ارزیابی فعالیت ضد میکروبی و ترکیبات فنولی عصاره الکلی لایه داخلی پوست بالنگ (Citrus medica l.)در نوشابه گازدار لیمویی

Evaluation of antimicrobial activity and phenolic compounds of alcoholic citron (Citrus medica l.) peel inner layer extract in the lemon carbonated drinks

Introduction:
Citrus fruits are rich sources of different vitamins, minerals, and fibers. In recent years, more attention has been paid to the antibacterial properties of non-edible parts of fruits (Tavasoli et al., 2009). Citron (Citrus medica L.) is one of the species of citrus fruits (Dalia, 2014). Flavored essential oils of this fruit (located in the outermost layer, pigmented layer of peel) are also regarded as an antibiotic. The citron juice along with honey is also known as an effective antidote to poisons. Citron peel contains citronellal, terpenes, aldehydes, ketones, esters, alcohols, and organic acids. Limonene neutralizes the effect of pathogenic microbes. Geraniol and other monoterpenes found in the extract of citron peel have also anti-cancer properties (Mina et al., 2011).
Carbonated beverages are products made by a mixture of water, carbon dioxide, additives, and sugar, or other sweeteners (Elhami Rad & Mohammadi, 2006). The harmful effect of consuming industrial soft drinks can be examined from different perspectives (Bawa, 2005). There are some reports that show benzene forms by a reaction between benzoate in soft drinks and ascorbic acid, which is a cancer-causing compound. Due to the health risks attributed to consumption of industrial soft drinks, many researchers are looking for ways to optimize the consumption of natural drinks, as synthetic materials make up most of the compounds used in industrial soft drinks (hunter, 2003). In this study, given the beneficial effects of citron, the ethanolic extract of citron peel was used as a preservative compound in the formulation of lemon soft drinks, and its antimicrobial activity against microorganisms contaminating soft drinks was studied in a culture medium and lemon carbonated beverages.
Materials and methods:
In this study, the ethanolic extract of citron peel with concentrations of 0, 0.8, 1, 1.5, and 2 mg/ml, as a preservative composition, was added to the formulation of lemon soft drinks, and its antimicrobial activity against the microorganisms contaminating the soft drinks, including Lactobacillus delbrueckii, Leuconostoc mesenteroides, Saccharomyces cerevisiae, Candida krusei, was investigated.
The total amount of phenolic compounds in the produced lemon soft drinks was also measured based on the Folin-Ciocalteu method (Lin & Tang, 2007). The characteristics of color, taste, and flavor, and the overall acceptability of the produced samples were evaluated using 10 evaluators by the hedonic method and through completing the assessment questionnaire (Karami et al., 2012). Data were analyzed based on a completely randomized design, with the help of ANOVA, and using SPSS ver. 19 software. The charts were plotted using Excel software. The statistical comparison of results was also conducted at a 95% confidence level.
Results and discussion:
The results showed that the ethanolic extract of citron peel exhibited an inhibitory effect on the microorganisms studied. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Lactobacillus delbrueckii bacteria against the citron peel extract were assessed as 0.6 mg/ml and 0.8 mg/ml, respectively, whereas, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Leuconostoc mesenteroides bacteria were the same, 1 mg/ml. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of yeast saccharomyces cerevisiae and candida krusei were evaluated as the same value of 0.8. The ethanolic extract of citron peel exhibited an inhibitory effect on microorganisms of Lactobacillus delbrueckii, Leuconostoc mesenteroides bacteria, saccharomyces cerevisiae, and yeast candida krusei.
The results of the total count of mesophilic aerobic bacteria (CFU/ml) indicated the antimicrobial activity of citron peel extract in lemon soft drinks. The citron peel extract, with its antibacterial properties, was able to reduce the microbial load of the samples containing the extract by approximately the same amount as the sample containing sodium benzoate.
The count of acid-resistant microorganisms showed that the citron peel extract has an antimicrobial activity in the lemon soft drink. In the control and T_4 (2 mg/ml) treatment samples, in which there was a high concentration of the citron peel extract, the count of acid-resistant bacteria dropped to zero over the time of storage, and no growth was observed in them.
The results indicated the antimicrobial activity of citron peel extract in the lemon soft drink (yeast and molds count (CFU/ml). There was no significant difference between the produced treatments and the control sample at day 0 (P>0.05). The T_1, T_2, and T_3 treatments, which had lower amounts of the extract, had a significant difference with other samples at day 30, 60, and 90 of the test, and they also had higher colony counts than the control and T_4 (2 mg/ml) treatment samples (p < 0.05). In all the studied treatments, expect for T_4 treatment, there was an increasing trend in the amount of yeast and molds, and then over time, the amount of yeast and molds dropped to zero in the control and T_4 (2 mg/ml) treatment samples, so that the yeast and molds count for these treatments at day 90 of the test reached the range declared by Institute of Standards and Industrial Research of Iran (negative), which shows the antibacterial properties of citron peel extract.
The results of studying the amounts of phenolic compounds of the produced soft drinks showed that by increasing the amount of citron peel extract, the amount of phenolic compounds in the produced soft drink also increased, which can be due to the rich content of phenolic compounds in the citron peel extract. Considering the acidic pH, the amount of phenolic compounds in the produced lemon soft drinks containing citron peel extract retained during the storage period, and significantly increased after 30 days, and then remained constant. There was a significant difference between the produced treatments and the control samples at day 0 and other studied times, and the amount of phenolic compounds measured in all the treatments containing the citron peel extract was significantly higher than that of the control sample (p < 0.05). By increasing the amount of the citron peel extract, the amount of phenolic compounds also increased.
The results of sensory evaluation showed that there was no significant difference between all the treatments containing the citron peel extract and the control sample in terms of taste, flavor, and color (P>0.05). By increasing the amount of citron peel extract, the rates of taste, flavor, and overall acceptability decreased compared to the control sample, however, this rate was not evaluated as significant by the evaluators (P>0.05). The evaluators were not able to detect a difference with the control sample in terms of color, and this parameter had no significant difference with the control sample (P>0.05).
Conclusion:
The results of this study indicated that the citron peel extract, by having antimicrobial, phenolic, and antioxidant properties, has an antimicrobial activity against the microorganisms contaminating soft drinks. The results showed that, by adding the citron peel extract with a rate of 2 mg/ml, lemon soft drinks with more desirable microbial quality and chemical properties can be produced that can compete in terms of sensory evaluation with the lemon soft drinks containing sodium benzoate.

Keywords: Lemon Soft Drink, Extract, Citron Peel, Phenolic Compounds, Antimicrobial Activity