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 American Journal of  
Food Science and Technology (AJFST)

Detection of  Adulteration in Besan (Cicer Arietinum) Using Microscopic Technique
Padmapriya Rajan1, Kyatanahalli S. Nagabhushana1*

Volume 3 Issue 2, Year 2024
ISSN: 2834-0086 (Online)

DOI: https://doi.org/10.54536/ajfst.v3i2.3786
https://journals.e-palli.com/home/index.php/ajfst

Article Information ABSTRACT

Received: September 11, 2024

Accepted: October 14, 2024

Published: November 30, 2024

Detection of  food adulteration in an unambiguous way requires a methodology that 
provides distinct differentiators between the authentic and adulterated samples. We present a 
simple method to detect the adulteration of  Besan (chickpea flour), an extensively used food 
ingredient for different food products in the Indian culinary industry. As many as 10 different 
samples, both branded and non-branded samples, were procured from the market and were 
evaluated for their purity. Polarized light Microscope was used as a simple evaluation tool to 
assess the purity of  the sample. The quality of  the image in light, polarized light, polarized 
light with analyzer, and dark mode bring about a clear distinction between pure besan and 
adulterated sample. Sample analyses were done in 50 X to 1000 X magnification. This non-
destructive analysis of  the sample brings about even a very small degree of  adulteration in 
the sample. From the analysis, it was clear that out of  10 samples tested, 5 samples were 
seen as adulterated. The chief  adulterants were yellow pea flour and corn flour. This simple 
method provides easy detection of  adulterants and adulteration in food samples.

Keywords

Birefringence, Besan, Cicer 
Arietinum, Food Adulteration, 
Polarized Light Microscope

1 Prayoga Institute of  Education Research, SY No. 133, Ravugodlu, Bolare Post, Off  Kanakapura Road, Bangalore, India
* Corresponding author’s e-mail: ksn@prayoga.org.in

INTRODUCTION
Food adulteration is one of  the major pervasive 
businesses which stimulate a lot of  health issues among 
consumers and is a very crucial thing to be considered 
from an economic point as well. This intentional addition 
of  foreign substances to food products tends to benefit 
the business but affects the product’s nutritional value. 
Though it has been a historical problem for centuries 
finding the simplest and authentic technique that could 
reveal the mixing of  foreign substances in food samples 
is still a challenging problem. Because the common 
adulterants are harmless cheap food products (or) 
dangerous chemicals which has the same physical nature 
as the original food sample. It also affects the quality 
of  food products, and the awareness about food safety 
procedures is yet to be explored by vendors (Oseyemi, 
2023). On the other hand, ensuring the quality of  food 
products also involves extensive experimental procedures 
(Júnior et al., 2023). So, finding the simplest non-
targeted method to identify adulterants would also act 
as a preliminary test to ensure the quality of  the food 
products. Many analytical techniques like HPLC, TLC, 
GC-MS, LC-MS, UV-Vis, NMR, and FTIR are available to 
detect adulteration but sample preparation and statistical 
procedures to analyze the spectral data are yet time-
consuming. Microscopy techniques are one of  the easiest 
ways to find adulterated substances but they are still to 
be explored as authenticated techniques for adulteration 
in the food industry. Chickpea (Cicer arietinum) flour or 
Besan is a rich source of  proteins and an indispensable 
flour used in most Indian traditional sweets and snacks. 
Because of  its high demand in India, it is rather being 
adulterated with low-cost foreign substances like corn 
flour and other legume flours. Besan is the safe choice for 

people suffering from diabetes as it contains high fiber 
and protein and less starch content. Rice and corn flour 
are not a good choice because of  its high starch content. 
The exact difference in the nutrients of  besan and corn 
flour has been well discussed in the literature (Vinod et 
al., 2023). The protein content of  besan is 22% and for 
corn flour, it is around 6.9%, mixing of  these two flours 
causes severe protein deficiency. The total carbohydrate 
content of  besan is 58% and for corn flour, it is 76%, 
and another important factor is the nature of  the starch. 
Besan has resistant starch (RS) which gets digested slowly, 
so it causes a lower spike in blood glucose levels. Corn 
flour has native starch which increases the spike in blood 
glucose levels. Mixing these two flours creates a severe 
nutritional imbalance in diabetic patients as well as others. 
Yellow peas (Pisum sativum) are another common foreign 
material that could be easily mixed with besan since it 
has the same physical nature as the besan. Pisum sativum 
has 70% carbohydrates, particularly starch content varies 
from 39% to 46% and it is also rich in proteins which is 
about 25% (Wu et al., 2023). From an economic point 
of  view, consumers are supposed to pay more money 
than the actual money for the besan when they mix these 
adulterants with the besan. Finding these substances in 
besan is yet a difficult task because of  its similar physical 
nature as other flours chemometric methods have 
become prominent in the food industry in analyzing 
data through spectroscopic methods. On the other hand, 
non-targeted method is a recent trend in identifying 
unknown components in the actual food sample (Gao et 
al., 2019). In the case of  besan, adulteration of  grass pea 
flour and pea flour in chickpea flour has been found by 
using Near-infrared reflectance spectroscopy along with 
chemometrics (Bala et al., 2022). Similarly, the addition 



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of  maize flour to chickpea flour has also been predicted 
through near-infrared spectroscopy followed by a partial 
least square regression method (Bala et al., 2022a). It was 
also mentioned that there are no chemical methods to 
identify the adulteration of  corn flour in besan, so the 
urgency to find the technique to differentiate besan from 
corn flour is increasing among researchers. Microscopy 
technique has been used to find the adulteration of  pea 
flour in chickpea flour based on seed testa macrosclereids 
(Dattatreya et al., 2010). Polarized light Microscopy is one 
of  the newly emerging authentic techniques to identify 
the foreign substances in food samples. Recently, it has 
been used to identify the percentage of  “wheat flour in 
Galician bread” (Fernández-Canto et al., 2022). In this 
work, we propose the microscopic technique to find 
the adulteration of  maize flour and yellow pea flour in 
chickpea flour based on the physical difference between 
the particles through stereo microscopy and the physical 
difference between the ‘Maltese cross’ pattern of  both 
the particles under polarized light microscopy. 

MATERIALS AND METHODS
Preparation of  Samples
To prepare the pure besan Bengal gram dal was grained 
and sieved with the appropriate consistency of  besan 
which is available in the market and corn flour has been 
purchased from the local market in Bangalore, Karnataka. 
A small amount of  besan which ranges from 0.005g to 
0.009g was placed on the microscopic slide and mixed 
with two to three drops of  water then the slide was 
allowed to dry at room temperature. Once dried, a small 
drop of  glycerin is added and it is spread over the surface 
of  the slide for better visualization. The same procedure 
has been followed for the corn flour, yellow pea flour, and 
all branded samples as well as with the mixture of  besan 
with adulterants. 

Methods
Polarizing Microscope “Leica DM750M” has been 
used to take the images of  the sample. All the images 
have been taken under four modes. This microscope is 
equipped with one polarizer, and one analyzer with the 
non-rotating mechanical stage to place the sample. 

First Mode
Once the Polarizer and Analyzer are removed it acts as 
a normal stereo microscope. So, the first image has been 
taken under normal conditions.

Second Mode
In this mode, the polarizer has been inserted before 
the sample stage and the images have been taken under 
polarized light.

Third Mode
The analyzer has been inserted above the sample stage 
and the image has been taken with both polarizer and 
analyzer.

Fourth Mode
In this mode, the polarizer has been rotated to get the 
dark field mode where the direction of  the polarizer is 
exactly perpendicular to the direction of  the analyzer. 
This has been verified by using cotton. Since cotton has 
the birefringence property by nature it reveals the colors 
when it is placed under exactly crossed polars. By doing 
this experiment the dark mode of  the microscope has 
been confirmed.

Birefringence Property of  Starch
Starch has a semi-crystalline nature which owns the 
property called birefringence by nature and can produce 
‘Maltese cross’ patterns under crossed polars. Many 
microscopy techniques have been utilized in the food 
industry so far, among them polarizing light microscopy 
has been specifically used to characterize the food 
products that have optical anisotropy (or) birefringence 
and the ‘Maltese cross’ pattern of  starch granules before 
and after heat treatment had been recorded earlier 
(Corradini & McClements, 2017). The clear-cut image of  
this pattern under 700X has also been reported in the 
literature (Sivak & Preiss, 1998). Along with that, it is also 
evident that the morphology, crystallinity, and thermal 
properties of  different types of  starch granules from 
wheat have been well discussed through light microscopy 
(Kumar et al., 2016). This property had been used to 
identify the presence of  starch in food components 

Figure 1: Besan under dark mode (500x)



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and particularly it was observed that corn starch had a 
polygon shape (McMahon, 2004). In this study, all three 
flours have starch but the nature of  the starch differs. 
While besan and yellow pea flour have resistant starch, 
corn flour possesses native starch. Accordingly, all three 
flours produce different interference patterns under 
crossed polars and this pattern also varies between the 
size, shape and nature of  the particles. Figure 1, Figure 
2, and Figure 3 have been taken under the dark mode 
of  the polarization microscope which provides clear-cut 
difference between the interference pattern of  all three 
samples. For besan, it is oval and corn flour has an exact 
‘Maltese cross’ pattern. Yellow pea flour has a slightly 
differentiated pattern with a dark patch in the center 
along with cracked lines in its sides.

RESULTS AND DISCUSSION
The microscopic images of  besan, corn flour, and yellow 
pea flour under all four modes provide unique features of  
each particle along with its size and shape. We intentionally 

mixed besan with corn flour and yellow pea flour in equal 
ratio and observation was made under all the modes, 
which helps us to differentiate all three samples through 
the naked eye easily. These images provide the clear-cut 
difference between the particle shape and size.

Besan
Besan particles are represented in Figure 4 and they are 
elliptical in shape, Figures 4a, 4b, 4c, and 4d represent 
besan particles in all four modes, Figure 4a is brighter as 
we pass light in all directions whereas Figure 4b and Figure 
4c are slightly dark as we pass polarized light. Particularly, 
Figure 4c provides a slight structural difference because 
of  the difference in the direction of  the polarizer and 
analyzer. Finally, dark mode provides the interference 
pattern of  besan particles which is an oval shape. It could 
be noted that a few besan particles have a thin line at their 
center but the size and shape of  the particles are different 
from yellow pea flour and corn flour.

Figure 2: Corn Flour under dark mode (500x)

Figure 3: Yellow pea flour under dark mode (500x)



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Figure 4a: First Mode

Figure 4b: Second Mode

Figure 4c: Third Mode

Figure 4d: Fourth Mode
Figure 4: Besan (500x)



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Corn Flour
Corn flour particles are represented by Figure 5 and 
they have irregular polygon shapes with a central black 
dot in it. As we mentioned, the second mode and third 

mode don’t provide much structural difference except for 
the variation in brightness. The fourth mode Figure 5d 
provides the exact ‘Maltese cross pattern’ under crossed 
polars because of  the nature of  the particles.

Figure 5a: First Mode

Figure 5b: Second Mode

Figure 5c: Third Mode



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Yellow Pea Flour
Yellow pea flour particles are bigger in size and the 
internal structure of  the particles varies based on their 
size. Also, some particles have a light crack inside them. 

These particles are represented by Figure 6, particularly, 
Figure 6c reveals the internal structure of  the particle 
which is different from besan. 

Figure 5d: Fourth Mode
Figure 5: Corn Flour (500x)

Figure 6a: First Mode

Figure 6b: Second Mode



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Besan with Adulterants
To see the possibilities of  identification of  adulterants 
in besan we mixed besan with corn flour and yellow pea 
flour with equal ratio and the samples were examined 
through the microscope under all four modes. Figure 7 
and Figure 8 represent the mixture of  besan with corn 
flour and the mixture of  besan with yellow pea flour 

respectively. Adulterant particles could be easily identified 
by seeing the images through the naked eye Figures 7a 
and 7d provide the unambiguous difference between 
besan and corn flour, similarly, Figures 8a,8c, and 8d 
provide the unambiguous difference between the besan 
and yellow pea flour.

Figure 6c: Third Mode

Figure 6d: Fourth Mode
Figure 6: Yellow Pea Flour(500x)

Figure 7a: First Mode



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Figure 7b: Second Mode

Figure 7c: Third Mode

Figure 7d: Fourth Mode
Figure 7: Besan and Corn Flour (500x)

Figure 8a: First Mode



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Figure 8b: Second Mode

Figure 8c: Third Mode

Figure 8d: Fourth Mode
Figure 8: Besan and Yellow Pea Flour(500x)

Market Samples
To see the practical applicability of  this technique 
ten market samples have been purchased from the 
supermarkets in Bangalore and they were examined 
under a microscope under all four modes. Among them, 

five samples were adulterated with corn flour, yellow pea 
flour, and unknown adulterants. Around 0.005g of  the 
samples were used from each pack and to prepare slides 
we followed the same procedure which we used for pure 
besan and adulterants. 



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Brand 1
This brand got adulterated with corn flour, Figure 9 
represents this brand and it could be easily noted in 9a 
that two to three particles have irregular polygon shapes 

with a central dot and those particles reveal a ‘Maltese 
cross pattern’ in a dark mode which could be seen in 
9d. This confirms the adulteration of  corn flour in the 
sample.

Figure 9a: First Mode

Figure 9b: Second Mode

Figure 9c: Third Mode



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Figure 9d: Fourth Mode
Figure 9: Brand 1 (500x)

Brand 2 
This brand is represented by Figure 10, it can be noted 
that a few particles are bigger, and from Figures 10c and 
10d it is clear that the internal structure and interference 

pattern of  these particles matches with yellow pea flour. 
So, it is confirmed that it is being adulterated with yellow 
pea flour.

Figure 10a: First Mode

Figure 10b: Second Mode



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Figure 10c: Third Mode

Figure 10d: Fourth Mode
Figure 10: Brand 2 (500x)

Brand 3
This brand is being adulterated with both corn flour and 
yellow pea flour, Figure 11 represents these particles and 
it could be noted in Figure 11a that a patch of  particles 
and a few lone particles are in irregular polygon shape, and 
around two particles are bigger in size and shape which 

is similar to yellow peas. In dark mode those irregular 
polygon-shaped particles reveal a ‘Maltese cross pattern’ 
and the interference pattern of  those bigger particles 
exactly matches with yellow pea particles. Hence, it could 
be confirmed that these particles are adulterated with 
both corn flour and yellow pea particles.

Figure 11a: First Mode



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Figure 11b: Second Mode

Figure 11c: Third Mode

Figure 11d: Fourth Mode
Figure 11: Brand 3 (500x)

Brand 4
This brand is represented by Figure 12 around six particles 
having irregular polygon shape with a central black dot at 

the center and it also reveals the ‘Maltese cross pattern’ 
in dark mode. So, it could be confirmed that it is being 
adulterated with corn flour.



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Figure 12a: First Mode

Figure 12b: Second Mode

Figure 12c: Third Mode



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Figure 12d: Fourth Mode
Figure 12: Brand 4 (500x)

Brand 5
This brand is represented by Figure 13, almost half  of  the 
particles have some weird black patches inside them which 
cannot be seen in the pure besan particles. In Figure 13d 
also it could be noted that those particles reveal different 
interference patterns because of  those dark patches 
which are not matching with the pure besan. Another 

important factor is these particles are not matching with 
corn flour particles and yellow pea flour particles also 
but it could be confirmed that it is being adulterated with 
some unknown foreign substances. So here this technique 
by using a polarization microscope acts as a non-targeted 
method to identify the adulteration hence it provides a 
clear-cut image of  pure besan particles.

Figure 13a: First Mode

Figure 13b: Second Mode



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Figure 13c: Third Mode

Figure 13d: Fourth Mode
Figure 13: Brand 5 (500x)

Brand 6
This brand has been represented by Figure 14 and it is 
pure besan, there is no shade of  corn flour particles and 

yellow pea flour in the image. Also, the particle shapes 
exactly match the pure besan.

Figure 14a: First Mode



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Figure 14b: Second Mode

Figure 14c: Third Mode

Figure 14d: Fourth Mode
Figure 14: Brand 6 (500x)

Brand 7
This brand is also pure besan which doesn’t have the 
shade of  corn flour and yellow pea flour but it could be 

noted in Figure 15d that a few particles have light cracks 
in the sides as yellow peas. But it is visible from 15a and 
15c that those particles match with pure besan particles.



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Figure 15a: First Mode

Figure 15b: Second Mode

Figure 15c: Third Mode



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Figure 15d: Fourth Mode
Figure 15: Brand 7 (500x)

Brands 8, 9 and 10
Figure 16, Figure 17, and Figure 18 represent brands 8, 
9 and 10 respectively. It could be seen there is no shade 

of  corn flour and yellow pea flour. These particles match 
exactly with besan particles. So, it can be confirmed that 
it is being non-adulterated.  

Figure 16a: First Mode

Figure 16b: Second Mode



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Figure 16c: Third Mode

Figure 16d: Fourth Mode
Figure 16: Brand 8 (500x)

Figure 17a: First Mode



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Figure 17b: Second Mode

Figure 17c: Third Mode

Figure 17d: Fourth Mode
Figure 17: Brand 9 (500x)



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Figure 18a: First Mode

Figure 18b: Second Mode

Figure 18c: Third Mode



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Figure 18d: Fourth Mode
Figure 18: Brand 10 (500x)

CONCLUSION                                                   
Food adulteration affects most of  the human beings 
without our knowledge and it is the main cause for most 
of  the health issues. So still the world needs to find 
authenticated and easy techniques from both targeted 
and non-targeted aspects. We provided the first clear-
cut image of  besan, corn flour, and yellow peas particles 
under a microscope as well as under crossed polars 
using a polarization microscope.  These images help us 
to differentiate adulterant particles without any chemical 
methods. One more advantage is a pinch of  sample 
is enough for this technique. Ten samples have been 
purchased from the market around Bangalore and tested 
for adulteration through this technique. It has been very 
successful in finding foreign substances in the sample.  

Abbreviations Used
HPLC High-Performing Liquid Chromatography
TLC Thin Film Liquid Chromatography
GC-MS Gas Chromatography-Mass Spectrometry
LC-MS Liquid Chromatography – Mass Spectrometry
UV-Vis Ultraviolet-visible Spectroscopy
NMR – Nuclear Magnetic Resonance Spectroscopy 
FTIR Fourier Infrared Transform Spectroscopy
PLM Polarized Light Microscope
RS Resistant Starch.

Acknowledgments
We thank Dr. HS Nagaraja, Chief  Mentor of  Prayoga for 
the useful discussions during the project period.

Supporting Information description
This supporting information contains all the images 
taken during the experiment.

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