Safety of lacto‐N‐neotetraose (LNnT) produced by derivative strains of E. coli BL21 as a novel food pursuant to Regulation (EU) 2015/2283

Abstract

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the change in the production process and specifications of lacto-N-neotetraose (LNnT) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharide (HiMO) LNnT but also contains lactose, lacto-N-triose II (LNT II), para-lacto-N-neo-hexaose (para-LNnH) and other related carbohydrates. The NF is produced by fermentation with two genetically modified strains of Escherichia coli BL21. LNnT when chemically synthesised or produced by microbial fermentation using another E. coli strain (K-12) is already authorised and included in the EU list of NFs. This application is limited to a change in the manufacturing process and specifications while target population, uses and use levels and consequently the anticipated intake do not change. The information provided on the manufacturing process, including the absence of DNA from the producing microorganisms, composition, identity and specifications of the NF do not raise safety concerns. Particularly, the proposed changes in the specifications are limited to a slightly higher ash content and limits for the presence of yeast and moulds, while specifications for methanol and LNnT fructose isomer have been removed. Food supplements are not intended to be used if other foods with the added NF or breast milk for young children are consumed on the same day. The Panel concludes that lacto-N-neotetraose (LNnT) as a NF when produced by fermentation with two genetically modified strains of E. coli BL21 is safe under the proposed conditions of use.

Safety of 3’‐sialyllactose (3’‐SL) sodium salt produced by derivative strains of Escherichia coli BL21 (DE3) as a Novel Food pursuant to Regulation (EU) 2015/2283

“…The Panel notes that a safety assessment of 3'-SL, when produced with a derivative strain of E. coli K-12 DH1, has been carried out by EFSA (EFSA NDA Panel, 2020a) and 3'-SL sodium salt is included in the Union list of authorised NFs. The Panel also notes that other HiMOs (LNnT and LNT) produced by fermentation with derivative strains of the same host strain E. coli BL21 (DE3) have been recently assessed with a positive outcome (EFSA NDA Panel, 2020c, 2022c.…”

“…Since 2015, several scientific opinions have been adopted by the EFSA NDA Panel on the safety of human‐identical milk oligosaccharides (HiMOs) as NFs pursuant to Regulation (EC) No 258/97 or Regulation (EU) 2015/2283: 2’‐fucosyllactose (2’‐FL) (EFSA NDA Panel, 2015a ), lacto‐N‐neotetraose (LNnT) (EFSA NDA Panel, 2015b ), LNnT and 2’‐FL in food supplements (FS) for children (EFSA NDA Panel, 2015c ), N‐acetyl‐ d ‐neuraminic acid (NANA) (EFSA NDA Panel, 2017 ), 2’‐FL/difucosyllactose (DFL) mixture (EFSA NDA Panel, 2019a ), lacto‐N‐tetraose (LNT) produced with a derivative strain of E. coli K‐12 DH1 (EFSA NDA Panel, 2019b ), 3’‐sialyllactose (3’‐SL) sodium salt produced with a derivative strain of E . coli K‐12 DH1 (EFSA NDA Panel, 2020a ), 6’‐sialyllactose (6’‐SL) sodium salt (EFSA NDA Panel, 2020b ), LNnT produced with derivative strains of E. coli BL21 (DE3) (EFSA NDA Panel, 2020c ), 3‐fucosyllactose (3‐FL) (EFSA NDA Panel, 2021 ), 2’‐FL/DFL mixture and LNT in FS for infants (EFSA NDA Panel, 2022a ), 2’‐FL and LNnT in FS for infants (EFSA NDA Panel, 2022b ) and LNT produced with derivative strains of E. coli BL21 (DE3) (EFSA NDA Panel, 2022c ).…”

“…coli K‐12 DH1, have been previously assessed by EFSA as NFs, with positive outcomes. In addition, 2’‐FL and LNnT (EFSA NDA Panel, 2020c ), produced with derivative strains of the same host strain E. coli BL21 (DE3), have been authorised as NFs in the European Union (Commission Implementing Regulation 2017/2470), and LNT produced with derivative strains of E. coli BL21 (DE3) has recently been assessed by EFSA with a positive outcome (EFSA NDA Panel, 2022c ).…”

Safety of 3‐fucosyllactose (3‐FL) produced by a derivative strain of Escherichia coli BL21 (DE3) as a Novel Food pursuant to Regulation (EU) 2015/2283

“…3‐FL is included in the Union list of authorised novel foods (NFs) (Commission Implementing Regulation (EU) 2017/2470 of 20 December 2017 4 ) when produced by fermentation with a genetically modified strain of Escherichia coli K‐12 MG1655. Since 2015, several scientific opinions have been adopted by the EFSA NDA Panel on the safety of human‐identical milk oligosaccharides (HiMOs) as NFs pursuant to Regulation (EC) No 258/97 or Regulation (EU) 2015/2283: 2’‐fucosyllactose (2’‐FL) (EFSA NDA Panel, 2015a ), lacto‐N‐neotetraose (LNnT) (EFSA NDA Panel, 2015b ), LNnT and 2’‐FL in FS for children (EFSA NDA Panel, 2015c ), N‐acetyl‐ d ‐neuraminic acid (NANA) (EFSA NDA Panel, 2017 ), 2’‐FL/difucosyllactose (DFL) mixture (EFSA NDA Panel, 2019a ), lacto‐N‐tetraose (LNT) produced with a derivative strain of E. coli K‐12 DH1 (EFSA NDA Panel, 2019b ), 3’‐sialyllactose (3’‐SL) sodium salt (EFSA NDA Panel, 2020a ), 6’‐sialyllactose (6’‐SL) sodium salt (EFSA NDA Panel, 2020b ), LNnT produced with derivative strains of E. coli BL21 (DE3) (EFSA NDA Panel, 2020c ), 3‐FL produced with a derivative strain of E. coli K‐12 MG1655 (EFSA NDA Panel, 2021 ), 2’‐FL/DFL mixture and LNT in FS for infants (EFSA NDA Panel, 2022a ), 2’‐FL and LNnT in FS for infants (EFSA NDA Panel, 2022b ) and LNT produced with derivative strains of E. coli BL21 (DE3) (EFSA NDA Panel, 2022c ).…”

Effect of Food Endotoxin on Infant Health

“…For pathogenic microorganisms, such as Salmonella, coliforms, Enterobacteriaceae, and Listeria monocytogenes, there are corresponding requirements and regulations in different countries [47][48][49]. In the composition standard of additives for infant formula milk powder, the limits of endotoxin in the fermentation process and adequate production of 2 -O-fucosyllactose and lacto-N-neotetraose (LNnT) are <10 EU/mg [50][51][52]. However, there is currently no standard for the limit of endotoxin in infant formula milk powder.…”