Full Length Research Paper - Global Journal of Environmental Science and Technology ( 2022) Volume 10, Issue 3

Glucose Production from Banana Peel using Aspergillus flavus and Aspergillus oryzae ATCC 17891

A Ajiboye¹, N Magaji¹ and R Olawoyin^{2* 1}Department of Biosciences and Biotechnology, Microbiology Unit, College of Pure and Applied Sciences, Kwara State University, Malete, Kwara State, Nigeria
²Department of Science Laboratory Technology, Federal Polytechnic, Offa, Kwara State, Nigeria
^*Corresponding Author:
R Olawoyin, Department of Science Laboratory Technology, Federal Polytechnic, Offa, Kwara State, Nigeria, Email: bosran@yahoo.com

Received: 11-Jun-2022, Manuscript No. GJEST-22-66420; Editor assigned: 14-Jun-2022, Pre QC No. GJEST-22-66420(PQ); Reviewed: 28-Jun-2022, QC No. GJEST-22-66420; Revised: 10-Aug-2022, Manuscript No. GJEST-22-66420(R); Published: 17-Aug-2022, DOI: 10.15651/GJEST.22.10.011

Introduction

Sugar is one of the basic needs of the Indonesian people, especially for consumption and food processing. Sugar demand in Indonesia is still dominated by sugar (sucrose). Glucose is a monosaccharide with the molecular formula C₆H₁₂O₆. Glucose is widely used in the food and pharmaceutical industries. Enzymatic hydrolysis produces glucose concentrations higher than the acid hydrolysis (Ayoola, et al., 2012).

Banana is a tropical fruit grown in over 122 countries worldwide. Until 2004, the culivated area of 3.8 million hectares and a total production of 56.4 million metric tonnes of the fruit were produced ranking it fourth behind rice, corn and milk (Chai, et al., 2004; Arumugam and Manikandan, 2011). In recent times, banana peel has been utilized for various industrial applications including bio-fuel production, bio-sorbents, pulp and paper, cosmetics, energy related activities, organic fertilizer, environmental cleanup and biotechnology related processes (Gunaseelan, 2004; Bori et al., 2007). Banana plantation occupies large part of the land, but it is a contamination source because after harvest, the tree is cut down and abandoned in the fields, which foments Sigatoka (Chillet, et al., 2009). All parts of the banana plant have medicinal applications (Amit and Shailandra, 2006): the flowers in bronchitis and dysentery and on ulcers; cooked flowers are given to diabetics; the astringent plant sap in cases of hysteria, epilepsy, leprosy, fevers, hemorrhages, acute dysentery and diarrhea, and it is applied on hemorrhoids, insect and other stings and bites; young leaves are placed as poultices on burns and other skin afflictions; the astringent ashes of the unripe peel and of the leaves are taken in dysentery and diarrhea and used for treating malignant ulcers (Girish and Satish, 2008); the roots are administered in digestive disorders, dysentery and other ailments; banana seed mucilage is given in cases of diarrhea in India (Bhat et al., 2010). Antifungal and antibiotic principles are found in the peel and pulp of fully ripe bananas (Brooks, 2008). The antibiotic acts against mycobacteria (Omojasola and Jilani, 2009). A fungicide in the peel and pulp of green fruits is active against a fungus disease of tomato plants (Ponnuswamy et al., 2011). Norepinephrine, dopamine, and serotonin are also present in the ripe peel and pulp (Ratule, et al., 2007). The first two elevate blood pressure; serotonin inhibits gastric secretion and stimulates the smooth muscle of the intestines (Anhwange et al., 2009). Some of the specific diseases known to be cured by banana are:

Anaemia: High in iron, bananas are believed to stimulate the production of haemoglobin in the blood and so help in cases of anaemia (Amit and Shailandra, 2006).

Blood pressure: Banana is extremely high in potassium yet low in salt, making it the perfect food for helping to beat blood pressure (Debabandya et al., 2010).

Depression: This is because bananas contain tryptophan, a type of protein that the body converts into serotonin known to make you relax, improve your mood and generally make you feel happier (Girish and Satish, 2008).

Materials and Methods

Sample collection and Identification bananas (Musa sapientum) samples were purchased from Ipata market, Ilorin, Kwara State. Nigeria. The banana sample were collected in sterile polythene bags each and labeled appropriately then immediately transported to the Microbiology laboratory (Makut, et al., 2021).

Test Organisms and Preparation of Spore Suspensions

The organisms used in this study were Aspergillus flavus and Aspergillus oryzae (ATCC 17891) was obtained from Federal Institute of Industrial Research Oshodi (FIIRO) Lagos state, Nigeria. The organisms were maintained on Potato Dextrose Agar (PDA) and stored at 4°C until use. For the preparation of the spore suspension, 10 ml of sterile water was added to 5-day old culture slants of the fungi, the surface of theculture was scratched with a sterilized loop andagitated thoroughly at 250 rpm on a shaker tosuspend the spores (Omojasola and Jilani, 2009).The number of the spores were counted by using theimproved Neubauer haemocytometer and adjusted toapproximately 2.0 x 10⁶ CFU/ml and 2.0 x 10⁵ CFU/mlof Aspergillus flavus and Aspergillus oryzae ATCC17891 respectively which were used as inoculatethroughout the study.

Substrate Pretreatment

The banana fruits were washed with clean water to remove dirt; after which they were peeled. The peel was then air-dried for 7 days and then pretreated using the alkali hydrolysis method (Omojasola and Jilani, 2009). It was also further kept in hot air oven at 60°C for 2 h to reduce the moisture content. Banana peel was blended into powdered form with a Binatone blender. It is then sieved through a mesh having a pore size of about 0.5 mm and stored in an air tight sample container for further use and dry place to avoid uptake of moisture (Nandini, et al., 2014).

Proximate Analysis of Banana Peel

The proximate analysis of the banana peel was determined. The parameters analyzed were; moisture content; ash; crude protein; lipid content; total carbohydrate and crude fibre (AOAC, 2019).

Submerged Fermentation

Mineral salt medium was prepared by using 40 g of sucrose (as a carbon source), 2.5 g yeast extract (as a nitrogen source), 1g KH₂PO₄, 0.5 g MgSO₄.7H₂0, and 0.5g KCl in 1 liter of deionized water. Aforementioned media being the high productive media was utilized by all referred scientists (Abd El-Aziz, 2013).

Ten grammes of the banana peel substrate were mixed with 90 ml of the prepared medium in 250 ml Erlenmeyer flask. These flasks were sterilized in the autoclave at 121°C for 15 minutes at 15 psi. It was then inoculated with 2.0 × 10⁵ spores/ml for A. flavus and control A. oryzae ATCC 17891 separately. These were incubated at 28 ± 2°C on a rotary shaker at 400 rpm (Meena et al., 2010). Glucose yield was estimated for at least 24 hours intervals using reducing sugar analysis/DNSA.

Optimization for Glucose Production

Different fermentation parameters were varied in order to increase the yield efficiency of banana peels under optimal conditions for glucose production. Fermentation conditions varied were: Substrate concentration (10.0 g-50.0 g); fermentation days (1-11 days). These conditions were varied by changing one variable while keeping the others constant. Optimal conditions were later combined in a single fermentation.

Data Analysis

The data were statistically processed to estimate the mean ± standard deviation (SD) and using the two-way analysis of variance (ANOVA). All data were analyzed according to the Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, IL). A P value of <0.05 was considered to be statistically highly significant.

Results

Proximate Analysis

The data from this study confirm the presence of nutrients which would serve as suitable substrate for the fermentative production of Glucose using A. flavus and A. oryzae ATCC 17891. The proximate analysis of the banana peel revealed that it contained carbohydrate 55.87%, crude protein 12.72%, crude fibre 8.37%, crude fat 8.17 %, ash 12.75 % and 11.65 % moisture. These nutrients serve as carbon, nitrogen and energy sources for glucose production (Table 1).

Morphological Characteristics of Fungal Isolates Isolated From Banana Peels.

The morphological characteristics of fungal isolates (F₁-F₃) are presented in Table 2.

Keys: F₁–F₃=Fungal Isolates

Physiochemical Parameters

The physicochemical parameters checked for include pH, TTA, and glucose produced for Aspergillus flavus and Aspergillus oryzae ATCC 17891 during fermentation of banana peel at different substrate concentration. The result for pH ranged from 3.69 ± 0.57^b to 6.87 ±1.98^a are presented in Table 3, total titrable acid values ranged from 96.70 ± 2.12^b to 1833.80 ± 278.03^a as presented in Table 4, the result for varying substrate concentration for glucose produced was determined using reducing sugar analysis/DNSA method and the result ranged from 1.50 ± 0.14^b to 46.60 ± 0.28^a are presented in Table 5.

pH, Total Titrable Acid of Banana Peel During Fermentation by Fungal Isolates at different Incubation Period (days).

The bar chart representation for pH, Total Titrable Acid During Fermentation of Banana Peels to Produce Glucose at Different Incubation Period (days) Using Fungal Isolates as shown in Figure 1 and Figure 2.

The result for glucose produced by Aspergillus flavus and Aspergillus oryzae at different incubation period (Days) ranged from 1.70 ± 0.00^a to 43.80 ±1.13^b aspresented in Table 6.

Discussion

Banana peel can yield glucose by the activities of cellulolytic organisms. Banana peel was used as a substrate for glucose production. Microorganisms have long been considered as harmful entities contributing towards diseases and food spoilage but also playing their role for the welfare of human being. Presently some of these microorganisms are being widely used in food industry for production of a large number of fermented food products and at the same time these are also very helpful for conversion of food industrial wastes into value added useful products such as enzymes, organic acids and glucose. (Michael, et al., 2013).

The proximate analysis of the banana peels was carried out to determine the percentage of the carbohydrate, protein, lipid, crude fiber, ash and moisture content present in the substrate. It was observed that the carbohydrate, protein and were high enough to serve as good carbon and energy sources for glucose production. The carbohydrate content of 55.87 % reported in this study exceeds the observation of Romelle et al., who reported 43.40%. This is an indication that banana peel is high in carbohydrates and provided a good carbon source for glucose production. The protein content would also serve as a good nitrogen source for microbial metabolism. Nitrogen is one of the most essential constituents of the medium for fungal fermentation and various studies have reported increased organic acid yields with high nitrogen content (Betiku et al., 2016); Omojasola and Okwechime, 2017). Banana peel contains appreciable level of lignocellulosic materials and other components such as carbohydrates, vitamins, bioactive compounds and minerals which qualify it for various bioconversion processes (Johann et al., 2007; Dzomeku et al., 2007). The results of this study further reinforce earlier observations confirming the suitability of agro industrial residues as fermentable substrates for organic acid production (Ncube, et al., 2012; Hajian and Yusoff, 2015). Omojasola and Adeniran reported yields of 112.67 g/L and 115.67 g/l with sweet potato peel by A. niger and A. terreus respectively. El-Imam, et al. produced 48.70 g/l with Jatropha curcas seedcake with A.terreus. Rao et al. recorded 24.46 g/l also fromJatropha seedcake. The variation in the IA yields fromthe different substrates maybe due to the differences inthe composition of the substrates, fermentingorganisms and conditions employed in thefermentation. Generally, it was observed that A. oryzae produced higher yields of Glucose than A. flavus andthis was observed in all the fermentations (Tables 3-5).

Aspergillus flavus was isolated from subjecting banana peel to submerged fermentation. Aspergillus flavus is found globally as a saprophyte in soils and causes disease on many important agriculture crops (Amaike and Nancy, 2011). Generally, excessive moisture conditions and high temperatures of storage grains and legumes increase the occurrence of A. flavus aflatoxin production. Aspergillus flavus is unique in that it is a thermotolerant fungus, so can survive at temperatures that other fungi cannot. A. flavus can contribute to the storage rots, especially when the plant material is stored at high moisture levels. A. flavus grows and thrives in hot and humid climates. (Hedayati, et al., 2007).

Evaluating the different fermentation parameters i.e. substrate concentration, incubation days for pH, total titratable acid and glucose produced of fermented banana peels to optimize the yield of glucose production. Most often, changes in fermentation conditions usually have a great influence on the production ability of a microbial strain. With the variation of substrate concentration, 50 g yielded maximum product of 42.00 g/l and 46.00 g/l by A. flavus and A. oryzae ATCC 17891 respectively on Day 7 of fermentation (Table 3).

Meena et al., (2010) also observed maximum production of Itaconic Acid at 120 h of fermentation although with much lower yields (maximum yield was 8.10 g/l) and this trend was observed when A. niger, A. nidulans, A. flavus (6.3, 5.6 and 4.8 g/l respectively) were used as fermenting organisms. Rafi et al., who observed highest Itaconic Acid yield of 28.88 g/l at 4 % (w/v) substrate concentration after which a decrease in yield with increase in substrate concentration was observed. This is in contrary with this work which therefore suggests that increase in substrate concentration improves the fermentation ability of the organisms (Aspergillus flavus and Aspergillus oryzae ATCC 17891.

The effect of pH is an important parameter in production of organic acid or bio-acids using fungal or any other microorganism. At pH 4.0, both isolates produced highest glucose concentrations. This is in support with Makut et al., who reported over 50 % yield of gluconic acid at pH range from 5 to 7. The best yield was at pH 6.0. However, the acid yield above and below this pH was poor (Dowdells, et al., 2010). The results confirm that banana peels is a good substrate for glucose production confirming the observations of Sharma et al., and Yi et al., about the suitability of agro-industrial as fermentable substrates.

Aspergillus flavus produced the highest TTA value at concentration 50 g/l and substrate concentration 10 g/l showed the lowest TTA while the control organism Aspergillus oryzae ATCC 17891 produced the highest TTA value at substrate concentration 50 g/l and lowest TTA value was at substrate concentration 10 g/l at day 7.

For Glucose Content (Reducing sugar), concentration 50 g/l showed the highest and 10 g/l showed the least value for Aspergillus flavus while 0concentration 40 g/l showed the highest value and 10 g/l showed the least value for Aspergillus oryzae ATCC 17891. This result is in line with Mafra, et al., who reported high titratable acidity at high substrate concentration used in the study. This acidity could be as a result of high substrate concentration used.

Conclusion

In conclusion, this study revealed that banana peel, which is a domestic and industrial agro waste, can serve as substrate to produce glucose when hydrolyzed by cellulolytic microorganisms, instead of being thrown away and left to rot and pollute the environment. It is also beneficial in the sense that by using cost efficient agro waste as alternate sources for higher production of acids and enzymes using Aspergillus flavus through submerged fermentation and Aspergillus oryzae ATCC 17891 a recommendable strain for industrial production of glucose. The study helps to scale up the glucose fermentation to a large-scale fermentor as grown on the optimization process.

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