MANUFACTURE OF CASEIN AND CASEINATES

Leonardo M. Calixto
FDST 429-151/Laboratory Report #1/January 20, 2015

1. Introduction
Approximately 3,5% of milk is composed by proteins, 80% of this protein is
represented by casein. Casein is defined as the protein precipitating from milk near
pH 4.6. It thus is not soluble at its isoelectric pH. Casein has several industrial
applications, either for food products or for no-food products.
This experiment had as objective to observe the effect of chymosin and lactic
acid upon casein regarding coagulation and separation.
2. Procedures
The procedure followed were in accordance with the handout available in the
course section in Blackboard. In addition to the instructions of the original procedure,
samples A and B were heated, after 40 minutes set undisturbed, to approximately 30
C.
3. Results
3.1. Sample A
After setting undisturbed for 40 minutes, sample A did not show signs of curd
or gel formation. However, after heating it to 31 C there was formation of a thick gel
at the bottom of the beaker. That happened because at room temperature the
enzyme (chymosin) did not had good proteolytic activity, different from what occurred
at 31 C, which is closer to the optimum temperature for proteolytic action of
chymosin.
3.2.

Sample B

After 3 minutes, undisturbed, sample B showed signs of curds. After 10

minutes, additionally to the curds there was formation of a slightly firm gel but still
easy to penetrate with the spatula. After 20 minutes, there were signs of phase
separation in the milk and the formation of a more viscous gel at the bottom of the
beaker. After 30 minutes, there was no more gel. After 40 minutes, the viscosity of
the solution was higher if compared to the original viscosity of milk; however, there
was no firm gel.
After heating sample B, there were more curds than in the not heated sample,
however there was no presence of gel. Even though temperature do not affect the
lactic acid in sample B, the increase of temperature still improved the coagulant
action of chymosin.
3.3.

Sample C

Table 1 includes the data relative to the first and to the second titration.

Table 1: pH recorded as a function of the acid added for both titrations.

First Titration
pH Lactic Acid (mmol)
6.79
0
6.40
1.0
5.98
2.0
5.69
3.0
5.46
4.0
5.23
5.0
5.09
6.0
4.88
7.0
4.79
7.5
4.71
8.0
4.67
8.3
4.65
8.5
4.57
9.0
4.53
9.4
4.50
9.6

Second Titration
pH Lactic Acid (mmol)
6.79
0
6.12
1.0
5.83
1.5
5.54
2.0
5.34
2.5
5.12
3.0
4.89
3.5
4.71
4.0
4.58
4.5
4.48
4.9

Using the data from the table above the following graph was plotted.

Figure 1: Titration curves

The reduction of pH to around 4,5, in both titrations, enabled the casein to
precipitate in the form of acid casein. That happened because casein is no longer
soluble in milk at pH near 4,6 (isoelectric point).
The density of milk at 20 C is 1,030 g/ml; therefore, 100 ml of milk corresponds
to 103 g of milk. Knowing that the yield can be calculated as the ratio between the

mass of casein obtained and the mass of milk, and that casein is equivalent to 2,86%
of the mass of milk, it was possible to calculate the theoretical yield of casein, as well
as, the yields on wet basis and dry basis. The results are shown in the table 2.
Table 2: Yield of Casein
Casein (g) Yield (%)
Wet Basis
19,34
18,78
Dry Basis
4,68
4,54
Theoretica
l
2,95
2,86
The difference between the theoretical yield and the dry yield indicates that there
was extraction of almost 59% casein over what was possible. Among the possible
explanations to what caused such discrepancy is the possibility of other components
of milk also have precipitated (other proteins included) or even residual water left in
the casein.
4. Study Questions
4.1. How did acidifying the milk (step 3) affect the chymosin coagulation time or
firmness?
The addition of acid in sample B resulted in quicker formation of curds. This is a
direct result of the combined action of the lactic acid and chymosin. Chymosin is an
enzyme naturally encountered in the stomach of ruminants therefore is more active in
an acid environment.
The acid will denature caseins, in other words, the micelle of casein will unfold,
beginning to link into longer and insoluble chains. The decrease in pH will help
activate chymosin that will attack and break some of the protein bonds, i.e., the
combined action of lactic acid and chymosin will precipitate casein forming curds.
(Baxter, 1995)
4.2.

Was there a difference between the first and second titration curves? If so
why?

Yes, the first titration required more addition of lactic acid to reach the desired
pH (4,5) when compared to the second titration. This occurred because the first
titration was performed in milk; meanwhile, the second was performed in casein
solubilized in NaOH. The buffering capacity of milk was responsible for the difference
in quantity of acid used in both titrations.
According to Walstra et al (2006) the quantity of acid needed to produce a
given decrease in pH depends on the buffering capacity of the milk, and hence on its
composition. Buffering primarily is due to protein and salts. Therefore, without the
other milk components to act as buffers the second titration was faster than the first
one.
4.3.

What types of products (food and non-food) are made using caseins?

In food products, casein is mainly used as an ingredient either to modify

physical properties of food or to provide nutritional complementation. The food
products made with casein as an ingredient include bakery, cheese products, coffee
whiteners and creamers, confectionery, cultured milk products, high fat powders,
shortenings and spreads, ice cream and frozen desserts, infant foods, instant
breakfasts and beverages, meat products, nutritional food bars, pasta
pharmaceuticals, soups and gravies, sports drinks, whipped toppings.
As for non-food products that uses casein in its production, some examples
are adhesive for wood, adhesive for foil laminates and paper, coatings for paper and
cardboard, horticultural spreaders, joint cements in wallboard, leather tanning, paints,
plastics in the form of buttons, buckles, imitation tortoiseshell (combs and hairclips),
imitation ivory (knife handles and piano keys), fountain pen barrels, shoehorns,
dominoes.
4.4.

Most casein is actually marketed in the form of sodium caseinate. What

steps would be necessary to convert acid casein (our final product) into
sodium caseinate?

To convert acid casein into protein is necessary to dissolute it in sodium

hydroxide and, in sequence, dry it, for example, in a spray dryer.
5. Conclusions
Under the right conditions, chymosin can easily attack K-casein, resulting in
the precipitation of casein. If combined with lactic acid, chymosin will have better
results in the formation of curds due to the acidification of milk, which provides better
conditions for the proteolytic action of this enzyme.
Acidification of milk to the isoelectric pH of casein will cause the precipitation
of such proteins. This principle is used in the manufacture of caseinates; in this
process, after obtaining acid casein, it is solubilized in an alkaline mean and then
subjected to a drying process.
This shows how the knowledge of milk chemistry and composition can be
applied in the dairy industry to produce diversified products, reducing waste.
6. References
Baxter, R. 1995. Say Cheese. ChemMatters. February Issue.
Walstra, P.; Wouters, J.T.M.; Geuters, T.J. 2006. Dairy Science and Technology. CRC
Press. 2nd Edition. 763 p.