Super Ohtaka® (fermented beverage of plant extracts) is prepared from approximately 50 vegetables and fruits. Natural fermentation is primarily performed using lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). An unidentified oligosaccharide was isolated from this beverage using carbon-Celite® column chromatography and high performance liquid chromatography. The oligosaccharide structure was confirmed by MALDI-TOF MS and NMR measurements. This oligosaccharide was identified as β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, it was newly found from natural source. We speculated that this oligosaccharide was generated or enhanced during the fermentation, and was barely degraded by artificial gastric juice and rat intestinal enzymes, although it was slightly hydrolyzed by pig pancreatic enzymes.
Sweet potato (Ipomoea batatas (L.) Lam) is a widely cultivated crop valued for its nutritional and functional properties. In Japan, consumers particularly prioritize the sweetness and texture of baked sweet potatoes. Among the free sugars in cooked sweet potatoes, maltose is predominant, generated by β-amylase-mediated hydrolysis of gelatinized starch during heating. This study investigated the relationship between starch gelatinization properties and β-amylase activity in ten soggy-type sweet potato cultivars (Aikomachi, Annouimo, Beniharuka, Benimasari, Fukumurasaki, Himeayaka, Karayutaka, Kenroku, Silksweet, and Tamayutaka) and one intermediate-type standard cultivar (Kokei 14), all cultivated in the same farm. We evaluated free sugar content, starch gelatinization characteristics, amylopectin chain-length distribution, and β-amylase activity. A positive correlation was observed between the proportion of DP13-24 chains in amylopectin and the onset (T o) and peak (T p) gelatinization temperatures, as measured by differential scanning calorimetry (DSC). Himeayaka, with the highest maltose content, also showed the highest β-amylase activity. In contrast, Silksweet had similar enzyme activity but lower maltose levels. These findings indicate that starch gelatinization behavior is key to the sweetness and texture of cooked sweet potatoes. Principal component analysis grouped the cultivars into three categories: typical soggy-type, slightly soggy-type, and near intermediate-type. These insights can guide the breeding and selection of cultivars aligned with consumer preferences.
Gum arabic (GA) is a highly branched arabinogalactan-protein complex widely used in food and pharmaceutical industries. Its complex side chains contain L-arabinofuranose residues linked via α-(1→3) and α-(1→4) bonds, which represent the final structural barrier to complete enzymatic degradation of GA. Here, we isolated and characterized two complementary α-L-arabinofuranosidases, FoAF2 and FoAF3, from Fusarium oxysporum 12S. FoAF2, a glycoside hydrolase (GH) family 54 enzyme, preferentially cleaves α-(1→3)-arabinosyl residues while exhibiting weak activity toward α-(1→4) linkages at high enzyme concentrations. In contrast, the GH43_34 enzyme FoAF3 displays strict specificity for α-(1→4)-arabinosyl residues but requires prior removal of neighboring α-(1→3) substituents for efficient catalysis. Structural modeling using AlphaFold 3 revealed that the constrained catalytic pocket of FoAF3 is highly sensitive to steric hindrance from adjacent branches, explaining its dependence on sequential FoAF2 action for complete debranching. This two-enzyme system functions similarly to the bifunctional Bifidobacterium BIAraE, but achieves the same effect using separate enzymes rather than fused domains. Combined with previously characterized F. oxysporum enzymes, FoAF2 and FoAF3 complete a comprehensive toolkit enabling systematic GA degradation from complex side chains to monosaccharides. These findings provide molecular insights into the mechanisms underlying α-L-arabinofuranosidase specificity and establish a foundation for the enzymatic modification of GA for industrial applications.
The influence of the genetic background of rice on its suitability for brewing shochu and sake (Japanese alcoholic beverages) has not been clarified. This study investigates the effects of starch biosynthesis-related genes (Wx and Alk) on the water absorption capacity of commercially available rice samples and thus examines the possibility of using rice genotype as a predictor of suitability for shochu and sake brewing. The 24 examined rice samples were classified into four genotypes: Alk/Wx a , alk/Wx a , alk/Wx b , and alk/wx. No significant differences were observed among these genotypes in terms of nonstarch properties such as grain morphology, as well as the water absorption ratio during the initial absorption phase. However, after 1 h of absorption, significant differences in terms of this ratio were observed (Alk/Wx a < alk/Wx a < alk/Wx b < alk/wx). Starch structural analysis revealed that amylose content increased in the order of alk/wx < alk/Wx b < alk/Wx a ≈ Alk/Wx a and the proportion of short amylopectin chains increased in the order of Alk/Wx a < alk/Wx a ≈ alk/Wx b . Correlation analysis revealed a significant negative correlation between the water absorption ratio at 20-120 min and amylose content. The Wx genotype, through its influence on amylose content, was found to play a major role in determining short-term and steamed-rice water absorption ratios regardless of rice type, thus being a promising descriptor of rice suitability for shochu and sake brewing. Thus, our work provides fundamental insights for cultivar breeding and developing brewing methods tailored to specific rice varieties.
A water-soluble sodium β-(1→4)-polyglucuronate (or sodium cellouronate, Na-CUA) was prepared from regenerated cellulose by catalytic oxidation in water at pH ~10. Digestive behavior of Na-CUA was evaluated in vitro using three enzymes, α-amylase, pepsin, and pancreatin in water at 80 or 40 °C for 30 or 60 min. The results showed that 98.95 % Na-CUA remained undigested, indicating that Na-CUA is categorized as water-soluble dietary fibers.
D-Glucosamine hydrochloride (GlcN), a monomer produced by the hydrolysis of chitosan, is a dietary supplement used worldwide to mitigate cartilage degeneration. Previous reports have shown that some dietary glucosamine migrates to the colon. However, the effect of glucosamine alone on colonic microbiota and bowel movements remains poorly understood. In this study, we evaluated the effect of glucosamine on the growth of 46 dominant human colonic bacterial species and 24 other important bacteria in vitro. Among the 70 gut bacterial species tested, the growth of 57 (81 %) was significantly enhanced by 0.5 g/L GlcN, with the most prominent growth activity (> 5-fold) observed in Anaerotruncus colihominis, Pseudoflavonifractor capillosus, and Roseburia hominis. These results indicate that a wide range of the tested gut bacteria can utilize GlcN, similar to the effect of conventional dietary fiber in improving bowel function. Next, we conducted an open-label, single-arm trial involving 29 healthy individuals to determine the effects of 1,500 mg GlcN/day, a commonly used dose. Stool color significantly changed during the 2 weeks of GlcN intake from brown to ocher (p < 0.01), suggesting enhanced colonic fermentation. The stool odor and the sensation of incomplete evacuation improved significantly (p < 0.05). Numerical measurements of bowel movements revealed significant increases in stool volume, defecation frequency, and the number of days of defecation during GlcN intake (p < 0.001). Thus, dietary glucosamine may stimulate gut microbiota growth in the colon and promote bowel movements. This study was registered with the University Hospital Medical Information Network (regd. no. UMIN000056757).
Glycoside hydrolase family 32 (GH32) enzymes play key roles in fructooligosaccharide metabolism in gut bacteria. In this study, a GH32 enzyme (GenBank code, GFO85652) containing carbohydrate binding module 66 (CBM66) from the gut bacterium Anaerostipes butyraticus (AbFEH) was heterologously expressed in Escherichia coli. We constructed an expression plasmid that does not contain sequences for the N-terminal signal peptide and the C-terminal region potentially involving cell-wall binding. The enzyme obtained (AbFEH∆C) was purified and characterized. Thin-layer chromatography and high-performance liquid chromatography analyses revealed that AbFEH∆C produced fructose from all the substrates, sucrose, 1-kestose, inulin, and levan, and intermediate oligosaccharide products were not observed. The ratio of activities towards sucrose, 1-kestose, nystose, inulin, and levan was 6:100:83:8:95 under the conditions of this study. A region containing M and CBM66 domains was further removed from AbFEH∆C, and the activities for both 1-kestose and levan of this mutant enzyme were about 400-fold lower than those of AbFEH∆C. Kinetic analysis indicated a low K m value for levan, while requiring higher substrate concentrations for 1-kestose and sucrose. Comparison of the predicted structure of AbFEH with crystal structures of some GH32 enzymes indicated that residues at subsite -1 were almost completely conserved, while some key residues found in GH32 enzymes were not present at subsites +1 and +2 in AbFEH. These observations suggest that AbFEH functions as fructan exohydrolase that exhibits low sucrose-hydrolyzing activity.
Perilla frutescens is a popular aromatic edible plant. The amount of Perilla residues produced by food or pharmaceutical industry is increasing because of the high demand for this plant. At present, most Perilla residues are incinerated, but there is increasing interest in using these materials as a biomass resource. In this study, an alkaline pretreatment to remove lignin from Perilla residues was optimized, and the ash, lignin, and total sugar contents of the treated materials were determined to evaluate their biomass potential. The optimum alkaline pretreatment for Perilla residues was 0.25 M NaOH at 121 °C for 60 min. The lignin and total sugar contents of the alkaline-pretreated Perilla residues were comparable to those reported for grain straw. These results suggest that alkaline-pretreated Perilla residues have high potential as biomass. With dual aims to reduce the volume of Perilla residues and to effectively use this resource, bacteria capable of decomposing Perilla seed shells after alkaline pretreatment were isolated from environmental samples. A total of 66 strains of degraders were isolated, of which one strain (strain SW8) was identified as Klebsiella aerogenes or Raoultella ornithinolytica with both cellulase and xylanase activities. Strain SW8 grew well at 25-35 °C with Perilla seed shells as the sole carbon source. Strain SW8 was identified as a useful bacterium to reduce the volume of, and effectively utilize, Perilla residues.
Filamentous fungi use various enzymes to degrade cellulose, some of which contain cellulose-binding domains (CBDs), most of which belong to carbohydrate-binding module family 1 (CBM1). We recently identified the novel fungal CBD, CBM104, from Gloeophyllum trabeum. Reportedly, CBM104 specifically binds to native crystalline cellulose, not to amorphous or artificially modified crystalline cellulose, exhibiting a unique adsorption characteristic. To gain further insights into CBM104, the adsorption properties of six different CBM104s, each appended to a different catalytic domain, were investigated. The adsorption tests illustrated that all CBM104s predicted to possess a three-dimensional structure in which two α-helices were crosslinked by disulfide bonds specifically adsorbed onto cellulose I. Conversely, CBM104 lacking these disulfide bonds failed to adsorb onto any form of cellulose used in this study, suggesting the importance of the fixed pair of α-helices for specific binding to cellulose I. To identify CBM104 homologs in which the disulfide bonds are conserved, a homology search was performed against fungal genomes, resulting in 144 hits. These CBM104 homologs were primarily appended to auxiliary activities (AA) family 9 or to domains that work cooperatively with AA9 enzyme. CBM104s were found only in certain orders of Agaricomycetes, and the majority of these fungi are suggested to have the ability to degrade plant cell walls. These results suggest that some Agaricomycetes utilize plant cell wall degradation systems involving CBM104-attached proteins. This study provides detailed insights into the structural factors involved in the adsorption capacity of CBM104, as well as its phylogenetic distribution.
Branching enzymes (BEs) are essential for defining the branching patterns of glycogen and starch by catalyzing the formation of α-1,6-glucosidic linkages. While most cyanobacteria accumulate glycogen, some species, such as Crocosphaera subtropica ATCC 51142, produce an insoluble branched α-glucan known as cyanobacterial starch. This strain possesses three BE isozymes: cceBE1, cceBE2, and cceBE3. Our previous studies demonstrated that cceBE1 and cceBE2 share similar enzymatic properties and that a "stopper structure" contributes to their preferential production of short chains with a degree of polymerization (DP) of 6 and 7. In contrast, cceBE3 produces small amounts of short (DP5-12) and long (DP30-40) chains and lacks the amino acid sequence corresponding to the stopper structure. To investigate the role of the stopper structure, we constructed a deletion mutant of cceBE1 lacking the stopper structure and characterized its enzymatic properties. The mutant retained catalytic activity but lost the ability to selectively produce glucan chains with DP6 and 7 (transferred chains), providing direct evidence for the stopper structure's role in regulating product chain length. Furthermore, we determined the crystal structure of cceBE3, confirming the absence of the stopper structure. We also identified a unique structural feature in cceBE3, termed subdomain B, located within the predicted substrate-binding site. Deletion of subdomain B led to increased production of short chains (DP3-7), suggesting its involvement in substrate binding and the determination of product specificity. These findings reveal structural determinants of product specificity in cyanobacterial BEs and offer a strategy for engineering BEs to produce novel starch-based materials.
Short linear maltodextrin (SLMD) is a novel maltodextrin synthesized from starch using the combined enzymatic actions. SLMD exhibits unique aggregating and solidifying properties. In this study, we prepared SLMD aggregates, solidified materials under various conditions, and investigated their crystallinity. Aggregates formed in the 50 % SLMD solution at 4 °C (AGG-4), 25 °C (AGG-25), and 50 °C (AGG-50) showed clear X-ray diffraction peaks. A B-type crystal diffraction pattern was observed for AGG-4, whereas an A-type pattern was observed for AGG-25 and AGG-50. Kneading SLMD with a limited quantity of water produced solidified slurries at 4 °C (SS-4) and 25 °C (SS-25). SS-4 exhibited a C-type structure with low crystallinity, whereas SS-25 showed an A-type structure with high crystallinity. In addition, B-type crystals were detected in the aggregates in the emulsions solidified with vegetable oil. Therefore, SLMD crystals occurred in different forms in the aggregates or solidified bodies under various conditions.
Alginate, a heteropolysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M), comprises poly-G, poly-M, and mixed poly-MG regions. Alginate lyases, classified within the polysaccharide lyase (PL) family, degrade alginate into unsaturated saccharides via β-elimination. Due to the abundance of alginate in brown algae, various marine bacteria produce alginate lyases for its assimilation. Recently, alginate lyases have also been identified in gut bacteria such as those of the genus Bacteroides. In this study, we purified an alginate lyase from enrichment culture supernatants containing alginate, using a human fecal sample, and isolated B. xylanisolvens strain MK6803, which can grow on alginate as a sole carbon source-unlike the type strain B. xylanisolvens XB1A. Draft genome sequencing of strain MK6803 revealed an alginate-metabolizing gene cluster encoding three alginate lyases belonging to PL6_1, PL17_2, and PL38, along with a putative oxidoreductase. This gene cluster was shared with B. ovatus CP926 and B. xylanisolvens CL11T00C41, but not with the type strain XB1A. Bacteroides species lacking this gene cluster exhibited no alginate assimilation, even if they possessed genes encoding one or more of the three alginate lyases. This suggests that the presence of the putative oxidoreductase, alongside the lyases, is essential for alginate assimilation in Bacteroides species. Phylogenetic analysis indicated horizontal gene transfer within the genus Bacteroides. These findings highlight the role of alginate metabolism in the adaptation of human gut microbiota.
Metagenomics can be used to obtain sequence information on putative genes in a microbial community. However, it is difficult to identify genes with specific functions among the numerous predicted genes. In this study, we attempted to identify genes induced in cultured microbes by the addition of saccharides using metagenomic and metatranscriptomic analyses. A mixture of arabinoxylan and its derived oligosaccharides was used as the inducer in this study. Some genes were highly induced in the presence of additive saccharides and formed gene clusters for the utilization of additive saccharides, suggesting that metatranscriptomic and metagenomic analyses are useful for analyzing carbohydrate-responsive genes in microbial communities and screening novel carbohydrate-active enzymes.
Rice bread, a gluten-free alternative to wheat bread, often suffers from poor texture due to inadequate viscoelasticity during baking. This study aimed to propose a baking method for pure rice bread by investigating the effects of hydroxypropylated potato starches (HPPS) with varying degrees of substitution on the baking performance of rice batter. First, the particle size distribution and thermal properties of HPPS were analyzed to characterize their fundamental attributes. Rice bread was then prepared using each type of HPPS, and their foaming properties were assessed. Additionally, dynamic viscoelastic measurements were performed during heating to assess rheological changes during baking. Results showed that a higher degree of substitution in HPPS reduced in the gelatinization onset temperature. Moreover, HPPS addition improved the cross-sectional structure of the rice bread. Notably, highly substituted HPPS suppressed the formation of large internal voids caused by bubble coalescence. These findings suggest that HPPS with a high degree of substitution enhances the overall quality of rice bread.
Root stubble represents a "hidden" (unrecovered) biomass in agriculture. This study investigated its potential as a source of fermentable sugars using the high-yielding rice cultivar Hokuriku 193. The ripened whole plant was disassembled into panicle, straw, and root stubble. The root stubble was further divided into the aboveground part (AP), basal part (BP), and root part (RP), with AP and BP together accounting for 81.8 % (w/w) of the root stubble. In comparison with the straw, AP contained more starch- and β-1,3-1,4-glucan; BP had more starch and ash; RP had more lignin and ash. The total amount of glucan and xylan in root stubble is equivalent to 61.8 % of that found in straw. Following gas-phase HCl pretreatment and the subsequent enzymatic saccharification, sugar yields from AP and BP exceeded 70 %. These results demonstrate that root stubble has strong potential as a new feedstock for saccharification substituting straw.
The purpose of this study was to clarify the effect of surfactant-added oils on the glass transition as a function of water content, fracture behavior, color, and crude oil content of the fried coatings (post-fried wheat flour-water mixture) obtained under various frying conditions (at 150-180 °C for 1.0-4.5 min). Polyglycerol oleic acid esters having hydrophile-lipophile balances of 7.4 (hydrophobic) and 13.3 (hydrophilic) were employed, and canola oils with and without 0.5 % (w/w) surfactants were used for frying. The samples obtained at 170 °C became glassy after frying times of 1.4 min, 1.9 min, and 2.4 min in the hydrophilic surfactant-added oil, hydrophobic surfactant-added oil, and surfactant-free oil, respectively. The glassy samples showed brittle fracture behavior, and the maximum fracture force for the glassy samples obtained using the surfactant-added oils was lower than that obtained using surfactant-free oil. The frying time to obtain glassy samples decreased with increasing frying temperature, and the frying time in the hydrophilic surfactant-added oil was reduced by 60-80 % compared to the surfactant-free oil. When the browning color of the glassy samples obtained for the shortest frying time was compared at each temperature, the samples fried in the hydrophilic surfactant-added oil showed less browning than those fried in the surfactant-free oil due to the reduction of frying time. There were no significant differences in the crude oil content between surfactant-free oil (69.9-105.7 g/100 g-defatted DM, dry matter) and the hydrophilic surfactant-added oil (78.6-115.5 g/100 g-defatted DM) at each frying temperature (except for 150 °C).
Cellouronate, β-1,4-glucuronan, is synthesized from regenerated cellulose via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical-mediated oxidation. Human intestinal bacteria were cultured in a medium containing cellouronate to evaluate its utilization. These experiments showed Bacteroides luhongzhouii to grow well in this medium. Several putative cellouronate lyases belonging to polysaccharide lyase family 38 from B. luhongzhouii were identified. Among these candidate enzymes, BlCUL1, which displayed the most similarity to authentic cellouronate lyases, was heterologously expressed and characterized. The recombinant BlCUL1 (rBlCUL1) showed the highest activity at pH 8.0 and was deactivated by treatment at pH 3.0 for 24 h or heating above 50 °C for 10 min. Moreover, the activity of rBlCUL1 was enhanced in the presence of Mg2+, Ca2+, or EDTA, but suppressed by Al3+ and completely inactivated by Fe3+. Analysis of the final reaction mixture generated from the rBlCUL1 mediated degradation of cellouronate revealed an oligomer as the main product, but the monomer was barely detectable. This study is the first to report and characterize a cellouronate lyase from human intestinal bacteria.
Western honeybee (Apis mellifera) α-glucosidase III (HBG-III), which is secreted from the hypopharyngeal glands of honeybees, plays a role in converting nectar into honey. Consequently, hypothesizing that HBG-III is a suitable marker of honey authenticity, we developed an analytical method to determine the HBG-III content and investigated its applicability to various commercial products. Following extraction from honey using phosphate-buffered saline, HBG-III was concentrated using an ultrafiltration membrane and subsequently fragmented with trypsin and lysyl endopeptidase mixture. The specific peptide fragments were used for quantitation by liquid chromatography-tandem mass spectrometry. The established method was validated for linearity, accuracy, precision, and the limit of quantitation (LOQ). As a result, the calibration curve was linear in the range of 0.01-0.3 μM, the mean recovery ranged from 73.8 to 89.2 %, the within-laboratory reproducibility (RSDwr) ranged from 3.9 to 6.5 %, and the LOQ was 1.9 mg/kg. An investigation of HBG-III concentrations in 65 honey products available on the Japanese market revealed that the HBG-III content of 15 low-priced honey products was below the LOQ. This suggested that these products may be adulterated with non-honey syrups. Therefore, this method can serve as an effective tool to verify the authenticity of honey products.
Super Ohtaka®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of vegetables and fruits is a naturally fermented mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). In this study, we separated water-soluble polysaccharides from Super Ohtaka® using dialysis and chromatography, yielding four polysaccharide fractions. The polysaccharide fraction designated as OEP3 exhibited hyaluronidase inhibitory activity. The half-maximal inhibitory concentration was 860 µg/mL. This polysaccharide not only stimulated macrophages but also inhibited hyaluronidase activity and showed weak 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.
