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Modification of Bovine β-Casein to Improve the Characteristics and Manufacturing Properties of Cow's Milk
by Gregory T. Bleck, Byung-Kwon Choi, Matthew B. Wheeler and Rafael Jiménez-Flores


Through the use of genetic engineering it has become possible to consider making substantial changes in the composition of milk utilizing transgenic animals. In this report we discuss the development of DNA vectors that will cause the production of modified versions of bovine ß-casein in the milk of transgenic mammals. We have produced and are continuing to make transgenic mice that contain these DNA vectors. These mice will serve as useful models to study the potential usefulness of these ß-casein modifications in the improvement of cow's milk.


Using genetic engineering it has become possible to modify proteins and then have them secreted into the milk of a transgenic mammal. For example, it has been shown that it is possible to express a wide variety of different proteins in the milk of mice, rabbits, pigs, goats, sheep and cows. Most of this work has been performed using proteins of pharmaceutical interest. In our studies, we have chosen to express proteins in milk of transgenic animals that have the potential to improve the quality of milk and be beneficial to the dairy industry.

The most common way of making transgenic animals is by microinjection of DNA encoding a gene of interest into the male pronucleus of a fertilized egg. The fertilized eggs are obtained using normal embryo transfer techniques. The genes are injected at the one cell stage of embryo development, and the genes become incorporated into all cells of the individual, including the germ cells, and are thus capable of being transmitted to one-half the offspring of that animal.

The genes encoding these foreign proteins are transmitted through normal Mendelian inheritance and the animals produce the proteins only in their milk during lactation. These new genes can be propagated in the dairy cattle population by use of embryo transfer, artificial insemination, in vitro fertilization and cloning.


Six proteins produced by the cow's mammary gland during lactation represent over 90% of the total protein produced in milk. These are the four caseins (as1, as2, ß and k) and the two major whey proteins, ß-lactoglobulin and a-lactalbumin.

Table 1: Approximate average milk composition of cattle (Eigel et al., 1984)

  Concentration or percentage in milk
Milk component Production level
Fat 3.7%
Lactose 4.6%
Protein 3.1%
Total Casein 27.3 g/l
as1-Casein 10 g/l
as2-Casein 3.4 g/l
ß-Casein 10 g/l
k-Casein 3.9 g/l
a-Lactalbumin 1.2 g/l
ß-Lactoglobulin 3.0 g/l

The caseins combine with calcium to form micellar structures that remain soluble in milk and are of functional importance in cheese making. Caseins form the curd in cheese and the amount of casein present in milk determines the cheese yield of the milk. The caseins have an open structure and are hydrated despite the high content of hydrophobic amino acids present in the molecule. These hydrophobic surfaces on the molecule are important in casein-casein interactions that are partially responsible for the high viscosity of casein solutions and for its foaming and emulsification properties.

It is the structure of the individual caseins, as well as their interaction with each other and with other constituents of milk such as calcium and whey proteins that makes this system of great interest to the food scientist. Although there has been considerable research on the biophysics and biochemistry of milk and milk proteins, the system is not completely understood. Biotechnology and genetic engineering offers the tool to systematically study the structure/function relationship of these proteins and how they can be manipulated to benefit the dairy industry.


The proposed modifications described in this section have been designed to improve the functional properties of milk during processing and improve the quality of milk protein. Bovine ß-Casein is 209 amino acids in length and is rich in the amino acid proline. It contains 5 phosphorylated serines clustered in the first 35 amino acids at the N-terminal end of the molecule and the rest of the protein is very hydrophobic. This hydrophobic nature of ß-casein has been correlated with excellent emulsifying, foaming and gelling characteristics in manufacturing processes.

One modification that we have made to ß-casein is the deletion of a plasmin cleavage site. Plasmin is one of the major milk proteases that is found at varying levels in the milk of all cows. The alteration made to the ß-casein molecule prevents plasmin from cutting it to form two smaller molecules, that have different properties than intact ß-casein. One of the peptides produced by this cleavage causes a bitter flavor in cheese.

The second alteration of ß-casein was the removal of the cleavage site for chymosin. Chymosin is the enzyme used in cheese making to start the formation of the curd. It cleaves a portion of the k-casein molecule exposing the inner contents of the casein micelle to the solution, thus causing precipitation of the a and ß-caseins and the formation of the cheese curd. Chymosin, however will also cleave ß-casein causing the loss of a portion of the molecule and disrupting its normal properties.

The last modification made to ß-casein in these studies was the addition of glycosylation sites to the molecule. A glycosylation site causes a carbohydrate to be attached to the casein molecule. The addition of a carbohydrate to a protein increases its hydrophilicity allowing it to be more soluble in water. This is a very important property of proteins used in many processed foods.


The mouse milk samples containing the bovine ß-casein variants are currently being analyzed. Some examples of the expected property changes in milk containing extra ß-casein or modified ß-casein are described below.

The addition of purified ß-casein to milk enhances curd firmness of the resulting cheese. Fortification of skim milk with ß-casein at a concentration 30% of that normally found in milk caused a 50% increase in curd firmness, indicating that the functional interactions of the caseins were increased possibly by additional electrostatic and hydrophobic interactions resulting from interactions of calcium and ß-casein. The addition of the purified ß-casein also causes a increase in the cheese yield of the milk.

Proteolysis susceptibility of ß-casein has been identified as the source of the bitter flavor in cheeses. The carboxylic end of ß-casein is extremely hydrophobic. When it is cleaved by enzymes such as chymosin and plasmin, it causes a bitter flavor. The modifications made to the ß-casein molecule will prevent this cleavage and prevent the formation of these bitter peptides in cheese as well as in milk.

The formation of a glycosylated ß-casein in milk should increase the solubility of ß-casein and modify other functional properties such as viscosity, water holding capacity, foaming and emulsification. Proof for an increase in solubility of ß-casein after glycosylation has been reported by a number of groups, but the behavior of this casein in milk has yet to be analyzed. The addition of a carbohydrate to the casein molecule will also effect casein micelle structure and may reduce the size of the micelles. The reduction in casein micelle size has been shown to be beneficial in a number of manufacturing processes.


Food chemists have not in the past had the opportunity to design the proteins that went into a food product. As we have shown by these studies it is now possible. This work shows that using genetic engineering it is now possible to design new proteins very easily and these proteins can be then produced in the milk of transgenic animals. We need to continue to define the potential effects of the modifications and their potential usefulness in the dairy industry.


These are some potential changes that may be beneficial to the dairy industry in the future. However, there are still many things we must understand before these modifications can be applied to dairy cattle. Doing these experiments in mice, allows us to examine the potential usefulness of the technology and create the best possible DNA vector to be used to benefit the dairy industry.

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