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DETERMINATION

OF DIETARY HABIT OF MALAYSIAN STINGLESS


BEE AND DETECTION OF ADULTERATION
BY USING EA-IRMS

by

AMIRUL SYAHIDAH ASMADI

A dissertation submitted in partial fulfilment of the requirements for the degree of Bachelor

of Technology (B. Tech.) in the field of Environmental Technology

School of Industrial Technology

UNIVERSITI SAINS MALAYSIA

December 2016
DECLARATION BY AUTHOR

This dissertation is composed of my original work, and contains no material previously

published or written by another person except where due reference has been made in the text.

The content of my dissertation is the result of work I have carried out since the

commencement of my research project and does not include a substantial part of work that

has been submitted to qualify for the award of any other degree or diploma in any university

or other tertiary institution.

___________________
Amirul Syahidah binti Asmadi
December 2016

ii
ACKNOWLEDGEMENTS

First of all, I would like to express my deepest gratitude to my supervisor, Dr.

Syahidah Akmal Muhammad for all the encouragement, guidance and support provided

during the research. I would also like to acknowledge Mr. Rusell Frew and teams from

University of Ottago, New Zealand for helping me to do some protein extraction analysis.

The project would not have been completed on time without their help.

I would like to thank the laboratory staff Mr. Ravi and Madam Aida and also not to

forget Dr. Syahidahs assistant from Analytical Research Biochemistry Centre, Madam Ainol

Syakira for providing necessary technical assistance during the research. I am also grateful

to Universiti Sains Malaysia and School of Industrial Technology for including the Final

Year Project in our short three and the half years studies. The effort is worthwhile as the

project makes us learn a lot. I would like to extend my sincere appreciation to lectures for

providing their kind assistance and advice during my hard time.

Last but not least, thank you to my very kind and helpful coursemates, especially Nur

Sabihah Ngah and Nurul Wahida A Ghani for willing to accompany and help me if I need

them. The project would not been completed on the time without their support. Although it

was a long process to complete the experiment and to collect necessary data within the short

period, I had much support from them, both physically and mentally.

Amirul Syahidah binti Asmadi

December 2016

iii
TABLE OF CONTENTS

Pages
ACKNOWLEGMENTS iii
TABLE OF CONTENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF SYMBOLS AND ABBREVIATION viii
ABSTRAK ix
ABSTRACT x

CHAPTER 1 : INTRODUCTION
1.1 Background of Study 2
1.2 Problem Statement 3
1.3 Research Questions 4
1.4 Objectives 4

CHAPTER 2 : LITERATURE REVIEW


2.1 Introduction of stable isotope 5
2.1.1 Isotope ratio 5
2.2 Principle of EA-IRMS 6
2.2.1 General notations used in IRMS 8
2.3 Malaysian stingless bee honey 8
2.3.1 C3 Plant and C4 Plant 9
2.4 Application of IRMS in detection of adulteration of honey 11

iv
CHAPTER 3 : METHODOLOGY
3.1 Experimental Design 14
3.1.1 Pure honey sample collection 14
3.1.2 Adulterated honey sample preparation 14
3.1.2a Type of adulterant 14
3.1.2b Syrup preparation 15
3.1.2c Adulterated honey samples 15
3.1.3 Preparation of chemical 15
3.1.3a Preparation of 10% sodium tungstate 15
3.1.3b Preparation of 0.335mol/L of sulphuric acid 15
3.1.4 Analysis Process 16
3.1.4a Honey Analysis Preparation 15
` 3.1.4b Honey Protein Analysis Preparation 15
3.2 Measurement by EA-IRMS Instrument 17
3.3 Data analysis 17

CHAPTER 4 : RESULTS AND DISCUSSIONS


4.1 Dietary habits of stingless bee 18
4.2 Detection of adulteration in honey samples 18
4.2.1 Stable carbon isotope in honey based on sample origin 20
4.2.2 Stable carbon isotope in adulterated honey 21

CHAPTER 5 : CONCLUSION AND RECOMMENDATIONS


5.1 Conclusion 24
5.2 Recommendations 24

REFERENCES 25

APPENDICES 28

v
LIST OF TABLES

Table Caption Page


2.1.1 Natural Abundance of Stable Isotopes and International 5
Standards Used in Isotope Ratio Analysis

4.1 Average results and standard deviation of 13C in 20


of honeys based on their sample origin.

4.2.1 Stable isotope ratios and standard deviation in the 21


stingless bee honey and their protein fractions, % of
adulteration and honey quality

4.2.2a Stable carbon isotope ratios in 60% sugarcane syrup, 22


55% corn syrup and 55% palm sugar syrup samples

4.2.2b Stable carbon isotope ratios in adulterated honeys and 22


their protein fractions

4.2.2c Stable carbon isotope ratio in stingless bee honey, 23


common honey, and Tualang honey that available in
market.

vi
LIST OF FIGURES

Figure Caption Page


1.1 The web chart of polluting substance diffusion in the 2
environment where the shaded area indicates the
environmental sectors that were visited by the bees

2.2 Simple schematic diagram of an EA IRMS for the 6


determination of 13C and 15N
4.1 The 13C mean values of stingless bee honey in 18
different region: Simpang Renggam, Merbok, Paloh,
Kemaman, Segamat, Kota Baharu, Pekan Nenas dan
Pontian.

vii
LIST OF SYMBOLS AND ABBREVIATIONS

Parts Per mil

Delta () Delta notation

EA Elemental Analyzer

IRMS Isotope Ratio Mass Spectrometry

EA-IRMS Elemental Analyzer- Isotope Ratio Mass Spectrometry

SCIRA Stable Carbon Isotope Ratio Analysis

ISIRA Internal Standard Isotope Ratio

AOAC Association of Official Analytical Chemistry

MARDI Malaysian Agricultural Research and Development Institute

HPLC High Performance (Pressure) Liquid Chromatography

viii
PENENTUAN TABIAT PEMAKANAN KE ATAS LEBAH KELULUT MALAYSIA
DAN PENGESANAN BAHANCAMPUR DENGAN MENGGUNAKAN EA-IRMS

ABSTRAK

Kajian ini mempunyai dua tujuan utama: (1) untuk menentukan tabiat pemakanan lebah

Kelulut Malaysia berdasarkan nilai isotop karbon stabil melalui sample madu 2) untuk

mengesan pencemaran madu yang ditambah dengan gula C4 dengan menggunakan Internal

Standard Isotope Ratio. Analisis 13C telah dijalankan ke atas sampel madu dengan

menggunakan jisim spektrometer nisbah isotop dalam kombinasi dengan penganalisis unsur

(EA-IRMS). Tumbuh-tumbuhan yang dikumpulkan oleh lebah kelulut melalui nektar dan

debunga digambarkan pada nilai 13C yang terkandung dalam madu, di mana nilai purata

13C yang dijumpai untuk madu lebah Kelulut tulen adalah dari nilai -26.33 sehingga -

28.06 (-27.04 0.21) bagi kategori tumbuhan C3 dan dari nilai -14.95 sehingga -

15.07 (-14.81 0.48) bagi kategori tumbuhan C4. Teori yang menyatakan bahawa madu

lebah Kelulut yang tulen dijangka mempunyai ciri-ciri tumbuhan C3 tidak boleh digunakan

untuk lebah spesies ini kerana nilai nisbah daripada madu kelulut dari Simpang Renggam

ditunjukkan ciri-ciri tumbuhan C4. Konsep analisis pengesanan pencemaran adalah di mana

perbezaan karbon isotop nisbah 13C dan 12C dalam madu akan memberi petunjuk kualitatif

dan kuantitatif daripada pencemaran madu. Dalam kajian ini, sampel madu dicemari bersedia

dengan tambahan sirap gula tebu, sirap jagung dan sirap gula melaka pada nisbah yang

berbeza 5%, 10%, 20% dan 50% mengikut berat. Hasilnya, bahancampur madu C3 dengan

gula C4 boleh dikesan manakala bahancampur dengan madu C3 dengan gula C3 tidak dapat

dikesan dan begitu juga sebaliknya.

ix
DETERMINATION OF DIETARY HABIT OF MALAYSIAN STINGLESS BEE
AND DETECTION OF ADULTERATION BY USING EA-IRMS

ABSTRACT

This study has two major purposes : (1) to determine the dietary habits of Malaysian stingless

bee based on the value of stable carbon isotope of the honey samples 2) to detect the

adulteration of honey added with C4 sugar by using internal standard isotope ratio. The 13C

analysis were conducted on these honey samples using an isotope ratio mass spectrometer in

combination with elemental analyser (EA-IRMS). The plants that stingless bees collected

nectar and pollen reflected in the 13C value contained in the honey, where the 13C average

values found for pure stingless bee honey were -26.33 to -28.06 (-27.04 0.21) for C3

plants and -14.95 to -15.07 (-14.81 0.48). A pure honey expected to have a C3 plant

characteristics are not applied to stingless bees species since the ratio value of stingless bee

honey from Simpang Renggam shown a C4 plant characteristics. The concept of analysis of

adulteration detection is where the difference of carbon isotopes ratio of 13C and 12C in honey

will give a qualitative and quantitave indication of honey adulteration. In this study,

adulterated honey samples were prepared with addition of sugar cane syrup, corn syrup and

palm sugar syrup at different ratio 5%, 10%, 20% and 50% by weight. As a result,

adulteration with C4 sugar can be detected while adulteration with C3 sugar with C3 honey or

C4 sugar with C4 sugar cannot be detected.

x
CHAPTER 1
INTRODUCTION

Honey is natural food that exhibit a wide range of biological effect and act as natural

antioxidant because of the presents of flavonoids and phenolic acid in the honey. Honey are

composed by sugars and others constituents such as aromatic substances, enzymes, organic

acid acids, vitamins, minerals and carotenoids (Alqarni et al., 2014). The chemical

composition of the honey in term of its protein, carbohydrate, enzyme, mineral and organic

acid content primarily depends on some biotic and abiotic factors around the bees colony for

example; floral sources (which has been distinguished as unifloral and multifloral) , climate

condition, soil and beekeeper practices (Kek et al., 2014, Gan et al., 2016).

According to Michener (2000), there are seven main families of bee species that have

been classified and Apidae spp. is one of these family. Apidae spp. has three subfamilies:

Xylocopinae, Nomadinae and Apinae. The subfamily Apinae has nineteen tribes including

Apini (honeybees), Meliponini (includes stingless bees), and Bombini. Trigona is a genus of

the Meliponini tribe which is found extensively in tropical regions. Trigona is the largest

genus of stingless bees and have many subgenera. Stingless bees are closely related to the

honeybees, bumblebees and orchid bees which are present in all tropical parts of the world

where it is estimated the between 400 to 500 different species of stingless bee are known

(Bradbear, 2009).

1
1.1 Background of Study

Bees is a good biological indicators because they can provide a numerous indicators

for each season. Almost all the environmental sectors such as soil, vegetation, water and air

(Figure 1) are sampled by the bees through foraging (Porrini et al., 2003). Materials such as,

nectar, pollen, honeydew, propolis and water that were collected by the bees are brought into

the hive then stored according to their verifiable criteria.

Fig 1.1 The web chart of polluting substance diffusion in the environment where the shaded
area indicates the environmental sectors that were visited by the bees. (adapted from Porrini
et al., 2003)

In related to this study, this mean that, which plants the bees collected the nectar and

pollen will be reflected in the 13C value contained in the honey and its protein (Cienfuegos

et al., 2015). The honey produce from the nectar and pollen and the propolis used in the hive,

all of these latter will in certain cases will accumulated the residues in the bees bodies. Thus,

they indicates the impairment of the environment they live in by stockpiling given

contaminants via a nutritional body balance whose input is greater than its output (Celli and

Maccagnani, 2003).

2
Since honey are consumed by us because of it health beneficial some of it may be less

effective or harmful if consumed in long term due to the adulteration of honey. It has become

a major problem nowadays and it has becoming a threat to the viability of the beekeeping

industry, stingless bee honey also become one of the product that are not exception that have

been adulterated. It is cheating the consumer who is expecting that they could get 100%

natural pure product with its organoleptic qualities and biological activity. In recent years

however, by using isotope ratio mass spectrometry where it has been accepted as a reliable

method in detection system as a tool for authenticity identification.

The concept is where the differences of stable carbon isotopes ratio of 13C and 12C in

honey and between its protein fraction will give a qualitative and quantitative indication of

honey adulteration. The different of stables carbon isotopes can be seen because of the

different of photosynthesis cycle will produce different carbon isotopes ratio that enable

stable carbon isotopes to detect any changes in carbon isotopes of honey (Padovan et al.,

2003).

1.2 Problem Statement

Currently, there is no study were made in order to measure the carbon composition

of Malaysian stingless bee, through 13C/12C the dietary habits of the stingless bee it can be

seen in the honey and the effectiveness of IRMS in detecting adulteration in the production

of stingless bee honey and others types of honey by addition of sugar syrup during or after

honey production. The IRMS method are used to observe the isotopic ratio carried into honey

has been accepted as an official method by AOAC for the detection of honey adulteration

(Padovan et al., 2003, Cabaero et al., 2006, Padovan et al., 2007, White et al., 1998,

International, 2005).

3
In addition of that, both adulterated honey and pure honey cannot be physically

differentiate in order to prove whether it is pure as it claim to be. Besides that, common honey

can be claim as stingless be honey after it adulterated with sweeteners. These is because the

taste and texture cannot be distinguish by the consumer. Therefore, it is necessary to carry

out an in-depth study in order to improve the knowledge in this field as to avoid other honey

producer taking advantage in lack of knowledge of consumer.

1.3 Research Questions

The research question are as follows:

1. What is the range of 13 C values of Malaysian stingless bee honey that will reflect

the foraging activities and dietary habit of the stingless bee?

2. Can the ISIRA technique used in detecting the low adulterated stingless bee honey

samples?

1.4 Objectives

This research attempts to fulfill the following objectives :

1. To measure the 13 C values of stingless bee honey in order to study the foraging

activities and dietary habit of stingless bee.

2. To find the effectiveness of ISIRA in detecting the lowest percentage of stingless bee

honey samples adulterated with different types of adulterant at different ratios.

4
CHAPTER 2
LITERATURE REVIEW

2.1 Introduction of stable isotope

Isotopes occupy the same position in the periodic table of the element. The nucleus

of each atom contains protons and neutrons where isotope may be defined as atoms

processing the same number of proton but different numbers of neutrons in their respective

nuclei (Kelly, 2003). For example, most of the carbon (approximately 99 %) has 6 protons

and 6 neutrons where it is written as 12C to reflect its atomic mass. However, about 1 % of

the carbon in the Earths biosphere has 6 protons and 7 neutrons, written as 13C forming the

heavy stable isotope of this important element. Stable isotopes do not decay into other

elements. In contrast, radioactive isotopes (e.g., 14C) are unstable and will decay into other

elements.

2.1.1 Isotope ratio

Isotopic ratios for the elements hydrogen, carbon, nitrogen, oxygen, and sulfur (Table

2.1.1) determined the demonstrated of the light isotopes to be by far most abundance, whereas

for heavier elements the ratio between light and heavy isotopes is more balance (Rossmann,

2001). These studies contributed to the understanding of the beginning and the development

of life on earth, which had not been accessible by other methods. At the same time, the

isotopic contents were no longer given as absolute abundances or abundance ratios, but as

deviations of the isotopic ratios from those of reference substances, calculated as -values

(Table 2.1.1). Thus, the very small differences in absolute abundances become more obvious

and the numbers were easier to handle.

5
Table 2.1.1 Natural Abundance of Stable Isotopes and International Standards Used in
Isotope Ratio Analysis
Element Isotope Abundance Standard Name Isotopic Ratio
(atom %)
1
Hydrogen H 99.985 Standard Mean Ocean 0.00015576
2
H 0.0145 Water (SMOW)
12
Carbon C 98.892 Pee Dee Belemnite 0.011237
13
C 1.108 (PDB)
14
Nitrogen N 99.6633 Air (air nitrogen) (AIR) 0.0036765
15
N 0.3663
16
Oxygen O 99.7587 Standard Mean Ocean 0.00200520
17
O 0.0375 Water
18
O 0.2039
32
Sulfur S 95.018 Canyon Diablo Troilite 0.0450045
33
S 0.750 (CDT)
34
S 4.215
36
S 0.02

The less abundant stable isotope(s) of an element have one or two additional neutrons

than protons, and thus are heavier than the more common stable isotope for those elements.

The chemical bonds and attractive forces of atoms with heavy stable isotopes are stronger

than those in the more common, lighter isotopes of an element. Heavy isotopes undergo all

of the same chemical reactions as light isotopes, but, simply because they are heavier, they

do it ever so slightly slower. These tiny differences in reaction rates cause the products of

reactions to have different isotope ratios than the source materials.

2.2 Principle of EA-IRMS

There two types of elemental analyser available; however for the analysis of the

carbon, C and nitrogen, N, the sample undergoes combustion in oxygen atmosphere or simply

known as EA-IRMS. The concept of EA-IRMS are where the solid samples are send in for

carbon and/or nitrogen measurements then they are analyzed using an elemental analyzer

plumbed into an isotope ratio mass spectrometer. The key components of the EA-IRMS

system are shown in Figure 2.2.

6
The analysis process of EA-IRMS can be divided into four steps which are; the

combustion of the sample material using elemental analyzer, introduction of the evolved

gases into the ion source of the mass spectrometer via the interface, ionization of the gas

molecules followed by separation and detection of the ions in the mass spectrometer and

finally is the raw data evaluation (Carter and Barwick, 2011). Each of the samples were tried

to specifically match samples with one of several in-house standards for checks on instrument

precision and linearity. These in-house standards include plant materials, animal tissues, soils,

sediments and chemical standards. In addition two standards are used to perform

normalization (linear regression) of data once the analysis is complete.

Fig. 2.2 Simple schematic diagram of an EA-RMS for the determination of 13C and 15N
(adapted from Carter and Barwick, 2011)

7
2.2.1 General notations used in IRMS

The stable isotope concentrations of a molecular compound or material are presented

in ratio form as the molar ratio of the heavy-to-light isotopes. Since this ratio is small, so the

stable isotope abundances relative typically presented to an international standard using

delta() notation as:

R sample
X = (R standard 1 ) x 1000,

where X is the delta value of the sample for element X (C, N, O, and etc.) in parts per

thousand (permil) () and R is the molar ratio of the heavy isotope (less common) to light

isotope (more common) in the sample and in an international standard, respectively.

2.3 Malaysian stingless bee honey

Stingless bees species that can be found in Malaysia is Trigona species or more

known as Kelulut bees among Malaysian. Based on Kelly et al. (2014) research, currently

Malaysia is a home for 17- 32 known species of stingless bees, however only two Trigona

itama and Trigona thorasica that are suitable and commonly used for commercial honey

production. According to MARDI, stingless bee honey contain many vitamins and essential

minerals that is twice as compared to ordinary honey due to it is ability to suck the nectar

from the flowers by going deep inside the flower. This is because of the small size of it is

body compared to ordinary bees species. As the medicinal values of the stingless bee honey

is higher than common honey, the demand of these stingless bee honey become much more

higher and also price value on market it is higher than the common honey.

A multifloral honey produced from stingless bee per hive basis is much more lesser

if compared common honey bee; Apis mellifera and because of this factor makes the

8
distribution of stingless bee is limited (Chuttong et al., 2016). Honey produced by stingless

bee are stored in the small clusters of small resin pots while Apis spp. honeys are stored in

the hexagonal-shaped honey combs (Kek et al., 2014). The total of the non-peroxide activities

derived from honey provide an excellent antibacterial potential, make it useful in medical

and therapy (MI et al., 2013). Honey from stingless bees has a distinct taste and aroma, more

fluid in texture, and undergoes slow crystallization process (Biluca et al., 2014). DeNiro and

Epstein (1978) stated that, the type of the animals tissue and the nature amount of the 13C

in their diet will affect the 13C values of the tissues and their product

2.3.1 C3 Plant and C4 Plant

C3 and C4 plants are using photosynthesis process to convert light to energy and

carbon dioxide, CO2 into plant food energy which are carbohydrates. The different between

this plants are in their leaf anatomy and enzymes that they are used to carry out

photosynthesis. These differences are important to their optimal growing conditions, nitrogen

and water-use efficiency, forage quality, and seasonal production profile. There are also

CAM (Crassulacean acid metabolism) plants, that are essentially are subset of the C4 plants.

But instead of performing C4 photosynthesis, kind of and outside cells and inside cells they

are doing it at night time and also at the day time.

C3 photosynthesis process only use the Calvin-Benson photosynthetic cycle in order

to fix CO2 that have been catalyzed by ribulose-1,5-bisphosphate carboxylase (Rubisco). The

process takes place inside of the chloroplast in mesophyll cell while C4 photosynthesis

process use the Hatch-Slack photosynthetic cycle where the photosynthetic activity of C4

plants such as maize and corn take place in two place which is in mesophyll cell and bundle

sheath cells where there are anatomically and biochemically distinct (Wang et al., 2012).

9
The use efficiency of the available light of C4 plants are more higher when compared

to their C3 relatives, particularly at high temperatures; and because of these considering factor

makes C4 plants particularly successful in areas with low water, low nutrients and high light.

In the full sunlight, the ability of the C3 plants to pull CO2 out of the air and turn it into

carbohydrates reaches its maximum since C3 plants only can use a fraction of the available

light energy. In contrast, C4 plants continue to turn CO2 into carbohydrate as available light

increases. In addition, the concentration of CO2 at which the CO2 produced by cellular

respiration exactly matches the amount fixed by photosynthesis (the CO2 compensation point)

is remarkably low.

Since a different photosynthesis cycle produce a different ratios of carbon isotopes,

so according to Calvin and Bassham (1962), plant with the C3 photosynthetic cycle and C4

photosynthesis cycle have ratio values between -21 to -32 and -12 to -19 of 13C

respectively. While for the CAM plant, Padovan et al. (2007) stated that the 13C value ranges

between -11 and -13.5. When the 13C values compared, C4 plant have higher of value

compared to C3 plant.. A pure honey is expected to have the characteristic properties of C3

plants since bees collect their nectar from flowering plants of dicotyledon as the carbon

isotope ratios of C4 and C3 plants in photosynthetic cycles are different (Simsek et al., 2012).

So this theory may be applied to the stingless bee to since bees collect their nectar from

flowering plant too.

10
2.4 Application of IRMS in detecting the adulteration of honey

Since early 1970s, in order to detect economy fraud in the food production, IRMS

technology has been used and more recently this online technique is culminating gained a

wider acceptance in food control laboratories as a reliable of another detection system.

Increasing competition in the world market and expectation of domestic consumer in high-

quality products at low price means there is a need for new tools for monitoring food quality

(Primrose et al., 2010).

Moreover, through the ages, the adulteration practices have become more

sophisticated and been refined because of improving knowledge in analytical sciences

(Oliveri and Downey, 2012). Adulteration by sweeteners is the most serious and important

authenticity issue where sugar syrups from corn, sugarcane and sugar beets and also syrups

of natural origin have been detected and can be commonly found in adulterated honeys

(Cengiz et al., 2014). Puscas et. al., (2013) stated that, adding sucrose within less than 1% of

its dried mass, or by overfeeding the bees with sugar or other types of sucrose are the most

commonly method used in the honey adulteration.

Thus, guaranteeing the quality of the honey has become a major concern in order to

ensure that the honey is authentic (Cengiz et al., 2014). Furthermore, in order to prosecute

dishonest traders, control laboratories and enforcement agencies, they need a tool of

unequivocal evidence of food adulteration which is can be ultimately offered by IRMS (Kelly,

2003). HPLC method is most widely used technique used in detecting the adulteration of

honey but since the HPLC only can detect the sugar content in the honey, it is unable to detect

the low levels of adulteration and it is inadequate technique in order to prove more

sophisticated falsification (Padovan et al., 2003).

11
The different carbon isotopes produced by different photosynthesis cycle enable the

SCIRA detect an adulteration of C4 sugar syrup in honey. However, according to the Winter

and White (1989), since the SCIRA only suitable for detection of honey adulterated with C4

plants sugar (for example sugarcane and corn syrup) but not the C3 plant sugars as the 13

C of studied honeys portrayed a C3 plant characteristics so they also developed an internal

standardization technique called ISIRA in order to improve the sensitivity with which an

adulterated honey could be detected . The technique is based on the determination of 13 C

of protein that have been isolated from the honey because the composition of protein does

not change even though the cane syrup or sugarcane syrup have been added to pure honey.

A honey can be determine whether it was adulterated or not through a laboratory

procedure where the process involved the analysis of the carbon isotopes ratios in the sugar

of honey and protein that have been extracted from the honey. Extraction of the protein

involved methods such as mechanical (filtration and centrifugation) and chemical

(precipitation) techniques. The percentage of the adulteration in the honey can be estimated
13
by determine the difference in C/12C ratios between sugar in the honey and its protein

(Padovan et al., 2003). Since the addition of adulterant syrups to pure honey will change its

carbon isotope ratio composition, but not its protein composition hence the isotope ratio of

the honey and its protein will have a very close values if its a pure honey and on the other

hand if the honey was adulterated.

Doner and White (1977) measured the mean value of 13C US honey and found

that, it was -25 0.98 Then in 1986, Burroughs and Otlet, established the relationship

between authentic UK honey with US honey, where the average 13C values of UK honeys

found, was -25.5 0.82 which is very similar to the US honey (Kelly, 2003). Even

tough, there is no significant correlation was observed, either between the origin and 13C

12
or between the plant species which nectar derived and 13C value, the average of Mexican

honey found was -25.6 0.9 where it is slightly more negative when compared to the

average 13C values of US honey (Cienfuegos et al., 2015).

The ratio value of the pure honey suggested by Simsek et al. (2012) and Anklam

(1998) should between +1.1 and -0.9 where White and Winters (1989) stated that, the

difference between of the 13C values the whole honey and protein fraction should not

exceed 1 or not more than 7% where it is the limit of acceptable extend of the adulterated

honey.

13
CHAPTER 3

METHODOLOGY

3.1 Experimental Design

This study is divided into several sections: (i) experimental design (ii) analysis using

EA-IRMS and (iii) simple statistical analysis on the significant different.

3.1.1 Pure honey sample collection

This study was conducted by 60 stingless bee honey samples that were provided by

the local beekeepers with guaranteed origin and made by traditional procedures in the honey-

producing region which are collected from the selected areas of Peninsular Malaysia. The

honey are from: Simpang Renggam, Johor (n=33), Merbok, Kedah (n=9), Paloh, Johor (n=6),

Kemaman, Terengganu (n=3), Pekan Nanas, Johor (n=1), Pontian, Johor (n=1), Kota Baharu,

Kelantan (n=1)

3.1.2 Adulterated honey sample preparation

Adulterated honey samples were prepared with addition of different types adulterant

at a ratio of 5%, 10%, 20% and 50% by weight.

3.1.2a Type of adulterant

Adulterant that have been used in this study were sugarcane, palm sugar (Gula

Melaka), and corn syrup. Each type of adulterant used were diluted at 60% of concentration

since with water since the sugarcane and palm sugar are in the solid and corn syrup were

diluted at 55% of concentration.

14
3.1.2b Syrup preparation

The sugarcane and palm sugar were weighed at 60 g then, two portion of 40 g of

water were weighed. Adulterants was added with water then diluted in the water bath at 35C

for 30 minutes. The syrup had been cooled before it were kept in the glass container at room

temperature. The corn syrup and water was weighed at 55 g and 45 g respectively. The

solution then mixed and diluted in the water bath at 35C for 30 minutes. The syrup had been

cooled before it were kept in the glass container at room temperature.

3.1.2c Adulterated honey samples

The syrups had been added into the honey samples at specified ratio and stirred for

one minute The samples were placed into a water bath at 35C for 20 min to let it mixed

completely. Then, the samples were let to cool.

3.1.3 Chemical preparation

3.1.3a Preparation of 10% sodium tungstate

Sodium tungstate oxide dihydrate 95% , Na2WO4.2H2O was weighed approximately

34 g and diluted with approximately 50 ml of deionised water in the beaker. The solution

was stirred until all the solute mixed completely. Then, the solution was transferred into a

100 ml volumetric flask and top up with deionised water until the graduated marking line.

3.1.3b Preparation of 0.335 mol/L of sulphuric acid

Roughly 50 mL of deionised water was poured into a 100 ml volumetric flask. Then

1.84 mL of concentrated sulfuric acid was dispensed to the volumetric flask using a pipette.

Dosing of the concentrated acid was done slowly with constant swirling of the flask. Once

15
the pipette is emptied, the volumetric flask contents was diluted further with deionised water

until the level reaches the 100 ml mark.

3.1.4 Stable Isotope Analysis Process

13C analyses on the honey samples were specified according to the AOAC Official

Method 998.12 (International, 2005) where the analysis procedure were explained in the

steps below:

3.1.4a Honey Analysis Preparation

Approximately 0.25 0.05 mg of honey sample and reference standard was weighed

into the tin capsules. The capsule was folded and compressed into a spherical ball to minimize

any air present and to contain the sample. The sample then placed into the auto sampler unit

of elemental analyzer (EA) for the measurement of carbon isotope ratio of total honey.

3.1.4b Honey Protein Analysis Preparation

Each of honey sample was weighed approximately 10 - 12 g and then was filtered

into a 50 ml centrifuge tube. A fresh mixture of 2 mL of sodium tungstate and 2 mL of 0.335

mol/L of sulfuric acid were added into the centrifuge tube and vortex for 30 seconds. The

final mixture was incubated in a water bath at 70C until visible floc forms with clear

supernatant. If no visible floc forms, or if the solution remains cloudy, 0.335 mol/L of sulfuric

was added once more into the solution. This step was repeated until a mixture of precipitate

and clear solution formed. Next, distilled water was added to the final solution, the solution

was mixed thoroughly and centrifuged at 3000 rpm for 5 minutes. The supernatant solution

was discarded and the precipitate was separated from the tube. The precipitate solution was

dried in an oven at 75C for 3 hours. Finally, approximately 0.25 0.05 mg of the dried

16
precipitate was weighed into the tin capsule and placed into the auto sampler unit of EA for

the measurement of carbon isotope ratio of honey protein.

3.2 Measurement by EA-IRMS Instrument

The total values of 13C of honey and protein samples were analyzed by Thermo

FLASH 2000 Organic Elemental Analyzer and Thermo DELTA V ADVANTAGE IRMS.

Urea and L-glutamic acid were used as reference standard for linear calibration curve.

3.3 Data Analysis

All data of the stable isotope ratio of 13C of the samples were grouped in the form

of table according to the origin of the honey. The percentage of adulteration of honey were

calculated based on formula described by White (1989).

13C () 13C (honey)


Adulteration = 13C (protein) 13C (adulterant) x 100

17
CHAPTER 4

RESULT AND DISCUSSION

The composition of honey samples and authentication of stingless bee honey, were

determine by using the technology of EA-IRMS where it was applied to analyse the carbon

isotope of honey and its protein.

4.1 Dietary habits of stingless bee

The data in the Figure 4.1 and Table 4.1 represents the stable carbon isotope of 13

for the honey sample from eight location around West Malaysia. The results of the standard

deviation shows that the data was accurate as the set of data are closed to the average. All

samples are classified according to their sample origin.

13C Mean Values of Stingless bee honey versus Sample Origin

Fig 4.1 The 13C mean values of stingless bee honey in different region; Simpang
Renggam, Merbok, Paloh, Kemaman, Segamat, Kota Baharu, Pekan Nenas dan Pontian.

18
Based on Figure 4.1, it can be seen that stingless bee honey from Kemaman has the

most depleted 13C (all data are presented as average s.d ) (-28.29 0.72 ) whereas

stingless bee from Simpang Renggam shown the highest value of 13C (-14.81 0.48 )

followed by stingless bee honey from Paloh (-26.33 0.07), Pekan Nanas ( -26.34 0.13),

Segamat (-26.51 0.12), Merbok (-26.67 0.21), Kota Bharu (-27.09 0.15) and Pontian

(-28.06 0.04).

Since, the range of ratios value of plant with the C3 photosynthetic cycle and C4

photosynthesis cycle are between -21 to -32 and -12 to -19 of 13C respectively

(Calvin and Bassham, 1962). The ratios value of 13C of stingless bee honey from all samples

origin except Simpang Renggam implies that the source of their of nectar and pollen comes

from C3 plants. While stingless bee honey from Simpang Renggam ratios value indicates that

their diet habit while foraging comes from the C4 plants.

However, according to the previous study conducted in Frances, Lichtfouse et al.

(2003) stated that, urban areas also negatively affected the carbon isotopic ratio of the plant

where study showed that the urban grasses have strikingly depleted average -35.08

compared with rural grasses, with an average value of -30.59 . In different cases studied

by Klom et al. (2006) using a C4 grasses (Eleusine indica) stated that, the highest values

of 13C values of C4 plant range found in low traffic zone were from -12 to -14, and

from -14 to -16, in high traffic zones. The arise changes occur either in the isotopic

composition of the atmospheric CO2 or in its concentration affected the variations in the plant

13C values.

From this statement, theory from Simsek et al. (2012) suggested that, a pure honey

expected to have a C3 plant characteristics are not applied to stingless bees species since the

ratio value of stingless bee honey from Simpang Renggam shown a C4 plant characteristics.

19
Various environments can be adapted by honeybees where they can feed on plants such as

nectar and pollen as their energy source, for protein and other nutrients (Taki et al., 2016).

These result suggested that stingless bee sensibly uses available food resources in various

environments where they are not selective to nectar and pollen from C3 plant only.

Table 4.1 Average results and standard deviation of 13C in of honeys based on their
sample origin.
Sample origin Average of 13CPDB, of honey Standard Deviation (S.D)
Simpang Renggam -14.81 0.48
Merbok -26.67 0.21
Kemaman -28.29 0.72
Paloh -26.33 0.07
Segamat -26.51 0.12
Kota Bharu -27.09 0.15
Pekan Nenas -26.34 0.13
Pontian -28.06 0.04

4.2 Detection of adulteration in honey samples

Since the authenticity and safety of the honey is of great importance to both

procedures and consumers. Adulterated foods affect the consumer negatively in terms of

nutrition, health and economics. The results of the analyses were given separately for the

honey samples based on sample origin, purposely adulterated honey and commercial honeys.

4.2.1 Stable carbon isotope in honey based on sample origin

According to the conducted 13C analysis adulteration was not detected in the pure

honey samples (Table 4.2.1). The 13C values found for pure stingless bee honey were -

26.33 to -28.06 (-27.04 0.21) for C3 plants and -14.95 to -15.07 (-14.81 0.48).

The 13C protein of the honey of C3 plant found was between -26.58 to -28.93 (-27.21

0.17) (Table 4.2.1). However, protein from all honey samples of C4 plants from Simpang

Renggam were unable to extract due to the small amount of protein. Honey contains a small

20
amount of proteins and according to Lee et al. (1998), Apis cerana honey only contains from

0.1% to 3.3% of protein while Apis mellifera honey contains between 0.2% and 1.6% of

protein.

Table 4.2.1 Stable isotope ratios and standard deviation in the stingless bee honey and their
protein fractions, % of adulteration and honey quality.
Sample 13CPDB, of 13CPDB, of Different of % Honey
origin honey ( S.D) protein ( S.D) protein- Quality
honey
Segamat -27.20 0.25 -26.68 0.21 0.52 0.04 0 Pure
Paloh -26.70 0.05 -26.90 0.08 0.20 0.03 0 Pure
Merbok -27.60 0.10 -27.60 0.28 0.00 0.18 0 Pure
Pekan Nenas -26.34 0.13 -26.58 0.10 -0.24 0.03 Pure
Pontian -28.06 0.04 -26.58 0.06 1.48 0.02 Pure**
Kemaman -28.29 0.72 -28.93 0.03 -0.64 0.69 Pure
Simpang -15.07 0.50 *no protein - - -
Renggam
The sample marked with *no protein means that they were unable to isolate the protein content in in the
honey.
The sample marked with **Pure means that the samples were adulterated by sweeteners, but under the
acceptable extend (% = 7)

4.2.2 Stable carbon isotope in adulterated honey

The value in Table 4.2.2a are described as mean ( s.d) where the sugarcane syrup

mean values was -12.19 ( 0.14), corn syrup -12.40 ( 0.04) and for palm sugar syrup

was -12.73 ( 0.40).

All of the samples that were deliberately contaminated with sugar had detectably

lower 13C ratio in sugar fraction of honey (Table 4.2.2b) except for the 5% corn syrup where

the sugar detected was higher 13C ratio in sugar fraction of honey where IRMS were

sensitive enough to detect low or high concentration of adulterating by C4 sugars. All

the samples prepared by adding sugarcane syrup, corn syrup and palm sugar syrup can be

detected as adulterated as the percentage of adulteration shown a value even though the ratio

of adulterant added was 5% of sugarcane syrup and palm sugar. However, 5% corn syrup

which were added into the samples, did not affect the 13C value of the honey or its protein.

21
The ratio value of the pure honey suggested should between +1.1 and -0.9 where

difference between of the 13C values the whole honey and protein fraction should not

exceed 1 or not more than 7% where it is the limit of acceptable extend of the adulterated

honey. This applied to the commercial honey (Table 4.2.2c) where it can be shown that the

Stingless bee honey 2 and Tualang honey were obviously adulterated with big difference

between their 13C values and their protein does exceed the 1. However common honey 2,

the adulteration cannot be detected since the value of honey was C3.

Table 4.2.2a Stable carbon isotope ratios in 60% sugarcane syrup, 55% corn syrup and 55%
palm sugar syrup samples
Reading 13C of sugarcane of corn syrup, 13C of palm sugar
syrup, syrup,
1 -12.09 -12.37 -13.01
2 -12.29 -12.42 -12.45
Mean -12.19 -12.40 -12.73
Standard deviation (s.d) 0.14 0.04 0.40

Table 4.2.2b Stable carbon isotope ratios in adulterated honeys and their protein fractions
Sample origin 13CPDB, 13CPDB, of Different of Percentage of
of honey protein protein-honey adulteration, %
5% Sugarcane -14.77 0.39 -15.24 0.04 -0.47 1.3
10% Sugarcane -14.83 0.22 -15.19 0.10 -0.38 2.1
20% Sugarcane -14.47 0.15 -15.10 0.02 -0.63 7.2
50% Sugarcane -14.63 0.89 *no protein - -
5% Palm Sugar -26.17 0.77 -27.41 0.05 -1.24 0.8
10% Palm Sugar -27.82 0.06 -28.28 0.01 -0.46 0.5
20% Palm Sugar -25.55 0.02 -26.20 0.07 -0.65 2.7
50% Palm Sugar -20.83 0.05 -21.54 0.04 -0.71 7.3
5% Corn Syrup -28.36 0.08 -28.32 0.03 +0.04 0.0
10% Corn Syrup -27.54 0.08 -27.70 0.04 -0.16 0.2
20% Corn Syrup -26.44 0.01 -26.83 0.19 -0.39 1.0
50% Corn Syrup -22.58 0.28 -23.17 0.31 -0.59 5.0

The sample marked with *no protein means that they were unable to isolate the protein content in in the
honey.

22
Table 4.2.2c Stable carbon isotope ratio in stingless bee honey, common honey, and
Tualang honey that available in market.
Sample 13CPDB, of 13CPDB, of Different of Honey
honey protein protein-honey Quality
Stingless bee honey 1 -26.90 0.13 -26.10 0.03 0.8 0.10 Pure
Stingless bee honey 2 -16.07 0.04 -28.09 0.52 -12.02 0.48 Adulterated
Common honey 1 -24.44 0.02 -24.89 0.22 -0.45 0.20 Pure
Common honey 2 -12.25 0.17 -12. 53 0.20 -0.28 0.03 Adulterated
Tualang honey -12.41 0.35 -21.93 0.00 -9.52 0.35 Adulterated

23
CHAPTER 5

CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

This study indicated that the stable carbon composition analysis from the stingless

bee honey show the foraging activities and dietary habits stingless bee honey from different

region. Various type of honey that were analyzed with the aim of discriminating the

authentication of the honey by determination of adulteration with the C4 sugar syrup can be

detected by the IRMS by using the ISIRA technique.

5.2 RECOMMENDATION

For the future research study on the stable isotope on honey, it will be better to

24
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27
APPENDICES
APPENDICES A
Carbon composition

Iso-trace Analysis Report


Results for: Russell Frew
Sample set: Whole Honey & Protein
Date: 17-11-16
Iso-trace Job Number: 161629
Average
Sample d13CVPDB, Carbon % d13CVPDB,
w/w Carbon ug %
Kelulut Segamat A - Whole Honey -26.73 31.99 242
Kelulut Segamat A - Whole Honey -27.22 30.08 216 -26.98
Kelulut Segamat A - Protein -26.47 38.17 283
Kelulut Segamat A - Protein -26.68 38.40 257 -26.58 -2.4
Kelulut Segamat B - Whole Honey -27.61 30.63 217
Kelulut Segamat B - Whole Honey -27.48 31.28 191 -27.55
Kelulut Segamat B - Protein -26.97 30.52 186
Kelulut Segamat B - Protein -27.00 39.60 239 -26.98 -3.3
Kelulut Segamat C - Whole Honey -26.96 37.26 230
Kelulut Segamat C - Whole Honey -27.37 29.28 231 -27.16
Kelulut Segamat C - Protein -26.78 38.60 262
Kelulut Segamat C - Protein -26.57 39.16 325 -26.68 -2.9
Kelulut Segamat D - Whole Honey -27.00 29.59 224
Kelulut Segamat D - Whole Honey -27.18 30.30 265 -27.09
Kelulut Segamat D - Protein -26.73 40.38 320
Kelulut Segamat D - Protein -26.25 40.09 313 -26.49 -3.6
Kelulut Paloh A - Whole Honey -26.95 28.61 184
Kelulut Paloh A - Whole Honey -26.78 29.02 185 -26.87
Kelulut Paloh A - Protein -26.78 34.98 213
Kelulut Paloh A - Protein -26.66 34.18 196 -26.72 -0.9
Kelulut Paloh B - Whole Honey -26.88 29.84 255
Kelulut Paloh B - Whole Honey -27.13 29.53 199 -27.00
Kelulut Paloh B - Protein -26.89 38.93 260
Kelulut Paloh B - Protein -26.44 38.83 260 -26.66 -2.0
Kelulut Paloh C - Whole Honey -26.96 29.53 233
Kelulut Paloh C - Whole Honey -26.68 29.52 277 -26.82
Kelulut Paloh C - Protein -26.56 40.85 402
Kelulut Paloh C - Protein -26.75 37.60 255 -26.66 -1.0
Kelulut Paloh D - Whole Honey -27.00 30.06 212
Kelulut Paloh D - Whole Honey -26.80 30.01 276 -26.90
Kelulut Paloh D - Protein -26.76 34.89 235
Kelulut Paloh D - Protein -26.77 35.13 233 -26.76 -0.8

28
Kelulut Merbok A - Whole Honey -27.72 30.85 180
Kelulut Merbok A - Whole Honey -27.64 30.69 198 -27.68
Kelulut Merbok A - Protein -26.03 35.76 316
Kelulut Merbok A - Protein -26.22 35.51 188 -26.12 -9.5
Kelulut Merbok B -Whole Honey -27.63 30.42 204
Kelulut Merbok B -Whole Honey -27.51 29.96 235 -27.57
Kelulut Merbok B - Protein -26.52 37.92 303
Kelulut Merbok B - Protein -26.34 38.47 345 -26.43 -6.8
Kelulut Merbok C - Whole Honey -27.74 29.30 181
Kelulut Merbok C - Whole Honey -27.63 29.43 234 -27.68
Kelulut Merbok C - Protein -26.62 38.11 236
Kelulut Merbok C - Protein -26.49 38.59 343 -26.56 -6.7
Kelulut Merbok D - Whole Honey -27.57 30.01 226
Kelulut Merbok D - Whole Honey -27.37 29.94 257 -27.47
Kelulut Merbok D - Protein -25.94 37.20 266
Kelulut Merbok D - Protein -25.98 36.50 246 -25.96 -9.3
Thorasica - Whole Honey -27.79 29.87 229
Thorasica - Whole Honey -27.86 29.42 189 -27.83
Thorasica - Protein -26.46 36.49 238
Thorasica - Protein -27.26 37.46 215 -26.86 -5.6
Commercial 1 - Whole Honey -26.80 30.59 208
Commercial 1 - Whole Honey -26.99 29.43 211 -26.90
Commercial 1 - Protein -26.12 35.68 253
Commercial 1- Protein -26.08 34.80 243 -26.10 -4.9
Commercial 2 - Whole Honey -16.04 30.71 209
Commercial 2 - Whole Honey -16.10 31.38 191 -16.07
Commercial 2 - Protein -27.73 41.65 222
Commercial 2 - Protein -28.46 38.37 54 -28.09 65.4
Tualang - Whole Honey -12.65 32.28 232
Tualang - Whole Honey -12.16 33.38 298 -12.41
Tualang - Protein -21.93 28.25 55 -21.93 77.9

29
Results protein extraction

Iso-trace Analysis Report


Results for: Russell Frew
Sample set: Whole Honey*
Date: 21-11-16
Iso-trace Job Number: 161629
Sample 13
CVPDB, Carbon % w/w Carbon ug
Kelulut Simpang Renggam A -15.38 31.45 227
Kelulut Simpang Renggam A -15.95 29.75 185
Kelulut Simpang Renggam B -14.69 31.21 227
Kelulut Simpang Renggam B -14.88 31.30 285
Kelulut Simpang Renggam C -15.29 31.43 224
Kelulut Simpang Renggam C -14.45 32.50 249
Kelulut Simpang Renggam D -15.56 30.21 211
Kelulut Simpang Renggam D -14.92 31.64 259
Kelulut A -14.85 31.81 261
Kelulut A -14.23 30.81 270
Kelulut B -15.21 30.15 215
Kelulut B -15.28 30.33 198
Kelulut C -15.23 30.60 208
Kelulut C -14.88 31.81 230
Kelulut D -14.46 30.97 291
Kelulut D -14.41 31.07 285
Kelulut E -14.93 30.56 230
Kelulut E -14.57 31.99 300
Kelulut F -15.45 30.33 219
Kelulut F -14.60 30.80 225
Kelulut G -14.91 30.97 264
Kelulut G -14.69 30.28 214
Kelulut H -15.81 29.76 187
Kelulut H -15.01 30.62 240
Kelulut I -15.08 29.77 248
Kelulut I -15.39 30.79 218
Kelulut J -15.18 30.09 231
Kelulut J -14.42 31.43 301
Kelulut K -15.48 30.64 233
Kelulut K -14.92 29.86 217
Kelulut L -16.34 29.07 192
Kelulut L -15.88 31.06 207

30
The following laboratory reference material(s) were used to determine isotopic and
elemental values:

VPDB,
Name 13C Carbon, %
Ref. 1: USGS-40 -26.24 40.80
Ref. 2: USGS-41 37.76 40.80

The following control material was used to determine precision and accuracy:

VPDB,
EDTA-OAS 13C Carbon, %
Accepted Values -38.93 0.20 41.10
Measured Values (n=5) -38.98 0.14 41.16 0.55

31
isotopic composition

Identifier 1 Rt Ampl 44 Elem Delta Corrected Delta Mean


Honey A1 287.8 7424 -15.034 -14.79
Honey A2 288.3 6754 -15.096 -14.86 -14.82
Honey B1 296.1 10116 -14.931 -14.68
Honey B2 287.9 15971 -14.875 -14.62 -14.65
Honey C1 287.9 11265 -14.966 -14.72
Honey C2 288.2 4816 -15.632 -15.44 -15.08
Honey D1 288.2 11651 -14.874 -14.63
Honey D2 288 9186 -15.004 -14.77 -14.70
Honey E1 287.6 16238 -14.878 -14.64
Honey E2 287.8 11677 -14.906 -14.67 -14.65
Honey F1 287.3 18413 -14.837 -14.60
Honey F2 287.6 8919 -15.015 -14.79 -14.70
Honey G1 288.1 9519 -14.94 -14.44
Honey G2 287.5 12526 -14.905 -14.39 -14.42
Honey H1 287.8 6443 -15.199 -14.70
Honey H2 288.4 8165 -15.563 -15.09 -14.90
Honey I1 288 8209 -15.03 -14.51
Honey I2 289.1 11947 -14.865 -14.33 -14.42
Honey J1 287.7 10248 -14.976 -14.45
Honey J2 288.1 6601 -15.099 -14.58 -14.51
Honey K1 288.4 6390 -15.146 -14.62
Honey K2 288.6 4097 -15.324 -14.81 -14.71
Honey L1 288.3 11537 -14.922 -14.37
Honey L2 288 10412 -14.926 -14.37 -14.37
Honey M1 287 5887 -15.134 -14.61
Honey M2 286.3 11532 -14.905 -14.35 -14.48
Honey N1 286.3 12665 -14.966 -14.41
Honey N2 286.5 12711 -14.909 -14.34 -14.37
Honey O1 286.6 5476 -15.041 -14.47
Honey O2 286.9 6835 -14.967 -14.38 -14.42
Honey P1 286.3 12868 -14.903 -14.30
Honey P2 286.5 9227 -15.121 -14.52 -14.41
Honey Q1 286.8 5289 -15.096 -14.48
Honey Q2 286.4 13394 -14.863 -14.23 -14.35
Honey R1 286.2 6693 -15.2 -14.56
Honey R2 286.2 9642 -15.001 -14.35 -14.45
Honey S1 286.2 10222 -15.988 -15.38
Honey S2 285.9 12617 -14.997 -14.34 -14.86
Honey T1 286.2 14522 -14.807 -14.13
Honey T2 286.5 7828 -15.088 -14.43 -14.28
Honey U1 288 8974 -14.933 -14.75
Honey U2 287.7 9697 -15.211 -15.03 -14.89
Honey V1 288 9093 -14.954 -14.75
Honey V2 288.1 8856 -15.817 -15.63 -15.19
Honey W1 288 8530 -15.014 -14.78
Honey W2 288 9928 -14.882 -14.63 -14.71
Honey X1 288.3 8120 -15.109 -14.86
Honey X2 444.9 6365 -19.2 -19.11 -16.98
Honey Y1 286.7 24272 -15.054 -14.78
Honey Y2 288 11842 -14.967 -14.67 -14.73

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Honey Terengganu 1 288.2 12095 -28.082 -28.33
Honey Terengganu 2 288.6 11282 -28.482 -28.75 -28.54
Honey Kota Baharu 1 288.2 10188 -26.985 -27.19
Honey Kota Baharu 2 288.5 8498 -26.785 -26.99 -27.09
Honey Merbok A1 287.6 11595 -26.426 -26.39
Honey Merbok A2 287.9 10826 -26.451 -26.39 -26.39
Honey Merbok B1 287.7 9409 -26.498 -26.42
Honey Merbok B2 287.6 11391 -26.471 -26.36 -26.39
Honey Merbok C1 288.8 8811 -26.637 -26.51
Honey Merbok C2 288 10627 -26.705 -26.56 -26.54
Honey Merbok D1 0.232 8406 -26.726 -26.82
Honey Merbok D2 0.24 9163 -26.861 -26.95 -26.89
Honey Merbok E1 0.249 12837 -26.719 -26.79
Honey Merbok E2 0.243 8619 -26.702 -26.76 -26.78
Honey Merbok F1 0.202 7484 -26.852 -26.91
Honey Merbok F2 0.257 11653 -26.939 -26.99 -26.95
Honey Merbok G1 0.209 7793 -26.894 -26.93
Honey Merbok G2 0.211 7420 -26.735 -26.76 -26.84
Honey Merbok H1 0.245 9020 -26.568 -26.57
Honey Merbok H2 0.265 9380 -26.576 -26.57 -26.57
Honey Merbok I1 0.23 8419 -26.808 -26.80
Honey Merbok I2 0.228 10204 -26.665 -26.64 -26.72
Kelulut Segamat I2 0.296 11122 -26.155 -26.46
Kelulut Segamat J1 0.239 7718 -26.153 -26.45 -26.43
Kelulut Segamat J2 0.289 9591 -26.112 -26.41 -26.95
Kelulut Paloh K1 0.292 10061 -26.113 -26.40
Kelulut Paloh K2 0.25 9875 -26.116 -26.40
Kelulut Paloh L1 0.237 9467 -26.148 -26.43 -26.40
Kelulut Paloh L2 0.254 9639 -26.11 -26.38
Kelulut Paloh M1 0.245 9222 -26.112 -26.38 -26.41
Kelulut Paloh M2 0.289 11174 -26.07 -26.33
Kelulut Paloh N1 0.212 8149 -26.108 -26.37 -26.36
Kelulut Paloh N2 0.223 8635 -26.141 -26.39
Kelulut Paloh O1 0.294 10806 -26.051 -26.25 -26.38
Kelulut Paloh O2 0.266 10664 -26.141 -26.33 -26.29
Kelulut Paloh P1 0.277 10603 -26.135 -26.30 -26.27
Kelulut Paloh P2 0.229 8601 -26.079 -26.23 -26.29
Kelulut S. Renggam Q1 0.272 10862 -15.481 -15.36
Kelulut S. Renggam Q2 0.203 8353 -15.312 -15.17 -15.27
Kelulut S. Renggam R1 0.232 9197 -15.203 -15.15
Kelulut S. Renggam R2 0.278 10694 -15.057 -14.99 -15.07
Kelulut S. Renggam S1 0.269 10107 -15.063 -15.00
Kelulut S. Renggam S2 0.24 8392 -15.169 -15.10 -15.05
Kelulut S. Renggam T1 0.287 11139 -15.006 -14.93
Kelulut S. Renggam T2 0.269 10778 -15.091 -15.02 -14.97
Kelulut S. Renggam U1 0.291 11315 -15.091 -15.01
Kelulut S. Renggam U2 0.241 9048 -15.201 -15.12 -15.07
Kelulut S. Renggam V1 0.263 10132 -15.147 -15.07
Kelulut S. Renggam V2 0.263 10523 -15.086 -15.00 -15.03
Kelulut S. Renggam W1 0.238 8841 -15.006 -14.90
Kelulut S. Renggam W2 0.27 9976 -15.108 -15.00 -14.95
Kelulut S. Renggam X1 0.213 8220 -15.12 -15.01
Kelulut S. Renggam X2 0.217 8039 -15.093 -14.98 -14.99
Madu As Syifa 1 0.29 11596 -24.37 -24.46 -14.97

33
Madu As Syifa 2 0.29 11517 -24.345 -24.43 -24.44
Madu Kelulut A.H.A 1 0.269 9935 -25.868 -25.98 -15.07
Madu Kelulut A.H.A 2 0.201 7558 -25.902 -26.01
Madu Tualang Pahang 1 0.252 9792 -12.024 -11.82
Madu Tualang Pahang 2 0.201 7943 -12.12 -11.91 -11.86
Madu Kelulut (Market) 1 0.285 11245 -15.015 -14.86 -14.95
Madu Kelulut (Market) 2 0.253 10188 -15.007 -14.85
Kelulut Terengganu 1 0.234 10104 -27.321 -27.44
Kelulut Terengganu 2 0.221 9592 -27.392 -27.51 -27.47
Madu Biasa Johor 1 0.247 9566 -12.346 -12.13
Madu Biasa Johor 2 0.226 9344 -12.582 -12.37 -12.25
Honey Thorasica A1 - - - -
Honey Thorasica A2 0.218 8967 -27.068 -27.02 -27.02
Honey Thorasica B1 0.267 10110 -26.372 -26.29
Honey Thorasica B2 0.281 9249 -26.514 -26.43 -26.36
Honey Thorasica C1 0.248 8754 -26.943 -26.87
Honey Thorasica C2 0.243 11267 -26.759 -26.67 -26.77
Honey + Sugarcane 50% - 1 0.299 10572 -13.726 -13.17
Honey + Sugarcane 50% - 2 0.283 9555 -13.614 -13.05 -13.11
Honey + Apple Cider 50% -1 0.253 5023 -19.512 -19.15
Honey + Apple Cider 50% -2 0.203 3580 -19.393 -19.02 -19.08
Common Honey 1 0.265 10509 -23.104 -22.85
Common Honey 2 0.274 10269 -23.155 -22.90 -22.87

34