QUALITY ASSESSMENT OF HONEY EXTRACTED USING TRADITIONAL METHODS FROM SOME HONEY BEARING COMMUNITIES OF KADUNA STATE

QUALITY ASSESSMENT OF HONEY EXTRACTED USING TRADITIONAL METHODS FROM SOME HONEY BEARING COMMUNITIES OF KADUNA STATE

ABSTRACT

Twelve commercial honey samples obtained using five different traditional methods of honey extraction, from some honey bearing communities of Kaduna State Nigeria, were analysed using laboratory analytical procedures and ANOVA statistical tool to determine the effects of traditional methods of honey extraction on the products quality parameters. The parameters determined alongside their range for the honey samples, included moisture content (15.50 – 22.00%), ash content (0.50 – 0.85%), pH (3.44 – 4.16), total acidity (0.12 – 0.37%), reducing sugar (50.08 – 54.20%), sucrose (26.61 – 28.80%), density (1322.35 – 1382.16g/L) and water insoluble content (1.30 – 1.70%) were obtained. For the microbiological characteristics; total bacteria plate count (1.7 x 102 – 9.8 x 103 cfu/g), total coliform count (1.3 x 102 – 7.7 x 103 cfu/g) mould count (2.0 x 101 – 1.5 x 103 cfu/g), while both yeast and E. coli were not detected in any of the samples. The ANOVA result revealed there were significant differences both within the methods of extraction and the percentage mean values of the quality parameters of commercial honey samples extracted and marketed in different retail outlets in Kaduna State. Although the honey samples had good shelf life and fairly – good physical properties, they, however, contained high water insoluble contaminants such as honey-wax;  had been adulterated with other substances like sugarcane sugar or corn syrup; and were also highly susceptible to attack by pathogenic micro organisms and fermentation. To address these challenges, it was recommended that commercial bee farmers and honey producers in the state be mobilized into forming a strong cooperative society to easily access cooperate supports via enlightenment; retraining, capital (grants or loans) support from the Nigerian Agriculture, Cooperative and Rural Development Bank (NACRDB) to acquire standard extraction equipment and facilities, and the establishment of honey monitoring and quality assessment centres by the state government, among others.


CHAPTER 1
INTRODUCTION
1.1             BACKGROUND OF THE STUDY
It has been reported that honey stored, processed and sealed by honey bees in the cells of their comb is always of good quality, but the different management methods and techniques of honey harvesting, extraction, and storage greatly influence the quality and marketing of the product (Mutsaers, 1991). Hence,  _honey as the most important product of beekeeping both from a quantitative and an economic points of view_has been defined as the natural sweet substance  produced by honey bees from nectar of plants (nectar honey), or from secretions of living parts of plants, or excretions of plant sucking insects on the living parts of plants (honeydew honey) which honey bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature (Bogdanov, et al., 2001). Thus, the importance and uses of honey is parallel to the history of man and in virtually every culture. Evidence can be found of some of its enormous uses as: an ancient valuable food and food derivative; as a medicinal aid for fighting infections (as an ointment for rashes, wounds and burns, and to help soothe sore throats) and diseases (as a comprehensive treatment of diabetic ulcers); and as a symbol employed in religious, magic and therapeutic ceremonies (Jennifer, 2007; Mark, 2008, Molan, 1999).

Consequently, owing to the vast applications and benefits derivable from honey, the current total annual world production of the product (honey) stands at, 1,134,000 tonnes; with the exception of some counties, like Nigeria_whose production level of honey is predominantly still on a subsistence and seasonally - small_scale commercial classification (Honey Travelers, 2011), and whose bulk of honey producers widely adopt the traditional methods of honey extraction. Mutsaers (1991), therefore, further added that some of these traditional methods of honey extraction can easily damage the quality of the product when poorly applied. For instance, he stated that methods which use heat, fire or boiling of honey will produce honey that is dark, dirty and has a reduced medicinal and health values. Thus, he concluded by quoting; “After having gone through so much care and trouble to produce high quality honey, it is a pity to spoil it by poor extraction methods”.

In addition, Honey Travelers (2011) remarked that unheated “raw” honey contains all the vital ingredients that give it its healthful properties and wonderful aroma; unfortunately, most commercial honey one sees in supermarkets and retail outlets in Kaduna State is not raw honey, this mass – produced honey is often heated by some traditional extraction methods, such as solar melting, to temperatures far above the normal temperatures of the bee hive (350C); in order to make the product (honey) easier to extract from the honey comb, to filter it and to package it among other reasons. This extraction method, thus changes honey’s essential composition and degrades its quality by partially destroying honey’s beneficial enzymes and boils off volatile compounds (vitamins such as Riboflavin,  Niacin and Pantothenic) that account for the unique delicate flora aroma of the honey (Gheldof, et al., 2002). Furthermore,  although, it is widely believed that honey, in its natural state in the honey comb, cannot spoil nor borne disease – causing – organisms, but some traditional methods of honey extraction, such as hand – squeezing  and sieving, easily expose the product to direct bacteria infestation mainly through dirty hands and unhygienic processing vicinity. Thus, increasing the risk of possible health hazards, such as Botulism and Honey intoxication, to its consumers (Food and Drug Administration, FDA, 2012 and The National Honey Board, 2012).

Unarguably, wholesomeness of food products that guarantee the safety of the consumers is the most important attribute of food quality (Oyinlola, 2004). Hence, in order to have a comprehensive quality assessment of any food product, it is of great necessity that the areas of purity, safety, economics, taste, appearance, prevailing legislative regulation, and consumer expectations are adequately examined (Ojeme, 1990). Although most marketers and consumers of honey in Kaduna state had been using some traditional physical techniques; such as match-stick-test to estimate the degree of moisture of honey, pouring honey into a transparent glass cup of cold water to determine its viscosity, putting drop of honey on the floor whether ants will approach it or not, to estimate its sugars level; to assess the quality of honey. All these techniques are highly questionable and unreliable. Unfortunately, not all marketers and consumers of these traditionally extracted honey can easily and quickly ascertain some of these intrinsic quality parameters of the product. Therefore, this project intends to identify and analyze the various traditional methods of honey extraction _ using some honey bearing communities of Kaduna state, Nigeria, as case study _ and any adverse effects such methods may have on the quality parameters of the product.

1.2       STATEMENT OF THE PROBLEM
Some of the most prominent problems associated with the need for the study are as follow:
1.2.1   Excessive moisture content of extracted honey; thereby enhancing rapid fermentation of the product.

1.2.2   Thinning of honey viscosity via the application of excessive heat, fire or boiling to ease extraction and packaging processes; thereby producing honey that is dark, dirty and has a reduced medicinal and health values.
1.2.3   Lack of personal hygiene (poor hand washing), and carrying out honey extraction in unhygienic environments; thereby making the product easily susceptible to germs infestation.
1.2.4   Use of unsuitable containers or extraction equipment that have not been properly sterilized or not compatible with the acidic nature of honey; thereby giving the product unpleasant odour and impure texture.
1.2.5   Adulteration of honey by the addition of corn or sugar-cane syrups, or any other contaminants to augment the quantity of the extracted product for much profitability; thereby posing grave danger to the health of ignorant consumers.



1.3       AIM AND OBJECTIVES OF THE STUDY
1.3.1   AIM OF THE STUDY
The study aims at carrying out a quality assessment of honey samples extracted, using traditional methods, from some honey bearing communities of Kaduna state, Nigeria.

1.3.2   OBJECTIVES OF THE STUDY
The study hopes to achieve the following objectives;
(i)                Collect samples of honey extracted using different traditional methods:
(ii)             Carryout laboratory analysis on the samples collected;
(iii)           Assess the results obtained and compare them with the quality template of honey, as laid down by the Nigerian Industrial Standard (NIS 473:2003).
(iv)           Ascertain its suitability for both human and industrial consumption
(v)              Make feasible recommendations on the results analysed, where necessary.

1.4       SIGNIFICANCE OF THE STUDY
The study, when concluded and all its recommendations carefully implemented, will assist in achieving the following:
(i)                Reveal the level of conformity of the quality of honey extracted using traditional methods and equipment with the standard laid down by the Nigerian Industrial Standard (NIS 473:2003)
(ii)             Ascertain doubts about the hygienic conditions of honey – extraction – containers and other processing equipment, so as to determine whether or not the product is safe for human consumption.
(iii)           Honey samples(s) whose overall quality parameters determined successfully meet up with the range recommended by the Nigerian Industrial Standard (NIS 473:2003), can be safely used as sugar substitute in confectioneries, and as health enhancer in medicine.
(iv)           Adequate information about the overall quality parameters of honey samples, at public domain, will enhance the marketability of the product, and also add value to the local honey businesses.

1.5       SCOPE OF THE STUDY
The study focuses on honey extracted using traditional methods Floating, Honey pressing, Hand squeezing, Solar melting (heating), and Sieving methods obtained from Ankwa, Doka, Giwa, Kafancha, Katari, Kukui, Mando and Sabon gari honey-bearing- communities of Kaduna State, Nigeria. The laboratory analysis of the quality parameters; the moisture content, ash content, total acidity (lactic acid), pH, sucrose content, reducing sugar level, water insoluble content, density, and microbiological characteristics of the samples collected will be carried out at the Departments of Agricultural Engineering; Food Technology; and Applied science; all of Kaduna Polytechnic. The result of the analysis will be compared with the Nigerian Industrial Standard (NIS 473;2003) for honey and feasible recommendations for further actions, presented.

CHAPTER II
LITERATURE REVIEW
2.0       HONEY EXTRACTION
            Mustsaers (1991) defined honey extraction as the central process in bee keeping of removing honey from honey comb, so that it is isolated in pure liquid form. This invariably, implies that whichever method of honey extraction that may be adopted by any honey producer be it the traditional method or modern method the resulting product is expected to be in a distinct and pure liquid form. Regretably, Mustsaers (1991) reported that most traditional methods and equipment employed in honey extraction do not concur with this definition: lack of personal hygiene (poor hand washing); use of unsuitable containers (non food-graded) that have not been properly sterilized; extraction of honey in the open/unhygienic environment; and eventual storing of the product in poorly sealed containers, cause the product (honey) to become easily adulterated and consequently, degenerated.

In view of the backlog of challenges posed by most traditional methods of honey extraction; with respect to the overall quality rating of the product, the centrifuge method was highlighted by Mustsaers (1991) as the most modern and widely used method of honey extraction by many large scale honey producers. In this method, as shown by Plate 1, the honey comb (uncapped) is placed inside frames and arranged in an apparatus (centrifuge extractor) consisting essentially of a compartment spun about a central axis to separate the honey from other materials of different specific gravities via the action and effect of a centrifugal force. This results in the formation of different layers by the particles in a colloidal solution, after which the honey, in liquid below the particles can be gently filtered-out. This method is quick and more efficient.


 









Plate 1: Centrifuge method of honey extraction

2.1       TRADITIONAL METHODS OF HONEY EXTRACTION
Honey extraction using traditional methods and equipment varies across different geographical locations and societies. Where as some societies view honey as a much revered, valuable and highly beneficial commodity that requires the utmost care and attention during its extraction processes, others see the product as a mere sweetener that does not deserve any special treatment during its extraction. As such, the latter handles the processes carelessly; thereby impacting negatively on the product’s overall quality rating, (Surendra, 2008).
           
However, Adjare (1990) and Mustsaers (1991) both presented a concise report on the most common traditional methods of honey extraction, which are found in most geographical locations around the world. They are as follow: 

2.1.1   FLOATING METHOD: In this method, the honey comb with capped honey (that is, honey comb filled with honey) is uncapped with the aid of a slightly heated knife. The combs are later chopped into pieces and put into an air – tight container(s) for about two days, to settle down; otherwise exposure to damp air increases honey’s moisture content (owing to honey’s hygroscopic nature) and the ease with which it ferments. After the two days period of being allowed to settle down elapses, the wax would have floated to the surface of the mixture leaving the honey below as shown by Plate 2. The wax is then skimmed off while the honey is filtered out via a clean cheese cloth or lace curtain material. The honey may be allowed to settle down for an extra day in order to remove further any remaining foam and wax particles.
            Although a simple method, floating has the following limitations:
·                    It is time consuming.
·                    The risk of honey being contaminated by hands is high.
·                    An already chopped comb can not be returned to the beehive for re-use by the bees. This reduces productivity level of the colony as more energy have to be subsequently spent on building another honey comb by the bees.

 








 
           
Plate 2: Floating method of honey extraction

2.1.2   HONEY PRESSING METHOD:  Here, the honey comb is first decapped and chopped into smaller pieces in a plastic container, after which it is transferred into a clean cloth or sack tightly wrapped_up or knotted at the opening, with netting at the bottom, which fits in a double pot positioned under the honey press. Thereafter, the filled sack is subjected to immense pressure by the press which expels the honey out from the sack and then drip on to trays during uncapping as shown in Plate 3. The scrap honey in the container (pot) is then sieved. A number of honey pressing machines are available depending on the desired capacity. They generally take the form of a simple hinged press or a press with a spindle which is used to move one plate down on to the other to squeeze the honey out from comb.
            The major limitations associated with the honey pressing method include:
·        Lost of the honey comb; meaning that the colony has to build a new comb.
·        Low extraction efficiency and low work rate; Ng’atiwa (1989) reported an extraction efficiency of about 58%, while Mutsaers (1991) concluded that, whereas the honey press can be used for extracting small quantity of honey
(2 kg/hr) a centrifuge is more efficient for quantities above 2kg/hr.
 








Plate 3: Honey pressing method of honey extraction

2.1.3   HAND – SQUEEZING METHOD: This method of honey extraction is common   with bee farmers and few honey producers who desire to consume the product themselves, or supply to consumers based on specific order. For instance, in Milkwood Bay, Port Elizabeth, South Africa, (Tim, 2012) reported that the locals, after collecting the honey combs from the hives, they cut the honey comb off the top-bars, after which all the honey comb are crushed and meshed in a plastic container as shown by Plate 4(a) & (b). For this harvest, they placed a big sieve on top of a honey bucket with a gate on the front, after which the crushed and meshed comb is poured or tipped into the sieve alongside some older stuff from a previous harvest. To speed_up the process, they will all squeeze the comb by grabbing great handfuls; this meant they expelled the honey from the wax much quicker. At the end of this process, about 5kg strained honey (from 3 combs, and about 0.5kg of bee wax in lumps) are obtained. These entire processes result to pure, cold-pressed organic honey that have got everything that was in the honey; pollen, propolis and lots more. They get honey that is not heated in anyway during the process, which means none of the delicate antibiotics and enzymes within the honey are destroyed. Finally, they will pour all into a jar without further filtration.  

Nonetheless, honey extraction by hand squeezing method has the following limitations;
·        High level of pollen content in extracted honey may pose an aesthetic concern in terms of marketability or long term storage.
·        Unhygienic hands easily contaminate the honey, and fermentation may occur after few days.












 






























Plate 4 (a) & (b): Hand-squeezing method of honey extraction


2.1.4       SOLAR MELTING METHOD:  This method is also referred to as the ‘Heating’ method because it involves the application of heat energy in melting- out honey from the honey comb. Unlike the crude means in which live embers are placed on a pile of capped honeycombs to extract the honey, this method adopts the use of a relatively safe solar radiation of the sun. In this method, the capped honey comb is first decapped and chopped into smaller pieces in a plastic container, after which it is transferred into a wide woven basket made of palm leaves. The woven basket is placed on top of a plastic basin into which the honey is collected as shown by Plate 5. As the intensity of the solar radiation from the sun increases, the viscosity of the content of the honey comb reduces, while both honey and wax trickle down into the plastic basin beneath until a great proportion of the content of comb is completely drained off by the heat. The substance collected is left untouched until the next day after which the bee wax must have hardened over the surface of the solution, thereafter scrapped – off. The clear honey is then collected separately in another plastic container.
Unfortunately, solar melting method of honey extraction has the following limitations:
·        Vasyl (2003) reported that exposure to excessive heat of over 370C results in the lost of health values of honey via reduced antibacterial strength and the destruction of important enzymes (such as diastase and invertase) activities
·        Lost of the honey’s nutritional values and quality as excessive heat also affects its appearance (darkens the natural honey colour, taste and fragrance (Subramanian et al., 2007).

·        The honey is contaminated by dust and dirts and easily absorbs moisture in the air thereby enhancing the rapid fermentation of the product.
 








Plate 5: Solar melting method of honey extraction
                                       
2.1.5       SIEVING METHOD: This method of honey extraction is very similar, in terms of set up, to the solar melting method. Unlike the solar melting method which makes use of woven basket made of palm leaves, and solar radiation from the sun to facilitate the ejection of honey from honey comb, the sieving method utilizes set of sieves of wired-net configurations ranging from coarse particle sized to very fine particles size openings. The sieves are arranged in a concentric form, the finest mesh being on the outside and coarser on the inside. The entire process is done in-door under room temperature (180 – 230c). Thus after chopping the decapped honey comb into smaller pieces, they are poured into the uppermost sieve with the biggest particle size configuration in the set of sieves set – up, in vertically downward descending order; in terms of particle size configurations. A plastic container is placed under the set of about four sieves, as shown by Plate 6, to collect the pure honey which falls solely by the force of gravity acting on the comb. This set – up is usually left undisturbed for at least three days depending on the viscosity of the product. This method of honey extraction poses less risk of contamination to the product, unlike the solar melting method.

 











Plate 6: Sieving method of honey extraction

However, the sieving method of honey extraction has its own limitations as follow.
·        It is time consuming
·        The sieve set up can accommodate only small quantity of honey comb for extraction process at a given time
·        The honey can easily absorb moisture from the air when left exposed for too long


2.2       QUALITY AND QUALITY CONTROL OF HONEY
Quality is defined as the degree of excellence which a material possesses (Guralink, 1975). Solabi (1985) in his own submission perceived quality as what is needed to satisfy consumers expectations at all times. Subsequently, Sanni (1997) viewed quality as the totality of features and characteristics of a product or services that bears on its quality to satisfy stated or implied needs. Thus, according to Oyinlola (2004), “Quality control” in food processing plants involves using quality inputs; raw and packaging materials that satisfy product specifications, standard manufacturing practices, proper laboratory analysis of samples taken at critical control points, good storage and shipping schedules for both the raw materials and the finished product.

The quality control of honey therefore, is built on two main principles, the first is to verify the (honey) genuineness, that is, to reveal possible frauds such as artificial honey and adulteration; and secondly, to determine its quality with respect to the need of the processor and the market (Krell, 1996). Undoubtedly, most of the challenges encountered with regards to the quality control of honey emanate from the harvesting and extraction processes adopted be it traditional or modern for the product. Hence, as a panacea to the possible risks to honey quality by the unwholesome practices of some traditional method of honey extraction, as highlighted earlier on by Adjare (1990) and Mutsaers (1991), the Devon Apicultural Research Group (DARG) based in the United Kingdom, suggested that all honey producing nations adopt a process of analyzing and dealing with possible hazard; called Hazard Analysis Critical Control Points (HACCP).Having a HACCP plan is compulsory for commercial honey producers in developed countries (DARG, 2012).

2.3       QUALITY PARAMETERS OF HONEY
Over the years, several researches have been conducted on the different parameters that govern the quality of honey. Some of the most important of these quality parameters are as follow:

2.3.1   MOISTURE CONTENT: As reported by Scott (2012), honey has a low availability of water for bacteria to use, being about 84% fructose and glucose. The 15 – 21% of water by weight in honey strongly interacts with the sugar molecules and thus, there is very little left available for anything else.
Hence, Krell (1996) stated that only osmophilic  (sugar – tolerant) yeasts can grow in high sugar concentration, with their rate of multiplication increased proportionally with increasing moisture content (usually above 18% moisture  in 100g of honey. Thus, while Lawal et al., (2009) reported  a moisture content of 19.6% for honey samples sourced from Yola, North-East – Nigeria, the amount of moisture of honey, as reported by other authors include 17.6% (SueBee, 2011); 34.7% in Jalingo, (Agbagwa, et al., 2011), and 21.0% (NIS, 2003).

2.3.2   pH: Honey pH is another property that makes the product such a potent anti-bacterial agent. According to Scott (2012), honey generally has a pH value between 3.2 – 4.5, making it a very acidic agent. Most bacteria, as further stated by Scott (2012) need a less acidic environment to thrive and propagate. For example, E. coli, Salmonella and Streptococci; all need a pH environment of between 4.0 – 4.5 to flourish. Thus, the pH of some honey samples as reported by some authors include 5.6 in Yola, (Lawal et al., 2009), 3.10 in Jalingo, (Agbagwa et al., 2011) and 3.42 – 6.10 (NIS, 2003).

2.3.3   Ash Content: Lawal et al., (2009) defined honey ash content as a measure of the mineral elements in honey. It indicates how good or bad the physical properties of honey are and it is also a good criterion  for determining honey botanical origin. Therefore, the ash content of some honey samples as reported by some authors include 0.80% in Yola, (Lawal et al., 2009), 0.953% in Jalingo, (Agbagwa et al., 2011), 0.17% (SueBee, 2011) and 0.60% (NIS, 2003).

2.3.4       Hydroxymethyl Furfural (HMF) Content: HMF content indicates honey freshness and overheating. HMF is a by-product of fructose decay which is practically absent in fresh honey, but it (HMF) increases upon storage, depending on the pH of honey, and on either the extracting or storage temperature. Hence, the presence of HMF is considered as the main indicator of honey deterioration (Krell, 1996). Thus, in international trade, a maximum value of 40mg per kilogram (kg) has proven satisfactory, while the codex proposal is a maximum of 60mg/kg (Bogdanov et al., 2001). Also, we have 0.38mg/100g for Yola, (Lawal et al., 2009) and 40.0mg/kg as maximum value by NIS (2003).

2.3.5       Total Acidity: Honey, according to (Wilkins and Lu, 1995), contains many kinds of acids, both organic and amino. Organic acids comprise most of the acids in honey, accounting for 0.17 – 1.17% of the mixture, and they include gluconic, formic, citric and lactic acids among a few. Honey, within the standard approved range for total acidity allows for an enhanced shelve stability of the product and thus, prevent spoilage by micro organisms (Williams et al., 2009). Total acidity as reported by other authors include 1.90g/ml (Agbagwa et al., 2011), 40.0mg/kg (NIS, 2003) and 29.12meq/kg (SueBee, 2011), and 0.04% lactic acid in Yola, (Lawal et al., 2009).

2.3.6       Sugar Content:  Honey is a mixture of sugars and other compounds. With respect to carbohydrates, honey is mainly fructose (about 38.5%) and glucose (about 31.0%), making it similar to the synthetically produced inverted (table) sugar syrup, which is approximately 48% fructose, 47% glucose, and 5% sucrose (National Honey Board, 2012). Simple sugar particularly fructose are responsible  for most of the physical and nutritional characteristics of honey (Krell, 1996) while the presence of sucrose  which constitute 1.3% of the dry matter in honey, provides information on adulteration and the botanical origin of honey. For other authors, Fructose content include 34% (Bidemi, 1999), 60% (Agbagwa et al., 2011), 36.35% in Jalingo, (Agbagwa, et al., 2011) and 38.38% (SueBee, 2011) .For Sucrose content: 1.8% (Agbagwa,et al.,2011),1.31%(SueBee, 2011) and maximum, 5.0% (NIS, 2003). For Glucose content: 39.18% in Jalingo (Agbagwa et al., 2011).

2.3.7       Microbiological Characteristics: The chief sources of micro – organisms in honey are the nectar of the flowers and the honey bee (Lawal et al., 2009). The microbiological parameters of honey include:

2.3.7.1Total Aerobic Mesophilic Bacteria Plate Count (TPC): There is a risk of introducing micro organisms into wounds, especially botulism (digestive system infection in children under one year of age), if honey is used as a dressing agent (Postmes et al., 1995). The risk can be avoided by sterilization via “Gamma Irradiation”. An international medical device ingredient microbiological requirement for manufacturing is a TPC of less than 500cfu/g (Activon, 2013). Other authors’ report on the TPC of some honey samples include: 1 x 103cfu/g (NIS, 2003) and 1.3 x 101cfu/ml in Yola (Lawal, et al., 2009).

2.3.7.2Mould/Yeast Counts: Yeasts have been shown to come from the nectar and from the intestinal content of bees (Frazier and Westhoff, 1994). Honey is often spoiled by some osmophyllic yeasts and moulds which are always present in honey, but they are unable to grow in it and to change its composition if the sugar concentration is about 83% or higher. The mould and yeast count as reported by some authors include: 1 x 103 cfu/g (NIS, 2003), and 1.3 x 101cfu/ml in Yola, (Lawal et al., 2009).

2.3.7.3E. coli and Coliform Count: Possible spore – forming bacteria contamination of honey by micro organisms (examples, coliform bacteria and pathogenic bacteria such as staphylococcus, salmonella and clostridium species) are indicative of poor sanitary quality of honey collection, extraction and storage conditions.  Thus, the coliform count in freshly processed honey, as presented by authors, is expected to read nil cfu/g (Activon, 2013), nil cfu/g in Yola, (Lawal et al., 2009) and nil cfu/g (NIS, 2003).

2.4             QUALITY STANDARDS AND REGULATIONS FOR HONEY
Quality standards and regulations for most commercial food products, as reported by Oyinlola (2004) is described as the total sum of activities, observations (physical and instrumental examinations) and other measures employed to ensure that a uniform standard of excellence is attained and maintained for all batches of the food products, manufactured at all times. Thus, Oyinlola (2004) further stated that a key tool to assure quality in any finished manufacture food such as honey is the “Product Standard Document”.

Consequently in Nigeria, however, the quality standards governing the regulation of honey production and marketing in the country as specified by NIS 473: 2003 is presented in Table 1:

Table 1: Quality Requirements of Nigerian Honey
S/NO
CHARACTERISTICS
SPECIFICATION
1
Moisture content (%) (max)
21.0
2
Ash content (%) (max)
0.6
3
Specific rotation (α)20 D
-20.4 to + 4.8
4
Total acidity
40 milli-equivalent per 1000 grammes (max)
5
pH
3.42 – 6.10
6
Sucrose content (%) (max)
5.0
7
Reducing sugar calculated as invert sugar (%) (max)
0.5
8
Fructose/dextrose ratio
106 – 119: 900
9
Water insoluble content (pressed honey) (%) (max)
0.5
10
Water insoluble content (unpressed honey) (%) (max)
0.1
11
Weight per volume (g/litre)
1352 – 1500
12
Diastase activity (diastase figure on gothe scale) (max)
8
13
Hydroxymethyl furfural content (mg/kg) (max)
40.0
14
Fiehes test (detection of technical sugar)
Negative
15
Proline content (mg/kg) (min)
180.0

Microbiology

a
Total aerobic mesophilic bacteria plate count (cfu/g) (max)
1.0 x 103
b
Mould/yeast count (cfu/g) (max)
5.0 x 101
c
Coliform count (cfu/g)
Nil
d
E. coli count (cfu/g)
Nil

Metallic contaminants


Arsenic (As) (mg/kg) (max)
1.0

Copper (Cu) (mg/kg) (max)
2.0

Lead (Pb) (mg/kg) (max)
0.5

Source: Standard Organization of Nigeria: Nigeria Industrial Standard (Standard for Honey) (NIS 473: 2003). ICS 67.180.10.

CHAPTER III
RESEARCH PROCEDURE
3.1       DESCRIPTION OF LOCATION
Twelve (12) different commercial honey samples extracted using five (5) different traditional methods, were sourced from eight (8) selected honey bearing communities within Kaduna State, North-Western Nigeria; namely Ankwa, Doka, Giwa, Kafancha, Katari, Kukui, Mando and Sabon Gari communities. The selected honey-bearing communities are situated in the following Local Government Areas (L.G.As) of Kaduna State:
Ankwa, Doka and Katari communities (Kachia L.G.A);  Kafancha community (Zango Kataf L.G.A); Kukui community (Kagarko L.G.A); Giwa community (Giwa L.G.A); Mando community (Igabi L.G.A) and Sabon Gari community (Sabon Gari L.G.A).

Thus, the sources of honey samples according to their sample labels were: Ankwa (sample label A2), Doka (sample labels D2 and E1), Kafancha (sample label E2), Katari (sample labels D1,F1 and F2), Kukui (sample label B2), Mando (sample label C2) and Sabon gari (sample labels A1 and C1), respectively.

3.2       HONEY SAMPLING METHODS
Twelve (12) different commercial honey samples labeled; A1,B1,C1,D1,E1,F1, A2,B2,C2,D2,E2 and F2, extracted from their respective honey combs using five different traditional methods of extraction; were obtained with the aid of questionnaires, in the following order:
(i)                   Floating method: (sample labels E2 and F2)
(ii)                Honey pressing Method: (sample labels A1,B1 and D2)
(iii)              Hand-squeezing method: (sample label F1).
(iv)              Solar melting (Heating) method: (sample labels A2, B2, C1andD1).
(v)                 Sieving method: (sample labels C2 and E1).
Having collected all the twelve (12) honey samples in neat, well sealed plastic containers, as shown by Plates 7, they were all stored on a wooden shelve in a dark room, under an average room temperature of 23oC, before being transferred to the laboratory for scientific analysis of the samples’ quality parameters – as had been established by several authors in chapter II. Therefore, the different laboratory analytical methods adopted in the determination of the various quality parameters for all the twelve commercial honey samples collected were as shown in Table 2.

Table 2: Laboratory Analytical Methods for Honey Sample Parameters
ANALYTICAL METHOD
QUALITY PARAMETER DETERMINED
        i.            Refractometry
      ii.            Furnacing (Decomposition)
    iii.            Glass electrode           
    iv.            Titration of acidity (Lactone hydrolysis)
      v.            Quantitative analysis
    vi.            Mathematical equivalence
  vii.            Mathematical difference
viii.            Microbiological Analysis,
-            Total plate count (TPC)
-          Mould / yeast count
-          Coliform count
-          E. coli count
Moisture content
Ash content
pH
Total titrable acidity (% Lactic acid)
Sugar (% glucose, fructose and sucrose) content
Density (weight per volume)
Water Insoluble content

Total aerobic mesophilic bacteria
Mould / Yeast
Coliform
E. coli                       





           
           
Plate 7: Honey sample collected for the study

3.3       LABORATORY ANALYSIS OF SAMPLES
Most of the laboratory analytical methods adopted for the determination of the quality parameters of all the twelve (12) commercial honey samples collected were according to the harmonized methods of the International Honey Commission, IHC (2009).

3.3.1   Determination Of Moisture Content: The moisture content of the samples were determined by the use of an Analogue Hand-Refractometer as shown by Plates 8a, and 8b which serves a dual purpose, for moisture content (Range 12.0-27.0%) and total sugar (Soluble solid) content (Range 58.0-90.0%) determination, under a maximum sample temperature of 200C. A broom stick-drop from one sample was placed on the glass slide of the refractometer after which the moisture level was viewed via a lens in the direction of the reflection of the sun light. A focusing ring on the refractometer was then employed in obtaining a real and clearer reading of the moisture content on the moisture scale. Thereafter, the residue of the observed sample was cleaned-off the glass slide of the refractometer, and the same procedure was repeated for each of the remaining samples.
 


           












 













           

Plate 8 (a) & (b): Analogue hand-refractometer
3.3.2   Determination Of Ash Content: An electronic digital VITCO Muffle Furnace with model number; TC344 (SELEC), as shown by Plate 9 was employed in the determination of the ash content of the samples. 5g of each sample was weighed-out separately, to the nearest 0.001g, into twelve quartz ash dishes (or crucibles) that had been pre-heated to remove any residual moisture from  the dish at low heat  rising to between 350-4000C ashing temperature. Thereafter, all the dishes were cooled in a dessicator to room temperature, and weighed to 0.001g. Thus, following the preliminary ashing, all the dishes were placed in the pre-heated furnace and then heated for at least 1hr before being cooled in the dessicator and weighed again. The ashing procedure continued until constant weights were reached for all the samples. The ash content of each sample was then calculated using the mathematical expression:
            Ash content (%)=  

           
Plate 9:Digital muffle furnace

3.3.3   Determination Of pH: The pH of the samples were determined by the use of an electronic digital pH metre with model number; ORION 310, and range of 0.00-14.00. Prior to the use of the PH device, it was calibrated using three (3) different Buffer solutions: buffer 4 (for acidity), buffer 7 (for neutrality), and buffer 9 (for alkalinity). Thereafter, 10ml of each honey sample was measured out separately into twelve (12) beakers and were each diluted with 20ml of distilled water. The dilution was to lower the viscosity of the samples so as to enable the sensitive device inside the glass electrode of the pH meter to easily read the pH level of each honey sample, as shown by Plates 10a and 10b, respectively.
           

           







Plate 10 (a) & (b): Digital pH meter

3.3.4   Determination Of Total Titrable Acidity (% Lactic Acid): 25ml of each honey sample was diluted with 25ml of distilled water, and each solution was then titrated separately against 0.1N NaoH (0.1 Normality sodium hydroxide solution) using 0.25ml phenolphthalein as an indicator as shown by Plate 11. The relative amount of lactic acid was determined using the mathematical formula:
                        Lactic acid (%)=
 

                                                                                                 









Plate 11: Determination of total titrable acidity

3.3.5   Determination Of Sugar (% Glucose, Fructose And Sucrose) Contents: The specific quantity of the various sugars (glucose, fructose and sucrose) present in each honey sample was determined using the method stipulated by the Association of Official Analytical Chemists (AOAC) in the year 2006. Having determined the total sugar (soluble solids) contents of each sample using the same refractometry method as highlighted in 3.3.1, the constant standard sugar concentration of each sugar (glucose, fructose and sucrose) content for all the honey samples was prepared by dissolving 10g of each industrially prepared fine sugar particles, separately, in 100ml of distilled water in three separate conical flasks. Thereafter, the specific quantity of each sugar content present in each honey sample was calculated using the following mathematic expressions:
Glucose content in sample=  x Total sugar content in sample

Fructose content in sample =  x Total sugar content in sample
           
Sucrose content in sample =  x Total sugar content in sample

Therefore, the Reducing sugar content of each honey sample was calculated using the mathematical expression; Reducing sugar content (%)= (Glucose content + fructose content) in sample

3.3.6   Determination Of Density (Weight Per Volume): The density of the honey samples was determined using the mathematical equivalent method. The weight of 120mL (0.12L) of each sample was equated to the weight of 1L of the same samples. The equivalent weight (g) per volume (L), that is density of each sample was determined with the aid of an OHAUS Brain weigh electronic digital Balance with model number: B500 and range of 0.00-550.00g as shown by Plate 12. Subsequently, the density of each sample was calculated using the format of the mathematical expression for sample A1:
Weight of sample A1: (g) per 0.12L = 164.74g
Thus, weight of sample A1 (g) per 1L=164.74 x 1L 
                                                                        0.12L
                                                                =  1372.83g
Therefore, the density of sample A1 was 1372.83 (g/L).hence, the sample procedure was repeated for each of the remaining samples.
 






                                                                                                        




Plate 12: Digital weighing balance

3.3.7   Determination Of Water Insoluble Content: The water insoluble content of the samples was determined by finding the mathematical difference between the values of the total solids and the soluble solids (total sugar content) of each sample. The value of the soluble for each sample was obtained using the same procedure of the refractometry method as highlighted in 3.3.1; the focusing_ring on the refractometer was employed in obtaining a real and clearer reading of the soluble solids on the soluble solids scale. Thus the water insoluble content of each sample was obtained using the mathematical expression:
            Total solids (%) =100 – Moisture content
            Water insoluble content (%) =Total solids – soluble solids

3.3.8   Determination Of Microbiological Characteristics: The microbiological analysis on the honey samples were carried out to determine the magnitude of the presence of four (4) different groups of micro organisms namely; Total aerobic mesophilic bacteria, Mould / Yeast, Total coliform and E. coli. The culture media, which were collected in separate conical flasks and also used for the analysis, were:
-                     Plate count Agar, PCA (10g in 444mL distilled water)
-                     Eosine methylene Blue Agar, EMB (8.89g in 250mL distilled water)
-                     MacConkey Agar, MA (10g in 200mL distilled water)
-                     Potato Dextrose Agar, PDA (9.81g in 250mL distilled water)
-                     Ringer tablet (1 piece in 500mL distilled water)
-                     Tartaric acid (2g in 9mL ringer solution + 11mL distilled water + 10g per 444mL PCA solution).
The procedures for the complete analysis were as follow:
3.3.8.1            All the culture media solutions and other glass wares to be used for the analysis were sterilized at a temperature of 1210C under a pressure of about 0.105MPa for 15 minutes in an Autoclave [SURGIFRIEND pressure steam sterilizer; Model number: SM 280E, Range: for temperature (36-1380C), for pressure (0.01 – 0.25 Mpa)] as shown by Plate 13.



 










Plate 13: Pressure – steam sterilizer

3.3.8.2            Thereafter, a serial dilution in two levels, 101 and 102, was carried out on all the honey samples by pipetting 1mL of each sample into 9mL each of the sterilized ringer solution in separate test tubes. The resulting solutions were thoroughly mixed and labeled as first dilutions such as: A1101; A2101; B1101; B2101; … F1101 and F2101.

3.3.8.3            Similarly, 1mL out of each first dilution was pipetted into another 9mL each of sterilized ringer solution in separate test tubes. The resulting solutions were thoroughly mixed and labeled as second dilutions such as: A1102,A2102,B1102,B2102…, F1102 and F2102.

3.3.8.4            Subsequently, 4mL was pitetted out of each first dilution out of which, also, 1mL each was poured into another set of four separate petric dishes labelled according to each honey sample such as: A1101 (dishes PDA, PCA, MA, and EMB), A2101 (dishes PDA, PCA, MA and EMB),…, F2101 (dishes PDA, PCA, MA and EMB).

3.3.8.5            Furthermore, 4mL was pipetted out of each second dilution out of which, also, 1mL each was poured into another set of four separate petric dishes labelled according to each honey sample such as:A1102 (PDA, PCA, MA and EMB), A2102 (dishes PDA, PCA, MA, and EMB),…, F2102 (dishes PDA, PCA, MA and EMB).

3.3.8.6            Thereafter, the volume of each of the four culture media_PDA, PCA, MA and EMB_was divided by 24 samples after which the resulting volume for each culture media was poured into its designated petric dishes for the first and second dilutions.

3.3.8.7            Afterwards, all the petric dishes containing the culture media were incubated appropriately as follow:
·                    PCA: The dishes (plates) were incubated at 370C for 24-48hr
·                    MA and EMB: The plates were incubated at 350C for 24-48hr
·                    PDA: The plates (dishes) were incubated at 300C for 24hr for yeast isolates and 3-5 days for moulds.
            Following the incubation period and the colony-forming unit counting processes for selected organism-carrying plates (dishes), the counted isolated organisms were identified using the Gram’s Staining Techniques, as follow:

3.3.8.8            A smear was prepared from a 24hour old culture of isolated organism, as shown by Plates 14a and 14b,  for each culture sample plate, on a clean slide and allowed to air dry, after which the back surface of the film was passed gently through heat (naked flame) thrice.
           







 











Plate 14(a) & (b): Identification of test media

3.3.8.9            Thereafter the film was placed on a staining rack and then flooded with crystal violet solution and was allowed to act for 1 minute before it was later rinsed with water and allowed to air dry.

3.3.8.10Gram iodine solution was then applied on the film and allowed to act for 1 minute before being rinsed again with water and allowed to air dry. Acetone was then applied on the film and allowed to act for 5 seconds before being rinsed immediately with water and allowed to air-dry.

3.3.8.11Sarfranine solution was applied on the film and allowed to act for 30 seconds before being rinsed with water and allowed to air dry, after the back surface of the slide had been blotted with blotting paper.

3.3.8.12Finally, a drop of oil immersion was applied on the film and the film was observed with the oil-immersion-objective (lens) of a microscope, thereby identifying the isolated organisms on the film, as shown by Plates 15a and 15b, respectively.
           






 

























            Plate 15 (a) and (b): Identification of isolated  organisms after gramm’s staining test using microscope

3.4       METHOD OF DATA ANALYSIS
To have a quick analysis of data and a better understanding of results, all data  collected were analysed using statistical tools namely; Percentages, and Analysis of Variance (ANOVA) for two-factor experiment for testing the formulated hypotheses, the two factors being the methods of extraction and the quality parameters of the honey samples under study; as stipulated by Murray and Larry (2011).

3.4.1   HYPOTHESES TESTING:
(a)       Null Hypothesis (Ho):
            Ho(1): There is no significant difference among the different methods of extraction.
            Ho(2): There is no significant difference among means of the quality parameters.
(b)       Alternative Hypothesis (Hi):
            Hi(1): There is significant difference among the different methods of extraction.
            Hi(2): there is significant difference among the means of the quality parameters.

3.4.2   LEVEL OF SIGNIFICANCE (¥)    
            5% or 0.05 level of significance (¥ = 0.05) was used for the hypotheses testing under a F- distribution table (Ftab).

3.4.3   DECISION RULE
            Reject null hypothesis, H0 (1) or H0(2) or both, if F-calculated value (Fcal) is greater than the F-table value (Ftab). Thereby, accepting the alternative hypothesis, Hi (1) or Hi(2)  or both, as the case may be. Otherwise, do not reject.
             

ANALYSIS OF VARIANCE (ANOVA) TABLE
            The ANOVA table for two-factor experiment, as stipulated by Murray and Larry (2011), was constructed in the format of Table 11 below:
           
            Table 11: ANOVA Table-Format For Two- Factor Experiment.
Variation
Degrees of freedom
Mean Square
F
Between rows (methods of extraction)
VR=  
a-1
/  with
(a – 1) and
(a – 1) (b – 1) degrees of freedom
Between columns (quality parameters)
VC=

b-1
/  with         (b-1) and              (a – 1)(b – 1) degrees of freedom
Residual or error
VE= V-VR-VC
(a-1) (b-1)


Total V= VR + VC+ VE
=

ab-1


Where:
a= number of factors (or treatments) on row
b= number of factors (or treatments) on column
J = entries on row (or methods of extraction)
K= entries on column (or quality parameters)
= mean square for rows
= mean square for columns
= mean square for residual or error
T= total of all entries
 Tj= total of entries in the jth row
T.k= total of entries in the kth column
= correction factor
V= total variation (or total sum of square)
VR= variation between rows (or methods of extraction)          
VC=variation between columns (or quality parameters)
VE= variation due to error (or residual)
X2= square of entries
å= summation of entries within a range
F= F-distribution calculated value (or F cal)











CHAPTER IV
RESULT AND DISCUSSION
4.1       PRESENTATION OF RESULTS
Honey samples extracted using Floating (labels E2 and F2), Honey Pressing (labels A1, B1 and D2), Hand- Squeezing (labels F1), Solar Melting (labels A2, B2, C1 and D1) and Sieving (labels C2 and E1) methods had percentage moisture content of 19.00, 20.40.17.40, 16.80, 19.00, 22.00, 17.50, 17.40, 17.70, 20.00,15.50, and 18.90, respectively (Table 3), while the total sugar content (soluble solids) of the samples were 79.50, 78.00, 81.00 81.50, 79.70, 76.70, 80.80, 81.00, 80.80, 78.40, 83.00 and 79.80 (Table 3).

The percentage total solids of the honey samples extracted using Floating, Honey Pressing, Hand-squeezing solar melting and Sieving methods were 81.00, 79.60, 82.60, 83.20, 81.00, 78.00, 82.50, 82.60, 82.30, 80.00, 84.50, and 81.10(Table 4), while the percentage water insoluble content of the samples were 1.60, 1.60, 1.60, 1.70, 1.30, 1.30, 1.70, 1.60, 1.50, 1.60, 1.50, and1.30 respectively (Table 4).
The percentage ash content of the honey samples extracted using Floating, Honey Pressing, Hand- squeezing, Solar Melting and Sieving methods were 0.60, 0.70, 0.65,0.70, 0.80, 0.75, 0.50, 0.85, 0.60, 0.70, 0.75 and 0.75 (Table 5). The honey sample also had pH values of 4.03, 3.94, 4.16, 4.09, 4.11, 3.86, 3.84, 3.77, 3.44, 4.01, 3.77 and 4.15, respectively  (Table 5). The total titrable acidity calculated as % lactic acid of the honey samples showed that samples extracted using Floating, Honey Pressing, Hand-Squeezing, Solar melting and Sieving methods had percentage lactic acid of 0.22, 0.20, 0.12, 0.14, 0.23, 0.32, 0.21, 0.23, 0.13, 0.14, 0.37 and 0.24 (Table 6), the density (grammes per liter) of the samples were1347.56, 1322.35, 1372.83, 1372.58, 1331.18, 1382.16, 1329.50, 1363.75, 1351.44, 1336.25, 1359.92 and 1341.85 respectively (Table 6). While the percentage sucrose content of the honey samples extracted using Floating, Honey Pressing, Hand- Squeezing, Solar-melting and sieving methods were 27.58, 27.06, 28.10, 28.27, 27.65, 26.61, 28.03, 28.10, 28.03, 27.20, 28.80 and 27.69 (Table 7); the percentage reducing sugar content of samples were 51.92, 50.94, 52.90, 53.22, 53.04, 50.08, 52.76, 52.90, 52.76, 51.20, 54.20 and 52.12 respectively (Table 7).

The microbiological analysis of the honey samples extracted using Floating, Honey Pressing, Hand-Squeezing Solar- Melting and sieving methods revealed that E. Coli counts were absent from all honey samples. Total aerobic mesophilic bacterial plate count (TPC) of samples were 4.5x103, 7.8x103, 9.0x103, 9.8x102, 4.1x103, 5.1x103, 8.0x103, 3.1x102, 1.7x102, 9.8x103, 7.2x102 and 6.2x103 colony forming units per gramme (cfu/g) (Table 8). The colony forming units per gramme of mould and yeast count of honey samples were 1.5x103, 7.0x101, 4.0x101, 3.0x102, 3.0x102, 4.0x102, 6.0x101, 3.0x102, 6.0x101, 2.0x101, 1.2x102 and 3.0x102,  respectively (Table 9), while the colony forming units per gramme of coli form count of honey samples were 3.5x102, 5.5x102, 6.0x102, 1.6x103, 1.5x102, 1.3 x122. 7.7x103, 4.7x102, 9.5x102, 7.2x102, 1.6x103, and 6.0x102 respectively (Table 10).

4.2       STATISTICAL ANALYSIS     
            All  data collected were analysed using Percentages and Analysis of Variance (ANOVA) for two- factor  experiments, as stipulated by Murray and Larry (2011), for testing the formulated hypotheses _ Null hypotheses, Ho(1), and Ho(2),  and the Alternative hypotheses, Hi(1) and Hi(2) _as was earlier stated under section 3.4.

4.2.1   TABLE OF DATA AND COMPUTATION OF RESULTS
            The percentage means of data of the two factors under study are shown in Table 12.
            Table 12: The Mean Results of the Methods of Extraction and The Quality Parameters of Honey Samples.
Quality  Parameter (% Mean)
Method of Extraction
Ash content
Lactic acid
Moisture content
Water insoluble content
Sucrose content
Reducing sugar
Total
Floating
0.65
0.21
19.70
1.60
27.32
51.43
100.91
Honey Pressing
0.72
0.16
17.73
1.53
28.01
53.05
101.20
Hand Squeezing
0.75
0.32
22.00
1.30
26.61
50.08
101.06
Solar-Melting
0.66
0.18
18.15
1.60
27.84
52.41
100.84
Sieving
0.75
0.31
17.20
1.40
28.25
53.16
101.07
Total
3.53
1.18
94.78
7.43
138.03
260.13
505.08


From Table 12 the following results can be computed:
a =5
b = 6
jk = (0.65)2 + (0.72)2 + (0.75)2 +…+ (53.16)2 = 1972.92
T = 100.91+ 101.20 + 101.06 + 100.84 + 101.07 =505.08
 = (100.91)2 (101.20)2 + (101.60)2 + (100.84)2 + (101.07)2 =10204.25
 = (3.53)2 + (1.18)2 + (94.78)2 + (7.43)2 + (138.03)2 + (260.13)2 = 15962.03
 = 8503.53
jk-  

 

 
 VE = V – VR – VC
\ VE = 10674.39 – 1700.72 – 7458.50 = 1515.17

Having computed the results, the ANOVA table is constructed according to the format presented in Table 11 under section 3.4.4, as shown in Table 13 below.

Table 13. Analysis of Variance Table For Computed Results
Variation
Degrees of freedom
Mean square
F
Between rows (methods of extraction)
VR = 1700.72
4
S2R =425.18
S2R / S2E =5.61 with 4 and  20 degrees of freedom 
Between columns (quality parameters)
VC = 7458.50
5
 = 1491.70
S2R / S2E 19.69 with 5 and 20 degrees of freedom
Error or residual
VE = 1515.17
20
 = 75.6

Total
 V= 10674.39

29


In order to obtain the table values of the F – distributions in table 13, the following expressions were employed:
Table value of F – distribution (F tab); from Table 14,
F tab = F¥, V1, V2
Where:
F tab = table value of F- distribution
F ¥ = level of significance for the F- distribution; where ¥ = 0.05
V1 = degrees of freedom for treatment (or factors)
V2 = degrees of freedom for error (or residual)

Therefore,
-         Between rows (methods of extraction).
F tab = F 0.05, 4, 20 = 2.87
-         Between columns (quality parameters);
Ftab = F 0.05, 5, 20 = 2.71

From the statistical analysis carried –out, the results revealed that the values of F – distribution calculated (F cal) for both factors (or treatments; methods of extraction and quality parameters) are greater than their table values (F tab) obtained. Consequently, these imply that significant difference exists among the methods of extraction in the analysis of variance with respect to the means of the quality parameters of honey samples which, likewise, did significantly differ.

Thus, the null hypotheses, Hi(1) and Hi(2)  are rejected while, the alternative hypotheses, H10  and H20  hold through. Hence, the traditional methods of honey extraction have significant impact on the quality parameters of the honey samples in this study.

4.3       DISCUSSION
Results of the moisture content of honey samples in this study, obtained using different traditional methods of honey extraction (Table 3), revealed that all the samples, except sample labelled F1 (Extracted using hand – squeezing method), were within the acceptable range (15.00 – 21.00%) as stipulated by NIS (2003) and Scott (2012). Honey samples labelled B1 (extracted using honey – pressing method) had the lowest moisture content (15.50%), while that of F1 (extracted using hand squeezing method) had the highest value (22.00%). High level of moisture in honey samples is indicative of either adulteration, mixing with water during extraction process or absorption of moisture during open storage (Lawal, et al, 2009). According to Agbagwa, et al., 2011), low moisture content of honey, as recorded for most samples in this study, forms the most important parameter of honey quality, because it defects the product’s storage life, processing characteristics and protects it from attack by micro-organisms. The water – insoluble content of the honey samples, extracted using different traditional methods (Table 4), were all outside of the acceptable range (0.1 – 0.5%) as required by NIS(2003). The results revealed water- insoluble content with a range of between 1.30 and 1.70% for all the honey samples in this study. Bogdanov, et al. (2001) reported that honey – wax is a major source of water – insoluble contamination and its high presence in all the samples suggests poor filtration techniques of the product by all the traditional methods of honey extraction in the area investigated.

According to NIS (2003), the maximum content of 0.6% is stipulated for honey ash level. This value is in contrast to the findings of this study which showed that, other than honey samples labeled A2 (0.5%) and C1 (0.6%) (both extracted using solar – melting method) and E2 (0.6%) (extracted using floating method,) all the honey samples extracted using different traditional methods (Table 5), had an ash content range of between 0.65 and 0.85%; beyond the acceptable limit by NIS (2003). The ash content of honey samples extracted using different traditional methods in Kaduna state indicate fairly good physical property. The pH value, however, were also within the acceptable range of between 3.42 and 6.10 (NIS, 2003) as revealed by Table 5. The significance of pH at acidic range in foods can not be over – emphasized. Lawal, et al. (2009) reported that such acidic range prevent the honey samples from constant infection by various species of micro- organisms and thus, help to ensure constant shelf – life for the honey samples. Thus, the acidic pH of all honey samples extracted using different traditional methods in Kaduna state, indicate they have good shelf – life.

Total titrable acidity, just like the pH at acidic range, allows for an enhanced shelve stability of honey and, thus, prevents spoilage by micro – organism (Williams, et al., 2009). The total titrable acidity of honey samples in this study, expressed in terms of percentage lactic acids (Table 6) was highest in sample labelled C2 (0.37%) (extracted using sieving method) and least in sample labelled A1 (0.12%) (extracted using honey – pressing method). However only four honey samples were within the acidic range (0.04-0.19%), as reported by Lawal, et al., (2009). The implication of this result is that, most of the honey samples extracted using different traditional methods in Kaduna   state are susceptible to attack by micro – organisms and spoilage, despite having good pH level. Results of the density (weight per volume) of honey samples in this study (Table 6), revealed half of all the samples across all the different traditional methods of extraction were below the acceptable range (1352 – 1500 g/L) as stipulated by NIS (2003). Low density of honey samples suggests high amount of water – insoluble particles (Bogdanov, et al., 2001); which implies one out of every two honey samples extracted using any type of traditional method in Kaduna state had been poorly filtered, or diluted with other substances such as sugar-cane sugar or corn syrup to increase its (honey’s) quantity for much profit during sales.                                                                                                        

Determination of sugars in honey is a quality criteria which is influenced by honey storage and heating; and thus, it is an indicator of honey freshness, adulteration and overheating (Agbagwa, et al., 2011). The results obtained for glucose, fructose and sucrose contents of honey samples in this study (Table 7) are in contrast to the range required by NIS (2003) and international standards. According to the proposal by the International Honey Commission (IHC), a minimum content of the sum of glucose and fructose (apparent reducing sugars) is 60g/100g (or 60%) for all blossom honeys, while sucrose has a maximum content of 5g/100g (or 5%), (Bogdanov et al., 2001). Thus, the non-  conformity of the sugars in the honey samples in this study with the stipulated standards suggests, that the honey samples extracted using different traditional methods in Kaduna  state have either been adulterated with sugar – cane sugars or possibly overheated during extraction processes.

Micro – organisms in honey may influence the stability of the product and its hygienic quality (Snowdon and Cliver, 1996). Microbes of concern in post – harvest handling of honey, as further reported, are those that are commonly found in the product (that is, yeasts and spore – forming bacteria), those that indicate the sanitary or commercial quality of honey (that is, Coliforms and yeasts), and those that are under certain conditions could cause human illness. The microbiological analysis of the honey samples in this study (Tables 8,9 and 10), therefore, revealed that non of the samples is completely free of adulteration. This can be seen clearly in the disparities between the results of enumeration of microbes in honey samples and the permissible limit prescribed by NIS(2003). Despite the absence of E. coli and yeast count in all the samples, only honey samples labelled B1 (extracted using honey – pressing method), C1 and B2 ( both extracted using solar-melting  method) and C2 (extracted using sieving method) were below the NIS (2003) maximum Total Bacteria Plate count (TPC) of 1.0 X 103 cfu/g. There was an overwhelming amount of the bacteria; Lactobacillus species, which was an indication of the high content of lactic acid in most of the honey samples. Other than honey samples labeled A1 (extracted using honey pressing method), all of the samples exceeded the NIS(2003) maximum mould count of 5.0 x101 cfu/g; recorded high presence of the mould species. Most worrisome situation was in Total coliform count (TCC) in which all the honey samples revealed presence of coliform species; which is in sharp contrast to a complete absence, as stipulated by NIS (2003). Thus, the poor Microbiological status of honey samples extracted using different traditional methods in Kaduna state is a clear indication of poor sanitary conditions and unhygienic quality of extraction processes. Consequently, such honey samples when consumed consistently over time could pose health risks; such as Botulism (digestive system disorder), and Honey intoxication (symptoms include dizziness, weakness, excessive perspiration, nausea, and vomiting), to humans (FDA, 2012 and The National Honey Board, 2012).
 





















CHAPTER V
CONCLUSSION AND RECOMMENDATION
5.1       CONCLUSION
            All the honey samples analyzed in this study had good shelf life and fairly good physical properties. Despite having these attributes, all of the honey samples contained high amount of water-insoluble contaminants (mainly honey-wax), the samples had been adulterated with other substances (possibly sugar-cane syrups), they had been overheated and were highly susceptible to attack by micro-organisms and spoilage. The presence of pathogenic organisms like the coliforms and lactobacillus bacteria in the honey samples indicated the poor sanitary and commercial qualities of the products. As such, these honey samples are not safe enough for human consumption, neither as sugar substitute in confectioneries nor as health enhancer in medicine. Therefore, the fact that the highest percentage of honey samples extracted and marketed in different retail outlets in Kaduna state are obtained using traditional methods of extraction, necessitates an urgent need to monitor and assess the quality of any batch of the product extracted, for the benefits of the health of the consuming public, and for the good of the honey industry in Kaduna state.      

5.2       RECOMMENDATION
            In order to holistically address the challenges posed by the traditional methods of honey extraction on the products quality status in Kaduna state, the following suggestions are recommended for prompt action by the authorities concerned;
5.2.1   All the commercial bee farmers and honey producers in Kaduna state should be mobilized by the state’s ministry of agriculture and water resources into forming a strong and formidable, Cooperative Society for proper identification, representation and easy access for/to cooperate supports.
5.2.2   More enlightenment and retraining on the current trends in apiculture and honey production should be given to the local commercial bee farmers and honey producers via their cooperative society by the state’s rural agriculture extension agency, to ensure the production of safe, wholesome and marketable honey.
5.2.3   Capital support, in form of grants or loans at low interest rates and good enough repayment period should be given to the local commercial bee farmers and honey producers, via their Cooperative society, by the Nigerian Agriculture, Cooperative  and Rural Development Bank (NACRDB) to acquire honey friendly and compatible extraction equipment, and build standard honey processing rooms or halls.
5.2.4   The Kaduna state government through a public - private partnership should intervene in the establishment of honey monitoring and quality assessment centers, alongside standard laboratories, in the major honey bearing communities of the state to render services to the local commercial bee farmers and honey producers at affordable cost.

REFERENCES

Activon (2013). Microbiological Quality control for Medical Grade Honey. Retrieved April 25, 2013, from Activon – Medical Manuka Honey Website: http://www.medicalhoney.com/quality-standard.

Adjare, S. (1990). BeeKeeping in Africa: Food and Agricultural Organization of the United Nations; Agricultural Services Bulletin 68/6, Chapter 5. Retrieved October 30, 2012, from http.//teca.fao.org/technology/beekeeping-africa-honey-and…

Agbagwa, O. E., Otokunefor, T. V, Frank – Peterside, & Nnenna (2011). Quality  assessment of Nigeria honey and Manuka honey. Journal of Microbiology and Biotechnology Research. J. Microbiol. Biotech. Res. 2011, 1 (3): 20 – 31. Retrieved September 27,  2012, from Scholars Research Library Website: http://scholarsresearchlibrary.com/archive.html.

Bagdanov, S., Lullmann, C., Martin, P. & Ohe W. (2001). Apiservices – Article-Honey Quality and International Regulatory Standards. Review by the International Honey Commission. Retrieved May 03, 2013, from http://www.beekeeping.com.articles/us/honey_quality.html

Devon Apicultural Reseach Group, DARG,  (2012) Bees for Development – Information Portal Search: Equipment, Harvesting and Handling Honey. Retrieved September 27, 2012 from Devon Apicultural Research Group, United Kingdom, Website: Http:///www.Beesfordevelopment.Org>Home>InfroamtionPortal

Food and Drug Administration, FDA (2012). Centre for  food safety and applied nutritions food borne pathogenic microorganisms and natural toxins handbook. “Grayanotoxin” Retrieved September 27, 2012, from http://www.fda.gov/food/foodsafety/foodborneillness/FoodbornepathogensNaturalToxins/BadBugBook/ucm071128.htm

Frazier, W. F. & Westhoff, D. C. (1994). Food Microbiology. (4th ed.) Singapore: McGraw Hill Book Company. Pp. 234 – 235.

Gheldorf, N., Wang X, & Engeseth N. (2002). Identification and quantification of antioxidant components of honeys from various floral sources.” J. Agric Food Chem 50 (21):5870 -7, PMID 12358452. Retrieved September 27, 2012, from http://www.ncbi.nlm.gov/pubmed/12358452).

Guralink, D. B. (1975). Webster’s New World Dictionary. New Delhi, India: Oxford and IBH Publishing Company.

 Harmonised Methods of the International Honey Commission. IHC (2009). Retrieved October 4, 2013, from http:///www.bee-hexagon.net/en/network.htm

Honey Travelers (2011). Raw Honey/Honey Traveler. Retrieved September 27, 2013 from http:///www.honeytraveler.com>honeytraveler>HoneyBuyersGuide.

Jennifer, E. (2007). UW Study tests topical Honey as a Treatment for diabetic Ulcers. UW Health’s Eau Claire family Medicine clinic. Retrieved September 27, 2012 from University of Wisconsin –Madison Website: http;//www.news.wisc.edu/releases/13738.

Krell, R. (1996) Value-added products from Beekeeping. FAO Agricultural Services Bulletin. No. 124, Food and Agricultural Organization of the United Nations, Rome, Italy, ISBN 92-5- 103819-8

Lawal, R. A.; Lawal, A. K., & Adelaku, J. B. (2009). Physico-chemical Studies on Adulteration of Honey in Nigeria. Pakistan Journal of Biological Sciences, 12: 1080 – 1084. Retrieved April 25, 2013, from http://www.scialert.net/fulltext/?doi=pjb.2009.1080.1084.

Mutsaers, M (1991). Processing Honey from Top – Bar Hives. Bees for Development (19th ed). Retrieved January 24, 2013, from http://www.beesfordevelopment.org>Home>InformationPortal>…Honeyextraction.

Mark, G. (2008). Roman cookery, London: Serif publishers. Retrieved September 27, 2012, from http://www.serifbooks.co.uk/books/cookery/...

Molan, P. C. (1999). The role of honey in the management of wounds. Journal of Wound Care. 8: 415 – 418.

Murray, R. S., & Larry, J. S. (2011). Schaum’s Outline of Statistics (4th ed.). McGraw- Hill Publishers. Pp. 403 – 427.

National Honey Board (2012). Carbohydrates and the Sweetness of Honey. Retrieved September 27, 2012 from http://www.honey.com/images/downloads/carb.pdf

NIS (2003). Nigerian Industrial Standard: Standard for Honey. 473: ICS 67.180.10. Price Group: SON.

Ng’atigwa V., & Yule, I. J. (1989). Agricultural Mechanization in Asia, Africa and Latin America: Increasing Honey Production in Tanzania through Mechanization. Vol. 27. Pp 36 - 37

Ojeme, F. (1990). Effective Quality Assurance System in Food and Drink Industries. Paper Presented at the National Workshop on Innovative Approach on Quality Control and Marketing of Food and Drinks in Nigeria.

Oyinlola, T. A. (2004). Challenges and Strategies of Quality Control in Food Manufacturing. Nigerian Journal of Engineering Management. Vol. 5. No. 4.

Postmes, T., Bogaard, A. E. & Hazen, M. (1995). The Sterilization of honey with cobalt 60 gramma radiation: A Study of honey spiked with spores of Clostridium botulinum and Bacillus subtilis. Experimentia 51 (9 – 10): 986 – 9 PMID 7556583. Retrieved September 27, 2012 from http://www.ncbi.lum.nih.gov/pubmed/7556583.

Sanni, L. O. (1997). Quality Assurance System in Food Industry. Abeokuta: Jedidah Publishers. Pp. 10 – 11.

Surendra, R. J. (2008). Honey: A Source of Nutrition, Medicine and Cash Income. Retrieved January 17, 2013, from International Centre for Integrated Mountain Development Website: http://www.icimod.org/%3Fq%3D1519

Subramanian, R., Hebbar, H. U., & Rastogi, N. K. (2007). Processing of Honey: A Review International Journal of Food Properties, 10: 127. Retrieved September 23, 2012, from http//dx.doi.org/10.1080%F10942910600981708.

Scott, (2012). Honey can be used for  a variety of medicinal purposes. Retrieved April 25, 2013, from http://www.todayifoundout.com/index.php/20...

Snowdon, J. A, & Cliver, D. O. (1996). Microorganisms in honey. International Journal of Food Microbiology, 31: 1 – 6.

SueBee (2011). Sioux Honey Association – SueBee Honey. Retrieved August 27, 2012 from http://www.suebee.com/sioux-honey-assn.

The National Honey Board (2012). Frequently asked questions. Retrieved  September 27, 2012 From http://www.honey.com/nhb/about-honey/frequently-asked-questions/category/honey-properties.

Tim, M. (2012). How to Harvest Honey from Natural Comb, Milkwood Permaculture. Retrieved January 17, 2013, from http://www.milkwood.net/2012/01/19/how-to-harvest-honey-from-natural-comb...

Vasyl, S. (2013). My Tender Sweet Honey. “Halytski kontrakty” Ukrainian Business Weekly Magazine. Retrieved September 27, 2012, from http://www.kontrakty.com.ua/ukr/gc/normer/2003/20/29.html.

Wilkins, A. L. & Lu, Y. (1995). Extractives from New – Zealand Honeys. 5. Aliphatic Dicarboxylic Acids in New-Zealand Rewarewa (Knightea excela) Honey. J. Agric. Food Chem., 43 (12): 3021. Retrieved September 27, 2012 from http://www.dx.doi.org/10.1021%2Fjf00060a006.

Williams, E. T. Jeffrey, J., Barminas, J. T.,  & Toma, I. (2009). Studies on the effects of the honey of two floral types (Zioziphus spp. and Acelia spp.) on organism associated with burn wound infections. Afr. J. Pure Appl. Chem. 3: 098 – 101.



























APPENDIX I
Table 3: Total sugar (soluble solids) content and moisture content of honey samples extracted using different traditional methods.
Sample
Method of Extraction
Total Sugar Content (%)
Moisture Content (%)
A1
Honey Pressing
81.00
17.40
B1
Honey Pressing
81.50
16.80
C1
Solar Melting
80.80
17.70
D1
Solar Melting
78.40
20.00
E1
Sieving
79.80
18.90
F1
Hand-Squeezing
76.70
22.00
A2
Solar Melting
80.80
17.50
B2
Solar Melting
81.00
17.40
C2
Sieving
83.00
15.50
D2
Honey Pressing
79.70
19.00
E2
Floating
79.50
19.00
F2
Floating
78.00
20.40


Table 4: Total solids and water insoluble content of honey samples extracted using different traditional methods.

Sample
Method of Extraction
Total Solids (%)
Water Insoluble Content (%)
A1
Honey Pressing
82.60
1.60
B1
Honey Pressing
83.20
1.70
C1
Solar Melting
82.30
1.50
D1
Solar Melting
80.00
1.60
E1
Sieving
81.10
1.30
F1
Hand-Squeezing
78.00
1.30
A2
Solar Melting
82.50
1.70
B2
Solar Melting
82.60
1.60
C2
Sieving
84.50
1.50
D2
Honey Pressing
81.00
1.30
E2
Floating
81.00
1.60
F2
Floating
79.60
1.60




APPENDIX II
Table5: Ash content and pH values of honey samples extracted using different traditional methods.

                   
Sample
Method of Extraction
Ash Content (%)
pH values
A1
Honey Pressing
0.65
4.16
B1
Honey Pressing
0.70
4.09
C1
Solar Melting
0.60
3.44
D1
Solar Melting
0.70
4.01
E1
Sieving
0.75
4.15
F1
Hand-Squeezing
0.75
3.86
A2
Solar Melting
0.50
3.84
B2
Solar Melting
0.85
3.77
C2
Sieving
0.75
3.77
D2
Honey Pressing
0.80
4.11
E2
Floating
0.60
4.03
F2
Floating
0.70
3.94


Table 5: Total Titrable acidity (% Lactic Acid) and density (Weight per volume) of honey samples extracted using different traditional methods.

Sample
Method of Extraction
Total titrable acidity (% Lactic Acid)
Density (Weight per volume, g/L )
A1
Honey Pressing
0.12
1372.83
B1
Honey Pressing
0.14
1372.58
C1
Solar Melting
0.13
1351.44
D1
Solar Melting
0.14
1336.25
E1
Sieving
0.24
1341.85
F1
Hand-Squeezing
0.32
1382.16
A2
Solar Melting
0.21
1329.50
B2
Solar Melting
0.23
1363.75
C2
Sieving
0.37
1359.92
D2
Honey Pressing
0.23
1331.18
E2
Floating
0.22
1347.56
F2
Floating
0.20
1322.35


APPENDIX III
Table 7: Sugar (% Glucose, Fructose and Sucrose) contents and reducing sugar content of honey samples extracted using different traditional methods.
                                                                      Sample Label
Method of Extraction
Standard Sugar Concentration in 100mL of Distilled Water
Sugar Content in Sample
                                                                    
Reducing sugar content (%)


Glucose (%)
Fructose (%)
Sucrose (%)
Glucose (%)
Fructose (%)
Sucrose (%)

A1
Honey Pressing
8.00
8.00
8.50
26.45
26.45
28.10
52.90
B1
Honey Pressing
8.00
8.00
8.50
26.61
26.61
28.27
53.22
C1
Solar Melting
8.00
8.00
8.50
26.38
26.38
28.03
52.76
D1
Solar Melting
8.00
8.00
8.50
25.60
25.60
27.20
51.20
E1
Sieving
8.00
8.00
8.50
26.06
26.06
27.69
52.12
F1
Hand-Squeezing
8.00
8.00
8.50
25.04
25.04
26.61
50.08
A2
Solar Melting
8.00
8.00
8.50
26.38
26.38
28.03
52.76
B2
Solar Melting
8.00
8.00
8.50
26.45
26.45
28.10
52.90
C2
Sieving
8.00
8.00
8.50
27.10
27.10
28.80
54.20
D2
Honey Pressing
8.00
8.00
8.50
26.02
26.02
27.65
53.04
E2
Floating
8.00
8.00
8.50
25.96
25.96
27.58
51.92
F2
Floating
8.00
8.00
8.50
25.47
25.47
27.06
50.94

APPENDIX IV
Table 8: Result of enumeration of Mesophilic Aerobic Bacteria Count (Total Plate Count, TPC) of honey samples extracted using different methods        
Sample Label
Method of extraction
Colonies counted
Colony forming unit per gramme (cfu/g)
Gram’s Reaction
Organisms isolated
A1
Honey Pressing
90x102
9.0x103
Gram positive short rods
Bacillus species

B1
Honey Pressing
98x101
9.8x102
Gram positive rods in chain
Lactobacillus species

C1
Solar Melting
17x101
1.7x102
Gram positive rods in chain
Lactobacillus species

D1
Solar Melting
98x102
9.8x103
Gram positive rods in chain
Lactobacillus species

E1
Sieving
62x102
6.2x103
Gram positive rods in chain
Lactobacillus species

F1
Hand-Squeezing
51x102
5.1x103
Gram positive rods in chain
Lactobacillus species

A2
Solar Melting
80x102
8.0x103
Gram positive oval-shaped cells
Yeast species

B2
Solar Melting
31x101
3.1x102
Gram positive rods in chain
Lactobacillus species

C2
Sieving
72x101
7.2x102
Gram positive rods in chain
Lactobacillus species

D2
Honey Pressing
41x102
4.1x103
Gram positive rods in chain
Lactobacillus species

E2
Floating
45x102
4.5x103
Gram positive rods in chain
Lactobacillus species

F2
Floating
78x102
7.8x103
Gram positive rods in chain
Lactobacillus species


APPENDIX V
Table 9: Result of enumeration of Total Coliform Count (TCC) of honey samples extracted using different traditional methods        
Sample Label
Method of extraction
Colonies counted
Colony forming unit per gramme (cfu/g)
Gram’s Reaction
Organisms isolated
A1
Honey Pressing
60x101
6.0x102
Gram negative rods
Coliform species

B1
Honey Pressing
160x101
1.6x103
Gram negative short rods
Coliform species

C1
Solar Melting
95x101
9.5x102
Gram negative short rods
Coliform species

D1
Solar Melting
72x101
7.2x102
Gram negative rods
Coliform species

E1
Sieving
60x101
6.0x102
Gram negative short rods
Coliform species

F1
Hand-Squeezing
13x101
1.3x102
Gram negative rods
Coliform species

A2
Solar Melting
77x102
7.7x103
Gram negative rods
Coliform species

B2
Solar Melting
47x101
4.7x102
Gram negative short rods
Coliform species

C2
Sieving
160x101
1.6x103
Gram negative rods
Coliform species

D2
Honey Pressing
15x101
1.5x102
Gram negative short rods
Coliform species

E2
Floating
35x101
3.5x102
Gram negative rods
Coliform species

F2
Floating
55x101
5.5x102
Gram negative rods
Coliform species



APPENDIX V
Table 10: Result of enumeration of Mould and Yeast (mould and yeast count) of honey samples extracted using different   traditional methods. 
Sample Label
Method of extraction
Colonies counted
Colony forming unit per gramme (cfu/g)
Culture media characteristics
Organisms isolated
A1
Honey Pressing
4.0x101
4x101
Whitish powdery colony
Mould species
B1
Honey Pressing
3.0x102
3x102
Whitish –swampy –powdery colony
Mould species
C1
Solar Melting
6.0x101
6x101
Whitish powdery colony
Mould species
D1
Solar Melting
2.0x101
2x101
Whitish powdery colony
Mould species
E1
Sieving
3.0x102
3x102
Whitish powdery colony
Mould species
F1
Hand-Squeezing
4.0x102
4x102
Blackish powdery colony
Mould species
A2
Solar Melting
6.0x101
6x101
Whitish powdery colony
Mould species
B2
Solar Melting
3.0x102
3x102
Greenish powdery colony
Mould species
C2
Sieving
1.2x102
12x101
Blackish powdery colony
Mould species
D2
Honey Pressing
3.0x102
3x102
Whitish swampy colony
Mould species
E2
Floating
1.5x103
15x102
Blackish and whitish –swampy – powdery colony
Mould species
F2
Floating
7.0x101
7x101
Blackish and greenish  powdery colony
Mould species






Location: Nigeria

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