Temperature impact on microbial and metabolic profiles in kimchi fermentation.

Kimchi is a traditional Korean fermented food and harbors diverse bacteria. Therefore, proper temperature management contributes to the fermentation and preservation of kimchi. In this study, we explored fermentation temperature influences the bacterial composition and metabolite variations in kimchi, employing pyrosequencing for bacterial community analysis and mass spectrometry for metabolite profiling. Elevated temperatures within the range of 10-15 °C had a significant impact on the community of lactic acid bacteria (LAB) compared to 4 °C, leading to increased bacterial diversity and richness. We observed a significant increase in Lactiplantibacillus plantarum and Latilactobacillus sakei, alongside a reduction in Lactococcus lactis, during fermentation at 10-15 °C. These changes occurred within a similar pH range across different kimchi fermentation periods. We performed a liquid extraction via the acetonitrile method, which involved the collection of kimchi samples, and performed LC-MS analysis. Our analysis revealed approximately 5000 metabolites. Notably, we observed a significant increase in metabolite counts, with 3048 metabolites increasing at 10 °C and 2853 metabolites exhibiting a similar trend at 15 °C. Metabolite analysis showed an increase in lactic and succinic acid with increased glucose and sucrose consumption at 10 and 15 °C. These results indicated that elevated temperatures significantly influenced the glycolysis and tricarboxylic acid cycle, leading to increased acidity during the fermentation process. These findings show the crucial role played by temperature in controlling the fermentation process, thereby influencing the bacterial succession and the resulting flavor and taste of the product. Therefore, proper temperature management can effectively control kimchi fermentation and yield the desired sensory properties.

Elemental Analysis of Kimchi Cabbage Leaves, Roots, and Soil and Its Potential Impact on Human Health.

In view of their rich mineral content and flavor, kimchi cabbage leaves and roots have high nutritional and medicinal values. In this study, we quantified major nutrient (Ca, Cu, Fe, K, Mg, Na, and Zn), trace (B, Be, Bi, Co, Ga, Li, Ni, Se, Sr, V, and Cr), and toxic (Pb, Cd, Tl, and In) elements in kimchi cabbage cultivation soil, leaves, and roots. The analysis method relied on inductively coupled plasma-optical emission spectrometry for major nutrient elements and inductively coupled plasma-mass spectrometry for trace and toxic elements and complied with the Association of Official Analytical Chemists (AOAC) guidelines. Kimchi cabbage leaves and roots featured high contents of K, B, and Be, while the contents of all toxic elements in all samples were below the WHO-stipulated threshold values and therefore did not pose any health risks. The distribution of elements was characterized by heat map analysis and linear discriminant analysis to reveal independent separation according to the content of each element. The analysis confirmed that there was a difference in content between the groups and that each group was independently distributed. This study may contribute to a better understanding of the complex relationships between plant physiology, cultivation condition, and human health.

Rapid Quantitative Analysis of Metabolites in Kimchi Using LC-Q-Orbitrap MS.

Kimchi is a traditional Korean salted spontaneous lactic acid bacteria (LAB)-fermented food made using various vegetables. Organic acids, free sugars, and amino acids are key metabolites produced during LAB fermentation that determine the taste and quality of kimchi. However, each metabolite is typically analyzed using an independent analytical method, which is time-consuming and expensive. Therefore, in this study, we developed a method based on LC-Q-Orbitrap MS using which 20 types of representative fermented kimchi metabolites were selected and simultaneously analyzed within 10 min. The established method was validated, and its detection and quantification limits, linearity, precision, and accuracy were found to satisfy the Association of Official Agricultural Chemists (AOAC) validation guidelines. The 20 metabolites were simultaneously extracted from kimchi with different degrees of fermentation and quantitatively analyzed using LC-Q-Orbitrap MS. These results were analyzed using linear discriminant analysis and heat mapping, and the metabolites were grouped into early, middle, and late stages of fermentation. Malic acid (6.518-7.701 mMol) was only present in the initial stage of fermentation, and l-phenylalanine rapidly increased from the middle stage (2.180 mMol) to late stage (4.770 mMol). Lactic acid, which is representative of the sour taste of kimchi, was detected in the middle stage and increased rapidly up to 74.452 mMol in the late stage. In summary, in this study, 20 major kimchi metabolites were accurately analyzed within 10 min and grouped based on the degree of fermentation. Therefore, the method established in this study accurately and rapidly provides information on kimchi consumption and fermentation that could be highly valuable to the kimchi industry and kimchi consumers.

Arginine metabolism and the role of arginine deiminase-producing microorganisms in kimchi fermentation.

In this study, we evaluated the correlation between the arginine deiminase (ADI) pathway and nitrogen cycle during cabbage kimchi fermentation. Nitrite used as a food additive can be converted to carcinogenic N-nitroso compounds via reactions with secondary amines under specific conditions; thus, high nitrate- and nitrite-containing foods present a potential risk to human health. We monitored the bacterial community, levels of ADI metabolites, and nitrogen compounds present in kimchi that contained bacteria that showed low ADI activity during fermentation. The dominant growth of microorganisms with weak ADI activity reduced arginine degradation and ornithine production. Furthermore, nitrite production in kimchi samples was affected by ADI activity. The ornithine and nitrite contents in the control kimchi were 1.7- and 2.6-fold higher at week 2 than at week 1. These results suggest that ADI-associated metabolism is correlated with the nitrogen cycle in kimchi and that the addition of bacteria with weak ADI activity may reduce nitrite production in kimchi.

Overexpression of Phosphoribosyl Pyrophosphate Synthase Enhances Resistance of Chlamydomonas to Ionizing Radiation.

The enzyme phosphoribosyl pyrophosphate synthase (PRPS) catalyzes the conversion of ribose 5-phosphate into phosphoribosyl diphosphate; the latter is a precursor of purine and pyrimidine nucleotides. Here, we investigated the function of PRPS from the single-celled green alga Chlamydomonas reinhardtii in its response to DNA damage from gamma radiation or the alkylating agent LiCl. CrPRPS transcripts were upregulated in cells treated with these agents. We generated CrPRPS-overexpressing transgenic lines to study the function of CrPRPS. When grown in culture with LiCl or exposed to gamma radiation, the transgenic cells grew faster and had a greater survival rate than wild-type cells. CrPRPS overexpression enhanced expression of genes associated with DNA damage response, namely RAD51, RAD1, and LIG1. We observed, from transcriptome analysis, upregulation of genes that code for key enzymes in purine metabolism, namely ribonucleoside-diphosphate pyrophosphokinase subunit M1, adenylate kinase, and nucleoside-diphosphate kinase. We conclude that CrPRPS may affect DNA repair process via regulation of de novo nucleotide synthesis.

Characterization of transcriptional response of Lactobacillus plantarum under acidic conditions provides insight into bacterial adaptation in fermentative environments.

Lactic acid bacteria (LAB) play an important role in kimchi fermentation by metabolizing raw materials into diverse metabolites. Bacterial adaptation is therefore a crucial element of fermentation. In this study, we investigated the transcriptional changes of Lactobacillus plantarum under acidic conditions to evaluate the elements of bacterial adaptation critical for fermentation. Differentially expressed genes (DEGs) have shown that transport function is primarily affected by acidic conditions. Five of the 13 significantly down-regulated genes and 7 of the 25 significantly up-regulated genes were found to have transport-related functions. We quantified the intracellular leucine content of bacteria grown at different pH ranges, determining that optimal bacterial leucine transport could be controlled by acidity during fermentation. Inhibition of L. plantarum growth was investigated and compared with other LAB at a pH range of 6.2-5.0. Interestingly, valinomycin inhibited L. plantarum growth from pH 6.2 to 5.0. This showed that L. plantarum had a wider range of transport functions than other LAB. These results suggested that L. plantarum had robust transport functions, and that this was the crucial factor for bacterial adaptation during fermentation.

Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis.

BACKGROUND: Jacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation. RESULTS: Time- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11. CONCLUSIONS: OsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage.

The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis.

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii.

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum.

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co(2+). Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co(2+) stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.

Improving lignocellulose degradation using xylanase-cellulase fusion protein with a glycine-serine linker.

The fungal hydrolytic system efficiently degrades lignocellulosics efficiently. We previously characterized two hydrolytic enzymes from Gloeophyllum trabeum, namely, endoglucanase (Cel5B) and xylanase (Xyl10g). To enhance lignocellulosic degradation, we designed a fusion protein (Xyl10g GS Cel5B) using a glycine-serine (GS) linker and expressed it in Pichia pastoris GS115, which produced a hydrolytic fusion enzyme for the degradation of lignocellulosics. Purified Xyl10g GS Cel5B protein has a molecular weight of approximately 97 kDa and shows a lower specific activity than Xyl10g or Cel5B. However, Xyl10g GS Cel5B can degrade popping-pretreated rice straw, corn stover, kenaf, and oak more efficiently than the mixture of Xyl10g and Cel5B, by about 1.41-, 1.37-, 1.32-, and 1.40-fold, respectively. Our results suggest that Xyl10g GS Cel5B is an efficient hydrolytic enzyme and a suitable candidate for degrading lignocellulosics to produce fermentable sugar.

Efficient approach for bioethanol production from red seaweed Gelidium amansii.

Gelidium amansii (GA), a red seaweed species, is a popular source of food and chemicals due to its high galactose and glucose content. In this study, we investigated the potential of bioethanol production from autoclave-treated GA (ATGA). The proposed method involved autoclaving GA for 60min for hydrolysis to glucose. Separate hydrolysis and fermentation processing (SHF) achieved a maximum ethanol concentration of 3.33mg/mL, with a conversion yield of 74.7% after 6h (2% substrate loading, w/v). In contrast, simultaneous saccharification and fermentation (SSF) produced an ethanol concentration of 3.78mg/mL, with an ethanol conversion yield of 84.9% after 12h. We also recorded an ethanol concentration of 25.7mg/mL from SSF processing of 15% (w/v) dry matter from ATGA after 24h. These results indicate that autoclaving can improve the glucose and ethanol conversion yield of GA, and that SSF is superior to SHF for ethanol production.

Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5' amplification promoting sequence.

Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.

Role of pCeMT, a putative metallothionein from Colocasia esculenta, in response to metal stress.

Metallothioneins (MTs) play a major role in metal homeostasis and/or detoxification in plants. In this study, a novel gene, pCeMT, was isolated from Colocasia esculenta and characterized. Our results indicate that Escherichia coli cells expressing pCeMT exhibited enhanced Cd, Cu, and Zn tolerance and accumulation compared with control cells. Furthermore, pCeMT-overexpressing tobacco seedlings displayed better growth under Cd, Cu, and Zn stresses and accumulated more Cd and Zn compared with the wild type. Interestingly, transgenic tobacco displayed markedly decreased hydrogen peroxide (H(2)O(2)) and lipid peroxidation levels under Cd, Cu, and Zn treatments. These results suggest that pCeMT could play an important role in the protection of plant cells from oxidative stress by reactive oxygen species (ROS) scavenging and in the detoxification of free metals by metal binding, leading to improved plant metal tolerance.

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants.

Cost-effective bioethanol production requires a supply of various low-cost enzymes that can hydrolyse lignocellulosic materials consisting of multiple polymers. Because plant-based enzyme expression systems offer low-cost and large-scale production, this study simultaneously expressed ß-glucosidase (BglB), xylanase (XylII), exoglucanase (E3), and endoglucanase (Cel5A) in tobacco plants, which were individually fused with chloroplast-targeting transit peptides and linked via the 2A self-cleaving oligopeptideex from foot-and-mouth disease virus (FMDV) as follows: [RsBglB-2A-RaCel5A], [RsXylII-2A-RaCel5A], and [RsE3-2A-RaCel5A]. The enzymes were targeted to chloroplasts in tobacco cells and their activities were confirmed. Similarly to the results of a transient assay using Arabidopsis thaliana protoplasts, when XylII was placed upstream of the 2A sequence, the [RsXylII-2A-RaCel5A] transgenic tobacco plant had a more positive influence on expression of the protein placed downstream. The [RsBglB-2A-RaCel5A] and [RsE3-2A-RaCel5A] transgenic lines displayed higher activities towards carboxylmethylcellulose (CMC) compared to those in the [RsXylII-2A-RaCel5A] transgenic line. This higher activity was attributable to the synergistic effects of the different cellulases used. The [RsBglB-2A-RaCel5A] lines exhibited greater efficiency (35-74% increase) of CMC hydrolysis when the exoglucanase CBHII was added. Among the various exoglucanases, E3 showed higher activity with the crude extract of the [RsBglB-2A-RaCel5A] transgenic line. Transgenic expression of 2A-mediated multiple enzymes induced synergistic effects and led to more efficient hydrolysis of lignocellulosic materials for bioethanol production.

Co-immobilization of three cellulases on Au-doped magnetic silica nanoparticles for the degradation of cellulose.

Three cystein-tagged cellulases co-immobilized on AuNP and Au-MSNP for the hydrolytic degradation of cellulose. The biochemical properties, stabilities, activities and reusability of these co-immobilized systems were compared to those of mixtures of free cellulases.

Fluorescent receptor-immobilized silica-coated magnetic nanoparticles as a general binding agent for histidine-tagged proteins.

The histidine-tagged protein binding capacity and purification efficiencies of TSMNPs (terpyridine receptor-immobilized silica-coated magnetic nanoparticles) is described.

Expression of thermostable bacterial beta-glucosidase (BglB) in transgenic tobacco plants.

Transgenic tobacco plants expressing the hyperthermostable beta-glucosidase BglB of Thermotoga maritima were generated with the goal of cost-effective production of the enzyme for the application in bioconversion of lignocellulosic biomass. The enzyme was targeted to the cytosol and chloroplasts, where it accumulated to level of 4.5% and 5.8% of total soluble protein, respectively. The optimal temperature and pH of the plant-expressed BglB was 80 degrees C and 4.5, respectively. BglB activity was preserved in leaves after lyophilization, but decreased by over 70% with drying at room temperature. When BglB was synergistically supplied in a 1% (w/v) rice straw with Cel5A for efficient cellulase conversion, a 37% increase in glucose was observed. This report demonstrates the potential of utilizing transgenic tobacco for mass production of BglB.

Rice phot1a mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth.

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 miccromol m(-2) s(-1)), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO(2) uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H(2)O(2) in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H(2)O(2) accumulation, leading to a reduction in plant growth.

Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of cry1AC-carrying Brassica rapa subsp. pekinensis.

The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.

Plant growth-promoting potential of endophytic bacteria isolated from roots of coastal sand dune plants.

Endophytic bacteria associated with the roots of coastal sand dune plants were isolated, taxonomically characterized, and tested for their plant growth-promoting activities. Ninety-one endophytic bacterial isolates were collected and assigned to 17 different genera of 6 major bacterial phyla based on partial 16S rDNA sequence analyses. Gammaproteobacteria represented the majority of the isolates (65.9%), and members of Pseudomonas constituted 49.5% of the total isolates. When testing for antagonism towards plant pathogenic fungi, 25 strains were antagonistic towards Rhizoctonia solani, 57 strains were antagonistic towards Pythium ultimum, 53 strains were antagonistic towards Fusarium oxysporum, and 41 strains were antagonistic towards Botrytis cinerea. Seven strains were shown to produce indole acetic acid (IAA), 33 to produce siderophores, 23 to produce protease, 37 to produce pectinase, and 38 to produce chitinase. The broadest spectra of activities were observed among the Pseudomonas strains, indicating outstanding plant growth-promoting potential. The isolates from C. kobomugi and M. sibirica also exhibited good plant growth-promoting potential. The correlations among individual plant growth-promoting activities were examined using phi coefficients, and the resulting data indicated that the production of protease, pectinase, chitinase, and siderophores was highly related.