Novel Food Information: Napin-rich Canola Protein Isolate

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• Background
1. Introduction
2. Development of the Product
3. Dietary Exposure
4. Chemistry
5. Microbiology
6. Nutrition
7. Toxicology
8. Allergenicity
• Conclusion

Background:

Health Canada has notified Merit Functional Foods Corporation that it has no objection to the food use of a napin-rich canola protein isolate (NRCPI) as a food ingredient and alternative protein source in unstandardized foods. The Department conducted a comprehensive safety assessment 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 Merit Functional Foods Corporation and the evaluation by Health Canada. This document contains no confidential business information.

The term "canola" refers to species of rapeseed (Brassica napus L. and Brassica rapa L.)and mustard (Brassica juncea L.) which produce oil that is low in erucic acid and glucosinolates. Canola species belong to the mustard or cabbage family of plants.

Napin refers to one of the two major storage proteins in canola seed.

1. Introduction

Merit has developed a napin-rich canola protein isolate (NRCPI) for use as an alternative protein source in unstandardized foods.

NRCPI is a high protein (≥90% dry basis) powder with a protein fraction containing a minimum of 80% napin. NRCPI is derived from the cold-pressed seeds of low erucic acid-low glucosinolate rapeseed (e.g., Brassica napus L. and Brassica rapa L) or canola-quality mustard (e.g., Brassica juncea). These seeds are from conventional (i.e., not genetically modified) canola species.

Merit indicated that the notified protein isolate (NRCPI) is substantially equivalent to another napin-rich canola protein isolate that is generally recognized as safe (GRAS) in the United States as described in GRAS Notice No. 327 (GRN No. 327). The specifications for NRCPI are the same as the protein isolate presented in GRN No. 327, and the manufacturing process is very similar except that the starting material in GRN No. 327 is heat and solvent treated canola meal that is derived from heat-treated seeds that are pressed and subsequently defatted by counter-current hexane extraction. Merit indicated that information presented in GRN No. 327 can be used to support the safety of NRCPI derived from cold-pressed canola meal.

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 NRCPI, the specifications and manufacturing methods, its intended use, nutritional composition; microbiological and toxicological information, as well as the presence of potential allergens. Merit Functional Foods Corporation provided data to support that NRCPI 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 of Part B of the Food and Drug Regulations (Novel Foods). Food use of protein fractions from canola has not been reported to any great extent. Therefore, NRCPI was considered a novel food 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".

2. Development of the Product

NRCPI is manufactured following the sequence below:

1. cold-pressed canola meal is mixed with salt water at a specific temperature and pH;
2. insoluble materials, including residual oil, are removed by decantation;
3. the resulting solution is concentrated by ultrafiltration, pasteurized, and cooled allowing for the precipitation and removal of non-napin proteins;
4. the supernatant, which contains the napin proteins, is further purified and subjected to a second ultrafiltration which retains the napin proteins and greatly reduces contaminant and anti-nutrient levels;
5. the retentate is heat treated, spray dried, and sieved to form the final napin-rich protein isolate.

3. Dietary Exposure

Merit estimated mean and 90th percentile intake of NRCPI for various age- and sex-groups based on data collected in the United States as part of the National Health and Nutrition Examination Survey (NHANES) 2013-2014. The exposure assessment assumed ingredient use of NRCPI in a wide variety of foods at levels ranging from 2-35 percent. Adolescent males (12-19 y) were estimated to have the greatest per capita intakes with mean and 90th percentile values of 33.43 and 58.85 g/day, respectively. Children (3-11 y) were estimated to have the greatest intake expressed on a per kilogram of body weight basis with mean and 90th percentile values of 0.84 and 1.44 g/kg/day, respectively.

Merit indicated that there is a high degree of similarity in the reference amounts for common foods between Canada and the United States, and indicated that market research found that Americans and Canadians reported very similar preferences for plant-based proteins. It is noted that the estimated intake of NRCPI exceeds those determined for soy protein from all food sources using data from the Canadian Community Health Survey—Nutrition. ¹ Thus, the exposure assessment is unlikely to underestimate exposure to NRCPI compared to the intake of a similar product by Canadians (i.e., soy protein isolate).

4. Chemistry

Merit analyzed multiple batches of NRCPI for trace elements (arsenic, cadmium, lead, and mercury), 11 mycotoxins, two polycyclic aromatic hydrocarbons, dioxins, and 17 pesticides deregistered in Canada and therefore considered to be environmental contaminants. The petitioner reported the majority of the trace elements were 'not detected' with the limits of detection (LOD) ranging from 0.001 to 0.01 parts per million. Using either the quantified analytical results or assuming the LOD values, it was determined that addition of NRCPI to the specified foods is not expected to result in increased concentration of chemical contaminants in the commodities to which it is added or would have a negligible impact on total dietary exposure to these contaminants.

NRCPI is a discreet ingredient that exists independent of any processing aids, additives, or other substances that the manufacturer may choose to use in processing it. Use of substances in manufacturing NRCPI must comply with relevant legal provisions where they exist, such as food additive provisions, and must not result in a violation of Section 4 of the Food and Drugs Act.

5. Microbiology

The temperature and filtration steps of the manufacturing process help to ensure the microbiological safety of NRCPI. To demonstrate compliance with microbiological specifications, Merit provided data on three non-consecutive production lots of NRCPI. The various microbiological tests, the tolerance limits, and methodologies were reviewed as they pertain to microbiological safety. The microbial analysis was conducted using validated methods from Health Canada's Compendium of Analytical Methods, and the specifications were considered acceptable. The results of the batch testing demonstrate that the microbiological specifications for the finished canola products are met consistently in a production run.

6. Nutrition

The reported proximate and amino acid composition of NRCPI derived from cold-pressed canola meal was substantially equivalent to the isolate obtained from heat-solvent treated canola meal as presented in GRN No. 327.

Merit provided evidence of digestion and absorption of NRCPI by measuring true fecal digestibility of its crude protein in rats. The results indicated that the digestibility of NRCPI was comparable to soy protein isolate.

Merit provided the Protein Digestibility-Corrected Amino Acid Score (PDCAAS) and the adjusted Protein Efficiency Ratio (PER) of NRCPI as measures of protein quality. Both the PDCAAS and PER were less than values for casein. However, the results indicated that the protein quality of NRCPI is within the range of common food proteins, and better than some other plant-based proteins.

As NRCPI is an incomplete protein of lower quality than casein, use as the only source of dietary protein would be inappropriate, similar to many other plant-based proteins. Merit proposed that NRCPI will be used as an alternative protein source as part of a mixed diet consumed ad libitum. In this context, individuals can easily meet metabolic demands by consuming a variety of food proteins which complement each other in terms of their amino acid profile. Only in an extreme scenario, in which high consumers exclude nearly all other sources of protein from their diet, is potential inadequacy possible. However, such a worst case scenario is unrealistic and implies the consumption of an unbalanced diet which is generally not recommended.

Merit indicated that the anti-nutrient and erucic acid composition of NRCPI derived from cold-pressed canola meal would be entirely comparable to the napin-rich canola protein isolate obtained from heat-solvent treated meal as presented in GRN No. 327. Based on the close similarities in nutritional composition and manufacturing method, this rationale was accepted for erucic acid and all measured anti-nutrients (glucosinolates, total phenolic acids, sinapic acid, phytic acid) except for the glucosinolate hydrolysis products (isothiocyanates, nitriles). The erucic acid content of NRCPI is expected to be negligible, whereas residual anti-nutrients are expected to be present in amounts comparable to other common foods with the exception of the glucosinolate hydrolysis products, whose levels are difficult to estimate and were not directly measured for NRCPI.

Glucosinolate hydrolysis products are formed from glucosinolates by the action of the enzyme myrosinase when they are brought into contact with each other following the rupture of plant cell walls. Exposure to these hydrolysis products is common following consumption of foods containing glucosinolates and myrosinase (e.g., cruciferous vegetables). Seed cooking during production of canola meal normally eliminates most myrosinase activity, but is not completely effective in eliminating exposure to glucosinolate hydrolysis products due to the action of myrosinases produced by the intestinal microflora. As heat treatment of seeds prior to pressing is not part of the production process for NRCPI, it is possible that glucosinolate hydrolysis products may be present in higher amounts than in the heat-solvent extracted napin-rich canola protein isolate presented in GRN No. 327. However, these levels are expected to be reduced by filtration steps during manufacturing, and exposure to endogenously produced hydrolysis products are limited by the total amount of glucosinolates which are expected to be present in amounts well below those found in broccoli.

Importantly, unacceptably high levels of glucosinolate hydrolysis products and the other anti-nutrients would be indicated by growth depression in animal feeding studies through potential impacts on protein digestibility, interference with mineral metabolism, or both. The petitioner measured the adjusted PER of NRCPI consistent with Health Canada's official method FO-1 (Determination of Protein Rating, October 15, 1981), which is a bioassay designed to identify differences between proteins in their capacity to promote weight gain in growing animals. The adjusted PER of NRCPI was within the range of common food proteins, and was greater than that of some other plant-based proteins. These results indicate that potential nutritional hazards posed by glucosinolate hydrolysis products and the other anti-nutrients in NRCPI are not a concern, as they are ultimately indistinguishable from the effects of eating any one of many non-novel food proteins of similar quality.

7. Toxicology

In order to allow bridging of the safety data from the napin-rich canola protein isolate presented in GRN No. 327 to NRCPI, Merit demonstrated substantial equivalency between isolate obtained from both cold-pressed and heat-solvent treated canola meal. Equivalency was based upon the constituents present (i.e., protein, moisture, ash, fat, amino acids), the proportions of those constituents (e.g., both the GRAS and notified formulations contained ≥ 90% protein), and the ratio of the storage protein (i.e., both the GRAS and notified formulations contained ≥ 80% napin, with the remainder being cruciferin). Based on this substantial equivalency, the data from GRN No. 327 was used to support the safety of NRCPI. All studies followed their respective Organisation for Economic Co-operation and Development (OECD) guidelines.

No genotoxicity was observed in the Ames assay, the in vivo bone marrow micronucleus assay, or the in vitro mammalian cell gene mutation test (mouse lymphoma assay). In a 90-day oral (dietary) rat study, statistically lower body weight gains were observed in the mid- and high-dose groups for males, and in the high-dose group for females; however, body weights (in all dose groups) were stated to be within historical control ranges. These reduced body weight gains were associated with reduced food consumption, due to the poor palatability of the dietary test material. Animals were otherwise determined to be in good health. The highest dose tested was considered to be the NOAEL, and is 11.24 g/kg bw/day.

Merit also noted that the canola species used for the production of canola protein isolates is bred to be low in anti-nutrients/toxins of concern, such as glucosinolates and erucic acid. Ultrafiltration steps during manufacturing help to remove these substances, and heat treatment during pasteurization and drying may further reduce the formation of toxic metabolites from glucosinolates. The petitioner submitted data for NRCPI, which demonstrated that the level of glucosinolates was < 1.95 µmol/g and erucic acid was ≤ 0.0016%; which is lower than levels found in common foods in the diet, and comparable to levels present in the test article from the aforementioned 90-day oral toxicity study.

Merit estimated a maximum dietary exposure of 1.44 g/kg bw per day, observed for the 90th percentile in children (3-11 years old). This is approximately 10-fold lower than the NOAEL obtained for the 90-day oral toxicity study in rats (11.24 g/kg bw per day; highest dose tested). This margin of exposure was determined to be sufficient, given the lack of toxicity in the safety study, as well as the lack of genotoxicity in several assays. It is also noted that other jurisdictions (Food Standards Australia New Zealand; European Food Safety Authority) that approved canola protein isolates assessed higher estimated maximum dietary exposures that ranged from 2.28 to 4.73 g/kg bw per day.

8. Allergenicity

Canola is a member of the mustard family Brassicaceae (also known as Cruciferae). Mustard is a priority allergen.

Merit provided an updated bioinformatics analysis for the allergenicity assessment of canola napin. The sequence was compared to known allergens using AllergenOnline.com (version 21.0; February, 2021), with full-length and sliding 80 amino acid (80-mer) segment FASTA comparisons. The highest identity matches were to mustard allergens from Brassica juncea and Sinapis alba; between 50% and 95% identical, based on full length FASTA alignments. The next highest matches were to tree nut allergens; full length sequence identity was below 50%, and with regard to the 80-mer sliding window, identities in the 35-41% range were observed.

The petitioner summarized an in vitro digestion assay conducted with the napin-rich canola protein isolate presented in GRN No. 327. The results demonstrate that the protein was stable for pepsin digestion at both pH 1.2 and 2.0, up to the time interval of 60 minutes (the maximum duration tested). Resistance to digestion is a common trait associated with food allergens. However, the relationship between digestibility and allergenicity is complex; it may be supportive, but it is not definitive.

The high identity matches to mustard allergens (between 50% and 95% for overall identity) and the observed resistance to digestibility in the aforementioned in vitro study, results in concern for potential cross-reactivity with mustard allergens. The homology to tree nuts was determined not to be a concern following an expert opinion from the Food Allergy and Food Intolerance Assessments Section.

Merit agrees that cautionary labelling for consumers with mustard allergy is warranted, and indicated that NRCPI will be labelled with an advisory warning indicating that it may be unsuitable for those with mustard allergy. It is noted that NRCPI manufactured from seeds of canola-quality mustard (e.g., Brassica juncea) would trigger mandatory "enhanced" food allergen labelling for mustard/mustard seeds.

Due to its concern regarding potential cross-reactivity, Health Canada will update its webpages to inform consumers that canola protein should not be consumed by those with mustard allergies. In addition, Health Canada will proactively contact representatives of Food Allergy Canada to explain Health Canada's position regarding consumption of canola protein by individuals with mustard allergies, and will inform Food Allergy Canada of Health Canada's plan to update its webpages with regard to its concern.

Conclusion:

Health Canada's review of the information presented in support of use of NRCPI as an alternative protein source has concluded that there are no food safety concerns for the general population. To address the allergenic risk to mustard allergic individuals, NRCPI will carry a label statement about this risk. Furthermore, Health Canada will update its webpages to inform consumers that canola protein should be not be consumed by those with mustard allergies, and will proactively inform Food Allergy Canada of this opinion.

It is the continuing responsibility of the manufacturers, sellers and distributors of NRCPI 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.

(Également disponible en français)

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