Novel food information: 2′-fucosyllactose (2′-FL) from Escherichia coli K-12 strain INB-2FL_03

Background

Health Canada has notified Inbiose N.V., that it has no objection to the use of 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K-12 INB-2FL_03, "the production strain" or "the strain", as an ingredient in infant formula, toddler formula, infant foods, other foods for special dietary use, as well as foods for young children and the general population. Infant formulas and human milk fortifiers containing 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K-12 INB-2FL_03, are further required to undergo pre-market review under Division 25.The Department conducted a comprehensive assessment of this oligosaccharide 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 Inbiose N.V., and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Inbiose N.V. has developed 2′-fucosyllactose from a genetically modified E. coli K-12 INB-2FL_03, strain, using standard molecular biology techniques. The bacterially synthesized 2′-FL has a purity of ≥ 94% and is expected to be used in infant formula for infants and toddler formulas to a maximum use level of 1.2 g 2′-FL per litre of formula (inclusive of overage). Various maximum use levels are proposed in infant foods, foods for young children and the general population, as well as other foods for special dietary use.

2′-FL is a naturally occurring oligosaccharide in human milk composed of fucose and lactose. The bacterially synthesized 2′-FL produced by Inbiose was confirmed to be chemically and structurally equivalent to 2′-FL isolated from human milk. This evaluation did not include an assessment of any potential claims that could be made about 2′-FL. Genetic modifications were made to INB-2FL_03 for the purpose of inserting the genes involved in the entire 2′-FL biosynthetic pathway. Strain INB-2FL_03 was engineered to: produce 2′-FL from lactose as a substrate, uptake and metabolize sucrose and secrete 2′-FL into the growth medium.

The safety assessment performed by Food and Nutrition 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: how 2′-FL was developed; how the composition and nutritional quality of 2′-FL compared to human milk oligosaccharides (HMOs); and what the potential is for 2′-FL to be toxic or cause allergic reactions.

The Food and Nutrition 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). As this 2′-FL is produced by a genetically modified bacterium, it is considered to be a novel food under the following part of the definition of novel foods:

"c) a food that is derived from a plant, animal, or microorganism that has been genetically modified such that
1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

Development of the modified microorganism

The production strain, INB-2FL_03, was derived from the well-characterized parent strain E. coli K-12 MG1655. E. coli K-12 MG1655 was genetically modified to introduce the entire 2′-FL biosynthetic pathway, which requires the two co-substrates, lactose and fucose [from guanosine diphosphate (GDP)-fucose], to be linked by α-1,2 fucosyltransferase, resulting in 2′-FL. This process depends on lactose and sucrose being imported into the cell from the growth medium. Using site-directed mutagenesis, twenty-three genes were fully or partially deleted and five genes were inserted in the genome. The purpose of the genomic deletions was to ensure optimal growth of the strain, better production of the enzymes, maintenance of the plasmid, and optimized efficiency of the 2′-FL production pathway. The five genes subsequently inserted into the genome were introduced to enable strain growth, uptake of the substrates sucrose and lactose, and the conversion of sucrose to the key intermediate, GDP-fucose, which together with lactose is further converted into 2'FL by an α-1,2 fucosyltransferase enzyme. The production strain is not present in the final 2′-FL product.

Characterization of the modified microorganism

Molecular characterization of the production strain was performed using whole genome sequencing (WGS). The DNA sequence, copy number and insertion site of each construct integrated into the chromosomal DNA was confirmed by WGS analysis after 60 generations in a serial flask cultivation experiment. Each construct was stably integrated into the chromosomal DNA. No chromosomal rearrangements were detected. WGS analysis also confirmed that the DNA sequence of the plasmid was correct. No vector backbone DNA was present in the genome of the production strain. In addition, optical cell density results for growth experiments as well as ultra-performance liquid chromatography (UPLC) detection of 2′-FL confirmed that the production organism was phenotypically stable after 60 generations.

An open reading frame (ORF) analysis was performed using WGS analysis and any new ORFs were evaluated by sequence comparison with known allergens and toxins. The analysis included all the insertions and deletions and their surrounding 1000 bp upstream/downstream sequences in addition to the whole sequence of the inserted plasmid. No significant matches to known allergens and toxins were found.

A single nucleotide polymorphism (SNP) analysis was also performed for all insertion sites in strain INB-2FL_03. None of the SNPs identified were in a coding region, and therefore, have no impact on the amino acid sequences of the produced proteins.

The list of donor organisms used during the development of production strain INB-2FL_03 was reviewed and deemed to be acceptable. All of the genes introduced into the production strain's genome, whether by chromosomal insertion or via plasmid, do not pose a safety concern regarding the unintended transfer of DNA from donor organisms since all genetic constructs were synthetically derived.

Based on the information provided, there are no safety concerns regarding the proposed use of 2′-FL produced by INB-2FL_03 from a molecular biological perspective.

Product information

The 2′-FL product is manufactured in a facility compliant with current Good Manufacturing Practices (cGMP) and/or Global Food Safety Initiative certifications and meets the requirements of the Safe Food for Canadians Act. The 2′-FL production process is divided into two main stages: fermentation and post-fermentation processing. The fermentation stage involves the inoculation of a small batch of sterilized fermentation medium with production strain INB-2FL_03, followed by sequential inoculations into commercially sized fermentation batches while temperature and pH conditions are controlled and optimized. The resulting fermentate is then purified during post-fermentation processing which involves the removal of cell biomass, residual fermentation media, by-products of the fermentation process, and other impurities via filtration steps, a decolouration step and ion removal step. Potential microbial contaminants are removed via sterile filtration. The filtrate is then concentrated by evaporation and nanofiltration to remove excess water. The resulting carbohydrate-rich filtrate is dried to obtain the final 2′-FL product.

Dietary exposure

2′-FL is intended for use in infant formula and toddler formulas, infant foods, foods for young children, other foods for special dietary use, and foods for the general population. Given that 2′-FL is a naturally occurring component of human milk, there is a history of exposure based on the natural quantity of 2′-FL ingested by infants, although intake is variable and can range between 0.7 to 8.4 g/L(^{Footnote 1},^{Footnote 2}).

The notification provided US population-based data from the National Health and Nutrition Examination Survey (NHANES 2017-2018) to determine estimated cumulative daily intake of 2′-FL based on intake of the proposed food categories at the proposed use levels. On a per-user basis, which calculates the maximum potential intake by assuming that individuals are consuming only products containing 2′-FL (not human milk), the 90^th percentile consumption of 2′-FL ranges from 2.5 g/day in infants 0-6 months of age up to 6.51 g/day in adults 50 years of age and above.

The Bureau of Data, Science and Knowledge Integration (BDSKI) conducted an exposure assessment using data from the 2004 and 2015 Canadian Community Health Survey (CCHS), also based on the proposed food categories at the proposed use levels. CCHS 2015 does not include data for infants less than one year of age; therefore, the 2004 CCHS data was used to determine exposure estimates in this age group. The 90^th percentile consumption of 2′-FL ranges from 3.49 g/day in infants 0-6 months of age up to 8.09 g/day in 9 to 13-year-old females.

When taking into consideration exposure by body weight, the 90^th percentile estimated intake using the 2017-2018 NHANES data ranges from 84 mg/kg bw/day for adults 50 years and above up to 579 mg/kg bw/day for infants 7 to 12 months of age. The 90^th percentile in the CCHS data ranges from 79 mg/kg bw/day for 51 to 70-year-old males up to 823 mg/kg bw/day for infants aged 7 to 12 months. Overall the estimated intakes in all age groups using the CCHS data are slightly higher yet within comparable magnitude to those provided by the petitioner using NHANES 2017-2018. For comparison, a 2023 European Food Safety Authority (EFSA) safety assessment of a proposed extension of use for 2′-FL indicates a maximum 95^th percentile intake of 1382 mg/kg bw/day for infants aged 0 to 6 months^{Footnote 3}. Both the NHANES and CCHS data are lower than the approved extension of use in the EFSA report.

Nutrition

2′-FL is a non-digestible oligosaccharide, with an energy yield of 200 kilocalories per 100 grams proposed for addition to various food categories for infants, children and adults. The intended effect is to serve as a nutrient that promotes the production of short-chain fatty acids, which colonocytes utilize for energy. Using the data provided by the petitioner, the energy contribution for any of the proposed uses was determined to be negligible, noting that the highest proposed level of 80 g/kg in a 40 g serving of the puffed cereal category would provide 6.4 kcal of energy per consumption.

For term infant and toddler formulas, the current submission proposes the same level of 2′-FL as in previously approved uses of 2′-FL manufactured by Chr. Hansen (formerly Jennewein Biotechnologie GmbH), DuPont Nutrition and Biosciences, and DSM/Glycom. Health Canada approved these uses at a maximum level of 1.2 g per litre of the liquid product as fed (including overage/manufacturing tolerances). The previous decisions regarding the nutritional safety of 2′-FL is applicable to the current submission for term infant formula and toddler formula on the basis that the 2′-FL from all four companies are chemically equivalent to 2′-FL in human milk.

2′-FL acts similarly to other fermentable oligosaccharides which are metabolized into short chain fatty acids or are excreted in the feces. It is not expected to have a negative effect on the absorption of nutrients.

Mild gastrointestinal symptoms such as bloating and flatulence may be experienced with an excessive consumption consistent with the effects of any incompletely absorbed carbohydrate. The most common adverse events reported were flatulence, stomach pain, diarrhoea and rumbling, which were only observed at the highest dose studied, i.e., 20 g/day in adults^{Footnote 4}. These gastrointestinal symptoms are expected to resolve with decreased consumption and are therefore self-regulated. The highest expected cumulative daily intake at the 90^th percentile for Canadian females ages 9 to 13 of 8.09 g/day is below the highest level causing mild adverse effects. Considering the latter comparison together with the history of exposure in infants from human milk up to levels of 8.4 g/L², it is not anticipated that the proposed food uses of the Inbiose 2′-FL will cause any adverse gastrointestinal effects in adults, young children, or infants.

As per Division 25 of the FDR, a premarket notification would be required for a new infant formula with the Inbiose 2′-FL at a maximum level of up to 1.2 g per litre of formula as fed inclusive of overage.

There are no nutritional safety concerns regarding the proposed levels of 2′-FL from strain INB-2FL_03 in the proposed food categories.

Microbiology

The host strain E. coli K-12 is a well-characterized, non-pathogenic strain which has been used extensively in industrial applications such as the production of food ingredients, food enzymes, food additives and biochemical products such as amino acids. In addition, the parent strain E. coli K-12 MG1655 is also well characterized, does not possess the fertility plasmid indicating that it is incapable of transferring its DNA to other organisms, is deficient in the O-antigen in its lipopolysaccharide, and is incapable of colonizing the human gastrointestinal system.

Microbiological specifications, validated testing methods, and Certificates of Analysis (CoA) were provided for 3 non-consecutive batches of the 2′-FL final product produced by INB-2FL_03. The test results were all within specification limits and were deemed acceptable. The production organism was successfully removed from the final 2′-FL product. The endotoxin specification for the 2′-FL final product was reviewed and was deemed to be acceptable. There are no safety concerns regarding endotoxins in the final 2′-FL product.

WGS analysis was used to determine if the genome of INB-2FL_03 contained any antimicrobial resistance genes. No antimicrobial resistance markers from helper plasmids were detected in the chromosome or in the plasmid. The absence of resistance markers from helper plasmids was further validated using PCR and replica plating. However, there were two antimicrobial resistance genes found in the chromosome of INB-2FL_03: formA (formaldehyde dehydrogenase) and mdfA (general efflux pump). These two genes are intrinsic to the parent E. coli K-12 MG1655. These endogenous resistance genes do not pose a safety concern since the final 2′-FL product is highly purified and does not contain the production organism, recombinant DNA (rDNA) or protein.

No signal peptides were detected for all of the genes which were introduced into the production strain INB-2FL_03 which means that no heterologous proteins were excreted out into the growth medium. Furthermore, test results were provided for three non-consecutive batches of the final 2′-FL product demonstrating an absence of protein.

To verify the absence of rDNA, three batches of the final 2′-FL product were tested using PCR with two primer sets, one that amplifies the heterologous gene from the plasmid and a heterologous gene on chromosomal DNA of the production strain. No rDNA was detected in three batches of final 2′-FL product which confirms the absence of the production strain and its rDNA.

Based on the information provided, there are no safety concerns with the use of 2′-FL produced by INB-2FL_03 from a microbiological perspective.

Chemistry

A list of substances used during the manufacturing of 2′-FL by fermentation of INB-2FL_03 was provided in the submission. Ingredients used in the fermentation media for cultures such as E. coli used to make 2′-FL are not typically regulated as food additives and usually there is no requirement under the Food and Drug Regulations (FDR) for their premarket review. However, Inbiose N.V. is responsible for ensuring that the use of any substance in manufacturing of 2′-FL does not result in a violation of section 4 of the Food and Drugs Act. In this regard, Inbiose N.V. stated that all materials used in the manufacture of 2′-FL are food grade.

Analytical data^{Footnote 5} reviewed by the Food Biomolecules Section (FBS) in the Bureau of Chemical Safety (BCS) demonstrated that 2′-FL produced by fermentation of INB-2FL_03 is chemically equivalent to 2′-FL isolated from human milk.

Inbiose N.V. referenced a previous submission from DuPont Nutrition & Biosciences for 2′-FL from E. coli K-12 sINB000846, which was previously reviewed and approved by the Food Directorate (now the Food and Nutrition Directorate), to support the safety of 2′-FL from the new production strain, INB-2FL_03. Information was provided to compare the manufacturing processes and specifications for two 2′-FL products. While there are minor differences in the product specifications and the substances used in manufacturing each 2′-FL product, FBS is of the opinion that 2′-FL from INB-2FL_03 is highly similar, from a chemistry perspective, to 2′-FL from sINB000846.

There are no chemical food safety concerns with the proposed uses of 2′-FL produced by INB-2FL_03.

Toxicology

A previous 2′-FL product produced via bacterial fermentation, using a genetically modified E. coli K-12 MG1655 strain (sINB000846) (DuPont, 2022^{Footnote 6}), was approved for use in infant and toddler formulas up to a maximum of 1.2 g/L (including overage), as well as a variety of foods for infants and toddlers, and foods and beverages for children and adults.

Inbiose N.V is using a new genetically modified bacterial strain to produce 2′-FL, E. coli K-12 MG1655 strain INB-2FL_03, and is proposing to also use their 2′-FL product in infant and toddler formulas up to a maximum of 1.2 g/L (including overage), as well as a variety of foods for infants and toddlers, and foods and beverages for children and adults.

Inbiose N.V stated that their 2′-FL product was highly similar to DuPont's 2′-FL product. Inbiose N.V. was formally authorised by the parent company of DuPont to use the information provided in the DuPont 2′-FL submission to support the submission of Inbiose's 2′-FL product. This included the toxicological studies provided for DuPont's 2′-FL product.

The Bureau of Nutritional Sciences (BNS) and FBS confirmed that the 2′-FL products would be considered nutritionally and chemically highly similar. Therefore, the toxicological studies using DuPont's 2′-FL product, which were previously evaluated by BCS' Pre-Market Toxicology Assessment Section (PTAS), could be used in support of the current submission.

BNS and FBS also confirmed that the 2′-FL component would be nutritionally and chemically equivalent to what is present in human breast milk.

The toxicological studies with DuPont's 2′-FL product showed no acute toxicity in rats (LD₅₀ > 5000 mg/kg bw), no treatment-related adverse effects in a 90-day dietary exposure study in rats up to the highest concentration tested (NOAEL equivalent to 5737 mg/kg bw/ day), and no genotoxicity (Ames and in vitro micronucleus test).

In addition, a toxin homology search was conducted on the new ORFs. The peptide sequences were compared with known toxins but no relevant matches were found.

Based on these results, no toxicity concerns were identified for Inbiose's 2′-FL product.

Using the cumulative Canadian exposure estimates provided by BDSKI, for "Eater's Only", 90^th percentile, the highest exposure was for older infants, 7 to 12 months, at 823 mg 2'-FL/kg bw/day. Using the NOAEL of 5737 mg/kg bw/day, from the 90-day oral rat toxicity study, there is about a 7-fold margin of exposure for 7 to 12-month-olds. Given the lack of toxicity for 2′-FL and the history of safe use in the diet from the consumption of breast milk, this margin of exposure was determined to be sufficient.

There are no toxicological concerns with the use of Inbiose's 2′-FL ingredient in infant and toddler formulas, foods for infants and toddlers, and foods and beverages for children and adults, at the proposed levels of use.

Allergenicity

To determine the allergenicity safety of 2′-FL produced by fermentation of INB-2FL_03, the petitioner conducted a homology search using the Access Food Allergy Research and Resource Program (FARRP) database (Version 22 released on May 25, 2023; http://www.allergenonline.org). Two types of searches were performed: one with an 80-amino acid sliding window looking for identities >35%, and another for 8-amino acid matches. The 80-amino acid search identified three matches: two against non-food allergens (Dermatophagoides pteronyssinus and Dermatophagoides farinae) and one against Juglans regia (English walnut), a priority allergen, which was determined to be of low significance at 36.90% as it is only slightly above the conservative cut-off of 35%.^{Footnote 7} The 8-amino acid search found two matches related to IS5 transposase, which is naturally present in E. coli K-12 MG1655. No relevant matches were found.

Moreover, previous evaluations of 2′-FL by the Food Directorate (now the Food and Nutrition Directorate) from other sources including 2′-FL from sINB000846 did not identify any allergenic concerns with this substance.

The specifications and analyses of the 2′-FL product for three non-consecutive batches included in the submission show that the presence of proteins is negligible in the 2′-FL product.

Based on the totality of evidence from 2′-FL's history of safe use with no reports of allergenic reactions from its consumption, the lack of significant matches in amino acid sequence homology searches, and its low dietary exposure, FBS of BCS is of the opinion that 2′-FL produced by fermentation of INB-2FL_03 is not expected to pose an allergenic concern when used in the proposed foods at the indicated levels of use.

However, the submission indicated that since lactose is used as a substrate in the 2′-FL production process and small amounts of residual lactose are present in the final product, the label "contains milk", in accordance with the requirements of the Food Allergy, Labelling and Consumer Protection Act of 2004, must be added to products containing 2′-FL.

Based on the information provided, there are no allergenicity concerns with the proposed uses of 2′-FL produced by INB-2FL_03.

Conclusion

Health Canada's review of the information presented in support of the use of 2′-FL derived from the production strain INB-2FL_03 in infant formula, toddler formula, infant foods, other foods for special dietary use, as well as foods for young children and the general population, does not raise concerns related to food safety.

Health Canada's opinion refers only to the use of 2′-FL as an ingredient in infant and toddler formulas, foods for young children, and other foods included in the submission. Infant formulas and human milk fortifiers containing 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K-12 INB-2FL_03, are further required to undergo pre-market review under Division 25.

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

Email: bmh-bdm@hc-sc.gc.ca

Footnotes

Footnote 1

McGuire, M.K., Meehan, C.L., McGuire, M.A., Williams, J.E., Foster, J., Sellen, D.W., Kamau-Mbuthia, E.W., Kamundia, E.W., Mbugua, S., Moore, S.E., Prentice, A.M., Kvist, L.J., Otoo, G.E., Brooker, S.L., Price, W.J., Shafii, B., Placek, C., Lackey, K.A., Robertson, B., Manzano, S., Ruíz, L., Rodríguez, J.M., Pareja, R.G., Bode, L., 2017. What's normal? Oligosaccharide concentrations and profiles in milk produced by healthy women vary geographically. Am. J. Clin. Nutr. 105, 1086–1100. https://doi.org/10.3945/ajcn.116.139980

Return to footnote 1 referrer

Footnote 2

Musumeci M, Simpore J, D'Agata A, Sotgiu S and Musumeci S, 2006. Oligosaccharides in colostrum of Italian and Burkinabe women. Journal of Pediatric Gastroenterology and Nutrition, 43, 372-378.

Return to footnote 2 referrer

Footnote 3

EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), Turck, D., Bohn, T., Castenmiller, J., De Henauw, S., Hirsch-Ernst, K. I., Maciuk, A., Mangelsdorf, I., McArdle, H. J., Naska, A., Pentieva, K., Siani, A., Thies, F., Tsabouri, S., Vinceti, M., Aguilera-Gomez, M., Cubadda, F., Frenzel, T., Heinonen, M., Knutsen, H. K. (2023). Safety of the extension of use of 20-fucosyllactose (20-FL) as a novel food pursuant to Regulation (EU) 2015/2283. EFSA Journal, 21(11), 1–13. https://doi.org/10.2903/j.efsa.2023.8334

Return to footnote 3 referrer

Footnote 4

Elison, E., Vigsnaes, L.K., Rindom Krogsgaard, L., Rasmussen, J., Sørensen, N., McConnell, B., Hennet, T., Sommer, M.O.A., Bytzer, P., 2016. Oral supplementation of healthy adults with 2′- O - fucosyllactose and lacto- N -neotetraose is well tolerated and shifts the intestinal microbiota. Br. J. Nutr. 116, 1356–1368. https://doi.org/10.1017/S0007114516003354

Return to footnote 4 referrer

Footnote 5

The petitioner referenced their ¹H-NMR data included in U.S. GRN 1091 for comparison of 2'-FL produced by fermentation of Escherichia coli strain K-12 INB-2FL_03 with 2'-FL isolated from human milk.

Return to footnote 5 referrer

Footnote 6

Health Canada (HC). Date modified 2022-06-16. Novel Food Information: 2′-fucosyllactose (2′-FL) from Escherichia coli K12 MG1655 strain (sINB000846). https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products/fucosyllactose-escherichia-coli/document.html.

Return to footnote 6 referrer

Footnote 7

The percent identity for the match was 36.90% which is only slightly above the conservative cut-off of 35% as defined by FAO/WHO (2009). The Expect value or E value for the match was 1.1e+002 (equivalent to 110). The E value specifies the number of hits one can "expect" to see by chance when searching a database of a particular size. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. The lower the E-value, or the closer it is to zero, the more "significant" the match is. As such, an E value of 110 likely represents a random alignment given the size of the allergen database. On this basis and given that no other hits with food allergens were noted in the 80 amino acid and 8 amino acid search, the match against this allergen is insignificant and does not pose an allergenic risk.

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