In addition, a wide range of other compounds, such as nitrate, ammonia, urea, nucleic acids, free amino acids, chlorophylls and alkaloids contain nitrogen. These compounds are called non-protein nitrogen and their relative contents are often higher in vegetables than in foods of animal origin [ 5 ]. Throughout the years, it has been proven that the conversion factor of 6.
In Table 1 the results of the amino acid analysis and the Kjeldahl analysis of the raw materials are shown. Both the traditional conversion factor of 6. For fish, shrimp and flour, the species-specific factors used in these calculations were the average conversion factors suggested by Mariotti et al. Protein content from amino acid analysis and Kjeldahl nitrogen analysis of cod, salmon, shrimp, white and whole wheat flour and dulse red seaweed. The conversion factors used for the Kjeldahl analysis are the commonly used conversion factor 6.
Different letters within the same row indicate significant differences between analyses within each species. More surprisingly, and except for the red algae, the species-specific conversion factors also gave significantly higher protein content than the amino acid analysis. One possible explanation may be that the concentration of some of the amino acids in the sample was reduced as a result of the hydrolysis process prior to the amino acid analysis and that the protein concentration calculated from this analysis was in fact underestimated.
Calculating conversion factors based on the results from this study gave factors of 4. There are risks associated with calculating protein from nitrogen and the resulting overestimation of protein content. One is the possibility of food adulteration, as a high protein content often raises the economic value of a product [ 23 ].
There have been some cases where the producer in order to increase the apparent protein content [ 24 ], and subsequently the economic value of the food product have added non-protein nitrogen, such as melamine. This may compromise food safety for consumers and hence, it is important that such food adulteration is rendered impossible.
Overestimation could thus give false premises for the establishment of new industries. For instance, the interest of increased industrial utilization of seaweeds has increased greatly the last decades. Such a difference could be crucial for the economy of small scale industries. The third common analytical technique for protein analysis is spectrophotometry. Here, the principle is that functional groups or regions within the protein absorb light in the ultraviolet or visible range of the electromagnetic spectrum — nm.
This absorbance is read and compared with known protein standards. Examples of such functional groups or regions are basic groups, aromatic groups, peptide bonds or aggregated proteins. While both nitrogen analysis and amino acid analysis may be performed without any pre-treatment of the raw material, extraction of proteins is a prerequisite before submitting the material to spectrophotometric analysis.
Also for protein extraction, the available methods are many. Most common protein extraction protocols are based on exposure of tissue to weak buffers or water, leading to collapse of cells with subsequent release of intracellular proteins as a response to the hypotonic shock that arises. This is very efficient for tissues containing cells without cell walls animal cells , but not as efficient for cells with cell walls plant cells. The latter being due to the cell walls protecting the cell against collapse [ 29 ].
In this study both protein sources of animal origin and protein sources of plant origin were included in order to evaluate these differences. These buffers contain chemicals with zwitterionic properties, along with one or more detergents and are known to be highly compatible to biological analyses. They are highly water soluble, have low salt-effects and minimal interference with biological functions [ 10 ]. The main drawback of these protocols is that they involve several expensive chemicals and some of the chemicals are suspected to be health damaging.
Proteins are often divided into four main classes, based on their solubility properties. Most foods are complex matrices, probably holding several of these protein classes and combining several solutes would probably optimize the extraction yield. In Table 2 , the extraction yields of the two different extraction methods are presented as protein content relative to the respective raw materials, calculated after amino acid analysis. The extraction yield was, however, lower in less processed and more complex plant materials, such as whole flour and dulse, the latter being comparable to a previous study [ 9 ].
Protein extraction yields calculated from amino acid analysis of cod, salmon, shrimp, white and whole wheat flour and dulse. Different letters within the same row indicate significant differences between the extraction yields of the different methods within each species. Extraction on ice probably protects the proteins against degradation, while heat treatment could possibly accelerate this. Thus, the extraction method should be chosen based on the purpose of further use.
However, in this study the goal was merely to examine the differences in protein extraction yield between methods, and thus, protein degradation was not analyzed. Following extraction, protein determination using two different spectrophotometric methods, the Bradford method [ 18 ] and a modified Lowry method [ 17 ], was tested and compared with amino acid analysis.
In the Bradford method, Coomassie G dye reacts with ionizable groups on the protein disrupting the proteins tertiary structure and exposing the hydrophobic pockets. This is followed by the dye binding to the hydrophobic amino acids forming stable complexes that can be read at nm [ 3 ].
The modified Lowry method is a combination of the Biuret method, where copper ions react with the peptide bonds within the protein, and a reaction between Folin-Ciocalteu reagent and the ring structure on aromatic amino acids. The total reaction forms a stable, dark blue complex that can be read at — nm [ 3 ].
The same was observed when using the modified Lowry method for plant proteins, while the Bradford method resulted in equal or lower protein estimates than the amino acid analysis. The Bradford method gave higher protein estimates than the amino acid analysis for all raw materials except for the dulse. Different letters within the same row indicate significant differences between methods within each species.
The Lowry method has been widely used for protein determination for many decades, due to its simplicity and availability. However, besides aromatic amino acids, a wide range of other compounds react with the Folin—Ciocalteu reagent [ 30 ]. Normally, the Bradford method has been recognized to be less prone to such interference. However, the results from this study show that also for several of the raw materials the protein estimates are very high compared to the amino acid analysis, indicating that there could be some kind of interference.
Differences in extraction efficiency of different amino acids could also be a possible explanation. In the Bradford analysis, the basic amino acids contribute more to the final color than do other amino acids [ 3 ], while aromatic amino acid contribute more to the color development in the modified Lowry method.
As seen in Table 5 , there were some significant differences in the extraction efficiency between hydrophobic proline, glycine, alanine, valine, isoleucine, leucine and phenylalanine , aromatic tyrosine, phenylalanine, tryptophan and histidine and basic lysine, histidine and arginine amino acids. As shown, there are many methods available for protein determination and all of them have their advantages and disadvantages. This great assortment of methods makes direct comparison between studies difficult and the choice of analytical method should thus be justified depending on the purpose of the study.
The results from this study show that protein determination based on nitrogen analysis for most food matrices overestimates the protein content compared to amino acid analysis, whether or not the species-specific conversion factors are used.
Spectrophotometric protein determination methods are often affected by interfering substances and could thus overestimate the protein content. Protein extraction often involves chemicals affecting both extraction yield and subsequent determination. This makes such protocols very dependent on the choice of buffers used and methods involving extraction should thus not be the primary choice for food purposes.
However, if the purpose is further analytical use, such methods could be a suitable alternative. All authors conceived and designed the experiments; H. National Center for Biotechnology Information , U. Journal List Foods v. Published online Jan 1. Hanne K. Edvinsen , Edel O.
Elvevoll , and Ida-Johanne Jensen. Author information Article notes Copyright and License information Disclaimer. Received Nov 15; Accepted Dec This article has been cited by other articles in PMC. Abstract The reported protein content of foods depends on the analytical method used for determination, making a direct comparison between studies difficult. Keywords: proteins, amino acids, analytical methods, extraction methods, Kjeldahl, Bradford, Lowry. Introduction Proteins have a major role in the growth and maintenance of the human body and are, along with carbohydrates and lipids, the energy giving nutrients in the diet.
Materials and Methods 2. Protein Extraction Methods 2. Indirect Protein Determinations 2. The Kjeldahl Method The Kjeldahl method was performed according to method The Modified Lowry Method The modified Lowry protein measurement was conducted according to the method described by Hartree [ 17 ]. The Bradford Method The Bradford assay was conducted according to the method described by Bradford [ 18 ]. Results and Discussion 3. Direct Protein Determination Amino Acid Analysis Amino acid analysis is one of the analytical principles for protein determination.
Indirect Protein Determination 3. Protein Content Based on Nitrogen Determination Some of the most frequently used methods for food protein determination are based on analysis of the total nitrogen content in the samples. Comparison between Amino Acid Analysis and the Kjeldahl Method In Table 1 the results of the amino acid analysis and the Kjeldahl analysis of the raw materials are shown. Table 1 Protein content from amino acid analysis and Kjeldahl nitrogen analysis of cod, salmon, shrimp, white and whole wheat flour and dulse red seaweed.
Open in a separate window. Spectrophotometric Methods and Protein Extraction The third common analytical technique for protein analysis is spectrophotometry. Table 2 Protein extraction yields calculated from amino acid analysis of cod, salmon, shrimp, white and whole wheat flour and dulse. Salmon Shrimp White flour wheat Whole flour wheat Dulse red seaweed Conclusions As shown, there are many methods available for protein determination and all of them have their advantages and disadvantages.
Author Contributions All authors conceived and designed the experiments; H. Conflicts of Interest The authors declare no conflict of interest. References 1. Amino acid nutrition in animals: Protein synthesis and beyond. Wolfe R.
The underappreciated role of muscle in health and disease. Wilson K. Principles and Techniques of Practical Biochemistry. Angell A. The protein content of seaweeds: A universal nitrogen-to-protein conversion factor of five. Imafidon G. Non-protein nitrogen contents of animal and plant foods. Food Chem. Jones D. Mariotti F. Converting nitrogen into protein—Beyond 6. Food Sci. Good N. Hydrogen ion buffers for biological research.
Maehre H. Enzymatic pre-treatment increases the protein bioaccessibility and extractability in dulse Palmaria palmata Mar. Alhamdani M. Single-step procedure for the isolation of proteins at near-native conditions from mammalian tissue for proteomic analysis on antibody microarrays. A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study.
An assay originally described by Bradford 1 has become the preferred method for quantifying protein in many laboratories. This technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and nonprotein components of biological samples see Note 1.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more. Skip to main content. This service is more advanced with JavaScript available. Advertisement Hide. The Bradford Method for Protein Quantitation. Authors Authors and affiliations Nicholas J. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in to check access. Bradford, M. Chial, H. Compton, S. Congdon, R. Friendenauer, S. CrossRef Google Scholar.
0コメント