The quality of protein and its type is important to determine its overall health impact upon consumption (1, 2). Protein provides energy, and it is also a vital component for other purposes, including enzyme activity and bio-chemicals passage in cellular membranes (3, 4). The accuracy in quantification of protein content is important to further determine its food’s economic value (5, 6). To determine protein’s quantity in food matrices, several methods are used and reported such as Kjeldahl nitrogen estimation, colorimetric assays (Bradford, Lowry, Bicinchoninic acid assay, and Biuret assay), total amino acid content analysis, chromatographic, and radiolabelling methods. Among them, Kjeldahl nitrogen method is the most widely used method for protein quantification in samples. The Kjeldahl method was discovered in 1883 by the Danish chemist Johan Gustav Christoffer Thorsager Kjeldahl and used to determine the nitrogen and protein content. Publicly, it was made available on March 7, 1883, during the meeting held by Danish Chemical Society (Kemisk Forening) (7–9). Initially, this method was designed to aid in grain’s protein changes at the time of fermentation and germination in brewing industries (10). This method indirectly quantifies the total content of protein present in food by direct measurement of nitrogen (5, 11). Today, the Kjeldahl method of protein quantification is universally accepted and used in many laboratories for various food samples. It consists of three major steps, i.e., digestion of organic nitrogen-containing samples with sulfuric acid to ammonium sulfate, distillation of digested sample solution at an elevated temperature and pH to release ammonia which is trapped in boric acid solution, and finally titration of boric acid solution with standard acid (10). Although the method has various advantages, such as its universality, high precision, and reproducibility, it is time-consuming to prepare the sample (digestion) for the analysis (12). There is also a limitation w.r.t. analytical selectivity as it cannot differentiate between protein-based nitrogen and non-protein nitrogen. Some modifications in the standard Kjeldahl method have enhanced the versatility and decreased the procedural time for analyzing different samples simultaneously (11, 13–15). Lee and collaborators (16) compared the standard Kjeldahl method with three modified Kjeldahl procedures by the addition of salicylic acid prior to digestion, the pre-reduction of nitrate to ammonium using CrK(SO₄)₂, and the addition of phenyl-acetate to the standard digestion mixture. This last procedure yielded the best nitrogen measuring results in plant tissue, but the salicylic acid method performed better in the presence of water. Amin and Flowers (17) reported the use of salicylic acid dissolved in concentrated sulfuric acid to recover other nitro compounds. A Kjeldahl method guide (18) suggests the use of salicylic acid followed by sodium thiosulfate for nitrate reduction. Further modifications made by researchers have improved the digestion process. Studies have reported the use of ultrasound and microwave energy in the Kjeldahl procedure. Domini and collaborators (19) concluded that a combination of ultrasound and microwave energy in the digestion process improves its performance by reducing digestion time to 7 min compared to 30 min for the classical Kjeldahl. Ultrasound energy has been used to substitute the distillation system in classical Kjeldahl for a purge-and-trap system in order to stimulate a chemical reaction between the alkaline reagent and the digestion mixture (20). Although these methods provide rapid analysis of protein in the sample; however, they may require an additional instrument. Routinely, in laboratory analysis, there is a need for a fast, robust, accurate, and reliable method for estimating protein content.

The introduction of novel analytical approach in laboratory analysis requires comparison and validation with standard methods being followed for quality assurance. In the validation process, two important and complementary stages involves single- and inter-laboratory validations (21). Additionally, the protein’s quantitative analysis is important for accurate labeling of food as well as its quality control (22). Primarily used methods for protein nitrogen determination in food samples has also been utilized for determining various other nitrogen forms in plant materials, soils, wastewater matrices, and biological tissues (23, 24). To date, many methods have been developed for determining protein in food samples. However, Kjeldahl digestion and distillation ’method is most frequently used method (25). For successful analysis, proper handling of samples and sample preparation are the major steps, affecting the overall analysis time of the sample, thus need to redefine the analysis by developing novel methods.

Generally, the method initially requires a flour matrix for the digestion process that needs lots of time, especially when dealing with a large sample set. Mainly, homogenizing oilseeds is difficult due to high oil content; flour is often sticky, cleaning the homogenizer is complex, and carries a high risk of cross-contamination compared to cereals and pulses. During digestion, frothing occurs in the oilseed crops when using standard digestion mixture and to avoid this issue, we developed a modified digestion mixture. To accomplish quick protein quantification, we have used this modified digestion mixture to digest the whole grain of five oilseed crops and compared it with their flour matrix. The aim of this research was to evaluate and validate the developed novel analysis method to reduce the homogenization time, cleaning, and avoid the frothing. The method demonstrated that the protein content from whole grain of five tested oilseed crops shows 100% non-significant differences when compared with their flour matrices in both the digestion methods. Several statistical analyses such as descriptive statistics, correlations, paired t-test, reliability tests, and different types of plot are employed to validate the results. We hope that this method if utilized can help save sample processing time, costs, and minimize the risk of cross-contamination with accurate results.

The present study was designed to modify the standard Kjeldahl method to quantify the protein content in five oilseed crops. Sample processing/preparation is the first major step required to successfully analyze chemical content. By directly digesting the whole grain of oilseed crops, protein quantification was accomplished quickly, and the results are comparable. The validation of this novel analysis demonstrated that the protein content from whole grain of oilseed crops showed 100% non-significant results compared with the flour matrix.

The health benefits of plant-based protein from different sources are recognized and much appreciated, particularly for the low cost of production and carbon footprint (34–36). The Alternatives to legumes are needed to cater to the increasing demand for plant based protein. The high protein content (15–50%) is reported in different oilseeds, making them ideal sources. Kjeldahl method is most commonly used for determining protein content in the food samples. As a standard practice, samples are ground before digestion. However, oilseeds often form cake while grinding due to high oil content; thus, cleaning grinding mills is challenging and increases the risk of sample cross contamination. The introduction of novel analytical approach in laboratory analysis requires validation of previous methods for assurance of its quality. Some modifications in the standard Kjeldahl method have enhanced the versatility and decreased the procedural time for analyzing different samples (16, 18–20). Hydrogen peroxide is added to digest the samples with high oil content to prevent foaming and aid rapid digestion. However, proper sample handling, their sampling, and analytical procedure followed are essential for accurate and precise analysis. In our research, we directly digested the whole grain of five different oilseed crops using two different digestion methods to accomplish quick protein quantification. And with the modification in digestion mixture and catalysts the process of protein quantification speeds up. There were two hypotheses in this study. First, for small-seeded oilseeds, whole grain (WG) gives equally precise results as with flour samples (FL). Therefore, using WG will speed up the protein quantification method. Second, the modified digestion mixture and traditional digestion mixture are equally effective and have no influence on nitrogen recovery. The descriptive statistical analysis shows that no major difference in the values of mean, standard deviation, variance, and skewness for the flour as well as whole grain samples. With paired sample test, which is used for determination of mean difference between two datasets, it has been found that the difference in the mean, as well as standard deviation is near to zero for each pair, i.e., pair 1 (WG1 and FL1), pair 2 (WG2 and FL2), pair 3 (WG1 and WG2), and pair 4 (FL1 and FL2). The Sig (2-tailed) Pearson correlation describes 100% significant result in whole grain and flour samples. Reliability test based on the results from 52 accessions belonging to five different oilseeds, demonstrated consistency of the results. The value of chi-square test for all the 52 accessions of oilseed crops was found to be 38.278 with 11 degree of freedom at zero significance, which shows the relationship between two categorical variables. Hotelling’s T-squared distribution (T2), a multivariate probability distribution, which is tightly related to the F-distribution and a generalization of ‘Student’s t-test, shows the significance of 0.186. Tukey’s test of non-additivity is a test that shows an interaction between different factors with no replication. Thus the validation of this novel analysis/modified approach of using whole grain in small-seeded oilseeds demonstrated non-significant differences with their respective flour samples.

For the determination of “food”s protein quantity, it is very essential to standardized analytical methods and shortens the overall analysis time. The correct quantification as well as determination of content of protein is important, and hence in the present study we analyze and compare the modification of Kjeldahl method for determination of protein content in five oilseed crops. Here, we directly digested whole grain of five oilseed crops instead of making flour to reduce processessing time and accomplish quick protein quantification. Further, to cope up with oil frothing during digestion time, we developed a modified digestion mixture. On the basis of validation parameters analyzed, it can be concluded that the protein content from whole grain of oilseed crops shows 100% non-significant results compared with the flour matrix indicating that there is no difference in the protein content if we digest the whole rain rather than that of the flour. Statistical analyses such as paired T-test, Pearson correlation, a range of reliability tests, and different types of plot analysis showed similar results in both flour and whole grain (reduce procedural time) for both the digestion mixtures. Hence, this method will help saving sample processing time, costs, and minimize the risk of cross-contamination with accurate results. This will enable a large data set to be finished in a very less duration of time.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

SL conceived the idea and contributed to experimentation and manuscript drafting. RB standardized digestion methods and proofread the manuscript. RB and SL contributed to statistical analysis. RY and JR provided diverse accessions of oilseeds. AK and SK contributed to resources, editing, and formal analysis. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.