Determination of the Effects of Food Preservatives Benzoic Acid and Sodium Nitrate on Lifespan, Fertility and Physical Growth in Caenorhabditis Elegans
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Journal of Survey in Fisheries Sciences 6(1) 49-63 2019 Determination of the effects of food preservatives benzoic acid and sodium nitrate on lifespan, fertility and physical growth in Caenorhabditis elegans Sarac H.1*; Sari M.2 Received: March 2019 Accepted: July 2019 Abstract Presently, the use of protective food additives such as benzoic acid and sodium nitrate is quite common. However, it was found that these additives, which initially appeared to be harmless, led to the emergence of a number of health problems. Cancer and diseases and deaths with no apparent causes are among the leading concerns. Therefore, the studies which can reveal the genotoxic potential of food preservatives and their negative effects on human health are very important in terms of ensuring food safety. Many model organisms are used to show these negative effects. Caenorhabditis elegans (C. elegans) is an important model organism, which is frequently used in determining the negative effects and toxic doses of substances that are toxic to or may have toxic effects on humans. In the present study, the aim was to determine the effects of different doses of benzoic acid and sodium nitrate, which are among the protective food additives known to cause certain diseases in humans, on lifespan, fertility and physical growth of C. elegans. Within the scope of the study; instead of the standard nutrient, C. elegans was supplemented with 5 different doses (0.006 g, 0.01 g, 0.02 g, 0.05 g, 0.1 g/10 mL) of benzoic acid and consequently, sodium nitrate, and lifespan, fertility and physical growth changes were examined in C. elegans which were exposed to different doses of benzoic acid and sodium nitrate. The findings were evaluated by reaching to comparisons with the control groups. At the end of the study, it has been determined that 0.01 g, 0.02 g, 0.05 g, 0.1 g/10 mL doses of benzoic acid, and all the administered doses of sodium nitrate (0.006 g, 0.01 g, 0.02 g, 0.05 g, 0.1 g/10 mL) have detrimental effects on lifespan, fertility and physical growth in C. elegans. On the other hand, 0.006 g/10 mL dose of benzoic acid did not cause any significant difference in lifespan and when compared to the control group. However, at a dose of 0.006 g/10 mL of benzoic acid, physical growth and fertility was found to be less than that of control group. As a result, it was determined that benzoic acid and sodium nitrate have negative effects on lifespan, fertility and physical growth due to dose increase. Downloaded from sifisheriessciences.com at 10:09 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2019.6.1.6 ] Keywords: Benzoic acid, Sodium nitrate, Caenorhabditis elegans, Lifespan, Fertility, Physical growth 1-Department of Crop and Animal Production, Vocational School of Sivas, Sivas Cumhuriyet University, Sivas, Turkey. 2- Department of Biology, Faculty of Science, Sivas Cumhuriyet University, Sivas, Turkey. *Corresponding author's Email: [email protected] 50 Sarac and Sari, Determination of the effects of food preservatives benzoic acid and sodium nitrate on … Introduction Studies have shown that in susceptible Preservatives commonly used in food individuals, benzoic acid even at low doses additives, are used to destroy bacteria, has side effects, such as brain injury, molds and yeasts, any microorganisms hypersensitivity, weight loss, triggering with or without pathogens, to prevent their asthma and nervous disorders, proliferation or activities, and thus delay hyperactivity and urticaria in children, skin deterioration (Surekha and Reddy, 2000; redness, swelling, itching and pain, Kucukoner, 2006; Altug, 2009; Guzel, increasing hormone oestrogen hormones 2013). Benzoic acid, which is widely used and tumour formation (Wibbertmann et al., in foods as preservative additives; a white 2000; Deshpande, 2002; Omaye, 2004; Qi colored, needle or leaflet-like, benzene et al., 2009; Erkmen, 2010; Ozpinar et al., ring, vapour volatile compound (Ucar, 2013). The symptoms in humans are 2004; Arslan, 2011) and the first chemical known to induce trigeminal innervation of preservatives to be legalized for use in taste cells in association with childhood food (Ogbadu, 1999; Guzel, 2013). hyperactivity and to cause temporary Benzoic acid is mostly used as sodium salt irritation and itching in the mouth and (sodium benzoate) in foods due to its low tingling and skin contact (Otero-Loseda, solubility in water. (Koyuncu, 2006; 2003; Zengin et al., 2011; Guzel, 2013). Yildiz, 2010; Guzel, 2013; Arslan, 2011; Sodium nitrate (NaNO3), which is the Ozturkcan and Acar, 2017). Its sodium salt of nitrate; is one of the antimicrobial activity is high on mold and preservative food additives used in foods yeasts, but it is not recommended for use (especially meat, meat products and fish) against bacteria. This is because pH values due to its characteristic flavour, natural above 4.5, where bacterial growth is high, colour preservation and antimicrobial reduce the mechanism of action of benzoic properties (Erkmen, 2010; Arslan, 2011). acid. (Robach, 1980; Baker et al., 1988; Although it varies by country, the Ozturkcan and Acar, 2017). A suitable pH incorporation of sodium nitrate or limit in which benzoic acid inhibits equivalent compounds of around 500 ppm microorganisms is an acidic pH ranging (mg/kg) into processed meat products has from 2.5 to 4.0. (Chichester and Tanner, become standard (Sanli and Kaya, 1998; 1972; Saldamli, 1985). Mostly found in Kaya, 2011). fruit juice, marmalade, margarine, jam, Nitrate ions have no direct toxic effect sodas, pickles, sauces, ketchup, jelly and (Bories ve Bories, 1995; Ozdestan and Downloaded from sifisheriessciences.com at 10:09 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2019.6.1.6 ] similar products in food industry (Fish et Uren, 2010). However, it was stated in al., 2000; Chayabutra and Ju, 2000; studies that nitrate taken together with food Kahraman and Geckil, 2005). The amount can be reduced to nitrite by bacterial nitrate prescribed for use in foods is between 0.2– reductase activity and nitrite, on the other 0.3% and is used only in French rennet. hand, is a precursor of nitrosamine (Arslan, 2011; Cotra, 2016). compounds that induce tumour formation in humans and animals, plays a role in the Journal of Survey in Fisheries Sciences 6(1) 2019 51 formation of cancers of the liver, lung, sensitive balance and 250 mL of distilled kidney, larynx, stomach and pancreas. water (distilled water, dH2O) was added. (Omaye, 2004; Erkmen and Bozoglu, The agar was thawed in a magnetic stirrer 2008; Erkmen, 2010; Aschebrook-Kilfoy and placed in an autoclave at 125°C for 15 et al., 2011). In addition, besides all these minutes. The medium exiting the autoclave negativities, dietary nitrate may cause was cooled to 55°C. The cooled TBX Agar methemoglobinemia, shortness of breath, medium was poured into approximately 60 dizziness, hypotension and circulatory mL petri dishes and allowed to solidify. collapse (Omaye; 2004; Erkmen, 2010). After solidification, TBX Agar media The present study aims to determine which were ready for use were added E. the effects of protective food additives coli OP50 strain with burner flame with the known to cause some diseases in humans; help of the loop and incubated at 37°C for of different doses of benzoic acid and 24 hours. After colony formation was sodium nitrate (0.006 g, 0.01 g, 0.02 g, observed, specific blue colonies belonging 0.05 g, 0.1 g/10 mL) on lifespan, fertility to the strain E. coli OP50 were identified. and physical growth in a model organism C. elegans. C. elegans is a model Preparation of liquid medium for E. coli organism, showing 60-80% gene OP50 strain homology with humans, providing After weighing 9.125 g Lauryl Sulfate experimental advantages because of its Tryptose Broth (LST Broth) on a precision basic features such as simplicity, balance, 250 mL of distilled water was transparency and short life cycle and added thereto and autoclaved at 120°C for frequently used in basic biological studies 15 minutes. The autoclave sterilized and toxicity studies for years. medium was cooled to 37°C and then a colony E. coli OP50 strains was taken from Material and methods TBX Agar medium with the help of a loop N2 wild type C. elegans and Escherichia and seeded into LST Broth in a laminar coli OP50 strain were used in the study and flow (sterile cabinet). After transfer to the stock cultures were obtained from liquid medium, it was incubated for 24 Caenorhabditis Genetic Center (CGC) of hours in an oven at 37°C. After the University of Minnesota. Stock reproduction, it was raised to 4 °C for use cultures were maintained in the dark and at in the study. appropriate temperatures (20-25°C for C. Downloaded from sifisheriessciences.com at 10:09 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2019.6.1.6 ] elegans; 4°C for Escherichia coli OP50 Preparation of Nematode Growth Media strain). (NGM) After weighing 2.5 g of Bacto-peptone, 3 g Purification of E. coli OP50 strain on TBX of NaCl and 20 g of Agar, it is dissolved in Agar medium 1 L of distilled water until it reaches the 9.125 g of TBX Agar (Tryptone Bile boiling temperature and autoclaved at 120 Glucuronide Agar) was weighed on a ºC for 15 minutes. After leaving the 52 Sarac and Sari, Determination of the effects of food preservatives benzoic acid and sodium nitrate on … autoclave, it was cooled to 55ºC. To ensure Lifespan analysis homogenisation of the cooled NGMs, Approximately 400 μl of E. coli OP50 previously prepared supplements were strain was added to the middle of the added to the medium; 1mL MgSO4 (1M), 1 NGMs containing FUDR and different mL Cholesterol (5 mg/mL), 1 mL CaCl2 doses of protective food additives were (1M) and 25 mL KPO4 buffer (pH: 7) were prepared for lifespan analysis and left to added to the medium by filtration through dry in sterile environment for 0.2 µm porous cellulose filters.