Optimal Inventory Strategies for Pharmaceutical Products Incorporating Carbon Emissions
Adekunle Odunayo Adejuwon1* and Victoria Anatolyivna Tsygankova2
1School of Health Information Management, University College Hospital
2Department for Chemistry of Bioactive Nitrogen-Containing Heterocyclic Compounds, Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine, Ukraine
Submission: April 02, 2019; Published: June 11, 2019
*Corresponding author: Adekunle Odunayo Adejuwon, School of Health Information Management, University College Hospital, Ibadan, Nigeria
How to cite this article: Adekunle O A, Victoria A T. α-Amylase Production Using Aspergilus vadensis Isolated from Pulverized Cocoa Seeds. Curr Trends
002 Biomedical Eng & Biosci. 2019; 19(2): 556010. DOI: 10.19080/CTBEB.2019.19.556010
Aim: α-Amylases are well known for their applications ranging from food and paper industries to pharmaceutical industries.
Materials and Methods: Aspergillus vadensis isolated from pulverised cocoa seed was employed in the submerged fermentation of plantain peel. The pH and temperature of the extract were measured at two days (48 hrs) interval using pH meter and sugar concentration was also determined using Fehling test. Protein content and α-amylase activity of the crude extract was determined.
Results:The pH of the crude extract decreased from 4.78 on the first day to 2.51 on day eight (192 hrs) while the temperature of the extract fluctuated between 27 oC and 28 oC. The sugar concentration in the extract increased from 3.36 mg/ml after 48 hrs to 26 mg/ml after 144 hrs and declined to 8.02 mg/ml after 192 hrs. The α-amylase activity also reached its peak (68.86 U/mg protein) on the 6th day (144 hrs) but declined to 50 U/mg protein on the 8th day (192 hrs).
Acknowledgements: It can be concluded that plantain peel can be employed as a cheap and readily available substrate in the production of α-amylase that is used for various biotechnologically-based industrial applications thereby, adding value to plantain and also decreasing the amount of this agro industrial waste in the environment.
α-Amylases are enzymes that are well known for their applications in starch and food, brewing, distilling, textile, paper and pharmaceutical industries [1-3]. This large range of applications is the triggering factor for the industrial production of this enzyme . Presently the enzyme is one of the mostly sought after as it has great significance in biotechnology; constituting a class of industrial enzymes that controls about 25% of the world’s total enzyme market [5,6]. Agro industrial wastes have been reported to be good substrates for the cost effective production of alpha amylases  and are thus attracting researchers for using agro industrial waste as a substrate for alpha amylase production. Agricultural wastes include but are not limited to plantain peel, wheat bran, rice husk, banana peel, vegetable waste and citrus waste. A wide range of microbes, such as bacteria and fungi are used for the industrial production of amylases . The use of microorganisms for the production of amylases is economical as microbes are easy to manipulate to obtain enzymes of desired characteristics . However, fungi are preferred over bacteria for enzyme production because of their filamentous nature, which helps in their penetration through solid substrate .
Plantain (Musa spp.) occupies a strategic position for rapid food production in Nigeria. It is ranked third among starchy staples . Total world production of plantain is estimated to be over 75 million metric tons . Twelve million metric tons are produced in Africa annually . Nigeria is one of the largest plantain producing countries in the world. It is the largest producer in West Africa with annual production of about 2.4 million metric tons mostly obtained from the Southern States . However, Nigeria does not feature among plantain exporting nations because it produces more for local consumption than for export . Plantain is a major source of carbohydrate for more than 50million people . Besides being the staple for many people in more humid regions, plantain is a delicacy and favoured snack for people even in other ecologies . In Nigeria, the ripe fruit are processed into different forms for consumption either by boiling, frying or roasting. The peel of the fruit is discarded as waste after the inner fleshy portion has been eaten, thereby constituting a menace to the environment, especially where its consumption is common . According to FAO  about 17,397,000 metric tonnes of plantain peels are generated in African countries.
The accumulation of the discarded peel may lead to the generation
of domestic waste which could constitute health hazards
in different parts of the country. Hence, this study aims at
generating additional value to plantain by utilizing the peel as
a substrate for the production of the enzyme amylase in a view
to converting this agricultural and potentially hazardous waste
The isolate of Aspergillus vadensis used for this investigation
was isolated from pulverised cocoa seeds. Potato dextrose agar
(PDA) media was prepared, autoclaved and poured in sterile Petri
dishes. One gram of the pulverised cocoa seeds was transferred
into 20 mL of sterile distilled water. 0.1 mL from the mixture
was transferred into the prepared PDA and the plates were
incubated at 27 oC for 72 hours. Nine different isolates were
observed on the incubated plates, Aspergillus vadensis used for
this study was identified based on its physical and microscopic
(Lactophenol cotton blue) characteristics . This was used
as a form of preliminary identification. Aspergillus vadensis was
further sub-cultured on a freshly prepared PDA and incubated at
27 oC for 72 hours .
The ripe plantain purchased from the local market was
peeled off and the pulp (fruit) was separated. The peels were
thoroughly washed using sterile distilled water and were cut
into small pieces followed by homogenization in a blender. Water
was added to facilitate the homogenization.
Fermentation was carried out using the modified method
of Khan & Yadav . Thirty gram (30 g) each of the paste of
plantain peel was dispensed into eight different 250 mL conical
flasks and about 70 mL of basal medium containing the following
in g/l (0.8 g NaCl, 0.8 g KCl , 0.1 g CaCl2, 2.0 g Na2HPO4, 0.2g
MgSO4, 0.1 g FeSO4, 8.0g Fructose, 2.0 g NH4Cl was added. The
contents in conical flasks were autoclaved, cooled at room temperature,
inoculated with 1 mL of 72 hours old grown culture
and incubated at 27 oC. The contents of one of the conical was
not inoculated with Aspergillus vadensis, this was used as the
control for comparison.
Different concentrations of glucose used as standard were
prepared from 5g of D-glucose dissolved in 45ml of distilled water.
The sugar contents of the D-glucose and crude extract were
determined, using Fehling’s sugar test method. 20ml of prepared Fehling solution “A” was mixed with 20 ml of Fehling solution “B”.
2ml of the Fehling solution mixture was added to 5 test tubes
containing the different concentrations of D glucose used as
standard, and the diluted crude. The test tubes were placed in a
water bath at 60 °C for 15min after which absorbance was read
by spectrophotometer at a wavelength of 500 nm.
Extraction of crude enzyme was done according to the modified
method of Adejuwon . Contents of the flask was filtered
using Whatman no. 1 filter paper in a cold chamber at 4 oC. The
extract was then subjected to cold centrifugation at 10,000 rpm
for 10 minutes at 4 oC using a high speed cold centrifuge. This
served as the crude enzyme. Amylase activity was determined
. The protein content of the preparation was determined
Concentration of protein in crude enzyme was determined
by the method of Bradford  in which crude enzyme was reacted
with Bradford reagent and the absorbance obtained was
compared with a standard graph plotted by reacting a standard
protein with known concentrations with the Bradford reagent
and plotting a graph between concentration of standard protein
(Bovine Serum Albumin) on X axis and absorbance at 595 nm
on Y axis
α-Amylase activity was assayed using the modified method
of Pfueller & Elliott  and Adejuwon et al. . The reaction
mixture was 2 mL of buffered (0.02m citrate phosphate buffer
pH 6.0), soluble starch (Sigma) and 0.5 mL enzyme. The control
consisted of only the prepared substrate. Incubation was at 35
°C for 30 minutes. The reaction was terminated with 3 mL of 1 N
HCl. 2 mL of the terminated reaction mixture was added to 3 mL
of 0.1N HCl. Colour was developed by adding 0.1ml iodine solution.
Controls consisted of only 2 mL of the prepared substrate.
One unit of α-amylase activity was defined as the amount of enzyme
which produced 0.01% reduction in the intensity of the
blue colour of the starch-iodine complex under assay conditions.
The pH of the crude extract from the fermenting media was
measured using a pH meter after every two days (48 hrs) and as
shown in Table 1, the pH decreased as fermentation progresses.
The temperature of the crude extract from the fermenting media was measured using a thermometer after every two days
(48 hrs) and as shown in Table 2, the temperature fluctuated between
27 oC and 28 oC as fermentation progressed. The sugar
concentration in the crude extract from the fermenting media
was determined using a Fehling test after every two days and as
shown in Table 3, the sugar concentration reached its peak on
the sixth day after fermentation and declined on the eight day.
The crude extract from the fermenting media was assayed for
α-amylase activity using the method of Pfueller & Elliott .
This was done after every two days (48 hrs) and as shown in
Table 4, α-amylase activity reached its peak on the sixth day (144
hrs) after fermentation and declined on the eight day (192 hrs).
As shown in Figure 1, the initial pH decreased from 4.78 on
the first day to 2.51 on day eighth (192 hrs). This may be due
to production of acid and alcohol as a result of prolonged fermentation.
As shown in Figure 2, the temperature in the crude extract varied between 27 oC and 28 oC. As shown in Figure 3,
the sugar concentration in the extract from the fermented peel
increased from 3.6 mg/ml after 48 hrs to 26 mg/ml after 144
hrs. It declined to 8.09 mg/ml on the 8th day. As shown in Figure
4, the α-amylase activity of the crude from the fermented
peel increased from 0 U/mg protein on the first day to 64 U/mg
protein on the sixth day. It declined to 50 U/ mg protein on the
Plantain peels are by-products of the plantain-processing
industry, which are normally dumped in landfills, rivers or un regulated grounds . The bio-accumulation of this waste may
pose serious environmental problem; hence, there is a need for
effective utilization of this waste. Agro industrial wastes such
as banana peel and cassava peel have been reported to be good
substrate for amylase production [4,24]. The expression of
α-amylase activity by Aspergillus vadensis with plantain peel as
the sole substrate is an indication of constitutive expression or
induction of the enzyme in the fungus. α-Amylase production by
microorganisms may be constitutive or inductive [25,26]. The
pH in the fermenting sample declined as the fermentation progressed;
this could be as a result of acid and alcohol production
due to prolonged fermentation.
The sugar concentration attained its peak 26 mg/ml on day
six after fermentation (144 hrs) and declined to 8.09 mg/ml on
the eight day, this showed that the fungus is able to optimally
utilize the sugar present in the fermenting sample on the 6th day
due to the complete hydrolysis of the substrate, this reflected in
the α-amylase activity which also reached its peak 64.86 U/mg
protein on the 6th day.
The decline in α-amylase activity from 64.86 U/mg protein
on the 6th day to 50 U/mg protein on the 8th day (192 hrs) could
be attributed to the decline in the sugar concentration in the fermenting
sample. It could also be attributed to depletion of nutrient
and also accumulation of by-products such as toxins and
Elmarzugi et al.  reported that amylase contributed
about 25-30% to a US$ 2.7 billion world enzyme market as at
2012 which is estimated to increase by 4% annually. Considering
the fact that Nigeria is one of the largest producers of plantain in
Africa , this study is important as it will not only add value
to plantain and help reduce environmental problems but could
also be a source of revenue for Nigeria as it looks to diversity in
It can be concluded that plantain peel can be employed as a
cheap and readily available substrate in the production of α-amylase
that is used for various industrial applications thereby,
adding value to plantain and also decreasing the amount of this
agro industrial waste in the environment. Future prospects of
the present study include optimization of pH and optimization
of the sugar concentration in order to maintain a constant production
for a longer period
Krishna PR, Srivastava AK, Ramaswamy NK, Suprasanna P, Souza SFD (2011) Banana peel as substrate for alpa-amylase production using Aspergillus niger NCIM 616 and process optimization. Indian Journal of Biotechnology 11: 314-319.
Khan JA, Yadav SK (2011) Production of alpha amylases by Aspergillus niger using cheaper substrates employing solid state fermentation. International Journal of Plant, Animal and Environmental Sciences 1(3): 100-108.
Reddy NS, Nigmmagadda A, Sambasiva Rio KRS (2003) An overview of the microbial α–amylase family. African J Biotechnol 2: 645-648.
Aiyer PV (2005) Amylases and their applications. Afr J Biotechnol 4: 1525-1529.
Ramachandran S, Patel AK, Nampoothiri KM, Francis F, Nagy V (2004) A potential raw material for the production of alpha-amylase. Bioresource Technol 93: 169-174.
IITA: International Institute of Tropical Agriculture (2014) A Reference Manual on Plantain Cultivation in West Africa 1-28.
John P, Marchal J (1995) Ripening and biochemistry of the fruit, In: Gowen SR (Eds.), Bananas and Plantains, Chapman and Hall, London, U.K.
Fakayode BS, Rahji MAY, Ayinde O, Nnom GO (2011) An economic assessment of plantain production in Rivers State, Nigeria. Int J Agric Econ Rural Dev 4(2).
FAO: Food and Agriculture Organization of the United Nations (2006) Production Yearbook 2004. FAO, Rome.
Fortaleza C (2012) The ultimate wealth guide to making millions of Naira yearly with plantain farming in Nigeria.
Adeolu AT, Enesi DO (2013) Assessment of proximate, mineral, vitamin and phytochemical compositions of plantain (Musa paradisiaca) bract - an agricultural waste. International Research Journal of Plant Science 4(7): 192-197.
Okareh OT, Adeolu AT, Adepoju OT (2015) Proximate and mineral composition of plantain (Musa paradisiaca) wastes flour; a potential nutrients source in the formulation of animal feeds. African Journal of Food Science and Technology 6(2): 53-57.
FAO: Food and Agriculture Organization of the United Nations (1988) Production Yearbook 42, FAO, Rome.
Cannon PF, Kirk PM (2007) Fungal Families of the World. CAB International Publishing, Wallingford, Oxfordshire, Pp 45-56.
Adejuwon AO, Oluduro AO, Agboola FK, Ajayi AA, Olutiola PO, et al. (2015) Expression of alpha-amylase by a tropical strain of Aspergillus niger: Effect of carbon source of growth. Nat Sci 13(8): 66-69.
Adejuwon AO (2010) Synthetic production of amylase from Aspergillus niger isolated from citrus fruit. African Journal of Basic and Applied Sciences 2(5-6): 158-160.
Adejuwon AO, Tsygankova VA, Alonge O (2018) Effect of cultivation conditions on activity of α-amylase from a tropical strain Aspergillus flavus Link. Journal of Microbiology, Biotechnology & Food Sciences 7(6): 571-575.
Osma JF, Herrera JLT, Couto Sr (2007) Banana skin: A novel waste forlactase production by Trametspubescens under solid-state conditions. Application to synthetic dye decolouration. Dye & Pigments 5: 32-37.
Brisibe EA, Bankgong H (2014) Biotechnological potential of alpha amylase production by Bacillus subtilis using cassava peel powder as a substrate. British Biotechnology Journal 4(11): 1201-1211.
Dixon M, Webb EC (1971) Enzymes. Longmans, London, Pp 950.
Dunn G (1974) A model for starch breakdown in higher plants. Phytochem 13: 1341-1346.
Mulimani VH, Patil G, Ramalingan N (2000) Alpha amylase production by solid state fermentation: A new practical approach to biotechnology sources. Biochem Adv 28: 161-163.
Teodoro CE, Martins LL (2000) Culture conditions for the production of thermostable amylase by Bacillus species. Braz J Microbiol 31: 298-2302.
Elmarzugi NA, El Enshasy HA, Abdul Hamid M, Hasham R, Aziz A, et al. (2014) A amylase economic and application value. World Journal of Pharmaceutical Research, 3(3): 4890-4906.