High-performance Liquid Chromatography With Evaporative Light Scattering Detection Research Articles

Determination of Saccharides in Cottonseed Processing Waste Liquid and By-Products by High Performance Liquid Chromatography with Evaporative Light Scattering Detection (HPLC-ELSD)

A method utilizing high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) has been developed and validated for the simultaneous determination of raffinose, glucose, sucrose, and stachyose in waste liquid from cottonseed processing and in byproducts including cottonseed meal. The separation was performed using a carbohydrate ES column (250 × 4.6 mm, 5 μm) and isocratic elution with acetonitrile and water (70:30, v/v). The temperature of the column oven was maintained at 35 °C. The drift tube temperature for evaporative light scattering detection (ELSD) was operated 40 °C, while the nitrogen flow was set to 1.5 L·min−1. The saccharides were separated by HPLC-ELSD within 20 min. The coefficients of determination (R 2 = 0.9986–0.9999) indicate that there was a linear relationship between saccharides and the response. The limits of detection were 0.004–0.023 mg·mL−1 and the quantification range was from 0.013 to 0.080 mg·mL−1. The intraday and interday precision were from 0.05 to 0.55% and 0.20 to 0.58%, respectively. The recovery was from 102.75 ± 2.03% to 106.76 ± 2.09% and the relative standard deviations were 1.87 to 1.98%. The method exhibited good stability in determining four saccharides in cottonseed processing waste liquid and the resultant byproduct cottonseed meal. This method exhibits high separation efficiency and sensitivity, excellent precision, and simplicity, which allows determination of the high value-added product, raffinose, in cottonseed processing waste liquid and meal.

Differentiation of High-Fructose Corn Syrup Adulterated Kelulut Honey Using Physicochemical, Rheological, and Antibacterial Parameters.

Kelulut (stingless bee) honey (KH) possesses a wide range of benefits for human consumption and can exhibit medical effects. Due to its high value, this premium honey is often adulterated with different types of cheaper sugars, causing low nutrients and potential food safety threats in the final product. This study aims to determine the physicochemical, rheological, and antibacterial properties of sugar-based adulterated KH from the stingless bee species Heterotrigona itama. Adulterated samples were prepared using pure honey mixed with different concentrations of high fructose corn syrup (HFCS), i.e., 10%, 20%, 30%, 40%, and 50%. Water activity, colour, total soluble solids, pH, turbidity, viscosity, and antimicrobial activity of KH were determined. In addition, the primary sugar composition (fructose, glucose, and trehalulose) was determined by high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). This study shows that the increasing percentage of HFCS addition in the KH samples significantly increases (p < 0.05) the total soluble solids, colour, pH, turbidity, viscosity, glucose, and fructose content; meanwhile, the water activity and trehalulose were reduced significantly (p < 0.05). Antimicrobial activity against S. aureus was reduced significantly (p = 0.006) by an increased percentage of HFCS compared to Control. Antimicrobial activity against P. aeruginosa was also found to be reduced significantly but showed non-significant effect from an increased percentage of HFCS in honey (p = 0.413). The bacterium S. aureus was more vulnerable to treatment with honey from both Control and adulterated groups compared to P. aeruginosa. In conclusion, HFCS-adulterated KH and authentic KH can be differentiated using all the parameters investigated. These data are vital for the governing bodies to ensure that KH sold in the markets is free from HFCS adulteration.

Separation, characterization and anti-inflammatory activities of galactoglycerolipids from Perilla frutescens (L.) Britton

The study was to optimize the separation procedures, characterize the galactoglycerolipids and explore their anti-inflammatory activities. Two monogalactosyldiacylglycerols (MGDGs) and three digalactosyldiacylglycerols (DGDGs) from Perilla frutescens (L.) Britton were obtained through one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The presence of additional MGDG (1-O-9Z,12Z,15Z-octadecatrienoyl-2-O-7Z,10Z,13Z-hexadecatrienoyl-3-O-(β-D-galactopyranosyl)-sn-glycerol) and DGDG (1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Z-octadecatrienoyl-3-O-(β-D-galactopyranosyl-(1'→6'')-α-D-galactopyranosyl)-sn-glycerol) was concluded for the first time in perilla, by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In lipopolysaccharide (LPS)-induced RAW264.7 cells, five galactoglycerolipids exhibited good inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner, suggesting that fatty acid chain length and unsaturation degree affected their anti-inflammatory activities.

A novel, rapid and robust HPLC-ELSD method for simultaneous determination of fructose, glucose and sucrose in various food samples: Method development and validation

The analysis of sugar compounds in food products requires rapid, accurate and cost-effective methods. For this purpose, a novel and robust high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) method was developed and validated for simultaneous determination of fructose, glucose and sucrose. Chromatographic separations were carried out on a Phenomenex Luna NH2 (5 μm particle size, 250 mm x 4.6 mm id, 100 Å) column with isocratic elution with acetonitrile:water (78:22, v/v). Drift tube temperature was set at 40 °C and nitrogen pressure was regulated to 350 kPa with Gain:7 and Filter:10 on the ELSD system. Within the test ranges for all sugar compounds (40-2000 μg mL−1), the regression equation revealed a good linear relationship (R2 = 0.9980−0.9992) with low LOD and LOQ values in the range of 0.10−0.16 and 0.30−0.49 μg mL−1. The method was successfully applied to 36 food samples including honey, molasses, jams and fruits.

Chemical Fingerprint Analysis and Quantitative Analysis of Saccharides in Morindae Officinalis Radix by HPLC-ELSD.

A method based on high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the quantitative analysis of three active compounds and chemical fingerprint analyses of saccharides in Morindae officinalis radix. Ten batches of Morindae officinalis radix were collected from different plantations in the Guangdong region of China and used to establish the fingerprint. The samples were separated with a COSMOIL Sugar-D column (4.6 mm × 250 mm, 5 μm) by using gradient elution with water (A) and acetonitrile (B). In addition, Trapped-Ion-Mobility (tims) Time-Of-Flight (tims TOF) was used to identify saccharides of Morindae officinalis radix. Fingerprint chromatogram presented 26 common characteristic peaks in the roots of Morinda officinalis How, and the similarities were more than 0.926. In quantitative analysis, the three compounds showed good regression (r = 0.9995–0.9998) within the test ranges, and the recoveries of the method were in the range of 96.7–101.7%. The contents of sucrose, kestose and nystose in all samples were determined as 1.21–7.92%, 1.02–3.37%, and 2.38–6.55%, respectively. The developed HPLC fingerprint method is reliable and was validated for the quality control and identification of Morindae officinalis radix and can be successfully used to assess the quality of Morindae officinalis radix.

Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application.

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.

Presence of Cholesterol in Non-Animal Organisms: Identification and Quantification of Cholesterol in Crude Seed Oil from Perilla frutescens and Dehydrated Pyropia tenera.

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida, and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3–0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

A Powerful HPLC-ELSD Method for Simultaneous Determination of Fecal Bile Acids in T2DM Rats Interfered by Sanhuang Xiexin Tang.

Bile acids (BAs) as important endogenous ligands can activate farnesoid X receptor (FXR) and G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) signaling to regulate glycolipid metabolism. In this study, a simple, reliable and sensitive analysis method for simultaneous determination of four BAs from rat feces based on high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was developed. Chromatographic analysis was performed with the mobile phases of acetonitrile and 0.2% formic acid. All the standard curves exhibited good linearity (R2≥0.99). The relative standard deviations of precision, stability and repeatability varied from 1.27 to 3.96%, 2.20 to 3.89% and 3.00 to 4.31%, respectively. The validated method was successfully applied to investigate the variation of four BAs in feces from T2DM rats after oral administration of Sanhuang Xiexin Tang (SXT). Data showed that SXT could remarkably increase the contents of conjunct BAs and decrease the contents of free BAs, which might contribute to ameliorate the symptoms of T2DM rats.

Comprehensive phytochemical analysis and sedative-hypnotic activity of two Acanthopanax species leaves.

Acanthopanax senticosus leaves (SCL) and Acanthopanax sessiliflorus leaves (SFL), which are usually made into functional teas, possess similar pharmacological activities. With the aim of revealing their chemical compositions and evaluating their sedative-hypnotic effects, comprehensive metabolite profiling analysis based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) and high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) as well as bioassay studies in mice were performed for the first time. Firstly, a total of 75 compounds (including 69 shared components) were identified or briefly characterized. Results indicated that the leaves of the two species were both rich in phytochemicals and contained similar structural types. Secondly, 20 and 7 chemical markers were identified from SCL and SFL, respectively. Five oleanane-type triterpene saponins (ciwujianoside C1, C3, D2, E and saniculoside N) and two lupine-type triterpene saponins (1-deoxychiisanoside and 24-hydroxychiisanoside) may be used for rapid identification of SCL and SFL. Thirdly, the contents of rutin, hederacoside D, ciwujianoside B, -C3, -E and ursolic acid in SCL (0.308%, 0.024%, 0.042%, 0.131%, 0.038%, and 0.255%, respectively) were higher than in SFL (0.067%, 0.005%, 0.012%, 0.015%, 0.002%, and 0.087%, respectively). Fourthly, an in vivo bioassay verified that both SCL and SFL could inhibit autonomous activity, shorten sleep latency and prolong sleep duration in a dose-dependent manner. To a certain degree, SCL showed a higher and more stable effect. The hypnotic effect could be inhibited by flumazenil (FLU). The two leaves not only had an obvious antagonism action of p-chlorophenoxyacetic acid (pCPA) but also showed a synergistic hypnotic effect with 5-hydroxytryptophan (5-HTP). The beneficial bioactivity may be mediated by 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA). Finally, network pharmacology analysis showed that the undifferentiated and differentiated compounds were the material basis for the similar and the different activities of two leaves. Some typical chemical markers (such as saniculoside N, hederacoside D, ciwujianoside C3, -E and ursolic acid, 24-hydroxychiisanoside and 1-deoxyisochiisanoside) were the potential active compounds and could be used as quality markers in the future. The present study furnished a basis for the further development and utilization of the leaves of these two Acanthopanax species.

Detection of galactooligosaccharides with high lactose interference in infant formula using a simple single epimer chromatography

Robust and more anti-interference enzymatic quantification of galactooligosaccharide (GOS) is important in consumer protection. However, many methods with harsh conditions could hardly separate GOS’s hydrolysates galactose from its structurally similar isomer glucose, since each of them has double epimers, especially to determinate a trace of GOS from large amounts of lactose in the food matrix. The investigation was designed to solve the problem by using High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD), applying friendly mobile phase and column. Result showed the content of galactose was seldom affected even by a high content of glucose by integrating the peak area of an excellent resolution single epimer. Moreover, the method existed a good linearity and stability (recovery rate at 90.5–105.1%), which met the statutory limit requirement for the quantitative analysis of concentrated GOS in infant formula. It was also helpful for separating and quantifying other sugar or epimers.

Chemical Fingerprint for Identification and Quality Control of Saccharides in Danhong Injection Based on HPLC-ELSD with Chemometrics

Chemical fingerprinting has been widely used for the quality control of complex natural products including traditional Chinese medicines and botanical drugs. However, there is still lack of appropriate method to monitor the batch-to-batch consistency of hydrophilic constituents, such as saccharides and oligosaccharides. In the present study, we developed a novel approach based on high performance liquid chromatography with evaporative light scattering detector(HPLC-ELSD) for establishing saccharide fingerprinting of Danhong Injection(DHI), which is a widely used botanical drug for treating cardiovascular diseases. Major saccharides in DHI were isolated and four compounds including fructose, glucose and two oligosaccharides were identified. The structures of two novel saccharides named glycerol-1-O-galactfpyranosyl-(1→4)-O-arabinofuranoside, and glycerol-1-O-galactpyranosyl-(1→4) [O-arabinfuranosyl-(1→3)]-O-arabinofuranoside were confirmed by NMR, HR-ESI-MS and GC-MS for the first time. The establishment approach was successfully applied to distinguishing 12 batches of DHI from other botanical drugs with the aid of principle component analysis as well as to evaluating batch-to-batch consistency with the help of calculating similarity of fingerprints. Our findings indicate that the chemical fingerprint of saccharides can be a useful tool for the quality control of complex natural products.

Changes and mechanisms of terrestroside B and terrestrosin K in stir-frying Tribuli Fructus

The contents of terrestroside B and terrestrosin K in Tribuli Fructus with different degree of stir-frying were determined by high performance liquid chromatography with evaporative light-scattering detector( HPLC-ELSD). The results showed that the contents of terrestroside B and terrestrosin K were increased at first and then decreased,and both of them had the highest content at the best time of heating. The results of simulated processing of Tribulus Terrestris saponins showed that when the processing time kept constant,the contents of terrestroside B and terrestrosin K were decreased gradually with the increase of processing temperature from 180 ℃ to240 ℃. At a certain temperature,the content of terrestrosin K was increased first and then decreased with the prolongation of processing time,and reached the highest level at 5 min. However,the content of terrestroside B was increased first and then decreased with the increase of processing time only at 180 ℃,and reached the highest level at 10 min. When the processing temperature was controlled at200,220 and 240 ℃ respectively,the content of terrestroside B was decreased gradually with the increase of processing time. The simulated processing products of tribuluside A,terrestroside B and terrestrosin K were qualitatively characterized by ultra-performance liquid chromatography-time of flight mass spectrometry( UPLC-TOF/MS). It was proved that tribuluside A and terrestrosin Ⅰ containing C-22-OH were dehydroxylated in the processing of Tribuli Fructus and transformed respectively into terrestroside B and terrestrosin K containing C-20-C-22 double bond. As a result,the contents of terrestroside B and terrestrosin K were increased. The sugar chains at C-3 and C-26 positions of terrestroside B and terrestrosin K could be deglycosylated and converted into monosaccharide chain saponins and short sugar chain saponins,so the contents of terrestroside B and terrestrosin K were reduced. The study provides reference for further revealing the processing principle of Tribuli Fructus.

Critical evaluation of a putative glucosamine excretion by Aspergillus niger CBS120.49 and Penicillium ochrochloron CBS123.824 under citric acid producing conditions

As one of the most frequently occurring monomers in the biosphere, glucosamine is a valuable metabolite for several applications. Although microbial glucosamine production is still in its infancy, it offers the possibility to circumvent problems associated with traditional production by hydrolysis. Of particular interest is a study with Aspergillus niger, which reports for the first time high glucosamine excretion in the early phase of citric acid production. These results have relevance for both the commercial glucosamine production and deeper insight into the regulation of organic acid excretion in fungi. To investigate glucosamine excretion, we performed bioreactor batch cultivations with Penicillium ochrochloron CBS123.824 and A. niger CBS120.49 using cultivation conditions which are known to trigger the production of citric acid. Glucosamine detection in culture filtrates was achieved by two photometric methods, High performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and HPLC with mass spectrometry detection (HPLC-MS). Surprisingly, we detected no glucosamine at all. Based on a critical review of published data for A. niger, we conclude that the reported high levels of excreted glucosamine might be an experimental artifact. However, growth experiments with glucosamine as a combined or single source for carbon or nitrogen showed that both organisms are in principle able to transport glucosamine across their plasma membrane, which is a prerequisite for the excretion of glucosamine.

A Simple Analysis Method for 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid by HPLC-ELSD with Column for Anion Analysis

4-Deoxy-l- erythro-5-hexoseulose uronic acid (DEH) is a rare deoxy sugar produced from alginate by the action of an exotype alginate lyase. A simple and rapid method for analyzing DEH using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed in this study. For chromatography, an isocratic elution of ammonium formate buffer including formic acid and a column for anion chromatography were used. In the developed method, DEH was detected at a retention time of 3.038 minutes and limits of detection (signal-noise ratio = 3) and quantification (signal-noise ratio = 10) were 37.5 and 124.9 µg/mL as a sodium DEH, respectively. In addition, separation and detection of alginate unsaturated oligosaccharides were also tested using the method. Within an analysis time of 10 minutes, it was possible to separate and detect unsaturated disaccharide, unsaturated trisaccharide, and unsaturated tetrasaccharide prepared using poly(β-d-mannuronate) lyase and sodium alginate of high mannuronate type. The HPLC-ELSD method established in this study will be applicable for quantitative analysis of DEH and measurement of exotype alginate lyase activity.

Development and Validation of an Analytical Method Based on HPLC-ELSD for the Simultaneous Determination of Rosmarinic Acid, Carnosol, Carnosic Acid, Oleanolic Acid and Ursolic Acid in Rosemary.

The safety, efficacy and stability of natural antioxidants have been the focus of research in the food industry, with the aim of rapidly analyzing and controlling the quality of rosemary and its extracts, a novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous determination of rosmarinic acid, carnosol, carnosic acid, oleanolic acid and ursolic acid in rosemary. Chromatographic separation was conducted with gradient elution mode by using a Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) with mobile phases of methanol and 0.6% acetic acid. The drift tube temperature of ELSD was 70 °C, and the pressure of nebulizer nitrogen gas was 40 Psi. The method developed has high sensitivity (with limits of detection from 1.3 to 8.6 μg/mL), acceptable linearity over the tested concentrations (with correlation coefficients from 0.991 to 0.999), good repeatability (with intra- and inter-day CV less than 3.1% for all analytes) and satisfactory accuracy (with recovery between 95.5% and 100.8%). The method has been demonstrated as a powerful tool for the functional ingredients analysis and quality control of rosemary and its extracts in a cost- and time-effective manner.

Determination of Propionylbrassinolide and Its Impurities by High-Performance Liquid Chromatography with Evaporative Light Scattering Detection.

The discovery of brassinolide in 1979, a milestone in brassinosteroids research, has sparked great interest of brassinolide analogs (BLs) in agricultural applications. Among these BLs, propionylbrassinolide has captured considerable attention because it shows plant growth regulating activity with an excellent durability. Two impurities of propionylbrassinolide were isolated and purified by semi-preparative high-performance liquid chromatography (HPLC), and the chemical structures were confirmed. For simultaneous separation and determination of propionylbrassinolide and impurities, an efficient analytical method based on HPLC with evaporative light scattering detector (HPLC-ELSD) was developed. The optimized analysis was performed on a C18 reversed phase column (250 mm × 4.60 mm, 5 μm) with isocratic elution of acetonitrile and water (90:10, v/v) as the mobile phase. The drift tube temperature of the ELSD system was set to 50 °C and the auxiliary gas pressure was 150 kPa. The regression equations demonstrated a good linear relationship (R2 = 0.9989–0.9999) within the test ranges. The limits of detection (LODs) and quantification (LOQs) for propionylbrassinolide, impurity 1 and 2 were 1.3, 1.2, 1,3 and 4.3, 4.0, 4.2 mg/L, respectively. The fully validated HPLC-ELSD method was readily applied to quantify the active ingredient and impurities in propionylbrassinolide technical concentrate. Moreover, the optimized separation conditions with ELSD have been successfully transferred to mass spectrometry (MS) detector for LC-MS determination.

Phospholipids composition and molecular species of large yellow croaker (Pseudosciaena crocea) roe

The research aims to study phospholipids (PL) classes and molecular species of large yellow croaker (Pseudosciaena crocea) roe. Both gas chromatographymass spectroscopy (GC-MS) and high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) were utilized to analyze and identify the PLs fatty acids compositions and classes in the P. crocea roe, respectively. Docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) account for 35.0% and 6.9% of the PLs. Phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE) and phosphatidylinositols (PI) account for 76.36 ± 0.62%, 12.30 ± 0.55%, 9.12 ± 0.02% and 1.09 ± 0.01% of the total PLs, respectively. In addition, the PLs molecular species were characterized by ultra-high performance liquid chromatography-electrospray ionization-quadruple-time of flight-mass spectrometry (UPLC-Q-TOF-MS). A total of 92 PLs molecular species was identified, including 49 PCs, 13 PEs, 10 phosphatidic acids (PAs), 13 phosphatidylserines (PSs), 3 phosphatidylglycerols (PGs), 2 sphingomyelins (SMs), and 2 PIs of the P. crocea roe.

Determination of soluble dietary fibre content of Okara treated with high hydrostatic pressure and enzymes: a comparative evaluation of two methods (AOAC and HPLC-ELSD).

Okara is a promising by-product from soybean and a rich source of dietary fibre, chiefly insoluble (IDF). To increase its solubility and functionality, a treatment with high hydrostatic pressure (HHP) assisted by food grade enzymes -Ultraflo® L or Viscozyme® L- has been performed. To monitor the effectiveness, an analysis of dietary fibre, mainly the soluble fraction (SDF), was accomplished by the AOAC enzymatic-gravimetric method with dialysis followed by both, spectrophotometric methods and High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD) analysis of the soluble fraction. A significant increase in SDF (≈1.5-times) to the expense of a decrease in IDF was shown and chromatograms revealed two peaks of 95 and 22kDa. Thus, treated Okara possessed a more balanced and convenient ratio of soluble to insoluble dietary fibre, which could have health benefits as prebiotic. Inbuilt interferences of the official AOAC's method for dietary fibre were confirmed and the direct HPLC-ELSD approach was about twice more sensitive than spectrophotometric methods. Consequently, the direct HPLC-ELSD analysis of the supernatant is proposed as a cheaper, faster and reliable method. Combined HHP-treatments plus specific enzymes represent a promising alternative for the valorisation and preservation of agrofood by-products.

Improvement in Thermal Stability of Sucralose by γ-Cyclodextrin Metal-Organic Frameworks.

To explain thermal stability enhancement of an organic compound, sucralose, with cyclodextrin based metal organic frameworks. Micron and nanometer sized basic CD-MOFs were successfully synthesized by a modified vapor diffusion method and further neutralized with glacial acetic acid. Sucralose was loaded into CD-MOFs by incubating CD-MOFs with sucralose ethanol solutions. Thermal stabilities of sucralose-loaded basic CD-MOFs and neutralized CD-MOFs were investigated using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and high performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Scanning electron microscopy (SEM) and powder X-ray diffraction (PXRD) results showed that basic CD-MOFs were cubic crystals with smooth surface and uniform sizes. The basic CD-MOFs maintained their crystalline structure after neutralization. HPLC-ELSD analysis indicated that the CD-MOF crystal size had significant influence on sucralose loading (SL). The maximal SL of micron CD-MOFs (CD-MOF-Micro) was 17.5 ± 0.9% (w/w). In contrast, 27.9 ± 1.4% of sucralose could be loaded in nanometer-sized basic CD-MOFs (CD-MOF-Nano). Molecular docking modeling showed that sucralose molecules preferentially located inside the cavities of γ-CDs pairs in CD-MOFs. Raw sucralose decomposed fast at 90°C, with 86.2 ± 0.2% of the compound degraded within only 1h. Remarkably, sucralose stability was dramatically improved after loading in neutralized CD-MOFs, with only 13.7 ± 0.7% degradation at 90°C within 24h. CD-MOFs efficiently incorporated sucralose and maintained its integrity upon heating at elevated temperatures.

Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava.

Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz) is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD). Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis). All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW), and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS), 10 trehalose-6-phosphate phosphatases (TPP), and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4) that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1) in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose under normal conditions. MeTPS1 was then transformed into tobacco (Nicotiana benthamiana). Results indicated that transgenic tobacco lines accumulated significant level of trehalose and possessed improved drought stress tolerance. In conclusion, cassava accumulated significantly high amount of trehalose under normal conditions due to multiplied trehalose biosynthesis gene families and constant expression of the active MeTPS1 gene. High levels of trehalose subsequently contributed to high drought stress tolerance.