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Quality Analysis of Different Types of Honey Using Refractometer
Wednesday, 15 May 2019 • 00:38 • 0 Cutey
Abstract:
Consumption
of honey and honey products has grown considerably during the last few decades.
Honey is susceptible to adulteration with cheaper sweeteners. Adulteration of
pure honey with synthetic honey has become much more prevalent in recent years.
Physicochemical
analysis is one of the methods used to measure the quality and authenticity of
honey. It
includes moisture content, total soluble solids and soluble sugars among others.
The
aims of this study are to explore the operating principle of refractometer and
how it works in detecting adulteration in honey, to examine the qualities of
different kinds of honey and to understand the application of food analysis
instruments in analysing the qualities of food product. Refractometric
method was used to determine the moisture, or conversely the soluble solids in
honey which in this study, ATAGO RX-CX 5000 Series Digital
Refractometer was used to determine the purity of a sample that
we used which are Kelulut Honey and Processed Honey (Happy Honey) by comparing its refractive
index to the value of pure substances. Through the application
of digital refractometer on different types of honey, several parameters, for
instances, total soluble solid, soluble sugar composition, moisture and refractive
index of the honey samples could be interpreted.
Keywords: Honey, refractometer, honey adulteration, total soluble solid, moisture, soluble sugars 1.0 Introduction
Honey
is a natural sweet substance produced by bees (Apis mellifera) from the nectar
of flowers or tree exudates (Liu et al, 2013). The composition of honey mainly
depends on climatic and environmental conditions and the diversity of the
plants from which they are harvested (Guler et al, (2007). Honey contains at
least 200 substances mainly carbohydrates and water. It also contains minerals,
proteins, free amino acids, enzymes, vitamins, organic acids, flavonoids,
phenolic acids, and other phytochemicals (Terrab et al, 2002). Most physical properties of honeys are defined
by the concentration and composition of carbohydrates. Moisture in honey is an
essential quality criterion in honey processing industries, as the probability
of fermentation of honey over storage increases with moisture content (Adebiyi
et al., 2004).
Consumption
of honey and honey products has grown considerably during the last few decades.
However, at the present time, the traceability of this food is limited to the
quality of each processor’s documentation. In case of doubt or fraud, there is
no standardized analysis available that can discriminate or determine the
botanical (floral or vegetable) and geographical (regional or territorial)
origin of the honey. Counterfeiting and product adulteration are now commonly
practiced in the global food marketplace. Honey adulteration is a
complex problem, which has a significant economic impact which it can be
occurred by the addition of different materials (Pilizota and Tiban , 2009). Adulteration,
or the addition of foreign substances to honey such as molasses, starch
solution, glucose, sucrose, water and inverted sugar (Sharbt and Abdel- Fattah,
1994. Ruoff and Bogdanov, 2004. Bogdanov, 2010). Because of its high
nutritional value and unique flavor, the price of natural bee honey is
relatively much higher than that of other sweeteners.
Honey is susceptible to adulteration with cheaper
sweeteners. Adulteration of pure honey with synthetic honey has become much more
prevalent in recent years. It should be emphasized that the adulteration of
pure honey is one issue and concern about the botanical and geographical origin
of honey or its authenticity is another, but the two can overlap, as in the
case of adulteration by honey of other geographical origin from a country where
quality measures are not as stringent and the honey price is much lower. Many
foods have the potential to be deliberately adulterated, but those that are
expensive and are produced under wide fluctuations in weather and harvesting
conditions are particularly susceptible which honey is one such material (Pilizota
and Tiban , 2009).
Physicochemical
analysis is one of the methods used to measure the quality and authenticity of
honey. Quality and authenticity of honey are crucial to ensure only a high
quality of natural honey is offered to consumers and to avoid any health
complications in the future. However, relatively few physicochemical data are
available on the Malaysian honey. Physicochemical analysis
is a set of tests to measure the quality and authenticity of honey. It includes
moisture content, pH, free acidity, electrical conductivity, ash content,
diastase, total soluble solids and soluble sugars among others (Codex
Alimentarius Commission, 2001). Multiple tests needed to be conducted on a single
honey sample because honey is
a complex mixture
of various substances
such as water, carbohydrates, proteins,
enzymes, minerals and
microorganisms (da Silva et
al., 2016).
Refractometric
method was used to determine the moisture, or conversely the soluble solids in
honey as previously described by the International Honey Commission. This
method is based on the principle that refractive index increases with solid
content. It is also an important tool that determines the
quality and grade of honey. Its accuracy has helped millions of beekeepers
around the world in producing high-quality honey. The nutrient grade of the
honey is measured in Brix. Higher Brix measurements mean higher nutrient grade.
So in food, the higher the Brix percentage, the more nutritious the food is. It
also applies in honey-making. So, the higher the Brix percentage in honey, the
better the quality is.
2.0 Objectives
The
objective of this experiment is to explore the operating principle of
refractometer and how it works in detecting adulteration in honey. Besides, to
examine the qualities of different kinds of honey, and to understand the
application of food analysis instruments in analyzing the qualities of food
product.
3.0 Materials and Apparatus
4.0 Methodology
5.0 Result and Graphic
5.1 Results
Through the application of digital
refractometer on different types of honey, several parameters, for instances,
total soluble solid, soluble sugar composition, moisture and refractive index
of the honey samples could be interpreted. In this study, these qualities of
honey were examined and could be important indicators of the occurrence of
fraud or adulteration in honey, which is not uncommon in the food market
nowadays.
Table 5.1.1 shows the measurement of
qualities of different types of honey using refractometer
5.2 Calculations
Obtaining
moisture content from total soluble solid (TSS) value
Kelulut honey
1st and 2nd trial
Moisture content = 100% - 67.6%
= 32.4%
3rd trial
Moisture content = 100% - 67.9%
= 32.1%
Happy honey
1st trial
Moisture content = 100% - 78.5%
= 21.5%
2nd trial
Moisture content = 100% - 76.1%
= 23.9%
3rd trial
Moisture content = 100% - 78.1%
= 21.9%
Calculating standard deviation and percentage error for
triplicates
Parameter 1 : Total soluble solid (%)
Kelulut honey
Percentage error = 0.173 x 100%
67.7
= 0.26%
Happy honey
Standard deviation =
Percentage error = 1.286 x 100%
77.6
= 1.66%
Parameter 2 : Moisture content
Kelulut honey
Standard deviation =
 = 0.713
Percentage error = 0.173 x 100%
32.3
= 0.54%
Happy honey
Standard deviation =
 = 1.286
Percentage error = 1.286 x 100%
22.4
= 5.74%
Parameter 3 : Refractive index
Kelulut honey
Standard deviation =
 = 0.00139
Percentage error = 0.00139 x 100%
1.459
= 0.10%
Happy honey
Standard deviation =
 = 0.00533
Percentage error = 0.00533 x 100%
1.485
= 0.36%
5.3 Graphics
Figure 5.3.1 shows the total soluble
solids, or known as sugar concentration in percentage of different types of
honey
Figure 5.3.2 shows the moisture
content of different types of honey
6.1 Principle of Refractometer and
Refractive Index
Refractometers are instruments to measure substances dissolved in
water and certain oils. The refractometer works using the principle of light
refraction through liquids. As light passes from air (Medium A) into a liquid (Medium
B) it slows down. Whenever light changes speed as it crosses a boundary from
one medium into another its direction of travel also changes, as it is
refracted (Figure 1). This phenomenon is what gives a "bent" look to
objects that are partially submerged in water. To put it simply, the more dissolved
solids water contains, the slower light travels through it, and the more
pronouced the "bending" effect on light. Refractometers use this principle
to determine the amount of dissolved solids in liquids by passing light through
a sample and showing the refracted angle on a scale (Mascosko, 1994).
Figure 1. Light crossing from any transparent medium into another in which
it has a different speed it is refracted which is bent from its original path.
A refractometer is
used to measure the refractive index of substances, usually liquids. Most
refractometers are based on the critical angle effect which defines the point
of balance, the shadow point or borderline, between refraction and total
internal reflection of light at a prism or sample interface (Jing Wang et al., 2018).
More generally, refractometers are used to measure a Refractive Index (RI) of
pure substances (liquid) as a unique characteristic, or used to measure the
concentration of one substance dissolved in another. Refractive index commonly
used to determine the purity of a sample by comparing its refractive index to
the value of pure substances.
The refractive
index of a substance, usually given the symbol n, is a measure of the speed of
light through the substance and is defined as the ratio of the speed of light
in the substance to the speed of light in a vacuum. For practical purposes the
speed of light in air rather than vacuum is used, the difference being very
small.
Refractive Index of given substance (n) = Speed of
light in vacuum
Speed of light in substance
Apart from the
fundamental RI scale, the Brix scale is the most widely used scale on a
refractometer. Brix, or sugar percetange is an internationally recognised
scale, which relates the concentration of sucrose in water at 20 °C to the RI of the solution (wt/wt). Most food products are more
complex than a sucrose solution which many other soluble ingredients may
contribute to the overall RI. However, the Brix scale is still used as the
standard. For non-sucrose based products the term ‘apparent Brix’ is strictly
more correct.
Refractometer
measures total soluble solids (TSS) concentration based on the principle of
refraction of light. When a ray of light travels obliquely from one medium to
another, it is bent or refracted. The refraction occurs because light travels
at slightly different velocities in different media, the extent being
proportional to the density of the solution or the soluble solids
concentration. The refractive index of a medium is defined as the ratio of the
sine of the angle of incidence to the sine of the angle of refraction when a ray
of monochromatic light is refracted from a vacuum (air) into the medium (S.K.
Mishra et al., 1989). In a Brix refiactometer, the refractive index is
calibrated into degrees Brix readings.
Total Soluble Solid
(TSS) content of a solution can be determined by the refractive index. This is
measure using a refractometer and is referred as the degree Brix. Brix is a
term used when a refractometer equipped with scale, based on the relationship
between refractive indices at 20 ֯ C and the percentage by mass of total soluble solids of a pure
aqueous sucrose solution. In principle, the unit Brix, which has been in common
use in industry for many years, represents the dry substance content of
solutions containing mainly sucrose (Dongare et al., 2014).
6.2 Physicochemical
Quality Based on Experiments Results
6.2.1 Moisture
Content
The
moisture content of honey (water-in-honey) is the quality aspect that
determines the ability of honey to remain fresh and to avoid spoilage by yeast
fermentation. Raw honey can have a moisture content of less than 14% and
the lower the water content the higher the perceived value of the honey.
It is internationally recognized that good quality honey should be processed at
less than 20% moisture content (Ajlouni & Sujirapinyokul, 2010). Low
moisture content is desirable because honey may begin to ferment and lose its
fresh quality if the water content is greater than 20%.
Unpasteurized
honey ferments because it contains wild yeast. However, due to honey's
high sugar concentration these yeasts are less likely to cause fermentation in
honey with low water content. The honey's low moisture content causes the
yeast to enter its dormant stage preventing the fermentation process. In
honey with high moisture content the yeast is more likely to cause fermentation
during storage resulting in higher acidity directly affecting the honey's
quality.
Honey
easily absorbs water from the air. This means that it can be difficult to
produce good quality honey with low moisture content in areas where humidity is
high (Alemu et al., 2013). Because honey's moisture content greatly
affects the possibility of fermentation during storage and thus the quality of
the honey, the measurement of the moisture content in honey is very
important. To control this factor, it is important that moisture content
be known with a good degree of accuracy. From the results obtained, the
moisture content for ‘happy’ honey (22.4 %) was lower than ‘kelulut’ honey
(32.3 %). Thus, it can be identified that ‘happy’ honey was greater in the
factor of storage stability and quality compared to ‘kelulut’ honey.
Honey
adulteration refers to the act of adding some foreign substances into pure
honey. This incident had existed since hundreds of years ago. It was recorded
in “The Virtues of Honey” the first book of Sir John Hill (1957) which alerted
readers: “Beware of honey with dishonest mixture or flour and other ingredients.”
(Bogdanov, 2010). One of the major problem in honey adulteration is the
moisture content and this had become a common problem in the market nowadays
due to the difficulties in identifying the adulterated honey. Moisture is the
third highest component of honey (National Honey Board, 1996). Moisture content
of honey may vary from 15 to 20 % (Bogdanov, 2010). This is the main criteria
used to determine the keeping quality and storage stability of honey. Besides that,
the origin of honey might also relate to the moisture content of honey.
Honey
from tropics might contain more moisture due to the humidity. Moisture content
of honey is also closely related to its fermentation. The higher the moisture
content of the honey, the higher the is possibility of fermentation happening
in the honey samples. Honey that contains more than 18 % of moisture is likely
to get fermented during prolonged storage (Ruoff & Bogdanov, 2004). The
higher the moisture content in honey sample, the higher the survival rate of
the yeast in honey. Thus, the higher the fermentation activity which occurs in
honey and indirectly leads to the increase of acidity of honey. Other than that, the moisture content of
honey might also be due to the time of honey collection. Moisture content of
honey that was collected during raining season is higher compared to the honey
collected in the dry season.
6.2.2
Total Soluble Solids (TSS)
From the results in Table
4.1.1, it showed that average of total soluble solids (TSS) for the Trigona sp.
of honey (Kelulut Honey) is 67.7 % while for Happy honey, the average of total
soluble solids (TSS) is 77.6 %. However, Kelulut honey has a least percentage
error of total soluble solids (TSS) which is 0.26 % compared to Happy honey
which is 1.66 %. Supposedly, the standard of the total soluble solids (TSS) for
the Trigona sp. of honey (Kelulut Honey) is ranged from 85.7 % to 83.6 % of TSS
(Nyau et al., 2013). However, in this experiment, we only get TSS which is 67.7
% that does not comply with the standard. From the research of Tarun K. D. et al., (2016), as per Alimentarius
Commision Standards (2001), a minimum TSS content of 65 % is required.
Meanwhile, the standard of TSS for Happy Honey that being processed should has
TSS in the range of 60% to 70% but from experiment, we get above 70% of TSS.
Total soluble solid is a
measure of dissolved solids in the honey samples. This is for testing the
quality of honey. The reason for
testing honey for quality control purposes is to verify the authenticity of the
product and to reveal the possible presence of artificial components or
adulterants, as well as to address processing and market needs (Krell, 1996).
This requires not only determining the total soluble solids (TSS) but also
moisture and mineral content (ash), the levels of hydroxymethylfurfural (HMF),
acidity, diastase activity, apparent sugars and water insoluble solids
(Bogdanov et al., 1999).
According research from
Francisco Anguebes et al. (2016), the
total soluble solids (TSS) can be a parameter to detect fraud in honey because
the Brix ͦ scale that used in the food industry is measuring the approximate
amount of sugars. The honey is mainly composed of sugars; about 25 different
oligosaccharides have been detected in the composition of honey. The fructose
and glucose are present in a higher concentration and provides the honey with
its extreme sweetness. The total soluble solids (TSS) which are directly
related to sugar content may be a reliable index of adulteration. Besides, the
soluble solids are also can serve as an indicator parameter of the rate
in solution
solids such as sugars, organic acids and minerals, nonetheless
directly related to sugars and the water levels in the samples.
In conclusion, in this
experiment, we get total soluble solids (TSS) for the Trigona sp. of honey
(Kelulut Honey) is lower than Happy honey that was being processed. If all the
honey samples, total soluble solids were generally more than 80 % and it can be
considered of high grade and highly stable upon storage. On the other hand,
honey with less than 80 % soluble solids is likely to ferment during storage.
According of the grading system of the United States Department of Agriculture
(USADA), honey with total soluble solids greater or equal to 81.4 % is
considered to be of higher grade (A and B), while that falling between 80 % and
81.3 % is considered to be of lower grade C.
6.2.3 Soluble sugar
In this experiment, the average refractive
index of kelulut honey that was 1.459 which is lower than happy honey which is
have sold in market that have 1.485. The
refractive index is a way to describe the speed in which light travels through
a substance relative to how quickly light travels through a vacuum. The
refractive index of honey can range anywhere between 1.3 to 1.7, and it is most
often between 1.474 and 1.504. The results obtained were in the range. The
reason why the refractive index of honey is not always the same is because of
the fact that the consistency of honey can vary due to its water content. The
higher the water content of honey, the lower the refractive index. This means
that the higher the water content of honey, the faster lighter will travel
through the honey. Dissolved sugar changes the refractive index of water
substantially. The refractive index of a carbohydrate solution increase with
the increases concentration. The percentages sugar, measured in degrees brix.
Degrees Brix (symbol °Bx) is the sugar content of an aqueous solution. One
degree Brix is 1 gram of sucrose in 100 grams (% w/w) of the solution. If the
solution contains dissolved solids other than pure sucrose, then the °Bx only
approximates the dissolved solid content.
6.3 Application or
other instrument in examining physicochemical properties of honey
Honey adulteration can also be detected
using several modern methods such as measuring stable carbon-isotope ratios,
NMR or differential calorimetry. Much attention has been paid to measuring
major sugars in honey with gas chromatography (GC) and liquid chromatography
coupled to various types of detectors (Abdel-Aal et al. 1993; Bogdanov et al.
2004).
Ruiz-Matute et al. (2010a) studied the
sugar composition of high-fructose corn syrup (HFCS) using gas chromatography
coupled with mass spectrometry (GC-MS). Sucrose syrups were analysed in
parallel as a control. HFCS was shown to contain fructosyl-fructose and other
unknown sugars that could be fructosyl-glucose. Honey produced using syrups for
bee-feeding and was also analysed to detect the effect of these syrups on sugar
composition. Fructosylfructose was detected in honey made by bees fed HFCS.
Fructosyl-fructose was detected also in honey from free-flying bees and bees
fed sucrose syrups, but at lower levels.
Guler et al. (2014) investigated the
sensitivity of methods for the analysis of carbon isotope ratios. They analysed
a total of 100 samples of unadulterated honey, honey made by bees fed with
different amounts (5, 20 and 100 litres/colony) of sugar syrups. These syrups
included corn syrups with high fructose-85 (HFC-85%), with moderate fructose-55
(HFC-55%), bee-feeding syrups (BFS), glucose syrups (GMS), and sucrose syrups
(SS). The honey samples were analysed for their D13C values for honey sugars
and proteins, the difference in the D13C values of the proteins and sugars
(Dd13C) and the percentage of C4 sugars. Adulteration in honey from colonies
fed syrups at 5 litres/colony was not detectable, but it was possible to detect
adulteration in colonies fed 20 and 100 litres/colony of HFC-85, and 100 litres/colony
of HFC-55.
Bertelli et al. (2010) published an
effective method for the detection of honey adulterated using sugar syrups. It
involves one-dimensional (1D) and two-dimensional (2D) nuclear magnetic
resonance (NMR) coupled with multivariate statistical analyses. They used 63
samples of honey from various botanical sources and seven different sugar
syrups marketed as specific bee-keeping products. They analysed 63
unadulterated honey samples and 63 samples of honey from colonies fed with
seven different sugar syrups commonly used in adulteration of honey. The best
discrimination model involved 1D-spectra and a cross verification analysis
showed a prediction capacity for this model of 95.2%. The 2D NMR analyses also
gave satisfactory results (cross-verification showed 90.5% accuracy).
Cordella et al. (2005) describe the
development of high performance anion exchange chromatography with Pulsed
Amperometric Detection (HPAEC-PAD) for the analysis of honey to detect
adulteration combined with chemometric techniques for processing chromatograms
for better discrimination of pure and adulterated honey. This method was
investigated using honey samples containing between 10% and 40% of different
industrial sugar syrups used for the feeding of honey bees.
7.0 Conclusion
In
conclusion, the operating principle of refractometer and how it works in
detecting adulteration in honey had been explored. A refractometer is used to
measure the refractive index of substances, usually liquids. The refractometer
works using the principle of light refraction through liquids. For example, refractometer
measures total soluble solids (TSS) concentration based on the principle of
refraction of light. When a ray of light travels obliquely from one medium to
another, it is bent or refracted. The refraction occurs because light travels
at slightly different velocities in different media, the extent being
proportional to the density of the solution or the soluble solids concentration.
Moreover, the qualities of different honey which are Kelulut Honey and
Processed honey were successfully examined by test the physicochemical quality like
the moisture content, total soluble solids and soluble sugars. Furthermore, the
application of food analysis instruments in analyzing the qualities of food
product which is the refractometer that we used in this study had been understand.
The qualities of the honey we used were successfully analysed by using the refractometer.
8.0 References
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9.0 Acknowledgement
In performing our assignment which is quality analysis of different types of honey using refractometer, we had to take the help and guideline of some respected persons, who deserve our greatest gratitude. The completion of this assignment gives us much pleasure. We would like to show our gratitude to Dr. Siti Fatimah Zaharah binti Mohamad Fuzi, Lecturer of Food Analysis 2 (BWD20603), Universiti Tun Hussein Onn Malaysia for giving us a good guideline for assignment throughout numerous consultations. We would also like to expand our deepest gratitude to all those who have directly and indirectly guided us in writing this assignment. In addition, a thank you to Mr. Ishak bin Ayub, Assistant Science Officer, Universiti Tun Hussein Onn Malaysia who introduced us to the methodology of using refractometer. Many people especially our classmates and team members itself, have made valuable comment suggestions on this proposal which gave us an inspiration to improve our assignment. We thank all the people for their help directly and indirectly to complete our assignment.
Appendix A
Figure shows the table of derivation of refractive indices at 20℃ corresponding to the
Brix value, as standards for honey regulated by United States Department of
Agriculture (USDA).
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B.Sc in Food Technology with Honour,UTHM
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This blog created for Food Analysis II subject which teach by our beautiful and cute lecturer which is Dr. Siti Fatimah Zaharah Binti Mohamad Fuzi.
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Muhammad Irfan Bin Zamri
(AW 170138)
Dr.Nina
Nurul Husnina Binti Rohazam
(AW 170101)
Dr.Amelia
 (AW 170173)
Dr.Puteri
 (AW 170060)
Dr.Bayah
 (AW 170020)
Dr.Sook Ling
Dr.Mawardi
 (CW 170009) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
