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Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 05 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.705.201 Evaluation of Antibacterial and Antifungal Activity of Cow Urine against Some Seed Borne Microflora Tanmay Ghosh1* and M.K. Biswas2 1 Department of Microbiology, Rabindra Mahavidyalaya, Champadanga, Hooghly, W.B., India 2 Department of Plant Protection, Palli Siksha Bhavana, Viswa-Bharati, Sriniketan, Birbhum, W.B., India *Corresponding author ABSTRACT Keywords Antibacterial, Antifungal, Cow Urine, Seed borne microflora Article Info Accepted: 16 April 2018 Available Online: 10 May 2018 Cow urine therapy and all traditional practices from Indian systems of medicine have a strong scientific base. The cow has proved to be a boon in the areas of agriculture, science and technology, industry, energy, medicine etc. for the development of any nation, in addition being eco-friendly in nature. In the present study the antibacterial and antifungal potentials of cow urine were investigated. Total 9 pathogenic and non-pathogenic bacterial and fungal cultures were used as test organism against three different cow urine samples. Fungal culture includes Aspergillus sp, Rhizopus sp, Mucor sp, Penicillium sp, Alternaria sp, Macrophomina sp and bacterial culture include Bacillus subtilis, Pseudomonas sp, Streptococcus sp. The highest zone of inhibition was shown against Aspergillus sp while the smallest zone of inhibition was shown against Macrophomina sp in fresh cow urine. The highest zone of inhibition was shown against Aspergillus sp while the smallest zone of inhibition was shown against Pseudomonas sp in photoactivated cow urine. The highest zone of inhibition was shown against Bacillus subtilis while the smallest zone of inhibition was shown against Pseudomonas sp in sterile cow urine. Based on cumulative effect against the test organism, the raw urine sample was found to be the most efficient inhibiting all the 9 test cultures but the activity was reported low against fungi compared bacteria. Introduction „The cow‟ is a mobile medical dispensary and cow urine is a panacea of all diseases (Pathak and Kumar, 2003). The cow urine, one of the ingredients of „Panchagawya‟ is capable of treating many curable as well as incurable diseases and has been used extensively in ayurvedic preparations since time immemorial as cited in ancient holy texts like Charaka Samhita, Sushruta Samhita, Vridhabhagabhatt, Atharva Veda, Bhavaprakash, Rajni Ghuntu, Amritasagar, etc (Pathak and Kumar, 2003). A lots of research has been conducted in Cow Urine Treatment and Research Centre, Indore over the past few years and it has been reported that gomutra is capable of curing blood pressure, blockage in arteries, arthritis, diabetes, heart attack, cancer, thyroid, asthma, psoriasis, eczema, prostrate, fits, AIDS, piles, 1714 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 migraine, ulcer, acidity, constipation, gynecological problems, ear and nose problems and several other diseases (Jain et al., 2010). The use of cow urine in India can be traced back to the Vedic and probably prevedic period also. Cow urine as such has been most widely referred, used and venerated animal urine owing to its immense therapeutic speciality. While externally it has been used as lotion, ointments and bath, but, internally it has been used in preparation of oral medications and drinks. There is existence of innumerable instances in various ancient medical texts of the curative properties of cow urine for a horde of human ailments. In ancient Indian system of medicine, urine of cow was accepted, used almost as a broad spectrum antibiotic quite akin to that of twenty first century. The cow urine not only used against ailments of diseases as therapeutic agents but also have several other uses as in agriculture and sericulture sectors. So this article attempts to bring forth the diversified use of this heretical potion as was in vogue in ancient Indian system of medicine as gleaned from the ancient medical texts and current scientific findings. In Veda, cow is considered the most valuable animal and is called Mother of all. Different products obtained from cow like urine, dung, milk, ghee and curd are used widely in number of Ayurvedic formulations (Shah, 1997) As per Ayurvedic literatures cow urine possess many medicinal properties and is used in curing number of diseases like skin diseases, kidney problems, epilepsy, anemia, constipation, respiratory disease etc (Krishnamurthi et al., 2004; Chauhan et al., 2001). Due to its therapeutic values majority of rural population in India use cow urine as a folklore remedy to get rid of various diseases. Nowadays, different preparations of cow urine like urine distillate, photo‐ activated urine, fresh urine, sterile urine have been marketed with cheap and affordable prices (Edwin et al., 2008). Thus the aim of the present work is to study antibacterial potential of photo‐ activated cow urine, fresh cow urine, cow‟s urine Fungi are one among the important aetiological agents of plant diseases. Fungi cause diseases in plants both in field and storage. The fungal infection of plants results in poor yield of crop and hence results in considerable economic loss. In severe cases, crop losses exceeding >50% can occur due to fungal infections. The management of mycotic diseases of plants mainly focuses the use of chemical agents. The use of synthetic fungicides appears promising but suffers from several drawbacks such as high cost, environmental pollution, adverse effects on non-target organisms and the emergence of resistant strains of pathogenic fungi. Natural products including cow urine and cow urine based formulations have been investigated for antifungal activity and the results appeared promising (Swami and Alane, 2013; Ashlesha et al., 2013; Gotora et al., 2014; Ashlesha and Paul, 2014; Kekuda et al., 2016). Cow urine is shown to exhibit potent inhibitory activity against a wide range of pathogenic microbes including phytopathogenic fungi (Akhter et al., 2006; Deshmukh et al., 2012; Kambar et al., 2013; Randhawa and Sharma, 2015; Aruna and Spadmapriya, 2016; Ruchira et al., 2016). Similarly, formulations based on cow urine and plants have shown to possess inhibitory effect against various pathogenic microorganisms (Yadav et al., 2008; Tiwari and Das, 2011; Shanthi et al., 2011; Rakesh et al., 2013; Mehta and Khan, 2014; Kekuda et al., 2014). The present study was carried out to investigate antifungal and antibacterial potential of cow urine against 6 phytopathogenic fungi and 3 bacteria. Biochemical analysis of cow urine The laboratory analysis of cow urine shows that it contains nitrogen, sulphur, phosphate, sodium, manganese, iron, silicon, chlorine, magnesium and mineral as its mineral content; malic, citric, titric, succinic, carbolic acids; 1715 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 calcium salts, Vitamins A, B, C, D, E; lactose, enzymes, creatinine, hormones and gold acids - all needed in smaller amounts by the human body to balance the "tridosha"- vata, pitta and Kapha. Any deficiency or excess of these substances inside the body causes disorders. Cow urine contains all of these substances with having a balanced proximate composition. Therefore, consumption of cow urine restores the balance of these substances and thus helps in curing from incurable diseases. The various mineral and chemical constituents present in cow urine are attributed with different biochemical roles in the body (World Intellectual Property Organization, 2013). For example nitrogen removes blood abnormalities, toxins and is a natural stimulant of urinary system; sulphur support peristaltic movement in large intestine and purified blood; copper control unwanted fat; iron help in production of haemoglobin and erythropoiesis; phosphorus helps in removal of stones from urinary tract; sodium purifies blood and acts as an antacid; potassium cures rheumatism and increase appetite, muscular strength, stamina and activeness; manganese prevents growth of germs, tissue nacrosis; calcium is a blood purifier and strengthens bones; gold is a germicidal and increases immunity. Our body contains many micronutrients that give us strength for life, but these micro-nutrients are flushed out of our body when we urinate. Cow urine meets the deficiency of these micronutrients in the body. It maintains the balance of these substances in our body and cures even the so called incurable diseases (Kirtikar and Basu, 2003). Mechanisms of action of cow urine Different fractions of CU possess antimicrobial activity due to the presence of certain components like volatile and nonvolatile ones (Jarald et al., 2008; Mohanty et al., 2014; Hu et al., 2007; Shaw et al., 2007). Presence of urea, creatinine, swarn kshar (aurum hydroxide), carbolic acid, phenols, calcium, and manganese has strongly explained the antimicrobial and germicidal properties of CU (Achliya et al., 2004; Jain et al., 2010; Kumar, 2001). Presence of amino acids and urinary peptides may enhance the bactericidal effect (Badadani et al., 2007) by increasing the bacterial cell surface hydrophobicity. CU enhances the phagocytic activity of macrophages. Higher amounts of phenols in fresh CU than CU distillate (CUD) makes it more effective against microbes. After photo-activation, few biogenic volatile inorganic and organic compounds such as CO2, NH3, CH4, methanol, propanol and acetone, and some metabolic secondary nitrogenous products are also formed (Upadhyay et al., 2010). Photo-activated CU (PhCU) becomes highly acidic in comparison to fresh CU. An increase in bactericidal action may be due to a significant decrease in pH, presence of inorganic phosphorus, chloride and dimethylamine may also play an important role (Naotoshi et al., 2007), along with increased formation of some reactive compounds like formaldehyde, sulfinol, ketones and some amines during photoactivation and long term storage (Türi et al., 1997). CU prevents the development of antibacterial resistance by blocking the Rfactor, a part of plasmid genome of bacteria (Chauhan and Singhal, 2006). CU contains phenolic acids (gallic, caffeic, ferulic, ocoumaric, cinnamic, and salicylic acids) which have antifungal characteristics (Singh et al., 2012). Antioxidant property of uric acid and allantoin present in CU correlates with its anticancer effect. CU reduces apoptosis in lymphocytes and helps them to survive better. This action may be due to the free radical scavenging activity of the urine components, and these components may prevent the process of aging (Jarald et al., 2008). It efficiently repairs the damaged DNA. Daily consumption of CU improves immunity due to the presence 1716 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 of swarn kshar and fastens the wound healing process, which is due to allantoin. CU enhances the immunocompetence by facilitating the synthesis of interleukin-1 and 2 (Chauhan, 2004; Singla and Garg, 2013), augments B - and T- lymphocyte blastogenesis, and IgA, IgM and IgG antibody titers (Kumar, 2013). Early morning first voided CU is more sterile and have more macro and micronutrients along with other enzyme/urea content could be more effective (Pescheck-Böhmer and Schreiber, 1999). Materials and Methods Collection and preparation of cow urine Cow urine was collected from the wellmaintained Goshala of local area. The cow selected for this research was a healthy Nagori cow, aged seven years being fed a uniform diet and undergoing regular vaccination schedule. Fresh cow urine was collected in sterile screw cap bottles and brought to the laboratory for testing. It was filtered by ordinary filter paper before being subjected to further testing. Sterile cow urine was prepared by sterilizing the urine sample maintained in an autoclave at a temperature of 121°C and 15 lb/in2 pressure for 15 minutes. Photo activated cow urine was prepared by keeping the urine in transparent sterile bottle for 72 hours in sunlight. Thus purified, fresh and photoactivated cow urine were stored at 4 °C for further future use as per experimental requirements. Different samples of cow urine thus obtained are: Fresh cow urine, Sterile cow urine, Photo activated cow urine, were incubated in Potato dextrose Agar (PDA) and Standard blotter paper for 5 to 7 days. Then the microbial colony on agar and paper plate were isolated and identified. Then they were made slant culture separately. Then they were stored in room temperature for further use. Medium PDA plates were used for the growth of fungal strain and MHA plates were used for the growth of Bacterial strain. 3.9 g of Potato dextrose agar and 3.8 g of Mueller Hinton Agar (MHA) was added to 100ml of distilled water in separate conical flask and autoclaved at 1210c for 15 minutes at 15 lbs and poured in sterile petri plates up to a uniform thickness of approximately 4mm and the agar is allowed to set at ambient temperature and used. A few slants were made for storage of Bacterial and fungal culture. Microbiological assay Well diffusion method The antimicrobial activity of cow urine was tested by preparing 5 different percentage of cow urine. Percentages were made by dissolving cow urine in sterile water. Five percentages 20%, 40%, 50%, 70%, 100% were taken for the analysis of antibacterial activity. A hollow tube was heated and pressed above the inoculated agar plate. It was removed immediately by making a well in the plate; two and three wells on each plate were made. Inoculums and incubation Collection of microorganisms All bacterial and fungal strains were isolated from different oil and cereal crops seed. Seeds 0.1mg of bacterial and fungal cultures was transferred to the agar plates. The inoculated plates were allowed to stand for 5 min, before making wells for different percentages to be tested. The percentages of cow urine were 1717 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 loaded at different concentrations in the well on agar plate. Then bacterial and fungal cultures and incubated at normal room temperature for 24-48 hours in an incubator. of cow urine) and with different groups (different percentage of cow urine) for different bacteria and fungi and statistical analysis was done Procedure of determining the efficacy of cow urine as antifungal agent Results and Discussion The five percentages (20%, 40%, 50%, 70% and 100% v/v) of cow urine were prepared. 5mL of different concentrations of cow urine was amended in 15mL of potato dextrose agar medium and mixed thoroughly by stirring. Control was maintained in which distilled water was used instead of cow urine. The medium was autoclaved and poured into sterilized Petri plates and left. The fungal discs of 5mm diameter were taken from actively growing cultures by using cork borer and the discs were transferred aseptically on PDA plates poisoned with cow urine. Plates were incubated at 28 ± 2∘C temperature in incubator for 7 days. After 7 days plates were observed and colony diameters were measured with the help of ruler. The percent of inhibition was calculated using the following formula given below: Percent inhibition of mycelia growth (%) 100 (𝐶 − 𝑇) I = ----------------𝐶 Where 𝐼 is inhibition percentage, 𝐶 is colony diameter in control plates, and 𝑇 is colony diameter in poisoned plates. Statistical analysis After incubation the diameter of zone of inhibition around the well was measured using zone reader. Corresponding 3 values of zones of inhibition for each percentage of cow urine were taken. The values so obtained were compared within the group (same percentage The results of antibacterial and antifungal potential of different cow urine preparation are shown in Table 1. The result of the present study shows that antibacterial and antifungal activity of fresh cow urine is more active than photoactivated urine. These observations are likely to be the result of the presence of certain volatile and non-volatile components present in urine (Fig. 1–6). The antibacterial activity of photoactivated urine may be due to its acidic pH. It may also be due to presence of more cations and formation of nitrosoamines. It was observed that gram positive organisms were more sensitive than gram negative organisms. These observations are likely to be the result of the differences in cell wall structure between gram positive and gram negative bacteria, with gram negative outer membrane acting as a barrier to many environmental substances. In the present studies six fungal species namely Aspergillus sp, Rhizopus sp, Mucor sp, Penicillium sp, Alternaria sp, Macrophomina sp and three bacterial cultures include Bacillus subtilis, Pseudomonas sp, Streptococcus sp are used. All percentages (20, 40, 50, 70, 100) of cow urine were effective against the growth of the fungus which exhibited the significant inhibition in the growth of fungal plant pathogens. With increase in concentration of cow urine there was corresponding increase in the inhibition of vegetative growth of the fungal pathogens. The diameter of the fungal colonies in poisoned plates was lesser when compared to control plates and it indicates the antifungal effect of cow urine. 1718 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 Table.1 The microbial zones of inhibition values Types of cow urine Fresh Photo activated Sterile Percent age of cow urine Diameter of zone of inhibition (mm) Name of the seed microflora (bacterial and fungal species) Aspergillus sp Rhizopus sp Mucor sp Penicillium sp Alternaria sp Macro Bacillus phomina sp subtilis Pseudomonas sp Streptococcus sp. 20% 15.26±1.0 12.22± 3.0 12.12±1.0 13.04± 0.0 13.63±2.0 11.42± 1.0 14.08± 1.0 12.38± 3.0 13.08±3.0 40% 17.23± 3.0 14.00± 0.0 13.25±3.0 15.48± 1.0 14.13±1.0 13.23± 3.0 16.45±1.0 13.22±1.0 16.07±1.0 50% 18.63±0.0 15.21±1.0 14.89±1.0 16.78± 3.0 15.24±3.0 14.22± 1.0 17.55± 3.0 15.02±1.0 17.10±1.0 70% 21.03±1.0 18.05± 0.0 17.05±3.0 19.12± 1.0 18.16±1.0 16.56± 0.0 20.62± 1.0 17.88±3.0 20.11±3.0 100% 24.42±3.0 21.02± 3.0 20.22±0.0 21.06± 2.0 21.72±1.0 19.66± 1.0 23.28± 1.0 19.72±2.0 22.21±1.0 20% 13.25±1.0 11.28± 1.0 11.57±1.0 12.37± 1.0 12.57±3.0 11.44± 1.0 13.64± 3.0 11.14±2.0 11.54±0.0 40% 15.35±3.0 13.80± 0.0 12.35±0.0 14.19± 0.0 13.87±0.0 12.00± 3.0 15.31± 0.0 12.01±1.0 15.34±2.0 50% 16.58±0.0 14.87± 1.0 13.18±1.0 15.25± 3.0 14.34±1.0 13.69± 2.0 16.59± 2.0 14.79±3.0 16.13±1.0 70% 19.79±1.0 17.69± 3.0 16.94±3.0 18.31± 2.0 17.31±2.0 15.70± 0.0 19.29± 1.0 16.47±2.0 19.51±3.0 100% 22.93±3.0 20.35± 1.0 19.58±0.0 20.21± 1.0 20.94±3.0 18.49± 1.0 22.19± 3.0 18.40±02 20.42±1.0 20% 11.48±1.0 10.29± 2.0 10.28±1.0 11.87± 0.0 11.22±2.0 10.57± 3.0 12.28± 1.0 10.16±3.0 11.11±0.0 40% 13.25±0.0 12.94± 1.0 11.25±1.0 12.83± 3.0 12.16±1.0 11.47± 0.0 14.42± 2.0 11.31±1.0 14.64±2.0 50% 14.72±2.0 13.05± 1.0 12.78±3.0 15.90± 0.0 13.05±3.0 13.43± 1.0 15.34± 3.0 13.66±0.0 17.31±3.0 70% 17.00±1.0 16.35± 0.0 15.34±1.0 17.05± 3.0 16.52±1.0 15.61± 3.0 18.49± 1.0 15.31±1.0 18.97±1.0 100% 20.94±3.0 19.85± 3.0 18.80±0.0 18.58± 1.0 19.31±3.0 18.54± 1.0 21.38± 3.0 17.84±3.0 20.28±3.0 Fig.1 Different types of cow urine 1719 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 1720 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 1721 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 1722 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 1714-1727 Fig.5 Efficacy of cow urine as antifungal agent Fig.6 Efficacy of cow urine as antifungal agent Result of determining the efficacy of cow urine as antifungal agent SL NO. 1 2 3 4 5 6 PERCENTAGE OF COW URINE 0%(control) 20% 40% 50% 70% 100% COLONY DIAMETER(mm) 59.30 37.30 33.70 31.30 28.70 25.00 1723 PERCENTAGE OF INHIBITIONOF FUNGAL GROWTH 0.0 37.09 43.17 47.21 51.60 57.84