DETERMINATION

OF THE NITRITE
CONTENT IN
CURED MEAT
By Group II
INTRODUCTIO
N
I. Introduction
Curing is the
addition to meats of
some combination of
salt, sugar, nitrite
and/or nitrate for the
purposes of
preservation, flavor
and color.
Nitric oxide (NO) combines with the
meat pigment, myoglobin, to form
nitrosomyglobin, dark red color.

Nitrite (NO2) is one of the two

primary, stable and nonvolatile
breakdown products of NO.
The main reasons for the addition
of nitrite to meat products are
anti-microbial action, color
fixation, preservation effect and a
significant indirect beneficial
effect on flavor .
Numerous methods have been proposed
for the determination of nitrite including
electroanalytical, chromatographic, and
spectrophotometric methods.

The widely used method involves the

use of the Griess diazotization reaction
or the Griess Test.
Griess test is an analytical chemistry
test which is the most frequently used
analytical approach to quantitate the
major metabolites of NO, i.e. nitrite and
nitrate, in a variety of biological fluids,
notably blood and urine.

The Griess reaction is specific for

nitrite.
The Griess Reagent System
is based on the chemical
reaction shown in Figure 1,
which uses sulfanilamide
and N-1-
napthylethylenediamine
dihydrochloride (NED)
under acidic (phosphoric
acid) conditions. This system
detects NO2 in a variety of
biological and experimental
liquid matrices such as
plasma, serum, urine and
tissue culture medium. The
nitrite sensitivity depends on
the matrix (Figure 2).
OBJECTIVE
II. Objective
To determine the nitrite content of cured
meats supplied at local eateries and cafeterias
in Miagao and its suitability for consumption.
SIGNIFICANC
E
III. SIGNIFICANCE
High concentrations of nitrite in food can cause toxicity in
the human body. According to the Agency for Toxic Substances
& Disease Registry, high levels of nitrite can cause a decrease
in blood pressure, increased heart rate, reduced ability of the
blood to carry oxygen to tissues, headaches, abdominal
cramps, vomiting, and even death. It is also suspected to cause
cancer of the gastrointestinal tract, though evidence is limited.
The significance of this study is that with the determination
of the nitrite content in cured meats, possible high level of
nitrite may be ascertained and the said effects may be
prevented.
METHODOLOGY
Materials:
Collection of samples

Collect at least 2
cuts of cooked Immediately
hotdog, chorizo, transfer to
and chicken bilog tightly sealed
from different plastic
eateries/ cafeterias
in Miagao. containers.

Methodology
Sample preparation: Nitrite
extraction
Weigh about 1 g of each cured meat sample.
Place inside test tubes (30 mL),then add 1 mL water.

Macerate meat using glass stirring rod.

Add 26 mL water to the test tube with the cured meat
sample.
Subject to a hot water bath for 20 minutes.
Let the solution to cool down, then filter. Keep the
filtrate for analysis.

Methodology
Preparation of reagents:
sulfanilamide solution
Dissolve 80 g of reagent
grade sulfanilamide in a
mixture of 200 ml
phosphoric acid and 700 ml
water.

Dilute solution to 1000 mL

mark.
Methodology
Preparation of reagents:
N-1-naphtylethylenediamine hydrochloride
Dissolve 0.56g of N-(1-
Naphthyl)-
ethylenediamine
dihydrochloride in 100
mL water.

Store in refrigerator.

Methodology
Nitrite analysis: preparation of
nitrite standard reference curve

Prepare 1 ml of
100 micromolar
Nitrite solution
by diluting 1 Dispense 50
microliter of 0.1 microliter of Perform 6 serial 2
M standard matrix per well fold dilution of
nitrite solution from B to H Standard Nitrite
to 1 ml matrix
used for the
experimental
samples water
Methodology
Click icon to add picture

Methodology
 Absorbance
plate:

Methodology
Determination of nitrite content in
sample
Measure 50 microliters of sample analyte
into the wells, creating triplicates.

Add 50 microliter of Sulphanilamide solution

into all wells including dilution series for
Standard curve.

Incubate for 5-10 mins at room temperature.

Methodology
Continuation...

Add 50 microliter of the NED solution to all

wells.

Incubate at room temperature for 5 -10

minutes or until magenta color appears,

Measure absorbance at a filter between 520

nm and 550 nm using Microplate reader.
Methodology
calculations
Generate a Nitrite Standard curve by plotting the
average absorbance value of each concentration of
the Nitrite Standard as a function of Y with nitrite
concentration as a function of X.

Determine average absorbance value of each

experimental sample. Determine its concentration
comparison to the Nitrite Standard reference curve.

Methodology
REFERENCES
The Ingredient Store.(n.d).Roles of Nitrites and/or Nitrates in Meat
Color.Retrieved from:
http://www.theingredientstore.com/foodpreservation/nitratesnitrates.htm.
Date retrieved: May 15, 2017.
Rashid, C.(2014).Spectrophotometric Determination of Nitrite in Curing
Meat Samples Using Diazo-Coupling Reaction).Retrieved from:
http://www.iasj.net/iasj?func=fulltext&aId=39668. Date retrieved: May
15, 2017.
Molecular Probes.(2003).Griess Reagent Kit for Nitrite
Determination.Retrieved from:
https://tools.thermofisher.com/content/sfs/manuals/mp07921.pdf. Date
retrieved: May 15, 2017.
Tsikas, D.(2007).Journal of Chromatography.Retrieved from:
http://www.sciencedirect.com/science/article/pii/S1570023206006398.
FIN