CHAPTER is the combination of nectar honeyCHAPTER is the combination of nectar honey





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2.1 Introduction

2.1.1 Honey

Honey is a
natural product that produced by both honey bees and stingless bees (Rao et al., 2016). Nectar collected from
flowering plants will be swallowed by bees through straw-like proboscis into a
special sac, which called the ‘honey stomach’. This is where an enzyme will be
added to cause inversion and transform the nectar’s complex sugars into simpler
ones. Bees will then spit out that mixture, other bees in the hive will consume
that mixture and spit out again into the honeycomb cells. The bees will then
fanning the honeycombs by flapping their wings. The water in the nectar will
evaporate and turning it into honey that will be stored as food for the bees (Long,

          Honey is valuable to human due to its
nutritional value and antimicrobials properties. Approximate 80 % of its weight
are made up of sugars (fructose and glucose) and small amounts of
oligosaccharides. Honey is a mixture where it can be different in terms of
composition and appearance due to its botanical origin.  It can be grouped into different types of
honey based on their origin which are nectar honey, honeydew honey and mixed
honey. Nectar honey is produced from plant nectar, while honeydew honey is
produced by honeybees and mixed honey is the combination of nectar honey and
honeydew honey. The honey collected from the same origin may have different
composition due to geographic area, bee species, season and harvest technology
and storage mode (Boukraâ, 2013). Honey is used not only to cure cough, asthma,
and hay fever, but also used for diarrhea and stomach ulcers caused by
infection with Helicobacter pylori
bacteria. Honey is also served as a source of carbohydrate during vigorous

2.1.2 Propolis

A sticky plant
substances known as resin is produced and secreted by plants as a result of
infection or injuries on parts of plant. Resin will then be collected by bees. Propolis,
a dark sticky material, is the material that collected by honeybees from living
plants. The propolis will be mixed with wax for construction and adaptation of
the bee nests. It is used to build, defend, disinfect, strengthen and isolate
their nests to fill holes and to preserve dead predators inside the hives (Vit et al., 2013). Not only that, it is also
used to maintain the low level of bacterial and fungal concentrations in the
bee hives (Hasan et al., 2008).

          Propolis is rich in bioflavonoids,
essential oils, minerals, vitamins and phenolic compounds. Other than that,
propolis contains high amount of antioxidant, antibiotic, antimicrobial and
anti- inflammatory properties. It is highly advantageous to human being due to
its benefits. It is used as natural antiseptic, antibiotics and antibacterial.
High level of flavonoids, antibiotics, antimicrobial and anti-inflammatory
properties of propolis prevents the spreading of bacteria and increase the
healing process of cuts and wounds. Propolis acts as antibacterial agents due
to its high amount of antimicrobial properties.

          Next, propolis also used to prevent
and treat cancers and respiratory diseases. High amount of antimicrobial in
propolis help to kill harmful bacteria and cancer cells in human body
especially mouth and colon. It is also used as supplement of chemotherapy
treatment on cancer patients. Respiratory diseases such as asthma and
bronchitis are treatable with the use of propolis. Lastly, the immune system of
human body can be strengthen by regular consuming propolis which helps to
increase antibiotics and antibacterial in human body. It also proven to
decrease the enzyme activity and enables the blood pressure in human body to

          According to Hasan et al. (2008), the activity and chemical
compositions of propolis are different depending on the plant origin, season of
propolis harvesting and the geographical location of the collected bee hive.
This reason is enough for the researchers to investigate the active compound of
new propolis. Not only that, pharmacological activities of propolis are more
abundant in tropical regions compared to temperate climates.

2.1.3 Bee bread

Bee bread has
the compositions of carbohydrates, proteins, amino acids, lipids, fats, sugars,
fibers, vitamins and mineral salts so it is considered as a functional food. Beebread
comes from bee pollen, honey and bee saliva and thus its composition is the
same as bee pollen, but it is three times higher in nutritional and
antimicrobial value compared to bee pollen. The formation of beebread starts
with the collection of pollen, mixed with flower nectar or honey and saliva,
then carried to the bee hive. At the bee hive, non-flying bees will fill the
mixture into honeycomb cells for 75 % of the cell volume. The remaining cell
volume will be filled up with honey which protects the pollen mass from oxygen.
After that, anaerobic lactic acid fermentation process will occurs and beebread
will be formed. The lactic acid fermentation of the beebread is the same as the
fermentation process in yoghurt and produces a more digestible end product
which enriched with new nutrients. The segregation of enzymes by bees through
their saliva has induce both fermentation and enzymatic processes. Both of
these process are important to break the wall of pollen known as exine. The
wall of pollen is made up of sporopollenin where it is a compound that provides
the characteristics of chemical resistance to pollen and preserves compound
inside the pollen while limit the capacity to absorb nutrients and bioactive
substances inside the pollen grain (Zuluaga et
al., 2015).

though the composition of beebread is the same as bee pollen, but it has a
lower pH of 3.8 to 4.3. It has less proteins and fats but have more
carbohydrates and lactic acid compared to pollen. The walls of bee pollen that
is hard to be destructed by gastrointestinal liquid have been partially
destructed during the fermentation process. Due to that, beebread has a
superior bioavailability and the rich content of pollen can be absorbed easily
in human body (Ivanišová et al.,
2015). Beebread can be used as food supplement due to wide range of nutrients
content, The presence of abundant amount of proteins, vitamins and phenolic
compounds as natural antioxidant in beebread has provide huge contributions to
improve human life. The antimicrobial and antioxidant properties of beebread
has provide protection for human body such as normalizes metabolism and enhance
the regeneration of tissue in human body.

2.2 Antimicrobial

2.2.1 Honey

possessed an antimicrobial properties that is used against pathogenic
microorganisms (Boukraâ, 2013). According to Cardoso and Silva (2016), there
are many factors which leads to the antimicrobial properties of honey. One of
it is high osmotic pressure and low water activity. High osmotic pressure in
honey is due to the high concentration of sugars in honey which reduces the
water activity and inhibit the growth of microorganisms because water is needed
by the microorganisms to grow. Honey is also hygroscopic, where it removes
moisture out to the environment and dehydrates the bacteria.

factor is low pH of honey (acidic environment) and glucose oxidase system which
forms gluconic acid. Honey is very
acidic as the pH of honey ranges in between 3 to 4. Low pH of honey is caused
by gluconic acid which derived from the glucose oxidase reaction (Cardoso and
Silva, 2016). The acidic condition is unfavourable for the growth of
microorganisms as the optimum pH for most microorganisms is in between 7.2 to
7.4 and cannot survive in pH levels lower than 4. Other than that, low protein
content of honey starves the bacteria by reducing the nitrogen needed by the
microorganisms to grow. Other factors such as high carbon to nitrogen ratio,
low redox potential due to high amount of reducing sugars and viscosity which
limits dissolved oxygen also contribute to the antimicrobial properties of
honey. Chemical agents such as pinocembrin, lysozyme, phenolic acids, terpenes,
benzyl alcohol and volatile substances contribute to the antimicrobial
properties of honey. Lysozyme, phenolic acids and flavonoids are the
non-peroxide factors that contribute to the antimicrobial properties of honey.

primary sources that contribute to the contamination of microbial include
digestive tracts of honey bees, dust, pollen and nectar which are hard to be
controlled. Secondary sources such as equipment and food handler also
contribute to microbial contamination. There are specific processing steps that
manipulate the microbial counts in most honey samples, thus it is important to
have the knowledge of moisture and temperature conditions where it can
influence the growth of microorganisms in honey while controlling the spoilage
of honey. Specific processing step can causes the low microbial counts in honey
samples. For example, high temperature of 71.1 °C was used to heat the honey
for 30 minutes where it should kill yeasts and vegetative bacteria. In
addition, strained honey injected and flows through a 150 µm screen and
filtered honey flows through a 1 µm filter. These steps may reduce or eliminate
most of the microorganisms.

honey has antimicrobial properties, thus it is capable in killing bacteria and
the major antimicrobial agent is hydrogen peroxide. Bacteria is very sensitive
to hydrogen peroxide. However, it has been determined that yeasts and molds
were not as sensitive to hydrogen peroxide as compared to bacteria. Yeasts and
molds are able to survive in honey. Under certain condition, there are some
vegetative and spore-forming bacteria are able to grow in honey for varying
period of time, but there are no bacteria known to be survive in honey (Doyle
and Sperber, 2009).

Figure 2.2.1: The chemical structure of hydrogen

peroxide is produced during glucose oxidation and it is the main inhibitor
which contributes to the antimicrobial properties of honey. According to Doyle
and Sperber (2009), glucose oxidase system forms peroxide, where glucose
oxidase system is an enzymatic reaction that is active in diluted honey was
determined by White, Subers and Schepartz in 1962. Glucose oxidase is the
enzyme that produce hydrogen peroxide and it is introduced to the honey when
bees harvest the nectar. This enzyme can be found in all honeys but the
concentration may vary from one honey to another due to the age and health
status of the hunting bees. Catalase originates from pollen and it hydrolyzes
hydrogen peroxide into oxygen and water. The amount of pollen grains in honey
determines the concentration the catalase and thus hydrogen peroxide levels may
differ in different honey. The antimicrobial properties of honey is correlated
with the level of hydrogen peroxide whereby it is determined by the glucose
oxidase and catalase level. When the glucose oxidase level is high, the
catalase level will be low thus the peroxide level will be high and vice versa.
The level of hydrogen peroxide is directly proportional to the inhibition of
bacterial growth in honey. High content of hydrogen peroxide in honey causes
bacteria to respond abnormally to proliferative signals and inhibit the growth
of bacteria (Hayashi et al., 2014).
Hydrogen peroxide also contributes to the healing properties of honey. The
concentration of hydrogen peroxide produced by diluted honey are lower than
those in 3 % hydrogen peroxide solutions. The low concentration of hydrogen
peroxide acts as an intracellular messenger and stimulates the wound healing.
It helps in both tissue repair and contributes to the antibacterial properties
of honey even on a heavily exuding wound (Boukraâ, 2013).

on the research result done by Mohapatra et
al. (2011), honey has both bacteriostatic and bactericidal activity against
many pathogens. The study showed that honey provides alternative treatment
against certain bacteria. The study suggested that, the active components of
honey extract need to be characterized and the benefits of honey in the
treatment of bacterial infections need to be investigated.