α-Glucan-type exopolysaccharides with varied linkage patterns: Mitigating post-prandial glucose spike and prolonging the glycemic response.

Microbial exopolysaccharides (EPSs) are traditionally known as prebiotics that foster colon health by serving as microbiota nutrients, while remaining undigested in the small intestine. However, recent findings suggest that α-glucan structures in EPS, with their varied α-linkage types, can be hydrolyzed by mammalian α-glucosidases at differing rates. This study explores α-glucan-type EPSs, including dextran, alternan, and reuteran, assessing their digestive properties both in vitro and in vivo. Notably, while fungal amyloglucosidase - a common in vitro tool for carbohydrate digestibility analysis - shows limited efficacy in breaking down these structures, mammalian intestinal α-glucosidases can partially degrade them into glucose, albeit slowly. In vivo experiments with mice revealed that various EPSs elicited a significantly lower glycemic response (p < 0.05) than glucose, indicating their nature as carbohydrates that are digested slowly. This leads to the conclusion that different α-glucan-type EPSs may serve as ingredients that attenuate post-prandial glycemic responses. Furthermore, rather than serving as mere dietary fibers, they hold the potential for blood glucose regulation, offering new avenues for managing obesity, Type 2 diabetes, and other related-chronic diseases.

Macromolecular α-glucans with α-1,3/α-1,4 branching structures produced using dual glycosyltransferases: Elucidation of physicochemical and slowly digestible properties.

Amylosucrase from Neisseria polysaccharea (NpAS) produces the linear amylose-like α-glucans by the elongation property from sucrose, and 4,3-α-glucanotransferase from Lactobacillus fermentum NCC 2970 (4,3-αGT) newly synthesizes the α-1,3 linkages after cleaving the α-1,4 linkages by the glycosyltransferring property. This study focused on the synthesis of high molecular α-1,3/α-1,4-linked glucans by combining NpAS and 4,3-αGT and analyzed their structural and digestive properties. The enzymatically synthesized α-glucans have a molecular weight of >1.6 × 107 g mol-1, and the α-4,3 branching ratios on the structures increased as the amount of 4,3-αGT increased. The synthesized α-glucans were hydrolyzed to linear maltooligosaccharides and α-4,3 branched α-limit dextrins (α-LDx) by human pancreatic α-amylase, and the amounts of produced α-LDx were increased depending on the ratio of synthesized α-1,3 linkages. In addition, approximately 80 % of the synthesized products were partially hydrolyzed by mammalian α-glucosidases, and the glucose generation rates decelerated as the amounts of α-1,3 linkages increased. In conclusion, new types of α-glucans with α-1,4 and α-1,3 linkages were successfully synthesized by a dual enzyme reaction. These can be utilized as slowly digestible and prebiotic ingredients in the gastrointestinal tract due to their novel linkage patterns and high molecular weights.

Structural analysis and prebiotic activity of exopolysaccharide produced by probiotic strain Bifidobacterium bifidum EPS DA-LAIM.

Exopolysaccharide (EPS)-producing Bifidobacterium bifidum EPS DA-LAIM was isolated from healthy human feces, the structure of purified EPS from the strain was analyzed, and its prebiotic activity was evaluated. The EPS from B. bifidum EPS DA-LAIM is a glucomannan-type heteropolysaccharide with a molecular weight of 407-1007 kDa, and its structure comprises 2-mannosyl, 6-mannosyl, and 2,6-mannosyl residues. The purified EPS promoted the growth of representative lactic acid bacteria and bifidobacterial strains. Bifidobacterium bifidum EPS DA-LAIM increased nitric oxide production in RAW 264.7 macrophage cells, indicating its immunostimulatory activity. Bifidobacterium bifidum EPS DA-LAIM also exhibited high gastrointestinal tract tolerance, gut adhesion ability, and antioxidant activity. These results suggest that EPS from B. bifidum EPS DA-LAIM is a potentially useful prebiotic material, and B. bifidum EPS DA-LAIM could be applied as a probiotic candidate. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01213-w.

Production of highly branched α-limit dextrins with enhanced slow digestibility by various glycogen-branching enzymes.

α-Limit dextrins (α-LDx) are slowly digestible carbohydrates that attenuate postprandial glycemic response and trigger the secretion of satiety-related hormones. In this study, more highly branched α-LDx were enzymatically synthesized to enhance the slowly digestible property by various origins of glycogen branching enzyme (GBE), which catalyzes the transglycosylation to form α-1,6 branching points after cleaving α-1,4 linkages. Results showed that the proportion of branched α-LDx in starch molecules increased around 2.2-8.1 % compared to α-LDx from starch without GBE treatment as the ratio of α-1,6 linkages increased after different types of GBE treatments. Furthermore, the enzymatic increment of branching points enhanced the slowly digestible properties of α-LDx at the mammalian α-glucosidase level by 17.3-28.5 %, although the rates of glucose generation were different depending on the source of GBE treatment. Thus, the highly branched α-LDx with a higher amount of α-1,6 linkages and a higher molecular weight can be applied as a functional ingredient to deliver glucose throughout the entire small intestine without a glycemic spike which has the potential to control metabolic diseases such as obesity and type 2 diabetes.

Potential Probiotic Properties of Exopolysaccharide-Producing Lacticaseibacillus paracasei EPS DA-BACS and Prebiotic Activity of Its Exopolysaccharide.

Exopolysaccharide (EPS)-producing Lacticaseibacillus paracasei EPS DA-BACS was isolated from healthy human feces and its probiotic properties, as well as the structure and prebiotic activity of the EPS from this strain were examined. EPS from L. paracasei EPS DA-BACS had a ropy phenotype, which is known to have potential health benefits and is identified as loosely cell-bounded glucomannan-type EPS with a molecular size of 3.7 × 106 Da. EPS promoted the acid tolerance of L. paracasei EPS DA-BACS and provided cells with tolerance to gastrointestinal stress. The purified EPS showed growth inhibitory activity against Clostridium difficile. L. paracasei EPS DA-BACS cells completely inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Aspergillus brasiliensis, as well as showed high growth inhibitory activity against Staphylococcus aureus and Escherichia coli. Treatment of lipopolysaccharide-stimulated RAW 264.7 cells with heat-killed L. paracasei EPS DA-BACS cells led to a decrease in the production of nitric oxide, indicating the anti-inflammatory activity of L. paracasei EPS DA-BACS. Purified EPS promoted the growth of Lactobacillus gasseri, Bifidobacterium bifidum, B. animalis, and B. faecale which showed high prebiotic activity. L. paracasei EPS DA-BACS harbors no antibiotic resistance genes or virulence factors. Therefore, L. paracasei EPS DA-BACS exhibits anti-inflammatory and antimicrobial activities with high gut adhesion ability and gastrointestinal tolerance and can be used as a potential probiotic.

Different physicochemical properties of entirely α-glucan-coated starch from various botanical sources.

Amylosucrase from Neisseria polysaccharea (NpAS) synthesizes α-1,4 glucan polymer from sucrose. In this study, we coated various botanical sources of raw starch with an α-glucan layer generated by NpAS to improve physicochemical properties. Field-emission scanning electron microscopy demonstrated that all surfaces of the starch granules were successfully coated via the NpAS reaction. X-ray diffraction analysis revealed that the crystallinity decreased and the crystal pattern changed to C-type as an amylose layer formed around the surface of the starch granules. Based on rapid viscosity and differential scanning calorimetry analyses, the gelatinization resistance of the α-glucan-coated starch increased owing to decreased viscosity and increased melting temperature. Therefore, the α-glucans coated the starches by enzymatic reactions of various botanical sources; these have applicability in the food and starch industries owing to various physicochemical properties such as enhanced thermostability. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01113-z.

New insights suggest isomaltooligosaccharides are slowly digestible carbohydrates, rather than dietary fibers, at constitutive mammalian α-glucosidase levels.

Isomaltooligosaccharides (IMOs) have been characterized as dietary fibers that resist digestion in the small intestine; however, previous studies suggested that various α-glycosidic linkages in IMOs were hydrolyzed by mammalian α-glucosidases. This study investigated the hydrolysis of IMOs by small intestinal α-glucosidases from rat and human recombinant sucrase-isomaltase complex compared to commonly used fungal amyloglucosidase (AMG) in vitro. Interestingly, mammalian α-glucosidases fully hydrolyzed various IMOs to glucose at a slow rate compared with linear maltooligosaccharides, whereas AMG could not fully hydrolyze IMOs because of its very low hydrolytic activity on α-1,6 linkages. This suggests that IMOs have been misjudged as prebiotic ingredients that bypass the small intestine due to the nature of the assay used. Instead, IMOs can be applied in the food industry as slowly digestible materials to regulate the glycemic response and energy delivery in the mammalian digestive system, rather than as dietary fibers.

Ultrastructure of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea.

Ultrastructural characteristics of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea were investigated using transmission and scanning electron microscopy. The spermiogenesis type of E. bruneus belongs to type I because of nuclear rotation occurrence. The spherical head of late spermatids and spermatozoa ultrastructurally consist of apple-shaped nucleus, nuclear notch (Y or branch-shaped) and nuclear fossa. The midpiece contains the proximal centriole (9+0 microtubule structure), two centrioles connection structure (three osmophilic filaments), the distal centriole (9+0 microtubule structure), an osmophilic basal foot, two necklaces (six or seven osmophilic particles) and osmophilic alar sheets. It contains six to nine spherical mitochondria showing two rows arrangement type. Also a flagellum (9+2 microtubule structure) presents six swelling shaped segments type lateral fins with regular interval. Consequently, ultrastructural features of E. bruneus spermatids and spermatozoa contribute to provide useful systematic characters for taxonomic purpose. It may contribute to study that whether ultrastructural features of cellular organelles (cytoplasmic canal, necklace and lateral fins) correlate to physiological function or not on the duration of motility and survival in fish spermatozoa.

Evaluation of Fertilizing Ability using Frozen Thawed Sperm in the Longtooth Grouper, Epinephelus bruneus.

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: 99.5±0.8%, DMSO 7.5%: 99.5±0.7%, and DMSO 10.0%: 99.6±0.6%). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: 96.2±2.3%, DMSO 7.5%: 95.3±3.6%, 10.0%: 96.6±1.8%) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm (98.4%±0.5) and DMSO concentration level of 5.0% (97.8±0.1%) were not significantly different. However, with 7.5% (97.2±0.6%) and 10.0% DMSO concentrations (95.9±0.2%) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; 66.8±1.8%, HR: 82.0±12.9%) and 2 years (FR; 78.5±14.8%, HR: 79.3±0.6%) cryopreserved sperm were lower than control (FR; 100%, HR: 91.1±3.6%) and 2 days cryopreserved sperm (FR; 99.6±0.6%, HR: 96.6±1.8%). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

Effect of Water Temperature and Salinity on the Fertilized Egg Development and Larval Development of Sevenband Grouper, Epinephelus septemfasciatus.

The fertilized eggs of E. septemfasciatus are spherical and transparent with buoyancy at 790 to 890 µm (average 821.8±2.0 µm) in diameter with 170 to 230 µm oil globules (average 192.9±0.93 µm). Hatching began approximately 46 and 35 hours after fertilization at 22.0°C and 25.0°C water temperature, respectively. The average total length of newly hatched larvae was 1.75±0.03 mm. Most of the yolk and oil globules were absorbed within 3 to 4 days after hatching. The larvae reached 2.48 to 2.72 mm in total length, and their mouths and anuses opened at 3 to 4 days after hatching. In this time, the mouth diameters of the larvae were 0.209 to 0.238 mm. The larvae reached 3.24 to 4.15 mm in total length at 11 to 17 days after hatching, and began to metamorphose at the time the second dorsal and pelvic spines appeared and elongated. The abdominal cavity was densely lined with melanophores. The larvae reached 5.12 mm in total length at 24 days after hatching.

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus).

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.