Pressurized Hot Water Extraction and Bio-Hydrogels Formulation with Aristotelia chilensis [Mol.] Stuntz Leaves.

Aristotelia chilensis is a plant rich in phenolics and other bioactive compounds. Their leaves are discarded as waste in the maqui berry industry. A new application of these wastes is intended by the recovery of bioactive compounds using pressurized hot water extraction with conventional or microwave heating. Both technologies have been selected for their green character regarding the type of solvent and the high efficiency in shorter operation times. Extractions were performed in the temperature range 140-200 °C with a solid/liquid ratio of 1:15 (w:w). The extracts' total phenolic content, antioxidant capacity, and saccharides content obtained with both heating methods were measured. Additionally, the thermo-rheological properties of the gelling matrix enriched with these extracts were analyzed. Optimum conditions for lyophilized extracts were found with conventional heating, at 140 °C and 20 min extraction; 250.0 mg GAE/g dry extract and 1321.5 mg Trolox/g dry extract. Close to optimum performance was achieved with microwave heating in a fraction of the time (5 min) at 160 °C (extraction), yielding extracts with 231.9 mg GAE/g dry extract of total phenolics and antiradical capacity equivalent to 1176.3 mg Trolox/g dry extract. Slightly higher antioxidant values were identified for spray-dried extracts (between 5% for phenolic content and 2.5% for antioxidant capacity). The extracts obtained with both heating methods at 200 °C contained more than 20% oligosaccharides, primarily glucose. All the formulated gelling matrices enriched with the obtained extracts displayed intermediate gel strength properties. The tested technologies efficiently recovered highly active antioxidant extracts, rich in polyphenolics, and valuable for formulating gelling matrices with potential applicability in foods and other products.

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid.

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

Study on a novel spherical polysaccharide from Fructus Mori with good antioxidant activity.

A novel polysaccharide (MFP1P) was isolated from Fructus Mori, followed by purification via DEAE-52 cellulose and 27 % ethanol fraction. The MFP1P had the molecular weight of 56.78 kDa and the total sugar content of 93.32±0.54 %. And the MFP1P is mainly composed of glucose, galactose, galacturonic acid and mannose with molar ratio of 66.62 %, 13.94 %, 18.24 % and 1.20 %, respectively. MFP1P was mainly composed of →3)-α-D-Gal (1→, ß-D-Man-(1→ and →6)-α-D-Glc (1→ glycosidic bond and showed a spherical chain conformation with uniform distribution in solution. The MFP1P exhibited great antioxidant activity with oxygen-free radical absorption capacity (ORAC) values of 291.63±6.81 µmol TE/g and MDA IC50 of 0.289±0.022 mg/mL.

Microwave-assisted extraction of arabinan-rich pectic polysaccharides from melon peels: Optimization, purification, bioactivity, and techno-functionality.

The effects of water to solids ratio (WSR, 10-30 mL/g), power (180-540 W), and irradiation time (IT, 5-15 min) in microwave-assisted extraction (MAE) were optimized to extract polysaccharides from melon peels (PMP). The maximum extraction yield (32.81 %) was obtained under 20.94 mL/g WSR, 414.4 W power, and 12.75 min IT. The main monosaccharide composition of purified PMP with an average molecular weight of 5.71 × 104 kDa were d-galacturonic acid, arabinose, glucose, and galactose. An ascending dose-dependent antiradical and antioxidant behavior for PMP (0-5.0 mg/mL) was found. The initial foaming capacity (38.6-110.3 %) and foaming stability (5.2-65.2 %) were significantly increased as a function of PMP concentration (1.0-5.0 %), while they reduced by increasing the mixing time (p < 0.05). The highest emulsifying activity index (44.1 m2/g) and emulsifying stability (69.3 %) at 5.0 % PMPs were determined. PMP gels with FTIR-identified functional groups can be formulated in new gluten-free functional products.

Pennelliisides A-C, 2,3,4-Trisubstituted Acyl Glucoses Isolated from Solanum pennellii.

Solanum species accumulate a variety of secondary metabolites in their trichomes, and it is well known that acyl sugars are specialized metabolites secreted by the trichomes. However, very few reports provide detailed information on the chemical structure of polyacylated glucose derivatives, due to the α and ß isomerization that can occur at the C-1 position. In this study, a strategy was established to isolate polyacylated glucose derivatives. According to the developed strategy, hydroxy groups were derivatized to a benzyloxy group using TriBOT. After isolation of the compounds in pure form and deprotection of the benzyloxy group, the chemical structures of pennelliisides A-C were determined as 2,3,4-O-triisobutyryl-d-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-d-glucose, and 3-O-decanoyl-2,4-O-diisobutyryl-d-glucose, respectively. Structural elucidation was performed using spectroscopic techniques, including 1D and 2D NMR, FD-MS, and GC-MS. It was also found that the fatty acid moiety contributes to the allelopathic properties of the isolated compounds.

Optimization, characterization, rheological study and immune activities of polysaccharide from Sagittaria sagittifolia L.

To improve the extraction efficiency of polysaccharides from Sagittaria sagittifolia L. (SPU) by ultrasonic assisted extraction (UAE), the optimal extraction conditions were optimized as follows: extraction temperature of 85℃, extraction time of 15 min and ratio of liquid to raw material 43 mL/g, under these conditions, the yield of SPU increased by about 168 % compared with hot water extraction (HWE). After separation and purification by DEAE-52 cellulose column and Sephadex G-50 column, the pure polysaccharide fraction (SPU70-W1) was obtained, and its structure, rheology and immune activity were analyzed. The results indicated that SPU70-W1 (7.70 kDa) contained mannose, glucose and galactose in the molar ratio of 2.06:93.58:4.36 with typical pseudoplasticity fluids behavior and possessed the backbone of →2,6)-α-D-Glcp-(1→, →3,6)-α-D-Glcp-(1→, α-D-Glcp-(1→ and 6)-α-D-Glcp-(1→. In addition, SPU70-W1 exhibited remarkable immunomodulatory activity. Thus, SPU70-W1 could contribute to the food, medicine, cosmetics as a functional additive.

A high-molecular weight exopolysaccharide from the Cs-HK1 fungus: Ultrasonic degradation, characterization and in vitro fecal fermentation.

This work was to examine the impact of power ultrasound (US) on the molecular properties of a high-molecular weight (MW) exopolysaccharide (EPS) from the Cs-HK1 medicinal fungus and the utilization, and prebiotic function of the US-treated EPS fractions in human fecal microflora in vitro. The US treatment caused notable reduction of intrinsic viscosity, average MW and aggregate size of EPS in water but no significant changes in the molecular structure. The US-treated EPS fractions were consumed more rapidly by the fecal microflora, resulting in a higher total level of short chain fatty acids. They also affected the relative abundance in the microflora more beneficially than the original EPS. The results suggest that power US is effective for modifying and improving the prebiotic properties of high-MW polysaccharides.

Preparation of Poly(glycidyl methacrylate) (PGMA) and Amine Modified PGMA Adsorbents for Purification of Glucosinolates from Cruciferous Plants.

Glucosinolates (GLs) are of great interest for their potential as antioxidant and anticancer compounds. In this study, macroporous crosslinked copolymer adsorbents of poly (glycidyl methacrylate) (PGMA) and its amine (ethylenediamine, diethylamine, triethylamine)-modified derivatives were prepared and used to purify the GLS glucoerucin in a crude extract obtained from a cruciferous plant. These four adsorbents were evaluated by comparing their adsorption/desorption and decolorization performance for the purification of glucoerucin from crude plant extracts. According to the results, the strongly basic triethylamine modified PGMA (PGMA-III) adsorbent showed the best adsorption and desorption capacity of glucoerucin, and its adsorption data was a good fit to the Freundlich isotherm model and pseudo-second-order kinetics; the PGMA adsorbent gave the optimum decolorization performance. Furthermore, dynamic adsorption/desorption experiments were carried out to optimize the purification process. Two glass columns were serially connected and respectively wet-packed with PGMA and PGMA-III adsorbents so that glucoerucin could be decolorized and isolated from crude extracts in one process. Compared with KCl solution, aqueous ammonia was a preferable desorption solvent for the purification of glucoerucin and overcame the challenges of desalination efficiency, residual methanol and high operation costs. The results showed that after desorption with 10% aqueous ammonia, the purity of isolated glucoerucin was 74.39% with a recovery of 80.63%; after decolorization with PGMA adsorbent, the appearance of glucoerucin was improved and the purity increased by 11.30%. The process of using serially connected glass columns, wet-packed with PGMA and PGMA-III, may provide a simple, low-cost, and efficient method for the purification of GLs from cruciferous plants.

Single cell oil production by a novel yeast Trichosporon mycotoxinivorans for complete and ecofriendly valorization of paddy straw

Background: Oleaginous yeasts can be grown on different carbon sources, including lignocellulosic hydrolysate containing a mixture of glucose and xylose. However, not all yeast strains can utilize both the sugars for lipogenesis. Therefore, in this study, efforts were made to isolate dual sugar-utilizing oleaginous yeasts from different sources. Results: A total of eleven isolates were obtained, which were screened for their ability to utilize various carbohydrates for lipogenesis. One promising yeast isolate Trichosporon mycotoxinivorans S2 was selected based on its capability to use a mixture of glucose and xylose and produce 44.86 ± 4.03% lipids, as well as its tolerance to fermentation inhibitors. In order to identify an inexpensive source of sugars, nondetoxified paddy straw hydrolysate (saccharified with cellulase), supplemented with 0.05% yeast extract, 0.18% peptone, and 0.04% MgSO4 was used for growth of the yeast, resulting in a yield of 5.17 g L−1 lipids with conversion productivity of 0.06 g L−1 h−1 . Optimization of the levels of yeast extract, peptone, and MgSO4 for maximizing lipid production using Box­Behnken design led to an increase in lipid yield by 41.59%. FAME analysis of single cell oil revealed oleic acid (30.84%), palmitic acid (18.28%), and stearic acid (17.64%) as the major fatty acids. Conclusion: The fatty acid profile illustrates the potential of T. mycotoxinivorans S2 to produce single cell oil as a feedstock for biodiesel. Therefore, the present study also indicated the potential of selected yeast to develop a zero-waste process for the complete valorization of paddy straw hydrolysate without detoxification

A Low-Cost Paper Glucose Sensor with Molecularly Imprinted Polyaniline Electrode.

For the hundreds of millions of worldwide diabetic patients, glucose test strips are the most important and commonly used tool for monitoring blood glucose levels. Commercial test strips use glucose oxidases as recognition agents, which increases the cost and reduces the durability of test strips. To lower the cost of glucose sensors, we developed a paper-based electrical sensor with molecularly imprinted glucose recognition sites and demonstrated the determination of various glucose concentrations in bovine blood solutions. The sensing electrode is integrated with molecular recognition sites in the conductive polymer. A calibration graph as a function of glucose concentration in aqueous solution was acquired and matched with a correlation coefficient of 0.989. We also demonstrated the determination of the added glucose concentrations ranging from 2.2 to 11.1 mM in bovine blood samples with a linear correlation coefficient of 0.984. This non-enzymatic glucose sensor has the potential to reduce the health care cost of test strips as well as make glucose sensor test strips more accessible to underserved communities.

Chemically modified carbon nitride-chitin-acetic acid hybrid as a metal-free bifunctional nanozyme cascade of glucose oxidase-peroxidase for "click off" colorimetric detection of peroxide and glucose.

Hybrid nanomaterials-based artificial enzymes with numerous utilities are necessary to develop future bionic devices in mimicking physiological processes. This paper demonstrates bifunctional enzyme mimicking roles of a metal-free nanozyme hybrid of chemically modified graphitic carbon nitride (MGCN), chitin and acetic acid (AcOH). The MGCN exhibited glucose oxidase-mimicking activity and chitin-AcOH mirrored peroxidase. MGCN-chitin-AcOH when in contact with glucose, oxidised glucose to gluconic acid and hydrogen peroxide (H2O2) while the chitin-AcOH decomposed the generated H2O2, as proved separately, by concurrent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). The super-sensitive colorimetric process produced linear regression equation for H2O2 as A = 0.00105C + 0.0630 (C:µM, R2 = 0.9961) with a detection limit of 0.052 µM, whereas for glucose, the linear relationship was A = 0.00084C + 0.0458 (C:µM, R2 = 0.9952) having a detection limit of 0.055 µM. The developed method was also successfully applied for assessment of H2O2 and glucose in human serum and urine samples. Non-enzymatic glucose test strips from MGCN-chitin-AcOH based hydrogel were reported and verified for semi-quantitative analysis of glucose. These compared well with results from standard enzyme-based colorimetric procedure. The developed hybrid nanozyme provided feasible alternatives to the two natural enzymes (peroxidase and glucose oxidase) realized through real sample analysis. The developed hybrid nanozyme can be successfully used for colorimetric detection of peroxide and glucose in medical diagnostics.

A Preliminary Study on Metabolome Profiles of Buffalo Milk and Corresponding Mozzarella Cheese: Safeguarding the Authenticity and Traceability of Protected Status Buffalo Dairy Products.

The aim of this study is to combine advanced GC-MS and metabolite identification in a robust and repeatable technology platform to characterize the metabolome of buffalo milk and mozzarella cheese. The study utilized eleven dairies located in a protected designation of origin (PDO) region and nine dairies located in non-PDO region in Italy. Samples of raw milk (100 mL) and mozzarella cheese (100 g) were obtained from each dairy. A total of 185 metabolites were consistently detected in both milk and mozzarella cheese. The PLS-DA score plots clearly differentiated PDO and non-PDO milk and mozzarella samples. For milk samples, it was possible to divide metabolites into two classes according to region: those with lower concentrations in PDO samples (galactopyranoside, hydroxybuthyric acid, allose, citric acid) and those with lower concentrations in non-PDO samples (talopyranose, pantothenic acid, mannobiose, etc.,). The same was observed for mozzarella samples with the proportion of some metabolites (talopyranose, 2, 3-dihydroxypropyl icosanoate, etc.,) higher in PDO samples while others (tagatose, lactic acid dimer, ribitol, etc.,) higher in non-PDO samples. The findings establish the utility of GC-MS together with mass spectral libraries as a powerful technology platform to determine the authenticity, and create market protection, for "Mozzarella di Bufala Campana."

Polymerization-Induced Coassembly of Enzyme-Polymer Conjugates into Comicelles with Tunable and Enhanced Cascade Activity.

Living organisms utilize spatially organized enzyme complexes to carry out signal transduction and metabolic pathways in an efficient and specific way. Herein, inspired by natural enzyme complexes, we report the polymerization-induced coassembly (PICA) of enzyme-polymer conjugates into comicelles with tunable and enhanced cascade activity by using the cascade reaction implemented by glucose oxidase (GOX) and horseradish peroxidase (HRP) as a model system. Notably, the cascade activity of GOX/HRP-polymer comicelles monotonically increases with the GOX/HRP ratio. The cascade activity of GOX/HRP-polymer comicelles is up to 4.9 times higher than that of free GOX and HRP mixtures at the same GOX/HRP ratio. We further demonstrate that our system can quickly detect glucose in contrast with a commercially available glucose assay kit. These findings provide a new and general method of PICA for the controlled construction of artificial enzyme complexes with tunable and enhanced activity in enzyme cascades for advanced biomedical applications.

Oligosaccharide and glucose esters from the roots of Polygala arillata.

The root of plant Polygala arillata has been used in the Oriental medicine as a tonic and for the treatment of certain diseases. Our current research on phytochemical profile of the roots of P. arillata led to the isolation of a new oligosaccharide ester (1, polygaloside), a new glucose ester (7, arillatoside), along with five known sucrose esters (2-6). Their structures were elucidated on the basis of extensive chemical and spectroscopic methods as well as comparison with those reported in the literature. The occurence of various oligosaccharide esters in P. arillata including unique compounds plays taxonomical impact and suggests potential in medicinal uses of the title plant.

Mammalian cell cultivation using nutrients extracted from microalgae.

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of "cultured meat." Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes.

Preparation of co-Co3 O4 /carbon nanotube/carbon foam for glucose sensor.

Co-Co3 O4 /carbon nanotube/carbon foam (Co-Co3 O4 /CNT/CF) nanocomposites were prepared by soaking melamine foam into a solution of Co(NO3 )2 ·6H2 O, followed by calcination in N2 and air in sequence. The obtained Co-Co3 O4 /CNT/CF nanocomposites were characterized with scanning electron microscopy and cyclic voltammetry. It was found that Co3 O4 nanoparticles were grown on the external of CF successfully, while CNTs were grown on the surfaces of CF in a large amount, which further improved the electrical conductivity of the. The prepared Co-Co3 O4 /CNT/CF nanocomposites were then used to construct nonenzymatic sensor to detect glucose in alkaline solution. The sensor showed detection range from 1.2 µM to 2.29 mM with a detection limit of 0.4 µM (S/N =3) and a high sensitivity of 637.5 µA-1 cm-2 . The developed sensor also showed an instant response, favorable reproducibility, and high selectivity. The results attest that Co-Co3 O4 /CNT/CF composites have great potential in the development of nonenzymatic sensors for glucose.

Isolations, characterizations and bioactivities of polysaccharides from the seeds of three species Glycyrrhiza.

In this paper, polysaccharides from the seeds of three species of genus Glycyrrhiza were extracted to investigate the physicochemical properties, structural characteristics and antioxidant activities. The polysaccharides were composed of xylose, mannose, galactose, and glucose with different molar ratio. Fourier transform infrared spectroscopy showed the presence of key functional groups of polysaccharides whereas scanning electron microscopy analysis revealed the characteristic morphology of different polysaccharides, and thermogravimetric analysis exhibited good thermal stability of all samples. The antioxidant activities of polysaccharides were evaluated in vitro. All the three polysaccharides demonstrated strong reducing power, as well as scavenging activity against DPPH, ABTS, and hydroxyl free radicals. Antioxidant assays indicated that all the polysaccharides have obvious antioxidant activities and possess a potential development and application value in food, cosmetics as well as pharmaceutical industries.

From in vitro to in silico: Modeling and recombinant production of DT-Diaphorase enzyme.

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni-NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done.

Mini-pillar microarray for individually electrochemical sensing in microdroplets.

High throughput and high sensitivity are two important aspects in multiple biomarker recognition, drug discovery and relevant biochemical sensing. Here, we integrate mini-pillar microarray with the circuit components toward high-throughput individual electrochemical sensing in microdroplets. On such droplet-microarray-based electrochemical platform, the high adhesion of the mini-pillar can hold a microdroplet (hundreds nanoliter to a few microliter) regardless of rotation and deformation. Each pillar as a unit has a three-electrode to achieve individual electrochemical sensing, and electrodes are integrated on one side to achieve the sequential electrochemical read-out. Qualitative and quantitative electrochemical assessments of multiple glucose concentrations in individual microdroplets are also achieved. Such mini-pillar-based individual electrochemical platform shows great potential in high-throughput and high-sensitive biomolecular recognitions, provides an opportunity to develop miniaturized sensing platform for emerging biological and pathological applications.

Bio-/Nanoimmobilization Platform Based on Bioinspired Fibrin-Bone@Polydopamine-Shell Adhesive Composites for Biosensing.

Inspired by blood coagulation and mussel adhesion, we report novel adhesive fibrin-bone@polydopamine (PDA)-shell composite matrix as highly efficient immobilization platform for biomacromolecules and nanomaterials. Fibrin, as a bioglue, and PDA, as a chemical adhesive, are integrated in a one-pot simultaneous polymerization consisting of biopolymerization of fibrinogen and chemical polymerization of dopamine. Fibrin fibers act as adhesive bones to construct scaffold, while PDA coat on the scaffold to form adhesive shell, generating 3D porous composite matrix with unique bone@shell structure. Two types of enzymes (glucose oxidase and acetylcholinesterase) and Au nanoparticles were adopted as respective model biomolecules and nanomaterials to investigate the immobilization capability of the matrix. The bionanocomposites showed high efficiency in capturing nanoparticles and enzymes, as well as significant mass-transfer and biocatalysis efficiencies. Therefore, the bionanocomposites exhibited significant potential in biosensing of glucose and paraoxon with limits of detection down to 5.2 µM and 4 ppt, respectively. The biological-chemical-combined polymerization strategy and composite platform with high immobilization capacity and mass-transfer efficiency open up a novel way for the preparation of high-performance bionanocomposites for various applications, in particular, biosensing.