CHAPTER ONE

1.0 INTRODUCTION

Fruits are very important components of the diet. According to this definition, fruits are a

sub-set of vegetables, as the term fruits refer to the mature ovary of a plant which encloses

the seed (International Agency for Research on Cancer, 2003).

Fruits are, in general, low in calories and fat but high in vitamins, minerals and dietary fiber.

Vegetables are categorized into five subgroups, based on their nutrient content and include:

dark green, starchy, orange, dried beans and peas, and other vegetables (Turcotte,

2010).Vegetables are sources of many nutrients, especially potassium, folate, the antioxidant

vitamins A and E, and dietary fiber. These nutrients help support body function in many

ways, which makes vegetables important components of a healthy diet. For example,

potassium helps to maintain healthy blood pressure, folate (folic acid) helps with red blood

cell production, vitamin A enhances immune function, and vitamin E protects cells from free

radicals. Major nutrient contributions of fruits include potassium, folate and dietary fiber.

Where vegetables are an excellent food source of vitamins A/beta-carotene and E, fruits offer

more substantial amounts of another antioxidant nutrient vitamin C. Vitamin C helps heal

cuts and wounds and keeps teeth and gums healthy. It also aids in iron absorption, protects

the body's cells from oxidative damage due to free radicals, and enhances immune system

function (Turcotte, 2010).

Fruits play a significant role in human nutrition, especially as sources of vitamins [C

(ascorbic acid), A, thiamine (B1), niacin (B3), pyridoxine (B6), folacin (also known as folic

acid or folate) (B9), E], minerals, and dietary fiber (Wargovich, 2000).Diets rich in fruits

have been shown to be correlated with positive health outcomes, including decreased

cardiovascular disease risk, lowered risk for certain cancers (Temple and Gladwin, 2003),

and lower body mass index (Charlton et al., 2003). A recent WHO/FAO expert consultation

1
report on diet, nutrition and prevention of chronic diseases, sets population nutrient goals and

recommends intake of a minimum of 400g of fruits(excluding potatoes and other starchy

tubers) per day for the prevention of chronic diseases such as heart diseases, cancer, diabetes

and obesity. The report states that there is convincing evidence that fruits decrease the risk for

obesity, and evidence that they probably decrease the risk of diabetes. Further, there is

convincing evidence that fruit and vegetables lower the risk for Cardiovascular

diseases(CVD), and also help to prevent and alleviate several micronutrient deficiencies,

especially in less developed countries (WHO 2003). Overall it is estimated that up to 2.7

million lives could potentially be saved each year if fruit and vegetable consumption was

sufficiently increased. Recommendations in this direction tend to complement and reinforce

other valid messages based on the long known health benefits of consuming vegetables and

fruit as dietary sources of fibre, vegetable proteins and protective micronutrients (Turcotte,

2010).

Pineapple (Ananas comosus L. Merr.) is a type of tropical plant originating from Brazil,

Bolivia, and Paraguay. This plant belongs to the Bromeliaceae family. Pineapple is a type of

fruit that is high in nutrients. One of the pineapples that are popular in Indonesia is honey

pineapple. This type of pineapple has a delicious and refreshing taste. The pulp of honey

pineapple has a thicker pulp than pineapples in general, is more extensive than regular

pineapples, and has a slightly yellowish color to orange. Pineapple fruit contains lots of

nutrients, including vitamin A, calcium, phosphorus, magnesium, iron, sodium, potassium,

dextrose, sucrose (cane sugar), and the enzyme bromelain, which is a 95% mixture of

cysteine proteases that can hydrolyze protein (proteolysis) and resistant to heat. Apart from

the pulp, pineapple peel also brings many benefits. Pineapple pulp contains many nutrients,

and pineapple peel is known to have antioxidants helpful in warding off free radicals.

Pineapple peel also contains nano cellulose which is helpful in the pharmaceutical, food and

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medical industries. Nan cellulose from pineapple peel has been used as a tooth-strengthening

material (Erukainure et al., 2011)

The various activities owned by the pineapple peel are inseparable from its compound

content. The content of compounds in each plant can be affected by differences in growing

places, such as soil conditions, temperature, light, and climate. In this case, one of the honey

pineapple producers in Central Java is the Pemalang area. Nevertheless, honey pineapple peel

has not been used optimally. In this study, phytochemical screening aimed to determine the

class of compounds in the ethanol extract of honey pineapple peel originating from the

Pemalang area, Central Java. Further, phytochemical screening is a simple, fast, and highly

selective method that can be employed to identify classes of compounds and determine the

presence of biologically active compounds distributed in plant tissues. Meanwhile, the

bacterial infection is a health problem caused by bacteria and can attack all body organs.

Bacteria enter the body in many ways, for example, through food or drink contaminated with

bacteria, contact with body fluids such as faeces, urine, and blood from people infected with

bacteria, and from using eating and drinking utensils. Contaminated food and drink from

cutlery are a cause of bacterial infection. Therefore, everyone should use eating and drinking

utensils that are clean from bacteria. An effort that can be made to get bacteria-free tableware

is to use antibacterial dish soap (Hatam et al., 2013)

Plant extracts have been widely used in antibacterial liquid soap formulations- antiseptic

liquid soap formulation for hand use using celery leaf extract. The chemical content of

celery leaf extract has been reported to contain essential oils, flavonoids, coumarin and

carbohydrates.

Apart from being used in liquid hand soap formulations, plant extracts have also been used in

antibacterial dishwashing soap formulations. Pineapple extract has also been used in making

dish soap. A product in the form of liquid dish soap that not only cleans the remaining dirt on

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food utensils but can also act as an antibacterial from the use of Pineapple extract. Based on

the study results, it was found that the concentration of 1 ml of Pineapple extract in 250 ml of

the sample could inhibit the development of E. Coli bacteria by 20.28 mm; this result is

greater than dishwashing soap products on the market. Based on the results of testing and

observations of product quality standards that have been carried out, it can be seen that the

product is following the SNI quality standard number 06-2048-1990. The formulation of dish

soap with coffee bean extract has also been carried out in the form of dish soap with a coffee

aroma and is active as an antibacterial. This liquid has a thick texture, suitable for

dishwashing liquids in general (Evelyna et al., 2019)

1.1 Background of the study

Pineapple (Ananas comosus) is a tropical plant with an edible and multiple fruits consisting

of coalesced berries named for resemblling of the pine cone (Nohon, (2009). It is the most

economically important plant in the Bromeliacea family. Pineapple maybe cultivated from a

crown cutting of the fruit. Total pineapple production worldwide is around 16 to 18 million

tons. There are several countries (e.g Thailand, Brazil, India, Philippines, China and Nigeria)

which contribute to the total production (Nohon, (2009). Pineapple is an important food

which can be eaten fresh or in a processed form. It is composed of nutrients which are good

for human health. This is due to researches carried out on the relationship between nutrients

in pineapple and human health. According to Elliot et al., (2009), pineapple contains

antioxidant flavonoids, vitamin A and C. These antioxidants reduce the oxidative damage

such as that caused by free radicals and chelating of metal. It also has the enzyme complex

protease (bromelain). Bromelain contains peroxidase, acid phosphatase, several protease

inhibitors and organically bound calcium. Vegetables are mostly annual crops belonging to

the group of plants called horticultural cropsthat are diverse in nature (Ardina and Suprianto,

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2017). Vegetables can be grouped into leafy and fruity vegetables depending on the nature of

their consumable products or parts.

The agricultural sector in Nigeria has suffered many reversals during the past couple of

decades. From era of booming of export trade in agricultural commodities, the Nigerian

agricultural sector has degenerated to an import dependent one (Nurlina et al., 2013).

Subsequently, it has failed to generate significant foreign exchange, feed agro-allied

industries, improve the living standards of farming households and rural dwellers and provide

effective demand for industrial goods and services. Increasing food production however is

vital for enhancing future food security in the country as this is no longer debatable but a

necessity. To achieve this, good knowledge of the current efficiency or inefficiency inherent

in the crop production sub-sector as well as factors responsible for the level of efficiency and

inefficiency must be critically examined. Rapid population growth and crippling economic

problems in many African countries including Nigeria and most recently the global economic

meltdown have reduced living standards and adversely affected eating habits causing

widespread malnutrition (Dimpudus, 2017).

1.2 Statement of the Problems

Fruits are important vegetable crop in Northern Nigeria. However, its production is hindered

or constrained by different biotic agents. Fungal pathogens are among the most important

organisms that attack fruits. Fruits are widely distributed in nature. One of the limiting

factors that influence the fruits economic value is the relatively short shelf-life period

caused by pathogens attacked. It is estimated that about20-25% of the harvested fruits

are decayed by pathogens during post-harvest handling even in developed countries

(Droby, 2006; Zhu, 2006).Despite these damages, little information is available with respect

to the fungal pathogens affecting fruits in Northern Nigeria. There is need to put efforts

towards production of crops and checking of these fungal pathogen as well for better fruits

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production.

1.3 Aims and Objective

The main aim of this study was to investigate the bacteriological examination and antibiotic

sensitivity pattern of bacterial isolation from pineapple (Ananas comosuss) from Dodoru

market in Northern Nigeria.

1.3.1 Objectives

The objectives of this research were;

i. Enumeration of bacterial load from pineapple obtained in Dodoru market.

ii. To isolate bacterial associated with spoilage of pineapple obtained in Dodoru market.

iii. To identify the bacteria responsible for the spoilage of pineapple obtained in Dodoru

market.

iv. To carry out antibiotic susceptibility test in the isolated bacteria.

1.4 Justification for the Study

Bacterial pathogens are the main cause of severe infections in humans and microbes

resistance to antibiotics has become a serious problem of public health. According to the

World Health Organization (WHO), 80% of the world's population uses medicinal plants as

the main primary health care source in the treatment of several diseases. In African countries

the rate is much higher (Chastelyna et al., 2017). Limited scientific evidence regarding safety

and efficacy to support the continued therapeutic application of some of these herbal

remedies exists compared to such evidence for synthetically formulated drugs (Chastelyna et

al., 2017). The utilization of the medicinal plants is often based on ancestral experience. With

the upsurge in the use of herbal remedies, there is a need for a thorough scientific evaluation

to validate or disprove the supposedly therapeutic effects of some of these medicinal plants.

However, a number of compounds extracted from traditional plants have not been thoroughly

studied for phytochemical and efficacy, hence, the need to study the phytochemical

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constituents of some medicinal plant seeds, and to determine their biological activities as well

as their efficacies.

1.5 Scope and Limitation

The scope of this study was to isolate, bacteria from Ananas comosuss identify and carry out

antibiotic susceptibility of the bacteria identified from Ananas comosuss obtained from

Dodoru market.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

The pineapple (Ananas comosus) is a tropical plant with an edible fruit and is the most

economically significant plant in the family Bromeliaceae. The pineapple is indigenous

to South America, where it has been cultivated for many centuries. The introduction of the

pineapple to Europe in the 17th century made it a significant cultural icon of luxury. Since

the 1820s, pineapple has been commercially grown in greenhouses and many tropical

plantations. Further, it is the third most important tropical fruit in world production. In the

20th century, Hawaii was a dominant producer of pineapples, especially for the US. However

by 2016, Costa Rica, Brazil, and the Philippines accounted for nearly one-third of the world's

production of pineapples (Jayani et al., 2018)

Pineapples grow as a small shrub; the individual flowers of the unpollinated plant fuse to

form a multiple fruit. The plant is normally propagated from the offset produced at the top of

the fruit or from a side shoot, and typically mature within a year (Rahardjo et al., 2017).

2.1 Scientific classification

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Monocots

Clade: Commelinids

Order: Poales

Family: Bromeliaceae

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Genus: Ananas

Species: A. comosus

Pineapple, whole and in longitudinal section

Fig. 2.1: Pineapple (sliced) (Chastelyna et al., 2017)

The flesh and juice of the pineapple are used in cuisines around the world. In many tropical

countries, pineapple is prepared and sold on roadsides as a snack. It is sold whole or in halves

with a stick inserted. Whole, cored slices with a cherry in the middle are a common garnish

on hams in the West. Chunks of pineapple are used in desserts such as fruit salad, as well as

in some savory dishes, including pizza toppings, or as a grilled ring on a hamburger.

Traditional dishes that use pineapple include hamonado, afritada, kaeng som pla,

and Hawaiian haystack. Crushed pineapple is used in yogurt, jam, sweets, and ice cream. The

juice of the pineapple is served as a beverage, and it is also the main ingredient

in cocktails such as the piña colada and in the drink tepache.

In the Philippines, a traditional jelly-like dessert called nata de piña has also been produced

since the 18th century. It is made by fermenting pineapple juice with Komagataeibacter

xylinus (Sridevi and Deswita, (2019)

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Pineapple vinegar is an ingredient found in Honduran, and Filipino cuisine, where it is

produced locally. In Mexico it is usually made with peels from the whole fruit, rather than the

juice, but in Taiwanese cuisine it is often produced by blending pineapple juice with grain

vinegar (Chastelyna et al., 2017).

The European Union consumed 50% of global total for pineapple juice in 2012–2016.

The Netherlands was the largest importer of pineapple juice in Europe. Thailand, Costa

Rica and the Netherlands are the major suppliers to the European Union market in 2012–

2016. Countries consuming the most pineapple juice in 2017 were Thailand, Indonesia and

the Philippines, having combined consumption of 47% of the world total. From 2007 to 2017,

the largest growth in pineapple juice consumption was by Angola. The consumption of

pineapple juice in China and India is low compared to their populations (Suerni et al., 2013)

2.2 Pineapple varieties/cultivars

Several varieties have been adopted by different countries including Smooth Cayenne, Queen,

Red spanish and MD2 (Ndungu, 2014). The life cycle of pineapple takes up to 22 months.

The flowering happens after attaining vegetative growth of 11-12 months after planting

(Chanda et al., 2010). The first harvest (plant crop) is done around 18 th month and the second

harvest from ratoon crop at the 22nd month (Chanda et al., 2010). In commercial plantations,

pineapple is generally produced as a monoculture with its roots originating adventitiously

(Harbone, 2006). The plant is a xerophyte and survives well throughout the year including

during drought periods (Balouiri et al., 2016). However, a well distributed annual rainfall of

at least 1000 mm and medium altitudes of 1350-1750 m above sea level are essential for it to

produce well. At high altitudes growth is slow and the fruit contains much acid. Deep sandy

loams with high organic matter are ideal. Other soil types can be used as long as they are not

water logged and organic matter is added (Balouiri et al., 2016; Reiza, et al., 2019). The

above and other characteristics contribute heavily to disease and nematode problems (Balouiri

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et al., 2016).

2.3 Economic Importance of Pineapple

Pineapple is cultivated predominantly for its fruit that is consumed fresh or as canned fruit

and juice. Pineapple is the only source of bromelain, a complex proteolytic enzyme used in

the pharmaceutical market and as a meattenderising agent (Davis and Stout 2017). The stems

and leaves of pineapple plant are also a source of fibre that is white, creamy and lustrous as

silk. Pineapple fibre has been processed into paper with remarkable qualities of thinness,

smoothness and pliability (Erukainure, et al., 2 0 11). Parts of the plant are used for silage

and hay for cattle feed. Processing wastes in the form of shell, core materials and centrifuged

solids from juice production are also used as animal feed. Alcoholic beverages can also be

made from juice. Most of the world production (about 70%), and most of the canned

pineapple (about 95%), comes from the cultivar ‘Smooth Cayenne’. Since ‘Smooth Cayenne’

does not provide the best quality fresh fruit all year round, there is pressure on

distributors/growers to switch to cultivars with superior quality fresh fruit than ‘Smooth

Cayenne’ (Hatam et al., 2013). According to Evelyna et al. (2019), Kenya is one of the main

exporters of the 10 % of pineapples that Africa produces in the world. Some reports indicate

that pineapple plants infected by M. javanica can present symptoms of nutritional deficiency,

low uptake to fertilizers, yellowish leaves, wilted leaf tips and stunted fruits (Elliot et al.,

2009).

2.3.1 Cultural practices

Identification of Meloidogyne spp. is the first step in deciding the most suitable control

measure (Nurlina et al., 2013). Different methods have been developed for managing the

effect of Meloidogyne spp. on crop yield (Dimpudus, 2017) with a number of these

techniques being approved for their efficacy. According to Chastelyna et al., (2017) use of

crop rotation with resistant crops and integration of fallow periods were proven effective in

11
managing nematode infection and populations. However, rotation alone is not effective due to

the wide host range of Meloidogyne spp. According to Jayani et al., (2018), use of resistant

plant cultivars has remained a challenge despite the fact that it is environmentally safe, due to

emergence of resistance- breaking Meloidogyne spp. Soil flooding has also been used to

reduce the density of nematodes in rice cultivation. However, soil flooding method is not

applicable in pineapple and other vegetable production due to the nature of the soil and the

agronomic changes caused in soil e.g. lack of oxygen, soil structure degradation that might

alter the overall production (Rahardjo et al., 2017).

Many previous studies have focused on the use of organic amendments such as animal and

green manure, neem (Azadirachta indica), Tithonia diversifoliar e.t.c as a control measure to

Meloidogyne spp. showing suppressive effects. Although the efficacy of these products under

controlled conditions is commonly recognized, results in field conditions are rather

inconsistent (Sridevi and Deswita, 2019). Some studies have reported no significant effect of

compost on nematode control. Suerni et al., 2013) reviewed several studies in which

Meloidogyne populations increased after the application of organic amendments. Moreover,

nematode control requires a large amount of organic amendment and therefore, it is quite

expensive and relatively difficult to implement especially in commercial pineapple

production.

2.4 Biological Control

Different antagonistic organisms such as soil fungi from the genera Trichoderma

(Hypocreaceae), Verticillium, Pochonia and Paecilomyces (now Purpureocillium) have

been tested against Meloidogyne spp. with only a few of them being developed into

commercial products for use in the field. found that some fungal biocontrol agents’ mode of

action to plant parasitic nematodes was direct pathogenicity, some produce substances that

inhibit nematode egg hatch or kill nematode juveniles, some degrade signalling compounds to

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which nematodes are attracted to, some induce plant resistance and some produce antagonistic

microbes to nematodes (Chanda et al., 2010).

There are a few challenges that limit the commercial use of biocontrol agents including: their

inconsistent performance in the field, some affect narrow range of soil pests; some act slower

to the pests than pesticides; and they are more expensive to produce than existing chemical

products. Attempts have been made to control Meloidogyne spp. using antagonistic bacteria

and fungi (Chanda et al., 2010).

2.5 Nutritional composition of pineapple

Pineapple (Ananas comosus L. Merr.) is one of the most economically important tropical

fruit crops in tropical and subtropical areas. The variety ‘Smooth Cayenne’ is the most widely

grown cultivar in the world, but now, this variety is progressively substituted by the

commercially highly successful ‘MD2’ sweet hybrid variety. Pineapple is greatly appreciated

by the consumer due to its pleasant and sweet taste, flavor and its juicy and fleshy pulp, in

addition to its nutritional and health-promoting properties. Pineapples are an important source

of sugars, organic acids (citric acid), essential minerals (Cu, Mg, Mn, K), fiber and vitamins

(A, C, B-group) for human nutrition. Sensorial characteristics and chemical composition of

pineapples (sugars, organic acids, minerals, fiber, vitamins, amino acids, aromatic

compounds, etc.) depends greatly on the variety and the geographical and climatological

plant growing conditions.

Pineapple Nutrition

One cup of fresh pineapple chunks has:

Calories: 82 grams

Protein: 0.89 grams

Fat: 0.20 grams

Carbohydrates: 22 grams

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Fiber: 2.3 grams

2.5.1 Traditional uses

The pineapple is native to South America and was brought to Europe by Spanish explorers.

Planting began on a large scale in Hawaii early in the 19th century. Growth of the industry

peaked in the 1950s, then declined slowly under the pressure of international competition.

Today, the bulk of the world's pineapple crop comes from Thailand, the Philippines, and

Brazil. Traditional uses include the brewing of pineapple wine, production of fiber, and

medicinal use to induce menstruation, induce abortion, kill parasitic amoebas, and expel

worms Harbone, (2006)

2.6 PHARMACOLOGICAL ACTIVITY OF PINEAPPLE

Several medical properties have been attributed to pineapple, therapeutic

activities and has been used by medical practitioners as an anti-diabetic,

hypolipidemic, anti-inflammatory, anti-diarrhoeal, hepatoprotective, anti-

asthmatic and anti-cancerous drug. Haridra is widely used in

cosmetology . The following section discusses its various therapeutic uses

in medicine (Elliot et al., 2014).

2.6.1 Gastrointestinal disorders

The fresh juice of Haridra is considered to be anthelmintic. The Curcumin

acts through nuclear factor (NF)-κB inhibition and it reduces the

production of adhesion molecules and inflammatory cytokines, resulting in

the amelioration of gastric injury in NSAIDs-induced gastropathy in rats. It

also improves gastric mucosal damage and decreases in leukocyte

adhesions, and intercellular adhesion molecule 1 and tumor necrosis

factor (TNF)-α production after curcumin administration. Curcuma longa

extract tablet decreased IBS prevalence and abdominal pain/discomfort

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score significantly between baseline and after treatment of eight-week

(Evelyna et al., 2019).

2.6.2 Respiratory disorders

The fresh juice of rhizome is given in bronchitis. In rhinitis and cough boil

Haridra in milk and mixed with jiggery given internally. In catarrhal cough,

sore throat, and throat infection the decoction of rhizome is used for

gargle and also the piece of rhizome is slightly burnt and given for

chewing. The chemical constituents of Curcuma longa like Tumerones,

curcuminoids, Curcumin and tetrahydrocurcumin has an anti-asthamatic

action. In asthma and congestion, fumes of Haridradidhumvarti (fumes

wick) is given (Evelyna et al., 2019).

2.6.3 Inflammatory disorders

Curcumin has been shown to inhibit a number of different molecules

involved in inflammation including phospholipase, lipooxygenase, COX-2,

leukotrienes, thromboxane, prostaglandins, nitric oxide, collagenase,

elastase, hyaluronidase, MCP-1, interferon-inducible protein, tumor

necrosis factor, and interleukin-12. Studies has proven

bisdemethylcurcumin (BDC) is more potent as an anti-inflammatory agent

as indicated by suppression of TNFinduced NF-κB activation, more potent

as an anti-proliferative agent, and more potent in inducing reactive

oxygen species(ROS). Hispolon analogues, which lacks one aromatic unit

in relation to curcumin, also exhibited enhanced anti-inflammatory and

anti-proliferative activities. The beneficial effect of curcumin(anti-

15
inflammatory compound) in sepsis appears to be mediated by the

upregulation of PPAR-γ, leading to the suppression of pro inflammatory

cytokine, TNF-α expression and release (Hatam et al., 2013).

2.6.4 Diabetes mellitus

Turmeric rhizome powder is very useful with Amla juice and Honey in

Madhumeha (diabetes mellitus). The ingestion of 6 g Curcuma longa

increased postprandial serum insulin levels, but did not seem to affect

plasma glucose levels or GI, in healthy subjects. The results indicate that

Curcuma longa may have an effect on insulin secretion. The active

principles in the rhizome of Turmeric plant viz; curcuminoids lower lipid

peroxidation by maintaining the activities of antioxidant enzymes like

superoxidedismutase, catalase and glutathione peroxidase at higher

levels. Antioxidant properties of curcuma longa is due to curcumin and its

three derivatives (demethoxycurcumin, bisdemethoxycurcumin and

diacetylcurcumin) (Hatam, et al., 2013).

2.6.5 Cardiovascular disorders

The antioxidants in turmeric also prevent damage to cholesterol, thereby

helping to protect against atherosclerosis. In fact, the ability of the

antioxidants in turmeric to decrease free radicals is similar to that in

vitamins C and E. Since the antioxidant activities of turmeric are not

degraded by heat (unlike most vitamins), even using the spice in cooking

provides benefits. Animal studies show that curcumin lowers cholesterol

and triglycerides, another fat that circulates in the blood stream and is a

risk factor for cardiovascular disease. In a recent study of atherosclerosis,

mice were fed a standard American diet, rich in refined carbohydrates and

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saturated fat, but low in fiber. Some of the mice, however, received this

diet plus turmeric mixed in with their food. After four months on these

diets, the mice that consumed the turmeric with their food had 20 percent

less blockage of the arteries than the mice fed the diet without the

turmeric. In another study, rabbits were fed turmeric plus a diet designed

to cause atherosclerosis. Several risk factors for the disease were

improved, including a decrease in cholesterol, triglycerides, and free-

radical damage (Erukainure et al., 2011).

2.6.6 Hepatoprotective

The powder of the rhizome mixed with amla juice is used in jaundice.

Corriliyum (Anjana) with Haridra, Red ochre (Gairika), and Amalaki

(Emblicaofficinalis) cures jaundice. Curcumin, the most common

antioxidant constituent of Curcuma longa rhizome extract, was reported

to enhance apoptosis of damaged hepatocytes which might be the

protective mechanism whereby curcumin down-regulated inflammatory

effects and fibrogenesis of the liver. The ethanolic extract of Curcuma

Longa rhizomes showed a significant hepatoprotective effect when orally

administrated in doses of 250 mg/kg and 500 mg/kg, and the protective

effect was dose dependent (Balouiri et al., 2016).

2.6.7 Neuroprotective activity

Curcuma oil significantly reduces the ill effect of ischemia by attenuating

nitrosative and oxidative stress. Ischemia induces collapse of

mitochondrial membrane potential, cytochrome c release, altering the

Bax: Bcl-2 ratio and subsequently caspases activation led to induction of

apoptosis in sequential fashion was reverse significantly by Curcuma oil.

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So there is an evidence for the high efficacy of Curcuma oil as a

neuroprotective, with an excellent therapeutic window for the prevention

of ischemic brain injury (Balouiri, et al., 2016).

2.6.8 Alzheimer’s disease

Curcumin when fed to aged mice with advanced plaque deposits similar

to those of Alzheimer’s disease, curcumin reduced the amount of plaque

deposition. It reduced oxidative damage and reversed the amyloid

pathology in an Alzheimer’s disease transgenic mouse. Alzheimer’s

disease symptoms characterized by inflammation and oxidation were also

eased by curcumin’s powerful antioxidant and anti-inflammatory

properties (Reiza et al., 2019).

2.6.9 Chemoprotective activity

Curcumin activate the DDR (DNA damage response), providing an

opportunity and rationale for the clinical application of these

nutraceuticals in the chemoprevention of prostate cancer.

Chemoprotective effects in esophageal epithelial cells exposed to bile

acids; Curcumin reverses bile acid suppression of gene expression of

SOD-1 and also able to inhibit bile acid induction of COX-2 gene

expression. Curcumin has demonstrated these chemopreventive

properties in cell cultures, animal models and human investigations (Reiza

et al., 2019).

2.6.10 Anti cancer activity

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Curcumin has been found to possess anticancer activities via its effect on

a variety of biological pathways involved in mutagenesis, oncogene

expression, cell cycle regulation, apoptosis, tumorigenesis and metastasis.

Curcumin has shown anti-proliferative effect in multiple cancers, and is an

inhibitor of the transcription factor NF-B and downstream gene products

(including c-myc, Bcl-2, COX- 2, NOS, Cyclin D1, TNF-a, interleukins and

MMP-9). In addition, curcumin affects a variety of growth factor receptors

and cell adhesion molecules involved in tumor growth, angiogenesis and

metastasis (Reiza et al., 2019).

2.6.11 Anti allergic activity

Curcumin suppressed compound 48/80-induced rat peritoneal mast cell

(RPMC) degranulation and histamine release from RPMCs. Curcumin

inhibited compound 48/80-induced systemic anaphylaxis in vitro and anti-

DNP immunoglobulin E (IgE) mediated passive cutaneous anaphylactoid

response in vivo. Curcumin has an ability to inhibit nonspecific and

specific mast cell-dependent allergic reactions (Chanda et al., 2010).

2.6.12 Antidermatophytic activity

Fresh juice of rhizome of Haridra is used as antiparastic in many skin

affections. Its rhizome powder mixed with cow’s urine is taken internally in

itching and dermatitis. Curcuma longa L. leaves have good promise as an

antifungal agent that could be used as a therapeutic remedy against

human pathogenic fungi on account of its various in vitro and in vivo

antifungal properties, viz., strong fungicidal action, long shelf-life, its

tolerability of heavy inoculum density, thermo stability, broad range of

antidermatophytic activity and absence of any adverse effects. Curcumin

19
obtained from the turmeric rhizome (Curcuma longa) have shown to

possess the ability to protect the skin from harmful UV-induced effects by

displaying antimutagen, antioxidant, free radical scavenging, anti-

inflammatory and anti-carcinogenic properties (Chanda et al., 2010).

2.6.13 Curcumin prevents drug resistance

The Curcumin is a potent drug resistance preventer. It exhibits novel

ability to prevent the upregulation of P-glycoprotein and its mRNA induced

by adriamycin (ADM). The prevention capacity is also functionally

associated with the elevated intracellular drug accumulation and parallel

enhanced ADM cytotoxicity (Sridevi and deswita, 2019).

2.6.14 Synergistic effect of Curcumin

Curcumin is known for its Synergistic effect as well. Curcumin (CCM) and

Docosahexaenoic acid (DHA) are dietary compounds known to antagonize

breast cancer cell proliferation. This study reveals that these compounds

in combination exert a variable antiproliferative effect across multiple

breast cell lines, which is synergistic in SK-BR-3 (Human Breast Cancer

Cell Line) cells. DHA enhanced cellular uptake of CCM in SK-BR-3 cells

without significantly enhancing CCM uptake in other cell lines. The

combination of DHA and CCM is potentially a dietary supplemental

treatment for some breast cancers, likely dependent upon molecular

phenotype. DHA enhancement of cellular curcumin uptake is one potential

mechanism for observed synergy in SK-BR-3 cells; hence, an event unique

to the combined presence of the two compounds. Human pancreatic

carcinoma cell lines BxPC-3 and Panc-1 were obtained from American

Type Culture Collection (Manassas, VA, USA). Authors observes that both

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curcumin and garcinol significantly (P<0.05) reduced cell viability in both

cell lines in a dose-dependent manner. Garcinol-(upper panel) or

curcumin-(lower panel) treated cytosolic extracts were used to evaluate

induction of apoptosis in PaCa cells. BxPC-3 and Panc-1 using ELISA-

Histone DNA Enrichment Assay. Results demonstrate a significant dose-

dependent increase in apoptotic cells in individual treatment with either

agent for 48 hours (Suerni et al., 2013).

CHAPTER THREE

3. MATERIALS AND METHODS

3.1 EQUIPMENT AND MATERIALS

MATERIALS

Equipment

Incubator

Filter paper

Petri dish

Test tube

Round Bottom flask

Microscope

Slide

Sterile loop

Cork borer

Sterile swab sticks

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3.1.2 REAGENTS

Ethanol

Grams iodine and decolorizer

Kovac’s reagent

Peptone water (medium)

Nutrient Agar

Manitol salt Agar

Oxidase reagent

Hydrogen peroxide

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3.2 Study Area

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3.3 Sample Collection

The samples (pineapple) was collected aseptically from Dodoru market into sterile containers

and transported to the laboratory for further analysis.

3.3.1 Sample Preparation

The samples was scraped and Five gram (5g) of the samples was dissolved in 20ml sterilized

water each, 1ml was pipette and severly diluted for the power of 10-4.

3.4 Media Preparation

3.4.1 Nutrient agar

5gram was collected and dissolved in 200ml of distilled water, in a conical flask it was then

sterilized for 15minute in autoclave. The prepared medium was used for the analysis

3.4.2 Eosin Methylene Blue agar

7.5g of medium was dissolved in 200mlof distilled water in conical flask, then sterilized for

15minutes.

3.4.5 Salmonella salt agar

12g of the medium was dissolved in 200ml of distilled water in a conical flask, and then

sterilized for 15minutes.

3.4.6 Manitol salt agar

8.2g of the medium was dissolved in 200ml of distilled water, it was then was sterilized and

used for for the analysis.

3.4.7 Cled medium

7.5g of the medium was dissolved in 200ml of distilled water and then was sterilized for 15

minutes.

3.5 Bacterial plate count

Pour plate method of inoculation was used 1ml of generally diluted sample was dispense in a

plate and about 15ml of nutrient agar was poured accordingly. The plate was allowed to

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solidified and then incubated for 24hr of 37 0C. the number of colonies were countered and

repeated as cfu/ml.

3.6 Sub-culturing of bacterial isolate

Isolative and differential media was used for the isolation. 1ml of the prepared sample was

inoculated in about 15ml of the prepared medium using pour plate method of inoculation.

The plate was allowed to solidify and then incubated for 24hrs at 37 0C. observation and

interpretation was made according to medium directives. The isolate was then sub-cultured

into agar medium for confirmation.

3.7 Smear Making

A drop of distilled water was place at the centre of the slide the loopfull of the bacterial

isolate was pick and emulsified on the water to make a thin smear. The smear was heat fixed

and allowed for a day.

3.8 Gram Staining and Microscopy

The Gram staining procedure modified by Ruckert and Morgan (2017) was used in this

research work. Clean glass slides was obtained and using the sterile technique a smear of

each of the microorganism from an 18-28hours culture (while micro-organism was still

young) was prepared, and heat-fixed. The smear was gently stained with basic dye crystal

violet and left for 1-2 minutes. This was then rinsed rapidly with water, followed by

treatment with gram’s iodine solution and left 1 minute which increased interaction between

cell and the dye so that the dye was more tightly bound or the cell was strongly stained. The

iodine was then poured off, blotted and slide or smear decolorized by washing with 95%

ethanol until no more stain ran from the side. The slide was then washed well with water and

stained with safranin for 30 seconds, which was then washed well and dried. Preliminary

characterization by Gram staining was done (using safranin) on each of the isolates using the

method of Dussault (2015) and observed under a light microscope at x 100 and this was

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observed under oil immersion. The Gram staining technique was to categorize the isolate into

Gram negative and Gram positive (Cappuccino and Sherman, 2020).

3.9 Biochemical Test for Pathogenic Bacteria

3.9.1 Catalase Test

A colony of 24hour old culture was picked using a sterile loop and then emulsified in a few

drop of hydrogen peroxide on a clean slide. Presence of effectiveness indicated positive

reaction whereas negative reaction showed no effervescence (Rueckert and Morgan, 2017).

3.9.2 Indole Test

The sterile wire loop was used to inoculate organism in a test tube containing 5ml of peptone

water (medium) and for 48h at 370C. after incubation, 0.5 ml of Kovac’s reagent was added

into the tube and allowed to stand for 15min. a rose spank indicated positive reaction

(Rueckert and Morgan, 2017).

3.9.3 Oxidase Test

A piece of filter was soaked oxidase reagent, a colony of test organism is then smeared on

filter paper deep purple color indicate that phenylenediamine in the reagent by the oxidase in

the test organism (Rueckert and Morgan, 2017).

3.9.4 Urease test

A well isolated colony was picked from the surface of the medium and inoculated as single

streak on the slant surface of Christenes’s urea agar. The pH shift was detected by the color

change of phenol red from light orange to magenta which indicated a positive result

(Chesbrough, 2016).

3.9.5 Citrate Test

Inoculated Simmons citrate agar lightly on the slant by touching the top of a needle to a

colony that is 18 to 24 hours old. Incubated at 350c to 370c for 18 to 24 hours. Some

26
organisms may require up to 7 days of incubation due to their limited rate of growth on

citrate medium. Observed the development of blue colour denoting alkalinization.

Positive: colour change (Prussian blue).

Negative: no colour change.

3.9.6 Methyl-Red Test

Inoculated two test tubes containing VP-MR broth with a pure culture of the organism under

investigation. Incubated at 35oc for 4 days.

Added 5 drops of MR indicate solution to the first tube (for VP test Barrit’s reagent to

another tube).

Positive: Red colouration.

Negative: No colour change.

3.9.7 Motility Test

A semisolid agar medium was prepared (Nutrient agar) and sterilized at 121 0C for 15 minutes

in a test tube

Using a straight wire, the samples were collected and inoculated into the agar by a single

straight stab down to the center of the tube, about half the depth of the medium. With the wire

still in place, the tubes were plugged and covered

The media were incubated at 37o C overnight

After incubation, the agar samples were observed for growth in or around the test tube. A

spread of growth away from the line of stab indicated motile organisms (Motility positive).

Growth only found along the line of stab indicates a motility negative result (Non-motile

organisms).

3.10 Sensitivity test

Bacterial isolated confirmed to be pathogens were subjected to sensitivity test, this was
carried out using Disc diffusion method. Both culture of the organisms was prepared by using
mc far land standard 0.5. swip stick was use to inoculate the organism into a prepared muller

27
hintan agar mate using separate method of inoculation. Sensitivity disc was then placed at the
center of the plate. Incubated for 24 hours at 37 oc. after the incubation period observe the
plate over to check weather the isolates are sensitive intermediate or resistant to the
antibiotics.

Antibiotics and their concentrations

Penicillin concentration: the mean concentration of penicillin reached in the serum of

females was three times higher than in males. The concentration of penicillin in the serum of
females increased with age and weight from the tenth to the seventeenth years. No such
increase was observed in the sera of males in relation to age and weight.

The differences in concentration of penicillin reached in the sera of the two sexes from 12
through 17 years of age were statistically significant (p less than 0.01). No significant
difference in concentration of penicillin in the serum was found in comparing obese and thin
girls of 10 through 15 years of age. The explanation for the differences between the two sexes
is not apparent.

Gentamicin concentration: the median serum gentamicin concentration at 60 minutes after

administration (C 60min) was 21.4 μg/mL with a distribution indicating that bacteria with
MIC ≥2 μg/mL were unlikely to be exposed to sufficient gentamicin for effective killing.
Approximately 90% of isolates from ambulatory practice and 36% of hospital isolates had
MICs at or below breakpoints for susceptibility with most of the remainder unlikely to be
responsive, even to higher IV doses.

Pefloxacin concentration: was determined by bioassay during 24 neutropenic periods. The

median diffusible fecal pefloxacin concentration was 187 micrograms/g. This concentration

was comparable with those found in volunteers following oral and intravenous administration

of pefloxacin (400 mg twice daily) (median of 171 and 155 micrograms/g, respectively).

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