Novel Food Information: 2′-Fucosyllactose, 3-Fucosyllactose, Lacto-N-Tetraose, 3′-Sialyllactose and 6′-Sialyllactose Blend Produced by Escherichia coli BL21(DE3) Strains

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 Chr. Hansen A/S (part of the Novonesis Group) that it has no objection to the use of their oligosaccharide blend composed of 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3′-sialyllactose sodium salt (3′-SL) and 6′-sialyllactose sodium salt (6′-SL) derived from genetically modified strains of Escherichia coli BL21(DE3) as an ingredient in infant and toddler formulas. Infant formulas and human milk fortifiers containing this novel food are further required to undergo pre-market review under Division 25. The Department conducted their comprehensive assessment 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 Chr. Hansen A/S, and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

The 2′-FL, 3-FL, LNT, 3′-SL, and 6′-SL was produced individually by genetically modified E. coli BL21(DE3) strains and then each purified oligosaccharide was combined to obtain the commercial blend.

Chr. Hansen A/S has developed the genetically modified E. coli BL21(DE3) production strains using standard molecular biology techniques. Genetic modifications were made to four E. coli BL21(DE3) strains for the purpose of inserting the genes involved in the oligosaccharide biosynthetic pathway for each oligosaccharide of interest. 3-FL was produced by strain JBT-3FL, LNT was produced by strain JBT-LNT, 3′-SL was produced by strain JBT-3SL and 6′-SL was produced by strain JBT-6SL. In addition, two optional "degradation" strains were assessed, JBT-LN(n)T-DS and JBT-SL-DS, which were genetically modified to uptake and metabolize/degrade specific residual sugar by-products and metabolites in the fermentation medium prior to the purification of the target oligosaccharide. During LNT production using strain JBT-LNT, the optional degradation strain JBT-LN(n)T-DS can be added at the end of fermentation to degrade residual carbohydrate by-products. Similarly, during the production of 3′-SL and 6′-SL using strain JBT-3SL and JBT-6SL, respectively, the optional degradation strain JBT-SL-DS can be added at the end of fermentation to degrade residual carbohydrate by-products.

Chr. Hansen A/S noted that the 2′-FL component within the blend will be derived from strain E. coli BL21(DE3) JBT-2FLΔlacZ, which was previously assessed and was authorized for use at an inclusion rate of 1.2 g/L for infant formula and 2.4 g/L for toddler formula, as consumed. Consequently, a re-assessment of the safety of the 2′-FL component was not required.

The intended use levels as consumed of each oligosaccharide are the following: 2′-FL (3.0 g/L), 3-FL (0.75 g/L), LNT (1.5 g/L), 3′-SL (0.23 g/L) and 6′-SL (0.28 g/L). The intended use level of 2′-FL is higher than the previously authorized inclusion rate. The Bureau of Chemical Safety (BCS) and the Bureau of Nutritional Sciences (BNS) have assessed the implications of adding a higher volume of the final 2′-FL product into infant and toddler formulas. Various maximum use levels are also proposed in infant and toddler formulas.

This assessment considered the safety of the final blended product and does not extend to the sale of the individual oligosaccharides. The only exception is the previously assessed 2′-FL ingredient that will continue to be permitted to be sold as a stand-alone product.

The bacterially synthesized milk oligosaccharides in the blend from Chr. Hansen A/S were confirmed to be chemically equivalent to oligosaccharides isolated from human milk. Oligosaccharides in human milk may support infant and toddler health as a prebiotic substrate for commensal bacteria as well as functioning as a modulator of the immune system. This evaluation did not include an assessment of any potential claims that could be made about oligosaccharides in human milk.

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 the oligosaccharide blend was developed; how the composition and nutritional quality of the bacterially synthesized milk oligosaccharides compared to human milk oligosaccharides; and what the potential is for the oligosaccharide blend to be toxic or cause allergic reactions. Chr. Hansen A/S has provided data which demonstrates that 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL are safe to use in infant and toddler formulas.

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 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL are produced by genetically modified bacteria, the blend 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

iii. One or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism".

Development of the modified microorganisms

The production strains E. coli BL21(DE3) JBT-3FL, JBT-LNT, JBT-3SL and JBT-6SL, as well as the optional degradation strains E. coli BL21(DE3) JBT-LN(n)T-DS and JBT-SL-DS, were derived from the well-characterized non-pathogenic E. coli BL21(DE3) parental strain. The parental strain was genetically modified by the insertion of biosynthetic pathways, resulting in strains with the following phenotypic characteristics:

1. JBT-3FL: uses lactose to produce 3-FL by efficiently importing lactose into the cell, ferments simple carbon sources like glycerol, expresses a heterologous α-1,3-fucosyltransferase which catalyzes the synthesis of 3-FL from lactose and GDP-fucose, overexpresses the genes involved in the synthesis of GDP-fucose and secretes 3-FL into the culture medium;
2. JBT-LNT: uses lactose to produce LNT by efficiently importing lactose into the cell, ferments simple carbon sources like glycerol, expresses a heterologous β-1,3-N-acetylglucosaminyltransferase which catalyzes LNT II from lactose, expresses a heterologous β-1,3-galactosyltransferase which catalyzes the synthesis of LNT from LNT II, overexpresses the genes involved in the synthesis of UDP-galactose and UDP-N-acetylglucosamine, and secretes LNT into the culture medium;
3. JBT-3SL: uses lactose to produce 3′-SL by efficiently importing lactose into the cell, ferments simple carbon sources like glycerol, expresses a heterologous α-2,3-sialyltransferase which catalyzes the synthesis of 3′-SL from lactose and CMP-sialic acid, overexpresses the genes involved in the synthesis of CMP-sialic acid, and secretes 3′-SL into the culture medium;
4. JBT-6SL: has the same metabolic traits as JBT-3SL except that it expresses a heterologous α-2,6-sialyltransferase (instead of α-2,3-sialyltransferase) which catalyzes the synthesis of 6′-SL from lactose and CMP-sialic acid;
5. JBT-LN(n)T-DS (optional degradation strain): degrades carbohydrate by-products in the culture medium at the end of fermentation with JBT-LNT by overexpressing the genes involved in carbohydrate uptake and carbohydrate metabolism; and, finally,
6. JBT-SL-DS (optional degradation strain): degrades carbohydrate by-products in the culture medium at the end of fermentation with JBT-3SL and JBT-6SL by overexpressing the genes involved in carbohydrate uptake and carbohydrate metabolism.

Characterization of the modified microorganisms

Whole genome sequencing of the production strains and degradation strains was performed to characterize the genetic modifications introduced into their respective genomes. No plasmid vector backbone was detected in all six strains. No genetic rearrangements were detected and all insertions and deletions were well-characterized. The genetic stability of genetic modifications was demonstrated for all production and degradation strains.

Open reading frame (ORF) and single nucleotide polymorphism (SNP) analysis reports were provided for all genetically modified loci within all six strains. A rationale was provided to explain why the few unintended ORFs and SNPs detected would not pose a safety risk. No putative fusion proteins or frame-shift mutations were identified. Additionally, there are no safety concerns regarding the putative expression of unintended proteins since extensive purification steps are applied during the manufacturing process to remove residual proteins and recombinant DNA.

The donor DNA sequences of the genetic constructs inserted into the chromosome of all four production strains and both degradation strains, were derived from the parental strain E. coli BL21(DE3) (amplified from genomic DNA), the closely related non-pathogenic and well-characterized E. coli K-12 (either amplified from genomic DNA or synthetic), and from well-characterised heterologous sources (all synthetically produced). There are no safety concerns regarding the potential carry-over of non-target DNA from heterologous sources since the genes of interest were synthetically produced. The regulatory elements were also synthetically produced from well-characterized donor organisms. Based on the information provided, there are no safety concerns regarding the donor organisms used during the development of the production strains and the degradation strains.

Based on the information provided regarding the molecular characterization of the six strains, there are no safety concerns, from a molecular biological perspective, regarding 2′-FL, 3-FL, LNT, 3′-SL and 6'-SL produced by genetically modified E. coli BL21(DE3) production strains, nor the use of the two optional degradation strains to improve the purity of the final product.

Product information

Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend is manufactured by the fermentation of production strains under sterile conditions in a facility compliant with cGMP and operating under Hazard Analysis Critical Control Point (HACCP) principles. Each of the five oligosaccharides (2′-FL, 3-FL, LNT, 3′-SL and 6′-SL) is first produced individually via separate microbial fermentation, is highly purified, and then mixed to produce the commercial blend. The processing steps and fermentation conditions are optimized for each oligosaccharide. Briefly the steps are the following:

1. Fermentation is performed according to a standard procedure using defined media containing no antimicrobials or inhibitors. During fermentation, each oligosaccharide is efficiently secreted into the medium;
2. During LNT production, the optional degradation strain coli BL21(DE3) JBT-LN(n)T-DS can be added at the end of fermentation to degrade residual carbohydrate by-products;
3. During the production of 3′-SL and 6′-SL, the optional degradation strain coli BL21(DE3) JBT-SL-DS can be added at the end of fermentation to degrade residual carbohydrate by-products;
4. Purification steps remove and inactivate the microbial biomass from the filtrate containing the oligosaccharide. Downstream purification steps remove impurities such as proteins, DNA, organic acids and inorganic salts; and, finally,
5. The final filtration steps ensure the removal of endotoxins and residual micro-impurities. The purified product is then dried into a powder that is ready for commercial use.

Dietary exposure

Infants on animal milk formulas without added sources of human milk oligosaccharides (HMOs) are exposed to less oligosaccharides than breast milk fed infants. Average HMO concentrations range from 20 g/L in the colostrum to 12 g/L in mature breast milk^{Footnote 1Footnote 2} compared to 1 g/L in the colostrum and about 20-fold less in mature bovine milk^{Footnote 3}.

The blend produced by Chr. Hansen A/S is composed of 5 individual oligosaccharides. The levels of the individual oligosaccharides each fall within their reported ranges in breast milk at the maximum proposed level of use for the blend (6.96 g/L). Using the maximum proposed use level (including overage), with the conservative consumption level of 260 mL/kg for infant formula^{Footnote 4}, the daily intake of the five oligosaccharides for infants (p95) and toddlers (p90) on a body weight basis is estimated to be 1.81 g/kg bw per day and 0.468 g/kg bw per day, respectively.

Nutrition

HMOs are non-digestible and structurally diverse complex carbohydrates that represent the third most abundant solid component of breast milk. More than 200 different HMO structures have been identified. The concentration and composition of HMOs in breast milk vary depending on factors such as the mother's genetic background (HMO secretor or non-secretor), environmental factors, geographical location, lactation stage, gestational age, and maternal health^{Footnote 5}.

HMOs do not function as direct energy sources for the infant. Most HMOs reach the colon where they are utilized by specific gut bacteria and only 1 % are absorbed. There are no indications that the 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL manufactured by Chr. Hansen A/S differs in absorption from similar components in breast milk. Based on the information submitted, it is not expected that the oligosaccharide blend will have a negative effect on the absorption of nutrients.

A randomized, controlled multicentre clinical growth and tolerance study of the 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend was submitted and reviewed^{Footnote 6}. Infants received a milk-based experimental formula that contained these oligosaccharides or a similar formula without these ingredients, which acted as a control. A breast milk fed group was also included as a reference group. As this study was conducted during the COVID-19 pandemic, additional statistical modelling was conducted to account for protocol deviations. Statistical aspects of the study were reviewed by the Bureau of Data, Science and Knowledge Integration (BDSKI). Overall, the study provides evidence for the safety and tolerance of 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend in infant formula which was extrapolated to toddler formula.

Based on the information provided, there are no nutritional concerns related to the proposed food use of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend at a maximum level up to 6.96 g per liter for term infant or toddler formulas as fed (inclusive of overage).

Microbiology

The host strain, E. coli BL21(DE3), is a well-characterized, non-pathogenic strain which has been used extensively as a host for the expression of recombinant proteins. It lacks the fertility plasmid and is, therefore, incapable of transferring its DNA to other organisms. This strain is incapable of colonizing the human gastrointestinal tract, does not produce antibiotics, nor does it possess antimicrobial resistance genes.

The microbiological specifications and Certificates of Analysis (CoA) for five non-consecutive batches of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend were provided. The specifications, methods and test results were all deemed to be acceptable. The novel food was tested for the presence of high-risk pathogens for this food type (such as Cronobacter spp., Salmonella spp. and Enterobacteriaceae) and all five batches complied with specified thresholds. The test results successfully demonstrated the absence of the production strains and degradation strains. There are no concerns regarding the microbiological specifications provided that they are met by the manufacturer.

The parent strain E. coli BL21(DE3) does not produce long-chain lipopolysaccharides (LPS) and the endotoxins it produces are considered non-toxic. Nevertheless, a filtration step is used at the end of the manufacturing process to remove residual endotoxins. The specifications include a limit for endotoxins and the CoA of 5 batches confirm that endotoxin test results fall well below the specified limit.

Some of the production or degradation strains possess antimicrobial resistance genes integrated into their genomes as a result of the strain development process. However, these antimicrobial resistance genes do not pose a safety concern since the oligosaccharide blend is devoid of transgenic DNA, as was demonstrated by real-time qPCR analysis of five non-consecutive batches. In addition, there are no concerns regarding the expression products of the antimicrobial resistance genes since fermentation occurs without the use of antimicrobials and the oligosaccharide blend is devoid of proteins, as was demonstrated by testing five batches.

Based on the information provided, there are no safety concerns regarding Chr. Hansen's blend containing 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL from a microbiological perspective.

Chemistry

The specifications for trace elements (arsenic, cadmium, lead and mercury) and the mycotoxin aflatoxin M1 established by the petitioner can be consistently met based on analytical results provided by the petitioner. The estimated concentrations of these contaminants in infant formula and toddler formula 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 formulae with the oligosaccharide blend at the maximum requested level of use was determined to contribute to negligible amounts of the overall dietary exposure for arsenic and lead, and are orders of magnitude lower than their respective toxicological reference values in the cases of cadmium and mercury. Overall, the potential exposure to arsenic, cadmium, lead and mercury from the proposed uses of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend are not expected to pose a chemical safety concern.

The Food Contaminants Section (FCS) notes that the analytical results for arsenic, cadmium and mercury are consistently non-detects at levels 2 – 20 times less than the specifications provided. In keeping with the principles of ALARA (as low as reasonably achievable), the FCS would encourage the petitioner to lower the specifications to more accurately reflect the concentrations of arsenic, cadmium and mercury found in the oligosaccharide blend.

A list of substances used during the manufacturing for each of the 3-FL, LNT, 3′-SL, and 6′-SL components produced by fermentation of E. coli BL21(DE3) strains was included in the submission. Ingredients used in the fermentation media for cultures such as E. coli BL21(DE3) production strains are not typically regulated as food additives and usually there is no requirement under the FDR for their premarket review. However, the petitioner is responsible for ensuring that the use of any substance in manufacturing does not result in a violation of section 4 of the Food and Drugs Act. The petitioner indicated that, all the materials used in the manufacturing process are of food grade quality and meet specifications set out in the Food Chemicals Codex (FCC) and/or Combined Compendium of Food Additives Specifications prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), where available.

The petitioner provided data to sufficiently demonstrate that 3-FL, LNT, 3′-SL, and 6′-SL components produced by genetically modified strains derived from E. coli BL21(DE3), on a molecular structure level, are equivalent to their counterparts found in human milk. These oligosaccharides also contain small residual amounts of other structurally related carbohydrates as impurities, which are all naturally present in breastmilk.

Based on the information provided, there are no chemical food safety concerns with the proposed uses of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend produced by fermentation of genetically modified strains derived from E. coli BL21(DE3).

Toxicology

The petitioner provided toxicological studies using Chr. Hansen's oligosaccharide blend containing 2′-FL (47.1%), 3-FL (16.0%), LNT (23.7%), 3′-SL sodium salt (4.1%) and 6′-SL sodium salt (4.0%). The studies were conducted in accordance with the available Organisation for Economic Co-operation and Development (OECD) guidelines, and in compliance with the principles of Good Laboratory Practice.

No evidence of mutagenicity/genotoxicity (bacterial reverse mutation assay, in vitro micronucleus test) was observed in the studies provided, suggesting that Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL are unlikely to be genotoxic. The No Observed Adverse Effect Level (NOAEL) for Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend in a 90-day dietary toxicity study in rats was concluded to be 10% in the diet (i.e., corresponding to mean intakes of 5.67 g/kg bw/day for males and 6.97 g/kg bw/day for females), the highest dose tested.

In a 21-day study in neonatal piglets, milk replacer containing Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend at up to 8.0 g/L (corresponding to ~3.6 g/kg bw/day) was well-tolerated. This dose is ~2-fold higher than the highest maximum requested use level in infants on a body weight basis (i.e., 1.81 mg/kg bw per day). No safety concerns were reported in a randomized, double-blinded, controlled clinical trial of the consumption of infant formula containing 3 g/L 2′-FL, 0.8 g/L 3-FL, 1.5 g/L LNT, 0.2 g/L 3′-SL and 0.3 g/L 6′-SL for 16 weeks, provided by the petitioner.

When comparing the NOAEL from the 90-day toxicity study with the highest estimated exposure per population category (P95 infants, P90 toddlers) the margins of exposure (MOE) are low, with a range from about 3 to 15. However, based on the absence of toxicologically relevant effects at the highest doses tested in rats and piglets, the lack of safety concerns in human clinical trials, as well as the conservatisms in the exposure estimate, the MOEs are considered sufficient from a safety perspective.

Based on the information provided, there are no toxicological concerns with the use of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend in infant and toddler formulas, at the proposed levels of use.

Allergenicity

HMOs are not proteinaceous and are not expected to elicit an immunoglobulin E (IgE) mediated allergic response in consumers. Furthermore, HMOs have a history of safe use in food for infants from their presence in breastmilk.

As determined by a modified version of the Bradford assay (Nanoquant), the overall protein content in Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend is low (specifications ≤ 100 µg protein/g preparation). Data from five batches of the oligosaccharide blend demonstrated it is essentially devoid of proteins, and thus it is not expected to pose an allergenic concern for consumers.

Based on the information provided, there are no allergenicity concerns with the proposed use of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend in infant and toddler formulas.

Conclusion

Health Canada's review of the information presented in support of the use of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend derived from genetically modified strains of E. coli BL21(DE3) in infant and toddler formulas, does not raise concerns related to food safety.

Health Canada's opinion refers only to the use of Chr. Hansen's 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL blend as an ingredient in infant and toddler formulas. Infant formulas and human milk fortifiers containing 2′-FL, 3-FL, LNT, 3′-SL and 6′-SL derived from genetically modified strains of E. coli BL21(DE3) 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
bmh-bdm@hc-sc.gc.ca