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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. (eighth sem. ) DATED- . 17-05-2012 CERTIFICATE
Licensed that this venture entitled “anti microbial exercise of various kinds of honey ” submitted by MOHIT KUMAR , college students of biotechnology Division, Beautiful Professioal College, Phagwara Punjab within the partial achievement of the requirement for the award of Bachelors of Expertise (biotechnology) Diploma of LPU, is a file of pupil’s personal research carried below my supervision & steerage. This report has not been submitted to another college or establishment for the award of any diploma.
Date: 17/5/2012 Title of Challenge Information Dr. Akshay Garg DECLARATION I, MOHIT KUMAR, pupil of B. Tech Biotechnology below Division of Biotechnology of Beautiful Skilled Univerersity, Punjab, hereby declare that every one the data furnished on this dissertation / capstone venture report relies alone intensive analysis and is real. This dissertation / report doesn’t, to the perfect of my data, comprise a part of my work which has been submitted for the award of my diploma both of this niversity or another college with out correct quotation. Date – 17/5/2012 Investigator- MOHIT KUMAR Regd. No. 10800037 Acknowledgement Any try at any stage cannot be satisfactorily accomplished with out the assist and steerage of discovered folks in my capstone venture“Antimicrobial exercise of various kinds of honey”.

I wish to specific my immense gratitude to my information Dr. Akshay Garg for his fixed assist and motivation that has inspired me to give you this venture. I additionally wish to thanks my group member who helped me in my venture. MOHIT KUMAR ABSTRACT Honey is a standard topical therapy for contaminated wounds. It may be efficient on antibiotic-resistant strains of micro organism. A number of native manufacturers of honey(Dabur) collected from totally different sources is used on this research. Additionally the honey is collected from varied apiaries from Pathankot(Punjab) and Saharanpur (Up).
The bacterial tradition have been taken from the laboratory of the Beautiful skilled College and was revived in nutrient broth media after which sub-cultured in nutrient agar media. The tactic used to check this antibiotic sensitivity of honey was performed with the assistance of Kirby Bauer methodology . Marked variations have been noticed within the antibacterial exercise of those honey samples. Bacterial species used have been Bacillus subtilis ,E. coli. , S. aureus and Burkholderia spp. The zone of inhibition produced towards totally different micro organism utilizing honey of various focus are as follows: E. oli (pathankot pattern): 100% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm; E. coli (dabur pattern): 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm; E. coli (saharanpur pattern): 100% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm; B. subtilis (pathankot): 100% = three. 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% = three. 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% =three. 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 various therapy teams have been measured by agar-well-diffusion assay and in contrast with management. The comparability of honey pattern with distilled water management had proved it vital.
CONTENTS PAGE 1. INTRODUCTION 7 1. 1) Honey as antibacterial agent 7 1. OBJECTIVE 9 2. LITERATURE REVIEWS 10 three. 1 TYPES OF HONEY 10 three. 2 COMPONENTS OF HONEY 11 three. MATERIALS AND METHODS 15 four. MATERIAL 15 four. 2 METHOD 16 four. RESULTS AND DISCUSSION 19 5. REFERENCES 32 1. ) INTRODUCTION Honey is a candy meals made by bees utilizing nectar from flowers. The Honey is a candy, viscous fluid produced by bees from the gathering of nectar, primarily from flowers. It’s thought-about to be a pure syrup. The Nectar is gathered by the bees and is slowly reworked into honey, by way of a protracted rocess involving the addition of enzymes and the gradual discount of moisture. Honey is a wealthy supply of carbohydrates primarily Fructose and Glucose. The chemical composition of honey varies relying on the plant supply, season and manufacturing strategies. Subsequently the Color, Focus and Compounds differ relying on the floral sources. Different compounds which will be present in Honey embrace Proteins and acids comparable to Gluconic Acid (C6H11O7, often known as 2,three,four,5,6- pentahydroxyhexanoic Acid), Minerals and Anti-Oxidants comparable to Hydrogen Peroxide (H202) and Nutritional vitamins (B6 and B12), (BD.
Yates et. al. 1996). Honey has a low pH and a low moisture content material, which is often on common about 17 %. The Gluconic Acid in honey is produced when bees secrete Glucose Oxidase, whereas processing the nectar, this give honey a low pH. There are various forms of honey from all over the world which are available in three major varieties that are liquid, whipped and comb. A number of native manufacturers of honey(Dabur) collected from totally different sources is used on this research. Additionally the honey is collected from varied apiaries from Pathankot(Punjab) and Saharanpur (Up) .
Every of those honey’s have been chosen as a result of they’re natural and available in well being meals shops 1. 1) The Antibacterial Results of Honey The honeys are proven to have antibacterial properties, particularly Manuka honey. Manuka Honey has had in depth analysis performed on it. It has been proven in lots of research that Manuka Honey has antimicrobial results (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, M A. 2004). The Mechanism of Antibacterial Properties Honey has many pure properties which allow it to inhibit micro organism.
These properties embrace, a low pH which is within the vary of pH three. 2 to four. 5, roughly three. 9 which is because of its content material of acidic compounds primarily Gluconic acid as acknowledged above. A low pH is inhibitory to most micro organism. Since most micro organism stay in environments round pH 7, the pH of honey might inhibit the micro organism (Barrett, J et. al. al 2005). It is because pH impacts the best way giant proteins comparable to enzymes work. Which causes the form of enzymes to vary, which then alters the general cost, this causes the protein to denature.
Honey incorporates small quantities of Hydrogen peroxide (H202) this varies relying on the honey, it’s produced because of the enzyme glucose oxidase exercise in producing Gluconic acid (Mundo, MA. et. al. 2004). C6H12O6 + H2O + O2 > C6H12O7 + H2O2 (glucose oxidase response) When honey is used topically (as, for instance, a wound dressing), hydrogen peroxide is produced by dilution of the honey with physique fluids. Because of this, hydrogen peroxide is launched slowly and acts as an antiseptic. Hydrogen peroxide is a robust oxidising agent (Free Radical) which has the flexibility to break cells.
In an aqueous answer hydrogen peroxide acts like an acid and might oxidise a wide range of compounds, by accepting free unpaired electrons. This permits the formation of different free radicals, which then causes a cascade impact. Subsequently altering organic buildings and due to this fact damaging cells. Honey is primarily a saturated combination of two monosaccharide, with a low water exercise; a lot of the water molecules are related to the sugars and few stay obtainable for microorganisms, so it’s a poor surroundings for his or her progress.
If water is blended with honey, it loses its low water exercise, and due to this fact now not possesses this antimicrobial property. Honey consists of assorted constituents comparable to water, carbohydrates, proteins, nutritional vitamins, amino acid, vitality and minerals. Apart from the most important ones, there should even be a number of minor constituents in honey, which can be taking part in a key function in figuring out the antimicrobial behaviour of honey. Previously, antimicrobial exercise of honey had been reported solely through the use of aqueous answer of honey.
It’s mentioned that honey possesses antibacterial property however it’s not clear whether or not it’s the bulk honey or some fraction of it. The antibacterial properties of honey pace up the expansion of recent tissue to heal the wound. The bactericidal impact of honey is reported to be depending on focus of honey used and the character of the micro organism Contemplating the truth that their may be some particular constituents which can be contributing to the antimicrobial behaviour, it was determined to carryout the research utilizing totally different solvents.
The current research due to this fact offers with the constituents within the totally different solvents adopted by analysis of extract for his or her antimicrobial behaviour towards sure species of micro organism. 2) OBJECTIVE Based mostly on the above details, the current research was outlined with following goal: 1) To examine the antibacterial impact of various honeys on. a. Gram-positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive Bacillus subtilis, and d. Gram- detrimental Burkholderia capicium 2) To examine the impact of honey on bacterial strains utilizing different-different dilutions. . 100% honey pattern b. 75% honey pattern c. 50% honey pattern d. 25% honey pattern three) To match the impact of honey and management (Distilled water) on a. Gram-positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive Bacillus subtilis, and d. Gram- detrimental Burkholderia capicium three) LITERATURE REVIEWS Honey has been used with medicinal functions since historical instances and there are studies of its subject use by Ayurvedic drugs, courting again to 2500 b. c. Hippocrates prescribed the usage of honey for a number of indications, together with therapy of wounds and gastritis.
Its therapeutic properties are talked about within the Koran and in Bible. Honey is part of conventional drugs for hundreds of years. The antimicrobial exercise of honey is due primarily to hydrogen peroxide produced enzymatically. Nevertheless, in undiluted honey the acidity can be a major antimicrobial issue. The pH, starting from three. 2 to four. 5, is low sufficient to inhibit the event of pathogens, whose optimum pH vary situates between 7. 2 and seven. four. When utilized to affected areas of tissue, acidity, osmotic impact and phytochemical elements lower with dilution; nonetheless, the exercise of hydrogen peroxide will increase from 2. to 50 instances. On these values, peroxide acts as an antiseptic, with out inflicting cell injury. The composition of sugars in honeys, from totally different floral origins, inhibits the event of assorted intestinal micro organism. All chemical and bodily elements make the therapeutic properties of honey distinctive: quick decline of infections and therapeutic of wounds, fast irritation restoration, minimizing of wounds, stimulation of angiogenesis, in addition to the event of epithelial and granular tissues. Honey for use with topical medicinal function, sure necessities are wanted, like being free f herbicides, pesticides, heavy metals and radioactive components and sterilized to stop secondary infections. three. 1) TYPES OF HONEY – ON BASIS OF FLORAL SOURCE- Blended- Most commercially obtainable honey is mixed, that means it’s a combination of two or extra honeys differing in floral supply, shade, taste, density or geographic origin. Polyfloral – Polyfloral honey, often known as wildflower honey, is derived from the nectar of many kinds of flowers. The style might differ from 12 months to 12 months, and the aroma and the flavour will be kind of intense, relying on which bloomings are prevalent.
Monofloral- Monofloral honey is made primarily from the nectar of 1 sort of flower. Totally different monofloral honeys have a particular taste and shade due to variations between their principal nectar sources. To supply monofloral honey, beekeepers hold beehives in an space the place the bees have entry to just one sort of flower. Honeydew honey- As an alternative of taking nectar, bees can take honeydew, the candy secretions of aphids or different plant sap-sucking bugs. Honeydew honey may be very darkish brown in shade, with a wealthy perfume of stewed fruit or fig jam, and isn’t candy like nectar honeys. . 2) COMPOSITION OF HONEY Carbohydrates Unsurprisingly, these comprise the most important portion of honey – about 82%. The carbohydrates current are the monosaccharide fructose (38. 2%) and glucose (31%); and disaccharides (~9%) sucrose, maltose, isomaltose, maltulose, turanose and kojibiose. There are additionally some oligosaccharides current (four. 2%), together with erlose, theanderose and panose, fashioned from incomplete breakdown of the upper saccharides current in nectar and honeydew. Proteins and Amino Acids.
Honey incorporates plenty of enzymes, together with invertase, which converts sucrose to glucose and fructose; amylase, which breaks starch down into smaller items; glucose oxidase, which converts glucose to gluconolactone, which in flip yields gluconic acid and hydrogen peroxide; catalase, which breaks down the peroxide fashioned by glucose oxidase to water and oxygen; and acid phosphorylase, which removes inorganic phosphate from natural phosphates. Honey additionally incorporates eighteen free amino acids, of which essentially the most ample is proline. Nutritional vitamins, Minerals and Antioxidants
Honey incorporates hint quantities of the B nutritional vitamins riboflavin, niacin, folic acid, pantothenic acid and vitamin B6. It additionally incorporates ascorbic acid (vitamin C), and the minerals calcium, iron, zinc, potassium, phosphorous, magnesium, selenium, chromium and manganese. The principle group of antioxidants in honey are the flavonoids, of which one, pinocembrin, is exclusive to honey and bee propolis. Ascorbic acid, catalase and selenium are additionally antioxidants. Typically talking, the darker the honey, the better its antioxidising properties. Different compounds
Honey additionally incorporates natural acids comparable to acetic, butanoic, formic, citric, succinic, lactic, malic, pyroglutamic and gluconic acids, and plenty of fragrant acids. The principle acid current is gluconic acid, fashioned within the breakdown of glucose by glucose oxidase. Honey additionally incorporates hydroxymethylfurfural, a pure product of the breakdown of straightforward sugars under pH 5. In line with research performed by Patricia E. Lusby et al Twelve of the 13 micro organism have been inhibited by all honeys used on this research with solely Serratia marcescens and the yeast Candida albicans not inhibited by the honeys.
Little or no antibacterial exercise was seen at honey concentrations 1%, with minimal inhibition at 5%. No honey was capable of produce full inhibition of bacterial progress. Though Medi honey and manuka had the general greatest exercise, the regionally produced honeys had equal inhibitory exercise for some, however not all, micro organism. He then postulated that honeys apart from these commercially obtainable as antibacterial honeys can have equal antibacterial exercise. These newly recognized antibacterial honeys might show to be a helpful supply of future therapeutic honeys. In line with Peter C.
Molan (2001), honey often is the “pure treatment” for many bacterial infections that might exchange man-made antibiotics. Researchers say that an enzyme within the honey turns right into a tiny quantity hydrogen peroxide when mixed with bodily fluids killing close by micro organism. Honey additionally causes a rise in lymphocyte and phagocyte exercise (Cooper et al. , 2011). The honeys are proven to have antibacterial properties, particularly Manuka honey. Manuka Honey has had in depth analysis performed on it. It has been proven in lots of research that Manuka Honey has antimicrobial results (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, MA. 2004).
On this research the antibacterial exercise of the 5 honeys have been in contrast, the Manuka honey was used as a constructive management. The nonperoxide antibiotic exercise is because of methylglyoxal (MGO) and an unidentified synergistic element. Most honeys comprise very low ranges of MGO, however manuka honey incorporates very excessive ranges. The presence of the synergist in manuka honey greater than doubles MGO antibacterial exercise. In vitro antibacterial exercise of uncooked and commercially obtainable honey was examined towards Gram-positive micro organism (Staphylococcus aureus) and Gram-negative micro organism (Escherichia coli, Pseudomonas aeruginosa, Shigella spp. nd Salmonella spp. ). Each kinds of honey confirmed antibacterial exercise towards take a look at organisms with the zone of inhibition starting from Eight. 13 to 30. 85 mm, whereas E. coli, S. aureus, and Shigella spp. confirmed sensibility in the direction of each kinds of Honey. Each kinds of honey confirmed no results on Salmonella spp. The efficiency of honey at 100%focus was discovered to be greater than all different concentrations examined. Nevertheless, no impact was noticed at focus of 6. 25% v/v honey within the case of each samples(Patricia E. Lusby 2004).
To check the antimicrobial exercise of honey, 60 samples of assorted botanical origin have been evaluated antimicrobial actions towards 16 scientific pathogens and their respective reference strains. The bioassay utilized for figuring out the antimicrobial impact employs the well-agar diffusion methodology and the estimation of minimal energetic dilution which produces a 1 mm diameter inhibition zone. Streptococcus pyogenes,Bacillus cereus and Bacillus subtilis have been confirmed to be as much as 60% extra resistant than their equal reference strains thus emphasizing the variability within the antibacterial impact of honey and the necessity for additional analysis (C.
Voidarou 2010). Pure merchandise, both as pure compounds or as standardized plant extracts, present limitless alternatives for brand new medication due to the prepared availability of chemical variety (Cos et al. , 2006). Honey and lemon-honey are conventional cures within the Center East and China and for a lot of centuries and have been used within the therapy and prevention of the widespread chilly and varied higher respiratory tract infections (Molan, 1992; Zulma ; Lulat, 1989). Antibacterial exercise of the honeys was assayed utilizing normal effectively diffusion strategies. oncluded that the sandal wooden from totally different sources and combination of of – and -santanols have been extremely efficient towards gram detrimental micro organism together with Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae and in addition to yeast Candida albicans. The current work provides robust proof of antibacterial actions of honey, sandal oil and black pepper (Sheikh Ahmad 2002). On the whole, all kinds of honey have excessive sugar content material in addition to low water content material and acidity, which forestall microbial progress.
Osmotic impact, impact of pH and hydrogen peroxide are represented as an “inhibition” consider honey (Postmes et al. , 1993). Most kinds of honey generate hydrogen peroxide when diluted due to the activation of the enzyme glucose oxidase, which oxidizes glucose to gluconic acid and hydrogen peroxide (Schepartz and Subers, 1964). Hydrogen peroxide is the most important contributor to the antimicrobial exercise of honey,and the totally different concentrations of this compound detached honeys consequence of their variable antimicrobial results (Molan, 1992).
Furthermore, non peroxide elements additionally play crucial function. The content material of non peroxide elements are associated to the floral supply and generally account for the most important a part of the antibacterial exercise in honey(Molan and Russell, 1988). Forty samples of various honey varieties (Acacia, Ziziphus , Brassica and Citrus) have been collected from totally different areas of Pakistan and analyzed for moisture, pH, complete acidity, ash, electrical conductivity, hydroxyl methyl furfural (HMF), sucrose, complete sugars, invert sugar, protein, proline contents in addition to macro and micro components.
The variation in composition of honey samples was noticed on account of various kinds of flora. Likewise, a major stage (P ; zero. 05) of ash, electrical conductivity, sucrose, complete sugar in addition to macro and micro components was additionally present in these honey varieties. Totally different formulations of honey has considerably inhibited progress of pathogenic microorganisms, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger when in comparison with management group, which is an proof that honey is a therapeutic agent getting used since historical time all through the world. Feng et. al. 1994) The discrepancy within the noticed antibacterial exercise will be on account of a number of causes. One chance may be associated to the variations in susceptibility of every species of microorganism to the antibacterial exercise of honey used. Related 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 exercise might clarify a few of the discrepancies of noticed with the antibacterial exercise of those honey.
The presence of unstable putative brokers and/or thermolabile antibacterial agent(s) is also inactivated through the experimental process and thus could also be thought-about as doable rationalization of the noticed insensitivity of some honey samples discovered within the preliminary research. four. ) MATERIALS AND METHOD four. 1) Supplies A number of native manufacturers of honey and Dabur honey collected from totally different sources is used on this research. Additionally the honey is collected from varied apiaries from- Pathankot(Punjab) and Saharanpur (Up). Within the antibacterial research, a number of bacterial species recognized to be pathogenic to human comparable to E. oli. , S. aureus and Burkholderia capicium and Bacillus subtilis was used. These strains have been obtained from biotechnology lab. in accordance to given requirement. Glass wares used:- Conical flask (500 milliliter), Conical flask (100 milliliter), Petri plates, glass rod, take a look at tubes spreader, glass beakers, Glass slides and canopy slip, Plastic wares used:- Beakers, Conical flask (500 milliliter), Conical flask (100 milliliter), dropper, appendrof tubes (2ml), auto pipette ideas (10? l, 100? l, and 1000? l) Devices used:- Scorching air oven, Autoclave machine, Laminar air circulation hood, Incubator, Microwave oven
Different materials used:- Sprit lamp, cork borer,take a look at tube stand, auto pipette (10? l, 100? l, and 1000? l), parafilm wax,,filter paper Chemical compounds used:- Nutrient agar, nutrient broth, Mueller Hinton agar, 70% Methanol, 100% methanol, ethyl alcohol ,sodium chloride. four. 2) Strategies Physicochemical research: Look of various honeys was noticed. pH is decided utilizing standard process like utilizing ph scale. Antibacterial research: Antibacterial research was carried out in steps. In step one, an in vitro screening might be carried out utilizing both disc diffusion or effectively diffusion methodology.
Properly diffusion was carried out utilizing plate diffusion, which I most well-liked. Preparation of take a look at supplies: Check supplies might be ready by diluting every honey at totally different dilutions, 25 ? l/100 ? l, 50 ? l/100 ? l, 75 ? l/100 ? l and one with no dilution. Furthermore, internet honey was additionally used as take a look at materials. All dilutions have been carried out with double distilled and deionised sterilized water. three. 2. 1) Supply of bacterial strains The E. coli. , S. aureus, Burkholderia capicium and Bacillus subtilis have been revived from the inventory obtainable within the varied Biotechnology laboratories of Beautiful Skilled College, Phagwara (Punjab), India.
Sub-culturing of bacterial strains in nutrient broth:- 100ml nutrient broth was ready and ten take a look at tubes have been taken, autoclaved, and after this the Bacterial strains was inoculated within the nutrient broth in several take a look at tubes after which incubated for 24 hours in incubator . Sub-culturing of the bacterial strains on strong media:- For All Bacterial strains:- 5. 6g of Nutrient agar was added to the 200ml of purified water. Then answer was heated and boiled for 1 minute to utterly dissolve the powder. Then media was autoclaved.
Then media(10-15 ml) was poured in Petri plates. Then Bacterial strains have been added to media. Then Petri plate have been stored in incubator for 18-48 hours at 37 diploma C. Saline preparation : Take . 58g of Nacl and dissolve it to 10ml of distled water and make saline earlier than 15 min of spreading and take the colonies from the nutrient agar plates and blend correctly within 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 finish dissolve medium then autoclaved .
After that pouring the petriplates and left it 20 minute for solidify medium and make wells , after solidify put the totally different Honeys within the wells and stored it to incubate for 18-48 hours at 37C. Testing of antibacterial exercise utilizing agar effectively diffusion methodology: The bacterial strains have been inoculated into 10 ml of sterile nutrient broth, and incubated at 37 °C for 18 h. Every tradition was then spreaded on the floor of sterile nutrient agar plate and likewise pour-plated in nutrient agar media to carry out the take a look at in triplet, one with the management and the opposite two ith the take a look at pattern. Making the wells With the assistance of a cork borer make wells within the agar rigorously with out tearing the gel. In two agar plate of all 4 units, 4 wells have been ready with the assistance of sterilized cork borer. Then with the hep of micropipette put 100 micro litre of honey pattern into the wellIn the wells of two plates of every set, samples of following concentrations: (1) 100% sterile Honey(2) 75% sterile Honey(three) 50% sterile Honey and (four) 25% sterile Honey; have been added through the use of micropipette. Additionally within the a effectively water was added as detrimental management.
Then with the assistance of micropipette put 100 micro litre of honey pattern into the effectively Substitute the lid of the plate between placing pattern into wells to attenuate publicity to air-borne contaminants. Cowl the lid tightly with paraffin tape to keep away from contamination. Incubation of the plates . A temperature vary of 35°C ± 2°C is required for 24-48 hours. Don’t incubate plates in carbon dioxide as it will lower the pH of the agar and lead to errors on account of incorrect pH of the media. Measuring zone of inhibition 1.
Following incubation, measure the zone sizes to the closest millimeter utilizing a ruler or caliper; embrace the diameter of the effectively within the measurement . 2. All measurements have been made with the unaided eye whereas viewing the again of the petri dish. Maintain the plate a couple of inches above a black, nonreflecting floor illuminated with mirrored mild . three. File the zone measurement on the recording sheet. Figure1: Testing of antibacterial exercise utilizing agar effectively diffusion methodology four. )RESULTS Sub-culturing of bacterial strains in nutrient broth:- Figure2: Tradition E. coli. , S. ureus and Burkholderia capicium and Bacillus subtilis in Nutrient broth media. Sub-culturing of the bacterial strains on strong media:- Fig. –three E. coli subcultured on nutrient agar Fig. – four B. Subtilis subcultured on nutrient agar Testing of antibacterial exercise utilizing agar effectively diffusion methodology: Fig 5 : No antibacterial exercise seen as no honey is poured (taken as detrimental management) through which E. coli is grown Fig 6 : No antibacterial exercise seen as no honey is poured (taken as detrimental management) through which Bacillus subtilis is grown ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST E. coli
FIG. 7 – ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY(pathankot pattern) IN E. coli: 100% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm FIG. Eight– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY (dabur pattern) IN E. coli : 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm FIG. 9– ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY (saharanpur pattern) 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 pattern): 100% = three. 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 pattern): 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% = three. 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 pattern): 100% = 2. 1cm, 75%= 1. 9cm, 50%= 1. 6cm, and 25%= 1. 4cm; FIG. four– 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% =three. 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 Look Look of every of honey model was examined and it was noticed that honey have been brown to darkish brown in shade, whereas different honey have been golden yellow in shade. Dabur honey- golden color,clear Pathankot pattern – golden color with ample transparency Saharanpur pattern – darkish yellow color with zero transparency The transparency is because of processing of honey,whereas much less clear samples are unprocessed and brought instantly from bee hive.
Desk 1- Diameter of the Zone of Inhibition by totally different focus of Pathankot honey pattern on totally different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Management(sterile DistilledWater)| 100%Honey pattern| 75%Honey pattern| 50%Honey pattern| 25%Honey pattern| E. coli. | zero. zero| 2. 6| 1. 9| 1. 7| 1. 2| Bacillus subtilis| zero. zero| three. zero| 2. 7| 2. 7| 2. 5| S. aureus| zero. zero| 2. 1| 1. 7| 1. four| 1. 2| Burkholderia capicium| zero. zero| 2. 1| 1. 9| 1. 5| 1. four| Table2- Diameter of the Zone of Inhibition by totally different focus of dabur honey pattern on totally different Bacterial strains.
Bacterial strains| Zone of Inhibition (in cm) | | Management(sterile DistilledWater)| 100%Honey pattern| 75%Honey pattern| 50%Honey pattern| 25%Honey pattern| E. coli. | zero. zero| 2. 2| 1. 6| 1. three| 1. 1| Bacillus subtilis| zero. zero| 2. 7| 2. 5| 2. four| 2. 2| S. aureus| zero. zero| three. zero| 2. 7| 2. 2| 1. Eight| Burkholderia capicium| zero. zero| 2. four| 1. 7| 1. 5| 1. 2| Desk 1- Diameter of the Zone of Inhibition by totally different focus of Saharanpur honey pattern on totally different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Management(sterile DistilledWater)| 100%Honey pattern| 75%Honey pattern| 50%Honey pattern| 25%Honey pattern| E. oli. | zero. zero| 2. Eight| 2. four| 2. three| 1. 7| Bacillus subtilis| zero. zero| three. 2| 2. 7| 2. three| 1. 5| S. aureus| zero. zero| 2. 9| 2. 5| 2. zero| 1. 6| Burkholderia capicium| zero. zero| 2. 5| 1. 9| 1. 7| 1. four| Within the preliminary screening course of was noticed that some honey manufacturers have extra whereas another have much less antibacterial exercise. Some honey confirmed antibacterial impact towards E. Coli and a few confirmed strongest exercise towards Bacillus subtilis ,S. aureus and Burkholderia capicium. Clear zones of inhibition have been produced by concentrated with out dilution.
By way of the evaluation of common values of the diameters of every honey, it’s doable to deduce a doable sample through which industrial honeys, comparable to DABUR honey, have a better common diameter whereas the do-it-yourself honeys from totally different areas, have a decrease common diameter even at much less concentrations, however the perfect antimicrobial exercise was proven by Saharanpur honey pattern with was collected instantly from the apiary with none processing and it confirmed clear and enormous inhibition zones for all bacterial strains which have been used for this take a look at.
Although after the dilutions have been made, the low concentrated samples of Saharanpur honey resulted in relatively poor antimicrobial exercise. The wells through which water was loaded shoed completely no antimicrobial exercise which was used as detrimental management. The outcomes additionally confirmed that decrease focus of honey made by way of dilution exhibits much less antimicrobial exercise than concentrated ones. Thus honey are positively efficient towards the bacterial strains in any respect concentrations. 6) REFERENCES Bibi S, Husain SZ, Malik RN (2008). Pollen evaluation and heavy metals detection in honey samples from seven chosen international locations. Pak. J. Bot. 40(2): 507-516 * Cooper R. A. , Molan P. C. , Harding Ok. G. (1999) Antibacterial exercise of honey towards strains of Staphylococcus aureus from contaminated wounds, J. R. Soc. Med. 92, 283–285. * Cooper R. How does honey heal wounds? In: Munn P, Jones R,editors. Honey and Therapeutic. UK: Worldwide Bee Analysis Affiliation; 2001. * de Jong H. J. 1999) The Land of Corn and Honey:The conserving of stingless bees (meliponiculture) within the ethno-ecological surroundings of Yucatan (Mexico) and El Salvador, Utrecht UniversityPress, Netherlands, 423 p. * Kamal A, Raza A, Rashid N, Hammed TG, Lami M, Gureshin MA,Nasim Ok (2002). Comparative research of Honey collected from flora of Pakistan. On Line J. Biol. Sci. 23(9): 626-627. * Khalil MI, Shahjahan M, Absar N (2006). Glycemic Response and Lycemic Index of Bangladeshi Honey in Sort 2 Diabetic Sufferers. Malaysian. J. Pharm. Sci. four(1): 13–19. * Lusby PE, Coombes AL, Wilkinson JM.
Bactericidal exercise of various honeys towards pathogenic micro organism. Arch Med Res 2005; 36: 464-467 * Molan P. C. (1997) The antibacterial exercise of honey. The character of the antibacterial exercise,Bee World 73, 5–28. * Mundo MA, Padilla-Zakour OI, Worobo RW. Development inhibition of foodborne pathogens and meals spoilage organisms by choose uncooked honeys. Int J Meals Microbiol 2004; 97: 1-Eight. * Tumin N, Halim NA, Shahjahan M, Noor Izani NJ, Sattar MA,Khan AH, et al. Antibacterial exercise of native Malaysian honey. Malaysian J Pharma Sci 2005; three: 1-10.

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