Novel Food Information: Fy Protein™ - A nutritional fungi protein from Fusarium sp. strain flavolapis
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Background
Health Canada has notified The Fynder Group, Inc. DBA: Nature's Fynd (herein referred to as Nature's Fynd) that it has no objection to the food use of Fy Protein™ from Fusarium sp. strain flavolapis (also referred to as Fusarium strain flavolapis in the literature) as a food ingredient and alternative protein source. The Department conducted a comprehensive assessment of Fy Protein™ 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 traits.
The following provides a summary of the notification from Nature's Fynd and the evaluation by Heath Canada. This document contains no confidential business information.
Introduction
Nature's Fynd developed Fy Protein™, which is a non-viable biomass of the non-genetically modified environmental isolate Fusarium sp. strain flavolapis, for use as a food ingredient or as an alternative protein source in food. Fy Protein™ is composed of approximately 48% protein (on a dry weight basis).
A safety assessment was conducted by Food Directorate evaluators 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 reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered the development of Fy Protein™, the specifications and manufacturing method, the intended use, nutritional composition, microbiological and toxicological information, as well as the presence of potential allergens. Nature's Fynd provided data to support that Fy Protein™ is safe for use as a food ingredient and alternative protein source in Canada.
The Food Directorate has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 (Novel Foods) of Part B of the Food and Drug Regulations. Food use of Fy Protein™ has not been reported to any great extent. Therefore, Fy Protein™ is considered novel under the following part of the definition for novel foods: "(a) a substance, including a microorganism that does not have a history of safe use as a food".
Development of the product
Fy Protein™ is manufactured using a proprietary fermentation process of Fusarium sp. strain flavolapis to form a mycelial biomat, i.e., a thin film of hyphae. At the end of the fermentation step, the mycelial biomat is physically separated from the fermentation medium and subjected to a heat inactivation step. The resulting non-viable biomat is then washed with water to remove any remaining media before being mechanically pressed to remove water to a specific moisture content, resulting in a semi-solid ingredient. Depending on the application, the Fy Protein™ ingredient can be used in semi-solid form or dried into flour through means of dehydration, grinding, and freezing steps.
Nature's Fynd indicated that Fy Protein™ is manufactured under current Good Manufacturing Practice conditions and involves various critical control points designed to limit the growth of common food pathogens and prevent the presence of viable source organism in the final product. Fy Protein™ undergoes further processing such as steaming or cooking depending on the finished product application prior to consumption.
Dietary exposure
Nature's Fynd estimated mean and 90th percentile intake of Fy Protein™ for various age- and sex-groups based on food consumption data from the Canadian Community Health Survey (CCHS) 2015. The dietary exposure estimates were prepared on the basis that current protein sources in proposed foods will be replaced with Fy Protein™, or Fy Protein™ will be an additional source of protein. The exposure assessment was based on the conservative assumption that Fy Protein™ will be added to all foods in each proposed food category at levels ranging from 6 to 25%.
Young children, ages 2 to 5 years, would have the greatest potential Fy Protein™ exposure per kilogram of body weight per day (g/kg bw/day) with mean and 90th percentile eaters-only values of 1.3 and 2.2 g/kg bw/day, respectively. This would equate to mean and 90th percentile eaters-only total daily Fy Protein™ intakes of 21.6 and 37.5 g/day in this subgroup. Male adolescents (12 to 19 years) would have the highest Fy Protein™ daily intakes with mean and 90th percentiles values of 32 and 64.1 g/day respectively.
Chemistry
Nature's Fynd analyzed multiple batches of the notified Fy Protein™ for trace elements (arsenic, cadmium, lead, and mercury), 14 mycotoxins, nitrates, and 17 pesticides deregistered in Canada (and therefore considered to be environmental contaminants). Nature's Fynd reported that Fusarium sp. strain flavolapis is known to produce fumonisins and beauvericin mycotoxins as secondary metabolites during cultivation. While fumonisins were not detected above the analytical limit of detection (LOD), beauvericin was detected. All other contaminants were reported as 'not detected' or were detected at very low concentrations. Using either the quantified analytical results, or assuming the LOD values, it was determined that potential dietary exposure to chemical contaminants, including mycotoxins, from the addition of Fy Protein™ to the specified foods, would result in adequate margins of exposure, or have a negligible impact on total dietary exposure. The levels of the reported contaminants in Fy Protein™ are therefore not expected to pose a concern to human health.
No food additives are used in the manufacture of Fy Protein™. In addition, based on information provided as part of the submission, substances from the growth and fermentation media are not expected to remain in the final ingredient. The use of any substance in manufacturing of Fy Protein™ must not violate of Section 4 of the Food and Drugs Act.
Microbiology
The source organism, Fusarium sp. strain flavolapis, was first isolated in August 2008 from a sample taken in Yellowstone National Park, Wyoming, U.S.A. Fusarium species are typically spore-forming ascomycetes, ubiquitously distributed in the environment, and frequently found in the gastrointestinal tract of healthy vegetarians. The notified Fusarium isolate is unable to form spores as it lacks the necessary genes required to produce aerial sexual spores.
The Public Health Agency of Canada's ePATHogen database classified the Fusarium genus as risk group 2, hence representing a pathogen that is able to cause serious disease in humans or animals but is unlikely to do so. Literature reports indicate that Fusarium infections are rare and opportunistic, developing in patients with trauma, injury, or in severely immunocompromised conditions, however, Fusarium spp. are of limited pathogenicity in otherwise healthy individuals. Further, the Fy Protein™ manufacturing process encompasses a heat inactivation kill step, which prevents viable Fusarium sp. strain flavolapis cells from being present in the final food. Fy Protein™ also undergoes further processing such as steaming or cooking, depending on the finished product application, prior to consumption. This information supports the lack of pathogenicity of the notified source organism when taken in the context of the manufacturing process used to develop the Fy Protein™ product.
The Fusarium sp. strain flavolapis genome has been fully sequenced and bioinformatics analyses were conducted to analyze the core biosynthesis enzymes and gene complexes, investigated for its potential to express mycotoxins or other harmful secondary metabolites, and used to perform a phylogenomic comparison against available related genomes. In silico analysis revealed that Fusarium sp. strain flavolapis has the capacity to express both fumonisins and beauvericin. This information has been addressed above in the Chemistry section.
The potential antimicrobial activity of Fusarium sp. strain flavolapis culture supernatant and biomass were investigated in three production batches and demonstrated an absence of antimicrobial activity.
The microbiological specifications, which accounted for absence of viable Fusarium sp. strain flavolapis, were provided. Certificates of analysis were provided for the final Fy Protein™ product for five non-consecutive batches demonstrating that all tested batches conform with microbiological specifications. The specifications, methods and test results were all deemed to be acceptable.
Nutrition
The Fy Protein™ composition consists mainly of protein (approximately 48%) containing all the essential amino acids, dietary fibre (approximately 35%), as well as total fat (approximately 5%), and ash (approximately 6%) on a dry weight basis. In addition, Fy Protein™ was reported to contain vitamin B1, pantothenic acid, vitamin B6, vitamin B12, calcium, zinc, manganese, phosphorous, and magnesium at levels consistent with those of other alternative protein sources. Based on the levels reported in Fy Protein™ and estimated daily intakes of Fy Protein™, the daily intakes of vitamins and minerals from Fy Protein™ consumption were calculated and further reviewed in the context of the corresponding Recommended Dietary Allowances (RDAs) for the children and adolescent groups. The daily vitamin and mineral intakes from Fy Protein™ consumption are lower than the corresponding RDAs and do not pose a nutritional safety concern.
Because Fy Protein™ is intended as a protein source, Nature's Fynd provided the Protein Digestibility-Corrected Amino Acid Score (PDCAAS) as a measure of protein quality for Fy Protein™, based on its indispensable amino acid contents and true protein digestibility of its crude protein in rats. The PDCAAS of Fy Protein™ was comparable to casein (91 vs 100, respectively). The PDCAAS of Fy Protein™ is within the range of common food proteins, and higher than some plant-based proteins. In addition, in terms of protein quantity and amino acid composition, Fy Protein™ is also comparable to some plant-based proteins.
Nature's Fynd indicated that the anti-nutrients lectins and oxalates are produced at low levels in fungi. The levels of oxalates analysed in Fy Protein™ were below the LOD. In addition, any lectin present is likely to be significantly reduced or removed through some of the Fy Protein™ manufacturing steps (e.g., heat inactivation step and wash steps). Depending on the final Fy Protein™ food application, steaming, and cooking steps are also likely to further reduce or eliminate lectins.
As mentioned previously, the composition of Fy Protein™ includes approximately 35% dietary fibre. Reports of adverse gastrointestinal reactions from consumption of mycelial biomass, which include bloating, gas, constipation, or diarrhea are likely due to the high fibre content. Fy Protein™ intakes at the 90th percentile can provide dietary fibre at levels comparable to the established Adequate Intakes for fibre. Fy Protein™ consumption is therefore not expected to result in gastrointestinal reactions beyond what would be expected from other dietary fibre sources.
Toxicology
To support the toxicological safety of Fy Protein™, Nature's Fynd provided a bacterial reverse mutation study, an in vitro micronucleus study, and a sub-chronic 90-day oral toxicity study. The studies were conducted according to the principles of Good Laboratory Practice (GLP) and their respective Organisation for Economic Co-operation and Development Testing Guidelines.
In the bacterial reverse mutation assay, Fy Protein™ was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535 or TA1537, or in Escherichia coli strain WP2 uvrA, in the presence or absence of metabolic activation with S9. In the in vitro micronucleus assay in cultured human lymphocytes, Fy Protein™ did not induce any increases in micronucleus frequency, either in cultures treated for a short period (4 h), both in the presence or absence of S9, or in cultures treated for a longer period (44 h), in the absence of S9 only.
A 90-day study was conducted in which Sprague-Dawley rats were fed 0, 50 000, 100 000 or 150 000 ppm of Fy Protein™, equivalent to 0, 2744, 5519 or 8115 mg/kg bw per day for males and 0, 3341, 6996 or 9892 mg/kg bw per day for females. No mortalities or signs of overt toxicity were observed, and there were no adverse effects related to clinical observations, ophthalmic findings, body weight, body weight gain, food consumption, food efficiency, oestrus cyclicity, hematological, blood chemical or clotting parameters, urinalysis results, organ weights (absolute and relative), or histological parameters.
In both male and female rats, there was an increase in mean serum thyroid-stimulating hormone (TSH) levels, and this increase was statistically significant in both male and female animals. Due to a lack of traditional dose-response, a lack of corresponding changes in T3 and T4 levels, a lack of correlating histopathological or organ weight changes in thyroid and pituitary glands, and the fact that all thyroid hormone values measured in this study (including TSH) fell within historical negative control ranges, it was concluded that Fy Protein™ did not have any adverse toxicological effects targeting the thyroid.
Under the conditions tested in the 90-day study, no signs of Fy Protein™ toxicity were identified. The No Observed Adverse Effect Level (NOAEL) was determined to be the highest dose, 150 000 ppm, corresponding to 8115 mg/kg bw per day for males and 9892 mg/kg bw per day for females. The margin of exposure (MoE) between the NOAEL and the estimated exposure (1.0 g/kg bw per day) is 8.1 or 9.9 for males and females, respectively. MoEs of this magnitude are generally considered acceptable for risk assessments of major macronutrients, which are present in foods in substantial quantities.
In addition to the full suite of toxicological studies conducted with Fy Protein™, the toxicological safety of this ingredient is further supported by reproductive, developmental, chronic, and carcinogenicity studies conducted with a very similar product, Quorn™ mycoprotein. These studies showed no adverse effects, and Quorn™ has an extensive history of safe food use worldwide.
Allergenicity
The amino acid sequence of each predicted Fusarium sp. strain flavolapis protein was queried against all the known allergens listed in the Allergen Online database. Using the homology threshold specified by Codex Alimentarius (2003), 262 Fusarium sp. strain flavolapis proteins were identified that possessed an 80-amino acid sliding window homology of >35% to allergens in the database. Given the conservative nature of the 35% threshold, Nature's Fynd refined the criteria to >50%, and took into account the statistical significance of the match by adding E-value criteria to their search.
The majority of the Fusarium sp. strain flavolapis protein matches were to fungal inhalation allergens, although a portion of Fusarium sp. strain flavolapis proteins showed homology to one or more food allergens. However, most of the proteins with homology to food allergens also shared strong homology with various other types of unrelated food allergens, as well as non-food allergens, suggesting that these homologies are more a result of genes that have been evolutionarily conserved across diverse species than an indicator of possible IgE cross-reactivity.
The identified Fusarium sp. strain flavolapis proteins shared significant sequence homology with at least one protein from Fusarium venenatum (i.e., the source organism for Quorn™ mycoprotein). The extensive history of safe food use of Quorn™ suggests that a considerable population has already been exposed to proteins that are very similar to the "high-homology" proteins in Fusarium sp. strain flavolapis. Cases of allergy to Quorn™ have been reported in the literature, primarily in individuals with a pre-existing allergy to some molds; however, these are extremely rare.
Fy Protein™ was tested in simulated gastric digestion and simulated intestinal digestion assays. The results suggest that Fy Protein™ is readily digestible; digestibility of the proteins confers a reduced risk for allergy. In addition, Nature's Fynd confirmed that the heat-inactivation step following the fermentation process in the manufacture of Fy Protein™ ensures that all proteins are denatured; denatured proteins are less allergenic because they tend to be more rapidly digested, and less likely to interact with the immune system.
Conclusion
Health Canada's review of the information presented in support of the use of Fy Protein™ as a food ingredient and alternative protein source has concluded there are no food safety concerns for the general population.
It is the continuing responsibility of the manufacturers, sellers, and distributors of Fy Protein™ to ensure that marketed products are in compliance with all statutory and regulatory requirements, including labelling and advertising.
This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food 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 Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca
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