ANALYSIS OF SUGAR AND PROTEIN IN MILK

Session date: 16.08.2019 Name: Sangarange Nirupuli Nishadi Perera

Submission date:23.08.2019 Partner:
Group: 20 Demonstrator: Paula De Thomas

Aim:
To calculate the concentration of Protein, contain in the skim milk and Whey
quantitatively by using Lowry method and analyse concentration of reducing sugar
content in whey by using Somogyi Nielson method.

Method:
Referred Clinical Bio Chemistry practical manual; Semester 02 – 2019, Session 02,
Page 46-49

Results:

Part C (i) Estimation of protein in skim milk and whey

Table 1:________________________________________________________ .

Tube 1 2 3 4 5 6 7 8
BSA (200 0 0 0.1 0.1 0.2 0.2 0.3 0.3
µg/mL),
H2O, mLmL 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2
Protein 0 0 40 40 80 80 120 120
(µg/ml)

Diluted skim - - - - - - - -
milk, mLwhey,
Diluted - - - - - - - -
mL
Absorbance 0.0588 0.0604 0.1707 0.1768 0.2972 0.289 0.3814 0.3893
(690 nm)
Absorbance - -.0008 0.0009 0.1112 0.1173 0.2377 0.2295 0.3219 0.3298
blank
Concentration 0 0 40 40 80 80 120 120
protein in
sample(µg/ml)
 Tube 9 10 11 12 13 14 15 16
BSA (200 0.4 0.4 0.5 0.5 0 0 0 0
µg/mL),
H2O, mLmL 0.1 0.1 0 0 0 0 0 0
[Protein] µg 160 160 200 200 - - - -
/mL
Diluted skim - - - - 0.5 0.5 - -
milk, mLwhey,
Diluted - - - - - - 0.5 0.5
mL
Absorbance 0.4826 0.4872 0.5977 0.5766 0.2557 0.2641 0.3526 0.3525
(690 nm)
Absorbance - 0.4231 0.4277 0.5382 0.5171 0.1962 0.2046 0.2931 0.293
blank

Concentration 160 160 200 200 67 71 102 103

of protein in
sample
(µg/mL)

 Plot the standard curve for BSA (tubes 1 - 12). Include all headings and axis labels,
and attach the graph to this proforma.

0.6

0.5

0.4
Absorbance ( 690nm)

Unknown - 1
0.3
Unknown - 2

0.2
Unknown - 3

Unknown - 4
0.1

0
0 50 100 150 200
Conentration of Protein (µg/ml)
Figure 01 : Standard Curve for Determination of Protein in Skim Milk and Whey by using
Lowry Method
Q1. Read off the concentration of protein (µg /mL) in the skim milk and whey (non-casein
protein) assay samples.
In the graph label tube 15 and 16 as unknown 3 and 4 respectively.

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑇𝑢𝑏𝑒 15 + 𝐶𝑜𝑛𝑐𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 16

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜 𝑜𝑓 𝑊ℎ𝑒𝑦 ( 𝜇𝑔/𝑚𝑙) =
2

(102 + 103)⁄
= 2 μg/ml

=102.5 μg/ml

In the graph label tube 13 and 14 as unknown 1 and 2 respectively.

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑇𝑢𝑏𝑒 13 + 𝐶𝑜𝑛𝑐𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 14

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜 𝑜𝑓 𝑆𝑘𝑖𝑚 𝑀𝑖𝑙𝑘 ( 𝜇𝑔/𝑚𝑙) =
2
67 + 71
= μg/ml
2
= 69 μg/ml

Q2. Calculate the concentration (g/100 mL) of total protein in the original skim milk and
whey (non-casein protein) samples. [Hint: remember that the skim milk underwent a
dilution in the process of making the whey. Also the untreated skim milk was diluted
for the assay].

𝜇𝑔 1𝑚𝑔 1𝑔
[𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝑔/100 𝑚𝑙)] = × × × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

69 𝜇𝑔 1𝑚𝑔 1𝑔
= × × × 400 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

= 2.76 g /100 ml

𝜇𝑔 1𝑚𝑔 1𝑔
[𝑊ℎ𝑒𝑦 (𝑔/100 𝑚𝑙)] = × × × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

102.5𝜇𝑔 1𝑚𝑔 1𝑔
= × × × 60 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

= 0.615 g/100 ml
 Skim milk Whey
Total Protein in original Skim milk Total Protein in Whey
2.76 g /100 ml 0.615 g/100 ml

Q3. Calculate the casein content as a percentage of the total protein in milk.

[𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 ] = [𝑊ℎ𝑒𝑦 (𝑛𝑚 𝐶𝑎𝑠𝑖𝑒𝑚)] + [ 𝐶𝑎𝑠𝑒𝑖𝑛]

[𝐶𝑎𝑠𝑒𝑖𝑛] = [𝑡𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛)] − [𝑤ℎ𝑒𝑦(𝑛𝑚 𝑐𝑎𝑠𝑒𝑖𝑛)]

[𝐶𝑎𝑠𝑒𝑖𝑛] = 2.76 𝑔 − 0.615 𝑔

= 2.145 𝑔/100 𝑚𝑙

𝐶𝑎𝑠𝑖𝑒𝑛
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑐𝑎𝑠𝑒𝑖𝑛 = × 100
𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛

𝑐𝑎𝑠𝑒𝑖𝑛 % = ( 2.145⁄2.76) × 100

= 77.7 %
Part C (ii) Estimation of reducing sugar in whey
Table 2:_____________________________________________________________

Tube 1 2 3 4 5 6 7
Lactose 200 (µg/mL), 0 0 0.1 0.1 0.2 0.2 0.3
mL
H2O, mL 0.5 0.5 0.4 0.4 0.3 0.3 0.2
[Lactose] µg /mL 0 0 40 40 80 80 120
Diluted whey, mL - - - - - - -
Absorbance (690 nm) 0.044 0.043 0.197 0.163 0.328 0.305 0.421
Absorbance - blank .0005 .0005 0.1535 0.1195 0.2845 0.2615 0.3775
Concentration of 0 0 40 40 80 80 120
reducing sugar in
sample (µg/mL)

Tube 8 9 10 11 12 13 14
Lactose 200 (µg/mL), mL 0.3 0.4 0.4 0.5 0.5 - -
H2O, mL 0.2 0.1 0.1 0 0 - -
[Lactose] µg /mL 120 160 160 200 200 - -
 Absorbance (690 nm) 0.430 0.522 0.533 0.553 0.407 0.396 0.477

Absorbance - blank 0.3865 0.4785 0.4895 0.5095 0.3635 0.3525 0.4335

Concentration of 120 160 160 200 200 110 135

reducing sugar in sample
(µg/mL)

• Plot the standard curve for lactose (tubes 1 - 12). Include all headings and axis labels,
and attach the graph to this proforma.

0.6

0.5

0.4
Absorbance (690nm)

Unknown - 1
0.3 Unknown - 2

0.2

0.1

0
0 50 100 150 200
Concentration of Reducing Sugar (µg/ml)
Figure 02 : Standard Curve for Determination of Reducing Sugar in Whey by using Somogyi -
Neilson Assay

Q4. Read-off the concentration of reducing sugar in the skim milk assay sample.
In the graph tube 13 and 14 label as unknown 1 and unknown 2 respectively.

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑜𝑓𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔𝑆𝑢𝑔𝑎𝑟(𝜇𝑔/𝑚𝑙)
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑇𝑢𝑏𝑒 13 + 𝐶𝑜𝑛𝑐𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 14
=
2
110(𝜇𝑔/𝑚𝑙) + 135(𝜇𝑔/𝑚𝑙)
=
2
= 122.5 𝜇𝑔/𝑚𝑙
Q5. Calculate the concentration (g/100 mL) of reducing sugar in the original skim milk
sample. [Hint: remember that the skim milk underwent a dilution in the process of
making the whey].

[𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑆𝑢𝑔𝑎𝑟 (𝑔/100 𝑚𝑙)]

𝜇𝑔 1𝑚𝑔 1𝑔
= × × × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

122.5 𝜇𝑔 1𝑚𝑔 1𝑔
= × × × 360 × 100 𝑚𝑙
𝑚𝑙 1000 𝜇𝑔 1000 𝑚𝑔

=4.41 g/100 ml

Q6. How do your results compare with those in Table 1, page 14?
Ans:
According to table 01(practical manual, page 43) the average Protein per 100 ml milk
is 3 - 4 g, and from 80 % which is casein. Also, average concentration sugar per 100
ml milk is 4 – 5g which is lactose.
According to my calculated results, samples contained 2.76 g /100ml of protein in the
skim milk and from that 77.7% is casein. Obtained values are slightly lower the
expected range. But for reducing sugar it gave the value 4.41g/100ml which is within
the considerable reference range given in the table 1.

Q7. Comment on the electrical charge and solubility of casein at its isoelectric point (pI =
4.7). Suggest how this may be associated with the souring of milk.
Ans
Casein is a protein it has many positive and negative electric charges. But, casein at
its isoelectric point 4.7 (at optimum pH 6.6) carry no net electric charge. Due to the
bacterial action on carbohydrate (lactose sugar), there is chance to change the pH
value of the milk (mostly tend to get the acidic, low pH).it will lead the change of the
isoelectric point of the milk and get imbalanced electrical charge. This will lead to form
lactic acid in milk and eventually it will lead to souring of milk.

Conclusion
In conclusion, the concentration of the skim milk 69 μg/ml and102.5 μg/ml was calculated by
performing Lowry assay. Also the reducing sugar concentration was 122.5 μg/ml was
calculated by using Somogyi – Nelson Assay. Calculated values were found to be less than
actual value given in the table (for protein)this might be happen due to the many reasons,
such as pipetting errors, samples not properly mix whiling diluting the samples.