Neonatal subchronic toxicity and in vitro genotoxicity studies of the human-identical milk oligosaccharide 3-fucosyllactose.

Human milk oligosaccharides, such as 3-fucosyllactose (3-FL), are bioactive components of breast milk associated with benefits for infant growth and development. Structurally identical compounds (human-identical milk oligosaccharides-HiMOs) can be produced using microbial fermentation, allowing their use in infant formula to increase its similarity with human milk. Toxicological studies are required to demonstrate safety of HiMOs and that of any impurities potentially carried over from the manufacturing process. Biotechnologically produced 3-FL was tested for potential genotoxicity (bacterial reverse mutation test and in vitro mammalian micronucleus test) and subchronic toxicity (90-day study with neonatal rats). In the 90-day study, 3-FL was administered by gavage to rats once daily from Day 7 of age, at doses up to 4000 mg/kg body weight (bw)/day (the maximum feasible dose), followed by a 4-week recovery period. Reference controls received 4000 mg/kg bw/day of oligofructose, an ingredient permitted for use in infant formula. Results for the genotoxicity studies were negative. In the 90-day study, there were no adverse effects of 3-FL on any of the parameters measured; thus, the no-observed-adverse-effect level (NOAEL) was 4000 mg/kg bw/day (the highest dose tested). These results support the safety of biotechnologically produced 3-FL for use in infant formula and other foods.

The Mean of Milk: A Review of Human Milk Oligosaccharide Concentrations throughout Lactation.

Human milk oligosaccharides (HMOs) are non-digestible and structurally diverse complex carbohydrates that are highly abundant in human milk. To date, more than 200 different HMO structures have been identified. Their concentrations in human milk vary according to various factors such as lactation period, mother's genetic secretor status, and length of gestation (term or preterm). The objective of this review is to assess and rank HMO concentrations from healthy mothers throughout lactation at a global level. To this aim, published data from pooled (secretor and non-secretor) human milk samples were used. When samples were reported as secretor or non-secretor, means were converted to a pooled level, using the reported mean of approximately 80/20% secretor/non-secretor frequency in the global population. This approach provides an estimate of HMO concentrations in the milk of an average, healthy mother independent of secretor status. Mean concentrations of HMOs were extracted and categorized by pre-defined lactation periods of colostrum (0-5 days), transitional milk (6-14 days), mature milk (15-90 days), and late milk (>90 days). Further categorizations were made by gestational length at birth, mother's ethnicity, and analytical methodology. Data were excluded if they were from preterm milk, unknown sample size and mothers with any known disease status. A total of 57 peer-reviewed articles reporting individual HMO concentrations published between 1996 and 2020 were included in the review. Pooled HMO means reported from 31 countries were analyzed. In addition to individual HMO concentrations, 12 articles reporting total HMO concentrations were also analyzed as a basis for relative HMO abundance. Total HMOs were found as 17.7 g/L in colostrum, 13.3 g/L in transitional milk, and 11.3 g/L in mature milk. The results show that HMO concentrations differ largely for each individual HMO and vary with lactation stages. For instance, while 2'-FL significantly decreased from colostrum (3.18 g/L ± 0.9) to late milk (1.64 g/L ± 0.67), 3-FL showed a significant increase from colostrum (0.37 g/L ± 0.1) to late milk (0.92 g/L ± 0.5). Although pooled human milk contains a diverse HMO profile with more than 200 structures identified, the top 10 individual HMOs make up over 70% of total HMO concentration. In mature pooled human milk, the top 15 HMOs in decreasing order of magnitude are 2'-FL, LNDFH-I (DFLNT), LNFP-I, LNFP-II, LNT, 3-FL, 6'-SL, DSLNT, LNnT, DFL (LDFT), FDS-LNH, LNFP-III, 3'-SL, LST c, and TF-LNH.

The forgotten role of food cultures.

Fermentation is one of if not the oldest food processing technique, yet it is still an emerging field when it comes to its numerous mechanisms of action and potential applications. The effect of microbial activity on the taste, bioavailability and preservation of the nutrients and the different food matrices has been deciphered by the insights of molecular microbiology. Among those roles of fermentation in the food chain, biopreservation remains the one most debated. Presumably because it has been underestimated for quite a while, and only considered - based on a food safety and technological approach - from the toxicological and chemical perspective. Biopreservation is not considered as a traditional use, where it has been by design - but forgotten - as the initial goal of fermentation. The 'modern' use of biopreservation is also slightly different from the traditional use, due mainly to changes in cooling of food and other ways of preservation, Extending shelf life is considered to be one of the properties of food additives, classifying - from our perspective - biopreservation wrongly and forgetting the role of fermentation and food cultures. The present review will summarize the current approaches of fermentation as a way to preserve and protect the food, considering the different way in which food cultures and this application could help tackle food waste as an additional control measure to ensure the safety of the food.

Genotoxicity and neonatal subchronic toxicity assessment of a novel mixture of the human-identical milk oligosaccharides lacto-N-fucopentaose I and 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) are a complex group of bioactive molecules largely observed in human breast milk but also occurring in limited amounts in other mammalian milks. Advances in biotechnology have enabled production of human-identical milk oligosaccharides (HiMOs), structurally identical molecules to HMOs found naturally in human milk, intended for addition to infant formula to more closely replicate breast milk. Biosynthesis of a novel mixture of two major HMOs, lacto-N-fucopentaose I and 2'-fucosyllactose (LNFP-I/2'-FL), recently became possible. To support the safety of LNFP-I/2'-FL for use in infant formula and other foods, it was subject to a safety assessment comprising a bacterial reverse mutation test, an in vitro mammalian cell micronucleus test, and a 90-day oral gavage study in neonatal rats. In the 90-day study (the first HiMO study to include the new endocrine-sensitive endpoints described in the 2018 version of OECD Test Guideline 408), LNFP-I/2'-FL was administered by oral gavage to neonatal rats once daily (from Day 7 of age) for 90 consecutive days, at doses up to 5000 mg/kg bw/day, followed by a 4-week recovery period. Concurrent reference controls received 5000 mg/kg bw/day of the approved infant formula ingredient oligofructose. LNFP-I/2'-FL was nongenotoxic in vitro. The highest dose tested (5000 mg/kg bw/day) was established as the no-observed-adverse-effect level in the 90-day study, as there were no test article-related adverse effects on clinical observations, body weight, food consumption, clinical pathology, and organ weights nor any noteworthy macroscopic or microscopic findings. This supports the safety of LNFP-I/2'-FL for its intended uses in food.

Uncovering carbohydrate metabolism through a genotype-phenotype association study of 56 lactic acid bacteria genomes.

Owing to their unique potential to ferment carbohydrates, both homo- and heterofermentative lactic acid bacteria (LAB) are widely used in the food industry. Deciphering the genetic basis that determine the LAB fermentation type, and hence carbohydrate utilization, is paramount to optimize LAB industrial processes. Deep sequencing of 24 LAB species and comparison with 32 publicly available genome sequences provided a comparative data set including five major LAB genera for further analysis. Phylogenomic reconstruction confirmed Leuconostoc and Pediococcus species as independently emerging from the Lactobacillus genus, within one of the three phylogenetic clades identified. These clades partially grouped LABs according to their fermentation types, suggesting that some metabolic capabilities were independently acquired during LAB evolution. In order to apply a genome-wide association study (GWAS) at the multigene family level, utilization of 49 carbohydrates was also profiled for these 56 LAB species. GWAS results indicated that obligately heterofermentative species lack 1-phosphofructokinase, required for D-mannose degradation in the homofermentative pathway. Heterofermentative species were found to often contain the araBAD operon, involved in L-arabinose degradation, which is important for heterofermentation. Taken together, our results provide helpful insights into the genetic determinants of LAB carbohydrate metabolism, and opens for further experimental research, aiming at validating the role of these candidate genes for industrial applications.

Production of HMOs using microbial hosts - from cell engineering to large scale production.

Human Milk Oligosaccharides (HMOs) constitute an important, highly abundant part of mothers' milk delivering many health benefits to the neonate. Until recently, limited availability of HMOs has prevented their use in infant nutrition and impeded research into their biological effects. The shift from chemical synthesis to biotechnological manufacturing has made them accessible in quantities and at prices that are within reach for commercial applications, including infant formula. It accelerated the studies in the field of pre-clinical and clinical HMO biology. This review gives a short overview of HMO manufacturing from the design and optimization of the microbial cell factory and the production of HMOs in the industrial fermentation process to the purification in the downstream process necessary to obtain a final product. Moreover, the transition from chemistry to biotechnology and the current regulatory landscape and commercialization progress are briefly reviewed.