Analysis of Vapours from Cinnamon Bark during Roasting
The research revealed the process of trapping roasted vapour and its chemical composition. Αlpha-Muurolene, Alpha-Patchoulene, (z)-2-Methoxy cinnamaldehyde, Alpha-Calacorene, Beta-Bergamotene, Isoledene 1,5-Decadiyne, Isoledene,5,7-diethyl and Butylcitrate were formed during the roasting of cinnamon bark. The result revealed that the vapour of roasted cinnamon bark has antioxidant properties. Roasted cinnamon bark vapours contain 70.14±0.03 DPPH,64.48±0.06ABTS and 6.56±0.02 FRAP. Vapours of the roasted cinnamon bark contain 78.98% cinnamaldehyde. The results also indicated that roasted vapour could be used in food preservation.
Spices have been used since antiquity. Although they have primarily been used as flavouring  and colouring agents, their role in food safety  and preservation  has been examined. The use of spices in treating and preventing a wide range of illnesses has been shown to have numerous health benefits including cancer, aging, metabolic, neurological, cardiovascular and inflammatory diseases. Spices’ role is connected to their components’ bioactivity, particularly the antioxidant activity [4, 5, 6].
The flavour is present in spices in its entirety. When spices are roasted, flavours with varying chemical compositions and antioxidant activity are released. These issues can be avoided by trapping roasted vapour during the roasting process . One experiment found that roasted clove powder in a bakery product (cake) increased the product’s shelf life . It has been reported  that enzymes are inactivated during the roasting process, resulting in nutrient loss and the destruction of undesirable microorganisms, toxins, allergens and food contaminants.
Cinnamon bark is used in both traditional and modern medicine, as well as in cooking. Since the flavour of cinnamon can be found in various foods, perfumes and medicinal products, it is widely used in the aromatherapy and essential oil industries. It was already reported that  cinnamaldehyde is responsible for spicy flavour and aroma. Cinnamon had higher antioxidant activity than other spices..Cinnamon is used to flavour chewing gums in addition to becoming a spice and seasonings agent . Cinnamon can also help to promote colon health, lowering the chance of colon cancer . Cinnamon is a coagulant that helps to stop bleeding . This essential oils of plants and compounds also have considerable anti-bacterial , anti-fungal , antioxidant  and anti-diabetic properties .
Numerous studies on the effect of roasting on the antioxidant activity of coffee brew  and clove  have been performed. However, there is no metadata on the elemental analysis and potent antimicrobial characteristics of roasted cinnamon bark vapour. As a result, the current study will characterize the chemical and antioxidant capacity of volatiles developed and accumulated during roasting using GC-MS and standard methods.
2. Materials and Methods
Cinnamon bark was purchased at a food store in Dumdum, West Bengal, India.
Reagents and Chemicals
In this investigation, all of the analytical quality reagents and solvents that are used were bought from MERCK in India.
2.1. Preparation of samples
Preparation of samples was followed by Dev et al., 2021  with minor alterations. Cinnamon bark was roasted at 80°C for 30 minutes in a round bottom pan. Then, vapours were condensed through a condenser. After that, volatile compounds were eventually captured by absorption in petroleum ether (b.p.40°C-60°C) and later removing the solvent.
2.2. Determination of Antioxidant Properties through Various Methods
2.2.1. Total Phenolic Assay
TPC of samples was determined spectrophotometrically using a minor alteration of the Folin-Ciocalteu reagent . A Gallic acid calibration curve was created. The findings were then demonstrated in terms of Gallic acid equivalents (mg GAE/gm). 100 μl of the extract was incubated for 5 minutes with 250 μl of Folin Ciocalteu’s reagent. After that, 1.5 mL of 20% sodium bicarbonate was added to the mixture. The same reagents were used to create a blank. After 25 minutes, absorbance at 765 nm was measured against a blank using a spectrophotometer.
2.2.2. DPPH Scavenging Activity
The scavenging activity of samples was determined using a slightly modified method . 1 mL of a 0.5 mM methanol solution of the DPPH radical was added to 2.0 mL of the sample, followed by 2.0 mL of 0.1 M sodium acetate buffer (pH 5.5). The mixtures were thoroughly mixed and retained at room temperature in the dark for 30 minutes. A double beam UV-VIS spectrophotometer to measure absorbance at 517 nm. As a negative control, methanol has been used.
The radical scavenging activity of samples was determined. 1 mL of sample was mixed with 1 mL of a 90 μM DPPH solution in methanol. After that, the final volume was made to 3 mL with methanol. The mixtures were thoroughly mixed and stored at 25°C in the dark for 1 hour. At 517 nm, the absorbance was measured.
2.2.3. ABTS Assay
The ABTS radical cation decolourization assay was used to determine the free radical scavenging activity of samples by Re et al.,1999 . The ABTS radical cation was created by the reaction of 2.45 mM potassium persulphate with 7 mM ABTS in water. Then the solution was kept in the dark place for 11-16 hours at room temperature before use. After diluting the solution with methanol, the absorbance at 734 nm was measured. The absorbance was measured 30 minutes after the addition of 10 µl of samples to 1 ml of diluted ABTS+ solution. The ABTS scavenging activity has been calculated by using the equation.
2.2.4. Ferric Reducing Antioxidant Power (FRAP) Assay
The sample’s reducing capacity was determined by using the ferric ion (Fe3 +) reducing antioxidant power (FRAP) method. This is explained by Benzie and Strain, 1996 . The FRAP reagent consists of 250 mM sodium acetate buffer (pH 3.6) and 10 mM TPTZ. 40 mM HCl and 20 mM FeCl3. The TPTZ solution, FeCl3 solution and acetate buffer were mixed at a ratio of 1: 1:10. A sample (100 µl) was mixed with 900 µl of FRAP reagent. The mixture was kept at 37°C for 4 minutes and the absorbance was measured at 593 nm.
2.3. GC-MS analysis
GC-MS was used to analyze the samples (Thermo Scientific). At a flow rate of 1 ml/min., a fused silica capillary column DB5-MS (30 m×25 mm, film thickness 0.25 μm) was used with helium as the carrier gas at a constant pressure of 100 kpa. The injector and detector temperatures were both 250°C. The components of the aromatic oil were determined by comparing their retention indices (RI), mass spectra (NIST library) and literature data.
2.4. Antimicrobial Activity
A well diffusion method was used to assess the antimicrobial activity of samples against various bacteria, as described by Dev et al.,2021 . 1 ml of sample was mixed with 100µl of tween 80 in distilled water (total volume 10 ml). Bacterial strains were cultured on nutrient agar plates at regular intervals. In the well diffusion assay, two Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes) were tested for antibacterial efficacy and these bacteria were inoculated into Nutrient Broth and kept for 24 hours at 30°C. In a flask, sterile Muller-Hinton agar (MHA) was prepared and then cooled to 45-50°C. The 20 ml agar was then poured through every inoculated Petri dish and rippled to equally distribute the medium. 10 µl of each bacterial strain was placed on these MHA-containing Petri plates. The 10 mm diameter of wells in the nutrient agar plate was made by a cork borer and after that, 10 µl of samples were placed. Then the Petri plates were kept in an incubator for 24 hours at 37°C temperature. After that, the plates were observed for the zone of inhibition and the diameter of the zone of inhibition was calculated for all plates. The assay was carried out in triplet forms.
2.5. Statistical Analysis
All values were measured in triplicate and data were noted as mean and standard deviation. The data for the various parameters were analyzed using the analysis of variance (ANOVA) technique and the post hoc Tukey HSD (Honestly Significant Difference) test was used to compare the means.
3. Results and discussion:
3.1. Reducing Power Assay (FRAP) of Cinnamon Bark Oil and its Roasted Vapour
The antioxidant activity of cinnamon bark oil and their roasted vapourizing reducing power assay were presented in Table 1. The ferric ions (Fe3+) reducing antioxidant power (FRAP) method was used to measure the reducing capacity of aromatic oil and its roasted vapours. During roasting, there was decreased reducing power capacity. Roasted vapour showed FRAP 6.56 µM/ml antioxidant activity, lower than the control. It was already reported  that the vapour sample had significantly lower antioxidant activity than the clove bud oil.
3.2. DPPH Assay of Cinnamon Bark Oil and its Roasted Vapour
The inhibitory effects of cinnamon bark oil and its roasted vapour sample on the DPPH activity were given in Table 1. It was already reported  that DPPH presented the highest scavenging power for cinnamon bark oil (80.15%) among vapour samples. Cinnamon bark oil, however, exhibited a very different profile wherein a progressive decrease in DPPH activity of roasted vapour sample 70.14% was observed. There were significant differences observed between the above two samples.
As a result, it can be concluded that roasted vapour exhibited DPPH activity.
3.3. ABTS+Assay of Cinnamon Bark Oil and their Roasted Vapours
As seen in Table 1, cinnamon bark oil showed ABTS+ radical scavenging activity (75.76%), which was already reported by Dev et al.,2021 . Furthermore, there were significant differences in ABTS+ scavenging potential between control cinnamon bark oil (unroasted) and roasted vapour cinnamon bark oil (64.48 %).
2A. 3.4. Total Phenolic Content (TPC) of Cinnamon Bark Oil and its roasted vapour
Table 2 showed total phenol content (mgGAE/ml) in unroasted cinnamon bark oil was 0.17 mgGAE/ml  which increased significantly in roasted vapour samples (1.27 mgGAE/ml).
The antioxidant activity of a vapour sample of roasted cinnamon bark could be associated with the presence of cinnamaldehyde, eugenol, coumarin  and Αlpha-Muurolene, which were analyzed through GC-MS.
3.5. GC-MS Analysis of Cinnamon Bark Oil and its roasted vapour
The running time of GC-MS for cinnamon bark oil and its roasted vapour was 45 minutes and Figure 1 shows the resulting spectrum. The mass spectrum was interpreted using the National Institute of Standards and Technology database (NIST). Table 3 shows the active principles and their retention time and concentration (percent). Thermal treatment significantly influenced the oils’ chemical constituents, flavour and percentage composition. Thermal treatment strongly influenced the oils’ chemical constituents, flavour and percentage composition. When comparing the vapour sample to the control, the retention time for eugenol is nearly the same. The results revealed that the oils contained a complex mixture of compounds, primarily monoterpene hydrocarbons, phytocompounds and sesquiterpenes. Cyclopropane,1-(2-methylene-3-butenyl)-1-(1-methylenepropyl), 1-chloro-pentadeca-5,10-diyne, (+)-2-Carene,4-α-isopropenyl, Bicyclo [4,10]-3-heptene, 2-isopropenyl-5-isopropyl-7,7-dimethyl, 1,5-Decadiyne, 5,6-Decadien-3-yne, 5,7-diethyl, Cubenene, Isoledene, Butylcitrate were formed when cinnamon barks were roasted. Some of the compounds were thought to be tentatively identified through comparative analysis in the NIST data and the absence of a specific standard molecule.
Table 3. GC-MS analysis of clove bud oil and its roasted vapour
3.6. Antimicrobial Activity of Cinnamon Bark Oil and its roasted vapour:
Figure 2 illustrates the results. The growth inhibition pattern showed vapour of cinnamon bark has antimicrobial Activity. The cinnamon bark oil had the highest inhibition zone against E.coli, with an IZD of 19.58 mm and the roasted vapour sample had an IZD of 17.28mm. The inhibition zone for cinnamon bark oil was developed against Bacillus subtilis with an IZD of 18.35 mm and the vapour sample was 15.34 mm. The inhibition zone for cinnamon bark oil was developed against S.aureus with an IZD of 14.14 mm and the vapour sample was 11.64 mm. The inhibition zone for cinnamon bark oil was created against Enterobacter aerogenes with an IZD of 11.23 mm and the vapour sample was 9.37 mm.
It was also reported by Farhoosh et al., 2011; Cravotto et al.,2006 ; Kim et al., 2015  that roasted vapour contains some compounds which have antimicrobial properties like cinnamaldehyde, eugenol, Cyclopropane and 1-(2-methylene-3-butenyl)-1-(1-methylenepropyl).
When aromatic spices are roasted, they release flavour molecules in a vapour state, which results in significant changes in composition and antioxidant activity upon recovery. However, the findings suggest that roasted vapours can be a flavouring agent with high antioxidant activity. These findings also concluded that the vapour of roasted cinnamon bark can be considered a potential alternative for synthetic antioxidants and can be utilized in the food industry.
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Rudraram, Telangana – 502329, India.
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