Comparison of taste characteristics between koji mold-ripened cheese and Camembert cheese using an electronic tongue system.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Garland chrysanthemum consumption ameliorates age-related hearing loss in C57BL/6 mouse; model system to explore hearing loss prevention foods in a short period.

Garland chrysanthemum (Glebionis coronaria L.) is an antioxidant-rich leafy vegetable. We found that garland chrysanthemum consumption ameliorated age-related hearing loss (AHL) in C57BL/6J mice, an early onset model. We also found that AHL progression was significantly ameliorated by three of ten products. Metabolome analysis of the 10 products using nuclear magnetic resonance (NMR) spectroscopy indicated that phytosterols may be involved in the amelioration of AHL. However, the direct inhibitory effect of phytosterol mixture on mouse AHL progression was not identified. These results suggest that garland chrysanthemum consumption delays AHL development in mice and its efficiency varies depending on the source of the product. Our findings also suggest that phytosterol content in garland chrysanthemum functions as an evaluation marker for the efficiency. Furthermore, to accelerate the search for foods that prevent AHL, we have used these data to develop an automatic threshold determination method for auditory brainstem response using machine learning.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria.

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation.

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.

In vitro and in silico characterisation of Lactobacillus paraplantarum D2-1, a starter culture for soymilk fermentation.

Soymilk contains several functional nutrients and is thus a promising ingredient for production of functional foods. The present research aimed to study starter properties, functional characteristics and safety of Lactobacillus paraplantarum D2-1, a promising starter culture for soymilk fermentation. Strain D2-1 actively fermented soymilk within 24 h but had weak activity of additional acid production after 7 d. Succinate and acetoin, which could be linked to flavour and taste, were accumulated in fermented soymilk. In vitro study revealed that the organism has several beneficial properties, including high survival ability in artificial gastric juice, high abilities of mucus adhesion and biofilm formation and production of γ-aminobutyric acid and conjugated linoleic acid, without any significant risks for consumption. Genome sequencing supported the desirable metabolic properties of the strain. These results indicate that L. paraplantarum D2-1 is a suitable starter for soymilk fermentation and is a promising probiotic candidate that can be used safely.

Comparison of external doses between radio-contaminated areas and areas with high natural terrestrial background using the individual dosimeter 'D-shuttle' 75 months after the Fukushima Daiichi nuclear power plant accident.

After the Fukushima Daiichi nuclear power plant accident, the air dose gradually decreases every year due to the physical decay of radioactive materials and environmental changes, as well as countermeasures. However, there is little information on personal behavioural patterns and individual dose from external exposure among the inhabitants around the nuclear power plant. To evaluate the dose from external exposure in Minamisoma city, and compare the differences with outside Fukushima Prefecture, we started the external dose assessment project in cooperation with city officials in Minamisoma and three other cities in Japan where the natural terrestrial background radiation level is relatively high. In these four cities, external dose was measured every hour for two weeks using an individual electronic dosimeter D-shuttle. The places of activity of participants were recorded every hour to compare and evaluate the dose from external exposure, and to clarify whether there is a difference in the exposure dose by behaviour. The annual effective doses from external exposure for 100 participants from four municipalities ranged from 0.566 to 1.295 with a mean value of 0.784 mSv, which was below the level where it is necessary to initiate further remedial actions. Mean external dose in Minamisoma city (0.820 mSv/year) was comparable to those in municipalities with a relatively high natural radiation background in Japan (0.793, 0.806, and 0.718 mSv/year in Fukuyama, Nanto, and Tajimi, respectively). The time spent at home and in the workplace accounted for most of the time of the participants, and this also contributed to the majority of the total dose from external exposure. The amount of exposure at times other than while at home or in the workplace was very small regardless of the indoor or outdoor location in the city. For future dose reduction and radiation protection, continuous dosimetry and countermeasures at home and in the workplace are important for individuals who present high values.

A survey of metabolic changes in potato leaves by NMR-based metabolic profiling in relation to resistance to late blight disease under field conditions.

Non-targeted nuclear magnetic resonance (NMR)-based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. 1 H NMR spectra of the foliar metabolites soluble in deuterium oxide- or methanol-d4 -based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l-malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field-grown plants in combination with targeted methods for quantifying marker levels, moving towards marker-assisted screening of new cultivars with durable late blight resistance. Copyright © 2016 John Wiley & Sons, Ltd.

GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum.

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.

Microbial composition and metabolic profiles during machine-controlled intra-factory fermentation of cocoa beans harvested in semitropical area of Japan.

Cocoa bean fermentation is typically performed in a spontaneous manner on farms in tropical countries or areas and involves several microbial groups. Metabolism by microbes markedly affects the quality of cocoa beans fermented and the chocolate produced thereof. The present study characterized the microbiota and their metabolic profiles in temperature- and humidity-controlled intra-factory cocoa fermentation in a semitropical area of Japan. Although environmental factors were uniform, the microbiota of cocoa beans subjected to intra-factory fermentation was not stable between tests, particularly in terms of the cell count levels and species observed. Fermentation was sometimes delayed, and fermenting microbes were present at very low levels after 24 hr of fermentation. Due to the unstable microbiota, the profiles of water-soluble compounds differed between tests, indicating the unstable qualities of the fermented cocoa beans. These results suggest the necessity of starter cultures not only in on-farm fermentation but also in machine-controlled intra-factory cocoa fermentation.

Characterization of a Lactobacillus gasseri JCM 1131T lipoteichoic acid with a novel glycolipid anchor structure.

We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131(T). The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.

The quest for probiotic effector molecules--unraveling strain specificity at the molecular level.

Pharmaceutical agents are widely applied for the treatment of gastrointestinal (and systemic) disorders and their role as modulators of host cell responses is relatively well characterized. By contrast, we are only beginning to understand the molecular mechanisms by which health-promoting, probiotic bacteria act as host cell modulators. The last decade has seen a rapid development of the genomics field for the widely applied probiotic genus Lactobacillus, and nowadays dozens of full genome sequences are available, as well as sophisticated post genomic and genetic engineering tools. This development has enabled comparative (functional) genomics approaches to identify the bacterial effector molecules involved in molecular communication with the host system that may underlie the probiotic effects observed. These efforts can also be complemented with dedicated mutagenesis approaches to eliminate or alter these effector molecules, followed by assessment of the host interaction consequences thereof, allowing the elucidation of the molecular mechanisms involved in probiotic health effects. Many of these approaches have pinpointed that the Lactobacillus cell envelope contains several effector molecules that are pivotal in the direct signaling capacity of these bacteria that underlies their immunomodulatory effects, including lipoteichoic acid, peptidoglycan, and (glyco)proteins. Moreover, the cell envelope contains several compounds such as wall teichoic acid and capsular polysaccharides that may not be involved in direct signaling to the host cell, but still affect signaling through shielding of other bacterial effector molecules. Initial structural studies revealed subtle strain- and species-specific biochemical differences in the canonical cell envelope compounds that are involved in these host interactions. These biochemical variations include the degree and positioning of d-alanyl and glycosyl substitution in lipoteichoic acids, and acetylation of peptidoglycan. Furthermore, specific peptides derived from peptidoglycan and envelope associated (glyco)proteins were recently identified as potent immunomodulators. The latter findings are exciting in the light of the possibility of more pharmacological application of these bioactive probiotic molecules, and especially cost-effective production and targeted delivery of bioactive peptides seems to emerge as a feasible strategy to harness this knowledge.

Determination of strain-specific wall teichoic acid structures in Lactobacillus plantarum reveals diverse α-D-glucosyl substitutions and high structural uniformity of the repeating units.

The structural diversity of wall teichoic acid (WTA) was investigated using biochemical and NMR analyses among 19 strains of Lactobacillus plantarum, of which seven were previously established to contain a glycerol-type backbone, whereas the remaining 12 strains possess ribitol-containing WTA. Despite the fact that the WTAs consisted of identical components, namely phosphoric acid, alditol (glycerol or ribitol) and glucose, comparative analysis of the (1)H and (13)C NMR spectra indicated the presence of six different structures, based on the observed differences in the anomeric signals of glucose residues. To determine the six WTA structures, their repeating units were prepared by alkaline hydrolysis, followed by fractionation on HPLC, and analysis by NMR spectroscopy using synthetic molecules as a reference. The structures of the six isolates were established as 1-α-D-glucosyl-sn-glycerol 3-phosphate, 1-α-D-kojibiosyl-sn-glycerol 3-phosphate, 1-α-D-nigerosyl-sn-glycerol 3-phosphate, 4-α-D-kojibiosylribitol 1-phosphate and 1,5-linked di-(2,4-di-α-D-glucosylribitol) phosphate. The backbone structures appeared to be 3,6'-linked poly(1-α-D-glucosyl-sn-glycerol phosphate) for the glycerol-type WTA and 1,5-linked poly(ribitol phosphate) for the ribitol-containing WTA. Moreover, in the analysis of the alkaline hydrolysates on HPLC, only single structures of repeating units were released from each WTA, indicating the high structural uniformity of the WTA in each strain. Notably, analyses of lipoteichoic acid isolated from representative strains harbouring the six different WTAs revealed the universal presence of a 1,3-linked poly(glycerol phosphate) chain, substituted at C-2 of the glycerol residues with glucose residues. These findings provide fundamental information on WTA structural variability in Lb. plantarum, which seems likely to play a pivotal role in the physiology of this bacterial species.

Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching.

BACKGROUND: Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. RESULTS: Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type. CONCLUSIONS: The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

A Case of Ascending Aortic Replacement and Aortic Valve Replacement in a Patient with Giant Pseudoaneurysm of the Ascending Aorta due to Prosthetic Valve Endocarditis after Transcatheter Aortic Valve Replacement Using Evolut PRO.

An 82-year-old man who underwent transcatheter aortic valve replacement (TAVR) using a 34-mm Evolut PRO (Medtronic, Minneapolis, MN, USA) for severe aortic stenosis 21 months previously presented with fever. Transesophageal echocardiography showed thickening of the valve leaflets of the transcatheter heart valve and movable structures such as verrucae on the upper edge of the Evolut PRO stent. Contrast-enhanced cardiac computed tomography showed a 72-mm saccular pseudoaneurysm on the dorsal aspect of the ascending aorta at the superior border of the Evolut PRO stent. Because of a suspected infected aortic aneurysm caused by prosthetic valve endocarditis (PVE), we performed aortic valve replacement using 23 mm Avalus (Medtronic, Minneapolis, MN, USA) and ascending aortic replacement using 26 mm Gelweave (Vascutek Terumo Inc, Scotland, UK). The postoperative course was uneventful. This report highlights that patients with PVE after TAVR may develop pseudoaneurysms of the ascending aorta.

Volatile and soluble metabolite profiles in surface-ripened cheeses with Aspergillus oryzae and Aspergillus sojae.

To determine the impact of traditional koji molds on chemical characteristics of soft-type natural cheese, novel surface mold-ripened cheeses with Aspergillus oryzae and Aspergillus sojae were studied by non-targeted metabolite profiling. Comprehensive water-soluble and volatile metabolite profiles of koji cheese were evaluated among five Aspergillus strains and other mold-ripened cheeses. Time-course changes in the metabolite profiles and degrading enzyme activities were also compared with those of an industrial Penicillium candidum starter culture. Koji cheeses differed from Camembert, Brie, and blue cheeses in higher lactic acid, amino acid, and acetoin levels and lower methyl ketone and volatile fatty acid levels. Time-course analysis revealed the associations of rapid accumulations of glutamic, aspartic, and 3-methylbutanoic acids and 3-methylbutanal with higher proteolytic activity, and methyl ketone and fatty acid derivative suppressions with lower lipolytic activity. Ethyl butanoate, diacetyl, and malic acid also characterized koji cheeses as strain-dependent metabolites. This study highlighted the key compositional difference derived from cheese ripening with Aspergillus strains. The findings could help quality improvements of koji cheese product.

Metabolomic evaluation of different starter culture effects on water-soluble and volatile compound profiles in nozawana pickle fermentation.

Metabolomic characterization of a lactic-fermented pickle of nozawana (Brassica rapa L. var. hakabura) was conducted to evaluate the effects of different starter culture strains on the chemical profiles. We compared the profiles of water-soluble and volatile compounds obtained by non-targeted nuclear magnetic resonance and solid-phase microextraction gas chromatography/mass spectrometry analyses. Principal component analyses indicated that the fermented samples differed significantly in terms of the levels of various compounds, including taste- and aroma-active components, such as water-soluble residual sugars, organic acids, mannitol, ethanol, dihydroxyacetone, ornithine, γ-aminobutyric acid, choline, volatile isothiocyanates, 3,4-epithiobutyl cyanide, 2,3-butanedione, acetoin, ethyl acetate, dimethyl trisulfide, and S-methyl thioacetate. Fermentation with a Latilactobacillus curvatus culture was associated with a unique metabolite profile characterized by higher levels of isothiocyanates and hexanoic acid and lower levels of lactic acid, acetic acid, acetoin, and 2,3-butanedione. These variations in the chemical profile might be associated with different qualities in fermented nozawana pickle products.

Comparison of components and synthesis genes of cell wall teichoic acid among Lactobacillus plantarum strains.

The contents, components, and synthesis genes of cell wall teichoic acid (WTA) in 18 strains of Lactobacillus plantarum were compared. The WTA of each strain was classified by its components as being either the glycerol- or the ribitol-type. The different strains in the WTA type showed marked differences also in two gene regions, tagD1-tagF2 and lp_1816-tagB2, as for the presence or absence, nucleotide sequences, and transcriptional activities. Our results clearly showed that the tagD1-tagF2 and lp_1816-tagB2 regions contained the synthesis genes of the WTA backbone of L. plantarum. We verified that the genes in the tagD1-tagF2 region were involved in the synthesis of the glycerol-type backbone. Furthermore, we propose that the genes in the lp_1816-tagB2 region were tarI, tarJ, tarK, and tarL, which are involved in the synthesis of the ribitol-type backbone.

Characterisation of the bacterial community structures of sunki, a traditional unsalted pickle of fermented turnip leaves.

The bacterial community structure in 29 naturally fermented samples of sunki, an unsalted lactic-fermented pickle in Japan, was determined by 16S rRNA gene-targeted metagenomic analysis. The data revealed that genus Lactobacillus was dominant in all samples and various bacterial species, related to Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus intestinalis, and Lactobacillus gasseri, showed a range of dominance depending on the samples. Comparative analysis of the bacterial composition by principal coordinate analysis and hierarchical clustering classified the varied bacterial composition of the 29 samples into three types of bacterial community structure. These types comprised lactobacilli belonging to different phylogenetic groups: type A had a certain ratio of Lactobacillus fermentum (70.3-22.1%, average 41.2%) in combination with several species belonging to Lactobacillus delbrueckii-phylogenetic group, type B comprised remarkably high levels of species Lactobacillus delbrueckii (average 89.5%), and type C had combinations of species belonging to Lactobacillus plantarum- and Lactobacillus buchneri-phylogenetic groups. Interestingly, these types differed in the compositional profiles of water-soluble and volatile compounds, and statistically significant differences were observed in the levels of acetic acid, succinic acid, ethanol, ethyl acetate, glutamic acid, γ-aminobutyric acid, and isovaleraldehyde. Furthermore, metabolomic analysis revealed a correlation of Lactobacillus fermentum dominance with pH value and lactic and acetic acid levels, with high R values of 0.643, -0.642, and 0.528, respectively. The data reported in this study showed the characteristics of the bacterial composition in the unsalted sunki pickle and its potential relationship with the compositional profile.

Structures of two monomeric units of teichoic acid prepared from the cell wall of Lactobacillus plantarum NRIC 1068.

The cell wall of Lactobacillus plantarum contains large amounts of cell wall teichoic acid (WTA). WTA was isolated from the cell wall of L. plantarum NRIC 1068 (= ATCC 8014 and 17-5) by extraction with trichloroacetic acid, and two monomeric units (F1 and F2) were prepared from the alkaline hydrolysate of WTA. Componential analysis by HPLC showed that these monomers were composed of ribitol, glucose, and phosphoric acid. Structural analyses of the monomers were performed by NMR spectroscopy with comparison to chemically synthesized monomers. The structures of F1 and F2 were determined to be 3,4-alpha-D-diglucosyl-2-phosphoryl ribitol and 3,4-alpha-D-diglucosyl-1-phosphoryl ribitol respectively. The unique structure of WTA in L. plantarum results from modification of the main chain with multiple glucose residues.

NMR- and GC/MS-based metabolomic characterization of sunki, an unsalted fermented pickle of turnip leaves.

This study revealed the compositional characteristics of sunki, a traditional, unsalted, lactic acid-fermented pickle produced using turnip leaf in Kiso district, Japan. Comprehensive compositional analysis by two metabolomic approaches based on NMR and solid-phase microextraction-GC/MS methods was used to determine its chemical composition by annotating 54 water-soluble and 62 volatile compounds. Principal component analysis showed that samples had different compositions, depending on the agricultural processing factory and production year. This variation potentially resulted from the differences in the lactic acid bacterial community produced during the spontaneous fermentation of sunki and in the initial nutritional composition of the turnip leaf. Partial least squares regression revealed that the acetic acid level showed a strong positive correlation with pH (R = 0.810), in contrast to the negative correlations of lactic acid and ethanol levels (R =  -0.533 and -0.547). This indicated the crucial impact of acetic acid-related metabolism on acidification during sunki fermentation.