Novel Food Information: 2′-fucosyllactose (2′-FL) from Escherichia coli K12 MG1655 strain (sINB000846)

On this page

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

Background:

Health Canada has notified DuPont Nutrition & Biosciences, that it has no objection to the use of 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K12 MG1655 (sINB000846), "the production strain" or "the strain", as an ingredient in infant formulas and foods for young children, and a variety of food and beverages for all age groups. Infant formulas and human milk fortifiers containing 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K12 MG1655 (sINB000846) 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 DuPont Nutrition & Biosciences (DuPont), and the evaluation by Health Canada. This document contains no confidential business information.

1. Introduction

DuPont, has developed 2′-fucosyllactose from a genetically modified E. coli K12 MG1655 (sINB000846) strain, using classical genetic modification methods. The bacterially synthesized 2′-FL has a purity of ≥ 96% and is expected to be used in formula for infants and young children to a maximum proposed use level of 1.2 g/L of prepared baby formula (including overage). Various maximum use levels are proposed in other foods, such as beverages, dairy products and their analogs, processed fruits and vegetables and juices, grain products intended for all ages, and oral and enteral tube feedings.

2′-FL is a naturally occurring oligosaccharide in human milk composed of L-fucose and D-lactose (a disaccharide of D-galactose and D-glucose). The bacterially synthesized 2′-FL produced by DuPont was confirmed to be chemically and structurally equivalent to 2′-FL isolated from human breast milk. This evaluation did not include an assessment of any potential claims that could be made about 2′FL. Genetic modifications were made to E. coli sINB000846 for the industrial production of oligosaccharides by the modification of several genes, which together, result in the uptake of sucrose and lactose and subsequent conversion of those materials to 2′-FL.

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

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). 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
  • i. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Microorganism

The production strain was optimized for industrial production of oligosaccharides by the modification of several genes which, together, result in the uptake of sucrose and lactose and subsequent conversion of those materials to 2′-FL. In total, nineteen 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. A plasmid was also introduced to the strain that enables the synthesis of 2′-FL out of GDP-fucose and lactose. The production strain is not present in the final 2′-FL product.

3. Characterization of the Modified Microorganism

Molecular characterization of the strain was performed with biochemical and molecular methods. Genetic modifications made to the host strain were verified using PCR, Sanger sequencing, and whole genome sequencing (WGS). Absence of helper plasmid markers was confirmed through PCR and biochemical phenotyping. Genetic stability was demonstrated using molecular methods over 60 generations of growth to show that no new mutations were generated. The WGS identified several mutations that were attributed to spontaneous natural variation. The majority of these were single point mutations that either occurred in intergenic regions (outside of the protein coding regions) or were silent and so would result in no amino acid changes in expressed proteins. No chromosomal re-arrangements were observed in the strain. No safety implications were raised in regard to identified single nucleotide polymorphisms and the strain appears to be stable.

Absence of recombinant DNA in the final product was also tested using quantitative PCR (qPCR) in three batches. The inserted genes in the strain do not express proteins that are excreted. There are no concerns with the effectiveness of the purification process at removing the host strain, endotoxins, DNA or protein from the final 2′-FL product.

The genome- and plasmid-inserted genes were all chemically synthesized and cloned into the production strain using standard molecular biology methods. This precluded the possibility of genetic material, other than the genes of interest, from being introduced into the production organism. There are no safety concerns about the use of these genes for carbohydrate metabolism, transport, and strain maintenance.

4. Product Information

DuPont's 2′FL product is manufactured at a site operating in accordance with good manufacturing practice (GMP) and/or Global Food Safety Initiative (GFSI) certifications and meet the requirements of the Safe Food for Canadians Act. DuPont's 2′-FL production process is divided into two main stages: fermentation and post‐fermentation processing. Post-fermentation includes steps of crude purification to remove the cell biomass, endotoxins and large molecules by microfiltration and ultrafiltration, followed by selective concentration, and crystallization to achieve a pure product. The pore size of the filtration membrane for the microfiltration ranges between 3,000 - 10,000 Daltons molecular cut-off to ensure sufficient endotoxin removal during cell mass removal. A nanofiltration membrane was used with a molecular cut-off in the range sufficient to ensure good process yield and concomitant removal of smaller molecules like salts, lactose and monomeric sugars.

5. Dietary Exposure

Human milk is composed of lactose, fat, and human milk oligosaccharides (HMOs). Of these components, HMOs are the third most abundant solid component and are present in breast milk in concentrations of 5–25 g/L (Chen, 2005; Thurl et al., 2010; Gabrielli et al., 2011). Most breast-fed infants are naturally exposed to 2′FL. In fact, out the 20 known HMOs in human breast milk, one of the most abundant is 2′FL (0.06 to 4.65 g/L) with an estimated intake from human milk ranging from 170–660 mg/kg to 1150 mg/kg body weight per day (Erney et al., 2000; EFSA, 2015).

Compared to human milk, the concentration of oligosaccharides found in the milk of other animals is approximate 100-1000-fold lower (Vandenplas et al., 2018). Importantly, since cow milk is often used for infant formula 2′FL is usually only present in formula in low concentrations or in some cases, not at all. This low concentration in current infant formula compositions motivated the development for an efficient method of large-scale synthetic 2′FL production.

The dietary exposure for infants consuming 2′-FL in infant formula would be lower or consistent with current exposure for breastfed infants, as the proposed rate of addition of 2′-FL in formulas is lower than that in breast milk. Breast milk contains on average 2.38 g of 2′FL/L (Erney et al. 2000), which is about two fold higher than the petitioner's proposed maximum specification of 1.2 g of 2′-FL/L (includes overage).

6. Nutrition

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. These gastrointestinal symptoms are expected to resolve with decreased consumption and to be amenable to self-regulation by manufacturers and consumers seeking to avoid formulation and consumption of products that are likely to elicit such symptoms. Elison et al. 2016 reported that the most common adverse events were flatulence, stomach pain, diarrhoea and rumbling, which were only observed at the highest dose studied, i.e., 20 g/day.

There are no nutritional safety concerns with the use of DuPont's 2′-FL in term infant formula at a maximum level up to 1.2 g per litre of formula as fed (inclusive of overage) and at various maximum levels in foods for young children, and other foods. Further pre-market assessment of any new infant formula containing 2′-FL as an ingredient would be required, as per Division 25 of the Food and Drug Regulations.

7. Microbiology

The host strain E. coli K12 is a well-characterized, non-pathogenic strain which has been used extensively in the biopharmaceutical industry as the host for expression of recombinant proteins. E. coli K12 is defective in at least three cell wall characteristics and is unable to colonize the human GI tract under normal conditions and is not considered to be pathogenic (Tourret et al., 2011; FDA, 1990; Curtiss, 1978). The National Institutes of Health considers K12 as an organism which does not present a significant risk to health or the environment (as per section III-F of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules).

The production strain sINB000846 does not contain any antibiotic resistance markers and has the same sensitivity to antibiotics as the wild type host, E. coli K12 strain MG1655. The removal of the helper plasmid, which contained an antibiotic resistance marker, was validated by PCR and replica plating on a plate containing the antibiotic for which the marker is present on the helper plasmid. Both the host organism and the production strain possess the same antimicrobial resistance genes and these do not pose a safety concern since the final product is absent of any remnants of the strain, protein or DNA.

The petitioner supplied specifications for food-grade 2′-FL, and the results of analyses of three non-consecutive batches that show that all batches conform with specifications. Microbial specifications were included and results from all three batches tested showed acceptable levels as compared to their specification. Microbial parameters tested included standard plate count, aerobic contaminants, total yeast and molds, Enterobacteriaceae, Salmonella, Listeria monocytogenes, Cronobacter sakazaki, Coagulase pos. staphylococci, C. perfringens, B. cereus, Enterococci, and Clostridia spores. It was confirmed by analytical testing of five manufacturing batches that the final 2′-FL product does not contain the production organism, residual DNA or recombinant proteins. The testing was done using standard methods and met specifications. There are no concerns regarding the specifications or microbial safety of the 2′-FL product.

8. Chemistry

The petitioner provided specifications for their 2′-FL including limits on trace elements, which they have established in the absence of specifications for this substance in the Food Chemicals Codex or Combined Compendium of Food Additives Specifications prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

The specifications for arsenic, cadmium, lead and mercury were found to be acceptable and can be consistently met based on analytical results for 2′-FL provided by DuPont. The estimated concentrations of these trace elements in the foods proposed would be well below the background levels typically seen in similar foods on the Canadian Market. Furthermore, potential exposure to these trace elements from the consumption of 2′-FL in foods proposed at the maximum requested levels of use was determined to contribute negligible amounts of arsenic and lead to overall dietary exposure and result in exposure to mercury and cadmium that are orders of magnitude lower than their respective toxicological reference values. Overall, the potential exposure to arsenic, cadmium, lead and mercury from the proposed uses of 2′-FL are not expected to pose a chemical safety concern.

A detailed list of all substances (chemical identity and CAS#) used during the manufacturing of 2′-FL was included in the submission. The acceptability of particular uses of substances for a technological purpose in manufacturing the 2′-FL is separate from the acceptability of the food itself. More generally, the petitioner is responsible for ensuring that the presence of contaminants in the product and the use of any substance in the manufacture of 2′-FL does not result in a violation of section 4 of the Food and Drugs Act. In this regard, the petitioner noted that all of the materials used in the production of 2′-FL are of food grade.

Tests also confirmed the absence of bacteria and bacterial endotoxins. The endotoxin specification (<300 EU/g) and validated test method used for testing of three batches of DuPont's 2′-FL were all deemed to be acceptable (<300 EU/g). There are no safety concerns regarding endotoxins in the final 2′-FL product.

The petitioner provided analytical data which demonstrated that 2′-FL produced by fermentation of sINB000846 is chemically and structurally equivalent to 2′-FL isolated from human milk. There are no chemical safety concerns with the proposed level of use of 2′-FL produced by the strain in infant formulas and foods for young children, and other foods.

9. Toxicology

DuPont intends to use this 2′-FL in a variety of food and beverages for all age groups, in addition to use in infant and toddler formulas up to a maximum of 1.2 g/L. The petitioner used nuclear magnetic resonance spectroscopy (1H NMR) and a reference standard to confirm the identity of the DuPont 2′-FL (≥ 96% purity), produced from E. coli strain sINB000846. Given that the reference standard was isolated from breast milk, this same analysis also confirmed that the DuPont 2′-FL is chemically equivalent to 2′-FL from human breast milk.

The petitioner referenced published studies to support the safety and tolerability of 2′-FL, which were reviewed previously for permitted uses of 2′-FL. The petitioner also provided unpublished toxicological studies, which tested DuPont's 2′-FL. An acute oral rat toxicity study determined the LD50 to be > 5000 mg/kg. An oral (dietary) rat subchronic 90-day toxicity study found the No Observed Adverse Effect Level (NOAEL) to be 5.737 g/kg bw per day (an equivalent dose from the highest dietary concentration of 10 %). Two in vitro genotoxicity assays (Ames-bacterial reverse mutation and mammalian micronucleus) were negative for mutagenicity and clastogenicity. All studies were compliant with their respective OECD guidelines. No adverse effects were observed in any study or assay. The results support the safety of the DuPont 2′-FL.

The petitioner referenced a clinical study (Elison et al., 2016) to support tolerability of 2′-FL in healthy adults (n=10 per group; male and female, aged 19-57 years). Treatment consisted of 5, 10 or 20 g per day of 2′-FL (chemically produced by Glycom A/S; 99.9 % purity) or 2 g glucose per day as a placebo, consumed daily with breakfast for two weeks. Only the highest dose (20 g per day) was associated with adverse events, which were considered to be mild (increased nausea, rumbling, bloating, passing of gas, diarrhoea, loose stools and urgency to pass stools). All participants completed the study, and no adverse effects were observed for clinical chemistry and haematological parameters, nor for clinical effects (e.g., heart rate, blood pressure). The petitioner estimated the maximum dietary exposure of 2′-FL in adults to be 5.9 g/day (90th percentile; adult users, 50+ years of age). At this level, no adverse effects were noted in the clinical study. EFSA (2015) evaluated synthetically produced 2′-FL for a similar extension of dietary use to adults, and expressed no concern with a similar maximum estimated dietary exposure for adults (5 g per day). For the new extension of use to older age groups, the highest estimated exposure is 0.14 g/kg bw per day for children 3-12 years. The NOAEL of 5.737 g/kg per day, from the 90-day oral toxicity rat study, is more than 40-fold greater than this highest estimated dietary intake. This margin of exposure was determined to be sufficient, given the lack of toxicity for 2′-FL, and the history of safe use in the diet from consumption of breast milk.

The petitioner conducted bioinformatics analyses on all modification sites and adjacent regions. The putative peptide sequences for altered regions of the production organism genome were compared with known toxins using UniProtKB/Swiss-Prot database. No relevant matches were found. Additionally, the maximum level of protein detected in the final product by a modified Bradford method was 3.64 µg/g (equivalent to 0.000364%), which is negligible. There are no toxicological concerns identified with 2′FL from strain INB000846 to be used in infant formulas and foods for young children, and other foods.

10. Allergenicity

The petitioner demonstrated that the final product is highly purified for 2′-FL (≥ 96%) and that the presence of protein is negligible. Additionally, the petitioner conducted bioinformatics analyses on all modification sites and adjacent regions. The putative peptide sequences for altered regions of the production organism genome were compared with known allergens using the Food Allergy Research and Resource Program (FARRP) database (Version 18B released on 23 March 2018; http://www.allergenonline.org). Two searches were performed; a search using a sliding window of 80 amino acid segments of each protein to find identities greater than 35 % (according to CODEX Alimentarius guidelines, 2003), and a search for 8 amino acid exact matches. No relevant matches were found.

Evaluators conducted an updated search of the literature, and did not identify any reports of allergenicity associated with 2′-FL. Overall, there were no allergenic concerns identified with the proposed use of 2′FL from E. coli strain INB000846 in infant formulas and foods for young children, and other foods.

Conclusion:

Health Canada's review of the information presented in support of the use of 2′-FL derived from the production strain in infant formulas and foods for young children, and other foods 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 formulas and 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 K12 MG1655 (sINB000846) 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 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 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