Eat your beets: Conversion of polysaccharides into oligosaccharides for enhanced bioactivity.

Bioactive oligosaccharides with the potential to improve human health, especially in modulating gut microbiota via prebiotic activity, are available from few natural sources. This work uses polysaccharide oxidative cleavage to generate oligosaccharides from beet pulp, an agroindustry by-product. A scalable membrane filtration approach was applied to purify the oligosaccharides for subsequent in vitro functional testing. The combined use of nano-LC/Chip Q-TOF MS and UHPLC/QqQ MS allowed the evaluation of the oligosaccharide profile and their monosaccharide complexity. A final product containing roughly 40 g of oligosaccharide was obtained from 475 g of carbohydrates. Microbiological bioactivity assays indicated that the product obtained herein stimulated desirable commensal gut bacteria. This rapid, reproducible, and scalable method represents a breakthrough in the food industry for generating potential prebiotic ingredients from common plant by-products at scale. INDUSTRIAL RELEVANCE: This work proposes an innovative technology based on polysaccharide oxidative cleavage and multi-stage membrane purification to produce potential prebiotic oligosaccharides from renewable sources. It also provides critical information to evidence the prebiotic potential of the newly generated oligosaccharides on the growth promotion ability of representative probiotic strains of bifidobacteria and lactobacilli.

Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Human milk oligosaccharide 2'-fucosyllactose supplementation improves gut barrier function and signaling in the vagal afferent pathway in mice.

2'-Fucosyllactose (2'-FL) is one of the predominant oligosaccharides found in human milk and has several well-established beneficial effects in the host. It has previously been shown that 2'-FL can improve the metabolic phenotype in high-fat (HF)-fed mice. Here we investigated whether dietary supplementation with 2'-FL was associated with improved intestinal barrier integrity, signaling in the vagal afferent pathway and cognitive function. Mice were fed either a low-fat (LF, 10% fat per kcal) or HF (45% fat per kcal) diet with or without supplementation of 2'-FL (10% w/w) in the diet for 8 weeks. Body weight, energy intake, fat and lean mass, intestinal permeability (ex vivo in Ussing chambers), lipid profiles, gut microbiome and microbial metabolites, and cognitive functions were measured. Vagal afferent activity was measured via immunohistochemical detection of c-Fos protein in the brainstem in response to peripheral administration of cholecystokinin (CCK). 2'-FL significantly attenuated the HF-induced increase in fat mass and energy intake. 2'-FL significantly reduced intestinal permeability and significantly increased expression of interleukin (IL)-22, a cytokine known for its protective role in the intestine. Additionally, 2'-FL led to changes in the gut microbiota composition and in the associated microbial metabolites. Signaling in the vagal afferent pathway was improved but there was no effect on cognitive function. In conclusion, 2'-FL supplementation improved the metabolic profiles, gut barrier integrity, lipid metabolism and signaling in the vagal afferent pathway. These findings support the utility of 2'-FL in the control of gut barrier function and metabolic homeostasis under a metabolic challenge.

Effects of Industrial Thermal Treatments on the Release of Bovine Colostrum Glycoprotein N-Glycans by Endo-ß-N-acetylglucosaminidase.

N-Glycans are structurally similar to human milk oligosaccharides, the gold standard prebiotics for infants. Bovine milk N-glycans released by endo-ß-N-acetylglucosaminidase (EndoBI-1) were shown to have similar prebiotic selectivity as human milk oligosaccharides, explaining the interest for N-glycan recovery for use as prebiotics. Industrial thermal treatments such as high-temperature short-time (HTST) and ultra-high-temperature (UHT) might favor the enzymatic deglycosylation of N-glycans through promoting protein denaturation. We investigated the effects of HTST (72 °C for 15 s) and UHT (135 °C for 3 s) on N-glycan release from bovine colostrum glycoproteins by nonimmobilized and amino-immobilized EndoBI-1. A total of 104 N-glycans including isomers/anomers were identified by high-resolution mass spectrometry. In both EndoBI-1 forms, HTST increased the release of N-glycans; however, the impact of UHT on releasing N-glycans was comparable to the nonthermal treatment. Although the amino-immobilized enzyme similarly released neutral N-glycans as the free form, it released fewer sialylated and fucosylated N-glycans.

2'-fucosyllactose Supplementation Improves Gut-Brain Signaling and Diet-Induced Obese Phenotype and Changes the Gut Microbiota in High Fat-Fed Mice.

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)-1ß, 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.

Fecal metatranscriptomics and glycomics suggest that bovine milk oligosaccharides are fully utilized by healthy adults.

Human milk oligosaccharides play a vital role in the development of the gut microbiome in the human infant. Although oligosaccharides derived from bovine milk (BMO) differ in content and profile with those derived from human milk (HMO), several oligosaccharide structures are shared between the species. BMO are commercial alternatives to HMO, but their fate in the digestive tract of healthy adult consumers is unknown. Healthy human subjects consumed two BMO doses over 11-day periods each and provided fecal samples. Metatranscriptomics of fecal samples were conducted to determine microbial and host gene expression in response to the supplement. Fecal samples were also analyzed by mass spectrometry to determine levels of undigested BMO. No changes were observed in microbial gene expression across all participants. Repeated sampling enabled subject-specific analyses: four of six participants had minor, yet statistically significant, changes in microbial gene expression. No significant change was observed in the gene expression of host cells exfoliated in stool. Levels of BMO excreted in feces after supplementation were not significantly different from baseline and were not correlated with dosage or expressed microbial enzyme levels. Collectively, these data suggest that BMO are fully fermented in the human gastrointestinal tract upstream of the distal colon. Additionally, the unaltered host transcriptome provides further evidence for the safety of BMO as a dietary supplement or food ingredient. Further research is needed to investigate potential health benefits of this completely fermentable prebiotic that naturally occurs in cow's milk.

Discovery of Novel High-Molecular Weight Oligosaccharides Containing N-Acetylhexosamine in Bovine Colostrum Whey Permeate Hydrolyzed with Aspergillus oryzae ß-Galactosidase.

Bovine milk oligosaccharides (BMOs) that resemble human milk oligosaccharides are found in whey permeate, indicating that dairy streams can be used as a potential source of bioactive oligosaccharides. Recovery of oligosaccharides from whey permeate is hindered by their low abundance and high concentration of lactose. In the present work, lactose in bovine colostrum whey permeate was hydrolyzed by Aspergillus oryzae ß-galactosidase to facilitate subsequent monosaccharide removal by membrane separation. Chromatographic separation coupled with high-resolution mass spectrometry revealed ß-galactosidase degradation of several ß-linkage-containing BMOs and production of novel oligosaccharides that ranged in size from 5 to 11 monosaccharide units containing several galactose repeating units and N-acetylhexosamine at their reducing ends. Optimization of BMO hydrolysis and separation methodology could generate high amounts of hetero-oligosaccharides for improved recovery of potentially biotherapeutic oligosaccharides.

Effect of Roasting on Oligosaccharide Abundance in Arabica Coffee Beans.

Emerging research into the bioactivities of indigestible carbohydrates is illuminating the potential of various foods and food streams to serve as novel sources of health-promoting compounds. Oligosaccharides (OS) are widely present in milks and some plants. Our previous research demonstrated the presence of OS in brewed coffee and spent coffee grounds. Armed with this new knowledge, the next step toward improving the utilization of these valuable components involved investigating the effect of roasting on the formation and abundance of coffee OS. In the present study, we used advanced mass spectrometry to analyze a variety of coffee samples and demonstrated that a great structural diversity and increased abundance of OS is associated with higher roasting intensity. The present investigation also evaluated methods for OS extraction and fractionation. A preparative-scale chromatographic method, based on activated carbon, was developed to isolate enough amounts of OS from coffee to enable future confirmation of prebiotic and other in vitro activities.

Milk Glycans and Their Interaction with the Infant-Gut Microbiota.

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants.

21st century toolkit for optimizing population health through precision nutrition.

Scientific, technological, and economic progress over the last 100 years all but eradicated problems of widespread food shortage and nutrient deficiency in developed nations. But now society is faced with a new set of nutrition problems related to energy imbalance and metabolic disease, which require new kinds of solutions. Recent developments in the area of new analytical tools enable us to systematically study large quantities of detailed and multidimensional metabolic and health data, providing the opportunity to address current nutrition problems through an approach called Precision Nutrition. This approach integrates different kinds of "big data" to expand our understanding of the complexity and diversity of human metabolism in response to diet. With these tools, we can more fully elucidate each individual's unique phenotype, or the current state of health, as determined by the interactions among biology, environment, and behavior. The tools of precision nutrition include genomics, metabolomics, microbiomics, phenotyping, high-throughput analytical chemistry techniques, longitudinal tracking with body sensors, informatics, data science, and sophisticated educational and behavioral interventions. These tools are enabling the development of more personalized and predictive dietary guidance and interventions that have the potential to transform how the public makes food choices and greatly improve population health.

Prebiotic milk oligosaccharides prevent development of obese phenotype, impairment of gut permeability, and microbial dysbiosis in high fat-fed mice.

Microbial dysbiosis and increased intestinal permeability are targets for prevention or reversal of weight gain in high-fat (HF) diet-induced obesity (DIO). Prebiotic milk oligosaccharides (MO) have been shown to benefit the host intestine but have not been used in DIO. We hypothesized that supplementation with bovine MO would prevent the deleterious effect of HF diet on the gut microbiota and intestinal permeability and attenuate development of the obese phenotype. C57BL/6 mice were fed a control diet, HF (40% fat/kcal), or HF + prebiotic [6%/kg bovine milk oligosaccharides (BMO) or inulin] for 1, 3, or 6 wk. Gut microbiota and intestinal permeability were assessed in the ileum, cecum, and colon. Addition of BMO to the HF diet significantly attenuated weight gain, decreased adiposity, and decreased caloric intake; inulin supplementation also lowered weight gain and adiposity, but this did not reach significance. BMO and inulin completely abolished the HF diet-induced increase in paracellular and transcellular permeability in the small and large intestine. Both BMO and inulin increased abundance of beneficial microbes Bifidobacterium and Lactobacillus in the ileum. However, inulin supplementation altered phylogenetic diversity and decreased species richness. We conclude that addition of BMO to the HF diet completely prevented increases in intestinal permeability and microbial dysbiosis and was partially effective to prevent weight gain in DIO.NEW & NOTEWORTHY This study provides the first report of the effects of prebiotic bovine milk oligosaccharides on the host phenotype of high-fat diet-induced obesity in mice.

Chemical Characterization of Potentially Prebiotic Oligosaccharides in Brewed Coffee and Spent Coffee Grounds.

Oligosaccharides are indigestible carbohydrates widely present in mammalian milk and in some plants. Milk oligosaccharides are associated with positive health outcomes; however, oligosaccharides in coffee have not been extensively studied. We investigated the oligosaccharides and their monomeric composition in dark roasted coffee beans, brewed coffee, and spent coffee grounds. Oligosaccharides with a degree of polymerization ranging from 3 to 15, and their constituent monosaccharides, were characterized and quantified. The oligosaccharides identified were mainly hexoses (potentially galacto-oligosaccharides and manno-oligosaccharides) containing a heterogeneous mixture of glucose, arabinose, xylose, and rhamnose. The diversity of oligosaccharides composition found in these coffee samples suggests that they could have selective prebiotic activity toward specific bacterial strains able to deconstruct the glycosidic bonds and utilize them as a carbon source.

Rapid Screening of Bovine Milk Oligosaccharides in a Whey Permeate Product and Domestic Animal Milks by Accurate Mass Database and Tandem Mass Spectral Library.

A bovine milk oligosaccharide (BMO) library, prepared from cow colostrum, with 34 structures was generated and used to rapidly screen oligosaccharides in domestic animal milks and a whey permeate powder. The novel library was entered into a custom Personal Compound Database and Library (PCDL) and included accurate mass, retention time, and tandem mass spectra. Oligosaccharides in minute-sized samples were separated using nanoliquid chromatography (nanoLC) coupled to a high resolution and sensitive quadrupole-Time of Flight (Q-ToF) MS system. Using the PCDL, 18 oligosaccharides were found in a BMO-enriched product obtained from whey permeate processing. The usefulness of the analytical system and BMO library was further validated using milks from domestic sheep and buffaloes. Through BMO PCDL searching, 15 and 13 oligosaccharides in the BMO library were assigned in sheep and buffalo milks, respectively, thus demonstrating significant overlap between oligosaccharides in bovine (cow and buffalo) and ovine (sheep) milks. This method was shown to be an efficient, reliable, and rapid tool to identify oligosaccharide structures using automated spectral matching.

Modeling lactose hydrolysis for efficiency and selectivity: Toward the preservation of sialyloligosaccharides in bovine colostrum whey permeate.

Enzymatic hydrolysis of lactose has been shown to improve the efficiency and selectivity of membrane-based separations toward the recovery of bioactive oligosaccharides. Achieving maximum lactose hydrolysis requires intrinsic process optimization for each specific substrate, but the effects of those processing conditions on the target oligosaccharides are not well understood. Response surface methodology was used to investigate the effects of pH (3.25-8.25), temperature (35-55°C), reaction time (6 to 58 min), and amount of enzyme (0.05-0.25%) on the efficiency of lactose hydrolysis by ß-galactosidase and on the preservation of biologically important sialyloligosaccharides (3'-siallylactose, 6'-siallylactose, and 6'-sialyl-N-acetyllactosamine) naturally present in bovine colostrum whey permeate. A central composite rotatable design was used. In general, ß-galactosidase activity was favored at pH values ranging from 3.25 to 5.75, with other operational parameters having a less pronounced effect. A pH of 4.5 allowed for the use of a shorter reaction time (19 min), lower temperature (40°C), and reduced amount of enzyme (0.1%), but complete hydrolysis at a higher pH (5.75) required greater values for these operational parameters. The total amount of sialyloligosaccharides was not significantly altered by the reaction parameters evaluated, suggesting specificity of ß-galactosidase from Aspergillus oryzae toward lactose as well as the stability of the oligosaccharides at pH, temperature, and reaction time evaluated.

Short communication: Quantification of carbohydrates in whey permeate products using high-performance anion-exchange chromatography with pulsed amperometric detection.

A method was developed for the characterization and quantification of the disaccharide lactose and 3 major bovine milk oligosaccharides (BMO) in dairy streams. Based on high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD), this method is advantageous because it requires minimal sample preparation and achieves good chromatographic separation of oligosaccharide isomers within 30min. The linear dynamic range and limit of detection were 0.1 to 10mg/L and 0.03 to 0.22mg/L, respectively. Mean recoveries of the BMO were excellent and ranged from 98.4 to 100.4%. Without complicated sample preparation procedures, this HPAE-PAD method measured BMO [3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and 6'-sialyllactosamine (6'SLN)] and lactose using a single instrument, therefore increasing the accuracy of the measurement and applicability for the dairy industry. In colostrum whey permeate, 3'SL, 6'SL, and 6'SLN were 94, 29, and 46mg/L, respectively. This work is the first to demonstrate that some commercial products, currently marketed for supporting a healthy immune system, contain significant amounts of bioactive BMO and therefore, carry additional bioactivities.

Kinetic characterization of a novel endo-ß-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

EndoBI-1 is a recently isolated endo-ß-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.

Characterizing the release of bioactive N-glycans from dairy products by a novel endo-ß-N-acetylglucosaminidase.

Endo-ß-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45-8.45), temperature (27.5-77.5°C), reaction time (15-475 min), and enzyme/protein ratio (1:3,000-1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (2(4)) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N-glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan-free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI-1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies.

A novel endo-ß-N-acetylglucosaminidase releases specific N-glycans depending on different reaction conditions.

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-ß-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip-Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions.

Quantitative analysis of gangliosides in bovine milk and colostrum-based dairy products by ultrahigh performance liquid chromatography-tandem mass spectrometry.

Milk gangliosides have gained considerable attention because they participate in diverse biological processes, including neural development, pathogen binding, and activation of the immune system. Herein, we present a quantitative measurement of the gangliosides present in bovine milk and other dairy products and byproducts. Ultrahigh performance liquid chromatography separation was used for high-throughput analysis and achieved a short running time without sacrificing chromatographic resolution. Dynamic multiple reaction monitoring was conducted for 12 transitions for GM3 and 12 transitions for GD3. Transitions to sialic acid fragments (m/z 290.1) were chosen for the quantitation. There was a considerable amount of gangliosides in day 2 milk (GM3, 0.98 mg/L; GD3, 15.2 mg/L) which dramatically decreased at day 15 and day 90. GM3 and GD3 were also analyzed in pooled colostrum, colostrum cream, colostrum butter, and colostrum buttermilk. The separation and analytical approaches here proposed could be integrated into the dairy industry processing adding value to side-streams.

Identification and characterisation of water and alkali soluble oligosaccharides from hazelnut skin (Corylus avellana L.).

Hazelnut skins are a good example of agricultural by-product with the potential to become a valuable source of functional ingredients. In this work, the fibre from hazelnut skins was extracted by using water and alkali solution and characterised by a suite of analytical tools (MALDI-FTICR, nano LC-Chip-Q-ToF and gas chromatography). Over thirty complex free oligosaccharides, composed mainly of galacturonic acid and N-acetylgalactosamine, were characterised for the first time in the present study. Their concentration ranged between 16 and 34mg per g of extract. The oligosaccharides isolated from this agricultural by-product are mainly hexose oligosaccharides (potentially galacto-oligosaccharides,) and xyloglucans. The identified composition could justify the bioactive activity of the extracts, namely prebiotic activity, previously demonstrated.