Novel food information: Rhiza mycoprotein – A non-viable dehydrated mycelium biomass of Neurospora crassa strain BSTR26

On this page

• Background
• Introduction
• Development of the Product
• Dietary Exposure
• Chemistry
• Microbiology
• Nutrition
• Toxicology
• Allergenicity
• Conclusion

Background

Health Canada has notified The Better Meat Co. that it has no objection to the food use of Rhiza Mycoprotein.

Health Canada conducted a comprehensive assessment of this product according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel characteristics.

The following provides a summary of the notification from The Better Meat Co. and the evaluation conducted by Health Canada. This document contains no confidential business information.

Introduction

The Better Meat Co. has developed a non-viable dehydrated mycelium biomass of a non-genetically modified, environmental isolate of Neurospora crassa for use as an alternative protein and a source of dietary fiber in meat, poultry, seafood, meat alternative, and dairy analog products. Rhiza Mycoprotein is composed of approximately 45% protein and 38% carbohydrates, of which 30% is total dietary fibre.

The safety assessment performed by Food and Nutrition Directorate (FND) evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how the product was developed, the specifications and manufacturing method, intended uses, including its predicted dietary exposure in the Canadian population, its nutritional composition, its microbiological safety, and whether it could be toxic or cause allergic reactions. The Better Meat Co. has provided data to support that this product is safe for use as food in Canada.

The FND has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Rhiza Mycoprotein is considered a novel food under the following part of the definition of novel foods: "a) a substance, including a microorganism, that does not have a history of safe use as a food".

Development of the Product

Rhiza Mycoprotein is manufactured by submerged fermentation using pure culture of N. crassa strain BSTR26. The company maintains master and working cell banks to guarantee the purity and stability of their cultures. The fermentation occurred under controlled parameters (cell density, temperature, agitation, etc.) using sterile growth medium.

Once the desired mycelium density is achieved, the fermentation is discontinued and the wet biomass is separated from the growth medium by means of filtration. The resulting moist cake is then diced into small pieces and dehydrated under controlled temperature and humidity (65-90°C for 60-90 minutes) until reaching a water activity lower than 0.65. This heating step represents a critical control point that contributes to render N. crassa cell non-viable. The resulting dried and shredded biomass is finally sieved and packaged.

Dietary exposure

The Better Meat Co. estimated intakes of Rhiza Mycoprotein based on the conservative assumption that it is added to all foods in each proposed food category at the maximum level (i.e., 17 to 30% on a dry basis) based on food consumption data from the 2015 Canadian Community Health Survey (CCHS). The petitioner estimated that the per capita intake of Rhiza Mycoprotein is approximately 15 g/day (mean) and 35 g/day (90^th percentile) on a dry basis.

The company's exposure estimate was not broken down by the Dietary Reference Intakes (DRI) age and sex groups, nor did they provide the exposure in mg/kg body weight/day. Colleagues in the Bureau of Data, Science and Knowledge Integration conducted a dietary exposure assessment of Rhiza Mycoprotein that corresponded to the proposed foods and use levels.

Based on the proposed uses, children aged 1-3 years have the highest estimated exposure (daily intake) to Rhiza Mycoprotein with mean and 90^th percentile intakes of 1.02 and 2.22 g/kg bw/day, respectively. Using the mean body weight (per 2015 CCHS), the mean and 90^th percentile intakes would be 14.2 g and 30.9 g Rhiza Mycoprotein per day, respectively. Males aged 19-30 years have the highest estimated 90^th percentile exposure (65.8 g/day) of Rhiza mycoprotein.

Chemistry

The Better Meat Co. provided specifications for Rhiza Mycoprotein, including limits on trace elements, which they have established in the absence of specifications for this ingredient in the Food Chemicals Codex and the Combined Compendium of Food Additive Specifications.

The company analyzed Rhiza Mycoprotein for trace elements (arsenic, cadmium, lead, and mercury) and eleven mycotoxins. The petitioner analyzed multiple batches of the product for these contaminants and reported either very low, or as was the case for many results, 'not detected' concentrations. The analytical limits of detection (LOD) employed for all contaminants evaluated were deemed to be suitably low. Using either the quantified analytical results or assuming the LOD values, the Bureau of Chemical Safety determined that the addition of Rhiza Mycoprotein to the specified foods is not expected to result in increased concentrations of chemical contaminants in the commodities to which it is added or would have a negligible impact on total dietary exposure to these contaminants.

Constituents of the fermentation media are not typically regulated as food additives and there is generally no requirement under the Food and Drug Regulations for their premarket review. However, the Food Additive Section conducted a review of the use of citric acid, sodium phosphate, dipotassium phosphate, and an antifoaming agent in the manufacture of the Rhiza Mycoprotein.

Citric acid, sodium phosphate, and dipotassium phosphate are used in the fermentation media. The use of these substances may be consistent with permitted food additive uses. Citric acid is permitted for use as a culture nutrient in unstandardized foods at a level of use consistent with Good Manufacturing Practice^{Footnote 1}. Similarly, sodium phosphate and dipotassium phosphate are permitted for use as acidity regulators in unstandardized foods at a level consistent with Good Manufacturing Practice^{Footnote 2}.

Regarding the antifoaming agent used in the fermentation medium, the petitioner provided a rationale, supported by data, demonstrating the removal of fat- and water-soluble substances during the downstream processing steps.

The manufacturer and distributor are responsible for ensuring that any substance used in the manufacture of Rhiza Mycoprotein complies with all regulatory requirements. This includes ensuring the food additives used in the manufacture of Rhiza Mycoprotein are in compliance with provisions set out in the Food and Drug Regulations and in the Lists of Permitted Food Additives; ensuring that all materials used in the manufacture of Rhiza mycoprotein are of food-grade quality, or equivalent, and meet appropriate food-grade specifications as set out in the most recent edition of the Food Chemicals Codex or in the Combined Compendium of Food Additive Specifications prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Additionally, the manufacturer and distributor are responsible for ensuring that Rhiza Mycoprotein and any substance used in its manufacture will not result in a violation of Section 4 of the Food and Drugs Act.

The Bureau of Chemical Safety has not identified any chemical food safety concerns with the proposed uses of Rhiza Mycoprotein.

Microbiology

The source microorganism, N. crassa strain BSTR26, is an environmental isolate, conidiating, obligate aerobe, saprophytic filamentous fungus that belongs to the ascomycete phylum. This species was first identified in 1843 as Monilia sitophila and is phenotypically recognizable by distinctive flushes of bright orange conidia on charred wood.

Taxonomical identification of strain BSTR26 was performed by sequencing and comparing the nuclear ribosomal internal transcribed spacer (ITS) sequences to the ITS Genebank database, allowing to narrow the identification to the Neurospora genus. The company then sequenced four targeted genetic loci (tmi, dmg, tml, and pho4) with known sequence variability across related species of the Neurospora genus (Dettman et al. 2003^{Footnote 3}, Luque et al. 2012^{Footnote 4}). These phylogenetic markers were compared against the GenBank non-redundant database to determine the most likely species, after which the sequences were aligned with those from a N. crassa reference strain and the closest related species. The sequences perfectly aligned with the N. crassa reference sequences while exhibiting variations when compared to the closest Neurospora species, hence demonstrating that the isolate belongs to the species N. crassa.

N. crassa is a model organism in genetics and molecular biology. Its genome was published in 2003 and remains one of the most well-studied and thoroughly annotated fungal genomes (Galagan et al., 2003^{Footnote 5}, Bartholomai et al. 2022^{Footnote 6}). The genetics, metabolisms, and physiology of this species are well understood.

As obligate aerobe, N. crassa is unable to survive or colonize the gastrointestinal tract. Despite its ubiquitous presence in the environment and more than a century of use in fundamental research, no evidence has been reported of this species being the causal agent of any disease or infection. Furthermore, no harmful secondary metabolite is known to be produced by any discovered strains of N. crassa and the scientific literature provides no evidence supporting that this species poses a risk of toxigenicity.

The genome of strain BSTR26 has been fully sequenced and aligned against the genome of the reference strain NC12, which genome is publicly available. Bioinformatic analyses were conducted to identify genes of potential concerns associated with antimicrobial resistances, virulence factors, toxigenicity and spore-forming capacity based on the methodologies published in the 2024 EFSA CEP Panel guidelines^{Footnote 7}. The search for antimicrobial resistance genes, toxins, and virulence factors yielded no biologically relevant hit.

The search for sporulation associated proteins yielded 30 biologically relevant hits, which share high sequence alignment with their counterpart in the reference strain NC12. This result was expected since N. crassa is known to conidiate. However, the sporulation capacity of N. crassa occurs under environmental stress, which is prevented during the fermentation stage of industrial settings. Further, the absence of viable cells and spores in the final product was demonstrated by the company. Therefore, the Bureau of Microbial and Toxicological Food Safety (BMTFS) has no safety concerns regarding the presence of sporulation associated genes in strain BSTR26.

The petitioner also performed an agar disk diffusion assay using as test article strain BSTR26 wet biomass following the methodology described by the Clinical and Laboratory Standards Institute^{Footnote 8}. Disks with imipenem/relebactam and without antibiotics were used as positive and negative controls, respectively. No inhibition zones were observed around the N. crassa wet biomass disks, hence demonstrating absence of antimicrobial activity in Rhiza Mycoprotein.

The microbiological specifications, which accounted for the absence of viable N. crassa strain BSTR26 cells and its spores, were provided. Certificates of analysis were provided for the final Rhiza Mycroprotein product for three non-consecutive batches demonstrating that all tested batches conform with microbiological specifications. The specifications, methods and test results were all deemed to be acceptable. Furthermore, the product is intended to be cooked prior to consumption, hence reinforcing the non-viability nature of the product.

Based on the totality of evidence, the BMTFS has no microbiological safety concerns regarding N. crassa strain BSTR26.

Nutrition

The Rhiza mycoprotein compositional analysis included proximates (moisture, protein, total fat, total dietary fibre, ash and carbohydrate (calculated)), amino acids, fatty acids, vitamins, minerals and anti-nutrients.

Based on data from three non-consecutive batches, Rhiza Mycoprotein consists of approximately 45% protein, 38% carbohydrates (of which 30% is total dietary fibre), 7% moisture, 3.6% total fat, and 7% ash on a dry basis.

Considering that Rhiza Mycoprotein is proposed as an alternative protein source, its protein quality and overall nutritional composition were compared to other plant-based protein sources on the Canadian market, as well as Fy Protein™, a similar mycoprotein approved as a novel food by Health Canada in 2023.

The company calculated the Protein Digestibility-Corrected Amino Acid Score (PDCAAS), as a measure of protein quality for Rhiza Mycoprotein. The PDCAAS of Rhiza Mycoprotein is 89, which is comparable to values reported for other protein sources (i.e., soy protein (100), beef (92), Fy Protein™ (91), pea protein concentrate (89), lentils (63) and chickpeas (52)).

The Recommended Dietary Allowance (RDA) for total protein is 1.05 g/kg/day for children aged 1-3 years (i.e., the subpopulation with the highest estimated exposure to Rhiza Mycoprotein). According to the intake estimates, the 90^th percentile consumption of Rhiza Mycoprotein among children aged 1-3 may contribute 14 g protein to the diet (representing 100% of the RDA)^{Footnote 9}. When considering the highest 90^th percentile estimated daily intake (among 19-30 year old males), Rhiza Mycoprotein would contribute approximately 30 g of protein to the diet. With an RDA of 0.80 g protein/kg body weight, consumption of Rhiza Mycoprotein could provide 47% of the RDA^{Footnote 10}.

While the protein content of Rhiza Mycoprotein could contribute the entire daily requirement for children aged 1-3 years, it is important to note that there is no Tolerable Upper Intake Level (UL) set for protein. Further, the National Academies of Sciences Engineering and Medicine (NASEM), formerly the Institute of Medicine, has reported that the risk of adverse events from excess protein intake from foods appears to be very low and applies to long-term consumption of high-protein diets. Rhiza Mycoprotein is not intended to be used as the only source of dietary protein, further mitigating the risk of any potential nutritional risk when used as proposed.

Rhiza Mycoprotein contains approximately 30% total dietary fiber comprised mainly of insoluble dietary fiber. The subpopulation with the highest estimated 90^th percentile intake (males 19-30 years) could consume up to 20 g total fiber per day from exposure to Rhiza Mycoprotein. While this represents approximately 53% of the Adequate Intake (AI) for dietary fiber, it is important to consider that Canadians do not consume enough dietary fiber. According to the CCHS (2015), the mean dietary fiber intakes among all age and sex groups are well below their respective AI. Lastly, there is no UL established for dietary fiber and excess consumption is self-limiting, largely due to adverse gastrointestinal effects. Consumption of Rhiza Mycoprotein is not expected to result in gastrointestinal adverse reactions beyond what would be expected from dietary fiber sources already in the Canadian food supply.

At the highest 90^th percentile intake level (males 19-30 years), Rhiza Mycoprotein provides <25% of the RDA of the following vitamins: thiamin, vitamin B12, vitamin C, vitamin A, vitamin D, vitamin E and vitamin K. Pantothenic acid, riboflavin and biotin provide >25% of the RDA; however, these nutrients do not have an established UL due to lack of suitable data demonstrating toxicity in humans. Finally, the highest intake of Rhiza Mycoprotein provides vitamin B6 and folate at levels of 8.7% and 10.9% of the UL, respectively. When the mean intake is considered for the same subpopulation, vitamin B6 and folate provide 4.1% and 5.2% of the UL, respectively. The exposure estimate is based on highly conservative assumptions of complete uptake in each requested food category; therefore, it is not expected that actual consumption will contribute these amounts to the diet and is not considered a nutritional safety concern.

Based on the information provided, the Bureau of Nutritional Sciences has not identified any safety concerns regarding the proposed use of Rhiza Mycoprotein from a nutritional perspective.

Toxicology

To demonstrate the toxicological safety of Rhiza Mycoprotein expressed by N. crassa strain BSTR26, the company submitted three genotoxicity studies and a 90-day oral toxicity study.

The toxicological studies submitted were conducted with a protein isolate that was very similar, but not identical, to the notified product. Like the notified product, the test material was produced using a wild type strain of N. crassa. The BMTFS confirmed that the strain used to produce the test item is representative of the notified product. Further, the Food Additive Section confirmed the safety of the manufacturing process for the notified product, including the raw materials used, based on information provided in the submission. For these reasons enumerated in this paragraph, the Pre-Market Toxicology Assessment Section (PTAS) read across the submitted safety studies to the notified product.

A published 90-day oral toxicity study^{Footnote 11} was conducted in accordance to OECD guideline 408. Male and female Sprague Dawley rats were dosed with up to 5,000 mg/kg bw/day of the test item by gavage for 90 consecutive days. Standard toxicological endpoints were recorded such as clinical signs, body weight, hematology, clinical chemistry, and gross necropsy/histopathology. No treatment-related adverse effects were observed, and the No Observed-Adverse-Effect Level (NOAEL) was established using the highest dose tested (5,000 mg/kg bw/day).

Three genotoxicity studies conducted with the same test item were submitted. These included an in-vitro Ames assay complaint with OECD guideline 471, an in-vivo micronucleus assay compliant with OECD guideline 474, and an in-vitro chromosome aberration test compliant with OECD guideline 473. The quality of the studies was considered sufficient to include in the evaluation, and no genotoxic effects were observed.

The most conservative exposure scenario on a body weight (bw) basis was daily intake for children aged 1 to 3 who would consume 2221 mg/kg bw/day at the 90^th percentile. Compared to the NOAEL of 5,000 mg/kg bw/day, this results in a margin of exposure (MOE) of 2.25. Although this margin is relatively low, other points are important to consider in the safety rationale.

First, the NOAEL was established using the highest dose tested in a toxicological animal study. No safety concerns were identified. Due to the nature of the test item and limitations of animal testing, it is not possible to test for a higher dose because doing so would result in nutritional imbalances, and thus adverse effects.

Second, this calculation represents an extreme scenario where it is assumed that all foods that can be made with Rhiza Mycoprotein will contain it, and that there will be daily exposure at levels consistent with the 90^th percentile for small children on a body weight basis for a lifetime. The MOE at the 50^th percentile, which is more representative of the average consumption patterns, is significantly higher at 7.7. Additionally, consumption on a body weight basis decreases significantly with age; for example the MOEs using daily intake values for males aged 14 and 30 would be 19 and 28, respectively.

Based on the nature of the test item, the constraints of animal testing, the lack of identified hazard, and the very conservative nature of the exposure assessment, the MOEs identified above are sufficient to account for uncertainties in the database.

The PTAS did not identify any toxicological safety concerns with Rhiza Mycoprotein.

Allergenicity

The allergenicity assessment integrated systematic literature review, human consumption trials, digestibility, proteomic analysis (in silico homology searches against the AllergenOnline database and BLASTP database), and epitope evaluation.

A literature review for pure N. crassa mycelium biomass as a food ingredient was performed by the company. It was concluded that oral consumption of the fungal protein is unlikely to cause an allergic reaction in consumers. Further, N. crassa fungal protein is well tolerated in human consumption trials, where no reports of major gastrointestinal issues, such as diarrhea or vomiting were observed.

Digestibility of the proteins contained in Rhiza Mycoprotein was demonstrated under stimulated gastric and intestinal conditions by comparison to pea proteins. Although not fully digested after 60 minutes under gastric conditions with pepsin, which was used to simulate the gastric phase, the proteins contained in the product were more digestible than pea proteins. Under intestinal conditions, in which the enzyme pancreatin was used to simulate the intestinal phase, both proteins contained in Rhiza Mycoprotein and pea proteins digested within 10 minutes.

N. crassa genomic protein sequences that were predicted to be allergenic using the Algpred 2.0 web server were then searched for significant sequence homology with manually annotated non-allergenic proteins from the UniprotKB/Swiss-Prot database. The remaining predicted allergenic proteins were further screened using the AllergyOnline (Version 21) database for allergenicity using at least an 80-amino acid sliding window and approach established by the FAO/WHO^{Footnote 12} for predicting cross-reactivity.

The AllergyOnline queries using default parameters identified 52 predicted allergenic proteins of which 22 shared >35% sequence identity to an allergen over 80 amino acids. The results were further refined to consider the significance of proteins sharing 50% homology over the full sequence with E-values <1x10^-7 identifying several putative N. crassa proteins with 50%-77% identity with known allergens from mold, fungus, yeasts and wheat.

Although N. crassa's proteins were considered potentially cross reactive with mold, fungus and yeast proteins, the sequence homology search against the non-redundant protein database revealed highly significant homology to proteins from many other species. Therefore, these proteins would be considered evolutionarily conserved and are therefore unlikely to pose a significant risk of allergenicity.

An hypothetical predicted protein from wheat presented sequence homologies with N. crassa protein sequences. These homologies required further analysis because wheat proteins are a source of major food allergens. Further, this hypothetical predicted wheat protein has no significant sequence homology identified with proteins from other taxa from the non-redundant protein search, hence suggesting they are unique to their own taxa.

The company demonstrated by protein BLAST searches that the homology of N. crassa protein sequences and the hypothetical predicted wheat protein was predominantly due to continuous glutamine (Poly Q) sequences of low complexity, and common feature of eukaryotic proteins, rather than true structural or evolutionary homology to wheat allergens. The removal of the poly-Q domain from the BLAST query yielded matches that belonged to Neurospora species while none were attributed to wheat. Additionally, evaluation of the entire amino acid sequence confirmed the complete absence of any established wheat IgE-binding allergenic epitopes.

Searches for 9-amino acid exact matches and full-length alignments to celiac-active peptides from the Celiac Database were conducted (on the originally identified 22 protein from the AllergyOnline database under default parameters). No exact matches meeting the criteria for a significant hit (>45% identity, ≥50% query coverage, and an E-value <1 × 10^-16) were identified.

Based on the totality of evidence, proteins from N. crassa are unlikely to pose a risk of eliciting an immunological response in consumers with wheat allergy and celiac disease.

The PTAS did not identify any priority food allergenic concerns with the proposed use of pure N. crassa biomass.

Conclusion

Health Canada's review of the information presented in support of the use of Rhiza Mycoprotein as an alternative protein and a source of dietary fiber does not raise concerns related to food safety.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food and Nutrition Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

For further information, please contact:

Novel Foods Section
Food and Nutrition Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmtfs.inquiries-requetes.bsamt@hc-sc.gc.ca