Original ArticleDetermination of total proteins in cow milk powder samples: a comparative study between the Kjeldahl method and spectrophotometric methods
Introduction
Milk is a mixture of several substances (lactose, lipids, proteins, amino acids, urea, creatinine, etc.) and its composition depends on several factors such as genetic breeding programs, feeding schemes and climate conditions, among others. In the last few years, an increase in cheese consumption occurred, so the determination of protein content of milk is an important factor for the price paid for by the industry (Bruhn & Franke, 1979; Depeters & Ferguson, 1992; Baker, Ferguson, & Chalupa, 1995; Fox, 1997; Coulon, Hurtaud, Remond, & Verite, 1998; Ferguson, 2000).
Determination of total proteins using spectrophotometric methods is commonly used in several areas such as clinical analysis, biochemistry, physiology, medical research as well as many other areas. Although there are two main problems with the Kjeldahl method (Helrich, 1990), namely, the long period of time needed to carry out the whole assay and the necessity to carry out two analyses to determine the difference between non-protein nitrogen (NPN) and total protein nitrogen (TPN), it is widely used in food science and technology and is the officially recognized standard reference method.
As pointed out by Zaia, Zaia, and Lichtig (1998) there are many studies of interfering substances in spectrophotometric methods for the determination of protein, however, there are not many comparative studies among spectrophotometric methods or between spectrophotometric methods and others, such as the Kjeldahl method.
In the present paper, a comparative study between the Kjeldahl method and several spectrophotometric methods (UV-280 and 220 nm (Stoscheck, 1990), biuret-340 and 550 nm (Gornall, Bardawill, & David, 1949), Bradford (Bradford, 1976), Lowry (Lowry, Rosebrough, Farr, & Randall, 1951), p-chloranil (Zaia, Verri, & Zaia, 1999)) was carried out to determine total proteins in cow milk powder samples (skim milk powder, whole milk powder, whey protein powder, buttermilk powder).
These methods were chosen because they are easy to carry out and they are based on different reactions. In the UV-220 nm (Stoscheck, 1990) and biuret (Gornall et al., 1949) methods, the absorbances are due to electronic transitions of peptide bond and electronic transitions of the complex copper/peptide bond, respectively, so in both methods peptides are measured. The absorbance in the UV-280 nm method (Stoscheck, 1990) is due to electronic transitions of a few amino acids. In the p-chloranil method (Birks & Slifikin, 1963; Zaia et al., 1999) the absorbance is due to a charge transfer complex amino acid/quinone. Hence, in both methods (UV-280 nm and p-chloranil), amino acids are measured. The absorbance of the samples in the Lowry method (Lowry et al., 1951; Chou & Goldstein, 1960; Legler, Müller-Platz, Meniges-Hetikamp, Pflieger, & Jülich, 1985) depends on the concentration of some amino acids and also on the amount of tetra peptides bonds because both are responsible for the reduction of the Folin–Ciocalteau reagent. Bradford method is based on the interaction between the dye BG-250 and proteins, small peptides or amino acids do not show any reaction with dye BG-250 (Bradford, 1976; Snyder & Desborough, 1978; Wei, Li, & Tong, 1997). Thus, it should be pointed out that among the spectrophotometric methods of this study, Bradford method is the only one that measured proteins. The p-chloranil was tested, because it was recently proposed for the determination of protein and as far as we know there is only one comparative study with it (Zaia, Verri, & Zaia, 2000). The Lowry method was chosen because among all the spectrophotometric methods, it is the most widely used and studied until today.
Section snippets
Materials and methods
Ultraviolet and visible spectrophotometries were carried out on spectrophotometer Shimadzu UV-1203.
Results and discussion
Table 1 shows straight-line equations, range of casein and BSA concentrations of work, and relative specific absorbance (RSA) [RSA# (specific absorbance of casein/specific absorbance of BSA) and (specific absorbance of casein or BSA for the X method/specific absorbance of casein or BSA for the biuret-550 nm method)]. The correlation coefficients for all the straight lines showed in Table 1 were at least 0.98. As shown in Table 1, the Bradford method showed the highest sensitivity for proteins
Conclusion
Among the studied methods, the Bradford method showed the highest sensitivity for proteins and the Lowry method showed the least variation of specific absorbance for casein and BSA. After the extraction of lipids, UV-220 nm could be used for the determination of total proteins because the values obtained were not statistically different from the TPN ones. The most important achievement of this paper was that the Bradford method could be used for the determination of total proteins in whole milk
Acknowledgements
This research was supported by a grant from CPG/UEL (No.413.024/99) and from CNPq No. 470087/01-3. The authors are grateful to CONFEPAR Company of Londrina, PR, Brazil, for donating the milk samples.
References (24)
- et al.
Responses in urea and true protein of milk to different protein feeding schemes for dairy cows
Journal of Dairy Science
(1995) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry
(1976)- et al.
Regional differences in nitrogen fractions in California herd milks
Journal of Dairy Science
(1979) - et al.
Nonprotein nitrogen and protein distribution in the milk of cows
Journal of Dairy Science
(1992) - et al.
Determination of serum proteins by means of the biuret reaction
Journal of Biological Chemistry
(1949) - et al.
On the chemical basis of the Lowry protein determination
Analytical Biochemistry
(1985) - et al.
Protein measurement with the folin phenol reagent
Journal of Biological Chemistry
(1951) - et al.
A linear regression method for the study of the coomassie brilliant blue protein assay
Talanta
(1997) - et al.
Spectrophotometric method for the simultaneous determination of proteins and amino acids with p-benzoquinone
Analytica Chimica Acta
(1993) - et al.
Determination of total proteinsA study of reaction between quinones and proteins
Talanta
(1999)
Determination of total proteins in several tissues of ratA comparative study among spectrophotometric methods
Microchemical Journal
Interaction of amino acids, proteins and amines with chloranil
Nature
Cited by (97)
Morphology, surface characteristics and tribological properties of whey protein/chitosan composite particles and their fat replacing effect in O/W emulsion
2024, International Journal of Biological MacromoleculesAdvancement of milk protein analysis: From determination of total proteins to their identification and quantification by proteomic approaches
2024, Journal of Food Composition and AnalysisParticle characteristics and tribo-rheological properties of soy protein isolate (SPI) dispersions: Effect of heating and incorporation of flaxseed gum
2023, International Journal of Biological Macromolecules