Aroma Chemicals Identification by Sophisticated Technique and Their Role Against Pathogens

,


Introduction
Citrus fruits were one of the most refreshing and nutrient rich fruits in the world.There are many different varieties of citrus fruits all over the world in tropical, sub-tropical and temperate regions.Citrus fruits include Oranges, Lemon, Lime, Mandarin, Grapefruit, Pomelos, Tangerines etc.They belong to Rutaceae family and rich in Vitamin C (Abobatta, 2019a).Citrus fruits have good nutritional value and they have been cultivated since 4000yrs ago.Citrus fruit production especially sweet oranges are mostly produced from Brazil, followed by USA and other countries.Important crops like oranges, lemon, limes, grapefruit, and pomelos are prominently grown around the world but its origin is from Southern Eastern Asian region.
Citrus fruit production has huge demand globally.In 2016 the production of citrus fruits was around 124.25 million tons from China which was followed by Brazil, India, USA, Spain, Mexico etc. (FAO, 2017).Among all the variants navel orange, blood orange and Valencia variety considered as sweet orange-Citrus sinensis L., Citrus aurantiumis named as bitter orange and it was sour in taste.Lemon (Citrus limon) and lime (Citrus aurantifolia) can be found all over the year in most of the places.There are citrus fruits like Mandarin (Citrus reticulate) harvested from August to September which can be peeled easily (Abobatta, 2019b).Most of the citrus fruits pulp is consumed and remaining 50% of the fruit is peel which cannot be consumed but it consists of oil glands to store the citrus essential.Cold pressed citrus oils have good aroma, and it can be used in food and beverage industries (Kapsaski-Kanelli et al., 2017).sesquiterpenes group (Bora et al., 2020).Flavonoids like, naringin, narirutin, naringenin,neohesperidin, nobiletin,hesperetin, hesperidin, luteolin, and tangerinepresent in citrus fruits act as anti-microbial agents and used in quality and food safety system to inhibit the microbial growth or as preservative (Damián-Reyna et al., 2016).
Citrus fruits are rich in polyphenols, vitamins, carotenoids, flavonoids, coumarins etc and they help to build up our immune system.Essential oils from citrus fruits can prevent food spoilage caused by food borne pathogens like Escherichia coli, Salmonella, Shigella, Vibrio cholera etc. and these oils are considered as preservatives or natural antimicrobial agents to overcome spoilage of food.Antimicrobial assay was performed to study the activity of essential oils against pathogens (Hasija et al., 2015).

Sample Collection
Minimum 5 to 6 kg of matured Citrus fruit variant were collected from (fruit market/orchids) different locations.Selection is mainly based on the color, texture of the skin and taste.Important parameter to identifyor select the citrus fruitsis to taste or check exact matured stage of the fruit without sourness/overripenscharacter.
Collected fruits were carefully bought to the lab and cleaned with sterile water and kept for air drying for 30min.After drying they are analyzed to identify the flavor molecules by performing GCMS.

Extraction of Flavor Molecules From Different Citrus Fruits by GCMS Analysis
Wash the fruits and keep them drying in room temperature before extraction.Peel of the selected citrus fruits like Narinja, Gondhorajlebu, lemon, Mosambiand Nagpurorange wereremoved carefully.10-20 g of each selected citrus fruitpeel were weighed and kept in separatebeaker.The peel of each fruit is mixed with cellulose powder.In next step transfer 60-70 ml of prepared sample into glass tube and fill the tube with floran liquid gas.Shake the contents using orbital shaker at 200 rpm for 2 hrs.After 2 hrs transfer the liquid gas into Dichloro methane solution and allow to stand for some time until liquid gas evaporates.After evaporation of liquid gas inject the sample into GCMS port and run the analysis.

Antimicrobial Activity of Citrus Essential Oils
Essential oils from selected citrus fruits are extracted through steam distillation and tested for their antimicrobial activity against both Gram-positive and Gram-negativeorganisms.Nutrient agar media was prepared as per the composition and poured in petri plates and allowed for solidification.After solidification spread plate technique was performed using E. coli, Pseudomonas, Bacillus and Staphylococcus aureus.Then incorporate the discs impregnated in essential oils for overnight and labeled as S1, S2, S3, S4, S5 and one blank disc was placed for comparison studies.After placing the discs in their respective places, the plates were incubated at 37 o C for 24-48 hrs.Zone of clearance or antimicrobial activity of selected citrus oils were measured after incubation.
In current era processed food and packed food was in high demand and to avoid contamination of food products people prefer natural ingredients as preservatives rather than synthetic chemicals.Among all the natural ingredients citrus essential oils play avital role to preserve foods.Citrus essential oils contain bioactive compounds which effectively inhibits or kills the pathogens.Present study states that selected citrus essential oils like Narinja oil exhibit high antimicrobial activity on Gram positive (Bacillius and Staphylococcus aureus) and Gram negative (Pseudomonas and E. coli) bacteria when compared to other oils.Antimicrobial activity of Narinja oil through πr 2 formula states that on E. coli the value was 153.86 mm 2 , Bacillus was 153.86 mm 2 , Staphylococcus aureus was 28.26 mm 2 and Pseudomonas was mm 2 .The values state that it was more effective on E. coli and Bacillus.
The πr 2 applied to check the antimicrobial activity of Lemon oil, itexhibited zone of inhibition on E. coli with πr 2 of 50.24 mm 2 , Bacillus was 50.24 mm 2 and there is no activity was found on Staphylococcus aureus and Pseudomonas.The values for Nagpur orange oil in πr 2 on E. coli was 28.26 mm 2 , Bacillus was 28.26 mm 2 and antagonistic activity of Gondhorajlebu oil showed only on E. coli and the value was 28.26 mm 2 .Grapefruit oil and lemon oil has excellent antimicrobial activity against Leuconostoc mesenteroides MS1, Escherichia coli ATCC 25922 and Lactobacillus plantarum ES147 and ATCC 8014 (Raspo et al., 2020).Citrus oil components terpineol and terpeneless fractions has more antagonistic activity on both Gram-positive and Gram-negative bacteria.Citrus essential oils and terpene derivatives has great antimicrobial activity on bacteria and candida strains (Mancuso et al., 2019).Death or Inhibition of microorganisms due to citrus oils was due to cytoplasmic membranedestruction (Kong et al., 2008).
There are reports stating citrus essential oils like finger citron oil exhibit antimicrobial activity on microbes like S. aureus with an inhibition zone 19.2±2.1 mm, on Bacillus subtilis the inhibition zone was 16.3±1.3mm, for Microcous luteus it was, 16.1±0.4mm and on E. coli it was 11.2±0.9mm (Li et al., 2019).Two main components of lemon oil like limonene and β-pinene exhibit high microbial activity on both Gram-positive and Gram-negative bacteria (Ben Hsouna et al., 2017).When we compare the above data with current results Limonene is major content in all citrus oils and other molecules in combination with Limonene exhibit great antimicrobial activity against Gram-positive and Gram-negative microbes.

Conclusions
In present research the oils extracted from citrus oils like C. aurantium (Narinja), C. hystrix (Gondhorajlebu), C. limon (Lemon), C. limetta (Mosambi) and Citrus reticulata Blanco (Nagur Orange) reveals they contains various aroma chemicals and has great potential activity to inhibit pathogenic microorganisms.Further research is to exploreand to study the selected citrus oils in medical, flavour and fragrance industry. Figu