Extraction and Profiling of Antifungal Compounds Produced by Azotobacter Species

Food loss and wastage occur in large quantities globally every year and it occurs in the entire supply chain right from the production to the processing stage. The loss of food is due to various factors like adopting traditional cultivation practices, low investment in the food sector, and more loss from poor pests and disease management of agricultural crops. The most important and major cause is due to microbial spoilage; fungi are most harmful to the consumers and also to the agriculture sector. Synthetic chemical strategies can prevent fungal growth and may reduce wastage but still causes accumulation of chemical substances in the environment and food chain in a long run. For these reasons, the use of bio-control technologies can be a great solution to agriculture and food sector as well. In view of this, the present study has been conducted using an efficient Azotobacter species, which belongs to the PGPR group. In this study, antifungal compounds produced by Azotobacter have been extracted by following solvent extraction protocols and identified using GCMS methods. The antifungal compounds were tested against the major fungal pathogens viz ., Aspergillus, Fusarium, and Penicillium species. The metabolites produced by Azotobacter species were efficient in controlling the growth of the fungal species. These compounds can be used as a potential bio-preservative in the food sector instead of synthetic chemicals. Thus, these compounds can further be analyzed and tested on the food sample, having a great scope in the future to replace the chemical preservatives.


INTRODUCTION
Globally, food insecurity and sustainable development to reduce hunger is a major challenge due to the ever-increasing population. 1 To achieve food security in a sustainable way, we need an improved food security system associated with economic, climatic, and economic shocks to the food system. 2,3Addressing the under-nutrition by considering the total food production can help in effectively optimizing food systems.The amount of food available for consumption even after exempting loss conversion must meet global needs, thus the nutrient required for the entire population must be considered. 4The quantity of food loss and wastage varies between various countries depending on income, urbanization, and economic growth. 5bout 25% of the global post-harvest loss is due to microbial spoilage. 6,7,8,1,9Microbial spoilage of fresh fruits and vegetables has been a problem globally as it causes human health risks.The source of contamination is water, soil, human handling, pests, storage, and transportation equipment. 8icrobial spoilage causes deterioration of the food and thus changes the property of the food right from the appearance, flavor, and odor of the product and thus makes it unfit for human consumption.
Among all the microorganisms, fungi are the most common and major spoilage-causing microbe as they can tolerate harsh climatic conditions and is capable of producing spores. 10ood spoilage occurs mainly due to four main groups of fungal species such as Zygomycetes, Penicillium, Aspergillus, and Fusarium. 9These fungal species produce mycotoxins which are dangerous to human health.To avoid food spoilage and mycotoxin residues in foods, different fungicides are being added to agricultural produce at various levels, which start from production to consumer. 11ersistent Organic Pollutants (POPs) or synthetic chemicals (herbicides, fungicides, and insecticides) used by food sectors mainly cause toxicity, and bioaccumulation in living beings and in the environment as well for a very long time. 12These chemicals are used as they are cheap and more effective in protecting the crop.
According to the UNEP (1993), many documents have reported various health issues, which include cancers, hematological morbidity, heart dysfunction, immune system deficiencies, and inborn deformities that can be attributed to the use of toxic chemicals.
A better way to reduce any harmful effect of toxic chemicals on humans and the environment is by using the bio-control method as it can be used at both pre and post-harvest stages. 10treptomyces natalensis produces Natamycin (E235), which is a commonly used antifungal food bio-preservative in the food industry. 10imilarly, many bacterial species produce different antifungal metabolites, which can be an effective method in preventing fungal contamination and accumulation of mycotoxin residues in food.Among the bacterial species, Azotobacter is one of the most versatile PGPR bacteria having multiple benefits. 13,14Azotobacter species produce phytohormones like auxins, cytokinins, gibberellic acid, indole acetic acid, and substances such as thionin, riboflavin, nicotin, giberalin, etc., that stimulate root development and plant growth, help in protection from phytopathogen, and improves nutrient uptake. 15n addition, Azotobacter produces an antifungal compound that protects the plant from various soil-borne diseases caused by various fungi such as Alterneria, Aspergillus, Fusarium, Curvularia, etc. Azotobacter produces several types of antifungal compounds such as azotobactin, azotochelin, aminochelin, HCN, testin, viscosinamide, zwittermicin A, etc. 2,4-DAPG is one of the efficient antibiotics that work well against various pathogens and possess antifungal, antihelminthic, and antibacterial properties. 14,16zotobacter has wide potential as an antibiotic, which can be made use in agriculture, and various food industrial applications as a bio-control agent as an alternative to chemical substances. 14

Azotobacter cultures
A total of 30 different previously isolated Azotobacter strains were used for the present study.The growth parameters have been checked for these isolates before conducting the experiments.[19]

Sub culturing of Azotobacter strain
7][18] The media was poured onto sterilized Petri plates and the bacteria was streaked onto the plates under sterile conditions.The plates were then kept for 5 days in incubator to observe the growth rate of different strains of Azotobacter species.

Fungal isolates
Aspergillus, Penicillium, and Fusarium cultures were used for the present study.The representative isolates were obtained from the University of Mysore and the identity of the isolates will be reconfirmed based on cultural and morphological characters by comparing standard strains.

Sub culturing of fungal species
The 3 fungal species; Aspergillus flavus, Fusarium verticillium, and Penicillium expansum were sub cultured on PDA Petri plates and was incubated at room temperature for 3 days to observe the complete growth.

Bio efficacy of Azotobacter species against fungal species
8][19] Bio-efficacy of different Azotobacter species against Aspergillus, Penicillium and Fusarium isolates were studied following dual culture method.The inoculated plates were incubated at 28±2°C for 3 to 4 days and after incubation period, the zone of inhibition will be measured from the edge of the bacterial colony up to the edge of fungal mycelia. 20The efficient Azotobacter species were used for the extraction of antifungal antibiotics; purification and characterization were done as per the standard protocols. 21

Preparation of solvent mixture
Solvent extraction mixture has been prepared by using equal volumes (1:1:1:1) of diethyl ether, ethyl acetate, hexane, and n-butanol as per the standard protocols. 21

Extraction and purification of antifungal compounds
The efficient Azotobacter strains were inoculated in 200mL each of Waksman broth and incubated at 32±2°C for 5 days for the maximum growth of the bacteria.After incubation, the cultured broth was centrifuged at 10,000 rpm for 25 mins.The Cell-Free Supernatant (CFS) was collected and the residue was discarded.In a separating funnel, an equal volume of CFS and solvent mixture has been taken and mixed continuously for 10 mins.After 10 mins of mixing, the reaction mixture was kept undisturbed for 30 mins to separate the solvent layer and compound mixture.The solvent eluted out and evaporated to 50% of the total solvent extract. 21,22he extract was concentrated by using a rotary evaporator at 55 o C vacuum.The extract with antifungal adherence was carried out for further purification through Thin Layer Chromatography (TLC).The concentrated extract was separated on silica gel sheets by TLC.Solvent system Toluene, ethyl acetate with concentration (7:3) for different Azotobacter species (6:4) was standardized and detected under UV at 254nm.The extract was dried and dissolved in ethyl acetate for further confirmation of antifungal activity.Active fractions of the extract were further scrapped and treated with acetone thrice and were reconfirmed through bio-autography.The active fractions were subjected to GC-MS (Thermo Scientific USA) as per the protocol. 23

Detection of metabolites
The analysis of the active fraction was performed by a Shimadzu QP-2010 Gas Chromatograph coupled to the Shimadzu GCMSQP-2010 Mass Spectrometer with a SGEBPX-5 column (30m length, 0.25µm film thickness).Helium was used as a carrier gas at a constant flow rate of 0.8 mL/min.The active fraction was dissolved in methanol and 1.0µL of the sample was injected using AOC5000 auto injector with a split ratio 100:1.The initial temperature was set to 50°C, and then increased at a rate of 3°C/min to 280°C and held isothermally for 5min.For MS detection, the ion source temperature was set to 200°C, and an electron ionization mode with ionizing energy of 70eV and a scan mass range of 100-1200amu was employed.The compounds were identified by comparing their relative retention times and fragmentation patterns of mass spectra with those reported in the literature as well as at the National Institute of Standards and Technology (NIST17.lib)data library.

Bio efficacy of Azotobacter species against fungal species
The bio-efficacy assay of Azotobacter species was tested against three different food-borne fungal species using a dual culture  method.For the dual culture method, a modified Waksmann and PDA were used to grow both cultures.After the incubation period (28±2°C for 3 to 4 days), the results were recorded by observing the extent of inhibition of fungal mycelia by Azotobacter strains. 20Zone of inhibition was measured from the edge of the bacterial colony up to the edge of fungal mycelia.Among 7 Azotobacter strains, 5 showed satisfactory results against the 3 selected fungal strains after a period of 3 days after inoculation (Figure 1).The efficacy of Azotobacter strains against the three fungal species has been discussed in Table 1; A. salinestris showed maximum inhibition against all three fungal species, followed by A. vinelandii, A. chroococcum, A. tropicalis, and A. nigricans.

DISCUSSION
In order to satisfy the consumer demand for less processed and preservative-free foods, bio-preservation has received growing interest for improving food quality and safety. 26Biological control of Fusarium spp.using sustainable, safe, and eco-friendly antagonistic bacteria is gaining importance in recent years. 27Some of the microorganisms that possess antifungal activity against food pathogens are; Lactic acid bacteria, Propionibacterium, Bacillus, and Azotobacter. 28,29,12,13Azotobacter is a prokaryote that fixes atmospheric nitrogen into ammonia, which can be easily assimilated in plants.
Azotobacter is a gram-negative, catalase and oxidase positive, non-spore forming bacteria. 12,131][32] Among them, A. chroococcum and A. vinelandii are found almost in all the rhizosphere soils. 18,31In the present study, different Azotobacter strains have been tested against fungal species, in order to confirm their bio control efficacy.The antifungal compounds responsible for the control of fungal contamination is then extracted and identified using GC -MS technique.Butanoic acid, butyl ester; 1-Nanodecene; diazene, [1-(2,2-dimethylhydrazino)-2-methylpropyl]ethyl-; hexadecane; dibutoxymethane are some of the compounds produced by the Azotobacter strains used in this study.The compounds (hexadecane Figure 3. Spectrum of the 9 major components produced by Azotobacter and octadecane) showed antifungal properties by inhibiting the mycelial growth in Verticillium dahlia, which causes vascular wilt disease in strawberries. 33Similarly, in the present study, tetradecene and nonadecene extracted from A. vinelandii were effective in inhibiting the growth of Fusarium verticillium which is a common source of contamination in cereals. 31 n o t h e r s t u d y r e p o r t e d t h a t , naphthalene, 1-Tetradecene, 2,4-Di-tertbutylphenol, hexadecane, octadecane, and 1-Nonadecene were found among the 37 compounds which could inhibit spore germination of Penicillium chrysogenum, Aspergillus niger, and Alternaria alternata.33,34 In the current study Octadecene, Hexadecane, and Naphthalene have also been produced by A. chroococcum and A. tropicalis.These compounds have been effective in controlling the growth of Aspergillus flavus in peanuts and Penicillium expansum commonly found in apples.In another study, 2,4-Di-tert-butylphenol compound isolated from Pseudomonas monteilii PsF84 from the tannery waste exhibited antifungal activity against F. oxysporum, which is a wilt causing soil-borne fungus.35 Similarly it was documented that, B. amyloliquefaciens produced formic acid, butyl ester, and acetic acid, butyl ester was produced by B. thuringiensis and identified using GC-MS analysis.36 Formic acid, butyl ester, and acetic acid have also been reported to be produced by A. vinelandii in the current study.Diacetyl and benzaldehyde produced by B. velezensis were effective in the growth of B. cinerea infection in grapes.37,38 Another study also showed that 3 methyl 1 butanol and 2-phenylethyl methyl ether produced by R. aquatilis were effective in controlling C. gloeosporioides which causes infection in the leaves and fruits of many plants.39,40 Therefore, nonadecene, octadecene, hexadecane, tetradecene, acetic acid, formic acid, and butyl ester are some of the compounds among the 39 antifungal compounds that have been identified in the current study, and which have also been proven to possess antifungal effects from previous studies.

CONCLUSION
The results of this study show that Azotobacter possesses certain antifungal compounds which have been extracted and purified.From the dual culture method, Azotobacter strains were found to be effective in controlling the growth of Aspergillus, Penicillium, and Fusarium species.Azotobacter strains were able to produce different antimicrobial compounds and these metabolites can be used in the food industry as an alternative to synthetic chemicals.The compounds that are produced by Azotobacter compounds that have minimum antifungal properties should be studied extensively.Proper research should be done on these compounds to recognize the bio-efficacy of each compound and accordingly utilize them in the food industries as a better alternative to synthesized chemicals.The metabolites produced by Azotobacter will be a novel strategy for the agriculture sector.

Figure 2 .
Figure 2. GC-MS chromatogram of the extract obtained from Azotobacter

Table 2 .
Compounds identified using GC-MS technique