Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Bifidobacterium Species and Its Physicochemical Properties.

α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from Bifidobacterium sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.

Lacticaseibacillus paracasei completely utilizes fructooligosacchrides in the human gut through ß-fructosidase (FosE).

The fecal microbiota of two healthy adults was cultivated in a medium containing commercial fructooligosaccharides [FOS; 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)]. Initially, the proportions of lactobacilli in the two feces samples were only 0.42% and 0.17%; however, they significantly increased to 7.2% and 4.8%, respectively, after cultivation on FOS. Most FOS-utilizing isolates could utilize only GF2; however, Lacticaseibacillus paracasei strain Lp02 could fully consume GF3 and GF4 too. The FOS operon (fosRABCDXE) was present in Lc. paracasei Lp02 and another Lc. paracasei strain, KCTC 3510T, but fosE was only partially present in the non-FOS-degrading strain KCTC 3510T. In addition, the top six upregulated genes in the presence of FOS were fosABCDXE, particularly fosE. FosE is a ß-fructosidase that hydrolyzes both sucrose and all three FOS. Finally, a genome-based analysis suggested that fosE is mainly observed in Lc. paracasei, and only 13.5% (61/452) of their reported genomes were confirmed to include it. In conclusion, FosE allows the utilization of FOS, including GF3 and GF4 as well as GF2, by some Lc. paracasei strains, suggesting that this species plays a pivotal role in FOS utilization in the human gut.

Optimized enzymatic synthesis of digestive resistant anomalous isoquercitrin glucosides using amylosucrase and response surface methodology.

Diverse flavonoid glycosides are present in the plant kingdom. Advanced technologies have been utilized to synthesize glycosyl flavonoids which exhibit good physicochemical characteristics. Previously, novel isoquercitrin (IQ) mono-, di-, and tri-glucosides (IQ-G1', IQ-G2', and IQ-G3'; atypical IQ-Gs (IQ-Gap)) were synthesized through the reaction of amylosucrase. Here, the regio-selective transglycosylation yields were predicted using response surface methodology for three variables (glucose donor (sucrose; 100-1500 mM), glucose acceptor (IQ; 100-400 µM), and pH (5.0-8.8)) using 1 unit/mL of enzyme at 45 °C; then, the optima were verified according to the experimental responses. Acidity (pH 5.0) was a major contributor for IQ-G1' production (> 50%), and high sucrose concentration (1500 mM) limited IQ-G3' production (< 15%). Low sucrose concentration (100 mM) at pH 7.0 promoted higher glycosyl IQ production (> 30%). Time-course production of IQ-Gap showed an exponential growth with different rates. IQ-Gap was stable under the simulated intestinal conditions compared with typical IQ-Gs. Digestive stable IQ-Gap can be effectively synthesized by modulating reaction conditions; thereby, atypical glycosyl products may contribute to the elucidation of nutraceutical potential of flavonoid glycosides. KEY POINTS: •Predictions of RSM were validated for the regio-selective IQ-Gap production. • Time course changes of IQ-Gap indicate non-processive glycosylation of DGAS. • IQ-Gap exceed typical IQ-G in digestive stability at simulated intestinal condition.

Genome Analysis of Enterococcus mundtii Pe103, a Human Gut-Originated Pectinolytic Bacterium.

Pectin exists in significant amounts in vegetables and fruits as a component of the plant cell wall. In human diet, pectin is not degraded by the human digestive enzymes due to its complex structure; only gut bacteria degrade pectin in the large intestine. To date, although pectin is one of the most important sources of dietary fiber in human diet, there have been only few reports on human gut-originated pectinolytic bacteria. In this study, the strain Enterococcus mundtii Pe103, a bacterium with pectin-degrading activity, was isolated from the feces of a healthy Korean adult female. Culture experiments revealed that it could grow on pectin as the sole carbon source by degrading pectin to approximately 35% within 13 h. We report the complete genome data of human gut E. mundtii Pe103. It consists of a circular chromosome (3,084,146 bps) and two plasmids (63,713 and 56,223 bps). Genomic analysis suggested that at least nine putative enzymes related to pectin degradation are present in E. mundtii Pe103. These enzymes may be involved in the degradation of pectin. The whole genome information of E. mundtii Pe103 could improve the understanding of the mechanism underlying the degradation of pectin by human gut microbiota.

Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation.

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans.

The effect of resistant starch (RS) on the bovine rumen microflora and isolation of RS-degrading bacteria.

Resistant starch (RS) in the diet reaches the large intestine without degradation, where it is decomposed by the commensal microbiota. The fermentation of RS produces secondary metabolites including short-chain fatty acids (SCFAs), which have been linked to a variety of physiological and health effects. Therefore, the availability of RS as a prebiotic is a current issue. The objectives of this study were (1) to use metagenomics to observe microbial flora changes in Bos taurus coreanae rumen fluid in the presence of RS and (2) to isolate RS-degrading microorganisms. The major microbial genus in a general rumen fluid was Succiniclasticum sp., whereas Streptococcus sp. immediately predominated after the addition of RS into the culture medium and was then drastically replaced by Lactobacillus sp. The presence of Bifidobacterium sp. was also observed continuously. Several microorganisms with high RS granule-degrading activity were identified and isolated, including B. choerinum FMB-1 and B. pseudolongum FMB-2. B. choerinum FMB-1 showed the highest RS-hydrolyzing activity and degraded almost 60% of all substrates tested. Coculture experiments demonstrated that Lactobacillus brevis ATCC 14869, which was isolated from human feces, could grow using reducing sugars generated from RS by B. choerinum FMB-1. These results suggest that Bifidobacterium spp., especially B. choerinum FMB-1, are the putative primary degrader of RS in rumen microbial flora and could be further studied as probiotic candidates.

Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.

Fluorescence detection of the transglycosylation activity of amylosucrase.

The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor. The result of HPLC (high performance liquid chromatography) show that the bioconversion of MUG with ASases was successfully accomplished using sucrose and MU. Kinetic studies of ASases were performed to determine kinetic parameter for sucrose and MU. The order of overall performance (kcat/Km) of transglycosylation activity for MU among DGAS, DGAS-B and NPAS was as follows: DGAS-B (8.1) > DGAS (5.0) > NPAS (0.4). The fluorescence-based transglycosylation assay using MU has a potential to be used as the detection of transglycosylation activity of ASase and to screen novel ASase variants, which may be improved in their transglycosylation activities.

Pyrodictium delaneyi sp. nov., a hyperthermophilic autotrophic archaeon that reduces Fe(III) oxide and nitrate.

A hyperthermophilic, autotrophic iron and nitrate reducer, strain Su06T, was isolated from an active deep-sea hydrothermal vent chimney on the Endeavour Segment in the north-eastern Pacific Ocean. It was obligately anaerobic, hydrogenotrophic and reduced Fe(III) oxide to magnetite and NO3- to N2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was more than 97 % similar to other species of the genera Pyrodictium and Hyperthermus. Therefore, overall genome relatedness index analyses were performed to establish whether strain Su06T represents a novel species. For each analysis, strain Su06T was most similar to Pyrodictium occultum PL-19T. Relative to this strain, the average nucleotide identity score for strain Su06T was 72 %, the genome-to-genome direct comparison score was 13-19 % and the species identification score at the protein level was 89 %. For each analysis, strain Su06T was below the species delineation cutoff. Based on its whole genome sequence and its unique phenotypic characteristics, strain Su06T is suggested to represent a novel species of the genus Pyrodictium, for which the name Pyrodictium delaneyi is proposed. The type strain is Su06T (=DSM 28599T=ATCC BAA-2559T).

Methanocaldococcus bathoardescens sp. nov., a hyperthermophilic methanogen isolated from a volcanically active deep-sea hydrothermal vent.

A hyperthermophilic methanogen, strain JH146(T), was isolated from 26 °C hydrothermal vent fluid emanating from a crack in basaltic rock at Marker 113 vent, Axial Seamount in the northeastern Pacific Ocean. It was identified as an obligate anaerobe that uses only H2 and CO2 for growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain is more than 97% similar to other species of the genus Methanocaldococcus . Therefore, overall genome relatedness index analyses were performed to establish that strain JH146(T) represents a novel species. For each analysis, strain JH146(T) was most similar to Methanocaldococcus sp. FS406-22, which can fix N2 and also comes from Marker 113 vent. However, strain JH146(T) differs from strain FS406-22 in that it cannot fix N2. The average nucleotide identity score for strain JH146(T) was 87%, the genome-to-genome direct comparison score was 33-55% and the species identification score was 93%. For each analysis, strain JH146(T) was below the species delineation cut-off. Full-genome gene synteny analysis showed that strain JH146(T) and strain FS406-22 have 97% genome synteny, but strain JH146(T) was missing the operons necessary for N2 fixation and assimilatory nitrate reduction that are present in strain FS406-22. Based on its whole genome sequence, strain JH146(T) is suggested to represent a novel species of the genus Methanocaldococcus for which the name Methanocaldococcus bathoardescens is proposed. The type strain is JH146(T) ( = DSM 27223(T) = KACC 18232(T)).

Identification and characterization of an archaeal kojibiose catabolic pathway in the hyperthermophilic Pyrococcus sp. strain ST04.

A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and ß-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and ß-glucose-1-phosphate (ß-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts ß-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from ß-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to ß-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.

Reaction kinetics of substrate transglycosylation catalyzed by TreX of Sulfolobus solfataricus and effects on glycogen breakdown.

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-ß-cyclodextrin (Glc4-ß-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-ß-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-ß-cyclodextrin [(Glc4)2-ß-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-ß-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.

Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Šis reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (ß/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or ß-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

Direct and simple detection of recombinant proteins from cell lysates using differential scanning fluorimetry.

A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT-PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins.

Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. of hyperthermophilic heterotrophs from deep-sea hydrothermal vents.

Two heterotrophic hyperthermophilic strains, ES1(T) and CL1(T), were isolated from Paralvinella sp. polychaete worms collected from active hydrothermal vent chimneys in the north-eastern Pacific Ocean. Both were obligately anaerobic and produced H2S in the presence of elemental sulfur and H2. Complete genome sequences are available for both strains. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains are more than 97% similar to most other species of the genus Thermococcus. Therefore, overall genome relatedness index analyses were performed to establish that these strains are novel species. For each analysis, strain ES1(T) was determined to be most similar to Thermococcus barophilus MP(T), while strain CL1(T) was determined to be most similar to Thermococcus sp. 4557. The average nucleotide identity scores for these strains were 84% for strain ES1(T) and 81% for strain CL1(T), genome-to-genome direct comparison scores were 23% for strain ES1(T) and 47% for strain CL1(T), and the species identification scores were 89% for strain ES1(T) and 88% for strain CL1(T). For each analysis, strains ES1(T) and CL1(T) were below the species delineation cut-off. Therefore, based on their whole genome sequences, strains ES1(T) and CL1(T) are suggested to represent novel species of the genus Thermococcus for which the names Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. are proposed, respectively. The type strains are ES1(T) ( =DSM 27261(T) =KACC 17923(T)) and CL1(T) ( =DSM 27260(T) =KACC 17922(T)).

Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04.

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13% sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (Pyrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-ß-cyclodextrin (G1-ß-CD) and ß-cyclodextrin (ß-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (Km) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-ß-cyclodextrin (G2-ß-CD) was 0.3 mM. The kcat values were 381.0 min(-1) for G3 and 1,545.0 min(-1) for G2-ß-CD. The enzyme was inhibited competitively by the reaction product G2, and the Ki constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.

Resistant starch utilization by Bifidobacterium, the beneficial human gut bacteria.

Resistant starch (RS) reaches the large intestine largely intact, where it is fermented by the gut microbiota, resulting in the production of short-chain fatty acids (SCFAs) that have beneficial effects on the human body. Bifidobacteria are a major species widely used in the probiotic field, and are increased in the gut by RS, indicating their importance in RS metabolism in the intestine. Bifidobacteria have a genetic advantage in starch metabolism as they possess a significant number of starch-degrading enzymes and extraordinary three RS-degrading enzymes, allowing them to utilize RS. However, to date, only three species of RS-degrading bifidobacteria have been reported as single isolates B. adolescentis, B. choerinum, and B. pseudolongum. In this review, we describe recent studies on RS utilization by Bifidobacterium, based on their biochemical characteristics and genetic findings. This review provides a crucial understanding of how bifidobacteria survive in specific niches with abundant RS such as the human gut.

Correction to: Versatile biotechnological applications of amylosucrase, a novel glucosyltransferase.

[This corrects the article DOI: 10.1007/s10068-019-00686-6.].

Application of Schizosaccharomyces japonicus in makgeolli fermentation and its brewing characteristics.

Recently, unconventional yeasts have become popular as fermentation starters in the brewing industry due to the growing consumer demand for aromatic diversity. Specifically, Schizosaccharomyces japonicus has been explored as a potential starter culture for beer and wine production because of its distinct brewing characteristics; however, its application in makgeolli fermentation has not been tested. Therefore, in the present study, two Sz. japonicus strains (SZJ-1 and SZJ-2) were isolated from natural sources, and their brewing characteristics for makgeolli fermentation were compared with those of commercial S. cerevisiae strain. Although the tested isolates showed a lower fermentation and carbon source consumption rate than control-, their overall alcohol fermentation characteristics were suitable for makgeolli production. Regarding flavor composition, Sz. japonicus-fermented makgeolli possessed more ester compounds (e.g., 2-phenylethyl acetate, ethyl acetate, and ethyl decanoate) than S. cerevisiae-fermented makgeolli. Therefore, Sz. japonicus can be used as an alternative culture starter in makgeolli fermentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01265-6.