Soy leghemoglobin (LegH) preparation as an ingredient in a simulated meat product and other ground beef analogues
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Health Canada has notified Impossible Foods, Inc. (California, U.S.) that it has no objection to the use of soy leghemoglobin (LegH) preparation as an ingredient in a simulated meat product (i.e., the Impossible™ Burger) and other ground beef analogues. The Department conducted a comprehensive 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.
Impossible Foods, Inc. has developed a soy leghemoglobin (LegH) preparation to be used as an ingredient in their ground beef simulated meat products including the Impossible™ Burger, a plant-based simulated meat product that emulates the taste and texture of a real beef burger. The LegH preparation is a mixture containing soy leghemoglobin protein, Pichia (yeast) proteins, and stabilizers (e.g., sodium chloride and sodium ascorbate). The preparation is added to these simulated meat products to provide nutrition, and the flavour and aroma of traditional animal-derived ground beef.
The soy leghemoglobin protein is the principal component of the LegH preparation. The source of the soy leghemoglobin protein is the soybean plant, Glycine max L. Soy leghemoglobin is a small 16 kDa holoprotein (i.e., a protein plus a heme cofactor) expressed within the nitrogen-fixing root nodules of the soybean plant. The root nodules are not part of the edible soybean tissues consumed by humans and thus there is no history of consumption for the soy leghemoglobin protein.
To manufacture the LegH preparation, the soy leghemoglobin protein is expressed by a yeast (Pichia pastorisFootnote 1) strain genetically modified to express the soy leghemoglobin protein. The expressed protein is subsequently isolated and purified to manufacture the final LegH preparation.
The LegH preparation will be marketed exclusively as a component of the company’s ground beef simulated meat products including the Impossible™ Burger, in Canada. Retail avenues include grocery store outlets and restaurants. For this submission, Impossible Foods, Inc. has indicated that the LegH preparation will not be marketed as an ingredient for general purchase and food use by other manufacturers.
The safety assessment performed by Food Directorate scientific 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 reflect international approach in this area (e.g., Codex Alimentarius). The safety 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.
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
The P. 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 G. 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 Pichia pastoris was recloned 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
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
A typical Impossible™ Burger weighs 113 g and has the following composition: 50-75 % water; 10-25 % proteins; 0-25 % oils; and 2 % miscellaneous components (i.e., salt, flavours, vitamins, essential amino acids). In addition to the LegH preparation, the Impossible™ Burger and the company’s other ground beef analogues may contain other proteins such as soy, pea, mung bean, lentil, corn, potato, and wheat.
5. Dietary Exposure
The LegH preparation will be added to the company’s ground beef simulated meat products including the Impossible™ Burger, to deliver not more than 0.8 % soy leghemoglobin protein, which is comparable to the myoglobin protein content of beef (i.e., 0.8-1.8 %). The use of the LegH preparation in simulated meat products is self-limiting as the imparted flavour would become unpleasant at higher levels.
In addition to the primary purposes of improving flavour and providing aroma, soy leghemoglobin protein has a nutritive value as a source of iron, analogous to the role of myoglobin as an iron source in meat. The petitioner has provided data to show that bovine hemoglobin and soy leghemoglobin protein have similar bioavailability when consumed as part of a food matrixFootnote 2. 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.
The average adult male requires 8 mg of iron to be absorbed from the diet on a daily basisFootnote 3. Adult women require 8-18 mg/day. Daily iron requirements for children, male adolescents, and female adolescents are 7-10, 8-11, and 8-15 mg, respectively. Pregnant women require 27 mg/day of iron.
Iron in the body is found primarily as hemoglobin, and the amount of iron stored in the body is directly related to serum ferritin levels. Mean corpuscular volume (i.e., average size of red blood cells) is a reliable indicator of reduced hemoglobin synthesis indicating iron-deficient erythropoiesis. Iron-deficiency anemia is often characterised by a reduction in the blood concentration of hemoglobin. In Canadians, 97 % of people aged 3-79 have hemoglobin levels at or above the reference values and 96 % of Canadians have sufficient serum ferritin levelsFootnote 4.
The estimated daily intake of soy leghemoglobin protein was calculated assuming 100 % of ground beef products will be replaced by ground beef simulated meat products containing soy leghemoglobin protein. Currently, the petitioner states that this ingredient is only intended for use in Impossible Foods, Inc.’s the Impossible™ Burger, however it may be used in the future for other ground beef simulated meat products. As such, the exposure assessment is considered very conservative.
Based on the estimated daily intake of ground beef (eaters only), the intake of soy leghemoglobin protein at the 90th percentile will be 800-1066 mg/day if all ground beef products were substituted for the soy leghemoglobin protein containing ground beef simulated meat products. The intake of soy leghemoglobin protein in one (1) Impossible™ Burger was estimated to be 678-904 mg/day. These values are comparable to the current consumption of myoglobin protein from meat and poultry sources.
The estimated exposure of soy leghemoglobin protein was used to determine the iron intake from soy leghemoglobin protein; namely 2.8-3.7 mg/day at the 90th percentile. The iron intakes from soy leghemoglobin protein consumption and from ground beef consumption are very similar and therefore no change in iron intake is expected from replacing ground beef with the soy leghemoglobin protein-containing simulated meat product.
The petitioner will be required to meet the provisions set out in Sections B.14.085 to B.14.090 of the Food and Drug Regulations for simulated meat products that include a minimum protein rating, an upper limit on fat content, a lower limit on protein and iron content, and lower limits for specific vitamins and minerals.
Based on the information submitted, the Nutrition Premarket Assessment Division (NPAD) has no safety concerns from a nutritional perspective with the use of the soy leghemoglobin protein at a level up to 0.8 % in ground beef simulated meat products (e.g., the Impossible™ Burger).
The petitioner provided the batch analyses of five (5) non-consecutive batches of LegH preparation. The microbiological specifications of the LegH preparation include an absence of enterohemorrhagic Escherichia coli (EHEC) including E. coli O157:H7, Salmonellaspp., and Listeria monocytogenes. The results of these batches demonstrated that the microbiological specifications for the finished LegH preparation are consistently met in a production run. All analytical methods are in accordance with AOAC International standards.
Based on the data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding the food use of the LegH preparation as an ingredient in ground beef simulated meat products such as the Impossible™ Burger from a microbiological perspective.
The food additive sodium ascorbate is added as a preservative during the final stages of the manufacturing process of the soy leghemoglobin protein and is permitted for such use (unstandardized foods) as per the List of Permitted Preservatives.
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. The LegH preparation consistently contains lower concentrations of these trace elements than the petitioner’s specifications, which are deemed to be suitably low. As well, the concentration of these trace elements in the final simulated meat product (i.e., the Impossible™ Burger), calculated based on the maximum level of use of soy leghemoglobin protein indicated by the petitioner (i.e., 0.8 %), are below background levels observed in ground beef sold in Canada. The addition of the LegH preparation to ground beef simulated meat products at the maximum proposed level of use is not expected to represent a health concern.
Based on the information provided, the CHHAD has not identified any chemical safety concerns with the proposed use of the soy leghemoglobin protein (and thus the LegH preparation) in ground beef simulated meat products such as the Impossible™ Burger.
The Pre-Market Toxicology Assessment Section (PTAS) evaluated the safety of the LegH preparation by considering information pertaining to the safety of the production organism and the potential toxicity of the soy leghemoglobin protein, as well as the preparation as a whole.
The petitioner provided information that showed soy leghemoglobin protein is structurally similar to other heme-proteins consumed in the diet, such as myoglobin present in animal meats. This supports the suggestion that the heme B molecule, a co-factor in the soy leghemoglobin protein and other hemoglobins, has a safe history of human food consumption.
A bioinformatics evaluation conducted by the petitioner did not identify the soy leghemoglobin protein, or the P. pastoris proteins present in the LegH preparation as having amino acid sequences homologous to any putative toxins. An in vitro simulated gastric fluid (SGF) assay demonstrated that soy leghemoglobin protein and the P. pastoris proteins would be susceptible to in vivo digestive processes.
The petitioner provided toxicology studies that 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 an in vitro reverse mutation assay and was not clastogenic in an in vitro chromosomal aberration test. Under conditions of the assays, the preparation was not considered genotoxic.
A No Observed Adverse Effects Level (NOAEL) of 1526 mg LegH preparation/kg body weight (bw) per day (equal to 750 mg soy leghemoglobin protein), the highest dose tested was established in a 28-day dietary toxicity study in Sprague Dawley rats (10 rats per group per sex). This subacute oral toxicity study was considered adequate to assess the toxicity of the soy leghemoglobin protein as protein toxins as a structural class can be defined as acute toxins.
The Food Additives Section (FAS) provided a conservative estimated dietary intake of the LegH preparation using data generated from the 2015 Canadian Community Health SurveyFootnote 5. FAS made the conservative assumptions that the maximum proposed inclusion level of LegH preparation (0.8 % based on soy leghemoglobin protein) was always used and that the simulated meat products containing the preparation will substitute all ground beef products. FAS estimated that children 1-3 years of age have the highest dietary intake on a body weight basis. The theoretical maximum daily intake (TMDI) of the LegH preparation for this age group is 117.2 mg/kg bw per day.
This dietary intake is 13-foldFootnote 6 less than the reported NOAEL, the highest dose tested in the 28-day toxicity study in rats. This difference is considered sufficient to protect consumers given the exposure estimate is extremely conservative and given the nature of the ingredient, as the dietary intake would be comparable to the current consumption of myoglobin protein from meat. The proteins in the preparation are digested under SGF assay conditions, which are similar to human digestive conditions. Based on this observation, the proteins are expected to be digested like other proteins in the diet.
Based on the available toxicity data, the PTAS has no safety concerns from a toxicological perspective with the use of the LegH preparation produced in P. pastoris for the proposed maximum inclusion of 0.8 % soy leghemoglobin protein in ground beef simulated meat products such as the Impossible™ Burger.
There are several known soy allergens (Gly m 4, Gly m 5, Gly m 6, and others) that are endogenous to the soybean. 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 protein and P. pastoris proteins present in the LegH preparation to known relevant allergens (i.e., >35 % sequence homology over a window of 80 amino acids; and sequence homology with 8 contiguous amino acids).
The soy leghemoglobin protein is denatured by thermal treatment leading to dissociation of the protein polypeptide from the heme co-factor. This information suggests the soy leghemoglobin protein is denatured when consumed in a cooked simulated meat product. Generally, denatured proteins are more accessible as substrates for proteolysis, and the protein would be expected to be digested.
A SGF assay suggests that the soy leghemoglobin protein and the P. pastoris proteins are readily digestible under conditions of normal digestion. Therefore, the proteins are not expected to be available to elicit an immune response.
The weight of evidence suggests that the soy leghemoglobin protein and the P. pastoris proteins present in the LegH preparation are not expected to pose an allergenic concern to consumers.
Based on the information reviewed, the PTAS has no safety concerns from an allergenic perspective regarding the with the use of the LegH preparation produced in P. pastoris for the proposed maximum inclusion of 0.8 % soy leghemoglobin protein in ground beef simulated meat products such as the Impossible™ Burger.
The safety assessment conducted by Health Canada has determined that the LegH preparation as an ingredient in the simulated meat product, the Impossible™ Burger and other ground beef analogues, 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:
Novel Food Section
Health Products and Food Branch
251 Sir Frederick Banting Driveway
Ottawa, Ontario, K1A 0K9
- Footnote 1
The current name for Pichia pastoris (Guillierm.) Phaff is Komagataella pastoris (Guillierm.) Y. Yamada, M. Matsuda, K. Maeda & Mikata, Biosc. Biotechn. Biochem. 59(3): 444 (1995), according to the synonymy summary of Species Fungorum(http://www.speciesfungorum.org/Names/SynSpecies.asp?RecordID=415539)
- Footnote 2
Proulx AK, Reddy MB (2006). Iron bioavailability of hemoglobin from soy root nodules using a Caco-2 cell culture model. J Agric Food Chem 54(4):1518-1522.
- Footnote 3
Institute of Medicine (2001) Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington DC, National Academies Press (US).
- Footnote 4
Cooper M, Greene-Finestone L, Lowell H, Levesque J, Robinson S (2015) Iron Sufficiency of Canadians. Health Reports 82-003-x, vol 23 (4).
- Footnote 5
Health Canada (2019). Food Consumption Table derived from Statistics Canada, Canadian Community Health Survey, Nutrition (2015), Share file. Ottawa.
- Footnote 6
MoE = 1536 mg/kg bw/day/117.2 mg/kg/bw/day = 13.
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