Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88)

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as it's anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

Dde as a protecting group for carbohydrate synthesis

Oligosaccharide synthesis using aminosugars requires the presence of a suitable amino protecting group. A number of protecting groups are currently used, and while many display favorable properties, most agents available still suffer from certain disadvantages. This report details the use of a hydrazine labile aminosugar protecting group, N -[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl] (Dde), which can be introduced and removed in a facile and cost-effective manner.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. neningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (Kd, in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (Kd, 1.1 × 10-7 mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [ 35S]PIF to triton solublized membranes from C2C 12 cells provided evidence for a binding protein of apparent M r of ∼40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target. ©2007 American Association for Cancer Research.

Tumor-host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-α2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a β3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site. © 2004 Wiley-Liss, Inc.

Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Functional application of lactic acid bacteria exopolysaccharide in complex food systems

Lactic acid bacteria expolysaccharides (LAB-EPS), in particular those formed from sucrose have the potential to improve food and beverage rheology and enhance their sensory properties potentially replacing or reducing expensive hydrocolloids currently used as improvers in food and beverage industries. Addition of sucrose not only enables EPS formation but also affects organic acid formation, thus influencing the sensory properties of the resulting food/beverage products. The first part of the study the organoleptic modulation of barley malt derived wort fermented using in situ produced bacterial polysaccharides has been investigated. Weisella cibaria MG1 was capable to produce exopolysaccharides during sucrosesupplemented barley malt derived wort fermentation. Even though the strain dominated the (sucrose-supplemented) wort fermentation, it was found to produce EPS (14.4 g l-1) with lower efficiency than in SucMRS (34.6 g l-1). Higher maltose concentration in wort led to the increased formation of oligosaccharide (OS) at the expense of EPS. Additionally, small amounts of organic acids were formed and ethanol remained below 0.5% (v/v). W. cibaria MG1 fermented worts supplemented with 5 or 10% sucrose displayed a shear-thinning behaviour indicating the formation of polymers. This report showed how novel and nutritious LAB fermented wort-base beverage with prospects for further advancements can be formulated using tailored microbial cultures. In the next step, the impact of exopolysaccharide-producing Weissella cibaria MG1 on the ability to improve rheological properties of fermented plant-based milk substitute plant based soy and quinoa grain was evaluated. W. cibaria MG1 grew well in soy milk, exceeding a cell count of log 8 cfu/g within 6 h of fermentation. The presence of W. cibaria MG1 led to a decrease in gelation and fermentation time. EPS isolated from soy yoghurts supplemented with sucrose were higher in molecular weight (1.1 x 108 g/mol vs 6.6 x 107 g/mol), and resulted in reduced gel stiffness (190 ± 2.89 Pa vs 244 ± 15.9 Pa). Soy yoghurts showed typical biopolymer gels structure and the network structure changed to larger pores and less cross-linking in the presence of sucrose and increasing molecular weight of the EPS. In situ investigation of Weissella cibaria MG1 producing EPS on quinoa-based milk was performed. The production of quinoa milk, starting from wholemeal quinoa flour, was optimised to maximise EPS production. On doing that, enzymatic destructuration of protein and carbohydrate components of quinoa milk was successfully achieved applying alpha-amylase and proteases treatments. Fermented wholemeal quinoa milk using Weissella cibaria MG1 showed high viable cell counts (>109 cfu/mL), a pH of 5.16, and significantly higher water holding capacity (WHC, 100 %), viscosity (> 0. 5 Pa s) and exopolysaccharide (EPS) amount (40 mg/L) than the chemically acidified control. High EPS (dextran) concentration in quinoa milk caused earlier aggregation because more EPS occupy more space, and the chenopodin were forced to interact with each other. Direct observation of microstructure in fermented quinoa milk indicated that the network structures of EPS-protein could improve the texture of fermented quinoa milk. Overall, Weissella cibaria MG1 showed favorable technology properties and great potential for further possible application in the development of high viscosity fermented quinoa milk. The last part of the study investigate the ex-situ LAB-EPS (dextran) application compared to other hydrocolloids as a novel food ingredient to compensate for low protein in biscuit and wholemeal wheat flour. Three hydrocolloids, xanthan gum, dextran and hydroxypropyl methylcellulose, were incorporated into bread recipes based on high-protein flours, low-protein flours and coarse wholemeal flour. Hydrocolloid levels of 0–5 % (flour basis) were used in bread recipes to test the water absorption. The quality parameters of dough (farinograph, extensograph, rheofermentometre) and bread (specific volume, crumb structure and staling profile) were determined. Results showed that xanthan had negative impact on the dough and bread quality characteristics. HPMC and dextran generally improved dough and bread quality and showed dosage dependence. Volume of low-protein flour breads were significantly improved by incorporation of 0.5 % of the latter two hydrocolloids. However, dextran outperformed HPMC regarding initial bread hardness and staling shelf life regardless the flour applied in the formulation.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Developing an index of dietary estimation of fermentable carbohydrate to allow dietary analysis for epidemiological studies

It has been proposed that long-term consumption of diets rich in non-digestible carbohydrates (NDCs), such as cereals, fruit and vegetables might protect against several chronic diseases, however, it has been difficult to fully establish their impact on health in epidemiology studies. The wide range properties of the different NDCs may dilution their impact when they are combined in one category for statistical comparisons in correlations or multivariate analysis. Several mechanisms have been suggested to explain the protective effects of NDCs, including increased stool bulk, dilution of carcinogens in the colonic lumen, reduced transit time, lowering pH, and bacterial fermentation to short chain fatty acids (SCFA) in the colon. However, it is very difficult to measure SCFA in humans in vivo with any accuracy, so epidemiological studies on the impact of SCFA are not feasible. Most studies use dietary fibre (DF) or Non-Starch Polysaccharides (NSP) intake to estimate the levels, but not all fibres or NSP are equally fermentable. It has been proposed that long-term consumption of diets rich in non-digestible carbohydrates (NDCs), such as cereals, fruit and vegetables might protect against several chronic diseases, however, it has been difficult to fully establish their impact on health in epidemiology studies. The wide range properties of the different NDCs may dilution their impact when they are combined in one category for statistical comparisons in correlations or multivariate analysis. Several mechanisms have been suggested to explain the protective effects of NDCs, including increased stool bulk, dilution of carcinogens in the colonic lumen, reduced transit time, lowering pH, and bacterial fermentation to short chain fatty acids (SCFA) in the colon. However, it is very difficult to measure SCFA in humans in vivo with any accuracy, so epidemiological studies on the impact of SCFA are not feasible. Most studies use dietary fibre (DF) or Non-Starch Polysaccharides (NSP) intake to estimate the levels, but not all fibres or NSP are equally fermentable. The first aim of this thesis was the development of the equations used to estimate the amount of FC that reaches the human colon and is fermented fully to SCFA by the colonic bacteria. Therefore, several studies were examined for evidence to determine the different percentages of each type of NDCs that should be included in the final model, based on how much NDCs entered the colon intact and also to what extent they were fermented to SCFA in vivo. Our model equations are FC-DF or NSP$ 1: 100 % Soluble + 10 % insoluble + 100 % NDOs¥ + 5 % TS** FC-DF or NSP 2: 100 % Soluble + 50 % insoluble + 100 % NDOs + 5 % TS FC-DF* or NSP 3: 100 % Soluble + 10 % insoluble + 100 % NDOs + 10 % TS FC-DF or NSP 4: 100 % Soluble + 50 % insoluble + 100 % NDOs + 10 % TS *DF: Dietary fibre; **TS: Total starch; $NSP: non-starch polysaccharide; ¥NDOs: non-digestible oligosaccharide The second study of this thesis aimed to examine all four predicted FC-DF and FC-NSP equations developed, to estimate FC from dietary records against urinary colonic NDCs fermentation biomarkers. The main finding of a cross-sectional comparison of habitual diet with urinary excretion of SCFA products, showed weak but significant correlation between the 24 h urinary excretion of SCFA and acetate with the estimated FC-DF 4 and FC-NSP 4 when considering all of the study participants (n = 122). Similar correlations were observed with the data for valid participants (n = 78). It was also observed that FC-DF and FC-NSP had positive correlations with 24 h urinary acetate and SCFA compared with DF and NSP alone. Hence, it could be hypothesised that using the developed index to estimate FC in the diet form dietary records, might predict SCFA production in the colon in vivo in humans. The next study in this thesis aimed to validate the FC equations developed using in vitro models of small intestinal digestion and human colon fermentation. The main findings in these in vitro studies were that there were several strong agreements between the amounts of SCFA produced after actual in vitro fermentation of single fibre and different mixtures of NDCs, and those predicted by the estimated FC from our developed equation FC-DF 4. These results which demonstrated a strong relationship between SCFA production in vitro from a range of fermentations of single fibres and mixtures of NDCs and that from the predicted FC equation, support the use of the FC equation for estimation of FC from dietary records. Therefore, we can conclude that the newly developed predicted equations have been deemed a valid and practical tool to assess SCFA productions for in vitro fermentation.

The Effects of Various Types of Feed additives on The Chemical Composition of Local Chicken Meat (Gallus domestica).

Abstract. This study aims to determine the effect of giving various types of feed additives to the chemical composition value of super chicken chicken (Gallus domestica). This research is an experimental research using 20 super chicken chickens that come from chicken growth research (growth study). The design used in this study was Completely Randomized Design (RAL), consisting of 4 treatments and 5 replications. The treatment given was (A0 = control (Vita chick 0.7 gram / liter; A1 = 20 ml / liter probio-FM; A2 = 0.08% MOS-oligosaccharide / kg of feed and A3 = herbal leuser KI 5 ml / liter). is a 90 day old super chicken breast Chicken Variable observed moisture content, protein content and fat content The data obtained were analyzed by using vocabulary and tested further by Duncan's Multiple Range Test The results showed that treatment (P> 0,05) to the value of water content and protein of super chicken fowl.Average value of water content at each treatment A0 (69,81%), A1 (70,74%), A2 (71,56%) and A3 (71,52%) while mean value of protein A0 (18,95%), A1 (19,61%), A2 (19,01%) and A3 (19,14%)) P <0,05) to the fat content of super chicken flesh, mean of fat content were A0 (2.02%), A1 (1.49%), A2 (1.37%) and A3 (2.0%).