EFSA’s opinion on Rhizomucor pusillus biomass powder, adopted on 29 September 2025, contains two protein figures for the same material. Section 3.9.1 states that the novel food “contains on average 50.8 (±0.9) g protein per 100 g, calculated as the total nitrogen measured by the Kjeldahl method multiplied by a nitrogen-to-protein conversion factor of 6.25 (crude protein), in accordance with Regulation (EU) No 1169/2011.” The next sentence: “Based on the sum of anhydrous amino acids, the applicant calculated the true protein content of the NF at 40.2 g/100 g.”

Both numbers describe the same powder. They are 10.6 g/100 g apart. Expressed against the crude figure that is 20.9% lower; expressed against the true figure the crude number is 26.4% higher. Those two percentages describe one gap and differ only in the denominator — which is itself a small demonstration of the problem this article is about. EFSA’s own convention, used in its insect opinions, is to divide by the crude figure.

Only one of the two numbers may appear on a European label. Annex I, point 10 of Regulation (EU) No 1169/2011 defines it without ambiguity: “‘protein’ means the protein content calculated using the formula: protein = total Kjeldahl nitrogen × 6,25.” The method is written into the legal definition. For a microbial biomass carrying substantial nitrogen in RNA and chitin, that formula returns the higher of the two figures, and it is the only one a manufacturer is permitted to declare.

This is not a labelling technicality. It is the numerator in every cost-per-kilogram-of-protein calculation in the sector, and for microbial biomass it is systematically overstated by an amount that nobody is required to disclose.

What the two numbers actually measure

Crude protein is an inference. Kjeldahl or Dumas returns total nitrogen; the 6.25 factor assumes that all of it sits in protein and that protein is 16% nitrogen by mass. Neither assumption holds for whole-cell biomass.

True protein, in the sense EFSA uses, is the sum of the anhydrous amino acid residues recovered by hydrolysis and quantified individually. It counts amino acids and nothing else.

The gap between them is non-protein nitrogen. The Rhizomucor dossier quantifies where it sits in this particular organism, though it never connects those measurements to the protein figure:

Component Reported value Basis in the opinion
Crude protein (Kjeldahl, N × 6.25) 50.8 ± 0.9 g/100 g, n = 5 industrial batches Table 1, “Total protein (%, w/w)”
True protein (sum of anhydrous amino acids) 40.2 g/100 g Section 3.9.1, attributed to the applicant
RNA “averaged 4.8 w/w (±0.1, n=5)” at industrial scale — printed without a percent sign Section 3.4
Chitin 9.13 ± 0.07 % w/w, n = 4 industrial batches Section 3.4, under antinutrients
Chitosan 9.2 ± 0.2 % w/w, small-scale batches only Section 3.4

Both pools carry nitrogen, and neither carries it at the density the 6.25 factor assumes. In the correction Geerits and colleagues apply to fungal biomass, RNA nitrogen is taken as 16.1% of RNA mass and chitin nitrogen at roughly 8.7% of chitin mass — against the 16% that 6.25 attributes to protein. Nitrogen from those two pools is counted as protein by the legal formula and excluded by the amino acid sum. The opinion reports the RNA and chitin figures and separately reports crude protein at N × 6.25, and at no point does the Panel remark on whether 6.25 is appropriate for a material composed this way. The phrase “non-protein nitrogen” does not appear anywhere in the text.

Practical consequence: when a supplier quotes a protein percentage for whole-cell microbial biomass, ask which of the two numbers it is and by which method. If the answer is “Kjeldahl” or “Dumas” or “N × 6.25”, it is the crude figure, and for a chitinous, RNA-bearing organism the amino acids will come in materially lower.

Most dossiers publish only the higher number

The Rhizomucor opinion is unusual in printing both. Across the recent EFSA record for microbial, fungal and algal biomass, it appears to be the only one that does.

Novel food Applicant Adopted Protein as reported Conversion factor stated True protein reported RNA limit in specification
Rhizomucor pusillus biomass powder The Protein Brewery B.V. 29 Sep 2025 50.8 ± 0.9 g/100 g; specification “crude protein (N*6.25) 44–68% w/w” Yes — Kjeldahl, N × 6.25 Yes — 40.2 g/100 g None
Fusarium sp. strain flavolapis biomass Nature’s Fynd 25 Jun 2025 11.3–13.4%, wet basis; specification “crude protein 11–15%” No factor stated anywhere for the novel food No None
Chlamydomonas reinhardtii THN 6 dried biomass Triton Algae Innovations 24 Mar 2025 35.8–36.1%, reported as “Protein (N × 6.25)”; specification “protein (crude) 30–70%” Yes — N × 6.25, AOAC 990.03 No — no amino acid data in the opinion None
Dried whole cell Euglena gracilis Kemin Foods L.C. 25 Mar 2020 17.8–23.2%; specification “protein ≥ 15%” No factor stated No None
Yarrowia lipolytica yeast biomass Skotan S.A. 17 Jan 2019 44.0, 55.3 and 44.4 g/100 g across three batches; specification 45–55 g/100 g No factor stated; method given as ISO 8968-1, a Kjeldahl method No None

Two things stand out. The first is that none of the five specifications carries an RNA limit, despite every one of these organisms being a single-cell or filamentous biomass of the type for which nucleic acid load has been a recognised constraint since the 1970s. The Rhizomucor opinion measures RNA at 4.8% and then omits it from the specification table.

The second is that the Fusarium dossier — a mycoprotein, the category where non-protein nitrogen is most consequential — states no conversion factor at all for the novel food. The figure 6.25 appears exactly once in that opinion, describing the casein control diet in a rat digestibility study. Its specification simply reads “crude protein 11–15%” on a wet basis, with two AOAC combustion methods named and no arithmetic disclosed. A buyer reading that specification cannot reconstruct what the number means.

Practical consequence: the specification sheet is not self-explanatory. For any whole-cell ingredient, the questions that resolve it are: which nitrogen method, which conversion factor, dry matter or as-is, and is there an amino acid sum. Four questions, and in most of these dossiers the public record answers fewer than half of them.

EFSA already solved this — for insects

The Panel has settled, precise language for exactly this problem. It sits in the insect opinions.

From the 2021 opinion on dried yellow mealworm: the novel food “contains on average 58.4 (± 2.2) g crude protein per 100 g, calculated using the conventional nitrogen-to-protein conversion factor of 6.25. The Panel notes that the use of the conventional factor overestimates the level of true protein content… Using this factor [4.76], the protein content of the NF amounts to 44.5 g/100 g (23.8% lower than with a conversion factor of 6.25). For regulatory purposes for nutrition labelling, protein is defined as the total nitrogen measured by the Kjeldahl method multiplied by a nitrogen-to-protein conversion factor of 6.25.”

The 2024-adopted opinion on frozen and dried whole yellow mealworm repeats the construction and applies it to both formats:

EFSA opinion Crude protein (N × 6.25) Alternative factor True protein Gap as stated by EFSA
Dried yellow mealworm, e6343 (2021) 58.4 ± 2.2 g/100 g 4.76 44.5 g/100 g 23.8% lower
Yellow mealworm, frozen, e9155 (adopted Nov 2024) 14.2 g/100 g 4.76 10.8 g/100 g Not stated numerically
Yellow mealworm, dried, e9155 (adopted Nov 2024) 46.5 g/100 g 4.76 35.4 g/100 g Not stated numerically

In the insect files EFSA names the source of the non-protein nitrogen (chitin), names a published alternative factor, prints both protein numbers, and states the percentage gap. In the fungal files it prints one number, or two without comment, and names no factor at all.

The asymmetry is hard to justify on chemistry. The Rhizomucor dossier reports 9.13% chitin plus 4.8% RNA — two nitrogen pools where mealworm has essentially one. If chitin alone warrants a Panel note in an insect opinion, chitin and nucleic acids together warrant one in a fungal opinion.

The guidance that was supposed to close this

EFSA’s own novel food guidance, adopted 27 June 2024, is explicit. Section 3.1.4: “The protein content of the novel food should be quantified using the 6.25 nitrogen-to-protein conversion factor. In case the protein content of the novel food is substantial, it should also be calculated as the sum of the anhydrous amino acids, to account for the presence of non-protein nitrogen, and the complete quantitative amino acid profile should be provided.”

That sentence is the reason the Rhizomucor opinion carries a true-protein figure at all. It also explains why the practice is patchy: the Chlamydomonas opinion was adopted nine months after the guidance and contains no amino acid data whatsoever, and the Fusarium opinion, adopted twelve months after it, contains no conversion factor. Both dossiers were submitted years before the guidance existed — 2023 and 2021 respectively — and neither was retrofitted.

Three limits are worth stating plainly. The guidance is generic to all novel foods, not written for microbial matrices; its own microorganism-specific section covers taxonomy and hazard identification rather than protein determination. It governs the authorisation dossier, not the label. And “substantial” is not defined.

What the rest of the rulebook says

The requirement to use 6.25 is not an EU peculiarity, and it is not new.

  • EU. Regulation (EU) No 1169/2011, Annex I, point 10: protein = total Kjeldahl nitrogen × 6,25. No derogation exists for high non-protein-nitrogen matrices.
  • United States. 21 CFR 101.9(c)(7): “Protein content may be calculated on the basis of the factor 6.25 times the nitrogen content of the food as determined by the appropriate method of analysis as given in the ‘Official Methods of Analysis of the AOAC International,’ except when official AOAC procedures described in this paragraph (c)(7) require a specific factor other than 6.25, that specific factor shall be used.” The escape hatch is narrow: the alternative factor must come from a referenced official AOAC procedure, not from a producer’s own derivation.
  • Codex. CXG 2-1985, section 3.3.2: “Protein = Total Kjeldahl Nitrogen x 6.25 unless a different factor is given in a Codex standard or in the Codex method of analysis for that food.” No Codex standard sets a factor for microbial, fungal or algal biomass.

Against that, the scientific position has been settled for a long time. The FAO/WHO JEMNU expert panel, meeting in July 2019 and reporting in 2020, recorded that “the conversion factor of 6.25 currently used in the standards for infant formula and follow-up formula has not been empirically determined and agreed that its application to a wide variety of proteins is highly inappropriate.” Its recommended alternatives — 6.1 for dairy-based and 5.7 for soy-based ingredients under its Option 1 — were expressly scoped to formulas, and the panel never examined microbial, fungal or algal matrices at all.

Older still: PAG/UNU Guideline No. 12 on single-cell protein for human consumption, published in 1983, states that “crude protein is total N x 6.25. Total N includes non-protein N and non-amino acid N, such as nucleic acid, urea, amines, and ammonia… Because of these variables, true protein should be determined by amino acid analysis.” A United Nations body identified the mechanism, named nucleic acids as the culprit and prescribed the fix forty-three years ago. No binding labelling instrument since has adopted any of it.

How large is the effect elsewhere

The Rhizomucor gap of 20.9% is not an outlier. Published work on adjacent matrices puts it in a consistent range:

Material Finding Source
Microalgae (21 new samples plus >50 literature profiles) 6.25 gives “an average of 40.8% overestimation of the protein content”; non-protein nitrogen averaged 26.2% of total N in algae against 15.9% in non-algal foods, reaching 54% in individual samples Templeton and Laurens, 2015
Filamentous fungal biomass, including Fusarium venenatum material from producers’ own GRAS dossiers Crude protein (N × 6.25) 52–59% of dry matter against lysine-derived protein 42–50% on the same material; chitin at 1–30% w/w of dry biomass implies “a protein overestimation of 0.6 to 13.1 %w/w” Geerits and colleagues, 2025
Edible insects 6.25 overestimates by an average of 17%; subtracting quantified chitin nitrogen still leaves “~13% overestimation” — “chitin is only responsible to a quarter for the discrepancy” Boulos, Tännler and Nyström, 2020
Dulse (red seaweed) 105.3 g protein/kg by amino acid analysis against 152.1 g/kg by Kjeldahl × 6.25 Mæhre and others, 2018
Foods generally Proposed default factor of 5.60, and 4.4 for vegetables and mushrooms; using 6.25 regardless of foodstuff means “‘protein’ is simply nitrogen expressed using a different unit” Mariotti, Tomé and Patureau Mirand, 2008

The insect result is the most useful corrective for anyone tempted by a simple fix. Subtracting chitin nitrogen closes only about a quarter of the gap; the remainder sits in free amino acids, nucleotides, amines and other small nitrogenous compounds that no single correction reaches. For fungal biomass, where RNA is a second large pool, an arithmetic adjustment is a worse answer than an amino acid analysis.

What it does to cost per kilogram of protein

The commercial consequence is arithmetic, and it lands on the buyer.

Take a powder purchased at an assumed €10 per kilogram — an illustrative figure, not a market price, used only to show the mechanism. Declared at 50.8% protein it implies €19.69 per kg of protein. The same powder at 40.2% implies €24.88 per kg of protein, 26.4% more. Nothing about the material changed. Only the basis of the numerator did.

That matters because cost per kilogram of protein is the unit the sector benchmarks in, and the incumbent it is benchmarked against is measured differently. Isolated dairy and plant proteins are highly purified, so their non-protein nitrogen fraction is small and 6.25 is closer to correct for them; whole-cell microbial biomass is not purified at all, and carries its cell wall and its ribosomes into the number. Comparing the two on declared protein flatters the biomass — which is the same class of error we found in the $4–6/kg cost-parity claim and in the choice of whey benchmark, arriving one level further upstream. In those cases the price was on the wrong basis. Here the protein is.

The economic point is made explicitly in the literature. Hayes, reviewing protein measurement methods in 2020, writes that overestimation “also overestimates the potential for their use and the economic feasibility and value of these new protein sources.” Mæhre and colleagues are blunter: “Overestimation could thus give false premises for the establishment of new industries.”

Practical consequence: when modelling cost per kilogram of protein for a biomass ingredient, use the amino acid sum if the supplier will provide one, and state which basis you used. A model built on declared protein and compared against an isolate is not comparing like with like, and the error runs in one direction.

The counter-argument

There is a serious case for the status quo, and it should not be dismissed.

Consistency has value. A single legal formula applied to every food makes label figures comparable across the entire market. Permitting product-specific factors invites manufacturers to select whichever factor flatters them, and a producer-derived factor is far harder for an enforcement authority to check than a Kjeldahl result. The 6.25 rule is crude precisely because it is difficult to game.

Non-protein nitrogen is not worthless. Nucleotides and free amino acids are absorbed and metabolised. Crude protein overstates amino acid supply, but the material is not inert filler.

Protein quality is handled separately, and honestly. The Rhizomucor opinion determined true ileal digestibility of each indispensable amino acid, found leucine limiting, calculated a DIAAS of 61% and concluded: “As this value is below the FAO threshold of 75%, the protein in the NF is not considered to be of ‘good quality’.” The Fusarium opinion reports a PDCAAS of 0.91 against 1.23 for its casein control. A formulator who reads the dossier rather than the specification sheet is not misled — the quality assessment rests on amino acid data throughout.

And the change is not costless. The dairy industry’s own estimate, reported in a trade association newsletter and cited in the BMJ in 2006, put the effect of moving European dairy from 6.38 to 6.25 at roughly €80 million. That is an industry estimate rather than an independent calculation, but it indicates why conversion factors are revised slowly.

The rebuttal is narrow. Nobody here is arguing that the label should change. The argument is that where a regulator has already obtained an amino acid sum, that number should appear in the specification alongside the crude figure — as it does in exactly one of the five opinions above.

What we could not establish

  • The basis of the Rhizomucor protein figures. Neither 50.8 nor 40.2 g/100 g is stated as dry matter or as-is anywhere in the opinion we read. The same table reports moisture separately at 4.8–6.5% w/w and section 3.3 gives dry matter as 96%, which suggests an as-received basis, but the opinion does not say so. We have not assumed one.
  • The RNA analytical method in that dossier is not named, and the value is printed in the running text as “4.8 w/w (±0.1, n=5)” without a percent sign. We read it as % w/w in the context of the surrounding table. The chitin figure is likewise given as “9.13 (±0.07, n = 4)” without a unit in section 3.9.3, and with “% w/w” in section 3.4.
  • Conventions for chitin nitrogen differ between sources. The fungal-biomass correction cited here uses roughly 8.7% nitrogen for chitin, derived from glucosamine; work on insect matrices reports 6.89% for the fully acetylated polymer. We have used the fungal figure and have not attempted to reconcile the two.
  • Whether any other EFSA opinion reports both figures. A full-text search of the EFSA Journal corpus for the phrase used in the Rhizomucor opinion returned no other hit, but the index does not yet cover the most recent 2026 outputs. We state this as a strong negative, not an exhaustive one.
  • Nitrogen-to-protein conversion factors for bacterial single-cell protein. We found no published factor for any bacterial SCP species. What exists is compositional: an ICI account of Pruteen from 1982 records amino acid nitrogen at 71% of total nitrogen and nucleic acid nitrogen at 19%, against 82% amino acid nitrogen for fish meal. That source is a scanned 1982 paper in which percent signs render unreliably; we have read the figures as percentages of total nitrogen because the surrounding sentence describes them that way.
  • The widely repeated “WHO 2% RNA limit.” PAG/UNU Guideline No. 12 sets a limit of 2 g of nucleic acid per day from single-cell protein for an adult. It contains no percentage composition threshold. The 2% figure circulating in the literature appears to be that daily limit divided by an unstated serving size. We could not locate any primary document setting a compositional limit, and we do not repeat it as one.
  • The Rhizomucor opinion is behind bot protection. The EFSA Journal page returns HTTP 403 to non-browser clients. The quotations above were read from the rendered article; the amended version of 29 January 2026 is the one cited, the original having been withdrawn.

What to watch

Three testable claims, so this piece can be checked later.

  1. Whether the next EFSA opinion on a microbial or fungal biomass reports both protein figures. The 2024 guidance requires the amino acid sum for dossiers where protein is “substantial.” Dossiers submitted after June 2024 should start producing it consistently. If they do not, the requirement is not being enforced.
  2. Whether an RNA limit ever enters a fungal biomass specification. None of the five examined carries one. For an organism measured at 4.8% RNA and intended for use “up to 35% w/w” in food products, that absence is worth tracking against the 1983 daily-intake guidance.
  3. Whether any producer voluntarily publishes a true-protein figure on a technical data sheet. It is not prohibited — the crude figure is mandatory on the nutrition declaration, but a specification sheet may carry both. The first supplier to do it will be able to defend its cost-per-kilogram-of-protein number in a way its competitors cannot, and that is a commercial argument rather than a regulatory one.

The Protein Brewery’s dossier, which took six years to clear EFSA while its US notice closed with no decision, is the one that printed both numbers. That is the more useful precedent it set.