Kjeldahl Analysis Protein Determination

Amino acids may contain more than one carboxyl or amine group in these cases, they may yield stepwise end points like other polyprotic acids (or bases), provided the different groups differ in iTs by 10 and are still strong enough to be titrated. 

The material is digested with sulfuric acid to decompose it and- convert the nitrogen to ammonium hydrogen sulfate  

The solution is cooled, concentrated-alkali is added to make the solution alkaline, and the volatile ammonia is distilled into a solution of standard acid, which is in excess. Following distillation, the excess acid is back-titrated with standard base. 

So there are one-half as many millimoles excess H2SO4 as NaOH that reacted with it. The reaction with the NH3 is 

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Alkali-extracted proteins from simflower oil cake (89% proteins, Nx6.25) and wheat gluten (76.5% proteins, Nx5.7) were reacted with n-octanol in the presence of an acid catalyst. Temperature, reaction time and catalyst concentration were varied according to an experimental design to maximize the esterification yield. The latter was determined by alkaline hydrolysis and subsequent analysis by gas chromatography. The hydrolysis of the peptide chain was traced by the determination of the amount of free amino groups in esterified proteins using 2,4,6-trinitrobenzene-sulfonic acid (TNBS) assays. The solubility curves of modified proteins in water as a function of pH were obtained by Kjeldahl analysis to determine the composition of the soluble and insoluble parts. 

The Kjeldahl total nitrogen determination method is not very sensitive, hut it suits well for analyzing insoluble samples without preceding disintegration. Automated Kjeldahl protein estimations are used especially in food analysis. 

Since late in the 19th century, the classical Kjeldahl method has been recognized and accepted universally as the authoritative method of analysis for determining the protein content in a wide variety of ingredients and finished products. As a result of technical innovations there are currently available and in use semiautomated or fully automated protein analysis systems that are based on the classical Kjeldahl procedure. 

Protein dispersibility index Dry product is dispersed in water by blending, then centrifuged at 880 x g for I0 min Protein dispersibility index (%) = (% water-dispersible prolein/% total protein) x 100 Advantages Tested in a collaborative study and found to be reproducible between laboratories. Disadvantages Limited to soy products protein is determined by Kjeldahl analysis, which can be a lengthy procedure. AOCS Method B a 10-65 (AOCS, 1999) AACC Method 46-24 (AACC, 2000)... 

As soy protein denatures, it forms insoluble aggregates. Two methods, NSI and PDI, are broadly used to evaluate protein solubility/dispersibility in soy protein products. The PDI (AOCS Official Method Ba-10-65, 1993) rapid stir method uses a blender to disperse the sample, and the NSI (AOCS Official Method Ba 11-65, 1993) slow stir method uses a laboratory stirrer. In both methods, the protein or nitrogen leached into the liquid phase is compared with the total protein or nitrogen in the sample as determined by Kjeldahl analysis. The NSI method usually gives lower values and is related to PDI by the formula (Central Soya Company, 1988) ... 

All protein determination methods described are not absolute and demand some form of calibration. The Kjeldahl s method remains the only official method currently available for calibration purposes and maintains its position as the most frequently used technique for the determination of organic nitrogen in food products. CE and immunochemical (enzyme-linked immunosorbent assay) techniques are most suitable for rapid separation and quantification of individual food proteins and are promising for widespread use in food protein analysis due to their high sensitivity, specificity, and simplicity of operation. There are numerous methods for the evaluation of the nutritional quality of food proteins. 

Quantitation. Various standard protein analytical methods can be nsed for quantitation of collagen. These include determination of the total nitrogen content by Kjeldahl analysis, followed by estimation of the collagen concentration... 

The hydrolysis of 0.06 M BAA by papain is a first-order reaction under the conditions of assay (100). Specific activity (Gi) is expressed as K (first-order rate constant calculated in decimal logarithms) per milligram of protein nitrogen per milliliter of reaction mixture. Protein nitrogen can be estimated by the turbidometric method of Btlcher (36). The nitrogen content of standard papain solutions is determined by micro-Kjeldahl analysis. 

The preparation time for a PER is extensive and involves several days for acquisition of rats and their acclimation before a feeding trial begins. Preparation of a consistent and uniform diet is not trivial. Adequate diet for each treatment for the course of the study should be prepared shortly in advance of the study. Testing of the diet involves chemical analysis for protein (i.e., Kjeldahl N takes several hours) and accurate determination of other diet constituents (e.g., ash, crude lipid, and dietary fiber) so that isocaloric diets can be formulated between treatment groups. These determinations take >1 day to complete. It is recommended that multiple samples from each diet be obtained for proximate analysis of diet before it is fed to ensure that each diet has the proper nitrogen content (and this is same between diet treatments) and that all diets are isocaloric. 

In order to improve the state of health and growth of domestic animals, their diet must be well balanced, therefore basic feed often needs to be enriched with respect to trace and minor elements. Such additions are made on the basis of results of analysis of the raw material, e.g. hay. A hay powder reference material has been considered necessary as being complementary to the rye grass material described in section 6.3. The EC Directive 79/373/EEC prescribes the determination of the nutrients Ca, P and Mg S and N are also important elements since they are used as indicators of the availability of proteins finally the administration of iodine to growing animals requires that this element be carefully monitored. Consequently, the hay powder CRM 129 was certified for its contents of Ca, K, Mg, P, S, Zn, I, N and Kjeldahl-N [10,11]. 

Proteins and Amino Acids Total protein in food and feed samples is commonly determined by Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis.14 FIPLC can separate major proteins and furnish protein profiles and speciation information. HPLC can be used to further characterize specific proteins via peptide mapping and amino acid sequence analysis. HPLC modes used for protein include IEC, SEC, RPC, and affinity chromatography with typical UV detection at 215 nm or MS analysis. Details on protein separations are discussed in the life sciences section. 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

Proteins in foods can be measured by NIR reflectance spectrometry with no sample preparation. This has replaced the standard Kjeldahl protein nitrogen determination, which required extensive sample preparation to convert protein nitrogen to ammonia, distillation of the released ammonia, and subsequent titration of the ammonia. The replacement of the Kjeldahl method for routine analysis by NIR has permitted online measurement of protein in food and beverage products. The Kjeldahl method is required for assaying the materials used to calibrate the NIR and for method validation. 

Historically, the quantitative determination of protein, particularly in the presence of large amounts of non-protein, was based on the estimation of the nitrogen content by the classical Kjeldahl method. Any other non-protein organic nitrogen was either assumed to be absent or was removed prior to analysis. The crude protein content was taken to be 6.25 times the % N found in the sample (this was based on the average N content of 16% in most proteins). 

Another critical food safety application is the determination of melamine in human and pet food. Melamine has been found in milk and pet food, deliberately added to watered-down or inferior products to boost the apparent protein concentration. The standard wet chemical method for protein in food is the Kjeldahl method, which actually measures nitrogen and is an indirect measure of protein. Melamine responds to the Kjeldahl method just like protein, but is not a protein and is toxic it combines with uric acid to form kidney stones. The USDA has set a maximum allowable limit of 2.5 ppm melamine for adults. Using their portable Nunavut Raman System, BaySpec, Inc., scientists demonstrated the measurement of melamine at levels down to 3 ppm in the field the analysis takes less than 10 s, is accurate, is repeatable, and is nondestructive. 

The determination of ammonia after the regular or modified Kjeldahl digestion presents rather less serious problems than those already dis cussed. The advantages of the micro-Kjeldahl distillation (69, 80, 81, 82, 83) as compared with the macro>method, or even the semimicro-method are now generally recognized. A comparative study of the macro-and microscale determination in the analysis of flour, wheat and com for their protein content was made by Robinson and Shellenberger (27). The micro-Kjeldahl method has been used for systematic plasma protein analysis (84, 85), saliva proteins (86), milk proteins (87), and cerebrospinal fluid protein (88),... 

Rodriguez-Otero et al. (46) tried to measure cheese composition by NIR in cheese. They tried to analyze cheese without any prior sample manipulation (not even grating) on the basis of increased knowledge in calibration techniques based on multivariate analysis. Repeatability of NIR moisture determination was approximately double in comparison with the reference method. Repeatability of determination of protein by reference (Kjeldahl) and NIR methods was higher for the NIR spectroscopy, probably because of the large sample size rather than to a lack of method precision. The repeatability of fat determination by reference (gravimetric extraction) and NIR methods was 0.31% for reference and 0.40% for NIRS. 

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