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LOVELY PROFESSIONAL UNIVERSITY CAPSTONE PROJECT REPORT TOPIC- ANTIMICROBIAL ACTIVITY OF DIFFERENT TYPES OF HONEY. PROJECT GUIDE- SUBMITTED BY- DR. AKSHAY GARG MOHIT KUMAR DEPT. OF BIOTECHNOLOGY REG. NO. – 10800037 ROLL NO- RB1R07B02 B. TECH BIOTECH. (8th sem. ) DATED- . 17-05-2012 CERTIFICATECertified that this project entitled â€Å"anti microbial activity of different types of honey † submitted by MOHIT KUMAR , students of biotechnology Department, Lovely Professioal University, Phagwara Punjab in the partial fulfillment of the requirement for the award of Bachelors of Technology (biotechnology) Degree of LPU, is a record of student’s own study carried under my supervision & guidance. This report has not been submitted to any other university or institution for the award of any degree.Date: 17/5/2012 Name of Project Guide Dr. Akshay Garg DECLARATION I, MOHIT KUMAR, student of B. Tech Biotechnology under Department of Biotechnology of Lovely Professional Unive rersity, Punjab, hereby declare that all the information furnished in this dissertation / capstone project report is based on my own intensive research and is genuine. This dissertation / report does not, to the best of my knowledge, contain part of my work which has been submitted for the award of my degree either of this niversity or any other university without proper citation. Date – 17/5/2012 Investigator- MOHIT KUMAR Regd. No. 10800037 Acknowledgement Any attempt at any level can't be satisfactorily completed without the support and guidance of learned people in my capstone projectâ€Å"Antimicrobial activity of different types of honey†.I would like to express my immense gratitude to my guide Dr. Akshay Garg for his constant support and motivation that has encouraged me to come up with this project. I also would like to thanks my group member who helped me in my project. MOHIT KUMAR ABSTRACT Honey is a traditional topical treatment for infected wounds. It can be effective on antibiotic-resistant strains of bacteria. Several local brands of honey(Dabur) collected from different sources is used in this study. Also the honey is collected from various apiaries from Pathankot(Punjab) and Saharanpur (Up).The bacterial culture were taken from the laboratory of the Lovely professional University and was revived in nutrient broth media and then sub-cultured in nutrient agar media. The method used to test this antibiotic sensitivity of honey was done with the help of Kirby Bauer method . Marked variations were observed in the antibacterial activity of these honey samples. Bacterial species used were Bacillus subtilis ,E. coli. , S. aureus and Burkholderia spp. The zone of inhibition produced against different bacteria using honey of different concentration are as follows: E. oli (pathankot sample): 100% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm; E. coli (dabur sample): 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm; E. coli (sahar anpur sample): 100% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm; B. subtilis (pathankot): 100% = 3. 0cm, 75%= 2. 7cm, 50%= 2. 7cm, and 25%= 2. 5cm; B. subtilis (dabur): 100% = 2. 7cm, 75%= 2. 5cm, 50%= 2. 4cm, and 25%= 2. 2cm; B. subtilis (saharanpur):100% = 3. 2cm, 75%= 2. 7cm, 50%= 2. 3cm, and 25%= 1. 5cm; Burkholderia spp. (pathankot): 100% = 2. 1cm, 75%= 1. 9cm, 50%= 1. 6cm, and 25%= 1. 4cm; Burkholderia spp. dabur): 100% = 2. 4cm, 75%= 1. 7cm, 50%= 1. 5cm, and 25%=1. 2cm; Burkholderia spp. (saharanpur):100% = 2. 5cm, 75%= 1. 9cm, 50%= 1. 7cm, and 25%=1. 4cm; S. aureus (pathankot):100%=2. 1cm, 75%= 1. 7cm, 50%= 1. 4cm, and 25%=1. 2cm; S. aureus (dabur):100% =3. 0cm, 75%= 2. 7cm, 50%= 2. 2cm, and 25%=1. 8cm; S. aureus (saharanpur): 100% = 2. 9cm, 75%= 2. 5cm, 50%= 2. 0cm, and 25%=1. 6cm; Zones of inhibition of different treatment groups were measured by agar-well-diffusion assay and compared with control. The comparison of honey sample with distilled water control had pro ved it significant.CONTENTS PAGE 1. INTRODUCTION 7 1. 1) Honey as antibacterial agent 7 1. OBJECTIVE 9 2. LITERATURE REVIEWS 10 3. 1 TYPES OF HONEY 10 3. 2 COMPONENTS OF HONEY 11 3. MATERIALS AND METHODS 15 4. MATERIAL 15 4. 2 METHOD 16 4. RESULTS AND DISCUSSION 19 5. REFERENCES 32 1. ) INTRODUCTION Honey is a sweet food made by bees using nectar from flowers. The Honey is a sweet, viscous fluid produced by bees from the collection of nectar, primarily from flowers. It is considered to be a natural syrup. The Nectar is gathered by the bees and is slowly transformed into honey, through a long rocess involving the addition of enzymes and the gradual reduction of moisture. Honey is a rich source of carbohydrates mainly Fructose and Glucose. The chemical composition of honey varies depending on the plant source, season and production methods. Therefore the Colour, Concentration and Compounds vary depending on the floral sources. Other compounds which can be found in Honey include Pr oteins and acids such as Gluconic Acid (C6H11O7, also known as 2,3,4,5,6- pentahydroxyhexanoic Acid), Minerals and Anti-Oxidants such as Hydrogen Peroxide (H202) and Vitamins (B6 and B12), (BD.Yates et. al. 1996). Honey has a low pH and a low moisture content, which is usually on average about 17 percent. The Gluconic Acid in honey is produced when bees secrete Glucose Oxidase, while processing the nectar, this give honey a low pH. There are many varieties of honey from around the world which come in three main types which are liquid, whipped and comb. Several local brands of honey(Dabur) collected from different sources is used in this study. Also the honey is collected from various apiaries from Pathankot(Punjab) and Saharanpur (Up) .Each of these honey’s were chosen because they are organic and readily available in health food stores 1. 1) The Antibacterial Effects of Honey The honeys are shown to have antibacterial properties, in particular Manuka honey. Manuka Honey has had extensive research done on it. It has been shown in many studies that Manuka Honey has antimicrobial effects (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, M A. 2004). The Mechanism of Antibacterial Properties Honey has many natural properties which enable it to inhibit bacteria.These properties include, a low pH which is in the range of pH 3. 2 to 4. 5, approximately 3. 9 which is due to its content of acidic compounds mainly Gluconic acid as stated above. A low pH is inhibitory to most bacteria. Since most bacteria live in environments around pH 7, the pH of honey could inhibit the bacteria (Barrett, J et. al. al 2005). This is because pH affects the way large proteins such as enzymes work. Which causes the shape of enzymes to change, which then alters the overall charge, this causes the protein to denature.Honey contains small amounts of Hydrogen peroxide (H202) this varies depending on the honey, it is produced as a result of the enzyme glucose oxidase activ ity in producing Gluconic acid (Mundo, MA. et. al. 2004). C6H12O6 + H2O + O2 > C6H12O7 + H2O2 (glucose oxidase reaction) When honey is used topically (as, for example, a wound dressing), hydrogen peroxide is produced by dilution of the honey with body fluids. As a result, hydrogen peroxide is released slowly and acts as an antiseptic. Hydrogen peroxide is a powerful oxidising agent (Free Radical) which has the ability to damage cells.In an aqueous solution hydrogen peroxide acts like an acid and can oxidise a variety of compounds, by accepting free unpaired electrons. This allows the formation of other free radicals, which then causes a cascade effect. Therefore altering biological structures and therefore damaging cells. Honey is primarily a saturated mixture of two monosaccharide, with a low water activity; most of the water molecules are associated with the sugars and few remain available for microorganisms, so it is a poor environment for their growth.If water is mixed with hone y, it loses its low water activity, and therefore no longer possesses this antimicrobial property. Honey consists of various constituents such as water, carbohydrates, proteins, vitamins, amino acid, energy and minerals. Besides the major ones, there must also be several minor constituents in honey, which may be playing a key role in determining the antimicrobial behaviour of honey. In the past, antimicrobial activity of honey had been reported only by using aqueous solution of honey.It is said that honey possesses antibacterial property but it is not clear whether it is the bulk honey or some fraction of it. The antibacterial properties of honey speed up the growth of new tissue to heal the wound. The bactericidal effect of honey is reported to be dependent on concentration of honey used and the nature of the bacteria Considering the fact that their might be some specific constituents which may be contributing to the antimicrobial behaviour, it was decided to carryout the studies u sing different solvents.The present study therefore deals with the constituents in the different solvents followed by evaluation of extract for their antimicrobial behaviour against certain species of bacteria. 2) OBJECTIVE Based on the above facts, the present study was defined with following objective: 1) To check the antibacterial effect of different honeys on. a. Gram-positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive Bacillus subtilis, and d. Gram- negative Burkholderia capicium 2) To check the effect of honey on bacterial strains using different-different dilutions. . 100% honey sample b. 75% honey sample c. 50% honey sample d. 25% honey sample 3) To compare the effect of honey and control (Distilled water) on a. Gram-positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive Bacillus subtilis, and d. Gram- negative Burkholderia capicium 3) LITERATURE REVIEWS Honey has been used with medicinal purposes since ancient tim es and there are reports of its topic use by Ayurvedic medicine, dating back to 2500 b. c. Hippocrates prescribed the use of honey for several indications, including treatment of wounds and gastritis.Its healing properties are mentioned in the Koran and in Bible. Honey is a part of traditional medicine for centuries. The antimicrobial activity of honey is due primarily to hydrogen peroxide produced enzymatically. However, in undiluted honey the acidity is also a significant antimicrobial factor. The pH, ranging from 3. 2 to 4. 5, is low enough to inhibit the development of pathogens, whose optimal pH range situates between 7. 2 and 7. 4. When applied to affected areas of tissue, acidity, osmotic effect and phytochemical factors decrease with dilution; however, the activity of hydrogen peroxide increases from 2. to 50 times. On these values, peroxide acts as an antiseptic, without causing cell damage. The composition of sugars in honeys, from different floral origins, inhibits the de velopment of various intestinal bacteria. All chemical and physical factors make the therapeutic properties of honey unique: fast decline of infections and healing of wounds, quick inflammation recovery, minimizing of wounds, stimulation of angiogenesis, as well as the development of epithelial and granular tissues. Honey to be used with topical medicinal purpose, certain requirements are needed, like being free f herbicides, pesticides, heavy metals and radioactive elements and sterilized to prevent secondary infections. 3. 1) TYPES OF HONEY – ON BASIS OF FLORAL SOURCE- Blended- Most commercially available honey is blended, meaning it is a mixture of two or more honeys differing in floral source, color, flavor, density or geographic origin. Polyfloral – Polyfloral honey, also known as wildflower honey, is derived from the nectar of many types of flowers. The taste may vary from year to year, and the aroma and the flavor can be more or less intense, depending on which bloomings are prevalent.Monofloral- Monofloral honey is made primarily from the nectar of one type of flower. Different monofloral honeys have a distinctive flavor and color because of differences between their principal nectar sources. To produce monofloral honey, beekeepers keep beehives in an area where the bees have access to only one type of flower. Honeydew honey- Instead of taking nectar, bees can take honeydew, the sweet secretions of aphids or other plant sap-sucking insects. Honeydew honey is very dark brown in color, with a rich fragrance of stewed fruit or fig jam, and is not sweet like nectar honeys. . 2) COMPOSITION OF HONEY Carbohydrates Unsurprisingly, these comprise the major portion of honey – about 82%. The carbohydrates present are the monosaccharide fructose (38. 2%) and glucose (31%); and disaccharides (~9%) sucrose, maltose, isomaltose, maltulose, turanose and kojibiose. There are also some oligosaccharides present (4. 2%), including erlose, theanderose and panose, formed from incomplete breakdown of the higher saccharides present in nectar and honeydew. Proteins and Amino Acids.Honey contains a number of enzymes, including invertase, which converts sucrose to glucose and fructose; amylase, which breaks starch down into smaller units; glucose oxidase, which converts glucose to gluconolactone, which in turn yields gluconic acid and hydrogen peroxide; catalase, which breaks down the peroxide formed by glucose oxidase to water and oxygen; and acid phosphorylase, which removes inorganic phosphate from organic phosphates. Honey also contains eighteen free amino acids, of which the most abundant is proline. Vitamins, Minerals and AntioxidantsHoney contains trace amounts of the B vitamins riboflavin, niacin, folic acid, pantothenic acid and vitamin B6. It also contains ascorbic acid (vitamin C), and the minerals calcium, iron, zinc, potassium, phosphorous, magnesium, selenium, chromium and manganese. The main group of antioxidants in hon ey are the flavonoids, of which one, pinocembrin, is unique to honey and bee propolis. Ascorbic acid, catalase and selenium are also antioxidants. Generally speaking, the darker the honey, the greater its antioxidising properties. Other compoundsHoney also contains organic acids such as acetic, butanoic, formic, citric, succinic, lactic, malic, pyroglutamic and gluconic acids, and a number of aromatic acids. The main acid present is gluconic acid, formed in the breakdown of glucose by glucose oxidase. Honey also contains hydroxymethylfurfural, a natural product of the breakdown of simple sugars below pH 5. According to studies done by Patricia E. Lusby et al Twelve of the 13 bacteria were inhibited by all honeys used in this study with only Serratia marcescens and the yeast Candida albicans not inhibited by the honeys.Little or no antibacterial activity was seen at honey concentrations 1%, with minimal inhibition at 5%. No honey was able to produce complete inhibition of bacterial g rowth. Although Medi honey and manuka had the overall best activity, the locally produced honeys had equivalent inhibitory activity for some, but not all, bacteria. He then postulated that honeys other than those commercially available as antibacterial honeys can have equivalent antibacterial activity. These newly identified antibacterial honeys may prove to be a valuable source of future therapeutic honeys. According to Peter C.Molan (2001), honey may be the â€Å"natural cure† for most bacterial infections that could replace man-made antibiotics. Researchers say that an enzyme in the honey turns into a tiny  amount hydrogen peroxide when combined with bodily fluids killing nearby bacteria. Honey also causes an increase in lymphocyte and  phagocyte activity (Cooper et al. , 2011). The honeys are shown to have antibacterial properties, in particular Manuka honey. Manuka Honey has had extensive research done on it. It has been shown in many studies that Manuka Honey has an timicrobial effects (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, MA. 2004).In this study the antibacterial activity of the five honeys were compared, the Manuka honey was used as a positive control. The nonperoxide antibiotic activity is due to methylglyoxal (MGO) and an unidentified synergistic component. Most honeys contain very low levels of MGO, but manuka honey contains very high levels. The presence of the synergist in manuka honey more than doubles MGO antibacterial activity. In vitro antibacterial activity of raw and commercially available honey was tested against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella spp. nd Salmonella spp. ). Both types of honey showed antibacterial activity against test organisms with the zone of inhibition ranging from 8. 13 to 30. 85 mm, while E. coli, S. aureus, and Shigella spp. showed sensibility towards both types of Honey. Both types of honey showed no effects on Salmonella spp. The potency of honey at 100%concentration was found to be higher than all other concentrations tested. However, no effect was observed at concentration of 6. 25% v/v honey in the case of both samples(Patricia E. Lusby 2004).To study the antimicrobial activity of honey, 60 samples of various botanical origin were evaluated antimicrobial activities against 16 clinical pathogens and their respective reference strains. The bioassay applied for determining the antimicrobial effect employs the well-agar diffusion method and the estimation of minimum active dilution which produces a 1 mm diameter inhibition zone. Streptococcus pyogenes,Bacillus cereus and Bacillus subtilis were proven to be up to 60% more resistant than their equal reference strains thus emphasizing the variability in the antibacterial effect of honey and the need for further research (C.Voidarou 2010). Natural products, either as pure compounds or as standardized plant extracts, provid e unlimited opportunities for new drugs because of the ready availability of chemical diversity (Cos et al. , 2006). Honey and lemon-honey are traditional remedies in the Middle East and China and for many centuries and have been used in the treatment and prevention of the common cold and various upper respiratory tract infections (Molan, 1992; Zulma ; Lulat, 1989). Antibacterial activity of the honeys was assayed using standard well diffusion methods. oncluded that the sandal wood from different sources and mixture of of – and -santanols were highly effective against gram negative bacteria including Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae and as well as yeast Candida albicans. The present work gives strong evidence of antibacterial activities of honey, sandal oil and black pepper (Sheikh Ahmad 2002). In general, all types of honey have high sugar content as well as low water content and acidity, which prevent microbial growth.Osmotic effect, effect of pH and hydrogen peroxide are represented as an â€Å"inhibition† factor in honey (Postmes et al. , 1993). Most types of honey generate hydrogen peroxide when diluted because of the activation of the enzyme glucose oxidase, which oxidizes glucose to gluconic acid and hydrogen peroxide (Schepartz and Subers, 1964). Hydrogen peroxide is the major contributor to the antimicrobial activity of honey,and the different concentrations of this compound indifferent honeys result in their variable antimicrobial effects (Molan, 1992).Moreover, non peroxide factors also play critical role. The content of non peroxide factors are related to the floral source and sometimes account for the major part of the antibacterial activity in honey(Molan and Russell, 1988). Forty samples of different honey types (Acacia, Ziziphus , Brassica and Citrus) were collected from different areas of Pakistan and analyzed for moisture, pH, total acidity, ash, electrical conductivity, hydroxyl methyl furfural (HMF), sucrose, total sugars, invert sugar, protein, proline contents as well as macro and micro elements.The variation in composition of honey samples was observed due to different types of flora. Likewise, a significant level (P ; 0. 05) of ash, electric conductivity, sucrose, total sugar as well as macro and micro elements was also found in these honey types. Different formulations of honey has significantly inhibited growth of pathogenic microorganisms, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger when compared to control group, which is an evidence that honey is a therapeutic agent being used since ancient time throughout the world. Feng et. al. 1994) The discrepancy in the observed antibacterial activity can be due to several reasons. One possibility might be related to the differences in susceptibility of each species of microorganism to the antibacterial activity of honey used. Similar observations are reported by others (Nzeako and Hamdi 2000; Ceyhan and Ugur 2001; Taormina et al. 2001). As reported by others (Melissa et al. 2004) dilution of honey enhances hydrogen peroxide mediated antibacterial activity may explain some of the discrepancies of observed with the antibacterial activity of these honey.The presence of unstable putative agents and/or thermolabile antibacterial agent(s) could also be inactivated during the experimental procedure and thus may be considered as possible explanation of the observed insensitivity of some honey samples found in the preliminary study. 4. ) MATERIALS AND METHOD 4. 1) Materials Several local brands of honey and Dabur honey collected from different sources is used in this study. Also the honey is collected from various apiaries from- Pathankot(Punjab) and Saharanpur (Up). In the antibacterial study, several bacterial species known to be pathogenic to human such as E. oli. , S. aureus and Burkholderia capicium and Bacillus subtilis was used. These strains were obtained from biote chnology lab. in accordance to given requirement. Glass wares used:- Conical flask (500 milliliter), Conical flask (100 milliliter), Petri plates, glass rod, test tubes spreader, glass beakers, Glass slides and cover slip, Plastic wares used:- Beakers, Conical flask (500 milliliter), Conical flask (100 milliliter), dropper, appendrof tubes (2ml), auto pipette tips (10? l, 100? l, and 1000? l) Instruments used:- Hot air oven, Autoclave machine, Laminar air flow hood, Incubator, Microwave ovenOther material used:- Sprit lamp, cork borer,test tube stand, auto pipette (10? l, 100? l, and 1000? l), parafilm wax,,filter paper Chemicals used:- Nutrient agar, nutrient broth, Mueller Hinton agar, 70% Methanol, 100% methanol, ethyl alcohol ,sodium chloride. 4. 2) Methods Physicochemical study: Appearance of different honeys was observed. pH is determined using conventional procedure like using ph scale. Antibacterial study: Antibacterial study was carried out in steps. In the first step, an i n vitro screening will be carried out using either disc diffusion or well diffusion method.Well diffusion was carried out using plate diffusion, which I preferred. Preparation of test materials: Test materials will be prepared by diluting each honey at different dilutions, 25 ? l/100 ? l, 50 ? l/100 ? l, 75 ? l/100 ? l and one with no dilution. Moreover, net honey was also used as test material. All dilutions were carried out with double distilled and deionised sterilized water. 3. 2. 1) Source of bacterial strains The E. coli. , S. aureus, Burkholderia capicium and Bacillus subtilis were revived from the stock available in the various Biotechnology laboratories of Lovely Professional University, Phagwara (Punjab), India.Sub-culturing of bacterial strains in nutrient broth:- 100ml nutrient broth was prepared and ten test tubes were taken, autoclaved, and after this the Bacterial strains was inoculated in the nutrient broth in different test tubes and then incubated for 24 hours in i ncubator . Sub-culturing of the bacterial strains on solid media:- For All Bacterial strains:- 5. 6g of Nutrient agar was added to the 200ml of purified water. Then solution was heated and boiled for 1 minute to completely dissolve the powder. Then media was autoclaved.Then media(10-15 ml) was poured in Petri plates. Then Bacterial strains were added to media. Then Petri plate were kept in incubator for 18-48 hours at 37 degree C. Saline preparation : Take . 58g of Nacl and dissolve it to 10ml of distled water and make saline before 15 min of spreading and take the colonies from the nutrient agar plates and mix properly in the saline. Nutrient agar medium for antimicrobial testing: 7g of nutrient agar medium was added to the 250 ml of distilled water and heated for 1 minute to complete dissolve medium then autoclaved .After that pouring the petriplates and left it 20 minute for solidify medium and make wells , after solidify put the different Honeys in the wells and kept it to incub ate for 18-48 hours at 37C. Testing of antibacterial activity using agar well diffusion method: The bacterial strains were inoculated into 10 ml of sterile nutrient broth, and incubated at 37  °C for 18 h. Each culture was then spreaded on the surface of sterile nutrient agar plate and also pour-plated in nutrient agar media to perform the test in triplet, one with the control and the other two ith the test sample. Making the wells With the help of a cork borer make wells in the agar carefully without tearing the gel. In two agar plate of all four sets, four wells were prepared with the help of sterilized cork borer. Then with the hep of micropipette put 100 micro litre of honey sample into the wellIn the wells of two plates of each set, samples of following concentrations: (1) 100% sterile Honey(2) 75% sterile Honey(3) 50% sterile Honey and (4) 25% sterile Honey; were added by using micropipette. Also in the a well water was added as negative control.Then with the help of micropi pette put 100 micro litre of honey sample into the well Replace the lid of the plate between putting sample into wells to minimize exposure to air-borne contaminants. Cover the lid tightly with paraffin tape to avoid contamination. Incubation of the plates . A temperature range of 35 °C  ± 2 °C is required for 24-48 hours. Do not incubate plates in carbon dioxide  as this will decrease the pH of the agar and result in errors due to incorrect pH of the media. Measuring zone of inhibition 1.Following incubation, measure the zone sizes  to the nearest millimeter  using a ruler or caliper; include the diameter of the well in the measurement . 2. All measurements were made with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light . 3. Record the zone size on the recording sheet. Figure1: Testing of antibacterial activity using agar well diffusion method 4. )RESULTS Sub-cultu ring of bacterial strains in nutrient broth:- Figure2: Culture E. coli. , S. ureus and Burkholderia capicium and Bacillus subtilis in Nutrient broth media. Sub-culturing of the bacterial strains on solid media:- Fig. –3 E. coli subcultured on nutrient agar Fig. – 4 B. Subtilis subcultured on nutrient agar Testing of antibacterial activity using agar well diffusion method: Fig 5 : No antibacterial activity seen as no honey is poured (taken as negative control) in which E. coli is grown Fig 6 : No antibacterial activity seen as no honey is poured (taken as negative control) in which Bacillus subtilis is grown ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST E. coliFIG. 7 – ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY(pathankot sample) IN E. coli: 100% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm FIG. 8– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY (dabur sample) IN E. coli : 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm FI G. 9– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY (saharanpur sample) IN E. coli: 100% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm; ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST B. subtilis FIG. 10– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY IN B. subtilis (pathankot sample): 100% = 3. cm, 75%= 2. 7cm, 50%= 2. 7cm, and 25%= 2. 5cm FIG. 11– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in B. subtilis (dabur sample): 100% = 2. 7cm, 75%= 2. 5cm, 50%= 2. 4cm, and 25%= 2. 2cm; FIG. 12– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in B. subtilis (saharanpur):100% = 3. 2cm, 75%= 2. 7cm, 50%= 2. 3cm, and 25%= 1. 5cm ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST Burkholderia capacium FIG. 13– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in Burkholderia capacium (pathankot sample): 100% = 2. 1cm, 75%= 1. 9cm, 50%= 1. 6cm, and 25%= 1. 4cm; FIG. 4– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY Burkholderia capacium (dabur): 100% = 2. 4cm, 75%= 1. 7cm, 50%= 1. 5cm, and 25%=1. 2cm FIG. 15– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY Burkholderia capacium (saharanpur):100% = 2. 5cm, 75%= 1. 9cm, 50%= 1. 7cm, and 25%=1. 4cm; ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST S. Aureus FIG. 16– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY S. aureus (pathankot):100%=2. 1cm, 75%= 1. 7cm, 50%= 1. 4cm, and 25%=1. 2cm FIG. 17– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY S. aureus (dabur):100% =3. 0cm, 75%= 2. 7cm, 50%= 2. 2cm, and 25%=1. cm FIG. 18– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY IN S. aureus (saharanpur): 100% = 2. 9cm, 75%= 2. 5cm, 50%= 2. 0cm, and 25%=1. 6cm Appearance Appearance of each of honey brand was examined and it was observed that honey were brown to dark brown in color, whereas other honey were golden yellow in color. Dabur honey- golden colour,transparent Pathankot sample – golden colour wit h sufficient transparency Saharanpur sample – dark yellow colour with zero transparency The transparency is due to processing of honey,while less transparent samples are unprocessed and taken directly from bee hive.Table 1- Diameter of the Zone of Inhibition by different concentration of Pathankot honey sample on different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Control(sterile DistilledWater)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. coli. | 0. 0| 2. 6| 1. 9| 1. 7| 1. 2| Bacillus subtilis| 0. 0| 3. 0| 2. 7| 2. 7| 2. 5| S. aureus| 0. 0| 2. 1| 1. 7| 1. 4| 1. 2| Burkholderia capicium| 0. 0| 2. 1| 1. 9| 1. 5| 1. 4| Table2- Diameter of the Zone of Inhibition by different concentration of dabur honey sample on different Bacterial strains.Bacterial strains| Zone of Inhibition (in cm) | | Control(sterile DistilledWater)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. coli. | 0. 0| 2. 2| 1. 6| 1. 3| 1 . 1| Bacillus subtilis| 0. 0| 2. 7| 2. 5| 2. 4| 2. 2| S. aureus| 0. 0| 3. 0| 2. 7| 2. 2| 1. 8| Burkholderia capicium| 0. 0| 2. 4| 1. 7| 1. 5| 1. 2| Table 1- Diameter of the Zone of Inhibition by different concentration of Saharanpur honey sample on different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Control(sterile DistilledWater)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. oli. | 0. 0| 2. 8| 2. 4| 2. 3| 1. 7| Bacillus subtilis| 0. 0| 3. 2| 2. 7| 2. 3| 1. 5| S. aureus| 0. 0| 2. 9| 2. 5| 2. 0| 1. 6| Burkholderia capicium| 0. 0| 2. 5| 1. 9| 1. 7| 1. 4| In the preliminary screening process was observed that some honey brands have more while some other have less antibacterial activity. Some honey showed antibacterial effect against E. Coli and some showed strongest activity against Bacillus subtilis ,S. aureus and Burkholderia capicium. Clear zones of inhibition were produced by concentrated without dilution.Through the analysis of average values of the diameters of each honey, it is possible to infer a possible pattern in which commercial honeys, such as DABUR honey, have a greater average diameter while the homemade honeys from different regions, have a lower average diameter even at less concentrations, but the best antimicrobial activity was shown by Saharanpur honey sample with was collected directly from the apiary without any processing and it showed clear and large inhibition zones for all bacterial strains which were used for this test.Though after the dilutions were made, the low concentrated samples of Saharanpur honey resulted in rather poor antimicrobial activity. The wells in which water was loaded shoed absolutely no antimicrobial activity which was used as negative control. 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