SCREENING OF GINGER AND MORINGA OLIFERA EXTRACTS FOR ANTIMICROBIAL ACTIVITY AGAINST COMMON POST-HARVEST BACTERIAL PATHOGENS ISOLATED FROM STORED YAM (DIOSCOREA ALATA)
ABSTRACT
Yam (Dioscorea spp.) is a tuber crop belonging to the family Dioscoreaceae with six economically important species. Dioscorea alata is the most widely cultivated species in West Africa especially Nigeria. However, pre- and post-harvest spoilage by microorganisms result in the loss of a significant percentage of cultivated yam. Therefore, this study is aimed at isolating and characterizing microbial pathogens of post-harvest rot in yam. Moreover, the antimicrobial effects of Moringaolifera and Ginger officinale extracts on the isolates was also targeted. Serially diluted samples from healthy, dry and soft rotted yam were inoculated onto nutrient agar and blood agar using spread plate technique. Pure cultures were obtained and identified using biochemical tests. These were further characterized by amplification of 16S rDNA. Isolates were tested for antimicrobial activities by measuring zones of inhibition on Mueller-Hinton agar. Results obtained, showed the presence of known bacterial pathogens of yam rot such as Streptococcus sp, Staphylococcus aureus, Aeromonas sp, Enterococcus sp and Micrococcus sp. The antimicrobial effect of M. olifera methanoic extracts was low in 12.5% isolates while 87.5% were completely resistant. Antimicrobial effect of G. officinale methanoic extracts was low in 12.5% isolates and intermediate in 37.5%. Also, 12.5% isolates showed complete resistance while another 12.5% were susceptible. Furthermore, 62.5% of the isolates were susceptible to the ethanoic extracts of G. officinale, while in 25% antimicrobial activity was intermediate and in 12.5%, there was complete resistance.Moringaolifera ethanoic extract had low antimicrobial effects in 50% of the isolates, intermediate in 25%, 12.5% were completely resistant, while 12.5% were susceptible. There was no antibacterial activity with water extracts of M olifera and G officinale against isolates. This study further confirms the efficacy of Molifera and Gofficinaleas antimicrobial agents of known pathogens associated with pre- and post-harvest spoilage of yam.
Keywords: Yam rot, Dioscorea alata, Ginger officinale, Moringer olifera, antimicrobial activity,
pathogen, spoilage.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgement iv
List of Tables vii
List of Figures viii
Abstract ix
Chapter 1: Introduction 1
1.0 Economic and social impact of yam and world production 1
1.0.2 Types in Yam 7
1.0.3 General Morphology and composition of yam tuber 7
1.0.4 Nutrients in Yam 8
1.0.5 Post-Harvest Food Loss 11
1.0.6Food Losses 12
1.0.7 Food waste 12
1.0.8 Post Harvesting in tuber yam 15
1.0.9 Agronomy of Yam and Storage 16
1.1.0 Plant extracts as antimicrobial agents 20
1.1.1 Moringa olifera 21
1.1.2 Ginger 23
1.2.0 Storage pathogens 26
1.2.1 Emergence of Post-Harvest Pathogen in yam 26
1.2.2 Wet rot 27
1.2.3 Dry rot 27
1.2.4 Soft rot 28
1.2.5 Post-harvest stored yam organisms 31
1.2.6 Management of post-harvest pathogens of Stored yam 31
1.3.0 Rationale for research 34
1.4.0 Aims and objectives of study 34
Chapter 2: Literature review
2.1 Sampling
36
2.2 Sterilization of materials 36
2.3 Sterilization of Sample 36
2.4 Preparation of Media 37
2.5 Isolation Microorganisms 37
2.6.1 Identification of microbial isolates 38
2.6.2 Biochemical tests for identification of bacteria isolates 38
2.7.0 Confirmation of microbial isolate by Molecular Techniques 41
2.7.1 Extraction of microbial DNA 41
2.7.2 Amplification of genetic material (16s RNA) 42
2.7.3 Plant extracts preparation 44
2.7.4 Plant extract chemical analysis by (FTIR) 44
2.8.0 Anti-microbial activities of plant extract 47
Chapter 3: Results
Identification of bacteria isolates48
Biochemical Test Results48
Identity of Bacterial Isolates48
Confirmation of microbial isolate by Molecular Techniques48
Amplification of genetic material (16s RNA)48
Plant extract chemical analysis (FTIR)49
Antimicrobial activities of plant extracts50
Chapter4: Discussion and conclusion 71
Chapter 5: References 76
LIST OF TABLES
Table 1: Data on world production trade in yam 3
Table 2: World production of yam 4
Table 3: Nutrient content of yam species 10
Table 4: Food supply chain and example 14
Table 5 Amplification of 16S RNA sub unit of the microbial genome 43
Table 6 Biochemical test results of pure culture isolates 53
Table 7 Distribution of microbial isolates by samples 55
Table 8 Result of FTIR of the plant extracts 63
Table 9 Result of UV-visible of plant extract 63
Table 10 Antibacterial activities of plant extracts on bacterial isolates from yam rot65 Table 11 Antibacterial effect of methanoic on plant extracts on bacterial isolates 66
Table 12 Antibacterial effect of ethanoic on plant extracts on bacterial isolates 68
LIST OF FIGURES
Figure 1: Change of yam production, cultivation area, yield and consumption……... 5
Figure 2: Yam and other root crops on sale in Tonga Kingdom 6
Figure3: Statistic report of food losses across the countries 13
Figure 4: Typical yam barn in West Africa 19
Figure 5: Moringa olifera plant 24
Figure 6: Ginger plant 25
Figure 10: Pictorial representation of a tuber yam showing lesions of dry rot 29
Figure 11: Pictorial representation of a tuber yam showing lesions of soft rot 30
Figure 12: The Fourier transformed infrared ray 45
Figure 13: The mechanism of action of FTIR 46
Figure 14: Representative showing the results of 16Srna PCR 56
Figure 15: FTIR of moringa methanoic extract 57
Figure 16: FTIR of moringa ethanoic extract 58
Figure 17: FTIR of ginger methanoic extract 59
Figure 18: FTIR of ginger ethanoic extract 60
Figure 19: FTIR of ginger water extract 61
Figure 20: FTIR of ginger water extract 62
Figure 21: Activities of some known antibiotic on streptococcus spp 64
Figure 22: Comparison of antibiotic and plant extract on staphylococcus spp 69
Figure 23:Comparison of antibiotic and plant extract on staphylococcus aurea 69
Figure 24:Comparison of antibiotic and plant extract on streptococcus spp 70
Figure 25:Comparison of antibiotic and plant extract on Aeromonas spp 70
Figure 26:Comparison of antibiotic and plant extract on Enterococcus spp 71
Figure 27:Comparison of antibiotic and plant extract on micrococcus spp 71
Figure 28:Comparison of antibiotic and plant extract on staphylococcus aurea 72
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.0 Economic and social impact of yam and world production
The crop yam (Dioscorea spp) is one of the common food crops in the tropics and plays vital roles in food security as a staple food in the regions where it is being cultivated (Maroya et al, 2012). Dioscorea sppoccur in Asia, East Africa, the Caribbean, India and Tonga Kingdom (Figure 1), south pacific as well as West Africa (Okigbo, 2004). (Maroya et al, 2012) estimated that yam consumption yearly is over 48million tones globally. As shown in Table 1, out of the 48million tons of yam (95% global supply) that are produced on 4million hectares annually, Nigeria alone produces 67.7% of global yam supply (FAO, 2010). This makes Nigeria the highest consumer of this staple crop after Cassava and Maize. However, demand for this commodity is increasing (Figure 2); as incomes increase consumers shift from substitutes to yam especially when the price of yam relative to price of its substitutes declines (Maroya et al, 2012).
Tuber yam, among many other staple foods, is susceptible to postharvest diseases caused by bacteria and fungi under poor storage conditions. In Nigeria, pathogens constitute the major factor to rot in yam production (Nari, 2004). Losses caused by pathogens attack vary from 20-30% generally in some crops (Park et al, 2008).
Yam plays an outstanding function in social cultural lives of some producing regions like the celebrated Moon festival and also the popular Yam festival in West Africa, an act that is well observed. In Nigeria for instance, the meals offered to
deity and associates consist mainly of mashed yam (Ogunleye, 2005). Yam storage in comparison with some other staple crops has relatively longer life span, so stored tuber symbolizes stored wealth, which can be sold throughout the year by the marketers. Also, tuber yam in West Africa particularly Nigeria can be converted into different staple transitional and end product forms (Okaka and Aajekwu, 1990; Okaka et al, 1991) which can be consumed by human beings and animals, used as the essential ingredient of snacks and flour that is now used in instant puree making (Coursey, 1983; Okaka and Okechukwu, 1987).
Yam can be eaten in a variety of ways as it can be roasted, fried, grilled, baked, barbecued, smoked and most commonly boiled. Yam is also used as food for livestock. Tuber yam can be dried, ground into flour and stored fpor use. However, in Nigeria, a country known to produce a large percentage of yam around the globe, it is believed that the supply of yam tuber is lower than its high demand, a problem currently facing the country (Ogundana, 1971; Okigbo et al,2000;Okigbo and Emoghene, 2004; Okigbo and Ogbonnaya, 2006).
Types of Yam
Over 300-600 types of yams are accessible, however, there are a few species that are grown principally for human consumption, while others might be grown for medicinal purposes (Okigbo, 2004). The cultured species in West Africa particularly Nigeria are the white yam (D. rotundata), yellow yam (D.cayenesis), water yam (D. alata),Cush cush yam(D.trifida), Bitter yam (D.dumetorum), Aerial yam (D.bulbifera), Chinese yam (D.esculenta) (Onwueme, 1978). Dioscorea cayenesis and D. rotundata are rare in West Africa while D. alata and D. esculenta are indigenous to Asia. In addition, there are various numbers of wild yams that are
also harvested as food (Wilson, 1982). D.alata, D. cayenesis, D. bulbifera and Ddumentorum function as medicinal and nutritional purposes, while others indicated in Table 3 are for nutritional purposes (Okwu,2006)
General Morphology and composition of yam tuber
Tuber yam shape ranges greatly due to environmental and genetic factors. Yam tubers are more or less cylindrical in shape and mostly weight 3-5kg (Omwueme and Charles, 1994).
A mature yam tuber has four concentric layers:
a. Corky periderm- the outer portion of the yam tuber that provides effective barrier against pathogenic attack and water loss.
b. Cortex- a layer located immediately beneath the cork that assists in storage of starch.
c. Meristematic layer – elongated thin-walled cells that give cover to the cortex
d. Ground tissue- the central portion of the tuber that house yam nutrients (Omwueme, 1978).
.