Chemical Composition and Effectiveness of Bay leaf and Cinnamon Essential Oils against Aflatoxigenic Fungi

: Spoilage of agricultural produce due to the action of Aspergillus spp. pose significant challenges to farmers and importer/exporter of agricultural produce. Aflatoxins are toxin produced by fungi and they contaminate food threatening food safety. Studies have shown that essential oils are capable of inhibiting the growth of Aspergillus spp. This study determined the chemical composition of the essential oils of Bay leaf and Cinnamon using Gas Chromatography-Mass Spectroscopy (GC-MS) techniques and evaluated the in vitro effect of the oils against Aflatoxigenic fungi ( Aspergillus flavus, Aspergillus niger and Aspergillus parasiticus ). Food poisoning technique was used to determine the inhibitory effect of the oils with radial mycelia growth of each observed and measured 24 hourly. The result of GC-MS analyses revealed cinnamaldehyde (5.83%), 3-Methyl-Benzofuran (14.49%), 2,6,6,9-tetramethyl-Tricycloundec-9-ene (10.59%) and other bioactive constituents as abundant in the essential oil of Cinnamon. Additionally, Bay leaf essential oil recorded Eucalyptol (12.30%), Eugenol (4.15%), and 2-octyl-Cyclopropaneoctanal (9.16%) among other bioactive compounds. Antifungal result revealed that both oils were active against the studied microorganisms as they were able to completely inhibit the growth of A. niger . Cinnamon oil had 92.69 and 91.27 % inhibition against A. flavus and A. parasiticus respectively at 72 hr of observation, while essential oil from Bay leaf recorded 23.50 % and 76.50 % inhibition for A. flavus and A. parasiticus after 72 hr. Results from this study have further confirmed the antimicrobial potentials of essential oils against the tested aspergillus spp. and their potential use in the development of natural antifungal products.


INTRODUCTION
Essential oils (EOs) are complex natural mixtures of volatile secondary metabolites obtained from diverse groups of plants (Sampietro et al., 2015), possessing hydrophobic character and are intricate mixtures of odoriferous, volatile, lipophilic liquid substances; fragrant products whose complex compositions are obtained from various parts of plants by hydro or steam distillation (Kar et al., 2018;Onyegbula et al., 2019).They exhibit antimicrobial and antioxidant properties, making them useful as natural food preservatives.They are also known for their antiseptic and medicinal properties such as analgesic, sedative, anti-inflammatory, spasmolytic, local anesthetic, and anti-carcinogenic (Pereira et al., 2019).The antimicrobial activity of plant oils and their extracts are the basis of various applications including raw and processed food preservation, pharmaceuticals, medicine, and natural therapies (Onyegbula et al., 2019).
Since ancient times, EOs have been used for their pesticidal potential against a wide variety of agricultural pests and as part of traditional practices.Their application as antimicrobial and antioxidant agent has been a growing trend reflecting the interest towards 'green consumerism' (Prakash & Kiran, 2016).Despite the use of various food preservation methods, microbial spoilage (bacteria, molds, and yeast) and oxidative deterioration (lipid peroxidation, nutritional and colour impairment) are still major challenges in the food industries (Prakash & Kiran, 2016).In tropical countries like Nigeria, especially the south and central regions, where rainfall, high temperature, and humidity are prevalent, these conditions encourage microbial growth and impair the quality and aesthetic value of Agricultural products (Agrios, 2000).According to Eskola et al. (2020) & Nazhand et al. (2020) and high incidence of mycotoxin (mycotoxins (Aflatoxin, Ochratoxin, Fumonisin, Zearalenone, Trichothecene, and Patulin) contamination on agricultural produce by different fungal species has been reported which surpass the 25% figure given by Food and Agricultural Organization (FAO).Of the major toxigenic mold contaminant of food, Aspergillus spp.are the most prevalent.They cause aspergillosis, which is an infection that results in illnesses ranging from mild to severe lung/ respiratory infections in both humans and animals (Shabeer et al., 2022).Reducing the postharvest loss of agricultural produce to spoilage microorganisms has been the focal point of several initiatives.Tolouee, et al. (2010), Marei et al. (2012) & Ju et al. (2017) among other authors have reported the successful use of essential oils in in vitro and in vivo studies to control food spoilage microorganisms.In line with public demand for quality food, assured food safety, control of the use of synthetic fungicides (which are associated with environmental and health risks), there is a need to study natural compounds as a suitable alternative to synthetic fungicides.This study aimed to characterize essential oils of Cinnamon and Bay leaf and assess their in vitro antifungal efficacy against some Aspergillus spp.

Sample collection and preparation
Bay leaf was purchased from Mandate market (8.45384 o N, 4.55627 o E) while the Cinnamon sticks were procured from Yoruba road market (8.48480 o N, 4.56507 o E), both in Ilorin, Kwara State.Both spices were milled using Waring commercial electric blender (Model 8011ESK) and kept airtight for further use.

Extraction of Essential Oil
Essential oil was extracted separately from both Cinnamon and Bay leaf using the hydro-distillation method with the aid of the Clevenger apparatus according to the method of Al-Hashemi ( 2014) with slight modifications.The blended Cinnamon bark and Bay leaf (2 kg each) were directly immersed separately in a 5 L capacity extraction flask and 4 L of water was added.The extraction flask was attached to the Clevenger apparatus with a condenser as shown in Figure 1 below.The mixture was set to boil and the extraction process was carried out for 1-2 hr.The condensed mixture of water and oil was separated in a Florentine flask due to their immiscibility and density difference.

GC-MS Analyses of Essential oils
A gas chromatography from Agilent USA (7890AGC system, 5675C inert MSD) hyphenated to a mass spectrophotometer (5975C) with triple axis detector equipped with an auto-injector (10ul syringe) was used; Helium gas was used as a carrier gas.All chromatographic separation was performed on capillary column having specifications: length; 30 m, internal diameter 0. MS Solution software provided by the supplier was used to control the system and to acquire the data; Identification of the compounds was carried out by comparing the mass spectra obtained with those of the standard mass spectra from the NIST library (NISTII).

Isolation and Identification of Aspergillus spp.
Isolate of Aspergillus spp.(A.niger, A. flavus, and A. parasiticus) were obtained from an old culture plate at the microbiology laboratory of the Nigerian Stored Products Research Institute.The isolates were identified through their morphological characteristic such as colour, hyphae pattern etc.A sterile inoculating loop was used to pick an innoculum from the identified old culture of each Aspergillus from the microbial plate and cultured it on a petri dish containing solidified potato dextrose agar.Continuous sub-culturing was done until pure cultures of each selected organism were obtained.The method described by Fawole & Oso (2004) was used to identify the fungal culture and a small portion of the fungal mycelia was picked and dropped on a glass slide with a drop of 70% alcohol.A drop of lacto-phenol cotton blue was added after which a coverslip was gently placed on the glass slide and viewed under a microscope with an x10 objective lens and later x40 objective lens for better magnification.Microscopic identification of fungi was determined based on the morphological appearance and the characteristic features as described by Standard Operating Procedures for Fungal Identification (2 nd Edition, 2019) (Barnett & Hunter 1999).

Antifungal Efficacy of Bay leaf and Cinnamon oils.
The antifungal effect of Bay leaf and Cinnamon oils against the isolated microorganisms was carried out using the modified food poisoning technique method as described by Adjou et al., (2012).A portion of 1 mL of the essential oils (Bay leaf and Cinnamon) were dispensed separately into different Petri dishes and 15 mL of Potato Dextrose Agar (PDA) was added to the solution, the Petri dishes were properly mixed to ensure the even distribution of the oils and then allowed to solidify.Purified fungal inoculums of the organisms with hemocytometer-spore count (1.0x10 6 I.O.Lawal et al.CFU/mL) standardized were used.A 6 mm diameter cork borer was used to create a well at the centre of the solidified media, then a pure inoculum of each organism were introduced into the wells separately and labeled accordingly.Media containing sterile water was used as a control.The treatments were done in duplicate and incubated at room temp (28±2 o C).Radial mycelia growth was measured 24 hourly using a ruler for 72 hours.
Where: R1= radial growth of the pathogen in control medium, R2= radial growth of the pathogen in the treated/test medium

RESULTS AND DISCUSSION
A GC-MS qualitative scan on both oils revealed 29 and 51 bioactive compounds for Cinnamon and Bay leaf EOs respectively.The significant compounds identified with their respective percentage abundance are highlighted in Tables 1 and 2, while the chromatograms are shown in Figures 1 and 2. Cinnamon essential oil revealed 3-methyl-Benzofuran as the most abundant at 14.49%, next to 2,6,6,9-tetramethyl-Tricycloundec-9-ene (10.59%).Cinnamaldehyde, benzofuran, 3,4-dihydro-Benzopyrimidine, 13-Octadecenal, 6-Octadecenoic acid, 3-(2-methoxyphenyl)-2-Propenal and A-Copaene were all identified to be present at 5.83%, 7.98%, 8.35%, 7.20%, 7.74%, 5.67%, and 4.15% respectively.Cinnamaldehyde has been reported in quite a number of studies as the main component of Cinnamon essential oil (Wijayanti et al., 2011).It is the compound responsible for its flavour and odour and according to Elumalai et al. (2011) & Wong et al. (2014), the presence of cinnamaldehyde contributes to the spicy taste as well as the fragrance of Cinnamon.Several factors such as environmental, climate, time of harvest and treatment, the plant's age, and post-harvest management of the plant contribute to the variation in the abundance of the compound in the essential oil of the spice (Anshory & Nugraha, 2017).
Bay leaf essential oil on the other hand revealed Eucalyptol (12.30%) as the most abundant compound, next to 2-Octyl-Cyclopropaneoctanal (9.16%), 7-Tetradecyne (6.68%), Cis-9-Hexadecenal (5.14) and Eugenol (4.15%).The oilsoluble terpenes, α-and β-pinene were as well identified, though in trace amounts, which account for 0.46 and 0.93% respectively of the essential oil.These compounds are known to contribute flavors and antimicrobial properties among others to essential oils (Salehi et al., 2019).According to Taban et al. ( 2018); Rincon et al., (2019), Bay leaf essential oils are known to have eucalyptol, eugenol, and linalool as the main compound, two (2) of which have been reported in this study, with eucalyptol being the most abundant.The in vitro antifungal effect of essential oils of Cinnamon and Bay leaf against the tested fungi are presented in Tables 3 and 4. It was observed generally that there was an increased mycelia growth in the treated plates over the observed period, hence a reduction in growth inhibition of the oils, which supports the claim of Pilar et al. (2015).Although, 100% inhibition was recorded for both essential oils against A. niger even after 72 hours of incubation, there was a decline in the inhibition power of Bay leaf essential oil from 81.96 to 23.46% for A. flavus within 24 -72 hours of incubation.Cinnamon essential oil on the other hand recorded above 90% growth inhibition for all microorganisms tested even after 72 hours incubation period.A similar trend was observed by (Thanaboripat et

CONCLUSION
This study has revealed that both essential oils of Cinnamon and Bay leaf possess rich bioactive compounds which are responsible for the distinct characteristic properties of the spices and also contribute to their effectiveness against aflatoxigenic fungi.The results from this study has further supports the potential application of essential oil from spices as natural substitute in the synthesis and development of bio-pesticides, to prevent aflatoxin contamination of agricultural products.This would offer a sustainable solution to the promise of food quality, safety and security.

Figure 1 :
Figure 1: Setup of Clevenger apparatus for hydro-distillation extraction 2 µm, thickness; 250 µm, treated with phenyl methyl silox.Other GC-MS conditions are ion source temperature (EI), 250 o C, interface temperature; 300 o C, pressure; 16.2 psi, out time, 1.8mm, 1 µL injector in Split mode with split ratio 1:50 and injection temperature of 300 o C. The column temperature started at 35 o C for 5 min and changed to 150 o Cat the rate of 4 o C/min.the temperature was raised to 250 o C at the rate of 20 o C/min and held for 5 min.The total elution was 43.75 min.

Figure 2 :
Figure 2: GC-MS chromatogram of Cinnamon essential oil

Table 2 :
Bioactive compounds of essential oil of Bay leaf

Table 3 :
In-Vitro Antifungal Efficacy of Cinnamon against Aspergillus spp.

Table 4 :
In-Vitro Antifungal Efficacy of Bay leaf against Aspergillus spp.
aResult shows mean ± standard error (SE) of duplicate readings.Means with different superscript on the same row for each organism are significantly (p<0.05)different.