Evaluation of Parasitic Body in Mango Fruit

Evaluation of Parasitic Body in Mango Fruit

Chapter One

Aims And Objectives

To determine the parasite composition of mango fruit
To determine whether washing mangoes with untreated water eliminates the parasites (pathogenic parasites) from them.

CHAPTER TWO

LITERATURE REVIEW

Parasites are organisms which have adapted themselves in or on another organism which is called a host, and lives at the expenses of the tissue and fluid of the host deriving their nutrient and protection from the host, thereby harming or being of no advantage to the host. (Crew, 1999).

They increase their fitness by exploiting host for food, habitat and dispersal. Parasites may be transmitted from animals to humans, from humans to humans, or from humans to animals. Several parasites have emerged as significant cause of food borne and water borne disease in the whole world.

This is achieved through consumption of contaminated food and water or by eating any raw fruits that has been contaminated with water or food. Parasites are of different types and ranges in size from tiny-single-celled, microscopic organisms (protozoa) to large multi-cellular worms, (helminthes) that may be seen with a microscope.

Some of the parasites are

Giardia lamblia (intestinalis) cryptosporidium parvum, Cyclospora-cayetanesis, Toxoplasma gonadii, and Trichnella Spiralis.

duodenalis formally called g.

Lamblia causes Giardiasis.

It is one celled microsiopic parasites that can live in the intestine of animals and people.

It is found in every region throughout the world and may cause chronic diarrhea, malabsorptoin, weight loss with symptoms for several months. (Hill, 1993). Giardiaisis is mainly acquired by transmission of cysts of G. Intestiinalis via soiled hands, contaminated with faeces (pentersen, 1988). Consumers get this disease by consuming food or water – contaminated with G. duodenalis cysts (infective stage of the organism) and by putting anything into the mouth that has touched the stool of a person or animal with giardiasis. This occur usually 1-2 weeks after ingestion of G. duodenalis cysts, which is the environmental survival form and infecting stages of the organism but may last for 4 to 6 weeks in healthy person.

There are cases of chronic illness lasting months or even years. People exposed to public places including those with HIV/AIDS infection are at risk for contracting giardiasis. (World Health Organization [WHO],2003).

Giardiasis can be prevented by washing hand with hot, soapy water before handling foods and eating, and after using the toilet diapering young children, and handling animals also by making sure infected individuals wash their hands frequently to reduce the spread of infection, and drinking water only from the treated municipal water supplies. When traveling to countries where the water supply may be unsafe to drink, either avoid drinking the water or boil to kill parasites. Drinking bottled beverages or hot coffee and tea are safe alternative. Do not swallow water while swimming especially in community pools where there might be people’s child or people suffring giardiasis. Drink only pasteurizes milk, juice or cider. Wash, peel, or cook raw fruits and vegetables before eating. Do not use untreated manure to fertilize fruits and vegetables.

Giardiasis is more prevalent in children than in adults, possibly because many individuals seem to have a lasting immunity after infection and is implicated in 25% of the cases of gastrointestinal diseases and may be present asymptomatically about 40% of those who are diagnosed with giardiasis demonstrate disaccharide intolerance during detectable infection and about six months after, the infection can no longer be detected. (John and Wily,1999).

Chronic cases of giardiasis in immunodeficient and normal individuals are frequently retractile to drug treatments (Nichole and Smith, 2002). Five outbreaks have been traced to food contamination by infected or infested food handlers, and the possibility of infections from contaminated fruits and vegetables that are eaten raw cannot be excluded.

Cryptosporidiosis; it is a celled microscopic parasite, and a significant cause of water borne illness worldwide. It is found in the intestine of many herd animals people get cryptosporidiosis by

Consuming food or water contaminated with parvum oocysts, (infective stage).The oocysts is the environmental resistant stage of the organism and are shed in the faeces of a host into soil.
By putting anything into the mouth (faecal oral route) that have touched stool of infected person. The symptoms of this disease are watery diarrhea, stomach cramp, upset stomach and light fever. Some case may be without symptoms. Symptoms appear to ten days after ingestion of parvum oocysts. The illness usually goes away without medical intervention in three to four days. But in some outbreaks in day care centres diarrhea has lasted one to four weeks. In people with weakened immune system, cryptosporidiosis can be serious, long lasting and sometimes fatal. However, there is no known effective drug or medication whatsoever for the treatment of cryptosporidiosis (Millard, Gensheimer and Addis, 1994).

CHAPTER THREE

MATERIALS AND METHOD

Preparation of spore suspensions

The mould Penicillium expansum used in this study was kindly provided by the ‘Instituto Nacional de Engenharia e Tecnologia Industrial’, in Lisbon, from its culture collection (strain 310 isolated from apple juice). In order to produce suﬃcient mature spores, P. expansum was inoculated into slants of potato dextrose agar (PDA) and incubated at 25–28 °C for 5 days. Spores were harvested from the slants by ﬂooding with 5–6 ml of sterile deionised water containing 0.1% Tween 80 and collecting the aliquots to an Erlenmeyer ﬂask, until 20–30 ml were obtained. A 10 min sonication treatment was followed by ﬁltration of the suspension through sterile glass wool to remove hyphae (branching formations in fungi). Microscopic examination of the ﬁltrate revealed free spores. The suspension was then diluted to give spores concentrations of 10⁵/ml and 10⁴/ml, measured by using a Newbauer chamber for counting spores.

Preparation and Inoculation of mango juice

Ripe edible mangoes of the cultivar Haden grown in Brazil and obtained from a commercial source were used for this study. The fruits were washed, peeled and their pulps macerated in a domestic juice extractor. The resulting puree was centrifuged (at 15000 r.p.m. during 15 min) to give the juice. Aliquots of 20.0 ml were poured into 22 sterile bottles of 100 ml, which were treated and stored for 132 h (5.5 days) in the following conditions: one group of six bottles was stored at 25 °C without any treatment, in order to follow the changes occurring during parasitic contamination of the juice, while the remaining 16 bottles were heated in a water bath at 80 °C during 15 min, in order to inactivate enzymes and to eliminate most of the juice natural microﬂora (Ejechi, Souzey, & Akpomedaye, 1998), so that the effects of inoculation could be investigated separately. Ten bottles of heated juice were inoculated with spores of P. expansum: ﬁve bottles were inoculated with 0.500 ml of suspension containing 10⁵ spores per ml, and ﬁve bottles were inoculated with 0.500 ml of suspension containing 10⁴ spores per ml, to give spores concentrations in the juice of about 2500/ml (batch A) and 250/ ml (batch B), respectively. The remaining six bottles of heated juice were stored at 25 °C and used as control samples.

CHAPTER FOUR

RESULTS AND DISCUSSION

Eﬀect of parasitic contamination

Figs. 1(a), 2(a) and 3 show, respectively, the highﬁeld, mid-ﬁeld and low-ﬁeld regions of the 1D spectra of natural juices, freshly prepared (time 0), and after storage during 132 h. Many diﬀerences may be observed in the whole spectral range, reﬂecting drastic changes in the juice composition (Table 1). In order to aid spectral interpretation, the information provided by 1D and 2D TOCSY spectra was combined and resonances were assigned by comparison of ¹H chemical shifts and spin– spin coupling constants with reference spectra of some commercial standards and literature values of metabolites commonly found in biological and food samples (Nicholson, Foxall, Spraul, Farrant, & Lindon, 1995; Fan, 1996; Gil et al., 2000). The pH of the samples was seen to decrease from 5.1 to 4.1 at 132 h, whereas soluble solids showed a slight increase from 14% to 16%. In the high-ﬁeld region (Fig. 1(a)), prominent increases are detected for several signals, namely a doublet at 1.14 ppm (isopropanol or 2,3-butanediol), a doublet at 1.32 ppm (lactic acid), singlet at 1.92 ppm (acetate), signals at 1.37 ppm and 2.22 ppm (acetoin), and singlet at 2.41 ppm (succinic acid). The relative variations of these compounds were measured by dividing the areas of each signal at diﬀerent storage times (A_t) by the area measured at time 0 (A₀). Absolute quantiﬁcation was not carried out because, although the same amount of TSP was added to all samples, the area of its signal showed signiﬁcant variations probably due to physical and/or chemical interactions with the juice components. As shown in Fig. 1(b), the largest increase is registered for isopropanol/2,3-butanediol (100· at 84 h). Acetate increases 25· at 132 h, while lactic acid, acetoin and succinic acid increase, respectively, 18·, 10· and 6· at 84 h. All these compounds are typical products of diﬀerent types of fermentation undergone by microorganisms present in the juice. In addition, alcoholic fermentation, typical of yeasts, does not seem to be important here since the amount of ethanol does not increase.

CHAPTER FIVE

CONCLUSIONS

High resolution ¹H NMR showed promising results in the detection of indicators of mango juice degradation, caused either by parasitic contamination or by deliberate contamination with the mould Peniccilium expansum.

Besides the utilization of the main sugars and the formation of typical fermentation products (namely acetate, lactic acid, acetoin and isopropanol/2,3-butanediol), changes in organic acids, amino acids, and less abundant components such as oligosaccharides and aromatic compounds were viewed. In particular, the naturally spoiled juice showed the unexpected formation of 3,4,5-trihydroxycyclohexane carboxylic acid (or a similar derived compound) which is, to our knowledge, a newly found product, possibly resulting from quinic and shikimic acids catabolism. In the P. expansum contaminated juice, speciﬁc changes regarded the utilization of citric and malic acids and the formation of a few minor compounds, still unassigned at this stage.

The practical use of the above changes as early indicators of mango juice parasitic contamination or P. expansum contamination requires further work involving the collection and analysis of samples at shorter intervals prior to 36 h of storage, and systematic comparison of the NMR timescale with that of selected alternative methods. Nevertheless, compared to the typical 3–5 days required by traditional plating procedures, it is clear that high resolution ¹H NMR enables earlier detection of the juice compositional degradation. This result, along with the ability to allow for structural characterisation of novel, unexpected compounds enlightening speciﬁc aspects of fruit biosynthesis, shows the potential usefulness of NMR in the area of food microbiological control.

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