CHAPTER 1

INTRODUCTION

1.1 Background

Honey is very famous as a dietary food as a supplement for

human. People now started to give their attention towards

the importance and benefits that can be gained from

consuming honey. Since the past 2,500 years, pure honey has

been consumed by human since there are a lot of benefits

from honey aside from acting as natural sweetener in food

and beverages.

The most well-known properties of honey are its antioxidant

and antimicrobial contents. The antimicrobial properties

makes honey useful to heal wounds. Different types of honey

contain different characteristics and properties. Hence, the

different sources of honey reflects the content and

characteristics.

In the Qur’an according to Sura 16 verse 68, it was stated

that honey is a miracle and it can be a healing sources for

mankind. Allah Almighty asked the bees: “Build habitats in

the mountains and the trees, and also under the human’s

residence. Then you can eat fruits and suck the nectar

freely from them, and travel the smooth way that been

prepared by your Lord. From the inside, there is extracts

from different colors and can act as medicine to human.

Also, it will be revealed only for the people who concern

and think”. With science and technology available today, the

benefits of honey can now been further investigated.

In Sarawak, Acacia mangium honey is abundant in Sarawak. It is

collected by the bees from the nectar of A. mangium tree

which is common in Southeast Asia. A. mangium honey is

considered as a raw honey because there are no additives or

preservatives added during production.

Another type of honey that is commonly available in the

market nowadays, is the monofloral Australian honey. As

these two types of honey; Australian and A. mangium honey are

very high available, thus it is good to investigate the

prebiotic ability of these honey.

1.2 Problem statement

There are several molecules that show prebiotic abilities

for example fructooligosaccharide (FOS),

galactooligosaccharide (GOS), and inulin as they increase

the population of commercial strains of Bifidobacteria (Kajiwara

et al. 2002). Prebiotic is one of the functional food that can

influence the microbiota composition of human. Prebiotic is

a source of food for beneficial bacteria that is present

inside the human’s gut known as a probiotic. Common

beneficial bacteria that available inside human’s gut are

Lactobacillus and Bifidobacterium.

According to Shamala et al. (2000), honey has been proved to

have the ability in supporting the growth of lactic acid

bacteria in rats’ small and large intestine both in vivo and in

vitro experiments. Honey has found to become one of the

sweetener that increase the production of lactic acid in

fermented milk (Ustunol et al. 2001). A lot of problems will

arise in the human gastrointestinal system if there is

shortage of probiotic inside the

human gut; such as gastritis, colitis, leaky gut, irritable

bowel (IBS), ulcers, constipation, diverticulosis,

diverticulitis, diarrhea and hemorrhoids.

Jan et al. (2010), did a research on several Malaysian local

honey from Tapah, Perak. These honey are wild Malaysian

Tualang honey, common wild honey and a commercial Tualang

honey. The result revealed that the commercial Tualang

support the higher population of B. longum BB 536 after

fructooligosachharides, followed by common wild honey and

wild Tualang honey.

By utilizing our own native honey and with the aim to

upgrade the Borneo’s food industry, this study can boost the

competitiveness of A. mangium honey here onwards. In order to

observe the A. mangium honey’s performance, this honey will be

compared with another honey; the Australian honey, because

it is cheap and highly available.

To further investigate the effect of A. mangium and

Australian honey, towards the growth of beneficial bacteria,

the in vitro fermentability model of honey oligosaccharide by

Lactobacillus and Bifidobacterium will be conducted. The amount of

short chain fatty acid and the concentration of beneficial

bacteria, Bifidobacteria and Lactobacilli will be used as the

parameter to determine the honey oligosaccharide’s

suitability as a potential prebiotic candidates.

1.3 Objectives

The objectives of this research project were:

1. To extract the oligosaccharide from A. mangium and

Australian Honey

2. To observe the growth of beneficial bacteria within in

vitro fermentation of honey oligosaccharides.

CHAPTER 2

LITERATURE REVIEW

2.1 The Honey

Pure honey without additive are good and promote human

health by proposing lots of beneficial ingredients to human

health such as carbohydrates, proteins, vitamin and trace

element (Bogdanov 2012). Table 2.1 summarized most all of

nutrients contained in honey. The percentage of carbohydrate

contained in honey is the highest compare to others elements

that is 300 kcal for each 100 g of pure honey (Bogdanov

2012).

There are lots of honey available in the market recently,

included the processed and unprocessed honey for example raw

honey and commercial honey. Previous research stated that

different honey will give different result on growth of

beneficial bacteria. According to Jan et al. (2010), wild

honey produced low population of bacterial growth compared

to commercial honey as can be referred in Table 2.2 is the

result of B. longum BB’s growth population in skimmed milk

with honey.

Instead of having prebiotics effect, honey also content

another properties such as antimicrobial, anti-inflammatory

and antioxidant properties. Thus, because of those abundant

good properties, honey becomes one of preferable food

nowadays since lots of people were concerned on benefits

that can be given by honey.

Table 2.1 The main nutrients that contain in common honey

Ingredient Recommended Daily Intake

Amount in 100 g

1-4 yearsold

4-15 years old

After 15 years old

Carbohydrates (kcal) 300 1000-1100 1400-2700 2400-3100

Proteins (g) 0.5 13-14 17-46 44-59

Fats (g) 0 - - -

Minerals

Sodium (mg) 1.6 – 1.7 300 410-550 550

Calcium (mg) 3 – 31 600 700-1200 1000-1200

Potassium (mg) 40 – 3500 1000 1400-1900 2000

Magnesium (mg) 0.7 – 13 80 120-310 300-400

Phosphorus (mg) 2 – 15 500 600-1250 700-1250

Zinc (mg) 0.05 – 2 3 5-9.5 7-10

Copper (mg) 0.02 – 0.6 0.5-1 0.5-1 0.5-1

Iron (mg) 0.03 – 4 8 8-15 10-15

Manganese (mg) 0.02 – 2 1-1.5 1.5-5 2-5

Chromium (mg) 0.01 – 0.3 0.02-0.06 0.02-0.1 0.03-1.5

Selenium (mg)

0.002 – 0.01

0.001-0,004

0.001-0.006 0.003-0.007

Source: Bogdanov (2012)

Table 2.2 Growth population of Bifidobacterium longum BB 536 in skimmed milk with honey and FOS (log10 CFU/mL) at 37˚C

Skimmed milk with honey/ FOS 0 h 24 h

Wild Tualang 5.50b ± 0.02 7.00c ± 0.01Common wild honey 5.40c ± 0.03 7.10c ± 0.02Commercial Tualang 5.30d ± 0.04 8.70b ± 0.05Fructooligosaccharid

es5.70a ± 0.01 8.80a ± 0.01

Control (skimmed

milk only)5.80a ± 0.07 7.00c ± 0.14

Source: Jan et al. (2010)On the other hand, honey comes out with a microbiological

composition which consist of concentrated sugar solution

thus, reflects the properties of honey as an impossible

medium for microorganism to grow. Honey is one of the

natural food product that contain less harmful bacteria,

plus honey also did not contain dangerous Bacillus species. So

far, total bacteria contain in honey are not dangerous to

human being and safe to be consumed directly without cook it

first (Bogdanov 2012).

Different honey has different level content of osmotolerant

yeasts. This type of yeast is one of the contributors

towards an undesirable fermentation in honey. Since moisture

content of honey quite high, this properties can enhance the

development of osmotolerant honey. So, in order to prevent

this undesirable fermentation, the special precaution should

be taken into special consideration towards all the honey

harvesters since the easiest way to control this problem is

by harvesting honey with low humidity. Plus, honey must be

stored carefully such as store it inside an air-tight

container.

There are several previous researches that stated honey can

maintain a healthy balance of gastrointestinal tracts of

human since it has the prebiotic properties as it contain

oligosaccharides molecules. In addition, human guts contain

millions of microorganism which all of them play an

important role in enhancing and provide normal digestion

together with the gut health by becoming a microorganism

resident on the guts surface (Wallace 2009). So, it is

necessary to control and preserve the bacteria inside the

gastrointestinal tract in order to maintain the performance

of gut’s health and digestion activities.

As been done by Ustunol (2001), it had been claimed that

honey increase the Bifidobacteria population while the other

sweetener that also been used did not stimulated the

Bifidobacteria growth. Clearly, honey can be a good and

preferable sweetener in milk.

2.2 The Oligosaccharide

Honey is a functional food in our dietary program, contain

carbohydrate which have 3 to 10 monosaccharide linked

together, known as oligosaccharide. The properties of

oligosaccharide that drawn recent attention as a prebiotic

is the indigestible characteristic, since it had been found

within the upper part of gastrointestinal track, as not been

digested, absorbed, nor hydrolyzed molecules and it improves

the consumer health by enhance the growth of beneficial

bacteria inside the host’s colon (Ustunol 2000).

The honey composed of monosaccharide, disaccharide and

oligosaccharide. However, monosaccharide and disaccharide

need to be expelled since the presence of these two types of

molecules will bring problem such as they also can become

the substrates toward the growth of bacteria as they are not

be able to ferment.

There are another non-digestible oligosaccharides that

commonly known and being used within this food industry’s

research namely melezitose, erlose, maltotriose, panose and

others. Sanz et al. (2005), conducted a study on how honey

oligosaccharide affects the bacteria population in human’s

gut intestinal track (GIT), and found honey which contain

higher amount of oligosaccharide resulted in large amount of

beneficial bacteria’s growth.

There are several ways to separates the oligosaccharides

from honey and removes the monosaccharides and disaccharides

compound. Those separation techniques of honey are by

nanofiltration, yeast treatment and activated charcoal

treatment. Nanofiltration is the process where honey is been

filtered using a special nano-sized membrane in repetitive

steps.

Yeast treatment is where Saccharomyces cerevisiae been used to

absorb the monosaccharide and disaccharide and left the

oligosaccharide to be used in the fermentation process. Next

technique is by activated charcoal treatment, using charcoal

in ethanol and water solution to absorb oligosaccharides

then undergoes several stirring stages in different level of

ethanol’s concentration to extract the absorbed

oligosaccharides from the activated charcoal.

Sanz et al. (2005) highlighted the comparison of separation

methods towards the population of Lactobacillus and Bifidobacteria.

The sample that gain the highest Prebiotic Index (PI) value

is from charcoal treatment because it contain the greatest

oligosaccharide content while for nanofiltration, the level

of oligosaccharide content is only at the average level. The

lowest amount of oligosaccharide content is the sample that

been treated by yeast treatment, since all of the

oligosaccharides had been hydrolyzed during the yeast

treatment process.

To prove this statement of oligosaccharide content in

samples after were treated by three techniques;

nanofiltration, yeast treatment and charcoal treatment, a

study on effect of different type of separation analysis of

honey towards population of both Lactobacillus and Bifidobacteria

had been conducted by Sanz et al. (2005).

The higher population changes on growth of both bacteria

subjected to charcoal treatment since the samples been

recorded to contain highest amount of oligosaccharides.

Table 2.3 sums the changes of Bifidobacteria and Lactobacillus

population due to different separation technique applied.

Table 2.3 The effect of different separation technique onpopulation changes of,,,,,,,,,,,,,,,,,,, Bifidobacteria andLactobacilli

Source: Sanz et al. (2005)

2.3 The Prebiotic

The usage of honey as prebiotics had been highlighted then

brought into the food industry, thus increase the demand and

popularity of honey to be included in human dietary program.

The reason why is because honey contain oligosaccharide that

Separation Method Nanofiltration Yeast Treatment Charcoal

TreatmentPopulation changeson growthof Lactobacilli

9.53 ± 0.08a 9.52 ± 0.17a 9.62 ± 0.23a

Population changeson growthof Bifidobacteria

8.55 ± 0.19c 8.21 ± 0.25b 8.61 ± 0.13c

poorly digested within the human gastrointestinal guts thus

become the source of food for hundreds of colon’s bacteria.

A prebiotic is actually a non-digestible food ingredient

that beneficially affects the host by selectively

stimulating the growth and activity of lactic acid bacteria

in the colon thus promoting the health of the host (Gibson

and Roberfroid 1995). Hence, by the action of providing the

good health condition towards the host, it is known as the

prebiotic effect.

There are three criteria that required for the foods to be

acknowledged as having a prebiotic effect, i.e., the food

must be able to ferment by the microbes that present inside

the colon, the food must be specifically selected and at the

same time it can stimulate the beneficial bacteria inside

the human gut, and the food must be resistant towards any

enzymatic activity, stomach acid and hydrolysis process (Loo

et al. 2010).

There are another types of prebiotic that contain

oligosaccharides and commonly known thus had been used so

far in this food industries research namely

fructooligosaccharides (FOS), high maize, nevelose, sago and

inulin. Loo et al. (2010), fructooligosaccharides (FOS) is the

commercial prebiotic since it will result on high population

of bacteria growth during the fermentation process. However,

the prebiotic effect of FOS not totally match and suitable

for all lactic acid bacteria species, as for FTCC 0291, L.

bulgaricus FTCC 0411, and B. bifidum BB12, the commercial

prebiotic gives the lowest cell count for these groups of

bacteria.

Futhermore, there are lots of prebiotics and come in a

various forms and types. Hussain et al. (2012), used inulin

containing chocolate to test the effects on in vitro growth of

four different strains of Bifidobacteria; B. longum BB536, B. breve

ATCC 15700, B. infantis ATCC 15697 and B. pseudocatenulatum G4. The

result stated that the inulin manage to promote all the

Bifidobacteria growths.

2.4 The Probiotic

Probiotic is the bacteria that present in human

gastrointestinal tract system and in the same time they

promote the good health for the host. This type of bacteria

known as beneficial bacteria, namely Bifidobacterium and

Lactobacillus. Non-pathogenic microorganism such as B. longum

that comes from species of Bifidobacteria is known as a

probiotic that exerts benefits on host health (Jan et al.

2010). Probiotic mostly can be found in dairy products such

as cultured milk and yogurt as well as it also abundant in

the form of health supplement, and generally known as a

functional food (Sahadeva et al. 2011).

Bacteria feed on fermentable carbohydrates, such as

oligosaccharide, and produce beneficial substance called

short-chain fatty acid (SCFAs). The major fermentation

products of prebiotic metabolism in large bowel are these

short-chain fatty acids (SCFAs), which had different effects

on colon morphology and function such as energy booster to

the intestinal mucosa, decrease the pH, and stimulate the

absorption of both sodium and water (Scheppach 1994). Those

types of fatty acid that commonly secreted during the

fermentation process are namely butyrate, acetate and

propionic acid.

The commercial probiotic strains were used in order to

promote a good health of human guts by boosting the

population of readily present probiotic inside the gut.

Despite of that, this commercial probiotic strains must have

a great tolerance with extreme pH so that they can always be

available in human gastrointestinal tract (GIT) after been

consumed. But so far, this beneficial bacteria always

founded to have the resistance towards pH and bile, that is

why it can be retained and survive in human GIT system and

capable to provide a good health condition of gut’s host.

According to Sahadeva et al. (2011), most of the commercial

probiotic that been tested their resistance towards pH and

bile; L. acidophilus, L. casei Shirota strains, Streptococcus

thermophilus, and Bifidobacterium, had met the requirement of

initial count population of WHO/FAO 2006. Moreover, the

highest viability of Lactobacillus spp., Bifidobacterium spp., and S.

thermophiles mostly are within the highest pH of 8.1 (Ting and

DeCosta 2009). Another properties of these three strains

that make them become most desirable strains are able to be

ingested, and always been used in food production (Ting and

DeCosta 2009).

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

3.1.1 Samples and Chemicals

Four fermentation substrates i.e., raw

organic A. mangium honey (Royal-B,

Malaysia), Australian honey (Capilano,

Malaysia), glucose (HmbG, Germany),

extracted A. mangium honey and extracted

Australian honey were used in this study.

Glucose was used as a positive control and

blank fermentation media as a negative

control.

Honey were extracted by separation analysis

by charcoal (MERCK, Germany), alcohol

(HmbG, Germany), filter papers (Whatman,

England), and distilled water. Samples

were sterilized by 0.22 µm membrane filter

(Bioflow PTFE, Malaysia).

3.1.2 Microorganisms

Mixed culture of B. animalis and L. acidophilus

from Blackmores Acidophilus Bifidus, US.

3.2 Methods

3.2.1 Separation Analysis

The separation analysis of honey was

adapted from Morales et al. (2006). The aim

of this method is to remove the

monosaccharide and disaccharide to retrieve

the oligosaccharide because monosaccharide

and disaccharide will become the substrate

towards the lactic acid bacteria (Morales,

2006). Honey of 0.5 g was dissolved in 20

mL of distilled water.

The 3 g of activated charcoal was stirred

in 250 mL of 10 % ethanol for 30 minutes to

eliminate the mono- and disaccharides. The

mixture then filtered, the charcoal residue

was left on filter paper. The charcoal

residue was washed with 25 mL of 10 %

ethanol then dissolved in 250 mL of 50 %

ethanol to extract the oligosaccharide that

was absorbed by the activated charcoal. The

mixture was then filtered again using

filter paper. The filtrate was vacuum

evaporated at 30 ˚C until it become

concentrated.

3.2.2 Inoculum and Seed Medium Preparation

The probiotic capsule that contain two

mixture strains; L. acidophilus and B. animalis

was growth inside the nutrient broth

(Oxoid, England) and incubated at 37 ˚C for

24 h. About 2.0 mL cultures was transferred

into 15 mL seed medium and incubated for 24

h at 37 ˚C to obtain inoculum of optical

density (OD) value that equal to 1.

Nutrient broth was used as a seed medium

and was autoclaved at 121 ˚C for 15

minutes. The inoculum was used for

fermentation purposes and the OD value of

bacteria cell mass must equivalent to 1 at

0 h of fermentation period. The OD value

for both 0 h and 24 h of fermentation

period was recorded.

3.2.3 Fermentation Preparation

The fermentation media was prepared using

the basic fermentation media (% / liter of

distilled water) NaCl 0.5 (MERCK, Germany),

peptone 1 (MERCK, Germany), and

carbohydrates (Australian honey, A. mangium

honey, extracted honey and glucose) 1. All

carbohydrates were sterilized using filter

sterilization with 0.2 µm pore size

filters. Then 50 mL of fermentation medium

was added into a falcon tube and 5 % (v/v)

of inoculum in fermentation media was

inserted. The control fermentation medium

was prepared with no added substrate.

The fermentation process was conducted

inside the 2.5 L anaerobic jar (MERCK,

Germany) and was left for 24 h at 37 ˚C.

Samples were removed at 0, 8, 12 and 24 h

of fermentation for enumeration of bacteria

using pour plating technique on Man, Rugosa

and Sharpe, MRS agar (HiMedia, India) while

the absorbance value was checked for 0 and

24 h fermentation period.

3.2.4 Enumeration of Bacteria

The enumeration for bacteria; L. acidophilus

was conducted by pour plating technique in

MRS agar and by checking the optical

density (OD) using a spectrophotometer.

All cultured media on MRS agar were

incubated for 2 days at 37 ˚C. Then, single

colonies and OD value were counted and

recorded after incubation.

3.2.5 Statistical Analysis

Fermentation were conducted in triplicate

for each sample. Analysis software of SAS

9.0 version was used in order to carry the

statistical analysis. The differences

between the means of the samples were

tested by ANOVA at the significance level

of P ≤ 0.05 and P ≤ 0.01, using the

Duncan’s Multiple Range Test.