restrepo et al. (2013). Specific activity was

determined based on the amount of protein (Ngo et al.,

2008). Molecular weight was analyzed by SDS - PAGE.

2.3 Data analysis

Using Statgraphics Centurion XVII.II program, data

were analyzed for the degree of variation and

significance of difference based on the analysis of

variance (ANOVA) to determine if significant

differences (p≤0.05) existed between treatments using

Least Significant Difference (LSD) or Duncan test. All

experiments were carried out in triplicate.

3. Results and discussion

3.1 Physical and chemical properties of rabbit lung

Healthy male crossbred rabbits in Vinh Long

province were used to get lungs as raw material for all

experiments. The angiotensin converting enzyme is a

protein component of rabbit lungs, so ACE extraction

from rabbit lungs is influenced by the physicochemical

properties of the lungs. At the same time, the basic

physicochemical properties of rabbit lung is needed to

determine the uniformity of the source of raw materials

for research. The results of basic physicochemical

composition analysis of rabbit lung showed in Table 1.

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Proximate parameters Value

Moisture (%) 81.89±1.24

Protein (%) 13.45±0.28

Lipid (%) 2.74±0.09

pH 6.54±0.03

Table 1. Chemical and physical properties of crossbred rabbit

lung

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The determination of the physical and chemical

components of the material is necessary because the

study of Kutrovic (2011) has shown that the enzyme

activity in the final product depends greatly on the

composition of the material. In particular, the presence of

protein, fat, pH and fatty acid components have a great

influence on enzyme collection efficiency. In addition,

the extraction process is also governed by moisture

content. The origin of enzymes is proteins; the results in

Table 1 showed that rabbit lungs were a source of protein

with relatively high protein content (13.45%). Many

studies have demonstrated a high activity of ACE

receptor from rabbit lungs, so the research resource

(crossbred rabbit lung) had a high potential for ACE

recovery (Cushman and Cheung, 1971; Shafiee et al.,

2005; Mansurah et al., 2013). Lipid content in the rabbit

lung was negligible (2.74%), which would be removed

through low-temperature centrifugation and filtration.

Moisture content and pH of 81.89% and pH6.54

respectively would be an important basis to calculate the

extraction, storage and purification process of ACE.

High protein and moisture content will have negative

impacts on the stability of ACE in lung rabbits,

especially in inappropriate conditions. Therefore, it is

necessary to keep the rabbit lung in low temperature

conditions. In addition, with a standard deviation of less

than 2% at all analytical criteria of the raw material, it

has been shown that homogeneity of research materials

has been ensured, Figure 1 also helped confirm this.

3.2 ACE extraction capability of different solvents

Enzymes are biological catalysts obtained mainly by

extraction from animals, plants or microorganisms. In

the study, crossbred rabbit lungs were used to perform

extraction of ACE with four solvents including distilled

water, Tris-HCl, acetone and ethanol. The results of

ACE extraction were shown in Figure 2 and Table 2.

The results in Figure 2 showed that the use of Tris-

HCl or distilled water to extract ACE from rabbit lungs

would help to obtain a high protein product. The extract

products of rabbit lungs by Tris-HCl and distilled water

had a protein content of 4.14% and 3.85%, respectively.

Meanwhile, the extract products after making

precipitation by acetone or ethanol had lower protein

content. This may be explained by the fact that

precipitation by organic solvents reduced the solubility

of the protein in the rabbit lungs so that the protein

content of the extract was low. However, in order to

determine the extraction efficiency of solvents, it was

also necessary to consider the specific activity of the

extract.

The analytical results in Figure 2 and Table 2

showed that the ACE activity and protein content of

extracted products by four solvents (acetone, ethanol,

distilled water and Tris-HCl) had the same variation

rules. High ACE activity obtained when the protein

content of extract products was high. Table 2 showed the

highest ACE activity of the product that used Tris-HCl

or distilled water to extract. The difference in enzyme

activity extracted by Tris-HCl or distilled water was not

significant, respectively 408.83 (U/L) and 398.57 (U/L).

The results were also consistent with the research of

Mansurah et al. (2013) and Abdulazeez and Kurfi

(2016), using sodium and Tris-HCl buffer solvent to

extract ACE from rat lung and rabbit lung.

From the above results, it can be confirmed, distilled

water and Tris-HCl gave much higher ACE extraction

efficiency when compared with extraction with acetone

and ethanol solvent. In addition, the difference in

extraction efficiency of Tris-HCl and distilled water was

not statistically significant, so distilled water could be

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Figure 1. Raw crossbred rabbit lungs

Figure 2. Protein content in products extracted by different

solvents. Different letters in the column indicate significant

differences in the test treatments at 95% confidence intervals.

Solvent Activity (U/L) Specific activity (U/g protein)

Acetone 53.53±9.36b 1.67±0.27b

Ethanol 12.47±10.22a 0.38±0.31a

Distilled water 398.57±14.65c 10.35±0.28c

Tris-HCl 408.83±17.05c 9.87±0.35c

Table 2. Specific activity and activity of the product extracted

by different solvents

Different letters in the column indicate significant differences

in the test treatments at 95% confidence intervals.

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selected as a more efficient extraction of ACE by

reducing cost and limiting the use of chemical

ingredients in extract.

The extraction of ACE from crossbred rabbit lung

with distilled water (Figure 3) obtained high activity and

specific activity, respectively, 398.57 U/L and 10.35 U/g

protein. However, research by Mansurah et al. (2013)

gave higher extraction efficiencies compared to this

research (specific activity of extract products reached 49

U/g protein). This difference may be due to the source of

rabbit lung used in the study. The obtained ACE was still

in its raw form (crude ACE) with low activity and there

were still some other protein components (Sallau et al.,

2008), so the use of ammonium sulfate to carry out the

process partial purification should be conducted to obtain

enzymes with higher purity.

3.3 The effect of frozen storage on changes in ACE

activity in rabbit lungs and crude ACE

Frozen storage is a solution to inhibit biological

processes or the growth of microorganisms in products,

which have been used in maintaining a stable source of

materials for this research. In addition, the freezing

process is a pre-treatment method that makes grinding

and extracting easy. However, a problem is the

degradation of enzyme activity due to the formation of

ice crystals in frozen products. The results of the ability

to maintain ACE activity in the rabbit lungs and the raw

ACE during frozen storage are shown in Figure 4.

Survey results showed that ACE activity of control

samples for rabbit lungs and crude ACE were almost the

same. The formation of ice crystals during freezing was a

factor that had an adverse effect on protein structure as

well as enzyme activity. However, Figure 4 shows that

this effect was negligible and ACE activity was reduced

after 24 hours of freezing but this difference is not

significant. In addition, ACE stability in rabbit lungs

could also be maintained up to 3 months of storage and

up to 5 months for extract products. The decrease in

enzyme activity during frozen storage was explained by

the effect of temperature fluctuations affecting the tissue

structure (Asgeirsson et al., 1995). Figure 4 also shows

that the structural components inside the rabbit lungs

may have changed drastically after slaughter and storage.

This transformation had an effect on ACE, which had

rapidly lost ACE activity that existed within the lung

structure.

From the above results, it is not appropriate to

storage ACE by freezing rabbit lungs, at -18±2°C only

can maintain ACE activity for 3 months of storage. This

change was also reflected in the appearance of the lung

(Figure 5), the discoloration has begun to occur in the

5th month of storage and become worse after 6 months

of storage. Although it is only an extract product, crude

ACE showed a better ability to maintain activity than the

lung (Figure 4). After 6 months of frozen storage, the

activity of crude ACE did not change much when

compared with storage rabbit lungs. In the first 5 months,

the difference in ACE activity was not statistically

significant.

Achieving activity stability as a result of the

experiment may be due to the fact that extraction had

eliminated some components that interact with ACE in

the product. Elimination of other components will help

ACE maintain better activity during frozen storage

(Sallau et al., 2008).

In short, rabbit lungs can only be stored until the 3rd

month while crude ACE can extend storage until the 5th

month that the activity was maintained. The ACE

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Figure 3. Crude ACE

Figure 4. Changes in ACE activity during frozen storage of

rabbit lungs and crude ACE

Figure 5. The appearance changes of rabbit lungs after frozen

storage

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activity decline of the extract in the 6th month of storage

was still not much.

3.4 The efficiency of ACE collection by ammonium

sulfate

In crude enzyme extracts, the presence of proteins

that were not enzymes and other components of the cell

has made the enzyme's specific activity low. To remove

these unwanted components, we often use protein

precipitation methods based on their solubility. In

precipitation agents, ammonium sulfate has been widely

used in enzymatic partial purification studies in

laboratories and also in industrial scale production due to

the effective precipitation and maintenance of enzyme

activity (Scopes, 1994), and the solubility is large as well

as the effect of temperature on its solubility is not

available (Dixon and Webb, 1964). In the scope of the

study, ACE precipitation was performed by the different

saturated concentration of ammonium sulfate to find the

appropriate concentration in ACE collection, the survey

results were shown in Table 3.

At the different concentration of salt

supplementation, the protein content of the product was

different. The addition of salt to 30% would help to

achieve the highest protein yield, this value would be

reduced to the lowest at 60-70% salt concentration.

However, the enzyme activity did not depend on protein

yield, making specific activity also varies (Table 3). This

made it possible to find a saturated salt concentration in

which the appropriate protein component precipitates.

The results of Table 3 showed that the precipitation

by ammonium sulfate helped to collect higher purity

enzyme, which was shown in increasing specific activity

at different salt concentrations, this was the initial step

for the purification process at a higher level. Although

the protein content was high when precipitation was at

30% saturation of ammonium sulfate, the specific

activity of the product was low. In contrast, precipitation

in the 50 to 60% ammonium sulfate fraction received

low protein content but had a very high specific activity,

which was 3 times higher than the 30% precipitate. The

increase in specific activity after precipitation may be

due to the removal of components that cause synergistic

interactions in the lungs (Sallau et al., 2008). The ACE-

active peptides would precipitate at certain

concentrations of amonium sulfate, which makes it easy

to collect ACE. However, there was still a significant

amounts of peptides similar to ACE and reduced the

activity of extract product. Both protein content and

specific activity decreased at higher concentration (60-

70%), indicating that the remaining protein content after

precipitation of 50-60% was not much and enzyme

activity was not high. Therefore, angiotensin converting

enzymes may be peptides that are susceptible to

precipitation in the fraction of 50-60% saturated

ammonium sulfate. The research results are similar to

those of Shafiee et al. (2005), using a saturation

concentration of 50% to 70% of ammonium sulfate to

preliminary purify ACE from rabbit lungs. In addition,

ACE yield of different precipitation concentrations

should also be considered. Research results Table 3

showed that different precipitation fractions yielded

different ACE, the highest yield efficiency was in the

precipitation fraction 50-60% saturated ammonium

sulfate (29.37%) and 28.01% (<30% saturated). But in

terms of purity, the 50-60% saturated precipitated

fraction had a purity of 4.22, much higher than that of

other fractions. This result was lower than that of

Quassinti et al. (1998) and Mansurah et al. (2013), the

results of these researches used saturated ammonium

sulfate of 65% and 70%, respectively, to collect the most

active ACE. Abdulazeez and Kurfi (2016) also used 70%

saturated ammonium sulfate to obtain ACE from rat

lungs, the specific activity obtained after dialysis was 50

U/g protein. All of these differences are likely due to the

actual conditions of the experiments and source of the

material. ACE after partial purification was analyzed by

SDS - PAGE to determine the molecular weight, the

results were shown in Figure 6.

Figure 6 shows that the 40-50% fraction contained

many protein components with different molecular

weight compared to 50-60% fraction. Moreover, the

results in Table 3 showed that the fraction of 50-60%

saturated ammonium sulfate had the highest specific

activity. Therefore, it can be concluded that 50-60%

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Concentration (%) Protein (%) Total activity (U/L) Specific activity (U/g protein) Purity Yield (%)

Control 3.94±0.09d 397.83±2.29c 10.11±0.22a 1 100

<30 8.61±0.05e 1114.30±27.89e 12.95±0.36a 1.28 28.01

30-40 1.93±0.09b 316.80±25.16b 16.49±1.66b 1.63 7.96

40-50 2.01±0.09b 480.70±33.89d 23.95±2.52c 2.37 12.08

50-60 2.74±0.05c 1168.57±14.65f 42.64±1.22d 4.22 29.37

60-70 0.91±0.05a 209.367±17.78a 23.19±2.24c 2.29 5.26

Table 3. Basic parameters of ACE collection efficiency by ammonium sulfate

Different letters in the column indicate significant differences in the test treatments at 95% confidence intervals

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fractions contain low levels of impurity proteins, which

helps to reduce the synergistic interactions in the

product, thus increasing the specific activity of the

product. This has also been demonstrated in the research

of Sallau et al. (2008). The result of Figure 6 also

showed that the molecular weight of ACE was

distributed around the line of 66.2 kDa. Therefore, the

extraction and partial purification collected low

molecular weight angiotensin converting enzyme (65 to

68 kDa) with amino terminal (N-terminal domain ACE).

This kind of enzyme has also been found in studies of

Deddish et al. (1994), Casarini et al. (1995) and Deddish

et al. (1996).

4. Conclusion

The research results have helped to obtain highly

active ACE from crossbred rabbit lungs for hypertension

research. The use of distilled water has brought high

extraction efficiency, the specific activity after extraction

was 10.35 (U/g protein). The results of frozen storage at

-18±2°C showed that the ability to maintain ACE

activity in rabbit lungs and crude ACE were 3 months

and 5 months, respectively. The partially purified ACE

by 50-60% saturated ammonium sulfate had a specific

activity of 42.64 U/g protein corresponding to a purity of

4.22 times and a yield of 29.37%. The molecular weight

of the product was about 66.2 kDa.

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors acknowledge Can Tho University in

providing facilities for the smooth running of this study.

References

AOAC. (1995). Official methods of analysis of AOAC

international. 15th ed. Washington, DC: Association

of Official Analytical Chemists.

Abdulazeez, M.A. and Kurfi, B.G. (2016). Isolation,

partial purification and characterization of

angiotensin converting enzyme from rat (Rattus

norvegicus) lungs. Bayero Journal of Pure and

Applied Sciences, 9(2), 24-29. https://

doi.org/10.4314/bajopas.v9i2.5

Asgeirsson, B., Hartemink, R. and Chlebowski, J.F.

(1995). Alkaline phosphatase from Atlantic cod

(Gadus morhua)-Kinetic and structural properties

which indicator adaptation to low temperatures.

Comparative Biochemistry and Physiology, 110(2),

315-329. https://doi.org/10.1016/0305-0491(94)

00171-P

Casarini, D.E., Carmona, A.K., Plavinik, F.L., Juliano, L.,

Zanella, M.T. and Ribeiro, A.B. (1995). Effects of

calcium blockers as inhibitors of angiotensin I–

converting enzyme. Hypertension, 26, 1145-1148.

https://doi.org/10.1161/01.HYP.26.6.1145

Cushman, D.W. and Cheung, H.S. (1971).

Spectrophotometric assay and properties of the

angiotensin -converting enzyme of rabbit lung.

Biochemical Pharmacology, 20(7), 1637-1648.

https://doi.org/10.1016/0006-2952(71)90292-9

Deddish, P.A., Wang, J., Michel, B., Morris, P.W.,

Davidson, N.O., Skidgel, R.A. and Erdös, E.G.

(1994). Naturally occurring active N-domain of

human angiotensin I-converting enzyme.

Proceedings of the National Academy of Sciences of

the United States of America, 91, 7807-7811. https://

doi.org/10.1073/pnas.91.16.7807

Deddish, P.A., Wang, L., Jackman, H.L., Michel, B.,

Wang, J., Skidgel, R.A. and Erdös, E.G. (1996).

Single domain angiotensin I converting enzyme

(kininase II): characteristics and properties. Journal

of Pharmacology and Experimental Therapeutics,

279(3), 1582-1589.

Dixon, M. and Webb, E.C. (1964). Enzyme. 2nd Ed. New

York: Academic Press.

Gupta, N., Srivastava, N. and Bhagyawant, S.S. (2018).

Vicilin—A major storage protein of mungbean

exhibits antioxidative potential, antiproliferative

effects and ACE inhibitory activity. PLoS One, 13

(2), e0191265. https://doi.org/10.1371/

journal.pone.0191265

Kutrovic, I., Marshall, S.N. and Zhao, X. (2011).

Hydrophobic immobilization of a bile salt activated

lipase from Chinook salmon (Oncorhynchus

tshawytscha). Journal of Molecular Catalysis B:

Enzymatic, 72 (3-4), 168-174. https://

doi.org/10.1016/j.molcatb.2011.06.001

Muñoz-Durango, N., Fuentes, C.A., Castillo, A.E.,

González-Gómez, L.M., Vecchiola, A., Fardella,

C.E. and Kalergis, A.M. (2016). Role of the Renin-

F
U

L
L

 P
A

P
E

R

Figure 6. The result of molecular weight analysis of fractions

40-50% and 50-60%

1087

https://doi.org/10.4314/bajopas.v9i2.5

https://doi.org/10.1016/0305-0491(94)00171-P

https://doi.org/10.1016/0305-0491(94)00171-P

https://doi.org/10.1161/01.HYP.26.6.1145

https://doi.org/10.1016/0006-2952(71)90292-9

https://doi.org/10.1073/pnas.91.16.7807

https://doi.org/10.1371/journal.pone.0191265

https://doi.org/10.1371/journal.pone.0191265

https://doi.org/10.1016/j.molcatb.2011.06.001

 Nguyen et al. / Food Research 4 (4) (2020) 1082 - 1088

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

Angiotensin-Aldosterone System beyond Blood

Pressure Regulation: Molecular and Cellular

Mechanisms Involved in End-Organ Damage during

Arterial Hypertension. International Journal of

Molecular Sciences, 17(7), 797. https://

doi.org/10.3390/ijms17070797

Murado, M.A., González, M.P. and Vázquez, J.A.

(2009). Recovery of proteolytic and collagenolytic

activities from viscera by-products of rayfish (Raja

clavata). Marine Drugs, 7(4), 803-815. https://

doi.org/10.3390/md7040803

Ngo, L.T.A., Pham, T.L. and Le, V.V.M. (2008).

Purification of Endopolygalacturonase from

Submerged Culture of Aspergillus awamori L1

Using a Two-step Procedure: Enzyme Precipitation

and Gel Filtration. International Food Research

Journal, 15(2), 135-140.

Quassinti, L., Miano, A., Bramucci, M., Maccari, E. and

Amici, D. (1998). Purification of swine serum

angiotensin converting enzyme with high recovery

of activity using lisinopril coupled to epoxy-

activated sepharose 6B. International Union of

Biochemistry and Molecular Biology Life, 44(5), 887

-889. https://doi.org/10.1080/15216549800201942

Restrepo, R.A., Loango, N., Moncada, M.V. and

Landazuri, P. (2013). Angiotensin-converting

enzyme inhibitory activity of Passiflora edulis f.

flavicarpa and Petroselinum crispum (mill) fuss.

British Journal of Pharmaceutical Research, 3(4),

776-785. https://doi.org/10.9734/BJPR/2013/3517

Sallau, A.B., Ibrahim, M.A., Salihu, A. and Patrick, F.U.

(2008). Characterization of phospholipase A2 from

Echis ocellatus venom. African Journal of

Biochemistry Research, 2, 98-101.

Scopes, R.K. (1994). Protein purification, principle and

practice. 3rd ed. New York: Springer-Verlag. https://

doi.org/10.1007/978-1-4757-2333-5

Shafiee, S.M., Keyhani, M., Shabani, M., Koochaki

Shalmani, I. and Kaviani, M. (2005). Extraction and

Development of Multi-Step Purification of

Angiotensin-I Converting Enzyme (ACE) from

Rabbit Lung. Razi Journal of Medical Sciences,11

(44), 979-985.

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 *Corresponding author.

Email: aftab_mb12@yahoo.com

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  Food Research 4 (4) : 1351 - 1356 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Microbiological analysis of different categories of food items in Dhaka city,

Bangladesh

*Uddin, M.A., Jabin, T., Siam, E.A., Afreen, M.T., Uddin, S.S. and Sayeda, U.

Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217,

Bangladesh

Article history:

Received: 5 March 2020

Received in revised form: 2

April 2020

Accepted: 8 April 2020

Available Online: 25 April

2020

Keywords:

Microbiological quality,

Pathogens,

Foods,

Antibiogram

DOI:
https://doi.org/10.26656/fr.2017.4(4).099

Abstract

Pathogenic microorganisms have been so far reported to contaminate a wide range of

foods triggering food borne infections or intoxications including the enteric complications,

abdominal pain, fever, bloodstream infection etc. The current study was attempted to

observe the microbiological quality of some popular foods collected from different places

of Dhaka city, Bangladesh. Conventional cultural, microscopic and biochemical tests were

followed for the detection and enumeration of bacterial isolates associated with these food

samples. The investigation encompassed detection of total viable bacterial count (TVBC)

and presumptive identification of other pathogenic bacteria from these samples. Higher

counts of TVBC, coliform, Staphylococcus spp. and fungal load were recorded as

1.46×107 CFU/g (yogurt 1), 4.5×106 CFU/g (yogurt 1), 5.6×105 CFU/g (raw meat) and

2.9×103 CFU/g (sea fish), respectively. Fecal coliform was detected only in one out of ten

samples. On the contrary Salmonella spp., Vibrio spp., Shigella spp. and Pseudomonas

spp. were completely absent. The antibiogram study showed that all the isolates were

sensitive against Kanamycin and Azithromycin. Better sensitivity was also observed

against Gentamycin and Ciprofloxacin. Varying degree of antibiotic resistance was also

detected against Cefixime, Amikacin and Neomycin. Our study emphasizes the need for

continuous monitoring of the various categories of food samples for the safety of public

health.

1. Introduction

Food is the basic and utmost need

for people and animals to survive. Sources of foods are

traditionally from animals and plants. It is consumed by

living beings for the constant supply of energy (Forbes,

2007). Foods are generally contaminated with various

bacterial species and fungi during harvesting; processing;

and through the use of associated machines, utensils,

water, etc. (Frazier and Westhoff, 2007; Alam et al.,

2015). The analysis of food products for the presence of

pathogenic microorganisms is one of the fundamental

procedures to maintain the safety and quality of food.

Though a number of advancement have been observed

recently in the field of food technology, foodborne

diseases are still considered as a major concern for

human health, in developing as well as in developed

countries. Everybody, especially very young or old and

immunologically weak people are vulnerable to

foodborne diseases and some severe complications can

happen as a result of infection (Sherin et al., 2015).

Foodborne illnesses are occurred due to the action of

microorganisms and/or their toxins, fungi with their

related toxins, and chemical contaminants (Khamis and

Hafez, 2011). The World Health Organization (WHO)

listed some hazards like potentially harmful bacteria,

viruses, toxins, parasites and chemicals that may be

found in food. According to the WHO, one person out of

10 people becomes ill and 420,000 people die every year

as a result of consuming contaminated food (World

Health Organization (WHO), 2018). The International

Commission on Microbiological Specifications for Food

(ICMSF), 2005 brought in the concept of Food Safety

Objectives (FSOs) as the maximum frequency and/or

concentration of a microbial hazard (micro-organisms or

toxins) in a food measured tolerable for consumer safety.

     Another current health related alarming issues is

the threat and existence of multidrug resistant pathogens

in foods. Antibiotic resistance is a feature of a

microorganism (CDC, 2018). The WHO defines

antimicrobial resistance as a microorganism's resistance

to an antimicrobial drug that was once able to treat an

infection caused by those microorganisms (WHO, 2014).

https://orcid.org/0000-0002-1733-4264

https://orcid.org/0000-0002-0151-0606

https://orcid.org/0000-0003-0032-8199

https://orcid.org/0000-0002-6152-1470

https://orcid.org/0000-0002-8393-0400

https://orcid.org/0000-0002-3618-3596

https://simple.m.wikipedia.org/wiki/People

https://simple.m.wikipedia.org/wiki/Animal

file:///C:/Users/LENOVO/Dropbox/Food%20Research/Manuscript%20Pending%20Production/FR-2020-099%20(1).docx#_ENREF_3#_ENREF_3

http://www.who.int/mediacentre/factsheets/fs194/en/

Uddin et al. / Food Research 4 (4) (2020) 1351 - 1356

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Antibiotic resistance is a global issue and the origin of

resistance is often found in the world’s tropical or hottest

regions (Collignon et al., 2018). As per the antibiotic

resistance report 2019, more than 2.8 million antibiotic-

resistant infections occur in the U.S. each year, and more

than 35,000 people die as a result (Edwards et al., 2019).

Along these lines of considerations, the current study

focused on the presumptive detection of some common

food borne pathogens from the daily consumable food

samples (fish, meat, yoghurt, milk, ice cream, juice). The

antibiogram profile of the suspected bacterial isolates

from the food samples was also tested in this study.

2. Materials and methods

2.1 Sample collection and processing

A total of eighteen food samples of various

categories such as fish, meat, yoghurt, ice cream, milk

and juice samples were collected from some popular

super shops located in various areas of Dhaka city such

as Mouchak, Shantinagar, and Badda areas. Collection of

samples was done in different time intervals and taken to

the laboratory as quickly as possible according to the

method suggested by FDA, 2013. Before calculating the

bacterial and fungal load, samples were subjected to

serial dilutions up to10-4.

2.2 Microbiological analyses

For the estimation of total viable bacterial count

(TVBC), 0.1 mL sample of each dilution was introduced

onto the nutrient agar (NA) plates following spread plate

technique. The nutrient agar plates were kept for

incubation at 37°C for 24 hrs. For the detection of

specific bacteria, from 10-2 and 10-3 dilutions of each

sample, 0.1 mL of suspension was spread onto

MacConkey agar, Mannitol salt agar (MSA) and

Pseudomonas agar media for the enumeration of

Coliform, Staphylococcus spp. and Pseudomonas spp.,

respectively (Cappuccino and Sherman, 2014; Afroz et

al., 2015; Uddin, 2018). All the plates were incubated at

37oC for 24 hrs. The characteristic pink colonies on

MacConkey agar, yellow colonies on MSA and colonies

with greenish fluorescent pigmentation on Pseudomonas

agar was indicative for the presumptive isolation of

coliform, Staphylococcus spp. and Pseudomonas spp.,

respectively.

2.3 Enrichment procedure.

For the identification of Salmonella spp. and Vibrio

spp., enrichment culture technique was followed. In this

procedure, 1 mL of the sample was inoculated into 9 mL

of selenite cystein broth (SCB) and alkaline peptone

water (APW) and incubated at 37oC for 6 hrs. Following

incubation, the samples were diluted up to 10-3 and then

0.1 mL of samples from 10-2 and 10-3 dilutions were

spread over Salmonella-Shigella agar (SS) and

Thiosulfate-Citrate-Bile-Sucrose Agar (TCBS) from the

respective enrichment media (Cappuccino and Sherman,

2014; Tabassum and Uddin, 2016). The appearance of

small blackish colonies after incubation for 24 hrs at

37oC was an indication of the presence of Salmonella

spp., while the large (2-41 mm) and slightly flattened,

yellow colonies on the TCBS agar referred to the

presence of Vibrio spp.

2.4 Biochemical tests for the confirmative identification

Biochemical tests Identification of the isolates was

carried out by major biochemical tests, for example-

Triple Sugar Iron (TSI), Motility Indole Urease (MIU),

Methyl-Red (MR), Voges-Proskauer (VP) and Citrate

Utilization were performed following the standard

methods (Cappuccino and Sherman, 2014).

2.5 Study of antibiogram

After the isolation of the bacterial isolates, antibiotic

susceptibility assay was carried out against different

groups of antibiotics in vitro by the Kirby-Bauer method

(Bauer et al., 1966). Drug resistance was observed

against Neomycin (30 µg), Cefixime (5 µg), Kanamycin

(30 µg), Gentamycin (10 µg), Amikacin (30 µg),

Ciprofloxacin (5 µg), and Azithromycin (25 µg). From

overnight culture plate, a small portion of a fresh colony

was transferred to Muller-Hinton broth and incubated at

37oC for 4 to 5 hrs until the growth reached to the

equivalent turbidity standard of McFarland (0.5

standards). Muller-Hinton agar plates were seeded

properly by spreading the inoculate using sterile cotton

swab. Discs (OXOID, UK) were placed gently at a

proportionate distance from each other using a sterile

needle. The plates were then incubated overnight at 37oC

and zones of inhibition (if any) were measured and

interpreted as susceptible, intermediate and resistant

categories by referring the recommended interpretative

standards (CLSI, 2006).

3. Results and discussion

Foodborne diseases are the leading global problem

causing considerable morbidity and mortality each year

(Hanson et al., 2012). The most frequent known causes

of foodborne diseases are pathogenic bacteria. In this

study, the goal was to analyze the microbiological

quality of different popular food items consumed by the

people of Dhaka metropolitan, Bangladesh. Therefore,

the current study attempted to check the presence of

microorganisms in such categories food items in terms of

total viable count as well as in the finding of different

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pathogenic organisms such as Vibrio spp., Salmonella

spp. and Shigella spp. along with the drug resistance

properties of the assumed bacterial isolates.

3.1 Isolation and enumeration of microorganisms

3.1.1 Total viable bacterial count (TVBC)

In this study, the total viable bacterial counts ranged

from 2.0×103 CFU/g to 1.64×107 CFU/g which was

found in commercial juice-4 and yoghurt-1 samples

respectively (Table 1). This finding was quite similar to

the study done by Oranusi et al. (2013) and Uddin, 2018.

According to FDA guideline (2013) the acceptable limit

is 5x104–105 CFU/g in case of total viable bacteria.  So,

14 out of 18 studied samples were very much within the

acceptable range as per FDA guideline.

3.1.2 Coliform, fecal coliform, Salmonella spp. and

Shigella spp. count

Coliform was detected in eight out of eighteen

samples of which the highest count as 4.5×106 CFU/g in

yoghurt-1 whereas the lowest count was 1.1×102 CFU/g

in ice cream-1 (Table 1). In this study, the detection of

coliform bacteria, especially E. coli and Klebsiella spp.

in these samples showed the possibility of the presence

of fecally contaminated microorganisms as also

suggested by Adams and Moss (2008). The lack of

hygiene of the food handlers may be a parameter for the

possible presence of coliforms in food items. In this

study, fecal coliform was noticed in a sea fish sample.

Fortunately, Salmonella spp., Shigella spp., Vibrio spp.

and Pseudomonas spp. were totally absent in the selected

food items. But this result was contradictory to the

findings of Tambekar et al. (2008) who identified these

organisms in some ready to eat street vendor food items.

3.1.3 Total Staphylococcal count

Staphylococcus spp. count was observed within the

range from 1.1×102 CFU/g – 4.5×106 CFU/g which was

found in ice cream-1 and yoghurt sample-1 respectively

(Table 1). The existence of S. aureus in food is a sign of

poor hygiene practices. S. aureus in food is connected

with cross contamination occurring during processing

and storage or through the contamination of raw

ingredients (Akindele and Ibrahim, 2016).

3.1.4 Total fungal load

Fungus presence is an indication of the availability

of various Mycotoxins in the food samples that may

render severe threat to human health (Habiba et al.,

2015). A study conducted by Șumălan et al. (2011)

showed variable fungal growth in between 3.8×104 -

5.9×104 CFU/g in the food samples. Overall fungal

growth was observed in only two samples which were

much lower than the previous report (Table 1). Fungi are

far and wide disseminated and seen wherever moisture is

present with sufficient nutrients that can prolong their

growth. Fungi are the foremost spoilage of foods and

feedstuffs. The propagation of various fungi in

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Sample

no
Sample

TVBC

(CFU/g or

CFU/mL)

TCC

(CFU/g or

CFU/mL)

TSC

(CFU/g or

CFU/mL)

TFC

(CFU/g or

CFU/mL)

Fecal coliform

(CFU/g or

CFU/mL)

1 Fish (raw) 4.6 ×104 1.6×104 0 0 0

2 Fish (frozen) 1.30×105 0 1.30×103 0 0

3 Sea fish (raw) 3.1×103 8.3×103 2.2×103 2.9×103 2×101

4 Fish (shrimp frozen) 1.94×106 8.1×104 2.6×103 0 0

5 Meat (raw) 7.2 ×104 1.5×104 5.6×105 0 0

6 Meat (frozen) 1.76×106 0 7.5×103 0 0

7 Sausage 8.2×102 1.2×102 4.5×101 4×101 0

8 Yogurt-1 1.64×107 4.5×106 2.2×105 0 0

9 Yoghurt-2 1.0×104 0 0 0 0

10 Yogurt-3 1.3×105 0 0 0 0

11 Ice cream-1 1×105 1.1×102 3.8×103 0 0

12 Ice cream-2 2×104 2.3×102 6.0×102 0 0

13 Pasteurized milk 4.4×105 0 0 0 0

14 Raw milk 8.24×106 2.5×105 4.8×104 0 0

15 Commercial juice-1 3.2×103 2×103 0 0 0

16 Commercial juice-2 3.7×103 0 0 0 0

17 Commercial juice-3 4.5×103 0 0 0 0

18 Commercial juice-4 2.0×103 0 0 0 0

Table 1. Microbiological analysis of the various categories of food items

TVBC: Total viable bacterial count, TCC: Total coliform count, TSC: Total staphylococcal count, TFC: Total fungal coliform.

Salmonella spp., Shigella spp., Vibrio spp., Pseudomonas spp. were completely absent in all the samples being studied.

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agricultural products leads to lessening in yield and

quality with noteworthy economic losses. The level of

contamination will vary with geographic location,

agricultural practices and the vulnerability of goods to

the incursion of fungi during storage and processing

periods (Adeyeye, 2016)

3.1.5 Antibiogram of the Isolates

Antibiotic resistance has augmented worldwide

causing to failures in the treatment of human infectious

diseases. Resistance against antibiotics by pathogenic

bacteria is a major threat in the anti-infective therapy of

both humans and animals. The Kirby-Bauer disk

diffusion method was used in this experiment to resolve

whether the isolated organisms were susceptible or

resistant towards a pool of commonly prescribed

antimicrobial agents. Antibiotic resistance pattern of E.

coli, Klebsiella spp. and Staphylococcus spp. has been

shown in Table 2. Both E. coli and Klebsiella spp.

showed a high degree of sensitivity against Neomycin,

Kanamycin, Gentamycin, Amikacin and Azithromycin.

40% of the Klebsiella spp. isolates were found to be

resistant against cefixime. On the contrary,

Staphylococcus spp. isolated from various samples

exhibited 100% resistance against Cefixime. Similar type

of resistance patten in  S. aureus isolates was also

detected by Taj et al. (2010). Researchers have found

that bacterial resistance to various antibiotics is encoded

by over 100 different genetic mechanisms, a number of

which are readily moveable to other bacteria through the

process of conjugal elements, transposons, plasmids etc

(Bhowmik et al., 2016).

4. Conclusion

It is obligatory that foods should be free of

contaminations as much as possible. The existence of E.

coli, Staphylococcus spp. and Klebsiella spp. indicates a

potential health risk as these organisms are pathogenic

and have been implicated in food borne diseases. Based

on FDA guideline (2013), the level of contaminations

was within unacceptable microbiological limits for four

samples; this could be due to improper processing, poor

handling practices and post-cross contamination which

can produce a risk to the health of the consumers.

Foodborne illness can be avoided by good hygiene

practices such as the use of Hazard Analysis Critical

Control Point (HACCP) application in the sequence of

food production, processing and storage. Ensuring proper

supervision and education to the food handlers/ food

vendors on food safety practices and stringent control of

various categories foods sold to the busy city residents

should be properly observed by the pertinent authorities

to ward off the food borne illness related epidemics

within the Dhaka city, Bangladesh. Besides recurrent use

of antibiotics should be prohibited as antibiotic defiant

strains are incessantly mounting.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

All the authors gratefully acknowledge Department

of Microbiology, Stamford University Bangladesh for

financial and technical support.

References

Adams, M.R. and Moss, M.O. (2008). Bacterial agents

of foodborne illness. In Food Microbiology, 3rd ed.

Cambridge, United Kingdom: The Royal Society of

Chemistry.

Adeyeye, S.A.O. (2016). Fungal mycotoxins in foods: A

review. Cogent Food and Agriculture, 2(1), 1-11.

https://doi.org/10.1080/23311932.2016.1213127

Afroz, H., Ahmed, T. and Uddin, M.A. (2015).

Microbiological analysis and antibacterial activity of

pear samples. Stamford Journal of Microbiology, 5

(1), 1-4. https://doi.org/10.3329/sjm.v5i1.26910

Akindele, P.O. and Ibrahim, K.A. (2016).

F
U

L
L

 P
A

P
E

R

Name of
Antibiotic disc

Zone of inhibition (mm) against test bacteria

Disc
Content

E. coli Klebsiella spp. Staphylococcus spp.

(N = 10) (N = 10) (N = 10)

R% S% R% S% R% S%

Neomycin 30 µg 0 100 10 90 3 70

Cefixime 5 µg 0 100 40 60 100 0

Kanamycin 30 µg 0 100 0 100 0 100

Gentamycin 10 µg 10 90 0 100 0 100

Amikacin 30 µg 15 85 0 100 10 90

Ciprofloxacin 5 µg 10 90 5 95 0 100

Azithromycin 25 µg 0 100 0 100 10 90

Table 2. Antibiogram profile of the suspected bacterial isolates

R% = Resistant percentage, S% = Sensitive percentage

1354

https://doi.org/10.1080/23311932.2016.1213127

https://doi.org/10.3329/sjm.v5i1.26910

Uddin et al. / Food Research 4 (4) (2020) 1351 - 1356

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

Microbiological analysis of ready-to-eat-foods

obtained from bukaterian within the Ekiti State

University and environment, Ado-Ekiti, Nigeria.

Journal of Advances in Microbiology, 1(2), 1-8.

https://doi.org/10.9734/JAMB/2016/30948

Alam, M.S., Feroz, F., Rahman, H., Das, K.K. and Noor,

R. (2015). Microbiological contamination sources of

freshly cultivated vegetables. Nutrition and Food

Science, 45 (4), 646-658. https://doi.org/10.1108/

NFS-04-2015-0032

Bauer, A.W., Kirby, W.M., Sherris, J.C. and Tierch, M.

(1966). Antibiotic susceptibility testing by a

standardized single disc method. American Journal

of Clinical Pathology, 45(4), 493-496. https://

doi.org/10.1093/ajcp/45.4_ts.493

Bhowmik, S., Chowdhury, R.A. and Uddin, M.A.

(2016). Microbiological analysis and detection of

anti-bacterial activity of Centella asiatica and Aloe

vera samples collected from different areas of Dhaka

city, Bangladesh. Stamford Journal of Microbiology,

6(1), 39-43. https://doi.org/10.3329/sjm.v6i1.33518

Cappuccino, J. and Sherman, N. (2014). Microbiology.

A laboratory manual. London, United Kingdom:

Pearson Education.

Centers for Disease Control and Prevention (CDC).

(2018). Antibiotic Prescribing and Use. Retrieved

April 29, 2015 from CDC website: https://

www.cdc.gov/antibiotic-use/index.html

Clinical Laboratory Standards Institute (CLSI). (2006).

Performance standards for antimicrobial disks

susceptibility tests: Approved standard-9th ed. CLSI

document. M2-A9, 26:1. Wayne, USA: CLSI.

Collignon, P., Beggs, J.J., Walsh, T.R., Gandra, S. and

Laxminarayan, R. (2018). Anthropological and

socioeconomic factors contributing to global

antimicrobial resistance: a univariate and

multivariable analysis. Lancet Planet Health, 2(9),

398–405. https://doi.org/10.1016/S2542-5196(18)

30186-4

Edwards, M., Hamilton, R., Oliver, N., Fitzgibbon, S.

and Samarasekera, R. (2019). Antibiotic Resistance:

Modelling the Impact on Mortality and Morbidity.

United Kingdom: Institute and Faculty of Actuaries.

Food and Drug Administration (FDA). (2013). Revised

guidelines for the assessment of microbiological

quality of processed foods. Retrieved from Food and

Drug Administration Philippines website: https://

ww2.fda.gov.ph/attachments/article/17218/FC2013-

010.pdf

Forbes, J.M. (2007). Voluntary food intake and diet

selection in farm animals. United Kingdom: CABI

Publishing. https://

doi.org/10.1079/9781845932794.0000

Frazier, W.C. and Westhoff, D.C. (2007). Food

Microbiology, 4th ed. New Delhi, India: Tata

McGraw-Hill.

Hanson, L.A., Zahn, E.A., Wild, S.R., Döpfer, D., Scott,

J. and Stein, S. (2012). Estimating global mortality

from potentially foodborne diseases: An analysis

using vital registration data. Population Health

Metrics, 10, 5. https://doi.org/10.1186/1478-7954-10

-5

ICMSF. (2005). Microorganisms in foods. 6. Microbial

ecology of food Commodities. 2nd ed., p. 25-40. New

York, USA: Kluwer Academic/Plenum Publishers.

Khamis, N. and Hafez, M. (2011): A microbial survey of

food served to oncohematology patients at a

university hospital. International Journal of

Infection Control, 7(1), 1-6. https://doi.org/10.3396/

ijic.v7i1.005.11

Oranusi, S.U., Oguoma, O.I. and Agusi, E. (2013).

Microbiological quality assessment of foods sold in

student’s cafeterias. Global Research Journal of

Microbiology, 3(1), 1 –7.

Șumălan, R.M., Doncean, A., Șimonea, L., Șumălan, R.,

Camen, D. and Beinsan, C. (2011). Study on fungal

load of pome fruits, hazardous factor for quality

maintaining and ensuring the consumer safety.

Journal of Horticulture, Forestry and Biotechnology,

15(2), 147- 151.

Sherin, A.S., Masry, E.L., Rashad, S.S., Hadia, H.B. and

Sorour, T.R. (2015). Survey of the Microbiological

Quality of Food Served at a University Hospital in

Egypt.  International Journal of Current

Microbiology and Applied Sciences, 4(9), 10-21

Tabassum, S. and Uddin, M.A. (2016). Microbiological

Analysis of Different Flavoured Milk Samples

Collected from Various Areas of Dhaka city,

Bangladesh. Bangladesh Journal of Microbiology,

33(1 and 2), 11-15. https://doi.org/10.3329/

bjm.v33i1.39597

Tambekar, D.H., Jaiswal, V., Dhanorkar, D., Gulhane, P.

and Dudhane, M. (2008). Identification of

microbiological hazards and safety of ready-to-eat

food vended streets of Amravati City, India. Journal

of Applied Bioscience, 7, 195-201.

Taj, Y., Abdullah, F.E. and Kazmi, S.U. (2010). Current

pattern of antibiotic resistance in Staphylococcus

aureus clinical isolates and the emergence of

vancomycin resistance. Journal of College of

Physicians and Surgeons Pakistan, 20(11), 728-732.

doi: 11.2010/JCPSP.728732.

Uddin, M.A. (2018). Microbiological analysis of ready

to eat foods collected from different places of Dhaka

F
U

L
L

 P
A

P
E

R

1355

https://doi.org/10.9734/JAMB/2016/30948

https://doi.org/10.1108/NFS-04-2015-0032

https://doi.org/10.1108/NFS-04-2015-0032

https://doi.org/10.1093/ajcp/45.4_ts.493

https://doi.org/10.3329/sjm.v6i1.33518

https://www.cdc.gov/antibiotic-use/index.html

https://www.cdc.gov/antibiotic-use/index.html

https://doi.org/10.1016/S2542-5196(18)30186-4

https://doi.org/10.1016/S2542-5196(18)30186-4

https://ww2.fda.gov.ph/attachments/article/17218/FC2013-010.pdf

https://ww2.fda.gov.ph/attachments/article/17218/FC2013-010.pdf

https://ww2.fda.gov.ph/attachments/article/17218/FC2013-010.pdf

https://doi.org/10.1079/9781845932794.0000

https://doi.org/10.1079/9781845932794.0000

https://doi.org/10.1186/1478-7954-10-5

https://doi.org/10.1186/1478-7954-10-5

https://doi.org/10.3396/ijic.v7i1.005.11

https://doi.org/10.3396/ijic.v7i1.005.11

https://doi.org/10.3329/bjm.v33i1.39597

https://doi.org/10.3329/bjm.v33i1.39597

https://www.ncbi.nlm.nih.gov/pubmed/?term=Taj%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=21078245

https://www.ncbi.nlm.nih.gov/pubmed/?term=Abdullah%20FE%5BAuthor%5D&cauthor=true&cauthor_uid=21078245

https://www.ncbi.nlm.nih.gov/pubmed/?term=Taj%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=21078245

https://www.ncbi.nlm.nih.gov/pubmed/?term=Abdullah%20FE%5BAuthor%5D&cauthor=true&cauthor_uid=21078245

Uddin et al. / Food Research 4 (4) (2020) 1351 - 1356

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

city, Bangladesh. Stamford Journal of Microbiology,

8(1), 30-33. https://doi.org/10.3329/sjm.v8i1.42436

World Health Organization (WHO). (2014).

Antimicrobial resistance Fact sheet. Retrieved March

7, 2015 from WHO Website: https://www.who.int/

news-room/fact-sheets/detail/antimicrobial-

resistance

World Health Organization (WHO). (2018). Food

Safety. Retrieved from WHO website: http://

www.who.int/news-room/facts-in-pictures/detail/

food-safety.

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https://doi.org/10.3329/sjm.v8i1.42436

http://www.who.int/mediacentre/factsheets/fs194/en/

https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

http://www.who.int/news-room/facts-in-pictures/detail/food-safety

http://www.who.int/news-room/facts-in-pictures/detail/food-safety


 *Corresponding author.

Email: tatikkhusni@gmail.com

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 1226 - 1233 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Microstructure and physicochemical characteristics of modified taro starch

after annealing, autoclaving-cooling and heat moisture treatment

1,2Setiarto, R.H.B., 1Kusumaningrum, H.D., 1Jenie, B.S.L., 2*Khusniati, T.,
2Widhyastuti, N. and 2Ramadhani, I.

1Departement of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agriculture

University (IPB University), Bogor 16680, Indonesia
2Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI) Main Street

Jakarta-Bogor Km 46, Cibinong Science Center, Cibinong, Bogor, 16911 West Java, Indonesia

Article history:

Received: 19 February 2020

Received in revised form: 20

March 2020

Accepted:  22 March 2020

Available Online: 15 April

2020

Keywords:

Annealing,

Autoclaving-cooling,

Heat moisture treatment,

Microstructure and

physicochemical

characteristics,

Modified taro starch

DOI:
https://doi.org/10.26656/fr.2017.4(4).079

Abstract

This study investigated the effects of annealing, autoclaving-cooling and heat moisture

treatment on the microstructure and physicochemical characteristics of taro starch. The

taro starch was treated by the annealing process (24 hrs, 50oC), the heat moisture

treatment (HMT) (moisture 25%, 3 hrs, 110oC), and the autoclaving (15 mins, 121oC) -

cooling (24 hrs, 4oC) with 1 and 2 cycles. The results show that the autoclaving-cooling 2

cycles (AC-2C) changed the microstructure of taro starch into a very compact and dense

structure because of formed double helix bound that cannot be hydrolyzed by pancreatic

enzymes so it can be converted became resistant starch as the prebiotic source. Pasting

properties analysis showed that AC-2C improved shear stress resistance, heat resistance

and low retrogradation modified taro starch (MTS). The AC-2C treatment increased water

binding capacity (73.84%), solubility (44.58%), and swelling power (16.71%) of MTS.

The water-binding capacity had a positive correlation with solubility and swelling power.

The AC-2C treatment increased amylose content (27.40%) and decreased reducing sugar

level (6.36%) of MTS, so it can encourage the formation of resistant starch to improve the

prebiotic properties of taro starch. Modified taro starch AC-2C is the best compared to

HMT and annealing based on microstructure and physicochemical characteristics.

1. Introduction

Colocasia esculenta known as taro was one of the

tubers from the Araceae family that is of the most widely

cultivated in tropical areas of Southeast Asia, South and

West Africa (Aboubakar et al., 2009). The taro starch

content is high (70-80%), so it can be applied as raw

materials for food processing (Arici et al., 2016). Taro

starch is highly digestible and it has a unique small size

of granules (3-5 µm) (Kaushal et al., 2012). Native taro

starch has limited use in food application as it has many

weaknesses such as narrow peak viscosity range, low

process tolerance, worse shear stress resistance, thermal

decomposition, high retrogradation and cooked starches

will form a weak, cohesive and rubbery paste (Yu et al.,

2018). These weaknesses increase the interest of many

researchers to modify taro starch so its physicochemical

characteristics could be improved (Sharlina et al., 2017).

Taro starch modification techniques could be carried out

by its physical, chemical or enzymatic properties

(Hazarika and Sit, 2016). Some physical modification

techniques could improve the functional properties of

taro starch including the autoclaving-cooling cycle, heat

moisture treatment (HMT) and annealing.

The autoclaving-cooling process was the physical

modification to convert the original taro starch into the

more resistant starch type III (RS3) (Dundar and

Gocmen, 2013). The repeated process of autoclaving-

cooling may cause in the formation of the retrograded

and crystallized amylose fraction (Ashwar et al., 2016).

The amylose fraction bound to other amylose fractions

via hydrogen bonds to form a double helix structure

(Simsek and El, 2015). The structure of the double helix

bound to the other double helix structures form crystals,

therefore there was a recrystallization process of the

amylose fraction, called RS3 formation process (Shah et

al., 2016).

The HMT process was conducted using low moisture

content (<35%) and heated at the temperature that

exceeds the gelatinization temperature (80-130oC) for 15

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mins to 16 hrs (Cheng et al., 2019). The amylose

recrystallization occurred during the cooling process.

The HMT method changed the characteristics of starch

gelatinization, which increased the tendency of

retrograded starch (Zheng et al., 2018). The association

of amylose-amylose chain in the amorphous area can

also be increased by the HMT process, resulted in the

granule material compaction (Anderson et al., 2002). In

addition, the amylose-amylopectin chain could also be

enhanced by the HMT process.

Annealing was a modification of starch achieved by

heating below the gelatinization temperature with the

water content addition (Xu et al., 2018). Modification of

starch with annealing tends to change the functional

properties and digestibility of starch without changing

the structure of starch granules (Wang et al., 2017).

Annealing treatment caused the gelatinization

temperature rise and the starch digestibility to fall (Wang

et al., 2018). Setiarto et al. (2018) report that

fermentation and two cycles of autoclaving-cooling can

improve prebiotic properties of modified taro flour and it

successfully increased the resistant starch (RS) content

by 2.8-fold compared to the control treatment. This study

is aimed to determine the microstructure and

physicochemical characteristics of modified taro starch

by implementing annealing, HMT and autoclaving-

cooling treatments.

2. Materials and methods

2.1 Materials

The main raw material used in this study was the

Bogor Taro of Pandan (Colocasia esculenta) with eight

months harvest age, from Cijeruk Bogor West Java,

Indonesia.

2.2 Taro starch extraction

Taro starch extraction was performed by applying

the technique from Airul et al. (2014) with a few

modifications. Taro tuber was peeled, washed, and

soaked in the mixture of 1% NaCl (3:4) for an hour to

remove oxalate crystals. It was then shredded and mixed

with distilled water (3:1) for one minute using a blender

(Phillips, Netherland). Double fold cotton cloth was

utilized to filter the taro pulp. The obtained taro pulp

filtrate was settled overnight to let the starch sink at the

base of the beaker glass. Taro pulp was centrifuged with

High Speed Centrifuge (Kubota, Japan) at 5000 rpm for

10 mins to obtain taro starch. Distilled water was used to

clean the taro starch three times to remove the

supernatant. After that, it was oven-dried at 50oC up to

the constant weight. Finally, the dry taro starch was

ground using the disk mill (China).

2.3 Annealing treatment

The annealing treatment of taro starch was

conducted by the technique by Wang et al. (2018). Taro

starch of 12 g was added to 60 mL of distilled water with

the ratio of taro starch: water (1:5) (b/v) was placed in a

polyethylene bag. The annealing treatment was carried

out at 50oC for 24 hrs by inserting a polyethylene bag

which had been tightly closed into a water bath (Hitachi,

Japan). Afterward, the taro starch was freeze dried,

crushed and filtered using the 100-mesh sieve. The

resulting taro starch from the annealing process was then

chilled at 4oC prior to further analysis.

2.4 Heat-moisture treatment

The taro starch modification using HMT was

obtained following Deka and Sit (2016). Forty-five

grams of taro starch (dry-based) was placed into a glass

container, and distilled water was added to it while

stirring until the water content reached 25%. Then, the

glass container was sealed, balanced for 48 hrs at room

temperature then heated at 110°C, 3 hrs in an electric

oven (Shimizu, Japan) for three hrs. The heated modified

taro starch was then dried at 40°C for overnight, milled

and sieved with a 100-mesh sieve.

2.5 Autoclaving-cooling treatment

The autoclaving-cooling method of taro starch

followed the procedure by Setiarto et al. (2018). The taro

starch was added with aquadest at the ratio of 1:2, heated

in an autoclave (Hitachi, Japan) at 121°C for 15 mins,

then chilled in refrigerator at 4°C for 24 hrs. Thereafter,

the treated taro starch was dried (70°C, 16 hrs) in an

oven (Shimidzu, Japan) until the moisture content

reached 12%, and milled using a pin disk mill

(Shimidzu, Japan). The starch was sieved to obtain the

100-mesh taro starch. The autoclaving-cooling treatment

was also completed with two cycles.

2.6 Microstructure analysis

Scanning Electron Microscopy (SEM) analysis was

conducted to establish the taro starch microstructure and

particle size. The sputter coater (Hitachi E 102 Ion

Sputter) which had a 20 nm thick gold layer with a

conductive two-sided carbon band was used to cover the

taro starch samples. The visualization of the surface and

the cross section of taro starch granules was performed

by placing a film on a piece of aluminum taped with

double-sided tape then covered with a 20-30 nm thick of

gold layer. An acceleration voltage of 20.0 kV was used

to analyze all samples of taro starch granules. The

Hitachi S 2400 scanning electron microscope was used

to record and analyze the taro starch granules. The taro

starch granules were captured with 10000×

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magnification.

2.7 Analysis pasting properties

Both the native and the modified starches were

analyzed to understand their gelatinization profile and

pasting properties implying the technique from Wang et

al. (2018) with the Brabender Viscograph (Brabender®

GmbH and Co. KG., Germany) modification. The

distilled water (450 mL) was added to 30 g of taro starch

sample (dry-based) and mixed to result in the

homogenous taro starch suspension (7% starch, b/b). The

sample was balanced at 50°C for 1 min prior to the

increasing temperature up to 95°C with 6°C/min speed.

The sample was then chilled at 50°C with 6°C/min

speed. These analyses were performed using the

Thermocline Software by Brabender® GmbH and Co.

KG. Germany.

2.8 Analysis of pH, water-binding capacity, solubility

and swelling power

A calibrated pH analyzer (Thermo Scientific Orion 5

-Star from the USA) was utilized to understand the pH

value of the MTS. MTS of 5 g was mixed with the

distilled water (20 mL) and then homogenized by a

magnetic stirrer for five mins according to the method

from Airul et al. (2014) to analyze the pH value.

Moreover, Airul et al. (2014) methods were also

implemented to understand the water-binding capacity of

the MTS. The mixture of dried taro starch and distilled

water (1 g and 15 mL, respectively) were shaken for an

hour. The mixture was then centrifuged at high speed

(3000 rpm) for 10 mins using the Kubota centrifuge,

Japan.

Furthermore, the other properties of MTS such as its

solubility as well as its swelling power were gathered

using the modified technique from Jiang et al. (2012).

The sample of dried taro starch (0.1 g) was immersed

with distilled water (10 mL) in the vial, mixed using the

magnetic stirrer for 30 mins, and heated gradually from

65, 75, 85 up to 95oC for an hour. The semiliquid or

slurry taro starch was chilled afterwards and centrifuged

with High Speed Centrifuge (Kubota, Japan) at 3000

rpm, 15 mins. The deposited wet starch was separated

from its supernatant, dried for 10 mins, and weighed.

The supernatant was finally dried in the oven (Hitachi,

Japan).  Finally, a drying oven (Hitachi, Japan) was

maintained at the temperature of 110oC to dry the

supernatant until it reached its constant weight.

2.9 Amylose, amylopectin and reducing sugar content

determinations

Spectrophotometer UV-Vis (Shimadzu UV-1800,

Japan) was used to calculate the contents of amylose,

amylopectin and reducing sugar. Both amylose and

amylopectin were determined by a method from Sharlina

et al. (2017) at 620 nm, while the reducing sugar level

was analyzed using a method from Miller (1959) at 540

nm.

2.10 Statistical analysis

There were three replications in this experiment,

where the statistical analyses were implemented to

process the research data. The Duncan statistical test was

implied to examine the considerable differences at the

level of p<0.05 utilizing the SPSS 18.0 statistical

software (SPSS, Inc., Chicago, IL, USA).

3. Results and discussion

3.1 Microstructure

The microstructures of the native, autoclaving-

cooling cycle, annealing, and heat-moisture treatment

(HMT) starches under the SEM microscope were

presented in Figures 1 A, B to C, D and E, respectively.

The SEM analysis was conducted using 1000x

magnification. As shown in Figure 1A various sizes and

shapes of granules were found in the native taro starch.

The microstructure of granules was irregular polygon,

approximately had 5 µm granule sizes. This result was

almost similar to Deka and Sit (2016) experiment. The

autoclaving-cooling taro starch showed a loss in its

physical integrity. The irregular shaped particles are

dominant as the granular structure transforms into a very

compact and dense structure (Figures 1B, 1C). This

process likely occurred due to the gelatinization during

the autoclaving treatments resulting in swelling, breakup,

and degradation of taro starch granule. The taro starch

granule particles were expanded and broken because of

the entry of water and the heating process at high

temperatures. The cooling process caused re-association

of hydrogen bonds, hence the amylose structure was

trapped in the amylopectin and forms a double helix

bond (Deka and Sit, 2016). Modified taro starch by

autoclaving-cooling cannot be hydrolyzed by pancreatic

enzymes so it can be converted became resistant starch

as the prebiotic source. Furthermore, it was also

observed that the swelling and agglomeration/fusion of

the granules were relatively higher in the autoclaving-

cooling case compared to the HMT and annealing

treatment.

Aggregated and fused starch granules were found in

modified starch by HMT and annealing (Figures 1D and

1E), although the starch gelatinization was not observed.

Aggregation and fusion of the starch granules were also

observed by Hazarika and Sit (2016). HMT and

annealing treatments may cause damage of granular

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integrity or the loss of birefringence without

gelatinization (Abbas et al., 2010). The annealing

treatment at 50oC temperature for 24 hrs, however, did

not show a remarkable change in the taro starch

microstructures. The annealing treatment in this study at

a temperature of 50oC for 24 hrs also did not cause a

significant change in the microstructure of taro starch.

This can be observed in Figure 1E where there were no

double helical bonds or crystallite structure formations

during the annealing treatment. The results were in

accordance with the research by Wang et al. (2017).

Wang et al. (2018) explained that the annealing

treatment plays a role in the increase of the amorphous

regions ordering in the starch granules through

increasing starch chains interaction.

3.2 Physicochemical characteristics

3.2.1 Pasting properties

Pasting properties of taro starch were determined

based on several parameters including gelatinization

temperature, gelatinization time, peak time, peak

temperature, viscosity peak, viscosity at 93oC, viscosity

at 93oC for 20 mins, final viscosity 50oC and setback

viscosity (Table 1). The autoclaving-cooling cycle

treatment reduced the gelatinization temperature of taro

starch. Meanwhile, HMT resulted in the increases

gelatinization temperature of taro starch while the

annealing treatment did not cause a significant change of

gelatinization temperature from the native taro starch

(Table 1). These results supported the research of Deka

and Sit (2016) which modified the starch using

microwave treatment and HMT. Simsek and El (2015)

reported starch gelatinization temperatures which were

indicated by the increase interactions between amylose-

amylose, amylose-amylopectin and amylopectin-

amylopectin, hydrogen bond formation between

molecules and formation of larger crystallite structures.

Peak viscosity of taro starch correlated with swelling

the powder of taro starch granules during the

gelatinization process. Annealing taro starch had the

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Figure 1. Microstructure of (A) Native taro starch, (B) AC-1C, (C) AC-2C, (D) Annealing, (E) HMT observed in SEM

microscope with 1000 magnification.

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highest PV (240 BU) than the other treatments. The

annealing treatment in this study increased the viscosity

of the pasting properties of taro starch. These results

supported the research reported by Wang et al. (2017).

The study by Wang et al. (2018) reported that the

annealing treatment increased the viscosity of the pasting

properties of potato starch. This was associated with a

decrease in deformability and an increase in stiffness of

annealing starch. The rise of the annealing potato starch

viscosity showed that it was highly resistant towards

heating and shearing. This occurred as the ordering of

amorphous regions in annealing potato starch granules

increases.

Starch viscosity at 93oC for 20 mins resulted in a

heat resistant taro starch. The viscosity of taro starch was

constant and then increases during the heating condition

at 93oC temperature for 20 mins, mainly HMT. It

indicated that the HMT taro starch had good heat

resistance (Kaushal et al., 2012). The native taro starch,

annealing, and AC-2C taro starches also had a good heat

resistance (Table 1). However, the HMT taro starch had

the highest increase in heat resistance compared to the

other treatments (Table 1). The modified taro starch by

autoclaving-cooling treatment (AC-1C and AC-2C) had

the lowest setback viscosity (+40 BU), which meant that

the retrograded rate of this starch was the lowest among

others. Annealing taro starch had the highest Setback

Viscosity value (+120 BU), indicating that this starch

was the most retrograded compared to the other

treatments (Table 1). The results of this study supported

the research from Deka and Sit (2016) which showed

that the taro starch modification using HMT (microwave,

autoclave and hot air oven) improved the heat resistance

and reduced the retrograded rate. Research on pasting

properties in taro flour by Arici et al. (2016) showed a

significant effect due to the drying process during flour

production. High carbohydrates and low protein levels in

the taro flour allowed the water to pass through the

starch granules, therefore the swelling power also

increased.

3.2.2 Solubility and swelling power

High temperature condition increased the solubility

and the swelling power of the native and the modified

taro starch (MTS) and peaked at 85oC to 95oC (Table 2).

These results supported the previous analyses by

Sharlina et al. (2017). Deka and Sit (2016) reported the

HMT taro starch solubility at 90oC ranged from 9.68% to

11.73%, with 10.25-13.25 (g/g) swelling power. Sharlina

et al. (2017) showed that the Dioscorea pyrifolia tubers

starch at 95oC had 26.72 (g/g) swelling power and

33.29% solubility. Moreover, Jiang et al. (2012) also

examine swelling power from D. opposite, D. alata, D.

nipponica, D. bulbifera, D. septemloba starch at 95oC

were 12.42, 16.92, 8.85, 12.79, 12.14 (g/g) respectively.

Meanwhile, the solubility of D. opposite, D. alata, D.

nipponica, D. bulbifera, D. septemloba starch at 95oC

was 16.27, 30.04, 11.14, 10.46, 14.40% respectively.

The solubility and swelling power improvements

after the autoclave treatment were also reported by

Dundar and Gocman (2013). The increasing value of the

MTS solubility and swelling power occurs due to the

weathering process of starch granules during HMT

treatment (Dundar and Gocman, 2013). The starches

resulted from annealing, HMT and autoclaving-cooling

cycle treatments showed significant swelling powers

compared to the native taro starch (Table 2). The taro

starch solubility was strongly affected by the interaction

between water with amylose and amylopectin, and the

amylose-amylose and amylose-amylopectin chains bonds

(Sharlina et al., 2017). Swelling power was heavily

influenced by amylose content, the starch ability to bind

water, the level of crystallinity, and the hydrogen bonds

(Jiang et al., 2012; Sharlina et al., 2017). Sharlina et al.

(2017) report that there was a negative correlation

between amylose starch content and swelling power.

This occurs as the swelling power capability was limited

by the increasing number of amylose in the starch

content.

The autoclaving-cooling cycle and HMT changed

the starch granules, increased the granule size, therefore

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Treatment

G
elatin

iza
tio

n

tim
e (m

in
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G
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n

te
m

p
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re
(°C

)

P
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 tim
e

(m
in

s)

P
eak

te

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p

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re

(°C
)

V
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 P

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U

)

V
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 9

3
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(B

U
)

V
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9
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/2

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’ (B

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)

F
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isco

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 (B

U
)

S
etb

ack

v
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 (B

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Native Taro starch 35 82.5 43 94 220 210 210 320 (+110)

AC-1C 24 66 37 85.5 170 160 140 180 (+40)

AC-2C 22 66 34 81 100 90 80 120 (+40)

Annealing 34 81 42 93 240 240 250 370 (+120)

HMT 39 88.5 - - - 50 110 190 (+80)

Table 1. Pasting properties as gelatinization time and temperature, peak time and temperature and viscosity profile of modified

taro starch

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lead to the swelling power improvement. The swelling

power of taro starch was also influenced by the structure

of amylose and amylopectin, and the magnitude

interactions in amorphous and crystalline regions. The

crystal structure in the amylopectin molecule played an

important role in the gelatinization and the swelling the

power of taro starch. The swelling power improvement

was affected by the long chain ratio of amylopectin (≥35

degrees polymerization), lipids and phosphorus (Naidoo

et al., 2015). Naidoo et al. (2015) showed that the

presence of lipid residues and amylose-lipid complexes

can reduce the swelling power of starch.

3.2.3 Water-binding capacity (WBC), pH, amylose

and amylopectin, reducing sugar content

The autoclaving-cooling cycle (AC-1C, AC-2C) and

HMT increased significantly (p<0.05) the WBC of taro

starch compared to the native taro starch. WBC of

modified taro starch AC-1C, AC-2C and HMT

treatments were not significantly different (p<0.05)

(Table 3). Modified taro starches (HMT, AC-1C, and AC

-2C) had WBC value between 70.94% and 73.84%

(p<0.05) (Table 3). D. pyrifolia starch was 2.45% WBC

value as reported by Sharlina et al. (2017). Moreover,

Jiang et al. (2012) reported the following WBC results:

D. opposite 1.41%, D. alata 1.56%, D. nipponica 4.43%,

D. bulbifera 2.53% and D. septemloba 5.57%. This study

revealed that the native taro starch and the MTS have

higher WBC values compared to D. pyrifolia, D.

opposite, D. alata, D. nipponica, D. bulbifera, D.

septemloba starches. The WBC (%) had a positive

correlation with solubility (%) and swelling power. As

the MTS bind with water, it got easier to expand and

dissolve in water. The WBC (%) was determined by the

number of hydrogen bonds formed between the water

molecule and the hydroxyl group in taro starch. In

addition, WBC (%) was also influenced by the degree of

starch polymerization and the diversification of the

structure of taro starch granules (Kaushal et al., 2012;

Sharlina et al., 2017).

Modified taro starches (MTS) had pH values

between 4.5 and 5.04 (Table 3). The HMT taro starch pH

(4.5) was not significantly different from the native taro

starch (4.56). On the other hand, the Annealing and the

autoclaving-cooling cycling treatments increased the pH

of taro starch. Sharlina et al. (2017) measured the pH

value of D. pyrifolia tuber starch of 3.43, which was

relatively lower than the pH from the native and the

MTS taro starches in this study. The heating process of

the HMT released the hydrogen ions (H+), therefore

increased the taro starch pH.

The experiments indicated that the amylose content

of native taro starch, HMT, AC-2C taro starch had

significant differences (p <0.05). The amylose content of

the native taro was 25.33%, the HMT taro starch was

16.01%, and the AC-2C taro starch was 27.40% (Table

3). The amylose value of the HMT taro starch relatively

decreased due to the partial gelatinization during the

heating process which damaging the amylose structure

(Zheng et al., 2018; Cheng et al., 2019). The autoclaving

-cooling 2 cycle (AC-2C) caused an increase in amylose

levels and a decrease in amylopectin level in taro starch.

The amylose content of modified taro starch (AC-2C)

revealed an increase significantly compared to native

taro starch (p <0.05) (Table 3). The improvement levels

of amylose in autoclaving-cooling (AC-2C) taro starch

were caused by depolymerization of long chain amylose

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 Treatment

Solubility (%) Swelling power (g/g)

65°C 75°C 85°C 95°C 65°C 75°C 85°C 95°C

Native taro starch 2.55±0.54a 6.43±0.44b 16.16±0.28e 42.06±0.24j 2.06±0.40a 13.12±0.46d 17.01±0.34e 23.75±0.84f

AC-1C 13.67±0.43d 16.80±0.23e 21.26±0.35f 39.99±0.73i 12.95±0.52c 14.34±0.82d 15.59±0.26d 24.99±0.60g

AC-2C 23.02±0.25f 23.66±0.14f 25.96±0.65g 44.58±0.59j 12.91±0.56c 14.99±0.64d 15.24±0.78d 16.71±0.50e

Annealing 2.17±0.49a 10.98±0.67c 34.07±0.43h 37.47±0.41i 3.35±0.38a 13.55±0.27d 16.74±0.63e 18.92±0.71e

HMT 6.81±0.58b 10.97±0.75c 17.34±0.29e 20.46±0.38f 8.71±0.89b 10.19±0.19c 13.83±0.74d 17.89±0.84e

Table 2. Solubility and swelling power of modified taro starch

Different superscript letters within a row were significantly different at p<0.05 level

Treatment
Water binding capacity

(%)
pH

Amylose
(% dry weight)

Amylopectin
(% dry weight)

Reducing sugar
(% dry weight)

Native taro starch 54.47±0.84a 4.56±0.02a 25.33±0.93b 74.67±0.72b 9.22±0.12c

AC-1C 71.21±0.68b 4.76±0.01a 26.01±0.88b 73.99±0.91b 6.12±0.31b

AC-2C 73.84±0.41b 5.04±0.01b 27.40±0.56c 72.60±0.58b 6.36±0.48b

Annealing 53.08±0.32a  4.76±0.02a 24.24±0.83b  75.76±0.82b 8.89±0.52c

HMT 70.94±0.41b  4.50±0.01a 16.01±0.57a  83.99±0.61c 2.53±0.71a

Table 3. Water binding capacity (WBC), pH, amylose, amylopectin, reducing sugar content of modified taro starch

Different superscript letters within a row were significantly different at p<0.05 level

1231

 Setiarto et al. / Food Research 4 (4) (2020) 1226 - 1233

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

molecules and amylopectin to form short chain amylose

fractions (Chen et al., 2018). In taro starch annealing,

there was a re-association of amylose chains formed by

the amylopectin breakdown (Wang et al., 2017). A study

by Moore et al. (2015) showed that the increased

amylose content and decreased reducing sugar level

resulted in a positive effect on the formation of resistant

starch (RS). Long-chain amylose was hydrolyzed either

by heating, acid or enzymatically to form retrograded

short chain amylose fractions (Chen et al., 2018).

The HMT and AC-1C, AC-2C starches showed a

significant decrease in reducing sugar levels compared to

the native taro starch (p<0.05) (Table 3). Annealing

treatment did not show a significant decrease in the

reducing sugar level of taro starch than the others

(p<0.05). The HMT showed the most significant

decrease by 2.53% in the reducing sugar content of taro

starch (Table 3). The decrease of reducing sugar levels in

HMT and autoclaving-cooling cycle treatments was

caused by the Maillard reaction. The Maillard reaction

occurs due to the interaction between the amino acids

and the reducing sugars at higher temperatures. Maillard

reaction was influenced by high temperature, heating

time and autoclaving-cooling cycles. The high reducing

sugar levels inhibited the occurrence of the retrograded

process, so it could reduce the RS level.

4. Conclusion

The autoclaving-cooling 2 cycles (AC-2C) changed

the microstructure of MTS transforms into a very

compact and dense, so it can be converted as resistant

starch as a prebiotic source. Despite, MTS by HMT and

annealing treatments showed similar microstructures

with their native taro starch. AC-2C improved shear

stress resistance, heat resistance and low retrogradation

modified taro starch (MTS). The AC-2C treatment

increased water binding capacity (73.84%), solubility

(44.58%), and swelling power (16.71%) of MTS. The

water-binding capacity had a positive correlation with

solubility and swelling power. The AC-2C treatment

increased amylose content (27.40%) and decreased

reducing sugar level (6.36%) of MTS, so it can

encourage the formation of resistant starch to improve

the prebiotic properties of taro starch. Modified taro

starch AC-2C is the best compared to HMT and

annealing based on microstructure and physicochemical

characteristics.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

The authors would like to express their deepest

gratitude to Research Center for Biology, Indonesian

Institute of Sciences, and Science and Technology

Postgraduate Scholarship from Ministry of Research and

Technology in the Republic of Indonesia which had

funded this research.

References

Abbas, K.A., Khalil, S.K. and Hussin, A.S.M. (2010).

Modified starches and their usages in selected food

products: a review study. Journal of Agricultural

Science, 2(2), 90-100. https://doi.org/10.5539/

jas.v2n2p90

Aboubakar, Njintang, N.Y., Scher, J. and Mbofung,

C.M.F. (2009). Texture, microstructure and

physicochemical characteristics of taro (Colocasia

esculenta) as influenced by cooking conditions.

Journal of Food Engineering, 91(3), 373-379.

https://doi.org/10.1016/j.jfoodeng.2008.09.030

Airul, A., Yusof, M.S.M., Jamil, M.S., Abdullah, A.,

Yusoff, S.F.M., Arip, M.N.M. and Lazim, A.M.

(2014). Physicochemical characterization of starch

extracted from Malaysian wild yam (Dioscorea

hispida Dennst.). Emirates Journal Food

Agriculture, 26(8), 652–658. https://doi.org/10.9755/

ejfa.v26i8.17098

Anderson, A.K., Guraya, H.S., James, C. and Salvaggio,

L. (2002). Digestibility and pasting properties of rice

starch heat-moisture treated at the melting

temperature. Starch/Stärke, 54, 401–409. https://

doi.org/10.1002/1521-379X(200209)54:9<401::AID

-STAR401>3.0.CO;2-Z

Arici, M., Yildirim, R.M., Ozülkü, G., Yasar, B. and

Toker, O.S. (2016). Physicochemical and nutritional

properties of taro (Colocasia esculenta L. Schott)

flour as affected by drying temperature and air

velocity. LWT - Food Science and Technology, 74,

434-440. https://doi.org/10.1016/j.lwt.2016.08.006

Ashwar, B.A., Gani, A., Wani, I.A., Shah, A., Masoodi,

F.A. and Saxena, D.C. (2016). Production of

resistant starch from rice by dual autoclaving

retrogradation treatment: In-vitro digestibility,

thermal and structural characterization. Food

Hydrocolloids, 56, 108-117. https://doi.org/10.1016/

j.foodhyd.2015.12.004

Chen, Y.F., Singh, J. and Archer, R. (2018). Potato

starch retrogradation in tuber: Structural changes and

gastro-small intestinal digestion in vitro. Food

Hydrocolloids, 84, 552-560. https://doi.org/10.1016/

j.foodhyd.2018.05.044

Cheng, K., Chen, S. and Yeh, A. (2019).

F
U

L
L

 P
A

P
E

R

1232

about:blank

about:blank

about:blank

about:blank

about:blank

about:blank

about:blank

about:blank

https://doi.org/10.1016/j.lwt.2016.08.006

https://doi.org/10.1016/j.foodhyd.2015.12.004

https://doi.org/10.1016/j.foodhyd.2015.12.004

about:blank

about:blank

 Setiarto et al. / Food Research 4 (4) (2020) 1226 - 1233

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

Physicochemical properties and in vitro digestibility

of rice after parboiling with heat moisture treatment.

Journal of Cereal Science, 85, 98-104. https://

doi.org/10.1016/j.jcs.2018.11.016

Deka, D. and Sit, N. (2016). Dual modification of taro

starch by microwave and other heat moisture

treatments. International Journal of Biological

Macromolecules, 92, 416-422. https://

doi.org/10.1016/j.ijbiomac.2016.07.040

Dundar, A.N. and Gocmen, D. (2013). Effects of

autoclaving temperature and storing time on resistant

starch formation and its functional and

physicochemical properties. Carbohydrate Polymers,

97(2), 764-771. https://doi.org/10.1016/

j.carbpol.2013.04.083

Hazarika, B.J. and Sit, N. (2016). Effect of dual

modification with hydroxypropylation and cross-

linking on physicochemical properties of taro starch.

Carbohydrate Polymers, 140, 269-278. https://

doi.org/10.1016/j.carbpol.2015.12.055

Jiang, Q., Gao, W., Li, X., Xia, Y., Wang, H., Wua, S.

and Xiao, P. (2012). Characterizations of starches

isolated from five different Dioscorea L. species.

Food Hydrocolloids, 29(1), 35-41. https://

doi.org/10.1016/j.foodhyd.2012.01.011

Kaushal, P., Kumar, V. and Sharma, H.K.  (2012).

Comparative study of physicochemical, functional,

antinutritional and pasting properties of taro

(Colocasia esculenta), rice (Oryza sativa) flour,

pigeon pea (Cajanus cajan) flour and their blends.

LWT - Food Science and Technology, 48(1), 59-68.

https://doi.org/10.1016/j.lwt.2012.02.028

Miller, G.L. (1959). Use of dinitrosalicylic acid reagent

for determination of reducing sugar. Journal

Analytical Chemistry, 31, 426-428. https://

doi.org/10.1021/ac60147a030

Moore, S.A., Ai, Y., Chang, F. and Jane, J. (2015).

Effects of alpha-amylase reaction mechanisms on

analysis of resistant starch contents. Carbohydrate

Polymers, 115, 465-471. https://doi.org/10.1016/

j.carbpol.2014.09.014

Naidoo, K., Amonsou, E.O. and Oyeyinka, S.A. (2015).

In vitro digestibility and some physicochemical

properties of starch from wild and cultivated

amadumbe corms. Carbohydrate Polymers, 125, 9-

15. https://doi.org/10.1016/j.carbpol.2015.02.066

Setiarto, R.H.B., Jenie, B.S.L., Faridah, D.N.,

Saskiawan, I. and Sulistiani. (2018). Effect of lactic

acid bacteria fermentation and autoclaving-cooling

for resistant starch and prebiotic properties of

modified taro flour. International Food Research

Journal, 25(4), 1691-1697.

Shah, A., Masoodi, F.A., Gani, A. and Ashwar, B.A.

(2016). In-vitro digestibility, rheology, structure, and

functionality of RS3 from oat starch. Food

Chemistry, 212, 749-758. https://doi.org/10.1016/

j.foodchem.2016.06.019

Sharlina, M.S.E., Yaacob, W.A., Lazim, A.M., Fazry, S.,

Lim, S.J., Abdullah, S., Noordin, A. and Kumaran,

M. (2017). Physicochemical Properties of Starch

from Dioscorea pyrifolia tubers. Food Chemistry,

220, 225-232. https://doi.org/10.1016/

j.foodchem.2016.09.196

Simsek, S. and El, S.N. (2015). In vitro starch

digestibility, estimated glycemic index and

antioxidant potential of taro (Colocasia esculenta L.

Schott) corm. Food Chemistry, 168, 257-261. https://

doi.org/10.1016/j.foodchem.2014.07.052

Wang, S., Wang, J., Wang, S. and Wang, S. (2017).

Annealing improves paste viscosity and stability of

starch. Food Hydrocolloids, 62, 203-211. https://

doi.org/10.1016/j.foodhyd.2016.08.006

Wang, X., Reddy, C.K. and Xu, B. (2018). A systematic

comparative study on morphological, crystallinity,

pasting, thermal and functional characteristics of

starches resources utilized in China. Food

Chemistry, 259, 81-88. https://doi.org/10.1016/

j.foodchem.2018.03.121

Xu, M., Saleh, A.S.M., Gong, B., Li, B., Jing, L., Gou,

M., Jiang, H. and Li, W. (2018). The effect of

repeated versus continuous annealing on structural,

physicochemical, and digestive properties of potato

starch. Food Research International, 111, 324–333.

https://doi.org/10.1016/j.foodres.2018.05.052

Yu, Z.Y., Jiang, S.W., Cai, J., Cao, X.M., Zheng, Z.,

Jiang, S.T., Wang, H.L. and Pan, L.J. (2018). Effect

of high pressure homogenization (HPH) on the

rheological properties of taro (Colocasia esculenta

(L). Schott) pulp. Innovative Food Science and

Emerging Technologies, 50, 160-168. https://

doi.org/10.1016/j.ifset.2018.09.002

Zheng, B., Wang, H., Shang, W., Xie, F., Li, X., Chen,

L. and Zhou, Z. (2018). Understanding the

digestibility and nutritional functions of rice starch

subjected to heat-moisture treatment. Journal of

Functional Foods, 45, 165-172. https://

doi.org/10.1016/j.jff.2018.03.041

F
U

L
L

 P
A

P
E

R

1233

https://doi.org/10.1016/j.jcs.2018.11.016

https://doi.org/10.1016/j.jcs.2018.11.016

https://doi.org/10.1016/j.ijbiomac.2016.07.040

https://doi.org/10.1016/j.ijbiomac.2016.07.040

https://doi.org/10.1016/j.carbpol.2013.04.083

https://doi.org/10.1016/j.carbpol.2013.04.083

https://doi.org/10.1016/j.carbpol.2015.12.055

https://doi.org/10.1016/j.carbpol.2015.12.055

about:blank

about:blank

about:blank

about:blank

about:blank

https://doi.org/10.1016/j.carbpol.2014.09.014

https://doi.org/10.1016/j.carbpol.2014.09.014

https://doi.org/10.1016/j.carbpol.2015.02.066

https://doi.org/10.1016/j.foodchem.2016.06.019

https://doi.org/10.1016/j.foodchem.2016.06.019

https://doi.org/10.1016/j.foodchem.2016.09.196

https://doi.org/10.1016/j.foodchem.2016.09.196

https://doi.org/10.1016/j.foodchem.2014.07.052

https://doi.org/10.1016/j.foodhyd.2016.08.006

https://doi.org/10.1016/j.foodhyd.2016.08.006

about:blank

about:blank

about:blank

https://doi.org/10.1016/j.ifset.2018.09.002

https://doi.org/10.1016/j.jff.2018.03.041


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November 16, 2016November 18, 2016
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