Food analysis (Unit -3)

Carbohydrate analysis:

1.colorimetric quantification
methods of mono and di-
saccharides:

Calorimetric methods are widely

used for the quantification of
mono- and di-saccharides due to
their simplicity, rapidity, and
sensitivity. These methods are
based on the ability of sugars to
reduce certain metal ions, such as
copper(II), to their lower oxidation
states. The reduced metal ions
can then react with colorimetric
reagents to produce colored
products, the intensity of which is
proportional to the amount of
sugar present.

**Common calorimetric methods

for sugar analysis include:**

* **Dinitrosalicylic acid (DNS)

method:** This method is based
on the reduction of DNS to 3-
amino-2,5-dihydroxybenzoic acid
(ADHB), which produces a red-
orange color. The intensity of the
color is measured
spectrophotometrically at 540
nm.
[Image of Dinitrosalicylic acid
(DNS) method]

* **Anthrone method:** This

method is based on the reaction
of anthrone with carbohydrates to
produce a blue-green color. The
intensity of the color is measured
spectrophotometrically at 620
nm.
[Image of Anthrone method]

* **Nelson-Somogyi method:**
This method is a modified version
of the DNS method that uses a
stronger alkali solution to
hydrolyze any non-reducing
disaccharides present in the
sample. The intensity of the color
is measured
spectrophotometrically at 540
nm.
[Image of Nelson-Somogyi
method]

**Calorimetric methods are

typically carried out in the
following steps:**
1. **Sample preparation:** The
sample is dissolved in a suitable
solvent, such as water or ethanol.

2. **Color development:** The

sample is mixed with the
colorimetric reagent and heated
to a specific temperature for a
specified time.

3. **Color measurement:** The

intensity of the colored product is
measured
spectrophotometrically.
4. **Quantification:** The
concentration of the sugar is
determined using a calibration
curve.

Advantages of calorimetric
methods:

* Simple and rapid

* Sensitive
* Relatively inexpensive

Disadvantages of calorimetric
methods:
* Not specific to a particular sugar
* Can be affected by the presence
of other interfering substances

Applications of calorimetric
methods:

* Food analysis
* Clinical chemistry
* Environmental analysis
* Pharmaceutical analysis

Here are some additional details

about the calorimetric
quantification of mono- and di-
saccharides:

* The DNS method is the most

widely used calorimetric method
for sugar analysis. It is simple,
rapid, and sensitive, and it can be
used to quantify a wide range of
sugars.

* The anthrone method is less

sensitive than the DNS method,
but it is more specific for certain
sugars, such as glucose and
fructose.
*The Nelson-Somogyi method is a
modification of the DNS method
that is used to quantify total
carbohydrates, including both
reducing and non-reducing sugars.

*Overall, calorimetric methods

are valuable tools for the
quantification of mono- and di-
saccharides in a variety of
applications.
2.HPLC methods of mono and di
saccharides using refractive index
detection :
High-performance liquid
chromatography (HPLC) with
refractive index (RI) detection is a
rapid, sensitive, and versatile
method for the separation and
quantification of mono- and
disaccharides. The method is
based on the principle that the
refractive index of a solution is
proportional to the concentration
of solute. As the sample solution
flows through the HPLC column,
the different sugars are separated
based on their interaction with
the stationary phase. The RI
detector measures the difference
in refractive index between the
eluent and the sample solution,
and this signal is used to generate
a chromatogram.

Equipment

HPLC system with RI detector

Analytical column
Sample preparation equipment
Centrifuge
Filter paper
Vials
HPLC software
Reagents
Mobile phase: Acetonitrile:water
(7:3)
Calibration standards: Fructose,
glucose, sucrose, maltose, and
lactose
Procedure

Prepare the mobile phase: Mix

acetonitrile and water in a 7:3
ratio. Degas the mobile phase for
at least 30 minutes before use.

Prepare the calibration standards:

Prepare a series of calibration
standards by diluting the stock
solutions of fructose, glucose,
sucrose, maltose, and lactose in
mobile phase. The concentration
range of the calibration standards
should cover the expected range
of sugar concentrations in the
samples.

Prepare the samples: Homogenize

the samples if necessary.
Centrifuge the samples to remove
any insoluble material. Filter the
samples through a 0.45 µm filter.

Inject the samples: Inject the

calibration standards and samples
onto the HPLC column. The
injection volume should be the
same for all standards and
samples.

Run the HPLC: Run the HPLC

program according to the
following conditions:

Mobile phase: Acetonitrile:water

(7:3)
Flow rate: 1.0 ml/min
Column temperature: Ambient
Elution mode: Isocratic
Run time: 25 min
Collect the data: The RI detector
will generate a chromatogram that
shows the elution of the sugars.
The peak area of each sugar is
proportional to its concentration
in the sample.

Quantify the sugars: Use the

calibration curve to quantify the
sugars in the samples. The
calibration curve is a plot of the
peak area of each sugar versus its
concentration in the calibration
standards.

Advantages of HPLC-RI for Mono-

and Disaccharide Analysis
Rapid: The analysis can be
completed in less than 30
minutes.
Sensitive: The RI detector is very
sensitive and can detect low
concentrations of sugars.
Versatile: The method can be used
to analyze a wide range of
samples, including food,
beverages, and biological fluids.
Specific: The RI detector is
specific for sugars and does not
detect other compounds in the
sample.
Disadvantages of HPLC-RI for
Mono- and Disaccharide Analysis
Requires specialized equipment:
HPLC systems with RI detectors
are expensive and require
specialized training to operate.
Sample preparation can be time-
consuming: The sample
preparation steps can be time-
consuming, especially for complex
samples.
Not suitable for all sugars: The RI
detector cannot detect all sugars,
such as sucrose and lactose.
Applications of HPLC-RI for Mono-
and Disaccharide Analysis
Quality control of food and
beverages
Analysis of sugar content in
biological fluids
Carbohydrate profiling of plants
Sugar analysis in pharmaceutical
formulations
Overall, HPLC-RI is a powerful tool
for the analysis of mono- and
disaccharides. The method is
rapid, sensitive, versatile, and
specific. However, it does require
specialized equipment and
training, and the sample
preparation steps can be time-
consuming.
3. Starch- enzymatic
quantification and determination
of uronic acid content and beta-
glucan content:

Starch-Enzymatic Quantification

Principle

Starch is a polysaccharide
composed of α-glucose units
linked in α-1,4 and α-1,6 glycosidic
bonds. It is the primary
carbohydrate storage molecule in
plants. Enzymatic quantification
of starch involves hydrolyzing the
starch into glucose using α-
amylase and amyloglucosidase
enzymes and measuring the
released glucose using a
colorimetric or enzymatic
method.

Materials

α-amylase enzyme
Amyloglucosidase enzyme
Glucose oxidase-peroxidase (GOP)
enzyme
Acetate buffer (pH 5.0)
Glucose standard solution
Color reagent (e.g., o-dianisidine)
Spectrophotometer
Microcentrifuge tubes
Incubator
Water bath
Procedure

Sample Preparation:

Grind the sample to a fine powder

using a mortar and pestle or a
grinding mill.
Weigh 50-100 mg of the sample
powder into a microcentrifuge
tube.
Starch Hydrolysis:

Add 500 µL of acetate buffer (pH

5.0) to the sample tube.
Add 2 µL of α-amylase enzyme
solution and mix thoroughly.
Incubate the tube at 37°C for 30
minutes.
Add 1 µL of amyloglucosidase
enzyme solution and mix
thoroughly.
Incubate the tube at 37°C for 30
minutes.
Glucose Quantification:
Prepare a glucose standard curve
using the glucose standard
solution.
To each sample tube, add 100 µL
of GOP enzyme solution and 100
µL of color reagent.
Incubate the tubes at 37°C for 15
minutes.
Measure the absorbance of each
sample and the glucose standard
solutions at 540 nm using a
spectrophotometer.
Calculation:
Calculate the glucose
concentration in each sample
using the glucose standard curve.
Multiply the glucose
concentration by 0.9 to obtain the
starch concentration.
Uronic Acid Determination

Principle

Uronic acids are acidic

monosaccharides with a carboxyl
group attached to the carbon
atom on the second position of
the sugar ring. They are major
components of pectic substances,
which are structural
polysaccharides in plant cell
walls. Uronic acid determination
involves decarboxylating the
uronic acids to carbon dioxide
using carbazole and sulfuric acid
and measuring the absorbance of
the resulting carbazole
chromophore.

Materials

Carbazole
Sulfuric acid
Boric acid
Sodium hydroxide
Uronic acid standard solution
Spectrophotometer
Microcentrifuge tubes
Water bath
Procedure

Sample Preparation:

Grind the sample to a fine powder

using a mortar and pestle or a
grinding mill.
Weigh 50-100 mg of the sample
powder into a microcentrifuge
tube.
Add 5 mL of sulfuric acid (75% v/v)
to the sample tube.
Heat the tube at 100°C for 1 hour
in a water bath.
Neutralize the solution by adding
boric acid solution (0.1 M) until
the pH reaches 7.0.
Uronic Acid Measurement:
Prepare a uronic acid standard
curve using the uronic acid
standard solution.
To each sample tube, add 200 µL
of carbazole solution (0.1% w/v) in
ethanol.
Incubate the tubes at 100°C for 10
minutes in a water bath.
Cool the tubes to room
temperature.
Add 500 µL of sodium hydroxide
solution (0.1 M) to each tube.
Measure the absorbance of each
sample and the uronic acid
standard solutions at 520 nm
using a spectrophotometer.
Calculation:

Calculate the uronic acid

concentration in each sample
using the uronic acid standard
curve.
β-Glucan Determination
Principle

β-glucans are polysaccharides

composed of β-glucose units
linked in β-1,3 and β-1,6 glycosidic
bonds. They are major
components of the cell walls of
cereals, such as oats, barley, and
rye.
The principle of beta-glucan
content determination is based on
the enzymatic hydrolysis of beta-
glucan to glucose. There are two
main methods for determining
beta-glucan content: the mixed-
linkage beta-glucan assay
(McCleary method) and the
enzymatic method.

Determination of β-glucan
Content

β-glucan is a type of non-starch

polysaccharide that is found in
the cell walls of cereal grains and
some other plants. It is a type of
dietary fiber, and it has a number
of health benefits. β-glucan
content can be determined using
an enzymatic assay that is based
on the reaction between β-glucan
and lichenase enzyme.

Materials:

Sample
Lichenase enzyme
Acetate buffer
Glucose oxidase enzyme
Peroxidase enzyme
4-aminoantipyrine
N,N-dimethylaniline
Spectrophotometer
Procedure:
Prepare a sample solution by
dissolving the sample in acetate
buffer.
Add lichenase enzyme to the
sample solution and incubate at
50°C for 60 minutes.
Add glucose oxidase enzyme and
peroxidase enzyme to the solution
and incubate at 37°C for 15
minutes.
Add 4-aminoantipyrine and N,N-
dimethylaniline to the solution
and incubate at room temperature
for 10 minutes.
Measure the absorbance of the
solution at 540 nm using a
spectrophotometer.

Calculation:

The β-glucan content of the

sample can be calculated using
the following equation:

β-glucan content (mg/g) =

(Absorbance * 0.067) / (Sample
weight (g))
These are just a few of the many
methods that can be used to
quantify starch, uronic acid, and
β-glucan. The best method for a
particular application will depend
on the specific requirements of
the assay.

There are two main methods for

determining β-glucan content:
Enzymatic method: This method is
based on the enzymatic hydrolysis
of β-glucan to glucose. The
amount of glucose released is
proportional to the amount of β-
glucan in the sample.

Colorimetric method: This method

is based on the reaction of β-
glucan with a dye to form a
colored complex. The intensity of
the color is proportional to the
concentration of β-glucan.

The enzymatic method is the more

accurate method, as it is specific
for β-glucan and does not measure
other carbohydrates. However,
the colorimetric method is
simpler and faster to perform.
Applications

The determination of uronic acid

and β-glucan content is important
for a number of applications,
including:

Food analysis: The uronic acid and

β-glucan content of foods can be
used to determine their dietary
fiber content.

4. Degree of methylation and

acetylation of pectin:
The degree of methylation and
acetylation of pectin are
important factors that affect its
physicochemical properties, such
as solubility, viscosity, and gelling
ability. These modifications are
introduced by specific enzymes,
and the extent of modification can
vary depending on the source of
the pectin and the extraction
conditions.

**Degree of methylation**

The degree of methylation (DM) of

pectin refers to the proportion of
carboxyl groups in the
galacturonic acid residues of the
pectin molecule that are esterified
with methanol. DM is typically
expressed as a percentage and
can range from 0% to 100%.
Higher DM values are associated
with increased solubility and
decreased gelling ability.

**Degree of acetylation**

The degree of acetylation (DA) of

pectin refers to the proportion of
hydroxyl groups in the
galacturonic acid residues of the
pectin molecule that are esterified
with acetic acid. DA is also
typically expressed as a
percentage and can range from
0% to 100%. Higher DA values are
associated with increased
solubility and decreased gelling
ability.

**Methods for determining the

degree of methylation and
acetylation**

There are several methods for

determining the degree of
methylation and acetylation of
pectin. Some of the most common
methods include:

* **Gas chromatography (GC)**:

GC is a versatile method that can
be used to quantify both
methanol and acetic acid released
from pectin upon hydrolysis.
* **High-performance liquid
chromatography (HPLC)**: HPLC
is another separation technique
that can be used to quantify
methanol and acetic acid.
* **Nuclear magnetic resonance
(NMR) spectroscopy**: NMR
spectroscopy can be used to
directly measure the number of
methyl and acetyl groups in the
pectin molecule.

**Factors affecting the degree of

methylation and acetylation**

The degree of methylation and

acetylation of pectin can be
affected by a number of factors,
including:

* **Source of pectin**: Different

sources of pectin can have
different degrees of methylation
and acetylation. For example,
citrus pectins typically have
higher DM and DA values than
apple pectins.
* **Extraction conditions**: The
extraction conditions can also
affect the degree of methylation
and acetylation. For example,
higher extraction temperatures
can lead to increased
demethylation and deacetylation.
* **Enzymatic modification**:
Specific enzymes can be used to
modify the degree of methylation
and acetylation of pectin. For
example, pectin methyl esterase
(PME) can remove methyl groups
from pectin, while pectin
acetylesterase (PAE) can remove
acetyl groups.

**Applications of pectin with

different degrees of methylation
and acetylation**

Pectin with different degrees of

methylation and acetylation has a
variety of applications in the food
industry. For example, low-
methoxyl pectin (LMP) is used as a
gelling agent in jams, jellies, and
confectionery products. High-
methoxyl pectin (HMP) is used as
a thickener and stabilizer in
beverages, sauces, and dressings.