beer; malt; protein determination; nitrogen determination; Kjeldahl

Chapter 9

The detectives refuse to get hung over protein determination in beer

📁 Case overview: An interesting case falls on the detectives’ table at an unlikely place, the beer garden. A client approaches them asking for help on how to perform compliant protein determination in beer. The detectives are happy to be of service and show him how to use the reference Kjeldahl method to determine nitrogen and protein content in beer and malt samples.

It’s a late summer evening and the beer garden is buzzing with happy chatter and the loud clinking of beer mugs. The last golden rays of the sun are stroking the leafy hair of Miss Mapple and making her blush with pleasure and warmth. The five detectives are comfortably lounging around their wooden table and reminiscing about the NIR calibration development workshop they just completed.

A person sitting at a table nearby overhears their lively discussion and eyes them curiously. He makes his way over to their table and politely ask their line of business. Shallot Holmes proudly explains that their agency handles cases on food analytics. The man is genuinely interested and asks if he can pose them a food analytics challenge of his own. If they successfully solve it, he will consider returning with more business for the detectives.

The detectives look at each other and unanimously nod their heads in agreement.

The man pulls up a chair and quickly states his case.

He is interested in opening up a brewery and delivering quality beer to beer gardens, such as the one they are sitting in. But he knows alcoholic beverages are subjected to strict regulations. He is particularly interested in protein determination in beer and malt samples to satisfy government requirements. He knows nothing about how to achieve this, but wants the most stringent, compliant method possible.

Miss Mapple, by now a deep shade of tanned red, offers to take the lead, as she is a big fan of reference methods. She pulls a pen out of one of her wasp holes and jots down on a napkin the steps for protein determination in beer:

– Homogenize sample by shaking or grinding
– Remove CO2 of the beer samples by shaking or stirring
– Digest sample using an appropriate Kjeldahl digestion instrument
– Distill and titrate the sample using a Kjeldahl system

She offers to show the man the robustness of the method based on beer and malt samples. They agree to meet again, same time, same place the following day. The five detectives finish their drinks and head on home for a good night sleep.

The next morning, the detectives meet at the office early, trying to keep their promised deadline. Miss Mapple has already been to the supermarket and presents them with the following samples:

  • Lager beer I with a labelled protein content of < 0.5 g/100 mL
  • Lager beer II with a labelled protein content of 0.5 g/100 mL
  • Malt I with a protein content of 12.5 g/100 g
  • Malt II with a protein content of 9.0 g/100 g

In no time, she chalks up the protocol for protein determination in beer and the detectives set out ready for action.

First, they will need to verify the digestion step of the process using acetanilide. They perform the following steps:

1. The detectives add 0.1 g of acetanilide in a 300 ml sample tube
2. To the tube, they add 1 Titanium tablet and 10 ml of sulfuric acid (conc. 98%)
3. They generate blanks of chemicals without any samples
4. Then they connect a scrubber to their Kjeldahl digestion unit to absorb acid fumes
5. They place the rack under a fume hood and affix the protection shield
6. Slowly, they add 6 mL of hydrogen peroxide (30%) with a dispenser down the glass wall of the sample tube
7. They wait until the fuming stops and the reaction subsides.
8. Finally, they insrt the rack together with the user protection shield and samples into cooling position
9. They mount the suction module onto the samples and start the digestion according to the following parameters:

StepTemperature (⁰C) Time (min)
13600
242065
Cooling25

Once digestion is completed, they let the samples cool down.

The detectives are now ready for digestion of samples for protein determination in beer. They place 10 mL of each beer sample and 0.4 g of each malt sample into a 300 mL sample tube. To each tube, they add 1 Titanium tablet and 10 mL of sulfuric acid (conc. 98%) and they ensure they use blanks once again. Then, they follow the exact same steps they used for the digestion of the acetanilide sample.

After the allocated digestion time is up, Nancy Beef observes that the liquid in one of the sample tubes is not yet a clear blue-green. Miss Mapple suggests digesting for an additional 15 minutes at 420⁰C and this solves the problem. After the samples have cooled off, Eggcule Poirot notices a darkening of the clear liquid samples.Miss Mapple explains that this phenomenon occurs due to hydrolysis of the matrix, for example of carbohdydrates. She assures Eggcule Poirot that the darkening effect will not influence their results.

They are now ready to move on to distillation and titration.

Miss Mapple suggests using the following parameters to set up their Kjeldahl system :

H2O volume50 mLTitration solutionH2SO4 0.05 mol/L
NaOH volume45 mLSensor typePotentiometric
Reaction time5 sTitration modeOnline
Distillation modeFixed timeTitration start time120 s
Distillation time180 sMeasuring modeEndpoint pH
Stirrer speed distillation5Endpoint pH4.65
Steam output100%Stirrer speed titration7
Titration typeBoric acidTitration start volume0 mL
Receiving solution volume60 mLTitration algorithmOptimal

Once the processes are complete, the detectives excitedly look at the data. But as Eggcule Poirot correctly points out, the results are calculated as a percentage of nitrogen. In order to determine the protein content of the sample, the nitrogen content must be multiplied by a sample-specific protein factor. Miss Mapple writes down the calculations they need to use for protein determination in beer:

beer; protein content; calculation

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 : titrant factor (for commercial solutions normally 1.000)

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

mSample : sample weight [g]

1000 : conversion factor [mL/L]

PF : sample-specific protein factor (6.25 for beer and malt)

For the reference substance, they determine the purity of the acetanilide with the following equation:

purity determinatino; acetanilide; reference sample

%N : percentage of weight of nitrogen

%NAc : percentage of weight of nitrogen corrected for the purity of reference substance acetanilide [%]

%P : percentage of weight of protein

P : purity of the reference substance acetanilide [%]

Firstly, the detectives obtain a table of their results for nitrogen determination and recovery for acetanilide analysis. They note down that the mean blank volume (VBlank) was 0.243 mL (n = 4) and look at their data.

AcetanilideSample mass (g)Sample volume (mL)% of weight of nitrogen (corrected for purity of reference substance)Recovery (%)
Sample 10.10097.64810.38100.2
Sample 20.10067.65510.43100.6
Sample 30.10918.26410.40100.4
Sample 40.10507.98810.45100.8
Average (%)10.42100.5
Rsd (%)0.30.3

They are pleased with the results as the nominal value of acetanilide is 10.36 % nitrogen and their recoveries are within the specification of 98 – 102 %.

Then they look at the beer sample with protein content of less than 0.5 g/100 ml:

Beer ISample mass (mL)Sample volume (mL)% N% P
Sample 1104.8990.06520.408
Sample 2104.8910.06510.407
Sample 3104.9030.06530.408
Average (%)0.06520.408
Rsd (%)0.10.1

And the beer sample with protein content of 0.5 g/100 ml:

Beer IISample mass (mL)Sample volume (mL)% N% P
Sample 1104.5560.06040.378
Sample 2104.5900.06090.381
Sample 3104.5920.06090.381
Average (%)0.06070.380
Rsd (%)0.50.5

Then, the malt sample with declared protein content of 12.5g/100 g

Malt ISample mass (g)Sample volume (mL)% N% P
Sample 10.46456.9252.01512.6
Sample 20.40796.1072.01412.6
Sample 30.39845.9271.99912.5
Average (%)2.00912.6
Rsd (%)0.50.5

And finally, the malt sample with declared protein content of 9g/100 g:

Malt IISample mass (g)Sample volume (mL)% N% P
Sample 10.42264.6551.4639.14
Sample 20.42454.6551.4569.10
Sample 30.39694.4171.4739.20
Average (%)1.4649.15
Rsd (%)0.60.6

The detectives slap each other on the backs. They have once again achieved reliable and reproducible results and right on schedule in their task of protein determination in beer. The results correspond well to the labelled values with low relative standard deviations (rsd) and a short digestion time of 65 minutes. The recovery of acetanilide was 100.5% (rsd = 0.3%), well within the specifications of 98 to 102%.

Miss Mapple is quick to give the Kjeldahl Tablet Titanium and hydrogen peroxide credit for speeding up their process, by helping them reduce digestion time and increase throughput. Using a highly-automated Kjeldahl system also tremendously cut down their processing times.

Happily, they finish off whatever is left from the beer samples, following the strict motto of no beer left behind. They meet the customer at the beer garden leaving him so impressed with their work, that he promises to come back with more cases. And Miss Mapple treats her teammates with an additional round of cider. She is a sweet apple after all 🍻.

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