Novel Food Information: Soy leghemoglobin (LegH) preparation as an ingredient in all simulated meat and poultry products

On this page

• Background
1. Introduction
2. Development of the production organism
3. Manufacturing of the LegH preparation
4. Product Information
5. Dietary Exposure
6. Nutrition
7. Microbiology
8. Chemistry
9. Toxicology
10. Allergenicity
• Conclusion

Background:

Health Canada has notified Impossible Foods Inc. (California, U.S.) that it has no objection to the food use of soy leghemoglobin (LegH) preparation as an ingredient in all simulated meat and poultry products at a maximum soy leghemoglobin protein level of 0.8 %. The Department conducted a comprehensive safety assessment of this ingredient 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 Impossible Foods Inc. and the evaluation by Health Canada. This document contains no confidential business information.

1. Introduction

Health Canada had previously conducted a pre-market safety assessment and issued a Letter of No Objection for the food use of the LegH preparation in Impossible Foods Inc.'s simulated meat product (i.e., the Impossible Burger) and other simulated ground beef analogues, at a maximum soy leghemoglobin protein level of 0.8 % as novel foods in Canada.

In October 2020, Health Canada received a new novel food submission from Impossible Foods Inc. The petitioner indicated that they intend on marketing additional simulated meat and poultry products (e.g., simulated ground pork and poultry) that would contain their LegH preparation. As such the petitioner requested an expansion in the use of the LegH preparation to all simulated meat and poultry products, at a maximum soy leghemoglobin protein level of 0.8 %. Updated dietary exposure data were provided to support the expanded food use of the LegH preparation.

In the new submission, the petitioner indicated that the manufacturing process and product specifications of the LegH preparartion have not significantly changed compared to the previous novel food submission. As such, the information provided in the previous pre-market assessment to support the safety of the LegH preparation is applicable to the assessment of this ingredient regarding its expanded food use. Additional information to support the safety of any manufacturing changes was also provided.

The safety assessment performed by the Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. The assessment considered: the safety of the Pichia pastoris production organism, the manufacturing process for the LegH preparation, the nutritional composition of the LegH preparation, and what the potential is for the preparation to present a toxic or allergenic concern. Impossible Foods Inc. has provided data to support that the LegH preparation is safe for use as an ingredient in all simulated meat and poultry products at a maximum soy leghemoglobin protein level of 0.8 % 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). The LegH preparation is considered to be 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;"

2. Development of the Production Organism

No changes were made to the production organism compared to what was previously assessed in 2018. The Pichia pastoris production organism was developed from a parental strain with an established history of safe use in the food industry. The LGB2 gene derived from soybean (Glycine max L.) encoding for the soy leghemoglobin protein was introduced into the P. pastoris genome along with the regulatory elements required for protein expression. Futhermore, as the soy leghemoglobin protein uses heme as a cofactor, the heme biosynthesis pathway of P. pastoris was subcloned into the yeast genome, resulting in an upregulation of heme production and the sufficient production of heme-bound soy leghemoglobin protein. The petitioner provided sufficient evidence to demonstrate that the parental strain is a non-pathogenic, non-toxigenic microorganism, and that the genetic changes made in developing the final production organism are well-characterised and do not introduce any element which would increase the production organism's potential pathogenicity, toxicity, or allergenicity

3. Manufacturing of the LegH Preparation

No significant changes were made to the manufacturing process compared to what was previously assessed in 2018. The manufacturing process of the LegH preparation consists of a controlled fermentation of the production organism which results in the expression of the soy leghemoglobin protein within the microorganism. After fermentation, the P. pastoris cells are lysed to release the desired soy leghemoglobin protein into the surrounding culture medium. This medium undergoes a series of filtration steps to isolate and concentrate the soy leghemoglobin protein, which is then formulated with suitable stabilizers to produce the standardized LegH preparation.

4. Product Information

The LegH preparation is a mixture containing soy leghemoglobin protein, P. pastoris (yeast) proteins, and stabilizers (e.g., sodium chloride and sodium ascorbate). The preparation is added to simulated meat and poultry products to provide nutrition, and the flavour and aroma of traditional animal-derived meat.

The soy leghemoglobin protein is the principal component of the LegH preparation. The source of soy leghemoglobin protein is the soybean plant (G. max L.) Leghemoglobin is a small 16-kDa holoprotein (i.e., protein plus a heme cofactor) expressed within the nitrogen-fixing root nodules of soy. The soy leghemoglobin protein that is the subject of this submission is expressed by a P. pastoris strain genetically modified to contain the soy gene encoding the leghemoglobin protein. The expressed leghemoglobin protein is subsequently isolated and purified to manufacture the final LegH preparation.

The LegH preparation is standardized to contain up to 9 % soy leghemoglobin on a wet weight basis and a soy leghemoglobin protein purity of at least 65 %. While the previous assessment was for the use of up to 0.8 % soy leghemoglobin protein in simulated ground beef products only, this assessment expands the use to all simulated meat and poultry products up to a maximum of 0.8 % soy leghemoglobin in each type of product. The LegH preparation will not be marketed to food manufacturers and therefore will be exclusively used in simulated meat and poultry products manufactured by Impossible Foods Inc. The petitioner's products are intended to be sold to restaurants as well as supermarket retail establishments.

5. Dietary Exposure

The LegH preparation will be added to Impossible Foods Inc.'s simulated meat and poultry products to deliver not more than 0.8 % soy leghemoglobin protein in each type of product. As in the previous assessment, the use of the LegH preparation in simulated meat and poultry products is self-limiting as the imparted flavour would become unpleasant at higher levels.

Health Canada performed an updated estimated dietary intake of the LegH preparation for the proposed new uses based on data generated from the 2015 Canadian Community Health Survey (CCHS)¹. The Department assumed that the maximum proposed level (0.8 % soy leghemoglobin protein) would be used in all simulated meat and poultry products, and that simulated meat and poultry products containing the ingredient would substitute all meat and poultry.

Based on this conservative dietary estimate, there are no safety concerns related to these simulated meat and poultry products containing the soy leghemoglobin protein at a maximum level of 0.8 % regarding dietary exposure to heme iron.

6. Nutrition

Soy leghemoglobin is a source of iron, analogous to the role of myoglobin being an iron source in meat. The petitioner provided data that demonstrate that heme iron from soy leghemoglobin has similar bioavailability compared to bovine hemoglobin when consumed as part of a food matrix². Currently, heme iron in the human diet is almost exclusively from animal sources, and its intake has been shown to have a positive correlation with iron status.

A worst-case exposure to soy leghemoglobin was estimated based on eater's only consumption information from the 2015 CCHS. Total meat consumption was used as proxy for consumption of all simulated meat and poultry products. It was assumed that this ingredient would be used at the maximum proposed level of use (0.8 % in all simulated meat and poultry products). Current intakes of iron³ by the Canadian population were added to the estimated iron exposure derived from soy leghemoglobin intake when used at a level of 0.8 % in simulated meat and poultry products. Total iron intakes were estimated to be well below (≤60 %) the tolerable upper intake level for iron at the 90th percentile⁴ . It is important to note that this estimate is considered very conservative and intakes will likely be much lower.

Based on the information submitted, Health Canada has not identified any nutritional concerns with the use of the LegH preparation in all simulated meat and poultry products, at a maximum soy leghemoglobin protein level of 0.8 %.

7. Microbiology

The petitioner confirmed that the manufacturing process and product specifications of the LegH preparation have not significantly changed compared to the previous novel food submission.

Based on the information previously provided, the Department has no safety concerns regarding the food use of the LegH preparation as an ingredient in all simulated meat and poultry products from a microbiological perspective.

8. Chemistry

According to the petitioner, there have been no significant changes to the manufacturing of the LegH preparation from the previous pre-market assessment. Health Canada has reviewed all the provided information and did not identify any food safety concerns.

Results of analyses of trace elements of greatest potential concern to human health, that is, arsenic, cadmium, lead and mercury, were provided by the petitioner in their previous submission and were deemed suitably low. The estimated concentrations of these trace elements in the final simulated meat and poultry products, calculated based on analytical results and the maximum level of use of soy leghemoglobin protein indicated by the petitioner of 0.8 %, are below background levels typically observed in various meat and poultry products sold in Canada. The addition of the LegH preparation to simulated meat and poultry products at the maximum proposed level of use is not expected to represent a health concern from a chemical contaminants perspective.

Based on the information provided, the Department has not identified any chemical safety concerns with the proposed use of the the LegH preparation in simulated meat and poultry products at a maximum soy leghemoglobin protein level of 0.8 %.

9. Toxicology

In 2019, Health Canada concluded that LegH preparation as an ingredient in simulated beef products was not expected to pose a toxicological concern when used at soy leghemoglobin protein levels up to 0.8 %. No additional toxicological data accompanied this submission. The LegH preparation composition has not changed, thus the toxicological studies previously provided by the petitioner can be used to support the proposed new uses.

The production organism (P. pastoris) is not toxigenic. Bioinformatics analysis conducted by the petitioner did not identify the soy leghemoglobin protein, or the P. pastoris proteins present in the preparation (at >1 %), to be significantly similar to any known toxins.

In vitro studies demonstrated that soy leghemoglobin and P. pastoris proteins are expected to be susceptible to in vivo digestive processes, similar to other dietary proteins.

Toxicology studies were conducted according to their respective Organization for Economic Co-operation and Development (OECD)guidelines for the Testing of Chemicals and under Good Laboratory Principles.

The LegH preparation was not mutagenic in a bacterial reverse mutation assay, and was not clastogenic in an in vitro chromosomal aberration test. Based on the negative results from these studies, the LegH preparation is not expected to be genotoxic.

No hazard was identified for the LegH preparation in an oral toxicity study in rats. A NOAEL of 1526 mg LegH preparation/kg bw per day (equal to 750 mg soy leghemoglobin/kg bw per day), the highest dose tested, was established in a 28-day dietary toxicity study. The subacute toxicity study was considered adequate to assess the toxicity of the soy leghemoglobin protein because protein toxins as a structural class can be defined as acute toxins, and therefore any effects would be observed within a shorter timeframe.

In order to assess the higher exposure level for the LegH preparation, Health Canada conducted an updated estimate of dietary intake based on data generated from the 2015 CCHS. The Department conservatively assumed a use level of 0.8 % soy leghemoglobin protein in all simulated meat and poultry products, and that simulated meat and poultry products containing the ingredient will substitute all meat and poultry.

The margins of exposure (MOEs) resulting from the comparison of the NOAEL of 1526 mg LegH preparation/kg bw/day in the rat oral toxicity study with the estimated dietary exposures for the highest consumers of meat and poultry (i.e., children 1-3 years old) were calculated to be 14 and 6 for the mean and 90th percentile consumers, respectively⁵. These MOEs are not considered to be of concern given that the critical point of departure was the highest dose tested (and did not show any adverse effects), as well as the conservative nature of the dietary exposure assessment. Additionally, there is an absence of genotoxicity based on the studies provided. Furthermore, the production organism is not toxigenic, and the soy leghemoglobin and P. pastorisproteins will be digested like other dietary proteins, and do not share any significant similarities to known toxins.

As of October 2020,⁶ more than 100 million quarter-pound servings of ground beef analogue products (i.e., the Impossible™ Burger) containing LegH preparation have been sold; no safety issues concerning the soy leghemoglobin preparation have been identified. The available evidence does not indicate a significant public health concern from consumption of the LegH preparation.

Based on the information provided, the Department did not identify any toxicological concerns with the use of soy leghemoglobin protein in the form of LegH preparation in all simulated meat and poultry products, at a maximum soy leghemoglobin protein level of 0.8 %.

10. Allergenicity

No additional information on potential for allergenicity was provided compared to what was previously assessed. Although there are several known soy allergens (Gly m 4, Gly m 5, Gly m 6 and others), these allergens are absent from the LegH preparation produced by P. pastoris.

Bioinformatics analyses confirmed the absence of significant similarity of the amino acid sequence of the soy leghemoglobin and P. pastoris proteins to known food allergens (i.e., >35 % sequence homology over a window of 80 amino acids; sequence homology with eight contiguous amino acids).

The petitioner demonstrated that soy leghemoglobin would be denatured at standard cooking temperatures, as well as in acidic conditions that are expected to mimic the stomach environment. Generally, denatured proteins are more accessible as substrates for proteolysis, and the protein would be expected to be digested. Furthermore, results from a simulated gastric fluid assay suggested that the soy leghemoglobin protein and the P. pastoris proteins are readily digestible under conditions of normal digestion.

Based on the information provided, the Department has no safety concerns from an allergenic perspective regarding the use of the LegH preparation produced in P. pastoris for the proposed maximum inclusion of 0.8 % soy leghemoglobin protein in all simulated meat and poultry products.

Conclusion:

The safety assessment conducted by Health Canada has determined that the LegH preparation as an ingredient in all simulated meat and poultry products, at a maximum soy leghemoglobin protein level of 0.8 %, is safe for human consumption.

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..

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For further information, please contact:

Health Canada
Novel Food Section
Food Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
PL2204E
Ottawa, Ontario, K1A 0K9
bmh-bdm@hc-sc.gc.ca