Sensitive lactate determination based on acclimated mixed bacteria and palygorskite co-modified oxygen electrode

A sensitive bacteria biosensor was prepared for the detection of trace lactate. The sensitive bioelement, Lactobacillus bulgaricus and Streptococcus thermophilus mixed cultrue, and palygorskite, a perfect matrix for bacteria, was co-immobilized on the surface of oxygen electrode. The biosensor possesses fine selective specificity, good sensitivity and longer operational life time, which were due to the mutual help relationship of symbiotic bacteria and 240 days acclimation with lactate as the carbon source. Hydrodynamic amperometry, an advanced electrochemical method, is suitable for on-line monitoring the concentration change of dissolved oxygen that is closely accompanied with the metabolism of lactate. Electrochemical data show that the current is very sensitive to the changes of the concentration of lactate. The response current was linear with lactic acid concentration in the range from 0 to 300 μmol L^? 1, where the response time is no more than 240 s (R = 0.9952), and the sensitivity was 1.87 mA mol^? 1 L. Experiments show the biosensor is also very useful for long time on-line monitoring of lactate, such as fermentation progress.     

Analyzing and monitoring of phage–bacteria interaction using CE

The utilization of CE for monitoring bacteria–phage interaction was investigated in this study. Streptococcus thermophilus and Lactobacillus bulgaricus strains and their phages were used as model bacteria and phages for the purpose of validation in this study. CE with heterogeneous polymer polyethylene oxide was utilized for the separation of intact bacteria and investigation of phage–bacteria interaction. An intact phage detection was carried out with CZE by adding SDS in the running buffer. Calibration graphs of bacteria and phages were obtained with R² values of 0.963 and 0.937, respectively. S. thermophilus strain was infected with its virulent phage B3‐X18 for investigation of phage–bacteria interaction. It was observed in capillary electropherogram that the culture was lysed depending on the multiplicity of infection value and it showed to be completely lysed when the multiplicity of infection value was 10. The interaction of S. thermophilus strain with L. bulgaricus phage was also investigated by using a CE and a microbiological method and it was observed that the L. bulgaricus phage attached itself to the cell wall of S. thermophilus strain without damaging the cell.     

Rapid differentiation of simple saccharides based on cluster ions by paper spray tandem mass spectrometry

Simple saccharides have a variety of biological functions, but their structural diversity and inherent structural features pose a major challenge for rapid analysis. In this work, we developed a derivative-free and ion mobility-free method for the rapid analysis of monosaccharides and disaccharides using paper spray tandem mass spectrometry. Trimeric cluster ions consisting of saccharide analytes, ligands and transition metal ions are used as precursor ions. We defined the R-value as the ratio of the intensity of the product ion that loses one molecule of ligand over the intensity of the product ion that loses one molecule of saccharide via collision induced dissociation (CID). The species and conformation of simple saccharides can be easily differentiated by calculating this R-value. With the capability of directly analyzing clinical samples using paper spray ionization, our method can be used to rapidly quantify the molar ratio of galactose to glucose in dried plasma samples to aid in the diagnosis of galactosemia. The analytical strategy provided herein has good potential to be applied to a wide range of saccharide analysis applications in the future.     

Comparison of two liquid-state NMR methods for the determination of saccharides in carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) roots

In order to determine the saccharide content of plant tissues, we studied a new non-destructive and fast analytical method that we call “direct quantitative proton nuclear magnetic resonance spectroscopy” (d q ¹H NMR): the application of quantitative proton nuclear magnetic resonance spectroscopy (q ¹H NMR) to non modified plant tissues along with capillary tubes containing a reference compound (for quantification) and deuterium oxide (for locking). Using this method, the saccharide content of samples of carrot (Daucus carota L.) roots was compared to the results given from similar samples by the formerly published q ¹H NMR determination of extracts obtained by the O'Donoghue/Davis method. The content in glucose and sucrose is significantly higher with the direct method than when an extraction step is included; the content in fructose is not significantly different. If a possible detection of saccharides included in glycosylated biological compounds is to be excluded, a more complete extraction of saccharides is probably obtained using the direct method.     

Fortification of Acidophilus-bifidus-thermophilus (ABT) Fermented Milk with Heat-Treated Industrial Yeast Enhances Its Selected Properties

The improvement of milk dairy products’ quality and nutritional value during shelf-life storage is the ultimate goal of many studies worldwide. Therefore, in the present study, prospective beneficial effects of adding two different industrial yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae pretreated by heating at 85 °C for 10 min to be inactivated, before fermentation on some properties of ABT fermented milk were evaluated. The results of this study showed that the addition of 3% and 5% (w/v) heat-treated yeasts to the milk enhanced the growth of starter culture, Lactobacillus acidophilus, Bifidobacteria, and Streptococcus thermophilus, during the fermentation period as well as its viability after 20 days of cold storage at 5 ± 1 °C. Furthermore, levels of lactic and acetic acids were significantly increased from 120.45 ± 0.65 and 457.80 ± 0.70 µg/mL in the control without heat-treated yeast to 145.67 ± 0.77 and 488.32 ± 0.33 µg/mL with 5% supplementation of Sacch. cerevisiae respectively. Moreover, the addition of heat-treated yeasts to ABT fermented milk enhanced the antioxidant capacity by increasing the efficiency of free radical scavenging as well as the proteolytic activity. Taken together, these results suggest promising application of non-viable industrial yeasts as nutrients in the fermentation process of ABT milk to enhance the growth and viability of ABT starter cultures before and after a 20-day cold storage period by improving the fermented milk level of organic acids, antioxidant capacity, and proteolytic activities.     

Optimization of Fermentation Process of Pomegranate Peel and Schisandra Chinensis and the Biological Activities of Fermentation Broth: Antioxidant Activity and Protective Effect Against H2O2-induced Oxidative Damage in HaCaT Cells

In this study, the lactobacillus fermentation process of pomegranate (Punica granatum L.) peel and Schisandra chinensis (Turcz.) Baill (PP&SC) was optimized by using the response surface method (RSM) coupled with a Box-Behnken design. The optimum fermentation condition with the maximal yield of ellagic acid (99.49 ± 0.47 mg/g) was as follows: 1:1 (w:w) ratio of pomegranate peel to Schisandra chinensis, 1% (v:v) of strains with a 1:1 (v:v) ratio of Lactobacillus Plantarum to Streptococcus Thermophilus, a 37 °C fermentation temperature, 33 h of fermentation time, 1:20 (g:mL) of a solid–liquid ratio and 3 g/100 mL of a glucose dosage. Under these conditions, the achieved fermentation broth (FB) showed stronger free radical scavenging abilities than the water extract (WE) against the ABTS⁺, DPPH, OH⁻ and O₂⁻ radicals. The cytotoxicity and the protective effect of FB on the intracellular ROS level in HaCaT cells were further detected by the Cell Counting Kit-8 (CCK-8) assay. The results showed that FB had no significant cytotoxicity toward HaCaT cells when its content was no more than 8 mg/mL. The FB with a concentration of 8 mg/mL had a good protective effect against oxidative damage, which can effectively reduce the ROS level to 125.94% ± 13.46% (p < 0.001) compared with 294.49% ± 11.54% of the control group in H₂O₂-damaged HaCaT cells. The outstanding antioxidant ability and protective effect against H₂O₂-induced oxidative damage in HaCaT cells promote the potential for the FB of PP&SC as a functional raw material of cosmetics.     

Comparative direct infusion ion mobility mass spectrometry profiling of Thermus thermophilus wild-type and mutant ∆cruC carotenoid extracts

The major carotenoid species isolated from the thermophilic bacterium Thermus thermophilus HB27 have been identified as zeaxanthin–glucoside–fatty acid esters (thermozeaxanthins and thermobiszeaxanthins). Most of the genes of the proposed T. thermophilus carotenoid pathway could be found in the genome, but there is less clarity about the genes which encode the enzymes performing the final carotenoid glycosylation and acylation steps. To get a further insight into the biosynthesis of thermo(bis)zeaxanthins in T. thermophilus, we deleted the megaplasmid open reading frame TT_P0062 (termed cruC) by both exchanging it with a kanamycin resistance cassette (ΔcruC:kat) and by generating a markerless gene deletion strain (ΔcruC). A fast and efficient electrospray ionization–ion mobility–time-of-flight mass spectrometry method via direct infusion was developed to compare the carotenoid profiles of wild type and mutant T. thermophilus cell culture extracts. These comparisons revealed significant alterations in the carotenoid composition of the ΔcruC mutant, which was found to accumulate zeaxanthin. This is the first experimental evidence that the ORF encodes the glycosyltransferase enzyme necessary for the glycosylation of zeaxanthin in the final modification steps of the thermozeaxanthin biosynthesis in T. thermophilus HB27. Also, the proposed method for direct determination of carotenoid amounts and species in crude acetone extracts represents an improvement over existing methods in terms of speed and sensitivity and may be applicable in high-throughput analyses of other terpenoids as well as other important bacterial metabolites like fatty acids and their derivatives.

高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物

建立了高效液相色谱(HPLC)同时测定生物质乳酸发酵液中有机酸及糖类的分析方法。使用Bio-Rad Aminex HPX-87H色谱柱,以5 mmol/L的H2SO4为流动相,在柱温55 ℃,流速0.6 mL/min条件下,采用示差折光检测器进行检测。结果表明,该方法可在17 min内实现发酵液中各种有机酸和糖类化合物等的完全分离与定量,6种有机酸和3种糖类化合物在0.15～5.19 g/L范围内的线性关系良好,回归方程的线性相关系数在0.9998以上。将该法用于米根霉发酵液的检测,两个水平的加标回收率为96.91%～103.11%,相对标准偏差(n=6)为0.81%～4.61%。该法适用于微生物发酵液中多种有机酸和糖类的快速、高效分离和定量测定。     

Rapid determination of 5-hydroxymethylfurfural by DART ionization with time-of-flight mass spectrometry

DART (direct analysis in real time), a novel technique with wide potential for rapid screening analysis, coupled with high-resolution time-of-flight mass spectrometry (TOF-MS) has been used for quantitative analysis of 5-hydroxymethylfurfural (5-HMF), a typical temperature marker of food. The DART/TOF-MS method was optimised and validated. Quantification of 5-HMF was achieved by use of a stable isotope-labelled 5-HMF standard prepared from glucose. Formation of 5-HMF from saccharides, a potential source of overestimation of results, was evaluated. Forty-four real samples (honey and caramelised condensed sweetened milk) and 50 model samples of heated honey were analysed. The possibility of using DART for analysis of heated samples of honey was confirmed. HPLC and DART/TOF-MS methods for determination of 5-HMF were compared. The correlation equation between these methods was DART?=?1.0287HPLC?+?0.21340, R ²?=?0.9557. The DART/TOF-MS method has been proved to enable efficient and rapid determination of 5-HMF in a variety of food matrices, for example honey and caramel.     

A biopolymer-based carbon nanotube interface integrated with a redox shuttle and a D-sorbitol dehydrogenase for robust monitoring of D-sorbitol

We describe the preparation and characterization of a glassy carbon electrode modified with a bionanocomposite consisting of a hyaluronic acid, dispersed carbon nanotubes, and electrostatically bound toluidine blue. The electrode was used to detect NADH in the batch and flow-injection mode of operation. The electrode was further modified by immobilizing sorbitol dehydrogenase to result in biosensor for D-sorbitol that displays good operational stability, a sensitivity of 10.6???A?mM^?1?cm^?2, a response time of 16?s, and detection limit in the low micromolar range. The biosensor was successfully applied to off-line monitoring of D-sorbitol during its bioconversion into L-sorbose (a precursor in the synthesis of vitamin C) by Gluconobacter oxydans. The sample assay precision is 2.5% (an average RSD) and the throughput is 65?h^?1 if operated in the flow-injection mode. The validation of this biosensor against a reference HPLC method resulted in a slope of correlation of 1.021?±?0.001 (R ²?=?0.99997).

Spectrophotometric determination of thiocyanate ions in stratal waters

The formation of [FeSCN]²⁺ complex in hydrochloric and sulfuric acid medium was studied by spectrophotometry using iron(III) sulfate and ammonium iron(III) sulfate solutions as reactants. A method for the determination of 10–200 μg SCN^? in 25 mL water solutions containing ammonium iron(III) sulfate in sulfuric acid medium was developed; its determination limit is 2.6 μg (P = 0.99, n = 9). The method was applied for the analysis of model water samples with macro- and micro-component compositions similar to that of water from the Arigol licensed area. Operational control of the accuracy rate was performed by the standard addition method. The developed method can be applied to analyze water samples containing 1–90 mg/L thiocyanate ions.     

Determination of erythromycins in fermentation broth using liquid phase extraction with back extraction combined with high performance liquid chromatography

Liquid phase extraction with back extraction (LPE-BE) combined with high performance liquid chromatography-diode array detection (HPLC-DAD) was applied for the extraction and determination of erythromycin A, B and C in fermentation broths. According to this procedure, the fermentation broth with the adjustment pH at a fixed value of 10 was first mixed with organic solvent (V_broth/V_org = 1.0). After shaking, the mixture was separated into two phases by microfuging at 13,000 rpm for 15 min. Then back extraction was performed into the acidic aqueous phase with pH 5.0 (V_org/V_aq = 1.0). After centrifugation at 3000, the two phases were separated and 50 μL of the acidic aqueous phase was injected into the HPLC. The effects of different variables such as the nature of extraction solvent and the pH of samples and buffer were investigated. At the most appropriate conditions, dynamic linear ranges of 0.5–8, 0.1–0.9 and 0.1–0.9 mg mL⁻¹ and limits of detection of 0.03, 0.003 and 0.002 mg mL⁻¹ were obtained for erythromycin A, B and C, respectively. Relative standard deviations (RSDs) of the proposed method were less than 9.5%. The mean recoveries were 99.5%. The proposed method is simple and sensitive with highly clean-up effect and it can be used for monitoring the progress of erythromycin fermentation.     

Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection

A rapid liquid-chromatography (LC) method is presented which uses fluorescence detection (FLD) for palytoxin analogues analysis in benthic dinoflagellates of the genus Ostreopsis. The amino-acidic reagent 6-aminoquinolyl-N-hydroxisuccinimidyl carbamate (AccQ) was used for fluorescence labelling followed by LC-FLD.The efficacy of the method is exemplified by comparison of the results of the quantification obtained by LC-FLD and the hemolytic assay performed for palytoxins for which a highly significant linear correlation was achieved (r² = 0.9118). The derivatized palytoxin analogues were determined in the range of 0.75-25 ng.The proposed method was successfully applied to the determination and quantification of palytoxin analogues in 14 samples from different strains of Ostreopsis from different locations (Western Mediterranean Sea, Canary Islands, Madeira Islands and Southern coasts of Brazil). To confirm the chemical structure of the toxins, samples were also analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) with a system that has a poorer sensitivity when compared with LC-FLD detection and the hemolytic assay. The successful use of this method with dinoflagellates is a good indicator of suitability for other types of marine samples.     

Rapid determination of 226Ra in environmental samples

A new rapid method for the determination of ²²⁶Ra in environmental samples has been developed at the Savannah River Site Environmental Lab (Aiken, SC, USA) that can be used for emergency response or routine sample analyses. The need for rapid analyses in the event of a Radiological Dispersive Device or Improvised Nuclear Device event is well-known. In addition, the recent accident at Fukushima Nuclear Power Plant in March, 2011 reinforces the need to have rapid analyses for radionuclides in environmental samples in the event of a nuclear accident. ²²⁶Ra (T1/2?=?1,620?years) is one of the most toxic of the long-lived alpha-emitters present in the environment due to its long life and its tendency to concentrate in bones, which increases the internal radiation dose of individuals. The new method to determine ²²⁶Ra in environmental samples utilizes a rapid sodium hydroxide fusion method for solid samples, calcium carbonate precipitation to preconcentrate Ra, and rapid column separation steps to remove interferences. The column separation process uses cation exchange resin to remove large amounts of calcium, Sr Resin to remove barium and Ln Resin as a final purification step to remove ²²⁵Ac and potential interferences. The purified ²²⁶Ra sample test sources are prepared using barium sulfate microprecipitation in the presence of isopropanol for counting by alpha spectrometry. The method showed good chemical recoveries and effective removal of interferences. The determination of ²²⁶Ra in environmental samples can be performed in less than 16?h for vegetation, concrete, brick, soil, and air filter samples with excellent quality for emergency or routine analyses. The sample preparation work takes less than 6?h. ²²⁵Ra (T1/2?=?14.9?day) tracer is used and the ²²⁵Ra progeny ²¹⁷At is used to determine chemical yield via alpha spectrometry. The rapid fusion technique is a rugged sample digestion method that ensures that any refractory radium particles are effectively digested. The preconcentration and column separation steps can also be applied to aqueous samples with good results.     

Volumetric properties and volumetric interaction parameters for the CsCl–monosaccharide (d-galactose,d-xylose and d-arabinose)–water systems at T=298.15 K

Density measurements have been carried out at T=298.15 K for the CsCl–monosaccharide (d-galactose, d-xylose and d-arabinose)–water systems. The apparent molar volume of saccharides V_φ,S in the ternary solutions, the corresponding infinite dilution apparent molar volume V_φ,S^∘, and the standard transfer volume Δ_tV_φ,S^∘ of saccharides from water to aqueous CsCl solutions have been determined. The McMillan–Mayer theory was employed to relate the excess thermodynamic functions to a series of interaction parameters to obtain the volumetric interaction parameters of CsCl with monosaccharide in water. These parameters are interpreted by the group additivity principle and the stereochemistry of these monosaccharide molecules.     

Multicomponent analysis of fermentation growth media using the electronic tongue (ET)

A potentiometric electronic tongue (ET) consisting of eight cross-sensitive chemical sensors and a standard pH electrode has been applied for analysis of simulated fermentation solutions typical for fermentation processes with Aspergillus niger. The electronic tongue has been found capable of simultaneous determination of ammonium, citrate and oxalate in complex media with good precision (typical error within 8%). The system preserved high sensitivity to the targeted substances also in the presence of sodium azide, which is commonly used for suppressing microbial activity in real-world fermentation samples. Sensor performance was fast and reproducible which promises well for routine application of the electronic tongue for fermentation process monitoring.     

Flavonoid-Rich Plants Used as Sole Substrate to Induce the Solid-State Fermentation of Laccase

High cost becomes the major obstacle for the industrial application of laccase. Many approaches have been applied to enhance the yield and decrease the cost of laccase. Since flavonoids are the natural inducers for laccase production, in this article, flavonoid-rich plants were taken as the sole substrate for the solid-state fermentation of Funalia trogii (Cui 3676). It indicated that flavonoid-rich plants can effectively promote the production of F. trogii laccase without the addition of inducers. The laccase activity was 42.5 IU g^?1 substrate when kudzu vine root was used as the substrate, which was enhanced by 4.46 times than that when bran was used as the substrate. Meanwhile, the solid-state fermentation of laccase could enrich flavonoids, benefiting their extraction. The content of flavonoids extracted from fermented kudzu vine root and Ginkgo biloba leaves was enhanced by 56.41 and 24.11 %, respectively, compared to the unfermented substrate, and the relative reductive ability and scavenging ability of hydroxyl radicals of flavonoids in the fermented residues were essentially unchanged. Thus, flavonoid-rich plants will become a kind of potential substrate for laccase fermentation which is beneficial in enhancing the yield and reducing the cost of laccase.     

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l^?1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol^?1 added cysteine and a productivity of 6.1 ± 0.0 g l^?1 h^?1. This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.     

H NMR and C-IRMS analyses of acetic acid from vinegar, O-IRMS analysis of water in vinegar: International collaborative study report

An international collaborative study of isotopic methods applied to control the authenticity of vinegar was organized in order to support the recognition of these procedures as official methods. The determination of the ²H/¹H ratio of the methyl site of acetic acid by SNIF-NMR (site-specific natural isotopic fractionation-nuclear magnetic resonance) and the determination of the ¹³C/¹²C ratio, by IRMS (isotope ratio mass spectrometry) provide complementary information to characterize the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic acetic acid. Both methods use the same initial steps to recover pure acetic acid from vinegar. In the case of wine vinegar, the determination of the ¹⁸O/¹⁶O ratio of water by IRMS allows to differentiate wine vinegar from vinegars made from dried grapes. The same set of vinegar samples was used to validate these three determinations.The precision parameters of the method for measuring δ¹³C (carbon isotopic deviation) were found to be similar to the values previously obtained for similar methods applied to wine ethanol or sugars extracted from fruit juices: the average repeatability (r) was 0.45 ‰, and the average reproducibility (R) was 0.91‰. As expected from previous in-house study of the uncertainties, the precision parameters of the method for measuring the ²H/¹H ratio of the methyl site were found to be slightly higher than the values previously obtained for similar methods applied to wine ethanol or fermentation ethanol in fruit juices: the average repeatability was 1.34 ppm, and the average reproducibility was 1.62 ppm. This precision is still significantly smaller than the differences between various acetic acid sources (δ¹³C and δ¹⁸O) and allows a satisfactory discrimination of vinegar types. The precision parameters of the method for measuring δ¹⁸O were found to be similar to the values previously obtained for other methods applied to wine and fruit juices: the average repeatability was 0.15‰, and the average reproducibility was 0.59‰. The above values are proposed as repeatability and reproducibility limits in the current state of the art.On the basis of this satisfactory inter-laboratory precision and on the accuracy demonstrated by a spiking experiment, the authors recommend the adoption of the three isotopic determinations included in this study as official methods for controlling the authenticity of vinegar.     

Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304

Streptomyces platensis NTU 3304, isolated from soil samples, produces extracellular L-glutamate oxidase in liquid culture. Strains of this species have never been reported to be able to produce this enzyme. The purified enzyme was immobilized onto a cellulose triacetate membrane which was held at an oxygen electrode. The sensor was specific to L-glutamate in accordance with the properties of the novel L-glutamate oxidase. The time required for each assay in batch operation was less than 3 min. A linear relationship is observed between the decrease in dissolved oxygen and the concentration of L-glutamate between 20 and 140 mg l^?1 (ca. 0.12 and 0.84 mM). The sensor retained 95% of its original activity after 400 assays over a period of 3 weeks. The sensor was applied to determine the concentration of L-glutamate in broth samples during L-glutamic acid fermentation. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.