Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60

alpha-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important alpha-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-alpha-glucanotransferase of high enzyme activity (60.8 U mg(-1)) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like alpha-glucan with (alpha 1 -> 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2 similar to+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB alpha-glucanotransferases, which might facilitate the production of tailored alpha-glucans by enzyme engineering in future.

The glycoside hydrolase family 70 (GH70) contains bacterial extracellular multidomain enzymes, synthesizing alpha-glucans from sucrose or starch-like substrates. A few dozen have been biochemically characterized, while crystal structures cover only the core domains and lack significant parts of auxiliary domains. Here we present a systematic overview of GH70 enzymes and their 3D structural organization and bacterial origin. A representative set of 234 permuted and 25 nonpermuted GH70 enzymes was generated, covering 12 bacterial families and 3 phyla and containing 185 predicted glucansucrases (GS), 15 branching sucrases (BrS), 8 "twin" GS-BrSs, and 51 alpha-glucanotransferases (alpha-GT). Analysis of AlphaFold models of all 259 entries showed that, apart from the core domains, the structural variation regarding auxiliary domains is far greater than anticipated, with nine different domain types. We analyzed the phylogenetic distribution and discuss the possible roles of auxiliary domains as well as possible correlations between enzyme specificity, auxiliary domain type, and bacterial origin.

The transglucosidase activity of GH31 α-glucosidases is employed to catalyze the synthesis of prebiotic isomaltooligosaccharides (IMOs) using the malt syrup prepared from starch as substrate. Continuous mining for new GH31 α-glucosidases with high stability and efficient transglucosidase activity is critical for enhancing the supply and quality of IMO preparations. In the present study, two α-glucosidases (MT31α1 and MT31α2) from Myceliophthora thermophila were explored for biochemical characterization. The optimum pH and temperature of MT31α1 and MT31α2 were determined to be pH 4.5 and 65 °C, and pH 6.5 and 60 °C, respectively. Both MT31α1 and MT31α2 were shown to be stable in the pH range of 3.0 to 10.0. MT31α1 displayed a high thermostability, retaining 60 % of activity after incubation for 24 h at 55 °C. MT31α1 is highly active on substrates with all types of α-glucosidic linkages. In contrast, MT31α2 showed preference for substrates with α-(1→3) and α-(1→4) linkages. Importantly, MT31α1 was able to synthesize IMOs and the conversion rate of maltose into the main functional IMOs components reached over 40 %. Moreover, MT31α2 synthesizes glucooligosaccharides with (consecutive) α-(1→3) linkages. Taken together, MT31α1 and MT31α2, showing distinct substrate and product specificity, hold clear potential for the synthesis of prebiotic glucooligosaccharides.

Solid-liquid thermo-reversibility, lacking in raw starch is an important property for gels allowing repeated thermal processing in food. In this work, the Thermoproteus uzoniensis 4-alpha-glucanotransferase (Tu alpha GT) was used to modify five different starches from potato, corn, rice, wheat, and pea and the thermo-reversibility of the modified starch gels was evaluated by rheological assay. Only modified potato starch (MPS) exhibited thermo-reversibility, which prompted us to perform detailed analyses of the structural and gel properties of this product. The enzymatic modification resulted in a marked decrease in amylopectin side chains with a degree of polmerization (DP) < 23, while the contents of side chains with DP 23-43 increased. These data indicated that Tu alpha GT preferentially extended the outer chains of amylopectin using amylose as a donor, which resulted in 17% increase in the amylopectin molecular weight to 3.0 x 10(7) g/mol. Notably, MPS displayed a delayed retrogradation and improved stability in four freeze-thaw cycles (-70 ?C and 25 ?C) as compared to the native starch. These analyses supported a model to explain the Tu alpha GT catalyzed conversion of potato starch to a thermo-reversible product. Importantly, the MPS thermo-reversible 6% (w/v) gel showed comparable rheological properties to a 1.5% (w/v) kappa-carrageenan hydrocolloid, highlighting the potential of MPS as a substitute to kappa-carrageenan and other hydrocolloids of emerging health concerns. This work offers a starch based thermo-reversible gel and a methodological framework to broadens the use of starch into novel application as a food ingredient.

Milk is often regarded as the gold standard for the nourishment of all mammalian offspring. The World Health Organization (WHO) recommends exclusive breastfeeding for the first 6 months of the life of the infant, followed by a slow introduction of complementary foods to the breastfeeding routine for a period of approximately 2 years, whenever this is possible ( Global Strategy for Infant and Young Child Feeding; WHO, 2003). One of the most abundant components in all mammals' milk, which is associated with important health benefits, is the oligosaccharides. The milk oligosaccharides (MOS) of humans and other mammals differ in terms of their concentration and diversity. Among those, goat milk contains more oligosaccharides (gMOS) than other domesticated dairy animals, as well as a greater range of structures. This review summarizes the biological functions of MOS found in both human and goat milk to identify the possible biological relevance of gMOS in human health and development. Based on the existing literature, seven biological functions of gMOS were identified, namely, MOS action as prebiotics, immune modulators, and pathogen traps; their modulation of intestinal cells; protective effect against necrotizing enterocolitis; improved brain development; and positive effects on stressor exposure. Overall, goat milk is a viable alternate supply of functional MOS that could be employed in a newborn formula.

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e., catechol, resorcinol, and hydroquinone) to form monoglucoside and diglucoside products. The production and yield of catechol glucosylated products were significantly higher than that of resorcinol and hydroquinone, revealing a preference for adjacent aromatic hydroxyl groups in glucosylation. Amino residues around acceptor substrate binding subsite +1 were targeted for semirational mutagenesis, yielding GtfZ-CD2 variants with improved resorcinol and hydroquinone glucosylation. Mutant L1560Y with improved hydroquinone mono-glucosylated product synthesis allowed enzymatic conversion of hydroquinone into α-arbutin. This study thus revealed the high potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. IMPORTANCE Glycosylation represents one of the most important ways to expand the diversity of natural products and improve their physico-chemical properties. Aromatic polyphenol compounds widely found in plants are reported to exhibit various remarkable biological activities; however, they generally suffer from low solubility and stability, which can be improved by glycosylation. Our present study on the glucosylation of benzenediol compounds by GH70 branching sucrase GtfZ-CD2 and its semirational engineering to improve the glucosylation efficiency provides insight into the mechanism of acceptor substrates binding and its glucosylation selectivity. The results demonstrate the potential of using branching sucrase as an effective enzymatic glucosylation tool.

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permutation of the catalytic domain, and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains. Despite these differences, the first crystal structure of a GtfC, GbGtfC-ΔC from Geobacillus 12AMOR1, and the first one representing a non-permuted GH70 enzyme, reveals high structural similarity in the core domains with most GtfBs, featuring a similar tunneled active site. We propose that GtfC (and related GtfD) enzymes evolved from starch-degrading α-amylases from GH13 by acquiring α-1,6 transglycosylation capabilities, before the events that resulted in circular permutation of the catalytic domain observed in other GH70 enzymes (glucansucrases, GtfB-type α-GTs). AlphaFold modeling and sequence alignments suggest that the GbGtfC structure represents the GtfC subfamily, although it has a so far unique alternating α-1,4/α-1,6 product specificity, likely determined by residues near acceptor binding subsites +1/+2.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase (Lr121 4,6-α-GTase), belonging to the glycosyl hydrolase (GH) 70 GtfB subfamily, converts starch and maltodextrins into linear isomalto/malto polysaccharides (IMMPs) with consecutive (α1 → 6) linkages. The recent elucidation of its crystal structure allowed identification and analysis of further structural features that determine its reaction and product specificity. Herein, sequence alignments between GtfB enzymes with different product linkage specificities (4,6-α-GTase and 4,3-α-GTase) identified amino acid residues in GH70 homology motifs, which may be critical for reaction and product specificity. Based on these alignments, four Lr121 GtfB-ΔN mutants (I1020M, S1057P, H1056G, and Q1126I) were constructed. Compared to wild-type Lr121 GtfB-ΔN, mutants S1057P and Q1126I had considerably improved catalytic efficiencies. Mutants H1056G and Q1126I showed a 9% decrease and an 11% increase, respectively, in the ratio of (α1 → 6) over (α1 → 4) linkages in maltodextrin-derived products. A change in linkage type (e.g., (α1 → 6) linkages to (α1 → 3) linkages) was not observed. The possible functional roles of these Lr121 GtfB-ΔN residues located around the acceptor substrate-binding subsites are discussed. The results provide new insights into structural determinants of the reaction and product specificity of Lr121 GtfB 4,6-α-GTase.

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.

Human milk oligosaccharides (hMOS) are associated with health benefits for newborns. We studied the composition of goat MOS (gMOS) from colostrum up to the 9th month of lactation to conceive an overview of the structures present and their fate. Potential correlations with factors such as age, parity, and lifetime milk production were examined. An effective method for gMOS extraction and ultra-high-performance liquid chromatography coupled to fluorescence detection (UPLC-FLD) analysis was established, following 2-aminobenzamide gMOS labeling. Considerable biological variability was highlighted among the 12 quantified gMOS and the 9 non-quantified structures in the individual milk samples. Most characteristic, 2′-fucosyllactose was present in 73.7% of the milk samples analyzed, suggesting the possibility of a secretor/non-secretor goat genotype, similar to humans. Contributing factors to the observed biological variability were goat age, parity, lifetime milk production, and the kids' sex. The results significantly contribute to the current understanding of (variations in) gMOS composition.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.

Scope: Cost-effective microbial biosynthesis of 2'-fucosyllactose (2'-FL) allows its application in infant formula. The specific effects of 2'-FL on the gastrointestinal immune barrier are still largely unknown. Methods/results: Here, we quantified and compared the effects of HMOs isolated from human milk, 2'-FL/lactose (Lac), and 2'-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfo-transferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2'-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2'-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2'-FL/Lac and 2'-FL all prevented A23187-induced barrier disruption in human T84 cells. Conclusion: Our findings indicate that 2'-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset, progression, and treatment of a broad spectrum of inflammatory conditions. In this study, we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form, as well as in its partially demannosylated and partially desialylated form, on HEK293 cells expressing TLR-8, and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells, N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent, i.e., chloroquine (CQN), that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8, it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold, p < 0.0001), partially demannosylated (0.3-fold, p < 0.0001) and partially desialylated (0.4-fold, p < 0.0001) N-glycans. Furthermore, the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN, the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune, infections or other inflammatory disorders.

Human milk is considered the golden standard in infant nutrition. Free oligosaccharides in human milk provide important health benefits. These oligosaccharides function as prebiotics, immune modulators, and pathogen inhibitors and were found to improve barrier function in the gut. Infant formulas nowadays often contain prebiotics but lack the specific functions of human milk oligosaccharides (hMOS). Milk from domesticated animals also contains milk oligosaccharides but at much lower levels and with less diversity. Goat milk contains significantly more oligosaccharides (gMOS) than bovine (bMOS) or sheep (sMOS) milk and also has a larger diversity of structures. This review summarizes structural studies, revealing a diversity of up to 77 annotated gMOS structures with almost 40 structures fully characterized. Quantitative studies of goat milk oligosaccharides range from 60 to 350 mg/L in mature milk and from 200 to 650 mg/L in colostrum. These levels are clearly lower than in human milk (5–20 g/L) but higher than in other domesticated dairy animals, e.g., bovine (30–60 mg/L) and sheep (20–40 mg/L). Finally, the review focuses on demonstrated and potential functionalities of gMOS. Some studies have shown anti-inflammatory effects of mixtures enriched in gMOS. Goat MOS also display prebiotic potential, particularly in stimulating growth of bifidobacteria preferentially. Although functional studies of gMOS are still limited, several structures are also found in human milk and have known functions as immune modulators and pathogen inhibitors. In conclusion, goat milk constitutes a promising alternative source for milk oligosaccharides, which can be used in infant formula.

The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhβ-gal) and Lactobacillus plantarum (Lp609Lhβ-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus, together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ∼90% of all GOS components. GOS mixtures obtained with β-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).

The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely, lactoferrin, lactoperoxidase, α-lactalbumin, immunoglobulin-G (IgG), and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (∼95%) of N-glycans present in the overall whey glycoprofile were attributed to three proteins: lactoferrin, IgG, and GlyCAM-1. We identified specific signature glycans for these main proteins; lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-β(1,4)-GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin, and GlyCAM-1 over time.

Galacto-oligosaccharides (GOS) have been reported to modulate the function of intestinal goblet cells and to improve mucus barrier function. However, GOS is available in many structurally different compositions and it is unknown how GOS structural diversity impacts this modulation of goblet cells. This study aims to investigate the effects of oligosaccharide content and glycosidic linkages of GOS on expression of genes associated with the secretory function of goblet cells. To investigate the effect of oligosaccharide content, LS174T cells were incubated with (β1 → 4)GOS of variable transgalactosylated oligosaccharides and lactose (Lac) composition. To investigate the effect of glycosidic linkages, we compared the effects of (β1 → 4)GOS with (β1 → 3)GOS, and with a mixture of α-linked oligosaccharides (lactose-derived oligosaccharides-LDO). The changes in mRNA expression of mucus-related genes were assessed by RT-PCR. GOS containing Lac significantly enhanced the expression of MUC2, TFF3 and RETNLB but not of Golgi sulfotransferases genes. In contrast, GOS without Lac did not impact these genes. Lac alone significantly enhanced MUC2, TFF3, RETNLB, CHST5, and GAL3ST2 genes suggesting that Lac might be responsible for goblet cell modulation in (β1 → 4)GOS preparations. (β1 → 3)GOS induced the expression of MUC2 and TFF3, and downregulated the RETNLB gene. Compared with the (β1 → 3) and GOS (β1 → 4)GOS, the α-linked LDO significantly upregulated the expression MUC2, TFF3, RETNLB and the Golgi sulfotransferases genes. We identify structural features of GOS that contribute to enhanced mucus integrity. Our study might lead to better GOS formulations for foods to prevent or treat different types of intestinal disorders.