Weighted analysis of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose concentrations in human milk.

Over 150 human milk oligosaccharides (HMOs) have been identified and their concentrations in human milk vary depending on Secretor and Lewis blood group status, environmental and geographical factors, lactation stage, gestational period, and maternal health. Quantitation of HMOs in human milk has been the focus of numerous studies, however, comprehensive and weighted statistical analyses of their levels in human milk are lacking. Therefore, weighted means, standard deviations, medians, interquartile ranges, and 90th percentiles for 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were calculated using random sampling and the levels of these HMOs in human milk reported in the literature. Probability distributions of the reported levels were also constructed. Although the levels reported in the published studies varied, the weighted means for 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL were calculated to be 2.58, 0.57, 0.94, 0.28, and 0.39 g/L, respectively, which are consistent with those that have been previously determined in other systematic analyses. Likely due to the use of weighting, the 90th percentiles were greater than the 95% confidence limits that have been previously calculated. Our study therefore provides accurate and important statistical data to help support the level of appropriate HMO supplementation in infant formula.

A safety evaluation of mixed human milk oligosaccharides in rats.

Human milk oligosaccharides (HMOs) are indigestible carbohydrates representing the third largest fraction of solutes in human breastmilk. They provide valuable prebiotic and anti-pathogenic functions in breastfed infants, but are not yet included in most infant formula products. Recent biotechnological advances now facilitate large-scale production of HMOs, providing infant formula manufacturers with the ability to supplement their products with HMOs to mimic human breastmilk. Although the safety of individual HMOs has been confirmed in preclinical toxicological studies, the safety of HMO mixtures has not been tested. We therefore performed bacterial reverse mutation and in vitro micronucleus tests and conducted a repeated-dose oral toxicity study in rats with a mixture of five HMOs (HMO MIX I), containing 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL). HMO MIX I was not genotoxic and did not induce adverse effects in the repeated dose study. The no-observed-adverse-effect-level (NOAEL) for HMO MIX I in this study is 10% in the diet (equivalent to 5.67 g HMO MIX I/kg bw/day for males and 6.97 g HMO MIX I/kg bw/day for females). Our results provide strong evidence for the safety of HMO MIX I in infant products and general foods.

Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters.

For the huge amount of chiral chemicals and precursors that can potentially be produced by biocatalysis, there is a tremendous need of enzymes with new substrate spectra, higher enantioselectivity, and increased activity. In this paper, a highly active alcohol dehydrogenase is presented isolated from Nocardia globerula that shows a unique substrate spectrum toward different prochiral aliphatic ketones and bulky ketoesters as well as thioesters. For example, the enzyme reduced ethyl 4-chloro-3-oxo butanoate with an ee >99% to (S)-4-chloro-3-hydroxy butanoate. Very interesting is also the fact that 3-oxobutanoic acid tert-butylthioester is reduced with 49.4% of the maximal activity while the corresponding tert-butyloxyester is not reduced at all. Furthermore, it has to be mentioned that acetophenone, a standard substrate for many known alcohol dehydrogenases, is not reduced by this enzyme. The enzyme was purified from wild-type N. globerula cells, and the corresponding 915-bp-long gene was determined, cloned, expressed in Escherichia coli, and applied in biotransformations. The N. globerula alcohol dehydrogenase is a tetramer of about 135 kDa in size as determined from gel filtration. Its sequence is related to several hypothetical 3-hydroxyacyl-CoA dehydrogenases whose sequences were derived by whole-genome sequencing from bacterial sources as well as known mammalian 3-hydroxyacyl-CoA dehydrogenases and ss-hydroxyacyl-CoA dehydrogenases from different clostridiae.