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

Document Type : Research Paper

Authors

azad university of ghazvin

Abstract

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

Keywords


الهامی راد ا ح و محمدی ع، 1385. بررسی فرمولاسیون نوشابه گازدار با استفاده از عرق بیدمشک و ارزیابی تغییرات فیزیکو شیمیایی و میکروبی آن در طی نگهداری. مجله پژوهشهای علوم و صنایع غذایی ایران،  دوره    2 شماره 1 . صفحات 39-27.
 پورامیر م و گلی ز، 1387. بررسی فعالیت آنتی اکسیدانی عصاره پوست نارنج و اثر بازدارنده آن بر اکسیداسیون لیپیدی در مدل بیولوژیک، هجدهمین کنگره ملی علوم و صنایع غذایی.
توسلی ص، ابراهیم زاده موسوی م ع، امام جمعه ز و رضوی ه، 1388.  بررسی خواص آنتی‌اکسیدانی و ضدمیکروبی اسانس رزماری در نوشابه لیمویی، پایان نامه، دانشکده صنایع غذایی، دانشگاه تهران.
حبشی م، میرزا م، مستوفی ی و جایمند ک، 1387. شناسایی و مقایسه ترکیبهای موجود در اسانس پوست میوه بالنگ (Citrus medica l) با دو روش استخراج ( تقطیر با آب و پرس سرد). فصلنامه تحقیقات گیاهان دارویی و معطر ایران،  جلد 24، شماره4. صفحات 436-428.
محمودی ر، پاک بین ب، وحیدی ر، 1395. تاثیر اسانس روغنی پوست بالنگ بر تغییرات کیفیت شیمیایی و میکروبی فیله ماهی قزل‌آلای رنگین‌کمان ذخیره شده در سرما. نشریه پژوهش های صنایع غذایی، دوره 26، شماره 3، صفحات 379-369. 
سلامی م، امام جمعه ز، ابراهیم زاده موسوی، م و رضایی ک، 1385. ﺍﻧﺪﺍﺯﻩ ﮔﻴﺮﻱ ﺟﺬﺏ ﻋﻄﺮ ﻭ ﻃﻌﻢ ﻧﻮﺷﺎﺑﻪ ﮔﺎﺯﺩﺍﺭ ﭘﺮﺗﻘﺎﻟﻲ ﺩﺭ ﻇﺮﻭﻑ ﺗﺮﻓﺘﺎﻻﺕ ﭘﻠﻲ ﺍﺗﻴﻠﻦ ﺑﺎ ﺍﺳﺘﻔﺎﺩﻩ ﺍﺯ ﻣﻴﻜﺮﻭ‌ﺍﺳﺘﺨﺮﺍﺝ ﺑﺎ ﻓﺎﺯ ﺟﺎﻣﺪ ـ ﮔﺎﺯ ﻛﺮﻭﻣﺎﺗﻮﮔﺮﺍﻓﻲ. ﻧﺸﺮﻳﻪ ﺷﻴﻤﻲ ﻭ ﻣﻬﻨﺪﺳﻲ شیمی ایران، دوره 25. شماره 3. صفحات 69-75.
شجاعی مهر م و ناظری س، 1392. بررسی اثر ضدمیکروبی و آنتی اکسیدانی عصاره اندام های مختلف و شناسایی فیتو شیمیایی برخی متابولیت های ثانویه بالنگ و دارابی، پایان نامه دانشگاه بوعلی سینا، دانشکده علوم کشاورزی.
شهابی ا، احمدی ع و حجازی م، 1387. بررسی خواص آنتی باکتریال ترکیبات فنولی هسته انگور و پوست انار در آب سیب بر باکتری Alicyclobacillus acidoterrestris، پایان نامه کارشناسی ارشد. دانشکده صنایع غذایی، دانشگاه تهران. صفحات 1-10.
شهابی قهفرخی ا، حجازی م ا، احمدی زنوز ع، قنبرزاده ب، 1390. کنترل باکتری Alicyclobacillus acidoterrestris در آب سیب به وسیله وسیله عصاره­های پوست انار. فصلنامه علوم و صنایع غذایی، شماره2 دوره 8.
کرمی ز، میرزائی ح، امام جمعه ز، صادقی ماهونک ع و خمیری م، 1391. ارزیابی فعالیت ضدمیکروبی عصاره اتانولی ریشه شیرین بیان در نوشابه پرتقالی، نشریه پژوهش های علوم و صنایع غذایی ایران، دوره 2 شماره 8، صفحات261-251.
موسسه استاندارد و تحقیقات صنعتی ایران، 1387. ویژگی ها و روش های آزمون میکروبیولوژی نوشابه های گازدار، چاپ اول، شماره استاندارد ایران 3846.
موسوی زرینی س، نجفی زرینی م و علوی م، 1394. ﺑﺮﺭﺳﯽ ﺍﺛﺮﺍﺕ ﺿﺪ‌ﻗﺎﺭﭼﯽ ﻋﺼﺎﺭﻩ ﻣﺘﺎﻧﻮﻟﯽ ﻭ ﺍﺗﺎﻧﻮﻟﯽ ﺍﻧﺪﺍﻡ ﻫﺎﯼ ﻫﻮﺍﯾﯽ ﺑﺎﻟﻨﮓ ﻭ ﻧﺎﺭﻧﺞ ﺑﺮﻋﻠﯿﻪ 2 ﻗﺎﺭﭺ ﺑﯿﻤﺎﺭﯾﺰﺍﯼ ﮔﯿﺎﻫﯽ. ﻫﻤﺎﯾﺶ ﻣﻠﯽ ﮔﯿﺎﻫﺎﻥ ﺩﺍﺭﻭﯾﯽ ﻭ ﮐﺸﺎﻭﺭﺯﯼ ﭘﺎﯾﺪﺍﺭ،  شماره 3. صفحات 25-32.
Abinu I, Adenopekun T, Adelowtan T, Ogunsanya T and Odugbemi T, 2007. Evaluation of the antimicrobial properties of different parts of Citrus Aurantifolia (Lime Fruit) as used locally. African Journal of Traditional, Complementary and Alternative Medicines 4(2): 185–190.
Adham AN, 2015. Phytochemical analysis and evaluation antibacterial activity of Citrus medica peel and juice growing in Kurdistan/Iraq. Journal of Applied Pharmaceutical Science 5(10): 136-141.
Arabshahi-Delouee S and Aalami M, 2007. Production of functional pineapple juice by incorporation of phenolic extracts from selected plants. International journal of food properties 10: 479-488.
Bawa S, 2005. The role of the consumption of beverages in the obesity epidemic. Journal of Royal Society for the promotion of Health 125(3), 124-128.
Dalia A A, 2014.preparation and characterize of pectin from peel of kabad (Citrus medica) Fruit in sulaimani city, Iraqi Kurdistan Region. International journal of current Research in chemistry and pharma ceutical Sciences 142-146.
Meena AK, Kandale Ajit, Rao MM, Panda P and Govind R, 2011. A review on citron-pharmacognosy, phytochemistry and medicinal uses. International Research Journal of Pharmacy 2(1):14.
Hunter ML, 2003. Development of low erosive carbonated fruit drinks, 2-Evaluation of an experimental orange drinks in vitro and situ. Journal of Dentistry (31), 253-260.
Kulisic T, Radonic A and Katalinic V, 2004. Use of different methods for testing antioxidative of oregano essential oil. Food chemistry (85), 633-640.
Kawanishi S, Hiraku Y, Murata M, and Oilawa S, 2002. The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Radical Biology Method 32, 822-832.
Laguerre M, Lecomte J and Villeneuve P, 2007. Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges. Progress in Lipid Research 46(5):244-82.
Lin JY and Tang CY, 2007. Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chemistry (101), 140-147.
Munwar S, Roy H, Rahaman SA, 2015. Antioxidant and Free Radical Scavenging Activity of Citrus Medica. International Journal of Pharma Research and Health Sciences 3 (4), 810-816.
Prompana K, Kandylis P, Tsakiris A, Kanellaki M and Kourkoutas Y, 2012. Application of alternative technologies for Elimination of artificial coloring in alcoholic beverages produced by citrus medica and potential impact in human health. Journal of Food and Nutrition Sciences 03(07):959-969.
Sah A, Vijay J and melkani A B, 2011. Antimicrobial Activity of six different parts of the plant citrus medical inn. Food Chemistry (123), 250-267.
Thronsberry C and McDougal, LK, 1983.  Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylocococci.Journal of Clinical Microbiology (18), 1084-91.
Vahidi R, Pourahmad R, Mahmoudi R, Hosseini SS, 2019. Chemical compounds and antibacterial and antioxidant properties of citron (Citrus medica L.) peel essential oil.   Journal of Food and Bioprocess Engineering 3(1): 83-88.