Microencapsulation of a synbiotic into PLGA/alginate multiparticulate gels

Probiotic bacteria have gained popularity as a defence against disorders of the bowel. However, the acid sensitivity of these cells results in a loss of viability during gastric passage and, consequently, a loss of efficacy. Probiotic treatment can be supplemented using ‘prebiotics’, which are carbohydrates fermented specifically by probiotic cells in the body. This combination of probiotic and prebiotic is termed a ‘synbiotic’. Within this article a multiparticulate dosage form has been developed, consisting of poly(d,l-lactic-co-glycolic acid) (PLGA) microcapsules containing prebiotic Bimuno™ incorporated into an alginate–chitosan matrix containing probiotic Bifidobacterium breve. The aim of this multiparticulate was that, in vivo, the probiotic would be protected against gastric acid and the release of the prebiotic would occur in the distal colon. After microscopic investigation, this synbiotic multiparticulate was shown to control the release of the prebiotic during in vitro gastrointestinal transit, with the release of galacto-oligosaccharides (GOS) initially occurred over 6 h, but with a triphasic release pattern giving further release over 288 h. Encapsulation of B. breve in multiparticulates resulted in a survival of 8.0 ± 0.3 log CFU/mL cells in acid, an improvement over alginate–chitosan microencapsulation of 1.4 log CFU/mL. This was attributed to increased hydrophobicity by the incorporation of PLGA particles.

Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine

The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and adjunctive treatment of neuropsychiatric disorders.

Rafinose na alimentação de leitões recém-desmamados

Pós-graduação em Zootecnia - FCAV

Effect of a whey-predominant starter formula containing LCPUFAs and oligosaccharides (FOS/GOS) on gastrointestinal comfort in infants.

Development of new infant formulas aims to replicate the benefits of breast milk. One benefit of breast milk over infant formulas is greater gastrointestinal comfort. We compared indicators of gastrointestinal comfort in infants fed a whey-predominant formula containing long-chain polyunsaturated fatty acids, galacto-oligo-saccharides and fructo-oligosaccharides, and infants fed a control casein-predominant formula without additional ingredients. The single-centre, prospective, double-blind, controlled trial randomly assigned healthy, full-term infants (n=144) to receive exclusively either experimental or control formula from 30 days to 4 months of age. A group of exclusively breast-fed infants served as reference (n=80). At 1, 2, 3, and 4 months, infants' growth parameters were measured and their health assessed. Parents recorded frequency and physical characteristics of infants' stool, frequency of regurgitation, vomiting, crying and colic. At 2-months, gastric emptying (ultrasound) and intestinal transit time (H2 breath test) were measured, and stool samples collected for bacterial analysis. Compared to the control (n=69), fewer of the experimental group (n=67) had hard stools (0.7 vs 7.5%, p<0.001) and more had soft stools (90.8 vs 82.3%, p<0.05). Also compared to the control, the experimental group's stool microbiota composition (mean % bifidobacteria: 78.1 (experimental, n=17), 63.7 (control, n=16), 74.3 (breast-fed, n=20), gastric transit times (59.6 (experimental, n=53), 61.4 (control, n=62), 55.9 (breast-fed, n=67) minutes) and intestinal transit times (data not shown) were closer to that of the breast-fed group. Growth parameter values were similar for all groups. The data suggest that, in infants, the prebiotic-containing whey-based formula provides superior gastrointestinal comfort than a control formula.

Feeding Dietary Mannan Oligosaccharides to Juvenile Nile Tilapis, Oreochromis niloticus, Has No Effect on Hematological Parameters and Showed Decreased Feed Consumption

Impaired immune system by environmental stressors can lead fishes to be more susceptible to diseases that limit the economic development of aquaculture systems. This study was set out to determine the effect of six levels of mannan oligosaccharides (MOS; ActiveMOS((R)); Biorigin, Lencois Paulista, Sao Paulo, Brazil) on the performance index and hematology of Nile tilapia, Oreochromis niloticus juveniles. Fish (13.62 g) were randomly distributed into 18 plastic aquaria (300 L; 20 fishes per aquarium) and fed during 45 d with a commercial diet supplemented with 0, 0.2, 0.4, 0.6, 0.8, and 1% dietary MOS, in a totally randomized design trial (n = 3); biometrical and hematological data were collected and analyzed. There were no significant differences in hematological parameters between fish fed control and MOS supplementation diets, and daily feed consumption (FC) decreased (P < 0.05) with increasing levels of dietary MOS. Dietary MOS did not increase leukocyte count and presented negative effects on FC of Nile tilapia. At 0.4% MOS supplementation, the individual weight gain was higher in absolute values but not different (P > 0.05) compared to control diet.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

A novel method for solid-phase synthesis of oligosaccharides using the N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker

The application of the N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker for the solid-phase synthesis of oligosaccharides is described. The oligosaccharide products can be cleaved from the resin by hydrazine, ammonia or primary amines, but the linker is stable under the conditions of oligosaccharide synthesis. The first sugar can be attached to the resin linker via a vinylogous amide bond, or by ether linkage using a p-aminobenzyl alcohol converter. (C) 2001 Elsevier Science Ltd. All rights reserved.

Parameter identification of the fermentative production of fructo-oligosaccharides by Aureobasidium pullulans

In this study, a mathematical model for the production of Fructo-oligosaccharides (FOS) by Aureobasidium pullulans is developed. This model contains a relatively large set of unknown parameters, and the identification problem is analyzed using simulation data, as well as experimental data. Batch experiments were not sufficiently informative to uniquely estimate all the unknown parameters, thus, additional experiments have to be achieved in fed-batch mode to supplement the missing information. © 2015 IEEE.

Production and extraction of polysaccharides and oligosaccharides and their use as new food additives

Polysaccharides and oligosaccharides can improve quality and enhance nutritional value of final food products due to their technological and nutritional features ranging from their capacity to improve texture to their effect as dietary fibers. For this reason, they are among the most studied ingredients in the food industry. The use of natural polysaccharides and oligosaccharides as food additives has been a reality since the food industry understood their potential technological and nutritional applications. Currently, the replacement of traditional ingredients and/or the synergy between traditional ingredients and polysaccharides and oligosaccharides are perceived as promising approaches by the food industry. Traditionally, polysaccharides have been used as thickening, emulsifying, and stabilizing agents, however, at this moment polysaccharides and oligosaccharides claim health and nutritional advantages, thus opening a new market of nutritional and functional foods. Indeed, their use as nutritional food ingredients enabled the food industry to develop a countless number of applications, e.g., fat replacers, prebiotics, dietary fiber, and antiulcer agents. Based on this, among the scientific community and food industry, in the last years many research studies and commercial products showed the possibility of using either new or already used sources (though with changed properties) of polysaccharides for the production of food additives with new and enhanced properties. The increasing interest in such products is clearly illustrated by the market figures and consumption trends. As an example, the sole market of hydrocolloids is estimated to reach $7 billion in 2018. Moreover, oligosaccharides can be found in more than 500 food products resulting in a significant daily consumption. A recent study from the Transparency Market Research on Prebiotic Ingredients Market reported that prebiotics' demand was worth $2.3 billion in 2012 and it is estimated to reach $4.5 billion in 2018, growing at a compound annual growth rate of 11.4% between 2012 and 2018. The entrance of this new generation of food additives in the market, often claiming health and nutritional benefits, imposes an impartial analysis by the legal authorities regarding the accomplishment of requirements that have been established for introducing novel ingredients/food, including new poly- and oligosaccharides. This chapter deals with the potential use of polysaccharides and oligosaccharides as food additives, as well as alternative sources of these compounds and their possible applications in food products. Moreover, the regulation process to introduce novel polysaccharides and oligosaccharides in the market as food additives and to assign them health claims is discussed.

The structures of the honeydew oligosaccharides synthesized by Claviceps africana

The structures of the principal oligosaccharides in the honeydew exudate of the sorghum ergot pathogen Claviceps africana, which has become epidemic in the Americas, have been characterized through linkage analysis using FAB-MS and GC-MS techniques, as 1,6-di-b-D-fructofuranosyl-D-mannitol and 1,5-di-b-D-fructofuranosyl-D-arabitol trisaccharides, 1-b-D-fructofuranosyl-D-mannitol and 5-b-D-fructofuranosyl-D-arabitol disaccharides and other minor disaccharides and trisaccharides. Their structural diversity is explained according to perceived biosynthetic interrelationships in pathways that appear to be unique amongst ergot fungi, particularly concerning intra-molecular reduction of fructose. The oligosaccharide, 1,6-di-b-D-fructofuranosyl-D-mannitol, which inhibits C. africana macrospore germination at a concentration in water of 1 g/mL or more, forms together with other slightly less bioactive oligosaccharides, the basis of a novel strategy to limit ergot disease losses in hybrid sorghum seed production.

Identifying and Characterizing New Ingredients in vitro for Prebiotic and Synbiotic use

The endogenous microbiota, constituting the microbes that live inside and on humans, is estimated to outnumber human cells by a factor of ten. This commensal microbial population has an important role in many physiological functions, with the densest microbiota population found in the colon. The colonic microbiota is a highly complex and diverse bacterial ecosystem, and a delicate balance exists between the gut microbiota and its host. An imbalance in the microbial ecosystem may lead to severe symptoms in and also beyond the gastrointestinal tract. Due to the important role of the gut microbiota in human health, means of its modification have been introduced in the dietary concepts of pro-, pre- and synbiotics. Prebiotics, which are usually carbohydrates, strive to selectively influence beneficial microbes resident in the colon with the aim of modifying the composition and functionality of the commensal microbial population towards a purportedly healthier one. The study of prebiotic effects on colonic micro-organisms is typically done by using human faecal material, though this provides relatively little information on bacterial populations and metabolic events in different parts of the colon. For this reason, several in vitro models have been developed to investigate the gut microbiota. The aim of this doctoral thesis was to screen through some of the promising prebiotic candidates, characterize their effects on the microbiota through the use of two in vitro methods (pure microbial cultures and a colon simulator model) and to evaluate their potential as emerging prebiotics or synbiotics when combined with the probiotic Bifidobacterium lactis . As a result of the screening work and subsequent colon simulation studies, several compounds with promising features were identified. Xylo-oligosaccharides (XOS), which have previously already shown promise as prebiotic compounds, were well fermented by several probiotic Bifidobacterium lactis strains in pure culture studies and in the following simulation studies utilizing the complex microbiota by endogenous B. lactis Another promising compound was panose, a trisaccharide belonging to isomalto-oligosaccharides (IMO) that also was also able to modify the microbiota in vitro by increasing the number of beneficial microbes investigated. Panose has not been widely studied previously and therefore, this thesis work provided the first data on panose fermentation in mixed colonic microbiota. Galacto-oligosaccharide (GOS) is an established prebiotic, and it was studied here in conjunction with another potential polygosaccharide polydextrose (PDX) and probiotic B. lactis Bi-07. In this final study, the synbiotics including GOS were more effective than the constituting pro- or prebiotics alone in modulating the microbiota composition, thus indicating a synergy resulting from the combination. The results obtained in this in vitro work can be, and have already been, utilized in product development aimed at the nutritional modification of the human colonic microbiota. Some of the compounds have entered the human clinical intervention phase to nvestigate in more detail the prebiotic and synbiotic properties seen in these in vitro studies.

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the glucooligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides

Prebiotics are nondigestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or activity of one, or a limited number of, bacteria present in the colon. The selected genera should have the capacity to improve host health (e.g. Bifidobacterium, Lactobacillus). To help identify preferred types, for inclusion into the diet, a quantitative equation [measure of the prebiotic effect (MPE)] is suggested. This will help evaluate, in vitro, the fermentation of dietary carbohydrates and compare their prebiotic effect. Although the approach is not meant to define health values, it is formulated to better inform the choice of prebiotic. It therefore, compares measurements of bacterial changes through the determination of maximum growth rates of predominant groups present in faeces, rate of substrate assimilation and the production of lactic, acetic, propionic and butyric acids. The equation will allow further in vitro comparisons of MPE, leading towards further studies (e.g. in humans) to determine the success of dietary intervention. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

In vitro investigation into the potential prebiotic activity of honey oligosaccharides

The effect of honey oligosaccharides on the growth of fecal bacteria was studied using an in vitro fermentation system. Prior to treatment, glucose and fructose (31.73 and 21.41 g/100 g of product, respectively) present in honey, which would be digested in the upper gut, were removed to avoid any influence on bacterial populations in the fermentations. Nanofiltration, yeast (Saccharomyces cerevisiae) treatment, and adsorption onto activated charcoal were used to remove monosaccharides. Prebiotic (microbial fermentation) activities of the three honey oligosaccharide fractions and the honey sample were studied and compared with fructooligosaccharide (FOS), using 1% (w/v) fecal bacteria in an in vitro fermentation system (10 mg of carbohydrate, 1.0 mL of basal medium). A prebiotic index (PI) was calculated for each carbohydrate source. Honey oligosaccharides seem to present potential prebiotic activity (PI values between 3.38 and 4.24), increasing the populations of bifidobacteria and lactobacilli, although not to the levels of FOS (PI of 6.89).

Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.