background The intestinal barrier serves as a vital defense system for maintaining gut health and immune homeostasis, with its dysfunction closely associated with various chronic diseases. Human milk oligosaccharides (HMOs), particularly 2’-fucosyllactose (2'-FL), demonstrate extensive biological activities in regulating gut microbiota, anti-inflammatory immunity, and preserving intestinal barrier integrity. Previous studies have shown that HMOs not only act as a "carbon source" for probiotic growth but also enhance epithelial barrier function through specific mechanisms via their metabolites. This study investigates how 2'-FL (produced by Hongmo Biotechnology Co., Ltd.) alleviates LPS-induced intestinal barrier damage in vitro by modulating surface proteins of Bifidobacterium DNG6, providing insightsits underlying mechanisms. Research design and methods This study employed a Caco-2 intestinal epithelial cell monolayer in vitro barrier model to simulate LPS-induced intestinal inflammation. Bifidobacterium DNG6 was cultured under three distinct carbon sources: lactose (Lac), galactooligosaccharide (GOS), and 2 -fucosylated lactose (2'-FL). Surface proteins (labeled as Sp-L, Sp-G, and Sp-F) were extracted, and the LPS-treated Caco-2 cells were subjected to intervention. The effects were evaluated through cell viability, cytotoxicity, inflammatory factor expression, and tight junction protein levels. Key findings 1. 2'-FL enhances cell viability and reduces cytotoxicity Studies demonstrate that LPS treatment significantly reduces Caco-2 cell viability. Notably, Sp-F (2'-FL treatment group) achieves a cell survival rate exceeding 88% at 800–1000 μg/mL, outperforming both Sp-L and Sp-G. Furthermore, Sp-F (2'-FL treatment group) markedly decreases lactate dehydrogenase (LDH) release levels, a biomarker of cell membrane integrity, indicating its potent efficacy in mitigating membrane damage. Figure 1: Effects of surface proteins on LPS-induced viability and LDH activity in Caco-2 cells 2. 2'-FL suppresses inflammatory responses and enhances immune regulation LPS significantly induced the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β, while Sp-F (2'-FL treated group) significantly suppressed their secretion levels, reducing them by 53.46%,35.91%, and 37.05% respectively. Meanwhile, the anti-inflammatory factor IL-10 levels increased to 140.13% of the LPS group, indicating its excellent immune-regulating function. Figure 2: Effects of surface proteins on the expression of TNF-α, IL-6, IL-1β, and IL-10 in LPS-induced monolayer barrier of Caco-2 cells 3. 2'-FL repairs barrier structures and enhances cell junctions In vitro experiments demonstrated that Sp-F (2'-FL treated group) significantly enhanced the mRNA expression (by 146.21%,268.53%, and 137.22% respectively) and protein levels of ZO-1, Claudin-1, and Occludin, which are tightly junction proteins in intestinal epithelial cells. These proteins form critical structures that maintain intestinal barrier integrity and prevent harmful substance permeation. The results indicated that Sp-F effectively repaired the intestinal epithelial barrier and improved intercellular junction integrity. Figure 3: Surface protein effects on protein mRNA and protein expression Discussion and outlook This study reveals the mechanism of 2'-FL (produced by Hongmo Biotechnology Co., Ltd.) in maintaining intestinal barrier homeostasis from the perspective of "bacterial surface proteins": By promoting the expression of surface proteins in Bifidobacterium DNG6, it significantly alleviates LPS-induced inflammatory damage and enhances tight junction protein expression. This discovery not only expands the understanding of HMOs molecular mechanisms but also provides theoretical support for the functional application of HMO in infant formula. Future research could further integrate animal models and clinical studies to explore its potential applications in populations such as preterm infants and inflammatory bowel disease patients. References [1] Zhao, J., et al. "Surface Proteins of Bifidobacterium Bifidum DNG6 Growing in 2'-FL Alleviating LPS-Induced Intestinal Barrier Injury in vitro." Journal of Dairy Science (2024): S0022-0302.

background Necrotizing enterocolitis (NEC) is the most common fatal intestinal disease in preterm infants, characterized by intestinal ischemia, necrosis, and systemic inflammation, with a mortality rate as high as 40%. Although breastfeeding has been proven to significantly reduce NEC risk, its mechanisms remain incompletely understood. Research has revealed that human milk oligosaccharides (HMO), particularly 2 -fucosyl lactose (2′-FL), play crucial roles in immune regulation and maintaining gut health. Evidence suggests that excessive activation of intestinal inflammatory responses inhibits the expression of endothelial nitric oxide synthase (eNOS), a key enzyme for vasodilation, leading to intestinal microcirculation disorders and exacerbating NEC pathology. Based on this, our study proposes that 2′-FL may alleviate NEC progression by upregulating eNOS expression and improving intestinal blood perfusion. Research design and methods The study employed a neonatal mouse necrotizing enterocolitis (NEC) model, simulating preterm infant intestinal injury through formula feeding combined with hypoxia stimulation. Four experimental groups were established: breastfed group, formula-fed NEC group, formula-fed with 2′-FL intervention group, and eNOS knockout (eNOS⁻/⁻) group. The protective effects of 2′-FL were evaluated through histopathological scoring, intestinal perfusion imaging, and qRT-PCR analysis of inflammatory factors and eNOS expression. Additionally, 16S rRNA sequencing was used to analyze changes in gut microbiota composition. HUVEC experiments further validated 2′-FLs direct regulatory effect on eNOS, with L-NIO/DPI, a pharmacological inhibitor of eNOS activity, serving as the negative control. Key findings 1.2′-FL significantly alleviates necrotizing enterocolitis (NEC) pathological damage In a neonatal mouse necrotizing enterocolitis (NEC) model, the addition of 2′-FL to formula milk significantly reduced NEC severity scores, with intestinal mucosal structural integrity approaching that of the breastfed group. Concurrently, the expression of pro-inflammatory factors IL-6 and IL-1β decreased by over 50% compared to baseline levels, demonstrating that 2′-FL effectively suppresses intestinal inflammatory responses and maintains tissue homeostasis. Figure 1: The addition of 2′-FL to infant formula alleviates the severity of necrotizing enterocolitis (NEC) in newborn mice. 2.2′-FL improves intestinal bleeding by upregulating eNOS expression Studies demonstrate that 2′-FL significantly restores intestinal microcirculation perfusion by upregulating endothelial nitric oxide synthase (eNOS) expression, resulting in a substantial increase in intestinal villus perfusion index comparable to the breastfed group. Evidence indicates that the protective effect of 2′-FL is completely abolished in eNOS knockout (eNOS⁻/⁻) mice or when eNOS inhibitors are used, confirming that its mechanism relies on eNOS-mediated vasodilation. Figure 2: Formula milk supplemented with 2′-FL enhances mesenteric perfusion in necrotizing enterocolitis (NEC) by upregulating eNOS expression. 3.2′-FL directly upregulates eNOS expression in signaling pathways In vitro experiments (HUVEC cells) further confirmed that 2′-FL reverses LPS-induced eNOS expression suppression and directly enhances eNOS mRNA synthesis. This finding provides molecular-level mechanistic support for in vivo studies, demonstrating that 2′-FL can fundamentally improve intestinal blood supply to combat necrotizing enterocolitis (NEC). Figure 3: 2′-FL enhances eNOS expression in HUVEC 2′-FLs impact on gut microbiota is a secondary effect Although 2′-FL treatment increased Ruminococcaceae abundance in the gut microbiota, β diversity analysis showed no direct correlation between microbial changes and NEC severity. This suggests that 2′-FLs core protective effect primarily stems from its regulation of mesenteric blood flow rather than microbial modulation. Figure 4: β diversity analysis of the microbiota from 2′-FL-treated mice Discussion and outlook This study demonstrates that 2′-FL improves intestinal blood flow by upregulating endothelial nitric oxide synthase (eNOS), a key vasodilator enzyme, thereby effectively alleviating necrotizing enterocolitis (NEC). This discovery not only expands the application scope of human milk oligosaccharides (HMOs) but also provides potential directions for optimizing infant formula. Future research could further explore the clinical value of 2′-FL. As the mechanisms of HMOs are better understood, more effective NEC prevention strategies may emerge, offering new hope for the health of premature infants. References [1] Good, Misty, et al. "The Human Milk Oligosaccharide 2-Fucosyllactose Attenuates the Severity of Experimental Necrotising Enterocolitis by Enhancing Mesenteric Perfusion in the Neonatal Intestine." British Journal of Nutrition, vol. 116, no. 7, 2016, pp. 1175-1187, doi:10.1017/S0007114516002944.

  background Ulcerative colitis (UC) is a gastrointestinal disorder characterized by chronic inflammation of the colon mucosa, often presenting with bloody diarrhea. Research indicates that UC patients exhibit significant dysbiosis in their gut microbiota: a reduction in beneficial bacteria like Bifidobacteria and an increase in harmful bacteria such as sulfate-reducing bacteria. This imbalance not only exacerbates inflammatory responses but also further weakens the bodys immune regulation. Human milk oligosaccharides (HMO), particularly 2′-fucosyllactose (2′-FL), act as natural prebiotics that selectively promote the growth of beneficial bacteria. By generating short-chain fatty acids (SCFAs), 2′-FL helps reduce inflammation and regulate immune responses, thereby maintaining gut health. This study aims to investigate the role of 2′-FL in improving the composition and metabolic activity of the gut microbiota in UC patients. Research design and methods This study employed an in vitro fermentation model to simulate the intestinal environment of ulcerative colitis (UC) patients and evaluate the regulatory effects of 2′-FL on gut microbiota. The experiment collected fecal samples from three healthy controls and three UC patients, with four fermentation containers set up: one negative control group and three treatment groups supplemented with 2′-FL, fructooligosaccharides (FOS), and galactooligosaccharides (GOS). All samples underwent fermentation within 48 hours to simulate the anaerobic environment of the distal colon. Bacterial population changes were analyzed using fluorescence in situ hybridization (FISH) combined with flow cytometry, while short-chain fatty acid (SCFAs) production was quantified via gas chromatography-mass spectrometry (GC-MS). The study comprehensively assessed the effects of 2′-FL from both microbial community and metabolite perspectives. Key findings 1. 2′-FL optimizes the gut microbiota structure in UC patients Research indicates that UC patients exhibit significantly lower total bacterial counts and Lactobacillus abundance at baseline compared to healthy controls, indicating microbial dysbiosis. The 2′-FL intervention significantly increased beneficial bacteria such as Bifidobacterium and Lactobacillus while reducing harmful bacteria like Desulfotrichum. Compared to other substrates (FOS and GOS), 2′-FL demonstrates superior efficacy in suppressing harmful bacterial proliferation and restoring microbial balance. Figure 1: The ratio of baseline microbial community changes in the control group and UC patients, measured using each oligonucleotide probe for negative controls and each substrate after 24 hours. 2. 2′-FL promotes the production of beneficial intestinal metabolites in UC patients In vitro fermentation demonstrated that 2′-FL significantly boosted short-chain fatty acids (SCFAs), particularly acetic acid, with concentrations rising dramatically from 0.952 to 49.9 mmol/L within 48 hours. Compared to other prebiotics, 2′-FL showed superior efficacy in elevating SCFAs such as acetic, propionic, and butyric acids. These fatty acids not only enhance intestinal barrier function but also play crucial roles in suppressing inflammation and regulating metabolism. Figure 2: Organic acid concentrations of each substrate in negative control and UC patient samples at baseline and post-inoculation at 8, 24, and 48 hours; acetyl esters (i), butyrate esters (ii), propionate esters (iii), and total SCFA (iv) Discussion and outlook This study demonstrates that 2 -fucosyl lactose (2′-FL) effectively alleviates ulcerative colitis (UC). By promoting the growth of beneficial bacteria (e.g., Bifidobacterium and Lactobacillus) while inhibiting harmful bacteria (e.g., Desulfotrichum) proliferation, 2′-FL significantly improves gut microbiota imbalance in UC patients. Additionally, 2′-FL markedly increases the production of short-chain fatty acids (SCFAs), particularly acetic acid, thereby enhancing intestinal barrier function and reducing inflammatory responses. These findings further establish 2′-FLs potential as a functional prebiotic in UC adjuvant therapy. Future research should investigate the safety and efficacy of long-term 2′-FL administration in vivo, and promote its practical application in prebiotic food development. References [1] Kennedy J M , Aminda D S , Walton G E ,[1] Kennedy J M , Aminda D S , Walton G E , et al.Comparison of Prebiotic Candidates in Ulcerative Colitis using an in vitro Fermentation Model[J].Journal of Applied Microbiology, 2024.DOI:10.1093.

background The early establishment of the gut microbiota is crucial for newborn health, playing a central role not only in immune system development but also in regulating metabolic functions. Studies have shown that human milk oligosaccharides (HMOs), particularly 2′-fucosylated lactose (2′-FL), significantly contribute to shaping beneficial gut microbiota and promoting the production of metabolites such as short-chain fatty acids. This study employed an in vitro human colon model (HCM) to simulate real intestinal environments, evaluating 2′-FLs regulatory effects on microbial abundance and metabolic balance. The findings provide scientific evidence supporting the functional applications of 2'-FL. Research design and methods This study employed an in vitro human colon model (HCM) to simulate the intestinal environment of infants and evaluate the regulatory effects of 2'-FL on gut microbiota abundance and metabolism. The HCM system was dividedthree regions: ascending colon, transverse colon, and descending colon, inoculated with eight representative infant intestinal bacteria to recreate a typical microecological system. By adding 2 grams of 2'-FL to the culture medium, real-time quantitative PCR (qPCR) was used to detect changes in microbial abundance, while liquid chromatography-mass spectrometry (LC/MS) was employed to measure the levels of metabolites such as short-chain fatty acids. Key findings 1. 2'-FL optimizes bacterial abundance in infant gut microbiota In the HCM model, 2'-FL significantly altered the abundance of representative microbial communities. PCR analysis revealed that beneficial bacteria such as Bacteroides distasonis increased markedly in the ascending, transverse, and descending colon, while harmful bacteria like Clostridium perfringens decreased. These results demonstrate that 2'-FL promotes the growth of beneficial bacteria and inhibits the proliferation of harmful bacteria, thereby contributing to the establishment of a healthy intestinal microecological environment. Figure 1: Fold changes in intestinal bacteria in (A) ascending colon, (B) transverse colon, and (C) descending colon following 2′-FL administration 2. 2'-FL promotes the production of beneficial intestinal metabolites The 2'-FL intervention significantly influenced free fatty acids (FFAs), the primary metabolites of gut microbiota. LC/MS analysis revealed marked variations in nine FFAs across colon regions. In the ascending colon, all FFAs except hexanoic acid showed significant elevation. The transverse colon exhibited reduced hexanoic acid levels with increased concentrations of other FFAs, while seven FFAs demonstrated marked elevation in the descending colon. These findings indicate that 2'-FL optimizes the gut metabolic environment by enhancing beneficial short-chain fatty acids, strengthening intestinal barrier function, and demonstrating anti-inflammatory effects. Figure 2: Schematic diagram of free fatty acid regulatory boxes in ascending colon (A), transverse colon (B), and descending colon (C) before and after 2′-FL addition 3. 2'-FL regulates the balance between gut microbiota and metabolites Spearman correlation analysis revealed that 2'-FL indirectly regulates intestinal microbial diversity, thereby modulating fatty acid production. In the ascending colon, Staphylococcus epidermidis showed negative correlations with propionic acid and Clostridium perfringens with valeric acid. The transverse colon exhibited positive correlations between Lactobacillus acidophilus and most free fatty acids (FFAs), while the descending colon demonstrated significant positive correlations between Lactobacillus acidophilus and FFAs, and negative correlations between Bifidobacterium adolescentis and most FFAs (excluding caproic and isovaleric acids). Notably, harmful bacteria like Clostridium perfringens showed significant negative correlations with key FFAs such as acetic acid. These findings suggest that 2'-FL supports gut health by optimizing the balance between microbial communities and metabolic processes. Figure 3: Correlation analysis of eight microbial species with nine free fatty acids in the ascending colon (A), transverse colon (B), and descending colon (C) Discussion and outlook This study utilized an in vitro human colon model (HCM) system to comprehensively reveal the crucial role of 2'-FL in regulating infant gut microbiota abundance and metabolism. The findings demonstrated that 2'-FL significantly promotes beneficial bacterial growth, inhibits harmful bacterial proliferation, and optimizes the production of metabolites such as short-chain fatty acids, thereby improving gut microbiota composition and metabolic balance. These results further establish 2'-FLs pivotal role in supporting early infant gut health. Future research should focus on evaluating the long-term efficacy of 2'-FL in vivo environments, particularly its performance under different feeding patterns, while exploring the development of functional foods based on 2'-FL to provide innovative solutions for infant gut health management. References [1] Zhang S, Chen L, Hu M, et al. 2-Fucosyllactose (2'-FL) changes infants gut microbiota composition and their metabolism in a host-free human colonic model[J]. Food Research International, 2023, 173: 113293.

  background After birth, newborns rapidly develop a gut microbiota that plays a vital role in intestinal development, immune system regulation, and the establishment of homeostasis. The early-life gut microbiota is influenced by multiple factors, with 2′-fucosyllactose(2′-FL) – a type of human milk oligosaccharide (HMOs) – proven to significantly promote infant gut health. This study aims to evaluate the effects of 2′-FL on the structure and metabolic activity of fecal microbiota in two-month-old infants, exploring its potential role in building a healthy gut environment. Research design and methods This study conducted fermentation experiments on fecal samples from two-month-old infants exclusively breastfed and formula-fed to evaluate the effects of 2′-FL on gut microbiota. Based on 2′-FL degradation capacity, fecal samples were categorizedrapid-degradants (consuming over 90% of 2′-FL within 24 hours) and slow-degradants (consuming only 5-6% of 2′-FL), aiming to investigate how degradation rates influence microbial communities. The experiment employed 16S rRNA gene sequencing to analyze microbial changes, while chromatographic methods were used to measure 2′-FL consumption, gas accumulation, and metabolite levels (such as lactic acid) for comprehensive evaluation of 2′-FLs effects. Key findings 1. 2′-FL optimizes the intestinal microbiota composition During the 2′-FL fermentation process, breastfed infants exhibited significantly increased bifidobacteria in their feces, establishing them as the dominant microbial community. In contrast, formula-fed infants showed more pronounced proliferation of lactobacilli. These beneficial bacteria produce lactic acid, which lowers intestinal pH levels and thereby inhibits harmful bacterial growth. This demonstrates that 2′-FL effectively promotes the growth of beneficial bacteria, contributing to the establishment of a healthy intestinal microecological environment. Figure 1: Heatmap of log-transformed relative abundance of fecal microbial groups in four groups after 24-hour culture with 2′-FL 2. 2′-FL promotes the production of beneficial metabolites in the gut microbiota Under the action of 2′-FL, breastfed infants exhibited higher accumulation of pyruvate and formate in stool cultures, while formula-fed infants showed a significant increase in lactic acid concentration. These metabolites, produced by gut-beneficial bacteria, serve as additional energy sources for the intestinal tract and help maintain an acidic environment, thereby further promoting the growth of beneficial bacteria. Figure 2: Changes in fecal lactate, pyruvate, formate, and pH levels relative to baseline (0 hour) after 24-hour culture with 2′-FL 3. 2′-FL improves the metabolic activity of intestinal microbiota Under the action of 2′-FL, both rapid-degrading and slow-degrading individuals exhibited enhanced metabolic activity in their fecal samples. Notably, rapid-degraders showed significantly increased glucose consumption, accompanied by elevated levels of acetic acid and other short-chain fatty acids (SCFAs). As key regulators of gut health, SCFAs enhance intestinal barrier function and possess anti-inflammatory properties. This demonstrates that 2′-FL can significantly improve intestinal metabolic activity characteristics, thereby maintaining the healthy balance of gut microbiota. Figure 3: 24-hour fecal cultures of fast-fermenting and slow-fermenting microbial communities show 2′-FL degradation and changes in glucose, fucose, acetic acid, and total short-chain fatty acids (SCFA) relative to baseline (0 h) levels. Discussion and outlook This study demonstrates that 2′-FL significantly optimizes the intestinal microbiota structure in infants, whether breastfed or formula-fed. It promotes the growth of beneficial bacteria, enhances the production of advantageous metabolites, and improves metabolic activity characteristics in the gut. As a functional ingredient, 2′-FL shows tremendous potential in regulating early-life gut microbiota, particularly in fostering intestinal health. With further research, the regulatory potential of 2′-FL in infant gut microbiota will be progressively validated, potentially offering an effective solution for improving gut health in non-breastfed infants. References [1]Nogacka, A.M., [1]Nogacka, A.M., et al. Influence of 2′-Fucosyllactose on the Microbiota Composition and Metabolic Activity of Fecal Cultures from Breastfed and Formula-Fed Infants at Two Months of Age. Microorganisms, 2021, 9(7), 1478.