1 SUGARS

1.1 Benedict test - reducing sugars

 Principle
To determinate if the sample contains reducing sugras, Benedict test can be used, which is a
qualitative sugar analysis. The Cu2+ containing solution can react with the reducing sugras
resulting Cu2O which is a green/brown compound.
Cu2+ (blue) reducing sugar
Cu2O (green-brown)

 Calculations and results

If the solution becomes green, reducing sugars are present in the sample, and if the solution
becomes brown it means that the sample contains big amount of reducing sugars. According
to the result of our solutions (fig 1.) there are some reducing sugars in our sample.

Figure 1: The result of the Benedict test (our sample is in the 4th test tube)

 Discussion
The green colour (positive test) of our samples solution is not intense which means that the
amount of reducing sugars (glucose, fructose, galactose) are not high, or there are more
reducing disaccharide (lactose), which has lower reducing capacity.
According to our GC and TLC result our sample contained 50% sucrose (non-reducing); 25%
lactose (reducing but disaccharide) and 25% glucose (reducing) which mainly support our
Benedict test result.

1.2 Luff-schoorl – reducing and non-reducing sugars

 Principle
The amount of reducing sugars can be determinated by boiling excess Cu2+-ions with the
aldose and ketose containing sample (1). The reacted Cu2+-ions can determinated with
iodometric titration (2). Carrez I and Carrez II solutions are used to deproteinate the sample to
avoid other reactions. To determinate the non-reducing sugars in the sample, acid hydrolysis
is applied to convert the saccharides to reducing monomers.
(1) R-CHO + 2 Cu2+ + 5 OH-  R-COOH + Cu2O + H2O
(2) 2 Cu2+ + 4I- (excess)  CuI + I2
2S2O32- + 2I  S4O62- + 2I-

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  Calculations and results
The amount of reacted sodiumthiosulphate can be determinated with the help of the blank:
mL blank - mL titration = X mL
25.4 mL – 23.8 mL = 1.6 mL for the reducing sugars (before hydrolysis)
25.4 mL – 24.5 mL = 0.9 mL for the reducing and non reducing (after hydrolysis)

Before the hydrolysis: the reducing sugars

o If glucose are the only reducing sugars present in the sample:
1.6 mL corresponds to 3.8 mg glucose in the Luff Schoorl table.
In the initial 100 mL of filtrate, the glucose content was then 76,8 mg (dilution factor is 20).
The initial weight of the sample was 1.0081g, which contains then 7.6% glucose.

o If lactose is the only reducing sugar present in the sample:

1.6 mL corresponds to 5.8 mg lactose in the Luff Schoorl table.
In the initial 100 mL of filtrate, the lactose content was then 116.0 mg (dilution factor is 20).
The initial weight of the sample was 1.0081g, which contains then 11.5% lactose.

After the hydrolysis: total sugar content

o If glucose are the only reducing sugars present in the sample:
0.9 mL corresponds to 2.2 mg glucose in the Luff Schoorl table.
In the initial 100 mL of filtrate, the glucose content was then 220.0 mg (dilution factor is 100).
The initial weight of the sample was 1.0081g, which contains then 21.8% glucose.

If glucose/fructose are the only reducing sugars present in the sample, the following equation
should be used:
Non-reducing sugar = RSafter hydrolyses x 0.95 – RSbefore hydrolyses x 0.95
= 21.8 x 0.95 – 7.6 x 0.95 = 13.5 %
If lactose/maltose are the only reducing sugars present in the sample:
Non-reducing sugar = RSafter hydrolyses . 0,95 – RSbefore hydrolyses
=21.8 x 0.95 – 11.5 = 9.21%

Table 1: The result of the Luff-Schoorl method

Reducing sugars (%) Non-reducing sugars

If only glucose 7.6 13.5
If only lactose 11.5 9.2
Average value 9.6 11.4

 Discussion
According to the recipe, the cookie’s sugar content was 50% sucrose, and 25-25% reducing
lactose and glucose. Since the sample total sugar content was 21.8%, the result of the ratio of
the reducing and non-reducing sugar correlates to the recipe.

1.3 Gas chromatography

 Principle
The different sugar molecules and their amount can be determinated with
gaschrolmatography. Since sugars are thermo-unstable, first derivatisation needs to be done.

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 This will help to carry out the oximation and silylation to reduce the original five peaks in the
chromatogram. Though reducing sugars will still result two peaks.

 Calculations and results

The different peaks are proportional to the ratio of the sugars in the sample. According to the
chromatogram of our sample (figure X.), there are four sugars: fructose, glucose, sucrose and
lactose in the order of polarity. For this gas chromatography external (Standard solution of
sugars) and internal standard were used to help with the calculation. First from the external
standard sugar solutions, the response factor (RF) should be calculated for each sugars and
then the mass of the sugars in the samples can be calculated. Finally the result have to be
converted according to the amount of sample was used (1.0081g) and also according to the
100 times dilution.
The calculation formulas are the following:
𝑚𝑠𝑢𝑔𝑎𝑟 × 𝐴𝐼𝑆
𝑅𝐹𝑠𝑢𝑔𝑎𝑟 =
𝑚𝐼𝑆 × 𝐴𝑠𝑢𝑔𝑎𝑟
𝑅𝐹 × 𝑚𝐼𝑆 × 𝐴𝑠𝑢𝑔𝑎𝑟
𝑚𝑠𝑢𝑔𝑎𝑟 =
𝐴𝐼𝑆

Table 2: Results and observations of the GC analysis

Sugar RF Area mass(mg/ml) %sugar(g/g w.b.)

IS - 5170.41699 -
fructose 0.67 1086.68566 0.085 0.84
glucose 0.68 7918.27808 0.630 6.25
sucrose 0.87 14685.3 1.499 14.87
lactose 0.84 6763.42005 0.660 6.55

Figure 2: Chromatogram of the sugar analysis

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  Discussion
The chromatogram shows that in our sample there is fructose, glucose, sucrose and lactose
present. However according to the recipe we should have only detected glucose, sucrose and
lactose. Their proportion is the similar (50% sucrose, 25% glucose and 25% lactose) as it was
in the recipe. The small amount of fructose could be a result of the heat treatment of baking: a
monomer coming from sucrose.

1.4 Thin Layer Chromatography

 Principle
The different kind of sugars can be determinated qualitatively with thin layer chromatography
(TLC) since sugars have different retention value. The standard solutions contain all the
possible sugars and because they have different attributes (solubility, size, charge, etc) they
can be separated. After the elution, the location of the spot can be visible by spraying a
reagent on the plate.

 Calculations and results

The results can be determinated by calculating the retention factors (RF) for the standards
and the sugars in the sample.
RF=distance from the start line to sugar spot/distance from the start line to the solvent line.
Table 3: The observations from GC analysis

Standard Our Sample

Sugar RF RF
glucose 0.239 0.239
sucrose 0.102 0.102
lactose 0.045 0.034

 Discussion
The chromatogram of the TLC is however not easily readable since the spots of the different
sugars were not separated properly, with the help of our GC analysis it is possible to read the
RF factors and determinate the different sugars present in our sample. The result of TLC is in
a correlation of the recipe of our sample, there is glucose sucrose and lactose present.

2 PROXIMAL ANALYSIS

2.1 Moisture and dry matter content of the cookie

2.2 Ash content

2.3 Salt content

2.4 Fat content by weibull

2.5 Protein content by Kjeldahl method

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3 MILK

In this laboratory practice we were determinating the fat content and the heat treatments of a direct
Ultra High Temperature (UHT) treated milk sample. The label of the milk can be read in the figure x.

Figure: The label of the milk product

3.1 Fat content by the Gerber Method

 Principle

The Gerber method is a volumetric fat content

determination procedure often used for dairy product
analysis. Sulphuric acid helps lowering the pH to destruct
the proteins and carbohydrates and destabilize the
emulsion to release the fat. Amylalcohol is used for
separating the fat phase and homogeneous the fat column.
Heating and centrifugation are also applied to enhance the
separation. The measurement is carried out in a special
glass flask, butyrometer, which is graduated in %fat.

 Calculations and results

The result could be red from the butyrometer directly,
which was 1.7g of fat in 100g milk sample, as it can be
seen on the x.th figure.

 Discussion
According to the label of the product, the milk sample
contains 1.5% fat. Our measurement resulted a slightly
higher value: 1.7% which difference could be because of
the difference in the methods.

1. Figure: The separated fat phase in the butyrometer

3.2 Fat content by the Röse-Gottlieb

 Principle
Röse-Gottlieb method is a gravimetric fat content determination procedure. Ammonia solution
is used to dissolve the casein micelles by destructing the bound between the fat and protein
molecules and also used for neutralizing the sample. Ethanol is added to prevent gelation by
enhancing the solubility of the proteins and also helps to separate the ether-water phase. A
mixture of diethyl ether and petroleum is used to carry out the extraction. With the help of the
diethylether polar lipids are also extracted and petroleum helps to avoid the co-extraction of

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 the lactose which would result an over estimation. Few drops of phenolphthalein is also used
to help make the separation line more visible.

 Calculations and results

After the procedure fat content can be determinated directly by measuring the fat containing
flask’s weight.
A = mass(g) of the extracted matter
M = mass (g) of the test portion
𝐴 0.1751𝑔
%𝐹𝐴𝑇 = × 100 = × 100 = 1,75%
𝑀 10𝑔
 Discussion
The result of the Röse-Gottlieb test is 1.75% which is higher than what we read from the label
of the product. However the difference is not much, the result is also similar to the obtained
value with the Gerber method. The difference could be the result of the not enough precise
laboratory practice or the difference in the method. If one would like to have a more precise
method, should choose the Röse-Gottlieb methods since it gives a percentage value with two
decimals. However the Gerber method is a faster and less equipment required method.

3.3 Turbidity Test

 Principle
The Turbidity test can give information about the used heat treatment to enhance the
microbiological stability of milk. Different temperature-time combinations result different
degree of denaturation of the whey proteins in milk. More intense heat treatment result more
denatured whey proteins which can be precipitate with ammonium-sulphate. This can be
separated by filtration and as a consequence it will not result turbid solution after additional
boiling.

 Calculations and results

The result of the turbidity test can be determinated visually by the solutions turbidity. We can
compare our sample (4th) also with other milks products result (figure X.).

1. 2. 3. 4. 5.
2. Figure: Turbidity test tubes(1.sterilized milk; 2.sterilized milk; 3.indirect UHT; 4.Direct UHT; 5.Raw milk)

 Discussion

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 As the picture of the result shows, more heat treatment cause less turbid solution. The two
sterilized milk samples are most transparent. The direct UHT is slightly more turbid than the
indirect UHT, though it should be the other way, since the direct UHT is a more intense heat
treatment.

3.4 The non enzymatic browning – 5-hydroximethyl-furaldehyde (HMF)

quantification

 Principle
The HMF quantification method can be used to determinate the used heat treatment on milk
Non enzymatic browning, Maillard reaction, results an intermediate product, the HMF which
can react with thiobarbituric acid (TBA) and form a yellow compound. The product can
quantified by spectrophotometer on 443nm wavelength. The absorbance is related to the
degree on the Maillard reaction. The sample has to be heat treated and acidified transform all
1-amino-2-deoxy-2-ketose (other intermediate product of non-enzymatic browning reaction)
into HMF. Trichloroacetic acid is used to precipitate the proteins in the sample to release the
bound HMF molecules.

 Calculations and results

The determination of the HMF content was made by the help of the standard curve (figure X.).
The calibration curve shows a satisfactory linear regression (R² = 0.98). According to the
trendline and with our sample’s absorbance (A=0.0585), we can determinate the HMF content
of the samples.

0.4
Standard curve of HMF
y = 8.4286x + 0.0069
R² = 0.9842
0.3
Absorbance Samples
Absorbance

0.2

0.1

3. figure: The standars curve of HMF

0
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
HMF concentration (mg/10mL)
4. figure

1. Table

Heat HMF Turbidity

Group Absorbance
treatment concentration(mg/10mL) test
Sterilized
1 0,1727 0,0197 +
milk
Sterilized
2 0,1664 0,0189 +
milk
Indirect
3 0,141 0,0159 ++
UHT
4 Direct UHT 0,0585 0,0061 ++
5 Raw milk 0,0185 0,0014 +++

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  Discussion
With the help of the standard curve’s linear equations we could determinate the concentration
of the HMF. The results are correlating with the turbidity test, and again, the opposite result
can be obtained about the two UHT treatments.
3.5 References

4 OILS

4.1 FAME

4.2 IV-value

4.3 Acid value and FFA-content

4.4 Polar compounds

4.5 PO-value

4.6 P-anisidine value

5 VITAMIN C

5.1 Peroxidase

 Principle:
To determinate the optimal blanching time, peroxidase
test can be used. Peroxidase is an indicator enzyme: if it
is still active, it converts the hydrogen peroxide to oxygen
and water, and as a result the oxygen can react with the
added guayacol and form a brown coloured component.
After a proper blanching of leek, enzymes are deactivated
and the brown colour will not appear on the surface of the
leaks. Different blanching times were tested and the
optimal time was determinate as the shortest blanching
time, after which the brown colour was not appearing.

 Results
Blanching time (s) Colour change in 3 minutes
45 yes
60 yes
90 yes
120 no Figure: The result of blanching
after 90 s.
 Conclusions:
The optimal blanching time was a bit difference for the white and for the green leaks, it seems
that white leaks could contain more peroxidase enzymes since the white ones become brown

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 even after one and a half minutes, but for the greens the optimal blanching time was less than
one minute.

5.2 Enzymatic browning

 Principle
During the processing of the fruits and vegetables when they are peeled or cutted, enzymes
will be released from the cells. If oxygen is present polyphenol oxidase catalyses the
conversion of phenolic compounds and results melanin. The speed of this reaction is
influenced by the temperature, the amount of oxygen present, and the pH as well. Certain
treatments can slow down the browning reaction, which is often used in the industry. The
purpose of this experiment is to examine the differences of the impact of the applied test
solutions.
Ascorbic acid lowers the pH and has an antioxidant activity. Potassium bisulphite (considered
as an allergen compound) and L-cysteine has a reducing activity due to the presence of
sulphur. Ethylenediamine-tetraacetic acid (EDTA) can bound the metal ions (iron and copper)
originate from the knife, which can also catalyse the enzymatic browning. Water acts as an
oxygen barrier and reduces the amount of oxygen present on the surface of the vegetables or
fruits. Honey also has an antioxidant activity, it has from 56.32 to 246.21 mg/100g honey as
Catechin equivalent by the Folin-Ciocalteu method (Al-Mamary et al. 2002).
To evaluate the impact of the test solutions, two slice of apple was placed in these solutions
for one hour and after that they were placed on a paper towel subjected to the air. The
changes in the colour were observed.

 Results

Figure: Apple slices trated with:

1) 0.1%Ascorbic acid 2) 100ppm Potassium bisulphite 3) 200ppm EDTA 4) 4mM Cysteine 5) Honey 6) Water

 Discussion
After 45 minutes on the open air, small changes could be observed of the surface of the apple
slices. The colour did not changed of most of the apples, only with EDTA and water treated
slices started browning a little, however after 30 minutes all of them remained the original
colour. Probably we could observe more changes after longer time. According to these results
we can state that the EDTA and the water are not enough strong solutions to avoid the
enzymatic browning.

5.3 Vitamin C

6 REFERENCES:

Al-Mamary, M., Al-Meeri, A. & Al-Habori, M., 2002. Antioxidant activities and total phenolics of different
types of honey. Nutrition Research, 22(9), pp.1041–1047. Available at:

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