CHAPTER ONE

1.0 INTRODUCTION/LITERATURE REVIEW

1.1 INTRODUCTION

Zobo drinks are traditional non- alcoholic beverage which is consumed in most

part of Nigeria, mostly in northern part of Nigeria (Osuntogun, 2014). The

economic and religious situation in Nigeria has made the zobo drink gain wide

acceptance in different occasions. It’s used as refreshment, entertainment in parties

or as appetizers before the main dish is served and it is also sold in market to

various consumers (Onuorah et al., 2017).

The zobo drink is a red liquid drink and taste like fruit punch, served as a fair

source of vitamin A, riboflavin, niacin, calcium and iron and is low in sugar

content (Qi et al., 2015). This drink also contains anthocyanins and Vitamins C,

among others and it is used in curing minor stomach ailments, sore throat and

strengthening the heart among other uses (Olawale, 2011).

Zobo drink is extracted from the dried reddish purple calyces of the plant Hibiscus

sabdriffa (Scott, 2013). The calyces are used to produce herbal teas and other food

products.The juice drink which is usually obtained by extraction of the calyx of

Hibiscus contains about 1% solid. The drink contains some microorganisms which

can cause food spoilage (Omemu et al., 2016). At present, the production
1
processes in neither mechanized nor standardized. Consequently, the shelf life of

the drink is less than two days (Samy, 2010). Furthermore, the mode of packaging

or dispensing of the juice in nylon or plastic container before retailing, that is taken

as Zobo i.e the largely unregulated nature of the trade, and poor hygienic practices

as well as lack of running water, toilet, proper storage and waste disposal facilities

at preparation and services point has resulted in poor unsanitary conditions

exposure to potential contaminants and an increased risk to public health (Omemu

and Aderoju, 2018).

Consequently, street drinks and foods safety has remained a major public health

concern globally, and more importantly in Nigeria were the regulation of this

critical sector is virtually non-existent or inadequate, making street foods and

drinks hazardous source of nutrition (Oyeyi and Lum-nwi, 2018). Foods frequently

serve as vehicle for spreading of several organisms some of which are pathogenic

(Singleton, 2019). In view of the facts, that Zobo is never subjected to any form of

post-production treatment that can eliminate or at least reduce the bacteria load in

the drink, it could be a potential source of health hazard. Also the activities

involved in the cooling and subsequent dispensing of the drink into containers also

represent potential source of health hazard (Singleton, 2019). Some researchers

Cruck and Shank (2014) have reported that some gastro intestinal illness

characterized by diarrhea, abdominal cramps, and vomiting which may be assumed

2
has been of unknown aetiology may arise from drinking drinks contaminated with

microorganisms.

1.2 AIM AND OBJECTIVES

To assess the microbial load found in zobo sold in Imo State University, Owerri.

Specific Objectives

1. To determine the microbial load in zobo sold in Imo State University

2. To isolate bacteria and fungi from zobo sold in Imo State University

3. To identify isolated bacteria and fungi from zobo sold in Imo State

University

4. To determine the percentage occurrence of bacteria and fungi isolated from

zobo sold in Imo State University

3
1.3 LITERATURE REVIEW

1.3.1 ZOBO

Zobo drink is one of the locally made beverages in Nigeria and Africa. It is

prepared through an indigenous technology from plant (roselle) flower,

scientifically known as Hibiscus sabdariffa which belongs to a plant

family Malvaceae (Wong, 2012).

People prefer zobo drink to the carbonated drinks because it is rich in natural

carbohydrate, protein, antioxidants, vitamin C, calcium, magnesium and zinc. It is

non-alcoholic, medicinal and has low glycemic index (Oboh et al., 2012).

Zobo is becoming acceptable in social gathering because it

is economically affordable and attractive to many people more than soda. Increase

in religious and health campaigns against alcoholic beverages in Nigeria and the

consequent decrease in the consumption of alcoholic beverages in certain areas has

afforded Zobo drink great potential as a local alternative to imported red wines in

particular and alcoholic beverages in general (Olayemi et al., 2011).

Recently, zobo drink has become a main source of income in many homes both in

rural communities and more in the urban areas where cottage business has

4
increased due to support from the government through the poverty alleviation

schemes, thereby alleviating poverty among the people (Essien et al., 2011).

Based on the numerous merits of zobo drink, many researchers had worked on

examining its nutritional value, sensory quality and medicinal properties. Zobo was

found to be very rich in vitamin C, other antioxidants and minerals (such

Potassium, Sodium and Phosphorus) but low protein (Olayemi et al., 2011). This

accounts for its limitation to solve protein energy malnutrition.

1.3.2 COMPOSITION OF ROSELLE (Hibiscus sabdariffa)

The leaf is reported to contain protein, fat, carbohydrate, fibre, ash, calcium,

phosphorus, iron, thiamine, B-carotene, riboflavin, niacin and ascorbic acid (Crane,

2019). The flower yields a yellow dye , the major pigment identified is

daphniphylline .The plant contains flavonoids such as hibiscitrin and hibiscetine

and dried calyces contain the flavonoids, gossypetine, hibiscetine and

sabadaretine, it also contain alkaloids, B-sistosterol, anthocyan, citricacid,

cyaniding-3- rutinose, delphanidin, steric acid and wax (Anocha et al.,2015). The

calyces are rich in acid and also contain crude protein and minerals such as iron

phosphorus, calcium, manganese, ascorbic acid, etc. The seeds contains protein,

fat, dietary fibre, nitrogen, fatty oil, cellulose pentosans and starch (Rao, 2016)

5
1.3.3 MEDICINAL BENEFITS OF ROSELLE (Hibiscus sabdariffa)

Hibiscus sabdariffa is used medically in the treatment of a variety of ailments

including hypertension, herperlipdemia, obesity, and diabetes (Perry, 2010).

Delphinidi 3-sambubioside, a hibiscus anthocyanin reduces leukemia cells through

oxygen reactive species-mediated mitochondrial pathway. The antihypertensive,

hyperlipdaemic, anti-obesity, and anti-obesity effects have been confirmed and the

possible mechanisms have also been delineated. These effects have been attributed

to various constituents of Hibiscus sabdariffa like flavonoids, anthocynins and

organic acids and Na+, vitamins A and C and Fe. Antibacterial activity of

gossypectin isolated from Hibiscus sabdariffa was carried out and results revealed

that the activity may be due to polyphenolic nature of the flavonoid gossypectin

(Kavimani et al., 2010).

1.3.4 NUTRITIONAL BENEFITS OF ZOBO DRINK

The fresh flower is rich in riboflavin, vitamin C, niacin, carotene, calcium and iron,

flavonoids which are nutritionally necessary. Flavonoids are responsible for the

deep red color in zobo beverage; it has been recognized as a powerful anti-oxidant

and a likely contributor to cancer health as well as immune function. In addition,

zobo is thought to have a mild diuretic effect and the ability to reduce high blood

pressure. Zobo is an excellent source of trace minerals and other nutritional

6
elements essential to health. It is rich in chromium, copper, manganese, iron,

selenium and phosphorus. Weight loss, medical thinkers said that, products like

zobo are realistic in weight loss regimes (Varro et al., 2011).

Roselle calyces contains nutrients like; protein, carbohydrate, lipid, minerals,

anthocyanins. Sugars like glucose, fructose and galactose and acids like; ascorbic

acid (vitamin C), phytic, oxalate, tannic and hydrocyanic. The aqueous extract of

the calyces is characterized as having high and low sugar content (Daramola and

Asuni, 2016).

1.3.5 UTILIZATION OF Hibiscus sabdariffa IN FOOD PRODUCTION

Roselle calyces in tropical Africa and neighbouring countries are used for many

purposes. Many parts of the Roselle plants, including seeds, leaves, fruits and roots

are used in various food products. The fleshy red calyces are the most popular

among them. The fresh calyces are used for making fruit juices, syrups, new wines,

puddings, jams, ice cream, jellies, gelatines, cakes and flavours whereas the dried

calyces are used to make spices, tea, sauces, butter, pies and other desserts. Roselle

tender stems/stalks and leaves are either consumed raw in salads or cooked alone

as vegetable soups/ stews or in combination with meat and other vegetables (Qi et

al., 2015; Atoagye, 2012).

7
The calyces are also gathered for sale, either fresh or dried. Dried vials are used in

Europe to flavour extracts for liqueurs. A drink is also produced from calyces’

infusion called "Zobo" in Nigeria, "Bissap” or Sobolo in Ghana, which is used for

refreshments and entertainment at home and public meeting (Qi et al., 2015).

1.3.6 PREPARATION OF ZOBO DRINK

Zobo drink is prepared by boiling the dry calyces of Hibiscus sabdariffa in water

for about 10-15minutes from which the pigments or flavor embedded is extracted.

After extraction, the filtrate may be taken as hot tea or allowed to cool; the sharp

taste of the raw extract is usually sweetened with sugar and also with ginger and

garlic. The sweetness of zobo drink does not last long due to spoilage by microbial

activities. Its shelf life is between 24-48 hours (Omemu et al., 2016).

8
Fig 1.3.6: Flow chart of zobo drink production

1.3.7 MICROBIAL CONTAMINATION

Coliforms biofilm can grow or regenerate in the distribution system. Age, low

residual disinfectant, warm climate, relatively high levels of total organic carbon,

9
the old iron pipe, and inadequate washing of the dead ends contribute to the growth

of the biofilm, sometimes bacteria, including E. coli, which it is released in the

water. Biofilm may support the regrowth of virulent bacteria, occurring in a factory

treatment failure (Payment, 2019).

Typically, the microorganisms’ grow on the water contact surface as biofilms and

water. Growth following treatment of drinking water is often called "regeneration"

or “regrowth”. The growth reflects generally higher water samples measuring

different bacteria plate count (HPC) values. High levels of HPC systems occur

mostly in the distribution pipeline, the pipeline in the country, in the portion in

water bottle and stagnant connection pipes in the apparatus, such as softeners,

carbon filters (Bartram et al., 2014). Contamination by microorganisms may also

happen through wrongly installed and/ or through concealed leaks in the piped

water system (Bartram et al., 2014).

In a research conducted by Amusa et al. (2015), the calyces of H. sabdariffa used

were found to harbour Aspergillums Niger, A. flatus, A. tamari, Penicillium

oxalicum, Fusarium oxysporum and Rhizopus spp. whereas the hawked Sobolo

drinks harbored Bacillus cereus, B. subtilis, Proteus spp., Pseudomonas

aeruginosa, Streptococcus pyogenes, Staphylococcus aureus and Escherichia coli;

and the freshly processed Sobolo drinks harbored no bacteria.

10
In a related research Musah et al. (2014), found seven different isolates in hawked

Sobolo drinks, namely Aspergillus spp., Bacillus spp., Klebsiella spp.,

Pseudomonas spp., Streptococcus spp., Staphylococcus aureus and Escherichia

coli.

1.3.7.1 Sources of Contamination of Roselle Drink

There are various sources of contamination of roselle drink; the main ones are the

calyces used, the processing environment including the method of preparation,

water and types of equipment used for the preparation of the drink, the packaging

material, and conditions of preservation and storage as discussed below.

The Roselle Calyces Used

The calyces have been reported to get infested with microorganisms through the

seed stock used, local growing conditions, and postharvest handling especially

during drying and storage and in the market. Studies confirm that the bulk of

contamination of the drink is through the calyces used as a major raw material. The

high content of water in fresh calyces predisposes the calyces to infection; even if

the calyces are later dried some of the microorganisms remain. At the local market,

roselle calyces are displayed in big containers (uncovered) or polyethene sheets for

sale; these expose calyces to microbial contamination (Izah et al., 2015). The

presence of contaminants and toxigenic fungi from roselle drink is a function of

11
contamination of the calyces used (Adebayo-Tayo and Samuel, 2019). These

authors reported that total bacteria counts, coliform counts, and total fungi counts

were in the range of 5.0–13.0 × 103, 0.00 − < 102 /g, and 3.4–7.3 × 104 CFU/g,

respectively, from dried H. sabdariffa calyces sold in a different market in Uyo,

Akwa Ibom State, Nigeria. The microbial counts in the zobo drinks sold in the

markets and hawked in streets ranged from 102 to 108 CFU/ml. These values

depend on the type of flavour, preservatives used, and storage duration (Adebayo-

Tayo and Samuel, 2019). For instance, bacteria counts of 8.1 × 10 4 CFU/ml, 1.21 × 

105 CFU/ml, 2.6 × 105 CFU/ml, 3.2 × 105 CFU/ml, and 2.5 × 106 CFU/ml were for

zobo drink prepared without sugar, with sugar, with no sugary spices, with sugary

spices, and with sugary spices + pineapple flavour, respectively (Seiyaboh et al.,

2013). Majority of the microbes found in the samples caused spoilage. Also, B.

subtilis, S. aureus, S. fecalis, L. brevis, P. putida, A. niger, A. flavus, S. cerevisiae,

P. citrinus, R. oligosporus, C. krusei, and Mucor spp. are microbial isolates found

in the dried roselle calyces sold in some market in Ogun State, Nigeria (Omemu et

al., 2016). The associated bacteria were Bacillus subtilis, Bacillus spp.,

Enterococcus faecalis, Micrococcus spp., Klebsiella spp. and Staphylococcus

aureus. The fungi isolates were Aspergillus flavus, A. terreus, A. glaucus, P.

expansum, Fusarium oxysporum, and Cladosporium spp. Aspergillus glaucus was

the dominant species among the six species. Odunfa (2018) reported that

12
Staphylococcus aureus levels of 108/mL were considered potentially hazardous to

consumers. The main fungi associated with roselle calyces were Aspergillus spp.

This may be due to the presence of their spores in a large number in the

atmosphere which can easily settle on the harvested calyces in the farm, during

processing, or in the market. It has been made clear that most contamination of the

drink was as a result of contamination of the calyces; this implies that some of the

calyces presently offered for sale in our markets are not acceptable for the

production of the drink for human consumption (Adebayo-Tayo and Samuel,

2019). Although during processing the calyces are often subjected to high

temperature, once the raw materials are contaminated, it becomes difficult to get

rid of all microorganisms through boiling; for example, boiling aflatoxin does not

reduce its potency (Mohammed et al., 2016).

Contamination during Preparation

Water is another raw material for the preparation of the drink. The quality of water

also determines the quality of the drink (Bolade et al., 2019). Clean and sterile

water is good for a quality drink as poor water introduces some microbes that are

dangerous to health. Other materials used such as sweeteners (sugar, sugar cane

juice, and pineapple juice), preservative materials such as garlic, lime, ginger, and

benzoic acid as well as equipment such as bowls, pots, and stirrer used during

preparation are possible means of contamination. Risiquat (2013) reported that

13
contamination may occur when the hot extract is left to cool, during the

introduction of flavour and sweeteners and bottling. The place of preparation if not

hygienic can be a source of contamination. In addition, the level of hygiene of the

processor and the activities of the handlers such as cleaning children after

defecating, cleaning mucus, sneezing, coughing, and excessive talking can be

means of introducing toxigenic microbes into the drink. Akhigbemidu et al. (2015)

affirmed that Streptococci spp. which are often found in the mouth enter the drink

during preparations via talking.

Contamination during Packaging

In developing countries, the drink is locally packaged by the use of polyethene

sachet/films and plastic bottles. These materials if already polluted can

contaminate the drink. Majority of the processors of the drink in the developing

countries are small-scale women and even some children who may not attach more

importance to hygiene. Due to ignorance and poverty level of some processors,

used bottles are reused for packaging; if such bottles were not thoroughly washed,

it can cause contamination of the drink. Besides, some processors use mouth to

blow air into polyethene sachets to open them; this is a means of introducing

microorganisms into the drink. All other unhygienic activities like singing during

packaging can cause contamination. Staphylococcus spp. and Pseudomonas spp.

14
may occur in the drink due to handling and other utensils used after processing

(Amusa et al., 2015).

Contamination during Storage

Conditions of a place of storage and duration of storage can also encourage

contamination. If the storage environment is unhygienic or favourable for

microbes’ multiplication, contamination can occur. The values of microbial counts

depend on the type of flavour, preservatives used, and storage duration (Izah et al.,

2015). Nevertheless, Risiquat (2013) found that the microbial population of the

drink seldom exceeds acceptable/level and tolerant level for ready-to-eat food the

day it is prepared.

1.3.8 PRECAUTION FOR ZOBO DRINK QUALITY AND DURABILITY

1.3.8.1 Proper Handling

If calyces are produced, packaged, and sold in aseptic conditions, contamination of

the drink will be minimal. Growers and processors of crops as well as market

women should be educated on how to handle their crops especially roselle calyces

to prevent contaminations. Thus, harvesting must be carefully done to reduce

contamination. Mohammed et al. (2012) reported that harvesting while raining can

downgrade the quality of the calyces and lower the output. Calyces should be

handled properly to avoid contact with the ground or filthy surfaces. Since calyces
15
contamination has been cited as the major point of contamination having sterile

calyces will ensure high durability and better quality drink (Adebayo-Tayo and

Samuel, 2019). Incidence of aflatoxin in dried calyces’ samples can be avoided

through proper handling, adequate storage facilities, and planting of Aspergillus

spp. resistant varieties (Adebayo-Tayo and Samuel, 2019).

1.3.8.2 Avoidance of Contamination during Preparation and Packaging

The drink should be prepared in hygienic environments and all materials and

equipment used should be sterile. Activities that will cause contamination such as

talking, sneezing, and scratching of the body should be avoided or minimized by

the use of mouth and nose guard and overall dress to serve as a precaution

(Adebayo-Tayo and Samuel, 2019). Also, to avoid contamination, the drink should

be aseptically poured into sterilized polythene sachets and then sealed back using a

thermoelectric sealing machine. Furthermore, thorough boiling should be

encouraged to kill the heat susceptible microbes that may be in the drink. The drink

is mainly prepared through boiling and steeping; however, steeping does not give

much heat influence on the microbes in the drink. So steeping method of

preparation should be discouraged (Adebayo-Tayo and Samuel, 2019).

16
1.3.8.3 Preservation

The following preservation methods could be used to prevent or reduce

contamination:

 Physical preservation: this is the preservation of materials or substance through

pasteurization, freezing, and refrigeration. Pasteurization is done when the bottled

drink is immersed in sterile water, heated to 70°C or more, and left for 20 minutes

(Adebayo-Tayo and Samuel, 2019). Egbere et al. (2017) recommended a

combination of pasteurization and sodium benzoate to extend the shelf life of the

drink to forty days. Freezing and refrigeration are achieved by subjecting the

microorganisms present in the drink to a very low temperature to make them

inactive and prevent their multiplication. Nevertheless, these cannot affect

microbes that thrive in freezing temperature. Besides, the electricity supply is not

adequate in some developing countries.

 Chemical preservation: this is carried out by the use of chemical preservatives.

Preservatives are chemical additives that are used to improve the keeping qualities

of food products to prevent or reduce the microorganism growth and sustain the

chemical quality of the food such as colour, taste, aroma, and texture (Bankole et

al., 2013). Chemical preservatives commonly utilized in the preparation of the

drink are acetic acid and sodium benzoate, although sorbate and propionate can

17
also be used. Acetic acid has been reported to have a superior effect on bacteria

than sodium benzoate and vice versa for fungi (Braide et al., 2012). Doughari et al.

(2017) reported that benzoic acid-treated samples sustained the taste until after 14

days of storage. The use of chemicals can reduce the qualities of the drink and can

be harmful to consumers of the drink. Studies have shown that the use of synthetic

chemical for food preservation could have adverse effects on human health. Thus,

acetic acid produced from biomass through a microbial method such as oxidative

fermentation was recommended as the best for food preservation this can be used

to elongate the shelf life of this nutritious drink (Izah et al., 2015).

 Biological method of preservation: this refers to the use of plant extracts

especially spices to preserve; those spices used include lime, ginger, garlic, pepper

fruit, and kola nut. Natural spices have been commonly used as food additives and

for improving flavour (Omoruyi and Emefo, 2012). Lime can reduce the microbial

load of the drink; this may be due to its acidic nature. Acidic foods inhibit the

survival of many pathogens but enhance the multiplication of acidic organisms,

hence the domination of Bacillus spp., Lactobacillus spp., and S. cerevisiae in the

drink (Egbere et al., 2017). The acidity of lime is organic and less corrosive to the

alimentary canal. Braide et al. (2012) further reported that lime was effective in

suppressing microbial load than garlic, ginger, and glove even better than sodium

benzoate. Although both ginger and garlic have antimicrobial effects, a mixture of

18
ginger and garlic is more effective than when used separately (Mahomoodally et

al., 2018). Izah et al. (2015) found that the ability of natural preservatives to

eliminate microorganisms is in the following order: lime > mixture of garlic and

ginger > ginger > garlic > clove > kola nut > cinnamon > nutmeg. These authors

recommended the use of these natural preservatives to enhance the quality of the

drink and to reduce spoilage.

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 CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 STUDY AREA

The samples were collected from students attending Imo State University. Imo

State University is located in Owerri capital territory of Imo State. It was

established in the year 1981 through lawNo_4passedby the Imo State House of

Assembly Owerri. It is situated along Okigwe Road Owerri Urban. It is bounded in

the East by Works layout, West by Aladimma Housing Estate, North by Orji and

South by lkenegbu. Imo state University is an autonomous public institution with

the general function of providing liberal education and encouraging the

advancement of learning. It has about a population of 25,000 students as of 2013,

comprising of both regular and part-time students. Imo State University lies

between latitude of 5030'13'N and longitude of 702’37'E.There are 11 faculties

with 73 department and other academic units in Imo State University. Imo State

University is made up of students who were engaged in full time undergraduate

studies and other social activities.

2.2 SAMPLE COLLECTION/SAMPLE SIZE

A total of 9 samples of commercially prepared zobo were collected from different

vendors in Imo State University. Samples were transported in a sterile

20
polyethylene bag to the microbiology laboratory, Imo State University, Owerri for

microbiological analysis.

2.3 STERILIZATION OF MATERIALS

The glass wares (Test tubes, pipettes, conical flasks, beakers, Petri-dishes and

universal bottles) were washed with soapy water and rinsed with distilled water;

they was allowed to air dry and wrapped with Kraft paper and was further

sterilized in a hot air oven at 180°C for 1hour and stored at 4°C.

2.4 MEDIA PREPARATION

Nutrient agar, MacConkey Agar and Sabouraud Dextrose agar was prepared

according to Manufacturer’s instruction, and sterilized by autoclaving at 121°C for

15 min. Salmonella-Shigella agar, which does not require autoclaving, was

sterilized by boiling for 15 min.

2.5 PREPARATION OF SAMPLES

10ml of each zobo samples were transferred into a test tube containing 90ml of

distilled water. The samples were further diluted following the procedure stated

below;

1. Set 10 test tubes on a test tube rack and label sequentially from 10-1 to 10-10

21
 2. Using sterile syringe, measure 9ml of normal saline or distilled water into
each test tube
3. Using a sterile pipette, transfer 1ml of the waste water into the first test tube

containing 9mls of distilled water and mix gently.

4. From the first test tube, collect 1ml using a fresh sterile pipette and transfer

to the second test tube and mix gently.

5. From the second test tube, 1ml will be transferred to the third test tube with

a fresh pipette. Further dilutions are to be made till the 10 -10 dilution, after

the ten dilutions, 1ml of the diluents is to be discarded from the test tube.

Making all the test tube 9mls again.

2.6 ENUMERATION OF TOTAL VIABLE BACTERIA

This was carried out using pour plate techniques. The already prepared media for

microbial count was allowed to cool to 450C. 15-20mls of the media was dispensed

into the petri-dish containing the 1ml of the aliquots from the dilution factor 10 -5

for microbial enumeration. The culture plate was gently rotated clockwise and

anticlockwise for even distribution of microorganisms. The media were allowed to

solidified, then inverted and incubated at 37oC for 24-48 hours and at 25OC for 3-5

days for bacteria and fungi respectively. At the end of the incubation, the numbers

of colonies were enumerated and the colony forming unit per ml (Cfu/ml) was

calculated.

22
2.7 ISOLATION AND IDENTIFICATION OF ISOLATES

At the end of the incubation, the morphology of the colonies should be observed

and record. All single colonies were sub-cultured on nutrient agar and sabouraud

dextrose agar and were incubated at 37 oC for 24-48 hours and at 25OC for 3-5 days

for bacteria and fungi respectively to obtain pure colonies of bacteria and fungi.

Sub-culturing continued until a single colony growth was achieved. All pure

colonies obtained were preserved in an agar slant containing nutrient agar and

sabouraud for further identification of isolates.

For further bacteria isolates identification, Gram staining, Citrate utilization test,

Coagulase test, Sugar fermentation test, Manitol salt test, Methyl red test, Voges

proskauer test, Indole test, and Catalase test were carried out (Cheesbrough, 2002).

Gram reaction test

Gram staining differentiates bacteria by the chemical and physical properties of

their cell walls by detecting peptidoglycan, which is present in the cell wall of

gram-positive bacteria. Gram-positive bacteria retain the crystal violet dye, and

thus are stained violet, while the gram-negative bacteria do not; after washing, a

counterstain is added (commonly safranin or fuchsine) that will stain these gram-

negative bacteria a pink color (Cheesbrough, 2010).

23
Procedure

Place slide with heat fixed smear on staining tray. Gently flood smear with crystal

violet and let stand for 1 minute. Tilt the slide slightly and gently rinse with tap

water or distilled water using a wash bottle. Gently flood the smear with Gram’s

iodine and let stand for 1 minute. Tilt the slide slightly and gently rinse with tap

water or distilled water using a wash bottle. The smear will appear as a purple

circle on the slide. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide

slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol

runs almost clear. Be careful not to over-decolorize. Immediately rinse with water.

Gently flood with safranin to counter-stain and let stand for 45 seconds. Tilt the

slide slightly and gently rinse with tap water or distilled water using a wash bottle.

Blots dry the slide with bibulous paper. View the smear using a light-microscope

under oil-immersion (Cheesbrough, 2010).

Motility Test

Motility test is performed by preparing a wet mount and is then observed under the

microscope. Motility of bacteria can also be tested by inoculating the bacteria in

the semi-solid motility medium (Cheesbrough, 2010).

24
Procedure

Prepare a semisolid agar medium in a test tube. Inoculate with a straight wire,

making a single stab down the center of the tube to about half the depth of the

medium. Incubate under the conditions favoring motility. Incubate at 37°C.

Examine at intervals, e.g. after 6 h, and 1 and 2 days (depends on generation time

of bacteria). Freshly prepared medium containing 1% glucose can be used for

motility tests on anaerobes (Cheesbrough, 2010).

Results: Hold the tube up to the light and look at the stab line to determine

motility.

Non-motile bacteria generally give growths that are confined to the stab-line, have

sharply defined margins and leave the surrounding medium clearly transparent.

While, motile Bacteria typically give diffuse, hazy growths that spread throughout

the medium rendering it slightly opaque (Cheesbrough, 2010).

Catalase Test

The catalase test facilitates the detection of the enzyme catalase in bacteria. It is

essential for differentiating catalase-positive Micrococcaceae from catalase

negative Streptococcaceae. While it is primarily useful in differentiating between

genera, it is also valuable in speciation of certain gram positives such as

25
Aerococcus urinae (positive) from Aerococcus viridians (negative) and gram

negative organisms such as Campylobacter fetus, Campylobacter jejuni, and

Campylobacter coli (all positive) from other Campylobacter species (Cheesbrough,

2010).

Procedure

Place a microscope slide inside a petri dish. Keep the petri dish cover available.

Using a sterile inoculating loop or wooden applicator stick, collect a small amount

of organism from a well-isolated 18- to 24-hour colony and place it onto the

microscope slide. Be careful not to pick up any agar. This is particularly important

if the colony isolate was grown on agar containing red blood cells. Carryover of

red blood cells into the test may result in a false-positive reaction. Using a dropper

or Pasteur pipette, place 1 drop of 3% H2O2 onto the organism on the microscope

slide. Do not mix. Immediately cover the petri dish with a lid to limit aerosols and

observe for immediate bubble formation (O 2 + water = bubbles). Observing for the

formation of bubbles against a dark background enhances readability

(Cheesbrough, 2010).

26
Coagulase Test

The coagulase test differentiates strains of Staphylococcus aureus from other

coagulase-negative species. S. aureus strains are capable of coagulating plasma in

the tube test and will produce clumps of cells in the slide test (Cheesbrough, 2010).

Procedure

The coagulase test can be performed using two different procedures - Slide test and

tube test. The slide test is simple, giving results within 10 seconds, but it can give

false negatives. The tube test is the definitive test, however, it can take up to 24

hours to complete. For both tests, clumping or clots of any size indicate a positive

response. While S. aureus is the most commonly isolated coagulase positive

organism, there are several other species of Staphylococcus which are positive for

coagulase activity. S. schleiferi and S. lugdunensis may give positive results in the

slide test for bound coagulase, and S. schleiferi and S. ntermedius may give

positive results in the tube coagulase test (Cheesbrough, 2010).

Oxidase Test

The oxidase test is a biochemical reaction that assays for the presence of

cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the

presence of an organism that contains the cytochrome oxidase enzyme, the reduced

colorless reagent becomes an oxidized colored product (Cheesbrough, 2010).

27
Procedure

Filter Paper Test Method: Soak a small piece of filter paper in 1% Kovács oxidase

reagent and let dry. Use a loop and pick a well-isolated colony from a fresh (18- to

24-hour culture) bacterial plate and rub onto treated filter paper. Observe for color

changes. Microorganisms are oxidase positive when the color changes to dark

purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when

the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase

negative if the color does not change or it takes longer than 2 minutes

(Cheesbrough, 2010).

Indole Test

The indole test screens for the ability of an organism to degrade the amino acid

tryptophan and produce indole. It is used as part of the IMViC (indole, MR-Vp

Citrate) procedures, a battery of tests designed to distinguish among members of

the family Enterobacteriaceae (Cheesbrough, 2010).

Procedure

Inoculate the tube of tryptone broth with a small amount of a pure culture. Incubate

at 37°C for 24 to 48 hours. To test for indole production, add 5 drops of Kovác's

reagent directly to the tube. A positive indole test is indicated by the formation of a

pink to red color (“cherry red ring”) in the reagent layer on top of the medium
28
within seconds of adding the reagent. If a culture is indole negative, the reagent

layer will remain yellow or be slightly cloudy (Cheesbrough, 2010).

Citrate Test

The citrate test screens a bacterial isolate for the ability to utilize citrate as its

carbon and energy source. A positive diagnostic test rests on the generation of

alkaline by-products of citrate metabolism. The subsequent increase in the pH of

the medium is demonstrated by the color change of a pH indicator. The citrate test

is often part of a battery of tests used to identify gram-negative pathogens and

environmental isolates (Cheesbrough, 2010).

Procedure

Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single

isolated colony and lightly streak the surface of the slant. A needle is the preferred

sampling tool in order to limit the amount of cell material transferred to the agar

slant. Avoid using liquid cultures as the inoculum source. Citrate utilization

requires oxygen and thus screw caps, if used, should be placed loosely on the tube.

Incubate at 35oC (+/- 2OC) for 18 to 48 hours. Some organisms may require up to 7

days of incubation due to their limited rate of growth on citrate medium

(Cheesbrough, 2010).

29
Citrate positive: growth will be visible on the slant surface and the medium will be

an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-

products of citrate catabolism raise the pH of the medium to above 7.6, causing the

bromothymol blue to change from the original green color to blue.

Citrate negative: trace or no growth will be visible. No color change will occur; the

medium will remain the deep forest green color of the uninoculated agar. Only

bacteria that can utilize citrate as the sole carbon and energy source will be able to

grow on the Simmons citrate medium, thus a citrate-negative test culture will be

virtually indistinguishable from an uninoculated slant (Cheesbrough, 2010).

Voges-Proskauer Test 

The Voges-Proskauer (VP) test is used to determine if an organism produces

acetylmethyl carbinol from glucose fermentation. If present, acetylmethyl carbinol

is converted to diacetyl in the presence of ∝- naphthol, strong alkali (40% KOH),

and atmospheric oxygen. The ∝- naphthol was not part of the original procedure

but was found to act as a color intensifier by Barritt and must be added first. The

diacetyl and quanidine-containing compounds found in the peptones of the broth

then condense to form a pinkish red polymer (Cheesbrough, 2010).

30
Procedure

Prior to inoculation, allow medium to equilibrate to room temperature. Using

organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.

Incubate aerobically at 37 degrees C. for 24 hours. Following 24 hours of

incubation, aliquot 2 ml of the broth to a clean test tube. Re-incubate the remaining

broth for an additional 24 hours. Add 6 drops of 5% alpha-naphthol, and mix well

to aerate. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.

Observe for a pink-red color at the surface within 30 min. Shake the tube

vigorously during the 30-min period (Cheesbrough, 2010).

Methyl Red Test

Methyl Red (MR) test is a biochemical test performed on bacterial species to detect

the ability of an organism to produce stable acids end products (Mixed-acid

fermentation) from supplied glucose. It is used as the part of the IMViC tests, a set

of four useful reactions that are commonly designed for the differentiation of

enteric (members of family Enterobacteriaceae) (Cheesbrough, 2010).

Some bacteria have ability to perform mixed acid fermentation of glucose in MR-

VP medium. The products of mixed-acid fermentation are a complex mixture of

acids, particularly lactate, acetate, succinate and formate as well as ethanol and

31
equal amounts of H2 and CO2. This causes the medium to acquire an acidic pH.

Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less

(Cheesbrough, 2010).

Procedure

By using sterile inoculating loop, inoculate the unknown microorganism into the

fresh, sterile medium. Leave the other broth uninoculated (this will be a control).

Incubate the inoculated tube at 35-37C for two to five days. After incubation,

obtain the broths from the incubator and add 5 drops of Methyl Red reagent to the

broth. Observe the color (Cheesbrough, 2010).

Triple Sugar Iron Agar

Triple sugar iron agar test is used to determine whether gram negative bacilli

utilize glucose and lactose or sucrose fermentative and produce hydrogen sulfide

(H2S).  It contains 10 parts of lactose: 10 parts of sucrose: 1 part of glucose and

peptone.  Phenol red and ferrous sulphate serves as an indicator for acidification of

medium and H2S production respectively (Cheesbrough, 2010).

Glucose is utilized first by a fermentative organism and the entire medium

becomes acidic (yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24

hours incubation period because of the presence of organic acids resulting from the

32
fermentation of glucose under anaerobic conditions in the butt of the tube. The

slant reverts to alkaline state that is indicated by red color as the fermentation

products gets oxidized to carbon dioxide (CO2) and water (H2O) and peptone in

aerobic condition the slant undergoes oxidation releasing alkaline amines (Phenol

red in alkaline pH turns red while in acidic pH turns yellow) (Cheesbrough, 2010).

Procedure:

Touch a well isolated colony with a sterile straight wire. Inoculate TSI by first

stabbing through the center of the medium to the bottom of the tube and then streak

the surface of the slant. Leave the cap loose and incubate the tube at 35 in ambient

air for 18 to 24 hours. Observe the reaction (Cheesbrough, 2010).

Lactophenol Cotton Blue Stain

Lactophenol Cotton Blue Stain is formulated with lactophenol, which serves as a

mounting fluid, and cotton blue. Organisms suspended in the stain are killed due to

the presence of phenol. The high concentration of the phenol deactivates lytic

cellular enzymes thus the cells do not lyse. Cotton blue is an acid dye that stains

the chitin present in the cell walls of fungi (Cheesbrough, 2002).

Procedure

Place a drop of Lactophenol Cotton Blue Stain in the center of a clean slide.

Remove a fragment of the fungus colony 2-3mm from the colony edge using an

inoculating or teasing needle or MycoMount™ adhesive strips (Cat. no. MM40).

33
Place the fragment in the drop of stain and tease gently. Apply a coverslip. Do not

push down or tap the cover slip as this may dislodge the conidia from the

conidiophores. Examine the preparation under low and high, dry magnification for

the presence of characteristic mycelia and fruiting structures. Consult appropriate

references for diagnostic features of fungi isolated in clinical and non-clinical

specimens (Cheesbrough, 2010).

34
 CHAPTER THREE

3.0 RESULTS

Table 3.1 shows the microbial cell count obtained from zobo sold in Imo State

University, Owerri. Total coliform count (MacConkey agar) of the zobo beverage

samples ranged from 0.2 x 105 Cfu/ml to 5 x 105 Cfu/ml, Total fungi count (SDA)

ranges from 0.6 x 105 Cfu/ml to 15.0 x 105 Cfu/ml, Total Staphylococcus count

(MSA) ranges from 0.2 x 105 Cfu/ml to 10.0 x 105 Cfu/ml, and Total heterotrophic

bacteria count (NA) ranges from 0.5 x 105 Cfu/ml to 10.0 x 105 Cfu/ml.

35
Table 3.1: Microbial load count of zobo sold in Imo State University, Owerri

Sample code MacConkey agar SDA (CFU/ml) MSA (CFU/ ml) NA (CFU/ ml)
(CFU/ml)
Z1 2.1 x 105 5.1 x 105 0.2 x 105 2.2 x 105
Z2 0.2 x 105 1.0 x 105 10.0 x 105 4.0 x 105
Z3 5.0 x 105 9.0 x 105 2.5 x 105 1.0 x 105
Z4 3.2 x 105 5.3 x 105 5.4 x 105 3.5 x 105
Z5 1.6 x 105 4.6 x 105 5.7 x 105 2.6 x 105
Z6 1.0 x 105 0.6 x 105 0.3 x 105 0.5 x 105
Z7 1.6 x 105 2.5 x 105 0.5 x 105 2.5 x 105
Z8 4.0 x 105 2.0 x 105 1.0 x 105 1.5 x 105
Z9 2.8 x 105 15.0 x 105 0.6 x 105 10.0 x 105
KEYS: Z = Zobo; SDA = Sabouraud Dextrose Agar; CFU/ml = Colony Forming Unit per milliter; MSA =
Mannitol Salt agar; NA = Nutrient agar

36
Table 3.2 shows the bacteriological and biochemical properties of the identified

bacteria from zobo beverage samples sold in Imo State University, Owerri. From

this table, the identified bacteria are Salmonella spp., Staphylococcus spp.,

Escherichia coli, Klebisella spp., and Bacillus spp.

37
Table 3.2: Bacterial and biochemical identification test of isolates from zobo sold in Imo State University,
Owerri.

BACTERIAL BIOCHEMICAL IDENTIFICATION TEST

IDENTIFIICATION TEST
Gram Cel. Mot Catalas Indol Oxidas Coagulas Citrat V. M G L S Probable
reactio arrangeme . e test e test e test e test e test P R organism
n test nt Test tes test
t
- Rod shape + + - - - - - + + - - Salmonella
spp.
+ Cocci - + - - + + + + + + + Staphylococc
us spp.
- Rod shape + + + - - - - + + + + Escherichia
coli
+ Rod shape + + - + - + + + + + + Bacillus spp.
- Rod shape - + - - - + + - + + - Klebisella
spp.
Keys: - = Negative; + = Positive; Cel. = Cellular; Mot. = Motility; V.P = Voges Proskuer; G = Glucose test; L =
Lactose test; S = Sucrose Test; spp. = Species

38
39
Table 3.3 shows the colonial and microscopic morphology of identified fungi

species using lactophenol cotton blue wet mount preparation of zobo sold in Imo

State University, Owerri. The identified fungi are; Aspergillus spp., Penicillium

spp., Rhizopus spp. and Yeast spp.

40
Table 3.3: Colonial and microscopic morphology of fungal isolates from zobo sold in Imo State University,
Owerri
Colonial morphology
Colony forms grey to whitish
greenish yellow coloration
Initially white to yellow but
becomes distinctly black as colony
develops
Cotton-like, white in color to gray
Creamy white colonies formed
KEY
spp. = Species
Microscopic morphology Probable organism
Conidiospores bearing conidia with Penicillium spp
brush like spores
The vesicle of the conidiospores is Aspergilus spp.
large and globase, bearing two series
of sterigmata over its entire surface.
The conidia are brown to black and
rough walled.
Simple and form apical, globular Rhizopus spp.
sporangia that are supported and
elevated by a column-shaped
columella.
Are bi-polar budding cells with Yeast spp.
lemon-shaped mother tips
41
Table 3.4 shows the prevalence incidence of the bacteria isolates on zobo sold in

Imo State University, Owerri. Escherichia coli had the highest rate of prevalence

of 9 (32.1%), Staphylococcus spp. 6 (21.4%), Klebsiella spp. 5 (17.9%), Bacillus

spp. and Salmonella spp. has the least prevalence of 4 (14.3%) each.

42
Table 3.4: The prevalence incidence of bacteria isolated from zobo sold in Imo State University, Owerri.

Isolates Sample code Percentage

occurrence
(%)
Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9
Salmonella + + - - + - - - + 14.3
spp.
Staphylococcu + + - + + + - - + 21.4
s spp.
Escherichia + + + + + + + + + 32.1
coli
Bacillus spp. - - + - - + + - + 14.3
Klebisella spp. + - - - + + + + - 17.9
KEYS: Z = Zobo; % = percentage; + = present; - = absent; Spp. = species

43
Table 3.5 show the percentage prevalence of fungi isolates from zobo sold in Imo

State University, Owerri. From the table, Yeast spp. has the highest prevalence of 7

(35.0%), Rhizopus spp. had 6 (30%), Aspergillus spp. had 5 (20%) and Penicillium

spp. had the least percentage prevalence of 4 (15%).

44
Table 3.5: The prevalence incidence of fungi isolated from zobo sold in Imo State University, Owerri.

Isolates Sample code Percentage

occurrence

(%)
Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9
Penicilliu - + - - - + + - - 15.0

m spp.
Aspergilus + - - + - + - - + 20.0

spp.
Rhizopus + - + + + + - - + 30.0

spp.
Yeast spp. + + + - + - + + + 35.0
KEYS: Z = Zobo; % = percentage; + = present; - = absent; Spp. = species

45
 CHAPTER FOUR

4.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

4.1 DISCUSSION

This study was carried out to assess microbial load on zobo sold in Imo State

University, Owerri. In this study, a high microbial load count was obtained from

zobo sold by vendors in the study area. Total coliform count (MacConkey agar)

of the zobo beverage samples ranged from 0.2 x 10 5 Cfu/ml to 5 x 105 Cfu/ml,

Total fungi count (SDA) ranges from 0.6 x 10 5 Cfu/ml to 15.0 x 105 Cfu/ml,

Total Staphylococcus count (MSA) ranges from 0.2 x 10 5 Cfu/ml to 10.0 x 105

Cfu/ml, and Total heterotrophic bacteria count (NA) ranges from 0.5 x 10 5

Cfu/ml to 10.0 x 105 Cfu/ml. In a similar estimation of total heterotrophic counts

in zobo, a count of 4.02 x 105 CFU/mL was obtained (Ibitoye et al., 2017).

Also, Salmonella spp., Staphylococcus spp., Escherichia coli, Klebisella spp.,

Bacillus spp., Aspergillus spp., Penicillium spp., Rhizopus spp. and Yeast spp.

were isolated microorganisms in this study. This is in agreement with the

findings of some earlier works carried out by Akinyosoye and Akinyele (2010)

and Braide et al. (2012) where they identified microorganisms associated with

zobo. The occurrence of the different bacterial isolates (Salmonella spp.,

Staphylococcus spp., Escherichia coli and Klebisella spp., Bacillus spp.,) in the

zobo drinks as obtained in this study is an indication of poor hygienic handling

46
of the beverage. These microorganisms are contaminants from contaminated

containers or from untreated water that is normally used in the preparation of

zobo.

Staphylococcus spp. in zobo could be possible through the processing methods

which usually involves the use of hands, since the organism is a common flora

of the skin. This organism is also responsible for Staphylococcal food poisoning

(Hobbs and Gilbert, 2018). Odunfa (2018) reported that Staphylococcus aureus

at levels of 108ml are considered potentially hazardous to consumers. The

occurrence of Pseudomonas spp also could be as a result of contamination from

food handlers or water since the organism is found in water and on skin surface

as flora to the skin (Foster, 2016). Generally, Escherichia coli are an indication

of water pollution and therefore the presence of the organism in zobo drink is

probably related to the source or quality of water used for the processing

(Foster, 2016). In addition, Escherichia coli isolated from water may have some

health implication (Akinyosoye and Akinyele, 2010).

The occurrence of Bacillus spp. could also be as a result of the prevalence of

their spores in the environment (Jay, 2018). Bacillus species forms spores which

could survive high temperature of processing. Bacillus have been isolated from

nonalcoholic beverages (Jay, 2018).

The occurrence of Yeast spp., Penicillium spp. and Rhizopus spp. might have

come from the soil and plant surface as they are cosmopolitan and ubiquitous

47
organisms found in the soil or growing on mature fruits, plant debris, decaying

vegetation, crops seeds, grains leaf surfaces and food stuffs. The fungal isolates

therefore found in the plant beverage could be traced to the time when the petals

were either being harvested or stored. This may have produced spores which

were attached to the petals and overcome adverse condition during the

preparation and finally germinated in the finished product. This is also

attributable to the prevalence of their spores in the atmosphere. The liberated

spores can easily settle on food and ceilings of rooms and then be germinated

(Okhuoya and Ayanlola, 2016). The isolation of Aspergillus spp. in this study is

of the highest concern because it is known to produce aflatoxin and can grow at

low water activities (Ilondu and Iloh, 2017). Therefore in order to avoid such

growth and possible production of toxic metabolites, care should be taken to dry

the product quickly before these moulds have the chance of establishing any

significant growth.

4.2 CONCLUSION AND RECOMMENDATIONS

Consumption of zobo drinks from local drink vendors poses a serious risk to

human health as has been demonstrated in this study due to the high prevalence

of pathogenic bacteria and fungi. Therefore it is recommended that government

and established authority should ensure greater regulatory control in the

production, distribution and consumption of zobo and indeed locally prepared

48
beverage drinks as their unregulated production and consumption portends a

grave public health concern

49
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