Quantitative estimation of carbohydrates Likhith K

CARBOHYDRATES
AND
QUANTITATIVE ESTIMATION
By
Mr. LIKHITH K
(Research Scholar)
Department of Biomedical Engineering
Manipal Institute of Technology
Eshwar Nagar, Manipal, Karnataka-576104

Contents
Introduction
Definition and classification of carbohydrates
 Monosaccharides
 Disaccharides
 Oligosaccharides
 Polysaccharides
Quantitative analysis of carbohydrates
 Anthrone method
 Nelson Somogyi method
 Dinitrosalicylic acid method (DNSA)
Conclusion
Reference

INTRODUCTION
Carbohydrates, which are made up of carbon, hydrogen, and oxygen, are organic compounds that serve as a
source of energy for microorganism, plants and animals.
The main monosaccharide is glucose (fig 1), which is utilized as an energy source by most living organisms.
Glucose can be derived from starch and sugars in the diet, from glycogen that is stored in the body, or synthesized
from the carbon skeleton of amino acids, lactate, glycerol, or propionate via gluconeogenesis (fig 2).
The brain preferentially uses glucose as its main source of energy, and glucose is the required energy source for
red blood cells and other cells with few or no mitochondria.
Figure 1

The fate of ingested carbohydrates in an animal is determined by the monomeric composition of the
carbohydrate, the types of linkages among monomers, and the degree of polymerization.
Digestible carbohydrates include monosaccharides, disaccharides, starch, and glycogen.
Only monosaccharides can be absorbed from the small intestine, but glycosidic linkages in
disaccharides, starch, and glycogen may be hydrolyzed by endogenous enzymes in the small intestine,
resulting in release of their constituent monosaccharides.
However, these enzymes show high specificity to their target sugar units (substrate), which
consequently results in only a limited number of carbohydrates in the feed that can be digested by the
animal.
Non-digestible carbohydrates that reach the large intestine may be digested by microbial enzymes
because intestinal microorganisms secrete glycoside hydrolases and polysaccharide lyases that humans
and other mammals do not express.
Non-digestible carbohydrates include oligosaccharides, resistant starch, and non-starch
polysaccharides and are collectively known as fiber.
The large differences in the physical properties of carbohydrates make it difficult to analyze fiber and
non-digestible carbohydrates.

Dietary fiber may be divided according to solubility.
Soluble dietary fiber (SDF) may be partially or completely fermented by the microbiota in the large intestine,
producing short-chain fatty acids (SCFA), which include acetate, propionate, and butyrate.
Insoluble dietary fiber (IDF) may also be fermented, but to a lesser extent than SDF.
Fermentation of dietary fiber is a major source of energy in ruminants and hindgut fermenters, but only to a lesser
extent in pigs and poultry.
The relationship between the host and the gut microbiota is symbiotic.
As microorganisms ferment non-digestible carbohydrates, endogenous mucosal secretions, and exfoliated
epithelial cells to utilize the carbon and N to sustain themselves, SCFA and lactate are produced and absorbed by
the animal.
Estimation of carbohydrates is very important in laboratory practices to quantify the amount of sugar in any give
biological sample.
It gives out preliminary data about the sugar composition in given sample
Carbohydrate estimation have a great role in medical, agriculture and food industry.

DEFINITION AND CLASSIFICATION OF CARBOHYDRATES
Classification(fig 3) based on molecular size or Degree of Polymerization, carbohydrates are grouped into
1. Monosaccharides
2. Disaccharides
3. Oligosaccharides
4. Polysaccharides
Figure 3

Monosaccharides are chiral, poly-hydroxylated aldoses or ketoses that cannot be hydrolyzed into smaller
carbohydrate units.
They can be classified according to the number of carbon atoms in their structure (fig 4), which range from three
to nine carbon atoms (i.e., triose, tetrose, pentose, hexose, heptose, octose, and nonose), by the type of carbonyl
group they contain (i.e., aldose or ketose), and by their stereochemistry (i.e., D or L (fig 1)), and they have the
general chemical formula (CH2O)n.
Figure 4

Aldoses are referred to as reducing sugars because of their reducing effect on certain ions or compounds,
oxidizing their aldehyde group to a carbonyl group.(fig 5)
The simplest aldose sugar with a chiral atoms glyceraldehyde, with its second C molecule attached to four
different groups, giving the ability for this C to have two spatial configurations, and glyceraldehyde therefore
exist in both the D- and the L- forms.(fig 6)
Chiral carbon atoms have each of their four tetrahedral bonds connected to a different group.
The chirality of sugars and amino acid are commonly designated by the D/L system and is named in relation to
the structure of glyceraldehyde.
Figure 5
Figure 6

1. Monosaccharides
The most common monosaccharides are the 6-C aldohexoses, which include the aldohexose D-glucose, and are
usually present in their ring structures called a pyranose ring rather than in open-chain structures (fig7).
Figure 7

In oligo- and polysaccharides, aldo-pentoses can occur as a 5-C ring structure known as a furanose ring(fig 8).
D-Glucose, considering all of its combined forms, is the most abundant monosaccharide that naturally occurs in
nature.
The most abundant ketose is D-arabino-hexulose, known more commonly by its trivial name, D-fructose. (fig 9)
The three trioses include ketose dihydroxyacetone and both enantiomeric forms of glyceraldehyde.
Erythrose and threose are examples of tetroses, and pentoses include ribose, arabinose, xylose, and apiose.
Figure 8 Figure 9

Sugars, such as glucose, galactose, mannose, and fructose, which have different structures, but have the same
chemical formula, C6H12O6, are called isomers. (fig 10)
Sugars that differ in configuration around only one carbon atom are called epimers, such as D-glucose and D-
mannose, which vary in their structures around C-2(fig 11).
Figure 10 Figure 11

A pair of enantiomers is a special type of isomerism where the two members of the pair are mirror images of
each other and are designated as being in the D- or L- structure (i.e., D-glucose or ʟ-glucose(fig 1)), depending
on the position of the –OH group linked to the asymmetric carbon farthest from the carbonyl group.
Other types of monosaccharides include alditols, or polyols, which are aldoses or ketoses that had their carbonyl
groups reduced to an alcohol.
An example of a naturally occurring alditol in plants and other organisms is D-glucitol, known commonly as
sorbitol(fig12), which is the product of the reduction of D-glucose.
Figure 12

Absorption and metabolism of polyols vary among types, but most are fermented in the large intestine.
Deoxy sugars are missing one or more hydroxyl groups attached to their carbon atoms, such as 6-deoxy-L-
mannose (L-rhamnose), which is commonly associated with pectin, 2-deoxy- D-ribose, the sugar component of
DNA, and 6-deoxy-L-galactose (L-fucose), a component of glycoproteins and glycolipids in cell walls and
mammalian cells. (Fig13)
Figure 13

Uronic acids are sugar acids in which the terminal –CH2OH group undergoes oxidation to yield a carboxylic
acid.(fig 14)
Uronic acids that contribute to dietary fiber include constituents of non-digestible polysaccharides of plants and
algae, such as D-glucuronic acid, D-galacturonic acid and D-mannuronic acid. (fig 15)
Sugar from the activated form of glucuronic acid is used in the synthesis of glycosaminoglycans in mammals, and
L-iduronic acid is synthesized from D-glucuronic acid after it has been incorporated into the carbohydrate chain.
Figure 14 Figure 15

2. Disaccharides
Two monosaccharide units joined by an acetal or ketal linkage is referred to as a disaccharide.
A glycosidic bond joins 2 monosaccharide units and it can either be an α-glycosidic bond (A) if the anomeric
hydroxyl group of the sugar is in the α configuration or a β-glycosidic bond (B) if it is in the β configuration. (fig
16)
A glycosidic bond is named according to the position of the carbon atom being linked, for example, an α-
glycosidic bond connecting C-1 of a glucose molecule and C-4 of another glucose molecule in maltose is called
an α-(1,4) glycosidic bond.
Figure 16
A B

The three most common disaccharides (fig 17)are
1. Maltose
2. Lactose
3. Sucrose
Maltose is a reducing sugar that is a product of the hydrolysis of starch by the enzyme α-amylase.
Lactose is a reducing sugar that consists of a D-glucosyl unit and an α-D-galactopyranosyl unit linked by a β-
(1,4) glycosidic bond and is present in milk and milk products such as skim milk and whey.
Sucrose is made up of a glucose and a fructose linked by an α-(1,2) glycosidic bond.
Contrary to the general head-to-tail linkage (anomeric carbon atom to carbon atom containing a hydroxyl group)
in the structure of oligo and polysaccharides, in sucrose the glycosidic bond linking an α-D-glucopyranosyl unit
and a β-D-fructofuranosyl unit is in a head-to-head fashion (anomeric carbon atom to anomeric carbon atom)
making it a non-reducing sugar.
Sucrose is synthesized through the process of photosynthesis to provide energy and carbon atoms for the
synthesis of other compounds in the plant.
Figure 17

Maltose, lactose, and sucrose are hydrolyzed into their constituent monosaccharide units by the enzymes maltase,
lactase, and invertase, respectively.(fig 18)
The α-glucosidases maltase-glucoamylase and sucrase-isomaltase complexes that are present in the
brush border of the small intestine cleave the glycosidic bonds in maltose and sucrose, respectively,
with most of the maltase activity coming from the sucrase-isomaltase complex.
The monosaccharides that result from the digestion of these disaccharides are readily absorbed in the
small intestine.
Lactase, a β-galactosidase, also is expressed by young mammals that digest lactose into its constituent
monosaccharides that are subsequently absorbed in the small intestine.
Figure 18

Other disaccharides that are present in nature include trehalose, cellobiose, and gentiobiose.(fig 19)
Trehalose is a nonreducing disaccharide made up of two α-D-glucopyranosyl units linked together by
an α-(1,1) (A) glycosidic bond.
Trehalose is found in small amounts in mushrooms, yeasts, honey, certain seaweeds, and invertebrates
such as insects, shrimps, and lobsters.
Trehalose is digested by the α-glucosidase enzyme trehalase, which is expressed in the small intestine
of humans and most animals.
Two glucose molecules are linked together by a β-(1,4) (B)and a β-(1,6) (C) glycosidic bonds to form
cellobiose and gentiobiose, respectively, and these disaccharides can be utilized only after microbial
fermentation because mammals lack the enzymes capable of digesting these bonds.
Figure 19
A B C

3. Oligosaccharides
Oligosaccharides consist (fig 20) of
1. Galacto-oligosaccharides
2. Fructo-oligosaccharides
3. Mannan-oligosaccharides
that cannot be digested by pancreatic or intestinal enzymes, but are soluble in 80% ethanol.
B
A
C
Figure 20 (A,B,C)

Galacto-oligosaccharides, or α-galactosides, that are present in large amounts in legumes, are comprised of
raffinose, stachyose, and verbascose, which have a structure consisting of a unit of sucrose linked to one, two, or
three units of D-galactose, respectively.
These oligosaccharides cause flatulence in humans due to the lack of an enzyme, α-galactosidase, that
hydrolyzes the glycosidic bonds linking the monosaccharides that constitute these α-galactosides and are,
therefore, utilized by bacteria in the large intestine.
In raffinose, (fig 21) D-galactose is linked to sucrose by an α-(1,6) bond, whereas two units and three units of D-
galactose are linked to sucrose, also via α-(1,6) glycosidic bonds, in stachyose and verbascose, respectively.
Figure 21

Transgalacto-oligosaccharides are another type of galacto-oligosaccharides that may have prebiotic effects in
humans and are commercially synthesized from the transglycosylation actions of β-glycosidases on lactose,
creating β-(1,6) polymers of galactose linked to a terminal glucose unit via an α-(1,4) glycosidic bond.
However, transgalacto-oligosaccharides are not naturally synthesized.
Fructo-oligosaccharides, or fructans, are chains of fructose monosaccharides with a terminal glucose unit and are
classified as inulins or levans. (fig 22)
Figure 22

Inulin is mostly found in dicotyledons, whereas levans are mainly found in monocotyledons.
Fructo-oligosaccharides are not hydrolyzed in the small intestine due to the β-linkages between their monomers,
but can be fermented to lactic acid and SCFA in the large intestine.
Inulin occurs naturally in onions, garlic, asparagus, bananas, wheat, and chicory as a storage carbohydrate.
Inulin is made up of β-D-fructofuranosyl units linked by β-(2,1) glycosidic linkages and have a Degree of
Polymerization that ranges from 2 to 60.
The polymer is composed of fructose residues present in the furanose ring form and often have a terminal
sucrose unit at the reducing end.
Levans are fructans that have an average length of 10 to 12 fructose units linked by β-(2,6) linkages, but can
have a Degree of Polymerization of more than 100,000 fructose units and are found in bacterial fructans and in
many monocotyledons.
Levans are derived from the transglycosylation reactions catalyzed by the enzyme levansucrase that is secreted
by certain bacteria and fungi that preferentially use the D-glycosyl unit of sucrose, thereby converting sucrose to
levans with β-(2,1) linked side-chains.
Polysaccharides containing a significant number of β-(2,1) linkages also can be referred to as “levan”.

A third type of fructans, called graminantype fructans, contain a combination of both β-(2,1) and β-(2,6) linkages
and are present in wheat and barley.
Mannan-oligosaccharides are composed of polymers of mannose that are derived from yeast cell walls, and are
located on the outer surface of yeast cell walls attached to β-glucans of the inner matrix via β-(1,6) and β-(1,3)
glycosidic linkages.
Mannan-oligosaccharides and fructo-oligosaccharides may behave as prebiotics due to their beneficial health
effects on the host by stimulating the growth or activity of certain bacteria in the large intestine.
4. Polysaccharides
Polysaccharides are high-molecular-weight carbohydrates that are polymers of monosaccharides.
 Polysaccharides are made up of sugar polymers that vary in size and may either be linear or branched.
The Degree of Polymerization varies with the type of polysaccharide and may range from 7,000 to 15,000 in
cellulose and up to more than 90,000 in amylopectin.
Polysaccharides can be classified as homo-polysaccharides if they contain only one type of sugar residue (e.g.,
starch, glycogen, and cellulose) or as hetero-polysaccharides if they contain two or more different kinds of sugar
residues in their structure (e.g., arabinoxylans, glucomannans, and hyaluronic acid).(fig 23)
In humans polysaccharides are present in large quantities, and are divided into starch and glycogen and non-
starch polysaccharides.

Starch can be linear or branched and is the storage form of carbohydrates in plants, whereas glycogen is highly
branched and is present only in animal tissue, primarily in the muscle and liver.
Starch is one of the most abundant carbohydrates in nature.
It is synthesized to store energy for plant growth and is stored in seeds, tubers, roots, stems, leaves, and some
fruits.
Starch is a polymer of D-glucose that is comprised of two types of molecules, amylose and amylopectin (fig 24).
Figure 23

Figure 24
 Amylose is a short linear polymer of glucose with an average Degree of Polymerization of 1,000 glucose units
linked via α-(1,4) bonds.
 Amylopectin contains larger chains of glucose with Degree of Polymerization of 10,000 to 100,000 with branch
points at the α-(1,6) linkages for every 20 to 25 glucose units.
 The total number of α-(1,6) linkages are only about 4 to 5 % of the total glycosidic bonds in amylopectin.
 Native starch contains both forms as semi-crystalline granules of varying proportions of amylose and
amylopectin, depending on the plant source.
 Starch granules have varying structural and chemical compositions depending on the plant species and the part
of the plant where it is located.
 The size of the starch granules influences the surface-to-volume ratio, and the smaller the granule, the larger the
surface-to-volume ratio resulting in more surface area for enzyme hydrolysis in the digestive tract.

Digestion of starch begins in the mouth where salivary α-amylase is secreted, which acts only on the α-(1,4)
linked linear chains of amylose and amylopectin, until this enzyme is deactivated by the low pH in the stomach.
Large quantities of pancreatic α-amylase specific only to α-(1,4) linkages are secreted into the duodenal lumen,
producing maltose and maltotriose as the products of luminal amylose and amylopectin digestion, along with the
branched oligosaccharide α-dextrin resulting from the partial hydrolysis of amylopectin due to the inability of α-
amylase to cleave α-(1,6) linkages.
Starch digestion is completed by oligosaccharidosis (i.e., α-glucosidases) expressed by glands in the small
intestine.
These α-glucosidases include sucrose-isomaltase and maltase-glucoamylase complexes.
Both complexes have differences in their degree of specificity for the products of α-amylase digestion and
cleave the α-(1,4) and α-(1,6) bonds in α-dextrins in a complementary manner, producing free glucose that is
transported into the enterocytes.

Glycogen, (fig 25) an α-(1,4)-D-glucan with α-(1,6) linked branches, has a higher degree of branching compared
with amylopectin and is present in animal tissues, mainly in skeletal muscle and the liver.
The branch points of glycogen occur after an average of 8 to 10 glycosyl units.
A polymer of glycogen may contain up to 100,000 units of glucose.
Digestion of glycogen is similar to that of amylopectin, which results in glucose absorption in the small intestine.
The extensive branching of glycogen enhances its solubility, which allows glucose to be mobilized more readily.
Figure 25

QUANTITATIVE ANALYSIS OF CARBOHYDRATES
Carbohydrates can be analyzed by both qualitative and quantitative methods.
In case of the qualitative analysis, the presence or absence of a carbohydrate in the given sample is determined,
whereas in the quantitative analysis, the concentration (mg/ml) of the carbohydrate in the given sample is
determined or compared with a suitable reference standard compound.
The most common mode of quantitative analysis are;
1. Anthrone Method
2. Nelson-Somogyi Method
3. Dinitrosalicylic Acid (DNSA) Method

1. Anthrone method
Aim: To estimate the concentration of total carbohydrates in a given sample by Anthrone method.
Principle:
Carbohydrates are first hydrolysed into simple sugars using sulphuric acid.
In hot acidic medium, glucose is dehydrated to hydroxymethyl furfural.
Anthrone reagent is used as a coloring agent that reacts with furfural derivative to form a blue-green complex.
Absorbance of these compounds is measured by a spectrophotometer at 630 nm.

Reagents:
 Test sample
 2.5 N HCl: Add 20.833mL of HCl in 100 mL of distilled water. (Prepare the solution fresh)
 Anthrone reagent: Dissolve 200 mg Anthrone in 100 ml of ice cold 95% H2SO4. Prepare fresh before use.
 Standard glucose Stock: Dissolve 100 mg of glucose in 100 ml water.
 Working standard: 10 ml of stock diluted to 100 ml with distilled water.
 Store at 4°C after adding a few drops of toluene
Procedure
 Take clean and dry test tubes and mark all the tubes as per the protocol.
 Pipette out 0.2-1.0 ml of glucose working standard solution in duplicate test tubes.
 Make up the volume to 1 ml in each test tube by adding distilled water.
 In one test tube take only 1 ml of distilled water and mark it as blank.
 Then add 3 ml of Anthrone reagent to each test tube and mix thoroughly.
 Heat the test tubes for 8 minutes in a boiling water bath.
 Cool rapidly and read the green to dark green color at 630 nm.
 Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis.
 From the standard graph calculate the amount of carbohydrate present in the sample

Standard graph for
Anthrone method at 630nm

2. Nelson-Somogyi Method
Aim: To estimate the concentration of reducing sugar in the given sample by Nelson-Somogyi method
Principle:
The reducing sugars when heated with alkaline copper tartrate reduce the copper from the cupric to cuprous state
and thus cuprous oxide is formed. (Reddish brown ppt)
When cuprous oxide is treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue
takes place.
The blue color developed is compared with a set of standards in a colorimeter at 620nm.

Reagents:
Alkaline Copper Tartrate
(a) Dissolve 2.5 g anhydrous sodium carbonate, 2 g sodium bicarbonate, 2.5 g potassium sodium tartrate and 20 g
anhydrous sodium sulphate in 80 mL water and make up to 100 mL.(A)
(b) Dissolve 15 g copper sulphate in a small volume of distilled water. Add one drop of sulphuric acid and make up
to 100 mL.(B)
Mix 4 mL of B and 96 mL of solution A before use.

Arsenomolybdate reagent:
(a)Dissolve 2.5 g ammonium molybdate in 45 mL water. Add 2.5 mL sulphuric acid and mix well. Then add 0.3
g disodium hydrogen arsenate dissolved in 25 mL water. Mix well and incubate at 37°C for 24–48 hours.
Standard glucose solution: Stock: 100 mg in 100 mL distilled water.
Working standard: 10 mL of stock diluted to 100 mL with distilled water [100 µg/mL].
Procedure:
Take clean and dry test tubes and mark all the tubes as per the protocol.
Pipette out 0.2-1.0 ml of glucose working standard solution in duplicate test tubes.
Make up the volume to 1 ml in each test tube by adding distilled water.
In one test tube take only 1 ml of distilled water and mark it as blank.
Add 1 mL of alkaline copper tartrate reagent to each tube.
Place the tubes in a boiling water for 10 minutes.
Cool the tubes and add 1 mL of arsenomolybolic acid reagent to all the tubes.

Make up the volume in each tube to 10 mL with distilled water.
Read the absorbance of blue color at 620 nm.
Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis.
From the standard graph calculate the amount of carbohydrate present in the sample
Standard graph for Nelson-
Somogyi Method at 620nm

3. Dinitrosalicylic Acid (DNSA) Method
Aim: To estimate the concentration of reducing sugar in a given sample by dinitrosalicylic acid (DNSA) method.
Principle:
For estimation of reducing sugar, dinitrosalicylic acid method is an alternative to Nelson-Somogyi method.
This method is simple, sensitive and adoptable for handling a large number of samples at a time.
This method tests for the presence of free carbonyl group (C=O), present in the so-called reducing sugars.
This involves the conversion of reducing sugar to furfural under alkaline conditions, which reduces one of the
nitro group (–NO2) of DNSA to amino group (–NH2) to produce orange brown color 3-amino-5-nitrosalicylic
acid, with absorbance maxima at 540 nm.

Reagents:
 Test sample
 Standard glucose solution: Dissolve 100 mg of glucose and make the final volume up to 100 ml with distilled water.
 Dinitrosalicylic acid reagent (DNSA Reagent): Dissolve by stirring 1 g dinitrosalicylic acid, 200 mg crystalline phenol
and 50 mg sodium sulphite in 100 ml 1% NaOH.
 Store at 4°C. Since the reagent deteriorates due to sodium sulphite, long storage is required, sodium sulphite may be
added at the time of use.
 40% Rochelle salt solution (potassium sodium tartrate): 40g of potassium sodium tartrate in 100 of distilled water.
 Procedure:
 Take clean and dry test tubes and mark all the tubes as per the protocol.
 Pipette out 0.2-1.0 ml of working standard solution in duplicate test tubes.
 Make up the volume to 1 ml in each test tube by adding distilled water.
 In one test tube take only 1 ml of distilled water and mark it as blank.
 Add 3 ml of DNSA reagent.
 Heat the contents in a boiling water bath for 5 min.
 Add 1 ml of 40% Rochelle salt solution when the contents of the tubes are warm.
 Cool and read the intensity of dark red color at 540 nm.

Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis.
From the standard graph calculate the amount of carbohydrate present in the sample
Standard graph for
Dinitrosalicylic acid method at
540nm

CONCLUSION
Carbohydrates are subdivided into several categories on the basis of the number of sugar units and
how the sugar units are chemically bonded to each other.
Categories include monosaccharides, disaccharides, oligosaccharides and polysaccharides
Sugars are intrinsic in plant products and milk products.
Sugars also are added to foods during processing and preparation or at the table.
These “added sugars” (or extrinsic sugars) sweeten the flavor of foods and beverages and improve
their palatability.
Sugars are also used in food preservation and for functional properties such as viscosity, texture, body,
and browning capacity.
They provide calories but insignificant amounts of vitamins, minerals, or other essential nutrients.
The Nutrition Facts label provides information on total sugars per serving but does not currently
distinguish between sugars naturally present in foods and added sugars.
So quantification of carbohydrate are very important in food and drug industry.
All the three above mention methods of quantification are major and most common in daily practices.
Quantification give an confirm idea about the amount of carbohydrate content in given test sample.

REFERENCE
L. Navarro ETAL.,(2019) A review: Structures and characteristics of carbohydrates in diets fed to pigs Navarro
et al. Journal of Animal Science and Biotechnology.10:39 https://doi.org/10.1186/s40104-019-0345-6
Joanne Slavin and Justin Carlson Carbohydrates Adv Nutr. 2014 Nov; 5(6): 760–
761. doi: 10.3945/an.114.006163
Somogyi, M. (1952). J. Biol. Chem., 200, 245.
Krishnaveni, S.; Theymoli Balasubramanian and Sadasivam, S. (1984). Food Chem., 15, 229.

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