Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae.

BACKGROUND: Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally. RESULTS: Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid. CONCLUSIONS: A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.

Effects of preheat treatment and syringic acid modification on the structure, functional properties, and stability of black soybean protein isolate.

This study investigated preheated (25-100°C) black soybean protein isolate (BSPI) conjugated with syringic acid (SA) (25 and 50 µmol/g protein) under alkaline conditions, focusing on the structure, functional properties, and storage stability. The results revealed that the SA binding equivalent and binding rate on BSPI increased continuously as the preheat temperature increased. Additionally, preheating positively impacted the surface hydrophobicity (H0) of BSPI, with further enhancement observed upon SA binding. Preheating and SA binding altered the secondary and tertiary structure of BSPI, resulting in protein unfolding and increased molecular flexibility. The improvement in BSPI functional properties was closely associated with both preheating temperature and SA binding. Specifically, preheating decreased the solubility of BSPI but enhanced the emulsifying activity index (EAI) and foaming capacity (FC) of BSPI. Conversely, SA binding increased the solubility of BSPI with an accompanying increase in EAI, FC, foaming stability, and antioxidant activity. Notably, the BSPI100-SA50 exhibited the most significant improvement in functional properties, particularly in solubility, emulsifying, and foaming attributes. Moreover, the BSPI-SA conjugates demonstrated good stability of SA during storage, which positively correlated with the preheating temperature. This study proposes a novel BSPI-SA conjugate with enhanced essential functional properties, underscoring the potential of preheated BSPI-SA conjugates to improve SA storage stability. PRACTICAL APPLICATION: Preheated BSPI-SA conjugates can be used as functional ingredients in food or health products. In addition, preheated BSPI shows potential as a candidate for encapsulating and delivering hydrophobic bioactive compounds.

[Archaeal diversity in sediment of Dunbasitawu salt lake, Xinjiang, China, estimated by 16S rRNA gene sequence analyses].

OBJECTIVE: We surveyed the composition and diversity of uncultured archaea in Xinjiang Dunbasitawu salt Lake sediment. METHODS: Environmental total DNA was directly extracted from the sediment. We constructed clone library of 16S rRNA gene amplified with archaea-specific primers. A total of 59 positive clones were randomly selected from the library and identified by restriction length polymorphism (RFLP) with enzyme Hae III. Clones with the unique RFLP pattern were sequenced, and then by phylogenetic analysis. RESULTS: The clone coverage C value was 89%, and Shannon-Wiener index was 2.69. In total, 21 Operational Taxonomic Units (OTUs) were obtained and affiliated with Euryarchaeota (92%) and Crenarchaeota (8%). The most of clones were affiliated to Halobacterium (24%), Haloarcula (18%), Natronorubrum (14%), and Halorubrum (8%), which belonged to family Halobacteriaceae (88%) with high similarity to that from thalassohaline environment. In addition, 11% of clones had less than 97% similarity with archaea sequences deposited in GenBank database. CONCLUSION: Compared with other similar Hypersaline environments, archaea diversity in Dunbasitawu salt lake was a little lower. The proportion of archara was different, but the composition is consistent. It was implied that some potential new species or lineages maybe exist in Dunbasitawu salt lake.

[A new method of microbial prospecting for oil and gas based on BIOLOG metabolic fingerprinting analysis].

OBJECTIVE: Establishing a new method of microbial prospecting for oil and gas based on microbial community metabolic function. METHODS: In this study, 45 shallow reservoir soil samples and 25 non-reservoir soil samples were collected. Carbon catabolic activity of the microbial community was measured by biolog microplate, and microbial anomaly of reservoir was judged by discriminant function. RESULT: We screened 10 typical carbon sources of 95 carbon sources which could reflect the differences of reservoir and non-reservoir soil microbial community structure and made the validation test set to experimental zone and controlled zone samples using the discriminant function. The discriminate rate of reservoir and non-reservoir were 97.8% and 100% respectively and the overall accuracy was 98.6%. CONCLUSION: Biolog technology provided a highly efficient and precise method for preliminarily predicting oil and gas reservoir.

Changes in phenolic compounds and antioxidant activities of "nine steaming nine sun-drying" black soybeans before and after in vitro simulated gastrointestinal digestion.

Black soybean (Glycine max (L.) Merr.) is rich in phenolic compounds, and processing technology has a significant effect on the content and activity of phenolic compounds. However, the mechanism of nine steaming and nine sun-drying processing technique is not fully understood. This paper presents the changes of phenolics content, phenolic acids composition and their influence on antioxidant activity before and after in vitro simulated gastrointestinal digestion of black soybeans (BS) under the process of nine steaming nine sun-drying. Results showed that the total phenolic content (TPC) and total flavonoids content (TFC) in BS were reduced by the heat treatment method, and exhibited a decreasing trend with more steaming and sun-drying cycle. During in vitro digestion, the contents and bioaccessibility of 12 phenolic acids (PA-12) in BS were highest in the stomach, followed by mouth and the intestine. The bioaccessibility of PA-12 in steamed and sun-dried BS was higher than that of raw black soybeans (S0D0) after digestion. It reached maximum after digestion at the third steaming and sun-drying cycle (i.e. S3D3), wherein the phenolic acids with the highest bioaccessibility were syringic acid, gallic acid, ferulic acid and chlorogenic acid. Syringic acid, in particular, increased significantly during digestion compared with that before digestion, which also increased during processing. The antioxidant activity of in vitro digested BS products with appropriate steaming and sun-drying degree increased compared with S0D0. Principal component analysis (PCA) showed that the in vitro digestion-induced properties of steamed and sun-dried BS could be well distinguished. The results confirm that the phenolic compounds and bioaccessibility of nine steamed nine sun-dried BS must be taken into account when assessing the improvement of human health.

Effect of selenium biofortification on bioactive compounds and antioxidant activity in germinated black soybean.

Biofortification using inorganic selenium has become an effective strategy to enhance selenium content in crops. In the present study, the effects of selenium biofortification on the chemical composition and antioxidant capacity of black soybean (BS) during germination were studied. The contents of selenium, total sugar, vitamin C, γ-aminobutyric acid, total polyphenols, and total flavonoids in selenium biofortified germinated black soybeans (GBS-Se) significantly increased compared to germinated black soybeans (GBS). However, the contents of soluble protein, fat, and reducing sugar were decreased, while fatty acid composition was not significantly different between GBS and BS. HPLC analysis showed that 12 phenolic acids of all samples, which mainly existed in free forms. Their contents increased at low concentration of selenium and decreased along with the rise of selenium concentrations. The antioxidant activity of GBS-Se as analyzed by Pearson correlation analysis positively correlated with the accumulation of phenolic substances. Principal component analysis (PCA) showed that GBS and GBS-Se were significantly different from BS. Moreover, the physicochemical indexes of GBS showed regularly changes with increasing selenium content, and those of GBS-Se50 and GBS-Se75 were significantly different from GBS. The results provide a systematic evaluation on the effect of selenium fortification on the germination of seeds and useful information for the development of Se-enriched functional foods. PRACTICAL APPLICATION: The organic selenium black soybean (BS) produced by the germination method can be directly processed and eaten to improve human health. In addition, complexes of organic selenium, vitamin C, and γ-aminobutyric acid of germinated BS can be developed into functional substances and applied to food or health products as functional ingredient and/or natural antioxidant supplements.

Effects of "nine steaming nine sun-drying" on proximate composition, protein structure and volatile compounds of black soybeans.

Nine steaming nine sun-drying is a traditional processing technology for food or medicinal materials. The dynamic changes of the proximate composition, protein structure and volatile compounds during nine-time steaming and sun-drying of black soybeans (BS) were studied. The proximate composition results showed that the content of protein, carbohydrate and fat of BS decreased after processing, whereas the relative content of amino acids remained basically unchanged. Protein structure was evaluated using Fourier transform infrared spectroscopy (FT-IR), Ultraviolet absorption spectroscopy (UV) and Fluorescence spectroscopy. FT-IR result revealed that the relative contents of ß-sheet and ß-turn of the secondary structure of black soybean protein isolate (BSPI) decreased but the relative contents of α-helix and random coil increased after steaming and sun-drying. The results of UV and fluorescence spectroscopy confirmed changes in the protein conformation. In addition, SPME-GCMS analysis demonstrated that hydrocarbons, alcohols and aldehydes were the main volatile compounds. The relative contents of 1-octen-3-ol and hexanal, which are the main sources of beany flavor decreased significantly compared with raw BS. Principal component analysis (PCA) results showed that the volatile compounds of nine steamed and nine sun-dried BS could be well distinguished during the process. These findings may therefore provide a scientific basis for the application of nine-time steamed and sun-dried BS in food industry and contribute to the understanding of process-induced chemical transformations in this ancient processing technique.

Impact of Germinated Brown Rice and Brown Rice on Metabolism, Inflammation, and Gut Microbiome in High Fat Diet-Induced Insulin Resistant Mice.

The constituents of germinated brown rice (GBR), brown rice (BRR), and white rice (WHR) and their impact on metabolism, inflammation, and gut microbiota in high fat (HF) diet-fed mice were examined. The contents of total fiber and γ-aminobutyric acid in BRR and GBR were higher than that in WHR (p < 0.05). Male C57 BL/6J mice received HF diet+26 g% of WHR, BRR, or GBR for 12 weeks. BRR and GBR comparably reduced HF diet-induced increases in fasting plasma glucose, lipids, insulin resistance, and inflammatory markers compared to WHR (p < 0.01). The abundance of fecal Bacteroidetes in mice fed HF+GBR or HF+BRR was higher than in HF+WHR-fed mice (p < 0.05). The abundance of fecal Lactobacillus gasseri in GBR-fed mice was greater than that in WHR- or BRR-fed mice (p < 0.05). The results indicated that GBR or BRR attenuated hyperglycemia, insulin resistance, and inflammation in mice. HF+GBR, but not HF+BRR, increased a probiotic bacteria in the gut.

Enhanced L-phenylalanine production by recombinant Escherichia coli BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach.

The L-phenylalanine (L-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved L-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in L-Phe production and a remarkable tolerance to 1 × 10(10) pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced L-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH-stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on L-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at µ (set) = 0.18 h(-1) in the first 20 h and a following linear varying rate feeding with F = (-0.55 × t + 18.6) ml/h, was developed to improve L-Phe production. With this two-stage feeding approach, a maximum L-Phe titer of 57.63 g/l with a high L-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential L-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).

Effects of "nine steaming nine sun-drying" on proximate composition, oil properties and volatile compounds of black sesame seeds.

Black sesame seeds (BSS) were processed by nine cycles of steaming and sun-drying, and the chemistry of their resulting products studied. That is, the shell color and structure, proximate composition, oil properties and volatile compounds of raw BSS were determined and compared with processed BSS. Various levels of shell color change and structure damage were observed. The proximate composition also differed, whereas the relative proportion of fatty acids and oil properties were unchanged. SPME-GCMS analysis revealed that aldehydes, hydrocarbons and alcohols were the main volatile compounds. And compared with raw BSS, four volatile substances were newly detected in the processed BSS. Principal component analysis (PCA) displayed the overall difference between samples and showed that repeated steaming and sun-drying process had a significant impact on the chemical composition of BSS.

Glutathione protects Lactobacillus sanfranciscensis against freeze-thawing, freeze-drying, and cold treatment.

Lactobacillus sanfranciscensis DSM20451 cells containing glutathione (GSH) displayed significantly higher resistance against cold stress induced by freeze-drying, freeze-thawing, and 4 degrees C cold treatment than those without GSH. Cells containing GSH were capable of maintaining their membrane structure intact when exposed to freeze-thawing. In addition, cells containing GSH showed a higher proportion of unsaturated fatty acids in cell membranes upon long-term cold treatment. Subsequent studies revealed that the protective role of GSH against cryodamage of the cell membrane is partly due to preventing peroxidation of membrane fatty acids and protecting Na(+),K(+)-ATPase. Intracellular accumulation of GSH enhanced the survival and the biotechnological performance of L. sanfranciscensis, suggesting that the robustness of starters for sourdough fermentation can be improved by selecting GSH-accumulating strains. Moreover, the results of this study may represent a further example of mechanisms for stress responses in lactic acid bacteria.

[Screening and evaluation of saline-alkali-tolerant and growth-promoting bacteria].

Salinity is the most important factor for the growth of crops. It is an effective method to alleviate the toxic effect caused by salt stress using saline-alkali-tolerant and growth-promoting bacteria in agriculture. Seven salt-tolerant bacteria were screened from saline-alkali soil, and the abilities of EPS production, alkalinity reduction and IAA production of the selected strains were investigated. A dominant strain DB01 was evaluated. The abilities of EPS production, alkalinity reduction and IAA production of strain DB01 were 0.21 g/g, 8.7% and 8.97 mg/L, respectively. The isolate was identified as Halomonas aquamarina by partial sequencing analysis of its 16S rRNA genes, and had the ability to inhibit the growth of Fusarium oxysporum f. sp., Alternaria solani, Phytophthora sojae and Rhizoctonia cerealis. It also could promote root length and germination rate of wheat seedlings under salt stress. Halomonas aquamarina can provide theoretical basis for the development of soil microbial resources and the application in saline-alkali soil improvement.

Assessment of binding interaction dihydromyricetin and myricetin with bovine lactoferrin and effects on antioxidant activity.

The binding interactions of bovine lactoferrin (BLF) with two flavonoids dihydromyricetin (DMY) and myricetin (MY) were investigated by the multi-spectroscopic, microscale thermophoresis (MST) techniques, molecular docking, and then their antioxidant activities were studied by detection of free radical scavenging activity against DPPH. Results of UV-vis and fluorescence spectroscopies showed that DMY/MY and BLF formed the ground state complex through the static quenching mechanism. Moreover, MY with more planar stereochemical structure had higher affinity for BLF than DMY with twisted stereochemical structure, according to the binding constant (Kb), free energy change (ΔG°), dissociation constant (Kd) and donor-acceptor distance (r). Thermodynamic parameters revealed that hydrogen bond and van der Waals force were major forces in the formation of BLF-DMY complex, while hydrophobic interactions played major roles in the formation of BLF-DMY complex. The circular dichroism (CD) study indicated that MY induced more conformational change in BLF than DMY. Furthermore, molecular modeling provided insights into the difference of binding interactions between BLF and two flavonoids. Finally, the radical scavenging activity assays indicated the presence of BLF delayed the decrease in antioxidant capacities of two flavonoids. These results were helpful to understand the binding mechanism and biological effects of non-covalent BLF-flavonoid interaction.

Production of 2,3-dihydroxyisovalerate by Enterobacter cloacae.

2,3-Dihydroxyisovalerate is an intermediate of the valine synthesis pathway. However, neither natural microorganisms nor valine producing engineered strains have been reported yet to produce this chemical. Based on the 2,3-butanediol synthesis pathway, a biological route of 2,3-dihydroxyisovalerate production was developed using a budA and ilvD disrupted Klebsiella pneumoniae strain in our previous research. We hypothesised, that other 2,3-butanediol producing bacteria could be used for 2,3-dihydroxyisovalerate production. Here a budA disrupted Enterobacter cloacae was constructed, and this strain exhibited a high 2,3-dihydroxyisovalerate producing ability. Disruption of ilvD in E. cloacae ΔbudA further increased 2,3-dihydroxyisovalerate level. The disruption of budA, encoding an acetolactate decarboxylase, resulted in the acetolactate synthesized in the 2,3-butanediol synthesis pathway to flow into the valine synthesis pathway. The additional disruption of ilvD, encoding a dihydroxy acid dehydratase, prevented the 2,3-dihydroxyisovalerate to be further metabolized in the valine synthesis pathway. Thus, the disruption of both budA and ilvD in 2,3-butanediol producing strains might be an universal strategy for 2,3-dihydroxyisovalerate accumulation. After optimization of the medium components and culture parameters 31.2 g/L of 2,3-dihydroxyisovalerate was obtained with a productivity of 0.41 g/L h and a substrate conversion ratio of 0.56 mol/mol glucose in a fed-batch fermentation. This approach provides an economic way for 2,3-dihydroxyisovalerate production.

Enhancement of glutathione production with a tripeptidase-deficient recombinant Escherichia coli.

Glutathione (GSH) degradation exists in the enzymatic synthesis of GSH by Escherichia coli, however, its degradation pathway is not very clear. This paper examines the key enzymes responding to GSH degradation in E. coli with the purpose of improving GSH production. The enzymes that are probably associated with GSH degradation were investigated by disrupting their genes. The results suggested that gamma-glutamyltranspeptidase (GGT) and tripeptidase (PepT) were the key enzymes of GSH degradation, and GGT contributed more to GSH degradation than PepT. Furthermore, GGT activity was affected greatly by culture temperature, and the effect of GGT on GSH degradation could be eliminated by shortening the culture time at 30 degrees C and extending the induction time at 42 degrees C. However, the effect of PepT on GSH degradation could be eliminated only by disrupting the PepT gene. Finally, GSH degradation was not observed in GSH biosynthesis by E. coli JW1113 (pepT(-), pBV03), which was cultured at 30 degrees C for 3 h and 42 degrees C for 5 h. GSH concentration reached 15.60 mM, which was 2.19-fold of the control. To the best of our knowledge, this is the first report of prohibiting GSH degradation with PepT-deficient recombinant E. coli. The results are helpful to investigate the GSH metabolism pathway and construct a GSH biosynthesis system.

Yeast extract promotes cell growth and induces production of polyvinyl alcohol-degrading enzymes.

Polyvinyl alcohol-degrading enzymes (PVAases) have a great potential in bio-desizing processes for its low environmental impact and low energy consumption. In this study, the effect of yeast extract on PVAases production was investigated. A strategy of four-point yeast extract addition was developed and applied to maximize cell growth and PVAases production. As a result, the maximum dry cell weight achieved was 1.48 g/L and the corresponding PVAases activity was 2.99 U/mL, which are 46.5% and 176.8% higher than the control, respectively. Applying this strategy in a 7 L fermentor increased PVAases activity to 3.41 U/mL. Three amino acids (glycine, serine, and tyrosine) in yeast extract play a central role in the production of PVAases. These results suggest that the new strategy of four-point yeast extract addition could benefit PVAases production.

Enhanced l-phenylalanine biosynthesis by co-expression of pheA(fbr) and aroF(wt).

A beta-2-thienylalanine-resistant E. coli K12 mutant carrying a Thr326Pro mutation in the regulation (R) domain of pheA (pheA(fbr)) was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. In the presence of 200mM l-phenylalanine (l-Phe), a recombinant E. coli WSH-Z06 (pAP-B03) carrying pheA(fbr) as well as wild-type aroF (aroF(wt)) exhibited more than 70% of the chorismate mutase-prephenate dehydratase (CM-PDT) activity as observed in the absence of this amino acid. The l-Phe titer of WSH-Z06 (pAP-B03) reached 35.38g/L in a 3-L fermentor, which was 2.81-fold higher than that of the original strain E. coli WSH-Z06. Furthermore, the l-Phe yield on glucose of WSH-Z06 (pAP-B03) (0.26mol/mol) was twice that of E. coli WSH-Z06. This recombinant E. coli WSH-Z06 (pAP-B03) is a potential strain for over-production of l-Phe.

Anti-oxidative stress and beyond: multiple functions of the protein glutathionylation.

Glutathionylation, covalently attaching glutathione(s) to cysteine residue(s) of a protein, has attracted great attention in recent years. The importance of glutathionylation was initially recognized for its role in protecting proteins from irreversible oxidation; however, more studies indicate that glutathionylation is also involved in redox regulation under both normal physiological conditions and oxidative stresses. Potential mechanisms for the formation of glutathionylated proteins have been proposed. Despite the differences among the details of these mechanisms, glutathionylation is generally induced by intermediates including glutathione disulfide, protein-sulfenic acids, and thiyl radical. Taking advantages of proteomics techniques, authors have established methods to identify glutathionylation utilizing (35)S-cysteine- or biotin-labeled glutathione, or anti-GSH antibodies. Glutathionylation serves multiple roles in cellular biochemistry, such as modulation of enzymatic activity, glutathione storage, and dynamic regulation of protein function. Development of more efficient methods for glutathionylation identification, systematic investigation of its roles in the context of cellular biochemistry, the interaction with other types of protein modification, and its relevance to some health-threatening diseases will be the wider focus of studies in protein glutathionylation.

A new strategy to enhance glutathione production by multiple H2O2 induced oxidative stresses in Candida utilis.

Effect of H(2)O(2)-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H(2)O(2) can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H(2)O(2) stresses with different concentrations (1 mmol/L at 4h, 2 mmol/L at 8h, and 4 mmol/L at 12h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H(2)O(2).