Kjeldahl; steam distillation; protein determination; plant-based meat

Chapter 19

The detectives plant and grow an idea for protein analysis in plant-based meat

📁 Case overview: The detectives look back on Vegenuary by working on a case of protein determination in plant-based meat. Will their Kjeldahl method for protein content analysis work well for three plant-based meat samples or will their customer be left starving for a better technique? Read on to find out.

The five detectives are relaxing on the sofa, drinking their afternoon tea and sharing what they did last weekend. Miss Mapple announces that she has successfully concluded Veganuary, a challenge to eat vegan food for the whole month of January. Shallot Holmes and Lieutenant Cornlumbo congratulate her warmly, but Nancy Beef and Eggcule Poirot stare at her in complete shock.

Eggcule Poirot is first to clear his throat. He expresses his dismay at how someone could follow a vegan diet without the precious presence of eggs in it. Nancy Beef seconds his opinion, saying no soya, red beet burger could substitute a juicy beef steak. Miss Mapple begs to disagree. She assures them her plant-based meat dishes were flavorful and satisfying. Eggcule Poirot is still shaking his head in disbelief.

Shallot Holmes watches in silent amusement. He gets up and walks over to their case cabinet. He pulls out a folder and suggests taking on a client case that is quite appropriate given their discussion. He clarifies that this customer is interested in establishing a nitrogen and protein determination method to control the quality of his plant-based meat products.

Nancy Beef immediately agrees. She does a quick search and shares her findings that a beefsteak contains 21 g protein / 100 g sample and a hardboiled egg contains 13 g protein / 100 g of sample. Eggcule Poirot catches her drift and proposes that they compare how the protein levels in their non-vegan food items compare to plant-based meat alternatives.

Shallot Holmes approves that this could be a side objective, but they still need to establish and validate a method for protein determination in plant-based meat. As the team’s vegan, he tasks Miss Mapple with leading the case. They head out for the evening, ready to jump on the case in the morning.

The next day, the private investigators seat themselves at their round table and look up at Miss Mapple. She has spent some time preparing herself by going over her Kjeldahl Proficiency Guide and its sequel, another Kjeldahl Practice Guide. She has also watched a webinar dedicated to protein and fat determination in plant-based meat and other vegan foods. And so of course, she suggests to use the classical reference method, steam distillation for protein analysis in plant-based meat. The detectives already have some experience with this method. In fact, they have used it and fat extraction to analyze protein and fat in plant-based food in their very first case!

To save the team some time, she has already purchased three different sample types:
• Vegan sausage – declared/labeled protein content (9 g/100 g)
• Quorn steak – declared/ labeled protein content (14 g / 100 g)
• Tofu – declared / labeled protein content (15 g / 100 g)

Nancy Beef is already smirking with satisfaction, but Eggcule Poirot casts a worried glance towards the values for quorn steak and tofu. Both have the potential to beat the hardboiled egg.

Miss Mapple hides her smile and continues by introducing the team to the three major steps they will need to perform for protein determination in plant-based meat:

1. Homogenization of the sample with a mixer.
2. Digestion of the sample using a digestion system.
3. Steam distillation and titration with a steam distillation unit.

The detectives take a last gulp of their coffees and shove the last pieces of breakfast in their mouths, then jump out of their seats to start with the proposed method.

As per protocol, they begin with sample homogenization. They cut their plant-based meat samples into small pieces and grind them to a fine paste with a mixer.

Once this is done, they move on to digestion. They decide to speed up their method by using a Kjeldahl Tablet as a catalyst. The detectives place each weighed portion of sample and reference standard in a 300 mL sample tube as follows:

SampleWeight (g)
Reference standard Glycine0.2
Vegan sausages1
Quorn steak1
Tofu1

The detectives proceed to add Kjeldahl tables and 15 ml of sulfuric acid (conc. 96% to each tube. They additionally prepare blanks (sample tube with chemicals but without the sample).

Miss Mapple insists on connecting a scrubber to their digester for absorbing the acid fumes created during digestion. This improves the safety of their process.

After this is done, the detectives mount the standard suction module onto the sample tubes for standard Kjeldahl digestion with Kjeldahl Tablets (as per the table below). They then insert the rack with the samples into cooling position and start the preheating step. Once preheating is completed, they shift the sample rack in the digestion position and immediately start the digestion of their plant-based meat samples and blanks according to the following parameters:

StepTemperature (°C)Time (min)
Preheating3500
142010
249090
Cooling-35

Lieutenant Cornlumbo reminds them, as they have already seen before in previous steam distillation experiments, that:

The first indication of a successful digestion is that the digested sample is clear and blue green with acceptable recoveries of a reference substance.

Once the detectives achieve this appearance to mark the end of digestion, they let their plant-based meat samples and blanks cool down in the cooling position.

The detectives move on to distillation and titration. They set up their Kjeldahl system as follows:

Method parameters (Steam Distillation System) Instrument Settings 
Reaction DetectionOffMaxAccuracy ModeOn
H2O Volume60 mLChiller / Tap WaterChiller
NaOH Volume63 mLChiller Temperature15 °C
Reaction Time5 sAutoDist ModeOn
Steam StepsFixed time
Steam Power100%
Level DetectionOff
Distillation time180 s
Stirrer Speed Distillation5
Titration TypeBoric acid titrationAutomated titration with Eco Titrator
H3BO3 Volume60 mL (4%)Method on Metrohm Eco TitratorBUCHI Blank / BUCHI Sample
Stirrer Speed Titration8TitrantH2SO4 0.1 mol/L
Titration Start Time180 sSensorPotentiometric (pH)
Sample Tube Aspiration30 sEndpoint pH4.65
Receiver Aspiration30 s

Miss Mapple happily notes that their Kjeldahl unit enables them to simply use one tab “PREP” to combine both preheating and priming steps. She also points out that she has turned on the AutoDist mode to prevent further preheating or priming steps with intermittent breaks in between the determinations. This should make their procedure faster and more convenient.

Once the results are in, Eggcule Poirot is eager to move on to the calculations. He is nearly sweating with anticipation over the final data.

Firstly, the detectives calculate their results as a percentage of nitrogen.

To calculate the protein content of their samples, they must multiple the nitrogen content with a sample-specific protein factor.

The calculations they use to obtain their results are as follows:

conversion calculation; nitrogen; protein; plant-based meat

Where:

wN: weight fraction of nitrogen

VSample: amount of titrant for the sample [mL]

VBlank: mean amount of titrant for the blank [mL]

z: molar valence factor (1 for HCl, 2 for H2SO4)

c: titrant concentration [mol/L]

f: titre value (for commercial solutions normally 1.000; refer to the product certificate)

MN: molecular weight of nitrogen (14.007 g/mol)

mSample: sample weight [g]

1000: conversion factor [mL to L]

%N: percentage weight of nitrogen

%NGly: percentage weight of nitrogen corrected for the purity of reference substance Glycine [%]

%P: percentage weight of protein

P: purity of the reference substance Glycine [%] as declared by the manufacturar

PF: sample-specific protein factor (6.25 for products with various protein sources, 5.7 for Tofu with only soy-based proteins)

In equation 4, the detectives consider the purity of a reference substance, glycine.

The detectives examine their data in nitrogen determination and recovery for glycine (assay 99.7%) so far, presented in three tables.

The first table examines the results for their first plant-based meat sample, vegan sausages:

They observe the data for vegan sausage:

Vegan sausageMass of sample (g)Volume of Sample (mL)%N%P
Sample 11.00385.2961.4048.773
Sample 21.01965.3721.4038.768
Sample 31.05795.5581.4018.758
Sample 41.01235.3551.4088.802
Sample 51.06035.5761.4038.768
Average--1.4048.774
RSD (%)--0.190.19

Here, the mean blank volume (VBlank) was 0.271 mL (n = 4, RSD = 0.7%).

Then, they obtain a table for quorn steak, where the mean blank volume (VBlank) was 0.268 mL (n = 4, RSD = 0.5%):

Quorn steakMass of sample (g)Volume of Sample (mL)%N%P
Sample 11.01827.9582.11813.236
Sample 21.08838.4292.10313.142
Sample 30.99847.8332.12513.279
Sample 41.09348.5162.11513.220
Sample 51.03127.8882.07212.951
Average--2.10713.166
RSD (%)--0.990.99

Lastly, they analyze their third plant-based meat sample, tofu:

TofuMass of sample (g)Volume of Sample (mL)%N%P
Sample 11.079510.6732.69915.386
Sample 21.016110.0372.69215.345
Sample 31.038410.2452.69115.336
Sample 41.040710.2732.69215.345
Sample 51.049310.3232.68315.295
Average--2.69115.341
RSD (%)--0.210.21

Where the mean blank volume (VBlank) was 0.282 mL (n = 4, RSD = 0.7%).

The detectives are more than satisfied with the results. Still, Shallot Holmes proposes that they do a method comparison to an official method before they present their data to their client. The private investigators agree that this is a good idea and compare their method to standard method AOAC 920.87:

 Method of the detectivesAOAC 920.87Notes/Impact
Catalyst2 x 3.7 g Tablets
Composition:
94.4% K2SO4
2.8% TiO2
2.8% CuSO4.5H2O
15 g K2SO4 + 0.7 g HgOEasy to handle for routine analysis. No toxic Hg. Amount of catalyst saved is ~50%
Sulfuric acid15 mL25 mLNo impact. Substantially less sulfuric acid, in turn less sodium hydroxide for alkalization.
Water60 mL200 mLNo impact. About 3 times less amount of water consumption per sample.
TitrationBoric acid titrationBack titrationNo impact
Protein factor6.25 for mixed protein sources, 5.7 for exclusively soy-based protiens5.7A general protein factor (6.25) is considered for mixed protein sources and protein factor 5.7 was used for soy-based proteins as the use of factor 6.25 is still debatable.

In conclusion, the detectives are pleased to note that their method for high speed digestion, steam distillation and titration provides reliable and reproducible results. Their data corresponds well to labelled values with low relative standard deviations. The average recovery for glycine in all sample sequences was 100 to 100.6% (RSD ≤ 0.1 % for all the presented configurations and digestion methods), which is within the specification of ≥ 98 % and RSD below 1%.

Shallot Holmes also shares that he will offer the client two solutions. Firstly, the client could couple his Kjeldahl unit to a titrator for easy automation that eliminates manual handling after distillation and titration. Alternatively, he could distill with a basic distillation unit and perform titration separately, for an economical solution with equally quantitative results.

The five detectives gather for a video conference and present their findings to the customer. The client is more than convinced and decides to go for the automated option for quick and reliable protein determination of his plant-based meat samples.

The detectives end the video chat and look around the room at each other. While most are grinning, in good spirits, Eggcule Poirot is sulking in the corner. Their work might have been well-done, but the quorn steak and tofu beat the protein values for hardboiled eggs. The others reassure him that food quality is not all about protein content. And eggs will undisputedly remain an indispensable part of vegetarian and meat-eaters’ diets. Eggcule Poirot cheers up slightly. And even cracks a smile when they decide to go for brunch and omelets the next day. Shallot Holmes also chuckles. With everyone in a good mood again, now the case is truly closed.