1.
3 Justification of the Study
This study will help give an idea on how contamination of these ready-to-eat vended fruits can
pose a threat to human health based on food poisoning. It will also create awareness to the role of
concerned government health officials in the control of microbial contamination in these read to-
eat vended fruits.
1.4 Aim of the Study
The study is aimed at isolation and identification of microorganisms associated with spoiled fruit
in Zungeru (pineapple, orange, tomatoes and pawpaw).
1.4.2 Objectives of the Study
i. Determination of total aerobic bacteria plate count and total fungal count.
ii. Isolation and identification of bacteria and fungi contaminants from spoilt fruits using
morphological and biochemical characterization.
iii. Determination of the percentage frequency distribution of the microbial isolates On the
from the spoilt fruits.
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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Microbial Contamination of spoiled Fruits
Most microorganisms that are initially observed on whole fruit or vegetable surfaces are soil
inhabitants, members of a very large and diverse community of microbes that collectively are
responsible for maintaining a dynamic ecological balance within most agricultural systems,
Vectors for disseminating these microbes include soil particles, airborne spores, and irrigation
water. Most bacteria and fungi that arrive on the developing crop plant either are completely
benign to the crop's health or, in many instances, provide a natural biological, barrier to
infestation by the subset of microorganisms responsible for crop damage (Janisiewicz. And
Korsten,) responsible for causing spoilage to the edible portion of the crop plant is the 2002
(Abano and Amoah.. 2011).
Spoilage microorganisms can be introduced to the crop on the seed itself, during crop growth in
the field, during harvesting and postharvest handling, or during storage and distribution. Those
same types of soil-borne spoilage microbes that occur on produce are the same spoilage
microorganisms that are present on harvesting, equipment, on handling equipment in the
packinghouse, in the storage facility, and on food contact surfaces throughout the distribution
chain. Therefore. Early intervention measures during crop development and harvesting through
the use of good agricultural practices (GAP) will provide dramatic reductions in yield loss due to
spoilage at all subsequent steps in the food-to-fork continuum (Abbott, 2012). Examples of
GAPs include foliar fungicide application in the field, cross-contamination prevention measures
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Tissue (Mandrell, et al., 2006). On plant structures other than the fruit, internalization can be
achieved through a number of specialized vessels and surface structures employed by the plant to
absorb and release water and to provide CO; and O; exchange (Avery et al., 2005).
However, the fruit of the plant lacks many of these structures, requiring the spoilage microbe to
employ other methods to become initialized (Baeza et al, 2007: Baranska et al, 2005). This may
partially explain the rather limited success of bacteria to spoil fruits and an improved ability to
spoil vegetables that are not the fruit of the plant. The natural acidity of most fruits also serves as
a barrier to many spoilage microbes, especially bacteria. By contrast. Spoilage fungi that
typically produce more diverse and greater amounts of extracellular depolymerizes successfully
attack and spoil both fruits and vegetables. Colonization and lesion development more typically
and more rapidly occurs within damaged or otherwise compromised plant tissue. External
damage such as bruising, cracks, and punctures creates sites for establishment and outgrowth of
the spoilage microbes. Lesion development, can be relatively rapid, occurring within days or
weeks. This presents the risk that rapidly reproducing spoilage microorganisms will arrive within
open wound sites at the packing facility and thereby through shedding from the asymptomatic
wound, present the potential for cross contamination within the facility during handling. Culling,
washing. Sorting, and packing before storage. Such cross contamination to some degree is
inevitable and. if not carefully managed with a robust facility sanitation program, could lead to
the establishment of a population of spoilage microbes endemic to the facility that may be
difficult to eradicate (Barro et al., 2006)
A further and potentially more serious complication is the introduction into the cold storage
facility of spoilage microorganisms already established in wound sites on product, whether the
product is in bins or boxed and palletized. Depending upon storage conditions and storage time
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Association with Foods
Cholera is regarded primarily as a waterborne infection, though food which has been in contact
with contaminated water can often serve as the vehicle. Consequently a large number of different
foods have been implicated in outbreaks, particularly products such as washed fruits and
vegetables which are consumed without cooking. Foods coming from a contaminated
environment may also carry the organism, for example sea foods and frog's rentlegspandemic. In
South the and CentralcurAmerica an uncooked fish marinade, in lime or lemon juice, ceviche has
been associated with some cases (Adams and Moss, 2008).
2.5.4 Escherichia coli
E. coli is an almost universal inhabitant of the gut of humans and other warm-blooded animals
where it is the predominant facultative anaerobe though only a minor component of the total
micro flora. Strains of E coli were first recognized as a cause of gastroenteritis by workers in
England investigating summer diarrhea in infants in the early 1940s. Until 1982, strains
producing diarrhea were classified into three types based on their virulence properties:
enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli
(ETEC). They are not very common causes of food-borme illness in developed countries, but an
important cause of childhood diarrhea in less developed countries (Adams and Moss. 2008).
Association with Foods
Faecal contamination of water supplies and contaminated food handlers have been most
frequently implicated in outbreaks caused by EPEC, EIEC and ETEC. A number of foods have
been involved, including a coffee substitute in Romania in 1961, vegetables, potato salad, and
sushi. In the United States, mould-ripened soft cheeses have been responsible for outbreaks in
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by ETEC (ST). E coli would not be expected to survive well in a fermented dairy product with a
pH below 5 but, where contamination is associated with mould-ripening, the local increase in pH
as a result of lactate utilization and amine production by the mould would allow the organism to
grow. Outbreaks caused by EHEC serotype Ol57:H7 have mostly involved undercooked ground
meat products and occasionally raw milk. Cattle seem to be an important reservoir of infection
and O157:H7 has been isolated from 0.9-8.2% of healthy cattle in the UK (Adams and Moss,
2008).
2.5.5 Klebsiella pneumoniae
Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In
particular, the medically most important Klebsiella species, Klebsiella pneumoniae, accounts for
a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias,
and soft tissue infections. The principal pathogenic reservoirs for transmission of Klebsiella are
the gastrointestinal tract and the hands of hospital personnel. Because of their ability to spread
rapidly in the hospital environment. These bacteria tend to cause nosocomial outbreaks
(Podschun and Ullmann. 2008).
Association with Foods
Klebsiella pneumoniae (K. pneumoniae) is one of the most Klebsiella genus in Enterobacteriacae
family, which is responsible for pneumonia (the destructive lung inflammation disease).
Vegetables are known as source of contamination with K. pneumonia. Raw vegetables are
usually consumed in salads and other dishes (Puspanadan et al., 2012).
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2.6 Decontamination of Fruits
Reduction of risk for human illness associated with raw produce can be better achieved through
controlling points of potential contamination in the field, during harvesting, during processing or
distribution; or in retail markets. Food-service facilities, or the home (70). To reduce and/or
eliminate the microbiological contamination of vegetables after harvest, Proper washing of fruits
and vegetables is essential for decontamination (Eni et al., 2010). Only clean potable water
should be used for irrigation, the drinking water should be used for bulk-soil removal and first
cleaning (Lyncher et al., 2009). The use of plastic sheets or other materials to prevent contact
with contaminated soil during harvest. Vigorous washing in potable water typically reduces the
number of microorganisms by 1-2 logs and is often as effective as treatment with 100 ppm
chlorinated water, the current industry standard (Eni et al., 2010).
Water supplemented with varying concentrations of organic acids, such as citric and sorbic,
acetic acids (vinegar), has been shown to reduce microbial populations on fruits and vegetables
(Eni et al., 2010). Washing and sanitizing agents can reduce the levels of surface contamination
of raw and processed fresh produce items, and therefore, can help reduce the likelihood that large
shigella agar were incubated at 37°C for 24hours while the petri-dishes that contained potato
dextrose agar were incubated at 25°C for 3days. The nutrient agar, macConkey agar, mannitol
salt agar and salmonella-shigella agar were used to check for total bacterial count, total coliform
count. Presence of Staphyiococcus aureus, Salmonella and Shigelia spp respectively. At the end
of the incubation period, the plates were brought out of the incubators and the colonies were
counted using a colony counter device and each count was expressed in colony forming unit per
g(CFU g) (Chees brough, 2006).
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3.4 Isolation of the cultured micro-organisms
The distinct colonies on nutrient agar and potato dextrose agar were carefully examined using
microscope for their morphological characteristics like color. Then these colonies were
subcultured on nutrient agar using streaking method and were incubated at 37°C for 24hours.
(Chees brough, 2006).
3.5 Identification of Isolates
Gram staining and other biochemical tests were carried out based on the method of Cheesbrough
(2006). The biochemical tests performed here included catalase test. Oxidase test, indole test and
coagulase test.
3.6 Biochemical tests
3.6.1 Catalase test: The discrete colonies of each of the isolates were collected with a wooden
stick and emulsified in a drop of hydrogen peroxide (H 2O2). Bubbles of gas indicated a positive
result according to Cheesbrough (2006).
3.6.2 Indole test: Here a little portion of each of the isolates was inoculated into 5ml of sterilized
prepared peptone water which was contained in different test tubes using a wire loop. And then,
the test tubes containing the organisms were left to incubate at 37°C for 48hours.
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CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 RESULTS
Table 4.1: Bacterial isolates from the sample
Table 4.1 shows the bacterial isolates from the 4 spoilt fruit sample analyzed, E coli and
klebsiella sp. were most predominant, spoilt tomatoes have the highest bacteria isolates
compared to the entire sample analyzed
Sample Isolate
1A E coli and Bacillus sp.
1B Klebsiella sp, Ecoli
1C E coli and Bacillus sp.
2A E coli and Klebsiella sp
2B E coli , Klebsiella sp and Stapylococcus sp
2C E coli , Klebsiella sp and Stapylococcus sp
3A Ecoli, Klebsiella sp
3B Bacillus sp., Klebsiella sp
3C Bacillus sp., Klebsiella sp
4A E coli , Klebsiella sp and Stapylococcus sp
4B E coli , Klebsiella sp ,Stapylococcus sp and Bacillus sp
4C E coli , ,Stapylococcus sp and Bacillus sp
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4.2 Discussion
The prevalence of the diverse group of microorganisms from the spoilt fruits under study could
be attributed to the statement made by Halablab et al., (2011) that fruits can be contaminated
with pathogenic microorganisms as harmful enteric bacteria and fungi pre- harvest, during
growing in the field through contact with soil, dust, irrigation water. The current findings
identified the following bacterial from the spoilt fruits; E. coli, Bacillus sp., Klebsiella sp. and
Staphylococcus aureus for the fungal isolates includes; Mucor sp., Mucor racemoss, C. ropicalis,
A. niger, Fusarium sp., A. flavus, A. stolonifer and Rhizopus stonlonifer suggesting that these
fungal organisms could be responsible for the fruit spoilage. This finding is in conformity with
previous works of Onyemata and Ibrahim (2018) to have examined six fruits spoilage, identified
the following Aspergillus niger, Aspergillus flavus, aspergillus niger complex, Mucor
racemosus, Mucor species and Fusurium species. High prevalence of A niger and Mucor sp.
from the current findings is consistent with the work of Onyemata and Ibrahim (2018) recording
52% for A. niger followed by Mucor species with frequency 339% and Fusarium species had the
least occurrence with the frequency of 13%.
High occurrence of E coli and Klebsiella sp. fiom the current finding is in agreement with the
work of Oji et al.,. (2016) to have conducted a research on microbial contamination of ready-to-
eat vended fruits in Abakpa Main market, From examination five (5) bacterial species were
isolated namely: Esehniehia coli, Staphylococcus aureus, Salmonella sp. Shigella sp and
Psencdomonas sp while one (1) fungal species, Mucor sp. was isolated from the vended fruit
samples. A similar study by Hassan and Zulkahar (2018) conducted a research on bacteriological
analysis of spoiled fruits samples comprising of pineapple and pawpaw bad higher occurrence in
spoiled fruits identified as Escherichia sp. Klebsiella sp with Bacillus sp and Staphyloccus sp.
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Spoilage of these fruit samples could be as a result of poor post-harvest activities which could
include human handling, have sting equipment, transport containers, wild and domestic animals,
insects, Dust, washing water, ice, transport vehicles, processing equipment, cutting slicing.
Packaging and shipping (Sunyoung, 2004: Issazacharia et al., 2010). Post harvesting processes
ranging from storage and rinsing to cutting are also possible sources of contamination. Cut
surfaces of leaves are a specific target for pathogenic bacteria such as Salmonella, which show a
specific tropism towards them. The number of recent food- poisoning outbreaks have been linked
to the consumption of fresh fruits and fruits and that unhygienic product handling is implicated
as the principal source of contamination (Robert. 2009, Tyrel et al, 2006). Poor hygienic
conditions in processing increase the risk of contamination with foodborne pathogens (Nguyen et
al., 2004).
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CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
This study has shown that bacterial species such as E. coli and Klebsiella sp. are common
Pathogens of fruits spoilage: are also pathogen of fruits spoilage. However. Spoilt fruits are
mostly contaminated by fungal species when compared to bacterial species from the result
obtained from the current research. With these we could say that E coli are the most common
microorganisms causing food spoilage most especially to fruits. These pathogenic E coli species
associated with fruits spoilage are of economical and public health significance.
5.2 Recommendations
Since fruits and vegetables have been very important in human nutritional qualities, this then
calls for proper handling of the produce from the pre-harvest to consumption. All the fruits are
advised to be eaten fresh or cooked either to avoid long term exposure to spoilage. Also, the high
moisture content of fruits and vegetables will be a serious limiting factor in their preservation.
Finally, the farmers who harvest the fruits into bags for transportation, the marketers and
consumers should take necessary precautions in preventing contamination and also try to create
an environment that would discourage the growth or multiplication of microorganisms. All the
above will help to reduce the risk to poisoning by aflatoxin and other mycotoxins which are
normally produced by those fungi isolated in this study. Hence, this is a great concern to all.
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