A kitchen tool to learn protein estimation

It is widely known that the Indian diet lacks adequate protein. Limited nutritional awareness remains a significant cause of protein deficiency, in addition to factors such as affordability, and local and cultural preferences for protein-rich foods. 

The internet and social media are abuzz with the benefits of protein-rich diets. But, how often do we check if the pulses and grains we routinely consume contain the same amounts of protein as nutrition charts show? 

Can we roughly estimate our daily protein consumption? Surprisingly, a kitchen test could help us find out!

Researchers from the University of Mumbai-DAE Centre for Excellence in Basic Sciences (CEBS), Mumbai have designed a ​‘laboratory-free’ method for school students to estimate the protein content of pulses and grains. Their study elaborates on a simple method for estimating protein in soaked grain extracts. It is based on the principle that liquids containing protein generate foam when shaken vigorously. 

Building on a module developed under the Vigyan Pratibha project at the Homi Bhabha Centre for Science Education (HBCSE), Mumbai, students can easily estimate the protein content in grains at home with minimal adult supervision. 

The idea to further develop this module into an educational tool gained momentum during the pandemic, when schools and laboratories were closed. ​“During COVID-19, we wanted to develop assays that students could perform even at home, so they could learn simple scientific methodology and understand how to develop controls in a scientific experiment”, says Subhojit Sen, Assistant Professor at CEBS, Mumbai. ​“It started as a small summer project with our first-year undergraduate students, Shyam and Neelima”, adds Sen, the corresponding author of the paper along with Rashmitha Madamakki, an alumna of the integrated B.Sc – M.Sc. programme at CEBS.

In their study, the team selected 13 grains and pulses commonly available in local markets and widely consumed in Indian homes. These included whole moong beans (whole green gram), chickpeas, rajma (kidney beans), ragi (finger millet), and wheat, among others. They soaked the grains in twice the amount of water overnight and either used the supernatant directly as soaked extracts, or boiled it. This was then added back to the soaked grains to create the ​‘boiled extracts’.

The team analysed several parameters, such as, grain pulse size, bubble dispersion, foam generation, and volume of water absorbed (VA) after soaking. Among these, the boiled extracts showed a promising correlation between protein content, foam stability, volume absorbed, and foamability (upon vigorous shaking for 15 seconds and resting it for 60 seconds). They used foam index (FI) – a ratio of the height of just the foam to the total height of the foam plus the liquid underneath as a quantitative parameter. 

Before testing these parameters, the team developed the foam index standards using known concentrations of casein — a milk protein — to develop a set of standards for FI. These values correlated well with the protein concentration of casein in the range of 20 – 100 μg/​ml, showing a linear fit. 

Next, they measured the foam index of the 13 grains under investigation. The aim was to determine the specific parameter(s) that correlated best across multiple grains when compared with laboratory standards – the Folin-Lowry method and UV absorbance for protein estimation. ​“By comparing it with gold-standard laboratory methods, the measurement of foam index can be a reliable estimate of the protein content of daals and grains”, says Sen. 

Challenges and solutions

The experiment involves vigorous shaking, foam generation, and a foam index that can vary across grain types, especially when students conducting the experiment are not adequately trained. This can be challenging if they deviate from the predetermined protocol. However, the experiment aims to introduce the idea of protein estimation using a simple, laboratory-free method. Hence, learning to measure volumes accurately with available resources and controlling the shaking of liquids will help in maintaining uniformity across samples. 

It is important to train students in ideas of controlled experimentation and learn the scientific method”,

says Sen.

He adds that methods such as these aim to develop ​‘scientific temper’, and should be accessible to all.

Secondly, foaming can also occur due to surfactants such as saponins present in certain seeds. To distinguish between the foam generated due to proteins versus saponins, the team designed a dye-diffusion assay. After shaking the soaked and boiled extracts, a small drop of Camlin® fountain pen ink was laid on top of the foam layer. The ink diffused immediately in samples with surfactant-foam, whereas it was distinctly retarded in samples with protein-foam, taking anywhere between five and 30 minutes to diffuse through— depending on protein content. 

Further, extracts from pulses with high protein content generate a thick foam upon shaking. The foam index of such extracts may not fall in the linear range of the standard curve. Hence, such extracts need to be diluted appropriately. Students trying the experiment may require guidance at this stage.

The way forward

The method is a user-friendly tool, particularly in light of the limited funding available to educational institutions nationwide. It serves as a stepping stone towards understanding the concept of estimation using resources in students’ immediate vicinity. 

What’s more, the technique is sustainable, and leads students to think about nutritional aspects of food, diversity of protein sources and local availability of protein-rich grains. 

One of the leading experts believe the method has strong potential to be developed into an educational tool, particularly, in view of the low intake of protein in India. ​“The research paper comparing the estimation tools of plant proteins along with foam index is interesting from a scientific point of view. The Folin-Lowry method along with the foam index can be correlated to the estimates of protein content using the micro Kjeldahl method. Thus, plant proteins being the primary protein source in India can be estimated by a simple tool”, says Pubali Dhar, Professor, Laboratory of Food Science Technology, Department of Home Science, University of Calcutta, who is an independent expert. 

At CEBS, Sen’s team has bigger plans. Their ambition is to transform this simple tool into ​‘educational kits’ that will be part of a larger citizen science project. The project will aim at collecting data on the diversity of protein-rich grains and mapping preferred protein sources across the country towards knowledge of cultivars with higher protein content. ​“Thanks to smartphones, and because more people are online, we can develop an app-based method to do all of this country-wide. Such a data-collection drive can be useful to governments not only to figure out which parts of the country need interventions for malnutrition, but also what kind of environments or agricultural practices yield higher protein in grains”, says Sen.