AResearch proposal submitted to the Department of Pure and Applied Sciences inpartial fulfilment of the requirement for the degree of Industrial Microbiologyand Biotechnology of the Technical University of Mombasa. NOVEMBER,2017 DECLARATIONThisResearch Proposal is my original work and has not been submitted for Degree inany other University. Signature………………………………………………Date……………………………………… ANTHONY MUSABTMB/023J/2014 SUPERVISORThisResearch Proposal has been submitted for Examination purpose with my approvalas the university supervisor.Signature………………………………………………………Date……………………………… MR. NZARO MAKENZIDepartment of Pure and AppliedSciences. ACKNOLEDGMENTI would wish to thank the Almighty God for enabling meto do this project proposal.
I would also like to appreciate the technicaluniversity of Mombasa especially my supervisor Mr makenzi and other lecturersfor equipping me with adequate knowledge and skills in this field.Lastly I thank my family and friends they have been agreat motivationa and a stepping shoulder in difficult times. Table of contentsTable of ContentsDECLARATION.. iiACKNOLEDGMENT. iiiTable of contents. ivABSTRACT. vCHAPTER ONE.
. 11.1BACKGROUND INFORMATION.. 11.
2 STATEMENT OF THE PROBLEMS. 21.3 JUSTIFICATION.. 21.
4 OBJECTIVES. 31.4.1 Main objectives.
31.4.2 Specific objectives. 31.5 HYPOTHESIS. 31.
5.1 Alternative hypothesis. 31.6 RESEARCH QUESTIONS. 31.7 SCOPE OF THE STUDY..
3CHAPTER 2. 42.0 LITERATURE REVIEW..
. 42.1 ONION AND GINGER.
. 42.2 BACTERIA.. 42.2.1 Gram negative. 42.
2.2 Gram positive. 52.3 MECHANISM OF ANTIBIOTIC RESISTANCE.. 62.
4 ACQUISTION OF RESISTANCE.. 7 ABSTRACTAntimicrobialpotentials of Allium cepa and Zingiberofficinale revealed that the extracts from fresh samples show different rangesof antimicrobial activities. Onion and ginger are widely used food spices forboth culinary and medicinal purpose .The study will aim at determiningantimicrobial activities of both onion and ginger in both individual extractsand blend form.
A blend of phytochemical extracts from onion and ginger will beevaluated against gram positive and gram negative bacteria for antimicrobialactivity .The study will use two gram negative bacteria and two gram positivebacteria such as; Escherichia coli, Salmonella typhi and Staphylococcus aureas ,Bacillus coagulans respectively . Water and methanol will used assolvents of the processes .The zone of inhibitions will tested and the diameterof the zones measured and compared. CHAPTER ONE1.1BACKGROUND INFORMATIONTheefficacy of traditional medicinal herbs is evident from their continued use bya significant portion of the world population.
Use of traditional medicine andmedicinal plants in most developing countries for maintenance of good healthhas been widely observed. Effort has been devoted over years to the search fornew antimicrobial materials from natural sources for food preservation. Naturalderived compounds and products may have application in controlling bacteria infood. Increasing reliance on the use of medicinal plants in industrializedsocieties has been traced to the extraction and development of several drugsand chemotherapeutics from this plants which are used for herbal remedies.Onion and ginger were consumed throughout in Europe in the Middle Ages and werelater thought to guard against evil spirit and plagues due to their strongodor. Folk healers traditionally used for food and medicinal applicationshaving anti-inflammatory antioxidant effects in both in vitro and in animalmodels. Foodborne diseases is gradually becoming a serious risk to public health with anumber of cases increasing yearly as people become infected by either eatingfood with microbes.
Proper control of microbes and effective sanitation willhelp to reduce the rate at which people become sick by eating food .spices ingeneral show both antimicrobial and anti-oxidants characteristics, inhibitingthe growth of bacteria and other pathogenic microbes. Scientists in the pasthave conducted numerous experiments testing foodborne pathogens withphytochemicals extracted from spices. The antimicrobial properties of activecompounds extracted from spices and other plants Have Shown to affect thebehavior of pathogenic bacteria and fungi in the agro food and medical sector.This study investigates the antimicrobial activity of phytochemical extractedfrom onion and ginger against E.coli ,Styphi, B coagulans and S. aureususing a well diffused agar. 1.
2 STATEMENT OF THE PROBLEMSMedicinalplants have been in use by both ancient and modern man of all cultures fortreating different ailments .A single plant processed in different formulationscan be used to cure a wide range of diseases and may show excellent resultsagainst different pathogens .The historical role of medicinal herbs in the treatmentand prevention of diseases do not assume their safety of uncontrolled use by aninformed public. Several cases of diarrhea by students have been reported dueto consumption of food with pathogens at neighbor hotels within the school inTudor area. This food pathogens has led to food borne diseases which has ledstudents suffer from diarrhea stomach ache and other disease effect. Onion andginger having some antimicrobialactivities which inhibit growth of some pathogenic microbes food in food, canthen be used as a source of inhibition to such pathogens such as S.
aureus and E.coli thus reducingthe food borne diseases caused by this microbes and other gram positive andgram negative bacteria. Onions and ginger been widely spread for sale withinthe school area,example: Buxton and Kongowea at very low prices and been spicesmostly used in food and drinks consumed by students daily will be then be acheapest way of controlling the food borne diseases by raw consumption or addedas spices in more concentration to increase its bio-availability in the GItract.1.
3 JUSTIFICATIONRampantfood contamination in many countries has been observed in research by WHO werelots of people are ill due to consumption of contaminated food and other die offood borne diseases. Food borne diseases impede socioeconomic development bystraining health care systems and interrupting national economies, trade andtourism. Plant extracts of onion and ginger have been proved to haveantimicrobial activity and can inhibit bacteria by different mechanism sincethey contain bioactive compounds, phenol molecule and inflammatory compoundswhich lead to anti-inflammatory effects which can be used for both preservationand medicinal purpose to inhibit the growth and replication of food pathogenthus reducing the bacterial load and food borne diseases.1.4 OBJECTIVES1.4.1 Main objectivesToinvestigate the antimicrobial activities of onion and ginger against grampositive and gram negative bacteria.1.
4.2 Specific objectives1.To determine the rate of inhibition using different solvents example water andethanol2.To find out the antimicrobial activity in individual extract and in combinedagainst gram negative and positive bacteria.3.
To determine the zone of inhibition diameter and compare.1.5 HYPOTHESIS1.5.1 Alternative hypothesis1.Gram negative are more resistant than gram positive against antibiotics becauseit contains outer membrane with lipopolysaccharide and protein which makes themembrane less permeable.
2.The extracts have different rates of activity in different solvents.1.6 RESEARCH QUESTIONS1 .What are the methodsused in onion and ginger plant extracts?2.
Will the onion andginger extract show antimicrobial activity? 1.7 SCOPE OF THE STUDYThestudy will be focused on the daily used spices used within Mombasa region andsome of the food pathogens which cause food borne diseases in the region.CHAPTER 22.0 LITERATUREREVIEW2.1 ONION ANDGINGEROnionhas been revered throughout time not only for its culinary use but also for itstherapeutic properties. Consumption of onion is beneficial to human health asit contains polyphenol molecules or phytonutrients which include; flavonoids,tannins, as well as allicin, quercetin binds to bacteria DNA gyrase whileallicin inhibits certain thiol containing enzymes in the microorganisms by therapid reaction of thiosulfinates.GINGERhas a long tradition of being very effective in alleviating symptoms ofgastro-intestinal distress. Ginger can also been used as herbal medicine sinceit possesses numerous therapeutic properties similar to onion includingantioxidant effects, an ability to inhibit the formation of inflammatorycompounds and direct anti-inflammatory effects.
The main antimicrobial agent isgingerol a natural occurring phenol which disrupt the cell wall of bacteriacausing cytoplasmic leakage.2.2 BACTERIA2.2.1 Gram negativeTheygive a negative results in gram stain. This type of bacteria retains the violetstain after the decolourisation step. Alcohol is used to degrade the outermembrane making the cell wall more porous. They have thin peptidoglycansandwiched in between inner cell membrane making them to take up thecounterstain and appear red or pink in a microscope.
220.127.116.11 Escherichia coliE.coliis a gram negative, straight rod bacteria of about 1.1-1.
5µm by 2.0-6.0µmoccurring either singly or in pairs. They belong to class proteobacteria, theorder enterobacteriales, the family enterobacteriaceae and genus Escherichia .They are motile by petrichous flagella and fulcatatively anaerobic having bothrespiratory and a fermentative type of metabolism. Biochemically they areoxidase negative and usually citrate negative.
E.coli occurs in normal flora in the lower parts of the intestine,optimal growth is about 37 degree Celsius but are known to grow at temperaturesof about 44.5 degree Celsius .E.colistrains containing enterotoxins are known to cause diarrheal diseases due totheir invasion and colonization characteristics .They also cause urinary tractinfections and nosocomial infections including; septicemia and meningitis.2.2.
1.2Salmonella typhi Isa subspecies of salmonella enterica, rod shaped, flagellated ,aerobic and agram negative bacteria which infect broad range of vertebrate hosts. They havea number of serovars which are divided into two; one which cause usually causeenteric infections and ones which usually cause invasive degradation ofanaerobic metabolic pathways. S typhi hasrecently been an emerging disease in Africa. It’s associated with HIV, malariaand malnutrition have contributed to spread of this disease and the need to useexpensive antimicrobial drugs in the poorest health services in the world.2.2.
2 Gram positive Itgives positive results to gram stain by taking up the crystal violet stain thenappear purple in microscope. This is due to their thick peptidoglycan layer inbacterial cell wall which retains the stain after wash.2.2.
2.1 Staphylococcus aureusS.aureusis a gram positive bacterium that is a member of firmicutes group .It alsobelongs to class Cocci, order Bacillus, family Staphylococcaceae and genusStaphylococcus. The cells are spherical about 0.5-1.5µm in diameter and inclusters or pairs .
The are non-motile non-sporing facultative anaerobic withboth respiratory and fermentative mechanisms. Colonies are usually white andcream on general purpose agar such as TSA or yellow on differential media suchas MSA .Biochemically they are catalase positive and can grow in about 10%sodium chloride with an optimum temperature of about 30-37 degrees Celsius.They are mainly associated with the skin and mucus membrane of warm bloodedanimal and serves as indicator of nasal contamination in foods. They are knownto be opportunistic pathogens in humans causing diarrhea by producing extracellular toxins .S. aureus is an important food borne pathogen causing food poisoning which leads todiarrhea. 2.
2.2.2 Bacillus coagulansItis gram positive lactic acid forming bacterial species. It mainly causes anoutbreak of coagulation in evaporated milk packed in a lower temperatures .
Itcomes from family of lactobacillae and bacillaceae .its catalase positive,spore forming, motile and a falcutative anaerobe. 2.3 MECHANISM OF ANTIBIOTICRESISTANCEBacteriacan resist the effect of antimicrobialsthrough a variety of mechanisms .
some species of bacteria are innatelyresistant to ? 1 class of antimicrobial agents.in some cases the resistance isinnate, but in many others it is acquired of greater concern are cases ofacquired resistance, where initially susceptible populations of bacteria becomeresistant to an antimicrobial agent and proliferate and spread under selectivepressure of use of the urgent .several mechanisms of antimicrobial resistanceare readily spread through a variety ofbacterial genera such as; drug inactivating enzyme were an organism acquiresgene encoding enzyme that destroys the antimicrobial agent before being effective ,increased eliminationof drugs where through efflux pump detrimental compound are transported orremoved out of the system an alternation in the target molecule where minorstructural changes of the target molecule prevents the drug from binding thus not effective.
2.4 ACQUISTION OF RESISTANCESeveralprocess in the body may lead to antimicrobial resistance through eitherspontaneous mutation which alters new gene and acquisition of new gene whichmay occur between different gene bacteria .spontaneous mutation may causeresistance due to the following; altering the target protein by ant-microbialagent by either modifying or eliminating the binding site ,production ofcompounds that will inactivate the ant-microbial agent, down regulation oraltering an outer membrane protein channel that the drug requires for cellentryexample; ompf in E.coli and upregulating pumps that expels the drug from the cell .Mechanisms of genetic exchange include conjugation ,transduction and transformation.
Each of these processes, transposons mayfacilitate then transfer and in-cooperation of the acquired resistance gene into the host genome. Conjugation among gram positive bacteria is usuallyinitiated by production of sex pheromones by the mating pair which facilitatethe dumping of donor and recipient organisms, allowing the exchange of DNA.Duringtransduction, resistance gene are transferred from one bacterium to another viaa bacteriophage. Finally in transformation bacteria acquire and incorporate DNA segments from otherbacteria that have released the DNA compliments in to environment after cell lysis, can move resistance gene in tosusceptible strains mutation an selection, together with mechanism of geneticexchange enable many bacteria species to adapt quickly to the introduction ofthe antimicrobial agents in to the environment . Although single mutation in akey bacteria slightly reduce its susceptibility of the host bacteria to thatantibacterial agent, it may just be enough to allow its initial survival untilit acquires additional mutation or additional genetic information resulting infull-fledged resistance to the antibacterial agent.
CHAPTER33.0METHODOLOGY3.1STUDY AREA Thisstudy will be carried in Mombasa county with a number of people population ofabout 1.2 million ,thus the study willbe beneficial to people living inMombasa county prevalence to onion and ginger antimicrobial activityantimicrobial activity to some of the food borne pathogens gram positive andgram negative bacteria 3.2 SAMPLINGMETHOD Theonion and ginger bought from Kongowea market near the university will be usedto get the plants extracts then taken to universities lab for experimentalanalysis.3.
3SAMPLE SIZE24 samples will beobtained for the study.3.4SAMPLE PROCESSING3.4.1Sample collection Onionand ginger will be bought in Kongowea market by randomly collecting them fromdifferent vendors .The samples from different vendors will be randomly pickedand grouped to one sample for ginger and onion ,then a random picked onion andginger will be mixed in one group that will form a blend .
3 grouped sampleseach of 1/4Kg will be used for sample exaction.3.4.
2 Sample preparationTheprocess of extraction is by hydrolysis, where 5g of onion and or ginger will behomogenized in different places using a pestle and a mortor.20ml of water willbe added to form a solution of each sample. The solution is then sterilized andfiltered to remove the residues. The solution of the extract will be evaporatedusing a rotary evaporator to form a dry residue which will be measured for eachof the individual extract and the blend extract then stored for antimicrobialanalysis.Thesame procedure will be used for extraction of both onion and ginger usingmethanol. The homogenate will be dissolved in 20ml of methanol.
Thereafter theextract will be sterilized filtered to remove residues. 3.5BACTERIAL STRAINS AND CULTURINGStaphylococcusaureus and Escherichia coli will be obtained from the microbiology laboratoryat the technical university of Mombasa.
The bacterial strain will be then besuspended in Triptych soy broth and incubated at 37 degree Celsius for24h.Diffusion method will be used using Mueller Hinton agar. Kirby-Bauer discfor Bacterial strain control will be used to show the susceptibility of theonion, ginger and onion-ginger blend extracts and their effectiveness on thetest microorganisms.3.6ANTIMICROBIAL TESTING Kirby-Bauerdisc diffusion method will be used for determination of antimicrobialactivities of onion and ginger extracts. Bacteria in a soft nutrient agarcultured for 24h at 37 degrees will be spread on the agar plate medium ofthickness between 5ml with a sterile glass spreader and rotated several times.The plate will be left for 30mins to ensure evenly distribution of theinoculum. The extract will be dissolved in three different measures; 0.
1g,0.01g and 0.001g for each of the three extracts to get nine samples dissolvedin 5ml 0f water and other nine samples dissolved in 5ml of methanol thus havingeighteen total dissolved extract samples.Theholes will be punched out of the agar by using a sterile cork borer of 6mmdiameter then will be filled with 100µl of the sterilized supernatants of eachextract. The plates are incubated until zones of inhibitions will be measuredto nearest millimeter using transparent ruler. The antibacterial activity ofthe extracts will be then compared to the activity of the antibiotics onKirby-Bauer disc.3.
7DATA ANALYSISTheresults will be recorded in a data entry book. The susceptibility results ofthe bacterial isolates will be classified into three; susceptible, intermediateand resistant based on the standard interpretation table. REFFERENCESMurray PR, Rosenthal KS,Pfaller MA 2009 Medical Microbiology 6th edition Nester, Anderson,Pearsall Microbiology, a Human perspective 4th editionD Acharya, S.Trakulsomboon, S.K Maldhup, and S.
Korbsrisate, (2012). Antibiotic susceptibilitypattern and indicator of decreased ciprofloxacin susceptibility of salmonellaenterica serovar typhi from Dhulikel Hospital, Nepal, jpn J Infect Dis, 65)264267.Serthi S, Dutta A, GuptaB, Gupta S (2013) Antimicrobial Activity of spices against isolated food bornepathogens. International Journal of Pharmacy and Pharmaceutical Sciences260-262.Facaultaviely AnaerobicGram-Negative rods (2000) In: Holt J (Ed), Bergeys Manuel of determinativeBacteriology (9th edn), Williams and Wilkins, Baltimore, USA,pp.532.
Gram positive cocci(2000) Bergeys Manuel of determinative Bacteriology (9th edn), In:Holt (Ed), Williams and Wilkins, Baltimore, USA, pp.532.Azu N ,Onyegha R (2006)Antimicrobial properties of Extracts of Alliumcepa (onion) and Zingiber officinale(ginger) on Escherichia coli, Salmonella typhi and Bacillus subitilis ,Internet scientificpublications 3(2).British Society ofAntimicrobial Chemotherapy, Disc diffusion method for antimicrobialsusceptibility testing, BR Soc Antimicrob Chemother; 2:1-46, (2002). 3.9BUDGET ACTIVITY/ITEM AMOUNT Raw materials 500 Project writing and Binding 2500 Reagent and media 6000 Transport 1000 Miscellaneous 2500 TOTAL 12500