Studying the Inhibition Effect of Some Food Additives Against Pathogenic Bacteria

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

Studying the inhibition effect of some food additives against pathogenic bacteria

Muhsin Hamad Edham1*, Nehan Bahaaldden Jafar2 and Zainab Hussein Fadhil2

1College of Pharmacy, University of Kirkuk – Iraq 2Department of Biology, College of Science, University of Kirkuk, Iraq.

*Corresponding authors: [email protected]

Abstract

Latterly, natural products have been used as antibiotics and have proven effective against a large number of microorganisms. The present study included the measurement of antibacterial activities of pomegranate juice or molasses, tamarind molasses, garlic oil with thyme and with chili pepper on the five selected bacteria (2 Gr+ and 3 Gr-) by disk and agar well diffusion assays on the Muller Hinton Agar (MHA) and Blood Agar (BA). Pomegranate and tamarind molasses exhibited a broad spectrum of anti-bacterial activity inhibiting both the groups of bacteria. Pomegranate and tamarind molasses has shown highest antimicrobial activity compared to garlic oil with herbs. Inter- selected of bacterial cultures, the highest antibacterial effect of pomegranate and tamarind molasses by using disk diffusion was recorded against E. coli, Staphylococcus aureus, Streptococcus mutans and Pseudomonas aeruginosa (20,18,20,12 mm) (20,20,20,17 mm) respectively while, when used agar well diffusion assay to tested antibacterial activities of pomegranate and tamarind molasses the result show rather increased inhibition zone of selected bacteria but, bacterial culture of Klepsiella pneumonia recorded resistance against food additives which can used in this study.

Key words: Food additives, Pomegranate molasses, Tamarind molasses, Garlic oil, Antibacterial activity. How to cite this article: Edham MH, Jafar NB, Fadhil ZH (2020): Studying the inhibition effect of some food additives against pathogenic bacteria, Ann Trop Med & Public Health; 23(IIb): S455. DOI: http://doi.org/10.36295/ASRO.2020.23231

1. Introduction Medicinal plants contribute at the present time in the treatment of many diseases, where the scientific interest and the increasing demand for these products contributed to encouraging the development of these products, as recent studies have shown that herbal medicines have a prominent role in the field of health and medicine (Gragg and newman, 2001). Medicinal plant products can be used to treat many difficult diseases, with the aim of combining the practices of modern medicine with folk medicine, which can treat many medical problems such as resistance to many pathogens to antibiotics and others (Afolayan, 2003; Olavi Pelkonen et al, 2014). Food additives can be used as anti-microbial preservatives, which inhibit the growth of bacteria and fungi; the food industry has been using a variety of non-antibiotic based antimicrobials, including additives and disinfectants for controlling food borne spoilage and pathogenic microorganisms. Food additives includes preservatives, sweeteners, color additives, spices and flavors, flavor enhancers, emulsifiers, nutrient supplements, texturing agents, acidulants and enzymes. The excessive and inappropriate use of antibiotics in medicine and agriculture has led to the emergence of antimicrobial resistant bacteria. (Meera et al, 2017). Pomegranate (Punicagranatum L.) is consumed both as fresh fruit and fruit juice. It is also being used in the production of jam, wine, liqueur, food coloring agent and flavor enhancer. The kernels are also used as a garnish for desserts and salads (Al-Maiman and Ahmad, 2002). The pomegranate molasses is commonly used in salads and many dishes in Asian countries (Altan and Maskan, 2005; Maskan, 2006). Pomegranate is also a rich source of anthocyanins, eilagitanins and other phenolic compounds that have been shown to have antioxidant and anti-tumor activity (Bige et al, 2010). There is a range of phytochemical compounds in pomegranate that have showed antimicrobial activity, but most of the researchers have found ©Annals of Tropical Medicine & Public Health S455

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

that ellagic acid and larger hydrolyzable tannins, such as punicalagin, have the most important activities. In many cases, the mixture of the pomegranate constituents offers the most advantage (Howell and Souza, 2013). Pomegranate fruits have also been used in traditional medicine to treat dysentery, diarrhea and respiratory diseases (Dey and Hazra, 2015). Recent study indicates that both pomegranate aril and peel extracts have an effective antimicrobial activity, as evidenced by the inhibitory effect on the bacterial growth of two important human pathogens, including Staphylococcus aureus and Escherichia coli, often involved in foodborne illness (Pagliarulo et al, 2016). In addition, experimental data strongly support the antibacterial activity of pomegranate extracts against oral pathogen such as S. mutans (Sabramaniam et al, 2012). Tamarind fruits are used in Indian spices as a tension agent to provide the acidity required in different meals and are widely used in foods around the world. It has been used for centuries as a medicinal plant, its fruits are the most important medicinal part (Gupta et al, 2014). Tamarind fruits have a proven hepato- protection activity associated with the presence of polyhydroxyl compounds, (El-Siddiget al., 2004; Meléndez and Carriles, 2006). Cavalletto and his co-workers reported in 1994 that allicin, the main antimicrobial compound in garlic, rapidly degrades into dialysis sulfide, and that garlic oil and dialysis sulfide (DADS) is the most abundant amount of sulfide in aqueous garlic extracts that have no antimicrobial effect. These early negative results, few studies of the post-antimicrobial activity of garlic oil and component sulfides, and some vegetable sulfur compounds, including DMTS and allele isothiocyanate (AITC), are more effective in inhibiting yeast growth than bacterial growth (Kyung and Fleming, 1997) .

2. Materials and Methods 2.1. Isolates and Cultivation The bacteriology and food microbiology laborator in Dept. of biology-Science College-Kirkuk University/ Iraq provided "5" selected bacteria (2 Gram+ and 3 Gram-) (these types of bacteria were taken from urinary tract, food poisoning samples, teeth infection, wounds, in Kirkuk Hospital). Bacterial isolates were identified by biochemical diagnostic depending on the method followed by (Collee et al. 1996). Bacteria E.coli and Klepsiella pneumonia were cultured and growth in Macconkey Agar, Staphylococcus aureus in Manitol Salt Agar (MSA), Streptococcus mutans in Blood Agar, and Pseudomonas aeruginosa in Nutrient Agar (NA) and incubation at 37C for 24 hr.

2.2. Preparation of Food Additives Pomegranate molasses was prepared traditionally by handpicking, washing, and peeling and the arils with seeds were hand-crushed and then squeezed in order to obtain the juice. The juice was concentrated to 30- 35% by heated for 4-5 hours until to be thickly then leave cooled. We used the similar methods to obtain Tamarind molasses without seeds. Garlic oil with herbs purchased from commercial market of the Kirkuk city, Iraq.

2.3. Determination of Antibacterial Activity The antimicrobial activity of the food additives which used in this study was measured by using agar disk and well diffusion assay in five pathogenic bacteria, two Gr+ bacteria (Staphylococcus aureus and Streptococcus mutans) and three Gr- bacteria (Pseudomonas aeruginosa, E.coli and Klepsiella pneumonia). According the pour plate method, (1 ml) of each bacterial culture (18-24 hr) was taken to the center of sterile Petri dish and then poured (20 ml) of (MHA) in each plates, which contained bacterial inoculums and mixed well, after the solidification of medium four wells of each plate were made by using cork borer (6mm). Afterward, distribute (20 µl) of food additives (Pomegranate, Tamarind molasses, Garlic oil with thyme and Garlic oil with chili pepper) into each well on the plates and allow it to diffuse for 15-20 minutes at lab temperature. The other assay of testing are disk diffusion method. In this method, standardized inoculums of all each bacteria speared on the surface of culture medium using L-shape then, placed filter paper discs (6 mm), containing the antibacterial agents as requested concentration and incubatation under suitable conditions (Mounyr et al, 2016). Commonly, the mechanism of antimicrobial agents is diffuses into the agar medium and inhibits growth of microorganism by measuring the diameters of inhibition zones as shown in (Fig. 1). Table 1 and 2. ©Annals of Tropical Medicine & Public Health S455

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

3. Result and Discussion Antibacterial activity of food additives against pathogenic bacteria The results are shown in fig. 1. Table (1,2) when using pomegranate molasses against E. coli bacteria on the medium (MHA) and in the case of the use of Disk and well where a diameter of 20 mm inhibitor was consistent with the results of a studies (Amy and Doris, 2013). (Sheilla et al, 2016) where they found that pomegranate molasses contain antioxidants, organic acids, antibiotics and enzymes such as Isocitric acid, Sulphate, Citric acid, Lactoferrin, Lactoperoxidase acting as antimicrobial (Bige et al, 2010). The results also showed that pomegranate molasses inhibited Staphylococcus aureus bacteria by 18 mm in the case of Disk and 20 mm in the case of well This explains the possibility of using these nutrients in the treatment of various infections caused by these pathogenic bacteria such as skin and pulmonary infections, tonsillitis, middle ear and urinary tract infections. These results are consistent with his findings (Valeria et al, 2019). The results also showed that the pomegranate molasses inhibitor Streptococcus mutants by 20 mm in the case of Disk and 25 mm in the case of well on the medium (MHA) and 20 and 22 mm in the case of Disk and well, respectively on the blood agar medium, fig. 2. Table 3 may be them substances that can prevent tooth decay, membrane pneumonia, peritonitis and peritonitis caused by Streptococcus mutants, which are rich in various compounds that inhibit bacteria and fungi (flavonoids, phenolic acid, tannins, moreover antioxidant) (Romeo et al,2015). These results are consistent with the results of both researchers ( Gianmaria et al, 2017) (Fischer et al, 2011). The results showed that pomegranate molasses inhibited the growth of Pseudomonas aerogun's bacteria 12 mm in the case of disk and 17 mm in the case of well on the medium (MHA). The difference in results in both cases may be due to the correlation of the active inhibitory material of these bacteria in pomegranate molasses with the paper material and less concentration than in the case of well. The results show that pomegranate molasses can be used in the treatment of many infections caused by Pseudomonas aerogenosa especially in burns and infections of the middle ear or other infections. These results are consistent with (Amy and Doris, 2013). The results also showed that Klepsiella pneumonia was resistant to pomegranate molasses on (MHA) in the case of tablets and pits. This explains the high resistance of these bacteria against the action of many plant extracts because of their possession of the capsule and a distinct cell wall and the presence of sugars that enhanced the presence of the capsule in this bacteria. Some researchers (Udayabdul-Reda et al, 2007) found that the aqueous and alcoholic extract of pomegranate inhibited the growth of Klepsiella pneumonia (14.10) mm respectively (fig. 1). The results showed in fig. 1. Table (1,2) that used Tamarind molasses on (MHA) medium by Disk method was inhibited against (E.coli, Staphylococcus aureus, Streptococcus mutants Pseudomonas aerogenosa) respectively (15,20,20,20) mm and well method (18,28,12,26) mm. Fig. 1. This results Enhances the high ability of Tamarind molasses to inhibit these pathogenic bacteria. Tamarind contains ions, minerals, proteins and organic acids such as Ascorbic acid, Tartaric acid, Citric acid, Terpenoids, Tannins, Riboflavin, which can be antioxidants and inhibitors of microorganisms such as bacteria and fungi (Dheerj etal, 2007). These results were consistent with the results of the researchers (jayaprakash et al, 2017) on E. coli, Staphylococcus aureus, and Pseudomonas aerogenosa, where recorded inhibition diameters respectively (22, 16, 15) mm and with the results of the researchers (Gupta et al 2014) on the bacteria E.coli, Staphylococcus aureus, Pseudomonas aerogenosa where recorded inhibition diameters respectively (16,18,13) mm. The present study did not any inhibition results of Tamarind molasses against Klepsiella pneumonia due to the presence of the capsule which was enhanced by the presence of different sugars in Tamarind molasses which became the protective agent for bacteria against the action of inhibitors in Tamarind molasses (Dheerj etal, 2007). The results as shown in tables (1, 2) fig. 1. when using Garlic oil (GO) with thyme, Garlic oil with chili peper on MHA medium and in two methods Disk and well are not inhibited for each of the pathogenic bacteria used in the current study (E .coli, Staphylococcus aureus, Streptococcus mutants Pseudomonas aerogenosa) except when using Garlic oil with peper against Staphylococcus aureus where was inhibition recorded a 15 mm. This may be due to the presence of oil that have inhibited and neutralized many active and inhibitory free radicals in the garlic plant, such as the Allicin compound in garlic, which inhibits the formation of Acetyl CoA and thus inhibits the construction of DNA, RNA and thus inhibits protein building (Khashan, 2014). Jay et al, 2004 indicated that (GO) did not have strong antibacterial effects because that allicin, the principal ©Annals of Tropical Medicine & Public Health S455

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

antimicrobial compound of garlic, was rapidly decomposed to diallyl sulfides, and that garlic oil, diallyl sulfides, and aqueous garlic extracts lacking allicin all had no antimicrobial effect. Because of these results, subsequent studies were conducted on the antimicrobial activity of garlic oil (GO) and its active sulfides compounds.

TABLE 1. Zone of inhibition (mm) for some food additives against different bacterial isolates (Disk diffusion testing) on MHA media

Sample Inhibition zone (mm)

Pomegranate Garlic oil with Tamarind Garlic oil with isolates molasses Thyme molasses chilli peper E. coli 20 - 20 - Staphylococcus aureus 18 - 20 15 Streptococcus mutans 20 - 20 - Pseudomonas aerogenosa 12 - 15 - Klepsiellapneumoniae - - - -

TABLE 2. Zone of inhibition (mm) for some food additives against different bacterial isolates (Agar well diffusion testing). On MHA media

Sample Inhibition zone (mm)

Pomegranate Garlic oil with Tamarind Garlic oil with isolates molasses (1) Thyme(2) molasses(3) chilli peper(4) E. coli 20 - 26 - Staphylococcus aureus 20 - 20 - Streptococcus mutans 25 - 28 - Pseudomonas aerogenosa 17 - 18 - Klepsiellapneumoniae - - - -

TABLE 3. Zone of inhibition (mm) for some food additives against Streptococcus mutans on Blood Agar

Diffusion (1) (2) (3) (4) samples

Disk diffusion 20 15 15 - Agar well diffusion 22 - 25 -

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

Disk

Well Figure 1. Streptococcus mutans, Pseudomonas aeruginosa, Staphylococcus aureus, Klepsiella pneumoniae, and E.coli on (MHA), disk and agar well diffusion assay.

Figure 2. Streptococcus mutans,on Blood agar, disk and agar well diffusion assay

4. Conclusion In recent study suggest that Pomegranate and Tamarind molasses are a much better antimicrobial agents so that to be a natural antimicrobials for food preservation showing large-scale as antibacterial activity against most pathogenic bacteria which used in this study than Garlic oils therefore, these substance can be considered a natural and inexpensive source of food preservation. We conclude in this present study that pomegranate and Tamarind molasses are also rich in phenols which act as antimicrobial agents and have high nutritional values in addition to containing antioxidant of great importance to human health.

References

1. Meera S. N., Indu U., Mary A. A. and Kumar V. 2017. Antimicrobial Food Additives and Disinfectants: Mode of Action and Microbial Resistance Mechanisms. Book: Food Borne Pathogens and Antibiotic Resistance. DOI: 10.1002/9781119139188.ch12. 2. Al-Maiman S.A. and Ahmad D. 2002. Changes in physical and chemical properties during pomegranate (Punicagranatum L.) fruit maturation. Food Chemistry, 76: 437-441. 3. Altan A. and Maskan M. 2005. Rheological behavior of pomegranate (Punicagranatum L.) juice and concentrate. Journal of Texture Studies, 36: 68-77. 4. Maskan M. 2006. Production of pomegranate (Punicagranatum L.) juice concentrate by various heating methods: colour degradation and kinetics. Journal of Food. Engineering, 72: 218-224. 5. Afolayan, A.J. 2003. Extracts from the shoots of Arctotisartoides inhibit the growth of bacteria and fungi .journal pharm.Bio.14,pp.22-25. 6. Olavi Pelkonen, Qihe Xu, and Tai-Ping Fan. 2014. Why is Research on Herbal Medicinal Products Important and How Can We Improve Its Quality J Tradit Complement Med. 4(1): 1–7. 7. Bige İ., Canan E. T. and Ömer U. Ç. 2010. A Research on the Composition of Pomegranate Molasses. Journal of Agricultural Faculty of Uludag University. Cilt 24, Sayı 2, 37-47.

Edham et al (2020): Inhibition effect of some additives January 2020 Vol. 23 (IIb)

8. Howell A. B. and ’Souza D. H. D. 2013. The pomegranate: Effects on bacteria and viruses that influence human health. Evidence- Based Complementary and Alternative Medicine, vol. 2013, Article ID 606212, 11 pages. 9. Dey D., Ray R. and Hazra B. 2015. Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and -lactamase producing Klebsiella pneumoniae, Journal of Pharmaceutical Biology, vol. 53, no. 10, pp. 1474–1480. 10. Pagliarulo C., De Vito V., Picariello훽 G. et al. 2016. Inhibitory effect of pomegranate (Punicagranatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli, Food Chemistry vol. 190, pp. 824–831. 11. Subramaniam P., Dwivedi S., Uma E. and GirishBabu K. L. 2012. Effect of pomegranate and aloevera extract on streptococcus mutans: An in vitro study, Dental Hypotheses, vol. 3, no. 3, pp. 99–105. 12. Gupta, C., Prakash, D. and Gupta, S. 2014. Studies on the antimicrobial activity of Tamarind (Tamarindusindica) and its potential as food bio-preservative. International Food Research Journal 21(6): 2437-2441. 13. El-SiddigK., Gebauer, J., Ebert, G., Ali, A.M. and Inanaga, S. 2004. Influence of salinity on emergence and early seedling growth of Tamarindusindica L. European Journal of Horticultural Science 69(2): 79-81. 14. Melιndez P. A. and Carriles, V.A. 2006. Antibacterial properties of tropical plants from Puerto Rico. Phytomedicine 13: 272-276. 15. Cavallito C. J. and Bailey J. H. 1944. Allicin, the antibacterial principle of Allium sativum. I: isolation, physical properties and antimicrobial action. J. Am. Chem. Soc.66:1950–1951. 16. Kyung K. H. and Fleming H. P. 1997. Antimicrobial activity ofsulfur compounds derived from cabbage. J. Food Prot. 60:67–71. 17. Collee J.G., Fraser A.G., Marmion B.P., Simmons A. M. and McCartney. 1996. Practical medical microbiology, 14th ed. UK: The Churchill Livingstone. Inc. pp. 329–41. 18. Mounyr B. , Moulay S. and Saad K. I. 2016. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis. 6: 7 19. Amy B. H. and Doris H. D. 2013. The Pomegranate: Effects on Bacteria and Viruses That Influence Human Health. Evidence-Based Complementary and Alternative Medicine. Article ID 606212, 11 pages. 20. Sheilla N., John H. M. and Ivan M. M. 2016. Effect of tamarind (Tamarindus indica L.) seed on antioxidant activity, phytocompounds, physicochemical characteristics, and sensory acceptability of enriched cookies and mango juice. Food Science & Nutrition . 4(4): 494–507 21. Valeria S., Cinzia Lucia R., Cinzia C. and Gabriele B., Flora V. R., Simona F., Nicolina T., Marco R. and Luca V. 2019. Beneficial Effects of Pomegranate Peel Extract and Probiotics on Pre-adipocyte Differentiation. Frontiers in Microbiology. April 2019 | Volume 10 | Article 660. 22. Romeo, F. V., Ballistreri, G., Fabroni, S., Pangallo, S., Nicosia, M. G., Schena, L., et al. 2015. Chemical characterization of different sumac and pomegranate extracts effective against botrytis cinerea rots. Molecules 20, 11941–11958. doi: 10.3390/molecules200711941. 23. Dheeraj S., Lobsang W. and Surendra K.M. 2007. Processed Products of Tamarind. Natural Product Radiance. 6(4): 315-321. 24. Gianmaria F. F., Elisa S., Daniela S., Gabiria P., Roberta C., Chiara P., Tiziana C., Brunella A., Marco C., Aniello I., Elena S., Annunziata G. C., et al. 2017. In Vitro Antibacterial Activity of Pomegranate Juice and Peel Extracts on Cariogenic Bacteria. Bio Med Research International.Volume 2017, Article ID 2152749, 7 pages. 25. Fischer, U. A., Carle, R., and Kammerer, D. R. 2011. Identification and quantification of phenolic compounds from pomegranate (Punicagranatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MS(n). Food Chem. 127, 807–821. 26. Uday Abdul- Reda H. , Bassam A. H. , Hind A. S., Ali T. A. and Sahar M. M. 2007. Antimicrobial activity of ethanolic and aqueous extracts of pomegranate peel against Extended Spectrum Beta-Lactamase producing bacteria. Journal of Thi-Qar University. 12(4):74-87. 27. Jayaprakash N., Judith J. V., Kaviyarasu K., Kombaiah K., John L. K., Jothi R. R., Murugan A. M. and Hamad A. 2017. Green synthesis of Ag nanoparticles using Tamarind fruit extract for the antibacterial studies. Journal of Photochemistry & Photobiology,B:Biology.S1011-1344(17)30151-3. 28. Jay W. K., Yeon S. K. and Kyu H. K. 2004. Inhibitory Activity of Essential Oils of Garlic and Onion against Bacteria and Yeasts. Journal of Food Protection, Vol. 67, No. 3, 2004, Pages 499–504. 29. Khashan A. A. 2014. Antibacterial Activity of Garlic Extract (Allium sativum) Against Staphylococcus aureus in Vitro. Global Journal of Bio-Science and Biotechnology. (G.J.B.B.), VOL.3 (4): 346-348.