CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Hazard Analysis and Critical Control Points (HACCP) as long been internationally recognized
and accepted as the system for effective food safety management (CAC, 2003). It is a systematic
preventive approach to food safety that addresses physical, chemical and biological hazards as a
means of prevention rather than finished product inspection. It is used in the food industry to
identify potential contamination and subsequently evaluating that the process is in control of
those points or steps of the food chain critical to food safety and key actions known as the
Critical Control Points(CCPs) is taken to eliminate risk of the hazards been realized. However,
its success and effectiveness in preventing food borne diseases and reducing food safety risks to
an acceptable level depends on its correct implementation and application (FAO and WHO,
2006; Lawley, 2007; Kok, 2009). HACCP was conceived in the 1960s when the US National
Aeronauts and Space Administration (NASA) asked Pillsbury to design and manufacture the first
foods for space flights. Since then, HACCP has been recognized internationally as a logical tool
for adapting traditional inspection methods to a modern, science based food safety system. Based
on risk-assessment, HACCP plans allow both industry and government to allocate their resources
efficiently in establishing and auditing safe food production practices (Okonko et al., 2009).

Pap known as koko in Hausa; a fermented, cereal gruel from millet is a staple food of several
communities in Nigeria. It is traditionally made from maize, sorghum or millet. Several reports
had identify steeping and souring as the two fermentation stages involved in the traditional
preparation of Ogi. It is prepared by steeping clean grains in water at room temperature(25±20C)
for 48- 72hours.The steeping water is decanted and the fermented grain is washed with clean
water and then wet-milled. The bran is removed by wet sieving and the sieved is allowed to settle
for another 24-48hours, a process referred to as souring during which time fermentation also
proceeds and solid starchy matter, Ogi sediments, gasara in Hausa is produced (Akingbala et al.,
1999). The wet Ogi usually has a smooth texture, a sour flavor resembling that of yoghurt and a
characteristics aroma that differentiates it from starch and flour. The color of the Ogi depends on
the type of cereal used, cream for maize, light brown for sorghum and whitish to grey for millet

1
(Banigo, 1993; Onyekwere et al., 1993). Fermentation of Ogi is by microorganism from the
environment and quality control is absent in the traditional method of preparation (Onyekwere et
al., 1997; Halm et al., 1990). Lactic acid bacteria, yeasts and mold are responsible for the
fermentation although Lactobacillus plantarium is the predominant microorganism. Yeasts of the
Saccharomyces and Candida species also contribute to the flavor development (Caplice and
Fitzgerald, 1999). A lot of nutrient losses occur during processing of cereals for Pap production
hence, several attempts have been made to improve the nutritional value status of Pap by
fortifying it with protein rich substrates. However, nutritional improvements of these fermented
cereal gruels with proteinous foods lowered their pasting viscosities and sometimes affected their
sensory attributes adversely. These factors are likely to influence acceptability (Osungabro,
2009). The pap is considered the most important weaning food for infants to compliment breast
milk from 4-6months of age in West Africa although it is consumed by adults (Banigo 1993;
Moses et al., 1993; Onyekwere et al., 1993).

Improper handling of food, preparation and consumption practices by consumers (FSAI, 1998;
Bryan,1988; Gorman et al.,(2002), inadequate hygiene practices such as hand washing (Corgan
et al., 2001),and use of unhygienic utensils and materials (Alterkruse et al.,1995; Knabel,1995;
Beumer and Giffel,1999), consumption of raw or unsafe food (CAST,1994; Redmond and
Griffiths,2003), as well as cross contamination through inanimate surfaces by raw food
(Roberts,1982; Ryan et al.,1996) are some of the factors and practices that have been implicated
in food borne outbreaks but fermented foods generally have a very good safety record even in the
developing world where food are manufactured by people without training in microbiology or
chemistry, often in unhygienic contaminated environments (Keith,1997). But while fermented
foods themselves are naturally safe, they do not solve the problem of contaminated drinking
water, environments heavily contaminated with human waste, improper personal hygiene in food
handlers and flies carrying disease organisms which implies that fermented food can be unsafe.
However, application of the principles that lead to the safety of fermented food would lead to an
improvement in the overall quality and the nutritional value of the food supply, reduction of
nutritional disease and thereby reducing transmission of infections agent through food. (Keith,
1997). Hazard analysis and critical control point (HACCP) approach appears to be the most
important strategy for improving the safety of foods in Africa. HACCP emphasizes monitoring

2
of critical control points by food handlers themselves. Critical risk factors can thus be rapidly
and easily identified and early corrective action can be taken (Etok, 1998).

1.2 Justification

Some microorganisms are biological hazards that are found in many sort of contaminated foods.
Such microorganisms include bacteria, fungi, virus, protozoans, and algae are detrimental to the
health of the food consumers and consequently can cause serious disease to them. Pap can
harbour such microbial hazards when contamination occurred particularly during its production
processes. Some of the microorganisms like moulds may be from contaminated raw ingredients,
e.g. contaminated millet grains used in the production process while some may be due to
contaminated water used in the production process, e.g. untreated water that is contaminated (for
example, with coliforms due to fecal contamination) been used. Many traditional foods became
contaminated due to poor hygienic production processes.

1.3 Statement of the Research Problem

Many students in the campus rely on some traditional foods sold by some food sellers, usually
old women in KUST Mini-market, as either their breakfast or lunch because of the fact that such
foods are very cheap compared to those sold in expensive restaurants outside the campus.
Unfortunately, such foods are prone to microbial contamination mostly due to improper personal
hygiene of the sellers, poor sanitation of the business environment or the cooking utensils, lack
of well treated water and contamination of the food ingredients during storage and transportation.
In this case, there is need to identify the possible source of hazards, possible hazards and possible
prevention and control measures for the identified hazards to ensure safe production of the foods
so as to ensure that the students are kept safe from various diseases that might have resulted from
eating contaminated foods.

1.4 Aim and Objectives

1.4.1 Aim

The aim of this research is to carryout microbiological as well as hazard analysis and critical
control points on traditional fermented food; Pap sold in KUST Wudil Mini-market.

3
1.4.2 Objectives

The objectives of this research are:

1. to isolate the microorganisms associated with spoilage of Pap

2. to identify the microorganisms isolated from the sample,
3. to identify microbiological hazards associated with the production of the food by hazard
analysis,
4. to identify critical control points in the production processes of the food.

4
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 General Overview on the Hazard Analysis and Critical Control Point, (HACCP)

2.1.1 History of HACCP

In the early 1960s, a collaborated effort between the Pillsbury Company, NASA, and the U.S.
Army Laboratories began with the objective to provide safe food for space expeditions. People
involved in this collaboration included Herbert Hollander, Mary Klicka, and Hamed El-Bisi of
the United States Army Laboratories in Natick, Massachusetts Paul A. Lachance of the Manned
Spacecraft Center in Houston, Texas , and Howard E. Baumann representing Pillsbury as its lead
scientist. To ensure that the food sent to space was safe, Lachance imposed strict microbial
requirements, including pathogen limits (including Escherichia coli, Salmonella and Clostridium
botulinum ) (Sperber et al., 2009). Using the traditional end product testing method, it was soon
realized that almost all of the food manufactured was being used for testing and very little was
left for actual use. Therefore, a new approach was needed (Sperber et al., 2009). NASA's own
requirements for critical control points (CCP) in engineering management would be used as a
guide for food safety. CCP derived from failure mode and effects analysis (FMEA) from NASA
via the munitions industry to test weapon and engineering system reliability. Using that
information, NASA and Pillsbury required contractors to identify "critical failure areas" and
eliminate them from the system, a first in the food industry then. Baumann, a microbiologist by
training, was so pleased with Pillsbury's experience in the space program that he advocated for
his company to adopt what would become HACCP at Pillsbury. Soon, Pillsbury was confronted
with a food safety issue of its own when glass contamination was found in farina, a cereal
commonly used in infant food. Baumann's leadership promoted HACCP in Pillsbury for
producing commercial foods, and applied to its own food production. This led to a panel
discussion at the 1971 National Conference on Food Protection that included examining CCPs
and good manufacturing practices in producing safe foods. Several botulism cases were
attributed to under-processed low-acid canned foods in 1970–71. The United States Food and
Drug Administration (FDA) asked Pillsbury to organize and conduct a training program on the
inspection of canned foods for FDA inspectors. This 21-day program was first held in September

5
1972 with 11 days of classroom lecture and 10 days of canning plant evaluations canned food
regulations were first published in 1969. Pillsbury's training program to the FDA in 1969, titled
"Food Safety through the Hazard Analysis and Critical Control Point System", was the first time
that HACCP was used(Sperber et al., 2009).

HACCP was initially set on three principles, now shown as principles one, two, and four in the
section below. Pillsbury quickly adopted two more principles, numbers three and five, to its own
company in 1975. It was further supported by the National Academy of Sciences (NAS) that
governmental inspections by the FDA go from reviewing plant records to compliance with its
HACCP system (Sperber et al., 2009). A second proposal by the NAS led to the development of
the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) in 1987.
NACMCF was initially responsible for defining HACCP's systems and guidelines for its
application and were coordinated with the Codex Committee for Food Hygiene, that led to
reports starting in 1992 and further harmonization in 1997. By 1997, the seven HACCP
principles listed below became the standard (Sperber et al., 2009). A year earlier, the American
Society for Quality offered their first certifications for HACCP Auditors, (First known as
Certified Quality Auditor-HACCP, they were changed to Certified HACCP Auditor (CHA) in
2004). HACCP expanded in all realms of the food industry, going into meat, poultry, seafood,
dairy, and has spread now from the farm to the fork (Sperber et al., 2009).

2.1.2 Definition of HACCP

HACCP is a management system in which food safety is addressed through the analysis and
control of biological, chemical, and physical hazards from raw material production, procurement
and handling, to manufacturing, distribution and consumption of the finished product
(NACCMF, 1997). A hazard is any biological, chemical, or physical property that may cause an
unacceptable consumer health risk (unacceptable contamination, toxin levels, growth, and/ or
survival of undesirable organisms) while a critical control point is any point, process step or
activity where a potential hazard for food safety can be eliminated, prevented or reduced to an
acceptable level (WHO, 1996).For successful implementation of a HACCP plan, management
must be strongly committed to the HACCP concept. A firm commitment to HACCP by top
management provides company employees with a sense of the importance of producing safe
food. HACCP is designed for use in all segments of the food industry from growing, harvesting,
6
processing, manufacturing, distributing, and merchandising to preparing food for consumption.
Prerequisite programs such as current Good Manufacturing Practices (cGMPs) are an essential
foundation for the development and implementation of successful HACCP plans. Food safety
systems based on the HACCP principles have been successfully applied in food processing
plants, retail food stores, and food service operations. The seven principles of HACCP have been
universally accepted by government agencies, trade associations and the food industry around
the world (NACCMF, 1997).

2.2 Public Health Risks in Street Fermented Foods

Most handlers of street-vended foods in Africa and the developing world at large are largely
ignorant of basic food safety issues. Consequently, street foods are commonly exposed to
dangerous abuses often at all stages of handling. Products (from the raw materials to the finished
stages) are often exposed to contamination like soil, dust and sand. Other common real risk
factors include time-temperature abused involving handling prepared foods under unsafe storage
temperatures and serving such foods cold or without sufficient reheating. As previously noted by
Bryan et al. (1988) for example, street vendor often use stands and cart of crude and inefficient
construction and running water is seldom available at the location. Utensils washing is usually
done in one or more buckets or pan and sometimes without soap, disinfection is rarely carried
out. Waste water is usually discarded in streets and garbage is at times discarded near the food
stand. This provides attraction to food by rodents and insects. In addition to all these, toilet
facilities are seldom readily available. This compels food handlers to pass out body wastes in
nearby hidden places and often they return to business without proper washing of hands. Most
traditional street foods are present and delivered openly, without proper protective packaging
(Akobundu, 1996).

2.3 Codex Alimentarius Commission, (CAC)

CAC is a body that was established in 1963 by the food and agriculture organization of the
United Nations (FAO), the Codex Alimentarius Commission (CAC) of the Food and Agriculture
Organisation/World Health Organisation has been responsible for developing standards,
guidelines and other recommendations on the quality and safety of food to protect the health of
consumers and to ensure fair practices in food trade. The mission of the CAC remains relevant,

7
buta number of factors have shown the need for new techniques to form the basis of food
standards, the most important of which is risk analysis. The authors give a brief description of
the role and work of the CAC and the efforts deployed by the Commission to respond to the
challenges posed by new approaches to government regulation, harmonisation of national
requirements based on international standards and the role of civil society. The Codex
Alimentarius (Latin for ‘Book of Food’) is a collection of internationally recognized standards,
codes of practice, guidelines and other recommendation relating to foods, food production and
food safety. Its name is derived from the Codex Alimentarius Austriacus (FAO, 2009). Its texts
are developed and maintained by the codex Alimentarius Commission.

2.4 Guidelines for Effective HACCP Implementation

The following guidelines as given by NACMCF, (1997) will facilitate the development and
implementation of effective HACCP plans. While the specific application of HACCP to
manufacturing facilities is emphasized here, these guidelines should be applied as appropriate to
each segment of the food industry under consideration.

2.4.1 Prerequisite programs

The production of safe food products requires that the HACCP system be built upon a solid
foundation of prerequisite programs. Each segment of the food industry must provide the
conditions necessary to protect food while it is under their control. This has traditionally been
accomplished through the application of cGMPs. These conditions and practices are now
considered to be prerequisite to the development and implementation of effective HACCP plans.
Prerequisite programs provide the basic environmental and operating conditions that are
necessary for the production of safe, wholesome food. Many of the conditions and practices are
specified in federal, state and local regulations and guidelines (e.g., cGMPs and Food Code). The
Codex Alimentarius General Principles of Food Hygiene describe the basic conditions and
practices expected for foods intended for international trade. In addition to the requirements
specified in regulations, industry often adopts policies and procedures that are specific to their
operations. Many of these are proprietary. While prerequisite programs may impact upon the
safety of a food, they also are concerned with ensuring that foods are wholesome and suitable for
consumption (Appendix A). HACCP plans are narrower in scope, being limited to ensuring food

8
is safe to consume. The existence and effectiveness of prerequisite programs should be assessed
during the design and implementation of each HACCP plan. All prerequisite programs should be
documented and regularly audited. Prerequisite programs are established and managed separately
from the HACCP plan. Certain aspects, however, of a prerequisite program may be incorporated
into a HACCP plan. For example, many establishments have preventive maintenance procedures
for processing equipment to avoid unexpected equipment failure and loss of production. During
the development of a HACCP plan, the HACCP team may decide that the routine maintenance
and calibration of an oven should be included in the plan as an activity of verification. This
would further ensure that all the food in the oven is cooked to the minimum internal temperature
that is necessary for food safety.

2.4.2 Education and training

The success of a HACCP system depends on educating and training management and employees
in the importance of their role in producing safe foods. This should also include information on
the control of foodborne hazards related to all stages of the food chain. It is important to
recognize that employees must first understand what HACCP is and then learn the skills
necessary to make it function properly. Specific training activities should include working
instructions and procedures that outline the tasks of employees monitoring each CCP.
Management must provide adequate time for thorough education and training. Personnel must be
given the materials and equipment necessary to perform these tasks. Effective training is an
important prerequisite to successful implementation of a HACCP plan.

2.4.3 Developing a HACCP plan

The format of HACCP plans will vary. In many cases the plans will be product and process
specific. However, some plans may use a unit operations approach. Generic HACCP plans can
serve as useful guides in the development of process and product HACCP plans; however, it is
essential that the unique conditions within each facility be considered during the development of
all components of the HACCP plan. In the development of a HACCP plan, five preliminary tasks
need to be accomplished before the application of the HACCP principles to a specific product
and process. The five preliminary tasks are:

1. Assemble a HACCP team

9
 2. Describe the food and its distribution
3. Describe the intended use and consumers of the food
4. Develop a flow diagram which describes the process
5. Verify the flow diagram

2.4.3.1 Assemble a HACCP team: The first task in developing a HACCP plan is to assemble a
HACCP team consisting of individuals who have specific knowledge and expertise appropriate
to the product and process. It is the team’s responsibility to develop the HACCP plan. The team
should be multi-disciplinary and include individuals from areas such as engineering, production,
sanitation, quality assurance, and food microbiology. The team should also include local
personnel who are involved in the operation as they are more familiar with the variability and
limitations of the operation. In addition, this fosters a sense of ownership among those who must
implement the plan. The HACCP team may need assistance from outside experts who are
knowledgeable in the potential biological, chemical and/or physical hazards associated with the
product and the process. However, a plan which is developed totally by outside sources may be
erroneous, incomplete, and lacking in support at the local level. Due to the technical nature of the
information required for hazard analysis, it is recommended that experts who are knowledgeable
in the food process should either participate in or verify the completeness of the hazard analysis
and the HACCP plan. Such individuals should have the knowledge and experience to correctly:
(a) conduct a hazard analysis; (b) identify potential hazards, identify hazards which must be
controlled; (d) recommend controls, critical limits, and procedures for monitoring and
verification; (e) recommend appropriate corrective actions when a deviation occurs; (f)
recommend research related to the HACCP plan if important information is not known; and (g)
validate the HACCP plan.

2.4.3.2 Describe the food and its distribution: The HACCP team first describes the food. This
consists of a general description of the food, ingredients, and processing methods. The method of
distribution should be described along with information on whether the food is to be distributed
frozen, refrigerated, or at ambient temperature.

2.4.3.3 Describe the intended use and consumers of the food: Describe the normal expected
use of the food. The intended consumers may be the general public or a particular segment of the
population (e.g., infants, immunocompromised individuals, the elderly, etc.).
10
2.4.3.4 Develop a flow diagram which describes the process: The purpose of a flow diagram is
to provide a clear, simple outline of the steps involved in the process. The scope of the flow
diagram must cover all the steps in the process which are directly under the control of the
establishment. In addition, the flow diagram can include steps in the food chain which are before
and after the processing that occurs in the establishment. The flow diagram need not be as
complex as engineering drawings. A block type flow diagram is sufficiently descriptive. Also, a
simple schematic of the facility is often useful in understanding and evaluating product and
process flow.

2.4.3.5 Verify the flow diagram: The HACCP team should perform an on-site review of the
operation to verify the accuracy and completeness of the flow diagram. Modifications should be
made to the flow diagram as necessary and documented.
After these five preliminary tasks have been completed, the seven principles of HACCP are
applied.

2.5 Seven Principles of HACCP

According to National Advisory Committee on Microbiological Criteria for Foods, NACMCF,

(1997), the following are the seven principles of HACCP:

 Principle 1: Conduct a hazard analysis

 Principle 2: Determine critical control points (CCPs)
 Principle 3: Establish critical limits
 Principle 4: Establish monitoring procedures
 Principle 5: Establish corrective actions
 Principle 6: Establish verification procedures
 Principle 7: Establish record-keeping and documentation procedures

2.5.1 Principle 1: Conduct a hazard analysis

After addressing the preliminary tasks discussed above, the HACCP team conducts a hazard
analysis and identifies appropriate control measures. The purpose of the hazard analysis is to
develop a list of hazards which are of such significance that they are reasonably likely to cause
injury or illness if not effectively controlled. Hazards that are not reasonably likely to occur

11
would not require further consideration within a HACCP plan. It is important to consider in the
hazard analysis the ingredients and raw materials, each step in the process, product storage and
distribution, and final preparation and use by the consumer. When conducting a hazard analysis,
safety concerns must be differentiated from quality concerns. A hazard is defined as a biological,
chemical or physical agent that is reasonably likely to cause illness or injury in the absence of its
control. Thus, the word hazard as used in this document is limited to safety. A thorough hazard
analysis is the key to preparing an effective HACCP plan. If the hazard analysis is not done
correctly and the hazards warranting control within the HACCP system are not identified, the
plan will not be effective regardless of how well it is followed.

The process of conducting a hazard analysis involves two stages. The first, hazard identification,
can be regarded as a brain storming session. During this stage, the HACCP team reviews the
ingredients used in the product, the activities conducted at each step in the process and the
equipment used, the final product and its method of storage and distribution, and the intended
use and consumers of the product. Based on this review, the team develops a list of potential
biological, chemical or physical hazards which may be introduced, increased, or controlled at
each step in the production process. Hazard identification focuses on developing a list of
potential hazards associated with each process step under direct control of the food operation. A
knowledge of any adverse health-related events historically associated with the product will be
of value in this exercise. After the list of potential hazards is assembled, stage two, the hazard
evaluation, is conducted.

In stage two of the hazard analysis, the HACCP team decides which potential hazards must be
addressed in the HACCP plan. During this stage, each potential hazard is evaluated based on the
severity of the potential hazard and its likely occurrence. Severity is the seriousness of the
consequences of exposure to the hazard. Considerations of severity (e.g., impact of sequelae, and
magnitude and duration of illness or injury) can be helpful in understanding the public health
impact of the hazard. Consideration of the likely occurrence is usually based upon a combination
of experience, epidemiological data, and information in the technical literature. When conducting
the hazard evaluation, it is helpful to consider the likelihood of exposure and severity of the
potential consequences if the hazard is not properly controlled. In addition, consideration should
be given to the effects of short term as well as long term exposure to the potential hazard. Such

12
considerations do not include common dietary choices which lie outside of HACCP. During the
evaluation of each potential hazard, the food, its method of preparation, transportation, storage
and persons likely to consume the product should be considered to determine how each of
thesefactors may influence the likely occurrence and severity of the hazard being controlled. The
team must consider the influence of likely procedures for food preparation and storage and
whether the intended consumers are susceptible to a potential hazard. However, there may be
differences of opinion, even among experts, as to the likely occurrence and severity of a hazard.
The HACCP team may have to rely upon the opinion of experts who assist in the development of
the HACCP.

2.5.1.1 Examples of Questions to be Considered When Conducting a Hazard Analysis

The hazard analysis consists of asking a series of questions which are appropriate to the process
under consideration. The purpose of the questions is to assist in identifying potential hazards.

a. Ingredients

1. Does the food contain any sensitive ingredients that may present microbiological hazards (e.g.,
Salmonella, Staphylococcus aureus); chemical hazards (e.g.,aflatoxin, antibiotic or pesticide
residues); or physical hazards (stones, glass, metal)?

2. Are potable water, ice and steam used in formulating or in handling the food?

3. What are the sources (e.g., geographical region, specific supplier)

b. Intrinsic Factors - Physical characteristics and composition (e.g., pH, type of acidulants,
fermentable carbohydrate, water activity, preservatives) of the food during and after processing.

1. What hazards may result if the food composition is not controlled?

2. Does the food permit survival or multiplication of pathogens and/or toxin formation in the
food during processing?

3. Will the food permit survival or multiplication of pathogens and/or toxin formation during
subsequent steps in the food chain?

13
4. Are there other similar products in the market place? What has been the safety record for these
products? What hazards have been associated with the products?

c. Procedures used for processing

1. Does the process include a controllable processing step that destroys pathogens? If so, which
pathogens? Consider both vegetative cells and spores.

2. If the product is subject to recontamination between processing (e.g., cooking, pasteurizing)

and packaging which biological, chemical or physical hazards are likely to occur?

d. Microbial content of the food

1. What is the normal microbial content of the food?

2. Does the microbial population change during the normal time the food is stored prior to
consumption?

3. Does the subsequent change in microbial population alter the safety of the food?

4. Do the answers to the above questions indicate a high likelihood of certain biological hazards?

e. Facility design

1. Does the layout of the facility provide an adequate separation of raw materials from ready-to-
eat (RTE) foods if this is important to food safety? If not, what hazards should be considered as
possible contaminants of the RTE products?

2. Is positive air pressure maintained in product packaging areas? Is this essential for product
safety?

3. Is the traffic pattern for people and moving equipment a significant source of contamination?

f. Equipment design and use

14
1. Will the equipment provide the time-temperature control that is necessary for safe food?

2. Is the equipment properly sized for the volume of food that will be processed?

3. Can the equipment be sufficiently controlled so that the variation in performance will be
within the tolerances required to produce a safe food?

4. Is the equipment reliable or is it prone to frequent breakdowns?

5. Is the equipment designed so that it can be easily cleaned and sanitized?

6. Is there a chance for product contamination with hazardous substances; e.g., glass?

7. What product safety devices are used to enhance consumer safety?

8. To what degree will normal equipment wear affect the likely occurrence of a physical hazard
(e.g., metal) in the product?

9. Are allergen protocols needed in using equipment for different products?

g. Packaging

1. Does the method of packaging affect the multiplication of microbial pathogens and/or the
formation of toxins?

2. Is the package clearly labeled “Keep Refrigerated” if this is required for safety?

3. Does the package include instructions for the safe handling and preparation of thefood by the
end user?

4. Is the packaging material resistant to damage thereby preventing the entrance of microbial
contamination?

5. Are tamper-evident packaging features used?

6. Is each package and case legibly and accurately coded?

7. Does each package contain the proper label?

8. Are potential allergens in the ingredients included in the list of ingredients on thelabel?
15
h. Sanitation

1. Can sanitation have an impact upon the safety of the food that is being processed?

2. Can the facility and equipment be easily cleaned and sanitized to permit the safe handling of
food

3. Is it possible to provide sanitary conditions consistently and adequately to assure safe foods?
i. Employee health, hygiene and education
1. Can employee health or personal hygiene practices impact upon the safety of the food being
processed?
2. Do the employees understand the process and the factors they must control to assure the
preparation of safe foods?

3. Will the employees inform management of a problem which could impact upon safety of
food?

j. Conditions of storage between packaging and the end user

1. What is the likelihood that the food will be improperly stored at the wrong temperature?
2. Would an error in improper storage lead to a microbiologically unsafe food?
k. Intended use
1. Will the food be heated by the consumer?
2. Will there likely be leftovers?

l. Intended consumer
1. Is the food intended for the general public?

2. Is the food intended for consumption by a population with increased susceptibility to illness
(e.g., infants, the aged, the infirmed, immunocompromised individuals)?

3. Is the food to be used for institutional feeding or the home?

2.5.2 Principle 2: Determine critical control points (CCPs)

A critical control point is defined as a step at which control can be applied and is essential to
prevent or eliminate a food safety hazard or reduce it to an acceptable level. The potential
16
hazards that are reasonably likely to cause illness or injury in the absence of their control must be
addressed in determining CCPs. Complete and accurate identification of CCPs is fundamental to
controlling food safety hazards. The information developed during the hazard analysis is
essential for the HACCP team in identifying which steps in the process are CCPs. One strategy
to facilitate the identification of each CCP is the use of a CCP decision tree. Although
application of the CCP decision tree can be useful in determining if a particular step is a CCP for
a previously identified hazard, it is merely a tool and not a mandatory element of HACCP. A
CCP decision tree is not a substitute for expert knowledge.

Critical control points are located at any step where hazards can be either prevented, eliminated,
or reduced to acceptable levels. Examples of CCPs may include: thermal processing, chilling,
testing ingredients for chemical residues, product formulation control, and testing product for
metal contaminants. CCPs must be carefully developed and documented. In addition, they must
be used only for purposes of product safety. For example, a specified heat process, at a given
time and temperature designed to destroy a specific microbiological pathogen, could be a CCP.
Likewise, refrigeration of a precooked food to prevent hazardous microorganisms from
multiplying, or the adjustment of a food to a pH necessary to prevent toxin formation could also
be CCPs. Different facilities preparing similar food items can differ in the hazards identified and
the steps which are CCPs. This can be due to differences in each facility’s layout, equipment,
selection of ingredients, processes employed, et cetra.

2.5.3 Principle 3: Establish critical limits

A critical limit is a maximum and/or minimum value to which a biological, chemical or physical
parameter must be controlled at a CCP to prevent, eliminate or reduce to an acceptable level the
occurrence of a food safety hazard. A critical limit is used to distinguish between safe and unsafe
operating conditions at a CCP. Critical limits should not be confused with operational limits
which are established for reasons other than food safety. Each CCP will have one or more
control measures to assure that the identified hazards are prevented, eliminated or reduced to
acceptable levels. Each control measure has one or more associated critical limits. Critical limits
may be based upon factors such as: temperature, time, physical dimensions, humidity, moisture
level, water activity, pH, titratable acidity, salt concentration, available chlorine, viscosity,
preservatives, or sensory information such as aroma and visual appearance. Critical limits must
17
be scientifically based. For each CCP, there is at least one criterion for food safety that is to be
met. An example of a criterion is a specific lethality of a cooking process such as a 5D reduction
in Salmonella. The critical limits and criteria for food safety may be derived from sources such
as regulatory standards and guidelines, literature surveys, experimental results, and experts.

2.5.4 Principle 4: Establish monitoring procedures

Monitoring is a planned sequence of observations or measurements to assess whether a CCP is

under control and to produce an accurate record for future use in verification. Monitoring serves
three main purposes. First, monitoring is essential to food safety management in that it facilitates
tracking of the operation. If monitoring indicates that there is a trend towards loss of control,
then action can be taken to bring the process back into control before a deviation from a critical
limit occurs. Second, monitoring is used to determine when there is loss of control and a
deviation occurs at a CCP, i.e., exceeding or not meeting a critical limit. When a deviation
occurs, an appropriate corrective action must be taken. Third, it provides written documentation
for use in verification. An unsafe food may result if a process is not properly controlled and a
deviation occurs. Because of the potentially serious consequences of a critical limit deviation,
monitoring procedures must be effective. Ideally, monitoring should be continuous, which is
possible with many types of physical and chemical methods. For example, the temperature and
time for the scheduled thermal process of low-acid canned foods is recorded continuously on
temperature recording charts. If the temperature falls below the scheduled temperature or the
time is insufficient, as recorded on the chart, the product from the retort is retained and the
disposition determined as in Principle 5. Likewise, pH measurement may be performed
continually in fluids or by testing each batch before processing. There are many ways to monitor
critical limits on a continuous or batch basis and record the data on charts. Continuous
monitoring is always preferred when feasible. Monitoring equipment must be carefully calibrated
for accuracy.

2.5.5 Principle 5: Establish corrective actions

The HACCP system for food safety management is designed to identify health hazards and to
establish strategies to prevent, eliminate, or reduce their occurrence. However, ideal
18
circumstances do not always prevail and deviations from established processes may occur. An
important purpose of corrective actions is to prevent foods which may be hazardous from
reaching consumers. Where there is a deviation from established critical limits, corrective actions
are necessary. Therefore, corrective actions should include the following elements: (a) determine
and correct the cause of non-compliance; (b) determine the disposition of non-compliant product
and (c) record the corrective actions that have been taken. Specific corrective actions should be
developed in advance for each CCP and included in the HACCP plan. As a minimum, the
HACCP plan should specify what is done when a deviation occurs, who is responsible for
implementing the corrective actions, and that a record will be developed and maintained of the
actions taken. Individuals who have a thorough understanding of the process, product and
HACCP plan should be assigned the responsibility for oversight of corrective actions. As
appropriate, experts may be consulted to review the information available and to assist in
determining disposition of non-compliant product.

2.5.6 Principle 6: Establish verification procedures

Verification is defined as those activities, other than monitoring, that determine the validity of
the HACCP plan and that the system is operating according to the plan. The NAS (1985) pointed
two out that the major infusion of science in a HACCP system centers on proper identification of
the hazards, critical control points, critical limits, and instituting proper verification procedures.
These processes should take place during the development and implementation of the HACCP
plans and maintenance of the HACCP system.

2.5.7 Principle 7: Establish record-keeping and documentation procedures

Generally, the records maintained for the HACCP System should include the following:

1. A summary of the hazard analysis, including the rationale for determining hazards and control
measures.

2. The HACCP Plan

i. Listing of the HACCP team and assigned responsibilities.

ii. Description of the food, its distribution, intended use, and consumer.
iii. Verified flow diagram.
19
iv. HACCP Plan Summary Table that includes information for:
a) Steps in the process that are CCPs.
b) The hazard(s) of concern.
c) Critical limits
d) Monitoring
e) Corrective actions
f) Verification procedures and schedule
g) Record-keeping procedures.

20
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area

Wudil local government area, Kano State of Nigeria is located on the latitude 11o 8.2127’N and
longitude 8o 5’E of the Greenwich Meridian. It is surrounded by Warawa local government to
the west and north, Gaya to the east, Garko and Albasu to the south. The 2006 census puts the
population of the area at 185,189 with an estimate land mass of 362km². Wudil town is the local
government’s headquarter which is strategically located on river Wudil and the river is the main
source of water supply in the town (NIPOST, 2006). KUST Mini-market is a market in Kano
university of Science and Technology, Wudil campus. The campus is located along Gaya road,
in Wudil town. The market was made for students and staffs shopping in the campus.

3.2 Sample Collection

Samples were collected from two different food processors after explaining to them that the
research will not be detrimental to their business to persuade them. The food processors were
individually surveyed some questions about how they prepare the food, how long does the
preparation take and the source of water they used. Samples of the food raw materials at different
stages of processing (namely; washing stage, cold steeping, after wet milling and sieving, raw
ogi), of processed ready-to-sell pap, after adding condiment (particularly sugar) to the pap, as
well as after storage of the pap for 6hours were collected from the first processor (sample A) and
were labeled as sample A1, A2, A3, A4, A5, A6, A7 (that is, washing stage (A1), cold steeping
(A2), after wet milling and sieving (A3), raw ogi (A4), processed ready-to-sell pap (A5), after
adding condiment (A6), after storage for 4-6hours (A7)). They were collected aseptically, placed
in sterile container, held in ice pack and were taken to the laboratory for analysis. For taking
sample at each step, the previously used sampling instrument is washed, dipped in 95% alcohol
and flamed, the latter two steps are repeated three times each.

The same samples were collected after two days from the second processor (sample B) and were
labeled as sampe B1, B2, B3, B4, B5, B6, B7 and were also taken to the laboratory for analysis.
Water samples used in the food preparation were collected for analysis and were labeled as

21
sample A and B for first and second processor respectively. Based on information obtained from
the answers given to the questions above by the processors and observing the production
processes of each processor, a flow diagram of the common preparation procedure employed by
the processors was prepared to provide a clear, simple, and complete description of all the steps
involved in the production process. Then control sample (sample C), was prepared in the
laboratory by adopting the preparation steps. Gas cooker was used for cooking of the pap. Faro
water was used for the food preparation.

The control sample was prepared by washing millet grains with portable water. The cleaned
millet grains are cold steeped in water at room temperature (25±20C) for 24hours. The steeping
water is decanted and the fermented grains were washed with clean water and then wet-milled.
The bran was removed by wet sieving and the sieved was allowed to settle for another 48hours, a
process referred to as souring during which fermentation also proceeds and solid starchy matter,
raw Ogi, gasara in Hausa was produced. Required quantity of the raw ogi was put in a sterilized
bowl and using sterilized metal spoon mixed with cold water to form a watery paste. The paste is
then added to a boiling water on an open fire, stirred well to form a semi-solid gruel and then
boiled further for 10-15 minutes. After this further boiling, the pap is formed, samples of ready
to sell pap and of storage for 6hours were collected at this step. However, in the control another
pap was prepared from the same raw ogi but, in the later prepared pap, condiment was added
while cooking the pap and condiment-added pap was sample was collected. The samples were
labeled as sample C1, C2, C3, C4, C5, C6 and C7 as in sample A and B above.

3.3 Sample and Media Preparation

Stock culture was prepared from each step ranging from the millet washing to the stored pap for
sample A, B and C. Serial decimal 10 fold dilution was done by transfering one millimeter of
initial suspension (10-1) into a tube containing 9ml of sterile 0.1% (w/v) peptone water. The
mixture was then homogenized to make 10 -2 dilution, these operations were repeated all through
to 10-5 dilutions. Different sterile syringes were used for each dilution as well as for each sample.

All the media used were prepared according to the manufacturer's instruction.

22
3.4 Hazard analysis

The hazard analysis method of Bryan (1992), was adopted. The hazard analysis consists of
observing the raw materials used, the surroundings processing environment, the preparation
processes and the storage practices of the pap to identify sources and modes of actual or potential
contamination. Different samples were collected and subsequently analyzed for the presence of
microorganisms with potential risk to human health.

3.5 Identification of Critical Control Point

The critical control point decision tree as developed by the Codex Alimentarius Committee on
Food Hygiene (CAC, 1993) and modified by (Dillon et al., 1995) was used to identify the critical
control points out of the trivial hazards points identified during the hazard analysis.

3.6 Microbiological Analysis

3.6.1 Isolation and enumeration of microorganisms

Pour plate method was used for sample inoculation. 1ml of the sample different dilution was
inoculated into a Petri dish, then a cooled molten agar medium was poured into the inoculated
plate. The mixture was mixed thoroughly by moving the Petri dish in a circular motion gently.
The mixture in the plates was allowed to solidify and the plates were inverted and incubated at
370C for 24 – 48 hours for bacteria. Fungi were incubated at room temperature for 48-96hours.

Total microbial counts were counted differently for each of the parameter used. The Aerobic
Plate Count was counted on Nutrient agar, Staphylococcal counted on Mannitol salt agar (MSA),
Coliform count on Eosin Methylene blue agar (EMB), Lactobacillus count on deManne Rogossa
& Sharpe Agar (MRS) while mold and yeast count was done on Sabouraud Dextrose agar
(SDA). The counts that were found to be less than 30 were termed too few to count (TFTC)
while those above 300 were termed too numerous to count (TNTC).

Colony forming Unit per milliliter of the sample (CFU/ml) was obtained by using the following
formula as

Colony Forming Unit per milliliter (CFU/ml) =CFU/ml

23
CFU/ml= (Average colony count/Volume plated) x Dilution factor

Colonies from a sector of incubated plates were picked, purified by subculturing to obtain pure
culture before being examined microscopically for Gram reaction, cell morphology, motility,
pigmentation and sporulation (Harrigan and McCance, 1995).

Confirmation of coliform organisms was carried out by inoculating colonies into lactose broth
with Durham tubes and incubating at 37oC and 44 oC for 24h and another 24h in the absence of
gas production. The presence of gas constituted a presumptive test and the broth was streaked out
on EMB agar incubated at 37oC for 42h. Typical colonies on EMB plates appearing bluish black
with greenish metallic sheen which are characteristics of Escherichia coli or brownish colonies
often convex and mucoid which are characteristics of Enterobacter aerogenes confirmed the
presence of coliform organisms (Cheesbrough, 2003).

Total coliforms count in the water samples was determined using MPN technique (Cheesbrough,
2006) as follows:

a. Presumptive test

1 ml of the water sample was inoculated into test tubes containing 9ml of Lactose broth with
Durham tubes which was incubated at 37°C for 24 to 48 h. Gas production within 24-48h
indicated a positive presumptive test while absence of gas production indicates negative
presumptive test. The appearance of air bubbles in Durham tubes before incubation was not
confused with the actual gas production. The presence of acid production within 48 h of
incubation is indicated by the change in color, showing a positive presumptive test.

b. Confirmatory test

A loopful each of the water sample in the positive presumptive test tubes was streaked on Eosin
Methylene Blue and incubated at 37°C for 24 h. Growth of bacterial colonies indicated that the
confirmed test is positive while absence of growth was considered as negative.

c. Completed test

Melted MacConkey agar was distributed into plates using pour plates technique and allowed to
solidify. A loopful of broth from each of the positive tubes was streaked on each of the
24
MacConkey agar plates. The plates were incubated at 37°C for 24 h. Gram stained preparation
from slant cultures corresponding to the positive tubes that showed acid and gas formation were
thoroughly examined.

3.6.2 Identification of isolates

3.6.2.1 Macroscopic and microscopic examination: The colonies were observed for
macroscopic characteristics such as colony size, shape, color, texture.

The colonies were then gram stained and observed microscopically for gram staining reaction,
cell morphology, sporulation, motility.

Gram staining method as described by Cheesbrough, (2006) was followed as:

a. Preparation of bacterial smear

h) Drop a sterile distilled water on center of a clean grease-free glass slide

i) Take a small portion of the colony and emulsify it on the slide make a thin
preparation on the slide.

j) When a broth culture is used, transfer a loopful to a slide to make the thin preparation.

k) Allow the smear to air-dry completely.

l) Rapidly pass the slide, smear uppermost, three times through the flame of a spirit
lamp or pilot flame of a Bunsen burner.

m) Allow the smear to cool before staining it.

b. Gram staining:

5. Cover the fixed smear with crystal violet stain for 30–60 seconds.

6. Rapidly wash off the stain with clean water.

7. Tip off all the water, and cover the smear with Lugol’s iodine for 30–60 seconds.

8. Wash off the iodine with clean water.

25
 9. Decolorize rapidly (few seconds) with acetone–alcohol. Wash immediately with clean
water.

10. Cover the smear with neutral red stain for 2 minutes.

11. Wash off the stain with clean water.

12. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-
dry.

13. Examine the smear microscopically using oil immersion objective lens (x100) to
observe the bacteria.

Gram positive bacteria appear purple while gram-negative bacteria appear reddish.

Fungal isolates were stained with cotton-blue lacto-phenol and observed microscopically for cell
shape, size and sporulatio. A portion of the colony was picked using wire loop and was
emulsified with cotton-blue lacto-phenol on a clean grease-free glass slide. The slide was
covered with cover-slip and observed using x10 objective lens, and x40 objective lens for
confirmation. Fungi were identified using fungal atlas (Banigo, 1993).

3.6.2.2 Biochemical tests: Bacteria were identified on the basis of the results obtained from
biochemical tests, analyzed using Bergey’s manual of systematic bacteriology (Sneath et al.,
1990). The tests include indole test, catalase test, oxidase test, coagulase test, citrate utilization
test, Methyl-Red Vogue's Parker (MR-VP) test. The tests are carried out as follows:

a. Indole test

The test demonstrates the ability of some bacteria especially Gram negative to decompose
tryptophan (an amino acid) to indole which is present in tryptone soya broth. Sterilized Tryptone
soya broth (TSB) was inoculated with bacterial isolates in test tubes and incubated at 37 oC for 72
hours. After the incubation period, 2ml of chloroform was added to the broth cultures in each test
tube and was shaken gently; thereafter 2ml of kovac’s reagent was added to each test tube and
also shaken gently. The test tubes were returned to the test tube rack and were allowed to stand
for about 20 minutes. A red colouration on the surface layer indicated indole production by the
bacterial isolate which is a positive result (Cheesbrough, 2006).
26
b. Catalase test

Most aerobic microorganisms produce catalase enzyme that is capable of reacting with hydrogen
peroxide (H2O2 ) to release oxygen. A few drops (i.e. 2 to 3 drops) of 3% hydrogen peroxide
were placed in the middle of each clean slide and sterilized inoculating loop was used to pick
bacterial isolate from pure culture. This was used to make a smear on the slide containing
hydrogen peroxide. The occurrence of effervescence caused by liberation of oxygen indicated
production of catalase by the bacteria (Cheesbrough, 2006).

c. Oxidase test

This test indicates the presence of cytochrome c oxidase that is able to reduce oxygen (O 2) and
artificial electron acceptors (Joanne et al., 2008). A drop of 1% tetra methyl-p-phenylenediamine
hydrogen chloride was dropped on a filter paper. Fresh culture of the isolate was then rubbed on
the filter paper and observed. A possible result was indicated by a purple colour change within
10 seconds.

d. Coagulase test

Smears of various bacterial isolates were made on different slides and a drop of plasma was
added. The slide was rocked for about 10 seconds and observed immediately for indications of
agglutination or clumping. The clumping of organism in about 10seconds indicated a positive
result (Cheesbrough, 2006).

e. Citrate Utilization test

The Simmon’s citrate agar slant in test tubes was used to carry out this test. Colony was picked
with a sterilized inoculating loop by flaming and was used to streak the slants in the test tubes.
The test tubes were incubated at 37 oC for 72 hours and the colour change from green to blue was
observed which indicated a positive result. No change indicated a negative result (Cheesbrough,
2006).

27
f. Methyl Red-Voges Proskauer (MR-VP) test

Test tubes containing sterilized MR-VP broth were inoculated with the isolate in duplicate and
incubated at 37oC for 72hours. After incubation, the MR-test and VP-test were performed on
each of the duplicate test tubes separately (Cheesbrough, 2006)

i. Methyl Red

To one of the test tubes, 5 drops of methyl red indicator was added and a change in colour was
watched out for. A bright red colour on the surface indicated a positive result while a yellow or
orange colour indicated a negative result.

ii. Voges Proskauer

To the second test tube, 1ml of 5% α-naphthol solution was added followed by 1ml of potassium
hydroxide (KOH) solution. The mixture was shaken and allowed to stand for some minutes and
observed. A red colour within 5 minutes was indicative of a positive reaction.

3.7 Statistical Analysis

Microbial load was determined using Log10 of Colony Forming Unit per milliliter (Log10CFU/ml)
of the samples. Data were subjected to analysis of variance (ANOVA) with the use of statistical
version for social siences (SPSS) version 22. The level of significance was taken as P=0.05.

28
 CHAPTER FOUR

4.0 RESULT AND DISCUSSION

4.1 Results

Table 4.1.1 shows the morphological, microscopic and biochemical characteristics of bacteria
isolated from the pap sample at stages of its preparation. The bacteria isolated include
Staphylococcus aureus, Streptococcus sp., Escherichia coli, Klebsiella sp., Pseudomonas sp.,
Lactobacillus spp.

Table 4.1.2 shows the macroscopic and microscopic characteristics of fungi isolated at different
stages of pap production. The fungi isolated include Penicillium spp., Aspergillus niger, A.
flavus, Mucor spp., Rhizopus spp., Fusarium sp. and Saccharomyces cereviciae.

Table 4.1.3 shows the distribution of the microorganisms at different stages of the pap
preparation steps.

Table 4.1.4 shows the mean microbial counts in Colony Forming Unit per milliliter (CFU/ml) at
different stages of the pap preparation for sample A, B and C.

A gradual reduction in pH was observed during fermentation for all the processors. The final pH
of the ready to raw uncooked raw ogi was 4.6, 4.3, and 3.9 for sample A, B, and C respectively.

Table 4.1.5 shows the mean microbial counts in log 10CFU/ml at different stages of the pap
preparation for sample A, B and C. The washed millet grains had mean TAPC ranging from
6.77log10CFU/ml for processor B to 7.03log10CFU/ml for processor A while that the TAPC for
control was 4.06log10CFU/ml. The ready to sell pap had TAPC ranging from 1.06log 10CFU/ml
for processor A to 2.10log10CFU/ml for processor B. Coliform counts of ready to sell pap was
1.33og10CFU/ml, 1.27log10CFU/ml and <1.00log10CFU/ml for processor A, B and Control
respectively. Lactic acid bacteria and yeasts were isolated at all stages of fermentation for all the
processors. Mould counts on the washed millet grains ranged from 1.20log 10CFU/ml to
3.21log10CFU/ml and 2.43log10CFU/ml for processor A, B and control respectively. After cold
steeping, the mould counts had reduced significantly (p=0.05) to <1x100log10CFU/ml for both
processor A, B and Control. Moulds were not isolated beyond steeping step for all the processors
as well as the control. Rapid reduction of pH during fermentation ensures the disappearance of
gram negative bacteria and other spoilage or pathogenic organisms which may be present in the
food due to the pH (Amoa- Awua et al., 2007).
Table 4.1.6 shows the hazards associated with each step in the production of the pap. Results
showed that the raw material (millet grains) is a source of contamination which can be due to
vegetative pathogens and moulds. Washing, cold steeping, wet milling and sieving are other
sources of contamination which can be due to physical contaminations, chemical and
microbiological quality of the water, handling and equipments used during steeping, wet milling
and sieving.

29
Flow charts showing hazards and CCP during production processes of pap was presented in
Fig.1

Table 4.1.7 shows the Coliforms count (MPN/100ml) determined using MPN technique as
described by Cheesbrough, (2006) of the water samples (sample A and B) collected from the
food processors. The results obtained showed that the water samples of all the food processors
are not within the range of satisfactory values of 1-3/100 ml of coliform count (but the most
acceptable limit is 0 coliform count which was stamped as Excellent) (Cheesbrogh, 2006). All
the water samples were unsatisfactory. This implies that they do not satisfy the World Health
Organization (W.H.O) standard requirements.

30
 Table 4.1.1 Morphological, microscopic and biochemical characteristics of the bacterial isolates

Characteristics 1st isolate. 2nd isolate 3rd isolate 4th isolate 5th isolate 6th isolate

Colony Moist and Low convex Pinkish on Large and moist Small rough, oval Small opaque
morphology yellow colony and discrete MacConkey and mucoid and greenish white colony on
on MSA agar agar and green colony on yellow colony on MRS agar
metallic sheen MacConkey agr MacConkey
colony on EMB
agar

Microscopy Cocci in cluster Cocci in Rod shaped Rod shaped Rod shaped Bacillus
chains

Gram reaction + + - - - +

Coagulase test + - - - - -

Catalase test + - + - + -

Indole test - - + + - -

MR/VP test +/+ -/- +/+ -/+ -/- -/-

Citrate test - - - + + -

Oxidase test - - + + -

Probable organism Staphylococcus Streptococcus Escherichia coli Klebsiella sp. Pseudomonas sp. Lactobacillus spp.
aureus sp.

____________________________________________________________________
Key + = Positive - = Negative, MSA=Mannitol Salt Agar, MRS Agar=de Mann Rogossa and Sharpe
Agar, EMB Agar=Eosine Methylene Blue Agar

31
Table 4.1.2 Morphology and Microscopy of the fungal Isolates.

S/N Morphological description Microscopic appearance Organism

1. Green or green-greyish color colonies. Brush-like conidiospore carries Penicillium sp.

conidia.

2. Yellow green surface with reddish- Green conidiospore with septate Aspergillus niger

brown underneath. hyphae.

3. Dark brown to black pigmentation. Thick septate hyphae with Aspergillus flavus

conidia borne in chains from

the sterigmata.

4. Black dusty and spongy colonies. Sporongiospores arise from Rhizopus sp.

long arching stolons opposite

rhizoids.

5. Colonies appears with pink or Spindlelike conidia, multicellular. Fusarium sp.

brown center and white edges.

6. Circular, white and creamy colonies. Single cells oval in shape with Saccharomyces cerevisiae

some cells showing budding.

7. White colonies that turns grey as it Sporongiospore arises singly Mucor sp.

grow older. from mycelium at any point, all

branches terminate at sporangia.

Table 4.1.3 Microorganisms distribution in the various stages of the pap production.

Preparation steps
32
 Isolates Washing Cold Wet milling Raw ogi Ready to Addition of Storage of
steeping & Sieving sell pap condiment pap for
≥6hours

Staphylococcus + + + + + + +
aureus

Streptococcus sp. + + + + + + +

Escherichia coli + + + + + + +

Pseudomonas sp. + + + + - + +

Klebsiella sp. + + + + - + +

Lactobacillus + + + + + + +
spp.

Saccharomyces + + + + - - -
cerevisiae

Aspergillus flavus + - - - - - -

Aspergillus niger + - - - - - -

Penicillium sp. + - - - - - -

Rhizopus sp. + - - - - - -

Fusarium sp. + - - - - - -

Mucor sp. + - - - - - -

____________________________________________________________________
Key + = present, - = absent

Table 4.1.4 Mean microbial counts in Colony Forming Unit per milliliter (CFU/ml) at different stages of
the pap preparation with pH value of each steps for sample A, B and C.

33
Sample Processing steps CFU/ml
TAPC SC CC LBC YC MC pH

A Washing 1.07x107 5.37x106 2.58x102 2.95x106 1.82x106 1.58x10¹ 6.3

Cold steeping 1.20x107 2.57x106 3.63x102 2.75x106 3.16x106 <1x100 4.9

Wet milling & 1.35x106 2.09x106 4.57x102 2.57x107 1.05x105 <1x100 5.5
sieving
1.05x105 4.37x105 8.71x104 7.08x108 4.37x105 <1x100 4.6
Raw ogi

Ready to sell pap

1.14x10¹ 1.58x10¹ 2.12x10¹ 8.51x105 7.41x103 <1x100 5.3
Addition of
condiment 2.29x103 3.55x103 8.51x102 1.29x107 1.32x104 <1x100 4.9

After storage for

6hours
5.25x106 3.31x106 3.47x104 9.55x107 4.47x104 <1x100 4.4

B Washing 5.89x106 1.07x105 1.92x102 2.19x106 5.25x106 1.62x103 6.7

Cold steeping 6.46x106 3.47x106 4.68x102 2.51x106 4.47x106 <1x100 5.9

Wet milling & 1.86x106 2.69x106 1.26x103 3.24x107 2.88x105 <1x100 6.1
sieving
1.29x105 5.37x104 7.59x103 1.02x109 1.45x106 <1x100 4.3
Raw ogi

Ready to sell pap

1.27x102 7.94x101 1.86x101 5.50x106 1.02x104 <1x100 5.2
Addition of
condiment 2.57x104 2.82x104 2.63x102 6.17x107 2.69x104 <1x100 4.8

After storage for

6hours
6.76x106 1.26x107 1.91x104 1.74x108 1.02x105 <1x100 4.5

C Washing 1.15x104 1.58x102 1.51x10¹ 1.26x105 1.32x104 2.69x102 6.4

Cold steeping 4.27.x103 6.61x10¹ 1.02x10¹ 1.58x106 1.48x105 <1.00x10¹ 5.6

Wet milling & 1.15x102 4.37x10¹ <1.00x10¹ 1.78x107 7.41x105 <1.00x10¹ 4.8
sieving
1.15x102 4.37x10¹ <1.00x10¹ 1.78x107 7.41x105 <1.00x10¹ 3.9
Raw ogi
<1.00x10¹ <1.00x10¹ <1.00x10¹1.15x105 1.51x103 <1.00x10¹ 4.7
Ready to sell pap
<1.00x10¹ <1.00x10¹ <1.00x10¹ 1.23x105 1.58x103 <1.00x10¹ 4.5
Addition of
condiment

for <1.00x10 <1.00x10 <1.00x10 1.00x10 1.29x10 <1.00x10 4.3

¹ ¹ ¹ 5 3 ¹
After storage

34
 6hours

KEY: TAPC=Total Aerobic Plate Count, SC=Staphylococcal count, CC=Coliform count,

LBC=Lactobacillus Count, YC=Yeast Count, MC=Mould Count

Table 4.1.5 Mean microbial counts in log10CFU/ml at different stages of the pap preparation with pH
value of each steps for sample A, B and C.

Sample Processing steps CFU/ml

35
 TAPC SC CC LABC YC MC pH

A Washing 7.03 6.73 2.41 6.47 6.26 1.20 6.3

Cold steeping 7.08 6.41 2.56 6.44 6.50 <1.00 4.9

Wet milling & sieving 6.13 6.32 2.66 7.41 5.02 <1.00 5.5

Raw ogi 5.02 5.64 4.94 8.85 5.64 <1.00 4.6

Ready to sell pap 1.06 1.20 1.33 5.93 3.87 <1.00 5.3

Addition of condiment 3.36 3.55 2.93 7.11 4.12 <1.00 4.9

After storage for 6hours 6.72 6.52 4.54 7.98 4.65 <1.00 4.4

B Washing 6.77 5.03 1.75 6.34 6.72 3.21 6.7

Cold steeping 6.81 6.54 2.67 6.40 6.65 <1.00 5.9

Wet milling & sieving 6.27 6.43 3.10 7.51 5.46 <1.00 6.1

Raw ogi 5.11 4.73 3.88 9.01 6.16 <1.00 4.3

Ready to sell pap 2.10 1.90 1.27 6.74 4.01 <1.00 5.2

Addition of condiment 5.41 4.45 2.42 7.79 4.43 <1.00 4.8

After storage for 6hours 7.83 7.10 4.28 8.24 5.01 <1.00 4.5

C Washing 4.06 2.20 1.18 5.10 4.12 2.43 6.4

Cold steeping 3.63 1.82 1.01 6.20 5.17 <1.00 5.6

Wet milling & sieving 3.98 2.01 1.22 6.37 4.98 <1.00 4.8

Raw ogi 2.06 1.64 <1.00 7.25 5.87 <1.00 3.9

Ready to sell pap <1.00 <1.00 <1.00 5.06 3.18 <1.00 4.7

Addition of condiment <1.00 <1.00 <1.00 5.09 3.20 <1.00 4.5

After storage for 6hours <1.00 <1.00 <1.00 5.00 3.11 <1.00 4.3

Table 4.1.6 Process identification of microbiological hazards for pap production

Processing Hazard Source

steps

36
1.Washing step Vegetative pathogens & moulds Storage of millet grains, food-

handlers, water.

2. Cold steeping Vegetative pathogens Water, environment, food-

handlers, utensil.

3. Wet milling & Vegetative pathogens Water, environment, milling-

sieving machine, food handlers.

4. Raw ogi Acid tolerant pathogens and Water, food handlers.

facultative anaerobes

5.Ready to sell Heat resistants pathogens Food handlers, environment,

pap milling machine, water.

6. After addition Vegetative pathogens Condiment added, food-

of condiment handlers, utensil.

7. After storage Vegetative pathogens Environment, food handlers.

for 6hours

Table 4.1.7 Estimation of number of coliforms using MPN standard (Monica Cheesbrough, 2006).

Samples Coliform count using MPN per Remarks

100ml

Sample A 11 Unsatisfactory

37
 Sample B 14 Unsatisfactory

4.2 Discussion

The HACCP approach determines quickly and relatively cheaply the points in food processes
that are critical to safety, while taking into account local habits and culture (Abdulsalam and
Kaferstein, 1994). All of the pap processors surveyed in this study are females and none of the
processors surveyed acquired the knowledge of pap production training through any formal
education. This agrees with the report of Motarjemi, (2002) that the developments in food
fermentation have been based on experience gained through trial and error by consecutive
38
generations of food producers and households who have used the technology for the domestic
preparation and preservation of foods. Though this lack of formal education by processors
presents a major pitfall because most of the processors lack a comprehensive understanding of
the underlying principles of the fermentation process and the requirements for ensuring quality
and safety. This may lead to unsafe products depending on the process, environmental conditions
and the condition of the raw materials (Motarjemi, 2002).

Water used for the washing, steeping, milling and sieving of the pap ingredient as well as
cooking the pap and washing cooking utensils by all the food processors was borehole water. In
several developing countries including Nigeria, the primary challenge is the lack of water, rather
than its quality (Ehiri et al., 2001). All the processors when asked about why borehole water was
their source of water simultaneously highlighting the advantage of tap water to them, said that
the taps often go dry for days and for that reason they just relies on borehole water always. As
part of good hygienic practice, safe water should be used in processing pap and for washing
hands and utensils so as to minimize the final bacterial load. The result of Coliforms count in
MPN/100ml determined using MPN technique as described by Cheesbrough, (2006) of the water
samples, Sample A and B collected from the first and second food processors respectively was
11/100ml and 14/100ml of coliform count. This showed that the water used by all the food
processors are not within the range of satisfactory values of 1-3/100 ml of coliform count (but
the most acceptable limit is 0 coliform count which was stamped as Excellent) (Cheesbrogh,
2006). All the water samples were unsatisfactory. This implies that they do not satisfy the World
Health Organization (W.H.O) standard requirements. The underground water supplies are
usually considered safe provided they are properly located, constructed and operated according
to the World Health Organization Guidelines for Drinking Water (WHO, 1976). Boreholes as a
low-cost technology option for domestic water supply in developing countries are generally
considered as ‘safe sources’ of drinking water. However, it is the collection, transportation,
storage and decanting of the water that can lead to subsequent contamination. Most pathogens
that can contaminate water supplies come from the feces of humans or animals (Edema and
Omemu, 2001).

The isolation of Escherichia coli and Staphylococcus aureus, streptococcus sp. at different stages
during processing and also in the raw ogi could be attributed to contamination from processors,

39
water used for steeping and sieving, utensils and probably the animals present in the environment
(Omemu and Aderoju, 2008). Several reports had shown that food handling personnel play
important role in ensuring food safety throughout the chain of food production, processing, and
storage (Umoh et al., 1999; Omemu et al., 2005; Omemu and Aderoju, 2008).

Isolation of Klebsiella sp. and Pseudomonas sp. at stages of the pap preparation could be
attributed to the water uses in the production process as well as the food handlers. One of the
species in genus Klebsiella is Klebsiella pneumoniae. It is an important opportunistic pathogen
that causes a variety of infectious diseases in humans, including septicaemia, liver abscesses,
diarrhea, and pneumonia (Cao et al., 2014; Guo et al., 2017). It is a well-known hospital-
acquired pathogen and associated with increased patient morbidity and mortality (Brisse et al.,
2009; Cabral et al., 2012). In addition to the clinical environment, K. pneumoniae is frequently
found in foods including raw vegetables, powdered infant formula, meat, fish, and street foods,
and has been considered as an important food-borne pathogen (Overdevest et al., 2014 ; Kim et
al., 2015 ; Davis and Price, 2016). In powdered infant formula, K. pneumoniae is included in the
hazard identification category “B” according to the FAO and WHO guidelines on
microorganisms (FAO-WHO, 2004). In recent years, an increasing number of food-borne
outbreaks caused by K. pneumoniae have been reported in different countries (Calbo et al., 2011;
Tambekar et al., 2011).

Faparusi, (1989), have also reported the presence of Sacchaomyces cerevisiae, Candida spp, and
Aspergillus spp isolated on cereal grains. The difference in the types isolated at different stages
of production by the different producers might likely be due to different environmental
conditions under which the pap was prepared and the type of organisms each individual producer
might be harbouring. The presence of moulds such as Aspergillus niger, Penicillium sp., Mucor
sp. and Rhizopus sp. on the surfaces of millet grains and during the early stage of fermentation
has been reported (Odunfa and Adeyele, 1985; Omemu et al., 2007a,b). They are most likely part
of the grains surface microflora that is undesirable in many foods because of their mycotoxin
producing potentials Jonsyn, 1991; Jespersen et al., 1994). However, the moulds were no longer
isolated after cold steeping the millet grains due to the reduction in the pH caused by
fermentation process. Aspergillus niger and Aspergilus flavus were the most isolated mould
during washing step at which the moulds were isolated. Sacharomyces cerevisiae dominate at the

40
later stage of the fermentation process. Anon, (1984), reported the same trend; this may likely be
as a result of increased acidity. The increased acidity may provide some assurance of
microbiological safety of the product, as most of the organisms count reduced with increased
acidity. However, the number of bacteria isolates at different stages from different producer are
higher than either the yeast or mould isolated, this could be due to environmental factors and the
extent of contamination together with the fact that bacteria grow faster than yeast and fungi.

Isolation of Lactic acid bacteria is due to the fermentation process taking place in the pap
production processes. Lactic acid bacteria (such as Lactobacillus plantarum) and yeast (such as
Saccharomyces cerevisiae) have been shown to predominantly involved in fermentation of ogi in
the production of pap, thus, playing roles in aroma development, microbial stability and flavour
enhancement (Omemu et al., 2008). Lactic acid fermentation also plays important roles in
reducing anti-nutritional factors, increasing nutrient density and antimicrobial antimicrobial
activities in fermented product (Osungbaro, 2009).

In the washing and cold steeping steps, the high microbial count observed for all the food
processors as compared to that of control could be due to contamination from environment,
water used in the processes as well as the food handlers. Also, the microbial load in the first
processor was higher than that of the second processor. This could be due to the different
processing environment of the processors as well as their personal hygiene and the water used
during the preparation (Onyekwere, 1997). However, the increase in microbial load in the cold
steeping step as compared to the load in washing step could be due to the environment and
prolonged period of storage in the step. Mould count after the steeping was drastically reduced in
all the processors as well as the control because of the fact that the reduction in the pH may not
support the growth of the moulds.

In wet milling and sieving step, coliform count had increased significantly (p=0.05) with
+0.10log10CFU/ml for processor A and +0.43log10CFU/ml for processor B as well as Total
aerobic plate count. This could be due to the water used in the processes. In the control, the
microbial load in the wet milling & sieving step increased as compared to the cold steeping step
of the control despite the fact that portable water was used during the steps but the contamination
could be due to the environment, the milling machine and the milling machine operator.

41
In the raw ogi formation step, an increase in the Lactobacillus and yeast count was observed with
increase in Lactobacillus count of +1.44log10CFU/ml for processor A and +1.50log10CFU/ml for
processor B due to the fermentation process in the step. The Total aerobic plate count,
staphylococcal count and coliform count has significantly reduced at this step. The increase in
the Lactobacillus count as well as the decrease in the Total aerobic plate count, staphylococcal
count and coliform count could be due to the increase in the acidity and the aerobic condition of
the fermenting medium, favoring the growth of only facultative aerobes and or aciduric
organisms. It may also be as a result of the inhibitory effect of antimicrobial products from the
lactic acid bacteria in the growth of unwanted harmful spoilage organisms (Motarjemi et al.,
2002). Toxic substances such as Diacetyl, organic acid and bactericin have been shown to be
released by lactic acid bacteria in the fermenting medium during food fermentation. These
substances are toxic to the pathogenic organisms that may be present in the fermenting substrates
(Omemu et al., 2008). However, a high reduction was observed in the control because of the fact
that the control was fermented for 48hours, thus the reduction of the pH and the increased
anaerobic condition will be higher as compared to the processors that carries out the fermentation
in less than 24hours.

In the ready to sell pap, the microbial load has drastically reduced in all the food processors as
compared to the microbial load in the raw ogi. This is due to the heat treatment of the raw ogi to
form the pap. The total aerobic plate count of 1.06log10CFU/ml and 2.10log10CFU/ml recorded
for the cooked ready to sell pap from processors A and B respectively could be attributed to the
survival of spores which could have come initially from the environment, food ingredients and
food handlers or by reduction of but not total elimination of large number of vegetative cells that
contaminated the raw foods and food ingredients (Obuekwe and Ogbimi, 1989). However, a very
drastic decrease in the microbial load was observed in the control because of the fact that the
heat treatment in the control was done in the range 10-15minutes as opposed to that of the two
processors whose heat treatment was done for less than 10 minutes. Thus, proper time and
temperature control in this step is critical in reducing the contamination of the food (Inabo et al.,
2000). It is highly recommended that after this step, the pap is served hot or kept under suitable
temperature, if it is not to be served at that time so as to avoid contamination. In case of the local
food processors, if the storage of the food at suitable temperature cannot be achieved, the food
should be reheated before serving.
42
In the addition of condiment to the ready to sell pap step, an increase in microbial load was
observed in all the food processors. Total aerobic plate count of producer A and B increased
significantly (p=0.05) ranging from 1.06log10CFU/ml and 2.10log10CFU/ml in ready to sell pap
to 3.36log10CFU/ml and 5.41log10CFU/ml after adding the condiment respectively, even though
the microbial load was drastically reduced during cooking of the pap. However, in the control, no
increased in the microbial load was observed because of the fact that the condiment was added
during cooking of the pap in contrast to its addition after the cooking. This implies that the
addition of the condiment after heat treatment could cause contamination of the food mainly due
the poor hygiene of the condiment or the food handlers adding the condiment. But when the
condiment is added during cooking of the pap if the pap is to be served with the condiment, the
microbial load associated with the condiment and the food handlers adding the condiment will be
eliminated by the heat treatment.

In the storage step, the stored samples (sample A and B) at ambient temperature for 6hours, had
the microbial load increased significantly (p=0.05). Total aerobic plate count increased with
+5.70log10CFU/ml and +5.73log10CFU/ml in processor A and B respectively as compared to the
Total aerobic plate count in the ready to sell pap of the two samples from the processors even
though no condiment was added to the stored pap. However, the microbial load in the stored
control pap had even decreased as compared to that of the ready to sell pap due to the fact that it
was stored in a refrigerator at temperature of -4oC. Prolonged period of storage of foods have
been shown to affect the level of contamination of the food. Where food is allowed to stand in
high ambient temperatures after being prepared (due to lack of refrigeration facilities)
considerable multiplication of pathogenic bacteria may occur, increasing the risk of diarrhea
(Black et al., 1999). This situation is particularly critical when the foods are consumed without
reheating and when reheating temperatures are typically well below levels capable of destroying
pathogens.

The Critical control points identified are cooking of the pap, addition of condiment after heat
treatment and storage at ambient temperature for ≥6hours. These CCPs were identified using
CCP decision tree. The first; cooking was identified as CCP because of the fact that there is
preventive measure of controlling hazard in the processes, that is; by cooking the pap at the right
temperature and time control. However, if it was done inappropriately, the contamination could

43
exceed the acceptable limits eventually why because the next steps could not reduce the hazards
been contaminated with to an acceptable level. Addition of condiment before heat treatment was
also identified as CCP because of the fact that the condiment added could introduce a hazardous
contaminants in to the food either from the condiment itself and or the food handlers eventually
such hazard might exceed the acceptable limits and there is no subsequent step that can reduce or
eliminate them. However, the control or preventive measure at this CCP is adding the condiment
during heat treatment to ensure total elimination or reduction of the hazards that might occur due
to the addition of the condiment. Storage at ambient temperature for ≥6hours was also identified
as CCP because of the fact that prolonged storage of food at ambient temperature affects its
contamination level. However, the control at this CCP is storing the food at temperature of about
-4OC so as to prevent the multiplying of some microorganisms that may be hazardous in the food.

CHAPTER FIVE

5.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS

44
5.1 Summary

HACCP is a management system in which food safety is addressed through the analysis and
control of biological, chemical, and physical hazards from raw material production, procurement
and handling, to manufacturing, distribution and consumption of the finished product
(NACCMF, 1997). Hazard Analysis and Critical Control Points (HACCP) as long been
internationally recognized and accepted as the system for effective food safety
management(CAC,2003).It is a systematic preventive approach to food safety that addresses
physical, chemical and biological hazards as a means of prevention rather than finished product
inspection. It is used in the food industry to identify potential contamination and subsequently
evaluating that the process is in control of those points or steps of the food chain critical to food
safety and key actions known as the Critical Control Points (CCPs). A critical control point is
defined as a step at which control can be applied and is essential to prevent or eliminate a food
safety hazard or reduce it to an acceptable level. The potential hazards that are reasonably likely
to cause illness or injury in the absence of their control must be addressed in determining CCPs.
CCP is taken to eliminate risk of the hazards been realized. Based on risk-assessment, HACCP
plans allow both industry and government to allocate their resources efficiently in establishing
and auditing safe food production practices (Okonko et al., 2009).

The hazard analysis consists of observing the raw materials used, the surroundings processing
environment, the preparation processes and the storage practices of the pap to identify sources
and modes of actual or potential contamination. Different samples were collected and
subsequently analyzed for the presence of microorganisms with potential risk to human health.

Pap known as koko in Hausa; a fermented, cereal gruel from millet is a staple food of several
communities in Nigeria. It is traditionally made from maize, sorghum or millet. Several reports
had identify steeping and souring as the two fermentation stages involved in the traditional
preparation of Ogi. The pap is considered the most important weaning food for infants to
compliment breast milk from 4-6months of age in West Africa although it is consumed by adults
(Banigo 1993; Moses et al., 1993; Onyekwere et al., 1993).

Most handlers of street-vended foods in Africa and the developing world at large are largely
ignorant of basic food safety issues. Consequently, street foods are commonly exposed to

45
dangerous abuses often at all stages of handling. Products (from the raw materials to the finished
stages) are often exposed to contamination like soil, dust and sand. Other common real risk
factors include time-temperature abused involving handling prepared foods under unsafe storage
temperatures and serving such foods cold or without sufficient reheating

5.2 Conclusion

Hazard analysis critical control point approach in quality management can be used in the
preparation of the traditional fermented foods. The identification of microbiological hazards and
critical control points in this study has revealed that many factors contributed to the
contamination of pap. The critical control points identified will be a point for all the processors
of this food and similar to take maximum care so that safe food could be produce, which will
increase consumer confidence and in turn more patronage. The presence of spoilage and
contamination organisms is an indication that the food is not produced under good hygiene
practices and poses a serious health risk to its direct consumers. The HACCP approach therefore,
has been shown to identify areas of concern where failure has not yet been experienced, making
it particularly useful for new operation. Water used in the production process can also be a major
source of microbial contamination of the food at various the stages of the food production.
Despite the unhygienic wet-milling and sieving processes involved in the traditional preparation
of pap, the low pH of the fermented food would make it safe for consumption. However,
appropriate safety measures and good manufacturing practices will best ensure good quality of
the food.

5.3 Recommendation

The following recommendations should be mapped out and embarked upon in order to reduce to
an acceptable level or totally eliminate microbial contamination in the production of pap, “koko”
and other traditional fermented foods. Some of these recommendations have been approved as
regulations by the Department of Health Education & Welfare, Public Health Services and Food
& Drug Administration (FDA) and local agency, National Agency for Food and Drug
Administration and Control (NAFDAC) over current trends in the manufacturing, processing,
packaging or holding of human food generally referred to as GMPs (Good Manufacturing
Practices). The recommendations are:

46
1. A strict microbiological safety measures should be employed during production period of
the food and post production processes, thus, contamination should be avoided in order to
ensure a safe food to the consumer. HACCP system should therefore be introduced and
enforced in the traditional preparation of fermented foods.
2. The local producers of pap should be enlightened on the importance of observing these
regulations – GMPs.
3. Infected personnel should be restricted from participating in the production of the food.
4. Portable water should be used during the production of the food.
5. Better preservative techniques should be used to prolong shelf life of the food.
6. The regulatory agencies especially the NAFDAC (National Agency for Food and Drugs
Administration and Control) should ensure strict compliance to the regulations by the
grass root.
7. The government of the federation should map out extensive public enlightenment
campaign especially in the rural areas to educate both the local producers and consumers
of the pap.
8. Finally, the government should also introduce the use of probiotics to the local producers
of pap and sell to them at a very subsidized rate. The probiotics are meant to mimic the
normal microbial flora in humans like the ones found on breast milk that offer protection
against diseases.

REFERENCES

47
Abdulsalam, M. and Kaferstein, F.K., (1994). Food safety in primary health care. World Health
Forum,15: 393–399.

Akingbala, J.O., Rooney, L.W. and Faubion, J.M. (1989). A Laboratory procedure for the
preparation of Ogi, a Nigerian fermented food. Journal of food science 46(5):
1523- 1526.

Akobundu, E.N.T. (1996).Packaging of street foods. Paper delivered at workshop on street foods
organized by Nigeria Institute of Food Science and Technology, University of
Agriculture, Umudike, Nigeria.

Altekruse, S.F., Street, D.A., Fein, S.B. and Levy, A.S. (1995). Consumer knowledge of Food-
borne microbial hazards and food handling practices. Journal of Food Protection
59:287- 294.

Amoa-Awua, W., Ngunjiri, P., Anlobe, J., Kpodo, K., Halm, M., Hayford, A.E., Jakobsen, M.,
(2007). The effect of applying GMP and HACCP to traditional food processing at
a semi-commercial kenkey production plant in Ghana. Food Control 18: 1449–
1457

Anon, I., (1984). Factors affecting life and death of microorganisms. New York: Academic
press. Microbiology of Foods, Vol 1.

Banigo, E.O.I. (1993). Nigerian Ogi in Handbook of indigenous fermented foods. K.H.
Steinkraus (Ed) pp 212-222. New York: Marcel Dekker.

Beumer, R.E. and Giffel, M.C. (1999).Pathogens in domestic kitchens, facts and fiction, In:
Tuijellers, A.C.T., Samson, R.A., Ronbouts, F.M., Notermans, S.(Eds0.Food
Microbiology and Food Safety into the next millennium. zest. The Netherlands,
pp.345-347.

Black, R.E., Lopez de Romana, K.H.G., Brrown, N. B., Bazalar, O.G. and Kanashiro, H.C.,
(1999). The incidence and etiology of infantile diarrhea and major routes of
transmission in Huascar, Peru. American Journal of Epidemiology 189: 785-799.

48
Bri, X., Xu, W.Y., (2013). An investigation of food poisoning caused by Klebsiella pneumoniae .
Chin. Pract. Med. 8, 275– 276. 10.3969/ j.issn.1673-7555.2013.26.224.

Brisse, S., Fevre, C., Passet, V., Issenhuthjean, S., Tournebize, R., Diancourt, L., (2009).
Virulent clones of Klebsiella pneumoniae : identification and evolutionary scenario based on
genomic and phenotypic characterization. PLoS ONE 4:e4982. 10.1371/journal.pone.0004982.

Bryan, F.L., Michanie, S.C., Alvarez, P. and Paniagua, A. (1988). Critical Control Points of
street vended food in the Dominican Republic. J. Food Prot. 51:373-383.

Bryan, F.L., Teupel, P., Riaz, S., Roohi, S., Quadar, F. and Malik, Z., (1992). Hazard analysis
and critical control points of food preparation and storage in homes in a village in
Pakistan. Journal of Food Protection 55 (9): 703-713.

CAC (Codex Alimentarius Commiission). (2003). General principles on food hygiene.

CAC/RCP 1-1969.

Calbo, E., Freixas, N., Xercavins, M., Riera, M., Nicolás, C., Monistrol, O., (2011). Foodborne
nosocomial outbreak of SHV1 and CTX-M-15-producing Klebsiella pneumoniae : epidemiology
and control. Clin. Infect. Dis. 52, 743–749. 10.1093/cid/ciq238 .

Cao, X., Xu, X., Zhang, Z., Han, S., Chen, J., Zhang, K., (2014). Molecular characterization of
clinical multidrug- resistant Klebsiella pneumoniae isolates. Ann. Clin. Microbiol. Antimicrob.
13, 1– 6. 10.1186/1476-0711-13-16.

Caplice, E. and Fitzgerald, G.F. (1999).Food fermentations: role of microorganisms in food

production and preservation. International Journal of Food Microbiology 50:131-
149.

CAST (Council for Agriculture, Science and Technology). (1994). Food borne pathogens: risks
and consequences. Task force report, Vol 122 AMES (IA). Washington, DC.

CODEX (1993). Codex Alimentarius Commission: Codex Committee on Food Hygiene.

Guideline for the application of hazard anlysis critical control point (HACCP) system (Alinorm
19/ 13A, Appendex B), Food and Agriculture Organization/WHO, Rome
http://www.codexalimentarius.net
49
Corgan, T.A., Bloomsfield, S., Humphery, T.J. (2001). Campylobacter and Salmonella
contamination levels on hands and kitchen surfaces following the preparation of
raw chicken in domestic kitchens and after cleaning. Annual symposium of the
society for Applied Microbiology, Swansea.

Davis, G.S., Price, L.B. (2016). Recent research examining links among Klebsiella pneumoniae
from food, food animals, and human extraintestinal infections. Curr. Environ. Health Rep. 3, 1–
8. 10.1007/s40572-016-0089-9.

Dillon, M. and Griffith, C., (1995). How to HACCP, an illustrated guide. MD Associate, 34a
Hainton Avenue, Grimsby, UK pp 18.

Ehiri, J.E., Azubuike, M.C., Ubbaonu, C.N., Anyanwu, E.C., Ibe, K.M., Ogbonna, M.O., (2001).
Critical control points of complementary food preparation and handling in eastern
Nigeria. Bulletin of the World Health Organization, 79: 423-433.

Etok, O.E. (1998). The street food trade in Africa: Safety and Socio- environmental issues. Food
Control 9(4):211- 215.

FAO&WHO (2006) FAO/WHO guidance to governments on the application of HACCP in small

and/or less- developed food business. FAO Food and Nutrition paper,no.86. ISSN
0254
4725,pp53,http//www.who.int/foodsafety/publications/fsmanagement/HACCP_S
LDB.pdf.

Faparusi, S.I., (1989). Sugar changes during production of burukutubeer. Journal of Science of
Agriculture. 21: 79-8

FSAI (Food Safety Authority of Ireland). (1998).Public awareness of knowledge and attitudes of
Food Safety in Ireland. In; Gorman, R., Bloomfield, S., Adley, C.C. (Eds).A study
of cross contamination of food Borne pathogens in the domestic kitchen in the
republic of Ireland. International Journal of Food Microbiology, Vol 76. Elsevier
Science, Amsterdam, pp.143-150.

50
FSAI (Food Safety Authority of Ireland). (1998).Public awareness of knowledge and attitudes of
Food Safety in Ireland. In; Gorman, R., Bloomfield, S., Adley, C.C. (Eds).A study
of cross contamination of food Borne pathogens in the domestic kitchen in the
republic of Ireland. International Journal of Food Microbiology, Vol 76.Elsevier
Science, Amsterdam, pp.143-150.

Gorman, R., Bloomsfield, S. and Adley, C.C. (2002).A study of cross contamination of food
borne pathogens in the kitchen in the Republic of Ireland. International Journal of
Food Microbiology 76:143-150.

Guo, Y., Wang, S., Zhan, L., Jin, Y., Duan, J., Hao, Z., et al. . (2017). Microbiological and
clinical characteristics of hypermucoviscous Klebsiella pneumoniae isolates associated with
invasive infections in China. Front. Cell. Infect. Microbiol. 7:24. 10.3389/ fcimb.2017.00024.

Halm, M., Lillie, A., Sorensen, A.K and Jakobsen, M. (1993).Microbiology and aromatic
characteristics of fermented maize dough for ken key production in Ghana.
International Journal of Food Microbiology 19:135-145.

Inabo, H.I., Ogbadu, L.J., Umoh, V.J., Ameh, J.B., (2000). Microbiological quality of selected
marketed condiments. Namoda Techscope J. 4: 20-30.

Jespersen, L., Halm, M., Kpodo, K., Jacobsen, M., (1994). Significance of yeasts and moulds
occurring in maize dough fermentation for kenkey production. International
Journal of Food Microbiology 24: 239-248.

Joanne, M., Linda, M., Christopher, J., (2008). Prescott, Harley and Klan's Microbiology, 7th
Edition. McGraw-Hill publishers, New York. ISBN:978-0-07-299291-5.

Jonsyn, F.E., (1991). Fungi associated with selected fermented foods in Sierria Leone. MIRCEN
Journal of Applied Microbiology and Biotechnology 5(4): 457-462.

Keith, H.S. (1997). Classification of fermented food. In: Worldwide review of household
fermentation techniques. Food Control Vol 8(5/6) pp 311-317.

Kim, H.S., Chon, J.W., Kim, Y. J., Kim D. H., Kim M. S., Seo K. H. (2015). Prevalence and
characterization of extended-spectrum-β-lactamase- producing Escherichia coli , and Klebsiella
51
pneumoniae , in ready-to-eat vegetables. Int. J. Food Microbiol. 207, 83–86.
10.1016/j.ijfoodmicro.2015.04.049.

Knabel, S.J. (1995).Food borne illness, role of home in food handling practices. Journal of Food
technology 49:119- 131.

Kok, M.S. (2009).Application of food safety management systems (ISO22000/HACCP) in the

Turkish poultry industry: a comparison based on enterprise size. Journal of food
Protection 72:2221-2225.

Lawley, R. (2007). Hygiene by design. Food Safety Watch.

http//www.foodsafetywatch.com/public/609.cfm.

Monica Cheesbrough (2006).District Laboratory Practice in tyggyg C66ountries Part 2 Second

Edition. Cambridge unversity press.page 38.

Moses, M.O., Mpuchane, S.F. and Murphy, O.M. (1993).Nigeria Ogi. In: handbook of
indigenous fermented foods K.H. Steinkraus(Ed) pp 212-222.New York: Marcel
Dekker.

Motarjemi, Y., Asante, A., (2002). Impact of small scale fermentation technology on food safety
in developing countries. International journal of Food Microbiology. 75, 213–
229.

Obuekwe, C.D., Ogbimi, A.O., (1989). Prevalence of Bacillus cereus and other Gram-positive
bacteria in Nigerian dried food condiments. Nig. Food J. 7: 11-19.

Odunfa, S.A., Adeyele, S., (1985). Microbiological changes during the traditional production of
ogi-baba, a West African fermented sorghum gruel. Journal of Cereal Science
3(2),173–180.

Okonko, I.O., Adejoye, O.D., Ogun, A.A., Ogunjobi, A.A., Nkang, A.O and Adebayo, T. (2009).
Hazard Analysis Critical Control Points and Microbiology qualities of sea foods as
affected by handlers’ hygiene in Ibadan and Lagos, Nigeria. African Journal of Food
Science Vol 3(2):035-050.

52
Omemu, A.M. and Aderoju, S.T., (2008). Food safety knowledge and practices of street food
vendors in the city of Abeokuta, Nigeria. Food Control 19, 396–402.

Omemu, A.M., Edema, M.O., Bankole, M.O., (2005). Bacteriological assessment of street
vended ready to eat (RTE) vegetables and pre-packed salad in Nigeria. Nigerian Journal
of Microbiology, 19(1–2), 497–504.

Omemu, A.M., Oyewole, O.B., Bankole, M.O., (2007a). Significance of yeasts in the
fermentation of maize for ogi production. Food Microbiology 24, 571–576.

Omemu, A.M., Oyewole, O.B., Bankole, M.O., Akintokun, A.K., (2007b). Yeasts and moulds
associated with ogi - a cereal based weaning food during storage. Research Journal of
Microbiology 2(2), 141-148.

Onyekwere, O.O., Akinrele, I.A and Koleoso, O.A.O. (1993). Industrialization of Ogi
fermentation. In: Industrialization of Indigenous Fermented Foods 33:329-362.

Onyekwere, O.O., Akinrele, I.A and Koleoso, O.A.O. (1997).In; K.H. Steinkraus(Ed).Handbook
of indigenous fermented foods pp 212-222.New York: Marcel Dekker.

Osungbaro, T.O. (2009). Physical and nutritive properties of fermented cereal foods. African
Journal of Food Science 3(2):023-027.

Overdevest, I. T., Heck, M., vander Zwaluw, K., Huijsdens, X., van Santen, M., Rijnsburger, M.,
et al. . (2014). Extended- spectrum β-lactamase producing Klebsiella spp. in chicken meat and
humans: a comparison of typing methods. Clin. Microbiol. Infect. 20, 251– 255. 10.1111/1469-
0691.12277.

Redmond, E.C and Griffiths, C.J. (2003). Consumer food handling in homes, a review of food
safety studies, Journal of food protection 66:130-161.

Roberts, D. (1982). Factors contributing to outbreaks of foods poisoning in England and Wales
between 1970-1999 .Journal of hygiene 89:491-498.

53
Ryan, M.J., Wall, P.G., Gilbert, R.J., Griffin, M. and Rowe, B. (1996). Risk factors for outbreaks
of infectious intestinal disease linked to domestic catering. Communicable
Disease report, CDR. Review, vol.13, pp. R179-R182.

Sperber, William, H., Stier, M., Richard, F., (December, 2009). "Happy 50th Birthday to
HACCP: Retrospective and Prospective". Food Safety magazine. pp.42-46.

Tambekar, D.H., Kulkarni R.V., Shirsa,t S.D., Bhadange, D.G., (2011). Bacteriological quality
of street vended food Pani Puri: a case study of Amravati city (MS) India. Biosci. Disc. 2, 350–
354.

54