Novel Food Information: 2′-fucosyllactose (2′-FL) from Escherichia coli K-12 (DH1) MDO MAP1001d strain

On this page

• Background
• Introduction
• Development of the Modified Microorganism
• Characterization of the Modified Microorganism
• Product Information
• Dietary Exposure
• Nutrition
• Microbiology
• Chemistry
• Toxicology
• Allergenicity
• Conclusion

Background

Health Canada has notified Glycom A/S (affiliated with DSM Nutritional Products, Ltd.), that it has no objection to the use of 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli K-12 (DH1) MDO MAP1001d, "the production strain" or "the strain", as an ingredient for use in term infant formulas and nutritional supplements (toddler formulas) intended for children 1 to 3 years of age. 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. Infant formulas and human milk fortifiers containing 2′-FL derived from a genetically modified strain of E. coli K-12 (DH1) MDO MAP1001d are further required to undergo pre-market review under Division 25 of Part B of the Food and Drug Regulations.

The following provides a summary of the notification from Glycom A/S, and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Intertek Health Sciences, Inc. (Intertek) submitted the notification on behalf of Glycom A/S. Glycom A/S has developed 2′-FL from the genetically modified E. coli K-12 (DH1) MDO MAP1001d strain, using classical genetic modification methods. The bacterially synthesized 2′-FL product has a specified purity of ≥ 94% and is expected to be used in formula for term infants and toddler formulas to a maximum proposed use level of 1.2 g/L of prepared formula (including overage).

2′-FL is a naturally occurring oligosaccharide in human milk and is composed of L-fucose and D-lactose (a disaccharide of D-galactose and D-glucose). Genetic modifications were made to E. coli K-12 (DH1) for the industrial production of oligosaccharides by the modification of several genes, which together, result in the fucosylation of lactose 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 an international approach in this area (e.g., Codex Alimentarius).

The safety assessment considered: how the Glycom 2′-FL product was developed; how the composition and nutritional quality of the 2′-FL product compared to human milk oligosaccharides (HMOs); and, what the potential is for the 2′-FL product to be toxic or cause allergic reactions. This evaluation did not include an assessment of any potential claims that could be made about 2′-FL.

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

• 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, E. coli K-12 (DH1) MDO MAP1001d (hereafter MAP1001d), was derived from the well-characterized E. coli K-12 (DH1) parent strain which was genetically modified to introduce the entire biosynthetic pathway for 2′-FL into the chromosome. As a result, production strain MAP1001d is capable of converting lactose into 2′-FL and over-expressing de novo pathway genes for GDP-fucose formation. Strain MAP1001d does not degrade lactose, nor does it produce secondary metabolites that hinder 2′-FL synthesis.

The host strain was genetically modified in order to produce the production strain MAP1001d. These genetic modifications include insertion of a heterologous gene expressing α-1,2-fucosyltransferase involved in 2′-FL biosynthesis which was synthetically derived from the donor Helicobacter pylori. The α-1,2-fucosyltransferase catalyzes the fucosylation of lactose to form 2′-FL, the final step in the engineered biosynthetic pathway. No safety concerns were identified with these genetic modifications, either individually or in terms of their net effect in creating an optimized biosynthetic pathway for 2′-FL.

Characterization of the Modified Microorganism

Whole genome sequencing of the production strain MAP1001d confirmed the genetic stability of all intended genetic modifications over 50 generations of subculturing. MAP1001d does not contain any antimicrobial resistance gene markers or fragments of the plasmid vector backbone within its genome.

Open reading frame (ORF), single nucleotide polymorphism (SNP) and structural variant analyses were performed to verify any unintended genetic modifications in MAP1001d. No chromosomal rearrangements, frame-shift mutations or novel ORFs were detected. Unintended effects, namely one gene inversion, one duplication of endogenous genes, and a deletion event, were well-characterized and do not pose a safety concern as they did not affect the synthesis or purity of 2′-FL. The highly purified 2′-FL final product was shown to be devoid of detectable proteins and recombinant DNA.

Based on the information assessed regarding the production strain design and characterization, there are no safety concerns regarding 2′-FL produced by E. coli K-12 (DH1) MDO MAP1001d, from a molecular perspective.

Product Information

Glycom's 2′-FL product is manufactured by fermentation of strain MAP1001d under sterile conditions in a facility compliant with current Good Manufacturing Practices (cGMP) and operating in accordance with Hazard Analysis Critical Control Point (HACCP) principles. Fermentation is performed according to a standard procedure using defined media containing no antimicrobials. During fermentation, 2′-FL is efficiently secreted into the medium. Purification steps remove and inactivate the microbial biomass from the 2′-FL containing filtrate. A series of polishing purification steps is followed by crystallization of 2′-FL by the controlled addition of acetic acid and seeding crystals. The 2′-FL crystals are isolated and washed with acetic acid to remove remaining traces of salts, biomolecules, and carbohydrate impurities, and then dried. Quality control measures are performed throughout the entire purification and isolation process to ensure that the 2′-FL batches to be released conform to product specifications.

Dietary Exposure

HMOs are the third most abundant solid component in human milk after lactose and fat and are present in breast milk at concentrations of 5–25 g/L (Chen, 2005; Thurl et al., 2010; Gabrielli et al., 2011). Most breastfed infants are naturally exposed to 2′-FL. Of the known HMOs in human breast milk, one of the most abundant is 2′-FL (0.06 to 4.65 g/L – Erney et al., 2000), with an average estimated intake from human milk of 170 to 660 mg/kg body weight per day (EFSA, 2015). Intake, however, may be up to 1150 mg/kg body weight per day (EFSA, 2015).

Compared to human milk, the concentration of oligosaccharides found in the milk of farm animals is approximately 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 biosynthesis of 2′-FL.

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 approximately 2-fold higher than the petitioner's proposed maximum use level of 1.2 g of 2′-FL/L (includes overage).

This maximum level of use is also being proposed in nutritional supplements (i.e., toddler formulas) solely intended for young children ages 1 to 3 years old. This concentration of 2′-FL has previously been approved by Health Canada in the same intended food products.

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.

From a nutritional perspective, Glycom's 2′-FL product contains 94.0 w/w % 2′-FL (assay, water-free), 3.0 w/w % D-lactose, 1.0 w/w % L-fucose, 1.0 w/w % difucosyl-D-lactose, and 1.0 w/w % 2′-fucosyl-D-lactulose. Analysis of independent production batches demonstrate consistency of the manufacturing process in yielding a high purity 2′-FL ingredient. Regarding the carbohydrate impurity profile, the saccharides structurally related to 2′-FL may be present in small quantities in the final isolated product and are largely limited to saccharides naturally present in human breast milk (i.e., D-lactose, L-fucose, and difucosyl-D-lactose) and an isomerization product of 2′-FL, 2′-fucosyl-D-lactulose. Specification limits have been established for these carbohydrates.

There are no nutritional safety concerns with the use of Glycom's 2′-FL product in term infant formulas, and toddler formulas (liquid, or powder to be reconstituted) for children 1 to 3 years of age, formulated as nutritional supplements up to a maximum level of 1.2 g of 2′-FL/L of formula as fed (inclusive of overage). 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.

Microbiology

The host strain, E. coli K-12 (DH1), 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. This strain is incapable of colonizing the human gastrointestinal tract and does not produce antibiotics.

Microbiological specifications for relevant bacterial pathogens, validated testing methods, and certificates of analysis were provided for 3 non-consecutive batches of the 2′-FL final product produced by MAP1001d. The test results were all within specification limits and were deemed acceptable. The production organism was demonstrated to be successfully removed from the final 2′-FL product. Given the information provided, there are no safety concerns regarding 2′-FL produced by E. coli K-12 (DH1) MDO MAP1001d, from a microbiological perspective.

Chemistry

The petitioner provided specifications for their 2′-FL product including limits on trace elements and residual carbohydrates, which they have established in the absence of specifications for this substance in the Food Chemicals Codex (FCC) 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 the petitioner. 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 do not pose a chemical safety concern.

The petitioner provided analytical data which sufficiently demonstrated that the 2′-FL produced by fermentation of E. coli K-12 MDO MAP1001d in Glycom's product is structurally equivalent to 2′-FL isolated from human milk.

The final Glycom product also contains a small amount of D-lactose, L-fucose, difucosyl-D-lactose, and 2'-fucosyl-D-lactulose. D-lactose, L-fucose, and difucosyl-d-lactose are natural constituents of human milk. 2'-Fucosyl-D lactulose is an isomerization product of 2'-FL during ultra-high temperature treatment of milk, including human donor milk. With consideration of the carbohydrate impurity profile, the petitioner provided supporting evidence that their 2′-FL product is highly similar to other 2′-FL products already approved for use in Canada.

A detailed list of substances used during the manufacturing of 2′-FL by fermentation of E. coli K-12 MDO MAP1001d was included in the submission. Ingredients used in the fermentation media for cultures such as E. coli-derived strains used to make 2′-FL are not typically regulated as food additives, and usually there is no requirement under the Food and Drug Regulations for their premarket review. However, the petitioner 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. The petitioner indicated that all of the materials used in the manufacture of 2′-FL are of food-grade quality or equivalent.

Acetic acid is used in the late stages of the 2′-FL manufacturing process to crystallize and purify the final product. The maximum residual level of acetic acid in the final product is 1.0%. This use, as described by petitioner, is considered to be a food additive use of this substance. Acetic acid is permitted for such use at a level consistent with Good Manufacturing Practice, as set out in the List of Permitted Food Additives with Other Accepted Uses, provided that acetic acid residues do not exceed 1.0 % in the 2′-FL product and 12 ppm in infant formula in which the product is used, based on the formula as consumed.

There are no chemical safety concerns with the proposed level of use of 2′-FL produced by the E. coli K-12 MDO MAP1001d strain in infant formulas and toddler formulas.

Toxicology

2′-FL, like other HMOs, is highly resistant to hydrolysis by digestive enzymes, reaching the large intestine mainly intact. Most of the 2′-FL is expected to be fermented by the microbiota into short-chain fatty acids in the large intestine, or to be excreted intact in the feces. A small fraction may be absorbed into the systemic circulation.

Glycom is requesting the use of this 2′-FL in term infant and toddler formulas up to a maximum of 1.2 g/L, which falls within the reported range for 2′-FL found in human milk mentioned above. Glycom's 2′-FL is structurally equivalent to the 2′-FL found in human milk. Glycom also provided supporting evidence that their 2′-FL product is highly similar to the 2′-FL already approved for use in Canada. Additionally, Glycom is requesting the same level of use of 2′-FL (i.e., 1.2 g/L) that has already been approved for term infant and toddler formulas in Canada. No toxicological safety concerns were identified in the assessment of the previously approved 2′-FL products.

Toxicology studies were submitted by Glycom to support the safety of the notified 2′-FL, at 1.2 g/L (including overages). These studies were conducted using Glycom's 2′-FL product from a different (but related) production organism; however, supporting evidence was provided to show that the 2′-FL product produced by the related strain is chemically and nutritionally highly similar to the 2′-FL product in the current submission (produced with E. coli K-12 DH1 MDO strain MAP1001d). Therefore, the toxicology studies were considered in the review of the current 2′-FL.

The in vitro bacterial reverse mutation assay indicated the 2′-FL was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in Escherichia coli strain WP₂uvrA, tested with and without exogenous metabolic activation. Additionally, the 2′-FL was not clastogenic or aneugenic when tested in human lymphocytes, with or without exogenous metabolic activation. The 2′-FL was not considered to be genotoxic under the conditions of the studies.

An OECD guideline 13-week gavage study was conducted using juvenile rats (7 days of age at start of study) administered doses of 2000, 4000, and 5000 mg of 2′-FL/kg body weight per day, along with a vehicle control (water), and a reference control (fructooligosaccharide; 5000 mg/kg body weight per day). A standard series of toxicological endpoints were evaluated, including clinical observations; body weight and food consumption; physical, sexual, and neurological development; clinical chemistry and haematological parameters; and pathology. No toxicologically relevant effects were identified. A No Observed Adverse Effect Level (NOAEL) of 5000 mg of 2'-FL/kg body weight per day (highest dose tested) was determined by the study authors and was confirmed by Health Canada.

A conservative estimate of consumption of 312 mg 2′-FL/kg body weight per day for infants, and of 80.47 mg 2′-FL/kg body weight per day in toddlers, was calculated by Health Canada based on the requested concentration of 1.2 g/L. This concentration falls within the cited range of 2′-FL found in human milk. No toxicologically relevant safety concerns were identified in the toxicology studies reviewed (which were representative of this 2′-FL).

Therefore, based on the evidence provided, there were no toxicological concerns with the addition of Glycom's 2′-FL product to infant formula, as well as nutritional supplements for children from 1 to 3 years of age, up to 1.2 g/L, including overage.

Allergenicity

2′-FL is a human milk oligosaccharide and would not be expected to elicit an allergic response. The petitioner demonstrated that the final product is highly purified for 2′-FL (≥ 94%), and that the presence of residual protein is negligible. The lack of protein detected (which includes potential protein allergens), indicates Glycom's 2′-FL product would be unlikely to pose an allergenicity concern.

Conclusion

Health Canada's review of the information presented in support of the use of Glycom's 2′-FL product derived from the production strain, in term infant formulas and nutritional supplements (toddler formulas) specifically intended for young children ages 1 to 3 years old, up to a maximum level of 1.2 g/L (as-consumed, inclusive of overage), does not raise concerns related to food safety.

Health Canada's opinion refers only to the use of Glycom's 2′-FL product as an ingredient in term infant formulas and toddler formulas at the level specified. Infant formulas and human milk fortifiers containing 2′-FL derived from a genetically modified E.coli K-12 (DH1) MDO MAP1001d strain are further required to undergo pre-market review under Division 25 of the Food and Drug Regulations.

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.

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