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Human milk oligosaccharides promote the growth and adhesion of Bifidobacteria , thus exerting multiple biological functions on intestinal epithelial cells. Bacterial surface proteins play an important role in bacterial–host intestinal epithelial interactions. In this study, we aimed to investigate the effects of surface proteins extracted from Bifidobacterium bifidum DNG6 (B. bifidum DNG6) consuming 2′-fucosyllactose (2′-FL) on Caco-2 cells monolayer barrier injury induced by lipopolysaccharide, compared with lactose and galacto-oligosaccharides. Our results indicated that 2′-FL may promote the surface proteins of B. bifidum DNG6 to improve intestinal barrier injury by positively regulating the NF-κB signaling pathway, reducing inflammation (TNF-α reduced by 50.34%, IL-6 reduced by 22.83%, IL-1β reduced by 37.91%, and IL-10 increased by 63.47%) and strengthening tight junction proteins (ZO,1 2.39×; claudin,1 2.79×; and occluding, 4.70×). The findings of this study indicate that 2′-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface proteins. The findings of this research will also provide theoretical support for the development of synbiotic formulations. The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Human milk oligosaccharide (HMO) is a unique component of breastmilk. To date, no study has investigated the correlation between HMO and infant nutritional status particularly through the lens of gut microbiota. Therefore, our study aims to investigate the relationships between 2'-Fucosyllactose (2'-FL) in HMO and Firmicutes/Bacteroidetes (F/B) ratio among stunted infants. METHODS: A case-control study was conducted among 103 mother-infant pairs in Malang City, Indonesia. The quantification of 2'-FL HMO was assessed using High-Performance Liquid Chromatography (HPLC). The F/B ratio was analyzed with real-time poly-chain reaction (RT-PCR). For bivariate analysis, we employed the Spearman correlation and Mann--Whitney tests, while for multivariate analysis, we utilized multiple linear regression. RESULTS: The findings showed that the stunted nutritional status was detected in 49 out of 103 infants. In this group, 40.81% of mothers of infants with a stunted nutritional status had a secretor-positive status, while all mothers of infants with appropriate nutritional status tested positive for the secretor status (100%). However, the association between maternal secretor status and infant nutritional status was not statistically significant (p>0.05). The average levels of 2'-FL HMO in breast milk were lower in the group with stunted infants compared to non-stunted infants (1.21 mg/L vs 1.40 mg/L). The regression analysis revealed a significant association of 2'-FL HMO levels with the presence of Bacteroidetes and value of the F/B ratio (p>0.05). CONCLUSIONS: The breast milk component 2'-FL HMO significantly influences the gut microbiota of stunted infants. Future research aimed at elucidating the mechanisms by which 2'-FL HMO modulates infant gut microbiota should consider not only concentration and specific bacterial taxa but also intake levels [ABSTRACT FROM AUTHOR]

Aging‐related metabolic disorders seriously affect the lifespan of middle‐aged and older people, potentially due to disruptions in the adaptive immune and gut microbial profiles. Dietary intervention offers a promising strategy for maintaining metabolic health. This study aimed to investigate the ameliorative effect of 2′‐fucosyllactose (2'‐FL) on aging‐induced metabolic dysfunction and the underlying mechanisms. The results revealed that 2'‐FL significantly relieved aging‐related metabolic disorders, including weight gain, lipid deposition, dyslipidemia, glucose intolerance, systemic inflammation, and abnormal hepatic metabolism. Flow cytometry analysis revealed a significant reduction in T cytotoxic (Tc), T helper (Th), and regulatory T (Treg) cells and a significant increase in Th17 cells in aged mice, while 2'‐FL relieved the aging‐induced proportional changes in Th and Th17 subtypes. The aging intestinal microecology was characterized by higher Th17/Treg ratios, impaired gut barrier function, lower gut bacterial diversity, decreased abundance of beneficial genera including Ligilactobacillus, Colidextribacter, Mucispirillum, and Lachnoclostridium, and increased abundance of harmful bacteria including Turicibacter and Desulfovibrio, which was ameliorated by 2'‐FL treatment. These findings highlight that 2'‐FL is an ideal dietary prebiotic for improving aging‐related metabolic disorders by modulating both the adaptive immune system and the gut microbial profile. [ABSTRACT FROM AUTHOR]

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In this study, the de novo synthesis pathway of 2’-FL was established in Escherichia coli BL21star(DE3). The β-galactosidase gene (lacZ) and the UDP-glucose lipid carrier transferase gene (wcaJ) were knocked out by the CRISPR/Cas9 system. The effects of various exogenous α-1,2-fucosyltransferases on 2’-FL synthesis were investigated. Subsequently, the transcriptional levels of genes involved in the synthesis pathway were regulated, and the fermentation conditions were optimized. The results showed that after fermentation for 72 h, a 2’-FL concentration of 0.34 g/L was obtained in shake flasks with overexpression of the de novo synthesis pathway genes in E. coli BL21star (DE3), and the concentration of 2’-FL was increased to 2.12 g/L by knockout of the lacZ and wcaJ genes. Expression of α-1,2-fucosyltransferase Wcfb from Bacteroides fragilis in shake flasks resulted in the highest concentration of 4.12 g/L. Under the optimized fermentation conditions: 28 ℃ and 0.2 mmol/L final isopropyl-β-D-thiogalactoside (IPTG), the 2’-FL concentration was increased to 5.01 and 31.2 g/L in a shake flask and in a 5 L fermenter under fed-batch condition, respectively.

ABSTRACT: Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2′-fucosyllactose (2′-FL) and 6′-sialyllactose (6′-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2′-FL and 6′-SL cotreatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal-inflamed suckling mice were treated with various mixtures of 2′-FL and 6′-SL. A 2′-FL:6′-SL ratio of 5:1 was identified as the most effective pretreatment HMO mixture in vitro; thus, this ratio was selected and used for low-, middle-, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as BW loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter, Mageeibacillus, and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus, and Saccharofermentans on short-chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggest that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development.

Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2′-fucosyllactose (2′-FL) and 6′-sialyllactose (6′-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2′-FL and 6′-SL cotreatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal-inflamed suckling mice were treated with various mixtures of 2′-FL and 6′-SL. A 2′-FL:6′-SL ratio of 5:1 was identified as the most effective pretreatment HMO mixture in vitro; thus, this ratio was selected and used for low-, middle-, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as BW loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter , Mageeibacillus , and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus , and Saccharofermentans on short-chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggest that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are key components of breast milk, playing a vital role in fostering the healthy development of infants and young children. Among the main HMOs components, 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) exhibit considerable benefits in terms of inhibiting pathogenic infections, regulating gut microbiota, and enhancing immunity. In recent years, the synthesis of 2′-FL and 3-FL has emerged as a prominent area of research in the field. Despite the potential advantages of chemical or enzymatic synthesis of 2′-FL and 3-FL, these methods are hindered by their inherent limitations, including intricate reaction procedures, high costs, and poor yield, which are not conducive to meeting the demand of the market. Therefore, more efficient microbial methods have been developed and applied in the commercial production of 2′-FL and 3-FL. This article systematically outlines the methods for preparing both 2′-FL and 3-FL, thoroughly expounds upon the engineering strategies for microbial synthesis of 2′-FL and 3-FL, and provides a compelling framework for further microbial production exploration of 2′-FL and 3-FL.

2′-Fucosyllactose (2′-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2′-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2′-FL. The 2′-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2′-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2′-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2′-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2′-FL or other HMOs.

Muscle mass and strength gradually decline from middle age, and appropriate physical activity and exercise habits during this period play a key role in preventing sarcopenia. Exercise is the most effective method for improving muscle health, and consuming appropriate dietary supplements along with exercise can boost its effects. Human milk is the most essential source for muscle growth during infancy, and 2′-fucosyllactose (2′-FL) is most abundant oligosaccharide in human milk. Therefore, we hypothesized that 2′-FL would exhibit muscle protective properties in 10-month-old aged male mice subjected to forced swimming exercises. Our findings revealed that 2′-FL administration significantly improved muscle mass, thickness, and strength of calf muscle compared to forced swimming exercise alone. Additionally, 2′-FL enhanced the hypertrophic changes in gastrocnemius and soleus. Furthermore, 2′-FL upregulated gene expression related to muscle protein synthesis (PI3K, AKT1) and muscle growth (A1R, TRPV4), while inhibiting gene expression related to muscle protein degradation (Atrogin-1, MuRF-1) and muscle growth inhibition (Myostatin, SIRT1). 2′-FL also ameliorated exercise-induced oxidative stress, restored antioxidant enzyme activity, and mitigated muscular cell damage. These findings suggest that 2′-FL may serve as a functional dietary supplement combined with exercise for muscle health.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp 11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2′-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP. • This study aimed to increase the production of 2′-FL by enhancing the solubility of α1,2-fucT. • The alpha helix rule and hydropathy contradiction rule were applied to make various mutants. • Increased solubility of the enzyme was screened through split-GFP using FACS. • The α1,2-fucT with improved solubility and higher production capability was selected. [ABSTRACT FROM AUTHOR]

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2′-fucosyllactose (2′FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3′-fucosyllactose (3′FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2′FL, 3′FL, and 1FL, with a higher proportion of 3′FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2′FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. Key points: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

2′‐Fucosyllactose (2′‐FL) is postulated to provide health benefits and promote the growth of probiotics. This work was undertaken to study the effects of 2′‐FL on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage. Yogurts were produced containing 2′‐FL (0 or 2 g/L) and Bifidobacterium strains of human origin (Bifidobacterium longum subsp. longum BB536 or Bifidobacterium longum subsp. infantis ATCC 15697) at a concentration of at least 109 CFU/mL. All yogurts were stored at 4°C for 5 weeks. Results showed that 2′‐FL was stable in yogurts for at least 5 weeks of cold storage, and the addition of 2′‐FL did not significantly alter yogurt fermentation parameters, associated metabolites, and the viability of mixed yogurt starter cultures and Bifidobacterium strains (p > 0.05). The addition of bifidobacteria had a negative impact (p < 0.05) on the survival rate of starter cultures, Streptococcus thermophilus and Lactobacillus delbureckii subsp. bulgaricus. Meanwhile, it is difficult to maintain a high survival rate of bifidobacteria in final yogurt products, and the addition of 2′‐FL could not enhance the viability of bifidobacteria. B. longum BB536 survived at a level higher than 106 CFU/g for 28 days, while B. infantis ATCC15697 maintained this level for only 7 days. In summary, this study has shown the impact of 2′‐FL and bifidobacterial species on yogurt properties, and results suggest that it is promising to use 2′‐FL in yogurt products as a prebiotic. Practical Application: Yogurt is known for its beneficial effects on human health and nutrition. This study reported the production of symbiotic yogurt containing bifidobacteria and 2′‐fucosyllactose (2′‐FL) as a functional food for specified health uses. The viability of yogurt starter cultures and probiotic bifidobacterial strains was analyzed in this study. Moreover, this research demonstrated that 2′‐FL could be added to yogurt without affecting the characteristics of yogurt significantly. [ABSTRACT FROM AUTHOR]

In the crucial early months of life, an infant's health, growth, and safeguarding are paramount. Breastfeeding stands out as the optimal and natural means of nourishment, offering profound benefits for their development and overall wellness. Within breast milk, a rich array of bioactive components, including hormones, oligosaccharides, and immunoglobulins, provide neonates with an ideal nutritional balance. Among these, Human Milk Oligosaccharides (HMOs) rank prominently, serving both as prebiotics and shields against various neonatal ailments. Particularly, compounds like 2'-Fucosyllactose (2'-FL) and Lacto-N-Neotetraose (LNnT) have been deemed safe for integration into infant formulas, amplifying their benefits. Extensive research underscores the manifold advantages of HMOs, from modulating gut microbiota to bolstering immune function and thwarting pathogenic invaders. HMOs also exhibit a nuanced interplay with maternal genetics, influencing their quantity and diversity. Infants nursed by secretor mothers tend to enjoy enhanced HMO benefits compared to non-secretor counterparts. Incorporating HMOs like Lacto-N-Neotetraose and 2'-Fucosyllactose into infant formulas represents a pivotal stride in optimizing child nutrition. This review offers a thorough exploration of recent research on HMOs, delving into their varied types, concentrations, and compositions, while emphasizing their profound impact on infant health and safety. By consolidating current research findings and recent progress, this study seeks to clarify the diverse effects of HMOs on different facets of human physiology, immune system maturation, modulation of gut microbiota, neurological functioning, and lifelong health implications. Emphasizing the pivotal role of HMOs in influencing human well-being from infancy to maturity, this review emphasize the necessity for continued research and potential utilization in clinical settings and nutritional science. [ABSTRACT FROM AUTHOR]

2′-fucosyllactose (2′-FL) is one of the most prevalent milk oligosaccharides. In this study, the anti-obesity effect of 2′-FL was explored in high-fat diet (HFD)-fed mice. Results showed that 2′-FL alleviated body weight gain, improved serum lipid profiles and increased energy expenditure. Under cold exposure, 2′-FL group showed higher body and rectal temperature, improving adaptive thermogenesis. 2′-FL alleviated lipid accumulation, increased mitochondrial DNA content, as well as upregulated the protein expression of thermogenic markers including UCP1, PRDM16, PGC-1α and the phosphorylation of AMPK in both white and brown adipose tissue. 2′-FL also increased the diversity of gut microbiota in HFD mice. The Bacteroides/Firmicutes ratio, and the Bifidobacterium abundance were increased, while Lachnospiraceae abundance was decreased after 2′-FL treatment. Moreover, 2′-FL to HFD mice altered the bile acid profiles at levels comparable to normal diet group. These results indicates that 2′-FL promotes thermogenesis and modulates gut microbiota to alleviate obesity.

Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is known about their effect on cells from the immune system. In this study, we investigated the direct effect of three structurally different HMOs on human derived macrophages before challenge with Staphylococcus aureus (S. aureus). The study demonstrates that individual HMO structures potently affect the activation, differentiation and development of monocyte-derived macrophages in response to S. aureus. 6´-Sialyllactose (6’SL) had the most pronounced effect on the immune response against S. aureus, as illustrated by altered expression of macrophage surface markers, pointing towards an activated M1-like macrophage-phenotype. Similarly, 6’SL increased production of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, IFN-γ and IL-1β, when exposing cells to 6’SL in combination with S. aureus compared with S. aureus alone. Interestingly, macrophages treated with 6’SL exhibited an altered proliferation profile and increased the production of the classic M1 transcription factor NF-κB. The HMOs also enhanced macrophage phagocytosis and uptake of S. aureus. Importantly, the different HMOs did not notably affect macrophage activation and differentiation without S. aureus exposure. Together, these findings show that HMOs can potently augment the immune response against S. aureus, without causing inflammatory activation in the absence of S. aureus, suggesting that HMOs assist the immune system in targeting important pathogens during early infancy.

Abstract Background The biosynthesis of human milk oligosaccharides (HMOs) using several microbial systems has garnered considerable interest for their value in pharmaceutics and food industries. 2′-Fucosyllactose (2′-FL), the most abundant oligosaccharide in HMOs, is usually produced using chemical synthesis with a complex and toxic process. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce 2′-FL, but the low stoichiometric yields (2′-FL/glucose or glycerol) are still far from meeting the requirements of industrial production. The sufficient carbon flux for 2′-FL biosynthesis is a major challenge. As such, it is of great significance for the construction of recombinant strains with a high stoichiometric yield. Results In the present study, we designed a 2′-FL biosynthesis pathway from fructose with a theoretical stoichiometric yield of 0.5 mol 2′-FL/mol fructose. The biosynthesis of 2′-FL involves five key enzymes: phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-d-mannose 4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG), and α-1,2-fucosyltransferase (FucT). Based on starting strain SG104, we constructed a series of metabolically engineered E. coli strains by deleting the key genes pfkA, pfkB and pgi, and replacing the original promoter of lacY. The co-expression systems for ManB, ManC, Gmd, WcaG, and FucT were optimized, and nine FucT enzymes were screened to improve the stoichiometric yields of 2′-FL. Furthermore, the gene gapA was regulated to further enhance 2′-FL production, and the highest stoichiometric yield (0.498 mol 2′-FL/mol fructose) was achieved by using recombinant strain RFL38 (SG104ΔpfkAΔpfkBΔpgi119-lacYΔwcaF::119-gmd-wcaG-manC-manB, 119-AGGAGGAGG-gapA, harboring plasmid P30). In the scaled-up reaction, 41.6 g/L (85.2 mM) 2′-FL was produced by a fed-batch bioconversion, corresponding to a stoichiometric yield of 0.482 mol 2′-FL/mol fructose and 0.986 mol 2′-FL/mol lactose. Conclusions The biosynthesis of 2′-FL using recombinant E. coli from fructose was optimized by metabolic engineering strategies. This is the first time to realize the biological production of 2′-FL production from fructose with high stoichiometric yields. This study also provides an important reference to obtain a suitable distribution of carbon flux between 2′-FL synthesis and glycolysis.

2′-Fucosyllactose (2′-FL) has great application value as a nutritional component and the whole cell biosynthesis of 2′-FL has become the focus of current research. Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation. In this study, systematic engineering of Y. lipolytica for efficient 2′-FL production was performed. By fusing different protein tags, the synthesis of 2′-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2′-FL production. By iterative integration of the related genes, increasing the precursor supply, and promoting NADPH regeneration, the 2′-FL synthesis was further improved. The final 2′-FL titer, 41.10 g/L, was obtained in the strain F5-1. Our work reports the highest 2′-FL production in Y. lipolytica, and demonstrates that Y. lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.

Background: The biosynthesis of human milk oligosaccharides (HMOs) using several microbial systems has garnered considerable interest for their value in pharmaceutics and food industries. 2′-Fucosyllactose (2′-FL), the most abundant oligosaccharide in HMOs, is usually produced using chemical synthesis with a complex and toxic process. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce 2′-FL, but the low stoichiometric yields (2′-FL/glucose or glycerol) are still far from meeting the requirements of industrial production. The sufficient carbon flux for 2′-FL biosynthesis is a major challenge. As such, it is of great significance for the construction of recombinant strains with a high stoichiometric yield. Results: In the present study, we designed a 2′-FL biosynthesis pathway from fructose with a theoretical stoichiometric yield of 0.5 mol 2′-FL/mol fructose. The biosynthesis of 2′-FL involves five key enzymes: phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-d-mannose 4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG), and α-1,2-fucosyltransferase (FucT). Based on starting strain SG104, we constructed a series of metabolically engineered E. coli strains by deleting the key genes pfkA, pfkB and pgi, and replacing the original promoter of lacY. The co-expression systems for ManB, ManC, Gmd, WcaG, and FucT were optimized, and nine FucT enzymes were screened to improve the stoichiometric yields of 2′-FL. Furthermore, the gene gapA was regulated to further enhance 2′-FL production, and the highest stoichiometric yield (0.498 mol 2′-FL/mol fructose) was achieved by using recombinant strain RFL38 (SG104ΔpfkAΔpfkBΔpgi119-lacYΔwcaF::119-gmd-wcaG-manC-manB, 119-AGGAGGAGG-gapA, harboring plasmid P30). In the scaled-up reaction, 41.6 g/L (85.2 mM) 2′-FL was produced by a fed-batch bioconversion, corresponding to a stoichiometric yield of 0.482 mol 2′-FL/mol fructose and 0.986 mol 2′-FL/mol lactose. Conclusions: The biosynthesis of 2′-FL using recombinant E. coli from fructose was optimized by metabolic engineering strategies. This is the first time to realize the biological production of 2′-FL production from fructose with high stoichiometric yields. This study also provides an important reference to obtain a suitable distribution of carbon flux between 2′-FL synthesis and glycolysis. [ABSTRACT FROM AUTHOR]

Background: As indigestible carbohydrates, milk oligosaccharides possess various benefits for newborns, mainly through intestinal microbiota, among which 2'‐fucosyllactose (2'‐FL) is the most predominant milk oligosaccharide. However, knowledge about the fermentative characteristics of 2'‐FL in the gut remains limited, especially in the small intestine. The aim of this study is to explore the differential fermentability of 2'‐FL by the small and large intestinal microbiota of piglets using fructo‐oligosaccharide (FOS) and lactose as controls in an in vitro batch fermentation experiment. During fermentation, microbial composition was characterized along with gas production and short‐chain fatty acid production. Results: 2'‐Fucosyllactose showed differential fermentability in jejunal and colonic fermentation. Compared with the colon, 2'‐FL produced less gas in the jejunum than in the FOS and lactose groups (P < 0.05). Meanwhile, 2'‐FL exhibited a different influence on the microbial composition and metabolism in the jejunum and colon compared with FOS and lactose. In the jejunum, compared with the FOS and lactose groups, the 2'‐FL group showed a higher abundance of Bacteroides, Prevotella, and Blautia, but a lower abundance of Streptococcus and Lactobacillus (P < 0.05), with a higher level of propionate and a lower level of lactate during fermentation (P < 0.05). In the colon, compared with the FOS and lactose groups, 2'‐FL increased the abundance of Blautia, Faecalibacterium, and Lachnospiraceae FCS020, but decreased the abundance of Prevotella_9, Succinivibrio, and Megasphaera (P < 0.05) with an increase in acetate production (P < 0.05). Conclusion: Overall, the results suggested that the small intestinal microbiota had the potential to ferment milk oligosaccharides. Meanwhile, in comparison with FOS and lactose, 2′‐FL selectively stimulated the growth of propionate‐producing bacteria in the jejunum and acetate‐producing bacteria in the colon. These results demonstrated the differences in fermentation properties of 2'‐FL by small and large intestinal microbiota and provided new evidence for the application of 2'‐FL in optimizing gut microbiota. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

2'-Fucosyllactose (2'-FL), the primary constituent of human milk oligosaccharides, has been identified as a potential regulator of inflammation in inflammatory bowel disease. Despite this recognition, the specific mechanisms through which 2'-FL alleviates ulcerative colitis (UC) remain ambiguous. This study seeks to investigate the potential anti-inflammatory properties of 2'-FL concerning intestinal inflammation and uncover the associated mechanisms. C57BL/6J mice were orally administered a daily dose of 500 mg/kg 2'-FL for 11 consecutive days, followed by the induction of colitis using 3 % (wt/vol) dextran sulfate sodium (DSS) for the final 6 days. Subsequently, a comprehensive range of techniques, including an Acyl–biotin exchange assay, fluorescein-isothiocyanate-labeled dextran assay, histopathology, ELISA, quantitative real-time PCR, Western blot, immunofluorescence staining, immunohistochemistry staining, Alcian blue-periodic acid schiff staining, TdT-mediated dUTP nick end labeling, transmission electron microscopy, iTRAQ quantitative proteomics, bioinformatics analysis, and the generation of signal transducer and activator of transcription 3 (STAT3) knockout mice, were employed to explore the relevant molecular mechanisms. Administration of 2'-FL significantly ameliorated DSS-induced colitis in mice and enhanced the integrity of the intestinal mucosal barrier. 2'-FL downregulated the phosphorylation of STAT3 and inhibited STAT3-related signaling pathways in colon tissues, which, in turn, reduced inflammatory responses. Interestingly, knockdown of STAT3 attenuated the protective effects of 2'-FL, highlighting that 2'-FL-mediated inflammatory attenuation is dependent on STAT3 expression. Additionally, 2'-FL could influence STAT3 activation by modulating the palmitoylation and depalmitoylation of STAT3. 2'-FL promotes the recovery of the intestinal mucosal barrier and suppresses inflammation in ulcerative colitis by inhibiting the palmitoylation and phosphorylation of STAT3. Treatment with 2'-FL leads to the reduction of LPS-binding protein (LBP) and Toll-like receptor 4 (TLR4). Moreover, 2'-FL treatment inhibits the expression of TNF receptor-associated factor 6 (TRAF6) and suppresses the palmitoylation and phosphorylation of the signal transducer and activator of transcription 3 (STAT3). TRAF6 might function upstream of STAT3. As a result, the production of inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1β (IL-1β), and interferon-gamma (IFN-γ), is inhibited. Furthermore, 2'-FL promotes the restoration of the intestinal mucosal barrier by upregulating the expression of tight junction proteins ZO-1 and Occludin, increasing the secretion of mucin protein Mucin, and restoring the integrity of tight junction structures. [Display omitted] [ABSTRACT FROM AUTHOR]

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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 lacto‐N‐fucopentaose I (LNFP‐I)/2′‐fucosyllactose (2'‐FL) mixture as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharides (HiMO) LNFP‐I and 2'‐FL, but it also contains d‐lactose, lacto‐N‐tetraose, difucosyllactose, 3‐fucosyllactose, LNFP‐I fructose isomer, 2′‐fucosyl‐d‐lactulose, l‐fucose and 2′‐fucosyl‐d‐lactitol, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli K‐12 DH1 MDO MP2173b) of E. coli K‐12 DH1 (DSM 4235). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF in a variety of foods, including infant formula (IF) and follow‐on formula, foods for infants and toddlers, foods for special medical purposes and food supplements (FS). The target population is the general population. The anticipated daily intake of LNFP‐I from use in IF is similar to the estimated natural mean highest daily intake in breastfed infants. Overall, the anticipated daily intake of LNFP‐I from the NF as a food ingredient at the maximum proposed use levels is unlikely to exceed the intake level of breastfed infants on a body weight basis. The intake in breastfed infants on a body weight basis is expected to be safe also for other population groups. The anticipated 2'‐FL intake is generally rather low. The use of the NF in FS is not intended if other foods with added NF components or human milk (for infants and young children) are consumed on the same day. The Panel concludes that the NF, a mixture of LNFP‐I and 2'‐FL, is safe under the proposed conditions of use. [ABSTRACT FROM AUTHOR]

ABSTRACTObservational evidence suggests that human milk oligosaccharides (HMOs) promote the growth of commensal bacteria in early life and adulthood. However, the mechanisms by which HMOs benefit health through modulation of gut microbial homeostasis remain largely unknown. 2′-fucosyllactose (2′-FL) is the most abundant oligosaccharide in human milk and contributes to the essential health benefits associated with human milk consumption. Here, we investigated how 2′-FL prevents colitis in adulthood through its effects on the gut microbial community. We found that the gut microbiota from adult mice that consumed 2′-FL exhibited an increase in abundance of several health-associated genera, including Bifidobacterium and Lactobacillus. The 2′-FL-modulated gut microbial community exerted preventive effects on colitis in adult mice. By using Bifidobacterium infantis as a 2′-FL-consuming bacterial model, exploratory metabolomics revealed novel 2′-FL-enriched secretory metabolites by Bifidobacterium infantis, including pantothenol. Importantly, pantothenate significantly protected the intestinal barrier against oxidative stress and mitigated colitis in adult mice. Furthermore, microbial metabolic pathway analysis identified 26 dysregulated metabolic pathways in fecal microbiota from patients with ulcerative colitis, which were significantly regulated by 2′-FL treatment in adult mice, indicating that 2′-FL has the potential to rectify dysregulated microbial metabolism in colitis. These findings support the contribution of the 2′-FL-shaped gut microbial community and bacterial metabolite production to the protection of intestinal integrity and prevention of intestinal inflammation in adulthood.IMPORTANCEAt present, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether human milk oligosaccharides could serve as effective therapeutics for gastrointestinal-related diseases. Results from the present study uncover 2′-FL-driven alterations in bacterial metabolism and identify novel B. infantis-secreted metabolites following the consumption of 2′-FL, including pantothenol. This work further demonstrates a previously unrecognized role of pantothenate in significantly protecting the intestinal barrier against oxidative stress and mitigating colitis in adult mice. Remarkably, 2′-FL-enhanced bacterial metabolic pathways are found to be dysregulated in the fecal microbiota of ulcerative colitis patients. These novel metabolic pathways underlying the bioactivities of 2′-FL may lay a foundation for applying individual oligosaccharides for prophylactic intervention for diseases associated with impaired intestinal homeostasis.

We present an electrochemical impedimetric-based biosensor for monitoring the variation in human milk oligosaccharide (HMO) composition. 2'-Fucosyllactose (2'FL) is an HMO associated with infant growth, cognitive development, and protection from infectious diarrhea, one of the major causes of infant death worldwide. Due to genetic variation, the milk of some women (non-secretors) contains no or very little 2'FL with potential implications for infant health and development. However, there is currently no technology to analyze the presence and concentration of HMOs in human milk at the point-of-care (POC). The lack of such technology represents a major impediment to advancing human milk research and improving maternal-infant health. Towards this unmet need, we report an impedimetric assay for HMOs with an α-1,2 linkage, the most abundant of which is 2'FL. The sensor uses a lectin for affinity, specifically Ulex europaeus agglutinin I (UEA), with electrochemical readout. In spiked studies, the sensor exhibited a high degree of linearity (R2 = 0.991) over 0.5 to 3.0 μM with a 330-nM detection limit. The sensor performance was clinically validated using banked human milk samples and correctly identified all secretor vs. non-secretor samples. Furthermore, despite the short 35-min assay time and low sample volume (25 μL), the assay was highly correlated with HPLC measurements. This bedside human milk testing assay enables POC, "sample-to-answer" quantitative HMO measurement, and will be a valuable tool to assess milk composition.

Abstract Introduction Human milk oligosaccharides (HMOs) are important components of human milk having diverse functions in the development of infants. Randomized controlled trials (RCTs) have demonstrated that infant formulas with the HMOs 2′-fucosyllactose (2′FL) and lacto-N-neotetraose (LNnT) are safe, well-tolerated, and support normal growth. This study aimed to generate real-world evidence (RWE) on growth and gastrointestinal (GI) tolerance in infants consuming a formula with 1 g/L 2′FL and 0.5 g/L LNnT, including a mixed feeding group not studied before in RCTs. Participants and methods This 8-week open-label prospective multicenter study was conducted in Germany and Austria, and included groups of healthy, exclusively breastfed infants (BF), exclusively formula-fed infants (FF) who received the HMO-formula, and infants mixed fed with both HMO formula and human milk (MF). Co-primary outcomes were anthropometry and gastrointestinal tolerance via validated Infant Gastrointestinal Symptom Questionnaire (IGSQ). Secondary outcomes included formula satisfaction and adverse events (AEs). Results One-hundred six infants completed the study (46 FF, 22 MF, and 38 BF). Mean anthropometric z-scores were comparable between groups and generally within ± 0.5 of WHO medians at week 8. IGSQ composite scores demonstrated good GI tolerance in all groups with no significant group differences at week 4 or 8. IGSQ composite scores in FF improved during the course of the study and parents provided high satisfaction ratings for the HMO-formula. Four potentially product-related AEs were reported in FF (no in MF). Conclusions In this RWE study examining an infant formula with HMOs, growth and GI tolerance outcomes were confirming the good tolerance and safety of this early feeding option previously reported in RCTs.

Abstract The most abundant human milk oligosaccharide 2′‐fucosyllactose (2′‐FL) is a valuable component that has gained significant attention from the food industry. To biosynthesize 2′‐FL, various Escherichia coli K12 derivatives have been genetically modified. To further enhance the application performance of E. coli K12, a novel E. coli K12 derivative BL27 was used as a chassis cell in this study, and modular pathway enhancement was performed to achieve de novo synthesis of 2′‐FL. The futC gene encoding α‐1,2‐fucosyltransferase was introduced, and the wcaJ gene was knocked out to prevent the conversion of GDP‐ l‐fucose to colanic acid. Next, the effects of overexpressing transcriptional regulators rcsA and rcsB and knocking out transcriptional regulators mcbR and waaF were evaluated to optimize the colanic acid pathway. The expression level, solubility, and activity of FutC were improved through genomic integration, TrxA‐tag fusion, and double mutation in F40S/Q237S. Fermentation conditions were optimized to achieve maximum 2′‐FL titers of 3.86 and 23.56 g/L in shake‐flask and fed‐batch cultivation, respectively. Over 85% of the products were successfully excreted into extracellular and almost no byproduct 2′,3‐difucosyllactose was generated. This study has explored a new microbial platform and modification strategies for the synthesis of 2′‐FL and provides opportunities for its commercial production.

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 lacto‐N‐fucopentaose I (LNFP‐I)/2′‐fucosyllactose (2’‐FL) mixture as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharides (HiMO) LNFP‐I and 2’‐FL, but it also contains d‐lactose, lacto‐N‐tetraose, difucosyllactose, 3‐fucosyllactose, LNFP‐I fructose isomer, 2′‐fucosyl‐d‐lactulose, l‐fucose and 2′‐fucosyl‐d‐lactitol, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli K‐12 DH1 MDO MP2173b) of E. coli K‐12 DH1 (DSM 4235). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF in a variety of foods, including infant formula (IF) and follow‐on formula, foods for infants and toddlers, foods for special medical purposes and food supplements (FS). The target population is the general population. The anticipated daily intake of LNFP‐I from use in IF is similar to the estimated natural mean highest daily intake in breastfed infants. Overall, the anticipated daily intake of LNFP‐I from the NF as a food ingredient at the maximum proposed use levels is unlikely to exceed the intake level of breastfed infants on a body weight basis. The intake in breastfed infants on a body weight basis is expected to be safe also for other population groups. The anticipated 2’‐FL intake is generally rather low. The use of the NF in FS is not intended if other foods with added NF components or human milk (for infants and young children) are consumed on the same day. The Panel concludes that the NF, a mixture of LNFP‐I and 2’‐FL, is safe under the proposed conditions of use.

2'-Fucosyllactose (2'-FL), the functional oligosaccharide naturally present in milk, has been shown to exert health benefits. This study was aimed to investigate the effect of 2'-fucosyllactose (2'-FL) on the browning of white adipose tissue in 3T3-L1 adipocytes and C3H10T1/2 cells. The results revealed that 2'-FL decreased lipid accumulations with reduced intracellular triglyceride contents in vitro. 2'-FL intervention increased the mitochondria density and the proportion of UCP1-positive cells. The mRNA expressions of the mitochondrial biogenesis-related and browning markers (Cox7a, Cyto C, Tfam, Ucp1, Pgc1α, Prdm16, Cidea, Elovl3, Pparα, CD137, and Tmem26) were increased after 2'-FL intervention to some extent. Similarly, the protein expression of the browning markers, including UCP1, PGC1α, and PRDM16, was up-regulated in the 2'-FL group. Additionally, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, compound C (1 μM), significantly decreased the induction of thermogenic proteins expressions mediated by 2'-FL, indicating that the 2'-FL-enhanced beige cell formation was partially dependent on the AMPK pathway. In conclusion, 2'-FL effectively promoted the browning of white adipose in vitro. [ABSTRACT FROM AUTHOR]

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 2′‐fucosyllactose (2'‐FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharide (HiMO) 2'‐FL, but it also contains d‐lactose, l‐fucose, fucosylgalactose, difucosyllactose, d‐glucose and d‐galactose, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli SGR5) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant applies for the same use and use levels as already authorised for 2'‐FL and included in the Union list of NFs, with the general population as target population. The Panel noted that the available intake estimate is not recent (2015) and based on a different database (2008–2010 UK data) than that used by EFSA. For this reason, the Panel decided to perform a new intake estimate according to the current EFSA approach. The Panel notes that the highest P95 daily intake of the NF from the use as food ingredient is higher than the estimated natural highest mean daily intake in breastfed infants and marginally higher in young children. The applicant also proposes to extend the use of 2'‐FL in food supplements (FS) for infants at the use level of 1.2 g/day. The resulting estimated intake in infants from the proposed use in FS is within the natural intake of 2'‐FL in breastfed infants. FS are not intended to be used if other foods with added 2'‐FL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use. [ABSTRACT FROM AUTHOR]

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 safety of the extension of use of 2′‐fucosyllactose (2′‐FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is already authorised as ingredient in several food categories, including infant formula (IF) and follow‐on formula (FOF). The applicant proposed to increase the maximum use levels of the NF in IF and FOF. EFSA estimated the anticipated daily intake of the NF from the proposed extension of use, including the already authorised conditions of use in other food categories. Additionally, a new intake estimate limited to the already authorised conditions of use of the NF was carried out following EFSA's current approach. The estimated daily intake of the NF from high consumption of IF alone at the proposed maximum use level in infants < 16 weeks of age is similar to the estimated natural highest mean daily intake of 2′‐FL from human milk in breastfed infants. The estimated highest P95 daily intakes of the NF from the proposed extension of use in IF and FOF (including the authorised uses in other food categories) and from the already authorised conditions of use, are comparable and both higher than the estimated natural highest mean daily intake of 2′‐FL from human milk in infants and to a lesser extent in young children. The Panel considers that the proposed extension of use of the NF in IF and FOF only marginally affects the highest P95 daily intake estimate from the authorised conditions of use, and therefore does not affect the safety of the NF. The Panel concludes that the NF, 2′‐FL, is safe under the proposed conditions of use. [ABSTRACT FROM AUTHOR]

2'-fucosyllactose（2'-FL）is the most abundant human milk oligosaccharide. Microbial fermentation is an important method to produce 2'-FL. Lactococcus lactis（L. lactis）, a food safety grade strain, can be directly used in dairy products, has not been reported for the production of human milk oligosaccharides. First, the 2'-FL salvage synthesis pathway was constructed by using a recombinant plasmid pNZ8148-2f containing gene fkp, futC, lacF in two usual L. lactis chassis NZ3900 and NZ9000. In the fermentation medium with 10 g/L fucose and 5 g/L lactose, the yields of 2'-FL in flasks were 0.16 g/L and 0.4 g/L, respectively. Combined with the growth assay, NZ9000 was considered as a better chassis. Second, 2'-FL de novo synthesis pathway was constructed in NZ9000, i.e., using homologous recombination technology and the unique knockout integration vector pNZ5319 in L. lactis，integrating the gene manA, manB, gmd and wcaG to the chromosome while knocking out unnecessary gene upp, and introducing the gene manC, futC and lacF to the cell by vector pNZ8148-1. Meanwhile, different promoter P32 and Pnis were used to optimize the expressions of the pathway enzymes. The best strain NZ9000 6 was obtained with a 2'-FL production of 0.28 g/L in flask fermentation medium with 20 g/L glucose and 5 g/L lactose. Finally, the concentrations of hemin, tryptone and yeast extract were optimized in the fermentation medium. With the addition of 2.5 μg/mL methemoferin chloride, 15 g/L tryptone, and 6 g/L yeast extract in the fermentation medium, the final 2'-FL production reached 1.58 g/L which was relatively high among the reported results. Thus, the feasibility of 2'-FL synthesis by L. lactis was proved, which provides a new idea for the production of 2'-FL. [ABSTRACT FROM AUTHOR]

2'-fucosyllactose（2'-FL）is the most abundant human milk oligosaccharide. Microbial fermentation is an important method to produce 2'-FL. Lactococcus lactis（L. lactis）, a food safety grade strain, can be directly used in dairy products, has not been reported for the production of human milk oligosaccharides. First, the 2'-FL salvage synthesis pathway was constructed by using a recombinant plasmid pNZ8148-2f containing gene fkp, futC, lacF in two usual L. lactis chassis NZ3900 and NZ9000. In the fermentation medium with 10 g/L fucose and 5 g/L lactose, the yields of 2'-FL in flasks were 0.16 g/L and 0.4 g/L, respectively. Combined with the growth assay, NZ9000 was considered as a better chassis. Second, 2'-FL de novo synthesis pathway was constructed in NZ9000, i.e., using homologous recombination technology and the unique knockout integration vector pNZ5319 in L. lactis，integrating the gene manA, manB, gmd and wcaG to the chromosome while knocking out unnecessary gene upp, and introducing the gene manC, futC and lacF to the cell by vector pNZ8148-1. Meanwhile, different promoter P32 and Pnis were used to optimize the expressions of the pathway enzymes. The best strain NZ9000 6 was obtained with a 2'-FL production of 0.28 g/L in flask fermentation medium with 20 g/L glucose and 5 g/L lactose. Finally, the concentrations of hemin, tryptone and yeast extract were optimized in the fermentation medium. With the addition of 2.5 μg/mL methemoferin chloride, 15 g/L tryptone, and 6 g/L yeast extract in the fermentation medium, the final 2'-FL production reached 1.58 g/L which was relatively high among the reported results. Thus, the feasibility of 2'-FL synthesis by L. lactis was proved, which provides a new idea for the production of 2'-FL. [ABSTRACT FROM AUTHOR]

2′-Fucosyllactose (2′-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2′-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and β-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2′-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2′-FL (0.98 g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2′-FL (18.22 g/L) was obtained, corresponding to a yield of 0.25 g/g glycerol and a substrate conversion of 0.88 g/g lactose. [ABSTRACT FROM AUTHOR]

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 safety of the extension of use of 2′‐fucosyllactose (2′‐FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is already authorised as ingredient in several food categories, including infant formula (IF) and follow‐on formula (FOF). The applicant proposed to increase the maximum use levels of the NF in IF and FOF. EFSA estimated the anticipated daily intake of the NF from the proposed extension of use, including the already authorised conditions of use in other food categories. Additionally, a new intake estimate limited to the already authorised conditions of use of the NF was carried out following EFSA's current approach. The estimated daily intake of the NF from high consumption of IF alone at the proposed maximum use level in infants

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 2′‐fucosyllactose (2’‐FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharide (HiMO) 2’‐FL, but it also contains d‐lactose, l‐fucose, fucosylgalactose, difucosyllactose, d‐glucose and d‐galactose, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli SGR5) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant applies for the same use and use levels as already authorised for 2’‐FL and included in the Union list of NFs, with the general population as target population. The Panel noted that the available intake estimate is not recent (2015) and based on a different database (2008–2010 UK data) than that used by EFSA. For this reason, the Panel decided to perform a new intake estimate according to the current EFSA approach. The Panel notes that the highest P95 daily intake of the NF from the use as food ingredient is higher than the estimated natural highest mean daily intake in breastfed infants and marginally higher in young children. The applicant also proposes to extend the use of 2’‐FL in food supplements (FS) for infants at the use level of 1.2 g/day. The resulting estimated intake in infants from the proposed use in FS is within the natural intake of 2’‐FL in breastfed infants. FS are not intended to be used if other foods with added 2’‐FL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Introduction: Human milk oligosaccharides (HMOs) are important components of human milk having diverse functions in the development of infants. Randomized controlled trials (RCTs) have demonstrated that infant formulas with the HMOs 2′-fucosyllactose (2′FL) and lacto-N-neotetraose (LNnT) are safe, well-tolerated, and support normal growth. This study aimed to generate real-world evidence (RWE) on growth and gastrointestinal (GI) tolerance in infants consuming a formula with 1 g/L 2′FL and 0.5 g/L LNnT, including a mixed feeding group not studied before in RCTs. Participants and methods: This 8-week open-label prospective multicenter study was conducted in Germany and Austria, and included groups of healthy, exclusively breastfed infants (BF), exclusively formula-fed infants (FF) who received the HMO-formula, and infants mixed fed with both HMO formula and human milk (MF). Co-primary outcomes were anthropometry and gastrointestinal tolerance via validated Infant Gastrointestinal Symptom Questionnaire (IGSQ). Secondary outcomes included formula satisfaction and adverse events (AEs). Results: One-hundred six infants completed the study (46 FF, 22 MF, and 38 BF). Mean anthropometric z-scores were comparable between groups and generally within ± 0.5 of WHO medians at week 8. IGSQ composite scores demonstrated good GI tolerance in all groups with no significant group differences at week 4 or 8. IGSQ composite scores in FF improved during the course of the study and parents provided high satisfaction ratings for the HMO-formula. Four potentially product-related AEs were reported in FF (no in MF). Conclusions: In this RWE study examining an infant formula with HMOs, growth and GI tolerance outcomes were confirming the good tolerance and safety of this early feeding option previously reported in RCTs. [ABSTRACT FROM AUTHOR]

2′-fucosyllactose (2′FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2′FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2′FL. EntFuc could catalyze the synthesis of 2′FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2′FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2′FL. [ABSTRACT FROM AUTHOR]

2′-Fucosyllactose (2′-FL) is known for its ability to provide various health benefits to infants, such as gut maturation, pathogen resistance, improved immunity, and nervous system development. However, the production of 2′-FL using α-L-fucosidases is hindered by the lack of low-cost natural fucosyl donors and high-efficiency α-L-fucosidases. In this work, a recombinant xyloglucanase from Rhizomucor miehei (RmXEG12A) was applied to produce xyloglucan-oligosaccharide (XyG-oligos) from apple pomace. Then, an α-L-fucosidase gene (PbFucB) was screened from the genomic DNA of Pedobacter sp. CAU209 and expressed in Escherichia coli. The capability of purified PbFucB to catalyze XyG-oligos and lactose to synthesize 2′-FL was further evaluated. The deduced amino acid sequence of PbFucB shared the highest identity (38.4%) with that of other reported α-L-fucosidases. PbFucB showed the highest activity at pH 5.5 and 35 °C. It catalyzed the hydrolysis of 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc, 20.3 U mg−1), 2′-FL (8.06 U mg−1), and XyG-oligos (0.43 U mg−1). Furthermore, PbFucB demonstrated a high enzymatic conversion rate in 2′-FL synthesis with pNP-Fuc or apple pomace–derived XyG-oligos as donors and lactose as acceptor. Under the optimized conditions, PbFucB converted 50% of pNP-Fuc or 31% of the L-fucosyl residue in XyG-oligos into 2′-FL. This work elucidated an α-L-fucosidase that mediates the fucosylation of lactose and provided an efficient enzymatic strategy to synthesize 2′-FL either from artificial pNP-Fuc or natural apple pomace–derived XyG-oligos. Key points: • Xyloglucan-oligosaccharide (XyG-oligos) was produced from apple pomace by a xyloglucanase from Rhizomucor miehei. • An α-L-fucosidase (PbFucB) from Pedobacter sp. CAU209 shared the highest identity (38.4%) with reported α-L-fucosidases. •PbFucB synthesized 2′-FL using apple pomace–derived XyG-oligos and lactose with a conversion ratio of 31%. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) shape the developing infant gut microbiota. In this study, a semi-continuous colon simulator was used to evaluate the effect of 2 HMOs—2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL)—on the composition of infant faecal microbiota and microbial metabolites. The simulations were performed with and without a probiotic Bifidobacterium longum subspecies infantis Bi-26 (Bi-26) and compared with a control that lacked an additional carbon source. The treatments with HMOs decreased α-diversity and increased Bifidobacterium species versus the control, but the Bifidobacterium species differed between simulations. The levels of acetic acid and the sum of all short-chain fatty acids (SCFAs) trended toward an increase with 2′-FL, as did lactic acid with 2′-FL and 3-FL, compared with control. A clear correlation was seen between the consumption of HMOs and the increase in SCFAs (−0.72) and SCFAs + lactic acid (−0.77), whereas the correlation between HMO consumption and higher total bifidobacterial numbers was moderate (−0.46). Bi-26 decreased propionic acid levels with 2′-FL. In conclusion, whereas infant faecal microbiota varied between infant donors, the addition of 2′-FL and 3-FL, alone or in combination, increased the relative abundance and numbers Bifidobacterium species in the semi-continuous colon simulation model, correlating with the production of microbial metabolites. These findings may suggest that HMOs and probiotics benefit the developing infant gut microbiota. [ABSTRACT FROM AUTHOR]

Cerebral ischemic stroke is one of the leading causes of death and disability worldwide. 2′-fucosyllactose (2′-FL), a human milk oligosaccharide, exerts anti-inflammatory effects and plays a protective role in arterial thrombosis; however, its role in ischemic stroke remains unclear. This study aimed to investigate the neuroprotective effects of 2′-FL and its potential mechanisms in a mouse model of ischemic stroke. Neurological score and behavior tests revealed that 2′-FL promoted the recovery of neurological deficits and motor function in middle cerebral artery occlusion (MCAO) mice, and that 2′FL led to a reduction in the size of cerebral infarct. Biochemical studies showed that administration of 2′-FL led to a reduction of reactive oxygen species (ROS)-related products in the brain of MCAO mice. 2′-FL upregulated IL-10 and downregulated TNF-α level. In addition, 2′-FL enhanced M2-type microglial polarization and upregulated CD206 expression at 7 days after MCAO. At 3 days after MCAO, 2′-FL increased IL-4 levels and activated STAT6. Our data show that 2′-FL reduced the neurological symptoms of ischemic stroke and ROS accumulation in the brain through IL-4/STAT6-dependent M2-type microglial polarization in MCAO mice. These results demonstrate that 2′-FL is a potentially effective therapeutic agent for ischemic stroke. [ABSTRACT FROM AUTHOR]

Obesity is characterized by fat accumulation, chronic inflammation and impaired satiety signaling, which may be due in part to gut microbial dysbiosis. Manipulations of the gut microbiota and its metabolites are attractive targets for obesity treatment. The predominant oligosaccharide found in human milk, acts as a prebiotic with beneficial effects on the host. However, little is known about the beneficial effects of 2'-FL in obesity. The aim of this study was to determine the beneficial effects of 2'-FL supplementation on the microbiota-gut-brain axis and the diet-induced obese phenotype in high fat (HF)-fed mice. Male C57/BL6 mice (n = 6/group; six weeks old) were counter-balanced into six weight-matched groups and fed either a low-fat (LF; 10% kcal as fat), HF (45% kcal as fat) or HF diet with 2'-FL (HF_2'-FL) at 1, 2, 5 and 10% (w/v) in drinking water for six weeks. General phenotypes (body weight, energy intake, fat and lean mass), cecal microbiome and metabolites, gut-brain signaling, intestinal permeability and inflammatory and lipid profiles were assessed. Only 10% 2'-FL, but not 1, 2 or 5%, decreased HF diet-induced increases in energy intake, fat mass and body weight gain. A supplementation of 10% 2'-FL changed the composition of cecal microbiota and metabolites compared to LF- and HF-fed mice with an increase in Parabacteroides abundance and lactate and pyruvate, respectively, whose metabolic effects corresponded to our study findings. In particular, 10% 2'-FL significantly reversed the HF diet-induced impairment of cholecystokinin-induced inhibition of food intake. Gene expressions of interleukin (IL)-1β, IL-6, and macrophage chemoattractant protein-1 in the cecum were significantly downregulated by 10% 2'-FL compared to the HF group. Furthermore, 10% 2'-FL suppressed HF diet-induced upregulation of hepatic peroxisome proliferator-activated receptor gamma, a transcription factor for adipogenesis, at the gene level. In conclusion, 10% 2'-FL led to compositional changes in gut microbiota and metabolites associated with improvements in metabolic profiles and gut-brain signaling in HF-fed mice. These findings support the use of 2'-FL for modulating the hyperphagic response to HF diets and improving the microbiota-gut-brain axis.

Ulcerative colitis is an inflammatory bowel disease (IBD) with relapsing and remitting patterns, and it is caused by varied factors, such as the intestinal inflammation extent and duration. We examined the preventative effects of human milk oligosaccharides (HMOs) on epithelial barrier integrity and intestinal inflammation in an interleukin (IL)-6-induced cell model and dextran sodium sulfate (DSS)-induced acute mouse colitis model. HMOs including 2′-fucosyllactose (FL) and 3-FL and positive controls including fructooligosaccharide (FOS) and 5-acetylsalicylic acid (5-ASA) were orally administrated once per day to C57BL/6J mice with colitis induced by 5% DSS in the administered drinking water. 2′-FL and 3-FL did not affect the cell viability in Caco-2 cells. Meanwhile, these agents reversed IL-6-reduced intestinal barrier function in Caco-2 cells. Furthermore, 2′-FL and 3-FL reversed the body weight loss and the remarkably short colon lengths in DSS-induced acute colitis mice. Moreover, 2′-FL and 3-FL obviously protected the decreasing expression of zonula occluden-1 and occludin in colon tissue relative to the findings in the DSS-treated control group. 2′-FL and 3-FL significantly reduced IL-6 and tumor necrosis factor-α levels in serum relative to the control findings. The summary of these results shows that HMOs prevent colitis mainly by enhancing intestinal barrier function and advancing anti-inflammatory responses. Therefore, HMOs might suppress inflammatory responses and represent candidate treatments for IBD that protect intestinal integrity. [ABSTRACT FROM AUTHOR]

Purpose: Pichia pastoris is well known for its ability to produce short and low-immunogenic humanized glycosyl chains onto recombinant glycoproteins, it was thus speculated to be applicable to synthesize oligosaccharides. In this study, generally recognized as safe (GRAS) microorganism Pichia pastoris GS115 was tested for its potential to be used as a new synthetic chassis to produce the most abundant human milk oligosaccharide 2′-fucosyllactose (2′-FL). Methods: To enable the de novo synthesis of 2′-FL, lactose transporter lac12, two enzymes of gmd, gmer, and fucosyltransferases futC were integrated into the genome of P. pastoris, under the control of constitutive PGAP promoter. Results: The resulting recombinant yeasts yielded up to 0.276 g/L through culture optimization in a 5 L bioreactor. Conclusion: To our knowledge, this is the first report of 2′-FL production in engineered Pichia pastoris. This work is a good starting point to produce 2′-FL using Pichia pastoris as a viable chassis. [ABSTRACT FROM AUTHOR]

The objective of the current study was to evaluate the potential of 2′-FL and GOS, individually and combined, in beneficially modulating the microbial composition of infant and toddler (12–18 months) feces using the micro-Matrix bioreactor. In addition, the impacts of GOS and 2′-FL, individually and combined, on the outgrowth of fecal bifidobacteria at (sub)species level was investigated using the baby M-SHIME® model. For young toddlers, significant increases in the genera Bifidobacterium, Veillonella, and Streptococcus, and decreases in Enterobacteriaceae, Clostridium XIVa, and Roseburia were observed in all supplemented fermentations. In addition, GOS, and combinations of GOS and 2′-FL, increased Collinsella and decreased Salmonella, whereas 2′-FL, and combined GOS and 2′-FL, decreased Dorea. Alpha diversity increased significantly in infants with GOS and/or 2′-FL, as well as the relative abundances of the genera Veillonella and Akkermansia with 2′-FL, and Lactobacillus with GOS. Combinations of GOS and 2′-FL significantly stimulated Veillonella, Lactobacillus, Bifidobacterium, and Streptococcus. In all supplemented fermentations, Proteobacteria decreased, with the most profound decreases accomplished by the combination of GOS and 2′-FL. When zooming in on the different (sub)species of Bifidobacterium, GOS and 2'-FL were shown to be complementary in stimulating breast-fed infant-associated subspecies of Bifidobacterium longum in a dose-dependent manner: GOS stimulated Bifidobacterium longum subsp. longum, whereas 2′-FL supported outgrowth of Bifidobacterium longum subsp. infantis. [ABSTRACT FROM AUTHOR]

Abstract Background The most abundant human milk oligosaccharide in breast milk, 2′-fucosyllactose (2′-FL), has been approved as an additive to infant formula due to its multifarious nutraceutical and pharmaceutical functions in promoting neonate health. However, the low efficiency of de novo synthesis limits the cost-efficient bioproduction of 2′-FL. Results This study achieved 2′-FL de novo synthesis in a generally recognized as safe (GRAS) strain Bacillus subtilis. First, a de novo biosynthetic pathway for 2′-FL was introduced by expressing the manB, manC, gmd, wcaG, and futC genes from Escherichia coli and Helicobacter pylori in B. subtilis, resulting in 2′-FL production of 1.12 g/L. Subsequently, a 2′-FL titer of 2.57 g/L was obtained by reducing the competitive lactose consumption, increasing the regeneration of the cofactor guanosine-5′-triphosphate (GTP), and enhancing the supply of the precursor mannose-6-phosphate (M6P). By replacing the native promoter of endogenous manA gene (encoding M6P isomerase) with a constitutive promoter P7, the 2′-FL titer in shake flask reached 18.27 g/L. The finally engineered strain BS21 could produce 88.3 g/L 2′-FL with a yield of 0.61 g/g lactose in a 3-L bioreactor, without the addition of antibiotics and chemical inducers. Conclusions The efficient de novo synthesis of 2′-FL can be achieved by the engineered B. subtilis, paving the way for the large-scale bioproduction of 2′-FL titer in the future.

Using 2′-fucosyllactose (2′-FL) as the sole carbon source can be an efficient way to screen bifidobacteria with superior probiotic capabilities since 2′-FL is a key element in promoting the growth of intestinal bifidobacteria in newborns. This approach was used in this work to screen eight bifidobacteria strains, including one strain of Bifidobacterium longum subsp. infantis BI_Y46 and seven strains of Bifidobacterium bifidum (BB_Y10, BB_Y30, BB_Y39, BB_S40, BB_H4, BB_H5 and BB_H22). Studies on their probiotic properties showed that BI_Y46 had a unique morphology with pilus-like structure, a high resistance to bile salt stimulation and a potent inhibitory action on Escherichia coli ATCC 25922. Similarly, BB_H5 and BB_H22 produced more extracellular polysaccharides and had a higher protein content than other strains. In contrast, BB_Y22 displayed considerable auto-aggregation activity and a high resistance to bile salt stimulation. Interestingly, BB_Y39 with weak self-aggregation ability and acid resistance had very excellent bile salt tolerance, extracellular polysaccharides (EPS) production and bacteriostatic ability. In conclusion, 2′-FL was used as sole carbon source to identify eight bifidobacteria with excellent probiotic properties. [ABSTRACT FROM AUTHOR]

As one of the most abundant components in human milk oligosaccharides, 2′‐fucosyllactose (2′‐FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine‐tuning the expression of pathway enzymes and achieved a striking increase of 2′‐FL production, directly facilitating the metabolic flux toward the key intermediate GDP‐l‐fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2′‐FL. Specifically, we first fine‐tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2′‐FL synthesis. Then, two key enzymes (GDP‐mannose 4,6‐dehydratase and GDP‐ l‐fucose synthase) were self‐assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD–RIDD (RI anchoring disruptor–RI dimer D/D domains), resulting in noticeable improvement of 2′‐FL production. Next, to further strengthen the metabolic flux toward 2′‐FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher–SpyTag or PDZ–PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2′‐FL biosynthesis and showed a 2.1‐fold increase of 2′‐FL production compared with a strain expressing free‐floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2′‐FL in shake flask fermentation and was capable of producing 25.1 g/L 2′‐FL by fed‐batch cultivation. This work provides novel approaches for further improvement and large‐scale production of 2′‐FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain. [ABSTRACT FROM AUTHOR]