EFFECT OF PAWPAW LEAVES EXTRACTS ON

BACTERIAL ISOLATES GOTTEN FROM POND WATER

BY

DANIYAN OLUWABUNMI LYDIA

SO4/SLT/17/2815

SUBIMTTED TO:
DEPARTMENT OF SCIENCE LABORATORY
TECHNOLOGY (MICROBIOLOGY OPTION), APPLIED
SCIENCES, RUFUS GIWA POLYTECHNIC, OWO.
ONDO STATE.
 CHAPTER ONE

1.0 INTRODUCTION

1.1. BACKGROUND INFORMATION

The Carica papaya (pawpaw) is a member of the small family “Carcaceae” allied to the

“Passifloraceae”. The family comprises of Jacarata, cyclicmorpha and carica genera (Oluma

et al; 2010). The only genus having species cultivated for their fruits is Carica. Until

recently, the Caricaceae was thought to comprise 31 species in three genera (namely; Carica,

Jacaratia and Jarilla) from tropical America and one genus, Cylicomorpha, from equatorial

Africa. However, a recent taxonomic revision proposed that some species formerly assigned

to Carica were more appropriate classified in the genus Vasconceilla (Badillo, 2002).

According, the family’s classification has been revised to comprise Cyclicomorpha and five

South and Central American genera (Carica, Jacaratia, Jarilla, Horovitzia and Vasconcella),

the Carica papaya the only species within the genus Carica (Badillo, 2002).

Although opinions differ on the origin of C.papaya in tropical American. It is likely that

C.papaya originates from the lower lands of eastern Central American, from Mexico to

Panama. Its seeds were distributed to the Carribean and south-east Asia during Spanish

exploration in the 16th Century, from where it spread rapidly to India, the pacific and Africa

(Villegas, 2000). Carica papaya is now grown in all tropical countries and many subtropical

regions of the world. It is one of the tropical plants grown regions of the world. It is one of

the tropical plants grown all over West African mainly for the local consumption. Caica

papaya is a soft-wooded perennial plant that lives for about 5-10 years, although commercial

plantations are usually replanted sooner (Chay-Prove et al; 2000). Fruit are ready to harvest

five to six months after flowering, which occurs five to eight months after seed germination.
The fruits range in size from 7- 30 cm long and vary in mass from about 250 to 3000g (Maffa

et al; 2003).

It is an interesting tree in that the male and female parts exist in different trees. Pawpaw

fruits, apart from being taken as food also have some medicinal importance. The fruits,

leaves, seeds and latex are used medicinally. Its main medicinal use is as a digestive agent; it

is prescribed for people who have difficulty digesting protein and is used to break up blood

clots after surgery, which is due to the presence of enzyme papain in the plant’s latex. The

latex from the trunk of the tree is also applied externally to speed the healing of wounds,

ulcers, boils and warts. The seed is used to expel worm and the flower may be taken in an

infusion to induce menstruation (Oduola et al; 2007). It has also been that reported that

annonaceous acetogenins derived from the extracts of the twigs of the pawpaw tree may be

good chemotherapeutic agents for cancer as these compounds inhibit enzymes necessary for

metabolic processes in tumour cells. Increasing interest in medicinal herbs has increased

scientific scrutiny of their therapeutic potentials and safety thereby providing physicians with

data to help patients make wise decisions about their use (Oduola et al; 2007).

Economically, Carica papaya is the most important species within the Caricaceae, being

cultivated widely for consumption as a fresh fruit and for use in drinks, jams, candies and as

dried and crystallised fruit. Green fruit and the leaves and flowers may also be used as a

cooked vegetable (Watson, 2008). Nutritionally, papaya is a good source of calcium and an

excellent source of vitamins A and C. Papaya also has several industrial uses. Biochemically,

its leaves and fruit are complex, producing several proteins and alkaloids with important

pharmaceutical and industrial applications (Moussaoui et al; 2001). Of these, however,

papain, is a particularly important proteolytic enzyme that is produced in the milky latex of

green, unripe papaya fruits (note that ripe papaya fruit contain no latex or papain). The latex

is harvested by scarifying the green skin to induce latex flow, which is allowed to dry before
collection for processing. Evolutionarily, papain may be associated with protection from the

frugivorous predators and wool and in the cosmetics industry, in soaps and shampoo.

The pawpaw fruit is a fleshy, juicy fruit usually green but turning yellow when ripe. The fruit

consist largely of water, sugar, vitamins A and C, protein and ash. It is one of the most

nutritious and cheapest fruits grown and consumed in Nigeria. The fruit can be freshly eaten

or cooked (Baiyewu, 2010). In the Southern part of Nigeria, pawpaw fruit production has

improved the diet of the local people, whose diet generally consisted of starch staples lacking

essential vitamin and minerals. These pawpaw fruits were usually displayed on benches and

in baskets for prospective customers in the open markets until sold, thereby exposing them to

further microbial infection (Baiyewu et al; 2007), beside those associated with the fruit

surface and those from adjacent infected fruits.

Pawpaw need a light, well-drained soil. They are easily killed by excess moisture. The soil

needs to be moist in hot weather and dry in cold weather. Since this is the opposite of

California's rain pattern, in addition to good drainage, plastic coverings to prevent

overwetting in winter may also be worthwhile. Pawpaw do not tolerate salty water or soil

requires a well-drained soil with high organic matter. Does not tolerate flooding even for

short duration. Recommended in regions with well distribution rainfall throughout the year

without flash floods, water-logging and strong winds (Agrolink, 2002). While doing best in

light, porous soils rich in organic matter, the plant will grow in scarified limestone, marl, or

various other soils if it is given adequate care. Optimum pH ranges from 5.5 to 6.7. Overly

acid soils are corrected by working in lime at the rate of 1-2 tons/acre (2.4- 4.8 tons/ha). On

rich organic soils the pawpaw makes lush growth and bears heavily but the fruits are of low

quality. It is recommended that no herbicides used due to the little woodyness of the pawpaw

tree stem which may end up damaged. Usually weeding is performed manually or
mechanically-aided, deep soil disturbances can damage the root system. It is advisable to use

black polyethylene film on the surface to avoid weed growth (Infoagro, 2002).

1.2 Objectives

This study was therefore initiated to investigate the effects of;

1. To determine the efficacy of pawpaw leaves extract on bacterial isolates gotten from pond

2. To access the photochemical components present in pawpaw that are responsible for its

antimicrobial features

3. To evaluate the type of bacterial infection that humans are susceptible to when they come

in contact with pond water

 CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 General Description and Origin of pawpaw
Pawpaw (Carica papaya L) is the most economically important fruit in the Caricaceae
Family. In some parts of the world, specially Australia and some islands of the West Indies, it
is known as pawpaw, names which are better limited to the very different, mainly wild
Asimina triloba Dunal, belonging to the Annonaceae. While the name pawpaw is widely
recognized, it has been corrupted to kapaya, kepaya, lapaya or tapaya in southern Asia and
the East Indies. In French, it is papaye (the fruit) and papayer (the plant), or sometimes
figuier des Iles. Spanish-speaking people employ the names melón zapote, lechosa, payaya
(fruit), papayo or papayero (the plant), fruta bomba, mamón or mamona, depending on the
country. In Brazil, the usual name is mamao. When first encountered by Europeans it was
quite naturally nicknamed "tree melon". (Morton, 2010).

The pawpaw is believed to be native to southern Mexico and neighboring Central America. It
is currently cultivated in Florida, Hawaii, Eastern British Africa, South Africa, Sri-Lanka,
India, Canary Islands, Malaysia and Australia. It is now present in every tropical and
subtropical country. Pawpaw was first described in 1526 by the Spanish chronicler Oviedo,
who found it first on Panamanian and Colombian coasts. The fruit was rapidly propagated in
the tropics, most likely due to the abundant and highly viable seeds. The crop has adapted
quite well to tropical areas with fertile soils and abundant rainfall. The history of pawpaw
spread was initiated approximately in 1500, when the Spanish conquerors carried seeds to
Panama and Dominican Republic. During the following century Spanish and Portuguese
sailors took the seeds to the Phillipines, Malaysia and India. For 1600 the fruit had been
produced in warm regions of South and Central America, Southern Mexico, the Antilles,
Bahamas, Bermuda y Florida. In the same century pawpaw seeds were taken from India to
Naples in Italy. The crop reached Hawaii between 1800 and 1820. Until 1900, pawpaw seeds
were taken to Florida, probably from Bahamas' plantations. The Solo variety has been
cultivated in Hawaii since 1911, probably brought from Barbados and Jamaica. The first
seeds of the Maradol variety were introduced into Mexico in 1978, through CONAFRUT, in
Xalapa, Veracruz (CRFG, 2012).

2.2 Cultivation of pawpaw

Pawpaw are well adapted to many soil types, like to be warm with both sunshine and
reflected heat, so the hottest place against the house where nothing else seems happy is an
ideal location. They also like to be as free from wind as possible, although this is not as
critical as their need for sun. Pawpaw can be grown successfully in shade, but the fruit is
rarely sweet. They are best planted in mounds or against the foundation of a building where
water can be controlled (CRFG, 2012)
Pawpaw need a light, well-drained soil. They are easily killed by excess moisture. The soil
needs to be moist in hot weather and dry in cold weather. Since this is the opposite of
California's rain pattern, in addition to good drainage, plastic coverings to prevent
overwetting in winter may also be worthwhile. Pawpaw do not tolerate salty water or soil
(CRFG, 2012)

Requires a well-drained soil with high organic matter. Does not tolerate flooding even for
short duration. Recommended in regions with well distribution rainfall throughout the year
without flash floods, water-logging and strong winds (Agrolink, 2002).

While doing best in light, porous soils rich in organic matter, the plant will grow in scarified
limestone, marl, or various other soils if it is given adequate care. Optimum pH ranges from
5.5 to 6.7. Overly acid soils are corrected by working in lime at the rate of 1-2 tons/acre
(2.44.8 tons/ha). On rich organic soils the pawpaw makes lush growth and bears heavily but
the fruits are of low quality (Morton, 2010).

It is recommended that no herbicides used due to the little woodyness of the pawpaw tree
stem which may end up damaged.

Usually weeding is performed manually or mechanically-aided, deep soil disturbances can

damage the root system. It is advisable to use black polyethylene film on the surface to avoid
weed growth (Infoagro, 2002).

Cultivation: To grow pawpaw successfully, the grower should make sure that the following
requirements are met.
The seed should preferably come from controlled crosses (female x bisexual or bisexual self,
or from bisexual trees known to have a high degree of self-pollination under field conditions.
The type of seed used is most important since it determines production potential and thus
restricts or enhances the value of other cultural methods

The soil should have good drainage. Pawpaw is very sensitive to even short periods of
flooding. If not killed, they lose their vigor and regain it very slowly. Irrigation should be
provided during dry spells because a fluctuating water supply may cause growth retardation,
flower abortion, and dropping of young fruits.

Pawpaw is a fast growing plants, which require an abundant supply of nutrients if they are to
be highly productive. Supplemental fertilization is particularly important in infertile soils.
Nitrogen and phosphorus are especially important. In Florida, young plants should be started
with 1/2 lb of a 10-10-10-5 mixture (with 30% of the nitrogen from natural organic sources)
at planting time or shortly after. This amount of fertilizer is applied every 2 weeks and should
be increased gradually to 1 1/2 lb until the plants are 7 to 8 months old. Thereafter, monthly
applications should be sufficient, unless unusually hard rains occur. Fertilizer should be
reapplied under these conditions.

2.3 Cultural practices of pawpaw

Seeds retain their viability for 2-3 years when kept air-dry in airtight containers. Seeds may
be sown in cold frames or boxes during January or in the open in March. Early planting is
much to be desired to make a vigorous plant before the beginning of following winter. Seeds
germinate in 2-3 weeks. When 2 or 3 true leaves have formed, seedlings should be
transplanted, spacing them 5-7.5 cm apart in seedbed. When plants are 7.5-10 m tall, they can
be set in their permanent places in the field. Usual planting distance is about 3-4 m apart each
way, giving about 1750 trees to the hectare. In selecting plants for field planting, the more
vigorous growing plants are usually the males and may be safely discarded except for a few.
By planting 2 or 3 plants in a hill, there is a chance for further selection and elimination of
excessive males when first flowers appear, about one male plant to each 25 or so females is
sufficient. Transplants must be watered and shaded. Mulch gives much better results than
clean culture, keeping down weeds, preserving moisture, shading the soil from hot summer
sun, and preventing the burning out of humus and nitrates in the top soil layer. Heavy
applications of stable manure or commercial fertilizers can often be used with profit.
Attempts at grafting and rooting shoots have not been successful on a commercial scale
(Morton, 2010).

Culture: Seeds may be planted directly in the field, or seedlings raised in beds or pots may be
transplanted when 6 weeks old or even up to 6 months of age, though there must be great care
in handling and the longer the delay the greater the risk of dehydrated or twisted roots; also,
transplanting often results in trunk-curvature in windy locations.

Small bottom perforated plastic containers can be used for seed. The small holes in the
bottom of plastic containers avoid the humidity inside of the containers.

The fertilizer is added to the containers with pawpaw seeds in order to obtain the
germination. This kind of containers offers advantages like: easy handling, easy release of the
plant and recycle containers.

2.4 Preparation and uses of pawpaw

Green, or unripe, pawpaw may be cooked like winter squash. Ripe pawpaws are terrific eaten
plain or with a dollop of yogurt. Simply cut in half and remove the seeds; the flesh is tender
enough to be scooped out with a spoon. Although pawpaw skin is not edible, its seeds are.
Pawpaw seeds resemble large peppercorns and also have a peppery taste. They may be
crushed and sprinkled on salads in the same way as crushed peppercorns. Left whole, they
make an interesting garnish for a fruit salad. Add pawpaw to fruit salads or pouree into a
thick juice (Healthnotes, 2001).

Unripe and mature pawpaw fruits

Pawpaw fruits should be harvested when the colour of the skin changes from dark green to
light green and when one yellow streak begins development from the base upwards. Fruits in
this condition will continue to ripen normally after harvest. Those fruits harvested before this
stage will fail to show complete ripening, and those harvested after, are more susceptible to
damage and bruising during handling (Medlicott, 2001).

2.5 Plant Extract

Majority of people on this planet relies on material medical (medicinal plants and other
materials) for their everyday health needs. 80% of the world population relies on plant
derived medicine for their health care (Gurib-Fakim, A., 2006). Plant derived medicine can be a
base for the development of new drugs (S.O. Asante, 2006). Plants contain a broad range of
bioactive compounds such as lipids, phytochemicals, pharmaceutics, flavors, fragrances and
pigments. Plant extracts are widely used in the food, pharmaceutical and cosmetics industries.
Extraction techniques have been widely investigated to obtain such valuable natural
compounds from plants for commercialization. However, the effect of conventional methods
of extraction: Soxhlet extraction method (Reuben, K.D, 2008) and cold methods on
antimicrobial assay has not been documented. This paper investigated the possible effect of
these methods of extraction on antimicrobial susceptibility testing of plant using Anchomanes
difformis (Blume) Engl. as a case study to maintain an appropriate standard of extraction
method in antimicrobial assay. Lack of such standard could lead to different results on
antimicrobial assay making it difficult to reproduce research work.

2.5.1 Use of plant-based extract

Farmers were using chemicals and synthetic compounds against bacteria to control it.
Unfortunately, these chemicals are not readily biodegradable and tend to persist for years in
the environment leading to the development of new physiological races of the bacteria
(Dwivedi and Neetu, 2012). This led to the urgent need for development of plant extract that
are more effective, economically feasible and ecofriendly than the conventional fungicides.
Recent efforts have focused on the development of environmentally safe, long-lasting, and
effective bio-control methods for management of anthracnose diseases. The utilization of
natural products, especially the plant extracts, has been has been shown to be effective
against many plant bacterial and considered to be safe for consumes and environments.

2.6 Pond water

Ponds are usually by definition quite shallow water bodies with varying abundances of
aquatic plants and animals. Ponds are frequently manmade or expanded beyond their original
depths and bounds by anthropogenic causes. Apart from their role as highly biodiverse,
fundamentally natural, freshwater ecosystems ponds have had, and still have, many uses,
including providing water for agriculture, livestock and communities, aiding in habitat
restoration, serving as breeding grounds for local and migrating species, decorative
components of landscape architecture, flood control basins, general urbanization (Keddy,
Paul A. 2010).

2.6.1 Economic benefits of a water pond

Economic benefits of a pond can be substantial depending upon use. Over time, the pond can
provide an income far in excess of construction costs. However, for many pond owners,
recreation or aesthetic pleasure and not income are the prime reasons for building a pond.
With knowledgeable management economic and recreational benefits can be enhanced and
sustained for many years. Benefits to the pond owner or land owner considering construction
of a pond include:

1. Irrigation

Ponds provide water to irrigate commercial crops, orchards and home gardens at reduced cost
to the land owner when compared to costs of pumping water from deep wells or buying it
from rural water districts. The Oklahoma Water Resources Board

regulate surface water use in our state and they indicate that, "In the state of Oklahoma all
surface water is considered the property of the state. It is different with ground water which is
considered a property owner's right. For all uses of surface water other than domestic, the
user must obtain a water use permit from the Oklahoma Water Resources Board. Domestic
use is limited to household, irrigation up to 3 acres and livestock up to the carrying capacity
of the land.

2. Swimming And Recreation

Many land owners use their ponds primarily for recreational activities, picnics, barbeques and
swimming. Sand beaches for swimming are easily constructed and maintained. Livestock
should not have direct access to ponds designed for swimming. Animal waste fouls water
with excess nutrients and creates noxious algal blooms that detract from the desired
recreational pleasures. A properly designed and managed pond can provide clean water and
beaches for swimming and an attractive landscape for outdoor activities.

3. Sport Fishing

Sport fishing is the most sought after benefit of most pond owners. However, many pond
owners are disappointed in their catch and become frustrated or lose interest in angling.
Without effective pond management, sport fishing is almost certain to decline in most ponds,
unmanaged fish ponds in Oklahoma often have few catchable fish and studies have found that
some ponds may contain as few as 57 total pounds of fish in the pond. Fish are often stunted
and many undesirable species can be present. Sport fishing can be improved both in total
pounds of fish caught and in size and numbers of fish caught. Sport fishing improvement is
obtained by managing fish, water, nutrients and the watershed. A well-managed sport fishing
pond can produce 500 or more lb/acre of high quality sport fish annually if proper
management strategies are followed. A new or newly renovated pond can produce catchable
sized channel catfish in one year and catchable pan fish and bass in 3 years. Contact your
state Fisheries Extension Specialist for detailed information about fish pond management
techniques tailored to your goals.

4. Commercial Aquaculture

Commercial Aquaculture Ponds that are suitable or drainable may have potential for
commercial aquaculture production. Many watershed ponds have been used to raise fish for
sale. Profit can range from $200 / surface acre of water or more depending on species and
stocking densities.

Small ponds are most profitable when used to raise high value fish. Fingerlings for farm pond
stocking are always in demand and bring more profit per acre to the small producer than the
production of commodity food or bait fish such as the channel catfish or golden shiner. Blue
gill, hybrid blue gill, largemouth bass and channel catfish fingerlings have the most potential
for small - scale production. Koi carp, aquarium fish and aquatic plants also can be raised
profitably in many ponds. Larger production potential can be achieved by renting suitable
ponds within your local area. Rent rates should be no more than $25 - $100/ acre. A rented
pond should have easy access but not of course, open to public fishing. Theft is always a
possibility and that makes obscure locations best. Automatic fish feeders can be used to
reduce travel frequency to rented ponds.

With sufficient ponds, commercial catfish production is possible, usually on a relatively small
- scale that makes direct marketing of fish the only profitable alternative. Quantity sales can
be made direct to individuals through advertising or word of mouth.

2.6.2 Micro-organisms found in the pond water

There are many thousands of types of bacteria and microorganisms found in aquatic
ecosystems, including your personal pond. The main aquatic groups include arthropods,
bacteria, protozoa, hydras, fungi, phytoplankton, and algae:
1. Arthropods include micro and macro invertebrates such as tardigrades (water
bears), crustaceans, some insect larvae, and water mites among others, and most
are visible without the use of a microscope. These unique and varied organisms
serve a variety of functions – food for other creatures, such as fish; soil aeration;
consumption of other microorganisms, like algae, and potentially problematic
creatures like mosquito larvae (though mosquito larvae are themselves considered
arthropods); and decomposition of organic matter.
2. Bacteria, as you likely know, there are good bacteria and bad bacteria. While this
group often gets a bad rep, ecosystems and the organisms within them (including
us) would die without them. They are among the tiniest and oldest
microorganisms on Earth, and can be found virtually everywhere – from a tiny
drop of rain to the cap of an ink pen. They are able to multiply exceedingly
quickly, making them exceptional at aiding in decomposition in ecosystems and
thus cycling nutrients. Specifically, there are heterotrophic bacteria and
autotrophic bacteria. The former are the ones responsible for decomposition and
nutrient cycling (and can also help to control algae populations by reducing the
nutrients that are available to them), while the latter are typically known as
cyanobacteria (often misleadingly called blue-green algae even though they are
not actually algae).
3. Protozoa are microscopic, unicellular organisms that resemble animals in that they
must consume other organisms in order to obtain the nutrients that they need to
live. For example, bacteria help break down organic matter, and protozoans in
turn feed on both the bacteria and the detritus (and in turn are eaten by
zooplankton, which in turn are consumed by invertebrates, which are in turn
devoured by fish, and so on). Without protozoans (which consume as much as
50% of bacteria), bacteria would be able to build up to potentially harmful levels,
and also without protozoans, an exceedingly critical link in the food web would be
removed, severely crippling the ecosystem as a whole and limiting the plants and
animals that would be able to survive in it. However, are parasitic and can cause
diseases such as giardia. Overall, though, having protozoa in your pond is a very
good and necessary thing without them, your pond ecosystem would have a very
difficult time functioning.
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