Multicellular coupling fermentation for 3'-sialyllactose conversion using N-acetyl-glucosamine and lactose / 生物工程学报

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg2+, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.

Disialyllacto-N-tetraose improves intestinal homeostasis of metabolic microenvironment to prevent the pathological development of necrotizing enterocolitis in neonatal rats / 中华实用儿科临床杂志

Objective:To investigate the effects of disialyllacto-N-tetraose (DSLNT) on low molecular weight metabolic profile of intestinal contents in neonatal rats with necrotizing enterocolitis (NEC), in an attempt to explore the protective mechanism of DLSNT on intestinal tract of neonates.Methods:Immediately after birth, SD rats were randomly divided into the control group, the NEC group and the NEC+ DSLNT group according to random number tale method.All rats were hand-fed by special formula milk.Rats in the NEC group and NEC+ DSLNT group were exposed to hypoxia (950 mL/L nitrogen, 10 min, thrice per day) and cold stress (4 ℃, 10 min, thrice per day) for continuous 3 days to establish rodent NEC model.Rats in the NEC+ DSLNT group were hand-fed with special formula containing 300 μmol/L DSLNT.All rats were sacrificed after 72 h, and intestinal contents were collected from ileum and colon, followed by untargeted metabolomic determination with the ultrahigh-performance liquid chromatography Q extractive mass spectrometry (UHPLC-QE-MS) method.The terminal ileum was examined by hematoxylin-eosin staining.The metabolome data were analyzed with multivariable analysis using SIMCA 14.1.The metabolites that met both variable importance in the projection (VIP) >1 in the orthogonal partial least squares analysis (OPLS-DA) model and P<0.05 in the t-test were screened as differential metabolites between groups. Results:DSLNT reduced the incidence of NEC and pathological scores of ileum tissue from neonatal rats with NEC [3.0(2.0, 3.0) scores vs.1.0(1.0, 2.0) scores, P<0.01], and also significantly suppressed inflammatory infiltration.OPLS-DA model based on the metabolome data determined by UHPLC-QE-MS could perform effective discrimination between the NEC group and the control group, as well as the NEC+ DSLNT group and the NEC group.There were 64 differential metabolites between the NEC group and the control group (VIP value>1 and P<0.05 for the OPLS-DA model). These metabolites included docosahexaenoic acid (+ 288.0%, P=0.028), xanthine (+ 372.1%, P=0.007), L-arginine (+ 233.1%, P=0.027), L-leucine (+ 232.7%, P=0.015), N-acetylneuraminic acid (-41.6%, P=0.014), and so forth.These metabolites were associated with 34 metabolic pathways.Among them, such 6 pathways as arginine biosynthesis, arginine and proline metabolism were the most disturbed pathways affected by NEC.There were 15 diffe-rential metabolites in between NEC+ DSLNT group and NEC group, which included D-mannose (-73.5%, P=0.032), xanthine (-63.4%, P=0.008), linoleic acid (+ 137.9%, P=0.047), nicotinamide adenine dinucleotide (+ 278.2%, P=0.005), and so forth.These metabolites were mapped to 7 metabolic pathways, among them, linoleic acid metabolism pathway was the most relevant differential pathway affected by DSLNT.There were 8 overlapped meta-bolites in both comparison strategies, and the variation trend of these overlapped metabolites in the NEC group was significantly reversed by DSLNT supplementation. Conclusions:DSLNT could significantly attenuate the NEC pathological damage caused by hypoxia/cold stress in neonatal rats.This protective effect is associated with the improvement of the metabolic profile of intestinal contents caused by NEC and the modulation of the linoleic acid metabolic pathway.The early preventive supplementation of DSLNT is of great significance in maintaining neonatal intestinal homeostasis and preventing the process of NEC.

Research progress on the effects of human milk oligosaccharides on infant intestinal flora / 中华实用儿科临床杂志

Infancy is the most critical period for the formation and development of intestinal flora, which is an important stage for the rapid succession and stable colonization of intestinal flora involved in the health and establishment and improvement of the immune system.Breast milk is rich in human milk oligosaccharides (HMOs), which can effectively promote the growth of beneficial bacteria in the infant intestine.It inhibits the invasion of pathogen, improves the composition of the intestinal flora, increases its diversity, and promotes the growth and development of the infant.This study mainly reviews the research status of HMOs and its influence on the infant intestinal flora.

The protective effects of disialyllacto-N-tetraose of neonatal intestinal barriers in the prevention of necrotizing enterocolitis / 中国新生儿科杂志

Objective:To study the expression of zonula occludens-1(ZO-1) in neonates with necrotizing enterocolitis (NEC) and to explore the effects of disialyllacto-N-tetraose (DSLNT), a compound extracted from human milk, on the intestinal barriers in rat model of NEC.Methods:(1) Human study: From Feb 2013 to Dec 2020, the pathological samples of ileum tissue from 21 neonates (12 patients with NEC and 9 with intestinal atresia) from Pathology Department of our hospital were collected. The expressions of ZO-1 in these samples were examined using immunohistochemistry (IHC) method. (2) Animal study: A total of 28 newborn rats were randomly assigned into control group ( n=8), NEC group ( n=10) and DSLNT+NEC group ( n=10). Experimental NEC model was established based on hypoxia (95%N 2 10 min) /cold exposure (4 ℃ 10 min) three times a day for consecutive 3 days. DSLNT+NEC group were fed with formula+DLSNT (300 μmol/L) during hypoxia/cold exposure. All the surviving rats were sacrificed at the end of the experiment (72 h) and the terminal ileum tissues were collected. Hematoxylin-Eosin (HE) staining was used to evaluate tissue damage and Western blotting was used to determine the expressions of ZO-1. (3) Cellular study: Bacterial lipopolysaccharide (LPS) was used to establish a cellular inflammation model in human intestinal epithelial cell lines (Caco-2) and DSLNT (300 μmol/L) was applied to this model. Thiazolyl blue assay was used to examine cell viabilities and immunofluorescence assay was used to detect ZO-1 expression. The effects of DSLNT on cell growth and tight junctions of Caco-2 cells were analyzed. Results:(1)Human study: The villi of mucous layer of the lesion were damaged in NEC patients. ZO-1 expressions at the epithelial junction of NEC patients were decreased compared with intestinal atresia patients and non-lesion intestines of NEC patients. (2)Animal study: Apical extrusion, necrosis and shedding of epithelial cell were seen at the lesions in NEC group. The expression of ZO-1 in NEC group was significantly lower than the control group and DSNLT+NEC group ( P<0.05).DSNLT+NEC group had higher survival rates (8/10 vs. 6/10) and lower ileum inflammatory pathological scores [2.0(1.0, 2.8) vs. 3.5(3.0, 4.0)] than NEC group. (3) Cellular study: Caco-2 cells exposed to LPS showed inhibited cell growth and decreased ZO-1 immunofluorescence staining. Caco-2 cells in the DSLNT+LPS group showed better viability than LPS group and were comparable with the control group. The expression of ZO-1 was significantly increased in the DSLNT+LPS group. Conclusions:Tight junction injury of the intestinal epithelial cell is an important characteristic of NEC. ZO-1 is a potential target for the prevention and treatment of NEC. DSLNT may protect the neonatal intestines by modulating the expression of ZO-1 and keeping tight junction integrity.

Prebiotics in the Infant Microbiome: The Past, Present, and Future / 대한소아소화기영양학회지

Recent advances in the bio-production of human milk oligosaccharides 2'-FL and 3-FL / 生物工程学报

Human milk oligosaccharides (HMO) are important immunoactive components found in breast milk. Scientific research proves that HMOs are significantly beneficial for infant health. 2'-fucosyllactose (2'-FL) is the major component of HMO, which obtained growing attentions from food industry. Besides, 3-fucosyllactose (3-FL) is another important fucosyllactose and it has a similar synthetic route comparing to 2'-FL. Thus, research of the two HMO components has interactive effects for each other. Recently, numerous publications are available for 2'-FL and 3-FL. The microbial cell factory is able to massively produce fucosyllactose via an efficient way, which will show considerable influences in dairy industry. In this paper, we review recent studies on 2'-FL and 3-FL, and discuss their prospects according to published literature and patents.

Hidratos de carbono complejos en la leche materna: los oligosacáridos (Parte 1) / Complex carbohydrates in breast milk: oligosaccharides (Part 1)

Los oligosacáridos de la leche materna (HMOs) son unas 200 moléculas distintas sintetizadas y secretadas por la glándula mamaria a partir de lactosa a la que diversos enzimas unen monosacáridos simples (glucosa, galactosa, n-acetil galactosamina, fucosa y ácido siálico). Estas uniones y sus diferentes orientaciones espaciales generan una gran diversidad de estructuras químicas y de funcionalidades. La concentración de los HMOs es mayor en el calostro (± 25 g/L), está relacionada con la duración del embarazo y la lactancia: disminuyen progresivamente hasta la mitad de los niveles iniciales. La genética materna influye en el perfil de algunos HMOs; el gen FUT2, que codifica la síntesis de la fucosiltransferasa 2 (FUT2) condiciona el llamado carácter secretor en 75-85% de las mujeres y hace que los antígenos del grupo ABO(H) sean secretados en los líquidos orgánicos (saliva, lágrimas, semen). La ausencia de actividad del gen FUT2 condiciona el carácter no-secretor (15-25% de las mujeres). La actividad del gen FUT3 condiciona la actividad de la fucosiltransferasa 3 (FUT3) que se asocia con el grupo sanguíneo Lewis+ mientras que su ausencia caracteriza a los portadores como Lewis 0. Los HMOs son absorbidos a nivel del intestino como trazas (1%) pero incluso en esas cantidades ejercerían efectos sistémicos.

Human milk oligosaccharides (HMOs) are a family of some 200 different molecules synthesized by the mammary gland. At the core is a molecule of lactose, which is linked by different enzymes to glucose, galactose, n-acetyl galactosamine, fucose or sialic acid. These linkages and their different spatial orientation generate, besides the possibilities of numerous chemical structures, the potential for different spatial isomers. The concentration of HMOs in human milk depends on pregnancy and breastfeeding duration. They are highest in colostrum (± 25 g/L) and decrease over time to half this initial level. Maternal genetics modifies the concentration and profile of some oligosaccharides. For example, the FUT2 gene codifies the synthesis of fucosyltransferase 2 (FUT2) whose activity generates the secretor status for antigens of the ABO(H) blood group in organic fluids (saliva, milk, tears, semen) among 75-85% of the carriers of the trait. The absence of activity of the FUT2 gene conditions the non-secretor status (15-25% of women). The FUT3 gene regulates the activity of the fucosyltransferase 3 (FUT3) that is associated with the Lewis blood group. Traces of HMOs (1%) are absorbed in the intestinal tract, however, they exert important systemic effects even at low concentrations.

Leche materna: características funcionales de los oligosacáridos de la leche materna (Parte 2) / Breast milk: functional characteristics of breast milk oligosaccharides (Part 2)

Durante los primeros meses de vida, los oligosacáridos de la leche materna (HMOs) aportados por la leche materna participan en procesos asociados con la maduración de tejidos y sistemas del tubo digestivo, modulan algunos de sus procesos metabólicos y ejercen efectos prebióticos y antimicrobianos. Otros efectos estudiados son su contribución a la instalación, desarrollo y estimulación de la microbiota residente con predomino de Bifidobacterium y Bacteroides, con efectos protectores frente a posibles colonizaciones o patologías por enteropatógenos (bacterianas, virus o parásitarias) que pueden actuar nivel local en el tubo digestivo, pero también pueden influir a nivel sistémico. Los HMOs modularían el desarrollo de la inmunidad innata y adaptativa, y probablemente previenen el desarrollo de fenómenos de atopia/alergia. Una patología propia de la etapa neonatal de los prematuros es la enterocolitis necrosante y algunos HMOs podrían disminuir el riesgo de su manifestación. Las actividades de los oligosacáridos de la leche materna contribuyen a la adaptación del lactante a los desafíos que plantea su entorno incluyendo la prevención de algunas patologías en edades posteriores, como es el caso de la diabetes tipo 1 y la obesidad.

During the first months of life, breast milk oligosaccharides (HMOs) stimulate development of the gastrointestinal tract in newborns and young infants; they modulate its metabolism and transport capabilities. Additionally, they exert prebiotic and antimicrobial activities and contribute to the development of the resident intestinal microbiota with a predominance of Bifidobacterium and Bacteroides and protect from colonization and infections by enteropathogens (bacteria, virus or parasites). It is highly probable that their activities extend beyond infancy and persist into adult life. HMOs stimulate the development of the innate and adaptive immune systems and decrease the risk of atopy/allergy. Their intake has been associated with a degree of protection against as necrotizing enterocolitis among premature infants. HMOs contribute to the long term adaptation and protection of newborn infants to unfavorable conditions of their environment and in this way may contribute to protect breastfed infants from type 1 diabetes and obesity.

Leche materna: efectos de los oligosacáridos de la leche materna en el crecimiento y desarrollo de los lactantes (Parte 3) / Breast milk: effects of breast milk oligosaccharides on the growth and development of infants (Part 3)

Actualmente existe la capacidad de sintetizar oligosacáridos de leche materna (HMOs) en cantidades importantes a partir de hidratos de carbono simples para emplear en estudios en lactantes e incluso en adultos. En los lactantes las fórmulas que contienen HMOs mantienen velocidades normales de incremento del peso, largo corporal y perímetro cefálico con variaciones del largo corporal, el peso y las masas magra y grasa característicos de ciertos HMOs. Algunos HMOs estimulan in vitro en monocitos estimulan en sangre periférica marcadores de inflamación semejantes a los observados con estímulos iguales en lactantes amamantados. Los HMOs están asociados con disminuciones del riesgo de enterocolitis necrosante en prematuros y en ratones. En seguimientos por cuatro meses, lactantes alimentados con una fórmula con 2' fucosil lactosa (2'FL) y lacto-N- neotetraosa (LNnT), mostraron patrones de crecimiento del peso, el largo corporal y el perímetro cefálico comparables a los de un grupo control que recibió la misma fórmula sin HMOs; tampoco hubo diferencias en sus patologías intercurrentes. Las concentraciones de HMOs en la leche pueden variar dependiendo de la localidad geográfica donde fueron obtenidas o el estado de la nutrición materna estos factores deben ser tenidos en cuenta al planificar estudios en grupos de población.

Human milk oligosaccharides (HMOs) are currently synthesized in amounts allowing studies with large numbers and longer follow ups of infants and adults. HMOs have been administered to adults in amounts of up to 20 grams per day without associated symptoms of gastrointestinal fermentation. The microbiota of these individuals presents changes considered positive: increases of Bifidobacterium and decreases of Firmicutes and Proteabacteria. A recent study in infants showed that specific HMOs modulate the growth of lean and fat mass or, on the contrary, decrease adipose tissue mass through not well characterized mechanisms. A study in infants fed for 4 months a formula containing both 2'-O-Fucosyllactose (2'-FL) and Lacto-N-neotetraose (LNnT) with a follow up of 8 months showed that body length, weight gain and head perimeter increased at rates comparable to those of breastfed infants or those fed a control formula. No differences in the incidence of upper and lower respiratory tract infections, skin allergies or use of antibiotics was observed. In the planning of population studies it is important to consider that in ethnically different populations breast milk may contain different profiles of HMOs depending on the area where live, suggesting that some of these profiles may be influenced by consanguinity.

Progress of human milk oligosaccharides and infant-related diseases / 国际儿科学杂志

Human milk oligosaccharides (HMOs) are the third largest solid component in human milk,followed by lactose and lipids.The importance of HMOs to infants has attracted more and more attention.The core structure of HMOs consists of galactose (Gal),glucose (Glc),N-acetylglucosamine (GlcAc),fucose (Fuc) and sialic acid (Sia) derived N-acetylneuraminic acid (Neu5Ac),which link with different groups that have different effects.HMOs could be used as prebiotics to regulate intestinal flora,as antiadhesives to resist pathogen adhesion,and as modulators of cell responses to regulate cellular inflammation.Through the mechanisms above,HMOs can affect many aspects of infant growth and development,such as relieving diarrhea,preventing respiratory infections,alleviating allergies,interfering with obesity,and even affecting the acquired immunodeficiency syndrome.This article will explain the structure of HMOs,the metabolism inside human body and the definite mechanism of action in process of infantile development and describe some related diseases.

Detection and comparison of 10 human milk oligosaccharides during lactational stage / 中国食品卫生杂志

Objective To detect 10 kinds of human milk oligosaccharides (HMOS) and compare their amounts during lactational stage.Methods Breast milk samples in different stages of lactation as colostrum (day 0-7 postpartum),transitional milk (day 8-15 postpartum),and mature milk (day 16-180 postpartum) were collected and 10 HMOS in those samples were detected and quantified by ultra high performance liquid chromatography-fluorescence detection after fluorescence labeling by using standard curves.Correlations between HMOS and lactation day were conducted by Person correlation analysis method,while the differences among three stages were calculated by ANOVA test.Results Ten HMOS were successfully separated and quantified under chosen chromatographic parameters.2'FL,3'SL,6'SL,LNT,LNnT and LNFP-I were negatively correlated and 3'FL was positively correlated with lactation days.They were different in three lactational stages (P ＜ 0.05),while P1,LNFP-V and LNnFP-V showed no correlation and difference (P ＞ 0.05).Conclusion The amount of HMOS changed during lactational stages.Seven HMOS were correlated with lactation days and different in three lactational stages (P ＜ 0.05).

Leite materno: ciência, mistérios e novas inspirações para fórmulas infantis / Maternal milk: science, misteries and new inspirations for infantile formulae

A Organização Mundial da Saúde (OMS) afirma que "aleitamento materno é uma forma inigualável de prover alimento ideal para o crescimento e desenvolvimento saudável das crianças". A utilização de fórmulas infantis é uma alternativa para lactentes que não podem ser ou são parcialmente amamentados, após terem sido utilizadas todas as técnicas possíveis, informativa e de suporte para garantir o aleitamento materno. Inúmeras fórmulas lácteas, com as mais diferentes composições e indicações, têm sido desenvolvidas numa busca incessante de encontrar a fórmula que se aproxime das características do leite materno. Esta multiplicidade de produtos tem exigido, por parte da OMS/FAO, das principais sociedades científicas mundiais e das agências reguladoras de vários países, a elaboração de normas técnicas para a produção e utilização destes alimentos. Sabe-se que a complexidade do leite materno, principalmente no que diz respeito aos seus componentes imunológicos, biodisponibilidade dos nutrientes e oscilação na composição ao longo do aleitamento materno, faz com que seja impossível mimetizar seus incríveis benefícios para o desenvolvimento e saúde do lactente a longo prazo. Porém, diversos avanços já ocorreram no desenvolvimento das fórmulas infantis, principalmente em relação a qualidade do perfil de aminoácidos e redução das concentrações de proteínas, quando comparados às formulações disponíveis no passado. No entanto, futuras inovações em relação a estrutura lipídica, assim como, do microbioma e dos oligossacarídeos do leite humano ainda são esperados nas fórmulas infantis das próximas gerações.