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LOVELY PROFESSIONAL UNIVERSITY CAPSTONE PROJECT REPORT TOPIC- ANTIMICROBIAL legal action OF disparate TYPES OF sweeten. 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 bulge entitled anti microbial essential action of various types of dulcify submitted by MOHIT KUMAR , students of biotechnology Department, Lovely Professioal University, Phagwara Punjab in the partial fulfillment of the requirement for the set apart of Bachelors of Technology (biotechnology) Degree of LPU, is a record of students own withdraw carried under my inadvertence & guidance. This report has not been submitted to any another(prenominal)(a) 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 Lovel y Professional Univerersity, Punjab, hereby decl ar that on the whole the information furnished in this dissertation / capstone project report is based on my own intensive investigate and is genuine. This dissertation / report does not, to the silk hat 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 with bulge neat citation. Date 17/5/2012 Investigator- MOHIT KUMAR Regd. No. 10800037 Acknowledgement Any attempt at any aim cant be satisfactorily completed without the patronize and guidance of learned people in my capstone projectAntimicrobial operation of different types of lamb.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 likewise would like to thanks my group ingredient who helped me in my project. MOHIT KUMAR ABSTRACT lamb is a traditional headal treatmen t for infected wounds. It can be issuanceive on antibiotic drug-resistant strains of bacterium. some(prenominal) local brands of love life(Dabur) collected from different firsts is apply in this study. Also the lamb is collected from non-homogeneous apiaries from Pathankot(Punjab) and Saharanpur (Up).The bacterial culture were taken from the laboratory of the Lovely professional University and was revived in alimentary stemma media and then sub-cultured in nutrient nutrient agar-agar media. The method acting used to test this antibiotic sensitivity of dulcorate was done with the help of Kirby Bauer method . Marked variations were observed in the bactericide drill of these making love archetypes. Bacterial species used were vitamin B complex subtilis ,E. coli. , S. aureus and Burkholderia spp. The regulate of inhibition produced against different bacteria employ love of different assiduousness be as follows E. oli (pathankot judge) ascorbic acid% = 2. 6cm, 75% = 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm E. coli (dabur take in) cytosine% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm E. coli (saharanpur exemplification) carbon% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm B. subtilis (pathankot) degree Celsius% = 3. 0cm, 75%= 2. 7cm, 50%= 2. 7cm, and 25%= 2. 5cm B. subtilis (dabur) one hundred% = 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 regularizes of inhibition of di fferent treatment groups were measured by agar- come up- diffusion assay and compargond with temper. The comparison of beloved sample with distilled water control had proved it significant.CONTENTS PAGE 1. INTRODUCTION 7 1. 1) Honey as bactericide agent 7 1. OBJECTIVE 9 2. LITERATURE REVIEWS 10 3. 1 TYPES OF dearest 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 move in by bees using ambrosia 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 passion, through a long rocess involving the addition of enzymes and the in small stages reduction of moisture. Honey is a rich source of carbohydrates mainly Fructose and Glucose. The chemical composition of sexual love varies depen ding on the plant source, season and production methods. Therefore the Colour, Concentration and Compounds vary depending on the floral sources. other(a) compounds which can be base in Honey include Proteins and venereal diseases such as Gluconic Acid (C6H11O7, in any end cognise 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 sweeten 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 honeys were chosen because they are organic and readily avail sufficient in health food stores 1. 1) The antibacterial drug Effects of Honey The honeys are shown to have antibacterial properties, in special(a) Manuka honey. Manuka Honey has had extensive research done on it. It has been shown in many studies that Manuka Honey has antimicrobic personal dumbfound (Barret J. , et. al, 2005 Coumbes A. L. , et. al. 2004 Mundo, M A. 2004). The Mechanism of bacteriacide Properties Honey has many natural properties which enable it to inhibit bacteria.These properties include, a low pH which is in the commit of pH 3. 2 to 4. 5, approximately 3. 9 which is payable to its content of acidic compounds mainly Gluconic acid as stated above. A low pH is repressive 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 elan large proteins such as enzymes work. Which causes the s hape of enzymes to change, which then alters the overall charge, this causes the protein to denature.Honey contains small amounts of Hydrogen gibe heat peroxide (H202) this varies depending on the honey, it is produced as a will of the enzyme glucose oxidase military action 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), henry 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 injure cells.In an aqueous solution hydrogen peroxide acts like an acid and can oxidise a mixed bag of compounds, by accepting free unpaired electrons. This allows the formation of other free radicals, which then causes a exhibitioner effect. Therefore altering biological structures and thence damaging cells. Honey is primarily a saturated sort of two monosaccharide, with a low water bodily process most of the water molecules are associated with the sugars and few bear available for microorganisms, so it is a poor environment for their growth.If water is mixed with honey, it loses its low water natural process, and therefore no longer possesses this healthful property. Honey consists of various constituents such as water, carbohydrates, proteins, vitamins, amino acid, energy and minerals. anyhow the major ones, there must excessively 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 entirely 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 element of it. The antibacterial properties of honey speed up the growth of spic-and-span ti ssue 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 fixed to carryout the studies using different solvents.The present study therefore deals with the constituents in the different solvents followed by evaluation of move out for their antimicrobial behaviour against original species of bacteria. 2) OBJECTIVE Based on the above facts, the present study was delimitate with following objective 1) To check the antibacterial effect of different honeys on. a. positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive vitamin B 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% h oney 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 times 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 better 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 sourness is as fountainhead as 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 a ffected 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 development of various intestinal bacteria. All chemical and physical factors make the redress 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 indirect 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 mo re honeys differing in floral source, emblazon, flavor, density or geographic origin. Polyfloral Polyfloral honey, alike known as wildflower honey, is derived from the nectar of many types of flowers. The taste may vary from year to year, and the sweetness 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 cook in color, with a rich sugariness of stewed fruit or fig jam, and is not sweet like nectar honeys. . 2) COMPOSITION OF HONEY Carbohydrates Unsurprisi ngly, these comprise the major portion of honey about 82%. The carbohydrates present are the monosaccharide laevulose (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 neither breakdown of the higher saccharides present in nectar and honeydew. Proteins and Amino Acids.Honey contains a form of enzymes, including invertase, which converts sucrose to glucose and levulose 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 honey 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 great 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 ba cteria 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 growth. 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 natural cure for most bacterial infections that could replace man-made antibiotics. inquiryers say that an enzyme in the honey turns into a tinyamount hydrogen peroxide when combined with bodily fluids killing nearby bact eria. Honey also causes an increase in lymphocyte andphagocyte 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 antimicrobial 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 front end 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 aerug inosa, 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 laterality 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 genus Cereus and Baci llus subtilis were proven to be up to 60% more resistant than their equal reference strains gum olibanum 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, provide 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 due 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 drug activity of the honeys was assayed using standard well diffusion methods. oncluded that the sandal timberland from different sources and mixture of of and -santanols were highly effective against gram negative bacteria including Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia e and as well as yeast Candida albicans. The present work gives strong enjoin of antibacterial activities of honey, sandal oil and black genus Capsicum (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 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 reader 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 t he 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 disparity in the observed antibacterial activity can be due to several reasons. One calamity might be related to the differences in susceptibility of all(prenominal) 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 hurlative 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 approach study. 4. ) MATERIALS AND METHOD 4. 1) Materials Several local brands of honey and Dabur honey collected from different sources is used in th is 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 biotechnology lab. in accordance to habituated requirement. glaze over wares used- Conical flask (500 milliliter), Conical flask (100 milliliter), Petri nursing homes, 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 existent used- Sprit lamp, cork borer,test tube stand, auto pipette (10? l, 100? l, and 1000? l), parafilm wax,,filter paper Chemicals used- nutritive agar, nutr ient caudex, 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. bactericide study Antibacterial study was carried out in steps. In the first step, an in 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 farm animal 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 incubator . Sub-culturing of the bacterial strains on consentient 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 turn 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 the right way in the saline. Nutrien t 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 leave it 20 minute for solidify medium and make surface , after solidify cat the different Honeys in the wells and kept it to incubate 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 mi cropipette 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 micropipette 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 immortalise to avoid contamination. Incubation of the plates . A temperature range of 35C 2C is required for 24-48 hours. Do not incubate plates in carbon dioxideas 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 sizesto the nearby millimeterusing a ruler or caliper include the diameter of the well in the measurement . 2. All mea surements 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. shapeure1 Testing of antibacterial activity using agar well diffusion method 4. )RESULTS Sub-culturing 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 HONEYS AGAINST E. coliFIG. 7 ZONES OF proscription 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 proscription BY VARIOUS DILUTIONS OF HONEY (dabur sample) IN E. coli 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm FIG. 9 ZONES OF proscription 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 HONEYS AGAINST B. subtilis FIG. 10 ZONES OF prohibition 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 HONEYS AGAINST Burkholderia cap acium 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 HONEYS 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 th at honey were brown to dark brown in color, whereas other honey were golden yellow in color. Dabur honey- golden saturation,transparent Pathankot sample golden colour with 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 stifling 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(st erile 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 vulgarize 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. The results also showed that lower concentration of honey made through dilution shows less antimicrobial activity than concentrated ones. Thus honey are definitely effective against the bacterial strains at all concentrations. 6) REFERENCES Bibi S, ibn Talal Hussein SZ, Malik RN (2008). Pollen analysis and heavy metals detection in honey samples from seven selected countries. Pak. J. Bot. 40(2) 507-516 * Cooper R. A. , Molan P. C. , Harding K. G. (1999) Antibacterial activity of honey against strains of Staphylococcus aureus from infected wounds, J. R. Soc. Med. 92, 283285. * Cooper R. How does honey heal wounds? In Munn P, Jones R,editors. Honey and Healing. UK International Bee Research Association 2001. * de Jong H. 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