Effect of Gamma Radiation on Oil Quality of Dried Mint Leaves

Corresponding Author: Abd El-Rahman Abd El-Raouf Ahmed Agricultural Engineering Research Institute, Agricultural Research Center, Egypt Email: abdo_aaaa2000@yahoo.com Abstract: The green mint crop is considered a medicinal and aromatic plant grown in most of Egypt's governors and it has multiple uses, whether it is used fresh or dried or mint oil is extracted from it and mint is also known as its medicinal importance as research has proven that it has many health benefits. This research was conducted to study the effect of gamma irradiation on the oil quality assessment of dried mint leaves. Dry mint leaves were treated by using doses of gamma radiation, 0.0, 5, 10, 15 and 20 kGy. The color properties as Chlorophyll a, Chlorophyll b and Carotenoids (mg/100g) were detected and also, the antioxidant activity, total phenols, total flavonoids and ascorbic acid of aqueous extract of mint leaves have been evaluated. The highest content of chlorophyll a, chlorophyll b, total chlorophyll and content of carotenoids in the dried mint leaves was observed in irradiated samples with 20 kGy. While, the highest content was in the irradiated sample with 5 kGy. The data demonstrated that the irradiated dried mint leaves at 5, 10, 15 KGy had lower phenolic, flavonoids, total antioxidant and ascorbic acid compounds than nonirradiated control in the mint extract. While, irradiated mint at 20 kGy had higher phenolic compounds. It was found significant differences in the content of chlorophyll (a), chlorophyll (b), phenol, flavonoids, antioxidants, ascorbic acid, while there was no significant difference in the carotene content. By using of irradiation with gamma rays of 20 kGy, the most influencing was the higher content of chlorophyll (a) and chlorophyll (b). While the use of the level of irradiation with gamma rays of 15 kGy had a greater impact on the high content of carotene, phenol, flavonoids. Also, the use of irradiation at 10 kGy levels had the best effect on the high content of antioxidant and ascorbic acid. This study indicated the Gamma irradiation of dried peppermint leaves may lead to improved quality of oil.


Introduction
Some herbs or crops are consumed in their fresh state and may deteriorate within a few days after harvest. One way to preserve these plant products is to dry them in either by traditional sun/shade drying or microwave drying/oven drying. The area of the mint farm in Egypt is about 4312 Feddan (1 hectare = 2.4 Feddan) during the winter and summer seasons, with a rate of 6.2% of the total area of medicinal and aromatic plants, amounting to 69154 Feddan in 2018. ASEAS (2018). In terms of foreign trade, the value of Egyptian mint oil reached about an amount of exports amounting to about 291 tons in 2018 and the rate of export growth increased with a growth rate in the amount of exports of about 87% between 2014(UNMS, 2018. Eliana et al. (2016) evaluated the effects of gamma irradiation on cytotoxicity and phenolic compounds of Thymus vulgaris L. and Menta piperita L., used in traditional medicine. Thirteen and fourteen phenolic compounds were detected in T. vulgaris and Mentha piperita, respectively, none of which was affected by the irradiation dose used (10 kGy). Amnah and Amal (2013) conducted to study the effect of gamma irradiation on the antioxidant activity, 84 quality assessment of microbial and sensory for fresh mint leaves. A high antioxidant activity when radioactive transactions gave a significant increase in the content of total phenols and total flavonoids compared to nonirradiated samples. Eric (2013) analyzed the effect of the drying methods on the nutritional characteristics of the spicy basil leave. Microwave-drying and oven drying were the methods that produced the best results for preserving most nutrients compared to the fresh herb, whereas ambient-air-drying, hot-air-drying and sun-drying brought about substantial losses in basil leave nutritional values. Gavahian et al. (2015) used that Steam Distillation (SD) for extraction of essential oils from Mentha piperita L. (peppermint) and the results were compared with those of the Hydro Distillation (HD). The results showed that although extraction with HD started sooner, both methods had the extraction time of about 1 h. Ghanooni et al. (2015) mentioned that the Mentha piperita L. Essential oils have different a bacterial activity. They investigated the effect of γ-irradiation on the bacterial activities of Mentha piperita L. Eessential oils in vitro and in vivo systems. The aerial parts of peppermint were irradiated in a cobalt 60 source with 0, 10 and 25 kGy absorbed doses. The oil was effective in reducing CFU caused by sepsis-induced CLP operation.
Generally, these processes may cause negative attribute to the final food product, Phenolic compounds possess a wide spectrum of biological effects including antioxidant and free radical scavenging properties. Phenolic is classified into two groups such as polyphenols and simple phenols according to (Marinova et al., 2005). Drying is the most common and effective method that increases the shelf life of spicy herbs by inhibiting the growth of microorganisms and preventing the onset of some biochemical reactions that may alter the organoleptic and nutritional characteristics of the dried leaf. However, drying must be performed carefully in order to preserve the aroma, appearance and nutritional characteristics of the raw herbs as much as possible (Crivelli et al., 2002). Yousif et al. (1999) mentioned that during the long hours of drying heat was conducted from the surface to the interior of the leaves and the rate of evaporation of water on the surface of the leaves was faster than the rate of diffusion to the surface. However, all dried leaves achieved moisture content less than 20%wt.
Exposing food material to radiation has great advantages in contrast to the conventional methods of preservation such as cold storage, fumigation, salting and drying as it does not lead to loss of flavor, odor, texture or quality. In addition, irradiation is a direct, simple and efficient one-time process. Irradiation treatment combined with proper refrigeration for storage can prolong the shelf-life of these food items without affecting the flavor and texture (Shurong et al., 2005).
Preserving food through radiation is considered a promising method to enhance the safety of the microbial quality of the food as it prolongs the period of validity.
Based on that, the radiation is considered a safe method of preserving food from microbial deterioration and consequently prolonging its marketing period (Haruvy and Deschenes, 2003). Boiling water extracted more polyphenols from dried plant materials than cool water, which means the heat of boiling water was the factor that led to higher total phenols being extracted from dried plant materials (Toor and Savage, 2004). Fatemi et al. (2010) showed that the γ-irradiation can be an effective way to treat problems in food supply pollution. The antibacterial activities of irradiated peppermint oil at two radiation doses of 10 kGy (the maximum permissible dose of radiation in food) and 25 kGy (sterile dose that all biological agents contaminating peppermint die) were studied to consider whether the γ-irradiation preserved the in vivo and in vitro antibacterial activities. Chlorophylls and carotenoids are frequent organic food components because they are naturally present in plants, giving them specific coloration. In vivo, these pigments play key roles in photosynthesis (Schoefs, 2002).
Among these tea pigments, pheophytins a and b were abundant, followed by chlorophylls a and b and carotenoids such as β-carotene and lutein in lower concentrations. All these pigments exhibited significant antioxidant activities against hydroperoxide generation, in the order chlorophyll a > lutein > pheophytin a > chlorophyll b > β-carotene > pheophytin b (Loranty et al., 2010). In particular, gamma irradiation was approved for disinfection and microbial control in various food products and supplements by the Food and Drug Administration (FDA), being a procedure technically and economically viable and physically safe with a powerful antimicrobial effect (Mizani et al., 2009). They reported that sun drying of green leafy vegetables caused a significant increase in TPC, reducing property and free radical scavenging activity, even though there was a significant decrease in vitamin C content according to (Oboh et al., 2009).
The aims of the current study were: (a) Drying of mint leaves using sun-drying method., (b) Select suitable dose of gamma radiation to irradiate dried mint leaves., (c) Studying some physical and chemical properties of extracting oil before and after irradiation and (d) Examine the influence of gamma radiations on oil mint quality.

Sample Preparation
The samples of fresh mint were purchased from a local market in Giza region, Egypt in July 2018. The leaves were carefully cleaned manually to remove dirt and damaged ones and were removed from the stalks. Then, the mint leaves were washed subjected to sun drying to achieve a percentage moisture content of < 20% at four days to dry in the Sun. Dried Mint leaves (moisture content < 20%), were sealed in low density polyethylene bags and stored at 4±1°C until exposed to gamma radiation.

Moisture Content
The moisture content of the Mint leaves was determined by the method of the difference between weights before and after exposing to hot air oven at 70°C for 72 h is considered as moisture. The percentages of moisture were calculated as follows (Anonymous, 1990): Where: W1 = Weight of the sample before drying, g W2 = Weight of sample after drying, g

Gamma Irradiation Treatments
Dried Mint leaves divided into 5 groups and treated with 60 Co (Cobalt 60) gamma rays, at the National Center for Radiation Research and Technology (NCRRT), Egypt. Dried Mint leaves were exposed to different doses of γrays; 0 (control), 5, 10, 15 and 20 (Gy). The control and irradiated samples were stored in plastic containers at room temperature (28-30°C).

Preparing of Dry Mint Extract Samples
Dried mint leaves which exposed to radiation in various doses were extracted by mixing them with distill water and stirring and turning for 15 min, then separated in Centrifugal Concentrators for 10 min (1000g). After filtering and extraction, a 110 mL were obtained and three replicates have been made after each test analysis (Pellegrini et al., 2003).

Determination of Chlorophyll a, Chlorophyll b and Carotenoids
The extraction process was done in triplicate. The solution was analyzed for Chlorophyll a (Ch a), Chlorophyll b (Ch b) and carotenoid content by a spectrophotometer JENWAY 6300 at various wavelengths 470, 645 and 662 nm is using a glass cuvette. The equations used for the quantification are given in Table 1. Results were expressed as mg g 1 of plant material.
The equations for calculation of chlorophyll a, chlorophyll b and the total carotenoid content (Sumanta et al., 2014

Total Antioxidant Assay
The antioxidant content has been estimated as an equivalent to Quercetin according to Meda et al. (2005).

Phenolic Analysis
The extracts obtained above were analyzed using an HPLC chromatograph (HewlettePackard 1100, Agilent Technologies, Santa Clara, CA, US) with a double online detection using a Diode Array Detector (DAD) with 280, 330 and 370 nm as preference wavelengths and a Mass Spectrometer (MS) equipped with an ESI source and a triple quadrupole-ion trap mass analyzer, which was connected to the HPLC system via the DAD cell outlet (Barros et al., 2013).
Total flavonoids assay: this is accomplished by using the method used by (Meda et al., 2005). DPPH (2, 2-Diphenyl-1-Picrylhydrazyl) radical scavenging activity assay Free radical scavenging activity against 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) radical was measured according to the modified method (Oboh, 2005). Ascorbic acid was used as the standard; while the inhibition ratio of DPPH scavenging activity was calculated from the equation: ,% / *100 AA Ac Ac Ac  Where: AA = Ascorbic acid Ac = Absorbance of control and As = Absorbance of the test sample.

Chlorophyll a and Chlorophyll b Contents
Data of Chlorophyll a and b were illustrated for all the tested mint samples in Table 1 and Fig 1 and 2. Other 86 scientists also reported that changes in the colour and the content of chlorophylls were related to the genotype but not to the growing conditions. The highest content of chlorophyll a, chlorophyll b and total chlorophyll in the mint leaves was observed 299.79, 289.72 and 589.511 mg/100 oil g in irradiated sample with 20 kGy, respectively. While, the lowest content of chlorophyll a, chlorophyll b and total chlorophyll in the mint leaves were observed 178.62, 162.95 and 341.57mg/100 oil g in irradiated sample with 5 kGy, respectively. These results were found, according to (Bekhradi et al., 2015).
In the tested mint leaves a ratio between chlorophyll a and chlorophyll b ranged from 1.035-1.566, meaning that chlorophyll a is the main form of chlorophyll in the leaves. These results were agreements with (Loranty et al., 2010).
Percentage of decreasing or increasing of chlorophyll a and b differed between all the tested mint samples compared with control samples in Fig. 3 and 4. For chlorophyll a, it was noticed that negative percentage of -39.006, -35.203 and -25.634% for irradiated samples of 5, 10 and 15 kGy while at the irradiated sample with 20 kGy has a positive percentage of 2.370% For chlorophyll b, it was found that negative percentage of-12.842 and -5.065% for irradiated samples of 5 and 10 kGy, while at irradiated samples with 15 and 20 kGy has a positive percentage of 1.150 and 54.964%.      88

Carotenoid Content
The basic pigments of green plants are chlorophylls, always accompanied by carotenoids. The highest content of carotenoids was observed in Mentha spicata leaves, 110.33 mg 100 oil g in irradiated sample with 20 kGy. While, the lowest content of carotenoids was observed in Mentha spicata 'leaves, 96.56 mg 100 oil g in irradiated sample with 5 kGy Table 1 and Fig. 5. Carotenoid content in other herbs like dills is significantly higher 30.3 mg 100 g 1 (Lisiewska et al., 2004). Percentage of decreasing or increasing of carotenoids differed between all the tested mint samples compared with control samples in Fig. 6. For carotenoids, it was noticed that negative percentage of -9.291, -6.097 and -3.138% of irradiated samples of 5, 10 and 15 kGy, while at the irradiated sample with 20 kGy has a positive percentage of 3.645%.

The Effects of γ-Irradiation and the Total Phenols and Total Flavonoids
The effects of γ-irradiation and the total phenols and total flavonoids are shown in Table 2 and Fig. 7 and 8.
The data demonstrated that the irradiated mint at 5, 10, 15 KGy had lower phenolic compounds than non-irradiated control in the mint extract. While, irradiated mint at 20 KGy had higher phenolic compounds. The significant decreases in the phenolic contents were 241.61, 249.02 and 251.30 mg/100g for samples irradiated at 5, 10 and 15 kGy, respectively, compared to the content in case of nonirradiated control (251.94 mg/100g). While, the increases in the phenolic contents were 258.82 mg/100g for sample irradiated at 20 kGy, compared to the content in case of non-irradiated (control)  The data demonstrated that the irradiated mint at 5, 10, 15 KGy had lower flavonoids, compounds than nonirradiated control in the mint extract. While, irradiated mint at 20 KGy had higher flavonoids compounds.
The significant decreases in the flavonoids contents were 1.14, 1.52 and 1.73 mg/100g for samples irradiated at 5, 10 and 15 kGy, respectively, compared to the content in case of non-irradiated control (1.84 mg/100g). While, the increases in in the phenolic contents were 3.28 mg/100g for sample irradiated at 20 kGy, compared to the content in case of non-irradiated (control). The maximum increase was obtained at 20 kGy dose.
Phenols are found in food either freely or as glycosides. There are more than 8000 kinds of natural  (Kim and Lee, 2004). Percentage of decreasing or increasing of phenolic differed between all the tested mint samples compared with control samples in Fig. 9. For phenolic, it was noticed that negative percentage of -4.100, -1.159 and -0.254%, for irradiated samples of 5, 10 and 15 kGy, compared to nonirradiated sample (control), while at the irradiated sample with 20 kGy has positive percentage of 2.731%, compared to non-irradiated sample (control).
Percentage of decreasing or increasing of flavonoids differed between all the tested mint samples compared with control samples in Fig. 10. For flavonoids, it was noticed that negative percentage of -38.133, -17.511 and -6.114%, for irradiated samples of 5, 10 and 15 kGy, compared to non-irradiated sample (control). While, at the irradiated sample with 20 kGy has a positive percentage of 78.003%, compared to non-irradiated sample (control). Flavonoids are vegetable dyes spread in different parts of the plants. They are divided into several various divisions that include Flavones. More precisely, Flavones are considered to be compounds that their color range varies from colorless to light yellow and can be found in citrus, cumin and mint (Alessandro et al., 2003).

The Effects of γ-Irradiation and the Total Antioxidant and Ascorbic Acid
The effects of γ-irradiation and the total antioxidant and ascorbic acid are shown in Table 3 and Fig. 11 and 12.

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The data demonstrated that the irradiated mint at 5, 10, 15 KGy had lower Total Antioxidant compounds than non-irradiated control in the mint extract. While, irradiated mint at 20 KGy had higher total Antioxidant compounds. The significant decreases in the Total Antioxidant contents were 600.87, 604.20 and 606.87 mg/100g for samples irradiated at 5, 10 and 15 kGy, respectively, compared to the content in case of nonirradiated control (609.75 mg/100g). While, the increases in the Total Antioxidant contents were 610.62 mg/100g for sample irradiated at 20 kGy, compared to the content in case of non-irradiated (control).
The data demonstrated that the irradiated mint at 5, 10, 15 KGy had lower Ascorbic acid compounds than non-irradiated control in the mint extract. While, irradiated mint at 20 KGy had higher Ascorbic acid compounds. The significant increase in the Ascorbic acid contents were 60.77, 61.15, 62.48 and 64.47 mg/100g for samples irradiated at 5, 10, 15 and 20 kGy, respectively, compared to the content in case of non-irradiated control (55.47 mg/100g). So, the lowest value of the Ascorbic acid contents was 55.47 mg/100g for sample non-irradiated. While, the highest value of the Ascorbic acid contents was 64.47 mg/100g for sample irradiated was obtained at 20 kGy dose. It was obvious that 20 kGy irradiation dose led to the highest increase in the total antioxidant.
The present findings are in good agreement with that of Taipina et al. (2009) who reported that there was no loss of the antioxidant content of pecan nuts as a result of gamma. While Huang and Mau (2006) found that the antioxidant content of the methanolic extract of Agaricusblazei was 45 mg/100g and this amount was increased to 106 mg/100g after 20 kGy of γ-irradiation.
Percentage of decreasing or increasing of total Antioxidants differed between all the tested mint samples compared with control samples in Fig. 13. For total Antioxidants, it was noticed that negative percentage of -1.456, -0.910 and -0.472% for irradiated samples of 5, 10 and 15 kGy, compared to non-irradiated sample (control), while at the irradiated sample with 20 kGy has a positive percentage of 0.143%, compared to non-irradiated sample (control).
Percentage of decreasing or increasing of Ascorbic acid differed between all the tested mint samples compared with control samples in Fig. 14. For Ascorbic acid, it was noticed that positive percentage of 9.555, 10.240, 12.637 and 16.225% for irradiated samples of 5, 10 and 15 kGy, compared to nonirradiated sample (control). So, the lowest increase was 9.555% in the irradiated sample with 5 kGy. While, the highest increase was 16.225% at the irradiated sample with 20 kGy.
Intense and/or prolonged thermal treatment may be responsible for a significant loss of natural antioxidants, as most of these compounds are relatively unstable. Yet in some cases, drying process causes little or no change to the content and activity of naturally occurring antioxidants, such as carotenoids and vitamin C (e.g., lycopene) according to Nicoli et al. (1999).

Statical Analysis
The economic benefit of using gamma irradiation with different radiation levels on the quality characteristics of dried mint oil. Table 1 showed that the use of irradiation with gamma rays at different radiation levels, which are 5, 10, 15 and 20 kGy, led to the effect on the quality specifications of oil extracted from dried mint leaves where the results showed that the highest percentage of chlorophyll A, chlorophyll B, the total chlorophyll, carotenoids in dried mint leaves were at a dose of irradiation of 20 kGy, as it was found that the highest value of phenolic compounds was at a dose of irradiation of 20 kGy.
By performing a variance analysis of the quality characteristics of the oil extracted from dried mint leaves and measured with the content of every 100 g of oil extracted from chlorophyll A, chlorophyll B, carotene, phenol, flavonoids, antioxidant compounds, ascorbic acid by mass at different irradiation levels 5, 10, 15, 20 kGy and it was found from the results shown in Table 3 that there were significant differences in the content of chlorophyll (A), chlorophyll (B), phenol, flavonoids, antioxidants, ascorbic acid, where the calculated value of F was about 459. 881, 17606.373, 632.746, 14.806, 88,844 and 4,666, respectively. While there was no significant difference in carotene content as the calculated F value reached 1.383.
By conducting LSD test to measure the least significant difference between the control sample and the radiation irradiated samples with gamma rays to know the fundamental differences between the different averages and to know the level of irradiation due to the greater impact on the quality specifications of peppermint oil, it was found from the results shown in Table 4 that the use of irradiation with gamma rays At 20 kGy, the most influencing was the higher content of chlorophyll (a) and chlorophyll (b). While the use of the level of irradiation with gamma rays at the level of 15 kGy had a greater impact on the high content of carotene, phenol, flavonoids. Also, the use of irradiation at 10 kGy level had the best effect on the high content of antioxidant and ascorbic acid.

Conclusion
The highest content of chlorophyll a, chlorophyll b, total chlorophyll and content of carotenoids in the dried mint leaves were observed in irradiated samples with 20 kGy. While, the highest content was in the irradiated sample with 5 kGy.
The irradiated dried mint leaves at 5, 10, 15 KGy had lower phenolic, flavonoids, Total antioxidant and Ascorbic acid compounds than non-irradiated control in the mint extract. While, irradiated mint at 20 KGy had higher phenolic compounds.
It was found significant differences in the content of chlorophyll (A), chlorophyll (B), phenol, flavonoids, antioxidants, ascorbic acid, while there was no significant difference in the carotene content.
By using of irradiation with gamma rays of 20 kGy, the most influencing was the higher content of chlorophyll (a) and chlorophyll (b). While the use of the level of irradiation with gamma rays of 15 kGy had a greater impact on the high content of carotene, phenol, flavonoids. Also, the use of irradiation at 10 kGy level had the best effect on the high content of antioxidant and ascorbic acid.
Therefore, the results indicate that the use of irradiation is a suitable method for dried mint leaf preservation,