Standard Kjeldahl Method Research Articles

Comparative Protein Quantification in Ultra Heat Treated Milk Using Kjeldahl Versus Dye Binding Methods

The goal of this study was to compare a Dye Binding Method with the standard Kjeldahl method in the context of protein determination in Ultra-Heat Treated (UHT) milk. Protein contents were determined in (25) samples of milk collected immediately post- ultra-heath treatment of milk and (25) samples collected after six-month storage at room temperature in Kjeldahl and Amido black methods. Samples digested using concentrated sulfuric acid at high temperatures and assayed using Automated Kjeldahl (Foss, Kjeltec™ 8200). Whereas, undigested samples were mixed with Amido Black a and density of color was measured against Amido black dilution series at wavelength of 620 nm. First stage: the average concentration of protein was (2.9256±0.01193 g/100 mL) versus (2.9396±0.01338 g/100 mL) using Kjeldahl versus Amido Black A respectively, with an average difference of (0,014). The concentrations ranged between (2.890-2.940 g/100 mL) and (2.910-2.960 g/100 mL) with a Coefficient of variation is (0.41%) and (0.46%) for Kjeldahl and Amido Black, respectively. (P) Value >0.0001. Therefore, there are no variances significantly different between two methods. Second stage: First stage: the average concentration of protein was (2.9224±0.03961 g/100 mL) versus (2.932±0.007071 g/100 mL) using Kjeldahl versus Amido Black A, respectively, with an average difference of (0.0096). The concentrations ranged between (2.770-3.000 g/100 mL) and (2.920- 2.940 g/100 mL) with a Coefficient of variation is (1.36%) and (0.24) for Kjeldahl and Amido Black respectively. (P) Value<0.0001. Therefore, there are variances significantly different between two methods.

Effect of Microwave Heat on the Nutritional Properties of Infected Red Kidney Beans

In this paper, the effect of microwave heat on the nutritional characteristics of affected red kidney beans, i.e., Rajma is studied. Evaluation of crude protein content in microwave heated red kidney beans are carried out using standard Kjeldahl method and Fourier transform infra-red (FTIR) spectroscopy. Red kidney beans affected by Pulse Beetle (Callosobruchus chinensis) have been analyzed throughout the experiments. The heat treatment of the samples is undertaken using a domestic microwave oven at 2.45 GHz. The samples are heated at different microwave powers, such as 90, 270, 450, 750, and 900 W, while the exposure time is varied from 30–90 s. The effective microwave power for maximum disinfestation is found to be 270–450 W at 30–60 s. Most of the red kidney beans are damaged at 900 W for 60 s. Using Kjeldahl method, a constant crude protein level of 22.5 % is obtained for untreated and treated samples at 90–450 W. Crude protein level of red kidney beans is changed at 750–900 W. From FTIR analysis, the absorbance spectrum of carbohydrate peaks is found at 900–1250 cm $^{-1}$ , a moisture peak at 1650 cm $^{-1}$ , and a protein peaks at 2800–3000 cm $^{-1}$ . Statistical analysis of the treated and untreated samples represents the lowest error of 0.0016 at 450 W power level for 60 s. Variances are determined to be the lowest with a value of 270 W and 450 W at 90 s and 60 s. These findings indicate the optimum microwave heat treatment for red kidney beans to retain its protein content.

Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb–β-casein-McAb–AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3′,5,5′-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL−1. The limit of detection (LOD) was 4.8ngmL−1 which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6–7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R2=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen–antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples.

Enzyme–antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2–560ngmL−1. The limit of detection (LOD) was 4.2ngmL−1 which was 10 times lower than that of traditional McAb–HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R2=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample.

Determination of ammonium and organic bound nitrogen by inductively coupled plasma emission spectroscopy

The continuous flow sample introduction technique with a hydride generator system in conjunction with an inductively coupled plasma emission spectrometer (ICP-AES-HG), is used in this study for quantitative determination of ammonium and organic bound nitrogen in aqueous and solid samples. Ammonia vapor released from ammonium salt after treatment with concentrated NaOH is transferred by argon to plasma for detection at 174.273 nm using axial argon plasma mode. The calibration curves were linear within a range of 25–1000 mg L −1 N as ammonium molybdate with correlation coefficients of better than 0.99 and limits of detection of about 10–25 mg L −1 N. The percent recovery of N (25–500 mg L −1 N) in soft (distilled) water and high salt content (1.7 mol L −1 NaCl) matrices was found to be in the range of about 97–102% with %RSD in the range of 4.6–0.62. The sensitivity, limit of detection, and blank contribution from the atmospheric nitrogen, were tremendously improved in this method compared with the available ICP-AES spray chamber counterpart. Furthermore, the ICP-AES-HG method gave results for real samples (soil, fertilizer, waste water) containing about 50–1800 mg L −1 N in good agreement with those obtained by the standard Kjeldahl method. No statistical differences at the 95% confidence level on applying the t-test were observed between the values obtained by the two methods. Thus, the ICP-AES-HG method is reliable and faster than the conventional tedious Kjeldahl method, superior to the ICP-AES spray chamber method, and almost free from matrix interference which is usually a critical factor in atomic emission spectroscopic techniques.

Suitability of NIRS analysis for estimating diet quality of free‐living red deer Cervus elaphus and roe deer Capreolus capreolus

In this study, we tested the efficiency of near infrared reflectance spectroscopy (NIRS) to assess nitrogen content in faeces of free‐living ruminants. Faecal nitrogen (FN) content was analysed in 168 pellet groups from red deer Cervus elaphus and roe deer Capreolus capreolus in the growing season and in winter using both the standard Kjeldahl method and NIRS analysis. Estimates of nitrogen content obtained by the two methods did not differ (P &gt; 0.1), and the correlation between FN values was significant (P &lt; 0.001). FN content ranged within 1.10–4.58% of dry matter and, as anticipated, it was higher in the growing season than in winter in both species (P &lt; 0.01). Faecal nitrogen values were also higher for roe deer than for red deer, although the difference was only significant in the growing season (P = 0.007). Our study confirmed that faecal nitrogen of free‐living ruminants can be accurately determined with NIRS analysis. NIRS represents a low‐cost analytical technique, which could replace conventional labourious methods and is highly promising for analyses of diet quality in free‐living ruminants.

A simple method for the quantitative recovery of nitrate‐N during Kjeldahl analysis of dry soil and plant samples

Abstract The standard Kjeldahl method does not recover nitrate‐N quantitatively. A simple and effective modification to the standard Kjeldahl procedure is proposed which includes nitrate‐N in the total nitrogen determination in dry soil and plant material, The method involves the nitration of a phenolic compound, such as phenyl acetate, followed by subsequent recovery of the nitrogen as ammonium by standard Kjeldahl analysis

ESTIMATION OF PROTEIN CONCENTRATION IN THREE PLANT PARTS AT FOUR GROWTH STAGES IN WHEAT BY NEAR INFRARED REFLECTANCE

Near Infrared Reflectance was examined to be used as a technique of estimating protein concentration in the head, flag leaf, and peduncle of Newana, hard red spring wheat (Triticum aestivum L.) at four growth stages. The plant samples were taken from a field experiment including three N levels. The standard Kjeldahl method was used to estimate protein concentration in these plant parts. Protein concentration in these samples ranged from 2.7% in the peduncle to 16.7% in the flag leaf. The NIR was calibrated using these plant parts. Using 3 of 19 available filters the R2 for the calibration between Kjeldahl and NIR was 0.98. The validity of the NIR calibration was tested using an independent set of plant parts of winter wheat taken at three growth stages. The r2 obtained between Kjeldahl and NIR was highly significant. It was concluded that NIR provides an accurate, rapid, and economical technique for estimating protein concentration in aerial plant tissues. This should facilitate the screening of wheats for genotypic variation in aerial plant protein.Key words: Wheat, Kjeldahl N, protein translocation, protein estimation, Triticum aestivum L.

Nitrogen determination using the14N/p, α/11C reaction at low energies

Nitrogen determination with the14N/p, α/11C reaction at 6.81 MeV, has been performed in organic compounds. Positron annihilation from11C allowed determination of nitrogen contents as low as 0.35% dried weight. Comparison of these results with those from the standard Kjeldahl method showed a 0.999 linear correlation coefficient.

Total Nitrogen Analysis of Soil and Plant Tissues

Abstract Progress of the Dumas and Kjeldahl procedures over the past century is reviewed. Many recent papers claim that various modifications of the standard Kjeldahl method enhance accuracy, precision, or speed, and reduce cost of analysis. Furthermore, several authors advocate use of Pyrex tubes heated in an aluminum block for digestion instead of traditional Kjeldahl flasks. A review of current semimicro-Kjeldahl methodology suggests that proceeding under the following conditions gives satisfactory results: sample size, 50–200 mg for plant material, 100–500 mg for soil; digestion acid, concentrated H2SO4; sample size ratio (mL/g) of 16:1 for soils and 22:1 for plant materials; digestion salt, minimum 0.33 g K2SO4 added/mL H2SO4; catalysts, add CuSO4 5H2O at rate of 10% (w/w) of K2SO4 added, HgO at 5% (w/w) of K2SO4, or add Se to K2SO4–CuSO4 5H2O mixtures at rate of 1% (w/w) K2SO4; digestion time, 1 and 3 h past clearing for plant materials and soils, respectively; pretreatments, use salicylic acid or reduced iron to recover nitrate from sample. Use of Pyrex tubes heated in an aluminum block appears suitable for digestions. Our results indicate that the 2 commercial tube digestion systems tested produce satisfactory recovery of total N from soils and plant materials. Ammonium in Kjeldahl digests may be quantitatively determined by distillationtitration, ammonia electrode, or colorimetric techniques.

Zirconium Dioxide, A New Catalyst, in the Kjeldahl Method for Total Nitrogen Determination

Abstract The standard Kjeldahl method for the determination of total nitrogen in fertilizers, feeds, foods, and beverages has been modified to eliminate the use of mercuric oxide, which is poisonous to the environment. A new mixed catalyst, consisting of 0.60 g cupric sulfate and 0.20 g zirconium dioxide in combination or 0.70 g zirconium dioxide alone, is recommended to replace the 0.70 g or more of mercuric oxide used in all official macro Kjeldahl methods.

Rapid determination of protein in soybean meals.

AbstractProtein content of soybean meal is determined by a modified biuret method. Samples are stirred with 0.5N sodium hydroxide containing normal or isopropyl alcohol and copper hydroxide at 70 C. Protein is determined from the absorbance of the filtered solution at 550 nm. Satisfactory correlation with the standard Kjeldahl method was obtained.

The use of automation in determining nitrogen by the Kjeldahl method, with final calculations by computer.

An account is given of the development of a system making maximum use of automated equipment in the routine determination of nitrogen in biological samples when the desired order of precision is better than 1 per cent. and the expected number of analyses is greater than 100 per day.An automatic digestion unit made by Technicon Instruments Company Ltd. tested for liquids did not achieve the accuracy desired. Results for biological fluids with this digestor tended to be low on occasion by up to 5 per cent. Recovery of nitrogen from solutions of the pure compounds creatine and arginine was as low as 70 per cent. and alterations in the digestion mixture and digestor heating conditions did not materially improve recovery. For this reason all samples were subsequently digested by a standard Kjeldahl method before automated determination of ammonia in the diluted digests.Studies were made of the indophenol-blue reaction used in the automated system for determining ammonia in aqueous solution. Conditions were most stable when the reaction coil was maintained at 22°± 1° C and dwell time in the coil was about 3 minutes. Effects of mineral content of test solutions, sampling rate and sampler cam irregularities on the reliability of results were investigated.Results for nitrogen determined in acid digests by the automated procedure and by a standard distillation method showed close agreement.The use of an I.B.M. 1130 computer allowed calculation of results to be carried out in half the time taken when using an electronic desk calculator.

Ultraviolet Spectrophotometric Determination of Protein Content in Milk

With the adoption of end-on type photo-multiplication system, optical densities of milk can be measured directly. Individual milks and skimmilks prepared from them were subjected to the ultraviolet spectrophotometric analysis. Two significant peaks around 230 and 280mμ, and the readings at the maximal absorbances were compared with the protein contents determined by the standard Kjeldahl method. Optical densities of both peaks showed a close correlation with values obtained by the standard Kjeldahl method, but the coefficient of correlation was slightly higher and the standard deviation a little lower with the readings at the longer wavelength (285mμ) than at the shorter one (243mμ). Coefficients of correlation at the shorter and longer wavelengths of 20-fold-diluted Holstein milks were 0.9965 and 0.9976; whereas, the standard deviations from the regression formulae were 0.019 and 0.016%, respectively. The protein content of Holstein milk can be measured as precisely as with the dye-binding method, using Diacid Light Green GS. Preservatives such as mercuric chloride, formalin, or chloroform did not affect the results significantly.