Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Albumin
Reexamination Certificate
2001-01-03
2004-09-28
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Albumin
C530S360000, C530S370000, C530S418000, C426S041000, C426S583000, C424S535000, C514S002600
Reexamination Certificate
active
06797810
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention concerns a method for isolation of proteins, especially from whey or soya, and for modification of the isolated proteins by bringing the proteins especially whey or soy or a concentrate thereof in contact with a reagent that forms sulfite ions in order to sulfonate the protein.
2. Description of the Related Art
Whey proteins are when compared to other proteins superior in their nutritional value especially for their lysine and methionine content. Processing whey proteins for human consumption and functional food products would augment the processing value of whey and thus increase the profitability of cheese production. Although whey proteins possess good opportunities for use as raw material for food stuffs, the biggest obstacles for their use are expensiveness of recovery and fraction isolation processes as well as varying and poor functional properties of protein concentrates and isolates such as poor solubility, emulsifying, gelling and foaming properties.
The isolation of whey proteins is complicated by their good solubility, which cannot be affected by change of pH at pH values 2-9 whilst the proteins are in their native form. Proteins may be isolated according to four principal methods; 1. denaturation by heat and precipitation, 2. ultrafiltration, 3. ion exchange, and 4. chemical modification and precipitation.
The best known method for isolation of whey proteins is denaturation by heat along with decrease of pH to acidic. This method accomplishes a protein that has lost almost all its main functional properties. This protein is used mainly in various spreads, e.g. processed cheese, as partial or total substitute for cheese [Hill et al.,
Can. Int. FoodSci. Technol. J
15(1982) 155-160].
Nowadays whey proteins are isolated mainly as protein concentrate by using ultrafiltration and drying or as protein isolate by using ion exchange adsorption techniques and drying. Both methods allow for isolation of functional proteins. The decisive factor when choosing between these production methods is functionality of the recovered product and its production costs.
However, there is great variation in composition, functionality and sensory properties amongst protein concentrates produced by methods described above which is why industry shuns from their use. The variation is due to varying compositions of the whey used and differences in pretreatment and production and handling conditions.
Even in protein isolates there are variations of various properties due to the factors described above. The ion exchange adsorption method used in their production evens out the variation somewhat, and gives eventually a product that differs in composition from the protein concentrate obtained by ultrafiltration. It has been noted that the isolates have clearly better quality and functional properties than the concentrates in terms of the protein and fat content as well as the protein's solubility, foaming expansion and stability, absence of protein denaturation and aggrecation as well as flavor. The relatively high lactose and mineral content as well as poor flavor of the concentrates are factors that limit their use by the food industry. The utility of whey protein isolates is in spite of their good properties limited by high product cost due to the method of production.
It is also well known that by changing the protein structure by chemical reaction one can affect the molecule's spatial structure/conformation, charge and hydrophobicity and thus even some other properties of the protein such as its solubility, viscosity, foaming and emulsification.
The most practical and simple chemical method for modifying the structure of the protein molecule is sulfonation, more particularly thiosulfonation, i.e. S-sulfonation that is accomplished by oxidative sulfitolysis. Thereby the sulfur bridges i.e. disulfide bonds between the protein's amino acid chains are cleaved which is accomplished by addition of sulfite ions that in turn initiates an oxidation-reduction reaction in which one sulfur is oxidized into sulfonate and the other is reduced to a sulfhydryl group.
By adding yet an oxidative factor the free sulfhydryl groups are reoxidized to 30 disulfide bonds which in turn continue in the reaction until all sulfhydryl groups have sulfonated or some other reaction factor becomes limiting. The principle of oxidative sultitolysis is described by the following equations:
2RS—SR+2HSO
3
−
< - - - >2RS—SO
3
−
+2RSH
2RSH+oxidizing agent - - - >RS—SR+2H-oxidizing agent
RS—SR+2HSO
3
−
+oxidizing agent - - - >2RS—SO
3
−
+2H-oxidizing agent (total
Here RS—SR stands for a protein molecule that consists of two amino acid chains R such that S—S is the disulfide bond between the two amino acid chains. It connects the amino acid chains and contributes to holding them locked in a certain position. The modified protein molecules may be precipitated out of the solution by lowering the pH from the sulfitolysis reaction pH to pH 3-5.
According to publication Kella, N .K. D., et al.,
J. Agr. Food Chem
. 37 (1989) 1203-1210, oxidative sulfitolysis is used for modification whey protein isolate molecules in order to affect the functional properties of the proteins such as solubility, viscosity, foaming expansion and stability. The property affecting factor was reduction of the number of disulfide bonds compared to the original number of the same. Certain properties were either improved or worsened along with the sinking amount of disulfide bonds. Amongst others, solubility lowered under 5% already by cleavage of 25% of the disulfide bonds whilst also the minimum solubility of the solubility of pH curve changed:
In the modification reaction the concentrations were protein isolates 1.0%, sulfite 0.1 M, urea 4 M and the pH 7.0 and temperature 25° C. As oxidizing agent was used oxygen blown through the solution and as catalyst CUSO
4
solubilized to a concentration of 800 mM. Protein isolates modified to varying degrees were isolated by precipitation with ammoniumsulfate which was added to the solution in such an amount that it formed a to 50% saturated solution. The changed solubility properties were not taken advantage of in protein isolation.
In the publication Gonzales, J. M., Damodaran, S.,
J Food Sci
. 55:6 (1990) 1559-1,563, oxidative sulfitolysis was used in order to isolate proteins in sweet raw whey in which the protein concentration was about 0.6% in almost similar experimental conditions as above; pH 7.0, sulfite concentration 0.1 M, temperature 25° C., oxygen as oxidizing agent and Cu
2+
ion of CuCO
3
as catalyst, in this case, however, as solid beads packed in a glass column. The product of sulfitolysis was oxidized to a sulfonate derivative by recycling it in the column packed with the aforementioned 35 beads. Then the remnants of the beads were removed from the liquid reaction mixture by centrifugation. The authors showed that in the conditions above, by mere sulfitolysis, i.e. by adding 0.1 M sulfite, only about 0.4 moles of the disulfide and sulfhydryl groups in relation to 43,000 g mol protein were sulfonated in 30 minutes and even this was thought to be due to the natural redox potential of whey. When oxidizing with oxygen using a catalyst in corresponding conditions about 1.5 moles of the disulfide and sulfhydryl groups were sulfonated in 3 minutes and about 2.3 moles in 30 minutes. The sulfonated and with copper chelated proteins were isolated as functional proteins by precipitation at pH 4.5. However, before precipitation from the solution the copper chelated in sulfonated whey protein had to be removed with EDTA treatment.
The above method was a complicated laboratory scale operation with non-concentrated whey protein. Here the reaction accelerating potential of increased temperature could not be taken advantage of because the solubility of oxygen and thus its concentration in the solution decrease such that this becomes the reaction limi
Hogan & Hartson LLP
Low Christopher S. F.
Mohamed Abdel A.
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