Experiment 3 : Milk

Aim
1. To study the effects of acid on milk protein coagulation.
2. To determine the effect of salt on milk protein coagulation.
3. To examine changes in colour and flavour of milk during heating.

Introduction
Milk is an essential component of the diet of ~6 billion people. The world production of milk
reaches 730 million tons/y (1, 2). Even though mammals produce milk to feed their offspring, in
many areas of the world humans continue to consume milk throughout their life. However, it
must be emphasized that lactose intolerance is widespread throughout the world and that a large
proportion of the world's population would not benefit from the putative benefits of milk.
In addition to milk, several dairy products such as cream, butter, yogurt, kefir, and cheese have
been produced and consumed worldwide for millennia. Therefore, the impact of milk and dairy
products on human health is quantitatively relevant and has been the subject of several
investigations, on both whole products and their isolated components. In particular, the fat
portion of milk (largely composed of SFAs) and some of its minor components, notably calcium
and oligosaccharides, are being actively researched for their potential health roles. (Visioli and
Strata, 2014)

In milk, the casein fractions associate with each other and with colloidal calcium phosphate to
form stable spherical structures known as casein micelles. The second protein fraction of milk is
the whey or serum. It makes up approximately 20% of milk protein and includes the
lactalbumins and lactoglobulins. Whey proteins are more hydrated than casein and are denatured
and precipitated by heat rather than by acid. Other protein components of milk include enzymes
such as lipase, protease, and alkaline phosphatase, which hydrolyze triglycerides, proteins, and
phosphate esters, respectively. Caseins in normal bovine milk form a self-assembled structure
together with micellar calcium phosphate. The α- and β-caseins are present in the interior and
throughout the structure, whereas the κ-caseins are mostly found on the surface of the micelles.
To some extent, the casein micelles (CMs) are considered as hard spheres protected by κ-casein
hairy layers. The CMs are extremely stable in normal conditions of milk. Moderate heating or
cooling does not cause the aggregation or the disruption of their internal structure. However, a
change in milk environmental conditions, such as the alteration of the pH of the milk, would
easily destabilize the micellar integrity. Hence, changing the milk pH by increasing the pH above
or lowering the pH below that of natural milk pH, may also lead to a change in CM size. (Sinaga,
Bansal and Bhandari, 2016). Chymosin also called rennet, which is a milk clotting enzyme
originally obtained from calf stomach. A protein subunit within the casein keeps the micelles
separated. When milk is exposed to rennin, this protein subunit is removed from the casein
micelles and they react with the free calcium ions. This reaction enables the micelles to clump
together, helping to form curd. The process of casein clumping, or coagulation is also called
curdling (Edelstein, 2014).
Alternatively, pH can be lowered to the isoelectric point for casein which also makes the caseins
coagulate. This process is known as acid coagulation. The factors affecting the milk protein
coagulation include the application of heat and the addition of acid, enzymes, polyphenolic
compounds, and salts (Brown, 2008). The increased acidity causes the micelles to lose their
negative charge and stop repelling one another therefore results in curdling (Edelstein, 2014).
The effect of salt on curdling is attributed to the fact that the ions of sodium and chloride from
the salt interact with the electrical charges on the surface of the milk proteins. (Sikan et al.,
2013). When the normally repulsive charges on the protein molecules are canceled by the ions
from the salt, the protein molecules can form aggregates as hydrogen bonds link them together,
causing coagulation.
Procedures
1. Effect of acid in milk protein coagulation
Materials
UHT full cream milk (40ml), vinegar (20ml), pH meter, distilled water (for rinsing pH meter
probe), glass rod (x1)

Procedure:
1. The pH and the initial appearance of the 40ml of milk contained within the 100ml beaker
is recorded.
2. The pH of vinegar in another beaker is recorded. The vinegar is then added into the milk
and stirred until well mixed.
3. The pH of the mixture is recorded.
4. The mixture is allowed to stand for 30 minutes, observations are recorded.

2.Effect of salt on milk protein coagulation

Materials
UHT full cream milk (40ml) (x2), sodium chloride (NaCl) (10g) , magnetic stirring hot plate, pH
meter, distilled water (for rinsing pH meter probe), glass rod (x1), time (x1)

Procedure:
1. 10g of sodium chloride is added into the 40ml milk. The mixture is stirred well with a
glass rod until the salt is completely dissolved. The pH and the appearance of the milk is
recorded. The milk is heated with the hot plate (medium heat No.5) until it boils. The
milk is boiled for 5 minutes before removing it from the heat.
2. The pH of the milk is checked after it has cooled to room temperature. Observations are
recorded.
3. Steps 1-3 are repeated using another 40ml of milk without the addition of sodium
chloride.
3.Changes of milk during heating
Materials
Sweetened condensed milk (-40ml), universal bottles (10ml, x4), test tube rack, timer, water
bath, pH meter, distilled water, glass pipette, colourflex spectrophotometer

1. 2ml of sweetened condensed milk is diluted into a universal bottle with 4ml of water
using a glass pipette. The initial pH of the diluted milk is recorded and the cap of the
sample is labelled.
2. 10ml of sweetened condensed milk is pipetted into a universal bottle. The bottle is then
closed with a lid. The initial pH of the sample is recorded and the cap of the sample is
labelled.
3. The colour and the flavour of the sample is recorded. The colour of the milk is measured
using a Colourflex spectrophotometer. The is conducted by filling the sample cup with ¼
of sample and measure the L*, a*, and b* readings. The flavour of the milk is determined
by sniffing it.
4. All the universal bottles are places on a rack and put in a 90 water bath.
5. The samples are take out of the water bath after 90 minutes, the changes in pH, colour
and flavour are checked when the sample is cooled to room temperature. The pH, colour
and flavour of the samples are recorded.
Results
Table 1.1 shows the results of various pH of milk and observation
pH Observation
Milk 6.85 White
Vinegar 1.87 Translucent
Milk + Vinegar 3.57 After 30 min, curd form
Milk + Salt (heated) 5.78 More precipitate formed,
yellowish , forms 2 layer
Milk (heated) 6.95 Less precipitate form, whitish

Table 1.2 shows the results of diluted and undiluted milk

Colour Flavour
L a b
Condense milk (diluted) 7.11 82.10 -2.34 11.55 Less sweet, caramel
Condense milk (undiluted) 6.93 74.06 -2.34 21.06 Sweet , caramel
Condense milk ( heated + diluted) 7.08 79.88 -1.56 13.34 Cooked flavour
Condense milk ( heated + 6.76 66.96 0.19 23.96 Cooked flavour
undiluted )

Discussion
In part 1 the pH of milk was 6.85 and the pH of the vinegar was pH1.87. After vinegar was
added to milk the pH of the milk is now 3.57. This is due to the increase of H+ from the vinegar
lowering the total pH of the milk. After 30 minutes there was a curd formed. This is because
milk contains proteins such as casein and its isoelectric point is at pH4.6. Thus when vinegar is
added to milk, pH of the milk is lower than the isoelectric point of the casein and this causes the
casein micelles to coagulate. The increased acidity causes the micelles to lose their negative
charge and stop repelling one another which results in curdling (Edelstein, 2014). It is because it
has a quantity of positive charges equal to the quantity of negative charges and the positive part
of each "micelle" is attracted by the negative part of the others so that the protein precipitates in
the form of demineralized casein and in the solution remain soluble calcium salts.
In part 2, after NaCl was added, the pH of the milk drop slightly to 5.78. The colour of the milk
becomes yellowish. The addition of NaCl causes milk to coagulate because the ions of sodium
and chloride from the salt interact with the electrical charges on the surface of the milk proteins.
When the normally repulsive charges on the protein molecules are canceled by the ions from the
salt, the protein molecules can form aggregates as hydrogen bonds link them together, causing
coagulation. The increase of temperature during heating, increase the kinetic energy lead to the
increasing frequency of collision and aggregation of casein micelles. The heated milk also
slightly forms precipitate because the denaturation of whey proteins forms soft curd during
heating. The white colour of milk is due to the casein micelle scatter light (Huppertz and Fox,
2006). The colour of milk with sodium chloride turns yellow because the decrease in casein
micelle

In part 3, the pH of diluted condense milk was 7.11 while undiluted condense milk was
6.93.Using the colourflex spectrophotometer, the L* determined was 82.10, a* -2.51 and b*
11.55 , while after heating it changes to 79.88,-1.56 and 13.34. For undiluted samples before
heating was 74.06, -2.36 and 21.06 and after heating it was 66.96, 0.19 and 23.96. Both of the
samples have a decrease in L* value which indicates both samples becomes darker.While for a*
value both sample have become less negative which indicate both samples becomes more red.
For b* value , both increases in value which means both samples becomes more yellow. Both of
the samples have a sweet milky smell before heating and cooked flavour after heating. This is
due to the Maillard browning occurred. Maillard reaction occurs between reducing sugar, amino
group in the whey protein during heating. The condensation of reducing sugar and amino acids
forms the browning compound, melanoidins that causes the colour changes in milk (Van Boekel,
1998). Denaturation of the whey protein also changes the flavour of the condense milk. Upon
prolonged heating, α-lactoglobulin and β-lactalbumin are denatured and precipitated. Denatured
proteins expose large number of sulfhydrl (-SH) groups, contributing to the flavours.

Factors affecting the experiment is when measuring pH of each condense milk, the probe might
not be cleaned thoroughly before immersing the probe to another solution. This could cause a
contamination of the solution and inaccuracy of the results. Next is the usage of colour
spectrophotometer, the sample might not fully cover the bottom of the sample cup.This causes
the results obtained to be in accurate. In addition, the number of readings of the colourflex
spectrophotometer was not taken 3 times but only taken once. If 3 readings was taken, this could
increases the chances of obtaining a more accurate results. Colourflex Spectrophotometry is a
widely used method in as the instrument is commontly available , simplicity of procedures as
well as the accuracy of the results. Thus this is a suitable method to conduct this experiment.
Discussion Questions
1.What effect did the vinegar have on the milk? Explain your answer
Adding vinegar to milk will decrease the existing ph of the milk. When pH decreases, the H+
ions increases and this will neutralize the negative charged on the casein micelle, making them
neutral.Thus there is no force to repell each other and separate them apart. They will then hit
each other and stick together, making a tangled mess of protein called curds. (Sinaga, Bansal and
Bhandari, 2016)

2.Explain the curdling process that may happen when salt is heated with milk
When milk is heated with salt, curdling process is speed up. This is because salt will reduce the
net-negative charge on the casein micelles and milk pH. pH of the solution will be lowered
because salt will lower the pH up to its isoelectric point which is pH 4.6 and this process is
known as salting out. The effect of salt on curdling is attributed to the fact that the ions of
sodium and chloride from the salt interact with the electrical charges on the surface of the milk
proteins.(Sikand et al., 2013). The normally repulsive charges on the protein molecules are
canceled by the ions from the salt causing the casein micelle is less stable and favours
coagulation of milk proteins.

3.Give one example of dairy product that is important to have curd formation
One example of dairy product that is important to have curd formation is cheese.
Part 2
1.What are the changes in terms of pH, colour and flavour of heated sweeteaned condense milk.
Explain ur answer.
The pH for diluted condense milk and undiluted condense milk both decreases from 7.11 to 7.08
and 6.93 to 6.76 respectively.The colour of both becomes more yellow from white yellow after
heating. The flavour of undiluted and diluted condense milk becomes cooked flavour from sweet
caramel flavour. This is because during heating denaturation of whey proteins expose the
sulfhydryl group. Its sulfhydryl (-SH) group are very active and liberate volatile sulphites include
H2S which mainly causes the cooked flavour of milk . The pH of the milk slightly decreases due
to the ionization in water increases and increase the amount of H+ and during heating, hydrolysis
of k-casein and dephosphorylation of αs1-, αs2 - and β-caseins. It also induces the precipitation
of primary and secondary calcium phosphate with the release of H+ that gives a result in
decreasing in the pH of milk.After determining the change of colour using the colourflex
spectrophotometer, the colour of the dilute and undiluted condense milk becomes darker and
more red due to decrease of L* value and increase in a* value. This is due to the Maillard
reaction happening between reducing sugar lactose, amino group in milk during heating. . The
condensation of reducing sugar and amino acids forms the browning compound, melanoidins
that causes the colour changes in milk (Van Boekel, 1998).

2.Name a compound that may attribute to the cooked flavour of heated milk
The cooked flavour of heated milk is due to Sulphur compound (H2S). During heating whey
proteins will denature. Denatured proteins will release Sulphur compounds that contributes to off
flavour. (Brown, 2008).
Conclusion
The addition of salt into milk favours the denaturation and coagulation of milk proteins. In milk,
casein associates with each other and with colloidal calcium phosphate to form casein micelles.
The addition of salt into milk favours the denaturation and coagulation of milk proteins. The
increased acidity causes the micelles to lose their negative charge and stop repelling one another
which results in curdling. Next, the salt ions interact with electrical charges on the surface of
milk proteins, resulting in coagulation. Heat will cause curdling if there is the present of salt or
acid. After a long duration of heating, the whey protein is denatured, releasing the sulfur
compounds which contribute to the flavours. Maillard reaction also has taken part during
heating. The colour changes of the milk, which is the brown pigment is caused due to the
prolonged heating in the final stage Maillard reaction.
References
Brown, A. (2008). Understanding Food: Principles and Preparation (pp. 200-201). Hawaii:
Thomson.
Edelstein, S. (2014). Food science (pp. 221-222). Burlington, MA: Jones & Bartlett Learning.

Huppertz, T. and Fox, P.F., 2006. Effect of NaCl on some physico-chemical properties of
concentrated bovine milk. International dairy journal, 16(10), pp.1142-1148

Sikand, V., Tong, P.S. and Walker, J., 2013. Effect of adding salt during the diafiltration step of
milk protein concentrate powder manufacture on mineral and soluble protein composition. Dairy
Science & Technology, 93(4-5), pp.401-413.

Sinaga, H., Bansal, N. and Bhandari, B. (2016). Effects of milk pH alteration on casein micelle
size and gelation properties of milk. International Journal of Food Properties, 20(1), pp.179-
197.
Van Boekel, M.A.J.S., 1998. Effect of heating on Maillard reactions in milk. Food
Chemistry, 62(4), pp.403-414.
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