Theoretical Studies for the Discovery of Potential Sucrase-Isomaltase Inhibitors from Maize Silk Phytochemicals: An Approach to Treatment of Type 2 Diabetes.

A theoretical analysis of the potential inhibition of human sucrase-isomaltase (SI) by flavonoids was carried out with the aim of identifying potential candidates for an alternative treatment of type 2 diabetes. Two compounds from maize silks, maysin and luteolin, were selected to be studied with the structure-based density functional theory (DFT), molecular docking (MDock), and molecular dynamics (MD) approaches. The docking score and MD simulations suggested that the compounds maysin and luteolin presented higher binding affinities in N-terminal sucrase-isomaltase (NtSI) than in C-terminal sucrase-isomaltase (CtSI). The reactivity parameters, such as chemical hardness (η) and chemical potential (µ), of the ligands, as well as of the active site amino acids of the NtSI, were calculated by the meta-GGA M06 functional in combination with the 6-31G(d) basis set. The lower value of chemical hardness calculated for the maysin molecule indicated that this might interact more easily with the active site of NtSI, in comparison with the values of the acarbose and luteolin structures. Additionally, a possible oxidative process was proposed through the quantum chemical calculations of the electronic charge transfer values (∆N) between the active site amino acids of the NtSI and the ligands. In addition, maysin displayed a higher ability to generate more oxidative damage in the NtSI active site. Our results suggest that maysin and luteolin can be used to develop novel α-glucosidase inhibitors via NtSI inhibition.

Intestinal Disaccharidase Deficiency in Adults: Evaluation and Treatment.

PURPOSE OF REVIEW: Disaccharidase deficiency in adults causes carbohydrate malabsorption, resulting in symptoms which significantly overlap with irritable bowel syndrome (IBS). This article discusses the diagnosis and treatment of disaccharidase deficiency within the context of recent literature. RECENT FINDINGS: Disaccharidase deficiency in adults is more common than previously thought, which includes lactase, sucrase, maltase and isomaltase enzymes. Deficiency in disaccharidases, which are produced by the intestinal brush border, will interfere with the breakdown and absorption of carbohydrates and may result in abdominal pain, gas, bloating and diarrhea. Patients deficient in all 4 disaccharidases are known as having "pan-disaccharidase" deficiency, which has a distinct phenotype with more reported weight loss than patients deficient in one enzyme. IBS patients who do not respond to low FODMAP dietary restriction may have undiagnosed disaccharidase deficiency and may benefit from testing. Diagnostic testing methods are limited to duodenal biopsies, which is the gold standard, and breath testing. Dietary restriction and enzyme replacement therapy have been shown to be effective treatments in these patients. Disaccharidase deficiency is an underdiagnosed condition in adults with chronic GI symptoms. Patients who do not respond to traditional treatment strategies for DBGI may benefit from testing for disaccharidase deficiency. Further studies delineating the distinctions between disaccharidase deficient patients and those with other motility disorders are needed.

Enzymatic glucosylation of polyphenols using glucansucrases and branching sucrases of glycoside hydrolase family 70.

Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.

Bensulfuron-Methyl, Terbutylazine, and 2,4-D Butylate Disturb Plant Growth and Resistance by Deteriorating Rhizosphere Environment and Plant Secondary Metabolism in Wheat Seedlings.

Frequent and improper use of herbicides disrupts a plant's metabolism, causing oxidative stress that degrades crop quality. However, few studies have examined the inhibitory effects of herbicides on plant growth and defense mechanisms in terms of their impact on soil quality and crop rhizosphere. Therefore, the current study investigated the detrimental impacts of six typical and multilevel herbicides on the microbial community and signal molecules in the soil as well as on the levels of hormones and secondary metabolites in wheat seedlings. Interestingly, bensulfuron-methyl, terbutylazine (TBA), and 2,4-D butylate significantly induced oxidative damage while reducing the number of phytohormones (salicylic acid and jasmonic acid) and secondary metabolites (tricin, quercetin, and caffeic acid) in the roots and leaves compared with the controls, isoproturon, fenoxaprop-p-ethyl, and pretilachlor. At twice the recommended levels (2×), they also decreased the microbial α diversity and, in particular, the abundance of Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Bacteroidia, Verrucomicrobia, Bacilli, Acidimicrobiia, Deltaproteobacteria, and Gemmatimonadetes by disrupting the level of enzymes (e.g., urease and sucrase) and metabolites (indole-3-acetic acid, salicylic acid, apigenin, 4-hydroxybenzoic acid, DIMBOA, and melatonin) in the rhizosphere soil. Overall, significant exposure to herbicides may inhibit wheat growth by disturbing the microbial composition in the rhizosphere soil and the distribution of secondary metabolites in wheat seedlings.

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection.

Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by α-amylases and α-glucosidases to produce monosaccharides for absorption. Inhibition of these enzymes is the major activity of the drugs acarbose and miglitol, which are used to manage diabetes. Furthermore, delaying carbohydrate digestion via inhibition of α-amylases and α-glucosidases is an effective strategy to blunt blood glucose spikes, a major risk factor for developing metabolic diseases. Here, we present an in vitro protocol developed to accurately and specifically assess the activity of α-amylases and α-glucosidases, including sucrase, maltase and isomaltase. The assay is especially suitable for measuring inhibition by compounds, drugs and extracts, with minimal interference from impurities or endogenous components, because the substrates and digestive products in the enzyme activity assays are quantified directly by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human α-amylase to assess starch hydrolysis with maltoheptaose as the substrate, and with brush border sucrase-isomaltase (with maltase, sucrase and isomaltase activities) derived from differentiated human intestinal Caco-2(/TC7) cells to assess hydrolysis of disaccharides. The wet-lab assay takes ~2-5 h depending on the number of samples, and the HPAE-PAD analysis takes 35 min per sample. A full dataset therefore takes 1-3 d and allows detection of subtle changes in enzyme activity with high sensitivity and reliability.

Soybean isoflavones modulate gut microbiota to benefit the health weight and metabolism.

Soybean isoflavones (SIs) are widely found in food and herbal medicines. Although the pharmacological activities of SIs have been widely reported, their effects on the intestinal microecology of normal hosts have received little attention. Five-week-old Kunming (KM) mice were administered SIs (10 mg/kg/day) for 15 days. Food intake, body weight, and digestive enzyme activity were measured. Small intestine microbiota, including lumen-associated bacteria (LAB) and mucosa-associated bacteria (MAB), were analyzed using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Short-chain fatty acids (SCFAs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that the mice that consuming SIs showed a higher food intake but a lower body weight gain rate than that of normal mice. Sucrase, cellulase, and amylase activities reduced, while protease activity increased after SIs intervention. Moreover, SIs increased the intestinal bacterial diversity in both LAB and MAB of normal mice. The composition of LAB was more sensitive to SIs than those of MAB. Lactobacillus, Adlercreutzia, Coprococcus, Ruminococcus, Butyricicoccus, and Desulfovibrio were the differential bacteria among the LAB of mice treated with SIs. In addition, acetic acid, valeric acid, isobutyric acid, isovaleric acid, and caproic acid decreased, while butyric acid and propionic acid increased in the mice treated with SIs. Taken together, SIs are beneficial for weight control, even in short-term interventions. The specific mechanism is related to regulating the gut microbiota, changing digestive enzyme activities, and further affecting carbohydrate absorption and metabolism.

Effects of urease-producing bacteria and eggshell on physiological characteristics and Cd accumulation of pakchoi (Brassica chinensis L.) plants.

Soil cadmium (Cd) contamination resulting from anthropogenic activity poses severe threats to food safety and human health. In this study, a pot experiment was performed to evaluate the possibility of using urease-producing bacterium UR21 and eggshell (ES) waste for improving the physiological characteristics and reducing Cd accumulation of pakchoi (Brassica chinensis L.) plants. UR21 has siderophore and IAA production ability. The application of UR21 and ES individually or in combination could improve the root and shoot length, and fresh and dry weight of pakchoi plants under Cd stress. In Cd + ES + UR21-treated plants, the dry weight of shoot and root were increased by 61.54% and 72.73%, respectively. The chlorophyll a, chlorophyll b, and carotenoid content were increased by 52.19%, 42.95%, and 95.56% in Cd + ES + UR21-treated plants. Meanwhile, the H2O2 and MDA content were decreased while the SOD and POD activity were increased, and an increase of soluble protein level in pakchoi plants was observed under Cd + ES + UR21 treatment. Importantly, eggshell and UR21 alone or in combination induced a decline of Cd content in pakchoi plants, especially that Cd + ES + UR21 treatment decreased Cd content in shoot and root by 26.96% and 42.91%, respectively. Meanwhile, the soil urease and sucrase activities were enhanced. Generally, the combined application of ureolytic bacteria UR21 and eggshell exhibited better effects than applied them individually in terms of alleviating Cd toxicity in pakchoi plants. Our findings may give a unique perspective for an eco-friendly and sustainable strategy to remediate heavy metal-polluted soils.

S-layer protein 2 of vaginal Lactobacillus crispatus 2029 enhances growth, differentiation, VEGF production and barrier functions in intestinal epithelial cell line Caco-2.

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.

Horizontal Gene Transfer and Gene Duplication of ß-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits.

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect ß-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of ß-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of ß-fructofuranosidase were found in Coleoptera and Hymenoptera. ß-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran ß-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of ß-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of ß-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens.

The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

Pre-weaning adaptation responses in piglets fed milk replacer with gradually increasing amounts of wheat.

Hyperprolific sows rear more piglets than they have teats, and to accommodate this, milk replacers are often offered as a supplement. Milk replacers are based on bovine milk, yet components of vegetable origin are often added. This may reduce growth, but could also accelerate maturational changes. Therefore, we investigated the effect of feeding piglets a milk replacer with gradually increasing levels of wheat flour on growth, gut enzyme activity and immune function compared with a diet based entirely on bovine milk. The hypothesis tested was that adding a starch component (wheat flour) induces maturation of the mucosa as measured by higher digestive activity and improved integrity and immunity of the small intestines (SI). To test this hypothesis, piglets were removed from the sow at day 3 and fed either a pure milk replacer diet (MILK) or from day 11 a milk replacer diet with increasing levels of wheat (WHEAT). The WHEAT piglets had an increased enzyme activity of maltase and sucrase in the proximal part of the SI compared with the MILK group. There were no differences in gut morphology, histopathology and gene expression between the groups. In conclusion, the pigs given a milk replacer with added wheat displayed immunological and gut mucosal enzyme maturational changes, indicatory of adaptation towards a vegetable-based diet. This was not associated with any clinical complications, and future studies are needed to show whether this could improve responses in the subsequent weaning process.

Extracellular polysaccharides produced by bacteria of the Leuconostoc genus.

Structurally diverse biopolymers, including extracellular polysaccharides (EPS), synthesized by bacteria can possess physicochemical and functional properties that make them important products of microbial synthesis with a broad and versatile biotechnological potential. Leuconostoc spp. belongs to the group of lactic acid bacteria as one of the predominant members and are relevant not only in varied food fermentations, but also can be employed in the production of extracellular homopolysaccharides (HoPS) such as α-glucans (dextran, alternan) and ß-fructans (levan,inulin) from the sucrose-containing substrates. EPS are synthesized by specific Leuconostoc spp. extracellular glycosyltransferases [dextran sucrase, alternansucrase (ASR)] and fructosyltransferases (levansucrase, inulosucrase) and enzymatic reactions can be performed in whole culture systems as well as using cell-free enzymes. Both α-glucans and ß-fructans have a wide range of properties, mostly depending on their pattern of linkages, which, although differing in some respects, make suitable prerequisites for their versatile application in many fields, especially in the food industry and biomedicine. As a rule, these properties (polymer type, molecular mass, rheological parameters), as well as the overall EPS yield, are strain-specific for the selected producers and depend to a large extent on the nutritional and growth conditions used, which in many cases remain not sufficiently optimized for Leuconostoc spp. This review summarizes the current knowledge on the potential of Leuconostoc spp. to produce commercially relevant EPS, including information on their applications in various fields, producer strains, production methods and techniques used, selected conditions, the productivity of bioprocesses as well as the possible use of renewable resources for their development.

Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose.

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with ß-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.

Development of Slowly Digestible Starch Derived α-Glucans with 4,6-α-Glucanotransferase and Branching Sucrase Enzymes.

Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

Sustained effects of resistant starch on the expression of genes related to carbohydrate digestion/absorption in the small intestine.

Resistant starch (RS) consumption has beneficial effects on health, such as reduced postprandial blood glucose levels. In this study, we evaluated the effect of a 14-day diet containing RS on α-glucosidase activity and the expression of genes related to carbohydrate digestion/absorption in rats. We examined whether the effects of RS persist when the rats were shifted to a control diet. The results suggest that RS consumption reduces α-glucosidase activity and Mgam, Si and Sglt1 mRNA levels in the proximal jejunum. In addition, RS consumption appeared to influence the serum GIP level, up to 2 days after the animals were shifted to a control diet. To our knowledge, this is the first report that RS has a sustained effect on gut hormone expression and the expression of genes related to carbohydrate digestion/absorption in the proximal jejunum.

The growth performance, intestinal digestive and absorptive capabilities in piglets with different lengths of small intestines.

The small intestine is an important digestive organ and plays a vital role in the life of a pig. We tested the hypothesis that the length of the small intestine is related to growth performance and intestinal functions of piglets. A total of 60 piglets (Duroc × Landrace × Yorkshire), weaned at day 21, were fed an identical diet during a 28-day trial. At the end of the study, all piglets were sacrificed, dissected and grouped according to small intestine lengths (SILs), either short small intestine (SSI), middle small intestine (MSI) or long small intestine (LSI), respectively. Positive relationships between SIL and BW, average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratios (G : F) were observed. Final BW, ADG, ADFI and G : F significantly increased (P < 0.05) in MSI and LSI piglets compared with SSI piglets. Short small intestine and MSI had greater jejunal mucosa sucrase and alkaline phosphatase activities (P < 0.05) than LSI piglets. The mRNA level of solute carrier family 2 member 2 (Slc2a2) in the jejunal mucosa of SSI piglets was the greatest. The MSI piglets had a greater (P < 0.05) ileal villus height than other piglets and greater (P < 0.05) villus height-to-crypt depth ratios than LSI piglets. However, the LSI piglets had a greater (P < 0.05) ileal crypt depth than SSI piglets. No significant differences in duodenal, jejunal, caecal and colonic morphologies were detected among the groups. Moreover, luminal acetate, propionate, butyrate and total short-chain fatty acid contents were greater (P < 0.05) in SSI and MSI piglets than those in LSI piglets. In addition, there was greater serum glucose concentration in MSI piglets than other piglets. Serum albumin concentration in SSI piglets was the lowest. In conclusion, these results indicate that SIL was significantly positively associated with growth performance, and in terms of intestinal morphology and mucosal digestive enzyme activity, the piglets with a medium length of small intestine have better digestion and absorption properties.

Whole pearl millet feeding does not impair performance and nutrient digestibility in 28-day-old broiler chickens.

The effects of varying inclusion levels of whole grain millet in millet-soya bean-based diets on growth performance, gizzard development, digesta characteristics and nutrient digestion in broiler chicken were investigated. Starter (0-14 days) and grower (15-28 days) broiler chicken diets containing pearl millet at 500 and 540 g/kg diet, respectively, were formulated. The diets comprised of 0%, 20%, 40%, 60%, 80% and 100% of millet incorporated as whole grain. One-day-old unsexed Arbor Acres Plus chicks (n = 540) were allotted to the experimental diets in a completely randomized design with the diets and water provided ad libitum for 28 days. Each treatment was replicated seven times, and each replicate had 12 chicks. Results showed that daily live weight gain and feed conversion ratio of chickens on the whole millet grain diets compared favourably with chicken on the control in both starter and grower phases, while feed intake reduced quadratically (p < .05) with increased whole grain millet levels in the starter phase. Morphological and structural characteristics of the gizzard and small intestine and intestinal digesta pH and viscosity were also unaffected (p > .05) by whole grain millet inclusion levels. However, the weight of intact millet grain in gizzard increased linearly (p < .001) with whole grain millet inclusion in the diets. Dietary whole grain millet inclusion also consistently lowered (p < .05) jejunal and ileal maltase and sucrase activities, but did not influence (p > .05) pancreatic amylase activity. Ileal crude protein and starch digestibility increased, while ileal energy digestibility decreased significantly (p < .05) with whole grain millet inclusion. Whole grain millet inclusion in broiler starter and grower diets up to 500-540 g/kg did not negatively impact on broiler chicken performance.

MiR-26a and miR-26b downregulate the expression of sucrase-isomaltase enzyme: A new chapter in diabetes treatment.

Type II diabetes is a metabolic disease that has affected 460 million people around the globe and become a heavy burden on health care system. Diabetic patients suffer from hyperglycemia and hyperinsulinemia which can damage vital organs in body like heart, kidneys, eyes and nervous system. Different strategies have been introduced to control or lessen these diabetic complications in which one of the most promising approaches is the inhibition of intestinal sucrase-isomaltase (SI). Inhibition of this enzyme will block the release of glucose into bloodstream and lead to reduced postprandial hyperglycemia. MicroRNAs are small regulatory molecules that play critical roles in different cellular pathways and molecular mechanisms. It is proved that microRNAs have significant effects on cellular mechanisms involved in diabetes and can be used as biomarkers for diagnosis of this metabolic disease. Based on bioinformatics analysis miR-26a and miR-26b can interact with a conserved 3'-UTR region of SI mRNA which lead to a hypothesis that these miRs may have negative regulatory effect on this enzyme. In this study, we investigated the impact of high glucose conditions on expression of sucrase-isomaltase, miR-26a and miR-26b in caco-2 cell line. It is proved that in a simulated diabetic condition there is a reverse correlation between the expression pattern of these miRs and SI. QRT-PCR method was used to evaluate the expression of our target molecules. Interestingly, transfection of miR-26a and miR-26b in caco-2 cell line reduced the transcription of SI mRNA and decreased the sucrase and maltase activity of its active sites. To sum up, our results demonstrate the first evidence of the significant effect of miR-26a and miR-26b on SI expression and activity. We proved that these microRNAs may directly inhibit this enzyme and can be used as a new scaffold in search of finding novel treatments for type II diabetes.

Detailed Structural Characterization of Glucans Produced by Glucansucrases from Leuconostoc citreum TMW 2.1194.

The water kefir organism Leuconostoc citreum TMW 2.1194 forms highly branched dextrans with O3- and O4-bound side chains. To obtain detailed information on the enzymatic synthesis of these polymers, the four glucansucrases encoded by Leuconostoc citreum TMW 2.1194 were cloned, heterologously expressed, and used for polysaccharide production. Molecular and macromolecular structure of the synthesized glucans were analyzed by methylation analysis, two-dimensional NMR spectroscopy, oligosaccharide analysis after partial hydrolysis, and asymmetric flow field-flow fractionation. It was demonstrated that two glucansucrases form insoluble glucans with variously branched dextran sections and varying portions of consecutive, 1,3-linked glucose units. In contrast, the other two glucansucrases synthesized O3- (Lc6255) and O4-branched (Lc1785) soluble dextrans. Analysis, isolation, and characterization of enzymatically liberated oligosaccharides showed that monomeric and elongated side chains are abundant in both polysaccharides. From the structures and size distributions it was concluded that Lc1785 is mainly responsible for synthesis of fermentatively produced soluble dextrans.