Chapter 2

The very first mysterious case of detecting protein and fat in plant-based food

📁 Case overview:The five detectives get to work on their very first case. A client is eager to detect fat and protein content accurately and compliantly in five different plant-based food samples. The private investigators put their heads together and decide to use automated fat extraction and the Kjeldahl method to solve the case. They discuss the methods, parameters and provide detailed results from the processes as they solve their first challenge.

Shallot Holmes looks around his team who are quietly sitting around the table and expectedly looking up at him. He slams down their very first case file on the tabletop.
He tells the eager investigators that today they will be helping their first client detect protein and fat in plant-based food. From the corner of his eyes, he notices Nancy Beef getting ready to object. Before she could do so, Shallot Holmes quickly explains the importance of plant-based food in environmental sustainability and health. Effectively silencing her objection, he then proceeds into the topic by getting more scientific.
A meat-substitute or meat analogue approximates certain qualities, such as texture, flavour and appearance or chemical characteristics of a specific meat, he explains. He consults his research notes and continues. The texture of the meat is imitated by processing pea, soy or wheat proteins, red beet root juice or plant heme for colour.

Protein ingredients are the most important component for product identity and product differentiation, whereas fats and oils contribute to the juiciness, tenderness, mouthfeel and flavor release of meat analogue formulations.

The client has hired the food detectives to help him determine fat and protein content in a variety of plant-based food samples. The client is interested in these analytes as means to control the production process of plant-based foods, as well as for quality control of the final product. After all, the nutritional label is not only a legal requirement, it is information carefully read by many health-conscious consumers.

Together, the team examines the plant-based food samples to be analyzed:

• soy schnitzel
• fish sticks based on wheat
• boiled sausage based on tofu and wheat protein
• steak based on soy and wheat
• cold cuts based on pea and soy protein

Shallot Holmes ask his colleagues for ideas on how to begin the investigation into the plant-based food samples. Immediately, Miss Mapple takes the lead and suggests using fat extraction for fat determination purposes. She tells the others that Soxhlet extraction is the most widely used method for fat determination, because it is a robust, easy method that satisfies official standards. She also suggests using a fat extractor that can perform Soxhlet extraction to analyze the samples.

Shallot Holmes points out the customer has been left dissatisfied by other agencies, as he felt not all fat has been fully detected in his plant-based food samples.
Miss Mapple has an easy explanation. Crude or fat free fat is determined by pure solvent extraction. But to determine total fat content, the team needs to perform an acid hydrolysis step prior to the solvent extraction. The acid hydrolysis step, she says, releases all the encapsulated or bound fat from the sample matrix of the plant-based foods. She suggests the use of an instrument that can perform the acid hydrolysis step, so that they can get the job done faster, quicker and with added convenience.

With this plan in hand, the team jumps into action. They process one set of samples with acid hydrolysis and one set of samples without acid hydrolysis. They extract the samples with petroleum ether 40 – 60 °C using Soxhlet extraction for 20 cycles. For the samples subjected to acid hydrolysis, they hydrolyze the samples with 4 M HCl for 30 minutes prior to extraction.

A few hours later, they are ready to analyze the results.

Free fat
Total fat
Soy schnetzel0.10 (10.2%)1.66 (4.55%)
Boiled sausage15.8 (0.64%)16.8 (0.59%)
Steak10.7 (0.92%)11.8 (0.77%)
Fish sticks12.9 (1.67%)14.1 (0.89%)
Cold cuts11.2 (0.30%)12.1 (0.64%)
fat extraction, fat determination, total fat, free fat, plant-based food

Indeed, in all five-plant based food samples, all determined fat contents were higher when the samples were hydrolyzed before the fat extraction. Miss Mapple and Shallot Holmes nod to each other satisfied.


Acid hydrolysis is indeed recommended for plant-based foods as a preparation step that releases bound fats from the matrix.


Certain that the client will be happy with the results, Shallot Holmes switches his attention to the protein determination part of the case. He proceeds to read from the file about the client’s need for a high level of certainty and compliance when determining protein amounts in his samples. The client already has dependable protein values on the nutritional label of his food products, but he would like to re-test the plant-based food samples and compare the results.

Eggcule Poirot springs into action and suggests using the three-step Kjedahl protein determination method. The technique, he adds, is widely used and accepted as a reference method according to most official norms worldwide. He quickly summarizes the three steps of the Kjeldahl process:

  1. Digestion to convert organic nitrogen to inorganic ammonium sulfate,
  2. Distillation preceded by alkalization to separate ammonia from the digest
  3. Titration for quantification of the ammonia

The team settles on using automated systems for digestion prior to Kjeldahl ,and for the actual Kjeldahl analysis to make the process safer and reduce the amount of chemicals they must use. Ready for action, the detectives download an app that helps them fully adapt the Kjeldahl process to their plant-based food samples. They work quickly and efficiently and soon achieve results that leave them all grinning:

Labelled content
Determined content
Soy schnetzel (n=3)4947.4 (0.04%)
Boiled sausage (n=3)1716.4 (0.11%)
Steak (n=3)2019.7 (0.95%)
Fish sticks (n=3)12.612.8 (0.44%)
Cold cuts (n=3)6.56.9 (1.32%)


Fantastic! The results for the protein content are in close accordance with the labelled values with only minimal deviations. The consistence of the results has a relative standard deviation of <2%. Shallot Holmes lauds the team for these exceptionally good results.


With both fat and protein content successfully determined in five different samples of plant-based foods, the team is ready to wrap it up. Shallot Holmes closes the file with a satisfied sigh and the five detectives head out into the spring evening to celebrate closing their first case with a cocktail from the local bar. Shallot Holmes locks up the office and decides he should treat his team to a workshop next.