MICROBIOLOGICAL QUALITY OF SPOILT FRUITS OBTAINED FROM SELECTED MARKETS WITHIN KADUNA METROPOLIS

MICROBIOLOGICAL QUALITY OF SPOILT FRUITS OBTAINED FROM SELECTED MARKETS WITHIN KADUNA METROPOLIS

CHAPTER ONE

1.1           Introduction

Fruits play a vital role in human nutrition by supplying the necessary growth factors such as vitamins and essential minerals in human daily diet and that can help to keep a good health. Fruits are widely distributed in nature. One of the limiting criteria is the relatively short shelf life period caused by pathogens attached (Essien et al., 2011).  It is estimated that about 20 – 25% of the harvested fruits are decayed by pathogens during post harvest handling even in developed countries.

According to Al-ttindi et al., (2011), it has been known that fruits constitute commercially and nutritionally important indispensable food commodity. Fruits are high highly perished product, the quality is affected by post harvest handling, transportation, storage and marketing. The improper handling, packaging, storage and transportation may result in decay and production of micro – organisms which become activated because of the changing physiological state of the fruits (Wilson et al., 2000). 

Al-Hindi et al., (2011) stated that extra care should be taken during personal handling of these fruits such as harvesting, clearing, sorting, packaging, transport and storage. Pathogenic organism can enter fruits and vegetables through damaged surfaces such as punctures, wounds, cuts and splits that occur during growing or harvesting (Durgesh et al., 2008).

Ayanda et al., (2013) stated that one of the factors influencing virulence of pathogens is their ability to produce enzymes capable of degrading their host’s tissue. Micro – organism especially bacterial and fungi have been identified as major organisms causing deterioration of various fruits by the secretion of extra cellular cell wall degrading enzymes.

The increase in food related out break prompted the united stated (U.S) Food and Drug administration in 1995 to request the national Advisory Committee on Microbiological criteria for food borne illness and fresh produce to provide recommendations to reduce the risk of food borne out break (National Advisory Committee on Microbiological Criteria for Foods, 1999).     

Bevchat (2000) stated that contamination of fruits and vegetables take place at all stages of fruit processing if proper sanitary and hygiene conditions are not maintained. Consumption of fruits products gad dramatically increased in the United State by more than 30% during the past few decades. It is also estimates that about 20% of all fruits produced is lost each year due to spoilage. During the period 1970 – 2004, United State per capital consumption of fruits increased by 19.9% to 694.3 pounds per capital per year (Ashok and Varum, 2011).

The focus of this chapter is to provide a general background on microbiological spoilage of fruit fresh – cut fruit, fermented or acidified fruits products.

1.2            Justification Of The Study

Microorganisms have attracted worldwide attention due to the significant losses associated with their impact on health and consequent national economy. The quality and safety of fruits is of importance, therefore market should not compromise for the sale of low quality of unsafe foods. The nutritional and economic importance of fruits when contaminated with fungi and other agents causing different types of disease.

1.3            Scope Of The Work                

The outcome the research will guide fruit users to know the various forms of organisms (bacteria and fungi) that are involved in the spoilage of the fruits. This will enable them device precautionary measures needed to prevent those specific types of organism from invading their fruits and causing spoilage.

1.4            Aims And Objective

                   i.                     To isolate the bacterial and fungal special associated with spoilage of fruits in some selected markets in Kaduna state.

                 ii.                     To identify the bacterial and fungal species isolated.

              iii.                     To study various fungal and bacterial pathogens responsible for the decay and deterioration of economically important fruits.

CHAPTER TWO

LITERATURE REVIEW

2.0     Introduction

Spoilage refers to any change in the condition of food in which the food become less palatable or even toxic, these changes may be accompanied by alteration in taste, smell, appearance or texture (Paul and Sainsbury, 2001).

Many fruits and vegetable present an ideal condition for the survival and growth of many types of microorganisms (Miedes and Lorences, 2004).   

Most of the reported out breaks have been associated with contamination, particularly members of the Enterobacteriaceae. A large number of lactic acid bacteria,  coli forms, moulds and yeast have been reportedly implicated in food spoilage as they use the carbohydrate content of the foods for undesirable fermentation processes (Essien et al, 2011)

However, about two-third of the spoilage of these items are caused by moulds of the genera Penicillum, Aspergillus, Botrytis, and Rhizopus (Frazier and westhoff, 1998). Fruits contain high level make them particularly desirable to fungi decay. According to Singh and Sharma (2007), generally, spoiling fungi are considered toxigenic or pathogenic. Toxigenic fungi have been isolated from spoilt fruits (Stinsen et al, 2000).

Use of untreated waste water and manure as fertilizers for the production of fruits and vegetable is a major contributing factor to contamination. Stated that in developing countries, food borne illnesses caused by frequent and in some areas they cause a large proportion of illness. (Meher and Reazi 2011).

Out break of salmonellosis have been associated with the consumption of cut water melon and cantaloupe in united state of America (CDC, 2009).

Salmonella species are important cause of gastro intestinal illness in humans, a wide range of salmonella serotype have been associated with out break involving fresh produce (fruit) (Banchat, 1995). Enteric pathogen such as Escherichia coli and salmonella are among the greatest concern during fruit related outbreak (Buck et al, 2003).

In recent years there has been an increase in the number of reported case of food borne illness linked to fresh fruit and vegetable (Mansah et al 2002; Buck et al, 2003; Eni et al, 2010).  Epidemiology data have shown that fruit crass contamination during preparation contribute remarkably to the occurrence of food borne disease (Gilling et al 2001, Kusumaningrum et al, 2004, CDC, 2009).  Listeria monocytogen is an important fruit borne pathogen capable of causing severe illness with high mortality rate (Sumathi and Slutsker, 2003).

Bacillus cerus have been isolated from spoiled mango and Bovicin have been tested to effect on the growth and spore germination. Spores of clostridium botulinum are commonly found on the fruit surface and multiply within the tissue (Burnett and Beuchat, 2001).  Contamination could be from human handling, transporting vehicle, dust, insect, rinse water, water used to apply fungicide, insecticide manure wild and domestic animal (Burnett and Beuchat, 2001, Buck et al, 2003). Several cases of typhoid fever outbreak have been associated with eating contaminated vegetable grown in or fertilized with contaminated soil or sewage (Beuchat, 2000).                         

2.1     Spoilage Microorganisms

Most microorganisms that are initially observed on whole fruits surface are soil in habitants, member of a very large and diverse community of microbes that collectively are responsible for maintaining a dynamic ecological balance within most agriculture system. Sources for disseminating these microbes include soil particles, air borne spores and irrigation water. Most bacteria and fungi that arrive on the developing crop plant either are completely stated that most bacteria and fungi that arrive on the developing crop plant either are completely benign to the crops health or in many instances, provide a natural biological barrier to infestation by the subset of microorganisms responsible for crop damage Janisiewicz et al, 2002), Janisiewicz and Korsten (2002).  According to Andrew the smaller subset of bacteria and fungi responsible for causing spoilage to the edible portion of the crop plant is the subset of this section.

Spoilage microorganisms can be introduced to the crop on the seed it self during crop growth in the field, harvesting and post harvest handling or storage and distribution some types of soil-borne spoilage microorganisms that are present on harvesting equipment (Amusa and Ashaye, 2009). On handling equipment in the packing house, in the storage facility an on food contact surface throughout the distribution chain. There fore, early intervention measures during crop development and harvesting through the use of good agricultural practice (GAP) will provide dramatic reduction in yield loss due to spoilage at all subsequent steps in the food to fork continuum (Eckert and Ogawa, 200). Examples of GAPS include foliar fungicide application in the field cross-contamination prevention measures (Stringent sanitation standard operating procedures) in the packing house and storage facility and use of post harvest fungicides (Lateef et al, 2006).

2.2     Fungal Contamination of Fruits

The colonization of fungi is a critical phase in the microbial spoilage of fruits (Snowdon, 2001).

The colonization process involves the ability of the microorganisms (Fungi) to establish itself with in the produce (Host). Susceptibility of the fruit and vegetable is largely due to differential chemical composition such as p^h, moisture contents are associated with their greater predisposition to fungal spoilage (Effiuvwerwere, 2000). The contamination of fruit and vegetable by fungi could also be as a result of poor handling practice in food supply chain, storage condition, distribution marketing practice and transportation (Effiuvwerwere, 2000) fungi affecting tomato (Lycopersium esculentus) includes Fusarim oxysporum, Fusarium moniliform, Aspergillus niger and Rhizopus stolonifer, they are responsible for tomato soft rot (Onuegbu, 2002). The percentage incidence and rot shows Rhizopus stolonifer caused the greatest rot in tomato fruits (Chuku,  et al., 2008)

2.3     Un-Usual Characteristics of Spoilage Microorganisms

Many fruits present nearly ideal conditions for the survival and growth of many types of microorganisms. The internal tissues are nutrient rich and many, especially vegetable, have ph near neutrality. Their structure is comprised mainly of the polysaccharides cellulose, hamicelluloses and pectin. The principal storage polymer is starch spoilage microorganisms exploit the host using extra cellular lytic enzymes that degrade these polymers to release water land the plant’s other intracellular constituents for use as nutrients for their growth. Miedes and Lorences (2004) stated that fungi in particular produce an abundance of extra cellular pechnasses and hemicellulases that are important factors for fungi spoilage. Some spoilage microbes are capable of colonizing and creating lesion on health, undamaged plant tissue (Tourna, 2005). Spoilage microorganisms also, can enter plant tissues during fruits development either through the calyxc flower end/or along the stem or through various specialized water and gas exchange structures of leafy matter. Successful establishment however, requires the spoilage microbe to over come multiple natural protective epidermis typically covered by a natural waxy cuticle layer containing the polymer cutin (Lequeu et al, 2003).

Adverse community of epiphytic microorganisms that present a further competitive barrier to the spoilage organisms also typically colonizes the outer most fruit surface. Over coming these barriers requires an exquisite and set of bio-chemical tools that allow the spoilage microorganisms to

                   i.            Identify and recognize the plant surface.

                 ii.            Employ one for more strategies to achieve irreversible attachment to the plant surface

              iii.            Initiate steps leading to internalization of the tissue (Mandrell et al, 2006).

2.4     Spillage of Fruits and Vegetables

One fourth of the harvested fruits and vegetables are spoiled before consumption. Spoilage of fresh fruits and vegetables and fruits reach the consumer as fresh, dried, frozen, fermented, pasteurized, or canned. Contamination may take place during harvesting, handling, transportation or storage unless proper hygienic conditions were not maintained. mechanical damage may increase the susceptibility to decay and the growth of microorganisms which may take plate washing process in contamination water may moisten surface enough to permit entry and growth of organisms. Storage in contaminated containers, use of contaminated dressing materials handling, fly infestation etc will also cause an accelerated rate of spoilage. (Essien et al, 2011),

The deterioration of raw vegetables and fruits may result from physical factors, action or combination of all these microbial spoilage in fruits and vegetable varies not only with the kind of fruits or vegetable but also to some extent with the variety. Microbial spoilage, leaves, flowers or root of the plant on the fruit or other special parts used as food; saprophytic organisms which may be secondary invaders after the action of plant pathogen or may enter a healthy fruit or vegetable as in the case of various rots or grow on its surface, as when bacteria multiply on moist, piled vegetable. At times a saprophyte may succeed the pathogen or a succession of saprophytes may be involved in the spoilage. The most commonly occurring types of microbial spoilage are as follows.

                   i.            Bacterial soft rot: these are caused by Erwinia crtatowa and related species which are fermenters of pectin’s, pseudomonas marginalize, clostridium and Bacillus spp have also been associated with these rots. It results water-soaked appearance, a soft mushy consistency and often a bed odor.

                 ii.            Gray mold rot: these are caused by species of Botrytis

              iii.            Rhizpus soft rot; these are caused by species Rhisopus e.g R. stolonifer. A rot results that often is soften and mushy. The cottony growth of the mold with small black dots of sporangia often covers masses of the foods.

              iv.            Anthracnose: this is usually caused by Colletotrichum lindemothianum, C. coccodes and other species. The defect is a spotting of leaves and fruit or seedpods (Lalieveld et al, 2003).

                 v.            Alternaria Rot: This is caused by Alternaria tenvis and other species areas become greenish – brown early in the growth of the mold and later turn to brown or black spots.

              vi.            Blue mold Rot: These are caused by species of Pencilfium digitations and other species. The bluish – green colour that gives the rot its name results from the masses of spores of the mold

            vii.            Downing mildew: These are caused by species of phytophothora, Bamia and other genera. The molds grow in white, wolly masses.

         viii.            Watery soft rot: This is caused chiefly by Sclerotinia selerotiorum found mostly in vegetables.

              ix.            Stem- end rots: These are caused by species of molds of several genera e.g Diplodia Alternaria, Phomopsis, Fusarium and others, involve the stem ends of fruits

                 x.            Black mold Rot: This is caused by Aspergillus niger. The rot gets its name from the dark brown to black masses of spores of the mold termed “smit”.

              xi.            Pink Rot: This is often caused by species of Alternaria but sometimes of Cera tostomella, Physalospora and other genera

            xii.            Pink Mold Rot: This is caused by pink – spored Trichothecium roseum

         xiii.            Fusarium Rot: This is a variety of types of rots caused by species of Fusarium

         xiv.            Green Mold Rot: This is caused usually by species of cladosporium but sometimes by other green – spored molds e.g  Trichoderma.

            xv.            Brown Rot: This is caused chiefly by sclerotinia (Monilinia fructicola) species.

         xvi.            Sliminess or souring: This is caused by saprophytic bacteria in piled, wet, heating vegetables.  (Priya and Paul 2014).

Fungal spoilage of vegetable soften results in water soaked mushy areas while fungal rots of fleshy fruits such as apples and peaches frequently show brown or cream – coloured areas in which mold mycella are growing in the tissue below the skin and aerial hyphae and spores may appear later. Some types of fungal spoilage appear as “dry roots” where the infected area is dry and hard and often discoloured. Rots of juicy fruits may result in leakage. (Samson,  et al., 2000).

The composition of the fruit and vegetable influences the likely type of spoilage. Thus, bacteria soft rot is widespread for the most part among the vegetables are some what acidic or are fairly dry at surface. Thus, the character of the spoilage will depend on the product attacked and the attacking organism (Chuku  et al., 2008).

CHAPTER THREE

MATERIALS AND METHODS

3.1     Study Area

The study was carried out in Kaduna state; Kaduna state is located in the North West zone of the current six (6) Geo political zones of Nigeria. Samples of spoilt fruit were collected from the market, Rail way market, Kasuwan Barchi, Bakin Dogo market and kakuri market respectively within Kaduna metropolis

3.2     Samples Collection

A total of hundred (100) samples of spoilt fruits were obtained from five (5) selected market 20 samples were collected randomly from each market comprising of 4 pineapples, 4 orange, 4 apples, 4 banana , and 4 water melon were collected each from Kawo market, rail way station market, Kasuwan Barchi  market  and Bakin dogo market. The samples were collected in a clean polythene bag and transported to the laboratory for fungal and bacterial analyses.

3.3     Media Preparation

Sabouraud’s dextrose agar (SDA) and nutrient agar were prepared according to the manufacture instruction and sterilized by autoclaving at 121^oc for 15  minutes to be use for bacteria and fungi isolation respectively.

3.4     Fungal Identification

Segment (1m) of tissue from the margins of the decayed fruits were cut with a sterile scalpel and placed on the sabouraud’s dextrose agar in petri-dishes and incubated at 28+1^oc for 5days for fungus isolation fungal isolates from plates were prepared into mounts on microscopic slides. These were examples under the microscope for comparison of fungal morphology with descriptions given by Samson and Reenen-Hoekstra, (2000).

3.5     Bacterial Identification

The samples were plated out using nutrient agar, Macconkey agar for bacterial identification. These were incubated for 24hours at 37^oc in the incubator. The discrete colonies from the sub cultured plates were identified by series of biochemical test for proper characterization and identification (Sherman N., and Cappuccino J.G., 2005)

3.6     Biochemical Test for the Characterization of Bacteria Isolates

The encountered bacteria were subjected to the following biochemical test according to Manga and Oyeleke, 2004

Catalase Test

Two millimeters (2ml) of 3% hydrogen peroxide solution was poured into a clean test-tube using sterile wooden stick (wire loop) colonies of the test organism were removed and immersed into the test tube containing hydrogen peroxide solution. The presence or absence of bubbles indicates a positive or negative result.

Coagulase Test

Cougulase is an enzymes that clot blood plasma this test is performed on Gram positive, catalase positive species to identify coagulase positive Staphylococcus aureus from other coagulase negative staphylococcus species. Two drop of physiological saline were dropped about 2cm apart on a clean grease free slide and a loop full the organism was emulsified in the physiological saline and a drop of human plasma added to one of the suspension, the other mixture with the palma served as the control, the slide were titled back and forth for one minutes. The presence of agglutination or clumping signifies a positive test.

Indole Test

This test was carried out to determine the amino acid produce as a result of hydrolytic degradation of protein, peptone nad peptides. The organism was grown in a 5ml of peptone water and nutrient broth for 24 hours. After the 24 hours of incubation 8 drop of Kovac indole reagent was added. The test tubes were shaken gently. A positive reaction is indicated by the development of a red colour in the reagent layer above the broth within one minute.

Motility Test

The motility test of the tube, it was then incubated at 35⁰c for 24 to 48 hours, at the end of the incubation the test tube was examined. The test was performed using glucose phosphate broth and culture on peptone broth. The line of inoculation would not be sharply defined and the rest of the medium would be somewhat cloudly if the organisms are motile, if the organisms are motility medium would be somewhat cloudy if the organisms are motile, if the organisms are motility medium was inoculated by making a fine stab with a needle to a depth of 1 – 2cm short of the botton not motile, the growth will be restricted to the line of inoculation which becomes sharply defined and the rest of the medium remains clear.   

                  

         

CHAPTER FOUR

4.0     Results

All the hundred (100) fruits samples obtained from the selected markets within Kaduna metropolis shows the presence of micro organisms, the bacteria species isolated from spoiled apple, fine apple, range, banana and water melon.

Table 4.1: Cultural characteristics of fungi on SDA and microscope

Colour	Microscopic	Inference
Back, green, circular entire flat	Colonies are faster growing black to	Alternaria species
	olivaceous black or grayish, branched acropetal chains of multicelled, conidia are produced sympodically from sample
Grey, green, white circular entire raised	Colonies were fast growing in shades of green sometime while mostly consisting of dense felt of conidioshores nad chains of single cellediconidia are produced in bisipetal succession from a phialide	Penicillium spp
Black, white, yellow, wrinkled undiluted flat	Colonies consist of a compact white or yellow basal felt covered by a dense layer of dark brown to black condial head, conidial head are large, globose becoming radiate and lending to split into severe loose columns	Aspergillus spp
Whitish cotton, lenticular undulate flat	Colonies are usually fast growingpaleor brightly coloured mycelium and are septate	Fusarium spp
White, grey, yellow, irregular lobate pulvinate	Rhizopus, colonies growing fast with pigmented hizoid and sporangiosphores mostly big whitish when young becoming blackish brown with age	Rhizopus spp

Table 4.2: Morphological characteristics of bacteria isolates on nutrient agar (NA)

Macroscope	Gram reaction	Inference
Golden yellow, smooth colonies	Purple, cocci in cluster gram positive	Staphylococcus aureus
Yellowish white smooth colonies	Gram negative/pink short rod	Escherichia coli
Smooth round colonies with greenish colour	Gram negative short rods	Salmonella typhimurium
Pink mucoid raised colonies	Gram negative short rod	Klebsiella pneumonia
Smooth round colonies with greenish colour	Gram negative short rods in pairs	Pseudomonas aeruginosa

Table 4.3: Percentage of fungal and bacterial isolates from fruit samples

Location	Fruits samples	No of sample	Fungal  isolates	Percentage of isolates	Bacteria isolate	Percentage of isolate
Bakin Dogo market	Apple Orange Watermelon Pineapple Banana	4 4 4 4 4	4 - 5 6 8	4 0 5 6 7	2 5 1 2 4	2 5 1 2 3
Kawo market	Apple Orange Watermelon Pineapple Banana	4 4 4 4 4	- 3 5 1 4	0 3 5 1 4	7 6 1 3 5	6 5 1 3 5
Kasuwan Barchi market	Apple Orange Watermelon Pineapple Banana	4 4 4 4 4	7 6 4 8 5	7 6 4 7 5	- - 10 8 12	0 0 9 7 10
Railway station market	Apple Orange Watermelon Pineapple Banana	4 4 4 4 4	7 4 - 7 6	7 4 0 7 6	4 11 5 3 2	3 9 5 3 2
Kakuri market	Apple Orange Watermelon Pineapple Banana	4 4 4 4 4	5 1 2 3 5	5 1 2 3 5	9 6 3 8 -	8 5 3 7 0

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1     Discussion

A total of 100 spoilt fruit samples were examined for the presence of micro organisms from the result obtained, the entire sample analyzed showed the presence of micro organisms, all the bacteria isolated in this study have previously been isolated from fruits and vegetable in other studies both in Nigeria and elsewhere (Adebolu and Hesan, 2001, Omemu and Bankole, 2005, Tambekar and Mundhada, 2006, Uzeh  et al., 2009). The high percentage of bacteria and fungi in this study are similar to those obtained in other studies in Nigeria (Uzeh  et al., 2009; Bukar,  et al.,  2010).

The micro organisms present in fruits are a direct reflection of sanitary quality of the cultivation water, harvesting, transportation, storage and processing of the produce (Bhunia, 2007).

Certain fungi such as Aspergillus, Fusarium and Penicillium spp are commonly occurring filamentous fungi and their growth may result in production of toxins known as mycotoxins, which can cause a variety of all effect in human from allergic responses to immunosuppression and cancer (Pitt, J. I A. D. Hocking, 2006).

The bacterial isolates identified in this study include Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcos aureus, Salmonella typhimurium. This is consistent with the findings of previous studies. The bacteria found to spoil the fruit were Klebsiella, Bacillus, Escherichia coli, Staphylococcus, Pseudomonas, Salmonella and Lactobacillus (Asok, Kumar, Vrun Bhushan, 2011). And Nwachukwu E. and Chukwu, C. M., 2013).

Presence  of E. coli indicates recent contamination by feacal matter and possible presence of other entric pathogens known to be causative agents of food borne gastro enteritis and bacterial diarrhoeal disease (Jiwa, S. F. H., Kiovacek, K., Wadstorm, T., 2011).

A number of studies from different countries have shown the presence of E. coli, coliforms and a variety of micro organisms like streptococcus pyogenes, straphylococcus spp, micro coccus spp e.t.c, (Nichols, G., Gillespie, I and D. Louvois J. 2000),  (Lateef, A., Oloke, J. K., Kana E. B and Pacheco, E. 2006) (Amusa, N. A., Ashaye, D. A., 2009).

5.2     Conclusion

Fruits are very important and have high dietary and nutritional qualities. They can also be used in the preparation of jellies, juice, jams and fruit salad.

Micro organisms are naturally present on all food stuff and can also be brought in by outside elements (wind, soil, water, insects, animals, human handling). They can become contaminated during growing, harvesting and transport of the raw materials and/or processing into edible products (Lelieveld, H. L. M., Mostert, J., Holeh and B. White, 2003).

The prevalence of some fungi and bacteria demand that appropriate control measures should be employed if farmers expect a beneficial outcome of their product. Adequate microbiological knowledge and handling practices of these products would therefore help to minimize wastes due to deterioration of fruits.

Proper measures should be adopted while handling fruits to limit the level of microbial contamination. It is therefore important that the farmers and stakeholders properly transport the fruits into bags, the marketers and consumers take necessary precaution to prevent contamination and also try to create an environment that will not encourage the growth and multiplication of micro organisms. This study has shown that micro organisms causing diseases are present in the decaying fruits and this is of public health importance.

5.3     Recommendation

It is recommended that proper washing of these fruits is essential before consumption. Regulation monitoring of the quality of fruits for human consumption should be introduced to avoid any bacterial pathogen outbreak. Handling and storage quality should also be improved.

A similar can be done on the microbiology quality of fruits.

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