Andre Smith

October 28, 2015

FST 101A-A06, Wednesday 9 AM
RH
LAB 5 Determination of Peroxides in Fish and Chips
I. Purpose/Objective
The purpose of this lab is to look at the peroxide formation in fish and chips by
using the ferrous oxidation-xylenol orange assay.
II. Introduction
Unsaturated fatty acids are susceptible to autoxidation by singlet or triplet oxygen,
and/or damage from free radical hydroxyl or superoxide. A free radical is a molecule
that has an unpaired electron which makes them very reactive. Edible oils can be
damaged by free radicals and oxygen can react with double bonds in the fatty acids to
make hydroperoxides. Oxygen free radicals can be generated by heat, photoactivation
of triplet oxygen, and the interaction of copper, iron, and other transition metals with
hydrogen peroxide. Many edible oils are packaged in dark containers to prevent the
light catalyzed reaction of singlet oxygen and unsaturated fats to form peroxides.
Fatty acid peroxides decompose into aldehydes and carboxylic acids responsible
for the rancid smell and sour taste oxidized food. Peroxide Value (PV) measures the
extent of peroxide formation. PV units are meq peroxide oxygen/ kg fat. The SI units
for PV is mmols peroxide/kg fat. Rancid taste is noticeable at greater than 15 SI PV
units. In replacement of a lengthy procedure to measure peroxides in the solid foods
used in this experiment, the ferrous oxidation xylenol orange assay (FOX Assay II)
will be used instead with hydrogen peroxide as the peroxide standard. In the presence
of peroxides, the iron from the xylenol orange will be oxidized from Fe2+ to Fe3+.
The solution will change color and shifts in color can be measured using a
spectrophotometer. The molar absorptivity of the sample is unknown, so a standard
curve can be used to find the concentration of hydrogen peroxide.
III. Procedure
The procedure for Lab 5 is found in Principles of Food Composition, Laboratory
Manual, FS&T 101A (2015) page 41. There were no modifications for this lab.
 IV. Data
Table 1: Initial and final weights for fish and chips.
Blended
Initial Final Weight
Weight of UV Fish (g) 45.2 5.8 39.4
Weight of Control Chips
(g) 45.7 5.4 40.3
Weight of Control Fish
(g) 45.6 5.7 39.9
Weight of UV Chips (g) 45.8 5.5 40.3

Table 2: Absorbance for standards and samples.

Type 1 Water 0.25 mM H2O2 Fox II Reagent nanomoles absorbance at 560

Tube # (ml) (ml) (ml) H2O2 nm
1 0.200 0.000 5 0.0 0.002
2 0.190 0.010 5 2.5 0.018
3 0.190 0.010 5 2.5 0.025
4 0.180 0.020 5 5.0 0.047
5 0.180 0.020 5 5.0 0.05
6 0.150 0.050 5 12.5 0.128
7 0.150 0.050 5 12.5 0.121
8 0.100 0.100 5 25.0 0.25
9 0.100 0.100 5 25.0 0.226
10 0.000 0.200 5 50.0 0.452
11 0.000 0.200 5 50.0 0.456
UV Fish 1 5 11.198 0.107
UV Fish 2 5 11.857 0.113
Control Chips
1 5 5.923 0.059
Control Chips
2 5 5.154 0.052
Control Fish 1 5 4.714 0.048
Control Fish 2 5 4.824 0.049
UV Chips 1 5 33.286 0.308
UV Chips 2 5 34.824 0.322
 Standard Curve for Hydrogen Peroxide measured by FOX II
Assay
0.5
y = 0.0091x + 0.0051
0.45
R = 0.9989
0.4
Absorbance at 560 nm

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
0 10 20 30 40 50 60
nmol H2O2

Fig. 1: Standard Curve for Hydrogen Peroxide using mean and standard deviation
error bars.

Table 3: Calculated mmol of peroxide, kilograms of fat in each, and PV for all fish
and chips samples.
mmol kg fat in Mean PV Std
Sample H2O2 sample Peroxide Value (mmol/kg) PV Dev
-3
UV Fish 1 5.599 x 10 0.003 1.866 1.921 0.078
-3
UV Fish 2 5.929 x 10 0.003 1.976
Control Chips
1 2.962 x 10-3 0.014 0.212 0.198 0.019
Control Chips
2 2.577 x 10-3 0.014 0.184
Control Fish 1 2.357 x 10-3 0.003 0.786 0.795 0.013
Control Fish 2 2.412 x 10-3 0.003 0.804
UV Chips 1 1.664 x 10-2 0.014 1.189 1.216 0.039
UV Chips 2 1.741 x 10-2 0.014 1.244
V. Calculations
Calculating nanomoles H2O2 in tube
The equation on the standard curve is y=0.0091x+0.0051. y=absorbance and
x=concentration. For UV Fish 1:
x= y-0.0051
0.0091
x= 0.107-0.0051 = 11.198 nanomoles H2O2
0.0091

Calculating milimoles of H2O2 in samples

Mmol H2O2= nanomoles H2O2 x 100 ml x 1 umol x 1 mmol
ml sample 1000 nmol 1000 umol

= 11.198 nmol H2O2 x 100 ml x 1 umol x 1 mmol = 5.599 x 10-3 mmol H2O2
0.2 ml sample 1000 nmol 1000 umol

Calculating kilograms of fat in samples:

kg of fat = grams of sample x grams of total fat content x 1 kg
grams in serving size 1000 g

= 39.4 g fish x 9 g of total fat x 1 kg = 0.003 kg of fat

106 g 1000 g

Calculating Peroxide Value:

The SI units for PV are millimoles peroxide/kg of fat.
PV= mmol H2O2
kg of fat
PV = 5.599 x 10-3 mmol H2O2 = 1.866 mmol H2O2/kg fat
0.003 kg fat
Calculating Mean Peroxide Value:
Using data for UV Fish Peroxide Values
1.866 +1.976 = 1.921
2
Calculating sample standard deviation for Peroxide Values:

Once the mean has been calculated, the standard deviation can begin to be calculated by
subtracting the mean from each number in the data set.
0.1.866-1.921= -0.055
0.1.976-1.921= 0.055
Square each of the differences
(-0.055)2 =3.025 x 10-3
(0.055)2 = 3.025 x 10-3
Add the squared numbers together and divide by n-1. In this case, 2-1=1. Then, take the
square root of the resulting number to calculate sample standard deviation.
3.025 x 10-3+3.025 x 10-3 = 6.05 x 10-3
1
6.05 10-3= 0.078

VI. Results/Discussion
Table 3 shows the peroxide values for the UV radiated food items and the control food
items. In both the UV group and control group, fish has the highest peroxide values,
although UV chips have a higher PV than the control fish. The sardines in olive oil have
9 grams of total fat and the corn chips have 10 grams of total fat. While there are more
antioxidants in fish than in chips, the fish, which has less unsaturated fat than the chips,
has a higher PV value. Peroxide formation can be catalyzed by certain transition metals
such as copper and iron. The fish has 8% iron per serving while the corn chips have 0%.
The temperature at which the samples were held could have also had an impact on
peroxide formation. Although total fat was used to determine peroxide formation,
 polyunsaturated fatty acids are more relevant than saturated and even monounsaturated
fatty acids. Polyunsaturated fatty acids have more than one double bond. This
characteristic of polyunsaturated fatty acids allows them to have more than 1 resonance
hybrid for radical formation.

VII. Conclusion
The purpose of this experiment was to quantify the extent of primary oxidation in the
fatty acids contained in sardines and corn chips. The spectrophotometer measured shifts
in color change with the FOX II assay reagents. The peroxide values of the foods were
calculated using the data received from the spectrophotometer and the standard curve.

VIII. Questions
2. Based on the information gathered from the Nutrition label for each sample, did your PV rank data
compare logically with label information?
They did not. The chips were expected to have a larger PV value because they have more unsaturated fat
and total fat than the fish, but the fish had a higher PV. The nutrition label also shows that the fish has
iron, which may have catalyzed the peroxide formation process in the fish.
3. Considering a SI PV>15 is considered rancid, do you think any of the samples you measured would
taste rancid?
The food samples would not taste rancid. A PV of >15 is needed to taste rancid and the largest PV is for
UV Fish 2 at 1.976.
4. Protection against light and controlling temperature is often used to inhibit peroxide formation in foods.
Can you think of anything else that may be helpful to increase shelf life of the edible products used in this
experiment?
The fish and chips are already packaged in such a way that they are protected from light, assuming the
fish are in a can. The fish could be vacuum packed to reduce the amount of oxygen that comes in contact
with it. This may not be effective for the chips as they may crack if they are tightly sealed.